Germline ETV6 mutation promotes inflammation and disrupts lymphoid development of early hematopoietic progenitors.

Germline mutations in ETV6 are associated with a syndrome of thrombocytopenia and leukemia predisposition, and ETV6 is among the most commonly mutated genes in leukemias, especially childhood B-cell acute lymphoblastic leukemia. However, the mechanisms underlying disease caused by ETV6 dysfunction are poorly understood. To address these gaps in knowledge, using CRISPR/Cas9, we developed a mouse model of the most common recurrent, disease-causing germline mutation in ETV6. We found defects in hematopoiesis related primarily to abnormalities of the multipotent progenitor population 4 (MPP4) subset of hematopoietic progenitor cells and evidence of sterile inflammation. Expression of ETV6 in Ba/F3 cells altered the expression of several cytokines, some of which were also detected at higher levels in the bone marrow of the mice with Etv6 mutation. Among these, interleukin-18 and interleukin-13 abrogated B-cell development of sorted MPP4 cells, but not common lymphoid progenitors, suggesting that inflammation contributes to abnormal hematopoiesis by impairing lymphoid development. These data, along with those from humans, support a model in which ETV6 dysfunction promotes inflammation, which adversely affects thrombopoiesis and promotes leukemogenesis.

Increased HDAC Activity and c-MYC Expression Mediate Acquired Resistance to WEE1 Inhibition in Acute Leukemia.

WEE1 is a cell cycle and DNA damage response kinase that is emerging as a therapeutic target for cancer. AZD1775 is a small molecule inhibitor of WEE1, currently in early phase clinical trials as a single agent and in combination with more conventional anti-neoplastic agents. As resistance to kinase inhibitors is frequent, we sought to identify mechanisms of resistance to WEE1 inhibition in acute leukemia. We found that AZD1775 resistant cell lines are dependent upon increased HDAC activity for their survival, in part due to increased KDM5A activity. In addition, gene expression analyses demonstrate HDAC dependent increase in MYC expression and c-MYC activity in AZD1775 treated resistant cells. Overexpression of c-MYC confers resistance to AZD1775 in cell lines with low baseline expression. Pharmacologic inhibition of BRD4, and thereby c-MYC, partially abrogated resistance to AZD1775. Thus, acquired resistance to WEE1 inhibition may be reversed by HDAC or BRD4 inhibition in leukemia cells.

Evidence for proton tunneling and a transient covalent flavin-substrate adduct in choline oxidase S101A.

The effect of temperature on the reaction of alcohol oxidation catalyzed by choline oxidase was investigated with the S101A variant of choline oxidase. Anaerobic enzyme reduction in a stopped-flow spectrophotometer was biphasic using either choline or 1,2-[2H4]-choline as a substrate. The limiting rate constants klim1 and klim2 at saturating substrate were well separated (klim1/klim2>9), and were >15-fold slower than for wild-type choline oxidase. Solvent deuterium kinetic isotope effects (KIEs) ~4 established that klim1 probes the proton transfer from the substrate hydroxyl to a catalytic base. Primary substrate deuterium KIEs ≥7 demonstrated that klim2 reports on hydride transfer from the choline alkoxide to the flavin. Between 15°C and 39°C the klim1 and klim2 values increased with increasing temperature, allowing for the analyses of H+ and H- transfers using Eyring and Arrhenius formalisms. Temperature-independent KIE on the klim1 value (H2Oklim1/D2Oklim1) suggests that proton transfer occurs within a highly reorganized tunneling-ready-state with a narrow distribution of donor-acceptor distances. Eyring analysis of the klim2 value gave lines with the slope(choline)>slope(D-choline), suggesting kinetic complexity. Spectral evidence for the transient occurrence of a covalent flavin-substrate adduct during the first phase of the anaerobic reaction of S101A CHO with choline is presented, supporting the notion that an important role of amino acid residues in the active site of flavin-dependent enzymes is to eliminate alternative reactions of the versatile enzyme-bound flavin for the reaction that needs to be catalyzed.

Characterization of the second conserved domain in the heme uptake protein HtaA from Corynebacterium diphtheriae.

HtaA is a heme-binding protein that is part of the heme uptake system in Corynebacterium diphtheriae. HtaA contains two conserved regions (CR1 and CR2). It has been previously reported that both domains can bind heme; the CR2 domain binds hemoglobin more strongly than the CR1 domain. In this study, we report the biophysical characteristics of HtaA-CR2. UV-visible spectroscopy and resonance Raman experiments are consistent with this domain containing a single heme that is bound to the protein through an axial tyrosine ligand. Mutants of conserved tyrosine and histidine residues (Y361, H412, and Y490) have been studied. These mutants are isolated with very little heme (≤5%) in comparison to the wild-type protein (~20%). Reconstitution after removal of the heme with butanone gave an alternative form of the protein. The HtaA-CR2 fold is very stable; it was necessary to perform thermal denaturation experiments in the presence of guanidinium hydrochloride. HtaA-CR2 unfolds extremely slowly; even in 6.8M GdnHCl at 37°C, the half-life was 5h. In contrast, the apo forms of WT HtaA-CR2 and the aforementioned mutants unfolded at much lower concentrations of GdnHCl, indicating the role of heme in stabilizing the structure and implying that heme transfer is effected only to a partner protein in vivo.