Combined inhibition of mTOR and CDK4/6 is required for optimal blockade of E2F function and long term growth inhibition in estrogen receptor positive breast cancer

Chrysiis Michaloglou, Claire Crafter, Rasmus Siersbaek, Oona Delpuech, Jon O Curwen, Larissa S Carnevalli, Anna D Staniszewska, Urszula M Polanska, Azadeh Cheraghchi-Bashi, Mandy Lawson, Igor Chernukhin, Robert McEwen, Jason S Carroll, Sabina C Cosulich

The cyclin dependent kinase (CDK) -retinoblastoma (RB) -E2F pathway plays a critical role in the control of cell cycle in estrogen receptor positive (ER+) breast cancer. Small molecule inhibitors of CDK4/6 have shown promise in this tumour type in combination with hormonal therapies, reflecting the particular dependence of this subtype of cancer on cyclin D1 and E2F transcription factors. mTOR inhibitors have also shown potential in clinical trials in this disease setting. Recent data has suggested cooperation between the phosphatidylinositol 3-kinase (PI3K)/mTOR pathway and CDK4/6 inhibition in preventing early adaptation and eliciting growth arrest, but the mechanisms of the interplay between these pathways have not been fully elucidated. Here we show that profound and durable inhibition of ER+ breast cancer growth is likely to require multiple hits on E2F mediated transcription. We demonstrate that inhibition of mTORC1/2 does not affect ER function directly, but does cause a decrease in cyclin D1 protein, RB phosphorylation and E2F mediated transcription. Combination of an mTORC1/2 inhibitor with a CDK4/6 inhibitor results in more profound effects on E2F dependent transcription, which translates into more durable growth arrest and a delay to the onset of resistance. Combined inhibition of mTORC1/2, CDK4/6 and ER delivers even more profound and durable regressions in breast cancer cell lines and xenografts. Furthermore, we show that CDK4/6 inhibitor resistant cell lines re-activate the CDK-RB-E2F pathway, but remain sensitive to mTORC1/2 inhibition, suggesting that mTORC1/2 inhibitors may represent an option for patients that have relapsed on CDK4/6 therapy.