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.     

JAK2 inhibition has different therapeutic effects according to myeloproliferative neoplasm development in mice

JAK2 inhibition therapy is used to treat patients suffering from myeloproliferative neoplasms (MPN). Conflicting data on this therapy are reported possibly linked to the types of inhibitors or disease type. Therefore, we decided to compare in mice the effect of a JAK2 inhibitor, Fedratinib, in MPN models of increasing severity: polycythemia vera (PV), post‐PV myelofibrosis (PPMF) and rapid post‐essential thrombocythemia MF (PTMF). The models were generated through JAK2 activation by the JAK2^V617F mutation or MPL constant stimulation. JAK2 inhibition induced a correction of splenomegaly, leucocytosis and microcytosis in all three MPN models. However, the effects on fibrosis, osteosclerosis, granulocytosis, erythropoiesis or platelet counts varied according to the disease severity stage. Strikingly, complete blockade of fibrosis and osteosclerosis was observed in the PPMF model, linked to correction of MK hyper/dysplasia, but not in the PTMF model, suggesting that MF development may also become JAK2‐independent. Interestingly, we originally found a decreased in the JAK2^V617F allele burden in progenitor cells from the spleen but not in other cell types. Overall, this study shows that JAK2 inhibition has different effects according to disease phenotypes and can (i) normalize platelet counts, (ii) prevent the development of marrow fibrosis/osteosclerosis at an early stage and (iii) reduce splenomegaly through blockage of stem cell mobilization in the spleen.     

The discovery of 2,5-isomers of triazole-pyrrolopyrimidine as selective Janus kinase 2 (JAK2) inhibitors versus JAK1 and JAK3

Members of the Janus kinase (JAK) family are potential therapeutic targets. Abnormal signaling by mutant JAK2 is related to hematological malignancy, such as myeloproliferative neoplasms (MPNs), and tyrosine kinase inhibitor (TKI)-resistance in non-small cell lung cancer (NSCLC). We discovered a potent and highly selective inhibitor of JAK2 over JAK1 and -3 based on the structure of 4-(2,5-triazole)-pyrrolopyrimidine. Among all triazole compounds tested, 2,5-triazole regioisomers more effectively inhibited JAK2 kinase activity than isomers with substitutions of various alkyl groups at the R₂ position, except for methyl-substituted 1,5-triazole, which was more potent than the corresponding 1,4- and 2,5-triazoles. None of the synthesized 1,4-isomers inhibited all three JAK family members. Compounds with phenyl or tolyl group substituents at the R₁ position were completely inactive compared with the corresponding analogues with a methyl substituted at the R₁ position. As a result of this structure–activity relationship, 54, which is substituted with a cyclopropylmethyl moiety, exhibited significant inhibitory activity and selectivity (IC₅₀ = 41.9 nM, fold selectivity JAK1/2 10.6 and JAK3/2 58.1). Compound 54 also exhibited an equivalent inhibition of wild type JAK2 and the V617F mutant. Moreover, 54 inhibited the proliferation of HEL 92.1.7 cells, which carry JAK2 V617F, and gefitinib-resistant HCC827 cells. Compound 54 also suppressed STAT3 phosphorylation at Y705.     

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.     

Activation of JAK2-V617F by Components of Heterodimeric Cytokine Receptors

The JAK2-V617F mutation is an important etiologic factor for the development of myeloproliferative neoplasms. The mechanism by which this mutated tyrosine kinase initiates deregulated signals in cells is not completely understood. It is believed that JAK2-V617F requires interactions with homodimeric cytokine receptors to elicit its transforming signal. In this study, we demonstrate that components of heterodimeric cytokine receptors can also activate JAK2-V617F. Expression of IL27Ra, a heterodimeric receptor component, enhanced the activation of JAK2-V617F and subsequent downstream signaling to activation of STAT5 and ERK. In addition, expression of components of the interleukin-3 receptor, IL3Ra and the common β chain, activated JAK2-V617F as well as STAT5 and ERK. Importantly, expression of IL27Ra functionally replaced the requirement of a homodimeric cytokine receptor to promote the activation and transforming activity of JAK2-V617F in BaF3 cells. Tyrosine phosphorylation of IL27Ra was not required to induce activation of JAK2-V617F or STAT5, or to enhance the transforming activity of JAK2-V617F. Expression of IL3Ra or the common β chain in BaF3 cells also enhanced the ability of JAK2-V617F to transform these hematopoietic cells. However, the heterodimeric receptor component IL12RB1 did not enhance the activation or transforming signals of JAK2-V617F in BaF3 cells. IL27Ra also activated the K539L and R683G JAK2 mutants. Together our data demonstrate that in addition to homodimeric receptors, some heterodimeric receptor components can support the activation and transforming signals of JAK2-V617F and other JAK2 mutants. Therefore, heterodimeric receptors may play unappreciated roles in JAK2 activation in the development of hematopoietic diseases including myeloproliferative neoplasms.     

