Crosstalk between metabolism and circadian rhythms is a fundamental building block of multicellular life, and disruption of this reciprocal communication could be relevant to disease. Here, we investigated whether maintenance of circadian rhythms depends on specific metabolic pathways, particularly in the context of cancer. We found that in adult mouse fibroblasts, ATP levels were a major contributor to signal from a clock gene luciferase reporter, although not necessarily to the strength of circadian cycling. In contrast, we identified significant metabolic control of circadian function across a series of pancreatic adenocarcinoma cell lines. Metabolic profiling of congenic tumor cell clones revealed substantial diversity among these lines that we used to identify clones to generate circadian reporter lines. We observed diverse circadian profiles among these lines that varied with their metabolic phenotype: The most hypometabolic line [exhibiting low levels of oxidative phosphorylation (OxPhos) and glycolysis] had the strongest rhythms, while the most hypermetabolic line had the weakest rhythms. Pharmacological enhancement of OxPhos decreased the amplitude of circadian oscillation in a subset of tumor cell lines. Strikingly, inhibition of OxPhos enhanced circadian rhythms only in the tumor cell line in which glycolysis was also low, thereby establishing a hypometabolic state. We further analyzed metabolic and circadian phenotypes across a panel of human patient-derived melanoma cell lines and observed a significant negative association between metabolic activity and circadian cycling strength. Together, these findings suggest that metabolic heterogeneity in cancer directly contributes to circadian function and that high levels of glycolysis or OxPhos independently disrupt circadian rhythms in these cells.

Abstract Background: CTLA-4 blockade is a highly effective therapeutic modality but only in a subset of cancer patients. We previously demonstrated that intratumoral administration of ultra-high concentration nitric oxide (UNO) augmented response to the immune checkpoint inhibitor, anti-mPD-1. Specifically, more than half of the mice treated with 10-minutes UNO (50,000 ppm) and anti-mPD-1 were primary and secondary tumor-free at Day 100 post-UNO treatment (vs 25% of control mice treated with anti-mPD-1 alone). In addition, the combination of UNO and anti-mCTLA-4 assessed in an immunocompetent breast cancer model (4T1) demonstrated prolonged survival in mice compared to single-agent anti-mCTLA-4. In CT26 and 4T1 mice, the immunodominant AH-1 antigen is associated with anti-tumor immune response. In this study, we assessed the systemic levels of CD8+ T-cells recognizing the AH-1 antigen following UNO treatment and the association with anti-tumor response. Methods: Here we explored the effects of UNO treatment in combination with anti-mCTLA-4 therapy. Colon carcinoma (CT26) tumor-bearing mice were treated with intra-tumoral 100,000 ppm UNO for 5 minutes in combination with 5 mg/kg (IP) anti-mCTLA-4. Effects on tumor growth and anti-tumor immune response were assessed. AH-1 specific CD8+ T cells were quantified by flow cytometry. Results: Combining 100,000 ppm UNO with anti-mCTLA4 in CT26 tumor-bearing mice resulted in considerable growth delay and regression of treated tumors. Furthermore, the combination induced a strong systemic response that led to the rejection of a distant metastasis-like tumor inoculated on the contralateral flank. Specifically, at Day 7 post-treatment, all UNO treated mice exhibited tumor-specific CD8+ T-cells in the blood and to a higher degree than in untreated mice. Moreover, systemic levels of tumor-specific CD8+ T-cells were higher in mice treated with the combination of UNO and anti-mCTLA4 compared to mice treated with monotherapy anti-mCTLA-4, indicating an increase in immune surveillance following UNO treatment. Finally, cured mice treated with combination therapy displayed higher systemic levels of antigen-specific CD8+ T-cells compared to untreated or naïve mice. Conclusion: UNO stimulates a higher tumor-specific T-cell response than anti-mCTLA-4 and synergizes with this drug to generate an even more significant targeted immune response. Citation Format: Yogev Sela, Matan Goldshtein, Jedidiah M. Monson, Hila Confino, Amir Avniel, Mark D. Pegram, Selena Chaisson. Ultra-high concentration nitric oxide (UNO) enhances anti-CTLA-4 treatment activity and induces a durable anti-tumor immune response [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C080.

