Palbociclib is a CDK4/6 inhibitor that received FDA approval for treatment of hormone receptor positive (HR+) HER2 negative (HER2neg) advanced breast cancer. To better personalize patients treatment it is critical to identify subgroups that would mostly benefit from it. We hypothesize that complex alterations of the Retinoblastoma (Rb) pathway might be implicated in resistance to CDK4/6 inhibitors and aim to investigate whether signatures of Rb loss-of-function would identify breast cancer cell lines resistant to palbociclib. We established a gene expression signature of Rb loss-of-function (RBsig) by identifying genes correlated with E2F1 and E2F2 expression in breast cancers within The Cancer Genome Atlas. We assessed the RBsig prognostic role in the METABRIC and in a comprehensive breast cancer meta-dataset. Finally, we analyzed whether RBsig would discriminate palbociclib-sensitive and -resistant breast cancer cells in a large RNA sequencing-based dataset. The RBsig was associated with RB1 genetic status in all tumors (p <7e-32) and in luminal or basal subtypes (p < 7e-11 and p < 0.002, respectively). The RBsig was prognostic in the METABRIC dataset (discovery: HR = 1.93 [1.5-2.4] p = 1.4e-08; validation: HR = 2.01 [1.6-2.5] p = 1.3e-09). Untreated and endocrine treated patients with estrogen receptor positive breast cancer expressing high RBsig had significantly worse recurrence free survival compared to those with low RBsig (HR = 2.37 [1.8 − 3.2] p = 1.87e−08 and HR = 2.62 [1.9− 3.5] p = 8.6e−11, respectively). The RBsig was able to identify palbociclib resistant and sensitive breast cancer cells (ROC AUC = 0,7778). Signatures of RB loss might be helpful in personalizing treatment of patients with HR+/HER2neg breast cancer. Further validation in patients receiving palbociclib is warranted.

CDK4/6 inhibitors represent a new treatment standard for hormone receptor-positive (HR+), HER2-negative advanced breast cancer (BC) patients. Although efficacious, resistance to these agents is universal. Here, we profiled a large panel of HR+ BC cell lines with conditioned resistance to the CDK4/6 inhibitor palbociclib, and analyzed cell cycle-related markers by gene expression profiles (GEP) and western blot (WB). GEP showed high molecular heterogeneity among the models, with E2F targets being significantly enriched both during treatment and at the time of resistance. By both WB and GEP, a common molecular feature at the time of palbociclib resistance was the concomitant overexpression of cyclin E1 and down-regulation of Rb. CCNE1 was the only significantly up-regulated gene among E2F targets at resistance with CCNE1 genomic amplification being observed in two resistant models. Rb was downregulated in all resistant models; a reduction of RB1 copy number was observed in three resistant cell lines. In silico analyses showed that CCNE1/RB1 ratio correlated with palbociclib IC50 in different datasets of both breast and non-breast cancer cell lines, performing better than CCNE1 or RB1 taken separately. Finally, the CCNE1/RB1 ratio was shown to be an adverse prognostic factor in patients with ER+ BC and to be able to discriminate palbociclib-sensitive versus resistant among patients enrolled in the NeoPalAna trial, a neoadjuvant trial testing palbociclib, performing better than CCNE1 or RB1 alone. Our data suggest that the CCNE1/RB1 ratio may be a viable biomarker of palbociclib resistance, warranting further clinical validation.

Randomized clinical trials demonstrated that CDK4/6 inhibitors are highly effective in patients with hormone receptor-positive (HR+), HER2-negative (HER2-) metastatic breast cancer in combination with endocrine therapy. The use of CDK4/6 inhibitors in clinics is becoming common for patients with HR+/HER2- metastatic breast cancer and will certainly increase in the near future. However, patients might show de novo or acquired resistance to these drugs. Molecular alterations have been suggested as determinants for de novo resistance to CDK4/6 inhibitors, but have never been validated in a clinical setting. In addition, molecular mechanisms of acquired resistance to palbociclib have been analyzed only in preclinical studies. Here we review the current knowledge on the available preclinical data about the mechanisms of de novo and acquired resistance to CDK4/6 inhibitors in breast cancer, and clinical data about potential biomarkers of response.

Purpose: Resistance to endocrine therapy (ET) and CDK4/6 inhibitors (CDK4/6i) is a clinical challenge in estrogen receptor (ER)-positive (ER+) breast cancer. Cyclin-dependent kinase 7 (CDK7) is a candidate target in endocrine-resistant ER+ breast cancer models and selective CDK7 inhibitors (CDK7i) are in clinical development for the treatment of ER+ breast cancer. Nonetheless, the precise mechanisms responsible for the activity of CDK7i in ER+ breast cancer remain elusive. Herein, we sought to unravel these mechanisms. Experimental Design: We conducted multi-omic analyses in ER+ breast cancer models in vitro and in vivo, including models with different genetic backgrounds. We also performed genome-wide CRISPR/Cas9 knockout screens to identify potential therapeutic vulnerabilities in CDK4/6i-resistant models. Results: We found that the on-target antitumor effects of CDK7 inhibition in ER+ breast cancer are in part p53 dependent, and involve cell cycle inhibition and suppression of c-Myc. Moreover, CDK7 inhibition exhibited cytotoxic effects, distinctive from the cytostatic nature of ET and CDK4/6i. CDK7 inhibition resulted in suppression of ER phosphorylation at S118; however, long-term CDK7 inhibition resulted in increased ER signaling, supporting the combination of ET with a CDK7i. Finally, genome-wide CRISPR/Cas9 knockout screens identified CDK7 and MYC signaling as putative vulnerabilities in CDK4/6i resistance, and CDK7 inhibition effectively inhibited CDK4/6i-resistant models. Conclusions: Taken together, these findings support the clinical investigation of selective CDK7 inhibition combined with ET to overcome treatment resistance in ER+ breast cancer. In addition, our study highlights the potential of increased c-Myc activity and intact p53 as predictors of sensitivity to CDK7i-based treatments.