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Supplementary Figure Legends Supplementary Figure 1. Schematic of LINCS library chemical screen. 384-well plates were seeded with (1) parental Ba/F3 cells cultured in the presence of 15% WEHI (as a source of IL-3), which were used as a control to eliminate inhibitors that exhibit offtarget toxicity and interfere with IL-3-mediated signaling, (2) Ba/F3-NRAS- G12D cells (growth factor-independent), or (3) OCI-AML3 cells (growth factor-independent). Included in the screen were cell-containing plates that only received LINCS library drugs (300 nM, determined to be the optimal screening concentration) for the purpose of assessing single agent activity of the LINCS library compounds, or plates that contained LINCS library compounds (300 nM) plus the MEK inhibitor, AZD6244 (20 nM, which was determined to be close to the IC50 against mutant NRAS-expressing cells). DMSO (vehicle) wells and AZD6244-only wells were included as controls in the plates. Following pintool administration of the library compounds, the 384-well plates were incubated for two days prior to administration of Cell Titer Glo (Promega, Madison, WI) and analysis of bioluminescence using a plate reader. Ideal candidates for further investigation were LINCS library compounds showing minimal activity as single agents against parental Ba/F3 cells, yet potentiation of the efficacy of AZD6244 against NRASdriven cells. "Hits" were validated for synergizing potential using several approaches, including cellular proliferation assays, signaling studies, and in vivo analysis. LINCS library compounds were anticipated to be useful, because of their limited spectrum of kinase targets, to more easily identify kinase mediators of RAS signaling that could be exploited for the purpose of drug development. Supplementary Figure 2. Selectivity of LINCS library kinase inhibitors toward mutant NRAS-expressing cells. (A) Preliminary chemical screen results: LINCS library kinase inhibitors identified as selectively displaying strong single agent activity against Ba/F3-NRAS-G12D and mutant NRAS-expressing OCI-AML3 cells, as compared to parental Ba/F3 cells. (B) Proliferation/validation studies comparing effects of LINCS library MEK inhibitors, PD0325901, GSK1120212, and AZD8330, against parental Ba/F3 versus Ba/F3-NRAS-G12D cells. (C) Proliferation/validation studies comparing the differential sensitivity of parental Ba/F3 and Ba/F3-NRAS-G12D cells to LINCS library PI3K inhibitors (PI-103, GSK2126458, and ZSTK474), which were identified as positively combining in the chemical screen with the Mek inhibitor, AZD6244, against Ba/F3-NRAS-G12D cells. Supplementary Figure 3. Proliferation studies showing positive combination effects between AZD6244 and LINCS library inhibitors. (A-C) Proliferation studies performed with AZD6244 combined with identified LINCS library PI3K and mTOR inhibitors against Ba/F3-NRAS-G12D cells. (D-F) Proliferation studies performed with AZD6244 combined with identified LINCS library PI3K inhibitors against OCI-AML3 cells. Shown in parentheses adjacent to figure legends are estimated IC50 values (nM) corresponding to individual drugs or drug combinations. Supplementary Figure 4. Treatment of Ba/F3-NRAS-G12D cells with GSK1904529A, AZD6244, or a combination of both in the presence of RPMI+10% FBS or RPMI+10%FBS supplemented with 15% WEHI-conditioned medium (used as a source of IL-3). Approximately 2-day treatments of Ba/F3-NRAS-G12D in the absence (A-B) or presence (C-D) of WEHI-conditioned medium. Supplementary Figure 5. Investigation of the combined effects of IGF-1R inhibition and MEK inhibition on RAS and IGF-1R protein expression in RAS-transformed cells. (A-D) Investigation of NRAS and IGF-1R protein expression in untreated Ba/F3NRAS-G12D cells and AZD6244+GSK1904529A-treated Ba/F3-NRAS-G12D cells. (E) Investigation of IGF-1R protein expression in AZD6244+GSK1904529A-treated Ba/F3KRAS-G12D cells. For these studies, drug treatments, alone and combined, were carried out for 1 hour prior to lysate preparation and immunoblotting. Supplementary Figure 6 (A-E). Induction of apoptosis in AZD6244 and GSK1904529A-treated wt and mutant RAS-expressing AML cell lines. (A-E) Approximately 72-hr treatments. HEL cells are shown as representatives of wt RASexpressing cells. Data shown are representative of two independent experiments in which similar results were observed. Supplementary Figure 6 (F-I). Cell cycle progression of AZD6244 and GSK1904529A-treated mutant RAS-expressing cells. (F) Cells were treated for approximately 48 hours prior to propidium iodide staining. (G-I) Cells were treated for approximately 72 hours prior to propidium iodide staining. Data shown are representative of two independent experiments in which similar results were observed. Supplementary Figure 6 (J-L). Effects of the combination of IGF-1R inhibitor, GSK1904529A (75 nM), and the Mek inhibitor, AZD6244 (20 nM), on cell cycle progression and induction of apoptosis of Ba/F3-NRAS-G12D cells.. (J) Approximately 24 hr cell cycle analysis using propidium iodide. (K) Approximately 48 hr cell cycle analysis using propidium iodide. (L) SubG1 fractions corresponding to cell cycle analysis performed