JAK2V617F Levels Research Articles

Endothelial JAK2V617F Expression Drives Inflammation and Cellular Senescence; New Evidence for the Roles of Endothelial Cells in MPN-Related Clotting Abnormalities?

JAK2V617F is the primary pathogenic mutation in patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), a heterogeneous group of haematological malignancies characterised by increases in one or more myeloid lineages. The most common cause of morbidity and mortality in patients with MPNs are thrombohemorrhagic incidents, although the events leading to these clotting abnormalities remain unclear. It is now known that a subset of MPN patients express JAK2V617F in endothelial cells (ECs) which has been linked with a poorer prognosis and an increase in the incidence of thrombosis. Furthermore, using transgenic mouse models we have recently shown that expression of JAK2V617F in ECs plays a critical role in aberrant thrombosis in vivo (PNAS, 2014. 111(6): p. 2295-300). Here, we describe the transcriptomic and phenotypic profiles of JAK2V617F-positive ECs.Lentivirally transduced human umbilical vein ECs (HUVECs) showed a sustained increase in levels of activated JAK2, STAT3 and STAT1 in JAK2V617F-expressing cells compared to WT JAK2. In addition, JAK2V617F also caused a significant increase in expression of total STAT1 protein. Subsequent RNAseq analysis revealed an upregulation of several mRNAs related to inflammation and permeability in JAK2V617F-expressing cells. Expression of interferon-related genes, including serine protease inhibitor B2 (SERPINB2) and early growth response protein 1 (EGR1), and chemokine ligand 2 (CCL2) were also increased in JAK2V617F cells. SERPINB2 and EGR1 have roles in initiating cellular senescence and CCL2 was previously reported to induce permeability. These data, combined with increased expression of total STAT1, suggest that JAK2V617F expression in ECs induces an inflammatory response in this cell type. Moreover, in vitro permeability assays show increased permeability in HUVECs transduced with JAK2V617Fcompared to WT JAK2, which was significantly amplified following stimulation with thrombin.We then determined whether any functional differences existed between patient-derived normal and JAK2V617F-expressing ECs. For this we obtained blood from JAK2V617F-positive polycythaemia vera patients undergoing therapeutic phlebotomy and isolated blood outgrowth endothelial cells (BOECs) by repeated culture of PBMCs. Allelic burden of JAK2V617F, determined by quantitative allele-specific amplification (QuASA) was shown to vary between patient samples, (0% to 59.8% (n=9)) and increased expression of JAK2V617F appeared to correlate with decreased proliferation and significant changes in cellular morphology.In order to quantify these differences, ptychography, a novel label-free imaging technology was used to generate quantitative phase images of BOECs in time-lapse, to measure phenotypic changes in cell morphology, motility and proliferation. These results confirmed that JAK2V617F allelic burden was inversely related to BOEC proliferation. Cells which expressed only WT JAK2 or low levels of JAK2V617F exhibited exponential growth. However, an increase in JAK2V617F resulted in a decrease in BOEC proliferation. Indeed, cells which expressed high JAK2V617F levels (30-60%) failed to proliferate over the 48 hour imaging period. Furthermore, JAK2V617F-high BOECs were also shown to be larger in size, multi-nucleated and less motile compared to JAK2V617F-low. The increase in cell size and decreases in cell motility and proliferation suggest that increased JAK2V617F expression induces senescence in BOECs.These data describe features of ECs which are commonly associated with dysfunctional endothelium: sustained inflammation/permeability, limited cell growth and senescence. The critical role of ECs in maintaining hemostasis and the observed features of EC dysfunction in cells which express JAK2V617F, implicate mutation burden in ECs as a contributing factor towards the thrombotic abnormalities observed in patients with MPNs. DisclosuresNo relevant conflicts of interest to declare.

Peripheral Blood JAK2V617F Quantitative Assessment in Clinical Practice: Correlations Between Allele Burden and Clinical Phenotype

▪Background: Peripheral blood (PB) JAK2V617F testing is ordered in routine clinical practice in several different clinical scenarios. Interpretation of the mutation level/allele burden information is however challenging given lack of a standardized assay and lack of adequate information to permit data interpretation in a non-research setting.Objectives: To describe: (i) disease distribution in unselected JAK2V617F-positive patients seen in routine clinical practice: (ii) disease-specific dispersion of JAK2V617F allele burden in these patients; and (iii) correlation of JAK2V617F allele burden with patient clinical characteristics both between and within specific diseases.Methods: The current study was approved by the Mayo Clinic institutional review board. Myeloid neoplasms were classified as per World Health Organization criteria. Clinical, laboratory and pathology results were centrally reviewed to confirm the reported diagnoses. JAK2V617F testing was done as part of routine clinical care; testing included allele burden measurement by a quantitative allele-specific PCR assay (sensitivity 0.01%) that is in clinical use at our institution (http://www.mayomedicallaboratories.com/test-catalog/Overview/31155); reporting of allele-burden data started in January 2012. The current analysis was restricted to PB samples from adult patients seen at our institution who consented to use of their medical records for research use; results of consecutive samples tested from January 2012 to April 2015 were collected. The PCR assay calibrator is a mixture of DNA from a JAK2V617F-positive cell line (HEL) and a -negative cell line (HL60). The final result is reported as % JAK2 V617F of total JAK2 (ie, [mutated/(mutated + wild-type)] x 100%).Results: We studied 349 patients (166 female; median age 65 years, range 18-92) who met eligibility criteria. The disease distribution was: polycythemia vera (PV) 146 patients (42%), primary myelofibrosis (PMF) 63 (18%), essential thrombocythemia (ET) 55 (16%) , post-PV MF 34 (10%), post-ET MF 12 (3%), myeloproliferative neoplasm-unclassified (MPN-u) 10 (3%), refractory anemia with ringed sideroblasts and thrombocytosis (RARS-T) 8 (2%), post-MPN acute myeloid leukemia (AML) 6 (2%), MPN-myelodysplastic syndrome-unclassified (MPN/MDS-u) 4 (1%), no identified hematological disease 2 (0.5%), and 'other' 9 (2.5%).The median (range) %JAK2V617F was as follows: PV 10.5% (0.5-86%), PMF 8% (0.5-88%), ET 2% (0.5-21%), post-PV MF 24% (0.8-95%), post-ET MF 14.5% (1-88%), MPN-u 4% (0.7-11%), RARS-T 2.5% (0.5-9%), post-MPN AML 31.5% (9-92%), MPN/MDS-u 14.5% (7-74%), no hematological disease 0.7% (n/a), and other 0.9% (0.5-19%) (p<0.0001).Distribution of patients in the following 7 JAK2V617Fallele burden strata: <1%, 1-2% , 3-5%, 6-10%, 11-20%, 21-50%, and 51-100% was: 42 patients (12%), 44 (13%), 61 (17%), 61 (17%), 49 (14%), 50 (15%) and 42 (12%), respectively. PV patients were identified across the entire allele burden spectrum, namely 3%, 12%, 16%, 19%, 14%, 20% and 16% in the aforementioned strata, respectively. Disease distribution in the 42 patients with <1% allele burden was: ET 14 patients, PMF 12, PV and 'other' 5 each, RARS-T and no hematological disease 2 each, and post-PV MF and MPN-u 1 each.JAK2V617F allele burden in ET (peak 21%) was significantly lower than PMF or PV (peak 86-88%) (p<0.0001). Mutation levels in post-ET (p<0.0001) and post-PV (p=0.004) MF were significantly higher than in ET and PV, respectively. Levels in post-PV/post-ET MF were also significantly higher than in PMF (p<0.0001). In contrast, JAK2V617F levels in ET and RARS-T were comparable (p=0.8). Finally, levels in post-MPN AML were significantly higher than in ET (p=0.0001) and PMF (p=0.007), marginally higher relative to PV (p=0.05), but not significantly higher relative to post-ET/post-PV MF.Conclusions: The current study provides guidance for the interpretation of peripheral blood JAK2 V617F allele burden, in routine clinical practice: i) levels below 1% are not infrequently reported, even in phenotypically classic PV, and do not imply low likelihood of a myeloid neoplasm, ii) allele burden per se is not diagnostically specific, but a threshold of greater than 20% might be used to question a diagnosis of ET or RARS-T, and iii) the highest allele burdens are seen in fibrotic and/or leukemic transformation of chronic-phase myeloproliferative neoplasm. DisclosuresPardanani:Stemline: Research Funding.

