Edwin Chen

JAK2 V617F impairs hematopoietic stem cell function in a conditional knock-in mouse model of JAK2 V617F-positive essential thrombocythemia

The JAK2 V617F mutation is found in most patients with a myeloproliferative neoplasm and is sufficient to produce a myeloproliferative phenotype in murine retroviral transplantation or transgenic models. However, several lines of evidence suggest that disease phenotype is influenced by the level of mutant JAK2 signaling, and we have therefore generated a conditional knock-in mouse in which a human JAK2 V617F is expressed under the control of the mouse Jak2 locus. Human and murine Jak2 transcripts are expressed at similar levels, and mice develop modest increases in hemoglobin and platelet levels reminiscent of human JAK2 V617F-positive essential thrombocythemia. The phenotype is transplantable and accompanied by increased terminal erythroid and megakaryocyte differentiation together with increased numbers of clonogenic progenitors, including erythropoietin-independent erythroid colonies. Unexpectedly, JAK2(V617F) mice develop reduced numbers of lineage(-)Sca-1(+)c-Kit(+) cells, which exhibit increased DNA damage, reduced apoptosis, and reduced cell cycling. Moreover, competitive bone marrow transplantation studies demonstrated impaired hematopoietic stem cell function in JAK2(V617F) mice. These results suggest that the chronicity of human myeloproliferative neoplasms may reflect a balance between impaired hematopoietic stem cell function and the accumulation of additional mutations.

Distinct clinical phenotypes associated with JAK2V617F reflect differential STAT1 signaling

The JAK2V617F mutation is associated with distinct myeloproliferative neoplasms, including polycythemia vera (PV) and essential thrombocythemia (ET), but it remains unclear how it generates disparate disorders. By comparing clonally-derived mutant and wild-type cells from individual patients, we demonstrate that the transcriptional consequences of JAK2V617F are subtle, and that JAK2V617F-heterozygous erythroid cells from ET and PV patients exhibit differential interferon signaling and STAT1 phosphorylation. Increased STAT1 activity in normal CD34-positive progenitors produces an ET-like phenotype, whereas downregulation of STAT1 activity in JAK2V617F-heterozygous ET progenitors produces a PV-like phenotype. Our results illustrate the power of clonal analysis, indicate that the consequences of JAK2V617F reflect a balance between STAT5 and STAT1 activation and are relevant for other neoplasms associated with signaling pathway mutations.

Janus Kinase Deregulation in Leukemia and Lymphoma

Genetic alterations affecting members of the Janus kinase (JAK) family have been discovered in a wide array of cancers and are particularly prominent in hematological malignancies. In this review, we focus on the role of such lesions in both myeloid and lymphoid tumors. Oncogenic JAK molecules can activate a myriad of canonical downstream signaling pathways as well as directly interact with chromatin in noncanonical processes, the interplay of which results in a plethora of diverse biological consequences. Deciphering these complexities is shedding unexpected light on fundamental cellular mechanisms and will also be important for improved diagnosis, identification of new therapeutic targets, and the development of stratified approaches to therapy.

Mouse models of myeloproliferative neoplasms: JAK of all grades

In 2005, several groups identified a single gain-of-function point mutation in the JAK2 kinase that was present in the majority of patients with myeloproliferative neoplasms (MPNs). Since this discovery, much effort has been dedicated to understanding the molecular consequences of the JAK2V617F mutation in the haematopoietic system. Three waves of mouse models have been produced recently (bone marrow transplantation, transgenic and targeted knock-in), which have facilitated the understanding of the molecular pathogenesis of JAK2V617F-positive MPNs, providing potential platforms for designing and validating novel therapies in humans. This Commentary briefly summarises the first two types of mouse models and then focuses on the more recently generated knock-in models.

