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The promising activity of BET protein inhibitors (BETi’s) is compromised by adaptive or innate resistance in acute myeloid leukemia (AML). Here, modeling of BETi-persister/resistance (BETi-P/R) in human postmyeloproliferative neoplasm (post-MPN) secondary AML (sAML) cells demonstrated accessible and active chromatin in specific superenhancers/enhancers, which was associated with increased levels of nuclear β-catenin, TCF7L2, JMJD6, and c-Myc in BETi-P/R sAML cells. Following BETi treatment, c-Myc levels were rapidly restored in BETi-P/R sAML cells. CRISPR/Cas9-mediated knockout of TCF7L2 or JMJD6 reversed BETi-P/R, whereas ectopic overexpression conferred BETi-P/R in sAML cells, confirming the mechanistic role of the β-catenin–TCF7L2–JMJD6–c-Myc axis in BETi resistance. Patient-derived, post-MPN, CD34+ sAML blasts exhibiting relative resistance to BETi, as compared with sensitive sAML blasts, displayed higher messenger RNA and protein expression of TCF7L2, JMJD6, and c-Myc and following BETi washout exhibited rapid restoration of c-Myc and JMJD6. CRISPR/Cas9 knockout of TCF7L2 and JMJD6 depleted their levels, inducing loss of viability of the sAML blasts. Disruption of colocalization of nuclear β-catenin with TBL1 and TCF7L2 by the small-molecule inhibitor BC2059 combined with depletion of BRD4 by BET proteolysis-targeting chimera reduced c-Myc levels and exerted synergistic lethality in BETi-P/R sAML cells. This combination also reduced leukemia burden and improved survival of mice engrafted with BETi-P/R sAML cells or patient-derived AML blasts innately resistant to BETi. Therefore, multitargeted disruption of the β-catenin–TCF7L2–JMJD6–c-Myc axis overcomes adaptive and innate BETi resistance, exhibiting preclinical efficacy against human post-MPN sAML cells.

The class I histone deacetylases are essential regulators of cell fate decisions in health and disease. While pan- and class-specific HDAC inhibitors are available, these drugs do not allow a comprehensive understanding of individual HDAC function, or the therapeutic potential of isoform-specific targeting. To systematically compare the impact of individual catalytic functions of HDAC1, HDAC2 and HDAC3, we generated human HAP1 cell lines expressing catalytically inactive HDAC enzymes. Using this genetic toolbox we compare the effect of individual HDAC inhibition with the effects of class I specific inhibitors on cell viability, protein acetylation and gene expression. Individual inactivation of HDAC1 or HDAC2 has only mild effects on cell viability, while HDAC3 inactivation or loss results in DNA damage and apoptosis. Inactivation of HDAC1/HDAC2 led to increased acetylation of components of the COREST co-repressor complex, reduced deacetylase activity associated with this complex and derepression of neuronal genes. HDAC3 controls the acetylation of nuclear hormone receptor associated proteins and the expression of nuclear hormone receptor regulated genes. Acetylation of specific histone acetyltransferases and HDACs is sensitive to inactivation of HDAC1/HDAC2. Over a wide range of assays, we determined that in particular HDAC1 or HDAC2 catalytic inactivation mimics class I specific HDAC inhibitors. Importantly, we further demonstrate that catalytic inactivation of HDAC1 or HDAC2 sensitizes cells to specific cancer drugs. In summary, our systematic study revealed isoform-specific roles of HDAC1/2/3 catalytic functions. We suggest that targeted genetic inactivation of particular isoforms effectively mimics pharmacological HDAC inhibition allowing the identification of relevant HDACs as targets for therapeutic intervention.

Histone deacetylase 3 (Hdac3) is a target of the FDA approved HDAC inhibitors, which are used for the treatment of lymphoid malignancies. Here, we used Cd19-Cre to conditionally delete Hdac3 to define its role in germinal center B cells, which represent the cell of origin for many B cell malignancies. Cd19-Cre-Hdac3−/− mice showed impaired germinal center formation along with a defect in plasmablast production. Analysis of Hdac3−/− germinal centers revealed a reduction in dark zone centroblasts and accumulation of light zone centrocytes. RNA-seq revealed a significant correlation between genes up-regulated upon Hdac3 loss and those up-regulated in Foxo1-deleted germinal center B cells, even though Foxo1 typically activates transcription. Therefore, to determine whether gene expression changes observed in Hdac3−/− germinal centers were a result of direct effects of Hdac3 deacetylase activity, we used an HDAC3 selective inhibitor and examined nascent transcription in germinal center-derived cell lines. Transcriptional changes upon HDAC3 inhibition were enriched for light zone gene signatures as observed in germinal centers. Further comparison of PRO-seq data with ChIP-seq/exo data for BCL6, SMRT, FOXO1 and H3K27ac identified direct targets of HDAC3 function including CD86, CD83 and CXCR5 that are likely responsible for driving the light zone phenotype observed in vivo.

Background Malignant gliomas have disproportionally high morbidity and mortality. Heterozygous mutations in the isocitrate dehydrogenase 1 (IDH1) gene are most common in glioma, resulting in predominantly arginine to histidine substitution at codon 132. Because IDH1R132H requires a wild-type allele to produce (D)-2-hydroxyglutarate for epigenetic reprogramming, loss of IDH1R132H heterozygosity is associated with glioma progression in an IDH1-wildtype-like phenotype. Although previous studies have reported that transgenic IDH1R132H induces the expression of nestin—a neural stem-cell marker, the underlying mechanism remains unclear. Furthermore, this finding seems at odds with better outcome of IDH1R132H glioma because of a negative association of nestin with overall survival. Methods Gene expression was compared between IDH1R132H-hemizygous and IDH1R132H-heterozygous glioma cells under adherent and spheroid growth conditions. The results were validated for (D)-2-hydroxyglutarate responsiveness by pharmacologic agents, associations with DNA methylation by bioinformatic analysis, and associations with overall survival. Bisulfite DNA sequencing, chromatin immunoprecipitation, and pharmacological approach were used. Findings Neural stem-cell marker genes, including CD44, NES, and PROM1, are generally downregulated in IDH-mutant gliomas and IDH1R132H-heterozygous spheroid growth compared respectively with IDH-wildtype gliomas and IDH1R132H-hemizygous spheroid growth, in agreement with their negative associations with patient outcome. In contrast, CD24 is specifically upregulated and apparently associated with better survival. CD24 and NES expression respond differentially to alteration of (D)-2-hydroxyglutarate levels. CD24 upregulation is associated with histone and DNA demethylation as opposed to hypermethylation in the downregulated genes. Interpretation The better outcome of IDH-mutant glioma is orchestrated exquisitely through epigenetic reprogramming that directs bidirectional expression of neural stem-cell marker genes.

