Suppression of triacylglyceride synthesis in target cells

Abstract

The present invention relates to a method for treating target cells, comprising administering to the target cells N-(2,2,2-Trifluoroethyl)-9-[4-[4-[[[4'-(trifluoromethyl)[1,1'-biphenyl]2-yl]carbonyl]amino]-1-piperidinyl]butyl]-9H-fluoren-9-carboxamide (lomitapide) or an analog thereof in an amount effective for suppressing synthesis of triacylglycerides (TGs) in the target cells. The present invention also relates to a method for treating target cells in a subject, comprising administering to the subject lomitapide or an analog thereof in an amount effective for suppressing synthesis of triacylglycerides (TGs) and / or formation of lipid droplets (LDs) in the target cells.

Description

[0001] 0073605-001157

[0002] SUPPRESSION OF TRIACYLGLYCERIDE SYNTHESIS IN TARGET CELLS

[0003] CROSS-REFERENCE TO RELATED APPLICATION

[0004] This application claims priority to United States Provisional Application No. 63 / 760,309, filed February 19, 2025, the contents of which are incorporated herein by reference in their entireties for all purposes.

[0005] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0006] This invention was made with government support under Grant No. CA256911 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0007] FIELD OF THE INVENTION

[0008] The invention relates to targetable lipid metabolism vulnerability in target cells, for example, tumor cells, including those adapted to cysteine limitation stress.

[0009] BACKGROUND OF THE INVENTION

[0010] Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with a 5-year survival rate less than 13%. The PDAC tumor microenvironment is characteristically nutrient deprived due to desmoplasia, poor vascularization and hyperactive cancer metabolism. The plasticity in cancer metabolism is critical for cancer cells to overcome nutrient limitation stress and to support tumor growth and progression. However, the roles of nutrient limitation stress in PDAC tumor growth and progression remained incompletely understood.

[0011] Dysregulated lipid metabolism is a critical hallmark of metabolic reprograming in cancer. High fat diet (HFD) and obesity among cancer patients contribute to dysregulated lipid metabolism in cancer cells by increasing lipid content in circulation. Furthermore, cancer cells increase de novo fatty acid (FA) synthesis and lipid uptakes due to mutations in oncogenes (e.g., KRAS, MYC) or tumor suppressors (e.g., VHL). The dysregulated lipid metabolism in cancer cells supports membrane biogenesis, which is required for tumor growth and cell proliferation. However, excess free FA (FFA) could lead to lipotoxicity, cell damage, and cell death. To protect cells from lipotoxicity, FFAs are esterified into triacylglycerides (TGs) by diacylglycerol O-acyltransferase 1 and 2 (DGAT1 and 2) and stored in lipid droplets (LDs). LDs in cancer cells serve as a reservoir for FA, which could be released through lipolysis to support energy metabolism and phospholipid synthesis when needed. LD accumulation correlates with tumor0073605-001157

[0012] aggressiveness and therapy resistance in many cancer types. There is evidence that elevated LD levels promote cancer cell migration, proliferation and drug resistance. In addition to serving as lipid and energy storage organelles, LDs are crucial for cell survival under stress. Stresses such as nutrient deprivation, hypoxia, acidosis, and chemotherapeutic agents promote lipolysis and / or lipogenesis. The storage of excess FA, polyunsaturated fatty acids, lipid peroxidation products, and acyl-ceramides in LDs allows cells to avoid stress-induced lipotoxicity and cell death.

[0013] Cystine (Cyso) and cysteine (Cys) are important amino acid nutrients for the antioxidant response and sulfur metabolism. In the oxidative blood circulation, Cys is oxidized into Cys2. SLC7A11, the functional subunit of the Cys2 / glutamate antiporter system xc', is responsible for cystine uptake. In the cell Cys2 is reduced to Cys and used for the synthesis of essential sulfur metabolites, such as glutathione (GSH) and iron-sulfur clusters (Fe-S). Although Cys2 and Cys are non-essential amino acids, cancer cells depend on exogenous Cys2 for antioxidant response and survival. Cys2 deprivation induces ferroptosis, a type of iron and lipid peroxidationdependent cell death in pancreatic cancer and other cancer. Intriguingly, Cys2 and Cys are among the most depleted amino acids in the PDAC tissue and tumor interstitial fluid. It is unclear how PDAC cells adapt to the chronic Cys2 limitation stress (CLS) and how such adaptation affects PDAC tumor growth and progression.

[0014] Cancer cells upregulate anabolic metabolism such as de novo synthesis of nucleotides and fatty acids (FA) to support cancer cell proliferation and tumor growth. The oxidative pentose phosphate pathway (OxPPP) provides ribose-5-phosphate (R5P) for the de novo synthesis of nucleotides and the NADPH for antioxidant response and de novo FA synthesis. G6PD (glucose-6-phosphate dehydrogenase), PGLS (6-phosphogluconolactonase) and PGD (phosphogluconate dehydrogenase) are the three enzymes in the OxPPP, which use G6P (glucose-6-phosphate) as precursor for the synthesis of R5P and the reduction of NADP+ to NADPH. G6PD catalyzes the rate limiting step of OxPPP and is upregulated in many cancer types including PDAC.

[0015] Targeting the dysregulated cancer metabolism is an area under intensive investigation. Drugs targeting nucleotide synthesis, such as gemcitabine and 5-FU have been successfully used in cancer treatment for decades. However, the development of cancer therapeutics targeting dysregulated lipid metabolism were more challenging. Recently the role of DGAT1 / 2 and LD emerged as critical players in tumor growth and progression. Several pharmacological inhibitors for DGAT1 / 2 have been used in clinical trial for diabetes, fatty liver disease and other metabolic0073605-001157

[0016] conditions. These inhibitors could potentially be useful in targeting the hyperactive lipid metabolism in cancer. Indeed, there is evidence supporting the anti-tumor activity of DGAT1 inhibitor A922500 in glioblastoma and clear cell renal cell carcinoma models. However, the doses of DGAT1 inhibitor required for anti -tumor activities in mouse models were 20- to 40-times higher than those typically used for other metabolic diseases (3mg / kg). Considering the well-documented gastrointestinal adverse effects of DGAT1 inhibitors in clinical trials, the feasibility of using such high doses in cancer patients is likely an issue.

[0017] There remains a need for safe and effective treatment of target cells such as tumor cells by targeting lipid metabolism vulnerability in target cells.

[0018] SUMMARY OF THE INVENTION

[0019] The present invention relates to a method for treating cells, for example, tumor cells. The invention is based on the inventors’ unexpected discovery that adaptation of pancreatic ductal adenocarcinoma (PDAC) cells to CLS (CLSA) promotes PDAC cell proliferation and tumor growth through translational upregulation of the oxidative pentose phosphate pathway (OxPPP). Through drug screening, the inventors have identified lomitapide as an inhibitor of CLSA PDAC tumor growth and a potent sensitizer of FOLFIRINOX chemotherapy. In particular, the inventors have discovered that lomitapide inhibits triacylglycerides synthesis to interfere with CLSA and chemotherapy -induced lipidomic reprograming.

[0020] A first method is provided for treating cells. The first method comprises administering to the target cells N-(2,2,2-Trifhioroethyl)-9-[4-[4-[[[4'-(trifhioromethyl)[l,l'-biphenyl]2-yl]carbonyl]amino]-l-piperidinyl]butyl]-9H-fluoren-9-carboxamide (lomitapide) or an analog thereof an amount effective for suppressing synthesis of triacylglycerides (TGs) in the target cells. As a result, the synthesis of TGs in the target cells is inhibited.

[0021] The first method may further comprise inhibiting proliferation of the target cells.

[0022] The first method may further comprise killing the target cells.

[0023] According to the first method, the lomitapide analog may be selected from the group consisting of 9-[4-[4-[[2-Chloro-5-(trifluoromethyl)benzoyl]amino]-l-piperidinyl]butyl]-N-(2,2,2-trifluoroethyl)-9H-fluorene-9-carboxamide, 9-[4-[4-[[2,6-Bis(trifluoromethyl)benzoyl]amino]-l-piperidinyl]butyl]-N-(2,2,2-trifluoroethyl)-9H-fluorene-9-carboxamide, monohydrochloride, N-(2,2,2-trifluoroethyl)-9-[4-[4-[[[4-(trifluoromethyl)[l,l-0073605-001157

[0024] biphenyl]-2-yl]carbonyl]amino]-l-piperidyl]butaneyl]-9H-fluorene-9-carboxamide, 9-[4-[4-[[[4-Chloro-4'-(trifluoromethyl)[l,r-biphenyl]-2-yl]carbonyl]amino]-l-piperidinyl]butyl]-N-(2,2,2-trifluoroethyl)-9H-fluorene-9-carboxamide, 9-[4-[4-[[2-fluoro-5-(trifluoromethyl)benzoyl]amino]-l-piperidinyl]butyl]-N-(2,2,2-trifluoroethyl)-9H-fluorene-9-carboxamide monohydrochloride, N-(2,2,2-Trifluoroethyl)-9-[4-[4-[[[4-(trifluoromethyl)[l,l-biphenyl]-2-yl]carbonyl]amino]-l-piperidinyl]butyl]-9H-fluorene-9-carboxamide, monohydrochloride, 9-[4-[4-[[2,5-bis(trifluoromethyl)benzoyl]amino]-l-piperidinyl]butyl]-N-(2,2,2-trifluoroethyl)-9H-fluorene-9-carboxamide monohydrochloride, and 9-[4-[4-[[[4-chloro-4'-(trifluoromethyl)[l,l-biphenyl]-2-yl]carbonyl]amino]-l-piperidinyl]butyl]-N-(2,2,2-trifluoroethyl)-9H-fluorene-9-carboxamide monohydrochloride.

[0025] According to the first method, the target cells may be tumor cells of a cancer selected from the group consisting of pancreatic cancer, colorectal cancer, glioblastoma, clear cell renal cell carcinoma, liver cancer, lung cancer, melanoma, prostate cancer, ovarian cancer, and acute myeloid leukemia.

[0026] The target cells may be selected from the group consisting of hepatocytes, enterocytes, and adipocytes.

[0027] The target cells may be adapted to stress selected from the group consisting of cystine limitation stress (CLS), hypoxia, acidosis, chemotoxic stress. The target cells may have an intracellular cysteine (Cys) concentration lower than that of control cells. The target cells may have an intracellular cystine (Cys ) concentration lower than that of control cells.

[0028] The first method may further comprise downregulating iron sulfur (Fe-S) cluster synthesis in the target cells.

[0029] The first method may further comprise downregulating DGAT1 and / or DGAT2 activity in the target cells.

[0030] The first method may further comprise switching mitochondrial oxidative phosphorylation to glycolytic metabolism in the target cells.

[0031] The first method may further comprise upregulating glycolysis, oxidative pentose phosphate pathway (PPP), lipid uptake or de novo lipid synthesis in the target cells.

[0032] The first method may further comprise inhibiting formation of lipid droplets (LD) in the target cells.0073605-001157

[0033] The first method may further comprise administering to the target cells a cancer drug, whereby the proliferation of the target cells is inhibited synergistically by the lomitapide or an analog thereof and the cancer drug. The cancer drug may be selected from the group consisting of nucleotide metabolism drugs, platinum drugs, topoisomerase inhibitors, poly(ADP-ribose) polymerase (PARP) inhibitors, anti-angiogenesis agents, and KRAS targeted therapies. The cancer drug may be selected from the group consisting of 5 fluorouracil (5-FU), gemcitabine, cytarabine, carboplatin, oxaliplatin, irinotecan, topotecan, etoposide, teniposide, doxorubicin, olaparib, talazoparib, rucaparib, and niraparib, bevacizumab, paclitaxel, albumin-bound or nab-paclitaxel, 4-[4-(3,8-diazabicyclo[3.2.1]oct-3-yl)-8-fluoro-2-[[(2R,7aS)-2-fluorotetrahydro-lH-pyrrolizin-7a(5H)-yl]methoxy]pyrido[4,3-d]pyrimidin-7-yl]-5-ethynyl-6-fluoro-2-naphthalenol (MRTX-1133), and sotorasib.

[0034] A second method is provided for treating target cells in a subject. The second method comprises administering to the subjectN-(2,2,2-Trifluoroethyl)-9-[4-[4-[[[4'-(trifluoromethyl)[l,r-biphenyl]2-yl]carbonyl]amino]-l-piperidinyl]butyl]-9H-fluoren-9-carboxamide (lomitapide) or an analog thereof in an amount effective for suppressing synthesis of triacylglycerides (TGs) in the target cells. The lomitapide analog may be selected from the group consisting of 9-[4-[4-[[2-Chloro-5-(trifluoromethyl)benzoyl]amino]-l-piperidinyl]butyl]-N-(2,2,2-trifluoroethyl)-9H-fluorene-9-carboxamide, 9-[4-[4-[[2,6-Bis(trifluoromethyl)benzoyl]amino]-l-piperidinyl]butyl]-N-(2,2,2-trifluoroethyl)-9H-fluorene-9-carboxamide, monohydrochloride, N-(2,2,2-trifluoroethyl)-9-[4-[4-[[[4-(trifluoromethyl)[l,l-biphenyl]-2-yl]carbonyl]amino]-l-piperidyl]butaneyl]-9H-fluorene-9-carboxamide, 9-[4-[4-[[[4-Chloro-4'-(trifluoromethyl)[l,r-biphenyl]-2-yl]carbonyl]amino]-l-piperidinyl]butyl]-N-(2,2,2-trifluoroethyl)-9H-fluorene-9-carboxamide, 9-[4-[4-[[2-fluoro-5-(trifluoromethyl)benzoyl]amino]-l-piperidinyl]butyl]-N-(2,2,2-trifluoroethyl)-9H-fluorene-9-carboxamide monohydrochloride, N-(2,2,2-Trifluoroethyl)-9-[4-[4-[[[4-(trifluoromethyl)[l,l-biphenyl]-2-yl]carbonyl]amino]-l-piperidinyl]butyl]-9H-fluorene-9-carboxamide, monohydrochloride, 9-[4-[4-[[2,5-bis(trifluoromethyl)benzoyl]amino]-l-piperidinyl]butyl]-N-(2,2,2-trifluoroethyl)-9H-fluorene-9-carboxamide monohydrochloride, and 9-[4-[4-[[[4-chloro-4'-(trifluoromethyl)[l,l-biphenyl]-2-yl]carbonyl]amino]-l-piperidinyl]butyl]-N-(2,2,2-trifluoroethy 1 )-9H-fluorene-9 -carb oxami de monohydrochi ori de .

[0035] The second method may further comprise inhibiting proliferation of the target cells.0073605-001157

[0036] The second method may further comprise killing the target cells.

[0037] According to the second method, the target cells may be tumor cells of cancer selected from the group consisting of pancreatic cancer, colorectal cancer, glioblastoma, clear cell renal cell carcinoma, liver cancer, lung cancer, melanoma, prostate cancer, ovarian cancer, and acute myeloid leukemia.

[0038] According to the second method, the target cells may be selected from the group consisting of hepatocytes, enterocytes, and adipocytes.

[0039] The target cells may be adapted to stress selected from the group consisting of cystine limitation stress (CLS), hypoxia, acidosis, and chemotoxic stress. The target cells may have an intracellular cysteine (Cys) concentration lower than that of control cells. The target cells may have an intracellular cystine (Cys ) concentration lower than that of control cells. The second method may further comprise downregulating iron sulfur (Fe-S) cluster synthesis in the target cells.

[0040] The second method may further comprise downregulating DGAT1 and / or DGAT2 activity in the target cells. The second method may further comprise switching mitochondrial oxidative phosphorylation to glycolytic metabolism in the target cells.

[0041] The second method may further comprise upregulating glycolysis, oxidative pentose phosphate pathway (PPP), lipid uptake or de novo lipid synthesis in the target cells.

[0042] The second method may further comprise inhibiting formation of lipid droplets (LD) in the target cells.

[0043] The second method may further comprise administering to the subject a cancer drug, whereby proliferation of the target cells is inhibited synergistically by the lomitapide or an analog thereof and the cancer drug. The cancer drug may be selected from the group consisting of nucleotide metabolism drugs, platinum drugs, topoisomerase inhibitors, poly(ADP -ribose) polymerase (PARP) inhibitors, anti-angiogenesis agents, and KRAS targeted therapies. The cancer drug may be selected from the group consisting of 5 fluorouracil (5-FU), gemcitabine, cytarabine, carboplatin, oxaliplatin, irinotecan, topotecan, etoposide, teniposide, doxorubicin, olaparib, talazoparib, rucaparib, and niraparib, bevacizumab, paclitaxel, albumin-bound or nab-paclitaxel, 4-[4-(3,8-diazabicyclo[3.2.1]oct-3-yl)-8-fluoro-2-[[(2R,7aS)-2-fluorotetrahydro-lH-pyrrolizin-7a(5H)-yl]methoxy]pyrido[4,3-d]pyrimidin-7-yl]-5-ethynyl-6-fluoro-2-naphthalenol (MRTX-1133), and sotorasib. The lomitapide or an analog thereof and the cancer drug may be0073605-001157

[0044] administered to the subject simultaneously. The lomitapide or an analog thereof and the cancer drug may be administered to the subject sequentially. Where the target cells are pancreatic ductal adenocarcinoma (PDAC) cells, the cancer drug may comprise leucovorin calcium, 5-fluorouracil, irinotecan and oxaliplatin, the second method may further comprise administering lomitapide to the target cells, for example, at a dosage of 5-60 mg daily.

