Effects of SP-2509 and OG-L002 on lipophagy using target or off-target molecules in glycolysis-suppressed pancreatic ductal adenocarcinoma cells

Effects of SP-2509 and OG-L002 on lipophagy using target or off-target molecules in glycolysis-suppressed pancreatic ductal adenocarcinoma cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Effects of SP-2509 and OG-L002 on lipophagy using target or off-target molecules in glycolysis-suppressed pancreatic ductal adenocarcinoma cells Shigeki Aoki, Zhiheng Zhang, Haruna Aoki, Keitaro Umezawa, Joshua Kranrod, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3282845/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Apr, 2024 Read the published version in Cell Death Discovery → Version 1 posted 10 You are reading this latest preprint version Abstract Although increased aerobic glycolysis is common in cancers, pancreatic ductal adenocarcinoma (PDAC) cells can survive glycolysis suppression. We aimed to identify potential therapeutic targets in glycolysis-suppressed PDAC cells. By screening anticancer metabolic compounds, we identified SP-2509, a selective lysine-specific demethylase (LSD) 1 inhibitor. SP-2509 lowered the viability of three distinct human PDAC cell lines (PANC-1, PK-1, and KLM-1 cells) under glycolysis suppression. The effects of three other LSD1 inhibitors (OG-L002, iadademstat, and T-3775440) on PDAC cell viability were investigated; OG-L002, but not iadademstat or T-3775440, lowered PDAC cell viability under glycolysis suppression, similar to SP-2509. However, knockdown of LSD1/LSD2 failed to lower the viability of PDAC cells subjected to glycolysis suppression. SP-2509 and OG-L002 lowered PDAC cell viability even when given to cells which already been depleted of LSD-1, subjected to glycolysis suppression. Proteomic analyses implied that glucose-starvation causes PDAC cells to switch to mitochondrial oxidative phosphorylation. We observed that fatty acid metabolism is important for the survival of PDAC cells following the suppression of glycolysis. SP-2509 and OG-L002 promoted lipid droplet accumulation in PDAC cells under glycolysis suppression by inhibiting lipophagy. This indicates the significant potential of SP-2509 and OG-L002 to impair oncogenic cell proliferation through regulation of lipophagic fluxes. SP-2509 showed anti-tumor effects of PDAC in 2-DG-treated mice with lipid droplet accumulation and alteration of the tumor microenvironment. Hence, there is potentially new therapeutic strategies for PDAC in the presence of dual inhibition of glycolysis and fatty acids metabolism. Biological sciences/Cancer/Cancer metabolism Biological sciences/Drug discovery/Target identification Pancreatic ductal adenocarcinoma LSD1 inhibitor Glycolysis-suppressed Fatty acid metabolism Lipid droplet accumulation Introduction Glycolysis is generally less efficient in producing ATP than mitochondrial oxidative phosphorylation (OXPHOS). However, tumors often display high levels of glucose uptake and lactate production via aerobic glycolysis, even with adequate oxygen, a phenomenon known as the Warburg effect [ 1 ]. The molecular mechanisms by which cancer cells upregulate glycolysis are increasingly researched. Notably, the AKT oncogene can enhance glycolytic flux by activating glucose transporters without affecting mitochondrial OXPHOS, thereby maintaining high levels of ATP [ 2 ]. Hypoxia-inducible factor 1α also promotes preferential dependence on glycolysis by inducing the transcription of glycolytic enzymes such as hexokinase (HK) and lactate dehydrogenase A (LDHA) [ 3 ]. The activation of pyruvate dehydrogenase (PDH) kinase-1 (PDK-1) inactivates PDH, preventing the entry of pyruvate into the mitochondrial tricarboxylic acid (TCA) cycle [ 4 ]. In contrast, inhibition of HK2, which catalyzes the rate-limiting and first obligatory step of glucose metabolism, can markedly reduce the proliferation of lung cancer cells [ 5 ]. Therefore, the glycolytic pathway, which includes glucose transporters and glycolytic enzymes, has been targeted for cancer therapy. Some cancer cells survive and even continue aggressive proliferation when glucose is depleted [ 6 ]. For example, breast cancer cells can survive suppression of glycolysis by modulating mitochondrial metabolism, including OXPHOS, by favoring glutamine utilization during glucose starvation [ 6 ]. Cancer cells can compensate for glucose depletion by metabolic pathways, such as fatty acid (FA) oxidation [ 7 ]. For example, breast cancer cells ubiquitously express carnitine palmitoyltransferase (CPT) 1C, which promotes energy production through increased FA oxidation. Consequently, these cells are resistant to glucose starvation [ 8 ]. FA metabolism includes complex molecular processes, including FA uptake and de novo synthesis of FAs. Colorectal and pancreatic cancer cells may favor de novo synthesis rather than uptake FAs from the tumor microenvironment, as they highly express the gene encoding FA synthase [ 9 ]. This suggests that cancer cells can proliferate under conditions of glycolysis suppression, if they can compensate for the use of other adaptive processes such as FA oxidation. This study examines novel therapeutic targets within the metabolic pathways that are common in pancreatic cancer, which may be lethal during its early stages [ 10 ]. Cancer recurrence and metastasis may occur even after successful surgery [ 10 ]. Approximately half of the patients with resected pancreatic ductal adenocarcinomas (PDAC) have a poor prognosis [ 10 ]. Patients with pancreatic cancer may also show resistance to chemotherapy such as gemcitabine [ 10 ]. Therefore, research is required into the molecular vulnerabilities of pancreatic cancer. Pancreatic adenocarcinoma is a major form of pancreatic cancer, with PDAC being the most prevalent [ 11 ]. PDAC arises from three precursors: pancreatic intraepithelial neoplasia, intraductal papillary mucinous neoplasms, and mucinous cystic neoplasms [ 11 ]. In over 90% of patients with PDAC, activating mutations in the KRAS oncogene exist. Therefore, KRAS may be a critical driver of tumorigenesis. G12C inhibitors, such as sotorasib, have shown remarkable efficacy against non-small-cell lung cancer. However, G12C mutation is rare in PDAC and there are no effective pharmacological KRAS inhibitors targeting KRAS G12D/V, which forms the majority of KRAS mutations in PDAC [ 12 ]. The metabolic pathways activated downstream of KRAS, which specifically regulates rate-limiting glycolytic enzymes, are increasingly researched as potential therapeutic targets. However, PDAC cells may be resistant to glycolysis inhibitors, such as the LDHA inhibitor [ 13 ]. Hence, there may be compensatory mechanisms enabling PDAC cell survival despite glycolysis suppression. In previous research, glucose starvation failed to lower the survival rate of PDAC cells that reprogrammed their glucose metabolism to OXPHOS [ 14 ]. Our study has explored the mechanism of how PDAC cells shift metabolism to OXPHOS when glycolysis is suppressed, and we have identified specific therapeutic targets possibly useful for the treatment of PDAC. Results SP-2509 dramatically lowered the viability of glycolytically-suppressed PDAC cells Cells were cultured in either low-glucose or galactose culture mediums to mimic glycolysis suppression [ 14 ]. To determine the therapeutic potential of compounds during these conditions, we screened a library of 130 compounds for agents that lowered the viability of glycolysis-suppressed pancreatic cancer cells. Two compounds, rotenone and SP-2509, were identified as potential candidates that reduced the viability of cells cultured in the presence of both low-glucose levels and galactose (Fig. 1 A, 1 B, and Supplementary Table 1). Our previous research indicated that glycolysis-suppressed PANC-1 cells were highly sensitive to rotenone [ 14 ], which is consistent with our findings here. Therefore, we chose to focus on SP-2509 and confirmed that it lowered the viability of PANC-1 cells under low-glucose conditions in a time-dependent manner (Fig. 1 C), consistent with the compound library screening results. We also confirmed that, under low-glucose conditions, SP-2509 lowered the viability of PK-1 and KLM-1, two other types of PDAC cells (Fig. 1 D and 1 E). Furthermore, SP-2509 markedly decreased ATP levels in low-glucose-treated PDAC cells (Fig. 1 F–H). 2-Deoxy-D-glucose (2-DG), an inhibitor of glycolysis that mimics glucose-deprivation, has been researched as an antitumor agent [ 15 ]. We confirmed that 2-DG significantly decreased lactate release in PDAC cells (Supplementary Fig. 1A–C), which was consistent with our previous reports [ 14 ], and combining 2-DG with oligomycin, a mitochondrial complex V inhibitor, significantly decreased intracellular ATP levels (Supplementary Fig. 1D–F). This suggests that residual 2-DG-resistant PDAC cells undergo a metabolic shift to mitochondrial OXPHOS. Therefore, we tested the effects of simultaneous co-treatments with 2-DG and SP-2509 on pancreatic cancer cells. SP-2509 lowered the viability of all three PDAC cell lines, especially in combination with 2-DG in a time-dependent manner (Fig. 1 I–K). Dual treatment decreased ATP production (Fig. 1 L–N). SP-2509 therefore exhibits anticancer activity, especially in PDAC cells, when glycolysis is suppressed. SP-2509 and OG-L002 exerted anticancer effects apart from their effects on LSD1/LSD2 SP-2509 is a potent and reversible inhibitor of LSD1 [ 16 ], which is a novel antitumor target [ 17 ]. RNA sequencing expression data from GEPIA2 tumor samples, including pancreatic adenocarcinoma, showed that LSD1 was expressed at higher levels in tumors than normal tissues (Supplementary Fig. 2). Therefore, we focused on LSD1 and its homolog LSD2 as targets of SP-2509 [ 18 ]. LSD1 acts as an activator or repressor of gene expression by removing methyl groups from mono- and dimethylated lysine 4 on histone H3 [ 18 ]. Our results showed that SP-2509 increased the protein expression of Tri-Methyl-Histone H3 (Lys4) (H3K4me3), suggesting that LSD1 was inhibited by SP-2509 (Fig. 2 A and Supplementary Fig. 3A). However, LSD1 or LSD2 depletion failed to lower the viability of PANC-1 cells under both glucose and low-glucose conditions (Fig. 2 B–E). Depletion of both LSD1 and LSD2 did not decrease the viability of PANC-1 cells under either high glucose or low-glucose conditions (Supplementary Fig. 4). Next, three selective LSD inhibitors were investigated (iadademstat, T-3775440, and OG-L002) for effects on the cell viability. Iadademstat and T-3775440 did not reduce the viability of PANC-1 cells under low-glucose conditions (Fig. 2 F and 2 G). In contrast, OG-L002 decreased the viability of PANC-1 cells cultured under low-glucose conditions compared with cells cultured under normal glucose conditions (Fig. 2 H). SP-2509 and OG-L002 lowered viability of LSD1 inhibited and glycolysis-suppressed cells (Fig. 2 I), suggesting that these two LSD1 inhibitors may have anticancer action beyond their specific effects on LSD1/LSD2. Quantitative proteomic analysis of