Abstract P3-03-09: Resistance to palbociclib depends on multiple targetable mechanisms highlighting the potential of drug holidays and drug switching to improve therapeutic outcome
Authors: Martin L-A , Pancholi S , Ribas R , Gao Q , Simigdala N , Nikitorowicz-Buniak J , Johnston SR , Dowsett M 
Published: March 2017

Abstract
Background: Combination of CDK4/6 inhibitors and endocrine therapy has been shown to improve clinical outcome in advanced estrogen receptor-positive (ER+) breast cancer (BC) patients. However, most patients will progress with acquired resistance, making the identification of new therapeutic strategies and potential biomarkers of great importance. Here, we show for the first time that certain resistance mechanisms are amenable to drug holidays and drug switching while others cause irreversible resistance to further perturbation of G1/S and highlight the potential of targeting these tumours by blockade of G2/M with inhibitors of Wee1 or CDK7.
Methods: To identify adaptive mechanisms associated with resistance to palbociclib, MCF7 and T47D cells adapted to long term oestrogen deprivation (LTED), which either retain or lose expression of ER respectively, were grown in the presence of palbociclib until they became resistant (LTED991R). Cell lines were subjected to exome sequencing, global gene expression analysis and siRNA knockdown of 709 cellular kinases to identify candidates associated with resistance. Candidate drug targets were evaluated in proliferation assays. Data was validated in vivo using MCF7-LTED xenografts.
Results: Exome data showed few genetic changes were associated with resistance to palbociclib. MCF7-LTED991R had a copy number (CN) gain of CCNE2 and ESR1 compared to MCF7-LTED while T47D-LTED991R showed loss of RB. Global gene expression analysis revealed increased expression of CDK4, CDK2, CDK7 and CCNE1 in MCF7-LTED991R and CCNE2 and CDK2 in T47D-LTED991R. In order to assess if these changes were drug specific, cell lines were treated with another CDK4/6 inhibitor, abemaciclib. Strikingly, MCF7-LTED991R cells showed sensitivity, possibly as a result of increased CDK4 expression, for which abemaciclib has increased potency. We next assessed the plasticity of the 991R phenotype by giving the cells a drug holiday followed by re-challenge. T47D-LTED991R cells remained resistant whilst the MCF7-LTED991R cells were re-sensitised, an observation we confirmed in a MCF7-LTED xenograft after long term treatment with palbociclib. Finally, siRNA kinome knockdown highlighted CDK4, CDK7 and Wee1 as associated with the resistant phenotypes suggesting targeting of G2/M in both RB+ and RB- 991R tumours may provide benefit; this possibility was confirmed by increased sensitivity to THZ1 (CDK7 inhibitor) or MK1775 (Wee1 inhibitor).
Conclusion: Few genetic changes are associated with resistance to palbociclib in ER+ BC in vitro but kinase re-wiring provides resistance. RB loss of function appears an irreversible mechanism of resistance while gain of cyclin E, and overexpression of CDK2 and CDK4 are amenable to a drug holiday, leading to re-sensitisation to palbociclib in vitro and in vivo. Of note, the palbociclib-resistant cells that retained RB expression were sensitive to abemaciclib possibly as a result of increased expression of CDK4 acting as compensatory mechanism. Finally, LTED cell lines retaining ER that were resistant to palbociclib also showed a gain in ESR1 CN, highlighting the potential for combination therapy with an alternate endocrine agent, such as fulvestrant.
Citation Format: Martin L-A, Pancholi S, Ribas R, Gao Q, Simigdala N, Nikitorowicz-Buniak J, Johnston SR, Dowsett M. Resistance to palbociclib depends on multiple targetable mechanisms highlighting the potential of drug holidays and drug switching to improve therapeutic outcome [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 P3-03-09.