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Purpose: Devimistat (CPI-613) is a novel inhibitor of tumoral mitochondrial metabolism. We investigated the effect of devimistat in vitro and in a phase Ib clinical trial in patients with advanced biliary tract cancer (BTC). Patients and Methods: Cell viability assays of devimistat ± gemcitabine and cisplatin (GC) were performed and the effect of devimistat on mitochondrial respiration via oxygen consumption rate (OCR) was evaluated. A phase Ib/II trial was initiated in patients with untreated advanced BTC. In phase Ib, devimistat was infused over 2 hours in combination with GC on days 1 and 8 every 21 days with a primary objective to determine the recommended phase II dose (RP2D). Secondary objectives included safety, overall response rate (ORR), progression-free survival (PFS), and overall survival (OS). Results: In vitro, devimistat with GC had a synergistic effect on two cell lines. Devimistat significantly decreased OCR at higher doses and in arms with divided dosing. In the phase Ib trial, 20 patients received a median of nine cycles (range, 3–19). One DLT was observed, and the RP2D of devimistat was determined to be 2,000 mg/m2 in combination with GC. Most common grade 3 toxicities included neutropenia (n = 11, 55%), anemia (n = 4, 20%), and infection (n = 3, 15%). There were no grade 4 toxicities. After a median follow-up of 15.6 months, ORR was 45% and median PFS was 10 months (95% confidence interval, 7.1–14.9). Median OS is not yet estimable. Conclusion: Devimistat in combination with GC is well tolerated and has an acceptable safety profile in patients with untreated advanced BTC.

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Purpose:Devimistat (CPI-613) is a novel inhibitor of tumoral mitochondrial metabolism. We investigated the effect of devimistat in vitro and in a phase Ib clinical trial in patients with advanced biliary tract cancer (BTC).Patients and Methods:Cell viability assays of devimistat ± gemcitabine and cisplatin (GC) were performed and the effect of devimistat on mitochondrial respiration via oxygen consumption rate (OCR) was evaluated. A phase Ib/II trial was initiated in patients with untreated advanced BTC. In phase Ib, devimistat was infused over 2 hours in combination with GC on days 1 and 8 every 21 days with a primary objective to determine the recommended phase II dose (RP2D). Secondary objectives included safety, overall response rate (ORR), progression-free survival (PFS), and overall survival (OS).Results:In vitro, devimistat with GC had a synergistic effect on two cell lines. Devimistat significantly decreased OCR at higher doses and in arms with divided dosing. In the phase Ib trial, 20 patients received a median of nine cycles (range, 3–19). One DLT was observed, and the RP2D of devimistat was determined to be 2,000 mg/m2 in combination with GC. Most common grade 3 toxicities included neutropenia (n = 11, 55%), anemia (n = 4, 20%), and infection (n = 3, 15%). There were no grade 4 toxicities. After a median follow-up of 15.6 months, ORR was 45% and median PFS was 10 months (95% confidence interval, 7.1–14.9). Median OS is not yet estimable.Conclusion:Devimistat in combination with GC is well tolerated and has an acceptable safety profile in patients with untreated advanced BTC.

Supplementary Figures S1-S5. Arginase does not regulate arginine levels in OVCAs and ECAs (S1); O-ASCs induce reduced viability of cancer cells deprived of arginine (S2); L-arginase depletes arginine within the media (S3); Cancer cells induce glucose uptake and lactate secretion of O-ASCs and vice versa (S4); NO induces resistance of cancer cells to paclitaxel (S5).

Supplementary Figure Legends. Legend for Supplementary Figures S1-S5.

Branched-chain amino acid transaminase 1 (BCAT1) is upregulated selectively in human isocitrate dehydrogenase (IDH) wildtype (WT) but not mutant glioblastoma multiforme (GBM) and promotes IDH(WT) GBM growth. Through a metabolic synthetic lethal screen, we report here that α-ketoglutarate (AKG) kills IDH(WT) GBM cells when BCAT1 protein is lost, which is reversed by re-expression of BCAT1 or supplementation with branched-chain α-ketoacids (BCKAs), downstream metabolic products of BCAT1. In patient-derived IDH(WT) GBM tumors in vitro and in vivo, co-treatment of BCAT1 inhibitor gabapentin and AKG resulted in synthetic lethality. However, AKG failed to evoke a synthetic lethal effect with loss of BCAT2, BCKDHA, or GPT2 in IDH(WT) GBM cells. Mechanistically, loss of BCAT1 increased the NAD(+)/NADH ratio but impaired oxidative phosphorylation, mTORC1 activity, and nucleotide biosynthesis. These metabolic alterations were synergistically augmented by AKG treatment, thereby causing mitochondrial dysfunction and depletion of cellular building blocks, including ATP, nucleotides, and proteins. Partial restoration of ATP, nucleotides, proteins, and mTORC1 activity by BCKA supplementation prevented IDH(WT) GBM cell death conferred by the combination of BCAT1 loss and AKG. These findings define a targetable metabolic vulnerability in the most common subset of GBM that is currently incurable.

The connections between metabolic state and therapy resistance in multiple myeloma (MM) are poorly understood. We previously reported that electron transport chain (ETC) suppression promotes sensitivity to the BCL-2 antagonist venetoclax. Here, we show that ETC suppression promotes resistance to proteasome inhibitors (PIs). Interrogation of ETC-suppressed MM reveals integrated stress response–dependent suppression of protein translation and ubiquitination, leading to PI resistance. ETC and protein translation gene expression signatures from the CoMMpass trial are down-regulated in patients with poor outcome and relapse, corroborating our in vitro findings. ETC-suppressed MM exhibits up-regulation of the cystine-glutamate antiporter SLC7A11 , and analysis of patient single-cell RNA-seq shows that clusters with low ETC gene expression correlate with higher SLC7A11 expression. Furthermore, erastin or venetoclax treatment diminishes mitochondrial stress–induced PI resistance. In sum, our work demonstrates that mitochondrial stress promotes PI resistance and underscores the need for implementing combinatorial regimens in MM cognizant of mitochondrial metabolic state.