PANC-1 cells in low-glucose conditions To clarify the influence of glycolytic suppression on PDAC cellular metabolism, proteomes of PANC-1 cells cultured under glucose and low-glucose conditions were analyzed. Quantitative proteomic analysis identified differentially expressed proteins in low-glucose and glucose cells. In total, 4 615 proteins were detected and quantified (Fig. 3 A). By taking a P -value ≤ 0.05 and fold change ≥ 1.2-fold as the cutoff criteria, 447 differentially expressed proteins were identified, with 199 proteins being upregulated and 248 proteins downregulated under low-glucose conditions (Fig. 3 A). To elucidate the functional roles of these proteins, we performed Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and WikiPathway analyses, with both analyses revealing that during low-glucose conditions, the pathways related to OXPHOS and the mitochondrial electron transport chain (ETC) were upregulated (Fig. 3 B and 3 C). Gene set enrichment analysis (GSEA) of KEGG and WikiPathways confirmed that proteins involved in mitochondrial OXPHOS and ETC pathways were significantly upregulated in PANC-1 cells under low-glucose conditions (Fig. 3 D and 3 E). By taking a P -value ≤ 0.01 and fold change ≥ 1.5-fold as the cutoff criteria, there were seven mitochondrial-related proteins among the 45 proteins with upregulated expression in PANC-1 cells cultured in low-glucose conditions (Fig. 3 F), suggesting that the cells reprogrammed energy metabolism to mitochondrial OXPHOS and ETC under conditions of glycolytic suppression. In general, acetyl-CoA enters the TCA cycle to generate NADH and FADH 2 , which are utilized by mitochondrial OXPHOS and ETC for ATP production [ 19 ]. Possible production of acetyl-CoA was promoted in PDAC cells cultured under low-glucose conditions compared to cells cultured under normal glucose conditions. Additionally, we speculated that SP-2509 and OG-L002 decreased ATP levels by inhibiting the acetyl-CoA production in glycolysis-suppressed PDAC cells (Fig. 3 G). SP-2509 decreased ATP production not directly attributed to acetyl-CoA derived from glycolysis Acetyl-CoA can be produced from glucose, FAs, or acetate via various metabolic pathways [ 20 ]. SP-2509 is regarded as a suppressor of glycolysis via its effects on LSD1 [ 21 ] [ 22 ]. Regarding the role of SP-2509 in the glycolysis of PDAC cells, we found that SP-2509 increased the mRNA levels of glycolytic enzymes (Fig. 4 A-C) and promoted lactate production (Fig. 4 D). Thus, implying that SP-2509 enhances glycolysis in PDAC cells. PDH is a gatekeeper enzyme that connects glycolysis to the TCA cycle; however, its phosphorylation by PDK decreases PDH activity [ 23 ]. PANC-1 cells cultured under low-glucose conditions showed enhanced PDH activity, as shown by the decreased phosphorylation level of PDH compared to cells cultured under normal glucose conditions (Fig. 4 E and Supplementary Fig. 3B). Notably, SP-2509 reversed the changes in PDH activity induced by glycolytic suppression (Fig. 4 E and Supplementary Fig. 3B). Hence, we speculated that SP-2509 limits the flux of pyruvate from the cytoplasm to the mitochondria for usage in the TCA cycle. Moreover, it lowered cell viability by reducing the level of acetyl-CoA derived from glycolysis. Dichloroacetate (DCA), an inhibitor of PDK, was used to activate PDH, thereby increasing the levels of acetyl-CoA derived from glycolysis. DCA markedly increased PDH activity by decreasing its phosphorylation level (Fig. 4 E and Supplementary Fig. 3B). However, DCA failed to reverse the reduction in ATP production following glycolysis-suppression in PANC-1 cells treated with SP-2509 (Fig. 4 F). Therefore, we postulate that SP-2509 activity may involve effects on glycolysis in PDAC cells; however, the effects on levels of acetyl-CoA derived from glycolysis may not explain why SP-2509 lowered ATP production by PDAC cells under low-glucose conditions. SP-2509 and OG-L002 significantly increased accumulation of intracellular lipid droplets (LDs) independent of LSD1 function Mitochondrial FA oxidation is another potentially significant source of acetyl-CoA in cells grown under glucose-deprived conditions [ 24 ]. Hence, we used perhexiline [ 25 ] which reduces the flux of FAs into the mitochondria by inhibiting CPT1 and CPT2. Perhexiline caused a marked decrease in intracellular ATP levels in PANC-1 cells grown under low-glucose conditions (Fig. 5 A) and increased the accumulation of LDs (Fig. 5 B). SP-2509 also induced LDs accumulation in three PDAC cell lines, especially in cells cultured under conditions of glycolytic suppression (Fig. 5 C and Supplementary Fig. 5). Other LSD1 inhibitors were investigated, with results indicating that OG-L002, but not iadademstat or T-3775440, induced the accumulation of LDs, whereas depletion of LSD1 failed to increase the intensity of LDs in PANC-1 cells cultured under low-glucose conditions (Fig. 5 D and Supplementary Fig. 3C). There were no significant differences in the gene expression of CPT1, CPT2, and acyl-CoA dehydrogenases between DMSO and SP-2509 treated PANC-1 cells (Supplementary Fig. 6), implying that SP-2509 did not target mitochondrial FA uptake and oxidation enzymes. SP-2509 and OG-L002 specifically inhibited lipophagy under conditions of glycolysis suppression Our previous research indicated that suppression of glycolysis activated autophagy, to maintain mitochondrial function and the survival of cancer cells [ 14 ]. Therefore, we explored whether SP-2509 regulates LDs accumulation through the autophagy pathway. Therefore, we treated glycolytic-suppressed PANC-1 cells with chloroquine (CQ), an autophagic flux inhibitor [ 26 ], and observed significant accumulation of LDs (Fig. 6 A). In general, FAs are stored in LDs upon reduction of nutrient levels in the tumor microenvironment, wherein they are hydrolyzed by autophagy, in a process known as lipophagy [ 27 ]. To assess the lipophagy process, we next performed dual staining using Lysotracker and BODIPY. There were many co-localizations of lysosomes (LysoTracker-red) and LDs (BODIPY-green) in Perherxilin cultured cells with an inhibitory effect on mitochondrial FAs uptake (Fig. 6 B). In contrast, there were a few co-localizations of lysosomes and LDs in SP-20509 or OG-L002 cultured cells (Fig. 6 B); thus, suggesting SP-2509 or OG-L002 inhibited the process before FAs uptake and LD fusion with lysosomes. In our previous research, glucose starvation increased autophagic flux in PANC-1 cells [ 14 ]. Here, we showed that SP-2509 nor OG-L002 did not stop autophagic flux; thereby, suggesting that SP-2509 nor OG-L002 did not influence bulk autophagy. (Fig. 6 C and Supplementary Fig. 3D). Collectively, these results suggest that SP-2509 and OG-L002 cause the disorder of LDs-related autophagosome fusion with lysosomes, an important step of lipophagy. SP-2509 showed anti-tumor effects in mice Next, we investigated the in vivo antitumor effects of 2-DG and SP-2509 co-treatment in xenografts from CB.17SCID mice implanted with PANC-1 cells. Simultaneous addition of both SP-2509 and 2-DG significantly reduced tumor size in mice compared to the other groups (Fig. 7 A), consistent with our in vitro results. CB.17SCID mice are severely immunodeficient and PANC-1 cells were subcutaneously implanted into the mice. Therefore, we employed an in vivo model of pancreatic cancer by implanting KPC-derived PDAC cells into the pancreas of C57/BL6 mice. SP-2509 lowered the viability and induced the accumulation of LDs in glycolysis-suppressed KPC cells (Supplementary Fig. 7). In mice, combination treatment generally improved median survival outcomes, despite relative lack of statistical significance (Fig. 7 B). After one week of dual treatment, we observed a significant increase in the accumulation of intra-tumoral LDs (Fig. 7 C). Interestingly, we found a decrease in the expression of alpha smooth muscle actin (a-SMA) (Fig. 7 D), and increased infiltration of CD4 + (Fig. 7 E) and CD8 + T cells (Fig. 7 F) into the center of the tumor. This indicated that dual treatment also showed improvement of tumor microenvironment. Taken together, these results indicate anti-tumor efficacy of 2-DG and SP-2509 combination treatment to PDAC tumors in mice. Discussion Previous studies have highlighted that LSD1 and LSD2 are implicated in tumor progression within various types of cancer and are potential targets for drug discovery research [ 28 – 31 ]. SP-2509 may slow tumor growth by inhibiting LSD1 [ 21 , 32 ]. The potential of SP-2509 as a therapeutic agent has been explored in phase I clinical testing of patients with Ewing sarcoma [ 33 ]. Here, we showed that SP-2509 has an antitumor effect on glycolysis-suppressed PDAC cells, whereas depletion of LSD1 and LSD2 did not have this effect, suggesting that SP-2509 targets other molecules in addition to its known effects on LSD1/LSD2. In support of our findings, SP-2509 decreased the viability of acute myeloid leukemia cells bearing an LSD1 knockout [ 34 ]. Moreover, both SP-2509 and OG-L002 showed potential anticancer effects in PDAC cells under low-glucose conditions, whereas other LSD1 inhibitors such as iadademstat and T-3775440 did not. Although the full extent of the potential target molecules of these inhibitors remains unclear, SP-2509 and OG-L002 are promising potential agents for cancer therapy and warrant further investigation. Abnormal glucose metabolism resulting from hyperglycemia or diabetes exacerbates pancreatic cancer [ 35 ]. Although antiglycolytic compounds are researched as therapies for pancreatic cancer cells, monotherapy with antiglycolytic compounds, such as 2-DG, failed to show antitumor effects in mouse experiments and patients [ 36 ]. In our previous research, we found that PANC-1 cells shifted their metabolism to mitochondrial OXPHOS when cultured under conditions of glycolysis suppression [ 14 ]. Here, inhibition of FA metabolism lowered ATP levels in PDAC cells cultured under conditions of glycolysis suppression, suggesting that FA metabolism played an essential role in the survival of PDAC cells during glucose starvation. FA metabolism can sustain rapid cell proliferation and provides an essential energy source for cancer cells during metabolic stress [ 37 ], such as when there is reduced availability of serum-derived lipids in the tumor microenvironment [ 38 ]. Excessive FA uptake and oxidation are associated with the development of liver cancer [ 39 ]. Upregulated FA synthesis, such as cholesterol synthesis, occurs in patients with glioblastoma and breast cancer [ 40 ]. Taken together, our results can suggest that FA metabolism plays an essential role in helping PDAC cells survive under low-glucose conditions. Released FAs are subsequently transported into the mitochondria and undergo FA oxidation, producing ATP that supports tumor survival [ 41 ]. Here, we showed that SP-2509 induced no change in the expression of enzymes related to FA uptake and oxidation. Nevertheless, SP-2509 and OG-L002 significantly induced the accumulation of LDs in PDAC cells under low-glucose