• Genetic-or compound CB-839-induced GAC inhibition reduces OXPHOS and has antileukemic activity in AML.• GAC inhibition synergizes with BCL-2 inhibition by compound ABT-199.Cancer cells require glutamine to adapt to increased biosynthetic activity. The limiting step in intracellular glutamine catabolism involves its conversion to glutamate by glutaminase (GA). Different GA isoforms are encoded by the genes GLS1 and GLS2 in humans. Herein, we show that glutamine levels control mitochondrial oxidative phosphorylation (OXPHOS) in acute myeloid leukemia (AML) cells. Glutaminase C (GAC) is the GA isoform that is most abundantly expressed in AML. Both knockdown of GLS1 expression and pharmacologic GLS1 inhibition by the drug CB-839 can reduce OXPHOS, leading to leukemic cell proliferation arrest and apoptosis without causing cytotoxic activity against normal human CD34 1 progenitors. Strikingly, GLS1 knockdown dramatically inhibited AML development in NSG mice. The antileukemic activity of CB-839 was abrogated by both the expression of a hyperactive GAC K320A allele and the addition of the tricarboxyclic acid cycle product a-ketoglutarate, indicating the critical function of GLS1 in AML cell survival. Finally, glutaminolysis inhibition activated mitochondrial apoptosis and synergistically sensitized leukemic cells to priming with the BCL-2 inhibitor ABT-199. These findings show that targeting glutamine addiction via GLS1 inhibition offers a potential novel therapeutic strategy for AML. (Blood. 2015;126(11):1346-1356

The pathophysiology of ineffective erythropoiesis in β-thalassemia is poorly understood. We report that RAP-011, an activin receptor IIA (ActRIIA) ligand trap, improved ineffective erythropoiesis, corrected anemia and limited iron overload in a mouse model of β-thalassemia intermedia. Expression of growth differentiation factor 11 (GDF11), an ActRIIA ligand, was increased in splenic erythroblasts from thalassemic mice and in erythroblasts and sera from subjects with β-thalassemia. Inactivation of GDF11 decreased oxidative stress and the amount of α-globin membrane precipitates, resulting in increased terminal erythroid differentiation. Abnormal GDF11 expression was dependent on reactive oxygen species, suggesting the existence of an autocrine amplification loop in β-thalassemia. GDF11 inactivation also corrected the abnormal ratio of immature/mature erythroblasts by inducing apoptosis of immature erythroblasts through the Fas-Fas ligand pathway. Taken together, these observations suggest that ActRIIA ligand traps may have therapeutic relevance in β-thalassemia by suppressing the deleterious effects of GDF11, a cytokine which blocks terminal erythroid maturation through an autocrine amplification loop involving oxidative stress and α-globin precipitation.

Key Points• Glutamine removal and knockdown of the glutamine transporter SLC1A5 have antileukemic activity in AML.• The glutaminase activity of L-asparaginase inhibits mTORC1 and protein synthesis and induces a strong autophagy in AML.Cancer cells require nutrients and energy to adapt to increased biosynthetic activity, and protein synthesis inhibition downstream of mammalian target of rapamycin complex 1 (mTORC1) has shown promise as a possible therapy for acute myeloid leukemia (AML). Glutamine contributes to leucine import into cells, which controls the amino acid/Rag/mTORC1 signaling pathway. We show in our current study that glutamine removal inhibits mTORC1 and induces apoptosis in AML cells. The knockdown of the SLC1A5 high-affinity transporter for glutamine induces apoptosis and inhibits tumor formation in a mouse AML xenotransplantation model. L-asparaginase (L-ase) is an anticancer agent also harboring glutaminase activity. We show that L-ases from both Escherichia coli and Erwinia chrysanthemi profoundly inhibit mTORC1 and protein synthesis and that this inhibition correlates with their glutaminase activity levels and produces a strong apoptotic response in primary AML cells. We further show that L-ases upregulate glutamine synthase (GS) expression in leukemic cells and that a GS knockdown enhances L-ase-induced apoptosis in some AML cells. Finally, we observe a strong autophagic process upon L-ase treatment. These results suggest that L-ase anticancer activity and glutamine uptake inhibition are promising new therapeutic strategies for AML. (Blood. 2013;122(20):3521-3532)

