Page 265 - Haematologica March 2020

The hydroxymethylome of multiple myeloma
expression in a cohort of 101 newly diagnosed MM patients (Figure 3C). PCLI was also significantly correlated to FOXM1 and CKS1B expression, but at a lower extent. No correlation was found between MM cell proliferation and ANP32E or ILF2 expression (Figure 3C). These data suggest that amplification of the 1q21 region, together with its hydroxymethylation, might affect cell prolifera- tion in MM through enhancing FAM72 expression.
FAM72D Is involved in MM cell proliferation
Examination of the top-12 FAM72 co-expressed (P<0.05) genes during NBC differentiation into PC, high- lighted FOXM1, a TF known to play a key role in MM cell proliferation40 (Figure 4A). Furthermore, a strong cor- relation between FOXM1 and FAM72 expression was evidenced in MM samples and derived HMCL (Figure 4A). ChIP-seq data from GM12878 lymphoblastoid cells indicated that FOXM1 binds to the FAM72 promoter (Online Supplementary Figure S6A), strongly suggesting that this TF directly regulates FAM72 expression. In sup- port of this hypothesis, FAM72 hydroxymethylation lev- els were positively correlated with FOXM1 expression (Online Supplementary Figure S6B). Comparison of FOXM1 and FAM72 coexpressed genes in patients from the prolif- eration molecular MM subgroup revealed a significant
A
overlap (86% of common genes) (Online Supplementary Figure S6C). Moreover, FAM72 coexpressed genes were significantly associated with M phase cell cycle annota- tions (Online Supplementary Figure S6D). FAM72 was sig- nificantly overexpressed in HMCL and MM cells com- pared to BMPC and monoclonal gammopathy of undeter- mined significance (MGUS), underlining the link with MM cell proliferation (Online Supplementary Figure S6E). Furthermore, gene set enrichment analysis (GSEA) of expression data from patients with high FAM72 expres- sion and a poor outcome revealed a significant enrich- ment of genes related to proliferation, overexpressed in proliferating PB compared to mature BMPC and stem cell genes (Online Supplementary Figure S7). To study the bio- logical function of FAM72 overexpression in MM, the XG21 and XG23 HMCL were selected for their low level of endogenous FAM72 expression (Online Supplementary Figure S8A-C). In fetal calf serum (FCS) free medium, FAM72D overexpression resulted in significant growth advantage and response to IL-6, a key MM growth factor (Figure 4B). Similar results were obtained with overex- pression of FAM72 in XG23 (Online Supplementary Figure S8D). These data support the recent characterization of FAM72B as an S/G2-M phase gene whose inactivation reduces cell proliferation in human fibroblasts.41 To test
BC
(ng/mL)
Figure 4. FAM72D regulates cell proliferation in multiple myeloma. (A) Analysis of FAM72 co-expressed genes during B-cell differentiation in naive B cells (NBC), centroblasts (CB), centrocytes (CC), memory B cells (MBC), pre-plasmablasts (prePB), plasmablasts (PB), early plasma cells (early PC), and bone marrow plasma cells (BMPC). Only the Top-12 co-regulated genes are shown. FOXM1 ranked at position 11. Graph on the right shows the correlation between FAM72 and FOXM1 gene expression in the multiple myeloma (MM) patients and derived cell lines for which 5hmCpG were mapped by SCL-exo. (B) Proliferation assay of XG21 (blue bars) and XG21-FAM72D (orange bars) cells in the presence of increasing concentrations of IL-6. (C) Impact of increasing concentrations of the FOXM1 inhibitor FDI-6 on the in vitro growth of bone marrow cells from MM patients (n=6).
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