Combination therapy for modulating lipid production

Abstract

The present invention relates to compositions and methods for inhibiting the supply of adipogenic lipids from internal and external sources in a subject. In some embodiments, the present invention relates to compositions and methods for treating cancer, including glioblastoma, by inhibiting lipogenic lipid supply in a subject.

Description

[0001] Statement Regarding Federally Funded Research

[0002] This invention was made with government support under Grant / Contract Nos. R01 NS112935, R01 CA240726, R01 CA227874, and R01 NS104332 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Related Applications

[0004] This application claims the benefit of U.S. Provisional Application No. 63 / 318,140 filed on March 9, 2022, U.S. Provisional Application No. 63 / 329,936 filed on April 12, 2022, U.S. Provisional Application No. 63 / 333,784 filed on April 22, 2022, and U.S. Provisional Application No. 63 / 338,595 filed on May 5, 2022, each of which is incorporated herein in its entirety. Technical Field

[0005] The present disclosure relates to compositions and methods for inhibiting lipid supply in a subject. In some embodiments, the present disclosure relates to compositions and methods for treating cancer. Background Art

[0006] Lipids constitute the basic structure of plasma membrane and many organelle membranes. Therefore, sufficient lipid supply is a prerequisite for cell growth and proliferation. Under physiological conditions, lipid levels are mainly regulated by sterol regulatory element binding protein (SREBP) of the transcription factor family that regulates numerous cellular processes. SREBP-1 is highly activated in several malignancies, including glioblastoma (GBM), liver cancer, breast cancer and colorectal cancer, but the specific activation and lipid metabolism mechanisms are still unclear. Since cells (e.g., cancer cells) that grow and divide rapidly require a large amount of fatty acids (FA) to carry out phospholipid and membrane biogenesis, the useful mode of cancer treatment represented by the therapy that reduces the availability of fatty acids. In addition, tumor cells contain a large amount of lipid droplets (LD), which together with lipoproteins such as LDL, HDL and VLDL serve as an important source of fatty acids (FA) and cholesterol that support tumor growth. LD and lipoproteins are hydrolyzed in lysosomes, releasing the FA and cholesterol stored in them that support tumor growth.

[0007] Therefore, there is a need for improved compositions and methods for inhibiting all lipid sources to treat cancer by inhibiting both lipogenesis and lipid release from LDs and lipoproteins in a subject. There is a need for improved compositions and methods for treating cancers including solid tumors and refractory cancers. There is a need for improved compositions and methods for treating glioblastoma and other forms of brain cancer. There is a need to increase the number of reactive oxygen species in a subject. There is a need to controllably cause mitochondrial damage to a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.

[0009] Figure 1A Glutamate, ammonia and lactate levels in H1299 or U87 cell culture media measured with a Bioprofile 100Plus analyzer are shown (mean ± SD; n = 3). H1299 or U87 cells were cultured in RPMI1640 or DMEM culture media supplemented with 5% FBS for 24 hours. The cells were then washed once with PBS and placed in fresh serum-free RPMI1640 or DMEM culture media containing or not containing glutamine (4mM) or glucose (5mM) for 12 hours before measurement. The cell culture conditions were the same before subsequent panel processing.

[0010] Figure 1B Shown are Western blot analyses of whole lysates of H1299 or U87 cells cultured for 12 h in serum-free medium in the presence or absence of glucose (5 mM), glutamine (4 mM), glutamate (4 mM), lactate (10 mM), or NH 4 Cl (4 mM).

[0011] Figure 1C Shown are Western blot analyses of whole lysates from H1299 or U87 cells after 12 h of NH4Cl stimulation at the indicated doses in the presence of glucose (5 mM) under serum-free culture conditions.

[0012] Figure 1D Shown are Western blot analyses of whole lysates from H1299 or U87 cells over time following stimulation with 4 mM NH4Cl in the presence of glucose (5 mM) under serum-free culture conditions.

[0013] Figure 1ERepresentative IF images of anti-SREBP-1 staining (red) of H1299 or U87 cells after 12 hours of stimulation with or without NH4Cl (4mM) are shown in serum-free medium in the presence of glucose (Gluc, 5mM). Nuclei were stained with DAPI (blue). Scale bar, 10 μm. The nuclear intensity of SREBP-1 of 30 cells was quantified by ImageJ (mean ± SEM) and is shown below. Significance was determined by unpaired Student's ttest. * p<0.05, $ p<0.001, # p<0.0001; NS not significant.

[0014] Figure 2A Shown are Western blot analyses of paired tumor (T) and adjacent normal (N) lung tissues from individuals with adenocarcinoma (Adeno), squamous cell carcinoma (Squamous), and large cell carcinoma (Large) lung cancer.

[0015] Figure 2B Representative immunohistochemistry (IHC) images of anti-GLS and anti-SREBP-1 staining in tumor and adjacent normal lung tissue from individuals with adenocarcinoma or squamous lung cancer are shown. Scale bar, 50 μm.

[0016] Figure 2C Ammonia levels in paired human lung tumors and adjacent normal lung tissues are shown. Significance was determined by unpaired Student's t-test; *p<0.05.

[0017] Figure 2D Representative IHC images of anti-GLS and anti-SREBP-1 staining from a lung cancer tissue microarray (TMA, n=99) containing 48 adenocarcinomas, 43 squamous, 5 large cell, and 3 small cell tumors and 50 paired adjacent normal lung tissues are shown. Scale bar, 100 μm.

[0018] Figure 2E Shown from Figure 2D GLS expression levels and SREBP-1 staining were quantified by ImageJ and shown with H-score. The red lines in the figures show the mean ± SEM. The data were analyzed by using one-way ANOVA followed by comparison with normal controls using Dunnett's multiple comparison adjustment. *p<0.001, **p<0.0001.

[0019] Figure 2F Shown from Figure 2CCorrelation between GLS expression and SREBP-1 levels in lung cancer TMA tissues shown. Correlation coefficient (R) and significance were determined by Pearson correlation test. p<0.0001.

[0020] Figure 2G Representative IHC images of anti-GLS and anti-SREBP-1 staining in tumor tissue from individuals with GBM are shown. Scale bar, 100 μm.

[0021] Figure 2H Representative IHC images of anti-GLS and anti-SREBP-1 staining in different types of gliomas in glioma TMAs (n=91) containing 12 A2, 8 AA, 45 GBM, 16 O2, and 10 AO are shown. Scale bar, 100 μm.

[0022] Fig.2I GLS expression and SREBP-1 staining from Figures 4G and 4H in TMA are shown quantified by ImageJ and H-score. Red lines in the figures show mean ± SEM. A2, astrocytoma grade II; AA, anaplastic astrocytoma, grade III. GBM, glioblastoma, grade IV; O2, oligodendroglioma, grade II and AO anaplastic oligodendroglioma, grade III.

[0023] Figure 2J Shows Figure 2H and 2I Correlation between GLS expression and SREBP-1 staining in glioma TMA tissues shown. Correlation coefficient and significance were determined by Pearson correlation test. p<0.0001.

[0024] Figure 2K Shown are patients with GLS expression (mean = 199.78) or SREBP-1 levels (mean = 200.02) ( Fig.2I ) for quantitative separation of TMA( Figure 2H Figure 2. Kaplan-Meier curves of overall survival of individuals (n=45) with GBM of ≥5% (n=45). Significance was determined by Log-rank test. For GLS, p=0.0042, and for SREBP-1 comparison, p=0.0001.

[0025] Figure 3A Shown are the effects of GFP-SCAP wild type or mutant D428A on SREBP-1 and SREBP-2 cleavage in H1299 cells compared to GFP control, as analyzed by Western blot.

[0026] Figure 3B Shown are the effects of GFP-SCAP wild type or mutant D428A on lung tumor growth as analyzed by bioluminescence imaging at day 50 after implantation via tail vein injection in mice (1×10 6 cells / mouse).

[0027] Figure 3C Shown are tumor growth rates from day 7 to day 50 quantified by bioluminescence imaging (mean ± SEM, n=5).

[0028] Figure 3D Shown are representative gross images (left panels) and lung sections (right panels) of mouse lungs after hematoxylin and eosin (H&E) staining on day 50 after implantation (middle panels; scale bar, 2 mm), as well as representative gross images and lung sections of IHC staining of SREBP-1 in tumor tissues from different groups (right panels; scale bar: 50 μm).

[0029] Figure 3E Shown are the percentage of tumor nodules per total lung area (upper panel) and the intensity of SREBP-1 staining (lower panel) quantified by ImageJ (mean ± SEM, n = 5).

[0030] Figure 3F Shown is the effect of GFP-SCAP wild type or mutant D428A on SREBP-1 and SREBP-2 cleavage in primary GBM30 cells compared with GFP control by western blot analysis.

[0031] Figure 3G Shown are the effects of GFP-SCAP wild type or mutant D428A on intracranial tumor growth in mice (3.5 x 103 cells / mouse), as analyzed by magnetic resonance imaging (MRI) (yellow circles). White arrows indicate injection sites.

[0032] Figure 3H Shown are brain sections stained with H&E (left panel; scale bar, 1 mm) and IHC of SREBP-1 (right panel, scale bar, 50 μm).

[0033] Fig. 3I Nuclear SREBP-1 staining in tumor tissues was quantified by ImageJ.

[0034] Figure 3J Mouse survival was assessed by Kaplan-Meier plots (n=7 / group). Significance was assessed by one-way ANOVA with Dunnett's multiple comparison adjustment ( Figure 3E , 3G and 3I) or two-way ANOVA ( Figure 3C ) to determine. Figure 3J ) Significance was determined by log-rank test. *p<0.05; **p<0.001; ***p<0.0001. NS not significant.

[0035] Figure 4A The relative levels of ammonia, glutamate and α-KG in H1299 cells measured with appropriate assay kits after treating cells with / without GPNA or CB-839 are shown. The results are expressed as mean ± SEM (n = 3). H1299 or U87 cells were cultured in RMPI 1640 or DMEM medium supplemented with 5% FBS for 24 hours. The cells were then washed once with PBS and treated with / without GPNA (5mM) or CB-839 (100nM) for 12 hours in fresh serum-free RMPI 1640 or DMEM medium in the presence of glutamine (4mM) and glucose (5mM) and then analyzed. Unless otherwise stated, the cell culture conditions before treatment are the same as in the subsequent small figures.

[0036] Figure 4B Shown are Western blot analyses of primary GBM30 cells treated with / without CB-839 (200 nM) in the absence or presence of glutamine, glutamate or NH4Cl (all 4 mM) for 12 hours under serum-free culture conditions.

[0037] Figure 4C Ammonia measurements were performed using an ammonia assay kit (n = 6) from tumor tissues derived from H1299 cell (4 x 106 implants / mouse) xenograft mouse models treated with vehicle (control, Ctrl) or CB-839 (30 mg / kg mouse body weight, intraperitoneally, twice daily for 3 days) according to the manufacturer's instructions. 3 Treatment was started at 4 pm. The middle panel shows representative IHC images of SREBP-1 in tumor tissues. Scale bar, 50 μm. Expression levels were quantified by analyzing 4 images per tumor and 3 tumors in each group using ImageJ.

[0038] Figure 5A Representative IHC images of anti-GLS and anti-SREBP-1 staining in tumors and adjacent normal tissues from individuals with adenocarcinoma (Adeno) or squamous lung cancer are shown. Scale bar, 50 μm.

[0039] Figure 5B Representative IHC images of anti-GLS and anti-SREBP-1 staining from lung cancer TMA are shown.

[0040] Figure 5C Representative images of different levels of anti-GLS or anti-SREBP-1 staining and scoring are shown.

[0041] Figure 5D Comparison of GLS expression and SREBP-1 levels in 50 paired tumor and adjacent normal lung tissues from lung cancer TMAs based on H-scores is shown. Significance was determined by unpaired Student's t-test. All p<0.0001.

[0042] Figure 5E It is shown that genetic inhibition of GLS or SREBP-1 significantly inhibits lung tumor growth in vivo. NSCLC H1299 cells were infected with lentivirus expressing shGLS or shSREBP-1 for 48 hours and then implanted into the flanks of nude mice (2x 106 cells / mouse). Tumors were isolated from mice 53 days after transplantation and imaged (left panel) and weighed (right panel) for comparison. Data are shown as mean ± SEM (n = 6). Significance was determined by one-way ANOVA with Dunnett's multiple comparison adjustment. *p < 0.002.

[0043] Fig. 5F Representative IHC images of anti-GLS, anti-SREBP-1, anti-ASPG, and anti-SDS staining in tumor tissue from a patient with GBM are shown. Scale bar, 50 μm.

[0044] Figure 5G Representative images of anti-GLS and anti-SREBP-1 staining from glioma TMAs are shown.

[0045] Figure 5H Representative images of different levels of anti-GLS or anti-SREBP-1 staining and scoring are shown.

[0046] Fig. 6A Shown are gross and macroscopic images of mouse lungs (a) and H&E staining of lung sections (b) at day 50 after mouse implantation with H1299 cells expressing GFP, wild-type (WT), or mutant GFP-SCAP D428A. Figure 3D The red box image is presented as a representative. Scale bar, 2 mm.

[0047] Figure 6B Quantification of the number of nodules on mouse lung sections by Image-J is shown (b, lower panel). Data are shown as mean ± SEM (n=5). Significance was determined by one-way ANOVA with Dunnett's multiple comparison adjustment. **p<0.001; ***p<0.0001.

[0048] Figure 6CMRI scans of mouse brains at day 12 after implantation of GBM30 cells stably transfected with GFP, wild-type or mutant (D428A) GFP-SCAP (3.5 x 103 cells / mouse) are shown. Yellow circles indicate tumor locations. White arrows indicate injection sites. Scatter plots show tumor volumes from MRI scans quantified from delineated regions of interest (ROI) (right panels). Significance was determined by unpaired Student's t-test. *p<0.05.

[0049] Fig.6D Shown as Figure 6C H&E staining of mouse brain sections resected on day 17 after implantation of GBM30 cells as described in . Figure 3H Rectangular frame images are used as representatives. Scale bar, 1 mm.

[0050] Figure 7 The relative growth of a GBM cell line (U373) in response to treatment with different antipsychotic drugs is shown, and pimozide is identified as the most potent drug to inhibit GBM cell proliferation. 4 U373 cells were cultured in 12-well plates in full DMEM medium containing 4.5 g / L glucose (25 mM) and 5% FBS for 24 hours, and then rinsed twice with PBS and replaced with fresh DMEM medium containing 1% FBS and 5 mM glucose, 4 mM glutamine and 1 mM pyruvate. Antipsychotic drugs were then added to each well at the indicated concentrations (0-100 μM), and viable cells were counted by trypan blue staining after 48 hours of treatment and normalized to the number of control cells treated with DMSO. PMZ, Pimozide; HAL, Haloperidol; IMI, Imipramine; CLO, Clozapine; OLA, Olanzapine; PER, Perphenazine; PRO, Promazine; SUL, Sulpiride; UA, U18666A. All drugs used DMSO as buffer solution.

[0051] Fig. 8AGBM30 (patient-derived primary GBM cells) are shown to be mesenchymal cells with mutant EGFRvIII (constitutive EGFR mutation lacking EGFR exons 2-7) and wild-type IDH. GBM30 were cultured in DMEM / F12 medium supplemented with B-27 (1×), heparin (2 μg / mL), EGF (20 ng / mL) and fibroblast growth factor (FGF, 20 ng / mL) in glass-bottom Geltrex matrix-coated 6 cm culture dishes, and U251 cells were cultured in full DMEM medium containing 5% FBS and 25 mM glucose in glass-bottom 6 cm culture dishes for 24 hours. After washing once with PBS and replacing with fresh DMEM medium containing 1% FBS and 5mM glucose, 4mM glutamine and 1mM pyruvate, after washing twice with PBS, the cells were then treated with / without pimozide (3 μM) for 24 hours, then stained with BODIPY 493 / 503 (green) / Lysotracker (red) / Hoechst 33342 (blue), and observed by confocal microscopy. The number of LDs stained with BODIPY was significantly reduced compared to the pretreatment condition without pimozide after culturing 24 hours with 1% FBS. In contrast, pimozide treatment blocked LD reduction, and the LDs accumulated in large quantities in the lysosomes stained with Lysotracker.

[0052] Figure 8B Shown are representative fluorescent imaging of lysosomes stained with lysotracker (red) (n=30 images in total) in GBM30 and U251 cells treated with / without pimozide (3 μM) for 24 hours under the same culture conditions as in (A). After pimozide treatment, lysosome size increased significantly.

[0053] Figure 8CLysosomal activity is determined by DQ-green BSA. GBM30 and U251 cells were treated with / without pimozide (3 μM) in 1% FBS, 5mM glucose, 4mM glutamine and 1mM pyruvate for 24 hours, washed twice with PBS, and then incubated with 10 μg / mL DQ-green BSA for 6 hours in fresh DMEM medium containing 1% FBS and 1% NEAA, 1% GlutaMax and 1% HEPES, and then observed by confocal microscopy. The nucleus was stained with Hoechst33342 (blue). In the control cells treated with DMSO, DQ-Green-BSA was endocytosed and delivered to the lysosome, where it was degraded by active acidic lysosomal hydrolases, which dequenched the fluorescence of the dye, thereby showing bright green spots in the lysosome. In contrast, when lysosomal activity was inhibited, DQ-Green-BSA could not be degraded, resulting in quenching of the dye's fluorescence (PMZ treatment). The significantly reduced green fluorescence in pimozide treatment compared to the control DMSO treatment demonstrated that lysosomal activity was significantly inhibited by pimozide.

[0054] Fig.8D Shown is the determination of lysosomal pH with ratio probe LysoSensor Yellow / Blue dextran.GBM30 and U251 cells were treated for 24 hours as inset C, and incubated for 24 hours with 1mg / ml LysoSensor Yellow / Blue dextran under dark conditions, then observed by confocal microscopy.Yellow fluorescence represents a stronger acidic lysosomal environment (pH about 4.5), and blue fluorescence represents a stronger neutral lysosomal environment (pH about 6.0).Data clearly show that pimozide treatment significantly improves lysosomal pH, causing it to rise from acidic to neutral levels.

[0055] Fig.9A Western blot analysis of whole lysates of GBM30 cells cultured in DMEM / F12 supplemented with B-27 (1×) serum-free supplement, heparin (2 μg / mL), EGF (20 ng / mL) and fibroblast growth factor (FGF, 20 ng / mL) and U251 cells cultured in DMEM medium containing 1% FBS, 5 mM glucose, 4 mM glutamine and 1 mM pyruvate after 24 hours of treatment with different doses of pimozide. P, precursor of SREBP; N, N-terminus of SREBP-1. C, C-terminus of SREBP-2. The data show that pimozide dose-dependently activates SREBP-1 / 2 cleavage and expression of its downstream fatty acid synthesis targets FASN and SCD1.

[0056] Fig. 9BReal-time PCR studies of gene expression of fatty acid and cholesterol synthesis pathways in GBM30 and U373 cells under the same culture / treatment conditions described above are shown. Results are shown as mean ± SD (n = 3). FASN, fatty acid synthase; LDLR, low-density lipoprotein receptor; SCD1, stearoyl-CoA desaturase 1; HMGCS1, 3-hydroxy-3-methylglutaryl-CoA synthetase 1; SREBF1, gene name for SREBP-1; ACLY, ATP citrate lyase; ACACA, acetyl-CoA carboxylase alpha; HMGCR, 3-hydroxy-3-methylglutaryl-CoA reductase. The data show that pimozide treatment significantly increased the expression of genes controlling fatty acid / cholesterol synthesis.

[0057] Fig. 9C Western blot analysis of membrane extracts (ASCT2) from GBM30 and U251 cells under the same culture / treatment conditions as in (A) is shown. The integral membrane protein transferrin receptor-1 (CD71), which mediates the uptake of transferrin-iron complexes, was used as a membrane loading control for ASCT2. The data show that pimozide treatment increases ASCT2 expression in a dose-dependent manner.

[0058] Fig.9D Representative confocal immunofluorescence images of anti-ASCT2 staining (green) in GBM30 and U251 cells under the same culture conditions as in (A) are shown. Cell nuclei were stained with DAPI (blue). The data show that pimozide treatment increased ASCT2 expression in a dose-dependent manner.

[0059] Fig.9E Western blot analysis of whole lysates and membrane proteins from GBM30 and U251 cells treated with / without pimozide (5 μM) and cholesterol (3 μg / ml) for 24 hours under the same culture conditions as panel (A) is shown. The data show that ASCT2 expression elevated by pimozide treatment is inhibited by cholesterol addition, while SREBP-1 activation is also inhibited.

[0060] Fig.9F Shown is a real-time PCR analysis of SREBP-1 associated with the ASCT2 gene promoter (also known as the SLC1A5 gene) by using an anti-SREBP-1 antibody after chromatin immunoprecipitation (ChIP). The top panel shows the putative SREBP-1 binding site (SRE) in the ASCT2 promoter by xxx promoter analysis. The data show that SREBP-1 binds to the ASCT2 gene promoter.

[0061] Figure 9GAnalysis of SREBP-1 transcriptional activity on the ASCT2 gene promoter by promoter-luciferase reporter assay is shown. U373 cells were transfected with pGL3-luc plasmids of different lengths of the ASCT2 gene promoter containing / without the putative binding site of SREBP-1 in DMEM medium containing 5% FBS for 24 hours, and then infected with adenovirus expressing empty, N-terminal ad-SREBP-1a, ad-SREBP-1c or ad-SREBP-2 for 24 hours before analysis. The data showed that N-terminal SREBP-1a is the main form that activates the ASCT2 gene promoter.

[0062] Figure 9H Western blot analysis of ASCT2 expression in GBM cells after overexpression of N-terminal active SREBP-1a, SREBP-1c or SREBP-2 forms is shown. GBM cells were infected with adenovirus expressing empty, N-terminal SREBP-1a, SREBP-1c or SREBP-2 for 48 hours in 5% FBS medium. The cells were then lysed and total cell lysates and membrane extracts were analyzed by Western blot. The data showed that N-terminal SREBP-1a is the major form that activates ASCT2 protein expression.

