Epigenetic Determinants of Pathogenesis and Resistance to Proteosome Inhibition in Mantle Cell Lymphoma.

Mantle Cell Lymphoma (MCL) is an aggressive tumor accounting for 5% of non Hodgkin’s lymphomas(NHL). Given the poor clinical outcomes in this disease with current therapy, it is of great importance to better understand disease pathogenesis. Since aberrant epigenetic gene regulation is a hallmark of cancer, we hypothesized that an unbiased genome-wide DNA methylation study would identify genes involved in MCL pathogenesis. Moreover, we hypothesized that aberrantly silenced/hypermethylated genes responsible for drug sensitivity in MCL could be pharmacologically manipulated for maximizing clinical benefit. To address these questions, we examined the abundance of DNA methylation in 25,000 promoter fragments using the HELP (HPA II Enzyme Ligation mediated PCR amplification) assay in MCL cell lines. This study revealed s substantial heterogeneity in genomic methylation between cell lines, possibly reflective of underlying biologic diversity in this disease. However, MCL cell lines did display consistent aberrant methylation when compared to naïve B cells extracted from normal human tonsils. Remarkably, among the 102 genes with a methylation fold change in >3 and p<0.0001 the majority (100) were hypermethylated in MCL vs. normal B cells. Amongst these genes we identified five tumor suppressor genes implicated in the pathogenesis of other cancers: Protocadherin 8,Paternally expressed gene 3 (PEG3),Myeloid leukemia factor 1, Transcription Factor AP-2 gamma and Homeobox D8. Interestingly. PEG3 and Protocadherin 8 are both known to be silenced by hypermethylation at their gene promoters in gastric and breast cancer respectively. These genes may also possibly function as tumor suppressor genes in MCL pathogenesis and are excellent candidates for future functional studies. We then integrated methylation and gene expression data to identify loci that were both significantly methylated and suppressed in MCL. To identify the pathways and biological processes most relevant to our data set of methylated genes, we queried the Ingenuity Pathway Analysis (IPA) Knowledge base with the top 560 hypermethylated genes (>2 Standard Deviation) across all MCL cell lines. An unsupervised core analysis from the IPA knowledge base using our gene set revealed gene networks revolving around tumor suppressor TP53 and the NPM1 transcription factor in MCL cell lines. Locus specific confirmation by MassArray confirmed methylation of the distal portion of the TP53 promotor in the MCL cell lines. Recently, the proteosome inhibitor Bortezomib(BZM) has been shown to have specific activity in MCL, with single agent response rates ~40% in heavily pretreated patients. We wondered whether aberrant epigenetic programming might contribute to resistance to this agent and whether reversal of DNA methylation could overcome cellular mechanisms of resistance to Bortezomib. Therefore, to determine whether pharmacologic re-expression of genes could overcome bortezomib resistance, we treated MINO (a Bortezomib-refractory MCL cell line) with two low doses of Decitabine (0.1 uM and 1 uM) for 48 hours and sequentially with 15 nM of Bortezomib (IC50) for an additional 48 hours. Pretreatment of BZM-resistant MINO cells with Decitabine significantly (p<0.001) reduced cell viability as compared to control, suggesting that Decitabine can overcome BZM resistance in this refractory cell line. We are currently determining which genes contribute to this effect and validating these results in patient samples from a prospective trial of BZM+EPOCH. We predict that this will provide the basis for future trials individualizing BZM based therapy based on methylation and gene expression signatures in MCL.