Metformin Inhibits JAK2V617F Activity By Activating AMPK and PP2A Containing B56 Alpha Subunits in MPN Cells

Metformin, which is a member of the biguanide family, suppresses the growth of a variety of malignant hematological cells. It is widely accepted that metformin inhibits the growth of these cells primarily by suppressing the mTOR pathway. In contrast, we previously found that metformin directly suppresses the activation of JAK2V617F in HEL and SET-2 cells. In the present study, we investigated the precise molecular mechanisms through which metformin inhibits the auto-phosphorylation of JAK2V617F. Initially, we speculated that AMPK, which is one of the main downstream molecules activated by metformin, might be involved in the process. Compound C, an inhibitor for AMPK, resumed the JAK2 activity in cells treated with metformin. For the next step, we introduced siRNA against an α subunit of AMPK and analyzed the phosphorylation levels of JAK2V617F. We found that knock-down of AMPK enhanced the basal phosphorylation levels of JAK2V617F but that metformin-induced JAK2 inhibition was only partially diminished. These results suggested that although AMPK was responsible for the metformin-induced JAK2V617F inhibition, other mechanisms are also required to achieve the full inhibition of JAK2V617F activity.Recently, several groups have revealed that PP2A-activationg drugs suppress the activity of JAK2V617F. In addition, recent studies have demonstrated that metformin activates PP2A. These reports inspired us to investigate the possibility that PP2A might be responsible for the metformin-mediated suppression of JAK2V617F. PP2A consists of three different subunits: A, B and C. The A subunit works as a scaffold, and the C subunit has catalytic activity. The B subunit contains a variety of members, and the diversity of the B units determines the substrate specificity. Initially, we analyzed whether metformin activates PP2A in JAK2V617F-positive cells. We found that metformin decreases the phosphorylation of the C-subunit of PP2A at Tyr 308, which negatively regulates PP2A function. For further study, we prepared the A subunit of PP2A knock-down cells using siRNA. We expected that the knock-down of PP2A would restore the JAK2V617F activity; instead, JAK2V617F was suppressed in these cells. To explain the discrepancy, we focused on the B subunits and hypothesized that only a specific type of PP2A complex is involved in the de-phosphorylation of JAK2V617F. To this end, we introduced a panel of siRNA targeting different B subunits and analyzed the effects on JAK2V617F activity. Knock-down of the B56α subunit resulted in the activation of JAK2V617F. In contrast, the JAK2V617F phosphorylation levels were decreased in the B56γ knock-down cells. Interestingly, we found that B56γ knock-down cells have enhanced AMPK activity. These results indicate that among the PP2A complexes, B56α containing complex works as negative regulator for JAK2 but that the B56γ subunit-containing complex may positively regulate JAK2 by inhibiting AMPK.In conclusion, both AMPK and PP2A-containing B56α subunits are involved in the metformin-induced JAK2V617F inhibition. In contrast, the B56γ subunit-containing PP2A may diminish metformin-induced JAK2 inhibition via the negative regulation of AMPK. These results suggest that the specific inhibition of the B56γ subunit containing PP2A may enhance the anti-leukemic action of metformin. DisclosuresKirito:Novartis Pharma KK: Honoraria.

In-Cell Detection By Flow Cytometry of JAK2 V617F Mutation Using Gold Nanoflares

Today’s conventional technology has to overcome many challenging aspects for integrating expressional analysis of a mutation into the cellular function of a living cell. It is very striking to detect the expressional status of a mutation in a living cell without compromising its cellular function as well as its viability that allows to perform subsequent functional analysis. This has been a long time goal for most of the biological studies.Myeloproliferative neoplasms (MPN) originate at the level of hematopoietic stem cell with a single nucleotide mutation in the cytosolic tyrosine kinase, Janus kinase 2 (JAK2) named as JAK2V617F which facilitates the constitutive intracellular activation of JAK2 and found in 95% of Polycythemia Vera (PV) patients. Detection of this mutation is a major criteria for diagnosis of the malignancy and changes in the expression levels of JAK2V617F is valued for the prognosis as well as response to the treatment.Here, we have utilized the nanotechnology’s new development, entitled as "multiplexed nanoflares", a gold nanoparticle agent that is capable of simultaneously detecting two distinct mRNA targets inside a living cell. These probes are highly oriented oligonucleotide sequences, which are hybridized to a reporter dye such as Cy3 and Cy5 and each complementary to its corresponding mRNA target. When multiplexed nanoflares are exposed to their targets, they provide a sequence specific signal in both extra and intracellular environments. Thus, this signal could be easily detected by flow cytometry or confocal microscopy.We have employed JAK2 V617F specific nanoflare probes labeled with Cy3 (JAK2V617F-Cy3) and JAK2 wild type (wtJAK2) nanoflare probe labeled with Cy5 (JAK2-Cy5) including negative and positive control probes to distinguish and isolate mRNA’s of JAK2V617F and wtJAK2 in live cells by measuring subsequent fluorescence with flow cytometry. The initial testing of the nanoflares has been performed by treating cell lines such as K562 (wtJAK2), SET-2 (JAK2V617F heterozygote), HEL (JAK2V617F homozygote) cells with nanoflare probes which did not alter cell viability and morphology with the applied concentrations during 18 hours of cell culture. We measured the levels of two mRNA targets, JAK2V617F and JAK2, and detected 90±6 % positivity with JAK2V617F-Cy3 treated HEL cells and no positivity in K562 cells, meanwhile wtJAK2 probe treated cells succeeded staining of SET and K562 cells. Real time reverse transcription PCR (RT-PCR) was performed on the same cells to compare the relative amounts of target RNA in each cell type by calculating relative changes in mRNA expression using the ΔΔCt (cycle threshold) method and comfirmed the results of flow cytometry with sensitivity of 0.001. Subsequently, we have tested mononuclear cells (MNC) received from peripheral blood of 5 patients with PV, one of which has JAK2V617F heterozygote allele (patient 5) and 4 patients had JAK2V617F homozygote allele (patients 1-4)which were tested with nested allel specific PCR. Flow cytomeyric analysis of the patients‘ MNC treated with the wtJAK2 and JAK2V617F probes demonstrated distinct populations of wtJAK2+ and JAK2V617F+. Populations of wtJAK2+/JAK2V617F+ (2.9%), wtJAK2-/JAK2V617F- ( 21.4%), wtJAK2+/JAK2V617F-( 0%), wtJAK2-/JAK2V617F+ ( 79%) of patient 5 and populations of JAK2V617F+ (36,8%± 5 ), JAK2V617F- of patients 1-4 were sorted by cell sorter with ~ 90% purity following detection and analysis of each populations for relative changes in mRNA expressions . The data revealed distinct ΔCt values between each populations.In this study, we have aimed for a new approach by utilizing nanotechnology aligned with the conventional techniques in order to save time, money and outcome. The capability of in-cell detection and isolation of the JAK2V617F and wtJAK2 first time by performing the expression analysis, changes the routine protocols backwards, enables the detecting direct effect of the mutation to the cells as well as other genes. This study creates a platform where functional analysis regarding to the more detailed understanding of the initiation of malignancy as well as responses to the therapies could be performed. DisclosuresNo relevant conflicts of interest to declare.

