Inhibiting Tumor Cell CDK9 Reprograms Cell Death Pathways to Enhance Efficacy of CAR T Cell Therapy

More than 50% of B cell malignancy patients who respond to chimeric antigen receptor (CAR) T cell therapies eventually relapse, and CAR T cell therapies for solid tumors have been largely ineffective. Clinical observations associate dysregulated expression of cell death molecules (e.g., FAS, FADD, BID and BCL2) in malignant B cells with poor outcomes in CAR-T cell-treated patients. Thus, innovative strategies that can enhance the sensitivity of tumor cells to CAR T cell cytotoxicity, potentially through modulation of cell death signaling, may improve efficacy of CAR T cell treatment. Cyclin Dependent Kinase 9 (CDK9) is a global regulator of gene transcription and repression. While it is often dysregulated in hematological malignancies, inhibition of CDK9 in lymphoma and leukemia cells abrogates pro-survival gene programs and induces apoptosis. Given the strong correlation between CDK9 inhibition and cell death induction, we hypothesized that CDK9 inhibition could sensitize B malignant cells to CAR T cell cytotoxicity, thereby improving the therapeutic efficacy. We report here that CDK9 inhibitor (CDK9-i) pre-treatment increased the sensitivity of B cell leukemia cells (Raji and Nalm6) to CD19-CAR T cell cytotoxicity. Furthermore, in vivo administration of CDK9-i to human B cell leukemia-bearing NOD/SCID/IL2Rg^−/− (NSG) mice significantly enhanced efficacy of CAR T cell therapy, by limiting the generation of PD-1⁺TIM3⁺ terminally exhausted CAR T cells, enhancing elimination of leukemia, and significantly improving overall survival. Bulk RNA-sequencing analysis revealed that CDK9-i pretreatment induced expression of genes associated with IFN-gamma response, TNFA signaling, inflammatory response and IFN-alpha response. Among these genes, cell death receptor signaling genes (TNFR1B, TNFRSF1A and IFNGR2) and cell death mediator genes (Caspase 8 (CASP8) and Gasdermin E (GSDME)) were significantly upregulated. Western Blot analysis revealed that CDK9-i priming and CAR T cell treatment significantly increased expression and cleavage of CASP8 and GSDME. These data suggest activation of cell apoptosis and pyroptosis in leukemia cells treated by CDK9-i. Competition assays showed that ablation of CASP8/MLKL or GSDME in CDK9-i-treated leukemia cells resulted in markedly increased resistance to CAR T cell killing in cultures. In vivo, NSG mice receiving CASP8/MLKL DKO cells were resistant to CDK9-i treatment, succumbing to disease a month before endpoints were reached for mice receiving WT Raji cells and CDK9-i treatment. Mechanistic studies revealed that CDK9-i regulated expression of CASP8 and GSDME were associated with activation of STAT5 and STAT3 signaling pathways in Raji cells. Inhibition of either STAT3 or STAT5 shows consistent decrease in expression of GSDME, CASP8 and FAS. These data indicate that CDK9-i treatment sensitizes leukemia cells to CAR T cell cytotoxicity via rewiring the expression and activity of cell death molecular pathways mediating apoptosis and pyroptosis. Importantly, we found that CDK9-i treatment-reprogrammed cell death pathways were associated with enhanced bystander killing of CD19-negative B cell leukemia cells likely through a mechanism of upregulating the expression of cell death signaling receptors such as FAS, TNFR1, TRAILR. Our findings demonstrate that CDK9-i-priming may potentiate elimination of tumor cells lacking specific antigen through a mechanism of enhanced bystander effect. In summary, combined CDK9-i treatment and CAR T cell therapy may have significant and broad implications in improving the efficacy of cellular immunotherapy against cancer.