GSK-3 inhibitor elraglusib (9-ing-41) modifies protein expression in the tumor microenvironment to enhance tumor-infiltrating immune cell activation in clinical tumor biopsies

Abstract

Immune checkpoint blockade (ICB) therapy has provided hope for treating various cancers. However, many tumors are resistant to ICB. Described herein are lifesaving compositions and anti-cancer methods including the administration of combination therapies of a small molecule GSK-3 inhibitor and a therapeutic agent effective for an immune checkpoint blockade (ICB) therapy to a subject in need thereof. In some aspects, the cancer response the the GSK-3 inhibitor can be utilized to guide the ICB therapy. In particular, tumors that are resistant to ICB are compromised by the GSK-3 inhibitor and become vulnerable to the combination therapies disclosed herein.

Description

GSK-3 INHIBITOR ELRAGLUSIB (9-ING-41) MODIFIES PROTEIN EXPRESSION IN THE TUMOR MICROENVIRONMENT TO ENHANCE TUMOR-INFILTRATING IMMUNE CELL ACTIVATION IN CLINICAL TUMOR BIOPSIESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to United States provisional application Serial No. 63 / 481 ,941 , filed January 27, 2023, the disclosure of which is incorporated by reference as if fully set forth herein in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with government support under grant number CA271636 awarded by the National Institutes of Health. The government has certain rights in the invention.FIELD OF THE INVENTION

[0003] The embodiments of the present invention relate to novel combination therapies for preventing and / or reversing serious and otherwise lethal cancer in subjects, which in some instances can appear to be unresponsive to an immune checkpoint blockade (ICB) therapy but are responsive to treatments using the combination therapies disclosed herein.BACKGROUND OF THE INVENTION

[0004] Checkpoint blockade immunotherapy is an innovative cancer treatment that uses a class of drugs known as immune checkpoint inhibitors. Much like other immunotherapies, the goal of checkpoint blockade immunotherapy is to strengthen the body's immune system and enhance its ability to fight off harmful growth (e.g., cancer) or foreign invaders. Checkpoint blockade immunotherapy or immune checkpoint blockade (ICB) has rapidly improved the treatment paradigm for various cancer types. Multiple single or combinations of ICB treatments have been approved by the US Food and Drug Administration (FDA), providing more options for patients with advancedcancer. However, there are certain tumors and classes of cancer that do not respond well to ICB treatments. In some cases, the response rates of different tumor types can be proportional to their corresponding tumor mutational burden (TMB), presumably from the immunogenic neoantigens that are recognized as foreign by cytotoxic T lymphocytes. In one example, poorly immunogenic tumors with low TMB, such as pancreatic and prostate cancers, are inherently more resistant to treatment with a checkpoint inhibition approach.

[0005] Glycogen synthase kinase 3 (GSK-3) is a serine / threonine protein kinase typically utilized in an addition of phosphate molecules onto serine and threonine amino acid residues on its target substrate. GSK-3 has, since 1980, been identified as a protein kinase for over 100 different proteins in a variety of different pathways. In mammals, including humans, GSK-3 exists in two isozymes encoded by two homologous genes GSK-3α (GSK3A) and GSK-3β (GSK3B). GSK-3 has been the subject of much research since it has been implicated in a number of diseases, including type 2 diabetes, Alzheimer's disease, inflammation, and even mental diseases. As a regulatory kinase for its namesake of glycogen synthase (GS), changes in GS brought about by GSK-3 can have broad metabolic effects. For example, a GS inhibition by GSK-3β leads to a decrease in glycogen synthesis in the liver and muscles, along with increased blood glucose or hyperglycemia. While GSK-3 is associated with the pathogenesis and progression of many diseases, what has not been effectively investigated is its potential effects on ICB therapies. The technology disclosed herein elucidates combinations of GSK-3 and ICB therapies that provide urgently needed treatments for patients in need, cancers, and diseases that are less responsive (or not responsive) to ICB therapies. BRIEF SUMMARY OF THE INVENTION

[0006] The following presents a simplified summary of the innovation in order to provide a basic understanding of some aspects of the invention. This summary is not an extensive overview of the invention. It is intended to neither identify key or critical elements of the invention nor delineate the scope of the invention. Its sole purpose is to present some concepts of the invention in a simplified form as a prelude to the moredetailed description that is presented later. Example embodiments of the present invention provide improved technology (e.g., patient care systems, combination therapies, deployments, and strategies) for use in preventing and / or in treating suspected or established cancers that can resist treatments.

[0007] Glycogen synthase kinase-3 (GSK-3) is a serine / threonine kinase that has been implicated in numerous oncogenic processes. GSK-3 inhibitor elraglusib (9-ING- 41) has shown promising preclinical and clinical antitumor activity across multiple tumor types. Despite promising early-phase clinical trial results, there have been limited efforts to characterize the potential immunomodulatory properties of elraglusib. Herein is disclosed that elraglusib promotes immune cell-mediated tumor cell killing of microsatellite stable colorectal cancer (CRC) cells. Mechanistically, elraglusib sensitized CRC cells to immune-mediated cytotoxicity and enhanced immune cell effector function.

[0008] Using western blots, it was found that elraglusib decreased CRC cell expression of NF-κB p65 and several survival proteins. Using microarrays, it was discovered that elraglusib upregulated the expression of proapoptotic and antiproliferative genes and downregulated the expression of cell proliferation, cell cycle progression, metastasis, TGFβ signaling, and anti-apoptotic genes in CRC cells. Elraglusib reduced CRC cell production of immunosuppressive molecules such as VEGF, GDF-15, and sPD-L1. Elraglusib increased immune cell IFN-γ secretion, which upregulated CRC cell gasdermin B expression to potentially enhance pyroptosis. Elraglusib enhanced immune effector function resulting in augmented granzyme B, IFN- γ, TNF-α, and TRAIL production.

[0009] Using a syngeneic, immunocompetent murine model of microsatellite stable CRC, we evaluated elraglusib as a single agent or combined with immune checkpoint blockade (anti-PD-1 / L1) and observed improved survival in the elraglusib and anti-PD-L1 group. Murine responders had increased tumor-infiltrating T cells, augmented granzyme B expression, and fewer regulatory T cells. Murine responders had reduced immunosuppressive (VEGF, VEGFR2) and elevated immunostimulatory (GM-CSF, IL-12p70) cytokine plasma concentrations.

[0010] To determine the clinical significance, we then utilized elraglusib-treated patient plasma samples and found that reduced VEGF and BAFF and elevated IL-1beta, CCL22, and CCL4 concentrations correlated with improved survival. Using paired tumor biopsies, we found that tumor-infiltrating immune cells had a reduced expression of inhibitory immune checkpoints (VISTA, PD-1, PD-L2) and an elevated expression of T-cell activation markers (CTLA-4, OX40L) after elraglusib treatment. These results address a significant gap in knowledge concerning the immunomodulatory mechanisms of the GSK-3 inhibitor elraglusib, provide a rationale for the clinical evaluation of elraglusib in combination with immune checkpoint blockade, and are expected to have an impact on additional tumor types, besides CRC.

[0011] Thus, in an example embodiment, the technology discloses a pharmaceutical composition comprising (a) and (b): (a) a small molecule GSK-3 inhibitor, or a pharmaceutically acceptable salt and / or solvate or hydrate thereof; and (b) a therapeutic agent effective for an immune checkpoint blockade (ICB) therapy; or a pharmaceutically acceptable salt and / or solvate or hydrate thereof; wherein the pharmaceutical composition is included in a simultaneous formulation comprising both (a) and (b); or included in two separate pharmaceutical formulations with the first formulation comprising (a) and the second formulation comprising (b), configured for an initial administration of (a) later followed by an administration of (b).

[0012] In some embodiments, the pharmaceutical composition can be wherein (a) above is comprising a compound of formula 1 (below) or a pharmaceutically acceptable salt and / or solvate or hydrate thereof:.

[0013] According to some aspects, the pharmaceutical compositions can be configured wherein (b) above includes an immune checkpoint inhibitor comprising an anti-PD-1 and / or anti-PD-L1 therapeutic agent, Pembrolizumab (Keytruda), Nivolumab (Opdivo), Cemiplimab (Libtayo), Atezolizumab (Tecentriq), Avelumab (Bavencio), Durvalumab (Imfinzi), a CTLA-4 inhibitor, Ipilimumab (Yervoy), tremelimumab (Imjuno), a LAG-3 inhibitor, Relatlimab, or a combination thereof.

[0014] In some embodiments, the pharmaceutical composition is provided wherein the first formulation is configured for an initial administration to a subject in need thereof; and the second formulation is configured for an administration to the subject within a period of time of less than one year after the initial administration.

[0015] According to some aspects, the pharmaceutical compositions disclosed above or herein can be further comprising a therapeutic agent or method (c) either in place of (b) or in addition to (b); wherein (c) is comprising an agent effective for a combination therapy, a targeted therapy, a chemotherapy, a treatment modality that affects a tumor microenvironment, an application of an electromagnetic radiation / radiation, or a combination thereof.

[0016] In some embodiments, the pharmaceutical compositions are provided wherein the therapeutic agent in (b) is determined after an administration of (a) by one or more changes in biomarkers observed from examining changes in a tumor microenvironment subsequent to the administration of (a).

[0017] According to some aspects, the pharmaceutical compositions disclosed herein can be provided wherein the first formulation is configured for a first route of administration in a subject in need thereof, and the second formulation is configured for a second different route of administration in the subject.

[0018] In some embodiments, a method for treating a subject diagnosed with a cancer or suspected of having a cancer is disclosed herein, the method comprising the steps of: (1) administering a therapeutically effective amount of a small molecule GSK-3 inhibitor, or a pharmaceutically acceptable salt and / or solvate or hydrate thereof; whereby a protein expression in a tumor microenvironment is changed to enhance a tumor-infiltrating immune cell activation; and (2) administering a therapeutically effect amount of an immune checkpoint inhibitor effective for treating the subject with animmune checkpoint blockade (ICB) therapy; whereby the ICB therapy is enhanced compared to the same ICB therapy, ceteris paribus, without an execution of step (1).

[0019] According to some aspects, the method disclosed above can be wherein the cancer comprises a primary tumor of colorectal cancer (CRC), pancreatic cancer, lung cancer or cancer of the pleural space, liver cancer, rectum cancer, or a combination thereof.

[0020] In some embodiments, the method is directed wherein the cancer comprises microsatellite stable (MSS) colorectal cancer (CRC). According to some aspects, the method is implemented wherein the subject has been previously diagnosed as not responding to an ICB (immune checkpoint blockade) therapy.

[0021] In some embodiments, the methods disclosed herein can be further comprising the step of: (1b) measuring one or more biomarkers derived from the cancer in the subject; wherein step (1b) is executed before step (1), after step (1), or executed repeatedly; and the execution of step (1b) is operative to provide a determination of an effectiveness of step (1). In this example, the method can be embodied wherein the determination is utilized to determine the immune checkpoint inhibitor (ICI) included in step (2), and the ICI is determined by one or more changes in biomarkers observed from examining changes in a tumor microenvironment after step (1).

[0022] According to some aspects, the method above is configured wherein the one or more biomarkers comprise one or more differentially expressed proteins.

[0023] For example, the method can be implemented wherein the one or more differentially expressed biomarkers or proteins comprise CD39; CD163 (or the antigen CD163); OX40L (CD134 or TNFRSF4); checkpoint molecules for VISTA; myeloid / neutrophil marker CD66b; myeloid markers IDO1, CD80, or CD11b; TAM / M2 macrophage marker CD68; myeloid marker CD40 or DC / myeloid marker CD11c; Nuclear factor-κB (NF-κB); NEK2 protein; T cell activation marker CTLA4; antigen PTEN; hematopoietic marker CD34; T cell activation marker CD44; antigen presentation B2M; immune cell activation marker HLA-DR; TAM / M2 macrophage marker ARG1; memory T cell marker CD45RO; proliferation marker Ki−67; NK cell marker CD56; T cell activation marker GITR; immune checkpoint molecule B7-H3; Treg marker CD127; expression of B cell marker CD20; antigen NY-ESO-1; progesterone receptor (PR);cytotoxic T cell marker CD8; antigen Her2; Treg marker Foxp3; T cell marker CD3; immune checkpoint protein expression of LAG3, PD-L2, PD-1; protein expression of mature B cell / DC marker CD35; antigen MART1; checkpoint PD-L1 and PR; immune checkpoint Tim-3; Treg marker CD25; checkpoint protein TIGIT or LAG-3; or a combination thereof.

[0024] In some embodiments, the method discussed above can be further comprising an administration of a combination therapy including an agent effective for a combination therapy, a targeted therapy, a chemotherapy, a treatment modality that affects a tumor microenvironment, an application of an electromagnetic radiation or a particle radiation, or a combination thereof.

[0025] According to some aspects, disclosed herein is a method to determine how a cancer patient will respond to an ICB therapy, the method comprising the steps of: (1) administering a small molecule GSK-3 inhibition treatment to the patient; (2) obtaining a post-treatment biopsy from the patient; and (3) measuring one or more biomarkers differentially expressed in the post-treatment biopsy; whereby the one or more biomarkers are indicative of how the patient will respond to the ICB therapy. In this example, in some embodiments, the method can be further comprising the steps of: (0) obtaining a pre-treatment biopsy from the patient; and (4) measuring the one or more differentially expressed proteins in the pre-treatment biopsy; whereby a comparison between a level of one or more differentially expressed proteins in the pre-treatment biopsy and in the post-treatment biopsy is enabled.

[0026] According to some aspects, the method disclosed above can be wherein a therapeutic development of a GSK-3 inhibitor is provided by the comparison of the levels of the one or more biomarkers and / or the one or more proteins.

[0027] In some embodiments, the method disclosed above can be further comprising the step of determining the ICB therapy based upon the one or more biomarkers and / or the one or move proteins. A system can be implemented using these example methods and embodiments.

[0028] In an alternative aspect, the present invention provides a long-term method of preventing a cancer or a recurrence of a cancer in a subject in need thereof,by administering a therapeutically effective amount of any of the compositions disclosed above or by implementing any of the methods disclosed above.

[0029] The invention contemplates that the above-described compounds or combinations can be derivatized or can be structurally altered, for example, by addition or substitution of one or more atoms using a radioisotope or using a different element (e.g., B or boron in place of C or carbon), by removal of an ester or by addition of a salt form, an amino acid, a sugar, or a peptide. Hydrates and / or solvates can be formed by 1) dissolving the molecule in water and / or solvent and slowly drying, whereby water and / or solvent remain hydrogen bonded with OH groups in the molecule or associated with the molecule. A formation of a hydrate or solvate can typically be confirmed by an attenuated total reflection (ATR) infrared spectrum acquired from the solid-state sample. The ATR spectrum of a hydrate or solvate will typically show increased broad bands (indicating hydrogen bonding) above about 3200cm-1, as compared to the non-hydrate or non-solvate solid sample. In some embodiments, the above-described compounds are attached to or associated with a targeting moiety. In some embodiments, the targeting moiety is a particle or an antibody with affinity for a specific type of cell.

[0030] Other implementations are also described and recited herein. These and other features and advantages will be apparent from a reading of the following detailed description and a review of the associated drawings. It is to be understood that both the foregoing general description and the following detailed description are explanatory only and are not restrictive of aspects as claimed. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Solely for the purpose of illustration, certain embodiments of the present invention are explained using examples in the drawings described below. It should be understood, however, that the invention is not limited to the precise arrangements, dimensions, and configurations shown. In the drawings:

[0032] FIG.1A-1O shows experiments determining how elraglusib enhances pyroptosis and sensitizes tumor cells to increase immune-mediated cytotoxicity in a co- culture model. Co-cultures were treated with drug concentrations as indicated. A 1:1 effector to target (E:T) ratio was used with a 24-h co-culture duration. EthD-1 was used to visualize dead cells; 10× magnification; scale bar indicates 100 µm. (FIG.1A)Representative SW480 and TALL-104 T cell co-culture assay images at the 24-h timepoint; 24 tumor cell pre-treatments with 5 µM elraglusib, followed by 24-h co- culture. (FIG.1B) Quantification of the co-culture experiment using the percentage of dead cells out of total cells (N = 3). (FIG.1C) Quantification normalized by cell death observed with drug treatment alone (N = 3). (FIG.1D) Representative SW480 and donor-derived CD8+ T cell co-culture assay images at the 24-h timepoint; 24-h tumor cell pre-treatment with 5 µM elraglusib, followed by a 24-h co-culture. (FIG.1E) Quantification of the co-culture experiment using the percentage of dead cells out of total cells (N = 3). (FIG.1F) Quantification normalized by cell death observed with drug treatment alone (N = 3). (FIG.1G) The number of HCT 116 GFP+ cells were quantified after 48 h of culture with DMSO, 5 μM elraglusib, and / or 5000 TALL-104 cells (N = 3). (FIG.1H) The number of HT-29 GFP+ cells was quantified after 48 h of culture with DMSO, 5 μM elraglusib, and / or 5000 TALL-104 cells (N = 3). (FIG.1I) The number of HCT 116 GFP+ cells was quantified after 48 h of culture with DMSO, 5 μM elraglusib, and / or 5000 NK-92 cells (N = 3). (FIG.1J) The number of HT-29 GFP+ cells was quantified after 48 h of culture with DMSO, 5 μM elraglusib, and / or 5000 NK-92 cells (N = 3). (FIG.1K) Representative 40 × images of HCT-116 GFP+ or HT-29 GFP+ CRC and TALL-104 T cell co-cultures. Black arrows indicate pyroptotic events. Representative images of western blot analysis of (FIG.1L) HCT-116 and (FIG.1M) HT-29 CRC cells for expression of indicated proteins after treatment with indicated cytokines or drugs. Quantification of IFN-γ secretion (pg / mL) post-DMSO or elraglusib treatment for 24 h in (FIG.1N) TALL-104 cells and (FIG.1O) NK-92 cells (N = 3). Error bars represent the mean + / − standard deviation. Statistical test: one-way ANOVA with Tukey’s test for multiple comparisons. p-value legend: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

[0033] FIG.2A-2I illustrate elraglusib treatment induces PD-L1 expression and suppresses survival pathways in tumor cells. Representative images of western blot analysis of (FIG.2A) HCT-116 and HT-29 CRC cells for expression of indicated proteins after increasing durations of elraglusib treatment (0–72 h). (FIG.2B) Representative images of western blot analysis of Mcl-1 expression in HCT-116 CRC cells after increasing durations of elraglusib treatment. CRC cells were treated with 1μM elraglusib for 24 h and treated versus untreated samples were compared in triplicate. Microarray analysis results were visualized using volcano plots for (FIG.2C) HCT-116, (FIG.2D) HT-29, and (FIG.2E) KM12C CRC cell lines (N = 3). The top down- and up-regulated genes post-elraglusib treatment as compared to controls are shown. Results were calculated using a fold-change cutoff of >1.5, <−1.5, and a p-value of <0.05. (FIG.2F) The number of genes up- or down-regulated in each of the three cell lines within several signaling pathways of interest (N = 3). Green indicates downregulation and red indicates upregulation of gene expression. (FIG.2G) A Venn Diagram was used to compare the 3124 genes that were differentially expressed post- treatment with elraglusib in the three-colon cancer (HCT-116, HT-29, KM12C) cell lines (N = 3). (FIG.2H) Tumor cells (HCT-116, HT-29) were treated with 1 μM elraglusib for 48 h and cell culture supernatant was analyzed with the Luminex 200. Fold-change is shown where red indicates a positive fold-change and green indicates a negative fold- change (N = 3). (FIG.2I) Tumor cells (HCT-116, HT-29) were treated with 5 μM elraglusib for 48 h and cell culture supernatant was analyzed with the Luminex 200. Fold-change is shown where red indicates a positive fold-change and green indicates a negative fold-change (N = 3).

[0034] FIG.3A-3J illustrate how elraglusib treatment increases effector molecule secretion and induces an energetic shift in cytotoxic immune cells. Representative images of western blot analysis of (FIG.3A) TALL-104 and (FIG.3B) HT-29 cytotoxic immune cells for expression of indicated proteins after increasing durations of elraglusib treatment (0–72 h). (FIG.3C) Proposed model for non-canonical NF-κB pathway activation: increased NIK expression indicates non-canonical NF-κB pathway activation which enhances the expression of chemokines and cytokines (CCL11, TNF-α, GM- CSF) and subsequently leads to increased recruitment and proliferation of cytotoxic immune cells (CD8+ T, CD4+ T, NK cells). Immune cells were treated with 1 μM elraglusib for 24 h and treated versus untreated samples were compared in triplicate. Microarray analysis results were visualized using volcano plots for (FIG.3D) NK-92 and (FIG.3E) TALL-104 immune cell lines (N = 3). (FIG.3F) A Venn diagram was used to compare the 124 genes that were differentially expressed post-treatment with elraglusib in the two immune (TALL-104, NK-92) cell lines (N = 3). (FIG.3G) 10 × single cellsequencing analysis of immune cells treated with elraglusib. TALL-104 and NK-92 cells were treated with 1 μM elraglusib for 24 h and aggregate data were visualized using a t- SNE plot. (FIG.3H) Immune cells show differential expression of mitochondria-encoded genes (MT) and ribosomal genes (RB) after elraglusib treatment. (FIG.3I) Heatmap comparing gene expression post-elraglusib treatment as compared to control. Fold- change is shown, where red indicates a positive fold-change and green indicates a negative fold-change. (FIG.3J) Immune cells (TALL-104, NK-92) were treated with 1 μM elraglusib for 48 h and cell culture supernatant was analyzed with the Luminex 200. Fold-change is shown, where red indicates a positive fold-change and green indicates a negative fold-change (N = 3).

[0035] FIG.4A-4M Illustrate that elraglusib enhances immune-cell tumor- infiltration to prolong survival in combination with anti-PD-L1 therapy in a syngeneic murine model of MSS colon carcinoma. (FIG.4A) Experimental model overview of the syngeneic murine colon carcinoma BALB / c murine model using MSS cell line CT-26. (FIG.4B) Kaplan-Meier estimator curves for all treatment groups as indicated. Statistical significance was determined using a Log-rank (Mantel-Cox) test. (FIG.4C) Overview of cell lineage markers used for flow cytometric immunophenotyping analysis. Two weeks after treatment initiation, immune cell subpopulations were analyzed in the spleen and tumor. (FIG.4D) Splenic T cells, (FIG.4E) tumor-infiltrating T cells, (FIG. 4F) splenic NK cells, and (FIG.4G) tumor-infiltrating NK cells were compared between responders (R, N = 3) and non-responders (NR, N = 18), regardless of treatment group. NK cell subsets based on the expression of CD11b and CD27 were compared in the spleen and visualized via (FIG.4H) bar graph and (FIG.4I) pie chart. NK cell subsets based on the expression of CD11b and CD27 were also compared in the tumor and visualized via (FIG.4J) bar graph and (FIG.4K) pie chart. T cell ratios were compared in the (FIG.4L) Spleen and (FIG.4M) Tumor. Statistical significance was determined using two-tailed unpaired T tests. p-value legend: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns: non-significant.

