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Synthetic lethality by targeting EZH2 methyltransferase activity in ARID1A-mutated cancers.

Bitler BG, Aird KM, Garipov A, Li H, Amatangelo M, Kossenkov AV, Schultz DC, Liu Q, Shih IeM, Conejo-Garcia JR, Speicher DW, Zhang R - Nat. Med. (2015)

Bottom Line: Here we show that inhibition of the EZH2 methyltransferase acts in a synthetic lethal manner in ARID1A-mutated ovarian cancer cells and that ARID1A mutational status correlated with response to the EZH2 inhibitor.We identified PIK3IP1 as a direct target of ARID1A and EZH2 that is upregulated by EZH2 inhibition and contributed to the observed synthetic lethality by inhibiting PI3K-AKT signaling.To our knowledge, this is the first data set to demonstrate a synthetic lethality between ARID1A mutation and EZH2 inhibition.

View Article: PubMed Central - PubMed

Affiliation: Gene Expression and Regulation Program, The Wistar Institute, Philadelphia, Pennsylvania, USA.

ABSTRACT
The gene encoding ARID1A, a chromatin remodeler, shows one of the highest mutation rates across many cancer types. Notably, ARID1A is mutated in over 50% of ovarian clear cell carcinomas, which currently have no effective therapy. To date, clinically applicable targeted cancer therapy based on ARID1A mutational status has not been described. Here we show that inhibition of the EZH2 methyltransferase acts in a synthetic lethal manner in ARID1A-mutated ovarian cancer cells and that ARID1A mutational status correlated with response to the EZH2 inhibitor. We identified PIK3IP1 as a direct target of ARID1A and EZH2 that is upregulated by EZH2 inhibition and contributed to the observed synthetic lethality by inhibiting PI3K-AKT signaling. Importantly, EZH2 inhibition caused regression of ARID1A-mutated ovarian tumors in vivo. To our knowledge, this is the first data set to demonstrate a synthetic lethality between ARID1A mutation and EZH2 inhibition. Our data indicate that pharmacological inhibition of EZH2 represents a novel treatment strategy for cancers involving ARID1A mutations.

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Response to the EZH2 inhibitor is dependent on ARID1A status. (a) Immunoblotting of ARID1A, EZH2, H3K27Me3 and loading control β-actin in the indicated cell lines. ARID1A mutation status is indicated as mutated (M) or wild type (W). (b-c) Immunoblotting of the indicated proteins following treatment with GSK126 for 72 hours. (d) Immunoblotting of the indicated proteins in RMG1 cells expressing shARID1A or control treated with or without 5 μM GSK126. Images of acini formed and the diameter of acini was measured. * P<0.0001. (e) Quantification of the diameter of acini formed by the indicated cells with or without 5 μM GSK126 treatment in 3D culture for 12 days. # P=0.914, * P<0.0001. ARID1A mutation status is indicated above the graph. (f) Quantification of cell numbers for the ARID1A mutated OVISE cells. n=6, * P<0.0001. (g) Immunoblotting of the indicated proteins in ARID1A mutated OVISE and TOV21G cells with or without wild type ARID1A restoration treated with or without 5 μM GSK126. (h) Acini formation was examined after 12 days in 3D culture and the diameter of acini was measured. # P>0.05, * P<0.0001. (i) Dose response curves of ARID1A mutated TOV21G cells with or without wild type ARID1A restoration treated with the indicated dose of GSK126 for 12 days in 3D cultures. Scale bars = 75 AU in NIH Image J software. Number of acini (n) is indicated on the graphs as the representative of three experimental repeats. Error bars represent s.e.m. P calculated with two-tailed t test.
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Figure 2: Response to the EZH2 inhibitor is dependent on ARID1A status. (a) Immunoblotting of ARID1A, EZH2, H3K27Me3 and loading control β-actin in the indicated cell lines. ARID1A mutation status is indicated as mutated (M) or wild type (W). (b-c) Immunoblotting of the indicated proteins following treatment with GSK126 for 72 hours. (d) Immunoblotting of the indicated proteins in RMG1 cells expressing shARID1A or control treated with or without 5 μM GSK126. Images of acini formed and the diameter of acini was measured. * P<0.0001. (e) Quantification of the diameter of acini formed by the indicated cells with or without 5 μM GSK126 treatment in 3D culture for 12 days. # P=0.914, * P<0.0001. ARID1A mutation status is indicated above the graph. (f) Quantification of cell numbers for the ARID1A mutated OVISE cells. n=6, * P<0.0001. (g) Immunoblotting of the indicated proteins in ARID1A mutated OVISE and TOV21G cells with or without wild type ARID1A restoration treated with or without 5 μM GSK126. (h) Acini formation was examined after 12 days in 3D culture and the diameter of acini was measured. # P>0.05, * P<0.0001. (i) Dose response curves of ARID1A mutated TOV21G cells with or without wild type ARID1A restoration treated with the indicated dose of GSK126 for 12 days in 3D cultures. Scale bars = 75 AU in NIH Image J software. Number of acini (n) is indicated on the graphs as the representative of three experimental repeats. Error bars represent s.e.m. P calculated with two-tailed t test.

