Pseudokinase Domain Of JAK2 Research Articles

Intrinsic Resistance to JAK2 Inhibition in Myelofibrosis

Abstract 2825Myeloproliferative neoplasms (MPN) are a group of clonal hematopoietic stem cell disorders with a variable clinical course: essential thrombocytosis (ET) and polycythemia vera (PV) are associated with excess clotting and bleeding but an overall normal or near-normal life expectancy, while myelofibrosis has a variable but generally poor prognosis. The disorders are unified by the constitutive activation of the JAK2 pathway, most commonly by a point mutation in the pseudokinase domain of JAK2 (JAK2 V617F). Small molecule tyrosine kinase inhibitors have been shown to be active in both preclinical and late-phase clinical testing, and yet notably pharmacodynamic validation of target (JAK2) inhibition has never been rigorously demonstrated in clinical studies. Clinical investigation of JAK2 inhibitors has focused on myelofibrosis, with some unexpected results: while reduction in spleen size and amelioration of constitutional symptoms are frequent, only modest changes in mutant clonal burden have been observed and complete responses by any criteria are exceedingly rare. In order to develop a feasible and informative pharmacodynamic assay for measuring JAK2 signaling, we used phospho-specific flow cytometry on whole peripheral blood to measure phosphorylation of STAT3 and STAT5, the canonical targets of JAK2 in hematopoietic cells. Treating whole blood with tyrosine kinase inhibitors currently in clinical development, we observed a dose-dependent decrease in phospho-STAT5 and STAT3 in granulocytes treated ex vivo in cells from normal donors, patients with ET, PV and to a lesser extent from patients with myelofibrosis. We found that phosphorylation of STAT3 and STAT5 in cells from patients with myelofibrosis were significantly less inhibited than in PV and ET as well as normal donors. Using quantitative RT-PCR, we found that sensitivity to inhibition did not correlate with JAK2 V617F clonal burden. To determine whether extracellular determinants conferred resistance, mixing studies showed that plasma from patients with myelofibrosis did not transfer resistance. Plasma cytokines that signal through JAK2 and/or are known to be altered in myelofibrosis were measured and no single cytokine appeared to account for the observed pattern of resistance. Taken together these observations suggest that there are cell intrinsic mechanisms that define a priori resistance to JAK2 inhibition in myelofibrosis, and the lesion is localized upstream of STAT3 and STAT5. Disclosures:Carroll:Sanofi Aventis Corporation: Research Funding; TetraLogic Pharmaceuticals: Research Funding; Agios Pharmaceuticals: Research Funding; Glaxo Smith Kline, Inc.: Research Funding.

New Insights on Assessing Intra-Family Selectivity for Jak2 Inhibitors

Abstract 5150Essential thrombocythemia (ET), polycythemia vera (PV) and myelofibrosis (MF) are myeloproliferative disorders (MPDs) characterized by a chronic over-production of cells of one or more blood cell lineages and/or bone marrow fibrosis which may, on occasion, progress to acute myeloid leukemia. The V617F gain of function mutation in the pseudokinase domain of Jak2, which results in constitutive activation of Jak2, is the most frequent mutation associated with MPD. Constitutively activated Jak2 can lead to dysregulated downstream signaling pathways (STAT, MAP kinase, and PI3 kinase) which in turn trigger abnormal growth, survival and differentiation of hematopoietic progenitors. Therefore, inhibition of constitutively activated Jak2 may offer therapeutic potential. Designing a Jak2V617F specific inhibitor encounters challenges due to the lack of enzymatic activity of the pseudokinase domain of Jak2. In lieu of a Jak2V617F mutant selective inhibitor, a highly selective inhibitor of Jak2 is likely an attainable goal.Jak2 is a member of the Jak family of kinases including Jak1, Jak3, and Tyk2. Highly selective Jak2 inhibitors may provide a better safety margin in chronic dosing settings in ET and PV patients since inhibiting other Jak family members could cause side-effects such as immunosuppression. Attaining the desired selectivity of Jak2 inhibition versus the other family members has been challenging and few compounds have been reported to date that have the desired Jak2 selectivity. This can be attributed to the high homology of the ATP binding pocket among Jak family members, but is also hampered by a lack of assays capable of distinguishing the Jak-selectivity profile in a physiologically relevant setting.We compared the potency and selectivity of compounds tested in a pSTAT5 AlphaScreen® assay panel consisting of isogenic Ba/F3 cell lines individually expressing translocated ETS leukemia (TEL) fusions of each Jak-family member (Ba/F3-TEL-Jak) with data from corresponding Jak enzyme assays. Here we report that the selectivity of inhibitor compounds illustrated in enzyme assays did not correlate with the selectivity profile in cell lines due to different shifts in potency for each family member between enzyme and cells (Figure 1). As a consequence the selectivity of compounds for Jak2 against Jak1 observed in enzyme assays may be reduced or reversed in cellular assays. On the other hand, Jak2 selectivity over Jak3 seen in the enzyme assays was conserved in the cellular assay. Thus, we propose that compounds that exhibit greater potency on Jak2 compared to Jak1 in the enzyme assays are needed and should be the main focus of medicinal chemistry efforts in order to attain Jak2 selectivity over Jak1 in a cellular context. [Display omitted] We also compared the potency and selectivity of compounds in the isogenic Ba/F3-TEL-Jak cell lines with data obtained with cytokine stimulated peripheral blood mononuclear cells (PBMCs). The potency and selectivity of compounds in PBMCs are determined by measuring the inhibition of phosphorylation of STAT5 in TPO or GM-CSF stimulated platelets or monocytes (mediated by Jak2) and in IL-2 stimulated lymphocytes (mediated by Jak1 and Jak3). We found that potency correlated well between PBMCs and Ba/F3-TEL-Jak2 cells, and the rank order of compounds based on IC50 values obtained with Ba/F3-TEL-Jak cell lines were conserved well in PBMCs; the compound selectivity profiles derived from the Ba/F3-TEL-Jak cell assays were predictive of Jak2 selectivity profiles obtained in the PBMC assays. Therefore, inclusion of Ba/F3-TEL-Jak pSTAT5 cellular assays may be useful for Jak family inhibitor development. Our results also suggest that relying solely on enzyme potency and selectivity data can be misleading, and that evaluating cellular selectivity in a biologically relevant context may provide a more meaningful understanding of selectivity and lead to the development of more selective Jak2 compounds. Disclosures:Liu:Amgen, Inc: Employment. Yu:Amgen: Employment. Pistillo:Amgen: Employment. Lee:Amgen: Employment. Schenkel:Amgen: Employment. Geuns-Meyer:Amgen: Employment. Archibeque:Amgen: Employment. Sinclair:Amgen: Employment. Emkey:Amgen: Employment. Molineux:Amgen: Employment.