[0063] Fig. 10A Shown are Western blot analyses of whole lysates or membrane extracts of GBM30, U373 or U251 cells cultured in DMEM / F12 supplemented with B-27 (1×) serum-free supplement, heparin (2 μg / mL), EGF (20 ng / mL) and fibroblast growth factor (FGF, 20 ng / mL), in 1% FBS culture conditions containing 5 mM glucose and 1 mM pyruvate, in the presence or absence of glutamine (4 mM) or NH3·H2O (4 mM), after treatment with / without pimozide (5 μM) for 24 h. The data showed that the absence of glutamine completely abolished pimozide-stimulated SREBP-1 activation, FASN / SCD1, and ASCT2 expression, whereas ammonia (NH4+) derived from NH3·H2O completely restored the expression, indicating that ammonia released from glutamine is an essential activator for pimozide-mediated activation of the SREBP-1 / adipogenic pathway and upregulation of ASCT2.

[0064] Fig. 10BShown are Western blot analyses of whole lysates or membrane extracts of GBM30, U373 or U251 cells cultured in DMEM / F12 supplemented with B-27 (1×) serum-free supplement, heparin (2 μg / mL), EGF (20 ng / mL) and fibroblast growth factor (FGF, 20 ng / mL), in 1% FBS culture conditions containing 5 mM glucose, 4 mM glutamine and 1 mM pyruvate, in the presence or absence of NH3·H2O (4 mM), after treatment with / without pimozide (5 μM), GPNA (1 mM, ASCT2 inhibitor) for 24 h. The data showed that pharmacological inhibition of glutamine uptake by GPNA completely abolished pimozide-stimulated SREBP-1 activation, FASN / SCD1, and ASCT2 expression, whereas ammonia (NH4+) derived from NH3·H2O completely restored the expression, further indicating that ammonia released from glutamine is an essential activator for pimozide-mediated activation of the SREBP-1 / lipogenesis pathway and upregulation of ASCT2.

[0065] Fig. 10C Shown are Western blot analyses of whole lysates or membrane extracts of GBM30 cells cultured in DMEM / F12 supplemented with B-27 (1×) serum-free supplement, heparin (2 μg / mL), EGF (20 ng / mL) and fibroblast growth factor (FGF, 20 ng / mL), in 1% FBS culture conditions containing 5 mM glucose, 4 mM glutamine and 1 mM pyruvate, in the presence or absence of NH3·H2O (4 mM), and after treatment with / without pimozide (5 μM) or CB-839 (100 nM, glutamine GLS inhibitor) (C) for 24 h. The data showed that CB-839 completely abolished pimozide-stimulated SREBP-1 activation, FASN / SCD1, and ASCT2 expression by pharmacological inhibition of GLS, whereas ammonia (NH4+) derived from NH3·H2O completely restored the expression, further indicating that ammonia released from glutamine is an essential activator for pimozide-mediated activation of the SREBP-1 / adipogenic pathway and upregulation of ASCT2.

[0066] Fig.11AShown are representative confocal fluorescence images of U251 cells stained with MitoTracker (staining mitochondria, green) and CellROX Deep Red (detecting reactive oxygen species, ROS) after treatment with / without pimozide (3 μM), GPNA (1 mM), DON (10 μM) or CB-839 (100 nM) alone or in combination for 24 hours in 1% FBS, 5 mM glucose, 4 mM glutamine, 1 mM pyruvate (n=12 images in total). ROS levels of more than 100 cells were quantified (mean ± SEM). Scale bar, 10 μm. Data show that the combination of pimozide and a glutamine metabolism inhibitor leads to mitochondrial fragmentation and a significant increase in ROS in mitochondria.

[0067] Fig. 11B Western blot analysis of whole lysates from GBM30, U373 or U251 cells treated for 48 hours with / without pimozide (5 μM), GPNA (1 mM), DON (10 μM) or CB-839 (100 nM) under the same culture conditions as in (A) is shown. The data show that the combination of pimozide and a glutamine metabolism inhibitor significantly increased the apoptosis markers caspase and PARP cleavage, indicating that the combination leads to significant tumor cell apoptosis.

[0068] Fig. 11C Proliferation curves of different cancer cells are shown, GBM30 was cultured in DMEM / F12 supplemented with B-27 (1×) serum-free supplement, heparin (2 μg / mL), EGF (20 ng / mL) and fibroblast growth factor (FGF, 20 ng / mL), with / without pimozide (3 μM), GPNA (1 mM), DON (10 μM) or CB-839 (100 nM) for 9 days. U373 and U251 were cultured in medium supplemented with 5% FBS for 24 hours. The cells were then washed twice with PBS and treated in fresh serum-free medium with / without pimozide (3 μM), GPNA (1 mM), DON (10 μM) or CB-839 (100 nM) in 1% FBS containing 5 mM glucose, 4 mM glutamine and 1 mM pyruvate for 4 days (mean ± SD, n = 3). The data showed that the combination of pimozide and a glutamine metabolism inhibitor significantly inhibited tumor cell proliferation.

[0069] Fig.11DRepresentative confocal fluorescence images of U251 cells stained with MitoTracker (stained mitochondria, green) and CellROX Deep Red (detected ROS levels) are shown in the presence or absence of GSH (3mM), as in (A), after treatment with / without pimozide (3μM), GPNA (1mM), DON (10μM) or CB-839 (100nM) alone or in combination. The data show that the antioxidant GSH reduces the ROS levels in mitochondria elevated by the combined treatment and restores the mitochondrial morphology to a level similar to that of untreated control cells, indicating that the combination of pimozide and glutamine metabolism inhibitors induces ROS that leads to mitochondrial fragmentation and damage. Scale bar, 10μm.

[0070] Fig.11E Representative cell micrographs of U251 after treatment with pimozide (3 μM), GPNA (1 mM), DON (10 μM) or CB-839 (100 nM) alone or in combination for 48 hours as in (A) are shown in the presence or absence of GSH (3 mM). The data showed that the antioxidant GSH significantly alleviated the combination-induced cell death.

[0071] Fig.11F Representative confocal fluorescence images of U251 cells stained with MitoTracker (staining mitochondria, green) and CellROX Deep Red (detecting ROS levels) after treatment with / without pimozide (3 μM), GPNA (1 mM), DON (10 μM) or CB-839 (100 nM) in combination as in (A) for 24 hours in the presence or absence of PA (palmitate, 20 nM) and OA (oleic acid, 20 μM) and cholesterol (3 μg / ml) are shown. The data show that the addition of fatty acids and cholesterol significantly reduces ROS caused by the combination of pimozide and glutamine metabolism inhibitors and restores mitochondrial morphology.

[0072] Fig.11G Western blots of GBM30, U251, and U373 cells treated with / without pimozide (3 μM), GPNA (1 mM), DON (10 μM), or CB-839 (100 nM) in combination as in (A) for 48 h in the presence or absence of PA (palmitate, 20 μM) and OA (oleic acid, 20 μM) and cholesterol (3 μg / mL) are shown. Cyto, cytosol; Mito, mitochondria. The data show that the addition of fatty acids and cholesterol significantly reduced the cleavage of apoptotic markers induced by the combination of pimozide and glutamine metabolism inhibitors, suggesting that the cell death caused by the combination is triggered by the limitation of fatty acid / cholesterol availability.

[0073] Fig.11H Micrographs are shown showing the growth of U251 treated with pimozide (3 μM) and GPNA (1 mM) as in (A) in the presence or absence of PA (palmitate, 20 μM) and OA (oleic acid, 20 μM) and cholesterol (3 μg / mL). Cells were cultured in 5% FBS full DMEM medium for 24 hours. Cells were then washed twice with PBS and treated in fresh serum-free medium in 1% FBS containing 5 mM glucose, 4 mM glutamine and 1 mM pyruvate for 72 hours. Scale bar, 100 μm.

[0074] Fig. 12A Figure 3 shows the dose-dependent inhibition of tumor growth by pimozide derived from GBM30 cells. 3×10 6 GBM30 cells. When the tumor volume reaches about 80mm 3 At 4 hr, mice were administered pimozide (15, 30, 60 mg / kg / day) or vehicle by intraperitoneal injection for 14 days. Fig. 12A Also shown are images of tumors after isolation from mice on the last day of treatment.

[0075] Fig. 12B Fold change of tumor growth curves normalized to tumor volume before treatment (day 0) is shown (left), and the right panel shows tumor volume (right). The 60 mg / kg dose was toxic, and mice died after 6-8 days of treatment.

[0076] Fig. 12C Colony formation assays are shown for studying the combined effects of pimozide and fatostatin in GBM cells. GBM30 was cultured in DMEM / F12 supplemented with B-27 (1×) serum-free supplement, heparin (2 μg / mL), EGF (20 ng / mL) and fibroblast growth factor (FGF, 20 ng / mL), and U251 and U373 were first cultured in 5% FBS full medium to grow for 6 days without treatment to form colonies. The cells were then washed twice with PBS and treated with / without pimozide (3 μM), fatostatin (5 μM) for 8 days in fresh medium containing 1% FBS, 5 mM glucose, 4 mM glutamine, 1 mM pyruvate medium, and the number of colonies was quantified. The data showed that single treatment with pimozide or fatostatin only slightly reduced the number of colonies, while their combination almost completely eradicated preformed colonies.

[0077] Fig.12DThe combination of pimozide and the SREBP fatostatin was shown to synergistically inhibit tumor growth in a GBM30-derived xenograft model. GBM30 cells were implanted into mice to form Fig. 12A The same tumor. When the tumor volume reaches about 80mm 3 At 14 days post-treatment, mice were treated with pimozide (15 mg / kg / day) and fatostatin (25 mg / kg / day) alone or in combination for 16 days. Tumors were isolated from mice and imaged on day 14 post-treatment. Data are shown as mean ± SD (n = 6).

[0078] Fig.12E The fold change of tumor growth curves normalized with the tumor volume on day 0 before treatment (left) and the unnormalized tumor volume is shown. Data are shown as mean ± SD (n = 6). Significance was determined by one-way ANOVA with Dunnett's multiple comparison adjustment. *p < 0.002. Tumor diameter was measured using a vernier caliper and calculated as V = L × S × S / 2 (S refers to the short diameter, L means the long diameter, and V is the tumor volume). The data show that the combination of pimozide and fatostatin synergistically inhibits tumor growth in a GBM30-derived xenograft model.

[0079] Fig.13A Colony formation assays are shown for studying the effects of pimozide treatment in GBM cells in the presence and absence of glutamine. GBM30 was cultured in DMEM / F12 supplemented with B-27 (1×) serum-free supplement, heparin (2 μg / mL), EGF (20 ng / mL) and fibroblast growth factor (FGF, 20 ng / mL), and U373, U251 cells were first cultured in 5% FBS full medium to grow for 6 days to form colonies. The cells were then washed twice with PBS and treated with pimozide (3 μM) in the presence or absence of glutamine (4 mM) in fresh medium containing 1% FBS and 5 mM glucose, 1 mM pyruvate. The data show that pimozide almost eradicates preformed colonies in the absence of glutamine.

[0080] Fig. 13BProliferation curves of GBM30, U373 and U251 cells cultured in DMEM / F12 supplemented with B-27 (1×) serum-free supplement, heparin (2 μg / mL), EGF (20 ng / mL) and fibroblast growth factor (FGF, 20 ng / mL) for 4 days in medium supplemented with 1% FBS containing / without pimozide (3 μM) or glutamine (4 mM) are shown (mean ± SD, n = 3). Cells were cultured in medium supplemented with 5% FBS for 24 hours. The cells were then washed twice with PBS and treated in fresh medium in 1% FBS containing 5 mM glucose and 1 mM pyruvate. The data show that pimozide kills GBM cells in the absence of glutamine.

[0081] Fig. 13C Colony formation assay is shown for studying the therapeutic effect of the combination of pimozide and glutamine metabolic inhibitors. Cells were first cultured in 5% FBS full medium for 6 days to form colonies. The cells were then washed twice with PBS and treated with pimozide (3 μM), GPNA (1 mM), DON (10 μM) or CB-839 (100 nM) alone or in combination in 1% FBS medium containing 5 mM glucose, 4 mM glutamine, 1 mM pyruvate for 8 days. The data show that the combination almost eradicates all colonies formed before treatment.

[0082] Fig.13D Proliferation curves of different cancer cells cultured in medium supplemented with 1% FBS containing / without Pimozide (3 μM), GPNA (1 mM), DON (10 μM) or CB-839 (100 nM) alone or in combination are shown. Cells were first cultured in medium supplemented with 5% FBS for 24 hours. The cells were then washed twice with PBS and treated for 4 days in 1% FBS containing 5 mM glucose and 1 mM pyruvate. The data show that the combination eradicated almost all of the tumor cells that were killed.

[0083] Fig.13E The combination of a glutamine metabolism inhibitor and pimozide was shown to synergistically inhibit tumor growth in a GBM30-derived xenograft model. 3×106 GBM30 cells were implanted in the flank of mice. When the tumor volume reached approximately 80 mm 3 At 14 days, mice were administered pimozide (15 mg / kg / day), GPNA (50 mg / kg / day), DON (0.2 mg / kg / day), or CB-839 (25 mg / kg / day) alone or in combination by intraperitoneal injection. Figure 22E also shows tumor images.

[0084] Fig.13FShown are tumor weights after isolation from mice on the last day of treatment.

[0085] Figure 13G Fold changes of tumor growth curves normalized with tumor volume before treatment (day 0) are shown. Data are shown as mean ± SEM (n=6). Significance was determined by one-way ANOVA with Dunnett's multiple comparison adjustment. *p<0.002. Data show that the combination synergistically inhibited tumor growth.

[0086] Fig.14 It is shown that inhibition of glutamine utilization sensitizes GBM cells to treatment with other antipsychotic drugs. Micrographs show the growth of U251 cells treated with / without pimozide (2, 3 μM), perphenazine (3, 5 μM), GPNA (1 mM) or CB-839 (100 nM). Cells were cultured in 5% FBS full medium for 24 hours. Cells were then washed twice with PBS and treated in fresh serum-free medium in 1% FBS containing 5 mM glucose, 4 mM glutamine and 1 mM pyruvate for 48 hours. Scale bar, 100 μm.

[0087] Fig.15 It is shown that inhibition of glutamine utilization sensitizes breast cancer cells to antipsychotic drug treatment. Micrographs show the growth of breast cancer cell lines (MDA-MB-231), liver cancer cell lines (Huh7) and lung cancer cell lines (H1299) treated with / without pimozide (2, 3 μM), perphenazine (3, 5 μM), GPNA (1 mM) or CB-839 (100 nM). Cells were cultured in 5% FBS full DMEM / RPMI1640 medium for 24 hours. The cells were then washed twice with PBS and treated in fresh serum-free medium in 1% FBS containing 5 mM glucose, 4 mM glutamine and 1 mM pyruvate for 48 hours. Scale bar, 100 μm.

[0088] Fig.16 It is shown that inhibition of glutamine utilization sensitizes lung cancer cells to antipsychotic drug treatment. Micrographs show the growth of lung cancer cell lines (H1299), liver cancer cell lines (Huh7) and lung cancer cell lines (H1299) treated with / without pimozide (2, 3 μM), perphenazine (3, 5 μM), GPNA (1 mM) or CB-839 (100 nM). Cells were cultured in 5% FBS full DMEM / RPMI1640 medium for 24 hours. Cells were then washed twice with PBS and treated in fresh serum-free medium in 1% FBS containing 5 mM glucose, 4 mM glutamine and 1 mM pyruvate for 48 hours. Scale bar, 100 μm.

[0089] Fig.17 It is shown that inhibition of glutamine utilization sensitizes lung cancer cells to antipsychotic treatment. Micrographs show the growth of lung cancer cell line (H1299) treated with / without pimozide (2, 3 μM), perphenazine (3, 5 μM), GPNA (1 mM) or CB-839 (100 nM). Cells were cultured in 5% FBS full DMEM / RPMI1640 medium for 24 hours. Cells were then washed twice with PBS and treated in fresh serum-free medium in 1% FBS containing 5 mM glucose, 4 mM glutamine and 1 mM pyruvate for 48 hours. Scale bar, 100 μm.

[0090] Fig.18A Representative MRI images are shown showing the effects of glutamine transporter ASCT2 inhibitor (GPNA, 50 mg / kg / day, intraperitoneal), glutaminase inhibitor (CB-839, 20 mg / kg / day, intraperitoneal) and SREBP-1 inhibitor (fatostatin, 25 mg / kg / day, intraperitoneal) in primary GBM30 cell-derived intracranial GBM model compared to vehicle control after 12 days of treatment. Tumors in mouse brains are marked with red circles. 5x 10 4 The cells were implanted into the mouse brains and drug treatment was started 7 days after implantation.

[0091] Fig.18B Mouse survival rates for the same mouse treatments as above were assessed by Kaplan-Meier plots (n=7 / group). Significance was determined by log-rank test. Statistical significance between Pimozide (PMZ) / Fatostatin (Fato) combination drug treatments. *p<0.01. All signal drug treatments were not significant compared to controls.

[0092] Fig.18C Mouse survival rates of the same mouse treatments as above were evaluated by Kaplan-Meier plots (n=7 / group). Significance was determined by log-rank test. Statistical significance between PMZ / GPNA, PMZ / CB-839 and vehicle and signal drug treatments was: *p<0.01. All signal drug treatments were not significant compared to controls.

[0093] Fig.19ARelative cell viability of patient-derived GBM30 (6 days) and U251 (3 days) cells after treatment with different antipsychotic drugs at indicated doses is shown (PMZ, pimozide; FLU, fluoxetine; HAL, haloperidol; IMI, imipramine; CLO, clozapine; OLA, olanzapine; PER, perphenazine; PRO, promazine; SUL, sulpiride).

[0094] Fig.19B Shown are representative micrographs showing the effects of PMZ treatment on normal human astrocytes (NHA, 6 days), GBM30 (6 days), and U251 (3 days); scale bar, 50 μm.

[0095] Fig.19C Quantification of the percentage of mortality following PMZ treatment as described above is shown. Statistical significance was analyzed by one-way ANOVA.

[0096] Fig. 20 Representative fluorescence imaging of octane-amine linked Pacific blue (20 μM) distribution in the plasma membrane of U251 cells after 24 hours is shown. Lysosomes and mitochondria were co-stained with LysoTracker (red) and MitoTracker (green).

[0097] Fig.21A Shown are representative fluorescence imaging of LDs and lysosomes in GBM30 cells stained with BODPIY493 / 503 (green) and Lysotracker (red) after 24 h of treatment with / without pimozide (PMZ) under 5% FBS or 1% FBS culture conditions.

[0098] Fig. 21B Shown are representative fluorescence imaging of LDL in GBM30 cells after treatment with / without pimozide (PMZ, 5 μM) for 24 hours and then supplementation with BODIPY-labeled LDL (green) for 4 hours followed by replacement with fresh medium without BODIPY-LDL for 16 hours. Scale bar, 10 μm.

[0099] Fig.22A Shown are the effects of 8-day treatment with / without pimozide (PMZ, 3 μM) in combination with ASCT2 (GPNA, G, 0.5 mM), GLS (CB-839, C, 100 nM), or general glutamine metabolism (6-diazo-5-oxo-1-norleucine, DON, D, 10 μM) inhibitors on GBM30 cell-derived colonies grown for 6 days prior to treatment.

[0100] Fig. 22BQuantification of colonies by ImageJ at day 8 after treatment is shown (mean ± sd, n = 3).

[0101] Fig.23 Representative fluorescence imaging of mitochondria in GBM U251 cells after 24 h of treatment with / without pimozide (3 μM), GPNA (0.5 mM), DON (10 M), and CB-839 (100 nM) are shown. Scale bar, 10 μm. DETAILED DESCRIPTION

[0102] Before disclosing and describing the method and system of the present invention, it should be understood that the method and system are not limited to specific synthetic methods, specific components or specific compositions. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments and is not intended to be limiting.

[0103] As used in the specification and the appended claims, the singular forms "a / an" and "the" include plural referents unless the context clearly indicates otherwise. In this article, ranges can be expressed as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from one particular value and / or to another particular value. Similarly, when a value is expressed as an approximation by using the antecedent "about", it is understood that the particular value forms another embodiment. It should be further understood that the endpoints of each range are significant relative to the other endpoint, as well as independently of the other endpoint.

[0104] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0105] Throughout the detailed description and claims of this specification, the word "comprise" and variations of the word, such as "comprising and comprises," means "including but not limited to," and is not intended to exclude, for example, other additives, ingredients, integers, or steps. "Exemplary" means "an example of..." and is not intended to convey an indication of a preferred or ideal embodiment. "Such as" is not used in a limiting sense, but for illustrative purposes.

[0106] Components that can be used to perform the disclosed methods and systems are disclosed. These and other components are disclosed herein, and it should be understood that when combinations, subsets, interactions, groups, etc. of these components are disclosed, although specific references to each different individual and collective combination and arrangement of these components may not be explicitly disclosed, each combination and arrangement is specifically carefully considered and described herein for all methods and systems. This applies to all aspects of the application, including but not limited to the steps in the disclosed methods. Therefore, if there are a variety of additional steps that can be performed, it should be understood that each of these additional steps can be performed using any specific embodiment or combination of embodiments of the disclosed methods.

[0107] The compounds described herein may include one or more asymmetric centers and may therefore exist in various isomeric forms, e.g., enantiomers and / or diastereomers. For example, the compounds described herein may be in the form of individual enantiomers, diastereomers, or geometric isomers, or may be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. Isomers may be separated from a mixture by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers may be prepared by asymmetric synthesis. See, e.g., Jacques et al., Enantiomers, Racemates, and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, EL, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, SH, Tables of Resolving Agents and Optical Resolutions, p. 268, EL Eliel, ed., Univ. of Notre Dame Press, Notre Dame, IN, 1972. The invention additionally encompasses the compounds as individual isomers substantially free of other isomers and alternatively as mixtures of various isomers.

[0108] Unless stated to the contrary, formulas with chemical bonds shown only as solid lines, rather than wedges or dashed lines, contemplate every possible isomer, such as each enantiomer, diastereomer, and meso compound, as well as mixtures of isomers, such as racemic or scalemic mixtures. Unless otherwise stated, formulas depicting one or more stereochemical features do not exclude the presence of other isomers.

