Brain targeted metabolic inhibition of cancer stem cells to destroy their stemness induced tumorigenicity

Abstract

Disclosed are compounds, nanoparticles, and compositions for effective delivery of metabolic inhibitors to disease state cells. Also disclosed are methods of treating a subject in need thereof, such as a subject with cancer.

Description

[0001] BRAIN TARGETED METABOLIC INHIBITION OF CANCER STEM CELLS TO DESTROY THEIR STEMNESS INDUCED TUMORIGENICITY

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims benefit of priority of U.S. Provisional Application No. 63 / 664,138, filed June 25, 2024, which is incorporated herein by reference.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with government support under CA240139 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0006] BACKGROUND

[0007] Glioblastoma (GBM) is the most common and lethal malignant brain tumor in adults. Recurrence and eventual death are inevitable as the vigorous invasiveness of GBM cells precludes gross total resection. Surgical removal of an entire involved cerebral hemisphere unfortunately still results in disease recurrence due to distant invasive microscopic residual cells that have migrated to the contralateral hemisphere. For this reason, standard of care therapy includes adjuvant radiotherapy and temozolomide-based chemotherapy following maximal safe surgical resection intended to treat microscopic residual tumor. However, as this protocol confers a median survival of only 14.6 months, advancements in understanding of the disease and the parameters which contribute to recurrence are urgently required. The compounds and methods disclosed herein address these and other needs.

[0008] SUMMARY

[0009] As described herein, there is a linear relationship between the metabolic dependency of the cancer initiating stem cells and their proliferative tumor initiating properties. Once the metabolic dependency is destroyed, these cells lose their ability to form tumors and / or become more vulnerable to apoptosis using therapeutic agents. Herein, different approaches for destroying the metabolic dependence of such cells are described, including genetic modification, pharmacological inhibitors, synthetic organic small molecules, and a prodrug of a widely used chemotherapeutic. Further, as described herein, these brain cancer initiating stem cells are metabolically plastic and inhibition of one pathway can result in more reliance on otherwise non-dominating metabolic substrate utilization.

[0010] Thus, described herein are evidence-based clues to develop combination therapeutic approaches for complete destruction of these cells to stop brain cancer recurrence. From a clinical point of view, therapeutics to attack the widely spread-out cancer stem cells in the brain are advantageously targeted across the blood brain barrier to achieve therapeutic efficacy. Thus, in some embodiments, the compounds described herein can be delivered using brain targeted nano delivery vehicles. As described herein, in some embodiments, the compounds described herein can be used in combination with a specific diet to stop or delay brain cancer recurrence. The types of cancers in this disclosure can be glioblastoma, glioma, metastatic brain cancer, and pediatric low grade glioma, and diffuse intrinsic pontine gliomas (DIPG) induced tumor.

[0011] By way of example, described herein are compounds comprising a structure represented by Formula I below

[0012] A^R1^L^Z

[0013] Formula I or a pharmaceutically acceptable salt thereof, wherein A comprises a mitochondrial targeting moiety; R1is a direct bond, or represents a substituted or unsubstituted C1-C10 alkylene; L is a substituted or unsubstituted alkylene, a carbonyl, an ester, an amide, a carbamate ester, an amine, an ether, a carbonate ester, a thioether, a thioester, or a urea; and Z comprises a lipid chain.

[0014] Also disclosed herein is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of the compounds or nanoparticles described herein.

[0015] Also disclosed herein are methods of treating a disease or disorder in a subject in need thereof, the methods comprising administering to the subj ect a therapeutically effective amount of any of the pharmaceutical compositions disclosed herein. In some examples, the disease comprises cancer.

[0016] BRIEF DESCRIPTION OF THE FIGURES

[0017] The accompanying figures, w hich are incorporated in and constitute a part of this specification, illustrate several aspects described below'. Figures 1A-1J. Analyses of signatory metabolic pathways in patient derived glioblastoma stem cells distinguishable from patient derived normal astrocytes. Tables showing the patient information for the (Figure 1 A) patient derived GSCs and (Figure IB) normal astrocyte samples. The normal astrocyte samples were obtained from the gene expression omnibus (GEO) database (GSE67835). (Figure 1C) Principal component analysis (PCA) and (Figure ID) correlation matrix between samples from the normal astrocytes and patient derived GSCs. (Figure IE) Volcano plot showing the significantly upregulated and downregulated genes in the patient derived GSCs compared to the normal human astrocytes. Genes with significance P < 0.05 were specified as either upregulated or downregulated in patient derived GSCs compared to the normal human astrocytes. (Figure IF) Hierarchical clustering heatmap showing the top 2500 differentially expressed genes (DEGs) between normal astrocytes and patient derived GSCs. (Figure 1G) Dot plot showing the top 10 enriched pathways based on KEGG pathway analyses. (Figure 1H) Clustered analysis of the top 100 differentially expressed genes showing the respective enriched pathways for each cluster. The clustered analysis was performed based on the / / -means clustering algorithm. Heatmaps showing the significantly different DEGs associated with (Figure II) unfavorable disease prognosis in GBM, and (Figure 1J) sternness potential. The significance level was maintained at P < 0.05. All sequencing analyses data in the heatmap is represented as normalized Zscore values.

[0018] Figures 2A-2J. In vitro validation of sequencing analyses for signatory metabolic pathways in patient derived GSCs. (Figure 2A) Western Blot images showing the relative expression of neural sternness marker: nestin (207-217 kDa), cancer sternness marker: CD 133 (133 kDa), proliferation marker: vimentin (57 kDa), and transcription factors responsible for sternness and self-renewal capacity: nanog (70 kDa) and SOX2 (34 kDa) in patient derived GSCs. Immunofluorescence-based visualization of (Figure 2B) cancer sternness marker, CD133 and (Figure 2C) proliferation marker, Ki67 in patient derived GSCs. Nuclei in the immunofluorescence images were stained using the DAPI stain. The images were taken at Ex / Em: 359 / 461 nm for DAPI channel at 700 V. Ex / Em: 499 / 520 nm for the green channel at 700 V. and Ex / Em: 553 / 568 nm for the red channel at 700 V. All scans were kept constant at 2 ws / pixel. (Figure 2D) Quantification of extent of glycolysis in patient derived GSC 1-4 as determined by Seahorse Glycostress assay using different glycolysis modulators. The glycolysis modulators used in the assay were glucose (10 mM) to initiate glycolysis, oligomycin (1 «m). an ATP synthase inhibitor, and 2-DG (50 mM). a structural analog of glucose which inhibits glycolysis. (Figure 2E) Dependence on fatty acid substrate for mitochondrial OXPHOS by patient derived GSCs 1-4. The inhibitors used in the assay were etomoxir (4 urn) to inhibit CPT1 A used in FAO, UK.5099 (2 «m) to inhibit mitochondrial pyruvate carrier for affecting glucose oxidation, and BPTES (3 um) to inhibit glutaminase for affecting the glutamine oxidation. (Figure 2F) Relative expression of fatty acid synthase, FASN (260-270 kDa), and fatty acid oxidation related proteins, HADHA (79 kDa) and ACADM (47 kDa) between recurrent and newly diagnosed patient derived GSCs, shown by Western Blot analyses. Mitochondrial and cytosolic reactive oxygen species (ROS) monitored qualitatively through (Figure 2G) live cell imaging under confocal microscope and quantified by (Figure 2H) normalized mean fluorescence intensity (MFI) using microplate reader. The live cell images for mitochondrial ROS were taken at Ex / Em: 553 / 568 nm for the red channel at 19% laser power and 750 V, and Ex / Em: 488 / 510 nm for the green channel at 2% laser power and 700 V. The live cell images for cytosolic ROS were taken at Ex / Em: 553 / 568 nm for the red channel at 9% laser power and 700 V, and Ex / Em: 488 / 510 nm for the green channel at 9.8% laser power and 700 V. All scans were kept constant at 2 rs / pixel. Mitochondrial ROS was determined using MitoSOX™ (5 «m). Cytosolic ROS was quantified using 2’, 7’ -dichlorofluorescin diacetate (DCFDA) (50 urn). Mitochondria of the cells were stained using Mito-GFP and Mito-RFP for Mitochondrial ROS and cytosolic ROS, respectively. (Figure 21) Heatmap showing the comparison between patient derived GSCs 1-4 based on the 13 differentially expressed mitochondrial protein coding genes and (Figure 2J) heatmap of the top differentially expressed genes associated with fatty acid metabolism between patient derived GSCs and normal astrocytes. The significance level for the top DEGs was maintained at P < 0.05. All sequencing analyses data are represented as normalized Zscore values. All statistical analyses were performed using ordinary one-way ANOVA at 95% CI and data represented as mean ± S.D.

[0019] Figures 3A-3M. Restricted fat partitioning to the brain decreases tumorigenicity of glioblastoma stem cells and increases survival. (Figure 3A) Schematic representation illustrating the study regimen for orthotopic glioblastoma PDX mouse model under ND or LCHFD to monitor the effect of dietary interventions on the growth and proliferation of Patient 1 GSCLuccells. Male and female BALB / c nude mice (N = 35) were divided into 2 groups, ND with n = 18 and LCHFD with n = 17. (Figure 3B) Average weekly calorie intake (kcal / animal / week) monitored by amount of food consumed by the animals per week, represented as a heatmap. (Figure 3C) Elevation of blood Lketone (mmol / L) level observed in LCHFD fed mice compared to ND fed mice at days 16, 66, and 80 of the study. The blood / kketone (mmol / L) level was monitored with Keto-Mojo® GK+ blood glucose and fl- ketone meter. (Figure 3D) Body weight (g) distribution of animals in the ND and LCHFD groups across all weeks of the study. Body weight measurements for the animals were collected in the afternoon. (Figure 3E) IVIS images showing n = 17 animals with aggressive tumor formation under ND and n = 2 animals with comparatively small tumor formation under LCHFD, respectively. Prior to imaging, luciferin was injected intraperitonially in each animal at 125 mg / kg concentration and the animal was kept under anesthetic chamber for 9 min. All images were taken keeping the animals under anesthesia through the nose pipes in the IVIS instrument. Data from the IVIS images revealed (Figure 3F) higher number of animals and higher percentage (%) of animals with tumor in ND group compared to the LCHFD group. (Figure 3G) Kaplan-Meier survival curve indicating a significant improvement in percent survival under LCHFD. The statistical significance was based on Log-rank (Mantel-Cox) test performed at 95% CI. (Figure 3H) MRI images of each animal under ND or LCHFD group taken using a 9.4 Tesla scanner equipped with a gradient (B- GA12S HP. 660mT / m). A mouse head transceiver volume coil was used for data acquisition. The animals were anesthetized using isoflurane 3% and 1-2% for anesthesia maintenance. Mice were controlled for temperature and monitored for respiration throughout the experiment. (Figure 31) Tumor volume (mm3) quantified by using the FMRIB software library’ at different timepoints from the MRI images reveal high rate of tumor progression in ND fed animals across the weeks post tumor cell inoculation. (Figure 3 J) Significantly increased blood ^ketone (mmol / L) levels and reduced blood glucose level (mg / dL) were observed at weeks 7 on special diet in animals fed with LCHFD compared to that of ND groups in the animals implanted with Patient 2 GSCLue. The blood ^-ketone (mmol / L) level and the blood glucose level was monitored with Keto-Mojo® GK+ blood glucose and J-ketone meter. (Figure 3K) IVIS images showing n = 15 animals with aggressive tumor under ND and no animals with tumor under LCHFD, respectively when these animals were impanted with Patient 2 GSCLuc. All IVIS images were obtained 9 min after injecting luciferin at 125 mg / kg concentration in each animal via the intraperitoneal route. (Figure 3L) Data from the IVIS images of Patient 2 GSCLucimplantation study- revealed most animals in the ND group experienced tumor formation but no animals showed any tumor formation in the LCHFD group and the corresponding graph with percentage of animals with and without tumor supports this observation. (Figure 3M) Immunofluorescence images of the coronal brain sections reveal presence of Ki67 positive Patient 2 GSCLuccells at the stereotactic site of implantation, whereas an absence of these cells away from site of implantation, 48 h and 72 h post implantation. Viable cells were confirmed based on Ki67 proliferation marker staining in CD133 positive cells. Images for 48 h post implantation were taken at Ex / Em: 359 / 461 nm for blue channel at 1% laser power and 700V, Ex / Em: 499 / 520 nm for green channel at 4% laser power and 710 V, Ex / Em: 553 / 568 nm for red channel at 12% laser power and 720 V. Images for 72 h post implantation were taken at Ex / Em: 359 / 461 nm for blue channel at 1% laser power and 700V, Ex / Em: 499 / 520 nm for green channel at 4% laser power and 710 V, Ex / Em: 553 / 568 nm for red channel at 12% laser power and 720 V. All scans were kept constant at 2 / zs / pixel. All statistical analyses were performed using two-tailed unpaired t test at 95% confidence interval (CI). Data is represented as mean ± S.D and considered significantly different when P < 0.05. Each point in the graph represents an animal.

[0020] Figures 4A-4D. Dietary' interventions alter signatory' tumor markers. H&E images of whole coronal brain sections and the images taken at the site of tumor cell implantation from (Figure 4A) LCHFD (n = 6) and (Figure 4B) ND (n = 4) fed mice reveal the absence and presence of tumor mass, respectively, at the stereotactic site of Patient 1 GSCLuccell implantation. The zoomed in images were taken by focusing specifically at the site of stereotactic orthotopic intracranial injection of the Patient 1 GSCLuccells. (Figure 4C) Immunofluorescence images of the coronal brain sections from both diet groups reveal a reduction in the expression of Ki67 in CD 133 positive cells at the site of implantation in LCHFD fed mice compared to the ND fed mice. All images were taken at Ex / Em: 359 / 461 nm for blue channel at 3% laser power and 750 V, Ex / Em: 499 / 520 nm for green channel at 4% laser power and 750 V, Ex / Em: 553 / 568 nm for red channel at 12% laser power and 620 V. All scans were kept constant at 2 ws / pixel. (Figure 4D) Quantification from immunofluorescence images confirm the statistically significant reduction in CD 133 and Ki67 in CD133 positive cells with respect to nuclei. For this quantification, multiple sections were used, and the statistical significance was determined using unpaired two- tailed t-test at 95% Cl and data is represented as mean ± S.D. Each data point represents an animal from the respective group.

