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Supplementary Figure 1 Select oncogenic target of interest Choose an oncogene-driven cell line and a cell line to be used as a counterscreen OCI-AML3 Ba/F3-NRAS-G12D.

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Presentation on theme: "Supplementary Figure 1 Select oncogenic target of interest Choose an oncogene-driven cell line and a cell line to be used as a counterscreen OCI-AML3 Ba/F3-NRAS-G12D."— Presentation transcript:

1 Supplementary Figure 1 Select oncogenic target of interest Choose an oncogene-driven cell line and a cell line to be used as a counterscreen OCI-AML3 Ba/F3-NRAS-G12D Ba/F3 Screen LINCS Chemical Library (at predetermined screening concentration) 384-well plate (Cell Titer Glo) Validation of LINCS Chemical Library “hits” Cellular proliferation assays 96-well plate (Cell Titer Glo) LINCS Kinase Inhibitor- Focused Chemical Library + AZD6244 Signaling studies Identification of LINCS Chemical Library “hits” Testing in murine models AZD6244 Choose selective inhibitor of downstream RAS signaling to test in combination with LINCS Chemical Library (use at no higher than the IC50) PI-103 (PIKK inhibitor) GSK2126458 (PI3K inhibitor) ZSTK474 (PI3K inhibitor) Torin 1 (mTOR inhibitor) GSK1904529A (IGF-1R inhibitor)

2 Percentage of viable cells PD0325901 (MEK inhibitor) GSK2126458 (PI3K Inhibitor) GSK1120212 (MEK Inhibitor) AZD8330 (MEK inhibitor) parental Ba/F3 Ba/F3-NRAS-G12D OCI-AML3 A [Drug, nM] Cell Number (% of Control) [Drug, nM] B C 018.7537.575150300 018.7537.575150300 0 20 40 60 80 100 120 0 20 40 60 80 100 120 0 20 40 60 80 100 120 Supplementary Figure 2

3 Cell Number (% of control) 02.5510204080 09.37518.7537.575150300 02.5+ 9.375 5+ 18.75 10+ 37.5 20+ 75 40+ 150 80+ 300 AZD6244 ZSTK474 Combination [Drug, nM] Cell Number (% of control) 02.5 5 10204080 01.252.55102040 02.5+ 1.25 5+ 2.5 10+ 5 20+ 10 40+ 20 80+ 40 AZD6244 GSK2126458 Combination [Drug, nM] Cell Number (% of control) 0510204080160 0 9.375 18.75 37.575150300 05+ 9.375 10+ 18.75 20+ 37.5 40+ 75 80+ 150 160+ 300 AZD6244 PI-103 Combination [Drug, nM] A B C Ba/F3-NRAS-G12D Cell Number (% of control) 02.5510204080 09.37518.7537.575150300 02.5+ 9.375 5+ 18.75 10+ 37.5 20+ 75 40+ 150 80+ 300 AZD6244 ZSTK474 [Drug, nM] OCI-AML3 D Cell Number (% of control) 0510204080160 09.37518.7537.575150300 05+ 9.375 10+ 18.75 20+ 37.5 40+ 75 80+ 150 160+ 300 AZD6244 P1013 Combination [Drug, nM] OCI-AML3 E Cell Number (% of control) 0510204080160 01.252.55102040 05+ 1.25 10+ 2.5 20+ 5 40+ 10 80+ 20 160+ 40 AZD6244 Torin1 Combination [Drug, nM] OCI-AML3 F ZSTK474 (150-300) AZD6244 (20-40) Combination (AZD=5-10; ZST=18.75-37.5) GSK2126458 (10-20) AZD6244 (20-40) Combination (AZD=5; GSK=2.5) PI-103 (37.5-75) AZD6244 (20-40) Combination (AZD=5-10; PI=9.375-18.75) ZSTK474 (150-300) AZD6244 (20-40) Combination (AZD=5-10; ZST=18.75-75) P1013 (40-80) AZD6244 (20) Combination (AZD=5-10; PI=9.375-18.75) Torin 1 (80-160) AZD6244 (20-40) Combination (AZD=10; Torin1=2.5) 0 20 40 60 80 100 120 0 20 40 60 80 100 120 0 20 40 60 80 100 120 0 20 40 60 80 100 120 0 20 40 60 80 100 120 0 20 40 60 80 100 120 Supplementary Figure 3

4 Cell Number (% of Control) Ba/F3-NRAS-G12D (RPMI+10% FBS) [Drug, nM] Cell Number (% of Control) Ba/F3-NRAS-G12D (RPMI+10% FBS) [Drug, nM] Cell Number (% of Control) Ba/F3-NRAS-G12D (15% WEHI (source of IL-3) [Drug, nM] Cell Number (% of Control) Ba/F3-NRAS-G12D (15% WEHI (source of IL-3) [Drug, nM] A B C D Supplementary Figure 4

