1. Volume 20, Issue 2, Pages 427-438 (July 2017)
A Role for Mitochondrial Translation in Promotion of Viability in K-Ras Mutant Cells 
Timothy D. Martin, Danielle R. Cook, Mei Yuk Choi, Mamie Z. Li, Kevin M. Haigis, Stephen J. Elledge 
Cell Reports 
Volume 20, Issue 2, Pages (July 2017)
DOI: /j.celrep
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2. Cell Reports 2017 20, 427-438DOI: (10.1016/j.celrep.2017.06.061)
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3. Figure 1
Genome-wide CRISPR Screens in Isogenic Colorectal Tumor Cells Identifies Synthetic Lethal Interactions with Mutant K-Ras
(A) Schematic of the CRISPR screens performed. Abundance of gRNAs was compared between the initial (PD0) and final (PD10) time points. Genes whose gRNAs are selectively depleted in the K-Ras mutant cells are putative synthetic lethal hits.
(B) Fold changes of genes in the DLD1 screen. The average log2 fold change for each gene was calculated and plotted for both WT/− and WT/K-Ras mutant DLD1 cells. Labeled genes are a selection of potential synthetic lethal partners of mutant K-Ras.
(C) Synthetic lethal genes that scored in both the HCT116 and DLD1 screens.
(D) Average fold changes for the top 55 K-Ras synthetic lethal genes identified in the DLD1 screen.
(E) Gene ontology terms enriched in putative K-Ras synthetic lethal genes.
For screen sequencing data and analyses, see Tables S1, S2, S3, and S4.
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4. Figure 2
Mitochondrial Genes and Pathways Are Synthetic Lethal with Mutant K-Ras
(A) Pathways enriched among the mitochondrial genes that are putatively synthetically lethal.
(B) Mitochondrial genes identified in the DLD1 screen.
(C) Depletion of the mitochondrial ribosome is synthetic lethal with mutant K-Ras. Components of the mitoribosome from the DLD1 screen and their associated false discovery rate (FDR) corrected p values.
(D) Inhibition of mitochondrial translation is more toxic to K-Ras mutant cells. Isogenic DLD1 cells were treated with the mitochondrial translation inhibitor tigecycline, and proliferation was measured.
(E) Inhibition of complex I of the respiratory chain is more toxic to K-Ras mutant cells. Colony formation of isogenic HCT116 cells treated with the complex I inhibitor rotenone.
(F) Inhibition of either the mitoribosome or complex I reduces the anchorage-independent growth of K-Ras mutant cells. Soft agar colony formation was measured in K-Ras mutant cells treated with the indicated mitochondrial inhibitors.
For all panels, ∗p < All cellular growth and allograft data are expressed as the mean ± SEM.
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5. Figure 3
An shRNA Screen Targeting Essential Genes Identifies Additional Synthetic Lethal Partners with Mutant K-Ras
(A) shRNA, but not CRISPR-based, screens can identify essential genes as synthetic lethal partners. Essential genes are universally lost in CRISPR-based screens, while shRNA-based screens can identify whether the reduced function of essential genes can be synthetically lethal.
(B) An shRNA screen to identify essential genes that are synthetic lethal with mutant K-Ras in isogenic DLD1 cells. Heatmap showing the log2 fold change at PD3, PD7, and PD10 of essential genes that are putative synthetic lethal partners of mutant K-Ras.
(C) Essential genes that are synthetic lethal with mutant K-Ras. Labeled genes are more strongly depleted in K-Ras mutant cells.
(D) Knockdown of CCT1/TCP1 is synthetic lethal with mutant K-Ras. Colony formation of isogenic DLD1 cells expressing tet-inducible shRNAs for TCP1.
(E) Knockdown of SAMM50 is synthetic lethal with mutant K-Ras. Colony formation of isogenic DLD1 cells expressing constitutively expressed shRNAs for SAMM50.
For all panels, ∗p < All cellular growth and allograft data are expressed as the mean ± SEM. For screen sequencing data and analysis, see Table S6.