Background: Immune checkpoint inhibitors have transformed clinical oncology. However, their use is limited as response is observed in only ~20–50% of patients. Previously, we demonstrated that treating CT26 tumor-bearing mice with ultra-high-concentration gaseous nitric oxide (UNO) followed by tumor resection stimulated antitumor immune responses. Accordingly, UNO may improve tumor response to immune checkpoint inhibitors. Here, we investigated the ability of UNO to improve the efficacy of a programmed cell death protein-1 (PD-1) antibody in vitro and in treating CT26 tumor-bearing mice. Methods: CT26 cells were injected into the flank of Balb/c mice (n = 15–16 per group). On day 6, CT26 cells were injected into the contralateral flank, and anti-mPD-1 injections commenced. Primary tumors were treated with intratumoral UNO on day 8. Tumor volume, response rates, toxicity, and survival were monitored. Results: (1) Short exposure to 25,000–100,000 parts per million (ppm) UNO in vitro resulted in significant upregulation of PD-L1 expression on CT26 cells. (2) UNO treatment in vivo consistently reduced cell viability in CT26 tumors. (3) Treatment reduced regulatory T-cell (Treg) levels in the tumor and increased levels of systemic M1 macrophages. UNO responders had increased CD8+ T-cell tumor infiltration. (4) Nine days after treatment, primary tumor growth was significantly lower in the combination arm vs. anti-mPD-1 alone (p = 0.0005). (5) Complete tumor regression occurred in 8/15 (53%) of mice treated with a combination of 10 min UNO and anti-mPD-1, 100 days post-treatment, compared to 4/16 (25%) of controls treated with anti-mPD-1 alone (p = 0.1489). (6) There was no toxicity associated with UNO treatment. (7) Combination treatment showed a trend toward increased survival 100 days post-treatment compared to anti-mPD-1 alone (p = 0.0653). Conclusion: Combining high-concentration NO and immune checkpoint inhibitors warrants further assessment especially in tumors resistant to checkpoint inhibitor therapy.

S1. DKK3 and Wnt signaling in CRC cells. S2. Derivation of murine colorectal tumoroids.

Supplementary Data from Bcl-xL Enforces a Slow-Cycling State Necessary for Survival in the Nutrient-Deprived Microenvironment of Pancreatic Cancer

Abstract Activation of Wnt signaling is among the earliest events in colon cancer development. It is achieved either via activating mutations in the CTNNB1 gene encoding β-catenin, the key transcription factor in the Wnt pathway, or most commonly by inactivating mutations affecting APC, a major β-catenin binding partner and negative regulator. However, our analysis of recent Pan Cancer Atlas data revealed that CTNNB1 mutations significantly co-occur with those affecting Wnt receptor complex components (e.g., Frizzled and LRP6), underscoring the importance of additional regulatory events even in the presence of common APC/CTNNB1 mutations. In our effort to identify non-mutational hyperactivating events, we determined that KRAS-transformed murine colonocytes overexpressing direct β-catenin target MYC show significant upregulation of the Wnt signaling pathway and reduced expression of Dickkopf 3 (DKK3), a reported ligand for Wnt co-receptors. We demonstrate that MYC suppresses DKK3 transcription through one of miR-17-92 cluster miRNAs, miR-92a. We further examined the role of DKK3 by overexpression and knockdown and discovered that DKK3 suppresses Wnt signaling in Apc-null murine colonic organoids and human colon cancer cells despite the presence of downstream activating mutations in the Wnt pathway. Conversely, MYC overexpression in the same cell lines resulted in hyperactive Wnt signaling, acquisition of epithelial-to-mesenchymal transition markers, and enhanced migration/invasion in vitro and metastasis in a syngeneic orthotopic mouse colon cancer model. Implications: Our results suggest that the MYC→miR-92a-|DKK3 axis hyperactivates Wnt signaling, forming a feed-forward oncogenic loop.

