Pathogenesis Of Myeloproliferative Disorders Research Articles

Frequency of <i>L1721W</i> Polymorphism in <i>TET2</i> Gene Among a Cohort of Sudanese Patients with Myeloproliferative Disorders: Possible Roles in Pathogenicity and Leukemic Transformation

Transformation of myeloproliferative disorders (MPDs) to acute leukemia is an evitable event that represents a stumbling block in the management of patients. The Janus Kinase-2 JAK2V617F mutation of MDP does not clarify the phenotypic variability observed in this disorder. But, a mutations in Ten-eleven-translocation-2 (TET2), a putative tumor suppressor gene, was recently implicated in MPDs and other hematologic malignancies. TET-2 is believed to play a role in leukemic transformation. This study aimed to determine the frequency of L1721W polymorphism in TET2 gene in a cohort of Sudanese patients with MPDs. Following informed consent, 25 (25/50, 50%) patients with polycythemia rubra vera (PRV), thirteen patients (13/50, 26%) with essential thrombocythemia (ET), eleven patients (11/50, 22%) with chronic myeloid leukemia (CML), and one patient (1/50, 2%) with primary myelofibrosis (PMF) were enrolled. None of the patients was in the transformation phase. Patients were diagnosed based on clinical picture, hematological parameters and JAK2V617F and BCR_ABL molecular aberrations. JAK2V617F was detected in Ph-negative-MPDs cases as (24/25, 96%) in PRV, (10/13, 76%) in ET, and (1/1, 100%) in PMF. BCR_ABL fusion was detected in all (11/11, 100%) cases of CML. DNA was extracted using the guanidine chloride method, followed by (PCR-RFLP) analysis. Only one patient showed the presence of L1721W polymorphism of the TET2. It was inferred that the low frequency of this transformation within the study cohort [all in chronic phase] probably indicates that it plays a minor role in MPD pathogenesis, while its role in blast transformation needs further studies in MPD patients.

Pathogenesis of Myeloproliferative Disorders.

Myeloproliferative neoplasms (MPNs) are a set of chronic hematopoietic neoplasms with overlapping clinical and molecular features. Recent years have witnessed considerable advances in our understanding of their pathogenetic basis. Due to their protracted clinical course, the evolution to advanced hematological malignancies, and the accessibility of neoplastic tissue, the study of MPNs has provided a window into the earliest stages of tumorigenesis. With the discovery of mutations in CALR, the majority of MPN patients now bear an identifiable marker of clonal disease; however, the mechanism by which mutated CALR perturbs megakaryopoiesis is currently unresolved. We are beginning to understand better the role of JAK2(V617F) homozygosity, the function of comutations in epigenetic regulators and spliceosome components, and how these mutations cooperate with JAK2(V617F) to modulate MPN phenotype.

Molecular pathogenesis of myeloproliferative disorders

Molecular pathogenesis of myeloproliferative disorders

Homeostasis of hematopoietic stem cells regulated by the myeloproliferative disease associated-gene product Lnk/Sh2b3 via Bcl-xL

Hematopoietic stem cells (HSCs) are maintained at a very low frequency in adult bone marrow under steady-state conditions. However, it is not fully understood how homeostasis of bone marrow HSCs is maintained. We attempted to identify a key molecule involved in the regulation of HSC numbers, a factor that, in the absence of Lnk, leads to HSC expansion. Here, we demonstrate that upon stimulation with thrombopoietin, expression of Bcl-xL, an antiapoptotic protein, was highly enhanced in Lnk-deficient HSCs compared to normal HSCs. As a result, Lnk-deficient HSCs underwent reduced apoptosis following exposure to lethal radiation. Downregulation of Bcl-xL expression in Lnk-deficient HSCs by short-hairpin RNA resulted in a great reduction of their capacity for reconstitution. These findings suggest that Lnk/Sh2b3 constrains the expression of Bcl-xL and that the loss of Lnk/Sh2b3 function enhances survival of HSCs by inhibiting apoptosis. Furthermore, our observations indicate that HSCs in patients with an Lnk/Sh2b3 mutation might become resistant to apoptosis due to thrombopoietin-mediated enhanced expression of Bcl-xL. Consequently, reduced apoptosis could facilitate accumulation of HSCs with oncogenic mutations leading to development of myeloproliferative disorders.