β-Thalassaemia major (β-TM) is an inherited haemoglobinopathy caused by a quantitative defect in the synthesis of β-globin chains of haemoglobin, leading to the accumulation of free α-globin chains that form toxic aggregates. Despite extensive knowledge of the molecular defects causing β-TM, little is known of the mechanisms responsible for the ineffective erythropoiesis observed in the condition, which is characterized by accelerated erythroid differentiation, maturation arrest and apoptosis at the polychromatophilic stage. We have previously demonstrated that normal human erythroid maturation requires a transient activation of caspase-3 at the later stages of maturation. Although erythroid transcription factor GATA-1, the master transcriptional factor of erythropoiesis, is a caspase-3 target, it is not cleaved during erythroid differentiation. We have shown that, in human erythroblasts, the chaperone heat shock protein70 (HSP70) is constitutively expressed and, at later stages of maturation, translocates into the nucleus and protects GATA-1 from caspase-3 cleavage. The primary role of this ubiquitous chaperone is to participate in the refolding of proteins denatured by cytoplasmic stress, thus preventing their aggregation. Here we show in vitro that during the maturation of human β-TM erythroblasts, HSP70 interacts directly with free α-globin chains. As a consequence, HSP70 is sequestrated in the cytoplasm and GATA-1 is no longer protected, resulting in end-stage maturation arrest and apoptosis. Transduction of a nuclear-targeted HSP70 mutant or a caspase-3-uncleavable GATA-1 mutant restores terminal maturation of β-TM erythroblasts, which may provide a rationale for new targeted therapies of β-TM.

Transglutaminase 2 is required for the development of IgA nephropathy.

Finding an effective treatment for acute myeloid leukemia (AML) remains a challenge, and all cellular processes that are deregulated in AML cells should be considered in the design of targeted therapies. We show in our current study that the LKB1/AMPK/TSC tumor suppressor axis is functional in AML and can be activated by the biguanide molecule metformin, resulting in a specific inhibition of mammalian target of rapamycin (mTOR) catalytic activity. This induces a multisite dephosphorylation of the key translation regulator, 4E-BP1, which markedly inhibits the initiation step of mRNA translation. Consequently, metformin reduces the recruitment of mRNA molecules encoding oncogenic proteins to the polysomes, IntroductionIn acute myeloid leukemia (AML), the oncogenic deregulation of mRNA translation markedly contributes to the malignant phenotype. However, the efficacy of clinically available therapies that target this process, such as rapamycin, remains moderate due to the induction of multiple resistance mechanisms, 1 and new approach should be considered to bypass these processes.The liver kinase B1 (LKB1) serine/threonine kinase is encoded by the tumor-suppressor gene, STK11, which harbors germ-line mutations in the inherited cancer predisposition, Peutz-Jeghers syndrome, and somatic mutations in sporadic cancers. 2 The search for substrates of LKB1 that mediate its tumor-suppressor function led to the identification of the LKB1/AMPK/TSC adenosine monophosphate-activated protein kinase (AMPK) as a direct LKB1 substrate. 3 AMPK is a heterotrimeric complex comprising a catalytic ␣-and 2 regulatory ␤-and ␥-subunits, and LKB1 enhances AMPK activity through the phosphorylation of the ␣-subunit at T 172 . 4 AMPK is allosterically activated by the accumulation of AMP molecules, due to metabolic stresses that inhibit adenosine triphosphate (ATP) production (eg, hypoxia, glucose deprivation) or stimulate ATP consumption 5 and is also activated by drugs used to treat type 2 diabetes, including metformin. 6 AMPK is therefore the main cellular energy sensor, acting as a central negative regulator of metabolic pathways, such as fatty acid oxidation and glucose consumption. 7 The LKB1/AMPK pathway also regulates the protein synthesis rate through the control of the serine/threonine kinase mammalian target of rapamycin (mTOR), 8 a process that is consistently deregulated in AML cells. 9 When activated, AMPK stimulates tuberous sclerosis complex 1/2 (TSC 1/2 ), which comprises the TSC1/hamartin and TSC2/tuberin proteins, through AMPK-mediated TSC2 phosphorylation at the T 1227 and S 1345 residues. 10 This stimulates the GTPase-activating protein (GAP) function of TSC2 toward the small G-protein Rheb (Ras homolog enriched in brain), and increases the pool of guanosine diphosphate (GDP)-bound Rheb molecules. Although the molecular mechanism underlying mTOR activation by Rheb-guanosine triphosphate (GTP) is still under debate, [11][12][13] it is well established that TSC2 activation switches off Rheb, resulting in the inhibition o...

