Identification and Characterization of Genes that Suppress BCR-ABL+ Leukemia

描述(申请人提供)：某些促进实体肿瘤的癌基因，如RAS和BRAF，在原代细胞中表达时会诱导衰老。癌基因诱导的衰老通过阻止有肿瘤转化风险的细胞的增殖而在抑制肿瘤发生方面发挥重要作用。因此，在许多情况下，参与癌基因诱导衰老的基因，如TP53和RB1，也是肿瘤抑制基因。Bcr-abl是一种来源于9号和22号染色体易位的致癌蛋白，它可以转化髓系祖细胞，并推动绝大多数慢性粒细胞白血病(CML)和20%-30%的成人急性淋巴细胞白血病(ALL)的发生。我们最近发现，BCR-ABL以及另外两种白血病融合蛋白Cbfb-MYH11和RUNX1-ETO，与RAS和BRAF一样，可以诱导原代人成纤维细胞和造血祖细胞衰老。我们的结果暗示，BCR-ABL+白血病的发展涉及到基因和/或表观遗传学改变，使一个或多个促进衰老的基因失活。与这一假说一致，在CML小鼠模型中，已知的促进衰老的基因如TP53和CDKN2A(p16INK4a/p14ARF)的失活可以与BCR-ABL协同作用，CDKN2A缺失在BCR-ABL+ALL患者中常见。值得注意的是，这些相同的基因改变也可以使人对伊马替尼产生抵抗力，伊马替尼是一种bcr-abl激酶活性的抑制剂， 慢性粒细胞白血病的一线治疗。因此，描绘BCR-ABL诱导原代细胞衰老的基因和调控途径将有助于识别与BCR-ABL协同促进白血病发生并导致伊马替尼耐药的基因改变。为了实现这一长期目标，我们开展了大规模的RNA干扰筛选，以确定其被敲除的基因使原代细胞能够克服BCR-ABL诱导的衰老。这些基因代表了bcr-abl+白血病的候选抑制基因。在这个应用中，我们提出了涉及CML和ALL的培养细胞和小鼠模型的实验，以确定这些候选基因在抑制bcr-abl+白血病中的作用。已证实的白血病抑制基因将被进一步研究，以深入了解它们如何促进衰老，并确定它们发挥作用的调控途径。最后，将测试已证实的白血病抑制基因在对伊马替尼和达沙替尼(主要的第二代酪氨酸激酶抑制剂)的耐药性发展中的作用。这些实验的结果将提高我们对bcr-abl+白血病发展的理解，揭示潜在的治疗干预新靶点，并有助于预测患者对治疗的反应性。

DESCRIPTION (provided by applicant): Certain oncogenes that promote solid tumors, such as RAS and BRAF, induce senescence when expressed in primary cells. Oncogene-induced senescence plays an important role in suppressing tumorigenesis by preventing proliferation of cells at risk for neoplastic transformation. Thus, in many instances, genes involved in oncogene-induced senescence, such as TP53 and RB1, are also tumor suppressors. BCR-ABL is an oncogenic kinase derived from the translocation between chromosomes 9 and 22 that can transform myeloid progenitor cells and drives the development of the vast majority chronic myeloid leukemia (CML) cases and 20%-30% of adult acute lymphoblastic leukemia (ALL) cases. We have recently found that BCR-ABL, as well as two other leukemogenic fusion-proteins, CBFB-MYH11 and RUNX1-ETO, can, like RAS and BRAF, induce senescence in primary human fibroblasts and hematopoietic progenitors. Our results imply that the development of BCR-ABL+ leukemias involves genetic and/or epigenetic alterations that inactivate one or more senescence-promoting genes. Consistent with this hypothesis, inactivation of genes known to promote senescence, such as TP53 and CDKN2A (p16INK4a/p14ARF), can cooperate with BCR-ABL in mouse models of CML, and CDKN2A deletions are frequently found in patients with BCR-ABL+ ALL. Significantly, these same genetic alterations can also confer resistance to imatinib, an inhibitor of BCR-ABL kinase activity that is the first-line treatment for CML. Thus, delineating the genes and regulatory pathways by which BCR-ABL induces senescence in primary cells will help identify genetic alterations that cooperate with BCR-ABL to promote leukemogenesis and are responsible for the emergence of imatinib resistance. Toward this long-term objective, we have carried out a large-scale RNA interference screen to identify genes whose knockdown enables primary cells to overcome BCR-ABL-induced senescence. These genes represent candidate suppressors of BCR-ABL+ leukemias. In this application, we propose experiments involving both cultured cells and mouse models of CML and ALL to determine the role of these candidates in suppression of BCR-ABL+ leukemias. The confirmed leukemia suppressor genes will be further studied to gain insights into how they promote senescence and to identify the regulatory pathways in which they function. Finally, confirmed leukemia suppressor genes will be tested for a role in the development of resistance to imatinib as well as dasatinib, the major second-generation tyrosine kinase inhibitor. The results of these experiments will improve our understanding of BCR-ABL+ leukemia development, reveal potential new targets for therapeutic intervention, and help predict patient responsiveness to treatment.