The glutaminase inhibitor CB-839 targets metabolic dependencies of JAK2-mutant hematopoiesis in MPN.

ABSTRACT: Hyperproliferation of myeloid and erythroid cells in myeloproliferative neoplasms (MPN) driven by the JAK2-V617F mutation is associated with altered metabolism. Given the central role of glutamine in anabolic and catabolic pathways, we examined the effects of pharmacologically inhibiting glutaminolysis, that is, the conversion of glutamine (Gln) to glutamate (Glu), using CB-839, a small molecular inhibitor of the enzyme glutaminase (GLS). We show that CB-839 strongly reduced the mitochondrial respiration rate of bone marrow cells from JAK2-V617F mutant (VF) mice, demonstrating a marked dependence of these cells on Gln-derived ATP production. Consistently, in vivo treatment with CB-839 normalized blood glucose levels, reduced splenomegaly and decreased erythrocytosis in VF mice. These effects were more pronounced when CB-839 was combined with the JAK1/2 inhibitor ruxolitinib or the glycolysis inhibitor 3PO, indicating possible synergies when cotargeting different metabolic and oncogenic pathways. Furthermore, we show that the inhibition of glutaminolysis with CB-839 preferentially lowered the proportion of JAK2-mutant hematopoietic stem cells (HSCs). The total number of HSCs was decreased by CB-839, primarily by reducing HSCs in the G1 phase of the cell cycle. CB-839 in combination with ruxolitinib also strongly reduced myelofibrosis at later stages of MPN. In line with the effects shown in mice, proliferation of CD34+ hematopoietic stem and progenitor cells from polycythemia vera patients was inhibited by CB-839 at nanomolar concentrations. These data suggest that inhibiting GLS alone or in combination with inhibitors of glycolysis or JAK2 inhibitors represents an attractive new therapeutic approach to MPN.

IL-1ß promotes MPN disease initiation by favoring early clonal expansion of JAK2-mutant hematopoietic stem cells.

ABSTRACT: JAK 2-V617F is the most frequent somatic mutation causing myeloproliferative neoplasm (MPN). JAK2-V617F can be found in healthy individuals with clonal hematopoiesis of indeterminate potential (CHIP) with a frequency much higher than the prevalence of MPNs. The factors controlling the conversion of JAK2-V617F CHIP to MPN are largely unknown. We hypothesized that interleukin-1ß (IL-1ß)-mediated inflammation can favor this progression. We established an experimental system using bone marrow (BM) transplantations from JAK2-V617F and GFP transgenic (VF;GFP) mice that were further crossed with IL-1ß-/- or IL-1R1-/- mice. To study the role of IL-1ß and its receptor on monoclonal evolution of MPN, we performed competitive BM transplantations at high dilutions with only 1 to 3 hematopoietic stem cells (HSCs) per recipient. Loss of IL-1ß in JAK2-mutant HSCs reduced engraftment, restricted clonal expansion, lowered the total numbers of functional HSCs, and decreased the rate of conversion to MPN. Loss of IL-1R1 in the recipients also lowered the conversion to MPN but did not reduce the frequency of engraftment of JAK2-mutant HSCs. Wild-type (WT) recipients transplanted with VF;GFP BM that developed MPNs had elevated IL-1ß levels and reduced frequencies of mesenchymal stromal cells (MSCs). Interestingly, frequencies of MSCs were also reduced in recipients that did not develop MPNs, had only marginally elevated IL-1ß levels, and displayed low GFP-chimerism resembling CHIP. Anti-IL-1ß antibody preserved high frequencies of MSCs in VF;GFP recipients and reduced the rate of engraftment and the conversion to MPN. Our results identify IL-1ß as a potential therapeutic target for preventing the transition from JAK2-V617F CHIP to MPNs.

Impact of Clonal Architecture on Clinical Course and Prognosis in Patients With Myeloproliferative Neoplasms.

Myeloproliferative neoplasms (MPNs) are caused by a somatic gain-of-function mutation in 1 of the 3 disease driver genes JAK2, MPL, or CALR. About half of the MPNs patients also carry additional somatic mutations that modify the clinical course. The order of acquisition of these gene mutations has been proposed to influence the phenotype and evolution of the disease. We studied 50 JAK2-V617F-positive MPN patients who carried at least 1 additional somatic mutation and determined the clonal architecture of their hematopoiesis by sequencing DNA from single-cell-derived colonies. In 22 of these patients, the same blood samples were also studied for comparison by Tapestri single-cell DNA sequencing (scDNAseq). The clonal architectures derived by the 2 methods showed good overall concordance. scDNAseq showed higher sensitivity for mutations with low variant allele fraction, but had more difficulties distinguishing between heterozygous and homozygous mutations. By unsupervised analysis of clonal architecture data from all 50 MPN patients, we defined 4 distinct clusters. Cluster 4, characterized by more complex subclonal structure correlated with reduced overall survival, independent of the MPN subtype, presence of high molecular risk mutations, or the age at diagnosis. Cluster 1 was characterized by additional mutations residing in clones separated from the JAK2-V617F clone. The correlation with overall survival improved when mutation in such separated clones were not counted. Our results show that scDNAseq can reliably decipher the clonal architecture and can be used to refine the molecular prognostic stratification that until now was primarily based on the clinical and laboratory parameters.

