Methods and compositions for treatment of branched-chain KETO acid (BCKA) accumulation in cancers

Abstract

Disclosed are methods and compositions for determining and treating a cancer and / or cancer-associated complications including, but not limited to Leptomeningeal Disease (LMD), neurodegeneration, and CAR-T cytotoxicity.

Description

[0001] Attorney Docket No. 10110-466WO1

[0002] METHODS AND COMPOSITIONS FOR TREATMENT OF BRANCHED-CHAIN KETO ACID (BCKA) ACCUMULATION IN CANCERS

[0003] I. CROSS REFERENCE TO RELATED APPLICATIONS

[0004] This Application claims the benefit of U.S. Provisional Application No. 63 / 754,731, filed on February 6, 2025, which is incorporated herein by reference in its entirety.

[0005] II. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0006] This invention was made with government support under grant no. CA274060 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0007] III. FIELDS

[0008] The present disclosure relates to methods and compositions for determining and treating branched-chain keto acid (BCKA) accumulation in the brain.

[0009] IV. BACKGROUND

[0010] Leptomeningeal disease (LMD) is a rapidly progressing and dreaded complication of cancer that is clinically detected in 5-15% of patients with late-stage solid tumors and is apparent at autopsy in up to 20-30% of cancer patients with metastatic disease and neurologic symptoms. LMD occurs when malignant cells seed to the leptomeninges (membrane coverings of the brain and spinal cord) and cerebral spinal fluid (CSF) compartments. Rapid and debilitating neurological symptoms drastically impact LMD patients' survival outcomes. The median survival time for treated LMD patients is 2-6 months, with death typically occurring due to progressive neurologic dysfunction. For the majority of the patients, the condition is rapidly terminal, and the primary goal of treatment is to improve patients' neurological function and quality of life through palliative and supportive care. Current LMD treatments include combination radiotherapy regimens with intrathecal chemotherapy (ex: cytarabine, methotrexate, thiotepa, etc) or intrathecal targeted / immune therapy. However, studies have shown variable / limited responses to treatment, which are frequently further complicated by the toxicity of the regimens. Systemic therapy can be added to control extracranial disease and potentially prolong patientAttorney Docket No. 10110-466WO1

[0011] survival. Non-Hodgkin B cell lymphomas (NHBCL) comprise 90% of all malignant lymphoma cases. For these patients, LMD may manifest either as metastatic dissemination of extracranial disease (occurring in 5-10% of NHBCL patients) or as the primary disease site, as in the case of 7-40% of primary central nervous system lymphomas. NHBCL, typically a systemic disease, is often well -controlled with combination chemoimmunotherapy. CNS recurrence, although uncommon, happens rapidly and frequently as the sole site of relapse when it occurs. Multiple studies have now demonstrated that CD19- targeting CAR T-cell therapies are effective at treating chemo-refractory NHBCL; however, the anti-tumor effects on responding LMD tumors are rarely long-lasting, despite clear evidence of CAR T-cell penetration into the CSF space.

[0012] What is needed are new therapeutics that effectively treat LMD, and other cancer-associated complications.

[0013] V. SUMMARY

[0014] Disclosed are methods relating to the treatment of Leptomeningeal disease (LMD), leptomeningeal lymphoma (LML), cancers, metastasis, and cancer-associated complication by administering a therapeutically effective amount of sodium phenylbutyrate alone or in conjunction with other anti-cancer therapies.

[0015] In one aspect disclosed herein are methods of treating, inhibiting, reducing, decreasing, ameliorating, and / or preventing Leptomeningeal disease (LMD), leptomeningeal lymphoma (LML) or a cancer (such as, for example, breast cancer or leptomeningeal metastatic disease), metastasis, and / or cancer associated complications, including but not limited to Leptomeningeal disease (LMD), CAR-T cytotoxicity, branched-chain keto acids (BCKA) accumulation the cerebrospinal fluid, and / or neurodegeneration comprising administering to a subject a therapeutically effective amount of a composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier. In some embodiments, the cancer-associated complication is Leptomeningeal Disease (LMD). In some embodiments, the composition is administered to the subject, intrathecally, orally, or systemically.

[0016] Also disclosed herein are methods of treating, inhibiting, reducing, decreasing, ameliorating, and / or preventing Leptomeningeal disease (LMD), leptomeningeal lymphoma (LML) or a cancer of any preceding aspect further comprises administering to the subject at least one anti-cancer agent such as, for example cytarabine, methotrexate, rituximab, and thiotepa.Attorney Docket No. 10110-466WO1

[0017] In some embodiments, LMD is associated with branched-chain keto acids (BCKA) accumulation in a sample of cerebrospinal fluid obtained from the subject, CAR-T cytotoxicity, and / or neurodegeneration.

[0018] Also disclosed herein are methods of treating, inhibiting, reducing, decreasing, ameliorating, and / or preventing Leptomeningeal disease (LMD), leptomeningeal lymphoma (LML) or a cancer wherein the administration of the composition comprising sodium phenylbutyrate and / or further administration of an anti-cancer agent reduces BCKA accumulation in a sample of cerebrospinal fluidCAR-T cytotoxicity, and / or neurodegeneration in the subject compared to an untreated control.

[0019] In some embodiments, the subject is a human.

[0020] In one aspect, disclosed herein, is a method of treating Leptomeningeal lymphoma (LML) in a subject comprising administering to a subject a composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier.

[0021] In one aspect, disclosed herein, are methods of enhancing T cell activity in a subject with Leptomeningeal Disease (LMD), comprising: determining a subject having an elevated concentration of Branched-Chain Keto Acids (BCKA) in the cerebrospinal fluid (CSF); and administering a therapeutic agent to reduce BCKA accumulation or inhibit BCKA-mediated signaling in the CSF, thereby reversing T cell exhaustion or inactivity.

[0022] Also disclosed herein are methods of enhancing T cell activity of any preceding aspect, wherein the therapeutic agent is a buffering agent to increase the pH of the CSF, a metabolic inhibitor that reduces the production of BCKAs, a BCAT1 inhibitor, or an agent to target the MAPK pathway, thereby restoring phosphorylation of p38α (MAPK14) or 4E-BP1 (EIF4EBP1) in T cells.

[0023] In one aspect, disclosed herein, are methods of treating, inhibiting, reducing, decreasing, ameliorating, and / or preventing leptomeningeal metastatic disease in a subject, comprising: administering to the subject a therapeutically effective amount of an agent that reduces BCKA accumulation in a cerebrospinal fluid compartment of the subject (such as, for example, phenylbutyrate and a pharmaceutically acceptable salt thereof). In some embodiments, treating comprises improving a neurological performance metric, prolonging survival, reducing leptomeningeal tumor burden, or a combination thereof. In some embodiments, administering the agent to reduce the BCKA accumulation enhances an immune favorable microenvironment (including, but not limited to increased T cell infiltration, increased T cell activation, decreased T cell exhaustion, or a combination thereof) in leptomeningeal metastatic disease relative to administration of the intrathecalAttorney Docket No. 10110-466WO1

[0024] dendritic cell therapy without the agent. In some embodiments, the agent to reduce the BCKA accumulation is administered intrathecally, orally, systemically, or by a combination thereof.

[0025] Also disclosed herein are methods of treating, inhibiting, reducing, decreasing, ameliorating, and / or preventing leptomeningeal metastatic disease of any preceding aspect further comprising administering to the subject a dendritic cell therapy to the subject (including, but not limited to intrathecal administration). In some embodiments, the dendritic cell therapy comprises administering dendritic cells loaded with MHC class II peptides comprising a HER2 peptide, a HER3 peptide, or both.

[0026] In some embodiments, the subject has breast cancer and / or leptomeningeal metastatic disease.

[0027] In one aspect, disclosed herein, is a method of treating, decreasing, reducing, inhibiting, ameliorating, and / or preventing breast cancer leptomeningeal metastatic disease (including, but not limited to HER2 positive breast cancer leptomeningeal metastatic disease and / or triple negative breast cancer leptomeningeal metastatic disease) in a subject, comprising: administering an agent to reduce branched chain keto acid accumulation in cerebrospinal fluid (such as, for example, phenylbutyrate and a pharmaceutically acceptable salt thereof), and a dendritic cell therapy (such as, for example, a peptide pulsed conventional dendritic cells).

[0028] In some embodiments, the peptide pulsed conventional dendritic cells comprise one or more MHC class II peptides that target HER2, HER3, or a combination thereof.

[0029] Also disclosed herein are methods of treating, decreasing, reducing, inhibiting, ameliorating, and / or preventing breast cancer leptomeningeal metastatic disease of any preceding aspect, wherein the method further enhances T cell infiltration into leptomeningeal tumor tissue, increases T cell activation, reduces T cell exhaustion, or a combination thereof. In some embodiments, the method further increases a CD4 Thl response, increases a B cell response, or both, in the cerebrospinal fluid compartment.

[0030] In some embodiments, the agent to reduce branched chain keto acid accumulation is administered prior to the dendritic cell therapy, concurrently with the dendritic cell therapy, or after the dendritic cell therapy. In some embodiments, administering the dendritic cell therapy comprises administering a plurality of doses.

[0031] VI. BRIEF DESCRIPTION OF DRAWINGSAttorney Docket No. 10110-466WO1

[0032] FIG. 1 is an illustration showing the seeding of lymphoma cells to the cerebrospinal fluid and the leptomeningeal membrane leading to Leptomeningeal Lymphoma (LML).

[0033] FIG. 2 shows poor survival and lack of effective therapies in LML with rapid neurological deterioration.

[0034] FIG. 3 shows an illustrated overview of how researchers investigated the tumor microenvironment of LML in human cerebrospinal fluid (top), an in-vivo mouse model (middle), and an in-vitro model (bottom).

[0035] FIGS. 4A and 4B display the cellular components of LML patient CSF in a t-distributed stochastic neighbor embedding (t-SNE) plot of single cell RNA-seq (scRNA-seq) results as shown in FIG. 4A. And a breakdown of the amount of different immune cells presents in LML patient CSF and non-LML CSF as shown in FIG. 4B.

[0036] FIGS. 5A, 5B, 5C show a quantification of the different types and states of T cells present in CSF from patients with LML that respond to CAR-T cell therapy and CSF of patients with LML that do not respond to CAR-T cell therapy as shown in FIG. 5 A. FIG.

[0037] 5B shows the characterization of tumor and CSF environment. FIG. 5B also shows levels of macrophages, T-NK cells and dendritic cells in liver tumors and in LML. FIG. 5C shows the composition of immune cells in CSF of patients with LML responding to CAR-T cell therapy, patients with LML that do not respond to CAR-T cell therapy, and non-LML patients (left). Also shown is the number of macrophages present in CSF of patients with LML that does or does not respond to CAR-T cell therapy (middle). And the number of macrophages present in the leptomeninges and liver tissue of LML-model mice (right).

[0038] FIGS. 6A and 6B show the percentage of total cells that are macrophages in the CSF of patients with and without LMD as shown in FIG. 6A. FIG. 6B shows the total percentage of cells that are macrophages from a breast cancer and melanoma-mouse model in different tissues.

[0039] FIG. 7 A and 7B show the number of interactions for each cell type in LML that does or does not respond to CAR-T cell therapy as shown in FIG. 7A. Additionally, FIG. 7B shows interactions of macrophages in LML patient CSF that do and do not respond to CAR- T cell therapy are shown.

[0040] FIG. 8 shows Multi-omic analysis of CSF.

[0041] FIGS. 9 A, 9B, and 9C show that Lipidomic / Proteomic analysis revealed neuronal degeneration in LML patients. FIG. 9A shows a volcano plot and the increase of lysophosphatidylcholine. FIG. 9B shows pathways analyses revealed the upregulation ofAttorney Docket No. 10110-466WO1

[0042] neurodegenerative signaling pathways. FIG. 9C shows the upregulation of neurodegenerative markers.

[0043] FIGS. 10A, 10B, and 10C show that metabolomic analysis of CSF identifies the dysregulation of branched-chain amino acid metabolism. FIGS. 10A and 10B show the concentrations of specific branched-chain a-keto acids (BCKA) in CSF from patients with and without LMD. FIG. 10C shows the concentrations of specific BCKAs from breast cancer LMD model mice, melanoma LMD model mice and control mice.

[0044] FIGS. 11A and 11B are schematics showing the hypothesis that the accumulation of BCKAs in the LML CSF are related to immune suppression & neuronal degeneration FIGS. 12A, 12B, 12C, 12D, and 12E show the results of neurological exams in LMD model-mice and control mice. FIG. 12A shows images of tail suspension of control and LMD mice. As shown in FIG. 12B, in the tail suspension test and as shown in FIG. 2D in the grip test LMD model-mice received significantly worse scores than control mice. FIG.

[0045] 12C shows images of control and LMD mice subjected to the grip test. As shown in FIG.

[0046] 12E, in the walking test there is no significant difference between control and LMD model¬ mice.

[0047] FIG. 13 shows markers of neurodegeneration and disassembly of the leptomeningeal membrane in micrographs comparing control and LML model brains stained with DAPI and MAP2, a marker of stable neuronal morphology.

[0048] FIG. 14 shows charts quantifying the percentage of fibroblasts from healthy pia and pia from LML mice.

[0049] FIGS. 15A and 15B show that BCKAs inhibit the metabolic activity of neurons and meningeal cells.

[0050] FIGS. 16A and 16B show the unique LMD microenvironment wherein tumor cells spread to the CSF and the meninges. FIG. 16A shows the parenchyma of the brain. FIG.

[0051] 16B shows spinal cord and cranial or peripheral nerves invaded by tumor cells.

[0052] FIG. 17 shows a schematic illustration of characterization of tumor and CSF environment.

[0053] FIGS. 18A and 18B show the result of proteomic analysis of LML patient CSF. FIG.

[0054] 18A shows the proteins of which expression is up- or down-regulated in LML CSF. FIG.

[0055] 18B shows a comparison of gene expression in LMD and non-LMD patients highlighting genes commonly impacted in neurodegeneration is shown as a heatmap.Attorney Docket No. 10110-466WO1

[0056] FIGS. 19A and 19B show lipidomic analysis of the CSF of patients with LML. FIG.

[0057] 19 A shows a volcano plot with data points that represent lipids that were significantly up-or down- regulated. FIG. 19B shows the accumulation of BCKA in mouse and human LMD.

[0058] FIG. 20 shows that Accumulation of BCKA in CNS is analogous to maple syrup urine disease

[0059] FIGS. 21A, 21B, 21C, 21D, 21E, 21F, 21G, and 21H show the impact of a mixture of BCKAs and a specific BCKA on different cells, as determined by cell markers, and over all cell viability. FIGS. 21A, 21B, 21C, and 2 ID (two samples) show the impact of different concentrations of BCKAs on amount of CD4, CD8 and total cells in CSF. FIG. 21 E shows the impact of increasing concentrations of BCKA on the overall concentration of immune signaling molecules - Granzyme B, Interferon-y (INFy), TNFa and IL-2. FIGS. 21F and 21G show the impact of increasing concentrations of a-ketoisocaprioc acid (KIC) and a BCKA mixture on cell viability. FIG. 21 H shows the average percentage of Granzyme B, INFy, and TNFa in CSF that has and has not been treated with a BCKA mixture. FIG. 21H further shows differences in percentage of average control of cytokines and signaling molecules present in CSF that is and is not treated with 50 pM BKAs.

[0060] FIG. 22 shows a difference in CSF when treated with BKAs. FIG. 22 also shows a difference in units of relative fluorescence when CSF is treated with varying amounts of BKAs.

[0061] FIG. 23 shows markers of neurodegeneration in healthy (left) and LMD (right) mouse brains. Micrographs are stained with DAPI and GFAP, a marker of neurodegeneration.

[0062] FIG. 24 A and 24B show the impact of BCKAs on neuronal as in FIG. 24A and meningeal as in FIG. 24B metabolic activity as determined by MTT assay.

[0063] FIG. 25 shows a schematic that Sodium Phenylbutyrate (PBA) can be used as a BCKA-lowering therapy in treatment of LMD.

[0064] FIGS. 26A, 26B, 26C, 26D, 26E, 26F, and 26G show the ability of sodium phenylbutyrate (PBA) to act as a therapeutic for the neurological symptoms of LMD. FIG.

[0065] 26A shows an illustration model of the treatments the mice received and how their efficacies were assessed. FIG. 26B shows the NeuroScore assessment scores at end point in the case of each therapeutic are displayed. Days in which no progression was observed for each NeuroScore criteria and treatment group are shown (bottom). Gray is no LMD control, blue is LMD no treatment, red is LMD with CAR-T cell therapy, purple is LMD with PBA treatment, and green is LMD with CAR-T cell therapy and PBA co-treatment. FIG. 26CAttorney Docket No. 10110-466WO1

[0066] shows that PBA as a mono- or co-therapy with CAR-T treatment extends the survival of LMD mice. No treatment, CAR-T cell therapy only, CAR-T cell therapy and PBA, PBA only, T cell therapy only are treatment groups shown. FIGS. 26D and 26E show that PBA improves survival and blocks neurological decline in lymphoma LMD. FIGS. 26F and 26G show that PBA improves CAR T efficacy in LMD

[0067] FIGS. 27A and 27B show that sodium phenylbutyrate (PBA) blocks neurodegeneration and improves quality of mice with LMD. FIG. 27 A shows micrographs of brains obtained from control, LMD, or LMD treated with PBA stained with DAPI and MAP2. FIG. 27B shows LMD mice treated with PBA have a higher endpoint weight than LMD mice with no treatment.

[0068] FIG. 28 shows mouse models of LMD and their probability of survival which having LML, Melanoma or Breast Cancer.

[0069] FIG. 29 shows that a sodium phenylbutyrate (PBA) and methotrexate (MTX) cotherapy improves survival in breast cancer LMD mice. Treatment groups include methotrexate treatment only, sodium phenylbutyrate and methotrexate co-therapy.

[0070] FIGS. 30A and 30B show the application of PBA to LMD treatment as monotherapy and with standard of care combination wherein the application of 3-methyl / 4-Methyl-2-Oxovaleric Acid as in FIG. 30A or 3-Methyl-2-oxobutanoic acid as in FIG. 30B blocks / improves CAR-T cytotoxicity for lymphoma patients.

[0071] FIGS. 31A-31D show the following: FIG. 31 A shows scRNAseq on patient CSF showing a decrease in early activation and an increase of T cells approaching exhaustion in LMD. FIG. 31B shows a mouse model of melanoma LMD confirming T cell exhaustion. FIG. 31C shows T cell exhaustion marker expression in mouse models of LMD compared to other sites of tumor in the same mice. FIG. 3 ID shows the proportion of active, proliferating T cells in patients with short (<5 mos) compared to long (>10 mos) survival.

[0072] FIGS. 32A-32B show the following: FIG. 32 A shows the relative abundance of KIC and KMV in patient CSF measured by mass spectrometry. FIG. 32B shows that KIC / KMV exhibited the greatest fold change in LMD compared to control CSF.

[0073] FIG. 33 shows BCAA / BCKA metabolism and opportunities for intervention. FIG. 34. shows Quantification of three BCKA (KIV, KIC and KMV) in patient CSF measured by mass spectrometry. KIC and KMV cannot be resolved spectrally and are therefore combined.

[0074] FIG. 35 shows correlation analysis of exhausted T cell clusters to BCKA concentration found in CSF of LMD patients.Attorney Docket No. 10110-466WO1

[0075] FIGS. 36A-36D show the following: FIG. 36A shows an ELISA assay of granzyme B and interferon-α in human T cells in physiological CSF with BCKA exposure. FIG. 36B shows a viability assay of human T cells in physiological CSF with BCKA exposure. FIG.

[0076] 36C shows a viability assay of tumor cells in physiological CSF with BCKA exposure. FIG.

[0077] 36D shows growth of a primary melanoma tumor in physiological CSF with BCKA exposure.

[0078] FIGS. 37A-37E show the following: FIG. 37 A shows Kaplan-Meier analysis in PBS injected mice versus tumor injected LMD mice. FIG. 37B shows mass spectrometry quantification of KIC in pia membranes of mice with A20 lymphoma LMD treated with PBA. FIG. 37C shows neurological assessments of mice from FIG. 37B. FIG. 37D shows Kaplan-Meier analysis in A20 lymphoma LMD mice with PBA treatment. FIG. 37E shows Kaplan-Meier analysis in A20 lymphoma LMD mice with CAR T and PBA treatment.

[0079] FIGS. 38A-38D show the following: FIG. 38A shows Pearson correlation analysis of KIC concentration in the pia versus survival time of mice. FIG. 38B shows scRNAseq showing expression of BCKA transporter and metabolic enzymes in different cell types from patient LMD tumors. FIG. 38C shows Compass2 metabolic analysis of previously published scRNAseq datal showing an enrichment of branched-chain amino acid (BCAA) metabolism and the TCA cycle in LMD tumors compared to extra-cranial disease. FIG. 38D shows relative expression of MAPK mediators in different clusters of tumor cells in scRNAseq of murine LMD and extra-cranial tumors.

[0080] FIGS. 39A-39D show the following: FIG. 39A shows IVIS bioluminescence imaging of luciferase-expressing SMI cells during formation of LMD in C57BL / 6 mice, with most mice succumbing to disease by 4 weeks. FIG. 39B shows Kaplan-Meier curves showing survival of three melanoma LMD models. FIG. 39C shows NeuroScoring showing neurological decline in 3 melanoma LMD models (higher score = worse performance). FIG.

[0081] 39D shows MRI of SMI LMD models in C57BL / 6 mice, in which mice exhibit ataxia characteristics and develop enlarged ventricles and hydrocephaly (arrows).

[0082] FIG. 40 shows an example of measuring acetyl CoA labelling in metabolic tracing experiment, in this case tracing labelled vitamin B5 and Cysteine.

[0083] FIG. 41 shows H& E staining and cell typing analysis from spatial RNAseq using 10X Visium platform (50 pm resolution) of fresh frozen tissue from melanoma LMD patient.

[0084] FIG. 42 shows iSTAT assay of pH level in patient CSF.Attorney Docket No. 10110-466WO1

[0085] FIGS. 43A-43B show the following: FIG. 43 A shows scRNAseq showing MAPK expression in T / NK cell clusters of human melanoma metastases to the LMD (14 samples) versus other sites of disease (21 samples). FIG. 43B shows a western blot showing pERK expression in T cells exposed to BCKA.

[0086] FIG. 44 shows compass analysis of previously published scRNAseq data shows a reduction in TCA cycle, glycolysis, fatty acid oxidation and ROS detoxification in T cells at LMD sites.

[0087] FIGS. 45A-45J show the immune landscape of leptomeningeal disease lacks active, proliferating T cells. FIG. 45A shows a schematic illustration demonstrates the CSF collection methods and the number of CSF samples collected from LMD and non-LMD control patients. The CSF-cellular compartment was used for scRNA-Seq, while the CSF-fluid compartment was used for multiomic analyses. Created in BioRender. Smalley, I. (2025). FIG. 45B shows a T-distributed stochastic neighbor embedding (t-SNE) plot of all cell populations in all CSF specimens. FIG. 45C shows a T-SNE plot showing clustering based on disease origin: breast cancer LMD (BC-LMD), lymphoma LMD (L-LMD), melanoma LMD (M-LMD), and No LMD controls. FIG. 45D shows a bar graph demonstrating the proportion of each functional group of T cells to the total T / NK cell compartment in LMD vs no-LMD. Colored boxes indicate the predicted activity (based on Supplemental Figure 1C). FIG. 45E shows pie charts show the distribution of each T / NK cluster in breast cancer LMD (BC-LMD), lymphoma LMD (L-LMD), melanoma LMD (M-LMD), and No LMD controls. The colored halo indicates the predicted functional / activation state of each T / NK cell based on gene expression profiles. FIG. 45F shows a schematic illustrating animal experiment to compare the composition of the pial membrane following PBS control or SMI tumor cell injection into CSF space via cisterna magna. Created in BioRender. Smalley, I. (2025). FIG. 45G shows a bar graph showing the average proportion of each T cell cluster in the pial membranes harvested from the animal experiment in FIG.

[0088] 45F, as determined using scRNAseq (n=3 for each cohort). FIG. 45H shows a schematic illustrating animal experiment to compare the composition of LMD tumors versus extracranial tumors after SMI melanoma, 4T1 breast cancer, and A20 lymphoma tumor cell injection into CSF space via cisterna magna or via tail vein (allowing lung / liver colonization). Created in BioRender. Smalley, I. (2025). FIG. 45I shows a bar graph showing the average proportion of CD8 T cells in LMD tumors versus extra-cranial tumors from animal experiment in FIG. 45H, as determined using scRNAseq (n=3 for each cohort).Attorney Docket No. 10110-466WO1

[0089] FIG. 45J shows bar graphs show the proportion of active proliferating T cells in patients with long (> 10 months) vs. short (< 5 months) survival.

[0090] FIGS. 46A-46I show accumulation of neurotoxic branched-chain keto acids in the LMD microenvironment. FIG. 46A shows a schematic illustration outlining the multi-omic analysis of patient CSF and the number of identified lipids, metabolites, and proteins in the CSF, in which percentages indicate the proportion of lipids, metabolites, and proteins in differential abundance between lymphoma LMD and non-LMD controls. Created in BioRender. Smalley, I. (2025). FIG. 46B shows pathway enrichment analysis using Ingenuity Pathway Analysis of differentially expressed proteins between control and LMD CSF. FIG. 46C shows a heatmap showing relative expression of neurodegenerative disease- associated proteins in lymphoma-LMD and non-LMD, in which Welch's t-test was used for statistical analysis. FIG. 46D shows volcano plots showing differentially abundant metabolites in the CSF of lymphoma LMD patients and no-LMD controls, in which P-keto-isocaproic / p-keto-a methyl valeric (KIC / KMV) isomers show the highest accumulation in LMD. FIG. 46E shows the absolute concentration of individual (KIV, KIC / KMV) and total branched-chain keto acids (BCKA) in CSF from lymphoma-LMD patients (n=18) compared to no-LMD (n=10) control CSF. FIG. 46F shows the absolute concentration of total branched-chain keto acids (BCKA) in CSF of patients with breast cancer LMD (n=12) and melanoma LMD (n=12) compared to no-LMD control CSF (n=10). FIG. 46G shows an MTT assay measuring metabolic activity of primary mouse neurons in response to BCKA exposure in neuronal culture media for seven days. FIG. 46H shows an MTT assay measuring metabolic activity of primary mouse neurons in response to BCKA exposure in physiological CSF for seven days. FIG. 461 shows correlation analysis of KIC levels in patient CSF to each patient’s KPS (Karnofsky Performance Status) score.

[0091] FIGS. 47A-47K show blocking BCKA accumulation and neurological progression in LMD. FIG. 47 A shows neurological assessment scores at the experimental endpoint for control and LMD mice following cisterna magna injection of breast cancer (EO771), melanoma (SMI, D4M, or YUMM3.2), lymphoma (A20), or PBS control. FIG. 47B shows Kaplan-Meier analysis for probability of survival of control and LMD mice following cisterna magna injection of breast cancer (EO771), melanoma (SMI, D4M, or YUMM3.2), lymphoma (A20), or PBS control. FIG. 47C shows immunofluorescent images of microtubule-associated protein 2 (MAP2, red) and DAPI (blue) in the brain hippocampus of the lymphoma LMD model, in which the top images show low magnification (the scale bar is 400 pm), and the bottom images show high magnification of outlined areas. FIG. 47DAttorney Docket No. 10110-466WO1

[0092] shows a violin plot showing MAP2 intensity quantification in control (n = 4) vs LMD (n = 5) mouse models. FIG. 47E shows a bar graph showing quantification of KIC levels in pia tissue of control (n = 14) and LMD mice (n = 20). FIG. 47F shows a schematic illustration for an in vivo experimental strategy for injecting A20 or PBS via cisterna magna and subcutaneously in the same animals, then treating the animals with 200 mg / kg sodium phenylbutyrate (PBA) or vehicle (PBS) daily. Created in BioRender. Smalley, I. (2025). FIG. 47G shows Kaplan-Meier graphs showing overall survival of mice with lymphoma LMD treated with vehicle control or PBA. FIG. 47H shows Kaplan-Meier graphs showing progression-free survival for mice with lymphoma LMD treated with vehicle control or PBA, in which progression was diagnosed in mice who scored a two or higher in any neurological assessment or experienced paralysis, > 10% weight loss, or death. FIG. 47I shows quantification of KIC levels in pia of LMD mice treated with vehicle control or PBA. FIG. 47J shows correlation analysis of KIC levels in pia with survival of phenylbutyrate-treated LMD mice. FIG. 47K shows dot plots showing scores for each neurological assessment at the endpoint or last assessment before death, in which data represent mean + SEM (FIGS. 47A and 47H), statistical significance was assessed using Student's t-test, statistical tests were two-sided, and ****P < 0.0001, and ns = not significant.

[0093] FIGS. 48A-48K show blocking BCKA-mediated T cell dysfunction. FIG. 48A shows correlation analysis for different T / NK clusters to the BCKA concentration found in CSF of LMD patients. FIG. 48B shows representative proliferation plots of cell-trace biolet labeled T cells after stimulation in CSF with exposure to different concentrations of BCKA or PBA control. FIG. 48C shows ELLA assays measuring the abundance of pro-inflammatory cytokines secreted from T-cells cultured in physiological CSF. FIG. 48D shows bar graph showing the viability of human T-cells measured by Calcein AM staining in response to different concentrations of KIC or the three branched-chain keto acids (BCKA, at physiological ratio) in physiological CSF. FIG. 48E shows the viability of human anti-CD19 CAR T-cells was measured by Calcein AM staining in response to different concentrations of branched-chain keto acids (BCKA) in physiological CSF for 72 hours. FIG. 48F shows ELLA assays measuring the abundance of pro-inflammatory cytokines secreted from human anti-CD19 CAR T cells cultured in physiological CSF. FIG. 48G shows schematic illustration for the in vivo experimental strategy for injecting A20 via cisterna magna and subcutaneously and treating with CART or CART + sodium phenylbutyrate (PBA). Created in BioRender. Smalley, I. (2025). FIG. 48H shows Kaplan-Meier graph showing the overall survival for CART-treated mice versus CART + PBAAttorney Docket No. 10110-466WO1

[0094] treated. FIG. 48I shows Kaplan-Meier graph showing progression-free survival for CART-treated mice versus CART + PBA-treated mice. Progression was diagnosed in mice who scored a two or higher in any neurological assessment or experienced paralysis, > 10% weight loss, or death. FIG. 48J shows quantification of MAP2 immunofluorescence intensity in the hippocampus of LMD mice with CART vs. CART + PBA treatment. FIG.

[0095] 48K shows correlation analysis of MAP-2 intensity in the hippocampus of LMD mice and survival time. Data includes mice treated with CART, CART + PBA, PBA only, and untreated controls. Error bars represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant.

[0096] FIG. 49 shows a graphical summary in which a multi-omic atlas of human and murine leptomeningeal disease (LMD) characterizes LMD by immune suppression and neurodegenerative processes, leading to rapid tumor progression and short survival, and further indicates that accumulation of toxic branched-chain keto acids (BCKA) directly inhibits neuron and T cell viability and function, and that BCKA-lowering therapy can block progression of neurological symptoms of LMD and facilitate efficacy of anti -tumor CAR T therapies, leading to improved survival. Created in BioRender. Smalley, I. (2025).

[0097] FIGS. 50A-50J show the following: FIG. 50A shows bar graphs showing the proportion of each cell type found in each control and LMD sample. FIG. 50B shows bar graphs showing the average number of each cell type found in 7mL of each control and LMD CSF sample. FIG. 50C shows bar graphs showing the average number and proportion of each non-tumor cell type found in 7mL of control or LMD CSF sample. FIG. 50D shows bar graphs showing the proportion of each T / NK cell cluster found in each control and LMD sample. FIG. 50E shows bar graphs showing the average number of each T / NK cell cluster found in 7mL of each control and LMD CSF sample. FIG. 50F shows bar graphs showing the average number and proportion of each T / NK cell cluster found in 7mL of control or LMD CSF sample. FIG. 50G shows bar graphs showing the proportion of each myeloid cell cluster found in each control and LMD sample. FIG. 50H shows bar graphs showing the average number of each myeloid cell cluster found in 7mL of each control and LMD CSF sample. FIG. 50I shows bar graphs showing the average number and proportion of each myeloid cell cluster found in 7mL of control or LMD CSF sample. FIG. 50J shows bar graphs showing the average number and proportion of each non-tumor cell type found in 7mL of control or LMD CSF sample separated by tumor type.

[0098] FIGS. 51A-51G show the following: FIG. 51A shows a heatmap showing the top markers associated with each myeloid cell cluster. FIG. 51B shows a heatmap showing theAttorney Docket No. 10110-466WO1

[0099] top markers associated with each macrophage cell cluster. FIG. 51C shows a Umap showing expression of MRC1, CD163, and CSF1R for three subclusters of macrophages identified. FIG. 51 D shows bar graphs showing the average number and proportion of each myloid cell type found in 7mL of control or LMD CSF sample separated by tumor type. FIG. 51E shows bar graphs showing the average number and proportion of each macrophage cluster found in 7mL of control or LMD CSF sample separated by tumor type. FIG. 5 IF shows bar graphs showing the average number and proportion of each T / NK cell cluster found in 7mL of control or LMD CSF sample, by tumor type. FIG. 51G shows a heat map showing expression of key T / NK cell markers used to assign functional / activation groups to each of the 12 T / NK cell clusters.

[0100] FIGS. 52A-52F show the following: FIG. 52A shows bar graphs showing the average proportion of each myloid cell type found in the leptomeninges of control (n=3) and SMI LMD mice (n = 3). FIG. 52B shows bar graphs showing the average proportion of macrophages found in the leptomeninges (n=3) and extra-cranial sites of metastases (n = 3) in mice. FIG. 52C shows violin plots showing expression of T cell exhaustion markers on T cells using scRNAseq analysis of leptomeningeal tumors and tumors at extra-cranial sites of metastasis in immune-competent murine models of 4T1 breast cancer, A20 lymphoma, and SMI melanoma. FIG. 52D shows bar graphs showing the average number and proportion of each myloid cell type found in 7mL of CSF from patients with long (> 10 months) vs. short (< 5 months) survival. FIG. 52E shows bar graphs showing the average number and proportion of each macrophage cluster found in 7mL of CSF from patients with long versus short survival. FIG. 52F shows bar graphs showing the number of CD8+ and CD4+ cells in CSF of patients with long versus short survival.