The pseudokinase domain of JAK2 is a dual-specificity protein kinase that negatively regulates cytokine signaling

Human JAK2 tyrosine kinase mediates signaling through numerous cytokine receptors. The JAK2 JH2 domain functions as a negative regulator and is presumed to be a catalytically inactive pseudokinase, but the mechanism(s) for its inhibition of JAK2 remains unknown. Mutations in JH2 lead to increased JAK2 activity, contributing to myeloproliferative neoplasms (MPNs). Here we show that JH2 is a dual-specificity protein kinase that phosphorylates two negative regulatory sites in JAK2: Ser523 and Tyr570. Inactivation of JH2 catalytic activity increased JAK2 basal activity and downstream signaling. Notably, different MPN mutations abrogated JH2 activity in cells, and in MPN (V617F) patient cells phosphorylation of Tyr570 was reduced, suggesting that loss of JH2 activity contributes to the pathogenesis of MPNs. These results identify the catalytic activity of JH2 as a previously unrecognized mechanism to control basal activity and signaling of JAK2.

JAK2V617F/TET2 mutations: does the order matter?

According to the World Health Organization classification, myeloproliferative neoplasms (MPN) include chronic myelogenous leukemia, also known as BCR-ABL1–positive MPN, classic BCR-ABL1-negative MPN including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), and non-classic forms (i.e. systemic mastocytosis, chronic eosinophilic leukemia not otherwise specified, chronic neutrophilic leukemia and unclassifiable MPN). All these subtypes are stem cell-derived clonal myeloproliferation, associated with the overproduction of mature blood elements and variable rates of transformation to acute myeloid leukemia (AML).1 JAK2V617F activating mutation is the most prevalent abnormality observed in BCR-ABL1-negative MPN, found in virtually all cases of PV and in about half of ET and PMF (96%, 55% and 65%, respectively). This mutation lies in the pseudokinase-domain of JAK2 and disrupts its regulatory activity. Another mutation affecting JAK2 exon 12 is observed in 3% of all PV cases. Mutations affecting W515 of the thrombopoietin receptor MPL are detected in PMF and ET patients. Additional mutations have been identified in MPN (reviewed in 1). Defects in the control of intracellular signaling involve mutations in LNK and CBL genes. Genetic abnormalities affecting epigenetic regulation, and possibly responsible for disease initiation, concern the ASXL1, EZH2 and TET2 genes. Finally, mutation in IKZF1 and IDH1/2 may be implicated in MPN transformation. In PV, among these additional mutations to JAK2V617F, TET2 mutations are those most frequently reported (16%); the others are only described in small subsets of patients.1 TET2 belongs to a family of three conserved genes in mammals: TET1, TET2 and TET3. The founding member of the family, TET1, has been identified as a fusion partner of MLL in the t(10;11)(q22;q23) translocation of acute leukemia.2,3 The involvement of TET3 in hematologic disorder has not yet been described. The TET proteins are members of the 2-oxoglutarate (2-OG)- and Fe(II)-dependent dioxygenase that are able to convert 5-methyl-cytosine (5-mC) to 5-hydroxymethylcytosine (hmC).4,5 Recent reports indicate an important role for TET1 and TET2 (and, therefore, hmC) in the control of ES cell self-renewal and differentiation.6 TET3 might be involved in genome reprogramming following fecundation.7

Identification of JAK2 mutations in canine primary polycythemia

Primary polycythemia in dogs is classified as a myeloproliferative syndrome with a chronic progressive course and unspecific symptoms. Diagnosis is based on exclusion criteria. In humans, the presence of an acquired recurrent mutation within the JAK2 gene has recently been identified in 90% of the patients with polycythemia vera. This mutation (V617F) is located in the pseudokinase domain of JAK2, leading to constitutive activation of the kinase responsible for the polycythemia. Detection of the mutation has now become a major diagnostic tool in humans for polycythemia vera diagnosis. As the canine JAK2 gene shares strong homology with its human counterpart, we looked for the presence of JAK2 mutations in dogs with an elevated hematocrit. Direct sequencing of the JAK2 exon 14 was performed on DNA extracted from the peripheral blood of five dogs suspected of primary polycythemia. Mutant subclones were expressed in interleukin-3-dependent BaF3 cells and tested for cytokine independency. One dog presented with a three-base change in codons 617 and 618 of JAK2 giving rise to V617F and C618L mutations. By polymerase chain reaction product subcloning, we demonstrated the coexistence of the wild-type sequence and a triple mutant sequence, while DNA from buccal swab contained the wild-type sequence only. Transfection of BaF3 cells with the triple mutant cDNA, but not with the wild-type complementary DNA, resulted in cytokine-independent growth and constitutive signal transducer and activation of transcription 5 phosphorylation. Identical mutations of the JAK2 gene occur in humans and dogs, giving rise to a constitutively active JAK2 kinase, suggesting a common mechanism for human and canine diseases. Thus, common diagnostic tools and therapeutic approaches may be relevant.

