NDRG1) As a Biomarker of Phosphatidylinositol-3-Kinase (PI3K)  Inhibitor Activity in a PTEN-Deﬁcient Glioma Cell Line

Abstract No. 5313 Phosphoproteomic analysis with reverse phase protein array identifies N-myc downstream regulated gene 1 (NDRG1) as a biomarker of phosphatidylinositol-3-kinase (PI3K)  inhibitor activity in a PTEN-deficient glioma cell line. Takeomi Inoue, Naoko Iwata, Eiji Nishiwaki, Yasuyuki Kirii Department of Research and Development, Carna Biosciences, Inc., BMA 3F 1-5-5 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047 Japan p-SGK (Ser422) p-SGK (Thr256) U-87 MG cells (PTEN-deficient) PIP2 PIP2

Abstract PTEN PI3Ka LN229 cells (PTEN-wt) PTENPTEN PI3Ka 2 LN229 U-87 * ** Aberrant activation of phosphoinositide 3-kinase (PI3K) signaling is commonly found in cancer, and leads to deregulation of PIP3 PIP3 1.5 mTORC2 PDK1 mTORC2 PDK1 2hr 6hr 24hr 2hr 6hr 24hr PDK1 PDK1 1 several intracellular processes, including cell survival, growth, proliferation and migration. PI3K activates AKT, 0 T308 P P S473 S422 P P T256 T308 P P S473 S422 P P T256 0.5 serum/glucocorticoid regulated kinase (SGK), phosphoinositide -dependent kinase 1 (PDK1), mammalian target of rapamy- AKT SGK1 AKT SGK1 -0.5 log conc (-) log conc 0 cin (mTOR), and several other molecules involved in cell progression and survival. Reports have also indicated that the dele- -1 TSC2 T346 TSC2 T346 -0.5 tion of the gene encoding the tumor suppressor phosphatase and tensin homologue (PTEN) is one of the key factors con- T246 P T246 P -1.5 P TSC1 NDRG1 P TSC1 NDRG1 2hr 6hr 24hr 2hr 6hr 24hr log conc (-) log conc * trolling activation of the PI3K pathway. Although many therapies to target the Ras-PI3K-mTOR axis in glioblastoma (GBM) PRAS40 PRAS40 -2 LN229 U-87 have been attempted, their eﬃcacies have not been pronounced, presumably because of the complexity of PI3K mediated Rheb Rheb -2.5 cont PIK90 cont PIK90 signaling. mTORC1 mTORC1

4EBP1 4EBP1 To elucidate the role of PTEN in PI3K signaling, the phosphorylation status of two glioma cell lines, LN229 (wild type PTEN) p70S6K p70S6K P S235/S236 P S235/S236 Figure 5. The time course of change in phosphorylation of SGK in LN229 and U-87 MG cells following PIK90 treatment. and U-87 MG (PTEN deficient) was investigated with phosphoproteomic reverse phase protein array (RPPA). When basal S6Rb S6Rb The phosphorylation of both SGK(Ser422) and SGK (Thr256) was inhibited by treatment with PIK90 in U-87 MG cells. How- phosphorylation was compared between both cell lines, the phosphorylation of N-myc downstream regulated gene 1 ever, the effect was weak comparing with inhibition of NDRG1 (Thr346) phosphorylation. The values are mean ± s.d. (n=8). (NDRG1) (Thr346), a physiological substrate of SGK1, was increased in U-87 MG cells compared with the levels in LN229 cells. Figure 2. PI3K signaling pathway in LN229 and U-87 MG cells. cont : untereated control, *: p < 0.05, **: p < 0.001 vs control. Following treatment with PIK-90, a PI3K inhibitor, phosphorylation of Akt (Ser473) and its direct substrate PRAS40 (Thr246) were decreased in both cell lines, however, NDRG1 (Thr346) phosphorylation was strongly inhibited only in U-87 MG cells. These data suggest that PTEN deletion affects the phosphorylation status of NDRG1 in U-87 MG cells and the differential ef- fects of PIK-90 on LN229 and U-87 MG cells is PTEN dependent. Therefore, detecting NDRG1 (Thr346) phosphorylation could (b) U-87 MG (a) (b) 7 7 LN229 U-87 MG p-Akt (Ser473) be a biomarker for PI3Kα inhibitor treatment in PTEN deleted cancer cells. Ongoing studies are focusing on elucidating the 0 (a) LN229 mechanism of action of PIK-90 in glioma cell lines. 6 p-Akt (Ser473) 6 -3 -2.5 -2 -1.5 -1 -0.5 0 p-PRAS40 (Thr246) PIK90 - + - + -0.5 5 5 p-NDRG1 (Thr346) -1 4 4 p-Akt (Ser473)

