Luspatercept Promotes Heme Biosynthesis and Erythroblast Island Formation in a Novel Low-Risk MDS Mouse Model

Myelodysplastic syndromes (MDS) are a group of diseases of hematopoietic stem cells (HSCs) that occur in the aging population and following therapy for unrelated cancers. Anemia-related symptoms caused by ineffective erythropoiesis and reduced RBC and hemoglobin levels are a serious burden reported by most MDS patients that has yet to be resolved by erythropoiesis-stimulating agents.

We previously reported the role of mitochondrial metabolism in MDS pathophysiology, whereby suppression of mitochondrial activity leads to the accumulation of oncometabolites that competitively inhibit α-ketoglutarate-dependent dioxygenases. This results in the hypermethylation of epigenomes and activation of hypoxia-inducible factor 1A (HIF1A), which causes a pseudohypoxia state that can ultimately drive MDS pathogenesis. Our recent informatics analyses of primary MDS samples identified a patient subset with high TGF-β signaling in which mitochondrial complex II (MC II) or succinate dehydrogenase (SDH) genes were downregulated. To investigate whether SDH dysregulation alone can contribute to MDS pathobiology, we generated a doxycycline-inducible mouse model that expresses shRNAs against the SDH subunits in blood cells. These mice showed reduced MC II, HIF1A upregulation, and buildup of intracellular succinate, a well-known oncometabolite in c-Kit⁺ bone marrow (BM) cells. We observed that knocking down Sdh is sufficient to induce hallmark features of MDS including pancytopenia, trilineage dysplasia, ring sideroblasts, and iron overload in the spleen while serial CFU plating and competitive transplantation assays revealed low multilineage differentiation and reconstitution potential of BM hematopoietic stem and progenitor cells (HSPCs). The anemic phenotype was primarily caused by a maturation block and accumulation of immature erythroid progenitors (EPs) and decreased downstream RBCs. Interestingly, we found that removing doxycycline and restoring Sdh expression rescues macrocytic anemia among other MDS phenotypes, and establishes the critical role of mitochondrial respiration in HIF1A activation and MDS disease progression. Finally, we found that treatment of SDH-KD mice with luspatercept, a TGF-β ligand trap recently approved by the FDA for MDS ameliorated anemia (but not EPO) and restored the BM HSC compartment. To investigate the mode of action of luspatercept, we performed scRNA-seq on drug-treated SDH-KD HSPCs and found a significant upregulation of OXPHOS and serine/glycine biosynthetic processes in EPs and a subset of macrophages characterized by erythroblast island (EBI) macrophage markers including EpoR, CD169, CD163, and Mertk. Thus, using our novel MDS model, we found that luspatercept alleviates anemia by promoting heme biosynthesis and EBI formation to support erythropoiesis in the BM and spleen. Flow cytometry and BM imaging of drug-treated SDH-KD mice revealed an increase of foamy central macrophages directly associated with BM sinusoids that were actively engulfing nucleated reticulocytes. This EPO-independent mode of action of luspatercept was further tested in a sickle cell mouse model and was found to be effective in treating anemia similarly by increasing the total percentage of EBIs.

In conclusion, our results indicate that pseudohypoxia and high TGF-β signaling-associated changes in respiration and/or metabolic regulation underlie MDS pathogenesis. Restoration of SDH or blocking TGF-β signaling by luspatercept treatment reverses anemia by promoting heme biosynthesis in EPs and expanding a subset of macrophages for EBI formation. Finally, elucidating the role of MC II in MDS may provide alternative targets that can be combined with the current standard of care HMA and luspatercept therapy tailored for different subtypes and stages of MDS.