Development of first-in-class AAK1 PROTACs for ovarian cancer

About This Grant

PROJECT SUMMARY Ovarian cancer remains the most lethal gynecologic cancer. Treatment strategies developed over the past four decades, however, have only achieved limited success. The current standard of care is surgical debulking followed by paclitaxel/platinum-based chemotherapy. Although patients respond initially, almost all will relapse due to rapid development of chemotherapy resistance, the mechanism of which is still largely unknown. Given the fact that protein kinases are critical in regulating pro-survival signaling, we conducted a kinome- wide shRNA screening, which identified AP2 associated kinase 1 (AAK1) as a “synthetic lethal” partner of paclitaxel. AAK1 is known for its role in regulating clathrin-dependent endocytosis. However, its role in cancer remains unexplored. TCGA datamining revealed that high AAK1 levels are associated with poor overall and progression-free survival in taxane-treated ovarian cancer patients, and AAK1 levels are higher in taxane non- responders than in responders. Consistently, silencing AAK1 sensitized paclitaxel to ovarian cancer cell lines and organoids, both in vitro and in vivo, demonstrating the critical role of AAK1 in promoting paclitaxel resistance. To investigate how AAK1 regulates paclitaxel sensitivity, we first discovered that caspase 7, activated by paclitaxel, cleaves AAK1 at D767, creating AAK1 (1-767) truncation form. We further identified CCR4-NOT complex as the key downstream effector for AAK1 (1-767) to promote paclitaxel resistance. Collectively, these findings position AAK1 as a promising target for enhancing paclitaxel efficacy in ovarian cancer. However, AAK1 inhibitors, despite their high potency in inhibiting cellular AAK1 kinase activity, require high concentrations to sensitize ovarian cancer cells to paclitaxel, indicating limited clinical translation potential for AAK1 inhibitors in ovarian cancer treatment. We hypothesize that the proteolysis targeting chimera (PROTAC) approach can more effectively target AAK1 for ovarian cancer treatment. Indeed, HC-332, a lead AAK1-targeting PROTAC, induces potent, selective, deep, and long-lasting degradation of AAK1 in ovarian cancer cells, in vitro and in vivo. HC-332 also sensitizes paclitaxel treatment in various ovarian cancer patient-derived organoids, primary cancer cells, and cell lines, including paclitaxel-resistant sublines, indicating its potential to improve paclitaxel response in both naive and recurred patients. Despite suboptimal PK properties, in vivo studies further demonstrated that HC-332 greatly improves paclitaxel response in ovarian cancer cell line and PDXs without obvious systemic toxicity. Collectively, our data supports the potential of AAK1 PROTACs in treating ovarian cancer. Thus, we plan to develop AAK1 PROTACS with high selectivity, potency, and druglike properties; and evaluate of the efficacy of AAK1 PROTAC/paclitaxel combination in ovarian cancer and elucidate the mechanism of action of PROTACs.