Janus Kinase 2 Mutation Research Articles

Idiopathic myelofibrosis and pyoderma gangrenosum involving a mutation of Janus kinase 2 (JAK2V617F), showing poor prognosis

ejd.2013.1956 Auteur(s) : Maejima Hideki1 hm4765@yahoo.co.jp, Takano Tetsurou1, Funatsu Sakae1, Katsuoka Kensei1, Oobe Makoto2, Kamata Toshinobu3 1 Department of Dermatology, 2 Department of Pathology, 3 Department of Internal Medicine, Kitasato University School of Medicine, Sagamihara, Kanagawa, 228-8555, Japan Janus kinase (JAK)-signal transducers and activators of transcription (STAT) signalling are activated by a wide array of cytokines, including colony-stimulating factors. The JAK-STAT pathway [...]

Dysfunction of the PI3 kinase/Rap1/integrin αIIbβ3 pathway underlies ex vivo platelet hypoactivity in essential thrombocythemia

AbstractPatients with myeloproliferative disorders (MPDs), such as essential thrombocythemia (ET) have increased risk of thrombosis and bleeding, which are major sources of morbidity and mortality. Most MPD patients have a gain of function mutation in Janus kinase 2 (JAK2V617F), but little is known how JAK2V617F affects platelet function. Here, we demonstrate that platelets from ET patients have impaired SFLLRN-mediated fibrinogen binding and have lost the potentiating effect of thrombopoietin (which couples to JAK2) on this pathway. In contrast, SFLLRN-mediated P-selectin expression, ATP secretion, phosphorylation of the PKC substrate pleckstrin, and Ca2+ mobilization were unaffected in JAK2V617F positive platelets. In addition, thrombopoietin-mediated JAK2 phosphorylation was unchanged, suggesting that signaling pathways activated downstream of JAK2 are impaired. Indeed, we found that platelets from JAK2V617F-positive ET patients have significantly reduced phosphorylation of the PI3 kinase substrate Akt, and have reduced activation of Rap1 in response to thrombopoietin, IGF-1, ADP, SFLLRN, and thrombin. This effect was independent of Giα P2Y12 purinergic receptor function as ADP-mediated inhibition of VASP phosphorylation was unchanged. These results demonstrate that the PI3 kinase/Rap1 pathway is intrinsically impaired in platelets from JAK2V617F-positive ET patients, resulting in diminished thrombin and thrombopoietin-mediated integrin αIIbβ3 activation.

Hypomorphic Janus kinase 3 mutations result in a spectrum of immune defects, including partial maternal T-cell engraftment

Mutations in Janus kinase 3 (JAK3) are a cause of severe combined immunodeficiency, but hypomorphic JAK3 defects can result in a milder clinical phenotype, with residual development and function of autologous T cells. Maternal T-cell engraftment is a common finding in infants with severe combined immunodeficiency but is not typically observed in patients with residual T-cell development. We sought to study in detail the molecular, cellular, and humoral immune phenotype and function of 3 patients with hypomorphic JAK3 mutations. We analyzed the distribution and function of T and B lymphocytes in 3 patients and studied the invitro and invivo responses of maternal T lymphocytes in 1 patient with maternal T-cell engraftment and residual production of autologous Tlymphocytes. B cells were present in normal numbers but with abnormal distribution of marginal zone-like and memory Bcells. B-cell differentiation to plasmablasts invitro in response to CD40 ligand and IL-21 was abolished. In 2 patients the T-cell repertoire was moderately restricted. Surprisingly, 1 patient showed coexistence of maternal and autologous T lymphocytes. By using an mAb recognizing the maternal noninherited HLA-A2 antigen, we found that autologous cells progressively accumulated invivo but did not compete with maternal cells invitro. The study of 3 patients with hypomorphic JAK3 mutations suggests that terminal B-cell maturation/differentiation requires intact JAK3 function, even if partially functioning T lymphocytes are present. Maternal T-cell engraftment can occur in patients with JAK3 mutations despite the presence of autologous T cells.

Adrenal incidentaloma and the Janus Kinase 2 V617F mutation: A case-based review of the literature

Adrenal incidentaloma was detected in an 81-year-old male patient and a 37-year-old female patient who had been diagnosed with essential thrombocytosis. Each patient's Janus Kinase 2 (JAK2) V617F mutation was positive, and they were evaluated as having non-functional adrenal incidentaloma. The JAK2 activates the signal transducers and activators of transcription (STAT) proteins which then activate the phosphoinositol-3 kinases, Ras, mitogen-activated protein (MAP) kinases, and transcription. Constitutive activation causes cell proliferation and dysregulation of apoptosis. It is thought that STAT3 activation-mediated JAK family kinases have a central role in the solid tumor cell series. Permanent activation of STAT3 and STAT5 causes tumor cell proliferation, survival, metastasis, and an increase in tumor-mediated inflammation in solid and hematologic tumors. According to our literature screening, irregular JAK signaling, seen at the pathogenesis of many solid and hematologic tumors, has not been previously evaluated with regard to adrenal tumors. As a result, our cases are the first coexistence of JAK V617F mutation with adrenal incidentaloma in the literature. Because of this, we think that JAK2 mutation must be evaluated to clarify the etiology of adrenal incidentalomas.

