Page 261 - Haematologica March 2020

The hydroxymethylome of multiple myeloma
by increasing DNA damage resistance.10 Nonetheless, other yet unidentified genes might participate in the path- ogenicity of 1q21 gain.
Tumor PC clones show different levels of differentia- tion,11 suggesting that MM could originate either from B cells that do not fulfill a complete differentiation program, or from PC that partially dedifferentiate. Cell differentia- tion relies on the selective engagement of small genomic regions called enhancers which are bound by transcription factors (TF) controlling cell-specific transcriptional pro- grams. As an early step of activation, enhancers undergo active DNA demethylation through iterative oxidation of 5mC into 5hmC, 5-formylcytosine (5fC) and 5-carboxyl- cytosine (5caC) by Ten-Eleven-Translocation (TET) enzymes and repair by the base excision repair machinery, including the T:G mismatch DNA glycosylase TDG which cleaves 5fC and 5caC.12 5hmC has been mapped genome-wide in several cell differentiation models, including in vitro differentiation of human naive B cells (NBC) into plasmablasts (PB), where 5hmC accumulates at PC identity genes, as well as in mouse germinal center B cells.13-15 These studies showed enrichment in 5hmC at poised/active enhancers as well as in the body of highly transcribed genes. Despite the wealth of information on the genetics of MM, the epigenetics of this disease is still poorly described. Nonetheless, a recent genome-wide investigation of active chromatin regions showed that opening of heterochromatin is a hallmark of MM.16 In addition, interrogation of DNA methylation in MM cells revealed that, despite a global hypomethylation, their genome shows specific hypermethylation of enhancers that normally undergo complete demethylation during B- cell commitment and are bound by B-cell TF.17 Interestingly, the methylation levels of these enhancers were anti-correlated with expression levels of B-cell-spe- cific TF in MM patients,17 suggesting that variations in tumor PC differentiation states could indeed be controlled through DNA methylation/demethylation mechanisms guided by specific TF. Here, we investigated the genome- wide distribution of 5hmC in tumor PC and, through the identification of MM-specific hydroxymethylated regions, evidenced new prognosis genes that might contribute to the understanding of this disease.
Methods
Primary multiple myeloma cells
Bone marrow samples were collected after patients’ written informed consent in accordance with the Declaration of Helsinki and institutional research board approval from Montpellier University hospital. Patients’ MM cells were purified using anti- CD138 MACS microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany). RNA and genomic DNA were extracted using Qiagen kits (Qiagen, Hilden, Germany) and their gene expression profile (GEP) obtained using Affymetrix U133 plus 2.0 microarrays as described.18 Plasma cell labeling index (PCLI)19 was investigated using BrdU incorporation and flow cytometry in 101 patients at diagnosis. Correlation between gene expression and PCLI was determined with a Spearman’s test. We used publicly available Affymetrix GEP (Gene Expression Omnibus, accession number GSE2658) of a cohort of 345 purified MM cells from previously untreated patients from the University of Arkansas for Medical Sciences (UAMS, Little Rock, AR), termed in the following UAMS-TT2 cohort. These patients were treated with total thera-
py 2 including high-dose melphalan (HDM) and autologous stem cell transplant (ASCT).20 We also used Affymetrix data from the total therapy 3 cohort (UAMS-TT3; n=158; E-TABM-1138)21 of 188 relapsed MM patients subsequently treated with bortezomib (GSE9782) from the study by Mulligan et al.22
FDI-6 treatment of primary MM cells from patients
Bone marrow of patients presenting with previously untreated MM (n=6) at the University Hospital of Montpellier was obtained after patients’ written informed consent in accordance with the Declaration of Helsinki and agreement of the Montpellier University Hospital Centre for Biological Resources (DC-2008- 417). Mononuclear cells were treated with or without graded con- centrations of FDI-6 and MM cells cytotoxicity was analyzed using anti-CD138-phycoerythrin monoclonal antibody (Immunotech, Marseille, France) as described previously.5
Genome-wide mapping of 5hmC and bioinformatics
5hmC was mapped by selective chemical labeling,23 coupled or not with exonuclease digestion,24,25 of 10 mg of sonicated (Bioruptor, Diagenode) genomic DNA from MM patients or from MCF-7 cells. Libraries were obtained using the TruSeq ChIP Sample Prep Kit (Illumina), quantified using the KAPA library quantification kit (KAPA Biosystems) and 50 bp single end sequenced with HiSeq 1500 (Illumina). Reads were mapped to hg19 and processed as described.25 SCL-exo signal was normalized to the input signal. Principal component analyses (PCA) were run online with Galaxy (http://deeptools.ie-freiburg.mpg.de/) with 5hmC signal bined by 10 kb windows. Heatmap clustering of hydrox- ymethylated CpG was run online (http://cistrome.org/).26 Search for transcription factor binding site (TFBS) motifs surrounding hydroxymethylated CpG used the online Centdist tool27 in 600 bp windows centered on 5hmCpG. Annotation of 5hmCpG used GREAT.28 Oxidative bisulfite modification of gDNA and hybridization to Illumina 450K arrays were run as previously described.29 ChIP-seq data for H3K27ac in primary MM cells16 were downloaded from the European Nucleotide Archive data- base (PRJEB25605). Data were deposited in the Gene Expression Omnibus database under accession number GSE124188.
A detailed description of additional methods is available in the
Online Supplementary Information.
Results
5hmC-enrichment partly recapitulates the molecular classification of MM
To better understand the relationship between DNA demethylation processes and cell identity in MM, we generated genome-wide maps of 5hmC in MM cells iso- lated from 11 patients belonging to different molecular subgroups, as well as for three human myeloma cell lines (HMCL), either by SCL-exo24,25 or SCL-seq23 (Figure 1A). In parallel, 10 MM samples were processed through the oxidative-bisulfite modification procedure and hybridized to Illumina 450K arrays (oxBS-450K).29 Annotation of 5hmC positive regions (40,586 CpG for oxBS-450K; 86,591 CpG for SCL-exo; 64,424 regions for SCL-seq) aggregated from all patients included in each procedure was run using GREAT.28 Results indicated that oxBS-450K did not generate meaningful information whereas SCL-exo and SCL-seq highlighted characteristics of PC biology such as endoplasmic reticulum stress, immune response, but also pathways which could be linked to MM, such as "IRF4 target genes"30 (Online
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