Although immunotherapy has revolutionized cancer care, patients with pancreatic ductal adenocarcinoma (PDA) rarely respond to these treatments, a failure that is attributed to poor infiltration and activation of T cells in the tumor microenvironment (TME). We performed an in vivo CRISPR screen and identified lysine demethylase 3A (KDM3A) as a potent epigenetic regulator of immunotherapy response in PDA. Mechanistically, KDM3A acts through Krueppel-like factor 5 (KLF5) and SMAD family member 4 (SMAD4) to regulate the expression of the epidermal growth factor receptor (EGFR). Ablation of KDM3A, KLF5, SMAD4, or EGFR in tumor cells altered the immune TME and sensitized tumors to combination immunotherapy, whereas treatment of established tumors with an EGFR inhibitor, erlotinib, prompted a dose-dependent increase in intratumoral T cells. This study defines an epigenetic-transcriptional mechanism by which tumor cells modulate their immune microenvironment and highlights the potential of EGFR inhibitors as immunotherapy sensitizers in PDA. SIGNIFICANCE: PDA remains refractory to immunotherapies. Here, we performed an in vivo CRISPR screen and identified an epigenetic-transcriptional network that regulates antitumor immunity by converging on EGFR. Pharmacologic inhibition of EGFR is sufficient to rewire the immune microenvironment. These results offer a readily accessible immunotherapy-sensitizing strategy for PDA.This article is highlighted in the In This Issue feature, p. 521.

Epithelial plasticity, reversible modulation of a cell's epithelial and mesenchymal features, is associated with tumor metastasis and chemoresistance, leading causes of cancer mortality. Although different master transcription factors and epigenetic modifiers have been implicated in this process in various contexts, the extent to which a unifying, generalized mechanism of transcriptional regulation underlies epithelial plasticity remains largely unknown. Here, through targeted CRISPR/Cas9 screening, we discovered two histone-modifying enzymes involved in the writing and erasing of H3K36me2 that act reciprocally to regulate epithelial-to-mesenchymal identity, tumor differentiation, and metastasis. Using a lysine-to-methionine histone mutant to directly inhibit H3K36me2, we found that global modulation of the mark is a conserved mechanism underlying the mesenchymal state in various contexts. Mechanistically, regulation of H3K36me2 reprograms enhancers associated with master regulators of epithelial-to-mesenchymal state. Our results thus outline a unifying epigenome-scale mechanism by which a specific histone modification regulates cellular plasticity and metastasis in cancer. Significance: Although epithelial plasticity contributes to cancer metastasis and chemoresistance, no strategies exist for pharmacologically inhibiting the process. Here, we show that global regulation of a specific histone mark, H3K36me2, is a universal epigenome-wide mechanism that underlies epithelial-to-mesenchymal transition and mesenchymal-to-epithelial transition in carcinoma cells. These results offer a new strategy for targeting epithelial plasticity in cancer. This article is highlighted in the In This Issue feature, p. 747

Cancer patients often harbor occult metastases, a potential source of relapse that is targetable only through systemic therapy. Studies of this occult fraction have been limited by a lack of tools with which to isolate discrete cells on spatial grounds. We developed PIC-IT, a photoconversion-based isolation technique allowing efficient recovery of cell clusters of any size - including single-metastatic cells - which are largely inaccessible otherwise. In a murine pancreatic cancer model, transcriptional profiling of spontaneously arising microcolonies revealed phenotypic heterogeneity, functionally reduced propensity to proliferate and enrichment for an inflammatory-response phenotype associated with NF-κB/AP-1 signaling. Pharmacological inhibition of NF-κB depleted microcolonies but had no effect on macrometastases, suggesting microcolonies are particularly dependent on this pathway. PIC-IT thus enables systematic investigation of metastatic heterogeneity. Moreover, the technique can be applied to other biological systems in which isolation and characterization of spatially distinct cell populations is not currently feasible.

Chromatin loops enable transcription-factor-bound distal enhancers to interact with their target promoters to regulate transcriptional programs. Although developmental transcription factors such as active forms of Notch can directly stimulate transcription by activating enhancers, the effect of their oncogenic subversion on the 3D organization of cancer genomes is largely undetermined. By mapping chromatin looping genome-wide in Notch-dependent triple-negative breast cancer and B cell lymphoma, we show that beyond the well-characterized role of Notch as an activator of distal enhancers, Notch regulates its direct target genes by instructing enhancer repositioning. Moreover, a large fraction of Notch-instructed regulatory loops form highly interacting enhancer and promoter spatial clusters termed "3D cliques." Loss- and gain-of-function experiments show that Notch preferentially targets hyperconnected 3D cliques that regulate the expression of crucial proto-oncogenes. Our observations suggest that oncogenic hijacking of developmental transcription factors can dysregulate transcription through widespread effects on the spatial organization of cancer genomes.

