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Evidence that synthetic lethality underlies the mutual exclusivity of oncogenic KRAS and EGFR mutations in lung adenocarcinoma.

Unni AM, Lockwood WW, Zejahu K, Lee-Lin SQ, Varmus H - Elife (2015)

Bottom Line: In transgenic mice programmed to express both mutant oncogenes in the lung epithelium, the resulting tumors express only one oncogene.Activation of ERK, p38 and JNK in the dying cells suggests that an overly active MAPK signaling pathway may mediate the phenotype.Together, our findings indicate that mutual exclusivity of oncogenic mutations may reveal unexpected vulnerabilities and therapeutic possibilities.

View Article: PubMed Central - PubMed

Affiliation: Cancer Biology Section, Cancer Genetics Branch, National Human Genome Research Institute, Bethesda, United States.

ABSTRACT
Human lung adenocarcinomas (LUAD) contain mutations in EGFR in ∼15% of cases and in KRAS in ∼30%, yet no individual adenocarcinoma appears to carry activating mutations in both genes, a finding we have confirmed by re-analysis of data from over 600 LUAD. Here we provide evidence that co-occurrence of mutations in these two genes is deleterious. In transgenic mice programmed to express both mutant oncogenes in the lung epithelium, the resulting tumors express only one oncogene. We also show that forced expression of a second oncogene in human cancer cell lines with an endogenous mutated oncogene is deleterious. The most prominent features accompanying loss of cell viability were vacuolization, other changes in cell morphology, and increased macropinocytosis. Activation of ERK, p38 and JNK in the dying cells suggests that an overly active MAPK signaling pathway may mediate the phenotype. Together, our findings indicate that mutual exclusivity of oncogenic mutations may reveal unexpected vulnerabilities and therapeutic possibilities.

No MeSH data available.


Related in: MedlinePlus

Co-expression of mutant KRAS and EGFR increases MAP kinase (MAPK) signaling.(A) Modified PC9 and H358 lung adenocarcinoma cells (see Figure 3) were cultured in the presence or absence of doxycycline (Dox) for 24 hr and analyzed for global gene expression changes using Affymetrix microarrays (see ‘Materials and methods’ for details). Microarray probes differentially expressed in each TetO cell line upon the addition of Dox were identified (corrected p < 0.01, compared to control without Dox), and those genes specifically induced by Dox in either TetO-KRAS or TetO-EGFR cells and not the TetO-GFP control cells were determined. The Venn diagram indicates the resulting number of gene probes identified in each cell line, including the 152 unique probes specifically modulated by expression of mutant KRAS and EGFR in both PC9 and H358 cells. (B) Gene Set Enrichment Analysis (GSEA) of the genes specifically regulated upon mutant KRAS and EGFR co-expression in both PC9 and H358 lung adenocarcinoma cells identified three oncogenic signatures that were significantly upregulated (FDR q-value <0.01) upon co-expression; the top two are indicative of KRAS signaling (see ‘Materials and methods’ and Supplementary file 5). The displayed enrichment plot is for the most significant gene set (q-value = 0, Normalized Enrichment Score = 2.29) demonstrating enrichment for genes related to the upregulation of mutant KRAS. (C) Ingenuity Pathway Analysis (IPA) of the KRAS + EGFR induced gene set was performed and the top ten significantly regulated canonical pathways in which these genes are involved are displayed (see ‘Materials and methods’). P38 MAPK signaling was identified as the most significant upregulated pathway from this analysis; ERK/MAPK signaling was the second. (D) A highly simplified diagram of the EGFR/RAS signaling pathway is illustrated; the components assessed by western blot highlighted in blue. (E) Increased MAPK signaling in cells co-expressing mutant oncogenes. The indicated cells were cultured for 3 days with or without Dox; lysates were assayed by western blotting for the indicated proteins and phospho-proteins. Where relevant, the phosphorylated Tyrosine (Y) or Threonine (T) residue being measured is shown. Data are representative of three independent experiments.DOI:http://dx.doi.org/10.7554/eLife.06907.008
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fig6: Co-expression of mutant KRAS and EGFR increases MAP kinase (MAPK) signaling.(A) Modified PC9 and H358 lung adenocarcinoma cells (see Figure 3) were cultured in the presence or absence of doxycycline (Dox) for 24 hr and analyzed for global gene expression changes using Affymetrix microarrays (see ‘Materials and methods’ for details). Microarray probes differentially expressed in each TetO cell line upon the addition of Dox were identified (corrected p < 0.01, compared to control without Dox), and those genes specifically induced by Dox in either TetO-KRAS or TetO-EGFR cells and not the TetO-GFP control cells were determined. The Venn diagram indicates the resulting number of gene probes identified in each cell line, including the 152 unique probes specifically modulated by expression of mutant KRAS and EGFR in both PC9 and H358 cells. (B) Gene Set Enrichment Analysis (GSEA) of the genes specifically regulated upon mutant KRAS and EGFR co-expression in both PC9 and H358 lung adenocarcinoma cells identified three oncogenic signatures that were significantly upregulated (FDR q-value <0.01) upon co-expression; the top two are indicative of KRAS signaling (see ‘Materials and methods’ and Supplementary file 5). The displayed enrichment plot is for the most significant gene set (q-value = 0, Normalized Enrichment Score = 2.29) demonstrating enrichment for genes related to the upregulation of mutant KRAS. (C) Ingenuity Pathway Analysis (IPA) of the KRAS + EGFR induced gene set was performed and the top ten significantly regulated canonical pathways in which these genes are involved are displayed (see ‘Materials and methods’). P38 MAPK signaling was identified as the most significant upregulated pathway from this analysis; ERK/MAPK signaling was the second. (D) A highly simplified diagram of the EGFR/RAS signaling pathway is illustrated; the components assessed by western blot highlighted in blue. (E) Increased MAPK signaling in cells co-expressing mutant oncogenes. The indicated cells were cultured for 3 days with or without Dox; lysates were assayed by western blotting for the indicated proteins and phospho-proteins. Where relevant, the phosphorylated Tyrosine (Y) or Threonine (T) residue being measured is shown. Data are representative of three independent experiments.DOI:http://dx.doi.org/10.7554/eLife.06907.008