Iron is a modifier of the phenotypes of JAK2-mutant myeloproliferative neoplasms.

JAK 2-V617F mutation causes myeloproliferative neoplasms (MPNs) that can manifest as polycythemia vera (PV), essential thrombocythemia (ET), or primary myelofibrosis. At diagnosis, patients with PV already exhibited iron deficiency, whereas patients with ET had normal iron stores. We examined the influence of iron availability on MPN phenotype in mice expressing JAK2-V617F and in mice expressing JAK2 with an N542-E543del mutation in exon 12 (E12). At baseline, on a control diet, all JAK2-mutant mouse models with a PV-like phenotype displayed iron deficiency, although E12 mice maintained more iron for augmented erythropoiesis than JAK2-V617F mutant mice. In contrast, JAK2-V617F mutant mice with an ET-like phenotype had normal iron stores comparable with that of wild-type (WT) mice. On a low-iron diet, JAK2-mutant mice and WT controls increased platelet production at the expense of erythrocytes. Mice with a PV phenotype responded to parenteral iron injections by decreasing platelet counts and further increasing hemoglobin and hematocrit, whereas no changes were observed in WT controls. Alterations of iron availability primarily affected the premegakaryocyte-erythrocyte progenitors, which constitute the iron-responsive stage of hematopoiesis in JAK2-mutant mice. The orally administered ferroportin inhibitor vamifeport and the minihepcidin PR73 normalized hematocrit and hemoglobin levels in JAK2-V617F and E12 mutant mouse models of PV, suggesting that ferroportin inhibitors and minihepcidins could be used in the treatment for patients with PV.

Inhibition of interleukin-1ß reduces myelofibrosis and osteosclerosis in mice with JAK2-V617F driven myeloproliferative neoplasm.

Interleukin-1ß (IL-1ß) is a master regulator of inflammation. Increased activity of IL-1ß has been implicated in various pathological conditions including myeloproliferative neoplasms (MPNs). Here we show that IL-1ß serum levels and expression of IL-1 receptors on hematopoietic progenitors and stem cells correlate with JAK2-V617F mutant allele fraction in peripheral blood of patients with MPN. We show that the source of IL-1ß overproduction in a mouse model of MPN are JAK2-V617F expressing hematopoietic cells. Knockout of IL-1ß in hematopoietic cells of JAK2-V617F mice reduces inflammatory cytokines, prevents damage to nestin-positive niche cells and reduces megakaryopoiesis, resulting in decrease of myelofibrosis and osteosclerosis. Inhibition of IL-1ß in JAK2-V617F mutant mice by anti-IL-1ß antibody also reduces myelofibrosis and osteosclerosis and shows additive effects with ruxolitinib. These results suggest that inhibition of IL-1ß with anti-IL-1ß antibody alone or in combination with ruxolitinib could have beneficial effects on the clinical course in patients with myelofibrosis.

Association of telomere length with diabetes mellitus and idiopathic dilated cardiomyopathy in a South Indian population: A pilot study.

Telomere shortening has been associated with ageing and with many age-related diseases including cancer, coronary artery disease, heart failure and diabetes. We sought to investigate the link between telomere shortening and age-related diseases like type 2 diabetes mellitus (DM) (without any complications: DM; with neuropathic complication: DN) and idiopathic dilated cardiomyopathy (IDCM) in south Indian population. We compared telomere lengths of blood lymphocytes taken from patients with associated age-related diseases, namely DM (n = 47), DN (n = 52) and IDCM (n = 34) and controls (n = 46). In addition, we evaluated the relationship between echocardiographic left ventricular ejection fraction (LVEF), left ventricular end diastolic and systolic diameters (LVEDd and LVESd) and telomere length in IDCM patients. Telomere length negatively correlated with age in the cohorts with diabetes and IDCM, and in controls. Average telomere length in diabetes and IDCM patients was significantly shorter than that of controls either before or after adjustments for age and sex. Duration of diabetes in patients with type 2 diabetes did not correlate with telomere length. No correlation was found between the length of telomeres and echocardiography parameters like LVEF, LVEDd and LVESd in IDCM patients. Though echocardiographic characteristics of IDCM did not correlate with telomere length, telomere shortening was found to be accelerated in diabetes (both DM and DN) and IDCM in a south Indian population. Neuropathic complication in diabetes had no effect on telomere shortening. While telomere shortening is a cause or a consequence of diabetic and cardiac pathology remains further investigation, the current study substantiates the usefulness of telomere length measurements as a marker in conjunction with other biochemical markers of age-related diseases.