[0021] Figures 5A-5D. Improvement in cognitive recognition functions of low carbohydrate high fat diet fed animals. (Figure 5A) Difference in locomotory function was observed from the foot placement analysis, represented by the trace path images between n = 9 tumor bearing mice from ND group and n = 10 mice without tumor from LCHFD group. (Figure 5B) Quantification based on the trace paths obtained from each animal of the respective groups, showing the distance walked (mm) and number of steps taken to cover the distance. Owing to the high tumor burden, 2 out of 9 tumor bearing mice, shown with red arrows in the ND group revealed circular locomotory pattern and inability to walk forward. (Figure 5C) Trace path images from the open field test to study the changes in sensorimotor cognition and anxiousness of animals. Data represents trace path from nude mice without exposure to tumor (n = 4). tumor bearing mice under ND (n = 6), and mice without tumor under LCHFD (n = 6) showing the increased trend of the tumor bearing mice to remain towards the walls of the open field. (Figure 5D) Data showing the number of beam breaks by each animal from the respective groups for every 5-min binblocks, across a total interval of 30 min. Each beam break is detected by the Photobeam Activity System (PAS) software when an animal crosses the photobeams that are configured across the PAS -open field.

[0022] Figures 6A-6E. Low carbohydrate high fat diet fed animals develop an improved gut-microbiome structure. Gut-microbiome structure was analyzed using fecal DNA samples isolated from n = 6 tumor bearing animals under ND and n = 6 animals without tumor under LCHFD. (Figure 6A) Heatmap showing the differences in percent (%) relative abundance of all microbial species expressed in the gut-microbiome betw een ND fed tumor bearing animals and LCHFD fed animals without tumor. %Relative abundance represented as heatmaps for all microbial (Figure 6B) phylum and (Figure 6C) family expressed between the tumor bearing animal under ND and animals without tumor under LCHFD. (Figure 6D) Difference in the bacterial composition based on the %relative abundance of all the microbial orders between tumor bearing ND fed animals and LCHFD fed mice without tumor. (Figure 6E) Improved Firmicutes and Bacteroidetes (F / B) ratio (top) and reduced %relative abundance of Bacteroidetes (bottom) in LCHFD fed animals compared to ND fed animals. All statistical significances were measured using two-tailed unpaired t test at 95% CI, and the data is represented as mean ± S.D. Each data point represents an animal from the respective group.

[0023] Figures 7A-7L Understanding the metabolic plasticity of GSCs through metabolomic mapping of liver and brain samples from ND and LCHFD fed animals which had tumor implantation. For the metabolomic mapping, n = 3 animals were used from each diet group. (Figure 7A) Significantly expressed metabolites represented as Venn diagram reveal 3 common metabolites were expressed between the brain and the liver samples. The level of significance was kept at P < 0.05. (Figure 7B) Left panel shows the Volcano plot to indicate presence of significantly upregulated or downregulated metabolites mapped between LCHFD and ND liver samples and the right panel shows the Volcano plot to indicate the presence of significantly upregulated or downregulated metabolites mapped betw een LCHFD and ND brain samples. Metabolites expressed at a significance level of P < 0.05 were demarcated in red. (Figure 7C) Heatmaps generated from the unsupervised clustering analysis of significantly expressed metabolites (P < 0.05) reveal remarkable difference in metabolomic profile of liver samples (left) and brain samples (right) between LCHFD and ND fed animals which had tumor implantation. (Figure 7D) Data revealing the pathways affected by the differential metabolite expression and their impact scores based on Kyoto-Encyclopedia Genes and Genomes (KEGG) pathway analysis in the liver (left) and brain (right) samples between LCHFD and ND fed animals which had tumor implantation. The level of significance for pathw ay analysis w as kept at / 3< 0.01. Flow chart showing the significantly impacted metabolites in red for (Figure 7E) tricarboxylic acid (TCA) cycle, (Figure 7F) pyruvate metabolism, (Figure 7G) arginine biosynthesis, (Figure 7H) vitamin B6 metabolism, and (Figure 71) phenylalanine, tyrosine, and tryptophan biosynthesis pathways due to the differential metabolomic mapping in brain samples of LCHFD and ND fed animals. The global metabolomic analysis for LCHFD vs ND samples were clustered with the complete method and Euclidian distance function. Any metabolite with / 3< 0.05 was considered significantly regulated (up or down). Heatmaps were generated with hierarchical clustering performed on the imputed matrix values utilizing the R library heatmap (1.0. 12). Volcano plots were generated by plotting log2foldchange vs -log) / 3) in GraphPad. Pathway analyses were done using metabolites with / 3< 0.01 in Metabo Analyst.

[0024] Figures 8A-8F. Dietary intervention regulates lipid distribution between peripheral organ and brain. For the following lipidomic analysis, n = 3 animals were used from each diet group. (Figure 8A) Significantly expressed lipids represented as Venn diagram reveal 30 common lipid species were expressed between the brain and the liver samples. The level of significance was kept at P < 0.05. (Figure 8B) Left panel shows the Volcano plot to indicate presence of significantly upregulated or downregulated hpid species between LCHFD and ND liver samples and the right panel shows the Volcano plot to indicate the presence of significantly upregulated or downregulated lipid species betw een LCHFD and ND brain samples. Lipids expressed at a significance level of / 3< 0.05 were demarcated in red. The different hpid species mentioned in the Volcano plots are, DG: diglycerides; PE: phosphatidylethanolamine; PC: phosphatidylcholine; PS: phosphatidylserine. Heatmaps generated based on the unsupervised clustered analysis of the significant lipids expressed (P < 0.05) between the (Figure 8C) liver samples and (Figure 8D) brain samples of LCHFD and ND fed animals. (Figure 8E) Number of lipid species under specific categories significantly upregulated in brain and liver between LCHFD and ND groups. (Figure 8F) The commonly expressed lipid species between LCHFD w ND liver and chow (top), and LCHFD vs ND brain and chow (bottom) reveal only 4 and 1 common lipid species, respectively. The global lipidomic analysis for LCHFD vs ND samples were clustered with the complete method and Euclidian distance function. Volcano plots were generated by plotting log2foldchange vs -log(L) in GraphPad prism. Lipids which were significantly present (P < 0.05) between LCHFD and ND were categorized based on the species.

[0025] Figures 9A-9E. Possible connection of low carbohydrate high fat dietary intervention with Circadian network. (Figure 9A) Heatmap from RNA sequencing analysis of patient derived GSCs 1-4 showing the DEGs of circadian rhythm related genes and GSEA plots with normalized enrichment score (NES) and false discovery rate (q value) for circadian rhythm and regulation of circadian rhythm. (Figure 9B) Heatmap showing the DEGs associated with circadian network between the patient derived GSCs 1-4 and patient derived normal astrocytes. Western blot data reveals the (Figure 9C) relative expression of LGMN (49 kDa) protein in patient derived GSCs compared to normal human astrocytes and brain endothelial hCMEC / D3 cells at the top and relative expression of CLOCK (95-110 kDa) in patient derived GSCs compared to endothelial cells at the bottom. (Figure 9D) Western blot performed in brain tissue homogenates and its quantification for change in expression of CLOCK and LGMN in tumor bearing animals fed with ND (n = 5) compared to both, BALB / c nude mice without tumor (n = 5) and LCHFD fed animals without tumor (n = 5). Statistical significance was quantified using ordinary one-way ANOVA at 95% CL Data is represented as mean ± S.D. Each point on the data represents an animal from the respective group. (Figure 9E) Representative immunofluorescence images confirmed the elevated CLOCK expression in CD133 positive cells in ND group compared to LCHFD group. All immunofluorescence images were taken under the confocal microscope with Ex / Em: 359 / 461 nm for blue channel at 1.7% laser power and 660 V, Ex / Em: 499 / 520 nm for green channel at 15% laser power and 603 V, Ex / Em: 553 / 568 nm for red channel at 1.2% laser power and 630 V. All scans were kept constant at 2 / s / pixel.

[0026] Figures 10A-10N. Effect of low carbohydrate high fat diet when tumorigenic patient derived GSCs are implanted peripherally. (Figure 10A) Schematic illustration showing the study design for glioblastoma flank PDX mouse model with either Patient 1 GSCX Ior Patient 2 GSCWTcells under normal or low carbohydrate high fat diet. Both male and female BALB / c nude mice were used for the study. Separate studies were performed for Patient 1 GSCs and Patient 2 GSCs. (Figure 10B) Food consumption data based on the average weekly calorie intake (Kcal / animal / week) represented as a heatmap to determine the differential calorie intake between the ND fed and the LCHFD fed mice. (Figure IOC) Statistically significant elevation in / / -ketone (mmol / L) level and a simultaneous reduction in glucose (mg / dL) levels observed in LCHFD fed mice blood compared to ND fed mice at 16 weeks on the respective diets. The blood / / -ketone (mmol / L) level and blood glucose level was monitored with Keto-Mojo® GK+ blood glucose and / / -ketone meter. (Figure 10D) Brightfield images showing the aggressive tumor formation in n = 7 and n = 12 mice out of a total of 15 mice / group in ND and LCHFD groups, respectively. The red circle determines the palpable tumor mass in the mice. Animals were kept under anesthesia while imaging. (Figure 10E) Percentage of animals with and without tumor till 90 days post cell implantation under ND and LCHFD groups. (Figure 10F) Tumor volume (mm3) of Patient 1 GSC tumor measured through Vernier Caliper readings of length and breadth for the peripheral tumor mass across days post cell implantation. (Figure 10G) Weekly body weight (g) measurement of all animals in ND and LCHFD groups. The arrow on the graph represents that at 5 weeks on special diet, Patient 1 GSCs were implanted subcutaneously at the right flank of all the mice. Body weight measurements and tumor volume measurements for the animals were taken in the afternoon. (Figure 10H) Heatmap of the food consumption pattern determined as average weekly calorie intake (Kcal / animal / week) showing differential calorie intake between the ND fed and the LCHFD fed mice for the Patient 2 GSCWTflank PDX study. (Figure 101) LCHFD mice show elevated blood / / -ketone (mmol / L) levels, and a simultaneous low blood glucose (mg / dL) level compared to ND fed mice at 5 weeks on the respective diets. The blood / / -ketone (mmol / L) and blood glucose level was monitored with Keto-Mojo® GK+ blood glucose and / / -ketone meter. (Figure 10J) Representative brightfield images showing the tumor formation in ND and LCHFD groups, respectively. The red circle determines the palpable tumor mass in the mice. Animals were kept under anesthesia while imaging. (Figure 10K) Percentage of animals with and without tumor formation till 90 days post cell implantation under ND and LCHFD groups. (Figure 10L) Probability of survival between ND and LCHFD fed mice determined through the Kaplan-Meier survival curve analysis. For the Kaplan-Meier survival curve analysis, Logrank (Mentel-Cox) test was used to determine level of significance. (Figure 10M) Tumor volume (mm3) of Patient 2 GSC PDX tumor measured through Vernier Caliper readings of length and breadth for the peripheral tumor mass across days post cell inoculation. (Figure ION) Weekly body weight (g) measurement for all animals in ND and LCHFD groups. The arrow on the graph represents that at 7 weeks on special diet, Patient 2 GSCs were implanted subcutaneously at the right flank of all the mice. Body weight measurements and tumor volume measurements for the animals were taken in the afternoon. All statistical analyses between the ND and LCHFD groups were performed based on two-tailed unpaired t test at P < 0.05. Each data point indicates an animal in the group and the data is represented as mean ± S.D.

[0027] Figure 11. Pictorial representation of the hypothesis that a low carbohydrate high fat diet based non-invasive therapeutic intervention affects glioblastoma progression. Schematics showing the invasiveness of the post-surgical residual GSCs leads to recurrence. Depiction of our hypotheses of metabolic modulation of invaded GSCs for no or delayed recurrence utilizing dietary intervention.

[0028] Figure 12. Hypothesis regarding the potential mechanism of action for newly synthesized FAO inhibitors.

[0029] Figures 13A-13C. Series of synthesized FAO inhibitors (Figure 13A), synthetic method (Figure 13A). and characterization by ’H NMR (Figure 13C).

[0030] Figures 14A-14B. Illustration of the inhibition of FAO in liver homogenate. Figure 15A-15B. Effect of TPP-palmitate on recurrent patient derived GSCs.

[0031] (Figure 15A) Top showing the structure of TPP-palmitate compound. Bottom showing the Western Blot analyses data and respective quantification for relative expression of ACADM and HADHA with respect to cyclophilin A control. (Figure 15B) Western blot data showing the relative expression of CD 133, vimentin, and SOX2 along with their respective quantifications. The recurrent patient 1 GSCs were treated with TPP-palmitate.

[0032] DETAILED DESCRIPTION

[0033] The materials, compounds, compositions, articles, and methods described herein may be understood more readily by reference to the following detailed description of specific aspects of the disclosed subject matter and the Examples and Figures included therein.

[0034] Before the present materials, compounds, compositions, and methods are disclosed and described, it is to be understood that the aspects described below are not limited to specific synthetic methods or specific reagents, as such may. of course, vary. It is also to be understood that the terminology’ used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

[0035] Also, throughout this specification, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which the disclosed matter pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.

[0036] General Definitions

[0037] In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings:

[0038] Throughout the description and claims of this specification the word “comprise” and other forms of the word, such as “comprising” and “comprises,” means including but not limited to. and is not intended to exclude, for example, other additives, components, integers, or steps.

[0039] As used in the description and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes mixtures of two or more such compositions, reference to “the compound” includes mixtures of two or more such compounds, reference to “an agent” includes mixture of two or more such agents, and the like.

[0040] “Optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0041] It is understood that throughout this specification the identifiers “first” and “second” are used solely to aid in distinguishing the various components and steps of the disclosed subject matter. The identifiers “first” and “second” are not intended to imply any particular order, amount, preference, or importance to the components or steps modified by these terms.