The Number Of Peripheral Blood CD4+CD25+FOXP3+ Regulatory T-Cells Is a Marker Of Response In Patients With JAK2 V617F Positive Myeloproliferative Neoplasms Treated With Pegylated-Interferon-α

Treatment of patients with polycythemia vera (PV) or essential thrombocythemia (ET) with pegylated interferon-a (Peg-Infa) frequently induces hematologic remission and may also induce a decrease in the JAK2V617Fallele burden and a reversion to polyclonal hematopoiesis. However, the mechanism(s) by which Peg-Infa generates these responses remain unclear. Possibilities include a direct suppression of clonal hematopoietic stem cells, a stimulation of quiescent residual normal stem cells or a beneficial alteration of the bone marrow immune microenvironment. We are currently exploring a variety of hypotheses and submit that Peg-Infa acts via a combination of these mechanisms. We have previously examined its role in the possible reversion to polyclonal hematopoiesis in patients treated in a Phase III trial for PV and ET with either Peg-Infa or hydroxyurea. Previous data has demonstrated that Peg-Infa treatment induces an increase in the number of circulating CD4+CD25+FOXP3+ regulatory T cells (Tregs). Tregs are an immunosuppressive T cell subset characterized by expression of FOXP3. They have an established influence on the state of the bone marrow immune microenvironment and appear to play a role in the suppression of anti-tumor immunity in a variety of malignant processes. Thus, they may have a previously unexplored influence on the pathophysiology of myeloproliferative neoplasms that is important in the context of Peg-Infa treatment. In this study, our aims were to serially monitor Tregs in a series of ET and PV patients treated with Peg-Infa to determine if treatment influenced Treg numbers and determine whether or not Tregs correlated with markers of response.Peripheral blood samples we collected at various time intervals and assessed for the number of circulating Tregs, the JAK2V617F allelic burden in granulocytes and, in females, the possible conversion of clonal to polyclonal hematopoiesis (detected by X-chromosome allelic usage ratio). The JAK2V617F allelic burden was measured by quantitative allele-specific PCR on an Applied Biosystems instrument in peripheral blood granulocytes enriched using density gradient centrifugation. The number of Tregs was measured in peripheral blood by flow cytometry using a cocktail of antibodies including CD4, CD25 and FOXP3. The proportion of Tregs among lymphocytes was measured. Using this information, and the absolute lymphocyte count from the CBC data, the absolute number of circulating Tregs was calculated and expressed as #Tregs/microliter. The number of Tregs/microliter was also correlated with the QT-PCR-determined transcript levels of FOXP3.In ongoing studies, 32 patients (15 females and 17 males) with PV and ET were treated with Peg-Infa. Following treatment, 15 of 23 patients with clinical follow-up demonstrated a 20% or greater decrease in the JAK2V617F allelic burden, indicating that Peg-Infa had an effect on the JAK2V617F-positive clone. Eight patients had no response or had an increase in JAK2V617F levels. Thirteen out of 15 female patients were informative for clonality testing. Two of these 13 patients developed polyclonal hematopoiesis during therapy. In one of these patients, the JAK2V617F allelic burden was unchanged, indicating that Peg-Infa had a selective effect on a pre-JAK2V617F PV clone, while in the other patients Peg-Infa clearly decreased the JAK2V617F-positive PV clone. There was a significant inverse correlation between the absolute number of peripheral blood Tregs and the JAK2V617F allelic burden. We also found that the changes in Treg numbers correlated with FOXP3 mRNA in circulating monuclear cells. Similar ongoing studies are being performed in the marrow of these patients and in patients treated with hydroxyurea.In conclusion, Peg-Infa induces changes in the number of circulating Tregs, in addition to the previously reported beneficial clinical effects including a decrease in the JAK2V617F allelic burden and, in some instances, re-establishment of polyclonal hematopoiesis. Furthermore, we found that the number of peripheral blood Tregs is inversely correlated to the JAK2V617Fallelic burden suggesting that it may serve as a marker of response and suggesting the Tregs may play a role in the pathophysiology of the disease.First and second authors contributed equallyThis work was supported by 1P01CA108671-O1A2 (NCI) MPD Consortium and the Leukemia & Lymphoma Society Disclosures:Swierczek:University of Utah : No financial compensation , No financial compensation Patents & Royalties. Prchal:University of Utah : No financial compensation , No financial compensation Patents & Royalties.

Single Cell Analysis Of JAK2V617F Positive MPN Stem/Progenitor Cells In Chronic Phase and Leukemic Transformation

IntroductionEssential thrombocytosis (ET), polycythemia vera (PV), and myelofibrosis (MF) share the JAK2V617F mutation, but differ with regard to clinical phenotype, rate of disease progression, and risk of leukemic transformation. Variation in the JAK2V617F neutrophil allele burden does not account for these observed differences in clinical behavior. We therefore investigated JAK2V617Fallele burden and genotype in the stem/progenitor populations of MPN patients in chronic and leukemic phases. MethodsWe studied 39 JAK2V617Fpositive MPN patients evaluated between 2005 and 2013, including 8 patients at leukemic transformation. CD34+ cells isolated from peripheral blood were flow sorted based on CD34, CD38, and the stem cell marker aldehyde dehydrogenase (ALDH). JAK2V617F allele percentages were calculated using an allele specific real time PCR assay. Cells were sorted into 96 well plates and single cell JAK2V617Fgenotypes were obtained using a nested PCR assay. Additional genomic lesions and chromosomal copy number variation were investigated in the sorted fractions when applicable. ResultsIn all MPN cases, the JAK2V617F mutation was detected in the CD34+CD38-ALDHhigh fraction – the same population in which the normal hematopoietic stem cell resides. Quantitative JAK2V617F allele burdens in this fraction were highest in MF > PV > ET. Single cell JAK2V617F genotyping revealed a higher proportion of JAK2V617F-/- cells in ET and PV than in MF, but JAK2V617F-/- cells were detectable in the CD34+CD38-ALDHhigh fraction of all cases. In most cases of PV and MF, this fraction contained a mixture of JAK2V617F-/-, JAK2V617F-/+, and JAK2V617F+/+ cells. Additional chronic phase lesions (including mutations of ASXL1 &TET2) were also found in the CD34+CD38-ALDHhighfraction.Two patterns of leukemic transformation were observed. The first pattern (in 7/8 patients) was identical to that of de novo AML (Gerber JM, Blood 2012), with emergence of a unique CD34+CD38-ALDHint fraction, which was clonal by JAK2V617F genotype and contained leukemia-specific lesions (e.g., 5q deletion). In contrast, the residual CD34+CD38-ALDHhigh population lacked the leukemic abnormalities and was oligoclonal with respect to JAK2V617F. In 3 of these AML cases, the CD34+CD38-ALDHint fraction was JAK2V617F-/-, while the JAK2V617F mutation remained detectable in the CD34+CD38-ALDHhigh fraction. Single cell genotyping performed during the leukemic phase of a PV patient revealed only JAK2V617F-/- CD34+CD38-ALDHint cells but identified JAK2V617F-/-, JAK2V617F-/+, and JAK2V617F+/+ CD34+CD38-ALDHhigh cells; JAK2V617F levels were barely detectable in the progenitors and neutrophils during this leukemic phase. Upon achievement of complete remission from AML, high JAK2V617F allele burdens were then found in the progenitors and neutrophils, as well as in the CD34+CD38-ALDHhigh fraction. A second pattern of leukemic transformation was seen in one patient, in whom no CD34+CD38-ALDHint population was present. The CD34+CD38-ALDHhigh population was expanded in this case and harbored JAK2V617F+/+ positive cells with the leukemia-specific lesion. ConclusionsUnlike CML, in which the BCR/ABL oncogene is typically present in the majority of CD34+CD38-ALDHhigh cells at diagnosis (Gerber JM, Am J Hematol 2011), the JAK2V617F mutation was present in only a minority of CD34+CD38-ALDHhigh cells in JAK2V617F positive ET and PV. Moreover JAK2V617F-/- cells were detected even in longstanding, advanced phase PV and MF. Lower JAK2V617F clonal burdens in the primitive CD34+CD38-ALDHhigh compartment as compared to neutrophils in most cases of MPN suggest that the JAK2V617F mutation does not confer a significant advantage at the stem cell level and that other genetic lesions may drive expansion of this population. JAK2V617F negative leukemias occur in about 35% of PV patients, apparently arising from the residual JAK2V617F negative CD34+CD38-ALDHhigh reservoir. We conclude that primitive stem/progenitor cells are mosaic with regard to JAK2V617F mutation status in the majority of MPN patients. Furthermore, acquisition of JAK2V617F, development of JAK2V617F homozygosity, and accrual of other acquired lesions in chronic phase MPN all occur in the primitive CD34+CD38-ALDHhigh compartment. Lesions specific to post MPN AML segregate to a distinct CD34+CD38-ALDHint population. Disclosures:Jones:Cytomedix: Patent holder for Aldefluor reagent, which is licensed by Cytomedix. This relationship is managed by the Johns Hopkins Office of Policy Coordination., Patent holder for Aldefluor reagent, which is licensed by Cytomedix. This relationship is managed by the Johns Hopkins Office of Policy Coordination. Patents & Royalties.

