Activation of the interferon signaling pathway is associated with resistance to CDK4/6 inhibitors and immune checkpoint activation in ER-positive breast cancer

Purpose: CDK4/6 inhibitors are highly effective against ER+/HER2- breast cancer (BC); however, intrinsic and acquired resistance is common. Elucidating the molecular features of sensitivity and resistance to CDK4/6i’s may lead to identification of predictive biomarkers and novel therapeutic targets, paving the way toward improving patient outcomes. Experimental design: Parental BC cells and their endocrine-resistant derivatives (EndoR) were used. Derivatives with acquired resistance to palbociclib (PalboR) were generated from parental and estrogen-deprivation resistant MCF7 and T47D cells. Transcriptomic and proteomic analyses were performed in palbociclib-sensitive and PalboR lines. Gene expression data from CDK4/6i neoadjuvant trials and publicly available datasets were interrogated for correlations of gene signatures and patient outcomes. Results: Parental and EndoR BC lines showed varying degrees of sensitivity to palbociclib. Transcriptomic analysis of these cell lines identified an association between high interferon (IFN) signaling and reduced CDK4/6i sensitivity; thus an ‘IFN-Related Palbociclib-Resistance Signature’ (IRPS) was derived. In two neoadjuvant trials of CDK4/6i plus endocrine therapy, IRPS and other IFN-related signatures were highly enriched in patients with tumors exhibiting intrinsic resistance to CDK4/6i. PalboR derivatives displayed dramatic activation of IFN/STAT1-signaling compared to their short-term treated or untreated counterparts. In primary ER+/HER2- tumors, the IRPS score was significantly higher in lumB than lumA subtype and correlated with increased gene expression of immune checkpoints, endocrine resistance, and poor prognosis. Conclusion: Aberrant IFN-signaling is associated with intrinsic resistance to CDK4/6i. Experimentally, acquired resistance to palbociclib is associated with activation of the IFN­ pathway, warranting additional studies to clarify its involvement in resistance to CDK4/6i. In this study, we sought to investigate the molecular profiles associated with intrinsic and acquired resistance to CDK4/6i in HR+/HER2- BC. To this end, we analyzed transcriptomic and proteomic profiles of endocrine-sensitive and -resistant BC cell lines tested for sensitivity to pharmacological or genetic inhibition of CDK4/6, studied tumor samples from patients with HR+/HER2- enrolled in neoadjuvant clinical trials evaluating CDK4/6i in combination with aromatase inhibitors, and examined preclinical models of acquired resistance to CDK4/6i. Integrating these data, we found intrinsic activation of IFN-signaling to be associated with reduced sensitivity to CDK4/6 inhibition in pre-clinical models, and with worse outcomes in patients. We also detected a remarkable up-regulation of IFN-signaling in CDK4/6i-resistant cell line derivatives. Taken together, our results suggest a role for activation of the IFN-signaling pathway in promoting both intrinsic and acquired resistance to CDK4/6 inhibitors in BC. The combination of CDK4/6 inhibitors (i) with endocrine therapy represents the standard of care for patients with ER+/HER2- advanced breast cancer. Despite marked improvement in patient outcomes with this combination, de novo and acquired resistance is common. Our study reveals an inverse association between response to CDK4/6i and interferon signaling pathway activation. We demonstrate that activation of the IFN signaling pathway is associated with intrinsic and acquired resistance to CDK4/6i. Clinically, enrichment for IFN-related gene signatures is associated with resistance to neoadjuvant CDK4/6i in early stage ER+ breast cancer. Furthermore, primary ER+/ HER2- breast cancers displaying intrinsic high interferon signaling are associated with poor prognosis. Overall, our findings suggest that the interferon pathway may offer predictive biomarkers to identify patients whose disease is less likely to respond to CDK4/6i, and potential actionable targets for the development of novel therapeutic approaches to prevent/reverse resistance to CDK4/6i.