Whole-Genome Analysis of Histone Modifications Reveals New Insights into the Biology and Clinical Behavior of Mantle Cell Lymphoma Subtypes

Introduction: Mantle cell lymphoma (MCL) is a mature B-cell neoplasm characterized by the t(11;14)(q13;q32) leading to cyclin D1 overexpression. Two major clinico-biological subtypes of MCL have been recognized, conventional (cMCL) and leukemic non-nodal MCL (nnMCL), being the former clinically more aggressive than the latter. Although these subtypes have been well characterized at the genomic level, the genome-wide landscape of histone modifications is widely unknown. Aim: To explore whether an integrative multiomic analysis of chromatin marks and gene expression may help us to better understand the biology and clinical behavior of cMCL and nnMCL. Methods: We carried out 2 profiling strategies. On the one hand, we performed ChIP-seq for H3K27ac, which marks active regulatory elements, in a series of 52 MCL cases, and we also profiled chromatin accessibility by ATAC-seq in 28 of these cases. On the other hand, we performed ChIP-seq for 6 histone modifications with complementary functions (H3K4me1, H3K4me3, H3K27ac, H3K36me3, H3K27me3 and H3K9me3) in purified neoplastic cells of 3 cMCL and 3 nnMCL, and we mined ChIP-seq data from normal B cell subpopulations. Additionally, we analyzed previously published expression data from 44 MCL profiled by Affymetrix U219 microarrays. Clinical data were available for most cases. Data were analyzed using a battery of computational and statistical approaches. Results: An initial unsupervised principal component analyses of the H3K27ac and ATAC-seq data revealed 2 main clusters corresponding to cMCL and nnMCL, indicating that the chromatin landscape is markedly different between the 2 MCL subtypes. A supervised differential analysis of H3K27ac data between cMCL and nnMCL showed extensive differences, mostly associated with increased H3K27ac in cMCL. Putting the data in the context of normal B cell subpopulations, we identified 746 de novo active regions in cMCL and 126 in nnMCL (LFC>1, FDR ≤0.05). Interestingly, more than 60% of these regions were enhancers according to the chromatin states analysis (derived from the integration of 6 histone modifications), and the majority of them presented an open chromatin configuration. This chromatin activation patterns correlated with transcriptional differences between cMCL and nnMCL. Analyzing the target genes of these regions, we identified 167 and 20 upregulated genes in cMCL and nnMCL, respectively. Upregulated genes in cMCL were linked to cellular processes related to cell cycle and cell proliferation according to gene ontology analysis. Analyzing the transcription factor binding motifs present in the ATAC-seq peaks among the de novo active H3K27ac regions in cMCL, we observed an enrichment in specific transcription factor families such as Krüppel-like family (KLF) and E2F. However, there was no significant enrichment in any transcription factor family among nnMCL de novo active regions. Moreover, although the cMCL-associated de novo H3K27ac signature was common to all cMCL cases, we did observe varying H3K27ac levels among cases. We then explored whether H3K27ac levels could be associated with clinical features in cMCL. In fact, we identified 100 specific regions whose increased H3K27ac levels were significantly associated with shorter overall survival, leading to the identification of two subgroups of cMCL with markedly different clinical behavior. Conclusions: The results obtained with this multilayer chromatin analysis confirm that MCL is a heterogeneous disease showing widespread differences between the 2 subtypes. cMCL present a clear de novo chromatin activation associated with upregulation of key genes involved in cMCL pathogenesis, while nnMCL cases seem to be characterized by the absence of this signature and an overall chromatin profile more similar to normal B cells. Moreover, the level of activation in regulatory regions in cMCL seems to be related with the clinical outcome of the patients, with more activation associated with shorter overall survival.