[0101] FIGS. 53A-53G show the following: FIG. 53A shows volcano plots showing differentially abundant lipids in CSF of lymphoma LMD vs. no-LMD. FIG. 53B shows the number of lipids identified to have significant changes in abundance in lymphoma LMD compared to no-LMD control for each lipid class. FIG. 53C shows volcano plots showing differentially abundant proteins in CSF of lymphoma patients with LMD compared to no- LMD controls. FIG. 53D shows a heat map showing the top upregulated and downregulated proteins in CSF from lymphoma LMD and their abundance in CSF from melanoma LMD. FIG. 53E shows a plot showing results from integration of proteomics and metabolomic analysis of CSF from patients with LMD comparared to no LMD controls. FIG. 53F shows a heatmap of relative expression of branched-chain amino acids and branched chain-keto acids in CSF of lymphoma-LMD patients and no-LMD control CSF, measured by massAttorney Docket No. 10110-466WO1

[0102] spectrometry. FIG. 53G shows pathway enrichment analysis of differentially abundant metabolites using the MetaboAnalyst 5.0 bioinformatics tool.

[0103] FIGS. 54A-54G show the following: FIG. 54A shows a Calcein AM flouresence assay reflecting the vaibility of primary mouse neurons cultured with 50pM BCKA in regular media for 7 days, in which data represent mean ± SEM and statistical analysis was done using student’s T-test. FIG. 54B shows an MTT assay measuring metabolic activity of primary rat neurons in response to BCKA exposure in neuronal media for 7 days, in which data represent mean + SEM and statistical analysis was done using student’s T-test. FIG.

[0104] 54C shows an MTT assay measuring metabolic activity of primary human meningeal cells in response to BCKA exposure for 72 hours, in which data represent mean ± SEM and statistical analysis was done using student’s T-test. FIG. 54D shows a violin plot showing relative expression of a reactive gliosis signature in glial cells of mice recieving injections of PBA control (control) or SMI melanoma cells via cisterna magna (LMD). FIGS. 54E-54G show Calcein AM assays on cell lines treated with increasing doses of BCKA at physiological ratio in physiological CSF for 48 hours, in which FIG. 54E shows human and murine lymphoma cell lines, FIG. 54F shows human and murine melanoma cell lines, and FIG. 54G shows human and murine breast cancer cell lines, and further in which data represent mean ± SD, statistical analysis was done using two-way ANOVA, and *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and no annotation = not significant.

[0105] FIGS. 55A-55H show the following: FIG. 55A shows a schematic illustartion of the neuroscoring system in the LMD mouse model. FIG. 55B shows individual graphs showing the scores for each neurological assessment at endpoint (or last assessment before death for each cell line model injected via cisterna magna). FIG. 55C shows images demonstrating hind limb splay in control and LMD mice. FIG. 55D shows images demonstrating the manifestation of kyphosis and loss of grooming in control PBS-injected mice and tumor- injected LMD mice. FIG. 55E shows images showing the deformity in skull shape of the animals with lymphoma LMD. FIG. 55F shows Kaplan-Meier analysis for probability of survival in control PBS-injected mice vs. those injected with SMI, D4M or YUMM3.2 mouse melanoma cells via ci sterna magna. FIG. 55G shows images showing a healthy, transparent pia membrane in the control animals (green arrow) and a lack of this membrane in the lymphoma LMD animals, in which LMD animals show significant tumor deposits instead (blue arrow). FIG. 55H shows immunofluorescent staining for GFAP (Opal 690, red) and a DAPI nuclear stain (blue) on brain tissues of animals injected with A20 or PBS intrathecally.Attorney Docket No. 10110-466WO1

[0106] FIGS. 56A and 56B show the following. FIG. 56A shows a Dot plot showing weight change of mice in different cohorts. FIG. 56B shows a Dot plots showing the number of days until mice displayed a score of 2+ for each assessment, paralysis, > 10% weight loss, or death.

[0107] FIG. 57 shows a correlation analysis for different lymphoid clusters and MO macrophages versus the BCKA concentration in CSF of LMD patients.

[0108] FIGS. 58A-58G show the following: FIG. 58A shows representative proliferation plots of cell-trace violet labeled T-cells after 0KT3, CD28, and IL-2 stimulation in full media with different concentrations of BCKAs or PBS (control), in which the experiment was repeated using three different PBMC donors. FIG. 58B shows the percentage of T cell division calculated from FIG. 58A using FlowJo. FIG. 58C shows bar graphs showing the percentage of T cell division following stimulation and exposure to BCKAs or PBS (control), in which the experiment was repeated using three individual PBMC donors. FIG.

[0109] 8D shows a bar graph showing expression of PD-1 and LAG3 on human CD4+ and CD8+ T cells in response to different concentrations of BCKA in physiological CSF for 5 days, in which frequency of PD1 and LAG3 positive cells was measured using flow cytometry, data represent mean ± SD, and data were repeated using different human PBMCs. FIG. 58E shows flow cytometry analysis for mouse anti-CD19 CART-mCherry-tagged positive cells compared to negative T cells at the day of mouse injection. FIG. 58F shows individual graphs showing scores for each neurological assessment at endpoint (or last assessment before death) for mice treated with CAR T or CAR T + PBA therapy. FIG. 58G shows individual graphs showing the number of days until mice displayed a score of 2+ for each assessment, paralysis, or death for mice treated with CAR T or CAR T + PBA therapy.

[0110] FIGS. 59A-59E show the following: FIG. 59A shows scRNAseq on patient CSF showing LMD results in a decrease in early activation and an increase of T cells approaching exhaustion. FIG. 59B shows pie charts of T cell proportions in each functional phenotype based on scRNAseq from LMD of each tumor type. FIG. 59C shows a mouse model of LMD confirming T cell exhaustion at the leptomeninges. FIG. 59D shows expression of T cell exhaustion markers in mouse models of breast (4T1) LMD compared to other sites of tumor in the same mice. FIG. 59E shows the proportion of active, proliferating T cells in patients with the typical short (< 5 months) survival compared to long (< 10 months) survival.

[0111] FIGS. 60A-60D show the following: FIG. 60A shows correlation analysis of KIC levels in patient CSF to each patient’s KPS score. FIG. 60B shows an MTT assay measuringAttorney Docket No. 10110-466WO1

[0112] metabolic activity of primary mouse neurons in response to BCKA exposure for seven days. FIG. 60C shows an MTT assay measuring metabolic activity of meningeal cells in response to BCKA exposure for seven days. FIG. 60D shows a violin plot showing expression of a reactive gliosis signature in glial cells of mice receiving injections of PBA control (control) or tumor cells via cisterna magna (LMD).

[0113] FIG. 61 shows Pearson correlation analysis of KIC concentration in pia versus survival time of mice.

[0114] FIG. 62 shows IT cDCl treatment prolongs survival and produces cures in HER2+ and TNBC-LMD. Kaplan-Meier analysis of mice with HER2+ TUBO LMD (left) and triple negative 4T1 LMD (right).

[0115] FIGS. 63A and 63B show a murine HER2 TUBO breast carcinoma cells were implanted subcutaneously in mice. FIG. 63A shows tumor growth curves in IFN-γ KO mice that were untreated (control), receiving HER2 peptide-pulsed cDC1 (HER2 DC1), receiving anti-semaphoring 4D MoAb (aSenia4D), or both treatments simultaneously (HER2 DCl+aSema4D). FIG. 63B shows identical experimental setup as “B” but in mice depleted of CD4 Th cells.

[0116] FIG. 64 shows ELISA detection of HER2 and HER3 specific IgG in the CSF was measured in LMD patients at various time points during IT cDCl therapy. Baseline sample was the CSF before treatment; negative control was the assay background (without IgG). Purified human recombinant anti-HER2 and anti-HER3 antibodies were used as positive controls.

[0117] VII. DETAILED DESCRIPTION

[0118] Before the present compounds, compositions, articles, devices, and / or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods or specific recombinant biotechnology methods unless otherwise specified or to particular reagents unless otherwise specified, as such may, of course vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0119] A. Definitions

[0120] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like.Attorney Docket No. 10110-466WO1

[0121] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed the “less than or equal to 10”as well as “greater than or equal to 10” is also disclosed. It is also understood that throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 1. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0122] In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings:

[0123] The term “administer,” “administering”, or derivatives thereof refer to delivering a composition, substance, inhibitor, or medication to a subject or object by one or more the following routes: oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation or via an implanted reservoir. The term “parenteral” includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injections or infusion techniques.

[0124] As used herein, the term “agent” refers to a living organism or biological substance, such as a bacterium, virus, protozoan, parasite, fungus, chemical, or toxin, that can be designed to purposefully fulfill a biological function or action.Attorney Docket No. 10110-466WO1

[0125] The terms “anticancer” and “anticarcinogen” refers to a substance, composition, or formula that counteracts the effects or inhibits the development of a cancerous cells and tissues.

[0126] As used herein, the term “buffer” refers to a solution consisting of a mixture of acid and its conjugate base, or vice versa. The solution is used as a means of keeping the pH at a nearly constant range to be used in a wide variety of chemical and biological applications.

[0127] As used herein, the term “carrier” encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations. The choice of a carrier for use in a composition will depend upon the intended route of administration for the composition. The preparation of pharmaceutically acceptable carriers and formulations containing these materials is described in, e.g., Remington's Pharmaceutical Sciences, 21st Edition, ed. University of the Sciences in Philadelphia, Lippincott, Williams & Wilkins, Philadelphia, PA, 2005. Examples of physiologically acceptable carriers include saline, glycerol, DMSO, buffers such as phosphate buffers, citrate buffer, and buffers with other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEENTM(ICI, Inc.; Bridgewater, New Jersey), polyethylene glycol (PEG), and PLURONICS™ (BASF; Florham Park, NJ). To provide for the administration of such dosages for the desired therapeutic treatment, compositions disclosed herein can advantageously comprise between about 0.1% and 99% by weight of the total of one or more of the subject compounds based on the weight of the total composition including carrier or diluent.

[0128] As used herein, the term “chemical compound” or “compound”, refers to a chemical substance consisting of two or more different types of atoms or chemical el ements in a fixed stoichiometric proportion. These compounds have a unique and defined chemical structure held together in a defined spatial arrangement by chemical bonds. Chemical compounds can be held together by covalent bonds, ionic bonds, metallic ions, or coordinate covalent bonds.Attorney Docket No. 10110-466WO1

[0129] A “chimeric antigen receptor” or CAR is an artificial T cell receptor used for immunotherapy. CAR are protein receptors that have been engineered to give T cells an enhanced ability to target a specific protein. CAR receptors are chimeric because the antigen binding and T cell activating functions have been combined into a single receptor.

[0130] “Composition” refers to any agent that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition (e.g., Leptomeningeal Disease (LMD), Leptomeningeal Lymphoma(-LML), cancer and / or cancer-associated complications). The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, a vector, polynucleotide, cells, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the term “composition” is used, then, or when a particular composition is specifically identified, it is to be understood that the term includes the composition per se as well as pharmaceutically acceptable, pharmacologically active vector, polynucleotide, salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc. In some aspects, the composition disclosed herein comprises sodium phenylbutyrate and a pharmaceutically acceptable carrier.

[0131] " Comprising" is intended to mean that the compositions, methods, etc. include the recited elements, but do not exclude others. " Consisting essentially of ' when used to define compositions and methods, shall mean including the recited elements, but excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. " Consisting of’ shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions provided and / or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0132] A “control” is an alternative subject or sample used in an experiment for comparison purposes. A control can be "positive" or "negative."

[0133] The term “cytotoxic” as used herein refers to the ability to kill a target cell. A cytotoxic T cell or NK cell may kill a target cell via target cell apoptosis using one or more different mechanisms including release of one or more cytotoxins or expression of a Fas ligand. In some embodiments, a cytotoxic T cell or NK cell kills a tumor cell via the releaseAttorney Docket No. 10110-466WO1

[0134] of one or more cytotoxins. “Cytotoxin” includes, but is not limited to, a perforin, a granzyme and a granulysin. Currently known granzymes are Granzyme A (GZMA), Granzyme B (GZMB), Granzyme H (GZMH), Granzyme K (GZMK), and Granzyme M (GZMM).

[0135] As used herein, cytotoxicity refers to the quality of being toxic to cells. Treating cells with a cytotoxic compound can result in a variety of cell fates, including necrosis (in which the cell membrane becomes compromised leading to cell lysis), senescence (in which the cell stops actively growing and dividing), or apoptosis (in which the cell activates a genetic program of controlled cell death).

[0136] A "decrease" can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also, for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant.

[0137] As used herein, “diagnose”, “diagnosed”, “diagnosing”, and any grammatical variations thereof as used herein, refers to the act of process of identifying the nature of an illness, disease, disorder, or condition in a subject by examination or monitoring of symptoms.

[0138] “Effective amount” of an agent refers to a sufficient amount of an agent to provide a desired effect. The amount of agent that is “effective” will vary from subject to subject, depending on many factors such as the age and general condition of the subject, the particular agent or agents, and the like. Thus, it is not always possible to specify a quantified “effective amount.” However, an appropriate “effective amount” in any subject case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an “effective amount” of an agent can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts. An “effective amount” of an agent necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.Attorney Docket No. 10110-466WO1

[0139] An "increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition or activity. An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount. Thus, the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase so long as the increase is statistically significant.

[0140] " Inhibit," "inhibiting," and "inhibition" mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

[0141] “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0142] A “pharmaceutically effective amount” of a drug necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0143] " Pharmaceutically acceptable" component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation of the invention and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained. When used in reference to administration to a human, the term generally implies the component has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U. S. Food and Drug Administration.

[0144] " Pharmaceutically acceptable carrier" (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic, and includes a carrier that is acceptable for veterinary and / or human pharmaceutical or therapeutic use. The terms "carrier" or "pharmaceutically acceptable carrier" can include, but are not limited to, phosphate buffered saline solution,Attorney Docket No. 10110-466WO1

[0145] water, emulsions (such as an oil / water or water / oil emulsion) and / or various types of wetting agents.

[0146] By “prevent” or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed.

[0147] By “reduce” or other forms of the word, such as “reducing” or “reduction,” is meant lowering of an event or characteristic (e.g., tumor growth). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, “reduces tumor growth” means reducing the rate of growth of a tumor relative to a standard or a control.

[0148] The term “subject” refers to any individual who is the target of administration or treatment, The subject can be a vertebrate, for example, a mammal. In one aspect, the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline. The subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.

[0149] A “T cell” refers to a type of lymphocyte that is one of the most important white blood cells of the immune system. T cells can be distinguished from other lymphocytes by the presence of a T-cell receptor (TCR) on their cell surface. The immune-mediated cell death function of T cells is carried by two major subtypes: CD8+“killer” T cells and CD4+“helper T cells.

[0150] The term “therapeutically effective” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.

[0151] A “therapeutic composition” refers to at least one substance, molecule, or compound suitable for administering to a subject, wherein the composition further includes aAttorney Docket No. 10110-466WO1

[0152] pharmaceutical carrier. A non-limiting example include a therapeutic composition comprises a nucleobase-poly-amino acid carrier and a sterile water-based solution.

[0153] As used herein, a “therapeutic benefit” refers to a benefit or effect obtained as a result of administering a treatment or therapeutic composition. Generally, the therapeutic benefit is a positive result that occurs as a result of a method used to treat or prevent a disease or disorder.

[0154] A “neurodegenerative disease” is caused by the progressive loss of structure or function of neurons or glial cells, which make up the nervous system. These diseases include but are not limited to amyotrophic lateral sclerosis (ALS), multiple sclerosis, Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, and prion diseases. Neurodegenerative diseases can lead to cognitive and physical impairments, neuroinflammation (inflammation of the brain and spinal cord), and deterioration of brain and spinal cord tissues.

[0155] “Therapeutic agent” refers to any composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition (e.g., a non-immunogenic cancer ). The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the terms “therapeutic agent” is used, then, or when a particular agent is specifically identified, it is to be understood that the term includes the agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.

[0156] “Therapeutically effective amount” or “therapeutically effective dose” of a composition (e.g. a composition comprising an agent) refers to an amount that is effective to achieve a desired therapeutic result. In some embodiments, a desired therapeutic result is the control of type I diabetes. In some embodiments, a desired therapeutic result is the control of obesity. Therapeutically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject. The term can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect, such as pain relief. The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the agent and / or agent formulation to be administered (e.g., the potency of theAttorney Docket No. 10110-466WO1

[0157] therapeutic agent, the concentration of agent in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art. In some instances, a desired biological or medical response is achieved following administration of multiple dosages of the composition to the subject over a period of days, weeks, or years.

[0158] As used herein, a “therapeutic regimen” refers to a structured treatment plan or strategy designed to improve and maintain health. Generally, a therapeutic regimen will be designed, prescribed, and / or administered by a licensed medical practitioner. The therapeutic regimen generally specifies the treatment dosage, the treatment scheduling, and the duration of the treatment. In some embodiments, the therapeutic regimen comprises one or more therapeutic compositions. In some embodiments, the therapeutic regimen comprises one or more therapeutic agents. In some embodiments, the therapeutic regimen comprises any combination of therapeutic compositions and therapeutic agents, such as for example the combination of an inhibitor and an antibody. In some embodiments, a therapeutic regimen comprises modifying, continuing, and / or initiating at least one therapeutic agent and / or therapeutic composition. In some embodiments, a therapeutic regimen comprises treating and / or preventing a disease, disorder, and / or condition.

[0159] The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

[0160] The terms “treat,” “treating,” and grammatical variations thereof as used herein, include partially or completely delaying, alleviating, mitigating or reducing the intensity of one or more attendant symptoms of a disorder or condition and / or alleviating, mitigating or impeding one or more causes of a disorder or condition. Treatments according to the disclosure may be applied preventively, prophylactically, palliatively or remedially. Treatments are administered to a subject prior to onset (e.g., before obvious signs ofAttorney Docket No. 10110-466WO1

[0161] Leptomeningeal lymphoma, Leptomeningeal disease, or cancer), during early onset (e.g., upon initial signs and symptoms of Leptomeningeal lymphoma, Leptomeningeal disease, or cancer), or after an established development of Leptomeningeal lymphoma, Leptomeningeal disease, or cancer.

[0162] Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.

[0163] Disclosed are methods relating to the treatment of Leptomeningeal disease (LMD), leptomeningeal lymphoma (LML), cancers, metastasis, and cancer-associated complication by administering a therapeutically effective amount of sodium phenylbutyrate alone or in conjunction with other anti-cancer therapies. The current application has the following innovation and novelty: 1. Patients with LMD have few therapeutic options and a dismal prognosis. This proposal addresses an urgent clinical need for novel therapeutic strategies for melanoma LMD. 2. Little is known about BCKA accumulation in the tumor microenvironment. Our group is the first to report this in patients and in vivo models. 3. The roles of BCKAs on tumor-promoting or immune-suppressive processes in the LMD microenvironment have never been explored and there are very few studies examining BCKA as signaling molecules rather than metabolic intermediates. BCKA-lowering therapies have never been applied to the treatment of LMD. 4. Combining metabolism-targeting agents with immune therapies is novel and has never been studied in LMD. 5. In vivo metabolic tracing and the use of physiological CSF, LMD-derived PDXs (the first validated melanoma PDX models of LMD known to be in existence), and CSF from LMD patients are cutting-edge approaches, enabling us to study the LMD tumor microenvironment in a more natural setting for the first time. 6. Our studies can also provide mechanistic insights into immune suppression in Maple Syrup Urine Disease. 7. We will leverage our unparalleled collection of Non-Animal Models (patient specimens, primary cultures, physiologically-matched CSF) and immune-competent murine models of LMD. 8. Our phospho-proteomics and chemical proteomics approaches are unbiased and innovative.

[0164] In one aspect disclosed herein is a method of treating Leptomeningeal disease (LMD) in a subject, comprising: administering to the subject a therapeutically effective amount of sodium phenylbutyrate; and a pharmaceutically acceptable carrier.Attorney Docket No. 10110-466WO1

[0165] In some embodiments, the therapeutically effective amount of sodium phenylbutyrate and the pharmaceutically acceptable carrier are administered intrathecally.

[0166] Some embodiments, further comprise administering to the subject a therapeutically effective amount of at least one anti-cancer agent. In some embodiments, the anti-cancer agent comprises cytarabine, methotrexate, thiotepa, rituximab, capmatinib, or tepotinib.

[0167] In some embodiments, LMD is associated with branched-chain keto acids (BCKA) accumulation in a sample of cerebrospinal fluid obtained from the subject, CAR-T cytotoxicity, or neurodegeneration.

[0168] In some embodiments, administering to the subject the therapeutically effective amount of sodium phenylbutyrate and the pharmaceutically acceptable carrier, reduces BCKA accumulation in a sample of cerebrospinal fluid obtained from the subject, compared to an untreated control.

[0169] In some embodiments, administering to the subject the therapeutically effective amount of sodium phenylbutyrate and the pharmaceutically acceptable carrier, reduces CAR-T cytotoxicity, in the subject, compared to an untreated control.

[0170] In some embodiments, administering to the subject the therapeutically effective amount of sodium phenylbutyrate and the pharmaceutically acceptable carrier, reduces neurodegeneration, in the subject, compared to an untreated control.

[0171] In one aspect, disclosed herein is a method of treating Leptomeningeal lymphoma (LML) in a subject, comprising: administering to the subject a therapeutically effective amount of sodium phenylbutyrate; and a pharmaceutically acceptable carrier.

[0172] In some embodiments, the therapeutically effective amount of sodium phenylbutyrate and the pharmaceutically acceptable carrier is administered intrathecally or systematically.

[0173] Some embodiments, further comprise administering to the subject a therapeutically effective amount of at least one anti-cancer agent.

[0174] In some embodiments, the anti-cancer agent comprises cytarabine, methotrexate, thiotepa, rituximab, capmatinib, or tepotinib.

[0175] In some embodiments, LML is associated with branched-chain keto acids (BCKA) accumulation in a sample of cerebrospinal fluid obtained from the subject.

[0176] In some embodiments, LML is associated with CAR-T cytotoxicity.

[0177] In some embodiments, LML is associated with neurodegeneration.

[0178] In some embodiments, administering to the subject the therapeutically effective amount of sodium phenylbutyrate and the pharmaceutically acceptable carrier reducesAttorney Docket No. 10110-466WO1

[0179] BCKA accumulation in a sample of cerebrospinal fluid obtained from the subject, compared to an untreated control.

[0180] In some embodiments, administering to the subject the therapeutically effective amount of sodium phenylbutyrate and the pharmaceutically acceptable carrier reduces CAR-T cytotoxicity in the subject compared to an untreated control.

[0181] In some embodiments, administering to the subject the therapeutically effective amount of sodium phenylbutyrate and the pharmaceutically acceptable carrier, reduces neurodegeneration, in the subject, compared to an untreated control.

[0182] In some embodiments, the subject is a human.

[0183] In one aspect, disclosed herein is a method of treating a subject with a cancer and / or a cancer-associated complication, comprising: administering to the subject a therapeutically effective amount of sodium phenylbutyrate; and a pharmaceutically acceptable carrier.

[0184] In some embodiments, the therapeutically effective amount of sodium phenylbutyrate and the pharmaceutically acceptable carrier is administered intrathecally.

[0185] Some embodiments, further comprise administering to the subject a therapeutically effective amount of at least one anti-cancer agent.

[0186] In some embodiments, the anti-cancer agent comprises cytarabine, methotrexate, thiotepa, rituximab, capmatinib, or tepotinib.

[0187] In some embodiments, the cancer-associated complication is Leptomeningeal Disease (LMD), Leptomeningeal metastasis (LML), or carcinomatosis (LMC).

[0188] In some embodiments, the cancer-associated complication is branched-chain keto acids (BCKA) accumulation in a sample of cerebrospinal fluid obtained from the subject.

[0189] In some embodiments, the cancer-associated complication is CAR-T cytotoxicity. In some embodiments, the cancer-associated complication is neurodegeneration. In some embodiments, administering the therapeutically effective amount of sodium phenylbutyrate and the pharmaceutically acceptable carrier to the subject reduces BCKA accumulation in a sample of cerebrospinal fluid obtained from the subject, compared to an untreated control.

[0190] In some embodiments, administering the therapeutically effective amount of sodium phenylbutyrate and the pharmaceutically acceptable carrier to the subject reduces CAR-T cytotoxicity in the subject compared to an untreated control.Attorney Docket No. 10110-466WO1

[0191] In some embodiments, administering the therapeutically effective amount of sodium phenylbutyrate and the pharmaceutically acceptable carrier to the subject reduces neurodegeneration in the subject compared to an untreated control.

[0192] In some embodiments, the subject is a human.

[0193] In one aspect, disclosed herein is a method for enhancing T cell activity in a subject, comprising: determining a subject having an elevated concentration of Branched-Chain Keto Acids (BCKA) in the cerebrospinal fluid (CSF); and administering a therapeutically effective amount of a therapeutic agent to reduce BCKA accumulation or inhibit BCKA-mediated signaling in the CSF, thereby reversing T cell exhaustion or inactivity.

[0194] In some embodiments, the therapeutic agent is a buffering agent to increase the pH of the CSF.

[0195] In some embodiments, the therapeutic agent is a metabolic inhibitor to reduce the production of BCKAs.

[0196] In some embodiments, the metabolic inhibitor comprises binimetinib, cobimetinib, trametinib, LY294002, wortmannin, BCAT1 inhibitors, or BCAT2 inhibitors.

[0197] In some embodiments, the BCAT1 or BCAT2 inhibitors comprise BAY-069, BT2, Bufalin, WQQ-345, Gabapentin, or ERG240.

[0198] In some embodiments, the therapeutic agent targets the MAPK pathway, thereby restoring phosphorylation of p38α (MAPK14) or 4E-BP1 (EIF4EBP1) in T cells.

[0199] In one aspect disclosed herein is a method of treating a cancer in a subject, comprising: administering to the subject a therapeutically effective amount of a therapeutic agent that reduces BCKA accumulation in a cerebrospinal fluid compartment of the subject.

[0200] In some embodiments, the therapeutic agent comprises phenylbutyrate and a pharmaceutically acceptable carrier thereof.

[0201] Some embodiments, further comprise administering an intrathecal dendritic cell therapy to the subject.

[0202] In some embodiments, the intrathecal dendritic cell therapy comprises administering dendritic cells loaded with MHC class II peptides that target HER2, HER3, or a combination thereof on cancer cells.

[0203] In some embodiments, administering the therapeutic agent that reduces BCKA accumulation enhances an immune favorable microenvironment in leptomeningeal metastatic disease relative to administration of the intrathecal dendritic cell therapy without the therapeutic agent.Attorney Docket No. 10110-466WO1

[0204] In some embodiments, the immune favorable microenvironment comprises increase in T cell infiltration, increase in T cell activation, decrease in T cell exhaustion, or a combination thereof.

[0205] In some embodiments, the cancer comprises a breast cancer induced leptomeningeal metastatic disease, breast cancer, leptomeningeal disease (LMD), leptomeningeal metastasis (EM), or carcinomatosis (EMC).

[0206] In some embodiments, treating the cancer comprises prolonging survival, reducing leptomeningeal tumor burden, or a combination thereof.

[0207] In some embodiments, the therapeutic agent to reduce BCKA accumulation is administered intrathecally, orally, systemically, or by a combination thereof.

[0208] In one aspect disclosed herein is a method of treating breast cancer leptomeningeal metastatic disease in a subject, comprising: administering a combination of a therapeutic agent to reduce branched chain keto acid accumulation in cerebrospinal fluid, and a dendritic cell therapy.

[0209] In some embodiments, the dendritic cell therapy comprises a peptide pulsed conventional dendritic cells.

[0210] In some embodiments, the therapeutic agent to reduce branched chain keto acid accumulation comprises sodium phenylbutyrate.

[0211] In some embodiments, the peptide pulsed conventional dendritic cells comprise one or more MHC class II peptides that target HER2, HER3, or a combination thereof on cancer cells.

[0212] In some embodiments, the method further enhances T cell infiltration into leptomeningeal tumor tissue, increases T cell activation, reduces T cell exhaustion, or a combination thereof.

[0213] In some embodiments, the method further increases a CD4 Thl response, increases a B cell response, or both, in the cerebrospinal fluid compartment.

[0214] In some embodiments, tire breast cancer leptomeningeal metastatic disease comprises HER2 positive breast cancer leptomeningeal metastatic disease, triple negative breast cancer leptomeningeal metastatic disease, or a combination thereof.

[0215] In some embodiments, the therapeutic agent to reduce branched chain keto acid accumulation is administered prior to the dendritic cell therapy, concurrently with the dendritic cell therapy, or after the dendritic cell therapy.

[0216] In some embodiments, administering the dendritic cell therapy comprises administering a plurality of doses.Attorney Docket No. 10110-466WO1

[0217] B. Method of treating Leptomeningeal disease (LMD)

[0218] Disclosed herein are methods of treating, inhibiting, reducing, decreasing, ameliorating, and / or preventing Leptomeningeal disease (LMD), and / or LMD associated complications, including but not limited to CAR-T cytotoxicity, and neurodegeneration, wherein the method comprises administration of a composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier.

[0219] In one aspect disclosed herein is a method of treating Leptomeningeal disease (LMD) in a subject comprising administering to a subject a composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier. In some embodiments, the composition is administered to the subject, intrathecally. In some embodiments, the composition is administered to the subject at a therapeutically effective amount. In some embodiments, the method further comprises administering to the subject at least one anticancer agent such as, for example cytarabine, methotrexate, rituximab, and thiotepa.

[0220] In one aspect, disclosed herein, is a method of treating a subject with a LMD comprising administering a composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier to the subject.

[0221] In some embodiments, the composition is administered to the subject, intrathecally. In some embodiments, the composition is administered to the subject at a therapeutically effective amount. In some embodiments, the method further comprises administering to the subject at least one anti-cancer agent such as, for example cytarabine, methotrexate, rituximab, and thiotepa.

[0222] In some embodiments, the LMD-associated complication is branched-chain keto acids (BCKA) accumulation in a sample of cerebrospinal fluid obtained from the subject. Branched-chain keto acids (BCKA) accumulation is also observed in patients with leptomeningeal disease from melanoma and breast cancer. It is observed that BCKA exerts an immunosuppressive and neurodegenerative microenvironment in patients with CNS leptomeningeal lymphoma. BCKA is also cytotoxic and inhibits T-cell proliferation thus the LMD CSF shows few active and proliferating T cells. BCKAs inhibit the secretion of pro- inflammatory cytokines from T-cells.

[0223] LMD associated complications result in a drastic reduction in quality of life. Examples of severe LMD complications include but are not limited to CAR-T cell cytotoxicity and neurodegeneration. LMD occurs when a cancer (such as, for example, breast cancer) spreads to the cerebral spinal fluid and surrounding meninges. Neurodegeneration associated with cancer can be a direct symptom of the cancer, aAttorney Docket No. 10110-466WO1

[0224] symptom of complications from cancer, i.e. LMD, or a symptom of cancer treatments. New evidence shows an increased concentration of branched-chain a-keto acids as a result of LMD. Thus, a method of reducing BCKA levels in LMD patients comprising treatment with sodium phenylbutyrate, was tested to ameliorate LMD-associated complications. As LMD affects the spinal column, traditional methods of drug administration are limited in therapeutic efficacy due to the presence of the blood-brain barrier. Intrathecal administration, directly into the CSF, removes the impediment of the blood-brain barrier to directly target LMD.

[0225] CAR-T cell therapy has been shown to successfully treat certain types of cancers, as a patient’s own T cells are engineered to recognize tumor-specific proteins - harnessing the patient’s immune system to fight off cancer. However, in some patients CAR-T cell therapy is not effective either due to tumor mutations or rejection of the CAR-T cells. Additionally, CAR-T cell therapy can have cytotoxic effects. In some embodiments, LMD is associated with CAR-T cytotoxicity. In some embodiments, LMD is associated with neurodegeneration.

[0226] In some embodiments, administering to the subject the composition of any of the preceding aspects, wherein the composition comprises sodium phenylbutyrate and a pharmaceutically acceptable carrier, reduces LMD, in a subject, compared to an untreated control. In some embodiments, administering to the subject the composition of any of the preceding aspects, reduces BCKA accumulation in a sample of cerebrospinal fluid obtained from the subject, compared to an untreated control. In some embodiments, administering to the subject the composition of any of the preceding aspects, reduces CAR-T cytotoxicity, in the subject, compared to an untreated control. In some embodiments, administering to the subject the composition of claim 1, reduces neurodegeneration, in the subject, compared to an untreated control. Sodium phenylbutyrate was tested as an anti-cancer therapeutic to improve CAR-T cell therapy efficacy and reduce associated cytotoxicity in a mouse model.

[0227] As shown here, sodium phenylbutyrate alone or in combination with other anti¬ cancer therapies has significantly better effects on subjects with cancer and / or cancer associated complications than current anti-cancer therapeutics alone. Sodium phenylbutyrate is the only proposed therapeutic for LMD and is shown here to increase quality of life of a subject treated with the composition of any of the preceding aspects wherein the composition comprises sodium phenylbutyrate and a pharmaceutically acceptable carrier and wherein, the subject is a human.Attorney Docket No. 10110-466WO1

[0228] The disclosed compositions can be used to treat any disease where uncontrolled cellular proliferation occurs such as cancers. A representative but non-limiting list of cancers that the disclosed compositions can be used to treat is the following: acoustic neuroma, adenocarcinoma, adrenal gland cancer, anal cancer, angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma), appendix cancer, benign monoclonal gammopathy, biliary cancer (e.g., cholangiocarcinoma), bladder cancer, breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast), brain cancer (e.g., meningioma; glioma, e.g., astrocytoma, oligodendroglioma; medulloblastoma), bronchus cancer, carcinoid tumor, cervical cancer (e.g., cervical adenocarcinoma), choriocarcinoma, chordoma, craniopharyngioma, colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma), epithelial carcinoma, ependymoma, endotheliosarcoma (e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma), endometrial cancer (e.g., uterine cancer, uterine sarcoma), esophageal cancer (e.g., adenocarcinoma of the esophagus, Barrett’s adenocarcinoma), Ewing's sarcoma, eye cancer (e.g., intraocular melanoma, retinoblastoma), familiar hypereosinophilia, gall bladder cancer, gastric cancer (e.g., stomach adenocarcinoma), gastrointestinal stromal tumor (GIST), head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma (OSCC), throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)), hematopoietic cancers (e.g., leukemia such as acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myelocytic leukemia (AML) (e.g., B-cell AML,, T-cell AML,), chronic myelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chronic lymphocytic leukemia (CLL,) (e.g., B-cell CLL, T-cell CLL); lymphoma such as Hodgkin lymphoma (HL) (e.g., B-cell LIL, T-cell HL) and non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma (DLBCL)), follicular lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas (e.g., mucosa-associated lymphoid tissue (MALT) lymphomas, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (i.e., “Waldenstrom's macroglobulinemia”), hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma and primary central nervous system (CNS) lymphoma; and T-cell NHL such as precursor T-lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T-Attorney Docket No. 10110-466WO1

[0229] cell lymphoma (CTCL) (e.g., mycosis fungiodes, Sezary syndrome), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplastic large cell lymphoma); a mixture of one or more leukemia / lymphoma as described above; and multiple myeloma (MM)), heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease), hemangioblastoma, inflammatory myofibroblastic tumors, immunocytic amyloidosis, kidney cancer (e.g., nephroblastoma a.k.a. Wilms' tumor, renal cell carcinoma), liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma), lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung), leiomyosarcoma (LMS), mastocytosis (e.g., systemic mastocytosis), myelodysplastic syndrome (MDS), mesothelioma, myeloproliferative disorder (MPD) (e.g., polycythemia Vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)), neuroblastoma, neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis), neuroendocrine cancer (e.g., gastroenteropancreatic neuroendocrine tumor (GEP-NET), carcinoid tumor), osteosarcoma, ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma), papillary adenocarcinoma, pancreatic cancer (e.g., pancreatic adenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors), penile cancer (e.g., Paget’s disease of the penis and scrotum), pinealoma, primitive neuroectodermal tumor (PNT), prostate cancer (e.g., prostate adenocarcinoma), rectal cancer, rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)), small bowel cancer (e.g., appendix cancer), soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma), sebaceous gland carcinoma, sweat gland carcinoma, synovioma, testicular cancer (e.g., seminoma, testicular embryonal carcinoma), thyroid cancer (e.g., papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC), medullary thyroid cancer), urethral cancer, vaginal cancer and vulvar cancer (e.g., Paget's disease of the vulva).

