Cancer Resistant Mice

项目总结 生殖系突变是否会对致命性癌症产生强大的抵抗力？某些生殖系基因类型可能 对肿瘤血管形成、营养需求或对免疫攻击的抵抗力支持较差 与宿主生存相容。特别有趣的是，一些突变可能会教唆宿主对neo- 抗原，甚至到自身抗原在同基因肿瘤中高表达。抗性鉴定 突变可能为癌症治疗提供新的方法和靶点。至少在人类群体中， 耐药突变将很难识别。人类生殖系遗传变异性，干性变异性 癌症基因组，以及一生中从未患上癌症的人的高频率 使得绘制新的人类抗性等位基因几乎是不可能的。在老鼠身上，找到这样的突变要容易得多。 许多近交系小鼠都存在同基因肿瘤系(具有相对稳定的基因组)。近亲交配 小鼠本身有一个明确的生殖系参考序列。每个个体在几乎所有的基因座上都是纯合的， 在基因上几乎与所有其他物种相同。在过去的几年里，我们利用这种情况 识别突变赋予抗癌能力的基因。使用随机胚系诱变剂ENU，我们 建立了第三代(G3)生殖系突变小鼠(C57BL/6J品系)。总计23,751台第三代(G3) 来自561个家系的小鼠，总共携带32,039个非同义编码/剪接改变，被登记到 每只小鼠皮下注射2E5 B16F10黑色素瘤细胞和抗PD-1的屏幕 分别在第5天、第8天和第11天注射抗体。第13天和第20天测量肿瘤体积。 对每个家系的创始人进行测序，以确定由 所有G3后代在所有诱变部位进行基因分型，并在筛选前进行基因分型。 自动减数分裂图谱允许快速检测甚至细微的表型并将其归类为致病原因 突变。这项筛查产生了几种突变，导致对可移植癌症产生抗药性。14.2%饱和度 在筛查中实现了常染色体基因组的部分(具有严重损害或 在纯合状态下测试的破坏性等位基因三次或更多)。因此，仍有许多未被发现的东西。 从我们已经知道的情况来看，有一个现实的机会将基因发现从这个屏幕转化为 人类癌症疗法。这项提议旨在延长癌症耐药性的筛查，并进一步推进 对两个抗药性突变的机制和翻译研究，每个突变都位于一个与人类同源的基因中， 测试围绕每个蛋白质靶点建立的治疗方法之间的协同作用，并为 临床应用。

PROJECT SUMMARY Can germline mutations cause strong resistance to otherwise lethal cancers? Certain germline genotypes might be poorly supportive of tumor vascularization, nutritional demands, or resistance to immune attack, yet compatible with host survival. Of particular interest, some mutations might abet the host response to neo- antigens, or even to self-antigens highly expressed in syngeneic tumors. The identification of resistance mutations could provide new approaches and targets for cancer therapy. At least in human populations, resistance mutations would be very difficult to identify. Human germline genetic variability, stem variability among cancer genomes, and the high frequency of humans who never develop cancer throughout their lives would make mapping novel human resistance alleles all but impossible. In mice, finding such mutations is much easier. Syngeneic tumor lines (with relatively stable genomes) exist for many inbred strains of Mus musculus. The inbred mice themselves have a defined germline reference sequence. Each individual is homozygous at nearly all loci, and almost genetically identical to all others. Over the past several years, we took advantage of this situation to identify genes in which mutations confer cancer resistance. Using the random germline mutagen ENU, we created third generation (G3) germline mutant mice (C57BL/6J strain). A total of 23,751 third-generation (G3) mice from 561 pedigrees, bearing a total of 32,039 non-synonymous coding/splicing changes were enrolled into a screen in which each mouse was injected subcutaneously with 2e5 B16F10 melanoma cells, and anti-PD-1 antibody was administered on days 5, 8, and 11. Tumor volume was measured on days 13 and 20. The G1 male founder of each pedigree was sequenced to identify all non-synonymous coding/splicing mutations induced by mutagenesis, and all G3 descendants were genotyped at all induced mutation sites in advance of screening. Automated meiotic mapping allowed quick detection of even subtle phenotypes and assignment to causative mutations. This screen yielded several mutations causing resistance to transplantable cancers. 14.2% saturation of the autosomal genome was achieved in screening (fraction of autosomal genes with severely damaging or destructive alleles tested in the homozygous state three times or more). Therefore, much remains undiscovered. From what we know already, there is a realistic chance of translating genetic discoveries from this screen to human cancer therapy. This proposal aims to extend screening for cancer resistance, and to further advance mechanistic and translational studies of two resistance mutations, each in a gene with a human orthologue, testing synergy between therapeutic approaches built around each protein target, and laying groundwork for clinical applications.