conditions, implying that SP-2509 and OG-L002 are more likely to target molecules related to FA storage. Lipid overload tends to provoke autophagy within cancer cells [ 40 ]. For example, cancer cells can employ LDs to modulate autophagy by providing lipid precursors for the formation of autophagic membranes [ 40 ]. In a previous study, we showed that autophagy is activated in PANC-1 cells in which glycolysis is suppressed [ 14 ]. Here, we showed that SP-2509 or OG-L002 inhibited the process of lipophagy, but did not significantly affect bulk autophagy. The involvement of lipophagy in lipid turnover is crucial for tumorigenesis and metastasis [ 42 ]. Poly-ubiquitination and LC3 may be important markers for recognition of LDs for lipophagy [ 42 ]. Our results suggest that SP-2509 and OG-L002 induced the accumulation of LDs by inhibiting lipophagy in PDAC cells under low-glucose conditions. Further research is needed to investigate how lipophagy is targeted by SP-2509 or OG-L002. In conclusion, we propose a strategy to limit the proliferation of PDAC cells under glycolytic suppression. Specifically, FA metabolism is important for the survival of PDAC cells under glycolysis-suppressed conditions. FAs metabolism play an important role when intracellular energy metabolism is reprogrammed to OXPHOS under glycolysis suppression. SP-2509 and OG-L002, LSD1 inhibitors, disturbed intracellular FAs metabolism by inhibiting lipophagy, and showed antitumor effects in PDAC, which is independent of LSD1/LSD2. We further propose that determining the cancer specific regulatory mechanism of lipophagy will expand the options for therapeutic strategies for PDAC. Materials and Methods Reagents 2-DG (FUJIFILM WAKO, Tokyo, Japan), CQ (Sigma-Aldrich, St. Louis, MO, USA), and iadademstat (ORY-1001) 2HCL (Selleckchem, Houston, TX, USA) were dissolved in milliQ water. SP-2509 (Merck, Darmstadt, Germany), OG-L002 (Selleckchem), T-3775440 HCL (Selleckchem), DCA (Tokyo Kasei Corp., Tokyo, Japan), perhexiline maleate salt (Cayman Chemical Company, Ann Arbor, MI, USA), and oligomycin (Sigma-Aldrich) were dissolved in dimethyl sulfoxide (DMSO; Nacalai Tesque, Kyoto, Japan). Cell culture PANC-1, PK-1, and KLM-1 cells were purchased from the RIKEN Cell Engineering Division (RIKEN BioResource Research Center, Tsukuba, Japan). Cells were maintained in RPMI-1640 (Nacalai Tesque, Inc., Kyoto, Japan) supplemented with 10% fetal bovine serum (Life Technologies, Grand Island, NY, USA) and antibiotics (Nacalai Tesque Inc., Kyoto, Japan). KPC cell line (C57/BL6 genetic background) was purchased from CancerTools (London, UK). Cells were maintained in DMEM (Nacalai Tesque) supplemented with 10% fetal bovine serum (Life Technologies, Carlsbad, CA, USA) and antibiotics (Nacalai Tesque). Cells were cultured at 37°C in a humidified chamber with 5% CO 2 . PANC-1 cells were cultured in medium supplemented with glucose (2 g/L), galactose (2 g/L), or low levels of glucose (0.2 g/L). PK-1 and KLM-1 cells were cultured in medium supplemented with glucose (2 g/L) or low levels of glucose (0.2 g/L). KPC cells were only cultured in 4.5 g/L glucose. Metabolic compound screening An Anti-Cancer Metabolism Compound library (96-well, catalog number L2130) was purchased from TargetMol (Wellesley Hills, MA, USA). PANC-1 cells were cultured in glucose (2 g/L), galactose (2 g/L), or low-glucose (0.2 g/L) medium with 130 different types of metabolic compounds for 48 h. To assess cell viability, the cells were then incubated with 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Dojindo Laboratory, Kumamoto, Japan) for 2 h. After incubation, the formazan crystals produced were dissolved in 200 µL DMSO. The absorbance of the resulting solution was measured at 540 nm to determine the amount of MTT-formazan formed. Measurement of intracellular ATP concentration Intracellular concentration of ATP was assessed using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI, USA) according to the manufacturer's protocol. Western blotting Cells were lysed on ice in lysis buffer [PBS (pH 7.4) containing 1% Triton X-100 (Nacalai Tesque) and a protease inhibitor cocktail (Roche, Mannheim, Germany)]. Equal amounts of protein from each sample were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore, Berlin, Germany). The membranes were blocked with 5% bovine albumin (Nacalai Tesque) or 5% nonfat dry milk (Cell Signaling Technology, Beverly, MA, USA) at 25 ℃ for 1 h and probed overnight with primary antibodies (Cell Signaling Technology) specific for H3K4me3, LC3B (D11), LSD1 (C69G12), phospho-PDH α1 (Ser293), PDH, or β-actin. Immunolabeled proteins were detected using horseradish peroxidase-conjugated secondary antibodies (GE Healthcare, Buckinghamshire, UK) and ECL Prime detection reagents (GE Healthcare). The signals were visualized using an ImageQuant LAS 4000 (Version 1.3) system (GE Healthcare). Transfection of siRNA siRNA oligonucleotides (Japan Bio Services Co. Ltd., Saitama, Japan) were transfected at final concentrations of 100 nM using Lipofectamine 2000 (Invitrogen, Waltham, MA, USA). Details of oligonucleotide sequences are presented in Supplementary Table 2. RNA isolation and quantitative real-time PCR Total RNA was isolated from PANC-1 cells using Sepasol-RNA I reagent (Nacalai Tesque) and reverse-transcribed using ReverTra Ace qPCR RT Master Mix (TOYOBO, Osaka, Japan). The resulting cDNA was mixed with THUNDERBIRD quantitative real-time PCR mix (TOYOBO). The mixture was then subjected to quantitative real-time PCR. Primers used in this study are listed in Supplementary Table 3. The cycling conditions were as follows: 95°C for 60 s, followed by 40 cycles (50 cycles for long-chain acyl-CoA dehydrogenase) of 95°C for 10 s and 60°C for 60 s. Relative expression of mRNA was calculated after normalization against the levels of 18S ribosomal RNA or β-actin. Measurement of cellular lactate Cellular lactate levels were measured using a Lactate Assay Kit-WST (Dojindo Laboratory). Proteomic analyses based on nano liquid chromatography-tandem mass spectrometry (NanoLC-MS/MS) and bioinformatics analysis PANC-1 cells were cultured in glucose or low-glucose medium for 6 days. Cells were lysed with ice-cold lysis buffer [PBS (pH 7.4) containing 1% Triton X-100, protease inhibitor, and phosphatase inhibitor cocktail (Roche)]. Proteins were precipitated from cell lysates using the ProteoExtract Protein Precipitation Kit (Millipore), according to the manufacturer’s guidelines. Precipitated proteins were dissolved in approximately 10 µL of 50 mM ammonium bicarbonate buffer (pH 8.1) containing 0.1% RapiGest SF (Waters Corporation, Milford, MA, USA). Total protein concentrations were determined using Pierce™ 660 nm protein assay reagent (Thermo Fisher Scientific), including Ionic Detergent Compatibility Reagent (Thermo Fisher Scientific, Waltham, MA, USA). To each 0.5 mL tube, 10 µg of protein was aliquoted and diluted with 25.5 µL of 50 mM ammonium bicarbonate buffer (pH 8.1) containing 0.1% RapiGest SF and 1.5 µL of dithiothreitol (100 mM in distilled water; Nacalai Tesque). The solution was heated at 60°C for 30 min. After cooling to 25°C, 3 µL of iodoacetamide (100 mM in distilled water; FUJIFILM WAKO) was added to the solution (final concentration was 10 mM), and the tubes were incubated at room temperature for 30 min in the dark. Next, the samples were digested with trypsin (mass spectrometry-grade, Promega; protein/enzyme = 20/1, w/w) at 37°C overnight. The solution was quenched with 10% TFA (pH < 3) and incubated at 37°C for 30 min. After centrifugation (13 300 × g , 10 min, 25°C), the solution was desalted using GL-Tip SDB (GL Science, Tokyo, Japan) according to the manufacturer’s instructions. The eluate was dried in vacuo and dissolved in distilled water containing 2% MeCN and 0.1% formic acid. For proteomic analysis, nanoLC-MS/MS analyses were performed on an Ultimate 3000 RSLCnano system (Thermo Fisher Scientific) coupled to a Q Exactive hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific) equipped with a nano-electrospray ionization source. The nanoLC system was equipped with a trap column (Thermo Fisher Scientific) and analytical column (Nikkyo Technos, Tokyo, Japan). Peptide separation was performed using a 90 min gradient between water containing 0.1% formic acid (mobile phase A) and acetonitrile containing 0.1% formic acid (mobile phase B) at a flow rate of 300 nL/min. The elution was set as follows: 0–3 min, 2% B; 3–63 min, 2–40% B; 93–95 min, 40–95% B; 95–105 min, 95% B; 105–107 min, 95–2% B; 107–120 min, 2% B. The mass spectrometer was operated in data-dependent acquisition mode. MS parameters were set as follows: spray voltage, 2.0 kV; capillary temperature, 275°C; S-lens RF level, 50; scan type, full MS; scan range, m/z 350–1500; resolution, 70 000; polarity, positive; automatic gain control target, 3 × 10 6 ; and maximum injection time, 100 ms. MS/MS parameters were set as follows: resolution, 17 500; automatic gain control target, 1 × 10 5 ; maximum injection time, 60 ms; normalized collision energy, 27; dynamic exclusion, 15 s; loop count, 10; isolation window, 1.6 m / z ; and charge exclusion, unassigned, 1, 8, > 8. Measurements were performed in duplicate for each sample and all replicated data were merged and used for quantitative analysis. Protein identification and relative quantitation were performed using the Proteome Discoverer 2.4 SP1 (Thermo Fisher Scientific). Search parameters were as follows: search engine, Sequest HT; protein database, SwissProt ( Homo sapiens ); enzyme name, trypsin (full); dynamic modification, oxidation (methionine, + 15.99 Da); static modification, carbamidomethyl (cysteine, + 57.02 Da); precursor mass tolerance of 10 ppm; and fragment mass tolerance of 0.02 Da. The label-free quantification parameters for the detected peptides were set as follows: precursor quantification, precursor abundance based on area, normalization mode, and total peptide amount. Gene symbols of 199 proteins upregulated under low-glucose conditions were used for pathway enrichment analysis using g:Profiler ( https://biit.cs.ut.ee/gprofiler/gost ). The raw data are presented in Supplementary Table 4. Ranking top eight pathways from KEGG and WikiPathways were displayed in the main figure. The abundance of proteins under glucose or low-glucose conditions was used for analysis with GSEA 4.3.2, using the following settings: number of permutations = 1000, permutation type = gene set, enrichment statistics = weighted, metric for ranking genes = ratios of classes. Immunofluorescence The cells were seeded in 35 mm glass bottom dishes (MATSUNAMI, Japan) and left overnight to attach. After CQ or SP-2509 treatment, cells were incubated with LysoTracker (1 µM) (Thermo Fisher Scientific, Hanover Park, IL, USA) for 1 hour. After that, cells were fixed in 4% paraformaldehyde for 10 min. Fixed cells were stained with 1 µg/mL BODIPY-493-/503 (Thermo Fisher Scientific) and 5 µg/mL Hoechst 33342 for 30 min at 37 ℃. All samples were imaged using a Carl Zeiss LSM780 laser scanning confocal microscope (Prenzlauer, Berlin, Germany). Images of five random fields of vision from each group were captured and analyzed using Image J. Ratio of average areas of LDs/cell number was used to indicate the level of LDs. Immunohistochemical For mouse samples, Immunofluorescence staining of α-SMA, CD4 and CD8 was done as previously described [ 43 ]. Rabbit anti-α-SMA (Abcam Cambridge, U.K.; dilution of 1:200), rabbit anti-CD4 (Abcam; dilution of 1:500), rat anti-CD8 (Abcam; dilution of 1:200), Alexa Fluor 488-conjugated secondary Ab (Abcam; dilution of 1:200), Alexa Fluor 546 conjugated secondary Ab (Thermo Fisher Scientific; dilution of 1:200). All samples were imaged using a Carl Zeiss LSM780 laser scanning confocal microscope (Prenzlauer, Berlin, Germany). Images of five random fields of vision from each group were captured and analyzed by Image J. Animal Treatment Protocols Female CB.17SCID mice (12 pups) and C57BL/6 mice (36 pups) aged 6 weeks were purchased from The Jackson Laboratory (Kanagawa, Japan) and maintained in an experimental animal facility at Chiba University (Chiba, Japan). For CB.17SCID mice, each mouse was subcutaneously administered with 1.5×10 6 PANC-1 cells suspended in 50 µL serum-free RPMI-1640 and 50 µL Cultrex™ Basement Membrane Extract, Type3, PathClear™ (bio-techne, Minneapolis, MN, USA). After 7 days, the tumor volume was over 100 mm 3 and the mice were randomly divided into the following four treatment groups: untreated control, 2-DG alone, SP-2509 alone, and a combination of 2-DG and SP-2509. SP-2509 (0.5 mg/mouse) was administered intraperitoneally to the mice twice per week, and 2-DG (10 mg/mouse) was administered intraperitoneally three times per week. Tumor volumes were calculated using the following standard formula: width 2 ×length×0.52 [ 44 ], and were measured every 2–3 days using Mitutoyo digital calipers (Mitutoyo, Tokyo, Japan). The tumor sizes were controlled so that their volumes did not exceed 2000 mm 3 . For C57BL/6 mice, each mouse had their pancreas orthotopically injected with 5 ×10 5 KPC cells suspended in 50 µL HBSS buffer. After 7 days, the mice were randomly divided into the following four treatment groups: untreated control, 2-DG alone, SP-2509 alone, and a combination of 2-DG and SP-2509. SP-2509 (0.5 mg/mouse) was administered intraperitoneally to the mice twice per week, and 2-DG (10 mg/mouse) was administered intraperitoneally three times per week. Oil Red O staining Oil Red O staining was conducted as previously described [ 45 ]. Samples were imaged using ECLIPSE Ci-L plus Upright Microscope (NIKON, Tokyo, Japan). Statistical analysis All data are presented as mean ± SD of at least three independent experiments, unless indicated otherwise. Statistical analysis was performed using an unpaired Student's t-test, Log-rank (Mantel-Cox) test or two-way ANOVA followed by Tukey’s test. P value < 0.05 was considered significant. All analysis were conducted using GraphPad Prism version 9 (Dotmatics, Boston, MS, USA) Declarations Significance: SP-2509 and OG-L002 potentially affect fatty acid metabolism and show anticancer activity towards glycolysis-suppressed PDAC cells, beyond their effects on LSD1 or LSD2. Data availability Additional data and materials supporting the findings of this study are available upon request from the corresponding authors. Supplementary information is available at https://www.nature.com/cddis/. Acknowledgments The authors would like to thank Editage (https://www.editage.com) for their assistance with English language editing. Conflict of interest The authors declare no potential conflicts of interest. Author contributions Z. Zhang : Investigation, data curation, formal analysis, funding acquisition, writing-original draft, writing-review, and editing. H. Aoki: Investigation, data curation, writing of the original draft. K. Umezawa: Investigation, data curation, and writing the original draft. J. Kranrod: Investigation, Data curation, formal analysis, writing-review, and editing. N. Miyazaki: Investigation. T. Oshima: Investigation. T. Hirao: Investigation. Y. Miura: Data curation. J. Seubert: Writing-review and editing. K. Ito: Writing-review and editing. S. Aoki: Conceptualization, data curation, formal analysis, investigation, visualization, methodology, funding acquisition, writing-original draft, writing-review, and editing. Ethics statement All animal procedures were approved by the Animal Care Committee of Chiba University. Financial support This work was supported by a Grant-in-Aid for Challenging Research (Exploratory) (22K19368, S. Aoki), Grant-in-Aid for Early-Career Scientists (19K16440, S. Aoki), Fund for the Promotion of Joint International Research (Fostering Joint International Research (A)) (19KK0399, S. Aoki), and JST SPRING (JPMJSP2109, Z. 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The combination of the glycolysis inhibitor 2-DG and sorafenib can be effective against sorafenib-tolerant persister cancer cells. Onco Targets Ther 12, 5359–5373 (2019) Mehlem A, Hagberg CE, Muhl L, Eriksson U & Falkevall A. Imaging of neutral lipids by oil red O for analyzing the metabolic status in health and disease. Nat Protoc 8, 1149–1154 (2013) Additional Declarations (Not answered) Supplementary Files Supplement1.png Supplement2.png Supplement3.pptx Supplement4.png Supplement5.png Supplement6.png Supplement7.png SupplementaryTable1.xlsx SupplementaryTable2.xlsx SupplementaryTable3.xlsx Cite Share Download PDF Status: Published Journal Publication published 05 Apr, 2024 Read the published version in Cell Death Discovery → Version 1 posted Unknown event 22 Jan, 2024 Editorial decision: Reject after peer review 20 Oct, 2023 Review # 2 received at journal 18 Oct, 2023 Reviewer # 2 agreed at journal 04 Oct, 2023 Review # 1 received at journal 18 Sep, 2023 Reviewer # 1 agreed at journal 09 Sep, 2023 Reviewers invited by journal 06 Sep, 2023 Submission checks completed at journal 22 Aug, 2023 First submitted to journal 21 Aug, 2023 Editor assigned by journal 21 Aug, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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However, tumors often display high levels of glucose uptake and lactate production via aerobic glycolysis, even with adequate oxygen, a phenomenon known as the Warburg effect [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The molecular mechanisms by which cancer cells upregulate glycolysis are increasingly researched. Notably, the AKT oncogene can enhance glycolytic flux by activating glucose transporters without affecting mitochondrial OXPHOS, thereby maintaining high levels of ATP [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Hypoxia-inducible factor 1α also promotes preferential dependence on glycolysis by inducing the transcription of glycolytic enzymes such as hexokinase (HK) and lactate dehydrogenase A (LDHA) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The activation of pyruvate dehydrogenase (PDH) kinase-1 (PDK-1) inactivates PDH, preventing the entry of pyruvate into the mitochondrial tricarboxylic acid (TCA) cycle [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In contrast, inhibition of HK2, which catalyzes the rate-limiting and first obligatory step of glucose metabolism, can markedly reduce the proliferation of lung cancer cells [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Therefore, the glycolytic pathway, which includes glucose transporters and glycolytic enzymes, has been targeted for cancer therapy.\u003c/p\u003e \u003cp\u003eSome cancer cells survive and even continue aggressive proliferation when glucose is depleted [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. For example, breast cancer cells can survive suppression of glycolysis by modulating mitochondrial metabolism, including OXPHOS, by favoring glutamine utilization during glucose starvation [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Cancer cells can compensate for glucose depletion by metabolic pathways, such as fatty acid (FA) oxidation [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. For example, breast cancer cells ubiquitously express carnitine palmitoyltransferase (CPT) 1C, which promotes energy production through increased FA oxidation. Consequently, these cells are resistant to glucose starvation [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. FA metabolism includes complex molecular processes, including FA uptake and \u003cem\u003ede novo\u003c/em\u003e synthesis of FAs. Colorectal and pancreatic cancer cells may favor \u003cem\u003ede novo\u003c/em\u003e synthesis rather than uptake FAs from the tumor microenvironment, as they highly express the gene encoding FA synthase [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. This suggests that cancer cells can proliferate under conditions of glycolysis suppression, if they can compensate for the use of other adaptive processes such as FA oxidation.\u003c/p\u003e \u003cp\u003eThis study examines novel therapeutic targets within the metabolic pathways that are common in pancreatic cancer, which may be lethal during its early stages [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Cancer recurrence and metastasis may occur even after successful surgery [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Approximately half of the patients with resected pancreatic ductal adenocarcinomas (PDAC) have a poor prognosis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Patients with pancreatic cancer may also show resistance to chemotherapy such as gemcitabine [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Therefore, research is required into the molecular vulnerabilities of pancreatic cancer. Pancreatic adenocarcinoma is a major form of pancreatic cancer, with PDAC being the most prevalent [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. PDAC arises from three precursors: pancreatic intraepithelial neoplasia, intraductal papillary mucinous neoplasms, and mucinous cystic neoplasms [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In over 90% of patients with PDAC, activating mutations in the KRAS oncogene exist. Therefore, KRAS may be a critical driver of tumorigenesis. G12C inhibitors, such as sotorasib, have shown remarkable efficacy against non-small-cell lung cancer. However, G12C mutation is rare in PDAC and there are no effective pharmacological KRAS inhibitors targeting KRAS G12D/V, which forms the majority of KRAS mutations in PDAC [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe metabolic pathways activated downstream of KRAS, which specifically regulates rate-limiting glycolytic enzymes, are increasingly researched as potential therapeutic targets. However, PDAC cells may be resistant to glycolysis inhibitors, such as the LDHA inhibitor [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Hence, there may be compensatory mechanisms enabling PDAC cell survival despite glycolysis suppression. In previous research, glucose starvation failed to lower the survival rate of PDAC cells that reprogrammed their glucose metabolism to OXPHOS [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Our study has explored the mechanism of how PDAC cells shift metabolism to OXPHOS when glycolysis is suppressed, and we have identified specific therapeutic targets possibly useful for the treatment of PDAC.