Aberration in DNA Methylation in B-Cell Lymphomas Has a Complex Origin and Increases with Disease Severity

Despite mounting evidence that epigenetic abnormalities play a key role in cancer biology, their contributions to the malignant phenotype remain poorly understood. Here we studied genome-wide DNA methylation in normal B-cell populations and subtypes of B-cell non-Hodgkin lymphoma: follicular lymphoma and diffuse large B-cell lymphomas. These lymphomas display striking and progressive intra-tumor heterogeneity and also inter-patient heterogeneity in their cytosine methylation patterns. Epigenetic heterogeneity is initiated in normal germinal center B-cells, increases markedly with disease aggressiveness, and is associated with unfavorable clinical outcome. Moreover, patterns of abnormal methylation vary depending upon chromosomal regions, gene density and the status of neighboring genes. DNA methylation abnormalities arise via two distinct processes: i) lymphomagenic transcriptional regulators perturb promoter DNA methylation in a target gene-specific manner, and ii) aberrant epigenetic states tend to spread to neighboring promoters in the absence of CTCF insulator binding sites

Precision Medicine for Relapsed Multiple Myeloma on the Basis of an Integrative Multiomics Approach.

PURPOSE: Multiple myeloma (MM) is a malignancy of plasma cells, with a median survival of 6 years. Despite recent therapeutic advancements, relapse remains mostly inevitable, and the disease is fatal in the majority of patients. A major challenge in the treatment of patients with relapsed MM is the timely identification of treatment options in a personalized manner. Current approaches in precision oncology aim at matching specific DNA mutations to drugs, but incorporation of genome-wide RNA profiles has not yet been clinically assessed. METHODS: We have developed a novel computational platform for precision medicine of relapsed and/or refractory MM on the basis of DNA and RNA sequencing. Our approach expands on the traditional DNA-based approaches by integrating somatic mutations and copy number alterations with RNA-based drug repurposing and pathway analysis. We tested our approach in a pilot precision medicine clinical trial with 64 patients with relapsed and/or refractory MM. RESULTS: We generated treatment recommendations in 63 of 64 patients. Twenty-six patients had treatment implemented, and 21 were assessable. Of these, 11 received a drug that was based on RNA findings, eight received a drug that was based on DNA, and two received a drug that was based on both RNA and DNA. Sixteen of the 21 evaluable patients had a clinical response (ie, reduction of disease marker ≥ 25%), giving a clinical benefit rate of 76% and an overall response rate of 66%, with five patients having ongoing responses at the end of the trial. The median duration of response was 131 days. CONCLUSION: Our results show that a comprehensive sequencing approach can identify viable options in patients with relapsed and/or refractory myeloma, and they represent proof of principle of how RNA sequencing can contribute beyond DNA mutation analysis to the development of a reliable drug recommendation tool

MALT1 Small Molecule Inhibitors Specifically Suppress ABC-DLBCL In Vitro and In Vivo

SummaryMALT1 cleavage activity is linked to the pathogenesis of activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL), a chemoresistant form of DLBCL. We developed a MALT1 activity assay and identified chemically diverse MALT1 inhibitors. A selected lead compound, MI-2, featured direct binding to MALT1 and suppression of its protease function. MI-2 concentrated within human ABC-DLBCL cells and irreversibly inhibited cleavage of MALT1 substrates. This was accompanied by NF-κB reporter activity suppression, c-REL nuclear localization inhibition, and NF-κB target gene downregulation. Most notably, MI-2 was nontoxic to mice, and displayed selective activity against ABC-DLBCL cell lines in vitro and xenotransplanted ABC-DLBCL tumors in vivo. The compound was also effective against primary human non-germinal center B cell-like DLBCLs ex vivo

TET1 is a tumor suppressor of hematopoietic malignancy

The methylcytosine dioxygenase TET1 (‘ten-eleven translocation 1’) is an important regulator of 5-hydroxymethylcytosine (5hmC) in embryonic stem cells. The diminished expression of TET proteins and loss of 5hmC in many tumors suggests a critical role for the maintenance of this epigenetic modification. Here we found that deletion of Tet1 promoted the development of B cell lymphoma in mice. TET1 was required for maintenance of the normal abundance and distribution of 5hmC, which prevented hypermethylation of DNA, and for regulation of the B cell lineage and of genes encoding molecules involved in chromosome maintenance and DNA repair. Whole-exome sequencing of TET1-deficient tumors revealed mutations frequently found in non-Hodgkin B cell lymphoma (B-NHL), in which TET1 was hypermethylated and transcriptionally silenced. Our findings provide in vivo evidence of a function for TET1 as a tumor suppressor of hematopoietic malignancy.National Institutes of Health (U.S.) (5RO1HD045022)National Institutes of Health (U.S.) (5R37CA084198

The Eph-receptor A7 is a soluble tumor suppressor for follicular lymphoma

Insights into cancer genetics can lead to therapeutic opportunities. By cross-referencing chromosomal changes with an unbiased genetic screen we identify the ephrin receptor A7 (EPHA7) as a tumor suppressor in follicular lymphoma (FL). EPHA7 is a target of 6q deletions and inactivated in 72% of FLs. Knockdown of EPHA7 drives lymphoma development in a murine FL model. In analogy to its physiological function in brain development, a soluble splice variant of EPHA7 (EPHA7(TR)) interferes with another Eph-receptor and blocks oncogenic signals in lymphoma cells. Consistent with this drug-like activity, administration of the purified EPHA7(TR) protein produces antitumor effects against xenografted human lymphomas. Further, by fusing EPHA7(TR) to the anti-CD20 antibody (rituximab) we can directly target this tumor suppressor to lymphomas in vivo. Our study attests to the power of combining descriptive tumor genomics with functional screens and reveals EPHA7(TR) as tumor suppressor with immediate therapeutic potential

