Molecular mechanisms promoting long-term cytopenia after BCMA CAR-T therapy in Multiple Myeloma

Abstract

ABSTRACTHematological toxicity is a common side effect of CAR-T therapies, being particularly severe in relapsed/refractory multiple myeloma (MM) patients. In this study, we analyzed a cohort of 48 patients treated with BCMA CAR-T cells to characterize the kinetics of cytopenia, identify predictive factors and determine potential mechanism underlying these toxicities. The overall incidence of cytopenia was 95.74%, and grade>3 thrombocytopenia and neutropenia one month after infusion was observed in 57% and 53% of the patients and was still present after 1 year in 4 and 3 patients respectively. Presence of cytopenia at baseline and high peak inflammatory markers highly correlated with cytopenia persisting up to three months. To determine potential mechanisms underpinning cytopenias, we evaluated the paracrine effect of BCMA CAR-T cells on the differentiation of HSPCs using anex-vivomyeloid differentiation model. Phenotypic analysis showed that supernatants from activated CAR-T cells (spCAR) halted HSPCs differentiation promoting more immature phenotypes, with reduced expression of granulocytic, monocytic and erythroid markers. Single-cell RNAseq demonstrated an upregulation of transcription factors associated with early stages of hematopoietic differentiation in the presence of spCAR (GATA2, RUNX1andCEBPA)and decreased activity of key regulons involved in neutrophil and monocytic maturation (ID2andMAFB). Our results suggest that CAR-T cell activation negatively influences hematopoietic differentiation through paracrine effects inducing arrest of HSPCs maturation and contributes to the understanding of severe cytopenia observed after CAR-T cell treatment in MM patients. These results may identify regulatory mechanisms involved in alter hematopoiesis and could lead to alternative therapeutic strategies.KEY POINTSLong-lasting cytopenia after BCMA CAR-T therapy correlates with baseline cytopenia and peak inflammatory markers.Supernatants from activated BCMA CAR-T cells induced an inhibition of ex-vivo myeloid differentiation and rewiring of transcriptional programs associated with hematopoietic differentiation.