Initial genome sequencing and analysis of multiple myeloma

Abstract

Multiple myeloma is an incurable malignancy of plasma cells, and its pathogenesis is poorly understood. Here we report the massively parallel sequencing of 38 tumour genomes and their comparison to matched normal DNAs. Several new and unexpected oncogenic mechanisms were suggested by the pattern of somatic mutation across the data set. These include the mutation of genes involved in protein translation (seen in nearly half of the patients), genes involved in histone methylation, and genes involved in blood coagulation. In addition, a broader than anticipated role of NF-κB signalling was indicated by mutations in 11 members of the NF-κB pathway. Of potential immediate clinical relevance, activating mutations of the kinase BRAF were observed in 4% of patients, suggesting the evaluation of BRAF inhibitors in multiple myeloma clinical trials. These results indicate that cancer genome sequencing of large collections of samples will yield new insights into cancer not anticipated by existing knowledge.

Figure 1. Evidence for transcription-coupled repair and functional importance (FI) of statistically significant mutations

(a) Intronic mutation rates subdivided by gene expression rates in MM. Rates of gene expression were estimated by proportion of Affymetrix Present (P) calls in 304 primary MM samples. Error bars indicate standard deviation. (b) FI scores were generated for all point mutations and divided into distributions for non-significant mutations (upper histogram) and significant mutations (lower). Comparison of distributions is via the Kolmogorov-Smirnov statistic.

Figure 2. Mutations likely to affect protein translation and/or homeostasis in MM

(a) Alignment of human, yeast, and bacterial RNB domain of DIS3. Positions of observed mutations are indicated with respect to the human sequence. Yeast equivalents are, respectively, S541, V568, G833, and R847. (b) 2D and 3D structures of yeast DIS3, with RNB domain colored in blue and mutations colored in red. (c) GSEA plot showing enrichment of ribosomal protein gene set amongst genes correlated with FAM46C expression in 414 MM samples.

Figure 3. HOXA9is a candidate oncogene in MM

(a) H3K27Me3 enrichment at the HOXA9 promoter in CD34 cells, CD19 cells, and MM cell lines relative to H3K27Me3 methylation at the BC site, known to be hypomethylated in all cells. (b) Relative HOXA9 expression vs. H3K27Me3 enrichment at the HOXA9 locus. (c) GFP competition assay in MM cell lines. Following lentiviral infection with seven HOXA9 shRNAs or a control shRNA targeting luciferase, GFP-positive cells were monitored by flow cytometry and compared to the proportion of GFP-positive cells present in the population 3 days post-infection (designated day 0). Error bars indicate standard error of the mean and represent a minimum of 3 independent experiments.