[0036] FIG.5A-5W illustrate that responders have a more immunostimulatory tumor microenvironment as compared to non-responders. IHC analysis of tumors two weeks post-treatment initiation or tumors from long-term mice. Non-responders (NR, N= 18) and responders (R, N = 3) were compared.20 × images, scale bar represents 100 μm. (FIG.5A, FIG.5B) CD3, (FIG.5C, FIG.5D) Granzyme B, (FIG.5E, FIG.5F) Ki67, (FIG.5G, FIG.5H) PD-L1, and (FIG.5I, FIG.5J) cleaved-caspase 3 (CC3) were compared at the two weeks after treatment initiation timepoint, and the long-term timepoint, respectively. Statistical significance was determined using two-tailed unpaired T tests (N = 6). Serum from long-term mice sacrificed was analyzed via cytokine profiling for (FIG.5K) CCL21, (FIG.5L) VEGFR2, (FIG.5M) CCL7, (FIG.5N) CCL12, (FIG.5O) BAFF, (FIG.5P) VEGF, (FIG.5Q) IL-1 β, (FIG.5R) IL-6, (FIG.5S) CCL22, (FIG.5T) GM-CSF, (FIG.5U) CCL4, (FIG.5V) TWEAK, and (FIG.5W) CCL2. Responders (red, N = 3) and non-responders (black, N = 18) were compared. A Kruskal-Wallis test was used to calculate statistical significance, followed by a Benjamini-Hochberg correction for multiple comparisons. p values are shown for analytes that were significantly different between responders and non-responders and are ordered by significance. p-value legend: * p < 0.05, ** p < 0.01, *** p < 0.001. ns: no significance.

[0037] FIG.6A-6D illustrate how patient plasma concentrations of cytokines correlate with progression-free survival (PFS), overall survival (OS), and in vivo response to therapy results. Plasma samples from human patients with refractory solid tumors of multiple tissue origins enrolled in a Phase 1 clinical trial investigating a novel GSK-3 inhibitor elraglusib (NCT03678883) were analyzed using a Luminex 200 (N = 19). (FIG.6A) Table summarizing patient demographics. (FIG.6B) Baseline and 24-h post-elraglusib plasma concentrations of cytokines, chemokines, and growth factors were plotted against PFS and OS. Simple linear regressions were used to calculate significance. p values less than 0.05 were reported as statistically significant. (FIG.6C) Cytokines grouped by function. Fold-change is shown where green indicates a negative (<0) fold-change compared to the baseline (pre-dose) value and red indicates a positive (>0) fold-change. (FIG.6D) Table comparing murine and human circulating biomarker trends. Red boxes indicate that an analyte concentration positively correlated with response to therapy / PFS / OS while green boxes indicate that an analyte concentration negatively correlated with response to therapy / PFS / OS.

[0038] FIG.7A-7H illustrate spatial profiling of patient tumor biopsies reveals a more immunostimulatory tumor microenvironment after elraglusib treatment. Patient samples were analyzed using NanoString GeoMx Digital Spatial Profiling (DSP) technology (N = 12 biopsies). (FIG.7A) Pie charts showing biopsy timepoint, primary tumor type, metastatic biopsy tissue type, and paired / unpaired biopsy sample information breakdowns. (FIG.7B) A representative region of interest (ROI) showing PanCK+ and CD45+ masking. Green indicates CK, red indicates CD45, and blue indicates DAPI staining. (FIG.7C) A Sankey diagram was used to visualize the study design where the width of a cord in the figure represents how many segments are in common between the two annotations they connect. The scale bar represents 50 segments. Blue cords represent CD45+ segments and yellow cords represent panCK+ segments. (FIG.7D) Heatmap of all areas of interest (AOIs). Patient IDs, immune cell locations, biopsy timepoint, biopsy tissue, primary tumor location, and segment identity information are color coded as indicated in the legend. (FIG.7E) PanCK+ ROI CD39 expression plotted against time-on-study (TOS). Points are color-coded by time on study (TOS) / time on treatment with darker blue points indicating a shorter TOS or time on treatment. (FIG.7F) CD45+ ROI CD163 expression plotted against TOS. (FIG.7G) Volcano plot showing a comparison of CD45+ region protein expression in post- treatment biopsies and pre-treatment biopsies regardless of timepoint. Grey points are non-significant (NS), blue points have p values <0.05, and red points have false discovery rate (FDR) values less than 0.05. The size of the point represents the log2 UQ Signal-to-noise ratio (SNR). (FIG.7H) Volcano plot showing a comparison of tumor infiltrating CD45+ immune cell segment protein expression in pre- versus post-treatment biopsies. Grey points are non-significant (NS), blue points have p values <0.05, and red points have false discovery rate (FDR) values less than 0.05. The size of the point represents the log2 UQ Signal-to-noise ratio (SNR).

[0039] FIG.8A-8J show experimental data for investigating elraglusib increases immune-mediated cytotoxicity in a co-culture model with CRC cells. (FIG.8A) Representative SW480 and TALL-104 T cell co-culture assay images at the 24-hour timepoint.24-hour tumor cell pre-treatment with 5 μM elraglusib, followed by 24-hour co-culture. EthD-1 was used to visualize dead cells, 10× magnification, scale barindicates 100 μm. (FIG.8B) Quantification of co-culture experiment using the percentage of dead cells out of total cells (N=3). (FIG.8C) Quantification normalized by cell death observed with drug treatment alone (N=3). (FIG.8D) Representative SW480 and donor-derived CD8+ T cell co-culture assay images at the 24-hour timepoint. (FIG.8E) Quantification of co-culture experiment using the percentage of dead cells out of total cells (N=3). (FIG.8F) Quantification normalized by cell death observed with drug treatment alone (N=3). A one-way ANOVA followed by a post-hoc Dunnett's multiple comparisons test was used to calculate statistical significance. (FIG.8G) HCT-116 GFP+ or HT-29 GFP+ cells were co-cultured with TALL-104 cells at a 1:1 E:T ratio and were treated with DMSO or 5 μM elraglusib. (FIG.8H) Representative 40× images were collected after 24 hours of DMSO or 5 μM elraglusib treatment. (FIG.8I) Representative 40× images of a co-culture of HCT-116 GFP+ cells and NK-92 cells at a 1:1 E:T ratio were collected after 36 hours of 5 μM elraglusib treatment. White arrows indicate pyroptotic events. (FIG.8J) Representative 40× images of a co-culture of HT-29 GFP+ CRC cells and NK-92 cells at a 1:1 E:T ratio were after 36 hours of 5 μM elraglusib treatment. White arrows indicate pyroptotic events. P-value legend: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

[0040] FIG.9A-9C show data supporting CRC cell lines selected represent diverse mutational backgrounds and exhibit varying elraglusib IC-50 values. Tumor cell lines (HCT-116, HT-29, SW480) and immune cell lines (NK-92, TALL-104) were treated as indicated for (FIG.9A) 24-hour or (FIG.9B) 72-hour cell viability was assessed to determine IC-50 values (N=3). (FIG.9C) Table of CRC cell lines included in the study and their diverse mutational profiles.

[0041] FIG.10A-10F provides internal controls for tumor cell microarray analysis. (FIG.10A) PCA mapping demonstrated clear mapping of HCT-116, HT-29, and KM12C cells (N=3). (FIG.10B) Quality control was determined satisfactory for further analysis. (FIG.10C) Hybridization controls. (FIG.10D) Labeling controls. (FIG.10E) Pos vs Neg Area Under the Curve (AUC). (FIG.10F) Signal box plot.

[0042] FIG.11A-11F show internal controls for immune cell microarray analysis. (FIG.11A) PCA mapping demonstrated clear mapping of NK-91 and TALL-104 cells (N=3). (FIG.11B) Quality control was determined satisfactory for further analysis.(FIG.11C) Hybridization controls. (FIG.11D) Labeling controls. (FIG.11E) Pos vs Neg Area Under the Curve (AUC). (FIG.11F) Signal box plot.

[0043] FIG.12A-12L provide data for the syngeneic murine colon carcinoma BALB / c murine model with MSS cell line CT-26 Kaplan Meier curves and mouse body weights grouped by treatment. Individual Kaplan Meier curves for isotype control (N=12) compared to (FIG.12A) elraglusib (N=12), (FIG.12B) anti-PD-1 (N=12), (FIG.12C) anti-PD-L1 (N=12), (FIG.12D) elraglusib + anti-PD-1 (N=12), and (FIG.12E) elraglusib + anti-PD-L1 (N=12). (FIG.12F) Bar graph indicating the percentage of responders (R) and non-responders (NR) per treatment group. Individual body weight plots for (FIG.12G) Isotype control (N=12), (FIG.12H) elraglusib (N=12), (FIG.12I) anti-PD-1 (N=12), (FIG.12J) anti-PD-L1 (N=12), (FIG.12K) elraglusib + anti-PD-1 (N=12), and (FIG.12L) elraglusib + anti-PD-L1 (N=12). P-value legend: * p < 0.05, ** p < 0.01.

[0044] FIG.13A-13H shows images and plots for immunohistochemistry analyses of tumor sections in responders as compared to non-responders. (FIG.13A) CD4, (FIG.13B) CD8, (FIG.13C) Foxp3, (FIG.13D) NKp46, (FIG.13E) TNF-related apoptosis-inducing ligand (TRAIL), (FIG.13F) PD-1, (FIG.13G) Vascular Endothelial Growth Factor (VEGF), and (FIG.13H) Transforming Growth Factor Beta 2 (TGFβ2) were compared at the 14 days post-treatment initiation timepoint and the end-of-study (EOS) timepoint, respectively. Non-responders (NR, N=18) and responders (R, N=3) were compared. Statistical significance was determined using two-tailed unpaired T tests (N=6).20× images, scale bar represents 100 μm.

[0045] FIG.14A-14S provide murine serum cytokine profiling assay results. Serum from end-of-study mice was analyzed via cytokine profiling for (FIG.14A) MIP-1 α, (FIG.14B) RANTES, (FIG.14C) Eotaxin, (FIG.14D) MIP-3 α, (FIG.14E) IP-10, (FIG.14F) CXCL12, (FIG.14G) Granzyme B, (FIG.14H) IFN-γ, (FIG.14I) IL-1 α, (FIG.14J) IL-2, (FIG.14K) IL-3, (FIG.14L) IL-4, (FIG.14M) IL-7, (FIG.14N) IL-10, (FIG.14O) IL-12 p70, (FIG.14P) IL-13, (FIG.14Q) IL-16, (FIG.14R) M-CSF, and (FIG.14S) Prolactin. Responders (red, N=3) and non-responders (black, N=18) were compared. A Kruskal-Wallis test was used to calculate statistical significance followed by a Benjamini-Hochberg correction for multiple comparisons.

[0046] FIG.15A-15C illustrate that a multiplex immunoassay reveals dynamic cytokine changes 8- and 24-hours post-elraglusib dose. Plasma samples from human patients with refractory solid tumors of multiple tissue origins enrolled in a Phase 1 clinical trial investigating GSK-3 inhibitor elraglusib (NCT03678883) were analyzed using a Luminex 200 (N=19). (FIG.15A) Raw analyte values are grouped by timepoint: baseline (pre-dose), 8-hour post-PK, and 24-hour post-PK and categorized by primary tumor location: appendix, adult T cell leukemia / lymphoma (ATLL), cholangiocarcinoma, colorectal, desmoid, hepatocellular carcinoma (HCC), leiomyosarcoma, non-small cell lung cancer (NSCLC), and pancreatic. The scale bar is in pg / mL. (FIG.15B) Raw analyte values are grouped by timepoint: 8-hour post-PK and 24-hour post-PK and categorized by elraglusib dose: 1, 2, 3.3, 5, 7, 9.3, or 12.37 mg / kg. (FIG.15C) Fold change heatmaps grouped by cytokine family.8- and 24-hours post-PK were compared to baseline (pre-PK) analyte values. Although colors can be sometimes shown in greyscale, in FIG.15C, green indicates downregulation and red indicates upregulation.

[0047] FIG.16 shows needle biopsies scanned by the GeoMx Digital Spatial Profiler (DSP). Whole slide scans are provided for all needle biopsies imaged by the GeoMx DSP (N=6 biopsies). PanCK is indicated by green, CD45 is indicated by red, and DAPI is indicated by blue staining. Slide IDs are provided on each image in white text.

[0048] FIG.17 shows tissue biopsies scanned by the GeoMx Digital Spatial Profiler (DSP). Whole slide scans are provided for all tissue biopsies imaged by the GeoMx DSP (N=6 biopsies). PanCK is indicated by green, CD45 is indicated by red, and DAPI is indicated by blue staining. Slide IDs are provided on each image in white text.

[0049] FIG.18A-18B provide images illustrating ROI selection and segmentation strategy using PanCK and CD45 markers. (FIG.18A) Representative image of whole tissue scan used for ROI identification. ROIs are outlined in red circles. (FIG.18B) Individual ROIs are ordered by number. Fluorescent channel settings: FITC / 525nm / SYTO 13 / DNA (Blue), Cy3 / 568nm / Alexa 532 / PanCK (Green), and Texas Red / 615nm / Alexa 594 / CD45 (Red). A yellow mask was used for PanCK+ tumor cell identification and a teal mask was used for CD45+ hematopoietic cell identification.

[0050] FIG.19A-19F illustrate that several proteins are differentially expressed when patients are segmented into two groups based on time-on-treatment. PCA plots showing how similar the different group levels are from one another in the (FIG.19A) total sample set (N=12) and (FIG.19B) at both biopsies timepoints [pre- (N=5), post- treatment, (N=7). Samples tend to cluster by tissue type and further separate by segment on PC2. Circles represent CD45+ segments and triangles represent pan CK+ segments. Biopsy tissue locations are colorcoded where blue indicates liver, yellow indicates lung, red indicates pleura, and green indicates rectum. (FIG.19C) Volcano plot showing the comparison of pre-treatment biopsy protein expression in CD45+ segments between long time-on-treatment (Long Tx) patients and short time-on- treatment (Short Tx) patients. Violin plots show statistically significant differentially expressed proteins including B cell marker CD20 (p=0.012) and myeloid activation marker CD80 (p=0.047). (FIG.19D) Volcano plot showing the comparison of CD45+ segments in post-treatment biopsies between Long Tx patients and Short Tx patients. Violin plots show statistically significant differentially expressed proteins including antigen NY-ESO-1 (p=0.021) and progesterone receptor (PR) (p=0.022). (FIG.19E) Volcano plot showing the comparison of pre-treatment protein expression in PanCK+ segments between Long Tx patients and Short Tx patients. Violin plots show statistically significant differentially expressed proteins including cytotoxic T cell marker CD8 (p=3.5E-3), antigen Her2 (p=0.033), Treg marker Foxp3 (p=0.033), T cell marker CD3 (p=0.035), B cell marker CD20 (p=0.046), LAG3 (p=0.023), PD-L2 (p=0.028), and PD-1 (p=0.046). (FIG.19F) Volcano plot showing the comparison of post-treatment protein expression in PanCK+ segments between Long Tx patients and Short Tx patients. Violin plots show statistically significant differentially expressed proteins including mature B cell / DC marker CD35 (p=8.5E-3), antigen NY-ESO-1 (p=8.7E-3), antigen Her2 (p=0.022), antigen MART1 (p=0.029), cytotoxic T cell marker CD8 (p=0.030), Treg marker Foxp3 (p=0.030), antigen PTEN (p=0.032), DC / myeloid marker CD11c (p=0.034), memory T cell marker CD45RO (p=0.036), checkpoint PD-L1 (p=0.047), and PR (p=0.049).

[0051] FIG.20A-20C provides images showing the immune cell localization categorization strategy. Immune cell locations were categorized as (FIG.20A) tumor-infiltrating, (FIG.20B) tumor-adjacent, or (FIG.20C) normal tissue. Representative ROI images are shown.

[0052] FIG.21A-21N show data for differential expression analyses of protein expression in human tumor biopsies pre- and post-elraglusib treatment. (FIG.21A) Volcano plot showing differential protein expression in PanCK+ regions between pre- (N=5) and post-treatment (N=7) biopsies in paired samples. (FIG.21B) Volcano plot showing differential protein expression in CD45+ regions between pre- and post- treatment biopsies in paired samples. (FIG.21C) Volcano plot showing differential protein expression between tumor-adjacent CD45+ segments and tumor-infiltrating CD45+ segments in pre-treatment biopsies. (FIG.21D) Volcano plot showing differential protein expression between tumor-adjacent CD45+ segments and tumor-infiltrating CD45+ segments in post-treatment biopsies. (FIG.21E) Volcano plot showing differential protein expression between CD45+ regions of pre-treatment and post- treatment biopsies. (FIG.21F) Volcano plot showing differential protein expression panCK+ regions of pre-treatment and post-treatment biopsies. (FIG.21G) Volcano plot showing differential post-treatment protein expression in tumor adjacent CD45+ immune cell segments in Long Tx patients as compared to Short Tx patients. (FIG.21H) Volcano plot showing differential post-treatment protein expression in CD45+ immune cell segments located in normal (non-tumor) tissue in Long Tx patients as compared to Short Tx patients. (FIG.21I) Volcano plot showing differential pre-treatment protein expression in CD45+ immune cell segments located in normal (non-tumor) tissue as compared to tumor-adjacent tissue. (FIG.21J) Volcano plot showing differential post- treatment protein expression in CD45+ immune cell segments located in normal (non- tumor) tissue as compared to tumor-adjacent tissue. (FIG.21K) Volcano plot showing differential pre-treatment protein expression in CD45+ immune cell segments located in normal (non-tumor) tissue as compared to those in tumor tissue. (FIG.21L) Volcano plot showing differential post-treatment protein expression in CD45+ immune cell segments located in normal (non-tumor) tissue as compared to those in tumor tissue. (FIG.21M) Volcano plot showing a comparison of tumor-adjacent CD45+ segments with tumor-infiltrating CD45+ segments in all biopsies regardless of timepoint. (FIG.21N) Volcano plot showing a comparison of post-treatment protein expression of tumorinfiltrating CD45+ immune cell segments in Long Tx patients as compared to Short Tx patients. Grey points are non-significant (NS), blue points have p values < 0.05, and red points have false discovery rate (FDR) values less than 0.05. The size of the point represents the log2 UQ Signal-to-noise ratio (SNR).

[0053] FIG.22 shows a table of HCT-116 CRC cell microarray analyses.

[0054] FIG.23 shows a table of HT-29 CRC cell microarray analyses.

[0055] FIG.24 shows a table of KM12C CRC cell microarray analyses.

[0056] FIG.25 shows a table of NK-92 immune cell microarray analyses.

[0057] FIG.26 shows a table of TALL-104 immune cell microarray analyses.

[0058] FIG.27 shows a table illustrating murine serum chemistry analysis does not show treatment-related toxicity.

[0059] FIG.28 shows a table illustrating individual patient information for human cytokine analyses.

[0060] All trademarks, images, likenesses, words, and depictions in the drawings and the disclosure are plainly in fair use and are provided solely for the purposes of illustration of the invention in view of need to treat diseases as further discussed in detail below, even if unintentionally placed herein. DETAILED DESCRIPTION OF THE INVENTION

[0061] The subject innovation is now described in some instances, when necessary, with reference to the drawings. In the following description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the present invention. It may be evident, however, that the present invention may be practiced without these specific details. In other instances, well-known structures, methods, and devices are shown in block diagram form or with illustrations in order to facilitate describing the present invention. It is to be appreciated that certain aspects, modes, embodiments, variations and features of the invention are described below in various levels of detail in order to provide a substantial understanding of the present invention.DEFINITIONS

[0062] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail. In general, chemical terminology is found in the International Union of Pure and Applied Chemistry GoldBook1. This disclosure is purposefully in commonly understood words, known to a person of skill in the art, but Merriam-Webster’s Online Dictionary is used, when appropriate, for terms not specifically demonstrated herein or not known in the art2.

[0063] As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the content clearly dictates otherwise. For example, reference to "a cell" includes a combination of two or more cells, and the like.

[0064] As used herein, the term "approximately" or "about" in reference to a value or parameter are generally taken to include numbers that fall within a range of 5%, 10%, 15%, or 20% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value). As used herein, reference to "approximately" or "about" a value or parameter includes (and describes) embodiments that are directed to that value or parameter. For example, description referring to "about X" includes description of "X".

[0065] As used herein, the term “or” means “and / or.” The term "and / or" as used in a phrase such as "A and / or B" herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A,B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0066] As used herein, the term "comprising" means that other elements can also be present in addition to the defined elements presented. The use of "comprising" indicates inclusion rather than limitation. The term “including” can be interchanged with “comprising”.

[0067] The term "consisting of" refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0068] As used herein the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention. For example, a pharmaceutical formulation can consist essentially of elraglusib and an immune checkpoint inhibitor, meaning that a variety of excipients or other additives can be present in the formulation, but no other active pharmaceutical ingredient (API) is present in the formulation, except in formulations wherein an intended synergistic effect is demonstrated by the claims or examples herein (e.g., a formulation consisting essentially of elraglusib and an immune checkpoint inhibitor, or pharmaceutically acceptable salts thereof). In another example, a pharmaceutical formulation can consist essentially of elraglusib and an immune checkpoint inhibitor, meaning that the formulation is provided in the form of a nasal spray, an inhaled formulation, an orally administered formulation, or an injection formulation, each of which is tailored for a fast-acting agent, therapeutic agent, or antidote but not tailored for long-term administration (e.g., as a simultaneous treatment). In another example, in the case of a preventative therapy to purposefully prevent a cancer, the opposite, long-term combination therapy, can be referred to with “consisting essentially of”. This example could be applied to a cancer patient who is best treated by an evolving combination therapy. In another example, the term “consisting essentially of” can also be exemplified by plain language provided in the claims.

[0069] The term "statistically significant" or "significantly" refers to statistical significance and generally means a two-standard deviation (2SD) or greater difference.

[0070] As used herein, the term "subject" refers to a mammal, including but not limited to a dog, cat, horse, cow, pig, sheep, goat, rodent, or primate. Subjects can be house pets (e.g., dogs, cats), agricultural stock animals (e.g., cows, horses, pigs, chickens, etc.), laboratory animals (e.g., mice, rats, rabbits, etc.), but are not so limited. Subjects particularly include human subjects in urgent treatment as described herein. The human subject may be a pediatric, adult, or a geriatric subject. The human subject may be of any sex.

[0071] As used herein, the terms "effective amount", “therapeutically effective amount”, and “pharmaceutically effective amount” include an amount sufficient to prevent or ameliorate a manifestation of or a suspected manifestation of a medical condition, such as a cancer. The manifestation can be a sign or symptom or otherwise. It will be appreciated that there will be many ways known in the art to determine the effective amount for a given application. For example, the pharmacological methods for dosage determination may be used in the therapeutic context. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will depend on the type and severity of the medical condition and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity, and type of medical condition. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. Examples of other factors can be route of administration and length of administration(s). The compositions can also be administered in combination with one or more additional therapeutic compounds.

[0072] The term “protecting group” refers to a group of atoms that, when attached to a reactive functional group in a molecule, mask, reduce or prevent the reactivity of the functional group. Typically, a protecting group may be selectively removed as desired during the course of a synthesis. Examples of protecting groups can be found in Greene and Wuts, Protective Groups in Organic Chemistry, 3rdEd.3and in Harrison, et al., Compendium of Synthetic Organic Methods, Vols.1-84. Examples of representative nitrogen protecting groups include, but are not limited to, formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl (“CBZ”), tert-butoxycarbonyl (“Boc”), trimethylsilyl (“TMS”), 2- trimethylsilyl-ethanesulfonyl (“TES”), trityl and substituted trityl groups, allyloxycarbonyl,9-fluorenylmethyloxycarbonyl (“FMOC”), nitro-veratryloxycarbonyl (“NVOC”) and the like. Examples of representative hydroxylprotecting groups include, but are not limited to, those where the hydroxyl group is either acylated (esterified) or alkylated such as benzyl and trityl ethers, as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers (e.g., TMS or TIPS groups), glycol ethers, such as ethylene glycol and propylene glycol derivatives and allyl ethers.

[0073] As used herein, a therapeutic that “prevents” a disorder or condition refers to a compound (or combination) that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample. For example, a compound (or combination) that prevents epilepsy may reduce the frequency of seizures and / or reduce the severity of seizures.

