Supplementary Figure 1 A APC B TP53 50 50 N/S N/S 40 40 30 30 20 20 10 10 Doubling time (h), SRB (h), Doublingtime Doubling time (h), SRB Doubling time n= 830n= n= 16n= 26 0 0 wt mut wt mut KRAS BRAF C 50 D 50 N/S 40 40 N/S 30 30 20 20 10 10 Doubling time (h), SRB Doubling time (h), SRB (h), time Doubling n= 19n= 29 n= 39n= 7 0 0 wt mut wt mut SMAD4 E F 60 N/S 40 20 27 12 (h), SRB time Doubling n= 20n= 5 0 wt mut G TCF7L2 H CTNNB1 60 50 N/S N/S 40 40 30 20 20 10 Doubling time (h),SRB n= 19n= 6 Doubling (h), SRB time n= 19n= 6 0 0 wt mut wt mut Supplementary Figure 1: Cell growth as a function of the mutational status of the most frequently mutated genes in colorectal tumors. The average doubling time of cell lines that are either wild type or mutant for APC (A), TP53 (B), KRAS (C), BRAF (D), PIK3CA (E), SMAD4 (F), TCF7L2 (G)andCTNNB1 (H) was calculated to assess the possible effects on tumor growth of the most frequent mutations observed in colorectal tumors. N/S: Student’s T‐test p>0.05. Supplementary Figure 2 A B 20000 GAPDH TYMS 4000 15000 3000 10000 2000 5000 1554696_s_at 1000 Pearson r =0.459 Pearson r =0.457 P value =0.024 AFFX-HUMGAPDH/M33197_5_at 0 P value =0.037 Relative expression (microarray) 0 0 20 40 60 80 100 Relative expression (microarray) expression Relative 0 20 40 60 80 100 Relative expression (qPCR) Relative expression (qPCR) C D PPOX CALCOCO2 80 80 60 60 40 40 238117_at 238560_at 20 20 Pearson r =0.590 Pearson r =0.772 P value =0.004 P value =0.025 0 0 Relative expression (microarray) 0 20 40 60 80 100 (microarray) Relative expression 0 100 200 300 400 500 Relative expression (qPCR) Relative expression (qPCR) E F CBX5 SMAD4 400 300 300 200 200 231862_at 202526_at 100 100 Pearson r =0.873 Pearson r =0.898 P value =0.015 0 P value =0.001 0 Relative expression (microarray) 0 100 200 300 400 500 Relative expression (microarray) 0 100 200 300 Relative expression (qPCR) Relative expression (qPCR) Supplementary Figure 2: Independent validation of mRNA expression levels. To independently validate the quantification of mRNA expression of the microarray experiments, the expression of GAPDH (A), TYMS (B), PPOX (C), CALCOCO2 (D), CBX5 (E)andSMAD4(F) was assessed by Real‐Time RT‐PCR. The Pearson’s correlation coefficient (r) and p value are shown. Supplementary Figure 3 A 6 Pearson r=0.54 5 p=0.0007 4 3 2 Percent mitotic cells Percent mitotic 1 0 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 Tumor PPOX mRNA levels B 6 Pearson r=0.34 5 p=0.04 4 3 2 Percent mitotic cells Percent mitotic 1 0 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 Tumor GAPDH mRNA levels Supplementary Figure 3: Correlation between PPOX or GAPDH expression and rates of proliferation in primary tumors. The correlation between the mRNA levels of PPOX (A)andGAPDH(B) expression in a cohort of 36 primary colorectal tumors (TCGA) and their proliferation rates (percentage of mitotic cells) is shown. The Pearson’s correlation coefficient (r) and p value are shown. Supplementary Figure 4 30 A Pearson r=0.027 p value=0.897 25 M) μ 20 15 10 GI50 5-FU ( 5 0 0 20 40 60 80 Doubling time (h) B 20 Pearson r=0.287 M) μ P value=0.321 15 10 5 GI50 NaGI50 iodoacetate ( 0 0 20 40 60 80 Doubling time (h) C 2000 Pearson r=-0.178 P value=0.561 M) μ 1500 1000 500 GI50 Acifluorfen ( Acifluorfen GI50 0 0 20 40 60 80 Doubling time (h) Supplementary Figure 4: Correlation between drug sensitivity and proliferation rates of colon cancer cells. The doubling time of colon cancer cell lines is plotted against the sensitivity (GI50) of these cell lines to the TYMS inhibitor 5‐FU (A), the GAPDH inhibitor Na iodoacetate (B) or the PPOX inhibitor acifluorfen (C). The Pearson’s correlation coefficient (r) and p value are shown. Supplementary Figure 5 ABHCT116 DLD1 HCT116 DLD1 100 100 ** 50 50 ** ** Colony formation (%) formation Colony Colony formation(%) ** 0 0 0 1200 01200 015 015 Acifluorfen (µM) Na iodoacetate (µM) CD Acifluorfen Na iodoactetate 100 100 0 μM 0 μM 400 μM 10 μM 80 800 μM 80 20 μM 1200μM 30 μM 60 60 40 40 20 20 Percentage apoptosis Percentage apoptosis 0 0 DLD1 RW2982 HCT116 T84 LIM2405 DLD1 HCT116 SW403 EF Supplementary Figure 5: Effects of acifluorfen and Na iodoacetate on the clonogenic potential of colon cancer cells and induction of apoptosis. (A‐B) The number of macroscopically visible colonies after treatment of HCT116 