Mentions: B.G.B designed and performed all the experiments and analyzed data and wrote the manuscript. K.M.A. contributed to Fig. 5g-i and manuscript writing. A.G. contributed to Fig. 2b,c, H.L. contributed to Supplementary Fig. 4g. M.A. contributed to Supplementary Fig. 2c, f. A.V.K. performed the analysis presented in Fig. 4a,c, D.C.S. contributed to the epigenetic set construction. Q.L. contributed to statistical design and analysis. I.M.S. contributed key reagents. J.R.C. and D.W.S. participated in experimental design. R.Z. conceived the study and wrote the manuscript.


Synthetic lethality by targeting EZH2 methyltransferase activity in ARID1A-mutated cancers.

Bitler BG, Aird KM, Garipov A, Li H, Amatangelo M, Kossenkov AV, Schultz DC, Liu Q, Shih IeM, Conejo-Garcia JR, Speicher DW, Zhang R - Nat. Med. (2015)

Response to the EZH2 inhibitor is dependent on ARID1A status. (a) Immunoblotting of ARID1A, EZH2, H3K27Me3 and loading control β-actin in the indicated cell lines. ARID1A mutation status is indicated as mutated (M) or wild type (W). (b-c) Immunoblotting of the indicated proteins following treatment with GSK126 for 72 hours. (d) Immunoblotting of the indicated proteins in RMG1 cells expressing shARID1A or control treated with or without 5 μM GSK126. Images of acini formed and the diameter of acini was measured. * P<0.0001. (e) Quantification of the diameter of acini formed by the indicated cells with or without 5 μM GSK126 treatment in 3D culture for 12 days. # P=0.914, * P<0.0001. ARID1A mutation status is indicated above the graph. (f) Quantification of cell numbers for the ARID1A mutated OVISE cells. n=6, * P<0.0001. (g) Immunoblotting of the indicated proteins in ARID1A mutated OVISE and TOV21G cells with or without wild type ARID1A restoration treated with or without 5 μM GSK126. (h) Acini formation was examined after 12 days in 3D culture and the diameter of acini was measured. # P>0.05, * P<0.0001. (i) Dose response curves of ARID1A mutated TOV21G cells with or without wild type ARID1A restoration treated with the indicated dose of GSK126 for 12 days in 3D cultures. Scale bars = 75 AU in NIH Image J software. Number of acini (n) is indicated on the graphs as the representative of three experimental repeats. Error bars represent s.e.m. P calculated with two-tailed t test.
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Figure 2: Response to the EZH2 inhibitor is dependent on ARID1A status. (a) Immunoblotting of ARID1A, EZH2, H3K27Me3 and loading control β-actin in the indicated cell lines. ARID1A mutation status is indicated as mutated (M) or wild type (W). (b-c) Immunoblotting of the indicated proteins following treatment with GSK126 for 72 hours. (d) Immunoblotting of the indicated proteins in RMG1 cells expressing shARID1A or control treated with or without 5 μM GSK126. Images of acini formed and the diameter of acini was measured. * P<0.0001. (e) Quantification of the diameter of acini formed by the indicated cells with or without 5 μM GSK126 treatment in 3D culture for 12 days. # P=0.914, * P<0.0001. ARID1A mutation status is indicated above the graph. (f) Quantification