Activation of ULK1 Kinase Mediates Clearance of Free Alpha-Globin in Human Beta-Thalassemic Erythroblasts

β- Thalassemiais a common, frequently debilitating, inherited anemia caused by HBB gene mutations that reduce or eliminate the expression of the β- globinsubunit of adult hemoglobin (HbA, α2β2). Consequently, excess free α- globinforms toxic precipitates in red blood cells (RBCs) and their precursors, leading to ineffective erythropoiesisand hemolytic anemia. Previously, we showed that free α- globinis eliminated by protein quality-control pathways, including the ubiquitin-proteasome system and autophagy (Khandros et al ., Blood 2012;119:5265). In β-thalassemic mice, disruption of the Unc-51-like autophagy activating kinase gene ( Ulk1) increased α- globinprecipitates and worsened the pathologies of β- thalassemia. Treatment of β-thalassemic mice with rapamycin to inhibit mTOR (an ULK1inhibitor) reduced α- globinprecipitates, lessened ineffective erythropoiesis, and increased the lifespan of circulating RBCs in an Ulk1-dependent fashion. To investigate the therapeutic potential of rapamycin in human β- thalassemia, we treated erythroid precursors generated by in vitro differentiation of patient-derived CD34 + hematopoietic stem and progenitor cells. Reverse-phase high-performance liquid chromatography (HPLC) analysis of hemoglobinized erythroblastsgenerated from transfusion-dependent (TD, n = 5) or non-transfusion-dependent (NTD, n = 5) β- thalassemiapatients revealed α-chain excesses (α-chain/β-like [β + γ + δ] chain) of approximately 40% and 15%, respectively (compared to 7 normal donors; P P P P P + cells (figure). We also observed decreases in the accumulation of autophagic markers, such as SQSTM1/p62 protein, by Western blotting. We observed no negative effects of rapamycin on the survival of patient-derived erythroblasts. Also of note, under our experimental conditions, rapamycin treatment of erythroblastsdid not induce fetal hemoglobinproduction, as has been previously reported, thereby excluding this potential mechanism for reducing globinchain imbalances. Overall, rapamycin treatment significantly reduced the accumulation of free α- globinin TD β- thalassemiaand almost fully corrected the imbalance in NTD β- thalassemiacells. Our findings identify a new drug-regulatable pathway for ameliorating β- thalassemia. Rapamycin is approved and well studied, and it has a generally manageable toxicity profile. Moreover, there are additional pharmacologic approaches to activating ULK via mTOR inhibition or other pathways. These approaches may lead to effective drug therapies for β- thalassemia, particularly NTD or intermittently TD forms of the disease. Disclosures Cappellini: Celgene Corporation: Membership on an entity9s Board of Directors or advisory committees; Vifor: Membership on an entity9s Board of Directors or advisory committees; Sanofi/Genzyme: Membership on an entity9s Board of Directors or advisory committees; Novartis: Honoraria.