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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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PIK3IP1 contributes to the observed synthetic lethality. (a) qRT-PCR analysis of PIK3IP1 mRNA in ARID1A mutated OVISE cells infected with lentivirus encoding the indicated shPIK3IP1s or controls. (n=3, # P=0.8149, *P<0.001). (b) Immunoblotting of PIK3IP1 and β-actin in the indicated OVISE cells. (c) Phase-contrast images of the indicated control or shPIK3IP1 (#3)-expressing OVISE cells treated with or without 5μM GSK126 for 12 days in 3D culture. (d) Quantification of (c). # P=0.628, *P<0.01. (e) Immunofluorescence staining for the apoptotic marker cleaved caspase 3 (green) in the acini formed by the indicated cells. Shown is shPIK3IP1 #3. Bars = 25 μm. (f) Quantification of (e). n=3, # P=0.642, * P<0.05. (g) Immunoblotting of phospho-AKT (p-AKT) and the indicated proteins in ARID1A mutated, PI3KCA wild type OVTOKO cells expressing a constitutively active myristoylated PI3KCA (I143V) mutant (Myr-PI3KCA) or controls. (h) Phase-contrast images of the indicated cells treated with or without 5μM GSK126 for 12 days in 3D culture. (i) Quantification of (h). # P>0.05 and * P<0.001. 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 5: PIK3IP1 contributes to the observed synthetic lethality. (a) qRT-PCR analysis of PIK3IP1 mRNA in ARID1A mutated OVISE cells infected with lentivirus encoding the indicated shPIK3IP1s or controls. (n=3, # P=0.8149, *P<0.001). (b) Immunoblotting of PIK3IP1 and β-actin in the indicated OVISE cells. (c) Phase-contrast images of the indicated control or shPIK3IP1 (#3)-expressing OVISE cells treated with or without 5μM GSK126 for 12 days in 3D culture. (d) Quantification of (c). # P=0.628, *P<0.01. (e) Immunofluorescence staining for the apoptotic marker cleaved caspase 3 (green) in the acini formed by the indicated cells. Shown is shPIK3IP1 #3. Bars = 25 μm. (f) Quantification of (e). n=3, # P=0.642, * P<0.05. (g) Immunoblotting of phospho-AKT (p-AKT) and the indicated proteins in ARID1A mutated, PI3KCA wild type OVTOKO cells expressing a constitutively active myristoylated PI3KCA (I143V) mutant (Myr-PI3KCA) or controls. (h) Phase-contrast images of the indicated cells treated with or without 5μM GSK126 for 12 days in 3D culture. (i) Quantification of (h). # P>0.05 and * P<0.001. 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)

PIK3IP1 contributes to the observed synthetic lethality. (a) qRT-PCR analysis of PIK3IP1 mRNA in ARID1A mutated OVISE cells infected with lentivirus encoding the indicated shPIK3IP1s or controls. (n=3, # P=0.8149, *P<0.001). (b) Immunoblotting of PIK3IP1 and β-actin in the indicated OVISE cells. (c) Phase-contrast images of the indicated control or shPIK3IP1 (#3)-expressing OVISE cells treated with or without 5μM GSK126 for 12 days in 3D culture. (d) Quantification of (c). # P=0.628, *P<0.01. (e) Immunofluorescence staining for the apoptotic marker cleaved caspase 3 (green) in the acini formed by the indicated cells. Shown is shPIK3IP1 #3. Bars = 25 μm. (f) Quantification of (e). n=3, # P=0.642, * P<0.05. (g) Immunoblotting of phospho-AKT (p-AKT) and the indicated proteins in ARID1A mutated, PI3KCA wild type OVTOKO cells expressing a constitutively active myristoylated PI3KCA (I143V) mutant (Myr-PI3KCA) or controls. (h) Phase-contrast images of the indicated cells treated with or without 5μM GSK126 for 12 days in 3D culture. (i) Quantification of (h). # P>0.05 and * P<0.001. 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 5: PIK3IP1 contributes to the observed synthetic lethality. (a) qRT-PCR analysis of PIK3IP1 mRNA in ARID1A mutated OVISE cells infected with lentivirus encoding the indicated shPIK3IP1s or controls. (n=3, # P=0.8149, *P<0.001). (b) Immunoblotting of PIK3IP1 and β-actin in the indicated OVISE cells. (c) Phase-contrast images of the indicated control or shPIK3IP1 (#3)-expressing OVISE cells treated with or without 5μM GSK126 for 12 days in 3D culture. (d) Quantification of (c). # P=0.628, *P<0.01. (e) Immunofluorescence staining for the apoptotic marker cleaved caspase 3 (green) in the acini formed by the indicated cells. Shown is shPIK3IP1 #3. Bars = 25 μm. (f) Quantification of (e). n=3, # P=0.642, * P<0.05. (g) Immunoblotting of phospho-AKT (p-AKT) and the indicated proteins in ARID1A mutated, PI3KCA wild type OVTOKO cells expressing a constitutively active myristoylated PI3KCA (I143V) mutant (Myr-PI3KCA) or controls. (h) Phase-contrast images of the indicated cells treated with or without 5μM GSK126 for 12 days in 3D culture. (i) Quantification of (h). # P>0.05 and * P<0.001. 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