Treatment Of Myeloproliferative Disorders Research Articles

Interference on the Abbott i-STAT creatinine assay caused by hydroxyurea

Abstract Serum creatinine is an important biomarker used for estimating glomerular filtration rate (eGFR). The present study was designed after a patient at our institution had a significantly elevated creatinine result taken from an i-STAT point of care test that utilizes electrochemical detection. The patient’s creatinine from the i-STAT was 3.8 mg/dL and was not consistent with their clinical history. The patient’s previous creatinine taken 2 days prior was 1.39 mg/dL and 1.27 mg/dL seven days prior. Both previous readings were measured with the Siemens Dimension Vista 1500 using a colorimetric enzymatic creatinine method. A subsequent creatinine measured on the Siemens Dimension Vista 1500 was 1.30 mg/dL. Upon chart review, the clinical team noted that the patient is taking 500 mg of hydroxyurea daily for treatment of a myeloproliferative disorder. Review of the i-STAT package insert revealed hydroxyurea as a known interfering substance and states for every 100 µmol/L hydroxyurea in the specimen, creatinine will be increased by approximately 1.85 mg/dL. Review of literature described the positive interference caused by hydroxyurea for the i-STAT creatinine and glucose assays but did not explain the mechanism of this positive interference. In order to characterize the mechanism of hydroxyurea interference, we investigated the situation further. A pool of plasma was spiked with hydroxyurea. The spiked pool was serial diluted (x2, x4, x8, x16, x32, x64, x128) to observe the creatinine results in the presence of and absence of hydroxyurea. The dilution series was analyzed using the Vista enzymatic creatinine and the Abbott i-STAT enzymatic creatinine methods. The i-STAT creatinine results show increasing creatinine concentration from interference as the concentration of hydroxyurea increased. The dilution series suggests increasing positive interference with hydroxyurea. Positive creatinine interference with hydroxyurea was not observed when analyzed on the Vista platform. Hydroxyurea is a widely used treatment for myeloproliferative disorders and also for managing sickle cell disease. Hydroxyurea is known to cause positive interference in the i-STAT enzymatic creatine method that uses electrochemical detection, with the degree of interference correlating with the concentration of hydroxyurea. The Vista colorimetric enzymatic creatinine method was not observed to have hydroxyurea interference. While both methods employ enzymatic reagent systems the final detection approach is important. Patients undergoing hydroxyurea treatment should avoid i-STAT measurement systems for creatinine.

Abstract 1847: Phenotypic evaluation and discrimination of clinical JAK inhibitors for myeloproliferative disorders

Abstract Small molecule inhibitors of Janus kinases (Jakinibs) represent a promising treatment for myeloproliferative disorders including polycythemia vera (PV), essential thrombocytosis (ET), and myelofibrosis (MF). Currently multiple JAK2 inhibitors (ruxolitinib, fedratinib, momelotinib, baricitinib) are either approved or in clinical development for the treatment of MF. The primary target of Jakinibs, the JAK-STAT pathway, plays a critical role in multiple cellular processes relevant for myelofibrosis, including survival, differentiation, and proliferation. However, all approved Jakinibs are reportedly promiscuous in that they inhibit kinases beyond the JAK family. Their overall clinical impact, therefore, reflects the sum of these target activities, as well as downstream secondary pharmacology relevant to both efficacy and safety. We evaluated these four Jakinibs using phenotypic profiling with the BioMAP® Diversity PLUS Panel to identify common versus differential activities. The BioMAP platform consists of human primary cell-based assay or systems designed to recapitulate key aspects of tissue and disease states. Distinct activity profiles were generated for each of the tested Jakinibs based on their impacts on 148 different biomarkers in 12 different model systems. Comparison of these profiles at concentrations relevant for clinical exposure levels revealed common activities including inhibition of T and B cell proliferation and decreased levels of inflammation and immune activation biomarkers. Together these activities represent a phenotypic signature related to shared primary JAK targets. In contrast, multiple differentiating activities were observed between the four drug profiles: baricitinib increased TNFα levels whereas momelotinib decreased TNFα in the LPS system modeling monocyte activation; fedratinib and momelotinib, but not ruxolitinib or baricitinib, inhibited endothelial cell proliferation; only momelotinib inhibited macrophage responses and decreased PGE2 levels. These data indicate that even within the specific JAK2 inhibitor drug class, individual entities have distinct modes of action relevant for clinical outcomes. Citation Format: Sharlene Velichko, Sheryl P. Denker, Alison O'Mahony. Phenotypic evaluation and discrimination of clinical JAK inhibitors for myeloproliferative disorders [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1847.

