BackgroundPatients with heterozygous germline mutations in phosphatase and tensin homolog deleted on chromosome 10 (PTEN) experience autoimmunity and lymphoid hyperplasia.ObjectivesBecause regulation of the phosphoinositide 3-kinase (PI3K) pathway is critical for maintaining regulatory T (Treg) cell functions, we investigate Treg cells in patients with heterozygous germline PTEN mutations (PTEN hamartoma tumor syndrome [PHTS]).MethodsPatients with PHTS were assessed for immunologic conditions, lymphocyte subsets, forkhead box P3 (FOXP3)+ Treg cell levels, and phenotype. To determine the functional importance of phosphatases that control the PI3K pathway, we assessed Treg cell induction in vitro, mitochondrial depolarization, and recruitment of PTEN to the immunologic synapse.ResultsAutoimmunity and peripheral lymphoid hyperplasia were found in 43% of 79 patients with PHTS. Immune dysregulation in patients with PHTS included lymphopenia, CD4+ T-cell reduction, and changes in T- and B-cell subsets. Although total CD4+FOXP3+ Treg cell numbers are reduced, frequencies are maintained in the blood and intestine. Despite pathogenic PTEN mutations, the FOXP3+ T cells are phenotypically normal. We show that the phosphatase PH domain leucine-rich repeat protein phosphatase (PHLPP) downstream of PTEN is highly expressed in normal human Treg cells and provides complementary phosphatase activity. PHLPP is indispensable for the differentiation of induced Treg cells in vitro and Treg cell mitochondrial fitness. PTEN and PHLPP form a phosphatase network that is polarized at the immunologic synapse.ConclusionHeterozygous loss of function of PTEN in human subjects has a significant effect on T- and B-cell immunity. Assembly of the PTEN-PHLPP phosphatase network allows coordinated phosphatase activities at the site of T-cell receptor activation, which is important for limiting PI3K hyperactivation in Treg cells despite PTEN haploinsufficiency.

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In β-thalassemia, accumulated free α-globin forms intracellular precipitates that impair erythroid cell maturation and viability. Protein quality control systems mitigate β-thalassemia pathophysiology by degrading toxic free α-globin, although the associated mechanisms are poorly understood. We show that loss of the autophagy-activating Unc-51–like kinase 1 (Ulk1) gene in β-thalassemic mice reduces autophagic clearance of α-globin in red blood cell precursors and exacerbates disease phenotypes, whereas inactivation of the canonical autophagy-related 5 (Atg5) gene has relatively minor effects. Systemic treatment with the mTORC1 inhibitor rapamycin reduces α-globin precipitates and lessens pathologies in β-thalassemic mice via an ULK1-dependent pathway. Similarly, rapamycin reduces free α-globin accumulation in erythroblasts derived from CD34+cells of β-thalassemic individuals. Our findings define a drug-regulatable pathway for ameliorating β-thalassemia.

Erythroid maturation is coordinated to maximize the production of hemoglobin A heterotetramers (α2β2) and minimize the accumulation of potentially toxic free α-or βglobin subunits. In β-thalassemia, mutations in the β-globin gene cause a build-up of free α-globin, which forms intracellular precipitates that impair erythroid cell maturation and 5 viability. Protein quality-control systems mitigate β-thalassemia pathophysiology by degrading toxic free α-globin. We show that loss of the Unc 51-like autophagy-activating kinase gene Ulk1 in β-thalassemic mice reduces autophagic clearance of α-globin in red cell precursors and exacerbates disease phenotypes, whereas inactivation of the canonical autophagy gene Atg5 has minimal effects. Systemic treatment with rapamycin 10 to inhibit the ULK1 inhibitor mTORC1 reduces α-globin precipitates and lessens pathologies in β-thalassemic mice, but not in those lacking Ulk1. Similarly, rapamycin reduces free α-globin accumulation in erythroblasts derived from β-thalassemic patient CD34 + hematopoietic progenitors. Our findings identify a new, drug-regulatable pathway for ameliorating β-thalassemia. 15 One Sentence Summary:Rapamycin alleviates -thalassemia by stimulating ULK1-dependent autophagy of toxic free -globin. 20

