Abstract 1995: DUSP4 loss enhances resistance to encorafenib plus cetuximab in BRAF mutant colorectal cancer
Authors: Needham Kristen , Chionh Fiona , Kaczmarczyk Stan , Kearney Connor , Jenkins Laura , Luk Ian , Mariadason John M. 
Published: March 2024

Abstract
The 5-year survival rate for metastatic colorectal cancer (CRC) is less than 14%. Hyperactivation of the ERK/MAPK pathway occurs at a high frequency in CRC, due to mutations (RAS, BRAF) or overexpression/amplification (EGFR) of its signalling components. Aberrant activation of this pathway drives tumour growth and survival and several inhibitors of this pathway are now clinically utilized. The BRAF V600E activating mutation occurs in ~10% of CRC patients and is correlated with an extremely poor prognosis. Use of the small molecule BRAF inhibitor encorafenib in combination with the EGFR inhibitor cetuximab is now approved for BRAF V600E mutant patients, however, the objective response rate is only ~20% and the treatment is rarely curative. Therefore, there is an urgent need to identify predictive biomarkers of response to encorafenib+cetuximab treatment. Dual-specificity phosphatase 4 (DUSP4) is rapidly induced upon activation of ERK/MAPK signaling upon which it acts as a critical negative regulator of signaling output by directly de-phosphorylating ERK. We have observed that DUSP4 is highly expressed in BRAF-mutant CRC cell lines and primary tumours, although there is a range of expression. Whether DUSP4 expression status impacts on response to encorafenib+cetuximab therapy is unknown. The aim of this study therefore was to determine the role of DUSP4 on sensitivity of BRAF-mutant CRC cells to encorafenib+cetuximab treatment in vitro. To examine this, we determined the impact of DUSP4 knock-down, knock-out and re-expression on sensitivity of BRAF-mutant CRC cancer cell lines to encorafenib+cetuximab treatment. Screening a panel of BRAF mutant CRC cell lines for sensitivity to encorafenib+cetuximab revealed a subset of resistant cell lines had either low or undetectable DUSP4 protein expression, while all sensitive cell lines had high DUSP4 expression. siRNA-mediated knockdown of DUSP4 in DUSP4 expressing BRAF-mutant CRC cell lines increased pERK and pFRA1, suggesting activation of ERK/MAPK signalling. DUSP4 knockdown also reduced sensitivity of BRAF mutant CRC cell lines to encorafenib+cetuximab compared to control. These findings were further validated using DUSP4 knock-out with CRISPR-Cas9. Conversely, DUSP4 re-expression in BRAF-mutant CRC cell lines lacking DUSP4 expression, decreased pERK and increased sensitivity to encorafenib+cetuximab treatment. These results demonstrate that DUSP4 regulates ERK/MAPK signalling in BRAF mutant CRC cell lines and that loss of DUSP4 promotes resistance to encorafenib plus cetuximab treatment. These findings warrant investigation of DUSP4 expression status as a predictive biomarker of encorafenib+cetuximab response in patient samples.
Citation Format: Kristen Needham, Fiona Chionh, Stan Kaczmarczyk, Connor Kearney, Laura Jenkins, Ian Luk, John M. Mariadason. DUSP4 loss enhances resistance to encorafenib plus cetuximab in BRAF mutant colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1995.