Efficacious intermittent dosing of a novel JAK2 inhibitor in mouse models of polycythemia vera

A high percentage of patients with the myeloproliferative disorder polycythemia vera (PV) harbor a Val617→Phe activating mutation in the Janus kinase 2 (JAK2) gene, and both cell culture and mouse models have established a functional role for this mutation in the development of this disease. We describe the properties of MRLB-11055, a highly potent inhibitor of both the WT and V617F forms of JAK2, that has therapeutic efficacy in erythropoietin (EPO)-driven and JAK2V617F-driven mouse models of PV. In cultured cells, MRLB-11055 blocked proliferation and induced apoptosis in a manner consistent with JAK2 pathway inhibition. MRLB-11055 effectively prevented EPO-induced STAT5 activation in the peripheral blood of acutely dosed mice, and could prevent EPO-induced splenomegaly and erythrocytosis in chronically dosed mice. In a bone marrow reconstituted JAK2V617F-luciferase murine PV model, MRLB-11055 rapidly reduced the burden of JAK2V617F-expressing cells from both the spleen and the bone marrow. Using real-time in vivo imaging, we examined the kinetics of disease regression and resurgence, enabling the development of an intermittent dosing schedule that achieved significant reductions in both erythroid and myeloid populations with minimal impact on lymphoid cells. Our studies provide a rationale for the use of non-continuous treatment to provide optimal therapy for PV patients.     

Erlotinib effectively inhibits JAK2V617F activity and polycythemia vera cell growth

JAK2(V617F), a mutant of tyrosine kinase JAK2, is found in most patients with polycythemia vera (PV) and a substantial proportion of patients with idiopathic myelofibrosis or essential thrombocythemia. The JAK2 mutant displays a much increased kinase activity and generates a PV-like phenotype in mouse bone marrow transplant models. This study shows that the anti-cancer drug erlotinib (Tarceva) is a potent inhibitor of JAK2(V617F) activity. In vitro colony culture assays revealed that erlotinib at micro-molar concentrations effectively suppresses the growth and expansion of PV hematopoietic progenitor cells while having little effect on normal cells. Furthermore, JAK2(V617F)-positive cells from PV patients show greater susceptibility to the inhibitor than their negative counterparts. Similar inhibitory effects were found with the JAK2(V617F)-positive human erythroleukemia HEL cell line. These data suggest that erlotinib may be used for treatment of JAK2(V617F)-positive PV and other myeloproliferative disorders.     

JAK1 and Tyk2 activation by the homologous polycythemia vera JAK2 V617F mutation: cross-talk with IGF1 receptor

The majority of polycythemia vera (PV) patients harbor a unique somatic mutation (V617F) in the pseudokinase domain of JAK2, which leads to constitutive signaling. Here we show that the homologous mutations in JAK1 (V658F) and in Tyk2 (V678F) lead to constitutive activation of these kinases. Their expression induces autonomous growth of cytokine-dependent cells and constitutive activation of STAT5, STAT3, mitogen-activated protein kinase, and Akt signaling in Ba/F3 cells. The mutant JAKs exhibit constitutive signaling also when expressed in fibrosarcoma cells deficient in JAK proteins. Expression of the JAK2 V617F mutant renders Ba/F3 cells hypersensitive to insulin-like growth factor 1 (IGF1), which is a hallmark of PV erythroid progenitors. Upon selection of Ba/F3 cells for autonomous growth induced by the JAK2 V617F mutant, cells respond to IGF1 by activating STAT5, STAT3, Erk1/2, and Akt on top of the constitutive activation characteristic of autonomous cells. The synergic effect on proliferation and STAT activation appears specific to the JAK2 V617F mutant. Our results show that the homologous V617F mutation induces activation of JAK1 and Tyk2, suggesting a common mechanism of activation for the JAK1, JAK2, and Tyk2 mutants. JAK3 is not activated by the homologous mutation M592F, despite the presence of the conserved GVC preceding sequence. We suggest that mutations in the JAK1 and Tyk2 genes may be identified as initial molecular defects in human cancers and autoimmune diseases.     