International audience; It is increasingly suggested that ecological and evolutionary sciences could inspire novel therapies against cancer but medical evidence of this remains scarce at the moment. The Achilles heel of conventional and targeted anticancer treatments is intrinsic or acquired resistance following Darwinian selection, i.e. treatment toxicity places the surviving cells under intense evolutionary selective pressure to develop resistance. Here, we review a set of data that demonstrate that Darwinian principles derived from the “smoke detector” principle can instead drive the evolution of malignant cells toward a different trajectory. Specifically, long term exposure of cancer cells to a strong alarm signal, generated by the DNA repair inhibitor AsiDNA, induces a stable new state characterized by a down‐regulation of the targeted pathways and does not generate resistant clones. This property is due to the original mechanism of action of AsiDNA, which acts by over‐activating a “false” signaling of DNA damage through DNA‐PK and PARP enzymes, and is not observed with classical DNA repair inhibitors such as the PARP inhibitors. Long‐term treatment with AsiDNA induces a new “alarm down” state in the tumor cells with decrease of NAD level and reactiveness to it. These results suggest that agonist drugs such as AsiDNA could promote a state‐dependent tumor cell evolution by lowering their ability to respond to high “danger” signal. This analysis provides a compelling argument that evolutionary ecology could help drug design development in overcoming fundamental limitation of novel therapies against cancer due to the modification of the targeted tumor cell population during treatment.

Histone deacetylase 3 (HDAC3) is the catalytic component of NCoR/SMRT corepressor complexes that mediate the actions of transcription factors implicated in the regulation of B-cell development and function. We crossed Hdac3 conditional knockout mice with Mb1-Cre knockin animals to delete Hdac3 in early progenitor B cells. The spleens of Hdac3F/-Mb1-Cre+/- mice were virtually devoid of mature B cells, and B220+CD43+ B-cell progenitors accumulated within the bone marrow. Quantitative deep sequencing of the Ig heavy chain locus from B220+CD43+ populations identified a defect in VHDJH recombination with a severe reduction in productive rearrangements, which directly corresponded to the loss of pre-B cells from Hdac3Δ/- bone marrow. For Hdac3Δ/- B cells that did show productive VDJ rearrangement, there was significant skewing toward the incorporation of proximal VH gene segments and a corresponding reduction in distal VH gene segment use. Although transcriptional effects within these loci were modest, Hdac3Δ/- progenitor cells displayed global changes in chromatin structure that likely hindered effective distal V-DJ recombination. Reintroduction of wild-type Hdac3 restored normal B-cell development, whereas an Hdac3 point mutant lacking deacetylase activity failed to complement this defect. Thus, the deacetylase activity of Hdac3 is required for the generation of mature B cells.

Philadelphia chromosome-positive (Ph+) B-cell precursor acute lymphoblastic leukemia (ALL) expressing BCR-ABL1 oncoprotein is a major subclass of ALL with poor prognosis. BCR-ABL1-expressing leukemic cells are highly dependent on double-strand break (DSB) repair signals for their survival. Here we report that a first-in-class HDAC1,2 selective inhibitor and doxorubicin (a hyper-CVAD chemotherapy regimen component) impair DSB repair networks in Ph+ B-cell precursor ALL cells using common as well as distinct mechanisms. The HDAC1,2 inhibitor but not doxorubicin alters nucleosomal occupancy to impact chromatin structure, as revealed by MNase-Seq. Quantitative mass spectrometry of the chromatin proteome along with functional assays showed that the HDAC1,2 inhibitor and doxorubicin either alone or in combination impair the central hub of DNA repair, the Mre11–Rad51–DNA ligase 1 axis, involved in BCR-ABL1-specific DSB repair signaling in Ph+ B-cell precursor ALL cells. HDAC1,2 inhibitor and doxorubicin interfere with DISC (DNA damage-induced transcriptional silencing in cis)) or transcriptional silencing program in cis around DSB sites via chromatin remodeler-dependent and -independent mechanisms, respectively, to further impair DSB repair. HDAC1,2 inhibitor either alone or when combined with doxorubicin decreases leukemia burden in vivo in refractory Ph+ B-cell precursor ALL patient-derived xenograft mouse models. Overall, our novel mechanistic and preclinical studies together demonstrate that HDAC1,2 selective inhibition can overcome DSB repair ‘addiction’ and provide an effective therapeutic option for Ph+ B-cell precursor ALL.

Simple Summary Targeting aberrant DNA repair in cancers in addition to transcription and replication is an area of interest for cancer researchers. Inhibition of DNA repair selectively in cancer cells leads to cytotoxic or cytostatic effects and overcomes survival advantages imparted by chromosomal translocations or mutations. In this review, we highlight the relevance of DNA repair-linked events in developmental diseases and cancers and also discuss mechanisms to overcome these events that participate in different cellular processes. Abstract Aberrant DNA repair pathways that underlie developmental diseases and cancers are potential targets for therapeutic intervention. Targeting DNA repair signal effectors, modulators and checkpoint proteins, and utilizing the synthetic lethality phenomena has led to seminal discoveries. Efforts to efficiently translate the basic findings to the clinic are currently underway. Chromatin modulation is an integral part of DNA repair cascades and an emerging field of investigation. Here, we discuss some of the key advancements made in DNA repair-based therapeutics and what is known regarding crosstalk between chromatin and repair pathways during various cellular processes, with an emphasis on cancer.

Histone deacetylases 1 and 2 (HDAC1,2) belong to the class I HDAC family, which are targeted by the FDA-approved small molecule HDAC inhibitors currently used in cancer therapy. HDAC1,2 are recruited to DNA break sites during DNA repair and to chromatin around forks during DNA replication. Cancer cells use DNA repair and DNA replication as survival mechanisms and to evade chemotherapy-induced cytotoxicity. Hence, it is vital to understand how HDAC1,2 function during the genome maintenance processes (DNA replication and DNA repair) in order to gain insights into the mode-of-action of HDAC inhibitors in cancer therapeutics. The first-in-class HDAC1,2-selective inhibitors and Hdac1,2 conditional knockout systems greatly facilitated dissecting the precise mechanisms by which HDAC1,2 control genome stability in normal and cancer cells. In this perspective, I summarize the findings on the mechanistic functions of class I HDACs, specifically, HDAC1,2 in genome maintenance, unanswered questions for future investigations and views on how this knowledge could be harnessed for better-targeted cancer therapeutics for a subset of cancers.

