Research Focus
Over the past several years our laboratory has focused on three interrelated areas; the mechanisms by which histone acetylation and methylation silences immune genes in cancer cells, studies on the design and application of agents that alter chromatin repression (epigenetic agents), and finally the role of microRNAs (miRNAs) in immune gene regulation and their potential use in the development of vaccines and treatments for tumors.

Mechanisms by which Histone Acetylation and Methylation
Silences Immune Genes in Cancer Cells

We first demonstrated that histone deacetylase inhibition can activate epigenetically silenced immune genes and have characterized  the histone modifications associated with IFN-γ induced (CIITA-dependent)  and HDACi (CIITA-independent) activation of MHC class II expression. 

Figure1.  Schematic of the potential mechanisms involved in the regulation of various histone modifications during distinct stages of DRα transcription.  In the presence of activation signals, the MHC class II enhanceosome forms at the DRα promoter and leads to histone acetylation at the LCR and promoter.  The H3K4 and H3K79 histone methyltransferases associate with the RNA pol II complex through interaction with PAF, leading to methylation of K4 and K79 at the DRα promoter and elicit transcription initiation.  As the transcription complex proceeds downstream, DRα exon 3 becomes enriched with methylated K36 and K79.  During the later stage of elongation at the 3′ end of the DRα gene, K79 HMT is released from the RNA pol II complex but K36 HMT remains bound to the transcription complex.  As transcription activation continues, the chaperone protein HIRA may mediate the exchange of the histone H3.3 variant for H3.1 in a replication-independent manner.

Using ChIP assays we demonstrated the location of acetylation and methylation of histone lysine in cancer cells and the differential spatial distribution of lysine alterations along the gene (locus control, promoter, exon3 and exon5). 

These studies suggest that these markers play a distinctive role during different phases of the transcription process.  Moreover, the HDACi trichostatin A (TSA), can replace the function of four histone acetyltransferases (CIITA, CBP, p300, PCAF) known to be recruited and required for activation of MHC class II. 

The Design and Application of Epigenetic Agents that alter Chromatin for Vaccines and Systemic Therapies

The HDACi TSA was shown to activate MHC class I and II, CD40, B7-1, B7-2 and NKG2D ligands in cancer cells in which these genes are repressed.  Based on this information, we developed an ‘epigenetic cancer vaccine’.  HDACi treated but not untreated cells can present antigens in vitro.  Cancer cells treated in vitro with either TSA or valproic acid, used in a single sc injection as a vaccine, were shown to establish durable immunity in two murine tumor models (J558 myeloma and B16 melanoma). 

Figure 2.  Epigenetic tumor cell vaccination: Tumor cells, treated with HDACi, were evaluated for the expression of immune molecules and antigen presentation in vitro and then inoculated subcutaneous. Tumor-free mice, after 40 days, were re-challenged with untreated cells and observed for tumor growth and CTLs.

Immunity was shown to be mediated by CD4, CD8 and also NK cells.  Studies in bone marrow chimeras (MHC class I and II, double-knockouts) suggests that the efficiency of the vaccine in vivo derives mainly from cross-presentation.  However, direct presentation of antigens by tumor cells may also contribute to immunity, although the magnitude of this contribution is as yet uncertain.  In our ongoing studies, attention to the type of HDACi, dosage, timing and various immunization protocols have improved the prevention statistics (to 100% with a single dose) and allowed initial positive results with a treatment model in which the tumor was allowed to reach palpable size. 

The recent finding that pretreatment of tumor cells with low dose HDACi can restore a robust response to IFN-γ with expression of MHC and costimulatory genes in an otherwise IFN-γ unresponsive tumor cell introduces a new dimension in treatment options with combined HDACi and IFN-γ.  These combinations are being explored in head and neck tumors.

The Role of microRNAs in Immune Gene Regulation

Figure 3.  Cellular Networks – Potential targets of miRNAs (Figure patterned after the neuronal pathways reported by Cui, Q et al., 2006).

Because of the repressive effects of miRNA we asked whether cancer cells might use miRs to repress immune genes.  We first carried out a computational investigation to determine the immune genes that are targeted by miRs.  We studied 613 genes known to regulate immunity and, utilizing predictive algorithms, identified 285 of these genes as microRNA (miRNA or miR) targets.  Of these, ~250 are newly predicted gene-miR interactions.  A statistical analysis of predicted miRNA binding sites in immune gene 3′UTRs indicated preferential targeting of immune genes compared to the genome.  Major targets include transcription factors, cofactors and chromatin modifiers whereas upstream factors, such as ligands and receptors (cytokines, chemokines and TLRs), were in general, non-targets. 

Figure 4.  Bioinfomatic Analysis of 3′UTR Binding Sites for miRNA in 613 Immune Gene Targets.

About 10% of the immune genes were ‘hubs’ with eight or more different miRNAs predicted to target their 3′UTRs.  Hubs focused on certain key immune genes, such as BCL6, SMAD7, BLIMP1, NFAT5, EP300 and others. 

MHC genes lacked miRNA target sites but two sites were identified in the CIITA gene and were shown experimentally to repress IFN-γ induced MHC class II activation.  Unexpectedly, factors involved in regulating message stability via AU-rich elements (ARE) were targets.  Moreover, multiple components involved in the generation and effector functions of miRNAs (Dicer and Argonautes) were themselves miRNA targets suggesting that a subset of miRNAs could indirectly control their own production as well as other miRNAs.  This prompted further studies on Dicer regulation.

Figure 5.  MicroRNAs, Chromatin, Stress and Expression.  Various stresses mediate multiple signaling pathways.  MiRs that regulate Dicer and chromatin are components of stress pathways that regulate gene expression patterns.

We found that Dicer levels of human cells vary substantially between cell types and several reports demonstrate that Dicer may be up or downregulated in different cancer types.  As mentioned above components of the miRNA machinery were themselves predicted targets of specific sets of miRNAs.  The RNase III enzyme, Drosha, and its RNA binding partner, Pasha/DGCR8, were non-targets.  However, Exportin-5, required for pre-miRNA transport from the nucleus to the cytoplasm, was the target of two miRNAs.  Strikingly, the DICER1 3′UTR was found to have 25 individual miRNA binding sites of which 6 were selected by all three algorithms.  Notably, an intense focus of miRNAs occurred at AGO1, with 62 potential miRNA binding sites in its 5682bp 3′UTR.  Of these, 20 high probability sites were selected by three algorithms.  AGO 2, 3 and 4 had 4, 1 and 12 miRNA binding sites respectively.  The let-7 miRNA targeted all four Argonaute family members and one algorithm (PicTar) also indicated a let-7 site in DICER1.  Other common miRNAs between DICER1 and AGO1 include miR-122, -103, -107, -124, -130, and 29.  These findings have broad implications and additional experiments, currently in progress, are necessary to clarify the functional significance of miRNA targets we identified in the RNAi machinery components.  One recent offshoot of the above finding has shown that several types of stress pathways, including dsRNA induced stress, regulate Dicer at the protein level. 

Research, currently in progress, focuses on attempts to produce ‘Dicer vaccines’ by upregulating silenced immune genes in tumor cells through transfection with siRNA or miRNA that represses Dicer.