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eSP-2509 dramatically lowered the viability of glycolytically-suppressed PDAC cells\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCells were cultured in either low-glucose or galactose culture mediums to mimic glycolysis suppression [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. To determine the therapeutic potential of compounds during these conditions, we screened a library of 130 compounds for agents that lowered the viability of glycolysis-suppressed pancreatic cancer cells. Two compounds, rotenone and SP-2509, were identified as potential candidates that reduced the viability of cells cultured in the presence of both low-glucose levels and galactose (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, and Supplementary Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOur previous research indicated that glycolysis-suppressed PANC-1 cells were highly sensitive to rotenone [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], which is consistent with our findings here. Therefore, we chose to focus on SP-2509 and confirmed that it lowered the viability of PANC-1 cells under low-glucose conditions in a time-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), consistent with the compound library screening results. We also confirmed that, under low-glucose conditions, SP-2509 lowered the viability of PK-1 and KLM-1, two other types of PDAC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Furthermore, SP-2509 markedly decreased ATP levels in low-glucose-treated PDAC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF\u0026ndash;H).\u003c/p\u003e \u003cp\u003e2-Deoxy-D-glucose (2-DG), an inhibitor of glycolysis that mimics glucose-deprivation, has been researched as an antitumor agent [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. We confirmed that 2-DG significantly decreased lactate release in PDAC cells (Supplementary Fig.\u0026nbsp;1A\u0026ndash;C), which was consistent with our previous reports [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], and combining 2-DG with oligomycin, a mitochondrial complex V inhibitor, significantly decreased intracellular ATP levels (Supplementary Fig.\u0026nbsp;1D\u0026ndash;F). This suggests that residual 2-DG-resistant PDAC cells undergo a metabolic shift to mitochondrial OXPHOS. Therefore, we tested the effects of simultaneous co-treatments with 2-DG and SP-2509 on pancreatic cancer cells. SP-2509 lowered the viability of all three PDAC cell lines, especially in combination with 2-DG in a time-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI\u0026ndash;K). Dual treatment decreased ATP production (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL\u0026ndash;N). SP-2509 therefore exhibits anticancer activity, especially in PDAC cells, when glycolysis is suppressed.\u003c/p\u003e\n\u003ch3\u003eSP-2509 and OG-L002 exerted anticancer effects apart from their effects on LSD1/LSD2\u003c/h3\u003e\n\u003cp\u003eSP-2509 is a potent and reversible inhibitor of LSD1 [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], which is a novel antitumor target [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. RNA sequencing expression data from GEPIA2 tumor samples, including pancreatic adenocarcinoma, showed that LSD1 was expressed at higher levels in tumors than normal tissues (Supplementary Fig.\u0026nbsp;2). Therefore, we focused on LSD1 and its homolog LSD2 as targets of SP-2509 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. LSD1 acts as an activator or repressor of gene expression by removing methyl groups from mono- and dimethylated lysine 4 on histone H3 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Our results showed that SP-2509 increased the protein expression of Tri-Methyl-Histone H3 (Lys4) (H3K4me3), suggesting that LSD1 was inhibited by SP-2509 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and Supplementary Fig.\u0026nbsp;3A). However, LSD1 or LSD2 depletion failed to lower the viability of PANC-1 cells under both glucose and low-glucose conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u0026ndash;E). Depletion of both LSD1 and LSD2 did not decrease the viability of PANC-1 cells under either high glucose or low-glucose conditions (Supplementary Fig.\u0026nbsp;4). Next, three selective LSD inhibitors were investigated (iadademstat, T-3775440, and OG-L002) for effects on the cell viability. Iadademstat and T-3775440 did not reduce the viability of PANC-1 cells under low-glucose conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). In contrast, OG-L002 decreased the viability of PANC-1 cells cultured under low-glucose conditions compared with cells cultured under normal glucose conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). SP-2509 and OG-L002 lowered viability of LSD1 inhibited and glycolysis-suppressed cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI), suggesting that these two LSD1 inhibitors may have anticancer action beyond their specific effects on LSD1/LSD2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eQuantitative proteomic analysis of PANC-1 cells in low-glucose conditions\u003c/h3\u003e\n\u003cp\u003eTo clarify the influence of glycolytic suppression on PDAC cellular metabolism, proteomes of PANC-1 cells cultured under glucose and low-glucose conditions were analyzed. Quantitative proteomic analysis identified differentially expressed proteins in low-glucose and glucose cells. In total, 4 615 proteins were detected and quantified (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). By taking a \u003cem\u003eP\u003c/em\u003e-value\u0026thinsp;\u0026le;\u0026thinsp;0.05 and fold change\u0026thinsp;\u0026ge;\u0026thinsp;1.2-fold as the cutoff criteria, 447 differentially expressed proteins were identified, with 199 proteins being upregulated and 248 proteins downregulated under low-glucose conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). To elucidate the functional roles of these proteins, we performed Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and WikiPathway analyses, with both analyses revealing that during low-glucose conditions, the pathways related to OXPHOS and the mitochondrial electron transport chain (ETC) were upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Gene set enrichment analysis (GSEA) of KEGG and WikiPathways confirmed that proteins involved in mitochondrial OXPHOS and ETC pathways were significantly upregulated in PANC-1 cells under low-glucose conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). By taking a \u003cem\u003eP\u003c/em\u003e-value\u0026thinsp;\u0026le;\u0026thinsp;0.01 and fold change\u0026thinsp;\u0026ge;\u0026thinsp;1.5-fold as the cutoff criteria, there were seven mitochondrial-related proteins among the 45 proteins with upregulated expression in PANC-1 cells cultured in low-glucose conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF), suggesting that the cells reprogrammed energy metabolism to mitochondrial OXPHOS and ETC under conditions of glycolytic suppression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn general, acetyl-CoA enters the TCA cycle to generate NADH and FADH\u003csub\u003e2\u003c/sub\u003e, which are utilized by mitochondrial OXPHOS and ETC for ATP production [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Possible production of acetyl-CoA was promoted in PDAC cells cultured under low-glucose conditions compared to cells cultured under normal glucose conditions. Additionally, we speculated that SP-2509 and OG-L002 decreased ATP levels by inhibiting the acetyl-CoA production in glycolysis-suppressed PDAC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG).\u003c/p\u003e\n\u003ch3\u003eSP-2509 decreased ATP production not directly attributed to acetyl-CoA derived from glycolysis\u003c/h3\u003e\n\u003cp\u003eAcetyl-CoA can be produced from glucose, FAs, or acetate via various metabolic pathways [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. SP-2509 is regarded as a suppressor of glycolysis via its effects on LSD1 [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Regarding the role of SP-2509 in the glycolysis of PDAC cells, we found that SP-2509 increased the mRNA levels of glycolytic enzymes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C) and promoted lactate production (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Thus, implying that SP-2509 enhances glycolysis in PDAC cells. PDH is a gatekeeper enzyme that connects glycolysis to the TCA cycle; however, its phosphorylation by PDK decreases PDH activity [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. PANC-1 cells cultured under low-glucose conditions showed enhanced PDH activity, as shown by the decreased phosphorylation level of PDH compared to cells cultured under normal glucose conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE and Supplementary Fig.\u0026nbsp;3B). Notably, SP-2509 reversed the changes in PDH activity induced by glycolytic suppression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE and Supplementary Fig.\u0026nbsp;3B). Hence, we speculated that SP-2509 limits the flux of pyruvate from the cytoplasm to the mitochondria for usage in the TCA cycle. Moreover, it lowered cell viability by reducing the level of acetyl-CoA derived from glycolysis. Dichloroacetate (DCA), an inhibitor of PDK, was used to activate PDH, thereby increasing the levels of acetyl-CoA derived from glycolysis. DCA markedly increased PDH activity by decreasing its phosphorylation level (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE and Supplementary Fig.\u0026nbsp;3B). However, DCA failed to reverse the reduction in ATP production following glycolysis-suppression in PANC-1 cells treated with SP-2509 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Therefore, we postulate that SP-2509 activity may involve effects on glycolysis in PDAC cells; however, the effects on levels of acetyl-CoA derived from glycolysis may not explain why SP-2509 lowered ATP production by PDAC cells under low-glucose conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eSP-2509 and OG-L002 significantly increased accumulation of intracellular lipid droplets (LDs) independent of LSD1 function\u003c/h3\u003e\n\u003cp\u003eMitochondrial FA oxidation is another potentially significant source of acetyl-CoA in cells grown under glucose-deprived conditions [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Hence, we used perhexiline [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] which reduces the flux of FAs into the mitochondria by inhibiting CPT1 and CPT2. Perhexiline caused a marked decrease in intracellular ATP levels in PANC-1 cells grown under low-glucose conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) and increased the accumulation of LDs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). SP-2509 also induced LDs accumulation in three PDAC cell lines, especially in cells cultured under conditions of glycolytic suppression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and Supplementary Fig.