Abstract
Abstract CD19-directed chimeric antigen receptor T-cell (CAR-T) therapies have shown breakthrough responses for relapsed or refractory diffuse large B-cell lymphoma (DLBCL), however, over half of patients still progress. Whole genome sequencing of CAR-T treated patients revealed RHOA (3p21.31) deletions highly enriched in patients with poor responses (p = 0.0013). RHOA is altered in approximately 25% of newly diagnosed DLBCL, yet no work has comprehensively defined its role in lymphoma biology or immune resistance. We created RHOA loss-of-function (LoF) cell lines and performed RNA-sequencing followed by drug viability assays to interrogate pathways of dependence in RHOA deficient DLBCL. We also created immunocompetent models of RHOA deficient lymphoma in vivo and conducted single-cell RNA sequencing to assess the resulting lymphoma microenvironments. CAR-T cells were engineered and used to challenge RHOA LoF tumors in vitro. Upon discovering highly upregulated gene sets involving PI3K/AKT/mTOR signaling, we employed drug viability assays and established a critical reliance on AKT signaling in RHOA LoF cell lines. Upregulated AKT was found to suppress JAK2-STAT1 mediated (IFN-γ) signaling and this effect was reversed with PI3K inhibition. Single-cell transcriptomics of RHOA deficient mouse lymphomas revealed decreased Cd8a expression and increased Cd4 expression of T-cells (p = 0.09 and p = 0.028, respectively), suggesting an immunosuppressive microenvironment as a result of abolished IFN-γ mediated immunorecognition. RHOA deficient cell lines were also found to downregulate surface CD19 expression (p < 0.0001) and demonstrated significantly decreased CAR-T cell killing in vitro (p = 0.0005). Future experiments will challenge RHOA deficient immunocompetent tumors in vivo with and without PI3K inhibitors to assess efficacy of combinatorial therapy. Citation Format: Austin D Newsam, Caroline A Coughlin, Yitzhar E Goretsky, Abdessumad Y Alaoui, Daniel E Tsai, David G Coffey, Anya K Sondhi, Evan R Roberts, Natalia Campos Gallego, Paola Manara, Daniel Bilbao, Bachisio Ziccheddu, Jay Y Spiegel, Michael D Jain, Frederick L Locke, Juan P Alderuccio, Francesco Maura, Jonathan H Schatz. RHOA inactivation subverts IFN-γ response and promotes antigen escape in CAR-T resistant diffuse large B-cell lymphoma [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P23.