Decreased expression of PIAS1 and PIAS3 in essential thrombocythemia patients

Gain of function mutation of Janus kinase 2 (JAK2V617F) has been identified in Philadelphia-negative myeloproliferative diseases; about half of essential thrombocythemia (ET) patients harbor this mutation. The activated JAK-STAT pathway promotes cell proliferation, differentiation and anti-apoptosis. We studied the role of negative regulators of the JAK-STAT pathway, PIAS, and SOCS in ET patients. Twenty ET patients and 20 healthy individuals were enrolled in the study. Thirteen of the ET patients harbored the JAK2V617F mutation based on mutation analysis. Quantitative-PCR was applied to assay the expression of SOCS1, SOCS3, PIAS1, PIAS3. The expression levels of PIAS1 and PIAS3 were significantly lower in ET groups than that in normal individuals. There was no significant difference between JAK2V617F (+) and JAK2V617F (-) patients. SOCS1 and SOCS3 expression did not differ between ET patients and normal individuals, or between JAK2V617F (+) and JAK2V617F (-) patients. We suggest that failed negative regulators of the JAK-STAT pathway take part in the pathomechanism of ET.

The Molecular and Cytokine Profile of Triple-Negative (JAK2 V617F, JAK2 exon 12, MPL negative) Myelofibrosis, a Myeloproliferative Neoplasm with Distinct Clinico-Pathologic Characteristics

AbstractAbstract 3805Myelofibrosis (MF) is a clonal Philadelphia chromosome negative myeloid neoplasm characterized by cytopenias with or without proliferative hematologic changes, extramedullary hematopoiesis and an increased propensity for transformation to acute myeloid leukemia. Identification of somatic mutations in Janus kinase 2 (JAK2) has revolutionized our understanding of the pathogenesis of some MF patients. However, there is a wide range of clinical heterogeneity in MF patients and somatic mutations of JAK2 (exon 14 or exon12) and MPL are only found in 50–60%/1–2% and 5–10% of MF patients respectively. Therefore, a large proportion of MF patients remain molecularly unaccounted after sequencing for these commonly analyzed mutations. We hypothesized that MF patients who are lack of JAK2 and MPL mutations, collectively called “triple-negative MF,” have a distinct clinicopathologic profile compared to their mutant counterparts.Our study included 76 MF patients (primary MF=48, post-essential thrombocythemic-MF=21, post-polycythemic-MF=7), with amedian age of 65 years (25–80) and predominantly intermediate 1 or 2 risk by the D-IPSS grouping (low=8, Int-1=20, Int-2=42, high=6). We compared hematologic and clinical variables between JAK2 mutant (N=40) and triple-negative MF (N=36) and found that triple-negative MF patients had a higher WBC count (14.4±23.6. vs 11±14.9) and higher platelet counts (365±434.1 vs 252±220.5) but similar hemoglobin levels, LDH levels and D-IPSS plus risk profiles. No difference in overall survival was noted (25.5 months in triple-negative vs 24 months in JAK2 positive).Newly discovered molecular mutations in myeloid malignancies have provided insight into the biology of MDS and some MPN cases. We performed direct sequencing on 9 genes including TET2 (Exon 3), DNMT3A (Exon 18/19, 20, 21, 22 and 23), IDH1/2 (Exon4), LNK (exon2–8), EZH2 (Exon 18/19, N/KRAS (Exon 1–2), CBL (Exon 8–9) and did not find any mutation in the triple-negative cohort. Recently, L3MBTL1 has been reported to be important in the pathogenesis of polycythemia vera, but sequencing of this gene in MF patients did not yield any mutations.Next generation sequencing technologies have been instrumental in the discovery of new genes important in disease pathogenesis in myeloid neoplasias. We performed whole exome sequencing in the triple-negative cases and found somatic mutations in the following genes, ODZ1 (cellular signal transducer), ZNFE391 (transcriptional regulator), DENND3 (protein-coding), ARHGEF11 (modulator of cellular process through G protein) and PIK3CD (Phosphorylate Inositol Lipids regulator involving in Immune response) in one patient and subsequently confirmed as somatic by direct sequencing. This is the first time, which these genetic mutations were reported in hematologic malignancies specifically in MF. However, these mutations did not recur when screening, a limited cohort of patients (N=20).For better understanding of this disease subtype, we compared the cytokine profile in these two groups based on the fact that, cytokines have been crucial in the pathogenesis of MF, and we found marked elevation in the levels of IL-1B (1.63 vs 0.42), IL-8 (2.89 vs 0.56), IL-17A (2.56 vs 1.12) and IFN-δ (2.07 vs 0.37) in the JAK2 positive patients compared to the triple-negative cases. Comparison of both groups showed similar responses to JAK inhibitors by spleen size shrinkage and constitutional symptoms.In conclusion, despite the discovery of JAK2 and MPL mutations, a subset of MF patients remains molecularly uncharacterized. We found that triple negative MF patients had higher leukocyte and platelet counts, but a similar prognosis. Moreover, we identified novel somatic mutations in 1 triple-negative MF patient, which has not been previously described interestingly, despite lower cytokine levels; responses to JAK-inhibitors were still noted. Whole exome sequencing and other immunogenetic studies may reveal novel genes which may elucidate the pathogenesis of these MF cases. Disclosures:No relevant conflicts of interest to declare.

