Abstract P1-09-03: Global knockdown of cellular kinases identifies MPS1 as a novel modulator of endocrine and palbociclib resistance highlighting a new role for MPS1 inhibitors
Authors: Nikitorowicz-Buniak J , Pancholi S , Simigdala N , Ribas R , Linardopoulos S , Dowsett M , Johnston SR , Martin L-A 
Published: February 2018

Background Estrogen-receptor positive (ER+) breast cancer (BC) accounts for over 75% of diagnosed cases. Despite treatment, a large proportion relapse with de novo or acquired endocrine resistant disease, making it one of the greatest challenges for BC research. Using a kinome siRNA library screen, we identified MPS1 that is required for recruitment of the spindle assembly checkpoint complex, as strongly associated with resistance to endocrine therapy and palbociclib. Until now, the target population for MPS1 inhibitors has focused on triple negative BC. Our unexpected finding shows, potential efficacy of MPS1 inhibitors in ER+ BC resistant to endocrine therapies and palbociclib, a prominent contemporary first-line combination for advanced disease.

Methods ER+ BC cell lines (MCF7, SUM44, ZR75.1, HCC1428 and T47D) adapted to estrogen independent growth (LTED) and sequential resistance to palbociclib (991R) were subjected to a siRNA screen targeting 709 kinases. Z-scores were used to identify the most robust candidates. Cell viability upon MPS1 inhibition with CCT289346 (MPS1i) was assessed 2D and 3D. The class effect was confirmed with other compounds targeting MPS1. Impact of MPS1i on ER co-localisation and ER-transactivation was assessed using confocal microscopy and reporter assays, respectively. Effect of MPS1i on chromosomal alignment and time spent in mitosis was established by time lapse and confocal microscopy. BrdU incorporation and cell cycle were assessed by FACS. PARP cleavage was used to measure apoptosis. Global gene expression analysis of MPS1 was carried out in two independent neoadjuvant studies of aromatase inhibitor (AI) treated patients.

Results Kinome knockdown identified targets associated with the G2/M checkpoint as strongly implicated in the LTED phenotype. In particular, MPS1 was the top common hit in LTED and 991R cell lines. Increase in MPS1 was evident in MCF7-LTED at both the transcript and protein level. Notably, the MPS1 inhibitor CCT289346 caused a significant reduction in viability of the majority of LTED and 991R cell lines tested.

Table. Sensitivity of ER+ BC cell lines to CCT289346Cell LineModelIC50 nMMCF7Wt90 LTEDESR1wt80 LTEDESR1Y537C50 TAMR55 ICIR50 LTEDICIR60 991R40 LTED991R45 LTED991R-ICIR25T47DWt110 LTED110 991R50 LTED991R60SUM44LTED40

Upon inhibition of MPS1, cells demonstrated shorter time in mitosis, aberration of cell cycle and amplified mitotic errors, resulting in increased apoptosis.

To evaluate the clinical relevance of MPS1 in ER+ BC treated with endocrine therapy, we interrogated publicly available datasets from patients treated with neoadjuvant AI therapy. In the anastrozole cohort, on-treatment gene expression of MPS1 (p&lt;0.0001) was significantly associated with poor response to anastrozole based on a 2-week residual Ki67 score &lt;10%. In the letrozole cohort, increased on-treatment expression of MPS1 (p=0.0118) was associated with poor response based of tumor shrinkage ≥50%.

Conclusion This novel finding shows MPS1 inhibitors are capable of inducing mitotic aberrations and apoptosis in ER+ BC models resistant to endocrine therapy and palbociclib providing a new therapeutic strategy.

Citation Format: Nikitorowicz-Buniak J, Pancholi S, Simigdala N, Ribas R, Linardopoulos S, Dowsett M, Johnston SR, Martin L-A. Global knockdown of cellular kinases identifies MPS1 as a novel modulator of endocrine and palbociclib resistance highlighting a new role for MPS1 inhibitors [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-09-03.