Phase II trial of panobinostat, an oral pan‐deacetylase inhibitor in patients with primary myelofibrosis, post–essential thrombocythaemia, and post–polycythaemia vera myelofibrosis

Myelofibrosis (MF) is a Philadelphia chromosome-negative stem cell myeloproliferative neoplasm (MPN) associated with cytopenias, splenomegaly, constitutional symptoms, and poor prognosis. MF patients commonly express JAK2 V617F mutation and activation of Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signalling. Agents targeting the JAK/STAT pathway have demonstrated efficacy in patients with MF. This study evaluated panobinostat, a pan-deacetylase inhibitor that depletes JAK2 V617F levels and JAK/STAT signalling in MPN cells, in patients with primary MF, post-essential thrombocythaemia MF, and post-polycythaemia vera MF. Patients received panobinostat 40 mg administered three times per week. Dose reductions were permitted for toxicities. The primary endpoint was response rate at 6 months using International Working Group for Myelofibrosis Research and Treatment (IWG-MRT) consensus criteria. Analyses of peripheral blood cells from treated patients revealed that panobinostat inhibited JAK/STAT signalling, decreased inflammatory cytokine levels, and decreased JAK2 V617F allelic burden. However, panobinostat was poorly tolerated at the dose and schedule evaluated, and only 16 of 35 patients completed ≥2 cycles of treatment. One patient (3%) achieved an IWG-MRT response. Common adverse events were thrombocytopenia (71.4%) and diarrhoea (80.0%). Although molecular correlative analyses suggested that panobinostat inhibits key intracellular targets, limited clinical activity was observed because of poor tolerance.

Ruxolitinib changes the natural course of myelofibrosis and its transplant outcome

THE AUTHORS' REPLY: In his letter referring to Will JAK1/2 inhibitors change the standard of care for myelofibrosis (MF)?, Dr. Raut raised two important issues about ruxolitinib: (1) its mechanism of action and (2) its beneficial effect on the outcome of hematopoietic stem cell transplantation (HSCT) in MF patients. The first issue was about how ruxolitinib improved survival without causing any significant effect on disease biology parameters such as the peripheral blast count, marrow fibrosis, cytogenetic response, and JAK2 V617F allele burden. At the last annual meeting of the American Society of Hematology, long-term outcome data of COMFORT-I and COMFORT-II trials that either showed significantly improved survival of ruxolitinib group compared to placebo (hazard ratio (HR) 0.58, 95% confidence interval (CI) 0.36-0.95) or best supportive care (HR 0.52, 95% CI, 0.27-1.00) were presented. Since peripheral blast counts, marrow fibrosis, and cytogenetics were not investigated in those trials, it is difficult to explain the survival advantage on those parameters. However, with the use of ruxolitinib, the two studies showed significant sustained reduction of the JAK2 V617F allele burden. In COMFORT-I trials, ruxolitinib resulted in reduction of JAK2 mutant allele burden by a mean of 10.9% at week 24 (P<0.0001) and -21.5% at week 48 (P<0.0002) that were independent of baseline JAK2 V617F levels [1]. COMFORT-II trials also showed significant reduction in allele burden (median reduction rate of -7.0% at 48 weeks and -8.7% at 72 weeks), which was associated with spleen response [2]. Verstovsek et al. reported that 50% reduction in palpable spleen length was associated with prolonged survival in ruxolitinib treated patients [3]. These observations suggest that ruxolitinib could alter MF prognosis through reduction of JAK2 V617F allele burden and spleen volume. However, we should note that survival improvement was not great and longer follow-ups are required. The relationship between JAK2 V617F allele burden and the survival is also unclear. The second issue was regarding pre-transplant ruxolitinib and the improvement of HSCT outcome by decreasing proinflammatory cytokines. Several convergent lines of evidence suggested that inflammatory cytokines act as mediators of acute GVHD and cause debilitating symptoms in MF. Ruxolitinib significantly decreased tumor necrosis factor-alpha and interleukin-6 levels for a prolonged time in patients with MF through anti-JAK1-mediated downregulation [1]. In a mouse allogeneic transplant model, ruxolitinib decreased alloreactive CD4+ T cells, expression of CXCR3 in activated T cells and GVHD, leading to improved survival [4]. Poor performance status is associated with poor outcome with HSCT in MF, whereas the reduction in symptomatic burden with JAK1/2 inhibitors may have the potential of improving transplant outcome. Massive splenomegaly is reported to be associated with delayed neutrophil and platelet recovery, suggesting that reduction of spleen volume may induce faster marrow recovery after HSCT [5]. Although these are indirect evidences, they support the suggestion that JAK1/2 inhibitors prior to HSCT may have a beneficial effect on the transplant outcome. Whether pre-transplant JAK1/2 inhibitors decrease GVHD and improve survival in HSCT should be addressed through a prospective trial.

Salutary Effect of Pegylated Interferona in PV and ET As Evaluated by Quantitation of Pre-JAK2V617F and JAK2V617F-Bearing Stem Cells and Granulocytes and Correlation with Circulating Regulatory T Cells and HSC Cell Cycle Status

AbstractAbstract ▪807▪This icon denotes a clinically relevant abstractThe discovery of JAK2 (JAK2V617F) mutation in polycythemia vera (PV) and essential thrombocythemia (ET) has revolutionized our understanding of PV and ET. However, JAK2V617F is not the disease-initiating mutation and constitutes only part of the clone. Pegylated interferona (PegInfα), a form of interferon-alpha with a longer half-life and more tolerable toxicity, can induce not only hematological remission but also reduction of JAK2V617F allelic burden and suppression of the pre-JAK2V617F PV clone, as demonstrated by the return of polyclonal hematopoiesis (Liu Blood, 03). It is not clear whether this salutary effect is achieved by stimulation of an immune response against the PV clone, preferential activation of normal dormant stem cells (HSCs), or suppression of the PV clone by PegInfα. Using X-chromosome based clonality (qTCA) and JAK2V617F allelic burden quantitative assays, we analyzed selected biomarkers in HSCs, granulocytes and platelets of 18 patients treated with PegInfα, and evaluated cell cycle activity in HSCs. Previous data have indicated that PegInfα modulates the number of CD4+FOXP3+ regulatory T cells (Tregs) in the periphery. This is perhaps an indicator that PegInfα alters immunity against the JAK2V617F-bearing clone. We hypothesized that PegInfα mobilizes marrow Tregs to the periphery, thus decreasing their immunosuppressive and tumor promoting influence on the marrow microenvironment. Therefore, we correlated the number of Tregs before, during and after treatment with JAK2 allelic burden in peripheral blood and marrow samples in ongoing studies.In ongoing studies, 18 patients (7 females and 11 males) with PV and ET were treated with PegInfα, with a median follow-up of 6 months. There were variable JAK2V617F allelic burdens at enrollment (1.2% – 99.8%). Following treatment, 7 of 18 patients demonstrated a decrease in JAK2V617F allelic burden, indicating that PegInfα had an effect on the JAK2V617F-positive clone, while 11 had no response or had an increase in JAK2V617F levels. Six out of 7 female patients were informative for at least one exonic polymorphism out of 5 X-chromosome genes, and all 6 had clonal hematopoiesis as determined by qTCA at the start of treatment. Two of these 6 patients developed polyclonal hematopoiesis during therapy, but in one the JAK2V617F allelic burden was unchanged, indicating that, in this patient, PegInfα had a selective salutary effect on a pre-JAK2V617F clone, while in other patients PegInfα clearly decreased the JAK2V617F-positive clone. In the 7 patients who demonstrated decreased JAK2V617F levels, there was an inverse relationship of JAK2V617F level with the number of circulating peripheral blood Tregs (examples shown in Figs 1 and 2). Statistical analysis of 18 patients showed a significant negative relationship between the number of circulating Tregs and JAK2V617F allelic burden (Spearman’s correlation coefficient -0.4308, p<0.001). This suggests that an immunomodulatory effect by PegInfα involving Tregs may, at least in part, be associated with suppression of pre-JAK2V617F and JAK2V617F-positive clonal hematopoiesis.We assessed total colony forming ability in methylcellulose culture media as a measure of hematopoietic progenitor abundance. In 5 patients treated with Pegasys, all 5 demonstrated an increase in colony-forming units. Furthermore, when cell cycle status of CD34+ HSCs was ascertained, we found a decrease in the percentage of HSCs in G0 (quiescence) in 4 out of 6 samples. These findings suggest that PegInfα therapy promotes normal HSC proliferation.For the first time we directly demonstrate that PegInfα therapy in some patients recruits dormant pre-clonal PV HSCs to active hematopoiesis, thereby resulting in production of polyclonal granulocytes and platelets. PegInfα also appears to mobilize Tregs to the circulation, an effect that may be associated with changes in the marrow microenvironment. We conclude that PegInfα can suppress both pre-JAK2V617F and JAK2V617F PV clones in some PV patients, as demonstrated by evaluation of HSCs, granulocytes and platelets, but this often occurs asynchronously. Our preliminary data suggest that PegInfα therapy is associated with an increase in cell cycle activity in HSCs and mobilization of Tregs to the circulation. This provides a possible mechanism by which polyclonal hematopoiesis is restored after PegInfα treatment. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Imetelstat Rapidly Induces and Maintains Substantial Hematologic and Molecular Responses in Patients with Essential Thrombocythemia (ET) Who Are Refractory or Intolerant to Prior Therapy: Preliminary Phase II Results