p-Akt (Ser473) RPPA (p-value) (p-value) -1.5 10 3 10 3 p-PRAS40 (Thr246) p-S6Rb (Ser235/236) -log Methods 2 -log 2 -2 p-S6Rb (Ser235/236) p-S6Rb (Ser235/236) R2=0.7037 Cell Culture : Two human GBM cell lines, LN229 and U-87 MG cells were incubated in DMEM or MEM respectively, contain- 1 1 -2.5 Immunoblong ing 10% FBS. Cells were treated with 0.5μM PIK-90 and harvested immediately and after 2, 6 and 24 hours. PIK-90 is reported p-NDRG1 (Thr346) 0 0 -2 -1 0 1 2 -2 -1 0 1 2 p-NDRG1 (Thr346) to inhibit PI3Kα (IC50 = 11nM), PI3Kβ (IC50 = 350nM), PI3Kδ (IC50 = 58nM) and PI3Kγ (IC50 = 18nM)[1]. Fold change Fold change (c) RPPA : Cells were collected, washed and homogenized in lysis buffer. Serially diluted lysates (1:1, 1:2, 1:4, 1:8) were spotted p-PKC(pan) βII (Ser660) p-NDRG1 (Thr346) 0 Figure 3. The change in phosphorylation of 180 phosphoproteins in (a) LN229 and (b) U-87 MG cells after 6-hour treatment -5 -3 -1 1 3 onto glass slides with an arrayer equipped with 32 pins in order to place the expression level of samples in a dynamic range -0.5 with PIK90. The phosphoproteins indicated in red exhibited significant change by PIK90 treatment. The change in phosphoryla- for signal detection. Each sample dilution series were then spotted in eight replicates. Signals generated from slides stained tion of NDRG1 (Thr346) was only found in U-87 MG (PTEN-deficient) cell line. γ-tubulin -1

with anti-phospho antibodies were analyzed employing SuperCurve algorithm to obtain a single value of relative concen- RPPA -1.5 tration for each lysate to draw the heatmap. For the statistical analysis, eight replicates were separately handled (n=8) and -2 two sample t-test was performed between untreated control and treated samples. The targets of the anti-phospho antibod- R2=0.8997 ies utilized in this study are shown in the website (http://www.carnabio.com/english/ images/rppa_antibody_en.pdf?121130) -2.5 Immunoblong and a paper[2]. Figure 6. Veriﬁcation of RPPA results by immunoblotting. (a) Immunoblott analysis of cell lysates prepared after 6-hour incu- [1] http://www.scbt.com/datasheet-364587-CASNumber-677338-12-4.html LN229 U-87 bation with or without PIK90. Relative concentrations as log value from RPPA results showed good correlations with immunob- [2] Masuda M et al. Mol Cell Proteomics, M113.033845, March 18, 2014. LN229p-Akt (Ser473) U-87 p-PRAS40 (Thr246) 2 (a)2hr 6hr 24hr 2hr 6hr 24hr (b) 2hr 6hr 24hr 2hr 6hr 24hr lotting data for (b) p-Akt (Ser473) and (c) p-NDRG1 (Thr346). The latter values are densitometric ratios of the phosphoproteins to 0 0 the loading control, γ-tubulin. -0.5 -0.5 Color Key -1 -1 -1.5