Mentions: To identify signaling pathways that may be responsible for the decreased cell viability and morphological changes induced by co-expression of mutant KRAS and EGFR, we generated gene expression profiles of the PC9 and H358 lung adenocarcinoma cells engineered to conditionally express mutant KRAS or EGFR. 24 hr after addition of Dox to PC9-TetO-KRASG12V and H358-TetO-EGFRL858R cells or to control lines with TetO-GFP we harvested RNA. Gene expression profiles were then compared to profiles from untreated cells. The 24 hr time point was chosen to measure changes in cell signaling likely to be caused directly by induction of the co-expressed oncogene rather than by reactive changes that could be attributed to secondary events occurring at later time points. Genes differentially expressed in cells treated or not treated with Dox were identified for each cell line. Those genes showing significant differences (ANOVA Corrected p < 0.01, compared to reciprocal no Dox control) were examined in similar tests with cells induced to express TetO-GFP to determine those specifically affected by co-expression of the two oncogenes (‘Materials and methods’). In total, 152 probe sets corresponding to 144 unique genes detected differential expression in both H358 and PC9 cells in response to mutant EGFR and KRAS (Figure 6A, Supplementary file 3).10.7554/eLife.06907.008Figure 6.Co-expression of mutant KRAS and EGFR increases MAP kinase (MAPK) signaling.


Evidence that synthetic lethality underlies the mutual exclusivity of oncogenic KRAS and EGFR mutations in lung adenocarcinoma.

Unni AM, Lockwood WW, Zejahu K, Lee-Lin SQ, Varmus H - Elife (2015)