and DLD1 cells with acifluorfen (A)andNaiodoacetate(B)wasassessed2‐3 weeks after 9h treatment with the indicated concentrations. Three independent experiments were carried out in triplicate and the average percentage (±SEM) relative to untreated controls is shown. (C‐D) The percentage of apoptotic cells after treatment of different colon cancer cell lines with the indicated concentrations of acifluorfen (C) or Na iodoacetate (D) was assessed by quantification of the number of cells with a sub‐diploid content of DNA as determined by propidium iodide staining and flow cytometry. The average (±SEM) of three independent experiments each in triplicate is shown. (E‐F)The indicated colon cancer cell lines were treated with acifluorfen (E) or Na iodoacetate (F) for 24h, and the presence of cleaved poly ADP ribose polymerase (PARP) was assessed by Western blotting. Supplementary Figure 6 A AcifluorfenB Na iodoacetate C DLD1 D RW2982 GHLIM2405 DLD1 100 100 100 100 75 75 75 75 50 50 50 50 25 25 Percent of cells Percent of cells 25 25 Percent of cells Percentcells of 0 0 0 0 0 400 800 1200 0 400 800 1200 0 102030 0 102030 Acifluorfen (μM) Acifluorfen (μM) Na iodoacetate (μM) Na iodoacetate (μM) F IJHCT116 E HCT116 T84 100 SW403 100 100 100 75 75 75 75 50 50 50 50 25 25 25 Percent cells of Percent of cells Percent of cells 25 Percent cells of 0 0 G0/G1 0 0 10 20 30 0 0 400 800 1200 0 400 800 1200 S 0102030 Acifluorfen (μM) Acifluorfen (μM) G2/M Na iodoacetate (μM) Na iodoacetate (μM) Supplementary Figure 6: Effects of acifluorfen or Na iodoacetate treatment on the cell cycle of colon cancer cells. The effects of acifluorfen (A)andNaiodoacetate(B) on the distribution of cells in the different phases of the cell cycle were assessed by propidium iodide and flow cytometry analysis. The results of a representative experiment are shownin(A‐B). The number of cells in the G0/G1, G2/M and S‐phase of the cell cycle (DNA content 2n, 4n and 2<n<4, respectively) after acifluorfen (C‐F) or Na iodoacetate (G‐J) in the indicated cell lines was quantified and the average (±SEM) of three independent experiments each carried out in triplicate is shown. Supplementary Figure 7 AB Oxadiazon CGP 3466B maleate 150 100 100 50 50 0 0 -50 -50 DLD1 (GI50=450.9μM) DLD1 (GI50=212.3μM) HCT116 (GI50=996.9μM) -100 HCT116 (GI50=190.1μM) -100 HCC2998 (GI50=890.1μM) HCC2998 (GI50=142.6μM) -150 Growth inhibition (% of (% control) Growth inhibition Growth of inhibition (% control) 1 2 3 4 0 1 2 3 4 Oxadiazon (Log μM) CGP 3466B maleate (Log μM) HCT116 HCT116 CD1.5 1.5 1.0 1.0 * 0.5 0.5 * Relative expression Relative expression 0.0 0.0 EFRNAi NT PPOX RNAi NT GAPDH 6 HCT116 HCT116 2.0×10 2.5×10 5 5 6 2.0×10 1.5×10 * * 1.5×10 5 1.0×10 6 1.0×10 5 Cell number Cell 5.0×10 5 number Cell 5.0×10 4 0 0 RNAi NT PPOX RNAi NT GAPDH Supplementary Figure 7: Effects of PPOX and GAPDH inhibition on the growth of colon cancer cells.(A‐B) Treatment with the PPOX inhibitor oxadiazon (A) or the GAPDH inhibitor CGP 3466B maleate (B) caused a dose‐ dependent reduction in the growth of colon cancer cells, as determined by sulforhodamine B staining. (C‐D) Transfection of RNAi oligos targeting PPOX (C)orGAPDH(D) resulted in a reduction in the levels of mRNA expression of these genes compared to untreated controls or cells transfected with a non‐target (NT) RNAi (all 10nM) as determined by qPCR. (E‐F) The number of cells 72h after control non‐target (NT), PPOX (E)orGAPDH(F)RNAi transfection was directly counted. The average (±SEM) of three independent experiments each in triplicate is shown. *Student’s T‐test p<0.05. Supplementary Figure 8 HT29 cells RKO cells 2000 2000 PBS PBS Na iodoacetate Na iodoacetate ) ) 3 3 1500 Acifluorfen 1500 Acifluorfen 1000 1000 500 500 Tumor size (mm Tumor size (mm Tumor 0 0 0 10 20 30 40 0 10 20 30 40 Time post cell injection (days) Time post cell injection (days) T84 cells Isreco1 cells 1500 500 PBS PBS Aciflorfen Acifluorfen * ) ) 3 * 3 * 400 * * * * * * * 1000 * * 300 * * * * * * * * * * * * 200 500 * 100 Tumor size (mm Tumor size Tumor (mm 0 0 0 10 20 30 40 0 10 20 30 Time post cell injection (days) Time post cell injection (days) HCC2998 cells 800 PBS Acifluorfen ) 3 600 400 200 Tumor size (mm 0 0 10 20 30 40 Time post cell injection (days) Supplementary Figure 8: Effects of GAPDH and PPOX inhibition on tumor growth using a xenograft model.