scholarly journals Histone methyltransferases EHMT1 and EHMT2 (GLP/G9A) maintain PARP inhibitor resistance in high-grade serous ovarian carcinoma

2019 ◽  
Vol 11 (1) ◽  
Author(s):  
Zachary L. Watson ◽  
Tomomi M. Yamamoto ◽  
Alexandra McMellen ◽  
Hyunmin Kim ◽  
Connor J. Hughes ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Dna Damage ◽  
Dna Repair ◽  
Ovarian Carcinoma ◽  
Immunofluorescent Staining ◽  
High Grade ◽  
Rna Seq ◽  
Serous Ovarian Carcinoma ◽  
Resistant Cells

Abstract Background Euchromatic histone-lysine-N-methyltransferases 1 and 2 (EHMT1/2, aka GLP/G9A) catalyze dimethylation of histone H3 lysine 9 (H3K9me2) and have roles in epigenetic silencing of gene expression. EHMT1/2 also have direct roles in DNA repair and are implicated in chemoresistance in several cancers. Resistance to chemotherapy and PARP inhibitors (PARPi) is a major cause of mortality in high-grade serous ovarian carcinoma (HGSOC), but the contribution of the epigenetic landscape is unknown. Results To identify epigenetic mechanisms of PARPi resistance in HGSOC, we utilized unbiased exploratory techniques, including RNA-Seq and mass spectrometry profiling of histone modifications. Compared to sensitive cells, PARPi-resistant HGSOC cells display a global increase of H3K9me2 accompanied by overexpression of EHMT1/2. EHMT1/2 overexpression was also observed in a PARPi-resistant in vivo patient-derived xenograft (PDX) model. Genetic or pharmacologic disruption of EHMT1/2 sensitizes HGSOC cells to PARPi. Cell death assays demonstrate that EHMT1/2 disruption does not increase PARPi-induced apoptosis. Functional DNA repair assays show that disruption of EHMT1/2 ablates homologous recombination (HR) and non-homologous end joining (NHEJ), while immunofluorescent staining of phosphorylated histone H2AX shows large increases in DNA damage. Propidium iodide staining and flow cytometry analysis of cell cycle show that PARPi treatment increases the proportion of PARPi-resistant cells in S and G2 phases, while cells treated with an EHMT1/2 inhibitor remain in G1. Co-treatment with PARPi and EHMT1/2 inhibitor produces an intermediate phenotype. Immunoblot of cell cycle regulators shows that combined EHMT1/2 and PARP inhibition reduces expression of specific cyclins and phosphorylation of mitotic markers. These data suggest DNA damage and altered cell cycle regulation as mechanisms of sensitization. RNA-Seq of PARPi-resistant cells treated with EHMT1/2 inhibitor showed significant gene expression changes enriched in pro-survival pathways that remain unexplored in the context of PARPi resistance, including PI3K, AKT, and mTOR. Conclusions This study demonstrates that disrupting EHMT1/2 sensitizes HGSOC cells to PARPi, and suggests a potential mechanism through DNA damage and cell cycle dysregulation. RNA-Seq identifies several unexplored pathways that may alter PARPi resistance. Further study of EHMT1/2 and regulated genes will facilitate development of novel therapeutic strategies to successfully treat HGSOC.

scholarly journals BRPF3 knockdown or inhibition moderately reverses olaparib resistance in high grade serous ovarian carcinoma, but depletion of H3K14 acetylation has no effect

2021 ◽  
Author(s):  
Benjamin G Bitler ◽  
Tomomi M Yamamoto ◽  
Alexandra McMellen ◽  
Hyunmin Kim ◽  
Zachary Levi Watson
Keyword(s):  
Dna Repair ◽  
Ovarian Carcinoma ◽  
Clinical Problem ◽  
Parp Inhibitors ◽  
High Grade ◽  
Rna Seq ◽  
Damage Resistance ◽  
Altered Expression ◽  
Serous Ovarian Carcinoma ◽  
H3k14 Acetylation

