Endocrine resistant estrogen receptor positive (ER+) breast cancers are dependent upon cyclin-dependent kinases (CDK) 4/6 for proliferation. As such, CDK4/6 inhibitors have been integrated into clinical practice for the treatment of advanced ER+ breast cancers. Despite initial efficacy, acquired resistance to CDK4/6 inhibitors is beginning to emerge. To identify mechanisms of resistance we have generated a palbociclib resistant (PalbR) MCF-7 breast cancer cell line. Using RNAseq, cell cycle analysis by flow cytometry and western blotting of cell cycle associated proteins we have identified that increased proliferation by PalbR cells is induced by an up-regulation of Janus kinases (JAK) 1, 2 and 3, which can be inhibited by the JAK 1 and 3 inhibitor tofactinib. Due to the dynamic upstream regulation of the cell cycle by the JAK/STAT pathway, our findings indicate that PalbR cells have reduced levels of the CDK inhibitor proteins p21Cip1 and p27Kip1. Mechanistically, loss of p21Cip1 and p27Kip1 de-represses CDK2 activity, and we find that PalbR cells have an enhanced sensitivity to the CDK2 inhibitor CYC065.
To complement this model, we are developing a novel panel of in vitro models that mimic the clinical treatment of patients. Here, palbociclib is combined with an endocrine therapy; tamoxifen, fulvestrant, or long-term estrogen deprivation to mimic clinical treatment with aromatase inhibitors. These models demonstrate differences in cellular morphology and growth trajectories compared to the PalbR cell line. In parallel we have generated an in vivo ER+ patient-derived xenograft model which is resistant to chronic fulvestrant-palbociclib treatment. We have performed comprehensive analyses to identify resistance mechanisms that are common to the treatment combinations, or are unique to each treatment regime. Our novel panel of resistant models provides a framework to identify the specific mechanisms of acquired resistance, and a vehicle for testing clinically relevant therapies that could counteract this resistance.