[0042] Chemical Definitions

[0043] As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described below. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms, such as nitrogen, can have hydrogen substituents and / or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds. Also, the terms "‘substitution" or “substituted with" include the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.

[0044] “Z1,” “Z2,” “Z3,” and “Z4” are used herein as generic symbols to represent various specific substituents. These symbols can be any substituent, not limited to those disclosed herein, and when they are defined to be certain substituents in one instance, they can, in another instance, be defined as some other substituents.

[0045] In general, the number of carbon atoms present in a given group may be designated as a range from “Cx-Cy”, where x and y are the lower and upper bounds of the range, respectively. The carbon number as used in the definitions herein refers to carbon backbone and carbon branching, but does not include carbon atoms of the substituents, such as alkoxy substitutions and the like. In some instances, a group may be referenced as including Co, which means that the group is absent or, in the case of a linking group, represents a direct bond whereby the groups at both ends of the linking group are directly linked. The term “direct bond,’’ as used herein, refers to an embodiment where the identified group is absent from the structure, and is replaced by a bond between other groups to which it is connected. For example, if the specification or claims recite A — D — E and D is defined as a direct bond, the resulting structure is A — E.

[0046] The term “aliphatic” as used herein refers to a non-aromatic hydrocarbon group and includes branched and unbranched, alkyd, alkenyl, or alkynyl groups.

[0047] The term “alkyl” as used herein is a branched or unbranched saturated hydrocarbon group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like. The alkyd group can also be substituted or unsubstituted. The alkyd group can be substituted with one or more groups including, but not limited to, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryd. aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described below.

[0048] Throughout the specification "alkyl” is generally used to refer to both unsubstituted alkyl groups and substituted alkyl groups; however, substituted alk l groups are also specifically referred to herein by identifying the specific substituent(s) on the alky l group. For example, the term "halogenated alkyl” specifically refers to an alkyl group that is substituted with one or more halides, e.g., fluorine, chlorine, bromine, or iodine. The term “alkoxyalkyd” specifically refers to an alkyl group that is substituted with one or more alkoxy groups, as described below. The term "alkydamino” specifically refers to an alkyd group that is substituted with one or more amino groups, as described below, and the like. When “alkyl” is used in one instance and a specific term such as "alkylalcohol” is used in another, it is not meant to imply that the term “alkyl” does not also refer to specific terms such as “alkylalcohol” and the like.

[0049] This practice is also used for other groups described herein. That is, while a term such as "cycloalkyl” refers to both unsubstituted and substituted cycloalkyl moieties, the substituted moieties can, in addition, be specifically identified herein; for example, a particular substituted cycloalkyl can be referred to as, e.g., an “alkylcycloalkyd.” Similarly, a substituted alkoxy can be specifically referred to as, e.g., a “halogenated alkoxy,” a particular substituted alkenyl can be, e.g., an “alkenylalcohol,” and the like. Again, the practice of using a general term, such as “cycloalkyl.” and a specific term, such as “alkylcycloalkyl,” is not meant to imply that the general term does not also include the specific term.

[0050] The term “alkoxy” as used herein is an alkyl group bound through a single, terminal ether linkage; that is, an “alkoxy” group can be defined as — OZ1where Z1is alkyd as defined above.

[0051] The term “alkenyl” as used herein is a hydrocarbon group of from 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon double bond. Asymmetric structures such as (Z1Z2)C=C(Z3Z4) are intended to include both the E and Z isomers. This can be presumed in structural formulae herein wherein an asymmetric alkene is present, or it can be explicitly indicated by the bond symbol C=C. The alkenyl group can be substituted with one or more groups including, but not limited to, alkyd, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo. sulfonyl, sulfone, sulfoxide, or thiol, as described below. The term "alkynyl" as used herein is a hydrocarbon group of 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon triple bond. The alkynyl group can be substituted with one or more groups including, but not limited to, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described below.

[0052] The term ‘"aryl” as used herein is a group that contains any carbon-based aromatic group including, but not limited to, benzene, naphthalene, phenyl, biphenyl, phenoxybenzene, and the like. The term “heteroaryl” is defined as a group that contains an aromatic group that has at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus. The term "non-heteroaryl." which is included in the term "aiyl." defines a group that contains an aromatic group that does not contain a heteroatom. The aryl or heteroaryl group can be substituted or unsubstituted. The aryl or heteroaryl group can be substituted with one or more groups including, but not limited to, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl. aldehyde, ammo, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol as described herein.

[0053] The term “cycloalkyf’ as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc. The term ‘'heterocycloalkyl” is a cycloalkyl group as defined above where at least one of the carbon atoms of the ring is substituted with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkyl group and heterocycloalkyl group can be substituted or unsubstituted. The cycloalkyl group and heterocycloalkyl group can be substituted with one or more groups including, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroary l, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol as described herein.

[0054] The term “cycloalkenyl” as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms and containing at least one double bound, z.e., C=C. Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, and the like. The term “heterocycloalkenyl” is a type of cycloalkenyl group as defined above, and is included within the meaning of the term ‘'cycloalkenyl,” where at least one of the carbon atoms of the ring is substituted with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkenyl group and heterocycloalkenyl group can be substituted or unsubstituted. The cycloalkenyl group and heterocycloalkenyl group can be substituted with one or more groups including, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo. sulfonyl, sulfone, sulfoxide, or thiol as described herein.

[0055] The term ‘"cyclic group’7is used herein to refer to either aryl groups, non-aryl groups (z.e., cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl groups), or both. Cyclic groups have one or more ring systems that can be substituted or unsubstituted. A cyclic group can contain one or more aryl groups, one or more non-ary l groups, or one or more aryl groups and one or more non-aryl groups.

[0056] The term ‘‘aldehyde” as used herein is represented by the formula — C(O)H. Throughout this specification “C(O)” or “CO” is a short hand notation for C=O, which is also referred to herein as a “carbonyl.”

[0057] The terms “amine” or “amino” as used herein are represented by the formula — NZ1 / 2, where Z1and Z2can each be substitution group as described herein, such as hydrogen, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyd, or heterocycloalkenyl group described above. “Amido” is — C(O)NZ1Z2.

[0058] The term “carboxylic acid” as used herein is represented by the formula — C(O)OH.

[0059] A “carboxylate” or “carboxyl” group as used herein is represented by the formula

[0060] — C(O)O'

[0061] The term “carbamide” means compounds having the group — N(Z') —

[0062] (CO)N(Z1)2 where each Z1can be, independently, an alkyl, alkenyl, alkynyl, aryl, arylalkyd, cycloalkyl, carbony l, ether, haloalkyl, heteroaryl and heterocyclyl.

[0063] The term “carbamate” means a group of the form — Z1OC(O)N(Z1) — , — Z1OC(O)N(Z1) Z1— , or — OC(O)N(Z1)2, where each Z1can be, independently, an alkoxy, aryloxy, alkyl, alkenyl, alkynyl, ary 1, arylalkyl, cycloalkyl, ether, formyl, haloalkyl, heteroaryl, and heterocyclyl. Carbamates include, e.g., arylcarbamates and heteroaryl carbamates.

[0064] The term “ester” as used herein is represented by the formula — OC(O)Z1or

[0065] — C OIOZ1, where Z1can be an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyd, or heterocycloalkeny 1 group described above. The term “ether” as used herein is represented by the formula Z'OZ2. where Z1and Z2can be. independently, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroar l, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

[0066] The term “formamide” refers to compounds comprising the — NC(O)H formamide group. Formamides include compounds having the formula HC(O)NZ1Z2wherein Z1and Z2can be, independently, hydrogen or an alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, carbonyl, ether, haloalkyl, heteroaryl and heterocyclyl.

[0067] The term “ketone” as used herein is represented by the formula Z'C(O)Z2. where Z1and Z2can be, independently, an alkyl, halogenated alky l, alkenyl, alkynyl, ar l, heleroar l. cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

[0068] The term “halide” or “halogen” as used herein refers to the fluorine, chlorine, bromine, and iodine.

[0069] The term “hydroxyl” as used herein is represented by the formula — OH.

[0070] The term “nitro” as used herein is represented by the formula — NO2.

[0071] The term “phosphonium group” refers to a compound of the formula — PR3 . wherein each R is independently hydrogen, alkyl, alkenyl, aryl, and aralkyl, as defined herein. The term “alkyl phosphonium” is a subset of phosphonium, wherein at least one R is an alkyl group. Alkyl phosphonium may be monoalkyl or dialkyl or trialkyl phosphonium. Similarly, the term “aryl phosphonium” is a subset of phosphonium, wherein at least one R is an aryl group. Aryl phosphonium may be monoaryl or diaryl or triaryl phosphonium. In addition, the term “arylalkyl phosphonium” is a subset of phosphonium, wherein the phosphorous atom comprises either at least one aryl group and at least one alkyl group.

[0072] The term “silyl” as used herein is represented by the formula — SiZ'Z2 / 3, where Z1, Z2, and Z3can be. independently, hydrogen, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

[0073] The term “sulfonyl” is used herein to refer to the sulfo-oxo group represented by the formula — SfOfiZ1. where Z1can be hydrogen, an alkyl, halogenated alkyl, alkenyl, alkynyl. aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

[0074] The term “sulfonylamino” or “sulfonamide” as used herein is represented by the formula — S(O)zNH — .

[0075] The term “thiol” as used herein is represented by the formula — SH.

[0076] The term “thio” as used herein is represented by the formula — S — . Unless stated to the contrary, a formula with chemical bonds shown only as solid lines and not as wedges or dashed lines contemplates each possible isomer, e.g. each enantiomer, diastereomer, and meso compound, and a mixture of isomers, such as a racemic or scalemic mixture.

[0077] Reference will now be made in detail to specific aspects of the disclosed materials, compounds, compositions, articles, and methods, examples of which are illustrated in the accompanying Examples and Figures.

[0078] Compounds

[0079] Disclosed herein are compounds and methods of making and use thereof. For example, disclosed herein are compounds comprising a structure represented by Formula I below

[0080] A'-R1-'L'^Z

[0081] Formula I or a pharmaceutically acceptable salt thereof, wherein A comprises a mitochondrial targeting moiety; R1is a direct bond, or represents a substituted or unsubstituted C1-C10 alkylene; L is a substituted or unsubstituted alkylene, a carbonyl, an ester, an amide, a carbamate ester, an amine, an ether, a carbonate ester, a thioether, a thioester, or a urea; and Z comprises a lipid chain.

[0082] In some examples of Formula I, the mitochondrial targeting moiety comprises a delocalized lipophilic cation. Delocalized lipophilic cations (DLCs) represent a group of compounds capable of penetrating plasma and mitochondrial membranes and accumulate in mitochondria. Suitable examples of delocalized lipophilic cations that penetrate the hydrophobic barriers of plasma and mitochondrial membranes include Rhodamine- 123, rhodacyanine MKT-077, dequalinium, triphenylphosphonium, guanidinium cations, and F16. In some examples of Formula I. the mitochondrial targeting moiety comprises an ar l phosphine or aryl phosphonium group (e.g., a monoaryl phosphonium, di ary l phosphonium, or triaryl phosphonium). In some examples of Formula I, the mitochondrial targeting moiety comprises a phosphonium group (e g., an aryl phosphonium group). In some examples of Formula I, the mitochondrial targeting moiety comprises a substituted or unsubstituted triphenylphosphonium (TPP). In some examples of Formula I, the mitochondrial targeting moiety comprises a mitochondria targeting peptide. In various examples according to Formula I, Z is a lipid chain. As used herein, the term "lipid chain" and the like, refers to saturated or unsaturated hydrocarbon chains derived from hydrophobic tails of lipids, for example alkyl, alkenyl or alkynyl chains, as described elsewhere herein. The lipid chain may be derived from di-aliphatic chain lipids, phospholipids, diglycerides, di-aliphatic glycolipids, sphingomyelin, glycosphingolipid, steroidal lipids, or hydrophilic polymer derivatized lipids. In some embodiments, the lipid chain comprises 2 to 40 carbons, e.g., 4 to 40 carbons, 2 to 20 carbons, 4 to 20 carbons, 10 to 40 carbons, 10 to 30 carbons, 10 to 20 carbons, or 20 to 30 carbons. In some examples of Formula I, Z is a substituted or unsubstituted alkyd. In some examples of Formula I, Z is a substituted or unsubstituted C5-C25 alkyl. For example. Z in various examples can be a C10- C25 linear alkyl (e.g., an unsubstituted C10-C25 linear alkyl). In some examples of Formula I, Z is C2-C25 alkyl. In some examples of Formula I, Z is C2-C16 alkyl. In some examples of Formula I, Z is a substituted or unsubstituted C2-C20 alky 1 (e.g., unsubstituted C2-C20 linear alkyl).

[0083] In various examples according to Formula I, R1is an unsubstituted C1-C10 alky lene, such as an unsubstituted C1-C4 alkylene. In certain according to Formula I, R1is C2 alkylene.

[0084] In various examples according to Formula I, L is an amide. In other examples according to Formula I, L is an ester.

[0085] In some embodiments, the compound comprises one of the following or a pharmaceutically acceptable salt thereof:

[0086] Also disclosed herein are nanoparticles (e.g., a population of nanoparticles) comprising any of the compounds disclosed herein. In some embodiments, the nanoparticles comprises a compound described herein and a polymer, such as a polyethylene glycol-lipid.

[0087] The nanoparticle can be of any shape, (e.g., a sphere, a rod, a quadrilateral, an ellipse, a triangle, a polygon, etc.). In some examples, the nanoparticle can have a regular shape, an irregular shape, an isotropic shape, an anisotropic shape, or a combination thereof. In some examples, the nanoparticles are substantially spherical in shape.