The performance of immune checkpoint inhibitors, which benefit only a subset of patients and can cause serious immune-related adverse events, underscores the need for strategies that induce T-cell immunity with minimal toxicity. The gut microbiota has been implicated in the outcomes of patients following cancer immunotherapy, yet manipulating the gut microbiome to achieve systemic antitumour immunity is challenging. Here, we show, in multiple murine tumour models, that inulin — a widely consumed dietary fibre — formulated as a colon-retentive orally administered gel can effectively modulate the gut microbiome in situ, induce systemic memory-T-cell responses, and amplify the antitumour activity of the checkpoint inhibitor anti-programmed-cell-death-protein-1 (anti-PD-1). Orally delivered inulin-gel treatments increased the relative abundances of key commensal microbes and their short-chain-fatty-acid metabolites, and led to enhanced recall responses for interferon-γ+ CD8+ T cells as well as to the establishment of stem-like T-cell factor-1+ PD-1+ CD8+ T cells within the tumour microenvironment. Gels for the in situ modulation of the gut microbiome may be applicable more broadly to treat pathologies associated with a dysregulated gut microbiome., One-sentence editorial summary: An orally administered gel that is retained in the colon modulates the gut microbiome of mice with murine tumours, inducing systemic memory-T-cell responses, and amplifying the antitumour activity of a checkpoint inhibitor.

Introduction Pancreatic ductal adenocarcinoma (PDAC) is predicted to be the second leading cause of cancer death in the US by 2030. Besides the late detection and lack of effective treatments, the greatest treatment hurdle to PDAC is the ineffectiveness of patient-derived tumor models. Precision medicine-based treatment decisions require the generation of consistent and trackable complex organoid models that is not achievable by tissue biopsies. Liquid biopsies such as blood, instead, contain circulating biomarkers released by the tumor, among which especially interested in for this study are circulating tumor cells (CTCs). This work develops microfluidic 3D organoids from CTCs to be used as a precision medicine tool to predict and guide the drug response of early stage I-IIB (PDAC) patients. Experimental Methods CTCs are isolated from PDAC patient blood samples using microfluidic Labyrinth following removal of red blood cells with 6% dextran. Collected cells post-isolation are immunofluorescence (IF) stained with DAPI (nuclei), CD45 (leukocyte), pancytokeratin (type I/II cytokeratin 1-8, 10, 14- 16 and 19), Vimentin, and Epithelial cell adhesion molecule (EpCAM) to identify CTCs and characterize their phenotypes. The mold of the microfluidic platform for CTC organoids is fabricated with the standard SU-8 photolithography. The PDMS-based device is tested with a low seeding number of a pancreatic cancer CTC cell line labeled fluorescently. Decellularization of cancer associated fibroblasts (CAFs) generates the in vitro matrix that supplies amino-acid precursors for PDAC metabolism, promoting CTC organoid formation. Serial images of cell growth are obtained throughout the course using a Ti-20 microscope. After each 7-day culture, organoids are dissociated from the device and IF stained using a similar panel as above (with Ki67 marker instead of CD45 for cell proliferation). Preliminary gemcitabine dosage-performance analysis is performed on-chip. Results CTC count from the tested patient panel (n=16) averages 17.0±8.4/mL of blood. CAFs are successfully decellularized on-chip and the embedded pancreatic CTC cell line growth curve is generated. IF staining of the dissociated cells from the organoids confirms the proliferation of the loaded pseudo-CTCs. Conclusion The work above demonstrates the successful isolation of CTCs from PDAC patient blood samples and an engineered microfluidic platform to expand low abundance of cells into organoids. It has a potential to be tested and employed to expand CTCs from PDAC into organoids for drug screening. Citation Format: Yu Zhang, Scott Smith, Minal Nenwani, Valerie Gunchick, Vaibhav Sahai, Sunitha Nagrath, Deepak Nagrath. A microfluidic platform for developing circulating tumor cells (CTCs) organoids for precision medicine in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB163.

Metabolic fluxes likely control cancer phenotypes and treatment responses but cannot be measured in cancers in human patients. Machine learning has become a popular tool to study cancer biology and can identify complex patterns in the data which may not be decipherable by traditional analyses. We propose an approach to combine 13C-tracer analysis with machine learning to quantify the metabolism of purines in glioblastoma patients. In recent years, multiple studies have been conducted on isotope tracing in cancer patients. While the data for the enrichment of metabolites in human tumors has improved our understanding of cancer metabolism, we lack methods to estimate metabolic fluxes from these enrichment values. A significant challenge in estimating the fluxes in human subjects is that we are limited to enrichment data from a single in-vivo measurement at isotopic non-steady state. We overcome this challenge by combining INST-MFA (Isotopic Non-Stationary-Metabolic Flux Analysis) with machine learning methods. We use the enrichment of circulating metabolites as an input and simulate the time-dependent metabolite enrichment profiles for randomized flux vectors. We use this data to train a convolutional neural network (CNN) model to predict the flux ratios. Apart from being able to predict the fluxes from a single time-point, a machine learning model drastically reduces the time required to fit a traditional INST-MFA model. We demonstrate the real-world validity of the model by predicting flux ratios in mice models and comparing them to results from traditional INST-MFA models. We apply the model to decipher the heterogeneity of the purine pathway in eight patients with brain tumors. Citation Format: Anjali Mittal, Andrew Scott, Abhinav Achreja, Vijay Tarnal, Wajd Al-Holou, Costas Lyssiotis, Daniel Wahl, Deepak Nagrath. A machine learning based method for in-vivo metabolic flux analysis of patient tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1160.