Hydroxyurea-induced cyclic thrombocytopenia in polycythemia vera

Hydroxyurea is an orally administered medication that is frequently used in the treatment of myeloproliferative disorders as well as sickle cell disease. Cyclical thrombocytopenia during hydroxyurea therapy is an unusual and largely under-recognized side effect. We herein describe a case of a 66-year-old man with polycythemia vera who was started on hydroxyurea, and who subsequently developed platelet count oscillations with peak platelet levels as high as 2900 × 109 /L, and nadirs as low as 8 × 109 /L. We also performed a literature review of the currently published cases of hydroxyurea-induced cyclic thrombocytopenia.

Therapeutic effect of JAK1/2 blockade on the manifestations of hemophagocytic lymphohistiocytosis in mice

AbstractHemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome, characterized by severe hyperinflammation and immunopathological manifestations in several tissues. These features result from organ infiltration by overactivated CD8 T-cells and macrophages, which produce high levels of pro-inflammatory cytokines, such as IFN-γ, TNF-α, IL-6, and IL-18. Recently, several Janus kinase 1/2 (JAK1/2) inhibitors, such as ruxolitinib, have been developed as immunosuppressive agents. They have proven beneficial effects in the treatment of myeloproliferative disorders and inflammatory conditions. To determine whether pharmacological inhibition of the JAK1/2 not only prevents the onset of HLH immunopathology but also is effective against existing HLH, cytotoxicity-impaired Prf1−/− and Rab27a−/− mice with full-blown HLH syndrome were treated with a clinically relevant dose of ruxolitinib. In vivo, ruxolitinib treatment suppressed signal transducer and activator of transcription 1 activation and led to recovery from HLH manifestations in both murine models. In the Prf1−/− mice, these beneficial effects were evidenced by a greater survival rate, and in both murine models, they were evidenced by the correction of blood cytopenia and a rapid decrease in serum IL-6 and TNF-α levels. During ruxolitinib treatment, liver tissue damage receded concomitantly with a decrease in the number of infiltrating inflammatory macrophages and an increase in the number of alternatively activated macrophages. In Rab27a−/− mice, central nervous system involvement was significantly reduced by ruxolitinib therapy. Our findings demonstrate that clinically relevant doses of the JAK1/2 inhibitor ruxolitinib suppresses the harmful consequences of macrophage overactivation characterizing HLH in 2 murine models. The results could be readily translated into the clinic for the treatment of primary, and perhaps even secondary, forms of HLH.

Fasudil, a clinically safe ROCK inhibitor, decreases disease burden in a Cbl/Cbl-b deficiency-driven murine model of myeloproliferative disorders