// Danielle P. Johnson 2,* , Gabriella S. Spitz 1,* , Shweta Tharkar 1 , Steven N. Quayle 3 , Jeffrey R. Shearstone 3 , Simon Jones 3 , Maria E. McDowell 1 , Hannah Wellman 1 , Jessica K. Tyler 4 , Bradley R. Cairns 2 , Mahesh B. Chandrasekharan 1 and Srividya Bhaskara 1,2 1 Department of Radiation Oncology, Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, UT 2 Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, UT 3 Acetylon Pharmaceuticals, Inc., Boston, MA 4 Department of Biochemistry and Molecular Biology, The University of Texas MD Anderson Cancer Center, Houston, TX * These authors contributed equally to the study Correspondence: Srividya Bhaskara, email: // Keywords : epigenetics, DNA repair, HDAC1,2, chromatin, EZH2 Received : November 01, 2014 Accepted : December 28, 2014 Published : December 31, 2014 Abstract Gain-of-function mutations in the catalytic site of EZH2 (Enhancer of Zeste Homologue 2), is observed in about 22% of diffuse large B-cell lymphoma (DLBCL) cases. Here we show that selective inhibition of histone deacetylase 1,2 (HDAC1,2) activity using a small molecule inhibitor causes cytotoxic or cytostatic effects in EZH2 gain-of-function mutant (EZH2 GOF ) DLBCL cells. Our results show that blocking the activity of HDAC1,2 increases global H3K27ac without causing a concomitant global decrease in H3K27me3 levels. Our data shows that inhibition of HDAC1,2 is sufficient to decrease H3K27me3 present at DSBs, decrease DSB repair and activate the DNA damage response in these cells. In addition to increased H3K27me3, we found that the EZH2 GOF DLBCL cells overexpress another chemotherapy resistance factor − B-lymphoma and BAL-associated protein (BBAP). BBAP monoubiquitinates histone H4K91, a residue that is also subjected to acetylation. Our results show that selective inhibition of HDAC1,2 increases H4K91ac, decreases BBAP-mediated H4K91 monoubiquitination, impairs BBAP-dependent DSB repair and sensitizes the refractory EZH2 GOF DLBCL cells to treatment with doxorubicin, a chemotherapy agent. Hence, selective HDAC1,2 inhibition provides a novel DNA repair mechanism-based therapeutic approach as it can overcome both EZH2- and BBAP-mediated DSB repair in the EZH2 GOF DLBCL cells.

There is an unmet need to develop new, more effective and safe therapies for the aggressive forms of triple negative breast cancers (TNBCs). While up to 20% of women under 50 years of age with TNBC harbor germline mutations in BRCA1, and these tumors are sensitive to treatment with poly(ADP) ribose polymerase inhibitors, a majority of TNBCs lack BRCA1 mutations or loss of expression. Findings presented here demonstrate that by attenuating the levels of DNA damage response and homologous recombination proteins, pan-histone deacetylase inhibitor (HDI) treatment induces 'BRCAness' and sensitizes TNBC cells lacking BRCA1 to lethal effects of PARP inhibitor or cisplatin. Treatment with HDI also induced hyperacetylation of nuclear hsp90. Similar effects were observed following shRNA-mediated depletion of HDAC3, confirming its role as the deacetylase for nuclear HSP90. Furthermore, cotreatment with HDI and ABT-888 induced significantly more DNA strand breaks than either agent alone, and synergistically induced apoptosis of TNBC cells. Notably, co-treatment with HDI and ABT-888 significantly reduced in vivo tumor growth and markedly improved the survival of mice bearing TNBC cell xenografts. These findings support the rationale to interrogate the clinical activity of this novel combination against human TNBC, irrespective of its expression of mutant BRCA1.

The Achilles heel of conventional and targeted anticancer treatments is intrinsic or acquired resistance following Darwinian selection, i.e. treatment toxicity place the surviving cells under intense evolutionary selective pressure to develop resistance. Here, we demonstrate that AsiDNA, the first antitumor drug with an agonist activity, can instead drive the evolution of malignant cells toward a different trajectory. Specifically, long term exposure of cancer cells to the strong alarm signal, generated by the AsiDNATM DNA repair inhibitors, did not promote resistance emergence. It induces a stable new state characterized by the down regulation of the targeted pathways that persists for months after treatments. This property is due to the original mechanism of action of AsiDNATM, which acts by over-activating a “false” signaling of DNA damage through DNA-PK and PARP enzymes, and is not observed with other DNA repair inhibitors such as the PARP inhibitors olaparib and talazoparib. Transcriptomic analysis of AsiDNATM treated independent populations derived from lung and breast cancers revealed the common evolution of independent AsiDNA-treated samples, resulting in a general down-regulation of enzymes involved in DNA repair and oncogenesis, and a specific down regulation of DNA-PK targets. Similar down regulation of DNA-PK targets was observed in xenografted tumor in animal models after three cycles of AsiDNA treatment. Chromatin analysis revealed a major change in AsiDNATM treated populations with an increased nucleosome stability that could be responsible for the epigenetic modification observed. Long term treatment with AsiDNATM induces a new “alarm down” state in the tumor cells that increase its efficacy. These results suggest that agonist drugs such as AsiDNATM could promote a state-dependent tumor cell evolution by lowering their ability to respond to damage signal as predicted by the ecological and evolutionary “smoke detector principle”. Citation Format: Wael Jdey, Maria Kozlac, Sergey Alekseev, Danielle Johnson, françoise Bono, Srividya bhaskara, frederic thomas, Marie Dutreix. AsiDNATM, a targeted therapy with no acquired resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2095.

Histone deacetylase 3 (HDAC3) contributes to the regulation of gene expression, chromatin structure, and genomic stability. Because HDAC3 associates with oncoproteins that drive leukemia and lymphoma, we engineered a conditional deletion allele in mice to explore the physiological roles of Hdac3 in hematopoiesis. We used the Vav-Cre transgenic allele to trigger recombination, which yielded a dramatic loss of lymphoid cells, hypocellular bone marrow, and mild anemia. Phenotypic and functional analysis suggested that Hdac3 was required for the formation of the earliest lymphoid progenitor cells in the marrow, but that the marrow contained 3-5 times more multipotent progenitor cells. Hdac3(-/-) stem cells were severely compromised in competitive bone marrow transplantation. In vitro, Hdac3(-/-) stem and progenitor cells failed to proliferate, and most cells remained undifferentiated. Moreover, one-third of the Hdac3(-/-) stem and progenitor cells were in S phase 2 hours after BrdU labeling in vivo, suggesting that these cells were impaired in transit through the S phase. DNA fiber-labeling experiments indicated that Hdac3 was required for efficient DNA replication in hematopoietic stem and progenitor cells. Thus, Hdac3 is required for the passage of hematopoietic stem/progenitor cells through the S phase, for stem cell functions, and for lymphopoiesis.