[0045] BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figs. 1A-1P show that adaptation to Cys2 limitation stress promotes PDAC cell proliferation and tumor growth. A and B, the levels of Cys and Cys2 in PDAC tissues (tumor) and paired para-tumor normal (normal) pancreatic tissues from KPC xenograft mice (n=10) (A) and PDAC patients (n=10) (B) were determined through LC-MS / MS after NEM derivation. C, The effect of CLSA on colony formation assay MiaPaCa-2 and Pan02 cells. CLSA-25 and CLSA-50 cells were cultured in Cys2-limited media containing 25 uM and 50 uM Cys2, respectively. Parental cells cultured in Cys2 replete (200 pM) medium were used as control. D, Cys levels in PDAC cells after being cultured in Cys2-limited (25 uM) media for 24-48 hours, or after adaptation to 25 uM Cys2-limitation for 4 weeks. E, The effects of CLSA on cell proliferation in PDAC cell lines. F and G, Representative bright-field microscopy (F) and quantitation (G) showing the effects of CLSA-I on tumoroid growth in two PDO models. H and I, the effect of CLSA-25, CLSA-50 (H) and CLSA-I on RSL3-induced ferroptosis in PDAC cell lines (H) and PDO (I) models. J, representative image (left) and quantitation of tumor weight of orthotopic xenograft tumors harvested from C57BL / 6 mice implanted with control or CLSA-50 Pan02 cells (n=10). K, ex vivo BLI imaging showing the liver metastatic lesions from mice in (J). Right, fraction of mice that developed liver metastasis in control or CLSA-50 group. L, quantitation of liver metastatic lesion numbers from mice in I-K (n=10). M, Representative BLI imaging from Albion BL6 mice implanted with control of CLSA-25 KPC tumor cells on day 1 and day 21. Left, quantitation of BLI imaging (phonton counts) over time. (n=10). N, ex vivo BLI imaging (left) and the weight (right) of pancreas harvested from M (n=10). O, representative MRI images (left) and quantitation of tumor volume of mice implanted with control or CLSA-I PDO1. (n=6 per group). P, photos (left) and weight of pancreas (right) (n=6) harvested from mice in O. Data in (A-B) were analyzed using two sample, two-tailed paired Stuent’s t-test; data in (D)- (E) and (J)-(P) were analyzed using two-tailed, two-sample unpaired Student’s t test. Data in (D)-(E) are representative results from at least three independent experiments (n = 30073605-001157

[0047] biological replicates per group for each experiment). *, **, *** and **** represents p<0.05, 0.01, 0.001 and 0.0001, respectively.

[0048] Figs. 2A-2J show that adaptation to Cys2 limitation stress promotes PDAC cell proliferation. A, the effects of Cys limitation (24 hours) on the levels of ATF4, p-eIF2a in MiaPaCa-2 cells. B, the effects of 72 hour CLS (50 uM or 25 uM) on PDAC cell death, as determined by PI staining and flow cytometry. C, the effects of 72 hour CLS (50 uM or 25 uM) on lipid peroxidation in PDAC cells, as determined by Cl 1-BODIPY staining and flow cytometry. D, the effects of DFO (100 uM) and ferrostatin- 1 (1 uM) treatment on cell death in PDAC cells after 72-hour CLS (25 uM) treatment. E and F, the effects CLS (25 or 50 uM) on colony formation in naive PDAC cells (E) or CLSA PDAC cells (F). G, The effects of CLSA on cell proliferation in Capan-2 and SW1990 cells. H, The effects of glutamine limitation (400 uM), arginine limitation (2 uM) and tryptophan limitation (10 uM) on colony formation (2 weeks) in MiaPaCa-2 cells. I, The effects of glutamine limitation (400 uM) and BSA supplementation (5%) on MiaPaCa-2 colony formation (2 weeks). J, MiaPaCa-2 cells were cultured in conventional DMEM media or Glu-limited DMEM (0.4 mM Glu plus 5% BSA) or Trp-limited DMEM (10 uM Trp) for 4 weeks before being used for cell proliferation assay in their respective nutrient replete or nutrient-limited cell culture media. Data in (B-D) were analyzed using two sample, two-tailed paired Student’s t-test. *** and **** represents p<0.001 and 0.0001, respectively.

[0049] Figs. 3A-3C show Cys metabolism in CLSA PDAC cells. A, GSH levels in PDAC cells after being cultured in Cys2-limited (25 uM) media for 24-48 hours, or after adaptation to 25 uM Cys2-limitation for 4 weeks. B, fractional labeling of M+4 GSH and M+4 or M+8 GSSG in control and CLSA-25 MiaPaCa-2 cells after incubation with [13Ce,15N2]-Cys2 for 2 or 4 hours. C, steady state labeling of Cys metabolites after incubation with [13Ce,13N2]-Cys2 for 24 hours. Data in (A and C) were analyzed using two sample, two-tailed paired Student’s t-test. *** and **** represents p<0.001 and 0.0001, respectively, ns, not significant.

[0050] Figs. 4A-4G show that CLSA cells downregulate Fe-S synthesis to promote glycolysis. A and B, fractional labeling of M+4 Cys (A) and Ala (B) in control and CLSA-25 MiaPaCa-2 cells after incubation with [13Ce,15N2]-Cys2 for 2 or 4 hours. C, the effect of CLSA-25 on aconitase 1 (ACO1) and 2 (ACO2) activities in PDAC cells. D, Western blotting showing the proteins levels of Fe-S proteins in mitochondrial complex I (NDUFS1), II (SDHB) and III (RISKE) in control or CLSA-25 PDAC cells. E and F, the effects of CLSA-25 on mitochondrial oxygen consumption0073605-001157

[0051] rate (OCR) or extracellular acidification rate (ECAR) in different PDAC cell lines. G, the effects of CLSA-25 on glucose uptake, as determined by 2-NBDG labeling and flow cytometry analysis, in PDAC cells. Data in (A-C) and (G) were analyzed using two sample, two-tailed unpaired Stuent’s t-test. Data in (D-F) are representative results from at least three independent experiments (n = 3 biological replicates per group for each experiment). *, **, *** and **** represents p<0.05, 0.01, 0.001 and 0.0001, respectively.

[0052] Figs. 5A-5E show A, Metabolic Set Enrichment Analysis showing the 25 most differentially regulated metabolic pathways in CLSA-25 MiaPaCa-2 and PANC-1 cells according to targeted metabolomics screening; B, heatmap showing the levels of different nucleotides in CLSA-25 PANC-1 and MiaPaCa-2 cells in metabolomics screening; C, Summary of 13C incorporation into glycolysis and PPP metabolites in control and CLSA-25 MiaPaCa2 cells using [U-13C] glucose as tracer. Red arrows indicate increases in 13C incorporation into the indicated metabolite; and D and E, fractional labeling of intermediate metabolites in glycolysis (E), PPP and nucleotide synthesis pathways after tracing with [U-13C] for indicated time.

[0053] Figs. 6A-6B show elevated nucleotide levels in CLSA PDAC cells. A and B, bar graph showing the nucleotide levels in metabolomics screening using control or CLSA-25 PANC-1 (A) or MiaPaCa-2 (B) cells. Data in (A-B) were analyzed using two sample, two-tailed paired Student’s t-test. * indicates p<0.05.

[0054] Figs. 7A-7M show that OxPPP is required for CLSA-mediated cell proliferation and ferroptosis resistance. A and B, the effects of OxPPP inhibitor G6PDil (50 uM) and reductive PPP inhibitor oxythiamine (50 uM) treatment on cell proliferation in control or CLSA-25 PANCI and MiaPaCa-2 cells. C, the effect of G6PDil treatment on cell viability in control or CLSA-25 MiaPaCa-2 and KPC cells. D, mRNA transcript levels of G6PD, PGLS and PGD in control and CLSA-25 MiaPaCa-2 cells. E, inhibition of global translation with cycloheximide (100 ug / mL) had no effect on the protein expression levels of G6PD in MiaPaCa-2 cells. F, correlation between G6PD staining intensity and overall survival (OS) and progression free survival (PFS) in a cohort of 100 PDAC patients. G, effects of G6PD siRNAl and 2 on the protein levels of G6PD in control or CLSA-25 MiaPaCa2 cells. H, NADPH and NADP+ in MiaPaCa-2 and PANCI cells used for the calculation of NADPH / NADP+ ratios in Fig. 8J. I, the effects of G6PD knockdown on sensitivities to RSL-3 induced ferroptosis in control and CLSA-0073605-001157

[0055] 25 PDAC cells. J, the effects of G6PD knockdown on cell proliferation in control and CLSA-25 PDAC cells. K, G6PD expression levels in PDAC cells stably expressing pLX304 vector control or pLX304-G6PD. L-M, the effects G6PD overexpression on RLS-3 induced ferroptosis sensitivities (L) or cell proliferation (M) in PDAC cells.

[0056] Figs. 8A-8J show that translational upregulation of OxPPP enzymes promotes nucleotide and NADPH synthesis in CLSA PDAC cells. A and B, Western blotting showing the expression of G6PD, PGLS and PGD, the three OxPPP enzymes in CLSA-25 PDAC cell lines (A) and CLSA-I PDOs (B). C and D, representative IHC staining (C) and quantitation (D) showing the expression of G6PD and PGD in PDAC tumor tissues and paired para-tumor normal tissues (n=18). E, AHA labeling of newly synthesized G6PD, PGD and GAPDH in Ctrl and CLSA-25 PDAC cells. F, sucrose gradient polysome profiling analysis showing a shift in polysome profiles in CLSA-25 MiaPaCa2 cells (F). G, qPCR analysis showing the distribution of the mRNA transcripts of G6PD, PGD, PGLS and GAPDH in CLSA-25 and control MiaPaCa2 cells. H and I, LC-MS / MS quantitation of the effects of CLSA-25 and G6PD KD on dNTP levels in MiaPaCa-2 (H) and PANC-1 (I) cells. J, LC-MS / MS analysis of the effects of CLSA-25 and G6PD KD on NADPH / NADP+ ratios MiaPaCa-2 and PANC-1 cells. Data in (D) were analyzed using two sample, two-tailed Mann Whitney U-test. Data in (H-J) were analyzed using two sample, two-tailed unpaired Student’s t-test. Data in (A-B) and (E-G) are representative results from at least three independent experiments. Data in (H-J) showed mean±SD of 4 biological repeats. *, **, *** and **** represents p<0.05, 0.01, 0.001 and 0.0001, respectively.

[0057] Figs. 9A-9M show that CLSA-mediated lipidomics reprograming in PDAC cells promotes triacylglycerides (TGs) synthesis and lipid droplet (LD) formation. A and B, volcano plot (A) and heatmap showing that CLSA-mediated lipidomics reprograming upregulates TG levels in MiaPaCa-2 cells. C and D, TLC analysis (C) and densitometry quantitation (D) showing the effects of CLSA-25 on TG levels in MiaPaCa-2 and PANC-1 cells. E-G, confocal microscopy imaging (E) and flow cytometry analysis (F and G)of LD staining showing upregulation of LD formation in CLSA-25 PDAC cells. Representative flow cytometry traces (left) and MFI (median fluorescence intensity) quantitation of flow cytometry data (right) are shown in F and G. H and I, TLC analysis (J) and densitometry quantitation (K) showing the effects of knockdown (KD) on TG levels in CLSA-25 MiaPaCa-2 and PANC-1 cells. J and K, the effect of CLSA-25 and G6PD inhibition (G6PDil 50 uM) on de novo FA synthesis of0073605-001157

[0058] palmitic acid (PA) and stearic acid (SA), as determined through [15N2,13Cs]-Glutamine tracing (24 hours) followed by LC-HRMS analysis of fractional13C incorporation. L and M, representative confocal micrograph (L) and quantitation (M) showing LD levels in PDAC tissues and paired para-tumor normal pancreas tissues. n=30. Data in (M) were analyzed using two sample, two-tailed Mann Whitney U-test. Data in (D), (F-G), (I-K) showed mean±SD of 3 biological repeats and were analyzed using two sample, two-tailed unpaired Student’s t-test. * and **** represents p<0.05 and 0.0001, respectively.

[0059] Figs. 10A-10E show LD staining in PDAC cells and patient tissues. A and B, TLC analysis (A) and densitometry quantitation (B) showing the effects of G6PD ectopic expression on TG levels in naive MiaPaCa-2 and PANC-1 cells. C and D, the effects of G6PD overexpression on LD levels in MiaPaCa-2 (A) and PANC-1 (B) cells. E, LD staining in tumor cells (CK19) and cancer associated fibroblasts (a-SMA) in PDAC patient tissue sections.

[0060] Figs. 11 A-l IP show that CLSA-mediated TG synthesis and LD formation is a metabolic vulnerability targetable by lomitapide. A, summary of drug screening results.. The grey line indicates 70% of cell death in the high throughput screening. B, dose response curves showing the effects of lipid metabolism drugs on the viability of control and CLSA-25 MiaPaCa-2 cells. C, dose response curves showing the effects of lomitapide on the viability of control and CLSA-I PDO#1 and 2. D and E, representative BLI imaging (D) and quantitation (E) (n=6, nude mice) showing the effects lomitapide treatment (10 mg / kg, oral gavage daily) on the orthotopic xenograft tumor growth of control or CLSA-25 MiaPaCa-2. F, the weight of xenograft tumors harvested from mice in D and E (n=6). G, volcano plot showing the effects of lomitapide treatment (1 uM) on lipidomics in CLSA-25 MiaPaCa-2 cells, as determined through targeted lipidomics screening.. H and I, TLC analysis (H) and densitometry quantitation (I) to show the effects of lomitapide treatment (4 uM) on TG levels in MiaPaCa-2 and PANC-1 cells. J and K, confocal microscopy imaging (J) and flow cytometry analysis (K) of LD staining to show the effects of lomitapide treatment (2 uM) on LD levels in PANC-1 and MiaPaCa-2 cells. L andM, TLC analysis (L) and densitometry quantitation (M) to show the effects of lomitapide treatment on the de novo TG synthesis using OA-alkyne probe. N, the effect of lomitapide treatment (0.5 uM) on palmitate-induced lipotoxicity in MiaPaCa-2 and PANC-1 cells. O, the inhibition of diacylglycerol O-acyltransferase activity in isolated liver microsomal membrane (5 ug) by lomitapide at different concentrations. The lower panel is the densitometry quantitation of TLC0073605-001157

[0061] analysis in the upper panel. P, the effects of DGAT1 and DGAT2 knockdown on cell viability in control and CLSA-25 PDAC cells. Data in (E-F) and (P) were analyzed using two sample, two-tailed unpaired Student’s t-test. Data in (B-C), (I), (K) and (N) show mean±SD from 3 biological repeats, ns, *, **, *** and **** represents not significant, p<0.05, 0.01, 0.001 and 0.0001, respectively.

[0062] Figs. 12A-12N show LD staining in PDAC cells and patient tissues. A, summary of cell viability data from positive hits in HTS drug screening. B, western blotting showing the expression levels of MTTP in human and murine PDAC cells. HepG2 and mouse liver lysate were used as positive control. C, principle component analysis of lipidomics screening data from vehicle or lomitapide (luM) treated CLSA-25 MiaPaCa-2 cells. D and E, summary of fold changes (D) and heatmap (E) of major lipid species from lipidomics screening of lomitapide treated CLSA-25 MiaPaCa-2 cells. F, lipidomics screening data showing the effects of lomitapide (luM) treatment on representative species in TG, CE, FFA and CAR. G, TLC analysis showing the dose-dependent effects of DGATli (A922500) and DGAT2i (PF-06424439) and lomitapide treatment on TG levels in MiaPaCa-2 cells. H, densitometry quantitation of TLC results in (G). I, the effects of G6PD overexpression on sensitivities to lomitapide treatment in MiaPaCa-2 and PANC-1 cells. J, TLC analysis showing the synthesis of NBD-TG in the presence of DOG (200 uM), NBD-PA-CoA (25 uM) and liver microsome (5 ug). K, the effects of pre-incubation time on the inhibitory activities of lomitapide in the in vitro DGAT activity assay in (K). Pre-incubation of lomitapide with microsome membrane for 1 hour or longer completely abrogated the inhibitory activities of 2 uM lomitapide. L, the effects of DGAT1 siRNA and DGAT2 siRNA on the mRNA transcript levels of DGAT1 and 2 in MiaPaCa-2 cells. M and N, the effects of DGAT1 and DGAT2 knockdown on LD levels in control or CLSA-25 MiaPaCa-2 (M) or PANC-1 (N) cells. Data in (F) and (L) were analyzed using two sample, two-tailed paired Stuent’s t-test. *, **, *** and **** represents p<0.05, 0.01, 0.001 and 0.0001, respectively, ns, not statistically significant.