Abstract Metastasis and chemoresistance—the two main reasons for the high mortality of cancer—are associated with a form of cellular plasticity known as epithelial-to-mesenchymal transition (EMT). Cancer cells undergoing EMT become invasive, facilitating metastasis, and undergo a shift in their vulnerability to antineoplastic drugs. In recent work, it has been shown that EMT does not involve a single mechanism but rather a diversity of programs, yielding a continuum of cell phenotypes along the epithelial-mesenchymal spectrum. We previously developed a lineage-traced model of pancreatic ductal adenocarcinoma (PDA) to study EMT in the context of stochastically-arising tumors. As expected, epithelial-mesenchymal plasticity in some tumors involves transcriptional repression of the epithelial state, resulting in a “classical EMT” (C-EMT) phenotype. Surprisingly, however, epithelial-mesenchymal plasticity in the majority of tumors involves post-transcriptional repression of the epithelial state, resulting in a “partial EMT” (P-EMT) phenotype. These two plasticity programs are associated with other aspects of tumor biology as well, including distinct modes of cellular invasion. Here, we identify calcium signaling in pancreatic cancer cells as a regulator of the P-EMT phenotype. Prolonged calcium flux induces PDA cells to remove E-cadherin (ECAD) and other epithelial proteins from the surface and relocalize it intracellularly. This loss of the epithelial phenotype occurs without changes in the abundance of mRNAs for these proteins, reminiscent of the P-EMT phenotype observed in tumors in vivo. In addition, inhibition of the calcium-signaling protein calmodulin blunts this EMT-inducing effect. These results implicate calcium signaling as a mediator of partial EMT phenotypes. Citation Format: Robert J. Norgard, Ravikanth Maddipati, Nicole M. Aiello, David Balli, Jason R. Pitarresi, Derick N. Rosario-Berrios, Jinyang Li, Salina Yuan, Taiji Yamazoe, Yogev Sela, Allyson J. Merrell, Maximilian D. Wengyn, Kathryn Sun, Anil K. Rustgi, Ben Z. Stanger. Calcium signaling induces a partial EMT in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B38.

Tumors contain a heterogeneous mixture of live cells that are either undergoing proliferation or reside in a nonproliferative state. Cells that do not divide are often associated with increased chemoresistance, challenging therapies that target rapidly proliferating cells. In pancreatic cancer, paucity of blood vessels frequently generates hypoperfused, “arid” regions that constrain tumor cell metabolism and proliferation. Nonetheless, experimental conditions for studying tumor cells in vitro typically mimic well-nourished, “fertile” environments optimized for supporting proliferating cells rather than the more prevalent quiescent state. Characterization of the poorly vascularized KPC model confirmed that the quiescent phenotype is common and is strongly correlated to reduced tissue perfusion. To formulate culture conditions that will emulate the arid microenvironment associated with the nonproliferative state in vitro, we systematically deprived various nutrients from tumor cells and established the necessary components for attaining cell quiescence. Deprivation of amino acids on top of glucose, oxygen, and serum levels was essential for obtaining a complete and reversible cell cycle arrest without loss of viability. Cell cycle arrest was accompanied by a number of physiologic adaptations. Metabolomic analysis indicated reduction in the levels of TCA cycle and mevalonate pathway intermediates indicative of increased reliance on nonglycolytic metabolism. In addition, free amino acid pools were diminished and tumor cells exhibited a significant increase in autophagic flux and micropinocytosis, suggesting that amino acid availability is limited under arid conditions. Concomitant with reduced proliferation and metabolic rewiring, tumor cells under arid conditions exhibited remarkable resistance to gemcitabine. To explore alternative strategies to target these nonproliferating pancreatic tumor cells, we performed a comprehensive genome-wide CRISPR/Cas9-based genetic screen and compared vulnerabilities under ”fertile” and “arid” conditions. Functional annotation of sgRNAs depleted under arid conditions indicated a striking dependence on oxidative phosphorylation for survival. Remarkably, our analysis also revealed that a number of targetable genetic and epigenetic programs driving cell cycle progression were not essential and, in some instances, even hazardous for cells under arid conditions. These results suggest that pancreatic tumor cells that cease proliferation in a nutrient-depleted environment are physiologically rewired and may exhibit a distinct drug response profile from rapidly proliferating cells. The experimental model we developed allows investigation of this dominant yet understudied cell population and may serve as a platform for identification of agents targeting the quiescent fraction of tumors. Citation Format: Yogev Sela, Jinyang Li, Miriam Doepner, Shivahamy Maheswaran, Clementina Mesaros, Ian Blair, Ophir Shalem, Ben Stanger. Dissecting vulnerabilities of pancreatic tumors’ silent fraction unravels secrets of an alternative cell state [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C50.