[0074] The term “treating” includes prophylactic and / or therapeutic treatments. The term “prophylactic or therapeutic” treatment is art-recognized and includes administration to the host of one or more of the subject compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic (i.e., it protects the host against developing the unwanted condition), whereas if it is administered after manifestation of the unwanted condition, the treatment is therapeutic, (i.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof).

[0075] The phrases “conjoint administration” and “administered conjointly” refer to any form of administration of two or more different therapeutic compounds such that the second compound is administered while the previously administered therapeutic compound is still effective in the body (e.g., the two compounds are simultaneously effective in the patient, which may include synergistic effects of the two compounds). For example, the different therapeutic compounds can be administered either in the same formulation or in a separate formulation, either concomitantly or sequentially. In certain embodiments, the different therapeutic compounds can be administered at the same time, within one minute, 2 minutes, 4 minutes, 6 minutes, 10 minutes, 30 minutes,or an hour or 90 minutes of one another. In some embodiments, the different therapeutic compounds can be administered within 1 year of one another. Thus, an individual who receives such treatment can benefit from a combined effect of different therapeutic compounds.

[0076] As used herein, the terms “treat,” “treatment,” “treating,” or “amelioration” when used in reference to a disease, disorder, or medical condition, refer to therapeutic treatments for a condition, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a symptom or condition. The term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a condition is reduced or halted. That is, “treatment” includes not just the improvement of symptoms or markers, but also a cessation or at least slowing of progress or worsening of symptoms that would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), sign(s), diminishment of extent of the deficit, stabilized (i.e., not worsening) state of a symptom or condition, delay or slowing of onset of symptoms or indications, and an increased lifespan as compared to that expected in the absence of treatment.

[0077] As used herein, the term "long-term" administration means that the therapeutic agent or drug is administered for a period of at least 12 weeks. This includes that the therapeutic agent, combination, or drug is administered such that it is effective over, or for, a period of at least 12 weeks and does not necessarily imply that the administration itself takes place for 12 weeks, e.g., if sustained release compositions or long-acting therapeutic agent or drug is used. Thus, the subject is treated for a period of at least 12 weeks. In many cases, long-term administration is for at least 4, 5, 6, 7, 8, 9 months or more, or for at least 1, 2, 3, 5, 7 or 10 years, or more.

[0078] The administration of the compositions contemplated herein may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation, application (e.g., topical, otic, or ocular), or transplantation. Administration can be accomplished by an implant. In some embodiments, compositions are administered parenterally. The phrases “parenteraladministration” and “administered parenterally” as used herein refers to modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. In one embodiment, the compositions contemplated herein are administered to a subject by direct injection into an artery, vein, lymph node, or organ (e.g., heart, muscle, organ).

[0079] The terms: “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction" or “decrease" or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g., the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.

[0080] The term “prodrug” is intended to encompass compounds which, under physiologic conditions, are converted into the therapeutically active agents of the present invention (e.g., any compound selected from this disclosure). A common method for making a prodrug is to include one or more selected moieties which are hydrolyzed under physiologic conditions to reveal the desired molecule. In other embodiments, the prodrug is converted by an enzymatic activity of the host animal. For example, esters or carbonates (e.g., esters or carbonates of alcohols or carboxylic acids) are preferred prodrugs of the present invention. In certain embodiments, some or all of the compounds selected from this disclosure in a formulation can be replaced with the corresponding suitable prodrug, e.g., wherein a hydroxyl in the parent compound ispresented as an ester or a carbonate or carboxylic acid present in the parent compound is presented as an ester. In some embodiments, a “prodrug” is made by using an absorbing particle that subsequently releases an active form after administration.

[0081] In some embodiments, the decrease in the one or more signs or symptoms is evaluated according to the DSM-55. In some embodiments, signs are observed or measured by a health care provider. Symptoms can be reported by the subject. In some embodiments, the decrease of signs or symptoms occurs in less than about 120 minutes, 90 minutes, less than about 60 minutes, less than about 30 minutes, less than about 15 minutes, less than about 10 minutes, or less than about 5 minutes, or less than about 3 minutes, or less than about 1 minute. In some embodiments, the decrease of signs or symptoms occurs in less than 1 day, less than 1 week, less than 1 month, or in less than 1 year.

[0082] The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4- fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, a “increase” is a statistically significant increase in such level.

[0083] As used herein, an agent or a therapeutic agent provided to a subject and suspected to be or involved in a treatment can be a small molecule less than 1000 MW or a large molecule not less than 1000 MW including biologics, oligonucleotides, peptides, oligosaccharides, and larger molecules. Any of the therapeutic agents disclosed herein can be used as or in combination with small molecules and / or large molecules as discussed herein.

[0084] As used herein, a subject may or may not be aware of suffering from a cancer. A health care provider may suspect cancer or may have confirmed cancer.

[0085] A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g., a cancer or related disorder) or one or more complications related to such a condition, and optionally, but need not have already undergone treatment for a condition or the one or more complications related to the condition. Alternatively, a subject can also be one who has not been previously diagnosed as having a condition in need of treatment or one or more complications related to such a condition. For example, a subject can be one who exhibits one or more risk factors for a condition, or one or more complications related to a condition or a subject who does not exhibit risk factors. A “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, suspected as having, or at risk of developing that condition. In another example, the subject has been brought into a treatment situation entirely without the subject’s knowledge and / or intent. For example, a subject can obviously be in need of treatment but not be responsive to an immune checkpoint inhibitor, and as described herein the present methods and formulations may save the subject’s life. PHARMACEUTICAL COMPOSITIONS

[0086] The compositions and methods of the present invention may be utilized to prevent a need for other treatment, to provide benefit when other treatment(s) fail, or to treat an individual in need thereof. In some embodiments, the individual is suspected of needing treatment. In certain embodiments, the individual is a mammal such as a human, or a non-human mammal. When administered to an animal, such as a human, the composition or the compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the invention and a pharmaceutically acceptable carrier. A compound can represent a combination therapy herein. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In some embodiments, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration (i.e., routes, such as injection or implantation, that circumvent transport or diffusion through an epithelial barrier), the aqueous solution is pyrogen-free, or substantially pyrogen-free. Theexcipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues, or organs. The pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, solution, syrup, suppository, injection or the like. The composition can also be present in a transdermal delivery system, e.g., a skin patch. The composition can also be present in a solution suitable for topical administration, such as a lotion, cream, or ointment.

[0087] A pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of a compound such as a compound of the invention. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition can be a self-emulsifying drug delivery system or a self-micro emulsifying drug delivery system. The pharmaceutical composition (preparation) also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a compound of the invention. Liposomes, for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.

[0088] The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0089] The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which canserve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible compositions employed in pharmaceutical formulations.

[0090] A pharmaceutical composition (preparation) can be administered to a subject by any of a number of routes of administration including, for example, orally, for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules including sprinkle capsules and gelatin capsules, boluses, powders, granules, pastes for application to the tongue; absorption through the oral mucosa (e.g., sublingually); subcutaneously; transdermal administration (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin). The compound may also be formulated for inhalation. Inhalation can include inhalation of a liquid (droplets or aerosol). Inhalation can include a micronized powder adhered to carrier particles or can be without carrier particles. In certain embodiments, a compound may be simply dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for same can be found in, for example, U.S. Patent Nos.6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896, as well as in patents cited therein.

[0091] The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient that can be combined with a carriermaterial to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.

[0092] Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound (or combination therapy) of the invention, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0093] Formulations of the invention suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), lyophile, powders, granules, or as a solution or a suspension in an aqueous or non- aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and / or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. Compositions or compounds may also be administered as a bolus, electuary or paste.

[0094] To prepare solid dosage forms for oral administration (capsules, including sprinkle capsules and gelatin capsules), tablets, pills, dragées, powders, granules and the like, the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and / or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8)absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents, such as, modified and unmodified cyclodextrins; and (11) coloring agents. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

[0095] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropyl methyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

[0096] The tablets, and other solid dosage forms of the pharmaceutical compositions, such as dragées, capsules (including sprinkle capsules and gelatin capsules), pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical- formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropyl methyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and / or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be usedinclude polymers and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.

[0097] Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophiles for reconstitution, micro-emulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, cyclodextrins and derivatives thereof, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

[0098] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0099] Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0100] Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.

[0101] The ointments, pastes, creams and gels may contain, in addition to an active compound, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

[0102] Powders and sprays can contain, in addition to an active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally containcustomary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0103] Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body. Such dosage forms can be made by dissolving or dispersing the active compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel.

[0104] The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intraocular (such as intravitreal), intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. Pharmaceutical compositions suitable for parenteral administration comprise one or more active compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

[0105] Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size inthe case of dispersions, and by the use of surfactants. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0106] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

[0107] In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

[0108] Injectable depot forms are made by forming microencapsulated matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissue.

[0109] For use in the methods of this invention, active compounds can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

[0110] Methods of introduction may also be provided by rechargeable or biodegradable devices. Various slow-release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinaceous biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of a compound at a particular target site.

[0111] Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0112] The selected dosage level will depend upon a variety of factors including the activity of the particular compound or combination of compounds employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound(s) being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compound(s) employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0113] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the pharmaceutical composition or compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. By “therapeutically effective amount” is meant the concentration of a compound that is sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of the compound will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent being administered with the compound of the invention. A larger total dose can be delivered by multiple administrations of the agent.Methods to determine efficacy and dosage are known to those skilled in the art. See, e.g., Isselbacher, et al., (1996)6.

[0114] In general, a suitable daily dose of an active compound used in the compositions and methods of the invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

[0115] If desired, the effective daily dose of the active compound may be administered as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In certain embodiments of the present invention, the active compound may be administered two or three times daily. In other embodiments, the active compound will be administered once daily.

[0116] The subject or patient receiving this treatment is any animal in need, including primates, in particular humans; and other mammals such as equines bovine, porcine, sheep, feline, and canine; bird such as poultry; and pets in general.

[0117] In certain embodiments, compounds of the invention may be used alone or conjointly administered with another type of therapeutic agent.

[0118] The present disclosure includes the use of pharmaceutically acceptable salts of compounds of the invention in the compositions and methods of the present invention. In certain embodiments, contemplated salts of the invention include, but are not limited to, alkyl, dialkyl, trialkyl or tetra-alkyl ammonium salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, L- arginine, benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, 1H-imidazole, lithium, L-lysine, magnesium, 4-(2- hydroxyethyl)morpholine, piperazine, potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, Na, Ca, K, Mg, Zn or other metal salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, 1-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid,adipic acid, l-ascorbic acid, l-aspartic acid, benzenesulfonic acid, benzoic acid, (+)- camphoric acid, (+)-camphor-10-sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, d-glucoheptonic acid, d-gluconic acid, d-glucuronic acid, glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, l-malic acid, malonic acid, mandelic acid, methanesulfonic acid , naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, proprionic acid, l-pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, l-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenic acid salts. In some embodiments, an N-H in elraglusib can become charged and a corresponding salt formed at or near the charge.

[0119] The pharmaceutically acceptable acid addition salts can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, and the like. Mixtures of such solvates can also be prepared. The source of such solvate can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent. The invention contemplates polymorphs, cocrystals, and amorphous forms of all substances discussed herein. As discussed above, solvates and / or hydrates can be formed by, for example, a slow evaporation whereby water and / or solvent remain hydrogen bonded with OH groups in the molecule. The formation of a solvate / hydrate can be quickly confirmed after the evaporation by using attenuated total reflectance Fourier transform infra-red spectroscopy wherein the solid solvate / hydrate is directly placed on the instrument and the subsequent IR spectrum is compared to the IR spectrum of the solid non-solvate, non-hydrate.

[0120] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0121] Examples of pharmaceutically acceptable antioxidants include: (1) water- soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0122] As discussed above, unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy;7The Encyclopedia of Molecular Cell Biology and Molecular Medicine;8Molecular Biology and Biotechnology: a Comprehensive Desk Reference;9Immunology;10Janeway's Immunobiology;11Lewin's Genes XI;12Molecular Cloning: A Laboratory Manual.;13Basic Methods in Molecular Biology;14Laboratory Methods in Enzymology;15Current Protocols in Molecular Biology (CPMB)16; Current Protocols in Protein Science (CPPS);17and Current Protocols in Immunology (CPI)18.

[0123] In the embodiments discussed and in any of the aspects, the disclosure described herein does not concern a process for cloning human beings, processes for modifying the germ line genetic identity of human beings, uses of human embryos for industrial or commercial purposes or processes for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such processes.

[0124] Other terms are defined herein within the description of the various aspects of the invention.GSK-3 INHIBITOR ELRAGLUSIB (9-ING-41) MODIFIES PROTEIN EXPRESSION IN THE TUMOR MICROENVIRONMENT TO ENHANCE TUMOR-INFILTRATING IMMUNE CELL ACTIVATION IN CLINICAL TUMOR BIOPSIES

[0125] 1. Introduction: Glycogen synthase kinase 3 (GSK-3) is a serine / threonine kinase with key roles in myriad biological processes, such as tumor progression, and inhibition of GSK-3 using novel small-molecule elraglusib has shown promising preclinical antitumor activity in multiple tumor types19. GSK-3 has two isoforms encoded in distinct genes in vertebrates, alpha (α) and beta (β), which share a 97% amino acid sequence identity within their catalytic domains. While the GSK-3 beta isoform has historically received more attention, several redundant activities exist between the two, including regulating Wnt / β-catenin signaling20. Although differential activity exists between the two isoforms, in regulating NF-κB activity for example, many GSK-3 inhibitors are non-specific and inhibit both isoforms21. A growing body of literature characterizes the immunomodulatory roles of GSK-3 in the context of anti- tumor immunity22. There are several known immunostimulatory functions of GSK-3 inhibition. In T cells, GSK-3, active in resting cells and inactivated upon T cell activation, controls T cell motility and T cell interactions with other cell types such as antigen- presenting cells (APCs)23. GSK-3 is also known to inhibit immune cell cytokine production24,25.

[0126] Moreover, GSK-3 is a central regulator of immune checkpoint protein expression in both tumor and immune cells. In the immune cell, inhibition of GSK-3 has been shown to downregulate programmed cell death protein 1 (PD-1) expression in CD8+ T cells to enhance CD8+ cytotoxic function in a murine model, resulting in reduced tumor growth25. Mechanistically, GSK-3 inactivation resulted in Tbx21 transcription, upregulating T-bet expression and the subsequent suppression of transcription of Pdcd1, which encodes PD-1. Additionally, several groups have shown that the inhibition of GSK-3 increases tumor cell PD-L1 expression26. Mechanistically, GSK-3 beta interacts with PD-L1 to induce phosphorylation-dependent proteasomal degradation of PD-L1 by β-transducin repeats-containing protein (β-TrCP), which serves as a substrate recognition site for E3 ubiquitin ligases26,27. Finally, aberrant overexpression of GSK-3 has been shown to promote tumor growth and epithelial-to- mesenchymal transition (EMT) through various mechanisms including via modulation ofpro-survival NF-κB signaling pathways28. Thus, GSK-3 is a promising target in the treatment of human malignancies, and GSK-3 inhibitors have immunomodulatory potential.

[0127] Globally, colorectal cancer (CRC) ranks third in terms of incidence and second in terms of mortality. Treatment options include surgery, chemotherapy, radiation therapy, targeted therapy, and immunotherapy. Immune checkpoint blockade (ICB) has now entered into clinical care for CRC with the recent U.S. Food and Drug Administration approvals of checkpoint inhibitors nivolumab and pembrolizumab for microsatellite instability-high (MSI-H) CRC cases after chemotherapy29. Thus far, ICB clinical trials have demonstrated efficacy in MSI-H CRC, however, the impressive durability of tumor regression stands in stark contrast with the lack of response observed in microsatellite stable (MSS) CRC29. Thus, there remains a substantial unmet need in the ~85% of patients with MSS CRC in whom ICB is less effective30. Moreover, the percentage of patients with MSS CRC dramatically increases to ~96% in Stage IV disease30.

[0128] We sought to evaluate elraglusib (9-ING-41), a small molecule that targets GSK-3, which has the potential to increase the efficacy of ICB. We chose to evaluate elraglusib, which inhibits both alpha and beta isoforms, because it is a clinically-relevant small molecule with superior pharmacokinetic properties and is significantly more potent than other GSK-3 inhibitors31,32. Although there are ongoing efforts to characterize the immunomodulatory impact of GSK-3 inhibitors, few utilize small-molecule elraglusib33,34,35. Here, we characterize the effects of elraglusib in vitro on tumor and immune cells, in vivo in combination with ICB in a syngeneic murine colon carcinoma BALB / c model using MSS cell line CT-26, and in human tumor biopsies and plasma samples from patients with refractory solid tumors of multiple tissue origins enrolled in a Phase 1 clinical trial investigating elraglusib (NCT03678883).

[0129] 2. Results; 2.1. Elraglusib Sensitizes Tumor Cells to Immune-Mediated Cytotoxicity: Additional methods are discussed in the Examples below. A co-culture of fluorescently labeled SW480 MSS CRC cells and TALL-104 CD8+ T cells treated with elraglusib led to an increase in tumor cell death after 24 h (FIG.8A). Treatment doses were significantly less than the 24- and 72-h IC-50s calculated for all cell lines evaluatedin the co-culture to ensure the majority of tumor cell death was immune-cell-mediated (FIG.9A, FIG.9B). We observed limited tumor cell death in SW480 monocultures treated with drug only (FIG.8B). In the co-culture with tumor and immune cells only, in the absence of the drug, we noted that the baseline percentage of dead cells out of total cells was approximately 45%, after normalization. Co-cultures of tumor cells and TALL- 104 T cells treated with 5 µM elraglusib had an average of 60% dead cells, while co- cultures treated with 10 µM of elraglusib had an average of 65% dead cells (FIG.8C).

[0130] Because TALL-104 cells are a human leukemic T cell line, we next wanted to determine the relevancy of these results using normal T cells. Donor-derived CD8+ T cells were isolated from a donor blood sample in accordance with an IRB-approved protocol. A co-culture of fluorescently labeled SW480 tumor cells and CD8+ donor derived CD8+ T cells was then treated with elraglusib and the percentage of dead cells out of total cells was quantified after 24 h (FIG.8D). We again observed limited tumor cell death in SW480 monocultures treated with drug only (FIG.8E). The data was then normalized, as previously described, and we noted even more robust immune cell- mediated tumor cell death in the co-cultures treated with elraglusib (FIG.8F). Co- cultures of tumor cells and donor derived CD8+ T cells treated with 5 µM elraglusib had an average of 65% dead cells, while co-cultures treated with 10 µM of elraglusib had an average of 75% dead cells.

[0131] Experimental data for investigating elraglusib increases in immune- mediated cytotoxicity in a co-culture model with CRC cells is presented in FIGs.8A-8J. (FIG.8A) Representative SW480 and TALL-104 T cell co-culture assay images at the 24-hour timepoint.24-hour tumor cell pre-treatment with 5 μM elraglusib, followed by 24-hour co-culture. EthD-1 was used to visualize dead cells, 10× magnification, scale bar indicates 100 μm. (FIG.8B) Quantification of co-culture experiment using the percentage of dead cells out of total cells (N=3). (FIG.8C) Quantification normalized by cell death observed with drug treatment alone (N=3). (FIG.8D) Representative SW480 and donor-derived CD8+ T cell co-culture assay images at the 24-hour timepoint. (FIG.8E) Quantification of co-culture experiment using the percentage of dead cells out of total cells (N=3). (FIG.8F) Quantification normalized by cell death observed with drug treatment alone (N=3). A one-way ANOVA followed by a post-hoc Dunnett's multiplecomparisons test was used to calculate statistical significance. (FIG.8G) HCT-116 GFP+ or HT-29 GFP+ cells were co-cultured with TALL-104 cells at a 1:1 E:T ratio and were treated with DMSO or 5 μM elraglusib. (FIG.8H) Representative 40× images were collected after 24 hours of DMSO or 5 μM elraglusib treatment. (FIG.8I) Representative 40× images of a co-culture of HCT-116 GFP+ cells and NK-92 cells at a 1:1 E:T ratio were collected after 36 hours of 5 μM elraglusib treatment. White arrows indicate pyroptotic events. (FIG.8J) Representative 40× images of a co-culture of HT-29 GFP+ CRC cells and NK-92 cells at a 1:1 E:T ratio were after 36 hours of 5 μM elraglusib treatment. White arrows indicate pyroptotic events. P-value legend: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

[0132] To determine if the increased amount of immune cell-mediated tumor cell killing was due to the drug’s impact on the tumor cells or the immune cells, we next pre- treated tumor cells with elraglusib for 24 h before the co-culture with immune cells began. We observed that pre-treatment with elraglusib sensitized SW480 tumor cells to TALL-104 cell-mediated tumor cell killing (FIG.1A). We again used the raw percentages of cell death to normalize the data and observed minimal amounts of drug cytotoxicity at the concentration and duration of treatment used (FIG.1B). Tumor cells pre-treated with 5 µM elraglusib for 24 h and then co-cultured with TALL-104 T cells had an average of 65% dead cells (FIG.1C). Once again, we sought to confirm these co- culture results using donor-derived CD8+ T cells instead of TALL-104 cells (FIG.1D). We observed similar results with the CD8+ T cells where elraglusib pre-treatment of tumor cells led to a statistically significant increase in tumor cell death after 24 h of co- culture (FIG.1B). Tumor cells pre-treated with 5 µM elraglusib for 24 h and then co- cultured with donor-derived CD8+ T cells had an average of 65% dead cells, while co- cultures treated with 10 µM of elraglusib for 24 h and then co-cultured with donor- derived CD8+ T cells had an average of 70% dead cells (FIG.1F).

