Abstract 4730: The selective WEE1 inhibitor azenosertib shows synergistic anti-tumor activity with encorafenib + cetuximab in multiple BRAFV600E models

Abstract BRAFV600E is a potent oncogenic driver which results in downstream hyperactivation of MAPK signaling and unchecked oncogenic growth, while simultaneously increasing replication stress (RS) and accumulation of DNA damage. The BRAFV600E-specific inhibitor encorafenib in combination with the anti-EGFR monoclonal antibody cetuximab (E + C) are approved in the second line plus (2L+) setting in metastatic colorectal cancer (mCRC) resulting in 20% overall response rate (ORR) in the confirmatory Phase 3 study. However, many patients do not respond, and alternative therapeutic options are still needed in this challenging patient population. WEE1 is a kinase involved in cell cycle progression, allowing cells to repair damaged DNA before entering the next cell cycle phase. Consequently, inhibition of WEE1 leads to premature mitotic entry, inducing replication stress (RS) and unchecked accumulation of DNA damage, ultimately resulting in mitotic catastrophe and cell death. Paradoxically, inhibition of oncogenic drivers like BRAFV600E have been demonstrated to drive high levels of chromosomal instability (CIN) and RS. We hypothesized that high levels of CIN and RS in BRAFV600E-mutant tumors before and after treatment with E + C present a vulnerability to WEE1 inhibition and provide rationale for combining azenosertib, a novel, selective, and orally bioavailable WEE1 inhibitor, with E + C. Here, we report the effectiveness of azenosertib combined with E + C as an anticancer treatment in BRAFV600E CRC models. In vitro combination of azenosertib with E + C increased cell growth inhibition over monotherapy azenosertib or doublet E+ C across multiple BRAFV600E cell lines, and was accompanied by a decrease in activity of MAPK and mediators of cell cycle checkpoints, highlighting possible mechanisms for synergy. In vivo treatment of BRAFV600E CRC cell-line derived xenograft (CDX) models with azenosertib demonstrated significant monotherapy tumor growth inhibition (TGI) in the LS411N model (69%) as well as synergistic TGI and tumor regression when combining azenosertib with E + C compared against E + C alone in two CDX models (LS411N: 108% vs 63%; HT-29: 101% vs 88%). Similarly, treatment of multiple BRAFV600E patient-derived xenograft (PDX, N=3) models of CRC resulted in synergistic triplet TGI (80%, 102%, 103%) and some tumor regression compared against monotherapy azenosertib (43%, 19%, 51%) or the E + C doublet (49%, 65%, 87%). Taken together, our results suggest that the in vitro and in vivo combination of azenosertib with E + C enhances tumor growth inhibition over single agent azenosertib or doublet E + C therapy, and may be an effective treatment option for patients with BRAFV600E mCRC. Citation Format: Nathan M. Jameson, Harshit Shah, Blake Skrable, Mona Abed, Hooman Izadi, Nam-phuong Nguyen, Olivier Harismendy, Mark R. Lackner. The selective WEE1 inhibitor azenosertib shows synergistic anti-tumor activity with encorafenib + cetuximab in multiple BRAFV600E models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4730.