Objectives: Mutations in Cbl or Cbl-b gene occur in 10% of myeloproliferative disorder (MPD) patients and are associated with poor prognosis. Hematopoietic Cbl/Cbl-b double knockout (DKO) leads to a disease in mice phenotypically similar to human MPDs. The aim of this study was to evaluate the anti-MPD activity of a clinically safe drug, Fasudil, identified in an in vitro kinase inhibitor as an inhibitor of proliferation of DKO mouse hematopoietic stem/progenitor cells (HSPCs).Methods: Fasudil exhibited relatively selective anti-proliferative activity against Cbl/Cbl-b DKO vs. control murine bone marrow HSPCs. We established a mouse model with uniform time of MPD onset by transplanting Cbl/Cbl-b DKO HSPCs into busulfan-conditioned NOD/SCID/gamma chain-deficient mice. Four weeks post-transplant, mice were treated with 100 mg/kg fasudil (13 mice) or water (control, 8 mice) daily by oral gavage, followed by blood cell count every 2 weeks.Results: By 2 weeks of treatment, total white cell and monocyte counts were significantly lower in mice treated with fasudil. We observed a trend towards improved survival in fasudil-treated mice that did not reach statistical significance. Notably, prolonged survival beyond 27 weeks was observed in two fasudil-treated mice, nearly twice the 16-week average life-span in the Cbl/Cbl-b DKO MPD model.Conclusions: Our results suggest a therapeutic potential for fasudil, a clinically safe drug with promising results in vascular diseases, in the treatment of MPDs or other mutant Cbl-driven myeloid disorders.

Fasudil, a Rho-Associated Protein Kinase (ROCK) Inhibitor, Decreases Disease Burden in a Cbl/Cbl-b Deficiency-Driven Murine Model for Myeloproliferative Disorders

Myeloproliferative disorders (MPDs) are heterogeneous clonal diseases with variable clinical courses. Mutations in the Cbl family genes have been reported in multiple independent studies to be present in about 10% of patients with MPDs and these patients tend of have a poorer prognosis. We have previously demonstrated that Cbl-flox/flox, Cbl-b-null mice rendered Cbl/Cbl-b double knockout (DKO) in the hematopoietic compartment with MMTV-Cre develop a disease phenotypically similar to human MPDs. An in vitro kinase inhibitor screen identified fasudil, a ROCK inhibitor, with relatively selective anti-proliferative activity against Cbl/Cbl-b DKO vs. control murine bone marrow cells. We established a mouse model with a uniform time of MPD onset by transplanting Cbl/Cbl-b DKO bone marrow cells (2x106 per recipient) into 21 busulfan-conditioned NOD/SCID/gamma chain-deficient (NSG) mice. All recipients showed donor cell engraftment. Four weeks post-transplant, we treated 13 mice with 100 mg/kg fasudil daily by gavage. By two weeks of treatment, total white cell and monocyte counts were significantly lower in mice treated with fasudil (p=0.02 and 0.04 respectively). For the entire group, we observed a trend towards improved survival in fasudil-treated mice that didn’t reach statistical significance (p=0.07). However, analysis of male recipients only (n=12) revealed a significant survival advantage in fasudil-treated group (p=0.04). The gender difference may stem from a currently unexplained more severe disease phenotype we observed in female recipients. Notably, while all untreated mice succumbed to MPD, prolonged survival was observed in 2 mice beyond 27 weeks, nearly twice the average life-span in the Cbl/Cbl-b DKO MPD model (16 weeks). The 2 long-term survivors had undetectable levels of myosin light chain (MLC), a downstream target of ROCK phosphorylation (figure) suggesting that inhibition of downstream signaling of ROCK is associated with improved disease control and survival. Taken together, our data suggest a therapeutic potential for fasudil in the treatment of MPDs or other mutant Cbl-driven myeloid disorders. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 2914: Phosphoproteomic and transcriptional biomarkers predict response to SAR302503, a JAK2 inhibitor, in human acute myeloid leukemia preclinical models