[0133] FIG.1A-1O demonstrate elraglusib enhances pyroptosis and sensitizes tumor cells to increase immune-mediated cytotoxicity in a co-culture model. Co-cultures were treated with drug concentrations as indicated. A 1:1 effector to target (E:T) ratio was used with a 24-h co-culture duration. EthD-1 was used to visualize dead cells; 10× magnification; scale bar indicates 100 µm. (FIG.1A) Representative SW480 and TALL-104 T cell co-culture assay images at the 24-h timepoint; 24 tumor cell pre-treatments with 5 µM elraglusib, followed by 24-h co-culture. (FIG.1B) Quantification of the co- culture experiment using the percentage of dead cells out of total cells (N = 3). (FIG.1C) Quantification normalized by cell death observed with drug treatment alone (N = 3). (FIG.1D) Representative SW480 and donor-derived CD8+ T cell co-culture assay images at the 24-h timepoint; 24-h tumor cell pre-treatment with 5 µM elraglusib, followed by a 24-h co-culture. (FIG.1E) Quantification of the co-culture experiment using the percentage of dead cells out of total cells (N = 3). (FIG.1F) Quantification normalized by cell death observed with drug treatment alone (N = 3). (FIG.1G) The number of HCT 116 GFP+ cells were quantified after 48 h of culture with DMSO, 5 μM elraglusib, and / or 5000 TALL-104 cells (N = 3). (FIG.1H) The number of HT-29 GFP+ cells was quantified after 48 h of culture with DMSO, 5 μM elraglusib, and / or 5000 TALL-104 cells (N = 3). (FIG.1I) The number of HCT 116 GFP+ cells was quantified after 48 h of culture with DMSO, 5 μM elraglusib, and / or 5000 NK-92 cells (N = 3). (FIG.1J) The number of HT-29 GFP+ cells was quantified after 48 h of culture with DMSO, 5 μM elraglusib, and / or 5000 NK-92 cells (N = 3). (FIG.1K) Representative 40 × images of HCT-116 GFP+ or HT-29 GFP+ CRC and TALL-104 T cell co-cultures. Black arrows indicate pyroptotic events. Representative images of western blot analysis of (FIG.1L) HCT-116 and (FIG.1M) HT-29 CRC cells for expression of indicated proteins after treatment with indicated cytokines or drugs. Quantification of IFN-γ secretion (pg / mL) post-DMSO or elraglusib treatment for 24 h in (FIG.1N) TALL-104 cells and (FIG.1O) NK-92 cells (N = 3). Error bars represent the mean + / − standard deviation. Statistical test: one-way ANOVA with Tukey’s test for multiple comparisons. p-value legend: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

[0134] To confirm these results, we repeated these experiments using a high- throughput GFP+ co-culture system with additional CRC cell lines HCT-116 and HT-29 (FIG.8G). We chose to evaluate both HCT-116 and HT-29 CRC cells in this co-culture model to determine if the elraglusib-mediated increase in immune cell-mediated SW480 cell killing could be reproduced in additional CRC cell lines. These cell lines were selected based on their varied mutational profiles, with both MSI-H and MSS statuses reflected (FIG.9C). When HCT-116 GFP+ cells were co-cultured with TALL-104 cells inthe presence or absence of 5 µM elraglusib we noted a significant decrease in GFP+ cells per low-powered field in the 5 µM elraglusib only, TALL-104 only, and combination therapy groups, as compared to the DMSO only control group (FIG.1G). We noted a significant decrease in the number of GFP+ cells per field in the combination therapy group of TALL-104 and 5 µM elraglusib co-culture condition as compared to TALL-104, which recapitulated the results observed in the first co-culture system. We observed a similar trend in the HT-29 cell line, where the combination therapy group showed increased tumor cell death as compared to the drug-only or T cell-only groups (FIG.1H). To determine if these results applied to other cytotoxic immune cell lines, we repeated the co-culture experiments with a natural killer cell line, NK-92. We observed similar trends in the co-culture of NK-92 cells with HCT-116 cells, where the combination of 5 µM elraglusib and NK-92 cells showed increased tumor cell death as compared to the drug-only treatment or immune-cell-only treatment (FIG.1I). In the HT- 29 cells, we noted increased tumor cell death in the combination therapy group as compared to immune cells only and as compared to DMSO only; however, the combination therapy group did not show statistical significance when compared with elraglusib only (FIG.1J).

[0135] 2.2. Elraglusib Enhances Tumor Cell Pyroptosis in a Co-Culture of Colorectal Cancer Cells and Immune Cells: To determine if pyroptosis-mediated immune cell activity played a role in the co-culture results, we examined higher-power co-culture images for evidence of pyroptosis. Indeed, we observed some pyroptotic events in the co-cultures involving tumor cells and TALL-104 cells only (FIG.1K). We did not observe any pyroptotic events in the DMSO or drug-only conditions, suggesting that tumor cell pyroptosis was mediated by an immune cell-secreted molecule as it was only observed in the co-culture wells with immune cells (FIG.1K and FIG.8H). Interestingly, in the co-culture of CRC (HCT-116, HT-29) and TALL-104 cells in the presence of 5 μM elraglusib treatment, we noted a significant increase in pyroptotic events (FIG.1K and FIG.8I, FIG.8J). To determine what immune-cell-secreted molecules were most likely contributing to tumor cell pyroptosis, we probed for a downstream mediator of pyroptotic death gasdermin B expression in tumor cells treated with a vehicle-only control (DMSO), 1 μM elraglusib, 100 ng / mL IFN-γ, 250 ng / mL IFN-γ, 1 ng / mL TNF-α, and 1 ng / mL TRAIL (FIG.1L). We observed an increase in gasdermin B expression with both concentrations of IFN-γ, used in both the HCT-116 cells and the HT-29 cells, suggesting that IFN-γ secreted by immune cells was a major contributor to the observed pyroptotic events (FIG.1M). To test whether immune cells secrete more IFN-γ post-treatment with elraglusib, we treated immune cell lines (TALL- 104, NK-92) with elraglusib for 24 h and indeed noted a significant increase in IFN-γ post-treatment in cell culture supernatants, although this effect was significantly greater in the TALL-104 cell line as compared to the NK-92 cell line (FIG.1N, FIG.1O).

[0136] 2.3. Elraglusib Upregulates Tumor Cell PD-L1 and Proapoptotic Pathway Expression as Well as Downregulates Immunosuppressive / Angiogenic Protein Expression and Pro-Survival Pathways: To help elucidate the mechanism behind the CRC cell sensitization to immune cell killing that we observed in the co-culture assays, we performed western blot analyses on CRC cells (HCT-116, HT-29) treated with elraglusib over a 72-h timecourse. Using the same low dose of elraglusib utilized in the co-culture assays, we observed little to no cleaved PARP (cPARP) in both cell lines analyzed until the 48-h timepoint, confirming that the tumor cell death observed in the co-culture assays was not a product of drug cytotoxicity (FIG.2A). Because GSK-3 is a known regulator of NF-κB signaling pathways, we also probed for NF-κB p65 and noted a decreased expression as the timecourse progressed. However, we observed increases in PD-L1 expression as the treatment duration increased. To further elucidate the elraglusib-mediated effects on tumor cell survival we probed for survival factors Bcl- 2 and Survivin and noted decreases in protein expression in both cell lines, especially at the later timepoints (48, 72 h). In HCT-116 cells, we also probed for survival factor Mcl-1 and again noted marked decreases in protein expression by the 24-h timepoint (FIG.2B). Although GSK-3 plays a role in the regulation of β-catenin, we did not focus on elraglusib-mediated effects on β-catenin because colon cancers often harbor mutations in β-catenin or adenomatous polyposis coli (APC), thus nullifying any impact GSK-3 inhibition would have on β-catenin expression. HCT116 cells are heterozygous for β-catenin, harboring one wild-type allele and one mutant allele with inactivation of one of the residues phosphorylated by GSK3β that is frequently mutated in tumors36. Moreover, HT-29, KM12C, and SW480 cells harbor APC mutations37(FIG.9C).

[0137] FIG.2A-2I demonstrate elraglusib treatment induces PD-L1 expression and suppresses survival pathways in tumor cells. Representative images of western blot analysis of (FIG.2A) HCT-116 and HT-29 CRC cells for expression of indicated proteins after increasing durations of elraglusib treatment (0–72 h). (FIG.2B) Representative images of western blot analysis of Mcl-1 expression in HCT-116 CRC cells after increasing durations of elraglusib treatment. CRC cells were treated with 1 μM elraglusib for 24 h and treated versus untreated samples were compared in triplicate. Microarray analysis results were visualized using volcano plots for (FIG.2C) HCT-116, (FIG.2D) HT-29, and (FIG.2E) KM12C CRC cell lines (N = 3). The top down- and up-regulated genes post-elraglusib treatment as compared to controls are shown. Results were calculated using a fold-change cutoff of >1.5, <−1.5, and a p-value of <0.05. (FIG.2F) The number of genes up- or down-regulated in each of the three cell lines within several signaling pathways of interest (N = 3). Green indicates downregulation and red indicates upregulation of gene expression. (FIG.2G) A Venn Diagram was used to compare the 3124 genes that were differentially expressed post- treatment with elraglusib in the three-colon cancer (HCT-116, HT-29, KM12C) cell lines (N = 3). (FIG.2H) Tumor cells (HCT-116, HT-29) were treated with 1 μM elraglusib for 48 h and cell culture supernatant was analyzed with the Luminex 200. Fold-change is shown where red indicates a positive fold-change and green indicates a negative fold- change (N = 3). (FIG.2I) Tumor cells (HCT-116, HT-29) were treated with 5 μM elraglusib for 48 h and cell culture supernatant was analyzed with the Luminex 200. Fold-change is shown where red indicates a positive fold-change and green indicates a negative fold-change (N = 3).

[0138] We then utilized microarray analysis to gain insights into gene expression changes in CRC cell lines post-GSK-3 inhibition with elraglusib. Several CRC cell lines (HCT-116, HT-29, KM12C) were treated with elraglusib at IC-50 concentrations or DMSO as vehicle control for 24 h, and treated versus untreated samples were compared in triplicate using microarray analysis (FIGs.10A-10F). Results were calculated using a fold change (FC) cutoff of >1.5, <−1.5, and a minimum p-value of <0.05. HCT-116 cells had 340 differentially expressed genes post-treatment (FIG.2C). The top differentially expressed genes of interest that were upregulated in HCT-116cells included many anti-proliferative (BTG2, TP53INP1, LYZ, GADD45A, CDKN1A, ATF3, SESN1, SUSD6) and proapoptotic (DRAM1, FAS, BLOC1S2, TNFRSF10B, KLLN, PLK3, MXD1, GADD45B, TRIM31, TP53I3, TNFRSF10A, BAK1) genes (FIG.22). Of note, several of the upregulated genes are known p53 targets (BTG2, MDM2, TP53INP1, DRAM1, GADD45A, CDKN1A, PMAIP1, ATF3, FAS, SESN1, TNFRSF10D, TNFRSF10B, AEN, PLK3, TP53I3, SUSD6, GDF15)38. Meanwhile, many of the downregulated genes included those that promote cell cycle progression (CDC25C, PRC1, ANLN, BARD1, PDK1, DHX32, CCNF, PRR11, TTK, FANCD2, AURKB, UHRF1), EMT (ENO2, MST1R) or cellular proliferation (FASN, ARHGEF39, FOXC1, CDCA3, MKI67). In FIG.22, HCT-116 CRC cells were treated with 1 μM elraglusib for 24 hours and treated versus untreated control samples were compared in triplicate via microarray analysis (N=3). Table showing differentially expressed genes of interest with their corresponding fold changes, p values, descriptions, and functions. Genes highlighted are known p53 targets. Genes are ordered by fold change and were calculated using a fold change cutoff of >1.5,

[0139] Internal controls for tumor cell microarray analysis are illustrated in FIG.10..(FIG.10A) PCA mapping demonstrated clear mapping of HCT-116, HT-29, and KM12C cells (N=3). (FIG.10B) Quality control was determined satisfactory for further analysis. (FIG.10C) Hybridization controls. (FIG.10D) Labeling controls. (FIG.10E) Pos vs Neg Area Under the Curve (AUC). (FIG.10F) Signal box plot.

[0140] Another upregulated (1.78-FC) gene of interest was PPP1R1C, and increased expression may increase tumor cell susceptibility to TNF-induced apoptosis39. Interestingly, CMTM4 expression was downregulated (−1.84-FC) post-treatment and is known to protect PD-L1 from being polyubiquitinated and targeted for degradation40. Furthermore, NEK2 was downregulated (−2.21-FC) post-treatment and NEK2 protein inhibition is known to sensitize PD-L1 blockade41.

[0141] In HT-29 cells, we observed 2307 differentially expressed genes post- treatment (FIG.2D). We also observed that many of the upregulated genes post- treatment were proapoptotic (AEN, TNFRSF12A, CCAR1, SFN) or anti-proliferative (SOCS7, CDKN1A, SMAD3, BCCIP, CRLF3) and many of the downregulated genes were involved with the promotion of cellular proliferation (TNIK, BRAF, EAPP, JAK1,PDS5B, CDCA3), cell cycle progression (MCIDAS, DYNC1H1, CDC45, UHRF1, CDK2, CDC25C, CCNE1, CDK1, BARD1, CCNE2), EMT (MTA3, AGGF1, E2F8, E2F7), or have antiapoptotic functions (PIM1, SGK1, BCL6, E2F7, TRIB1) (FIG.23). Interestingly, NCR3LG1 (B7-H6) was upregulated (1.78-FC) post-treatment and is known to trigger NCR3-dependent NK cell activation and cytotoxicity42. In FIG.23, HT-29 CRC cells were treated with 1 μM elraglusib for 24 hours and treated versus untreated control samples were compared in triplicate via microarray analysis (N=3). Table showing differentially expressed genes of interest with their corresponding fold changes, p values, descriptions, and functions. Genes highlighted are known p53 targets. Genes are ordered by fold change and were calculated using a fold change cutoff of >1.5, <0.05.

[0142] Finally, KM12C cells had 1032 differentially expressed genes post- treatment (FIG.2E). We observed an upregulation of proapoptotic genes (TNFRSF12A, BIK) while we observed a downregulation of genes involved in the promotion of EMT (CXCL1, AGGF1, IRF2BP2, MET, NRP1, GDF15, E2F8), the promotion of cell cycle progression (CDCA2, IGFBP2, CDC25C, CCNE1, CCND2, CDK1, CCNE2, BARD1), cellular proliferation (MKI67, BRAF), and the regulation of TGFβ signaling (TGFBR2, LTBP1, TGFBR3, CD109) in KM12C cells post-treatment as compared to control (FIG. 24). Of note, we noticed an upregulation (1.53-FC) of GZMA (granzyme A) expression post-treatment, which is known to cleave gasdermin B to induce pyroptosis43. In FIG. 24, KM12C CRC cells were treated with 1 μM elraglusib for 24 hours and treated versus untreated control samples were compared in triplicate via microarray analysis (N=3). The table shows differentially expressed genes of interest with their corresponding fold changes, p values, descriptions, and functions. Genes highlighted are known p53 targets. Genes are ordered by fold change and were calculated using a fold change cutoff of >1.5,

[0143] Several relevant signaling pathways had differentially expressed genes post-elraglusib in all three cell lines including the VEGFA-VEGFR2, TGFβ, IL-18, CCL18, EGF / EGFR, miR-targeted genes in lymphocytes, apoptosis, and cell cycle signaling pathways (FIG.2F). The most significant commonly downregulated signaling pathway was VEGFA-VEGFR2, which had 29 downregulated genes in HCT-116 cells,37 downregulated genes in HT-29 cells, and 48 downregulated genes in KM12C cells. A Venn diagram was used to compare the 3124 genes that were differentially expressed post-treatment with elraglusib as compared to control in the three colon cancer cell lines (HCT-116, HT-29, KM12C) (FIG.2G). HCT-116 cells had 241 (7.7%), HT-29 cells had 1805 (57.8%) and KM12C cells had 549 (17.6%) differentially expressed genes post- treatment as compared to control. HCT-116 and HT-29 cells shared 46 differentially expressed genes (1.5%), HCT-116 and KM12C shared 27 (0.86%), KM12C and HT-29 shared 430 (13.8%), and all three cell lines shared 26 (0.83%). All three cell lines showed a post-treatment differential expression of NF-κB regulators with increased expression of many negative regulators of NF-κB (NFKBIA, TNFAIP3, TRAIP, IL32) and decreased expression of several positive regulators of NF-κB (IRAK1BP1, FADD, IL17RA, MYD88, ERBB2IP, IL17RB, TNFSF15, NFKBIZ, NFKBIA, MAP3K1, TRAF5, TRAF6, TAB3, TNFRSF11A, MTDH, TLR3) (FIG.22, FIG.23, FIG.24).

[0144] We previously found that elraglusib treatment of human CRC cell lines (HCT-116, HT-29, KM12C) with varied mutational profiles modified cytokine, chemokine, and growth factor secretion into cell culture media44. Here, we treated tumor cells (HCT-116, HT-29) with 1 μM or 5 μM elraglusib for 48 h and subsequently analyzed the cell culture supernatant using Luminex 200 technology (FIG.2H, FIG.2I). Several cytokines, chemokines, and growth factors associated with angiogenesis and / or EMT were downregulated in both cell lines (HCT-116, HT-29) at both concentrations of elraglusib. Notably, GDF-15, GM-CSF, and VEGF all had decreased secretion post- treatment in both cell lines and at both concentrations of elraglusib. Likewise, several cytokines, chemokines, and growth factors associated with immunosuppression were also downregulated post-treatment, including CCL5 / RANTES, DcR3, Fas, and soluble PD-L1 (sPD-L1).

[0145] 2.4. Elraglusib Enhances Immune Cell Effector Function: we next analyzed immune cell lines (TALL-104, NK-92) using western blot analysis. Interestingly, when we probed for the same proteins in the cytotoxic immune cell lysates, we observed many opposing trends to those observed in the tumor cells. In TALL-104 cells, we did not notice significant changes in NF-κB or survival protein Bcl-2 as treatment duration increased (FIG.3A). Because of the differential impact ofelraglusib on tumor and immune cells that we observed via western blot, we next sought to compare the levels of another survival protein Mcl-1 in NK-92 natural killer cells and we did not observe a significant decrease in Mcl-1 protein expression through the 72-h endpoint (FIG.3B). Surprisingly, we noted increases in survival protein Survivin and NF-κB-inducing kinase (NIK), a protein commonly associated with activation of the non- canonical NF-κB signaling pathway, which led us to create a working model of NIK- mediated immune cell recruitment (FIG.3C).

[0146] How elraglusib treatment increases effector molecule secretion and induces an energetic shift in cytotoxic immune cells is shown in FIG.3A-3J. Representative images of western blot analysis of (FIG.3A) TALL-104 and (FIG.3B) HT-29 cytotoxic immune cells for expression of indicated proteins after increasing durations of elraglusib treatment (0–72 h). (FIG.3C) Proposed model for non-canonical NF-κB pathway activation: increased NIK expression indicates non-canonical NF-κB pathway activation which enhances the expression of chemokines and cytokines (CCL11, TNF-α, GM-CSF) and subsequently leads to increased recruitment and proliferation of cytotoxic immune cells (CD8+ T, CD4+ T, NK cells). Immune cells were treated with 1 μM elraglusib for 24 h and treated versus untreated samples were compared in triplicate. Microarray analysis results were visualized using volcano plots for (FIG.3D) NK-92 and (FIG.3E) TALL-104 immune cell lines (N = 3). (FIG.3F) A Venn diagram was used to compare the 124 genes that were differentially expressed post-treatment with elraglusib in the two immune (TALL-104, NK-92) cell lines (N = 3). (FIG.3G) 10 × single-cell sequencing analysis of immune cells treated with elraglusib. TALL-104 and NK-92 cells were treated with 1 μM elraglusib for 24 h and aggregate data were visualized using a t-SNE plot. (FIG.3H) Immune cells show differential expression of mitochondria-encoded genes (MT) and ribosomal genes (RB) after elraglusib treatment. (FIG.3I) Heatmap comparing gene expression post-elraglusib treatment as compared to control. Fold-change is shown, where red indicates a positive fold-change and green indicates a negative fold-change. (FIG.3J) Immune cells (TALL- 104, NK-92) were treated with 1 μM elraglusib for 48 h and cell culture supernatant was analyzed with the Luminex 200. Fold-change is shown, where red indicates a positive fold-change and green indicates a negative fold-change (N = 3).

[0147] Next, microarray analysis was used to obtain insights into gene expression changes in immune cell lines post-GSK-3 inhibition with elraglusib. Immune cell lines (TALL-104, NK-92) were treated with elraglusib at IC-50 concentrations or DMSO as vehicle control for 24 h, and treated versus untreated samples were compared in triplicate using microarray analysis (FIGs.11A-11F). Results were calculated using a fold-change (FC) cutoff of >1.5, <−1.5, and a minimum p-value of <0.05. NK-92 cells had 61 differentially expressed genes post-treatment (FIG.3D). We observed an increase in genes that promote immune cell proliferation (TNFSF14, RAB38) and control immune cell adhesion and migration (WNK1) post-elraglusib treatment (FIG.25). In FIG.25, K-92 immune cells were treated with 1 μM elraglusib for 24 hours and treated versus untreated control samples were compared in triplicate via microarray analysis (N=3). The table shows differentially expressed genes of interest with their corresponding fold changes, p values, descriptions, and functions. Genes are ordered by fold change and were calculated using a fold change cutoff of >1.5, <0.05.

[0148] Internal controls for immune cell microarray analysis are shown in FIG.11. (FIG.11A) PCA mapping demonstrated clear mapping of NK-91 and TALL-104 cells (N=3). (FIG.11B) Quality control was determined satisfactory for further analysis. (FIG.11C) Hybridization controls. (FIG.11D) Labeling controls. (FIG.11E) Pos vs Neg Area Under the Curve (AUC). (FIG.11F) Signal box plot.

[0149] We also noted decreases in proapoptotic genes (MIR186, S100A12) and genes involved in the activation of latent TGFβ to suppress immune cell function (ITGB8). TALL-104 cells had 64 differentially expressed genes post-treatment (FIG.3E). We observed an increased expression of genes involved in the modulation of NF-κB activity (RNY4, RNY5), cytotoxic granule exocytosis (STX19, VAMP8), and anti- apoptotic gene BCL2-related protein A1 (BCL2A1) (FIG.26). In FIG.26, TALL-104 immune cells were treated with 1 μM elraglusib for 24 hours and treated versus untreated control samples were compared in triplicate via microarray analysis (N=3). The table shows differentially expressed genes of interest with their corresponding fold changes, p values, descriptions, and functions. Genes are ordered by fold change and were calculated using a fold change cutoff of >1.5, <0.05.

[0150] We also saw an upregulation (1.56-FC) of kinesin family member 7 (KIF7), which is required for T cell development and MHC expression, as well as an increased expression (1.52-FC) of chemokine C-C motif ligand 3 (CCL3) which is known to recruit and enhance the proliferation of CD8+ T cells45. In contrast, we observed a decreased expression of genes involved in TGFβ signaling pathways (ACVR1B, PTPN14) and proapoptotic genes (HSPA1A, UBE3A). We also saw a decreased expression (-1.56- FC) of inhibitory immune checkpoint protein tyrosine phosphatase, non-receptor type 3 (PTPN3). In total, there were 124 differentially expressed genes post-treatment and only 1, an unnamed gene (probe set ID TC22000564.hg.1, coding), was shared between both cell lines (FIG.3F).

[0151] To determine if there was any heterogeneity in response to drug treatment, we employed a 10 × single-cell sequencing analysis on both immune cell lines (TALL-104, NK-92) treated with low-dose 1 μM elraglusib or vehicle control (DMSO) for 24 h. As expected, samples clustered by cell type when aggregate data were visualized using a t-SNE plot (FIG.3G). Interestingly, immune cells showed a differential expression of mitochondrial-encoded genes (MT) and ribosomal genes (RB) post-treatment with elraglusib, suggesting a metabolic shift in line with the extensive metabolic reprogramming undertaken in immune cells post-activation46to support immune cell activities such as cytokine production (FIG.3H). Several genes showed the same trends post-treatment in both cell lines (FIG.3I). In both cell lines, we observed an increase in immune cell activation marker CD69 and a decrease in the immunosuppressive marker CHI3L1. Finally, we noted an increase in immune cell attractant CCL4 in the NK-92 cells and an increase in immune cell chemoattractant CXCR4 in the TALL-104 cells.

[0152] Because the previously observed non-canonical NF-κB pathway activation is known to enhance the expression of immune cell chemotactic chemokines and cytokines, we sought to determine how elraglusib treatment impacts the immune cell secretome. TALL-104 and NK-92 cells were treated with 1 µM elraglusib for 48 h before cell culture supernatant was collected for cytokine profile analysis. TALL-104 cells treated with elraglusib showed increases in effector molecules IFN-γ, granzyme B, and TRAIL concentrations, as measured in picogram per milliliter (FIG.3J). In contrast, NK-92 cells treated with elraglusib showed increases in IFN-γ and TRAIL but decreases in the concentration of secreted granzyme B.