Abstract Pancreatic ductal adenocarcinoma (PDA) has a poor prognosis, and new strategies for prevention and treatment are urgently needed. We previously reported that histone H4 acetylation is elevated in pancreatic acinar cells harboring Kras mutations prior to the appearance of premalignant lesions. Because acetyl-CoA abundance regulates global histone acetylation, we hypothesized that altered acetyl-CoA metabolism might contribute to metabolic or epigenetic alterations that promote tumorigenesis. We found that acetyl-CoA abundance is elevated in KRAS-mutant acinar cells and that its use in the mevalonate pathway supports acinar-to-ductal metaplasia (ADM). Pancreas-specific loss of the acetyl-CoA–producing enzyme ATP-citrate lyase (ACLY) accordingly suppresses ADM and tumor formation. In PDA cells, growth factors promote AKT–ACLY signaling and histone acetylation, and both cell proliferation and tumor growth can be suppressed by concurrent BET inhibition and statin treatment. Thus, KRAS-driven metabolic alterations promote acinar cell plasticity and tumor development, and targeting acetyl-CoA–dependent processes exerts anticancer effects. Significance: Pancreatic cancer is among the deadliest of human malignancies. We identify a key role for the metabolic enzyme ACLY, which produces acetyl-CoA, in pancreatic carcinogenesis. The data suggest that acetyl-CoA use for histone acetylation and in the mevalonate pathway facilitates cell plasticity and proliferation, suggesting potential to target these pathways. See related commentary by Halbrook et al., p. 326. This article is highlighted in the In This Issue feature, p. 305

Abstract Pancreatic adenocarcinoma (PDA) is highly refractory to immunotherapy, a consequence of T-cell exclusion from the tumor microenvironment (TME). Based on a pathway analysis of human PDAs, we hypothesized that the receptor tyrosine kinase ephrin A2 (EPHA2) drives this immunosuppressive TME, as its expression negatively correlated with CD8A, CD3, PRF1, GZMB, and patient survival (EPHA2low/EPHA2high log rank hazard ratio 0.115, 95% CI of ratio 0.0315-0.416, TCGA dataset). Deletion of Epha2 in tumor cells increased T-cell influx, decreased the number of infiltrating myeloid suppressor cells, and sensitized tumors to therapy. Treatment of Epha2-deficient tumors with combination of chemo and immunotherapy resulted in suppressed tumor growth or tumor regression in up to 85% of cases. Examination of Epha2-dependent gene expression nominated Ptgs2 as a downstream mediator of T-cell exclusion. Like EPHA2, PTGS2 exhibited a negative correlation with intratumoral T cells, cytolytic activity, and patient survival (PTGS2low/PTGS2high log rank hazard ratio 0.152, 95% CI of ratio 0.054-0.430, TCGA dataset). KPCY mice (mutant KrasG12D (K), dominant negative p53R172H (P), Cre recombinase (C), YFP protein (Y)) deficient in pancreatic ductal cell Ptgs2 had significantly increased overall survival compared to Ptgs2 sufficient KPCY mice (Ptgs2def/Ptgs2suff log rank hazard ratio 0.5001, 95% CI of ratio 0.294-0.851). Ptgs2 deletion promoted T-cell influx in both autochthonous and implanted tumors. Inversely, overexpression of Ptgs2 decreased the number of tumor-infiltrating T cells, increased the proportion of suppressor myeloid cells, and conferred resistance to the combination therapy. Remarkably, pharmacologic inhibition of PTGS2 sensitized the tumors to immunotherapy, suppressing the growth of implanted tumors and increasing the survival of treated KPCY mice (median survival of untreated and treated mice 151 and 199 days, respectively; survival curve log rank p-value= 0.017). These studies suggest that a tumor cell-intrinsic EPHA2-PTGS2 signaling axis regulates the immune TME in PDA and suggests that a two-step approach targeting T-cell exclusion and exhaustion holds promise for this treatment-refractory disease. Citation Format: Nune Markosyan, Jinyang Li, Yu Sun, Lee Richman, Jeffrey Lin, Fangxue Yan, Liz Quinones, Yogev Sela, Taiji Yamazoe, Naomi Gordon, John Tobias, Katelyn Byrne, Andrew Rech, Garret FitzGerald, Ben Stanger, Robert Vonderheide. Tumor cell-intrinsic EPHA2 suppresses antitumor immunity by regulating PTGS2 (COX-2) in pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B33.

