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PDF file - 421K, Supplementary Table S1A. Results from mRNA expression profiling in PDAC cells. Supplementary Table S1B. Results from mRNA expression profiling in CAFs. Supplementary Table S2A. Highest ranked gene sets from GSEA of the PDAC expression data. Supplementary Table S2B. Highest ranked gene sets from GSEA of the CAF expression data.

PDF file - 261K, Reductions in global genomic 5mC in stromal myofibroblasts and malignant epithelial cells in progression from early to late PanIN. DNMT1 levels are reduced in human pancreatic CAFs and are dependent on cellular proliferation. The proliferation index assessed by Ki67 IHC is lower in CAFs than in malignant epithelial cells and is reduced in both components following DAC treatment of the KPC-Brca1 tumors. DAC leads to induction of mRNA expression of interferon response pathway genes. Large areas of sarcomatoid change and loss of CAFs in tumors that have escaped from growth inhibition by DAC.

The tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC) is a complex ecosystem that drives tumor progression; however, in-depth single cell characterization of the PDAC TME and its role in response to therapy is lacking. We performed single-cell RNA sequencing on freshly collected human PDAC samples either before or after chemotherapy. Overall, we found a heterogeneous mixture of basal and classical cancer cell subtypes, along with distinct cancer-associated fibroblast and macrophage subpopulations. Strikingly, classical and basal-like cancer cells exhibited similar transcriptional responses to chemotherapy, and did not demonstrate a shift towards a basal-like transcriptional program among treated samples. We observed decreased ligand-receptor interactions in treated samples, particularly TIGIT on CD8+ T cells and its receptor on cancer cells, and identified TIGIT as the major inhibitory checkpoint molecule of CD8+ T cells. Our results suggest that chemotherapy profoundly impacts the PDAC TME and may promote resistance to immunotherapy.

The tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC) is a complex ecosystem that drives tumor progression. Improving our understanding of the PDAC TME and its role in response to therapy via in-depth single cell characterization will have broad clinical implications for biomarker development and therapeutic design. In this study, we performed single-cell RNA sequencing on freshly collected human PDAC samples of primary (n=16) or metastatic (n=11) origin, either before (n=20) or after (n=7) chemotherapy. We found a heterogeneous mixture of basal and classical Moffitt cancer cell subtypes in all samples, along with distinct cancer-associated fibroblast (CAF) and tumor-associated macrophage (TAM) subpopulations. We identified the major CAF subpopulations as inflammatory CAFs (iCAFs) and myofibroblastic CAFs (myCAFs); within these subpopulations were a very few cells expressing immunogenic features which have previously been associated with antigen presenting CAFs (apCAFs). Tumor-associated macrophages (TAMs) could be categorized into two major subpopulations, C1QC+ TAMs or SPP1+ TAMs, each with distinct functional characteristics. For example, phagocytosis-associated gene sets were enriched in C1QC+ TAMs, while angiogenesis-associated gene sets were enriched in SPP1+ TAMs. Comparison of naïve and chemotherapy treated primary PDAC samples revealed that classical and basal-like cancer cells exhibited similar transcriptional responses to chemotherapy; this contrasts with some previous reports which posited a shift towards a basal-like transcriptional program among treated samples. We further noted that treated samples evinced fewer ligand-receptor interactions, particularly between TIGIT on CD8+ T cells and its ligand on cancer cells. We also identified TIGIT, not PD1, as the major inhibitory checkpoint molecule of CD8+ T cells in the PDAC TME. Altogether, our results suggest that chemotherapy impacts the PDAC TME and may further reduce response to immunotherapy. Citation Format: Daniel Weissinger, Emily A. Kawaler, Gregor Werba, Ende Zhao, Despoina Kalfakakou, Surajit Dhara, Grace Oh, Xiaohong Jing, Nina Beri, Lauren Khanna, Tamas Gonda, Paul E. Oberstein, Cristina Hajdu, Cynthia Loomis, Adriana Heguy, Mara H. Sherman, Amanda W. Lund, Theodore H. Welling, Igor Dolgalev, Aristotelis Tsirigos, Diane M. Simeone. Single-cell sequencing elucidates the effects of chemotherapy on cancer cell heterogeneity and the tumor microenvironment of human pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr PR010.