log conc (-) log conc -1.5 -2 ** ** log conc (-) log conc * -2 -2.5 * Time ** ** ** * -2.5 ** -3 * * Conclusion −1 −0.5 0 0.5 1 ** Value cont PIK90 cont PIK90 1. In LN-299 cells, basal phosphorylation levels were higher than U-87 MG cells. However, PI3K and mTORC2 LN229−cont−0hr LN229 cells pathway signals were attenuated. LN229−cont−2hr (PTEN-wt) LN229−cont−6hr 2. In contrast, in U-87 MG cells, PI3K and mTORC2 pathways were activated. LN229−cont−24hr LN229 U-87 LN229 U-87 U87−cont−0hr 2hr 6hr 24hr 2hr 6hr 24hr 2hr 6hr 24hr 2hr 6hr 24hr 3. By treatment with PIK90, among PI3K and mTORC2 pathway, the phosphorylation of NDRG1 (Thr346) U-87 MG cells p-S6Rb (Ser235/236) U87−cont−2hr 0 0 p-NDRG1 (Thr346) (PTEN-deﬁcient) was strongly inhibited in U-87 MG cells. U87−cont−6hr -0.5(c) (d) -0.5 -1 U87−cont−24hr -1 * 4. These results suggest that NDRG1 (Thr346) phosphorylation could be an eﬃcacy biomarker of PI3Kα in- -1.5 -1.5 ** * p.p53..Ser9. p.p53..Ser6. hibitor treatment. p.NPM..Ser4. p.p53..Ser46. p.p53..Ser15. p.p53..Ser20. p.p53..Ser37. p.Bim..Ser69. p.Rb..Ser795. p.Rb..Ser780. p.Src..Tyr527. p.Ret..Tyr905. p.Akt..Thr308. p.Syk..Tyr323. p.c.Kit.Tyr703. p.Akt..Ser473. p.Shc..Tyr239. p.Shc..Tyr317. p.Bcl.2..Thr56. p.Cofilin..Ser3. p.Bcl.2..Ser70. p.p53..Ser392. p.Chk2..Thr68. p.Bad..Ser112. p.Bad..Ser136. p.Chk2..Ser19. p.Bad..Ser155. p.c.Jun..Ser73. p.c.Jun..Ser63. p.ATF.2..Thr71. p.c.Fos..Ser32. p.ATR..Ser428. p.PAK2..Ser20. p.PLCg.Tyr783. p.FLT3..Tyr591. p.PLCg.Tyr759. -2 p.SGK..Thr256. p.Stat.1.Tyr701. p.SGK..Ser422. p.RelB..Ser552. p.Gab1..Tyr627. p.Gab2..Tyr452. p.IRS.1.Ser612. p.IRS.1.Ser307. p.c.Raf..Ser338. p.c.Raf..Ser259. p.Chk1..Ser345. p.Chk1..Ser296. p.Chk1..Ser317. p.Stat.5..Tyr694. p.Stat.6..Tyr641. p.Stat.3..Tyr705. p.Stat.2..Tyr690. ** p.GSK.3ß..Ser9. p.LKB1..Ser428. p.A.Raf..Ser299. p.B.Raf..Ser445. p.PKM2..Tyr105. p.ATM..Ser1981. p.PLCg.Tyr1217. p.BAP1..Ser592. p.PKCd..Thr505. p.Wee1..Ser642. p.VASP..Ser239. p.VASP..Ser157. p.MYPT..Thr853. p.PDK1..Ser241. p.RSK2..Ser227. p.HSP27..Ser82. p.Stat.3..Ser727. p.SHP.2..Tyr580. p.MYPT..Ser507. p.MYPT..Ser668. p.eIF4E..Ser209. p.PTEN..Ser380. p.eIF4B..Ser422. p.MSK1..Thr581. p.4E.BP1..Thr70. p.CREB..Ser133. p.4E.BP1..Ser65. p.PKCth..Thr538. p.eEF2k..Ser366. p.FoxO1..Ser256. p.GSK.3a..Ser21. p.Raptor..Ser792. p.LRP6..Ser1490. p.IkB.a..Ser32.36. p.TACC3..Ser558. p.Chk2..Ser33.35. p.Rb..Ser807.811. p.SHIP2..Tyr1135. p.AP2M1..Thr156. p.eNOS..Ser1177. p.eIF4G..Ser1108. p.AMPKa..Thr172. Non.p.Src..Tyr416. Non.p.Src..Tyr527. p.Syk..Tyr525.526. p.NDRG1..Thr346. p.mTOR..Ser2448. p.FoxO3a..Ser253. p.ß.catenin..Ser45. p.p90RSK..Thr573. p.p90RSK..Ser380. p.Histone.H3..Thr3. p.PRAS40..Thr246. p.Aurora.A..Thr288. p.BRCA1..Ser1524. p.AMPKß1..Ser108. p.4E.BP1..Thr37.46. p.IRS.1.Ser636.639. p.Cyclin.D1..Thr286. log conc (-)log conc p.TAK1..Thr184.187. p.Histone.H3..Thr11. p.Mnk1..Thr197.202. p.Histone.H3..Ser10. p.Histone.H3..Ser28. p.S6Rb..Ser235.236. p.Src.Family..Tyr416. p.NFkB.p65..Ser468. p.NFkB.p65..Ser536. p.Met..Tyr1234.1235. log conc (-) log conc p.ALK..Tyr1282.1283. p.TBK1.NAK..Ser172. p.Tyk2..Tyr1054.1055. Non.p.4E.BP1..Thr46. p.Jak2..Tyr1007.1008. p.PKD.PKCμ..Ser916. p.PKCz.l..Thr410.403. p.Smad2..Ser465.467. p.PKCa.ß..Thr638.641. p.FoxO3a..Ser318.321. p.HER4.ErbB4..Tyr984. p.MEK1.2..Ser217.221. p.SEK1.MKK4..Thr261. p.MAPKAPK.2..Thr222. p.MAPKAPK.2..Thr334. p.PKCd.th..Ser643.676. p.PKC.pan..ßII..Ser660. p.RSK3.Thr356.Ser360. p.Smad1.5..Ser463.465. p.EGF.Receptor..Tyr992. p.HER2.ErbB2..Tyr1248. p.Tuberin.TSC2..Ser939. p.IGF.I.Receptor..Tyr980. p.c.Raf..Ser289.296.301. p.MARCKS..Ser167.170. p.p70.S6.kinase..Thr389. p.Tuberin.TSC2..Tyr1571. p.EGF.Receptor..Tyr1068. p.EGF.Receptor..Tyr1045. p.Histone.H2A.X..Ser139. p.Tuberin.TSC2..Thr1462. p.PKD.PKCμ..Ser744.748.