Interest in the structure, function, and evolutionary relations of circulating and intracellular globins dates back more than 60 years to the first determination of the three-dimensional structure of these proteins. Non-erythrocytic globins have been implicated in circulatory control through reactions that couple nitric oxide (NO) signaling with cellular oxygen availability and redox status. Small artery endothelial cells (ECs) express free α-globin, which causes vasoconstriction by degrading NO. This reaction converts reduced (Fe2+) α-globin to the oxidized (Fe3+) form, which is unstable, cytotoxic, and unable to degrade NO. Therefore, (Fe3+) α-globin must be stabilized and recycled to (Fe2+) α-globin to reinitiate the catalytic cycle. The molecular chaperone α-hemoglobin-stabilizing protein (AHSP) binds (Fe3+) α-globin to inhibit its degradation and facilitate its reduction. The mechanisms that reduce (Fe3+) α-globin in ECs are unknown, although endothelial nitric oxide synthase (eNOS) and cytochrome b5 reductase (CyB5R3) with cytochrome b5 type A (CyB5a) can reduce (Fe3+) α-globin in solution. Here, we examine the expression and cellular localization of eNOS, CyB5a, and CyB5R3 in mouse arterial ECs and show that α-globin can be reduced by either of two independent redox systems, CyB5R3/CyB5a and eNOS. Together, our findings provide new insights into the regulation of blood vessel contractility.

Most cells can eliminate unstable or misfolded proteins through quality control mechanisms. In the inherited red blood cell disorder β-thalassemia, mutations in the β-globin gene (HBB) lead to a reduction of the corresponding protein and the accumulation of cytotoxic free α-globin, which causes maturation arrest and apoptosis of erythroid precursors and reductions in the lifespan of circulating red blood cells. We showed previously that excess α-globin is eliminated by ULK1-dependent autophagy and that stimulation of this pathway by systemic mTORC1 inhibition alleviates β-thalassemia pathologies. We show here that disruption of the bi-cistronic microRNA locus miR-144/451 alleviates β-thalassemia by reducing mTORC1 activity and stimulating ULK1-mediated autophagy of free α-globin through two mechanisms. Loss of miR-451 upregulated its target mRNA, Cab39, which encodes a cofactor for LKB1, a serine-threonine kinase that phosphorylates and activates the central metabolic sensor, AMPK. The resultant enhancement of LKB1 activity stimulated AMPK and its downstream effects, including repression of mTORC1 and direct activation of ULK1. Additionally, loss of miR-144/451 inhibited the expression of erythroblast transferrin receptor 1 (TfR1), causing intracellular iron restriction, which has been shown to inhibit mTORC1, reduce free α-globin precipitates and improve hematological indices in β-thalassemia. The beneficial effects of miR-144/451 loss in β-thalassemia were inhibited by disruption of the Cab39 or Ulk1 genes. Our findings link the severity of a common hemoglobinopathy to a highly expressed erythroid microRNA locus and to a fundamental, metabolically regulated protein quality control pathway that is amenable to therapeutic manipulation.

β-Thalassemia is a common, frequently debilitating, inherited anemia caused by HBB gene mutations that reduce or eliminate the expression of the β-globin subunit of adult hemoglobin (HbA, α2β2). Consequently, excess free α-globin forms toxic precipitates in red blood cells (RBCs) and their precursors, leading to ineffective erythropoiesis and hemolytic anemia. Previously, we showed that free α-globin is 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-globin precipitates and worsened the pathologies of β-thalassemia. Treatment of β-thalassemic mice with rapamycin to inhibit mTOR (an ULK1 inhibitor) reduced α-globin precipitates, 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 erythroblasts generated from transfusion-dependent (TD, n = 5) or non-transfusion-dependent (NTD, n = 5) β-thalassemia patients revealed α-chain excesses (α-chain/β-like [β + γ + δ] chain) of approximately 40% and 15%, respectively (compared to 7 normal donors; P < 0.001). Rapamycin (10µM or 20µM) or the proteasome inhibitor MG132 (2.5µM) was added to day 13 cultures, which contained mid- to late-stage erythroblasts, and α-globin accumulation was determined by HPLC 2 days later. As expected, proteasome inhibition by MG132 raised free α-globin levels in thalassemic erythroblasts (P < 0.01) and induced cell death (P < 0.01). In contrast, rapamycin reduced free α-globin in a dose-dependent manner by 40% and 85% in TD (P < 0.0001) and NTD β-thalassemia (P < 0.001), respectively, but had no effect on erythroblasts derived from normal CD34+ 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 erythroblasts did not induce fetal hemoglobin production, as has been previously reported, thereby excluding this potential mechanism for reducing globin chain imbalances. Overall, rapamycin treatment significantly reduced the accumulation of free α-globin in TD β-thalassemia and almost fully corrected the imbalance in NTD β-thalassemia cells. 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 entity's Board of Directors or advisory committees; Vifor: Membership on an entity's Board of Directors or advisory committees; Sanofi/Genzyme: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria.