ALBERT

Description: During the last years, the study of molecular alterations associated with multiple myeloma (MM) has been mostly focused on the analysis of the genome, transcriptome and DNA methylome. These analyses are showing that (epi)genetic heterogeneity and extensive perturbation of the transcriptional landscape are hallmarks of MM. Our previous analysis of the whole DNA methylome of MM revealed that this epigenetic mark globally shows a poor correlation with gene expression, and therefore did not allow us to better understand gene deregulation in MM. In contrast, the chromatin structure and histone modifications are emerging as essential epigenetic layers to understand the mechanisms underlying gene expression changes in cancer, but remain widely unexplored in MM. We have now performed a deep ChIP-seq profiling of CD138+ sorted cells from bone marrow samples obtained from four MM patients and three biological replicates of normal plasma cells (NPCs) using antibodies against H3K4me3, H3K4me1, H3K27ac, H3K36me3, H3K27me3 and H3K9me3. Different combinations of these marks allow us to segment the MM and NPC genome into functional chromatin states, including active, weak or poised promoters, active or weak enhancers, transcriptional elongation, polycomb-repressed regions and heterochromatic regions. The initial unsupervised exploration of the data showed that the chromatin landscape of MM is widely altered as compared to NPCs. A supervised analysis of chromatin states revealed that MM globally shows a more active chromatin structure than NPCs. From over 40,000 regions identified with differential chromatin structure between MM and NPCs, 88% were de novo activated in neoplastic plasma cells. Analyzing the chromatin of individual genes, we observed that there were roughly ten times more genes gaining activity upon neoplastic transformation than those acquiring repressed chromatin marks. Interestingly, the genes showing more activate chromatin were enriched with biosynthesis and metabolic processes, while genes with repressed chromatin were related to gene ontology terms related to B cell signaling. Among those genes gaining de novo activity in MM, we selected several candidates and we are currently performing functional in vitro assays to explore their implication in MM pathogenesis. Furthermore, as extensive chromatin activation is a hallmark of MM, we are currently analyzing additional 15 MM cases and NPCs by ChIP-seq for H3K27ac (marking active promoters and enhancers) and ATAC-seq (marking active regulatory regions) to validate our initial findings and explore chromatin heterogeneity in MM. Collectively, our initial exploration of histone modification profiles in MM has revealed that MM cells acquire a more active chromatin landscape, with thousands of regions gaining activation as compared to NPCs. Reversing this global activation by epigenetic drugs, such as BET inhibitors, may represent an attractive therapeutic option for MM. During the meeting, updated information will be presented, including data from all 19 MM patients studied as well as functional data from new candidate genes involved in MM pathogenesis. Disclosures Paiva: Celgene: Honoraria, Research Funding; Janssen: Honoraria; Takeda: Honoraria, Research Funding; Sanofi: Consultancy, Research Funding; EngMab: Research Funding; Amgen: Honoraria; Binding Site: Research Funding.

Description: Introduction SOX11 is a transcription factor (TF) aberrantly expressed in the majority of mantle cell lymphomas (MCLs), which is generally associated with aggressive clinical behaviour. No mutations, genetic aberrations or direct correlations with differential DNA methylation at the promoter related to its expression have been found in MCL. Deeper insights into its regulation can be found by considering the three-dimensional (3D) chromatin structure. It is becoming clear that the genome can be partitioned into 3D building blocks, topologically associated domains (TADs) and that enhancer regions likely regulate genes within their TADs by 3D contacts, but do not affect genes outside their own TADs. By mapping the 3D chromatin structure, we previously identified a distant putative SOX11 enhancer showing enhancer activity and 3D contacts with the SOX11 gene in the SOX11-positive MCL cell line Z-138, but not in the SOX11-negative MCL cell line JVM-2. Aims We aimed to deepen our understanding of the differential 3D contacts and enhancer activity previously observed at the putative SOX11 enhancer in SOX11-positive versus SOX11-negative MCL cell lines by addressing the following questions: (i) Do TAD boundaries around the SOX11 locus change between SOX11-positive and -negative MCLs? (ii) How do the 3D contacts and chromatin states at this region behave in primary MCL cases and normal B cells? (iii) Is the putative SOX11 enhancer involved in SOX11 expression in other tissues? Methods We have extended our experimental analyses of the putative SOX11 enhancer by performing (i) HiC-sequencing and 3D fluorescence in situ hybridization (3D FISH) in MCL cell lines Z-138 and JVM-2, (ii) 4C-sequencing, chromatin inmmunoprecipiation followed by deep sequencing (ChIP-seq) of 6 histone marks, an Assay for Transposase-Accessible Chromatin with deep sequencing (ATAC-seq) and chromatin state modeling by chromHMM (using the 6 histone marks) in primary MCL cases and normal naive and memory B-cells. Furthermore, we have explored the activity of this region in other SOX11 expressing cell lines studied within the ENCODE Consortium. Results HiC-sequencing in the cell lines Z-138 (SOX11-positive) and JVM-2 (SOX11-negative) showed that the SOX11 locus and its putative enhancer are located within the same TAD in both samples. Hence, shifts in TAD boundaries do not seem to underlie the differential 3D chromatin interactions between the SOX11 locus and its putative enhancer in these two cell lines. By ChIP-seq and chromatin state modeling we observed that the promoter of SOX11 is poised, i.e., carrying histone marks H3K4me3 and H3K27me3, in normal naive and memory B-cells and the SOX11-negative MCL primary case. Furthermore, we observed weak enhancer activity at the putative SOX11 enhancer in normal naive and memory B-cells and the SOX11-negative MCL primary case, but strong enhancer activity, marked by the presence of H3K27ac, only in SOX11-positive samples. In addition, by ATAC-seq we identified two specific chromatin accessible regions that potentially represent the transcription factor binding sites responsible for activation of this enhancer region in SOX11-positive MCLs. By 4C-sequencing we observed that the SOX11 locus and its putative enhancer show high 3D contacts in two other SOX11-positive MCL cell lines (GRANTA-519 and JEKO-1) and in a SOX11-positive primary MCL case, but not in a SOX11-negative primary MCL case. Furthermore, the differential 3D contacts at these regions in Z-138 and JVM-2 were confirmed by 3D FISH, which is currently being performed in primary MCL cases. Interestingly, no 3D contacts were observed in normal naive and memory B cells, indicating that although the SOX11 promoter is poised within these normal B-cell subpopulations, primed looping at these regions does not exist and seems not to explain the 3D contacts we observed in SOX11-positive MCL cell lines and primary cases. When investigating chromatin states in cell lines studied by ENCODE with an active SOX11 promoter (H1-hESC, HSMM and NHLF) none of them show activity in the identified region, suggesting that the putative SOX11 enhancer is de novo activated only in the context of MCL lymphomagenesis. Conclusions We provide new evidence that the activation of a distant SOX11 putative enhancer and its 3D contacts to the SOX11 gene, is a de novo event in SOX11-positive MCL cell lines and primary cases that is likely specific for this malignancy. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Modulation of the DNA methylation landscape during cell differentiation is a well-established phenomenon. The B-cell lineage represents a paradigmatic cellular model to study the dynamic epigenome during cell development and specification because major B-cell maturation stages are well defined and display differential phenotypic and gene expression features. Furthermore, different B-cell subpopulations show different proliferation abilities, microenvironmental influences and life spans, providing a window of opportunity to study the epigenome in the context of multiple processes. Methods: We performed whole-genome bisulfite sequencing (WGBS), high-density methylation microarrays and gene expression profiling of ten purified human B-cell subpopulations spanning the entire differentiation program, ranging from uncommitted progenitors to terminally-differentiated plasma cells. Results: The results of both WGBS and methylation microarrays indicate that B-cell ontogenesis involves an extensive and gradual reconfiguration of the DNA methylome. We uncovered that non-CpG methylation at CpApC trinucleotides is present in progenitor cells and disappears upon B-cell commitment independently of CpG demethylation. CpG methylation, in contrast, changed extensively during the entire B-cell maturation program, with one quarter of all measured CpGs showing dynamic methylation. B-cell enhancers suffered more extensive methylation changes than promoter regions, especially in the early differentiation steps up to the germinal center B-cell (gcBC) stage, and their demethylation seemed to be mediated by binding of lineage-specific transcription factors. Enhancers with dynamic methylation were related to genes involved in a large B-cell network that showed high gene expression variability throughout differentiation. In highly proliferative gcBCs, we observed a shift of dynamic methylation from regulatory towards non-functional elements; gcBCs start to undergo global demethylation of late-replicating heterochromatic regions and methylation of polycomb-repressed regions. This signature becomes particularly extensive in long-lived memory B cells and plasma cells, indicating that these changes start in highly proliferative cells and then accumulate in non-proliferative cells with extended lifespan. Conclusion: Our epigenomic analysis of the B-cell differentiation program extends our knowledge on how the DNA methylome is modulated during cell specification and maturation and offers a resource for researchers in the field, both at global and single gene levels. Disclosures No relevant conflicts of interest to declare.

Description: Analyzing the DNA methylome of multiple myeloma (MM), a plasma cell neoplasm, by whole-genome bisulfite sequencing and high-density arrays, we observed regional DNA hypermethylation embedded in extensive global hypomethylation. In contrast to the widely reported DNA hypermethylation of promoter-associated CpG islands (CGIs) in cancer, hypermethylated sites in MM as compared to normal plasma cells were located outside CpG islands and were unexpectedly associated with intronic enhancer regions active in normal B cells. Both RNA-seq and in vitro reporter assays indicated that enhancer hypermethylation is globally associated with downregulation of its host genes. ChIP-seq and DNAseI-seq further revealed that DNA hypermethylation in these regions was related to enhancer decommissioning. Hypermethylated enhancer regions overlap with binding sites of B-cell specific transcription factors (TFs) and the degree of enhancer methylation inversely correlated with expression levels of these TFs in MM. Furthermore, hypermethylated regions in MM were methylated in stem cells and gradually became demethylated during normal B-cell differentiation suggesting that MM cells reacquire epigenetic features of undifferentiated cells upon loss of expression of B-cell specific TFs. Overall, we have identified DNA hypermethylation of developmentally-regulated enhancers as a new type of epigenetic modification associated with the pathogenesis of MM. Disclosures No relevant conflicts of interest to declare.

Description: In CLL, subsets of patients carrying stereotyped B cell receptors (BcR) share similar biological and clinical features independently of IGHV gene somatic hypermutation status. Although the chromatin landscape of CLL as a whole has been recently characterized, it remains largely unexplored in stereotyped cases. Here, we analyzed the active chromatin regulatory landscape of 3 major CLL stereotyped subsets associated with clinical aggressiveness. We performed chromatin-immunoprecipitation followed by sequencing (ChIP-Seq) with an antibody for the H3K27ac histone mark in sorted CLL cells from 19 cases, including clinically aggressive subsets #1 (clan I genes/IGKV(D)1-39, IG-unmutated CLL (U-CLL)(n=3)], #2 [IGHV3-21/IGLV3-21, IG-mutated CLL (M-CLL)(n=3)] and #8 [IGHV4-39/IGKV1(D)-39, U-CLL(n=3)] which we compared to non-stereotyped CLL cases [5 M-CLL|5 U-CLL]. In addition, a series of 15 normal B cell samples from different stages of B-cell differentiation were analyzed [naive B cells from peripheral blood (n=3), tonsillar naive B cells (n=3), germinal centre (GC) B cells (n=3), memory B cells (n=3), tonsillar plasma cells (n=3)]. Initial unsupervised principal component analysis (PCA) disclosed a distinct chromatin acetylation pattern in CLL, regardless of stereotypy status, versus normal B cells. CLL as a whole was found to be closer to naive and memory B cells rather than GC B cells and plasma cells. Detailed analysis of individual principal components (PC) revealed that PC4, which accounts for 5% of the total variability, segregated subset #8 cases and GC B cells from other CLLs and normal B cell subpopulations. Although PC4 accounts for only a small part of the total variability (5%), this suggests that subset #8 cases may share some chromatin features with proliferating GC B cells, in line with the fact that subset #8 BcR are IgG-switched. We also investigated whether stereotyped CLLs have different chromatin acetylation features compared to non-stereotyped CLLs matched by IGHV somatic hypermutation status and identified 878 Differential Regions (DR) in subset #8 vs. U-CLL, 84 DR in subset #1 vs. U-CLL and 66 DR in #2 compared vs. M-CLL. As subset #8 cases seemed to have the most distinct profile, we further characterized the detected regions. The 435 and 443 regions gaining and losing activation, respectively, mostly targeted promoters (29.5%) and regulatory elements located in introns (31%) and distal intergenic regions (21.8%). Hierarchical clustering based on the 878 DRs enabled the clear discrimination of subset #8 cases from U-CLL and normal B cells; however, it is worth noting that for several of these 878 DRs the acetylation patterns were shared between subset #8 and normal B cell subpopulations rather than subset #8 and U-CLL. Of note, 11/435 regions gaining activity on subset #8 were found within the gene encoding for the EBF1 transcription factor (TF); additional regions were associated with genes significant to CLL pathogenesis, e.g. TCF4 and E2F1. Moreover, 3 DRs losing activity in subset #8 were located within the CTLA4 gene and 2 DRs within the IL21R gene, which we have recently reported as hypermethylated and not expressed in subset #8. Next, we performed TF binding site analysis by MEME/AME suit, separately for regions gaining or losing activity, and identified significant enrichment (adj-p