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A preclinical evaluation of the PI3K alpha/delta dominant inhibitor BAY 80-6946 in HER2-positive breast cancer models with acquired resistance to the HER2-targeted therapies trastuzumab and lapatinib

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Abstract

The PI3K pathway is a key mechanism of trastuzumab resistance, but early attempts to indirectly target this pathway with mTOR inhibitors have had limited success. We present the results of a preclinical study of the selective alpha/delta isoform dominant PI3K inhibitor BAY 80-6946 tested alone and in combination with HER2-targeted therapies in HER2-positive cell lines, including models with acquired resistance to trastuzumab and/or lapatinib. A panel of HER2-positive breast cancer cells were profiled for their mutational status using Sequenom MassARRAY, PTEN status by Western blot, and anti-proliferative response to BAY 80-6946 alone and in combination with the HER2-targeted therapies trastuzumab, lapatinib and afatinib. Reverse phase protein array was used to determine the effect of BAY 80-6946 on expression and phosphorylation of 68 proteins including members of the PI3K and MAPK pathways. The Boyden chamber method was used to determine if BAY 80-6946 affected cellular invasion and migration. BAY 80-6946 has anti-proliferative and anti-invasive effects when used alone in our panel of cell lines (IC50s 3.9–29.4 nM). BAY 80-6946 inhibited PI3K signalling and was effective in cells regardless of their PI3K, P53 or PTEN status. The combination of HER2-targeted therapies and BAY 80-6946 inhibited growth more effectively than either therapy used alone (with clear synergism in many cases), and can restore sensitivity to trastuzumab and lapatinib in cells with acquired resistance to either trastuzumab and/or lapatinib. The addition of BAY 80-6946 to HER2-targeted therapy could represent an improved treatment strategy for patients with refractory metastatic HER2-positive breast cancer, and should be considered for clinical trial evaluation.

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Acknowledgments

We thank Dr. Scott Wilhelm and Bayer Pharmaceuticals for providing us with BAY 80-6946. We also thank Dr. Norma O’Donovan, Dublin City University, for her gift of SKBR3-T, SKBR3-L, SKBR3-TL and HCC1954-L, and Dr. Neil O’Brien, University of California Los Angeles, for BT474-PAR and BT474-Res. We also thank St James University Hospital pharmacy for providing us with trastuzumab.

Conflict of interest

The authors state that they have no conflicts of interest in relation to this article or the funding bodies.

Financial information

This work was supported by Irish Cancer Society Research (CRS11ELS), Health Research Board (HRA/POR2012/054), BREAST-PREDICT, NECRET, the North Eastern Cancer Research and Education Trust and the Royal Irish Academy Mobility Grant 2013.

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Authors and Affiliations

  1. Department of Medical Oncology, Beaumont Hospital, Royal College of Surgeons in Ireland, Dublin, Ireland

    N. Elster, M. Cremona, C. Morgan, S. Toomey, A. Carr, B. T. Hennessy & A. J. Eustace

  2. Department of Pathology, Beaumont Hospital, Royal College of Surgeons in Ireland, Dublin, Ireland

    A. O’Grady

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  1. N. Elster
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  2. M. Cremona
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  3. C. Morgan
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Corresponding author

Correspondence to B. T. Hennessy.

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B. T. Hennessy and A. J. Eustace, joint senior authors have contributed equally to this work.

Electronic supplementary material

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10549_2014_3239_MOESM1_ESM.docx

Supplementary Fig. 1: Effect of 5nM BAY 80-6946 on (a) invasion and (b) migration in MDAMB453 and BT474 cells over a 24 h period. Error bars represent standard deviations of triplicate experiments. ‘*’ represents p < 0.05 as calculated by Student’s t-test. Supplementary material 1 (DOCX 56 kb)

10549_2014_3239_MOESM2_ESM.docx

Supplementary Fig. 2: Percentage changes in protein expression or phosphorylation as calculated from RPPA analysis following treatment of cells with 1nM BAY 80-6946 for 6 h relative to untreated controls in A) panel of HER2 positive BC cells, B) SKBR3 parental and resistant models C) HCC1954 and BT474 parental and resistant models. Standard deviations are calculated using propagation of error from triplicate independent biological experiments. Supplementary material 2 (DOCX 126 kb)

10549_2014_3239_MOESM3_ESM.docx

Supplementary Fig. 3: Percentage changes in protein expression or phosphorylation as calculated from RPPA analysis following treatment of cells with ‘●‘1nM BAY 80-6946, ‘▲‘lapatinib or ‘□‘a combination of lapatinib and BAY 80-6946 for 30 min relative to untreated controls SKBR3 and HCC1954 parental and lapatinib resistant cells. Standard deviations are calculated using propagation of error from triplicate independent biological experiments. Supplementary material 3 (DOCX 110 kb)

10549_2014_3239_MOESM4_ESM.docx

Supplementary Fig. 4: Efficacy of lapatinib or afatinib (-◊-), BAY 80-6946 (-□-) and a combination of lapatinib or afatinib with BAY 80-6946 (–∆–) in MDAMB453 cell lines which have de novo resistance to HER2-inhibitors. Error bars are representative of standard deviations across triplicate experiments. The ratio of lapatinib: BAY 80-6946 in this assay is fixed at 1:1. Supplementary material 4 (DOCX 50 kb)

Supplementary material 5 (DOCX 34 kb)

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Elster, N., Cremona, M., Morgan, C. et al. A preclinical evaluation of the PI3K alpha/delta dominant inhibitor BAY 80-6946 in HER2-positive breast cancer models with acquired resistance to the HER2-targeted therapies trastuzumab and lapatinib. Breast Cancer Res Treat 149, 373–383 (2015). https://doi.org/10.1007/s10549-014-3239-5

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