HLTF gene silencing: a novel determinant of sensitivity to autophagy inhibition

描述(申请人提供)：自噬是一种溶酶体依赖的降解过程，保护癌细胞免受代谢和治疗压力的影响。自噬在大多数晚期癌症中上调，并已被确定为癌症治疗的新靶点。氯喹(CQ)衍生物抑制自噬增强了许多抗癌治疗的疗效。许多临床试验正在测试多种抗癌药物的组合 使用羟基氯喹(HCQ)的药物，但目前还没有生物标记物可以将HCQ组合或方案的开发集中到最有可能从这一策略中受益的患者亚群中。为了确定对自噬抑制敏感的候选生物标记物，差异表达的微阵列分析 对HCQ敏感和耐药的人癌细胞系进行了基因分析。在HCQ敏感细胞中下调最多的基因是解旋酶样转录因子(HLTF)，这是一种未被充分研究的肿瘤抑制基因，参与了复制应激过程中维持基因组完整性的多个方面。在20-40%的肺癌、结肠癌和胃癌中，HLTF的表达被启动子甲基化抑制。在一大组细胞系中，几乎只在对HCQ敏感的细胞系中发现了HLTF基因沉默。在HLTF沉默的细胞中强制表达HLTF可产生对HCQ的抵抗。自噬抑制和HLTF之间的联系可能是通过在HCQ处理后不久观察到的DNA氧化损伤来实现的。甲基化特异性聚合酶链式反应能够检测黑色素瘤和乳腺癌患者血清中HLTF基因甲基化状态，表明HLTF基因沉默在多种恶性肿瘤中是常见的，血清检测可能能够将患者归类为HLTF基因沉默或表达。这项建议将检验以下假设：a)HLTF基因沉默通过使氧化DNA损伤无法修复而增加对自噬抑制剂的敏感性~b)临床分级检测患者血清或肿瘤中的HLTF启动子甲基化可以是一种敏感而特异的HLTF基因沉默检测方法c)HLTF基因沉默在许多恶性肿瘤中常见，并可预测接受HCQ治疗的患者的临床疗效。完成这些特定目标所获得的知识将建立一个新的机制框架，将自噬抑制与DNA损伤反应联系起来。这项工作将阐明通常沉默的肿瘤抑制基因HLTF的功能，并将确定哪些恶性肿瘤应该成为自噬抑制剂开发的靶点。最后，由于目前正在开发新的自噬抑制剂用于癌症患者的临床试验，这些目标的完成将为未来开发CLIA批准的预测试验提供必要的支持数据，该试验可用于登记最有可能受益于自噬抑制剂的患者。

DESCRIPTION (provided by applicant): Autophagy is a lysosome-dependent degradative process that protects cancer cells from metabolic and therapeutic stress. Autophagy is up regulated in most advanced cancers and has identified as a new target for cancer therapy. Autophagy inhibition with chloroquine (CQ) derivatives augments the efficacy of many anticancer therapies. Numerous clinical trials are testing the combination of variety of anticancer agents with hydroxychloroquine (HCQ), but there currently is no biomarker that can focus the development of HCQ combinations or regimens involving novel emerging autophagy inhibitors into patient subsets that will most likely benefit from this strategy. To identify a candidate biomarker of sensitivity to autophagy inhibition, a microarray analysis of differentially expressed genes in HCQ-sensitive and HCQ-resistant human cancer cell lines was conducted. The most down regulated gene in HCQ-sensitive cells was helicase- like transcription factor (HLTF), an understudied tumor suppressor gene involved in multiple aspects of maintaining genomic integrity during replication stress. HLTF expression is silenced by promoter methylation in 20-40% of lung, colon, and gastric carcinomas. In a large panel of cell lines HLTF gene silencing was found almost exclusively in cell lines that were sensitive to HCQ. Forced expression of HLTF in HLTF silenced cells conferred resistance to HCQ. The link between autophagy inhibition and HLTF may be through oxidative DNA damage that was observed soon after HCQ treatment. A methylation-specific PCR assay was able to detect HLTF methylation status of tumors in the serum samples of patients with melanoma and breast cancer indicating that HLTF gene silencing is common across multiple malignancies and a serum assay may be able to classify patients as HLTF gene silenced or expressed. This proposal will test the hypotheses that a) HLTF gene silencing confers sensitivity to autophagy inhibitors by allowing oxidative DNA damage to go unrepaired~ b) A clinical grade assay for HLTF promoter methylation in the serum or tumors of patients can be a sensitive and specific assay for HLTF gene silencing c) HLTF gene silencing is common in a number of malignancies and predicts of clinical response in patients treated with HCQ. Knowledge gained from completion of these specific aims will establish a new mechanistic framework that links autophagy inhibition to the DNA damage response. This work will shed light on the functions of commonly silenced tumor suppressor gene HLTF and will determine which malignancies should be a target for the development of autophagy inhibitors. Finally, as novel autophagy inhibitors are currently being developed for clinical trials in cancer patients, completion of these aims will provide the supporting data necessary for future development of a CLIA approved predictive assay that could be used to enroll the patients most likely to benefit from autophagy inhibitors.