Dissecting the Epigenetic Landscape of Smoldering, Newly Diagnosed and Relapsed Multiple Myeloma Revealed IRAK3 As a Marker of Disease Progression
Authors: Rahmat Mahshid , Haradhvala Nicholas , Sklavenitis-Pistofidis Romanos , Park Jihye , Huynh Daisy , Bustoros Mark , Berrios Brianna , Reidy Mairead , Perilla-Glen Adriana , Rivotto Bradley , Hornburg Kalvis , Dorfman David M. , Licht Jonathan D. , Getz Gad , Ghobrial Irene M. 
Published: October 2019

Abstract
Introduction. Multiple myeloma (MM) is a complex and heterogeneous malignancy of plasma cells that has two precursor states: monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM). MGUS and SMM are asymptomatic states that eventually give rise to overt MM, with some patients progressing, while others do not. Recent studies in MM pathobiology have highlighted epigenetic alterations that contribute to the onset, progression and heterogeneity of MM. Global hypomethylation of DNA, including tumor suppressor genes, and hypermethylation of B-cell specific enhancers, abnormal histone methylation patterns due to the overexpression of histone methyltransferases such as MMSET, and deregulation of non-coding RNAs along with mutations in different classes of chromatin modulators underline a potential for epigenetic biomarkers in disease prognosis and treatment. This study aimed to define epigenetic pathways that lead to the dynamic regulation of gene expression in MM pathogenesis.
Methods. We performed ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) and RNA-seq on 10 MM cell lines and CD138+ plasma cells isolated from bone marrow aspirates of 3 healthy donors, 9 SMM, 8 newly diagnosed MM (NDMM) and 9 relapsed (RRMM) patients. ATAC-seq reads were trimmed of adapters, aligned to hg19 using bowtie2, and filtered for mapping quality >=Q30 using the ENCODE ATAC-seq pipeline. Reads mapping to promoter regions, defined as -400 to +250 bases from a refseq transcription start site, were counted using bedtools for each sample. Promoter read counts were then normalized by the total number of reads in promoters in the sample, scaled to 1 million total reads, and converted to log10(x+1) space.
Results. To characterize the epigenetic contribution to disease progression in MM, we first identified accessible promoter regions in normal plasma cells (NPC), SMM, NDMM and RRMM patients and found regions displaying differential accessibility in MM progression. Next, we intersected the list of differential accessible regions (DARs) with matched transcriptome data and observed two main clusters: genes with unaltered transcription profiles and genes in which the dynamics of open chromatin regions (OCRs) correlated with gene expression. Transcriptomic analysis revealed that a large portion of the differentially expressed (DE) genes in SMM remain DE in NDMM as compared to NPCs (882 genes out of 1642 and 1150 DE genes in SMM and NDMM, respectively). Those genes were significantly enriched for pathways like epithelial mesenchymal transition, cell cycle checkpoints and mitosis, KRAS signaling and interleukin-JAK-STAT pathways. To investigate the genes that behaved differently among the stages of disease, we looked at differential accessibility and expression in NDMM and SMM samples, and integrated them with Whole-Genome Bisulfite-Sequencing and 450K DNA-methylation data from MM patients and healthy donors (BLUEPRINT). This analysis led to the identification of novel genes in MM progression, such as the transcriptional repressor ZNF254 and IRAK3, a negative regulator of the TLR/IL1R signaling pathway. Although gene expression data for these genes showed comparable mRNA levels in SMM and NPCs, followed by a significant decrease in NDMM/ RRMM, ATAC-seq revealed a striking drop in promoter accessibility in SMM, NDMM and RRMM cases. Comparison of ATAC-seq peaks to DNA methylation and ChIP-seq data revealed that the altered OCR of IRAK3 is actually hypermethylated in MM patients and marked by H3K4me3, a marker of active promoters, in MM cell lines. Hypermethylation of IRAK3 has been described in hepatocellular carcinoma, where it is associated with poor prognosis. Together, our data suggest that the identified IRAK3 OCR may act as a bivalent domain that loses accessibility in the precursor states and gains DNA methylation in MM progression. Hence, IRAK3 methylation could be a novel prognostic marker in MM.
Conclusion. We have generated a global epigenetic map of primary tumors from patients at the smoldering, newly diagnosed and relapsed/refractory stage of multiple myeloma. Integrative analysis of ATAC-seq data with DNA methylome, transcriptome and whole-genome map of active and repressive histone marks in our study led to the identification of IRAK3 as a novel epigenetic biomarker of disease progression.

Disclosures
Licht: Celgene: Research Funding. Ghobrial:Takeda: Consultancy; BMS: Consultancy; Celgene: Consultancy; Janssen: Consultancy.