MLL1 Complex Is a Critical Regulator of Fetal Hemoglobin Repression

Increasing fetal-type hemoglobin (HbF) expression in adult erythroid cells represents a promising strategy for treating β-globinopathies including sickle cell disease and β-thalassemia. However, the mechanisms responsible for the repression of HbF expression in adult erythroid cells remain unclear. We have identified MLL1 complex as a critical regulator of HbF repression. Knockdowns of MEN1 and KMT2A encoding critical components of the complex by lentiviral shRNAs caused dramatic increases in γ- and ε-globin mRNA levels (up to 15- and 22-fold respectively) without causing major changes in β-globin expression in HUDEP-2 cells. Interestingly, we found that MEN1 and KMT2A knockdowns also induced a significant downregulation of BCL11A mRNA levels in these cells, suggesting that BCL11A may be a direct target of MLL1 complex. In supporting this notion, significant binding of MEN1 and KMT2A were readily detected at the promoter and a critical enhancer of BCL11A. We further tested these findings in normal human CD34⁺ hematopoietic stem and progenitor cells (HSPCs) induced to undergo erythroid differentiation in culture. Consistent with regulation of BCL11A transcription by MLL1 complex, both MEN1 and KMT2A were detected at high levels during early phase of differentiation and their expression declined gradually during terminal differentiation, in a similar pattern as the expression of BCL11A. MEN1 knockdowns in these cells also significantly increased γ- and ε-globin mRNA levels (up to 13- and 38-fold respectively) and γ-globin protein levels while decreasing BCL11A levels. Furthermore, MEN1 knockdowns did not block differentiation of the cells as enucleated red blood cells were readily detected and the number of F cells significantly increased in the knockdown cultures. Since MENIN inhibitors blocking the interaction between MEN1 and KMT2A showed mild side effects in clinical trials for acute myeloid leukemia (AML), we further tested MENIN inhibitor MI-3454 in inducing fetal and embryonic globin expression in human CD34⁺ HSPCs. Remarkably, treatment with MI-3454 significantly stimulated β- and ε-globin gene expression in these cells with γ-globin levels reaching over 30% of total β-globin levels. In summary, these results suggest that MLL1 complex is a critical regulator of fetal and embryonic globin expression and treatment with MENIN inhibitors could represent an effective therapeutic strategy for sickle cell disease and β-thalassemia.