Tumour TypeClassificationCell LineCell Line OriginID Testing Method Date ID Test (month-year). Prostate LNCaPATCC CRL 1740STR AnalysisAug-11 Breast BT-474cDrs.J Albanell and J Baselga, Laboratorio Ricerca Oncologica, Barcelona, Spain. STR AnalysisMarch-12 Prostate PC3ATCC CRL 1435STR AnalysisJul-12 Breast MDA-MB-468ATCC HTB 132STR AnalysisOct-12 Prostate DU-145Applied Cell Sciences STR AnalysisApril-11 Breast HCC70ATCC CRL 2315STR AnalysisAug-11 Breast MDA-MB-157ATCC HTB 24STR AnalysisNov-12 FibrosarcomaFibrousHT1080ECACC STR AnalysisSept-13 Supplementary Table 1 Summary of cell line origins and verification.

Supplementary Table 2 Antibody Target Size (kDa) Order CodeSpecies Dilution Primary Dilution Secondary Vinculin115Sigma V9131Mouse1:5000 CST :2000 Total AKT60CST 9272Rabbit1:500 CST : 2000 pAKT Ser47360CST 9271Rabbit1:500 CST : 2000 pAKT Thr30860CST 2965Rabbit1:1000 CST : 2000 pS6 Ser235/23632CST 2211Rabbit1:1000 CST : 2000 S632CST 2217Rabbit1:1500 CST : 2000 pPRAS40 Thr24640 Invitrogen G Rabbit1:1000 CST : 2000 Total ERK 1/242/44CST 9102Rabbit1:1000 CST : 2000 pERK1/2 Thr202/Tyr20442/44CST 9101Rabbit1:1000 CST : 2000 Total GSK3b46BD Rabbit1:500 CST : 2000 pGSK3b Ser946 CST 9336 Rabbit1:1000 CST : 2000 FOXO178-82CST 2880Rabbit1:1000 CST : 2000 pFOXO1 Thr24 /pFOXO3a Thr CST 9464Rabbit1:1000 CST : 2000 FOXO3a78-82CST 2497Rabbit1:1000 CST : 2000 mTOR289CST 2972Rabbit1:1000 CST : 2000 pmTOR Ser CST 2971Rabbit1:1000CST : 2000 Summary of antibodies used in all experimental analysis.

Supplementary Figure 1 A B C D JEKO cells AZD8186 inhibits PI3K pathway in PTEN null but not PTEN WT PIKC3A MT cells (A) AZD8186 compared to pan-PI3K inhibitor GDC0941. Exposure to 500nM AZD8186 (2 hours) inhibits protein phosphorylation in PTEN null HCC70 breast cell line but not in PTEN WT BT474 breast cell line. Exposure to 500nM GDC-0941 pan PI3K inhibitor inhibits protein phosphorylation in both PTEN null and WT breast cell lines. Equal amounts of protein were loaded on each gel. Western blots were analysed for the phospho- proteins or total-proteins as indicated. (B) Inhibition of protein phosphorylation in PTEN null LNCAP prostate cell line determined by Western blot analysis; cell lysates generated following a 2 hour exposure to AZD8186 at range of concentrations from 3 to 0.01 μM. No inhibition of pMAPK. (C) Inhibition of protein phosphorylation in human mantle cell lymphoma JEKO B cells; IgM stimulated PI3Kδ activity measured by Western Blot analysis of AKT and MAPK signaling. (D) Representative images at 20X magnification illustrating the induction of nuclear accumulation of FOXO3a in PTEN WT PIK3CA mutant BT474 cells exposed to GDC0941 and BYL719 for 2 hours.

Supplementary Figure 2 A B - C AZD8186 inhibits LPA, EGF and rac dependent Ser473 AKT phosphorylation in serum starved prostate and breast cancer cells. (A) MDA-MB-468 and PC3 cells were serum starved (MDA-MB-468 overnight, PC3 6 hours) and incubated with Vehicle (0.1% DMSO) or various concentrations of AZD8186 or BYL719 for 1 hour then treated with LPA (10 μM) or EGF (166ng/ml) for 15 minutes. Mesoscale assay analyses and phosphoprotein quantification methods were as described in Materials and Methods. Results are representative of three independent experiments. (B) HT1080 (RAC1 N92I) and MDA-MB- 157 (RAC1 P29S) cells were starved overnight, incubated with Vehicle (0.1% DMSO) or 250 nM AZD8186 for 1 hour then treated with LPA for 15 minutes. Western blot analyses were as described in Materials and Methods. Results are representative of three independent experiments. (C) HT1080 (RAC1 N92I) and MDA-MB-157 (RAC1 P29S) cells grown in RPMI medium 10%FCS were incubated with Vehicle (0.1% DMSO) or 250 nM AZD8186 for 1 hour then cell lysates were prepared. Western blot analyses were as described in Materials and Methods. Results are representative of three independent experiments.