Background: PARP inhibitors (PARPi) kill cancer cells by stalling DNA replication and preventing DNA repair, resulting in a critical accumulation of DNA damage. Resistance to PARPi is a growing clinical problem in the treatment of high grade serous ovarian carcinoma (HGSOC). Acetylation of histone H3 lysine 14 (H3K14ac) and associated histone acetyltransferases (HATs) have known functions in DNA repair and replication, but their expression and activities have not been examined in the context of PARPi-resistant HGSOC. Results: Using mass spectrometry profiling of histone modifications, we observed altered H3K14ac enrichment in PARPi-resistant HGSOC cells relative to isogenic PARPi-sensitive lines. By RT-qPCR and RNA-Seq, we also observed altered expression of numerous HATs in PARPi-resistant HGSOC cells and a PARPi-resistant PDX model. Knockdown of HATs only modestly altered PARPi response, although knockdown and inhibition of PCAF significantly increased resistance. Pharmacologic inhibition of HBO1 severely depleted H3K14ac but did not affect PARPi response. However, knockdown and inhibition of BRPF3, which is known to interact in a complex with HBO1, did reduce PARPi resistance. Conclusions: This study demonstrates that severe depletion of H3K14ac does not affect PARPi response in HGSOC. Our data suggest that bromodomain functions of HAT proteins such as PCAF, or accessory proteins such as BRPF3, may play a greater role in PARPi response than acetyltransferase functions.

scholarly journals Oncogenic B-Myb Is Associated With Deregulation of the DREAM-Mediated Cell Cycle Gene Expression Program in High Grade Serous Ovarian Carcinoma Clinical Tumor Samples

2021 ◽  
Vol 11 ◽  
Author(s):  
Audra N. Iness ◽  
Lisa Rubinsak ◽  
Steven J. Meas ◽  
Jessica Chaoul ◽  
Sadia Sayeed ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Ovarian Carcinoma ◽  
Treatment Response ◽  
Target Genes ◽  
Gene Expression Signature ◽  
Cell Cycle Gene ◽  
High Grade ◽  
Serous Ovarian Carcinoma ◽  
Cell Cycle Gene Expression

Cell cycle control drives cancer progression and treatment response in high grade serous ovarian carcinoma (HGSOC). MYBL2 (encoding B-Myb), an oncogene with prognostic significance in several cancers, is highly expressed in most HGSOC cases; however, the clinical significance of B-Myb in this disease has not been well-characterized. B-Myb is associated with cell proliferation through formation of the MMB (Myb and MuvB core) protein complex required for transcription of mitotic genes. High B-Myb expression disrupts the formation of another transcriptional cell cycle regulatory complex involving the MuvB core, DREAM (DP, RB-like, E2F, and MuvB), in human cell lines. DREAM coordinates cell cycle dependent gene expression by repressing over 800 cell cycle genes in G0/G1. Here, we take a bioinformatics approach to further evaluate the effect of B-Myb expression on DREAM target genes in HGSOC and validate our cellular model with clinical specimens. We show that MYBL2 is highly expressed in HGSOC and correlates with expression of DREAM and MMB target genes in both The Cancer Genome Atlas (TCGA) as well as independent analyses of HGSOC primary tumors (N = 52). High B-Myb expression was also associated with poor overall survival in the TCGA cohort and analysis by a DREAM target gene expression signature yielded a negative impact on survival. Together, our data support the conclusion that high expression of MYBL2 is associated with deregulation of DREAM/MMB-mediated cell cycle gene expression programs in HGSOC and may serve as a prognostic factor independent of its cell cycle role. This provides rationale for further, larger scale studies aimed to determine the clinical predictive value of the B-Myb gene expression signature for treatment response as well as patient outcomes.

Abstract GMM-031: CLINICAL PATHOLOGIC EXPRESSION OF CELL CYCLE REGULATORY COMPLEXES IN HIGH GRADE SEROUS OVARIAN CARCINOMA

2019 ◽  
Author(s):  
Audra N. Iness ◽  
Lisa Rubinsak ◽  
Jessica Chaoul ◽  
Mikhail Dozmorov ◽  
Cora Uram-Tuculescu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Ovarian Carcinoma ◽  
High Grade ◽  
Serous Ovarian Carcinoma

scholarly journals Digital Immune-Related Gene Expression Signatures In High-Grade Serous Ovarian Carcinoma: Developing Prediction Models For Platinum Response

2019 ◽  
Vol Volume 11 ◽  
pp. 9571-9583
Author(s):  
Fabian Mairinger ◽  
Agnes Bankfalvi ◽  
Kurt Werner Schmid ◽  
Elena Mairinger ◽  
Pawel Mach ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ovarian Carcinoma ◽  
Prediction Models ◽  
High Grade ◽  
Related Gene ◽  
Serous Ovarian Carcinoma ◽  
Immune Related Gene ◽  
Gene Expression Signatures

scholarly journals Cells with DNMT3A Mutations Are More Sensitive to Cytarabine-Induced DNA Damage