Histone H3K4 methylation is connected to gene transcription from yeast to humans, but its mechanistic roles in transcription and chromatin dynamics remain poorly understood. We investigated the functions for Set1 and Jhd2, the sole H3K4 methyltransferase and H3K4 demethylase, respectively, in S. cerevisiae. Here, we show that Set1 and Jhd2 predominantly co-regulate genome-wide transcription. We find combined activities of Set1 and Jhd2 via H3K4 methylation contribute to positive or negative transcriptional regulation. Providing mechanistic insights, our data reveal that Set1 and Jhd2 together control nucleosomal turnover and occupancy during transcriptional co-regulation. Moreover, we find a genome-wide co-regulation of chromatin structure by Set1 and Jhd2 at different groups of transcriptionally active or inactive genes and at different regions within yeast genes. Overall, our study puts forth a model wherein combined actions of Set1 and Jhd2 via modulating H3K4 methylation−demethylation together control chromatin dynamics during various facets of transcriptional regulation., Histone H3K4 methylation affects gene transcription from yeast to humans. Here, the authors show that Set1 and Jhd2, the only H3K4 methyltransferase and H3K4 demethylase, respectively, in budding yeast, co-regulate both positive and negative transcription mediated by nucleosomal turnover and occupancy.

Background and purpose This single institution phase I trial determined the maximum tolerated dose (MTD) of concurrent vorinostat and capecitabine with radiation in non-metastatic pancreatic cancer. Material and methods Twenty-one patients received escalating doses of vorinostat (100–400mg daily) during radiation. Capecitabine was given 1000mg q12 on the days of radiation. Radiation consisted of 30Gy in 10 fractions. Vorinostat dose escalation followed the standard 3+3 design. No dose escalation beyond 400mg vorinostat was planned. Diffusion-weighted (DW)-MRI pre- and post-treatment was used to evaluate in vivo tumor cellularity. Results The MTD of vorinostat was 400mg. Dose limiting toxicities occurred in one patient each at dose levels 100mg, 300mg, and 400mg: 2 gastrointestinal toxicities and one thrombocytopenia. The most common adverse events were lymphopenia (76%) and nausea (14%). The apparent diffusion coefficient (ADC) increased in most tumors. Nineteen (90%) patients had stable disease, and two (10%) had progressive disease at time of surgery. Eleven patients underwent surgical exploration with four R0 resections and one R1 resection. Median overall survival was 1.1years (95% confidence interval 0.78–1.35). Conclusions The combination of vorinostat 400mg daily M–F and capecitabine 1000mg q12 M–F with radiation (30Gy in 10 fractions) was well tolerated with encouraging median overall survival.

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA. Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples.

High grade epithelial ovarian cancers are relatively sensitive to DNA damaging platinum-based chemotherapy, suggesting that the dependencies of ovarian tumors on DNA damage response pathways can be harnessed for therapeutic purposes. Our goal was to determine if the DNA damage mark gamma-H2AX phosphorylation (pH2AX) could be used to identify suitable cytotoxic histone deacetylase inhibitors (HDACi) for ovarian cancer treatment. Nineteen chemically diverse HDACi compounds were tested in 7 ovarian cancer cell lines. Fluorescent, biochemical and cell-based assays were performed to assess DNA damage by induction of pH2AX and to measure cell viability and apoptosis. The relationships between pH2AX and the cellular effects of cell viability and apoptosis were calculated. Selected HDACi were tested in combination with cisplatin and other DNA damaging agents to determine if the HDACi improved upon the effects of the DNA damaging agents. The HDACi compounds induced differing levels of pH2AX expression. High levels of pH2AX in HDACi-treated ovarian cancer cells were tightly associated with decreased cell viability and increased apoptosis. Consequently, a ketone-based HDACi was chosen and found to enhance the effects of cisplatin, even in ovarian cancer cells with extreme resistance to DNA damaging drugs. In conclusion, a fluorescent-based assay for pH2AX can be used to determine cellular responses to HDACi in vitro and may be a useful tool to identify potentially more effective HDACi for the treatment of ovarian cancer. In addition, these results lend support to the inclusion of ketone-derived HDACi compounds for future development.

SummaryHdac3 is essential for efficient DNA replication and DNA damage control. Deletion of Hdac3 impaired DNA repair and greatly reduced chromatin compaction and heterochromatin content. These defects corresponded to increases in histone H3K9,K14ac; H4K5ac; and H4K12ac in late S phase of the cell cycle, and histone deposition marks were retained in quiescent Hdac3-null cells. Liver-specific deletion of Hdac3 culminated in hepatocellular carcinoma. Whereas HDAC3 expression was downregulated in only a small number of human liver cancers, the mRNA levels of the HDAC3 cofactor NCOR1 were reduced in one-third of these cases. siRNA targeting of NCOR1 and SMRT (NCOR2) increased H4K5ac and caused DNA damage, indicating that the HDAC3/NCOR/SMRT axis is critical for maintaining chromatin structure and genomic stability.

Histone lysine methylation is a dynamic process that plays an important role in regulating chromatin structure and gene expression. Recent studies have identified Jhd2, a JmjC domain-containing protein, as an H3K4-specific demethylase in budding yeast. However, important questions regarding the regulation and functions of Jhd2 remain unanswered. In this study, we show that Jhd2 has intrinsic activity to remove all three states of H3K4 methylation in vivo and can dynamically associate with chromatin to modulate H3K4 methylation levels on both active and repressed genes and at the telomeric regions. We found that the plant homeodomain (PHD) finger of Jhd2 is important for its chromatin association in vivo. However, this association is not dependent on H3K4 methylation and the H3 N-terminal tail, suggesting the presence of an alternative mechanism by which Jhd2 binds nucleosomes. We also provide evidence that the JmjN domain and its interaction with the JmjC catalytic domain are important for Jhd2 function and that Not4 (an E3 ligase) monitors the structural integrity of this interdomain interaction to maintain the overall protein levels of Jhd2. We show that the S451R mutation in human SMCX (a homolog of Jhd2), which has been linked to mental retardation, and the homologous T359R mutation in Jhd2 affect the protein stability of both of these proteins. Therefore, our findings provide a mechanistic explanation for the observed defects in patients harboring this SMCX mutant and suggest the presence of a conserved pathway involving Not4 that modulates the protein stability of both yeast Jhd2 and human SMCX.