Modification of CellSensor irf1-bla TF-1 and irf1-bla HEL Assays for Direct Comparison of Wild-Type JAK2 and JAK2 V617F Inhibition

The Janus kinase (JAK)-signal transducer and activator of transcription pathway is an important therapeutic target because of its role in the regulation of cell growth. Aberrant, constitutive activation of JAK2 signaling has been implicated in myeloproliferative disorders with a single, activating somatic V617F mutation in the JH2 pseudokinase domain of JAK2 as the prevalent molecular lesion. Invitrogen has developed the CellSensor(®) cell lines interferon regulatory factor-1 (irf1)-beta-lactamase (bla) TF-1 and irf1-bla HEL for use in evaluating inhibitors of wild-type JAK2 and mutant JAK2 V617F, respectively. Both contain a bla reporter gene downstream of the irf1 response element stably integrated into either TF-1 or HEL cells. A fluorescence resonance energy transfer-based bla substrate is utilized to give a robust detection of JAK2 activity. Examination of Invitrogen's protocols for the two cell lines revealed significant differences that are not conducive to direct comparison of inhibitor activities against wild-type and mutant JAK2. Systematic changes to standardize the two assays were incorporated and evaluated for effects on assay response ratio, assay quality, and potency for a diverse series of inhibitors.

Requirement of Lipid Raft Integrity for Signaling by JAK2-V617F

AbstractAbstract 4191JAK2 is a cytoplasmic tyrosine kinase that plays an important role in signaling following activation of various cytokine receptors. JAK2 activation promotes growth, survival, and differentiation of various cell types. Mutation of JAK2 is seen in numerous hematopoietic diseases, most notably in myeloproliferative neoplasms (MPNs). JAK2-V617F is a frequent mutation found in the classical MPNs: polycythemia vera, essential thrombocythemia, and primary myelofibrosis. The single amino acid change of valine to phenylalanine occurs in the pseudokinase domain of JAK2, relieving auto-inhibition of the kinase domain and allowing constitutive kinase activity. Numerous mouse models have demonstrated that JAK2-V617F can induce MPN-like disorders in mice. Thus, this point mutation, as well as other less common JAK2 mutations, is believed to play an important etiologic role in the development of MPNs in humans. The development and use of JAK2 inhibitors in clinical trials has shown promising results, again demonstrating the important role JAK2 plays in MPNs. While the JAK2-V617F mutation, as well as other JAK2 mutations, decreases auto-inhibition of JAK2 kinase activity, it is clear that mutated JAK2 still requires the expression of cytokine receptors to induce activation of transforming signals in hematopoietic cells. Normally, JAK2 binds to homodimeric and heterodimeric cytokine receptors through specific receptor motifs and is activated by various structural changes induced by cytokine stimulation. Following activation it utilizes receptor tyrosines as substrates for phosphorylation, leading to recruitment of downstream signaling molecules, such as STAT5, among others. JAK2 then activates STAT5 via phosphorylation and STAT5 then translocates to the nucleus to regulate transcription of target genes. JAK2-V617F does not require ligand for activation, but still requires the scaffolding function of cytokine receptors to facilitate its full activation and activation of downstream signaling via phosphorylation. Lipid rafts are microdomains of the plasma membrane that are enriched in cholesterol and sphingolipids. They have gained appreciation in signal transduction as sites of localization of signaling mediators, including membrane-bound receptors. Congregation of signaling proteins in lipid rafts within the plasma membrane promotes complex formation and signaling cascade activation. We have recently demonstrated that JAK2 is present in lipid rafts during erythropoietin signaling and that lipid raft integrity is required for erythropoietin-mediated signal transduction (Blood 2009, 114: 292). In our current study, we demonstrate that constitutive JAK-STAT signaling driven by JAK2-V617F is sensitive to lipid raft disruption. Human erythroleukemia (HEL) cells express constitutive activation of JAK-STAT signaling due to the presence of JAK2-V617F. Treatment of these cells with methyl-beta-cyclodextrin to disrupt lipid rafts abolished JAK2, STAT5, and STAT3 activation. Similar results are obtained in other cell lines harboring JAK2-V617F and that exhibit JAK-STAT activation that is dependent on this activated form of JAK2. We also demonstrate that JAK2-V617F co-localizes with lipid rafts, as shown by immunofluorescence, and that this co-localization is abolished by lipid raft disruption. This suggests the loss of JAK2-V617F-mediated JAK-STAT activation we observe following lipid raft disruption may be due to an inhibition of properly localized protein complex formation in the plasma membrane that is necessary for JAK2-V617F signaling. Lipid rafts may provide a site for an accumulation of JAK2-V617F-containing signaling complexes and may be necessary for the cellular signals initiated by JAK2-V617F. Our data show JAK2-V617F-driven JAK-STAT pathway activation is vulnerable to lipid raft disrupting agents and suggest lipid raft integrity as a potential therapeutic target for JAK2-V617F positive neoplasms. Targeting lipid rafts in combination with JAK2 kinase inhibitors may allow for more effective kinase inhibition at lower doses, potentially decreasing undesirable side effects associated with kinase inhibitor treatment. Disclosures:No relevant conflicts of interest to declare.

