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A chemical-genetic screen reveals a mechanism of resistance to PI3K inhibitors in cancer.

Muellner MK, Uras IZ, Gapp BV, Kerzendorfer C, Smida M, Lechtermann H, Craig-Mueller N, Colinge J, Duernberger G, Nijman SM - Nat. Chem. Biol. (2011)

Bottom Line: However, there has been no systematic approach for analyzing gene-drug interactions in human cells.Applying this approach to breast cancer revealed various synthetic-lethal interactions and drug-resistance mechanisms, some of which were known, thereby validating the method.These data reveal a new mechanism of resistance to PI3K inhibitors with direct clinical implications.

View Article: PubMed Central - PubMed

Affiliation: CeMM-Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.

ABSTRACT
Linking the molecular aberrations of cancer to drug responses could guide treatment choice and identify new therapeutic applications. However, there has been no systematic approach for analyzing gene-drug interactions in human cells. Here we establish a multiplexed assay to study the cellular fitness of a panel of engineered isogenic cancer cells in response to a collection of drugs, enabling the systematic analysis of thousands of gene-drug interactions. Applying this approach to breast cancer revealed various synthetic-lethal interactions and drug-resistance mechanisms, some of which were known, thereby validating the method. NOTCH pathway activation, which occurs frequently in breast cancer, unexpectedly conferred resistance to phosphoinositide 3-kinase (PI3K) inhibitors, which are currently undergoing clinical trials in breast cancer patients. NOTCH1 and downstream induction of c-MYC over-rode the dependency of cells on the PI3K-mTOR pathway for proliferation. These data reveal a new mechanism of resistance to PI3K inhibitors with direct clinical implications.

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c-MYC induction confers resistance to PI3K/mTOR inhibition(a) Western blot analysis of ICN1 or control MCF10A cells treated with BEZ-235 (pg/μl) as indicated for 24 hours. Total lysates were probed with an antibody against phosphorylated ribosomal S6 kinase (Thr371) and total mTOR as a loading control (see Supplementary Fig. 20 for an uncropped version). (b) Data from the screen shows c-MYC as a significant hit for resistance to BEZ-235. (c) Relative c-MYC mRNA levels in ICN1 and c-MYC cells as determined by qRT-PCR. Shown is the fold change compared to wild-type MCF10A cells and standard deviations of 3 replicates. (d) Dose-response curve of c-MYC or control MCF10A cells treated with BEZ-235. Cells were treated for 5 days as indicated and relative cell number was measured. The data represent four independent experiments were performed in triplicate and error bars indicate standard deviations. (e) Quantitative RT-PCR of c-MYC expression in wild-type MCF10A or ICN1 cells transfected with Luciferase siRNA and ICN1 cells transfected with c-MYC siRNA pool (ICN1+si). Standard deviations of 3 replicates are indicated. (f) Dose-response curve of cells in (e) treated with BEZ-235 or vehicle for 5 days. Three replicates were performed; error bars indicate standard deviation. (g) Oncomine analysis (see Methods) of c-MYC gene copy number in PI3K/mTOR inhibitor sensitive or resistant cell lines. The red-boxed area indicates cell lines with c-MYC gene amplification (Chi square P value is indicated).
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Figure 4: c-MYC induction confers resistance to PI3K/mTOR inhibition(a) Western blot analysis of ICN1 or control MCF10A cells treated with BEZ-235 (pg/μl) as indicated for 24 hours. Total lysates were probed with an antibody against phosphorylated ribosomal S6 kinase (Thr371) and total mTOR as a loading control (see Supplementary Fig. 20 for an uncropped version). (b) Data from the screen shows c-MYC as a significant hit for resistance to BEZ-235. (c) Relative c-MYC mRNA levels in ICN1 and c-MYC cells as determined by qRT-PCR. Shown is the fold change compared to wild-type MCF10A cells and standard deviations of 3 replicates. (d) Dose-response curve of c-MYC or control MCF10A cells treated with BEZ-235. Cells were treated for 5 days as indicated and relative cell number was measured. The data represent four independent experiments were performed in triplicate and error bars indicate standard deviations. (e) Quantitative RT-PCR of c-MYC expression in wild-type MCF10A or ICN1 cells transfected with Luciferase siRNA and ICN1 cells transfected with c-MYC siRNA pool (ICN1+si). Standard deviations of 3 replicates are indicated. (f) Dose-response curve of cells in (e) treated with BEZ-235 or vehicle for 5 days. Three replicates were performed; error bars indicate standard deviation. (g) Oncomine analysis (see Methods) of c-MYC gene copy number in PI3K/mTOR inhibitor sensitive or resistant cell lines. The red-boxed area indicates cell lines with c-MYC gene amplification (Chi square P value is indicated).

Mentions: Ribosomal S6 Kinase (S6K) and the eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) are main effector molecules of mTORC1 and their phosphorylation stimulates protein translation 29. Interestingly, S6K and 4EBP1 phosphorylation was equally inhibited in ICN1 expressing cells as in control cells (Fig. 4a, Supplementary Fig. 21). This suggests that ICN1 uncouples mTORC1 signaling from proliferation by a downstream mechanism.


