JAK2-mutant hematopoietic cells display metabolic alterations that can be targeted to treat myeloproliferative neoplasms

Increased energy requirement and metabolic reprogramming are hallmarks of cancer cells. We show that metabolic alterations in hematopoietic cells are fundamental to the pathogenesis of mutant JAK2-driven myeloproliferative neoplasms (MPNs). We found that expression of mutant JAK2 augmented and subverted metabolic activity of MPN cells, resulting in systemic metabolic changes in vivo, including hypoglycemia, adipose tissue atrophy, and early mortality. Hypoglycemia in MPN mouse models correlated with hyperactive erythropoiesis and was due to a combination of elevated glycolysis and increased oxidative phosphorylation. Modulating nutrient supply through high-fat diet improved survival, whereas high-glucose diet augmented the MPN phenotype. Transcriptomic and metabolomic analyses identified numerous metabolic nodes in JAK2-mutant hematopoietic stem and progenitor cells that were altered in comparison with wild-type controls. We studied the consequences of elevated levels of Pfkfb3, a key regulatory enzyme of glycolysis, and found that pharmacological inhibition of Pfkfb3 with the small molecule 3PO reversed hypoglycemia and reduced hematopoietic manifestations of MPNs. These effects were additive with the JAK1/2 inhibitor ruxolitinib in vivo and in vitro. Inhibition of glycolysis by 3PO altered the redox homeostasis, leading to accumulation of reactive oxygen species and augmented apoptosis rate. Our findings reveal the contribution of metabolic alterations to the pathogenesis of MPNs and suggest that metabolic dependencies of mutant cells represent vulnerabilities that can be targeted for treating MPNs

Nonenzymatic β‑Carotene Degradation in Provitamin A‑Biofortified Crop Plants

Provitamin A biofortification, the provision of provitamin A carotenoids through agriculture, is regarded as an effective and sustainable intervention to defeat vitamin A deficiency, representing a global health problem. This food-based intervention has been questioned in conjunction with negative outcomes for smokers and asbestos-exposed populations of the CARET and ATBC trials in which very high doses of β-carotene were supplemented. The current notion that β-carotene cleavage products (apocarotenoids) represented the harmful agents is the basis of the here-presented research. We quantitatively analyzed numerous plant food items and concluded that neither the amounts of apocarotenoids nor β-carotene provided by plant tissues, be they conventional or provitamin A-biofortified, pose an increased risk. We also investigated β-carotene degradation pathways over time. This reveals a substantial nonenzymatic proportion of carotene decay and corroborates the quantitative relevance of highly oxidized β-carotene polymers that form in all plant tissues investigated

Plant-type phytoene desaturase: Functional evaluation of structural implications

<div><p>Phytoene desaturase (PDS) is an essential plant carotenoid biosynthetic enzyme and a prominent target of certain inhibitors, such as norflurazon, acting as bleaching herbicides. PDS catalyzes the introduction of two double bonds into 15-<i>cis</i>-phytoene, yielding 9,15,9'-tri-<i>cis</i>-ζ-carotene via the intermediate 9,15-di-<i>cis</i>-phytofluene. We present the necessary data to scrutinize functional implications inferred from the recently resolved crystal structure of <i>Oryza sativa</i> PDS in a complex with norflurazon. Using dynamic mathematical modeling of reaction time courses, we support the relevance of homotetrameric assembly of the enzyme observed <i>in crystallo</i> by providing evidence for substrate channeling of the intermediate phytofluene between individual subunits at membrane surfaces. Kinetic investigations are compatible with an ordered ping-pong bi-bi kinetic mechanism in which the carotene and the quinone electron acceptor successively occupy the same catalytic site. The mutagenesis of a conserved arginine that forms a hydrogen bond with norflurazon, the latter competing with plastoquinone, corroborates the possibility of engineering herbicide resistance, however, at the expense of diminished catalytic activity. This mutagenesis also supports a “flavin only” mechanism of carotene desaturation not requiring charged residues in the active site. Evidence for the role of the central 15-<i>cis</i> double bond of phytoene in determining regio-specificity of carotene desaturation is presented.</p></div

Calreticulin and JAK2V617F driver mutations induce distinct mitotic defects in myeloproliferative neoplasms

Abstract Myeloproliferative neoplasms (MPNs) encompass a diverse group of hematologic disorders driven by mutations in JAK2, CALR, or MPL. The prevailing working model explaining how these driver mutations induce different disease phenotypes is based on the decisive influence of the cellular microenvironment and the acquisition of additional mutations. Here, we report increased levels of chromatin segregation errors in hematopoietic cells stably expressing CALRdel52 or JAK2V617F mutations. Our investigations employing murine 32DMPL and human erythroleukemic TF-1MPL cells demonstrate a link between CALRdel52 or JAK2V617F expression and a compromised spindle assembly checkpoint (SAC), a phenomenon contributing to error-prone mitosis. This defective SAC is associated with imbalances in the recruitment of SAC factors to mitotic kinetochores upon CALRdel52 or JAK2V617F expression. We show that JAK2 mutant CD34 + MPN patient-derived cells exhibit reduced expression of the master mitotic regulators PLK1, aurora kinase B, and PP2A catalytic subunit. Furthermore, the expression profile of mitotic regulators in CD34 + patient-derived cells allows to faithfully distinguish patients from healthy controls, as well as to differentiate primary and secondary myelofibrosis from essential thrombocythemia and polycythemia vera. Altogether, our data suggest alterations in mitotic regulation as a potential driver in the pathogenesis in MPN

Stereoconfiguration of PDS products.

<p>(A) Phytofluene isomers: trace a represents phytofluene from a PDS assay. The peak marked with * represents the ζ-carotene formed. Only the correct 9,15-di-<i>cis</i>-phytofluene isomer is formed as revealed by comparison with authentic standards isolated from sources where <i>cis</i>-configurations are known, such as trace b, phytofluene from the <i>tangerine</i> mutant of tomato fruit [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187628#pone.0187628.ref031" target="_blank">31</a>] and trace c, phytofluene from <i>Dunaliella bardawil</i> grown in the presence of norflurazon [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187628#pone.0187628.ref032" target="_blank">32</a>]. The synthetic standards all-<i>trans</i> and 15-<i>cis</i>-phytofluene are shown in trace d. (B) ζ-carotene isomers: trace e, from PDS assays. Only the correct 9,15,9’-tri-<i>cis-</i>ζ-carotene is formed, as revealed by the effect of illumination of the PDS assay (trace f) whereby the photolabile central double bond is isomerized to <i>trans</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187628#pone.0187628.ref004" target="_blank">4</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187628#pone.0187628.ref024" target="_blank">24</a>] yielding the 9,9’-di-<i>cis</i> species accompanied by small amounts of the 9-<i>cis</i> and all-<i>trans</i> species. Trace g, extract from <i>tangerine</i> tomato fruit containing 9,9’-di-<i>cis</i>-ζ-carotene. The peak marked with * represents β-carotene, detected because of spectral overlap. HPLC traces (HPLC system 2) were recorded at 400 nm. UV/VIS spectra are given as insets.</p