Binding

0 %

0.1 %

0.1-1 %

1-5 %

5-10 %

10-35 %

100 %

MUTANTS ATYPICAL A6 ABC1 ALK ABL1 Alpha BRD BRAF PDHK PIKK EGFR RIQ TAF FGFR3 TIF1

FLT3 LIPID Class I PI3K Class II PI3K KIT Class III PI3K Type III PI4K LRR2 Type II PI4K MET Type I PI5K PIK3CA Type II PI5K RET Type III PI5K

Supplemental Figure 1: GS-9829 binds selectively to class I PI3K. GS-9829 specificity was tested in a kinase selectivity profiling assay on a panel of 442 kinases. GS-9829 (10 μM) was tested in an ATP site competition assay. GS-9829 was considered active for a kinase when the kinase fraction that remained bound to ATP was less than 30%. Schemes show the kinases tested; the rounded scheme shows a phylogenetic tree. Red circles indicate the kinases for which GS-9829 shows binding activity. GS-9829 showed no significant binding activity outside the PI3K family. CD8+ B cells CD11b+ CD8+ B cells CD11b+ A (CD3+ (CD3- (CD3+ (CD3- CD8+) B220+) CD8+) B220+)

-6 20 50 15

10

Cell percent 5 Cell number x 10 0 0 - ++ - ++ - ++ - ++ - ++ - ++ < p110δ > Ctr Ctr Ctr Ctr Ctr Ctr 3m 3m 3m 3m 3m

inh 3m 3.5m 3.5m 3.5m 3.5m 3.5m 3.5m

B CD4+ CD8+ CD4+ CD8+ Central Central Effector Central Naive Naive Naive Naive -6 memory memory memory memory

30 10 20

Cell percent 10 Cell number x 10 0 0 - + + - + + --+ + + + - + + < p110δ > - + + - + + - + + inh Ctr Ctr Ctr Ctr Ctr Ctr Ctr Ctr 3m 3m 3m 3m 3m 3m 3m 3m 3.5m 3.5m 3.5m 3.5m 3.5m 3.5m 3.5m 3.5m

C G-CSF IL-5 IL-10 IL-13 1500 40 400 3000 300 1000 30 2000 20 200 pg/ml 500 10 100 1000 0 0 0 0

Ctr δ inh Ctr δ inh Ctr δ inh Ctr δ inh

p110 p110 p110 p110

IL-12 IL-15 IP10 KC 600 20000 1500 600 15000 400 1000 400 10000 pg/ml 200 500 200 5000 0 0 0 0

Ctr δ inh Ctr δ inh Ctr δ inh Ctr δ inh

p110 p110 p110 p110

MCP-1 MIP1a MIP1b RANTES 200 250 800 150 150 200 600 100 150 100 400

pg/ml 100 50 50 50 200 0 0 0 0

Ctr δ inh Ctr δ inh Ctr δ inh Ctr δ inh

p110 p110 p110 p110

Supplemental Figure 2: Serum cytokines in GS-9829-treated MLR/lpr mice. (A) Reanalysis of some of cell populations included in Figure 4C with the y-axis magnified. (B) Reanalysis of some of cell populations of Figure 5A as above. (C) Levels of different cytokines in GS-9829-treated MLR/lpr mouse serum were tested using a multiplex assay in flow cytometry. GS-9829 treatment began at month 3 or 3.5 and was terminated at 5.5 months. GM-CSF, IL-2, IFNγ, IL-1α, IL-1β, IL-2, IL-4, IL-9, IL-7, IL-17 and MIP-2 were also examined and were undetectable in most mice. A 125 GS-9829 100 IL-8 Cytokine EC50 (nM), n = 3 75 IL-17 53.7

50 α 57.4 INFγ TNF vs control IL-5 IL-15 25 IL-4 IL-12 59 TNFαIL-17 0 69.5 Percent cytokine production 1 2 3 IL-4 1 10 10 10 GS-9829 (nM) IL-13 72.5 125 IL-5 100 100 IL-10 102 75 IL-1 181 50 IL-2 vs control IFNγ 192 25 IL-1 IL-10 IL-2 233 0

Percent cytokine production 1 2 3 1 10 10 10 IL-8 >5000 GS-9829 (nM)