[0230] It is understood and herein contemplated that the disclosed treatment regimens can used alone or in combination with any anti-cancer therapy known in the art including, but not limited to Abemaciclib, Abiraterone Acetate, ABITREXATE® (Methotrexate),Attorney Docket No. 10110-466WO1

[0231] ABRAXANE® (Paclitaxel Albumin- stabilized Nanoparticle Formulation), ABVD, AB VE, ABVE-PC, AC, AC-T, ADCETRIS® (Brentuximab Vedotin), ADE, Ado-Trastuzumab Emtansine, ADRIAMYCIN® (Doxorubicin Hydrochloride), Afatinib Dimaleate, AFINITOR® (Everolimus), AKYNZEO® (Netupitant and Palonosetron Hydrochloride), ALDARA® (Imiquimod), Aldesleukin, ALECENSA® (Alectinib), Alectinib, Alemtuzumab, ALIMTA® (Pemetrexed Disodium), ALIQOPA® (Copanlisib Hydrochloride), ALKERAN™ for Injection (Melphalan Hydrochloride), ALKERAN™ Tablets (Melphalan), ALOXI® (Palonosetron Hydrochloride), ALUNBRIG® (Brigatinib), AMBOCHLORIN® (Chlorambucil), AMBOCLORIN® (Chlorambucil), Amifostine, Aminolevulinic Acid, Anastrozole, Aprepitant, AREDIA® (Pamidronate Disodium), ARIMIDEX® (Anastrozole), AROMASIN® (Exemestane), ARRANON® (Nelarabine), Arsenic Trioxide, ARZERRA® (Ofatumumab), Asparaginase Erwinia chrysanthemi, Atezolizumab, AVASTIN® (Bevacizumab), Avelumab, Axitinib, Azacitidine, BAVENCIO® (Avelumab), BEACOPP, BECENUM® (Carmustine), BELEODAQ® (Belinostat), Belinostat, Bendamustine Hydrochloride, BEP, BESPONSA® (Inotuzumab Ozogamicin), Bevacizumab, Bexarotene, BEXXAR® (Tositumomab and Iodine I 131 Tositumomab), Bicalutamide, BICNU® (Carmustine), Bleomycin, Blinatumomab, BLINCYTO® (Blinatumomab), Bortezomib, BOSULIF® (Bosutinib), Bosutinib, Brentuximab Vedotin, Brigatinib, BuMel, Busulfan, BUSULFEX® (Busulfan), Cabazitaxel, CABOMETYX® (Cabozantinib-S-Malate), Cabozantinib-S-Malate, CAF, CAMPATH® (Alemtuzumab), CAMPTOSAR® (Irinotecan Hydrochloride), Capecitabine, CAPOX, CARAC® (Fluorouracil-Topical), Carboplatin, CARBOPLATIN-TAXOL, Carfilzomib, CARMUBRIS® (Carmustine), Carmustine, Carmustine Implant, CASODEX® (Bicalutamide), CEM, Ceritinib, CERUBIDINE® (Daunorubicin Hydrochloride), CERVARIX® (Recombinant HPV Bivalent Vaccine), Cetuximab, CEV, Chlorambucil, CHLORAMBUCIL -PREDNISONE, CHOP, Cisplatin, Cladribine, CLAFEN® (Cyclophosphamide), Clofarabine, CLOFAREX® (Clofarabine), CLOLAR® (Clofarabine), CMF, Cobimetinib, COMETRIQ® (Cabozantinib-S-Malate), Copanlisib Hydrochloride, COPDAC, COPP, COPP-ABV, COSMEGEN® (Dactinomycin), COTELLIC® (Cobimetinib), Crizotinib, CVP, Cyclophosphamide, CYFOS® (Ifosfamide), CYRAMZA® (Ramucirumab), Cytarabine, Cytarabine Liposome, CYTOSAR-U® (Cytarabine), CYTOXAN® (Cyclophosphamide), Dabrafenib, Dacarbazine, DACOGEN® (Decitabine), Dactinomycin, Daratumumab, DARZALEX® (Daratumumab), Dasatinib, Daunorubicin Hydrochloride, Daunorubicin Hydrochloride and Cytarabine Liposome,Attorney Docket No. 10110-466WO1

[0232] Decitabine, Defibrotide Sodium, DEFITELIO® (Defibrotide Sodium), Degarelix, Denileukin Diftitox, Denosumab, DEPOCYT® (Cytarabine Liposome), Dexamethasone, Dexrazoxane Hydrochloride, Dinutuximab, Docetaxel, DOXIL® (Doxorubicin Hydrochloride Liposome), Doxorubicin Hydrochloride, Doxorubicin Hydrochloride Liposome, DOX-SL® (Doxorubicin Hydrochloride Liposome), DTIC-DOME® (Dacarbazine), Durvalumab, EFUDEX® (Fluorouracil-Topical), ELITEK® (Rasburicase), ELLENCE® (Epirubicin Hydrochloride), Elotuzumab, ELOXATIN® (Oxaliplatin), Eltrombopag Olamine, EMEND® (Aprepitant), EMPLICITI® (Elotuzumab), Enasidenib Mesylate, Enzalutamide, Epirubicin Hydrochloride, EPOCH, ERBITUX® (Cetuximab), Eribulin Mesylate, ERIVEDGE® (Vismodegib), Erlotinib Hydrochloride, ERWINAZE® (Asparaginase Erwinia chrysanthemi), ETHYOL® (Amifostine), Etopophos® ETOPOPHOS® (Etoposide Phosphate), Etoposide, Etoposide Phosphate, EVACET® (Doxorubicin Hydrochloride Liposome), Everolimus, EVISTA® (Raloxifene Hydrochloride), EVOMELA® (Melphalan Hydrochloride), Exemestane, 5-FU® (Fluorouracil Injection), 5-FU® (Fluorouracil-Topical), FARESTON® (Toremifene), FARYDAK® (Panobinostat), FASLODEX® (Fulvestrant), FEC, FEMARA® (Letrozole), Filgrastim, FLUDARA® (Fludarabine Phosphate), Fludarabine Phosphate, FLUOROPLEX® (Fluorouracil— Topical), Fluorouracil Injection, Fluorouracil-Topical, Flutamide, FOLEX® (Methotrexate), FOLEX PFS® (Methotrexate), FOLFIRI, FOLFIRI-BEVACIZUMAB, FOLFIRI-CETUXIMAB, FOL FIRINOX, FOLFOX, FOLOTYN® (Pralatrexate), FU-LV, Fulvestrant, GARDASIL® (Recombinant HPV Quadrivalent Vaccine), GARDASIL 9® (Recombinant HPV Nonavalent Vaccine), GAZYVA® (Obinutuzumab), Gefitinib, Gemcitabine Hydrochloride, GEMCITABINE-CISPLATIN, GEMCITABINE-OXALIPLATIN, Gemtuzumab Ozogamicin, GEMZAR® (Gemcitabine Hydrochloride), GILOTRIF® (Afatinib Dimaleate), GLEEVEC® (Imatinib Mesylate), GLIADEL® (Carmustine Implant), GLIADEL WAFER® (Carmustine Implant), Glucarpidase, Goserelin Acetate, HALAVEN® (Eribulin Mesylate), HEMANGEOL® (Propranolol Hydrochloride), HERCEPTIN® (Trastuzumab), HPV Bivalent Vaccine, Recombinant, HPV Nonavalent Vaccine, Recombinant, HPV Quadrivalent Vaccine, Recombinant, HYCAMTIN® (Topotecan Hydrochloride), HYDREA® (Hydroxyurea), Hydroxyurea, Hyper-CVAD, IBRANCE® (Palbociclib), Ibritumomab Tiuxetan, Ibrutinib, ICE, ICLUSIG® (Ponatinib Hydrochloride), IDAMYCIN® (Idarubicin Hydrochloride), Idarubicin Hydrochloride, Idelalisib, IDHIFA® (Enasidenib Mesylate), IFEX® (Ifosfamide), Ifosfamide, IFOSFAMIDUM® (Ifosfamide), IL-2 (Aldesleukin), ImatinibAttorney Docket No. 10110-466WO1

[0233] Mesylate, IMBRUVICA® (Ibrutinib), IMFINZI® (Durvalumab), Imiquimod, IMLYGIC® (Talimogene Laherparepvec), INLYTA® (Axitinib), Inotuzumab Ozogamicin, Interferon Alfa-2b, Recombinant, Interleukin-2 (Aldesleukin), INTRON A® (Recombinant Interferon Alfa-2b), Iodine I 131 Tositumomab and Tositumomab, Ipilimumab, IRESSA® (Gefitinib), Irinotecan Hydrochloride, Irinotecan Hydrochloride Liposome, ISTODAX® (Romidepsin), Ixabepilone, Ixazomib Citrate, IXEMPRA® (Ixabepilone), JAKAFI® (Ruxolitinib Phosphate), JEB, JEVTANA® (Cabazitaxel), KADCYLA® (Ado-Trastuzumab Emtansine), KEOXIFENE® (Raloxifene Hydrochloride), KEPIVANCE® (Palifermin), KEYTRUDA® (Pembrolizumab), KISQALI® (Ribociclib), KYMRIAH® (Tisagenlecleucel), KYPROLIS® (Carfilzomib), Lanreotide Acetate, Lapatinib Ditosylate, LARTRUVO® (Olaratumab), Lenalidomide, Lenvatinib Mesylate, LENVIMA® (Lenvatinib Mesylate), Letrozole, Leucovorin Calcium, LEUKERAN® (Chlorambucil), Leuprolide Acetate, LEUSTATIN® (Cladribine), LEVULAN® (Aminolevulinic Acid), LINFOLIZIN® (Chlorambucil), LIPODOX® (Doxorubicin Hydrochloride Liposome), Lomustine, LONSURF® (Trifluridine and Tipiracil Hydrochloride), LUPRON® (Leuprolide Acetate), LUPRON DEPOT® (Leuprolide Acetate), LUPRON DEPOT-PED® (Leuprolide Acetate), LYNPARZA® (Olaparib), MARQIBO® (Vincristine Sulfate Liposome), MATULANE® (Procarbazine Hydrochloride), Mechlorethamine Hydrochloride, Megestrol Acetate, MEKINIST® (Trametinib), Melphalan, Melphalan Hydrochloride, Mercaptopurine, Mesna, MESNEX® (Mesna), METHAZOLASTONE® (Temozolomide), Methotrexate, METHOTREXATE LPF® (Methotrexate), Methylnaltrexone Bromide, MEXATE® (Methotrexate), MEXATE-AQ® (Methotrexate), Midostaurin, Mitomycin C, Mitoxantrone Hydrochloride, MITOZYTREX® (Mitomycin C), MOPP, MOZOBIL® (Plerixafor), MUSTARGEN® (Mechlorethamine Hydrochloride), MUTAMYCIN® (Mitomycin C), MYLERAN® (Busulfan), MYLOSAR® (Azacitidine), MYLOTARG® (Gemtuzumab Ozogamicin), NANOPARTICLE PACLITAXEL® (Paclitaxel Albumin-stabilized Nanoparticle Formulation), NAVELBINE® (Vinorelbine Tartrate), Necitumumab, Nelarabine, NEOSAR® (Cyclophosphamide), Neratinib Maleate, NERLYNX® (Neratinib Maleate), Netupitant and Palonosetron Hydrochloride, NEULASTA® (Pegfilgrastim), NEUPOGEN® (Filgrastim), NEXAVAR® (Sorafenib Tosylate), NILANDRON® (Nilutamide), Nilotinib, Nilutamide, NINLARO® (Ixazomib Citrate), Niraparib Tosylate Monohydrate, Nivolumab, NOLVADEX® (Tamoxifen Citrate), NPLATE® (Romiplostim), Obinutuzumab, ODOMZO® (Sonidegib), OEPA, Ofatumumab, OFF, Olaparib, Olaratumab, Omacetaxine Mepesuccinate, ONCASPAR® (Pegaspargase),Attorney Docket No. 10110-466WO1

[0234] Ondansetron Hydrochloride, ONIVYDE® (Irinotecan Hydrochloride Liposome), ONTAK® (Denileukin Diftitox), OPDIVO® (Nivolumab), OPPA, Osimertinib, Oxaliplatin, Paclitaxel, Paclitaxel Albumin-stabilized Nanoparticle Formulation, PAD, Palbociclib, Palifermin, Palonosetron Hydrochloride, Palonosetron Hydrochloride and Netupitant, Pamidronate Disodium, Panitumumab, Panobinostat, PARAPLAT® (Carboplatin), PARAPLATIN® (Carboplatin), Pazopanib Hydrochloride, PCV, PEB, Pegaspargase, Pegfilgrastim, Peginterferon Alfa-2b, PEG-INTRON® (Peginterferon Alfa-2b), Pembrolizumab, Pemetrexed Disodium, PERJETA® (Pertuzumab), Pertuzumab, PLATINOL® (Cisplatin), PLATINOL-AQ® (Cisplatin), Plerixafor, Pomalidomide, POMALYST® (Pomalidomide), Ponatinib Hydrochloride, PORTRAZZA® (Necitumumab), Pralatrexate, Prednisone, Procarbazine Hydrochloride, PROLEUKIN® (Aldesleukin), PROLIA® (Denosumab), PROMACTA® (Eltrombopag Olamine), Propranolol Hydrochloride, PROVENGE® (Sipuleucel-T), PURINETHOL® (Mercaptopurine), PURIXAN® (Mercaptopurine), Radium 223 Dichloride, Raloxifene Hydrochloride, Ramucirumab, Rasburicase, R-CHOP, R-CVP, Recombinant Human Papillomavirus (HPV) Bivalent Vaccine, Recombinant Human Papillomavirus (HPV) Nonavalent Vaccine, Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine, Recombinant Interferon Alfa-2b, Regorafenib, RELISTOR® (Methylnaltrexone Bromide), R-EPOCH, REVLIMID® (Lenalidomide), RHEUMATREX® (Methotrexate), Ribociclib, R-ICE, RITUXAN® (Rituximab), RITUXAN HYCELA® (Rituximab and Hyaluronidase Human), Rituximab, Rituximab and, Hyaluronidase Human,, Rolapitant Hydrochloride, Romidepsin, Romiplostim, RUBIDOMYCIN® (Daunorubicin Hydrochloride), RUBRACA® (Rucaparib Camsylate), Rucaparib Camsylate, Ruxolitinib Phosphate, RYDAPT® (Midostaurin), Sclerosol Intrapleural Aerosol (Talc), Siltuximab, Sipuleucel-T, SOMATULINE DEPOT® (Lanreotide Acetate), Sonidegib, Sorafenib Tosylate, SPRYCEL® (Dasatinib), STANFORD V, Sterile Talc Powder (Talc), STERITALC® (Talc), STIVARGA® (Regorafenib), Sunitinib Malate, SUTENT® (Sunitinib Malate), SYLATRON® (Peginterferon Alfa- 2b), SYLVANT® (Siltuximab), Synribo SYNRIBO® (Omacetaxine Mepesuccinate), TABLOID® (Thioguanine), TAC, TAFINLAR® (Dabrafenib), TAGRISSO® (Osimertinib), Talc, Talimogene Laherparepvec, Tamoxifen Citrate, TARABINE PFS® (Cytarabine), TARCEVA® (Erlotinib Hydrochloride), TARGRETIN® (Bexarotene), TASIGNA® (Nilotinib), TAXOL® (Paclitaxel), TAXOTERE® (Docetaxel), TECENTRIQ® (Atezolizumab), TEMODAR® (Temozolomide), Temozolomide, Temsirolimus, Thalidomide, THALOMID®Attorney Docket No. 10110-466WO1

[0235] (Thalidomide), Thioguanine, Thiotepa, Tisagenlecleucel, TOLAK® (Fluorouracil- Topical), Topotecan Hydrochloride, Toremifene, TORISEL® (Temsirolimus), Tositumomab and Iodine I 131 Tositumomab, TOTECT® (Dexrazoxane Hydrochloride), TPF, Trabectedin, Trametinib, Trastuzumab, TREANDA® (Bendamustine Hydrochloride), Trifluridine and Tipiracil Hydrochloride, TRISENOX® (Arsenic Trioxide), TYKERB® (Lapatinib Ditosylate), UNITUXIN® (Dinutuximab), Uridine Triacetate, VAC, Vandetanib, VAMP, VARUBI® (Rolapitant Hydrochloride), VECTIBIX® (Panitumumab), VelP, VELBAN® (Vinblastine Sulfate), VELCADE® (Bortezomib), VELSAR® (Vinblastine Sulfate), Vemurafenib, VENCLEXTA® (Venetoclax), Venetoclax, VERZENIO® (Abemaciclib), VIADUR® (Leuprolide Acetate), VIDAZA® (Azacitidine), Vinblastine Sulfate, VINCASAR PFS® (Vincristine Sulfate), Vincristine Sulfate, Vincristine Sulfate liposome, Vinorelbine Tartrate, VIP, Vismodegib, VISTOGARD® (Uridine Triacetate), VORAXAZE® (Glucarpidase), Vorinostat, VOTRIENT® (Pazopanib Hydrochloride), VYXEOS® (Daunorubicin Hydrochloride and Cytarabine Liposome), WELLCOVORIN® (Leucovorin Calcium), XALKORI® (Crizotinib), XELODA® (Capecitabine), XELIRI, XELOX, XGEVA® (Denosumab), XOFIGO® (Radium 223 Dichloride), XTANDI® (Enzalutamide), YERVOY® (Ipilimumab), YONDELIS® (Trabectedin), ZALTRAP® (Ziv-Aflibercept), ZARXIO® (Filgrastim), ZEJULA® (Niraparib Tosylate Monohydrate), ZELBORAF® (Vemurafenib), ZEVALIN® (Ibritumomab Tiuxetan), ZINECARD® (Dexrazoxane Hydrochloride), Ziv-Aflibercept, ZOFRAN® (Ondansetron Hydrochloride), ZOLADEX® (Goserelin Acetate), Zoledronic Acid, ZOLINZA® (Vorinostat), ZOMETA® (Zoledronic Acid), ZYDELIG® (Idelalisib), ZYKADIA® (Ceritinib), and / or ZYTIGA® (Abiraterone Acetate). The treatment methods can include or further include checkpoint inhibitors including, but are not limited to antibodies that block PD-1 (such as, for example, Nivolumab (BMS-936558 or MDX1106), pembrolizumab, cemiplimab, CT-011, MK-3475), PD-L1 (such as, for example, atezolizumab, avelumab, durvalumab, MDX-1105 (BMS-936559), MPDL3280A, or MSB0010718C), PD-L2 (such as, for example, rHIgM12B7), CTLA-4 (such as, for example, Ipilimumab (MDX-010), Tremelimumab (CP-675,206)), IDO, B7-H3 (such as, for example, MGA271, MGD009, omburtamab), B7-H4, B7-H3, T cell immunoreceptor with Ig and ITIM domains (TIGIT)(such as, for example BMS-986207, OMP-313M32, MK-7684, AB-L54, ASP-8374, MTIG7192A, or PVSRIPO), CD96, B- and T-lymphocyte attenuator (BTLA), V-domain 1g suppressor of T cell activation (VISTA)(such as, for example, JNJ-61610588, CA-170), TIM3 (such as, for example, TSR-022, MBG453, Sym023, INCAGN2390, LY3321367, BMS-986258, SHR-1702,Attorney Docket No. 10110-466WO1

[0236] RO7121661), LAG-3 (such as, for example, BMS-986016, LAG525, MK-4280, REGN3767, TSR-033, BI754111, Sym022, FS118, MGD013, and Immutep).

[0237] “Concurrent administration”, “administration in combination”, “simultaneous administration” or “administered simultaneously” as used herein, means that the compounds are administered at the same point in time or essentially immediately following one another. IN the latter case, the two compounds are administered at times sufficiently close that the results observed are indistinguishable from those achieved when the compounds are administered at the same point in time.

[0238] “Systemic administration” refers to the introducing or delivering to a subject an agent via a route which introduces or delivers the agent to extensive areas of the subject’s body (e.g. greater than 50% of the body), for example through entrance into the circulatory or lymph systems.

[0239] By contrast, “local administration” refers to the introducing or delivery to a subject an agent via a route which introduces or delivers the agent to the area or area immediately adjacent to the point of administration and does not introduce the agent systemically in a therapeutically significant amount. For example, locally administered agents are easily detectable in the local vicinity of the point of administration but are undetectable or detectable at negligible amounts in distal parts of the subject’s body. Administration includes self-administration and the administration by another.

[0240] The disclosed composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier may be administered in such amounts, time, and route deemed necessary in order to achieve the desired result. The exact amount of the disclosed composition will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease (such as, for example, LMD), the particular composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier, disclosed herein, its mode of administration, its mode of activity, and the like. The disclosed composition sodium phenylbutyrate and a pharmaceutically acceptable carrier is preferably formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disease (such as, for example, LMD) being treated and the severity of the disease (such as, for example, LMD); the activity of the disclosedAttorney Docket No. 10110-466WO1

[0241] composition employed; the specific composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier, employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier employed; the duration of the treatment; drugs used in combination or coincidental with the specific composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier employed; and like factors well known in the medical arts.

[0242] The disclosed composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier may be administered by any route. In some embodiments, the composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier is administered via a variety of routes, including intrathecal, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intra vaginal, intraperitoneal, topical (as by powders, ointments, creams, and / or drops), mucosal, nasal, buccal, enteral, sublingual; by intratracheal instillation, bronchial instillation, and / or inhalation; and / or as an oral spray, nasal spray, and / or aerosol. In general, the most appropriate route of administration will depend upon a variety of factors including the nature of the disclosed composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier (e.g., its stability in the environment of the gastrointestinal tract), the condition of the subject (e.g., whether the subject is able to tolerate oral administration), etc.

[0243] The exact amount of the disclosed composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier required to achieve a therapeutically or prophylactically effective amount will vary from subject to subject, depending on species, age, and general condition of a subject, severity of the side effects, identity of the particular compound(s), mode of administration, and the like. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.

[0244] The concentration of active agent(s) can vary widely and will be selected primarily based on activity of the active ingredient(s), body weight and the like in accordance with the particular mode of administration selected and the patient's needs. Concentrations, however, will typically be selected to provide dosages ranging from about 0.1 or 1Attorney Docket No. 10110-466WO1

[0245] mg / kg / day to about 50 mg / kg / day and sometimes higher. Typical dosages range from about 3 mg / kg / day to about 3.5 mg / kg / day, preferably from about 3.5 mg / kg / day to about 7.2 mg / kg / day, more preferably from about 7.2 mg / kg / day to about 11.0 mg / kg / day, and most preferably from about 11.0 mg / kg / day to about 15.0 mg / kg / day. In certain preferred embodiments, dosages range from about 10 mg / kg / day to about 50 mg / kg / day. In certain embodiments, dosages range from about 20 mg to about 50 mg given orally twice daily. It will be appreciated that such dosages may be varied to optimize a therapeutic and / or prophylactic regimen in a particular subject or group of subjects.

[0246] In one aspect, disclosed herein is composition of any preceding aspect comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier selected from an excipient, a diluent, a salt, a buffer, a stabilizer, a lipid, an emulsion, a nanoparticle, and a cream. One or more active agents (e.g. sodium phenylbutyrate) can be administered in the “native” form or, if desired in the form of salts, esters, amides, prodrugs, or a derivative that is pharmacologically suitable. Salts, esters, amides, prodrugs, and other derivatives of the active agents can be prepared using standards procedures known to those skilled in the art of synthetic organic chemistry and described, for example, by March (1992) Advanced Organic Chemistry; Reactions, Mechanisms, and Structure, 4thEd. N. Y. Wiley-Interscience.

[0247] In some embodiments, the disclosed composition comprising sodium phenyl butyrate and a pharmaceutically acceptable carrier can be prepared as a “concentrate”, e.g. in a storage container of a premeasure volume and / or a predetermined amount ready for dilution, or in a soluble capsule ready for addition to a specified volume of water, saline, alcohol, hydrogen peroxide, or other diluent.

[0248] In some embodiments, the disclosed composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or more times. In some embodiments, the disclosed composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier is administered daily. In some embodiments, the disclosed composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier is administered every day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, or more. In some embodiments, the disclosed composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrierAttorney Docket No. 10110-466WO1

[0249] is administered every week, every 2 weeks, every 3 weeks, every 4 weeks, or more. In some embodiments, the disclosed composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier is administered every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, every 12 months, or more. In some embodiments, the disclosed composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier is administered every year, every 2 years, every 3 years, every 4 years, every 5 years, or more.

[0250] It is understood and herein contemplated that while a single administration of the compounds of the disclosed anti-cancer combination therapies (i.e., sodium phenylbutyrate and / or CAR-T cells) would be ideal, not every patient will respond in the same manner. Thus, in one aspect, disclosed herein are anti-cancer combination therapies methods treating, preventing, reducing, and / or inhibiting a cancer; wherein the sodium phenylbutyrate is administered at least, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20, 21, 22, 23, or 24 times a day or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 times per week for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks. Also disclosed herein are anti-cancer combination therapies methods treating, preventing, reducing, and / or inhibiting a cancer of any preceding aspect; wherein the at least one anti-cancer agent is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20, 21, 22, 23, or 24 times a day or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 times per week for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks. It is further understood and herein contemplated that the order and duration of the administered components can vary as appropriate for the subject being treated. In one aspect, disclosed herein are anti-cancer combination therapies methods treating, preventing, reducing, and / or inhibiting a cancer; wherein the sodium phenyl butyrate is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36 hours, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, 30, 31, 45 days, 2, 3, 4, 5, 6 months prior to administration of the pulsed dendritic cells.

[0251] C. Method of treating Leptomeningeal Lymphoma (LML)

[0252] Disclosed herein are methods of treating, inhibiting, reducing, decreasing, ameliorating, and / or preventing Leptomeningeal lymphoma (LML), and / or LML-associated complications, including but not limited to CAR-T cytotoxicity, and neurodegeneration, wherein the method comprises administration of a composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier.Attorney Docket No. 10110-466WO1

[0253] In one aspect disclosed herein is a method of treating Leptomeningeal lymphoma (LML) in a subject comprising administering to a subject a composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier. In some embodiments, the composition is administered to the subject, intrathecally. In some embodiments, the composition is administered to the subject at a therapeutically effective amount. In some embodiments, the method further comprises administering to the subject at least one anticancer agent such as, for example cytarabine, methotrexate, rituximab, and thiotepa.

[0254] In one aspect, disclosed herein, is a method of treating a subject with a LML comprising administering a composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier to the subject.

[0255] In some embodiments, the composition is administered to the subject, intrathecally. In some embodiments, the composition is administered to the subject at a therapeutically effective amount. In some embodiments, the method further comprises administering to the subject at least one anti-cancer agent such as, for example cytarabine, methotrexate, rituximab, and thiotepa.

[0256] In some embodiments, the LML-associated complication is branched-chain keto acids (BCKA) accumulation in a sample of cerebrospinal fluid obtained from the subject. Branched-chain keto acids (BCKA) accumulation is also observed in patients with leptomeningeal disease from melanoma and breast cancer. It is observed that BCKA exerts an immunosuppressive and neurodegenerative microenvironment in patients with CNS leptomeningeal lymphoma. BCKA is also cytotoxic and inhibits T-cell proliferation thus the LML CSF shows few active and proliferating T cells. BCKAs inhibit the secretion of pro-inflammatory cytokines from T-cells.

[0257] LML-associated complications result in a drastic reduction in quality of life. Examples of severe LML complications include but are not limited to CAR-1’ cell cytotoxicity and neurodegeneration. LML occurs when a cancer (such as, for example, breast cancer) spreads to the cerebral spinal fluid and surrounding meninges. Neurodegeneration associated with cancer can be a direct symptom of the cancer, a symptom of complications from cancer, i.e, LML, or a symptom of cancer treatments. New evidence shows an increased concentration of branched-chain a-keto acids as a result of LML. Thus, a method of reducing BCKA levels in LML. patients comprising treatment with sodium phenylbutyrate, was tested to ameliorate LML-associated complications. As LML affects the spinal column, traditional methods of drug administration are limited in therapeutic efficacy due to the presence of the blood-brain barrier. IntrathecalAttorney Docket No. 10110-466WO1

[0258] administration, directly into the CSF, removes the impediment of the blood-brain barrier to directly target LML.

[0259] CAR-T cell therapy has been shown to successfully treat certain types of cancers, as a patient’s own T cells are engineered to recognize tumor-specific proteins - harnessing the patient’s immune system to fight off cancer. However, in some patients CAR-T cell therapy is not effective either due to tumor mutations or rejection of the CAR-T cells. Additionally, CAR-T cell therapy can have cytotoxic effects. In some embodiments, LML is associated with CAR-T cytotoxicity. In some embodiments, LML is associated with neurodegeneration.

[0260] In some embodiments, administering to the subject the composition of any of the preceding aspects, wherein the composition comprises sodium phenylbutyrate and a pharmaceutically acceptable carrier, reduces LML, in a subject, compared to an untreated control. In some embodiments, administering to the subject the composition of any of the preceding aspects, reduces BCKA accumulation in a sample of cerebrospinal fluid obtained from the subject, compared to an untreated control. In some embodiments, administering to the subject the composition of any of the preceding aspects, reduces CAR-T cytotoxicity, in the subject, compared to an untreated control. In some embodiments, administering to the subject the composition of claim 1, reduces neurodegeneration, in the subject, compared to an untreated control. Sodium phenylbutyrate was tested as an anti-cancer therapeutic to improve CAR-T cell therapy efficacy and reduce associated cytotoxicity in a mouse model.

[0261] As shown here, sodium phenylbutyrate alone or in combination with other anti-cancer therapies has significantly better effects on subjects with cancer and / or cancer associated complications than current anti-cancer therapeutics alone. Sodium phenylbutyrate is the only proposed therapeutic for LML and is shown here to increase quality of life of a subject treated with the composition of any of the preceding aspects wherein the composition comprises sodium phenylbutyrate and a pharmaceutically acceptable carrier and wherein, the subject is a human.

[0262] The disclosed compositions can be used to treat any disease where uncontrolled cellular proliferation occurs such as cancers. A representative but non-limiting list of cancers that the disclosed compositions can be used to treat is the following: acoustic neuroma, adenocarcinoma, adrenal gland cancer, anal cancer, angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma), appendix cancer, benign monoclonal gammopathy, biliary cancer (e.g., cholangiocarcinoma), bladder cancer, breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast,Attorney Docket No. 10110-466WO1

[0263] mammary cancer, medullary carcinoma of the breast), brain cancer (e.g., meningioma; glioma, e.g., astrocytoma, oligodendroglioma; medulloblastoma), bronchus cancer, carcinoid tumor, cervical cancer (e.g., cervical adenocarcinoma), choriocarcinoma, chordoma, craniopharyngioma, colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma), epithelial carcinoma, ependymoma, endotheliosarcoma (e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma), endometrial cancer (e.g., uterine cancer, uterine sarcoma), esophageal cancer (e.g., adenocarcinoma of the esophagus, Barrett’s adenocarcinoma), Ewing's sarcoma, eye cancer (e.g., intraocular melanoma, retinoblastoma), familiar hypereosinophilia, gall bladder cancer, gastric cancer (e.g., stomach adenocarcinoma), gastrointestinal stromal tumor (GIST), head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma (OSCC), throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)), hematopoietic cancers (e.g., leukemia such as acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chronic lymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL); lymphoma such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma (DLBCL)), follicular lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas (e.g., mucosa-associated lymphoid tissue (MALT) lymphomas, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (i.e., “Waldenstrom's macroglobulinemia”), hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma and primary central nervous system (CNS) lymphoma; and T-cell NHL such as precursor T-lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T- cell lymphoma (CTCL) (e.g., mycosis fungiodes, Sezary syndrome), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplastic large cell lymphoma); a mixture of one or more leukemia / lymphoma as described above; and multiple myeloma (MM)), heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease), hemangioblastoma, inflammatory myofibroblastic tumors, immunocytic amyloidosis, kidney cancer (e.g., nephroblastoma a.k.a. Wilms' tumor, renal cellAttorney Docket No. 10110-466WO1

[0264] carcinoma), liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma), lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung), leiomyosarcoma (LMS), mastocytosis (e.g., systemic mastocytosis), myelodysplastic syndrome (MDS), mesothelioma, myeloproliferative disorder (MPD) (e.g., polycythemia Vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)), neuroblastoma, neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis), neuroendocrine cancer (e.g., gastroenteropancreatic neuroendocrine tumor (GEP-NET), carcinoid tumor), osteosarcoma, ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma), papillary adenocarcinoma, pancreatic cancer (e.g., pancreatic adenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors), penile cancer (e.g., Paget's disease of the penis and scrotum), pinealoma, primitive neuroectodermal tumor (PNT), prostate cancer (e.g., prostate adenocarcinoma), rectal cancer, rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)), small bowel cancer (e.g., appendix cancer), soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma), sebaceous gland carcinoma, sweat gland carcinoma, synovioma, testicular cancer (e.g., seminoma, testicular embryonal carcinoma), thyroid cancer (e.g., papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC), medullary thyroid cancer), urethral cancer, vaginal cancer and vulvar cancer (e.g., Paget's disease of the vulva).