Resistance to immunotherapy is one of the biggest problems of current oncotherapeutics. WhileT cell abundance is essential for tumor responsiveness to immunotherapy, factors that define the T cell inflamed tumor microenvironment are not fully understood. We conducted an unbiased approach to identify tumor-intrinsic mechanisms shaping the immune tumor microenvironment(TME), focusing on pancreatic adenocarcinoma because it is refractory to immunotherapy and excludes T cells from the TME. From human tumors, we identified EPHA2 as a candidate tumor intrinsic driver of immunosuppression. Epha2 deletion reversed T cell exclusion and sensitized tumors to immunotherapy. We found that PTGS2, the gene encoding cyclooxygenase-2, lies downstream of EPHA2 signaling through TGFβ and is associated with poor patient survival. Ptgs2 deletion reversed T cell exclusion and sensitized tumors to immunotherapy; pharmacological inhibition of PTGS2 was similarly effective. Thus, EPHA2-PTGS2 signaling in tumor cells regulates tumor immune phenotypes; blockade may represent a novel therapeutic avenue for immunotherapy-refractory cancers. Our findings warrant clinical trials testing the effectiveness of therapies combining EPHA2-TGFβ-PTGS2 pathway inhibitors with anti-tumor immunotherapy, and may change the treatment of notoriously therapy-resistant pancreatic adenocarcinoma.

Resistance to immunotherapy is one major problem of current clinical care for cancer patients. While T-cell abundance is essential for tumor responsiveness to immunotherapy, factors that dictate T-cell infiltration in tumor microenvironments are not fully understood. To understand the tumor cell-intrinsic factors underlying the heterogeneity of tumor immunity and sensitivity to immunotherapy, we established a new experimental system by generating a library of congenic pancreatic tumor cell clones from a genetic mouse model driven by mutant Kras and p53. These tumor cell clones robustly formed implanted tumors that recapitulated the T cell-inflamed and non-T cell-inflamed tumor microenvironments in human patients, associated with distinct patterns of infiltration by T cells and myeloid cells. We found that the non-T cell-inflamed phenotype was dominant over the T cell-inflamed phenotype in the local tumor microenvironment. Both quantitative and qualitative features, specifically expression of markers of prior TCR activation, of intratumoral CD8+ T cells predicted the response to immunotherapies. An integrated transcriptomic and epigenetic analysis revealed that tumor cell-intrinsic expression of the chemokine CXCL1 as a major determinant of the non-T cell-inflamed microenvironment, and ablation of tumor cell-intrinsic CXCL1 promoted T-cell infiltration and sensitivity to a combination of chemotherapies, CD40 agonist, and checkpoint blockades. Similarly, we identified tumor cell-intrinsic EPHA2 and PTGS2 as key regulators of immune infiltration and immunotherapy response in our experimental system. Ablation of tumor cell-intrinsic EPHA2 or PTGS2 enhanced T-cell infiltration and suppressed myeloid cell infiltration in implanted pancreatic tumors, and increased sensitivities of tumors to the combined immunotherapy. These results demonstrated that heterogeneity of tumor immune phenotypes is driven by tumor cell-intrinsic factors that can be manipulated to influence the outcome of immunotherapies. The observation that non-T cell-inflamed phenotype is dominant emphasized the importance of targeting mechanisms driving T-cell low phenotype for improving immunotherapy response. Citation Format: Jinyang Li, Katelyn T Byrne, Nune Markosyan, Taiji Yamazoe, Fangxue Yan, Zeyu Chen, Yu H. Sun, Jeffrey Lin, Yogev Sela, Robert J. Norgard, Salina Yuan, Allyson J. Merrell, John W. Tobias, Robert H. Vonderheide, Ben Z. Stanger. Investigation of tumor-cell-intrinsic factors regulating immune infiltration and response to immunotherapy in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A28.