CRLF2 and JAK2 Mutations: Occurrence and Function In Pre-B ALL Cell Lines

AbstractAbstract 5109Thymic stromal lymphopoietin (TSLP), a cytokine produced by epithelial cells promotes early B-cell development and activates dendritic cells. It has recently been reported that a subset of B-cell precursor acute lymphoblastic leukemia (pre-B ALL) overexpresses the TSLP receptor CRLF2. CRLF2 overexpression is linked to translocations between sex chromosomes – localizing CRLF2 – and the immunoglobulin heavy chain locus on chromosome 14, or to an interstitial deletion on the gonosomes. Both events, translocation and deletion juxtapose CRLF2 to a different promoter (IgH or P2RY8). Performing quantitative real-time PCR we tested pre-B ALL and acute myeloid leukemia (AML) cell lines for overexpression of CRLF2. AML cell lines were included in the screening because we knew from an earlier TSLP project that the AML cell line MUTZ-3 is TSLP-responsive, and thus positive for the cytokine receptor. Three of 63 (5%) pre-B ALL cell lines tested (INC, MHH-CALL4, MUTZ-5) overexpressed CRLF2 mRNA. CRLF2-high cell lines carry a t(14;Y). With respect to the 58 AML cell lines tested: some expressed CRLF2 mRNA, but none of them rivalled the aforementioned pre-B cell lines. Pre-B ALL cell lines show the association between chromosomal CRLF2 aberrations and JAK2 pseudokinase domain mutations that has been described for primary pre-B ALL cells: cell lines MHH-CALL4 (JAK2I682F) and MUTZ-5 (JAK2R683G) and – newly described - also the CRLF2-high pre-B ALL cell line INC express a mutated version of Janus kinase 2 (JAK2R683G). We established a PCR based assay system that allowed for the rapid detection of the JAK2R683G mutation: none of the CRLF2-low or –negative pre-B ALL cell lines exhibited this mutation. All three CRLF2-high/JAK2mu cell lines showed high phosphorylation levels of the JAK2 downstream target STAT5. Inhibition of the JAK kinase led to dephosphorylation of STAT5. However, repression of 3H-thymidine uptake and induction of apoptosis by inhibition of the JAK2/STAT5 pathway was weaker in the JAK2mu pre-B ALL cell lines than in the JAK2V617F positive essential thrombocythemia-derived cell line SET-2. Provided that these results reflect the situation in primary cells, mutated JAK2 seems to be of lesser importance for growth and survival of pre-B ALL cells than for cells from myeloproliferative neoplasms. The CRLF2-high/JAK2mu cell lines INC, MHH-CALL4 and MUTZ-5 are promising model systems for the study of the roles of high-level CRLF2 expression and of JAK2 mutations in pre-B ALL. Disclosures:No relevant conflicts of interest to declare.

SS6-2 Inhibition of RNAse L and PKR by the anti-cancer drug sunitinib impairs innate immunity in virus-infected mice

Kinases control virtually all aspects of biology. Forty-eight human proteins have a kinase-like domain that lacks at least one of the conserved catalytic residues; these proteins are therefore predicted to be inactive and have been termed pseudokinases. Here, we describe exciting work suggesting that pseudokinases, despite lacking the ability to phosphorylate substrates, are still pivotal in regulating diverse cellular processes. We review evidence that the pseudokinase STRAD controls the function of the tumour suppressor kinase LKB1 and that a single amino acid substitution within the pseudokinase domain of the tyrosine kinase JAK2 leads to several malignant myeloproliferative disorders. We also discuss the emerging functions of other pseudokinases, including HER3 (also called ErbB3), EphB6, CCK4 (also called PTK7), KSR, Trb3, GCN2, TRRAP, ILK and CASK.

Perspectives for the use of structural information and chemical genetics to develop inhibitors of Janus kinases

Gain-of-function mutations in the genes encoding Janus kinases have been discovered in various haematologic diseases. Jaks are composed of a FERM domain, an SH2 domain, a pseudokinase domain and a kinase domain, and a complex interplay of the Jak domains is involved in regulation of catalytic activity and association to cytokine receptors. Most activating mutations are found in the pseudokinase domain. Here we present recently discovered mutations in the context of our structural models of the respective domains. We describe two structural hotspots in the pseudokinase domain of Jak2 that seem to be associated either to myeloproliferation or to lymphoblastic leukaemia, pointing at the involvement of distinct signalling complexes in these disease settings. The different domains of Jaks are discussed as potential drug targets. We present currently available inhibitors targeting Jaks and indicate structural differences in the kinase domains of the different Jaks that may be exploited in the development of specific inhibitors. Moreover, we discuss recent chemical genetic approaches which can be applied to Jaks to better understand the role of these kinases in their biological settings and as drug targets.

Abstract A20: Development and validation of a novel acute myeloid leukemia xenograft model that is dependent on the JAK2V617F mutation for growth in vivo

