• admin | April 30, 2021


    Benjamin Ebert, MD & Ross L. Levine, MD

    Harvard Medical School & Memorial Sloan-Kettering Cancer Center
    Project Title: “Whole Genome Sequencing to Identify Germline and Somatic Disease Alleles Which Contribute to MPD Pathogenesis

    Two central goals for elucidating the biology of myeloproliferative disease are the characterization of the full complement of somatic mutations that cause MPD, and the identification inherited alleles that predispose individuals to the development of MPD. The improved technologies, decreasing costs, and computational tools for whole-genome and whole-exome sequencing promise to transform both of these endeavors. We propose to identify genes with somatic mutations in MPD by sequencing neutrophils and matched normal DNA from the same patients to identify somatically acquired mutations. To discern driver from passenger mutations, we will sequence all candidate genes in a larger set of 150 patient samples. To identify inherited predisposition alleles, we will perform genome sequencing on a set of Ashkenazi Jewish families with familial MPD, and we will examine any identified alleles in a set of 150 individuals from 20 pedigrees. We will functionally validate disease genes in murine models, leveraging a suite of models generating in the labs of the co-PI’s bearing, respectively, the JAK2V617F mutation, the MPL515L mutation, TET2loss, and ASXL1 loss. A more complete genetic understanding of MPD promises to lead to insights into the biology of the disease and the identification of novel therapeutic targets.

    Robert Kralovics, PhD

    University of Vienna, Austria
    “Deciphering the genetic complexity of myeloproliferative disorders”

    Despite the considerable increase in knowledge of the genetic basis of MPD, over one third of MPD patients remain negative for any of the known mutations. The JAK2-V617F-negative and MPL-negative MPDs are the main focus group of this proposal. In Specific aim 1, we aim at identification of somatic mutations in JAK2-V617F-negative and MPL-negative patients by in depth analysis of the entire gene-coding genome. We will apply “next-generation” (or “deep”) sequencing technology to detect mutations specifically in cells derived from the MPD stem cell clone, and after functional validation, analyze the mutations for their clinical relevance in large number of MPD patients. In Specific aim 2, we will focus on genetic lesions associated with leukemic transformation of MPD. We previously identified defects targeting the p53 and JAK/STAT pathways and we plan to investigate their role in a multicenter study. We plan to closely study two transcription factors (CUX1, IKZF1) frequently deleted in post-MPD leukemia. We also aim at finding novel leukemia associated lesion by deep sequencing of the genome of transformed MPD patients. Novel diagnostic markers, therapeutic targets, and prognostic markers for post-MPD leukemia are expected to emerge from the results of the proposed studies.

    Shaoguang Li, MD, PhD

    University of Massachusetts
    “Identification of Alox5 as a potential target gene for the treatment of Polycythemia Vera”

    Polycythemia vera (PV) is one major form of myeloproliferative diseases (MPDs) and is a stem cell-derived clonal disorder. Recent discovery of the JAK2V617F mutation in approximately 100% of PV patients promoted the development of targeted therapy of this MPD. Besides developing JAK2 inhibitors, we believe that it will be critical to fully understand how JAK2V617F signals in order to identify key downstream targets of JAK2V617F for PV therapy. Toward this goal, we identified a novel gene Alox5 functioning downstream of JAK2V617F. Our preliminary data show that loss of Alox5 impedes the development of JAK2V617F-induced PV in mice, that JAK2V617F activates Alox5, and that inhibition of the function of Alox5 by a PDA-approved anti-asthma drug Zileuton improves the disease situation in mice. Based on these findings, we propose to investigate the functional relationship between JAK2V617F and Alox5, and to further test whether Alox5 serves as an effective target gene for PV using mouse PV model and human PV cells. The proposed studies in this application will help to build a solid foundation for treating human PV by inhibiting Alox5 and related pathways in future clinical trials.

