Harnessing the CRL4 ubiquitin ligase for antagonizing colorectal carcinogenesis

结直肠癌（CRC）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，134，490人将被诊断， 2016年将有49，190人死于CRC。虽然生物制剂受到了广泛关注，但一线 治疗转移性CRC的化疗仍然基于细胞毒性联合化疗 FOLFIRI（亚叶酸+5-FU+伊立替康）或FOLFOX（亚叶酸+5-FU+奥铂）的骨架。是 值得注意的是，只有18-25%的CRC患者对伊立替康有良好的反应。虽然FDA批准 伊立替康临床应用已有20年，至今仍无诊断性生物标志物帮助肿瘤学家判断 两种方案中哪一种更可能对给定的CRC患者有效。我们的研究表明， CULLIN 4 B（CUL 4 B）泛素连接酶是一种有前途的预测生物标志物，也是一种治疗靶点， 肿瘤对伊立替康的反应。已经发现CUL 4 B基因在大范围的细胞中扩增或过表达。 实体瘤，包括结肠直肠癌、乳腺癌、肺癌、卵巢癌和前列腺癌。在用 喜树碱家族化疗药物CUL 4 B，而不是其副产物CUL 4A，靶向拓扑异构酶I （Top1）用于癌细胞中的降解。因此，具有高水平CUL 4 B表达的CRC诱导了 Top1的过度破坏，有效地减弱了这种化疗药物的细胞毒性。我们 假设CUL 4 B过度降解Top1是CRC成为 伊立替康难治性。重要的是，我们发现CUL 4 B的失活，而不是CUL 4A的失活， 通过Top1导向的化疗药物使伊立替康耐药CUL 4 Bhigh肿瘤对细胞毒性杀伤敏感。 这些数据表明，CUL 4 B是一个有吸引力的干预靶点，可能导致致敏的细胞因子。 75-82%既往对Top1导向化疗药物治疗耐药的CRC患者 (e.g. FOLFIRI方案）。在三个具体目标中，我们将（1）评估CUL 4 B作为以下生物标志物的价值： 使用我们独特的572例注释良好的CRC患者样本集， 与伊立替康或FOLFIRI;（2）进一步开发和验证有效的小分子CUL 4 B抑制剂， 通过高通量筛选进行鉴定，以增加其效力和药理学性质;（3）检查 我们的主要CUL 4 B抑制剂与伊立替康协同作用，以增强其在体内使用CRC的杀肿瘤活性 细胞系异种移植物、遗传性WNT/APC诱导的肠和结肠腺瘤以及患者源性肿瘤 CRC的异种移植（PDTX）模型。我们为两个实验室到诊所的研究做好了独特的准备， 基于伊立替康的化疗的预测性生物标志物，可立即用于告知决策- 在临床中进行CRC管理，以及旨在验证CUL 4 B作为 临床前CRC模型中可行的药物靶点，并开发新的治疗剂，使CUL 4 B高 CRC患者，约占所有CRC的75%，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.