Recurrent loss-of-function deletions are prevalent genomic alterations in tumors; nonetheless, these deletions occasionally create conditional therapeutic vulnerabilities in tumors. Our previous study used The Cancer Genome Atlas High-Grade Serous Ovarian Carcinoma (TCGA-HGSOC) dataset to identify the 19p13.3 locus as the most pervasive deletion in the high-grade serous ovarian carcinoma (HGSOC). Subsequently, the machine learning integrated-precision oncology platform identified that the loss of UQCR11, a gene located within the chromosome 19p13.3 locus, created a therapeutic collateral target, the MTHFD2. Furthermore, pharmacological inhibition of MTHFD2 in the UQCR11-deleted cells led to complete tumor remission in our in vivo mouse model (Nature metabolism, 2022). Encouraged by the promising anti-tumor effect of the MTHFD2 inhibitor, we extended our research to other tumor types. By analyzing the TGCA datasets, we found that UQCR11 deletion was a common occurrence among humans' most prevalent solid tumors, especially in pan breast and lung cancer. Within the pan breast cancer dataset, we found that over 50% of patients with triple-negative breast cancer (TNBC), the most difficult-to-treat breast cancer subtype in women, displayed either heterozygous or homozygous deletions in the UQCR11 gene. Moreover, the TGCA-TNBC dataset analysis revealed that the genomic copy loss of UQCR11 positively correlates with the deceased mRNA expression of the gene, bringing our interest to validate the collateral lethal targets in this breast cancer subtype. We first analyzed The Cancer Cell Line Encyclopedia (CCLE) database and further explored the correlation between the copy number alteration and mRNA expression of UQCR11 in several breast cancer cell lines. Then, we selected seven human TNBC cells with copy number neutral or deletion UQCR11 and validated the copy number and gene expression of UQCR11 and MTHFD2 using qPCR and western blot. We next treated these cells with a selective MTHFD2 inhibitor, DS18561882, and found that UQCR11-deleted cells showed a more favorable response than the UQCR11-intact cells. Finally, to test the therapeutic potential of targeting MTHFD2-null tumors in vivo, we generated isogenic mouse breast cancer cell lines using shRNA-mediated UQCR11 knockdown. Consistent with our previous findings, the MTHFD2 expression level was upregulated upon UQCR11 knockdown, suggesting a compensation function of MTHFD2 in UQCR11-deleted cells. Furthermore, the UQCR11 knocked-down cells showed better responses to MTHFD2 inhibitor treatment than the parental control cells. In summary, our study provides evidence that targeting MTHFD2 holds strong therapeutic potential in over 50% of the TNBC patients with UQCR11 deletion and highlights the broad efficacy of targeting MTHFD2 for individualized therapeutic strategy in different tumor types co-occurring with UQCR11 deletion. Citation Format: Samantha Sharma, Tao Yu, Abhinav Achreja, Xinna Zhang, Deepak Nagrath, Xiongbin Lu. Genomic loss of UQCR11 creates therapeutic vulnerability in triple-negative breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3913.

Chromosomal alterations that occur frequently in cancers confer selective advantages for tumor progression by deleting tumor-suppressing genes or amplifying oncogenic drivers. However, the collateral effect of these, i.e., deletion of essential genes or upregulation of metabolic regulators, exposes cancers to biological pressures by suppressing essential metabolic pathways. Cancer cells rely on paralogous metabolic pathways to compensate for this loss of function, which in turn opens the door to exploit metabolic vulnerabilities arising as a direct consequence of chromosomal alterations. These pathways are collateral lethal (CL) targets and offer a unique precision medicine approach for metabolic therapeutics. We have developed an integrated machine learning and systems biology platform, collateral lethal gene identification via metabolic fluxes (CLIM) to identify metabolic targets across multiple cancers. Results: In ovarian cancers with 19p13.3 deletion, a loss of Complex III subunit-encoding gene UQCR11 occurs, leading to suppressed electron transport chain (ETC) activity. CLIM predicted that MTHFD2 acts noncanonically to oxidize NADH to NAD+ to compensate for reduced NAD+ recycling via the ETC. We demonstrate selective death of ETC deficient ovarian cancers by targeting MTHFD2 and provide mechanistic validation of oxidative MTHFD2 flux. We observed significant shrinkage of ovarian tumors with ETC deficiency upon MTHFD2 knockdown in mouse models. In triple negative breast cancers (TNBC), we discovered a chromosomal amplification leading to reprogramming of the serine biosynthesis pathway. CLIM predicts a target in the tryptophan metabolism. Methods: Patients are stratified using machine learning to identify tumors with distinct chromosomal alterations. Multiobjective metabolic flux analysis is employed to predict CL pathways in curated genome-scale metabolic models. Validation of metabolic rewiring predicted by CLIM were examined using 3-2H-glucose, 4-2H-glucose and 2,3,3-2H-serine to probe the one carbon metabolism. Oxidative MTHFD2 flux was quantified using innovative combinatorial tracer study with 2H-formate and 4-2H-glucose. We utilized gain- and loss-of-function assays in multiple cell-lines to show utility of the predicted CL targets in TNBCs with chromosomal amplifications. Conclusion: Our integrated platform successfully identifies CL gene pairs to decipher novel metabolic targets specific to chromosomal alterations across many cancer types. Importantly, targets identified by CLIM are based on quantitative flux analysis. This approach reveals functional connection between CL pairs, thereby providing a mechanistic understanding of emerging lethality. This is critical in developing therapeutic interventions that selectively target cancer cells, with minimal off-target or side effects. Citation Format: Abhinav Achreja, Jin Heon Jeon, Mark Slayton, Tao Yu, Olamide Animasahun, Minal Nenwani, Fulei Wuchu, Anjali Mittal, Xiongbin Lu, Sofia D. Merajver, Deepak Nagrath. Identification of collateral lethal targets in cancers using integrated machine learning and flux analysis platform for personalized metabolic therapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6037.

One of the most challenging aspects of PDAC is intense therapeutic resistance towards radiotherapy. Cellular signaling pathways are tightly regulated to protect and withstand constant DNA-damaging insults. Similarly, pancreatic cancer cells are known to activate important metabolic events in the advent of cellular stress. Fuel sources obtained via the high metabolic flexibility of PDAC cells confer on them enhanced survival and the ability to escape therapy-induced cell death. To this end, we aim to dissect the role of altered metabolic reprogramming in radioresistant PDAC cells. Investigation of systemic metabolic changes post-radiation of 8Gy in in vitro systems revealed reduced total NAD/NADH ratio and hinted at the increased dependency of PDAC cells on the one-carbon metabolic pathway. Using combinatorial stable-isotope tracing techniques, we confirmed the role of methylene tetrahydrofolate dehydrogenase (MTHFD1/2) enzymes as key regulators in maintaining total NAD/NADH ratio in irradiated cells. Moreover, inhibition of the one-carbon metabolic pathway hindered the ability of PDAC cells to resolve radiation-induced DNA damage. In summary, we propose that PDAC cells modulate the flux of MTHFD1/2 enzymes to reprogram their metabolic state and generate intermediates for DNA repair and cell survival, thus establishing MTHFD1/2 enzymes as potential therapeutic targets to tackle radioresistance. Citation Format: Minal Nenwani, Abhinav Achreja, Olamide Animasahun, Itisam Sarangi, Jyotirmoy Roy, Srinadh Choppara, Fulei Wuchu, Miya Paserba, Anjali Mittal, Sergio Quispe, Aradhana Mohan, Meredith Morgan, Theodore S. Lawrence, Deepak Nagrath. Targeting one-carbon metabolism radiosensitizes pancreatic ductal adenocarcinoma cells (PDAC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1092.