. FOXO3a CHROM. BOUND TOTALCYTONUC. SOL. Supplementary Figure 3 Vinculin Total AKT AKT-P (Ser473) Control Western blot analysis of showing induction of FOXO3a association with the chromatin fraction following treatment with AZD8186. Control cells were treated for 2hrs with vehicle (0.1% DMSO). To induce FOXO3A translocation cells were treated with 500nM AZD8186 (AZD8186). (A) Treatment with 500nM AZD8186 reduced pAKT (Ser 473). (B) Control and AZD8186 treated cells were subjected to cell fractionation to generate cytosolic (CYTO), a soluble nuclear (NUC. SOL.), and chromatin bound fractions (CHROM. BOUND) and Western blotted for FOXO3a. Total cell lysate from Control and AZD8186 treatred lysates are included for reference. AB AZD8186 ControlAZD8186ControlAZD8186ControlAZD8186ControlAZD8186

PTEN deficient cell lines PTEN WT cell lines Supplementary Figure 4 AZD8186 GI50 (μM) Cell lines (multiple tumour types) AZD8186 inhibits proliferation in a subset of cancer cell lines. AZD8186 was tested in a broad cell panel, the cell lines are indicated on the Y axis and the GI50 on the x axis. Red bars indicate cell lines with a mutation or deletion in PTEN. Green bars represent cell lines with WT PTEN. Sensitive cell lines indicated below GI50 1μM.

Supplementary Table 3 GI50 of all cell lines showing sensitivity to AZD8186.

Supplementary Figure 5 PTEN T47DMDA-MB-231MDA-MB-134MDA-MB-468MDA-MB-453MCF7KPL4HCC1569BT20HCC1395MDA-MB-415HCC1187AHCC1419HCC70SKBR3CAMA 1SUM52PEBT474HCC1954MDA-MB-436HCC1187BMDA-MB-157 LNCAPPC3LNCAP-CR22RV1VCAPDU145LNCAP-AIRWPEIPC346-FluIBPH1 PTEN ZR DU145 HCC70 U87MG MDAMB 468 BT549 Western blot protein analysis of PTEN status across a panel of breast and prostate cell lines. Original Western blots used to indicate the PTEN protein status shown in Figure 2E and 2F. Cell lines included in the figure are highlighted in bold.

Supplementary Figure 6 pAKT: Ser473pPRAS40 : Thr246 % normalised control 50mg/kg 25mg/kg 4h24h4h24h 50mg/kg 25mg/kg 4h24h4h24h Con 50mg/kg 2h8h Con 50mg/kg 2h8hCon % normalised control pAKT: Ser473pPRAS40: Thr246 A B 30mg/kg + ABT 4h 24hCon 4h 24hCon mg/kg + ABT % normalised control pAKT: Ser473pPRAS40: Thr246 C Pathway biomarker suppression in PC3 and HID28 tumours. (A) Mice bearing PC3 tumours treated with AZD8186 for 4 and 24 hours with 50 and 25mg/kg AZD8186, the inhibition of pAKT and pPRAS-40 are shown. (B) Mice bearing HID28 tumours treated with AZD8186 for 2 and 8 hours at 50mg/kg were analysed for suppression of the pathway biomarkers AKT (Ser473) and pPRAS40. (C) Mice bearing PC3 tumours were treated with 30mg/kg AZD8186 in the presence of 100 mg/kg ABT and analysed for pathway biomarkers. The mean percentage inhibition relative to control treated tumours is shown. Bars represent the SD.

Tumour Lung % normalised control AZD8186 GDC-0941 HCC70 PC3 Days of treatment Tumour volume (cm 3 ) Days of treatment Tumour volume (cm 3 ) Control AZD8186 GDC-0941 Control % normalised control AZD8186 GDC-0941 Control AZD8186 GDC-0941 Control pAKT Ser473 at 2 hr AZD8186 :30mg/kg bid + ABT bid GDC-0941 :150mg/kg qd Supplementary Figure 7 AB C D AZD8186 selectively suppresses pathway biomarkers in tumour versus lung (A) Mice bearing HCC70 tumours were treated with 30mg/kg AZD8186 and 150mg/kg GDC0941. Mean tumour volume is shown for each group, bars represent SEM. (B) Tumour and lung samples taken from each mouse2 hours following treatment were analysed for suppression of pAKT (Ser473). The mean percentage inhibition relative to control tumours is represented. Bars represent the SEM. (C) Mice bearing PC3 tumours were treated with 30mg/kg AZD8186 and 150mg/kg GDC0941. Mean tumour volume is shown for each group, bars represent SEM. (B) Tumour and lung samples taken from each mouse 2 hours following treatment were analysed for suppression of pAKT (Ser473). The mean percentage inhibition relative to control tumours is represented. Bars represent the SEM.