Akt activation through the phosphorylation of erythropoietin receptor at tyrosine 479 is required for myeloproliferative disorder-associated JAK2 V617F mutant-induced cellular transformation

The disruption of Janus kinase 2 (JAK2) signaling regulation by its point mutation, V617F, is involved in various myeloproliferative disorders (MPDs). JAK2 V617F mutant induced constitutive activation of Akt when erythropoietin receptor (EpoR) was coexpressed; however, the physiological role of Akt activation in MPDs has not been elucidated. LY294002, a phosphoinositide 3-kinase (PI3K) inhibitor, inhibited Akt activation and induced apoptotic cell death in cells expressing JAK2 V617F mutant and EpoR. Previously, it has been shown that the phosphorylation at Y479 in EpoR is critical for the interaction with PI3K, an upstream molecule of Akt. Hence, EpoR mutant with a point mutation of Y479F, which fails to activate Akt, is useful for addressing the role of Akt activation in JAK2 V617F mutant-induced tumorigenesis. Interestingly, under the expression of EpoR Y479F mutant, JAK2 V617F mutant failed to exhibit potent anti-apoptotic activity. In addition, JAK2 V617F mutant-induced phosphorylation of CREB and GSK-3β was significantly decreased in cells expressing EpoR Y479F mutant, resulting in the downregulation of Bcl-XL and Mcl-1 expression. Furthermore, compared with when nude mice were inoculated with cells expressing JAK2 V617F mutant and EpoR, the lifespan of nude mice inoculated with cells expressing JAK2 V617F mutant and EpoR Y479F mutant was effectively prolonged. Taken together, it was clarified that PI3K-Akt activation through the phosphorylation of EpoR at Y479 is required for oncogenic signaling of JAK2 V617F mutant and that targeted disruption of this pathway has therapeutic utility.     

Splenectomy Normalizes Hematocrit in Murine Polycythemia Vera

Splenic enlargement (splenomegaly) develops in numerous disease states, although a specific pathogenic role for the spleen has rarely been described. In polycythemia vera (PV), an activating mutation in Janus kinase 2 (JAK2^V617) induces splenomegaly and an increase in hematocrit. Splenectomy is sparingly performed in patients with PV, however, due to surgical complications. Thus, the role of the spleen in the pathogenesis of human PV remains unknown. We specifically tested the role of the spleen in the pathogenesis of PV by performing either sham (SH) or splenectomy (SPL) surgeries in a murine model of JAK2^V617F-driven PV. Compared to SH-operated mice, which rapidly develop high hematocrits after JAK2^V617F transplantation, SPL mice completely fail to develop this phenotype. Disease burden (JAK2^V617) is equivalent in the bone marrow of SH and SPL mice, however, and both groups develop fibrosis and osteosclerosis. If SPL is performed after PV is established, hematocrit rapidly declines to normal even though myelofibrosis and osteosclerosis again develop independently in the bone marrow. In contrast, SPL only blunts hematocrit elevation in secondary, erythropoietin-induced polycythemia. We conclude that the spleen is required for an elevated hematocrit in murine, JAK2^V617F-driven PV, and propose that this phenotype of PV may require a specific interaction between mutant cells and the spleen.     

Fullerene derivative prevents cellular transformation induced by JAK2 V617F mutant through inhibiting c-Jun N-terminal kinase pathway

The constitutively activated mutation (V617F) of tyrosine kinase Janus kinase 2 (JAK2) is found in the majority of patients with myeloproliferative neoplasms (MPNs). The development of a novel chemical compound to suppress JAK2 V617F mutant-induced onset of MPNs and clarification of the signaling cascade downstream of JAK2 V617F mutant will provide clues to treat MPNs. Here we found that a water-soluble pyrrolidinium fullerene derivative, C(60)-bis (N, N-dimethylpyrrolidinium iodide), markedly induced apoptosis of JAK2 V617F mutant-induced transformed cells through a novel mechanism, inhibiting c-Jun N-terminal kinase (JNK) activation pathway but not generation of reactive oxygen species (ROS). Pyrrolidinium fullerene derivative significantly reduced the protein expression level of apoptosis signal-regulating kinase 1 (ASK1), one of the mitogen-activated protein kinase kinase kinases (MAPKKK), resulting in the inhibition of upstream molecules of JNK, mitogen-activated protein kinase kinase 4 (MKK4) and mitogen-activated protein kinase kinase 7 (MKK7). Strikingly, the knockdown of ASK1 enhanced the sensitivity to pyrrolidinium fullerene derivative-induced apoptosis, and the treatment with a JNK inhibitor, SP600125, also induced apoptosis of the transformed cells by JAK2 V617F mutant. Furthermore, administration of both SP600125 and pyrrolidinium fullerene derivative markedly inhibited JAK2 V617F mutant-induced tumorigenesis in nude mice. Taking these findings together, JAK2 V617F mutant-induced JNK signaling pathway is an attractive target for MPN therapy, and pyrrolidinium fullerene derivative is now considered a candidate potent drug for MPNs.     

