Molecular mechanisms promoting long-term cytopenia after BCMA CAR-T therapy in multiple myeloma

Abstract

Hematologic toxicity is a common side effect of chimeric antigen receptor T-cell (CAR-T) therapies, being particularly severe among patients with relapsed or refractory multiple myeloma (MM). In this study, we characterized 48 patients treated with B-cell maturation antigen (BCMA) CAR-T cells to understand kinetics of cytopenia, identify predictive factors, and determine potential mechanisms underlying these toxicities. We observed that overall incidence of cytopenia was 95.7%, and grade >3 thrombocytopenia and neutropenia, 1 month after infusion, was observed in 57% and 53% of the patients, respectively, being still present after 1 year in 4 and 3 patients, respectively. Baseline cytopenia and high peak inflammatory markers were highly correlated with cytopenia that persisted up to 3 months. To determine potential mechanisms underlying cytopenias, we evaluated the paracrine effect of BCMA CAR-T cells on hematopoietic stem and progenitor cell (HSPC) differentiation using an ex vivo myeloid differentiation model. Phenotypic analysis showed that supernatants from activated CAR-T cells (spCAR) halted HSPC differentiation, promoting more immature phenotypes, which could be prevented with a combination of interferon γ, tumor necrosis factor α/β, transforming growth factor β, interleukin-6 (IL-6) and IL-17 inhibitors. Single-cell RNA sequencing demonstrated upregulation of transcription factors associated with early stages of hematopoietic differentiation in the presence of spCAR (GATA2, RUNX1, CEBPA) and a decrease in the activity of key regulons involved in neutrophil and monocytic maturation (ID2 and MAFB). These results suggest that CAR-T activation induces HSPC maturation arrest through paracrine effects and provides potential treatments to mitigate the severity of this toxicity.

Graphical abstract

Figure 1.

Long-lasting severe cytopenia developed in patients treated with BCMA CAR-T therapy. (A) Prevalence of grade ≥3 anemia, thrombocytopenia, and neutropenia at baseline (0) and at 1, 2, 3, and 6 months after infusion of CAR-T treatment. Evolution of aggregate hemoglobin (B), neutrophil (C), and platelet (D) counts from CAR-T infusion up to 1 year of follow-up.

Figure 2.

HSPCs differentiated in the presence of the supernatant of activated CAR-T cells presented less mature phenotypes. (A) Schematic representation of the ex vivo myeloerythroid differentiation model employed. CD34⁺ HSPCs were harvested and subjected to differentiation under 3 conditions, namely the addition of supernatant produced by the coculture of untransduced lymphocytes (spUTD), BCMA CAR-T cells (spCAR) to the MM tumoral cell line U266 for 48 hours, and control condition without the addition of supernatant. Cytokine production was measured in the supernatant. The phenotype obtained after 12 and 24 days of ex vivo differentiation of the HSPCs was studied using next generation flow cytometry. scRNA-seq was performed after 24 days of ex vivo differentiation. (B) Concentration of IFN-γ, TNF-α, IL-2, and IL-6 cytokines in the spUTD (blue) and activated BCMA CAR-T cells (red) after 48 hours of coculture with the MM cell line U266 at a 1:1 effector-to-target ratio (n = 3). (C) Proportion of HSPCs differentiated (n = 3) in the control (green), spUTD (blue), and spCAR (red) conditions. Analysis of less differentiated (upper panel) and more differentiated (lower panel) cells is shown for the 3 lineages, namely neutrophilic (CD10⁻; CD10⁺CD16⁺), monocytic (CD14⁻CD64⁺; CD14⁺CD35⁺), and erythroid (CD71⁺CD36⁻; CD71⁺CD36⁺) lineages. The proportion of cells that achieved mature myeloerythroid phenotypes was significantly lower in the spCAR group. (D) FACS gating results of HSPCs that were differentiated in the presence of spUTD (blue) or spCAR (red) at day 24 of differentiation. Gates of more differentiated cells are shown for neutrophilic (CD10⁺CD16⁺), monocytic (CD14⁺CD35⁺), and erythroid (CD71⁺CD36⁺) lineages, respectively. (E) The proportion of HSPCs after differentiated for 24 days (n = 2) in the control (green), spUTD (blue), spCAR (red), and spCAR with inhibitors mix (yellow) conditions. The mix included IL-6 inhibitor at working concentration of 0.1 mM, TGF-β inhibitor at 1 mM, IFN-γ inhibitor at 1 mg/mL, IL-17a inhibitor at 1 mM; and TNF-α-TNF-β inhibitor at 0.1 mg/mL. Welch tests for panel B and unpaired t tests for panel C were used. ∗P < .05; ∗∗P > .01.

Figure 3.

Characterization of differentiated CD34⁺ cells at single-cell level. scRNA-seq of ex vivo liquid culture differentiation samples of healthy CD34⁺ cells at day 24 after addition of spCAR or spUTD as control was performed. (A) An overview of the 8259 cells that passed quality control and filtering for subsequent analyses in this study. On the left, UMAP plot showing the 11 clusters that were analyzed and annotated. On the right, UMAP plot showing the distribution of cells from each condition (spUTD or spCAR). (B) Dot plot with the expression of canonical markers. (C) Violin plots of cell markers for mature neutrophils (MPO, PRTN3, ELANE, LTF, and MMP8) and precursors of neutrophils (RUNX1, GATA2, CXCR4, and CEBPA). (D) Violin plots of cell markers for mature monocytes (CD14, CXCL16, IL18, and CD74) and monocyte precursors (CLU, CCL23, VSTM1, and PADI4). (E) UMAP plot showing the expression of KIT and TPSAB1, which are mainly distributed within erythroid precursors. (F) Gene ontology analysis of granulocytes and erythroid precursor clusters corresponding to cells exposed to spUTD and spCAR, respectively, showed pathways that confirm these phenotypes. (G) Expression of the cytokine TGF-β, TNF, and IL-1 receptors families in neutrophils, precursors of neutrophils, monocytes, precursors of monocytes, and erythroid precursors.

Figure 4.

Analysis of GRNs in cells differentiated in presence of spCAR or spUTD. SimiC was applied to infer GRNs associated with more mature phenotypes or with those still in the differentiation process. (A) Heat map showing the regulatory dissimilarity score between cells exposed to spUTD and those exposed to spCAR of the different regulons within neutrophil lineage cells and monocytic lineage cells. (B) Histograms showing the activity score of KLF6 and CEBPB regulons, which are associated with neutrophilic lineage, and the IKZF1, MEF2C, and MAFB regulons, associated with monocytic lineage, for cells exposed to spUTD and those exposed to spCAR. (C) Representative scheme of cytokine-related genes that belong to the regulons KLF6, CEBPB, IKZF1, MEF2C, and MAFB. (D) Networks of the TFs KLF6, CEBPB, IKZF1, MEF2C, and MAFB and their top related genes.