Molecular Pathogenesis of MDS and CMML

MDS 和 CMML 的分子发病机制

• 批准号: 7767246
• 负责人: Jaroslaw P Maciejewski
• 金额: $ 39.25万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-29 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7767246
• 关键词: Acquired uniparental disomy; Acute Myelocytic Leukemia; Adopted; Affect; Area; Blood Cells; Bone Marrow; CD3 Antigens; Cells; Chromosome abnormality; Chronic Myelomonocytic Leukemia; Clinical; Complement; Cytogenetic Analysis; Cytogenetics; DNA; Defect; Detection; Development; Diagnosis; Diagnostic; Disease; Disease Progression; Dysmyelopoietic Syndromes; Dysplasia; Evolution; Fingers; Frequencies; Gene Mutation; Genes; Genome Scan; Genomics; Germ Lines; Hematopoiesis; Hematopoietic stem cells; Heterogeneity; In Vitro; Individual; Investigation; Karyotype determination procedure; Knock-in Mouse; Lead; Lesion; Link; Loss of Heterozygosity; Maps; Marrow; Mediating; Metaphase; Methods; Molecular; Molecular Target; Mutation; Myelodysplastic/Myeloproliferative Disease; Oncogenes; Oncogenic; Outcome; Pancytopenia; Pathogenesis; Pathologic; Pathway interactions; Patients; Play; Production; Receptor Protein-Tyrosine Kinases; Recurrence; Resolution; Ring Finger Domain; Risk; Role; Sampling; Scheme; Signal Transduction; Signal Transduction Pathway; Single Nucleotide Polymorphism; Sorting - Cell Movement; Testing; Translations; Tumor Suppressor Proteins; Unbalanced Translocation; Uniparental Disomy; Variant; alpha-Thalassemia; attenuation; base; clinical phenotype; clinical practice; cytopenia; design; improved; leukemia; mutant; new therapeutic target; outcome forecast; prognostic; proto-oncogene protein c-cbl; public health relevance; src-Family Kinases; theories; therapeutic target; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): In myelodysplastic syndrome (MDS) chromosomal abnormalities play an important diagnostic and prognostic role, pointing towards possible disease-causing pathways. However, using traditional metaphase cytogenetics (MC), chromosomal lesions can be found in only about 50% of patients; higher resolution methods may allow for detection of cryptic chromosomal lesions in patients with normal and abnormal MC. Detection of such defects could facilitate identification of novel therapeutic targets, aid diagnosis and improve current prognostic schemes by explaining phenotypic heterogeneity within established MDS subtypes. Single nucleotide polymorphism arrays (SNP-A) are a new method for high resolution karyotyping and detection of unbalanced DNA defects including somatic uniparental disomy (UPD). We have demonstrated that somatic UPD is very frequent in MDS and hypothesized that shared regions of UPD may point towards mutations of genes involved in the development of MDS. In particular areas of somatic UPD have been linked to the presence of biallelic mutations, such as Jak2 (UPD9p) and Ftl3 (UPD13q). Based on this theory we mapped invariant UPD and deletions in a large group of patients with MDS and found that UPD11q was particularly frequent in patients with chronic myelomonocytic leukemia. By sequencing the c-Cbl gene, an E3 ubiquitin ligase in this chromosomal region, a mutation involving a critical part of this gene was identified. In this proposal we plan to test the clinical utility of SNP-A karyotyping and establish its impact on prognosis, clinical phenotype and the discovery of new molecular targets in MDS. In SA1 we will analyze paired samples (bone marrow and sorted CD3+ cells as a clonal control) from patients with MDS using the Affymetrix 6.0 SNP array. New invariant clonal lesions, in particular somatic UPD, will be identified, mapped and their impact on clinical phenotypes, survival and progression analyzed. The influence of newly detected lesions will be assessed in the context of IPSS to improve prognostic accuracy. In SA2, as a proof of concept that recurrent somatic UPD points towards important mutations, we will analyze in detail patients with UPD11q and c-Cbl mutations and determine the frequency and clinical outcomes of this mutation associated with this lesion. We will perform in vitro studies to delineate the effect of c-Cbl mutations on tyrosine kinase receptors and Src family kinases and explain whether pathologic mechanisms are mediated by mutant c-Cbl. For example, we will compare functional consequences of ring finger mutant knock-in vs. c-Cbl protein knockdown. Improved cytogenetic diagnostics using SNP-A karyotyping may contribute to a better understanding of the molecular pathogenesis of MDS and has the potential of identification of genes involved in the pathogenesis of MDS, perhaps pointing towards therapeutic targets for development of molecular therapies. Clinically, SNP-A-based karyotyping may complement cytogenetic diagnosis and, through the detection of previously cryptic chromosomal lesions (including UPD), may allow for better prognostic assignment. PUBLIC HEALTH RELEVANCE: Myelodysplastic syndromes (MDS) is a heterogeneous group of bone marrow failure states characterized by dysplastic hematopoiesis, deficient blood cell production and a propensity to progression to acute myelogenous leukemia (AML); this heterogeneity has greatly impeded investigations into the molecular pathogenesis and potential therapies for these diseases. We propose that single nucleotide polymorphisms arrays (SNP-A) can be applied, complementary to metaphase cytogenetics for the identification of chromosomal abnormalities, including a newly recognized class of lesion, somatic uniparental disomy. We will precisely map these lesions, identifying genes that may play a role in the disease and potentially act as targets of therapy.

