Abstract B014: Investigating universal mechanisms of leukemic cell resistance to CAR-T cell therapy

Abstract Chimeric antigen receptor T cell (CAR-T) therapy is a promising therapeutic for the treatment of relapsed/refractory leukemias of varying cell origins, including T-cell acute lymphoblastic leukemia (T-ALL), B-cell acute lymphoblastic leukemia (B-ALL), and acute myeloid leukemia (AML). Despite the potential of CAR-T cell therapy, many patients will fail to reach remission, and approximately 70% of T-ALL (Pan et al., Nat Med, 2025), 50% of B-ALL (Pasquini et al., Blood Adv, 2020; Maude et al., Leukemia, 2018), and 85% of AML patients (Bhagwat et al., Nat Med, 2024) treated with CD5-, CD19-, and CD123-CAR-T, respectively, will fail their therapy. Multiple studies have explored the mechanisms conferring resistance to CAR-T cell therapy, including impaired target antigen recognition, pro-tumorigenic secreted factors, defective immunological synapse formation, and loss of CAR-T cell efficacy and persistence. However, the target cell–intrinsic mechanisms underlying CAR-T resistance remain unclear (Bhagwat et al., Nat Med, 2024; Richards et al., Cancer Discovery, 2020; Zheng et al., Blood, 2021; Yan et al., Blood Adv, 2022)[WM1] . We hypothesize that leukemia-intrinsic factors underpin CAR-T efficacy irrespective of leukemia cell type. To address this, we transduced human T-ALL and AML cell lines with an MSCV-based retroviral construct co-expressing GFP and full-length human CD19, allowing surface CD19 expression at levels comparable to those observed in human B-ALL cell lines. CD19+ leukemic cells were then co-cultured with CD19-targeting CAR-T cells (4-1BB co-stimulatory and CD3-zeta signaling domain) for seven days and subsequently defined as “low sensitivity” or “high sensitivity” based on leukemic cell viability post-co-culture. Each CD19+ cell line was subjected to RNA-sequencing and mass spectrometry to identify specific leukemia intrinsic factors associated with CAR sensitivity. Here, we found that high protein (but not mRNA) levels of RIPK1 - a regulator of cell extrinsic apoptosis – correlate with elevated CAR-T sensitivity, independent of antigen and leukemic cell origin. Future studies aim to increase the breadth of CD19-expressing human leukemic cell lines interrogated using ectopic expression of a truncated surface CD19 protein, thus avoiding non-physiological pro-survival CD19-directed signaling (e.g., PI3K/AKT activation). These preliminary findings suggest that leukemia-intrinsic protein profiles, independent of antigen or subtype, may govern CAR-T sensitivity and offer novel therapeutic targets effective across leukemia cell types. Citation Format: Isaac G. Vigil, Julian Grandvallet Contreras, Kole R. DeGolier, James P. Scott-Browne, Matthew T. Witkowski, Terry J. Fry. Investigating universal mechanisms of leukemic cell resistance to CAR-T cell therapy [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr B014.