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F. Chatonnet et al.
Supplementary Figure S1A). Based on these annotation data, only SCL-exo and SCL-seq identified regions were further analyzed. Results were first compared to genome- wide 5hmC maps of NBC and PB previously generated by SCL-seq.14 PCA of the signal showed dispersion of the samples, suggesting variability between tumor hydrox- ymethylomes. Nonetheless, most MM hydroxymethy- lomes grouped closer to plasmablasts (PB than to NBC) (Figure 1B). When running PCA only with MM patients from the MMSET, CCND1 and Proliferation groups, 3 clusters were observed (C1 to C3 Figure 1B). These clus- ters gathered together patients from similar molecular groups, although two patients did not follow this rule (E12097 and E6068), indicating that molecular groups are probably heterogeneous in nature and that 5hmC maps can help refine molecular clustering. We next generated the union of significantly hydroxymethylated CpG (P< 5e-2) overlapping between samples within each cluster. These 6,385 individual CpG were clusterized according to their 5hmC levels (Figure 1C). The resulting heatmap evidenced two groups of CpG that were selectively more hydroxymethylated either in C1 patients (C'1 cluster) or in C2 patients (C'2 cluster). Analysis of motif enrichment for transcription factor binding sites in C'1 and C'2 and their comparison with motifs found in NBC and PB hydroxymethylated regions suggested that MM cells from C2 patients remain more differentiated than those from C1 patients. Indeed, the BLIMP1 (a major regulator of PC differentiation) motif was significantly enriched in C'2 but not in C'1 regions (Online Supplementary Figure
S1B). Accordingly, functional annotation through GREAT showed that C'2 regions associated with endoplasmic reticulum stress gene signature or IL-6 signaling, features of mature PC, whereas C'1 regions did not (Figure 1C). Conversely, NOTCH, MYC, Cell Cycle and BCR signal- ing, which are more characteristic of proliferating and/or undifferentiated B cells, were significantly more associat- ed with C'1 regions than with C'2 regions (Figure 1C). Of note, the SUH and GLI motifs were also selectively enriched in C'1 regions (Online Supplementary Figure S1B). Hence, C1 patients from the proliferation group probably have active NOTCH and SHH pathways, both important for proliferation of CD138+ MM cells.31-32 In addition, the binding motif for MEIS1, a known regulator of hematopoietic progenitor self-renewal,33 was also selec- tively enriched in C'1 regions. Collectively, these data indicate that MM cells from the proliferation group have an under-differentiated phenotype and proliferation is likely to rely on a MEIS1/SUH/GLI transcription factor cocktail as well as MYC activity. These results also show that 5hmC is indeed indicative of the biological and clin- ical traits of patients and could be used to delineate groups with different characteristics.
5mC oxidation targets MM plasma cell enhancers
Investigating the genomic location of high confidence MM 5hmCpG (86,591 CpG, p<1e-5) showed that they were mostly distributed in introns and distal intergenic regions and, as previously observed in NBC, PB and mouse activated B cells,14,15 genes which were close to a 5hmC
AC
B
Figure 1. Analysis of the hydroxymethylome of multiple myeloma (MM). (A) Molecular classification of the patient samples analyzed in this study. HMCL indicates the names of cell lines derived from patient samples. The molecular groups are based on Zhan et al.1 MM: multiple myeloma, BM: bone marrow, PCL: plasma cell leukemia. (B) Principal component analysis (PCA) of 5-hydroxymethylcytosine (5hmC) distribution either in all MM patients, compared to naive B cells (NBC) and plas- mablast (PM) (left), or in the subset of MM patients from the CCND1, MMSET and proliferation groups. Clusters 1, 2 and 3 (C1, C2 and C3) group samples with sim- ilar 5hmC distribution. (C) Heatmap clustering of the SCL-seq signal in C1 to C3 patients at the union of overlapping 5hmCpG and functional annotation with GREAT of genes associated with C'1 and C'2 5-hydroxymethylated CpG(5hmCpG). For each annotation, the corresponding P value is indicated.
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