5 Supplementary Figure 5 BCA DE F

6 OCI-AML3 Percentage NB4 Percentage NOMO-1 Percentage SKM-1 Percentage HEL AB CDE Supplementary Figure 6 (A-E)

7 Supplementary Figure 6 (F-I) F Percentage NB4 SKM-1 Percentage NOMO-1 Percentage HI G OCI-AML3

8 Percentage G0G1 G2M S 0 10 20 30 40 50 60 K J ControlGSK1904529AAZD6244GSK1904529A+ AZD6244 ControlGSK1904529A AZD6244GSK1904529A+ AZD6244 Percentage subG1 0 5 10 15 20 25 30 35 40 45 ControlGSK1904529AAZD6244GSK1904529A+ AZD6244 L Percentage Supplementary Figure 6 (J-L) 24 hr 48 hr

9 ControlGSK1904529A AZD6244 GSK1904529A+ AZD6244 Percentage M72 hr Supplementary Figure 6 (M)

10 Supplementary Figure 6 (N) N MOLM14 NB4 OCI-AML3 Control AZD6244 (300 nM) GSK1904529A (300 nM) AZD6244+ GSK1904529A

11 SKM-1 (RPMI+10%FBS) Cell Number (% of Control) [Drug, nM] SKM-1 (95% HS-5 SCM) Cell Number (% of Control) [Drug, nM] SKM-1 (95% HS27a SCM) Cell Number (% of Control) [Drug, nM] NB4-luc+ (RPMI+10%FBS) Cell Number (% of Control) [Drug, nM] NB4-luc+ (95% HS-5 SCM) Cell Number (% of Control) [Drug, nM] NB4-luc+ (95% HS27a SCM) Cell Number (% of Control) [Drug, nM] Supplementary Figure 7 (part 1)

12 OCI-AML3 (RPMI+10%FBS) Cell Number (% of Control) [Drug, nM] OCI-AML3 (95% HS-5 SCM) Cell Number (% of Control) [Drug, nM] NOMO-1 (RPMI+10%FBS) Cell Number (% of Control) [Drug, nM] NOMO-1 (95% HS27a SCM) Cell Number (% of Control) [Drug, nM] MOLM14 [PKC412, nM] Cell Number (% of Control) Supplementary Figure 7 (part 2)

13 Supplementary Figure 8 Normalized growth Treatment

14 Supplementary Figure 9 Cell Number (% of Control) GSK1904529A (37.5) ZSTK474 (150) Combination (9.4-18.75) 0 20 40 60 80 100 120 140 04.79.418.7537.575150300 ZSTK474 [Drug, nM] 0 4.7 9.4 18.7537.575150 300 160 Ba/F3-KRAS-G12D Cell Number (% of Control) Ba/F3-NRAS-G12D GSK1904529A (>300) ZSTK474 (150-300) Combination (37.5) 0 20 40 60 80 100 120 140 04.79.418.7537.575150300 ZSTK474 [Drug, nM] 0 4.7 9.4 18.7537.575150300 160 Cell Number (% of Control) Parental Ba/F3 GSK1904529A (>300) ZSTK474 (>300) Combination (>300) 0 20 40 60 80 100 120 140 04.79.418.7537.575150300 ZSTK474 04.7 9.4 18.7537.575150300 160 [Drug, nM] A BC GSK1904529A

15 Supplementary Figure 10 C D Phospho-IGF1R  / Total IGF1R  2.1 0.71.21.80.891.39 AB OCI-AML3 NOMO-1 NB4 SKM-1 HEL MOLM14 OCI-AML3 NOMO-1 NB4 SKM-1 HEL MOLM14 OCI-AML3NOMO-1 NB4SKM-1HELMOLM14 Ba/F3-KRAS-G12DBa/F3-NRAS-G12D Ba/F3

16 Supplementary Figure 11 B A

17 Supplementary Figure 12 A B

18 Supplementary Figure 13 ControlAZD6244 (37.5 nM) GSK- 1904529A (37.5 nM) Combi- nation A B Control NVP- AEW541 (300 nM) GSK- 1904529A (300 nM) AZD6244 (300 nM) AZD6244 + NVP- AEW541 AZD6244 + GSK- 1904529A

19 Supplementary Figure 14 Control 300 nM NVPAEW541 300 nM GSK1904529A 300 nM AZD6244 AZD6244+NVPAEW541 AZD6244+ GSK1904529A (Ser 65)

20 Absorbance (560 nm) AB E Cell Number (% of no treatment control) NB4 Supplementary Figure 15 NB4 C Absorbance (560 nm) HEL D Cell Number (% of no treatment control) F GFP Control IGF-1R hp1 IGF-1R hp2 0 GSK1904529A (18.75 nM) AZD6244 (18.75 nM) Combination 0 GSK1904529A (37.5 nM) AZD6244 (37.5 nM) Combination 0 GSK1904529A (75 nM) AZD6244 (75 nM) Combination GFP Control IGF-1R hp1 IGF-1R hp2 0 GSK1904529A (18.75 nM) AZD6244 (18.75 nM) Combination GFP Control IGF-1R hp1 IGF-1R hp2 GFP Control IGF-1R hp1 IGF-1R hp2 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80 0 100 20 120 40 60 80 0 20 40 60 80 100 120

21 Supplementary Figure 16 A B Grams NVPAEW541AZD6244NVPAEW541+ AZD6244 NVPAEW541AZD6244 NVPAEW541+ AZD6244

22 Supplementary Figure 17 Cell Number (% of Control) NVPAEW541 (300 nM) AZD6244 (300 nM) NVPAEW541 (300 nM) + AZD6244 (300 nM) *

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