AbstractAbstract 179 Background:Myeloproliferative neoplasms (MPNs), such as essential thrombocythemia (ET), are driven by neoplastic progenitor cells. The JAK2 V617F mutation can be detected in approximately 50% of patients (pts) with ET, and the JAK2 V617F allele burden can be used to measure the treatment-induced molecular response (MR) over time. Telomerase is upregulated in neoplastic progenitor cells and sustains indefinite replication. Imetelstat is a first in class, potent, specific inhibitor of telomerase which selectively distributes to bone marrow and inhibits thrombopoiesis. In vitro studies demonstrate that imetelstat selectively inhibits spontaneous megakaryocytic colony-forming unit (CFU-Meg) growth from the blood of pts with ET but not from healthy individuals. Phase I studies have demonstrated that imetelstat inhibits telomerase activity in pts at doses of 7.5 mg/kg and above.Therefore, unlike conventional cytoreductive therapy and JAK2 kinase inhibitors, imetelstat may be uniquely able to selectively inhibit proliferation of neoplastic clonogenic cells in pts with ET and modify the biology and progression of the disease. Methods:A phase II study enrolled pts with ET who had failed or were intolerant to at least one prior therapy, or who refused standard therapy. Pts were treated with imetelstat 7.5 mg/kg or 9.4 mg/kg IV weekly. After attainment of best platelet response in the induction phase, maintenance dosing with imetelstat was commenced with dosing based upon platelet count. Primary endpoint was best overall hematologic response (HR) with complete response (CR) defined as platelet count <400 × 103/μl maintained for at least 4 consecutive weeks in the absence of new thromboembolic events. A key secondary endpoint was rate of MR in patients with JAK2 V617F molecular mutations. JAK2 V617F allele burden was measured by allele-specific quantitative real-time PCR with a limit of detection of 0.1%. CFU-Meg growth pre- and post-treatment and tolerability were also assessed. Results:As of July 9, 2012, 13 pts were treated. Median age was 60 yrs (range 21–83) with a median of 2 prior treatments (range 1–3). Median years since initial diagnosis were 5.8 (range 0.3 to 24.9) and initial platelet count was 809 × 103/μl (range 601 to 1359 × 103/μl).Best overall HR was 100%, with 11 of 13 pts achieving a confirmed CR after a median of 6.1 weeks (range 5.1 to 14.1 wks). Twelve of 13 pts remain on maintenance therapy (median time on study 26.1 weeks) and despite transient elevations of platelets above best response, pts continue to be responsive to imetelstat. Four pts have reached 1 year of therapy and continue to be treated with ongoing HR. Dosing frequency on maintenance therapy was generally reduced with time.A substantial decrease in JAK2 V617F allele burden was demonstrated in all 5 JAK2 V617F-positive pts (mean allele burden reduction of 82%; range of 59–94%, see table below). Four pts who were eligible for MR assessment by LeukemiaNetcriteria (initial JAKV617F allele burden >10%) reached molecular partial responses (PR): one pt after 12 weeks, which has been maintained through 1 year, and 3 other pts at 24, 36 and 48 weeks of therapy. One additional pt with JAK2 V617F levels of 4.8% prior to therapy has also had a 75% reduction after 12 weeks of treatment.A reduction in the spontaneous growth of CFU-Meg was also observed in the 2 pts tested, with 93% and 96% reduction from baseline, respectively.Long-term administration of imetelstat was generally well tolerated. Common adverse events reported on therapy were mild to moderate gastrointestinal toxicities, reductions in neutrophil counts, and fatigue.JAK2 V617F Allele BurdenPt #Baseline3 mo6 mo9 mo12 mo126.4%7.7%6.1%1.8%3.4%253.3%52.2%43.7%44.5%21.8%310.8%11.2%14.3%2.4%431.1%14.8%2.0%54.8%1.2% Conclusions:Imetelstat rapidly induces and maintains hematologic responses in pts with ET who have failed or are intolerant to conventional therapies. Importantly, substantial MR is observed in all JAK2 V617F-positive pts and inhibition of the neoplastic clonogenic growth ex-vivo is demonstrated. The reduction in JAK2 V617F allele burden and cytokine-independent growth of CFU-Meg suggests that imetelstat has a relatively selective inhibitory effect on the growth of the neoplastic clone(s) which drive ET and has the potential to modify the underlying biology of MPNs. Additional data will be presented from this ongoing study. Disclosures:Baerlocher:Geron Corporation: Research Funding. Oppliger Leibundgut:Geron Corporation: Research Funding. Ayran:Geron Corporation: Employment. Blaney:Geron Corporation: Employment. Burington:Geron Corporation: Employment. Morfeld:Geron Corporation: Employment. Odenike:Sanofi Aventis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees. Reddy:Geron Corporation: Employment. Roeth:Geron Corporation: Research Funding. Stuart:OncoMed Pharmaceuticals: Consultancy; Geron Corporation: Consultancy, Employment.

Does increasing the JAK2V617F assay sensitivity allow to identify more patients with MPN?

Does increasing the JAK2V617F assay sensitivity allow to identify more patients with MPN?

Heat Shock Protein 90 Inhibitor Is Synergistic with JAK2 Inhibitor and Overcomes Resistance to JAK2-TKI in Human Myeloproliferative Neoplasm Cells

We determined the activity of hsp90 inhibitor, and/or Janus-activated kinase 2 (JAK2) tyrosine kinase inhibitor (TKI), against JAK2-V617F-expressing cultured mouse (Ba/F3-JAK2-V617F) and human (HEL92.1.7 and UKE-1) or primary human CD34(+) myeloproliferative neoplasm (MPN) cells. Following exposure to the hsp90 inhibitor AUY922 and/or JAK2-TKI TG101209, the levels of JAK2-V617F, its downstream signaling proteins, as well as apoptosis were determined. Treatment with AUY922 induced proteasomal degradation and depletion of JAK2-V617F as well as attenuated the signaling proteins downstream of JAK2-V617F, that is, phospho (p)-STAT5, p-AKT, and p-ERK1/2. AUY922 treatment also induced apoptosis of HEL92.1.7, UKE-1, and Ba/F3-hJAK2-V617F cells. Combined treatment with AUY922 and TG101209 caused greater depletion of the signaling proteins than either agent alone and synergistically induced apoptosis of HEL92.1.7 and UKE-1 cells. Cotreatment with AUY922 and TG101209 also induced significantly more apoptosis of human CD34(+) MPN than normal hematopoietic progenitor cells. As compared with the sensitive controls, JAK2-TKI-resistant HEL/TGR and UKE-1/TGR cells exhibited significantly higher IC(50) values for JAK2-TKI (P < 0.001), which was associated with higher expression of p-JAK2, p-STAT5, p-AKT, and Bcl-xL, but reduced levels of BIM. Unlike the sensitive controls, HEL/TGR and UKE/TGR cells were collaterally sensitive to the hsp90 inhibitors AUY922 and 17-AAG, accompanied by marked reduction in p-JAK2, p-STAT5, p-AKT, and Bcl-xL, with concomitant induction of BIM. Findings presented here show that cotreatment with hsp90 inhibitor and JAK2-TKI exerts synergistic activity against cultured and primary MPN cells. In addition, treatment with hsp90 inhibitor may overcome resistance to JAK2-TKI in human MPN cells.

