Imatinib mesylate (IM), a potent inhibitor of the BCR/ABL tyrosine kinase, has become standard first-line therapy for patients with chronic myeloid leukemia (CML), but the frequency of resistance increases in advancing stages of disease. Elimination of BCR/ABL-dependent intracellular signals triggers apoptosis, but it is unclear whether this activates additional cell survival and/or death pathways. We have shown here that IM induces autophagy in CML blast crisis cell lines, CML primary cells, and p210 BCR/ABL -expressing myeloid precursor cells. IM-induced autophagy did not involve c-Abl or Bcl-2 activity but was associated with ER stress and was suppressed by depletion of intracellular Ca 2+ , suggesting it is mechanistically nonoverlapping with IM-induced apoptosis. We further demonstrated that suppression of autophagy using either pharmacological inhibitors or RNA interference of essential autophagy genes enhanced cell death induced by IM in cell lines and primary CML cells. Critically, the combination of a tyrosine kinase inhibitor (TKI), i.e., IM, nilotinib, or dasatinib, with inhibitors of autophagy resulted in near complete elimination of phenotypically and functionally defined CML stem cells. Together, these findings suggest that autophagy inhibitors may enhance the therapeutic effects of TKIs in the treatment of CML.

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Key Points• TFR2, a gene mutated in hemochromatosis and a partner of the EPO receptor, limits erythropoiesis expansion in mice.• Iron deficiency mimics TFR2 deletion in the erythroid compartment.Transferrin receptor 2 (TFR2) contributes to hepcidin regulation in the liver and associates with erythropoietin receptor in erythroid cells. Nevertheless, TFR2 mutations cause iron overload (hemochromatosis type 3) without overt erythroid abnormalities. To clarify TFR2 erythroid function, we generated a mouse lacking Tfr2 exclusively in the bone marrow (Tfr2 BMKO ). Tfr2 BMKO mice have normal iron parameters, reduced hepcidin levels, higher hemoglobin and red blood cell counts, and lower mean corpuscular volume than normal control mice, a phenotype that becomes more evident in iron deficiency. In Tfr2 BMKO mice, the proportion of nucleated erythroid cells in the bone marrow is higher and the apoptosis lower than in controls, irrespective of comparable erythropoietin levels. Induction of moderate iron deficiency increases erythroblasts number, reduces apoptosis, and enhances erythropoietin (Epo) levels in controls, but not in Tfr2 BMKO mice.Epo-target genes such as Bcl-x L and Epor are highly expressed in the spleen and in isolated erythroblasts from Tfr2 BMKO mice. Low hepcidin expression in Tfr2 BMKO is accounted for by erythroid expansion and production of the erythroid regulator erythroferrone. We suggest that Tfr2 is a component of a novel iron-sensing mechanism that adjusts erythrocyte production according to iron availability, likely by modulating the erythroblast Epo sensitivity. (Blood.

SummaryEx vivo gene therapy based on CD34+ hematopoietic stem cells (HSCs) has shown promising results in clinical trials, but genetic engineering to high levels and in large scale remains challenging. We devised a sorting strategy that captures more than 90% of HSC activity in less than 10% of mobilized peripheral blood (mPB) CD34+ cells, and modeled a transplantation protocol based on highly purified, genetically engineered HSCs co-infused with uncultured progenitor cells. Prostaglandin E2 stimulation allowed near-complete transduction of HSCs with lentiviral vectors during a culture time of less than 38 hr, mitigating the negative impact of standard culture on progenitor cell function. Exploiting the pyrimidoindole derivative UM171, we show that transduced mPB CD34+CD38− cells with repopulating potential could be expanded ex vivo. Implementing these findings in clinical gene therapy protocols will improve the efficacy, safety, and sustainability of gene therapy and generate new opportunities in the field of gene editing.

Inappropriately low expression of the key iron regulator hepcidin (HAMP) causes iron overload in untransfused patients affected by ␤-thalassemia intermedia and Hamp modulation provides improvement of the thalassemic phenotype of the Hbb th3/؉ mouse. HAMP expression is activated by iron through the bone morphogenetic protein (BMP)-son of mothers against decapentaplegic signaling pathway and inhibited by ineffective erythropoiesis through an unknown "erythroid regulator." The BMP pathway is inactivated by the serine protease TMPRSS6 that cleaves the BMP coreceptor hemojuvelin. Here, we show that homozygous loss of Tmprss6 in Hbb th3/؉ mice improves anemia and reduces ineffective erythropoiesis, splenomegaly, and iron loading. All these effects are mediated by Hamp up-regulation, which inhibits iron absorption and recycling. Because Hbb th3/؉ mice lacking Tmprss6 show residual ineffective erythropoiesis, our results indicate that Tmprss6 is essential for Hamp inhibition by the erythroid regulator. We also obtained partial correction of the phenotype in Tmprss6 haploinsufficient Hbb th3/؉ male but not female mice and showed that the observed sex difference reflects an unequal balance between iron and erythropoiesis-mediated Hamp regulation. Our study indicates that preventing iron overload improves ␤-thalassemia and strengthens the essential role of Tmprss6 for Hamp suppression, providing a proof of concept that Tmprss6 manipulation can offer a novel therapeutic option in this condition. IntroductionThe liver antimicrobial peptide hepcidin (HAMP) is the central regulator of systemic iron homeostasis. HAMP controls the surface expression of the iron exporter ferroportin on duodenal enterocytes and macrophages, modulating iron absorption and recycling. HAMP is activated by the bone morphogenetic proteins (BMP)-son of mothers against decapentaplegic (SMAD) signaling pathway, in response to increased body iron and by the IL-6-signal transducer and activator of transcription (STAT)3 pathway in inflammation. 1 The glycosylphosphatidylinositol (GPI)-anchored protein hemojuvelin (HJV) is a BMP coreceptor and homozygous mutations of HAMP or HJV cause juvenile hemochromatosis in humans 2,3 and severe iron overload in mice. 4,5 The BMP pathway is inhibited by matriptase-2 (MT-2), a type II transmembrane serine protease encoded by the transmembrane proteaSe serine 6 (TMPRSS6) gene and mainly expressed in the liver. 6,7 MT-2, by cleaving HJV from the hepatocyte surface, attenuates the BMP-SMAD signaling and down-regulates HAMP expression. 8 Mice deficient for both Tmprss6 and Hjv show markedly decreased Hamp mRNA levels and systemic iron overload, as do Hjv deficient mice, 9 in agreement with Hjv being the serine protease substrate.TMPRSS6 plays an essential role for erythropoiesis: homozygous inactivation of the Tmprss6 gene leads to excessive Hamp production, impaired dietary iron absorption and microcytic anemia in mice, 10,11 and iron-refractory iron deficiency anemia (IRIDA) in humans. [12][13][14][15][16][17] The importan...

