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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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) GSK (PI3K inhibitor) ZSTK474 (PI3K inhibitor) Torin 1 (mTOR inhibitor) GSK A (IGF-1R inhibitor)

Percentage of viable cells PD (MEK inhibitor) GSK (PI3K Inhibitor) GSK (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 Supplementary Figure 2

Cell Number (% of control) AZD6244 ZSTK474 Combination [Drug, nM] Cell Number (% of control) AZD6244 GSK Combination [Drug, nM] Cell Number (% of control) AZD6244 PI-103 Combination [Drug, nM] A B C Ba/F3-NRAS-G12D Cell Number (% of control) AZD6244 ZSTK474 [Drug, nM] OCI-AML3 D Cell Number (% of control) AZD6244 P1013 Combination [Drug, nM] OCI-AML3 E Cell Number (% of control) AZD6244 Torin1 Combination [Drug, nM] OCI-AML3 F ZSTK474 ( ) AZD6244 (20-40) Combination (AZD=5-10; ZST= ) GSK (10-20) AZD6244 (20-40) Combination (AZD=5; GSK=2.5) PI-103 ( ) AZD6244 (20-40) Combination (AZD=5-10; PI= ) ZSTK474 ( ) AZD6244 (20-40) Combination (AZD=5-10; ZST= ) P1013 (40-80) AZD6244 (20) Combination (AZD=5-10; PI= ) Torin 1 (80-160) AZD6244 (20-40) Combination (AZD=10; Torin1=2.5) Supplementary Figure 3

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

Supplementary Figure 5 BCA DE F

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

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

Percentage G0G1 G2M S K J ControlGSK AAZD6244GSK A+ AZD6244 ControlGSK A AZD6244GSK A+ AZD6244 Percentage subG ControlGSK AAZD6244GSK A+ AZD6244 L Percentage Supplementary Figure 6 (J-L) 24 hr 48 hr

ControlGSK A AZD6244 GSK A+ AZD6244 Percentage M72 hr Supplementary Figure 6 (M)

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

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)

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)

Supplementary Figure 8 Normalized growth Treatment

Supplementary Figure 9 Cell Number (% of Control) GSK A (37.5) ZSTK474 (150) Combination ( ) ZSTK474 [Drug, nM] Ba/F3-KRAS-G12D Cell Number (% of Control) Ba/F3-NRAS-G12D GSK A (>300) ZSTK474 ( ) Combination (37.5) ZSTK474 [Drug, nM] Cell Number (% of Control) Parental Ba/F3 GSK A (>300) ZSTK474 (>300) Combination (>300) ZSTK [Drug, nM] A BC GSK A

Supplementary Figure 10 C D Phospho-IGF1R  / Total IGF1R  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

Supplementary Figure 11 B A

Supplementary Figure 12 A B

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

Supplementary Figure 14 Control 300 nM NVPAEW nM GSK A 300 nM AZD6244 AZD6244+NVPAEW541 AZD6244+ GSK A (Ser 65)

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 GSK A (18.75 nM) AZD6244 (18.75 nM) Combination 0 GSK A (37.5 nM) AZD6244 (37.5 nM) Combination 0 GSK A (75 nM) AZD6244 (75 nM) Combination GFP Control IGF-1R hp1 IGF-1R hp2 0 GSK A (18.75 nM) AZD6244 (18.75 nM) Combination GFP Control IGF-1R hp1 IGF-1R hp2 GFP Control IGF-1R hp1 IGF-1R hp

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

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