scholarly journals Clinical, Hematological, and Biologic Characteristics in Chronic Myelomonocytic Leukemia Patients with a JAK2 V617F Mutation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3189-3189
Author(s):  
Klaus Geissler ◽  
Agnes Barna ◽  
Eva Jäger ◽  
Temeida Alendar ◽  
Elmir Ljubuncic ◽  
...  
Keyword(s):  
Research Funding ◽  
Real Life ◽  
Jak2 V617f ◽  
Advisory Board ◽  
Chronic Myelomonocytic Leukemia ◽  
World Health ◽  
Jak2 V617f Mutation ◽  
V617f Mutation ◽  
Myelomonocytic Leukemia

Abstract Background:Chronic myelomonocytic leukemia (CMML) is a hematopoietic malignancy of the elderly with a heterogenous molecular pathophysiology. Whereas mutations in components of the RAS pathways are among the most common somatic mutations in CMML the JAK2 V617F mutation which is a typical finding in polycythemia vera and around 50% of patients with essential thrombocythemia and primary myelofibrosis, respectively, is by far less frequently detected in CMML but can be consistently found in a subgroup of patients in larger series. Due to the fact that JAK2 V617F-positive CMML is a rare disease the clinical, hematological and in vitro growth characteristics of this entity are poorly investigated. In the "Austrian Biodatabase for Chronic Myelomonocytic Leukemia (ABCMML)" we retrospectively and prospectively collect clinical, biologic, and molecular information of patients with CMML from different centers in a real life setting. Aims:Our aim was to characterize the clinical, hematological, molecular and biologic features of CMML patients harboring a JAK2 V617F mutation. Methods:The diagnosis of CMML was established according to diagnostic criteria of the World Health Organization (WHO) classification of 2008 (Vardiman et al, Blood 2009). Clinical and hematological data were obtained from patients records. For molecular characterization we used next-generation sequencing with amplicon-based target enrichment of 39 CMML associated genes. Only mutations with an allele burden of >10% were considered positive in this analysis. Autonomous colony-forming units granulocyte/macrophage (CFU-GM) growth in the absence of exogenous cytokines was assessed using semisolid cultures as previously described (Geissler et al, J Exp Med 1996). Results:Up to now targeted NGS data are available in 116 patients and in vitro culture data in 75 patients respectively. We identified 13 CMML patients who had a JAK2 V617F mutation with an allele frequency >10%. Clinical, hematological, and biologic characteristics in these patients were compared with 103 patients who had NGS sequencing and were negative for the JAK2 V617F mutation. As shown in Table 1 JAK2 V617F-positive CMML patients had significantly higher WBC counts, higher hemoglobin values, higher platelet counts and more pronounced splenomegaly as compared to JAK2 V617F-negative patients. On the other hand the percentage on monocytes in peripheral blood and the numbers of CFU-GM growing in vitro without addition of exogenous growth factors were lower in CMML patients with the JAK2 V617F mutation as compared to patients without this mutation. The majority of JAK2 V617F-positive patients had additional mutations that can be also found in JAK2 V617F-negative patients, in particular mutations in genes of epigenetic regulation and RNA-splicing, respectively. As shown in Figure 1 there was a trend towards a better survival of patients with the JAK2 V617F mutation as compared to JAK2 V617F-negative patients (p=0.05). In a JAK2 V617F-positive CMML patient with splenomegaly, who was treated with the JAK1/2 inhibitor ruxolitinib off label, we were able to demonstrate the disappearance of constitutional symptoms and a durable spleen response lasting for over 56 months (Fig. 2). Conclusion:Out data show that CMML patients with the JAK2 V617F mutation have hematological, biologic and clinical characteristics different from JAK2 V617F-negative CMML patients. These findings suggest that JAK2 V617F-positive CMML patients should be regarded as a distinct subgroup which may benefit from specific targeted treatments. Disclosures Geissler: Novartis: Honoraria. Pfeilstöcker:Novartis: Consultancy, Speakers Bureau. Burgstaller:Novartis: Consultancy, Honoraria. Zach:Novartis: Other: Honoraria for Advisory Board. Hörmann:Novartis: Other: Honoraria for Advisory Board. Jäger:Roche: Other: Personal fees, Research Funding. Sperr:Amgen: Honoraria, Research Funding; Novartis: Honoraria. Kusec:Novartis: Other: Honoraria for lectures. Valent:Novartis: Honoraria, Research Funding; Amgen: Honoraria; Celegene: Honoraria, Research Funding.

scholarly journals Prevalence of JAK2 V617F in Individuals That Meet World Health Organization Erythrocytosis Criteria for Polycythemia Vera

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1775-1775 ◽  
Author(s):  
Miguel Piris-Villaespesa ◽  
Gloria Muñoz-Martin ◽  
Ricardo Sanchez ◽  
Claudia Nuñez-Torron ◽  
Adolfo Saez-Martin ◽  
...  
Keyword(s):  
General Population ◽  
Sensitivity And Specificity ◽  
Research Funding ◽  
High Sensitivity ◽  
Jak2 V617f ◽  
World Health ◽  
Jak2 V617f Mutation ◽  
Pcr Assay ◽  
V617f Mutation ◽  
Health Organization