\u0026nbsp;5). Other LSD1 inhibitors were investigated, with results indicating that OG-L002, but not iadademstat or T-3775440, induced the accumulation of LDs, whereas depletion of LSD1 failed to increase the intensity of LDs in PANC-1 cells cultured under low-glucose conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and Supplementary Fig.\u0026nbsp;3C). There were no significant differences in the gene expression of CPT1, CPT2, and acyl-CoA dehydrogenases between DMSO and SP-2509 treated PANC-1 cells (Supplementary Fig.\u0026nbsp;6), implying that SP-2509 did not target mitochondrial FA uptake and oxidation enzymes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eSP-2509 and OG-L002 specifically inhibited lipophagy under conditions of glycolysis suppression\u003c/h3\u003e\n\u003cp\u003eOur previous research indicated that suppression of glycolysis activated autophagy, to maintain mitochondrial function and the survival of cancer cells [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Therefore, we explored whether SP-2509 regulates LDs accumulation through the autophagy pathway. Therefore, we treated glycolytic-suppressed PANC-1 cells with chloroquine (CQ), an autophagic flux inhibitor [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], and observed significant accumulation of LDs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). In general, FAs are stored in LDs upon reduction of nutrient levels in the tumor microenvironment, wherein they are hydrolyzed by autophagy, in a process known as lipophagy [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. To assess the lipophagy process, we next performed dual staining using Lysotracker and BODIPY. There were many co-localizations of lysosomes (LysoTracker-red) and LDs (BODIPY-green) in Perherxilin cultured cells with an inhibitory effect on mitochondrial FAs uptake (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). In contrast, there were a few co-localizations of lysosomes and LDs in SP-20509 or OG-L002 cultured cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB); thus, suggesting SP-2509 or OG-L002 inhibited the process before FAs uptake and LD fusion with lysosomes. In our previous research, glucose starvation increased autophagic flux in PANC-1 cells [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Here, we showed that SP-2509 nor OG-L002 did not stop autophagic flux; thereby, suggesting that SP-2509 nor OG-L002 did not influence bulk autophagy. (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC and Supplementary Fig.\u0026nbsp;3D). Collectively, these results suggest that SP-2509 and OG-L002 cause the disorder of LDs-related autophagosome fusion with lysosomes, an important step of lipophagy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eSP-2509 showed anti-tumor effects in mice\u003c/h3\u003e\n\u003cp\u003eNext, we investigated the \u003cem\u003ein vivo\u003c/em\u003e antitumor effects of 2-DG and SP-2509 co-treatment in xenografts from CB.17SCID mice implanted with PANC-1 cells. Simultaneous addition of both SP-2509 and 2-DG significantly reduced tumor size in mice compared to the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA), consistent with our \u003cem\u003ein vitro\u003c/em\u003e results. CB.17SCID mice are severely immunodeficient and PANC-1 cells were subcutaneously implanted into the mice. Therefore, we employed an \u003cem\u003ein vivo\u003c/em\u003e model of pancreatic cancer by implanting KPC-derived PDAC cells into the pancreas of C57/BL6 mice. SP-2509 lowered the viability and induced the accumulation of LDs in glycolysis-suppressed KPC cells (Supplementary Fig.\u0026nbsp;7). In mice, combination treatment generally improved median survival outcomes, despite relative lack of statistical significance (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). After one week of dual treatment, we observed a significant increase in the accumulation of intra-tumoral LDs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Interestingly, we found a decrease in the expression of alpha smooth muscle actin (a-SMA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD), and increased infiltration of CD4\u003csup\u003e+\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE) and CD8\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF) into the center of the tumor. This indicated that dual treatment also showed improvement of tumor microenvironment. Taken together, these results indicate anti-tumor efficacy of 2-DG and SP-2509 combination treatment to PDAC tumors in mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePrevious studies have highlighted that LSD1 and LSD2 are implicated in tumor progression within various types of cancer and are potential targets for drug discovery research [\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. SP-2509 may slow tumor growth by inhibiting LSD1 [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The potential of SP-2509 as a therapeutic agent has been explored in phase I clinical testing of patients with Ewing sarcoma [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Here, we showed that SP-2509 has an antitumor effect on glycolysis-suppressed PDAC cells, whereas depletion of LSD1 and LSD2 did not have this effect, suggesting that SP-2509 targets other molecules in addition to its known effects on LSD1/LSD2. In support of our findings, SP-2509 decreased the viability of acute myeloid leukemia cells bearing an LSD1 knockout [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Moreover, both SP-2509 and OG-L002 showed potential anticancer effects in PDAC cells under low-glucose conditions, whereas other LSD1 inhibitors such as iadademstat and T-3775440 did not. Although the full extent of the potential target molecules of these inhibitors remains unclear, SP-2509 and OG-L002 are promising potential agents for cancer therapy and warrant further investigation.\u003c/p\u003e \u003cp\u003eAbnormal glucose metabolism resulting from hyperglycemia or diabetes exacerbates pancreatic cancer [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Although antiglycolytic compounds are researched as therapies for pancreatic cancer cells, monotherapy with antiglycolytic compounds, such as 2-DG, failed to show antitumor effects in mouse experiments and patients [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In our previous research, we found that PANC-1 cells shifted their metabolism to mitochondrial OXPHOS when cultured under conditions of glycolysis suppression [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Here, inhibition of FA metabolism lowered ATP levels in PDAC cells cultured under conditions of glycolysis suppression, suggesting that FA metabolism played an essential role in the survival of PDAC cells during glucose starvation. FA metabolism can sustain rapid cell proliferation and provides an essential energy source for cancer cells during metabolic stress [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], such as when there is reduced availability of serum-derived lipids in the tumor microenvironment [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Excessive FA uptake and oxidation are associated with the development of liver cancer [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Upregulated FA synthesis, such as cholesterol synthesis, occurs in patients with glioblastoma and breast cancer [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Taken together, our results can suggest that FA metabolism plays an essential role in helping PDAC cells survive under low-glucose conditions.\u003c/p\u003e \u003cp\u003eReleased FAs are subsequently transported into the mitochondria and undergo FA oxidation, producing ATP that supports tumor survival [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Here, we showed that SP-2509 induced no change in the expression of enzymes related to FA uptake and oxidation. Nevertheless, SP-2509 and OG-L002 significantly induced the accumulation of LDs in PDAC cells under low-glucose conditions, implying that SP-2509 and OG-L002 are more likely to target molecules related to FA storage. Lipid overload tends to provoke autophagy within cancer cells [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. For example, cancer cells can employ LDs to modulate autophagy by providing lipid precursors for the formation of autophagic membranes [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In a previous study, we showed that autophagy is activated in PANC-1 cells in which glycolysis is suppressed [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Here, we showed that SP-2509 or OG-L002 inhibited the process of lipophagy, but did not significantly affect bulk autophagy. The involvement of lipophagy in lipid turnover is crucial for tumorigenesis and metastasis [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Poly-ubiquitination and LC3 may be important markers for recognition of LDs for lipophagy [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Our results suggest that SP-2509 and OG-L002 induced the accumulation of LDs by inhibiting lipophagy in PDAC cells under low-glucose conditions. Further research is needed to investigate how lipophagy is targeted by SP-2509 or OG-L002.\u003c/p\u003e \u003cp\u003eIn conclusion, we propose a strategy to limit the proliferation of PDAC cells under glycolytic suppression. Specifically, FA metabolism is important for the survival of PDAC cells under glycolysis-suppressed conditions. FAs metabolism play an important role when intracellular energy metabolism is reprogrammed to OXPHOS under glycolysis suppression. SP-2509 and OG-L002, LSD1 inhibitors, disturbed intracellular FAs metabolism by inhibiting lipophagy, and showed antitumor effects in PDAC, which is independent of LSD1/LSD2. We further propose that determining the cancer specific regulatory mechanism of lipophagy will expand the options for therapeutic strategies for PDAC.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cb\u003eReagents\u003c/b\u003e \u003c/p\u003e \u003cp\u003e2-DG (FUJIFILM WAKO, Tokyo, Japan), CQ (Sigma-Aldrich, St. Louis, MO, USA), and iadademstat (ORY-1001) 2HCL (Selleckchem, Houston, TX, USA) were dissolved in milliQ water. SP-2509 (Merck, Darmstadt, Germany), OG-L002 (Selleckchem), T-3775440 HCL (Selleckchem), DCA (Tokyo Kasei Corp., Tokyo, Japan), perhexiline maleate salt (Cayman Chemical Company, Ann Arbor, MI, USA), and oligomycin (Sigma-Aldrich) were dissolved in dimethyl sulfoxide (DMSO; Nacalai Tesque, Kyoto, Japan).