Combined epigenetic and immunotherapy for blastic and classical mantle cell lymphoma.

Classical MCL (cMCL) constitutes 6-8% of all B cell NHL. Despite recent advances, MCL is incurable except with allogeneic stem cell transplant. Blastic mantle cell lymphoma (bMCL) is a rarer subtype of cMCL associated with an aggressive clinical course and poor treatment response, frequent relapse and poor outcomes. We treated 13 bMCL patients with combined epigenetic and immunotherapy treatment consisting of vorinostat, cladribine and rituximab (SCR). We report an increased OS greater than 40 months with several patients maintaining durable remissions without relapse for longer than 5 years. This is remarkably better then current treatment regimens which in bMCL range from 14.5-24 months with conventional chemotherapy regimens. We demonstrate that the G/A870 CCND1 polymorphism is predictive of blastic disease, nuclear localization of cyclinD1 and response to SCR therapy. The major resistance mechanisms to SCR therapy are loss of CD20 expression and evasion of treatment by sanctuary in the CNS. These data indicate that administration of epigenetic agents improves efficacy of anti-CD20 immunotherapies. This approach is promising in the treatment of MCL and potentially other previously treatment refractory cancers.

Distinct epigenetic features of differentiation-regulated replication origins.

BACKGROUND: Eukaryotic genome duplication starts at discrete sequences (replication origins) that coordinate cell cycle progression, ensure genomic stability and modulate gene expression. Origins share some sequence features, but their activity also responds to changes in transcription and cellular differentiation status. RESULTS: To identify chromatin states and histone modifications that locally mark replication origins, we profiled origin distributions in eight human cell lines representing embryonic and differentiated cell types. Consistent with a role of chromatin structure in determining origin activity, we found that cancer and non-cancer cells of similar lineages exhibited highly similar replication origin distributions. Surprisingly, our study revealed that DNase hypersensitivity, which often correlates with early replication at large-scale chromatin domains, did not emerge as a strong local determinant of origin activity. Instead, we found that two distinct sets of chromatin modifications exhibited strong local associations with two discrete groups of replication origins. The first origin group consisted of about 40,000 regions that actively initiated replication in all cell types and preferentially colocalized with unmethylated CpGs and with the euchromatin markers, H3K4me3 and H3K9Ac. The second group included origins that were consistently active in cells of a single type or lineage and preferentially colocalized with the heterochromatin marker, H3K9me3. Shared origins replicated throughout the S-phase of the cell cycle, whereas cell-type-specific origins preferentially replicated during late S-phase. CONCLUSIONS: These observations are in line with the hypothesis that differentiation-associated changes in chromatin and gene expression affect the activation of specific replication origins.

The DNA repair endonuclease Mus81 facilitates fast DNA replication in the absence of exogenous damage.

The Mus81 endonuclease resolves recombination intermediates and mediates cellular responses to exogenous replicative stress. Here, we show that Mus81 also regulates the rate of DNA replication during normal growth by promoting replication fork progression while reducing the frequency of replication initiation events. In the absence of Mus81 endonuclease activity, DNA synthesis is slowed and replication initiation events are more frequent. In addition, Mus81-deficient cells fail to recover from exposure to low doses of replication inhibitors and cell viability is dependent on the XPF endonuclease. Despite an increase in replication initiation frequency, cells lacking Mus81 use the same pool of replication origins as Mus81-expressing cells. Therefore, decelerated DNA replication in Mus81-deficient cells does not initiate from cryptic or latent origins not used during normal growth. These results indicate that Mus81 plays a key role in determining the rate of DNA replication without activating a novel group of replication origins.

Allele-specific genome-wide profiling in human primary erythroblasts reveal replication program organization.

We have developed a new approach to characterize allele-specific timing of DNA replication genome-wide in human primary basophilic erythroblasts. We show that the two chromosome homologs replicate at the same time in about 88% of the genome and that large structural variants are preferentially associated with asynchronous replication. We identified about 600 megabase-sized asynchronously replicated domains in two tested individuals. The longest asynchronously replicated domains are enriched in imprinted genes suggesting that structural variants and parental imprinting are two causes of replication asynchrony in the human genome. Biased chromosome X inactivation in one of the two individuals tested was another source of detectable replication asynchrony. Analysis of high-resolution TimEX profiles revealed small variations termed timing ripples, which were undetected in previous, lower resolution analyses. Timing ripples reflect highly reproducible, variations of the timing of replication in the 100 kb-range that exist within the well-characterized megabase-sized replication timing domains. These ripples correspond to clusters of origins of replication that we detected using novel nascent strands DNA profiling methods. Analysis of the distribution of replication origins revealed dramatic differences in initiation of replication frequencies during S phase and a strong association, in both synchronous and asynchronous regions, between origins of replication and three genomic features: G-quadruplexes, CpG Islands and transcription start sites. The frequency of initiation in asynchronous regions was similar in the two homologs. Asynchronous regions were richer in origins of replication than synchronous regions.

Methylation of histone H3 on lysine 79 associates with a group of replication origins and helps limit DNA replication once per cell cycle.