Abstract Mutant KRAS is thought to initiate pancreatic tumorigenesis, orchestrating a program that leads to cell de-differentiation, proliferation, and symbiotic cooperation with neighboring cells, enabling the cancer cells to thrive in a particularly harsh microenvironment. Recent studies have highlighted the role of metabolites in regulating the epigenome. Although oncogenic KRAS is known to reprogram cellular metabolism, the role of metabolic control of the epigenome in pancreatic tumorigenesis is poorly understood. We showed that expression of KRASG12D in mouse pancreas promotes elevated histone acetylation levels in pancreatic acinar cells, and that this precedes tumor development. We hypothesized that augmented acetyl-CoA metabolism may play a role in facilitating pancreatic tumorigenesis. To test this, we generated mice deficient for Acly (acetyl-CoA producing enzyme) in pancreas (Pdx1-Cre; Aclyf/f mice). In the context of KRASG12D expression, ACLY deficiency reduces histone acetylation levels in pancreatic acinar cells and impairs formation of neoplastic lesions. ACLY deficiency also impairs pancreatitis-induced tumor development. In testing roles for acetyl-CoA-dependent processes in ADM, we found that targeting either histone acetylation by BET inhibition or cholesterol synthesis with statins suppressed tumor onset. In vivo, response to BET and cholesterol synthesis blockade is associated with recruitment of CD8+ T-cells. The findings indicate that ACLY-dependent metabolic and epigenetic remodeling promote tumor development and point to the potential to target acetyl-CoA metabolism for pancreatic cancer. Potential role of tumor-infiltrating leukocytic cells in modulating acetyl-CoA metabolism in vivo, cancer cell-autonomous mechanisms for acetyl-CoA levels regulation, as well as consequences for therapeutic targeting and dietary interventions, will be discussed. Citation Format: Alessandro Carrer, Sophie Trefely, Steven Zhao, Sydney Campbell, Yogev Sela, Simone Sidoli, Benjamin A. Garcia, Nathaniel W. Snyder, Ben Z. Stanger, Kathryn E. Wellen. All the roads bring to Rome: How acetyl-CoA metabolism supports multistep pancreatic carcinogenesis [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A03.

The biological and functional heterogeneity between tumors-both across and within cancer types-poses a challenge for immunotherapy. To understand the factors underlying tumor immune heterogeneity and immunotherapy sensitivity, we established a library of congenic tumor cell clones from an autochthonous mouse model of pancreatic adenocarcinoma. These clones generated tumors that recapitulated T cell-inflamed and non-T-cell-inflamed tumor microenvironments upon implantation in immunocompetent mice, with distinct patterns of infiltration by immune cell subsets. Co-injecting tumor cell clones revealed the non-T-cell-inflamed phenotype is dominant and that both quantitative and qualitative features of intratumoral CD8+ T cells determine response to therapy. Transcriptomic and epigenetic analyses revealed tumor-cell-intrinsic production of the chemokine CXCL1 as a determinant of the non-T-cell-inflamed microenvironment, and ablation of CXCL1 promoted T cell infiltration and sensitivity to a combination immunotherapy regimen. Thus, tumor cell-intrinsic factors shape the tumor immune microenvironment and influence the outcome of immunotherapy.