Abstract 4938: Elucidation and pharmacologic targeting of master regulator proteins representing mechanistic determinants of macrophage state and immunoevasive potential

Abstract PURPOSE: Macrophages (mΦ) exhibit extensive transcriptional plasticity within the tumor microenvironment (TME). While M1 mΦ promote anti-tumor immunity, regulatory M2 mΦ drive tumor progression and resistance to immune checkpoint therapy. In a recent study, Obradovic et al. (Cell 2021) used VIPER, a network-based algorithm that infers protein activity from transcriptomic data, to identify a highly immunosuppressive TAM subset in clear cell renal carcinoma (ccRCC) characterized by TREM2+/C1Q+/APOE+ (TCA+) expression. TCA+ mΦ were associated with poor prognosis, metastasis, and immune evasion. We aim to identify Master Regulator (MR) proteins that mechanistically control the TCA+ program and may serve as actionable targets for selective depletion or reprogramming of these cells toward neutral, antitumor or pro-inflammatory states. METHODS: We performed pooled single-cell CRISPR interference (CRISPRi) via Perturb-seq targeting 50 candidate MRs identified by VIPER from genes differentially expressed in TCA+ versus M0/M1 mΦs, followed by time-resolved scRNA-seq. THP-1 monocytes were differentiated and polarized to the M2 state using IL-4 and IL-13 for 48 hours, then profiled across seven time points spanning M2 polarization (0, 6, 12, 24, 48, 96, 192 hr). Approximately 100,000 cells per time point were analyzed, along with ∼10,000 unperturbed THP-1-derived M0, M1, and M2 mΦs as references. We aim to generate perturbational RNA-seq profiles of TCA+ mΦs with >350 drugs using PLATE-seq to identify compounds that either target individual MRs (OncoTarget) or invert the global MR-activity signature (OncoTreat), thus phenocopying validated genetic perturbations. UNPUBLISHED DATA: We generated a comprehensive dataset comprising ∼700,000 time-resolved (∼100,000 cells per time point), genetically perturbed mΦs, enabling high-resolution characterization of MR-specific transcriptional responses across the full course of M2/TCA+ polarization. The dataset captures early, intermediate, and late transcriptional changes induced by targeted repression of candidate MRs, providing a dynamic view of the regulatory architecture underlying the acquisition and maintenance of the TCA+ state. We will integrate with >350 drug-perturbation profiles to establish a matched pharmacologic resource for downstream identification of compounds capable of modulating MR activity or globally shifting the TCA+ transcriptional program. CONCLUSION: This study provides a large-scale, time-resolved map of regulatory programs in immunosuppressive TCA+ mΦs, with the aim of identifying and targeting Master Regulators to invert their immunosuppressive phenotype. This framework offers a path to neutralize mΦ-mediated immunosuppression and improve responses to immune checkpoint therapy. Citation Format: Gaetano Viscido, Mikko Turunen, Zhouzerui Liu, Justyn Chang, Meghna S. Raman, Leo B. Dupire, Hanrui Zhang, Tim Olsen, Jeremy Worley, Aleksandar Obradovic, Andrea Califano. Elucidation and pharmacologic targeting of master regulator proteins representing mechanistic determinants of macrophage state and immunoevasive potential [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4938.