How Epidemiology of Polycythemia Vera Has Changed in the Last 10 Years: Results From the Whole Prospective Cohort of Patients in Cyto-PV Trial As Compared with Eclap Prospective Cohort

AbstractAbstract 1748 Introduction:Polycythemia vera (PV) is a chronic myeloproliferative neoplasms characterized by erythrocytosis, vasomotor disturbances, pruritus, risk of disease progression into acute myeloid leukemia or myelofibrosis and cardiovascular events, the last representing the main cause of morbidity and mortality. Since 2005 the V617F point mutation in Janus Kinase 2 (JAK2) gene gained a dominant role in determining the molecular basis and the diagnosis of PV. We compared the clinical epidemiology of the 1638 patients included in the ECLAP trial in the years 1997 to 2001, with that of a “modern” cohort of 365 PV, JAK2-positive patients included in the Italian CYTO-PV randomized clinical trial and followed from the year 2008 to 2012. Methods:Patients were eligible in CYTO-PV trial and in ECLAP study if they met WHO-2008 diagnostic criteria and the criteria established by the PVSG or Pearson/Messinezy respectively. Clinical characteristics have been compared. The incidence of major cardiovascular events (CV death plus major thrombosis [stroke, acute coronary syndrome, transient ischemic cerebral attack, peripheral arterial thrombosis, pulmonary embolism, abdominal thrombosis, deep vein thrombosis) and total CV events incidence has been evaluated. The median follow up was 31.0 months (range 0– 48.13 months) and 33.1 months (range 0–63.6) for patients included CYTO-PV and in ECLAP respectively. Results:In CYTO-PV 49.3% patients with recent PV diagnosis were included (within 2 years prior inclusion) while in ECLAP the proportion was 35.5%. Mean age at recruitment was similar for patients in CYTO-PV (64.5 yrs) and ECLAP (65.4 yrs). History of thrombosis was reported in 28.9 % vs 38.6% patients in the CYTO-PV and in ECLAP, respectively (p<0.05). Consistently higher proportions of arterial and venous thrombotic events were found in ECLAP as compared to CYTO-PV. History of major bleeding was reported in 1.7% vs 4.8% of the patients in CYTO and ECLAP, respectively. Medical treatment at recruitment was more intensive in CYTO-PV vs. ECLAP: phlebotomy 72.3% vs 63.5% (ns), hydroxyurea (HU) 54.2 vs 48.4, antiplatelet drugs 84.9% vs 58.3% (p<0.05), aspirin 77.0% vs 50.2% (p<0.05), anti-hypertensive and hypocholesterolemic medications were administered respectively in 48.5% and 13% of CYTO–PV patients vs. 39% and 3.5% of ECLAP population (p<0.05) As compared with ECLAP, the incidence of risk of major thombosis in CYTO-PV was 2.7 vs 4.4 and of total CV events was 3.4 vs 5.5 per 100 person/years, respectively. The incidence of total CV events in CYTO-PV for the subgroups of patients with age <65 and no previous thrombosis (PT), age > 65 and no PT, age <65 and PT, age > 65 and PT at randomization was 2.2, 4.8, 3.5 and 3.4 per 100 person/years, respectively. Conclusions:The comparison of these two cohorts of PV patients followed 10 years apart suggests that JAK-2 PV patients are currently better managed for the control of classical CV risk factors, are more frequently administered aspirin, and HU with better control of their disease, and eventually have a risk of thrombosis approximately half than in the past. Disclosures:No relevant conflicts of interest to declare.

High Percentage of JAK2 exon 12 Mutation in Polycythemia Vera and Essential Thrombocythemia Among Populations of Qatar

AbstractAbstract 5064The chronic myeloproliferative Neoplasm (NPM) are clonal hematopoietic stem cell malignancies with 3 main subtypes: polycythemia vera (PV), essential thrombocytosis, and idiopathic myelofibrosis. PV is characterized by increased RBC proliferation in the absence of erythropoietin and proliferation of myeloid lineages usually is noted, A gain-of-function mutation of Janus kinase 2 (JAK2) V617F, is identified in about 95% of patients with PV and about 50% of patients with essential thrombocytosis and idiopathic myelofibrosis.It has been shown that JAK2 exon 12 mutations can activate erythropoietin signaling pathways while these findings have been confirmed by many studies from Western countries, there are no reports from Asian countries in general and Arab countries in particular about the prevalence of the JAK2 exon 12 mutation in patients with PV and ET. In the present study, we determined the prevalence of JAK2V617F and JAK2 exon 12 mutations in patients with PV and ET in Qatar. Materials and MethodsWe enrolled patients with a diagnosis of PV and ET at National Centre for Cancer Care and Research in Qatar from January till June 2012. The diagnosis of PV and ET was established according to the 2008 World Health Organization criteria. The study included 82 patients. Clinical information and the CBC data at diagnosis were obtained from medical records. Pretreatment serum erythropoietin levels. Total DNA was isolated from buffy coat cells taken from peripheral blood using a kit (QIAamp DNA Mini Kit, Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Allele-specific polymerase chain reaction (PCR) was performed using 80 ng of genomic DNA as the template in a35-cycle PCR reaction at an annealing temperature of 58°C, as previously described. The mutant allele yields a 203-base-pair (bp) PCR product (sensitivity of mutant allele detection <1%).For exon 12 mutation screening, 80 ng of genomic DNA was amplified by specific primers designed to amplify a region of 453 bp containing the 128 bp of the exon 12 sequence of JAK2. PCR products were directly sequenced in both directions on an ABI 3730 DNA Analyzer using the BigDye Terminator Sequencing kit. ResultsWe examined the occurrence of JAK2V617F and JAK2 exon 12 mutations in a clinical cohort of 82 patients with polycythemia vera (PV) and Essential thrombocythemia (ET) Of which 42 patients had PV aged 25 to 53, 13 (31%) females and 29 (69 %) males and V617F mutation was detected in all of them exon 12 mutation was detected in 38 (90. 47%) patients. We found 2 different exon 12 mutations:3 N542-E543del, 1 F537-K539delinsL, and among 40 ET patients aged 25 to 59, 22 (55 %) males and 18 (45%) females, 35 patients (87. 5%) were JAK2 V617F and JAK 2 exon12 positive and 5 (12. 5%) were JAK2V617F as well as exon 12 negative patients with V617F and exon 12 mutations showed significantly higher WBC and platelet counts at diagnosis than patients with exon V617F mutation alone (P =. 021 and P =. 038, respectively). We report a surprisingly high incidence of exon 12 mutations in MPN patients with PVand ET in Qatar, a result quite different from reports in the Western literature (P =. 001). ConclusionOur data suggest that exon 12 mutation of JAK2 in patients with PV and ET may have an uneven geographic distribution. A clinical laboratory providing the V617F test alone may risk missing a substantial number of patients with PV in areas with a high incidence of exon 12 mutation. the importance of such associations may need further studies and evaluations. Disclosures:Yassin:Qatar National Research Fund: Patents & Royalties, Research Funding. Rafii El-Ayoubi:Qatar National Research Fund: Patents & Royalties, Research Funding. Al-Dewik:Qatar National Research Fund: Patents & Royalties, Research Funding.