Abstract Research to develop new anti-cancer treatments has recently shifted focus to identifying and targeting molecules and pathways essential for cancer stem cell survival. However, preclinical models that rely on cell lines for drug testing do not capture the heterogeneity of response typically seen in the clinic that likely reflects the underlying genetic and functional heterogeneity of the tumors. Moreover, the use of cell lines makes it difficult to develop companion biomarker tools for patient stratification. We have taken a novel approach that combines drug testing of a large cohort of patient samples in xenograft assays, with multiplexed phosphoflow cytometric and RNA-Seq analysis of each patient sample to develop biomarkers that predict drug response. We applied this approach to study the efficacy of SAR302503 (Sanofi), a small molecule inhibitor of JAK2, against leukemia stem cells (LSCs) in acute myeloid leukemia (AML). JAK2 inhibitors have demonstrated efficacy in clinical trials for treatment of myeloproliferative disorders. Activated JAK2 signaling has been reported in AML, however it is not clear whether JAK2 inhibitors are effective in this disease, particularly against the disease-sustaining LSCs. SAR302503 treatment reduced leukemic engraftment in 22 of 34 (65%) AML patient samples of multiple subtypes with heterogeneous cytogenetic and molecular abnormalities. Phosphoflow analysis showed that AML samples that were sensitive to JAK2 inhibition in xenotransplantation assays exhibited high basal levels of pSTAT5 that were rapidly decreased by SAR302503 treatment in vitro, whereas non-responding samples showed low levels of pSTAT5, indicating that pSTAT5 is a useful drug response biomarker. This biomarker has now been validated in an independent cohort of AML patient samples. Phosphoflow cytometric profiling also enabled the rational design and testing of a novel drug combination regimen (SAR302503+Dasatinib) with efficacy against LSCs. Additionally, RNA-Seq analysis of paired vehicle- and drug-treated patient samples revealed that samples that were responsive to JAK2 inhibition in vivo had distinct transcriptional profiles compared to those that were not, suggesting that a molecular signature predictive of response to JAK2 inhibition can be identified. Overall, our approach, involving large-scale analysis of patient samples using state-of-the-art xenograft assays, captures the heterogeneity of response typically seen in the clinic that likely reflects the underlying genetic and functional heterogeneity of the tumors and offers a new paradigm for development of both novel agents that effectively target LSCs and biomarker tools to identify the patients most likely to benefit from targeted treatment. Citation Format: Weihsu Claire Chen, Julie S. Yuan, Nathan Mbong, Andreea C. Popescu, Yan Xing, Gitte Gerhard, Wei Zhang, Yussanne Ma, Richard Moore, Marco Marra, Mark D. Minden, Donna E. Hogge, Cynthia Guidos, John E. Dick, Jean C.Y. Wang. Phosphoproteomic and transcriptional biomarkers predict response to SAR302503, a JAK2 inhibitor, in human acute myeloid leukemia preclinical models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2914. doi:10.1158/1538-7445.AM2014-2914

Computational identification of JAK2 inhibitors: a combined pharmacophore mapping and molecular docking approach

The Janus-associated kinase 2 (JAK2) V617F mutation is believed to play a critical role in the pathogenesis of polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis. The discovery of activating mutations associated with inhibition of cascade of events mediated by JAK2 target became an attractive approach for the treatment of myeloproliferative disorder. In this study, we performed a ligand-based pharmacophore modeling to explore the important chemical features of JAK2 inhibitors. The top ten hypotheses were generated based on 47 known inhibitors of JAK2 using PHASE module of Schrodinger software. The best pharmacophore hypothesis was found to be AADDR.212 which consists of two acceptors, two donors and one ring aromatic group. The selected model was validated by survival score, selectivity, and GH score. Two types of validation studies were done which includes potency validation by virtual screening against set of decoys, and selectivity validation by screening against set of inhibitors of JAK1, JAK2, JAK3, and Tyk2 (all tyrosine kinase family proteins). The selected model was utilized as a 3D query to screen against ZINC natural and chemical database, and subsequently the screened compounds were filtered by applying the Lipinski’s rule of five, ADME properties and molecular docking. Finally, fifteen compounds were obtained as novel virtual hits to inhibit the JAK2 enzyme.