描述（由申请人提供）：在骨髓增生异常综合征（MDS）中，染色体异常发挥着重要的诊断和预后作用，指出了可能的致病途径。然而，使用传统的中期细胞遗传学（MC），只有约50%的患者能够发现染色体病变；更高分辨率的方法可以检测正常和异常 MC 患者的隐蔽染色体病变。检测此类缺陷可以通过解释已建立的 MDS 亚型内的表型异质性，促进新治疗靶点的识别、帮助诊断并改善当前的预后方案。 单核苷酸多态性芯片 (SNP-A) 是一种用于高分辨率核型分析和检测不平衡 DNA 缺陷（包括体细胞单亲二体性 (UPD)）的新方法。我们已经证明体细胞 UPD 在 MDS 中非常常见，并假设 UPD 的共享区域可能指向参与 MDS 发展的基因突变。体细胞 UPD 的特定区域与双等位基因突变的存在有关，例如 Jak2 (UPD9p) 和 Ftl3 (UPD13q)。基于这一理论，我们绘制了一大群 MDS 患者的不变 UPD 和缺失图谱，发现 UPD11q 在慢性粒单核细胞白血病患者中特别频繁。通过对 c-Cbl 基因（该染色体区域中的一种 E3 泛素连接酶）进行测序，鉴定出了涉及该基因关键部分的突变。 在本提案中，我们计划测试 SNP-A 核型分析的临床实用性，并确定其对 MDS 预后、临床表型和新分子靶点发现的影响。在 SA1 中，我们将使用 Affymetrix 6.0 SNP 阵列分析 MDS 患者的配对样本（骨髓和分选的 CD3+ 细胞作为克隆对照）。新的不变克隆病变，特别是体细胞 UPD，将被识别、绘制，并分析它们对临床表型、生存和进展的影响。新检测到的病变的影响将在 IPSS 的背景下进行评估，以提高预后准确性。在 SA2 中，作为复发性体细胞 UPD 指向重要突变的概念证明，我们将详细分析 UPD11q 和 c-Cbl 突变的患者，并确定与该病变相关的该突变的频率和临床结果。我们将进行体外研究来描述 c-Cbl 突变对酪氨酸激酶受体和 Src 家族激酶的影响，并解释病理机制是否由突变型 c-Cbl 介导。例如，我们将比较无名指突变体敲入与 c-Cbl 蛋白敲低的功能后果。 使用 SNP-A 核型分析改进细胞遗传学诊断可能有助于更好地了解 MDS 的分子发病机制，并具有鉴定参与 MDS 发病机制的基因的潜力，或许可以为分子疗法的开发指明治疗靶点。临床上，基于 SNP-A 的核型分析可以补充细胞遗传学诊断，并且通过检测先前隐匿的染色体病变（包括 UPD），可以实现更好的预后分配。 公共卫生相关性：骨髓增生异常综合征 (MDS) 是一组异质性骨髓衰竭状态，其特征是造血发育不良、血细胞生成不足以及有进展为急性髓性白血病 (AML) 的倾向；这种异质性极大地阻碍了对这些疾病的分子发病机制和潜在疗法的研究。我们建议可以应用单核苷酸多态性阵列（SNP-A），作为中期细胞遗传学的补充，用于鉴定染色体异常，包括新认识的一类病变，体细胞单亲二体性。我们将精确地绘制这些病变的图谱，识别可能在疾病中发挥作用并可能作为治疗靶点的基因。