Cholangiocarcinoma is the second most common primary liver cancer with extremely low survival rates. However, a combination of gemcitabine and cisplatin is the only treatment for advanced unresectable cholangiocarcinoma currently, and the cancer can become resistant to the treatment, contributing to the high mortality. CPI-613 is a lipoic acid analog, which can inhibit dehydrogenases and disrupt mitochondrial metabolism. It is regarded as a promising anti-cancer drug and has been tested in multiple clinical trials. Efficacy and working mechanisms of CPI-613 have not been established in cholangiocarcinoma. In this study, we aim to evaluate the performance of CPI-613 in combination with other therapeutic drugs and unravel the metabolic underpinnings. We treated cholangiocarcinoma cell lines with different genetic backgrounds with standard of care chemotherapy (gemcitabine and cisplatin) alone, CPI-613 alone, or the combination of the two. Results from viability assay showed that CPI-613 had synergistic (not simply additive) effects with standard of care chemotherapy. CPI-613 has a relatively short half-life, so longer infusion may be more beneficial even if the total dose remains the same. To mimic longer infusion, we treated the cells with multiple times of drug addition (fractional dosing), and compared the results to single addition. Oxygen consumption rate (an indicator of mitochondrial function) of cells under fractional dosing regimen was significantly lower in both short and long term, as measured by Agilent Seahorse and Resipher respectively. The results provided mechanistic support for longer infusion in clinical practice. We also collected plasma from patients before, during and after CPI-613 treatment in a clinical trial, and global metabolomics was performed. Global metabolomic changes induced by CPI-613 were identified and specific alternations in patients with complete response could be exploited as treatment targets or prediction markers. High frequency of isocitrate dehydrogenase 1 (IDH1) mutation is a characteristic of cholangiocarcinoma. Ivosidenib, an IDH1 inhibitor, has been shown to improve survival in chemotherapy-refractory cholangiocarcinoma patients in a phase 3 clinical trial. However, ivosidenib can increase alpha-ketoglutarate concentration, hence increasing the flux of TCA cycle and boosting mitochondrial fitness of the cancer cells. We hypothesized that the combination of CPI-613 and Ivosidenib could negate this effect and have better anti-cancer activities. Indeed, results from viability assay demonstrated synergy between CPI-613 and Ivosidenib. Metabolic flux analysis and global metabolomics were performed to illustrate the metabolic mechanisms of the combination of CPI-613 and Ivosidenib. Our study provides the mechanistic support for the use of CPI-613 in combination with other therapeutic agents and the results should be translated to clinical studies. Citation Format: Fulei Wuchu, Olamide Animasahun, Srinadh Choppara, Valerie Gunchick, Minal Nenwani, Jyotirmoy Roy, Miya Paserba, Abhinav Achreja, Vaibhav Sahai, Deepak Nagrath. The role of CPI-613 in cholangiocarcinoma treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2236.

Background & Hypothesis: Triple-negative breast cancer (TNBC) is a difficult to treat and deadly form of breast cancer. Pyruvate is an essential intermediate component of cellular metabolism which cancerous cells, including breast cancer cells, rely upon. Importantly, pyruvate and phosphoenolpyruvate are important nodes in the glycolytic pathway that support serine synthesis, lactate production and the TCA cycle. As a result of this, inhibition of the metabolic enzymes which regulate pyruvate production is currently used for cancer therapy. Apart from the glycolytic pathway, malic enzyme 2 (ME2) contributes to pyruvate and NADPH production through the oxidation of malate. Therefore, ME2 is a critical pathway supporting multiple metabolic needs of TNBC. Here, we investigate TNBC cell lines and assess their reliance on ME2 to drive accelerated growth, and thus potentially making ME2 a therapeutic target in TNBC. Methods: We investigated four TNBC lines (HCC1806, Hs578T, BT20, MDAMB468) with distinct serine synthesis activity assessed via western blotting of serine metabolism enzymes. To knockdown ME2 in TNBC cell lines, we used siRNA transient transfection and shRNA-encoding lentiviral vectors. The growth rates of TNBC cells were determined by cell counting in the BioTek BioSpa 8. Lastly, we used immunohistology to analyze the expression of ME2 and serine metabolic genes in a TNBC tumor microarray. Results: Modulation of the serine synthesis pathway revealed an unexpected synergy between serine metabolism and ME2. Knockdown of ME2 reduced the expression of serine metabolism genes but did not affect the expression of ME1 or ME3. The proliferative rate of cells with elevated serine synthesis, BT20 and MDAMB468 - was significantly reduced under ME2 knockdown, while Hs578T and HCC1806 experienced a modest reduction. Immunohistologic staining of TNBC tumors corroborated the link between ME2 and serine metabolism. Conclusions: We observed that TNBC cell lines with elevated, but not reduced, expression of serine synthesis genes were growth-inhibited in the absence of ME2. Our results indicate that ME2 is required for optimal growth in some forms of TNBC and may serve as a therapeutic target for the treatment of cancers with elevated serine biosynthesis. Ongoing studies are aimed at discerning additional biomarkers of sensitivity to ME2 inhibition in TNBC and other aggressive cancers. Citation Format: Mark D. Slayton, Jin Heon Jeon, Abhinav Achreja, Zackariah A. Farah, Brisilda Nilaj, Yi-Hsien Eu, Alisa Lui, Mason Collard, Liwei Bao, Celina Kleer, Deepak Nagrath, Sofia D. Merajver. Malic enzyme 2 identified as metabolic target in triple-negative breast cancers with increased serine biosynthesis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3921.

Branched-chain amino acids (BCAAs) supply both carbon and nitrogen in pancreatic cancers, and increased levels of BCAAs have been associated with increased risk of pancreatic ductal adenocarcinomas (PDACs). It remains unclear, however, how stromal cells regulate BCAA metabolism in PDAC cells and how mutualistic determinants control BCAA metabolism in the tumour milieu. Here, we show distinct catabolic, oxidative and protein turnover fluxes between cancer-associated fibroblasts (CAFs) and cancer cells, and a marked reliance on branched-chain α-ketoacid (BCKA) in PDAC cells in stroma-rich tumours. We report that cancer-induced stromal reprogramming fuels this BCKA demand. The TGF-β-SMAD5 axis directly targets BCAT1 in CAFs and dictates internalization of the extracellular matrix from the tumour microenvironment to supply amino-acid precursors for BCKA secretion by CAFs. The in vitro results were corroborated with circulating tumour cells (CTCs) and PDAC tissue slices derived from people with PDAC. Our findings reveal therapeutically actionable targets in pancreatic stromal and cancer cells.