Substitution of pseudokinase domain residue Val-617 by large non-polar amino acids causes activation of JAK2

Explaining the uniqueness of the acquired somatic JAK2 V617F mutation, which is present in more than 95% of polycythemia vera patients, has been a challenge. The V617F mutation in the pseudokinase domain of JAK2 renders the unmutated kinase domain constitutively active. We have performed random mutagenesis at position 617 of JAK2 and tested each of the 20 possible amino acids for ability to induce constitutive signaling in Ba/F3 cells expressing the erythropoietin receptor. Four JAK2 mutants, V617W, V617M, V617I, and V617L, were able to induce cytokine independence and constitutive downstream signaling. Only V617W induced a level of constitutive activation comparable with V617F. Also, only V617W stabilized tyrosine-phosphorylated suppressor of cytokine signaling 3 (SOCS3), a mechanism by which JAK2 V617F overcomes inhibition by SOCS3. The V617W mutant induced a myeloproliferative disease in mice, mainly characterized by erythrocytosis and megakaryocytic proliferation. Although JAK2 V617W would predictably be pathogenic in humans, the substitution of the Val codon, GTC, by TTG, the codon for Trp, would require three base pair changes, and thus it is unlikely to occur. We discuss how the predicted conformations of the activated JAK2 mutants can lead to better screening assays for novel small molecule inhibitors.     

Evaluation of the JAK2-V617F gene mutation in Turkish patients with essential thrombocythemia and polycythemia vera

An activating mutation of Janus kinase 2 (JAK2-V617F) was previously described in chronic myeloproliferative disorders (MPD). In previously published studies, the frequency of the JAK2-V617F mutation was determined to be 80-90 % for patients with polycythemia vera (PV) and 40-70 % for essential thrombocythemia (ET). In this study, we analyzed the relationship between the JAK2-V617F mutation and clinical-hematological parameters in Turkish patients with MPD and compared these findings with published studies from other geographic regions. A total of 148 patients were studied; of which, 70 were diagnosed with PV and 78 with ET. The mutation status of JAK2 was determined using a tetra-primer polymerase chain reaction. We found that 80 % of the PV group and 42 % of the ET group were positive for the JAK2-V617F mutation. When all patients were analyzed, the levels of white blood cells, hemoglobin and splenomegaly were significantly different in patients with the JAK2-V617F mutation (p < 0.05). To our knowledge, this study is the first to evaluate the relationship between MPD and JAK2-V617F in Turkish patients. The JAK2-V617F mutation is frequently detected in the Turkish patients with MPD, and especially in patients with PV. Hence, it would be useful to include JAK2 mutation screening in the initial evaluation of patients suspected to have MPD.     

Impact of JAK2 V617F Mutation on Hemogram Variation in Patients with Non-Reactive Elevated Platelet Counts

Background

Non-reactive platelet counts elevation occurs mainly in myeloproliferative disorders (MPDs), which have been reported to be closely associated with JAK2 V617F mutation. Complete blood cell count (CBC) is essential in diagnosis of MPDs, however, the impact of JAK2 V617F mutation on the patients’ hemogram variation remains not clear.

Methods

JAK2 V617F mutation was detected by allele specific real-time quantitative fluorescence PCR (AS-qPCR).

Results

Of the 402 non-reactive platelet elevating patients, JAK2 V617F mutation was detected in 222 (55.2%) patients. RBC counts, WBC counts, platelet-large contrast ratio (P-LCR), platelet distribution width (PDW) and mean platelet volume (MPV) were much higher in JAK2 V617F mutated patients, except platelet counts. In addition, when the patients were classified into subgroups by blood cell counts, it was found that JAK2 V617F mutation rate increased progressively with the increase of RBC counts and WBC counts, other than platelet counts. Furthermore, trilineage hyperplasia group showed highest JAK2 V617F mutation rate (93.26%), followed by the bilineage hyperplasia groups. Lastly, JAK2 V617F mutant allele burden was found much higher in polycythemia vera (PV) patients [median(P₂₅–P₇₅): 45.02%(35.12%–54.22%)] than in essential thrombocythemia (ET) patients [median(P₂₅–P₇₅): 28.23%(17.77%–41.66%)], and that it increased with WBC counts (r = 0.393, p = 0.000) and RBC counts(r = 0.215, p = 0.001), other than platelet counts (r = −0.051, p = 0.452). Further analysis revealed that in ET patients, JAK2 V617F mutant allele burden correlated with WBC counts and platelet counts positively, other than RBC counts, while in PV patients, it correlated with WBC counts and RBC counts positively, but not platelet counts.