[0109] Some compounds disclosed herein may exist in the form of one or more tautomers. Tautomers are interconvertible structural isomers that differ in the position of one or more protons or another variable atom. As an example:

[0110]

[0111] Whether one tautomeric form is more common than the other will depend both on the specific compound and its local chemical environment. Unless otherwise stated, a depiction of a tautomeric form encompasses all possible tautomeric forms.

[0112] The compounds disclosed herein can be provided in the form of pharmaceutically acceptable salts. Examples of such salts are acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid and nitric acid; salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid and polygalacturonic acid; salts formed by elemental anions such as chloride, bromide and iodide; salts formed by metal hydroxides such as sodium hydroxide, potassium hydroxide, calcium hydroxide, lithium hydroxide and magnesium hydroxide; salts formed by metal carbonates such as sodium carbonate, potassium carbonate, calcium carbonate and magnesium carbonate; salts formed by metal bicarbonates such as sodium bicarbonate and potassium bicarbonate; salts formed by metal sulfates such as sodium sulfate and potassium sulfate; and salts formed by metal nitrates such as sodium nitrate and potassium nitrate. Unless it is explicitly stated that it is a free base or free acid, the compound capable of forming a salt includes both pharmaceutically acceptable salts and free bases / acids.

[0113] On the one hand, disclosed herein are methods and compositions for treating cancer, diseases associated with tumor cell growth, or diseases associated with increased lipid utilization. On the one hand, disclosed herein is a method for treating cancer in a patient in need, the method comprising administering to the patient a single at least one ammonia inhibitor or a combination with one or more lipid metabolism inhibitors or their related agents. On the one hand, disclosed herein are methods and compositions for targeting and / or inhibiting multiple pathways in a lipogenic pathway. In some embodiments, the ammonia inhibitor and lipid metabolism inhibitor are synergistically combined to enhance therapeutic efficacy. In some embodiments, the one or more lipid metabolism inhibitors include at least one inhibitor that inhibits the utilization / release of lipids from extracellular uptake (LDL) and internal storage (lipid droplets, LD). In some embodiments, the compositions and methods include one or more ammonia inhibitors and / or one or more inhibitors that inhibit the utilization / release of lipids from extracellular uptake (LDL) and internal storage (lipid droplets, LD).

[0114] In some embodiments, the inhibitor that inhibits the utilization / release of lipids from extracellular uptake and internal storage comprises a SREBP inhibitor, a fatty acid synthesis inhibitor, a cholesterol synthesis pathway inhibitor, a lysosomal dysregulator, or a combination thereof.

[0115] In some embodiments, the method comprises administering at least one ammonia inhibitor and at least one lysosomal dysregulator. In some embodiments, the method comprises administering at least one ammonia inhibitor and at least one SREBP inhibitor. In some embodiments, the method comprises administering at least one ammonia inhibitor and at least one fatty acid synthesis inhibitor. In some embodiments, the method comprises administering at least one ammonia inhibitor and at least one cholesterol synthesis pathway inhibitor.

[0116] In some embodiments, the method comprises administering at least one ammonia inhibitor, at least one lysosomal dysregulator, and at least one SREBP inhibitor. In some embodiments, the method comprises administering at least one ammonia inhibitor, at least one lysosomal dysregulator, and at least one fatty acid synthesis inhibitor. In some embodiments, the method comprises administering at least one ammonia inhibitor, at least one lysosomal dysregulator, and at least one cholesterol synthesis pathway inhibitor.

[0117] In some embodiments, the lysosomal dysregulation agent increases lysosomal pH.

[0118] In some embodiments, the lysosomal disorder agent comprises an antibiotic, an antipsychotic, an antimalarial, an antiamoebic, a chemical chaperone, an antidepressant, an antiparasitic, a mucolytic, an isoflavone, a monosaccharide analog, a calcium channel agonist or activator, a potassium channel agonist or activator, a micropeptide, an antiepileptic, an immunosuppressant, an antiviral / anticancer inhibitor, a cathepsin inhibitor, a protease inhibitor or a peptidase inhibitor, an alumina compound or a derivative thereof, a kinase inhibitor, a fatty acid synthesis inhibitor, a cholesterol synthesis inhibitor, a serotonin or dopamine inhibitor, an exosome-related inhibitor, a β-galactosidase inhibitor, a heat shock protein (HSP) inhibitor, a piperidine, a bone disease-related inhibitor, or a combination thereof.

[0119] In some embodiments, the antibiotic comprises bafilomycin A, concanamycin, salicylihalamide, oximidine, or a combination thereof. In some embodiments, the antipsychotic comprises pimozide, haloperidol, clozapine, olanzapine, perphenazine, promazine, sulpiride, penfluridol, olanzapine, chlorpromazine, or a combination thereof. In some embodiments, the antimalarial comprises chloroquine, hydroxychloroquine, or a combination thereof. In some embodiments, the chemical partner comprises migalasatat, N-octyl-β-valine amine, NCGC607, or a combination thereof. In some embodiments, the antidepressant comprises fluoxetine. In some embodiments, the antiparasitic comprises pyrimethamine.

[0120] In some embodiments, the mucolytic agent comprises N-acetylcysteine, ambroxol, monensin, or a combination thereof. In some embodiments, the isoflavone comprises genistein, 3,4,7-trihydroxyisoflavone, or a combination thereof. In some embodiments, the monosaccharide analog comprises afegostat. In some embodiments, the calcium channel agonist or activator comprises ML-SA1, MK6-83, or a combination thereof. In some embodiments, the potassium channel agonist or activator comprises ICA-069673. In some embodiments, the micropeptide comprises humanin, SD1002, or a combination thereof. In some embodiments, the antiepileptic drug comprises retigabine. In some embodiments, the immunosuppressant comprises rapamycin, sirolimus, P140, or a combination thereof.

[0121] In some embodiments, the antiviral / anticancer inhibitor comprises apilimod, BRD 1240, saliphenylhalamide, or a combination thereof. In some embodiments, the cathepsin inhibitor comprises RO5461111, odanacatib, CA030, CA-074, CLIK-164, CLIK-181, CLIK-195, SB-357114, L-006235, LHVS (also known as Mu-Leu-HphVSPh), or a combination thereof. In some embodiments, the protease or peptidase inhibitor comprises pepstatin A, α1-antichymotrypsin, CLIK-148, or a combination thereof. In some embodiments, the alumina compound comprises SD1003 or a derivative thereof. In some embodiments, the kinase inhibitor comprises Ly294002, YM-201636, YM-201636, or a combination thereof. In some embodiments, the fatty acid synthesis inhibitor comprises eliglustat, ibiglustat, lucrerastat, or a combination thereof.

[0122] In some embodiments, the cholesterol synthesis inhibitor comprises U18666A, lonafarnib, tipifarnib, or a combination thereof. In some embodiments, the serotonin or dopamine inhibitor comprises SF-22. In some embodiments, the exosome inhibitor comprises GW4869. In some embodiments, the galactosidase inhibitor comprises deoxygalactonojirimycin. In some embodiments, the HSP inhibitor comprises VER-155008. In some embodiments, the piperidine comprises miglustat. In some embodiments, the bone disease-related inhibitor comprises SB-242784, FR167356, or a combination thereof. In some embodiments, the lysosomal dysregulator comprises α-logeline, 5N, 6S-(N'-butylimidomethylene)-6-thio-1-deoxygalactonojirimycin, PADK, or a combination thereof.

[0123] In some embodiments, the lysosomal dysregulator is a synthetic peptide, P140 peptide, currently in Phase III trials for lupus. In some embodiments, the lysosomal dysregulator has the following formula:

[0124]

[0125]

[0126]

[0127]

[0128] In some embodiments, the ammonia inhibitor comprises an ASCT2 inhibitor. In some embodiments, the ammonia inhibitor comprises an ASCT2 inhibitor, and the ASCT2 inhibitor includes V-9302, GPNA, benzylserine (BenSer),

[0129]

[0130] 2-amino-4-bis(aryloxybenzyl)aminobutyric acid (AABA) or a combination thereof. In some embodiments, the 2-amino-4-bis(aryloxybenzyl)aminobutyric acid, for example, a compound has the following formula:

[0131] wherein R is independently selected from C0-4 alkaryl and C0-4 alkylheteroaryl in each case. When C is 0, the substituent is simply aryl or heteroaryl, and when C is 1, the substituent may be specified as benzyl, etc. Exemplary aryl groups include unsubstituted and monosubstituted aryl groups, wherein the substitution is selected from C1-4 alkyl, C1-4 haloalkyl, C1-4 alkoxy, F, Cl, COOH, CN. Specific substituents (when C is 0) include 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, 2-trifluorophenyl, 3-trifluorophenyl, 4-trifluorophenyl, 2-chlorophenyl, 3-chlorophenyl, 4-chlorophenyl, 2-fluorophenyl, 3-fluorophenyl, and 4-fluorophenyl. In other embodiments, C is 1; specific substituents include 2-methylbenzyl, 3-methylbenzyl, 4-methylbenzyl, 2-methoxybenzyl, 3-methoxybenzyl, 4-methoxybenzyl, 2-chlorobenzyl, 3-chlorobenzyl, 4-chlorobenzyl, 2-fluorobenzyl, 3-fluorobenzyl and 4-fluorobenzyl. Exemplary heteroaryl includes pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, preferably when C is 0 or 1.

[0132] In some embodiments, the ACST2 inhibitor is one of:

[0133]

[0134] In some embodiments, the ACST2 inhibitor is

[0135]

[0136] In some embodiments, the ammonia inhibitor comprises a glutaminase inhibitor. In some embodiments, the ammonia inhibitor comprises a GLS1 inhibitor. In some embodiments, the ammonia inhibitor comprises a GLS2 inhibitor.

[0137] In some embodiments, the ammonia inhibitor comprises a glutaminase inhibitor comprising 6-diazo-5-oxonorleucine (also known as "DON"), bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide ("BPES"), 5-(3-bromo-4-(dimethylamino)phenyl)-2,2-dimethyl-2,3,5,6-tetrahydrobenzo[a]phenanthridin-4(1H)-one, telaglenastat ("CB-839"), 2-(2-amino-4-methylpentanamido)-6-diazo-5-oxonorleucine, IPN60090, e.g., GK921; UPGL00004; BPTES1 JHU-083, or a combination thereof, and a compound having the formula:

[0138]

[0139] In some embodiments, the SREBP inhibitor comprises an SRBEP-2 inhibitor. In some embodiments, the SREBP inhibitor is an S2P inhibitor, an S1P inhibitor, an SQLE inhibitor, a fatty acid synthesis pathway inhibitor, an SCD1 inhibitor, an HMG-CoA inhibitor, a FASN inhibitor, or a combination thereof. In some embodiments, the SREBP inhibitor comprises fatostatin, tocotrienol, artesunate, ursolic acid, archazolidB, PF-429242, nelfinavir, cinobufotalin, 24-pyridine; 1-(4-bromophenyl)-3-(25-pyridin-3-yl)urea, fesocostat, YTX-7739, TVB-2640, PF-05221304; ND646; PF-05175157, CP640186, NB-598, terbinafine, or a combination thereof.

[0140] Exemplary HMG-CoA inhibitors include cerivastatin, itavastatin, pitavastatin, simvastatin, simvastatin acid, mevastatin, 3'-hydroxysimvastatin acid, 6'-hydroxymethylsimvastatin acid, lovastatin, atorvastatin, fluvastatin, pravastatin, and rosuvastatin.

[0141] In some embodiments, the SREBP inhibitor comprises a compound having the formula:

[0142]

[0143] In some embodiments, the at least one ammonia inhibitor, the SREBP inhibitor, the fatty acid synthesis inhibitor, or the cholesterol synthesis pathway inhibitor and the lysosomal dysregulatory agent are administered simultaneously. In some embodiments, the first ammonia inhibitor, SREBP inhibitor, fatty acid synthesis inhibitor, or cholesterol synthesis pathway inhibitor is administered during a first time period, and the lysosomal dysregulatory agent is administered during a second time period.

[0144] In some embodiments, the first ammonia inhibitor, the SREBP inhibitor, the fatty acid synthesis inhibitor, or the cholesterol synthesis pathway inhibitor is administered for a period of 1-28 days, 7-28 days, 14-28 days, 21-28 days, 1-21 days, 7-21 days, 14-21 days, 1-14 days, 7-14 days, 1-10 days, 2-10 days, 5-10 days, 1-7 days, 2-7 days, 1-5 days, 1-4 days, 1-3 days, 1-2 days, or 1 day.

[0145] In some embodiments, the lysosomal dysregulation agent is administered for a period of 1-28 days, 7-28 days, 14-28 days, 21-28 days, 1-21 days, 7-21 days, 14-21 days, 1-14 days, 7-14 days, 1-10 days, 2-10 days, 5-10 days, 1-7 days, 2-7 days, 1-5 days, 1-4 days, 1-3 days, 1-2 days, or 1 day.

[0146] In some embodiments, the method further comprises administering to the subject at least one additional anticancer agent. In some embodiments, the method further comprises administering to the subject at least one additional anticancer agent comprising Abemaciclib, Abiraterone Acetate, Abitrexate (Methotrexate), Abraxane (Paclitaxel Albumin-stabilized Nanoparticle Formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (Brentuximab Vedotin), ADE, Ado-Trastuzumab Emtansine, Adriamycin (Doxorubicin Hydrochloride), Afatinib Maleate (Afatinib Dimaleate, Afinitor (Everolimus), Akynzeo (Netupitant and Palonosetron Hydrochloride), Aldara (Imiquimod), Aldesleukin, Alecensa (Alectinib), Alectinib, Alemtuzumab, Alimta (Pemetrexed Disodium), Aliqopa (Copanlisib Hydrochloride), Alkeran for Injection (Melphalan Hydrochloride), Alkeran Tablets (Melphalan Hydrochloride), Tablet) (Melphalan), Aloxi (Palonosetron Hydrochloride), Alunbrig (Brigatinib), Ambochlorin (Chlorambucil), AmboclorinChlorambucil, Amifostine, Aminolevulinic Acid, Anastrozole, Aprepitant, Aredia (Pamidronate Disodium), Arimidex (Anastrozole), Aromasin (Exemestane), Arranon (Nelarabine), Arsenic Trioxide, Arzerra (Ofatumumab), Asparaginase Erwinia chrysanthemi), Atezolizumab, Avastin (Bevacizumab), Avelumab, Axitinib, Azacitidine, Bavencio (Avelumab), BEACOPP, Becenum (Carmustine), Beleodaq (Belinostat), Belinostat, Bendamustine Hydrochloride, BEP, Besponsa (Inotuzumab Ozogamicin), Bevacizumab, Bexarotene, Bexxar (Tositumomab), and Iodine I 131 Tositumomab 131Tositumomab), Bicalutamide, BiCNU (Carmustine), Bleomycin, Blinatumomab, Blincyto (Blinatumomab), Bortezomib, Bosulif (Bosutinib), Bosutinib, BrentuximabVedotin, Brigatinib, BuMel, Busulfan, Busulfex (Busulfan), Cabazitaxel, Cabometyx (Cabozantinib-S-Malate), Cabozantinib-S Malate, CAF, Campath (Alemtuzumab), Camptosar, (Irinotecan Hydrochloride), Capecitabine, CAPOX, Carac (Fluorouracil - Topical), Carboplatin, CARBOPLATIN-TAXOL, Carfilzomib, Carmubris (Carmustine), Carmustine Implant (Carmustine Implant), Casodex (Bicalutamide), CEM, Ceritinib, Cerubidine (Daunorubicin Hydrochloride), Cervarix (recombinant HPV bivalent vaccine), Cetuximab, CEV, Chlorambucil, Chlorambucil-PREDNISONE, CHOP, Cisplatin, Cladribine, Clafen (Cyclophosphamide), Clofarabine, Clofarex (Clofarabine), Clolar (Clofarabine), CMF, Cobimetinib, Cometriq (Cabozantinib-S-Malate), Copanlisib Hydrochloride, COPDAC, COPP, COPP-ABV, Cosmegen (Dactinomycin), Cotellic (Cobimetinib), Crizotinib, CVP, Cyclophosphamide, Cyfos (Ifosfamide), Cyramza (Ramucirumab), Cytarabine, Cytarabine liposomeLiposome, Cytosar-U (Cytarabine), Cytoxan (Cytoxan), Dabrafenib, Dacarbazine, Dacogen (Decitabine), Dactinomycin, Daratumumab, Darzalex (Daratumumab), Dasatinib, Daunorubicin Hydrochloride, Daunorubicin Hydrochloride and Cytarabine Liposome, Decitabine, Defibrotide Sodium, Defitelio (Defibrotide Sodium), Degarelix, Denileukin Diftitox, Denosumab, DepoCyt (Cytarabine Liposome), Dexamethasone, Dexrazoxane Hydrochloride, Dinutuximab, Docetaxel, Doxil (Doxorubicin Hydrochloride Liposome), Doxorubicin Hydrochloride, Doxorubicin Hydrochloride Liposome Liposome), Dox-SL (liposome doxorubicin hydrochloride), DTIC-Dome (dacarbazine), Durvalumab, Efudex (fluorouracil - topical), Elitek (Rasburicase), Ellence (Epirubicin Hydrochloride), Elotuzumab, Eloxatin (Oxaliplatin), EltrombopagOlamine, Emend (Aprepitant), Empliciti (Elotuzumab), Enasidenib Mesylate, Enzalutamide, Epirubicin Hydrochloride, EPOCH, Erbitux (Cetuximab), Eribulin Mesylate, Erivedge (Vismodegib), Erlotinib Hydrochloride, Erwinaze (Asparaginase Erwinia chrysanthemi), Ethyol (Amifostine), Etopophos (Etoposide Phosphate), Phosphate), Etoposide, Etoposide Phosphate, Evacet (Doxorubicin Hydrochloride Liposomal), Everolimus, Evista (Raloxifene Hydrochloride), Evomela (Melphalan Hydrochloride), Exemestane, 5-FU (Fluorouracil Injection), 5-FU (Fluorouracil Topical), Fareston (Toremifene), Farydak (Panobinostat), Faslodex (Fulvestrant), FEC, Femara (Letrozole), Filgrastim, Fludara (Fludarabine Phosphate), Phosphate), Fludarabine Phosphate, Fluoroplex (Fluorouracil - Topical), Fluorouracil Injection, Fluorouracil - Topical, Flutamide, Folex (Methotrexate), FolexPFS (methotrexate), FOLFIRI, FOLFIRI-BEVACIZUMAB, FOLFIRI-CETUXIMAB, FOLFIRINOX, FOLFOX, Folotyn (Pralatrexate), FU-LV, Fulvestrant, Gardasil (recombinant H PV quadrivalent vaccine), Gardasil 9 (recombinant HPV nine-valent vaccine), Gazyva (Obinutuzumab), Gefitinib, Gemcitabine Hydrochloride, Gemcitabine-CISPLATIN, Gemcitabine-Oxaliplatin, Gemtuzumab Ozogamicin Ozogamicin, Gemzar (gemcitabine hydrochloride), Gilotrif (afatinib dimaleate), Gleevec (imatinib mesylate), Gliadel (carmustine implant), Gliadel Wafer (carmustine implant), Glucarpidase, Goserelin Acetate, Halaven (eribulin mesylate), Hemangeol (propranolol hydrochloride), Herceptin (trastuzumab), Recombinant HPV Bivalent Vaccine, Recombinant HPV Nine-valent Vaccine, Recombinant HPV Four-valent Vaccine, Hycamtin (topotecan hydrochloride), Hydrochloride), Hydrea (Hydroxyurea), Hydroxyurea, Ultra CVAD, Ibrance (Palbociclib), Ibritumomab Tiuxetan, Ibrutinib, ICE, Iclusig (Ponatinib Hydrochloride), Idamycin (Idarubicin Hydrochloride),Hydrochloride), Idarubicin Hydrochloride, Idelalisib, Idhifa (Ensidipine Mesylate), Ifex (Ifosfamide), Ifosfamide, Ifosfamidum (Ifosfamide), IL-2 (Aldesleukin), Imatinib Mesylate, Imbruvica (Ibrutinib), Imfinzi (Durvalumab), Imiquimod, Imlygic (Talimogene Laherparepvec), Inlyta (Axitinib), Ointuzumab, Interferon Alpha-2b, Recombinant Interleukin-2 (Aldesleukin), Intron A (Recombinant Interferon Alpha-2b), Iodobenzylguanidine I 131 Tositumomab and Tositumomab, Ipilimumab, Iressa (gefitinib), Irinotecan hydrochloride, Irinotecan hydrochloride liposome, Istodax (romidepsin), Ixabepilone, Ixazomib Citrate, Ixempra (ixabepilone), Jakafi (ruxolitinib phosphate Phosphate), JEB, Jevtana (Cabazitaxel), Kadcyla (Adotrastuzumab Emtansine), Keoxifene (Raloxifene Hydrochloride), Kepivance (Palifermin), Keytruda (Pembrolizumab), Kisqali (Ribociclib), Kymriah (Tisagenlecleucel), Kyprolis (Carfilzomib), Lanreotide Acetate, Lapatinib Ditosylate, Lartruvo (Olarumab), Lenalidomide, Lenvatinib Mesylate Mesylate), Lenvima (lenvatinib mesylate), Letrozole, Leucovorin Calcium, Leukeran (chlorambucil), Leuprolide acetate (Acetate, Leustatin (Cladribine), Levulan (Aminolevulinic Acid), Linfolizin (Chlorambucil), LipoDox (Doxorubicin Hydrochloride Liposomal), Lomustine, Lonsurf (Trifluridine and Tipiracil Hydrochloride), Lupron (Luprolide Acetate), Lupron Depot (Luprolide Acetate), Lupron Depot-Ped (Luprolide Acetate), Lynparza (Olaparib), Marqibo (Vincristine Sulfate Liposome), Matulane (Procarbazine Hydrochloride), Hydrochloride), Mechlorethamine Hydrochloride, Megestrol Acetate, Mekinist (Trametinib), Melphalan, Melphalan Hydrochloride, Mercaptopurine, Mesna, Mesnex (Mesna), Temozolomide (Methazolastone or Temozolomide), Methotrexate, Methotrexate LPF (Methotrexate), Methylnaltrexone Bromide, Mexate or Methotrexate, Mexate-AQ (Methotrexate), Midostaurin, Mitomycin C (Mitomycin C), Mitoxantrone Hydrochloride, Mitozytrex (Mitomycin C), MOPP, Mozobil (Plerixafor), Mustarden (Mechlorethamine HydrochlorideHydrochloride), Mutamycin (Mitomycin C), Myleran (Busulfan), Mylosar (Azacitidine), Mylotarg (Ointuzumab), Nanoparticle Paclitaxel (Albumin-Stable Nanoparticle Formulation of Paclitaxel), Navelbine (Vinorelbine Tartrate), Necitumumab, Nelarabine, Neosar (Cyclophosphamide), Neratinib Maleate, Nerlynx (Neratinib Maleate), Netupitant, and Palonosetron Hydrochloride Hydrochloride), Neulasta (Pegfilgrastim), Neupogen (Filgrastim), Nexavar (Sorafenib Tosylate), Nilandron (Nilutamide), Nilotinib, Nilutamide, Ninlaro (Ixazomib Citrate), Niraparib Tosylate Monohydrate, Nivolumab, Nolvadex (Tamoxifen Citrate), Citrate), Nplate (Romiplostim), Obinutuzumab, Odomzo (Sonidegib), OEPA, Ofatumumab, OFF, Olaparib, Olamab, Omacetaxine Mepesuccinate, Oncaspar (Pegaspargase), Ondansetron Hydrochloride, Onivyde (Irinotecan Liposomal Hydrochloride), Ontak (DenileukinDiftitox), Opdivo (Nivolumab), OPPA, Osimertinib, Oxaliplatin, Paclitaxel, Paclitaxel Albumin-Stable Nanoparticle Formulation, PAD, Palbociclib, Palifermin, Palonosetron Hydrochloride, Palonosetron Hydrochloride and Netupitant, Pamidronate Disodium, Panitumumab, Panobinostat, Paraplat (Carboplatin), Carboplatin (Paraplatin or Carboplatin), Pazopanib Hydrochloride Hydrochloride), PCV, PEB, Pegaspargase, Pegfilgrastim, Peginterferon Alfa-2b, PEG-Intron, Pembrolizumab, Pemetrexed Disodium, Perjeta (Pertuzumab), Pertuzumab, Cisplatin, Cisplatin-AQ, Plerixafor, Pomalidomide, Pomalyst (Pomalidomide), Ponatinib Hydrochloride, Portrazza (Nexituzumab), Pralatrexate, Prednisone, Procarbazine Hydrochloride Hydrochloride), Proleukin (Aldesleukin), Prolia (Denosumab), Promacta (Eltrombopag Olamine), Propranolol Hydrochloride, Provenge (Sipuleucel-T), Purinethol (Mercaptopurine), Purixan (Mercaptopurine), Radium 223 Dichloride, Raloxifene HydrochlorideHydrochloride), Ramucirumab, Rasburicase, R-CHOP, R-CVP, Recombinant Human Papillomavirus (HPV) Bivalent Vaccine, Recombinant Human Papillomavirus (HPV) Nine-Valent Vaccine, Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine, Recombinant Interferon α-2b, Regorafenib, Relistor (Methylnaltrexone Bromide), R-EPOCH, Revlimid (Lenalidomide), Rheumatrex (Methotrexate), Ribociclib, R-ICE, Rituxan (Rituximab), Rituxan Hycela (Rituximab and Hyaluronidase Human), Rituximab, Rituximab and Human Hyaluronidase, Rolapitant Hydrochloride, Romidepsin, Romiplostim, Rubidomycin (Daunorubicin Hydrochloride), Rubraca (Rucaparib Camsylate), Rucaparib Camsylate, Ruxolitinib Phosphate, Rydapt (Midostaurin), Sclerosol Intrapleural Aerosol (Talc), Siltuximab, Sipuleucel-T, Somatuline Depot (Lanreotide Acetate), Sonidegib, Sorafenib Tosylate Tosylate), Sprycel (dasatinib), STANFORD V, Sterile Talc Powder (talc), Steritalc (talc), Stivarga (regorafenib), Sunitinib Malate, Sutent (sunitinib malate), Sylatron (peginterferon alfa-2b), Sylvant (siltuximab), Synribo (homacetaxineMepesuccinate), Tabloid (Thioguanine), TAC, Tafinlar (Dabrafenib), Tagrisso (Osimertinib), Talc, Talilav, Tamoxifen Citrate, Tarabine PFS PFS (Cysarabinoside), Tarceva (Erlotinib HCl), Targretin (Bexarotene), Tasigna (Nilotinib), Taxol (Paclitaxel), Taxotere (Docetaxel), Tecentriq (Atezolizumab), Temodar (Temozolomide), Temozolomide, Temsirolimus, Thalidomide, Thalomid (Thalidomide), Thioguanine, Thiotepa, Tisagenlecleucel, Tolak (Fluorouracil - Topical), Topotecan HCl, Toremifene, Torisel (Temsirolimus), Tositumomab, and Iodine I 131 Tositumomab, Totect (Dexrazoxane Hydrochloride), TPF, Trabectedin, Trametinib, Trastuzumab, Treanda (Bendamustine Hydrochloride), Trifluridine and Tipiracil Hydrochloride, Trisenox (Arsenic Trioxide), Tykerb (Lapatinib Ditosylate), Unituxin (Datuximab), Uridine Triacetate, VAC, Vandetanib, VAMP, Varubi (Rolapitant Hydrochloride), Vectibix (Panitumumab), VeIP, Velban (Vinblastine Sulfate)Sulfate, Velcade (bortezomib), Velsar (vinblastine sulfate), Vemurafenib, Venclexta (Venetoclax), Venetoclax, Verzenio (Abemaciclib), Viadur (leuprorelin acetate), Vidaza (azacitidine), Vinblastine sulfate, Vincasar PFS (vincristine sulfate), Vincristine sulfate, Vincristine sulfate liposomal, Vinorelbine Tartrate, VIP, Vismodegib, Visogard (Uridine triacetate) Triacetate), Voraxaze (Glucarpidase), Vorinostat (Vorinostat), Pazopanib (Pazopanib HCl), Vyxeos (Daunorubicin HCl and Liposomal Cytarabine), Leucovorin (Wellcovorin or Leucovorin Calcium), Xalkori (crizotinib), Xeloda (capecitabine), XELIRI, XELOX, Xgeva (denosumab), Xofigo (radium 223 dichloride), Xtandi (enzalutamide), Yervoy (ipilimumab), Yondelis (trabectedin), Zaltrap (ziv-aflibercept), Zarxio (filgrastim), Zejula (niraparib tosylate monohydrate), Zelboraf (vemurafenib), Zevalin (ibritumomab tiuxetan), Zinecard (dexrazoxane hydrochloride), Ziv-aflibercept, Zofran (ondansetron hydrochloride), Hydrochloride), Zoladex (goserelin acetate), Zoledronic Acid, Zolinza (vorinostat), Zometa (zoledronic acid), Zydelig (idelalisib), Ceritinib, and / or Zytiga (abiraterone acetate).

