Harnessing the CRL4 ubiquitin ligase for antagonizing colorectal carcinogenesis

结直肠癌是第四大常见癌症，也是癌症相关死亡的第二大原因。 在美国。美国癌症协会估计，将有134,490人被诊断出 2016年将有49,190人死于CRC。尽管生物制剂受到了极大的关注，但一线 以细胞毒联合化疗为主的化疗治疗转移性结直肠癌 FOLFIRI(亚叶酸+5-FU+伊立替康)或FOLFOX(亚叶酸+5-FU+奥沙拉铂)的主干。它是 值得注意的是，只有18%-25%的结直肠癌患者对伊立替康有好感。尽管FDA批准了 伊立替康临床应用已有20年之久，目前尚无生物标志物可帮助肿瘤学家作出判断 对于给定的结直肠癌患者，这两种方案中哪一种更有可能有效。我们的研究表明， Cullin 4B(CUL4B)泛素连接酶是一种很有前途的预测生物标志物，也是治疗的靶点。 肿瘤对伊立替康的反应。CUL4B基因已被发现在广泛的 实体肿瘤，包括结直肠癌、乳腺癌、肺癌、卵巢癌和前列腺癌。在接受治疗后， 喜树碱家族化疗药物CUL4B，而不是其类似的CUL4A，靶向拓扑异构酶I (Top1)用于癌细胞的降解。因此，具有高水平CUL4B表达的CRC诱导 Top1的过度破坏，有效地减弱了该化疗药物的细胞毒性。我们 假设CUL4B过度降解Top1是CRC成为 伊立替康难治。重要的是，我们证明了CUL4B的失活，而不是CUL4A的有效失活 Top1定向化疗药物对伊立替康耐药CUL4B高肿瘤的细胞毒杀伤增敏作用。 这一数据表明，CUL4B是一个有吸引力的干预目标，可能导致对 75-82%的结直肠癌患者以前对Top1定向化疗药物耐药 (例如FOLFIRI方案)。在三个具体目标中，我们将(1)评估CUL4B作为生物标志物的价值 使用我们独特的572例已治疗的结直肠癌患者样本做出治疗决定 伊立替康或FOLFIRI；(2)进一步开发和验证有效的小分子CUL4B抑制剂 通过高通量筛选鉴定以提高其效力和药理特性；(3)检查 我们的领先CUL4B抑制剂与伊立替康协同作用，在体内使用CRC增强其杀瘤活性 异种细胞系移植、遗传性WNT/APC诱导的肠腺瘤和结肠腺瘤以及患者来源的肿瘤 结直肠癌异种移植模型。我们为实验室到临床研究的发展做好了独特的准备 一种基于伊立替康的化疗的预测生物标记物，立即适用于指导决策- 临床结直肠癌处理的制定，以及旨在验证CUL4B作为一种 临床前结直肠癌模型中可行的药物靶点，并开发一种新的治疗剂使CUL4B升高 结直肠癌患者约占所有结直肠癌患者的75%，他们对伊立替康治疗敏感。

Colorectal cancer (CRC) is the 4th most common cancer and the 2nd leading cause of cancer–related deaths in the United States. The American Cancer Society estimates that 134,490 people will be diagnosed and 49,190 will die from CRC in 2016. Although biologic agents have received much attention, the first-line chemotherapy for treatment of metastatic CRC remains based on a cytotoxic combination chemotherapy backbone of either FOLFIRI (folinic acid+5-FU+irinotecan) or FOLFOX (folinic acid+5-FU+oxilaplatin). It is noteworthy that only 18-25% of CRC patients respond favorably to irinotecan. Although the FDA-approved irinotecan has been used in clinic for 20 years, there is still no diagnostic biomarker to help oncologists decide which of the two regimens is more likely to be effective for a given CRC patient. Our studies suggest that the CULLIN 4B (CUL4B) ubiquitin ligase is a promising predictive biomarker, as well as a therapeutic target for tumor response to irinotecan. The CUL4B gene has been found amplified or overexpressed in a wide range of solid tumors, including colorectal, breast, lung, ovarian, and prostate cancers. Upon treatment with the camptothecin family of chemotherapy drugs, CUL4B, but not its paralog CUL4A, targets topoisomerase I (Top1) for degradation in cancer cells. As a result, CRCs with high levels of CUL4B expression induce excessive destruction of Top1, effectively attenuating the cytotoxicity of this chemotherapy drug. We hypothesize that excessive Top1 degradation by CUL4B is a major mechanism by which CRCs become refractory to irinotecan. Importantly, we showed that inactivation of CUL4B, but not CUL4A, effectively sensitized irinotecan-resistant CUL4Bhigh tumors to cytotoxic killing by Top1-directed chemotherapy drugs. This data suggest that CUL4B is an attractive target for intervention, potentially leading to sensitization of the 75-82% CRC patients who were previously resistant to treatment with a Top1-directed chemotherapeutic agent (e.g. FOLFIRI regimen). In three specific aims, we will (1) assess the value of CUL4B as a biomarker for treatment decisions of CRC using our unique collection of 572 well annotated CRC patient samples treated with irinotecan or FOLFIRI; (2) further develop and validate potent small molecule CUL4B inhibitors we identified via high throughput screening to increase their potency and pharmacological properties; (3) examine our lead CUL4B inhibitors that synergize with irinotecan to enhance its tumoricidal activity in vivo using CRC cell line xenografts, genetic WNT/APC-induced intestinal and colon adenomas, and patient-derived tumor xenograft (PDTX) models of CRC. We are uniquely prepared for both Laboratory-to-Clinic studies to develop a predictive biomarker for irinotecan-based chemotherapy that is immediately applicable for informing decision- making of CRC management in the clinic, and Clinic-to-Laboratory studies aimed at validating CUL4B as a feasible drug target in pre-clinical CRC models, and developing a new therapeutic agent to render CUL4Bhigh CRC patients, which represent approximately 75% of all CRCs, sensitive to irinotecan-based therapy.