Abstract Introduction: The diagnostic criteria for polycythemia vera (PV) has recently been updated by the World Health Organization (WHO). The criterion for erythrocytosis has been modified downwards: hemoglobin (hb)> 16.5 g/dL or hematocrit (hto)> 49% in men and hb> 16 g/dL or hto> 48% in women. This reduction increases the potential number of patients that would be test for JAK2 V617F mutation if PV is suspected. The V617F mutation in the JAK2 gene is present in 95% of cases of PV. It is estimated that the prevalence of this mutation in the general population is around 0.2%. Our aims are to determine the prevalence of JAK2 V617F in individuals with erythrocytosis according to WHO2016 criteria and to find prognostic factors that could help to identify patients with PV. Methods: We prospectively studied all hemograms performed in our laboratory during 7 nonconsecutive days. Variables studied were hb, hto, leukocytes, neutrophils, platelets, MCV, MCH, MCHC and RDW. JAK2 V617F mutation was studied in all males that had hb> 16.5 g/dl or hto> 49% or females that had hb> 16 g/dl or hto> 48%. JAK2 V617F mutation was studied by PCR assay in which an amplification control fragment and the JAK2 mutant allele were simultaneously amplified. All positive samples were confirmed by quantitative real-time PCR in a reference laboratory. Positive results were considered when the JAK2 V617F allele ratio was ≥ 0.7. The variables collected were correlated with the result of the JAK2 test in a univariate way. The T-Student test was used for the quantitative variables and the Chi-square test for the categorical variables. For the cell count variables, the Mann-Whitney U test was used. Results: A total of 15366 HG were analyzed. 1271 (8.3%) met the inclusion criteria for erythrocytosis. JAK2 V617F was performed on 1001 samples (270 samples were not suitable for the PCR assay due to low quality). Twelve samples (1.2%) were positive for JAK2 V617F mutation. However, 5 samples were excluded due to a known diagnosis of myeloproliferative neoplasm. Therefore, finally prevalence of JAK2 V617 mutation in 996 patients that met WHO erythrocytosis criteria was 0.8% (8/996). Medians for all parameter studied for each group are shown in table 1. In order to find out parameters that could increase the incidence probabilities to identify patients with JAK2 V617F we performed an univariate analysis of the variables included, according to JAK2 mutational status. We found that patients with JAK2 V617F had higher levels of leukocytes, neutrophils, platelets and RDW than patients with negative JAK2 (p <0.001), while the levels of MCV (p = 0.033) and MCH (p = 0.015) were lower in patients with JAK2 V617F (table 2). For the variables that have been statistically significant, the area under the ROC curve (AUC) and the optimal cut-off point that maximizes sensitivity and specificity were calculated (Youden index). Of interest, neutrophils, platelets and RDW show high AUC (≥ 0.79), with high sensitivity and specificity (table 3). Finally, in order to find out the potential interest of these findings, we studied medical records of the 8 patients with JAK2 V617F mutations, finding that 4 patients (50%) had previously suffered a vascular event. Conclusion: The prevalence of JAK2 V617F mutation in subjects with elevated Hb or hto according to WHO2016 criteria is increased with respect to that of the general population. Among this group of subjects, those with JAK2 V617F show significantly different levels of leukocytes, neutrophils, platelets, MCV, CMH and RDW. Interestingly, neutrophils, platelets and RDW show high sensitivity and specificity. Therefore, it is necessary to explore combinations of these parameters and their optimal cut-off points to elaborate efficient strategies for an early diagnostic approach. Disclosures Martinez Lopez: Bristol Myers Squibb: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Janssen: Research Funding, Speakers Bureau. García Gutiérrez:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Incyte: Honoraria, Research Funding.

In Vitro Expansion of Polycythemia Vera Progenitors Favors Expansion of Erythroid Precursors without JAK2 V617F Mutation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3506-3506 ◽  
Author(s):  
Josef T. Prchal ◽  
Ko-Tung Chang ◽  
Jaroslav Jelinek ◽  
Yongli Guan ◽  
Amos Gaikwad ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Polycythemia Vera ◽  
Peripheral Blood ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Erythroid Progenitors ◽  
Erythroid Precursors ◽  
In Vitro Expansion ◽  
V617f Mutation

Abstract A single acquired point mutation of JAK2 1849G&gt;T (V617F), a tyrosine kinase with a key role in signal transduction from growth factor receptors, is found in 70%–97% of patients with polycythemia vera (PV). In the studies of tyrosine kinase inhibitors on JAK2 1849G&gt;T (see Gaikwad et all abstract at this meeting) we decided to study the possible therapeutic effect of these agents using native in vitro expanded cells from peripheral blood. To our surprise, the in vitro expansion of PV progenitors preferentially augmented cells without JAK2 1849G&gt;T mutation. We used a 3 step procedure to amplify erythroid precursors in different stages of differentiation from the peripheral blood of 5 PV patients previously found to be homozygous or heterozygous for the JAK2 1849G&gt;T mutation. In the first step (days 1–7), 106/ml MNCs were cultured in the presence of Flt-3 (50 ng/ml), Tpo (100 ng/ml), and SCF (100 ng/ml). In the second step (days 8–14), the cells obtained on day 7 were re-suspended at 106/ml in the same medium with SCF (50 ng/ml), IGF-1 (50 ng/ml), and 3 units/ml Epo. In the third step, the cells collected on day 14 were re-suspended at 106/ml and cultured for two more days in the presence of the same cytokine mixture as in the step 2 but without SCF. The cultures were incubated at 37oC in 5% CO2/95% air atmosphere and the medium renewed every three days to ensure good cell proliferation. The expanded cells were stained with phycoerythrin-conjugated anti-CD235A (glycophorin) and fluorescein isothiocyanate-conjugated anti-human-CD71 (transferrin receptor) monoclonal antibodies and analyzed by flow cytometry. The cells were divided by their differential expression of these antigens into 5 subgroups ranging from primitive erythroid progenitors (BFU-Es and CFU-Es) to polychromatophilic and orthochromatophilic erythroblasts; over 70% of harvested cells were early and late basophilic erythroblasts. The proportion of JAK2 1849G&gt;T mutation in clonal PV granulocytes (GNC) before in vitro expansion and in expanded erythroid precursors was quantitated by pyrosequencing (Jelinek, Blood in press) and is depicted in the Table. These data indicate that in vitro expansion of PV progenitors favors expansion of erythroid precursors without JAK2 V617F mutation. Since three PV samples were from females with clonal granulocytes, erythrocytes, and platelets, experiments were underway to determine if the in vitro expanded erythroid cells were clonal PV cells without JAK2 V617F mutation, or derived from polyclonal rare circulating normal hematopoietic progenitors. The Proportion of JAK2 T Allele Patients GNC T Allele (%) Expanded Cells T Allele (%) PV1 (Female) 81 10 PV2 (Male) 77 28 PV3 (Male) 44 42 PV4 (Female) 78 19 PV5 (Female) 78 28

