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Supplementary Figure 1

Supplementary Figure 1. Select oncogenic target of interest. Choose selective inhibitor of downstream RAS signaling to test in combination with LINCS Chemical Library (use at no higher than the IC50). Choose an oncogene-driven cell line and a cell line to be used as a

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Supplementary Figure 1

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

  2. A Supplementary Figure 2 parental Ba/F3 120 Ba/F3-NRAS-G12D OCI-AML3 100 80 60 Percentage of viable cells 40 20 0 PD0325901 (MEK inhibitor) GSK2126458 (PI3K Inhibitor) GSK1120212 (MEK Inhibitor) AZD8330 (MEK inhibitor) C B 120 120 100 100 80 80 Cell Number (% of Control) Cell Number (% of Control) 60 60 40 40 20 20 0 0 0 18.75 37.5 75 150 300 0 18.75 37.5 75 150 300 [Drug, nM] [Drug, nM]

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

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

  5. Supplementary Figure 5 A B C F D E

  6. Supplementary Figure 6 (A-E) A B OCI-AML3 NB4 Percentage Percentage C D E HEL SKM-1 NOMO-1 Percentage Percentage Percentage

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

  8. Supplementary Figure 6 (J-L) J 24 hr 60 50 40 G0G1 Percentage 30 G2M S 20 10 0 Control GSK1904529A AZD6244 GSK1904529A+ AZD6244 48 hr 48 hr K L subG1 45 40 35 Percentage 30 Percentage 25 20 15 10 5 0 Control GSK1904529A Control GSK1904529A AZD6244 GSK1904529A+ AZD6244 AZD6244 GSK1904529A+ AZD6244

  9. Supplementary Figure 6 (M) M 72 hr Percentage Control GSK1904529A GSK1904529A+ AZD6244 AZD6244

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

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

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

  13. Supplementary Figure 8 Normalized growth Treatment

  14. Supplementary Figure 9 A 160 Parental Ba/F3 140 120 100 80 Cell Number (% of Control) 60 GSK1904529A (>300) 40 ZSTK474 (>300) Combination (>300) 20 0 0 4.7 GSK1904529A 9.4 18.75 37.5 75 150 300 0 4.7 9.4 18.75 37.5 75 150 300 ZSTK474 [Drug, nM] B C 160 160 Ba/F3-NRAS-G12D Ba/F3-KRAS-G12D 140 140 GSK1904529A (>300) ZSTK474 (150-300) GSK1904529A (37.5) 120 120 Combination (37.5) ZSTK474 (150) Combination (9.4-18.75) 100 100 Cell Number (% of Control) Cell Number (% of Control) 80 80 60 60 40 40 20 20 0 0 4.7 300 9.4 0 18.75 37.5 75 150 GSK1904529A 4.7 GSK1904529A 9.4 0 18.75 37.5 75 150 300 0 4.7 9.4 18.75 37.5 75 150 300 ZSTK474 0 4.7 9.4 18.75 37.5 75 150 300 ZSTK474 [Drug, nM] [Drug, nM]

  15. Supplementary Figure 10 Ba/F3-NRAS-G12D Ba/F3-KRAS-G12D A B Ba/F3 OCI-AML3 NOMO-1 MOLM14 SKM-1 NB4 HEL C D OCI-AML3 NOMO-1 MOLM14 OCI-AML3 SKM-1 MOLM14 NB4 HEL NOMO-1 SKM-1 NB4 HEL Phospho-IGF1Rb/ Total IGF1Rb 2.1 0.7 1.2 1.8 0.89 1.39

  16. Supplementary Figure 11 A B

  17. Supplementary Figure 12 A B

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

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

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

  21. Supplementary Figure 16 A NVPAEW541+ AZD6244 NVPAEW541 AZD6244 B Grams NVPAEW541 AZD6244 NVPAEW541+ AZD6244

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

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