Abstract Background: The Janus-associated kinase (JAK) proteins are a family of non-receptor tyrosine kinases (JAK1, JAK2, JAK3, and TYK2) that play an important role in cellular survival, proliferation, and differentiation. A gain of function mutation (V617F) in the pseudo-kinase domain of JAK2 is detected at high frequency in Philadelphia-negative myeloproliferative disorders such as polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis. To further elucidate the role of JAK2V617F on cell growth in vivo we established and validated a novel xenograft model using the acute myeloid leukemia cell line, SET2, which is heterozygous for the mutation. Materials and Methods: SET2 cells containing stable doxycycline-inducible JAK2V617F shRNA clones were generated by lentiviral transduction. Cell proliferation and viability were assessed by incorporation of 3H-Thymidine or by ATP quantitation. Cell-cycle and apoptosis markers were evaluated by Western blotting using antibodies against cyclin D1, PIM1, cleaved caspase 3 and 7 and PARP. Phospho-STAT5 and total STAT5 were measured using both western blotting and ELISA. A SET2 shRNA clone was selected for in vivo growth in SCID beige female mice and treated with doxycycline or a JAK2 inhibitor (JAK2i). Plasma and tumor drug levels were measured by LC/MS. Results: Genetic knockdown using a doxycycline-inducible JAK2V617F shRNA confirmed SET2 cells are dependent on this gain of function mutation for growth based on inhibition of cell proliferation in the presence of doxycycline. The latter was corroborated with a selective JAK2i (Ki= 4nM) that potently inhibited SET2 cell viability (EC50 = 355nM). The JAK2i rapidly suppressed phosphorylation of STAT5 (EC50 = 313nM) and induced cycle arrest at the G0–G1 phase based on downregulation of cyclin D1 expression. The JAK2i also effectively inhibited expression of PIM-1 kinase, a STAT5 target gene, and activated cleavage of procaspase 3 and 7 and DNA-repair enzyme PARP to induce apoptosis in a time- and dose-dependent manner. A SET2 xenograft model was established by in vivo selection of a variant of the doxycycline- JAK2V617F shRNA parental cell line. Its dependence on JAK2V617F was confirmed by treatment with doxcycline and oral administration of a JAK2i, which resulted in significant tumor growth inhibition in vivo. Investigation of the PK/PD relationship underlying the activity of JAK2i provided direct evidence of phospho-STAT5 suppression and induction of apoptosis for its antitumor effect in vivo. Plasma drug levels correlated with the duration and magnitude of suppression. Conclusion: A JAK2V617F dependent SET2 xenograft model has been established and validated by shRNA gene knockdown and pharmacologically with a JAK2i in vivo. JAK2V617F inhibition results in suppression of STAT5 phosphorylation, cell-cycle arrest and induction of apoptosis in vitro and in vivo. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A20.

Mutation Analysis of JAK-1, 2 and 3 in Children with Down Syndrome and Acute Leukemia.

Abstract 5035Children with Down Syndrome (DS) have an increased risk of developing leukemia, including both acute myeloid (ML-DS), as well as acute lymphoblastic leukemia (DS-ALL). ML-DS can be preceded by a pre-leukemic clone in newborns (transient leukemia-TL), which in most cases resolves spontaneously. Janus Kinase (JAK) 1-3 belongs to a family of intracellular non-receptor protein tyrosine kinases that transduce cytokine-mediated signals via the JAK-STAT pathway. JAK plays an important role in regulating the processes of cell proliferation, differentiation and apoptosis in response to cytokines and growth factors. Mullighan et al. described JAK 1-3 mutations in non-DS high-risk childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL; PNAS, 2009). In T-ALL, JAK-1 mutations are a frequent event (∼25%) as reported among others by Jeong et al (Clinical Cancer Research, 2008). Mutations in JAK-2 and JAK-3 have been described in TL and ML-DS. Bercovich et al. recently reported mutations within the pseudokinase domain of JAK-2 in DS-ALL patients (Lancet 2008). This activating JAK-2 mutation differs from the V617F exon 14 mutation found in myeloproliferative diseases. However, JAK-1 has never been investigated in Down syndrome leukemias. Therefore we performed mutational analysis of the pseudokinase and kinase domains of JAK-1, 2 and 3 by direct sequencing in 8 TL, 16 ML-DS and 35 DS-ALL samples taken at initial diagnosis. The TL and ML-DS samples were unpaired. In the ML-DS group, 12 patients were classified as FAB M7, 3 as FAB M0 and 1 as FAB M6; all 35 DS-ALL patients were classified as BCP-ALL. Mutations in JAK-1 were found in 1 ML-DS patient (D625R) and in 1 DS-ALL patient (V651M). These mutations were localised in the same region of the pseudokinase domain, but not identical to the activating mutations in JAK1 described in high-risk ALL (Mullighan et al., PNAS 2009). The JAK-1 mutated ML-DS patient had a complex karyogram, and the DS ALL patient a normal karyotype. No events occurred in either of the patients with a follow-up of 2.4 and 3.1 years, respectively. JAK-2 activating mutations at position R683 were found in 5/35 (14.3%) of the DS-ALL patients. These patients had diverse cytogenetic aberrations, and had no events at a median follow up of 4.4 years. In the TL and ML-DS patients no mutations were identified in JAK-2. For JAK-3, 1 TL-patient (13%) and 1 ML-DS patient (6.3%) harboured the A573V-mutation. This activating mutation is previously described in ML-DS patients and the megakaroyblastic cell line CMY ((Kiyoi et al, Leukemia 2007). Because the mutations occur in both TL and ML-DS, this suggests that they do not play a role in the clonal progression model from TL to ML-DS. A mutation at JAK3 R1092C, which to our knowledge has never been reported before, was found in 1 DS-ALL patient. This patient had a deletion on chromosome 12 (p11p13), and was in CCR with a follow up of 5 years. In conclusion, JAK-mutations are rare in DS-leukemias, except for JAK-2 mutations in DS-ALL, which occur in approximately 15% of cases. The rarity of JAK-1 mutations in DS is in accordance with the rarity of T-ALL in DS. Of interest, none of the DS ALL cases with a JAK-2 mutation relapsed so far, which differs from the patients with JAK-2 mutations that were recently in high-risk BCP-ALL. Hence, JAK-2 may be an interesting novel therapeutic target. DisclosuresNo relevant conflicts of interest to declare.