Significant Activity of the JAK2 Inhibitor, INCB018424 in Patients with Secondary, Post-Myeloproliferative Disorder (MPD) Acute Myeloid Leukemia (sAML): Results of An Exploratory Phase II Study.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 631-631 ◽  
Author(s):  
Farhad Ravandi ◽  
Srdan Verstovsek ◽  
Zeev Estrov ◽  
Jan A. Burger ◽  
Solly George ◽  
...  
Keyword(s):  
Phase Ii ◽  
Stable Disease ◽  
Research Funding ◽  
Phase Ii Study ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Significant Activity ◽  
Stat Signaling ◽  
V617f Mutation ◽  
Grade 3

Abstract Abstract 631 Background: Mutations of JAK2 gene have been identified in a significant proportion of patients with MPDs with the selective JAK2 inhibitors demonstrating significant activity. Patients with AML following prior MPD (sAML) respond poorly to standard cytotoxic chemotherapy and have a poor outcome. Abnormalities of the Jak-Stat signaling pathway have also been identified in a number of other hematological malignancies; chromosomal translocations resulting in TEL-JAK2 constructs lead to the constitutive activation of STAT5, IL-3-independent cellular proliferation, and leukemogenesis. Similarly, infection with oncogenic viruses such as human T-cell lymphotrophic virus, type I, and Abelson murine leukemia viruses results in enhanced kinase activity of Jaks, possibly accounting for their leukemogenic potential. Furthermore, disrupted Jak-Stat signaling has been reported in a number of leukemias. Aim: To identify potential activity of INCB018424 in patients with advanced hematological cancers. Methods: We are conducting a phase II study of INCB018424 in patients with relapsed/refractory leukemias for which no standard therapies are anticipated to result in a durable remission. Patients with performance status 0,1,and 2 with adequate organ function and no active, uncontrolled intercurrent illness or infection receive INCB018424 orally at 25 mg BID daily for 4 weeks (cycle #1). Response is assessed after 2 cycles of treatment. Responding patients or patients with stable disease are allowed to continue until progression. Predetermined dose modifications to 15 mg or 10 mg BID are allowed for drug related toxicities. Results: Eighteen patients [median age, 68 years; (range, 53-88] with relapsed and refractory leukemias (9 de novo AML, 3 sAML, 2 ALL, 1 MDS, 2 CMML, 1 CML) have been treated. The median number of prior therapies is 2 (range,1 to 6). Five patients (1 with AML, 2 with sAML, and 3 with CMML) had the JAK2 V617F mutation. Cytogenetic abnormalities include diploid in 7, chromosome 5 and 7 in 5, t(2;9) in 1, and the Philadelphia chromosome in 2. Pts have received a median of 1 cycle of therapy (range, 1-5 cycles) with 8 pts having stable disease (3 for 2 cycles, 2 for 3 cycles, 1 for 4 cycles, and 2 for 5 cycles). Three patients (including 2 with sAML and 1 with CMML, all with JAK2 mutation) have had significant declines in their bone marrow blasts (to <5%) associated with significant decrease in the size of the spleen and clinical improvement. The regimen has been very well tolerated with only grade 3 side effects being elevation of liver enzymes in 2 patients (thought not to be related to the study drug) and grade 3 thrombocytopenia in 1 patient. Conclusion: INCB018424 has significant activity in sAML and CMML associated with JAK2 V617F mutation. Clinical studies combining it with chemotherapy in sAML are warranted. Disclosures: Ravandi: Incyte Corporation: Research Funding. Verstovsek:Incyte: Research Funding. Garrett:Incyte Corporation: Employment. Newton:Incyte Corporation: Employment.