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2643-2643
Author(s):  
Kartika Venugopal ◽  
Jianping Li ◽  
Daniil E Shabashvili ◽  
Luisa M Posada ◽  
Yang Feng ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Dna Damage ◽  
Dna Repair ◽  
Dna Replication ◽  
Mouse Model ◽  
Comet Assay ◽  
Continuous Infusion ◽  
Annexin V ◽  
Bolus Administration

Abstract Mutations in the DNA methyltransferase 3A (DNMT3A) gene are recurrent in de novo acute myeloid leukemia (AML) (20-35%) and are associated with poor prognosis. Although studies demonstrated survival benefit after daunorubicin dose intensification for DNMT3A mutant AML, these patients tend to be older (median age at diagnosis 67 y.o. in DNMT3A-mutated cases compared to 60 y.o. in DNMT3A-WT) and more likely to be unfit for high-dose chemotherapy. In older patients, cytarabine monotherapy, a nucleoside analog chain terminator that stalls DNA replication, may be preferred due to lower toxicity. However, its efficacy in DNMT3A mutant AML has not been evaluated. Previous gene expression studies in large AML cohorts and in a mouse model of Dnmt3a-mutant hematopoiesis demonstrated negative enrichment of the cell cycle related signatures, including G2/M checkpoint and E2F targets. Hence, we hypothesized cell cycle-specific drugs cytarabine and fludarabine may be effective in DNMT3A-mutant context. DNMT3A mutant AML cell lines (MUT) SET-2 and KO-52 were more sensitive to cytarabine and fludarabine than DNMT3A wild-type (WT) cells KU-812 and K-562 in vitro (cytarabine IC50 46.1 and 165.8 μM in MUT SET-2 and KO-52 compared to 465.2 μM and not reached in WT KU-812 and K-562, respectively; fludarabine IC50 1.1 and 1.3 μg/ml in MUT vs 16 μg/ml and not reached in WT, Figure 1A). Higher proportion of cells with mutant DNMT3A were undergoing apoptosis 24 hours after cytarabine exposure (26.5±2.3% and 13.1±2.3% Annexin V+ cells in SET-2 and KO-52 vs 6.9±2.6% and 4.6±2.5% in KU-812 and K-562, respectively, p<0.05). Analysis of the DNA damage signaling revealed increased levels of phospho-CHK1, γH2A.X, and cleaved PARP (stalled replication forks, DNA damage, and apoptosis markers). To investigate the mechanism of differential sensitivity to cytarabine-induced DNA damage, we overexpressed WT or R882 mutant forms of DNMT3A in U2OS cells, a well-established model for DNA damage studies. MUT cells were more sensitive to cytarabine compared to WT (IC50 38 μM vs 213 μM after 48 hours of exposure, Figure 1B), which was accompanied by increased apoptosis (22.3±7.6% in MUT vs 7.2±0.3% in WT Annexin V+ cells, p<0.03). DNMT3A-MUT cells showed persistence of the phospho-CHK1 levels over 12 hours of continuous cytarabine exposure, whereas WT showed resolution of the CHEK1 signaling after initial activation. Consistently, MUT cells accumulated significantly more DNA damage over 36 hours of continuous exposure compared to WT, as evidenced by γH2A.X immunofluorescent staining, and by Comet assay (Figure 1C). Fewer MUT cells were able to complete replication and progress to the G2 phase of the cell cycle (5.3±0.2% in MUT vs 11.2±0.6% in WT, flow-cytometric analysis by DNA content, p<0.01). Notably, both WT and MUT cells were proficient in DNA repair by homologous recombination (HR, tested by RAD51 foci formation visualized by immunofluorescence) and by non-homologous end joining (NHEJ, 53BP1 foci). These data demonstrate that enhanced sensitivity to cytarabine in cells expressing mutant DNMT3A is due to increased susceptibility to DNA damage during replication, and not to defects in DNA repair. Consistently, even though MUT cells showed more DNA damage by Comet assay after 12 hours of cytarabine treatment, it was largely repaired 60 minutes after drug wash-out, similar to WT, Figure 1D). These observations support continuous infusion of cytarabine as a delivery method of choice in the clinic, rather than bolus administration. Finally, bone marrow cells derived from Dnmt3a-mut conditional knock-in mice showed impaired clonogenic survival in MethoCult when exposed to 25 nM cytarabine ex vivo, compared to Dnmt3a-WT (p<0.003). We are currently investigating gene expression, DNA methylation, and chromatin accessibility profiles in WT and MUT cells; and cytarabine efficacy in a pre-clinical mouse model of Dnmt3a(mut):Flt3(ITD):Npm1(c) leukemia. In conclusion, our studies show cells with DNMT3A mutations may be sensitive to DNA damage induced by clinically relevant nucleoside analogs such as cytarabine. Our data support continuous infusion of cytarabine rather than bolus administration, and establish its mechanistic basis. These results demonstrate, in addition to its role in epigenetic control, DNMT3A contributes to preserving genome integrity during DNA replication. Figure 1. Figure 1. Disclosures Licht: Celgene: Research Funding.