Conclusions

JAK2 V617F mutation occurs frequently in patients with non-reactive elevated platelet counts. The presence of JAK2 V617F mutation has great impact on hemogram variation, including RBC counts, WBC counts, platelet parameters and lineage hyperplasia, but not on platelet counts. Besides, JAK2 V617F mutant allele burden affects the blood cell proliferation pattern.     

Critical Roles of Myc-ODC Axis in the Cellular Transformation Induced by Myeloproliferative Neoplasm-Associated JAK2 V617F Mutant

The acquired mutation (V617F) of Janus kinase 2 (JAK2) is observed in the majority of patients with myeloproliferative neoplasms (MPNs). In the screening of genes whose expression was induced by JAK2 (V617F), we found the significant induction of c-Myc mRNA expression mediated by STAT5 activation. Interestingly, GSK-3β was inactivated in transformed Ba/F3 cells by JAK2 (V617F), and this enhanced the protein expression of c-Myc. The enforced expression of c-Myc accelerated cell proliferation but failed to inhibit apoptotic cell death caused by growth factor deprivation; however, the inhibition of GSK-3β completely inhibited the apoptosis of cells expressing c-Myc. Strikingly, c-Myc T58A mutant exhibited higher proliferative activity in a growth-factor-independent manner; however, this mutant failed to induce apoptosis. In addition, knockdown of c-Myc significantly inhibited the proliferation of transformed cells by JAK2 (V617F), suggesting that c-Myc plays an important role in oncogenic activity of JAK2 (V617F). Furthermore, JAK2 (V617F) induced the expression of a target gene of c-Myc, ornithine decarboxylase (ODC), known as the rate-limiting enzyme in polyamine biosynthesis. An ODC inhibitor, difluoromethylornithine (DFMO), prevented the proliferation of transformed cells by JAK2 (V617F). Importantly, administration of DFMO effectively delayed tumor formation in nude mice inoculated with transformed cells by JAK2 (V617F), resulting in prolonged survival; therefore, ODC expression through c-Myc is a critical step for JAK2 (V617F)-induced transformation and DFMO could be used as effective therapy for MPNs.     

Co‐targeting the PI3K/mTOR and JAK2 signalling pathways produces synergistic activity against myeloproliferative neoplasms

Aberrant JAK2 signalling plays a central role in myeloproliferative neoplasms (MPN). JAK2 inhibitors have proven to be clinically efficacious, however, they are not mutation‐specific and competent enough to suppress neoplastic clonal haematopoiesis. We hypothesized that, by simultaneously targeting multiple activated signalling pathways, MPN could be more effectively treated. To this end we investigated the efficacy of BEZ235, a dual PI3K/mTOR inhibitor, alone and in combination with the JAK1/JAK2 inhibitor ruxolitinib, in different preclinical models of MPN. Single‐agent BEZ235 inhibited the proliferation and induced cell cycle arrest and apoptosis of mouse and human JAK2V617F mutated cell lines at concentrations significantly lower than those required to inhibit the wild‐type counterpart, and preferentially prevented colony formation from JAK2V617F knock‐in mice and patients' progenitor cells compared with normal ones. Co‐treatment of BEZ235 and ruxolitinib produced significant synergism in all these in‐vitro models. Co‐treatment was also more effective than single drugs in reducing the extent of disease and prolonging survival of immunodeficient mice injected with JAK2V617F‐mutated Ba/F3‐EPOR cells and in reducing spleen size, decreasing reticulocyte count and improving spleen histopathology in conditional JAK2V617F knock‐in mice. In conclusion, combined inhibition of PI3K/mTOR and JAK2 signalling may represent a novel therapeutic strategy in MPN.     

A tetra-primer polymerase chain reaction approach for the detection of JAK2 V617F mutation

Recently, an acquired somatic point mutation (p.V617F) in a highly conserved residue of the pseudokinase domain of the JAK2 tyrosine kinase was shown to be associated with myeloproliferative disorders. Because of the clinical importance of this mutation in diagnosing myeloproliferative disorders and its relevance for disease progression, we have developed a tetra-primer polymerase chain reaction (PCR) assay to detect JAK2 p.V617F. Titration studies showed that the assay could reliably detect one copy of the mutant allele in a mix of 50 wild-type alleles suggesting that the lower detection limit of this assay is estimated to be 2%. This study demonstrates that genotyping and quantifying of the JAK2 V617F mutation can be performed by tetra-primer PCR using both freshly isolated and formalin-fixed tissues. Our tetra-primer PCR assay is sensitive, low-cost, and easy to use method for the detection of JAK2 p.V617F, which could be used even in low-tech laboratories.     

