RHOA Loss of Function Impairs the IFNγ Response and Promotes CD19 Antigen Escape to Drive CAR-T Resistance in Diffuse Large B-cell Lymphoma

• Published in: bioRxiv
• Main Authors: Newsam, Austin D, Ziccheddu, Bachisio, Gowda Saralamma, Venu Venkatarame, Coughlin, Caroline A, Goretsky, Yitzhar E, Youssfi, Abdessamad A, Russo, Marco Vincenzo, Gallego, Natalia Campos, Fattakhov, Nikolai, Coffey, David G, Tsai, Daniel E, Carmona-Berrio, David, Suissa, David M, Manara, Paola, Sondhi, Anya K, Roberts, Evan R, Sheffield-Veney, Isaiah, Spiegel, Jay Y, Amador, Catalina, Alderuccio, Juan Pablo, Bilbao, Daniel, Jain, Michael D, Maura, Francesco, Locke, Frederick L, Schatz, Jonathan H
• Format: Journal Article Paper
• Language: English
• Published: United States Cold Spring Harbor Laboratory Press 04.03.2025; Cold Spring Harbor Laboratory
• Subjects: AKT protein; Antigens; B-cell lymphoma; CD19 antigen; Cell activation; Chimeric antigen receptors; Cytotoxicity; Immunotherapy; Intellectual property; Lymphocytes B; Lymphocytes T; Lymphoma; Macrophages; Metastases; Microenvironments; Pathogenesis; Patients; Research funding; RhoA protein; Tumors; Whole genome sequencing; γ-Interferon
• ISSN: 2692-8205; 2692-8205
• DOI: 10.1101/2025.02.27.640687

CD19-directed chimeric antigen receptor (CAR)-T cells are breakthrough therapies for aggressive B-cell lymphomas, but less than half of patients achieve durable responses. We previously showed through whole-genome sequencing of tumors from CAR-T-treated patients that deletions of (3p21.31) are enriched in cases progressing after treatment. 's roles in resistance and pathogenesis are poorly defined, despite loss-of-function alterations that occur in ~20% of newly diagnosed diffuse large B-cell lymphoma (DLBCL) cases. To evaluate mechanisms of CAR-T resistance, we created RHOA-deficient DLBCL systems and confirmed cell-intrinsic loss of response to CAR-19 in vitro and in vivo. RHOA loss promotes AKT activation that impairs cell-intrinsic responses to interferon gamma (IFNγ). Moreover, expression of the CAR target CD19 is consistently down-regulated accompanied by a drive toward plasmablast differentiation. RHOA deficient tumors demonstrate greatly increased sensitivity to AKT-pathway inhibitors, which reverse impaired IFNγ responses. Lymphoma microenvironments in vivo in immunocompetent mice reveal that RHOA loss promotes decreased infiltration by cytotoxic T cells and enrichment of M2-polarized macrophages, known markers of CAR-T resistance in lymphoma clinical cases. Overall, we characterize RHOA deficiency as an AKT-mediated CAR-T resistance driver and implicate avoidance of T-cell mediated killing as a likely reason for RHOA's frequent loss in DLBCL pathogenesis.