Abstract P3-03-12: CDK4/6 inhibitor resistant ER-positive cells remain dependent on estrogen signalling and retain sensitivity to endocrine therapy
Authors: Lenihan C , Bouchekioua-Bouzaghou K , Abdulghani R , Chupin J , Shia A , Schmid P 
Published: May 2019

Background: Dysregulation of the cyclin D-CDK4/6-Rb pathway is a frequent feature of ER positive breast cancers and has been linked with endocrine resistance. Several randomised trials have shown that CDK4/6 inhibitors in combination with endocrine treatment can substantially improve the outcome of patients with metastatic ER positive breast cancer. With increased clinical use of CDK4/6 inhibitors acquired resistance is emerging as a major clinical challenge. It is critical to understand the sensitivity and responsiveness of these resistant cells to estrogen to consider continued use of endocrine therapy in combination with CDK4/6 inhibition.

Results: Long-term culture of the ER positive cell lines T47D and MCF7 with increasing doses of drug generated distinct clones with acquired resistance to either palbociclib or ribociclib. ESR1 expression was stable across all resistant clones. All clones continued to be reliant on estrogen for cell growth, with estrogen deprivation inducing cell death. Resistant clones still responded to estrogen stimulation. Estradiol treatment induced at least 2-fold transcriptional upregulation of pS2 and c-myc; the induction was inhibited by tamoxifen and fulvestrant treatment. Both palbociclib and ribociclib resistant clones remained sensitive to endocrine therapy, with tamoxifen IC50 values equivalent to the parental T47D and MCF7 cell lines.

Mechanisms of resistance were different between the different CDK4/6 inhibitors and within the two cell lines. Palbociclib resistant MCF7 and T47D clones all increase cyclin E protein levels, but only T47D transcriptionally upregulated cyclin E. MCF7 clones transcriptionally upregulated cyclin D, with corresponding protein increase. Therefore, CDK2/cyclin E complexes seem to drive progression in MCF7 cells, whilst CDK2/cyclin D complexes function in T47D cells. Ribociclib resistant clones presented with unchanged or even decreased CDK2 expression, but demonstrated notable increase in E2F1, indicating the requirement of a non-canonical CDK-cyclin pairing independent of CDK2.

Cell lines did not exhibit complete cross resistance to all CDK4/6 inhibitors. All ribociclib-resistant T47D and MCF7 clones conferred cross-resistance to palbociclib. The majority of clones were also resistant to abemaciclib, although one T47D clone retained abemaciclib sensitivity. All palbociclib-resistant clones were equally resistant to ribociclib. Conversely, whilst one T47D ribociclib clone exhibited marked cross resistance to abemaciclib, the majority of clones remained sensitive to treatment with abemaciclib. This highlights potential different resistant mechanisms for abameciclib independent characterised cell cycle changes for palbociclib and ribociclib.

Conclusions: Cells resistant to CDK4/6 inhibitors remain dependent on estrogen signalling and retain sensitivity to endocrine therapy. We continue to show that resistance is mediated by non-canonical CDK-cyclin pairings. Palbociclib and ribociclib resistant clones confer cross-resistance to both Palbociclib and ribociclib but incomplete cross-resistance to abemaciclib.

Citation Format: Lenihan C, Bouchekioua-Bouzaghou K, Abdulghani R, Chupin J, Shia A, Schmid P. CDK4/6 inhibitor resistant ER-positive cells remain dependent on estrogen signalling and retain sensitivity to endocrine therapy [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-03-12.