Reducing Acute Myeloid Leukemia resistance to CAR T cell therapy by epigenetic activation of the tumor inflammasome-pyroptosis signaling Free

Acute myeloid leukemia (AML) faces major clinical challenges characterized by high relapse and low cure rates. Chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment for B cell malignancies, but it has limited efficacy in treating AML. Poor anti-tumor efficacy is often attributed to tumor resistance mechanisms and T cell dysfunction. The dysregulation of cell death in tumor cells plays a crucial role in promoting tumor resistance to immunotherapies. However, effective strategies that can disrupt this resistance mechanism remain an unmet need. Immunogenic cell death, such as pyroptosis, promotes effective innate and adaptive immunity. Pyroptosis is triggered by inflammasome activation and executed by caspase (CASP) and gasdermin (GSDM) proteins. Here we demonstrate that a previously unrecognized role of the NLRP3 inflammasome-pyroptosis signaling in sensitizing AML cells to CAR T cell killing. To test the hypothesis that epigenetic therapy agents that can activate the inflammasome-pyroptosis signaling in target tumor cells could potentially enhance therapeutic efficacy of CAR T cells against AML, we used a hypothesis-driven drug screening assay and identified that the DNA methylation inhibitor decitabine (DAC) induced high levels NLRP3 in murine AML C1498 cells. Bulk RNA-sequencing (RNA-seq) analysis revealed that DAC pre-treatment of murine C1498 AML cells induced expression of genes that were associated with inflammasomes and cell death molecules (Nlrp3, Il1b, Casp1, Casp4, Fas). Pretreatment of murine C1498 AML cells expressing human CD19 (C1498^hCD19 cells) with DAC significantly enhanced their sensitivity to murine BBz-CAR T cells directed against human CD19 (hCD19-CAR T cells). Furthermore, DAC pretreatment of immunocompetent mice bearing C1498^hCD19 cells, resulted in significantly enhanced in vivo antitumor activity of hCD19-CAR T cells, improving the overall disease-free survival. This beneficial effect was achieved in a second AML mouse model induced by MLL-AF9 fusion gene (MAF9). Mechanism studies showed that DAC priming of leukemia mice promoted the expansion of memory-like CAR T cells but restrained the generation of exhaustion-like CAR T cells. As a result, DAC-primed C1498 leukemia-bearing mice generated and maintained memory CAR T cells with robust self-renewal capability and long-term immunosurveillance capability. In the canonical NLRP3-CASP1-GSDMD pathway, damage-associated molecular patterns (DMAPs) induce NLRP3 oligomerization to activate CASP1. We observed that pharmacological inhibition of NLRP3 inflammasome-pyroptosis signaling, including NLRP3, GSDMD and pan-caspases, in DAC-primed tumor cells increases their resistance to CAR T cell cytotoxicity. GSDMD is the down-stream effector of the NLRP3 inflammasome pathway. Intriguingly, ablating GSDMD in C1498^hCD19 cells enabled them to resist CAR T cell therapy in vivo, indicating that the NLRP3-GSDMD-mediated pyroptosis plays crucial roles in CAR T cell eradication of tumors in DAC-primed AML-bearing mice. Finally, we found that DAC pretreatment of human AML THP-1 cells activated their expression of inflammasome gene program and enhanced their sensitivity to human CD33-CAR T cell cytotoxicity. In vivo DAC-priming of human xenograft THP-1 AML-bearing immunodeficient NSG mice resulted in significantly augmented therapeutic efficacy of human CD33-CAR T cells, leading to significantly overall survival rates of these leukemia mice. Collectively, our findings identified that the inflammasome-pyroptosis pathway in tumor cells to make them more vulnerable to CAR T cell cytotoxicity, thereby reducing tumor resistance and its-induced CAR T cell dysfunction. They also suggest that novel and translational strategies that can selectively activate the inflammasome-pyroptosis signaling in tumor cells could potentially enhance therapeutic efficacy of CAR T cells.