PLEKHA7 A Novel Target for Mutant KRAS Therapy

PLEKHA7 突变 KRAS 治疗的新靶点

• 批准号: 9485728
• 负责人: GARTH POWIS
• 金额: $ 8.28万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-06 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9485728
• 关键词: Adherens Junction; Anchorage-Independent Growth; Automobile Driving; Binding; Binding Proteins; CNKSR1 gene; Cancer Cell Growth; Cancer Patient; Cell Line; Cell membrane; Cell physiology; Cells; Characteristics; Colon Carcinoma; Crystallization; Cytoskeleton; Disease; E-Cadherin; Epithelial Cells; Event; Family; GTP Binding; Genes; Growth; Guanosine Triphosphate; Human; Immunoprecipitation; KRAS2 Gene Mutation; KRAS2 gene; Lead; Legal patent; Ligands; Link; Lipids; Malignant Neoplasms; Medical; Membrane; Membrane Proteins; Microtubules; Molecular; Molecular Target; Monomeric GTP-Binding Proteins; Mutate; Mutation; Neoplasm Metastasis; New Agents; Normal Cell; Oncogenes; Oncogenic; PH Domain; Parents; Patients; Pharmaceutical Preparations; Pharmacology; Phenotype; Phosphatidylinositols; Plasma Proteins; Play; Point Mutation; Positioning Attribute; Property; Proteins; RAS inhibition; Regulation; Roentgen Rays; Role; Signal Pathway; Signal Transduction; Signaling Protein; Site; Small Interfering RNA; Structure; Surface; Techniques; Tight Junctions; Validation; Work; Zinc Fingers; base; cancer cell; cancer type; carcinogenesis; cell motility; colon cancer cell line; design; effective therapy; genetic regulatory protein; imaging study; in vivo; inhibitor/antagonist; knock-down; member; mutant; novel; novel strategies; novel therapeutics; preclinical study; protein folding; protein function; public health relevance; scaffold; small molecule; small molecule inhibitor; therapy resistant; tumor; tumor growth

 DESCRIPTION (provided by applicant): The KRAS oncogene is found in 25% of patient tumors across many cancer types. Despite extensive effort since its discovery over 40 years ago there is no effective treatment for mutated KRAS, and an estimated 350,000 patients in the US in 2014 with mutated KRas in their tumors will die of their disease. Mutated KRAS plays a critical role in driving tumor growth and resistance to therapy. Its effects are so powerful that i overrides the activity of many of the new molecularly targeted signaling drugs being developed for cancer today. Thus, finding new agents that inhibit the effects of mutated KRas is a critical unmet need in cancer today. However, intracellular signaling by wild type KRas protein controls many aspects of normal cell function, so that a therapy directed at inhibiting mutant KRas should ideally leave wild type KRas function unaffected. Using a global siRNA functional screen and isogenic cell line pairs of mutant or wild type KRas we identified PLEKHA7 (pleckstrin homology domain containing, family A7) as a protein that when knocked down inhibits the proliferation of colon cancer cells with mutant KRas, but remarkably not wild type KRas cells. Other aspects of the mutant KRas phenotype were also inhibited including anchorage independent (3D) growth, cell invasion, and in vivo tumor growth. We have also shown that PLEKHA7 knockdown decreases the active (GTP bound) form of mutant but not of wild type KRas, with inhibition of downstream mutant KRas signaling. PLEKHA7 is normally found in the adherens junction of normal epithelial cells. In cancer cells it is found in plasma membrane tight junctions but its function is not known. PLEKHA7 is a member of a group of signaling proteins containing a distinctive 3D protein fold, the pleckstrin homology (PH)-domain, that binds to membrane phosphoinositides to position the parent proteins at specific sites on the membrane important for their function. Our previous studies have shown that PH domains can be selectively drugged by small molecules, thus inhibiting the signaling function of the proteins. We have evidence that in cancer cells PLEKHA7 is associated with proteins of the plasma membrane-bound KRas signaling nanocluster. We thus hypothesize that PLEKHA7 selectively regulates mutated KRas activity in the signaling nanocluster, and therefore that the PH- domain of PLEKHA7 is a target for small molecules inhibitors as pharmacological probes for mechanistic studies of PLEKHA7 function, as well as selectively blocking the growth of mutated KRas cancer cells as potential therapy. This represents a new paradigm for attacking Ras via inhibition of associated regulatory signaling nanocluster proteins. The objectives of our study are: 1) to investigate the mechanism for PLEKHA7's ability to selectively inhibit mutated KRas; 2) to conduct structural studies of the PLEKHA7 PH domain and two potential "hinge" regions, to delineate PLEKHA7's function in associating with the plasma membrane and other proteins of the KRas signaling nanocluster; and 3) to identify small molecule inhibitors of the PLEKHA7 PH- domain as pharmacological probes to study the role of PLEKHA7 in KRas regulation, and as leads for potential agents to treat mutated KRas tumors.