[0265] It is understood and herein contemplated that the disclosed treatment regimens can used alone or in combination with any anti-cancer therapy known in the art including, but not limited to Abemaciclib, Abiraterone Acetate, ABITREXATE® (Methotrexate), ABRAXANE® (Paclitaxel Albumin- stabilized Nanoparticle Formulation), ABVD, AB VE, ABVE-PC, AC, AC-T, ADCETRIS® (Brentuximab Vedotin), ADE, Ado-Trastuzumab Emtansine, ADRIAMYCIN® (Doxorubicin Hydrochloride), Afatinib Dimaleate, AFINITOR® (Everolimus), AKYNZEO® (Netupitant and Palonosetron Hydrochloride), ALDARA® (Imiquimod), Aldesleukin, ALECENSA® (Alectinib), Alectinib, Alemtuzumab, ALIMTA® (Pemetrexed Disodium), ALIQOPA® (Copanlisib Hydrochloride), ALKERAN™ for Injection (Melphalan Hydrochloride), ALKERAN™Attorney Docket No. 10110-466WO1

[0266] Tablets (Melphalan), ALOXI® (Palonosetron Hydrochloride), ALUNBRIG® (Brigatinib), AMBOCHLORIN® (Chlorambucil), AMBOCLORIN® (Chlorambucil), Amifostine, Aminolevulinic Acid, Anastrozole, Aprepitant, AREDIA® (Pamidronate Disodium), ARIMIDEX® (Anastrozole), AROMASIN® (Exemestane), ARRANON® (Nelarabine), Arsenic Trioxide, ARZERRA® (Ofatumumab), Asparaginase Erwinia chrysanthemi, Atezolizumab, AVASTIN® (Bevacizumab), Avelumab, Axitinib, Azacitidine, BAVENCIO® (Avelumab), BEACOPP, BECENUM® (Carmustine), BELEODAQ® (Belinostat), Belinostat, Bendamustine Hydrochloride, BEP, BESPONSA® (Inotuzumab Ozogamicin), Bevacizumab, Bexarotene, BEXXAR® (Tositumomab and Iodine 1 131 Tositumomab), Bicalutamide, BICNU® (Carmustine), Bleomycin, Blinatumoniab, BLINCYTO® (Blinatumoniab), Bortezomib, BOSULIF® (Bosutinib), Bosutinib, Brentuximab Vedotin, Brigatinib, BuMel, Busulfan, BUSULFEX® (Busulfan), Cabazitaxel, CABOMETYX® (Cabozantinib-S-Malate), Cabozantinib-S-Malate, CAF, CAMPATH® (Alemtuzumab), CAMPTOSAR® (Irinotecan Hydrochloride), Capecitabine, CAPOX, CARAC® (Fluorouracil-Topical), Carboplatin, CARBOPLATIN-TAXOL, Carfilzomib, CARMUBRIS® (Carmustine), Carmustine, Carmustine Implant, CASODEX® (Bicalutamide), CEM, Ceritinib, CERUBIDINE® (Daunorubicin Hydrochloride), CERVARIX® (Recombinant HPV Bivalent Vaccine), Cetuximab, CEV, Chlorambucil, CHLORAMBUCIL -PREDNISONE, CHOP, Cisplatin, Cladribine, CLAFEN® (Cyclophosphamide), Clofarabine, CLOFAREX® (Clofarabine), CLOLAR® (Clofarabine), CMF, Cobimetinib, COMETRIQ® (Cabozantinib-S-Malate), Copanlisib Hydrochloride, COPDAC, COPP, COPP-ABV, COSMEGEN® (Dactinomycin), COTELLIC® (Cobimetinib), Crizotinib, CVP, Cyclophosphamide, CYFOS® (Ifosfamide), CYRAMZA® (Ramucirumab), Cytarabine, Cytarabine Liposome, CYTOSAR-U® (Cytarabine), CYTOXAN® (Cyclophosphamide), Dabrafenib, Dacarbazine, DACOGEN® (Decitabine), Dactinomycin, Daratumumab, DARZALEX® (Daratumumab), Dasatinib, Daunorubicin Hydrochloride, Daunorubicin Hydrochloride and Cytarabine Liposome, Decitabine, Defibrotide Sodium, DEFITELIO® (Defibrotide Sodium), Degarelix, Denileukin Diftitox, Denosumab, DEPOCYT® (Cytarabine Liposome), Dexamethasone, Dexrazoxane Hydrochloride, Dinutuximab, Docetaxel, DOXIL® (Doxorubicin Hydrochloride Liposome), Doxorubicin Hydrochloride, Doxorubicin Hydrochloride Liposome, DOX-SL® (Doxorubicin Hydrochloride Liposome), DTIC-DOME® (Dacarbazine), Durvalumab, EFUDEX® (Fluorouracil-Topical), ELITEK® (Rasburicase), ELLENCE® (Epirubicin Hydrochloride), Elotuzumab, ELOXATIN® (Oxaliplatin),Attorney Docket No. 10110-466WO1

[0267] Eltrombopag Olamine, EMEND® (Aprepitant), EMPLICITI® (Elotuzumab), Enasidenib Mesylate, Enzalutamide, Epirubicin Hydrochloride, EPOCH, ERBITUX® (Cetuximab), Eribulin Mesylate, ERIVEDGE® (Vismodegib), Erlotinib Hydrochloride, ERWINAZE® (Asparaginase Erwinia chrysanthemi), ETHYOL® (Amifostine), Etopophos® ETOPOPHOS® (Etoposide Phosphate), Etoposide, Etoposide Phosphate, EVACET® (Doxorubicin Hydrochloride Liposome), Everolimus, EVISTA® (Raloxifene Hydrochloride), EVOMELA® (Melphalan Hydrochloride), Exemestane, 5-FU® (Fluorouracil Injection), 5-FU® (Fluorouracil-Topical), FARESTON® (Toremifene), FARYDAK® (Panobinostat), FASLODEX® (Fulvestrant), FEC, FEMARA® (Letrozole), Filgrastim, FLUDARA® (Fludarabine Phosphate), Fludarabine Phosphate, FLUOROPLEX® (Fluorouracil— Topical), Fluorouracil Injection, Fluorouracil-Topical, Flutamide, FOLEX® (Methotrexate), FOL, EX PFS® (Methotrexate), FOLFIRI, FOLFIRI-BEVACIZUMAB, FOLFIRI-CETUXIMAB, EOT, FIRINOX, FOLFOX, FOLOTYN® (Pralatrexate), FU-LV, Fulvestrant, GARDASIL® (Recombinant HPV Quadrivalent Vaccine), GARDASIL 9® (Recombinant HPV Nonavalent Vaccine), GAZYVA® (Obinutuzumab), Gefitinib, Gemcitabine Hydrochloride, GEMCITABINE-CISPLATIN, GEMCITABINE-OXALIPLATIN, Gemtuzumab Ozogamicin, GEMZAR® (Gemcitabine Hydrochloride), GILOTRIF® (Afatinib Dimaleate), GLEEVEC® (Imatinib Mesylate), GLIADEL® (Carmustine Implant), GLIADEL, WAFER® (Carmustine Implant), Glucarpidase, Goserelin Acetate, HALAVEN® (Eribulin Mesylate), HEMANGEOL® (Propranolol Hydrochloride), HERCEPTIN® (Trastuzumab), HPV Bivalent Vaccine, Recombinant, HPV Nonavalent Vaccine, Recombinant, HPV Quadrivalent Vaccine, Recombinant, HYCAMTIN® (Topotecan Hydrochloride), HYDREA® (Hydroxyurea), Hydroxyurea, Hyper-CVAD, IBRANCE® (Palbociclib), Ibritumomab Tiuxetan, Ibrutinib, ICE, ICLUSIG® (Ponatinib Hydrochloride), IDAMYCIN® (Idarubicin Hydrochloride), Idarubicin Hydrochloride, Idelalisib, IDHIFA® (Enasidenib Mesylate), IFEX® (Ifosfamide), Ifosfamide, IFOSFAMIDUM® (Ifosfamide), IL-2 (Aldesleukin), Imatinib Mesylate, IMBRUVICA® (Ibrutinib), IMFINZI® (Durvalumab), Imiquimod, IMLYGIC® (Talimogene Laherparepvec), INLYTA® (Axitinib), Inotuzumab Ozogamicin, Interferon Alfa-2b, Recombinant, Interleukin-2 (Aldesleukin), INTRON A® (Recombinant Interferon Alfa-2b), Iodine I 131 Tositumomab and Tositumomab, Ipilimumab, IRESSA® (Gefitinib), Irinotecan Hydrochloride, Irinotecan Hydrochloride Liposome, ISTODAX® (Romidepsin), Ixabepilone, Ixazomib Citrate, IXEMPRA® (Ixabepilone), JAKAFI® (Ruxolitinib Phosphate), JEB, JEVTANA® (Cabazitaxel), KADCYLA® (Ado-TrastuzumabAttorney Docket No. 10110-466WO1

[0268] Emtansine), KEOXIFENE® (Raloxifene Hydrochloride), KEPIVANCE® (Palifermin), KEYTRUDA® (Pembrolizumab), KISQALI® (Ribociclib), KYMRIAH® (Tisagenlecleucel), KYPROLIS® (Carfilzomib), Lanreotide Acetate, Lapatinib Ditosylate, LARTRUVO® (Olaratumab), Lenalidomide, Lenvatinib Mesylate, LENVIMA® (Lenvatinib Mesylate), Letrozole, Leucovorin Calcium, LEUKERAN® (Chlorambucil), Leuprolide Acetate, LEUSTATIN® (Cladribine), LEVULAN® (Aminolevulinic Acid), LINFOLIZIN® (Chlorambucil), LIPODOX® (Doxorubicin Hydrochloride Liposome), Lomustine, LONSURF® (Trifluridine and Tipiracil Hydrochloride), LUPRON® (Leuprolide Acetate), LUPRON DEPOT® (Leuprolide Acetate), LUPRON DEPOT-PED® (Leuprolide Acetate), LYNPARZA® (Olaparib), MARQIBO® (Vincristine Sulfate Liposome), MATULANE® (Procarbazine Hydrochloride), Mechlorethamine Hydrochloride, Megestrol Acetate, MEKINIST® (Trametinib), Melphalan, Melphalan Hydrochloride, Mercaptopurine, Mesna, MESNEX® (Mesna), METHAZOLASTONE® (Temozolomide), Methotrexate, METHOTREXATE LPF® (Methotrexate), Methylnaltrexone Bromide, MEXATE® (Methotrexate), MEXATE-AQ® (Methotrexate), Midostaurin, Mitomycin C, Mitoxantrone Hydrochloride, MITOZYTREX® (Mitomycin C), MOPP, MOZOBIL® (Plerixafor), MUSTARGEN® (Mechlorethamine Hydrochloride), MUTAMYCIN® (Mitomycin C), MYLERAN® (Busulfan), MYLOSAR® (Azacitidine), MYLOTARG® (Gemtuzumab Ozogamicin), NANOPARTICLE PACLITAXEL® (Paclitaxel Albumin-stabilized Nanoparticle Formulation), NAVELBINE® (Vinorelbine Tartrate), Necitumumab, Nelarabine, NEOSAR® (Cyclophosphamide), Neratinib Maleate, NERLYNX® (Neratinib Maleate), Netupitant and Palonosetron Hydrochloride, NEULASTA® (Pegfilgrastim), NEUPOGEN® (Filgrastim), NEXAVAR® (Sorafenib Tosylate), NILANDRON® (Nilutamide), Nilotinib, Nilutamide, NINLARO® (Ixazomib Citrate), Niraparib Tosylate Monohydrate, Nivolumab, NOLVADEX® (Tamoxifen Citrate), NPLATE® (Romiplostim), Obinutuzumab, ODOMZO® (Sonidegib), OEPA, Ofatumumab, OFF, Olaparib, Olaratumab, Omacetaxine Mepesuccinate, ONCASPAR® (Pegaspargase), Ondansetron Hydrochloride, ONIVYDE® (Irinotecan Hydrochloride Liposome), ONTAK® (Denileukin Diftitox), OPDIVO® (Nivolumab), OPPA, Osimertinib, Oxaliplatin, Paclitaxel, Paclitaxel Albumin-stabilized Nanoparticle Formulation, PAD, Palbociclib, Palifermin, Palonosetron Hydrochloride, Palonosetron Hydrochloride and Netupitant, Pamidronate Disodium, Panitumumab, Panobinostat, PARAPLAT® (Carboplatin), PARAPLATIN® (Carboplatin), Pazopanib Hydrochloride, PCV, PEB, Pegaspargase, Pegfilgrastim, Peginterferon Alfa-2b, PEG-INTRON® (Peginterferon Alfa-Attorney Docket No. 10110-466WO1

[0269] 2b), Pembrolizumab, Pemetrexed Disodium, PERJETA® (Pertuzumab), Pertuzumab, PLATINOL® (Cisplatin), PLATINOL-AQ® (Cisplatin), Plerixafor, Pomalidomide, POMALYST® (Pomalidomide), Ponatinib Hydrochloride, PORTRAZZA® (Necitumumab), Pralatrexate, Prednisone, Procarbazine Hydrochloride, PROLEUKIN® (Aldesleukin), PROLIA® (Denosumab), PROMACTA® (Eltrombopag Olamine), Propranolol Hydrochloride, PROVENGE® (Sipuleucel-T), PURINETHOL® (Mercaptopurine), PURIXAN® (Mercaptopurine), Radium 223 Dichloride, Raloxifene Hydrochloride, Ramucirumab, Rasburicase, R-CHOP, R-CVP, Recombinant Human Papillomavirus (HPV) Bivalent Vaccine, Recombinant Human Papillomavirus (HPV) Nonavalent Vaccine, Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine, Recombinant Interferon Alfa-2b, Regorafenib, RELISTOR® (Methylnaltrexone Bromide), R-EPOCH, REVLIMID® (Lenalidomide), RHEUMATREX® (Methotrexate), Ribociclib, R-ICE, RITUXAN® (Rituximab), RITUXAN HYCELA® (Rituximab and Hyaluronidase Human), Rituximab, Rituximab and, Hyaluronidase Human,, Rolapitant Hydrochloride, Romidepsin, Romiplostim, RUBIDOMYCIN® (Daunorubicin Hydrochloride), RUBRACA® (Rucaparib Camsylate), Rucaparib Camsylate, Ruxolitinib Phosphate, RYDAPT® (Midostaurin), Sclerosol Intrapleural Aerosol (Talc), Siltuximab, Sipuleucel-T, SOMATULINE DEPOT® (Lanreotide Acetate), Sonidegib, Sorafenib Tosylate, SPRYCEL® (Dasatinib), STANFORD V, Sterile Talc Powder (Talc), STERITALC® (Talc), STIVARGA® (Regorafenib), Sunitinib Malate, SUTENT® (Sunitinib Malate), SYLATRON® (Peginterferon Alfa- 2b), SYLVANT® (Siltuximab), Synribo SYNRIBO® (Omacetaxine Mepesuccinate), TABLOID® (Thioguanine), TAC, TAFINLAR® (Dabrafenib), TAGRISSO® (Osimertinib), Talc, Talimogene Laherparepvec, Tamoxifen Citrate, TARABINE PFS® (Cytarabine), TARCEVA® (Erlotinib Hydrochloride), TARGRETIN® (Bexarotene), TASIGNA® (Nilotinib), TAXOL® (Paclitaxel), 'TAXOTERE® (Docetaxel), TECENTRIQ® (Atezolizumab), TEMODAR® (Temozolomide), Temozolomide, Temsirolimus, Thalidomide, THALOMID® (Thalidomide), Thioguanine, Thiotepa, Tisagenlecleucel, TOLAK® (Fluorouracil- Topical), Topotecan Hydrochloride, Toremifene, TORISEL® (Temsirolimus), Tositumomab and Iodine I 131 Tositumomab, TOTECT® (Dexrazoxane Hydrochloride), TPF, Trabectedin, Trametinib, Trastuzumab, TREANDA® (Bendamustine Hydrochloride), Trifluridine and Tipiracil Hydrochloride, TRISENOX® (Arsenic Trioxide), TYKERB® (Lapatinib Ditosylate), UNITUXIN® (Dinutuximab), Uridine Triacetate, VAC, Vandetanib, VAMP, VARUBI® (Rolapitant Hydrochloride), VECTIBIX® (Panitumumab), VelP,Attorney Docket No. 10110-466WO1

[0270] VELBAN® (Vinblastine Sulfate), VELCADE® (Bortezomib), VELSAR® (Vinblastine Sulfate), Vemurafenib, VENCLEXTA® (Venetoclax), Venetoclax, VERZENIO® (Abemaciclib), VIADUR® (Leuprolide Acetate), VIDAZA® (Azacitidine), Vinblastine Sulfate, VINCASAR PFS® (Vincristine Sulfate), Vincristine Sulfate, Vincristine Sulfate Liposome, Vinorelbine Tartrate, VIP, Vismodegib, VISTOGARD® (Uridine Triacetate), VORAXAZE® (Glucarpidase), Vorinostat, VOTRIENT® (Pazopanib Hydrochloride), VYXEOS® (Daunorubicin Hydrochloride and Cytarabine Liposome), WELLCOVORIN® (Leucovorin Calcium), XALKORI® (Crizotinib), XELODA® (Capecitabine), XELIRI, XELOX, XGEVA® (Denosumab), XOFIGO® (Radium 223 Bichloride), XTANDI® (Enzalutamide), YERVOY® (Ipilimumab), YONDELIS® (Trabectedin), ZALTRAP® (Ziv-Aflibercept), ZARXIO® (Filgrastim), ZEJULA® (Niraparib Tosylate Monohydrate), ZELBORAF® (Vemurafenib), ZEVALIN® (Ibritumomab Tiuxetan), ZINECARD® (Dexrazoxane Hydrochloride), Ziv-Aflibercept, ZOFRAN® (Ondansetron Hydrochloride), ZOLADEX® (Goserelin Acetate), Zoledronic Acid, ZOLINZA® (Vorinostat), ZOMETA® (Zoledronic Acid), ZYDELIG® (Idelalisib), ZYKADIA® (Ceritinib), and / or ZYTIGA® (Abiraterone Acetate). The treatment methods can include or further include checkpoint inhibitors including, but are not limited to antibodies that block PD-1 (such as, for example, Nivolumab (BMS-936558 or MDX1106), pembrolizumab, cemiplimab, CT-011, MK-3475), PD-L1 (such as, for example, atezolizumab, avelumab, durvalumab, MDX-1105 (BMS-9365.59), MPDL3280A, or MSB0010718C), PD-L2 (such as, for example, rHIgM12B7), CTLA-4 (such as, for example, Ipilimumab (MDX-010), Tremelimumab (CP-675,206)), IDO, B7-H3 (such as, for example, MGA271, MGD009, omburtamab), B7-H4, B7-H3, T cell immunoreceptor with Ig and ITIM domains (TIGIT)(such as, for example BMS-986207, OMP-313M32, MK-7684, AB-154, ASP-8374, MTIG7192A, or PVSRIPO), CD96, B- and T-lymphocyte attenuator (BTLA), V-domain Ig suppressor of T cell activation (VISTA)(such as, for example, JNJ-61610588, CA-170), TIM3 (such as, for example, TSR-022, MBG453, Sym023, INCAGN2390, LY3321367, BMS-986258, SHR-1702, RO7121661), LAG-3 (such as, for example, BMS-986016, LAG525, MK-4280, REGN3767, TSR-033, BI754111, Sym022, FS118, MGD013, and Immutep).

[0271] “Concurrent administration”, “administration in combination”, “simultaneous administration” or “administered simultaneously” as used herein, means that the compounds are administered at the same point in time or essentially immediately following one another. IN the latter case, the two compounds are administered at times sufficiently close that theAttorney Docket No. 10110-466WO1

[0272] results observed are indistinguishable from those achieved when the compounds are administered at the same point in time.

[0273] “Systemic administration” refers to the introducing or delivering to a subject an agent via a route which introduces or delivers the agent to extensive areas of the subject’s body (e.g. greater than 50% of the body), for example through entrance into the circulatory or lymph systems.

[0274] By contrast, “local administration” refers to the introducing or delivery to a subject an agent via a route which introduces or delivers the agent to the area or area immediately adjacent to the point of administration and does not introduce the agent systemically in a therapeutically significant amount. For example, locally administered agents are easily detectable in the local vicinity of the point of administration but are undetectable or detectable at negligible amounts in distal parts of the subject’s body. Administration includes self-administration and the administration by another.

[0275] The disclosed composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier may be administered in such amounts, time, and route deemed necessary in order to achieve the desired result. The exact amount of the disclosed composition will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease (such as, for example, LML), the particular composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier, disclosed herein, its mode of administration, its mode of activity, and the like. The disclosed composition sodium phenylbutyrate and a pharmaceutically acceptable carrier is preferably formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disease (such as, for example, LML) being treated and the severity of the disease (such as, for example, LML); the activity of the disclosed composition employed; the specific composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier, employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier employed; the duration of the treatment; drugs used in combination or coincidental with the specific composition comprising sodiumAttorney Docket No. 10110-466WO1

[0276] phenylbutyrate and a pharmaceutically acceptable carrier employed; and like factors well known in the medical arts.

[0277] The disclosed composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier may be administered by any route. In some embodiments, the composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier is administered via a variety of routes, including intrathecal, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and / or drops), mucosal, nasal, buccal, enteral, sublingual; by intratracheal instillation, bronchial instillation, and / or inhalation; and / or as an oral spray, nasal spray, and / or aerosol. In general, the most appropriate route of administration will depend upon a variety of factors including the nature of the disclosed composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier (e.g., its stability in the environment of the gastrointestinal tract), the condition of the subject (e.g., whether the subject is able to tolerate oral administration), etc.

[0278] The exact amount of the disclosed composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier required to achieve a therapeutically or prophylactically effective amount will vary from subject to subject, depending on species, age, and general condition of a subject, severity of the side effects, identity of the particular compound(s), mode of administration, and the like. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.

[0279] The concentration of active agent(s) can vary widely and will be selected primarily based on activity of the active ingredient(s), body weight and the like in accordance with the particular mode of administration selected and the patient's needs. Concentrations, however, will typically be selected to provide dosages ranging from about 0.1 or 1 mg / kg / day to about 50 mg / kg / day and sometimes higher. Typical dosages range from about 3 mg / kg / day to about 3.5 mg / kg / day, preferably from about 3.5 mg / kg / day to about 7.2 mg / kg / day, more preferably from about 7.2 mg / kg / day to about 11.0 mg / kg / day, and most preferably from about 11.0 mg / kg / day to about 15.0 mg / kg / day. In certain preferred embodiments, dosages range from about 10 mg / kg / day to about 50 mg / kg / day. In certain embodiments, dosages range from about 20 mg to about 50 mg given orally twice daily. ItAttorney Docket No. 10110-466WO1

[0280] will be appreciated that such dosages may be varied to optimize a therapeutic and / or prophylactic regimen in a particular subject or group of subjects.

[0281] In one aspect, disclosed herein is composition of any preceding aspect comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier selected from an excipient, a diluent, a salt, a buffer, a stabilizer, a lipid, an emulsion, a nanoparticle, and a cream. One or more active agents (e.g. sodium phenylbutyrate) can be administered in the “native” form or, if desired in the form of salts, esters, amides, prodrugs, or a derivative that is pharmacologically suitable. Salts, esters, amides, prodrugs, and other derivatives of the active agents can be prepared using standards procedures known to those skilled in the art of synthetic organic chemistry and described, for example, by March (1992) Advanced Organic Chemistry; Reactions, Mechanisms, and Structure, 4thEd. N. Y. Wiley-Interscience.

[0282] In some embodiments, the disclosed composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier can be prepared as a “concentrate”, e.g. in a storage container of a premeasure volume and / or a predetermined amount ready for dilution, or in a soluble capsule ready for addition to a specified volume of water, saline, alcohol, hydrogen peroxide, or other diluent.

[0283] In some embodiments, the disclosed composition comprising sodium phenyl butyrate and a pharmaceutically acceptable carrier is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or more times. In some embodiments, the disclosed composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier is administered daily. In some embodiments, the disclosed composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier is administered every day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, or more. In some embodiments, the disclosed composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier is administered every week, every 2 weeks, every 3 weeks, every 4 weeks, or more. In some embodiments, the disclosed composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier is administered every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, every 12 months, or more. In some embodiments, the disclosed composition comprising sodium phenylbutyrate and aAttorney Docket No. 10110-466WO1

[0284] pharmaceutically acceptable carrier is administered every year, every 2 years, every 3 years, every 4 years, every 5 years, or more.

[0285] It is understood and herein contemplated that while a single administration of the compounds of the disclosed anti-cancer combination therapies (i.e., sodium phenylbutyrate and / or CAR-T cells) would be ideal, not every patient will respond in the same manner. Thus, in one aspect, disclosed herein are anti-cancer combination therapies methods treating, preventing, reducing, and / or inhibiting a cancer; wherein the sodium phenylbutyrate is administered at least, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20, 21, 22, 23, or 24 times a day or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 times per week for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks. Also disclosed herein are anti-cancer combination therapies methods treating, preventing, reducing, and / or inhibiting a cancer of any preceding aspect; wherein the at least one anti-cancer agent is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20, 21, 22, 23, or 24 times a day or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 times per week for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks. It is further understood and herein contemplated that the order and duration of the administered components can vary as appropriate for the subject being treated. In one aspect, disclosed herein are anti-cancer combination therapies methods treating, preventing, reducing, and / or inhibiting a cancer; wherein the sodium phenylbutyrate is administered at least 1, 2, 3, 4,, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36 hours, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, 30, 31, 45 days, 2, 3, 4, 5, 6 months prior to administration of the pulsed dendritic cells.

[0286] D. Method of treating cancer and / or cancer associated complications

[0287] In one aspect, disclosed herein, is a method of treating a subject with a cancer and / or a cancer-associated complication comprising administering a composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier to the subject.

[0288] In some embodiments, the composition is administered to the subject, intrathecally. In some embodiments, the composition is administered to the subject at a therapeutically effective amount. In some embodiments, the method further comprises administering to the subject at least one anti-cancer agent such as, for example cytarabine, methotrexate, rituximab, and thiotepa.

[0289] In some embodiments, the cancer-associated complication is Leptomeningeal Disease (LMD). In some embodiments, the cancer-associated complication is branched-chain keto acids (BCKA) accumulation in a sample of cerebrospinal fluid obtained from theAttorney Docket No. 10110-466WO1

[0290] subject. Branched -chain keto acids (BCKA) accumulation is also observed in patients with leptomeningeal disease from melanoma and breast cancer. It is observed that BCKA exerts an immunosuppressive and neurodegenerative microenvironment in patients with CNS leptomeningeal lymphoma. BCKA is also cytotoxic and inhibits T-cell proliferation thus LMD CSF shows few active and proliferating T cells. BCKAs inhibit the secretion of pro- inflammatory cytokines from T-cells.

[0291] Cancer associated complications result in a drastic reduction in quality of life. Examples of severe cancer complications include but are not limited to neurodegeneration and Leptomeningeal Disease. LMD occurs when cancer spreads to the cerebral spinal fluid and surrounding meninges. Neurodegeneration associated with cancer can be a direct symptom of the cancer, a symptom of complications from cancer, i.e. LMD, or a symptom of cancer treatments. New evidence shows an increased concentration of branched-chain a-keto acids as a result of LMD and certain cancer treatments. Thus, a method of reducing BCKA levels in cancer and LMD patients, treatment with sodium phenylbutyrate, was tested to ameliorate cancer associated complications. As LMD affects the spinal column, traditional methods of drug administration are limited in therapeutic efficacy due to the presence of the blood-brain barrier. Intrathecal administration, directly into the CSF, removes the impediment of the blood -brain barrier to directly target LMD.

[0292] C AR-T cell therapy has been shown to successfully treat certain types of cancers, as a patient’s own T cells are engineered to recognize tumor-specific proteins - harnessing the patient’s immune system to fight off cancer. However, in some patients CAR-T cell therapy is not effective either due to tumor mutations or rejection of the CAR-T cells. Additionally, CAR-T cell therapy can have cytotoxic effects. In some embodiments, the cancer-associated complication is CAR-T cytotoxicity. In some embodiments, the cancer-associated complication is neurodegeneration.

[0293] In some embodiments, administering to the subject the composition of any of the preceding aspects, wherein the composition comprises sodium phenylbutyrate and a pharmaceutically acceptable carrier, reduces LMD, in a subject, compared to an untreated control. In some embodiments, administering to the subject the composition of any of the preceding aspects, reduces BCKA accumulation in a sample of cerebrospinal fluid obtained from the subject, compared to an untreated control. In some embodiments, administering to the subject the composition of any of the preceding aspects, reduces CAR-T cytotoxicity, in the subject, compared to an untreated control. In some embodiments, administering to the subject the composition of claim 1, reduces neurodegeneration, in the subject, compared toAttorney Docket No. 10110-466WO1

[0294] an untreated control. Sodium phenylbutyrate was tested as an anti-cancer therapeutic to improve CAR-T cell therapy efficacy and reduce associated cytotoxicity in a mouse model.

[0295] As shown here, sodium phenylbutyrate alone or in combination with other anti¬ cancer therapies has significantly better effects on subjects with cancer and / or cancer associated complications than current anti-cancer therapeutics alone. Sodium phenylbutyrate is the only proposed therapeutic for LMD and is shown here to increase quality of life of a subject treated with the composition of any of the preceding aspects wherein the composition comprises sodium phenylbutyrate and a pharmaceutically acceptable carrier and wherein, the subject is a human.

[0296] The disclosed compositions can be used to treat any disease where uncontrolled cellular proliferation occurs such as cancers. A representative but non-limiting list of cancers that the disclosed compositions can be used to treat is the following: acoustic neuroma, adenocarcinoma, adrenal gland cancer, anal cancer, angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma), appendix cancer, benign monoclonal gammopathy, biliary cancer (e.g., cholangiocarcinoma), bladder cancer, breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast), brain cancer (e.g., meningioma; glioma, e.g., astrocytoma, oligodendroglioma; medulloblastoma), bronchus cancer, carcinoid tumor, cervical cancer (e.g., cervical adenocarcinoma), choriocarcinoma, chordoma, craniopharyngioma, colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma), epithelial carcinoma, ependymoma, endotheliosarcoma (e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma), endometrial cancer (e.g., uterine cancer, uterine sarcoma), esophageal cancer (e.g., adenocarcinoma of the esophagus, Barrett's adenocarcinoma), Ewing's sarcoma, eye cancer (e.g., intraocular melanoma, retinoblastoma), familiar hypereosinophilia, gall bladder cancer, gastric cancer (e.g., stomach adenocarcinoma), gastrointestinal stromal tumor (GIST), head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma (OSCC), throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)), hematopoietic cancers (e.g., leukemia such as acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chronic lymphocytic leukemia (CLL) (e.g., B-cell CI. L, T-cell CLL); lymphoma such as Hodgkin lymphoma (HL) (e.g., B-cell TIL,, T-cell HL) and non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large ceil lymphomaAttorney Docket No. 10110-466WO1

[0297] (DLCL) (e.g., diffuse large B-cell lymphoma (DLBCL)), follicular lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas (e.g., mucosa-associated lymphoid tissue (MALT) lymphomas, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (i.e., “Waldenstrom's macroglobulinemia”), hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B -lymphoblastic lymphoma and primary central nervous system (CNS) lymphoma; and T-cell NHL such as precursor T-lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g., mycosis fungiodes, Sezary syndrome), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplastic large cell lymphoma); a mixture of one or more leukemia / lymphoma as described above; and multiple myeloma (MM)), heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease), hemangioblastoma, inflammatory myofibroblastic tumors, immunocytic amyloidosis, kidney cancer (e.g., nephroblastoma a.k.a. Wilms' tumor, renal cell carcinoma), liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma), lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung), leiomyosarcoma (LMS), mastocytosis (e.g., systemic mastocytosis), myelodysplastic syndrome (MDS), mesothelioma, myeloproliferative disorder (MPD) (e.g., polycythemia Vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)), neuroblastoma, neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis), neuroendocrine cancer (e.g., gastroenteropancreatic neuroendocrine tumor (GEP-NET), carcinoid tumor), osteosarcoma, ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma), papillary adenocarcinoma, pancreatic cancer (e.g., pancreatic adenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors), penile cancer (e.g., Paget's disease of the penis and scrotum), pinealoma, primitive neuroectodermal tumor (PNT), prostate cancer (e.g., prostate adenocarcinoma), rectal cancer, rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)), small bowel cancer (e.g., appendix cancer), soft tissue sarcoma (e.g., malignant fibrousAttorney Docket No. 10110-466WO1

[0298] histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma), sebaceous gland carcinoma, sweat gland carcinoma, synovioma, testicular cancer (e.g., seminoma, testicular embryonal carcinoma), thyroid cancer (e.g., papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC), medullary thyroid cancer), urethral cancer, vaginal cancer and vulvar cancer (e.g., Paget's disease of the vulva).