    Saghi Ghaffari, MD, PhD

    Mt. Sinai School of Medicine
    “Understanding Molecular Mechanisms of Regulation of Myeloproliferative Disorders in Mouse and Human”

    Identification of transcription factors that are downstream targets of Myeloproliferative disorders (MPDs) signaling pathways will be an important step for success of targeted therapy. We have recently shown that loss of FoxO3 Forkhead transcription factor causes a myeloproliferative syndrome with increased accumulation of reactive oxygen species (ROS) in primitive hematopoietic progenitors leading to relative deficiency of Lnk, a negative regulator of cytokine receptor signaling that mediates the overactivation of intracellular signaling through the AKT/mTOR signaling pathway. In vivo scavenging of ROS corrects these biochemical abnormalities and relieves the myeloproliferation. Based on these studies and other findings we hypothesize that (a) increased ROS specifically enhances myeloid expansion of MPDs (b) that may be mediated by abnormal regulation of FoxO (in particular FoxO3) in primitive hematopoietic stem/progenitor compartment of MPDs. To test this hypothesis, we propose to investigate (1) whether FoxO (FOXO3a) regulation of ROS-LnkmTOR is involved in the pathogenesis of human polycythemia vera and (2) how FoxO are regulated in hematopoietic stem/progenitors; what factors may cooperate with FoxO to induce progression to leukemia in MPDs. We believe findings generated by this proposal will provide valuable information regarding our understanding of MPDs myeloid expansion and its treatment.

    Toshiaki Kawakami, MD, PhD

    La Jolla Institute for Allergy and Immunology
    “Project Title: SPS Complex in MPD”

    We recently found that phospholipase C (PLC)-β3 is a novel tumor suppressor. PLC-β3-deficient mice develop myeloproliferative disease (MPD) and lymphoma. The mutant mice have increased numbers of hematopoietic stem cells with increased proliferative, survival, and myeloid-differentiative abilities. These properties are dependent on increased activity of the transcription factor Stat5 and can be antagonized by the tyrosine phosphatase SHP-1. The C-terminal non-catalytic domain of PLC-β3 directly interacts with SHP-1 and Stat5 to form the multimolecular SPS complex and to facilitate Stat5 dephosphorylation by SHP-1. Stat5-dependent transformation mechanism caused by PLC-β3 deficiency seems to be operative in human MPD, as our preliminary data indicate that a subset of human MPD patients have drastically reduced levels of PLC-β3 expression and high levels of STAT5 phosphorylation. In this project, we will investigate the possibility that Lyn (Src family kinase) might interact with the SPS complex and act as a regulator of SHP-1 phosphatase activity in both in vitro and in vivo settings. Since the JAK2V617F mutation is found in many MPD patients, we will investigate the effect of PLC-β3 downregulation on JAK2V617F-induced MPD in mice. This study will reveal novel mechanisms for MPD pathogenesis and identify novel therapeutic targets for MPD treatment.

    Wei Tong, PhD

    University of Pennsylvania School of Medicine
    “K63 ubiquitination in JAK2 Signaling and Myeloproliferative Neoplasms”

    JAK2 is a central kinase for cytokine signaling and hematopoiesis. Constitutive active mutation, JAK2V617F, is found frequently in myeloproliferative neoplasms (MPNs). We previously identified the adaptor protein Lnk as a key negative regulator of JAK2 in hematopoietic stem and progenitor cells. We further demonstrated that loss of Lnk accelerates and exacerbates oncogenic JAK2-induced MPNs in mice. Importantly, loss-of-function mutations in Lnk are found in MPN patients with elevated STAT signaling. To explore mechanisms of Lnk functions we identified novel Lnk interacting proteins, one of which is BRISC. BRISC (Brcc36 isopeptidase complex) is a deubiquitinating enzyme complex that specifically removes K63 ubiquitin (Ub) conjugates. Unlike K48-Ub chains that are the principal targeting signal for proteosomal degradation, K63-Ub mediates non-degradative events such as signal transduction. Our preliminary data demonstrates that JAK2 is K63- ubiquitinated; therefore we aim to test the hypothesis that Lnk docks BRISC to JAK2 thereby modulating JAK2 K63-ubiquitination and activation. We will also assess the role of BRISC and JAK2 K63-ubiquitination in JAK2VFmediated cell proliferation, signaling, and MPN development in vivo. Thus, our studies implicate previously unappreciated post-translational modifications of Lnk and JAK2 that represent a novel regulatory mechanism of JAK2 signaling in hematopoiesis and MPNs.