[0063] Figs. 13A-13N show that Lomitapide inhibits chemotoxic stress-induced LD formation and enhances the efficacy of FOLFIRINOX regimen in PDAC. A and B, volcano plot and heatmap showing the effects of mFOLFIRINOX (10 uM) treatment on the levels of TG, phospholipids (PL) and lysophospholipids (LPL) in MiaPaCa-2 cells, as determined LC-MS / MS targeted lipidomics screening. mFOLFIRINOX cocktail consists of 5-FU (8 uM), Oxaliplatin (80073605-001157

[0064] uM) and SN-38 (4 uM). Grey line indicates p=0.05. C, TLC analysis showing the effects of mFOLFIRINOX (8 uM) and lomitapide (4 uM) treatment on TG levels in MiaPaCa-2 and PANC-1 cells. D, the effects of low dose lomitapide (50 or 200 nM) treatment on sensitivities to mFOLFIRINOX treatment in PDO1 and PDO2. E, schematic illustration of the establishment of PDX mouse models and treatment regimen of these mice with lomitapide, mFOLFIRINOX or the combination of both. F-I, the effects lomitapide, mFOLFIRINOX or the combination treatment on tumor growth (F), tumor weight (G) and18F-DG PET-CT scan (H and I). (H) shows the representative images of18F-DG PET-CT scan from PDX1 group and (I) shows the quantitation of standardized uptake value (S.U.V.) from PDX1 and PDX2 groups. Arrowhead in (H) indicates PDX xenograft tumor and * indicates bladder. n=6 mice per group. J and K, representative images (J) and quantitation (K) of LD staining using cryosections of tumor tissues harvested from PDX experiments in (F-I). L-N, the effects lomitapide, mFOLFIRINOX or the combination treatment on the tumor growth and survival in KPC genetically engineered PDAC mouse model. Representative ultrasound scan images 4 weeks after treatment (L), quantitation of tumor volume determined through ultrasound imaging (M) and survival of the mice (N) are shown. n=6 mice per group. Data in (F-G), (I), (K) and (M) were analyzed using two sample, two-tailed unpaired Student’ s t-test. Data in (N) were analyzed using two-tailed log-rank test, ns, *, H ***anj ****represents not significant, p<0.05, 0.01, 0.001 and 0.0001, respectively.

[0065] Figs. 14A-14E show that lomitapide enhances the efficacy of FOLFIRINOX regimen in PDAC. A, the effects of mFOLFIRINOX (8 uM) and lomitapide (2 uM) treatment on LD levels in MiaPaCa2 and PANC-1 cells. B, the effects of DGAT1 and 2 knockdowns on mFOLFIRINOX sensitivities in naive MiaPaCa-2 and PANC-1 cells. C, Tumors harvested from PDX-1 and PDX-2 mice treated with vehicle control, lomitapide (10 mg / kg) mFOLFIRINOX or the combination of both. D, representative images of18F-DG PET-CT scan from PDX2 mice treated with vehicle control, lomitapide, mFOLFIRINOX or the combination of both in Figs. 13F-13I. E, bodyweight of KPC mice receiving vehicle control, lomitapide, mFOLFIRINOX or the combination of both in Figs. 13J-13L.

[0066] Fig. 15 shows the effects of lomitapide and other DGAT1 inhibitors (AZD7687, PF04620110, A922500) on cell viability in 4 ccRCC cell lines (A498, ACHN, 769-P and OSRC-2.0073605-001157

[0067] DETAILED DESCRIPTION OF THE INVENTION

[0068] The present invention relates to method for treating target cells, for example, tumor cells. The invention is based on the inventor’s discovery that lomitapide could be used to target lipid metabolism vulnerability in tumor cells, including those adapted to cysteine limitation stress or those subjected to chemotoxic stress. The inventors have discovered that CLSA-mediated metabolic and lipidomics reprograming promotes PDAC tumor growth and lomitapide could be used to target the dysregulated lipid metabolism in PDAC. The investors’ discovery showed that lomitapide targets lipid metabolism vulnerability by inhibiting the synthesis of TGs and / or formation of LDs in cancer cells.

[0069] Upon investigation of the effects of chronic CLS in PDAC, the inventors have discovered that adaptation to CLS (CLSA) promotes PDAC cell proliferation, ferroptosis resistance and tumor growth. CLSA PDAC cells translationally upregulate OxPPP enzymes G6PD, PGD and / or PGLS to promote biosynthesis of NADPH, nucleotides and FAs. Through screening of a metabolism-focus drug library, the inventors have also identified that lomitapide could potently induce lipotoxicity in CLSA PDAC by suppressing TG synthesis, LD formation and increasing toxic lipid species. The inventors have further showed that the FOLFIRINOX chemotherapy regimen induces lipidomics reprograming and lomitapide could target FOLFIRINOX-induced LD formation to increase FOLFIRINOX efficacy. In patient derived xenograft models and the KrasLSL-Kras G12D / +; Trp53LSL- R172H / +; Pdxl-Cre (KPC) genetically engineered mouse model, lomitapide inhibited PDAC tumor growth and robustly improved the efficacy of FOLFIRINOX regimens. These data have shed new light on the role of nutrient stress in PDAC progression and showed that lomitapide could be used to exploit stress-induced vulnerability in PDAC lipid metabolism.

[0070] The term “cystine limitation stress (CLS)” as used herein refers to a stress induced by limitation of cystine uptake. The CLS may be either due to reduction of cystine concentration in a tumor microenvironment, which is very common in PDAC tumor microenvironment, or due to pharmacological treatment with SLC7A11 inhibitors.

[0071] The term “cells adapted to stress” as used herein refers to cells capable of overcoming stress. The stress may be death and cell cycle arrest induced by CLS, hypoxia, acidosis and chemotherapy.0073605-001157

[0072] The term “cells adapted to cystine limitation stress (CLS)” as used herein refers to cells capable of overcoming CLS -induced cell death and cell cycle arrest to resume proliferation under CLS.

[0073] The term “intracellular cysteine (Cys) concentration” as used herein refers to the concentration of cysteine (Cys) within a cell. The intracellular Cys concentration may be determined by using techniques known in the art.

[0074] The term “intracellular cystine (Cys ) concentration” as used herein refers to the concentration of cystine (Cys ) within a cell. The intracellular Cys? concentration may be determined by using techniques known in the art.

[0075] The term “synergistically” as used herein refers to a way involving synergy, where the combined effect of two agents is greater than the sum of the individual effects of the two agents.

[0076] The present invention provides a first method for treating target cells. The first method comprises administering to the target cells N-(2,2,2-Trifluoroethyl)-9-[4-[4-[[[4'-(trifluoromethyl)[l,T-biphenyl]2-yl]carbonyl]amino]-l-piperidinyl]butyl]-9H-fluoren-9-carboxamide (lomitapide) or an analog thereof in an amount effective for suppressing synthesis of triacylglycerides (TGs) in the target cells. As a result, the synthesis of TGs in the target cells is inhibited. The synthesis of TGs in the target cells may be inhibited by at least about 70%, 80%, 90%, 95%, 99% or 100%.

[0077] The first method may further comprise inhibiting proliferation of the target cells. The proliferation of the target cells may be inhibited by at least about 70%, 80%, 90%, 95%, 99% or 100%.

[0078] The first method may further comprise killing the target cells. At least about 70%, 80%, 90%, 95%, 99% or 100% of the target cells may be killed.

[0079] According to the first method, the analog of lomitapide may be selected from the group consisting of 9-[4-[4-[[2-Chloro-5-(trifluoromethyl)benzoyl]amino]-l-piperidinyl]butyl]-N-(2,2,2-trifluoroethyl)-9H-fluorene-9-carboxamide, 9-[4-[4-[[2,6-Bis(trifluoromethyl)benzoyl]amino]-l-piperidinyl]butyl]-N-(2,2,2-trifluoroethyl)-9H-fluorene-9-carboxamide, monohydrochloride, N-(2,2,2-trifluoroethyl)-9-[4-[4-[[[4-(trifluoromethyl)[l,l-biphenyl]-2-yl]carbonyl]amino]-l-piperidyl]butaneyl]-9H-fluorene-9-carboxamide, 9-[4-[4-[[[4-Chloro-4'-(trifluoromethyl)[l,r-biphenyl]-2-yl]carbonyl]amino]-l-piperidinyl]butyl]-N-(2,2,2-trifluoroethyl)-9H-fluorene-9-carboxamide, 9-[4-[4-[[2-fluoro-5-0073605-001157

[0080] (trifluoromethyl)benzoyl]amino]-l-piperidinyl]butyl]-N-(2,2,2-trifluoroethyl)-9H-fluorene-9-carboxamide monohydrochloride, N-(2,2,2-Trifluoroethyl)-9-[4-[4-[[[4-(trifluoromethyl)[l,l-biphenyl]-2-yl]carbonyl]amino]-l-piperidinyl]butyl]-9H-fluorene-9-carboxamide, monohydrochloride, 9-[4-[4-[[2,5-bis(trifluoromethyl)benzoyl]amino]-l-piperidinyl]butyl]-N-(2,2,2-trifluoroethyl)-9H-fluorene-9-carboxamide monohydrochloride, and 9-[4-[4-[[[4-chloro-4'-(trifluoromethyl)[l,l-biphenyl]-2-yl]carbonyl]amino]-l-piperidinyl]butyl]-N-(2,2,2-trifluoroethyl)-9H-fluorene-9-carboxamide monohydrochloride.

[0081] According to the first method, the target cells may be tumor cells of a cancer. The cancer may be selected from the group consisting of pancreatic cancer, colorectal cancer, glioblastoma, clear cell renal cell carcinoma, liver cancer, lung cancer, melanoma, prostate cancer, ovarian cancer, and acute myeloid leukemia. The tumor cells of colorectal cancer may be SW480 or HT-29 cells. The tumor cells of melanoma may be WM793 or 1205Lu cells. The tumor cells of liver cancer may be HepG2 cells. In one embodiment, the tumor cells are pancreatic ductal adenocarcinoma (PDAC) cells.

[0082] According to the first method, the target cells may be selected from the group consisting of hepatocytes, enterocytes, and adipocytes.

[0083] According to the first method, the target cells may be adapted to stress selected from the group consisting of cystine limitation stress (CLS), hypoxia, acidosis, and chemotoxic stress.

[0084] According to the first method, the target cells may have an intracellular cysteine (Cys) concentration lower than that of control cells. The control cells may be non-target cells from the same tissue. The intracellular Cys concentration of the target cells may be at least about 0.5%, 1%, 5%, 10%, 20% or 50% lower than that of the control cells.

[0085] According to the first method, the target cells may have an intracellular cystine (Cys ) concentration lower than that of control cells. The control cells may be non-target cells from the same tissue. The intracellular Cys? concentration of the target cells may be at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 99% lower than that of the control cells.

[0086] The first method may further comprise downregulating iron sulfur (Fe-S) cluster synthesis in the target cells. The synthesis of the Fe-S cluster may be measured by using techniques known in the art.0073605-001157

[0087] The first method may further comprise downregulating DGAT1 and / or DGAT2 activity in the target cells. The downregulation of the DGAT1 and / or DGAT2 activity may be measured by using techniques known in the art.

[0088] The first method may further comprise switching mitochondrial oxidative phosphorylation to glycolytic metabolism in the target cells. The switching may be measured by using techniques known in the art.

[0089] The first method may further comprise upregulating glycolysis in the target cells. The upregulation of glycolysis may be measured by using techniques known in the art.

[0090] The first method may further comprise upregulating oxidative pentose phosphate pathway (PPP) in the target cells. The upregulation of oxidative PPP may be measured by using techniques known in the art.

[0091] The first method may further comprise upregulating lipid uptake in the target cells. The upregulation of the lipid uptake may be measured by using techniques known in the art. The first method may further comprise upregulating de novo lipid synthesis in the target cells. The upregulation of de novo lipid synthesis may be measured by using techniques known in the art.

[0092] The first method may further comprise inhibiting formation of lipid droplets (LD) in the target cells. The inhibition of the LD formation may be measured by using techniques known in the art.

[0093] The first method may further comprise administering to the target cells a cancer drug such that the proliferation of the target cells is inhibited synergistically by the lomitapide or an analog thereof and the cancer drug. The cancer drug may be selected from the group consisting of nucleotide metabolism drugs, platinum drugs, topoisomerase inhibitors, poly(ADP-ribose) polymerase (PARP) inhibitors, anti-angiogenesis agents, and KRAS targeted therapies. The nucleotide metabolism drugs, also referred to as nucleotide analogues, may be selected from the group consisting of 5-FU, gemcitabine, and cytarabine. The platinum drugs may be selected from the group consisting of carboplatin, and oxaliplatin. The topoisomerase inhibitors may be selected from the group consisting of irinotecan, topotecan, etoposide, teniposide, and doxorubicin. The poly(ADP-ribose) polymerase (PARP) inhibitors may be selected from the group consisting of olaparib, talazoparib, rucaparib, and niraparib. The anti-angiogenesis agent may be bevacizumab. The KRAS targeted therapies may be MRTX-1133 or sotorasib. The cancer drug may be selected from the group consisting of 5 fluorouracil (5-FU), gemcitabine,0073605-001157

[0094] cytarabine, carboplatin, oxaliplatin, irinotecan, topotecan, etoposide, teniposide, doxorubicin, olaparib, talazoparib, rucaparib, and niraparib, bevacizumab, paclitaxel, albumin-bound or nab-paclitaxel, 4-[4-(3,8-diazabicyclo[3.2.1]oct-3-yl)-8-fluoro-2-[[(2R,7aS)-2-fluorotetrahydro-lH-pyrrolizin-7a(5H)-yl]methoxy]pyrido[4,3-d]pyrimidin-7-yl]-5-ethynyl-6-fluoro-2-naphthalenol (MRTX-1133), and sotorasib. Where the target cells are pancreatic ductal adenocarcinoma (PDAC) cells and the cancer drug comprise leucovorin calcium, 5 -fluorouracil, irinotecan and oxaliplatin, the first method may comprise administering the lomitapide to the target cells.

[0095] The present invention also provides a second method for treating cells in a subject. The second method comprises administering to the subject N-(2,2,2-Trifluoroethyl)-9-[4-[4-[[[4'-(trifluoromethyl)[l,r-biphenyl]2-yl]carbonyl]amino]-l-piperidinyl]butyl]-9H-fluoren-9-carboxamide (lomitapide) or an analog thereof in an amount effective for suppressing synthesis of triacylglycerides (TGs) in target cells. As a result, the synthesis of TGs in the target cells is inhibited. The synthesis of TGs in the target cells may be inhibited by at least about 70%, 80%, 90%, 95%, 99% or 100%.

[0096] The second method may further comprise inhibiting proliferation of the target cells. The proliferation of the target cells may be inhibited by at least about 70%, 80%, 90%, 95%, 99% or 100%.

[0097] The second method may further comprise killing the target cells. At least about 70%, 80%, 90%, 95%, 99% or 100% of the target cells may be killed.

[0098] According to the second method, the analog of lomitapide may be selected from the group consisting of 9-[4-[4-[[2-Chloro-5-(trifluoromethyl)benzoyl]amino]-l-piperidinyl]butyl]-N-(2,2,2-trifluoroethyl)-9H-fluorene-9-carboxamide, 9-[4-[4-[[2,6-Bis(trifluoromethyl)benzoyl]amino]-l-piperidinyl]butyl]-N-(2,2,2-trifluoroethyl)-9H-fluorene-9-carboxamide, monohydrochloride, N-(2,2,2-trifluoroethyl)-9-[4-[4-[[[4-(trifluoromethyl)[l,l-biphenyl]-2-yl]carbonyl]amino]-l-piperidyl]butaneyl]-9H-fluorene-9-carboxamide, 9-[4-[4-[[[4-Chloro-4'-(trifluoromethyl)[l,T-biphenyl]-2-yl]carbonyl]amino]-l-piperidinyl]butyl]-N-(2,2,2-trifluoroethyl)-9H-fluorene-9-carboxamide, 9-[4-[4-[[2-fluoro-5-(trifluoromethyl)benzoyl]amino]-l-piperidinyl]butyl]-N-(2,2,2-trifluoroethyl)-9H-fluorene-9-carboxamide monohydrochloride, N-(2,2,2-Trifluoroethyl)-9-[4-[4-[[[4-(trifluoromethyl)[l,l-biphenyl]-2-yl]carbonyl]amino]-l-piperidinyl]butyl]-9H-fluorene-9-carboxamide,0073605-001157

[0099] monohydrochloride, 9-[4-[4-[[2,5-bis(trifluoromethyl)benzoyl]amino]-l-piperidinyl]butyl]-N-(2,2,2-trifluoroethyl)-9H-fluorene-9-carboxamide monohydrochloride, and 9-[4-[4-[[[4-chloro-4'-(trifluoromethyl)[l,l-biphenyl]-2-yl]carbonyl]amino]-l-piperidinyl]butyl]-N-(2,2,2-trifluoroethyl)-9H-fluorene-9-carboxamide monohydrochloride.