The BCL-2 antagonist venetoclax is highly effective in multiple myeloma (MM) patients exhibiting the 11;14 translocation, the mechanistic basis of which is unknown. In evaluating cellular energetics and metabolism of t(11;14) and non-t(11;14) MM, we determine that venetoclax-sensitive myeloma has reduced mitochondrial respiration. Consistent with this, low electron transport chain (ETC) Complex I and Complex II activities correlate with venetoclax sensitivity. Inhibition of Complex I, using IACS-010759, an orally bioavailable Complex I inhibitor in clinical trials, as well as succinate ubiquinone reductase (SQR) activity of Complex II, using thenoyltrifluoroacetone (TTFA) or introduction of SDHC R72C mutant, independently sensitize resistant MM to venetoclax. We demonstrate that ETC inhibition increases BCL-2 dependence and the ‘primed’ state via the ATF4-BIM/NOXA axis. Further, SQR activity correlates with venetoclax sensitivity in patient samples irrespective of t(11;14) status. Use of SQR activity in a functional-biomarker informed manner may better select for MM patients responsive to venetoclax therapy., Venetoclax monotherapy is effective in 40% of t(11:14) positive multiple myeloma (MM). Here, the authors show that electron transport chain complex I (CI) and complex II (CII) activity predict MM sensitivity to venetoclax, and inhibition of CI with IACS-010759 or CII with TTFA increase sensitivity.

Childhood posterior fossa group A ependymomas (PFAs) have limited treatment options and bear dismal prognoses compared to group B ependymomas (PFBs). PFAs overexpress the oncohistone-like protein EZHIP (enhancer of Zeste homologs inhibitory protein), causing global reduction of repressive histone H3 lysine 27 trimethylation (H3K27me3), similar to the oncohistone H3K27M. Integrated metabolic analyses in patient-derived cells and tumors, single-cell RNA sequencing of tumors, and noninvasive metabolic imaging in patients demonstrated enhanced glycolysis and tricarboxylic acid (TCA) cycle metabolism in PFAs. Furthermore, high glycolytic gene expression in PFAs was associated with a poor outcome. PFAs demonstrated high EZHIP expression associated with poor prognosis and elevated activating mark histone H3 lysine 27 acetylation (H3K27ac). Genomic H3K27ac was enriched in PFAs at key glycolytic and TCA cycle–related genes including hexokinase-2 and pyruvate dehydrogenase. Similarly, mouse neuronal stem cells (NSCs) expressing wild-type EZHIP (EZHIP-WT) versus catalytically attenuated EZHIP-M406K demonstrated H3K27ac enrichment at hexokinase-2 and pyruvate dehydrogenase, accompanied by enhanced glycolysis and TCA cycle metabolism. AMPKα-2, a key component of the metabolic regulator AMP-activated protein kinase (AMPK), also showed H3K27ac enrichment in PFAs and EZHIP-WT NSCs. The AMPK activator metformin lowered EZHIP protein concentrations, increased H3K27me3, suppressed TCA cycle metabolism, and showed therapeutic efficacy in vitro and in vivo in patient-derived PFA xenografts in mice. Our data indicate that PFAs and EZHIP-WT–expressing NSCs are characterized by enhanced glycolysis and TCA cycle metabolism. Repurposing the antidiabetic drug metformin lowered pathogenic EZHIP, increased H3K27me3, and suppressed tumor growth, suggesting that targeting integrated metabolic/epigenetic pathways is a potential therapeutic strategy for treating childhood ependymomas.

Cellular metabolism sits at the forefront of growth and has been identified as an essential element for the progression of cancer. Large-scale chromosomal alterations and mutations incurred by cancerous cells can predispose cancer cells to dysfunctional metabolic programming independent of microenvironmental pressure. Cancer cells are adaptable to this reprogramming and find alternative measures to sustain elevated metabolic demands. However, this process reduces the number of functional metabolic pathways. Dysregulated metabolic pathways within cancerous cells create an opportunity for therapeutic intervention by targeting genetic chokepoints within these pathways. We analyzed metabolic landscape of breast cancers by combining genomic and transcriptomic data from cancer patient datasets, to identify metabolic pathways prone to dysregulation due to genomic influence. Our platform identified disruption in the serine biosynthesis, which some cancerous cells rely upon to sustain elevated growth in a subset of breast carcinoma. We utilized gain- and loss-of-function techniques in multiple breast cancer cell lines to recapitulate metabolic disruption observed in tumors. These in vitro screens are complemented by metabolic flux analysis and stable-isotope tracer analysis that reveal the vulnerable metabolic pathways in breast cancers with dysregulated serine metabolism. Our data demonstrate that specific metabolic targets can be identified through this integrated screening and validated empirically to confirm their significance as avenues for potential therapeutics. Citation Format: Mark Daniel Slayton, Abhinav Achreja, Jin Heon Jeon, Liwei Bao, Alisa Liu, Mason Collard, Deepak Nagrath, Sofia Merajver. Integrated in vitro and in silico screening of breast cancer reveals serine metabolism as a precision oncology target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3030.

Immunoaffinity based EV isolation technologies use antibodies targeting surface markers on EVs to provide higher isolation specificity and purity compared to existing approaches. One standing challenge for researchers is how to release captured EVs from the substrate to increase downstream and biological studies. The strong binding between the antibody and antigen or the antibody and substrate is commonly unbreakable without operating at conditions outside of the critical physiological range, making the release of EVs problematic. Additionally, immuno-affinity approaches are usually low-throughput due to their low flow velocity to ensure adequate time for antibody-antigen binding. To overcome these limitations, we modified the OncoBean chip, a previously reported circulating tumor cell isolation microfluidic device. The OncoBean chip is a radial flow microfluidic device with bean-shape microposts functionalized with biotin-conjugated EPCAM antibody through biotin-avidin link chemistry. It was demonstrated that the high surface area and varying shear rate provided by the bean-shaped posts and the radial flow design in the chip, enabled efficient capture of CTCs at high flow rate. We replace the anti-EPCAM with antibodies that recognize common EV surface markers to achieve high-throughput EV isolation. Moreover, by incorporating desthiobiotin-conjugated antibodies, EVs can be released from the device after capture, which offers a significant improvement over the existing isolation. The released EVs were found to be functional by confirming their uptake by cells using flow cytometry and fluorescent microscopy. We believe the proposed technology can facilitate both the study of EVs as cell-to-cell communicators and the further identification of EV markers.

