Pomalidomide Modulates Transcription Networks Regulating Human Erythropoiesis and Globin Switching: Implications for Treatment of Hemoglobinopathies

Sickle cell disease (SCD) represents a major challenge in hematology, with approximately 100,000 Americans afflicted and the annual number of newborns with SCD set to rise over the next 40 years worldwide. Current treatment approaches rely on increasing levels of fetal hemoglobin (HbF) to prevent painful vaso-occlusive crises and hemolysis secondary to red cell sickling. Hydroxyurea remains the only pharmacologic intervention approved for SCD; however, it has limited efficacy and carries significant side effects such as myelosuppression. Thus, there is a critical need to develop drugs that enhance HbF production without similar dose limiting side effects. Second generation immunomodulatory drugs, such as pomalidomide, are a class of emerging HbF inducers both in vitro and in vivo. Recent work from our laboratory revealed that hydroxyurea and pomalidomide differentially regulate HbF production in CD34+ cells undergoing erythroid differentiation using a 3-phase culture system. Pomalidomide, but not hydroxyurea, was found to decrease BCL11A expression through a yet to be defined mechanism. In the present study, we sought to characterize erythropoiesis and the expression of key transcription factor networks in this 3-phase culture system to determine the mechanisms underlying pomalidomide’s effect. Following a four day expansion period, isolated CD34+ cells from the peripheral blood of SCD or normal individuals were differentiated along erythroid lineage in the presence of pomalidomide (1μM) or DMSO (vehicle) for 14 days. As an additional control, CD34+ cells were also treated with hydroxyurea (10μM). Proliferation and erythroid differentiation were assessed at 7, 11 and 14 days of culture. Although a 50% decrease in cell growth was noted in cells treated with hydroxyurea, no such decrement was found in control, DMSO and pomalidomide-treated cells. Moreover, pomalidomide produced a transient delay in erythroid differentiation between days 6 and 11 of culture phenotypically documented by flow cytometric analysis using glycophorin-A, α-4 integrin and band 3 as surface markers monitoring erythroid differentiation as well as morphologically by May-Grunwald Giemsa staining. In contrast, cells treated with hydroxyurea demonstrated accelerated differentiation, compared to the control cultures. However, by day 14 of culture, no significant difference was observed under any condition, suggesting that the delayed cells eventually finished terminal differentiation. In terms of HbF induction, we confirmed elevated production in the cultures with pomalidomide by measuring the number of F-cells by flow cytometry. We also evaluated the production of γ-globin chains by qRT-PCR and western blot at D4 and D11 and found a dramatic increase in the production of γ-globin, in both SCD and normal samples treated with pomalidomide.

We posited that pomalidomide might foster changes in transcription factors known to play a role in both erythropoiesis and globin switching. To this end, we evaluated the expression kinetics of BCL11A, SOX6, KLF1, MI2β, GATA1 and FOG1 via qRT-PCR and western blot analyses. In DMSO-treated cultures the above transcription factors were maximally expressed between days 6-8, and their levels diminished during the remainder of the culture. Conversely, pomalidomide markedly decreased BCL11A, SOX6, KLF1 and MI2β between days 4 and 6 in cultures of both SCD and normal samples. In line with our results, MI2β acts as a positive regulator of BCL11A and KLF1, and previous studies have shown that its knock down in CD34 cells leads to decreased levels of BCL11A and KLF1. Western blot analyses confirmed the qRT-PCR data. Further, the divergent expression patterns correlated temporally with the differentiation delay suggesting that pomalidomide modulates expression of members of the BCL11Atranscription factor complex, thereby augmenting γ-globin production.

Taken together, these data provide evidence that pomalidomide influences erythropoiesis by modulating transcription factor expression in CD34+ cells differentiated in the 3-phase culture system, leading to a decrease in BCL11A and activation of γ-globin production. Importantly, further exploration of these pathways that function to regulate erythropoiesis and promote HbF silencing, may help elucidate the mechanism of action of pomalidomide as well as identify additional druggable molecules.