1. Supplementary Figure S1 Inhibition of AKT and S6 phosphorylation. After 4 h treatment with 1 µM of GDC-0941, EVSA-T parental, resistant Clone1, Clone2, Clone3, Clone4, and Clone5 were lysed and analyzed by Western blot. +-+-+-+-+-+- ParentalClone1Clone2Clone3Clone4Clone5 AKT S6 pS235/236 S6 pS473 AKT pT308 AKT p240/244 S6

2. GDC-0349 (TORC1/2) Cell viability (% of DMSO) Drug conc. (µM) GDC-0068 (AKT) Supplementary Figure S2 Cell growth inhibition by a panel of compounds. EVSA-T parental, resistant clone1, clone2, clone3, clone4, and clone5 cells were seeded in 384-well plates and treated for 3 days with various concentrations of AKT or TORC1/2 inhibitor. Subsequently, cell viabilities were measured with CellTiter-Glo® Luminescent Cell Viability Assay Kit.

3. Supplementary Figure S3 Effect of GDC-0980 on AKT and S6 phosphorylation. After a 4 h treatment with serial concentrations of GDC-0980, EVSA-T parental and resistant clone1 cells were lysed and analyzed by Western blot. 0 0.10.3 1 3 GDC-0980 (µM) EVSA-T Parental EVSA-T Clone1 pS235/236 S6 S6 pS473 AKT AKT 10 0 0.10.3 1 3 10

4. Supplementary Figure S4 BYL719 (PI3Kα selective) Cell viability (% of DMSO) Drug conc. (µM) GDC-0032 (PI3Kß sparing) GDC-0980 (PI3K-mTOR) Cell viability (% of DMSO) Drug conc. (µM) TGX-221 (PI3Kß selective) GSK2636771 (PI3Kß selective) CH5132799 (Pan-PI3K) Cell viability (% of DMSO) AZD-6482 (PI3Kß selective) Cell viability (% of DMSO) Drug conc. (µM) Supplementary Figure S4 Cell growth inhibition by a panel of compounds. EVSA-T parental, resistant Clone1, Clone2, Clone3, Clone4, and Clone5 cells were seeded in 384-well plates and treated for 3 days with various concentrations of PI3K inhibitors. Cell viability was measured with the CellTiter-Glo® Luminescent Cell Viability Assay Kit.

5. AKT pS473 AKT S6 EVSA-T Parental EVSA-T Clone1 pS235/236 S6 DMSO GDC-0941GDC-0032GDC-0068GDC-0349TGX-221 BYL719lapatinibcrizotiniblinsitinibCH5183284DAFDMSO GDC-0941GDC-0032GDC-0068GDC-0349TGX-221 BYL719lapatinibcrizotiniblinsitinibCH5183284DAF Supplementary Figure S5 Inhibition of AKT phosphorylation. After a 4 h treatment with 1 µM GDC-0941, GDC-0032, GDC-0068, TGX-221, lapatinib, crizotinib, or CH5183284, 3 µM of GDC-0349, BYL719, or linsitinib, or 10 µg/mL of DAF, EVSA-T parental and resistant clone1 cells were lysed and analyzed by Western blot.

6. crizotinib (MET/ALK) erlotinib (EGFR) PD173074 (FGFR) lapatinib (HER2/EGFR) PD0325901 (MEK)VX-11e (ERK) linsitinib (IGF1R/InsR) Cell viability (% of DMSO) Drug conc. (µM) Cell viability (% of DMSO) Supplementary Figure S6 docetaxelpaclitaxeldoxorubicinSAHA fluvestrant Cell viability (% of DMSO) Drug conc. (µM) Cell viability (% of DMSO) Drug conc. (µM) Cell viability (% of DMSO) Supplementary Figure S6 Cell growth inhibition by a panel of anti-cancer agents. EVSA-T parental, resistant clone1, clone2, clone3, clone4, and clone5 cells were seeded in 384-well plates and treated for 3 days with various concentrations of compounds. Cell Viability was measured with CellTiter-Glo® Luminescent Cell Viability Assay Kit.

