Mutations of E3 Ubiquitin Ligase Cbl Family Members Constitute a Novel Common Pathogenic Lesion in Myeloid Malignancies

Purpose Acquired somatic uniparental disomy (UPD) is commonly observed in myelodysplastic syndromes (MDS), myelodysplastic/myeloproliferative neoplasms (MDS/MPN), or secondary acute myelogenous leukemia (sAML) and may point toward genes harboring mutations. Recurrent UPD11q led to identification of homozygous mutations in c-Cbl, an E3 ubiquitin ligase involved in attenuation of proliferative signals transduced by activated receptor tyrosine kinases. We examined the role and frequency of Cbl gene family mutations in MPN and related conditions. Methods We applied high-density SNP-A karyotyping to identify loss of heterozygosity of 11q in 442 patients with MDS, MDS/MPN, MPN, sAML evolved from these conditions, and primary AML. We sequenced c-Cbl, Cbl-b, and Cbl-c in patients with or without corresponding UPD or deletions and correlated mutational status with clinical features and outcomes. Results We identified c-Cbl mutations in 5% and 9% of patients with chronic myelomonocytic leukemia (CMML) and sAML, and also in CML blast crisis and juvenile myelomonocytic leukemia (JMML). Most mutations were homozygous and affected c-Cbl; mutations in Cbl-b were also found in patients with similar clinical features. Patients with Cbl family mutations showed poor prognosis, with a median survival of 5 months. Pathomorphologic features included monocytosis, monocytoid blasts, aberrant expression of phosphoSTAT5, and c-kit overexpression. Serial studies showed acquisition of c-Cbl mutations during malignant evolution. Conclusion Mutations in the Cbl family RING finger domain or linker sequence constitute important pathogenic lesions associated with not only preleukemic CMML, JMML, and other MPN, but also progression to AML, suggesting that impairment of degradation of activated tyrosine kinases constitutes an important cancer mechanism.

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Cbl and TET2 Mutations Are Present in Refractory Ph+ Disorders Including Accelerated and Blast Crisis CML and ALL.

Blood ◽

10.1182/blood.v114.22.2173.2173 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2173-2173

Author(s):

Hideki Makishima ◽

Anna M. Jankowska ◽

Heather Cazzolli ◽

Bartlomiej P Przychodzen ◽

Courtney Prince ◽

...

Keyword(s):