Absence of JAK2 V617F mutation in thalassemia intermedia patients

JAK2 is a cytoplasmic tyrosine kinase that has a vital role in signal transduction from several hemopoietic growth factor receptors. The JAK2 V617F mutation has been implicated in a variety of diseases mainly related to myeloproliferative disorders including polycythemia Vera, essential thrombocythemia, and idiopathic Myelofibrosis but has not been previously described in Thalassemia patients. We studied 36 Lebanese patients diagnosed with thalassemia intermedia and assessed the presence or absence of the JAK2 V617F mutation using JAK2 activating mutation assay (In VivoScribe Technologies) and Polymerase Chain Reaction (PCR). None of the thalassemia intermedia patients were positive for this mutation. To our knowledge, this study is the first to determine the status of JAK2 V617F mutation in thalassemia intermedia patients and expands the international published literature on JAK2. The latter’s V617F mutation does not seem to play a role in this hematologically important clinical entity.     

Stimulation of the amino acid transporter SLC6A19 by JAK2

JAK2 (Janus kinase-2) is expressed in a wide variety of cells including tumor cells and contributes to the proliferation and survival of those cells. The gain of function mutation ^V617FJAK2 mutant is found in the majority of myeloproliferative diseases. Cell proliferation depends on the availability of amino acids. Concentrative cellular amino acid uptake is in part accomplished by Na⁺ coupled amino acid transport through SLC6A19 (B(0)AT). The present study thus explored whether JAK2 activates SLC6A19. To this end, SLC6A19 was expressed in Xenopus oocytes with or without wild type JAK2, ^V617FJAK2 or inactive ^K882EJAK2 and electrogenic amino acid transport determined by dual electrode voltage clamp. In SLC6A19-expressing oocytes but not in oocytes injected with water or JAK2 alone, the addition of leucine (2 mM) to the bath generated a current (I_le), which was significantly increased following coexpression of JAK2 or ^V617FJAK2, but not by coexpression of ^K882EJAK2. Coexpression of JAK2 enhanced the maximal transport rate without significantly modifying the affinity of the carrier. Exposure of the oocytes to the JAK2 inhibitor AG490 (40 μM) resulted in a gradual decline of I_le. According to chemiluminescence JAK2 enhanced the carrier protein abundance in the cell membrane. The decline of I_le following inhibition of carrier insertion by brefeldin A (5 μM) was similar in the absence and presence of JAK2 indicating that JAK2 stimulates carrier insertion into rather than inhibiting carrier retrival from the cell membrane. In conclusion, JAK2 up-regulates SLC6A19 activity which may foster amino acid uptake into JAK2 expressing cells.     

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.     

2,7-Pyrrolo[2,1-f][1,2,4]triazines as JAK2 inhibitors: modification of target structure to minimize reactive metabolite formation

The JAK2/STAT pathway has important roles in hematopoiesis. With the discovery of the JAK2 V617F mutation and its presence in many patients with myeloproliferative neoplasms, research in the JAK2 inhibitor arena has dramatically increased. We report a novel series of potent JAK2 inhibitors containing a 2,7-pyrrolotriazine core. To minimize potential drug-induced toxicity, targets were analyzed for the ability to form a glutathione adduct. Glutathione adduct formation was decreased by modification of the aniline substituent at C2.     

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.     

Identification of oncostatin M as a JAK2 V617F-dependent amplifier of cytokine production and bone marrow remodeling in myeloproliferative neoplasms