LY2784544, a Novel JAK2 Inhibitor, Decreases In Vitro Growth of Hematopoietic Human Progenitors From JAK2 V617F Positive Polycythemia Vera Patients

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 5054-5054 ◽  
Author(s):  
Lourdes Florensa ◽  
Beatriz Bellosillo ◽  
Leonor Arenillas ◽  
Liandong Ma ◽  
Richard Walgren ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Dependent Manner ◽  
In Vitro Growth ◽  
Vitro Assay ◽  
V617f Mutation

Abstract Abstract 5054 Introduction: The discovery of JAK2 V617F mutation in patients with myeloproliferative disorders (MPD) has opened new perspectives for the development of targeted therapies. We have studied the efficacy of a novel molecule LY2784544 with JAK2 inhibitory activity in the in vitro growth of myeloid progenitors from JAK2 V617F-positive polycythemia vera (PV) patients. Objectives: To investigate the efficacy of LY2784544 in the inhibition of endogenous(e)BFU-E and CFU-GM growth in PV patients. Methods: In vitro cultures in semisolid media were performed from peripheral blood mononuclear cells (PBMC) of 6 PV patients who had never received cytoreductive treatment (4 patients with homozygous JAK2 V617F and 2 patients with heterozygous JAK2 V617F). PBMC were suspended in methylcellulose (Methocult. StemCell, Vancouver, Canada) without the addition of EPO and containing 0–30.0 μM LY2784544 drug. Concurrent plates containing EPO were plated as control cultures. The medium was distributed in multidishes and they were incubated at 37° with 5% CO2 and 95% humidity. Hemoglobinized colonies and granulomonocytic colonies were counted on day 14 by standard criteria (BFU-E defined by an aggregate of >50 hemoglobinized cells or three or more erythroid subcolonies and CFU-GM was defined by an aggregate of >50 cells). Each in vitro assay was performed in duplicate. DNA was obtained from peripheral blood granulocytes from each patient to quantify the JAK2 V617F allele burden at the time of culture assay. Results: LY2784544, at concentrations ranging from 0.03–30.0 μM, inhibited growth of unselected peripheral blood eBFU-E and CFU-GM from PV patients carrying the JAK2 V617F mutation in a dose-dependent manner, although without achieving complete inhibition of all colonies (fig.1). Conclusions: In vitro studies show that LY2784544 decreases the eBFU-E and CFU-GM growth in therapy-naive JAK2 V617F positive PV patients. Our data suggest that LY2784544 may be a candidate for the treatment of MPD carrying the JAK2 V617F mutation. Disclosures: Ma: Eli Lilly and Company: Employment. Walgren:Eli Lilly and Company: Employment.

Short Survival and High Rates of Transformation Prevail in Chronic Myelomonocytic Leukemia Despite Hypomethylating Agent Therapy.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2800-2800
Author(s):  
Emily J. Vannorsdall ◽  
Vu H. Duong ◽  
Xinyi Ng ◽  
Dan P. Zandberg ◽  
Michael L. Tidwell ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Clinical Trials ◽  
Research Funding ◽  
Response Rates ◽  
Chronic Myelomonocytic Leukemia ◽  
World Health ◽  
Free Survival ◽  
Entire Cohort ◽  
Myelomonocytic Leukemia

Abstract Abstract 2800 Background: Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic stem cell disorder categorized as a mixed myeloproliferative/myelodysplastic disorder in the World Health Organization classification system. Diagnostic criteria include a persistent peripheral blood monocytosis >1 × 109/L and bone marrow dysplasia. Our recent review of SEER Medicare data (ASH 2011 abstract 2784) demonstrated that CMML has a shorter overall survival (OS) and more frequent progression to acute myeloid leukemia (AML), compared to myelodysplastic syndromes (MDS). Due to the heterogeneity of this disease and its differences from MDS, efforts to identify prognostic factors have been ongoing. The MD Anderson prognostic score was previously validated, but was derived from patients treated prior to the availability of the hypomethylating agents (HMAs) azacitidine and decitabine. HMAs have now emerged as standard therapy, with reported response rates of 37–69%, but their impact on survival and AML transformation is unclear. The OS of CMML patients has been reported at 12–18 months and transformation rates have varied between 15–52%. We reviewed our own single-center experience with CMML over the past 12 years. Methods: We conducted a retrospective review of CMML patients evaluated at the University of Maryland Greenebaum Cancer Center between January 2000 and August 2012. Patient and disease characteristics, treatments, complications, progression to AML, and OS were recorded and analyzed. Descriptive statistics were used for baseline characteristics and Kaplan-Meier analysis was performed for all time-to-event data. Statistical analyses were performed using SPSS version 20.0. Results: We identified 35 patients with CMML, 71% were male and 71% white, with a median age of 69 (range 34–86) years; 75% had <10% bone marrow (BM) blasts and 68% had low-risk cytogenetic findings (normal karyotype or -Y). Most patients treated prior to 2005 received hydroxyurea and/or erythropoiesis-stimulating agents or were enrolled on clinical trials, while patients treated since 2005 received HMAs as primary therapy. The median OS of the entire cohort was 19.5 months, with 49% of patients progressing to AML with a median time to progression (TTP) of 16.9 months. Of the entire cohort, patients with <10% and ≥10% BM blasts had an estimated OS of 19.4 and 11.7 months respectively (p=.021). Patients with low-, intermediate-, and high-risk (complex karyotype, +8, or chromosome 7 abnormalities) cytogenetic findings had an estimated OS of 23.3, 16.5, and 12.0 months respectively (p<0.001). Twenty-two patients received HMAs. Their estimated OS was 16.5 months, compared to 23.0 months for patients who did not receive HMAs (p =.683); 50% of patients treated with HMAs had known progression to AML, with TTP varying from 3–28 months. AML-free-survival was 16 months in patients receiving HMAs, compared to 14 months in patients not treated with HMAs (p=0.960). The majority of patients receiving HMA therapy (63%) were treated with ≥ 6 cycles; 57% of these patients transformed to AML despite initial response, often in a sudden and unpredictable manner. Conclusions: Published trials using HMAs in CMML have been limited by small patient numbers, short median follow-up, and paucity of data on AML transformation. Our study had a median follow-up period of 41.1 months. We found a high rate of AML transformation and short OS even in patients who received HMAs. HMA treatment had no statistically significant impact on AML-free survival or OS. Although the results may be confounded by some selection bias, treatment with HMAs was largely based on the date of diagnosis rather than prognostic variables or performance status. Therefore, the favorable response rates previously reported with these agents, and also seen in our patients, do not appear to translate into an OS or AML-free-survival advantage. Our study underscores the continued need for novel agents and the need to prioritize clinical trials for this group of patients. Additionally, based on our data, early bone marrow transplantation should be strongly considered for CMML patients when feasible. Disclosures: Davidoff: Novartis: Research Funding; Celgene: Research Funding; GlaskoSmithKline: Research Funding. Baer:Novartis, Inc.: Research Funding; Celgene, Inc.: Research Funding.

