Supplementary Figures from Upregulation of IGF1R by Mutant <i>RAS</i> in Leukemia and Potentiation of <i>RAS</i> Signaling Inhibitors by Small-Molecule Inhibition of IGF1R
Authors: Weisberg Ellen , Nonami Atsushi , Chen Zhao , Nelson Erik , Chen Yongfei , Liu Feiyang , Cho HaeYeon , Zhang Jianming , Sattler Martin , Mitsiades Constantine , Wong Kwok-Kin , Liu Qingsong , Gray Nathanael S. , Griffin James D. 
Published: April 2023

<p>Supplementary Figure 1. Schematic of LINCS library chemical screen. Supplementary Figure 2. Selectivity of LINCS library kinase inhibitors toward mutant NRAS-expressing cells. Supplementary Figure 3. Proliferation studies showing positive combination effects between AZD6244 and LINCS library inhibitors. 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�S supplemented with 15% WEHI-conditioned medium (used as a source of IL-3). 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. Supplementary Figure 6 (A-E). Induction of apoptosis in AZD6244 and GSK1904529A-treated wt and mutant RAS-expressing AML cell lines. Supplementary Figure 6 (F-I). Cell cycle progression of AZD6244 and GSK1904529A-treated mutant RAS-expressing cells. 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. 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. 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 OCI-AML3 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. 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. Supplementary Figure 8. Heightened response of mutant RAS-expressing HL60 cells to IGF as compared to Hel cells. 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. 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. Supplementary Figure 11. MEK inhibition as a predictor of response to IGF-1R and MEK inhibitor combination treatment. 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. 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. Supplementary Figure 17. Combined effects of IGF-1R and MEK inhibition against mutant NRAS-positive AML patient cells.</p>