Buccal Mucosa Cells Are Unsuitable As a Control Tissue to Distinguish Somatic Hematopoietic Mutations From Germline Mutations: Nail DNA Is a Suitable Surrogate

Abstract 5156Blood is an easily obtainable source of genomic DNA (gDNA) for genetic analysis of blood neoplasms. However, germline and non-hematopoietic cell gDNA, essential for distinguishing tumor-specific acquired mutations from constitutional genetic variants, is more difficult to obtain, an issue magnified with the advent of whole genome sequencing. Skin fibroblasts or fibroblasts isolated from marrow biopsies are a source of non-hematopoietic cell gDNA, and in some instances, T-lymphocytes formed before the clonal event have been used as the control tissue. However, such samples require invasive and time-consuming procedures, trained personnel and additional in vitro culturing or other manipulations. Consequently, buccal mucosa cells have been employed as a convenient alternative source of non-hematopoietic cell gDNA. Unfortunately, the appropriateness of buccal mucosa cells for this purpose has come into question in both genomic sequencing studies and bone marrow transplant studies in which the donor genotype was rapidly evident in the recipient's buccal mucosa cell gDNA (Endler et al., Bone Marrow Transplant., 1999).To determine if buccal mucosa cell gDNA adequately represented the germline genotype in myeloproliferative neoplasms (MPN), we compared JAK2 V617F levels in granulocyte and buccal mucosa cell gDNA using an allele-specific, quantitative real-time polymerase chain reaction assay (Moliterno et al., Blood, 2006). JAK2 V617F, a well documented somatic mutation, was identified in six of six buccal mucosa cell samples and the quantity was 55–100% of that seen in patient granulocyte gDNA. Cytospin preparations failed to reveal non-buccal mucosa cell contamination (>95%) and neither extensive pre-washing of the cells (3 to 4 washes in PBS) nor DNAase treatment before gDNA extraction eliminated or even noticeably reduced the quantity of measurable JAK2 V617F. Furthermore, in keeping with the known presence of circulating JAK2 V617F, DNA isolated from patient cell-free saliva also demonstrated equally high quantities of JAK2 V617F.In an independent study of a two-year old child with the clinical phenotype of PV and a high JAK2 V617F allele burden, buccal mucosa cells were analyzed to exclude congenital PV and a significant JAK2 V617F allele burden was observed. However, subsequent analysis of gDNA extracted from cultured fibroblasts, isolated peripheral blood lymphocytes and nails were all negative for the JAK2 V617F mutation.We therefore investigated the potential of using nails as an alternative noninvasive source of gDNA. Using a forensic nail DNA extraction kit (QIAamp DNA Investigator kit, QIAGEN cat. #56504), we found that gDNA from the nails of two JAK2 V617F-positive patients were negative for the JAK2 V617F mutation both by allele-specific quantitative PCR and by a JAK2 V617F-specific pyrosequencing assay. In addition, when we encountered a previously unreported JAK2 mutation, T514M, we were able to demonstrate that it was an acquired mutation by using nail gDNA for direct PCR sequencing, as shown below. [Display omitted] While this method is very convenient and suitable for PCR-based studies, it is limited for use in genome-wide studies because the isolated nail gDNA is highly degraded (<400 bp). However, using a NaOH-based technique (Cline et al., J. Forensic Sci., 2003) we have been able to consistently obtain gDNA fragments ranging from 1000 – 3000 bp from either toenails (∼10 ng/mg), or fingernails (∼40 ng/mg). Thus, it should be possible to generate enough gDNA (4–5 μg) with two rounds of nail clippings for genomic sequencing studies. This technique should also lend itself to clonality studies in genetically appropriate female subjects where lymphocytes have been the traditional gDNA source.We conclude that whether from blood cells, saliva or an unknown source, buccal mucosa cells are heavily contaminated with and inseparable from DNA of hematopoietic cell origin. We also demonstrate that both fingernails and toenails are a suitable source of non-hematopoietic cell gDNA, which can be used to distinguish between acquired and constitutional genetic variants in hematopoietic tissue. In addition, we have identified a protocol that can generate nail gDNA of a potentially suitable quality and quantity for genome-wide sequencing studies. Disclosures:No relevant conflicts of interest to declare.

A Phase II Trial of Panobinostat, an Orally Available Deacetylase Inhibitor (DACi), in Patients with Primary Myelofibrosis (PMF), Post Essential Thrombocythemia (ET), and Post Polycythemia Vera (PV) Myelofibrosis

AbstractAbstract 630 Introduction:Medical treatment options for myelofibrosis (MF) are palliative in nature. Short of allogeneic stem cell transplantation, no therapies are available that are curative or confer a survival benefit. Panobinostat (PAN; LBH589) is a potent pan-deacetylase inhibitor that belongs to a structurally novel cinnamic hydroxamic acid class of compounds. Preclinical studies demonstrated that treatment with PAN alone depletes the levels of JAK2V617F (JAK2) and signaling downstream through STAT3 and 5, AKT, and c-RAF/MAPK in human MPN progenitor cells, including those from patients (pts) with MF (Blood 2009; 114:5024). In a prior phase IA/II study of PAN in pts with advanced hematologic malignancies, among 12 pts with MF evaluable for response, 4 pts demonstrated durable clinical improvement. Methods:In this phase II trial, approximately 50 adult pts with primary MF, post essential thrombocythemia, or post polycythemia vera MF who have an International Prognostic Scoring System -MF score of 2 or 3 along with either symptomatic splenomegaly (>10 cm below the costal margin [BCM]) or anemia, enrolled. Initial PAN dose is 40 mg three times a week until disease progression or unacceptable toxicity with dose adjustments as required. Response is assessed using the International Working Group criteria. Peripheral blood samples are collected for correlative science studies at 7 time points during treatment; pre-dose on the first day of the study, 6 and 24 hours after the first dose, pre-dose on the first day of cycles 2, 4 and 6, and at the end of the study. The correlative studies include the determination of JAK2V617F allelic burden, immunoblot analyses of the protein expression of p-STAT3, p-STAT5, p-AKT, p-ERK1/2, p-PIM, as well as of their un-phosphorylated counterparts. Also, protein levels of HSP70 and acetylated (Ac)-α-tubulin, as well as the mRNA expression of JAK2 and PRV-1 genes were determined. Results:As of August 12, 2010, 31 pts (24 JAK2 mutated, 7 JAK2 wild type) have been enrolled in the trial. The majority of pts required dose reduction. Adverse events observed are consistent with the known safety profile of PAN. Correlative studies have been performed on MPN cells from19 patients. As compared to the pre-treatment levels, treatment with PAN (for 6 and/or 24 hours) resulted in varying levels of depletion of p-STAT3, p-STAT5, p-AKT, p-ERK1/2 and p-PIM protein. A decline in JAK2 and PRV1 mRNA levels (20 to 80%) was also observed. Conversely, PAN treatment induced Ac-α-tubulin and HSP70 levels, consistent with HDAC6 and HSP90 inhibition. This was associated with decline in the protein levels of AKT, PIM1 and STAT3. PAN treatment also reduced MCL-1 and Bcl-xL, while inducing BIML levels. Notably, of the 10 patients in whom it has been determined, PAN treatment depleted the allelic burden of JAK2V617F by an amount ranging between 10 to 90%. Conclusions:Consistent with its role as a pan-deacetylase inhibitor, PAN exerts in vivo anti-MPN cell activity, as was highlighted by the decrease in the allelic burden of JAK2V617F. Further patient enrollment is ensuing, and more mature clinical toxicity and efficacy data will be presented. Disclosures:DeAngelo:Novartis: Membership on an entity's Board of Directors or advisory committees. Off Label Use: Panobinostat is an investigational agent currently being evaluated for the treatment of hematologic and solid malignancies. Paley:Novartis: Employment. Snyder:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees. Ondovik:Novartis: Employment. Rine:Novartis: Employment.

