Sotatercept (ACE-011) is an activin type IIA receptor (ActRIIA) fusion protein which antagonizes activin and is currently being evaluated in clinical trials for the treatment of cancer-related bone loss and anemia. Administration of sotatercept has been associated with an increase in the hemoglobin (Hb) and hematocrit (Ht) levels in preclinical and clinical studies, yet the mechanisms underlying the effects of this drug on erythropoiesis remain unknown. We, therefore, addressed the potential mechanisms underlying the effects of sotatercept on erythroid lineage by investigating its direct and indirect influences on in vitro erythropoiesis.
The direct effects of sotatercept on the ability of human primary CD34+ cells to form erythroid (Ery) colonies and to generate Ery cells in liquid cultures was examined. Neither the number, the size nor the morphology of BFU and CFU-E colonies were affected by sotatercept treatment. Likewise, the percentage of CD36+/Glycophorin (GPA)+ cells generated in liquid cultures treated with sotatercept was similar to that observed in control cultures. We further showed that treatment with activin induced Ery differentiation of K562 erythroleukemic cells and this effect was counteracted by sotatercept, which is in agreement with its function as activin trap. By contrast, activin alone or in combination with sotatercept did not stimulate Ery differentiation of primary CD34+ cells, suggesting that sotatercept does not affect in vitro erythropoiesis directly or through its interaction with activin.
Since stromal cells (SC) are both a major source of activin and important players in the regulation of hematopoiesis we next examined if sotatercept might influence the ability of BM-derived SC to affect erythropoiesis. Conditioned media (CM) produced by a heterogeneous population of SC inhibited Ery differentiation of CD34+ cells (i.e. the percentage of CD36+/GPA+ cells was 5-fold lower in cultures containing SC-CM as compared to control) and skewed their phenotype towards the myeloid lineage. SC-mediated inhibition of erythropoiesis was associated with the maintenance of a CD34+ cells phenotype (i.e. twice as many cells were CD34+in cultures differentiated in the presence of SC-CM as compared to control) and with the abrogation of the Ery gene signature, including a drastic suppression of most Hb and glycophorin encoding genes as well as genes involved in Hb stabilization and heme metabolism.
When identical experiments were performed in the presence of CM produced by SC treated with sotatercept, the ability of SC-derived CM to inhibit erythropoiesis was significantly diminished. CD34+ cells differentiated in media conditioned by sotatercept-treated SC, generated 1.7-fold more CD36+/GPA+ Ery progenitors (p value ≤ 0.005) and 1.3-fold more mature GPA+ eryrthroblasts (p value ≤ 0.05) as compared to those generated in the presence of CM from untreated SC. In addition, they formed 1.4 times greater number of BFU-E colonies than that observed in the presence of CM derived from untreated SC. Importantly, CM from sotatercept-treated SC did not affect other hematopoietic lineages (e.g. expression of CD33, CD14 or CD61). These observations indicated that treatment with sotatercept modulated SC function to create a milieu of soluble factors which are more permissive for erythropoiesis.
To indentify these potential factors we performed gene expression profiling of SC cultured in the presence or absence of sotatercept. The number of genes significantly affected by the drug was relatively small, but included genes encoding proteins which are known for their erythropoiesis-stimulatory roles (e.g. Angiotensin II, SDF1, BMP6 and IGFBP2), proteins which are potent inhibitors erythropoiesis (e.g. VEGF) and proteins with less well defined effects on erythroid lineage (e.g. BMP2, Oncostatin and Il-6). We validated these findings by measuring the levels of these secreted proteins, as well as of activin levels in the SC-CM, and demonstrated that sotatercept modifies the secretory profile of SC.
We propose that SC-derived negative signals may represent an important component which contributes to the regulation of erythropoiesis. The stimulatory effects of sotatercept on red cell production, therefore, appear to be a consequence of its ability to counteract such inhibitory signals and to induce the production of a variety of Ery-promoting factors by the BM microenvironment.
Iancu-Rubin:Celgene: Research Funding. Hoffman:Celgene: Research Funding.
Asterisk with author names denotes non-ASH members.