Molecular Mechanisms Promoting Long-Term Cytopenia after BCMA CAR-T Therapy in Multiple Myeloma
Authors: Palacios-Berraquero Maria Luisa , Rodriguez-Marquez Paula , Calleja-Cervantes Maria Erendira , Berastegui Nerea , Zabaleta Aintzane , Burgos Leire , Alignani Diego , San-Martin Patxi , Vilas-Zornoza Amaia , Rodríguez Diaz Saray , Inogés Susana , López-Díaz De Cerio Ascensión , Huerga Sofia , Tamariz-Amador Luis-Esteban , Rifon Jose J. , Pierola Ana Alfonso , Lasarte Juan J , Paiva Bruno , Hernaez Mikel , Rodríguez-Otero Paula , San-Miguel Jesús F. , Ezponda Teresa , Rodriguez-Madoz Juan Roberto , Prosper Felipe 
Published: December 2024

Hematologic toxicity, specifically prolonged cytopenia, is a common side effect associated with CAR-T therapies, being particularly severe in patients with relapsed/refractory Multiple Myeloma (MM) (1). However, to date, its etiology is poorly understood. Thus, the main objective of this study was to identify and understand the underlying mechanisms of prolonged cytopenias associated with CAR-T therapy targeting BCMA.

In our work, we combined the retrospective analysis of a cohort of 48 patients treated with BCMA CAR-T cells at Clinica Universidad de Navarra, with ex vivo transcriptomic analysis using single-cell RNA sequencing, 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&gt;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 (2). Thus, supernatants from CAR-T cells or control T lymphocytes, co-cultured with MM tumor cell line U266, were added to the differentiation process. Phenotypic analysis by flow cytometry showed that supernatants from activated CAR-T cells (spCAR) halted HSPCs differentiation, promoting more immature phenotypes, with reduced expression of granulocytic, monocytic, and erythroid maturation markers, suggesting a paracrine effect related to CAR-T cells activation. Interestingly, this immature phenotype could be prevented by a combination of IFNγ, TNFα/β, TGFβ, IL-6, and IL-17 inhibitors, indicating the essential role of these cytokines and providing potential targets to prevent these cytopenias. Transcriptomic analyses at single cell level (scRNA-seq), performed using 10X Genomics technology, revealed that cells differentiated in the presence of spCAR were enriched in transcription factors related to early stages of hematopoietic differentiation, such as GATA2 RUNX1 and CEBPA. Additionally, gene regulatory network (GRN) analysis performed with SimiC (3), identified decreased activity of key regulons involved in neutrophil and monocytic maturation, such as MAFB and ID2, providing insights into the molecular mechanisms responsible for the observed phenotypic changes.

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 mechanisms promoting severe cytopenia observed after CAR-T therapy in MM and provides potential targets to prevent or decrease its severity.

References: Brudno JN., et al. Nature reviews. (2024). Berastegui N., et al. Nature Communications (2022). Peng J., et al. Communications Biology (2022).