For 2017, the estimated lifetime risk of developing colorectal cancer was 1 in 22. Even though preventative colonoscopy screening and standard-of-care surgery, radiation, and chemotherapy have decreased the death rate from colorectal cancer, new therapies are needed for metastatic colorectal cancer. Here, we developed a novel small molecule, compound 2, that inhibited proliferation and viability of human colorectal cancer cells (HCT-116, DLD-1, SW480, and 10.1). Compound 2 inhibited cell migration, invasion, and epithelial–mesenchymal transition processes and potently increased cell apoptosis in human colorectal cancer cells. Compound 2 also modulated mitotic stress signaling, leading to both inhibition of Wnt responsiveness and stabilization and activation of p53 to cause cell-cycle arrest. In mouse xenografts, treatment with compound 2 (20 mg/kg/day, i.p.) induced cell death and inhibited tumor growth more than four-fold compared with vehicle at day 34. Neither acute cytotoxicity nor toxicity in animals (up to 1,000 mg/kg, i.p.) were observed for compound 2. To our knowledge, compound 2 is the first reported potent small molecule that inhibits Wnt/β-catenin signaling, activates p53 signaling regardless of p53 mutation status, and binds microtubules without detectable toxicity. Thus, compound 2 offers a novel mechanism of action and a new strategy to treat colorectal cancer. Significance: These findings identify a potent small molecule that may be therapeutically useful for colon cancer that works by inhibiting Wnt/β-catenin signaling, activating p53, and binding microtubules without detectable toxicity. Cancer Res; 78(17); 5072–83. ©2018 AACR.

Prostate cancer (PCa) is the second leading cause of cancer-related death for men in the United States. Approximately 35% of PCa recurs and is often transformed to castration-resistant prostate cancer (CRPCa), the most deadly and aggressive form of PCa. However, the CRPCa standard-of-care treatment (enzalutamide with abiraterone) usually has limited efficacy. Herein, we report a novel molecule (PAWI-2) that inhibits cellular proliferation of androgen-sensitive and androgen-insensitive cells (LNCaP and PC-3, respectively). In vivo studies in a PC-3 xenograft model showed that PAWI-2 (20 mg/kg per day i.p., 21 days) inhibited tumor growth by 49% compared with vehicle-treated mice. PAWI-2 synergized currently clinically used enzalutamide in in vitro inhibition of PCa cell viability and resensitized inhibition of in vivo PC-3 tumor growth. Compared with vehicle-treated mice, PC-3 xenograft studies also showed that PAWI-2 (20 mg/kg per day i.p., 21 days) and enzalutamide (5 mg/kg per day i.p., 21 days) inhibited tumor growth by 63%. Synergism was mainly controlled by the imbalance of prosurvival factors (e.g., Bcl-2, Bcl-xL, Mcl-1) and antisurvival factors (e.g., Bax, Bak) induced by affecting mitochondrial membrane potential/mitochondria dynamics. Thus, PAWI-2 utilizes a distinct mechanism of action to inhibit PCa growth independently of androgen receptor signaling and overcomes enzalutamide-resistant CRPCa. SIGNIFICANCE STATEMENT Castration-resistant prostate cancer (CRPCa) is the most aggressive human prostate cancer (PCa) but standard chemotherapies for CRPCa are largely ineffective. PAWI-2 potently inhibits PCa proliferation in vitro and in vivo regardless of androgen receptor status and uses a distinct mechanism of action. PAWI-2 has greater utility in treating CRPCa than standard-of-care therapy. PAWI-2 possesses promising therapeutic potency in low-dose combination therapy with a clinically used drug (e.g., enzalutamide). This study describes a new approach to address the overarching challenge in clinical treatment of CRPCa.

Today, pancreatic cancer (PC) remains a major health problem in the US. The fact that cancer stem cells (CSCs) become enriched in humans following anti-cancer therapy implicates CSCs as key contributors to tumor dormancy, metastasis, and relapse in PC. A highly validated CSC model (FGβ₃ cells) was used to test a novel compound (PAWI-2) to eradicate CSCs. Compared to parental bulk FG cells, PAWI-2 showed greater potency to inhibit cell viability and self-renewal capacity of FGβ₃ cells. For FGβ₃ cells, dysregulated integrin β₃-KRAS signaling drives tumor progression. PAWI-2 inhibited β₃-KRAS signaling independent of KRAS. This is clinically relevant. PAWI-2 targeted the downstream TBK1 phosphorylation cascade that was negatively regulated by optineurin phosphorylation via a feedback mechanism. This was confirmed by TBK1 genetic knockdown or co-treatment with TBK1-specific inhibitor (MRT67307). PAWI-2 also overcame erlotinib (an EGFR inhibitor) resistance in FGβ₃ cells more potently than bortezomib. In the proposed working model, optineurin acts as a key regulator to link inhibition of KRAS signaling and cell cycle arrest (G2/M). The findings show PAWI-2 is a new approach to reverse tumor stemness that resensitizes CSC tumors to drug inhibition.

Proximity-enabled crosslinking techniques facilitate the formation of covalent bonds within and between proteins, contributing to the elucidation of protein dynamics and function.