Introduction: The Philadelphia (Ph) chromosome was the first reported chromosomal rearrangement linked to a human malignancy. Ph-positive precursor B-cell acute lymphoblastic leukemic (Ph+Pre-B-ALL) expressing the BCR-ABL1 oncoprotein is a major subclass of ALL with poor prognosis and high relapse rates in adults. BCR-ABL1 drives an aberrantly hyperactive DNA repair that confers survival advantages to leukemic cells and serves as its ‘Achilles' heel'. We used HDAC1,2 selective inhibitor in isolation or in combination with doxorubicin (a chemotherapy regimen component used for treating pre-B-ALL) to combat the aberrant DSB repair processes. Experimental design: We used a combination of molecular and cell biology, proteomics and functional genomics-based approaches and primary patient-derived xenograft mouse models to understand the genome stability mechanisms targeted by HDAC1,2 inhibition to overcome oncogenic signals promoted by the BCR-ABL1 fusion protein. Results: Mass-spectrometry analysis and DNA repair assays showed that HDAC1,2 inhibition and doxorubicin treatments impact common as well as distinct DNA repair and genome maintenance networks including decreased chromatin association of Mre11-Rad51-DNA ligase 1, the central hub of DNA repair. Translating this mechanistic knowledge, we then showed that the HDAC1,2 inhibitor either alone or when combined with a low dose doxorubicin caused cytotoxicity in primary patient Ph+ Pre-B-ALL cells, and decreased leukemia burden without adverse side-effects in vivo in refractory Ph+ Pre-B-ALL PDX models that we created. We recently found that HDAC1,2 targets H4K91ac mark in Ph+Pre-B-ALL cells. H4K91ac occurs within the body of nucleosome core and thus directly controls nucleosome or chromatin integrity. Increased H4K91ac following HDAC1,2 inhibition occurs around DSB sites genome-wide could cause a delay in the rate at which DSBs are repaired in Ph+Pre-B-ALL cells as a result of defective nucleosome structure around DSB sites. Conclusion: Our comprehensive study using a multipronged approach showed that HDAC1,2 inhibition as a monotherapy or in combination with a low-dose doxorubicin can override DSB repair ‘addiction' in Ph+ Pre-B-ALL. We are currently using the genome-wide ChIP-seq approach and an homing endonuclease DSB reporter system to examine how HDAC1,2 inhibition changes the chromatin landscape in Ph+Pre-B-ALL cells. Citation Format: Danielle Johnson, Shwetha Tharkar, Simon Jones, Matt Jarpe, Mahesh Chandrasekharan, Srividya Bhaskara. Targeting DNA repair in Philadelphia positive B-ALL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5844.

Cytochrome P450 monooxygenases or CYPs, a family of endobiotics and xenobiotics metabolizing enzymes, are found in all organisms. We reported earlier that the promoters of Drosophila Cyp6a2 and Cyp6a8 genes are induced by caffeine. Since caffeine antagonizes adenosine receptor (AdoR) and inhibits cAMP phosphodiesterase (PDE), we used luciferase reporter gene to examine whether in SL-2 cells and adult Drosophila, induction of the two Cyp6 genes is mediated via AdoR and/or PDE pathway. Results showed that AdoR is not involved because AdoR agonists or antagonists do not affect the Cyp6 promoter activities. However, inhibition of PDE by specific inhibitors including caffeine causes induction of both Cyp6 gene promoters. We also found that flies mutant for dunce gene coding for cAMP-PDE, have higher Cyp6a8 promoter activity than the wild-type flies. We demonstrate that caffeine treatment increases intracellular cAMP levels, and cAMP treatment induces the Cyp6 gene promoters. Since both Cyp6 genes have multiple sites for JUN transcription factors, which generally play a positive role in cAMP pathway, effect of Drosophila jun (D-jun) on the Cyp6a8 promoter activity was examined. Results showed that the expression of D-jun sense plasmid causes downregulation rather than activation of the Cyp6a8 promoter. Conversely, expression of antisense plasmid increased the promoter activity. Interestingly, caffeine treatment decreased the D-JUN protein level in SL-2 cells as well as in adult flies. These results suggest that D-jun acts as a negative regulator, and caffeine induction of Cyp6a8 and Cyp6a2 genes is mediated by the upregulation of cAMP pathway and downregulation of the D-JUN protein level.

Histone deacetylases (HDAC) are enzymes that modify key residues in histones to regulate chromatin architecture, and play a vital role in cell survival, cell cycle progression, and tumorigenesis. To understand the function of Hdac3, a critical component of the N-CoR/SMRT repression complex, a conditional allele of Hdac3 was engineered. Cre-recombinase-mediated inactivation of Hdac3 led to a delay in cell cycle progression, cell-cycle dependent DNA damage, and apoptosis in mouse embryonic fibroblasts (MEFs). While no overt defects in mitosis were observed in Hdac3−/− MEFs, including normal H3Ser10 phosphorylation, DNA damage was observed in Hdac3−/−interphase cells, which appears to be associated with defective DNA double strand break repair. Moreover, we noted that Hdac3−/− MEFs were protected from DNA damage when quiescent, which may provide a mechanistic basis for the action of histone deacetylase inhibitors on cycling tumor cells.

Histone deacetylase 3 (Hdac3) is an enzymatic component of transcriptional repression complexes recruited by the nuclear hormone receptors. Inactivation of Hdac3 in cancer cell lines triggered apoptosis, and removal of Hdac3 in the germ line of mice caused embryonic lethality. Therefore, we deleted Hdac3 in the postnatal mouse liver. These mice developed hepatomegaly, which was the result of hepatocyte hypertrophy, and these morphological changes coincided with significant imbalances between carbohydrate and lipid metabolism. Loss of Hdac3 triggered changes in gene expression consistent with inactivation of repression mediated by nuclear hormone receptors. Loss of Hdac3 also increased the levels of Ppar gamma2, and treatment of these mice with a Ppar gamma antagonist partially reversed the lipid accumulation in the liver. In addition, gene expression analysis identified mammalian target of rapamycin signalling as being activated after deletion of Hdac3, and inhibition by rapamycin affected the accumulation of neutral lipids in Hdac3-null livers. Thus, Hdac3 regulates metabolism through multiple signalling pathways in the liver, and deletion of Hdac3 disrupts normal metabolic homeostasis.