B Cell death of BMDM treated with GS9829 or AS6052 (1μM, 72h) on BMDM Control (DMSO) p110δ Inhibitor (GS9829) p110γ Inhibitor (AS605240) 103 103 103 0.39 % 0.76 % 0.52 % 1.74 % 0.25 % 3.02 %

102 102 102

101 101 101 7-AAD 100 100 100

94.8 % 4.09 % 92.9 % 4.83 % 90.6 % 6.12 % 0 1 2 0 1 2 0 1 2 10 10 10 103 10 10 10 103 10 10 10 103

Annexin-V

p110δ Inhibitor (GS9829) p110γ Inhibitor (AS605240) 0.1μM 100 0.1μM 100 1μM 1μM μ 90 10μM 90 10 M

80 80

70 70 Cell viability (%) Cell viability (%) 60 60 24 48 72 24 48 72 Time, h Time, h

Supplemental Figure 3:GS-9829 inhibits Th1 and Th2 cytokine secretion by activated T cells and lack of toxicity for BMDM at low GS-9829 and AS6052 doses. (A) Cytokines produced by activated T cells were analyzed using PBMC preincubated with serial dilutions of GS-9829 (60 h) and activated via CD3 (see Methods). Cytokine levels in culture supernatants were assessed by ELISA. Graphs show percent inhibition of cytokine production at different GS-9829 concentrations compared to maximum cytokine levels (without GS-9829; 100%). The table shows the apparent IC50 for the inhibitor. (B) To assess the cytotoxic effects of PI3K inhibitors on macrophages, day 7 BMDM differentiated from C57BL/6J mice were cultured with DMSO, GS-9829 or AS-605240 (PI3Kγ-specific inhibitor) at 10, 1 or 0.1 μM for up to 3 days. Cultures were supplemented with PI3K inhibitors (or DMSO) every 12 h. Cells were harvested at 24, 48 and 72 h and viability was assessed by flow cytometry using annexin-V-FITC and 7-AAD staining. Upper plots show a representative experiment. Graphs show the percent of viable cells in different conditions. Supplemental Table 1: GS-9829 kinase selectivity Kinase selectivity profiling for GS-9829 was tested at 10 µM in an ATP site competition assay for 442 kinases as described in methods. The table indicates the percent of enzyme that remained bound to the immobilized ATP probe in the presence of GS-9829. % % % Ambit Gene Symbol Ambit Gene Symbol Ambit Gene Symbol Binding Binding Binding AAK1 100 AURKA 100 CAMK2D 100 ABL1(E255K)- 100 AURKB 73 CAMK2G 100 phosphorylated ABL1(F317I)- 98 AURKC 96 CAMK4 100 nonphosphorylated ABL1(F317I)-phosphorylated 92 AXL 91 CAMKK1 100 ABL1(F317L)- 78 BIKE 68 CAMKK2 94 nonphosphorylated ABL1(F317L)-phosphorylated 100 BLK 100 CASK 68 ABL1(H396P)- 95 BMPR1A 100 CDC2L1 96 nonphosphorylated ABL1(H396P)- 100 BMPR1B 72 CDC2L2 100 phosphorylated ABL1(M351T)- 89 BMPR2 73 CDC2L5 69 phosphorylated ABL1(Q252H)- 70 BMX 100 CDK11 100 nonphosphorylated ABL1(Q252H)- 68 BRAF 82 CDK2 100 phosphorylated ABL1(T315I)- 89 BRAF(V600E) 80 CDK3 87 nonphosphorylated ABL1(T315I)-phosphorylated 88 BRK 95 CDK4-cyclinD1 100 ABL1(Y253F)- 100 AURKA 100 CDK4-cyclinD3 74 phosphorylated ABL1-nonphosphorylated 100 AURKB 73 CDK5 100 ABL1-phosphorylated 100 AURKC 96 CDK7 83 ABL2 99 AXL 91 CDK8 100 ACVR1 100 BIKE 68 CDK9 98 ACVR1B 90 BLK 100 CDKL1 100 ACVR2A 100 BMPR1A 100 CDKL2 87 ACVR2B 100 BMPR1B 72 CDKL3 100 ACVRL1 100 BMPR2 73 CDKL5 83 ADCK3 97 BMX 100 CHEK1 95 ADCK4 93 BRAF 82 CHEK2 100 AKT1 100 BRAF(V600E) 80 CIT 89 AKT2 67 BRK 95 CLK1 96 AKT3 85 BRSK1 75 CLK2 77 ALK 96 BRSK2 100 CLK3 87 AMPK-alpha1 98 BTK 68 CLK4 100 AMPK-alpha2 95 CAMK1 100 CSF1R 79 ANKK1 86 CAMK1D 100 CSK 94 ARK5 99 CAMK1G 100 CSNK1A1 100 ASK1 100 CAMK2A 100 CSNK1A1L 90 ASK2 100 CAMK2B 100 CSNK1D 74 CSNK1E 95 ERBB4 88 IRAK4 100 CSNK1G1 81 ERK1 99 ITK 91 CSNK1G2 73 ERK2 91 JAK1(JH1 D-catalytic) 98 CSNK1G3 61 ERK3 100 JAK1(JH2D-pseudokin.) 93 CSNK2A1 100 ERK4 95 JAK2(JH1 D-catalytic) 84 CSNK2A2 77 ERK5 99 JAK3(JH1 D-catalytic) 89 CTK 100 ERK8 94 JNK1 87

