Treatment with luspatersept exerts a pleiotropic effect on the molecular signatures of bone marrow cell subsets Free

Luspatercept (LUSPA) a transforming growth factor-beta (TGF-β) superfamily inhibitor is a novel agent for treating the anemia of patients with lower-risk Myelodysplastic Neoplasms (LR-MDS). Although late erythroblasts (Ery) are considered as the target cells of LUSPA (Suragani RNVS et al, Nat Med 2014) limited data exist regarding its impact on the transcriptome of other cell types.

We investigated the effect of LUSPA therapy on the molecular signature of FACS sorted bone marrow hematopoietic stem and progenitor cells (HSPCs), erythroblasts (Ery) and CD3+ T cells from five patients with LR-MDS, by utilizing single cell transcriptomic analysis (10x Genomics). All patients harbored SF3B1 mutations; two patients had mutations in TET2, one in DNMT3A and one in RUNX1.

In pretreatment samples cells were partitioned into 12 cell clusters and annotated based on their distinct gene expression profile. Pathway enrichment analysis (EnrichR) of each cluster's molecular signature identified enhanced TGF-β signaling in HSPCs and myeloid-primed progenitor clusters (My P), rather than erythroblast (Ery) or megakaryocyte-erythroid progenitors (MEP), indicating that HSPCs could also be targeted by LUSPA.

After treatment with LUSPA we observed a downregulation of the expression of genes associated with TNF signaling via NF-κB and interferon-γ (IFN-γ) signaling in all clusters except from the late erythroblast (Late Ery) cluster. Downregulation of genes associated with cholesterol homeostasis, a pathway associated with proliferation and myeloid bias of hematopoietic progenitors (Mitroulis et al, Cell, 2018), was also observed in the clusters of HSPC, multipotent progenitors (MPP) and myeloid-biased progenitors (My P). Of note, downregulation of genes associated with TGF-β signaling was observed in the clusters of HSCs, myeloid progenitor (My P), and megakaryocyte-erythroid progenitors (MEP), but not in erythroblasts (Ery). Increased cell proliferation signatures, such as the G2M cell cycle pathway and E2F targets, were also observed after Luspatercept in MEPs and myeloid progenitor clusters (My P).

Members of the TGF-β superfamily are known to suppress anti-tumor immunity by actin on T cells (Pinjusic K et al, J Immunother Cancer 2022), therefore we also analyzed T cells.

In pretreatment samples, cells were partitioned into 11 cell clusters, including 5 clusters of CD8+ T cells, 4 clusters of CD4+ T cells, a cluster of proliferating cells and a cluster of progenitor T cells. After treatment with LUSPA we observed an increase in the cytotoxic score in three cytotoxic T lymphocyte (CTL) cell clusters (IFN-CTL, KLRB1-CTL and NKG7-CTL). Specifically, there was a significant upregulation in the expression of GZMA, GZMH, NKG7, CX3CR1, GNLY in NKG7-CTL cluster, of GZMA, GZMK in KLRB1-CTL and GZMA and GZMH in IFN-CTL. Additionally, there was a downregulation in the exhaustion score of the NKG7-CTL cluster. Regarding cell exhaustion score, genes that were downregulated after treatment with LUSPA included TNFRSF1B, TNFRSF9, FAM3C, IFNG, LYST, and GBP2. IFN signaling has been previously associated with cytotoxic activity in bone marrow CD8+ T cells in MDS (Tasis et al, Cancer Res Commun, 2024)To this direction, we observed an increase in the IFN score of the NKG7-CTL cluster, which was associated with an upregulation in the expression of IFITM1, IFIT3, OASL, XAF1, LY6E, IRF9, MX1, and EIF2AK2. Also, in the Treg-like cluster pathway analysis showed that LUSPA treatment enhanced the IFN response signature associated with the upregulation in the expression of SAMD9L, EIF2AK2, OASL and downregulated the apoptosis signature associated with a decrease in the expression of DNAJA1, JUN, DDIT3, SAT1, BIRC3. In the other CD4+ T cell clusters, the upregulation of IFN response signature was also observed in IL7R-CD4+ and CCR7-CD4+ clusters.

Taken together our preliminary findings point to a direct effect of LUSPA on HSPC subsets by modulating the expression of genes associated with inflammatory, cholesterol, TGF-β and cell proliferation pathways, but also on T cells by enhancing the cytotoxic signature of CTL, paired with the induction of an IFN related response in several T cell clusters. Our results are consistent with the reported trilineage response after LUSPA (Garcia-Manero G et al, Blood 2022) and suggest a pleiotropic effect of LUSPA potentially including a beneficial immunomodulation in patients with MDS.