Importance Evolutionary medicine may provide insights into human physiology and pathophysiology, including tumor biology. Objective To identify mechanisms for cancer resistance in elephants and compare cellular response to DNA damage among elephants, healthy human controls, and cancer-prone patients with Li-Fraumeni syndrome (LFS). Design, Setting, and Participants A comprehensive survey of necropsy data was performed across 36 mammalian species to validate cancer resistance in large and long-lived organisms, including elephants (n = 644). The African and Asian elephant genomes were analyzed for potential mechanisms of cancer resistance. Peripheral blood lymphocytes from elephants, healthy human controls, and patients with LFS were tested in vitro in the laboratory for DNA damage response. The study included African and Asian elephants (n = 8), patients with LFS (n = 10), and age-matched human controls (n = 11). Human samples were collected at the University of Utah between June 2014 and July 2015. Exposures Ionizing radiation and doxorubicin. Main Outcomes and Measures Cancer mortality across species was calculated and compared by body size and life span. The elephant genome was investigated for alterations in cancer-related genes. DNA repair and apoptosis were compared in elephant vs human peripheral blood lymphocytes. Results Across mammals, cancer mortality did not increase with body size and/or maximum life span (eg, for rock hyrax, 1% [95% CI, 0%-5%]; African wild dog, 8% [95% CI, 0%-16%]; lion, 2% [95% CI, 0%-7%]). Despite their large body size and long life span, elephants remain cancer resistant, with an estimated cancer mortality of 4.81% (95% CI, 3.14%-6.49%), compared with humans, who have 11% to 25% cancer mortality. While humans have 1 copy (2 alleles) ofTP53, African elephants have at least 20 copies (40 alleles), including 19 retrogenes (38 alleles) with evidence of transcriptional activity measured by reverse transcription polymerase chain reaction. In response to DNA damage, elephant lymphocytes underwent p53-mediated apoptosis at higher rates than human lymphocytes proportional toTP53status (ionizing radiation exposure: patients with LFS, 2.71% [95% CI, 1.93%-3.48%] vs human controls, 7.17% [95% CI, 5.91%-8.44%] vs elephants, 14.64% [95% CI, 10.91%-18.37%];P Conclusions and Relevance Compared with other mammalian species, elephants appeared to have a lower-than-expected rate of cancer, potentially related to multiple copies ofTP53. Compared with human cells, elephant cells demonstrated increased apoptotic response following DNA damage. These findings, if replicated, could represent an evolutionary-based approach for understanding mechanisms related to cancer suppression.

Histone H3 lysine 4 (H3K4) methylation is a dynamic modification. In budding yeast, H3K4 methylation is catalyzed by the Set1-COMPASS methyltransferase complex and is removed by Jhd2, a JMJC domain family demethylase. The catalytic JmjC and JmjN domains of Jhd2 have the ability to remove all three degrees (mono-, di-, and tri-) of H3K4 methylation. Jhd2 also contains a plant homeodomain (PHD) finger required for its chromatin association and H3K4 demethylase functions. The Jhd2 PHD finger associates with chromatin independent of H3K4 methylation and the H3 N-terminal tail. Therefore, how Jhd2 associates with chromatin to perform H3K4 demethylation has remained unknown. We report a novel interaction between the Jhd2 PHD finger and histone H2A. Two residues in H2A (Phe-26 and Glu-57) serve as a binding site for Jhd2 in vitro and mediate its chromatin association and H3K4 demethylase functions in vivo. Using RNA sequencing, we have identified the functional target genes for Jhd2 and the H2A Phe-26 and Glu-57 residues. We demonstrate that H2A Phe-26 and Glu-57 residues control chromatin association and H3K4 demethylase functions of Jhd2 during positive or negative regulation of transcription at target genes. Importantly, we show that H2B Lys-123 ubiquitination blocks Jhd2 from accessing its binding site on chromatin, and thereby, we have uncovered a second mechanism by which H2B ubiquitination contributes to the trans-histone regulation of H3K4 methylation. Overall, our study provides novel insights into the chromatin binding dynamics and H3K4 demethylase functions of Jhd2.

To examine whether caffeine, the most widely used xenobiotic compound, would induce insect cytochrome P450 or CYP gene expression, upstream DNA fragments of Cyp6a2 (0.12, 0.26, 0.52 and 0.98-kb) and Cyp6a8 (0.06, 0.1, 0.2, 0.5 and 0.8-kb) genes of Drosophila melanogaster were individually fused to the firefly luciferase (luc) reporter gene. Promoter activities of these constructs were examined in Drosophila SL-2 cells using luciferase assays. Activity of 0.2- and 0.8-kb upstream DNA of Cyp6a8 was also measured in transgenic female flies. When these flies were treated with 2 mM pure caffeine or Vivarin caffeine, both DNA fragments showed a 4–5-fold induction of promoter activity. Endogenous Cyp6a8 and Cyp6a2 genes in these flies also showed caffeine-induced expression. In addition, both 0.2- and 0.8-kb DNAs showed differential basal and caffeine-induced activity in head, ovaries, gut, cuticle plus fat body and malpighian tubules. However, in all tissues 0.8-kb DNA always showed higher basal and caffeine-induced activities compared to the 0.2-kb DNA, suggesting that the additional DNA present in the 0.8-kb fragment has sequences that enhance both activities. In SL-2 cells, all reporter constructs of each Cyp6 gene showed significantly higher basal activity than the empty vector. Sequences that boost basal activity are located in − 265/− 129 and − 983/− 522 DNA of Cyp6a2, and − 199/− 109 and − 491/− 199 DNA of Cyp6a8 genes. While the 0.12- and 0.1-kb upstream DNAs of Cyp6a2 and Cyp6a8 genes respectively did not show caffeine-inducibility in SL-2 cells, the longest upstream DNA of each gene gave the highest level of induction. Caffeine-responsive sequences are not clustered at one place; they appear to be dispersed in − 983/− 126 and − 761/− 109 regions of Cyp6a2 and Cyp6a8 genes which also contain many binding sites for activator protein 1 (AP1) and cyclic AMP response element binding protein (CRE-BP). Significance of these binding sites in caffeine-inducibility has been discussed.