[0088] The nanoparticles can have an average particle size. “Average particle size” and “mean particle size” are used interchangeably herein, and generally refer to the statistical mean particle size of the particles in a population of particles. For example, the average particle size for a plurality of particles with a substantially spherical shape can comprise the average diameter of the plurality of particles. For a particle with a substantially spherical shape, the diameter of a particle can refer, for example, to the hydrodynamic diameter. As used herein, the hydrodynamic diameter of a particle can refer to the largest linear distance between two points on the surface of the particle. Mean particle size can be measured using methods known in the art, such as evaluation by scanning electron microscopy, transmission electron microscopy, and / or dynamic light scattering. The nanoparticles can, for example, have an average particle size of 50 nanometers (nm) or more (e.g., 60 nm or more, 70 nm or more, 80 nm or more, 90 nm or more, 100 nm or more, 1 10 nm or more, 120 nm or more, 130 nm or more, 140 nm or more, 150 nm or more, 160 nm or more, 170 nm or more, 180 nm or more, 190 nm or more, 200 nm or more, 225 nm or more, 250 nm or more, 275 nm or more, 300 nm or more, 325 nm or more, 350 nm or more. 375 nm or more. 400 nm or more, 425 nm or more, 450 nm or more, or 475 nm or more). In some examples, the nanoparticles can have an average particle size of 500 nm or less (e g., 475 nm or less, 450 nm or less, 425 nm or less, 400 nm or less, 375 nm or less, 350 nm or less, 325 nm or less, 300 nm or less, 275 nm or less, 250 nm or less, 225 nm or less. 200 nm or less, 190 nm or less, 180 nm or less, 170 nm or less, 160 nm or less. 150 nm or less, 140 nm or less, 130 nm or less, 120 nm or less. 110 nm or less, 100 nm or less, 90 nm or less, 80 nm or less, 70 nm or less, or 60 nm or less). The average particle size of the nanoparticles can range from any of the minimum values described above to any of the maximum values described above. For example, the nanoparticles can have an average particle size of from 50 nm to 500 nm (e.g., from 50 nm to 275 nm, from 275 nm to 500 nm, from 50 nm to 200 nm. from 200 nm to 350 nm, from 350 nm to 500 nm, from 60 nm to 500 nm, from 50 nm to 475 nm, from 60 nm to 475 nm, from 100 nm to 200 nm, from 120 nm to 140 nm, or from 150 nm to 200 nm).

[0089] With respect to particle size distribution characterization, a parameter used to define the size range of the nanoparticles is called the “poly dispersity index” (PDI). The term “poly dispersity” (or “dispersity” as recommended by IUPAC) is used to describe the degree of non-uniformity of a size distribution of particles. PDI is basically a representation of the distribution of size populations within a given sample. The numerical value of PDI ranges from 0.0 (for a perfectly uniform sample with respect to the particle size) to 1.0 (for a highly polydisperse sample with multiple particle size populations).

[0090] In some examples, the nanoparticles can have a poly dispersity index of 0.3 or less (e.g., 0.29 or less, 0.28 or less, 0.27 or less, 0.26 or less, 0.25 or less, 0.24 or less, 0.23 or less. 0.22 or less, 0.21 or less, 0.20 or less, 0.19 or less, 0.18 or less, 0.17 or less, 0.16 or less. 0.15 or less, 0.14 or less, 0.13 or less, 0.12 or less, 0.11 or less. 0.10 or less, 0.09 or less, 0.08 or less, 0.07 or less, 0.06 or less, 0.05 or less, 0.04 or less, 0.03 or less, 0.02 or less, or 0.01 or less).

[0091] In some examples, the nanoparticles can be substantially monodisperse. “Monodisperse” and “homogeneous size distribution,” as used herein, and generally describe a population of particles where all of the particles are the same or nearly the same size. As used herein, a monodisperse distribution refers to particle distributions in which 80% of the distribution (e.g., 85% of the distribution, 90% of the distribution, or 95% of the distribution) lies within 25% of the median particle size (e.g.. within 20% of the median particle size, within 15% of the median particle size, within 10% of the median particle size, or within 5% of the median particle size).

[0092] In some examples, the polymer comprises a biodegradable polymer. Such polymers are recognizable and identifiable by one or ordinary skill in the art. Non-limiting examples of synthetic, biodegradable polymers include: poly(amides) such as poly(amino acids) and poly(peptides); poly(esters) such as poly(lactic acid), poly(glycolic acid), poly(lactic-co- glycolic acid) (PLGA), and poly(caprolactone); poly(anhydrides); poly(orthoesters); poly(carbonates); and chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), fibrin, fibrinogen, cellulose, starch, collagen, and hyaluronic acid, copolymers and mixtures thereof. The properties and release profiles of these and other suitable polymers are known or readily identifiable.

[0093] In some embodiments, the polymer comprises a polyethylene gly col-lipid. In some embodiments, the polymer (e g., the polyethylene gly col-lipid) includes a mitochondrial targeting moiety. The mitochondrial targeting moiety can comprise a delocalized lipophilic cation. Delocalized lipophilic cations (DLCs) represent a group of compounds capable of penetrating plasma and mitochondrial membranes and accumulate in mitochondria. Suitable examples of delocalized lipophilic cations that penetrate the hydrophobic barriers of plasma and mitochondrial membranes include Rhodamine-123, rhodacyanine MKT-077, dequalinium, triphenylphosphonium, guanidinium cations, and Fl 6. In some examples, the mitochondrial targeting moiety comprises an aryl phosphine or and phosphonium group (e.g., a monoaryl phosphonium, diaryl phosphonium, or triaryl phosphonium). In some examples, the mitochondrial targeting moiety comprises a phosphonium group (e.g., an aryl phosphonium group). In some examples, the mitochondrial targeting moiety comprises a substituted or unsubstituted triphenylphosphonium (TPP).

[0094] In some examples, the poolymer includes a poly(D,L-lactic-co-glycolic acid)-block- poly(ethylene glycol)-triphenylphosphonium (PLGA-Z?-PEG-TPP) polymer. Pharmaceutical Compositions and Methods of Use

[0095] Also disclosed herein is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of a compound or nanoparticles described herein.

[0096] The compounds disclosed herein, and compositions comprising them, can also be administered utilizing liposome technology, slow release capsules, implantable pumps, and biodegradable containers. These delivery methods can. advantageously, provide a uniform dosage over an extended period of time. The compounds can also be administered in their salt derivative forms or crystalline forms.

[0097] The compounds disclosed herein can be formulated according to known methods for preparing pharmaceutically acceptable compositions. Formulations are described in detail in a number of sources which are well known and readily available to those skilled in the art. For example, Remington ’s Pharmaceutical Science by E.W. Martin (1995) describes formulations that can be used in connection with the disclosed methods. In general, the compounds disclosed herein can be formulated such that an effective amount of the compound is combined with a suitable excipient in order to facilitate effective administration of the compound. The compositions used can also be in a variety of forms. These include, for example, solid, semi-solid, and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspension, suppositories, injectable and infusible solutions, and sprays. The preferred form depends on the intended mode of administration and application. The compositions can also include conventional pharmaceutically-acceptable carriers and diluents which are known to those skilled in the art.

[0098] Examples of carriers or diluents for use with the compounds include ethanol, dimethyl sulfoxide, glycerol, alumina, starch, saline, and equivalent earners and diluents. To provide for the administration of such dosages for the desired application, compositions disclosed herein can comprise between about 0.1% and 100% by weight of the total of one or more of the subject compounds based on the weight of the total composition including carrier or diluent.

[0099] The pharmaceutical earner employed can be. for example, a solid, liquid, or gas. Examples of solid carriers include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Examples of liquid carriers are sugar syrup, peanut oil, olive oil, and water. Examples of gaseous carriers include carbon dioxide and nitrogen. Formulations suitable for administration include, for example, aqueous sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions, which can include suspending agents and thickening agents. The formulations can be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a freeze dried (lyophilized) condition requiring only the condition of the sterile liquid carrier, for example, water for injections, prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powder, granules, tablets, etc. It should be understood that in addition to the excipients particularly mentioned above, the compositions disclosed herein can include other agents conventional in the art having regard to the type of formulation in question.

[0100] Compounds disclosed herein, and compositions comprising them, can be delivered to a cell either through direct contact with the cell or via a carrier means. Carrier means for delivering compounds and compositions to cells are known in the art.

[0101] For the treatment of oncological disorders, the compounds or compositions disclosed herein can be administered to a patient in need of treatment in combination with other antitumor or anti cancer substances and / or with radiation and / or photodynamic therapy and / or with surgical treatment to remove a tumor. These other substances or treatments can be given at the same as or at different times from the compounds or compositions disclosed herein. For example, the compounds or compositions disclosed herein can be used in combination with mitotic inhibitors such as taxol or vinblastine, alkylating agents such as cyclophosamide or ifosfamide, antimetabolites such as 5-fluorouracil or hydroxyurea, DNA intercalators such as adriamycin or bleomycin, topoisomerase inhibitors such as etoposide or camptothecin, antiangiogenic agents such as angiostatin, antiestrogens such as tamoxifen, and / or other anti-cancer drugs or antibodies, such as, for example, GLEEVEC (Novartis Pharmaceuticals Corporation) and HERCEPTIN (Genentech, Inc.), respectively, or an immunotherapeutic such as ipilimumab and bortezomib.

[0102] In certain examples, compounds and compositions disclosed herein can be locally administered at one or more anatomical sites, such as sites of unwanted cell growth (such as a tumor site or benign skin grow th, e.g., injected or topically applied to the tumor or skin growth), optionally in combination with a phamiaceutically acceptable carrier such as an inert diluent. Compounds and compositions disclosed herein can be systemically administered, such as intravenously or orally, optionally in combination with a pharmaceutically acceptable carrier such as an inert diluent, or an assimilable edible carrier for oral delivery. They can be enclosed in hard or soft shell gelatin capsules, can be compressed into tablets, or can be incorporated directly with the food of the patient’s diet. For oral therapeutic administration, the active compound can be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, aerosol sprays, and the like.

[0103] The tablets, troches, pills, capsules, and the like can also contain the following: binders such as gum tragacanth, acacia, com starch or gelatin; diluents such as dicalcium phosphate; a disintegrating agent such as com starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of Wintergreen, or cherry’ flavoring can be added. When the unit dosage form is a capsule, it can contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials can be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules can be coated with gelatin, wax. shellac, or sugar and the like. A syrup or elixir can contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry' or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound can be incorporated into sustained-release preparations and devices.

[0104] Compounds and compositions disclosed herein, including pharmaceutically acceptable salts thereof, can be administered intravenously, intramuscularly, or intraperitoneally by infusion or injection. Solutions of the active agent or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary' conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms.

[0105] The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient, which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. The ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. Optionally, the prevention of the action of microorganisms can be brought about by various other antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal. and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the inclusion of agents that delay absorption, for example, aluminum monostearate and gelatin.

[0106] Pharmaceutical compositions disclosed herein suitable for injectable use include sterile aqueous solutions or dispersions. Furthermore, the compositions can be in the form of sterile powders for the extemporaneous preparation of such sterile injectable solutions or dispersions. In some examples, the final injectable form can be sterile and can be effectively fluid for easy syringability. In some examples, the pharmaceutical compositions can be stable under the conditions of manufacture and storage; thus, they can be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof.

[0107] Sterile injectable solutions are prepared by incorporating a compound and / or agent disclosed herein in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile- filtered solutions.

[0108] Pharmaceutical compositions disclosed herein can be in a form suitable for topical use such as. for example, an aerosol, cream, ointment, lotion, dusting powder, mouth washes, gargles, solution, tincture, and the like. In some examples, the compositions can be in a form suitable for use in transdermal devices. In some examples, it will be desirable to administer them topically to the skin as compositions, in combination with a dermatologically acceptable carrier, which can be a solid or a liquid. Compounds and agents and compositions disclosed herein can be applied topically to a subject’s skin. These formulations can be prepared, utilizing any of the compounds disclosed herein or pharmaceutically acceptable salts thereof, via conventional processing methods.

[0109] Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol / glycol blends, in which the compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers, for example.

[0110] Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.

[0111] Pharmaceutical compositions disclosed herein can be in a form suitable for rectal administration wherein the carrier is a solid. In some examples, the mixture forms unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. The suppositories can be conveniently formed by first admixing the composition with the softened or melted carriers) followed by chilling and shaping in molds.

[0112] In addition to the aforementioned carrier ingredients, the pharmaceutical formulations described above can include, as appropriate, one or more additional carrier ingredients such as diluents, buffers, flavoring agents, binders, surface-active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like. Furthermore, other adjuvants can be included to render the formulation isotonic with the blood of the intended recipient. Compositions containing any of the compounds disclosed herein, and / or pharmaceutically acceptable salts thereof, can also be prepared in powder or liquid concentrate form.

[0113] Useful dosages of the compounds and agents and pharmaceutical compositions disclosed herein can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art.

[0114] The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms or disorder are affected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days.

[0115] In various examples, composition includes a second compound or composition comprising a therapeutic agent. In some examples, the therapeutic agent comprises an anticancer agent.

[0116] Also disclosed herein is a method of treating a disease or condition in a subject. These methods can comprise administering to the subject a therapeutically effective amount of a compound described herein.

[0117] Also provided herein are methods of preventing or reducing the recurrence of cancer in a subject following surgical resection of a tumor that comprise administering to the subject a therapeutically effective amount of a compound described herein.

[0118] Also provided herein are methods of preventing or reducing the recurrence of brain cancer in a subject following surgical resection, the method comprising silencing or reducing expression of CPT1 A in the subject.

[0119] In some examples, the disease comprises a cancer. Exemplary cancers suitable for the present method include prostate cancer, lung cancer, breast cancer, brain cancer, ovarian cancer, lymphoma cancer, leukemia cancer, head and neck cancer, pancreatic cancer, cervical cancer, colon cancer and rectal cancer, endometrial cancer, esophagus cancer, liver cancer, penile cancer, skin-melanoma, skin-nonmelanoma, stomach cancer, testicular cancer, vaginal cancer, uterine cancer, vulvar cancer, paranasal cancer, orophary ngeal cancer, or laryngeal cancer. In some examples, the cancer is breast cancer. In some examples, the cancer is brain cancer, such as glioblastoma, glioma, metastatic brain cancer, a pediatric low grade glioma, or a diffuse intrinsic pontine gliomas (DIPG) induced tumor.