Hepatocyte nuclear factor 4 alpha (HNF4α) is a transcription factor that plays a critical role in hepatocyte function, and HNF4α‐based reprogramming corrects terminal liver failure in rats with chronic liver disease. In the livers of patients with advanced cirrhosis, HNF4α RNA expression levels decrease as hepatic function deteriorates, and protein expression is found in the cytoplasm. These findings could explain impaired hepatic function in patients with degenerative liver disease. In this study, we analyzed HNF4α localization and the pathways involved in post‐translational modification of HNF4α in human hepatocytes from patients with decompensated liver function. RNA‐sequencing analysis revealed that AKT‐related pathways, specifically phospho‐AKT, is down‐regulated in cirrhotic hepatocytes from patients with terminal failure, in whom nuclear levels of HNF4α were significantly reduced, and cytoplasmic expression of HNF4α was increased. cMET was also significantly reduced in failing hepatocytes. Moreover, metabolic profiling showed a glycolytic phenotype in failing human hepatocytes. The contribution of cMET and phospho‐AKT to nuclear localization of HNF4α was confirmed using Spearman's rank correlation test and pathway analysis, and further correlated with hepatic dysfunction by principal component analysis. HNF4α acetylation, a posttranslational modification important for nuclear retention, was also significantly reduced in failing human hepatocytes when compared with normal controls. Conclusion: These results suggest that the alterations in the cMET‐AKT pathway directly correlate with HNF4α localization and level of hepatocyte dysfunction. This study suggests that manipulation of HNF4α and pathways involved in HNF4α posttranslational modification may restore hepatocyte function in patients with terminal liver failure., Although drug induced liver injury (DILI) is a rare clinical event, it carries significant morbidity and mortality, leaving it as the leading cause of acute liver failure in the United States. It is one of the most challenging diagnoses encountered by gastroenterologists. DILI is also the most common single adverse event that has led to withdrawal of drugs from the marketplace, drug attrition and failure of implicated drugs to obtain FDA approval. The development of various drug injury networks have played a vital role in expanding our knowledge regarding drug, herbal and dietary supplement related liver injury. In this review, we discuss what defines liver injury, epidemiology of DILI, it's biochemical and pathologic patterns, and management.

Extracellular vesicles (EVs) are ubiquitous nanoscale particles released from many different types of cells. They have been shown to contain proteins, DNA, RNA, miRNA, and most recently, metabolites. These particles can travel through the intercellular space and bloodstream to have regulatory effects on distant recipients. When an EV reaches a target cell, it is taken up and degraded to release its contents for utilization within the cell. In addition to regulatory effects, EVs have been shown to supplement the high metabolic demands of recipient cells in a nutrient-deprived tumor microenvironment. We developed an integrated empirical and computational platform to quantify metabolic contribution of source cell-derived EVs to recipient cells. The versatile Exo-MFA software tool utilizes (13)C stable-isotope tracing data to quantify the metabolic contributions of EVs from a source cell type on a recipient cell type. This is accomplished by creating EV-depleted culture medium, producing isotope-labeled EVs from the source cells, isolating the labeled EVs from the culture supernatant, culturing the recipient cells in the presence of the labeled EVs, and measuring the resulting metabolite levels across several time points.

Extracellular vesicles (EVs), including exosomes, have a key role in the paracrine communication between organs and compartments. EVs shuttle virtually all types of biomolecules such as proteins, lipids, nucleic acids, metabolites and even pharmacological compounds. Their ability to transfer their biomolecular cargo into target cells enables EVs to play a key role in intercellular communication that can regulate cellular functions such as proliferation, apoptosis and migration. This has led to the emergence of EVs as a key player in tumor growth and metastasis through the formation of "tumor niches" in target organs. Recent data have also been shown that EVs may transform the microenvironment of primary tumors thus favoring the selection of cancer cells with a metastatic behavior. The release of EVs from resident non-malignant cells may contribute to the metastatic processes as well. However, cancer EVs may induce malignant transformation in resident mesenchymal stem cells, suggesting that the metastatic process is not exclusively due to circulating tumor cells. In this review, we outline and discuss evidence-based roles of EVs in actively regulating multiple steps of the metastatic process and how we can leverage EVs to impair metastasis.