[0299] It is understood and herein contemplated that the disclosed treatment regimens can used alone or in combination with any anti-cancer therapy known in the art including, but not limited to Abemaciclib, Abiraterone Acetate, ABITREXATE® (Methotrexate), ABRAXANE® (Paclitaxel Albumin- stabilized Nanoparticle Formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, ADCETRIS® (Brentuximab Vedotin), ADE, Ado-Trastuzumab Emtansine, ADRIAMYCIN® (Doxorubicin Hydrochloride), Afatinib Dimaleate, AFINITOR® (Everolimus), AKYNZEO® (Netupitant and Palonosetron Hydrochloride), ALDARA® (Imiquimod), Aldesleukin, ALECENSA® (Alectinib), Alectinib, Alemtuzumab, ALIMTA® (Pemetrexed Disodium), ALIQOPA® (Copanlisib Hydrochloride), ALKERAN™ for Injection (Melphalan Hydrochloride), ALKERAN™ Tablets (Melphalan), ALOXI® (Palonosetron Hydrochloride), ALUNBRIG® (Brigatinib), AMBOCHLORIN® (Chlorambucil), AMBOCLORIN® (Chlorambucil), Amifostine, Aminolevulinic Acid, Anastrozole, Aprepitant, AREDIA® (Pamidronate Di sodium), ARIMIDEX® (Anastrozole), AROMASIN® (Exemestane), ARRANON® (Nelarabine), Arsenic Trioxide, ARZERRA® (Ofatumumab), Asparaginase Erwinia chrysanthemi, Atezolizumab, AVASTIN® (Bevacizumab), Avelumab, Axitinib, Azacitidine, BAVENCIO® (Avelumab), BEACOPP, BECENUM® (Carmustine), BELEODAQ® (Belinostat), Belinostat, Bendamustine Hydrochloride, BEP, BESPONSA® (Inotuzumab Ozogamicin), Bevacizumab, Bexarotene, BEXXAR® (Tositumomab and Iodine I 131 Tositumomab), Bicalutamide, BICNU® (Carmustine), Bleomycin, Blinatumomab, BLINCYTO® (Blinatumomab), Bortezomib, BOSULIF® (Bosutinib), Bosutinib, Brentuximab Vedotin, Brigatinib, BuMel, Busulfan, BUSULFEX® (Busulfan), Cabazitaxel, CABOMETYX® (Cabozantinib-S-Malate), Cabozantinib-S-Malate, CAP, CAMPATH® (Alemtuzumab), CAMPTOSAR® (Irinotecan Hydrochloride), Capecitabine, CAPOX, CARAC® (Fluorouracil-Topical), Carboplatin, CARBOPLATIN-TAXOL, Carfilzomib, CARMUBRIS® (Carmustine), Carmustine, Carmustine Implant, CASODEX® (Bicalutamide), CEM, Ceritinib, CERUBIDINE® (Daunorubicin Hydrochloride), CERVARIX® (Recombinant HPV Bivalent Vaccine), Cetuximab, CEV,Attorney Docket No. 10110-466WO1

[0300] Chlorambucil, CHLORAMBUCIL-PREDNISONE, CHOP, Cisplatin, Cladribine, CLAFEN® (Cyclophosphamide), Clofarabine, CLOFAREX® (Clofarabine), CLOLAR® (Clofarabine), CMF, Cobimetinib, COMETRIQ® (Cabozantinib-S-Malate), Copanlisib Hydrochloride, COPDAC, COPP, COPP-ABV, COSMEGEN® (Dactinomycin), COTELLIC® (Cobimetinib), Crizotinib, CVP, Cyclophosphamide, CYFOS® (Ifosfamide), CYRAMZA® (Ramucirumab), Cytarabine, Cytarabine Liposome, CYTOSAR-U® (Cytarabine), CYTOXAN® (Cyclophosphamide), Dabrafenib, Dacarbazine, DACOGEN® (Decitabine), Dactinomycin, Daratumumab, DARZALEX® (Daratumumab), Dasatinib, Daunorubicin Hydrochloride, Daunorubicin Hydrochloride and Cytarabine Liposome, Decitabine, Defibrotide Sodium, DEFITELIO® (Defibrotide Sodium), Degarelix, Denileukin Diftitox, Denosumab, DEPOCYT® (Cytarabine Liposome), Dexamethasone, Dexrazoxane Hydrochloride, Dinutuximab, Docetaxel, DOXIL® (Doxorubicin Hydrochloride Liposome), Doxorubicin Hydrochloride, Doxorubicin Hydrochloride Liposome, DOX-SL® (Doxorubicin Hydrochloride Liposome), DTIC-DOME® (Dacarbazine), Durvalumab, EFUDEX® (Fluorouracil-Topical), ELITEK® (Rasburicase), ELLENCE® (Epirubicin Hydrochloride), Elotuzumab, ELOXATIN® (Oxaliplatin), Eltrombopag Olamine, EMEND® (Aprepitant), EMPLICITI® (Elotuzumab), Enasidenib Mesylate, Enzalutamide, Epirubicin Hydrochloride, EPOCH, ERBITUX® (Cetuximab), Eribulin Mesylate, ERIVEDGE® (Vismodegib), Erlotinib Hydrochloride, ERWINAZE® (Asparaginase Erwinia chrysanthemi), ETHYOL® (Amifostine), Etopophos® ETOPOPHOS® (Etoposide Phosphate), Etoposide, Etoposide Phosphate, EVACET® (Doxorubicin Hydrochloride Liposome), Everolimus, EVISTA® (Raloxifene Hydrochloride), EVOMELA® (Melphalan Hydrochloride), Exemestane, 5-FU® (Fluorouracil Injection), 5-FU® (Fluorouracil-Topical), FARESTON® (Toremifene), FARYDAK® (Panobinostat), FASLODEX® (Fulvestrant), FEC, FEMARA® (Letrozole), Filgrastim, FLUDARA® (Fludarabine Phosphate), Fludarabine Phosphate, FLUOROPLEX® (Fluorouracil— Topical), Fluorouracil Injection, Fluorouracil-Topical, Flutamide, FOLEX® (Methotrexate), FOLEX PFS® (Methotrexate), FOLFIRI, FOLFIRI-BEVACIZUMAB, FOLFIRI-CETUXIMAB, FOLFIRINOX, FOLFOX, FOLOTYN® (Pralatrexate), FU-LV, Fulvestrant, GARDASIL® (Recombinant HPV Quadrivalent Vaccine), GARDASIL 9® (Recombinant HPV Nonavalent Vaccine), GAZYVA® (Obinutuzumab), Gefitinib, Gemcitabine Hydrochloride, GEMCITABINE-CISPLATIN, GEMCITABINE-OXALIPLATIN, Gemtuzumab Ozogamicin, GEMZAR® (Gemcitabine Hydrochloride), GILOTRIF® (Afatinib Dimaleate), GLEEVEC® (Imatinib Mesylate),Attorney Docket No. 10110-466WO1

[0301] GLIADEL® (Carmustine Implant), GLIADEL WAFER® (Carmustine Implant), Glucarpidase, Goserelin Acetate, HALAVEN® (Eribulin Mesylate), HEMANGEOL® (Propranolol Hydrochloride), HERCEPTIN® (Trastuzumab), HPV Bivalent Vaccine, Recombinant, HPV Nonavalent Vaccine, Recombinant, HPV Quadrivalent Vaccine, Recombinant, HYCAMTIN® (Topotecan Hydrochloride), HYDREA® (Hydroxyurea), Hydroxyurea, Hyper-CVAD, IBRANCE® (Palbociclib), Ibritumomab Tiuxetan, Ibrutinib, ICE, ICLUSIG® (Ponatinib Hydrochloride), IDAMYCIN® (Idarubicin Hydrochloride), Idarubicin Hydrochloride, Idelalisib, IDHIFA® (Enasidenib Mesylate), IFEX® (Ifosfamide), Ifosfamide, IFOSFAMIDUM® (Ifosfamide), IL-2 (Aldesleukin), Imatinib Mesylate, IMBRUVICA® (Ibrutinib), IMFINZI® (Durvalumab), Imiquimod, IMLYGIC® (Talimogene Laherparepvec), INLYTA® (Axitinib), Inotuzumab Ozogamicin, Interferon Alfa- 2b, Recombinant, Interleukin-2 (Aldesleukin), INTRON A® (Recombinant Interferon Alfa- 2b), Iodine I 131 Tositumomab and Tositumomab, Ipilimumab, IRESSA® (Gefitinib), Irinotecan Hydrochloride, Irinotecan Hydrochloride Liposome, ISTODAX® (Romidepsin), Ixabepilone, Ixazomib Citrate, IXEMPRA® (Ixabepilone), JAKAFI® (Ruxolitinib Phosphate), JEB, JEVTANA® (Cabazitaxel), KADCYLA® (Ado-Trastuzumab Emtansine), KEOXIFENE® (Raloxifene Hydrochloride), KEPIVANCE® (Palifermin), KEYTRUDA® (Pembrolizumab), KISQALI® (Ribociclib), KYMRIAH® (Tisagenlecleucel), KYPROLIS® (Carfilzomib), Lanreotide Acetate, Lapatinib Di tosylate, LARTRUVO® (Olaratumab), Lenalidomide, Lenvatinib Mesylate, LENVIMA® (Lenvatinib Mesylate), Letrozole, Leucovorin Calcium, LEUKERAN® (Chlorambucil), Leuprolide Acetate, LEUSTATIN® (Cladribine), LEVULAN® (Aminolevulinic Acid), LINFOLIZIN® (Chlorambucil), LIPODOX® (Doxorubicin Hydrochloride Liposome), Lomustine, LONSURF® (Trifluridine and Tipiracil Hydrochloride), LUPRON® (Leuprolide Acetate), LUPRON DEPOT® (Leuprolide Acetate), LUPRON DEPOT-PED® (Leuprolide Acetate), LYNPARZA® (Olaparib), MARQIBO® (Vincristine Sulfate Liposome), MATULANE® (Procarbazine Hydrochloride), Mechlorethamine Hydrochloride, Megestrol Acetate, MEKINIST® (Trametinib), Melphalan, Melphalan Hydrochloride, Mercaptopurine, Mesna, MESNEX® (Mesna), METHAZOLASTONE® (Temozolomide), Methotrexate, METHOTREXATE LPF® (Methotrexate), Methylnaltrexone Bromide, MEXATE® (Methotrexate), MEXATE-AQ® (Methotrexate), Midostaurin, Mitomycin C, Mitoxantrone Hydrochloride, MITOZYTREX® (Mitomycin C), MOPP, MOZOBIL® (Plerixafor), MUSTARGEN® (Mechlorethamine Hydrochloride), MUTAMYCIN® (Mitomycin C), MYLERAN® (Busulfan), MYLOSAR® (Azacitidine),Attorney Docket No. 10110-466WO1

[0302] MYLOTARG® (Gemtuzumab Ozogamicin), NANOPARTICLE PACLITAXEL® (Paclitaxel Albumin-stabilized Nanoparticle Formulation), NAVELBINE® (Vinorelbine Tartrate), Necitumumab, Nelarabine, NEOSAR® (Cyclophosphamide), Neratinib Maleate, NERLYNX® (Neratinib Maleate), Netupitant and Palonosetron Hydrochloride, NEULASTA® (Pegfilgrastim), NEUPOGEN® (Filgrastim), NEXAVAR® (Sorafenib Tosylate), NILANDRON® (Nilutamide), Nilotinib, Nilutamide, NINLARO® (Ixazomib Citrate), Niraparib Tosylate Monohydrate, Nivolumab, NOLVADEX® (Tamoxifen Citrate), NPLATE® (Romiplostim), Obinutuzumab, ODOMZO® (Sonidegib), OEPA, Ofatumumab, OFF, Olaparib, Olaratumab, Omacetaxine Mepesuccinate, ONCASPAR® (Pegaspargase), Ondansetron Hydrochloride, ONIVYDE® (Irinotecan Hydrochloride Liposome), ONTAK® (Denileukin Diftitox), OPDIVO® (Nivolumab), OPPA, Osimertinib, Oxaliplatin, Paclitaxel, Paclitaxel Albumin-stabilized Nanoparticle Formulation, PAD, Palbociclib, Palifermin, Palonosetron Hydrochloride, Palonosetron Hydrochloride and Netupitant, Pamidronate Disodium, Panitumumab, Panobinostat, PARAPLAT® (Carboplatin), PARAPLATIN® (Carboplatin), Pazopanib Hydrochloride, PCV, PEB, Pegaspargase, Pegfilgrastim, Peginterferon Alfa-2b, PEG-INTRON® (Peginterferon Alfa- 2b), Pembrolizumab, Pemetrexed Disodium, PERJETA® (Pertuzumab), Pertuzumab, PLAI’INOL® (Cisplatin), PLATINOL-AQ® (Cisplatin), Plerixafor, Pomalidomide, POMALYST® (Pomalidomide), Ponatinib Hydrochloride, PORTRAZZA® (Necitumumab), Pralatrexate, Prednisone, Procarbazine Hydrochloride, PROLEUKIN® (Aldesleukin), PROLIA® (Denosumab), PROMACTA® (Eltrombopag Olamine), Propranolol Hydrochloride, PROVENGE® (Sipuleucel-T), PURINETHOL® (Mercaptopurine), PURIXAN® (Mercaptopurine), Radium 223 Dichloride, Raloxifene Hydrochloride, Ramucirumab, Rasburicase, R-CHOP, R-CVP, Recombinant Human Papillomavirus (HPV) Bivalent Vaccine, Recombinant Human Papillomavirus (HPV) Nonavalent Vaccine, Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine, Recombinant Interferon Alfa-2b, Regorafenib, RELISTOR® (Methylnaltrexone Bromide), R-EPOCH, REVLIMID® (Lenalidomide), RHEUMATREX® (Methotrexate), Ribociclib, R-ICE, RITUXAN® (Rituximab), RITUXAN HYCELA® (Rituximab and Hyaluronidase Human), Rituximab, Rituximab and, Hyaluronidase Human,, Rolapitant Hydrochloride, Romidepsin, Romiplostim, RUBIDOMYCIN® (Daunorubicin Hydrochloride), RUBRACA® (Rucaparib Camsylate), Rucaparib Camsylate, Ruxolitinib Phosphate, RYDAPT® (Midostaurin), Sclerosol Intrapleural Aerosol (Talc), Siltuximab, Sipuleucel-T, SOMATULINE DEPOT® (Lanreotide Acetate), Sonidegib, Sorafenib Tosylate,Attorney Docket No. 10110-466WO1

[0303] SPRYCEL® (Dasatinib), STANFORD V, Sterile Talc Powder (Talc), STERITALC® (Talc), STIVARGA® (Regorafenib), Sunitinib Malate, SUTENT® (Sunitinib Malate), SYLATRON® (Peginterferon Alfa-2b), SYLVANT® (Siltuximab), Synribo SYNRIBO® (Omacetaxine Mepesuccinate), TABLOID® (Thioguanine), TAG, TAFINLAR® (Dabrafenib), TAGRISSO® (Osimertinib), Talc, Talimogene Laherparepvec, Tamoxifen Citrate, TARABINE PFS® (Cytarabine), TARCEVA® (Erlotinib Hydrochloride), TARGRETIN® (Bexarotene), TASIGNA® (Nilotinib), TAXOL® (Paclitaxel), TAXOTERE® (Docetaxel), TECENTRIQ® (Atezolizumab), TEMODAR® (Temozolomide), Temozolomide, Temsirolimus, Thalidomide, THALOMID® (Thalidomide), Thioguanine, Thiotepa, Tisagenlecleucel, TOLAK® (Fluorouracil- Topical), Topotecan Hydrochloride, Toremifene, TORISEL® (Temsirolimus), Tositumomab and Iodine I 131 Tositumomab, TOTECT® (Dexrazoxane Hydrochloride), TPF, Trabectedin, Trametinib, Trastuzumab, TREANDA® (Bendamustine Hydrochloride), Trifluridine and Tipiracil Hydrochloride, TRISENOX® (Arsenic Trioxide), TYKERB® (Lapatinib Ditosylate), UNITUXIN® (Dinutuximab), Uridine Triacetate, VAC, Vandetanib, VAMP, VARUBI® (Rolapitant Hydrochloride), VECTIBIX® (Panitumumab), VelP, VELBAN® (Vinblastine Sulfate), VELCADE® (Bortezomib), VELSAR® (Vinblastine Sulfate), Vemurafenib, VENCLEXTA® (Venetoclax), Venetoclax, VERZENIO® (Abemaciclib), VIADUR® (Leuprolide Acetate), VIDAZA® (Azacitidine), Vinblastine Sulfate, VINCASAR PFS® (Vincristine Sulfate), Vincristine Sulfate, Vincristine Sulfate Liposome, Vinorelbine Tartrate, VIP, Vismodegib, VISTOGARD® (Uridine Triacetate), VORAXAZE® (Glucarpidase), Vorinostat, VOTRIENT® (Pazopanib Hydrochloride), VYXEOS® (Daunorubicin Hydrochloride and Cytarabine Liposome), WELLCOVORIN® (Leucovorin Calcium), XALKORI® (Crizotinib), XELODA® (Capecitabine), XELIRI, XELOX, XGEVA® (Denosumab), XOFIGO® (Radium 223 Dichloride), XTANDI® (Enzalutamide), YERVOY® (Ipilimumab), YONDELIS® (Trabectedin), ZALTRAP® (Ziv-Aflibercept), ZARXIO® (Filgrastim), ZEJULA® (Niraparib Tosylate Monohydrate), ZELBORAF® (Vemurafenib), ZEVALIN® (Ibritumomab Tiuxetan), ZINECARD® (Dexrazoxane Hydrochloride), Ziv-Aflibercept, ZOFRAN® (Ondansetron Hydrochloride), ZOLADEX® (Goserelin Acetate), Zoledronic Acid, ZOLINZA® (Vorinostat), ZOMETA® (Zoledronic Acid), ZYDELIG® (Idelalisib), ZYKADIA® (Ceritinib), and / or ZYTIGA® (Abiraterone Acetate). The treatment methods can include or further include checkpoint inhibitors including, but are not limited to antibodies that block PD-1 (such as, for example, Nivolumab (BMS-936558 or MDX1106), pembrolizumab, cemiplimab, CT-011, MK-Attorney Docket No. 10110-466WO1

[0304] 3475), PD-L1 (such as, for example, atezolizumab, avelumab, durvalumab, MDX-1105 (BMS-936559), MPDL3280A, or MSB0010718C), PD-L2 (such as, for example, rHIgM12B7), CTLA-4 (such as, for example, Ipilimumab (MDX-010), Tremelimumab (CP-675,206)), IDO, B7-H3 (such as, for example, MGA271, MGD009, omburtamab), B7- H4, B7-H3, T cell immunoreceptor with Ig and ITIM domains (TIGIT)(such as, for example B MS-986207, OMP-313M32, MK-7684, AB-154, ASP-8374, MTIG7192A, or PVSRIPO), CD96, B- and T-lymphocyte attenuator (BTLA), V-domain Ig suppressor of T cell activation (VISTA)(such as, for example, JNJ-61610588, CA-170), TIM3 (such as, for example, TSR-022, MBG453, Sym023, INCAGN2390, LY3321367, BMS-986258, SHR-1702, RO7121661), LAG-3 (such as, for example, BMS-986016, LAG525, MK-4280, REGN3767, TSR-033, BI754111, Sym022, FS118, MGD013, and Immutep).

[0305] “Concurrent administration”, “administration in combination”, “simultaneous administration” or “administered simultaneously” as used herein, means that the compounds are administered at the same point in time or essentially immediately following one another. IN the latter case, the two compounds are administered at times sufficiently close that the results observed are indistinguishable from those achieved when the compounds are administered at the same point in time.

[0306] “Systemic administration” refers to the introducing or delivering to a subject an agent via a route which introduces or delivers the agent to extensive areas of the subject’s body (e.g. greater than 50% of the body), for example through entrance into the circulatory or lymph systems.

[0307] By contrast, “local administration” refers to the introducing or delivery to a subject an agent via a route which introduces or delivers the agent to the area or area immediately adjacent to the point of administration and does not introduce the agent systemically in a therapeutically significant amount. For example, locally administered agents are easily detectable in the local vicinity of the point of administration but are undetectable or detectable at negligible amounts in distal parts of the subject’s body. Administration includes self-administration and the administration by another.

[0308] The disclosed composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier may be administered in such amounts, time, and route deemed necessary in order to achieve the desired result. The exact amount of the disclosed composition will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease (such as, for example, cancer, and / or cancer-associated complication (such as, for example, LMD or LML)), the particularAttorney Docket No. 10110-466WO1

[0309] composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier, disclosed herein, its mode of administration, its mode of activity, and the like. The disclosed composition sodium phenylbutyrate and a pharmaceutically acceptable carrier is preferably formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disease (such as, for example, cancer, and / or cancer-associated complication (such as, for example, LMD or LML)) being treated and the severity of the disease (such as, for example, cancer, and / or cancer-associated complication (such as, for example, LMD or LML)); the activity of the disclosed composition employed; the specific composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier, employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier employed; the duration of the treatment; drugs used in combination or coincidental with tire specific composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier employed; and like factors well known in the medical arts.

[0310] The disclosed composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier may be administered by any route. In some embodiments, the composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier is administered via a variety of routes, including intrathecal, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and / or drops), mucosal, nasal, buccal, enteral, sublingual; by intratracheal instillation, bronchial instillation, and / or inhalation; and / or as an oral spray, nasal spray, and / or aerosol. In general, the most appropriate route of administration will depend upon a variety of factors including the nature of the disclosed composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier (e.g., its stability in the environment of the gastrointestinal tract), the condition of the subject (e.g., whether the subject is able to tolerate oral administration), etc.Attorney Docket No. 10110-466WO1

[0311] The exact amount of the disclosed composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier required to achieve a therapeutically or prophylactically effective amount will vary from subject to subject, depending on species, age, and general condition of a subject, severity of the side effects, identity of the particular compound(s), mode of administration, and the like. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.

[0312] The concentration of active agent(s) can vary widely and will be selected primarily based on activity of the active ingredient(s), body weight and the like in accordance with the particular mode of administration selected and the patient's needs. Concentrations, however, will typically be selected to provide dosages ranging from about 0.1 or 1 mg / kg / day to about 50 mg / kg / day and sometimes higher. Typical dosages range from about 3 mg / kg / day to about 3.5 mg / kg / day, preferably from about 3.5 mg / kg / day to about 7.2 mg / kg / day, more preferably from about 7.2 mg / kg / day to about 11.0 mg / kg / day, and most preferably from about 11.0 mg / kg / day to about 15.0 mg / kg / day. In certain preferred embodiments, dosages range from about 10 mg / kg / day to about 50 mg / kg / day. In certain embodiments, dosages range from about 20 mg to about 50 mg given orally twice daily. It will be appreciated that such dosages may be varied to optimize a therapeutic and / or prophylactic regimen in a particular subject or group of subjects.

[0313] In one aspect, disclosed herein is composition of any preceding aspect comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier selected from an excipient, a diluent, a salt, a buffer, a stabilizer, a lipid, an emulsion, a nanoparticle, and a cream. One or more active agents (e.g. sodium phenylbutyrate) can be administered in the “native” form or, if desired in the form of salts, esters, amides, prodrugs, or a derivative that is pharmacologically suitable. Salts, esters, amides, prodrugs, and other derivatives of the active agents can be prepared using standards procedures known to those skilled in the art of synthetic organic chemistry and described, for example, by March (1992) Advanced Organic Chemistry; Reactions, Mechanisms, and Structure, 4thEd. N. Y. Wiley-Interscience.

[0314] In some embodiments, the disclosed composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier can be prepared as a “concentrate”, e.g. in a storage container of a premeasure volume and / or a predetermined amount ready for dilution, or in a soluble capsule ready for addition to a specified volume of water, saline, alcohol, hydrogen peroxide, or other diluent.Attorney Docket No. 10110-466WO1

[0315] In some embodiments, the disclosed composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or more times. In some embodiments, the disclosed composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier is administered daily. In some embodiments, the disclosed composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier is administered every day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, or more. In some embodiments, the disclosed composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier is administered every week, every 2 weeks, every 3 weeks, every 4 weeks, or more. In some embodiments, the disclosed composition comprising sodium phenylbutyrate and a pharmaceutically acceptable carrier is administered every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, every 12 months, or more. In some embodiments, the disclosed composition comprising sodium phenyl butyrate and a pharmaceutically acceptable carrier is administered every year, every 2 years, every 3 years, every 4 years, every 5 years, or more.

[0316] It is understood and herein contemplated that while a single administration of the compounds of the disclosed anti-cancer combination therapies (i.e., sodium phenylbutyrate and / or CAR-T cells) would be ideal, not every patient will respond in the same manner. Thus, in one aspect, disclosed herein are anti-cancer combination therapies methods treating, preventing, reducing, and / or inhibiting a cancer; wherein the sodium phenyl butyrate is administered at least, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20, 21, 22, 23, or 24 times a day or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 times per week for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks. Also disclosed herein are anti-cancer combination therapies methods treating, preven ting, reducing, and / or inhibiting a cancer of any preceding aspect; wherein the at least one anti-cancer agent is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20, 21, 22, 23, or 24 rimes a day or at least I, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 times per week for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks. It is further understood and herein contemplated that theAttorney Docket No. 10110-466WO1

[0317] order and duration of the administered components can vary as appropriate for the subject being treated. In one aspect, disclosed herein are anti -cancer combination therapies methods treating, preventing, reducing, and / or inhibiting a cancer; wherein tire sodium phenyl butyrate is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36 hours, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, 30, 31, 45 days, 2, 3, 4, 5, 6 months prior to administration of the pulsed dendritic cells.

[0318] VIII. EXAMPLES

[0319] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and / or methods claimed herein are made and evaluated and are intended to be purely exemplary and are not intended to limit the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is, at or near atmospheric.

[0320] Example 1: Branched-chain keto acids promote an immune-suppressive and neurodegenerative microenvironment in leptomeningeal disease.

[0321] Leptomeningeal disease (LMD), also known as leptomeningeal lymphoma (LML), occurs with a frequency of 5-20% associated with different cancers. Currently, there are limited effective therapeutics for patients with LMD as many LMD patients do not respond to CAR-T cell therapy. There is a need for new treatments effective against LMD. A study investigated the tumor microenvironment of LML in cerebral spinal fluid (CSF) from B Cell lymphoma patients and in tissue from an in vivo LMD mouse model to identify new therapeutics. Single cell RNA-seq and multi-omics (lipidomics and proteomics) were conducted on patient CSF and mouse brain tissue (FIG. 3). A therapeutic for LMD was tested in in-vitro and in the in-vivo mouse model (FIG. 3).

[0322] First, scRNA-seq was conducted on the CSF of patients with LML responding to CAR-T cell therapy, patients with LML not responding to CAR-T cell therapy, and patients without LML. scRNA-seq revealed that the microenvironment of LML consists of B cells, CD4+ T cells, CD8+ T cells, Natural Killer cells (NK), macrophages, monocytes, and plasmacytoid dendritic cells (FIG. 4). Compared to CSF from non-LML patients, LML CSF contained a larger number of B cells, CD4+ T cells, and CD8+ T cells (FIG. 4).Attorney Docket No. 10110-466WO1

[0323] Furthermore, CSF from LML patients showed few active and proliferating T cells (FIG.

[0324] 5A). The majority of T cells present in the CSF of patients with LML responding or not responding to CAR-T cell therapy were naive or exhausted T cells (FIG. 5A). Additional analysis comparing responding versus non-responding CSF revealed that CSF from non¬ responding patients contained a larger number of macrophages indicating that the LML microenvironment is enriched with macrophages (FIG. 5C). These findings were confirmed in the LMD mouse model as leptomeningeal tissue from these mice had a greater number of macrophages than liver tissue from the same mouse (FIG. 5C). The percentage of total cells that were macrophages was quantified in LMD and non-LMD melanoma patient CSF and in different tissues from the mouse LMD model (FIG. 6). Additionally, scRNA-seq revealed higher macrophage cell-cell interaction in LML poor responders (FIG. 7). Thus, scRNA-seq identifies an immunosuppressive cellular microenvironment in LML patient CSF and in an LML mouse model.

[0325] The study next investigated the macromolecular composition of CSF from patients with LML compared to the CSF of non-LML patients via multi-omics. In general, a difference was observed between lipids, metabolites, and proteins in LML CSF. Furthermore, lipidomic and proteomic analysis revealed markers of neurodegeneration in LML patients. Proteomics data revealed consistent up- and down-regulation of hallmarks of neurodegeneration (FIG. 18). For example, in LMD CSF, transferrin was significantly upregulated while CDH4 was significantly downregulated (FIG. 18). Thus, improvement of neurodegenerative physiological symptoms is an indicator of improved LMD.

[0326] Metabolomic analysis of LMD CSF identified dysregulation of branched-chain a-keto acid (BCKA) metabolism. Specific BCKAs such as ketoisovaleric acid (KIV), a-ketoisocaprioc acid (KIC), and α-keto-β-methylvaleric acid (KMV) were upregulated in LMD CSF (FIG. 19A, 10B-10C). Targeted metabolomics revealed significantly increased concentrations of KIC / KMV and KIV in LMD CSF (FIG. 19 A, 10B-10C). The increase of BCKAs indicates that an accumulation of BCKAs is responsible for immune suppression and neuronal degeneration in LMD patients.

[0327] To test the cytotoxicity of BCKAs, healthy CSF was treated with different concentrations of a BCKA mix. Treating CSF with a BCKA mix resulted in a decrease in CD4+, CD8+, and total cells in a dose dependent response (FIGS. 21C-21D). Excess BCKA has an immunosuppressive response as treating CSF with a BCKA mix resulted in a decrease in immune cell signaling molecules, specifically pro-inflammatory signaling molecules (FIGS. 21E and 21H). Increasing concentrations of the BCKA mix and KIC are toxic as 1Attorney Docket No. 10110-466WO1

[0328] mM of KIC and 1 mM of the BCKA mixture reduced cell viability by 25% (FIGS. 21F-21G). Additionally, BCKAs inhibit CAR-T cell activation and viability (FIG.22). Thus, an increased concentration of BCKAs in LML inhibits the secretion of proinflammatory molecules and results in cytotoxicity.

[0329] Morphological neurodegenerative symptoms were observed in both LML patients and in an LML mouse model. LML mice received a score of 3 in neurological exams compared to healthy mice, 0. The tail suspension test resulted in a significantly increased NeuroScore in LML mice (FIG.12). Additionally, a significant decrease in the grip strength of LML mice compared to control mice was observed (FIG.12). In general, LML took longer to walk 75 cm than healthy mice (FIG.12). Physiologically, immunohistochemistry on healthy and LML mouse brains reveals neuronal degradation and a loss of leptomeningeal integrity. Brains from mice with LML exhibited less microtubule associate protein 2 (MAP2), a marker of neuronal stability (FIG.13-14). Staining of healthy and LMD mouse brains for glial fibrillary acid protein (GFAP), a protein in which the dysregulation of is implicated in multiple neurological disorders, revealed a substantial increase in GFAP in LMD brains (FIG.23). Additionally, the pia mater of LML mice exhibited a much lower percentage of fibroblasts compared to healthy pia (FIGS. 13-14). Overall, neurodegeneration is a symptom of LML. To identify the cause of neurodegeneration in LML, primary neurons and meningeal cells were treated with various concentrations of BCKAs and a metabolic assay was performed (FIG.24). BCKAs inhibit the metabolic activity of neurons and meningeal cells in a dose-dependent response (FIG.24). Thus, BCKA accumulation promotes neurological decline in LMD.

[0330] Since BCKA accumulation is responsible for the symptoms of LMD, BCKA-lowering therapies likely play a role in ameliorating the effects of LMD. Sodium phenyl butyrate is known to play a role in branched-chain amino acid synthesis, so it was tested as an LMD therapeutic. Treatment of LMD mice with a combination of sodium phenylbutyrate and CAR-T cell therapy significantly improved NeuroScore assessments and reduced the number of progression free days (FIGS. 26A-26B). Additionally, treatment of LMD mice with sodium phenylbutyrate alone or in combination with CAR-T cell therapy greatly improved survival when compared to no treatment, T-cell therapy, or CAR-T cell therapy alone (FIG.26C). Sodium phenylbutyrate also improved methotrexate therapy as the probability of survival increased with a combination therapy of methotrexate and sodium phenylbutyrate compared to methotrexate treatment alone (FIG.29). Additionally, treatment with sodium phenylbutyrate alone increased mouse quality of life as determined by endpointAttorney Docket No. 10110-466WO1

[0331] weight (FIG.27). Furthermore, sodium phenyl butyrate blocks neurodegeneration as LMD mice heated with sodium phenyl butyrate brains displayed comparable amounts of MAP2 to healthy mice (FIG.27). Sodium phenylbutyrate as a mono- or combination therapy improves symptoms, quality of life, and probability of survival in LMD mice.

[0332] LMD is characterized by a dysfunctional T cell landscape, lack of dendritic cells, and an accumulation of pro-tumorigenic macrophages. CSF from LML patients shows -omics signatures associated with neurodegeneration and an accumulation of BCKAs. In this model, BCKAs suppress T cell activation, proliferation, and viability, inhibit the normal metabolic function of neuronal cells, and disrupt the leptomeningeal layer. Treatment with therapies to reduce BCKAs can increase survival and reduce neurodegenerative symptoms in an LMD mouse model. Sodium phenylbutyrate, which impacts the regulation of branched-chain amino acid biogenesis, can be used as a monotherapy for LMD as well as a combination therapy with the current standards of care. Sodium phenylbutyrate can be used as a therapy for any conditions that result in CAR-T cytotoxicity.

[0333] Example 2: To determine if BCKA accumulation supports tumor growth, metabolism, and survival at the leptomeninges.

[0334] We hypothesize that BCKA support melanoma growth and survival in the CSF environment by fueling the TCA cycle and promoting growth signaling. We will leverage our unique cellular and organoid models of LMD using live-cell imaging, viability, proliferation, and cytotoxicity in physiological CSF with BCKA exposure. For mechanistic insights, we will examine signaling stimulated by BCKA exposure and validate it using knockdown experiments. We will utilize in vitro and in vivo stable isotope tracing and targeted Seahorse assays to examine how tumor cells utilize exogenous BCKA. Finally, we will use in vivo models of LMD to determine if inducible disruption of BCKA utilization would block tumor growth. At the end of this aim, we will establish if BCKA accumulation supports melanoma cell growth, metabolism, and survival in the leptomeningeal environment.

[0335] Leptomeningeal metastasis is therapy resistant: Despite promising advances in targeted and checkpoint inhibitor therapies for systemic disease, melanoma tumors at the leptomeninges are uniquely resistant to most therapeutic interventions, highlighting a great clinical need for a better understanding of LMD biology.

[0336] The cellular microenvironment of LMD is immune-suppressed: Our previous analysis of patient specimens showed fewer infiltrating T cells in melanoma LMD compared to tumors at other sites of disease. To define the comprehensive cellular landscape of LMD,Attorney Docket No. 10110-466WO1

[0337] scRNA-seq was performed on 34 CSF specimens from 22 patients with LMD and 6 patients without LMD. LMD CSF showed a significant decrease in T cells that were naive or in early activation states, an increase in T cells approaching exhaustion (p < 0.05, FIG. 31 A), and was dominated by T cells that were inactive, exhausted, or approaching exhaustion (not shown). Validation in the SMI mouse model of LMD confirmed a shift from predominantly naive T cells towards an accumulation of exhausted CD8 T cells (p < 0.01, FIG. 31B). To assess if immune suppression is specifically accentuated in LMD tumors, a second cohort of mouse models was used to compare LMD tumors to extra-cranial metastatic sites. There was a large increase in T cells that expressed high levels of exhaustion markers in LMD compared to T cells at other disease sites (FIG. 31C). Analysis of patient specimens further identified that patients with the typical short survival (< 5months) had much fewer active and proliferating T cells (FIG. 31D). Together, these results put forward suppression of T cell activity as a major feature of LMD and suggest that the rapid progression of LMD is in part caused by the lack of anti-tumor immunity within this location.