[0100] According to the second method, target cells may be tumor cells of a cancer. The cancer may be selected from the group consisting of pancreatic cancer, colorectal cancer, glioblastoma, clear cell renal cell carcinoma, liver cancer, lung cancer, melanoma, prostate cancer, ovarian cancer, and acute myeloid leukemia. The tumor cells of colorectal cancer may be SW480 or HT-29 cells. The tumor cells of melanoma may be WM793 or 1205Lu cells. The tumor cells of liver cancer may be HepG2 cells. In one embodiment, the tumor cells are pancreatic ductal adenocarcinoma (PDAC) cells.

[0101] According to the second method, target cells may be selected from the group consisting of hepatocytes, enterocytes, and adipocytes.

[0102] According to the second method, the target cells may be adapted to stress selected from the group consisting of cystine limitation stress (CLS), hypoxia, acidosis, and chemotoxic stress.

[0103] According to the second method, the target cells may have an intracellular cysteine (Cys) concentration lower than that of control cells. The control cells may be non-target cells from the same tissue. The intracellular Cys concentration of the target cells may be at least about 0.5%, 1%, 5%, 10%, 20% or 50% lower than that of the control cells.

[0104] According to the second method, the target cells may have an intracellular cystine (Cys2) concentration lower than that of control cells. The control cells may be non-target cells from the same tissue. The intracellular Cys concentration of the target cells may be at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 99% lower than that of the control cells.

[0105] The second method may further comprise downregulating iron sulfur (Fe-S) cluster synthesis in the target cells. The synthesis of the Fe-S cluster may be measured by using techniques known in the art.

[0106] The second method may further comprise downregulating DGAT1 and / or DGAT2 activity in the target cells. The downregulation of the DGAT1 and / or DGAT2 activity may be measured by using techniques known in the art.0073605-001157

[0107] The second method may further comprise switching mitochondrial oxidative phosphorylation to glycolytic metabolism in the target cells. The switching may be measured by using techniques known in the art.

[0108] The second method may further comprise upregulating glycolysis in the target cells. The upregulation of glycolysis may be measured by using techniques known in the art.

[0109] The second method may further comprise upregulating oxidative pentose phosphate pathway (PPP) in the target cells. The upregulation of oxidative PPP may be measured by using techniques known in the art.

[0110] The second method may further comprise upregulating lipid uptake in the target cells. The upregulation of the lipid uptake may be measured by using techniques known in the art.

[0111] The second method may further comprise upregulating de novo lipid synthesis in the target cells. The upregulation of de novo lipid synthesis may be measured by using techniques known in the art.

[0112] The second method may further comprise inhibiting formation of lipid droplets (LD) in the target cells. The inhibition of the LD formation may be measured by using techniques known in the art.

[0113] The second method may further comprise administering to the subject a cancer drug such that the proliferation of the target cells is inhibited synergistically by the lomitapide or an analog thereof and the cancer drug. The cancer drug may be selected from the group consisting of nucleotide metabolism drugs, platinum drugs, topoisomerase inhibitors, poly(ADP-ribose) polymerase (PARP) inhibitors, anti-angiogenesis agents, and KRAS targeted therapies. The nucleotide metabolism drugs, also referred to as nucleotide analogues, may be selected from the group consisting of 5-FU, gemcitabine, and cytarabine. The platinum drugs may be selected from the group consisting of carboplatin, and oxaliplatin. The topoisomerase inhibitors may be selected from the group consisting of irinotecan, topotecan, etoposide, teniposide, and doxorubicin. The poly(ADP-ribose) polymerase (PARP) inhibitors may be selected from the group consisting of olaparib, talazoparib, rucaparib, and niraparib. The anti-angiogenesis agent may be bevacizumab. The KRAS targeted therapies may be MRTX-1133 or sotorasib. The cancer drug may be selected from the group consisting of 5 fluorouracil (5-FU), gemcitabine, cytarabine, carboplatin, oxaliplatin, irinotecan, topotecan, etoposide, teniposide, doxorubicin, olaparib, talazoparib, rucaparib, and niraparib, bevacizumab, paclitaxel, albumin-bound or nab-0073605-001157

[0114] paclitaxel, 4-[4-(3,8-diazabicyclo[3.2.1]oct-3-yl)-8-fluoro-2-[[(2R,7aS)-2-fluorotetrahydro-lH-pyrrolizin-7a(5H)-yl]methoxy]pyrido[4,3-d]pyrimidin-7-yl]-5-ethynyl-6-fluoro-2-naphthalenol (MRTX-1133), and sotorasib. The lomitapide or an analog thereof and the cancer drug may be administered to the subject simultaneously or sequentially. Where the target cells are pancreatic ductal adenocarcinoma (PDAC) cells and the cancer drug comprise leucovorin calcium, 5-fluorouracil, irinotecan and oxaliplatin, the second method may comprise administering the lomitapide to the subject. The second method may further comprise administering to the subject lomitapide at a dosage of 5-60 mg daily.

[0115] Example 1. Adaptation to cystine limitation stress confers a targetable lipid metabolism vulnerability in pancreatic ductal adenocarcinoma (PDAC) cells.

[0116] Cystine / cysteine is critical for antioxidant response and sulfur metabolism in cancer cells and is one of the most depleted amino acids in the PDAC microenvironment. The effects of cystine limitation stress (CLS) on PDAC progression are poorly understood. Here we report that adaptation to CLS (CLSA) promotes PDAC cell proliferation and tumor growth through translational upregulation of the oxidative pentose phosphate pathway (OxPPP). OxPPP activates the de novo synthesis of nucleotides and fatty acids to support tumor growth. On the other hand, CLSA-mediated lipidomic reprogramming depends on triacylglycerides synthesis to mitigate lipotoxicity. Through drug screening, we identified lomitapide as an inhibitor of CLSA PDAC tumor growth and a potent sensitizer of FOLFIRINOX chemotherapy. Mechanistically, lomitapide inhibits triacylglycerides synthesis to interfere with CLSA and chemotherapy -induced lipidomic reprograming. Taken together, CLSA-mediated metabolic and lipidomics reprograming promotes PDAC tumor growth and lomitapide could be used to target the dysregulated lipid metabolism in PDAC.

[0117] Adaptation to cystine limitation stress promotes PDAC cell proliferation, ferroptosis resistance and tumor growth.

[0118] To determine whether PDAC cells might be subjected to CLS, we determined the levels Cys and Cys2 in PDAC and para-tumor normal pancreatic tumor tissue from C57BL / 6 mice orthotopically implanted with a PDAC line derived from the KPC (KrasLSL'G12D / Trp53LSL'R172H / Pdxl-Cre) model. The levels of Cys and Cys in tumor tissues were approximately 90% (for Cys) to 50% (for Cys ) lower when compared to para-tumor normal pancreatic tissues (Fig. 1 A).0073605-001157

[0119] The Cys to Cys2 ratios were higher in adjacent pancreas tissues than in tumors (Fig. 1 A).

[0120] Similarly, we observed a significant decrease in Cys levels in patient PDAC tissues when compared to para-tumor normal pancreas control in a cohort of 10 PDAC patients (Fig. IB). These data suggest that PDAC cells are subjected to chronic CLS.

[0121] Most cell culture media (e.g. DMEM or RPMI) contain 200 pM Cys2, which is significantly higher than Cys2 levels in the human plasma (25-50 pM). Cells cultured in vitro have higher demand for Cys2 due to direct exposure to ambient O2. As shown in Fig. 2A, decreasing Cys2 concentrations to 50 pM or lower in MiaPaCa-2 mediated to increased phosphorylation of eIF2a and expression of ATF4, which suggested the activation of the integrated stress response by Cys2 limitation. As expected, acute reduction of Cys2 from 200 pM to 50 or 25 pM led to lipid peroxidation and ferroptosis-like cell death in PDAC cells in in 72 hours (Figs. 2B-2D). Approximately 5 to 30 percent of PDAC cells were able to adapt to CLS at 25 pM and resume cell growth and proliferation after 3-4 weeks exposure to CLS (Fig. 1C and Fig. 2E). The proportion of PDAC cells adapted to 50 pM CLS were much higher (Fig. 2E). Here we use CLSA-25 and CLSA-50 to refer to PDAC cells adapted to 50 or 25 pM Cys2, respectively. Culturing PDAC cells in medium containing 25 pM Cys2 reduced the intracellular Cys levels by approximately 75% to 90% in 24-48 hours (Fig. ID). Although Cys level modestly recovered in CLSA-25 MiaPaCa-2 and PANC-1 cells after 4-week adaptation, it was still ~60-50 % lower than in naive control cells cultured in approximately 75% to 90% in 24-48 hours (Fig. ID). Although Cys level modestly recovered in CLSA-25 MiaPaCa-2 and PANC-1 cells after 4-week adaptation, it was still -60-50 % lower than in naive control cells cultured in Cys2 replete medium (Fig. ID). No recovery of intracellular Cys was observed in CLSA-25 Pan02 and KPC cells (Fig. ID). Interestingly, CLSA PDAC cells proliferate much faster than parental cells, despite severe reduction of intracellular Cys (Fig. IE). Given the considerable interest in using Cys2 deprivation agents as potential cancer therapeutics, we asked whether CLS induced by pharmacological agents might have similar effects on PDAC cells. We previously reported that imidazole ketone erastin (IKE) induced ferroptosis in PDAC cell lines and patient-derived organoids (PDOs). As shown in Figs. 1F-1G, PDAC organoids adapted to IKE-induced CLS (CLSA-I, for inhibitor induced adaptation) also grew faster than naive control PDOs.

[0122] GPX4 is the sole glutathione peroxidase responsible for scavenging lipid peroxide and preventing ferroptosis. To determine the effect of CLSA on ferroptosis sensitivities, we0073605-001157

[0123] treated CLSA PDAC cells or PDOs with GPX4 inhibitor RSL-3. As shown in Figs. 1H and II, the IC50 of RSL-3 in CLSA-25 cells and CLSA-I PDOs increased dramatically by 10 to 500-fold when compared to naive controls. CLSA-50 cells also developed ferroptosis resistance, although to a lesser extent (Fig. II).

[0124] To determine whether CLSA affects tumor growth, we implanted CLSA Pan02 and KPC cells and their naive control orthotopically in the pancreas of C57BL / 6 mice. Mice implanted with CLSA-50 Pan02 cells grew modestly larger tumors, with increased numbers of liver metastases when compared to the control group (Figs. 1J-1L). The liver metastasis incident increased from 30% in control to 90% in CLSA-50 group (Fig. IK). In the KPC xenograft experiment, the growth of orthotopically implanted CLSA-25 KPC tumors were dramatically faster than control tumors, although no metastasis was detected in either group (Fig. IN).

[0125] Similarly, orthotopically implanted CLSA-I PDOs developed larger tumors when compared to naive control (Figs. 10- IP). Taken together, our data showed that CLSA promotes PDAC cell proliferation, ferroptosis resistance and tumor growth.

[0126] To determine whether nutrient limitation of other amino acids might induce similar adaptation in PDAC cells, we cultured MiaPaCa-2 cells in medium containing reduced amounts of glutamine (400 pM), tryptophan (10 pM) or arginine (2 pM), respectively. These amino acids and concentrations were selected based on previous reports showing their depletion in PDAC. MiaPaCa-2 cells were able to proliferate in Trp-limited media, but not in Glu- or Arg-limited conditions for 2-4 weeks (Fig. 2H). BSA supplementation allowed PDAC cell adaptation to glutamine limitation, as previously reported (Fig.21). However, Glu-limitation adapted cells (in the presence of 5% BSA) proliferate slower than naive control (Fig. 2J). Although MiaPaCa-2 cells were able to proliferation under Trp limitation conditions for up to 4 weeks, those cells stop proliferating after 4 weeks (Fig. 2J). Therefore, our data suggested that PDAC CLSA induced a unique adaptation mechanism to promote PDAC cell proliferation and tumor growth.

[0127] CLSA cells downregulate iron sulfur cluster synthesis to promote glycolysis.

[0128] A major function of Cys is to be used for GSH synthesis, which is required for antioxidant response and ferroptosis resistance. Acute Cys limitation (25 pM) markedly reduced GSH levels by fifty to ninety percent in PDAC cells (Fig. 3A), which is consistent with the induction of ferroptosis by acute CLS. However, in CLSA-25 PDAC cells GSH levels were comparable to that of naive control cells (Fig. 3A), despite little to no recovery in intracellular0073605-001157

[0129] Cys. To understand the effects of CLSA on Cys metabolism, we used13N2,13Ce-cystine to label Cys metabolites in MiaPaCa-2 cells. Control or CLSA-25 cells were incubated with 200 pM (for control) or 25 pM (for CLSA-25)15N2,13Ce- Cys2 for 0, 2 or 4 hours. The fractional labeling of M+4 Cys reached 68% at 2 hours and plateau after that in the control group (Fig. 4A). The M+4 Cys fractions in CLSA-25 group were 54% and 38% at 2 and 4 hours, respectively. Despite lower fractional labeling of M+4 Cys in CLSA-25 cells, the fractional labeling of GSH and GSSG levels in the two group were similar (Fig. 3B), which suggested faster turnover of GSH / GSSG in CLSA-25 cells. Interestingly, the steady-state labeling of Cys, GSH, GSSG and CoA all decreased in CLSA-25 cells after 24 hours incubation with15N2,13Ce- Cys2 (Fig. 3C), which suggested that CLSA-25 cells might increase its Cys uptake from alternative sources, such as macropinocytosis and the transsulfuration pathway.

[0130] We noted that the labeling of M+4 Alanine was dramatically reduced in CLSA-25 cells (Fig. 4B). Alanine is a by-product of Cys desulfurase NFS1, which uses Cys as substrate for the synthesis of Fe-S. Fe-S is a critical co-factor for oxidoreductases such as aconitase (ACO) and mitochondrial respiratory complexes I, II and III. We confirmed that the activities of ACO1 and ACO2 were markedly reduced in CLSA-25 PDAC cells (Fig. 4C). The protein levels of NDUFS1, a Fe-S binding subunit in complex I, was downregulated in all the CLSA-25 PDAC cells. The down regulation of Fe-S proteins in complex II (SDHB) and complex III (RISKE) was also observed in some CLSA cells (Fig. 4D). These observations are consistent with the notion that loss of Fe-S destabilized these proteins. Mitochondria stress test showed that CLSA cells markedly downregulates mitochondrial oxygen consumption rates (OCR) while upregulating extracellular acidification rate (ECAR) (Figs. 4E-4F). Glucose uptake was also dramatically upregulated in CLSA cells (Fig. 4G). Our data suggest that by downregulating Fe-S synthesis, CLSA cells promote a switch from mitochondrial oxidative phosphorylation to glycolytic metabolism.

[0131] Metabolic reprogramming in CLSA cells activates PPP metabolism and nucleotide synthesis.

[0132] To gain comprehensive understanding of the CLSA-induced metabolic reprograming, we performed metabolomics screening using control and CLSA-25 PDAC cells (MiaPaCa2 and PANCI). As shown in Fig. 5A, metabolic set enrichment analysis (MSEA) showed that Warburg effects, Glycolysis, Pentose phosphate pathway (PPP) and nucleotide metabolisms (both Purine0073605-001157

[0133] and Pyrimidine) were among the most overrepresented metabolic pathways in CLSA-25 MiaPaCa-2 and / or PANC-1 cells.

[0134] Nucleotides are critical building blocks required for the synthesis of rRNA, mRNA and DNA in proliferating cells. PPP is a glycolysis shunt pathway that provides R5P and PRPP (Phosphoribosyl pyrophosphate), which are essential precursors for the synthesis of both purine and pyrimidine nucleotides. CLSA-25 cells have increased levels of multiple ribonucleotides and deoxyribonucleotides (Fig. 5B and Figs. 6A-6B). To determine whether CLSA activates PPP and de novo nucleotide synthesis, we used [U-13C]-glucose and LC-MS / MS to analyze the effects of CLSA on the glucose entry into glycolysis and PPP metabolism (Figs. 5C-5E). As shown in Figs. 5D and 5E, CLSA increases glucose flux into glycolytic metabolites (Fig. 5E), PPP metabolites (R5P, PRPP) and nucleotides (IMP, UMP) (Fig. 5E). Our data suggest that CLSA-mediated metabolic reprograming promotes glycolysis, PPP and de no nucleotide synthesis to support cancer cell proliferation and tumor growth.

[0135] OxPPP is translationally upregulated in CLSA PDAC cells to promote nucleotides and NADPH synthesis

[0136] G6PD and TKT (transketolase) are the first and rate limiting enzymes for the oxidative and reductive PPP, respectively. Given the importance of de novo nucleotides synthesis in cell proliferation, we evaluated the effects of a G6PD inhibitor (G6PDil) and a TKT inhibitor (Oxythiamine) on proliferation of CLSA25 and naive PDAC cells. As shown in Figs. 7A-7B, G6PDil abrogated CLSA-mediated cell proliferation in MiaPaCa-2 and PANCI cells, while Oxythamine had no effect. CLSA PDAC cells were also more sensitive to G6PDil -induced cell death (Fig. 7C). To determine whether OxPPP might be upregulated by CLSA, we evaluated the protein expression levels of G6PD, PGLS and PGD in CLSA-25 PDAC cells and CLSA-I PDOs. As shown in Fig. 8A, G6PD protein levels were increased in all the CLSA-25 cells and CLSA-I PDOs when compared to their naive controls. The expression of PGLS and PGD were upregulated in PDAC cells and PDOs except for MiaPaCa2 (for PGLS) and Capan-2 (for PGD) (Fig. SA).

