Introduction:
Next generation sequencing of bladder cancer has revolutionized our understanding of the disease and promises to move the field towards better risk stratification, therapeutic target identification, and more personalized therapies for patients. Specifically, genes involved in epigenetic modifications have been shown to be frequently mutated in bladder cancer. In fact, over 90% of NMIBC harbor inactivating mutations in at least one chromatin modifying enzyme including ARID1A, KDM6A, and KMT2C/D. Among these, AT Rich Interactive Domain 1A (ARID1A), a member of the SWI/SNF chromatin modifying complex, harbors truncating/inactivating mutations in about 20% of both NMIBC and MIBC, making it the most frequently mutated epigenetic gene in bladder cancer and suggesting its mutation as an early event in bladder cancer tumorigenesis.
Previously, Dr. Varambally and others have shown that the histone methyltransferase Enhancer Of Zeste Homolog 2 (EZH2), which is responsible for generating a transcriptionally repressive chromatin mark, is over expressed and required for growth of multiple aggressive cancers, including bladder cancer. EZH2 functions as the catalytic subunit of the polycomb repressive complex 2 (PRC2) which methylates lysine 27 on histone 3 (H3K27me), resulting in chromatin condensation and transcriptional silencing.
Herein, we show that ARID1A mutations sensitize bladder cancer cells in vitro and in vivo to EZH2 inhibition with the small molecule GSK-126. We hope that these findings will help to elucidate the epigenetic molecular underpinnings of bladder cancer, and will result in new epigenetic therapeutic targets for patients.
Methods:
In silico analysis using the TCGA dataset compared disease-free survival between ARID1A mutant (ARID1Amut) and wildtype (wt) tumors.
Western blot was used to compare EZH2, ARID1A, and H3K27me3 protein levels between matched pairs of bladder cancer and normal urothelium from cystectomy specimens at our institution.
Cell proliferation, viability, and colon formation assays were performed in the presence and absence of EZH2 inhibitor GSK-126 in bladder cancer cell lines with and without ARID1A mutations. Stable ARID1A knockdown cell lines were also tested to investigate causation.
Murine xenograft experiments were performed using bladder cancer cell lines with and without ARID1A mutations to compare tumor growth inhibition by intraperitoneal GSK-126.
In order to understand the molecular mechanisms behind differential GSK-126 sensitivity, RNA microarray was used to evaluate differentially expressed genes in bladder cancer cell lines with and without ARID1A mutations after treatment with GSK-126.
Western blot and chromatin-immunopreciptation analysis was performed to confirm the microarray findings.
Results:
In silico analysis revealed that ARID1Amut bladder cancers show worse disease-free survival compared to ARID1Awt tumors (p-value: 0.007) [Fig1]. Western blot analysis revealed that EZH2 and resultant H3K27me3 levels are dramatically increased in bladder tumors compared with normal urothelium, with a concomitant decrease in ARID1A protein levels. Using 3 ARID1Awt bladder cancer cell lines (T-24, 5637, RT-112) and 3 ARID1Amut cell lines (HT-1197, HT-1376, VM-CUB1) we showed that the proliferation of only ARID1Amut cell lines is inhibited by EZH2 inhibitor GSK-126 [Fig2]. ARID1A knockdown in 5637 cells resulted in de novo GSK-126 sensitivity in proliferation assays. These findings were also recapitulated in vivo using murine xenograft models with intraperitoneal GSK-126 treatment for 3 weeks. To determine the molecular mechanisms behind the synthetic lethality of EZH2 inhibition in ARID1mut cell lines, we performed transcriptomic analysis comparing ARID1Awt and mut cell lines in the presence and absence of GSK-126. Several genes were differentially expressed that could explain the differential sensitivity to GSK-126 inhibition including MTSS1 (or “missing in metastasis”), optineurin (OPTN), an autophagy regulator, and the tumor suppressor Protein Tyrosine Phosphatase, Receptor Type, R (PTPRR). The GSK-126 mediated induction of these proteins only in ARID1Amut cell lines was confirmed by western blot. ChIP analysis using antibodies to EZH2 and H3K27me3 in combination with PCR templates to the promoters of MTSS1, OPTN, and PTPRR showed that GSK-126 inhibition abrogated EZH2 binding to and methylation of those foci only in ARID1Amut cell lines.
Conclusion:
ARID1A mutation is a reliable biomarker for EZH2 inhibitor sensitivity in bladder cancer cells. As EZH2 inhibitors are currently in phase I clinical trials with tolerable side effect profiles, these findings warrant further validation and future consideration for therapeutic intervention in early and late stage bladder cancer patients. Future experiments will include further investigation of the candidate genes MTSS1, OPTN, and PTPRR to determine if their induction is sufficient for the GSK-126 sensitivity of ARID1Amut cells. CRISPR-CAS9 correction of ARID1A mutations in ARID1Amut cell lines will be used to determine if this sensitivity to GSK-126 can be rescued. Furthermore, shRNA knockdown of other SWI-SNF components will be performed to determine if other mutations can similarly sensitize to GSK-126 treatment, and broaden the eligibility to future therapy. Xenograft studies are currently underway to compare the differential sensitivity of ARID1A mut and wt PDX models to GSK-126. Finally, we are investigating the utility of urinary cell free DNA in the detection of ARID1A mutations as a non-invasive eligibility screen for future therapeutics.
Funding: N/A
INVESTIGATING THE SYNTHETIC LETHALITY OF EZH2 INHIBITION IN ARID1A MUTANT BLADDER CANCER
Category
Bladder Cancer > Muscle Invasive Bladder Cancer
Description
Poster #10 / Podium #
Poster Session I
12/4/2019
2:00 PM - 5:30 PM
Presented By: James Ferguson
Authors:
James Ferguson
Hasib Rehman
Darshan Chandrashekar
Guru Sonpavde
George Netto
Soory Varambally