Abstract 673
Mantle Cell Lymphoma (MCL) is an aggressive and incurable malignancy arising from naïve B cells (NBC) in the mantle zone of lymph node follicles. Murine models over-expressing Cyclin D1, ...the putative oncogene implicated in the majority of MCL, do not fully recapitulate the disease phenotype. We therefore hypothesize that there are additional mechanisms contributing to MCL pathogenesis and undertook an integrative approach by studying genome-wide DNA methylation and gene expression in primary MCL to uncover additional genes and pathways involved in MCL pathogenesis.
We therefore compared and contrasted the DNA methylation levels of 14,000 gene promoters in MCL patients and normal tonsillar NBC controls using the HELP (HPA II tiny fragment Enrichment by Ligation mediated PCR) assay. All patient samples were obtained prior to any treatment from peripheral blood or apheresis specimens from newly diagnosed patients with histologically confirmed MCL. We found significant hypo-methylation of gene promoters in the MCL patients as compared to normal NBCs. Integrating genomic methylation data from HELP and gene expression data from Affymetrix U133 arrays, we determined a signature of differentially methylated genes with reciprocal changes in mRNA levels. Using pathway analysis and gene ontogeny, we selected genes for validation by choosing loci that were differentially methylated and fulfilling the following characteristics (i) demonstrating a clear methylation state change (from hypomethylated in normal B cells to methylated in MCL or vice-versa) using a threshold of 0.0 on the log ratio scale, (ii) Genes that function as tumor suppressors and were hypermethylated and suppressed in MCLs in our data (iii) Overexpressed/ hypomethylated genes with existing therapeutic options available or in clinical trial (iv) involved in pathways controlling biological processes with known relevance to MCL i.e. cell cycle control, apoptosis. Our panel included four differentially hypermethylated genes CDKN2B, MLF-1, PCDH8, HOXD8 and four differentially hypomethylated genes CD37, HDAC1, NOTCH1 and CDK5. MassArray Epityper analysis confirmed the presence of differential methylation at the promoter region of these genes, which was consistent between MCL patients and cell lines in all 8 genes studied. Remarkably, PCDH8 and CDKN2B have previously been shown to be silenced by methylation at their gene promoters and transfection of PCDH8 and CDKN2B have been shown to reduce tumor growth in breast cancer and MCL cell line models respectively.
Based on these data, we hypothesized that the aberrantly hypermethylated and thereby silenced tumor suppressor genes in MCL could be pharmacologically induced by DNA hypomethylating agents and HDAC inhibitors for therapeutic benefit. We therefore next treated MCL cell lines MINO and Z138 with the DNA methyltransferase inhibitor Decitabine alone and in combination with the HDAC inhibitor SAHA. HELP analysis of MINO and Z138 cells treated with hypomethylating doses of decitabine (0.5uM × 3days) showed widespread reversal of aberrant gene promoter hypermethylation. Hypomethylation in these cell lines was accompanied with 3-7 fold increase in mRNA levels of tumor suppressor genes CDKN2B, MLF-1, PCDH8 and HOXD8. Concurrent treatment with SAHA (1 uM x 1 dose) synergized with Decitabine leading to 5-15 fold increase in mRNA of these tumor suppressor genes in Z138 cells. Importantly, treatment with Decitabine and SAHA as single agents decreased MCL cell viability by 60% and 40% respectively and the combination synergised in anti-MCL cytotoxicity with > 90% decrease in cell viability.
In conclusion, our analysis shows prominent aberrant gene promoter methylation patterns in MCL genome and identifies novel differentially methylated and expressed genes in MCL cell lines and primary MCLs. Furthermore, we demonstrate that reversal of aberrant hypermethylated and silenced genes can be targeted for therapeutic benefit using epigenetic drugs in MCL. We are currently modeling our combination epigenetic drug therapy in primary MCL cells in culture and murine xenograft systems. We expect these results to support the development of clinical trials investigating the prospective use of combination epigenetic therapy in MCL.
No relevant conflicts of interest to declare.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Bortezomib is more active against mantle cell lymphoma (MCL) than against most other lymphoma subtypes. Nevertheless, up to half of patients with MCL have bortezomib resistant disease. Factors ...contributing to intrinsic resistance to bortezomib have not been determined. Here we used a panel of eight bortezomib sensitive (median IC50 5.9 nM) and three relatively bortezomib resistant cell lines (median IC50 12.9 nM) to investigate differences in tumor biology that could determine sensitivity to bortezomib. Bortezomib effectively inhibited high baseline proteasome activity and induced a comparable degree of proteasome inhibition in both sensitive and resistant cells. At 10 nM, bortezomib induced the proapoptotic BH3-only protein Noxa in sensitive but not resistant cells. At higher concentrations of bortezomib, however, Noxa was also upregulated in resistant cells and this effect was sufficient to induce apoptosis. Silencing of Noxa with siRNA rescued these cells from apoptosis, arguing against a defect in Noxa regulation or function as the basis of bortezomib resistance. Bortezomib was equally effective against cells with high and low constitutive NF-κB signaling. Also, sensitive and resistant MCL cell lines showed comparable activation of the AKT pathway. We conclude that bortezomib can overcome classic mechanisms of resistance to apoptosis and that determinants of bortezomib sensitivity in MCL are due to differences in signaling or stress pathways upstream of Noxa.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
L'origine des pharmacopées traditionnelles L'élaboration des pharmacopées Les médicaments du XXIe siècle Comment les connaissances des savoirs thérapeutiques se sont-elles transmises au travers des ...différentes cultures ? Cet ouvrage innovant, qui réunit les travaux présentés au 4e Congrès européen d'ethnopharmacologie, fait remonter à la préhistoire les sources des connaissances thérapeutiques. Si les pharmacopées écrites jalonnent l'histoire des grandes médecines savantes, d'autres modes d'accès à la connaissance semblent exister dans l'univers chamanique des sociétés de tradition orale ainsi que dans la façon dont les animaux malades se soignent par les plantes. L'évaluation des propriétés pharmaco-toxicologiques et chimiques des plantes d'usage traditionnel devrait par ailleurs favoriser le développement futur des médicaments à base de plantes, l'un des thèmes porteurs abordés dans cet ouvrage. Mais l'objectif de ce livre est aussi de susciter, partout dans le monde, de nouvelles thématiques de recherche dans le domaine de la préhistoire du médicament et de la compréhension de l'acquisition et de la transmission du savoir. Le développement du phytomédicament non toxique destiné à l'homme et à l'animal figure également parmi les enjeux majeurs de demain.