HELP (HpaII Tiny Fragment Enrichment by Ligation-Mediated PCR) Assay for DNA Methylation Profiling of Primary Normal and Malignant B Lymphocytes

The role of cytosine methylation in the regulation of gene expression during normal development and malignant transformation is currently under intense investigation. An ever increasing body of evidence demonstrates that carcinogenesis is associated with aberrant DNA methylation of the promoters of tumor suppressor genes (Chin Med J (Engl) 111:1028-1030, 1998; Leukemia 17:2533-2535, 2003), hypomethylation of oncogenes (Toxicol Appl Pharmacol 206:288-298, 2005; Toxicology 50:231-245, 1988), and concurrent loss of methylation in the intergenic areas and gene bodies, which may lead to genomic instability and chromosomal fragility (Cytogenet Cell Genet 89:121-128, 2000). Single locus methylation assays have focused largely on specific known tumor suppressor genes or oncogenes (Chin Med J (Engl) 111:1028-1030, 1998; Cancer Res 57:594-599, 1997; Hum Genet 94:491-496, 1994; Mol Cell Biol 14:4225-4232, 1994; Gastroenterology 116:394-400, 1999). Such approaches, while being useful, have clear limitations. With the advent of genome-wide microarray-based techniques, it has become possible to perform genome-wide exploratory studies to better understand genomic patterning of DNA methylation and also to discover new potential disease-specific epigenetic lesions (J Cell Biochem 88:138-143, 2003; Genome Res 16:1075-1083, 2006). In order to capture this type of information from primary human tissues, we have adopted and optimized the HELP assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR) to compare and contrast the abundance of cytosine methylation of genomic regions that are relatively enriched for CpG dinucleotides. While we have mainly used a custom NimbleGen-Roche high-density oligonucleotide microarray containing 25,626 HpaII amplifiable fragments, many other microarray platforms or high throughput sequencing strategies can be used with HELP

DNA Methyltransferase 1 Contributes to Epigenetic Signatures and Biological Phenotype during Normal B-Cell Differentiation and Lymphomagenesis

Abstract Normal hematopoiesis requires incremental changes in gene expression in order to establish cellular phenotypes with specialized functions. We are particularly interested in the transcriptional and epigenetic programming of germinal center (GC) B-cells, which acquire unusual biological features normally associated with cancer. Specifically, GC B-cells (i.e. centroblasts - CB) undergo rapid DNA replication while at the same time undergoing genetic recombination, and give rise to a majority of B-cell lymphomas. We hypothesized that epigenetic programming would play a critical role in the CB stage of development, and that gene-specific and genome-wide DNA methyltransferase activity is critical for these cells. We first examined the CpG methylation levels of 24,000 gene promoters in five sets of primary human B-cells just prior to (i.e. naïve B-cells - NBC) and upon entering the GC reaction (i.e. CBs). This was achieved using the HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR) assay, which relies on differential digestion of genomic DNA by the isoschizomer enzymes HpaII and Msp. HELP is a robust and reproducible method that provides accurate and quantitative measurement of DNA methylation levels throughout the genome. Remarkably, we found that the DNA methylation profile of B-cells undergoes a significant shift as readily appreciated by hierarchical clustering. The epigenetic signatures of NBC and CB are differentiation-stage dependent and do not vary significantly between individuals. The coefficient of correlation between individuals was 0.98, as compared to the NBC vs. CB fractions 0.92–0.95. Supervised analysis demonstrated that 266 genes (P<0.001) were differentially methylated upon entry of NB-cells into the GC reaction. We further correlated the methylation status of these genes with their gene expression level. The most heavily affected pathways by differential methylation and concordant expression in naïve B-cells were the Jak/STAT and MAP3K signaling pathways, while in CBs the p38 MAPK pathway and Ikaros family of genes were most affected. Given the epigenetic reprogramming observed in CBs vs. NBCs, along with the need for maintenance of methylation during rapid replication, we predicted that DNA methyltransferase (DNMT) enzymes play a critical role in centroblasts. By performing QPCR and Western blots on isolated fractions of human tonsilar lymphocytes and anatomical localization by immunohistochemistry, we found that DNMTs have a complex temporal and combinatorial expression pattern whereby DNMT1 was the main methyltransferase detectable in centroblasts. Additionally we studied 10 DLBCL cell lines and a panel of primary DLBCL (n=176 for mRNA and 70 for protein) for DNMTs expression. Spearman Rank correlation analysis revealed that DNMT1 was preferentially highly expressed in GCB vs. ABC primary DLBCLs, as well as in BCR vs. OxPhos DLBCLs. Taken together, our data suggest that i) dynamic changes in epigenetic programming contribute to formation of GCs, ii) that DNMT1 may play both a de novo and maintenance methylation role in GC cells, iii) that DNMT1 is markedly upregulated in normal centroblasts and in DLBCLs with the BCR or GCB gene expression profiles and iv) specific therapeutic targeting of DNMT1 rather than non-specific global inhibition of DNA methylation could be a useful anti-lymphoma strategy for germinal center-derived DLBCLs