7. p110beta 1051 ESWTTKVNWMAHTVRKDYRS----- 1070 p110delta 1025 ESWKTKVNWLAHNVSKDNRQ----- 1044 p110alpha 1049 GGWTTKMDWIFHTIKQHALN----- 1068 p110gamma 1078 KGWTVQFNWFLHLVLGIKQGEKHSA 1102 Supplementary Figure S7 The alignment of C-terminus of Human p110 family

8. Parental Clone3 Clone4 Clone5 PIK3CB D1067Y (amplitude) PIK3CB WT (amplitude) EVSA-T Supplementary Figure S8 Droplet digital PCR (ddPCR) detection of PIK3CB D1067Y mutation.10 ng genomic DNA from EVSA-T parental and three GDC-0941-resistant derivative clones were analyzed by ddPCR for presence and allele frequency of the D1067Y mutation. PIK3CB D1067Y allele frequency was 0% and 29.7%, 32.7%, and 28% in EVSA-T parental and resistant clones 3,4, and 5, respectively. Data represent three independent ddPCR experiments. Supplementary Figure S8

9. Supplementary Figure S9 Copy number Supplementary Figure S9 Detection of copy number variation. Copy number variation of 35 genes were analyzed by quantitative PCR assay in EVSA-T parental cells, resistant clone1, clone2, and clone3.

10. SUM-52PE HCC-1569 ZR-75-1 Drug conc. (µM) Cell viability (% of DMSO) Drug conc. (µM) Parental cell Resistant cell Supplementary Figure S10 A. Cell growth inhibition by GDC-0941. ZR-75-1 parental, ZR-75-1 resistant pool, HCC-1569 parental, HCC-1569 resistant pool, SUM-52PE parental, and SUM-52PE resistant pool cells were seeded in 384-well plates and treated for 3 days with various concentrations of cpmpounds. Subsequently, cell viabilities were measured with CellTiter-Glo® Luminescent Cell Viability Assay Kit. B. Droplet digital PCR (ddPCR) analysis of PTEN-null GDC-0941 resistant cell lines. 10ng genomic DNA from GDC-0941-resistant SUM52PE and HCC1569 derivative pool cell lines was analyzed by ddPCR for presence of PIK3CB D1067A, V, and Y mutations. Synthetic oligos were used as positive controls for each mutation. Data are representative of three independent replicate ddPCR reactions. Supplementary Figure S10 SUM52PE. 0941.Res. HCC1569. 0941.Res. D1067A. oligo SUM52PE. 0941.Res. HCC1569. 0941.Res. D1067V. oligo SUM52PE. 0941.Res. HCC1569. 0941.Res. D1067Y. oligo A B

11. A498 PIK3CB D1067V (amplitude) PIK3CB WT (amplitude) Human scramble PIM4973g Patient A498Human scramble PIM4973g Patient Supplementary Figure S11 Droplet digital PCR detection of PIK3CB D1067V mutation. 10 ng genomic DNA from A498, Control human scramble, and tumor tissue in PIM4973g study were analyzed by ddPCR for presence and allele frequency of the D1067V mutation. Data represent three independent ddPCR experiments. Supplementary Figure S11

12. GDC-0941 (Pan-PI3K)GDC-0032 (ß sparing)TGX-221 (ß selective) % of survival Transients Stables % of survival Supplementary Figure S12 PIK3CB mutant expressing EVSA-T cells are less sensitive to PI3K inhibitors. EVSA-T parental cells and cells transiently (upper panel) or stably (lower panel) transfected with PIK3CB WT, PIK3CB D1067A, PIK3CB D10167V, or PIK3CB D1067Y mutants were seeded in 384-well plates and treated for 3 days with various concentrations of GDC-0941, GDC- 0032 or TGX-221. Subsequently, cell viabilities were measured with CellTiter-Glo® Luminescent Cell Viability Assay Kit. Supplementary Figure S12 PIK3CB WT Mock PIK3CB D1067A PIK3CB D1067V PIK3CB D1067Y PIK3CB WT Mock PIK3CB D1067A PIK3CB D1067V PIK3CB D1067Y