Combination treatment for myeloproliferative neoplasms using JAK and pan‐class I PI3K inhibitors

Current JAK2 inhibitors used for myeloproliferative neoplasms (MPN) treatment are not specific enough to selectively suppress aberrant JAK2 signalling and preserve physiological JAK2 signalling. We tested whether combining a JAK2 inhibitor with a series of serine threonine kinase inhibitors, targeting nine signalling pathways and already used in clinical trials, synergized in inhibiting growth of haematopoietic cells expressing mutant and wild‐type forms of JAK2 (V617F) or thrombopoietin receptor (W515L). Out of 15 kinase inhibitors, the ZSTK474 phosphatydylinositol‐3′‐kinase (PI3K) inhibitor molecule showed strong synergic inhibition by Chou and Talalay analysis with JAK2 and JAK2/JAK1 inhibitors. Other pan‐class I, but not gamma or delta specific PI3K inhibitors, also synergized with JAK2 inhibitors. Synergy was not observed in Bcr‐Abl transformed cells. The best JAK2/JAK1 and PI3K inhibitor combination pair (ruxolitinib and GDC0941) reduces spleen weight in nude mice inoculated with Ba/F3 cells expressing TpoR and JAK2 V617F. It also exerted strong inhibitory effects on erythropoietin‐independent erythroid colonies from MPN patients and JAK2 V617F knock‐in mice, where at certain doses, a preferential inhibition of JAK2 V617F mutated progenitors was detected. Our data support the use of a combination of JAK2 and pan‐class I PI3K inhibitors in the treatment of MPNs.     

Evaluation of clinical and laboratory findings with JAK2 V617F mutation as an independent variable in essential thrombocytosis

Essential thrombocythemia (ET) is an entity of classic Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), characterized by thrombocytosis with megakaryocytic hyperplasia and thrombocytes are increased with abnormal functions. Discovery of the protein tyrosine kinase JAK2 V617F allele contributed to better understanding of the pathogenetic mechanisms of MPNs. Acquired single point mutation in the JAK2 V617F was determined approximately 50–60 % of patients with ET. In this study we aimed to investigate the relationship between JAK2 V617F gene mutation, hematologic, biochemical markers and the complications in the ET patients. A total of 268 patients diagnosed with ET and 219 of those studied for JAK2 gene mutation were followed at the hematology clinics of three major hospitals between 2008 and 2013 were screened retrospectively. Laboratory, clinical and hematologic parameters were compared for JAK2 V617F positive and JAK2 V617F negative patients with ET. 102 (46 %) patients were positive with the JAK2 V617F mutation. The complications were observed in 61 (28 %) patients and 38 (62 %) of them had JAK2 V617F mutation. The levels of white blood cells, neutrophil, basophil, red blood cells, hemoglobin, hematocrit, mean platelet volume, thrombocytes, eosinophil; urea, creatinine were significantly different in patients with the JAK2 V617F mutation (P < 0.05). Presence of the JAK2 V617F mutation supports the diagnosis of ET. It would be useful to investigate the JAK2 V617F mutation and the hematologic and biochemical markers at diagnosis with respect to consider the risk of developing complications and to take the precautions against these complications.     

JAK2 in Myeloproliferative Disorders Is Not Just Another Kinase

Myeloproliferative disorders (MPD) represent a subcategory of hematological malignancies and are characterized by a stem cell-derived clonal proliferation of myeloid cells including erythrocytes, platelets, and leucocytes. Traditionally, the term ‘MPD’ included chronic myeloid leukemia (CML), polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis with myeloid metaplasia (MMM). At present, these four disorders are referred to as ‘classic’ MPD and are distinguished from a spectrum of other MPD-like clinico-pathologic entities that are operationally classified as ‘atypical’ MPD. The oncogenic mutations(s) in classic MPD are unknown except for CML, which is associated with an activating mutation (Bcr/Abl) of the gene encoding for the Abl cytoplasmic protein kinase (PTK). In the last 3 months, a somatic point mutation of JAK2 (JAK2^V617F), the gene encoding for another cytoplasmic PTK was reported in the majority of patients with PV and approximately half of those with either ET or MMM. The same mutation was also found in a small number of patients with either atypical MPD or the myelodysplastic syndrome but not in normal controls, germline tissue including T lymphocytes, and patients with secondary erythrocytosis. In vitro, JAK2^V617F was associated with constitutive phosphorylation of JAK2 and its downstream effectors as well as induction of erythropoietin hypersensitivity in cell lines. In vivo, murine bone marrow transduced with a retrovirus containing JAK2^V617F induced erythrocytosis in the transplanted mice. Taken together, these observations suggest that JAK2^V617F is an acquired myeloid lineage-specific mutation that engenders a pathogenetic relevance for the PV phenotype in MPD.