A phase II trial of oral panobinostat (LBH589), a novel deacetylase inhibitor (DACi) in patients with primary myelofibrosis (PMF), post-essential thrombocythemia (ET) myelofibrosis, and post-polycythemia vera (PV) myelofibrosis.

TPS283 Background: Medical treatment options for myelofibrosis (MF) are palliative in nature. Short of allogeneic stem cell transplantation, no therapies are available that are curative or confer a survival benefit. Panobinostat (PAN; LBH589) is a potent pan-deacetylase inhibitor that belongs to a structurally novel cinnamic hydroxamic acid class of compounds. Preclinical studies demonstrated that treatment with PAN alone depletes the levels of JAK2V617F (JAK2) and signaling downstream through STAT3 and 5, AKT, and c-RAF/MAPK in human MPN progenitor cells, including those from patients (pts) with MF (Blood 2009;114:5024). In a prior phase IA/II study of PAN in pts with advanced hematologic malignancies, among 12 pts with MF evaluable for response, 4 pts demonstrated durable clinical improvement. We present a phase II, multicenter, open-label, 2-stage study to further evaluate the efficacy of PAN as a single agent in pts with MF. Methods: Adult pts with PMF, post ET/PV MF who have an IPSS- MF score of 2 or 3 along with either symptomatic splenomegaly (>10 cm below the costal margin [BCM]) or anemia, are eligible. Pts will receive PAN orally 40 mg TIW until disease progression or unacceptable toxicity. There will be 2 cohorts of 20 pts (20 JAK2 mutated, 20 JAK2 wild-type). The study is a 2- stage trial for each independently powered cohort. If no responses are seen in the first 13 pts of either cohort within 6 cycles of treatment, the cohort will be closed to further accrual. Primary endpoint is overall response (CR, PR, and clinical improvement). Secondary endpoints include comparison of responses in pts who are JAK2 mutated to those who are JAK2 wild-type; the symptomatic improvement of MF pts using the Myelofibrosis Symptom Assessment Form, and safety and tolerability of PAN in MF pts. Exploratory endpoints include correlative biomarker studies. As of January 12, 2010, 8 pts have been enrolled. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Novartis Novartis Novartis Novartis Novartis

Cotreatment with panobinostat and JAK2 inhibitor TG101209 attenuates JAK2V617F levels and signaling and exerts synergistic cytotoxic effects against human myeloproliferative neoplastic cells

AbstractThe mutant JAK2V617F tyrosine kinase (TK) is present in the majority of patients with BCR-ABL–negative myeloproliferative neoplasms (MPNs). JAK2V617F activates downstream signaling through the signal transducers and activators of transcription (STAT), RAS/mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3 (PI3)/AKT pathways, conferring proliferative and survival advantages in the MPN hematopoietic progenitor cells (HPCs). Treatment with the pan-histone deacetylase (HDAC) inhibitor panobinostat (PS) is known to inhibit the chaperone function of heat shock protein 90, as well as induce growth arrest and apoptosis of transformed HPCs. Here, we demonstrate that PS treatment depletes the autophosphorylation, expression, and downstream signaling of JAK2V617F. Treatment with PS also disrupted the chaperone association of JAK2V617F with hsp90, promoting proteasomal degradation of JAK2V617F. PS also induced apoptosis of the cultured JAK2V617F-expressing human erythroleukemia HEL92.1.7 and Ba/F3-JAK2V617F cells. Treatment with the JAK2 TK inhibitor TG101209 attenuated JAK2V617F autophosphorylation and induced apoptosis of HEL92.1.7 and Ba/F3-JAK2V617F cells. Cotreatment with PS and TG101209 further depleted JAK/STAT signaling and synergistically induced apoptosis of HEL92.1.7 and Ba/F3-JAK2V617F cells. Cotreatment with TG101209 and PS exerted greater cytotoxicity against primary CD34+ MPN cells than normal CD34+ HPCs. These in vitro findings suggest combination therapy with HDAC and JAK2V617F inhibitors is of potential value for the treatment of JAK2V617F-positive MPN.

Correlation of Novel Molecular Markers in Patients with Myeloproliferative Neoplasms.

Abstract 4968Elucidation of the molecular aberrations underlying the development of myeloproliferative neoplasms (MPNs) has progressed rapidly during the previous years. In addition to the JAK2V617F mutation, which is found in over 90% of patients with polycythemia vera (PV) and in around 50% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF), several novel markers have recently been described. These include mutations in the Ten-Eleven-Translocation-2 (TET-2) gene, as well as overexpression of the hematopoietic transcription factor nuclear factor erythroid-2 (NF-E2). While the individual frequency of these abnormalities is well known, so far, studies that evaluate the relationship between these markers and their correlation with hematological parameters have not been conducted. The International Myeloproliferative Research Consortium (MPD-RC) has instituted a Tissue Bank (MPD-RC Trial 106) allowing the collection of MPN patient samples from member institutions in the US and Europe. Using this resource, we have investigated the relationship among the following molecular markers and hematological parameters in a series of 66 MPN patients: TET-2 mRNA expression, JAK2V617F allele burden, NF-E2 mRNA expression, granulocyte clonality (female patients) as well as hematocrit, hemoglobin concentration, platelet numbers and leukocyte counts. Our crossectional cohort included 37 PV patients, 14 ET, 4 PMF and one post PV MF patient. Sixtyeight percent of the patients were treated, medication including hydroxyurea, anagrelide, ASA, interferon and one patient treated with imatinib mesylate. We acknowledge that treatment may affect the molecular markers being investigated. In addition, the desired effect of treatment on hematological parameters may not be paralleled by a similar effect on molecular markers. Therefore, investigation of treated patients may not reveal biological relationships present in untreated diseases states. We thus tested the effect of cytoreductive treatment on the expression of molecular markers by comparing treated and untreated patients in our cohort. The JAK2V617F allele burden was significantly lower in treated patients than in untreated patients (p = 0.008). The same difference was noted when PV patients were analyzed alone (p = 0.006) In contrast, neither TET-2 nor NF-E2 mRNA expression were affected by treatment.In the 66 MPN patients evaluated, a significant inverse Spearman correlation of -0.25 was noted between NF-E2 mRNA expression and hemoglobin concentration (p = 0.05). This relationship was more pronounced when PV patients were analyzed alone (Spearman's r = -0.41, p = 0.01). In addition, a correlation of 0.51 between JAK2V617F allele burden and NF-E2 mRNA expression was noted (p = 0.0007). Occurrence of the recently discovered TET-2 gene mutations, which are present in around 15% of MPN patients, was measured indirectly by determining TET-2 mRNA expression. None of the other markers and parameters assessed was significantly correlated with the amount of TET-2 mRNA expressed.We report a previously unrecognized inverse relationship between NF-E2 mRNA expression and hemoglobin concentration. These data complement several recent findings in MPN patients. While NF-E2 is overexpressed in granulocytes of all three MPN subtypes, PV, ET and PMF, its overexpression in CD34+ hematopoietic stem cells has so far only been observed in PMF. We have recently shown that ex vivo overexpression of NF-E2 in CD34+ hematopoietic stem cells drastically reduces erythroid colony formation. It was previously noted that mean JAK2V617F allele burdens in PMF patients are higher than in PV or ET. Here, we confirm the previously noted positive correlation between JAK2V617F allele burden and NF-E2 mRNA expression. Our data therefore suggest that high levels of JAK2V617F in PMF patients directly or indirectly augment NF-E2 overexpression, which may contribute to the anemia of Primary Myelofibrosis. DisclosuresNo relevant conflicts of interest to declare.

Establishment of Mouse PMF Model by the Introduction of Constitutive STAT5a Into Purified Hematopoietic Stem Cells.

