The hydroxymethylome of multiple myeloma identifies FAM72D as a 1q21 marker linked to proliferation

Abstract

Cell identity relies on the cross-talk between genetics and epigenetics and their impact on gene expression. Oxidation of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) is the first step of an active DNA demethylation process occurring mainly at enhancers and gene bodies and, as such, participates in processes governing cell identity in normal and pathological conditions. Although genetic alterations are well documented in multiple myeloma (MM), epigenetic alterations associated with this disease have not yet been thoroughly analyzed. To gain insight into the biology of MM, genome-wide 5hmC profiles were obtained and showed that regions enriched in this modified base overlap with MM enhancers and super enhancers and are close to highly expressed genes. Through the definition of a MM-specific 5hmC signature, we identified FAM72D as a poor prognostic gene located on 1q21, a region amplified in high risk myeloma. We further uncovered that FAM72D functions as part of the FOXM1 transcription factor network controlling cell proliferation and survival and we evidenced an increased sensitivity of cells expressing high levels of FOXM1 and FAM72 to epigenetic drugs targeting histone deacetylases and DNA methyltransferases.

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. 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 similar 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.

Figure 2

Genomic and epigenomic characterization of sites of 5-methylcytosine (5mC) oxidation in multiple myeloma plasma cells (MMPC). (A) CEAS analysis of the distribution of high confidence multiple myeloma (MM) 5hmCpG (n=86,591) and box plot comparing expression levels of genes associated or not with 5-hydrox-ymethylcytosine (5hmC) peaks in MM patients. (B) Heatmap representation of H3K4me1 and H3K27ac ChIP-seq signals at MM 5hmCpG. NBC and PC H3K4me1 data were from the Blueprint Consortium (http://dcc.blueprint-epigenome.eu/#/files), and MM H3K27ac data were downloaded from the European Nucleotide Archive (PRJEB25605). (C) Integrated genome browser (IGB) visualization of H3K4me1 from normal plasma cells (PC H3K4me1), 5hmC from in vitro naive B cells (NBC) and plasma cells (PC) differentiated plasmablasts (PB 5hmC), MM 5hmCpG and SCL-exo signal from E10025 at selected super enhancers active in MM1.S cells. The survival of patients from the Arkansas cohort classified as high (n=71) or low (n=343) GAS2 expression is shown on the right.

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 indicated 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.

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).

Figure 5

FAM72 expression levels predict multiple myeloma cell sensitivity to drugs. (A) 12 HMCL were cultured with graded concentrations of Boterzomib for four days and IC50 were calculated with mean values of five experiments determined on sextuplet culture wells. High FAM72 expression (Affymetrix microarrays) was significantly correlated with resistance to bortezomib. (B) Histone deacetylase/decitabine inhibitors (HDACi/DNMTi) induce a significant downregulation of FAM72 and FOXM1 in multiple myeloma (MM). Nine HMCL were treated for seven days with decitabine (DNMTi) and TSA during the last 24 hours and gene expression was assessed using Affymetrix U133P microarrays. (C) FAM72 expression predicts 5azacitidine/SAHA combination sensitivity of primary myeloma cells of patients. Mononuclear cells from tumor samples of 17 patients with MM were cultured for 4 days in the presence of IL-6 (2 ng/mL) with or without 2 μM 5azacitidine and 300 nM SAHA. At day 4 of culture, the count of viable MMC was determined using CD138 staining by flow cytometry. Samples from Figure 6C and Online Supplementary Figure S10C were grouped as FAM72^high or low and FOXM^high or low and correlation coefficients between FAM72 or FOXM1 expression and the percentage of living MM cells were calculated with GraphPad Prism.

Figure 6

5-methylcytosine (5mC) oxidation at FAM72D enhancer and GAS2, DEPTOR and MYC super enhancers associate with multiple myeloma. 5mC oxidation in multiple myeloma (MM) cells occurs at super-enhancers (SE) and normal enhancers and as such participates in the establishment of an MM-specific transcription program and the maintenance of plasma cell identity. Several scenarios may lead to FAM72D overexpression in MM, including 1q21 gain/amplification, FOXM1 upregulation and DNA demethylation, and might combine in high risk MM to enhance survival and proliferation.