1. Abemaciclib (µM) LY3009120 (µM) 0 0.005 0.01 0.02 0.05 0.1 0.2 0.25
0.25
0.5
Abemaciclib (µM) 1 2 4
Supplementary Figure S1. RAF inhibition by LY sensitizes KRAS mutant cancer cells to abemaciclib. HCT116 cells were in treated for 5 days with increasing concentrations of LY and abemaciclib. At the end of the experiment, cells were fixed and stained with crystal violet solution.

2. A Calu-6 (KRAS Q61K) B HCT116 (KRAS G13D) C A375 (BRAF V600E) D
SK-MEL 30 (NRAS Q61K) E
HT-29 (BRAF V600E)
Supplementary Figure S2. Heatmaps of LY and abemaciclib combination in different tumor cell lines. Plots show the observed percent inhibition minus the expected additive response (EAR). The EAR is estimated using Bliss Independence for each combination dose. The numbers on the left side are concentrations of abemaciclib, and the numbers on top are concentrations of LY The red color indicates additive or synergistic effects, and the darker the red indicates the more synergistic effect by combination.

3. A B
APC
D-Cyclin activation
Geometric Mean Abs. IC50 (mM)
Geometric Mean Abs. IC50 (mM)
Cell Lines
Cell Lines C D
RB1
PI3K pathway mutations
Geometric Mean Abs. IC50 (mM)
Geometric Mean Abs. IC50 (mM)
Cell Lines
Cell Lines E
CDKN2A
Geometric Mean Abs. IC50 (mM)
Cell Lines
Supplemental Figure S3. In vitro activities of LY and abemaciclib combination in cancer cell screen panel (CCSP) and their sensitivity association with APC mutations (A), D-cyclin activation (B), RB mutations (C), PI3K pathway mutations (D) and CDKN2A mutations (E). Green color indicates cell lines with mutation or alteration of specified gene or pathway in individual panels.

4. A. Calu-6 (KRAS Q61K)
B. HCT116 (KRAS G13D)
C. SKMel-30 (NRAS Q61K)
D. A375 (BRAF V600E)
E. WM (BRAF V600E )
F. HT-29 (BRAF V600E)
G. HCT116 (intermittent dosing)
Supplemental Figure S4. Body weight changes of rats bearing the indicated xenograft tumors treated with LY , abemaciclib or their combination from the same studies shown in Figure 3.

5. A. SK-Mel30
Ctrl
pan-RAFi
CDK4/6i
Combo
%G1= 40.02
%S = 45.85
%G2/M = 14.13
%Debris = 14.46
%G1= 55.16
%S = 32.91
%G2/M = 11.94
%Debris = 18.76
%G1= 62.38
%S = 23.34
%G2/M = 14.28
%Debris = 13.26
%G1= 74.94
%S = 11.85
%G2/M = 13.21
%Debris = 13.30
B. A375.
%G1= 67.07
%S = 25.49
%G2/M = 7.44
%Debris: 2.29
%G1= 71.75
%S = 19.57
%G2/M = 8.67
%Debris: 1.49
%G1= 75.73
%S = 14.80
%G2/M = 9.47
%Debris: 3.71
%G1= 91.31
%S = 2.09
%G2/M = 6.61
%Debris: 2.13
Supplementary Figure S5. Combinatory treatment targeting Raf and CDK4/6 enhances cell cycle G1 arrest, A, cell cycle analysis of SK-Mel-30 cells treated with DMSO, LY (0.01 µM), abemaciclib (0.5 µM), or the combination for 48 h. B, cell cycle analysis of A375 cells treated with DMSO, LY (0.01 µM), abemaciclib (1 µM), or the combination for 72 h. After treatment, cells were subjected to PI staining and subsequent flow cytometry analysis for cell cycle distribution. Dead cells are indicated as debris. Representative histograms were shown from at least two independent experiments. Also see Figure 4.

6. Supplementary Table S1: Mutational status of cancer cell lines tested in this study
Genetic mutation
Cancer
type
BRAF
KRAS
NRAS
TP53
CDKN2A
PIK3CA
Calu-6
lung WT
Q61K
R196*
HCT116
colon
G13D
del31
H1047R
HT-29
V600E
R273H
P449T
SKMel-30
skin
T284fs*21
P114L
A375
E61*, E69*, W66R
WM-266-4