Bousquet Michelle S, Ma Jia Jia, Ratnayake Ranjala, Havre Pamela A, Yao Jin, Dang Nam H, Paul Valerie J, Carney Thomas J, Dang Long H, Luesch Hendrik
Institute of Molecular and Cell Biology (IMCB), A*STAR, Proteos , Singapore 138673, Singapore.
Smithsonian Marine Station , 701 Seaway Drive, Fort Pierce, Florida 34949, United States.
ACS Chem Biol. 2016 May 20;11(5):1322-31. doi: 10.1021/acschembio.5b00860. Epub 2016 Mar 3.
Colorectal cancer (CRC) is a genetic disease, due to progressive accumulation of mutations in oncogenes and tumor suppressor genes. Large scale genomic sequencing projects revealed >100 mutations in any individual CRC. Many of these mutations are likely passenger mutations, and fewer are driver mutations. Of these, activating mutations in RAS proteins are essential for cancer initiation, progression, and/or resistance to therapy. There has been significant interest in developing drugs targeting mutated cancer gene products or downstream signaling pathways. Due to the number of mutations involved and inherent redundancy in intracellular signaling, drugs targeting one mutation or pathway have been either ineffective or led to rapid resistance. We have devised a strategy whereby multiple cancer pathways may be simultaneously targeted for drug discovery. For proof-of-concept, we targeted the oncogenic KRAS and HIF pathways, since oncogenic KRAS has been shown to be required for cancer initiation and progression, and HIF-1α and HIF-2α are induced by the majority of mutated oncogenes and tumor suppressor genes in CRC. We have generated isogenic cell lines defective in either oncogenic KRAS or both HIF-1α and HIF-2α and subjected them to multiplex genomic, siRNA, and high-throughput small molecule screening. We have identified potential drug targets and compounds for preclinical and clinical development. Screening of our marine natural product library led to the rediscovery of the microtubule agent dolastatin 10 and the class I histone deacetylase (HDAC) inhibitor largazole to inhibit oncogenic KRAS and HIF pathways. Largazole was further validated as an antiangiogenic agent in a HIF-dependent manner in human cells and in vivo in zebrafish using a genetic model with activated HIF. Our general strategy, coupling functional genomics with drug susceptibility or chemical-genetic interaction screens, enables the identification of potential drug targets and candidates with requisite selectivity. Molecules prioritized in this manner can easily be validated in suitable zebrafish models due to the genetic tractability of the system. Our multidimensional platform with cellular and organismal components can be extended to larger scale multiplex screens that include other mutations and pathways.
结直肠癌（CRC）是一种遗传性疾病，归因于癌基因和肿瘤抑制基因中突变的逐步积累。大规模基因组测序项目显示，任何单个结直肠癌中都存在超过100种突变。其中许多突变可能是过客突变，而驱动突变较少。其中，RAS蛋白的激活突变对于癌症的起始、进展和/或对治疗的抗性至关重要。开发针对突变癌基因产物或下游信号通路的药物引起了极大的兴趣。由于涉及的突变数量以及细胞内信号传导中固有的冗余性，针对一种突变或信号通路的药物要么无效，要么导致快速耐药。我们设计了一种策略，通过该策略可以同时针对多种癌症信号通路进行药物发现。为了进行概念验证，我们靶向致癌性KRAS和HIF信号通路，因为致癌性KRAS已被证明是癌症起始和进展所必需的，并且HIF-1α和HIF-2α在大多数结直肠癌中由突变的癌基因和肿瘤抑制基因诱导产生。我们已经构建了致癌性KRAS缺陷或HIF-1α和HIF-2α均缺陷的同基因细胞系，并对它们进行了多重基因组、siRNA和高通量小分子筛选。我们已经确定了用于临床前和临床开发的潜在药物靶点和化合物。对我们的海洋天然产物文库进行筛选，导致重新发现了微管药物多拉司他汀10和I类组蛋白去乙酰化酶（HDAC）抑制剂拉戈唑，它们可抑制致癌性KRAS和HIF信号通路。使用具有激活的HIF的遗传模型，拉戈唑在人细胞和斑马鱼体内以HIF依赖的方式进一步被验证为抗血管生成剂。我们将功能基因组学与药物敏感性或化学-遗传相互作用筛选相结合的总体策略，能够识别具有必要选择性的潜在药物靶点和候选物。由于该系统的遗传易处理性，以这种方式优先选择的分子可以很容易地在合适的斑马鱼模型中得到验证。我们具有细胞和生物体成分的多维平台可以扩展到更大规模的多重筛选，包括其他突变和信号通路。