[0137] To determine whether CLS in the PDAC patient tumor microenvironment might also lead to PPP upregulation, we stained G6PD and PGD in PDAC tissues and paired para-tumor normal tissue from 18 patients. As shown in Figs. 8C-8D, the expression levels of G6PD and PGD were robustly upregulated in PDAC tissues when compared to paired para-tumor pancreatic tissues.0073605-001157

[0138] To investigate whether OxPPP upregulation might contribute to PDAC progression, we further determined the expression levels of G6PD in PDAC tissues from a cohort of 100 patients. As shown in Table 1, G6PD high patients had larger tumor size, more advanced TNM stages and higher incidence of lymph node metastasis (Table 1). G6PD high patients also had lower overall survival and relapse free survival (Fig. 7F), which indicates a critical role for OxPPP in PDAC tumor growth and progression.

[0139] Next, we investigated the mechanism underlying CLSA-mediated OxPPP upregulation in PDAC. We found no significant changes in the mRNA transcript levels of the three genes in this pathway (G6PD, PGLS and PGD) in CLSA-25 cells (Fig. 7D). G6PD is a very stable proteins, with no detectable degradation even after 24 hours cycloheximide (CHX) treatment (Fig. 7E). Therefore, we hypothesized that OxPPP enzymes might be translationally regulated in CLSA PDAC cells. We used L-Azidohomoalanine (AHA) to label newly translated proteins in CLSA-25 and naive MiaPaCa2 and PANC-1 cells. The labeled peptides were tagged using a biotin-alkyne click-chemistry probe. After pull-down with streptavidin, the abundance of AHA-labeled G6PD, PGLS and PGD was determined by Western blotting. The levels of newly translated G6PD and PGD were 2- to 5-fold higher in CLSA MiaPaCa-2 and PANCI cells when compared to control (Fig. 8E). AHA-tagged PGLS only increased in CLSA-25 PANC-1 cells, but not in MiaPaCa-2 cells (Fig. 8E), which is consistent with the PGLS expression pattern in these cells.

[0140] To further investigate the translational regulation of PPP enzymes, a sucrose gradient polysome profding experiment was carried out (Figs. 8F and 8G). Polysome profiles showed an overall shift of mRNA into heavier polysomes in CLSA25 MiaPaCa-2 cells (Fig. 9F), which indicated upregulation of translational initiation. The mRNA peak of G6PD and PGD shifted from fraction 3 in control cells to fraction 6 and 4, respectively, in CLSA25 cells showing that the number of ribosomes associated with those mRNAs is higher in CLSA25 compared with control cells. In contrast, the peak of PGLS and GAPDH shifted toward lighter fractions, which is consistent with AHA labeling results in MiaPaCa-2. The shift toward heavier polysomes for the mRNAs of G6PD and PGD suggests that OxPPP is translationally upregulated in CLSA cells.

[0141] To investigate the role of OxPPP in CLSA-mediated phenotypes, we used siRNA to knockdown G6PD in PDAC cells. Both nucleotide levels (Figs. 8H-8I) and NADPH / NADP+0073605-001157

[0142] ratios (Fig. 8J) were increased in CLSA25 PDAC cells, which was suppressed by siRNA depletion of G6PD (Figs. 8H-8J and Fig. 7H). G6PD siRNA also inhibited the CLSA-mediated increase in ferroptosis resistance and cell proliferation (Figs. 7I-7J). To determine whether activation of OxPPP is sufficient to mediate CLSA phenotypes, we overexpressed G6PD in naive PDAC cells (Fig. 7K). G6PD overexpression increased ferroptosis resistance in PDAC cells, while cell proliferation was not affected by ectopic G6PD (Figs. 7L-7M). Taken together, our data suggested that activation of OxPPP is at least partially responsible for CLSA-mediated nucleotide synthesis, cell proliferation and ferroptosis resistance.

[0143] CLSA promotes lipogenesis and lipid droplet formation in PDAC.

[0144] OxPPP has been implicated in promoting lipogenesis in adipocytes and cancer cells1736. NADPH, along with malonyl-CoA, is required for de novo fatty acid synthesis by fatty acid synthase and suppression of OxPPP inhibits lipogenesis in cancer cells17,18. To determine the effects of CLSA on PDAC lipid metabolism, we performed untargeted lipidomic screening using naive and CLSA-25 MiaPaCa2 cells (Figs. 9A-9B). Triacylglycerides (TGs) and ceramides (Cer) were the most upregulated and most downregulated lipid species, respectively, in CLSA-25 cells (Figs. 9A-9B). TGs are the main component of LDs that serve as energy and lipid storage in the cell. We confirmed through thin layer chromatography (TLC) that TGs were upregulated in CLSA-25 PANC-1 and MiaPaCa2 cells (Figs. 9C-9D). Confocal microscopy and flow cytometry quantitation indicated that CLSA promotes LD formation (Figs. 9E-9G). G6PD overexpression (OE) in naive MiaPaCa-2 and PANC-1 cells was sufficient to increase TG and LD levels (Figs.

[0145] 10A-10D). Conversely, G6PD knockdown in CLSA PDAC cells significantly decreased TG levels (Figs. 1 OH- 101). These data suggested that CLSA promotes lipogenesis and LD formation through activation of the OxPPP. To further critically investigate this notion, we used Relabeled glutamine labeling to determine the effects of CLSA and G6PD inhibition (with G6PDil) on de novo FA synthesis. As shown in Figs. 9J-9K, the13C labeled palmitate (PA, C16:0) and stearate (SA, C18:0), especially those species with more than 813C atom incorporations (M+x, x>8), were increased in CLSA-25 PANC-1 cells when compared to naive control cells. These results indicated activation of de novo FA synthesis in CLSA PDAC cells. The inhibition of the OxPPP pathway with G6PDil suppressed de novo PA and SA synthesis and largely abrogated the effects of CLSA-25, especially in PA and SA species with greater than 1213C atom0073605-001157

[0146] incorporations (M+x, x>12). Taken together, our data strongly indicated that CLSA-25 promotes de novo lipid synthesis in an OxPPP-dependent manner.

[0147] To determine whether LD formation is upregulated in PDAC, we perform LD staining in PDAC tissue cryosections from 30 patients. LD staining in PDAC patient tissues and paired para-tumor normal tissues revealed dramatic increase in LD formation in tumor tissues (Figs. 9L-9M). In PDAC tissues LD almost exclusively colocalize with CK19 positive PDAC cells, but not with a-SMA positive cancer-associated fibroblast cells (Fig. 10E). Taken together, our data indicate that OxPPP upregulation in CLSA PDAC promotes lipogenesis and LD formation.

[0148] CLSA PDAC cells are hypersensitive to the lipid metabolism drug lomitapide

[0149] To investigate whether CLSA could expose exploitable metabolic vulnerabilities, we performed a drug screening using a metabolism-focus library of more than 800 compounds. 76 compounds showed more than 70% inhibition of cell viability in CLSA group (Fig. 11 A).

[0150] Among the most effective drugs were glycolysis / glucose uptake inhibitors (e.g. PFK158, BAY-876) and nucleotide synthesis inhibitors (e.g. Leflunomide, Pemetrexed acid) (Figs. 11 A and 12A), which is consistent with upregulation of glycolysis and nucleotide in CLSA cells.

[0151] Approximately 30% of the positive hits were compounds targeting lipid metabolism, including many FDA-approved drugs for cardiovascular diseases (e.g. Lomitapide, Mevastatin) and type II diabetes (e.g. Troglitazone, Pioglitazone) (Fig. 11 A and 12A). Considering the excellent safety profdes of lipid metabolism drugs, several drugs within this group were further investigated in naive and CLSA PDAC cells (Fig. 1 IB). Among these drugs, lomitapide showed excellent efficacies toward CLSA PDAC cells and PDOs with IC50 in the high nanomolar to low micromolar range (Figs. 1 IB-11C). Lomitapide is an orphan drug approved by the FDA for the treatment of homozygous familial hypercholesterolemia (HoFH). Developed as an inhibitor for microsomal triacylglycerides transfer protein (MTTP), lomitapide inhibits the transfer of TG to ApoB in hepatocytes and enterocytes to suppress the secretion of ApoB-containing lipoprotein into the bloodstream. Since our data showed that TGs were upregulated in CLSA PDAC cells, we further determined the effects of lomitapide on tumor growth in an orthotopic xenograft model. As shown in Figs. 1 ID-1 IF, orthotopically implanted CLSA-25 MiaPaCa-2 xenograft grew faster than naive control tumors. The CLSA-25 group was more sensitive to the anti-tumor effect of lomitapide and treatment with 10 mg / kg lomitapide completely abrogated the CLSA0073605-001157

[0152] effects on promoting tumor growth (Figs. 1 ID-1 IF). These observations are consistent with data in cell culture models showing that CLSA sensitizes PDAC cells to lomitapide treatment.

[0153] Lomitapide induce lipotoxicity by suppressing triacylglycerides synthesis and LD formation.

[0154] In an effort to determine the mechanism underlying lomitapide cytotoxicity, we found no detectable expression of MTTP in PDAC cells (Fig. 12B). Therefore, we performed targeted lipidomics screening to investigate the effect of lomitapide treatment on lipid metabolism in PDAC cells. As shown in Fig. 1 II and Figs. 12C-12F, lomitapide treatment (1 pM) significantly decreased the levels of TGs and cholesterol ester (CE) while increasing the levels free fatty acids (FFAs) and acyl-carnitines (CARs), two toxic lipid species. The levels of structural lipids also increased after lomitapide treatment. TLC analysis (Figs. 1 IF- 11G and 12G-12H) showed that lomitapide treatment suppressed TG levels in PDAC cells in a dose-dependent manner, while the levels of DG were not affected. LD staining and confocal imaging indicated that lomitapide robustly inhibited LD formation in PDAC cells (Figs. 11H-1 II). It is interesting to note that when compared to DGAT1 inhibitor (DGATli) A922500 and DGAT2 inhibitor (DGAT2i) PF-06424439, two DGAT inhibitors in clinical trials, lomitapide was able to achieve larger reduction of TG levels at much lower concentrations (Figs. 12G-12H).

[0155] To understand the mechanism by which lomitapide regulates TGs, we used oleic acid-alkyne (OA-alkyne), a click-chemistry fatty acid probe, to evaluate the effects of lomitapide treatment on de novo TG synthesis. MiaPaCa2 and PANC-1 cells were pre-treated with various concentrations of lomitapide for 2 hours and then pulsed with OA-alkyne. After 30 minutes labeling, the lipids were extracted, labeled with 3-azido-7-hydroxycoumarin through clickchemistry reaction, and the OA-alkyne labeled lipids were resolved through TLC. As shown in Figs. 11 J-l IK, lomitapide inhibited de novo TG synthesis with an IC50 of approximately 0.5 pM. Accompanying the dose-dependent decrease in OA-labeled TGs, the free, unincorporated OA levels were increased in lomitapide treated PDAC cells (Figs. 11 J-l IK), which is consistent with observations in targeted lipidomic screening. TG synthesis is essential for cells to neutralize FFA-induced lipotoxicity. Therefore, we determined the effects of lomitapide treatment on sensitivity to palmitate-induced lipotoxicity in PDAC cells. As shown in Fig. 1 IN, co-treatment with 0.5 pM lomitapide dramatically sensitized PDAC cells to palmitate-induced cell death. Since G6PD overexpression promotes TG synthesis and LD formation, we further determined0073605-001157

[0156] the effects of OxPPP activation on lomitapide sensitivity. As shown in Fig. 121, G6PD overexpression reduced the lomitapide IC50 by approximately two-fold, which indicated that activation of OxPPP was sufficient to sensitize PDAC cells to lomitapide.

[0157] Mutations in oncogenes and tumor suppressors (e.g. KRAS, MYCN, VHL) have been previously shown to promote TG synthesis and LD formation. For example, elevated TGs and LDs have been well documented in clear cell renal cell carcinoma (ccRCC), MYCN-applified neuroblastoma, glioblastoma and colorectal cancer. Given our data showing that lomitapide is a more potent inhibitor of TGs and LDs, we hypothesized that lomitapide could also be used to exploit lipid metabolism vulnerability in cancers other than PDAC. To test this hypothesis, we tested lipotoxi city-induction effects of lomitapide in comparison to other known DGAT inhibitors in a panel of ccRCC cells. Our data showed that lomitapide is at least 10 times more effective at inducing lipotoxicity than existing DGAT inhibitors such as AZD7687, PF04620110 andA922500 (Fig. 15).

[0158] The inhibition of TG synthesis and accumulation of FFA in lomitapide-treated PDAC cells is reminiscent of similar phenotypes in DGAT1 and DGAT2 knockout mice39. Therefore, we investigated the effects of lomitapide on DGAT activities following a previously reported protocol. As shown in Fig. 12J, microsomal membranes isolated from mouse liver were able to synthesize TG using dioleyl glycerol (DOG) and NBD-palmitoyl-CoA as substrate. Using this assay, we determined that lomitapide could inhibit DGAT activation with an apparent IC50 of approximately 1 pM (Fig. 110). It has been previously reported that lomitapide could be processed and degraded by cytochrome P45037, which is enriched in the microsomal membrane used for the in vitro DGAT activity assay. Pre-incubation of lomitapide with microsomal membranes for greater than 1 hour completely abrogated its inhibition of DGAT activities (Fig.

[0159] 12K). Therefore, the inhibitory effect of lomitapide on DGAT activity in this in vitro assay is likely underestimated.

[0160] To determine whether inhibition of DGAT 1 and 2 might be responsible for the cytotoxicity of lomitapide, we used siRNA to deplete DGAT 1 and 2 in MiaPaCa-2 and PANC-1 cells. As shown in Figs. 12L-12N, DGAT1 or 2 knockdown significantly reduced the LD staining in control and CLSA-25 PDAC cells. Moreover, CLSA-25 PDAC cells were more sensitive to DGAT1 or 2 siRNA induced cell death than naive control cells (Fig. 1 IP), which is consistent with our previous observations with lomitapide. Taken together, our data suggested0073605-001157

[0161] lomitapide inhibits TG synthesis and LD formation by targeting DGAT activities in PDAC cells, and CLSA cells are hypersensitive to lomitapide-induced lipotoxicity, likely due to CLSA-mediated activation of lipogenesis.

[0162] Lomitapide inhibits chemotherapy-induced LD-formation and TG synthesis and sensitizes PDAC tumors to the FOLFIRINOX in PDX and KPC models.

[0163] TG synthesis and LD formation is critical for cells to avoid stress-induced lipotoxicity. Targeted lipidomics analysis of MiaPaCa-2 cells treated with modified FOLFIRINOX (mFOLFIRINOX) chemotherapy showed that mFOLFIRINOX treatment induced dramatic lipidomics reprograming that increased TG levels while reducing the levels of phospholipids and lysophospholipids (Figs. 13A-13B). This observation is consistent with the notion that chemotoxic stress induces lipolysis and storing excess FFA in TG and LD to mitigate lipotoxicity. We confirmed that mFOLFIRINOX treatment increased TG and LD levels (Fig. 13C and Fig. 14A). mFOLFIRINOX-induced TG synthesis and LD formation was abrogated by lomitapide treatment (Fig. 13C and Fig. 14A), which suggested that lomitapide could be used to enhance the efficacy of mFOLFIRINOX chemotherapy. Indeed, as shown in Fig. 13D, cotreatment of PDOs with mFOLFIRINOX and 200 nM lomitapide effectively reduced mFOLFIRINOX IC50 by approximately two to five-fold in control or CLSA-1 PDOs, respectively. Chemo-sensitization effects of lomitapide could be observed at a concentration as low as 50 nM (Fig. 13D). The knockdown of DGAT1 or DGAT2 also dramatically reduced the mFOLFIRINOX IC50 (Fig. 14B), which is consistent with the notion that lomitapide sensitizes PDAC to chemotherapy by targeting DGAT-mediated TG synthesis and LD formation.

[0164] To determine whether lomitapide could be used to improve the efficacy of FOLFIRINOX therapy, we used PDX models derived from two PDAC patients (Fig. 13E). NSG mice bearing PDX tumor fragments were administered with vehicle control, lomitapide, mFOLFIRINOX or the combination treatment when the tumor size reached 3 mm in diameters (Fig. 13E). As shown in Figs. 13F-13G, mFOLFIRINOX treatment inhibited the tumor growth in PDX1 and PDX2 mice by 52% and 16%, respectively. Lomitapide treatment alone inhibited PDX1 and PDX2 growth by 40% and 35%, respectively. When combined with mFOLFIRINOX, the inhibition of tumor growth was further enhanced to 78% and 77% (Figs. 13F-13G and Fig. 14C). The inhibitory effects of single and combination treatments on tumor growth were further confirmed with positron emission tomography scan using18F -Deoxyglucose as tracer (Figs. 13H-13I and0073605-001157

[0165] Fig. 14D). LD staining of tumor tissues collected from PDX1 showed that mFOLFIRINOX treatment significantly increased LD levels in tumor sections, which could be inhibited by lomitapide treatment (Figs. 13J-13K). Similar changes in LD levels were observed in PDX2 tumor sections, although the differences were not statistically significant except in the lomitapide single treatment group (Figs. 13J-13K).

