Adaptive regulation of cancer cell fate following oncogene inhibition

癌基因抑制后癌细胞命运的适应性调节

• 批准号: 8868342
• 负责人: Mohammad Fallahi-Sichani
• 金额: $ 12.02万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-17 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8868342
• 关键词: Accounting; Affect; Antineoplastic Agents; Apoptosis; Apoptotic; Autocrine Communication; Award; Biochemical; Biochemistry; Biological Assay; Biological Markers; Biology; Cell Death; Cell Line; Cell Surface Receptors; Cells; Coculture Techniques; Collaborations; Colon Carcinoma; Combined Modality Therapy; Computer Simulation; Dana-Farber Cancer Institute; Data; Disease; Disease remission; Drug Targeting; Drug resistance; Effectiveness; Engineering; Environment; Feedback; Foundations; Future; Geminin; Genotype; Goals; Gray unit of radiation dose; Growth; Health; Heterogeneity; Immunofluorescence Immunologic; Individual; Inflammatory; JUN gene; Knowledge; Life; Ligands; Link; MAPK8 gene; MEK inhibition; MEKs; Macrophage Activation; Mass Spectrum Analysis; Measurement; Measures; Mediating; Melanoma Cell; Memorial Sloan-Kettering Cancer Center; Mentors; Mentorship; Methods; Microscopy; Modeling; Molecular; Oncogene Proteins; Oncogenes; Oncogenic; Pathway interactions; Patients; Pharmaceutical Preparations; Phase; Phenotype; Protein Array; Proteins; Regulation; Research Personnel; Resistance; Ribosomal Protein S6; Role; Signal Transduction; Statistical Models; Systems Biology; Techniques; Testing; Therapeutic; Time; Training; Training Activity; Transcriptional Activation; Tumor Biology; Work; base; cancer cell; cancer pharmacology; cancer type; career; cell fixing; cell killing; cell type; cellular engineering; cellular imaging; design; drug efficacy; drug sensitivity; improved; inhibitor/antagonist; innovation; macrophage; medical schools; melanoma; neoplastic cell; novel; novel strategies; prevent; receptor; resistance mutation; response; senescence; single cell analysis; skills; success; therapeutic effectiveness; therapeutic target; transcription factor; tumor; tumor microenvironment

 DESCRIPTION (provided by applicant): The overall goal of this proposal is to develop a single-cell, and network-level understanding of cell signaling mechanisms involved in adaptive drug responses through the application of engineering and systems biology approaches. I have chosen melanoma and drugs targeting the BRAFV600E oncoprotein, since adaptation is well known to be important in this type of cancer. Treatment of BRAFV600E melanomas with drugs, such as vemurafenib, that inhibit RAF/MEK signaling is effective in the short term, but remission is not durable. Drug resistance is thought to involve short-term adaptive responses that up-regulate compensatory pro-growth and/or anti-apoptotic mechanisms. The discovery and analysis of adaptive responses in melanoma represents a breakthrough in tumor biology and reveals hitherto unsuspected plasticity in signaling biochemistry. However, systematic data comparing BRAFV600E tumor cells is generally lacking and important questions are unanswered. It is not known whether adaptive mechanisms in different cell types are fundamentally similar or they are different from one cell type to the next or even one single cell to the next. Further, it is not clear how different adaptive responses are related to each other, how they are affected by tumor microenvironment, and how they are integrated to determine the fate of an individual cell. Answering these questions is critical for developing single or multi-component biomarkers of drug responsiveness and for designing rational and effective combination therapies to overcome drug adaptation and ultimately drug resistance. Our previous studies show that adaptive responses are diverse across melanomas, involving different combinations of signaling cascades. In particular, we identified an adaptive mechanism involving JNK/c-Jun that diminishes drug efficacy. RAF and JNK inhibitors induce synergistic cell killing in melanoma cells in which c-Jun mediated adaptive response occurs. Single-cell studies show that JNK inhibition enhances suppression of phospho-S6 ribosomal protein, promotes apoptosis in a subset of cells that would otherwise become quiescent and apoptosis-resistant in the presence of vemurafenib alone, and increases drug maximal effect (Emax). This work identified involvement of different pathways in adaptive responses, their diversity with genotype and time, and suggested that it would be critical to examine the diverse phenotypes induced by BRAFV600E inhibition at a single-cell level. Therefore, in this proposal I will couple high-throughput measurement, fixed and live single- cell analysis, and a combination of statistical and mechanistic computational modeling techniques to: (1) identify key molecules (ligands, receptors and transcription factors) linked to JNK/c-Jun mediated adaptive response and crosstalk with other adaptive responses in a set of BRAFV600E melanoma cell lines and primary patient-derived melanoma cells having different genotypes, (2) develop network-level models of adaptive response which discriminate among key adaptive network states observed across different cell types and their association with phenotypic responses and drug sensitivity, (3) assess the diversity and magnitude of adaptive responses across individual cells and determine their association with individual cell phenotypes (proliferation, quiescence, senescence, cell death, etc.), (4) investigate the contribution of other cell types within the tumo microenvironment, in particular tumor-associated macrophages, in drug-induced adaptive and phenotypic responses, and (5) utilize these data to identify mechanism-based biomarkers for different pathway adaptations, use these biomarkers to design and test novel combination therapeutics that take into account malignant cells, the tumor microenvironment, and the dynamics exerted by the treatment itself. The success of these studies is directly linked to the proposed training activities I intend to undertake during the mentored phase of this award. I believe that with my extensive engineering and computational background, being awarded a K99/R00 award will allow me to deepen my understanding of tumor biology (concentrating initially on melanoma) and to obtain advanced training in a highly supportive and innovative training environment of Harvard Medical School. In addition, to support me in my training, and in my transition to the independent phase of my career, I will be benefitting from the mentorship and collaboration with leading experts in the fields I propose studying. This includes my mentor Dr. Peter Sorger (Harvard Medical School), and collaborators Dr. Neal Rosen (Memorial Sloan Kettering Cancer Center), Dr. Nathanael Gray (Dana Farber Cancer Institute), and Dr. Steve Gygi (Harvard Medical School). The skills and knowledge acquired during the mentored phase of this award will be instrumental for the above proposed studies and future studies, and for successfully launching my career as an independent investigator.