 描述（由申请人提供）：KRAS癌基因存在于多种癌症类型中25%的患者肿瘤中。尽管自40多年前发现以来进行了广泛的努力，但对于突变的KRAS没有有效的治疗方法，2014年美国估计有350，000名肿瘤中突变的KRAS患者将死于他们的疾病。突变的KRAS在驱动肿瘤生长和对治疗的抵抗中起着关键作用。它的作用是如此强大，以至于我超越了许多新的分子靶向信号药物的活性，这些药物目前正在开发用于癌症。因此，寻找抑制突变的KRas作用的新药物是当今癌症中一个关键的未满足的需求。然而，野生型KRas蛋白的细胞内信号传导控制正常细胞功能的许多方面，使得针对抑制突变型KRas的疗法理想地应使野生型KRas功能不受影响。使用全局siRNA功能筛选和突变型或野生型KRas的同基因细胞系对，我们鉴定了PLEKHA 7（含有普列克底物同源结构域，家族A7）作为一种蛋白质，其在被敲低时抑制具有突变型KRas的结肠癌细胞的增殖，但显著地不抑制野生型KRas细胞的增殖。突变KRas表型的其他方面也受到抑制，包括锚定非依赖性（3D）生长，细胞侵袭和体内肿瘤生长。我们还表明，PLEKHA 7敲低降低了突变型KRas的活性（GTP结合）形式，但不降低野生型KRas的活性（GTP结合）形式，抑制了下游突变型KRas信号传导。PLEKHA 7通常存在于正常上皮细胞的粘附连接中。在癌细胞中，它存在于质膜紧密连接中，但其功能尚不清楚。PLEKHA 7是一组信号蛋白的成员，其含有独特的3D蛋白折叠，即普列克底物蛋白同源（PH）结构域，其结合膜磷酸肌醇以将亲本蛋白定位在对其功能重要的膜上的特定位点。我们以前的研究表明，PH结构域可以被小分子选择性地药物化，从而抑制蛋白质的信号传导功能。我们有证据表明，在癌细胞中，PLEKHA 7与质膜结合的KRas信号纳米簇的蛋白质相关。因此，我们假设PLEKHA 7选择性地调节信号传导纳米簇中突变的KRas活性，因此PLEKHA 7的PH结构域是小分子抑制剂的靶标，作为PLEKHA 7功能的机制研究的药理学探针，以及选择性地阻断突变的KRas癌细胞的生长作为潜在的治疗。这代表了通过抑制相关的调节信号纳米簇蛋白来攻击Ras的新范例。本研究的目的是：1）研究PLEKHA 7选择性抑制突变型KRas的机制; 2）对PLEKHA 7的PH结构域和两个潜在的“铰链”区域进行结构研究，以阐明PLEKHA 7与质膜和KRas信号纳米簇的其他蛋白的结合功能;和3）鉴定PLEKHA 7 PH-结构域的小分子抑制剂作为药理学探针以研究PLEKHA 7在KRas调节中的作用，以及作为治疗突变的KRas肿瘤的潜在药剂的先导。