Context-Dependent Requirements for Mitochondrial Apoptosis in CAR T Killing of Cancer Cells

Abstract

Chimeric antigen receptor (CAR) T cell therapy is now an FDA approved treatment for several hematologic malignancies, yet not all patients respond to this treatment. While some resistance mechanisms have been identified, the possibility that cell death pathways in target cancer cells could impact response to CAR T therapy remains underexplored. The goal of my thesis work was to understand how CAR T cells kill target cancer cells, and whether defects in cell death pathways in target cancer cells could alter their response to CAR T treatment. To address these questions, I generated a HeLa in vitro model system. HeLa cells with intact (HeLa-19) and deficient Bak/Bax (HeLa-DKO-19) expressing CD19 were co-cultured with CD19 CAR T cells. Viability of target cells was then quantified using a) Annexin V / Hoechst staining and b) an impedance-based readout of cell death. Bak/Bax deficiency, which blocks the mitochondrial pathway of apoptosis, conferred resistance to CAR T killing. Compared to the wildtype cells, Bak/Bax null cells required exposure to ten times as many CAR T cells to achieve a similar level of killing. To confirm that mitochondrial apoptosis contributes to CAR T killing, I forced the expression of Bcl-2 and Bcl-XL in HeLa-19 cells, and observed that both of these anti-apoptotic proteins conferred protection from CAR-T killing in a similar manner to Bak/Bax knockout. To assess whether these findings were generalizable to additional target cancer cells, I generated mantle cell lymphoma cell lines (JeKo-1) expressing increased levels Bcl-2 and Bcl-XL. Despite being protected from cell death induced by etoposide, the Bcl-2 and Bcl-XL JeKo-1 cell lines were not protected from CD19 CAR T killing, in contrast to my previous results. In conclusion, defects in the mitochondrial pathway of apoptosis conferred protection from CAR T killing in a solid tumor (HeLa) cell line, but not in one of hematopoietic origin (JeKo-1). While increasing target cell susceptibility to apoptosis could be a strategy to enhance CAR T killing, in some settings target cells may not depend on the mitochondrion for their demise. Understanding how CAR T cells kill target cells, particularly apoptotically resistant target cells, could help us develop immunotherapies that have the potential to help the most patients, given that defects in apoptosis are a hallmark of cancer. Future work will be necessary to understand why some target cells are protected by mitochondrial resistance and others are not; and to identify the precise mechanism by which CAR T cells can kill mitochondrially resistant cells at high E:T ratios.