JNK pathway suppression drives resistance to combination endocrine therapy and CDK4/6 inhibition in ER+ breast cancer
Authors: Alexandrou Sarah , Lee Christine S. , Fernandez Kristine J. , Wiharja Celine E. , Eshraghi Leila , Reeves John , Reed Daniel A. , Portman Neil , Phan Zoe , Milioli Heloisa H. , Nikolic Iva , Cadell Antonia L. , Croucher David R. , Simpson Kaylene J. , Lim Elgene , Hickey Theresa E. , Millar Ewan K. A. , Alves Carla L. , Ditzel Henrik J. , Caldon C. Elizabeth 
Published: January 2025

ABSTRACTPurposeEndocrine therapy in combination with CDK4/6 inhibition doubles the progression-free survival of patients with advanced ER+ breast cancer, but resistance is inevitable, leaving patients with limited treatment options.Experimental DesignWe performed unbiased genome-wide CRISPR/Cas9 knockout screens using ER+ breast cancer cells to identify novel drivers of resistance to combination endocrine therapy (tamoxifen) and CDK4/6 inhibitor (palbociclib) treatment. Screen hits were validated by CRISPR/Cas9 knockout models, mechanistic analyses and evaluation of patient samples.ResultsOur screens identified the inactivation of JNK signalling, including loss of the kinaseMAP2K7, as a key driver of combination resistance. We developed multiple CRISPR/Cas9 knockout ER+ breast cancer cell lines (MCF-7 and T-47D) to investigate the effects ofMAP2K7, MAPK8andMAPK9loss.MAP2K7knockout increased metastatic burdenin vivoand led to impaired JNK-mediated stress responses, as well as promoting cell survival and reducing senescence entry following endocrine therapy and CDK4/6 inhibitor treatment. Mechanistically, this occurred via loss of the AP-1 transcription factor c-JUN, leading to an attenuated response to combination endocrine therapy plus CDK4/6 inhibition.Furthermore, we analysed ER+ advanced breast cancer patient cohorts and found that inactivation of the JNK pathway was associated with increased metastatic burden, and low pJNKT183/Y185activity correlated with a poorer response to systemic endocrine and CDK4/6 inhibitor therapies.ConclusionsOverall, we demonstrate that suppression of JNK signalling enables persistent growth during combined endocrine therapy and CDK4/6 inhibition. Furthermore, our data provide a pre-clinical rationale to screen patients’ tumours for JNK signalling deficiency prior to receiving combined endocrine therapy and CDK4/6 inhibition.STATEMENT OF TRANSLATIONAL RELEVANCEResistance to CDK4/6 inhibitors in the context of endocrine therapy resistance presents an urgent clinical challenge for the management of estrogen receptor positive (ER+) breast cancer. However, the mechanisms driving resistance to this therapeutic combination remain poorly understood. Here, we have identified inactivation of JNK signalling, specifically loss of the JNK kinaseMAP2K7, as a major determinant of resistance to combined endocrine therapy and CDK4/6 inhibition in ER+ breast cancer. We uncovered thatMAP2K7loss augments tumour survivalin vitroandin vivo.This occurs via disruption of activator protein-1 (AP-1) transcription factors, and thus prevents the induction of therapy-induced senescence and JNK-induced stress response. These findings reveal a critical tumour suppressor role for the JNK pathway in ER+ breast cancer, highlighting the importance of identifying patients with deficient JNK signalling and cautioning against the development of JNK inhibitors for this setting.