Acute Budd Chiari and Underlying JAK 2 Mutation Requiring TIPS Placement

Purpose: The JAK2V617F mutation is strongly implicated in the pathogenesis of myeloproliferative disorders (MPD) which account for at least 50% of cases of Budd-Chiari Syndrome (BCS). MPD is a common cause of splanchnic vein thrombosis in young women. Patients with BCS and underlying JAK2 mutation have severe hepatic outflow obstruction with worse Child-Pugh scores, and often require decompressive procedures such as transjugular intrahepatic portosystemic shunts (TIPS) placement or liver transplantation compared to those without the mutation. A 19 year old Caucasian woman with chronic abdominal pain, nausea and vomiting was found to have splenic vein thrombus on ultrasound. An initial hypercoagulable workup was negative. She was instructed to discontinue her oral contraceptives and started on anticoagulation with warfarin. Imaging studies two months later showed resolution of the thrombus. She was noncompliant with her anticoagulation and presented six months later with similar symptoms. A repeat ultrasound of the abdomen showed thrombosis of two of her hepatic veins, right portal vein and splenic vein. She was immediately restarted on anticoagulation. She was noted to have a mildly elevated ALT in the 50-60 range but her other liver function tests remained normal. She also had a normal CBC except for mild anemia and a normal INR. Further workup revealed a positive JAK2 mutation. She was admitted one month later with worsening abdominal pain, new large ascites and occlusion of all three hepatic veins despite anticoagulation. She was diagnosed with acute BCS. Hepatic venography demonstrated “spider web” collaterals of the right and left hepatic veins consistent with BCS. She underwent a large volume paracentesis and successful TIPS placement with decrease in her hepatic venous pressure gradient from 27 to 5 mm Hg. Liver biopsy showed evidence of moderate to severe sinusoidal dilation in zone 3 consistent with BCS. She recovered well with resolution of her ascites and abdominal pain. She was discharged home with the anticipation of lifelong anticoagulation. Patients with newly diagnosed BCS should be routinely screened for JAK2 mutations. Unlike the majority of patients with MPD, this subgroup of patients have normal hemoglobin levels, platelet counts and erythropoietin levels making diagnosis difficult with laboratory values alone. These patients present with severe hepatic outflow obstruction which portends a worse clinical outcome, and thus should be aggressively intervened upon at time of presentation to ensure long-term survival.

Differential expression of apoptosis-related genes from death receptor pathway in chronic myeloproliferative diseases

BackgroundChronic myeloproliferative disorders (MPDs) are clonal haematopoietic stem cell malignancies characterised by an accumulation of mature myeloid cells in bone marrow and peripheral blood. Deregulation of the apoptotic machinery may...

The analysis of JAK2 and MPL mutations and JAK2 single nucleotide polymorphisms in MPN patients by MassARRAY assay

Recent studies have shown that JAK2 V617F, MPL W515L/K and JAK2 exon 12 mutations underlie the major molecular pathogenesis of myeloproliferative disorders (MPN). Allele-Specific Polymerase Chain Reaction (AS-PCR), direct sequencing and MassARRAY assay were used to ascertain the real prevalence of these mutations and the influence of genetic susceptibility in Chinese MPN patients. The positive rate of JAK2 V617F in polycythaemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF) was 82.0%, 36.6% and 51.1% respectively. One ET patient and two PMF patients harboured the MPL W515L mutation and three PV patients harboured JAK2 exon 12 mutations. All of these patients were confirmed as JAK2 V617F negative. Clinical data demonstrated that PV patients with JAK2 exon 12 mutations were younger, had higher haemoglobin levels and white blood cell counts than PV patients with JAK2 V617F. In addition, through analysis of 4 polymorphic loci of JAK2 gene, no significant difference of distribution frequency was found among PV, ET and PMF patients. Distribution frequency of haplotype also was not significantly different among PV, ET and PMF patients. We conclude that JAK2 V617F is a major molecular pathogenesis in Chinese MPN patients. MPL W515L mutation and JAK2 exon 12 mutations can also be found in JAK2 V617F negative MPN patients.

Human-induced pluripotent stem cells from blood cells of healthy donors and patients with acquired blood disorders

AbstractHuman induced pluripotent stem (iPS) cells derived from somatic cells hold promise to develop novel patient-specific cell therapies and research models for inherited and acquired diseases. We and others previously reprogrammed human adherent cells, such as postnatal fibroblasts to iPS cells, which resemble adherent embryonic stem cells. Here we report derivation of iPS cells from postnatal human blood cells and the potential of these pluripotent cells for disease modeling. Multiple human iPS cell lines were generated from previously frozen cord blood or adult CD34+ cells of healthy donors, and could be redirected to hematopoietic differentiation. Multiple iPS cell lines were also generated from peripheral blood CD34+ cells of 2 patients with myeloproliferative disorders (MPDs) who acquired the JAK2-V617F somatic mutation in their blood cells. The MPD-derived iPS cells containing the mutation appeared normal in phenotypes, karyotype, and pluripotency. After directed hematopoietic differentiation, the MPD-iPS cell-derived hematopoietic progenitor (CD34+CD45+) cells showed the increased erythropoiesis and gene expression of specific genes, recapitulating features of the primary CD34+ cells of the corresponding patient from whom the iPS cells were derived. These iPS cells provide a renewable cell source and a prospective hematopoiesis model for investigating MPD pathogenesis.