1 DAPK1 100 ERN1 99 JNK2 89 DAPK2 91 FAK 90 JNK3 92 DAPK3 97 FER 100 KIT 89 DCAMKL1 96 FES 89 KIT(A829P) 95 DCAMKL2 100 FGFR1 100 KIT(D816H) 91 DCAMKL3 100 FGFR2 77 KIT(D816V) 88 DDR1 63 FGFR3 100 KIT(L576P) 79 DDR2 100 FGFR3(G697C) 84 KIT(V559D) 75 DLK 86 FGFR4 98 KIT(V559D,T670I) 90 DMPK 56 FGR 97 KIT(V559D,V654A) 98 DMPK2 98 FLT1 100 LATS1 91 DRAK1 100 FLT3 99 LATS2 100 DRAK2 99 FLT3(D835H) 100 LCK 95 DYRK1A 68 FLT3(D835Y) 100 LIMK1 64 DYRK1B 100 FLT3(ITD) 100 LIMK2 69 DYRK2 100 FLT3(K663Q) 91 LKB1 100 EGFR 80 FLT3(N841I) 85 LOK 90 EGFR(E746-A750del) 94 FLT3(R834Q) 98 LRRK2 93 EGFR(G719C) 97 FLT4 100 LRRK2(G2019S) 88 EGFR(G719S) 100 FRK 97 LTK 100 EGFR(L747-E749del,A750P) 66 FYN 78 LYN 100 EGFR(L747-S752del,P753S) 87 GAK 100 LZK 100 EGFR(L747-T751del,Sins) 100 GCN2(Kin.Dom.2,S808) 84 MAK 85 EGFR(L858R) 52 GRK1 100 MAP3K1 94 EGFR(L858R,T790M) 100 GRK4 73 MAP3K15 100 EGFR(L861Q) 100 GRK7 70 MAP3K2 74 EGFR(S752-I759del) 100 GSK3A 80 MAP3K3 100 EGFR(T790M) 100 GSK3B 54 MAP3K4 93 EIF2AK1 81 HCK 92 MAP4K2 72 EPHA1 88 HIPK1 76 MAP4K3 100 EPHA2 91 HIPK2 81 MAP4K4 97 EPHA3 91 HIPK3 100 MAP4K5 100 EPHA4 100 HIPK4 82 MAPKAPK2 84 EPHA5 90 HPK1 95 MAPKAPK5 60 EPHA6 100 HUNK 85 MARK1 100 EPHA7 82 ICK 100 MARK2 84 EPHA8 98 IGF1R 98 MARK3 99 EPHB1 100 IKK-alpha 71 MARK4 100 EPHB2 100 IKK-beta 72 MAST1 90 EPHB3 100 IKK-epsilon 95 MEK1 73 EPHB4 100 INSR 100 MEK2 58 EPHB6 100 INSRR 95 MEK3 97 ERBB2 99 IRAK1 100 MEK4 92 ERBB3 66 IRAK3 83 MEK5 91 MEK6 100 PCTK1 74 PRKD3 100 MELK 75 PCTK2 90 PRKG1 88 MERTK 100 PCTK3 96 PRKG2 85 MET 86 PDGFRA 92 PRKR 100 MET(M1250T) 83 PDGFRB 77 PRKX 78 MET(Y1235D) 95 PDPK1 100 PRP4 100 MINK 75 PFCDPK1(P.falciparum) 98 PYK2 100 MKK7 100 PFPK5(P.falciparum) 100 QSK 77 MKNK1 100 PFTAIRE2 92 RAF1 100 MKNK2 100 PFTK1 95 RET 100 MLCK 100 PHKG1 66 RET(M918T) 95 MLK1 100 PHKG2 90 RET(V804L) 96 MLK2 100 PIK3C2B 21 RET(V804M) 96 MLK3 100 PIK3C2G 70 RIOK1 88