Preclinical Evaluation of the BET-Bromodomain (BET-BRD) Inhibitor OTX015 in Leukemia Cell Lines Harboring the JAK2 V617F Mutation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 873-873
Author(s):  
Maria Eugenia Riveiro ◽  
Lucile Astorgues-Xerri ◽  
Charlotte Canet-jourdan ◽  
Mohamed Bekradda ◽  
Esteban Cvitkovic ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cell Lines ◽  
Research Funding ◽  
Leukemia Cell ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Mrna Levels ◽  
Protein Levels ◽  
V617f Mutation

Abstract Background: Exposure of cancer cells to BET-BRD protein inhibitors has been associated with a significant downregulation of C-MYC expression, leading to suppression of the transcriptional program linked to proliferation and survival. C-MYC mRNA expression, mediated by STAT5 activation, is induced by the JAK2 (V617F) mutation (JAK2mu) in transfected BA/F3 cells (Funakoshi-Tago, et al. 2013). We selected JAK2mu leukemia-derived cell lines for preclinical evaluation of OTX015 (Oncoethix, Switzerland), a selective orally-bioavailable inhibitor of BET-BRD proteins with promising early results in an ongoing phase I study in hematologic malignancies (Herait et al, AACR 2014, NCT01713582). Material and Methods: Antiproliferative effects of OTX015 and JQ1 were evaluated in three established JAK2mu human myeloid leukemia cell lines (SET2, MUTZ8, HEL 92.1.7). GI50 (OTX015 concentration inducing 50% growth inhibition) and Emax (% cell proliferation at 6 µM OTX015) values were determined by MTT assay after 72h exposure. Protein levels were analyzed by Western blot, and RT-PCR was performed with Fast SYBR Green Master Mix on a StepOnePlus Real-Time PCR System. For cell cycle analysis, cells were stained with propidium iodide and analyzed with a FACScan flow cytometer. Induction of apoptosis was evaluated by Annexin-V. Simultaneous schedules of OTX015 combined with ruxolitinib, a JAK2 inhibitor, were evaluated. Combination index (CI) was determined using the Chou & Talalay method; CI<1 reflects synergy, CI=1 additivity and CI>1 antagonism. Results: After 72h exposure, SET2 was the most sensitive cell line (GI50=0.12 µM and Emax=15%), and HEL92.1.7 cells had a GI50=1.9 µM with an Emax=23%. MUTZ8 was the most resistant cell line with an Emax=61%. Similar GI50 and Emax values are observed with JQ1. A significant increase in the fraction of apoptotic cells was observed in SET2 cells after 72h 500 nM OTX015 exposure. Non-significant increases in Annexin-positive cells were seen in HEL92.1.7 and MUTZ8 cells. Cell cycle analysis revealed a significant increase in the percentage of SET2 cells in subG0/G1 after 24, 48, and 72h 500 nM OTX015, correlating with the increase in apoptosis. Conversely, an increase in the percent cells in the G1 phase was observed in HEL 92.1.7 cells. After 4h 500 nM OTX015, BRD2 mRNA levels were significantly increased in all three cell lines, whereas BRD3 levels were not modified. BRD4 mRNA levels increased significantly after 48h in SET2 cells. OTX015 treatment induced a transitory reduction of C-MYC mRNA levels after 4h with an increase at 24h in all cell lines. At the protein level, C-MYC decreased substantially in SET2 cells after 4h, with complete disappearance after 48h without recovery, while in the less sensitive MUTZ8 cell line, the decrease in C-MYC protein levels was transitory. Conversely, this proto-oncogene was not modified in HEL92.1.7 cells. In addition, p-STAT5 protein was downregulated by OTX015 in SET2 cells, but was increased in MUTZ8 cells after longer exposure time. Furthermore, BCL2 mRNA and protein levels decreased in SET2 cells, correlating with the apoptosis induction seen with OTX015 treatment. In HEL92.1.7 cells, P21 mRNA levels and cyclin D1 protein levels increased after 4h and 48h OTX015 treatment, respectively. Moreover, concomitant combination of OTX015 with ruxolitinib showed a highly antagonist effect (CI>7) in SET2 cells, the most sensitive cell line to both agents. On the other hand, very strong synergy was observed in HEL92.1.7 (CI=0.19) and MUTZ8 (CI=0.41), despite their low sensitivity to single agent OTX015. Conclusions. Our findings demonstrate that OTX015 exhibits potent activity against cultured leukemic cells expressing the JAK2 V617F mutation, inducing apoptosis or cell cycle arrest at submicromolar concentrations. This activity correlates with modulation of C-MYC, p-STAT5, BCL2, P21 and cyclin D1 mRNA and protein levels following OTX015 treatment. Our study highlights the novel and synergistic activity of the combination of a BRD antagonist and a JAK inhibitor in human leukemic cells harboring the JAK2 V617 F mutation, supporting the rationale for in vivo testing of OTX015 in combination with JAK inhibitors in leukemic JAK2mu models. Disclosures Riveiro: Oncoethix SA: Research Funding. Astorgues-Xerri:Oncoethix SA: Research Funding. Canet-jourdan:Oncoethix SA: Research Funding. Bekradda:Oncoethix SA: Research Funding. Cvitkovic:Oncoethix SA: Membership on an entity's Board of Directors or advisory committees, Shareholder and CSO Other. Herait:Oncoethix SA: CMO and Shareholder Other. Raymond:Oncoethix SA: Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals Germline-Somatic Interactions in Myelofibrosis Susceptibility