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6. Figure 4
CRISPR/Cas9-Mediated Genome Engineering of K-Ras Mutant Isogenic Immortalized Primary Cells
(A) Schematic of the KRAS locus and the targeting vector used to generate conditional K-Ras mutant alleles. A gRNA located near exon 2 and an AAV-delivered homology donor was used to generate LSL conditional HA-tagged K-Ras mutant cell lines.
(B) Cre treatment leads to K-Ras mutant expression and activation. K-Ras activation was measured by Raf-RBD pull-downs in isogenic cells treated with either control GFP or Cre-expressing adenovirus.
(C) Expression of mutant K-Ras leads to increased MAPK and PI3K signaling. LSL-K-Ras G12C AALE cells were treated with GFP or Cre-expressing adenovirus, and 7 days later, cell lysates were immunoblotted with the indicated antibodies to determine the activation of the PI3K and MAPK pathways, two effector pathways downstream of active K-Ras.
(D) Endogenous mutant K-Ras expression leads to cellular transformation. LSL-K-Ras G12C AALE cells from (C) were placed in soft agar, and colony growth was quantified 4 weeks later. ∗p < 0.05.
(E) K-Ras mutant expressing cells are more sensitive to complex I inhibition by rotenone. LSL-K-Ras G12C AALE cells from (C) were treated with rotenone to compare the sensitivity of K-Ras-mutant- and WT-expressing cells to oxidative phosphorylation inhibition. Cell viability was measured 2, 4, and 6 days after rotenone treatment.
(F) Mitoribosome inhibition by tigecycline is more toxic to K-Ras-mutant-expressing cells. LSL-K-Ras G12C AALE cells from (C) were treated with tigecycline to compare the sensitivity of K-Ras-mutant- and WT-expressing cells to mitochondrial translation inhibition. Cell viability was measured 2, 4, and 6 days after tigecycline treatment.
All cellular growth data are expressed as mean ± SEM.
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7. Figure 5
Knockout of Synthetic Lethal Genes or Inhibition of Complex I and the Mitoribosome Reduces K-Ras Mutant Cell Growth In Vivo
(A) Knockout of Ndufb10 reduces the proliferation of CT26 tumor cells. Cas9 and gRNAs targeting either control Rosa26 or Ndufb10 were expressed in CT26 cells and immunoblotted for the indicated proteins (above) and measured for proliferation (below). NT, non-treated.
(B) Mutation of Mrpl52 reduces the proliferation of CT26 tumor cells. Cas9 and gRNAs targeting either control Rosa26 or Mrpl52 were expressed in CT26 cells and immunoblotted for the indicated proteins (above) and measured for proliferation (below). NT, non-treated.
(C) Mutation of either Ndufb10 or Mrpl52 reduces the anchorage-independent growth of CT26 tumor cells in soft agar. Cells from (A) and (B) were tested for anchorage -independent growth in soft agar, and colony number was quantified.
(D) Mutation of either Ndufb10 or Mrpl52 reduces the in vivo tumor growth of CT26 tumor cells. CT26 cells expressing Cas9 and control Rosa26 gRNA or gRNAs targeting either Ndufb10 or Mrpl52 were subcutaneously injected as allografts in BALB/c mice, and tumor growth was measured over time.
(E) CT26 cells are sensitive to complex I inhibition by VLX-600 and mitoribosome inhibition by tigecycline. CT26 cells were treated with increasing concentrations of VLX-600 or tigecycline to determine sensitivity to oxidative phosphorylation or mitochondrial translation inhibition, respectively. Cell viability was measured 72 hr after drug treatment, and IC50 values for each compound were determined.
(F) Combined mitochondrial inhibition by VLX-600 and tigecycline reduces tumor growth. CT26 subcutaneous allografts were treated with the indicated inhibitors and concentrations, and tumor growth was monitored.
All cellular growth and allograft data are expressed as mean ± SEM.
Cell Reports  , DOI: ( /j.celrep )
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