Combination Treatment with JAK2 and PI3K Inhibitors in Myeloproliferative Neoplasms

Abstract 180 Background and Aims.The main pathogenic molecular events associated with myeloproliferative neoplasms (Polycythemia Vera, Essential Thrombocytosis, and Primary Myelofibrosis) are mutations in Janus kinase 2 (JAK2) or in the thrombopoietin receptor that arise in the hematopoietic stem/progenitor cells. Both type of mutations lead to constitutive activation of the JAK2 signaling pathways. The approved JAK2 inhibitor (Ruxolitinib) is not expected to be selective for the mutant JAK2/receptor signaling or to completely suppress the multiple signaling pathways activated by the aberrant JAK2 signaling.We postulate that myeloproliferative neoplasms can be treated more effectively if we target the constitutive JAK2 signaling by a JAK2 inhibitor together with another kinase inhibitor targeting a specific pathway that is co-activated by the aberrant JAK2 signaling. This should increase targeting specificity, reduce JAK2 inhibitor dosages, and minimize potential side effects of these drugs. To this end, we constructed cell line models of myeloproliferative neoplasms and tested the models using a JAK2 inhibitor in combination with a panel of kinase inhibitors to identify combination pairs that give the best synergism. The synergistic pair was further confirmed in mouse models of myeloproliferative neoplasms. Methods.Mouse Ba/F3 cells were engineered to express either JAK2 WT, or JAK2 V617F, or TpoR W515L, or TpoR JAK2 WT, or TpoR JAK2 V617F, or Bcr-Abl. The effect of two JAK2 inhibitors (Ruxolitinib and TG101348) in combination with a panel of 15 various kinase inhibitors (one JNK, one B-Raf, one ROCK-1, one TIE-2, one PI3K, two CDK, two MAPK, three p38, and three mTOR inhibitors). An 8×8 constant ratio Latin square design were used for testing inhibition of cell proliferation/survival in these cell line models. Calculations were carried out using the Chou-Talalay method to determine which drug-pair demonstrated synergism in inhibiting cell growth. Further eight PI3K inhibitors were acquired and tested when we found strong synergism between the JAK2 inhibitors and the PI3K inhibitor ZSTK474 in the first panel.The engineered Ba/F3 cells were also inoculated into female BALB/c nude mice to generate the JAK2 mutant mouse model. These mice were treated intravenously with Ruxolitinib and the PI3K inhibitor GDC0941. Blood profile and physical parameters of the mice were measured for 14 days post treatment.Bone marrow cells from mice reconstituted with bone marrow from JAK2 V617F knock-in mice were plated for colony formation in the presence or absence of Ruxolitinib and the PI3K inhibitor GDC0941. Primary Epo-independent colonies from CD34+ cells of one PV patient were assessed in two independent experiments in the presence or absence of combination drugs. Results.Out of 15 kinase inhibitors tested, three PI3K inhibitors (ZSTK474, GDC0941 and BEZ235), synergized with JAK2 inhibitors (Ruxolitinib and TG101348) in inhibiting cell growth. The combination index was less than 0.5 in all 8×8 dose combination ratios. The JAK2-PI3K inhibitors combination was specific for JAK2 signaling as growth of Ba/F3 cells expressing Bcr-Abl (at equivalent STAT5 activation levels) was unaffected by this combination treatment.Balb/c mice inoculated with Ba/F3 cells expressing TpoR JAK2 V617F were found to have increased spleen weight due to proliferation of autonomous cells. Our combination treatment using Ruxolitinib and GDC0941 could drastically reduce spleen weight compared to treatment with either compound alone.Endogenous erythroid colony forming unit (CFU-E) and burst forming unit (BFU-E) formation from JAK2 V617F knock-in bone marrow cells was reduced significantly by the combined use of Ruxolitinib and GDC0941 compared to individual drugs. Similarly, Epo-independent BFU-E colony formation from peripheral CD34+ cells of one JAK2 V617F-positive PV patient was reduced significantly by the drug combination. Conclusions.Our findings of strong synergy between the JAK2 inhibitors and PI3K inhibitors suggested that we may be able to administer these drugs at lower concentrations than when the drugs are used individually. It provides a framework for combination trials using compounds in these two classes in patients with myeloproliferative neoplasms. Disclosures:No relevant conflicts of interest to declare.