[0153] 2.5. Elraglusib Significantly Prolongs Survival in Combination with Anti- PD-L1 Therapy in a Syngeneic MSS CRC Murine Model: because elraglusib activated immune cells and increased tumor cell PD-L1 expression, we sought to evaluate the potential for elraglusib to increase the efficacy of ICB and utilized a syngeneic murine colon carcinoma BALB / c murine model using a MSS cell line CT-26 (FIG.4A). Mice were randomly assigned to one of seven groups: isotype (N = 12), elraglusib (N = 12), elraglusib + isotype (N = 12), anti-PD-1 (N = 12), anti-PD-L1 (N = 12), elraglusib + anti- PD-1 (N = 12), and elraglusib + anti-PD-L1 (N = 12). In this MSS CRC model, we observed a significantly improved survival curve in the elraglusib and anti-PD-L1 combination therapy group (FIG.4B). We also observed statistically significant improved survival in the elraglusib, anti-PD-1, and anti-PD-L1 alone groups as compared to the control (FIGs.12A-12E). However, we saw the most sustained response in the elraglusib and anti-PD-L1 combination therapy group (FIG.12F). Murine body weights did not differ significantly regardless of the treatment group (FIGs. 12G-12L).

[0154] Data for the syngeneic murine colon carcinoma BALB / c murine model with MSS cell line CT-26 Kaplan Meier curves and mouse body weights grouped by treatment is shown in FIG.12A-12L. Individual Kaplan Meier curves for isotype control (N=12) compared to (FIG.12A) elraglusib (N=12), (FIG.12B) anti-PD-1 (N=12), (FIG.12C) anti-PD-L1 (N=12), (FIG.12D) elraglusib + anti-PD-1 (N=12), and (FIG.12E) elraglusib + anti-PD-L1 (N=12). (FIG.12F) Bar graph indicating the percentage of responders (R) and non-responders (NR) per treatment group. Individual body weight plots for (FIG.12G) Isotype control (N=12), (FIG.12H) elraglusib (N=12), (FIG.12I) anti-PD-1 (N=12), (FIG.12J) anti-PD-L1 (N=12), (FIG.12K) elraglusib + anti-PD-1 (N=12), and (FIG.12L) elraglusib + anti-PD-L1 (N=12). P-value legend: * p < 0.05, ** p < 0.01.

[0155] Additionally, the mice did not show evidence of significant treatment- related toxicity on complete blood count or serum chemistry analysis (FIG.27). In FIG. 27, murine serum chemistry analysis does not show treatment-related toxicity. Wholeblood from long-term mice sacrificed was submitted for serum chemistry analysis. Results are shown from the complete metabolic panel and complete blood count with differential. ALB: Albumin, ALP: Alkaline Phosphatase, ALT: Alanine aminotransferase, AMY: Amylase, ANIS: Anisocytosis, AST:Aspartate aminotransferase, ATYP: Atypical Lymphs, BAS: Absolute Basophils, BAS%: % Basophils, BUN: Urea Nitrogen, CA: Calcium, CHOL: Cholesterol, CL: Chloride, CPK: Creatine kinase, CPLT: Clumped Platelets, CREA: Creatinine, DBIL: Direct Bilirubin, EOS: Absolute Eosinophils, EOS% : % Eosinophils, GGT: Gamma-glutamyl Transferase, GLU: Glucose, HB: Hemoglobin, HCT: Hematocrit, HJB: Howell-Jolly Bodies, HYPO: Hypochromasia, HYPR: Hyperchromasia, K: Potassium, LDH: Lactate Dehydrogenase, LIP: Lipase, LPLT: Large Platelets, LUC: Absolute Large Unstained Cells, LUC%: % Large Unstained Cells, LYM: Absolute Lymphocytes, LYM%: % Lymphocytes, MAC: Macrocytosis, MCH: Mean Corpuscular Hemoglobin, MCHC: Mean Corpuscular Hemoglobin Count, MCV: Mean Corpuscular Volume, MG: Magnesium, MIC: Microcytosis, MON: Absolute Monocytes, MON%: % Monocytes, NA: Sodium, NEU: Absolute Neutrophils, NEU%: % Neutrophils, PHOS: Inorganic Phosphorus, PLT: Platelet Count, POLK: Poikilocytosis, RBC: Red Blood Cell Count, TBIL: Total Bilirubin, TPRO: Total Protein, TRIG: Triglyceride, WBC: White Blood Cell Count.

[0156] Both the elraglusib individual treatment and dual treatment groups maintained normal renal function, as evidenced by normal blood urea nitrogen (BUN) and creatinine, and were free of significant electrolyte perturbations. Liver function tests did not reveal any evidence of liver toxicity and the dual-treatment mice did not have any elevations in AST, ALT, or bilirubin. As can be expected in mice with significant tumor burdens, mice across treatment groups had decreased albumin levels and evidence of mild marrow hypoplasia resulting in mild anemia, and reduced white blood cell and platelet counts. This effect was independent of the treatment group and likely related to tumor burden at the time of sacrifice.

[0157] FIG.4A-4M Illustrates that elraglusib enhances immune-cell tumor- infiltration to prolong survival in combination with anti-PD-L1 therapy in a syngeneic murine model of MSS colon carcinoma. (FIG.4A) Experimental model overview of the syngeneic murine colon carcinoma BALB / c murine model using MSS cell line CT-26.(FIG.4B) Kaplan-Meier estimator curves for all treatment groups as indicated. Statistical significance was determined using a Log-rank (Mantel-Cox) test. (FIG.4C) Overview of cell lineage markers used for flow cytometric immunophenotyping analysis. Two weeks after treatment initiation, immune cell subpopulations were analyzed in the spleen and tumor. (FIG.4D) Splenic T cells, (FIG.4E) tumor-infiltrating T cells, (FIG. 4F) splenic NK cells, and (FIG.4G) tumor-infiltrating NK cells were compared between responders (R, N = 3) and non-responders (NR, N = 18), regardless of treatment group. NK cell subsets based on the expression of CD11b and CD27 were compared in the spleen and visualized via (FIG.4H) bar graph and (FIG.4I) pie chart. NK cell subsets based on the expression of CD11b and CD27 were also compared in the tumor and visualized via (FIG.4J) bar graph and (FIG.4K) pie chart. T cell ratios were compared in the (FIG.4L) Spleen and (FIG.4M) Tumor. Statistical significance was determined using two-tailed unpaired T tests. p-value legend: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns: non-significant.

[0158] 2.6. Murine Responders Have More T Cell Tumor-Infiltration and Elevated Tumoral CD8+ / Treg and CD4+ / Treg Ratios: to begin to evaluate our hypothesis that elraglusib increases immune cell activation and recruitment, we utilized multi-color flow cytometry to characterize the natural killer (NK) and T cell populations two weeks after treatment initiation, and immune cell subpopulations were analyzed in both the spleen and the tumor (FIG.4C). Two weeks after treatment initiation, mice were grouped as responders (R) or non-responders (NR) based on a tumor volume less than or greater than 100 mm3, respectively. Compared to non-responders, regardless of treatment group, responders two weeks after treatment had statistically significantly reduced levels of splenic CD4+ and CD8+ T cells and had increased percentages of CD69+ activated T cells and Foxp3+ regulatory T cells (Tregs) (FIG.4D). Meanwhile, responders had increased percentages of tumor-infiltrating CD3+ and CD4+ T cells (FIG.4E). We also observed that responders had increased percentages of splenic KLRG1+ mature NK cells and tumor-infiltrating CD11b- / CD27- immature NK cells, and decreased percentages of tumor-infiltrating CD11b+ / CD27-activated NK cells two weeks after treatment initiation (FIG.4F, FIG.4G). We did not observe striking differences between non-responders and responders in the splenic immature naturalkiller cell subsets (CD11b- / CD27-, CD11b- / CD27+, CD11b+ / CD27+, CD11b+CD27-) (FIG.4H, FIG.4I). In contrast, we did observe significant differences between non- responders and responders in the tumor-infiltrating immature natural killer cell subsets (FIG.4J, FIG.4K). We observed that responders had a greater proportion of immature (CD11b- / CD27-) NK cells and a reduced proportion of mature (CD11b+CD27-) NK cells two weeks after treatment initiation. When comparing the T cell ratios, compared to non- responders, responders had a reduced splenic CD8+ / Treg and CD4+ / Treg ratio (FIG.4L). The CD8+ / Treg ratio is commonly used as an index of TIL’s effector function47. Additionally, responders had an elevated intra-tumoral CD8+ / Treg and CD4+ / Treg ratio (FIG.4M). Overall, the observed changes in immune cell subsets in responders are consistent with the increased infiltration of cytotoxic immune cells into the tumor.

[0159] 2.7. Murine Responders Show an Immunostimulatory Tumor Microenvironment by IHC: to further interrogate the tumor microenvironment (TME), we conducted immunohistochemistry (IHC) on tumor sections from the two weeks post- treatment initiation timepoint or from the end-of-study (EOS) timepoint. We compared non-responders (NR) and responders (R) and stained for T cell marker CD3 and observed that responders had significantly more CD3+ T cells as compared to non- responders at both timepoints. (FIG.5A, FIG.5B). To determine if there were any differences in immune cell activation, we stained for Granzyme B and again observed that responders had significantly more Granzyme B+ staining at both timepoints as compared to non-responders (FIG.5C, FIG.5D). We stained for Ki67 as a marker of tumor cell proliferation and observed that responders had less tumor cell proliferation as compared to responders at both timepoints (FIG.5E, FIG.5F). As we found that elraglusib upregulated tumor cell PD-L1 expression and observed an improvement in survival when elraglusib was combined with anti-PD-L1 therapy as compared to anti- PD-1 therapy, we next looked at PD-L1 staining in the tumor sections (FIG.5G, FIG.5H). We observed that responders had more PD-L1+ tumor cells than non- responders at both timepoints. To examine tumor cell apoptosis, we then stained for cleaved-caspase 3 (CC3) and noted that there was no difference in CC3 expression at the mid-timepoint (two weeks after treatment initiation); however, responders did havesignificantly elevated CC3 expression than non-responders at the EOS timepoint (FIG.5I, FIG.5J).

[0160] FIG.5A-5W illustrate that responders have a more immunostimulatory tumor microenvironment as compared to non-responders. IHC analysis of tumors two weeks post-treatment initiation or tumors from long-term mice. Non-responders (NR, N = 18) and responders (R, N = 3) were compared.20 × images, scale bar represents 100 μm. (FIG.5A, FIG.5B) CD3, (FIG.5C, FIG.5D) Granzyme B, (FIG.5E, FIG.5F) Ki67, (FIG.5G, FIG.5H) PD-L1, and (FIG.5I, FIG.5J) cleaved-caspase 3 (CC3) were compared at the two weeks after treatment initiation timepoint, and the long-term timepoint, respectively. Statistical significance was determined using two-tailed unpaired T tests (N = 6). Serum from long-term mice sacrificed was analyzed via cytokine profiling for (FIG.5K) CCL21, (FIG.5L) VEGFR2, (FIG.5M) CCL7, (FIG.5N) CCL12, (FIG.5O) BAFF, (FIG.5P) VEGF, (FIG.5Q) IL-1 β, (FIG.5R) IL-6, (FIG.5S) CCL22, (FIG.5T) GM-CSF, (FIG.5U) CCL4, (FIG.5V) TWEAK, and (FIG.5W) CCL2. Responders (red, N = 3) and non-responders (black, N = 18) were compared. A Kruskal-Wallis test was used to calculate statistical significance, followed by a Benjamini-Hochberg correction for multiple comparisons. p values are shown for analytes that were significantly different between responders and non-responders and are ordered by significance. p-value legend: * p < 0.05, ** p < 0.01, *** p < 0.001. ns: no significance.

[0161] We also analyzed the expression of CD4, CD8, Foxp3+, NKp46, TRAIL, PD-1, VEGF, and TGFβ2 to gain additional insights into the tumor immune microenvironment at both the two weeks after treatment initiation timepoint and the EOS timepoint, respectively (FIG.13). To examine helper T cell presence, we stained for CD4 and observed that responders had more CD4+ T cells than non-responders at both timepoints (FIG.13A). Interestingly, we saw the same trends when we examined CD8 expression, where responders had more CD8+ T cells than non-responders, which differed from the flow cytometry results but could be explained by the large variability in CD8a+ T cells we observed using flow cytometry in the non-responder group (FIG. 13B). We did not observe statistically significant differences in Foxp3+ Treg expression between responders and non-responders at either timepoint (FIG.13C). When weexamined NK cell tumor-infiltration by IHC, we noted more NKp46+ NK cells in responders at the two weeks after treatment initiation timepoint, but this difference was not significant at the EOS timepoint (FIG.13D). We chose to examine another cytotoxic mediator, TRAIL, and observed no difference between responders and non-responders at the mid-timepoint but, interestingly, observed reduced TRAIL expression in the responders as compared to the non-responders at the EOS timepoint (FIG.13E). We also examined PD-1 expression and did not note any significant differences between responders and non-responders at either of the timepoints (FIG.13F). Again, we noted a similar lack of significance when we examined immunosuppressive and angiogenic VEGF expression (FIG.13G). Finally, we examined immunosuppressive TGFβ2 expression and noted no differences between responders and non-responders at the mid-timepoint but noted that responders had significantly reduced expression at the EOS timepoint (FIG.13H).

[0162] 2.8. Murine Responders Have Reduced Tumorigenic and Elevated Immunomodulatory Cytokine Concentrations: we next analyzed murine serum samples from EOS mice for cytokine profiles and noted interesting trends between responders and non-responders. Responders were more likely to have reduced serum concentrations of CCL21 (p = 0.000213), VEGFR2 (p = 0.000282), CCL7 (p = 0.000633), CCL12 (p = 0.0092), BAFF (p = 0.0116), and VEGF (p = 0.0396) compared to non-responders (FIGs.5K-5P). In contrast, responders had elevated serum concentrations of IL-1 β (p = 0.00135), IL-6 (p = 0.0022), CCL22 (p = 0.00803), GM-CSF (p = 0.0108), CCL4 (p = 0.0127), TWEAK (p = 0.02), and CCL2 (p = 0.0291) compared to non-responders (FIGs.5Q-5W).

[0163] Analytes that were statistically significant between responders and non- responders at both timepoints (two weeks after treatment initiation, EOS) included CCL7 / MCP-3 / MARC (p = 2.19 × 10−5), CCL12 / MCP-5 (p = 0.000606), TWEAK / TNFSF12 (p = 0.00112), BAFF / TNFSF13B (p = 0.00469), IL-1 β / IL-1F2 (p = 0.00507), CCL21 / 6Ckine (p = 0.00539), VEGF (p = 0.00646), IFN-γ (p = 0.00817), CCL4 / MIP-1 β (p = 0.0133), IL-6 (p = 0.229), and GM-CSF (p = 0.0257). The entire panel of cytokines, chemokines, and growth factors analyzed by multiplex immunoassay in murine serum from the EOS timepoint included BAFF, MCP-1, MIP-1 α, MIP-1 β,RANTES, MCP-3, Eotaxin, MCP-5, VEGFR2, MIP-3 α, CCL21, MDC, IP-10, CXCL12, GM-CSF, Granzyme B, IFN-γ, IL-1 α, IL-18, IL-2, IL-3, IL-4, IL-6, IL-7, IL-10, IL-12 p70, IL-13, IL-16, VEGF, M-CSF, Prolactin, and TWEAK (FIGs.14A-14S). FIG.14A-14S provide murine serum cytokine profiling assay results. Serum from end-of-study mice was analyzed via cytokine profiling for (FIG.14A) MIP-1 α, (FIG.14B) RANTES, (FIG.14C) Eotaxin, (FIG.14D) MIP-3 α, (FIG.14E) IP-10, (FIG.14F) CXCL12, (FIG.14G) Granzyme B, (FIG.14H) IFN-γ, (FIG.14I) IL-1 α, (FIG.14J) IL-2, (FIG.14K) IL-3, (FIG.14L) IL-4, (FIG.14M) IL-7, (FIG.14N) IL-10, (FIG.14O) IL-12 p70, (FIG.14P) IL-13, (FIG.14Q) IL-16, (FIG.14R) M-CSF, and (FIG.14S) Prolactin. Responders (red, N=3) and non-responders (black, N=18) were compared. A Kruskal- Wallis test was used to calculate statistical significance followed by a Benjamini- Hochberg correction for multiple comparisons.

[0164] 2.9. Patient Plasma Concentrations of Cytokines from a Phase 1 Clinical Trial Investigating Elraglusib Correlate with Progression-Free Survival, Overall Survival, and In Vivo Response to Therapy Results: to determine the clinical relevance of the biomarkers of response identified in our murine model, we next employed Luminex 200 technology to analyze plasma samples from patients with refractory solid tumors of multiple tissue origins enrolled in a Phase 1 clinical trial investigating elraglusib (NCT03678883). Patients included in this analysis represented multiple tumor types, including appendix (N = 3, 15.8%), adult T-cell leukemia / lymphoma (ATLL) (N = 1, 5.3%), cholangiocarcinoma (N = 1, 5.3%), colorectal (N = 7, 36.8%), desmoid (N = 1, 5.3%), hepatocellular carcinoma (HCC) (N = 1, 5.3%), leiomyosarcoma (N = 1, 5.3%), non-small cell lung cancer (NSCLC) (N = 2, 10.5%), and pancreas (N = 2, 10.5%) cancer (FIG.6A). The median PFS was 75.9 days, and the median OS was 101 days (FIG.28). In FIG.28, (individual patient information for human cytokine analysis), tumor type, elraglusib dose (mg, mg / kg), number of cycles, age at enrollment, sex, race, ethnicity, median progression, free survival and median overall survival data is shown (N=19).

[0165] Baseline and 24 h post-elraglusib plasma concentrations of cytokines, chemokines, and growth factors were plotted against PFS and OS and simple linear regressions were used to calculate significance. A heatmap was then used to visualizelinear regression values (FIG.6B). We found that baseline concentrations of several analytes (IL-12, CXCL11, Fas Ligand, IL-8, VEGF, IL-1 β, M-CSF, IL-2) correlated with progression-free survival (PFS). Likewise, concentrations of several analytes (IL-12, IL- 1 β, IL-21, IL-8, IFN-α, IFN-γ, M-CSF, CCL4, Fas Ligand, IL-2, IL-10, CCL11, IL-15, IL- 4, Granzyme B, CXCL11) 24 h post dose also correlated with PFS. We next analyzed overall survival (OS) data and noted that baseline concentrations of several analytes (IL-8, CXCL11, CCL11, IFN-α, TNF-α, Fas Ligand, TRAIL R2, IL-1 β) correlated with OS. Next, 24 h post-dose concentrations of several analytes (IFN-α, Fas Ligand, TRAIL R2, CCL11) also correlated with OS. Importantly, Fas Ligand and CCL11 were the only two analytes that were statistically significant in all four comparisons (pre-dose, post- dose, OS, PFS) and were found to be positivity correlated with improved PFS and OS.

[0166] FIG.6A-6D illustrate how patient plasma concentrations of cytokines correlate with progression-free survival (PFS), overall survival (OS), and in vivo response to therapy results. Plasma samples from human patients with refractory solid tumors of multiple tissue origins enrolled in a Phase 1 clinical trial investigating a novel GSK-3 inhibitor elraglusib (NCT03678883) were analyzed using a Luminex 200 (N = 19). (FIG.6A) Table summarizing patient demographics. (FIG.6B) Baseline and 24-h post-elraglusib plasma concentrations of cytokines, chemokines, and growth factors were plotted against PFS and OS. Simple linear regressions were used to calculate significance. p values less than 0.05 were reported as statistically significant. (FIG.6C) Cytokines grouped by function. Fold-change is shown where green indicates a negative (<0) fold-change compared to the baseline (pre-dose) value and red indicates a positive (>0) fold-change. (FIG.6D) Table comparing murine and human circulating biomarker trends. Red boxes indicate that an analyte concentration positively correlated with response to therapy / PFS / OS while green boxes indicate that an analyte concentration negatively correlated with response to therapy / PFS / OS.

[0167] Many of the analytes were upregulated at 8 and 24 h after treatment as compared to baseline (FIG.6C). When the cytokines, chemokines, and growth factors were grouped by timepoint and raw values were visualized with a heatmap, we noticed several interesting trends (FIG.15A). When grouped by primary tumor location (appendix, adult T cell leukemia / lymphoma [ATLL], cholangiocarcinoma, colorectal,desmoid, hepatocellular carcinoma [HCC], leiomyosarcoma, non-small cell lung cancer [NSCLC], pancreatic), we noted that the patient with a desmoid tumor had elevated expression of many of the analytes included in the panel. When cytokines were grouped by elraglusib dose (1, 2, 3.3, 5, 7, 9.3, 12.37) in milligrams per kilogram, we noted that patients receiving a 7 mg / kg dose had increased expression of many of the analytes included in the panel at both the 8- and 24-h post-dose timepoints (FIG.15B). Finally, when cytokines were grouped by cytokine, chemokine, or growth factor family we noted that TNF family molecules (BAFF, Fas Ligand, Fas, TNF-α, TRAIL R2, TRAIL R3, TRAIL, TRANCE) has a decreased expression at the 8-h post-dose timepoint as compared to baseline and had increased over baseline levels by the 24-h timepoint (FIG.15C).

[0168] FIG.15A-15C illustrate that a multiplex immunoassay reveals dynamic cytokine changes 8- and 24-hours post-elraglusib dose. Plasma samples from human patients with refractory solid tumors of multiple tissue origins enrolled in a Phase 1 clinical trial investigating GSK-3 inhibitor elraglusib (NCT03678883) were analyzed using a Luminex 200 (N=19). (FIG.15A) Raw analyte values are grouped by timepoint: baseline (pre-dose), 8-hour post-PK, and 24-hour post-PK and categorized by primary tumor location: appendix, adult T cell leukemia / lymphoma (ATLL), cholangiocarcinoma, colorectal, desmoid, hepatocellular carcinoma (HCC), leiomyosarcoma, non-small cell lung cancer (NSCLC), and pancreatic. The scale bar is in pg / mL. (FIG.15B) Raw analyte values are grouped by timepoint: 8-hour post-PK and 24-hour post-PK and categorized by elraglusib dose: 1, 2, 3.3, 5, 7, 9.3, or 12.37 mg / kg. (FIG.15C) Fold change heatmaps grouped by cytokine family.8- and 24-hours post-PK were compared to baseline (pre-PK) analyte values. Although colors can be (depending on where viewed) in greyscale, in FIG.15C, green indicates downregulation and red indicates upregulation.

[0169] To compare both murine and human circulating biomarker trends, we created a table to visualize major trends (FIG.6D). EOS analyte concentrations that positively correlated with OS in the mouse model included IL-1 β, CCL22, CCL4, TWEAK, GM-CSF, and IL-6. Those that negatively correlated with OS in the mouse model included CCL21, VEGFR2, CCL12, BAFF, and VEGF. Interestingly, we observedthat many of these trends held when analyzing the human data. IL-1 β, CCL22, and CCL4 all were positively correlated with PFS and OS in the human cohort; likewise, BAFF and VEGF were negatively correlated with OS and PFS. GM-CSF and IL-6 had opposing correlations in the human cohort as compared to the murine cohort.

