Volume 67, Issue 6, Pages 986-990 (June 2015) Combination AZD5363 with Enzalutamide Significantly Delays Enzalutamide-resistant Prostate Cancer in Preclinical Models  Paul Toren, Soojin Kim, Thomas Cordonnier, Claire Crafter, Barry R. Davies, Ladan Fazli, Martin E. Gleave, Amina Zoubeidi  European Urology  Volume 67, Issue 6, Pages 986-990 (June 2015) DOI: 10.1016/j.eururo.2014.08.006 Copyright © 2014 European Association of Urology Terms and Conditions

Fig. 1 Targeting the Akt pathway with AZD5363 in enzalutamide (ENZ)-resistant models. (A) MR49C and MR49F Akt pathway signalling protein levels following treatment with 1μM AZD5363 for 24h. (B) Cell proliferation of MR49C and MR49F cells assessed with crystal violet assay after 48h of treatment with indicated doses of AZD5363. Pooled means of triplicate experiments are plotted plus or minus the standard error of the mean. (C) AZD5363 induces apoptosis in MR49C and MR49F cells as assessed using caspase-3 activity assay. (D) Change in weekly mean tumour volume (left) and serum prostate-specific antigen (PSA) level (right) in mice treated with AZD5363 100mg/kg twice daily versus vehicle. Treatment was started when tumours reached 200mm3; ENZ 10mg/kg given daily since tumour inoculation was stopped when treatment started. Treatments were given by oral gavage 5 d on and 2 d off. (E) Representative images of Ki67 staining of collected vehicle- and AZD5363-treated MR49F xenografts. Complete results are shown in Supplementary Figure 2c. (F) Waterfall plots showing individual responses in tumour volume (left) and serum PSA change from baseline (right) between groups after 3 wk of treatment. CTL=control. European Urology 2015 67, 986-990DOI: (10.1016/j.eururo.2014.08.006) Copyright © 2014 European Association of Urology Terms and Conditions

Fig. 2 Combination targeting of Akt and androgen receptor with AZD5363 and enzalutamide (ENZ) delays ENZ resistance. (A) Western blots demonstrating Akt downstream effectors and apoptotic and cell cycle markers following treatment with dimethyl sulfoxide control, 1μM AZD5363, 10μM ENZ, or 1μM AZD5363 plus 10μM ENZ in LNCaP and C4-2 cells. (B) Apoptosis in LNCaP and C4-2 cells after 24h of treatment as assessed using caspase-3 activity assay. (C) Cell proliferation as assessed using crystal violet assay after 48h of treatment with AZD5363, ENZ, or AZD5363 plus ENZ in LNCaP and C4-2 cells at indicated doses. Pooled means of triplicate experiments are shown. *Synergistic effect of the combination calculated using the bliss independence model. (D) Mean tumour volume (left) and serum prostate-specific antigen (PSA) level (right) values in LNCaP castrate-resistant prostate cancer (CRPC) xenografts treated with vehicle, 37.5mg/kg AZD5363 twice daily, daily 10mg/kg, or AZD5363 37.5mg/kg twice daily plus daily 10mg/kg ENZ. Mice started treatment at the onset of CRPC and were treated for 5 d on and 2 d off. The inset in the left-side chart demonstrates observation of tumour volume in combination-treated mice up to 19 wk (five mice reached 19 wk). (E) Immunohistochemical Ki67 staining of representative samples from each group of xenografts collected at the end of study. Complete results from tumour microarrays are shown in Supplementary Figure 6c. (F) Tumour volume (left) and serum PSA level (right) of mice treated following castration with vehicle or AZD5363 37.5mg/kg twice daily plus daily 10mg/kg ENZ given at time of castration for 5 d on and 2 d off. (G) Mean weekly PSA values following treatment with AZD5363 plus ENZ given at time of castration or CRPC from both studies. * p<0.05; ** p<0.01; *** p<0.001. CTL=control. European Urology 2015 67, 986-990DOI: (10.1016/j.eururo.2014.08.006) Copyright © 2014 European Association of Urology Terms and Conditions

Supplementary Fig. 1 – Additional in vitro results of AZD5363 treatment of enzalutamide (ENZ)-resistant cells. (a) Time course demonstrating increase of pAkt protein levels with 10μM ENZ treatment relative to dimethyl sulfoxide control. Bar graphs (right) show densitometric intensity of the bands with pAkt levels normalized to vinculin levels. (b) Cleaved poly ADP ribose polymerase protein levels in MR49C and MR49F cells after 24h of treatment with 1μM AZD5363. (c) Cell cycle fraction of ENZ-resistant cells following AZD5363 treatment. MR49C and MR49F cells were treated with AZD5363 for 24h and then fixed and stained with propidium iodide and analysed by flow cytometry. European Urology 2015 67, 986-990DOI: (10.1016/j.eururo.2014.08.006) Copyright © 2014 European Association of Urology Terms and Conditions

