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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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EZH2 inhibitor triggers apoptosis of ARID1A mutated cells. (a) qRT-PCR analysis of EZH2 untranslated region (UTR) and open reading frame (ORF) in TOV21G cells expressing a UTR targeting shEZH2 together with a wild type EZH2 or a SET domain deleted EZH2 mutant (EZH2 ΔSET). n=3, * P<0.01. (b) Immunoblotting of EZH2 and GAPDH in the indicated cells. (c) Images of acini formed in 3D culture for 12 days. Scale bars = 75 AU in NIH image J software. (d) Quantification of (c), *P<0.01 and #P>0.05. (e) Immunofluorescence staining of Ki67 (red), H3K27Me3 (green) and DAPI (blue) for acini formed by ARID1A mutated OVISE and TOV21G cells cultured in 3D treated with 5 μM GSK126 or vehicle control for 12 days. Scale bars = 25μM. Note the different scale bars in different panels due to growth suppression by GSK126 treatment. (f-g) Quantification of (e). 200 cells from each of the indicated groups were examined for expression of Ki67 and H3K27Me3. n=3, 4, 5 and 4, *P <0.01. (h) Same as (e), but stained for cleaved caspase 3 (green) and DAPI (blue) after 8 days of GSK126 treatment. Scale bars = 25μM. (i-j) Quantification of (h). 200 cells from each of the indicated groups were examined for cleaved caspase 3 positivity. n=6 and 5,* P <0.01. 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 3: EZH2 inhibitor triggers apoptosis of ARID1A mutated cells. (a) qRT-PCR analysis of EZH2 untranslated region (UTR) and open reading frame (ORF) in TOV21G cells expressing a UTR targeting shEZH2 together with a wild type EZH2 or a SET domain deleted EZH2 mutant (EZH2 ΔSET). n=3, * P<0.01. (b) Immunoblotting of EZH2 and GAPDH in the indicated cells. (c) Images of acini formed in 3D culture for 12 days. Scale bars = 75 AU in NIH image J software. (d) Quantification of (c), *P<0.01 and #P>0.05. (e) Immunofluorescence staining of Ki67 (red), H3K27Me3 (green) and DAPI (blue) for acini formed by ARID1A mutated OVISE and TOV21G cells cultured in 3D treated with 5 μM GSK126 or vehicle control for 12 days. Scale bars = 25μM. Note the different scale bars in different panels due to growth suppression by GSK126 treatment. (f-g) Quantification of (e). 200 cells from each of the indicated groups were examined for expression of Ki67 and H3K27Me3. n=3, 4, 5 and 4, *P <0.01. (h) Same as (e), but stained for cleaved caspase 3 (green) and DAPI (blue) after 8 days of GSK126 treatment. Scale bars = 25μM. (i-j) Quantification of (h). 200 cells from each of the indicated groups were examined for cleaved caspase 3 positivity. n=6 and 5,* P <0.01. 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: To establish that the observed effects are specifically due to inhibition of EZH2 activity, we knocked down EZH2 expression in ARID1A mutated cells in combination with GSK126 treatment. Knockdown of EZH2 mimicked the growth inhibition by GSK126 (Supplementary Fig. 2a-f). GSK126 did not significantly further affect the growth of EZH2 knockdown ARID1A mutated cells (Supplementary Fig. 2d-f). The observed growth inhibition by EZH2 knockdown was rescued by wild type EZH2 but not by an enzymatically inactive SET domain deleted EZH2 mutant (Fig. 3a-d) 7. This supports the notion that the observed growth inhibition depends upon EZH2 methyltransferase activity.


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)

EZH2 inhibitor triggers apoptosis of ARID1A mutated cells. (a) qRT-PCR analysis of EZH2 untranslated region (UTR) and open reading frame (ORF) in TOV21G cells expressing a UTR targeting shEZH2 together with a wild type EZH2 or a SET domain deleted EZH2 mutant (EZH2 ΔSET). n=3, * P<0.01. (b) Immunoblotting of EZH2 and GAPDH in the indicated cells. (c) Images of acini formed in 3D culture for 12 days. Scale bars = 75 AU in NIH image J software. (d) Quantification of (c), *P<0.01 and #P>0.05. (e) Immunofluorescence staining of Ki67 (red), H3K27Me3 (green) and DAPI (blue) for acini formed by ARID1A mutated OVISE and TOV21G cells cultured in 3D treated with 5 μM GSK126 or vehicle control for 12 days. Scale bars = 25μM. Note the different scale bars in different panels due to growth suppression by GSK126 treatment. (f-g) Quantification of (e). 200 cells from each of the indicated groups were examined for expression of Ki67 and H3K27Me3. n=3, 4, 5 and 4, *P <0.01. (h) Same as (e), but stained for cleaved caspase 3 (green) and DAPI (blue) after 8 days of GSK126 treatment. Scale bars = 25μM. (i-j) Quantification of (h). 200 cells from each of the indicated groups were examined for cleaved caspase 3 positivity. n=6 and 5,* P <0.01. 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 3: EZH2 inhibitor triggers apoptosis of ARID1A mutated cells. (a) qRT-PCR analysis of EZH2 untranslated region (UTR) and open reading frame (ORF) in TOV21G cells expressing a UTR targeting shEZH2 together with a wild type EZH2 or a SET domain deleted EZH2 mutant (EZH2 ΔSET). n=3, * P<0.01. (b) Immunoblotting of EZH2 and GAPDH in the indicated cells. (c) Images of acini formed in 3D culture for 12 days. Scale bars = 75 AU in NIH image J software. (d) Quantification of (c), *P<0.01 and #P>0.05. (e) Immunofluorescence staining of Ki67 (red), H3K27Me3 (green) and DAPI (blue) for acini formed by ARID1A mutated OVISE and TOV21G cells cultured in 3D treated with 5 μM GSK126 or vehicle control for 12 days. Scale bars = 25μM. Note the different scale bars in different panels due to growth suppression by GSK126 treatment. (f-g) Quantification of (e). 200 cells from each of the indicated groups were examined for expression of Ki67 and H3K27Me3. n=3, 4, 5 and 4, *P <0.01. (h) Same as (e), but stained for cleaved caspase 3 (green) and DAPI (blue) after 8 days of GSK126 treatment. Scale bars = 25μM. (i-j) Quantification of (h). 200 cells from each of the indicated groups were examined for cleaved caspase 3 positivity. n=6 and 5,* P <0.01. 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: To establish that the observed effects are specifically due to inhibition of EZH2 activity, we knocked down EZH2 expression in ARID1A mutated cells in combination with GSK126 treatment. Knockdown of EZH2 mimicked the growth inhibition by GSK126 (Supplementary Fig. 2a-f). GSK126 did not significantly further affect the growth of EZH2 knockdown ARID1A mutated cells (Supplementary Fig. 2d-f). The observed growth inhibition by EZH2 knockdown was rescued by wild type EZH2 but not by an enzymatically inactive SET domain deleted EZH2 mutant (Fig. 3a-d) 7. This supports the notion that the observed growth inhibition depends upon EZH2 methyltransferase activity.

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