Targeted degradation of CDK4 and 6 to overcome treatment resistance in breast cancer

Abstract Although CDK4/6 inhibitors (in combination with an endocrine therapy) are the preferred systemic therapy for treatment of patients with metastatic, Estrogen Receptor-positive (ER+) breast cancer (BC), development of resistance to CDK4/6 inhibitors is universal. Therefore, development of novel therapeutic strategies to target CDK4/6 inhibitor resistance remains an unmet clinical needs. To probe the dependency of ER+ BC cells and tumors on CDK4 and 6 for growth, we profiled a panel of ER+ BC cell lines, patient-derived xenografts (PDXs) and primary tumors from ER+ BC patients for expression of these proteins. We observed near universal expression of CDK4 but no detectable expression of CDK6 in ER+ BC samples. However, overexpression of CDK6 (and not CDK4) is a common mechanism of resistance to CDK4/6 inhibitors in ER+ BC patients. Using a panel of ER+ BC cell lines engineered to overexpress CDK6, we showed that CDK6 overexpression confers resistance to palbociclib although palbociclib completely blocked phosphorylation of pRB in these cells. Furthermore, overexpression of a kinase-dead (K43M) mutant of CDK6 (referred to as CDK6-KD) was sufficient to confer palbociclib resistance both in vitro and in vivo. Our mechanistic investigations revealed that CDK6 overexpression promotes transcriptional reprogramming of BC cells in a kinase-independent fashion and that the non-kinase function of CDK6 is a key driver of palbociclib resistance. Finally, we showed that CDK4/6-D, a CDK4/6-selective proteolysis targeting chimera (PROTAC), which causes targeted degradation of CDK6 (and CDK4), and eliminates both kinase and non-kinase functions of CDK6, exerts potent tumor growth inhibition in CDK6 high, palbociclib-resistant ER+ BC xenograft models. Supported by our preliminary data, we hypothesize that high CDK6 in ER+ BC cells causes CDK4/6 inhibitor resistance, at least in part, through kinase-independent transcriptional reprogramming, and that pharmacological targeting of both the kinase and non-kinase of CDK6 is necessary to inhibit the growth of these tumors. Specific Aim 1: To define the roles of kinase and non-kinase functions of CDK6 in conferring palbociclib resistance in a panel of CDK6-high, palbociclib-resistant cell line- and patient-derived xenograft (PDX) models of ER+ BC using advanced 3D genomic and machine learning approaches. Specific aim 2: To evaluate the in vivo efficacy of a CDK4/6-selective degrader (CDK4/6-D) in a panel of CDK6- high, palbociclib-resistant ER+ BC xenografts, both as a single agent and in combination with endocrine therapy. Specific Aim 3: To evaluate the efficacy of CDK4/6-D in a panel of CDK6-high, palbociclib-resistant PDXs engrafted in a unique NeoThy humanized mouse model, both as a single agent and in combination with endocrine therapy and checkpoint inhibition. Our novel therapeutic approach has the potential to improve clinical outcomes in many BC patients.