INTRODUCTION Proteasome inhibitors (PI) such as bortezomib (Velcade), carfilzomib (Kyprolis) and ixazomib (Ninlaro)) have been shown to be efficacious in multiple myeloma (MM) therapy. However, despite being an effective first line therapy, resistance to PI usually develops, leading to relapse and refractory disease. Previous studies have implicated a role of OXPHOS, glycolysis, antioxidants and serine metabolism in PI resistance. Mitochondrial metabolism plays a central role in malignant progression not only by generating ATP but also by providing precursors for synthesis of several biomolecules such as proteins, nucleotides, fatty acids and antioxidants that can influence the efficacy of MM therapies. We previously determined reduced mitochondrial electron transport chain (ETC) activity promotes sensitivity to the BCL-2 antagonist, venetoclax. However, the relationship between the metabolic state of the mitochondria and proteasome inhibitor (PI) sensitivity is not fully understood. Unexpectedly, we found ETC inhibition or reduced ETC activity to promote resistance to PIs. Here, we investigate the mechanistic basis for divergent effects of mitochondrial stress on sensitivity to PIs and venetoclax. METHODS We have used RNA-Seq and carbon isotope tracing using labeled U 13C-glucose or U 13C-glutamine, flow cytometry and western blot analysis in MM cell lines treated with mitochondrial Complex I inhibitor, IACS-010759 (IACS) +/- Bortezomib; and immunostaining of MM patient samples and interrogation of Multiple Myeloma Research Foundation's CoMMpass Study (NCT01454297, Interim Analysis 15). RESULTS We find that mitochondrial ETC (complex I-V) inhibition antagonizes bortezomib (BTZ) and carfilzomib (CFZ) induced cell death in MM in contrast to promoting sensitivity to venetoclax. Additionally, cell lines exhibiting intrinsically reduced ETC activity were more resistant to PI in comparison to cell lines with higher ETC activity. Evaluation of CoMMpass MM trial (NCT0145429, IA15) and serial samples from 50 patients before PI treatment and after relapse, show pathways related to OXPHOS and TCA cycle to be downregulated in poor survival patients, corroborating our in vitro observations on reduced ETC activity promoting resistance to PI. To elucidate the mechanistic basis of ETC-inhibition induced PI resistance we performed RNA-Seq and U 13C-glucose and glutamine tracing in L363 cells treated with the ETC Complex I inhibitor IACS +/- BTZ. RNA-Seq analysis and further confirmation by western blot analysis reveals integrated stress response (ISR) upregulation, ATF4 induction, and suppression of protein translation and global protein ubiquitination levels, likely responsible for resistance to proteasome inhibition in cells co-treated with IACS and BTZ compared to BTZ alone. Stable isotope tracing reveals an upregulation of reductive carboxylation; while RNA-Seq data and flow cytometry demonstrate increase in the cystine/glutamate transporter SLC7A11. The ensuing metabolic rewiring in mitochondrially suppressed MM induces several metabolic vulnerabilities including sensitivity to the SLC7A11 inhibitor, erastin. We also show that knockdown of ATF4 re-sensitizes ETC-inhibited cells to BTZ while ablating sensitivity to venetoclax (previously reported). Furthermore, examination of patient samples demonstrates inter-cellular heterogeneity in ATF4 expression. Our results thus, support the role ATF4 as key determinants of PI and BCL-2 antagonist efficacy. CONCLUSION We show mitochondrial ETC inhibition induces ISR mediated resistance to PI. These ETC-inhibited cells are however sensitive to BCL-2 antagonists and afford additional metabolic vulnerabilities that can be capitalized upon to target metabolic heterogeneity in MM. Our study underscores the need for implementing combinatorial regimens in MM cognizant of mitochondrial metabolic heterogeneity-mediated resistance. Disclosures Lonial: Janssen: Consultancy, Honoraria, Research Funding; BMS/Celgene: Consultancy, Honoraria, Research Funding; AMGEN: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria. Boise: AstraZeneca: Consultancy, Research Funding; Abbvie: Consultancy. Jaye: Stemline Therapeutics: Honoraria. Nooka: Janssen Oncology: Consultancy, Research Funding; GlaxoSmithKline: Consultancy, Other: Travel expenses; Sanofi: Consultancy; Bristol-Myers Squibb: Consultancy; Takeda: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Oncopeptides: Consultancy; Adaptive technologies: Consultancy; Karyopharm Therapeutics: Consultancy.

Large-scale chromosomal alterations, particularly chromosomal deletions in cancer genomes confer functional advantages to cancer cells via the loss of tumor suppressor genes (TSGs). However, due to the nature of these focal and arm-level deletions, essential house-keeping genes in the neighborhood of TSGs are potentially lost. We explore the emergence of metabolic adaptations and vulnerabilities that arise due to the collateral loss of essential metabolic genes. In our previous work, we showed that genomic loss in the locus containing SMAD4 and ME2 in pancreatic ductal adenocarcinomas forces these cells to rely on ME3 to compensate for the collateral loss of ME2; thereby revealing a highly selective metabolic target in these cells. Cancer cells can not only exploit such genetic redundancies but also rely on redundancies built into their complex metabolic network to compensate for the loss of metabolic function. Importantly, there is an unexplored landscape of these genomic loss events beyond well-characterized TSGs. To address these challenges, we have developed a platform to identify patient-specific metabolic vulnerabilities emerging due to distinct patterns of genomic loss events across tumors. Our platform presents opportunities for precision-based therapeutic intervention by targeting metabolic vulnerabilities in cancer patients. It uses genomic and clinical data available in cancer patient databases to obtain candidate metabolic genes that are lost to genomic deletions in an unbiased manner. To delineate metabolic redundances and tackle the complexity of genome-scale metabolic models, we employ an innovative multi-objective metabolic flux analysis approach. The utility of this platform is demonstrated via the discovery of a novel metabolic target in a cohort of ovarian cancer patients. The predicted collateral lethal target is validated in vitro using RNA interference and small-molecule inhibitors. Furthermore, we verify the metabolic mechanism of vulnerability predicted by the algorithm using deuterium tracing experiments. The target is also validated in vivo with mice containing ovarian tumors derived from cancer cells with and without the genomic deletion. Surprisingly, the collateral lethal metabolic target was also found to exist in a subset of aggressive endometrial cancers. Finally, we developed a multi-layer machine learning model to predict occurrence of the particular genomic deletion in ovarian and endometrial cancer patients with minimal molecular information to remove the need for whole-genome sequencing data. The model was trained and tested using the publicly-available molecular data from TCGA and AACR GENIE datasets. Citation Format: Abhinav Achreja, Tao Yu, Anjali Mittal, Srinadh Choppara, Noah Meurs, Olamide Animasahun, Jin Heon Jeon, Aradhana Mohan, Anusha Jayaraman, Reva Kulkarni, Justin Reinhold, Michele Cusato, Analisa Difeo, Xiongbin Lu, Deepak Nagrath. Genomic loss in cancers enable discovery of metabolic targets for precision cancer therapy via multiobjective flux analysis and machine learning [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 236.

Even though pancreatic ductal adenocarcinoma (PDAC) is associated with fibrotic stroma, the molecular pathways regulating the formation of cancer associated fibroblasts (CAFs) are not well elucidated. An epigenomic analysis of patient-derived and de-novo generated CAFs demonstrated widespread loss of cytosine methylation that was associated with overexpression of various inflammatory transcripts including CXCR4. Co-culture of neoplastic cells with CAFs led to increased invasiveness that was abrogated by inhibition of CXCR4. Metabolite tracing revealed that lactate produced by neoplastic cells leads to increased production of alpha-ketoglutarate (aKG) within mesenchymal stem cells (MSCs). In turn, aKG mediated activation of the demethylase TET enzyme led to decreased cytosine methylation and increased hydroxymethylation during de novo differentiation of MSCs to CAF. Co-injection of neoplastic cells with TET-deficient MSCs inhibited tumor growth in vivo. Thus, in PDAC, a tumor-mediated lactate flux is associated with widespread epigenomic reprogramming that is seen during CAF formation.