Organizational resilience and social-economic sustainability: COVID-19 perspective.

COVID-19 has affected the global economy like no other crisis in the history of mankind. It forced worldwide lockdown and economic shutdown to the point from where the recovery process has been very difficult. It has affected demand, supply, production and consumption in such a way that the entire economic development cycle has gone to its lowest levels. COVID-19 has also affected the social and economic sustainability structure which has led from one crisis to another and the developing countries have been the worst hit. Economic crisis resulted in unemployment leading to labour migrations, inevitable casualties and rising poverty etc. However, at a certain level, a few industries and organizations have shown resilience with better anticipation and survivability which may lead them to a quicker recovery. The current study aims at presenting a holistic view of organizational resilience which leads to the overall sustainable development. The study considers three aspects of organizational resilience as crisis anticipation, organizational robustness and recoverability. It assesses the impact of the aspects of resilience on social sustainability and economic sustainability. The study uses empirical analysis of primary data which is analysed to verify the hypothesized relationships by using a structural equation modelling approach. The study finds out that predicting the crisis and disruptions, building robustness and recoverability have a positive effect on both the social and economic aspects of sustainability. Findings of the study have their practical implications for industry, researchers and society.

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.

Tracking of epigenetic changes during hematopoietic differentiation of induced pluripotent stem cells.

BACKGROUND: Differentiation of induced pluripotent stem cells (iPSCs) toward hematopoietic progenitor cells (HPCs) raises high hopes for disease modeling, drug screening, and cellular therapy. Various differentiation protocols have been established to generate iPSC-derived HPCs (iHPCs) that resemble their primary counterparts in morphology and immunophenotype, whereas a systematic epigenetic comparison was yet elusive. RESULTS: In this study, we compared genome-wide DNA methylation (DNAm) patterns of iHPCs with various different hematopoietic subsets. After 20 days of in vitro differentiation, cells revealed typical hematopoietic morphology, CD45 expression, and colony-forming unit (CFU) potential. DNAm changes were particularly observed in genes that are associated with hematopoietic differentiation. On the other hand, the epigenetic profiles of iHPCs remained overall distinct from natural HPCs. Furthermore, we analyzed if additional co-culture for 2 weeks with syngenic primary mesenchymal stromal cells (MSCs) or iPSC-derived MSCs (iMSCs) further supports epigenetic maturation toward the hematopoietic lineage. Proliferation of iHPCs and maintenance of CFU potential was enhanced upon co-culture. However, DNAm profiles support the notion that additional culture expansion with stromal support did not increase epigenetic maturation of iHPCs toward natural HPCs. CONCLUSION: Differentiation of iPSCs toward the hematopoietic lineage remains epigenetically incomplete. These results substantiate the need to elaborate advanced differentiation regimen while DNAm profiles provide a suitable measure to track this process.

Evaluation of DNA damage in Type 2 diabetes mellitus patients with and without peripheral neuropathy: A study in South Indian population.

BACKGROUND: The increasing incidence of Type 2 diabetes mellitus globally has collaterally increased the incidence of diabetes-associated complications such as neuropathy. Oxidative stress induced DNA damage is one of the mechanisms implicated in the pathogenesis of diabetic complications. Here we aimed to evaluate the extent of DNA damage in diabetes patients with and without clinical neuropathy using the Cytokinesis Block Micronucleus Cytome assay, in a group of South Indian population. MATERIALS AND METHODS: The Cytokinesis Block Micronucleus Cytome assay was performed in lymphocyte cultures of 42 type 2 diabetes patients (22 with neuropathy and 20 without neuropathy) and 42 age and sex matched controls. Nuclear aberrations like Nuclear Buds, Nucleoplasmic Bridges and Micronuclei were analyzed. RESULTS: The frequency of nuclear aberrations in diabetes patients with neuropathy was higher than compared to diabetes patients without neuropathy. The mean frequencies of nuclear aberrations per cell in diabetes patients with neuropathy and without neuropathy were 0.02 ± 0.02 and 0.01 ± 0.01, respectively. This was significantly higher than in the controls (0.002 ± 0.002) (P < 0.0001). An increasing trend of nuclear aberrations in correlation with the duration of diabetes was observed. CONCLUSION: This study highlights the use of the Cytokinesis Block Micronucleus Cytome assay as a potent tool for the identification of DNA damage, which may prove to be useful biomarker to assess the severity diabetes-associated complications such as neuropathy. Implementation of this technique at the clinical level would potentially enhance the quality of management of patients with diabetes and its complications like neuropathy.