Chimeric antigen receptor T cell (CART) therapy has revolutionized cancer treatment by modifying patient T cells to target and destroy cancer cells. Despite its promise, treatment relapse is a prevalent issue. FDA-approved CART products against B cell malignancies utilize either CD137 or CD28 costimulatory domains. Preliminary studies in our lab have found that CD137-costimulated CARTs exhibit the ability to target cells lacking the conventional CD19 antigen under certain conditions, hinting at an alternative cytotoxic pathway. 

A genome-wide CRISPR-Cas9 knock-out screen was performed on target-negative K562 cells, a model myeloid leukemia cell line, to characterize this unknown mechanism. K562 cells were edited to express Cas9 nuclease, and a genome-wide gRNA library was transduced to knock out individual genes. Concurrently, CART cells were created by inserting either CD137 or CD28 costimulatory domains into T lymphocytes and activated by target-presenting cells. 

Cytotoxic killing assays were performed on K562 cells with activated CART cells and remaining K562 genomic DNA was sequenced. MAGeCK analysis revealed gRNAs that persisted when comparing CD137-exposed cells to CD28-exposed control, with several top hits being mitochondrial related, including MRPL4, COASY, and MPV17L2. Overrepresentation of these genes indicate their significance in K562 cell survival and thus the novel killing mechanism performed by CD137-co-stimulated CART cells. Further experiments involving single gene KOs and common pathways will be done to confirm the contribution of selected genetic factors. These insights uncover potential genetic mutations conferring resistance to treatment, allowing for enhanced patient stratification and personalized treatment plans to improve cancer patient outcomes.