Conditional Expression of Heterozygous or Homozygous Jak2V617F From Its Endogenous Promoter Induces a Polycythemia Vera-Like Disease.

Abstract 437A somatic point mutation (V617F) in the JAK2 tyrosine kinase was found in a majority of patients with polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). However, the contribution of JAK2V617F in these three clinically distinct myeloproliferative disorders (MPDs) remained unclear. To investigate the actual role of JAK2V617F in the pathogenesis of MPDs, we generated an inducible Jak2V617F knock-in mouse, in which the expression of Jak2V617F is under control of the endogenous Jak2 promoter. Expression of heterozygous Jak2V617F evoked all major features of human PV, which included marked increase in hemoglobin and hematocrit, increased red blood cells, leukocytosis, thrombocytosis, splenomegaly, reduced serum levels of erythropoietin (Epo) and Epo-independent erythroid colonies. Homozygous Jak2V617F expression resulted in a more severe form of PV associated with markedly elevated leukocytosis, neutrophilia and thrombocytosis, and a majority of these mice succumbed due to massive cardiac thrombosis. Activation of Stat5, Akt and Erk was significantly enhanced in erythroblast cells expressing homozygous Jak2V617F compared to heterozygous Jak2V617F, suggesting that the degree of activation of downstream signaling pathways would be affected by the Jak2V617F gene dosage. This may also partly explain the severe phenotype in mice expressing homozygous Jak2V617F. We conclude that heterozygous Jak2V617F is sufficient to cause PV, whereas homozygous Jak2V617F increases the severity of PV disease and the risk of thrombosis. Our results also provide strong evidence that Jak2V617F gene dosage does not play a defining role in determining PV versus ET phenotype in this mouse model of MPD. Disclosures:No relevant conflicts of interest to declare.

Hematopoiesis of Human Induced Pluripotent Stem Cells Derived From Blood Cells of Healthy Donors and Patients with the JAK2-V617F Somatic Mutation.

Abstract 812Human induced pluripotent stem (iPS) cells derived from somatic cells hold promise to develop novel patient-specific cell therapies and research models for inherited and acquired diseases. We and others previously reprogrammed human adherent cells such as postnatal fibroblasts to iPS cells that resemble adherent human embryonic stem (hES) cells. It is also highly desirable to reprogram blood cells that are easily accessible and less exposed to environmental mutagens. The large numbers of umbilical cord blood (CB) cells that are collected and stored in multiple cell banks are examples that could be used as a source of either autologous or allogeneic but histo-compatible iPS cell lines. As in vitro expansion of hematopoietic stem/progenitor cells from CB and adult sources remains a challenge, unlimited expansion of derived iPS cells in combination with further optimized hematopoietic differentiation methods should provide a vital alternative to amplify histo-compatible blood stem cells for blood/BM transplantation purposes. More critically, the ability to reprogram blood cells is essential if one wishes to generate iPS cells containing somatic mutations that are restricted to the blood cells and found in acquired hematological disorders, such as myeloproliferative disorders (MPDs), in order to investigate their pathogenesis. The BCR/ABL-negative MPDs, which include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), are a heterogeneous group of diseases characterized by increased proliferation of erythroid, megakaryocytic and myeloid lineages alone or in combination. The acquired common somatic mutation JAK2-V617F is present in >95% of PV, and ∼50% of ET and PMF patients. The discovery of this mutation in 2005 has significantly improved our understanding of MPD mechanisms and has intensified the search for drug(s) that may effectively inhibit this aberrant kinase activation. However, new disease models are needed to answer questions it raised such as how gene dosages of JAK2-V617F and other pre-disposing mutations affect the MPD pathogenesis. Here we report the derivation of iPS cells from postnatal human blood cells and the potential of these pluripotent cells for hematopoietic differentiation and disease modeling. Multiple human iPS cell lines were generated from previously frozen cord blood, adult peripheral blood and marrow CD34+ cells of healthy donors. The hematopoietic differentiation potential of these human iPS cells was examined by an improved method of EB formation and differentiation under a feeder- and serum-free condition. After two weeks of treatment, the cells were harvested and analyzed by both hematopoietic colony-forming assays and FACS for the presence of hematopoietic markers. Both myeloid and erythroid colonies were detected as we have observed using hES cells. By FACS analysis, CD45+ (27%-64%) and CD43+ (36%-60%) hematopoietic cells co-expressing undetectable to intermediate levels of CD34 marker were also observed. Multiple iPS cell lines were also generated from peripheral blood CD34+ cells of two patients with myeloproliferative disorders (MPDs) who acquired the JAK2-V617F somatic mutation in their blood cells. The MPD-derived iPS cells containing the mutation appeared normal in phenotypes, karyotype and pluripotency. To determine if these MPD iPS cell lines could be used as a model to study abnormal human hematopoiesis, we used the same serum-free differentiation protocol to direct them into hematopoietic lineages. Similar to the increased erythropoiesis of hematopoietic progenitor (CD34+) cells isolated from PV patients, including one subject whose blood-derived iPS cells were used in this study, re-differentiated hematopoietic progenitor (CD34+CD45+) cells from the PV-iPS cells showed enhanced erythropoiesis as compared to those from the iPS cells derived from normal CD34+ cells. They also showed a gene expression pattern similar to the primary CD34+ cells from the PV patient. These iPS cells thus provide a renewable cell source and a prospective model for investigating MPD pathogenesis. In combination with the gene targeting technology we recently described, human iPS cell lines derived from patients and subsequent hematopoietic differentiation technologies provide a novel model for investigations of various blood diseases with either acquired or inherited mutations. Disclosures:No relevant conflicts of interest to declare.