[0166] To further evaluate the chemo-sensitization effects of lomitapide, we tested the effects of lomitapide and mFOLFIRINOX treatments in the genetically engineered KPC (LSL-Kras12D / +; LSL-Trp53R172H / +:Pdxl-Cre) PDAC model. The PDAC tumor formation in KPC mice were monitored by ultrasound imaging. Mice were randomly assigned into one of the four treatment groups (vehicle, lomitapide, mFOLFIRINOX or combination) when the tumor volume reached 20-50 mm3. The mice were treated for 4 weeks, and the tumor growth was monitored through ultrasound during this period. After stopping treatment at week 4, the mice were further monitored for survival. As shown in Figs. 13L-13M, after 4 weeks of treatments, lomitapide reduced KPC tumor volume by 37%, while the effects of mFOLFIRINOX on tumor size were not statistically significant. The combination treatment reduced tumor size by 78% (Fig. 13M). As a monotherapy, lomitapide improve the median survival from 52 days (in control group) to 70 days (p=0.04). The median survival in the mFOLFIRINOX group was 65 days (p=0.20). The combination treatment further increased the median survival to 121 days (p=0.001). Therefore, lomitapide could inhibit tumor growth and improve animal survival in KPC model as a single agent. When combined with mFOLFIRINOX, lomitapide robustly increased the chemotherapy efficacy and prolonged animal survival (Fig. 13N). Importantly, although mFOLFIRINOX treatment modestly reduced the body weight of KPC mice, lomitapide treatment, alone or in combination with mFOLFIRINOX, had no additional effects on weight loss (Fig. 14E). These data indicated that lomitapide and its combination with mFOLFIRINOX were well tolerated in mice.

[0167] Taken together, our data suggested that lomitapide has potent chemo-sensitization effects, potentially through inhibiting TG synthesis, LD formation and enhancing chemotherapy-induced lipotoxicity.

[0168] Discussion

[0169] Cys and Cys are among the most depleted amino acids in the PDAC tissues and there is intense interest in developing Cys2 deprivation agents as potential anti-cancer therapies.0073605-001157

[0170] However, the effects of chronic CLS adaptation on cancer cells and the underlying mechanisms are poorly understood. Our data provide important insights into the CLS A in PDAC. First, CLS A PDAC cells can maintain GSH synthesis despite severe reduction in intracellular Cys. This is remarkable since GSH levels in the PDAC cell are 20-300 fold higher than Cys (Fig. ID and Fig.

[0171] 3 A). Among the major Cys metabolites Fe-S was the most severely downregulated in CLSA cells, which resulted in the downregulation of mitochondrial OXPHOS and upregulation of glycolytic metabolism.

[0172] Second, all the CLSA PDAC cells and PDOs proliferated faster in the culture and grew into larger xenograft tumors when implanted orthotopically. Consistent with the elevated cell proliferation, CLSA PDAC cells increase de novo nucleotide synthesis and lipogenesis. This is at least in part due to the translational upregulation of the OxPPP pathway, which supplies R5P and NADPH for nucleotide and lipid anabolism, respectively. While genetic or pharmacological inhibition of G6PD abrogated the CLSA-mediated phenotypes, ectopic expression of G6PD could only partially recapitulate some of the CLSA phenotypes (e.g. ferroptosis resistance, lipogenesis), but not others (e.g. cell proliferation). These findings implicated the involvement of other metabolic pathways in addition to OxPPP during CLSA. It was recently reported that PDAC cells that adapted to glucose and glutamine limitation also exhibited enhanced cell proliferation. However, we found very few surviving PDAC cells under glutamine limitation conditions in the absence BSA supplementation, which is consistent with earlier reports.

[0173] Tryptophan and arginine are two other amino acids severely depleted in the PDAC miroenvironment. We found that although PDAC cells were able to adapt to tryptophan limited cell culture medium over a period of 2-4 weeks, adapted cells proliferated slower in their respective nutrient limited media and eventually stop growing beyond 4 weeks. These findings indicate that CLS might induced unique adaptation mechanisms not shared by the deprivation of other amino acids.

[0174] Third, in a drug screening we found that CLSA cells were hypersensitive to drugs targeting glycolysis, nucleotide metabolism and lipid metabolism, which was consistent with the upregulation of these metabolic pathways in CLSA PDAC cells. Among FDA-approved lipid metabolism drugs, lomitapide was selected for further investigation. Instead of targeting MTTP, our data showed that lomitapide inhibits TG synthesis and LD formation to sensitize PDAC cells to lipotoxicity. The inhibitory effects on DGAT activities were further confirmed in a0073605-001157

[0175] reconstituted in vitro activity assay. Lomitapide has been previously reported to inhibit the mTORCl and ZDHHC5. However, the lomitapide concentrations required for such inhibitions (25 pM) are at least one order of magnitude higher than required for the induction of cytotoxicity in PDAC cells and PDOs. In contrast, the IC50 of lomitapide toward TG synthesis (approximately 500 nM in OA-alkyne tracing experiment) were comparable to concentrations required for its cytotoxic effects in PDAC cell lines and tumoroids. Moreover, lomitapide treatment markedly increased the sensitivity of PDAC cells to palmitate-induced lipotoxicity. Collectively, our data showed that lomitapide sensitizes PDAC cells to stress-induced lipotoxicity by targeting TG synthesis and LD formation.

[0176] Targeting the dysregulated cancer metabolism is an area under intensive investigation. Drugs targeting nucleotide synthesis, such as gemcitabine and 5-FU have been successfully used in cancer treatment for decades. However, the development of cancer therapeutics targeting dysregulated lipid metabolism were more challenging. Recently the role of DGAT1 / 2 and LD emerged as critical players in tumor growth and progression. Several pharmacological inhibitors for DGAT1 / 2 have been used in clinical trial for diabetes, fatty liver disease and other metabolic conditions. These inhibitors could potentially be useful in targeting the hyperactive lipid metabolism in cancer. Indeed, there is evidence supporting the anti -tumor activity of DGAT1 inhibitor A922500 in glioblastoma and clear cell renal cell carcinoma models. However, the doses of DGAT1 inhibitor required for anti -tumor activities in mouse models were 20- to 40-times higher than those typically used for other metabolic diseases (3mg / kg). Considering the well-documented gastrointestinal adverse effects of DGAT1 inhibitors in clinical trials, the feasibility of using such high doses in cancer patients is likely an issue.

[0177] Importantly, lomitapide robustly inhibited tumor growth and improved the efficacy of FOLFIRINOX chemotherapy regimen when used at a clinically relevant dose. The lomitapide dose used in our pre-clinical animal experiments (10 mg / kg) is equivalent to approximately 0.8 mg / kg in humans, which has been previously shown to be safe for HoFH patients for at least 78 weeks in clinical trials. Considering that a typical FOLFIRINOX chemotherapy session lasts only 2 weeks, it is possible that higher doses of lomitapide could be tolerable as chemosensitizing agents. Therefore, lomitapide could serve as a powerful agent for targeting the dysregulated lipid metabolism in PDAC and other cancers. Since the anti-tumor activity of0073605-001157

[0178] lomitapide doesn’t depend on inhibition of MTTP, modifications to enhance its DGAT inhibitory activity while minimizing its activity toward MTTP would be desirable.

[0179] Materials and Methods

[0180] Antibodies, key reagents and inhibitors

[0181] Information for antibodies, key reagents and inhibitors are included in Tables 2 and 3. Cell culture

[0182] The PDAC cell lines (MiaPaCa-2, PANC-1, SW-1990, Capan-2) were obtained from the ATCC. KPC cells were derived from Kras,sl~Kras GI2D / +- Tp53i,: 2i!; Pdxl-Cre (KPC) genetically engineered mouse model. Pan02 cell was a gift from Dr. Shari Pilon-Thomass (Moffitt Cancer Center). All ATCC cell lines were characterized or authenticated by the ATCC using short tandem repeat profiling and passaged in our laboratory for fewer than 6 months before use. All cell lines used were free of microbial (including mycoplasma) contamination. We routinely perform test for mycoplasma contamination in the lab at least monthly. All pancreatic cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% Penicillin-Streptomycin and maintained at 37 °C in a humidified 5% CO2 incubator.

[0183] Induction of CLSA

[0184] CLSA-25 and CLSA-50 cell culture media were prepared by supplementing pyruvate, glutamine, methionine and cystine-free DMEM media (Gibco, 21013-024) with 1 mM Sodium Pyruvate, 4 mM glutamine, 200 uM L-Methionine and 25 uM L-cystine (for CLSA25) or with 50 uM L-cystine (for CLSA50). To induce CLSA, PDAC cells were cultured in indicated L-cystine limited media supplemented with 10% FBS and 1% Penicillin-Streptomycin for 4 weeks. The media were changed every other day during this period. After 4 weeks adaptation, cells were frozen and stored in liquid nitrogen. After recovery from liquid nitrogen cells were cultured in their respective induction media and used for experiments within 6 passages.

[0185] Patient derived organoid (PDOs) culture

[0186] PDO were derived and cultured following a previosly described protocol. To induce CLSA-I PDO, two PDOs were cultured in medium containing 0.5 uM IKE (Imidazole ketone erastin) for 4 weeks. For PDO xenografts, CLSA-I or control PDOs were digested into single cells using TrypLE Express and then neutralized with Human Complete Feeding Medium (hCPLT) contained Human Wash Medium with BSA (Advanced DMEM / F-12, lOmM HEPES, 1 x GlutaMAX Supplement, lOOpg / ml Primocin and 0.1% BSA). 1 *105of PDO cells were0073605-001157

[0187] resuspected into Matrigel and injected orthotopically into pacreas. The growth of tumors were monored through MRI scan using 9.4 T MR scanner (Bruker BioSpec 94 / 30 USR) at Multimodality Preclinical Molecular Imaging Center in Tianjin Medical University General Hospital. The tumor volume was calculated with the formula: volume = length x width x width / 2.

[0188] Treatment of PDAC cells and PDOs with modified FOLFIRINOX (mFOLFIRINOX) mFOLFIRINOX Treatment of PDAC cells and PDO cultures were carried out. The mFOLFIRINOX cocktail consisted of 5-FU, SN-38 (metabolic product of Irenocan responsible for its inhibitory activity toward DNA topoisomerase I) and oxaliplatin at a 2: 1 :2 ratios. The indicated mFOLFIRINOX concentrations were the concentrations of 5-FU and oxaliplatin.

[0189] Cell proliferation assay

[0190] IxlO5Cells per well were seeded onto a 12-well plate in triplicate. The media were changed every two days. At indicate time points, the cells were fixed with 10% formalin for 15 min and stained with 0.5 mL 10% crystal violet solution for 20 min with shaking. After staining, the cell were washed with water for five times and dried overnight. The crystal violet staining was solubilized with 1 ml 10% acetic acid absorbance at OD 590 nm was determined with a plate reader.

[0191] Cell viability assay

[0192] 1 xlO4cells per well were seeded onto 96-well plates (Corning, 3610) and treated with different drugs at indicated concentrations for two to three days. Cell viability were tested using CellTiter-Glo Cell Viability Assay (Promega, G7572) and recorded as relative luminescence units (RLU) using FlexStation 3 (Molecular Devices).

[0193] 2-NBDG Glucose uptake assay

[0194] PDAC cells were seeded at 2 x 105cells / well in al2 well plate. The cells were incubated with 500 pL of culture medium containing 50 pM 2-NBDG and incubated at 37 °C for 2 h. The reaction was terminated by washing the cells twice with ice cold PBS. The cell was re-suspended through trypsinization and analyzed through flow cytometry using the excitation / emission at 467 / 542 nm.

[0195] Mitochondria stress test and glycolysis stress test.

[0196] Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were determined using Seahorse XFe24 Extracellular Flux Analyzer (Agilent Seahorse Bioscience)0073605-001157

[0197] following protocols recommended by the manufacturer. Cells were seeded on XF24 cell culture microplates (2.5*10A5 cells / well). Cells were maintained in a XF DMEM assay medium (Agilent, 103575-100) in a non-CCh incubator for 30 min before the assay. The XF Cell Mito Stress Test Kit (Agilent, 103015-100), was used for the OCR assay. XF Glycolysis Stress Test Kit was used for the ECAR assay (Agilent, 103020-100). For mitochondria stress test, the baseline recording was followed by sequential injection the following reagent to the final concentration: 1.5 uM oligomycin, 1 uM FCCP, and 0.5 uM rotenone / antimycin A. For glycolysis stress test, the baseline recording was followed by sequential injection the following reagents: 10 mM D-glucose, 1 uM oligomycin, and 50 mM 2-DG.

[0198] RNA interference and other constructs

[0199] The knockdowns of G6PD, DGAT1 and DGAT2 was achieved using the 27mer duplexes DsiRNA system from IDT. The targeting sequences are listed in Table 4. pLX304-G6PD was constructed by subcloning the human G6PD cDNA into pLX304 vector through Gateway subcloning. The lentiviral particles were packaged in HEK293 cells using the PEI transfection method, and concentrated. After selection with blasticidine, the surviving cells were pooled together and used for assays.

[0200] Western blotting

[0201] Cells were lysed in SDS-NP40 buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% NP40, 1% SDS, ImM protease inhibitors cocktail) on ice for 1 min. Cells were scraped from the plate and sonicated briefly for three times. Then lysates were centrifuged at 20,000 xg, 4 °C for 10 min. 20-40 pg proteins were separated by SDS-PAGE and transferred onto PVDF membrane. The membranes were incubated in blocking buffer (5% (w / v) nonfat dry milk in Tris-buffered saline, 0.05% Tween 20 (TBS-T)) for 30 min at RT., and incubated with primary antibodies for 20 h at 4 °C, followed by incubation with secondary antibodies for 60 min at RT. Antibody information are listed in Table 4.

[0202] Reverse transcription-polymerase chain reaction

[0203] 1 x 106cells were washed with ice-cold PBS and total RNA was extracted using RNeasy kit (Qiagen, Cat#74106). 1 pg RNA was used for the reverse transcription using High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, 4368814). cDNA was diluted 1 : 10 then used for qPCR with ABI qPCR master mix (Thermo, Cat#4367659). Each RT-PCR0073605-001157

[0204] experiment was performed independently at least three times. The PCR primers used are listed in Table 5.

[0205] L- Azidohomoalanine (AHA) labeling of OxPPP enzymes.

[0206] 7xl06cells on a 10 cm dish were rinsed twice with 5 ml IxPBS and incubated with L-Met free DMEM medium for 1 hour in 37°C, 5% CO2 incubator. After 1 hour, L-AHA were added to the final concentration of 1 mM. After 1 hour labeling with AHA, cells were lysed. To label newly-synthesized proteins, lysate was mixed with 2x click-chemistry reaction buffer (2 mM Copper Sulfate, 20 uM Biotin-Alkyne, 2 mM TCEP in IxPBS) and incubated for 60 minutes at room temperature with shaking. The reaction was terminated by adding TCA to the final concentration of 10% and incubated on ice for 30 minutes. After 10,000 g centrifugation at 4°C 10 minutes, the precipitate was washed with 1ml ice-cold acetone for 3 times, and air-dried at room temperature for 30 minutes. The pellet was re-dissolved in 500 ul RIPA buffer with proteinase inhibitor cocktail and sonicated for 10 seconds on ice 3 times. The biotin-labeled proteins were precipitated with 40 ul NeutrAvidin resin and affinity -purified OxPPP enzymes were detected with Western blotting using antibodies for G6PD, PGD, PGLS and with GAPDH as control.

[0207] Polysome profiling

[0208] IxlO7cells were treated with cycloheximide (100 ug / ml) in the cell culture media for 2 minutes and the cells were placed on ice. After removal of media, the cells were washed twice with ice-cold IxPBS / cycloheximide (100 ug / ml). Cells were scraped in 600 ul Polysome Buffer (50 mM HEPES, pH 7.4, 250 mM KC1, 5 mM MgCh, 250 mM sucrose) containing 1% Triton x-100, 1.3% NaDOC, 100 ug / ml cycloheximide, 0.1 U / ul SUPERase. In RNAase inhibitor (AM2694, Invitrogen). After 10 minutes lysis at 4°C with rocking, the lysates were centrifuged at 3,000 g for 15 minutes at 4°C. 600 ul supernatant was loaded to a sucrose gradient (20%-47%, w / w) and centrifuged in a Beckman SW41Ti rotor at 34,000rpm for 160 minutes at 4°C. After centrifugation, 8 fractions were collected from each. The RNA from each fraction was extracted using TRlzol (15596018, Invitroen) and used for qPCR analysis.