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Blast Crisis ◽

Uniparental Disomy ◽

Ring Finger ◽

Chronic Phase ◽

Homozygous Deletion ◽

Juvenile Myelomonocytic Leukemia ◽

Tet2 Mutation ◽

Myelomonocytic Leukemia

Abstract Abstract 2173 Poster Board II-150 Loss of heterozygosity (LOH) due to acquired uniparental disomy (UPD) is a commonly observed chromosomal lesion in myeloproliferative neoplasms (MPN) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN) including chronic myelomonocytic leukemia (CMML). Most recurrent areas of LOH point towards genes harboring mutations. For example, UPD11q23.3 and UPD4q24 were found to be associated with c-Cbl and TET2 mutations, respectively. Cbl family mutations (c-Cbl and Cbl-b) have been associated with atypical MDS/MPN including CMML and juvenile myelomonocytic leukemia (JMML) as well as more advanced forms of MDS and secondary AML (sAML). Ring finger mutants of Cbl abrogate ubiquitination and thereby tumor suppressor function related to inactivation of phosphorylated receptor tyrosine kinases, Src and other phosphoproteins. TET2 mutations are present in a similar disease spectrum. The TET family of proteins is involved in conversion of methylcytosine to methylhydroxycytosine which cannot be recognized by DNMT1. Thereby, the proteins seem to counteract maintenance hypermethylation. In our screen of MDS/MPN, we found c-Cbl and Cbl-b ring finger mutations in 5/58 (9%) of CMML and AML derived from CMML, 2/39 (5%) MDS/MPNu, 4/21 (19%) JMML and 14/148 (9%) RAEB/sAML. In the same cohort, TET2 mutations were present in 37% and 14% of patients with MDS/MPN and MDS, respectively. Of note we did not find any TET2 mutations in JMML. We and others have also noted that TET2 and c-Cbl mutations were also detected in atypical chronic myeloid leukemia. While translocations resulting in BCR/ABL fusion characterize CML, we stipulated that in analogy to other chronic myeloproliferative diseases, TET2 and c-Cbl mutations may be also present in CML and contribute to phenotypic heterogeneity within BCR/ABL associated disorders. In particular, progression of CML to accelerated phase (AP) or blast crisis (BC) could be associated with acquisition of additional lesions. When 22 patients with CML chronic phase (CP) were screened, no TET2 and c-Cbl mutations were found. However, we identified 1 c-Cbl, 2 Cbl-b (6%) and 6 TET2 (12%) mutations in 51 patients with CML-AP (N=18) and CML-BC (N=33) with myeloid and lymphoid/mix 24 and 9 phenotype, respectively. These mutations were mutually exclusive. We also noted that TET2 mutations were present in 1/9 CML in BC with lymphoid phenotype. We subsequently screened Ph+ ALL cases (N=9) and found a TET2 mutation in 1 case but no Cbl family mutations. In contrast when 9 Ph- ALL cases were screened as controls, neither TET2 or Cbl mutations were found. SNP-A analysis revealed 2 cases of LOH involving chromosome 4 (UPD4q24 and del4) in a patient with lymphoid blast crisis and Ph+ ALL, respectively. However, UPD was not found in Cbl family gene regions (11q23.3 or 3q13.11). A homozygous deletion of Cbl-b region was seen in a CP patient. Cbl family mutations were associated with a more complex karyotype than TET2 mutations (67% vs. 17% cases with abnormal phenotype). Patients with Cbl family mutations were resistant to imatinib which was effective in only 2 out of 6 patients with TET2mutations. Dasatinib was effective in 2 patients with TET2 mutation. Median over all survival of progressed CML was 47, 49 and 48 months in patients with Cbl, TET2 or no mutations, respectively. In conclusion, our results indicate that Cbl family mutations can occur as secondary lesions in myeloid type aggressive CML (AP and myeloid BC), but not in lymphoid types. TET2 mutations were identified in both lymphoid BC and Ph1+ALL, as well as myeloid BC and AP. In contrast to CMML or JMML in which a vast majority of mutations are homozygous, all Cbl family mutations were heterozygous (no LOH). Similarly, all but two TET2 mutations were heterozygous (1 hemizygous in del4 and 1 homozygous case in UPD4q), suggesting that additional cooperating lesions affecting corresponding pathways may be present. These mutations likely represent secondary lesions which contribute to more either progression (CML) or more aggressive features (Ph+ ALL) and characterize disease refractory to therapy with imatinib. Disclosures: No relevant conflicts of interest to declare.

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Incidence and Patient Survival of Myeloproliferative Neoplasms (MPNs) and Myelodysplastic/Myeloproliferative Neoplasms (MDS/MPNs) in the United States: A Population-Based View of the Modern Diagnostic Era

Blood ◽

10.1182/blood.v126.23.2806.2806 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2806-2806

Author(s):

Samer A Srour ◽

Susan S Devesa ◽

Lindsay M Morton ◽

David P Check ◽

Rochelle E Curtis ◽

...

Keyword(s):

