Abstract
Background: Front-line chemoimmunotherapy for diffuse large B-cell lymphoma (DLBCL), the most common non-Hodgkin lymphoma, is effective, however at least 40% of patients relapse or are refractory (r/r). Of this r/r DLBCL cohort, approximately 10% will be cured with conventional second-line treatments consisting of salvage chemotherapy and autologous stem cell transplant. CD19-directed chimeric antigen receptor T-cell (CAR-19) therapies are promising new second-line options in diffuse large B-cell lymphoma (DLBCL) and show exciting 30-40% durable complete responses in heavily pretreated patients. Recent whole-genome sequencing data published by our lab revealed that deletion of Ras homolog family member A (RHOA) is strongly associated with poor response to CAR-19 therapy in r/r DLBCL patients (p = 0.0013). In newly diagnosed DLBCL, 20% of patients harbor a RHOA deletion and another 5% have loss-of-function mutations. To date, no work has comprehensively identified the role of RHOA in DLBCL or how its inactivation may protect CAR-T resistant tumors.
Methods: We created a novel in vivo model of RHOA deficient lymphoma by transplanting Eμ-Myc transgenic hematopoietic stem cells (HSCs) transduced with a shRhoa vector. We also created a syngeneic model of RHOA-deficient lymphoma with MYC-driven BL3750 cells. In vitro flow cytometry and Xcelligence real-time cell analysis experiments were utilized to assess cytotoxicity of CAR-19 against RHOA-deficient lymphoma cell lines. RNAseq analysis was conducted on Rhoa knockdown cell lines to elucidate cell-intrinsic mechanisms of lymphomagenesis. Cell proliferation and migration assays were performed to characterize the phenotype of RHOA-deficient DLBCL cell lines.
Results: 18 mice were transplanted (11 shRhoa, 7 shRenilla control) and are being monitored for lymphoma development. In vitro CAR-T experiments demonstrated that while RHOA-deficient cells demonstrate a similar degree of killing initially, a population of RHOA-deficient cells persists and enrich at later time points. RNA-seq analysis of A20 shRhoa murine lymphoma cells uncovered enrichment of gene sets associated will cell-cycle progression and interferon (INF) signaling pathways. Cell proliferation assay and Western blot of RHOA inactivated cell lines revealed increased proliferation in shRhoa cells through the AKT/mTOR pathway compared to controls.
Conclusions: RHOA-deficient lymphomas, while not entirely evading CAR-19 cells in vitro, demonstrate resistance mechanisms that allows a small population to persist. RNA-seq analysis reveals upregulated oncogenic and immunoinhibitory pathways, consistent with in vitro cell proliferation and Western blot assays. Further research will challenge in vivo RHOA-deficient tumors with CAR-19 cells and employ scRNA-seq to reveal transcriptomic changes within RHOA-deficient lymphoma responsible for CAR-19 resistance.
Citation Format: Austin Newsam, Yitzhar Goretsky, Evan Roberts, Daniel Bilbao, Jonathan Schatz. Characterization of RHOA inactivation as a driver of CAR-T therapy resistance in diffuse large B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1771.