Abstract 3900Poster Board III-836 BackgroundPolycythemia vera (PV), essential thrombocythemia (ET), and primary myelofiblosis (PMF) are pathologically related and now classified under myeloproliferative neoplasm (MPN). Subsequent studies revealed that MPN is a group of clonal hematopoietic stem cell disorders characterized by proliferation of one or more of the myeloid lineages. The somatic activating mutation in the JAK2 tyrosine kinase, JAK2V617F, is now broadly recognized as a mutation responsible for MPN (Levine R.L. and Gilliland D.G. Blood 2008). Indeed, Most of PV patients, and half of patients with ET or PMF possess this mutation. Recent studies revealed that PV phenotype can be generated in homozygous JAK2V617F transgenic mice, while ET or atypical CML-like marked leukothrombocytosis with mild myelofibrosis can be observed in heterozygous JAK2V617F mice (Tiedt et al, Blood 2008, Shide et al Leukemia 2008). These results indicate that expression levels of JAK2V617F may influence PV and ET phenotypes. On the other hand, typical PMF phenotype has not been generated by the introduction of JAK2V617F. According to the WHO criteria, PMF could be defined as “spent phase of hematopoiesis” with fibrosis formation followed by increased bone marrow cellularity as consequences of granulocytic proliferation and megakaryocyte changes with ineffective hematopoiesis. In this study, we focused on STAT5a, a direct downstream molecule of JAK2, because we previously reported that upon transplantation, purified CD34- lineage- sca-1+ c-Kit+ (CD34-KSL) hematopoietic stem cells (HSCs) transduced with constitutive active form of STAT5A acted as MPN initiating cells causing granulocytosis without erythrocytosis/thrombocytosis (Kato Y. et al, J Exp Med 2005). Based on these observations, we attempted to make PMF model through mimicking typical PMF dynamics; hyper proliferation of HSCs by the introduction of constitutive active STAT5a and following early HSC exhaustion. Materials and MethodsCD34-KSL HSCs or CD34+KSL hematopoietic progenitor cells (HPCs) were purified from bone marrow (BM) of C57BL/6 (B6)-Ly5.1 mice. Then, the cells were retrovirally transduced with STAT5a wild-type (wt) or its constitutive active mutant, STAT5a(1*6). The prepared cells were used for methylcellulose assay and were transplanted into lethally irradiated B6-Ly5.2 recipient mice together with 5 × 105 B6-Ly5.1/5.2 competitor BM cells. Peripheral blood (PB) of transplanted mice was monitored biweekly for donor chimerism and lineage deviation using flow cytometry. Subsequently, histrogical analyses of bone marrow and spleen were performed to determine myelofiblosis grade and detecting extramedullar hematopoiesis. Finally, immunohistochemical staining of bone marrow with anti-TGF-b antibody was performed to detect effector cells of myelofibrosis. ResultsTransplantation of STAT5a (1*6) transduced HSCs resulted in generation of 57 MPN mice (total 83 mice), while no MPN mouse was obtained by STAT5a (1*6) transduced HPCs (total 12 mice). Pathological analysis revealed that majority (70%) of MPN mice had PMF phenotype as defined by leukoerythroblastosis and dacryocytosis without leukothrombocytosis. These mice with PMF phenotype showed marked splenomegaly with extramedullary hematopoiesis, and granulocytic proliferation with megakaryocyte change. In BM, granulocytic proliferation advanced to severe myelofibrosis and osteomyelosclerosis in very short period of time (4 to 8 weeks). Those mice died of hemorrhage induced by pancytopenia within a few months, much faster than the mice with JAK2V617F based PV/ET models. Immunohistological analysis revealed that dominance of Gr-1 / Mac-1 positive granulocytes and CD41 positive small megakaryocytes strongly expressing TGF-beta, a putative inducer of fibroblastosis in BM of PMF mice. ConclusionBy transplanting STAT5a(1*6) transduced HSCs, we were able to develop mice with phenotype closely resembling human PMF. Because PMF is rare disease, this animal model should be useful for understanding etiology of PMF, for evaluating existing treatment, and for developing therapeutics targeting STAT5a or its downstream pathway. Disclosures:No relevant conflicts of interest to declare.

Co-Treatment with JAK2 Inhibitor TG101209 and Panobinostat or hsp90 Inhibitor AUY922 Attenuates Mutant JAK2-V617F Levels and Activity in Human Myeloproliferative Disorder Cells

The mutant JAK2-V617F is present and constitutively active in myeloproferative disorders (MPD), including polycythemia vera (PV), essential thrombocytosis and primary myelofibrosis (MF). JAK2-V617F activates the signal transduction through the STAT, RAS/MAPK and PI3K/AKT pathways, conferring proliferative and survival advantage in the MPD hematopoietic progenitor cells (HPCs). We have previously reported that the panhistone deacetylase (HDAC) inhibitors e.g., panobinostat (Novartis Pharmaceuticals Inc), induces growth arrest and apoptosis of neoplastic HPCs. Panobinostat (PS), by inhibiting HDAC6, induces hyperacetylation of heat shock protein (hsp) 90, which attenuates the ATP binding and chaperone function of hsp90. This is associated with polyubiquitylation and degradation of hsp90 client protein signaling kinases, e.g, FLT-3, Bcr-Abl, AKT and c-RAF. Additionally, the mutant versions of signaling oncoprotein kinases, e.g., FLT-3, Bcr-Abl, B-RAF and c-KIT, may be even more dependent on hsp90 chaperone function for maintaining their proper folding and active conformation. This makes the mutant oncoprotein kinase even more susceptible to misfolding and proteasomal degradation upon inhibition of hsp90 function. In the present studies we determined the effects of PS or the non-geldanamycin analogue hsp90 inhibitor (AUY922, Novartis) and/or the JAK-2 inhibitor TG101209 (TG) (TargeGen, San Diego, CA) in the cultured (human erythroleukemia HEL and mouse BaF3 cells expressing JAK2-V617F) and primary human MPD cells (from two patients with PV and MF. Treatment with PS (10 to 30 nM) dose-dependently increased % of cells in G1 and decreased % of cells in S phase of cell cycle, as well as induced apoptosis of HEL and BaF3/JAK2-V617F cells. This was associated with attenuation in the levels of JAK2-V617F, p-JAK2, p-STAT3, p-STAT5, p-AKT (serine 473), AKT, p-ERK1/2, p-GATA1 (serine 310), GATA1 and Bcl-xL in HEL cells. PS induced lysine acetylation of hsp90 but not of JAK2. Similar effects were observed, following treatment with AUY922 (AUY). Both PS and AUY induced significantly less apoptosis of BaF3/hEpoR versus BaF3/JAK2-V617F cells. PS and AUY also reduced the chaperone association of JAK2-V617F with hsp90. Treatment with TG (0.2 to 2.0 uM) also dose-dependently attenuated the levels of p-STAT3, p-STAT5, p-AKT (serine 473), AKT, p-ERK1/2, p-GATA1 (serine 310) and Bcl-xL, which was associated with apoptosis of HEL and BaF3/JAK2-V617F cells. Co-treatment with PS and TG induced more apoptosis of the cultured and primary MPD cells than treatment with either agent alone. AUY also increased apoptosis of HEL cells induced by TG. Co-treatment with PS and AUY synergistically induced apoptosis of HEL cells (combination indices < 1.0). Co-treatment with 20 nM of PS or 15 nM of AUY augmented TG mediated attenuation of p-STAT3, p-STAT5 and Bcl-xL levels in HEL and BaF3/JAK2-V617F cells. These findings demonstrate for the first time that pan-HDAC inhibitor PS and hsp90 inhibitor AUY attenuate the levels and signaling of JAK2-V617F and enhance the anti-JAK2-V617F activity of TG in human MPD cells. Our findings also support the rationale to determine the in vivo activity of PS or AUY in combination with JAK2 inhibitors against human MPD.

Rapid decline of JAK2V617F levels during hydroxyurea treatment in patients with polycythemia vera and essential thrombocythemia

In polycythemia vera (PV) and essential thrombocythemia (ET) the discovery of the presence of JAK2 V617F mutations indicates that tyrosine kinase activation is a common pathogenetic mechanism in Ph- MPD.[1][1] The main therapeutic goal is to diminish the risk of clinical complications without