[0147] In some embodiments, the cancer comprises acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), cancer in adrenocortical carcinoma, adrenal cortex cancer, AIDS-related cancer, Kaposi sarcoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendix cancer, carcinoid, astrocytoma, atypical teratoid / rhabdoid tumor, basal cell carcinoma, skin cancer (non-melanoma), bile duct cancer, extrahepatic bladder cancer, bladder cancer, bone cancer (including Ewing sarcoma), sarcoma) and osteosarcoma and malignant fibrous histiocytoma), brain tumor, breast cancer, bronchial tumor, Burkitt lymphoma (non-Hodgkin), carcinoid tumor, cardiac tumor, atypical teratoid / rhabdoid tumor, embryonal tumor, germ cell tumor, lymphoma, primary cervical cancer, bile duct cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative neoplasm, colon cancer, colon cancer, craniopharyngioma, cutaneous T-cell lymphoma, ductal carcinoma in situ (DCIS), embryonal tumor, central nervous system tumor, endometrial cancer, Ependymoma, esophageal tumor, olfactory neuroblastoma, Ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, intraocular melanoma, retinoblastoma, fallopian tube cancer, bone fibrous histiocytoma, malignant and osteosarcoma, gallbladder cancer, gastric (gastric or stomach) cancer, gastric cancer, gastrointestinal carcinoid, gastrointestinal stromal tumor (GIST), gastrointestinal stromal tumor (GIST), germ cell tumor, central nervous system tumor, extracranial tumor, extragonadal tumor, ovarian testicular cancer, gestational trophoblastic disease, glioma, hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer, histiocytosis, Langerhans cell carcinoma (Langerhans Cell, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, pancreatic neuroendocrine tumor, Kaposi's sarcoma, renal-Langerhans cell histiocytosis, laryngeal cancer, laryngeal cancer and papillomatosis, leukemia, lip and oral cancer, liver cancer (primary), lung cancer, lung cancer, lymphoma-macroglobulinemia, Waldenstrom-non-Hodgkin lymphoma ( -Non-Hodgkin lymphoma), male breast cancer, malignant fibrous histiocytoma of bone and osteosarcoma, melanoma, intraocular (eye) cancer, Merkel cell carcinoma, mesothelioma, malignant mesothelioma, occult primary metastatic squamous neck cancer, midline tract cancer involving the NUT gene, oral cancer, multiple endocrine neoplasms syndrome, multiple myeloma / plasma cell neoplasms, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasms and chronic myeloproliferative neoplasms, chronic myeloid leukemia (CML), acute myeloid leukemia (AML), nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, lip and oral cavity cancer and oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian Cancer, pancreatic cancer and pancreatic neuroendocrine tumors (islet cell tumors), papillomatosis, paraganglioma, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pheochromocytoma, pituitary tumor, plasma cell neoplasms / multiple myeloma, pleuropulmonary blastoma, pregnancy and breast cancer, primary central nervous system (CNS) lymphoma, primary peritoneal cancer, prostate cancer, rectal cancer, renal cell (kidney) cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, salivary gland tumors, Ewing sarcoma, Kaposi sarcoma, osteosarcoma, rhabdomyosarcoma, uterine sarcoma, vascular tumors, Sézary syndrome (Sézary syndrome syndrome), skin cancer, small cell lung cancer, small intestinal cancer, soft tissue sarcoma, squamous cell carcinoma, primary metastatic squamous neck cancer, stomach (stomach or gastric) cancer, gastric cancer, T-cell lymphoma, skin cancer, testicular cancer, throat cancer, thymoma and thymic cancer, thyroid cancer, ureter and renal pelvis cancer, transitional cell carcinoma, urethral cancer, uterine cancer, endometrial and uterine sarcoma, vaginal cancer, vaginal cancer, hemangioma, vulvar cancer, Waldenstrom's macroglobulinemia ( Macroglobulinemia) or Wilms Tumor.

[0148] It should be understood that the method comprises administering the composition, compound or formulation in the amount, time and route required to achieve the desired effect. The exact amount will vary from subject to subject, depending on the species, age and general condition of the subject, the severity of the cancer, the specific composition, compound or formulation, its mode of administration, its mode of activity, etc. For ease of administration and uniformity of dosage, the composition, compound or formulation is preferably formulated in dosage unit form. However, it should be understood that the total daily dosage of the composition, compound or formulation will be determined by the attending physician within the scope of reasonable medical judgment. The specific therapeutically effective dosage level for any particular subject will depend on a variety of factors, including: the cancer being treated and the severity of the cancer; the activity of the composition, compound or formulation used; the specific composition, compound or formulation used; the age, weight, general health, sex and diet of the subject; the time of administration, route of administration and excretion rate of the specific composition, compound or formulation used; the duration of treatment; the drugs used in combination or simultaneously with the specific composition, compound or formulation used, and similar factors well known in the medical field.

[0149] The composition, compound or formulation can be administered by any route. In some embodiments, the composition, compound or formulation is administered by a variety of routes, including oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (such as by powder, ointment, cream and / or drops), mucosal, nasal, oral, intestinal, sublingual, by intratracheal instillation, bronchial instillation and / or inhalation and / or as an oral spray, nasal spray and / or aerosol. The most appropriate route of administration will generally depend on various factors, including the properties of the composition, compound or formulation (e.g., its stability in the environment of the gastrointestinal tract), the condition of the subject (e.g., whether the subject can tolerate oral administration), etc.

[0150] The exact amount of the composition, compound or formulation required to achieve a therapeutically effective amount will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of side effects or symptoms, the identity of the particular composition, compound or formulation, the mode of administration, etc. The amount to be administered to, for example, a child or adolescent can be determined by a medical practitioner or technician in the field, and can be less than or the same as that administered to an adult.

[0151] In one aspect, disclosed herein is a kit comprising: a first agent comprising an ammonia inhibitor; and another agent comprising one or more lipid metabolism inhibitors or related agents thereof.

[0152] In some embodiments, the lipid metabolism inhibitor according to any of the preceding aspects comprises at least one inhibitor that inhibits the utilization / release of lipids from extracellular uptake (LDL) and internal storage (lipid droplets, LD).

[0153] In some embodiments, the inhibitor of inhibiting utilization / release of lipids from extracellular uptake and internal storage according to any of the preceding aspects comprises a SREBP inhibitor, a fatty acid synthesis inhibitor, a cholesterol synthesis pathway inhibitor, or a lysosomal dysregulator.

[0154] In some embodiments, the kit comprises at least one ammonia inhibitor and at least one SREBP inhibitor according to any one of the preceding aspects.

[0155] In some embodiments, the kit comprises at least one ammonia inhibitor and at least one fatty acid synthesis inhibitor according to any one of the preceding aspects.

[0156] In some embodiments, the kit comprises at least one ammonia inhibitor and at least one cholesterol synthesis pathway inhibitor according to any one of the preceding aspects.

[0157] In some embodiments, the kit comprises at least one ammonia inhibitor and at least one lysosomal disorder agent according to any of the preceding aspects.

[0158] In some embodiments, the kit comprises at least one ammonia inhibitor, at least one lysosomal dysregulator, and at least one SREBP inhibitor according to any one of the preceding aspects.

[0159] In some embodiments, the kit comprises at least one ammonia inhibitor, at least one lysosomal dysregulator, and at least one fatty acid synthesis inhibitor according to any of the preceding aspects.

[0160] In some embodiments, the kit comprises at least one ammonia inhibitor, at least one lysosomal dysregulator, and at least one cholesterol synthesis pathway inhibitor according to any of the preceding aspects.

[0161] In one aspect, disclosed herein is a pharmaceutical composition comprising at least one ammonia inhibitor alone or in combination with one or more lipid metabolism inhibitors or related agents thereof.

[0162] In some embodiments, the pharmaceutical composition comprises the one or more lipid metabolism inhibitors, comprising at least one inhibitor of inhibiting the utilization / release of lipids from extracellular uptake (LDL) and internal storage (lipid droplets, LD) according to any one of the preceding aspects.

[0163] In some embodiments, the inhibitor that inhibits utilization / release of lipids from extracellular uptake and internal storage comprises a SREBP inhibitor, a fatty acid synthesis inhibitor, a cholesterol synthesis pathway inhibitor, or a lysosomal dysregulator according to any one of the preceding aspects.

[0164] In some embodiments, the pharmaceutical composition comprises at least one ammonia inhibitor and at least one SREBP inhibitor according to any one of the preceding aspects.

[0165] In some embodiments, the pharmaceutical composition comprises at least one ammonia inhibitor and at least one fatty acid synthesis inhibitor according to any one of the preceding aspects.

[0166] In some embodiments, the pharmaceutical composition comprises at least one ammonia inhibitor according to any one of the preceding aspects and at least one cholesterol synthesis pathway inhibitor.

[0167] In some embodiments, the pharmaceutical composition comprises at least one ammonia inhibitor according to any one of the preceding aspects and at least one lysosomal disorder agent.

[0168] In some embodiments, the pharmaceutical composition comprises at least one ammonia inhibitor, at least one lysosomal dysregulator, and at least one SREBP inhibitor according to any one of the preceding aspects.

[0169] In some embodiments, the pharmaceutical composition comprises at least one ammonia inhibitor, at least one lysosomal dysregulator, and at least one fatty acid synthesis inhibitor according to any of the preceding aspects.

[0170] In some embodiments, the pharmaceutical composition comprises at least one ammonia inhibitor according to any one of the preceding aspects, at least one lysosomal dysregulator, and at least one cholesterol synthesis pathway inhibitor.

[0171] In one aspect, disclosed herein is a method of treating a solid tumor in a patient in need thereof, the method comprising administering to the patient at least one ammonia inhibitor, SREBP inhibitor, fatty acid synthesis inhibitor, or cholesterol synthesis pathway inhibitor alone or in combination with an inhibitor that inhibits the utilization / release of lipids from extracellular uptake and internal storage.

[0172] In one aspect, disclosed herein is a method of inhibiting lipogenesis in a patient in need thereof, the method comprising administering to the patient at least one ammonia inhibitor, SREBP inhibitor, fatty acid synthesis inhibitor, or cholesterol synthesis pathway inhibitor alone or in combination with an inhibitor that inhibits the utilization / release of lipids from extracellular uptake and internal storage.

[0173] In one aspect, disclosed herein is a method of increasing reactive oxygen species in a patient in need thereof, the method comprising administering to the patient at least one ammonia inhibitor, SREBP inhibitor, fatty acid synthesis inhibitor, or cholesterol synthesis pathway inhibitor alone or in combination with an inhibitor that inhibits utilization / release of lipids from extracellular uptake and internal storage.

[0174] In one aspect, disclosed herein is a method of causing mitochondrial damage to a patient in need thereof, the method comprising administering to the patient at least one ammonia inhibitor, SREBP inhibitor, fatty acid synthesis inhibitor, or cholesterol synthesis pathway inhibitor alone or in combination with an inhibitor that inhibits the utilization / release of lipids from extracellular uptake and internal storage.

[0175] Examples

[0176] The following examples are set forth below to illustrate compositions, devices, methods and results according to the disclosed subject matter. These examples are not intended to include all aspects of the subject matter disclosed herein, but are intended to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention that are apparent to those skilled in the art.

[0177] Example 1: Ammonia is a key activator that stimulates SCAP / INSIG dissociation and SREBP-1 activation to promote adipogenesis and tumor growth

[0178] Experimental Procedure

[0179] Reagents. Antibodies to SCAP (9D5) (no. sc-69836), PDI (H-17) (no. sc-30932), and lamin A (H-102) (no. sc-20680) were purchased from Santa Cruz Biotechnology. Antibody to SCAP (no. A303-554A) was purchased from Bethyl Laboratories, Inc. Antibodies to SREBP-2 (no. 557037) and SREBP-1 (IgG-2A4) (557036) for western blotting were purchased from BD Pharmingen. Antibodies to SREBP-1 (2A4) (no. ab3259), GLS (no. ab93434), and transmembrane protein (no. ab24586) for immunofluorescence (IF) were from Abcam. Antibodies against ASPG (No. HPA069761) and SDS (No. LS-C173534) were from Sigma and Lifespan Biosciences, respectively. Antibodies against GFP (No. 11814460001), FLAG-tag (No. F3165), p-EGFR Y1086 (No. 369700) and EGFR (No. 05-1047) were purchased from Roche, Sigma, Invitrogen and Millipore, respectively. Antibodies to FASN (No. 3180S), SCD1 (M38) (No. 2438S), HA-tag (C29F4) (No. 3724S), p-Akt Thr308 (No. 9275S), Ser473 (587F11) (No. 4051S), Akt (pan) (C67E7) (No. 4691S), BiP (C50B12) (3177s) and Grp94 (20292S) were purchased from Cell Signaling. Antibody to ribosome-binding glycoprotein I (PIPA527562) was purchased from Fisher. Antibody to ERGIC-53 (rat homolog, p58) (E1031) was purchased from Sigma. Glucose (No. G8644), sodium L-lactate (No. L7022), α-ketoglutarate sodium salt (No. K1875), L-glutamic acid monosodium salt monohydrate (No. 49621) and ammonium hydroxide solution (No. K318612) were from Sigma. L-glutamine (No. 25030-081) and sodium pyruvate (No. 11360-070) were from Life Technologies.Ammonium chloride (No. 12125-02-9), GPNA (γ-L-glutamyl-p-nitroaniline hydrochloride) (No. 151495) and RPMI1640 containing 2 g / L sodium bicarbonate and without L-glutamine and glucose (No. 091646854) were from MP Biomedicals. CB-839 (No. A14396-5) was from AdooQ Bioscience, and pepstatin A (No. P5318), leupeptin (No. L2884) and human EGF (No. E9644) were purchased from Sigma. Dulbecco's modified Eagle's medium (DMEM) without glucose, pyruvate, glutamine (No. 17-207-CV) and DPBS (21-030-CV) was purchased from Corning. Water-soluble cholesterol (No. C4951), 25-hydroxycholesterol (25-HC) (No. H1015) and GTP (10106399001) were purchased from Sigma. Hanks' Balanced Salt Solution (HBSS) (No. 14170) was purchased from Life Technologies. Octyl-α-KG (SML2205), L-histidine monohydrochloride (H5659), L-isoleucine (I7403), L-leucine (L8912), L-lysine monohydrochloride (L8662), L-methionine (M5308), L-phenylalanine (P5482), L-threonine (T8441), L-tryptophan (T8941), L-valine (V0513), L-aspartic acid (A7219), L-arginine monohydrate (A8381), L-arginine hydrochloride (A6969), L-tyrosine (T8566) and L-cystine dihydrochloride (C6727) were purchased from Sigma. Ammonia assay kit (ab83360), glutamate assay kit (ab138883) and α-ketoglutarate (α-KG) assay kit (ab83431) were purchased from Abcam. ATG5 siRNA (sc-41445) was purchased from Santa Cruz. siRNAs for GDH1 (catalog number L-004032-00-0005), GDH2 (catalog number L-009067-01-0005), ASPG (catalog number E-030336-00-0005) and SDS (catalog number L-008214-01-0005) were purchased from Dharmacon.Creatine kinase (CK) (10127566001), creatine phosphate sodium tetrahydrate (27920), sorbitol (56755), adenosine 5'-triphosphate disodium salt hydrate (A7699), and hexyl β-D-glucopyranoside (53180) were from Sigma.