[0209] LC-MS / MS analysis of NEM-cysteine, GSH, dNTP and NADP+ / NADPH

[0210] NEM-cysteine', The measurement of NEM-derivatized cysteine in pancreatic cancer cells was carried out following our previously reported protocol (1). For preparation of human and mouse tissue samples, 10 mg PDAC or para-tumor normal tissues were immersed in 10 ul NEM0073605-001157

[0211] Tissue Buffer (25 mM N-Ethylm al eimide, 10 mM Ammonium Formate, pH7.0) spiked with 10 uM NEM-15N,13C3-Cysteine as interior control) and homogenized with a glass Dounce tissue homogenizer on ice. The homogenate was centrifuged at 20,000 rpm, 4°C for 5 minutes and 5 ul supernatant were diluted in 45 ul NEM Tissue Buffer and used for LC-MS / MS analysis.

[0212] GSH'. 2xl05cells in 12 well plate (70% confluency) were washed with ice cold IxPBS three times and the residual PBS was thoroughly removed. 225 ul ice-cold 0.1% formic acid and 25 ul [13C2,15N] GSH (lug / ml in 0.1% formic acid) was added to the well. The cells were scrapped in to a 1.5 ml Eppendorf tube. Cells were vortexed for 15 seconds, snap-frozen in liquid nitrogen for 1 minute and thawed in a room temperature water bath. After three freeze-thaw cycle, cells were centrifuged at 13,000 g for 3 minutes and the supernatant were collected for LC-MS / MS analysis.

[0213] dNTP: IxlO6cells (in 6-well plate) were washed with ice-cold PBS and residual PBS was removed. 490 ul ice-cold 80% MeOH spiked with 10 ul lOOnM [13Cio,15N5]-dATP (646237, Sigma) internal standard were added and cells were scrapped into a 1.5 mL tube. After vortex, the lysates were centrifuged at 21,130 g, 4°C for 10 min. The supernatant was transferred to a new tube and mixed with 1 ml 50 mM NH4AC, pH 4.5 and used for subsequent SPE. Oasis WAX 1 cc, 30 mg, 30 um cartridge (Waters, Milford, MA, USA) was used for SPE. The cartridge was activated with 1 ml methanol, and equilibrated with 1 ml dH2O prior to sample loading. After the samples loading by gravity, the cartridge was washed by 1 mL 50 mM NH4AC, pH 4.5, and 1 mL 0.5 % (25 % NH4OH) in methanol. The analytes were finally eluted with 1 ml buffer containing 80 % methanol, 15 % water and 5 % (25% NH4OH). The eluent was dried by Speed-vac and reconstituted with 50 ul reconstitution solution for LC-MS / MS analysis.

[0214] NADP+ / NADPH: IxlO6cells in 6-well plate were washed with ice-cold PBS and extracted with 0.4 ml 80% MeOH pre-chilled to -20°C. The plates are transferred to -80°C for 15 minutes and cells were scrapped into a 1.5 ml tube. After centrifugation at 15,000 g for 10 minutes at 4°C, the supernatant was used directly of LC-MS / MS analysis.

[0215] LC-MS / MS: All LC-MS / MS analysis were carried out using Sciex QTRAP 6500+ mass spectrometry coupled with a Sciex EXion HPLC separation system. The multiple reaction monitoring mode (MRM) was used to analyze and quantify metabolites. All peaks were integrated and quantified by Sciex OS 3.0 software.

[0216] High throughput drug screening0073605-001157

[0217] Cells were seeded onto 96-well plates at an optimal density (?? Cells per well in ?? ul) in their respective medium incubated at 37°C in a 5% CO2 environment for 16 hours. Following incubation, compounds from a metabolic disease focus library (L5200, TOPSCIENCE) were added to the cells at a final concentration of 5pM (with DMSO serving as the vehicle control). After incubation with the compounds for 48 hours, cell viability was measured using the CCK-8 assay.. Compounds that significantly inhibited cell viability (more than 70% inhibition) in Mia-PaCa2-CLSA cells were identified as potential hits.

[0218] Patients tissue specimens

[0219] Tumor samples and para-tumor normal tissues were obtained from 100 patients with PDAC treated at the Tianjin Cancer Hospital from 2014 and 2018. All patients underwent radical pancreaticoduodenectomy with R0 margins confirmed by two pathologists. None of the patients had received chemotherapy or radiotherapy at the time of sample collection. All patients were treated with systemic gemcitabine-based chemotherapy after operation for six cycles after surgery. No radiotherapy was given before or after surgery. Postoperative follow-up of patients were conducted every 3 months initially. Overall survival (OS) was defined as the time interval from the date of surgery to that of death due to any cause or that of the last follow-up. Relapse-free survival (RFS) was calculated from the date of surgery to that of local recurrence or metastasis. Local recurrence or metastasis was diagnosed by radiological examination (contrast-enhanced CT or MRI scanning). All patients provided written informed consent. The study protocols were approved by the Ethics Committee of the Tianjin Cancer Institute and Hospital.

[0220] Orthotopic xenograft and bioluminescence imaging (BLI)

[0221] The orthotopic PDAC xenograft and BLI imaging were carried out following previously reported procedures(l, 3, 4) according to IACUC protocols approved by the IACUC committee at the Penn State College of Medicine or Tianjin Cancer Hospital. Briefly, Luciferase labeled PDAC cells (IxlO5for KPC and Pan02 cells, IxlO6for MiaPaCa-2 cells) were resuspended in 20 ul Matrigel (diluted in DMEM media) and orthotopically injected into carefully exposed pancrease of 6-7 weeks old female mice (C57BL / 6J for KPC and Pan02 cells, BALB / c Nude mice for MiaPaCa-2 cells). The wound was closed with suture and wound clip. Non-invasive BLI imaging was carried out using Xenogen IVIS 200 1 week after the surgery to monitored the orthotopic tumor growth. At the end of the experiment, liver were inspected for visible metastatic lessions and ex vivo BLI imaging was carried out to monitor liver metastasis.0073605-001157

[0222] PDX model experiments

[0223] The experimental protocol was approved by the Ethics Committee of Tianjin Medical University Cancer Institute and Hospital, following the NIH Guide for the Care and Use of Laboratory Animals. Pathogen-free NSG mice (6-7 weeks old, female) were used for the primary tumor xenograft (PDX) model. Tumor specimens were collected from pancreatic cancer patients who underwent surgical resection, and the tissues were transplanted into 8-week-old NSG mice under a sterile environment. After three successful passages, the PDX tumor was excised and cut into 2 mmfragments and inoculated into NSG mice. Once the transplanted tumors reached 3 mm, mice were randomly assigned into 4 groups (6 mice per group) to receive different treatments. The treatment groups included: (A) vehicle control (saline), (B) FOLFIRINOX (Oxaliplatin: 10 mg / kg, intravenous injection once weekly; 5-FU: 10 mg / kg, intraperitoneal injection once weekly; Irinotecan: 5 mg / kg, intraperitoneal injection once weekly), (C) Lomitapide treatment at 10 mg / kg via daily oral gavage, and (D) combination therapy with FOLFIRINOX and Lomitapide. Tumor size was measured weekly using calipers, and the tumor volume was calculated with the formula: volume = length x width x width / 2. At the end of the experiment, tumors were harvested from the mice for further processing and analysis.

[0224] For PET-CT scan, mice were fasted for 6 hours prior to the experiment but had free access to water. Before the injection of the imaging agent, the mice were weighed, and18F-FDG was administered via the tail vein at a dosage of 5 pCi / g, with the injection time recorded.

[0225] One hour after the tail vein injection of the 18F-FDG tracer, a micro-PET / CT scan was performed. The mice were anesthetized using isoflurane gas and positioned prone on the small animal PET / CT bed, with infrared beams used to center the scanning field. Static imaging was then conducted, starting with the PET scan followed by the CT scan, for a total duration of approximately 10 minutes. The CT and PET images were transmitted to the IRIS workstation for image reconstruction and fusion. The equipment we used is the French Inviscan small animal imaging system, IRIS PET / CT, and the18F-FDG radioactive tracer was provided by the Molecular Imaging and Nuclear Medicine Department of Tianjin Cancer Hospital.

[0226] KPC mouse model

[0227] KPC mice (C57BL / 6Smoc-Trp53em4(R172H)Kras em4(LSL G12D)Tg(Pdxl-cre)Sm0Cmice, 8 weeks old, male) were purchased from Shanghai Model Organisms Center. The development of pancreatic cancer in these mice were monitored by ultrasound scans(Canon Aplio i800, Japan).0073605-001157

[0228] Mice with tumor volumes of 20-60 mm3were randomly assigned into four groups: (A) vehicle control (saline), (B) mFOLFIRINOX , (C) Lomitapide and (D) combination therapy with mFOLFIRINOX plus Lomitapide and treated as described above. The treatments continued for 4 weeks, during which the tumor volume was monitored biweekly via ultrasound scans. The survival of mice continued to be monitored after stoping the treatment.

[0229] IHC analysis

[0230] Sections of formalin-fixed, paraffin-embedded tumors were subjected to IHC analysis for G6PD and PGD as using a DAB (3,3 ’-diaminobenzidine) substrate kit (Maixin) according to the manufacturer’s instructions. Sections were incubated with anti-G6PD (sc-373886, Santa Cruz Biotechnology, at 1:200 dilution) or anti-PGD (sc-398977, Santa Cruz Biotechnology, at 1:200 dilution) antibodies overnight at 4°C. After wash, the sections were stained with a secondary antibody for 30 minutes at room temperature. PBS was substituted for each primary antibody as a negative control. Five random fields were examined under a light microscope.

[0231] Immunoreactivity was semiquantitatively scored according to the estimated percentage of positive tumor cells as described previously. IHCstaining results were blindly and independently performed by two pathologists who were blinded to the clinical data. Staining intensity was scored 0 (negative), 1 (low), 2 (medium), and 3 (high). Staining extent was scored 0 (0% stained), 1 (1%- 25% stained), 2 (26%-50% stained), and 3 (51%- 100% stained). The final score was determined by multiplying the intensity scores with staining extent and ranged from 0 to 9. Final scores (intensity score x percentage score) less than or equal 4 were considered as low staining, more than 4 were high staining. Antibodies used in IHC staining can be found in Table 5.

[0232] Lipid droplets staining

[0233] For the staining of lipid droplets in tissues, we subjected fresh cancer / cancer-adjacent tissues from patients or mouse tumor fragments to rapid freezing, followed by embedding in OCT compound and sectioning. The sections were stored at -20°C. Prior to staining, the sections were gradually thawed at 4°C for 20 minutes, then permeabilized with ice-cold acetone for 20 minutes. Afterward, sections were blocked with 3% BSA for 30 minutes. Subsequently, BODIPY™ 493 / 503 NHS ester (succinimidyl ester) dye was added at a final concentration of 5 pM for the staining of adiposomes. The sections were incubated at room temperature for 600073605-001157

[0234] minutes, washed three times with PBS, and counterstained with DAPI. For quantitation, The average LD numbers per cell from 5 random fields (20x objective) were counted.

[0235] For lipid droplet staining in cells, 3xl05cells were seeded onto a 3.5 cm glass bottom cell culture dish (50% confluency). The glass bottom was coated with 0.2% gelatin. Cells were stained with 1.5 ul LipidSpot™-488 dye diluted in 1 ml cell culture medium in a 37°C CO2 incubator for 30 minutes. After staining, the cells were washed with IxPBS and fixed with 4% paraformaldehyde for 10 minutes, washed with IxPBS twice and counterstained with DAPI. The lipid droplets were imaged with Leica SP8 laser scanning confocal microscope equipped with an 63x oil immersion objective.

[0236] Thin layer chromatography (TLC)

[0237] 5xl06cells in a 10 cm dish were washed with IxPBS twice and harvested with a cell scrapper. The cell pellets were wash with IxPBS twice and the lipids were extracted with 750 ul m ethanol :choloroform (4:1, v / v). After centrifugation at 13,300 rpm for 5 minutes at room temperature, the supernatant was mixed with 600 ul 0.1% aqueous acetic acid and 300 uL chloroform and vortexed for 10 seconds. The mixture was centrifuged at 13,300 rpm for 5 minutes and the lower organic phase was transferred to a glass vassel and dried under nitrogen. The dried lipids were reconsituted in choloroform and loaded onto Silica gel 60 TLC plate (1.11845.0001, Merck). The TLC plates were dervloped using hexane / di ethyl ether / acetic acid 60:40: 1 (v / v / v). The lipids were visualized using iodine vapor and imaged with a scanner.

[0238] Oleate-Alkyne tracing

[0239] 2xl06PDAC cells in a 6 cm dish were incubated with media containing 25 uM Oleic acid-Alkyne and 1% fatty acid free BSA for 30 minutes. To determien the effects of lomitapide, cells were pre-treated with lomitapide at various concentrations for 2 hours prior to Oleic acid-alkyne labeling. After labeling, lipids were extracted and reacted with 3-azido-7-hyoxyconmarin. The Oleic acid-alkyne labeled lipids were spoted onto a TLC plate (Merk, 1118450001) and developed with hexane / di ethyl ether / acetic acid 60:40:1 (v / v / v).

[0240] DGAT activity assay

[0241] DGAT activity assay was performed using 1,2 dioleoyl-sn-glycerol (DOG, D0138, Sigma)and NBD-palmitoyl CoA (810705P, Avanti Polar Lipids) as substrate and crude miccrosomal membrane isolated from mouse liver following a previously reported protocol (8).

[0242] 5 ug liver membrane (in 50 ul buffer containing 50 mM Tris-HCl,pH 7.6, 250 mM sucrose) was0073605-001157

[0243] incubated with 150 ul reaction master mix (20 ul IM Tris-HCL, pH 7.6, 1 ul IM MgCL, 10 ul 4mM DOG, 10 ul 12.5 mg / ml FFA-free BSA, 10 ul 500 uM NBD-palmitoyl CoA and 99 ul dH2O) with or without lomitapide, mixed by vortexing and incubated at 37°C for 10 minutes with shaking. The reaction was terminated by the addition of 750 ul methano / chloroform 4:1 v / v followed by vortexing. After 17,000 g centrifugation at room temperature for 5 minutes, the 400 ul lower organic phase was transferred to a glass tube, dried with SpeedVac. After reconstituion in chloroform, the NBD-labeled lipids were resolved through TLC.

[0244] Metabolomics and lipidomics screening

[0245] Metabolomics screening was performed through LC-MS / MS. The MSEA analysis was performed using Metaboanalyst 5.0. Targeted quantitative lipidomics assay was performed with assistance from Metware Bio (Woburn, MA). 1x107cells were harvested using a cell scrapper. The cell pellets were resuspended in 100 ul dH2O containing proteinase inhibitors. 50 ul cell suspensions were used for lipid extraction while the remainding samples were used for protein assay. The data acquisition instruments consisted of Ultra Performance Liquid Chromatography (UPLC) (NexeraLC-40, Shimadzu) equipped with Thermo Accucore™C30 (2.6 pm, 2.1 mmxlOO mm i.d.) column and tandem mass spectrometry (MS / MS) (Triple Quad 6500+, AB / SCIEX). Liquid phase conditions: Mobile phase: A phase was acetonitrile / water (60 / 40, V / V) (0.1% formic acid added, 10 mmol / Lammonium formate); B phase was acetonitrile / Isopropyl alcohol (10 / 90, V / V) (0.1% formic acid added, 10 mmol / L ammonium formate); Gradient program: 80:20(V / V) at 0 min, 70:30(V / V) at 2 min, 40:60(V / V) at 4 min , 15:85(V / V) at 9 min, 10:90(V / V) at 14 min, 5:95(V / V) at 15.5 min, 5:95(V / V) at 17.3 min, 80:20(V / V) at 17.5 min, 80:20(V / V) at 20 min; Flow rate: 0.35 ml / min; Column temperature: 45°C; Injection volume: 2 pL. Mass spectrometry conditions:

[0246] LIT and triple quadrupole (QQQ) scans were acquired on a triple quadrupole-linear ion trap mass spectrometer (QTRAP), QTRAP® 6500+ LC-MS / MS System, equipped with an ESI Turbo Ion-Spray interface, operating in positive and negative ion mode and controlled by Analyst 1.6.3 software (Sciex). The ESI source operation parameters were as following: ion source, turbo spray; source temperature 500 °C; ion spray voltage (IS) 5500 V(Positive),-4500 V(Neagtive); Ion source gas 1 (GS1), gas 2 (GS2), curtain gas (CUR) were set at 45, 55, and 35 psi, respectively. Instrument tuning and mass calibration were performed with 10 and 100 pmol / L polypropylene glycol solutions in QQQ and LIT modes, respectively. QQQ scans were acquired0073605-001157

[0247] as MRM experiments with collision gas (nitrogen) set to 5 psi. DP and CE for individual MRM transitions was done with further DP and CE optimization. A specific set of MRM transitions were monitored for each period according to the lipids eluted within this period.