No Evidence for the JAK2 (V617F) or JAK2 Exon 12 Mutations in Primary Mediastinal Large B-cell Lymphoma

Dysregulated JAK2 signaling has been shown to play a significant role in the pathogenesis of myeloproliferative disorders. Recently, our work comparing gene expression signatures of primary mediastinal large B-cell lymphomas (PMLBCL) versus nodal diffuse large B-cell lymphomas revealed a relative increase in JAK2 transcripts in the former, suggesting a role for increased JAK2 signaling in a subset of these tumors. Given the likelihood of increased JAK2 signaling in PMLBCL, we sought to determine whether JAK2 activating mutations were an alternative mechanism for increased JAK2 signaling in untreated PMLBCLs. We performed amplification refractory mutation analysis for the JAK2 (V617F) mutation and bidirectional sequencing for the recently described JAK2 exon 12 mutations on genomic DNA isolated from a well-characterized cohort of PMLBCLs. No evidence of the mutant JAK2 (V617F) allele or JAK2 exon 12 mutations was detected in 31 PMLBCL cases tested. Analysis using cell lines derived from PMLBCLs (n = 1) and from the molecularly similar classic Hodgkin lymphoma (n = 4) also failed to reveal involvement of a mutant JAK2 allele. Taken together, these results suggest that JAK2 signaling in PMLBCLs occurs by mechanisms distinct from JAK2 (V617F) or JAK2 exon 12 activating mutations.

Molecular and Genetic Bases of Myeloproliferative Disorders: Questions and Perspectives

The discovery of the JAK2V617F mutation followed by the discovery of JAK2 exon 12 and MPLW515 mutations has completely modified the understanding, diagnosis, and management of the classic myeloproliferative disorders (MPDs), which include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Nonetheless, genetic defects have not yet been identified in about 40% of ET and PMF. There is now strong evidence that these mutations are the oncogenic events that drive these disorders and are responsible for most biologic and clinical abnormalities. In addition, there are convincing data indicating that the number of JAK2V617F copies (homozygosity vs. heterozygosity) is important in explaining how a single mutation can be associated with several disorders. However, it is still uncertain whether these mutations are sufficient to explain the full development, heterogeneity, and progression of MPD, or if other genetic or epigenetic events are also necessary. In this review, we discuss different hypothetical models of MPD pathogenesis supported by recent findings. Further characterization of the molecular events operating in these disorders will be essential in fully understanding their pathogenesis and in developing new therapeutic approaches.