 描述（由申请人提供）：本提案的总体目标是通过应用工程和系统生物学方法，对适应性药物反应中涉及的细胞信号传导机制进行单细胞和网络水平的理解。我选择了黑色素瘤和靶向BRAFV 600 E癌蛋白的药物，因为众所周知，适应在这类癌症中很重要。用抑制RAF/MEK信号传导的药物（如vemurafenib）治疗BRAFV 600 E黑色素瘤在短期内有效，但缓解不持久。耐药性被认为涉及上调补偿性促生长和/或抗凋亡机制的短期适应性反应。黑色素瘤适应性反应的发现和分析代表了肿瘤生物学的突破，并揭示了迄今为止未被怀疑的信号生物化学可塑性。然而，比较BRAFV 600 E肿瘤细胞的系统数据通常缺乏，重要的问题尚未得到解答。目前尚不清楚不同细胞类型的适应机制是否基本相似，或者它们在一种细胞类型与另一种细胞类型之间或甚至在一个细胞与另一个细胞之间是不同的。此外，目前还不清楚不同的适应性反应如何相互关联，它们如何受到肿瘤微环境的影响，以及它们如何整合以决定单个细胞的命运。解决这些问题对于开发药物反应性的单一或多组分生物标志物以及设计合理有效的联合治疗以克服药物适应性并最终克服耐药性至关重要。我们以前的研究表明，适应性反应是不同的黑色素瘤，涉及不同的信号级联组合。特别是，我们确定了一种涉及JNK/c-Jun的适应性机制，这种机制会降低药物疗效。RAF和JNK抑制剂诱导黑色素瘤细胞中的协同细胞杀伤，其中发生c-Jun介导的适应性反应。单细胞研究表明，JNK抑制增强了对磷酸化-S6核糖体蛋白的抑制，促进了在单独使用维罗非尼的情况下原本会变得静止和抗凋亡的细胞亚群中的凋亡，并增加了药物最大效应（Emax）。这项工作确定了适应性反应中不同途径的参与，它们随基因型和时间的多样性，并表明在单细胞水平上检查BRAFV 600 E抑制诱导的不同表型至关重要。因此，在本提案中，我将结合高通量测量、固定和活单细胞分析以及统计和机械计算建模技术的组合，以：（1）识别关键分子（配体，受体和转录因子）与JNK/c-Jun介导的适应性反应以及与一组BRAFV 600 E黑色素瘤细胞系和原发性患者中的其他适应性反应的串扰相关。衍生的具有不同基因型的黑色素瘤细胞，（2）开发适应性反应的网络水平模型，其区分在不同细胞类型中观察到的关键适应性网络状态及其与表型反应和药物敏感性的关联，（3）评估个体细胞之间适应性反应的多样性和幅度，并确定它们与个体细胞表型的关联（增殖、静止、衰老、细胞死亡等），(4)研究肿瘤微环境中的其它细胞类型，特别是肿瘤相关巨噬细胞，在药物诱导的适应性和表型应答中的作用，和（5）利用这些数据来鉴定不同途径适应的基于机制的生物标志物，使用这些生物标志物来设计和测试考虑恶性细胞，肿瘤微环境，以及治疗本身所产生的动力。这些研究的成功与我打算在该奖项的指导阶段开展的拟议培训活动直接相关。我相信，凭借我广泛的工程和计算背景，被授予K99/R 00奖将使我加深对肿瘤生物学的理解（最初专注于黑色素瘤），并在哈佛医学院高度支持和创新的培训环境中获得高级培训。此外，为了支持我的培训，并在我过渡到我的职业生涯的独立阶段，我将受益于指导和与我建议学习的领域的领先专家的合作。这包括我的导师Peter Sorger博士（哈佛医学院），以及合作者Neal罗森博士（纪念斯隆凯特琳癌症中心），Nathanael Gray博士（达纳法伯癌症研究所）和Steve Gygi博士（哈佛医学院）。在该奖项的指导阶段获得的技能和知识将有助于上述拟议的研究和未来的研究，并成功地启动我的职业生涯作为一个独立的调查员。