[0338] Accumulation of branched-chain keto acids in LMD: Using untargeted metabolomic profiling of patient CSF, we found a tremendous accumulation of keto acids a-ketoisocaproate (KIC), a-keto-p-methylvalerate (KMV), in the CSF of patients with LMD from multiple tumor types, with melanoma showing the greatest accumulation (FIG. 32A). The third branched-chain keto acid a-ketoisovalerate (KIV) was not detected in the untargeted metabolomic dataset, but KIC and KMV showed the largest fold increase of any metabolite (FIG. 32B). KIC, KMV, and KIV, collectively referred to as branched-chain keto acids (BCKA), result from the first step of branched-chain amino acid (BC AA) metabolism, when the ammonia group is removed from leucine, isoleucine, and valine, respectively. Pathway enrichment analysis of the 49 altered metabolites highlighted BCAA / BCKA metabolism as highest enriched pathway (enrichment ratio = 9.524, p-value = 0.003). Downstream, BCKA are converted to coenzyme A and succinate, feeding the TCA cycle (FIG. 33). BCKAs are strong metabotoxins, neurotoxins, and acidogens. BCKA accumulation is typically associated with a severe metabolic disorder termed Maple Syrup Urine Disease, where BCKA accumulation causes neurological dysfunction. Likewise, the major symptoms of LMD result from neurological dysfunction. Given these preliminary findings, we quantified the absolute concentration of all three BCKA (total BCKA) in LMD patient CSF (by lumbar puncture) and found an accumulation of ~20-50pM, as high as ~80pM in some patients (FIG. 34). With obstructed CSF flow' frequently observed withAttorney Docket No. 10110-466WO1

[0339] LMD due to bulky disease foci, the direct tumor microenvironment likely experiences even higher BCKA concentration. Importantly, the BCKA concentration in CSF of melanoma LMD patients is analogous to the plasma concentration of BCKA in patients with symptomatic BCKA-accumulating metabolic disorders who benefit from BCKA-lowering therapy’0. In our LMD patients, the relative abundance of BCKA negatively correlated with Karnofsky Performance Status (a measure of cancer patients' general well-being and ability to participate in daily life activities, p = 0.002, n = 34, data not shown) and is positively correlated with the abundance of exhausted T cells in patient CSF (FIG. 35). BCKA accumulation may occur with increased activity of branched-chain amino acid transferase (BCAT) enzymes or with decreased activity of the branched-chain keto acid dehydrogenase complex (BCKD, FIG. 33). We show that BCKA can directly affect T cell viability, proliferation, and cytokine secretion upon stimulation in a physiological CSF environment (FIG. 36A and 36B). However, BCKA does not harm tumor cell viability, and at ~25-50pM concentrations BCKA enhances tumor cell line growth instead (FIG. 36C). Primary melanoma tumor cells isolated from a CNS metastasis likewise show enhanced growth in physiological CSF with BCKA exposure (FIG. 36D). Overall, BCKA accumulation in the CSF of LMD patients is correlated with worse performance status, fosters an immune-suppressive and tumor-promoting microenvironment, and may be a unique, targetable metabolic vulnerability of LMD.

[0340] Inhibition of BCKA: Pharmacological inhibition of BCKA accumulation is widely used for metabolic disorders like Maple Syrup Urine Disease. BCKA-lowering therapy is well tolerated and may have additional health benefits. Therefore, BCKA-lowering therapies have tremendous untapped potential for patients with LMD. Several strategies can target BCKA accumulation, including blocking the production of BCKA through inhibition of BCAT enzymes and by promoting the downstream metabolism of BCKA to Coenzyme A molecules (FIG. 33). The FDA-approved sodium phenylbutyrate (PBA) to treat BCKA accumulation in Maple Syrup Urine Disease in 1996. PBA is an allosteric inhibitor of the branched-chain alpha-ketoacid dehydrogenase kinase (BCKDK), a negative regulator of the BCKD complex, thereby promoting the metabolism of BCKA to coenzyme A or succinate. PBA has exceptional CNS penetration and is safe for long-term use, making it an ideal therapeutic candidate with a high potential for rapid clinical translation. In our preliminary A20 lymphoma LMD models, PBA effectively reduced BCKA levels in the tumor microenvironment (FIG. 37B), diminished neurological decline (FIG. 37C), extended theAttorney Docket No. 10110-466WO1

[0341] overall survival (median survival 23 vs. 35 days, p ~ 0.0037, FIG. 37D), and extended the progression-free survival (Neuroscore <2) compared to the untreated group (median PF'S 21 vs 34 days, p = 0.0048). All animals had both LMD tumors and flank tumors to model concurrent extra-cranial disease. Notably, three animals from the PBA treatment cohort reached the endpoint on their flank tumors while showing no progression of LMD. Interestingly, PBA also sensitized the A20 LMD model to CAR T cell therapy (median survival 29 vs. 49 days, p = 0.0612, FIG. 37E, and progression-free median survival 29 vs 43 days, p = 0.0424). The overall abundance of KIC in the meningeal tissues (pia mater) of mice correlated to the overall survival time (FIG. 38A). We show that targeting BCKA accumulation in LMD has great potential in improving the quality of life, survival, and efficacy of immune-based therapies.

[0342] Despite disease control at extra-cranial sites and parenchymal brain metastasis, tumors grow especially quickly at the leptomeninges. Healthy CSF typically lacks much protein and micronutrients, and little is known about the mechanisms supporting rapid tumor growth in this harsh microenvironment. Our global metabolomics analysis of CSF from patients with and without LMD has identified BCKAs as one of the most differentially abundant metabolites elevated in CSF of LMD patients (FIG.2B). ScRNAseq data of LMD tumors shows that tumor cells highly express the BCKA uptake transporters SLC 16A1 and SLC16A4, the branched-chain keto acid dehydrogenase enzymes (BCKDHA and BCKDHB), and the activator of BCKDHA, PPM1K (FIG.3, FIG.8B). On the other hand, the branched-chain alpha-ketoacid dehydrogenase kinase (BCKDK, which inhibits the branched-chain keto acid dehydrogenase complex) is low in expression, suggesting that LMD tumor cells can readily utilize exogenous BCKA. BCKA can replenish the TCA metabolite pools, fueling the TCA cycle that is upregulated in tumor cells during distant metastasis. We utilized Compass, an algorithm to characterize cellular metabolic states based on scRNAseq, on our data from patient melanoma specimens and showed that the TCA cycle and branched-chain amino acid (BCAA) metabolism pathway (which includes downstream BCKA metabolism) were higher in tumor cells in LMD compared to other disease sites (FIG.8C). Consistent with these results, integrated proteomic and metabolomic analysis of differentially abundant molecules in CSF from patients with and without LMD showed significant enrichment of the TCA cycle, including differences in succinate levels (7 matched molecules out of 42 total molecules, p = 0.00014, FDR = 0.006). Producing acetyl-CoA from BCKA metabolism can also sustain tumor survival through its function as a cofactor for histone acetyltransferase, facilitating rapid epigenetic adatations. InAttorney Docket No. 10110-466WO1

[0343] preliminary experiments, we see an increase in tumor cell growth with exposure to BCKA (FIG. 36C and 36D). We show that melanoma upregulates BCKA metabolism and the TCA cycle in LMD and are well suited to utilize exogenous BCKA. This data is supported by the observed increase in tumor growth with BCKA exposure in CSF.

[0344] Models of melanoma leptomeningeal metastasis: We established five primary cultures from CSF-circulating tumor cells from patients with melanoma LMD, two LMD patient-derived xenograft (PDX) models from human LMD tumors, and five immune- competent syngeneic mouse models of melanoma LMD using cisterna magna injection of SMI, Yumm 3.2, Yummer 1.7, D4M-UV2, and B16-F10 cells. These models fully recapitulate human disease in the rate of progression (FIGS. 7A, 9A-B), accumulation of BCKA (FIG. 37B), immune suppression (FIG. 32B) and neurological decline (FIGS. 7C, 9C). These models also recapitulate the anatomic features of LMD, colonizing the leptomeningeal space and CSF, frequently leading to hydrocephalus similarly as in patients, and can be fitted with our murine Ommaya-like reservoirs for delivery of intrathecal therapies analogous to patients (FIGS. 9 A and 9D). We have collected -200 CSF specimens from ~50 patients with melanoma LMD. We have an active rapid autopsy tissue collection program and already banked patient-matched LMD and extra-cranial tissues from 7 patients with melanoma LMD. We have an unparalleled collection of in vitro models, mouse models, tissues, and CSF, enabling us to make powerful, clinically relevant discoveries for this devastating disease.

[0345] This aim is based on our observations that rapid LMD progression is coupled to drastic accumulation of BCKA in patients (FIGS. 2A, 2B, 8A), inhibition of BCKA accumulation attenuates disease progression in mouse models (FIGS. 7B-7D), and that BCKA exposure increases tumor growth in vitro (FIG. 36C). Recent discoveries indicate that metabolic intermediates may have secondary functions as signaling molecules, shifting the perspective on the metabolism-centric role of metabolites. BCKA exposure has been shown to mediate changes in the insulin-induced AKT phosphorylation and regulate multiple proteins in the MAPK pathway. In our patient data, we observe upregulated signaling through the PI3K / AKT and MAPK pathways, including increased expression of MAPK and mTOR targets in the tumor cells at the leptomeninges and we have confirmed higher MAPK mediator expression in LMD compared to other sites of metastasis (FIG.

[0346] 38D). Based on this and our preliminary data showing the upregulation of BCKA metabolism and TCA cycle (FIG. 38B-C), we hypothesize that the accumulation of BCKAAttorney Docket No. 10110-466WO1

[0347] ill LMD promotes tumor growth and survival by fueling the TCA cycle and inducing MAPK signaling.

[0348] Test if BCKA promotes tumor growth and viability. To test how BCKA accumulation promotes the viability and growth of melanoma in the harsh CSF environment, we will utilize eight human melanoma cell lines (WM163, WM9, 1205Lu, WM793, WM1366, IPC298, A375, Sk-Mel28), 2 LMD patient-derived primary tumor cultures and physiological CSF (Na+150 mM, K+3 mM, Ca2+1.4 mM, Mg2+0.8mM, P 1mM, Cl-155mM, NaHCO326mM, Osmolarity -279 mOsm / L, pH 7.3) aerated with 5% CO2 / 95% O2and supplemented with glucose 60 mg / dl, and 1% bovine serum albumin (protein ~35mg / dL) to mimic the normal physiological conditions of human CSF. We will expose the tumor cells to increasing doses of BCKA (range 0-250 pM) in physiological CSF. We will assess viability using Calcein AM (live) / SYTOX (dead) staining and confirm using flow cytometry assessment of Annexin V and TMRM staining. We will measure the dynamics of tumor cell growth over time (14 days) using live cell imaging in an Incucyte. We will repeat these experiments with CSF from 10 LMD and 10 non-LMD patients. To further mimic the LMD environment, we will repeat these experiments in direct and indirect co-culture with primary human meningeal cells ( co-cul tures optimized) using transwell plates and iBidi p-Slides, and by plating dTomato-labeled SMI, D4M and Yumm3.2 murine melanoma cells on short-term ex vivo culture on the pia membrane from C57BL6 mice. We will monitor the viability of non-tumor cells in case BCKA promotes tumor growth indirectly by promoting non-tumor cell death and nutrient release. Genetic models: We will examine how the doxycycline-inducible knockdown of Branched Chain Keto Acid Dehydrogenase El Subunit Alpha (BCKDHA) or overexpression of BCKDK (a negative regulator of the dehydrogenase complex) in the tumor cells will affect survival and disease progression in two LMD mouse models. We cannot modulate BCKA transporters as they transport other metabolites as well. Briefly, dTomato-tagged SMI and Yumm3.2 tumor cells will be transduced in vitro and implanted in C57BL / 6 mice via cisterna magna injection (5-50,000 cells, depending on the cell line, in 5 pL PBS). Tumors will establish for three days, and then mice will be randomized to doxycycline-formulated or control chow. Mouse cohort calculation is above. An additional three animals will be included in each treatment group to collect pial tissues at the 21 -day time point for histological assessment of tumor burden based on the d'T'omato tag. These data will determine if tumor cells utilize accumulatedAttorney Docket No. 10110-466WO1

[0349] BCKA in the leptomeningeal space to sustain cell growth and survival in these harsh conditions.

[0350] Signaling transduction effects of BCKA on tumor cells. Since BCKA have been shown to also act as signaling molecules promoting activation of AKT and MAPK pathways and we see an upregulation of PI3K and MAPK targets in human LMD tumors and in tumor cells exposed to LMD CSF, we will check phosphorylation and total expression of MEK1 / 2, ERK1 / 2, AKT, and mTOR in response to BCKA exposure by Western Blot. We will then test if depleting BCKA from human LMD CSF samples using purified BCKDH along with its cofactors (ImM NAD+, 0.5mM CoA, 0.2 mM TPP, 1 mM Mg2+) will diminish the activation of PI3K / AKT and MAPK pathways when tumor cells when exposed to patient CSF. BCKA depletion will be confirmed by mass spectrometry, as previously (FIG. 4). This will determine if BCKA exposure promotes alterations in signal transduction that support tumor growth and survival in LMD and identify targetable mediators of this mechanism.

[0351] Trace the utilization of BCKA in tumor metabolism. Rapid metabolic adaptations are essential for tumor survival during distant metastasis, but we know very little about the metabolic landscape of LMD in the unique, nutrient-scarce CSF environment. To determine if these tumors utilize BCKA to fuel the TCA cycle, we will perform stable isotope tracing of13C6-labeled KIC and KMV and13C5-labeled KIV in two melanoma cell lines cultured in physiological media, physiological CSF, and LMD patient-derived CSF. We will prioritize the analysis of the stable isotope tracers through the BCKA metabolism pathway and the TCA cycle. We will validate these findings in vivo in the immune-competent SMI LMD mouse model and one patient-derived xenograft model of LMD following an established protocol. We will inject tumor cells via cisterna magna as above, via tail vein for lung and liver colonization or at the “'primary site” (intradermally). Animals will fast for 5 hours; then, we will continuously infuse stably labeled KIC (81 nmol / min) mixed with unlabeled KMV (40 nmol / min) and KIV (76 nmol / min) at a constant rate of 0.0836 pL / g / min for four hours. Following jugular vein infusion using a swivel tether coupled to an infusion pump, we will collect the tumors in CSF, pia mater, and extra-cranial sites. The cells in CSF will be analyzed in batches of 4-5 mice each (-20 L per animal) to isolate the tumor cells. The CSF samples will be placed on ice, and the tumor cells will be enriched separately with a rapid magnetic bead sort using Miltenyi mouse tumor cell isolation kit (20-minute negative selection on ice). We will analyze metabolites using liquid chromatography-high resolution mass spectrometry (LC-HRMS) as previously described. Comparisons: We will compareAttorney Docket No. 10110-466WO1

[0352] LMD tumors to tumors at the extra-cranial sites. Metabolites traced: we will trace the metabolism of13C- labeled BCKA and incorporation of13C- label to acetyl CoA and its entry into the TCA and labeling of citrate, a-ketoglutarate, succinate, malate, and fumarate. While most of these metabolites have moderate to good stability, Acetyl-CoA has low stability. However, we have already optimized extraction and quenching protocols and are capable of detecting labeled acetyl-CoA in metabolic tracing experiments (FIG. 40) Controls: We will include serum (cheek bleed 1 minute prior to euthanasia), a non-target highly metabolic tissue (e.g., liver in intrathecally-injected animals and brain in tail-vein injected animals), and analogous tissues from a control animal not infused with tracers. Cohort size: to accommodate a 5% attrition during tumor implantation, 10% attrition during catheter placement, and 5% attrition for infusion failure / tracer leak, we will include seven animals for all non-tumor cohorts / controls and 14 animals for all tumor cohorts to successfully obtain data on a minimum of five control animals and ten tumor-bearing animals (accounting for larger variability in metabolism of tumor models). These data will allow us to examine BCKA metabolism in the whole LMD tumor and isolated tumor cells to determine how the CSF environment affects tumor metabolism and if the tumor cells utilize BCKA to fuel their TCA cycle at the leptomeninges.

[0353] Process-specific validation of BCKA-fueled metabolism: Seahorse Mito Stress Test, Glycolysis Stress Test, and real-time ATP Rate assays will examine mitochondrial respiration, glycolysis, and ATP production in the tumor cells in response to BCKA exposure in CSF (physiological CSF will be modified to accommodate specific assays) and Seahorse media conditions. We will further validate these findings using a spatial RNAseq dataset on a cohort of 9 leptomeningeal tissues harvested from melanoma patients with LMD (data on hand, one tissue exemplified in FIG. 41). We will examine the gene expression of enzymes mediating BCAA / BCKA metabolism and TCA relative to the tumor position in these tissues. These experiments will provide additional tumor-specific and metabolic processspecific validation for the stable isotope tracing and confirm these processes in patient LMD tissues.

[0354] Results: We expect exogenous BCKA to enhance tumor viability, growth, and signaling through MAPK / AKT pathways. Knockdown of BCKDHA or overexpression of BCKDK in tumor cells is expected to blunt tumor growth in mouse LMD. Metabolic tracing will likely show the utilization of branched-chain keto acids to fuel the TCA cycle by replenishing acetyl CoA. This change will be confirmed by targeted Seahorse assaysAttorney Docket No. 10110-466WO1

[0355] showing a higher mitochondrial respiratory capacity and rate of ATP production in physiologic CSF with BCK.

[0356] Example 3: To define the mechanisms by which BCKA promote T cell dysfunction.

[0357] We hypothesize that BCKA reversibly inhibit T cell function multi-modally, through acidosis, metabolic control, and by serving directly as signaling molecules regulating 4E-BPl-mediated translation and inhibiting p38a mitogen-activated protein kinase signaling. We will utilize transgenic T cells with peptide-specific TCR in physiological CSF to confirm whether BCKA exposure inhibits T cell-mediated tumor killing and if it is a reversible effect. We will measure the effect of BCKA on T cell metabolism and production of reactive oxygen species in CSF through Seahorse T cell metabolic profiling, MitoSOX green, and aconitase activity assays. In these studies, we will utilize HEPES and sodium bicarbonate buffering to quantify the contribution of BCKA-induced acidosis to the observed T cell dysfunction. Finally, we will utilize immunoprecipitation to examine the effects of BCKA on TCR complex integrity. At the end of this aim, we will elucidate the mechanism of T cell dysfunction induced by BCKA exposure in the CSF environment.

[0358] This aim is based on our observation that the environment of melanoma LMD has a dysfunctional T cell landscape and that there is a positive correlation between the concentration of BCKA in patient CSF and the presence of exhausted, inactive T cells in their LMD compartment (FIG. 35). We also show that activation of T cells in physiological CSF containing BCKA leads to suppressive effects on T cell proliferation, viability, and cytokine secretion (FIGS. 36Aand 36B). The duration, extent, and mechanisms of the T cell suppression remain unclear. Acidosis has clear inhibitory effects on T cell function, and the buffering capacity of CSF is weaker than that of blood due to lack of serum protein and cellular hemoglobin buffers. BCKA are strong acidogens, and our preliminary analysis of patient CSF indicates a significant decrease in the pH of CSF in patients with LMD (FIG.

[0359] 42). Importantly, we only see a 2-fold increase in lactic acid in LMD (data not shown) but a 50-fold increase in BCKA (FIG. 42A). BCKA are stronger acidogens than lactate (pKA= 2.651 for KIC versus 3.9 for lactate). Therefore, the pH difference is likely driven by BCKA rather than lactate. Metabolic intermediates may also have secondary functions as signaling molecules. BCKA exposure can mediate insulin-induced AKT phosphorylation, total protein synthesis through phosphorylation of translational repressor 4E-BP1, and signaling in the MAPK pathway. In our previously published scRNAseq data, we observed a decreaseAttorney Docket No. 10110-466WO1

[0360] ill the expression of several MAPK signaling mediators in T cells from LMD compared to other sites of melanoma metastasis, including 4E-BP1 (EIF4EBP1) and p38a (MAPK14, FIG. 43 A). This result was particularly striking in the activated T cells (FIG. 13A, right). In vitro, we see decreased ERK phosphorylation with BCKA exposure in T cells. Therefore, we hypothesize that BCKA reversibly inhibit T cell function multi-modally, through acidosis and metabolic control and by serving directly as signaling molecules regulating 4E-BP1 -mediated translation and inhibiting of p38a.

[0361] Determine whether BCKA directly inhibit T cell killing and if these effects are reversible. To determine whether BCKA accumulation directly decreases T cell-mediated tumor killing and whether these suppressed phenotypes are reversible, we will perform in vitro T cell killing assays in physiological CSF. We will use SMI, Yummer 1.7, Yum 3.2, Bl 6, and D4M-UV2 melanoma cell lines and autologous mouse CD4 and CD8 T cells (hereafter referred to as T cells) expressing TCRs reactive against melanoma antigens gpl 00, tyrosinase, and TRP1. We will stimulate T cells in presence of 0-250,uM BCKA and with an 8-hour to 7-day pre-treatment and then removal of BCKA from T cells prior to tumor cell plating. T cells will be used at effector-to-target ratios of 10: 1, 1:1, and 1:10 to evaluate the serial killing capacity of T cells. Target killing will be monitored using a real-time killing assay and flow cytometry analysis of caspase 3. Concurrently, T cells will be analyzed for cytolytic potential by monitoring CD107a membrane surface expression. After BCKA exposure, T cells will be extensively washed and co-cultured with target cells after the last wash, Oh,.511, Ih, 3h, 5h. Target killing assays will be conducted as above. T cells will be examined for activation using CD3, CD8 CD4, CD44, CD25, CD45RA to CD45RO conversion, exhaustion by TIM-3, PD-1, LAG-3, CD39, TOX, TCF1, and TCF7, and apoptosis with propidium iodide and Annexin V. We will measure secretion of granzyme B by ELISA and cytokines using the Proteome Profiler Mouse XL, Cytokine Array. These data will allow us to determine if BCKA inhibit T cell killing and if these effects are reversible or if the T cells are terminally dysfunctional following BCKA exposure.

[0362] Establish if BCKA affects T cell function via acidosis. To determine if acidosis is the primary mechanism of T cell inhibition in LMD, we will perform in vitro T cell killing assays as in section 2.1 with and without increasing doses of HEPES and sodium bicarbonate added to buffer the acidogenic effects of BCKA (base CSF contains NaHCO3 at a low 26mM dose). The pH of all conditions will be recorded. To determine if BCKA exposure accounts for the majority of the suppressed and dysfunctional T cell phenotypesAttorney Docket No. 10110-466WO1

[0363] ill the LMD microenvironment and if buffering would reverse these effects, we would perform bulk RNAseq on T cells following activation in CSF from patients with and without LMD and in physiological CSF containing BCKA, with and without buffering. We would examine whole transcriptome changes in T cells in response to these conditions and compare them to the T cell transcriptional states we recently observed in CSF of breast cancer (data on hand but unpublished), lymphoma and melanoma LMD patients. Finally, we would test if CSF buffering using HEPES / sodium bicarbonate injected in synthetic CSF directly into the ventricle daily via our murine Ommaya-like reservoir would affect the tumor infiltration and tumor cell killing using adoptive T cell transfer of tyrosinase reactive transgenic T cells into RAG2 knockout animals implanted with SMI melanoma tumor cells. One set of animals would contain luciferase-labeled T cells with unlabeled tumor cells, and another would contain luciferase-labeled tumor cells with unlabeled T cells. Mouse cohort calculation is above. These data will (1) determine if exposure to BCKA in physiological CSF during activation would fully recapitulate the “dysfunctional” transcriptional states observed with activation in CSF from LMD patients, (2) whether the addition of BCKA at physiological levels found in LMD CSF would promote significant CSF acidification, and (3) whether buffering BCKA-induced acidosis would reverse BCKA-induced T cell dysfunction and promote tumor cell killing.

[0364] Assessing the impact of BCKA on T cell signaling. To test for signal transduction effects of BCKA, CD4 and CD8 T cells isolated from healthy human donors will be activated (anti-CD3, anti-CD28 mAbs, IL-2) and exposed to BCKA in physiological CSF. Post-exposure, cells will be reactivated with anti-CD3 mAb only for 0, 1, and 5 min. This procedure will be repeated with buffering to rule out the indirect effects of acidosis on signal transduction. Lysates will undergo phospho-tyrosine, serine, and threonine proteomic analysis. Tyrosine-phosphorylated tryptic peptides will be immunoprecipitated with immobilized antibodies, and phosphoserine and phosphothreonine-containing peptides will be fractionated and enriched with immobilized metal affinity chromatography and analyzed using a nanoflow liquid chromatograph interfaced with an electrospray Orbitrap mass spectrometer (RSLCnano and Exploris 480, Thermo). Data analysis and validation: Analysis will be carried out as we have done previously. Differentially phosphorylated proteins will be validated by Western Blot. We will prioritize hits surrounding TCR, 4E-BP1, and MAPK signaling for functional validation using siRNA-mediated silencing of targets and enzymes responsible for target phosphorylation and dephosphorylation using electroporation and subsequent re-assessment of T cell function. G-chain cytokineAttorney Docket No. 10110-466WO1

[0365] signaling: Another key mechanism mediating T cell function is signaling in response to IL- 2 and IL- 15. Classically, when these cytokines engage with their receptor, the JAK-STAT pathway is recruited. Studies have shown that failure of this pathway has severe effects on T cell functions, such as proliferation. Therefore, we will also examine the status of JAK- STAT signaling in these analyses. These data would reveal the direct effects of BCKA exposure on post-translational modifications and critical signal transduction in T cells independent of acidosis and those resulting from acidosis.

[0366] The effect of BCKA on TCR complex integrity. The α / β TCR in complex with CD3 signaling subunits are the key regulators of T cell responses. The fidelity of the complex is essential for T cell activation. A recent study showed that exposure of T cells to lactic acid affected genes involved in TCR signaling. To determine if BCKA disrupt TCR-CD3 complex formation, we will perform immunoprecipitation of bead-immobilized anti-CD3ε on T cell lysates and check the co-precipitation of the other components of the TCR complex. Human T cells will be plated on OKT3-coated plates and stimulated with CD28 and IL2 in the context of increasing doses of BCKA (0-250 uM). TCR complex will be pulled down from lysates using 5 pg of biotinylated anti-CD3e and streptavidin-conjugated DynaBeads. The eluted product will be resolved on an SDS-page and blotted using antibodies against CD3δ, CD3γ, CD3ζ, 'I’CRa, and TCRp. Based on the initial results, we will perform systematic immunoprecipitation of the other individual subunits of the TCR complex to confirm alterations in the binding of these components to the complex. Successful TCR engagement results in phosphorylation of the immunoreceptor tyrosinebased activation motif (ITAM) on CD3ζ, mediated by recruitment of Lek and Fyn tyrosine kinases to the TCR. This phosphorylation then serves as a docking site for signaling molecules and induces the downstream signal transduction. To determine if BCKA affects the fidelity or strength of ITAM phosphorylation, we will perform aWestern Blot analysis of total and tyrosine 142-phosphorylated CD3ζ on the T cell lysates from the immunoprecipitation. Together, these data will show if BCKA exposure affects the initiation of TCR signal transduction or the integrity of the TCR complex.

[0367] The effect of BCKA on T cell metabolism and production of reactive oxygen species (ROS). It is now well established that T cell metabolism is the driving force behind T cell function and is a critical determinant of anti-tumor efficacy. ROS are critical mediators of T cell-mediated immunity, with transiently elevated ROS acting as critical second messengers in T cell receptor (TCR) signaling. COMPASS2analysis of scRNAseq data from patientAttorney Docket No. 10110-466WO1

[0368] specimens shows that T cell metabolism at the leptomeningeal sites is very different from that at extra-cranial sites of disease or from metastases to the brain parenchyma (FIG. 44), resulting in a global downregulation of most metabolic processes, including ROS detoxification, glycolysis, and the TCA cycle. We will test if BCKA exposure leads to a critical disruption of normal T cell metabolism and ROS regulation.

[0369] Metabolic profiling of BCKA-exposed T cells. To test if BCKA accumulation disrupts T cell function via metabolic dysregulation, we will profile human T cell metabolism using the Seahorse XF T cell Metabolic Profiling Kit with minor modifications. Instead of Base Media, we will utilize the physiological CSF and the glucose concentration will be adjusted to 60mg / dl to mimic human LMD CSF conditions (data not shown). The fourth injection port will release BCKA for the T Cell Fitness Assay and the T Cell Persistence Assay. The T Cell Fitness Assay will monitor ATP production and the kinetics of T cell glycolysis following BCKA exposure and with consecutive stimulation with oligomycin, BAM15, and Rotenone / Antimycin A (in BCKA-exposed and BCKA-naive T cells). The T Cell Persistence assay will measure the ratio of ATP production from oxidative respiration versus glycolysis and determine the cells’ spare respiratory capacity, which is known to determine T cell persistence and memory development. These data will determine how BCKA affects normal T cell glycolytic and mitochondrial metabolism kinetics, the production of ATP, respiratory capacity, and the proportion of ATP production from glycolysis

[0370] ROS induction in BCKA-exposed T cells. To determine if BCKA exposure promotes ROS dysregulation (specifically the formation of superoxide radical O2⁻), we will plate human T cells on OKT3-coated plates (5 pg / mL) and stimulate them with CD28 (2 pg / mL) and IL2 (200 IU / L) in the presence of increasing BCKA doses. We will then assess ROS using the MitoSOX Green (Invitrogen) superoxide indicator and aconitase enzymatic activity assay (inactivated by oxidative stress) using a kit from Abeam. For negative and positive controls, we will use T cells treated with 1 pM MitoTEMPO for reduction and 30pM menadione for oxidation (since SOD knockout or overexpression may be ideal for this experimental setup). In parallel, we will also include antioxidant / superoxide dismutase controls using Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), a metalloporphyrin-mimetic compound with superoxide dismutase activity. We will examine changes in the superoxide dismutase-sensitive reduction of aconitase inactivation and MitoSOX indicator fluorescence intensity in response to T cell activation in normal media,Attorney Docket No. 10110-466WO1

[0371] physiological CSF, and with exposure to 0-250 pM BCKA in physiological CSF conditions. These data will establish if BCKA disrupts normal ROS kinetics in response to T cell stimulation in the CSF environment.

[0372] Results: We anticipate a reduction in T cell-mediated tumor cell killing with exposure to BCKA, that are reversible with short-term exposure but lead to terminal exhaustion and apoptosis with long-term exposure. It is likely that some of the inhibitory effects on T cells are mediated through acidosis, affecting TCR complex interactions. However, BCKA accumulation is expected to also promote TCA metabolism and constitutively high ROS production. Activated T cells must upregulate glycolysis even in the presence of oxygen to support effector functions; therefore, a BCKA-mediated decrease in glycolytic capacity and glycolysis-mediated ATP production is expected. We anticipate BCKA will affect 4E-BP1 -mediated translational regulation and MAPK activation. We do not anticipate strong MAPK inhibitory effects on the tumor cells since they frequently dysregulate normal MAPK activity, amplifying signaling strength.

[0373] Example 4: To therapeutically target BCKA accumulation in LMD.

[0374] We hypothesize that melanoma LMD tumors are uniquely sensitive to BCKA-inhibiting therapies through direct effects on the tumor cells and induction of an immune-favorable environment in LMD. In this aim, we will utilize four immune-competent murine models of melanoma LMD and one patient-derived LMD model to determine the most effective strategy for inhibiting BCKA accumulation for the treatment of LMD. We will identify the mechanism of action for PBA through cytotoxicity and histone acetylation assays, activity-based protein profiling, immunophenotyping of the in vivo LMD environment, and in vivo assessment of T cell infiltration using adoptive T cell transfer models. We will confirm the direct tumor effects of BCKA inhibition (initial priority on using PBA) using immune-deficient NSG mice. Finally, we will test if targeting BCKA accumulation will sensitize melanoma LMD to checkpoint inhibitor therapy. At the completion of this aim, we will identify the most efficacious strategy to target BCKA accumulation, characterize the mechanism of action, and determine if targeting BCKA can sensitize LMD to immune therapy.

[0375] Targeting BCKA accumulation in LMD, We found that targeting BCKA accumulation using PBA showed promising efficacy in the A20 murine model of lymphoma LMD (FIGS. 37B-37E). PBA significantly improved overall survival (FIG. 37D) and reduced neurological deterioration, leading to longer progression-free survival andAttorney Docket No. 10110-466WO1

[0376] diminished neurological symptoms at the endpoint (FIG. 37C). It is unclear if PBA will prove to be similarly effective in LMD from solid tumors or whether it is the most effective BCKA-lowering strategy for LMD. Although we see a clear reduction in BCKA levels with PBA therapy (FIG. 37B), the full mechanism of action, including direct effects on tumor cells or the immune microenvironment, is unknown. We observed PBA to sensitize lymphoma LMD to CAR T therapies (FIG. 37E), suggesting a possible utility of PBA (or BCKA reduction) in sensitizing LMD to other immune -based standard-of-care therapies. We hypothesize that PBA can effectively target the unique metabolic vulnerabilities of LMD from melanoma through efficient reduction of BCKA accumulation, direct cytotoxic effects on tumor cells, and by promoting an anti-tumor immune microenvironment. We believe it can sensitize melanoma LMD tumors to immune-based therapies.

[0377] Inhibiting BCKA in murine models of LMD. We will examine if PBA is effective in melanoma LMD models or if alternative targeting of processes leading to BCKA accumulation may improve this efficacy. We will compare PBA to an alternative, more specific BCKDK inhibitor (BT2), and to individual and dual inhibition of BCAT1 / 2, enzymes that convert BC AA to BCKA. We will determine if selective targeting of either or both BCAT1 and BCAT2 would show better anti -LMD efficacy than targeting BCKDK (FIG. 33). Uncoupling possible HDAC inhibitor mechanisms from BCKA-lowering mechanisms of PBA. The full mechanism of action for PBA is not entirely clear, but it may possess a broad spectrum of molecular functions. In addition to being an inhibitor of BCKDK and an ammonia scavenger, it has also been shown to be a chemical chaperone and a pan HDAC inhibitor. HDAC inhibition is tested as a potential anti-cancer therapeutic in multiple tumor types. The chemical proteomics experiment (3.5 below) will allow us to identify definitive targets of PBA in vivo. Including the more specific BT2 and BCAT1 / 2 inhibitors and histone acetylation assays will allow us to separate the possible HDAC- modulating effects of PBA that may compound the interpretation of the results.