United States ◽

Patient Survival ◽

Myeloproliferative Neoplasms ◽

The United States ◽

Population Based ◽

Mutation Testing ◽

Myelogenous Leukemia ◽

World Health ◽

Juvenile Myelomonocytic Leukemia ◽

Myelomonocytic Leukemia

Abstract Introduction: Epidemiologic information on myeloproliferative neoplasms (MPNs) and myelodysplastic (MDS)/MPNs is largely derived from single institution and European population-based studies. Further, descriptive data encompassing the era of the World Health Organization classification (WHO) of hematopoietic neoplasms and JAK2 V617 mutation testing are sparse. Therefore, we utilized population-based data to comprehensively assess incidence and patient survival of MPNs and MDS/MPNs in the United States (US) during 2001-2012. Methods: Using data from the National Cancer Institute's Surveillance, Epidemiology and End Results Program (SEER) Program, we assessed the incidence rates (IRs), IR ratios (IRRs), and 95% confidence intervals (CI) for each MPN and MDS/MPN entity. We assessed IRs overall and according to sex, race/ethnicity, calendar year of diagnosis, and method of diagnostic confirmation. We utilized the SEER*Stat Survival Session to calculate 5-year relative survival (RS) and 95% CI for each disease entity overall and according to sex and age at diagnosis. Results: A total of 44,912 patients were diagnosed with MPNs and MDS/MPNs during 2001-2012. IRs were highest for polycythemia vera (PV) (n=10,812; IR=10.9 per 1,000,000 person-years) and essential thrombocythemia (ET) (n=9,394; IR=9.6). Except for ET and mastocytosis, the overall IRs for all entities were significantly higher among males than females, with male-to-female IRRs ranging from 1.4-2.3. PV incidence peaked in 2003-2004 and progressively decreased thereafter in contrast to IRs for ET which markedly increased after 2003-2004, with a suggestion of decrease after 2008. BCR-ABL1-positive chronic myelogenous leukemia (CML) IRs increased progressively over the study period while CML, NOS decreased over the study decade. All evaluable MPNs were associated with lower IRs among Hispanic whites than non-Hispanic whites (NHWs), with the exception of BCR-ABL1-positive CML, chronic eosinophilic leukemia (CEL), and juvenile myelomonocytic leukemia which occurred similarly among both groups. Except for CEL, Asians/Pacific Islanders had significantly lower MPN IRs than NHWs. ET, MPN-unclassifiable, and CEL IRs were 18%, 19%, and 60% higher, respectively, among blacks than NHWs. Among males and females, for all evaluable MPNs and MDS/MPNs, five-year RS was more favorable for younger (<60 years) than older (>60 years) individuals. Patients with PV or ET had the most favorable RS among both sexes and age groups, ranging from 92.0%-96.7% among those <60 years and 79.1%-87.9% among those >60 years. Patients with chronic neutrophilic leukemia, chronic myelomonocytic leukemia, and atypical BCR-ABL1 -negative CML patients had the least favorable 5-year RS (<35%). Females generally had more favorable survival than males, except for older males with PV who had significantly better survival than older women (RS IRR=1.08, 95%CI=1.03-1.13). Conclusion: MPNs are a heterogeneous group of diseases and varying age, sex, and racial/ethnic incidence patterns support distinct etiologies and/or susceptible populations. The introduction of JAK2 V617 mutation testing in 2005 may have had a differential impact on IRs of PV and ET. Less favorable RS among older ages for all MPN subtypes suggests the need for inclusion of these individuals in clinical trials as new treatments become available. Disclosures No relevant conflicts of interest to declare.

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Myelodysplastic/Myeloproliferative Neoplasms

Hematology ◽

10.1182/asheducation-2011.1.264 ◽

2011 ◽

Vol 2011 (1) ◽

pp. 264-272 ◽

Cited By ~ 29

Author(s):

Mario Cazzola ◽

Luca Malcovati ◽

Rosangela Invernizzi

Keyword(s):

Clinical Laboratory ◽

Myeloproliferative Neoplasms ◽

Chronic Myelomonocytic Leukemia ◽

World Health ◽

Refractory Anemia ◽

Molecular Diagnostic ◽

Juvenile Myelomonocytic Leukemia ◽

Lymphoid Tissues ◽

Ring Sideroblasts ◽

Myelomonocytic Leukemia

Abstract According to the World Health Organization (WHO) classification of tumors of hematopoietic and lymphoid tissues, myelodysplastic/myeloproliferative neoplasms are clonal myeloid neoplasms that have some clinical, laboratory, or morphologic findings that support a diagnosis of myelodysplastic syndrome, and other findings that are more consistent with myeloproliferative neoplasms. These disorders include chronic myelomonocytic leukemia, atypical chronic myeloid leukemia (BCR-ABL1 negative), juvenile myelomonocytic leukemia, and myelodysplastic/myeloproliferative neoplasms, unclassifiable. The best characterized of these latter unclassifiable conditions is the provisional entity defined as refractory anemia with ring sideroblasts associated with marked thrombocytosis. This article focuses on myelodysplastic/myeloproliferative neoplasms of adulthood, with particular emphasis on chronic myelomonocytic leukemia and refractory anemia with ring sideroblasts associated with marked thrombocytosis. Recent studies have partly clarified the molecular basis of these disorders, laying the groundwork for the development of molecular diagnostic and prognostic tools. It is hoped that these advances will soon translate into improved therapeutic approaches.

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Aggressive Transformation of Juvenile Myelomonocytic Leukemia Associated with Duplication of Oncogenic KRAS due to Acquired Uniparental Disomy

The Journal of Pediatrics ◽

10.1016/j.jpeds.2013.01.003 ◽

2013 ◽

Vol 162 (6) ◽

pp. 1285-1288.e1 ◽

Cited By ~ 15

Author(s):

Motohiro Kato ◽

Naoko Yasui ◽

Masafumi Seki ◽

Hiroshi Kishimoto ◽

Aiko Sato-Otsubo ◽

...

