Hematological 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 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. Single-cell RNAseq demonstrated an upregulation of transcription factors associated with early stages of hematopoietic differentiation in the presence of spCAR (GATA2, RUNX1 and CEBPA) and decreased activity of key regulons involved in neutrophil and monocytic maturation (ID2 and MAFB). 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 POINTS

Long-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.

The authors have declared no competing interest.

This study was supported by the Instituto de Salud Carlos III co-financed by European Regional Development Fund-FEDER A way to make Europe (PI19/00726, PI19/00922, PI20/01308, PI22/01044 and PMPTA22/00109). Red de Terapia Celular TERCEL (RD16/0011/0005). Red de Terapias Avanzadas TERAV (RD21/0017/0009). Centro de Investigacion Biomedica en Red de Cancer CIBERONC (CB16/12/00489 and CB16/12/00369). Ministerio de Ciencia e Innovacion co-financed by European Regional Development Fund-FEDER A way to make Europe (PID2022-137914OB-I00). European Commission (H2020-JTI-IMI2-2019-18: Contract 945393; SC1-PM-08-2017: Contract 754658; and H2020-MSCA-IF-2019: Grant Agreement 898356). Gobierno de Navarra (DESCARTHeS: 0011-1411- 2019-000079 and 0011-1411-2019-000072; AGATA: 0011-1411-2020-000011 and 0011-1411-2020- 000010; SOCRATHeS: 0011-1411-2022-000053 and 0011-1411-2022-000088; DIAMANTE: 0011- 1411-2023-000105 and 0011-1411-2023-000074; and alloCART-LMA: PC011-012). Asociacion Espanola Contra el Cancer-AECC (INVES19059EZPO). Cancer Research UK [C355/A26819], FC AECC and AIRC under the Accelerator Award Program. Paula and Rodger Riney Foundation. Supported by PhD fellowships from Gobierno de Navarra (0011-0537-2019-000001: N.B.) and from Ministerio de Ciencia, Innovacion y Universidades (FPU19/06160: P.R-M).

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Ethics Committee of the University of Navarra gave ethical approval for this work

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All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. The scRNA-seq data generated in this study have been deposited in the GEO database (GSE250444).