scholarly journals Integrated Analyses of microRNAs Demonstrate Their Widespread Influence on Gene Expression in High-Grade Serous Ovarian Carcinoma

PLoS ONE ◽  
2012 ◽  
Vol 7 (3) ◽  
pp. e34546 ◽  
Author(s):  
Chad J. Creighton ◽  
Anadulce Hernandez-Herrera ◽  
Anders Jacobsen ◽  
Douglas A. Levine ◽  
Parminder Mankoo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ovarian Carcinoma ◽  
High Grade ◽  
Serous Ovarian Carcinoma

scholarly journals Development and Validation of the Gene Expression Predictor of High-grade Serous Ovarian Carcinoma Molecular SubTYPE (PrOTYPE)

2020 ◽  
Vol 26 (20) ◽  
pp. 5411-5423 ◽  
Author(s):  
Aline Talhouk ◽  
Joshy George ◽  
Chen Wang ◽  
Timothy Budden ◽  
Tuan Zea Tan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ovarian Carcinoma ◽  
Molecular Subtype ◽  
High Grade ◽  
Serous Ovarian Carcinoma ◽  
Development And Validation

scholarly journals CtBP1/2 differentially regulate genomic stability and DNA repair pathway in high-grade serous ovarian cancer cell

Oncogenesis ◽  
2021 ◽  
Vol 10 (7) ◽  
Author(s):  
YingYing He ◽  
Zhicheng He ◽  
Jian Lin ◽  
Cheng Chen ◽  
Yuanzhi Chen ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Dna Damage ◽  
Dna Repair ◽  
Dna Replication ◽  
Genomic Stability ◽  
High Grade ◽  
Serous Ovarian Cancer ◽  
Repair Pathway ◽  
Serous Ovarian Carcinoma ◽  
Skov3 Cells

AbstractThe C-terminal binding proteins (CtBPs), CtBP1 and CtBP2, are transcriptional co-repressor that interacts with multiple transcriptional factors to modulate the stability of chromatin. CtBP proteins were identified with overexpression in the high-grade serous ovarian carcinoma (HGSOC). However, little is known about CtBP proteins’ regulatory roles in genomic stability and DNA repair in HGSOC. In this study, we combined whole-transcriptome analysis with multiple research methods to investigate the role of CtBP1/2 in genomic stability. Several key functional pathways were significantly enriched through whole transcription profile analysis of CtBP1/2 knockdown SKOV3 cells, including DNA damage repair, apoptosis, and cell cycle. CtBP1/2 knockdown induced cancer cell apoptosis, increased genetic instability, and enhanced the sensitivity to DNA damage agents, such as γ-irradiation and chemotherapy drug (Carboplatin and etoposide). The results of DNA fiber assay revealed that CtBP1/2 contribute differentially to the integrity of DNA replication track and stability of DNA replication recovery. CtBP1 protects the integrity of stalled forks under metabolic stress condition during prolonged periods of replication, whereas CtBP2 acts a dominant role in stability of DNA replication recovery. Furthermore, CtBP1/2 knockdown shifted the DSBs repair pathway from homologous recombination (HR) to non-homologous end joining (NHEJ) and activated DNA-PK in SKOV3 cells. Interesting, blast through TCGA tumor cases, patients with CtBP2 genetic alternation had a significantly longer overall survival time than unaltered patients. Together, these results revealed that CtBP1/2 play a different regulatory role in genomic stability and DSBs repair pathway bias in serous ovarian cancer cells. It is possible to generate novel potential targeted therapy strategy and translational application for serous ovarian carcinoma patients with a predictable better clinical outcome.

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