Keyword(s):

Uniparental Disomy ◽

Juvenile Myelomonocytic Leukemia ◽

Myelomonocytic Leukemia

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Correlation of Clinical Features With the Mutational Status of GM-CSF Signaling Pathway-Related Genes in Juvenile Myelomonocytic Leukemia

Pediatric Research ◽

10.1203/pdr.0b013e3181961d2a ◽

2009 ◽

Vol 65 (3) ◽

pp. 334-340 ◽

Cited By ~ 53

Author(s):

Nao Yoshida ◽

Hiroshi Yagasaki ◽

Yinyan Xu ◽

Kazuyuki Matsuda ◽

Ayami Yoshimi ◽

...

Keyword(s):

Signaling Pathway ◽

Clinical Features ◽

Juvenile Myelomonocytic Leukemia ◽

Gm Csf ◽

Mutational Status ◽

Myelomonocytic Leukemia

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Myelodysplastic and Myeloproliferative Disorders of Childhood: A Study of 167 Patients

Blood ◽

10.1182/blood.v93.2.459 ◽

1999 ◽

Vol 93 (2) ◽

pp. 459-466 ◽

Cited By ~ 149

Author(s):

Sandra Luna-Fineman ◽

Kevin M. Shannon ◽

Susan K. Atwater ◽

Jeffrey Davis ◽

Margaret Masterson ◽

...

Keyword(s):

Clinical Features ◽

Age At Onset ◽

Myeloproliferative Disorders ◽

Juvenile Myelomonocytic Leukemia ◽

Genetic Associations ◽

Adult Type ◽

Hemoglobin F ◽

Monosomy 7 ◽

Prognostic Features ◽

Myelomonocytic Leukemia

Abstract Myelodysplastic syndromes (MDS) and myeloproliferative syndromes (MPS) of childhood are a heterogeneous group of clonal disorders of hematopoiesis with overlapping clinical features and inconsistent nomenclature. Although a number of genetic conditions have been associated with MDS and MPS, the overall contribution of inherited predispositions is uncertain. We report a retrospective study examining clinical features, genetic associations, and outcomes in 167 children with MDS and MPS. Of these patients, 48 had an associated constitutional disorder. One hundred one patients had adult-type myelodysplastic syndrome (A-MDS), 60 had juvenile myelomonocytic leukemia (JMML), and 6 infants with Down syndrome had a transient myeloproliferative syndrome (TMS). JMML was characterized by young age at onset and prominent hepatosplenomegaly, whereas patients with A-MDS were older and had little or no organomegaly. The most common cytogenetic abnormalities were monosomy 7 or del(7q) (53 cases); this was common both in patients with JMML and those with A-MDS. Leukemic transformation was observed in 32% of patients, usually within 2 years of diagnosis. Survival was 25% at 16 years. Favorable prognostic features at diagnosis included age less than 2 years and a hemoglobin F level of less than 10%. Older patients tended to present with an adult-type MDS that is accommodated within the French-American-British system. In contrast, infants and young children typically developed unique disorders with overlapping features of MDS and MPS. Although the type and intensity of therapy varied markedly in this study, the overall outcome was poor except in patients with TMS.

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Genotypic Representation of Myelodysplastic/Myeloproliferative Neoplasms in Nrg, Nrg-3GS and Srg-W41 Mice with Transgenic Expression of Human Cytokines

Blood ◽

10.1182/blood-2018-99-111728 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2038-2038

Author(s):

Hein Than ◽

Naoto Nakamichi ◽

Anthony D. Pomicter ◽

John O'Shea ◽

Orlando Antelope ◽

...