p.p90RSK..Thr359.Ser363. * p.VEGF.Receptor.2..Tyr951. p.PDGF.Receptor.ß..Tyr771. p.PDGF.Receptor.ß..Tyr751. p.PDGF.Receptor.ß..Tyr740. p.MKK3.MKK6..Ser189.207. p.SAPK.JNK.Thr183.Tyr185. p.ß.catenin..Ser33.37.Thr41. p.VEGF.Receptor.2..Tyr1175. p.VEGF.Receptor.2..Tyr1059. p.PDGF.Receptor.ß..Tyr1021. p.PDGF.Receptor.ß..Tyr1009. p.p38.MAPK..Thr180.Tyr182.. p.HER2.ErbB2..Tyr1221.1222. p.Zap.70..Tyr319..Syk..Tyr352. p.PAK1..Thr423..PAK2..Thr402. p.IGF.I.Receptor..Tyr1135.1136. p.PAK1..Ser144..PAK2..Ser141.

p.FoxO1..Thr24..FoxO3a..Thr32. -2 p.Acetyl.CoA.carboxylase..Ser79.

p.p44.42.MAP.kinase..Thr202.Tyr204. ** -2.5 p.GSK.3a.ß..Ser21.9..GSK.3a.preferred p.PAK1..Ser199.204..PAK2..Ser182.197. p.Smad2..Ser465.467..Smad3..Ser423.425. p.FoxO1..Thr24..FoxO3a..Thr32..FoxO4..Thr28. p.Ezrin..Thr567..Radixin..Thr564..Moesin..Thr558. p.IGF.I.Receptor..Tyr1131..Insulin.Receptor..Tyr1146. p.Aurora.A..Thr288..Aurora.B..Thr232..Aurora.C..Thr198. -2.5 * -3 5. The down-regulation of SGK1 by PIK90 is relatively weak comparing with the suppression of NDRG1 p-NDRG1 (Thr346) cont PIK90 cont PIK90 p-PKC (pan) βII (Ser660) phosphorylation, indicating that NDRG1 phosphorylation would be also controlled by unknown signal- p-SGK (Thr256) Figure 4. The time course of phosphoprotein expressions in LN229 and U-87 MG cells after exposure to PIK90. ing pathways. p-S6Rb (Ser235/236) The phosphorylation of (a) Akt (Ser473), (b) PRAS40 (Thr246) and (c) S6 ribosomal protein (S6Rb) (Ser235/236) was decreased in 6. RPPA results showed good correlations with immunoblotting data and RPPA is an appropriate tool for Figure 1. Basal phosphoprotein content in untreated LN229 (PTEN-wt) and U-87 MG (PTEN-deﬁcient) cells. both cell lines in response to PIK90 treatment. In contrast, (d) NDRG1 (Thr346) phosphorylation was inhibited only in U-87 MG Most phosphoprotein expressions are higher in LN229 cells except for several molecules in PI3K and mTOR cells. The inhibition was time dependent-manner up to 24 hours. The values are mean ± s.d. (n=8). cont : untreated control, *: p surveying phosphoproteome. complex 2 (mTORC2) pathway. < 0.05, **: p < 0.001 vs control.