Pneumopathie induite par l’hydroxyurée

Hydroxyurea is an antimetabolite drug used in the treatment of myeloproliferative disorders. Common adverse effects include haematological, gastrointestinal cutaneous manifestations, and fever. Hydroxyurea-induced pneumonitis is unusual. A female patient was treated with hydroxyurea for polycythemia vera. She was admitted 20 days after commencing treatment with a high fever, productive cough, clear sputum and nausea. A chest CT-scan showed diffuse ground-glass opacities. Microbiological investigations were negative. The symptoms disappeared a few days after discontinuation of the drug and rechallenge led to a relapse of symptoms. Our case and 15 earlier cases of hydroxyurea-induced pneumonitis are reviewed. Two patterns of this disease may exist: an acute febrile form occurring within 1 month of introduction of hydroxyurea and a subacute form without fever. Even if uncommon, one should be aware of this complication of hydroxyurea.

Activation of the Janus kinase/signal transducer and activator of transcription pathway in multiple myeloma is not related to point mutations in kinase and pseudokinase domains of JAK1

Considering the recent impact of tyrosine kinase inhibitors in the treatment of myeloproliferative disorders carrying a recurrent JAK2 mutation not identified in multiple myeloma (MM), this study aimed to search for mutations in kinase and pseudokinase domains of the JAK1 gene in an attempt to define any critical and recurring change that can be used as a therapeutic target. We obtained CD138 + purified cells from 27 bone marrow aspirates of untreated MM, four normal controls and four MM cell lines. After amplification of kinase and pseudokinase domains of JAK1 in cDNA samples, the fragments were automatically sequenced. Seventy-eight percent of MM cases showed at least one polymorphism, all being synonymous single nucleotide polymorphisms (SNPs), with allele frequencies consistent with previous studies in normal European, African American and Asian populations. The four cell lines also showed only synonymous SNPs. Mutations in the kinase and pseudokinase domains of the JAK1 gene do not seem to be important for activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway because we were not able to find any recurrent mutation in a case series of 27 patients and four MM cell lines.

Abstract 907: Efficacy of SAR302503, a JAK2 inhibitor, in primary human acute myeloid leukemia xenografts.

Abstract Small molecule inhibitors of Janus kinase 2 (JAK2) such as Ruxolitinib and SAR302503 have demonstrated efficacy in clinical trials for treatment of myeloproliferative disorders. Activated JAK2 signaling has been reported in some acute myeloid leukemia (AML) samples even though JAK2 mutations are relatively rare in AML. Whether JAK2 inhibitors are effective in AML, particularly against the disease-sustaining leukemia stem cells (LSC), is not clear. We report that SAR302503 (Sanofi, Cambridge MA), an orally administered small molecule JAK2 inhibitor, shows efficacy in a xenograft model of human AML established by intrafemoral injection of primary AML cells into sublethally irradiated NOD.SCID mice. The AML samples tested were of multiple subtypes with heterogeneous cytogenetic and molecular abnormalities. Starting 2 weeks post transplantation to permit establishment of an AML graft, mice received twice daily oral gavage with 60 mg/kg SAR302503 or vehicle alone (0.5% methylcellulose) for 14 consecutive days. In 17 of 34 AML samples, SAR302503 treatment reduced leukemic engraftment in the injected femur (56-94% relative reduction, RR, compared to controls; p<0.05) as well as non-injected bones (30-95% RR; p<0.05). 5 additional samples exhibited a partial response (<50% RR in injected femur and 31-64% RR in non-injected bones, p<0.05). In preliminary serial transplantation studies, AML cells harvested from SAR302503-treated primary mice showed reduced ability to generate a leukemic graft in untreated secondary mice compared to controls, suggesting that JAK2 inhibition reduces LSC function and/or survival. Given the heterogeneous response to SAR302503 treatment observed in vivo, we carried out phosphoflow cytometric analysis of patient samples to identify biomarkers that could predict drug response. AML samples that were sensitive to JAK2 inhibition in xenotransplantation assays exhibited high basal levels of pSTAT5, often in only a small subset of cells, that were rapidly decreased by SAR302503 treatment in vitro, whereas non-responding samples showed low levels of pSTAT5, suggesting that pSTAT5 is a useful drug response biomarker. Our results demonstrate the potential of SAR302503 to target AML cells including LSCs in a broad cross section of AML patients, and warrant further studies to identify responders and non-responders and better characterize proteomic biomarkers of drug response. The approach we have taken, which focuses on large-scale analysis of primary samples using state-of-the-art xenograft assays, offers a new paradigm for preclinical drug development to identify both novel agents that effectively target LSCs and the patients most likely to benefit from targeted treatment. Citation Format: Weihsu C. Chen, Andreea C. Popescu, Yan Xing, Gitte Gerhard, Julie Yuan, Mark Minden, Cynthia Guidos, Donna E. Hogge, John E. Dick, Jean CY Wang. Efficacy of SAR302503, a JAK2 inhibitor, in primary human acute myeloid leukemia xenografts. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 907. doi:10.1158/1538-7445.AM2013-907

