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Approximately 50% of patients with hematologic malignancies relapse after chimeric antigen receptor (CAR) T cell treatment; mechanisms of failure include loss of CAR T persistence and tumor resistance to apoptosis. We hypothesized that both of these challenges could potentially be overcome by overexpressing one or more of the Bcl-2 family proteins in CAR T cells to reduce their susceptibility to apoptosis, both alone and in the presence of BH3 mimetics, which can be used to activate apoptotic machinery in malignant cells. We comprehensively investigated overexpression of different Bcl-2 family proteins in CAR T cells with different signaling domains as well as in different tumor types. We found that Bcl-xL and Bcl-2 overexpression in CAR T cells bearing a 4-1BB costimulatory domain resulted in increased expansion and antitumor activity, reduced exhaustion, and decreased apoptotic priming. In addition, CAR T cells expressing either Bcl-xL or a venetoclax-resistant Bcl-2 variant led to enhanced antitumor efficacy and survival in murine xenograft models of lymphoma and leukemia in the presence or absence of the BH3 mimetic venetoclax, a clinically approved BH3 mimetic. In this setting, Bcl-xL overexpression had stronger effects than overexpression of Bcl-2 or the Bcl-2(G101V) variant. These findings suggest that CAR T cells could be optimally engineered by overexpressing Bcl-xL to enhance their persistence while opening a therapeutic window for combination with BH3 mimetics to prime tumors for apoptosis.

Intrahepatic, perihilar, and distal cholangiocarcinoma (iCCA, pCCA, dCCA) are highly malignant tumours with increasing mortality rates due to therapy resistances. Among the mechanisms mediating resistance, overexpression of anti‐apoptotic Bcl‐2 proteins (Bcl‐2, Bcl‐xL, Mcl‐1) is particularly important. In this study, we investigated whether antiapoptotic protein patterns are prognostically relevant and potential therapeutic targets in CCA. Bcl‐2 proteins were analysed in a pan‐cancer cohort from the NCT/DKFZ/DKTK MASTER registry trial (n = 1140, CCA n = 72) via RNA‐sequencing and transcriptome‐based protein activity interference revealing high ranks of CCA for Bcl‐xL and Mcl‐1. Expression of Bcl‐xL, Mcl‐1, and Bcl‐2 was assessed in human CCA tissue and cell lines compared with cholangiocytes by immunohistochemistry, immunoblotting, and quantitative‐RT‐PCR. Immunohistochemistry confirmed the upregulation of Bcl‐xL and Mcl‐1 in iCCA tissues. Cell death of CCA cell lines upon treatment with specific small molecule inhibitors of Bcl‐xL (Wehi‐539), of Mcl‐1 (S63845), and Bcl‐2 (ABT‐199), either alone, in combination with each other or together with chemotherapeutics was assessed by flow cytometry. Targeting Bcl‐xL induced cell death and augmented the effect of chemotherapy in CCA cells. Combined inhibition of Bcl‐xL and Mcl‐1 led to a synergistic increase in cell death in CCA cell lines. Correlation between Bcl‐2 protein expression and survival was analysed within three independent patient cohorts from cancer centers in Germany comprising 656 CCA cases indicating a prognostic value of Bcl‐xL in CCA depending on the CCA subtype. Collectively, these observations identify Bcl‐xL as a key protein in cell death resistance of CCA and may pave the way for clinical application. [ABSTRACT FROM AUTHOR]