Abstract
Background: CD19-directed chimeric antigen receptor T-cell (CAR-19) therapies are promising new options in relapsed or refractory diffuse large B-cell lymphoma (DLBCL), but over half of patients still progress. Recent whole-genome sequencing data published by our lab revealed that deletion of RHOA (3p21.31) is strongly associated with poor response (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-19 resistant tumors. Methods: RHOA loss-of-function (LoF) lymphoma cell lines were generated using shRNA and CRISPR approaches. Diminished RHOA activity was confirmed via active RHO pulldowns. RNA-sequencing in conjunction with western blotting and flow cytometry was conducted to uncover deregulated pathways in RHOA-deficient lymphoma. Cell proliferation, migration, and viability were assessed to phenotype RHOA LoF and uncover signaling pathways activated in RHOA-deficient lymphoma. CD19-directed murine and human CAR-T cells were co-cultured with corresponding RHOA LoF systems to assess CAR-T engagement and killing. Results: RNA-seq analysis of RHOA-deficient cell lines revealed enrichment of gene sets associated with cell-cycle progression and interferon signaling pathways, and cell proliferation and chemotaxis assays both demonstrated increased activation. Western blotting revealed upregulated pAKT, but strikingly RHOA LoF systems showed enhanced sensitivity to PI3K, AKT, and mTOR inhibition. We assessed if RHOA LoF associates with previously described decreased CD19 driving CAR-19 resistance and found consistent downregulation by flow cytometry in both A20 shRhoa (p = 0.0002) murine lymphomas and RIVA human cell line CRISPR het RHOA knockouts (p = 0.0031), confirmed by western blotting in both systems. RNA-seq analysis of 74 DLBCL patients (PCAWG data) revealed a strong positive correlation between expression CD19 and RHOA (p < 0.001, R2 = 0.236). Impedance-based serial cell viability assessments and flow cytometry cytotoxicity assays with CAR-19 and RHOA LoF cells revealed a statistically significant decrease in CAR-19 killing of RHOA-deficient lymphomas (p = 0.0005 and p < 0.0001). Conclusion: RHOA-deficient lymphoma relies on PI3K/AKT/mTOR signaling for proliferation that works in parallel with decreased CD19 to decrease CAR-19 killing. Upregulation of interferon signaling pathways gives evidence of increased immunoinhibitory ligands that could promote exhausted CAR19 products. 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. Keywords: Aggressive B-cell non-Hodgkin lymphoma, Immunotherapy, Microenvironment No conflicts of interests pertinent to the abstract.
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