Lnk inhibits myeloproliferative disorder-associated JAK2 mutant, JAK2V617F

The JAK2 mutation JAK2V617F is found frequently in patients with myeloproliferative disorders (MPD) and transforms hematopoietic cells to cytokine-independent proliferation when expressed with specific cytokine receptors. The Src homology 2 (SH2) and pleckstrin homology (PH) domain-containing adaptor protein Lnk (SH2B3) is a negative regulator of hematopoietic cytokine signaling. Here, we show that Lnk is a potent inhibitor of JAK2V617F constitutive activity. Lnk down-regulates JAK2V617F-mediated signaling and transformation in hematopoietic Ba/F3-erythropoietin receptor cells. Furthermore, in CFU assays, Lnk-deficient murine bone marrow cells are significantly more sensitive to transformation by JAK2V617F than wild-type (WT) cells. Lnk, through its SH2 and PH domains, interacts with WT and mutant JAK2 and is phosphorylated by constitutively activated JAK2V617F. Finally, we found that Lnk levels are high in CD34(+) hematopoietic progenitors from MPD patients and that Lnk expression is induced following JAK2 activation. Our data suggest that JAK2V617F is susceptible to endogenous negative-feedback regulation, providing new insights into the molecular pathogenesis of MPD.

Do we know more about essential thrombocythemia because of JAK2V617F?

The discovery of the JAK2V617F mutation has triggered enormous interest and some progress in our understanding of the pathogenesis of myeloproliferative disorders. This article reviews its impact upon our knowledge regarding essential thrombocythemia. The discovery of JAK2V617F has led to the proposal that essential thrombocythemia, polycythemia vera, and primary myelofibrosis be discarded as separate diagnoses, and the rationale for this change is discussed. Simplified diagnostic criteria based upon testing for JAK2V617F are proposed. Interesting data are emerging regarding disease progression and risk of complications, specifically thrombosis, pregnancy loss, and perhaps progression to myelofibrosis. The JAK2V617F allele burden is emerging as a potentially important risk factor, although its measurement is not yet standardized. It also may serve as a tool for monitoring the effects of emerging novel therapies, which hold the potential to revolutionize treatment, ranging from reducing risk of complications to potential cure. As with many important scientific discoveries, however, although the discovery of the JAK2V617F mutation has helped us to move forward, there are many new questions and many old questions still unanswered.

Gene Alterations and Expression of Hematopoietic Transcription Factors in Pediatric Acute Megakaryoblastic Leukemia (AMKL): A Comparison of Down Syndrome (DS)-Associated AMKL with Non-DS-AMKL.

Morphological and cytogenetic studies have demonstrated there is a diverse heterogeneity for acute megakaryoblastic leukemia (AMKL). In children, there are 2 major AMKL subgroups, with the disease developing in patients with and without Down syndrome (DS-AMKL and non-DS-AMKL, respectively). Somatic mutations of the GATA1 gene are found in children with DS-AMKL. Recently, activating mutations of the JAK3 gene have been found in both DS-AMKL and non-DS-AMKL patients. Furthermore, JAK2V617F plays a critical role in the pathogenesis of myeloproliferative disorders that are found in adults with non-DS-AMKL. To examine the biological characteristics of the DS-AMKL and non-DS-AMKL blast cells, we compared the morphology, immunophenotypes, gene alterations (GATA1, JAK2, JAK3) and the expression of the hematopoietic transcription factors in blast cells derived from 29 DS-AMKL and 21 non-DS-AMKL children. AMKL was defined by the presence of blast cells that expressed at least one platelet-associated antigen (CD36, CD41, CD42, or CD61). Written informed consent for banking and molecular analysis of the blast cells was obtained from the parents of each of the patients. Blast cell morphology was categorized as follows: type 1, completely undifferentiated blasts; type 2, intermediately differentiated blasts with cytoplasmic blebs; type 3, blasts with dysmegakaryocytopoiesis; and type 4, blasts with deep blue cytoplasm. The respective distributions of the morphology of the blasts in DS-AMKL and non-DS-AMKL patients were as follows: type 1 (41%, 39%), type 2 (4%, 39%), type 3 (11%, 22%), and type 4 (44%, 0%). Type 4 blasts were only seen in DS-AMKL. Atypical expression of lymphoid-associated antigen CD7 was detected in 90% and 53% of the immunophenotyped-patients with DS-AMKL and non-DS-AMKL, respectively (p = 0.003). Glycophorin A was only detected on the blasts of 34% of the patients with DS-AMKL (p = 0.012). Interestingly, 78% of the type 4 blasts were positive for glycophorin A. The GATA1, JAK2 and JAK3 mutations were analyzed in bone marrow or peripheral blood samples. High-molecular weight DNA was extracted from the samples using standard methods. We amplified the genomic DNA corresponding to exon2 of GATA1, exon14 of JAK2, and all 23 exons of the JAK3 gene, respectively, with the amplified products sequenced directly on a DNA sequencer (310; Applied Biosystems, Foster City, CA) using a BigDye terminator cycle sequencing kit (Applied Biosystems). The frequencies of the gene mutations in DS-AMKL and non-DS-AMKL were distributed as follows: GATA1 (100%, 8%), JAK2V617F (6%, 0%) and JAK3 (12%, 8%). To define the differentiation stage based on the molecular level, we quantified the expression of the transcription factors related to the megakaryo-erythroid cell lineages. The expression of the transcription factors (GATA1, GATA2, FOG-1, NF-E2, SCL, PU.1, C/EBPa, c-mpl, Epo-R, CD41b, bglobin) was analyzed by real time RT-PCR in blasts that were sorted by flow cytometry. Total RNA was extracted from the purified blasts. Synthesis of cDNA was performed using a Thermoscript RT-PCR system (Invitrogen, San Diego, CA) according to the manufacturer's instructions. Real-time RT-PCR was performed using an ABI Prism 7000 Sequence Detection System (Applied Biosystems, Branchburg, NJ). Normalized gene expression levels are given as the ratio between the mean value for the target gene and that for the GAPDH gene in each sample. Median bglobin transcript levels in DS-AMKL blasts were significantly higher than those seen for the non-DS-AMKL blasts (p = 0.006). Particularly, median bglobin and GATA1 transcript levels in the type 4 blasts, which were only found in DS-AMKL, were significantly higher than those found in the blasts of other types (p = 0.033 and p = 0.021, respectively). Median GATA1, NF-E2 and Epo-R transcript levels in type 3 blasts, which differentiated into mature megakaryocytes, were significantly lower than those found in the blasts of other types (p = 0.030, p = 0.036, and p = 0.028, respectively). In conclusion, blasts of DS-AMKL differentiated not only into the megakaryocyte but also the erythroid lineage because of the overexpression of glycophorin A and bglobin under the GATA1 gene mutations. Considering the totally different gene alteration patterns and the levels of transcription factors that were observed, DS-AMKL needs to be classified into a category that differs from non-DS-AMKL.

