Pomalidomide and Dexamethasone Regulate Human Erythroid Progenitor Signaling through Two Distinct Pathways

With the exception of cytokines, corticosteroids such as dexamethasone (Dex) and the immunomodulatory drugs (IMiDs) such as pomalidomide (Pom) are the primary pharmacologic agents that directly improve red cell production in patients with disordered erythropoiesis. Corticosteroids are the standard of care for patients with Diamond Blackfan anemia (DBA) while the IMiDs are used for patients with myelodysplastic syndrome (MDS) to improve the profound anemia seen in these patients. While it is generally accepted that loss of erythroid progenitors such as burst forming unit-erythroid (BFU-E) and colony forming unit-erythroid (CFU-E) underlie congenital and acquired bone marrow failure syndromes such as DBA and MDS, our limited understanding of the regulatory programs governing erythroid progenitor self-renewal and differentiation hinder development of more specific therapies aimed at increasing erythroid progenitor numbers in these diseases. To investigate the dynamics of erythroid progenitors in normal and diseased states, we used an in vitro culture system consisting of 3 phases to study human erythropoiesis in the presence of Pom or Dex.

Our earlier work provided evidence that Pom transcriptionally reprograms adult erythroid progenitors, and delays the BFU-E to CFU-E transition; this delay is through a yet to be characterized mechanism. To elucidate the effects of Pom on erythroid progenitors we differentiated adult CD34+ hematopoietic stem and progenitor cells (HSPCs) and performed gene expression microarray analyses on erythroid cells at day 4. We found that Pom induced significant expression changes in 20 genes including TEA domain transcription factor 2 (TEAD2), a transcription factor involved in stem cell self-renewal and hippo signaling. To date, a function for TEAD2 in human erythropoiesis has not been identified. qRT-PCR and western blot studies validated that Pom induces a greater than 100-fold increase in TEAD2 expression compared to vehicle control. Previous studies have shown that members of the TEAD family cooperate with cytokines such as transforming growth factor-β (TGF-β) and Bone Morphogenic Protein 4 (BMP4), two known modulators of erythropoiesis. We observed that Pom did not affect the levels of phospho-SMAD2 by western blot analysis, a downstream effector of TGF-β. In marked contrast, Pom-treated cells exhibited higher levels of p-SMAD1/5/9 suggesting that Pom may enhance or activate the BMP4 pathway. These data suggest that Pom leads to the self-renewal of BFU-E through the activation of TEAD2. We are currently investigating the downstream signaling pathways involved.

The role of Dex in human erythropoiesis is more controversial. Previous studies, particularly in murine models have suggested that the effects of Dex occur at the BFU-E stage. However, in our studies, we found that Dex acts at the level of CFU-E in models of human erythropoiesis. We found that Dex promotes both the self-renewal as well as the differentiation of CFU-E. We found that treatment with dexamethasone resulted in a 4-fold increase in the percentage of CFU-E in culture when compared to vehicle control. Interestingly, these effects vary with the type of HSPC used (peripheral blood vs. cord blood). Indeed, while Dex induced extensive proliferation of adult CFU-Es, it had no effects on CFU-E from cord blood. These results suggest a specificity for corticosteroids during development and suggest that these drugs may prove more useful in certain types of anemia. Finally, clinical data from three different DBA patients demonstrated a response to steroids within a week of treatment, as measured by an increase in reticulocytes suggesting that the population responding to treatment is indeed the CFU-E, reinforcing our in vitro findings. We are now investigating the molecular mechanisms involving the effects of dexamethasone on erythroid progenitors.

Together, these results suggest that Pom and Dex may influence erythroid progenitor self-renewal and differentiation through independent mechanisms and that combination therapy may be beneficial for a variety of diseases characterized by disordered erythropoiesis.