[0170] 2.10. PanCK+ Expression of Immunosuppressive CD39 Negatively Correlated with Time-on-Treatment (Tx time) While CD45+ Expression of Monocyte / Macrophage Marker CD163 Positively Correlated with Tx Time: to gain insights into the human TME post-elraglusib, we utilized GeoMx Digital Spatial Profiling (DSP) technology to profile the expression of 59 proteins in tumor biopsies (N = 12) from patients treated with elraglusib (N = 7). A total of 42% (N = 5) of the tumor biopsies analyzed were collected near or before treatment start (pre-treatment) and 58% (N = 7) of the biopsies analyzed were collected from post-treatment (average time-on-treatment [Tx time] at post-treatment biopsy: 270 days) (FIG.7A). Primary tumor types included CRC (N = 4, 33%) and pancreatic cancer (N = 8, 67%), while metastatic biopsy tissue sites included lung (N = 2, 17%), liver (N = 7, 58%), rectum (N = 2, 17%), and pleura (N = 1, 8%). We analyzed five paired tumor biopsies (N = 10 slides total, 83%), and two unpaired biopsies (N = 2 slides total, 17%). Half (N = 6, 50%) of the tumor sections were needle biopsies (FIG.16) and half (N = 6, 50%) were tissue biopsies (FIG.17). All patients included in this analysis were considered responders based on the definition used in the Phase 1 trial that treatment response is equal to disease control greater than 16 weeks. Our region of interest (ROI) selection strategy focused on mixed tumor and immune cell segments within FFPE tissue. ROIs were segmented based on panCK+ and CD45+ morphology stains to compare tumor versus immune cells protein expression (FIG.7B, FIG.18A-18B). We utilized a PCA plot to visualize dimensionality reduction and, as expected, samples tended to cluster by tissue type (liver, lung, pleura, rectum) and further separated by segment (CD45, panCK) on PC2 (FIG.19A, FIG.19B). We utilized a Sankey diagram to visualize the study design, where the width of a cord in the figure represents how many segments are in common between the two annotations they connect (FIG.7C). This visualization is useful for showing the flow of samples and how they relate to the biological annotations. For example, approximately half of the ROIs from the entire experiment were CD45+ and the other half werepanCK+. As expected, samples tended to cluster together based on patient ID, primary tumor location, biopsy timepoint, metastatic biopsy tissue site, immune cell location, or segment (CD45, panCK) type when visualized on an aggregate heatmap (FIG.7D). As we were interested in the ability to predict a patient’s time-on-treatment (Tx time), we sought to correlate pre-treatment protein expression levels among the responders with Tx time data and found that PanCK+ segment expression of immunosuppressive CD39 negatively correlated with Tx time (FIG.7E), while CD45+ segment monocyte / macrophage marker CD163 expression positively correlated with Tx time (FIG.7F)48.

[0171] FIG.18A-18B provide images illustrating ROI selection and segmentation strategy using PanCK and CD45 markers. (FIG.18A) Representative image of whole tissue scan used for ROI identification. ROIs are outlined in red circles. (FIG.18B) Individual ROIs are ordered by number. Fluorescent channel settings: FITC / 525nm / SYTO 13 / DNA (Blue), Cy3 / 568nm / Alexa 532 / PanCK (Green), and Texas Red / 615nm / Alexa 594 / CD45 (Red). A yellow mask was used for PanCK+ tumor cell identification and a teal mask was used for CD45+ hematopoietic cell identification.

[0172] FIG.7A-7H illustrate spatial profiling of patient tumor biopsies reveals a more immunostimulatory tumor microenvironment after elraglusib treatment. Patient samples were analyzed using NanoString GeoMx Digital Spatial Profiling (DSP) technology (N = 12 biopsies). (FIG.7A) Pie charts showing biopsy timepoint, primary tumor type, metastatic biopsy tissue type, and paired / unpaired biopsy sample information breakdowns. (FIG.7B) A representative region of interest (ROI) showing PanCK+ and CD45+ masking. Green indicates CK, red indicates CD45, and blue indicates DAPI staining. (FIG.7C) A Sankey diagram was used to visualize the study design where the width of a cord in the figure represents how many segments are in common between the two annotations they connect. The scale bar represents 50 segments. Blue cords represent CD45+ segments and yellow cords represent panCK+ segments. (FIG.7D) Heatmap of all areas of interest (AOIs). Patient IDs, immune cell locations, biopsy timepoint, biopsy tissue, primary tumor location, and segment identity information are color coded as indicated in the legend. (FIG.7E) PanCK+ ROI CD39 expression plotted against time-on-study (TOS). Points are color-coded by time onstudy (TOS) / time on treatment with darker blue points indicating a shorter TOS or time on treatment. (FIG.7F) CD45+ ROI CD163 expression plotted against TOS. (FIG.7G) Volcano plot showing a comparison of CD45+ region protein expression in post- treatment biopsies and pre-treatment biopsies regardless of timepoint. Grey points are non-significant (NS), blue points have p values <0.05, and red points have false discovery rate (FDR) values less than 0.05. The size of the point represents the log2 UQ Signal-to-noise ratio (SNR). (FIG.7H) Volcano plot showing a comparison of tumor- infiltrating CD45+ immune cell segment protein expression in pre- versus post-treatment biopsies. Grey points are non-significant (NS), blue points have p values <0.05, and red points have false discovery rate (FDR) values less than 0.05. The size of the point represents the log2 UQ Signal-to-noise ratio (SNR).

[0173] 2.11. Tumor-Infiltrating Immune Cells Have Reduced Inhibitory Checkpoint Expression and Elevated Expression of T Cell Activation Markers after Elraglusib Treatment: when comparing all samples, CD45+ regions of post-treatment biopsies had increased protein expression of T cell activation marker OX40L (p = 0.016) and decreased protein expression of checkpoint molecules VISTA (p = 2.0 × 10−24), PD-L1 (p = 3.2 × 10−13), PD-L2 (p = 2.0 × 10−9), LAG3 (p = 5.1 × 10−4), and PD−1 (p = 5.6 × 10−9). CD45+ regions of post-treatment biopsies also had a decreased protein expression of myeloid / neutrophil marker CD66b (p = 7.5 × 10−15), myeloid markers IDO1 (p = 4.8 × 10−6), CD80 (p = 5.4 × 10−6), and CD11b (p = 6.7 × 10−3), TAM / M2 macrophage marker CD68 (p = 3.8 × 10−4), myeloid / T cell activation marker OX40L (p = 0.016), myeloid marker CD40 (p = 0.020), and DC / myeloid marker CD11c (p = 0.022) as compared to pre-treatment samples (FIG.7G). Because we were interested in the differential expression of proteins based on immune cell location in relation to the tumor, we annotated CD45+ ROI locations as tumor-infiltrating, tumor-adjacent, or normal tissue (FIG.20A, FIG.20B, FIG.20C). When comparing tumor-infiltrating CD45+ immune cell segments in pre- versus post-treatment biopsies, it was found that post- treatment tumor-infiltrating CD45+ immune cell segments had a reduced protein expression of immune checkpoints VISTA (p = 1.6 × 10−14), PD-L1 (p = 1.1 × 10−6), PD- L2 (p = 7.6 × 10−4), and PD−1 (p = 1.6 × 10−3) and elevated protein expression of T cell activation markers CTLA4 (p = 3.1 × 10−5) and OX40L (p = 1.6 × 10−3) (FIG.7H). Wealso noted that post-treatment tumor-infiltrating CD45+ immune cell segments had a reduced protein expression of myeloid marker CD66b (p = 1.4 × 10−13), antigen PTEN (p = 1.5 × 10−11), hematopoietic marker CD34 (p = 3.3 × 10−9), T cell activation marker CD44 (p = 3.1 × 10−7), antigen presentation B2M (p = 2.5 × 10−6), immune cell activation marker HLA-DR (p = 3.1 × 10−5), TAM / M2 macrophage marker ARG1 (p = 7.6 × 10−5), memory T cell marker CD45RO (p = 1.1 × 10−4), proliferation marker Ki−67 (p = 2.7 × 10−4), TAM / M2 macrophage marker CD68 (p = 5.8 × 10−4), myeloid marker IDO1 (p = 6.6 × 10−4), myeloid marker CD80 (p = 2.4 × 10−3), NK cell marker CD56 (p = 6.9 × 10−3), DC / myeloid marker CD11c (p = 9.5 × 10−3), and T cell activation marker GITR (p = 0.021) and had an elevated protein expression of immune checkpoint molecule B7-H3 (p = 0.012) and Treg marker CD127 (p = 0.012) as compared to pre-treatment tumor- infiltrating CD45+ immune cell segments.

[0174] 2.12. Patients with a Long Time-on-Treatment Have Decreased B Cell and Myeloid Marker Expression in Immune Cell Regions and Have Decreased Immune Checkpoint Expression in Tumor Cell Regions: next, we sought to compare pre- treatment biopsy protein expression in CD45+ segments between patients who were undergoing treatment for a longer duration of time, called “Long Tx patients”, and patients who participated in the study for a shorter duration of time, called “Short Tx patients”, and observed that Long Tx patients had a reduced protein expression of B cell marker CD20 (p = 0.012) and myeloid activation marker CD80 (p = 0.047) (FIG.19C). Long Tx was defined as a Tx time greater than 275 days (~39 weeks). Then, we compared protein expression in CD45+ segments in post-treatment biopsies between Long Tx patients and Short Tx patients. We observed that Long Tx patients had reduced protein expression of antigen NY-ESO-1 (p = 0.021) and progesterone receptor (PR) (p = 0.022) (FIG.19D). We then compared pre-treatment biopsy protein expression in PanCK+ segments between Long Tx patients and Short Tx patients and observed that Long Tx patients had reduced protein expression of cytotoxic T cell marker CD8 (p = 3.5 × 10−3), antigen Her2 (p = 0.033), Treg marker Foxp3 (p = 0.033), T cell marker CD3 (p = 0.035), and B cell marker CD20 (p = 0.046). Long Tx patients also had reduced immune checkpoint protein expression of LAG3 (p = 0.023), PD-L2 (p = 0.028), and PD-1 (p = 0.046) (FIG.19E). We carried out the same analysis with afocus on panCK+ segments in post-treatment biopsies. Long Tx patients had reduced protein expression of mature B cell / DC marker CD35 (p = 8.5 × 10−3), antigen NY-ESO- 1 (p = 8.7 × 10−3), antigen Her2 (p = 0.022), antigen MART1 (p = 0.029), cytotoxic T cell marker CD8 (p = 0.030), Treg marker Foxp3 (p = 0.030), antigen PTEN (p = 0.032), DC / myeloid marker CD11c (p = 0.034), memory T cell marker CD45RO (p = 0.036), checkpoint PD-L1 (p = 0.047), and PR (p = 0.049) as compared to Short Tx patients (FIG.19F). Several additional comparisons were made between pre- and post- treatment biopsies, immune cell location in proximity to the tumor, and paired and unpaired biopsies (FIGs.21A-21N).

[0175] FIG.21A-21N show data for differential expression analyses of protein expression in human tumor biopsies pre- and post-elraglusib treatment. (FIG.21A) Volcano plot showing differential protein expression in PanCK+ regions between pre- (N=5) and post-treatment (N=7) biopsies in paired samples. (FIG.21B) Volcano plot showing differential protein expression in CD45+ regions between pre- and post- treatment biopsies in paired samples. (FIG.21C) Volcano plot showing differential protein expression between tumor-adjacent CD45+ segments and tumor-infiltrating CD45+ segments in pre-treatment biopsies. (FIG.21D) Volcano plot showing differential protein expression between tumor-adjacent CD45+ segments and tumor-infiltrating CD45+ segments in post-treatment biopsies. (FIG.21E) Volcano plot showing differential protein expression between CD45+ regions of pre-treatment and post- treatment biopsies. (FIG.21F) Volcano plot showing differential protein expression panCK+ regions of pre-treatment and post-treatment biopsies. (FIG.21G) Volcano plot showing differential post-treatment protein expression in tumor-adjacent CD45+ immune cell segments in Long Tx patients as compared to Short Tx patients. (FIG.21H) Volcano plot showing differential post-treatment protein expression in CD45+ immune cell segments located in normal (non-tumor) tissue in Long Tx patients as compared to Short Tx patients. (FIG.21I) Volcano plot showing differential pre-treatment protein expression in CD45+ immune cell segments located in normal (non-tumor) tissue as compared to tumor-adjacent tissue. (FIG.21J) Volcano plot showing differential post- treatment protein expression in CD45+ immune cell segments located in normal (non- tumor) tissue as compared to tumor-adjacent tissue. (FIG.21K) Volcano plot showingdifferential pre-treatment protein expression in CD45+ immune cell segments located in normal (non-tumor) tissue as compared to those in tumor tissue. (FIG.21L) Volcano plot showing differential post-treatment protein expression in CD45+ immune cell segments located in normal (non-tumor) tissue as compared to those in tumor tissue. (FIG.21M) Volcano plot showing a comparison of tumor-adjacent CD45+ segments with tumor-infiltrating CD45+ segments in all biopsies regardless of timepoint. (FIG.21N) Volcano plot showing a comparison of post-treatment protein expression of tumor- infiltrating CD45+ immune cell segments in Long Tx patients as compared to Short Tx patients. Grey points are non-significant (NS), blue points have p values < 0.05, and red points have false discovery rate (FDR) values less than 0.05. The size of the point represents the log2 UQ Signal-to-noise ratio (SNR).

[0176] 3. Discussion: ICB is a promising treatment strategy for many cancer patients, including MSI-H CRC patients. However, the response rate to ICB in MSS CRC patients is very limited, especially as the tumor stage advances, thus, there is a clear need for improved treatment strategies for this patient population. Evaluating the combination of ICB with small molecules in oncology represents one of the ways we might improve the efficacy of ICB in MSS CRC patients. Here, we focus on the small- molecule inhibitor of GSK-3 elraglusib and characterize several immunomodulatory mechanisms that provide a clinical rationale for the combination of GSK-3 inhibitors such as elraglusib in combination with ICB.

[0177] We demonstrate that the small-molecule inhibition of GSK-3 using elraglusib leads to increased natural killer and T-cell-mediated CRC cell killing in a co- culture model. Moreover, elraglusib acts on tumor cells to sensitize them to immune- cell-mediated killing. This tumor cell sensitization could be resultant of drug-induced modifications in the tumor cell secretome, such as decreased VEGF expression, decreased soluble PD-L1, and increased CXCL14, as we previously described44,49. VEGF has been shown to inhibit T cell activation50, while CXCL14 is a known NK cell chemoattractant51. It has been shown that the soluble or shed version of PD-L1 can retain the ability to bind PD-1 and function as a decoy receptor to negatively regulate T cell function, despite being a truncated version lacking the membrane domain of the protein52. Therefore, the increase in efficacy, in combination with ICB, that we observedin the murine model could be due to the concomitant downregulation of sPD-L1 and upregulation of cell-surface-expressed PD-L1.

[0178] Elraglusib-mediated immunostimulation may also function, in part, by inducing pyroptosis in cancer cells. Pyroptosis is a lytic and pro-inflammatory type of programmed cell death that results in cell swelling and membrane perforation. Although the role of pyroptosis in cancer is controversial, it has been suggested that pyroptosis may contribute to anti-tumor immunity53. Since we observed gasdermin B expression post-IFN-γ treatment in CRC cells, and because we found that elraglusib treatment upregulated immune cell IFN-γ secretion, we hypothesize that the IFN-γ released from CD8+ T cells and NK cells is responsible for triggering pyroptosis, which may contribute to elraglusib-mediated immunostimulation. Future experiments will seek to further characterize the role of pyroptosis in the co-culture system after treatment with elraglusib.

[0179] Another mechanism behind elraglusib-mediated immunomodulation is the suppression of inflammatory NF-κB signaling and survival pathways in the tumor cells. We demonstrated that elraglusib treatment of CRC cells decreased Survivin, NF-κB p65, Bcl-2, and Mcl-1expression while increasing PD-L1 expression. This is in accordance with previous studies that have shown that GSK-3 is a positive regulator of NF-κB54. Microarray data showed an increased expression of antiproliferative, proapoptotic, and NF-κB regulator genes and decreased expression of genes involved in cell cycle progression, antiapoptotic, and EMT genes in CRC cell lines. Multiplex immunoassay data showed a decreased tumor cell secretion of proteins involved in angiogenesis, EMT, and immunosuppression.

[0180] Meanwhile, we observed the opposite effect on NF-κB signaling in immune cells, where we observed that drug treatment increased NF-κB-inducing kinase (NIK) expression. NIK is the upstream kinase that regulates the activation of the non- canonical NF-κB signaling pathway and may suggest a role for non-canonical NF-κB signaling in immune cells after elraglusib treatment, which future studies could evaluate. It is known that the increased expression of NIK leads to an enhanced expression of chemokines and cytokines such as CCL3, TNF-α, and MCP-1, thus leading to the increased recruitment and proliferation of cytotoxic immune cells55. Moreover, thetreatment of immune cells with elraglusib increased effector molecule secretion and increased expression of genes involved in cytotoxic granule exocytosis, cellular proliferation, and the modulation of NF-κB activity. Moreover, elraglusib treatment resulted in the decreased gene expression of proapoptotic molecules and regulators of TGFβ signaling which may also contribute to the tumor-suppressive and anti-angiogenic effects of elraglusib that have been previously described56.

[0181] In a syngeneic murine colon carcinoma BALB / c murine model using MSS cell line CT-26, we observed significantly improved survival in mice treated with elraglusib and anti-PD-L1 therapy. We also demonstrated increased survival in mice treated with elraglusib alone as compared to the control group. We also observed statistically significant improved survival in the anti-PD-1 and anti-PD-L1 alone groups as compared to the control. Responders had reduced percentages of splenic CD4+ T cells and splenic CD8+ T cells and increased percentages of CD69+ activated T cells and Foxp3+ Tregs. The increased splenic percentages of both activated and end-stage T cells in the responder groups could be indicative of an anti-tumor immune response that was mounted earlier in the course of treatment. Future studies could analyze the changes in these immune cell populations during the course of therapy in greater detail, especially as we could have missed important changes in immune cell subtypes due to the limited timepoints. Compared to non-responders, responders also had more CD3+ and CD4+ tumor-infiltrating lymphocytes. Further studies could evaluate the contribution of CD4+ versus CD8+ tumor-infiltrating T cells to the observed response to elraglusib and anti-PD-L1 therapy, especially considering the recent interest in the contribution of CD4+ helper T cells to anti-tumor immunity57. We did not observe many significant differences in splenic NK cell subpopulations in either the tumor or the spleen, although perhaps the timepoint we chose to analyze was not representative of NK cell subpopulation changes that may have occurred earlier or later in the course of treatment. One limitation of this model is that it is a heterotopic flank tumor model as opposed to an orthotopic colon tumor model, which may be more representative of the CRC TME. Follow-up experiments could examine the contribution of CD4+ T cells, CD8+ T cells, and NK cells to response to therapy in the murine MSS CRC model by blocking the function of each cell population in individual experiments.

[0182] We observed that murine responders had reduced serum concentrations of BAFF, CCL7, CCL12, VEGF, VEGFR2, and CCL21. BAFF is a cytokine that belongs to the TNF ligand superfamily, which may promote tumorigenesis indirectly through the induction of inflammation in the TME and directly through the induction of EMT58. Meanwhile, CCL7 has been shown to enhance both cancer progression and metastasis via EMT, including in CRC cells59. Similarly, others have demonstrated that CXCR4 plays a critical role in the promotion of the progression of inflammatory CRC60. It is commonly known that the expression of VEGF-1 in CRC is associated with disease localization, stage, and long-term survival61. We previously observed the suppression of VEGF in a panel of CRC cell lines post-elraglusib treatment and saw a similar suppression of VEGF in the murine responders. Moreover, we noted a decrease in VEGFR2 in murine responders, a protein that is highly expressed in CRC and promotes angiogenesis62. Finally, CCL21 has been shown to play a role in colon cancer metastasis63. Since many of the downregulated analytes in responders play a role in EMT, future studies of elraglusib could include metastatic CRC models.

[0183] We observed that responders had elevated serum concentrations of CCL4, TWEAK, GM-CSF, CCL22, and IL-12p70 as compared to non-responders. Others have demonstrated that CCL4 is an important chemokine in the TME, determining response to ICB, and that a lack of CCL4 can lead to the absence of CD103+ dendritic cells (DCs)64. DCs are an important cell population influencing the response to ICB, and although we did not monitor their levels in this study, it is conceivable that they played a role in influencing response to therapy. For this reason, further studies could monitor DC populations during the course of therapy. TWEAK is commonly expressed by peripheral blood monocytes and upregulates its expression after exposure to IFN-γ65. TWEAK has also been shown to promote the nuclear translocation of both classical and alternative NF-κB pathway subunits66. GM-CSF is a well-known immunomodulatory factor that has immunostimulatory functions but is also predictive of poor prognosis in CRC67. Finally, we observed increased levels of IL-12p70 in murine responders. IL-12 is a potent, pro-inflammatory cytokine that has been shown to increase the activation and cytotoxicity of both T and NK cells as well as to inhibit immunosuppressive cells such as TAMs68and myeloid-derived suppressor cells(MDSCs)69. We demonstrated that GSK-3 inhibitors such as elraglusib represent a possible combination strategy to increase the efficacy of ICB in patients with MSS CRC. The elraglusib-mediated increase in tumor surface-cell-expressed PD-L1 presumably makes this an ideal small molecule to combine with anti-PD-L1 therapies. As this study was concerned solely with CRC, future studies could evaluate the combination of GSK- 3 inhibitors with ICB in other malignancies of interest, such as pancreatic cancer.

[0184] The cytokine analysis of plasma samples from patients with refractory solid tumors of multiple tissue origins enrolled in a Phase 1 clinical trial investigating elraglusib (NCT03678883) revealed that elevated baseline plasma levels of proteins such as IL-1 β and reduced levels of proteins such as VEGF correlated with improved PFS and OS. PFS was also found to be positively correlated with elevated plasma levels of immunostimulatory analytes such as Granzyme B, IFN-γ, and IL-2 at 24 h after elraglusib treatment. Several of these secreted proteins correlated with results from the in vivo study, where the expression of proteins such as IL-1 β, CCL22, CCL4, and TWEAK was positively correlated with improved response to therapy while the expression of proteins such as BAFF and VEGF negatively correlated with response to therapy70. These results introduce novel circulating biomarkers for correlations with response to therapy, which could provide significant clinical utility.

[0185] DSP analysis of paired FFPE tumor biopsies from patients with CRC or pancreatic cancer before and after treatment revealed that CD39 expression in PanCK+ segments was negatively correlated with duration of treatment, while CD163 expression in CD45+ segments was positively correlated with duration of treatment and potential therapeutic benefit. It is known that CD39 can inhibit costimulatory signaling, increase immunosuppression during T cell priming, and its expression is associated with TAMs, Tregs, and inhibited cytotoxic immune cell function71. CD39 has been shown to suppress pyroptosis, impair immunogenic cell death, and CD39 expression on endothelial cells regulates the migration of immune cells and promotes angiogenesis71. Moreover, CD163 is a marker of cells from the monocyte / macrophage lineage therefore future studies could evaluate the impact of monocyte / macrophages on response to elraglusib. We also noted that immune cell segments showed differential protein expression based on the proximity to the tumor where tumor-infiltrating immune cellshad decreased expression of immune checkpoints (PD-L1, Tim-3, PD-1) and Treg markers (CD25, CD127) as compared to tumor-adjacent immune cells, regardless of timepoint. While the downregulation of immune checkpoint proteins PD-1, TIGIT, and LAG-3 by elraglusib has been previously described in melanoma models72, our findings regarding VISTA and PD-L2 have not yet been reported. Interestingly, we observed a downregulation of PD-L1 expression in PanCK+ segments at the post-treatment biopsy as compared to the pre-treatment biopsies. This was unexpected as we, and others, have shown that GSK-3 inhibition upregulates tumor cell PD-L1 expression26,73. The observed decrease in PanCK+ PD-L1 expression may be a result of the late timepoint of the post-treatment biopsies, as the average time-on-study at the post-treatment timepoint was 270 days. These novel observations regarding emerging immune checkpoint inhibitors should be included in future correlative studies regarding GSK-3 inhibition.

