Operationalizing DNMT1-Targeting to Treat Chemorefractory Pancreatic Cancer

胰腺导管腺癌（PDAC）是一个棘手的、致命的问题。大多数PDAC患者接受 积极的多药物化疗，但中位总生存期仍<1年。牢固的基础 有限的化疗反应是突变和缺失的主要调节细胞凋亡p53在大多数 PDAC。因此，需要不依赖于p53的替代治疗方式。关键的表观遗传调节因子 DNA甲基转移酶1（DNMT 1）是一个科学验证的分子靶点，可以减少化疗难治性肿瘤的发生。 包括PDAC在内的恶性肿瘤，因为它影响癌细胞周期，通过p53非依赖性上皮形成退出。 DNMT 1可以被嘧啶核苷类似物前药地西他滨（Dec）或5-氨基-1，2-二氢嘧啶核苷（5-氨基-1，2-二氢嘧啶核苷）抑制/耗尽。 氮杂胞苷（5Aza），被批准用于治疗骨髓恶性肿瘤。然而，几个完整的临床结果 使用Dec、5Aza或类似物治疗PDAC和其它实体瘤的试验令人失望。其原因 相关研究表明，Dec未能引起DNMT 1抑制药效学 在>75%的实体癌组织中有效。我们在临床前和临床研究中进一步探讨了原因 我们在PDAC细胞中发现了嘧啶代谢的固有和适应性结构， 加工成DNMT 1耗尽核苷酸。幸运的是，有一些实际的治疗方法可以 对抗Dec/5Aza对DNMT 1靶向的这些抗性机制。具体来说，我们假设， 对Dec，5Aza的抗性来自嘧啶代谢的适应性反应， 利用替代方案，并通过与嘧啶代谢的无毒改性剂组合。 目的1：评价Dec和5Aza给药方案，旨在利用 嘧啶代谢适应和增加S期依赖性DNMT 1消耗。我们的初步数据 表明安排Dec/5Aza给药以利用关键嘧啶的反应性峰和谷， 代谢酶和频繁的分布式给药以增加恶性细胞S期进入和治疗暴露之间的重叠，增加了体内的实质性益处-我们将扩展这些数据以指导 机械上合理的，易于实施的临床治疗时间表改进。目标2：增强 通过将Dec和5Aza与嘧啶的非细胞毒性修饰剂组合的非细胞毒性DNMT 1靶向 新陈代谢.我们已经发现胞苷脱氨酶（CDA）的自动上调， Dec/5Aza，以及与Dec/5Aza竞争掺入DNA的从头嘧啶合成， 对抵抗力有着至关重要的作用。因此，我们将联合收割机Dec/5 Aza与临床非细胞毒性抑制剂结合使用 分解代谢和从头嘧啶代谢途径，以鉴定增加DNMT 1靶向的组合， 响应率和持续时间。我们在这里评估的治疗概念对于临床翻译是实用的，例如， 我们已经开发出临床口服形式的CDA-抑制剂+Dec/5Aza， 从头嘧啶合成抑制剂，将临床前原理证明快速转化为临床治疗。

Pancreatic ductal adenocarcinoma (PDAC) is a recalcitrant, deadly problem. Most PDAC patients receive aggressive multi-agent chemotherapy but median overall survivals remain at <1 year. A well-established basis for limited chemotherapy response is mutation and deletion of the master regulator of apoptosis p53 in most PDAC. Thus, alternative treatment modalities that do not rely on p53 are needed. The key epigenetic regulator DNA methyltransferase 1 (DNMT1) is a scientifically validated molecular target to cytoreduce chemo-refractory malignancies including PDAC, since it effects cancer cell cycling exits by p53-independent epithelialization. DNMT1 can be inhibited/ depleted by the pyrimidine nucleoside analog pro-drugs decitabine (Dec) or 5- azacytidine (5Aza), approved to treat myeloid malignancies. However, results from several completed clinical trials using Dec, 5Aza, or analogs to treat PDAC and other solid tumors have disappointed. A reason for this was suggested by correlative studies that revealed failure of Dec to elicit DNMT1-inhibiting pharmacodynamic effect in >75% of solid cancer tissues. We explored reasons for this further, in both pre-clinical and clinical studies we found inherent and adaptive configurations of pyrimidine metabolism in PDAC cells that forestall Dec or 5Aza processing into DNMT1-depleting nucleotide. Fortunately, there are practical modifications to therapy that can counter these mechanisms of resistance to DNMT1-targeting by Dec/5Aza. Specifically, we hypothesize that resistance to Dec, 5Aza emerges from adaptive responses of pyrimidine metabolism that can be anticipated and exploited using alternative schedules, and by combination with non-toxic modifiers of pyrimidine metabolism. Aim 1: Evaluate schedules of Dec and 5Aza administration designed to exploit peaks/troughs in pyrimidine metabolism adaptation and increase S-phase dependent DNMT1-depletion. Our prelim data indicates that scheduling Dec/5Aza administration to exploit reactive peaks and troughs in key pyrimidine metabolism enzymes, and frequent, distributed administration to increase overlap between malignant cell S-phase entries and treatment exposures, adds substantial benefit in vivo – we will extend this data to guide mechanistically-rational, readily implementable schedule improvements to clinical therapy. Aim 2: Enhance non-cytotoxic DNMT1-targeting by combining Dec and 5Aza with non-cytotoxic modifiers of pyrimidine metabolism. We have found that auto-upregulation of cytidine deaminase (CDA) that rapidly catabolizes Dec/5Aza, and of de novo pyrimidine synthesis that competes with Dec/5Aza for incorporation into DNA, contributes critically to resistance. We will therefore combine Dec/5Aza with clinical non-cytotoxic inhibitors of catabolic and de novo pyrimidine metabolism pathways, to identify combinations that increase DNMT1-targeting, response rates and durations. The treatment concepts we evaluate here are practical for clinical translation, e.g., we have developed into the clinic oral forms of CDA-inhibitor+Dec/5Aza that are readily combined with clinical inhibitors of de novo pyrimidine synthesis, to rapidly translate pre-clinical proof-of-principle into clinical therapy.