Miscreant myeloproliferative disorder stem cells

Myeloproliferative disorders (MPDs), typified by robust marrow and extramedullary hematopoiesis, have a propensity to progress to acute leukemia. Although the hematopoietic stem cell (HSC) origin of MPDs was suggested over 30 years ago, only recently the HSC-specific effects of MPD molecular mutations have been investigated. The pivotal role of BCR-ABL in chronic myeloid leukemia (CML) development provided the rationale for targeted therapy, which greatly reduced mortality rates. However, BCR-ABL inhibitor-resistant CML HSCs persist that may be a reservoir for relapse. This has provided the impetus for investigating molecular mechanisms governing the production of recalcitrant HSC. Comparatively little was known about the molecular events driving BCR-ABL-negative MPDs until seminal studies revealed that a large proportion of MPD patients harbor a JAK2-activating point mutation, JAK2V617F. Although JAK2 activation appears to be central to BCR-ABL-negative MPD pathogenesis, its effects may be cell type and context specific. Recent evidence suggests that acquired mutations misdirect differentiation and survival of the MPD-initiating stem cell resulting in the production of aberrant self-renewing progenitors that subvert the microenvironment leading to leukemia stem cell generation and leukemic transformation. Thus, combined therapies targeting aberrant molecular pathways may be required to redirect miscreant MPD stem cells.

Adaptor Protein Lnk Negatively Regulates Mutant MPL and JAK2 Alleles Associated with Myeloproliferative Disorders

Activating mutations in the cytokine receptor/JAK2 signaling axis are found at high frequency in myeloproliferative disorders (MPD). Lnk, an SH2-containing adaptor protein, is a negative regulator of several hematopoietic cytokine receptors including MPL and EpoR. Here, we assessed whether Lnk can attenuate the activity of mutant MPLW515L, JAK2V617F and JAK2K539L found in MPD patients. Lnk overexpression in Ba/F3-MPLW515L cells inhibited cytokine-independent growth, while suppression of Lnk in UT7-MPLW515L cells enhanced proliferation. Lnk-mediated growth inhibition was associated with downregulation of JAK/STAT, MAPK and PI3K signaling pathways. Similarly, Lnk inhibited cytokine-independent growth conferred by JAK2V617F and JAK2K539L in Ba/F3-EpoR cells. Following thrombopoietin stimulation, Lnk became tyrosyl-phosphorylated and associated with activated wild-type (WT) MPL and MPLW515L at the plasma membrane of Ba/F3 cells. An SH2 mutant Lnk (R392E) failed to bind and inhibit WT MPL and MPLW515L, demonstrating that the SH2 domain is essential for Lnk down-modulation of the receptors. The Lnk-MPL interaction was also detected with endogenously expressed proteins from cultured bone marrow cells. A series of C-terminally truncated Lnk constructs were used to determine which Lnk regions are required for Lnk inhibition of JAK2. Experiments in 293T cells indicated that Lnk SH2 domain binds to phosphorylated JAK2. In addition, other Lnk regions associated with non-phosphorylated JAK2, and these interactions were critical for Lnk inhibition of JAK2V617F and JAK2K539L constitutive activation. Our data suggest a model wherein Lnk downregulation of the receptor/JAK2 signaling involves two mechanisms; one is inhibition of the cytokine receptor utilizing JAK2, the second is direct suppression of JAK2 kinase activity. Furthermore, while the receptor mediated inhibition requires the SH2 domain, direct inhibition of JAK2 mutants, V617F and K539L, relies on other Lnk domains. Further elucidating the molecular mechanisms underlying Lnk inhibition of signaling pathways abnormally activated by oncogenic alleles, will provide insight into the pathogenesis of MPD and may have therapeutic value.