Keyword(s):

Bone Marrow ◽

Myeloid Leukemia ◽

Myeloproliferative Neoplasms ◽

Model Systems ◽

Juvenile Myelomonocytic Leukemia ◽

Functional Screening ◽

Human Cd34 ◽

Cd34 Cells ◽

Significant Difference ◽

Myelomonocytic Leukemia

Abstract Myelodysplastic/myeloproliferative neoplasms (MDS/MPN) are complex clonal hematopoietic stem cell malignancies with overlapping dysplastic and proliferative features. Genomic analyses have charted the somatic mutation spectrum of MDS/MPN and revealed a major role for epigenetic dysregulation in their pathogenesis. No disease-modifying therapies are currently available, as progress has been hampered by a lack of genetically faithful in vivo model systems suitable for the preclinical development of new strategies. Yoshimi et al (Blood. 2017;130:397-407) recently showed that patients' chronic myelomonocytic leukemia (CMML) and juvenile myelomonocytic leukemia (JMML) cells transplanted into NOD/SCID-IL2Rγ-/-mice expressing human IL3, GM-CSF and SCF transgenes (NSG-3GS mice) produced xenografts that had mutations characteristic of the input cells. Since we had demonstrated a superior level of chimerism achieved from transplants of normal human CD34+cord blood cells in SirpaNOD/Rag1-/-/IL2rγc-/-/W41/41mice with c-KIT deficiency (with an otherwise mixed NOD-C57Bl/6 background - SRG-W41 mice) compared to conventional NSG or NRG hosts (Miller et al. Exp Hematol. 2017;48:41-49), it was of interest to explore their use as hosts of samples from patients with MDS/MPN: CMML, atypical chronic myeloid leukemia (aCML) and secondary acute myeloid leukemia (sAML) progressed from CMML or aCML. Heparinized blood or bone marrow samples were obtained from patients treated at Huntsman Cancer Institute after informed consent. Diagnoses included CMML (n=5), aCML (n=2), and sAML (n=2). Unseparated cells were shipped by overnight courier to Vancouver and CD34+cells isolated on the same day were injected intravenously into sub-lethally irradiated female NRG mice or SRG-W41 mice, or in some cases the same sex and strains also carrying the human 3GS transgenes (NRG-3GS or SRG-W41-3GS mice) in accordance with British Columbia Cancer Agency institutional guidelines. Occasionally when mice were not immediately available, or large numbers of cells were available, cells were viably cryopreserved and transplanted later after thawing. Mice were observed for up to 36 weeks after xenotransplantation with .05 to 1.1x106 human CD34+cells. Engraftment of human CD45+cells in xenografts was evaluated by immunophenotyping, and a median of 90% human chimerism (range: 1% - 95%) was achieved at the time of bone marrow harvest from xenografts. Variant allele frequencies (VAF) were determined in genomic DNA extracted from both the patient samples (CD34+cells) and matching fluorescence-activated cells (FACS)-sorted human CD45+cells (hCD45+cells) purified from xenografts (1-5 xenografts per patient sample). DNA samples were subjected to PCR amplification with extension primers and analyzed using a MALDI-TOF mass spectrometer (MassArray, Agena Bioscience, San Diego, CA). Each mutation call was assigned by the software based on the molecular weight of the extended primer. Analysis of hCD45+cells from eight xenograft samples so far demonstrated a strong correlation of VAF between the patient samples and hCD45+cells from xenografts, in both SRG-W41-3GS (R2=0.94, p<0.01) and NRG-3GS (R2=0.97, p<0.01) models (Figure 1). This tight correlation of VAF was illustrated in hCD45+cells from xenografts transplanted with CMML, aCML or sAML cells. The majority of mutations detected were those in epigenetic regulator genes, such as ASXL1, EZH2 and TET2. No significant difference in VAF was observed between CD34+and CD34- compartments within the hCD45+cells. Additional samples, including specimens from patients with the related myeloproliferative neoplasm, chronic neutrophilic leukemia (CNL) are being analyzed and will be presented. These findings demonstrate the utility of SRG-W41-3GS as well as NRG-3GS as receptive hosts of primary human MDS/MPN cells with genetic evidence of their growth in these mice closely recapitulating the mutational profiles of the transplanted cells. These new strains may facilitate the development of functional screening and pre-clinical testing of novel therapeutic strategies for a range of human MDS/MPN and related myeloid disorders. Disclosures Deininger: Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Blueprint: Consultancy.

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Correlation of Clinical Features with the Mutational Status of GM-CSF Signaling Pathway-Related Genes in Children with Juvenile Myelomonocytic Leukemia

Blood ◽

10.1182/blood.v110.11.1528.1528 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1528-1528 ◽

Cited By ~ 1

Author(s):

Nao Yoshida ◽

Hiroshi Yagasaki ◽

Ayami Yoshimi ◽

Yoshiyuki Takahashi ◽

Yinyan Xu ◽

...

