Leukemia-intrinsic determinants of CAR-T response revealed by in vivo genome-wide CRISPR screening [scRNA-seq]

CAR-T cells are a promising new treatment for B cell malignancies. However, the majority of patients inevitably go on to experience disease relapse through largely unknown means. To investigate leukemia-intrinsic CAR-T resistance mechanisms, we performed genome-wide CRISPR-Cas9 loss-of-function screening in an immunocompetent murine model of B cell acute lymphoblastic leukemia (B-ALL). We identified IFN??/JAK/STAT signaling and components of the antigen processing and presentation pathway as key mediators of resistance to anti-CD19 CAR-T therapy in vivo, but not in vitro. Transcriptional characterization of this model demonstrated an upregulation of these pathways in CAR-T treated relapsed tumors, and examination of data from CAR-T treated B-ALL patients revealed an association between poor outcomes and increased expression of JAK/STAT/MHC-I in leukemia cells. Together, our data identify an unexpected mechanism of resistance to immunotherapy, in which tumor cell interaction with IFN??-secreting CAR-T cells in vivo activates the expression of an MHC-I inhibitory T cell program. Overall design: Single cell RNAseq comparison of 3 biological replicates each of mouse B-ALL cells isolated from either spleen or bone marrow challenged with CAR-T cells targeting either CD19 or a EGFRvIII