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 313-313
Author(s):  
Derek W Brown ◽  
Youjin Wang ◽  
Andrew St. Martin ◽  
Stephen R. Spellman ◽  
Shu-Hong Lin ◽  
...  
Keyword(s):  
Telomere Length ◽  
Board Of Directors ◽  
Research Funding ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Risk Haplotype ◽  
Advisory Committees ◽  
Genome Wide ◽  
Increased Risk ◽  
V617f Mutation

Abstract Introduction: Myelofibrosis (MF) is a rare myeloproliferative neoplasm (MPN) characterized by bone marrow fibrosis, progressive bone marrow failure, and increased risk of acute myeloid leukemia. While MF arises from somatic driver mutations in JAK2, MPL, and CALR, some MPN patients may have a heritable component. To comprehensively examine the genetic etiology of MF, we performed the first integrative analysis of SNP array genotyping (using Infinium Global Screening Array), targeted long-read sequencing (using PacBio SMRT sequencing) and telomere length (TL, using qPCR assay). Methods: Our study included 937 MF patients who received an allogeneic hematopoietic cell transplant (HCT) between 2000 and 2016 and had an available pre-HCT blood sample at the Center for International Blood and Marrow Transplant Research Repository. Somatic mosaic chromosomal alterations (mCAs, including deletions, duplications, or copy-neutral losses-of-heterozygosity (CNLOH)) were called with the Mosaic Chromosomal Alteration (MoChA) algorithm using raw genotyping intensity data. A genome-wide association study (GWAS) was restricted to include 827 MF patients of European ancestry and utilized 4,135 genetically-matched healthy controls. Results: GWAS identified six independent MF susceptibility loci at genome-wide significance (P&lt; 5×10 -8); four of which replicate prior MPN susceptibility loci [9p24.1(JAK2), 5p15.33(TERT), 3q25.33(IFT80), and 4q24(TET2)] and two novel MF loci [6p21.35(HLA-DQB1-AS1) and 17p13.1(TP53)] (Figure 1). A transcriptome-wide association analysis using whole blood GTEx data highlighted the 9p24.1 locus with increased JAK2 expression associated with elevated risk of MF (P= 2.18×10 -19). A strong colocalization statistic further indicated shared genetic component between eQTL and this JAK2 locus (HyPrColoc Posterior Probability= 0.6) (Figure 2). Based on the strong signal identified at TERT (Figure 1), we investigated the relationship between MF risk and genetically-inferred telomere length using a panel of 19 germline variants previously found to be associated with telomere length. Of the 19 telomere-length associated variants investigated, 7 were found to be associated with MF risk (binomial P= 2.31×10 -5, linear trend P= 5.48×10 -4) (Figure 3). Both Mendelian randomization and genome-wide genetic correlation analyses further indicated that increased risk of MF was associated with longer inherited telomere length. Utilizing available clinical mutation data on a subset of 185 patients, MF cases carrying the germline risk haplotype of the 9p24.1(JAK2) susceptibility locus were observed to more frequently have the JAK2 V617F mutation (71% vs 59%; P= 0.02). Targeted PacBio long-read sequencing around JAK2 provided further evidence of linkage between the germline risk allele and the JAK2 V617F mutation. Detectable autosomal mCAs were also abundant in MF cases with 67.4% having at least one mCA (compared to ~3% in the general population) and 27.6% having an mCA spanning JAK2 (mostly CNLOH) (Figure 4). In addition, using a binomial test for biased allelic imbalance, a cis relationship was identified at 9p24.1 in which the MF risk haplotype was predominantly duplicated by CNLOH (binomial P=1.36×10 -9). Regional sequencing of JAK2 further confirmed duplication of JAK2 V617F by CNLOH. Finally, we observed an inverse association between autosomal mCAs and qPCR measured telomere length (OR= 0.22, 95% CI= 0.07-0.65, P= 6.40×10 -3). These results were consistent by mCA chromosomal region and copy number state. Conclusion: Our results suggest a molecular framework for the genetic etiology of MF in which both genetically-inferred telomere length and germline variation at JAK2 are associated with increased MF risk. The 9p24.1 risk haplotype predisposes to the acquisition of a somatic JAK2 V617F mutation in cis and subsequent duplication of JAK2 V617F by mCAs (usually CNLOH). This process leads to aberrant JAK2 activity and increased clonal proliferation, accelerating telomere length shortening and increasing genomic instability in patients with MF. Figure 1 Figure 1. Disclosures Gupta: AbbVie: Consultancy, Honoraria; Constellation Pharma: Consultancy, Honoraria; Roche: Consultancy; Pfizer: Consultancy; BMS-Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Honoraria, Research Funding. Lee: Janssen: Other; Incyte: Research Funding; AstraZeneca: Research Funding; Kadmon: Research Funding; National Marrow Donor Program: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; Syndax: Research Funding; Takeda: Research Funding; Amgen: Research Funding. Saber: Govt. COI: Other.