Mammalian DNA replication starts at distinct chromosomal sites in a tissue-specific pattern coordinated with transcription, but previous studies have not yet identified a chromatin modification that correlates with the initiation of DNA replication at particular genomic locations. Here we report that a distinct fraction of replication initiation sites in the human genome are associated with a high frequency of dimethylation of histone H3 lysine K79 (H3K79Me2). H3K79Me2-containing chromatin exhibited the highest genome-wide enrichment for replication initiation events observed for any chromatin modification examined thus far (23.39% of H3K79Me2 peaks were detected in regions adjacent to replication initiation events). The association of H3K79Me2 with replication initiation sites was independent and not synergistic with other chromatin modifications. H3K79 dimethylation exhibited wider distribution on chromatin during S-phase, but only regions with H3K79 methylation in G1 and G2 were enriched in replication initiation events. H3K79 was dimethylated in a region containing a functional replicator (a DNA sequence capable of initiating DNA replication), but the methylation was not evident in a mutant replicator that could not initiate replication. Depletion of DOT1L, the sole enzyme responsible for H3K79 methylation, triggered limited genomic over-replication although most cells could continue to proliferate and replicate DNA in the absence of methylated H3K79. Thus, prevention of H3K79 methylation might affect regulatory processes that modulate the order and timing of DNA replication. These data are consistent with the hypothesis that dimethylated H3K79 associates with some replication origins and marks replicated chromatin during S-phase to prevent re-replication and preserve genomic stability.

Genome-wide depletion of replication initiation events in highly transcribed regions.

This report investigates the mechanisms by which mammalian cells coordinate DNA replication with transcription and chromatin assembly. In yeast, DNA replication initiates within nucleosome-free regions, but studies in mammalian cells have not revealed a similar relationship. Here, we have used genome-wide massively parallel sequencing to map replication initiation events, thereby creating a database of all replication initiation sites within nonrepetitive DNA in two human cell lines. Mining this database revealed that genomic regions transcribed at moderate levels were generally associated with high replication initiation frequency. In genomic regions with high rates of transcription, very few replication initiation events were detected. High-resolution mapping of replication initiation sites showed that replication initiation events were absent from transcription start sites but were highly enriched in adjacent, downstream sequences. Methylation of CpG sequences strongly affected the location of replication initiation events, whereas histone modifications had minimal effects. These observations suggest that high levels of transcription interfere with formation of pre-replication protein complexes. Data presented here identify replication initiation sites throughout the genome, providing a foundation for further analyses of DNA-replication dynamics and cell-cycle progression.

Preferential localization of human origins of DNA replication at the 5'-ends of expressed genes and at evolutionarily conserved DNA sequences.

BACKGROUND: Replication of mammalian genomes requires the activation of thousands of origins which are both spatially and temporally regulated by as yet unknown mechanisms. At the most fundamental level, our knowledge about the distribution pattern of origins in each of the chromosomes, among different cell types, and whether the physiological state of the cells alters this distribution is at present very limited. METHODOLOGY/PRINCIPAL FINDINGS: We have used standard λ-exonuclease resistant nascent DNA preparations in the size range of 0.7-1.5 kb obtained from the breast cancer cell line MCF-7 hybridized to a custom tiling array containing 50-60 nt probes evenly distributed among genic and non-genic regions covering about 1% of the human genome. A similar DNA preparation was used for high-throughput DNA sequencing. Array experiments were also performed with DNA obtained from BT-474 and H520 cell lines. By determining the sites showing nascent DNA enrichment, we have localized several thousand origins of DNA replication. Our major findings are: (a) both array and DNA sequencing assay methods produced essentially the same origin distribution profile; (b) origin distribution is largely conserved (>70%) in all cell lines tested; (c) origins are enriched at the 5'ends of expressed genes and at evolutionarily conserved intergenic sequences; and (d) ChIP on chip experiments in MCF-7 showed an enrichment of H3K4Me3 and RNA Polymerase II chromatin binding sites at origins of DNA replication. CONCLUSIONS/SIGNIFICANCE: Our results suggest that the program for origin activation is largely conserved among different cell types. Also, our work supports recent studies connecting transcription initiation with replication, and in addition suggests that evolutionarily conserved intergenic sequences have the potential to participate in origin selection. Overall, our observations suggest that replication origin selection is a stochastic process significantly dependent upon local accessibility to replication factors.

Inhibition of histone deacetylase in cancer cells slows down replication forks, activates dormant origins, and induces DNA damage.