The JAK2 mutation V617F is detectable in a majority of patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). Enforced expression of JAK2 V617F in mice induces myeloproliferation and bone marrow (BM) fibrosis, suggesting a causal role for the JAK2 mutant in the pathogenesis of MPNs. However, little is known about mechanisms and effector molecules contributing to JAK2 V617F-induced myeloproliferation and fibrosis. We show that JAK2 V617F promotes expression of oncostatin M (OSM) in neoplastic myeloid cells. Correspondingly, OSM mRNA levels were increased in the BM of patients with MPNs (median 287% of ABL, range 22-1450%) compared to control patients (median 59% of ABL, range 12-264%; P < 0.0001). OSM secreted by JAK2 V617F+ cells stimulated growth of fibroblasts and microvascular endothelial cells and induced the production of angiogenic and profibrogenic cytokines (HGF, VEGF, and SDF-1) in BM fibroblasts. All effects of MPN cell-derived OSM were blocked by a neutralizing anti-OSM antibody, whereas the production of OSM in MPN cells was suppressed by a pharmacologic JAK2 inhibitor or RNAi-mediated knockdown of JAK2. In summary, JAK2 V617F-mediated up-regulation of OSM may contribute to fibrosis, neoangiogenesis, and the cytokine storm observed in MPNs, suggesting that OSM might serve as a novel therapeutic target molecule in these neoplasms.     

Stimulation of the glucose carrier SGLT1 by JAK2

JAK2 (Janus kinase-2) overactivity contributes to survival of tumor cells and the ^V617FJAK2 mutant is found in the majority of myeloproliferative diseases. Tumor cell survival depends on availability of glucose. Concentrative cellular glucose uptake is accomplished by Na⁺ coupled glucose transport through SGLT1 (SLC5A1), which may operate against a chemical glucose gradient and may thus be effective even at low extracellular glucose concentrations. The present study thus explored whether JAK2 activates SGLT1. To this end, SGLT1 was expressed in Xenopus oocytes with or without wild type JAK2, ^V617FJAK2 or inactive ^K882EJAK2 and electrogenic glucose transport determined by dual electrode voltage clamp experiments. In SGLT1-expressing oocytes but not in oocytes injected with water or JAK2 alone, the addition of glucose to the extracellular bath generated a current (I_g), which was significantly increased following coexpression of JAK2 or ^V617FJAK2, but not by coexpression of ^K882EJAK2. Kinetic analysis revealed that coexpression of JAK2 enhanced the maximal transport rate without significantly modifying the affinity of the carrier. The stimulating effect of JAK2 expression was abrogated by preincubation with the JAK2 inhibitor AG490. Chemiluminescence analysis revealed that JAK2 enhanced the carrier protein abundance in the cell membrane. The decline of I_g during inhibition of carrier insertion by brefeldin A was similar in the absence and presence of JAK2. Thus, JAK2 fosters insertion rather than inhibiting retrieval of carrier protein into the cell membrane. In conclusion, JAK2 upregulates SGLT1 activity which may play a role in the effect of JAK2 during ischemia and malignancy.     

Effects of the JAK2 inhibitor, AZ960, on Pim/BAD/BCL-xL survival signaling in the human JAK2 V617F cell line SET-2

The Janus-associated kinase 2 (JAK2) V617F mutation is believed to play a critical role in the pathogenesis of polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis. We have characterized a novel small molecule JAK2 inhibitor, AZ960, and used it as a tool to investigate the consequences of JAK2 V617F inhibition in the SET-2 cell line. AZ960 inhibits JAK2 kinase with a K(i) of 0.00045 microm in vitro and treatment of TEL-JAK2 driven Ba/F3 cells with AZ960 blocked STAT5 phosphorylation and potently inhibited cell proliferation (GI(50)=0.025 microm). AZ960 demonstrated selectivity for TEL-JAK2-driven STAT5 phosphorylation and cell proliferation when compared with cell lines driven by similar fusions of the other JAK kinase family members. In the SET-2 human megakaryoblastic cell line, heterozygous for the JAK2 V617F allele, inhibition of JAK2 resulted in decreased STAT3/5 phosphorylation and inhibition of cell proliferation (GI(50)=0.033 microm) predominately through the induction of mitochondrial-mediated apoptosis. We provide evidence that JAK2 inhibition induces apoptosis by direct and indirect regulation of the anti-apoptotic protein BCL-xL. Inhibition of JAK2 blocked BCL-XL mRNA expression resulting in a reduction of BCL-xL protein levels. Additionally, inhibition of JAK2 resulted in decreased PIM1 and PIM2 mRNA expression. Decreased PIM1 mRNA corresponded with a decrease in Pim1 protein levels and inhibition of BAD phosphorylation at Ser(112). Finally, small interfering RNA-mediated suppression of BCL-xL resulted in apoptotic cell death similar to the phenotype observed following JAK2 inhibition. These results suggest a model in which JAK2 promotes cell survival by signaling through the Pim/BAD/BCL-xL pathway.