Multiomics Data Integration in the Complete Myeloma Genome Reveals Frequent Centromeric Rearrangements and Their Epigenomic Consequences

Introduction: Myeloma has the most complex genomic architecture among blood cancers. Central to this complexity are structural variants (SVs), large-scale genomic changes with the capacity to disrupt chromatin organization. SVs can modify the epigenomic landscape near DNA breakpoints, leading to gene dysregulation without directly altering coding sequences or DNA copy number. Here, we elucidate SVs involving the previously hidden regions of the human genome, namely compact heterochromatin including centromeric regions. These dense and repetitive regions, traditionally dismissed as “junk DNA”, emerge as critical areas of investigation.

Methods: Short-read whole-genome sequencing (srWGS) data from 23 CoMMpass cases were mapped to the CHM13v2 genome assembly by BWA-MEM. RNA-seq CoMMpass (N = 928) and control GSE148924 (N = 12) sets were processed by DESeq2 normalization, variance-stabilizing transformation, and limma batch correction. Gene under-/over-expression was assessed by Z-score (>2/<-2) and log2-fold change (>1/<-1). Patient-derived xenografts (PDXs, N = 13) and KMS27 and PCM6 cell lines were studied by multiomics: srWGS (Illumina), long-read HiFi WGS (PacBio), and Micro-C (CantanaBio); including cases with t(11;14), t(4;14), t(14;16), and hyperdiploidy. Raw reads were aligned to the CHM13v2 assembly. HiFi alignments were phased by WhatsHap. In-house algorithms were built for the analysis of SVs involving repetitive regions in srWGS and DNA methylation within the HiFi reads. Copy-number variants were assessed by CNVRobot. Micro-C data were processed with Juicer tools.

Results: SVs affecting compact heterochromatin were detected in 61% of CoMMpass cases (14/23) and frequently involved (peri)centromeres of chromosomes 8, 12, 15, and 16. Breakpoints at partner loci often disrupted tumor-suppressor genes (CYLD, GAS8, UNC5D) and recombined near proto-oncogenes (MYC, NFKB1), which increased gene expression compared to control plasma cells.

In a PDX case, we resolved the +1q as “jumping translocations” between 1q12 pericentromeric heterochromatin and 6q15, 15p11.2, and 18q11.2. The previous hg38 assembly could not resolve t(1;6) and t(1;15) due to DNA breakpoints being present in gaps of this reference genome. HiFi reads enabled breakpoint mapping at 1q (impossible by srWGS), and Micro-C data confirmed derivative chromosome der(1)t(1;18), der(6)t(1;6), and der(15)t(1;15) interactions, and were associated with loss of the partner chromosome arm. DNA methylation analysis of HiFi phased data showed significant hypomethylation of partner loci juxtaposed to the 1q pericentromere. We identified this pattern as a mark of compact heterochromatin rearrangements, confirming an "open chromatin" state, consistent with increased transcription in the CoMMpass data.

Allele-specific DNA methylation changes were also observed in key chromosomal SVs involving super-enhancers, such as t(11;14) CCND1-IGH, t(4;14) NSD2-IGH, t(14;16) IGH-MAF, and t(6;8) TXNDC5-MYC. For example, in a t(11;14) PDX case, we detected hypomethylation (~2 kb) at the chr11 breakpoint, followed by 225 kb hypermethylation and subsequent hypomethylation spanning CCND1. Micro-C data showed interactions between these hypomethylated regions on chr11 and the IGH promoter through introduction of a de novo translocation-specific regulatory TAD loop absent in non-t(11;14) samples, allowing close interaction of the translocation breakpoint with CCND1. No methylation changes were observed in other SVs lacking super-enhancers or compact heterochromatin involvement.

The level of compact heterochromatin breakage was not linked to global hypomethylation. However, global methylation analysis showed a higher proportion of methylated CpG sites in the t(4;14) group (71.8%, N = 4) compared to other molecular groups: t(11;14) (44.8%, N = 4), t(14;16) (46.1%, N = 2), and hyperdiploidy (34.1%, N = 4). This supports the role of NSD2 in forming H3K36me2 histone marks, essential for maintaining DNA methylation.

Conclusions: Utilizing novel computational algorithms, complete genome assembly, and multiomics data integration, we provide the first genomic evidence of SVs affecting compact heterochromatin as common changes in the myeloma genome. These rearrangements alter the DNA topology, epigenomic code and gene expression in juxtaposed loci, suggesting a driving role in myeloma genome instability.