BET Bromodomain Inhibition Targets Both c-Myc and IL7R in Acute Lymphoblastic Leukemia

Abstract 672Long-term disease-free survival is achieved in over 80% of children with B-cell acute lymphoblastic leukemia (B-ALL) but only 40% of adults. Particular genomic alterations in B-ALL, including rearrangements of MLL and the cytokine receptor subunit CRLF2, confer a poor prognosis in both children and adults. In addition, current therapies for B-ALL are associated with significant short- and long-term toxicities, highlighting the critical need for new therapeutics. The novel compound JQ1 inhibits the BET class of human bromodomain proteins from mediating the assembly of macromolecular protein complexes that are required for transcriptional activation and polymerase elongation. In hematologic and epithelial tumors, JQ1 can downregulate the expression of c-MYC and thereby suppress malignant growth and survival. We investigated the therapeutic potential of JQ1 across multiple genetically-defined subsets of B-ALL. JQ1 potently induced apoptotic cell death (IC50∼30–300 nM) in all B-ALL cell lines (697, CEMO-1 NALM-6, MHH-CALL4, MUTZ-5, Reh, RS4;11, SEMK2) studied. Among the most sensitive lines (IC50<50 nM) were MUTZ-5 and MHH-CALL4, which both harbor IGH@-CRLF2 translocations as well as activating mutations in JAnus Kinase 2 (JAK2). CRLF2 heterodimerizes with the IL7 receptor (IL7R) subunit in response to thymic stromal lymphopoietin, which induces JAK/STAT, MAP kinase and AKT signaling. To identify mechanisms through which JQ1 induces cell death in MUTZ-5 and MHH-CALL4 cells, we quantified transcript and protein levels for relevant targets in the presence of JQ1 500 nM or vehicle (DMSO). Chromatin immunoprecipitation was also performed with antibodies against BRD4 followed by PCR to determine the effects of JQ1 on BRD4 binding at relevant promoters. As previously observed, JQ1 induced the downregulation of MYC mRNA, loss of BRD4 at the MYC promoter, and reduced the expression of c-Myc target genes. Immunoblotting with phospho-specific antibodies demonstrated almost complete loss of JAK2 and STAT5 phosphorylation in cells treated with JQ1, suggesting that JQ1 also blocks signaling downstream of CRLF2/IL7R. While the levels of CRLF2 mRNA were unaffected by JQ1 in MUTZ-5 and MHH-CALL4 cells, JQ1 markedly downregulated IL7R mRNA and depleted BRD4 from the IL7R promoter in both lines. The reduction in IL7R mRNA levels led to dramatic decreases in IL7R surface expression. Genome-wide expression profiling demonstrated a highly restricted effect of JQ1, with IL7R and MYC being the 7th and 23rd most downregulated genes, respectively. In fact, IL7R was the only cytokine receptor in both CRLF2-rearranged B-ALL lines that was significantly downregulated by JQ1 treatment. In addition, JQ1 potently reduced IL7R mRNA across other B-ALL cell lines with diverse cytogenetics. To determine whether JQ1 could suppress the growth of human B-ALL in vivo, we xenografted a human CRLF2-rearranged B-ALL primary sample into Nod.SCID.IL2RG−/− mice. Upon the development of >30% bone marrow involvement by human CD45+/CRLF2+ B-ALL cells, mice were randomized to receive JQ1 (50mg/kg intraperitoneally daily) or vehicle (DMSO). After 5 days of treatment, sentinel mice were sacrificed for pharmacodynamic endpoints. Spleens from mice treated with JQ1 had markedly reduced c-Myc expression and STAT5 phosphorylation compared with spleens from vehicle-treated mice. In survival cohorts (n=9 per arm), treatment with JQ1 significantly prolonged overall survival (p=0.0002) compared with vehicle. These results demonstrate that BET bromodomain inhibition is a promising therapeutic strategy for patients with B-ALL, including subsets with high-risk cytogenetics. Moreover, the surprising finding that JQ1 also targets IL7R expression suggests that bromodomain inhibitors may be especially useful in malignant and nonmalignant disorders dependent on IL7R. Disclosures:Bradner:Tensha Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Acetylon Pharmaceuticals: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; SHAPE Pharmaceuticals: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Weinstock:Novartis: Consultancy, Research Funding.