Mutant Calreticulin Requires Both Its Mutant C-terminus and the Thrombopoietin Receptor for Oncogenic Transformation

UNLABELLED Somatic mutations in calreticulin (CALR) are present in approximately 40% of patients with myeloproliferative neoplasms (MPN), but the mechanism by which mutant CALR is oncogenic remains unclear. Here, we demonstrate that expression of mutant CALR alone is sufficient to engender MPN in mice and recapitulates the disease phenotype of patients with CALR-mutant MPN. We further show that the thrombopoietin receptor MPL is required for mutant CALR-driven transformation through JAK-STAT pathway activation, thus rendering mutant CALR-transformed hematopoietic cells sensitive to JAK2 inhibition. Finally, we demonstrate that the oncogenicity of mutant CALR is dependent on the positive electrostatic charge of the C-terminus of the mutant protein, which is necessary for physical interaction between mutant CALR and MPL. Together, our findings elucidate a novel paradigm of cancer pathogenesis and reveal how CALR mutations induce MPN. SIGNIFICANCE The mechanism by which CALR mutations induce MPN remains unknown. In this report, we show that the positive charge of the CALR mutant C-terminus is necessary to transform hematopoietic cells by enabling binding between mutant CALR and the thrombopoietin receptor MPL.

Genetic variation at MECOM , TERT , JAK2 and HBS1L-MYB predisposes to myeloproliferative neoplasms

Clonal proliferation in myeloproliferative neoplasms (MPN) is driven by somatic mutations in JAK2, CALR or MPL, but the contribution of inherited factors is poorly characterized. Using a three-stage genome-wide association study of 3,437 MPN cases and 10,083 controls, we identify two SNPs with genome-wide significance in JAK2V617F-negative MPN: rs12339666 (JAK2; meta-analysis P=1.27 × 10−10) and rs2201862 (MECOM; meta-analysis P=1.96 × 10−9). Two additional SNPs, rs2736100 (TERT) and rs9376092 (HBS1L/MYB), achieve genome-wide significance when including JAK2V617F-positive cases. rs9376092 has a stronger effect in JAK2V617F-negative cases with CALR and/or MPL mutations (Breslow–Day P=4.5 × 10−7), whereas in JAK2V617F-positive cases rs9376092 associates with essential thrombocythemia (ET) rather than polycythemia vera (allelic χ2 P=7.3 × 10−7). Reduced MYB expression, previously linked to development of an ET-like disease in model systems, associates with rs9376092 in normal myeloid cells. These findings demonstrate that multiple germline variants predispose to MPN and link constitutional differences in MYB expression to disease phenotype.

Rps14 haploinsufficiency causes a block in erythroid differentiation mediated by S100A8 and S100A9.

Impaired erythropoiesis in the deletion 5q (del(5q)) subtype of myelodysplastic syndrome (MDS) has been linked to heterozygous deletion of RPS14, which encodes the ribosomal protein small subunit 14. We generated mice with conditional inactivation of Rps14 and demonstrated an erythroid differentiation defect that is dependent on the tumor suppressor protein p53 (encoded by Trp53 in mice) and is characterized by apoptosis at the transition from polychromatic to orthochromatic erythroblasts. This defect resulted in age-dependent progressive anemia, megakaryocyte dysplasia and loss of hematopoietic stem cell (HSC) quiescence. As assessed by quantitative proteomics, mutant erythroblasts expressed higher levels of proteins involved in innate immune signaling, notably the heterodimeric S100 calcium-binding proteins S100a8 and S100a9. S100a8—whose expression was increased in mutant erythroblasts, monocytes and macrophages—is functionally involved in the erythroid defect caused by the Rps14 deletion, as addition of recombinant S100a8 was sufficient to induce a differentiation defect in wild-type erythroid cells, and genetic inactivation of S100a8 expression rescued the erythroid differentiation defect of Rps14-haploinsufficient HSCs. Our data link Rps14 haploinsufficiency in del(5q) MDS to activation of the innate immune system and induction of S100A8-S100A9 expression, leading to a p53-dependent erythroid differentiation defect.

