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.

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.