Analysis of Oncogenic Signaling Pathway Induced by a Myeloproliferative Neoplasm-associated Janus Kinase 2 (JAK2) V617F Mutant

Janus kinase 2 (JAK2) is an essential non-receptor type tyrosine kinase for various cytokine signals. In 2005, a somatic JAK2 mutation (V617F) was found in the majority of myeloproliferative neoplasm (MPN) patients. It has been shown that the V617F mutation caused the constitutive activation of JAK2, exhibiting the cytokine-independent survival and proliferation of Ba/F3 cells. In addition, tumorigenesis was induced after a transplantation of Ba/F3 cells expressing JAK2 V617F mutant in nude mice, suggesting that JAK2 V617F mutant behaves as a potent oncogene product. We found that JAK2 V617F mutant causes aberrant activation of a transcription factor c-Myc, which is critical for the JAK2 V617F mutant-caused oncogenic activities. In the screening of genes which expression was induced by JAK2 V617F mutant, we detected the significant induction of target genes of c-Myc such as Aurora kinase A (Aurka) and ornithine decarboxylase (ODC). Interestingly, JAK2 V617F mutant enhanced resistance to cisplatin (CDDP)-induced DNA damage and ectopic expression of Aurka in Ba/F3 cells exhibited similar resistance to CDDP. Conversely, knockdown and inhibition of Aurka in cells expressing JAK2 V617F mutant abolished the resistance to CDDP, suggesting that Aurka is most likely critical for resistance to DNA damage in cells transformed by JAK2 V617F mutant. In addition, we found that ODC inhibitor, DL-α-difluoromethylornithine (DFMO) prevented the proliferation of the JAK2 V617F mutant-induced transformed cells. Taking these observations together, c-Myc plays an essential role in JAK2 V617F mutant-induced hematopoietic disorder and would be a good target for the treatment of MPN.

Advances in the management of myelofibrosis.

Myelofibrosis (MF) is a rare and serious hematologic malignancy classified as a Philadelphia chromosome-negative myeloproliferative neoplasm (MPN). The disease is more common in males and in older individuals. Of the MPNs, MF presents with the most severe morbidity and greatest mortality. Although the cause of MF is unknown, it is thought to occur from acquired mutations that target the hematopoietic stem cell. We reviewed the current literature pertaining to the pathophysiology, clinical presentation, diagnosis, risk stratification, and treatment of MF. The strengths and limitations of present treatment options as well as the emerging clinical experience with Janus kinase 2 (JAK2) inhibitors are explored. Diagnosis is often one of exclusion and is facilitated using the World Health Organization or International Working Group for Myelofibrosis Research and Treatment criteria, depending on whether primary or secondary MF is suspected. Treatment is complicated by a lack of disease familiarity of general practitioners and the advanced age of presenting patients. Although allogeneic stem cell transplant offers a potential cure, most treatments for this condition are limited to symptomatic management, with little to no effect on survival. Appropriate patient assessment and risk stratification are essential for predicting outcomes and allowing treating physicians to tailor therapy accordingly. Significant advances have been made in understanding the pathophysiology of MF, leading to novel therapeutic approaches. The discovery of the JAK2 mutation and the development of JAK2 inhibitors provide clinicians with a new effective treatment option. Ruxolitinib is the first JAK1/2 inhibitor approved by the Food and Drug Administration (FDA) for the treatment of patients with intermediate- or high-risk MF. In clinical studies, ruxolitinib produced a significantly greater reduction in spleen size and improved quality of life compared with placebo or best available therapy. Several future therapies, including combination therapies with ruxolitinib, are currently under investigation.

Advances in the Management of Myelofibrosis

Background: Myelofi brosis (MF) is a rare and serious hematologic malignancy classifi ed as a Philadelphia chromosome-negative myeloproliferative neoplasm (MPN). The disease is more common in males and in older individuals. Of the MPNs, MF presents with the most severe morbidity and greatest mortality. Although the cause of MF is unknown, it is thought to occur from acquired mutations that target the hematopoietic stem cell. Methods: We reviewed the current literature pertaining to the pathophysiology, clinical presentation, diagnosis, risk stratifi cation, and treatment of MF. The strengths and limitations of present treatment options as well as the emerging clinical experience with Janus kinase 2 (JAK2) inhibitors are explored. Results: Diagnosis is often one of exclusion and is facilitated using the World Health Organization or International Working Group for Myelofi brosis Research and Treatment criteria, depending on whether primary or secondary MF is suspected. Treatment is complicated by a lack of disease familiarity of general practitioners and the advanced age of presenting patients. Although allogeneic stem cell transplant offers a potential cure, most treatments for this condition are limited to symptomatic management, with little to no effect on survival. Appropriate patient assessment and risk stratifi cation are essential for predicting outcomes and allowing treating physicians to tailor therapy accordingly. Conclusions: Signifi cant advances have been made in understanding the pathophysiology of MF, leading to novel therapeutic approaches. The discovery of the JAK2 mutation and the development of JAK2 inhibitors provide clinicians with a new effective treatment option. Ruxolitinib is the fi rst JAK1/2 inhibitor approved by the Food and Drug Administration (FDA) for the treatment of patients with intermediate- or high-risk MF. In clinical studies, ruxolitinib produced a signifi cantly greater reduction in spleen size and improved quality of life compared with placebo or best available therapy. Several future therapies, including combination therapies with ruxolitinib, are currently under investigation.

