Reawakening of Human Fetal Hemoglobin and an Epigenetic Path to the Clinic for Sickle Cell Disease and Beta-Thalassemia: Identification of an Orally-Available, Potent, and Selective Euchromatic Histone Lysine Methyltransferase 1 and 2 (EHMT1/2) Inhibitor

Expression of genes in the β-globin locus switch via epigenetic control at key stages of early gestational development from an embryonic (HbE, α₂ε₂) to fetal form (HbF, α₂γ₂) and again around birth to the adult form (HbA, α₂β₂). Sickle cell disease (SCD) is a multi-organ genetic disorder caused by a point mutation in the adult β-globin gene producing sickled hemoglobin (HbS, α₂β^s₂). It is well-established that SCD patients carrying genetic variations in HbF-regulatory loci have a concomitant increase in blood levels of HbF (ranging from 5-25% of total Hb). These individuals exhibit fewer major crises or no clinical symptoms, suggesting that re-expression of the γ-gene and increasing HbF is a potential disease modifying treatment approach for SCD patients. Hydroxyurea (HU) and L-glutamine are currently the only FDA-approved drugs for the treatment of SCD, with HU working in-part, through the re-induction of HbF. Safety and efficacy responses to HU vary widely, and there remains a strong need for more robust HbF induction strategies with improved safety profiles.

Previous groups have shown that inhibitors to various epigenetic modifiers are able to overcome γ-gene silencing and induce HbF; however, associated defects in hematopoiesis have been reported. Recently, histone methytransferase enzymes EHMT1 and EHMT2, responsible for the repressive chromatin mark dimethyl-lysine 9 on histone H3 (H3K9me2), have been shown to be essential for robust repression of γ-globin expression. To-date, an in vitro tool compound UNC0638 has demonstrated activation of γ-globin expression in human adult erythroid progenitor cells but no evaluation of in vivo efficacy of EHMT1/2 inhibition on HbF induction has been possible due to a lack of drug-like properties.

Here we describe the generation and characterization of a potent, selective, and orally available small molecule inhibitor of EHMT1/2, EPZ035544. EHMT2 enzymatic activity in a biochemical assay, as measured by the half-maximal inhibitory concentration (IC₅₀) is 10nM and cellular inhibition of H3K9me2 correlated well with an IC₅₀ of 55nM. Treatment of EPZ035544 in primary human CD34⁺ progenitor cells led to dose-dependent decreases in H3K9me2 with concomitant induction of γ-globin relative to β-globin expression of 10-30% as measured by mass spectrometry and qPCR leading to 100% of F-positive cells. Chromatin immunoprecipitation and sequencing studies performed on EPZ035544-treated CD34⁺ cells demonstrated that H3K9me2 was reduced throughout the genome with an associated increase in H3K9Ac. The γ-globin gene locus displayed a reduction of H3K9me2 and an increase of H3K9Ac and RNA Polymerase II occupancy leading to significantly increased γ-globin gene expression upon EPZ035544 treatment.

The drug-like properties of EPZ035544 enabled the first prolonged in vivo investigation of an EHMT1/2 inhibitor by continuous oral dosing over 90 days in C57BL/6 mice. As mice do not have HbF, we measured the levels of embryonic hemoglobin (Hbb-εy) which is normally repressed after birth. We performed twice-daily (BID) oral dosing in two dose groups: 25 and 50 mg per kilogram of body weight (mg/kg) and once-daily (QD) oral dosing in a single dose group of 75 mg/kg. Dose-dependent increases in the absolute levels of Hbb-εy mRNA in peripheral blood mononuclear cells (PBMCs) were observed with maximal induction of ~ 100 fold achieved at the 50 mg/kg BID and 75 mg/kg QD doses. Using quantitative mass spectrometry, we also observed drug-induced increases in Hbb-εy at the protein level. A corresponding reduction in H3K9me2 levels in PBMCs and bone marrow, as measured by flow cytometry and ELISA, strongly suggests that these effects were a direct consequence of EHMT1/2 inhibition. EPZ035544 was well tolerated throughout the study with no significant changes observed in body weights, spleen weights, or complete blood cell counts. In summary, we have developed an orally bioavailable EHMT1/2 inhibitor with demonstrated on-target in vivo induction of mouse Hbb-εy expression with up to 90 days of continuous dosing. To our knowledge this is the first study to test the in vivo effects of an EHMT1/2 inhibitor on hemoglobin induction. We believe this approach holds promise as a novel treatment option for β-globinopathies, and we continue to advance our program towards the clinic.