Co-expression of mutant KRAS and EGFR increases MAP kinase (MAPK) signaling.(A) Modified PC9 and H358 lung adenocarcinoma cells (see Figure 3) were cultured in the presence or absence of doxycycline (Dox) for 24 hr and analyzed for global gene expression changes using Affymetrix microarrays (see ‘Materials and methods’ for details). Microarray probes differentially expressed in each TetO cell line upon the addition of Dox were identified (corrected p < 0.01, compared to control without Dox), and those genes specifically induced by Dox in either TetO-KRAS or TetO-EGFR cells and not the TetO-GFP control cells were determined. The Venn diagram indicates the resulting number of gene probes identified in each cell line, including the 152 unique probes specifically modulated by expression of mutant KRAS and EGFR in both PC9 and H358 cells. (B) Gene Set Enrichment Analysis (GSEA) of the genes specifically regulated upon mutant KRAS and EGFR co-expression in both PC9 and H358 lung adenocarcinoma cells identified three oncogenic signatures that were significantly upregulated (FDR q-value <0.01) upon co-expression; the top two are indicative of KRAS signaling (see ‘Materials and methods’ and Supplementary file 5). The displayed enrichment plot is for the most significant gene set (q-value = 0, Normalized Enrichment Score = 2.29) demonstrating enrichment for genes related to the upregulation of mutant KRAS. (C) Ingenuity Pathway Analysis (IPA) of the KRAS + EGFR induced gene set was performed and the top ten significantly regulated canonical pathways in which these genes are involved are displayed (see ‘Materials and methods’). P38 MAPK signaling was identified as the most significant upregulated pathway from this analysis; ERK/MAPK signaling was the second. (D) A highly simplified diagram of the EGFR/RAS signaling pathway is illustrated; the components assessed by western blot highlighted in blue. (E) Increased MAPK signaling in cells co-expressing mutant oncogenes. The indicated cells were cultured for 3 days with or without Dox; lysates were assayed by western blotting for the indicated proteins and phospho-proteins. Where relevant, the phosphorylated Tyrosine (Y) or Threonine (T) residue being measured is shown. Data are representative of three independent experiments.DOI:http://dx.doi.org/10.7554/eLife.06907.008
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fig6: Co-expression of mutant KRAS and EGFR increases MAP kinase (MAPK) signaling.(A) Modified PC9 and H358 lung adenocarcinoma cells (see Figure 3) were cultured in the presence or absence of doxycycline (Dox) for 24 hr and analyzed for global gene expression changes using Affymetrix microarrays (see ‘Materials and methods’ for details). Microarray probes differentially expressed in each TetO cell line upon the addition of Dox were identified (corrected p < 0.01, compared to control without Dox), and those genes specifically induced by Dox in either TetO-KRAS or TetO-EGFR cells and not the TetO-GFP control cells were determined. The Venn diagram indicates the resulting number of gene probes identified in each cell line, including the 152 unique probes specifically modulated by expression of mutant KRAS and EGFR in both PC9 and H358 cells. (B) Gene Set Enrichment Analysis (GSEA) of the genes specifically regulated upon mutant KRAS and EGFR co-expression in both PC9 and H358 lung adenocarcinoma cells identified three oncogenic signatures that were significantly upregulated (FDR q-value <0.01) upon co-expression; the top two are indicative of KRAS signaling (see ‘Materials and methods’ and Supplementary file 5). The displayed enrichment plot is for the most significant gene set (q-value = 0, Normalized Enrichment Score = 2.29) demonstrating enrichment for genes related to the upregulation of mutant KRAS. (C) Ingenuity Pathway Analysis (IPA) of the KRAS + EGFR induced gene set was performed and the top ten significantly regulated canonical pathways in which these genes are involved are displayed (see ‘Materials and methods’). P38 MAPK signaling was identified as the most significant upregulated pathway from this analysis; ERK/MAPK signaling was the second. (D) A highly simplified diagram of the EGFR/RAS signaling pathway is illustrated; the components assessed by western blot highlighted in blue. (E) Increased MAPK signaling in cells co-expressing mutant oncogenes. The indicated cells were cultured for 3 days with or without Dox; lysates were assayed by western blotting for the indicated proteins and phospho-proteins. Where relevant, the phosphorylated Tyrosine (Y) or Threonine (T) residue being measured is shown. Data are representative of three independent experiments.DOI:http://dx.doi.org/10.7554/eLife.06907.008
Mentions: To identify signaling pathways that may be responsible for the decreased cell viability and morphological changes induced by co-expression of mutant KRAS and EGFR, we generated gene expression profiles of the PC9 and H358 lung adenocarcinoma cells engineered to conditionally express mutant KRAS or EGFR. 24 hr after addition of Dox to PC9-TetO-KRASG12V and H358-TetO-EGFRL858R cells or to control lines with TetO-GFP we harvested RNA. Gene expression profiles were then compared to profiles from untreated cells. The 24 hr time point was chosen to measure changes in cell signaling likely to be caused directly by induction of the co-expressed oncogene rather than by reactive changes that could be attributed to secondary events occurring at later time points. Genes differentially expressed in cells treated or not treated with Dox were identified for each cell line. Those genes showing significant differences (ANOVA Corrected p < 0.01, compared to reciprocal no Dox control) were examined in similar tests with cells induced to express TetO-GFP to determine those specifically affected by co-expression of the two oncogenes (‘Materials and methods’). In total, 152 probe sets corresponding to 144 unique genes detected differential expression in both H358 and PC9 cells in response to mutant EGFR and KRAS (Figure 6A, Supplementary file 3).10.7554/eLife.06907.008Figure 6.Co-expression of mutant KRAS and EGFR increases MAP kinase (MAPK) signaling.

Bottom Line: In transgenic mice programmed to express both mutant oncogenes in the lung epithelium, the resulting tumors express only one oncogene.Activation of ERK, p38 and JNK in the dying cells suggests that an overly active MAPK signaling pathway may mediate the phenotype.Together, our findings indicate that mutual exclusivity of oncogenic mutations may reveal unexpected vulnerabilities and therapeutic possibilities.

View Article: PubMed Central - PubMed

Affiliation: Cancer Biology Section, Cancer Genetics Branch, National Human Genome Research Institute, Bethesda, United States.

ABSTRACT
Human lung adenocarcinomas (LUAD) contain mutations in EGFR in ∼15% of cases and in KRAS in ∼30%, yet no individual adenocarcinoma appears to carry activating mutations in both genes, a finding we have confirmed by re-analysis of data from over 600 LUAD. Here we provide evidence that co-occurrence of mutations in these two genes is deleterious. In transgenic mice programmed to express both mutant oncogenes in the lung epithelium, the resulting tumors express only one oncogene. We also show that forced expression of a second oncogene in human cancer cell lines with an endogenous mutated oncogene is deleterious. The most prominent features accompanying loss of cell viability were vacuolization, other changes in cell morphology, and increased macropinocytosis. Activation of ERK, p38 and JNK in the dying cells suggests that an overly active MAPK signaling pathway may mediate the phenotype. Together, our findings indicate that mutual exclusivity of oncogenic mutations may reveal unexpected vulnerabilities and therapeutic possibilities.

No MeSH data available.


Related in: MedlinePlus