Limits...
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.

Show MeSH

Related in: MedlinePlus

GSK126, an EZH2 inhibitor, is selective against ARID1A knockdown cells compared with controls. (a) Flow-diagram of the evaluation for a panel of epigenetic inhibitors. ARID1A wild type OCCC RMG1 cells were transduced with lentivirus encoding a shARID1A or control. Following drug selection, cells were plated onto Matrigel and treated with 15 individual small molecules and vehicle control using IC50 concentrations as detailed in Supplementary Table 2. (b) Immunoblotting of ARID1A, EZH2, H3K27Me3 and loading control β-actin in the indicated cells. (c) Quantification of the average acini diameter (each symbol represents a small molecule) graphed as a scatter plot. The x-axis indicates the acini size formed by control ARID1A wild type treated cells, while the y-axis indicates the acini size formed by the same small molecule treated shARID1A-expressing RMG1 cells. * P <0.0001 calculated with two-tailed t test using GraphPad Prism 5 software. Number of acini (n) for each of the small molecules used for analysis is listed in Supplementary Table 1. Error bars represent s.e.m. (d) Representative images of acini from indicated small molecules. Scale Bars = 75 of measurable units (AU) using the NIH Image J software. GSK126 (100nM) represents screening hit that selectively inhibits the growth of ARID1A knockdown cells compared with controls. ITF2357 and AR-42 represent small molecule inhibitors that showed no significant effects in growth inhibition and significantly suppressed cell growth regardless of ARID1A status, respectively.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4352133&req=5

Figure 1: GSK126, an EZH2 inhibitor, is selective against ARID1A knockdown cells compared with controls. (a) Flow-diagram of the evaluation for a panel of epigenetic inhibitors. ARID1A wild type OCCC RMG1 cells were transduced with lentivirus encoding a shARID1A or control. Following drug selection, cells were plated onto Matrigel and treated with 15 individual small molecules and vehicle control using IC50 concentrations as detailed in Supplementary Table 2. (b) Immunoblotting of ARID1A, EZH2, H3K27Me3 and loading control β-actin in the indicated cells. (c) Quantification of the average acini diameter (each symbol represents a small molecule) graphed as a scatter plot. The x-axis indicates the acini size formed by control ARID1A wild type treated cells, while the y-axis indicates the acini size formed by the same small molecule treated shARID1A-expressing RMG1 cells. * P <0.0001 calculated with two-tailed t test using GraphPad Prism 5 software. Number of acini (n) for each of the small molecules used for analysis is listed in Supplementary Table 1. Error bars represent s.e.m. (d) Representative images of acini from indicated small molecules. Scale Bars = 75 of measurable units (AU) using the NIH Image J software. GSK126 (100nM) represents screening hit that selectively inhibits the growth of ARID1A knockdown cells compared with controls. ITF2357 and AR-42 represent small molecule inhibitors that showed no significant effects in growth inhibition and significantly suppressed cell growth regardless of ARID1A status, respectively.

Mentions: Since epigenetic mechanisms may play a critical role in ARID1A mutated OCCC, we evaluated a panel of 15 commercially available small molecule inhibitors known to target epigenetic regulators to identify “hits” that selectively inhibit the growth of ARID1A inactivated cells (Supplementary Table 1). Over 90% of the ARID1A mutations observed in OCCC are frame-shift or nonsense mutations that result in loss of ARID1A protein expression 4,5,12. To mimic loss of ARID1A protein expression caused by the vast majority of ARID1A mutations 4 and ensure the same genetic background, we performed the screen using ARID1A wild type OCCC RMG1 cells with or without shRNA-mediated ARID1A knockdown (Fig. 1a,b and Supplementary Fig. 1a). We performed the screen in 3 dimensional (3D) cultures using Matrigel to more closely mimic the tumor microenvironment 13. Notably, ARID1A knockdown itself did not significantly affect the growth of RMG1 cells in 3D culture (Supplementary Fig. 1b). We used the doses of each small molecule based on their previously established IC50 concentrations (Supplementary Table 2). Diameters of acini formed in 3D culture were measured as a surrogate for cell growth (Fig. 1c). We identified three small molecule inhibitors that significantly and selectively inhibited the growth of ARID1A knockdown cells compared to controls (Supplementary Table 1). GSK126 was the hit with the highest selectivity against ARID1A knockdown cells (Fig. 1c,d and Supplementary Table 1). We observed a decrease in acini size by GSK126 using two individual shARID1As (Supplementary Fig. 1c-e). GSK126 is a highly selective and potent small molecule inhibitor of EZH2 methyltransferase activity 9. Notably, ARID1A knockdown did not alter the expression levels of EZH2 or H3K27Me3 (Fig. 1b).