[0120] In some embodiments, the compound is delivered in a biocompatible nanocarrier, such as a nanocarrier targeted to the brain. In certain embodiments, the nanocarrier comprises polymeric nanoparticles described herein, such as polymeric nanoparticles comprising PLGA-b-PEG functionalized with a mitochondrial targeting moiety, such as a triphenylphosphonium (TPP) cation.

[0121] In some embodiments, the methods described herein can further comprise administering a glycolysis inhibitor to the subject, administering a chemotherapeutic agent to the subject, administering a probiotic to the subject, administering a high fat diet to the subject, administering a low carbohydrate diet to the subject, or a combination thereof.

[0122] Also provided herein are methods of preventing or reducing the recurrence of brain cancer in a subject following surgical resection of a tumor. These methods can comprise administering a high fat, low carbohydrate diet to the subject. In certain embodiments, the cancer comprises brain cancer, such as glioblastoma, glioma, metastatic brain cancer, a pediatric low grade glioma, or a diffuse intrinsic pontine gliomas (DIPG) induced tumor.

[0123] EXAMPLES

[0124] To further illustrate the principles of the present disclosure, the following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compositions, articles, and methods claimed herein are made and evaluated. They are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc ); however, some errors and deviations should be accounted for. Unless indicated otherwise, temperature is °C or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of process conditions that can be used to optimize product quality and performance. Only reasonable and routine experimentation will be required to optimize such process conditions.

[0125] Example 1. Low Carbohydrate High Fat Diet Curbs Glioblastoma by Shifting Lipids and Limiting Cancer Stem Cell Metabolic Flexibility.

[0126] Summary

[0127] Metabolic rewiring fuels tumorigenicity of cancer stem cells contributing to universal glioblastoma (GBM) recurrence. Tumorigenic fueling of glioblastoma stem cells (GSCs) which have invaded into eloquent brain areas become unresectable and resistant to standard therapy. We found that lipid utilization through fatty acid oxidation governs sternness and tumorigenicity of patient derived GSCs using a pool of recurrent and newly diagnosed GBM patients. Transcriptomic analyses of patient derived GSCs and normal astrocytes from epilepsy patients revealed signatory fatty' acid oxidation subty pe in GSCs highlighting a unique metabolic subty pe. Our findings open an avenue to uncover the importance of and the mechanisms whereby lipid metabolism controls GBM recurrence through dietary intervention focusing around GSCs. When the patient derived GSCs are implanted in mouse brain, tumor recurrence can be modulated by controlling fat partitioning to the brain resulting beneficial effects from low carbohydrate high fat diet (LCHFD). The lipid partitioning and metabolism contributing to GSC mediated tumor recurrence was confirmed using metabolomic and lipidomic analyses. Gut microbiome analyses revealed that LCHFD played a significant role in shaping the microbiome and provided clues to further support our data that circadian network was affected. Utilization of dietary modification as a non -pharmacologic treatment for GBM, particularly during the postoperative window, could be of enormous benefit by attacking the preferred metabolism of residual cancer stem cells. This study reveals clues which can be used to understand the signal transduction pathways which can play role in delaying or stopping GBM recurrence by dietary restrictions during the wound healing postoperative period.

[0128] Introduction

[0129] The treatment failure of microscopic residual disease and the resultant dismal survival of GBM is driven in part by intratumoral heterogeneity; cells sensitive to chemoradiation are rapidly repopulated by treatment-resistant descendants of glioblastoma stem cells (GSCs), leading to tumor recurrence. GBM intratumoral heterogeneity is maintained by GSCs, which are characterized by multilineage differentiation, enduring selfrenewal, and promotion of malignant growth. GSCs facilitate therapeutic resistance, tumor invasion, immune evasion, and tumor angiogenesis.

[0130] We started an exploratory work to understand whether metabolic profiles of these treatment resistant invading GSCs can contribute to their sternness, tumorigenicity, and recurrence potential. A report utilizing patient derived disintegrated tumor tissue in xenograft model demonstrated potential beneficial effect of low carbohydrate-high fat diet. A study utilizing commercially available patient derived glioblastoma cell lines concluded negative effect of high fat diet due to participation of hydrogen sulfide. There are few completed and active clinical trials investigating the benefits of diet changes for GBM patients and some of these documented potential benefits of high fat diets; in contrast, lab studies documented conflicting findings in terms of tumor growth in rodents. This current work is geared towards understanding the signal transduction pathways which can be driven by different diets to find a correlation betw een metabolic preferences of GSCs in response to dietary restrictions to prevent or delay tumor recurrence. In addition to understand the clinical significance of diet effect on glioblastoma recurrence, our present work can potentially help to shine some light in the dichotomy between what so far is known in this area. Our work utilizing GSCs from a pool of newly diagnosed and recurrent patients indicated that these cells utilize fatty acid-based metabolism which contribute to tumor progression. We found that in GBM, GSCs utilize fatty’ acids via fatty acid oxidation (FAO) for growth and survival. Thus, we set out to understand whether dietary fat content and peripheral fat utilization can contribute to proliferative potential of GSCs and whether such dietary' intervention can be therapeutically beneficial. In this example, we describe our results to shed light into the utilization of dietary modification as a non-pharmacologic treatment for GBM particularly in the immediate postoperative period when patients are not treated with chemoradiation to allow for surgical wound healing and to suggest that our findings may be worthwhile to investigate in humans (Fig. 11).

[0131] Results and Discussion

[0132] Analyses of Signatory Metabolic Pathways in Patient Derived Glioblastoma Stem Cells Distinguishable from Patient Derived Normal Astrocytes. Developing dietary intervention for inhibiting the metabolic fueling of GSCs post-surgery during the wound healing period will require an in-depth understanding about the substrate utilization characteristics of these cells to generate ATP for their growth and proliferation. Thus, we focused on gaining knowledge about the metabolic preferences in patient derived GSCs from two recurrent and two newly diagnosed patients (Fig. 1A) and compared their genomic profiles with normal astrocytes, the glial cells that undergo malignant transformation and gives rise to GBM. The gene sequence for patient derived GSCs 1-4 is submitted in the Gene Expression Omnibus (GEO) database with accession no. GSE274672. We analyzed normal astrocyte samples from brain of 6 living subjects data from GEO database with an Accession no. GSE67835. The brain samples for the astrocytes were obtained from living subjects at the time of epilepsy surgeries, and the tissues were confirmed as normal through electroencephalogram (EEG) and pathological examinations (Fig. IB). Principal component analyses (PC A) showing significant separation between normal astrocytes and patient derived GSCs hinted strong gene expression differences between the cancer versus normal glia cell types (Fig. 1C). The correlation matrix with a negative correlation between the astrocytes and patient derived GSCs suggested that the variables follow a opposite directions, if one variable increases, the other tends to decrease, and vice versa (Fig. ID) Based on the gene alignment, 5381 genes were significantly (P < 0.05) upregulated, and 684 genes were significantly (P < 0.05) downregulated in patient derived GSCs compared to the normal astrocytes (Fig. IE). The upregulated genes that had the highest fold change difference between the normal astrocytes and GSCs were annexin A2 (ANXA2). transgelin 2 (TAGLN2 matrix metalloproteinase 16 (MMP 16). chloride intracellular channel 1 (CLIC1), midkine (MDK), p53 apoptosis effector related to PMP22 (PERP), which are prognostic markers for GBM. These genes are mainly associated with regulation of cell growth, signal transduction, invasiveness, and self-renewal properties in GBM, specifically in GSCs. Elevation in MDK is associated with temozolomide (TMZ) resistance and promoting cancer stem-cell like property'. Among the downregulated genes, ethanolamine-phosphate phosphor-lyase (ETNPPL) is known to be expressed in astrocytes for regulating metabolism of phosphatidylinositol and phosphatidylserine. Solute carrier family 1 member 2 (SLC1A2) is characterized to be less expressed in glioblastoma and more in healthy brain tissue. Neurexin-1 (NRXN1) facilitates neuron like differentiation while suppressing tumor progression. We also observed downregulation of glial fibrillary acidic protein (GFAP) gene in the patient derived GSCs (Fig. IE). Depleted GFAP in the patient GSCs compared to normal astrocytes indicated persistent invasion of GSCs into the brain parenchyma migratory dynamics. Next, we focused our analyses on the potential and confirmed prognostic markers which contribute to poor outcomes in GBM and observed a significantly high differential expression of these genes in GSCs compared to the astrocytes. For example, an elevated expression of AKT serine / threonine kinase 1 (AKT1). disheveled- 3 (1)VL3). inhibitor of differentiation 3 (11)3). and nestin (NES) confirmed the sternness characteristics of the patient derived GSCs (Fig. IE). High vimentin (VIM) expression on the patient derived cells suggested that the GSCs highly invasive and therapeutic resistance characteristics (Fig. IE). A landscape of genetic differences between the astrocytes and the patient derived GSCs can be observed in the hierarchical clustering heatmap of the top 2500 differentially expressed genes (DEGs) (Fig. IF). By utilizing the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses on the top 500 DEGs between the normal astrocytes and the patient derived GSCs, we focused on the pathways that showed enrichment between the astrocytes and GSCs to dissect any potential metabolic signatures for the GSCs (Fig. 1G). These analyses revealed a significant enrichment of genes associated with tricarboxylic acid cycle (TCA cycle) and oxidative phosphorylation (OXPHOS) in the GSCs (Fig. 1G). From these observations, we hypothesized that the GSCs may be utilizing either glucose, glutamine, long chain fatty acids for oxidation in the mitochondria to gather energy for their growth and proliferation. Further analysis indicated that the carbon metabolism is enriched significantly in the patient derived GSCs compared to normal astrocytes suggesting utilization of carbon substrate such as acetyl-CoA potentially from fatty acids and utilization by the TCA cycle (Fig. 1G). For further validation of our hypothesis that fatty acids may be utilized by the mitochondria for oxidation to promote an energy efficient ATP production pathway, we relied on the Volcano plot represented in Fig. IE which suggested elevated levels of fatty acid synthase (FASN) and acetyl-CoA acetyltransferase 2 (ACA / 2) involved in endogenous fatty acid synthesis and lipid metabolism, respectively. These observ ations suggested the potential role of fatty acid metabolism in fueling tumorigenic potential of the GSCs. When clustering analyses were performed on top 100 DEGs between the patient derived GSCs and human astrocytes utilizing C-means clustering algorithm, we observed that out of the 5 gene clusters, fatty acid metabolism related pathways were enriched in cluster 2 and asymmetric stem cell division was enriched in cluster 3 (Fig. 1H). These observations suggested that the GSCs possess a metabolic preference towards utilizing fatty acids as substrates for their high energy demand. We also observed significantly upregulated genes with respect to unfavorable prognosis in glioblastoma (Fig. II) and sternness potential (Fig. 1J) in patient derived GSCs in contrast to human astrocytes. These distinct genetic differences and contrasts between normal astrocytes in the brain and patient derived GSCs from the varied background highlights that a change in metabolic preference towards fatty acid oxidation (FAO) potentially drives the invasiveness and tumorigenicity of the GSCs.

[0133] To validate our sequencing analyses, we further confirmed the sternness of the cell lines derived from the patients by performing Western Blot analyses for Nestin, SOX2, Vimentin, Nanog. and CD133. These data indicated prominent cancer sternness properties in GSCs derived from recurrent patients 1 and 2 compared to the newly diagnosed patients (Fig. 2A). These data were further supported by immunofluorescence analyses of the cancer specific sternness marker CD133 (Fig. 2B) and all the other sternness markers. Proliferation marker Ki67 indicated aggressive nature of the recurrent patients’ cells compared to the ones derived from newly diagnosed patients (Fig. 2C). We also characterized these cells at multiple passages. The sternness markers at passage number 9, 12, 13, 14 and every 5 passages thereafter for patients 1, 2, 3, and 4, indicating that these cells maintain their sternness and tumorigenic properties.

[0134] We then analyzed the state of glycolysis in GSCs by studying the changes in extracellular acidification rate (ECAR) in response to the sequential administration of D-glucose, oligomycin, and 2-deoxy-D-glucose (2-DG). The data indicated that the glycolysis level was low7in patients' cells (Fig. 2D). Analyses of OXPHOS confirmed our RNA sequencing analyses that the GSCs rely on mitochondrial respiration. We then investigated glutamine, glucose, and FAO based metabolic profiles of the GSCs (Fig. 2E for FAO, glucose and glutamine not shown). Our studies confirmed our hypothesis based on RNA sequencing analyses that GSCs rely mostly on FAO for their energy need. We next asked whether the enhanced FAO in patient derived GSCs could be a result of cell culture condition. Thus, we compared the FAO, glucose, and glutamine pathways in patient 1 GSCs grown in B27 and N2 supplement, in 10% fetal bovine serum (FBS), or in supplement and absence of 10% FBS. Under all these conditions, GSCs utilize FAO as a major pathway for their ATP need. Quantification by Western Blot analyses of FASN which catalyzes fatty acid synthesis by Western Blot analyses indicated greater expression of this protein in recurrent patients compared to the newly diagnosed patients (Fig. 2F). Quantification of medium-chain acyl- CoA dehydrogenase (ACADM), the enzyme which catalyzes the first step of mitochondrial FAO and hydroxy acyl-CoA dehydrogenase / 3-ketoacyl-CoA thiolase / enoyl-CoA hydratase subunit A (HADHA) which catalyzes the last three steps of mitochondrial FAO indicated higher expression of these proteins in the recurrent patients compared to two newly diagnosed patients (Fig. 2F). The increased FAO based metabolism in these patient GSCs was further supported by an enhanced mitochondrial ROS in patients with recurrent disease compared to newly diagnosed patients (Fig. 2G, Fig. 2H); whereas cytosolic ROS was at the similar level across these GSCs (Fig. 2G, 2H) Our analyses of the expressions of the genes involved in mitochondrial respiration revealed that these genes are upregulated in the recurrent patient 1 compared to the other patients (Fig. 21). Patient 1 had significantly upregulated the expressions of MT-ND2, MT-ND3, MT-ND4. MT-ND4L, MT-ND5 an MT- ND6 coding for complex I, gene MT-CYB coding for complex III; gene MT-CO1. MT-CO2 and MT-CO3 coding for complex IV and gene MT-ATP6 and MT-ATP8 coding for complex V. A heatmap of differentially expressed genes for FAO between normal astrocytes and the patient derived GSCs further confirmed that FAO is a signatory pathway which can be utilized for dietary interventions of GBM patients (Fig. 2J). The gene sequence enrichment analyses (GSEA) graphs and gene ontology (GO) plots for all the analyses were determined.

