(OA-01) TET2 and EZH2 Genetic Alterations Promote Malignant CAR-T Transformation in Anti-BCMA CAR-T Therapy

B-cell maturation antigen (BCMA)-directed chimeric antigen receptor (CAR) T cell therapy is an effective treatment option for patients with multiple myeloma (MM). Although rare, the development of secondary malignancies, including myeloid and T-cell neoplasms, is a cause for concern. Sporadic cases of T-cell malignancies have been reported in MM patients who have previously received CAR-T therapy. It remains unclear to what extent various factors, including CAR vector insertion, pre-existing somatic or germline mutations, and immune dysfunction, contribute to CAR-T-cell lymphomagenesis. Here we dissect the underlying mechanism of a CAR+ peripheral T-cell lymphoma (PTCL) with skin, peripheral blood (PB), bone marrow (BM), and lymph node involvement following anti-BCMA CAR-T therapy.

Methods:

We isolated CAR+ peripheral T-cell lymphoma (PTCL) from PB and performed single cell sequencing, RNA sequencing, and functional experiments to characterize the secondary malignancy post anti-BCMA CAR-T therapy. To dissect which genomic feature contributed to the secondary malignancy we generated CAR-T cells and knocked down mutational targets. We used BCMA expressing cell lines to elicit an antigen specific response of lab generated CAR-T cells and isolated tumor CAR-T cells.

Results:

Six months post-infusion, a 51-year-old male presented with a facial lesion and lymphocytosis due to a CD4-/CD8- PTCL involving skin, blood, and bone marrow. Our comprehensive genomic analysis revealed the development of new TET2 and EZH2 mutations associated with clonal hematopoiesis of indeterminate potential (CHIP) and T-cell lymphomas. We generated CAR-T cells from the patient at the time of apheresis and isolated malignant CAR-T cells from the PB for comparison of function. Antigen specific stimulation with BCMA cell lines revealed loss of function in cytokine production capacity with no response seen in malignant CAR-T cells. The cytotoxic capability of malignant tumor CAR-T cells against BCMA expressing cells was decreased by 98% compared to cells generated from PB harvested prior to CART therapy (P < 0.01). To quantify the individual effects of genomic mutations we generated EZH2 and TET2 CAR-T mutants. EZH2 inhibition in CAR-T cells increased CD69, CD38 and CD127 while reducing FAS expression resisting apoptosis. TET2 knock down in CAR-T cells resulted in enhanced proliferation and enhanced expansion compared to control CAR-T cells. Knock down of TET2 and EZH2 resulted in a decrease in functional killing. EZH2 knockdown mutant resulted in a 28% reduction of cytotoxicity while TET2 knock down reduced killing by 53% when compared to control CAR-T cells (p < 0.05).

Conclusions:

TET2 and EZH2 mutations result in epigenetic changes such as open chromatin, leaving the genome more vulnerable to additional alterations. TET2 and EZH2-loss of function mutations in CAR-T cells provide advantages in proliferation and regeneration promoting malignant CAR-T transformation following anti-BCMA CAR-T therapy.