A preclinical evaluation of the PI 3 K alpha / delta dominant 3 inhibitor BAY 80-6946 in HER 2-positive breast cancer models 4 with acquired resistance to the HER 2-targeted therapies 5 trastuzumab and lapatinib 6

11 Abstract The PI3K pathway is a key mechanism of 12 trastuzumab resistance, but early attempts to indirectly target 13 this pathway with mTOR inhibitors have had limited suc14 cess. We present the results of a preclinical study of the 15 selective alpha/delta isoform dominant PI3K inhibitor BAY 16 80-6946 tested alone and in combination with HER2-tar17 geted therapies in HER2-positive cell lines, including mod18 els with acquired resistance to trastuzumab and/or lapatinib. 19 A panel of HER2-positive breast cancer cells were profiled 20 for their mutational status using Sequenom MassARRAY, 21 PTEN status byWestern blot, and anti-proliferative response 22 to BAY 80-6946 alone and in combination with the HER223 targeted therapies trastuzumab, lapatinib and afatinib. 24 Reverse phase protein array was used to determine the effect 25 of BAY 80-6946 on expression and phosphorylation of 68 26 proteins including members of the PI3K and MAPK path27 ways. The Boyden chamber method was used to determine if 28 BAY80-6946 affected cellular invasion andmigration. BAY 29 80-6946 has anti-proliferative and anti-invasive effects when 30 used alone in our panel of cell lines (IC50’s 3.9–29.4 nM). 31 BAY 80-6946 inhibited PI3K signalling and was effective in 32 cells regardless of their PI3K, P53 or PTEN status. The 33 combination of HER2-targeted therapies and BAY 80-6946 34 inhibited growth more effectively than either therapy used 35 alone (with clear synergism in many cases), and can restore 36 sensitivity to trastuzumab and lapatinib in cells with acquired 37 resistance to either trastuzumab and/or lapatinib. The addi38 tion of BAY 80-6946 to HER2-targeted therapy could rep39 resent an improved treatment strategy for patients with 40 refractory metastatic HER2-positive breast cancer, and 41 should be considered for clinical trial evaluation. 2