13. PIK3CB WT EVSA-T Stables PIK3CB D1067A PIK3CB D1067V PIK3CB D1067Y pS235/236 S6 S6 pS473 AKT AKT Actin DMSO TGX-221 DMSO TGX-221 DMSO TGX-221 GDC-0941 GDC-0032 GDC-0941GDC-0032GDC-0941GDC-0032 DMSO TGX-221 GDC-0941GDC-0032 Supplementary Figure S13 Inhibition of AKT and S6 phosphorylation. After a 4 h treatment with 1 µM GDC-0941, TGX-221, or GDC-0032, EVSA-T cells stably transfected with WT PIK3CB or PIK3CB D1067A/V/Y mutants were lysed and analyzed by Western blot. Supplementary Figure S13

14. Supplementary Figure S14 p110β dependency of A498. A. 10 nM of PIK3CA or PIK3CB siRNAs were treated to A498 for 4 days and cell viabilities were measured with CellTiter-Glo® Luminescent Cell Viability Assay Kit. B. 10 nM of PIK3CA or PIK3CB siRNAs were treated to A498 for 4 days and cells were lysed and analyzed by Western blot with indicated antibodies. AB

15. p110α p110ß ERK AKT 0 0.10.3 1 30 0.10.3 1 3 GDC-0941 (µM) EVSA-T Parental EVSA-T Clone1 Supplementary Figure S15 ATP competition assay. Cell lysate of EVSA-T parental or resistant clone1 was incubated with GDC-0941 at a serial concentration for 30 min followed by 10 min incubation with desthiobiotin-ATP probe. Then, biotinylated proteins were pulldowned with avidin agarose beads. Pulldowned proteins were then applied to Western blot with indicated antibodies.

16. Intensity Total AKTpS473 AKTpT308 AKT Supplementary Figure S16 Relative quantification of baseline AKT phosphorylation levels in EVSA-T parental cells and resistant clones. Total protein lysates were collected from EVSA-T parental cells (red) and GDC-0941 resistant clones (blue) and subjected to reverse phase protein array (RPPA) hybridization (Theranostics Health, Rockville, MD). Relative intensity values (y-axis) were calculated based on fluoresent signal intensity derived from the target primary antibody normalized to signal intensity from the fluorescent total protein dye Sypro Ruby.

17. AKT pS473 AKT S6 pS235/236 S6 pT308 AKT p110β MockPIK3CB WTPIK3CB D1067Y Supplementary Figure S17 Signal profile in Rat-2 stable cell lines. The Mock, PIK3CB WT, or PIK3CB D1067Y stably expressing Rat-2 cell lines were lysed 4 days after seeding to 96U plate and analyzed by Western blot. Supplementary Figure S17

18. Supplementary Table S1 Compound nameMode of actionVendor GDC-0941Pan-PI3K inhibitorGenentech, Inc. CH5132799Pan-PI3K inhibitorSelleck Chemicals GDC-0032PI3Kß sparing inhibitorGenentech, Inc. GDC-0980PI3K-mTOR inhibitorGenentech, Inc. TGX-221PI3Kß inhibitorSelleck Chemicals AZD-6482PI3Kß inhibitorSelleck Chemicals GSK2636771PI3Kß inhibitorSelleck Chemicals BYL719PI3Kα inhibitorSelleck Chemicals GDC-0068AKT inhibitorGenentech, Inc. GDC-0349TORC1/2 inhibitorGenentech, Inc. VX-11eERK inhibitorChemietek PD0325901MEK inhibitorGenentech, Inc. PD173074Pan-FGFR inhibitorLC laboratories CH5183284Pan-FGFR inhibitorActive Biochem crizotinibALK/MET inhibitorGenentech, Inc. lapatinibHER2/EGFR inhibitorGenentech, Inc. erlotinibEGFR inhibitorGenentech, Inc. linsitinibIGF1R/InsR inhibitorToronto Research Chemicals fluvestrantER degraderSelleck Chemicals docetaxelMicrotubule stabilizerGenentech, Inc. paclitaxelMicrotubule stabilizer inhibitorGenentech, Inc. doxorubicinDNA intercalatorGenentech, Inc. SAHAHDAC inhibitorGenentech, Inc. DAFAnti-EGFR/HER3 antibodyGenentech, Inc. Supplementary Table S1 Compound list

19. Supplementary Table S2