Deregulated expression of cytokine receptor gene, CRLF2, is involved in lymphoid transformation in B-cell precursor acute lymphoblastic leukemia

AbstractWe report 2 novel, cryptic chromosomal abnormalities in precursor B-cell acute lymphoblastic leukemia (BCP-ALL): a translocation, either t(X;14)(p22;q32) or t(Y;14)(p11;q32), in 33 patients and an interstitial deletion, either del(X)(p22.33p22.33) or del(Y)(p11.32p11.32), in 64 patients, involving the pseudoautosomal region (PAR1) of the sex chromosomes. The incidence of these abnormalities was 5% in childhood ALL (0.8% with the translocation, 4.2% with the deletion). Patients with the translocation were older (median age, 16 years), whereas the patients with the deletion were younger (median age, 4 years). The 2 abnormalities result in deregulated expression of the cytokine receptor, cytokine receptor-like factor 2, CRLF2 (also known as thymic stromal-derived lymphopoietin receptor, TSLPR). Overexpression of CRLF2 was associated with activation of the JAK-STAT pathway in cell lines and transduced primary B-cell progenitors, sustaining their proliferation and indicating a causal role of CRLF2 overexpression in lymphoid transformation. In Down syndrome (DS) ALL and 2 non-DS BCP-ALL cell lines, CRLF2 deregulation was associated with mutations of the JAK2 pseudokinase domain, suggesting oncogenic cooperation as well as highlighting a link between non-DS ALL and JAK2 mutations.

Comparison of two homogeneous cell‐based kinase assays for JAK2 V617F: SureFire™ pSTAT5 and GeneBLAzer® FRET assays

The Janus kinase (JAK)/Signal Transducers and Activators of Transcription (STAT) signaling pathway plays an important role in cellular responses to cytokines and growth factors. Aberrant, constitutive activation of JAK2 signaling has been implicated in myeloproliferative disorders (MPDs) with a single, activating somatic V617F mutation in the JH2 pseudokinase domain of JAK2 as the prevalent molecular lesion. In order to discover small molecule inhibitors for this target, cell‐based kinase assays are important for identifying potential inhibitors. Here, we present two homogeneous 384‐well plate cell‐based assays utilizing Invitrogen's CellSensor® JAK2 V617F irf1‐bla HEL cell line: (1) SureFire(tm) pSTAT5 AlphaScreen® assay from PerkinElmer; and (2) GeneBLAzer® fluorescence resonance energy transfer (FRET) assay from Invitrogen. The SureFire(tm) assay measures levels of phosphorylated STAT5 while the GeneBLAzer® assay monitors ?‐lactamase activity in cells. The S/B ratios for the SureFire(tm) and GeneBLAzer® assays were 10 and 3, respectively. The experimental procedures will be compared, as well as the potency of select compounds. In conclusion, both assays are suitable for compound profiling.

Specific JAK2 mutation (JAK2R683) and multiple gene deletions in Down syndrome acute lymphoblastic leukemia

AbstractChildren with Down syndrome (DS) have a greatly increased risk of acute megakaryoblastic leukemia (AMKL) and acute lymphoblastic leukemia (ALL). Both DS-AMKL and the related transient myeloproliferative disorder (TMD) have GATA1 mutations as obligatory, early events. To identify mutations contributing to leukemogenesis in DS-ALL, we undertook sequencing of candidate genes, including FLT3, RAS, PTPN11, BRAF, and JAK2. Sequencing of the JAK2 pseudokinase domain identified a specific, acquired mutation, JAK2R683, in 12 (28%) of 42 DS-ALL cases. Functional studies of the common JAK2R683G mutation in murine Ba/F3 cells showed growth factor independence and constitutive activation of the JAK/STAT signaling pathway. High-resolution SNP array analysis of 9 DS-ALL cases identified additional submicroscopic deletions in key genes, including ETV6, CDKN2A, and PAX5. These results infer a complex molecular pathogenesis for DS-ALL leukemogenesis, with trisomy 21 as an initiating or first hit and with chromosome aneuploidy, gene deletions, and activating JAK2 mutations as complementary genetic events.

Functional Determination of the Interaction Interface Between the Kinase and Pseudokinase Domains of JAK2 V617F

