Identification of molecular mechanisms promoting long-term cytopenia after BCMA CAR-T therapy in Multiple Myeloma
Hematological toxicity is a common side effect of CAR-T cell therapies, being particularly severe in relapsed/refractory multiple myeloma (MM) patients treated with CAR-T cells. In this work, 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 thrombopenia and neutropenia persisting one month after infusion was reported in 57% and 53% of the 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 an ex-vivo myeloid differentiation model. Phenotypic analysis showed that supernatants from activated CAR-T cells (spCAR) halted HSPCs differentiated 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, RUNX1 or CEBPA) and a decrease activity of key regulons involved in neutrophil and monocytic maturation (ID2 and MAFB). Overall, our study contributes to the understanding of severe cytopenia observed after CAR-T cell treatment of MM patients, providing potential molecular drivers. Our results suggest that CAR-T cell activation would negatively influence hematopoietic differentiation through paracrine effects inducing arrest of HSPCs maturation. Identification of regulatory mechanism involved in alter hematopoiesis could led to new therapeutic strategies. Overall design: In the current study, we characterized the kinetics of cytopenia in a cohort of patients with R/R MM receiving BCMA CAR-T cell treatment, evaluating their correlation with clinical and laboratory parameters. To dwell into the mechanisms of these cytopenias, we studied the effect of activated CAR-T cell supernatant on ex-vivo hematopoietic differentiation. Our results using phenotypic and single cell transcriptional studies allowed us to identify abnormal transcriptional and regulatory programs involved in hematopoietic differentiation, providing molecular mechanistic insights driving prolonged cytopenia.