Page 264 - Haematologica March 2020

F. Chatonnet et al.
A MM-specific 5hmC signature identifies prognosis genes
We next delineated a MM-specific 5hmC signature, independently of molecular subgroups, through first call- ing CpG that were hydroxymethylated (P<1e-5) in at least 33% of the myeloma samples and not falling into genomic regions hydroxymethylated in PB (p<1e-2), and second by iterative clustering of these CpG according to their hydroxymethylation signal both in MM patients and in PB. Hence, a cluster of 415 CpG uniquely hydroxymethy- lated in MM was obtained (Online Supplementary Figure S3A). A list of 29 genes, including BMP6 and FAM72D (Online Supplementary Figure S3B), associating with at least 3 of these hydroxymethylated CpG was next established and investigated for prognostic value in the UAMS-TT2 (n=256) and UAMS-TT3 (n=158) Arkansas cohorts (http://genomicscape.com/). Interestingly, around a quarter (7/29) of the 29 genes significantly associated with 5hmCpG were located at 1q21.1 (P=7.5e-4) whereas none were at 1q21.2 (Figure 3A and Online Supplementary Figure S3C-D). Results showed that among these 29 genes, only six had no prognostic value (P>0.05) in both analyzed cohorts (Figure 3A). Others could be classified as either of good (n=4) or of poor (n=7) prognosis in both cohorts, as exemplified for FAM72D (Figure 3B). Investigating the expression of these genes during the differentiation of NBC, poor prognostic genes were mostly expressed in
A Cohort P value
pre-PB, whereas good prognostic genes were mainly expressed in non-proliferating cells (Online Supplementary Figure S4). Among the poor prognostic genes located on 1q21.1, FAM72D is a gene of unknown function which implication in MM biology has not yet been addressed. Of note, recent duplication events of the ancestral FAM72A gene led to the presence of four highly conserved FAM72 genes in human (FAM72A ,B , C, D)39 which cannot be dis- criminated at the mRNA level (99% identity). Accordingly, "FAM72" is used thereafter when referring to expression data. FAM72 expression levels correlated with 1q21 copy number, which is associated with poor out- come in MM (Online Supplementary Figure S5A-B). Importantly, FAM72 expression levels correlated with 5hmC enrichment at the FAM72D locus (Online Supplementary Figure S5C) which was not biased by 1q21 amplification (Online Supplementary Figure S5D) and was associated with the presence of the enhancer histone modification H3K4me1 in normal plasma cells (Online Supplementary Figure S5E). Conversely to BMP6 (Online Supplementary Figure S4 and S5F), expression of FAM72 was found to be higher in proliferative B cells, i.e. centrob- lasts and pre-PB (Online Supplementary Figure S4), as well as in the proliferation group (Online Supplementary Figure S5G). In accordance with a putative role of FAM72 in stimulating MM cell proliferation, plasma cell labeling index (PCLI)19 was significantly correlated with FAM72
B
C
P<0.0001
Gene Correlation (R2) P-value
Figure 3. A multiple myeloma-specific hydroxymethylation signature uncovers new prognostic genes. (A) Genes associated with at least three MM-specific 5hmCpG were analyzed with Genomic-Scape 2.0 for their prognostic value in two cohorts of patients (Arkansas TT2 and TT3). Genes associated with good prognosis are indi- cated in green and genes associated with poor prognosis in red. The associated P-values are also indicated with the same color code. Cutoff was set at P=0.05. (B) Overall survival of patients from the TT2 Arkansas cohort expressing low (blue curves) or high (red curves) levels of FAM72D (lower panels). (C) Correlations between plasma cell labeling index (PCLI) scores and gene expression values in a cohort of 101 patients.
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