Despite extensive efforts, reproducible assessment of pancreatic ductal adenocarcinoma (PDA) heterogeneity and plasticity at the single cell level remains elusive. Systematic, network-based analysis of single cell RNA-seq profiles showed that most PDA tumors comprise three coexisting lineages whose aberrant transcriptional state is mechanistically controlled by distinct regulatory programs. These lineages were characterized by the aberrant activation of either gastrointestinal lineage markers (GLS), transcriptional effectors of morphogen pathways (MOS) and acinar to ductal metaplasia markers (ALS). Each lineage was characterized by cells in two different cell states determined by the differential activation of MEK signaling (M+/M-) and high cellular plasticity. These states were confirmed in multiple cohorts, cell lines, PDX models and harmonized with bulk profile analyses. Master regulators (MRs) of GLS and MOS state were predictive of patient’s survival in bulk profiles. Cross-state plasticity was confirmed by lineage tracing assays, while pooled CRISPR/Cas9 assays confirmed the essentiality of identified MR proteins. Finally, mechanistic MR-mediated cell state control was confirmed by MR expression-mediated reprogramming of MOS cells to a GLS state. Our work provided a mechanistic model of pancreatic cancer heterogeneity and testable hypothesis to target cell state-specific pancreatic cancer dependencies. Citation Format: Pasquale Laise, Mikko Turunen, Hans Carlo Maurer, Alvaro Curiel Garcia, Ela Elyada, Bernhard Schmierer, Lorenzo Tomassoni, Jeremy Worley, Mariano J. Alvarez, Jordan Kesner, Xiangtian Tan, Somnath Tagore, Ester Calvo Fernandez, Kelly Wong, Alexander L. E. Wang, Sabrina Ge, Alina C. Iuga, Aaron T. Griffin, Winston Wong, Gulam A. Manji, Faiyaz Notta, David A. Tuveson, Kenneth P. P. Olive, Andrea Califano. Pancreatic cancer comprises co-existing transcriptional states regulated by distinct master regulator programs [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 A007.
Regulatory T cells (Tregs) are highly attractive targets for immunotherapy development, however, there remains a critical need for clinically actionable targeting strategies that specifically inhibit human tumor-infiltrating Tregs (TI-Tregs) while preserving function of cytotoxic effectors and peripheral non-tumor Tregs (P-Tregs). To this end, we sought to identify, validate, and target novel master regulators of TI-Tregs by leveraging a suite of next-generation bioinformatic tools and rigorous ex vivo and in vivo screening and validation methodologies. Specifically, we performed VIPER (Virtual Inference of Protein Activity) analysis on a large dataset of T cell transcriptional profiles from matched peripheral blood and tumors of 36 human patients, yielding 17 master regulator (MR) proteins predicted to uniquely drive the TI-Treg phenotype across cancers. To identify putative therapeutics that modulate TI-Treg MRs, we performed a systematic ex vivo drug screen with an unbiased panel of 1,554 FDA-approved and experimental compounds coupled to RNA sequencing (PLATE-Seq) on human TI-Tregs and P-Tregs. Drug candidates with preferential cytotoxic activity on TI-Tregs versus P-Tregs that also reversed MR transcriptional activity were thoroughly validated in vivo in the MC38 tumor model. In parallel, we performed a pooled in vivo CRISPR/Cas9 screen via the CHIME (CHimeric IMmune Editing) system to identify candidate MRs that regulate TI-Treg recruitment to and/or retention within MC38 tumors. By these approaches, we successfully validated an as-yet poorly described TI-Treg MR, TRPS1 (Transcriptional Repressor GATA Binding 1), which after genetic deletion across the hematopoietic compartment diminished TI-Tregs while preserving P-Tregs in MC38-bearing mice, and led to enhanced spontaneous control of MCA205 sarcomas. In addition, we found the widely used nucleoside analog chemotherapeutic Gemcitabine exhibits preferential inhibitory activity against human TI-Tregs versus P-Tregs, and validated in mice that at sub-clinical dose levels Gemcitabine exhibits immune-dependent therapeutic activity that significantly potentiates checkpoint blockade control of late-stage MC38 tumors. By single cell RNA sequencing of TI- and P-Tregs flow-sorted from Gemcitabine-treated mice, we observe specific depletion of a TI-Treg subset enriched for expression of TI-Treg MRs, including TRPS1. Together, these studies reveal new putative regulators of human TI-Tregs and identify a readily available clinical therapeutic that exhibits inhibitory activity against them. These findings specifically warrant future investigation into the role of TRPS1 in TI-Treg activity and clinical evaluation of low-dose Gemcitabine as a Treg-targeting strategy capable of enhancing checkpoint blockade immunotherapy. Citation Format: Casey R. Ager, Aleksandar Obradovic, Mikko Turunen, Mohsen Khosravi-Maharlooei, Charles Karan, Andrea Califano, Charles G. Drake. Elucidating and targeting master regulators of tumor infiltrating regulatory T cells [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 3612.
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