A chemical-genetic screen reveals a mechanism of resistance to PI3K inhibitors in cancer.

Muellner MK, Uras IZ, Gapp BV, Kerzendorfer C, Smida M, Lechtermann H, Craig-Mueller N, Colinge J, Duernberger G, Nijman SM - Nat. Chem. Biol. (2011)

c-MYC induction confers resistance to PI3K/mTOR inhibition(a) Western blot analysis of ICN1 or control MCF10A cells treated with BEZ-235 (pg/μl) as indicated for 24 hours. Total lysates were probed with an antibody against phosphorylated ribosomal S6 kinase (Thr371) and total mTOR as a loading control (see Supplementary Fig. 20 for an uncropped version). (b) Data from the screen shows c-MYC as a significant hit for resistance to BEZ-235. (c) Relative c-MYC mRNA levels in ICN1 and c-MYC cells as determined by qRT-PCR. Shown is the fold change compared to wild-type MCF10A cells and standard deviations of 3 replicates. (d) Dose-response curve of c-MYC or control MCF10A cells treated with BEZ-235. Cells were treated for 5 days as indicated and relative cell number was measured. The data represent four independent experiments were performed in triplicate and error bars indicate standard deviations. (e) Quantitative RT-PCR of c-MYC expression in wild-type MCF10A or ICN1 cells transfected with Luciferase siRNA and ICN1 cells transfected with c-MYC siRNA pool (ICN1+si). Standard deviations of 3 replicates are indicated. (f) Dose-response curve of cells in (e) treated with BEZ-235 or vehicle for 5 days. Three replicates were performed; error bars indicate standard deviation. (g) Oncomine analysis (see Methods) of c-MYC gene copy number in PI3K/mTOR inhibitor sensitive or resistant cell lines. The red-boxed area indicates cell lines with c-MYC gene amplification (Chi square P value is indicated).
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Figure 4: c-MYC induction confers resistance to PI3K/mTOR inhibition(a) Western blot analysis of ICN1 or control MCF10A cells treated with BEZ-235 (pg/μl) as indicated for 24 hours. Total lysates were probed with an antibody against phosphorylated ribosomal S6 kinase (Thr371) and total mTOR as a loading control (see Supplementary Fig. 20 for an uncropped version). (b) Data from the screen shows c-MYC as a significant hit for resistance to BEZ-235. (c) Relative c-MYC mRNA levels in ICN1 and c-MYC cells as determined by qRT-PCR. Shown is the fold change compared to wild-type MCF10A cells and standard deviations of 3 replicates. (d) Dose-response curve of c-MYC or control MCF10A cells treated with BEZ-235. Cells were treated for 5 days as indicated and relative cell number was measured. The data represent four independent experiments were performed in triplicate and error bars indicate standard deviations. (e) Quantitative RT-PCR of c-MYC expression in wild-type MCF10A or ICN1 cells transfected with Luciferase siRNA and ICN1 cells transfected with c-MYC siRNA pool (ICN1+si). Standard deviations of 3 replicates are indicated. (f) Dose-response curve of cells in (e) treated with BEZ-235 or vehicle for 5 days. Three replicates were performed; error bars indicate standard deviation. (g) Oncomine analysis (see Methods) of c-MYC gene copy number in PI3K/mTOR inhibitor sensitive or resistant cell lines. The red-boxed area indicates cell lines with c-MYC gene amplification (Chi square P value is indicated).
Mentions: Ribosomal S6 Kinase (S6K) and the eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) are main effector molecules of mTORC1 and their phosphorylation stimulates protein translation 29. Interestingly, S6K and 4EBP1 phosphorylation was equally inhibited in ICN1 expressing cells as in control cells (Fig. 4a, Supplementary Fig. 21). This suggests that ICN1 uncouples mTORC1 signaling from proliferation by a downstream mechanism.

Bottom Line: However, there has been no systematic approach for analyzing gene-drug interactions in human cells.Applying this approach to breast cancer revealed various synthetic-lethal interactions and drug-resistance mechanisms, some of which were known, thereby validating the method.These data reveal a new mechanism of resistance to PI3K inhibitors with direct clinical implications.

View Article: PubMed Central - PubMed

Affiliation: CeMM-Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.

ABSTRACT
Linking the molecular aberrations of cancer to drug responses could guide treatment choice and identify new therapeutic applications. However, there has been no systematic approach for analyzing gene-drug interactions in human cells. Here we establish a multiplexed assay to study the cellular fitness of a panel of engineered isogenic cancer cells in response to a collection of drugs, enabling the systematic analysis of thousands of gene-drug interactions. Applying this approach to breast cancer revealed various synthetic-lethal interactions and drug-resistance mechanisms, some of which were known, thereby validating the method. NOTCH pathway activation, which occurs frequently in breast cancer, unexpectedly conferred resistance to phosphoinositide 3-kinase (PI3K) inhibitors, which are currently undergoing clinical trials in breast cancer patients. NOTCH1 and downstream induction of c-MYC over-rode the dependency of cells on the PI3K-mTOR pathway for proliferation. These data reveal a new mechanism of resistance to PI3K inhibitors with direct clinical implications.

Show MeSH
Related in: MedlinePlus