[0378] Targeting BCKA accumulation. To identify the optimal strategy for targeting BCKA accumulation, we will use cisterna magna injection of SMI and D4M cells in C57BL6 mice (FIG. 39B) and PDX in NSG mice. On day 3, mice will be treated with PBA (200 mg / kg, IP) another inhibitor of BCKDK (BT2, 20 mg / kg, IP), selective inhibitor of BCAT1 (BCAT-IN-2, 100mg / kg, PO), selective inhibitor of BCAT2 (BCATc Inhibitor 2, 30mg / kg, subQ) or a dual BCAT1 / 2 inhibitor (BAY-069, 0.6 mg / kg, PO) administered every other day. Mouse cohort calculation is above. Three additional animals will be included in each group to collect pial tissues on day 21 to quantify BCKA. We will determine which modality ofAttorney Docket No. 10110-466WO1

[0379] targeting BCKA accumulation lowers the BCKA the most and prolongs survival to the greatest extent. We will perform immunostaining for acetylated histones H3 and H4 on the LMD tumors. These experiments would determine if promoting BCKA metabolism using PBA or BT2 or inhibiting BCKA production using either individual or dual inhibitors of BCAT1 / 2 would be an effective therapeutic strategy for LMD, and which modality would show the strongest therapeutic effects.

[0380] Inhibiting Neurological Progression. For these same mice, we will perform neurological assessment using NeuroScores adapted from models of Huntington's disease and amyotrophic lateral sclerosis to quantify disease progression and neurological decline. These assessments will evaluate hindlimb function, including paralysis, motor task deficits, and gait abnormalities. Limb weakness, paralysis, and mobility deficits are common symptoms of progressi ve LMD in patients and our murine models of LMD (FIG. 37C, FIG.

[0381] 39C). The brain tissues will be analyzed by immunohistochemistry for gliosis (GFAP staining) and neuronal degradation (loss of MAP2 reactivity). These experiments will allow us to determine which BCKA-lowering treatment strategy would most effectively reduce the severity of neurological progression in murine models of LMD.

[0382] The direct effects of PBA on tumor cells. In our lymphoma LMD models, we observed an increase in survival (23 weeks vs 35 weeks) with PBA (FIG. 37D). Of the 13 mice treated with PBA, three relapsed only at the subcutaneous sites and did not exhibit symptoms of LMD progression, suggesting promising efficacy against LMD tumors. Here, we will determine if PB A’s mechanism of action includes direct effects on the tumor cells in the LMD space and if the cells at the leptomeninges (in CSF) are uniquely more sensitive to PBA treatment, independent of the immune system. We initially plan to use PBA for these experiments since it is FDA-approved and had promising preliminary efficacy in lymphoma LMD models.

[0383] In vitro effects of PBA on tumor cells. We will first compare the effects of PBA on tumor cells in CSF compared to Human Plasma-Like Medium and normal culture media. We will treat eight human melanoma and lymphoma cell lines with increasing doses of PBA (0.1-5mM) in normal cell culture media (5% FBS in RPMI), Human Plasma-Like Medium, physiologic synthetic CSF, and human CSF from LMD patients. We will assess the viability as above. We will measure the effects on tumor cell growth over 14 days using live cell imaging. To further mimic the LMD microenvironment, we will repeat these experiments with direct and indirect co-culture of tumor cell lines with primary human meningeal cells using transwell plates and the iBidi p-Slides, respectively, and by plating murine SMI andAttorney Docket No. 10110-466WO1

[0384] D4M dTomato-labeled cells on short-term ex vivo culture of the pia membrane from C57BL6 mice in physiological CSF. For both culture systems, we would perform Histone H3 and H4 acetylation assays (Abeam) on the tumor cells. These experiments will determine if PBA has direct, immune -independent effects on the tumor cells and if they may be mediated through HDAC-inhibitory mechanisms.

[0385] Uncoupling effects on the tumor from effects on the immune system. To uncouple PBA’s effects on tumor cells from those on immune cells and determine if in vivo efficacy of PBA requires an active immune system, we will repeat our PBA experiment with LMD models in an immune-deficient setting using luciferase-expressing SMI and D4M in NSG mice. We will compare tumors injected at the leptomeninges via cisterna magna to those injected intra-dermally or via tail-vein to colonize the livers / iungs. Each extra-cranial injection cohort will have both the extra-cranial and cisterna magna injections in the same mouse. We will measure neurological performance and survival and analyze changes in disease burden through bioluminescent imaging. We will compare the efficacy of PBA in NSG mice to that of immune-competent animals and compare the tumor reduction / control at the leptomeninges to other sites of disease. Mouse cohort calculation is above. These experiments will confirm if PBA has direct, immune-independent effects on the tumor cells in vivo and if the tumors at the leptomeninges are more sensitive to PBA than other sites of disease.

[0386] BCKA inhibition promote an immune-favorable environment in LMD We initially plan to use PBA for these experiments but may adjust based on section 3.1. We show that PBA treatment promotes the efficacy of CAR T cell therapy in murine models of lymphoma LMD (Fig. 37E). To confirm if inhibition of BCKA accumulation affects immune remodeling, we will harvest the pial membranes from the mouse experiments and assess the numbers and activation status of the CD4 and CD 8 T cells using flow cytometry. We will track the extent of T cell infiltration into the LMD microenvironment using luciferase-labeled transgenic T cells in two models of LMD. We will inject Pmel and Trpl -expressing SMI and D4M murine melanoma cell lines into the leptomeningeal space via cisterna magna of 12-week-old B6. Cg-Rag2mil lC8’VJ (RAG2 knockout) mice and allow tumors to establish for seven days. Then, animals will start treatment with vehicle control or PBA, as above. On the fourth day of treatment, we will infuse 4 x 106pre-activated, luciferase-expressing Pmel CD8+ T cells and Trpl CD4+ T cells via the tail vein. The mice will then be injected with D-luciferin (150 mg / kg, retro-orbitally) and imaged with an IVIS2000 machine daily. Mouse cohort calculation is above. These experiments will determine ifAttorney Docket No. 10110-466WO1

[0387] BCKA-lowering PBA will increase T cell infiltration and activation in the LMD microenvironment.

[0388] BCKA inhibition sensitize LMD to immune therapies We initially plan to use PBA for these experiments. In combining PBA with anti-CD19 CAR T cell therapy, we observed a “cure” in two animals, an outcome not previously achieved in murine models of LMD (Fig. 37E). We expect that PBA can augment other immune-based therapies. As a proof of concept, we will test if PBA can re-sensitize melanoma LMD to checkpoint inhibitor therapy. Although exceptionally effective for extra-cranial unresectable melanoma, checkpoint inhibition, administered either systemically, intrathecally, or in combination, shows limited response rates and provides only a minor increase in overall survival in LMD patients who respond. The latest advancement in checkpoint inhibition for treating unresectable extra-cranial melanoma is the combination of anti-PDl and anti-LAG3 antibodies, showing enhanced progress! on -free survival over anti-PDl monotherapy (10.1 months vs 4.6 months, respectively). We will use three immune-competent models of melanoma LMD using SMI, D4M, and B16-F10. Mice will receive either the mouse anti- PDl plus mouse anti-LAG3 antibodies (lOmg / kg plus lOmg / kg respectively, intraperitoneal) or the combination of the two antibodies with PBA (200 mg / kg, intraperitoneal). Mouse anti-IgG antibodies, with and without PBA combination, will be used as controls. We will record overall survival and use neurological assessment NeuroScores). At the endpoint, the pia membranes will be analyzed for immune cell infiltration and activation. Mouse cohort calculation is above. These experiments will determine if PBA treatment can re-sensitize melanoma LMD to immune checkpoint inhibition.

[0389] Identify the molecular targets of phenylbutyrate in vivo. We will utilize Moffitt’s Chemical Biology Core to synthesize a PBA-linked probe for activity-based protein profiling of PBA across 3 LMD models (SMI, D4M, and Yumm3.2). The probe will react with the active sites of proteins inhibited by PBA to identify the functional targets of PBA by mass spectrometry. To ensure CSF penetration, we will administer the probe via the murine Ommaya reservoir. Pia from 4 mice treated for 3 days will be analyzed individually following probe pull-down. This data will elucidate the functional molecular targets of PBA in vivo (mechanism of action), thereby identifying druggable targets for LMD.

[0390] Results: We expect a robust anti -tumor activity of BCKA reduction in murine models of LMD from solid tumors, leading to a “hot” immune microenvironment. We anticipate that PBA’s exceptional efficacy stems from both BCKA-lowering properties and inhibitionAttorney Docket No. 10110-466WO1

[0391] of HDAC activity in tumor cells. Due to PBA’s excellent CNS penetration, safety profile, and likely multi-modal mechanism of action, we believe it would be the best BCKA-reducing treatment strategy for LMD. PBA is expected to sensitize melanoma cells to the anti-PDl / LAG3 combination.

[0392] Considerations for not using more genetic models: Our chosen models are the BEST tools in existence for LMD. 1) our priority is to move therapies to clinic rapidly, so we prioritize FDA approved inhibitors. 2) The BCKA are likely coming from the tumor microenvironment (as discussed in alternative strategies) and depletion of BCKA transporters would not be an appropriate strategy because they also transport other metabolites. 3) Knock-in or BCKA supplementation are not ideal as BCKA-low, immune intact, mouse LMD tumors do not exist.

[0393] We will establish the role of BCKA in the LMD microenvironment and provide a preclinical rationale for adopting BCKA-lowering therapies for the treatment of melanoma LMD. Successful completion of this work has the potential to transform the clinical management of LMD from melanoma and other tumor types.

[0394] Material and Methods

[0395] Animals- As both male and female patients develop leptomeningeal disease (LMD) with equal prevalence, we will use equal numbers of male and female mice for all strains. Except in the case of breast cancer LMD models, since breast cancer LMD predominantly affects female patients, in which case we will utilize only female animals for these models. The mice will be bred and maintained in our AAALAC-approved Animal Facility at the Moffitt Cancer Center. The C57BL / 6J strain will provide an optimal immunocompetent system for the study of SMI, D4M, Yumm3.2, and B16F10 cells. All animals are provided with food and water ad libitum and they are housed at 3-5 animals (depending on sex) per cage. The mice are used starting 10-12 weeks of age.

[0396] Test if BCKA promotes tumor growth and viability. We will examine how the doxycycline-inducible knockdown of Branched Chain Keto Acid Dehydrogenase El Subunit Alpha (BCKDHA) or overexpression of BCKDK (a negative regulator of the dehydrogenase complex) in the tumor cells will affect survival and disease progression in three LMD murine models. We cannot modulate BCKA transporters as they transport other metabolites as well. Briefly, dTomato-tagged SMI and D4M tumor cells will be transduced in vitro and implanted in C57BL / 6 mice via cisterna magna injection (5-50,000 cells, depending on the cell line, in 5 μL PBS). Tumors will establish for three days, and then miceAttorney Docket No. 10110-466WO1

[0397] will be randomized to doxycycline-formulated or control chow. We will assess survival and disease progression based on neurological performance. Based on our preliminary results, we will include 12 mice per group to achieve a 95% statistical power to detect differences between the median overall survival time using one-way ANOVA and an F test with a significance level of 0.01, assuming the probability of type-I error of 5% and attrition rate of 5% for animals not surviving surgery (typical rate observed with cisterna magna injections). An additional three animals will be included in each treatment group to collect pial tissues at the 21-day time point for histological assessment of tumor burden based on the dTomato tag. These data will determine if tumor cells utilize accumulated BCKA in the leptomeningeal space to sustain cell growth and survival in these harsh conditions.

[0398] Trace the utilization of BCKA in tumor metabolism. Rapid metabolic adaptations are essential for tumor survival during distant metastasis, but we know very little about the metabolic landscape of LMD in the unique, nutrient-scarce CSF environment. To determine if these tumors utilize BCKA to fuel the TC A cycle, we will perform stable isotope tracing of13C6-labeled KIC and KMV and13C5-labeled KIV in two melanoma cell lines cultured in physiological media, physiological CSF, and LMD patient-derived CSF. We will prioritize the analysis of the stable isotope tracers through the BCKA metabolism pathway and the TCA cycle. We will validate these findings in vivo in the immune-competent SMI LMD mouse model and one patient-derived xenograft model of LMD following an established protocol. We will inject tumor cells via cisterna magna as above, via tail vein for lung and liver colonization or at the “primary site” (intradermally). Animals will fast for 5 hours; then, we will continuously infuse stably labeled KIC (81 nmol / min) mixed with unlabeled KMV (40 nmol / min) and KIV (76 nmol / min) at a constant rate of 0.0836 pL / g / min for four hours. Following jugular vein infusion using a swivel tether coupled to an infusion pump, we will collect the tumors in CSF, pia mater, and extra-cranial sites. The cells in CSF will be analyzed in batches of 4-5 mice each (~20 pL per animal) to isolate the tumor cells. The CSF samples will be placed on ice, and the tumor cells will be enriched separately with a rapid magnetic bead sort using Miltenyi mouse tumor cell isolation kit (20-minute negative selection on ice). We will analyze metabolites using liquid chromatography-high resolution mass spectrometry (LC-HRMS) as previously described. Comparisons: We will compare LMD tumors to tumors at the extra-cranial sites. Metabolites traced: we will trace the metabolism of13C- labeled BCKA and incorporation of13C- label to acetyl CoA and its entry into the TCA and labeling of citrate, a-ketoglutarate, succinate, malate, and fumarate.Attorney Docket No. 10110-466WO1

[0399] While most of these metabolites have moderate to good stability, Acetyl-CoA has low stability. However, we have already optimized extraction and quenching protocols and are capable of detecting labeled acetyl-CoA in metabolic tracing experiments (FIG. 40) Controls: We will include serum (cheek bleed 1 minute prior to euthanasia), a non-target highly metabolic tissue (e.g., liver in intrathecally-injected animals and brain in tail-vein injected animals), and analogous tissues from a control animal not infused with tracers. Cohort size: to accommodate a 5% attrition during tumor implantation, 10% attrition during catheter placement, and 5% attrition for infusion failure / tracer leak, we will include seven animals for all non-tumor cohorts / controls and 14 animals for all tumor cohorts to successfully obtain data on a minimum of five control animals and ten tumor-bearing animals (accounting for larger variability in metabolism of tumor models). These data will allow us to examine BCKA metabolism in the whole LMD tumor and isolated tumor cells to determine how the CSF environment affects tumor metabolism and if the tumor cells utilize BCKA to fuel their TCA cycle at the leptomeninges.

[0400] Establish if BCKA affects T cell function via acidosis. We would test if CSF buffering using HEPES / sodium bicarbonate injected in synthetic CSF directly into the ventricle daily via our murine Ommaya-like reservoir would affect the tumor infiltration and tumor cell killing using adoptive T cell transfer of tyrosinase reactive transgenic T cells into RAG2 knockout animals implanted with SMI melanoma tumor cells. One set of animals would contain luciferase-labeled T cells with unlabeled tumor cells, and another would contain luciferase-labeled tumor cells with unlabeled T cells. We would measure T cell infiltration (one set) and tumor burden (second set) daily using an IVIS instrument and record survival and neurological progression. Based on preliminary results, we will include 12 mice per group. These data will (1) determine if exposure to BCKA in physiological CSF during activation would fully recapitulate the “dysfunctional” transcriptional states observed with activation in CSF from LMD patients, (2) whether the addition of BCKA at physiological levels found in LMD CSF would promote significant CSF acidification, and (3) whether buffering BCKA-induced acidosis would reverse BCKA-induced T cell dysfunction and promote tumor cell killing.

[0401] Targeting BCKA accumulation. To identify the optimal strategy for targeting BCKA accumulation in LMD, we will utilize cisterna magna injections, as before. SMI and D4M melanoma cells will be used in C57BL6 mice. PDX cells will be in NSG animals. On day three, mice will be treated with PBA (200 mg / kg, IP), another inhibitor of BCKDK (BT2, 20 mg / kg, IP), selective inhibitor of BCAT1 (BCAT-IN-2, 100mg / kg, PO), selectiveAttorney Docket No. 10110-466WO1

[0402] inhibitor of BCAT2 (BCATc Inhibitor 2, 30mg / kg, subQ) or a dual BCAT1 / 2 inhibitor (BAY-069, 0.6 mg / kg, PO) administered every other day. Based on preliminary results, we will include 12 mice per group. Three additional animals will be included in each group to collect pial tissues on day 21 to quantify BCKA. We will determine which modality of targeting BCKA accumulation lowers the BCKA the most and prolongs survival to the greatest extent. We will perform immunostaining for acetylated histones H3 and H4 on the LMD tumors. These experiments will allow us to determine if BCKA-lowering PBA treatment would prolong survival in murine melanoma LMD models. These experiments would also determine if promoting BCKA metabolism using PBA or BT2 or inhibiting BCKA production using either individual or dual inhibitors of BCAT1 / 2 would be a more effective therapeutic strategy for LMD.

[0403] Inhibiting Neurological Progression. For these same mice, we will perform neurological assessment using NeuroScores adapted from models of Huntington's disease and amyotrophic lateral sclerosis to quantify disease progression and neurological decline. These assessments will evaluate hindlimb function, including paralysis, motor task deficits, and gait abnormalities. Limb weakness, paralysis, and mobility deficits are common symptoms of progressive LMD in patients and our murine models of LMD (FIG. 37C) The brain tissues will be analyzed by immunohistochemistry for gliosis (GFAP staining) and neuronal degradation (loss of MAP2 reactivity). These experiments will allow us to determine which BCKA-lowering treatment strategy would most effectively reduce the severity of neurological progression in murine models of LMD.

[0404] In vitro effects of PBA on tumor cells. To determine if PBA’s mechanism of action includes direct effects on the tumor cells in the LMD space and if the cells at the leptomeninges (in CSF) are uniquely more sensitive to PBA treatment, independent of the immune system, we will first compare the effects of PBA on tumor cells in CSF compared to Human Plasma-Like Medium and normal culture media. We will treat eight human melanoma and lymphoma cell lines with increasing doses of PBA (0. l-5mM) in normal cell culture media (5% FBS in RPMI), Human Plasma-Like Medium, physiologic synthetic CSF, and human CSF from LMD patients. We will assess the viability as above. We will measure the effects on tumor cell growth over 14 days using live cell imaging. To further mimic the LMD microenvironment, we will repeat these experiments with direct and indirect co¬ culture of tumor cell lines with primary human meningeal cells using transwell plates and the iBidi q-Slides, respectively, and by plating murine SMI and D4M dTomato-labeled cellsAttorney Docket No. 10110-466WO1

[0405] on short-term ex vivo culture of the pia membrane from C57BL6 mice in physiological CSF. For both culture systems, we would perform Histone H3 and H4 acetylation assays (Abeam) on the tumor cells. These experiments will allow us to determine if PBA has direct, immune-independent effects on the tumor cells in vitro and if they may be mediated through HD AC- inhibitory mechanisms.

[0406] Uncoupling effects on the tumor from effects on the immune system. If PBA proves to be the best BCKA-reducing strategy, treatment of our animal models with PBA can complicate the interpretation of results as PBA can have an effect on both the tumor and the immune cells. To uncouple these effects and determine if the in vivo efficacy observed previously results from direct tumor effects or if it requires an active immune system, we will repeat our PBA experiment with SMI and D4M LMD models in an immune-deficient setting by injecting luciferase-expressing cells into NSG mice. We will compare tumors injected at the leptomeninges via cisterna magna to those injected intra-dermally or via tailvein to colonize the livers / lungs. Each extra-cranial injection cohort will have both the extra¬ cranial and cisterna magna injections in the same mouse. We will measure neurological performance and survival and analyze changes in disease burden through bioluminescent imaging of luciferase activity on IVIS2000. We will compare the efficacy of PBA in NSG mice to that of immune-competent animals and compare the tumor reduction / control at the leptomeninges to other sites of disease. The animal experiment results will guide the cohort size calculations for this animal experiment. Provisionally, we plan to include 12 mice per cohort. These experiments will confirm if PBA has direct, immune-independent effects on the tumor cells in vivo and if the tumors at the leptomeninges are more sensitive to PBA than other sites of disease.

[0407] BCKA inhibition promote an immune-favorable environment in LMD. The experiments show that PBA treatment promotes the efficacy of CAR T cell therapy in murine models of lymphoma LMD (FIG. 37E). To confirm if inhibition of BCKA accumulation affects immune remodeling, we will harvest the pial membranes from the mouse experiments and assess the numbers and activation status of the CD4 and CD8 T cells using flow cytometry. We will track the extent of I' cell infiltration into the LMD microenvironment using luciferase-labeled transgenic T cells in two models of LMD. We will inject Pmel and Trpl -expressing SMI and D4M murine melanoma cell lines into the leptomeningeal space via cisterna magna of 12-week-old B6.Cg-Rag2tm1.1Cgn / J (RAG2 knockout) mice and allow tumors to establish for seven days. Then, animals will start treatment with vehicle control or PBA, as above. On the fourth day of treatment, we willAttorney Docket No. 10110-466WO1

[0408] infuse 4 x 106pre-activated, luciferase-transduced Pmel CD8+ T cells and Trpl CD4+ T cells via the tail vein. The mice will then be injected with D-luciferin (150 mg / kg, retro-orbitally) and imaged with an IVIS2000 machine daily. The animal experiment results from the cohort size calculations. Provisionally, we plan to include 12 mice per cohort. These experiments will determine if BCKA-lowering PBA will increase T cell infiltration and activation in the LMD microenvironment.

[0409] BCKA inhibition sensitize LMD to immune therapies. In combining PBA with anti¬ CD 19 CAR T cell therapy, we observed a “cure” in two of the animals, an outcome not previously achieved in murine models of LMD (FIG. 37E). We expect that PBA can augment other standard-of-care therapies, especially immune-based therapies. As a proof of concept, we will test if PBA can re-sensitize melanoma LMD to checkpoint inhibitor therapy. Although exceptionally effective for extra-cranial unresectable melanoma, checkpoint inhibition, administered either systemically, intrathecally, or in combination, shows limited response rates and provides only a minor increase in overall survival in LMD patients who respond. The latest advancement in checkpoint inhibition for treating unresectable extra-cranial melanoma is the combination of anti-PDl and anti-LAG3 antibodies, showing enhanced progression-free survival over anti-PDl monotherapy (10.1 months vs 4.6 months, respectively). We will use three immune-competent models of melanoma LMD using SMI, D4M, and B16-F10. Mice will receive either the mouse anti-PDl plus mouse anti-LAG3 antibodies (lOmg / kg plus lOmg / kg respectively, intraperitoneal) or the combination of the two antibodies with PBA (200 mg / kg, intraperitoneal). Mouse anti-lgG antibodies, with and without PBA combination, will be used as controls. We will record overall survival and use neurological assessment NeuroScores). At the endpoint, the pia membranes will be analyzed for immune cell infiltration and activation. The animal experiment results will guide the cohort size calculations. Provisionally, we plan to include 12 mice per cohort. These experiments will determine if PBA treatment can re-sensitize melanoma LMD to immune checkpoint inhibition.

[0410] Identify the molecular targets of phenylbutyrate in vivo. We will utilize Moffitt’s Chemical Biology Core to synthesize a PBA-linked probe for activity-based protein profiling of PBA across 3 LMD models (SMI, D4M, and Yumm3.2). The probe will react with the active sites of proteins inhibited by PBA to identify the functional targets of PBA by mass spectrometry. To ensure CSF penetration, we will administer the probe via theAttorney Docket No. 10110-466WO1

[0411] murine Ommaya reservoir. This data will elucidate the functional molecular targets of PB A in vivo (mechanism of action), thereby identifying druggable targets for LMD.

[0412] Drug dosing- For small molecule inhibitor of GLS1, cyclophosphamide and sodium phenyl butyrate will be administered by intraperitoneal injection by laboratory personnel with expertise in this procedure (or formulated chow when available). Animals will be observed carefully for signs of drug toxicity. We will euthanize the animals if they appear to show signs of sickness or a 20% change in body weight. Stable isotope-labeled metabolites will be administered to animals via jugular vein catheter, placed surgically under anesthesia by the comparative medicine technician with extensive expertise in this procedure. CAR T therapy will be infused via tail vein injection.

[0413] Euthanasia- The chosen method of euthanasia is CO2 inhalation followed by cervical dislocation, except in the cases of in vivo stable isotope tracing where the CO2 inhalation will affect the metabolism and therefore cervical dislocation is the chosen method of euthanasia. These methods are consistent with the recommendations of the American Veterinary Medical Association Guidelines of Euthanasia.

[0414] Minimization of pain and distress: Animals on study will be assessed three times a week by the Pi’s staff and vivarium staff for any complications arising due to surgery or tumor growth. The survival surgeries will be performed under anesthesia (isoilurane 2%) under aseptic conditions. Every effort will be taken to limit the pain and discomfort of the animals during the proposed studies. Analgesics (carprofen) will be given prior to and after the intrathecal inoculation of the tumor cells into recipient animals and catheter placement surgery. The animals on study will be monitored for signs of infection, discomfort, distress (ulceration, hunched posture, weight loss, failure to groom) or paralysis and will be euthanized accordingly. Mice that lose more than 20% (emaciated appearance; rapid weight loss over two to four days; or progressive weight loss over a few weeks) or gain more than 20% of their original body weight, or show signs of morbidity or distress (e.g. hypoactivity, cyanosis, pale mucous membranes, head pressing, head tilt, circling, impaired locomotion, non-purposeful movements, failure to groom, failure to respond to stimuli, isolation from cage mates, shivering, ataxia, shallow, rapid and / or labored breathing, soiled anogenital area, vocalization, lack of inquisitiveness, and / or a hunched posture) will be euthanized.

[0415] Example 5: A pan-cancer atlas of leptomeningeal disease reveals branched chain keto acids drive immunosuppression and neurodegeneration.

[0416] Leptomeningeal disease (LMD) is a rapidly fatal complication of systemic cancer that is clinically detected in 5-15% of patients with late-stage cancers and is apparent atAttorney Docket No. 10110-466WO1

[0417] autopsy in up to 20-30% of patients. LMD occurs when malignant cells seed to the membrane coverings of the brain and spinal cord and the cerebral spinal fluid (CSF) compartments1. Rapid and debilitating neurological symptoms drastically impact the survival outcomes and quality of life for LMD patients. The median survival time for LMD patients with treatment is 2-6 months, with death typically occurring due to progressive neurologic dysfunction. Current LMD treatments include combination radiotherapy regimens with intrathecal chemotherapy (cytarabine, methotrexate, rituximab, and thiotepa, etc.) or intrathecal targeted or immune therapies. Systemic therapy can be added to control extracranial disease and potentially prolong patient survival. However, these approaches rarely achieve durable responses. This limited therapeutic success reflects a central gap in our understanding of LMD biology.

[0418] Despite decades of research, fundamental questions about LMD pathophysiology remain unanswered, creating critical barriers to therapeutic advances. While the anatomical challenges of drug delivery across the blood-brain barrier are well recognized, the unique features of the leptomeningeal space that promote tumor growth and treatment resistance are poorly understood. Additionally, the mechanisms driving rapid neurological decline -the primary cause of mortality - have not been determined. The field notably lacks insight into how the distinct metabolic environment of the cerebrospinal fluid may influence tumor progression and treatment efficacy. Understanding these aspects is critical for developing effective therapies, as current treatments rarely provide durable responses. This knowledge gap is especially relevant for emerging immunotherapies like CAR-T cells, which show promise but face unique challenges in the central nervous system environment. Indeed, though multiple studies have shown that CD19-targeting CAR T-cell therapies are effective at treating chemo-refractory Non-Hodgkin B Cell Lymphomas (NHBCL), retrospective analyses of ten studies shows that the anti-tumor effects are not as long-lasting for CNS tumors as they are for extra-cranial disease, despite clear evidence of CAR T-cell penetration into the CSF space. Outcomes are frequently further complicated by the neurotoxicity of these regimens.

[0419] To gain insights into the biology of this anatomical site and the mechanisms that promote tumor growth, drug resistance, and neurological deterioration, we used single-cell transcriptomics and bulk multi-omics techniques to profile the cellular and fluid compartments of the tumor microenvironment in LMD of solid tumors and hematological malignancies. Using patient specimens, in vivo models, and in vitro functional studies, the marked accumulation of branched-chain keto acids manifest in LMD was shown to promoteAttorney Docket No. 10110-466WO1

[0420] an immune suppressive and neurodegenerative tumor microenvironment (FIG. 49). Importantly, this study provides the first preclinical evidence for adapting branched-chain keto acid-lowering treatments to improve the quality of life and survival outcomes for LMD patients.

[0421] Leptomeningeal disease harbors an immune-suppressed microenvironment with few active, proliferating T cells.

[0422] To define the cellular landscape of LMD, single-cell RNA sequencing analysis (scRNA-seq) was performed on thirty-four CSF specimens from twenty-two patients with breast cancer, melanoma, and lymphoma LMD, including CSF specimens from six patients without leptomeningeal involvement (FIG. 45A). Cell profiling analysis identified substantial infiltration of tumor and immune cells, with tumor type-specific differences in the degree of myeloid and T / NK cell infiltration (FIG. 45B, C and Extended Data Figs. 2A-J). There was an increased accumulation of immune-suppressive myeloid cell populations in the CSF of LMD patients, including M2 macrophages and MDSC-like cells, which was most prominent in LMD from solid tumors (FIGS. 51A-E). On the other hand, lymphoma LMD had the highest infiltration of T cells (FIG. 5 IF). Analysis of markers associated with T cells allowed predictions of the function of each identified T / NK cell cluster (FIG. 51G). Compared to patients with no CNS involvement, the CSF environment of patients with LMD showed a significant decrease in T cells that were naive or in early activation states and an increase in T cells approaching exhaustion (p < 0.05, FIG. 45D). Overall, CSF from all LMD types was dominated by T ceils that were inactive, exhausted, or approaching exhaustion (FIG. 45E). To validate these findings, we profiled the immune compartment in our SMI murine model of LMD (FIG. 45F). Compared to control animals, the pial tissues of LMD mice confirmed a shift from predominantly naive T cells towards a significant accumulation of exhausted CD8 T cells (p < 0.01, FIG. 45G). Moreover, these tissues demonstrated a shift towards a more immune-suppressive myeloid compartment harboring macrophages, M2 macrophages, and MDSC-like cells (FIG. 52A), mirroring human disease.

[0423] To assess if immune suppression is specifically accentuated in LMD tumors, a second cohort of mouse models was used to compare the T cells and macrophages infiltrating breast cancer, melanoma, and lymphoma LMD tumors versus extracranial metastatic sites (FIG. 45H). There were large increases in the proportion of total macrophages and M2 macrophages in the LMD tumors (FIG. 52B), as well as increased inAttorney Docket No. 10110-466WO1

[0424] infiltrates of T cells (p - 0.05, FIG. 451) that expressed high levels of exhaustion markers compared to T cells at other disease sites (FIG. 52C).

[0425] Finally, stratifying by survival times, the immune-suppressed microenvironment was associated with short survival in LMD, where the greatest expansion of M2, macrophages, MDSC-like cells, and monocytes occurred in patients with the shortest survival time (< 5 months versus < 10 months, FIG. 52D, E). Although we saw no differences in the overall number of CD4+ or CD8+T cells, patients with a shorter overall survival also had much lower proportions of active and proliferating T cells (p < 0.01, Fig 1J, FIG. 52F). Together, these results put forward suppression of T cell activity as a major feature of LMD and suggest that the rapid progression of LMD is in part caused by the lack of anti-tumor immunity within this location.

[0426] Accumulation of branched-chain keto acids is linked to neurodegeneration in the LMD microenvironment

[0427] In addition to profound immune suppression, we hypothesized that additional mechanisms must contribute to the aggressive nature, profound neurological impairment, and poor outcomes of LMD. To gain mechanistic insights, multi-omic analyses of the acellular CSF fluid from patients with and without LMD was performed. Overall, 27-32% of proteins, metabolites, and lipids were significantly altered in the CSF of patients with LMD (FIG. 46A). Amongst these changes were significant decreases in four lipids of the lysophosphatidylcholine class, which have been shown to induce demyelination. At the same time, we see significant increases in seven lipids of the sphingomyelin class, whose presence in CSF indicate myelin loss in patients with demyelinating neuropathies (FIG.

[0428] 53A, B), which intriguingly suggested a connection between LMD and neurodegeneration. In support of this premise, of the few proteins significantly depleted from CSF in LMD patients, several are involved in critical processes of normal neuron function, including GRIA4 (learning and memory), ARSA (an essential enzyme regulating myelination), THY 1 (axon regeneration), CDH4 (neuronal outgrowth), NPTXR (synaptic activity), and the neurotransmitters TAC1 and TAC3 (behavioral responses) (FIG. 53C, D). Pathway enrichment analysis of differentially expressed proteins also highlighted processes associated with innate immune responses (e.g., macrophage function and immunodeficiency) and neurodegenerative disorders, including Alzheimer's and multiple sclerosis (FIG. 46B).

[0429] Given the strong associations with neurodegenerative processes in LMD, we examined the abundance of proteins whose accumulation or depletion is associated withAttorney Docket No. 10110-466WO1

[0430] neurodegenerative diseases such as Alzheimer's and Parkinson's. Immunoglobulin G, transferrin, vitamin-D binding protein, PARK7, and DJ-1 were significantly enriched in CSF from LMD patients, while there were significant reductions in amyloid β precursor, neurosin (KLK6), and BDNF (FIG. 46C, FIG. 53D). The enrichment and depletion of many of these markers were statistically significant over the course of LMD progression in a second CSF cohort of serial specimens from LMD patients (Spearman correlation).

[0431] Integrating proteomic and metabolomic data revealed that the most significant alterations were in complement and coagulation cascades, cell adhesion processes, and multiple metabolic processes, including central carbon metabolism, the citrate cycle, the pentose phosphate pathway, and pyruvate and glutathione metabolism; thus, there are an extensive metabolic alteration in the CSF microenvironment of LMD patients (FIG. 53E).

[0432] Analysis of individual differentially abundant metabolites identified a striking accumulation of the branched-chain keto acids (BCKA) a-ketoisocaproate (KIC) and a-keto-P-methylvalerate (KMV) in the CSF of LMD patients (p < 0.01, FIG. 46D, FIG. 53F). Branched-chain keto acids are abnormal metabolites that result from an incomplete metabolism of branched-chain amino acids and are known as metabotoxins, neurotoxins, and acidogens. BCKAs are clinically associated with a group of severe metabolic disorders termed Maple Syrup Urine Disease, where BCKA and branched-chain amino acid accumulation are linked to neurological dysfunction.

