Key Points• IDH1 promotes leukemogenesis in vivo in cooperation with HoxA9.• Pharmacologic inhibition of mutant IDH1 efficiently inhibits AML cells of IDH1-mutated patients but not of normal CD34 1 bone marrow cells.Mutations in the metabolic enzymes isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) are frequently found in glioma, acute myeloid leukemia (AML), melanoma, thyroid cancer, and chondrosarcoma patients. Mutant IDH produces 2-hydroxyglutarate (2HG), which induces histone-and DNA-hypermethylation through inhibition of epigenetic regulators. We investigated the role of mutant IDH1 using the mouse transplantation assay. Mutant IDH1 alone did not transform hematopoietic cells during 5 months of observation. However, mutant IDH1 greatly accelerated onset of myeloproliferative disease-like myeloid leukemia in mice in cooperation with HoxA9 with a mean latency of 83 days compared with cells expressing HoxA9 and wild-type IDH1 or a control vector (167 and 210 days, respectively, P 5 .001). Mutant IDH1 accelerated cell-cycle transition through repression of cyclindependent kinase inhibitors Cdkn2a and Cdkn2b, and activated mitogen-activated protein kinase signaling. By computational screening, we identified an inhibitor of mutant IDH1, which inhibited mutant IDH1 cells and lowered 2HG levels in vitro, and efficiently blocked colony formation of AML cells from IDH1-mutated patients but not of normal CD341 bone marrow cells. These data demonstrate that mutant IDH1 has oncogenic activity in vivo and suggest that it is a promising therapeutic target in human AML cells. (Blood.

A major obstacle to curing chronic myeloid leukemia (CML) is residual disease maintained by tyrosine kinase inhibitor (TKI)-persistent leukemic stem cells (LSC). These are BCR-ABL1 kinase independent, refractory to apoptosis, and serve as a reservoir to drive relapse or TKI resistance. We demonstrate that Polycomb Repressive Complex 2 is misregulated in chronic phase CML LSCs. This is associated with extensive reprogramming of H3K27me3 targets in LSCs, thus sensitizing them to apoptosis upon treatment with an EZH2-specifi c inhibitor (EZH2i). EZH2i does not impair normal hematopoietic stem cell survival. Strikingly, treatment of primary CML cells with either EZH2i or TKI alone caused signifi cant upregulation of H3K27me3 targets, and combined treatment further potentiated these effects and resulted in signifi cant loss of LSCs compared to TKI alone, in vitro , and in long-term bone marrow murine xenografts. Our fi ndings point to a promising epigenetic-based therapeutic strategy to more effectively target LSCs in patients with CML receiving TKIs. SIGNIFICANCE:In CML, TKI-persistent LSCs remain an obstacle to cure, and approaches to eradicate them remain a signifi cant unmet clinical need. We demonstrate that EZH2 and H3K27me3 reprogramming is important for LSC survival, but renders LSCs sensitive to the combined effects of EZH2i and TKI. This represents a novel approach to more effectively target LSCs in patients receiving TKI treatment. Cancer Discov; 6(11); 1248-57.

Molecular genetics may influence outcome for patients with myelofibrosis. To determine the impact of molecular genetics on outcome after allogeneic stem cell transplantation, we screened 169 patients with primary myelofibrosis (n = 110), post-essential thrombocythemia/polycythemia vera myelofibrosis (n = 46), and myelofibrosis in transformation (n = 13) for mutations in 16 frequently mutated genes. The most frequent mutation was JAK2V617F (n = 101), followed by ASXL1 (n = 49), calreticulin (n = 34), SRSF2 (n = 16), TET2 (n = 10), U2AF1 (n = 11), EZH2 (n = 7), MPL (n = 6), IDH2 (n = 5), IDH1 (n = 4), and CBL (n = 1). The cumulative incidence of nonrelapse mortality (NRM) at 1 year was 21% and of relapse at 5 years 25%. The 5-year rates progression-free (PFS) and overall survival (OS) were and 56%, respectively. In a multivariate analysis CALR mutation was an independent factor for lower NRM (HR, .415; P = .05), improved PFS (HR, .393; P = .01), and OS (HR, .448; P = .03). ASXL1 and IDH2 mutations were independent risk factors for lower PFS (HR, 1.53 [P = .008], and HR, 5.451 [P = .002], respectively), whereas no impact was observed for "triple negative" patients. Molecular genetics, especially CALR, IDH2, and ASXL1 mutations, may thus be useful to predict outcome independently from known clinical risk factors after allogeneic stem cell transplantation for myelofibrosis.

