MYC Translocations In Multiple Myeloma Involve Recruitment Of Enhancer Elements Resulting In Over-Expression and Decreased Overall Survival

Myc deregulation is an important and recurrent molecular event in myeloma. MYC is located at chromosome 8q24 and gene-mapping analyses of copy number abnormalities (CNA) within the myeloma genome have identified a hot spot of activity at this locus. We have previously found evidence of CNA at 8q24 in 9% of 114 presenting cases. The presence of recurrent copy number variants at 8q24 suggest either an important underlying molecular mechanism active at this locus, that is important in the pathogenesis of myeloma, or that deregulation of MYC is an important recurrent molecular event in myeloma. In order to address the prevalence and mechanism of rearrangements at the 8q24 locus and their association with chromosomal translocations into the Ig gene loci we used a massively parallel sequencing approach combined with pull down of the region surrounding the MYC locus as well as the Ig loci to define the molecular mechanism active at this locus and their association with other better described translocations.

CD138-positive bone marrow plasma cells were selected to a purity >95% using magnetic assisted cell sorting (Miltenyi Biotech). We developed a targeted capture using the SureSelect (Agilent) system by tiling RNA baits across the MYC, IGH, IGK and IGL loci. The region captured surrounding MYC spanned from 127.5-129.8 Mb on chromosome 8, which is roughly 1 Mb either side of MYC which is located at 128.75 Mb. This region includes POU5F1B and PVT1 which are common sites of translocations in myeloma. Repetitive sequences in the MYC region were masked resulting in an overall capture of 66% of the sequence. DNA from 104 samples were assayed using 150 ng of DNA and a modified capture protocol with eight cycles of pre-hybridization PCR and 11 cycles of post-hybridization PCR. Samples were barcoded using Illumina indexes and up to 27 samples were sequenced per lane on a HiSeq2000 generating 76-bp paired-end reads. After base calling and quality control metrics, the raw fastq reads were aligned to the reference human genome (build GRCh37) resulting in a median depth of 289X per sample after de-duplication for the captured region. Translocation breakpoints were identified in the sequencing data using SVDetect. Bioinformatically called breakpoints were further filtered based on depth, unique mappability, number of supporting reads and whether or not they were detected in non-tumor samples.

Of the 104 samples interrogated, 8q24 breakpoints were found in 22 (21%). These samples comprised of t(11;14) (n=6), t(14;16) (n=10), t(14;20) (n=1), t(6;14) (n=1), t(7;14) (n=1), and those with no IGH translocation (n=3). There was an enrichment for t(14;16) (10/17) and t(11;14) (5/27) and a depletion of t(4;14) (0/13) and hyperdiploid (3/28) with an 8q24 breakpoint. Eight of the 22 samples had an 8q24 translocation involving IGH, IGK or IGL. The remaining samples with 8q24 breakpoints had partner genes scattered throughout the genome, but with a preponderance for those associated with B cell biology or known myeloma-associated genes. These genes included CCND1, FAM46C, XBP1, KRAS and FOXO3. In all samples the position of the breakpoints on 8q24 were spread out over 1.2 Mb. The majority were situated telomeric of PVT1 (51.5%) with 12% within PVT1, 15% between MYC and PVT1 and 15% between POU5F1B and MYC. Of the 3 samples with breakpoints within PVT1 2 have potential fusion genes, one with FOXO3 and one with LINC00309. Nine samples had two breakpoints on 8q24, often with two different chromosome partners. Several samples had complex rearrangements resulting in multiple chromosomal segments being joined together, often involving MYC, an Ig enhancer and the primary Ig partner oncogene (e.g. CCND3). Given that 8 samples have translocations involving MYC and Ig loci, which presumably results in overexpression of MYC driven by the Ig loci enhancers, we looked for enhancer sequences in the partner chromosomes of the other samples. Most samples had easily identifiable B cell H3K27Ac marks (taken from ENCODE), identifying active regulatory elements. 55 samples were taken from the MRC Myeloma IX trial and a comparison of those with and without MYC breakpoints showed a significant decrease in overall survival (P=0.04) in those with a breakpoint (median of 21.5 versus 55.8 months).

We conclude that MYC translocations occur in up to 21% of presenting myeloma samples and involve the sequestration of active enhancer elements, resulting in increased expression of MYC.