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1.
Figure 7

Figure 7. A model for context specific KRAS dependency in colon cancers. From: TAK1 (MAP3K7) inhibition promotes apoptosis in KRAS-dependent colon cancers.

In KRAS-independent colon cancers, APC loss of function results in hyperactivation of canonical Wnt signaling through stabilization of β-catenin in cooperation with upstream Wnt activators. TAK1 can be a negative regulator of canonical Wnt signaling in these cells. In KRAS-dependent cells, oncogenic KRAS upregulates BMP-7 expression/secretion, activating the BMP receptor resulting in TAK1 activation. KRAS and TAK1 in these cells are activators of Wnt signaling by promoting β-catenin nuclear localization, which is stabilized by virtue of APC loss of function mutations. KRAS-mediated anti-apoptotic signaling could also be facilitated by NF-κB activation. Dashed lines represent unknown molecular interactions.
See also Figure S6.

Anurag Singh, et al. Cell. ;148(4):639-650.
2.
Figure 4

Figure 4. Associations between the KRAS dependency gene set, TAK1 dependence and KRAS-driven canonical Wnt signaling in colon cancer patients. From: TAK1 (MAP3K7) inhibition promotes apoptosis in KRAS-dependent colon cancers.

(A) Heat map representation of gene expression most correlated with TAK1 dependence from the KRAS dependency gene set across a panel of colon cancer cell lines of various genotypes. Cell lines are ordered by IC50 values for 5Z-7-oxozeaenol, leftmost being the highest and rightmost being the lowest. Clustering of genes was performed with Euclidean distance as a similarity metric. Values are presented as log2 median-centered intensities. Genes highlighted in orange text are putative or bona fide TCF4 target genes.
(B) Basal normalized TCF4 luciferase reporter activity (TOP-FLASH) in nominal units for a panel of KRAS-independent and KRAS-dependent colon cancer cell lines. Data are represented as the means of 3 independent experiments +/- SEM.
(C) Average expression of non-TCF4 or TCF4 target genes depicted in Figure 4A in colon cancer patients genotyped as either APC mutant/KRAS-wild-type (red circles) or APC mutant plus KRAS mutant (green squares). P-values represent a comparison of mean expression scores of genes for each class.
See also Figure S3.

Anurag Singh, et al. Cell. ;148(4):639-650.
3.
Figure 1

Figure 1. Classification of KRAS mutant colon cancer cells into KRAS-independent and KRAS-dependent groups. From: TAK1 (MAP3K7) inhibition promotes apoptosis in KRAS-dependent colon cancers.

(A) Representative 6-day 96-well viability assays in 4 KRAS mutant colon cancer cell lines transduced with either control or 2 independent KRAS-directed lentiviral shRNAs (A and B), at 2 viral MOIs. Cell lines in red text are KRAS-independent, and those in green text are KRAS-dependent. Quantitation and transformation of relative cell density values yields the Ras Dependency Index depicted in Fig. 1B.
(B) Ras Dependency Index plot for a panel of 21 KRAS mutant colon cancer cell lines. Dashed line represents the “Dependency Threshold” of 2.0. Data are presented as the mean of three independent experiments +/- SEM.
(C) KRAS protein depletion 4 days post-infection with KRAS-directed shRNAs and effects on apoptosis, as assessed by caspase-3 and PARP cleavage, in a representative panel of KRAS-dependent versus-KRAS independent cell lines. Lanes 1, 2 and 3 are as in panel A. Data is representative of two independent experiments.
(D) Activating phosphorylations of the Erk (p-Erk1/2) and Akt (p-Akt) kinases, following KRAS depletion in SW837 KRAS-independent versus SW620 2 KRAS-dependent cells, 4 days post-infection with 3 different viral titres (MOIs of 1, 2 and 4) of shKRAS-B. Total protein levels (t-Erk1 and and t-Akt) are shown as gel loading controls. Note: different exposure times were used for the individual panels. Data is representative of two independent experiments.

Anurag Singh, et al. Cell. ;148(4):639-650.
4.
Figure 3

Figure 3. Validation of MAP3K7/TAK1 as a pro-survival mediator in KRAS-dependent colon cancers. From: TAK1 (MAP3K7) inhibition promotes apoptosis in KRAS-dependent colon cancers.

(A) IC50 values (μM) for effects on cellular proliferation and viability with the TAK1 kinase inhibitor 5Z-7-oxozeaenol in a panel of colon cancer cell lines that have been genotyped as KRAS mutant (KRAS-independent – red circles or KRAS-dependent – green squares), BRAF mutant (blue triangles) or wild-type for both KRAS and BRAF (OTHER – grey diamonds). Effects on growth were measured 3 days post-treatment. Data are represented as the mean of 3 independent experiments and error bars indicate the median ± interquartile range. *denotes p<0.00001; n.s. – not significant.
(B) Effects of TAK1 inhibition on apoptosis and signaling in a representative panel of KRAS-independent and KRAS-dependent cell lines, 24h after treatment. PARP and caspase-3 cleavage are shown as indicators of apoptosis, and AMPK threonine 172 (T172) phosphorylation is shown as a downstream indicator of TAK1 signaling activity. GAPDH serves as a gel loading control.
(C) TAK1 inhibition in mice with xenografted human tumors derived from the HCT8/SW837 (KRAS-independent) and SK-CO-1/SW620 (KRAS-dependent) cell lines. Cells expressing firefly luciferase were injected subcutaneously into the flanks of nude mice. Tumors are shown as imaged by IVIS detection of luminescence counts (in photons/sec) following 14 days of tumor growth followed by 6 days of treatment with either 15mg/kg 5z-7-oxozeaenol or vehicle (5% DMSO in arachis oil), IP delivery q.d. Quantitation of tumor volume (mm3) is also shown. Tumor volume data are represented as the mean of 4 tumors in 2 mice for each group +/- SEM.
See also Figure S2 and Table S2.

Anurag Singh, et al. Cell. ;148(4):639-650.
5.
Figure 5

Figure 5. KRAS and TAK1 regulate canonical Wnt signaling in KRAS-dependent cancer cells. From: TAK1 (MAP3K7) inhibition promotes apoptosis in KRAS-dependent colon cancers.

(A) TOP-FLASH luciferase reporter activity as a function of lentiviral shRNA-mediated KRAS depletion at increasing MOIs in LS174T/SW1463 (KRAS-independent) versus SW620/SK-CO-1 (KRAS-dependent) cells. Cell lines were transduced to stably express luciferase under the control of TCF4 response elements. Reporter activity is plotted relative to shGFP (vector) expressing cells. Data are represented as the mean of triplicate experiments +/- SEM.
(B) TOP-FLASH activity in KRAS-independent and KRAS-dependent cell lines following TAK1 inhibition with increasing concentrations of 5Z-7-oxo (μM). Data are represented as means of triplicate experiments ± SEM.
(C) Protein expression levels of the endogenous Wnt target gene Axin 2 following treatment of cells with the indicated concentrations of 5Z-7-oxo. GAPDH serves as a loading control.
(D) Laser confocal micrographs of SW1116 KRAS-dependent cells treated with either DMSO vehicle or 5μM 5Z-7-oxo for 24h. E-cadherin localization is shown in the red channel, β-catenin in green and DAPI-stained nuclei are in blue. Scale bar = 20μM.
(E) Forced overexpression of epitope-tagged oncogenic G12V mutated RAS protein isoforms in HT29 cells and sensitivity to TAK1 pharmacological inhibition with 5Z-7-oxozeaenol. Expression levels of exogenous and endogenous Ras proteins are shown by immunoblotting with a pan-ras monoclonal antibody. NRAS/KRAS4B are HA-tagged and KRAS4A is V5-tagged.
(F) Overexpression of mutant KRAS(12V) followed by TAK1 inhibition in HT29 cells and effects on TOP-FLASH reporter activity. Data are presented as the means of three independent experiments +/- SEM.
(G) Overexpression of KRAS(12V) in HT29 cells and effects on TAK1 and Erk phosphorylation (p-TAK1/p-Erk) as well as Axin 2 levels. Total TAK1 and Erk1 serve as loading controls.
(H) Confocal micrographs showing E-cadherin or KRAS (red) and β-catenin (green) localization in vector control or oncogenic HA tagged KRAS-4B(12V) expressing HT29 cells. KRAS expression is visualized using an HA polyclonal antibody. Scale bar = 25μm.
See also Figure S4.

Anurag Singh, et al. Cell. ;148(4):639-650.
6.
Figure 2

Figure 2. Analysis of kinases from a “KRAS dependency signature” in colon cancer cell lines. From: TAK1 (MAP3K7) inhibition promotes apoptosis in KRAS-dependent colon cancers.