PI3Kδ Inhibitor Idelalisib Inhibits AKT Signaling In Myelofibrosis Patients On Chronic JAK Inhibitor Therapy

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4065-4065
Author(s):  
Sarah A Meadows ◽  
Huong (Marie) Nguyen ◽  
Christophe Queva ◽  
Brian J. Lannutti ◽  
Adam Kashishian ◽  
...  
Keyword(s):  
Cell Lines ◽  
Research Funding ◽  
Jak2 V617f ◽  
Akt Signaling ◽  
Jak2 V617f Mutation ◽  
Employment Equity ◽  
Cell Lysates ◽  
Cd34 Cells ◽  
Equity Ownership ◽  
V617f Mutation

Abstract Background Myelofibrosis (MF) is characterized by activation of the JAK-STAT pathway, with the JAK2 V617F mutation found in 50-60% of patients. Although JAK inhibitors, such as FDA-approved ruxolitinib, have been effective in reducing splenomegaly and mitigating symptoms, patients uniformly exhibit “disease persistence” which is equated with a lack of hematologic or molecular remissions, or with loss of clinical improvement over time. Prior studies using cell lines or primary patient samples have shown that the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway is dysregulated in MPNs and is a potential therapeutic target (Kamishimoto et al, Cell Signal 2011; Huang et al, ASH 2009 Abstract 1896; Vannucchi et al, ASH 2011 Abstract 3835; Khan et al, Leukemia 2013). In CLL and other B-cell malignancies, the PI3K pathway is constitutively upregulated and is dependent on PI3Kδ. Idelalisib is a δ-isoform-specific PI3K inhibitor that is efficacious in patients with CLL and indolent NHL. Herein, the specific aims of our study were: 1) to determine whether the PI3Kδ isoform is expressed in progenitor cells from MF patients, and 2) to evaluate the inhibitory effects of idelalisib on basal and thrombopoietin (TPO)-stimulated AKT/S6RP phosphorylation (p-AKT/p-S6RP) in cell lines and in primary samples from MF patients who were either on chronic ruxolitinib (RUX) therapy or were not exposed to ruxolitinib (RUX-naïve or off-therapy at the time of sample collection). Methods To evaluate isoform expression, CD34+ cells from the peripheral blood of MF patients were sorted by FACSAria and cell lysates were analyzed by Simple Western using Peggy (ProteinSimple) with recombinant protein as a positive control. For cell line studies, BaF3/MPL W515L and UT-7/TPO cells were stimulated with recombinant human TPO and incubated with idelalisib. Whole cell lysates were analyzed by Western blot to quantify the % of p-AKT and p-S6RP levels compared to idelalisib-untreated cells. For MF patient samples, PBMCs were isolated from the whole blood of MF patients who were either RUX-naïve or on chronic RUX therapy and treated for 2 hours with idelalisib. Antibodies specific to p-AKT Ser473 and pS6RP Ser235/236 were used to quantify the proportion of p-AKT and pS6RP in basal and TPO-stimulated CD34+/CD3-/CD14-/CD19-/CD66- gated cells. Results The PI3Kδ isoform was found to be the predominant isoform expressed in 3 of 3 RUX-naïve and 4 of 4 chronic RUX patients tested; PI3Kβ was expressed at lower levels and no PI3Kα or γ was detected (Figure 1). In BaF3/MPL cells, p-AKT levels decreased by 51%, 64% and 67%, with 0.1, 1.0, 2.0 µM idelalisib, respectively, when compared to idelalisib-untreated cells; p-S6RP levels decreased by 24%, 27%, and 41%, respectively. Similarly, for UT-7/TPO cells, p-AKT decreased by 11%, 44%, and 55%, and p-S6 decreased by 13%, 28% and 48%, respectively. In CD34+ cells from RUX-naïve patients (n=3), p-AKT and p-S6RP levels decreased with increasing concentrations of idelalisib (0.02, 0.2, 2 µM). All patients on chronic RUX treatment demonstrated decreased p-AKT (n=3) and p-S6RP (n=4 basal, n=3 TPO-induced; patient 4 was only tested for basal) levels with increasing concentrations of idelalisib in both basal (Figure 2A) and TPO-stimulated (Figure 2B) assays. All 4 chronic RUX and 2 of 3 RUX-naïve patients tested carried the JAK2 V617F mutation. Conclusions The PI3Kδ isoform was identified as the predominant isoform expressed in CD34+ cells from MF patients. In both cell lines and patient samples, idelalisib inhibits the PI3K/AKT pathway, with a dose-dependent decrease of p-AKT and p-S6RP. Inhibition was observed for both RUX-naïve and chronic RUX-treated patients. Studies are underway to evaluate the effects of idelalisib on progenitor colony formation and induction of cell cycle arrest and apoptosis. * Meadows and Nguyen are first co-authors Disclosures: Meadows: Gilead: Employment, Equity Ownership. Queva:Gilead: Employment, Equity Ownership. Lannutti:Gilead, Acetra, Effector: Consultancy, Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Kashishian:Gilead: Employment, Equity Ownership. Jun:Gilead: Employment, Equity Ownership. Coutre:Gilead: Research Funding. Dansey:Gilead: Employment, Equity Ownership. Gotlib:Gilead: Consultancy, Research Funding.

