Developing ATAC-array as a novel epigenetic biomarker to guide personalized therapy in pancreatic cancer

摘要：尽管化疗仍然是全身治疗的主要手段，但大量的癌症 患者对此无反应。约一半接受手术切除的胰腺导管腺癌(PDAC)患者 尽管辅助性化疗，但一年内复发。利用全基因组ATAC-Seq全面 分析切除的PDAC的肿瘤细胞固有染色质可及性模式，我们发现了 1092个染色质基因座在无病生存患者之间显示不同的可及性 一年，DFS和GT；患者一年。基于这一特征，我们开发了一种新型的临床应用ATAC-ARRAY 适用的微阵列平台，无需与NEXT相关的时间和成本即可读取染色质 世代排序。使用这个新的平台，我们开发了一个基于染色质的预后评分，推导出 来自我们训练集样本的ATAC阵列结果，这在一个独立的队列上得到了进一步的验证 PDAC患者衍生的3D器官。通过分析过度表达的转录因子结合位点 在DFS延长的患者中发现染色质基因座可访问，我们还确定HNF1B为转录 显示短DFS患者与长DFS患者之间核定位差异的因素。将这两者结合起来 简单的方法学(即ATAC阵列“预后评分”和免疫组织化学测定或 HNF1B核定位)，我们开发了一种算法，在诊断时对PDAC进行分层 患者分为DFS差异超过7倍的预后组。我们的分层算法 提供了一个简单的和临床上可实现的预测有利和不利的表观遗传状态在PDAC。 基于这项工作，我们现在假设，基于ATAC阵列的染色质可及性分析将提供 一种预测局部和转移性胰腺癌预后的临床相关方法，并进一步 允许合理选择患者进行表观遗传治疗的临床试验。通过这笔R21赠款，我们现在的目标是 优化ATAC-ARRAY用于临床，并将其扩展到胰腺癌的所有阶段。我们的目标是优化 允许ATAC-ARRAY应用于从针刺活检中提取的稀少肿瘤标本的方法。我们 此外，计划在患者衍生的3D有机化合物中筛选体外表观遗传药物，以确定是否有不良反应 染色质的可及性模式可能适用于表观遗传的“重新编程”疗法，从而使ATAC- ARRAY可提供选择患者进行表观遗传治疗的治疗性生物标志物。这样做的长期目标是 该项目旨在加强全基因组评估染色质可及性作为临床相关指标的可行性。 PDAC患者预后的指标，并促进将表观遗传药物合理纳入 表现出预后不良的表观遗传学特征的患者的治疗方案。

ABSTRACT: Although chemotherapy remains the mainstay of systemic therapy, a large number of cancer patients fail to respond to. About half of surgically resected pancreatic ductal adenocarcinoma (PDAC) patients relapse within a year despite adjuvant chemotherapy. Using genome-wide ATAC-Seq to comprehensively analyze tumor cell-intrinsic chromatin accessibility patterns of resected PDAC, we discovered a signature of 1092 chromatin loci displaying differential accessibility between patients with disease free survival (DFS) < 1 year and patients with DFS > 1 year. Based on this signature, we developed “ATAC-array” – a novel, clinically applicable microarray platform that reads chromatin accessibility without the time and cost associated with Next Generation Sequencing. Using this novel platform, we developed a chromatin-based Prognosis Score, derived from the ATAC-array results from our training set samples, which was further validated on an independent cohort of PDAC patient-derived 3D organoids. By analyzing over-represented transcription factor binding sites among chromatin loci found to be accessible in patients with prolonged DFS, we also identified HNF1b as a transcription factor displaying differential nuclear localization between patients with short vs. long DFS. Combining these two simple methodologies (i.e., the ATAC-array “Prognosis Score” and immunohistochemical determination or HNF1b nuclear localization), we have developed an algorithm that at the time of diagnosis stratifies PDAC patients into prognostic groups with more than seven-fold differences in DFS. Our stratification algorithm provides a simple and clinically achievable prediction of favorable vs unfavorable epigenetic states in PDAC. Based on this work, we now hypothesize that ATAC-array based analysis of chromatin accessibility will provide a clinically relevant means of predicting prognosis in both localized and metastatic pancreatic cancer, and further allow rational selection of patients for clinical trials of epigenetic therapy. Through this R21 grant, we now aim to optimize ATAC-array for clinical use and expand it to all stages of pancreatic cancer. Our goal is to optimize methodologies allowing ATAC-array to be applied to scant tumor specimens retrieved from needle biopsies. We additionally plan to screen epigenetic drugs in vitro in patient-derived 3D organoids to determine whether adverse patterns of chromatin accessibility might be amenable to epigenetic “reprogramming” therapy, such that ATAC- array could provide a therapeutic biomarker selecting patients for epigenetic therapy. The long-term goal of this project is to enhance the feasibility of genome-wide assessment of chromatin accessibility as a clinically relevant indicator of outcome in PDAC patients, and to facilitate the rational inclusion of epigenetic drugs into the therapeutic regimens of patients displaying poor-prognosis epigenetic signatures.