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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.