The Janus kinases (JAKs) are cytosolic protein tyrosine kinasess mediating cytokine receptor function essential for blood formation and the immune response. The V617F mutation in the pseudokinase domain of JAK2 is present in over 95% of Polycythemia Vera patients and in 50% of Essential Thrombocythemia and Primary Myelofibrosis patients. The V617F mutation leads to constitutive kinase activity, which activates downstream STAT5, STAT3, MAP-kinase and phosphatidylinositol-3-kinase pathways. In the absence of a complete three-dimensional structure of JAK2, the structural mechanism of activation by the V617F mutation has remained elusive. Position 617 of JAK2 is located within one of the two predicted interfaces between the pseudokinase and the kinase domains of JAK2. We have shown that large hydrophobic residues such as Trp, Leu, Ile and Met also activate JAK2 when substituted for Val at position 617. These V617 mutations might either remove an inhibitory interaction exerted by the pseudokinase domain or stabilize an active conformation of the kinase domain. To investigate the nature of the conformational change promoted by the V617 mutations, we employed cysteine-scanning mutagenesis of residues in the activation loop of JAK2, and their predicted interaction partners on the pseudokinase side, and characterized their constitutive and ligand-dependent activity in proliferation and dual luciferase assays. The solved structure of the JAK2 kinase domain in its active form places residues Q1003, D1004 and K1005 on the tip of the activation loop. These residues were individually mutated to cysteine and stably expressed in murine pro-B Ba/F3 cells expressing the erythropoietin receptor. We hypothesized that cysteine residues placed in the activation loop might promote interactions with the pseudokinase domain or form disulfide bonds with naturally occurring Cys616 and Cys618 of the pseudokinase domain, if, as predicted, close proximity would occur between activation loop residues and the pseudokinase domain loop beta4-beta5 that contains V617F. GFPsorted cells expressing the K1005C mutant were able to proliferate in a medium lacking cytokines, similar to cells expressing V617F, but to a lower degree. This proliferation advantage could be explained by the formation of a disulfide bond between C1005 and C618 of JAK2, or by a conformational change induced by placing the smaller Cys residue at position 1005, which would lock the activation loop in an active conformation. Substitution of V617 to Cys abolished the constitutive signaling by JAK2 K1005C, and decreased the weak activity of JAK2 V617C, suggesting that the active conformations of V617 mutants and K1005C are different. Cysteine mutants at positions 1003 or 1004 could not synergize with V617C or induce constitutive activation. We also investigated whether JAK2 V617F activation simply required dimerization, or rather a particular dimer orientation of EpoR cytosolic domains. We took advantage of fusion proteins where a 28 amino acid coiled coil was used to replace the extracellular domain of EpoR in order to impose discrete dimeric orientations to the transmembrane and cytosolic domains of the EpoR. Two out of the seven fusion proteins were constitutively active when co-expressed with WT JAK2 in STAT5-dependent luciferase assays. In contrast, all dimers were active in the presence of JAK2 V617F, suggesting that dimerization and not a particular dimeric orientation promotes JAK2 V617F activation.

Differential Biological Properties and Signaling Effects of Different JAK2 Alleles

JAK2, a member of the Janus family of non-receptor protein tyrosine kinases, acts as an important mediator of cytokine receptor signaling and plays a critical role in hematopoiesis. Somatic mutations in JAK2 have been identified in a spectrum of myeloproliferative disorders (MPD). A specific point mutation (V617F) in the pseudokinase domain (JH2) of JAK2 has been identified in most patients with polycythemia vera (PV) and 50–60% patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF). Although JAK2V617F is the predominant allele associated with PV, ET and PMF, other activating alleles of JAK2 also have been identified in distinct MPD. Somatic mutations in the exon 12 (linker region between JH2 and JH3) of JAK2 are associated with a distinct type of PV characterized by isolated erythrocytosis. Another activating mutation (T875N) in the kinase domain (JH1) of JAK2 has been identified in a cell line derived from acute megakaryoblastic leukemia (AMKL) patient, and expression of JAK2T875N in murine bone marrow induces a MPD with features of AMKL. Thus, different activating JAK2 alleles are associated with different disease phenotypes. The basis for the differences in the in vivo effects of different JAK2 alleles remains unclear. To understand the biological and signaling effects of these JAK2 mutants, we have generated Ba/F3-EpoR cell lines expressing JAK2WT, JAK2V617F, JAK2 exon 12 mutant K539L or JAK2T875N. Expression of JAK2V617F, JAK2K539L or JAK2T875N in Ba/F3-EpoR cells resulted in IL-3 independence, whereas JAK2WT failed to grow in absence of IL-3. We observed significantly increased proliferation of JAK2T875N expressing cells compared to cells expressing either JAK2V617F or JAK2K539L in the absence of cytokine. Moreover, expression of JAK2T875N caused increased transformation of Ba/F3-EpoR cells in a colony assay. Biochemical analysis revealed that the kinase activity of JAK2K539L was higher than JAK2V617F but comparable to JAK2T875N. More importantly, significant differences in signaling downstream of these JAK2 mutants were observed. Expression of JAK2T875N resulted in increased phosphorylation of Stat3, Stat5, Gab2, Akt, and Erk/MAPK and induction of Pim1 and Pim2 compared to JAK2V617F and JAK2K539L mutants. Although JAK2K539L exhibited more kinase activity than JAK2V617F but it was less potent in activation of downstream signaling molecules, such as Stat5, Gab2, Erk/MAPK. Variable association of mutant JAK2 with EpoR was also observed, with JAK2K539L being the most and JAK2T875N being the least associated with EpoR. These results strongly suggest that the biological properties and signaling effects of JAK2 alleles are different, and they may interact differentially with cytokine receptor, which might contribute to the differences in the disease phenotype.

Characterization of JAK2 V617F Allele Burden in Advanced Myelofibrosis (MF) Patients: No Change in V617F:WT JAK2 Ratio in Patients with High Allele Burdens despite Profound Clinical Improvement Following Treatment with the JAK Inhibitor, INCB018424