of cell numbers for the ARID1A mutated OVISE cells. n=6, * P<0.0001. (g) Immunoblotting of the indicated proteins in ARID1A mutated OVISE and TOV21G cells with or without wild type ARID1A restoration treated with or without 5 μM GSK126. (h) Acini formation was examined after 12 days in 3D culture and the diameter of acini was measured. # P>0.05, * P<0.0001. (i) Dose response curves of ARID1A mutated TOV21G cells with or without wild type ARID1A restoration treated with the indicated dose of GSK126 for 12 days in 3D cultures. Scale bars = 75 AU in NIH Image J software. Number of acini (n) is indicated on the graphs as the representative of three experimental repeats. Error bars represent s.e.m. P calculated with two-tailed t test.
Mentions: B.G.B designed and performed all the experiments and analyzed data and wrote the manuscript. K.M.A. contributed to Fig. 5g-i and manuscript writing. A.G. contributed to Fig. 2b,c, H.L. contributed to Supplementary Fig. 4g. M.A. contributed to Supplementary Fig. 2c, f. A.V.K. performed the analysis presented in Fig. 4a,c, D.C.S. contributed to the epigenetic set construction. Q.L. contributed to statistical design and analysis. I.M.S. contributed key reagents. J.R.C. and D.W.S. participated in experimental design. R.Z. conceived the study and wrote the manuscript.

Bottom Line: Here we show that inhibition of the EZH2 methyltransferase acts in a synthetic lethal manner in ARID1A-mutated ovarian cancer cells and that ARID1A mutational status correlated with response to the EZH2 inhibitor.We identified PIK3IP1 as a direct target of ARID1A and EZH2 that is upregulated by EZH2 inhibition and contributed to the observed synthetic lethality by inhibiting PI3K-AKT signaling.To our knowledge, this is the first data set to demonstrate a synthetic lethality between ARID1A mutation and EZH2 inhibition.

View Article: PubMed Central - PubMed

Affiliation: Gene Expression and Regulation Program, The Wistar Institute, Philadelphia, Pennsylvania, USA.

ABSTRACT
The gene encoding ARID1A, a chromatin remodeler, shows one of the highest mutation rates across many cancer types. Notably, ARID1A is mutated in over 50% of ovarian clear cell carcinomas, which currently have no effective therapy. To date, clinically applicable targeted cancer therapy based on ARID1A mutational status has not been described. Here we show that inhibition of the EZH2 methyltransferase acts in a synthetic lethal manner in ARID1A-mutated ovarian cancer cells and that ARID1A mutational status correlated with response to the EZH2 inhibitor. We identified PIK3IP1 as a direct target of ARID1A and EZH2 that is upregulated by EZH2 inhibition and contributed to the observed synthetic lethality by inhibiting PI3K-AKT signaling. Importantly, EZH2 inhibition caused regression of ARID1A-mutated ovarian tumors in vivo. To our knowledge, this is the first data set to demonstrate a synthetic lethality between ARID1A mutation and EZH2 inhibition. Our data indicate that pharmacological inhibition of EZH2 represents a novel treatment strategy for cancers involving ARID1A mutations.

Show MeSH
Related in: MedlinePlus