Keyword(s):

Prognostic Factors ◽

Clinical Outcome ◽

Clinical Features ◽

Juvenile Myelomonocytic Leukemia ◽

Gm Csf ◽

Ptpn11 Mutation ◽

Mutational Status ◽

Hbf Level ◽

Mutation Age ◽

Myelomonocytic Leukemia

Abstract Juvenile myelomonocytic leukemia (JMML) is a rare clonal myeloproliferative disorder that affects young children. It is characterized by specific hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF) in vitro. Mutations in RAS, NF1, or PTPN11 positioned in the GM-CSF signal pathway, are thought to be involved in the pathogenesis of JMML. However, no information is available on the relationship between these mutations and clinical features of JMML. The impacts of these mutations on clinical outcome also remain unclear. We tested 49 Japanese children with JMML for N-RAS, K-RAS, and PTPN11 mutations and evaluated their clinical significance. We also assessed correlations between mutational status and clinical and laboratory findings, including age at diagnosis, fetal hemoglobin (HbF), platelet count, and cytogenetic abnormality, all which have been proposed as prognostic factors for JMML. Of the 49 JMML patients, cytogenetic abnormalities were detected in 13, including 8 with monosomy 7. For 2 patients, a clinical diagnosis of neurofibromatosis type 1 (NF1) was confirmed. PTPN11 and N-/K-RAS mutations were found in 22 (45%) and 8 (16%) patients, respectively. Neither PTPN11 nor RAS mutations nor NF1 were present in 17 (35%) patients, and no simultaneous aberrations in these genes were found. In patients with the PTPN11 mutation, age at diagnosis was older (35 vs 11 months; P=0.001, or 12 months; P<0.01) and HbF level was higher (31 vs 10%; P=0.03, or 16%; P<0.01) than for patients with the RAS mutation or without any aberration, suggesting that the clinical outcome for patients with the PTPN11 mutation might be poorer, because a higher HbF level and older age have been reported to be poor prognostic factors. In fact, overall survival (OS) at 5 years was lower for patients with the PTPN11 mutation than for those without (20±9% vs 58±9%; P=0.02). In addition to PTPN11 mutation, age older than 24 months (P<0.01) and abnormal karyotype (P=0.02) were also associated with poor prognosis for OS. Of the 49 patients, 33 received stem cell transplantation (SCT). OS probabilities for patients with and without a mutation in PTPN11 at 5 years after SCT were 25±10% and 64±12%, respectively (P=0.04). More importantly, mutation in PTPN11 was the only unfavorable factor for relapse after SCT (P<0.01). Seven patients died owing to relapse and 12 from complications. All patients who died after relapse had a PTPN11 mutation. In summary, our results suggest that PTPN11-mutated JMML might be a distinct subgroup with specific clinical characteristics and a poor outcome.

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The Transmembrane Domain of PDGFR-β Plays An Important Role in ETV6-PDGFR-β Activation

Blood ◽

10.1182/blood.v112.11.5320.5320 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5320-5320

Author(s):

Federica Toffalini ◽

Jean-Baptiste Demoulin

Keyword(s):

Tyrosine Kinases ◽

Transmembrane Domain ◽

Myeloproliferative Neoplasms ◽

Chromosomal Translocations ◽

Wild Type ◽

Sam Domain ◽

Helix Loop Helix ◽

Hybrid Proteins ◽

Tm Domain ◽

Myelomonocytic Leukemia

Abstract The ETV6-PDGFRβ hybrid protein (EPβ) is found in a subset of patients with chronic myelomonocytic leukemia (CMML) associated with eosinophilia. EPβ is the archetype of a larger group of hybrid receptors that are produced by chromosomal translocations of PDGFR genes and cause atypical myeloproliferative neoplasms. In EPβ, the N-terminal portion of the ETV6 transcription factor replaces the PDGFRβ ligand-binding domain and induces the activation of the chimera through its pointed domain (PNT domain, also called helix-loop-helix or SAM domain). This domain mediates ligand-independent EPβ oligomerization, resulting in constitutive tyrosine kinase activity. In addition, we showed that EPβ and other hybrid receptor tyrosine kinases are much more stable than wild-type receptors and that the deletion of the PNT domain induced EPβ protein degradation, suggesting a link between the clustering and the stabilization of the EPβ protein. The PDGFRβ transmembrane domain (TM domain) is retained in EPβ and in most PDGFRβ hybrid proteins that have been described. We observed that the deletion of the TM domain (EPβ-ΔTM mutant) strongly impaired the ability of EPβ to sustain growth factor-independent proliferation of Ba/F3 and 32D cells. The phosphorylation of the mutant protein was also markedly reduced. We confirmed that EPβ is not inserted in membranes but resides in the cytosol, indicating that the PDGFRβ TM domain does not act as a transmembrane domain in EPβ but has a completely different function. The EPβ-ΔTM mutant retained the ability to self-associate in co-immunoprecipitation experiments, but showed a decreased level of polymerization when using cross-linking agents, suggesting that this domain is required for optimal clustering of EPβ. In line with our findings on the PNT domain, the EPβ-ΔTM protein was less stable and, as a result, was expressed at a lower level. In conclusion, we demonstrate that the TM domain plays a role in EPβ activation by promoting the clustering of the protein and by preventing its degradation in cooperation with the PNT domain.

