Wanke Zhao

JAK2V617F and p53 mutations coexist in erythroleukemia and megakaryoblastic leukemic cell lines.

BackgroundJAK2V617F, a gain-of-function mutant form of tyrosine kinase JAK2, is found in the majority of patients with Ph- myeloproliferative neoplasms (MPNs), a group of chronic hematological diseases that often lead to acute leukemia. The current study is intended to find other gene mutations that collaborate with JAK2V617F to cause leukemic transformation.MethodsTotal RNA and genomic DNA were isolated from two JAK2V617F-positive cell lines, namely, erythroleukemic HEL and megakaryoblastic leukemic SET-2 cells. Candidate genes were amplified by PCR and further sequenced.ResultsHomozygous mutations of the TP53 gene which encodes tumor suppressor p53 were found in HEL and SET-2 cells. While HEL cells, which have homozygous JAK2V617F, contain a rare M133K p53 mutation, SET-2 cells, which have a heterozygous JAK2V617F mutation, contain a common R248W p53 alteration. Western blot analyses revealed high levels of p53 expression in both cells. M133K and R248W are located in the DNA binding domain of p53. Structural analyses revealed that they potentially disrupt the interaction of p53 with DNA, thereby causing loss of p53 function.ConclusionsJAK2V617F and p53 mutations coexist in leukemia cells. We believe that JAK2V617F is able to drive leukemic transformation when the function of tumor suppressor p53 is lost. The interplay of JAK2V617F with p53 may affect the progression of MPNs.

Elevated Levels of Mast Cells Are Involved in Pruritus Associated with Polycythemia Vera in JAK2V617F Transgenic Mice

Pruritus occurs frequently in patients with polycythemia vera (PV), and the pathophysiology of PV-associated pruritus is unclear. We have previously demonstrated that transgenic mice expressing JAK2V617F displayed clear PV-like phenotypes. In the current study, we found frequent occurrence of pruritus with aged JAK2V617F transgenic mice and further investigated the underlying mechanisms by studying mast cells, key players in allergic reactions and anaphylaxis. Massive accumulations of mast cells were observed in the skin of pruritic JAK2V617F transgenic mice. In vitro culture yielded much higher mast cell counts from the bone marrow, spleen, peripheral blood, and peritoneal cavity of JAK2V617F transgenic mice than from controls. Cultured mast cells from JAK2V617F transgenic mice exhibited enhanced proliferative signals, relative resistance to cell death upon growth factor deprivation, and a growth advantage over control cells under suboptimal growth conditions. However, these mast cells displayed normal morphology and contained normal levels of mast cell proteases before and after degranulation. Finally, the JAK2 inhibitor G6 effectively reduced mast cell numbers and alleviated pruritus in JAK2V617F transgenic mice. Collectively, these data demonstrate that mast cells are involved in PV-associated pruritogenesis and that JAK2 inhibitors are potential antipruritus drugs.

Generation of a New Congenic Mouse Strain with Enhanced Chymase Expression in Mast Cells

Mast cells are effector cells best known for their roles in IgE-associated allergy, but they also play a protective role in defense against pathogens. These cells express high levels of proteases including chymase, tryptase and carboxypeptidase. In the present study, we identified a congenic strain of C57BL/6 mice expressing an extraordinarily high level of chymases Mcp-2 and Mcp-4 in mast cells. The overexpression was associated with variant Mcp-2 and Mcp-4 genes originated from DBA/2 mice that also expressed high levels of the two enzymes. Real time PCR analysis revealed that Mcp-2 and Mcp-4 were selectively overexpressed as tryptases, Cpa3 and several other chymases were kept at normal levels. Reporter gene assays demonstrated that single-nucleotide polymorphisms (SNPs) in the promoter region of Mcp-2 gene may be partly responsible for the increased gene transcription. Our study provides a new model system to study the function of mast cell chymases. The data also suggest that expression of chymases differs considerably in different strains of mice and the increased chymase activity may be responsible for some unique phenotypes observed in DBA/2 mice.