Genetic Profiling of BCR/ABL Negative Myeloproliferative Disorders Using High-Resolution Microarrays

Recently, the identification of the gain of function mutation JAK2V617F delivered important insights into the pathogenesis of BCR/ABL negative myeloproliferative disorders (MPD). JAK2V617F is detectable in more than 90% of polycythemia vera (PV) patients (pts) and in approximately 50% of pts with essential thrombocythemia (ET) or primary myelofibrosis (PMF), representing the genetic hallmark of BCR/ABL negative disease. However, about 30% of MPD pts lack the JAK2V617F mutation and previous studies on ET and PV demonstrated that clonality exceeds the percentage of V617F mutated cells. These findings suggest that additional genetic alterations are involved in the pathogenesis of MPD, in both JAK2 mutated and unmutated pts. To identify novel genetic aberrations and to determine whether specific lesions are associated with disease phenotype, genomic DNA from granulocytes of 72 MPD pts classified according to the WHO criteria was analyzed using high-resolution, genome-wide microarray techniques [disease, number analyzed, JAK2 mutation status: PMF, n=14, 9/14; post-ET MF, n=5, 3/5; post-PV MF, n=5, 5/5; PV, n=37, 37/37; ET, n=11, 11/11]. In a first approach, all cases were investigated by comparative genomic hybridization to 8k arrays (array CGH) with an average probe spacing of less than 1 Mb. While no genomic imbalances were found in ET, 11% of PV pts (n=4) exhibited large (>10 Mb) deletions on 20q (n=2) or gains on 9p and 1q (n=1, each). In addition, small (<1 Mb) recurrent gains in 1q21.1 (n=2) and 22q11.23 (n=2) were identified. In MF pts the incidence of large genomic imbalances was 25% (n=6) with trisomy 9 (n=3) being the most frequent aberration followed by loss of 20q, 5q, and 13q in single cases. Furthermore, in one pt with post-PV MF small genomic losses in 17q11.2 (2 Mb) and 17p13.2 (0.8 Mb) were identified harbouring NF1 but not TP53. Deletion of the NF1 allele without concomitant loss of TP53 was confirmed by FISH. To further increase resolution and to investigate the role of uniparental disomy (UPD), single nucleotide polymorphism (SNP) analysis using the Affymetrix 250k Nsp SNP array was performed in all MF cases. Copy number estimation and loss of heterozygosity probability were analyzed using a set of 117 remission samples from acute myeloid leukemia pts as a common reference. SNP analysis confirmed all anomalies detected by array CGH. In addition, SNP analysis revealed small genomic losses (1.6–2.6 Mb) in 1q21.2 (n=3), 5q13.2, and 3p13 (n=1, each), and in one secondary MF pt another microdeletion in 17q11.2 (1.2 Mb). UPDs recurrently affected 9p (n=5) in a region harbouring the JAK2 locus. In single cases, large UPDs of 1q (25 Mb), 2p (14 Mb), 5q (4 Mb), 6p (11 Mb), and 7q (11 Mb) were identified. Of note, all JAK2V617F mutated post-PV and post-ET MF cases exhibited 9p abnormalities represented either by trisomy 9 or UPD of 9p. In conclusion, using a combined microarray approach we were able to detect novel submicroscopic alterations in addition to known abnormalities. Parallel analysis of both techniques clearly demonstrated the superiority of array-SNP mapping. Further analyses on larger pt populations and correlation with global gene expression data will facilitate the identification of disease-related genes that are involved in the pathogenesis of BCR/ABL negative MPD.

JAK3, JAK2 and GATA1 Mutations in Childhood Acute Megakaryoblastic Leukemia with and without Down Syndrome.