JAK Kinase Inhibitors as Possible Treatment for Myeloproliferative Disorders and Cancer: Patent Highlight.

JAK Kinase Inhibitors as Possible Treatment for Myeloproliferative Disorders and Cancer: Patent Highlight.

Hydroxyurea: a key player in cancer chemotherapy

Hydroxyurea (HU) is a simple organic compound currently used as a cancer chemotherapeutic agent. It acts specifically on the S-phase of the cell cycle by inhibiting the enzyme ribonucleoside diphosphate reductase, thereby hindering the reductive conversion of ribonucleotides to deoxyribonucleotides and thus limiting de novo DNA synthesis. HU is employed in hemotological settings as a first-line treatment of myeloproliferative disorders, such as polycythemia vera, essential thrombocythemia and primary myelofibrosis, apart from having a vital role in combination therapy for management of malignant melanoma, head and neck cancers and brain tumors. It offers an advantage that the patient may take this drug on an ambulatory basis with minimum clinical toxicity, while some of its limitations include gastrointestinal disturbance and bone marrow depression. This review will summarize and present the overall effects of HU and its combination therapy as an anticancer agent.

Janus kinase 2 inhibitors in myeloproliferative disorders

Importance of the field: JAK2 is an obligatory kinase for the proliferation and differentiation of erythroid cells and megakaryocytes thus representing a relevant therapeutic target for agents that specifically inhibit its activity particularly in myeloproliferative disorders (MPD) harboring JAK2V617F mutations.Areas covered in this review: We discuss the physiopathology of the JAK2 signaling pathway and review clinical trials of JAK2 inhibitors for the treatment of MPD using papers and meeting abstracts published up to September 2010.What the reader will gain: This review helps in understanding the potential role of JAK2 inhibitors in MPD clinical trials and provides a comprehensive review regarding their efficacy and safety in these disorders.Take home message: JAK2 inhibitors may prove to be useful only for suppressing disease manifestations. However, unlike drugs such as IFN which are capable of eliminating the malignant clone, JAK2 inhibitors are unable to eradicate the disease. In fact, results to date indicate that although these inhibitors reduce splenomegaly and alleviate constitutional symptoms irrespective of JAK2 mutational status, most have only a modest impact on the JAK2V617F allele burden. Considering the relevant risk of serious complications in patients undergoing splenectomy, these drugs could find a suitable indication in patients with myelofibrosis awaiting bone marrow transplantation.