Comprehensive review of JAK inhibitors in myeloproliferative neoplasms

Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem-cell disorders, characterized phenotypically by the abnormal accumulation of mature-appearing myeloid cells. Polycythemia vera, essential thrombocythemia, primary myelofibrosis (also known as 'BCR-ABL1-negative' MPNs), and chronic myeloid leukemia (CML) are the primary types of MPNs. After the discovery of the BCR-ABL1 fusion protein in CML, several oncogenic tyrosine kinases have been identified in 'BCR-ABL1-negative' MPNs, most importantly, JAK2V617F mutation. The similarity in the clinical characteristics of the BCR-ABL1-negative MPN patients along with the prevalence of the Janus kinase mutation in this patient population provided a strong rationale for the development of a new class of pharmacologic inhibitors that target this pathway. The first of its class, ruxolitinib, has now been approved by the food and drug administration (FDA) for the management of patients with intermediate- to high-risk myelofibrosis. Ruxolitinib provides significant and sustained improvements in spleen related and constitutional symptoms secondary to the disease. Although noncurative, ruxolitinib represents a milestone in the treatment of myelofibrosis patients. Other types of JAK2 inhibitors are being tested in various clinical trials at this point and may provide better efficacy data and safety profile than its predecessor. In this article, we comprehensively reviewed and summarized the available preclinical and clinical trials pertaining to JAK inhibitors.

Prevalence and clinical outcomes of the 46/1 haplotype, Janus kinase 2 mutations, and ten-eleven translocation 2 mutations in budd-chiari syndrome and their impact on thrombotic complications post Liver Transplantation

Latent myeloproliferative disorders (MPDs) can be identified by Janus kinase 2 (JAK2) mutations in patients with idiopathic Budd-Chiari syndrome (BCS). The incidence and clinical outcomes of JAK2 mutations, novel ten-eleven translocation 2 (TET2) mutations, and the 46/1 haplotype in BCS are unknown for liver transplantation (LT). We undertook molecular studies of 66 patients presenting with BCS and correlated the results with the clinical outcomes. An overt MPD was present in 20% of the cases, and a latent MPD confirmed by the presence of a JAK2 mutation was detected in 45%. Testing for a TET2 mutation identified MPDs at the molecular level in another 7% of the subset of patients with BCS who were evaluated. The 46/1 haplotype frequency was significantly greater in BCS patients versus the general population (P < 0.001). The presence of JAK2 and TET2 mutations had no impact on 1-year survival. Thirty-six patients underwent LT, and 12 developed liver-related thrombotic complications (33%). Ten of these 12 patients required retransplantation. Retransplantation was more likely in those patients who developed liver-related thrombotic complications (P < 0.001). A JAK2 mutation was highly associated with the development of thrombotic complications after LT (P = 0.005). In conclusion, the presence of JAK2V617F predicts hepatic and extrahepatic thrombotic complications after LT. Testing for TET2 mutations can identify another 7% of idiopathic BCS patients with molecular MPDs.

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.

The HDAC inhibitor Givinostat modulates the hematopoietic transcription factors NFE2 and C-MYB in JAK2V617F myeloproliferative neoplasm cells

We investigated the mechanism of action of the histone deacetylase inhibitor Givinostat (GVS) in Janus kinase 2 (JAK2)(V617F) myeloproliferative neoplasm (MPN) cells. GVS inhibited colony formation and proliferation and induced apoptosis at doses two- to threefold lower in a panel of JAK2(V617F) MPN compared to JAK2 wild-type myeloid leukemia cell lines. By global gene expression analysis, we observed that at 6 hours, GVS modulated 293 common genes in the JAK2(V617F) cell lines HEL and UKE1, of which 19 are implicated in cell cycle regulation and 33 in hematopoiesis. In particular, the hematopoietic transcription factors NFE2 and C-MYB were downmodulated by the drug specifically in JAK2(V617F) cells at both the RNA and protein level. GVS also inhibited JAK2-signal transducer and activator of transcription 5-extracellular signal-regulated kinase 1/2 phosphorylation, but modulation of NFE2 and C-MYB was JAK2-independent, as shown using the JAK2 inhibitor TG101209. GVS had a direct effect on the NFE2 promoters, as demonstrated by specific enrichment of associated histone H3 acetylated at lysine 9. Modulation by GVS of NFE2 was also observed in freshly isolated CD34(+) cells from MPN patients, and was accompanied by inhibition of their proliferation and differentiation toward the erythroid lineage. We conclude that GVS acts on MPN cells through dual JAK2-signal transducer and activator of transcription 5-extracellular signal-regulated kinase 1/2 inhibition and downmodulation of NFE2 and C-MYB transcription.

The interplay between inhibition of JAK2 and HSP90

A recent article by Weigert et al. published in The Journal of Experimental Medicine described the in vitro generation of synthetic mutations in Janus kinase 2 (JAK 2) that decreased the potency of JAK2 (or JAK1/JAK2) inhibitors in artificial systems. The authors found that heat shock protein 90 (HSP90) inhibitors circumvented the potency shift and suggested that HSP90 inhibition may abrogate JAK inhibitor resistance in these experimental systems. However, the clinical relevance of these laboratory-generated JAK2 mutations, which have not been identified to-date in patients treated with JAK inhibitors, and the therapeutic potential of HSP90 inhibitors in diseases involving aberrant JAK-STAT signaling remain to be determined.