Class I family enzymes, histone deacetylases 1 and 2 (HDAC1,2), are targets of FDA-approved small molecule inhibitors currently used in cancer therapy. HDAC inhibitors impair DNA repair and replication in cancer cells to trigger genotoxic stress and cause cell death. How HDAC1,2 function in genome maintenance processes, DNA replication and repair, in mammalian cells is far from fully understood. Using small molecules to selectively inhibit HDAC1,2 activities, we have systematically defined their direct functions in DNA replication and repair. Our studies show that HDAC1,2 inhibition reduces replication fork velocity and activates the replication stress associated DNA damage independent of its effect on transcription. HDAC1,2 inhibition increases histone H4K16 acetylation (H4K16ac) and nuclease sensitivity of nucleosomes associated with newly replicated DNA, suggesting that HDAC1,2 are required for chromatin compaction during DNA replication. Thus, mechanistically, HDAC1,2 activities are required for maintaining nascent chromatin structure and dynamics required for efficient progression of the replication fork. In addition to H4K16ac, we have found that HDAC1,2 inhibition activities increases H3K27ac and H4K91ac present on the histone tail and core regions, respectively. Increase in these histone acetyl marks perturbs the acetyl-methyl or the acetyl-ubiquityl switch during DNA repair leading to impaired EZH2-mediated H3K27me3-dependent DNA repair signaling and the H4K91ubiquityl-dependent 53BP1-mediated DNA repair signaling in chemoresistant EZH2 gain-of-function diffuse large B-cell lymphoma cells. We are currently investigating the therapeutic benefits of HDAC1,2 inhibition in mouse models of aggressive chemoresistant diffuse large B-cell lymphoma and in primary patient samples. Overall, our studies have uncovered novel functions for HDAC1,2 in genome maintenance processes and provide insight into the mode-of-action of HDAC1,2 inhibitors in overcoming chemoresistance in cancer cells (via targeting the DNA repair process) and in inducing DNA damage and death in the rapidly cycling cancer cells (via targeting DNA replication). Note: This abstract was not presented at the conference. Citation Format: Danielle Johnson, Simon Jones, Steven Quayle, Jeff Shearstone, Srividya Bhaskara. Inhibition of histone deacetylases 1 and 2 (HDAC1,2) perturbs DNA replication and DNA repair in cancer cells: Implications in mechanism-based therapeutic strategies. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B29.

Histone deacetylase (HDAC) 3, as a cofactor in co-repressor complexes containing silencing mediator for retinoid or thyroid-hormone receptors (SMRT) and nuclear receptor co-repressor (N-CoR), has been shown to repress gene transcription in a variety of contexts. Here, we reveal a novel role for HDAC3 as a positive regulator of IL-1-induced gene expression. Various experimental approaches involving RNAi-mediated knockdown, conditional gene deletion or small molecule inhibitors indicate a positive role of HDAC3 for transcription of the majority of IL-1-induced human or murine genes. This effect was independent from the gene regulatory effects mediated by the broad-spectrum HDAC inhibitor trichostatin A (TSA) and thus suggests IL-1-specific functions for HDAC3. The stimulatory function of HDAC3 for inflammatory gene expression involves a mechanism that uses binding to NF-κB p65 and its deacetylation at various lysines. NF-κB p65-deficient cells stably reconstituted to express acetylation mimicking forms of p65 (p65 K/Q) had largely lost their potential to stimulate IL-1-triggered gene expression, implying that the co-activating property of HDAC3 involves the removal of inhibitory NF-κB p65 acetylations at K122, 123, 314 and 315. These data describe a novel function for HDAC3 as a co-activator in inflammatory signaling pathways and help to explain the anti-inflammatory effects frequently observed for HDAC inhibitors in (pre)clinical use.

Successful DNA replication and packaging of newly synthesized DNA into chromatin are essential to maintain genome integrity. Defects in the DNA template challenge genetic and epigenetic inheritance. Unfortunately, tracking DNA damage responses (DDRs), histone deposition, and chromatin maturation at replication forks is difficult in mammalian cells. Here we describe a technology called iPOND (isolation of proteins on nascent DNA) to analyze proteins at active and damaged replication forks at high resolution. Using this methodology, we define the timing of histone deposition and chromatin maturation. Class 1 histone deacetylases are enriched at replisomes and remove predeposition marks on histone H4. Chromatin maturation continues even when decoupled from replisome movement. Furthermore, fork stalling causes changes in the recruitment and phosphorylation of proteins at the damaged fork. Checkpoint kinases catalyze H2AX phosphorylation, which spreads from the stalled fork to include a large chromatin domain even prior to fork collapse and double-strand break formation. Finally, we demonstrate a switch in the DDR at persistently stalled forks that includes MRE11-dependent RAD51 assembly. These data reveal a dynamic recruitment of proteins and post-translational modifications at damaged forks and surrounding chromatin. Furthermore, our studies establish iPOND as a useful methodology to study DNA replication and chromatin maturation.

While a number of DNA binding transcription factors have been identified that control hematopoietic cell fate decisions, only a limited number of transcriptional corepressors (e.g., the retinoblastoma protein [pRB] and the nuclear hormone corepressor [N-CoR]) have been linked to these functions. Here, we show that the transcriptional corepressor Mtg16 (myeloid translocation gene on chromosome 16), which is targeted by t(16;21) in acute myeloid leukemia, is required for hematopoietic progenitor cell fate decisions and for early progenitor cell proliferation. Inactivation of Mtg16 skewed early myeloid progenitor cells toward the granulocytic/macrophage lineage while reducing the numbers of megakaryocyte-erythroid progenitor cells. In addition, inactivation of Mtg16 impaired the rapid expansion of short-term stem cells, multipotent progenitor cells, and megakaryocyte-erythroid progenitor cells that is required under hematopoietic stress/emergency. This impairment appears to be a failure to proliferate rather than an induction of cell death, as expression of c-Myc, but not Bcl2, complemented the Mtg16(-/-) defect.