Acute myeloid leukemia (AML) in children with Down syndrome (DS) exhibits unique characteristics, which includes the predominance of megakaryoblastic leukemia (AMKL). In about 10% of DS infants, transient myeloproliferative disorder (TMD) develops and then spontaneously resolves in most cases. However, 20% of TMD cases develop AMKL within 3 years, suggesting that additional genetic alterations might occur during the progression from TMD to AMKL. GATA1 mutations contribute to both the pathogenesis of TMD and AMKL, suggesting that the acquisition of additional genetic alterations might be necessary for the progression from TMD to AMKL. Recently, activating mutations of the JAK3 gene have been found in each of 16 non-DS and 3 DS-AMKL patients. These results suggest that the activating JAK3 mutations are involved in the pathogenesis of AMKL both with and without DS. In this study, we simultaneously analyzed JAK3, JAK2V617F which plays a critical role in the pathogenesis of myeloproliferative disorders and GATA1 mutations in non-DS children with AMKL and in DS children with AMKL or TMD. The study population included 11 non-DS children with AMKL and 20 AMKL and 6 TMD children with DS. After obtaining informed consent from the parents of the children for the purpose of sample banking and molecular analyses, bone marrow (BM) or peripheral blood (PB) samples were obtained from all of the patients with AMKL and TMD at the time of diagnosis. High-molecular weight DNA was extracted from the samples using standard methods. For the screening of the JAK3 mutations, we amplified genomic DNA that corresponded to all 23 exons of the JAK3 gene by using polymerase chain reaction (PCR) that employed 22 primer pairs. Amplified products were subjected to the denaturing HPLC (DHPLC) analysis using a WAVE Maker System (Transgenomic Inc., San Jose, CA). The DHPLC gradients and temperatures were determined using WAVE Maker System software. When heterozygous profiles were identified after a visual inspection of the chromatograms, amplified products were cloned into the pGEM-T Easy vector (Promega, Madison, WI) and sequenced on a DNA sequencer (310; Applied Biosystems, Foster City, CA) using a BigDye terminator cycle sequencing kit (Applied Biosystems). For screening of GATA1 mutations and JAK2V617F mutations, we amplified the genomic DNA that corresponded to exon 2 of GATA1 and exon12 of JAK2 by using PCR that employed 1 primer pair respectively. Amplified products were cloned into the pGEM-T Easy vector and sequenced on a DNA sequencer using a BigDye terminator cycle sequencing kit. Mutations of JAK3 were found in 2 of the DS-AMKL patients and in 1 DS-TMD patient. A573V and A593T substitutions in the JH2 pseudokinase domain along with Q501H substitutions in the JH3 domain were observed in the DS-AMKL patients and an I87T substitution in the JH7 domain of the receptor-binding domain was observed in DS-TMD. In one of the DS-AMKL patients, A573V and A593T substitutions were found in the same allele. GATA1 mutations were observed in 19 of the 20 DS-AMKL patients, in all of the DS-TMD patients and in 1 non-DS-AMKL patient. JAK2V617F mutation was only found in 1 DS-AMKL patient who had also JAK3 mutation (Q501H). The fact that the JAK3 mutations were found in both the AMKL and TMD patients indicates that this is an early event that occurs during the development of AMKL in DS patients and that other genetic changes may contribute to the development of AMKL.

Adaptor protein Lnk negatively regulates the mutant MPL, MPLW515L associated with myeloproliferative disorders

AbstractRecently, activating myeloproliferative leukemia virus oncogene (MPL) mutations, MPLW515L/K, were described in myeloproliferative disorder (MPD) patients. MPLW515L leads to activation of downstream signaling pathways and cytokine-independent proliferation in hematopoietic cells. The adaptor protein Lnk is a negative regulator of several cytokine receptors, including MPL. We show that overexpression of Lnk in Ba/F3-MPLW515L cells inhibits cytokine-independent growth, while suppression of Lnk in UT7-MPLW515L cells enhances proliferation. Lnk blocks the activation of Jak2, Stat3, Erk, and Akt in these cells. Furthermore, MPLW515L-expressing cells are more susceptible to Lnk inhibitory functions than their MPL wild-type (MPLWT)–expressing counterparts. Lnk associates with activated MPLWT and MPLW515L and colocalizes with the receptors at the plasma membrane. The SH2 domain of Lnk is essential for its binding and for its down-regulation of MPLWT and MPLW515L. Lnk itself is tyrosine-phosphorylated following thrombopoietin stimulation. Further elucidating the cellular pathways that attenuate MPLW515L will provide insight into the pathogenesis of MPD and could help develop specific therapeutic approaches.