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IntroductionChronic myelogenous leukemia (CML) is a clonal myeloproliferative disorder arising from neoplastic transformation of hematopoietic stem/progenitor cells 1 by the p210 BCR/ABL protein that is translated from bcr/abl transcripts generated by the bcr/abl chimeric gene of the Philadelphia chromosome. [2][3][4][5] The oncogenic potential of p210 BCR/ABL depends on its tyrosine kinase activity and rests on its ability to interact with and phosphorylate several substrates, which in turn activate cytoplasmic and nuclear effectors. 6 Numerous studies on the mechanisms of p210 BCR/ABL leukemogenesis have focused on the activation of cytoplasmic effectors such as RAS and PI-3K, 7,8 but less is known about the nuclear effectors, the mechanisms of their activation or loss of function, and their contribution to the phenotype of transformed cells. The nuclear effectors of p210 BCR/ABL include transcription factors whose activity is also modulated by the products of other leukemia-specific translocation or directly by mutation. 6 One of the transcription factors whose expression in enhanced by p210 BCR/ABL and may have an important role in BCR/ABLdependent leukemogenesis is c-Myb. 9 The importance of c-Myb in normal hematopoiesis is supported by studies showing that ablation of c-Myb expression leads to impaired liver hematopoiesis, 10 reduced erythroid and myeloid colony formation, 11 and defective B-and T-cell lymphopoiesis. 12,13 The function of c-Myb may depend on its ability to modulate levels of genes (ie, CD34, c-Kit, Flt3) expressed by early progenitor cells and involved in the regulation of cell proliferation and survival. [14][15][16] A target of c-Myb in hematopoietic cells is also In CML cells, the c-Myb gene is intact 18,19 but expression is often increased, in part, via enhanced protein stability regulated by the PI-3K/Akt pathway 9 The c-Myb gene has been proposed as a therapeutic target in acute myelogenous leukemia (AML) and in CML based on the differential requirement of c-Myb by normal and leukemic cells. 20,21 However, these studies were based on the use of antisense oligodeoxynucleotides in dose-response comparisons of normal and leukemic colony formation and on clonogenic assays of mixed normal and leukemic cells in which the phenotype of surviving cells was assessed by measuring bcr/abl transcripts. Since a genetic approach for assessing the role of c-Myb in BCR/ABL-dependent leukemogenesis has not been taken, we used hematopoietic progenitor cells from c-Myb knockout mice with 1 or 2 functional alleles to assess their reliance on c-Myb levels for p210 BCR/ABL -dependent in vitro transformation and leukemogenesis. We show here that p210 BCR/ABL -transduced Lin Ϫ Sca-1 ϩ and Lin Ϫ Sca-1 ϩ Kit ϩ marrow progenitors are more dependent than their normal counterpart on optimal c-Myb levels for primary and secondary colony formation. Moreover, in a mouse model of CML-blast crisis, p210 BCR/ABLexpressing wild-type c-Myb marrow cells induced primary and secondary leukemia with a shorter latency of tha...

In eukaryotes, initiation of DNA replication requires the activity of the origin recognition complex (ORC). The largest subunit of this complex, Orc1p, has a critical role in this activity. Here we have studied the subnuclear distribution of the overexpressed human Orc1p during the cell cycle. Orc1p is progressively degraded during S-phase according to a spatio-temporal program and it never colocalizes with replication factories. Orc1p is resynthesized in G1. In early G1, the protein is distributed throughout the cell nucleus, but successively it preferentially associates with heterochromatin. This association requires a functional ATP binding site and a protein region partially overlapping the bromo-adjacent homology domain at the N-terminus of Orc1p. The same N-terminal region mediates the in vitro interaction with heterochromatin protein 1 (HP1). Fluorescence resonance energy transfer (FRET) experiments demonstrate the interaction of human Orc1p and HP1 in vivo. Our data suggest a role of HP1 in the recruitment but not in the stable association of Orc1p with heterochromatin. Indeed, the subnuclear distribution of Orc1p is not affected by treatments that trigger the dispersal of HP1.