MYC Rearrangements in Multiple Myeloma Are Complex, Can Involve More Than Five Different Chromosomes, and Correlate with Increased Expression of MYC and a Distinct Downstream Gene Expression Pattern

Introduction: The proto-oncogene MYC (locus 8q24.21) is a key transcription factor in multiple myeloma (MM) resulting in significant gene deregulation and impacting on many biological functions, including cell growth, proliferation, apoptosis, differentiation, and transformation. Chromosomal rearrangement and copy number change at the MYC locus are secondary events involved in MM progression, which are thought to lead to aggressive disease. Current analyses of the MYC locus have not been large and have reported rearrangements in 15% of new-diagnosed MM. However, more recent studies using advanced genomic techniques suggest that the frequency of MYC rearrangements may be much higher, and that a full reassessment of the role of MYC in MM pathogenesis may be critical. In this study, we analyzed 1280 MM patients to provide a better understanding of the role of this important genomic driver in MM pathogenesis.

Methods: In total, 1280 tumor normal pairs of CD138 sorted bone marrow plasma cells and their germline control samples were analyzed by: 1. Targeted sequencing of 131 genes and 27 chromosome regions (n=100) with 4.5 Mb captured region surrounding MYC ; 2. Exome sequencing (n=461) with 2.3 Mb captured region surrounding MYC ; 3. Whole genome sequencing (n=719). Normalized tumor/germline depth ratio in targeted-sequencing cases and MANTA were used for detection of somatic copy number and structural variants. Expression analysis was performed using RNA-seq or microarrays.

Results: MYC translocations were found in 25% (323/1280) of patients and occurred most frequently as inter-chromosomal translocations involving 2-5 chromosomes (90%, 291/323). Of the remaining cases, 5% (17/323) of the translocations involved inversion of chromosome 8 and 5% (15/323) were complex, affecting more than 5 chromosomal loci. The proportion of MYC translocations involving 2, 3, 4, and 5 loci was 62% (200/323), 23% (74/323), 8% (26/323) and 3% (8/323), respectively. Using abnormal rearranged cases (29/100), we found copy number imbalances >14.2 kb in size associated with a MYC translocation in 76% (22/29). Another 7% (2/29) of cases with translocations showed complex intra-chromosomal rearrangement. A region of 2.0 Mb surrounding MYC was identified as a translocation breakpoint hot-spot incorporating 96% of breakpoints. This region also contained two hotspots for chromosomal gain and tandem duplications. MYC rearrangements were not randomly distributed across the spectrum of MM with an excess being seen in hyperdiploidy (76% of rearranged samples, P <0.0001). Importantly, 67% (207/308) of cases with a MYC translocation involving 5 or less chromosomes had one of the commonly known super-enhancers involved in the translocation. Gene expression analysis was used to explore the impact of these events on downstream gene expression patterns. The results showed that inter- and/or intra-chromosomal rearrangements were associated with a significantly (P <0.0001) higher MYC expression (4.1-fold). In patients where rearrangements were associated with additional copies of MYC there was higher expression of MYC in comparison to cases with a translocation but lacking copy number gain (P=0.04). To identify downstream genes deregulated by MYC rearrangements we compared gene expression between those with and without a translocation, independently of hyperdiploidy. Genes that showed >2-fold change in expression (P <0.01) included MYC and the non-protein coding oncogene PVT1 that is located next to MYC . Genes with significantly lower levels of expression were involved in B-cell biology including CD79A and AHR, or were associated with cell proliferation, migration, adhesion, apoptosis and/or angiogenesis (FGF16, ADAMTS1, FBXL7, HRK, PDGFD, and PRKD1) .

Conclusions: This study confirms the central role of MYC in the pathogenesis of clinical cases of MM, and as such defining it as a critical therapeutic target. We will be able to target MYC better if we understand how it is deregulated and in this respect we show that the MYC locus rearrangements are complex and it is a hot-spot for heterogeneous inter- as well intra-chromosomal rearrangements, including complex rearrangements involving >5 chromosomes. These events lead to increased MYC expression consistent with it being a driver of disease progression, particularly in the hyperdiploid subset of MM.