There is tremendous interest in graphene-based membranes as protective molecular barriers or molecular sieves for separation technologies. Graphene oxide (GO) films in the dry state are known to be effective barriers for molecular transport and to expand in the presence of moisture to create enlarged intersheet gallery spaces that allow rapid water permeation. Here we explore an application for GO membranes as water-breathable barrier layers for personal protective equipment, which are designed to allow outward perspiration while protecting the wearer from chemical toxicants or biochemical agents in the local environment. A device was developed to measure permeation rates of small-molecular toxicants in the presence of counter-current water flow simulating active perspiration. The technique was applied to trichloroethylene (TCE) and benzene, which are important environmental toxicants, and ethanol as a limiting case to model very small, highly water-soluble organic molecules. Submicron GO membranes are shown to be effective TCE barriers, both in the presence and absence of simulated perspiration flux, and to outperform current barrier technologies. A molecular transport model is developed, which suggests the limited toxicant back-permeation observed occurs not by diffusion against the convective perspiration flow in hydrophobic channels, but rather through oxidized domains where hydrogen-bonding produces a near-stagnant water phase. Benzene and ethanol permeation fluxes are higher than those for TCE, likely reflecting the effects of higher water solubility and smaller minimum molecular dimension. Overall, GO films have high water breathability relative to competing technologies and are known to exclude most classes of target toxicants, including particles, bacteria, viruses, and macromolecules. The present results show good barrier performance for some very small-molecule species, but not others, with permeation being favored by high water solubility and small minimum molecular dimension.

Canonical mutations in IDH1 and IDH2 produce high levels of the R-enantiomer of 2-hydroxyglutarate (R-2HG), which is a competitive inhibitor of αKG-dependent enzymes and a putative oncometabolite. Mutant IDH1 collaborates with HoxA9 to induce monocytic leukemia in vivo. We employed two mouse models and a patient derived AML xenotransplantation (PDX) model to evaluate the in vivo transforming potential of R-2HG, S-2HG, and αKG independent of the mutant IDH1 protein. We show that R-2HG, but not S-2HG or αKG, is an oncometabolite in vivo that does not require the mutant IDH1 protein to induce hyperleukocytosis and to accelerate the onset of murine and human leukemia. Thus, circulating R-2HG acts in a paracrine fashion and can drive the expansion of many different leukemic and preleukemic clones that may express wildtype IDH1, and therefore can be a driver of clonal evolution and diversity. In addition we show that the mutant IDH1 protein is a stronger oncogene than R-2HG alone when comparable intracellular R-2HG levels are achieved. We therefore propose R-2HG independent oncogenic functions of mutant IDH1 that may need to be targeted in addition to R-2HG production to exploit the full therapeutic potential of IDH1 inhibition.

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Mutations in isocitrate dehydrogenase 1 (IDH1) are found in 6% of AML patients. Mutant IDH produces R-2-hydroxyglutarate (R2HG), which induces histone-and DNA-hypermethylation through inhibition of epigenetic regulators, thus linking metabolism to tumorigenesis. Here we report the biochemical characterization, in vivo antileukemic effects, structural binding and molecular mechanism of the inhibitor HMS-101, which inhibits the enzymatic activity of mutant IDH1 (IDH1mut). Treatment of IDH1mut primary AML cells reduced 2-hydroxyglutarate levels (2HG) and induced myeloid differentiation in vitro. Co-crystallization of HMS-101 and mutant IDH1 revealed that HMS-101 binds to the active site of IDH1mut in close proximity to the regulatory segment of the enzyme in contrast to other IDH1 inhibitors. HMS-101 also suppressed 2HG production, induced cellular differentiation and prolonged survival in a syngeneic mutant IDH1 mouse model and a patient-derived human AML xenograft model in vivo. Cells treated with Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

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