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

Hematological toxicity is a common side effect of CAR-T therapies, 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 95.7%, and grade>3 thrombocytopenia and neutropenia, one month after infusion, was observed in 57% and 53% of the patients, being still present after one 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 HSPCs differentiation using an ex-vivo myeloid 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, which could be prevented with a combination of IFNγ, TNFα/ß, TGFß, IL-6 and IL-17 inhibitors. Single-cell RNA-seq demonstrated upregulation of transcription factors associated with early stages of hematopoietic differentiation in the presence of spCAR (GATA2, RUNX1, CEBPA) and decreased activity of key regulons involved in neutrophil and monocytic maturation (ID2, MAFB). Our results suggest that CAR-T cell activation negatively influences hematopoietic differentiation through paracrine effects inducing HSPCs maturation arrest. Moreover, our study contributes to the understanding of severe cytopenia observed after CAR-T therapy in MM and provides potential treatments to prevent or decrease its severity.

The transcription factor DDIT3 is a potential driver of dyserythropoiesis in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are hematopoietic stem cell (HSC) malignancies characterized by ineffective hematopoiesis, with increased incidence in older individuals. Here we analyze the transcriptome of human HSCs purified from young and older healthy adults, as well as MDS patients, identifying transcriptional alterations following different patterns of expression. While aging-associated lesions seem to predispose HSCs to myeloid transformation, disease-specific alterations may trigger MDS development. Among MDS-specific lesions, we detect the upregulation of the transcription factor DNA Damage Inducible Transcript 3 (DDIT3). Overexpression of DDIT3 in human healthy HSCs induces an MDS-like transcriptional state, and dyserythropoiesis, an effect associated with a failure in the activation of transcriptional programs required for normal erythroid differentiation. Moreover, DDIT3 knockdown in CD34+ cells from MDS patients with anemia is able to restore erythropoiesis. These results identify DDIT3 as a driver of dyserythropoiesis, and a potential therapeutic target to restore the inefficient erythroid differentiation characterizing MDS patients.