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)

GSK126, an EZH2 inhibitor, is selective against ARID1A knockdown cells compared with controls. (a) Flow-diagram of the evaluation for a panel of epigenetic inhibitors. ARID1A wild type OCCC RMG1 cells were transduced with lentivirus encoding a shARID1A or control. Following drug selection, cells were plated onto Matrigel and treated with 15 individual small molecules and vehicle control using IC50 concentrations as detailed in Supplementary Table 2. (b) Immunoblotting of ARID1A, EZH2, H3K27Me3 and loading control β-actin in the indicated cells. (c) Quantification of the average acini diameter (each symbol represents a small molecule) graphed as a scatter plot. The x-axis indicates the acini size formed by control ARID1A wild type treated cells, while the y-axis indicates the acini size formed by the same small molecule treated shARID1A-expressing RMG1 cells. * P <0.0001 calculated with two-tailed t test using GraphPad Prism 5 software. Number of acini (n) for each of the small molecules used for analysis is listed in Supplementary Table 1. Error bars represent s.e.m. (d) Representative images of acini from indicated small molecules. Scale Bars = 75 of measurable units (AU) using the NIH Image J software. GSK126 (100nM) represents screening hit that selectively inhibits the growth of ARID1A knockdown cells compared with controls. ITF2357 and AR-42 represent small molecule inhibitors that showed no significant effects in growth inhibition and significantly suppressed cell growth regardless of ARID1A status, respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4352133&req=5

Figure 1: GSK126, an EZH2 inhibitor, is selective against ARID1A knockdown cells compared with controls. (a) Flow-diagram of the evaluation for a panel of epigenetic inhibitors. ARID1A wild type OCCC RMG1 cells were transduced with lentivirus encoding a shARID1A or control. Following drug selection, cells were plated onto Matrigel and treated with 15 individual small molecules and vehicle control using IC50 concentrations as detailed in Supplementary Table 2. (b) Immunoblotting of ARID1A, EZH2, H3K27Me3 and loading control β-actin in the indicated cells. (c) Quantification of the average acini diameter (each symbol represents a small molecule) graphed as a scatter plot. The x-axis indicates the acini size formed by control ARID1A wild type treated cells, while the y-axis indicates the acini size formed by the same small molecule treated shARID1A-expressing RMG1 cells. * P <0.0001 calculated with two-tailed t test using GraphPad Prism 5 software. Number of acini (n) for each of the small molecules used for analysis is listed in Supplementary Table 1. Error bars represent s.e.m. (d) Representative images of acini from indicated small molecules. Scale Bars = 75 of measurable units (AU) using the NIH Image J software. GSK126 (100nM) represents screening hit that selectively inhibits the growth of ARID1A knockdown cells compared with controls. ITF2357 and AR-42 represent small molecule inhibitors that showed no significant effects in growth inhibition and significantly suppressed cell growth regardless of ARID1A status, respectively.
Mentions: Since epigenetic mechanisms may play a critical role in ARID1A mutated OCCC, we evaluated a panel of 15 commercially available small molecule inhibitors known to target epigenetic regulators to identify “hits” that selectively inhibit the growth of ARID1A inactivated cells (Supplementary Table 1). Over 90% of the ARID1A mutations observed in OCCC are frame-shift or nonsense mutations that result in loss of ARID1A protein expression 4,5,12. To mimic loss of ARID1A protein expression caused by the vast majority of ARID1A mutations 4 and ensure the same genetic background, we performed the screen using ARID1A wild type OCCC RMG1 cells with or without shRNA-mediated ARID1A knockdown (Fig. 1a,b and Supplementary Fig. 1a). We performed the screen in 3 dimensional (3D) cultures using Matrigel to more closely mimic the tumor microenvironment 13. Notably, ARID1A knockdown itself did not significantly affect the growth of RMG1 cells in 3D culture (Supplementary Fig. 1b). We used the doses of each small molecule based on their previously established IC50 concentrations (Supplementary Table 2). Diameters of acini formed in 3D culture were measured as a surrogate for cell growth (Fig. 1c). We identified three small molecule inhibitors that significantly and selectively inhibited the growth of ARID1A knockdown cells compared to controls (Supplementary Table 1). GSK126 was the hit with the highest selectivity against ARID1A knockdown cells (Fig. 1c,d and Supplementary Table 1). We observed a decrease in acini size by GSK126 using two individual shARID1As (Supplementary Fig. 1c-e). GSK126 is a highly selective and potent small molecule inhibitor of EZH2 methyltransferase activity 9. Notably, ARID1A knockdown did not alter the expression levels of EZH2 or H3K27Me3 (Fig. 1b).

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