Distinct effects of concomitant Jak2V617F expression and Tet2 loss in mice promote disease progression in myeloproliferative neoplasms

Signaling mutations (eg, JAK2V617F) and mutations in genes involved in epigenetic regulation (eg, TET2) are the most common cooccurring classes of mutations in myeloproliferative neoplasms (MPNs). Clinical correlative studies have demonstrated that TET2 mutations are enriched in more advanced phases of MPNs such as myelofibrosis and leukemic transformation, suggesting that they may cooperate with JAK2V617F to promote disease progression. To dissect the effects of concomitant Jak2V617F expression and Tet2 loss within distinct hematopoietic compartments in vivo, we generated Jak2V617F/Tet2 compound mutant genetic mice. We found that the combination of Jak2V617F expression and Tet2 loss resulted in a more florid MPN phenotype than that seen with either allele alone. Concordant with this, we found that Tet2 deletion conferred a strong functional competitive advantage to Jak2V617F-mutant hematopoietic stem cells (HSCs). Transcriptional profiling revealed that both Jak2V617F expression and Tet2 loss were associated with distinct and nonoverlapping gene expression signatures within the HSC compartment. In aggregate, our findings indicate that Tet2 loss drives clonal dominance in HSCs, and Jak2V617F expression causes expansion of downstream precursor cell populations, resulting in disease progression through combinatorial effects. This work provides insight into the functional consequences of JAK2V617F-TET2 comutation in MPNs, particularly as it pertains to HSCs.

JAK2V617F homozygosity drives a phenotypic switch in myeloproliferative neoplasms, but is insufficient to sustain disease

Genomic regions of acquired uniparental disomy (UPD) are common in malignancy and frequently harbor mutated oncogenes. Homozygosity for such gain-of-function mutations is thought to modulate tumor phenotype, but direct evidence has been elusive. Polycythemia vera (PV) and essential thrombocythemia (ET), 2 subtypes of myeloproliferative neoplasms, are associated with an identical acquired JAK2V617F mutation but the mechanisms responsible for distinct clinical phenotypes remain unclear. We provide direct genetic evidence and demonstrate that homozygosity for human JAK2V617F in knock-in mice results in a striking phenotypic switch from an ET-like to PV-like phenotype. The resultant erythrocytosis is driven by increased numbers of early erythroid progenitors and enhanced erythroblast proliferation, whereas reduced platelet numbers are associated with impaired platelet survival. JAK2V617F-homozygous mice developed a severe hematopoietic stem cell defect, suggesting that additional lesions are needed to sustain clonal expansion. Together, our results indicate that UPD for 9p plays a causal role in the PV phenotype in patients as a consequence of JAK2V617F homozygosity. The generation of a JAK2V617F allelic series of mice with a dose-dependent effect on hematopoiesis provides a powerful model for studying the consequences of mutant JAK2 homozygosity.

ID1 promotes expansion and survival of primary erythroid cells and is a target of JAK2V617F-STAT5 signaling

The discovery of JAK2V617F as an acquired mutation in the majority of patients with myeloproliferative disorders (MPDs) and the key role of the JAK2-STAT5 signaling cascade in normal hematopoiesis has focused attention on the downstream transcriptional targets of STAT5. Despite evidence of its vital role in normal erythropoiesis and its ability to recapitulate many of the features of myeloid malignancies, including the MPDs, few functionally validated targets of STAT5 have been described. Here we used a combination of comparative genomics and chromatin immunoprecipitation assays to identify ID1 as a novel target of the JAK2-STAT5 signaling axis in erythroid cells. STAT5 binds and transactivates a downstream enhancer of ID1, and ID1 expression levels correlate with the JAK2V617F mutation in both retrovirally transfected fetal liver cells and polycythemia vera patients. Knockdown and overexpression studies in a well-characterized erythroid differentiation assay from primary murine fetal liver cells demonstrated a survival-promoting action of ID1. This hitherto unrecognized function implicates ID1 in the expansion of erythroblasts during terminal differentiation and suggests that ID1 plays an important role in the pathogenesis of polycythemia vera. Furthermore, our findings contribute to an increasing body of evidence implicating ID proteins in a wider range of cellular functions than initially appreciated.