A preclinical evaluation of the MEK inhibitor refametinib in HER2-positive breast cancer cell lines including those with acquired resistance to trastuzumab or lapatinib

// John O’Shea 1 , Mattia Cremona 1 , Clare Morgan 1 , Malgorzata Milewska 1 , Frankie Holmes 2 , Virginia Espina 3 , Lance Liotta 3 , Joyce O’Shaughnessy 4 , Sinead Toomey 1 , Stephen F. Madden 5 , Aoife Carr 1 , Naomi Elster 1 , Bryan T. Hennessy 1, * and Alex J. Eustace 1, * 1 Department of Medical Oncology, Royal College of Surgeons in Ireland, Beaumont Hospital, Beaumont, Ireland 2 Texas Oncology-Memorial Hermann Memorial City, US Oncology Research, Houston, TX, USA 3 George Mason University, Manassas, VA, USA 4 Baylor-Sammons Cancer Center, Texas Oncology, US Oncology, Dallas, TX, USA 5 Data Science Centre, Royal College of Surgeons in Ireland, Dublin, Ireland * These authors have contributed equally to this work Correspondence to: Bryan T. Hennessy, email: bryanhennessy74@gmail.com Keywords: MEK inhibitor, HER2-positive breast cancer, acquired resistance to HER2-targeted therapies, reverse phase protein array Received: August 25, 2016     Accepted: June 02, 2017     Published: July 22, 2017 ABSTRACT Purpose: The MEK/MAPK pathway is commonly activated in HER2-positive breast cancer, but little investigation of targeting this pathway has been undertaken. Here we present the results of an in vitro preclinical evaluation of refametinib, an allosteric MEK1/2 inhibitor, in HER2-positive breast cancer cell lines including models of acquired resistance to trastuzumab or lapatinib. Methods: A panel of HER2-positive breast cancer cells were profiled for mutational status and also for anti-proliferative response to refametinib alone and in combination with the PI3K inhibitor (PI3Ki) copanlisib and the HER2-targeted therapies trastuzumab and lapatinib. Reverse phase protein array (RPPA) was used to determine the effect of refametinib alone and in combination with PI3Ki and HER2-inhibitors on expression and phosphorylation of proteins in the PI3K/AKT and MEK/MAPK pathways. We validated our proteomic in vitro findings by utilising RPPA analysis of patients who received either trastuzumab, lapatinib or the combination of both drugs in the NCT00524303/LPT109096 clinical trial. Results: Refametinib has anti-proliferative effects when used alone in 2/3 parental HER2-positive breast cancer cell lines (HCC1954, BT474), along with 3 models of these 2 cell lines with acquired trastuzumab or lapatinib resistance (6 cell lines tested). Refametinib treatment led to complete inhibition of MAPK signalling. In HCC1954, the most refametinib-sensitive cell line (IC 50 = 397 nM), lapatinib treatment inhibits phosphorylation of MEK and MAPK but activates AKT phosphorylation, in contrast to the other 2 parental cell lines tested (BT474-P, SKBR3-P), suggesting that HER2 may directly activate MEK/MAPK and not PI3K/AKT in HCC1954 cells but not in the other 2 cell lines, perhaps explaining the refametinib-sensitivity of this cell line. Using RPPA data from patients who received either trastuzumab, lapatinib or the combination of both drugs together with chemotherapy in the NCT00524303 clinical trial, we found that 18% (n=38) of tumours had decreased MAPK and increased AKT phosphorylation 14 days after treatment with HER2-targeted therapies. The combination of MEK inhibition (MEKi) with refametinib and copanlisib led to synergistic inhibition of growth in 4/6 cell lines tested (CI @ED 75 = 0.39-0.75), whilst the combinations of lapatinib and refametinib led to synergistic inhibition of growth in 3/6 cell lines (CI @ED 75 = 0.39-0.80). Conclusion: Refametinib alone or in combination with copanlisib or lapatinib could represent an improved treatment strategy for some patients with HER2-positive breast cancer, and should be considered for clinical trial evaluation. The direct down-regulation of MEK/MAPK but not AKT signalling by HER2 inhibition (e.g. by lapatinib or trastuzumab), which we demonstrate occurs in 18% of HER2-positive breast cancers may serve as a potential biomarker of responsiveness to the MEK inhibitor refametinib.