Overcoming intrinsic mechanisms of cell cycle inhibitor resistance in estrogen receptor-positive (ER+) breast cancer

Estrogen receptor-positive breast cancer (ER+BC) represent about 70% of all breast cancer cases. Approximately 30-50% of ER+BCs eventually develop resistance to primary endocrine therapy and progress to metastatic disease. The addition of cyclin-dependent kinases 4 and 6 inhibitors (CDK4/6i) to endocrine therapy has improved disease control in advanced and metastatic ER+BC. However, progression on CDK4/6i is common and further research is required to fully understand and overcome the mechanisms of CDK4/6i resistance in ER+BC. We performed single-cell RNA sequencing (scRNA-seq) of temporal tumor samples from the FELINE clinical trial (NCT02712723) to uncover emergent resistance phenotypes in ER+BC patients receiving a combination of ribociclib with letrozole or letrozole alone in a neoadjuvant setting. In the majority of patient tumors, resistant cancer cells activated ErbB growth factor signaling during treatment, with an upregulation in ERBB2 and ERBB4 in post-treatment cells, as well as increased activity of transcription factors downstream of ErbB signaling. This indicates that endocrine therapy plus CDK4/6i activated compensatory ErbB growth factor signaling in resistant cells, supporting an established estrogen-independent state of proliferation. To further understand this shift in cell state, we used afatinib, an ErbB family blocker, in combination with ribociclib to target this resistance mechanism. We developed multiple in vitro three-dimensional ER+BC cell culture models that are sensitive or resistant to ribociclib. Samples were collected regularly during monotherapy or combination of ribociclib and afatinib treatment and bulk RNA-sequencing measured the dynamical changes in the cell state over short and long timescales. Generalized additive models quantitatively evaluated cancer cell plasticity and synergy of combination therapy using the RNA-seq data. Our models revealed how activity of signaling pathways drive transcriptional changes in cell cycle regulators that promote or inhibit cell cycle progression. We found that ribociclib plus afatinib synergize to promote early and sustained cell cycle arrest, and later apoptosis, in both sensitive and resistant cells. Combination therapy simultaneously restored G1/S phase regulation by inhibiting the cyclin E/CDK2 complex and enforced G2/M phase control by inhibiting the cyclin B/CDK1 complex, thereby blocking entry into mitosis. This dual inhibition strategy not only controlled cancer proliferation, but also induced caspase-dependent apoptosis. Overall, our study revealed a key CDK4/6i resistance mechanism, and demonstrated that a combination of ribociclib plus afatinib overcomes intrinsic mechanisms of resistance in ER+BC.