Decitabine and Gemtuzumab Ozogamicin In Patients with Advanced Myelofibrosis

AbstractAbstract 5061 Background:While hypomethylating agents, azacitidine and decitabine, are approved for use in treatment of myelodysplastic syndromes (MDS), their role in treatment of myeloproliferative disorders is evolving. The modified dosing schedule of decitabine (20 mg/m2 IV for 5 days) in MDS has shown overall response rates of approximately 40%. A clinical study of azacitidine in myelofibrosis (MF) has shown a response rate of 32%. We investigated the activity of the combination of decitabine and gemtuzumab ozogamicin (GO) (anti-CD33 antibody), a combination with activity in patients with MDS and acute myelogenous leukemia (AML), in patients with advanced myelofibrosis. Patients and Method:We reviewed the records of patients with MF treated with the combination of decitabine and GO. Result:Seven patients were treated till the decisions by FDA to withdraw GO from the market. Age ranged from 60–69 years (median 65 years), 5 patients were male and no of prior therapies ranged 0–4 (median2). Three patients had MF progressed to acute myeloid leukemia. Prior treatments included, hydroxyura, thalidomide, prednisone, pomolidamide etc. All patients were positive for JAK 2 V617F mutation. Cytogenetic abnormalities were seen in 4 patients (hyperdiploid, complex or -5/-7) and 3 were with diploid cytogenetics. Five patients received 3 cycles of Decitabine plus myelotarg while 2 patients had only one cycle. Four patients had stable disease, one had clearance of marrow blasts (40%>0%), two had no response. Three patients showed decreases in splenic size. Clinically significant infectious complications were encountered in 4 patients. Conclusions:The combination of decitabine and GO showed early signs of activity in patients with MF but future investigation of this combination will be limited due to lack of access to GO. Disclosures:Borthakur:eisai: Research Funding.

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

AbstractAbstract 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. [Display omitted] Disclosures:Ma:Eli Lilly and Company: Employment. Walgren:Eli Lilly and Company: Employment.

ChemInform Abstract: The Role of Interferon-Alpha in the Treatment of Myeloproliferative Disorders

The interferons are cytokines with a wide array of biological properties. In hematological malignancies the most used IFN class is -alpha; it has been used for thirty years but the mode of action is still not absolutely clear. Nevertheless, the benefits of IFN-alpha for the treatment of CMD have been described in particular for CML and less for PV, ET and MMM. IFN-alpha is presently considered the golden standard of therapy for CML patients not eligible for SCT; the antileukemic effect has been well documented by hematological and cytogenetic response. The survival advantage for IFN treated patients is remarkable in comparison with patients treated with conventional chemotherapy. Recently, the combination IFN-alpha plus Ara-C has demonstrated to increase the rate of major cytogenetic response and to prolong survival. To date, there is not a generally accepted treatment for ET, PV and MMM, which can reduce the risk of thromboembolism and/or hemorragic events. In several subsets of ET and PV patients, IFN-alpha can be considered as first line therapy. IFN-alpha is usually associated with the development of early and later side effects, that reduce the enthusiasm for its use. In the future PEG-IFN-alpha would improve the quality of life of IFN-treated CMD patients.

The current status and the future of JAK2 inhibitors for the treatment of myeloproliferative diseases

Janus kinases (JAKs) are critical components of cytokine signaling pathways which regulate immunity, inflammation, hematopoiesis, growth, and development. The recent discovery of JAK2-activating mutations as a causal event in the majority of patients with Philadelphia chromosome negative (Ph-) myeloproliferative disorders (MPDs) prompted many pharmaceutical companies to develop JAK2-selective inhibitors for the treatment of MPDs. JAK2 inhibitors effectively reduce JAK2-driven phosphorylation of signal transducer and activator of transcription 5, and cell proliferation and cell survival in JAK2-activated cells in vitro and in vivo. Most inhibitors are currently being evaluated in patients with one form of MPD, myelofibrosis. Patients treated with these inhibitors experienced a rapid reduction of splenomegaly, significant improvement of constitutional symptoms, and increased daily activity with few adverse events. A partial reduction of JAK2V617F disease burden during the treatment with JAK2 inhibitors was also observed. The inhibitors appear to have a therapeutic benefit in the treatment of these disorders. The results of ongoing clinical trials will allow further evaluation of clinical benefits and safety of these compounds. In this review, the authors summarize the status of JAK2 inhibitors in development and discuss their benefits and challenges.