Background: Activating mutations in the pseudokinase domain of JAK2 occur at a high frequency in Philadelphia chromosome-negative myeloproliferative disorders (MPDs). Increasing JAK2 V617F allele burden has been shown to correlate with disease severity (bone marrow dysfunction, organomegaly and constitutional symptoms), which is consistent with exaggerated JAK2 signaling playing a central role in MPDs. INCB018424 is a potent and selective JAK1/2 inhibitor currently being evaluated in the clinic for the treatment of MPDs including primary myelofibrosis (PMF), post-polycythemia vera and essential thrombocythemia myelofibrosis (Post-PV/ET MF).Methods: The percentage of mutant JAK2 V617F relative to wild-type JAK2 was determined in peripheral blood using a quantitative single-nucleotide extension assay. The assay is based on the single nucleotide extension of a DNA primer that anneals adjacent to the V617F mutation. The percentage of JAK2 V617F in bone marrow was determined using pyrosequencing as previously described. The relationship between JAK2 V617F allele burden and clinical phenotype was assessed with samples obtained at study entry. JAK2 V617F allele burden was assessed in nineteen PMF and Post-PV/ET MF patients treated with 25 mg BID INCB018424 for a minimum of three months using peripheral blood and bone marrow.Results: There is an excellent correlation in the JAK2 V617F allele burden between samples obtained from bone marrow and peripheral blood, suggesting that the fraction of cells bearing the mutant clone remains stable during hematopoiesis in this patient group. The mean %V617F values were 59%, 80% and 86% for Post-ET MF (n=2), Post-PV MF (n=8) and PMF (n=9) patients. V617F allele burden correlated with spleen size, absolute neutrophil count (ANC) and Body Mass Index (BMI) in this patient population (R2 values if 0.61, 0.17 and 0.44, respectively), with a higher allele burden predicting larger spleen size, a higher ANC and a lower BMI. Eighteen of the nineteen evaluable JAK2 V617F-positive patients had baseline JAK2 V617F values above 50%, with the mean %V617F value for all evaluable patients being 81%, implying that the majority of hematopoietic stem cells contain the mutant allele in this patient population. During INCB018424 therapy (at least 3 months of treatment) in these patients, profound improvements in clinical endpoints including reduction or resolution of splenomegaly and constitutional symptoms are observed, yet only a modest decrease in JAK2 V617F allele burden is seen in both bone marrow and peripheral blood (13% and 9 % decrease from baseline, respectively, at 3 months).Conclusions: While the patient group is small and heterogeneous in nature, clinical correlates of V617F allele burden observed in myelofibrosis patients in this study are similar to those reported in PV. In addition, despite profound clinical improvements in these patients, V617F allele burdens do not change dramatically suggesting that the clinical activity of INCB018424 might involve inhibition of downstream signaling in cells harboring activating JAK2 mutations rather than changing the allele burden.

Activation Loop Tyrosines Allow the JAK2(V617F) Mutant to Attain Hyperactivation

A gain-of-function mutation (V617F) in the pseudokinase domain of JAK2 is frequently present in patients with myeloproliferative disorders such as polycythemia vera, essential thrombocythemia, and primary myelofibrosis. This mutation might serve as an important diagnostic biomarker for these uncommon diseases and may represent a target for novel therapy. It is imperative that a well-defined molecular mechanism be provided to account for the gain of function. This manuscript focuses on whether the V617F mutation is sufficient to cause constitutive activation of the enzyme. The evidence presented suggests that the V617F mutation would not cause constitutive activation because its hyperactivating effect is not observed when the mutation is combined with the YY1007,1008FF mutations. The phosphorylation of these two tyrosines within the activation loop is generally accepted as an essential step in the enzyme's normal transition from a basal state of activity to a fully active catalytic state following cytokine receptor stimulation. These observations are consistent with an interpretation that V617F-induced hyperactivation does not supersede the requirement for receptor-mediated activation, as others have shown by combining the V617F mutation with critical mutations in the enzyme's FERM domain. Thus, JAK2(V617F) should be considered as a hyperactive kinase rather than a constitutively active kinase.

Mutations of JAK2 in acute lymphoblastic leukaemias associated with Down's syndrome

Children with Down's syndrome have a greatly increased risk of acute megakaryoblastic and acute lymphoblastic leukaemias. Acute megakaryoblastic leukaemia in Down's syndrome is characterised by a somatic mutation in GATA1. Constitutive activation of the JAK/STAT (Janus kinase and signal transducer and activator of transcription) pathway occurs in several haematopoietic malignant diseases. We tested the hypothesis that mutations in JAK2 might be a common molecular event in acute lymphoblastic leukaemia associated with Down's syndrome. JAK2 DNA mutational analysis was done on diagnostic bone marrow samples obtained from 88 patients with Down's syndrome-associated acute lymphoblastic leukaemia; and 216 patients with sporadic acute lymphoblastic leukaemia, Down's syndrome-associated acute megakaryoblastic leukaemia, and essential thrombocythaemia. Functional consequences of identified mutations were studied in mouse haematopoietic progenitor cells. Somatically acquired JAK2 mutations were identified in 16 (18%) patients with Down's syndrome-associated acute lymphoblastic leukaemia. The only patient with non-Down's syndrome-associated leukaemia but with a JAK2 mutation had an isochromosome 21q. Children with a JAK2 mutation were younger (mean [SE] age 4.5 years [0.86] vs 8.6 years [0.59], p<0.0001) at diagnosis. Five mutant alleles were identified, each affecting a highly conserved arginine residue (R683). These mutations immortalised primary mouse haematopoietic progenitor cells in vitro, and caused constitutive Jak/Stat activation and cytokine-independent growth of BaF3 cells, which was sensitive to pharmacological inhibition with JAK inhibitor I. In modelling studies of the JAK2 pseudokinase domain, R683 was situated in an exposed conserved region separated from the one implicated in myeloproliferative disorders. A specific genotype-phenotype association exists between the type of somatic mutation within the JAK2 pseudokinase domain and the development of B-lymphoid or myeloid neoplasms. Somatically acquired R683 JAK2 mutations define a distinct acute lymphoblastic leukaemia subgroup that is uniquely associated with trisomy 21. JAK2 inhibitors could be useful for treatment of this leukaemia. Israel Trade Ministry, Israel Science Ministry, Jewish National Fund UK, Sam Waxman Cancer Research Foundation, Israel Science Foundation, Israel Cancer Association, Curtis Katz, Constantiner Institute for Molecular Genetics, German-Israel Foundation, and European Commission FP6 Integrated Project EUROHEAR.