[0186] When we analyzed differential protein expression between Long Tx patients and Short Tx patients, we found that Long Tx patients had a reduced post- treatment expression of mature B cell / DC marker CD35, antigen NY-ESO-1, antigen Her2, antigen MART1, cytotoxic T cell marker CD8, Treg marker Foxp3, antigen PTEN, DC / myeloid marker CD11c, memory T cell marker CD45RO, checkpoint PD-L1, and PR in PanCK+ segments as compared to Short Tx patients, which introduces several novel potential biomarkers of response to GSK-3 therapy, which should be validated in further studies. Moreover, when we compared post-treatment protein expression in tumor- infiltrating CD45+ immune cell segments in Long Tx patients and Short Tx patients, we found that Long Tx patients had a decreased expression of antigens NY-ESO-1, PTEN, and PR as compared to Short Tx patients. Interestingly, these three antigens (NY-ESO- 1, PTEN, and PR) had decreased expression in Long Tx patients post-treatment, regardless of tumor or immune cell region.

[0187] There are several potential limitations of this study. One such limitation is that we tested the combination of elraglusib and ICB therapy in a mouse model using only one MSS CRC cell line. Future studies could determine how other MSS CRC cell lines, and perhaps MSI-H cell lines, will respond to this combination treatment. We also had sample size limitations for the number of mice that were included in each treatmentgroup at each flow cytometry timepoint throughout the course of the study, due to the feasibility of the mouse work. Future studies could include larger numbers of mice per flow cytometry timepoint or include a comparison of both male and female mice to determine if there are any sex-specific effects. Furthermore, given access to an expanded cohort of tumor biopsies from patients treated with elraglusib, it would be interesting to analyze pre-treatment biopsies between responders and non-responders using DSP technology to aid in identifying predictive biomarkers.

[0188] This work demonstrates that the small-molecule inhibition of GSK-3 using elraglusib may be a potential means to increase the efficacy of ICB and improve response in patients with MSS CRC, and possibly other tumor types. These findings support further studies and the clinical development of elraglusib in combination with ICB, anti-PD-L1 therapy in particular. Moreover, this study, to our knowledge, represents the first digital spatial analysis of tumor biopsies from patients treated with elraglusib and very few oncology drugs have been evaluated using GeoMx technology to date. The novel circulating biomarkers of response to GSK-3 inhibition identified using the cytokine profiling data could provide significant clinical utility and the spatial proteomics data provide us with novel insights into the immunomodulatory mechanisms of GSK-3 inhibition.

[0189] In some embodiments, the technology herein solves the problem that: MSS CRC represents about 85% of the total patient population, but checkpoint blockade therapy does not effectively work for MSS CRC. The results herein introduce novel biomarkers for correlations with response to therapy which could provide significant clinical utility and suggest that elraglusib, and other GSK-3 inhibitors, should be further evaluated in combination with immune checkpoint blockade.

[0190] According to some aspects, the technology disclosed herein provides a pharmaceutical composition comprising (a) and (b): (a) a small molecule GSK-3 inhibitor, or a pharmaceutically acceptable salt and / or solvate or hydrate thereof; and (b) a therapeutic agent effective for an immune checkpoint blockade (ICB) therapy; or a pharmaceutically acceptable salt and / or solvate or hydrate thereof; wherein the pharmaceutical composition is included in a simultaneous formulation comprising both (a) and (b); or included in two separate pharmaceutical formulations with the firstformulation comprising (a) and the second formulation comprising (b), configured for an initial administration of (a) later followed by an administration of (b).

[0191] According to some aspects, the pharmaceutical composition can be wherein (a) above is comprising a compound of formula 1 or a pharmaceutically acceptable salt and / or solvate or hydrate thereof: (formula 1, or elraglusib, or salt / hydrate / solvate).

[0192] According to some aspects, an example of an acceptable hydrochloride salt is shown below: [(3-(5-Fluoro-1-benzofuran-3-1,3-dioxa-5-aza-s-indacen-7- yl)-3-pyrrolin-1-ium-2,5-dione—chloride(1-) (1 / 1)]

[0193] In any of the embodiments herein, an example of a hydrate for implementation, with hydrogen bonding illustrated by two dots, is shown below:

[0194] In some embodiments, the pharmaceutical compositions can be configured wherein (b) above includes an immune checkpoint inhibitor comprising an anti-PD-1 and / or anti-PD-L1 therapeutic agent, Pembrolizumab (Keytruda), Nivolumab (Opdivo), Cemiplimab (Libtayo), Atezolizumab (Tecentriq), Avelumab (Bavencio), Durvalumab (Imfinzi), a CTLA-4 inhibitor, Ipilimumab (Yervoy), tremelimumab (Imjuno), a LAG-3 inhibitor, Relatlimab, or a combination thereof.

[0195] According to some aspects, the pharmaceutical composition is provided wherein the first formulation is configured for an initial administration to a subject in need thereof; and the second formulation is configured for an administration to the subject within a period of time of less than one year after the initial administration.

[0196] In some embodiments, the pharmaceutical compositions disclosed above or herein can be further comprising a therapeutic agent or method (c) either in place of (b) or in addition to (b); wherein (c) is comprising an agent effective for a combination therapy, a targeted therapy, a chemotherapy, a treatment modality that affects a tumor microenvironment, an application of an electromagnetic radiation / radiation, or a combination thereof.

[0197] According to some aspects, the pharmaceutical compositions are provided wherein the therapeutic agent in (b) is determined after an administration of (a) by one or more changes in biomarkers observed from examining changes in a tumor microenvironment subsequent to the administration of (a).

[0198] In some embodiments, the pharmaceutical compositions disclosed herein can be provided wherein the first formulation is configured for a first route of administration in a subject in need thereof, and the second formulation is configured for a second different route of administration in the subject.

[0199] According to some aspects, a method for treating a subject diagnosed with a cancer or suspected of having a cancer is disclosed herein, the method comprising the steps of: (1) administering a therapeutically effective amount of a small molecule GSK-3 inhibitor, or a pharmaceutically acceptable salt and / or solvate or hydrate thereof; whereby a protein expression in a tumor microenvironment is changed to enhance a tumor-infiltrating immune cell activation; and (2) administering a therapeutically effect amount of an immune checkpoint inhibitor effective for treating the subject with an immune checkpoint blockade (ICB) therapy; whereby the ICB therapy is enhanced compared to the same ICB therapy, ceteris paribus, without an execution of step (1).

[0200] In some embodiments, the method disclosed above can be wherein the cancer comprises a primary tumor of colorectal cancer (CRC), pancreatic cancer, lung cancer or cancer of the pleural space, liver cancer, rectum cancer, or a combination thereof.

[0201] According to some aspects, the method is directed wherein the cancer comprises microsatellite stable (MSS) colorectal cancer (CRC). According to some aspects, the method is implemented wherein the subject has been previously diagnosed as not responding to an ICB (immune checkpoint blockade) therapy.

[0202] According to some aspects, the methods disclosed herein can be further comprising the step of: (1b) measuring one or more biomarkers derived from the cancer in the subject; wherein step (1b) is executed before step (1), after step (1), or executed repeatedly; and the execution of step (1b) is operative to provide a determination of an effectiveness of step (1). In this example, the method can be embodied wherein the determination is utilized to determine the immune checkpoint inhibitor (ICI) included in step (2), and the ICI is determined by one or more changes in biomarkers observed from examining changes in a tumor microenvironment after step (1).

[0203] In some embodiments, the method above is configured wherein the one or more biomarkers comprise one or more differentially expressed proteins.

[0204] For example, the method can be implemented wherein the one or more differentially expressed biomarkers or proteins comprise CD39; CD163 (or the antigen CD163); OX40L (CD134 or TNFRSF4); checkpoint molecules for VISTA; myeloid / neutrophil marker CD66b; myeloid markers IDO1, CD80, or CD11b; TAM / M2 macrophage marker CD68; myeloid marker CD40 or DC / myeloid marker CD11c; Nuclear factor-κB (NF-κB); NEK2 protein; T cell activation marker CTLA4; antigen PTEN; hematopoietic marker CD34; T cell activation marker CD44; antigen presentation B2M; immune cell activation marker HLA-DR; TAM / M2 macrophage marker ARG1; memory T cell marker CD45RO; proliferation marker Ki−67; NK cell marker CD56; T cell activation marker GITR; immune checkpoint molecule B7-H3; Treg marker CD127; expression of B cell marker CD20; antigen NY-ESO-1; progesterone receptor (PR); cytotoxic T cell marker CD8; antigen Her2; Treg marker Foxp3; T cell marker CD3; immune checkpoint protein expression of LAG3, PD-L2, PD-1; protein expression of mature B cell / DC marker CD35; antigen MART1; checkpoint PD-L1 and PR; immune checkpoint Tim-3; Treg marker CD25; checkpoint protein TIGIT or LAG-3; or a combination thereof.

[0205] According to some aspects, the method discussed above can be further comprising an administration of a combination therapy including an agent effective for a combination therapy, a targeted therapy, a chemotherapy, a treatment modality that affects a tumor microenvironment, an application of an electromagnetic radiation or a particle radiation, or a combination thereof.

[0206] In some embodiments, disclosed herein is a method to determine how a cancer patient will respond to an ICB therapy, the method comprising the steps of: (1) administering a small molecule GSK-3 inhibition treatment to the patient; (2) obtaining a post-treatment biopsy from the patient; and (3) measuring one or more biomarkers differentially expressed in the post-treatment biopsy; whereby the one or more biomarkers are indicative of how the patient will respond to the ICB therapy. In this example, in some embodiments, the method can be further comprising the steps of: (0) obtaining a pre-treatment biopsy from the patient; and (4) measuring the one or moredifferentially expressed proteins in the pre-treatment biopsy; whereby a comparison between a level of one or more differentially expressed proteins in the pre-treatment biopsy and in the post-treatment biopsy is enabled.

[0207] In some embodiments, the method disclosed above can be wherein a therapeutic development of a GSK-3 inhibitor is provided by the comparison of the levels of the one or more biomarkers and / or the one or more proteins.

[0208] According to some aspects, the method disclosed above can be further comprising the step of determining the ICB therapy based upon the one or more biomarkers and / or the one or move proteins. A system can be implemented using these example methods and embodiments.

[0209] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0210] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within thescope of the disclosure. The methods, kits, formulations, and devices disclosed herein can be combined in any way into systems to address the current public health emergency.

[0211] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting. The Examples are provided to demonstrate examples of future planned work, which in some experiments is emergency work. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. EXAMPLES

[0212] The invention now being generally described, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present invention and are not intended to limit the invention. EXAMPLE 1 DISCUSSION OF EXPERIMENTAL PROCEDURES

[0213] The experimental results discussed above involved the following methods and procedures.

[0214] 4. Methods; 4.1. Cell Culture Maintenance: human CRC cells SW480 (RRID: CVCL_0546), HCT-116 (RRID: CVCL_0291), HT-29 (RRID: CVCL_0320), and KM12C (RRID: CVCL_9547) were used in this study. SW480 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS and 1% Penicillin–Streptomycin HCT-116 and HT-29 were cultured in McCoy’s 5A (modified) Medium supplemented with 10% FBS and 1% Penicillin–Streptomycin. KM12C cells were cultured in Eagle’s Minimal Essential Medium supplemented with 10% FBS and 1% Penicillin–Streptomycin. Human immune cells NK-92 (RRID: CVCL_2142), TALL- 104 (RRID: CVCL_2771), and patient-derived CD8+ T cells were also used in this study. NK-92 cells were cultured in Alpha Minimum Essential medium supplemented with 2 mM L-glutamine, 1.5 g / L sodium bicarbonate, 0.2 mM inositol, 0.1 mM 2- mercaptoethanol, 0.02 mM folic acid, 12.5% horse serum, and 12.5% FBS. TALL-104 cells (CD2+; CD3+; CD7+; CD8+; CD56+; CD4−; CD16−) and patient-derived T cells(CD3 +; CD8 +) were cultured in RPMI-1640 containing 20% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. Recombinant human IL-2 (Miltenyi cat# 130-097744) with a final concentration of 100 units / mL was added to all immune cell culture media. All cell lines were incubated at 37°C in a humidified atmosphere containing 5% CO2. Cell lines were authenticated and tested to ensure the cultures were free of mycoplasma infection. FIGs.9A-9C are data supporting CRC cell lines selected represent diverse mutational backgrounds and exhibit varying elraglusib IC-50 values. Tumor cell lines (HCT-116, HT-29, SW480) and immune cell lines (NK-92, TALL-104) were treated as indicated for (FIG.9A) 24-hour or (FIG.9B) 72-hour cell viability was assessed to determine IC-50 values (N=3). (FIG.9C) Table of CRC cell lines included in the study and their diverse mutational profiles.

[0215] 4.2. Measurement of Cell Viability: cells were seeded at a density of 3 × 103cells per well in a 96-well plate (Greiner Bio-One, Monroe, NC, USA). Cell viability was assessed using the CellTiter Glo assay (Promega, Madison, WI, USA). Cells were mixed with 25 μL of CellTiter-Glo reagents in 100 μL of culture volume, and bioluminescence imaging was measured using the Xenogen IVIS imager (Caliper Life Sciences, Waltham, MA). The percent of cell viability was determined by normalizing the luminescence signal to control wells. Dose-response curves were generated, and the half-maximal inhibitory concentration (IC-50) was calculated using Graph-Pad Prism (RRID: SCR_002798) version 9.2.0. For IC50 generation, concentrations were log- transformed, and data were then normalized to control, and a log (inhibitor) versus response (three parameters) test was used.

[0216] 4.3. Pyroptosis Assay: recombinant Human TNF-α (Cat #300-01A, PeproTech, Rocky Hill, NJ, USA) and Recombinant Human IFN-γ (Cat # 300-02, Peprotech, Rocky Hill, NJ, USA) were purchased for use in western blot analysis while rhTRAIL was generated in-house74. Example Reagents are shown below: Example Reagent Table 1. Rea ent Source Identifier ConcentrationReagent Source Identifier Concentration Anti-GSDMB antibody Sigma-Aldrich Cat# HPA052407 1:1000

[0217] 4.4. Isolation of Donor-Derived CD8+ T Cells: an Easy Step Human CD8+ T Cell Isolation Kit was used to isolate CD8+ T cells from a donor PBMC sample via negative selection (Cat #, 17913, Stem Cell Technologies, Vancouver, Canada).

[0218] 4.5. Collection of Cell Culture Supernatants Used in Cytokine Measurements: cells were plated at 3.5 × 104cells in a 48-well plate (Thermo Fisher Scientific, Waltham, MA, USA) in complete medium and incubated at 37 °C with 5% CO2. At 24 h after plating, almost all the tumor cells were adherent to the bottom of the flask and the complete medium was replaced with the drug-containing medium. Subsequently, the culture supernatants were collected after 48 h of incubation and were frozen at −80 °C until the measurement of cytokines was performed. On the day of analysis, samples were thawed and centrifuged to remove cellular debris.

[0219] 4.6. Human Cytokine Profiling: human cell line culture supernatants were analyzed using an R&D systems Human Premixed Multi-Analyte Kit (R&D Systems, Inc., Minneapolis, MN, USA) and a Luminex 200 (RRID: SCR_018025) Instrument (LX200-XPON-RUO, Luminex Corporation, Austin, TX, USA) according to the manufacturer’s instructions. Sample levels of TNF-α, 4-1BB / TNFRSF9 / CD137, IL- 8 / CXCL8, Ferritin, IFN-β, IL-10, CCL2 / JE / MCP-1, VEGF, CXCL13 / BLC / BCA-1, IFN-γ, CCL20 / MIP-3 α, CCL3 / MIP-1 α, CCL22 / MDC, CCL4 / MIP-1 β, Fas Ligand / TNFSF6, IL- 17 / IL-17A, IL-2, BAFF / BLyS / TNFSF13B, GM-CSF, CXCL5 / ENA-78, TRANCE / TNFSF11 / RANK L, CXCL9 / MIG, G-CSF, IFN-γ R1 / CD119, VEGFR3 / Flt-4, C- Reactive Protein / CRP, CXCL11 / I-TAC, IL-21, CXCL14 / BRAK, IL-6, Fas / TNFRSF6 / CD95, TRAIL R3 / TNFRSF10C, IL-4, CCL5 / RANTES, PD-L1 / B7-H1, CCL7 / MCP-3 / MARC, Chitinase 3-like 1, CXCL10 / IP-10 / CRG-2, IL-1 β / IL-1F2, IL-7, Prolactin, CCL8 / MCP-2, TRAIL R2 / TNFRSF10B, M-CSF, IL-15, Granzyme B, IFN-α, TREM-1, IL-12 / IL-23 p40, TRAIL / TNFSF10, CCL11 / Eotaxin, and IL-18 / IL-1F4. Quantitative analysis with 6 standards and a minimum of 50 counts per bead region was used with the Luminex to generate analyte values reported as picograms / milliliter(pg / mL). Sample concentrations less than the lower limit of detection for each particular analyte were recoded as the lower limit value divided by 10. Sample concentrations above the upper limit of detection for a particular analyte were recoded as the upper limit of detection.

[0220] 4.7. Murine Cytokine Profiling: whole blood from mice was collected, allowed to clot, and serum was isolated using a serum separator tube (SST) according to manufacturer instructions. Murine serum samples were analyzed using an R&D systems Murine Premixed Multi-Analyte Kit (R&D Systems, Inc., Minneapolis, MN, USA) and a Luminex 200 (RRID: SCR_018025) Instrument (LX200-XPON-RUO, Luminex Corporation, Austin, TX, USA) according to the manufacturer’s instructions. Sample levels of GM-CSF, IL-7, IL-12 p70, CCL2 / JE / MCP-1, IL-1 β / IL-1F2, VEGF, IL-2, IL-4, VEGFR2 / KDR / Flk-1, IL-6, IL-10, IL-13, IFN-γ, IL-3, IL-16, CXCL10 / IP-10 / CRG-2, CCL5 / RANTES, CCL7 / MCP-3 / MARC, CCL12 / MCP-5, Prolactin, M-CSF, CCL3 / MIP-1 α, IL-1 α / IL-1F1, CCL20 / MIP-3 α, CCL4 / MIP-1 β, TWEAK / TNFSF12, CXCL12 / SDF-1 α, BAFF / BLyS / TNFSF13B, Granzyme B, CCL21 / 6Ckine, CCL11 / Eotaxin, and CCL22 / MDC. Sample values are reported in picograms per milliliter (pg / mL). Quantitative analysis with 6 standards and a minimum of 50 counts per bead region was used with the Luminex to generate analyte values reported as picograms / milliliter (pg / mL). Sample concentrations less than the lower limit of detection for each particular analyte were recoded as the lower limit value divided by 10. Sample concentrations above the upper limit of detection for a particular analyte were recoded as the upper limit of detection. Data analysis and visualization were generated using R (RRID: SCR_001905) software (R Development Core Team, 2020). When comparing responders and non-responders, a Kruskal-Wallis test was used to calculate statistical significance, followed by a Benjamini-Hochberg correction for multiple comparisons.

[0221] 4.8. GFP+ Cell Line Generation: a total of 50,000 HT-29 or HCT 116 cells were seeded in a 12-well tissue culture plate and allowed to adhere overnight. They were then transduced with lentivirus containing the plasmid pLenti_CMV_GFP_Hygro (pLenti CMV GFP Hygro (656-4) was a gift from Eric Campeau & Paul Kaufman (Addgene viral prep # 17446-LV; RRID: Addgene_17446)) at a multiplicity of infection of 10 with 8 μg / mL polybrene (hexadimethrine bromide (Cat # 107689, Sigma Aldrich, St.Louis, MO, USA) for 24 h, before washing with PBS and replacing with fresh medium

[0057] . The cells were then sorted for GFP-positivity using a BD FACSAria™ III Cell Sorter (RRID: SCR_016695).

[0222] 4.9. Multicolor Immune Cell Co-Culture Experiments: a total of 10,000 HCT-116, SW480, or HT-29 colorectal cancer cells were plated per well in a clear- bottom, black-walled 48-well tissue culture plate and were allowed to adhere overnight. Cells were subsequently treated with DMSO, 5 μM or 10 μM elraglusib, and / or 10,000 TALL-104, donor-derived CD8+ T cells, orNK-92 cells (for an effector-to-tumor ratio of 1:1) for 24 h. Immune cell monocultures were treated with DMSO, 5 μM or 10 μM elraglusib and cell viability was monitored to ensure that the concentrations of drug used were not cytotoxic to immune cells. CRC cells were labeled using CellTrackerTMGreen CMFDA (5-chloromethyl fluorescein diacetate), immune cells (NK- 92, TALL-104) were labeled using CellTracker™ Blue CMAC Dye (7-amino-4- chloromethylcoumarin), and ethidium homodimer-1 (EthD-1) was used as a marker of cell death (Invitrogen, Waltham, MA, USA).10 × images were captured using a Nikon Ti-U Inverted Fluorescence Microscope and NIS-Elements F Package imaging software 3.22.00 Build 710 (Nikon Instruments Inc., New York, NY, USA). The number of red / green color cells in random fields was determined using thresholding and particle analysis in the Fiji modification (RRID: SCR_002285) of ImageJ and expressed as a dead / live cell ratio. Normalization was carried out by subtracting the percentage of cell death due to drug or vehicle control (DMSO) from the percentage of dead cells observed in the co-culture of tumor and immune cells treated with the drug. At least 100 cells were evaluated per sample, with 3 independent replicates. Statistical analysis was carried out using GraphPad Prism 9 (RRID: SCR_002798).

[0223] 4.10. Single-Color Immune Cell Co-Culture Experiments: a total of 5000 HT-29 GFP+ or HCT 116 GFP+ cells were plated per well in a clear-bottom, black- walled 96-well tissue culture plate and were allowed to adhere overnight. Cells were subsequently treated with DMSO, 5 μM elraglusib, and / or 5000 TALL-104 or NK-92 cells (for an effector-to-tumor ratio of 1:1) for 48 h. Nine images were taken per well at 10 × magnification using a Molecular Devices ImageXpress®Confocal HT.ai High- Content Imaging System and quantified for the number of GFP+ objects using theMetaXpress (RRID: SCR_016654) software (Molecular Devices, San Jose, CA, USA). 40 × images were also taken at 24 h for representative images of cellular morphology changes. Statistical analysis was carried out using GraphPad Prism 9 (RRID: SCR_002798).

[0224] 4.11. Generation of Single-Cell Suspensions: spleens were strained, filtered, and washed while tumors were collected, washed, and digested before lymphocytes were collected using a Percoll gradient (Cat # P1644-100ML, Sigma Aldrich, St. Louis, MO, USA).