Phenotype risk scores identify patients with unrecognized Mendelian disease patterns

Hidden effects of Mendelian inheritance Identifying the determinate factors of genetic disease has been quite successful for Mendelian inheritance of large-effect pathogenic variants. In these cases, two non- or low-functioning genes contribute to disease. However, Mendelian effects of lesser strength have generally been ignored when looking at genomic consequences in human health. Bastarache et al. used electronic records to identify the phenotypic effects of previously unidentified Mendelian variations. Their analysis suggests that individuals with undiagnosed Mendelian diseases may be more prevalent in the general population than assumed. Because of this, genetic analysis may be able to assist clinicians in arriving at a diagnosis. Science, this issue p. 1233 Electronic health records coupled with exome sequencing identify disease phenotypes linked to Mendelian inheritance. Genetic association studies often examine features independently, potentially missing subpopulations with multiple phenotypes that share a single cause. We describe an approach that aggregates phenotypes on the basis of patterns described by Mendelian diseases. We mapped the clinical features of 1204 Mendelian diseases into phenotypes captured from the electronic health record (EHR) and summarized this evidence as phenotype risk scores (PheRSs). In an initial validation, PheRS distinguished cases and controls of five Mendelian diseases. Applying PheRS to 21,701 genotyped individuals uncovered 18 associations between rare variants and phenotypes consistent with Mendelian diseases. In 16 patients, the rare genetic variants were associated with severe outcomes such as organ transplants. PheRS can augment rare-variant interpretation and may identify subsets of patients with distinct genetic causes for common diseases.

Myotubularin family phosphatase ceMTM3 is required for muscle maintenance by preventing excessive autophagy in Caenorhabditis elegans

BackgroundAutophagy is a ubiquitous cellular process responsible for the bulk degradation of cytoplasmic components through the autophagosomal-lysosomal pathway. In skeletal muscle, autophagy has been regarded as a key regulator for muscle mass maintenance, and its imbalance leads to sarcopenia. However, the underlying mechanism is poorly understood.ResultsIn this study, we demonstrate that ceMTM3, a FYVE-domain containing myotubalarin family phosphatase, is required for the maintenance of muscle fibers by preventing excessive autophagy in Caenorhabditis elegans. Knockdown of ceMTM3 by using feeding-based RNA interference caused loss of muscle fibers accompanied by shortening of muscle cell and body size in aged C. elegans worms. This was preceded by the occurrence of excessive autophagy in the muscle and other tissues, which subsequently resulted in increased lysosomal activity and necrotic cell death. However, knockdown of ceMTM3 did not aggravate the abnormalities of muscle wasting in autophagy-deficient atg-18 mutant worms.ConclusionsOur data suggest an important role of ceMTM3 in regulating autophagy and maintaining muscle fibers. This study may have clinical implications for prevention and treatment of sarcopenia.

Tyrosine kinase inhibitors targeting FLT3 in the treatment of acute myeloid leukemia

Acute myeloid leukemia (AML) is a cancer of the myeloid lineage of blood cells. Although significant progress has been made in treating many types of cancers during recent years, AML remains a deadly disease with survival rate lagging behind other blood cancers. A combination of toxic chemotherapies has been the standard AML treatment for more than 40 years. With intensive efforts to define the pathogenesis of AML, novel therapeutic drugs targeting key molecular defects in AML are being developed. Mutated in nearly 30% of AML, FMS-like tyrosine kinase 3 (FLT3) represents one of the most attractive targets. FLT3 mutants resulted from either internal tandem duplication (ITD) or point mutations possess enhanced kinase activity and cause constitutive activation of signaling. To date, several small molecule inhibitors of FLT3 have been developed but their clinical efficacy is limited due to a lack of potency and the generation of drug resistance. Therefore, next-generation FLT3 inhibitors overcoming these limitations are urgently in need. This review focuses on the pathological role of mutant FLT3 in the development of AML, the current status of FLT3 inhibitor development, and mechanisms underlining the development of resistance to existing FLT3 inhibitors.

Cardiac hypertrophy associated with myeloproliferative neoplasms in JAK2V617F transgenic mice

BackgroundMyeloproliferative neoplasms (MPNs) are blood malignancies manifested in increased production of red blood cells, white blood cells, and/or platelets. A major molecular lesion associated with the diseases is JAK2V617F, an activation mutation form of tyrosine kinase JAK2. Cardiovascular events represent the leading cause of morbidity and mortality associated MPNs, but the underlying mechanism is not well understood.MethodsPreviously, we generated JAK2V617F transgenic mice which displayed MPN-like phenotypes. In the present study, we further characterized these mice by analyzing the time course of MPN phenotype development and associated cardiac abnormalities. We performed detailed histochemical staining of cardiac sections.ResultsJAK2V617F transgenic mice developed cardiomegaly as a subsequent event of increased blood cell production during the course of MPN phenotype development. The cardiomegaly is manifested in increased ventricular wall thickness and enlarged cardiomyocytes. Trichrome and reticulin staining revealed extensive collagen fibrosis in the heart of JAK2V617F transgenic mice. Thrombosis in the coronary artery and inflammatory cell infiltration into cardiac muscle were also observed in JAK2V617F transgenic mice, and the latter event was accompanied by fibrosis.ConclusionJAK2V617F-induced blood disorders have a major impact on heart function and lead to cardiac hypertrophy. JAK2V617F transgenic mice represent an excellent model system to study both hematological malignancies and cardiovascular diseases.