[0248] 13Cg,15N2-cystine tracing

[0249] Cystine isotopologue tracing was performed following a previously described protocol with modifications. Cystine, methionine and glutamine free DMEM media (Gibco, 21013-024) were supplemented with 400 uM (for control group) or 50 uM (for CLSA-25 group)13Ca,15N-cysteine, 100 uM methionine, 4 mM glutamine and 10% dialyzed FBS and stored at 4°C for 48 hours for the oxidation of cysteine to cystine. 7xl06control or CLSA-25 MiaPaCa-2 cells were pre-conditioned in the same medium containing12C,14N-cystine for 12 hours. After preconditioning, cells were labeled with their respective media containing isotopologue cystine for indicated time (2, 4 or 24 hours). The metabolites were extracted with 2 ml 80% MeOH. After centrifugation, the supernatant was dried under nitrogen flow and analyzed through LC-MS / MS.

[0250] 13Ce -glucose tracing to glycolosis and PPP / nucleotide metabolites

[0251] 13Ce -glucose tracing was performed as we described previously with modifications (9, 11). 7xl06control or CLSA-25 MiaPaCa2 cells were incubated with DMEM medium (Life technology, D98002710) containing 200 uM (for control group) or 25 uM cystine (for CLSA-25 group), 10 mM13Ce -glucose and 10% dialyzed FBS for indciated time (from 0-360 minutes). The supernatant was collected and dried using SpeedVac and re-dissolved in 50 pL of methanol / water (50 / 50) for LC-MS / MS analysis.

[0252] 13Cs,15N2 -glutamine tracing for de novo fatty acid synthesis

[0253] IxlO6PANC-1 cells on a 6 cm dish was washed with PBS and incubated with isotopic labeling media containing 4mM13Cs,15N2 -glutamine containing 200 uM (for control cells) or 25 uM (for CLSA-25 cells) L-Cys2 and dialyzed FBS. After 24-hour incubation, cells were washed with IxPBS twice and scrapped into a 1.5 ml tube on ice. The cells were centrifuged at 500 g at 4°C for 3 minutes and the cell pellets were vortexed in 1 ml 50% methanol with 0.05 M HC1 for 10 seconds. 500 ul chloroform was added to each tube and vortexed vigorously to extract lipids. After 16,000 g centrifugation for 5 minutes, the lower organic phase was transferred into a glass vial. The upper aqueous phase was extracted with chloroform again and the organic phase was combined and dried under nitrogen flow. The dried lipids were reconstituted in 90% methanol solution containing 0.3 M KOH and incubated in a 2 ml glass vial at 80 °C for 1 hour. After0073605-001157

[0254] saponification, the 100 ul formic acid was added to the reaction and the mixture was extracted with 900 ul hexane twice. The hexane extracts were combined and dried under nitrogen flow. After reconstitution in 50 ul methanol / chloroform 4: 1 v / v, the saponified fatty acids were used for mass spectrometry analysis. The LC / MS system consisted of a Dionex Ultimate 3000 XRS pump (Thermo Scientific, San Jose, CA), a Dionex Ultimate 3000 XRS open autosampler (Thermo Scientific, San Jose, CA), a Dionex Ultimate 3000 RS column compartment (Thermo Scientific, San Jose, CA), and an Exactive Plus orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA). Data was collected using Xcalibur 3.0.63 software (Thermo Scientific, San Jose, CA) and analyzed using FreeStyle 1.8.63.0 software (Thermo Scientific, San Jose, CA). A binary gradient of solvent A: 97 / 3 water / methanol with 10 mM tributylamine and 15 mM acetic acid (pH 4.5) and solvent B: 100% methanol was as follows: 80 to 99% B from 0 to 20 min, remaining at 99% B from 20 to 40 min, from 99% B to 80% B to 41 min, and remaining steady at 80% B to 50 min. The separation was performed on a Luna C8(2) reversed-phase column (150 x 2.0mm, 3pm particle size, 100 A pore size, Phenomenex, Torrance, CA). The flow rate was 200 pL / min, the autosampler temperature was 5°C, injection volume 10 pL, and column temperature 25 °C. The mass spectrometer was operated in negative mode. The electrospray settings were set as follows sheath gas flow rate 45 (arbitrary units), auxiliary gas flow rate 10 (arbitrary units), sweep gas flow rate 2 (arbitrary units), spray voltage 2.5kV, capillary temperature 250°C, S-lens RF level 50, and auxiliary gas temperature 400°C. The mass spectrometer resolution was set to 140,000 and the automatic gain control was set to 3e6 with a maximum injection time of 100ms. The scan range was 200-400 m / z from 0-20 min and 300-575 m / z from 20-50 min.

[0255] The term “about” as used herein when referring to a measurable value such as an amount, a percentage, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate.

[0256] All documents, books, manuals, papers, patents, published patent applications, guides, abstracts, and / or other references cited herein are incorporated by reference in their entirety. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended0073605-001157

[0257] that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

[0258] Table 1. Correlations between clinicopathological features and G6PD expression in pancreatic cancer

[0259] G6PD

[0260] Spearman rank Low High p-values r value Gender

[0261] Male 32 25 0.397 0.032 Female 18 25

[0262] Age

[0263] > 60 27 28 0.755 0.086

[0264] <60 23 22

[0265] Tumor size (cm)b

[0266] <3 27 19 0.041 0.205

[0267] >3 23 31

[0268] LN metastasis

[0269] 41 35 <0.001 0.480

[0270] 9 15

[0271] stage

[0272] 37 24 <0.001 0.376

[0273]

[0274] 13 26

[0275] Table 2. Antibodies and their dilutions

[0276]

[0277] 0073605-001157

[0278]

[0279] Table 3. Key Reagents

[0280]

[0281] 0073605-001157

[0282] >

[0283]

[0284] 0073605-001157

[0285]

[0286] 0073605-001157

[0287] Table 4. siRNA sequence

[0288]

[0289] Table 5. qPCR sequence

[0290]

Claims

1. 0073605-001157WHAT IS CLAIMED:

1. A method for treating target cells, comprising administering to the target cells N-(2,2,2-Trifluoroethyl)-9-[4-[4-[[[4'-(trifluoromethyl)[l,l'-biphenyl]2-yl]carbonyl]amino]-l-piperidinyl]butyl]-9H-fluoren-9-carboxamide (lomitapide) or an analog thereof in an amount effective for suppressing synthesis of triacylglycerides (TGs) in the target cells.

2. The method of claim 1, further comprising inhibiting proliferation of the target cells.

3. The method of claim 1, further comprising killing the target cells.

4. The method of any one of claims 1-3, wherein the lomitapide analog is selected from the group consisting of 9-[4-[4-[[2-Chloro-5-(trifluoromethyl)benzoyl]amino]-l-piperidinyl]butyl]-N-(2,2,2-trifluoroethyl)-9H-fluorene-9-carboxamide, 9-[4-[4-[[2,6-Bis(trifluoromethyl)benzoyl]amino]-l-piperidinyl]butyl]-N-(2,2,2-trifluoroethyl)-9H-fluorene-9-carboxamide, monohydrochloride, N-(2,2,2-trifluoroethyl)-9-[4-[4-[[[4-(trifluoromethyl)[l,l-biphenyl]-2-yl]carbonyl]amino]-l-piperidyl]butaneyl]-9H-fluorene-9-carboxamide, 9-[4-[4-[[[4-Chloro-4'-(trifluoromethyl)[l,r-biphenyl]-2-yl]carbonyl]amino]-l-piperidinyl]butyl]-N-(2,2,2-trifluoroethyl)-9H-fluorene-9-carboxamide, 9-[4-[4-[[2-fluoro-5-(trifluoromethyl)benzoyl]amino]-l-piperidinyl]butyl]-N-(2,2,2-trifluoroethyl)-9H-fluorene-9-carboxamide monohydrochloride, N-(2,2,2-Trifluoroethyl)-9-[4-[4-[[[4-(trifluoromethyl)[l,l-biphenyl]-2-yl]carbonyl]amino]-l-piperidinyl]butyl]-9H-fluorene-9-carboxamide, monohydrochloride, 9-[4-[4-[[2,5-bis(trifluoromethyl)benzoyl]amino]-l-piperidinyl]butyl]-N-(2,2,2-trifluoroethyl)-9H-fluorene-9-carboxamide monohydrochloride, and 9-[4-[4-[[[4-chloro-4'-(trifhioromethyl)[l,l-biphenyl]-2-yl]carbonyl]amino]-l-piperidinyl]butyl]-N-(2,2,2-trifluoroethyl)-9H-fluorene-9-carboxamide monohydrochloride.

5. The method of any one of claims 1-4, wherein the target cells are tumor cells of a cancer selected from the group consisting of pancreatic cancer, colorectal cancer, glioblastoma, clear cell renal cell carcinoma, liver cancer, lung cancer, melanoma, prostate cancer, ovarian cancer, and acute myeloid leukemia.

6. The method of any one of claims 1-4, wherein the target cells are selected from the group consisting of hepatocytes, enterocytes, and adipocytes.0073605-0011577. The method of any one of claims 1-6, wherein the target cells are adapted to stress selected from the group consisting of cystine limitation stress (CLS), hypoxia, acidosis, and chemotoxic stress.

8. The method of any one of claims 1-7, wherein the target cells have an intracellular cysteine (Cys) concentration lower than that of control cells.

9. The method of any one of claims 1-7, wherein the target cells have an intracellular cystine (Cys ) concentration lower than that of control cells.

10. The method of any one of claims 1-9, further comprising downregulating iron sulfur (Fe-S) cluster synthesis in the target cells.

11. The method of any one of claims 1-10, further comprising downregulating DGAT1 and / or DGAT2 activity in the target cells.

12. The method of any one of claims 1-11, further comprising switching mitochondrial oxidative phosphorylation to glycolytic metabolism in the target cells.

13. The method of any one of claims 1-12, further comprising upregulating glycolysis, oxidative pentose phosphate pathway (PPP), lipid uptake or de novo lipid synthesis in the target cells.

14. The method of any one of claims 1-13, further comprising inhibiting formation of lipid droplets (LD) in the target cells.

15. The method of any one of claims 1-14, further comprising administering to the target cells a cancer drug, whereby the proliferation of the target cells is inhibited synergistically by the lomitapide or an analog thereof and the cancer drug.

16. The method of claim 15, wherein the cancer drug is selected from the group consisting of nucleotide metabolism drugs, platinum drugs, topoisomerase inhibitors, poly(ADP-ribose) polymerase (PARP) inhibitors, anti-angiogenesis agents, and KRAS targeted therapies.

17. The method of claim 15 or 16, wherein the cancer drug is selected from the group consisting of 5 fluorouracil (5-FU), gemcitabine, cytarabine, carboplatin, oxaliplatin, irinotecan, topotecan, etoposide, teniposide, doxorubicin, olaparib, talazoparib, rucaparib, and niraparib, bevacizumab, paclitaxel, albumin-bound or nab-paclitaxel, 4-[4-(3,8-diazabicyclo[3.2.1]oct-3-yl)-8-fluoro-2-[[(2R,7aS)-2-fluorotetrahydro-lH-pyrrolizin-7a(5H)-yl]methoxy]pyrido[4,3-d]pyrimidin-7-yl]-5-ethynyl-6-fluoro-2-naphthalenol (MRTX-1133), and sotorasib.0073605-00115718. A method for treating target cells in a subject, comprising administering to the subject N-(2,2,2-Trifluoroethyl)-9-[4-[4-[[[4'-(trifluoromethyl)[l,r-biphenyl]2-yl]carbonyl]amino]-l-piperidinyl]butyl]-9H-fluoren-9-carboxamide (lomitapide) or an analog thereof in an amount effective for suppressing synthesis of triacylglycerides (TGs) in the target cells.

19. The method of claim 18, further comprising inhibiting proliferation of the target cells.

20. The method of claim 18, further comprising killing the target cells.

21. The method of any one of claims 18-20, wherein the lomitapide analog is selected from the group consisting of 9-[4-[4-[[2-Chloro-5-(trifluoromethyl)benzoyl]amino]-l-piperidinyl]butyl]-N-(2,2,2-trifluoroethyl)-9H-fluorene-9-carboxamide, 9-[4-[4-[[2,6-Bis(trifluoromethyl)benzoyl]amino]-l-piperidinyl]butyl]-N-(2,2,2-trifluoroethyl)-9H-fluorene-9-carboxamide, monohydrochloride, N-(2,2,2-trifluoroethyl)-9-[4-[4-[[[4-(trifluoromethyl)[l,l-biphenyl]-2-yl]carbonyl]amino]-l-piperidyl]butaneyl]-9H-fluorene-9-carboxamide, 9-[4-[4-[[[4-Chloro-4'-(trifluoromethyl)[l,T-biphenyl]-2-yl]carbonyl]amino]-l-piperidinyl]butyl]-N-(2,2,2-trifluoroethyl)-9H-fluorene-9-carboxamide, 9-[4-[4-[[2-fluoro-5-(trifluoromethyl)benzoyl]amino]-l-piperidinyl]butyl]-N-(2,2,2-trifluoroethyl)-9H-fluorene-9-carboxamide monohydrochloride, N-(2,2,2-Trifluoroethyl)-9-[4-[4-[[[4-(trifluoromethyl)[l,l-biphenyl]-2-yl]carbonyl]amino]-l-piperidinyl]butyl]-9H-fluorene-9-carboxamide, monohydrochloride, 9-[4-[4-[[2,5-bis(trifluoromethyl)benzoyl]amino]-l-piperidinyl]butyl]-N-(2,2,2-trifluoroethyl)-9H-fluorene-9-carboxamide monohydrochloride, 9-[4-[4-[[[4-chloro-4'-(trifluoromethyl)[l,l-biphenyl]-2-yl]carbonyl]amino]-l-piperidinyl]butyl]-N-(2,2,2-trifluoroethyl)-9H-fluorene-9-carboxamide monohydrochloride.

22. The method of any one of claims 18-21, wherein the target cells are tumor cells of a cancer selected from the group consisting of pancreatic cancer, colorectal cancer, glioblastoma, clear cell renal cell carcinoma, liver cancer, lung cancer, melanoma, prostate cancer, ovarian cancer, and acute myeloid leukemia.

23. The method of any one of claims 18-21, wherein the target cells are selected from the group consisting of hepatocytes, enterocytes, and adipocytes.0073605-00115724. The method of any one of claims 18-23, wherein the target cells are adapted to stress selected from the group consisting of cystine limitation stress (CLS), hypoxia, acidosis, and chemotoxic stress.

25. The method of any one of claims 18-24, wherein the target cells have an intracellular cysteine (Cys) concentration lower than that of control cells.

26. The method of any one of claims 18-24, wherein the target cells have an intracellular cystine (Cys ) concentration lower than that of control cells.

27. The method of any one of claims 18-26, further comprising downregulating iron sulfur (Fe-S) cluster synthesis in the target cells.

28. The method of any one of claims 18-27, further comprising downregulating DGAT1 and / or DGAT2 activity in the target cells.

29. The method of any one of claims 18-28, further comprising switching mitochondrial oxidative phosphorylation to glycolytic metabolism in the target cells.

30. The method of any one of claims 18-29, further comprising upregulating glycolysis, oxidative pentose phosphate pathway (PPP), lipid uptake or de novo lipid synthesis in the target cells.

31. The method of any one of claims 18-30, further inhibiting formation of lipid droplets (LD) in the target cells.

32. The method of any one of claims 18-31, further comprising administering to the subject a cancer drug, whereby proliferation of the target cells is inhibited synergistically by the lomitapide or an analog thereof and the cancer drug.

33. The method of claim 32, wherein the cancer drug is selected from the group consisting of nucleotide metabolism drugs, platinum drugs, topoisomerase inhibitors, poly(ADP-ribose) polymerase (PARP) inhibitors, anti-angiogenesis agents, and KRAS targeted therapies.

34. The method of claim 32 or 33, wherein the cancer drug is selected from the group consisting of 5 fluorouracil (5-FU), gemcitabine, cytarabine, carboplatin, oxaliplatin, irinotecan, topotecan, etoposide, teniposide, doxorubicin, olaparib, talazoparib, rucaparib, and niraparib, bevacizumab, paclitaxel, albumin-bound or nab-paclitaxel, 4-[4-(3,8-diazabicyclo[3.2.1]oct-3-yl)-8-fluoro-2-[[(2R,7aS)-2-fluorotetrahydro-lH-pyrrolizin-7a(5H)-yl]methoxy]pyrido[4,3-d]pyrimidin-7-yl]-5-ethynyl-6-fluoro-2-naphthalenol (MRTX-1133), and sotorasib.0073605-00115735. The method of any one of claims 32-34, wherein the lomitapide or an analog thereof and the cancer drug are administered to the subject simultaneously.

36. The method of any one of claims 32-34, wherein the lomitapide or an analog thereof and the cancer drug are administered to the subject sequentially.

37. The method of any one of claims 32-34, wherein the target cells are pancreatic ductal adenocarcinoma (PDAC) cells, and the cancer drug comprises leucovorin calcium, 5-fluorouracil, irinotecan and oxaliplatin, the method comprising administering the lomitapide.

38. The method of claim 37, further comprising administering to the subject the lomitapide at a dosage of 5-60 mg daily.

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