[0225] 4.12. Flow Cytometry: flow cytometry viability staining was conducted by suspending murine spleen and tumor single cell suspensions in Zombie Violet fixable viability kit (Cat # 423114, BioLegend, San Diego, CA, USA) according to manufacturer instructions for 30 min at room temperature. Staining for membrane surface proteins was conducted using conjugated primary antibodies for 1 h on ice, according to manufacturer instructions. Cells were fixed and permeabilized using the eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set according to manufacturer instructions (Cat# 00-5523-00, Invitrogen, Waltham, MA, USA). Cells were resuspended in Flow Cytometry Staining Buffer (R&D Systems, Minneapolis, MN, USA) and analyzed using a BD Biosciences LSR II (RRID: SCR_002159) and FlowJo (RRID: SCR_008520) version 10.1 (FlowJo, Ashland, OR, USA). Exemplary reagents are shown below: Example Reagent Table 2. Reagent Source Identifier Concentration Zombie Violet™ Fixable Viability Kit BioLegend 423114 1:1000 CD45 Monoclonal Antibody (30-F11), eVolve 83-0451-PE Rat Anti-Mouse CD3 Molecular Complex, BD Clone 17A2 (RUO)Biosciences555275 0.125 µg / testCD335 (NKp46) Monoclonal Antibody (29A1.4), APCeBioscience™17-3351- 820.125 µg / testAPC / Cy7 anti-mouse / human CD11b, clone: M1 / 70BioLegend 101226 0.125 µg / testCd27 Monoclonal Antibody (LG.7F9), FITC eBioscience™11-0271-82 0.5 µg / testReagent Source Identifier Concentration Klrg1 Monoclonal Antibody (2F1), PE-eBiosci25-5893- Cyanine7ence™820.25 µg / testAnti-mouse CD45, eBioscience, eVolve 605, clone: 30-F11Invitrogen83-0451- 420.5 µg / testCD4 Monoclonal Antibody (RM4-5), PE- Cyanine7Invitrogen25-0042- 820.25 µg / testPE Rat Anti-Mouse CD8a, Clone 53-6.7 BD (RUO)Biosciences553032 0.125 µg / testCD69 Monoclonal Antibody (H1.2F3), FITC eBioscience™11-0691-81 0.5 µg / testFOXP3 Monoclonal Antibody (FJK-16s), APC Manufacturer17-5773-82 1 µg / test

[0226] 4.13. Natural Killer Cell Immunophenotyping: the NK cell flow cytometry panel included the following directly conjugated primary antibodies: anti-mouse CD45, eBioscience eVolve 605 clone: 30-F11 (Ref # 83-0451-42, Invitrogen), PE anti-mouse CD3 molecular complex (17A2) (mat. #: 555275, BD biosciences), anti-mouse NKp46 APC (Ref # 17-3351-82), APC / Cy7 anti-mouse / human CD11b clone: M1 / 70 (cat# 101226, BioLegend), anti-Cd27 Monoclonal Antibody (LG.7F9) FITC (eBioscience™, Thermo Scientific, cat # 11-0271-82), and (Klrg1 Monoclonal Antibody (2F1) PE- Cyanine7 (eBioscience, Thermo Scientific, cat # 25-5893-82). Gating strategies are as follows: NK cell: live / CD45 / CD3- / NK1.1+ Mature NK cell: live / CD45 / CD3- / NK1.1+ / KRLG1+ Activated NK cell: live / CD45 / CD3- / NK1.1+ / CD11b+ NK cell subset 1: live / CD45 / CD3- / NK1.1+ / CD11b-CD27- NK cell subset 2: live / CD45 / CD3- / NK1.1+ / CD11b-CD27+ NK cell subset 3: live / CD45 / CD3- / NK1.1+ / CD11b+CD27+ NK cell subset 4: live / CD45 / CD3- / NK1.1+ / CD11b+CD27-

[0227] 4.14. T Cell Immunophenotyping: the T cell flow cytometry panel included the following directly conjugated primary antibodies: anti-mouse CD45 superbright 600 clone: 30-511 (ref# 63-0451-82, eBioscience), anti-CD3 APC-Cy7 clone 17A2(BD Biosciences, cat # 560590), eBioscience anti-mouse CD4 PE-Cy7 clone: RM4-5 (Ref # 25-0042-82, Invitrogen), PE anti-mouse CD8a (Ly-2)(53-6.7) (cat # 553032, BD), anti- mouse CD69 FITC clone: H1.2F3 (Ref# 11-0691-81, eBioscience), and Foxp3 (FJK- 16s) APC (eBioscience). Gating strategies are as follows: CD4+ T cell: live / CD45+ / CD3+ / CD4+ / Foxp3- CD8+ T cell: live / CD45+ / CD3+ / CD8+ Treg: live / CD45+ / CD3+ / CD4+ / Foxp3+ Activated CD8+ T cell: live / CD45+ / CD3+ / CD8+ / CD69+

[0228] 4.15. Western Blot Analysis: cells were plated in a 6-well plate and incubated overnight before the spent media was replaced with drugged media. Drug treatment lasted for indicated durations. Protein was extracted using radioimmunoprecipitation (RIPA) assay buffer (Cat # R0278, Sigma-Aldrich, St. Louis, MO, USA) containing cOmplete™, Mini, EDTA-free Protease Inhibitor Cocktail (Cat # 4693159001, Roche, Basel, Switzerland) from sub-confluent cells. Denaturing sample buffer was added, samples were boiled at 95 degrees for 10 min, and an equal amount of protein lysate was electrophoresed through NuPAGE™ 4 to 12%, Bis-Tris, 1.5 mm, Mini Protein Gels (Invitrogen, Waltham, MA, USA) then transferred to PVDF membranes. The PVDF membrane was blocked with 5% non-fat milk (Sigma-Aldrich, St. Louis, MO, USA) in 1 × TTBS. Primary antibodies were incubated with the transferred PVDF membrane in blocking buffer at 4 °C overnight. Secondary antibodies included Goat anti-Rabbit IgG (H + L) Secondary Antibody, HRP (Cat # 31460, Invitrogen, Waltham, MA, USA), and Goat anti-Mouse IgG (H + L) Secondary Antibody, HRP (Cat # 31430, Invitrogen, Waltham, MA, USA). The signal was detected using Pierce™ ECL Western Blotting Substrate (Cat # 32106, Thermo Scientific, Waltham, MA, USA) and a Syngene Imaging System (RRID: SCR_015770). Exemplary reagents are shown below:Example Reagent Table 3. Source Identifier ConcentrationPARP Antibody Cell Signaling Cat# 9542S 1:1000

[0229] 4.16. In Vivo Studies: the experimental in vivo protocol (Protocol # 19-01- 003) was approved by the Institutional Animal Care and Use Committee of Brown University (Providence, RI, USA). Six- to seven-week-old female BALB / c mice (RRID: IMSR_JAX:000651) were purchased from Taconic. A total of 50,000 cells were suspended in 50 µL ice-cold PBS and 50 µL Matrigel (Catalog # 354234, Corning, New York, NY, USA), and 100 uL was injected subcutaneously into the rear flanks. Once tumor volume reached at least 100 mm3, mice were randomly assigned to one of seven groups (12 mice / group): Control (isotype), elraglusib, elraglusib + Isotype, anti-PD-1, anti-PD-L1, elraglusib + anti-PD-1, and elraglusib + anti-PD-L1. All treatments were delivered by IP injection using the following dosing schedule: Isotype (70 mg / kg, twice a week), elraglusib (70 mg / kg, twice a week), anti-PD-1 (10 mg / kg, twice a week), anti- PD-L1 (10 mg / kg, twice a week). Treatment dosing was determined based on prior studies of elraglusib75and anti-PD-1 / anti-PD-L176,77. The solvent for elraglusib was DMSO and the formulation buffer for elraglusib was made using 75% PEG400, 7% Tween 80, and 18% Ethanol. The treatment continued until mice developed signs of discomfort from excessive tumor growth. Mice were weighed once a week to monitor signs of drug toxicity. The length (L) and width (W) of the masses were measured threetimes per week with a digital caliper, and the tumor volume was calculated by applying the formula: 0.5LW2. Collection of whole blood and serum was performed by cardiac puncture and sent to Antech Diagnostics GLP (Morrisville, NC, USA) for blood cell count and chemistry tests, or in-house cytokine profiling. Tumors and organs were dissected and harvested for analysis by IHC and flow cytometry.

[0230] 4.17. Immunohistochemistry: excised tissues were fixed with 10% neutral buffered formalin and paraffin-embedded. Five-micrometer tissue sections were cut with a microtome and mounted on glass microscope slides for staining. Hematoxylin and eosin staining was completed for all tumor specimens. Paraffin embedding and sectioning of slides were performed by the Brown University Molecular Pathology Core Facility. Slides were dewaxed in xylene and subsequently hydrated in ethanol at decreasing concentrations. Antigen retrieval was carried out by boiling the slides in 2.1 g citric acid (pH 6) for 10 min. Endogenous peroxidases were quenched by incubating the slides in 3% hydrogen peroxide for 5 min. After nuclear membrane permeabilization with Tris-buffered saline plus 0.1% Tween 20, slides were blocked with horse serum (Cat# MP-7401-15, Vector Laboratories, Burlingame, CA, USA), and incubated with primary antibodies overnight in a humidified chamber at 4C. After washing with PBS, a secondary antibody (Cat# MP-7401-15 or MP-7402, Vector Laboratories, Burlingame, CA, USA) was added for 30 min, followed by diaminobenzidine application (Cat# NC9276270, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. Samples were counterstained with hematoxylin, rinsed with distilled water, dehydrated in an increasing gradient of ethanol, cleared with xylene, and mounted with Cytoseal mounting medium (Thermo Fisher Scientific, catalog no.8312- 4). Images were recorded on a Zeiss Axioskop microscope (RRID: SCR_014587), using QCapture (RRID: SCR_014432). QuPath software (RRID: SCR_018257) was used to automatically count positive cells. For each IHC marker, five 20 × images per group were analyzed, and results were represented as the absolute number of positive cells per 20 × field. The signal was quantified by converting randomly sampled 20 × images into 16-bit images and then utilizing Fiji to employ MaxEntropy thresholding.Example Reagent Table 4. Reagent Source Identifier Concentration CD4 (D7D2Z) Rabbit mAb CellSignaling25229S 1:200CD8α (D4W2Z) XP®Rabbit mAb (Mouse Cell Specific)Signaling98941 1:800Anti-TRAIL antibody Abcam ab231265 20 µg / mlIHC Formulated)Signaling12202 1:800PD-1 / CD279 Polyclonal antibody Proteintech18106-1-1:1000MA5-a × KM12C) were plated in a 6-well plate and allowed to adhere overnight before 24-h treatment, as indicated. Then, 1 × 106immune cells (NK92, TALL-104) were plated and treated with elraglusib as indicated for 24 h. RNA was isolated from cell pellets in batches of 6 using an RNeasy Plus Mini Kit (Cat # 74134, Qiagen, Hilden, Germany). Acceptable RNA concentration and quality were verified with Nanodrop and Bioanalyzer measurements. GeneChip™ Human Transcriptome Array 2.0 assays were conducted according to manufacturer instructions in two batches using randomized samples to limit batch effects (Cat# 902162, Applied Biosystems, Waltham, MA, USA). Applied Biosystems Transcriptomic Analysis Console (TAC) software (RRID: SCR_016519) wasused to calculate fold changes in gene expression relative to the untreated control cells. Values were considered statistically significant for p values < 0.05.

[0232] 4.19. Single-Cell RNA Sequencing: single cells were captured and 3′ single-cell gene expression libraries were conducted (Next GEM v3.1) using the 10 × Genomics Chromium system by SingulOmics (SingulOmics, New York, NY, USA). Gene expression libraries were sequenced with ~200 million PE150 reads per sample on Illumina (RRID: SCR_016387) NovaSeq (Illumina, Inc., San Diego, CA, USA). After sequencing, clean reads were then analyzed with human reference genome GRCh38 using Cell Ranger v6.1.2 ([RRID: SCR_017344],10 × Genomics, Pleasanton, CA, USA). Data were analyzed and visualized using Loupe Browser ([RRID: SCR_018555], 10 × Genomics, Pleasanton, CA, USA).

[0233] 4.20. Digital Spatial Profiling: an Agilent Technologies hybridization oven was used for baking tissue onto slides (Agilent, Santa Clara, CA, USA). A NanoString GeoMx®Digital Spatial Profiler (DSP) instrument (NanoString, Seattle, WA, USA) was used to scan slides, identify regions of interest (ROIs), and collect photocleavable barcodes according to manufacturer instructions. A custom panel was designed to include the following proteins: Ms IgG1, Ms IgG2a, Rb IgG, GAPDH, Histone H3, S6, Beta-2-microglobulin, CD31, CD45, Ki-67, ARG1, CD11b, CD11c, CD14, CD163, CD39, CD40, CD68, HLA-DR, GZMB, CD20, CD3, CD34, CD4, CD56, CD66b, CD8, Foxp3, Fibronectin, 4-1BB, B7-H3, CTLA4, GITR, IDO1, LAG3, OX40L, STING, Tim-3, VISTA, Bcl-2, ER-α, EpCAM, Her2, MART1, NY-ESO-1, PR, PTEN, PanCk, SMA, CD127, CD25, CD27, CD44, CD45RO, CD80, ICOS, PD-1, PD-L1, and PD-L2. An Eppendorf MasterCycler Gradient Thermal Cycler was used to generate the Illumina sequencing libraries from the photocleaved tags. (Eppendorf, Hamburg, Germany). An Agilent Fragment Analyzer (RRID: SCR_019417) was used for library size distribution analysis with a high-sensitivity NGS Fragment Kit (Cat# DNF-474-0500, Agilent, Santa Clara, CA, USA). qPCR for quantification was run using an Illumina-compatible KAPA Library Quantification Kits (ROX Low) (cat# KK4873) on an Applied Biosystems ViiA 7 Real-Time qPCR / PCR Thermal Cycler System (Applied Biosystems, San Francisco, CA, USA) and was analyzed using QuantStudio software (RRID: SCR_018712). Sequencing was performed using a NextSeq 500 / 550 High Output Kit v2.5 (75 Cycles)kit (cat# 20024906) on an Illumina Sequencing NextSeq 550 System ([RRID: SCR_016381], Illumina, San Diego, CA, USA). The initial annotated dataset went through quality control (QC) to check if housekeeper genes and background (isotype) control molecules were themselves correlated with the predictors of interest. Every ROI was tested for raw sequencing reads (segments with <1000 raw reads were removed), % sequencing saturation (defined as [1-deduplicated reads / aligned reads]%, segments below ~50% were not analyzed), and nuclei count per segment (>100 nuclei per segment is generally recommended). Both immunoglobulins (IgGs) and housekeeper genes were highly correlated with one another. Signal to noise (SNR) ratio was calculated using background probes and all probes were detected above the background in at least one ROI. Finally, data were normalized based on background IgG expression and all normalization factors were well distributed. Data analysis and visualization were generated using R ([RRID: SCR_001905], R Development Core Team, 2020).

[0234] 4.21. Clinical Specimens: archival tumor specimens and peripheral blood samples were collected from patients enrolled in the Phase I study of Elraglusib (9-ING- 41), a small-molecule-selective glycogen synthase kinase-3 beta (GSK-3b) inhibitor, as monotherapy or combined with cytotoxic regimens in patients with relapsed or refractory hematologic malignancies or solid tumors (Clinicaltrials.gov NCT03678883) who received treatment at the Lifespan Cancer Institute (Providence, RI, USA). The study was conducted in accordance with the Declaration of Helsinki and the International Conference on Harmonization Good Clinical Practice guidelines. The study protocol was approved by the Institutional Review Board (IRB) of Rhode Island Hospital under protocol number 1324888-120. The patients also participated in a Lifespan Cancer Institute research protocol designed to investigate molecular and genetic features of tumors and mechanisms of resistance (Rhode Island Hospital IRB protocol number 449060-38). All patients provided written informed consent. EXAMPLE 2 PRODRUG AND PRECURSOR DEVELOPMENT

[0235] Chemical derivatives and mixtures of the elraglusib are developed as precursors (or prodrugs). The prodrug can be converted (to active) by an enzymatic activity of the host subject, a release from a carrier, action of a cancer cell, a digestionsuch as by an amylase, or can be converted by a hydrolysis, wherein a hydroxyl in the parent compound is presented as an ester or a carbonate or carboxylic acid present in the parent compound is presented as an ester. Particles (such as silica) or even a food may be used as carriers / nanocarriers of the therapeutic agent(s) for prodrug / precursor experiments. A precursor or prodrug can be metabolized to the active parent compound (therapeutic agent) in vivo (e.g., the ester is hydrolyzed to the corresponding hydroxyl, or carboxylic acid). Formulation of a precursor / prodrug is accomplished by mixing the subject elraglusib with a carrier / nanocarrier, by mixing with an acid and / or an alcohol (then testing hydrolysis at various pH), or by attaching an enzymatically cleavable functional group. The design of prodrugs requires consideration of the following: (1) can the therapeutic agent be modified, (2) what effect will the modification have on the ADMET (absorption, distribution, metabolism, excretion, and toxicity), and (3) can the parent therapeutic agent be regenerated efficiently without producing toxic by-products. Chemical modification of the proposed elraglusib requires a suitable functional group that allows reaction to form the targeted pro-moiety that bestows the desired ability for hydrolysis back to the therapeutic agent. Example groups include hydroxyls, amines, carboxylic acids, esters, thiols, and carbonyls. The purpose of the targeted promoiety generally dictates its targeted functions in formulation. For example, increased membrane permeability of a hydrophilic drug can be accomplished through lipidation (decreasing hydrophilicity), typically via ester bond formation. In doing so, the prodrug offers improved bioavailability through enhanced absorption from the gastrointestinal tract into systemic circulation or via topical application. Other routes to improve ADMET properties include the addition of ionizable groups to increase solubility (increasing hydrophilicity). The masking of metabolically labile groups is also investigated to prevent their premature breakdown. Future work contemplates the conjugation of peptidic epitopes for the active targeting of specific tissue / cell surface receptors and the site- sensitive activation of the precursor / prodrug for selective release.

[0236] Elraglusib is further examined without the ester (in a charged form) to determine various penetrations into cells and if blood brain barrier (BBB) penetration can be controlled using either de-esterification, saponification, charge, or a prodrug as described above.EXAMPLE 3 EXAMPLE STATISTICAL ANALYSIS

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[0238] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[0239] The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the present aspects and embodiments. The present aspects and embodiments are not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect and other functionally equivalent embodiments are within the scope of the disclosure. Various modifications in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects described herein are not necessarily encompassed by each embodiment. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be encompassed by the following exemplary claims.

Claims

CLAIMS We claim:

1. A pharmaceutical composition comprising (a) and (b): (a) a small molecule GSK-3 inhibitor, or a pharmaceutically acceptable salt and / or solvate or hydrate thereof; and (b) a therapeutic agent effective for an immune checkpoint blockade (ICB) therapy; or a pharmaceutically acceptable salt and / or solvate or hydrate thereof; wherein the pharmaceutical composition is included in a simultaneous formulation comprising both (a) and (b); or included in two separate pharmaceutical formulations with the first formulation comprising (a) and the second formulation comprising (b), configured for an initial administration of (a) later followed by an administration of (b).

2. The pharmaceutical composition of claim 1, wherein (a) comprises a compound of formula 1 or a pharmaceutically acceptable salt and / or solvate or hydrate thereof:.

3. The pharmaceutical composition of claim 1, wherein (b) includes an immune checkpoint inhibitor comprising an anti-PD-1 and / or anti-PD-L1 therapeutic agent, Pembrolizumab (Keytruda), Nivolumab (Opdivo), Cemiplimab (Libtayo), Atezolizumab (Tecentriq), Avelumab (Bavencio), Durvalumab (Imfinzi), a CTLA-4 inhibitor, Ipilimumab(Yervoy), tremelimumab (Imjuno), a LAG-3 inhibitor, Relatlimab, or a combination thereof.

4. The pharmaceutical composition of claim 1, wherein the first formulation is configured for an initial administration to a subject in need thereof; and the second formulation is configured for an administration to the subject within a period of time of less than one year after the initial administration.

5. The pharmaceutical composition of claim 1, further comprising a therapeutic agent or method (c) either in place of (b) or in addition to (b); wherein (c) is comprising an agent effective for a combination therapy, a targeted therapy, a chemotherapy, a treatment modality that affects a tumor microenvironment, an application of an electromagnetic radiation / radiation, or a combination thereof.

6. The pharmaceutical composition of claim 1, wherein the therapeutic agent in (b) is determined after an administration of (a) by one or more changes in biomarkers observed from examining changes in a tumor microenvironment subsequent to the administration of (a).

7. The pharmaceutical composition of claim 1, wherein the first formulation is configured for a first route of administration in a subject in need thereof, and the second formulation is configured for a second different route of administration in the subject.

8. A method for treating a subject diagnosed with a cancer or suspected of having a cancer, the method comprising the steps of: (1) administering a therapeutically effective amount of a small molecule GSK-3 inhibitor, or a pharmaceutically acceptable salt and / or solvate or hydrate thereof; whereby a protein expression in a tumor microenvironment is changed to enhance a tumor-infiltrating immune cell activation; and(2) administering a therapeutically effect amount of an immune checkpoint inhibitor effective for treating the subject with an immune checkpoint blockade (ICB) therapy; whereby the ICB therapy is enhanced compared to the same ICB therapy, ceteris paribus, without an execution of step (1).

9. The method of claim 8, wherein the cancer comprises a primary tumor of colorectal cancer (CRC), pancreatic cancer, lung cancer or cancer of the pleural space, liver cancer, rectum cancer, or a combination thereof.

10. The method of claim 8, wherein the cancer comprises microsatellite stable (MSS) colorectal cancer (CRC).

11. The method of claim 8, wherein the subject has been previously diagnosed as not responding to an ICB (immune checkpoint blockade) therapy.

12. The method of claim 8, further comprising the step of: (1b) measuring one or more biomarkers derived from the cancer in the subject; wherein step (1b) is executed before step (1), after step (1), or executed repeatedly; and the execution of step (1b) is operative to provide a determination of an effectiveness of step (1).

13. The method of step 12, wherein the determination is utilized to determine the immune checkpoint inhibitor (ICI) included in step (2), and the ICI is determined by one or more changes in biomarkers observed from examining changes in a tumor microenvironment after step (1).

14. The method of claim 13, wherein the one or more biomarkers comprise one or more differentially expressed proteins.

15. The method of claim 14, wherein the one or more differentially expressed biomarkers or proteins comprise CD39; CD163 (or the antigen CD163); OX40L (CD134 or TNFRSF4); checkpoint molecules for VISTA; myeloid / neutrophil marker CD66b; myeloid markers IDO1, CD80, or CD11b; TAM / M2 macrophage marker CD68; myeloid marker CD40 or DC / myeloid marker CD11c; Nuclear factor-κB (NF-κB); NEK2 protein; T cell activation marker CTLA4; antigen PTEN; hematopoietic marker CD34; T cell activation marker CD44; antigen presentation B2M; immune cell activation marker HLA- DR; TAM / M2 macrophage marker ARG1; memory T cell marker CD45RO; proliferation marker Ki−67; NK cell marker CD56; T cell activation marker GITR; immune checkpoint molecule B7-H3; Treg marker CD127; expression of B cell marker CD20; antigen NY- ESO-1; progesterone receptor (PR); cytotoxic T cell marker CD8; antigen Her2; Treg marker Foxp3; T cell marker CD3; immune checkpoint protein expression of LAG3, PD- L2, PD-1; protein expression of mature B cell / DC marker CD35; antigen MART1; checkpoint PD-L1 and PR; immune checkpoint Tim-3; Treg marker CD25; checkpoint protein TIGIT or LAG-3; or a combination thereof.

16. The method of claim 8, further comprising an administration of a combination therapy including an agent effective for a combination therapy, a targeted therapy, a chemotherapy, a treatment modality that affects a tumor microenvironment, an application of an electromagnetic radiation / radiation, or a combination thereof.

17. A method to determine how a cancer patient will respond to an ICB therapy, the method comprising the steps of: (1) administering a small molecule GSK-3 inhibition treatment to the patient; (2) obtaining a post-treatment biopsy from the patient; and (3) measuring one or more biomarkers differentially expressed in the post- treatment biopsy; whereby the one or more biomarkers are indicative of how the patient will respond to the ICB therapy.

18. The method of claim 17, further comprising the steps of:(0) obtaining a pre-treatment biopsy from the patient; and (4) measuring the one or more differentially expressed proteins in the pre- treatment biopsy; whereby a comparison between a level of one or more differentially expressed proteins in the pre-treatment biopsy and in the post-treatment biopsy is enabled.

19. The method of claim 18, wherein a therapeutic development of a GSK-3 inhibitor is provided by the comparison of the levels of the one or more biomarkers and / or the one or more proteins.

20. The method of claim 18, further comprising the step of determining the ICB therapy based upon the one or more biomarkers and / or the one or move proteins.

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