Establishing in vitro carcinoma models of the Alternative Lengthening of Telomeres pathway to elucidate molecular mechanisms and identify therapeutic vulnerabilities

项目摘要/摘要 端粒酶非依赖性交替延长的细胞和分子机制 端粒(ALT)端粒维持机制是一个活跃的研究领域，尤其是在非上皮性疾病中 ALT常见的癌症(如胶质瘤、肉瘤、胰腺神经内分泌肿瘤)。对许多人来说 在这些癌症中，预后很差，治疗选择有限。最近的一次调查结果令人鼓舞 临床前研究表明，DNA损伤反应(DDR)蛋白的一种药理抑制剂ATR， 对骨肉瘤和胶质母细胞瘤癌细胞表现出选择性毒性，这些细胞利用ALT途径 端粒维持，而不是上调酶，端粒酶。不太受赏识，也不够- 被调查的是使用ALT途径的腺癌。我们假设ALT阳性 腺癌与ALT阳性的非上皮源性癌症有共同的分子特征，这些 相似性将导致对ATR抑制的类似敏感性。 这项提议的首要目标是用前列腺癌细胞研究腺癌中的ALT。 LINES作为一个初始的体外模型系统，研究是否有类似的药物抑制策略 ALT阳性腺癌中的DDR蛋白，如ATR，也将显示出疗效。在目标1中，我们将 建立并验证第一个使用ALT的腺癌细胞系。我们以前的实验室 发现ALT阳性肿瘤与体细胞失活突变之间存在很强的相关性 ATRX和DAXX基因。到目前为止，还没有鉴定出显示ALT的前列腺癌细胞系 表型。使用CRISPR Cas9基因组编辑技术，我们将创建等基因前列腺癌细胞 ATRX或DAXX基因突变失活的品系。在目标2中，我们将描述分子特征 通过比较端粒酶阳性/ALT阴性的前列腺癌细胞的ALT过程 亲本系端粒酶组分的相对表达、端粒酶活性、核 结构，以及同源导向的重组的活动水平，这被认为是ALT的关键 路径。最后，在目标3中，我们将评估ALT阳性前列腺癌细胞对DDR的反应 抑制力。初步证据显示，ALT阳性细胞中有一种戏剧性的DDR激活，这表明 这些已经受到压力的细胞DNA损伤水平的增加或修复能力的降低可能是 足以诱导细胞凋亡。DDR蛋白将使用RNA干扰策略以及 丙氨酸氨基转移酶阳性前列腺癌细胞系中现有的药理抑制剂。委员会的研究结果 拟议中的研究将通过增加我们对分子的理解而具有真正的生物学和治疗价值 腺癌中ALT的潜在机制，并通过确定这一独特的分子亚群是否 癌症容易通过抑制DDR蛋白而增强杀伤力。

PROJECT SUMMARY/ABSTRACT The cellular and molecular mechanisms underlying the telomerase-independent Alternative Lengthening of Telomeres (ALT) telomere maintenance mechanism is an area of active research, particularly in non-epithelial cancers where ALT is commonly found (e.g. gliomas, sarcomas, pancreatic neuroendocrine tumors). For many of these cancers the prognosis is poor and therapeutic options are limited. Encouraging results from a recent pre-clinical study show a pharmacological inhibitor of the DNA Damage Response (DDR) protein, ATR, exhibits selective toxicity against osteosarcoma and glioblastoma cancer cells that utilize the ALT pathway for telomere maintenance, instead of up-regulating the enzyme, telomerase. Less appreciated and under- investigated are adenocarcinomas that employ the ALT pathway. We hypothesize that ALT-positive adenocarcinomas share molecular features with ALT-positive cancers of non-epithelial origin, and that these similarities will result in a comparable sensitivity to ATR inhibition. The overarching goal of this proposal is to study ALT in adenocarcinomas, using prostate cancer cell lines as an initial in vitro model system, to investigate whether similar strategies of pharmacologically inhibiting DDR proteins, such as ATR, in ALT-positive adenocarcinomas will also show efficacy. In Aim 1, we will generate and validate the first adenocarcinoma cell lines that employ ALT. Our laboratory previously discovered a strong correlation between ALT-positive tumors and somatic inactivating mutations in either the ATRX and DAXX genes. To date, no prostate cancer cell line has been identified that displays the ALT phenotype. Using the CRISPR cas9 genome editing technique, we will create isogenic prostate cancer cell lines with inactivating mutations in ATRX or DAXX genes. In Aim 2, we will characterize the molecular features underlying the ALT process in prostate cancer cells by comparing the telomerase-positive/ALT-negative parental lines for differences in the relative expression of telomerase components, telomerase activity, nuclear architecture, and activity levels of homology-directed recombination, which is thought to be crucial to the ALT pathway. Finally, in Aim 3, we will evaluate the response of ALT-positive prostate cancer cells to DDR inhibition. Preliminary evidence shows a dramatically activated DDR in ALT-positive cells, suggesting that an increase in the level of DNA damage or a reduction in repair capacity in these already stressed cells may be sufficient to induce apoptosis. DDR proteins will be inhibited using RNA interference strategies, as well as existing pharmacological inhibitors in ALT-positive prostate cancer cell lines. The research findings of the proposed study will have real biological and therapeutic value by increasing our understanding of the molecular mechanisms underlying ALT in adenocarcinomas, and by determining if this unique molecular subset of carcinomas is susceptible to enhanced killing by inhibiting DDR proteins.