scholarly journals Increased death receptor resistance and FLIPshort expression in polycythemia vera erythroid precursor cells

Blood ◽  
2006 ◽  
Vol 107 (9) ◽  
pp. 3495-3502 ◽  
Author(s):  
Ann Zeuner ◽  
Francesca Pedini ◽  
Michele Signore ◽  
Giusy Ruscio ◽  
Carlo Messina ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Death Receptor ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Erythroid Cells ◽  
Jak2 Mutation ◽  
Erythroid Precursor ◽  
V617f Mutation ◽  
Induced Apoptosis

Polycythemia vera (PV) is a clonal myeloproliferative disorder characterized by excessive erythrocyte production. Most patients with PV harbor an activating JAK2 mutation, but the molecular links between this mutation and erythrocyte overproduction are unknown. The interaction between death receptors and their ligands contributes to the physiological regulation of erythropoiesis through the inhibition of erythroblast proliferation and differentiation. With the use of an in vitro culture system to generate differentiating erythroid cells, we found that erythroblasts derived from patients with PV harboring the JAK2 V617F mutation were able to proliferate and generate higher numbers of mature erythroid cells in the presence of inhibitory signals delivered by CD95 (Fas/Apo-1) and TRAIL receptor stimulation. JAK2-mutated PV erythroblasts showed lower levels of CD95-induced caspase activation and incomplete caspase-mediated cleavage of the erythroid transcription factor GATA-1, which was entirely degraded in normal erythroblasts on CD95 stimulation. JAK2 mutation was associated in PV erythroblasts with cytokine-independent activation of the JAK2 effectors Akt/PKB and ERK/MAP and with a deregulated expression of c-FLIPshort, a potent cellular inhibitor of death receptor–induced apoptosis. These results show the presence in PV erythroblasts of proliferative and antiapoptotic signals that may link the JAK2 V617F mutation with the inhibition of death receptor signaling, possibly contributing to a deregulation of erythropoiesis.

Modulated Expression of BCL-xL and GATA-1 Genes Is a Common Feature in Myeloproliferative Disorders (MPD) Both in JAK2-V617F Positive and Negative Patients

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1533-1533
Author(s):  
Sarah Pozzi ◽  
Francesca Bertolotti ◽  
Silvio Parodi ◽  
Raffaella Ponassi ◽  
Barbara Biasotti ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Statistical Significance ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Myeloproliferative Disorders ◽  
Expression Level ◽  
Jak2 V617f Mutation ◽  
Mutational Status ◽  
V617f Mutation

Abstract JAK2-V617F mutation has been identified in more than 90% of patients with Polycythemia Vera (PV) and in 50 to 60% of patients with Essential Thrombocythemia (ET) or Primary Myelofibrosis (PMF). The mutation has reinforced the original idea that ET, PV, and PMF have a common background; however, some key questions remain open: why, in JAK2-V617F patients, only a proportion of progenitors is bearing the mutation and the other is wild type (wt); why patients with the same mutation have a different disease; what have in common patients JAK2-V617F positive (mutated) and wt with the same disease. We observed that a new synthetic peptide (072RB) able to bind Bcl-xL molecule, exerting an apoptotic effect, inhibited the in vitro cord blood (CB) mononuclear cells (MNC) growth. Moreover, this effect correlated with a high expression level of Bcl-xL messenger (RQ-PCR). Since Bcl-xL was involved in erythropoiesis, we extended the expression studies to bone marrow (BM) MNC from16 PV (13/16 mutated),15 ET (9/15 mutated) and peripheral blood (PB) MNC from 18 PMF (9/18 mutated). JAK2 mutational status was assigned by allele-specific-PCR (AS-PCR). MNC from PV-BM and PMF-PB showed a Bcl-xL level of expression significantly higher than in MNC from healthy donors (NBM and NPB) both in mutated and in wt patients (PV: p=0.01 and p=0.004; PMF: p=0.005 and p=0.05 respectively). In ET, the expression level of Bcl-xL tended to be elevated compared to controls but did not reach the statistical significance. Since other factors can modulate Bcl-xL expression independently from the constitutive activation of JAK2/STATs pathway induced by JAK2-mutated, we analysed GATA-1 gene, a transcription factor that binds the Bcl-xL promoter and that is involved in erythropoiesis and megakariocytopoiesis. We observed that GATA-1 was highly expressed in PV-BM and PMF-PB MNC both in mutated and in wt patients (PV: p=0.01 and p=0.05; PMF: p=0.001 and p=0.03). In ET-BM MNC, GATA-1 followed the Bcl-xL pattern of expression. The highest messengers levels were observed PMF-PB MNC and CB MNC that, after in vitro 072RB peptide treatments, showed a 25%–50% of cells growth inhibition with respect to untreated controls. The protein expression was confirmed by cytofluorimetric analyses. Our finding may indeed be compatible with reduced apoptosis both in mutated and wt patients. Thus, the elevated expression levels of Bcl-xL and GATA-1 genes may represent a common feature in MPD, independent from the presence of the JAK2-V617F mutation and supports the hypothesis of a “phenotypic continuum” in MPD. It is noteworthy that in patients bearing the mutation, a variable proportion of hematopoietic progenitors in PV, ET and PMF have been documented to be wt. In this context, our findings may explain why wt hematopoiesis is not overtaken by the mutated counterpart. These results could open a new insight in the field of MPD molecular characterization and may lead to new therapeutic approaches.

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