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

Abstract

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 tumor 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 have suggested cooperation between the 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 in 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 reactivate 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. Mol Cancer Ther; 17(5); 908-20. ©2018 AACR.

Figure 1. Vistusertib (AZD2014) inhibits E2F mediated gene transcription.

Effects of vistusertib (AZD2014) treatment (24 hours) on RB and mTORC1/2 substrates measured by Western blotting in the ER+ cell lines MCF-7 (A) and HCC-1428 (B). (C) Targeted gene expression profiling of 96 genes in MCF-7 cells, including 43 E2F dependent genes. Heatmap shows the 27 E2F dependent genes with a significant fold change modulation vs DMSO (-2 < fold change <2) and a p-value <0.05.

Figure 2. Combined vistusertib (AZD2014) and palbociclib treatment results in more profound growth inhibition and enhanced CDK-RB-E2F pathway modulation.

(A) The effect of vistusertib (AZD2014) and palbociclib, alone or in combination, on the growth of MCF-7 cells over 120 hours, measured using an Incucyte Zoom. (B) The effect of vistusertib (AZD2014, 100nM) and palbociclib (300nM), alone or in combination, on cell number (6 days). Dotted line represents cell number at day 0. ***<0.0001 by Student t-test. (C) Analysis of mTOR pathway and CDK-RB-E2F pathway biomarkers following 24 hours treatment with increasing concentrations of vistusertib (AZD2014) or palbociclib, alone and in combination. (D) qPCR analysis of six E2F dependent genes. Bar charts represent log fold change mRNA expression relative to DMSO control following 6 and 24 hours exposure to 100nM AZD2014, 300nM palbociclib or the combination. (E) E2F dependent protein levels measured by western blotting, following 24 hours treatment using stated concentrations of vistusertib (AZD2014) with or without palbociclib.

Figure 3. Combined vistusertib (AZD2014) and palbociclib treatment results in significant tumour growth inhibition in ER+ breast cancer xenografts.

(A) Efficacy combination study of vistusertib (AZD2014, 7.5mg/kg once daily p.o.) and palbociclib (50 mg/kg once daily p.o.) compared to either agent alone in MCF-7 xenograft grown in male SCID mice. (B) Biomarker analysis of MCF-7 xenografts treated with vistusertib (AZD2014) and palbociclib. Tumours were excised at the end of the study and protein expression was analysed by Western blotting. (C) Efficacy combination study of vistusertib (AZD2014, 10mg/kg twice daily p.o., 2 days on/5 days off) and palbociclib (50 mg/kg once daily p.o.) compared to either agent alone in MCF-7 xenograft grown in male SCID mice. p values were calculated using two-sided Student’s t test. * p<0.05; ** p<0.01; *** p<0.001.

Figure 4. Inhibition of mTORC1/2 and CDK4/6 signalling causes long term growth arrest via inhibition of E2F signalling.

(A) MCF-7 cells were treated with vistusertib (AZD2014) and/or palbociclib and cell number was assessed at the indicated times using a sytox green endpoint. (B) β-galactosidase activity (green) and the nucleus (blue) were stained following treatment of MCF-7 cells as indicated (8 days). (C) Bar charts representing the proportion of cells expressing low or high levels of β-galactosidase activity. Each bar represents the mean +/- s.e.m from three independent experiments. (D) MCF-7 cells were treated with vistusertib (AZD2014) and/or palbociclib as indicated and % confluency was measured using an Incucyte Zoom. For some treatments, parallel wells were set up and after 19 days compound was removed from half the wells (washout) and % confluency measured for a further 8 days. (E) MCF-7 cells were treated with 100nM vistusertib (AZD2014) plus 300nM palbociclib for 21 days. Cells were then left in media containing the drugs (long term treated; LTT) or washed a returned to drug free media (release; Rel) for a further 72 hours before being lysed and subject to immunoblot with the antibodies indicated; (F) Efficacy combination study of vistusertib (AZD2014, 10mg/kg twice daily p.o., 2 days on/5 days off), palbociclib (50 mg/kg once daily p.o.) and fulvestrant (5 mg/kg/ week, s.c.) compared to each agent alone in MCF-7 xenograft grown in male SCID mice. p values were calculated using two-sided Student’s t test. * p<0.05; ** p<0.01; *** p<0.001

Figure 5. Inhibition of mTOR signalling does not affect binding of ER to chromatin.

(A) ChIP-seq signal in a 5 Kb window around 23,500 ER binding sites that are common between MCF-7 cells treated with vehicle, RAD001, or vistusertib (AZD2014, 500 nM) for two hours (see Supplementary Figure S2B for Venn diagram of the differential binding analysis of the three conditions). (B) Examples of ER binding at well-known ER binding sites from the UCSC genome browser (http://genome.ucsc.edu/). (C) Selected enriched motifs in the 17,561 ER binding sites identified by MACS2 (49) in vehicle treated MCF-7 cells. Similar motifs were identified in ER binding sites identified in MCF-7 cells treated with RAD001 or vistusertib (AZD2014).

Figure 6. Palbociclib resistant cells reactivate CDK-RB-E2F signalling and retain sensitivity to vistusertib (AZD2014).

(A) MCF-7 cells, (parental or palbociclib resistant PC1,5,6,7) were treated with 1000nM palbociclib for 24 hours before lysis and Western blotting. (B) Parental MCF-7 cells and palbociclib resistant MCF-7 cells (PC6) were treated with increasing concentrations of vistusertib (AZD2014) in the presence or absence of 1000nM palbociclib before lysis and Western blotting.