Introduction: We have previously shown using the KPC (KrasLSL G12D/+; p53 r172H/+; Pdx1-Cre) mouse model of PDAC that sequential treatment with the DNA hypomethylating agents (HMA) followed by anti-PD-1 led to increased tumor necrosis, slowed tumor growth, increased tumor-infiltrating CD8+ cells, and significantly increased mean survival. However, acquired treatment resistance occurred, with emergence of a specific subtype of M2-polarized putatively immunosuppressive Chi3l3+ macrophages. In this study, we characterize the mechanism of polarization of these cells, define their function, and identify a potential therapeutic strategy to combine with epigentic therapy to prevent unfavourable macrophage polarization and improve the efficacy of epigenetic priming of immunotherapy against PDAC. Methods: Studies were conducted with primary macrophages (PM)e isolated from bone marrow (BMDM) or the peritoneum of 6-8 weeks mice and RAW264.7 cells were also used and exposed to conditioned media from primary KPC cell lines with and without previous treatment with the HMAs decitabine or azacytidine. The effect was compared with polarization by direct HMA, IFN-γ and IL-4. Gene set enrichment analysis was done on RNASeq data from myeloid cells and validated by RT-PCR. We used cytokine arrays and Western blot validation to identify secreted cytokines from KPC cells. Results: We profiled expression of RAW264.7 cells, BMDM and PMs following exposure to IL-4 or IFN-γ and identified a signature associated with each treatment (CD206, Fizz1, Tlr8, IGF1, Mgl2 associated with IL-4 and TNFα, CD86, CD64, CD40, NOS2 associated with IFN-γ). Direct exposure of myeloid cells to hypomethylating agents did not result in a significant polarization. We next used conditioned media of KPC cells to identify a hypomethylation specific effect and found a significant IL-4 like enrichment (Chi3l3, Arg1, Il4i1, Raet1a, Lgals4) and a HMA-specific myeloid signature (CD137, IL1a, Ccl2, Ccl7, Spp1) with treatment. To assess which cytokines might trigger this polarization, we employed cytokine arrays and identified a small number of candidate cytokines that are specific to hypomethylation induced polarization of myeloid cells, including CXCL1/2, CCL2, GM-CSF, FGF-21, IGFBP-6 and ICAM-1. Conclusions: Our results show that paracrine secretion of cytokines from cancer cells treated with HMA drugs, rather than direct effect of hypomethylating therapy on macrophages, is responsible for polarizing macrophages into an immunosuppressive subtype. We further identified several candidate cytokines secreted by cancer cells following hypomethylating therapy that may be relevant for this immunosuppressive polarization of macrophages and might be specific therapeutic targets in combination with hypomethylating therapy. Citation Format: Arturo Orlacchio, Daniel Weissinger, Catherine Do, Benjamin Tycko, Diane M. Simeone, Tamas Gonda. Hypomethylating therapy induces a potential immuno-suppressive myeloid phenotype by altering cancer cell cytokine secretion in PDAC [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C041.

4158 Background: Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related death with a 5-year survival rate of 10%. Novel strategies for advanced PDAC are a critical need. A DNA hypomethylating agent (HMA) increased tumor-infiltrating effector T cells and significantly prolonged survival in combination with immune checkpoint blockade (ICB) in a PDAC mouse model (Gonda et al 2020). We hypothesized that combining HMA with ICB will lead to therapeutic benefit in pts with advanced PDAC. Methods: This is an open-label, single-arm, single-center, Phase 2 trial of AZA plus pembro in pts with unresectable or metastatic PDAC. Pts were treated with AZA 50 mg/m2 subcutaneous daily for 5 days Q4W beginning week 1 day 1 followed by pembro 200 mg IV Q3W starting week 3 day 1. Key eligibility criteria included documented progression on or following first-line systemic treatment, ECOG PS 0-1, and adequate organ function. The primary objective was progression-free survival (PFS). Thirty-one evaluable pts were required to detect an improvement in PFS from 2 mo to 4 mo with a one-sided p-value of 0.05 and 80% power. Secondary endpoints included safety and tolerability, overall response rate (ORR), disease control rate (DCR), and overall survival (OS). Data cutoff was February 10, 2022. Results: Between Oct 2017 and Sept 2021, 36 pts were enrolled (median age 62.5, 75% white, 72% male). At data cutoff, 34 and 31 pts received at least 1 dose of AZA and pembro, respectively. Median PFS for ≥1 dose of AZA was 1.48 mo (95% CI: 1.35, 1.74) and for ≥1 dose of pembro was 1.51 mo (95% CI, 1.38, 3.42). The median OS was 4.67 months. Among the 34 pts, 3 (8.8%) experienced a partial response (PR) by RECIST with a DCR of 32.4%. Of the 3 pts with PR, 2 received 35 doses of pembro and continued beyond 2 years with an ongoing PR. One pt remains on pembro 20 months after study completion. None of the pts with a PR were microsatellite instability-high or tumor mutation burden high, and next-generation sequencing of the 3 cancers identified a BRCA1 variant and POLE variant. Treatment-related AEs (TRAEs) occurred in 20/34 pts (59%), the most common of which were diarrhea and fatigue. Grade ≥3 occurred in 7/34 (21%) including 1 immune-related grade 5 event (encephalitis). Conclusions: AZA plus pembro demonstrated a tolerable safety profile but no PFS benefit compared to historical controls. Our data suggests combined epigenetic therapy and ICB may expand therapeutic options in a subset of pts with PDAC. Further investigation is needed to identify biomarkers to predict response and elucidate effective timing, sequencing, and combination of epigenetic agents. Follow-up for OS is ongoing. Correlative analysis of epigenetic effects and characterization of the tumor immune microenvironment will be reported. Clinical trial information: NCT03264404.