Volume 67, Issue 6, Pages 1177-1185 (June 2015) High Efficacy of Combination Therapy Using PI3K/AKT Inhibitors with Androgen Deprivation in Prostate Cancer Preclinical Models  Rute B. Marques, Ashraf Aghai, Corrina M.A. de Ridder, Debra Stuurman, Sander Hoeben, Agnes Boer, Rebecca P. Ellston, Simon T. Barry, Barry R. Davies, Jan Trapman, Wytske M. van Weerden  European Urology  Volume 67, Issue 6, Pages 1177-1185 (June 2015) DOI: 10.1016/j.eururo.2014.08.053 Copyright © 2014 European Association of Urology Terms and Conditions

Fig. 1 In vitro activity of an AKT inhibitor (AZD5363) and a PI3Kβ/δ inhibitor (AZD8186) in prostate cancer cell lines. (A) Half maximal inhibitory concentration (IC50) in androgen-depleted medium and in the presence of 0.1nM of the synthetic androgen R1881. Correlation with PTEN, phospho-AKT (S473), and androgen receptor (AR) expression is shown in the heat map under the graph. Light orange=no expression; middle orange=positive; dark orange=high expression. Cell line characteristics are summarized in Table 1. (B) Analysis of the PI3K/AKT pathway activity by Western blot of phosphoprotein targets in PC346Flu1. Conc=concentration; IC50=half maximal inhibitory concentration; AR=androgen receptor. European Urology 2015 67, 1177-1185DOI: (10.1016/j.eururo.2014.08.053) Copyright © 2014 European Association of Urology Terms and Conditions

Fig. 2 Preclinical evaluation of AZD5363 and AZD8186 as single agents and in combination with surgical castration in (A) PTEN-negative PC346C and (B) PTEN-positive PC310 patient-derived xenografts. Xenografts were transplanted subcutaneously in male NMRI nude mice. Mice were randomized over six groups: placebo, castration, AKT inhibitor (AKTi), PI3K inhibitor (PI3Ki), AKTi plus castration, and PI3Ki plus castration. They were dosed orally with AKTi (AZD5363: 100mg/kg once daily), PI3Ki (AZD8186: 75mg/kg twice daily), or placebo control. T0 marks the start of treatment. Results represent tumor volume (mean plus standard error of the mean; n=13 or 14 mice per group). In the combination-treated PC346C groups, treatment with PI3K/AKT inhibitors was stopped after 60 d and mice (n=5) were supplemented with testosterone via Silastic implants (Dow Corning Corp., Midland, MI, USA) to check for the presence of viable tumor cells. AKTi=AKT inhibitor; cas=castration; PI3Ki=PI3K inhibitor. European Urology 2015 67, 1177-1185DOI: (10.1016/j.eururo.2014.08.053) Copyright © 2014 European Association of Urology Terms and Conditions

Fig. 3 Biomarker analysis of PI3K/AKT and AR pathway activity after treatment with AZD5363 and AZD8186. Plasma and tumor material were collected from the PC346C xenograft experiment 4h after the last doses. (A) Western blot analysis of PI3K/AKT targets’ phosphorylation (n=3 representative mice per group). (B) Quantification of phospho/total protein ratios for AKT (by electrochemiluminescence assay), PRAS40 (by enzyme-linked immunosorbent assay [ELISA]), and GSK3 (by Western blot intensity). Data are given as mean plus standard error of the mean (SEM) (n=5–7). (C) AR pathway analysis: expression of AR (by Western blot) and AR targets PSA (ELISA on plasma), TMPRSS2, and FKBP5 (by reverse-transcription-polymerase chain reaction) (data given as mean plus SEM). * p<0.05; ** p<0.005, by Mann-Whitney two-sided test. ELISA=enzyme-linked immunosorbent assay; no.=number; SEM=standard error of the mean. European Urology 2015 67, 1177-1185DOI: (10.1016/j.eururo.2014.08.053) Copyright © 2014 European Association of Urology Terms and Conditions

Fig. 4 Reintroduction of wild-type PTEN in a LNCaP-inducible model. (A) Western blot analysis of PTEN, phospho-AKT (Ser473), and phospho-PRAS40 (Thr246) expression. LNCaP-PTEN cells (500 000 cells per well) were seeded in six-well plates and steroid-starved in RPMI-1640 medium plus 5% dextran-coated, charcoal-treated fetal bovine serum (FBS) for 48h before the stimulations. PTEN expression was induced with 350 ng/ml doxycycline in the presence or absence of 1nM R1881 androgen. Cell lysates were collected after 24h incubation. (B) Methylthiazolyldiphenyl-tetrazolium bromide (MTT) viability assay: LNCaP-PTEN cells were treated for 7 d with 100 ng/ml doxycycline in the presence or absence of 0.1nM R1881. Results are presented as percent growth relative to the androgen (R1881)-stimulated control. Doxycycline treatment (leading to PTEN re-expression and PI3K/AKT inhibition) inhibited growth of LNCaP-PTEN cells. Maximum impact was achieved upon inhibition of both PI3K/AKT and AR pathways (+doxycycline, −R1881). (C) LNCaP-PTEN cells were grown in RPMI-1640 medium plus 5% FBS and treated with 100 ng/ml doxycycline, in the presence or absence of 1μM of the antiandrogen MDV3100 (enzalutamide). MTT was assayed after 1, 3, 5, and 7 d of stimulation. Results are presented as mean MTT absorbance (570nm) plus standard deviation (SD). Doxycycline itself had no impact on cell viability, as demonstrated by treatment of control-transfected LNCaP-TetON cells lacking the PTEN vector (Supplementary Fig. 6). (D) Reverse-transcription-polymerase chain reaction quantification of AR and its target genes KLK3 (PSA), TMPRSS2, and FKBP5 (data given as mean normalized expression plus SD). LNCaP-PTEN cells were treated for 24h with R1881 and doxycycline, as described in Figure 4A. * p<0.05. Dox=doxycycline; FBS=fetal bovine serum; MTT=methylthiazolyldiphenyl-tetrazolium bromide; SD=standard deviation. European Urology 2015 67, 1177-1185DOI: (10.1016/j.eururo.2014.08.053) Copyright © 2014 European Association of Urology Terms and Conditions