[0135] Restricted Fat Partitioning to the Brain Decreases Tumorigenicity of Glioblastoma Stem Cells and Increases Survival. Our understanding that metabolic need of GSCs can be satisfied through mitochondrial OXPHOS using FAO as a major source of energy production led us to hypothesize that the clinical unmet needs in the treatment strategy to stop or delay GBM recurrence are following: (1) addressing how to utilize dietary' modification as a non-pharmacologic treatment for GBM, particularly during the postoperative window in the immediate postoperative period when patients are not treated with chemoradiation, and (2) understanding signal transduction pathways how dietary restrictions can affect the circadian rhythm, gut microbiome, and other cell signaling pathways for a specific GBM subtype to design specific dietary needs which might be suitable during two-week postoperative period to initiate destruction of the residual cancer stem cells by attacking their preferred metabolism which promote their sternness and tumorigenicity and injunction with traditional therapies after to stop or delay recurrence.

[0136] To probe our hypotheses, we first studied the effects of dietary intervention on GBM tumor progression on an orthotopic patient derived xenograft (PDX) model of patient 1 GSCs. A cohort of female and male BALB / c nude mice were divided into two groups: in group 1, 18 mice were kept under normal diet (ND) and in group 2, 17 mice were kept under low carbohydrate high fat diet (LCHFD); the normal diet comprised 24.0% protein, 18.0% fat, and 58.0% carbohydrate and LCHFD comprised 18.3% protein, 60.3% fat. and 21.4% carbohydrate (Fig. 3A). The calorie intake and blood / ^ketone levels were monitored regularly (Figs. 3B and 3C). Although the two groups were placed on different diet ty pes, we observed comparable calorie consumption per animal (Fig. 3B). Once the mice in the LCHFD group showed significantly higher blood Lketone levels compared to the ND groups and the ketosis level was maintained, we implanted luciferase expressing recurrent patient 1 GSCs, Patient 1 GSCLucorthotopically in the brain of these animals (Fig. 3A for details and for intracranial axes). The quality and transduction efficiency of luciferase expression in patient 1 GSCs was validated using in vivo imaging system (IVIS) as well as quantifying the luminescence and normalizing the data with respect to protein content of the cells to validate that the transfection efficiency was maximum. These validations are important particularly when studying whether dietary interventions can prevent or promote tumor formation in anatomically challenging intracranial space, if not all cells in the population are expressing luciferase, tumor growth or size estimations from the bioluminescence signal may be inaccurate. The distinctive dietary regimen for both the groups continued throughout the study where the ND group continued to receive normal diet and the LCHFD group continued the low carbohydrate high fat diet. All animals in each group maintained comparable body weight ranges (Fig. 3D). The tumor progression in all mice was monitored using IVIS imaging. We observed that tumor occurrence was significantly more in ND group compared to the LCHFD group (Fig. 3E, 3F). Quantitatively, out of 18 animals from the ND group, 17 exhibited significantly high tumor burden (Fig. 3E, 3F). In contrast, only 2 of 17 animals in the LCHFD group showed tumor formation and the tumor volume for these two animals were significantly lower compared to the ND group (Fig. 3E, 3F). Our observations supported that fat metabolism under ketosis in the LCHFD group enhances peripheral fat metabolism, increasing fatty acid utilization by peripheral organs such as by the liver. This altered metabolic pathway limits fatty acid availability for brain cells, restricting the preferred metabolic substrate supply for GSCs to support their ATP production by FAO. Consequently, in the animals of LCHFD group, we see less tumor formation.

[0137] We also observed significantly higher survival of mice in the LCHFD group compared to the ND groups (Fig. 3G). To visualize the tumors in the brain in the animals in the ND group more directly, we employed magnetic resonance imaging (MRI) of 6 representative animals from ND and 6 from the LCHFD group using a 9.4 Tesla scanner equipped with a gradient and a mouse head transceiver volume coil for data acquisition. Anatomical T2-weighted RARE images indicated large tumor volume in all the animals in ND group and no such tumor area was observed in the LCHFD group (Fig. 3H). For each animal which showed tumor, tumor masks were manually defined in each slice in the coronal view of the brain and the total volume was then calculated using the ‘-V’ option of the command line tool fslstats of FSL. The quantification of tumor volume (mm3) measured for the tumor bearing animals under ND demonstrated aggressive tumor progression (Fig. 31). As a control, a pilot study analyzing the effects of low-fat diet (LFD) on the grow th of patient 1 GSCs following the same experimental protocol did not offer any beneficial outcome on tumor grow th and survival further supporting our hypothesis that lipid restriction to the brain is the key for controlling the growth inhibition and proliferation of these GSCs.

[0138] Orthogonal Validation of Beneficial effects of LCHFD using Less Proliferative Patient 2 GSCs. The patient 1 GSCs are highly proliferative as evidenced form their Ki67 score of 79% (Fig. 1A) and these cells are aggressive to result in rapid tumor formation and out data demonstrated the ability of dietary intervention can be beneficial for such highly proliferative GBM subtype. In contrast, patient 2 GSCs are less proliferative with a Ki67 value of only 10-15% (Fig. 1A). Such less proliferative GBM subtype are difficult to treat since their infiltration is much more aggressive compared to highly proliferative subtype. Thus, we next investigated whether the LCHFD diet can be beneficial for less proliferative GBM subtype utilizing luciferase expression patient 2 GSCs. The luciferase expressing patient 2 GSCs w ere characterized, and the transfection efficiency was quantified as it w as done with patient 1 GSCs to make sure that the conclusions and comparisons drawn from the two cells lines are rigorously carried out. Patient 2 GSCLucmediated orthotopic brain PDX tumor formation and growth analyses was performed using ND and LCHFD groups following the same protocol as mentioned for Patient 1 GSCs. The blood / -ketone levels were monitored prior to tumor implantation, and a significantly high blood / -ketone level and less glucose level in the LCHFD group indicated onset of ketosis in the animals as well as during the entire duration of experiment (Fig. 3J, data shown for week 7). In this less proliferative GBM subtype as well we observed aggressive tumor formation under ND, but no such tumor formation in the mice fed with LCHFD (Fig. 3K). In this study, more than 72% of the animals in ND group demonstrated aggressive tumor formation, whereas none of the animals in the LCHFD group formed tumor until 90 days post cell implantation (Fig. 3L). These data suggested that dietary interventions using LCHFD can be utilized as a non- pharmacological modality for both highly proliferative and less proliferative GBM subtypes.

[0139] Effects of LCHFD on Adherence and Proliferation of GSCs at the Implantation Site. During these studies, we questioned whether the Patient GSCs when implanted intracranially under LCHFD interventions experience different local brain environment which might contribute to initial adherence and survival of the GSCs immediately after implantation and whether this parameter is playing role in tumor formation and eventual growth. To address these issues, we analyzed brain coronal sections from mice either 48 h or 72 h post implantation of Patient 2 GSCLucunder LCHFD and observed the presence of Ki67 positive viable cells in the brain region. Our data indicated that CD133 positive Patient 2 GSCs are proliferative as evident from their high Ki67 expression (Fig. 3M, top), in contrast the cells away from the implantation site did not have any appreciable Ki67 expression (Fig. 3M, bottom) further adding another layer of rigor in our data that LCHFD does not impair initial GSC adherence or survival but prevents tumor formation through metabolic alterations.

[0140] Dietary Interventions Alters Signatory Tumor Metabolic Markers. The results obtained from qualitative imaging to support that LCHFD has beneficial effects for tumor control of GBM were further confirmed by ex vivo tumor analyses. To focus on the correct region of the brain area, we used the coordination axes which were used for intracranial tumor implantation (Fig. 3A) and further analyzed the injection site by using MRI (Fig. S14). Keeping this information in mind, we performed H and E imaging of representative whole brain tissues from LCHFD and ND groups (Fig. 4A and 4B). The whole brain images further confirmed the absence of tumor region in the brains of the LCHFD group and presence of extensive tumor region in the brains of the ND group (Fig. 4A and 4B). We further collected the H and E images zooming at the injection site (Fig. 4A and 4B) and using these areas of the tissues, immunofluorescence imaging was carried out to quantify the cancer sternness marker CD 133 and proliferation marker Ki67. Extensive imaging using tissues from different animals in the group and various tissue regions of the same animal, we found that the ND group contain significantly higher presence of CD133 positive GSCs and the Ki67 marker was also significantly higher compared to the LCHFD group (Fig. 4C and 4D).

[0141] We also observed a higher expression of vimentin in the surrounding cells in the LCHFD group indicating tissue repair. Our analyses also indicated reduced expression of fatty acid transporting CD36 in the brain of the LCHFD group supporting that peripheral fat utilization probably results in restriction of fat transport to the brain. The LCHFD group showed reduced expression of fatty acid synthesis FASN and HADHA for fatty acid oxidation indicating that restricting fat access by the brain result in metabolic changes to the tumor cells. Our studies also documented that nutrient restriction results in overall metabolic collapse as evidenced from reduced expression of the glucose transporter GLUT1 and other glycolytic markers. These observations further supported that the metabolic restriction results in overall destruction of tumor cells as evidenced from increased caspase 9 expression in LCHFD group.

[0142] Improved Cognitive Recognition Functions of LCHFD Fed Animals. One of the important parameters which should be considered for treatment or lifestyle changes in GBM patients is the quality of daily living activities. We investigated the neurobehavioral pattern of the animals from the above study to understand whether the lowered tumor burden arising from the diet manipulation can improve neurocognitive behavior of these animals. Using foot placement test and open field test we were able to study the differences in the walking pattern, exploratory behavior, and locomotory functions between the tumor bearing animals under ND and LCHFD fed animals without tumor. Foot placement analysis trace path demonstrates walking impairment is correlated to tumor burden. Tumor bearing animals cannot walk straight across the platform, instead, they tend to walk towards the edges of the platform or make circular movements (Fig. 5A). Animals under LCHFD, with no tumor, reveal no walking impairment. Quantification of the number of steps taken by the animals in different groups it was observed that for the same distance, the number of steps taken by animals with tumor under ND are higher compared to animals under LCHFD, suggesting neurological dysfunctions (Fig. 5B). Open field trace path and the respective quantification revealed that the tumor bearing animals in the ND group have a lower tendency to explore the field compared to the non-tumor bearing animals in the LCHFD group (Figs. 5C and 5D). Trace path LCHFD fed animals resembled nude mice with no brain PDX. These data suggested that the mice under LCHFD with less or no tumor burden showed no motor function impairment compared to the animals under ND group with high tumor burden. All data suggested that LCHFD can be a potential aid to slow the tumor recurrence from the GSCs with improved neurocognitive behavior pattern.

[0143] Improved Gut-microbiome Structure of LCHFD Fed Animals. In addition to understanding the major metabolic alterations in tumor progression by LCHFD dietary intervention, it is important that we explore the effects of this non-pharmacological intervention on the health and well-being of the animals. Since, the gut-microbiome can potentially shape human health and lifestyle, we utilized fecal DNA samples isolated from the animals under ND and LCHFD to analyze any shift in gut-microbiome structure due to the diet, presence of tumor, or both. We also analyzed the gut microbiome of animals maintained on a normal diet without tumor cell inoculation, serving as the normal diet control group. A principal component analysis (PCA) was carried out for integration of our microbiome sequencing. The prognosis of GBM is known to be affected due to an altered gut-microbiome, which emphasizes the importance of learning whether LCHFD has the ability7to show' an improved outcome. We were able to quantify the difference in %relative abundance of all the microbial species between the ND and LCHFD groups and observed an elevated abundance of Lactococcus lactis in the gut-microbiome of all animals under LCHFD, which highlights a probiotic effect (Fig. 6A). We further analyzed the difference in %relative abundance with respect to phylum demonstrating a remarkable increase in %relative abundance of phylum Bacteroidetes in ND fed animals with tumor (Fig. 6B). This phylum is known to be enriched in tumor tissues. The LCHFD group showed significantly lower %relative abundance of the Bacteroidetes phylum but higher %relative abundance of phylum Firmicutes compared to both ND group and control animals (Fig. 6B). The low' abundance of Bacteroidetes and high abundance of Firmicutes under LCHFD validated the importance for an intervention to be able to improve the gutmicrobiome structure for an effective clinical outcome. When we compared the ND and LCHFD groups based on the %relative abundance at the hierarchical level of microbial family, we observed all animals in the LCHFD group revealed a high abundance of Streptococcaceae (Fig. 6C), suggesting a shift towards anti-tumor activity and improved immune surveillance. Upon further analyses of the bacterial composition based on the hierarchical order of the microbiome, we observed an elevation in %relative abundance of Lactobacillales in LCHFD, thus, supporting our observation at the species level and highlighting the importance of LCHFD in improving the gut-microbiome structure (Fig. 6D). Utilizing the results from %relative abundance, we also determined two additional factors that indicate a shift towards improved outcome. First, we quantified the Firmicutes:Bacteroidetes (F / B) ratio where a significant upregulation in LCHFD group indicated a shift towards achieving a healthy stage (Fig. 6E). Second, corresponding to the F / B ratio, a significantly less %relative abundance of Bacteroidetes in LCHFD highlight an improved microbiome (Fig. 6E). It is important to note here that when we compared to ND fed animals that never received any tumor implantation, we observed a significantly high level in the F / B ratio that supports the effectiveness of LCHFD on providing a shift towards a healthy outcome. These findings together suggest that the microbiome of the animals with tumor in ND group and normal diet control animals possess similar gut microbiome compared to the animals in the LCHFD group. Consumption of LCHFD reshapes the gut microbiome, promoting the establishment of a distinct microbial niche that may contribute to the suppression of tumorigenesis and consequently decrease cancer risk.