Genetic resistance to JAK2 enzymatic inhibitors is overcome by HSP90 inhibition

Enzymatic inhibitors of Janus kinase 2 (JAK2) are in clinical development for the treatment of myeloproliferative neoplasms (MPNs), B cell acute lymphoblastic leukemia (B-ALL) with rearrangements of the cytokine receptor subunit cytokine receptor-like factor 2 (CRLF2), and other tumors with constitutive JAK2 signaling. In this study, we identify G935R, Y931C, and E864K mutations within the JAK2 kinase domain that confer resistance across a panel of JAK inhibitors, whether present in cis with JAK2 V617F (observed in MPNs) or JAK2 R683G (observed in B-ALL). G935R, Y931C, and E864K do not reduce the sensitivity of JAK2-dependent cells to inhibitors of heat shock protein 90 (HSP90), which promote the degradation of both wild-type and mutant JAK2. HSP90 inhibitors were 100-1,000-fold more potent against CRLF2-rearranged B-ALL cells, which correlated with JAK2 degradation and more extensive blockade of JAK2/STAT5, MAP kinase, and AKT signaling. In addition, the HSP90 inhibitor AUY922 prolonged survival of mice xenografted with primary human CRLF2-rearranged B-ALL further than an enzymatic JAK2 inhibitor. Thus, HSP90 is a promising therapeutic target in JAK2-driven cancers, including those with genetic resistance to JAK enzymatic inhibitors.

Tumor Genetic Testing for Patient Selection in Phase I Clinical Trials: The Case of PI3K Inhibitors

Much progress has been made in the development of targeted therapies in cancer during the last few years. As a result of whole-scale sequencing of cancer genomes, a comprehensive view of somatic mutational landscapes in cancer and potential therapeutic targets has emerged. Important findings of genome-scale sequencing include the discovery of mutations in BRAF and phosphatidylinositol 3-kinase (PI3K), just to cite some notable examples. The identification of driver genetic mutations has opened the gates to the development of targeted therapies that are specifically aimed at these mutations. This approach seems to be successful in some circumstances. For instance, an inhibitor that blocks a specific mutant of the serine/threonine kinase B-RAF (V600E-B-RAF) has recently demonstrated dramatic efficacy in treating metastatic melanoma Likewise, an inhibitor of the anaplastic lymphoma kinase (ALK) proved highly effective against a subset of non–small-cell lung cancers that had been prescreened for an oncogenic EML4-ALK translocation. Also, a Janus kinase 2 (JAK2) inhibitor has proved beneficial against myelofibrosis harboring an activating JAK2 mutation. In B cell hematological malignancies, which selectively express the class I PI3K isoform, a PI3K -specific inhibitor has shown remarkable activity in refractory tumors. In basal-cell carcinomas and in medulloblastomas harboring mutations in hedgehog pathways genes, hedgehog pathway inhibitors have also shown activity in patients with advanced disease. These results, together with prior identification of sensitivity to epidermal growth factor receptor (EGFR) agents in tumors with EGFR mutations, have resulted in a growing need to determine the presence of actionable mutations in tumors. This is not an easy task. To begin with, since many of these mutations are uncommon, there is a need to screen a large number of tumors in order to identify the presence of therapeutically relevant mutations. For example, even a common cancer such as non–small-cell lung cancer requires testing a large number of cases to identify those with EGFR mutations (10% of all cases), EML4-ALK rearrangements (4%), or MET, HER2, and B-RAF abnormalities (1% to 2% each). In the clinical trial of crizotinib for EML4-ALK-translocated lung cancer, 1,500 patients were prescreened to identify only 82 cases with the translocation The challenge of screening for a large number of mutations that are present only in a small number of patients may be eased by high-throughput genotyping platforms that perform well with nucleic acid derived from formalin fixation and paraffin-embedding tissue and can assess over a hundred mutations (and growing) with potential therapeutic implications. These early successes and the need to select patients with given mutations in order to observe clinical benefit is impacting on the way patients are being selected for early clinical trials with targeted agents. In a target dependent fashion we need to decide how early and in which situations should tumor gene mutational analysis be incorporated in the process. In this regard, the study by Janku et al in the article that accompanies this editorial illustrates some of the issues that the field is facing in targeting the PI3K gene, a frequently mutated gene in a variety of tumors. In the present study, the presence of PIK3CA mutations was prospectively analyzed in 140 patients referred to the phase I program at MD Anderson. In patients with breast, cervical, endometrial and ovarian cancer, they detected 25 tumors (17%) with PIK3CA mutations in the helical and kinase domain. In 23 patients harboring PIK3CA mutations who were then treated with inhibitors of the PI3K pathway, Response Evaluation Criteria in Solid Tumors Group–defined responses were observed in seven (30%). In comparison, only seven responses were observed in 70 patients with tumors without PIK3CA mutations (10% response rate). The authors conclude that the presence of PIK3CA mutations is associated with increased sensitivity to regimens that include PI3K/mammalian target of rapamycin (mTOR)/AKT inhibitors. Janku et al should be commended for their genotyping efforts and commitment to enrich their clinical trial population with patients with PI3K mutations. However, the interpretation of their results is challenging for a number of reasons. Laboratory studies to date have not conclusively shown that tumor cells harboring PI3K mutations have increased sensitivity to PI3K/mTOR and pan-PI3K inhibitors. We cannot rule out at this time what some of the more isotype specific compounds may display differential sensitivity, depending on which PI3K isoform is involved. The lack of a strong correlation between PI3K mutations and sensitivity to mTOR and pan-PI3K inhibitors contrasts with the tight correlation between the high sensitivity to B-RAF, ALK and EGFR inhibitors in cell lines harboring mutations in these genes. In addition to the laboratory findings not supporting a strong correlation, the lack of a stronger correlation between PI3K mutations and response to therapy could also be a result of additional reasons. As acknowledged by Janku et al, some of the JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L S