\u003c/p\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003ePANC-1, PK-1, and KLM-1 cells were purchased from the RIKEN Cell Engineering Division (RIKEN BioResource Research Center, Tsukuba, Japan). Cells were maintained in RPMI-1640 (Nacalai Tesque, Inc., Kyoto, Japan) supplemented with 10% fetal bovine serum (Life Technologies, Grand Island, NY, USA) and antibiotics (Nacalai Tesque Inc., Kyoto, Japan). KPC cell line (C57/BL6 genetic background) was purchased from CancerTools (London, UK). Cells were maintained in DMEM (Nacalai Tesque) supplemented with 10% fetal bovine serum (Life Technologies, Carlsbad, CA, USA) and antibiotics (Nacalai Tesque). Cells were cultured at 37\u0026deg;C in a humidified chamber with 5% CO\u003csub\u003e2\u003c/sub\u003e. PANC-1 cells were cultured in medium supplemented with glucose (2 g/L), galactose (2 g/L), or low levels of glucose (0.2 g/L). PK-1 and KLM-1 cells were cultured in medium supplemented with glucose (2 g/L) or low levels of glucose (0.2 g/L). KPC cells were only cultured in 4.5 g/L glucose.\u003c/p\u003e\n\u003ch3\u003eMetabolic compound screening\u003c/h3\u003e\n\u003cp\u003eAn Anti-Cancer Metabolism Compound library (96-well, catalog number L2130) was purchased from TargetMol (Wellesley Hills, MA, USA). PANC-1 cells were cultured in glucose (2 g/L), galactose (2 g/L), or low-glucose (0.2 g/L) medium with 130 different types of metabolic compounds for 48 h. To assess cell viability, the cells were then incubated with 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Dojindo Laboratory, Kumamoto, Japan) for 2 h. After incubation, the formazan crystals produced were dissolved in 200 \u0026micro;L DMSO. The absorbance of the resulting solution was measured at 540 nm to determine the amount of MTT-formazan formed.\u003c/p\u003e\n\u003ch3\u003eMeasurement of intracellular ATP concentration\u003c/h3\u003e\n\u003cp\u003eIntracellular concentration of ATP was assessed using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI, USA) according to the manufacturer's protocol.\u003c/p\u003e\n\u003ch3\u003eWestern blotting\u003c/h3\u003e\n\u003cp\u003eCells were lysed on ice in lysis buffer [PBS (pH 7.4) containing 1% Triton X-100 (Nacalai Tesque) and a protease inhibitor cocktail (Roche, Mannheim, Germany)]. Equal amounts of protein from each sample were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore, Berlin, Germany). The membranes were blocked with 5% bovine albumin (Nacalai Tesque) or 5% nonfat dry milk (Cell Signaling Technology, Beverly, MA, USA) at 25 ℃ for 1 h and probed overnight with primary antibodies (Cell Signaling Technology) specific for H3K4me3, LC3B (D11), LSD1 (C69G12), phospho-PDH α1 (Ser293), PDH, or β-actin. Immunolabeled proteins were detected using horseradish peroxidase-conjugated secondary antibodies (GE Healthcare, Buckinghamshire, UK) and ECL Prime detection reagents (GE Healthcare). The signals were visualized using an ImageQuant LAS 4000 (Version 1.3) system (GE Healthcare).\u003c/p\u003e\n\u003ch3\u003eTransfection of siRNA\u003c/h3\u003e\n\u003cp\u003esiRNA oligonucleotides (Japan Bio Services Co. Ltd., Saitama, Japan) were transfected at final concentrations of 100 nM using Lipofectamine 2000 (Invitrogen, Waltham, MA, USA). Details of oligonucleotide sequences are presented in Supplementary Table\u0026nbsp;2.\u003c/p\u003e\n\u003ch3\u003eRNA isolation and quantitative real-time PCR\u003c/h3\u003e\n\u003cp\u003eTotal RNA was isolated from PANC-1 cells using Sepasol-RNA I reagent (Nacalai Tesque) and reverse-transcribed using ReverTra Ace qPCR RT Master Mix (TOYOBO, Osaka, Japan). The resulting cDNA was mixed with THUNDERBIRD quantitative real-time PCR mix (TOYOBO). The mixture was then subjected to quantitative real-time PCR. Primers used in this study are listed in Supplementary Table\u0026nbsp;3. The cycling conditions were as follows: 95\u0026deg;C for 60 s, followed by 40 cycles (50 cycles for long-chain acyl-CoA dehydrogenase) of 95\u0026deg;C for 10 s and 60\u0026deg;C for 60 s. Relative expression of mRNA was calculated after normalization against the levels of 18S ribosomal RNA or β-actin.\u003c/p\u003e\n\u003ch3\u003eMeasurement of cellular lactate\u003c/h3\u003e\n\u003cp\u003eCellular lactate levels were measured using a Lactate Assay Kit-WST (Dojindo Laboratory).\u003c/p\u003e\n\u003ch3\u003eProteomic analyses based on nano liquid chromatography-tandem mass spectrometry (NanoLC-MS/MS) and bioinformatics analysis\u003c/h3\u003e\n\u003cp\u003ePANC-1 cells were cultured in glucose or low-glucose medium for 6 days. Cells were lysed with ice-cold lysis buffer [PBS (pH 7.4) containing 1% Triton X-100, protease inhibitor, and phosphatase inhibitor cocktail (Roche)]. Proteins were precipitated from cell lysates using the ProteoExtract Protein Precipitation Kit (Millipore), according to the manufacturer\u0026rsquo;s guidelines. Precipitated proteins were dissolved in approximately 10 \u0026micro;L of 50 mM ammonium bicarbonate buffer (pH 8.1) containing 0.1% RapiGest SF (Waters Corporation, Milford, MA, USA). Total protein concentrations were determined using Pierce\u0026trade; 660 nm protein assay reagent (Thermo Fisher Scientific), including Ionic Detergent Compatibility Reagent (Thermo Fisher Scientific, Waltham, MA, USA). To each 0.5 mL tube, 10 \u0026micro;g of protein was aliquoted and diluted with 25.5 \u0026micro;L of 50 mM ammonium bicarbonate buffer (pH 8.1) containing 0.1% RapiGest SF and 1.5 \u0026micro;L of dithiothreitol (100 mM in distilled water; Nacalai Tesque). The solution was heated at 60\u0026deg;C for 30 min. After cooling to 25\u0026deg;C, 3 \u0026micro;L of iodoacetamide (100 mM in distilled water; FUJIFILM WAKO) was added to the solution (final concentration was 10 mM), and the tubes were incubated at room temperature for 30 min in the dark. Next, the samples were digested with trypsin (mass spectrometry-grade, Promega; protein/enzyme\u0026thinsp;=\u0026thinsp;20/1, w/w) at 37\u0026deg;C overnight. The solution was quenched with 10% TFA (pH\u0026thinsp;\u0026lt;\u0026thinsp;3) and incubated at 37\u0026deg;C for 30 min. After centrifugation (13 300 \u0026times; \u003cem\u003eg\u003c/em\u003e, 10 min, 25\u0026deg;C), the solution was desalted using GL-Tip SDB (GL Science, Tokyo, Japan) according to the manufacturer\u0026rsquo;s instructions. The eluate was dried \u003cem\u003ein vacuo\u003c/em\u003e and dissolved in distilled water containing 2% MeCN and 0.1% formic acid.\u003c/p\u003e \u003cp\u003eFor proteomic analysis, nanoLC-MS/MS analyses were performed on an Ultimate 3000 RSLCnano system (Thermo Fisher Scientific) coupled to a Q Exactive hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific) equipped with a nano-electrospray ionization source. The nanoLC system was equipped with a trap column (Thermo Fisher Scientific) and analytical column (Nikkyo Technos, Tokyo, Japan). Peptide separation was performed using a 90 min gradient between water containing 0.1% formic acid (mobile phase A) and acetonitrile containing 0.1% formic acid (mobile phase B) at a flow rate of 300 nL/min. The elution was set as follows: 0\u0026ndash;3 min, 2% B; 3\u0026ndash;63 min, 2\u0026ndash;40% B; 93\u0026ndash;95 min, 40\u0026ndash;95% B; 95\u0026ndash;105 min, 95% B; 105\u0026ndash;107 min, 95\u0026ndash;2% B; 107\u0026ndash;120 min, 2% B. The mass spectrometer was operated in data-dependent acquisition mode. MS parameters were set as follows: spray voltage, 2.0 kV; capillary temperature, 275\u0026deg;C; S-lens RF level, 50; scan type, full MS; scan range, \u003cem\u003em/z\u003c/em\u003e 350\u0026ndash;1500; resolution, 70 000; polarity, positive; automatic gain control target, 3 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e; and maximum injection time, 100 ms. MS/MS parameters were set as follows: resolution, 17 500; automatic gain control target, 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e; maximum injection time, 60 ms; normalized collision energy, 27; dynamic exclusion, 15 s; loop count, 10; isolation window, 1.6 \u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e; and charge exclusion, unassigned, 1, 8, \u0026gt;\u0026thinsp;8. Measurements were performed in duplicate for each sample and all replicated data were merged and used for quantitative analysis.\u003c/p\u003e \u003cp\u003eProtein identification and relative quantitation were performed using the Proteome Discoverer 2.4 SP1 (Thermo Fisher Scientific). Search parameters were as follows: search engine, Sequest HT; protein database, SwissProt (\u003cem\u003eHomo sapiens\u003c/em\u003e); enzyme name, trypsin (full); dynamic modification, oxidation (methionine, +\u0026thinsp;15.99 Da); static modification, carbamidomethyl (cysteine, +\u0026thinsp;57.02 Da); precursor mass tolerance of 10 ppm; and fragment mass tolerance of 0.02 Da. The label-free quantification parameters for the detected peptides were set as follows: precursor quantification, precursor abundance based on area, normalization mode, and total peptide amount. Gene symbols of 199 proteins upregulated under low-glucose conditions were used for pathway enrichment analysis using g:Profiler (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://biit.cs.ut.ee/gprofiler/gost\u003c/span\u003e\u003cspan address=\"https://biit.cs.ut.ee/gprofiler/gost\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The raw data are presented in Supplementary Table\u0026nbsp;4. Ranking top eight pathways from KEGG and WikiPathways were displayed in the main figure. The abundance of proteins under glucose or low-glucose conditions was used for analysis with GSEA 4.3.2, using the following settings: number of permutations\u0026thinsp;=\u0026thinsp;1000, permutation type\u0026thinsp;=\u0026thinsp;gene set, enrichment statistics\u0026thinsp;=\u0026thinsp;weighted, metric for ranking genes\u0026thinsp;=\u0026thinsp;ratios of classes.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence\u003c/h3\u003e\n\u003cp\u003eThe cells were seeded in 35 mm glass bottom dishes (MATSUNAMI, Japan) and left overnight to attach. After CQ or SP-2509 treatment, cells were incubated with LysoTracker (1 \u0026micro;M) (Thermo Fisher Scientific, Hanover Park, IL, USA) for 1 hour. After that, cells were fixed in 4% paraformaldehyde for 10 min. Fixed cells were stained with 1 \u0026micro;g/mL BODIPY-493-/503 (Thermo Fisher Scientific) and 5 \u0026micro;g/mL Hoechst 33342 for 30 min at 37 ℃. All samples were imaged using a Carl Zeiss LSM780 laser scanning confocal microscope (Prenzlauer, Berlin, Germany). Images of five random fields of vision from each group were captured and analyzed using Image J. Ratio of average areas of LDs/cell number was used to indicate the level of LDs.\u003c/p\u003e\n\u003ch3\u003eImmunohistochemical\u003c/h3\u003e\n\u003cp\u003eFor mouse samples, Immunofluorescence staining of α-SMA, CD4 and CD8 was done as previously described [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Rabbit anti-α-SMA (Abcam Cambridge, U.K.; dilution of 1:200), rabbit anti-CD4 (Abcam; dilution of 1:500), rat anti-CD8 (Abcam; dilution of 1:200), Alexa Fluor 488-conjugated secondary Ab (Abcam; dilution of 1:200), Alexa Fluor 546 conjugated secondary Ab (Thermo Fisher Scientific; dilution of 1:200). All samples were imaged using a Carl Zeiss LSM780 laser scanning confocal microscope (Prenzlauer, Berlin, Germany). Images of five random fields of vision from each group were captured and analyzed by Image J.