[0180] Clinical samples. Individual lung tumors and adjacent normal tissues, lung tumor tissue microarrays (TMA) containing 50 paired (tumors and matching adjacent normal lung tissues) and 49 unpaired lung tumor tissues and individual GBM tumor tissues are from the Department of Pathology at The Ohio State University. All human tissues were collected from Ohio State University Hospitals (Ohio State University Hospitals) according to the scheme approved by the Institutional Review Board (IRB) and HIPPA, and confirmed by histology. The glioma TMA with 91 tumors is from the University of Kentucky (University of Kentucky), and the IRB approval of the United Kingdom has been obtained before the study began. The HIV and hepatitis B test results of all samples are negative. ScanScope is used to scan TMA slides, and ImageScope v11 software (Aperio Technologies, Vista, CA, USA, in Vista, California, USA) is used for analysis. The staining intensity of the tissue is divided into 0, 1+, 2+ or 3+. The H-score was calculated using the following formula: H-score = [1 x (% of cells with 1+) + 2 x (% of cells with 2+) + 3 x (% of cells with 3+)] x 100.

[0181] Plasmids. pCMV-Myc-Insig-1, pcDNA3.1-2xFlag-SREBP-1a (full length) and -1c (full length), pcDNA3.0-HA-SREBP-2 (full length), and pcDNA3.0-GFP-SCAP (QQQ) plasmids were obtained or cloned as previously described. pcDNA3.0-GFP-SCAP wild-type plasmid was a gift from Dr. Peter Espenshades at Johns Hopkins University. pcDNA3.0-GFP-SCAP (D428A) was constructed by PCR from pcDNA3.1-SCAP D428A plasmid provided by Drs. Brown and Goldstein at the University of Texas Southwestern Medical Center. Four other single-point mutants, including pcDNA3.0-GFP-SCAP-(D428E), -(D428N), -(D428K), -(S326A), -(S330A), -(S326A / S330A), and -(V353G), were constructed using site-directed mutagenesis (Q5 Site-Directed Mutagenesis Kit, No. E0554S, NEB).

[0182] Cell culture and transfection. U87, U87EGFR, LN229, T98, M233, HepG2, HEK293T and MDA468 were maintained in DMEM (No. 15-013-CV, Cellgro). H1299, H1975, HCC4006 and H1299-luc cell lines were cultured in RPMI-1640 culture medium (No. 15040CV, Cellgro). All culture media were supplemented with 5% HyClone fetal bovine serum (FBS, No. SH30071.03, GE Healthcare) and 4mM glutamine (No. 25030-081, Life Technologies). GBM30, primary GBM patient-derived cells were maintained in DMEM / F12 (No. MT90090PB, Fisher) containing B-27 serum-free supplement (1×), heparin (2 mg / ml), EGF (50 ng / ml), glutamine (2 mM) and fibroblast growth factor (FGF, 50 ng / ml). All cell lines were cultured at 37° C. in a humidified atmosphere of 5% CO 2 . Transfection of plasmids was performed using X-tremeGENE HP DNA transfection reagent (No. 06366236001, Roche) according to the manufacturer's instructions.

[0183] Cell proliferation. A total of 2×104 cells were seeded into 12-well plates and washed with PBS after 24 hours, followed by addition of fresh medium containing 1% dialyzed FBS (No. 35-071-CV, Cellgro) and supplemented with or without 5 mM glucose or / and 4 mM glutamine for 4 days. After trypan blue staining, live cells were counted using a hemocytometer at the specified time.

[0184] Western Blot. Cells were lysed with RIPA buffer containing a protease inhibitor cocktail and phosphatase inhibitors. Proteins were separated on 12% SDS-PAGE and transferred to ECL nitrocellulose membranes (No. 1620112, Bio-Rad). After blocking for 1.5 hours in 5% skim milk powder diluted in Tris-buffered saline containing 0.1% Tween 20, the membranes were incubated with various primary antibodies followed by incubation with appropriate secondary antibodies conjugated to horseradish peroxidase. Immunoreactivity was revealed using an ECL kit (No. RPN2106, GE Healthcare).

[0185] Quantitative real-time PCR. Total RNA was isolated with TRIzol according to the manufacturer's protocol, and cDNA was synthesized with the iScript cDNA synthesis kit. Quantitative real-time PCR was performed using the Applied Biosystems (ABI) 7900HT Real-Time PCR System with iQ SYBRGreen Supermix. Expression was normalized to the 36B4 housekeeping gene and calculated using the comparative method (2-ΔΔCt).

[0186] The pLKO.1-puro lentiviral vector containing shRNA of SREBP-1 (No. 1: TRCN0000414192; No. 2: TRCN0000421299), shSREBP-2 (TRCN0000020665), shGLS (No. 1: TRCN0000051135; No. 2: TRCN0000051137) and non-mammalian shRNA control (No. SHC002) was purchased from Sigma. 293FT cells were transfected with shRNA vectors and packaging plasmid psPAX2 (No. 12260, Addgene) and encapsulation plasmid pMD2.G (No. 12259, Addgene) using polyethyleneimine (No. 23966; Polysciences). Supernatants were harvested after 48 and 72 hours and concentrated using Lenti-X concentrators (No. 631232; Clontech) according to the protocol. Viral titers were quantified by real-time PCR using a qPCR lentiviral titration kit. Lentiviral transduction was performed with polybrene (8 μg / ml) according to the Sigma protocol. Cells were infected with shRNA at the same multiplicity of infection (MOI).

[0187] siRNA knockdown. After the cells were seeded and cultured in full medium supplemented with 5% FBS for 24 hours, the relevant siRNAs targeting ATG5, GDH1 / 2, ASPG or SDS were transfected into H1299 cells using lipofectamine RNAiMAX (13778-150, Invitrogen) for 24 hours. The cells were then washed once with PBS and treated for 12 hours as described in each experiment. The treated cells were harvested and extracted for real-time qRT-PCR and Western blot analysis.

[0188] RNA sequencing. Total RNA from treated H1299 cells was extracted using a total RNA purification plus kit (No. 48300, NORGEN BIOTEK CORP., Canada), followed by quality assessment by NanoDrop One (No. 70-105-8111, Thermo Fisher Scientific, USA). For mRNA library generation, 200 ng of total RNA was treated with the NEBNext PolymRNA Magnetic Separation Module (No. E7490L, New England Biolabs, USA) according to the manufacturer's protocol. Subsequently, the isolated mRNA was fragmented for 10 minutes. cDNA was synthesized and amplified for 12 PCR cycles using the NEBNext Ultra II Directional (Stranded) RNA Library Preparation Kit (No. E7760L, New England Biolabs, USA) and the NEBNext Multilex Oligos Indexes Kit from Illumina according to the manufacturer's instructions. The distribution of template length and adapter-dimer contamination was evaluated using an Agilent 2100 bioanalyzer (No. G2939BA, Agilent Technologies, Inc.) and a high sensitivity DNA kit (No. 5067-4626, Agilent Technologies, Inc.). The average template length was about 150 bp. The contamination of adapter-dimers was negligible. The concentration of the cDNA library was determined using Invitrogen's Qubit dsDNA HS reagent (No. 32851, Invitrogen), and read on a Qubit fluorometer (No. Q33238, Thermo Fisher), and paired-end sequencing was performed on the cDNA library on NovaSeq6000 SP 300 cycles (≤2x 150 bp) (Illumina, USA, USA). The raw data was mapped to the human reference genome (GRCh38p12) by HISAT2 v2.1.0. Differentially expressed genes (DEGs) were called using the limma-voom method. Fold changes in gene expression, false discovery rate (FDR), and p-values ​​were calculated. Pathway analysis of highly significant DEGs (p-value < 10-6) was performed by Ingenuity Pathway Analysis (IPA) (QIAGEN Bioinformatics). Enriched metabolomics pathways were ranked by Z-score. Heatmaps generated by MeV4.9 visualized the top 20 pathways. RNA-seq was performed by the OSUCCC Genomics Shared Resource.

[0189] Immunoprecipitation. Immunoprecipitation was performed as previously described28. Briefly, HEK293T cells were transiently transfected with pcDNA3.0-GFP, pcDNA3.0-GFP-SCAP wild type or pcDNA3.0-GFP-SCAP (D428A) with / without pCMV-Myc-Insig-1 using X-tremeGENE HPDNA transfection reagent. At 24 hours post-transfection, cells were washed once with ice-cold PBS and lysed with 0.5 ml of immunoprecipitation (IP) lysis buffer (50 mM HEPES-KOH, pH 7.4, 100 mM NaCl, 1.5 mM MgCl2, 0.1% Nonidet P-40, 1 μg / ml pepstatin A, 10 μg / ml leupeptin and 2 μg / ml aprotinin). Cell lysates were passed through a 22-gauge needle 15 times and incubated at 4°C for 1 hour. Cell extracts were clarified by centrifugation at 20,000xg for 30 minutes at 4°C. Supernatant was pre-cleared for 1 hour (No. 11243233001, Roche Applied Science) by rotating at 4°C with 30 μl of pre-equilibrated protein G-agarose beads. The pre-clarified lysate was incubated at 4°C for 1 hour with 2 μg of anti-GFP antibody, then 30 μl of pre-equilibrated protein G-agarose beads were added, and rotated at 4°C for 16 hours. After centrifugation, beads were washed three times with 1 ml of ice-cold IP lysis buffer. Bead-bound proteins were eluted by boiling in SDS-PAGE sample buffer, and SDS-PAGE and subsequent Western blot analysis were performed.

[0190] Metabolite analysis in cell culture medium. Metabolite levels in culture medium containing glucose, glutamine, lactate, glutamate and NH4+ were measured using a Nova Bioprofile 100Plus bioanalyzer (Nova Biomedical). H1299 (4×105) or U87 (3×105) cells were inoculated in a 60 mm culture dish for 24 hours. After the cells were washed with PBS, they were switched to 2.5 ml serum-free medium containing glucose (5 mM) and glutamine (4 mM) for 12 hours. The culture or control medium (without cultured cells) was centrifuged at 12,000 rpm for 1 minute and run on a bioanalyzer. After trypan blue staining, the number of cells was determined by using a hemocytometer. Under each experimental condition, the consumption of glucose and glutamine or the production of lactate, glutamate and NH4+ was calculated by subtracting its level in the control medium and normalizing it to the number of cells.

[0191] Measure ammonia levels in tissues and cells and bind to SCAP. To measure ammonia levels in tumors and normal tissues, 10 mg of tissue was collected and lysed by homogenization on ice in an ammonia assay buffer (100 μl) from a commercial kit. The lysate was centrifuged at 20,000 x g for 5 minutes at 4 ° C, and the ammonia level in the supernatant was measured by an ammonia assay kit according to the manufacturer's instructions (number ab83360, Abcam). To measure the ammonia bound by SCAP, a total of 1.3x107 HEK293T cells were inoculated in a 15 cm culture dish for 24 hours. The cells were transfected with GFP, GFP-SCAP wild type or D428A mutant and myc-Insig1 plasmid for 24 hours, and then washed with PBS, followed by adding fresh DMEM medium containing glucose (5 mM) and NH4CI (4 mM) for 2 hours in the absence of glutamine. The cells were then washed with ice-cold PBS and lysed with 1 ml of a buffer containing a protease inhibitor cocktail 50 (25 mM Tris, pH 8.0, 150 mM NaCl, 1% (w / v) LMNG (DL14035, Biosynth Carbosynth). The cell lysate was passed through a 22-gauge needle 30 times and incubated at 4°C for 1 hour. The cell extract was clarified by centrifugation at 17,000 x g for 10 minutes at 4°C. The supernatant was incubated for 1 hour at 4°C by rotating with 50 μl of pre-equilibrated GFP-Trap agarose beads (No. gta, ChromoTek). The precipitated protein complex was washed twice with 1 ml buffer (25 mM Tris, pH 8.0, 150 mM NaCl, 0.005% (w / v) LMNG) and then added to 50 μl ammonia assay buffer to measure ammonia according to the kit instructions. Ammonia, glutamate and α-KG levels in cells were measured using an ammonia assay kit, a glutamate assay kit (ab138883) and an α-KG assay kit (ab83431), respectively, according to the manufacturer's instructions.

[0192] Immunohistochemistry. Tissue sections were cut from biopsy paraffin blocks. Tissue slides were placed in an oven at 60°C for 30 minutes and then deparaffinized by incubating three times with xylene for 5 minutes each, followed by immersion three times in graded alcohols (100%, 95%, 80% and 70%) for 2 minutes each. Slides were washed 3 times with distilled water (dHO) for 5 minutes and then immersed in 3% hydrogen peroxide for 10 minutes, followed by washing with dHO. Slides were transferred to preheated 0.01M citrate buffer (pH 6.0) in a steamer for 30 minutes and then washed with dHO and PBS after cooling. Slides were blocked with 3% BSA / PBS at room temperature for 1 hour and then incubated overnight at 4°C with primary antibodies, followed by incubation at room temperature for 30 minutes with appropriate secondary antibodies containing biotinylated anti-rabbit IgG and biotinylated anti-mouse IgG. After incubation with avidin-biotin complex, followed by washing with PBS (3×5 minutes) and staining with NovaRed solution, the slides were washed with tap water, counterstained with hematoxylin, and briefly immersed in graded alcohols (70%, 80%, 95%, and 100%) and xylene for 2×5 minutes. Finally, the slides were fixed and imaged with SPOT 5.2 (SPOT IMAGING) or scanned with AperioScanscope XT scanner (Leica).

[0193] Immunofluorescence. Cells grown on coverslips were washed twice with PBS and fixed with 4% formaldehyde for 15 minutes, then permeabilized with 0.1% Triton X-100 / PBS for 5 minutes and blocked with 3% bovine serum albumin for 30 minutes at room temperature. Cells were stained with primary antibodies overnight at 4°C or stained at 37°C for 30 minutes, followed by incubation with Alexa Fluor 568-labeled goat anti-rabbit IgG (H+L) (No. A11036, Invitrogen) at 37°C for 30 minutes. Cells were washed three times with PBS in a dark room. Coverslips were washed, inverted, mounted on slides using ProLong Gold antifading reagent and DAPI (No. 2188179, Invitrogen) as described above, and observed with a Zeiss LSM510 Meta confocal microscope.

[0194] Preparation of cell membrane fraction and nuclear extract. The cells were washed once with PBS, scraped into 1 ml PBS, and centrifuged at 1000 x g for 5 minutes at 4°C. The cells were then suspended on ice in an ice-cold buffer containing 10 mM HEPES-KOH (pH 7.6), 10 mM KCl, 1.5 mM MgCl2, 1 mM sodium EDTA, 1 mM sodium EGTA, 250 mM sucrose, and a protease inhibitor cocktail (5 μg / ml pepstatin A, 10 μg / ml leupeptin, 0.5 mM PMSF, 1 mM DTT, and 25 μg / ml ALLN) for 30 minutes. The extract was then passed through a 22G x 11 / 2 needle 30 times and centrifuged at 890 x g for 5 minutes at 4°C to isolate the nuclei. The nuclear pellet was resuspended in 0.1 ml of buffer C (20 mM HEPES / KOH pH 7.6, 0.42 M NaCl, 2.5% (v / v) glycerol, 1.5 mM MgCl2, 1 mM sodium EDTA, 1 mM sodium EGTA) and a protease inhibitor cocktail (5 μg / ml pepstatin A, 10 μg / ml leupeptin, 0.5 mM PMSF, 1 mM DTT, and 25 μg / ml ALLN). The suspension was rotated at 4°C for 60 minutes and centrifuged at 20,000 x g for 20 minutes at 4°C in a microcentrifuge. The supernatant was designated as the "nuclear extract". The nuclear extract was heated at 100°C for 10 minutes with 5x loading buffer prior to SDS-PAGE.

[0195] The supernatant from the 890xg spin was centrifuged at 20,000xg for 20 minutes at 4°C, and the pellet was dissolved in 0.1 ml of SDS lysis buffer (10 mM Tris-HCl pH 6.8, 100 mM NaCl, 1% (v / v) SDS, 1 mM sodium EDTA and 1 mM sodium EGTA), incubated at 37°C for 30 minutes, and designated as the "membrane fraction". Protein concentration was determined by pierce rapidgold BCA protein assay kit (A53225, Thermo Scientific). Bromophenol blue solution (1 μl 100x) was added to each sample before SDS-PAGE and subsequent Western blot analysis.

[0196] Preparation of rat liver cytosol. Male Sprague-Dawley rats (350-400 x g) were anesthetized by isoflurane (1349003, Sigma) inhalation following intraperitoneal injection of buprenorphine (1078700, Sigma) and carprofen (PHR1452, Sigma), and their livers were perfused via the portal vein with 0.9% (w / v) NaCl (R5201-01, B. Braun). The liver was excised and broken in 2 ml / g of ice-cold buffer A (50 mM HEPES-KOH (pH 7.2), 250 mM sorbitol, 70 mM KOAc, 5 mM EGTA potassium, 2.5 mM Mg (OAc) 2 and protease inhibitors) supplemented with 1 mM dithiothreitol (43819, Sigma), and then subsequently 10 impacts were performed in a Dounce homogenizer equipped with a Teflon pestle. The homogenate was centrifuged at 1000 x g for 10 minutes. The supernatant was centrifuged at 20,000 x g for 20 minutes, at 186,000 x g for 1 hour, and at 186,000 x g for 45 minutes. After spinning at 186,000 x g, the fat layer was carefully removed before collecting the aqueous supernatant. All steps were performed at 4 ° C. The final supernatant, considered as cytosol (total protein concentration: 20-35 mg / ml), was divided into aliquots, snap-frozen in liquid nitrogen, and stored at -80° C. For experiments, cytosol was thawed in a 37° C. water bath and on ice for use.

[0197] Microsomal membranes were isolated from H1299 cells for ER budding assays. H1299 cells were washed and scraped from duplicate 15 cm culture dishes into 2 ml of ice-cold DPBS containing protease inhibitors. The cells were centrifuged at 1000 x g for 5 minutes at 4°C. The tubes were snap frozen in liquid nitrogen and stored at -80°C after aspirating the supernatant. When needed, the tubes were thawed in a 37°C water bath for 50 seconds and placed on ice. Each cell pellet was resuspended in 0.4 ml of buffer B (10 mM HEPES-KOH (pH 7.2), 250 mM sorbitol, 10 mM KOAc, 1.5 mM Mg(OAc)2 and protease inhibitors), passed through a 22-gauge needle 20 times, and centrifuged at 1000 x g for 5 minutes at 4°C. The supernatant was transferred to a siliconized microcentrifuge tube (No. 1212M66, Thomas scientific) and centrifuged at 16,000 x g for 3 minutes at 4°C. Subsequently, each pellet was resuspended in 0.5 ml of buffer A and centrifuged again at 16,000 x g for 3 minutes at 4°C. Microsomes for in vitro vesicle formation assays were obtained by dissolving the remaining pellet in 60-100 μl of buffer A. The protein concentration was determined after adding 5 μl of the microsomal suspension to 5 μl of a 20% (w / v) hexyl-β-D-pyranoglucoside solution.

[0198] In vitro vesicle formation assay. Each reaction in a final volume of 80 μl contained 50 mM HEPES-KOH, pH 7.2, 250 mM sorbitol, 70 mM KOAc, 5 mM EGTA potassium, 2.5 mM Mg(OAc)2, 1.5 mM ATP, 0.5 mM GTP, 10 mM creatine phosphate, 4 units / ml of creatine kinase, protease inhibitors, 37-80 μg H1299 microsomal protein, and 600 μg of rat liver cytosol. The reaction was carried out in siliconized 1.5 ml microcentrifuge tubes at 37°C for 15 minutes, stopped by transferring the tubes to ice, and then subsequently centrifuged at 16,000×g for 3 minutes at 4°C to obtain a mid-speed pellet (membrane fraction) and a mid-speed supernatant. The intermediate supernatant was collected from each sample and centrifuged again at 61,000 rpm for 40 minutes at 4°C in a Beckman TLA120.1 rotor to obtain a high-speed pellet (vesicle fraction). The vesicle and membrane fractions were each resuspended in 60 μl of buffer (10 mM Tris-HCl, 100 mM NaCl, 1% (w / v) SDS plus protease inhibitors at pH 7.6, supplemented with 15 μl of buffer: 150 mM Tris-HCl, 15% SDS, 25% (v / v) glycerol, 0.02% (w / v) bromophenol blue and 12.5% ​​(v / v) 2-mercaptoethanol at pH 6.8) and heated at 100°C for 10 minutes. The vesicle and membrane fractions were subjected to 10% SDS-PAGE and analyzed by immunoblotting.

[0199] Lipid synthesis determination. Cells were grown in serum-free medium (containing 5mM glucose and 2mM glutamine) for 24 hours, and stimulated with or without glutamine (4mM) for 10 hours, and the serum-free medium was then replaced with a fresh serum-free medium containing 5mM glucose. After switching to a new serum-free medium (containing 2mM glucose alone or with 2mM glutamine), 0.5μCi 14C-glucose was added to the culture medium and incubated for 2 hours. Cells were washed twice with PBS, and lipids were extracted with 500μl hexane: isopropanol (3: 1) for 1 hour. The liquid phase was collected in a 1.5ml test tube, placed overnight to air dry, and then lipids were dissolved in 200μl chloroform for 0.5-1 hour, and then analyzed with a scintillation counter (Beckman Coulter).

[0200] Xenograft mouse model. Athymic nu / nu female mice (6-8 weeks old) were used. For the lung cancer model, H1299-luc cells were transfected with pC3.0-GFP, pC3.0-GFP-SCAP wild type or pC3.0-GFP-SCAP D428A for 24 hours. The cells were selected with 600ng / ml G418 for two weeks and implanted into mice by tail vein injection (1x 106 cells / mouse suspended in 0.1ml of PBS). After seven weeks, the mice were sacrificed and the lungs were collected, fixed with 4% formaldehyde, and embedded in paraffin. The sections (5μm) were cut and stained with H&E and IHC. For the intracranial xenograft model, GBM30 cells (3.5x103 cells in 4μl of PBS) that stably expressed GFP, GFP-SCAP wild type or GFP-SCAP D428A mutant were stereotactically implanted into the mouse brain. The mice were observed and scanned by magnetic resonance imaging (MRI) until they became moribund, at which time they were sacrificed. The Research Animal Care Subcommittee of the Ohio State University Medical Center approved all animal procedures.