(A) Schematic representation of the methodology used to derive a colon cancer KRAS dependency gene expression data set. Gene expression microarray data for 4 indicated KRAS-independent versus KRAS-dependent cell lines were analyzed for significantly underexpressed (IND) or overexpressed (DEP) genes by student T-test analysis (two-tailed, homoscedastic) followed by selection of probe sets whose average expression was 2-fold higher or lower, yielding 687 IND genes and 832 DEP genes.
(B) Hierarchical clustering of gene expression for 47 DEP “druggable” protein, lipid or other ATP-dependent kinase genes or kinase regulatory genes. Heat map shows log2 median-centered intensity values and similarly expressed genes are clustered using Euclidean distance as a similarity metric. MAP3K7 (encoding TAK1) is highlighted with an asterisk.
(C) Protein expression levels of indicated kinases in a panel of KRAS-independent and KRAS-dependent cell lines. GAPDH serves as a loading control.
(D) Depletion of DEP kinase genes in SW620 versus SW837 cells. Each colored bar represents an individual shRNA sequence per gene, with the same color-coding as in Figure S1F. Fold growth inhibition per shRNA per kinase was computed by dividing the relative cell density of SW837 by that of SW620 cells and using a weighted average to account for viral titre. The plot shows cumulative log2 fold growth inhibition for each shRNA per kinase; i.e., a value of 1 on the plot indicates a 2-fold greater growth inhibitory effect for a given shRNA in SW620 compared to SW837 cells. The log2 fold growth inhibition for each individual shRNA was then cumulated for each kinase gene. Data are represented as the mean value corresponding to each shRNA from three independent experiments.
(E) Knockdown of TAK1 with increasing viral titres of shTAK1-D encoding lentiviruses (MOI) and associated apoptotic effects assessed by PARP cleavage. GAPDH serves as a loading control. Data are representative of two independent experiments.
See also Figure S1 and Table S1.

Anurag Singh, et al. Cell. ;148(4):639-650.
7.
Figure 6

Figure 6. Oncogenic KRAS regulates a BMP-7/BMPR1A signaling. From: TAK1 (MAP3K7) inhibition promotes apoptosis in KRAS-dependent colon cancers.

(A) Depletion of KRAS in two KRAS-independent (LS-174T and SW837) and two KRAS-dependent cell lines (SW620 and SK-CO-1) and subsequent effects on expression of BMP-7 as well as downstream effects on Smad1/TAK1 phosphorylation (p-Smad1/p-TAK1). The 20kD secreted form of BMP7 is shown. Phospho-TAK1 represents the TAK1 autophosphorylation site and is a measure of TAK1 activity. Total Smad1/5/8 and total TAK1 (t-Smad1/5/8/t-TAK1) proteins are shown as gel loading controls. Data are representative of two independent experiments.
(B) Effects of BMP7 depletion on proliferation and viability of SW620 KRAS-dependent cells. Plot shows cell density 6 days post-infection with either shGFP control or 5 different BMP7-directed lentiviral shRNAs. Data are represented as the mean of three independent experiments ± SEM. Western blots on the right panel show BMP-7 levels and apoptotic effects as measured by PARP and Caspase3 cleavage following BMP-7 depletion with two independent lentiviral shRNAs (D and E).
(C) Effects on BMP7 protein and transcript levels following induced activation of ER-KRAS(12V) fusion protein with various doses of 4-HT in HT29 cells. Left panel shows levels of total and secreted BMP-7 following ER-KRAS(12V) induction with 4-HT. Levels of Axin 2 and phosphorylated Erk (p-Erk1/2) are also shown following ER-KRAS(12V). Total Erk (t-Erk1) serves as a loading control.
(D) TOP-FLASH reporter activity following 4-HT induced activation of ER-KRAS(12V) and depletion of the indicated genes via lentiviral shRNA delivery at various viral titres. Reporter activity is shown relative to shGFP control.
(E) Introduction of a V5-tagged constitutively activated (CA) mutant of the BMP receptor, BMPR1A (Q233D) or control vector in HT29 cells and effects on 5Z-7-oxozeaenol sensitivity in terms of IC50 values.
(F) Signaling and apoptotic effects of TAK1 inhibition using 5Z-7-oxozeaenol at the indicated concentrations 24h post-treatment in BMPR1A-CA expressing cells. Caspase3 and PARP cleavage are indicators of apoptotic cell death. Axin 2 levels are shown as a readout of Wnt signaling. Phosphorylated smad1/5/8 levels serve as a readout of BMP signaling. GAPDH serves as a gel loading control. BMPR1A-CA expression is visualized using a monoclonal V5 antibody.
(G) Effects of BMPR1A-CA expression on β-catenin localization (red) in HT-29 cells following treatment with 5μM 5Z-7-oxozeaenol or vehicle control for 24h, as assessed by immunofluorescence confocal microscopy. DAPI-stained nuclei are shown in blue. Scale bar = 10μM.
See also Figure S5.

Anurag Singh, et al. Cell. ;148(4):639-650.

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