BackgroundIn the SOD1G93A mutant rat model of amyotrophic lateral sclerosis (ALS), neuronal death and rapid paralysis progression are associated with the emergence of activated aberrant glial cells that proliferate in the degenerating spinal cord. Whether pharmacological downregulation of such aberrant glial cells will decrease motor neuron death and prolong survival is unknown. We hypothesized that proliferation of aberrant glial cells is dependent on kinase receptor activation, and therefore, the tyrosine kinase inhibitor masitinib (AB1010) could potentially control neuroinflammation in the rat model of ALS.MethodsThe cellular effects of pharmacological inhibition of tyrosine kinases with masitinib were analyzed in cell cultures of microglia isolated from aged symptomatic SOD1G93A rats. To determine whether masitinib prevented the appearance of aberrant glial cells or modified post-paralysis survival, the drug was orally administered at 30 mg/kg/day starting after paralysis onset.ResultsWe found that masitinib selectively inhibited the tyrosine kinase receptor colony-stimulating factor 1R (CSF-1R) at nanomolar concentrations. In microglia cultures from symptomatic SOD1G93A spinal cords, masitinib prevented CSF-induced proliferation, cell migration, and the expression of inflammatory mediators. Oral administration of masitinib to SOD1G93A rats starting after paralysis onset decreased the number of aberrant glial cells, microgliosis, and motor neuron pathology in the degenerating spinal cord, relative to vehicle-treated rats. Masitinib treatment initiated 7 days after paralysis onset prolonged post-paralysis survival by 40 %.ConclusionsThese data show that masitinib is capable of controlling microgliosis and the emergence/expansion of aberrant glial cells, thus providing a strong biological rationale for its use to control neuroinflammation in ALS. Remarkably, masitinib significantly prolonged survival when delivered after paralysis onset, an unprecedented effect in preclinical models of ALS, and therefore appears well-suited for treating ALS.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0620-9) contains supplementary material, which is available to authorized users.

The serine/threonine kinase mammalian target of rapamycin (mTOR) is crucial for cell growth and proliferation, and is constitutively activated in primary acute myeloid leukemia (AML) cells, therefore representing a major target for drug development in this disease. We show here that the specific mTOR kinase inhibitor AZD8055 blocked mTORC1 and mTORC2 signaling in AML. Particularly, AZD8055 fully inhibited multisite eIF4E-binding protein 1 phosphorylation, subsequently blocking protein translation, which was in contrast to the effects of rapamycin. In addition, the mTORC1-dependent PI3K/Akt feedback activation was fully abrogated in AZD8055-treated AML cells. Significantly, AZD8055 decreased AML blast cell proliferation and cell cycle progression, reduced the clonogenic growth of leukemic progenitors and induced caspase-dependent apoptosis in leukemic cells but not in normal immature CD34 þ cells. Interestingly, AZD8055 strongly induced autophagy, which may be either protective or cell death inducing, depending on concentration. Finally, AZD8055 markedly increased the survival of AML transplanted mice through a significant reduction of tumor growth, without apparent toxicity. Our current results strongly suggest that AZD8055 should be tested in AML patients in clinical trials.