2 MRCKA 89 PIK3CA 43 RIOK2 57 MRCKB 100 PIK3CA(C420R) 61 RIOK3 92 MST1 87 PIK3CA(E542K) 33 RIPK1 87 MST1R 79 PIK3CA(E545A) 39 RIPK2 100 MST2 100 PIK3CA(E545K) 38 RIPK4 98 MST3 81 PIK3CA(H1047L) 36 RIPK5 100 MST4 93 PIK3CA(H1047Y) 63 ROCK1 73 MTOR 100 PIK3CA(I800L) 95 ROCK2 78 MUSK 88 PIK3CA(M1043I) 61 ROS1 100 MYLK 80 PIK3CA(Q546K) 64 RPS6KA4(Kin.D.1-N-t) 100 MYLK2 100 PIK3CB 3.7 RPS6KA4(Kin.D.2-C-t) 60 MYLK4 79 PIK3CD 0 RPS6KA5(Kin.D.1-N-t) 91 MYO3A 91 PIK3CG 1.2 RPS6KA5(Kin.D.2-C-t) 100 MYO3B 85 PIK4CB 74 RSK1(Kin.Dom.1-N-t) 74 NDR1 88 PIM1 100 RSK1(Kin.D.2-C-t) 94 NDR2 80 PIM2 100 RSK2(Kin.D.1-N-t) 97 NEK1 100 PIM3 100 RSK3(Kin.D.1-N-t) 100 NEK11 75 PIP5K1A 71 RSK3(Kin.D.2-C-t) 85 NEK2 100 PIP5K1C 35 RSK4(Kin.D.1-N-t) 70 NEK3 97 PIP5K2B 91 RSK4(Kin.D.2-C-t) 98 NEK4 96 PIP5K2C 100 S6K1 94 NEK5 100 PKAC-alpha 100 SBK1 100 NEK6 95 PKAC-beta 95 SgK110 87 NEK7 100 PKMYT1 100 SGK3 76 NEK9 96 PKN1 82 SIK 100 NIM1 100 PKN2 81 SIK2 100 NLK 76 PKNB(M.tuberculosis) 97 SLK 64 OSR1 94 PLK1 100 SNARK 100 p38-alpha 96 PLK2 88 SNRK 100 p38-beta 85 PLK3 93 SRC 100 p38-delta 86 PLK4 79 SRMS 77 p38-gamma 91 PRKCD 96 SRPK1 96 PAK1 100 PRKCE 93 SRPK2 99 PAK2 100 PRKCH 100 SRPK3 88 PAK3 87 PRKCI 76 STK16 37 PAK4 100 PRKCQ 96 STK33 100 PAK6 74 PRKD1 100 STK35 97 PAK7 80 PRKD2 75 STK36 96 STK39 100 TNIK 77 ULK2 74 SYK 100 TNK1 78 ULK3 78 TAK1 95 TNK2 90 VEGFR2 100 TAOK1 73 TNNI3K 97 VRK2 51 TAOK2 64 TRKA 64 WEE1 98 TAOK3 79 TRKB 95 WEE2 100 TBK1 87 TRKC 78 YANK1 88 TEC 100 TRPM6 34 YANK2 98 TESK1 94 TSSK1B 76 YANK3 72 TGFBR1 100 TTK 100 YES 89 TGFBR2 100 TXK 95 YSK1 100 TIE1 100 TYK2(JH1D-catalytic) 92 YSK4 100 TIE2 85 TYK2(JH2D- 100 ZAK 100 pseudokinase) TLK1 100 TYRO3 60 ZAP70 74 TLK2 100 ULK1 84

3