The BCL6 transcriptional repressor is required for development of germinal center (GC) B-cells and diffuse large B-cell lymphomas (DLBCL). The BCL6 BTB domain corepressor interaction is an important mediator of BCL6 actions and a potential therapeutic target. BTB point mutations that disrupt corepressor recruitment inactivate BTB domain repressor function. A similar effect can be achieved using specific BCL6 BTB groove binding peptides or small molecules. Although BCL6 can recruit multiple corepressors, its transcriptional repression mechanism of action in normal and malignant B-cells is unknown. In this study we addressed the question of how BCL6 mediates transcriptional repression in normal and malignant B-cells through its association with SMRT or NCOR corepressor complexes. We combined extensive genomic binding (ChIP-seq) analysis of BCL6, its corepressors SMRT and NCOR and epigenetic chromatin patterns both in DLBCL and in primary human GC B-cells, with functional and biochemical experiments performed in vitro, in cell-based assays and in vivo to reveal the biological and transcriptional mechanism of BCL6. We find that in B-cells, virtually all SMRT/NCOR binding sites overlap with BCL6 binding sites indicating that in these cells BCL6 uniquely sequesters SMRT/NCOR complexes from other factors to mediate cell specific functions. Interestingly, these BCL6-SMRT/NCOR complexes preferentially localize at B cell enhancers. Moreover, BCL6-SMRT/NCOR complexes can inactivate these enhancers by deacetylating H3K27. The SMRT/NCOR-dependent H3K27 de-acetylation is mediated by HDAC3 and opposed by p300 histone acetyltransferase. Furthermore, we show that BCL6 BTB targeted inhibitors may reverse the enhancer silencing effect by disrupting the association of BCL6 with SMRT/NCOR complexes. Taken together these results point to an enhancer “toggling” mechanism that might provide the basis for B-cells to undergo rapid transcriptional changes in response to environmental cues. This mechanism might be particularly relevant to the shuttling of B-cells from the dark to the light zone during affinity maturation facilitating the rapid on/off gene expression changes necessary for this transition. For example genes associated with BCL6-SMRT/NCOR enhancers were highly downregulated in the dark zone compared to the light zone and they were also significantly enriched in genes upregulated by CD40 signaling which occurs in the light zone during B-cell activation by Tfh cells and disrupts the association of BCL6 and SMRT/NCOR. In summary we describe a novel mechanism of enhancer regulation. Reversible enhancer toggling may be critical for dynamic modulation of the BCL6 transcriptional program during the GC reaction as well for the therapeutic effects of BCL6 inhibitors. This mechanism is likely a general biological phenomenon that applies to enhancer regulation in a variety cell types and tumors. Citation Format: Katerina Chatzi, Yanwen Jiang, Chuanxin Huang, Paul Zumbo, Srividya Bhaskara, Alexander D. MacKerell, Jr., Fengtian Xue, Christopher E. Mason, Scott W. Hiebert, Leandro Cerchietti, Vivian J. Bardwell, Olivier Elemento, Melnick Ari. A biochemical basis for toggling enhancers from the active to poised state in B-cells. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B14.

The majority of epithelial ovarian carcinomas harbor molecular alterations in DNA-damage response genes. Epithelial ovarian cancers are surprisingly sensitive to DNA-damaging platinum-based (cisplatin or carboplatin) chemotherapy, suggesting that the dependencies of ovarian cancers on impaired DNA-damage response pathways can be harnessed for therapeutic purposes. Our group and others have shown that histone deacetylase (HDAC) inhibitors are promising anti-tumor agents, particularly when combined with DNA-damaging drugs in ovarian cancer cells. In this study, H2AX phosphorylation (pH2Ax), a known histone mark of DNA damage and other histone marks related to DNA damage and repair pathways, including the newly recognized acetylated lysine K56 on histone 3 (H3K56ac) were used to distinguish the biological effects of a unique and diverse panel of clinically utilized and novel HDAC inhibitors. Ovarian cancer cell lines with diverse molecular alterations, including mutations in p53, Brca1, Akt, Ras, and overexpression of MDR1, were utilized in these screens. The HDAC inhibitors that induced the highest levels of pH2Ax produced the greatest anti-tumor effects (growth inhibition and apoptosis). Furthermore, a screen of DNA-damage response histone marks led to the selection of novel HDAC inhibitors that exerted synergistic anti-tumor effects when combined with cisplatin and other DNA-damaging drugs. Taken together, our results support a role of HDAC inhibitors in the treatment of ovarian cancer and suggest that pH2Ax is a potential surrogate biomarker of the efficacy of HDAC inhibitor combination therapy with DNA-damaging agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2985.

Summary The BCL6 transcriptional repressor is required for the development of germinal center (GC) B cells and diffuse large B cell lymphomas (DLBCLs). Although BCL6 can recruit multiple corepressors, its transcriptional repression mechanism of action in normal and malignant B cells is unknown. We find that in B cells, BCL6 mostly functions through two independent mechanisms that are collectively essential to GC formation and DLBCL, both mediated through its N-terminal BTB domain. These are (1) the formation of a unique ternary BCOR-SMRT complex at promoters, with each corepressor binding to symmetrical sites on BCL6 homodimers linked to specific epigenetic chromatin features, and (2) the "toggling" of active enhancers to a poised but not erased conformation through SMRT-dependent H3K27 deacetylation, which is mediated by HDAC3 and opposed by p300 histone acetyltransferase. Dynamic toggling of enhancers provides a basis for B cells to undergo rapid transcriptional and phenotypic changes in response to signaling or environmental cues.

Background Histone deacetylases (HDACs) play a critical role in the maintenance of genome stability. Class I HDACs, histone deacetylase 1 and 2 (Hdac1 and Hdac2) are recruited to the replication fork by virtue of their interactions with the replication machinery. However, functions for Hdac1 and Hdac2 (Hdacs1,2) in DNA replication are not fully understood. Results Using genetic knockdown systems and novel Hdacs1,2-selective inhibitors, we found that loss of Hdacs1,2 leads to a reduction in the replication fork velocity, and an increase in replication stress response culminating in DNA damage. These observed defects are due to a direct role for Hdacs1,2 in DNA replication, as transcription of genes involved in replication was not affected in the absence of Hdacs1,2. We found that loss of Hdacs1,2 functions increases histone acetylation (ac) on chromatin in S-phase cells and affects nascent chromatin structure, as evidenced by the altered sensitivity of newly synthesized DNA to nuclease digestion. Specifically, H4K16ac, a histone modification involved in chromatin decompaction, is increased on nascent chromatin upon abolishing Hdacs1,2 activities. It was previously shown that H4K16ac interferes with the functions of SMARCA5, an ATP-dependent ISWI family chromatin remodeler. We found SMARCA5 also associates with nascent DNA and loss of SMARCA5 decreases replication fork velocity similar to the loss or inhibition of Hdacs1,2. Conclusions Our studies reveal important roles for Hdacs1,2 in nascent chromatin structure maintenance and regulation of SMARCA5 chromatin-remodeler function, which together are required for proper replication fork progression and genome stability in S-phase.