Abstract 3612: Elucidating and targeting master regulators of tumor infiltrating regulatory T cells
Authors: Ager Casey R. , Obradovic Aleksandar , Turunen Mikko , Khosravi-Maharlooei Mohsen , Karan Charles , Califano Andrea , Drake Charles G. 
Published: June 2022

Abstract
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.