Supplementary Fig. 2 – Additional in vivo results of AZD5363 treatment of enzalutamide (ENZ)-resistant xenografts. (a) Western blot showing basal levels of pAkt and Akt and downstream effectors S6, 4eBP-1 in ENZ-resistant MR49C and MR49F cell lines and parenteral LNCaP. Densitometry results of the representative Western blots (right) represent values normalized to vinculin levels. (b) A microarray of collected tumours (10 vehicle and 8 AZD5363 samples with triplicates) was stained with 1/500 Ki67, 1/25 pAkt (Ser473), and 1/150 Akt. Representative images (left) of mean staining intensity scores (right) by a blinded pathologist at ×20 magnification. Means are plotted plus or minus the standard error of the mean. European Urology 2015 67, 986-990DOI: (10.1016/j.eururo.2014.08.006) Copyright © 2014 European Association of Urology Terms and Conditions

Supplementary Fig. 3 – Treatment of enzalutamide (ENZ)-resistant cells with combination ENZ and AZD5363. (a) Cancer-specific survival (left) and overall survival of MR49F xenografts treated with vehicle or AZD5363 100mg/kg twice daily once total tumour volume reached >200mm3. (b) Mean weight of mice during treatment in each group. (c) Mean tumour volume (left) and mean serum PSA level (right) in mice treated with AZD5363 37.5mg/kg twice daily and AZD5363 75mg/kg twice daily. Daily ENZ 10mg/kg started at tumour inoculation was continued in control and treatment arms. All in vivo treatments were given as oral gavage 5 d on, 2 d off. (d) Waterfall plots of tumour volume (left) and PSA level (right) after 3 wk of treatment with AZD5363 37.5mg/kg twice daily and AZD5363 75mg/kg twice daily with concomitant daily ENZ 10mg/kg. (e) Cancer-specific survival of MR49F xenografts treated with AZD5363 37.5mg/kg twice daily and AZD53 63 75mg/kg twice daily with daily ENZ 10mg/kg. (f) Mean body weight of mice during indicated treatments. European Urology 2015 67, 986-990DOI: (10.1016/j.eururo.2014.08.006) Copyright © 2014 European Association of Urology Terms and Conditions

Supplementary Fig. 4 – Effects of AZD5363 plus enzalutamide (ENZ) on cell cycle analysis and 22RV1 cells. (a) LNCaP, C4-2, and 22RV1 cells were treated with monotherapy or combination AZD5363 1μM and ENZ 10μM for 24h. Cells were fixed and stained with propidium iodide and analysed by flow cytometry. Pooled mean results of at least two separate experiments are shown plus or minus the standard error of the mean. (b) Western blots demonstrating Akt downstream effectors and apoptotic and cell cycle markers following treatment for 24h with control dimethyl sulfoxide, 1μM AZD5363, 10μM ENZ, or 1μM AZD5363 plus 10μM ENZ in 22RV1 cells. (c) Cell proliferation assessed using crystal violet assay after 48h of treatment with AZD5363, ENZ, or AZD5363 plus ENZ in 22RV1 cells. Pooled results from three separate experiments with triplicates are shown. *=synergistic effect of the combination using the bliss independence model. (d) Apoptosis in 22RV1 cells after 24h of treatment as assessed using caspase-3 activity assay. European Urology 2015 67, 986-990DOI: (10.1016/j.eururo.2014.08.006) Copyright © 2014 European Association of Urology Terms and Conditions

Supplementary Fig. 5 – Additional results of AZD5363 plus enzalutamide (ENZ) in LNCaP in vivo models. (a) Study design for LNCaP in vivo models. (b) Kaplan-Meier survival curves for mice treated with vehicle, AZD5363 37.5mg/kg twice daily, daily ENZ 10mg/kg, or both at time of castrate-resistant prostate cancer (CRPC): overall survival (left), time to tumour doubling (centre), and cancer-specific survival (right). (c) Plot showing individual maximal percent growth and regression from baseline tumour volume for all mice during 12 wk of treatment. (d) Mean body weight of mice treated at time of CRPC. (e) Reasons mice euthanized prior to tumour size end point in mice treated at time of CRPC. No mice in study of AZD5363 plus ENZ given at time of castration were euthanized during study period. (f) Mean body weight of mice treated with vehicle or AZD5363 37.5mg/kg twice daily plus daily ENZ 10mg/kg at time of castration. European Urology 2015 67, 986-990DOI: (10.1016/j.eururo.2014.08.006) Copyright © 2014 European Association of Urology Terms and Conditions

Supplementary Fig. 6 – Immunohistochemical changes in LNCaP castrate resistant prostate cancer xenograft microarray. A tissue microarray was constructed from five xenograft samples collected at the end of the study among LNCaP xenografts treated at time of castration-resistant prostate cancer. Staining intensity was graded by a pathologist blinded to group. (a) Mean intensity staining is shown for p-Akt and Akt with representative images (right). (b) Mean intensity staining for p-4e-BP1/4e-BP-1. (c) Mean intensity staining for p-S6/S6. (d) Mean intensity staining for Ki67. European Urology 2015 67, 986-990DOI: (10.1016/j.eururo.2014.08.006) Copyright © 2014 European Association of Urology Terms and Conditions