[0201] Hematoxylin and eosin staining. Paraffin tissue sections were deparaffinized in xylene and rehydrated in degraded ethanol dilutions (100%, 95% and 70% ethanol). After washing with dH2O, the slides were stained with hematoxylin and eosin (H&E) solution in sequence, followed by washing with dH2O. The slides were then dehydrated in degraded ethanol and immersed in xylene, followed by mounting in Permount (VECTOR, No. H-5000-60).

[0202] Mouse luminescence imaging. Mice implanted with luciferase-expressing H1299 cells were injected intraperitoneally with luciferin (No. 122796, Perkin Elmer) solution (15 mg / ml in PBS, dose 150 mg / kg). Bioluminescence images were acquired using the IVIS Lumina system and analyzed by LivingImage software.

[0203] Magnetic resonance imaging. The animals were anesthetized with 2.5% isoflurane mixed with 1 liter / minute carbogen (95% O2 and 5% CO2), and then maintained with 1% isoflurane. Physiological parameters including respiration and temperature were monitored using a small animal monitoring system (Model 1025, Small Animals Instruments, Inc. Stony Brook, NY). Air pillows were used to monitor respiration. Core temperature was maintained using circulating warm water in the animal stand. Animals were injected intraperitoneally with 0.1M gadolinium-based contrast agents at a rate of 100 μl per 25 g body weight. Imaging was performed using a Bruker BioSpec94 / 30USRMRI system (Bruker BioSpin, Karlsruhe, Germany) and a mouse brain circular polarization (CP) surface coil and an 86 mm diameter CP volume coil as receiver and transceiver coils, respectively. Data were collected using a T1-weighted rare sequence with the following acquisition parameters: TR = 1200 ms, TE = 7.5 ms, rare factor = 4, NA = 3, FOV = 20 mm × 20 mm, matrix size = 256 × 256, slice thickness = 1 mm, number of slices = 18. Following the T1-weighted acquisition, a T2-weighted rare sequence was also used (parameters: TR = 2500 ms, TE = 33 ms, rare factor = 8, NA = 4, FOV = 20 mm × 20 mm, matrix size = 256 × 256, slice thickness = 1 mm, number of slices = 18). For data analysis, the region of interest (ROI) containing the tumor (high-density area) was outlined. Tumor volume was calculated based on the outlined ROI. All imaging experiments were performed at the OSU Small Animal Imaging Core.

[0204] Molecular dynamics simulations. The cryo-EM structure of the Insig / SCAP complex (PDB ID: 6M49) 50 was used as the initial structure for the simulations. The SCAP structure without 25-HC was prepared by replacing the partially unfolded S4 helix (residues 354-358) in the inactive conformation with a fully folded S4 helix built with Modeller V10.1 using NPC1 (PDB code: 6W5S) 67 as a template. The CHARMM-GUI membrane builder was used to build a membrane bilayer consisting of 366 hydrated palmitoyl-oleoyl-phosphatidylcholine (POPC) molecules 68, 69. Each system was solvated with approximately 34,000 TIP3P water molecules (a type of water used for simulations, representing a 3-position rigid water molecule with charges and Lennard-Jones parameters assigned to each of the 3 atoms (HOH)) and 0.15 M NaCl 70. The CHARMM 36 force field was used for proteins, lipids and ions, while the ligand (25-HC) was parameterized using SwissParam71. All simulations were performed at 310 K and the temperature was adjusted using the v-rescale scheme72. Solutes (proteins, membranes and ligands) and solvents (water and ions) were coupled separately with a relaxation time constant of 0.1 ps. The Parrinello-Rahman barostat was used to maintain the pressure at 1 bar with a coupling constant of 0.2 ps. The isothermal compressibility was 4.5×10-5 bar-1. The pressure was semi-isotropically coupled, with the x and y directions coupled together and the z direction coupled independently. All bonds were constrained using the LINCS algorithm. The integration time step was 2 fs. Non-bonded long-range electrostatic interactions were calculated using the particle mesh Ewald method with a cutoff of The van der Waals interaction is also used All simulations were performed using Gromacs 202073. Each system was first energy minimized using the steepest descent method with a maximum step size of 50,000 or when the maximum force in the system was less than After energy minimization, 500 ps equilibrium simulations were performed with positional restraints on proteins, lipids, and ligands, followed by six 1 ns simulations with progressively fewer positional restraints. Finally, a ~1 microsecond long generation simulation without any restraints was run for each system, with trajectories saved every 100 ps (~10,000 frames total per simulation) for subsequent analysis.

[0205] Quantitative and statistical analysis. Unless otherwise stated, all data including Western blots, metabolite analysis, and mouse experiments represent at least two biological replicates. Data analysis was performed using GraphPad Prism 7. Statistical significance was obtained using paired or unpaired Student's t-test or one-way or two-way ANOVA, depending on the data. Multiplicity was adjusted by Dunnett's or Turkey's method. The Kaplan-Meier method was used to generate patient and mouse overall survival curves, and differences in survival were tested using the log-rank test.

[0206] result

[0207] Ammonia released from glutamine is a key activator of SREBP activation and lipogenesis. Glutaminolysis is known to be highly activated in many cancers to promote rapid growth. In this process, glutamine is first deaminated by glutaminase (GLS) to release the polar molecule ammonia (NH3) and produce glutamate. Glutamate is further converted to α-ketoglutarate (α-KG) which is incorporated into the tricarboxylic acid (TCA) cycle in mitochondria to produce energy. Using the Bioprofile100Plus analyzer, the levels of major metabolites derived from glutaminolysis and glycolysis were measured in the culture medium of H1299 and U87 cells. NH3 and glutamate converted to NH4+ in aqueous solution were detected in the culture medium of cells cultured in the presence of glutamine without glucose (12 hours) ( Figure 1A , middle panel). Thereafter, NH3 and NH4+ are referred to as ammonia. In contrast, when cells were cultured in the presence of glucose but not glutamine, lactate and glutamate ( Figure 1A , top panel), indicating that glutamate can be derived from glucose when glutamine is absent. When glutamine and glucose were combined, all three metabolites, NH4+ / glutamate / lactate ( Figure 1A , bottom small image).

[0208] Next, metabolites involved in SREBP activation were investigated. Western blot analysis showed that glutamate, ammonia (derived from added NH4Cl), or lactate alone or glucose in combination with glutamate or lactate failed to activate SREBP-1 or -2 cleavage and promote FASN and SCD1 expression ( Figure 1B Interestingly, in the presence of glucose, ammonia induced SREBP-1 and -2 cleavage, as well as FASN and SCD1 expression, to a similar extent as the combination of glutamine and glucose ( Figure 1B , Lane 9 vs Lane 6). In addition, the effect of ammonia was dose- and time-dependent ( Figure 1C and Figure 1DImmunofluorescence imaging further revealed that ammonia significantly stimulated SREBP-1 translocation into the nucleus in the presence of glucose but not in the absence of glutamine ( Figure 1E ), whereas glutamate, lactate, or α-KG stimulation could not do so.

[0209] Together, these data suggest that ammonia released from glutamine is a key activator of SREBP activation and lipogenesis and that the presence of glucose is required to maintain SCAP stability through its N-glycosylation, a prerequisite for SREBP activation by either glutamine or ammonia.

[0210] Suppression of ammonia release from glutamine by inhibition of GLS abolished SREBP activation and lipogenesis. Next, the role of ammonia released from glutamine in stimulating SREBP activation and lipogenesis by inhibiting glutamine uptake and glutaminolysis was verified. Glutamine uptake was inhibited by γ-glutamyl-p-nitroaniline (GPNA), an inhibitor of SLC1A5 (also known as ASCT2), which has been characterized as the major glutamine transporter, and glutaminolysis was blocked with CB-839, a GLS inhibitor. Metabolite analysis showed that both GPNA and CB-839 treatment significantly reduced the levels of glutamate, ammonia, and α-KG in cells and cell culture media ( Figure 4A ), which was associated with a significant reduction in glutamine consumption by both H1299 and U87 cells. The cells were then supplemented with ammonia (by adding NH4Cl), glutamate, or α-KG to determine which metabolite could restore SREBP activation inhibited by CB-839. Western blot analysis clearly showed that only ammonia, and not glutamate or α-KG, could strongly restore SREBP activation and FASN / SCD1 expression ( Figure 4B ). We further investigated whether inhibition of GLS by CB-839 had an effect on SREBP-1 activation in an H1299-derived xenograft mouse model. Consistent with the reduction of ammonia levels in tumor tissue ( Figure 4C , left panel), immunohistochemistry (IHC) staining showed that SREBP-1 levels were significantly reduced in tumors from mice treated with CB-839 compared with the vehicle-treated group ( Figure 4C , right panel). GLS was also genetically inhibited using lentiviral-mediated shRNA to inhibit ammonia release from glutamine.

[0211] Together, these data demonstrate that ammonia is released from glutamine and, together with glucose, activates SREBPs and lipogenesis, revealing a novel glutamine-GLS-ammonia-SREBP activation axis that links glutaminolysis and lipogenesis, two metabolic pathways that are highly upregulated in various cancers.

[0212] In clinical samples of lung cancer and glioma, high GLS expression was significantly associated with strong SREBP-1 activation. The next step was to investigate whether the above molecular link between glutaminolysis and lipogenesis could be verified in human tissues. Seven paired frozen tissues from individuals with adenocarcinoma (Adeno), squamous cell and large cell lung cancer, i.e., tumor and adjacent normal human lung tissue, were measured by Western blotting. The data showed that all seven tumor tissues contained high levels of GLS and strong SREBP-1 expression and cleavage, as well as significantly increased FASN protein ( ) compared with adjacent normal lung tissue. Figure 2A ). Then, multiple paraffin-embedded tumor tissues and adjacent normal lung tissues from patients with adenocarcinoma and squamous cell lung cancer were studied by IHC staining. Consistent with the Western blot results, IHC staining showed that GLS expression and cytoplasmic and nuclear SREBP-1 staining were highly elevated in tumor tissue (T), while both were low in adjacent normal tissue (N) ( Figure 2B and Figure 5A Then, ammonia levels in ten paired human lung tumors and adjacent normal lung tissues were measured using an ammonia assay kit. Elevated GLS expression and SREBP-1 activation ( Figure 2A and Figure 2B , Figure 5A ), the data showed that ammonia levels in tumors were significantly higher than those in paired normal tissues ( Figure 2C ). Tissue microarrays (TMA) containing 99 tumors and 50 matched adjacent normal lung tissues from individuals with different types of lung cancer were then studied. IHC staining showed that more than 90% of lung tumor tissues contained high levels of GLS and strong SREBP-1 staining compared to adjacent normal lung tissue ( Figure 2D and Figure 2E , Figures 5B-5D ). Pearson correlation analysis showed that GLS expression was closely correlated with SREBP-1 staining in these lung cancer tissues ( Figure 2F ). Thus, genetic knockdown of GLS in a xenograft model gave the same results as SREBP-1 knockdown, significantly inhibiting tumor growth in an H1299 cell-derived xenograft mouse model ( Figure 5E ). Multiple GBM tissues and a TMA containing 91 glioma samples were also studied, ranging from low-grade astrocytomas (A2) and oligodendrogliomas (O2) to high-grade anaplastic astrocytomas (AA), GBMs, and anaplastic oligodendrogliomas (AO). As in lung cancer tissues, IHC staining showed that high GLS expression was associated with strong SREBP-1 staining in tumor tissues from low-grade to high-grade gliomas ( Figure 2G-2I , Figure 5F-5H). Pearson correlation analysis showed that GLS expression was closely correlated with SREBP-1 staining in these glioma tissues ( Figure 2J ). Kaplan-Meier plot analysis further demonstrated that higher GLS expression and stronger SREBP-1 staining were associated with worse survival in individuals with GBM ( Figure 2K The presence of other amino acid deaminases, specifically ASPG and SDS, was determined by IHC. The staining results showed that no enzymes were detected in GBM tumor samples ( Fig. 5F ), confirming a specific positive correlation between GLS and SREBP-1 in patient tissues. In conclusion, these large clinical sample analyses showed that GLS expression is significantly associated with SREBP-1 activation in human cancers, providing strong evidence supporting a molecular link between glutaminolysis and lipogenesis under physiological conditions.

[0213] Disruption of SCAP interaction with ammonia by D428 mutation inhibits lung cancer and glioblastoma growth. We next investigated whether disruption of the SCAP-ammonia interaction by changing D428 in SCAP to alanine (D428A) would affect tumor growth. GFP, GFP-SCAP wild-type, or D428A mutant were transfected into H1299 lung cancer cells stably expressing luciferase. Western blot analysis showed that in the presence of glucose and glutamine, wild-type SCAP expression significantly increased SREBP-1 and -2 cleavage, which was abolished by the D428A mutation ( Figure 3A These stably transfected cells were then implanted into mice by tail vein injection and studied by bioluminescent imaging, which clearly showed at day 50 post-implantation that wild-type SCAP expression significantly increased tumor growth in the lung area compared to the control GFP group, while the D428A mutation abolished this increase ( Figure 3B and Figure 3C ). The overall lung images showed that the number of tumor lesions on the lung surface was greater in the wild-type SCAP group compared with the GFP group, while the lung tumor lesions in the D428A mutant group were significantly less than those in the wild-type SCAP group ( Figure 3D , left small picture, Fig. 6A ).

[0214] Hematoxylin and eosin (H&E) staining confirmed that the number of tumor lesions in the lungs of the wild-type SCAP group was significantly increased compared with the GFP group and the SCAP D428A group ( Figure 3D and Figure 3E , Figure 6B ). IHC staining showed that SREBP-1 staining in lung tumor tissues in the wild-type SCAP group was significantly elevated compared with the GFP group, while the D428A mutation completely abolished this increase ( Figure 3D and Figure 3E These experiments were repeated using primary GBM30 cells53-55. As in H1299 cells ( Figure 3A ), wild-type SCAP expression significantly increased SREBP-1 and -2 cleavage in GBM30 cells, which was abolished by the D428A mutation ( Figure 3F ). These stably transfected GBM cells were implanted into the mouse brains, and tumor growth was studied by magnetic resonance imaging (MRI) on days 12 and 17. Imaging results showed that tumor volumes in the wild-type SCAP group were significantly larger than those in the GFP control group, and the D428A mutation reduced tumor growth to a volume similar to that of the GFP control group ( Figure 3G and Figure 6C H&E staining of the brains of GBM-bearing mice further showed that the tumor sizes in the different groups at day 17 were consistent with those detected by MRI imaging ( Figure 3H , left small picture and Fig.15 D) IHC staining showed that SREBP-1 staining in the wild-type SCAP group was significantly stronger than that in the other two groups ( Figure 3H and Fig. 3I In addition, the survival time of mice implanted with wild-type SCAP-expressing cells was significantly shorter than that of mice in the GFP group, and the D428 mutation significantly prolonged the survival time of mice to a level similar to that of mice in the control group ( Figure 3J Together, these data from two cancer models demonstrate that disruption of the SCAP-NH4+ interaction by mutating the D428 residue significantly inhibits SREBP-1 activation and tumor growth.

[0215] SREBP is a master transcription factor that plays a key role in regulating lipid metabolism. Interestingly, it is spatially confined to the ER membrane after synthesis. The mechanism that triggers SREBP to exit from the ER for subsequent nuclear translocation and activation of lipogenesis remains unclear. In this example, ammonia released by glutamine plays an unprecedented role as a key activator of SREBP activation and lipid synthesis. By showing a molecular link between GLS expression and SREBP-1 activation in human lung cancer and glioma tissues, physiological evidence between glutaminolysis and lipogenesis is also shown. In addition, it has been demonstrated that in addition to lung cancer and GBM, glutamine / ammonia and glucose synergistically activate SREBP and lipogenesis in melanoma, liver cancer and breast cancer cells, indicating that this is a common mechanism that works in a wide range of cancer types. In summary, ammonia plays an unexpected role in regulating SREBP activation and lipid metabolism.

[0216] When glucose and glutamine levels are low, SREBP-1 activation and lipogenesis are reduced, regardless of oncogenic signaling, allowing tumor cells to conserve limited amounts of nutrients to maintain basic cellular activity and survival. Thus, tumors function as well-organized organs that coordinate oncogenic signaling with nutrient availability to dynamically control the activity of anabolic pathways as tumors grow. From this perspective, combining targeting oncogenic signaling with nutrient restriction is an effective approach for cancer therapy.

[0217] In conclusion, this example reveals that ammonia released from glutamine serves as a key signaling molecule to activate lipid metabolism. Developing techniques or methods that can directly detect the interaction or binding of ammonia with specific targets is essential to reveal the largely unexplored functions of ammonia. In addition, there is a need to develop sensors to directly measure ammonia levels in tissues and microenvironments to explore its physiological functions. Effective cancer therapies are needed to limit ammonia signaling to prevent it from stimulating tumor growth.

[0218] Ammonia released from glutamine acts synergistically with glucose to promote lipogenesis by activating sterol regulatory element binding protein (SREBP), a transcription factor that binds to the endoplasmic reticulum (ER) and plays a central role in lipid metabolism. Ammonia activates glucose-regulated N-glycosylated SREBP cleavage-activating protein (SCAP) to dissociate from the ER retention protein Insig by binding to SCAP aspartate 428 (D428) and serine 326 / 330 residues, thereby triggering sequential conformational changes in SCAP, ultimately leading to SREBP translocation and lipogenic gene expression. Interestingly, 25-hydroxycholesterol prevents ammonia from entering its binding site on SCAP, thereby blocking binding to SCAP and inhibiting SCAP / Insig dissociation. Mutating D428 to alanine (D428A) also prevents ammonia binding to SCAP and the ensuing conformational changes, abrogating SREBP-1 activation and inhibiting tumor growth.

[0219] SREBP is synthesized as an inactive precursor (about 125 kD) retained in the endoplasmic reticulum (ER) membrane and activated by a strictly controlled ER-Golgi-nuclear translocation process. SREBP first binds to SREBP-cleavage activation protein (SCAP), which further binds to COPII-coated vesicles that transport the SCAP / SREBP complex from the ER to the Golgi. In the Golgi, SREBP is sequentially cleaved by site-1 and -2 proteases, which release its N-terminal form (about 65 kD), which then enters the nucleus to activate lipogenic gene expression. However, the transport of the SCAP / SREBP complex is inhibited by the ER retention protein, the insulin-induced gene protein (Insig), which contains two subtypes, Insig-1 and -2. Insig binds to SCAP and retains the SCAP / SREBP complex in the ER. It was previously revealed that cholesterol or 25-hydroxycholesterol (25-HC) can bind to SCAP or Insig to enhance their association, thereby mediating a negative feedback loop to regulate SREBP activation. However, the key step for activated SCAP to dissociate from Insig for subsequent translocation remains unclear.

[0220] It is demonstrated herein that glucose stimulates SREBP activation and lipogenesis by promoting SCAP N-glycosylation and stability. In this example, it was unexpectedly found that when glutamine is deficient, despite low cholesterol levels and stable SCAP N-glycosylation, glucose alone cannot activate SREBP and lipogenesis. It is revealed here that N-glycosylated SCAP requires the stimulation of ammonia released by glutamine to undergo continuous conformational changes in order to dissociate from Insig and promote SREBP translocation and lipogenesis. A binding site for ammonia was identified in the central position of the SCAP transmembrane domain, which contains D428 and serine S326 / S330 residues, indicating that the function of ammonia is blocked by 25-hydroxycholesterol (25-HC), which blocks access to its binding site on SCAP, thereby inhibiting SCAP / Insig dissociation and SREBP activation. This example further demonstrates that targeting the key molecular chain between glutamine, glucose and lipid metabolism is a strategy for treating malignancies and metabolic syndrome. Therefore, inhibiting lipid supply from both internal storage lipid droplets and external lipoproteins by inhibiting lysosomal function may achieve a synergistic effect.

[0221] Example 2: In vitro use of pimozide

[0222] Glioblastoma (GBM) is the most lethal primary brain tumor in adults, and the prognosis for patients has remained largely unchanged over the past two decades, with the median survival of patients remaining at 12-16 months after initial diagnosis, even with extensive treatment. Therefore, there is an urgent need to identify new and effective therapies for GBM. At the time of diagnosis, many GBM cells have typically invaded the surrounding normal brain tissue, making complete surgical resection of the tumor unlikely. In addition, the blood-brain barrier (BBB) ​​significantly limits the penetration of many anti-tumor drugs into GBM tissue. Even if the drugs can be effectively delivered intracranially, GBM cells quickly develop resistance to these drugs, compromising their efficacy and affecting the overall outcome. Therefore, GBM is one of the most difficult cancers to treat.

[0223] Fatty acids (FA) and cholesterol are two essential lipids for cell growth and proliferation. FA constitutes the hydrophobic tail of phospholipids, and cholesterol is inserted between phospholipids, regulating membrane fluidity and permeability. Studies have shown that GBM cells obtain abundant cholesterol from external sources by upregulating the low-density lipoprotein receptor (LDLR)-mediated uptake of LDL, the main cholesterol carrier in the bloodstream. LDL contains abundant cholesterol esters (CE) that are hydrolyzed in lysosomes to release free cholesterol and FA for GBM growth. Lipid droplets (LD) are a hallmark of adipocytes and contain abundant CE and triacylglycerols (TAG). Studies have also shown that patient-derived GBM tissue contains a large number of LDs that are also present in other cancers such as breast cancer, prostate cancer, liver cancer, pancreatic cancer, colon cancer, and kidney cancer. LD hydrolysis in lysosomes and the release of stored cholesterol and FA promote GBM growth. Recent studies have shown that ammonia (NH4+) released by glutamine activates sterol regulatory element binding protein 1 (SREBP-1), a key transcription factor that regulates lipogenic gene expression to promote FA and cholesterol synthesis. Together, these studies demonstrate that GBM cells actively acquire multiple lipid sources to ensure an adequate supply of FAs and cholesterol to support their rapid growth. Therefore, limiting GBM cells' access to lipid sources that include LDL, LD hydrolysis, and de novo synthesis is an effective approach to targeting this deadly cancer. However, it is very challenging to block all three lipid sources simultaneously, specifically in a clinically relevant manner.