    Alison Moliterno, MD

    Johns Hopkins
    “Proteomic Approach to the Diagnosis of Chronic MPD’s.”

    The long-term goal of this project is to define the protein signature of platelets in patients with Chronic MPD’s. By comparing different profiles we hope to identify characteristics associated with each particular disease, clotting risk, or disease transformation. Platelet proteins are easily obtained from patients in a clinical setting.

    Mingjiang Xu, MD, PhD

    University of Illinois at Chicago
    “Exploration of a unique phosphatase as a potential therapeutic target for treatment of PV.”

    Investigation of abnormal functioning of protein tyrosine phosphates(PTP) patients which is activated by PTP-MEG2. Investigate the role of PTP-MEG2 and whether it can serve as a good target for drugs which can be used to treat PV. This study would target a potential molecular mechanism underlying the pathobiology of PV.

    Jose Lopez, MD

    Baylor College of Medicine
    “Macrophage-derived Prothrombotic Microparticles and Thrombosis in Myeloproliferative Disorders.”

    One of the most striking and unexplained features of MPDs are clots at unusual sites, such as in the veins coming out of the liver. Because platelets arise from the abnormal cells causing the MPDs, they have been blamed for the abnormal clotting. However, platelets normally do not participate in the formation of clots in veins. We propose that the clots observed in patients suffering from MPDs are caused by the production of a clotting factor released from cells in the liver, called Kupffer cells. Because Kupffer cells derive from blood cells, they too are affected by mutations causing the MPDs. We hypothesize that these mutations render Kupffer cells hyper-reactive to compounds present in very high concentrations in the blood arriving from the gut. These compounds stimulate Kupffer cells to produce large amounts of the clotting factor, which may then produce clots in the veins coming out of the liver, as well as in other parts of the body. We expect this work to shed light on the causes of excessive clotting patients with MPDs and to lead to improvements in its therapy.


    Xiao-Feng Yang, MD, PhD

    Baylor College of Medicine
    “Novel Antigen targets for Immunotherapy in the Myeloproliferative Diseases”

    Polycythemia Vera (PV) is a malignant blood cell disease similar to chronic myeloid leukemia (CML). Despite progress, PV therapeutics still needs to be improved. Good responses to the drug interon-a (IFN-a) indicate that host immune system plays an important role in controlling PV. Our proposal will investigate, in the context of IFN-a therapy, the hypothesis that PV tumor cells express a set of specific proteins molecules (tumor antigens) that are capable of eliciting immune responses leading to remission of PV, which may be similar to, if not the same as, the antigens that mediate anti-tumor immunity in other MPD like CML. We have found two new tumor antigens called CML66 and CML28. These two antigens cannot be found in normal tissues but can be easily found in a variety of tumors suggesting they are tumor-specific. The goal of this proposal focuses on the potential for novel tumor antigens and other potential PV-associated antigens to serve as targets for immunotherapy of PV and other blood tumors.

    Ruben Mesa, MD

    Mayo Clinic, Rochester
    “Novel therapies for Myelofibrosis with Myeloid Metaplasia”

    Myelofibrosis with myeloid metaplasia (MMM) is a bone marrow stem cell disease that results in anemia, splenic enlargement, fatigue, acute leukemia, and death. There is no curative or palliative therapy for MMM, nor is there tissue culture or animal model for investigation of new agents. We have previously examined the effects of potential new therapies on hematopoietic progenitors from MMM patients in vitro. Based on these studies, a Phase II multi-center clinical trial of R115777 in MMM is ongoing. We now propose a three-pronged approach to identify potential new therapies for the disease. First, we will screen a spectrum of investigational drugs to identify agents that might be appropriate for future clinical trials. Second, we will investigate the reason a novel drug, 17-allylamino-17-demethoxygeldanamycin (17-AAG), is effective in killing MMM-derived cells in vitro. Finally, we will use the new techniques of “proteonimcs” (a method of studying all of the proteins in a cell at once) to identify proteins that are uniquely increased or decreased in MMM cells compared to normal counterparts to gain further insight into MMM pathogenesis. Support of this project will hopefully lead directly to clinical trials of new and targeted therapies for MMM patients.