Efficacy of ALK5 inhibition in myelofibrosis

Myelofibrosis (MF) is a bone marrow disorder characterized by clonal myeloproliferation, aberrant cytokine production, extramedullary hematopoiesis, and bone marrow fibrosis. Although somatic mutations in JAK2, MPL, and CALR have been identified in the pathogenesis of these diseases, inhibitors of the Jak2 pathway have not demonstrated efficacy in ameliorating MF in patients. TGF-β family members are profibrotic cytokines and we observed significant TGF-β1 isoform overexpression in a large cohort of primary MF patient samples. Significant overexpression of TGF-β1 was also observed in murine clonal MPLW515L megakaryocytic cells. TGF-β1 stimulated the deposition of excessive collagen by mesenchymal stromal cells (MSCs) by activating the TGF-β receptor I kinase (ALK5)/Smad3 pathway. MSCs derived from MPLW515L mice demonstrated sustained overproduction of both collagen I and collagen III, effects that were abrogated by ALK5 inhibition in vitro and in vivo. Importantly, use of galunisertib, a clinically active ALK5 inhibitor, significantly improved MF in both MPLW515L and JAK2V617F mouse models. These data demonstrate the role of malignant hematopoietic stem cell (HSC)/TGF-β/MSC axis in the pathogenesis of MF, and provide a preclinical rationale for ALK5 blockade as a therapeutic strategy in MF.

Identification of an orally available compound with potent and broad FLT3 inhibition activity

FLT3 internal tandem duplication (FLT3-ITD) is an activating mutation found in 20–30% of patients with acute myeloid leukemia (AML), which makes FLT3 an attractive target for the treatment of AML. Although FLT3-mutant patients respond to current FLT3 inhibitors, relapse usually happens because of the acquisition of resistant secondary mutations at the FLT3 catalytic domain, which is mainly on D835. In the search for compounds with broad FLT3 inhibition activities, we screened a kinase inhibitor library by using our unique FLT3 substrate and identified JAK3 inhibitor VI (designated JI6 hereafter) as a novel FLT3 inhibitor, which selectively targets FLT3 D835 mutants as well as FLT3-ITD. JI6 effectively inhibited FLT3-ITD-containing MV4-11 cells and HCD-57 cells transformed with FLT3-ITD and D835 mutants. Furthermore, administration of JI6 effectively targeted FLT3 signaling in vivo and suppressed the myeloproliferative phenotypes in FLT3-ITD knock-in mice, and significantly prolonged the survival of immunodeficient mice implanted with the transformed HCD-57 cells. Therefore, JI6 is a promising candidate for the development of next-generation anti-AML drugs.

Quantitative analyses of myelofibrosis by determining hydroxyproline

BACKGROUND Myeloproliferative neoplasms (MPNs) are blood malignancies manifested in increased production of red blood cells, white blood cells, and/or platelets. Myelofibrosis is a subtype of MPNs characterized by the formation of scar-like tissues in the bone marrow due to abnormal hematopoiesis. It is considered a disease of both hematopoietic stem cells and stem cell niches. Patients with myelofibrosis have very poor prognosis, and there is no effective treatment so far. Myelofibrosis has routinely been detected by using histochemical staining methods which produce qualitative rather than quantitative results. In this study, we developed a quantitative assay of bone marrow myelofibrosis in JAK2V617F transgenic mice by determining hydroxyproline. METHODS The JAK2V617F transgenic mices tissue was collected to detect the bone marrow myelofibrosis. Statistical analyses were performed using the GraphPad Prism program. Differences of samples between two groups were accessed using t tests. P values less than 0.05 (2-tailed) were considered significantly different. RESULTS We developed a quantitative method for detecting myelofibrosis by analyzing the content of hydroxyproline, a modified amino acid largely restricted to collagen which forms the fibrotic structure in bone marrow tissues. Our study also demonstrated age-dependent development of bone marrow myelofibrosis in JAK2V617F transgenic mice. CONCLUSIONS In the present study, we have developed a new method for detecting bone marrow myelofibrosis by analyzing hydroxyproline contents. The method is highly sensitive and accurate. It provides more accurate, representative, and quantitative information than histochemical analyses. We believe that this method should find wide applications for analyzing the progression of myelofibrosis and efficacy of drug treatment.