Genomic alterations in TET2 and EZH2 serve as critical drivers of clonal malignant evolution in anti-BCMA CAR-T therapy for multiple myeloma.

Abstract Background: B cell maturation antigen (BCMA) targeted Chimeric antigen receptor (CAR) T cell therapy has emerged as a transformative treatment for patients with relapsed or refractory multiple myeloma (RRMM), demonstrating deep and durable responses. Nevertheless, emerging reports of secondary hematologic malignancies, particularly of T-cell origin, introduce a new dimension of complexity to the safety profile of these engineered cell therapies. We recently reported a patient with CD4 and CD8 negative PTCL responding to targeted therapy including anti-CCR4 antibody (Aleman et al, NEJM, 2025). This and other sporadic reports of T-cell neoplasms following CAR-T infusion in MM patients necessitate deeper investigation into the pathophysiology, which likely involves a multifactorial interplay between vector integration, clonal hematopoiesis and immune perturbations induced by lymphodepletion or viral infections. Here, we delineate the pathogenesis in our patient with CAR+ peripheral T-cell lymphoma with involvement of the skin, peripheral blood (PB), and bone marrow (BM) emerging in the aftermath of anti-BCMA CAR T cell 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 the genomic drivers that contributed to the secondary malignancy we generated CAR-T cells from healthy donors and from the PTCL patient and knocked down mutational targets. We characterized the functional result of TET2 and EZH2 in single and double knock down mutants byusing BCMA expressing cell lines to elicit an antigen specific response of lab generated CAR-T cells. Results A 51-year-old male presented with a facial lesion and lymphocytosis due to a CD4-/CD8- PTCL involving skin, blood, and bone marrow six months post anti-BCMA CAR-T infusion, Single cell sequencing and bulk RNA sequencing of PTCL tumor revealed the development of new mutations in TET2 and EZH2 which are associated with clonal hematopoiesis of indeterminate potential (CHIP) and T-cell lymphomas. We generated CAR-T cells from a healthy donor and the PTCL patient at the time of apheresis and compared functionality to isolated malignant CAR-T cells from the PB. Antigen specific stimulation with BCMA expressing cell lines revealed loss of function in cytokine production capacity in malignant CAR-T cells not seen in control CAR-T. 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 discern the contribution of genomic mutations we generated EZH2 and TET2 CAR-T mutants from healthy donor and PB harvested prior to CART therapy from our PTCL patient. 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 increased proliferation and doubled the expression of in CCR4+ compared to control. CCR4+ is a skin homing chemokine which was expressed in all our patient's malignant CAR-T tumor cells and served as an indicator for treatment with mogamulizumab. EZH2 and TET2 double mutant almost tripled the expression of CCR4 compared to control and caused a loss of CD7 in 73% of cells. Loss of CD7 is hallmark of T cell lymphomas. 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). Double knock down of TET2 and EZH2 further decreased overall cytotoxicity by 67% (p<0.01). 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 while promoting malignant CAR-T transformation following anti-BCMA CAR-T therapy.