[0433] Process enrichment analysis of differentially abundant (p <0.05) metabolites showed branched-chain amino acid metabolism pathways were enriched in patients with LMD (FIG.

[0434] 53G). The accumulation of KIC / KMV and a-ketoisovalerate (KIV) was confirmed in the CSF of patients with lymphoma (n = 18, p <0.0001), breast cancer (n = 12, p < 0.05) and melanoma (n = 12, p < 0.01) LMD compared to CSF of patients without CNS involvement (H = 9) using targeted mass spectrometry analysis with stable isotope -labeled standards for individual BCKAs (Figs. 2E and 2F). Combined levels of BCKAs as high as 79.73 pM were observed in patients with LMD and almost undetectable in the CSF from patients without LMD, Thus, a marked accumulation of BCKA in CSF is a hallmark of LMD from multiple tumor types.

[0435] The toxic effects of high-dose (1-50 mM) BCKAs on neurons, glial cells, and astrocytes have been demonstrated. However, it is unclear if these doses represent physiological levels of BCKAs found in human CSF associated with any BCKA-accumulating disorders, as absolute concentrations of these metabolites have not beenAttorney Docket No. 10110-466WO1

[0436] reported. Exposure of neurons to BCKA levels found in the leptomeningeal environment compromised neuronal metabolic activity and viability in complete neuronal media (FIG.

[0437] 46G, FIG. 54A, B). Similarly, the metabolism of primary human meningeal cells is sensitive to BCKA exposure (FIG. 54C). Further, treatment of primary neurons to levels of BCKA found in LAID patients (e.g., 50 pM) in the context of physiological CSF impaired neuronal metabolic activity (p < 0.01, FIG. 46H). Moreover, consistent with spatial transcriptomics analysis showing increased expression of GFAP and MT2, known gliosis markers, at the interface between tumor and stromal cells in LMD tissues, low levels of BCKAs also induced a reactive gliosis, a common feature of neurodegenerative disease, in the SM1 murine models of LMD (p < 0.001, FIG. 54D). Finally, in accord with a role in LMD-associated neurodegenerative processes, the abundance of KIC, the most neurotoxic BCKA, negatively correlated with the Karnofsky Performance Status in our LMD patient cohort (R = -0.3367, p < 0.05, FIG. 461). In contrast, established human and murine lymphoma, melanoma, and breast cancer cell lines were not sensitive to BCKA exposure, with some cell lines even showing increased viability with lower-dose BCKA exposure (FIG. 54E-G). These data suggest that accumulation of BCKA in LMD promotes a neurodegenerative microenvironment favoring tumor growth and survival.

[0438] Targeting BCKA accumulation blocks neurological progression in murine models of LMD

[0439] To recapitulate the neurological progression of LMD, five immune-competent murine models of LMD were established through orthotopic injection of A20 lymphoma, EO771 breast cancer, SMI melanoma, D4M melanoma, and YLJMM3.2 melanoma cells into the CSF space via the cisterna magna. To evaluate these animal models, neurological assessments commonly utilized in the study of neurodegenerative diseases were performed, including tail suspension test (hind limb splay), grip strength test, motor function test, grooming, and kyphosis (FIG. 55 A), Consistent with clinical manifestations of LMD in humans, there was a profound reduction in the ability of animals with LMD to perform a normal hind limb splay, a loss in neuro-motor coordination, and kyphosis (excessive curve of the spine, FIG. 47A, Extended Data Figs. 7B-D). These symptoms were paired with a substantial reduction in grooming and a deformity in skull shape (FIG. 55D, E).

[0440] Most mice with LMD reach the endpoint based on neurological complications three to four weeks after tumor engraftment (FIG. 47B, FIG. 55F). At necropsy, significant degradation of the pial membrane was noted in the lymphoma LMD animals (FIG. 55G). InAttorney Docket No. 10110-466WO1

[0441] Parkinson's disease, Alzheimer’s disease, schizophrenia and other neurodegenerative processes, neurological degeneration is associated with a loss of cortical microtubule-associated protein 2 (MAP2) reactivity, gliosis, and increases in GFAP expression. A loss of cortical MAP2 reactivity was manifest in animals with LMD, within the non-tumor tissues adjacent to the tumor-containing CSF space (p < 0.01, FIG. 47C, D). This loss was coupled with increased GFAP staining of the meningeal surfaces of the brain, suggesting reactive gliosis (FIG. 55H). Together, these data establish models of LMD that recapitulate this central feature of human LMD and consequently enable the mechanistic determination of drivers of LMD progression.

[0442] Analysis of the digested pia tissues confirmed an accumulation of BCKAin animals with LMD (p < 0.05, FIG. 47E). To test if BCKA-reducing therapy can slow neurological deterioration or improve overall survival in LMD mouse models, they were treated with sodium phenylbutyrate (PBA), an FDA-approved drug that is used to treat inborn metabolic diseases that result in the accumulation of BCKAs and branched-chain amino acids, such as Maple Syrup Urine Disease and amyotrophic lateral sclerosis. Mice were injected with tumors subcutaneously and intrathecally (FIG. 47F). PBA as a monotherapy effectively extended both the overall survival (median survival 23 vs. 35 days, p = 0.0037) and the progression-free survival (NeuroScore < 2) compared to the untreated group (median PFS 21 vs. 34 days, p = 0.0048) and reduced LMD-associated weight loss (p < 0.05, FIG. 47G, H and FIG. 56A). PBA therapy led to reductions in BCKA accumulation and the extent of BCKA reduction correlated with overall survival in PBA-treated animals (R = -0.8669, p < 0.05, FIG. 471, J). PBA treatment also delayed the onset of neurological symptoms and reduced the severity of neurological symptoms at endpoint (FIG. 47K and FIG. 56B). These data suggest that reducing BCKA accumulation may preserve neurological function and improve quality of life and survival.

[0443] BCKA promotes T cell dysfunction

[0444] BCKA accumulation results in a constellation of symptoms for patients with Maple Syrup Urine Disease, including immune dysfunction, but the mechanisms of immune suppression are unknown. In LMD patient CSF the accumulation of BCKA directly correlated with the numbers of Tregs and dysfunctional CD8+T cells and NK cells (FIG.

[0445] 48A, FIG. 57). To test if BCKA accumulation affects T cell viability and function, primary human CD4+and CD8+T cells were treated with BCKA in regular T cell media. BCKA treatment reduced T cell proliferation following stimulation (FIG. 58A, B) and treating T cells with BCKA in the context of physiological CSF blunted T cell proliferation even atAttorney Docket No. 10110-466WO1

[0446] 50 μM doses of BCKAs (FIG. 48B, FIG. 8C). BCKA treatment of T cells in physiological CSF severely reduced the secretion of pro-inflammatory effector cytokines, including interferon-y (p < 0.0001), granzyme B (p < 0.01), and interleukin-2 (p < 0.001, FIG. 48C). Treatment with KIC alone or combined BCKAs reduced the viability of healthy donor T cells even at 25-50 pM doses after a 48-hour exposure in physiological CSF (Fig 4D). There were also modest increases in the expression of the LAG3 checkpoint protein on BCKA treated CD4+ and CD8+ T cells, yet this was not statistically significant (FIG. 58D). This change was consistent with the elevated expression of LAG3 observed in CD8 T cell cluster 4, an exhausted cluster of T cells whose numbers correlated with BCKA accumulation in patient CSF (FIG. 48A and 51G). Similar effects on viability and cytokine secretion were observed in BCKA-treated CD 19- targeting CAR T cells (FIG. 48E and 48F), highlighting a vulnerability of CAR T cells to the LMD environment. This data suggests that BCKA accumulation directly promotes a dysfunctional T cell landscape in LMD.

[0447] BCKA-reducing therapy enhances the efficacy of CAR-T cell therapy against LMD

[0448] Despite the effectiveness of CD19-targeting CAR T-cell therapy in treating chemo¬ refractory B-cell lymphoma, the responses in LMD patients are short-lived. Given that BCKA directly affects CAR T cell viability and function, we tested if BCKA-reducing PBA augments the efficacy CAR T therapy in animal LMD models (FIG. 48G). Intriguingly, combination therapy prolonged overall survival (median survival 29 vs.49 days, p = 0.0612) and progression-free survival (median survival 29 vs. 43 days, p = 0.0424) in the lymphoma LMD model (FIGS. 48H, 481, and 58E). Mice treated with the combination had improved neurological function scores at endpoint, highlighting the potential use of PBA to improve neurological function and the quality of life with LMD, even with eventual progression (FIG. 58F, G). Significant neurotoxicity has been a major challenge with anti-CD19 CAR T therapies. Consistent with the enhanced neurologic function in treated animals, PBA treatment blocked the loss of MAP2 reactivity in the cortex of animals with LMD, further validating the effect of BCKA reduction on preserving neuronal integrity in the LMD mouse model (p < 0.05, FIG. 48J). Finally, MAP2 reactivity correlated with survival time across all treatment cohorts (FIG. 48K). Together these data provide preclinical rationale for enhancing CAR T cell efficacy using BCKA-reducing therapy.

[0449] DiscussionAttorney Docket No. 10110-466WO1

[0450] This study provides fundamental insights into the pathobiology and therapeutic opportunities for LMD patients. Our findings establish metabolic rewiring of the cerebrospinal fluid as a key driver of disease progression and challenge the long-standing paradigm that anatomical barriers alone account for dismal treatment outcomes in LMD. The discovery of BCKA accumulation in LMD as a central mechanism linking immune dysfunction and neurodegeneration has broad implications, informing our understanding of the brain tumor microenvironment and neurodegenerative conditions more broadly.

[0451] Particularly striking is the identification of BCKAs as metabolic mediators that simultaneously suppress anti-tumor immunity and promote neurological decline. While metabolic regulation of immune function is a hallmark of cancer, the studies presented herein reveal how specific metabolites can create a unique microenvironment that shapes both immune and neurological outcomes. This finding bridges the traditionally separate fields of cancer metabolism, neuro-oncology, and immunotherapy, and suggest new therapeutic strategies that may improve both survival and quality of life for LMD patients.

[0452] The analysis of the fluid microenvironment of LMD showed significant alterations in the abundance of -30% of lipids, proteins, and metabolites, indicating demyelination, disruption of normal neuronal maintenance, and neurodegenerative processes. The most profound discovery stemmed from the analysis of the fluid metabolomics, where there is a ~45-60-fold increase in BCKAs in LMD patients. BCKAs are derived from the deamination of the branched-chain amino acids, and their accumulation is linked to the neurotoxicity identified in patients with Maple Syrup Urine disease (MUSD, a rare inborn metabolism disorder). Although limited studies have investigated the role of BCKAs in cancer, the elevation of BCKAs has never been reported in LMD, and the effects of these toxic metabolites on T-cell function or neurodegeneration with cancer have never been explored. Our data show that BCKAs can significantly reduce the viability, proliferation, and effector function of endogenous T cells and anti-CD19 CAR T cells, providing new opportunities to engineer metabolically resilient CAR-T cells and / or develop combination therapies that protect immune cell function. Rapid and debilitating neurological symptoms are the leading cause of poor survival and impaired quality of life in LMD patients, and these were also manifest in LMD mouse models. Preserving neurological function is extremely important to LMD patients and their caregivers. BCKA-lowering therapy, such as PBA, has improved neurological symptoms in MUSD patients and importantly, PBA monotherapy shows a profound improvement in the neurological functions and the survival in LMD mouse models, confirming a role for BCKAs in LMD pathogenesis. Furthermore, PBAAttorney Docket No. 10110-466WO1

[0453] treatment also enhanced the efficacy of anti-CD 19 CAR T cell therapy in a lymphoma LMD model, with the combination showing the highest median overall survival, highest progression-free survival, and most improved neurological symptoms. The identification of BCKAs as central mediators of LMD progression opens multiple therapeutic avenues beyond phenylbutyrate, such as dietary modification or other BCKA-reducing strategies, all of which have the potential to improve the quality of life, survival outcomes, and efficacy of immunotherapies in patients with LMD.

[0454] Methods

[0455] Patient specimens

[0456] This study was conducted according to recognized ethical guidelines (e.g., Declaration of Helsinki, CIOMS, Belmont Report, U. S. Common Rule). All CSF specimens were obtained during routine clinical care and procured under protocols approved by the Institutional Review Board (MCC#19332). CSF was spun down to separate the fluid and cellular compartments.

[0457] Cell cultures

[0458] Cell Lines: The murine lymphoma A20 cell line was purchased from ATCC. Human lymphoma cell lines 0CI-LY3, Toledo, SUDHL4, and JEKO were a generous gift from Dr. Fredrick Locke (Moffitt Cancer Center). All lymphoma cell lines were maintained using RPMI-164 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS, Sigma-Aldrich), ImM sodium pyruvate (Corning), lx MEM Nonessential Amino Acid (CORNING), and 0.05 mM 2-mercaptoethanol (Sigma- Aldrich). Human melanoma cell lines 1205Lu, WM164, WM793, A374, and MBM01, and murine melanoma cell lines SMI and D4M were a generous gift from Dr. Keiran Smalley (Moffitt Cancer Center). Murine melanoma cell line YUMM3.2 was generously gifted by Dr. Sheri Holmen (Huntsman Cancer Institute, UT). All melanoma cell lines were maintained in RPMI-164 with 5% heat- inactivated FBS. Dr. Eric Lau (Moffitt Cancer Center) generously shared the human breast cancer cell line MDA-MB2 1. Murine cell lines 4T1 and E0771 were purchased from ATCC. All breast cancer cell lines were maintained in 10% heat-inactivated fetal bovine serum. 'The identities of all cell lines were confirmed through short tandem repeat validation analysis, and all cell cultures were routinely tested for mycoplasma contamination.

[0459] Primary T cells: Human T cells were isolated from peripheral blood mononuclear cells (PBMC) using the EasySep Human T Cell Iso Kit (STEMCELL), while mouse T cells were isolated from the spleens of Balb / C mice using the EasySep Mouse T Cell Isolation Kit (STEMCELL) following the manufacturer's instructions. Then, T cells were cultured inAttorney Docket No. 10110-466WO1

[0460] 10% FBS-RPMI with 1% sodium pyruvate, 1% nonessential amino acids, and 1% Pen- Strep.

[0461] CD19–28z CART generation: T cells were stimulated with Dynabeads T-Activator CD3 / CD28 (Gibco) and 200IU / mL of IL-2 (PeproTch) in complete media. The next day, in retronectin-coated 6 -well plates, stimulated T cells were cultured with retro viral supernatant collected from Phoenix E (mouse) or RD 114 (human) producer cells (gift from Dr. Marco Davila, Moffitt Cancer Center) to transduce mouse or human T cells as previously described. Transduction efficiency was detected using flow cytometry as a percentage of mCherry+or Ametrine+cells.

[0462] Primary human meningeal cells: Primary human meningeal cells (ScienCell) were grown in Meningeal Cell Media containing 2% FBS, 1% growth supplements, and 1% antibiotics, per the manufacturer instructions.

[0463] Primary neurons: Primary mouse neurons were isolated from 3 days postnatal mouse brains and digested in papain / DNase solution. Primary Rat Cortex Neurons were purchased from Gibco (A36511). Murine neurons were maintained in a polylysine-coated 96-well plate. The culture media consisted of Neurobasal Plus Medium containing IX B27 (50X, Gibco), 200mM Glutamax, and 20mM HEPES.

[0464] Physiological cerebrospinal fluid (CSF):

[0465] To prepare CSF with physiological properties, synthetic CSF (TOCRIS) was supplemented with 1% FBS, 60mg / dL D-Glucose (Sigma- Aldrich), lx MEM Nonessential Amino Acids (CORNING), and 15mM HEPES (LONZA). CSF was then filtered with a 0.22 mM filter and was used within one day of preparation.

[0466] B ranch ed-chain keto acids:

[0467] 3-methyl-2-oxovaleric acid sodium salt (KMV) (Toronto Research Chemical), 4-methyl-2-oxovaleric acid (KIC) (Sigma), and Sodium 3-methyl -2-oxobutyrate (KIV) (Sigma) were used to prepare branched-chain ketoacid (BCKA) solutions (in PBS) in the ratio 1:2.2: 1.6 to recapitulate the physiological concentrations.

[0468] T cell proliferation assay:

[0469] 48-well plates were coated with OKT3 (5 pg / mL). 'Then, 2 x 105fresh PBMC, prelabeled with cell trace violet (CTV), were cultured in 500 pL of full media containing CD28.2 (2 pg / mL) and IL2 (200 IU / L) for 24 hours. The next day, 400 pL of the media was carefully removed, and 350 pL of either full media or physiological CSF was added with 50Attorney Docket No. 10110-466WO1

[0470] | L BCKA at 10X the required concentration or PBS vehicle control. The experiments were repeated with technical and biological triplicates using three different PBMC donors.

[0471] Cell viability tests:

[0472] In black 96-well plates, pre-coated with 0KT3 (5 pg / mL), 3 x 104T-cells were cultured in complete media and IL-2 for one day, and then the media was changed to either fresh full media or CSF with BCKA or PBS vehicle control. For cancer and CAR T cells, ~3xl04cells were plated in black 96-well plates in 40 μL full media. Then, 140 pL of physiological CSF and 20 pl, of 10X BCKAs (PBS for controls) were added to each well. After 48 hours, Calcein Am (Invitrogen) was added for one hour at 37 °C, and the fluorescence intensity was measured at 485nm excitation and 520 nm emission wavelengths. The experiments were repeated using two different PBMC donors.

[0473] The viability of lymphoma cells was validated with propidium iodide staining. In 24-well plates, lymphoma cell lines were cultured at density 2. 5x 105in 100 pL full media with 350 μL CSF and 50 μL 10X BCKA. After 48 hours, cells were collected, stained with propidium iodide, and analyzed by flow cytometry.

[0474] For neuron viability assay, 4xl04neurons were plated in a polylysine-coated 96-well plate in neurobasal media or physiological CSF with 0-50 pM BCKA exposure. After 7 days, Calcein Am (Invitrogen) was added for one hour at 37 °C, and the fluorescence intensity was measured at 485nm excitation and 520 nm emission wavelengths.

[0475] MTT assays

[0476] A 96-well plate was coated with poly-L-lysine, and then primary mouse neurons were cultured in neurobasal media or physiological CSF. Then, 50 pM BCKA or PBS vehicle control was added. On the third day, the media was changed with fresh media with BCKA or PBS vehicle control. On the 7thday, 20 pL of MTT (5 mg / mL) solution was added to each well and incubated at 37 °C for four hours, then 200 μL of DMSO was added to dissolve formazan crystals. Absorbance was read at 550 nm. Primary meningeal cells were cultured in a 96 well-plate in full media for 72 hours. Then, the same protocol for the MTT assay was performed.

[0477] ELLA multiplex cytokine assay and checkpoint expression:

[0478] Forty-eight well plates were coated with OKT3 (5 pg / mL), then 2 x IO5fresh PBMC were cultured in the presence of CD28 (2 pg / mL) and IL2 (200 IU / L) for 24 hours, and 500 pL of complete media. The next day, 400 μL of the media was carefully removed, and 350 μL of either full media or physiological CSF was added with 50 μL BCKA (10X the requiredAttorney Docket No. 10110-466WO1

[0479] concentration) or PBS vehicle control. On the fifth day of culturing, 300 pL of the supernatant was collected to measure Granzyme B, Interferon-y, and IL-2 using an Ella multiplex assay plate (Bio-techne). The cells were stained using the following anti-human antibodies: CD4 (BUV395), CD8 (FITC), PD-l(PE), andLAG3 (BV421). Live / Dead (NIR) stain was used for viability. Samples were analyzed on an LSR II flow cytometer (Beckman Coulter), and all data were analyzed using FlowJo. The experiments were repeated with technical and biological triplicates using three different PBMC donors (endogenous T cells) or two different donors (CAR T cells).

[0480] Immunohistochemistry:

[0481] Mouse brains were collected immediately after euthanasia. Tissue was fixed in 10% neutral buffered formalin for ~ 24 hours, transferred to aqueous 70% ethanol, and kept at 4 °C until sectioning. Tissues were embedded in paraffin blocks and sectioned (4-micron thickness) at the Tissue Core of Moffitt Cancer Center. Sections were rehydrated, then blocked with goat serum (Gibco) for one hour, followed by incubation with rabbit anti-GFAP (1:1000) or rabbit anti-MAP2 (1:1000) antibody overnight at 4 °C. Alexa Fluor 680 goat IgG (Invitrogen) was the secondary antibody. Slides were mounted using SlowFade Diamond mounting media containing DAPI and imaged on the Akoya slide scanning microscope. Images were visualized using Phenochart 1.2.0 software and quantified using ImageJ software.

[0482] Animals:

[0483] All mouse work was conducted following recognized ethical guidelines and IACUC approval. Female BALB / c and C57BL / 6 mice at approximately 12 weeks of age were used to establish murine models of LMD. A20 lymphoma cells (5 x 104cells in 5 pL PBS), SMI melanoma (2.5 x 104cells in 5 pL PBS), and E0771 breast cancer cells (104cells in 5 pL PBS) were injected via the cisterna magna. LMD animals utilized in drug treatment studies were also injected in the left side lymph node of the leg flank (concentration 5 x 105cells in 30 pLPBS) or subcutaneously (5 x 106cells in 100 pl, PBS). The control group was injected with only PBS in both the cisterna magna (5 pL) and the left side lymph node of the leg flank (30 pL) or subcutaneously (lOOpL PBS). The extracranial metastasis group was injected intravenously in the tail vein with either tumor cells (concentration 5 x 104cells in 100 pL PBS) or 100 pL of PBS as a control. Mice were anesthetized during the procedure and received analgesics for three days to alleviate any pain from the surgery. Mice were weighed twice a week and evaluated using neurological function assessments. For theAttorney Docket No. 10110-466WO1

[0484] neurological evaluation, we used a group of tests previously described in neurodegenerative diseases, including 1) hind limb splay, which assesses the ability of the mouse to control the whole-body muscles and perform a hind leg splay during a tail suspension, 2) motor function, which assesess the neuro-motor coordination and function, 3) Grooming, which is a behavioral test for the overall health status of the mouse, 4) Gripping of the wire lid is an indicator of the strength of the fore limb, and 5 ) Kyphosis, which is used to observe spine curvature or deformity and has been used to evaluate neurodegenerative diseases in mouse models. Anti-CD19 CAR T-treated mice had lymphodepletion using cyclophosphamide (150 pg / gm body weight, IP injection) 24 hours before CAR T injection. CAR T transfection efficiency was 60% (confirmed by mCherry -positive cells), and mice received -0.5 x 106CAR T cells via tail vein injection. Sodium phenylbutyrate (PBA)-treated mice received daily IP injection (0.2 mg / gm body weight in PBS). Mice were euthanized by cervical dislocation under anesthesia when they lost more than 20% of their original weight or showed severe neurological decline such as paralysis or ataxia. For subsequent single-cell RNAseq analysis, pia mater membranes and extra-cranial injection site tumors were harvested at the endpoint. Tissues were digested using a tumor dissociation kit (Miltenyi Biotec). For BCKA analysis, the pia membrane was directly collected in 80% Methanol and then stored at -80 until analysis.

[0485] Single-cell RNA-seq

[0486] Cell number and viability were assessed using Countess II FL automated cell counter. Cells were resuspended in cold 0.2% BSA / PBS at an optimum concentration (500 cells / pl). The cell suspension was directly loaded for scRNA-seq library preparation using the Chromium Single Cell Reagent Kit following the manufacturer’s protocol (lOx Genomics, USA) as previously described74. The cDNA libraries were prepared using the Single-Cell 3' Library Prep Kit (10X Chromium). The resulting libraries were sequenced on the Illumina NextSeq 500 instrument using v2.5 flow cells. Approximately 80,000 to 1,000,000 mean sequencing reads per cell were generated. scRNA-seq sequencing data were demultiplexed, aligned, and quantified using the 10X Genomics Cell Ranger Single-Cell Software Suite against the human reference genome. Quality control and cell type analysis were performed as previously described.

[0487] Proteomic, metabolomic, and lipidomic analyses

[0488] Proteomic, metabolomic, and lipidomic analyses of cell-free CSF samples were performed at Moffitt Cancer Center’s Proteomics & Metabolomics Core. LC-MS gradeAttorney Docket No. 10110-466WO1

[0489] solvents and additives, including water, methanol, acetonitrile, and formic acid, were purchased from Burdick and Jackson, Honeywell (sourced through VWR), and Thermo Scientific. Neat stand...

Claims

Attorney Docket No. 10110-466W01IX. CLAIMSWhat is claimed is:

1. A method of treating Leptomeningeal disease (LMD) in a subject, comprising:administering to the subject a therapeutically effective amount of sodium phenylbutyrate; anda pharmaceutically acceptable carrier.

2. The method of claim 1, wherein the therapeutically effective amount of sodium phenylbutyrate and the pharmaceutically acceptable carrier are administered intrathecally.

3. The method of claim 1, further comprises administering to the subject a therapeutically effective amount of at least one anti-cancer agent.

4. The method of claim 3, wherein the anti-cancer agent comprises cytarabine, methotrexate, thiotepa, rituximab, capmatinib, or tepotinib.

5. The method of claim 1, wherein LMD is associated with branched-chain keto acids (BCKA) accumulation in a sample of cerebrospinal fluid obtained from the subject.

6. The method of claim 1, wherein LMD is associated with CAR-T cytotoxicity.

7. The method of claim 1, wherein LMD is associated with neurodegeneration.

8. The method of any one of claims 1-7, wherein administering to the subject the therapeutically effective amount of sodium phenylbutyrate and the pharmaceutically acceptable carrier of claim 1, reduces BCKA accumulation in a sample of cerebrospinal fluid obtained from the subject, compared to an untreated control.

9. The method of any one of claims 1-8, wherein administering to the subject the therapeutically effective amount of sodium phenylbutyrate and the pharmaceutically acceptable carrier of claim 1, reduces CAR-T cytotoxicity, in the subject, compared to an untreated control.Attorney Docket No. 10110-466W01 10. The method of any one of claims 1-9, wherein administering to the subject the therapeutically effective amount of sodium phenylbutyrate and the pharmaceutically acceptable carrier of claim 1, reduces neurodegeneration, in the subject, compared to an untreated control.

11. The method of any one of claims 1-10, wherein the subject is a human.

12. A method of treating Leptomeningeal lymphoma (LML) in a subject, comprising:administering to the subject a therapeutically effective amount of sodium phenylbutyrate; anda pharmaceutically acceptable carrier.

13. The method of claim 12, wherein the therapeutically effective amount of sodium phenylbutyrate and the pharmaceutically acceptable carrier is administered intrathecally or systematically.

14. The method of any one of claim 12 or claim 13, further comprising administering to the subject a therapeutically effective amount of at least one anti-cancer agent.

15. The method of any one of claims 12-14, wherein the anti-cancer agent comprises cytarabine, methotrexate, thiotepa, rituximab, capmatinib, or tepotinib.

16. The method of any one of claims 12-15, wherein LML is associated with branched-chain keto acids (BCKA) accumulation in a sample of cerebrospinal fluid obtained from the subject.

17. The method of any one of claims 12-16, wherein LML is associated with CAR-T cytotoxicity.

18. The method of any one of claims 12-17, wherein LML is associated with neurodegeneration.

19. The method of any one of claims 12-18, wherein administering to the subject the therapeutically effective amount of sodium phenylbutyrate and the pharmaceutically acceptable carrier of claim 12, reduces BCKA accumulation in a sample of cerebrospinal fluid obtained from the subject, compared to an untreated control.

20. The method of any one of claims 12-19, wherein administering to the subject the therapeutically effective amount of sodium phenylbutyrate and the pharmaceutically acceptable carrier of claim 12, reduces CAR-T cytotoxicity, in the subject, compared to an untreated control.Attorney Docket No. 10110-466W01 21. The method of any one of claims 12-20, wherein administering to the subject the therapeutically effective amount of sodium phenylbutyrate and the pharmaceutically acceptable carrier of claim 12, reduces neurodegeneration, in the subject, compared to an untreated control.

22. The method of any one of claims 12-21, wherein the subject is a human.

23. A method of treating a subject with a cancer and / or a cancer-associated complication, comprising:administering to the subject a therapeutically effective amount of sodium phenylbutyrate; anda pharmaceutically acceptable carrier.

24. The method of claim 23, wherein the therapeutically effective amount of sodium phenylbutyrate and the pharmaceutically acceptable carrier is administered intrathecally.

25. The method of any one of claim 23 or claim 24, further comprising administering to the subject a therapeutically effective amount of at least one anti-cancer agent.

26. The method of any one of claims 23-25, wherein the anti-cancer agent comprises cytarabine, methotrexate, thiotepa, rituximab, capmatinib, or tepotinib.

27. The method of any one of claims 23-26, wherein the cancer-associated complication is Leptomeningeal Disease (LMD), Leptomeningeal metastasis (LML), or carcinomatosis (LMC).

28. The method of any one of claims 23-27, wherein the cancer-associated complication is branched-chain keto acids (BCKA) accumulation in a sample of cerebrospinal fluid obtained from the subject.

29. The method of any one of claims 23-28, wherein the cancer-associated complication is CAR-T cytotoxicity.

30. The method of any one of claims 23-29, wherein the cancer-associated complication isneu rodegener ation.

31. The method of any one of claims 23-30, wherein administering the therapeutically effective amount of sodium phenylbutyrate and the pharmaceutically acceptable carrier to the subjectAttorney Docket No. 10110-466W01 reduces BCKA accumulation in a sample of cerebrospinal fluid obtained from the subject, compared to an untreated control.

32. The method of any one of claims 23-31, wherein administering the therapeutically effective amount of sodium phenylbutyrate and the pharmaceutically acceptable carrier to the subject reduces CAR- T cytotoxicity in the subject compared to an untreated control.

33. The method of any one of claims 23-32, wherein administering the therapeutically effective amount of sodium phenylbutyrate and the pharmaceutically acceptable carrier of claim 23 to the subject reduces neurodegeneration in the subject compared to an untreated control.

34. The method of any one of claims 23-33, wherein the subject is a human.

35. A method for enhancing T cell activity in a subject of claim 23, comprising:determining a subject having an elevated concentration of Branched-Chain Keto Acids (BCKA) in the cerebrospinal fluid (CSF); andadministering a therapeutically effective amount of a therapeutic agent to reduce BCKA accumulation or inhibit BCKA-mediated signaling in the CSF, thereby reversing T cell exhaustion or inactivity.

36. The method of claim 35, wherein the therapeutic agent is a buffering agent to increase the pH of the CSF.

37. The method of claim 35 or 36, wherein the therapeutic agent is a metabolic inhibitor to reduce the production of BCKAs.

38. The method of any one of claims 35-37, wherein the metabolic inhibitor comprises binimetinib, cobimetinib, trametinib, LY294002, wortmannin, BCAT1 inhibitors, or BCAT2 inhibitors.

39. The method of any one of claims 35-38, wherein the BCAT1 or BCAT2 inhibitors comprise BAY-069, BT2, Bufalin, WQQ-345, Gabapentin, or ERG240.Attorney Docket No. 10110-466W01 40. The method of any one of claims 35-39, wherein the therapeutic agent targets the MAPK pathway, thereby restoring phosphorylation of p38α (MAPK14) or 4E-BP1 (EIF4EBP1) in T cells.

41. A method of treating a cancer of claim 23 in a subject, comprising:administering to the subject a therapeutically effective amount of a therapeutic agent that reduces BCKA accumulation in a cerebrospinal fluid compartment of the subject.

42. The method of claim 41, wherein the therapeutic agent comprises phenylbutyrate and a pharmaceutically acceptable carrier thereof.

43. The method of claim 41 or claim 42, further comprising administering an intrathecal dendritic cell therapy to the subject.

44. The method of any one of claims 41-43, wherein the intrathecal dendritic cell therapy comprises administering dendritic cells loaded with MHC class II peptides that target HER2, HER3, or a combination thereof on cancer cells.

45. The method of any one of claims 41-44, wherein administering the therapeutic agent that reduces BCKA accumulation enhances an immune favorable microenvironment in leptomeningeal metastatic disease relative to administration of the intrathecal dendritic cell therapy without the therapeutic agent.

46. The method of any one of claims 41-45, wherein the immune favorable microenvironment comprises increase in T cell infiltration, increase in T cell activation, decrease in T cell exhaustion, or a combination thereof.

47. The method of any one of claims 41-46, wherein the cancer comprises a breast cancer induced leptomeningeal metastatic disease, breast cancer, leptomeningeal disease (LMD), leptomeningeal metastasis (LM), or carcinomatosis (EMC).

48. The method of any one of claims 41-47, wherein treating the cancer comprises prolonging survival, reducing leptomeningeal tumor burden, or a combination thereof.Attorney Docket No. 10110-466W0149. The method of any one of claims 41-48, wherein the therapeutic agent to reduce BCKA accumulation is administered intrathecally, orally, systemically, or by a combination thereof.

50. A method of treating breast cancer leptomeningeal metastatic disease of claim 47 in a subject, comprising:administering a combination of a therapeutic agent to reduce branched chain keto acid accumulation in cerebrospinal fluid, and a dendritic cell therapy.

51. The method of claim 50, wherein the dendritic cell therapy comprises a peptide pulsed conventional dendritic cells.

52. The method of claim 50 or claim 51, wherein the therapeutic agent to reduce branched chain keto acid accumulation comprises sodium phenylbutyrate.

53. The method of any one of claims 50-52, wherein the peptide pulsed conventional dendritic cells comprise one or more MHC class II peptides that target HER2, HER3, or a combination thereof on cancer cells.

54. The method of any one of claims 50-53, wherein the method further enhances T cell infiltration into leptomeningeal tumor tissue, increases T cell activation, reduces T cell exhaustion, or a combination thereof.

55. The method of any one of claims 50-54, wherein the method further increases a CD4 Thl response, increases a B cell response, or both, in the cerebrospinal fluid compartment.

56. The method of claim 52, wherein the breast cancer leptomeningeal metastatic disease comprises HER2 positive breast cancer leptomeningeal metastatic disease, triple negative breast cancer leptomeningeal metastatic disease, or a combination thereof.Attorney Docket No. 10110-466W01 57. The method of any one of claims 50-56, wherein the therapeutic agent to reduce branched chain keto acid accumulation is administered prior to the dendritic cell therapy, concurrently with the dendritic cell therapy, or after the dendritic cell therapy.

58. The method of any one of claims 50-57, wherein administering the dendritic cell therapy comprises administering a plurality of doses.

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