[0144] Metabolomic Mapping of Liver and Brain Samples from ND and LCHFD GBM Models. To understand the metabolites produced by the brain and peripheral nutrient utilization by the liver, we conducted metabolomic analyses of the liver and brain samples from both ND and LCHFD groups. Liver and brain samples from 3 animals of ND and 3 animals of LCHFD groups were analyzed using hybrid LC-MS-based metabolomic analyses. Untargeted metabolomic analyses of brain samples identified 403 named metabolites in at least 3 of 12 samples, with 325 detected across all samples. Additionally, 1,876 unnamed metabolite features were detected in at least 3 samples, and 1,486 were present in all samples. In liver samples, 502 named metabolites were identified in at least 3 samples, with 414 detected in all 12 samples, alongside 2,301 unnamed metabolite features in at least 3 samples and 1,865 in all samples. Data were analyzed using principal component analysis (PCA), unsupervised hierarchical clustering, volcano plots, and statistical comparisons to identify tissue-specific metabolic signatures. For both tissue types, the PCA of all samples showed separation of groups with the brain samples showing distinct group separation along the PCI axis. The Venn diagram comparison of significant metabolites in brain and liver samples with a significance of P < 0.05 is represented in Fig. 7A where we observed 3 commonly expressed metabolites between the brain and liver. These metabolites were pantothenic acid, betaine, and 2-Mercaptopyridine N-oxide. The global Volcano plots suggested most of the metabolites are downregulated in the brain samples of LCHFD compared to the ND group brains (Fig. 7B). We performed unsupervised clustering analyses between ND, and LCHFD liver and brain samples depicting the metabolites that are significantly up or downregulated between ND and LCHFD groups. This analysis confirmed 142 named metabolites which are significantly different between the brain sample groups, and 28 named metabolites are significantly different between the liver sample groups. The 3 common metabolites between brain and liver samples were observed to be increased in the liver of LCHFD group and decreased in the brain of LCHFD group. (Fig. 7C). The decreased levels of pantothenic acid in the brain suggested less involvement of brain cells in the synthesis of fatty acids and phospholipids (Fig. 7C). Additionally, reduced levels of betaine in the brain samples of LCHFD potentially indicate downregulation of sternness of GSCs, as betaine has inhibitory effects on stem-like properties (Fig. 7C). These data also directed us to a striking observation that glutathione (GSH) ( = 0.0131, Log2 fold change = 4.984) was upregulated in the LCHFD group liver samples compared to the ND group inferring upregulated lipid metabolism in the liver of LCHFD group (Fig. 7C). The loss of GSH did not cause liver failure, but it decreased lipogenic enzyme expression, circulating triglyceride levels, and fat storage. Further, we observed upregulation of nicotinic acid in liver of LCHFD group as the nicotinic acid regulates low density' lipoprotein, cholesterol, and low-density' lipoprotein levels (Fig. 7C). The distinguished metabolic features observed in the animals in the LCHFD group did not induce any hepatoxicity as evident from the plasma aspartate aminotransferase (AST) level indicating comparable values among the groups.

[0145] Further, we investigated the pathway analyses for brain and liver samples from these groups to understand how the pathways which are mostly affected by the differentially expressed metabolites. For the analyses, we considered the metabolites which are significantly present in one of the groups compared to the other with aP < 0.01 and we found that arginine biosynthesis, vitamin B6 metabolism, and phenylalanine, tyrosine and try ptophan biosynthesis were highly impacted (Fig. 7D). Amino acid synthetic precursors were decreased along with components of TCA cycle and pyruvate metabolism in the brain samples of LCHFD compared ND group. Components of TCA cycle and pyruvate metabolism such as malate (P = 0.0006, Log2 fold change = -2. 149) and fumarate (P = 0.0013, Log2 fold change = -0.967) were found to be decreased in LCHFD group brain samples compared to the ND group which may be due to the reduced energy production requirement in the brain due to the absence of tumor in the LCHFD brain samples (Fig. 7E and Fig. 7F). Arginine biosynthesis was highly impacted as 3 of the major metabolites were significantly less in the LCHFD brain samples compared to ND brain samples (Fig 7G). We also investigated vitamin B6 metabolism as it is important for normal brain development and for keeping the nervous system and the immune system healthy. Metabolites of biosynthesis of Vitamin B6 such as pyridoxal was significantly higher (P = 0.0045, Log2 fold change = 3.864) in brain samples of the LCHFD group (Fig. 7H). Aromatic amino acid synthesis was also affected significantly with lower presence of tyrosine (P = 0.0145, Log2 fold change = -1.642) in the brain samples of LCHFD group compared the brain samples of ND group (Fig. 71). These observations suggested that there is reduced OXPHOS and ATP production in the LCHFD group brain samples. A comparison of LCHFD group brain metabolism with ND group indicated downregulation of amino acid biosynthesis (Fig, 71).

[0146] Altogether metabolomic mapping in the brain and liver samples from two groups indicated that LCHFD restricted fat availability to the brain through utilization of the fat by peripheral liver metabolism which resulted in less metabolic activity of the GSCs to survive and / or grow. Elevated levels of metabolites in ND animals with tumor suggested that the normal diet animal with tumor have higher energy and amino acid requirements compared LCHFD normal mouse. These profiles revealed distinct brain metabolic alterations under LCHFD, potentially contributing to the suppression of GSC tumorigenesis observed in our intracranial implantation model.

[0147] Dietary Intervention Regulates Lipid Distribution between Peripheral Organ and Brain in LCHFD Glioblastoma Model. To understand the lipid production and regulation in LCHFD and ND groups, we performed lipidomic analyses on the samples from brain and liver. Samples were analyzed using LCMS method after scaling the lipid extraction for each sample. Detected lipids were first identified and classified into specific lipid classes. For the brain samples, 741 putatively annotated lipids across 17 classes were found in at least 3 samples, and 2667 unnamed features were found in at least 3 samples. For the liver samples, 819 putatively annotated lipids across 13 classes were found in at least 3 samples, and 2547 unnamed features were found in at least 3 samples. The Venn diagram comparison of significant lipids in brain and liver samples with a significance of P < 0.05 is represented in Fig. 8A, where we observed higher number of lipid species detected in the brain compared to that of the liver. Out of these lipid species, 30 lipids were common between brain and liver, however, further analyses are required to determine whether they play a crucial role in driving metabolism (Fig. 8A). A full PCA of the 2 groups showed distinct separation between LCHFD and ND samples along the PCI axis for both sample sets. In the brain samples. 264 annotated lipid features were found to be significantly different between the two groups with a majority showing increased levels in the brain samples of LCHFD group (Fig. 8B). In the liver samples, 110 annotated lipids features were found to be significantly different between the two groups, with most of the lipids show ing increased in the liver samples of LCHFD group (Fig. 8B). Among the significantly upregulated lipids in the liver samples between LCHFD and ND, determined through the volcano plot, we observed the presence of various diglyceride species like DG(36: 1) (P = 0.0003, Log2 fold change = 1.855), DG(38:6) (P = 0.0006, Log2 fold change = 1.614), DG(38:5) (P = 0.0011, Log2 fold change = 1.415) and DG(36:2) (P = 0.0019, Log2 fold change = 1.884) suggesting elevated lipid metabolism in the liver of LCHFD fed animals (Fig. 8B). Among the significantly upregulated lipids in the brain samples of LCHFD group, we observed the presence of phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE) namely PC(40:5) (P = 0.0034, Log2 fold change=1.843) , PE(38: 1) (P = 0.0019, Log2 fold change = 1.378), PS(44: 10) (P = 0.0141, Log2 fold change = 2.396), PS(42:9) (P = 0.0102, Log2 fold change = 2.576), PE(40:6) (P = 0.0029, Log2 fold change = 1.018) and PE(36:2) (P = 0.0043, Log2 fold change = 1.070) which are considered to be structural lipids w ith involvement in lipid bilayer formation (Fig. 8B). Similar observation was revealed through the unsupervised hierarchical clustering analysis of the lipids in the liver and brain samples of LCHFD (Fig. 8C) and ND (Fig. 8D) fed animals. High abundance of PS in the LCHFD brain samples indicate improved cell-to-cell communication and healthy brain functioning. It also indicates the ability of the brain cells to utilize PS as an indicator of apoptotic signaling when required. Comparing the significantly upregulated lipids in brain and liver, we w ere able to highlight the increased number of PC and PE in brain (Fig. 8E). Presence of PC and PE in the brain indicated improved neurotransmitter function and a shift tow ards healthy state of the brain. Despite the extensive results from lipidomic analyses suggesting LCHFD’ s role in partitioning lipids betw een peripheral organ and brain, the origin of the detected lipid species remains unclear. To answer this, we analyzed whether the elevated lipid species in the brain and the liver directly come from the chow that the animals are fed with. The results reveal only 4 lipids were common bet een the liver samples of LCHFD vs ND groups and LCHFD vs ND chow; with the lipid species being different from the ones we observed in the volcano plots and unsupervised clustering (Fig. 8F). Results based on the comparison between brain samples of LCHFD vs ND groups and LCHFD vs ND chow, we only observed a single lipid to be common which was again different from the ones we observed in the volcano plots and unsupervised clustering (Fig. 8F). These data validated that the lipids upregulated in the LCHFD liver are metabolic by-products and these lipids are restricted in the brain. Our data also suggested that presence of polyunsaturated fatty acid upon the LCHFD dietary intervention is a promissory way to improve metabolism of the host.

[0148] In summary , the lipodomic analyses supported that LCHFD induces distinct lipid profdes in the brain and liver samples in the context of orthotopic GBM PDX model compared to ND regimen. In the brain. LCHFD brings upregulation of structural phospholipids leting us to conclude enhanced neurotransmiter function, cellular networking, and probably enhanced neuroprotection resulting halted GBM progression. In contract, in the LCHFD group liver there is increased diglycerides suggesting elevated lipid metabolism. There is no significant overlap between tissue and dietary chow lipids further strengthening the conclusion that these changes are fueled by metabolism not due to dietary uptake. Altogether, these data supported LCHFD ’s therapeutic potential for GBM by redistributing the lipids between peripheral organs and the brain.

[0149] In summary, the lipodomic analyses supported that LCHFD induces distinct lipid profiles in the brain and liver samples in the context of orthotopic GBM PDX model compared to ND regimen. In the brain, LCHFD brings upregulation of structural phospholipids leting us to conclude enhanced neurotransmiter function, cellular networking, and probably enhanced neuroprotection resulting halted GBM progression. In contract, in the LCHFD group liver there is increased diglycerides suggesting elevated lipid metabolism. There is no significant overlap between tissue and dietary chow lipids further strengthening the conclusion that these changes are fueled by metabolism not due to dietary' uptake. Altogether, these data supported LCHFD ’s therapeutic potential for GBM by redistributing the lipids between peripheral organs and the brain.

[0150] Possible Connection with Circadian Network. Conclusions from our metabolomic and lipidomic led us to hypothesize whether the LCHFD-induced lipidomic changes in our patient GSC derived GBM model with upregulated diglycerides in the liver and phospholipids in the brain, these alterations might intersect with the circadian network. We studied role of circadian clock in the lipid metabolism of these tumor bearing animals under different diet supplements. Disrupted circadian regulation of tumor initiating cancer stem cells contribute to tumor recurrence. Endogenous circadian rhythms are established by transcriptional factors BMAL1 and CLOCK contributing to transcriptional output linked to metabolism, immune regulation, and other cellular pathways. Thus, we hypothesized that under the dietary changes, the GSCs which rely on lipid metabolism for their growth and tumongenicity might show alteration of circadian networks which will open new- mechanistic pathways for non-pharmacological or pharmacological treatment options for GBM. Studies have documented higher expression levels of the core circadian clock gene such as CLOCK and LGMN in the high-grade glioma. A recent study documented disrupted circadian clock in GSCs. Thus, we questioned whether there is a relation between lipid metabolism and circadian clock under the influence of different diet since we documented changes in gut microbiome composition. We thus, analyzed the circadian rhythm associated DEGs between patients 1-4 and observed a positive NES based on regulation of circadian rhythm and circadian rhythm related enriched gene set in the recurrent vs newly diagnosed patient derived GSCs indicating tumorigenic GSCs disrupt the circadian network (Fig. 9A). Critical circadian network related genes were upregulated between the normal astrocytes and patient derived GSCs (Fig. 9B). We compared the levels of LGMN and CLOCK in all the four-patient derived GSCs in comparison with normal brain endothelial or normal human astrocytes indicated that LGMN and CLOCK levels are higher in patient derived GSCs suggesting their potential role in leading cancer aggressiveness (Fig. 9C). We supported our observations using brain homogenates from the in vivo special diet study. Tumor bearing brains from animals under ND and non-tumor brains from the LCHFD group were compared to the brain tissues harvested from BALB / c nude mice without brain PDX. Western Blot data showed a significant difference in CLOCK expression between tumor bearing and non-tumor bearing animals (Fig. 9D). No difference in CLOCK was observed between the control mice and LCEIFD fed mice. Similar expression pattern of LGMN w as observed across the 3 groups (Fig. 9D). Immunofluorescence imaging in tumor implantation regions of the brain sections of ND and LCHFD groups revealed high CLOCK expression in CD 133 positive cells in ND group compared to LCHFD group (Fig. 9E). Brain sections from the LCHFD group showed presence of scarcely populated CD 133 positive cells, however, these cells have possibly reached a state of vulnerability and are not able to proliferate into aggressive tumors due to the less proximity.

