Abstract 5306: IRAK1: A novel TOLLway to target ovarian cancer
Authors: Standing David , Gunewardena Sumedha , Sayed Afreen A. , Pritchard Michele T. , Pathak Harsh B. , Godwin Andrew K. , Petersen Shariska , Khabele Dineo , Roy Jensen A. , Dandawate Prasad , Weir Scott J. , Anant Shrikant 
Published: June 2022

Abstract
Ovarian cancer (OvCa) is the leading cause of gynecologic cancer-related deaths. While the overall response rate to first line therapy is encouraging (~80%), the majority of women develop recurrent disease that is characterized by resistance to chemotherapy. It is believed that cancer stem cells (CSCs) may contribute, in part, to resistance and recurrence of OvCa. To understand the pathways involved in enhancing this stem-like phenotype, we performed RNA sequencing. We identified that the TLR-ILR1 (TIR) pathways are highly activated in cisplatin resistant OvCa and in CSC-enriched 3-dimensional culture models. To further understand the role of the TIR pathway, we mined the Cancer Genome Atlas database and observed that interleukin receptor-associated kinase 1 (IRAK1), a critical mediator of TIR signaling is upregulated in cancer tissues. In addition, the locus surrounding the IRAK1 gene is amplified in 10% of OvCa patients. We confirmed that IRAK1 expression is upregulated in a majority of OvCa samples by immunohistochemistry of a tumor microarray consisting of 100 patient and paired non-cancerous fallopian tube tissues. Furthermore, this upregulation correlated with early cancer onset and shorter overall survival. To study the specific role of IRAK1 in OvCa, we knocked down its expression using specific shRNA. This significantly impaired cancer growth both in vitro in 2-dimension (2D) and 3-dimensional (3D) spheroid cultures, and in vivo in peritoneal disease models. Moreover, IRAK1 knockdown resulted in decreased expression of CSC marker genes, including MYC, ALDH1A1, DCLK1, and KLF4 suggesting a critical role in maintenance of stemness programming. Since IRAK1 is an upstream kinase that is activated by TIR receptors, we were intrigued by mechanisms driving its activation. In this regard, we have observed that low molecular weight hyaluronic acid (LMW HA) is present at high levels (100-200 ng/ml) in malignant ascites following peritoneal metastasis. Treatment of OVCAR8, A2780 and A1847 cells with LMW HA (50-200 ng/ml) induced IRAK1 phosphorylation at 80 ng/ml that was further enhanced at 200 ng/ml. In addition, LMW HA induced stemness and multidrug resistance genes. With additional studies using specific inhibitors, we identified that the increased spheroid formation occurred via a CD44-PKC-IRAK1-STAT3 signaling axis. Finally, using molecular modeling and in silico screening, coupled with Eurofin’s ScanMAX platform, we identified TCS2210 as a novel highly specific IRAK1 inhibitor. Also, TCS2210 abrogated LMW HA induced activation of IRAK1 and STAT3, and CSC marker genes MYC and DCLK1. Moreover, TCS2210 effectively suppressed OvCa cell growth in in vitro 2D and 3D cultures, and in peritoneal disease models alone and in combination with cisplatin. These data, taken together, strongly suggest that IRAK1 is a valid therapeutic target for OvCa.
Citation Format: David Standing, Sumedha Gunewardena, Afreen A. Sayed, Michele T. Pritchard, Harsh B. Pathak, Andrew K. Godwin, Shariska Petersen, Dineo Khabele, Jensen A. Roy, Prasad Dandawate, Scott J. Weir, Shrikant Anant. IRAK1: A novel TOLLway to target ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5306.