The mechanisms by which steatosis of the liver progresses to non-alcoholic steatohepatitis, and end-stage liver disease remain elusive. Metabolic derangements in hepatocytes controlled by SIRT1 plays a role in the development of fatty liver in inbred-animals. The ability to perform similar studies using human tissue has been limited by the genetically variability in man. We generated human induced pluripotent stem (iPS)-cells with controllable expression of SIRT1. By differentiating edited-iPS cells into hepatocytes and knocking down SIRT1, we found increased fatty acid biosynthesis that exacerbates fat accumulation. To model human fatty livers, we repopulated decellularized rat livers with human mesenchymal cells, fibroblasts, macrophages, and human SIRT1-knockdown iPS-derived hepatocytes and found that the human iPS-derived liver tissue developed macrosteatosis, acquired proinflammatory phenotype and shared a similar lipid and metabolic profiling to human fatty livers. Biofabrication of genetically-edited human liver tissue may become an important tool for investigating human liver biology and disease.

Advanced ovarian cancer usually spreads to the omentum. However, the omental cell-derived molecular determinants modulating its progression have not been thoroughly characterized. Here, we show that circulating ITLN1 has prognostic significance in patients with advanced ovarian cancer. Further studies demonstrate that ITLN1 suppresses lactotransferrin’s effect on ovarian cancer cell invasion potential and proliferation by decreasing MMP1 expression and inducing a metabolic shift in metastatic ovarian cancer cells. Additionally, ovarian cancer-bearing mice treated with ITLN1 demonstrate marked decrease in tumor growth rates. These data suggest that downregulation of mesothelial cell-derived ITLN1 in the omental tumor microenvironment facilitates ovarian cancer progression., Advanced ovarian cancer usually spreads to the omentum. Here, the authors show that circulating intelectin-1 (ITLN1) has prognostic significance in patients with advanced ovarian cancer, and that mesothelial cell-derived ITLN1 in the omental tumor microenvironment suppresses ovarian cancer progression.

Branched chain amino acids (BCAAs) in cancer serve as requisite precursors for protein synthesis, maintaining metabolite pools in the tricarboxylic acid (TCA) cycle, and sustaining production of nucleotides and lipids. However, the role of stromal cancer associated fibroblasts (CAFs) in support of BCAA metabolism in tumors is still poorly understood. Since most studies in pancreatic cancers have focused on systemic or cancer cell autonomous BCAA metabolism, understanding cancer-stromal ecosystem requires insight into the intersection of cancer-associated transformations in the stroma with reprogramming of their BCAA metabolism. Deciphering the precise role of various cellular components in BCAA metabolism of tumors is complicated by conflicting evidence from past studies and the challenging nature of the intricate tumor microenvironment (TME). Neither systemic in vivo BCAA metabolism nor cancer cells’ BCAA metabolism alone is sufficient to dissect the stromal role. The difficulty in understanding BCAA metabolism in the tumor milieu is exacerbated by nutrient-scarcity, exchange reactions, and metabolite sharing between cancer and stromal cells. Both, the fibrotic environment and nutrient scarcity are difficult to mimic in aggressive murine PDAC models. The metabolic fates of the BCAAs, leucine, valine, and isoleucine, are cell- and tissue-dependent. BCAA transaminases (BCAT1/2), first deaminate BCAAs to branched chain a-ketoacids (BCKAs). The second step in BCAA metabolism involves irreversible BCKA oxidation catalyzed by the mitochondrial BCKA dehydrogenase (BCKDH) complex. Further, oxidation of BCKAs results in succinyl-CoA and acetyl-CoA that act as anaplerotic or ketogenic sources for the TCA cycle. Our recent study revealed differential BCAA metabolism in cancer and stromal compartments of PDAC tumors. We identified a strikingly higher BCAA catabolic flux in CAFs but increased BCKA oxidative flux in cancer cells. Further, CAF-secreted BCKAs were used for maintaining protein synthesis, augmenting TCA cycle metabolite pools, and increasing oxidative phosphorylation in cancer cells. To corroborate the mechanistic underpinnings discovered in our human CAF and cancer cell-line model, we employed two patient-derived models: circulating tumor cells (CTCs) and tumor slice cultures. Collectively, we elucidated an undiscovered metabolic-signaling crosstalk between PDAC and stromal cells and demonstrated that targeting BCAA metabolism in PDAC tumors could mitigate PDAC aggression. Ziwen Zhu, Abhinav Achreja, Noah Meurs, Olamide Animasahun, Sarah Owen, Anjali Mittal, Pooja Parikh, TingWen Lo, Janusz Franco-Barraza, Jiaqi Shi, Mara Sherman, Edna Cuikerman, Andrew Pickering, Anirban Maitra, Vaibhav Sahai, Meredith Morgan, Sunitha Nagrath, Thedore Lawrence, Deepak Nagrath, "Tumor Reprogrammed Stromal BCAT1 Fuels Branched Chain Ketoacid Dependency in Stromal-Rich PDAC Tumors", Nature Metabolism, 2 (8), 2020. Citation Format: Deepak Nagrath. Stromal BCAT1 drives branched-chain ketoacid dependency in stromal-rich PDAC tumours [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT009.

Omental adipose stromal cells (O-ASC) are a multipotent population of mesenchymal stem cells contained in the omentum tissue that promote endometrial and ovarian tumor proliferation, migration, and drug resistance. The mechanistic underpinnings of O-ASCs' role in tumor progression and growth are unclear. Here, we propose a novel nitric oxide (NO)–mediated metabolic coupling between O-ASCs and gynecologic cancer cells in which O-ASCs support NO homeostasis in malignant cells. NO is synthesized endogenously by the conversion of l-arginine into citrulline through nitric oxide synthase (NOS). Through arginine depletion in the media using l-arginase and NOS inhibition in cancer cells using NG-nitro-l-arginine methyl ester (l-NAME), we demonstrate that patient-derived O-ASCs increase NO levels in ovarian and endometrial cancer cells and promote proliferation in these cells. O-ASCs and cancer cell cocultures revealed that cancer cells use O-ASC–secreted arginine and in turn secrete citrulline in the microenvironment. Interestingly, citrulline increased adipogenesis potential of the O-ASCs. Furthermore, we found that O-ASCs increased NO synthesis in cancer cells, leading to decrease in mitochondrial respiration in these cells. Our findings suggest that O-ASCs upregulate glycolysis and reduce oxidative stress in cancer cells by increasing NO levels through paracrine metabolite secretion. Significantly, we found that O-ASC–mediated chemoresistance in cancer cells can be deregulated by altering NO homeostasis. A combined approach of targeting secreted arginine through l-arginase, along with targeting microenvironment-secreted factors using l-NAME, may be a viable therapeutic approach for targeting ovarian and endometrial cancers. Cancer Res; 75(2); 456–71. ©2014 AACR.