for “K.” Supplementary Figure 6 (M). Effects of the combination of GSK1904529A (37.5 nM) and AZD6244 (37.5 nM) on cell cycle progression of Ba/F3-KRAS-G12D cells. Approximately 72 hr cell cycle analysis using propidium iodide. Supplementary Figure 6 (N). Effects of the combination of GSK1904529A (300 nM) and AZD6244 (300 nM) on soft agar colony growth of wt RAS-expressing MOLM14 cells, mutant KRAS-expressing NB4 cells, and mutant NRAS-expressing OCIAML3 cells. Supplementary Figure 7 (part 1). Treatment of human AML cells with GSK1904529A, AZD6244, or a combination of both in the presence of RPMI+10% FBS, 95% HS-5 SCM, or 95% HS27a SCM. Approximately 2-day treatments of SKM1 (upper panel) or NB4-luc+ (lower panel). Supplementary Figure 7 (part 2). Treatment of human AML cells with GSK1904529A, AZD6244, or a combination of both in the presence of RPMI+10% FBS, 95% HS-5 SCM, or 95% HS27a SCM. Approximately 2-day treatments of OCIAML3 (upper panel) or Nomo-1 (lower panel). Shown as a positive control for HS-5 or HS27a SCM cytoprotection are MOLM14 cells treated with PKC412 in the absence or presence of HS-5 or HS27a SCM (right side). Supplementary Figure 8. Heightened response of mutant RAS-expressing HL60 cells to IGF as compared to Hel cells. Compared in this figure are 0, 1, and 3 ng/mL IGF. Supplementary Figure 9. Investigation of the combined effects of GSK1904529A and the PI3K inhibitor, ZSTK474, against wild-type or mutant RAS-expressing Ba/F3 cells. (A-C) Approximately 72-hr proliferation studies performed with GSK1904529A 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. Shown in parentheses adjacent to figure legends are estimated IC50 values (nM) corresponding to individual drugs or drug combinations. Supplementary Figure 10. Investigation of phospho- and total-Erk1/Erk2 and phospho and total IGF-1R levels in wt and mutant RAS-expressing human AML cell lines. (A-B) Phospho- and total Erk1/Erk2 protein expression in wt and mutant RAS- expressing AML cells. (C-D) Phospho- and total IGF-1R protein expression in human wt and mutant Ras-expressing AML cells. The phospho-IGF1Rbeta/Total IGF1Rbeta ratio for HL60 was determined in an independent experiment to be 77.6% of the phosphoIGF1Rbeta/Total IGF1Rbeta ratio for OCI-AML3 (data not shown). Supplementary Figure 11. MEK inhibition as a predictor of response to IGF-1R and MEK inhibitor combination treatment. Investigation of IGF-1R inhibitor, MEK inhibitor, and drug combination effects on phosphorylation of Erk1/Erk2 in wt and mutant NRAS-expressing cells (A) or wt and mutant KRAS-expressing cells (B). Cells were treated for approximately 1 hour prior to lysate preparation and immunoblotting. Supplementary Figure 12. Effects of NVPAEW541 on IGF-1R (A) and Erk1/Erk2 (B) phosphorylation. Supplementary Figure 13. Investigation of the combined effects of IGF-1R inhibition and MEK inhibition on phosphorylation of Erk1/Erk2 (A) and AKT (B) in mutant RAS-expressing cells. Supplementary Figure 14. Investigation of the combined effects of IGF-1R inhibition and MEK inhibition on phospho-4E-BP1 (Serine 65) expression in mutant RAS-expressing cells. Supplementary Figure 15. Effects of IGF-1R KD in the mutant KRAS-expressing human cell line, NB4, and wt RAS-expressing human cell line, Hel. (A) Comparison of growth of NB4 control and IGF-1R KD cells. (B) Comparison of growth of NB4 control and IGF-1R KD cells following an approximately 72-hr treatment with GSK1904529A, AZD6244, or a combination of both. (C) Comparison of growth of Hel control and IGF-1R KD cells. (D) Comparison of growth of Hel control and IGF-1R KD cells following an approximately 72-hr treatment with GSK1904529A, AZD6244, or a combination of both. (E-F) Investigation of levels of IGF-1R in GFP control and IGF-1R knockdown cells. Supplementary Figure 16. Mouse spleen measurements from in vivo leukemia study investigating the effects of NVPAEW541, AZD6244, or NVPAEW541+AZD6244 treatment of mice tail vein-injected with OCI-AML3-luc+ cells. (A) Shown are spleens dissected from two representative mice per treatment group one week after the final imaging (which was performed following 7 days of drug treatment). (B) Bar graph showing composite average spleen weights for each treatment group. Supplementary Figure 17. Combined effects of IGF-1R and MEK inhibition against mutant NRAS-positive AML patient cells. Approximately 72-hr proliferation studies performed with NVPAEW541 and AZD6244, alone or combined, against mutant NRASexpressing AML patient cells (n=3). Two independent experiments were performed with four concentration points (0, 75, 150, 300 nM), for which similar results were observed. Shown here are composite results for 300 nM of each drug alone or combined. *p=0.0373 (one-tailed unpaired student’s t-test). Patient characteristics: Mutant NRAS-positive AML3 cells were derived from a 35 year-old patient with AML, NOS, harboring NRASG13V, with 92% bone marrow blasts. Mutant NRAS-positive AML4 cells were derived from a 62 year-old patient with AML, NOS, harboring NRAS-G12D, with 83% blasts. Mutant NRAS-positive AML6 cells were derived from a 69 year-old patient with AML (acute myelomonocytic leukemia), harboring NRAS-G13D, with 43% bone marrow blasts.