[0224] Recently, many studies have shown that various antipsychotic drugs exhibit antitumor activity. The proposed antitumor mechanisms of these drugs are very broad, including damaging lysosomes, stimulating autophagy, inhibiting the function of different oncogenes, activating tumor suppressors, etc. Some reported mechanisms are controversial, such as the presence of autophagy stimulation despite the induction of lysosomal damage, because it is expected that autophagic flux will be blocked when lysosomal activity is inhibited. In summary, the main mechanism of the antitumor effect of these antipsychotic drugs remains unclear. In addition, these agents have only shown mild to moderate antitumor effects in preclinical animal studies involving GBM. The question of why tumor cells are insensitive to these drugs and / or how tumor cells become resistant to these drugs has not been studied clearly, which limits the reuse of these drugs in cancer treatment. Nevertheless, these neuron-based antipsychotic drugs remain of great interest, especially for brain tumors, because the drugs can effectively cross the BBB.

[0225] Nine FDA-approved antipsychotic drugs were screened for antitumor effects. Pimozide, used to treat schizophrenia and motor and speech tics associated with Tourette's syndrome, was the most potent drug for killing GBM cells in vitro ( Figures 19A-19C ). In a Phase II clinical trial in the 1980s, pimozide was tested for the treatment of melanoma (n=30), but only 6 (17%) patients responded well. Recent studies have reported that pimozide treatment can cause lysosomal membrane permeabilization, resulting in the release of the protease cathepsin into the cytosol, while overactivation of autophagy causes GBM cell death. In contrast, another study showed that pimozide treatment alone had no significant effect in an intracranial model of GBM and produced only a modest antitumor effect when combined with radiation therapy. These data suggest that GBM cells become resistant to pimozide, but the underlying mechanism is unclear. The data presented in this article show that pimozide treatment effectively inhibits the hydrolysis of both LD and LDL in GBM cells, causing them to accumulate in lysosomes ( Fig.21A and Fig. 21B ). Supplementation of GBM cells with free FA and cholesterol significantly rescued cell viability inhibited by pimozide. These results question the conclusion of others that pimozide-induced GBM cell death is caused by autophagy activation and protease leakage in lysosomes, because FA / cholesterol addition did not rescue autophagy and protease-mediated cell killing. In addition, the activity of lysosomal proteases is mainly dependent on the acidic environment (pH 4.5 to 5.0). Once exposed to the neutral environment in the cytosol (pH about 7.0), lysosomal proteases will rapidly lose their activity.

[0226] Stable isotope 13C-glutamine tracking experiments were performed and found that glutamine uptake, glutaminolysis, and citrate synthesis mediated by reductive carboxylation derived from glutamine were greatly increased after pimozide treatment in vitro. Furthermore, pharmacological inhibition of the glutamine transporter ASCT2 or glutaminase (GLS), which controls the first step of glutaminolysis, in combination with pimozide to inhibit glutamine uptake or glutaminolysis, respectively, resulted in almost complete elimination of GBM cell-derived colonies, whereas monotherapy of each drug showed only a slight inhibitory effect ( Figures 22A-22B Additionally, it was observed that the combined treatment induced significant mitochondrial fragmentation ( Fig.23 ).

[0227] Among nine antipsychotics, pimozide was the most potent drug for killing GBM cells in vitro. Nine antipsychotics, including two antidepressants (imipramine and fluoxetine), were reported to have antitumor effects in vitro or in vivo. Pimozide was found to be the most potent drug for reducing GBM cell viability ( Fig.19A The drug almost completely killed primary GBM30 cells and U251 cells derived from patients at 5 μM ( Figures 19B-19C ), while having no toxic effects on normal human astrocytes (NHA), even at doses up to 10 μM.

[0228] Fluorescently labeled pimozide enters the lysosomes of GBM cells. The structure of pimozide shows that the pKa of the amide residue located in the center of the molecule is about 8.63 ( Fig.19A ), which can be quickly protonated in an acidic environment. Lysosomes are the most acidic organelles in cells (pH 4.5 to 5.0), and the activity of lysosomal hydrolases is completely dependent on this acidic environment. It is speculated that pimozide enters the lysosomes of GBM cells and is protonated, and is retained in the lysosomal cavity in the form of protonated pimozide, which is positively charged and cannot pass through the lysosomal membrane. This process consumes a large amount of lysosomal protons (H+), which increases the lysosomal pH and inhibits the lysosomal hydrolysis function, thereby blocking the hydrolysis of LD and LDL. However, there is no direct evidence that antipsychotic drugs can enter the lysosome to destroy the acidic environment.

[0229] To test this, the fluorescent compound Pacific blue was used to label pimozide ( Fig. 20), and cells treated with labeled pimozide were observed by confocal microscopy to determine its distribution and investigate whether it enters lysosomes. Lysosomes were co-stained with LysoTracker (red). The data showed that pimozide directly enters lysosomes, while octanamide-linked Pacific blue alone could not enter the cells and could only bind to the plasma membrane of GBM cells, which ruled out the influence of the fluorophore dye on the passage of labeled pimozide across the plasma membrane ( Fig. 20 ). Therefore, the trafficking and subcellular localization of fluorescently labeled pimozide in the cytoplasm of GBM cells faithfully reflects the trafficking and subcellular localization of the unlabeled pimozide compound.

[0230] Pimozide inhibits LD and LDL hydrolysis in GBM cells. To explore how pimozide affects GBM cells, its effect on LD and LDL hydrolysis was studied. Pimozide treatment significantly inhibited LD and LDL hydrolysis, causing them to accumulate in lysosomes ( Figures 21A-21B Fluorescence imaging also showed that lysosomes became enlarged, swollen, and aggregated after 24 hours of treatment ( Figures 21A-21B ,arrow).

[0231] Pharmacological targeting of glutamine metabolism in combination with pimozide almost eradicated all preformed GBM colonies. The data herein further showed that combining pimozide (3 μM) with pharmacological targeting of the glutamine transporter ASCT2 and γ-glutamyl-p-nitroaniline (GPNA, 0.5 mM) or the key glutaminolytic enzyme GLS and CB-839 (100 nM) or the broad antagonist of glutamine metabolism 6-diazo-5-oxo-I-norleucine (DON, 5 μM) almost completely eliminated preformed GBM colonies, whereas each drug alone had only a slight inhibitory effect ( Figures 22A-22B ). These data strongly suggest that such a combination approach may be a promising strategy for GBM.

[0232] Targeting glutamine metabolism in combination with pimozide resulted in significant mitochondrial damage. We explored how combining pimozide with ASCT2 or GLS inhibitors or DON kills GBM cells. Using fluorescent imaging, each combination resulted in significant mitochondrial fragmentation, whereas the therapeutic effects of the individual drugs were not significant ( Fig.23 ), which strongly suggests that these combinations lead to mitochondrial damage.

[0233] In summary, combining lipogenesis inhibition with lysosomal inhibition allows for simultaneous inhibition of all three lipid sources: de novo lipid synthesis, internal LD ​​storage, and external lipoproteins. By significantly reducing lipid levels, a highly efficient method of killing tumor cells is provided, thereby becoming an effective method for cancer therapy.

[0234] It will be apparent to those skilled in the art that various modifications and variations may be made in the present disclosure without departing from the scope or spirit of the present invention. Other embodiments of the present disclosure will be apparent to those skilled in the art in view of the description and practice of the methods disclosed herein. The description and examples are intended to be considered exemplary only, with the true scope and spirit of the present invention being indicated by the appended claims.

Claims

1. A method of treating cancer in a patient in need thereof, the method comprising administering to the patient at least one ammonia inhibitor alone or in combination with one or more lipid metabolism inhibitors or related agents thereof.

2. A method of treating cancer in a patient in need thereof, the method comprising administering to the patient at least one lipogenesis inhibitor or in combination with one or more lysosomal destruction inhibitors.

3. The method of claim 1, wherein the one or more lipid metabolism inhibitors include at least one inhibitor that inhibits the utilization / release of lipids from extracellular uptake (LDL) and internal storage (lipid droplets, LD).

4. The method according to any one of the preceding claims, wherein the inhibitors that inhibit lipogenesis include SREBP inhibitors, fatty acid synthesis inhibitors, cholesterol synthesis pathway inhibitors.

5. The method according to any one of the preceding claims, wherein the inhibitor that inhibits the utilization / release of lipids from extracellular uptake and internal storage comprises a SREBP inhibitor or a lysosomal dysregulator.

6. The method according to any one of the preceding claims, comprising administering at least one ammonia inhibitor and at least one lysosomal dysregulatory agent.

7. The method according to any one of the preceding claims, comprising administering at least one ammonia inhibitor and at least one SREBP inhibitor.

8. A method according to any one of the preceding claims comprising administering at least one ammonia inhibitor and at least one fatty acid synthesis inhibitor.

9. The method according to any one of the preceding claims, comprising administering at least one ammonia inhibitor and at least one cholesterol synthesis pathway inhibitor.

10. The method according to any one of the preceding claims, comprising administering at least one ammonia inhibitor, at least one lysosomal dysregulator and at least one SREBP inhibitor.

11. The method of any one of the preceding claims comprising administering at least one ammonia inhibitor, at least one lysosomal dysregulator and at least one fatty acid synthesis inhibitor.

12. The method of any one of the preceding claims comprising administering at least one ammonia inhibitor, at least one lysosomal dysregulator, and at least one cholesterol synthesis pathway inhibitor.

13. The method of any one of the preceding claims, wherein the lysosomal dysregulation agent increases lysosomal pH.

14. The method of any one of the preceding claims, wherein the lysosomal disorder agent comprises an antibiotic, an antipsychotic, an antimalarial, a chemical chaperone, an antidepressant, an antiparasitic, a mucolytic, an isoflavone, a monosaccharide analog, a calcium channel agonist or activator, a potassium channel agonist or activator, a micropeptide, an antiepileptic, an immunosuppressant, an antiviral / anticancer inhibitor, a cathepsin inhibitor, a protease inhibitor or a peptidase inhibitor, an alumina compound or a derivative thereof, a kinase inhibitor, a fatty acid synthesis inhibitor, a cholesterol synthesis inhibitor, a serotonin or dopamine inhibitor, an exosome-related inhibitor, a β-galactosidase inhibitor, a heat shock protein (HSP) inhibitor, a piperidine, a bone disease-related inhibitor, or a combination thereof.

15. The method of claim 14, wherein the antibiotic comprises bafilomycin A, concanamycin, salicylihalamide, oximidine, or a combination thereof.

16. The method of claim 14, wherein the antipsychotic comprises pimozide, haloperidol, clozapine, olanzapine, perphenazine, promazine, sulpiride, penfluridol, olanzapine, chlorpromazine, or a combination thereof.

17. The method of claim 14, wherein the antimalarial drug comprises chloroquine, hydroxychloroquine, or a combination thereof.

18. The method of claim 14, wherein the chemical chaperone comprises migalasatat, N-octyl-β-valienamine, NCGC607, or a combination thereof.

19. The method of claim 14, wherein the antidepressant comprises fluoxetine.

20. The method of claim 14, wherein the antiparasitic drug comprises pyrimethamine.

21. The method of claim 14, wherein the mucolytic agent comprises N-acetylcysteine, ambroxol, monensin, or a combination thereof.

22. The method of claim 14, wherein the isoflavones comprise genistein, 3,4,7-trihydroxyisoflavone, or a combination thereof.

23. The method of claim 14, wherein the monosaccharide analog comprises afegostat.

24. The method of claim 14, wherein the calcium channel agonist or activator comprises ML-SA1, MK6-83, or a combination thereof.

25. The method of claim 14, wherein the potassium channel agonist or activator comprises ICA-069673.

26. The method of claim 14, wherein the micropeptide comprises humanin, SD1002, or a combination thereof.

27. The method of claim 14, wherein the anti-epileptic drug comprises retigabine.

28. The method of claim 14, wherein the immunosuppressant comprises rapamycin, sirolimus, P140, or a combination thereof.

29. The method of claim 14, wherein the antiviral / anticancer inhibitor comprises apilimod, BRD 1240, saliphenylhalamide, or a combination thereof.

30. The method of claim 14, wherein the cathepsin inhibitor comprises RO5461111, odanacatib, CA030, CA-074, CLIK-164, CLIK-181, CLIK-195, SB-357114, L-006235, LHVS (also known as Mu-Leu-HphVSPh), or a combination thereof.

31. The method of claim 14, wherein the protease or peptidase inhibitor comprises pepstatin A, α1-antichymotrypsin, CLIK-148, or a combination thereof.

32. The method of claim 14, wherein the alumina compound comprises SD1003 or a derivative thereof.

33. The method of claim 14, wherein the kinase inhibitor comprises Ly294002, YM-201636, YM-201636, or a combination thereof.

34. The method of claim 14, wherein the fatty acid synthesis inhibitor comprises Fatostatin, FT113, BI99179, BI99179, FASN-IN-4 tosylate, IPI-9119, TVB-3166, TVB-3663, TVB-2640, PF429242, GSK2194069, Pseudoprotodioscin, , dihydrochloride, betulin, C75, lycorine, GSK837149A, ACC1 / 2-IN-1, ACC1 / 2-IN-2, JA-ACC, PF-05175157, Firsocostat, MK-4074, CP-610431, 7ACC2, CP-640186, CP-640186, Olumacostat glasaretil, 7ACC1, hACC2-IN-1, CMS-121, fesocostat, Moiramide B, ND-646, acetyl-CoA carboxylase-IN-1, A-908292, Cerulenin, 28UCM, GSK2194069, Orlistat, TOFA, PF-05221304, YTX-465, T-3764518, MF-438, A939572, XEN723, CVT-12012, CAY10566, YTX-7739, MK-8245, GSK1940029, SCD1-IN-1, SCD1 inhibitor-4, eliglustat, ibiglustat, lucrerastat, or a combination thereof.

35. The method of claim 14, wherein the cholesterol synthesis inhibitor comprises a statin, NB-598, Terbinafine, AY9944, BM 15766 sulfate, Triparanol, U18666A, lonafarnib, tipifarnib, or a combination thereof.

36. The method of claim 14, wherein the serotonin or dopamine inhibitor comprises SF-22.

37. The method of claim 14, wherein the exosome inhibitor comprises GW4869.

38. The method of claim 14, wherein the galactosidase inhibitor comprises deoxygalactonojirimycin.

39. The method of claim 14, wherein the HSP inhibitor comprises VER-155008.

40. The method of claim 14, wherein the piperidine comprises miglustat.

41. The method of claim 14, wherein the bone disease-related inhibitor comprises SB-242784, FR167356, or a combination thereof.

42. The method of any of the preceding claims, wherein the lysosomal dysregulatory agent comprises α-logeline, 5N,6S-(N'-butylimidomethylene)-6-thio-1-deoxygalactonojirimycin, PADK, or a combination thereof.

43. The method of any one of the preceding claims, wherein the ammonia inhibitor comprises an ASCT2 inhibitor.

44. The method of any of the preceding claims, wherein the ammonia inhibitor comprises an ASCT2 inhibitor comprising V-9302, GPNA, benzylserine (BenSer), 2-amino-4-bis(aryloxybenzyl)aminobutyric acid (AABA), or a combination thereof.

45. The method of any one of the preceding claims, wherein the ammonia inhibitor comprises a glutaminase inhibitor.

46. ​​The method of any one of the preceding claims, wherein the ammonia inhibitor comprises a GLS1 inhibitor.

47. The method of any one of the preceding claims, wherein the ammonia inhibitor comprises a GLS2 inhibitor.

48. The method of any one of the preceding claims, wherein the ammonia inhibitor comprises a glutaminase inhibitor, the glutaminase inhibitor comprising 6-diazo-5-oxonorleucine (also known as "DON"), bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (also known as "BPES"), 5-(3-bromo-4-(dimethylamino)phenyl)-2,2-dimethyl-2,3,5,6-tetrahydrobenzo[a]phenanthridin-4(1H)-one (also known as "Compound 98"), telaglenastat (also known as "CB-839"), 2-(2-amino-4-methylpentanamido)-6-diazo-5-oxonorleucine, IPN60090, for example GK921; UPGL00004; BPTES; JHU395; JHU-083 or a combination thereof.

49. The method of any one of the preceding claims, wherein the SREBP inhibitor comprises a SRBEP-2 inhibitor.

50. The method of any of the preceding claims, wherein the SREBP inhibitor is a S2P inhibitor, a S1P inhibitor, a SQLE inhibitor, a fatty acid synthesis pathway inhibitor, a SCD1 inhibitor, a HMG-CoA inhibitor, a FASN inhibitor, or a combination thereof.

51. The method of any of the preceding claims, wherein the SREBP inhibitor comprises fatostatin, tocotrienol, artesunate, ursolic acid, archazolid B, PF-429242, nelfinavir, cinobufotalin, 58-pyridine; 1-(4-bromophenyl)-3-(58-pyridin-3-yl)urea, fesocostat, YTX-7739, TVB-2640, PF-05221304; ND646; PF-05175157, CP640186, NB-598, terbinafine, or a combination thereof.

52. The method of any one of the preceding claims, wherein the at least one ammonia inhibitor, the SREBP inhibitor, the fatty acid synthesis inhibitor or the cholesterol synthesis pathway inhibitor and the lysosomal dysregulatory agent are administered simultaneously.

53. The method of any of the preceding claims, wherein a first ammonia inhibitor, a SREBP inhibitor, a fatty acid synthesis inhibitor, or a cholesterol synthesis pathway inhibitor is administered during a first time period and a lysosomal dysregulatory agent is administered during a second time period.

54. The method of any of the preceding claims, wherein the first ammonia inhibitor, the SREBP inhibitor, the fatty acid synthesis inhibitor, or the cholesterol synthesis pathway inhibitor is administered for a period of 1-28 days, 7-28 days, 14-28 days, 21-28 days, 1-21 days, 7-21 days, 14-21 days, 1-14 days, 7-14 days, 1-10 days, 2-10 days, 5-10 days, 1-7 days, 2-7 days, 1-5 days, 1-4 days, 1-3 days, 1-2 days, or 1 day.

55. The method of any one of the preceding claims, wherein the lysosomal dysregulation agent is administered for a period of 1-28 days, 7-28 days, 14-28 days, 21-28 days, 1-21 days, 7-21 days, 14-21 days, 1-14 days, 7-14 days, 1-10 days, 2-10 days, 5-10 days, 1-7 days, 2-7 days, 1-5 days, 1-4 days, 1-3 days, 1-2 days, or 1 day.

56. The method of any of the preceding claims, further comprising administering to the subject at least one additional anti-cancer agent.

