Protein Phosphatase PP2A and DNA damage in cell fate decisions of acute myeloid leukemic cells

项目摘要 急性髓系白血病（AML）是一种以造血成熟的协同阻滞为特征的癌症 和增殖优势。它几乎无法治愈，5年生存率仅为25%， 65岁以上患者<1年。目前的化疗耐受性非常差，大多数患者会复发。 克服分化阻滞是一种有前途的治疗途径，但对于AML主要缺乏 没有已知的驱动致癌基因的亚型。我们的研究探讨了针对这两个主要方面的新途径 白血病细胞存活，分化阻滞和增殖能力增加， 在多种亚型之间。 可逆的蛋白质磷酸化控制着这些细胞命运决定的许多方面，而蛋白质磷酸化过程中的不平衡也是一个重要因素。 激酶和磷酸酶的正常活性有助于AML的发病机制。然而，尽管异常的 在AML中常见的激酶的活化被充分研究并用于治疗目的， 在AML中也普遍存在的磷酸酶常常被忽视。该提案侧重于丝氨酸/苏氨酸 磷酸酶肿瘤抑制蛋白磷酸酶2A（PP 2A）及其在影响细胞增殖与 AML中的差异化决策。对于F99期，我们确定了PP 2A在驱动髓样细胞增殖中的新作用。 分化和细胞周期停滞。根据我们的初步数据，我们的总体假设是PP 2A 通过细胞周期调节因子p21驱动分化和细胞周期停滞。我们特别寻求建立 PP 2A介导p21诱导的机制及潜在下游因子的作用 蛋白和CEBP蛋白在AML中使用基因过表达和敲低驱动分化 系统、CHIP-Seq和使用质谱流式细胞术的单细胞分析。 K 00阶段集中于鉴定介导DNA损伤诱导分化的因子。而DNA 损伤已显示诱导AML和造血祖细胞的分化， 机制知之甚少。有趣的是，DNA修复机制包括ATM和ATR丝氨酸苏氨酸， 激酶保护白血病细胞免受损伤诱导的分化，并在AML中过度活化。这就提出 抑制DNA修复可能促进基因组胁迫诱导的分化。我的假设是 K 00期是指PP 2A能拮抗DNA修复激酶并促进对DNA应答的分化 AML中的损伤。我将确定PP 2A和根据初步数据确定的其他潜在候选人的角色 和文献，如GADD 45蛋白和p21在DNA损伤诱导的AML分化中使用遗传学方法， PP 2A的药理学激活，GADD 45的过表达、敲低和敲除，以及GADD 45的整体表达。 体外和体内RNAi和全基因组CRISPER筛选分析。成功完成本提案 将产生新的见解，细胞途径能够诱导生长停滞和终末分化， 成熟阻断了诸如AML的癌症。

PROJECT SUMMARY Acute Myeloid Leukemia (AML) is a cancer characterized by a co-operative block in hematopoietic maturation and proliferative advantage. It is near incurable, with a 5-year survival rate of only 25% and median survival <1year in patients over 65 years. Current chemotherapy is very poorly tolerated and majority of patients relapse. Overcoming the differentiation block is a promising therapeutic avenue, however is majorly lacking for AML subtypes without a known driver oncogene. Our study examines new pathways to target the two major aspects of leukemic cell survival, the differentiation block and increased proliferative ability, that can prove effective across multiple subtypes. Reversible protein phosphorylation controls many aspects of such cell fate decisions, and an imbalance in the normal activities of kinases and phosphatases contribute to pathogenesis of AML. However, while the aberrant activation of kinases, frequent in AML, is well studied and exploited for therapeutic purposes, inactivation of phosphatases, also prevalent in AML, is often overlooked. This proposal focuses on the serine threonine phosphatase tumor suppressor Protein Phosphatase 2A (PP2A) and its role in influencing proliferation vs differentiation decisions in AML. For the F99 phase, we identified a novel role for PP2A in driving myeloid differentiation and cell cycle arrest in AML. Based on our preliminary data, our overall hypothesis is that PP2A drives differentiation and cell cycle arrest through the cell cycle regulator p21. We specifically seek to establish the mechanism of PP2A mediated p21 induction, and the role of potential downstream factors Retinoblastoma protein and CEBP proteins in driving differentiation in AML using genetic overexpression and knockdown systems, CHIP-Seq and single cell analysis using Mass Cytometry. The K00 phase focuses on identification of factors mediating DNA damage induced differentiation. While DNA damage has been shown to induce differentiation in AML and hematopoietic progenitors, the molecular mechanism is poorly understood. Interestingly, DNA repair machinery including ATM and ATR serine threonine kinases protect leukemic cells from damage induced differentiation and are hyperactivated in AML. This raises the possibility that inhibiting DNA repair can promote genomic stress induced differentiation. My hypothesis for the K00 phase is that PP2A can antagonize DNA repair kinases and promote differentiation in response to DNA damage in AML. I will establish the role of PP2A and other potential candidates identified from preliminary data and literature such as GADD45 proteins and p21 in DNA damage induced differentiation in AML using genetic and pharmacological activation of PP2A, overexpression, knockdown and knockout of GADD45 as well as global in-vitro and in-vivo RNAi and genome wide CRISPER screen analyses. Successful completion of this proposal will yield new insight into cellular pathways capable of inducing growth arrest and terminal differentiation of maturation blocked cancers such as AML.