Vorinostat interferes with Wnt and NF-κB pathways in the M-07e cell line

The wingless-type (Wnt) signalling pathway is involved in leukemogenesis and the glycogen-synthase kinase 3 has been reported to control both -catenin and nuclear factor (NF)-B.1 Thus, if NF-B and Wnt pathways are really controlled by common genes, drugs able to modulate one or both of these signals would represent innovative therapeutic tools in treatment of myeloproliferative disorders.

Treatment of Progression of Myeloproliferative Disorders(MPD) to MDS/AML by Azacytidine (AZA) : A Preliminary Report on 17 Patients (pts)

Background: MDS or AML occurring during the course of MPD carry a very poor prognosis and current treatments, with the exception of allogeneic SCT, have very limited efficacy. AZA significantly improves survival in higher risk MDS (ASH 2007, abstr n° 817) and in some AML, but its use has not been extensively reported so far in MDS and AML post MPD.Methods: The French health agency (AFSSAPS) designed a still ongoing pt named program (ATU) of AZA in higher risk MDS and poor risk AML. 17 pts with MDS or AML post MPD, included in this program in 5 centers (Paris-Avicenne, Paris St Louis, Paris-Cochin, Tours and Toulouse), before April 08 and having completed ≥ 1 cycle of AZA (75 mg/m2/d during 7 days per 28 d cycle) are analysed here. Response was evaluated according to IWG 2006 criteria for MDS and IWG-AML 2003 criteria for AML.Results: median age was 68y (range 38–83), M/F: 7/10. The initial MPD was Polycythemia Vera (PV) in 6 pts, Essential Thrombocytopenia (ET) in 7 pts, primary myelofibrosis (MF) in 2 pts and unclassified in 2 pts. Median interval from diagnosis to progression was 10 years (range 7 months-22 years). 10/16 (62%) pts had JAK2-V617F mutation. Treatment of MPD had consisted of HU in 7 pts, pipobroman in 4 pts, both in 4 pts, imatinib in 1 pt and Interferon Alfa in 1 pt. At the time of inclusion, WHO classification was AML in 8 pts, and MDS in 9 pts (RAEB-2 in 8pts, RAEB-1 in 1pt) Karyotype was normal (n=2), isolated −7/7q- (n=3), isolated del 5q (n=1), + 8 (n=2), isolated −17 (n=1), t[1;7] (n=2), complex (n=3) including −7 (3), −5(2) and +8(1), and a failure (n=3). Before AZA, 2 pts had received intensive chemotherapy (1 CR, 1 failure) followed by allo SCT in 1 of them (who subsequently relapsed).The median number of cycles of AZA administered was 4 (range 1–9). 2 pts received only 1 cycle due to early death (from aspergillosis and unknown cause). The overall response rate (ORR, including CR, PR, CRi) was 10/17 (59%). In the 15 pts who received more than 1 cycle, 4/8 MDS achieved CR (including one cytogenetic CR) and 2/8 achieved PR. RBC and platelet transfusion independence were achieved in 4/8 and 6/8 respectively (resp). In AML, 1/7 pts achieved CR and 3/7 achieved CRi. 2 of the 10 responders relapsed (after 5 and 12 months resp) and the other 8 remained responders after 1+ to 12 + months (median 3.5). 9/10 responders were still alive after 2 to 16 months (median 5.5). Interestingly, in 3 pts with a very long history of MPD (15, 16 and 22 years) treated with HU or pipobroman, who at progression had MDS and cytopenias, response to AZA was associated to recurrence of MPD (2 TE and 1 PV) requiring restart of cytoreductive therapy in 2 of them.Conclusion: 5 AZA gives encouraging results in MDS or AML occurring in the course of MPD with an overall response rate of 59%. An update, with cases recently included in the program, will be presented.