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Unique Gain-of-Function of Mutated c-CBL Tumor Suppresor in Myeloid Neoplasms.

Blood ◽

10.1182/blood.v114.22.2970.2970 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2970-2970

Author(s):

Masashi Sanada ◽

Takahiro Suzuki ◽

Lee-Yung Shih ◽

Makoto Otsu ◽

Motohiro Kato ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Tyrosine Kinases ◽

E3 Ubiquitin Ligase ◽

Tumour Suppressor ◽

Wild Type ◽

Gain Of Function ◽

Hematopoietic Stem ◽

Myeloid Neoplasms ◽

Ubiquitin Ligase Activity ◽

Type C

Abstract Abstract 2970 Poster Board II-946 Acquired uniparental disomy (aUPD) is a common feature of myeloid neoplasms, especially myelodysplastic syndromes (MDS) / myeloploriferative neoplasms (MPN). aUPDs preferentially affected several chromosomal arms in distinct subsets of patients, and frequently associated with mutated oncogenes and tumour suppressor genes. Among these, the most common aUPDs are those involving 11q, which defined a unique subset of myeloid neoplasms that were clinically characterized by frequent diagnosis of chronic myelomonocytic leukaemia (CMML) with normal karyotypes. Recently, we and other groups reported that 11qUPD are genetically defined by the presence of homozygous mutations of C-CBL. C-CBL proto-oncogene is the cellular homolog of the v-Cbl transforming gene of the Cas NS-1 murine leukemia virus. C-CBL is thought to be involved in the negative modulation of tyrosine kinase signalling, primarily through their E3 ubiquitin ligase activity that is responsible for the down-regulation of activated tyrosine kinases. As expected from the latter function, we demonstrated that wild-type C-CBL has tumour suppressor functions; c-Cbl null mice showed expanded hematopoietic progenitor pools, promoted blastic crisis induced by a bcr/abl transgene, and spontaneous development of late-onset invasive cancers in complete penetrance. On the other hand, mutated C-CBL showed clear oncogenic potential; all tested mutants strongly transformed NIH3T3 fibroblasts, and prolonged replating capacity of hematopoietic progenitors. All reported C-CBL mutations involved the linker-RING finger domains that are central to the E3 ubiquitin ligase activity. We demonstrated that mutated C-CBL not only lost their E3 ubiquitin ligase activity, but also inhibited that of wild-type C-CBL, leading to prolonged activation of a broad spectrum of tyrosine kinases after ligand stimulations in fibroblasts and hematopoietic cells. In accordance with this, c-Cbl−/− hematopoietic stem/progenitor cells (HSPCs) showed enhanced sensitivity to a variety of cytokines, but unexpectedly, transduction of C-CBL mutants into c-Cbl−/− HSPCs further augmented the sensitivity to a broader spectrum of cytokines, indicating the presence of gain-of-function in mutated C-CBL that is not simply mediated by inhibition of wild-type C-CBL functions. The gain-of-function effects of C-CBL mutants on cytokine sensitivity of HSPCs largely disappeared in the c-Cbl+/+ background or by co-transduction of wild-type C-CBL, which may suggest the pathogenic importance of loss of wild-type c-Cbl alleles found in most cases of C-CBL-mutated myeloid neoplasms. Our findings provide a novel insight into a role of gain-of-function mutations of a tumour suppressor associated with aUPD in the pathogenesis of some of myeloid cancer subsets. Currently, further functional studies regarding the molecular mechanism of the gain-of-function are ongoing. Disclosures: Omine: Alexion: Consultancy, Research Funding.

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