Hypoxia-induced mutagenesis and resistance to targeted therapy

 描述（由申请人提供）：发现癌细胞含有其生存所必需的突变蛋白，这导致了特异性抑制癌细胞的靶向治疗的发展。这些高度特异性的药物已经显示出令人鼓舞的临床结果，并且在某些情况下几乎可以完全缓解肿瘤。然而，一个不可避免的和知之甚少的限制是癌细胞产生耐药性的能力，绕过或阻断治疗效果。在这个提议中，我们试图从机制上理解肿瘤如何能够快速进化并在治疗中存活。通过机械地探测肿瘤微环境对肿瘤细胞的影响，特别是低氧应激现象，发现了两种在低氧应激期间增加突变的新途径。这两种途径都作用于DNA修复机制，降低高保真同源重组（HR）修复，并促进低保真度、易错的非同源末端连接（NHEJ）。这种向NHEJ的转换与新突变和拷贝数变异的发展有关，这两种突变和拷贝数变异都是基因组不稳定性的特征和治疗耐药性的潜在机制。本研究提案的机制目标是恢复HR修复，从而降低致突变性NHEJ对肿瘤进展的影响。在靶向治疗的背景下，这可以减少导致耐药突变的发展。这将通过研究缺氧刺激HR/NHEJ诱变转换的两种潜在机制来完成。(Aim 1）我们已经发现，缺氧的主要转录调节因子HIF-1介导了关键HR蛋白Rad 51的蛋白酶体降解，同时促进了NHEJ蛋白的活化和表达。将阐明Rad 51降解的机制，并使用HR功能和基因组不稳定性测定进行拯救实验。(Aim 2）我们对多个全基因组RNA测序筛选的生物信息学分析发现了3个新的HIF-1靶基因，它们被认为是HR的负调控因子。有趣的是，它们都是线粒体代谢酶，即精胺合成酶、6-磷酸果糖-2-激酶/果糖-2，6-二磷酸酶4和甘露糖磷酸异构酶。这类新的代谢酶，被称为SIMME，或应激诱导的代谢突变酶，提供了线粒体中的应激反应和细胞核中增加的诱变之间的新联系，既促进细胞存活和应激后的适应;线粒体通过调节能量使用，细胞核通过促进新突变的积累。这些酶减少HR修复的机制将使用siRNA沉默，蛋白质过表达和CRISPR介导的基因编辑来探索，以创建敲除和点突变体。最后，将通过在小鼠中植入SIMME突变体肿瘤，同时用靶向治疗来探索挽救HR对靶向治疗敏感性的影响。该项目的长期目标是利用这些靶点和机制来预防微环境诱导的诱变，开发药物来预防或延迟接受靶向治疗的患者的耐药性。

 DESCRIPTION (provided by applicant): The discovery that cancer cells harbor mutated proteins necessary for their survival has led to the development of targeted therapies to specifically inhibit them. These highly specific drugs have shown encouraging clinical results, and almost total tumor remission is possible in some instances. However, an inevitable and poorly- understood limitation is the ability of cancer cells to develop resistance, bypassing or blocking the therapies effect. In this proposal, we seek to mechanistically understand how tumors are able to quickly evolve and survive therapy. By mechanistically probing the effects of tumor microenvironment on tumor cells, specifically the phenomenon of hypoxic stress, two novel pathways that increase mutagenesis during low-oxygen stress were discovered. Both of these pathways act on DNA-repair mechanisms, decreasing high-fidelity homologous recombination (HR) repair, and promoting low-fidelity, error-prone non-homologous end joining (NHEJ). This switch to NHEJ is associated with the development of new mutations and copy number variations, both traits of genomic instability and potential mechanisms of therapy resistance. The mechanistic goal of this research proposal is to restore HR repair, thus decreasing the impact of mutagenic NHEJ on tumor progression. In the context of targeted therapy, this could reduce the development of resistance-causing mutations. This will be done by investigating two potential mechanisms by which hypoxia stimulates the HR/NHEJ mutagenic switch. (Aim 1) We have found that the master transcriptional regulator for hypoxia, HIF-1, mediates the proteasomal degradation of Rad51, a key HR protein, while promoting the activation and expression of NHEJ proteins. The mechanism of the Rad51 degradation will be elucidated, and rescue experiments will be performed using assays for HR function and genomic instability. (Aim 2)Our bioinformatics analysis of multiple genome-wide RNA sequencing screens has identified 3 novel HIF-1 target genes that are putative negative regulators of HR. Intriguingly, these are all mitochondrial metabolic enzymes, namely spermine synthase, 6-phosphofructo-2- kinase/fructose-2,6-biphosphatase 4, and mannose phosphate isomerase. This novel class of metabolic enzymes, dubbed SIMME, or stress-induced metabolic mutator enzymes, provides a novel connection between stress response in the mitochondria and increased mutagenesis in the nucleus, both promoting cell survival and adaptation following stress; the mitochondria by modulating energy usage, and the nucleus by promoting the accumulation of new mutations. The mechanism by which these enzymes decrease HR repair will be probed using siRNA silencing, protein overexpression, and CRISPR-mediated genetic editing to create knock-outs and point mutants. Finally, the effect of rescuing HR on the sensitivity to targeted therapy will be explored by implanting SIMME mutant tumors in mice, while treating with targeted therapy. The long-term goal of this project is to use these targets and mechanisms to prevent microenvironment-induced mutagenesis, developing agents to prevent or delay the onset of resistance in patients receiving targeted therapy.