57. The method of any of the preceding claims, further comprising administering to the subject at least one additional anticancer agent, the at least one additional anticancer agent comprising Abemaciclib, Abiraterone Acetate, Abitrexate (Methotrexate), Abraxane (Paclitaxel Albumin-stabilized Nanoparticle Formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (Brentuximab Vedotin), ADE, Ado-Trastuzumab Emtansine, Adriamycin (Doxorubicin Hydrochloride), Afatinib Maleate, Dimaleate, Afinitor (Everolimus), Akynzeo (Netupitant and Palonosetron Hydrochloride), Aldara (Imiquimod), Aldesleukin, Alecensa (Alectinib), Alectinib, Alemtuzumab, Alimta (Pemetrexed Disodium), Aliqopa (Copanlisib Hydrochloride), Alkeran for Injection Injection (Melphalan Hydrochloride), Alkeran Tablet (Melphalan), Aloxi (Palonosetron Hydrochloride), Alunbrig (Brigatinib), Ambochlorin (Chlorambucil), AmboclorinChlorambucil, Amifostine, Aminolevulinic Acid, Anastrozole, Aprepitant, Aredia (Pamidronate Disodium), Arimidex (Anastrozole), Aromasin (Exemestane), Arranon (Nelarabine), Arsenic Trioxide, Arzerra (Ofatumumab), Asparaginase Erwinia chrysanthemi), Atezolizumab, Avastin (Bevacizumab), Avelumab, Axitinib, Azacitidine, Bavencio (Avelumab), BEACOPP, Becenum (Carmustine), Beleodaq (Belinostat), Belinostat, Bendamustine Hydrochloride, BEP, Besponsa (Inotuzumab Ozogamicin), Bevacizumab, Bexarotene, Bexxar (Tositumomab), and Iodine I 131 Tositumomab 131Tositumomab), Bicalutamide, BiCNU (Carmustine), Bleomycin, Blinatumomab, Blincyto (Blinatumomab), Bortezomib, Bosulif (Bosutinib), Bosutinib, BrentuximabVedotin), Brigatinib, BuMel, Busulfan, Busulfex (Busulfan), Cabazitaxel, Cabometyx (Cabozantinib-S-Malate), Cabozantinib-S Malate, CAF, Campath (Alemtuzumab), Camptosar, (Irinotecan Hydrochloride), Capecitabine, CAPOX, Carac (Fluorouracil - Topical), Carboplatin, CARBOPLATIN-TAXOL, Carfilzomib, Carmubris (Carmustine), Carmustine Implant (Carmustine Implant), Casodex (Bicalutamide), CEM, Ceritinib, Cerubidine (Daunorubicin Hydrochloride), Cervarix (recombinant HPV bivalent vaccine), Cetuximab, CEV, Chlorambucil, Chlorambucil-PREDNISONE, CHOP, Cisplatin, Cladribine, Clafen (Cyclophosphamide), Clofarabine, Clofarex (Clofarabine), Clolar (Clofarabine), CMF, Cobimetinib, Cometriq (Cabozantinib-S-Malate), Copanlisib Hydrochloride, COPDAC, COPP, COPP-ABV, Cosmegen (Dactinomycin), Cotellic (Cobimetinib), Crizotinib, CVP, Cyclophosphamide, Cyfos (Ifosfamide), Cyramza (Ramucirumab), Cytarabine, Cytarabine liposomeLiposome, Cytosar-U (Cytarabine), Cytoxan (Cytoxan), Dabrafenib, Dacarbazine, Dacogen (Decitabine), Dactinomycin, Daratumumab, Darzalex (Daratumumab), Dasatinib, Daunorubicin Hydrochloride, Daunorubicin Hydrochloride and Cytarabine Liposome, Decitabine, Defibrotide Sodium, Defitelio (Defibrotide Sodium), Degarelix, Denileukin Diftitox、Denosumab、DepoCyt(Cytarabine Liposome)、Dexamethasone、Dexrazoxane Hydrochloride、Dinutuximab、Docetaxel、Doxil(Doxorubicin Hydrochloride Liposome)、Doxorubicin Hydrochloride、Doxorubicin Hydrochloride Liposome Liposome, Dox-SL (Doxorubicin Hydrochloride Liposomal), DTIC-Dome (Dacarbazine), Durvalumab, Efudex (Fluorouracil - Topical), Elitek (Rasburicase), Ellence (Epirubicin Hydrochloride), Elotuzumab, Eloxatin (Oxaliplatin), Eltrombopag Olamine, Emend (Aprepitant), Empliciti (Elotuzumab), Enasidenib MesylateMesylate, Enzalutamide, Epirubicin Hydrochloride, EPOCH, Erbitux (Cetuximab), Eribulin Mesylate, Erivedge (Vismodegib), Erlotinib Hydrochloride, Erwinaze (Asparaginase Erwinia chrysanthemi), Ethyol (Amifostine), Etopophos (Etoposide Phosphate), Etoposide, Etoposide Phosphate, Evacet (Doxorubicin Hydrochloride Liposomal), Everolimus, Evista (Raloxifene Hydrochloride) Hydrochloride), Evomela (Melphalan Hydrochloride), Exemestane, 5-FU (Fluorouracil Injection), 5-FU (Fluorouracil Topical), Fareston (Toremifene), Farydak (Panobinostat), Faslodex (Fulvestrant), FEC, Femara (Letrozole), Filgrastim, Fludara (Fludarabine Phosphate), Fludarabine Phosphate, Fluoroplex (Fluorouracil Topical), Fluorouracil Injection, Fluorouracil Topical, Flutamide, Folex (Methotrexate), Folex PFS (methotrexate), FOLFIRI, FOLFIRI-BEVACIZUMAB, FOLFIRI-CETUXIMAB, FOLFIRINOX, FOLFOX, Folotyn (Pralatrexate), FU-LV, Fulvestrant, Gardasil (recombinant HPV quadrivalent vaccine), Gardasil 9 (recombinant HPV nine-valent vaccine), Gazyva (Obinutuzumab), Gefitinib, GemcitabineHydrochloride), Gemcitabine-CISPLATIN, Gemcitabine-OXALIPLATIN, Gemtuzumab Ozogamicin, Gemzar (Gemcitabine Hydrochloride), Gilotrif (Afatinib Dimaleate), Gleevec (Imatinib Mesylate), Gliadel (Carmustine Implant), Gliadel Wafer (Carmustine Implant), Glucarpidase, Goserelin Acetate, Halaven (Eribulin Mesylate), Hemangeol (Propranolol Hydrochloride), Hydrochloride), Herceptin (Trastuzumab), Recombinant HPV Bivalent Vaccine, Recombinant HPV Nine-valent Vaccine, Recombinant HPV Quadrivalent Vaccine, Hycamtin (Topotecan Hydrochloride), Hydrea (Hydroxyurea), Hydroxyurea, Super CVAD, Ibrance (Palbociclib), Ibritumomab Tiuxetan, Ibrutinib, ICE, Iclusig (Ponatinib Hydrochloride) Hydrochloride), Idamycin (Idarubicin Hydrochloride), Idarubicin Hydrochloride, Idelalisib, Idhifa (Ensidipine Mesylate), Ifex (Ifosfamide), Ifosfamide, Ifosfamidum (Ifosfamide), IL-2 (Aldesleukin), Imatinib Mesylate, Imbruvica (Ibrutinib), Imfinzi (Durvalumab), Imiquimod, Imlygic (Talimogene Laherparepvec), Inlyta (Axitinib), Ointuzumab, Interferon Alpha-2b, Recombinant Interleukin-2 (Aldesleukin), Intron A (Recombinant Interferon Alpha-2b), Iodobenzylguanidine I131 Tositumomab and Tositumomab, Ipilimumab, Iressa (gefitinib), Irinotecan hydrochloride, Irinotecan hydrochloride liposome, Istodax (romidepsin), Ixabepilone, Ixazomib Citrate, Ixempra (ixabepilone), Jakafi (ruxolitinib phosphate Phosphate), JEB, Jevtana (Cabazitaxel), Kadcyla (Adotrastuzumab Emtansine), Keoxifene (Raloxifene Hydrochloride), Kepivance (Palifermin), Keytruda (Pembrolizumab), Kisqali (Ribociclib), Kymriah (Tisagenlecleucel), Kyprolis (Carfilzomib), Lanreotide Acetate, Lapatinib Ditosylate, Lartruvo (Olarumab), Lenalidomide, Lenvatinib Mesylate Mesylate), Lenvima (Lenvatinib Mesylate), Letrozole, Leucovorin Calcium, Leukeran (Chlorambucil), Leuprolide Acetate, Leustatin (Cladribine), Levulan (Aminolevulinic Acid), Linfolizin (Chlorambucil), LipoDox (Liposomal Doxorubicin Hydrochloride), Lomustine, Lonsurf (Trifluridine and Tipiracil Hydrochloride), Lupron (Leuprolide Acetate), Lupron Depot (Leuprolide Acetate), Lupron Depot-Ped (Lupron Acetate), Lupron Depot-Ped (Lupron Acetate), Lupron Depot-Ped (Lupron Acetate), Lupron Depot-Ped (Lupron Acetate), Lupron Depot-Ped (Lupron Acetate), Lupron Depot-Ped (Lupron Acetate), Lupron Depot-Ped (Lupron Acetate), Lupron Depot-Ped (Lupron Acetate), Lupron Depot-Ped (Lupron Acetate), Lupron Depot-Ped (Lupron Acetate), Lupron Depot-Ped (Lupron Acetate), Lupron Depot-Ped (Lupron Acetate), Lupron Depot-Ped (Lupron Acetate), Lupron Depot-Ped (Lupron Acetate), Lupron Depot-Ped (Lupron Acetate), Lupron Depot-Ped (Lupron Acetate), Lupron Depot-Ped (Lupron Acetate), Lupron Depot-PedDepot-Ped (Leuprolide Acetate), Lynparza (Olaparib), Marqibo (Vincristine Sulfate Liposome), Matulane (Procarbazine Hydrochloride), Mechlorethamine Hydrochloride, Megestrol Acetate, Mekinist (Trametinib), Melphalan, Melphalan Hydrochloride, Mercaptopurine, Mesna, Mesnex (Mesna), Methazolastone or Temozolomide, Methotrexate, Methotrexate LPF (Methotrexate), Methylnaltrexone Bromide (Methylnaltrexone Bromide), Bromide), Methotrexate (Mexate or Methotrexate), Methotrexate-AQ (Mexate-AQ) (Methotrexate), Midostaurin, Mitomycin C, Mitoxantrone Hydrochloride, Mitozytrex (Mitomycin C), MOPP, Mozobil (Plerixafor), Mustargen (Mechlorethamine Hydrochloride), Mutamycin (Mitomycin C), Myleran (Busulfan), Mylosar (Azacitidine), Mylotarg (Ointozumab), Nanoparticle Paclitaxel (Paclitaxel Albumin-Stable Nanoparticle Formulation), Navelbine (Vinorelbine Tartrate) Tartrate), Necitumumab, Nelarabine, Neosar (cyclophosphamide), NeratinibMaleate), Nerlynx (Neratinib Maleate), Netupitant and Palonosetron Hydrochloride, Neulasta (Pegfilgrastim), Neupogen (Filgrastim), Nexavar (Sorafenib Tosylate), Nilandron (Nilutamide), Nilotinib, Nilutamide, Ninlaro (Ixazomib Citrate), Niraparib Tosylate Monohydrate (Niraparib Tosylate Monohydrate), Nivolumab, Nolvadex (Tamoxifen Citrate), Nplate (Romiplostim), Obinutuzumab, Odomzo (Sonidegib), OEPA, Ofatumumab, OFF, Olaparib, Olazumab, Omacetaxine Mepesuccinate, Oncaspar (Pegaspargase), Ondansetron Hydrochloride Hydrochloride), Onivyde (liposomal irinotecan hydrochloride), Ontak (denileukin Diftitox), Opdivo (nivolumab), OPPA, Osimertinib, Oxaliplatin, Paclitaxel, Paclitaxel albumin-stabilized nanoparticle formulation, PAD, Palbociclib, Palifermin, Palonosetron Hydrochloride, Palonosetron Hydrochloride and Netupitant, Pamidronate Disodium, Panitumumab, Panobinostat, Paraplat (Carboplatin), Carboplatin (Paraplatin or Carboplatin), Pazopanib HydrochlorideHydrochloride), PCV, PEB, Pegaspargase, Pegfilgrastim, Peginterferon Alfa-2b, PEG-Intron, Pembrolizumab, Pemetrexed Disodium, Perjeta (Pertuzumab), Pertuzumab, Cisplatin, Cisplatin-AQ, Plerixafor, Pomalidomide, Pomalyst (Pomalidomide), Ponatinib Hydrochloride, Portrazza (Nexituzumab), Pralatrexate, Prednisone, Procarbazine Hydrochloride Hydrochloride), Proleukin (Aldesleukin), Prolia (Denosumab), Promacta (Eltrombopag Olamine), Propranolol Hydrochloride, Provenge (Sipuleucel-T), Purinethol (Mercaptopurine), Purixan (Mercaptopurine), Radium 223 Dichloride, Raloxifene Hydrochloride Hydrochloride), Ramucirumab, Rasburicase, R-CHOP, R-CVP, Recombinant Human Papillomavirus (HPV) Bivalent Vaccine, Recombinant Human Papillomavirus (HPV) Nine-Valent Vaccine, Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine, Recombinant Interferon α-2b, Regorafenib, Relistor (Methylnaltrexone Bromide), R-EPOCH, Revlimid (Lenalidomide), Rheumatrex (Methotrexate), Ribociclib, R-ICE, Rituxan (Rituximab), Rituxan Hycela (Rituximab and HyaluronidaseHuman), Rituximab, Rituximab and Human Hyaluronidase, Rolapitant Hydrochloride, Romidepsin, Romiplostim, Rubidomycin (Daunorubicin Hydrochloride), Rubraca (Rucaparib Camsylate), Rucaparib Camsylate, Ruxolitinib Phosphate, Rydapt (Midostaurin), Sclerosol Intrapleural Aerosol (Talc), Siltuximab, Sipuleucel-T, Somatuline Depot (Lanreotide Acetate), Sonidegib, Sorafenib Tosylate Tosylate), Sprycel (Dasatinib), STANFORD V, Sterile Talc Powder (Talc), Steritalc (Talc), Stivarga (Regorafenib), Sunitinib Malate, Sutent (Sunitinib Malate), Sylatron (Peginterferon alpha-2b), Sylvant (Siltuximab), Synribo (Omacetaxine Mepesuccinate), Tabloid (Thioguanine), TAC, Tafinlar (Dabrafenib), Tagrisso (Osimertinib), Talc, Talilav, Tamoxifen Citrate, Tarabine PFS (PFS (Cysarabinoside), Tarceva (Erlotinib HCl), Targretin (Bexarotene), Tasigna (Nilotinib), Taxol (Paclitaxel), Taxotere (Docetaxel), Tecentriq (Atezolizumab), Temodar (Temozolomide), Temozolomide, Temsirolimus, Thalidomide, Thalomid (Thalidomide), Thioguanine, Thiotepa, Tisagenlecleucel, Tolak (Fluorouracil - Topical), Topotecan HCl, Toremifene, Torisel (Temsirolimus), Tositumomab, and Iodine I 131 Tositumomab, Totect (Dexrazoxane Hydrochloride), TPF, Trabectedin, Trametinib, Trastuzumab, Treanda (Bendamustine Hydrochloride), Trifluridine and Tipiracil Hydrochloride, Trisenox (Arsenic Trioxide), Tykerb (Lapatinib Ditosylate), Unituxin (Datuximab), Uridine Triacetate, VAC, Vandetanib, VAMP, Varubi (Rolapitant Hydrochloride), Vectibix (Panitumumab), VeIP, Velban (Vinblastine Sulfate) Sulfate, Velcade (bortezomib), Velsar (vinblastine sulfate), Vemurafenib, Venclexta (Venetoclax), Venetoclax, Verzenio (Abemaciclib), Viadur (leuprorelin acetate), Vidaza (azacitidine), Vinblastine sulfate, Vincasar PFS (vincristine sulfate), Vincristine sulfate, Vincristine sulfate liposomal, Vinorelbine Tartrate, VIP, Vismodegib, Visogard (Uridine triacetate)Triacetate), Voraxaze (Glucarpidase), Vorinostat (Vorinostat), Pazopanib (Pazopanib HCl), Vyxeos (Daunorubicin HCl and Liposomal Cytarabine), Leucovorin (Wellcovorin or Leucovorin Calcium), Xalkori (crizotinib), Xeloda (capecitabine), XELIRI, XELOX, Xgeva (denosumab), Xofigo (radium 223 dichloride), Xtandi (enzalutamide), Yervoy (ipilimumab), Yondelis (trabectedin), Zaltrap (ziv-aflibercept), Zarxio (filgrastim), Zejula (niraparib tosylate monohydrate), Zelboraf (vemurafenib), Zevalin (ibritumomab tiuxetan), Zinecard (dexrazoxane hydrochloride), Ziv-aflibercept, Zofran (ondansetron hydrochloride), Hydrochloride), Zoladex (goserelin acetate), Zoledronic Acid, Zolinza (vorinostat), Zometa (zoledronic acid), Zydelig (idelalisib), Ceritinib, and / or Zytiga (abiraterone acetate).

58. The method of any one of the preceding claims, wherein the cancer comprises acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), cancer in adrenocortical carcinoma, adrenal cortex cancer, AIDS-related cancers, Kaposi sarcoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendix cancer, carcinoid, astrocytoma, atypical teratoid / rhabdoid tumor, basal cell carcinoma, skin cancer (non-melanoma), bile duct cancer, extrahepatic bladder cancer, bladder cancer, bone cancer (including Ewing sarcoma and osteosarcoma and malignant fibrous histiocytoma), brain tumor, breast cancer, bronchial tumor, Burkitt lymphoma, lymphoma (non-Hodgkin), carcinoid tumor, cardiac (cardiac or heart) tumor, atypical teratoid / rhabdoid tumor, embryonal tumor, germ cell tumor, lymphoma, primary cervical cancer, bile duct cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative neoplasm, colon cancer, colon cancer, craniopharyngioma, cutaneous T-cell lymphoma, ductal carcinoma in situ (DCIS), embryonal tumor, central nervous system tumor, endometrial cancer, ependymoma, esophageal tumor, olfactory neuroblastoma, Ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell Tumors, eye cancer, intraocular melanoma, retinoblastoma, fallopian tube cancer, bone fibrous histiocytoma, malignant and osteosarcoma, gallbladder cancer, gastric (gastric or stomach) cancer, gastric cancer, gastrointestinal carcinoid, gastrointestinal stromal tumor (GIST), gastrointestinal stromal tumor (GIST), germ cell tumor, central nervous system tumor, extracranial tumor, extragonadal tumor, ovarian testicular cancer, gestational trophoblastic disease, glioma, hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer, histiocytosis, Langerhans cell carcinoma (LangerhansCell), Hodgkin's lymphoma (Hodgkin's lymphoma), hypopharyngeal cancer, intraocular melanoma, islet cell tumor, pancreatic neuroendocrine tumor, Kaposi's sarcoma, renal-Langerhans cell histiocytosis, laryngeal cancer, laryngeal cancer and papillomatosis, leukemia, lip and oral cancer, liver cancer (primary), lung cancer, lung cancer, lymphoma-macroglobulinemia, Waldenstrom-non-Hodgkin lymphoma ( -Non-Hodgkin lymphoma, male breast cancer, malignant fibrous histiocytoma of bone and osteosarcoma, melanoma, intraocular (eye) cancer, Merkel cell carcinoma cellcarcinoma), mesothelioma, malignant mesothelioma, occult primary metastatic squamous neck cancer, midline tract cancer involving the NUT gene, oral cancer, multiple endocrine neoplasm syndrome, multiple myeloma / plasma cell neoplasms, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasms and chronic myeloproliferative neoplasms, chronic myeloid leukemia (CML), acute myeloid leukemia (AML), nasal and paranasal sinus cancer, nasopharyngeal cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, lip and oral cavity cancer and oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer, pancreatic cancer and pancreatic neuroendocrine tumors (islet cell tumors), papillomatosis, paraganglioma, paraganglioma, paranasal sinus and nasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pheochromocytoma, pituitary tumor, plasma cell Neoplasms / Multiple Myeloma, Pleuropulmonary Blastomycin, Pregnancy and Breast Cancer, Primary Central Nervous System (CNS) Lymphoma, Primary Peritoneal Cancer, Prostate Cancer, Rectal Cancer, Renal Cell (Kidney) Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Salivary Gland Tumors, Ewing Sarcoma, Kaposi Sarcoma, Osteosarcoma, Rhabdomyosarcoma, Uterine Sarcoma, Vascular Tumors, Sézary Syndrome, Skin Cancer, Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Cell Carcinoma, Primary Metastatic Squamous Neck Cancer, Stomach (Stomach or Gastric) Cancer, Gastric Cancer, T-Cell Lymphoma, Skin Cancer, Testicular Cancer, Laryngeal Cancer, Thymoma and Thymic Cancer, Thyroid Cancer, Ureter and Renal Pelvis Cancer, Transitional Cell Carcinoma, Urethra Cancer, Uterine Cancer, Endometrial and Uterine Sarcoma, Vaginal Cancer, Vaginal Cancer, Hemangioma, Vulvar Cancer, Waldenstrom Macroglobulinemia ( Macroglobulinemia or Wilms Tumor.

59. A kit comprising: A first agent comprising an ammonia inhibitor; and another agent comprising one or more lipid metabolism inhibitors or related agents.

60. The kit according to any one of the preceding claims, wherein the lipid metabolism inhibitor comprises at least one inhibitor that inhibits the utilization / release of lipids from extracellular uptake (LDL) and internal storage (lipid droplets, LD).

61. The kit of any of the preceding claims, wherein the inhibitor that inhibits utilization / release of lipids from extracellular uptake and internal storage comprises a SREBP inhibitor, a fatty acid synthesis inhibitor, a cholesterol synthesis pathway inhibitor, or a lysosomal dysregulator.

62. A kit according to any one of the preceding claims, comprising at least one ammonia inhibitor and at least one SREBP inhibitor.

63. A kit according to any one of the preceding claims, comprising at least one ammonia inhibitor and at least one fatty acid synthesis inhibitor.

64. A kit according to any one of the preceding claims, comprising at least one ammonia inhibitor and at least one cholesterol synthesis pathway inhibitor.

65. A kit according to any one of the preceding claims comprising at least one ammonia inhibitor and at least one lysosomal dysregulatory agent.

66. A kit according to any one of the preceding claims comprising at least one ammonia inhibitor, at least one lysosomal dysregulator and at least one SREBP inhibitor.

67. A kit according to any one of the preceding claims comprising at least one ammonia inhibitor, at least one lysosomal dysregulator, and at least one fatty acid synthesis inhibitor.

68. The kit of any one of the preceding claims comprising at least one ammonia inhibitor, at least one lysosomal dysregulator, and at least one cholesterol synthesis pathway inhibitor.

69. A pharmaceutical composition comprising at least one ammonia inhibitor alone or in combination with one or more lipid metabolism inhibitors or related agents thereof.

70. A pharmaceutical composition according to any one of the preceding claims, wherein the one or more lipid metabolism inhibitors include at least one inhibitor that inhibits the utilization / release of lipids from extracellular uptake (LDL) and internal storage (lipid droplets, LD).

71. A pharmaceutical composition according to any of the preceding claims, wherein the inhibitor that inhibits utilization / release of lipids from extracellular uptake and internal storage comprises a SREBP inhibitor, a fatty acid synthesis inhibitor, a cholesterol synthesis pathway inhibitor, or a lysosomal dysregulator.

72. A pharmaceutical composition according to any one of the preceding claims, comprising at least one ammonia inhibitor and at least one SREBP inhibitor.

73. A pharmaceutical composition according to any one of the preceding claims, comprising at least one ammonia inhibitor and at least one fatty acid synthesis inhibitor.

74. A pharmaceutical composition according to any of the preceding claims, comprising at least one ammonia inhibitor and at least one cholesterol synthesis pathway inhibitor.

75. A pharmaceutical composition according to any one of the preceding claims comprising at least one ammonia inhibitor and at least one lysosomal dysregulator.

76. A pharmaceutical composition according to any one of the preceding claims comprising at least one ammonia inhibitor, at least one lysosomal dysregulator, and at least one SREBP inhibitor.

77. A pharmaceutical composition according to any one of the preceding claims comprising at least one ammonia inhibitor, at least one lysosomal dysregulator, and at least one fatty acid synthesis inhibitor.

78. A pharmaceutical composition according to any one of the preceding claims comprising at least one ammonia inhibitor, at least one lysosomal dysregulator, and at least one cholesterol synthesis pathway inhibitor.

79. A method of treating a solid tumor in a patient in need thereof, the method comprising administering to the patient at least one ammonia inhibitor, SREBP inhibitor, fatty acid synthesis inhibitor, or cholesterol synthesis pathway inhibitor alone or in combination with an inhibitor that inhibits the utilization / release of lipids from extracellular uptake and internal storage.

80. A method of inhibiting lipogenesis in a patient in need thereof, the method comprising administering to the patient at least one ammonia inhibitor, SREBP inhibitor, fatty acid synthesis inhibitor, or cholesterol synthesis pathway inhibitor alone or in combination with an inhibitor that inhibits utilization / release of lipids from extracellular uptake and internal storage.

81. A method of increasing reactive oxygen species in a patient in need thereof, the method comprising administering to the patient at least one ammonia inhibitor, SREBP inhibitor, fatty acid synthesis inhibitor, or cholesterol synthesis pathway inhibitor alone or in combination with an inhibitor that inhibits utilization / release of lipids from extracellular uptake and internal storage.

82. A method of causing mitochondrial damage to a patient in need thereof, the method comprising administering to the patient at least one ammonia inhibitor, SREBP inhibitor, fatty acid synthesis inhibitor, or cholesterol synthesis pathway inhibitor alone or in combination with an inhibitor that inhibits utilization / release of lipids from extracellular uptake and internal storage.

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