TP53-mutant myeloid neoplasms are distinct clinicogenomic entities characterized by poor survival. Chimeric antigen receptor (CAR) T-cell therapy might be a promising therapeutic option for TP53mut AML/MDS. However, the AML-intrinsic determinants of efficacy of T-cell-based approaches are largely unknown. We here address the highly relevant question as to whether, and if so, how TP53 deficiency in AML cells might confer resistance to CAR T-cell therapy.

Taking advantage of a recently developed, CRISPR/Cas9-engineered TP53 isogenic AML cell line harboring TP53 null, missense, or wildtype alleles as well as making use of CD33- and CD123-directed second generation CAR T-cells, we use a combination of in vitro flow cytometry-based co-incubation assays, live-cell imaging, gene expression profiling and an in vivo therapeutic model to address our objective.

We observed increased resistance of TP53mut AML cells to CAR T-cells in vitro (Panel A). CAR T-cells engaging TP53mut AML cells upregulated exhaustion markers resulting in longer time-to-killing (Panel B-C). TP53mut AML xenografted immunodeficient mice treated with CAR T-cells exhibited shorter survival compared to wildtype controls (Panel D). Transcriptional profiling revealed upregulation of the cholesterol pathway in TP53mut AML cells under CAR T-cell attack (Panel E). Simultaneously, CAR T-cells engaging TP53mut AML demonstrated a downregulated Wnt pathway (Panel F). Rational pharmacological targeting of either of these pathways - using simvastatin and BIO-acetoxime, respectively - rescued TP53mut AML cell sensitivity to CAR T-cell-mediated killing (Panel G). Similarly, CRISPR/Cas9-engineering of CAR T-cells to upregulate Wnt pathway signaling constitutively via Regnase-1 knockout rescued TP53mut AML cell sensitivity (Panel H).

We demonstrate that TP53 deficiency in AML cells confers resistance to CAR T-cell therapy by inducing CAR T-cell exhaustion. Furthermore, we identified the cholesterol pathway as a potential therapeutic vulnerability of TP53mut AML cells, and the Wnt pathway as a promising avenue to enhance the efficacy of CAR T-cell therapy. Our data suggest that pharmacological co-interventions or genetic engineering of CAR T-cell therapies may be a strategy towards more efficacious and tolerable cellular therapies for patients with TP53mut myeloid neoplasms.