Protein acetylation is a reversible process regulated by histone deacetylases (HDAC) that is often altered in human cancers. Suberoylanilide hydroxamic acid (SAHA) is the first HDAC inhibitor to be approved for clinical use as an anticancer agent. Given that histone acetylation is a key determinant of chromatin structure, we investigated how SAHA may affect DNA replication and integrity to gain deeper insights into the basis for its anticancer activity. Nuclear replication factories were visualized with confocal immunofluorescence microscopy and single-replicon analyses were conducted by genome-wide molecular combing after pulse labeling with two thymidine analogues. We found that pharmacologic concentrations of SAHA induce replication-mediated DNA damage with activation of histone gammaH2AX. Single DNA molecule analyses indicated slowdown in replication speed along with activation of dormant replication origins in response to SAHA. Similar results were obtained using siRNA-mediated depletion of HDAC3 expression, implicating this HDAC member as a likely target in the SAHA response. Activation of dormant origins was confirmed by molecular analyses of the beta-globin locus control region. Our findings demonstrate that SAHA produces profound alterations in DNA replication that cause DNA damage, establishing a critical link between robust chromatin acetylation and DNA replication in human cancer cells.

Human uterine smooth muscle and leiomyoma cells differ in their rapid 17beta-estradiol signaling: implications for proliferation.

Uterine leiomyomas, benign uterine smooth muscle tumors that affect 30% of reproductive-aged women, are a significant health concern. The initiation event for these tumors is unclear, but 17beta-estradiol (E2) is an established promoter of leiomyoma growth. E2 not only alters transcription of E2-regulated genes but also can rapidly activate signaling pathways. The aim of our study is to investigate the role of rapid E2-activated cytoplasmic signaling events in the promotion of leiomyomas. Western blot analysis revealed that E2 rapidly increases levels of phosphorylated protein kinase C alpha (PKC alpha) in both immortalized uterine smooth muscle (UtSM) and leiomyoma (UtLM) cell lines, but increases levels of phosphorylated ERK1/2 only in UtLM cells. Our studies demonstrate a paradoxical effect of molecular and pharmacological inhibition of PKC alpha on ERK1/2 activation and cellular proliferation in UtLM and UtSM cells. PKC alpha inhibition decreases levels of phosphorylated ERK1/2 and proliferation in UtLM cells but raises these levels in UtSM cells. cAMP-PKA signaling is rapidly activated only in UtSM cells with E2 and inhibits ERK1/2 activation and proliferation. We therefore propose a model whereby E2's rapid activation of PKC alpha and cAMP-PKA signaling plays a central role in the maintenance of a low proliferative index in normal uterine smooth muscle via its inhibition of the MAPK cascade and these pathways are altered in leiomyomas to promote MAPK activation and proliferation. These studies demonstrate that rapid E2-signaling pathways contribute to the promotion of leiomyomas.

Bloom's syndrome helicase and Mus81 are required to induce transient double-strand DNA breaks in response to DNA replication stress.

Perturbed DNA replication either activates a cell cycle checkpoint, which halts DNA replication, or decreases the rate of DNA synthesis without activating a checkpoint. Here we report that at low doses, replication inhibitors did not activate a cell cycle checkpoint, but they did activate a process that required functional Bloom's syndrome-associated (BLM) helicase, Mus81 nuclease and ataxia telangiectasia mutated and Rad3-related (ATR) kinase to induce transient double-stranded DNA breaks. The induction of transient DNA breaks was accompanied by dissociation of proliferating cell nuclear antigen (PCNA) and DNA polymerase alpha from replication forks. In cells with functional BLM, Mus81 and ATR, the transient breaks were promptly repaired and DNA continued to replicate at a slow pace in the presence of replication inhibitors. In cells that lacked BLM, Mus81, or ATR, transient breaks did not form, DNA replication did not resume, and exposure to low doses of replication inhibitors was toxic. These observations suggest that BLM helicase, ATR kinase, and Mus81 nuclease are required to convert perturbed replication forks to DNA breaks when cells encounter conditions that decelerate DNA replication, thereby leading to the rapid repair of those breaks and resumption of DNA replication without incurring DNA damage and without activating a cell cycle checkpoint.

DNA-PK is involved in repairing a transient surge of DNA breaks induced by deceleration of DNA replication.

Cells that suffer substantial inhibition of DNA replication halt their cell cycle via a checkpoint response mediated by the PI3 kinases ATM and ATR. It is unclear how cells cope with milder replication insults, which are under the threshold for ATM and ATR activation. A third PI3 kinase, DNA-dependent protein kinase (DNA-PK), is also activated following replication inhibition, but the role DNA-PK might play in response to perturbed replication is unclear, since this kinase does not activate the signaling cascades involved in the S-phase checkpoint. Here we report that mild, transient drug-induced perturbation of DNA replication rapidly induced DNA breaks that promptly disappeared in cells that contained a functional DNA-PK whereas such breaks persisted in cells that were deficient in DNA-PK activity. After the initial transient burst of DNA breaks, cells with a functional DNA-PK did not halt replication and continued to synthesize DNA at a slow pace in the presence of replication inhibitors. In contrast, DNA-PK deficient cells subject to low levels of replication inhibition halted cell cycle progression via an ATR-mediated S-phase checkpoint. The ATM kinase was dispensable for the induction of the initial DNA breaks. These observations suggest that DNA-PK is involved in setting a high threshold for the ATR-Chk1-mediated S-phase checkpoint by promptly repairing DNA breaks that appear immediately following inhibition of DNA replication.