Targeting Interleukin‐1 Receptor‐Associated Kinase 1 (IRAK1) In Ovarian Cancer

Abstract

Ovarian cancer (OvCa) is the leading cause of gynecologic cancer-related deaths, due largely to late-stage diagnoses and the development of chemoresistant disease. Nearly 70% of affected women develop recurrent disease, characterized by resistance to standard chemotherapy despite initial response. Cancer stem cells (CSCs) are a subset of cells that have an innate ability to self-renew and enter a dormant state and have been further implicated in resistance to chemotherapy due to high expression of the ATP-binding Cassette (ABC) transporters. To understand the pathways that may be implicated in enhancing this stem-like phenotype, RNA sequencing was performed on cisplatin sensitive and isogenic cisplatin-resistant OvCa cell lines, as well as cells grown in 2- dimensional (2-D) or 3-dimensional (3-D) cultures. OvCa cells grown as spheroids, a 3-D growth model, are enriched with CSCs, and therefore represent a surrogate for stemness. Interestingly, the Toll-like (TLR) or interleukin-1 (IL-1) receptor pathways were highly enriched in resistant cancer cells and spheroid cultures. A critical factor in these signaling pathways is the interleukin receptor associated kinase 1 (IRAK1). IRAK1 is also highly expressed in chemoresistant OvCa and is responsible for activating numerous downstream pathways involved in tumor progression, such as NF-kB, P38/MAPK, and C-Jun. High expression of IRAK1 is associated with poor overall survival based on analysis of the Cancer Genome Atlas (TCGA) publicly available datasets. Due to the important role IRAK1 has in OvCa growth signaling and patient outcomes, IRAK1 represents an attractive target for CSC-targeted drug therapy. The present study is aimed at identifying small molecule inhibitors of IRAK1 by using in-silico molecular docking-based screening. FDA-approved compound libraries were screened using Autodock Vina software. Compounds were assessed for binding within the c-terminal kinase domain. The compounds are ranked-based on their binding energies (BE) and hydrogen bonding patterns. Our screen identified multiple compounds, one of which included mebendazole, an anthelminthic with a BE of -10.3 kCal/mol. This suggests potent interaction with the kinase domain of IRAK1. To confirm the binding in vitro, we performed cellular thermal shift stability assays (CETSA). We observed that mebendazole treatment protected IRAK1 protein from thermal denaturation, confirming the in silico docking data. Lastly, we performed hexosaminidase proliferation assays to assess the effectiveness of mebendazole on inhibiting cancer cell growth. Cisplatin sensitive OvCar3, A1847, A2780 OvCa and cisplatin resistant C30 OvCa cell lines were treated with increasing doses of mebendazole. The inhibitory concentration 50 (IC50) was determined to be approximately 300 nM at 72h for all cell lines. This suggests that mebendazole has potent anti-cancer activity that may be, in part, mediated through the inhibition of IRAK1.