Supplementary Figure Legends s1

Supplementary Figure Legends

Supplementary Figure 1. Effect of MK-1775 combined with AZD8055, WYE-

125132, and Torin 2, respectively, on cell cycle progression of parental Ba/F3 cells and mutant NRAS-expressing Ba/F3 cells. (A) Drug treatment of Ba/F3 cells for 48 hours prior to staining with propidium iodide for cell cycle analysis. (B) Drug treatment of Ba/F3-NRAS-G12D for 48 hours prior to staining with propidium iodide for cell cycle analysis.

Supplementary Figure 2. Effect of MK-1775 combined with AZD8055 on phosphorylation of MAPK in mutant RAS-expressing Ba/F3 cells. (A-B) Effect of approximately two-hr treatment of Ba/F3-NRAS-G12D cells (A) or Ba/F3-KRAS-G12D cells (B) with MK-1775 (75 nM), alone and combined with AZD8055 (20 nM), on phosphorylation of MAPK.

Supplementary Figure 3. Effect of MK-1775 combined with AZD8055 on phosphorylation of histone H2A.X, a marker of DNA damage, in wt and mutant

NRAS-expressing Ba/F3 cells. Effect of approximately 24-hr treatment of cells with

MK-1775 (75 nM) or AZD8055 (20 nM), alone or combined, on phosphorylation of histone H2A.X.

Supplementary Figure 4. Wee1 kinase is the main target of MK1775 that mediates synergy with TORC inhibitors. Measurement of cell proliferation following approximately 24-hr treatment of NB4 cells with MK-1775, Torin 2, MK-1775+Torin 2, or MK-1775+Torin 2 in the presence of a CDK1 inhibitor at the indicated concentrations.

Supplementary Figure 5. Investigation of the combined effects of MK-1775 and mTOR inhibitors against mutant NRAS-expressing primary AML patient samples.

Approximately 72-hr proliferation studies. Experiments shown are representative of two independent experiments for which similar results were observed.

Supplementary Figure 6. Comparison of effects of MK-1775+/-mTOR inhibitors against Ba/F3-NRAS-G12D cells and Ba/F3-NRAS-G12D cells expressing constitutively active AKT (Ba/F3-NRAS-G12D-AKT (myr)). Approximately 72-hr treatments of Ba/F3-NRAS-G12D cells (A, C) or Ba/F3-NRAS-G12D-AKT (myr) cells

(B, D) with MK-1775 alone, mTOR inhibitor alone, or a combination of MK-

1775+mTOR inhibitor. (E) Calcusyn combination indices derived from four-point concentration proliferation experiments. The cut-off for nearly additive effects (C.I.: 1.1) is marked by a dashed line.

Supplementary Figure 7. Investigation of the combined effects of MK-1775 and the

PI3K inhibitor, ZSTK474, against wild-type or mutant RAS-expressing Ba/F3 cells.

(A-C) Approximately 72-hr proliferation studies performed with MK-1775 and ZSTK474 against parental Ba/F3 cells, Ba/F3-NRAS-G12D cells, or Ba/F3-KRAS-G12D cells.

Each experiment shown is representative of two independent experiments for which similar results were observed. Supplementary Figure 8. Proliferation studies performed against NRAS-driven melanoma cell lines, M13, K033, and K023, treated with MK-1775+/- mTOR inhibitor, AZD8055. (A-F) Dose-response curves showing single agent versus drug combination effects following approximately 48 hours of treatment.

Supplementary Figure 9. Proliferation studies performed against KRAS-driven colorectal cancer cell lines, HCT116, DLD1, and SW480, treated with MK-1775+/- mTOR inhibitor, AZD8055. (A-F) Dose-response curves showing single agent versus drug combination effects following approximately 72 hours of treatment.

Supplementary Figure 10. Proliferation studies performed with MK-1775 and mTOR inhibitors against active KRAS-expressing pancreatic cancer cell lines. (A-

D) Dose-response curves showing single agent versus drug combination effects following approximately 72-hr assays.

Supplementary Figure 11 (A-D). Proliferation studies performed with MK-1775 and mTOR inhibitors (WYE125132, AZD8055) against human active KRAS-expressing lung cancer cells. (A-D) Dose-response curves showing single agent versus drug combination effects following approximately 72-hr assays.

Supplementary Figure 11 (E-H). Proliferation studies performed with MK-1775 and mTOR inhibitors (WYE125132, AZD8055) against human active KRAS-expressing lung cancer cells. (E-H) Dose-response curves showing single agent versus drug combination effects following approximately 72-hr assays.

Supplementary Figure 11 (I-L). Proliferation studies performed with MK-1775 and

Torin 2 against human active KRAS-expressing lung cancer cells. (I-L) Dose- response curves showing single agent versus drug combination effects following approximately 72-hr assays.

Supplementary Figure 12 (A-C). Proliferation studies performed with MK-1775 and

Torin 2 against active KRAS-expressing cells derived from murine models of mutant KRAS-driven lung cancer. (A-B) Dose-response curves showing single agent versus drug combination effects following approximately 48-hr assays. These data are representative of two independent experiments in which similar results were observed. T2

(Lkb-1-p53-kd/KRAS active) and T5 (Lkb-1-p53-kd/KRAS active). (C) Calcusyn combination indices derived from seven point concentration proliferation experiments.

The cut-off for nearly additive effects (C.I.: 1.1) is marked by a dashed line.

Supplementary Figure 12 (D-G). Proliferation studies performed with MK-1775 and

Torin 1 against active KRAS-expressing cells derived from murine models of mutant KRAS-driven lung cancer. (D-G) Dose-response curves showing single agent versus drug combination effects following approximately 48-hr assays. 634: p53- kd/KRAS active. 857: p53-kd/KRAS active. T2: Lkb-1-p53-kd/KRAS active. T5: Lkb-1- p53-kd/KRAS active. Supplementary Table I. Combination indices. MK-1775 is abbreviated as “MK.”

Values less than 0.9 indicate synergy and are in shades of red (darker shades mean higher synergy). Values greater than 0.9 do not indicate synergy and are colored white. Shown are 634 (p53-kd/KRAS active), T2 (Lkb-1-p53-kd/KRAS active) and T5 (Lkb-1-p53- kd/KRAS active) lung cancer cell lines.

Supplementary Figure 13. In vivo model of inducible mutant KRAS-positive lung cancer. Mouse strains harboring conditional activating mutation (G12D) at the endogenous Kras locus are activated by nasal instillation of 5x106 pfu of adenovirus encoding CRE recombinase between 6-8 weeks of age under isoflurane anesthesia.

Expression of cre recombinase in infected lung epithelial cells will remove loxp flanked stop cassette located before ATG start codon of the mutant Kras gene. Induced KRAS mice express activating KRAS G12D mutant in lung epithelial cells, and have a disease latency of 13-15 weeks after induction.