\u003c/p\u003e\n\u003ch3\u003eAnimal Treatment Protocols\u003c/h3\u003e\n\u003cp\u003eFemale CB.17SCID mice (12 pups) and C57BL/6 mice (36 pups) aged 6 weeks were purchased from The Jackson Laboratory (Kanagawa, Japan) and maintained in an experimental animal facility at Chiba University (Chiba, Japan). For CB.17SCID mice, each mouse was subcutaneously administered with 1.5\u0026times;10\u003csup\u003e6\u003c/sup\u003e PANC-1 cells suspended in 50 \u0026micro;L serum-free RPMI-1640 and 50 \u0026micro;L Cultrex\u0026trade; Basement Membrane Extract, Type3, PathClear\u0026trade; (bio-techne, Minneapolis, MN, USA). After 7 days, the tumor volume was over 100 mm\u003csup\u003e3\u003c/sup\u003e and the mice were randomly divided into the following four treatment groups: untreated control, 2-DG alone, SP-2509 alone, and a combination of 2-DG and SP-2509. SP-2509 (0.5 mg/mouse) was administered intraperitoneally to the mice twice per week, and 2-DG (10 mg/mouse) was administered intraperitoneally three times per week. Tumor volumes were calculated using the following standard formula: width\u003csup\u003e2\u003c/sup\u003e\u0026times;length\u0026times;0.52 [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], and were measured every 2\u0026ndash;3 days using Mitutoyo digital calipers (Mitutoyo, Tokyo, Japan). The tumor sizes were controlled so that their volumes did not exceed 2000 mm\u003csup\u003e3\u003c/sup\u003e. For C57BL/6 mice, each mouse had their pancreas orthotopically injected with 5 \u0026times;10\u003csup\u003e5\u003c/sup\u003e KPC cells suspended in 50 \u0026micro;L HBSS buffer. After 7 days, the mice were randomly divided into the following four treatment groups: untreated control, 2-DG alone, SP-2509 alone, and a combination of 2-DG and SP-2509. SP-2509 (0.5 mg/mouse) was administered intraperitoneally to the mice twice per week, and 2-DG (10 mg/mouse) was administered intraperitoneally three times per week.\u003c/p\u003e\n\u003ch3\u003eOil Red O staining\u003c/h3\u003e\n\u003cp\u003eOil Red O staining was conducted as previously described [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Samples were imaged using ECLIPSE Ci-L plus Upright Microscope (NIKON, Tokyo, Japan).\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of at least three independent experiments, unless indicated otherwise. Statistical analysis was performed using an unpaired Student's t-test, Log-rank (Mantel-Cox) test or two-way ANOVA followed by Tukey\u0026rsquo;s test. \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant. All analysis were conducted using GraphPad Prism version 9 (Dotmatics, Boston, MS, USA)\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSignificance:\u003c/strong\u003e SP-2509 and OG-L002 potentially affect fatty acid metabolism and show anticancer activity towards glycolysis-suppressed PDAC cells, beyond their effects on LSD1 or LSD2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdditional data and materials supporting the findings of this study are available upon request from the corresponding authors. Supplementary information is available at https://www.nature.com/cddis/.\u003cbr\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Editage (https://www.editage.com) for their assistance with English language editing.\u003cbr\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no potential conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eZ.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eZhang\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Investigation, data curation, formal analysis, funding acquisition, writing-original draft, writing-review, and editing. \u003cstrong\u003eH. Aoki:\u003c/strong\u003e Investigation, data curation, writing of the original draft. \u003cstrong\u003eK. Umezawa:\u003c/strong\u003e Investigation, data curation, and writing the original draft. \u003cstrong\u003eJ. Kranrod:\u003c/strong\u003e Investigation, Data curation, formal analysis, writing-review, and editing. \u003cstrong\u003eN. Miyazaki:\u0026nbsp;\u003c/strong\u003eInvestigation. \u003cstrong\u003eT. Oshima:\u003c/strong\u003e Investigation. \u003cstrong\u003eT. Hirao:\u003c/strong\u003e Investigation. \u003cstrong\u003eY. Miura:\u003c/strong\u003e Data curation. \u003cstrong\u003eJ. Seubert:\u0026nbsp;\u003c/strong\u003eWriting-review and editing. \u003cstrong\u003eK. Ito:\u003c/strong\u003e Writing-review and editing. \u003cstrong\u003eS. Aoki:\u003c/strong\u003e Conceptualization, data curation, formal analysis, investigation, visualization, methodology, funding acquisition, writing-original draft, writing-review, and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal procedures were approved by the Animal Care Committee of Chiba University.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFinancial support\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by a Grant-in-Aid for Challenging Research (Exploratory) (22K19368, S. Aoki), Grant-in-Aid for Early-Career Scientists (19K16440, S. Aoki), Fund for the Promotion of Joint International Research (Fostering Joint International Research (A)) (19KK0399, S. Aoki), and JST SPRING (JPMJSP2109, Z. Zhang) from the Japan Society for the Promotion of Science. \u0026nbsp; The authors thank the Yasuda Medical Foundation (S. Aoki), the SGH Foundation (S. Aoki), and the Uehara Memorial Foundation (S. Aoki).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKim JW \u0026amp; Dang CV. Cancer's molecular sweet tooth and the Warburg effect. Cancer Res 66, 8927\u0026ndash;8930 (2006)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElstrom RL, Bauer DE, Buzzai M, Karnauskas R, Harris MH, Plas DR, et al. Akt stimulates aerobic glycolysis in cancer cells. Cancer Res 64, 3892\u0026ndash;3899 (2004)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu CJ, Wang L-Y, Chodosh LA, Keith B \u0026amp; Simon MC. Differential roles of hypoxia-inducible factor 1alpha (HIF-1alpha) and HIF-2alpha in hypoxic gene regulation. Mol Cell Biol 23, 9361\u0026ndash;9374 (2003)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeidemann A, \u0026amp; Johnson RS. Biology of HIF-1alpha. 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Nat Protoc 8, 1149\u0026ndash;1154 (2013)\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-discovery","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddiscovery","sideBox":"Learn more about [Cell Death Discovery](http://www.nature.com/cddiscovery/)","snPcode":"41420","submissionUrl":"https://mts-cddiscovery.nature.com/","title":"Cell Death Discovery","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Pancreatic ductal adenocarcinoma, LSD1 inhibitor, Glycolysis-suppressed, Fatty acid metabolism, Lipid droplet accumulation","lastPublishedDoi":"10.21203/rs.3.rs-3282845/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3282845/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAlthough increased aerobic glycolysis is common in cancers, pancreatic ductal adenocarcinoma (PDAC) cells can survive glycolysis suppression. We aimed to identify potential therapeutic targets in glycolysis-suppressed PDAC cells. By screening anticancer metabolic compounds, we identified SP-2509, a selective lysine-specific demethylase (LSD) 1 inhibitor. SP-2509 lowered the viability of three distinct human PDAC cell lines (PANC-1, PK-1, and KLM-1 cells) under glycolysis suppression. The effects of three other LSD1 inhibitors (OG-L002, iadademstat, and T-3775440) on PDAC cell viability were investigated; OG-L002, but not iadademstat or T-3775440, lowered PDAC cell viability under glycolysis suppression, similar to SP-2509. However, knockdown of LSD1/LSD2 failed to lower the viability of PDAC cells subjected to glycolysis suppression. SP-2509 and OG-L002 lowered PDAC cell viability even when given to cells which already been depleted of LSD-1, subjected to glycolysis suppression. Proteomic analyses implied that glucose-starvation causes PDAC cells to switch to mitochondrial oxidative phosphorylation. We observed that fatty acid metabolism is important for the survival of PDAC cells following the suppression of glycolysis. \u0026nbsp;SP-2509 and OG-L002 promoted lipid droplet accumulation in PDAC cells under glycolysis suppression by inhibiting lipophagy. This indicates the significant potential of SP-2509 and OG-L002 to impair oncogenic cell proliferation through regulation of lipophagic fluxes. SP-2509 showed anti-tumor effects of PDAC in 2-DG-treated mice with lipid droplet accumulation and alteration of the tumor microenvironment. Hence, there is potentially new therapeutic strategies for PDAC in the presence of dual inhibition of glycolysis and fatty acids metabolism.\u003c/p\u003e","manuscriptTitle":"Effects of SP-2509 and OG-L002 on lipophagy using target or off-target molecules in glycolysis-suppressed pancreatic ductal adenocarcinoma cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-09-22 19:35:28","doi":"10.21203/rs.3.rs-3282845/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"transferred","content":"Cell Death Discovery","date":"2024-01-22T13:40:42+00:00","index":"","fulltext":""},{"type":"decision","content":"Reject after peer review","date":"2023-10-20T09:04:35+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2023-10-18T10:08:05+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2023-10-04T19:08:26+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2023-09-18T13:44:56+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2023-09-09T08:10:40+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2023-09-06T07:09:47+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-08-22T09:47:08+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Disease","date":"2023-08-21T14:28:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-08-21T14:28:02+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-discovery","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddiscovery","sideBox":"Learn more about [Cell Death Discovery](http://www.nature.com/cddiscovery/)","snPcode":"41420","submissionUrl":"https://mts-cddiscovery.nature.com/","title":"Cell Death Discovery","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3618e0e4-9641-469b-8b1b-856678980c16","owner":[],"postedDate":"September 22nd, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":24205328,"name":"Biological sciences/Cancer/Cancer metabolism"},{"id":24205329,"name":"Biological sciences/Drug discovery/Target identification"}],"tags":[],"updatedAt":"2024-04-06T07:06:10+00:00","versionOfRecord":{"articleIdentity":"rs-3282845","link":"https://doi.org/10.1038/s41420-024-01933-4","journal":{"identity":"cell-death-discovery","isVorOnly":false,"title":"Cell Death Discovery"},"publishedOn":"2024-04-05 04:00:00","publishedOnDateReadable":"April 5th, 2024"},"versionCreatedAt":"2023-09-22 19:35:28","video":"","vorDoi":"10.1038/s41420-024-01933-4","vorDoiUrl":"https://doi.org/10.1038/s41420-024-01933-4","workflowStages":[]},"version":"v1","identity":"rs-3282845","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3282845","identity":"rs-3282845","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}