    Richard D’Andrea, MD

    Child Health Research Institute
    “Identification of Growth Factor Receptor Mutations in Polycythemia Vera”

    Polycythemia vera (PV) is a mature onset, myeloproliferative disorder, characterized by increased numbers of red blood cells and white blood cells. In PV, blood precursor cells in the bone marrow are hypersensitive to several growth factors or hormones, a mechanism thought to contribute to their increased growth. We have previously shown that alteration to a cell surface receptor for on of these growth factors can lead to similar altered growth factor responses and to a PV-like disease in mouse models. We have been screening PV patients for genetic alterations in the gene for this receptor and have detected a sequence change within a critical region of the gene. To investigate this further, we propose to screen larger numbers of PV patients and normal subjects for the presence of this altered sequence to determine its importance for the disease. We then have several assays in which to test the receptor responses affected by this change. In a second approach to identifying other genetic changes that contribute to PV, we propose to use a novel cloning technique to isolate genes from a PV patient that confer altered growth factor responses on normal cells.

    Vahid Afshar-Kharghan, MD

    Baylor College of Medicine
    “Continuation of 2002 Grant”

    One of the most important complications of myeloproliferative disorders is the organ damage caused by occlusion of blood vessels such as stroke or heart attack. Bleeding is also a common problem among patients with myeloproliferative disorders. Currently, we cannot predict which patient will develop a thrombotic (occlusion of blood flow) or hemorrhagic complication. The number and function of blood cells (including platelets) are abnormal in patients with myeloproliferative disorders. Platelets play an important role in the formation of blood clots and cessation of bleeding in normal individuals. In patients with myeloproliferative disorders, in addition to having an abnormal platelet count, platelet function is frequently altered. The change in platelet function is not similar among all patients with myeloproliferative disorders. Some of these patients have platelets that have less activity compared to normal platelets (hypoactive), while others have platelets that are hyperactive. We propose that the level of platelet activity determines which patients are at a higher risk of thrombosis and which ones are at risk of bleeding complication. We will identify the inherited and acquired factors that modulate platelet function in patients with myeloproliferative disorders, and will try to determine new risk factors that can be used to predict the risk of developing thrombotic or bleeding problems.


    Dr. Josef Prchal

    Baylor College of Medicine
    “Continuation of 2001 Grant”

    The principal goal of the Research Proposal is to find the cause of Essential Thrombocythemia (ET) by identifying the gene(s) and its (their) mutation(s) that cause Essential Thrombocythemia (ET.) Additionally, we will attempt to identify the mutations of genes causing congenital thrombocythemia. We believe that successful fulfillment of this goal is an essential prerequisite for a) accurate and rapid diagnosis of ET, b) design of rational specific therapeutic strategies, and a) eventual cure of ET.


    Dr. Josef Prchal

    Baylor College of Medicine
    “Continuation of 2000 Grant”


    Dr. Josef Prchal

    Baylor College of Medicine
    “Locate and identify the gene or genes that lead to the development of the Polycythemia Vera phenotype.”

    Polycythemia Vera (PV) is an acquired clonal myeloproliferative disorder. Our ultimate aim in identification of the gene or genes that lead to the development of PV phenotype – an essential requirement for the development of specific PV therapy and ultimate PV cure. Based on our on-going evaluation of the accumulating data, it appears that the first acquired mutation is a loss of inhibitory function by another genetic event(s) necessary for full PV (and ET) phenotype. There is frequent overlap of PV and ET. Some PV Patients first present with thrombocytosis without elevation of red cell mass that may precede by even several years the development of full PV phenotype; yet these subjects already have a PV specific defect, i.e. the presence of EPO independent crythroid progenitors when tested in vitro in the presence of scrum. We believe that the delineation of ET lesion will help to clarify the clinical complexity of PV phenotype.