Microhomology-mediated end joining drives complex rearrangements and over-expression of MYC and PVT1 in multiple myeloma

Abstract

MYC is a widely acting transcription factor and its deregulation is a crucial event in many human cancers. MYC is important biologically and clinically in multiple myeloma, but the mechanisms underlying its dysregulation are poorly understood. We show that MYC rearrangements are present in 36.0% of newly diagnosed myeloma patients, as detected in the largest set of next generation sequencing data to date (n=1267). Rearrangements were complex and associated with increased expression of MYC and PVT1, but not other genes at 8q24. The highest effect on gene expression was detected in cases where the MYC locus is juxtaposed next to superenhancers associated with genes such as IGH, IGK, IGL, TXNDC5/BMP6, FAM46C and FOXO3.We identified three hotspots of recombination at 8q24, one of which is enriched for IGH-MYC translocations. Breakpoint analysis indicates primary myeloma rearrangements involving the IGH locus occur through non-homologous end joining, whereas secondary MYC rearrangements occur through microhomology-mediated end joining. This mechanism is different to lymphomas, where non-homologous end joining generates MYC rearrangements. Rearrangements resulted in over-expression of key genes and ChIP-seq identified that HK2, a member of the glucose metabolism pathway, is directly over-expressed through binding of MYC at its promoter.

Epigenomic translocation of H3K4me3 broad domains over oncogenes following hijacking of super-enhancers

“…GRCh37 human genome assembly was used for sequence mapping using BWA-MEM (v0.7.12). Chromosomal rearrangements were called using Manta v.0.29.6 (Mikulasova et al 2019; X. Chen et al 2016).…”

“…Patient derived xenografts were generated by passaging primary patient CD138+ selected cells through the previously described SCID-rab myeloma mouse model (Yata and Yaccoby 2004; Mikulasova et al 2019). Detailed methods are described previously (Mikulasova et al 2019). Seven patient derived xenografts were used in this study ( Supplementary Table 4 ).…”

“…ChIP-seq was performed as previously reported (Mikulasova et al 2019), for the myeloma cell lines KMS11 and MM1S as well as seven myeloma patient derived xenograft samples. ChIP-seq for the histone marks H3K4me1, H3K4me3, H3K9me3, H3K27me3, H3K27Ac, and H3K36me3 (Active Motif, Carlsbad, CA, USA) were included in this study.…”

“…RNA was prepared using the TruSeq stranded total RNA Ribo-zero gold kit (Illumina, San Diego, CA, USA) and libraries were sequenced using 75 bp paired end reads on a NextSeq500 (Illumina). RNA-seq data was analysed as previously reported (Mikulasova et al 2019). Briefly, raw data were aligned to the human genome assembly GRCh38 with gene transcript quantification being processed by Star (v2.5.1b) and Salmon (v0.6.0) algorithms.…”

Linear and circular PVT1 in hematological malignancies and immune response: two faces of the same coin

“…Elevated PVT1 levels were detected in MM BM cells compared with normal tissue [68] and primarily in MYCrearranged MM cases [69]. In particular, high PVT1 was associated with MM carrying recurrent MYC fusion genes (with IGH, IGK, IGL, TXNDC5/BMP6, FOXO3 and FAM46C partner genes) or complex rearrangements involving more than five loci, or hyperdiploid cases, that also overexpressed MYC [69].…”