[0151] Effect of Low Carbohydrate High Fat Diet When Tumorigenic Patients GSCs are Implanted Peripherally. To summarize the data and draw stronger conclusions supporting the hypothesis that lipid partitioning to the brain plays a pivotal role in controlling tumorigenicity of GSCs via dietary- intervention, we focused on a control experiment where we implanted the GSCs peripherally under the influence of two dietary interventions, ND and LCHFD. As indicated that diet supplementation has significant role in formation and progression of orthotopically implanted tumor. To understand the role of metabolic substrate partitioning between brain and rest of the body, we performed a flank tumor xenograft study with patient 1 GSCs and patient 2 GSCs. For this, BALB / c nude mice were divided into two groups for each study with patient 1 GSC and patient 2 GSC. In each group 15 mice were kept under ND and LCHFD groups for patient 1 GSC implantation study (N = 30) (Fig. 10A). The average weekly calorie intake (kcal)Zanimal throughout the experimental timeline (i.e. , 17 weeks), blood / 5-Ketone levels (mmol / L) and glucose levels (mg / dL) were monitored regularly (Figs. 10B and 10C). Animals in LCHFD have consumed higher amount calorie compared to ND fed animals (Fig. 10B). After 4 weeks, animals in LCHFD have shown significantly higher blood / LKetone levels (mmol / L) and significantly lower blood glucose levels (mg / dL) suggesting that animals are under ketosis. All animals from both groups were inoculated with patient 1 GSCs on the right flank, and their tumor initiation and progression were monitored (Fig 10D). We continued providing the respective dietary supplementation to both groups throughout the experimental timeline and found that out of 15 animals inoculated with patient 1 GSC cells 7 of them (47%) formed tumor in ND fed group compared to 12 (80%) in the LCHFD fed group (Fig 10E). We monitored the tumor progression as well as body weight, and all animals across both dietary groups maintained comparable body weight ranges throughout the study period (Fig. 10F & G). Similar study was carried out in N = 20 BALB / c nude mice with patient 2 GSC after dividing them into two dietary groups (n = 10 for each group). The average weekly calorie intake (kcal) / animal data revealed the similar observation where LCHFD fed mouse consumed higher amount calorie compared ND fed mice (Fig. 10H). After 5 weeks on diet, LCHFD fed animals possessed significantly higher blood kKetone levels (mmol / L) and significantly lower glucose levels (mg / dL) suggesting that LCHFD fed animals are undergoing ketosis (Fig. 101). After 7 weeks on diet, animals were inoculated with patient 2 GSC for tumor formation and 90% of animals in the LCHFD group formed tumor within 90 days of implantation whereas 50% of animals formed tumor in the ND fed group (Fig. 10J and K). Survival rate was not significantly different between the group until 90 days post cell inoculation (Fig. 10L). The tumor volume was steadily increasing for the animals which formed tumor and body weight was in similar range for both groups despite the difference in the tumor formation (Fig. 10M and N). These results showed that in the case of glioblastoma stem cells inoculated at peripheral location, animals fed with LCHFD group shown higher possibility of tumor formation in both PDX models suggesting that the metabolized fat from diet might possibly be easily accessible for tumor cells in the peripheral region for growth and progression.

[0152] Conclusions

[0153] Tumorigenicity of invaded treatment resistant GSCs continues to play an important role in GBM recurrence. In this study, we successfully demonstrate a direct correlation between the sternness potential and preferred metabolic pathways which promote tumorigenicity in the GSCs, the highly resistant cell population in GBM. Leveraging our understanding of the preferred metabolic substrate utilization by the GSCs we focus on developing a strategy that can potentially restrict substrate availability' in the brain by altering peripheral metabolism. In this context, we highlight the ability of a non- pharmacological intervention, a LCHFD supplementation, on regulating the proliferation of the GSC. We achieve the prevention in GSC population by hindering the metabolism which eventually contributes to tumor grow th suppression. Results from in vivo orthotopic glioblastoma mouse model using recurrent patient derived GSCs, indicate continued LCHFD supplementation has the potential to curb GSC growth and survival. In addition to shedding light on the hpidomic and metabolomic structural nuances of the brain and liver tissues of tumor bearing animals under ND and non-tumor bearing animals under LCHFD, our study was able to document an improvement in shaping the gut microbiome and neurological behavior in the animals where metabolic substrate restricts GSCs to grow and proliferate, implying a positive shift towards a healthy state. We observed that LCHFD was associated with a higher incidence of tumor formation in peripherally implanted GSCs, suggesting that metabolized dietary' fats may enhance tumor cell growth in peripheral tissues. Our results strongly support our root hypothesis that lipid availability influences GSC tumorigenicity’, highlighting the role of metabolic substrate partitioning in tumor progression. Our findings suggest that dietary interventions can be considered as a valuable non-pharmacological therapeutic strategy' to prevent or delay GBM particularly in the critical post-surgical period, when chemoradiation is delayed allowing for wound healing. Additionally, dietary interventions could serve as a complementary supplement to conventional therapies following surgery, further supporting efforts to reduce or delay GBM recurrence.

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[0202] 52. Q. Cao et al., Circadian clock cryptochrome proteins regulate autoimmunity. Proc. Natl. Acad. Sci. U. S. A. 114, 12548-12553 (2017).

[0203] 53. Z. Wang et al., Circadian clock genes promote glioma progression by affecting tumour immune infiltration and tumour cell proliferation. Cell Prolif. 54, el2988 (2021).

[0204] 54. Z. Dong et al. , Targeting Glioblastoma Stem Cells through Disruption of the Circadian Clock. Cancer Discov. 9, 1556-1573 (2019).

[0205] Example 2. Small Molecule Inhibitors for FAO in GSCs.

[0206] A series of compounds were synthesized to inhibit FAO in GSCs in a different fashion. Typically, FAO inhibitors include ncompounds which bind to protein targets. In this series of compounds, we synthesized triphenylphosphine (TPP)- modified fatty acid ester compound with the idea that these saturated alkyl chain to the fatty acid could help the TPP-modified fatty acid ester compounds be taken by the cells as fatty’ acid, but the modification with TPP at the end could avoid the intracellular beta-oxidation, resulting in the disorder of cellular mechanism and dysregulation of functional proteins in GSCs (Fig. 12). The inhibited FAO and fatly acid synthesis pathways could reduce the production of ATP and acetyl-CoA, which will greatly suppress the growth of cancer cells. Compared with the classic cisplatin drugs or etomoxir, the heavy metal-free alkyl-TPP compounds should have less nephrotoxicity and neurotoxicity to the normal cells. Therefore, this discovery led us to hypothesize that the FAO mechanism in cancer cells can be inhibited by the alkyl-TPP compounds by affecting the expression of enzymes which are involved in FAO or fatty acid synthesis pathways. Due to the presence of the long alkyl chain and TPP hydrophobic cations, the TPP-modified fatty acid ester may easily cross the mitochondrial membrane and enter the mitochondrial matrix where the FAO process takes place. The uptake of alkyl-TPP will make an impact on the expression of some crucial enzy mes related to FAO pathway, such as long-chain acyl-CoA synthetase (LACS), CPT-la, and FASN. The dysregulation of these crucial enzy mes will effectively inhibit the FAO process in aggressive cancer cells and decrease the production of ATP and acetyl-CoA. The grow th and proliferation of aggressive GSCs will be effectively inhibited because of bioenergy shortage.

[0207] A series of alkyl-TPP with different alkyl chains were synthesized according to the modified synthesis route and characterized with NMR,13C NMR,31P NMR and mass spectra (Figs. 13A-13C). In addition, TPP-ethanol and fatty acids with different alky l chain were also prepared as the control groups. From theXH NMR spectra, all of the compounds had similar peaks at 7.5-8.0 ppm, which belong to the protons of triphenylphosphine moiety and 4.5-4.5 ppm, which could be assigned to the methylene groups adjacent to the P and O atoms. The peaks at 2.3 ppm and 0.8 ppm shown in all theJH NMR spectra were the protons adjacent to the carboxyl group and the methyl groups at the end of the alkyl chain, respectively. The only difference was the proton numbers between 1. 1-1.8 ppm. reflecting the different numbers of methylene in the alkyl chains.

[0208] Due to the same long alkyl chain as fatty7acid and the TPP hydrophobic cations, alkyl-TPP compounds should be easy to cross mitochondrial membrane. The redox stability' is crucial for their FAO inhibition capability. If the alkyl-TPP compounds could also be oxidized in mitochondria like fatty acid, their FAO inhibition will be invalid. Therefore, fatty7acid P-oxidation detection will be performed in vitro by mixing fresh liver homogenate with fatty acid / TPP-modified fatty' acid esters. The P-oxidation of fatty' acids and alky l-TPP was researched according to the concentration changes of ketone bodies, which are mitochondrial ketolysis products of acetyl-CoA. After treatment with lauric acid, the concentrations of the ketone bodies were higher than the other groups. This indicated that the lauric acid could be oxidized by the enzy mes in the liver homogenate, and TPP-ethanol and lauric-TPP cannot be oxidized (Figs. 14A-14B)

[0209] The series of organic small molecules that we synthesized in the lab were studied to investigate their efficacy in inhibiting fatty acid oxidation-based ATP production and the subsequent effects on the cancer sternness and overall sternness. Out of all the organic small molecules, we observed that TPP-palmitate showed the highest reduction in fatty acid oxidation and ATP production in breast cancer brain metastatic MDA-MB-231-BR cells. Utilizing this understanding, to support our findings that suggest a correlation between metabolic modulation and reduced tumorigenicity of the patient derived GSCs, we treat the recurrent patient 1 GSCs with 10 pM TPP-palmitate for 24 h. Our results from Western Blot analyses revealed reduced expression of acyl-CoA dehydrogenase (ACADM) and the trifunctional protein complex of hydroxy acyl -Co A dehydrogenase, 3- ketoacyl-CoA thiolase, and enoyl-CoA hydratase (HADHA), suggesting TPP-palmitate effectively inhibits the initialization and final breakdown of the substrate (Fig. 15A, top showing the TPP- palmitate chemical structure and bottom showing the western blot data). Further, results also demonstrated the correlation between inhibition of fatty acid oxidation and sternness features of the patient-derived GSCs. We observe a reduced expression of cancer sternness marker, CD 133; proliferation marker, vimentin; and SOX2, the transcription factor which provides the cancer initiating stem cells their self-renewal capacity’ (Fig. 15B).

[0210] Taken together, this shows that TPP-palmitate. a mitochondrion targeting organic small molecule can potentially lead to reduced sternness of cancer initiating stem cells by altering their metabolic substrate utilization. This further corroborates with our findings, signifying metabolic modulation induces vulnerability in cancer initiating stem cells, affects their tumorigenicity, and finally holds potential in stopping tumor recurrence.

Claims

CLAIMSWhat is claimed is:

1. A compound defined by Formula I belowA'-R^^Z Formula I or a pharmaceutically acceptable salt thereof, wherein A comprises a mitochondrial targeting moiety:R1is a direct bond, or represents a substituted or unsubstituted C1-C10 alkylene;L is a substituted or unsubstituted alkylene, a carbonyl, an ester, an amide, a carbamate ester, an amine, an ether, a carbonate ester, a thioether, a thioester, or a urea; andZ comprises a lipid chain.

2. The compound of claim 1, wherein the mitochondrial targeting moiety comprises a lipophilic delocalized cation.

3. The compound of any one of claims 1-2, wherein the mitochondrial targeting moiety comprises an aryl phosphine or aryl phosphonium group (e.g.. a monoaryl phosphonium, diaryl phosphonium, or triaryl phosphonium).

4. The compound of any one of claims 1-3, wherein the mitochondrial targeting moiety comprises a phosphonium group (e.g., an aryl phosphonium group).

5. The compound of any one of claims 1-4, wherein the mitochondrial targeting moiety comprises a substituted or unsubstituted tripheny lphosphonium (TPP).

6. The compound of any one of claims 1-5, wherein R1is an unsubstituted Ci-Cio alkylene, such as a C2 alkylene.

7. The compound of any one of claims 1-6, wherein L is an amide.

8. The compound of any one of claims 1-6, wherein L is an ester.

9. The compound of any one of claims 1-8, wherein Z is a substituted or unsubstituted alkyl.

10. The compound of any one of claims 1-9, wherein Z is a substituted or unsubstituted C2-C20 alkyl.

11. The compound of any one of claims 1-10, wherein Z is an unsubstituted C2-C20 linear alkyl.

12. The compound of any one of claims 1-11, wherein the compound comprises one of the following or a salt thereof13. A method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of a compound defined by any one of claims 1-12.

14. A method of preventing or reducing the recurrence of cancer in a subject following surgical resection of a tumor, the method comprising administering to the subject a therapeutically effective amount of a compound defined by any one of claims 1-12.

15. The method of any one of claims 13-14, wherein the cancer comprises brain cancer, such as glioblastoma, glioma, metastatic brain cancer, a pediatric low grade glioma, or a diffuse intrinsic pontine gliomas (DIPG) induced tumor.

16. The method of any one of claims 13-15, wherein the compound is delivered in a biocompatible nanocarrier.

17. The method of claim 16, wherein the nanocarrier is targeted to the brain.

18. The method of any one of claims 16-17, wherein the nanocarrier comprises polymeric nanoparticles comprising PLGA-b-PEG functionalized with a mitochondrial targeting moiety, such as a triphenylphosphonium (TPP) cation.

19. A method of preventing or reducing the recurrence of brain cancer in a subject following surgical resection, the method comprising silencing or reducing expression of CPT1 A in the subject.

20. The method of claim 19, wherein the cancer comprises brain cancer, such as glioblastoma, glioma, metastatic brain cancer, a pediatric low grade glioma, or a diffuse intrinsic pontine gliomas (DIPG) induced tumor.

21. The method of any one of claims 13-20, wherein the method further comprises administering a glycolysis inhibitor to the subject.

22. The method of any one of claims 13-21, wherein the method further comprises administering a chemotherapeutic agent to the subject.

23. The method of any one of claims 13-22, wherein the method further comprises administering a probiotic to the subject.

24. The method of any one of claims 13-23, wherein the method further comprises administering a high fat diet to the subject.

25. The method of any one of claims 13-23, wherein the method further comprises administering a low carbohydrate diet to the subject.

26. The method of any one of claims 13-25, wherein the method further comprises administering a high fat, low carbohydrate diet to the subject.

27. A method of preventing or reducing the recurrence of brain cancer in a subject following surgical resection of a tumor, the method comprising administering a high fat diet to the subject, a low carbohydrate diet to the subject, or a combination thereof.

28. The method of claim 27, wherein the cancer comprises brain cancer, such as glioblastoma, glioma, metastatic brain cancer, a pediatric low grade glioma, or a diffuse intrinsic pontine gliomas (DIPG) induced tumor.

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