Essential genes shape cancer genomes through linear limitation of homozygous deletions.

The landscape of somatic acquired deletions in cancer cells is shaped by positive and negative selection. Recurrent deletions typically target tumor suppressor, leading to positive selection. Simultaneously, loss of a nearby essential gene can lead to negative selection, and introduce latent vulnerabilities specific to cancer cells. Here we show that, under basic assumptions on positive and negative selection, deletion limitation gives rise to a statistical pattern where the frequency of homozygous deletions decreases approximately linearly between the deletion target gene and the nearest essential genes. Using DNA copy number data from 9,744 human cancer specimens, we demonstrate that linear deletion limitation exists and exposes deletion-limiting genes for seven known deletion targets (CDKN2A, RB1, PTEN, MAP2K4, NF1, SMAD4, and LINC00290). Downstream analysis of pooled CRISPR/Cas9 data provide further evidence of essentiality. Our results provide further insight into how the deletion landscape is shaped and identify potentially targetable vulnerabilities.

Author Correction: Identification of multiple risk loci and regulatory mechanisms influencing susceptibility to multiple myeloma.

The original version of this Article contained an error in the spelling of a member of the PRACTICAL Consortium, Manuela Gago-Dominguez, which was incorrectly given as Manuela Gago Dominguez. This has now been corrected in both the PDF and HTML versions of the Article. Furthermore, in the original HTML version of this Article, the order of authors within the author list was incorrect. The PRACTICAL consortium was incorrectly listed after Richard S. Houlston and should have been listed after Nora Pashayan. This error has been corrected in the HTML version of the Article; the PDF version was correct at the time of publication.

Identification of multiple risk loci and regulatory mechanisms influencing susceptibility to multiple myeloma.

Genome-wide association studies (GWAS) have transformed our understanding of susceptibility to multiple myeloma (MM), but much of the heritability remains unexplained. We report a new GWAS, a meta-analysis with previous GWAS and a replication series, totalling 9974 MM cases and 247,556 controls of European ancestry. Collectively, these data provide evidence for six new MM risk loci, bringing the total number to 23. Integration of information from gene expression, epigenetic profiling and in situ Hi-C data for the 23 risk loci implicate disruption of developmental transcriptional regulators as a basis of MM susceptibility, compatible with altered B-cell differentiation as a key mechanism. Dysregulation of autophagy/apoptosis and cell cycle signalling feature as recurrently perturbed pathways. Our findings provide further insight into the biological basis of MM.

The multiple myeloma risk allele at 5q15 lowers ELL2 expression and increases ribosomal gene expression.

Recently, we identified ELL2 as a susceptibility gene for multiple myeloma (MM). To understand its mechanism of action, we performed expression quantitative trait locus analysis in CD138+ plasma cells from 1630 MM patients from four populations. We show that the MM risk allele lowers ELL2 expression in these cells (Pcombined = 2.5 × 10-27; ßcombined = -0.24 SD), but not in peripheral blood or other tissues. Consistent with this, several variants representing the MM risk allele map to regulatory genomic regions, and three yield reduced transcriptional activity in plasmocytoma cell lines. One of these (rs3777189-C) co-locates with the best-supported lead variants for ELL2 expression and MM risk, and reduces binding of MAFF/G/K family transcription factors. Moreover, further analysis reveals that the MM risk allele associates with upregulation of gene sets related to ribosome biogenesis, and knockout/knockdown and rescue experiments in plasmocytoma cell lines support a cause-effect relationship. Our results provide mechanistic insight into MM predisposition.

Identification of sequence variants influencing immunoglobulin levels.

Immunoglobulins are the effector molecules of the adaptive humoral immune system. In a genome-wide association study of 19,219 individuals, we found 38 new variants and replicated 5 known variants associating with IgA, IgG or IgM levels or with composite immunoglobulin traits, accounted for by 32 loci. Variants at these loci also affect the risk of autoimmune diseases and blood malignancies and influence blood cell development. Notable associations include a rare variant at RUNX3 decreasing IgA levels by shifting isoform proportions (rs188468174[C>T]: P = 8.3 × 10-55, ß = -0.90 s.d.), a rare in-frame deletion in FCGR2B abolishing IgG binding to the encoded receptor (p.Asn106del: P = 4.2 × 10-8, ß = 1.03 s.d.), four IGH locus variants influencing class switching, and ten new associations with the HLA region. Our results provide new insight into the regulation of humoral immunity.

Direct evidence for a polygenic etiology in familial multiple myeloma.

Although common risk alleles for multiple myeloma (MM) were recently identified, their contribution to familial MM is unknown. Analyzing 38 familial cases identified primarily by linking Swedish nationwide registries, we demonstrate an enrichment of common MM risk alleles in familial compared with 1530 sporadic cases (P = 4.8 × 10-2 and 6.0 × 10-2, respectively, for 2 different polygenic risk scores) and 10 171 population-based controls (P = 1.5 × 10-4 and 1.3 × 10-4, respectively). Using mixture modeling, we estimate that about one-third of familial cases result from such enrichments. Our results provide the first direct evidence for a polygenic etiology in a familial hematologic malignancy.

Genome-wide association study identifies multiple susceptibility loci for multiple myeloma.

Multiple myeloma (MM) is a plasma cell malignancy with a significant heritable basis. Genome-wide association studies have transformed our understanding of MM predisposition, but individual studies have had limited power to discover risk loci. Here we perform a meta-analysis of these GWAS, add a new GWAS and perform replication analyses resulting in 9,866 cases and 239,188 controls. We confirm all nine known risk loci and discover eight new loci at 6p22.3 (rs34229995, P=1.31 × 10(-8)), 6q21 (rs9372120, P=9.09 × 10(-15)), 7q36.1 (rs7781265, P=9.71 × 10(-9)), 8q24.21 (rs1948915, P=4.20 × 10(-11)), 9p21.3 (rs2811710, P=1.72 × 10(-13)), 10p12.1 (rs2790457, P=1.77 × 10(-8)), 16q23.1 (rs7193541, P=5.00 × 10(-12)) and 20q13.13 (rs6066835, P=1.36 × 10(-13)), which localize in or near to JARID2, ATG5, SMARCD3, CCAT1, CDKN2A, WAC, RFWD3 and PREX1. These findings provide additional support for a polygenic model of MM and insight into the biological basis of tumour development.

Variants in ELL2 influencing immunoglobulin levels associate with multiple myeloma.

Multiple myeloma (MM) is characterized by an uninhibited, clonal growth of plasma cells. While first-degree relatives of patients with MM show an increased risk of MM, the genetic basis of inherited MM susceptibility is incompletely understood. Here we report a genome-wide association study in the Nordic region identifying a novel MM risk locus at ELL2 (rs56219066T; odds ratio (OR)=1.25; P=9.6 × 10(-10)). This gene encodes a stoichiometrically limiting component of the super-elongation complex that drives secretory-specific immunoglobulin mRNA production and transcriptional regulation in plasma cells. We find that the MM risk allele harbours a Thr298Ala missense variant in an ELL2 domain required for transcription elongation. Consistent with a hypomorphic effect, we find that the MM risk allele also associates with reduced levels of immunoglobulin A (IgA) and G (IgG) in healthy subjects (P=8.6 × 10(-9) and P=6.4 × 10(-3), respectively) and, potentially, with an increased risk of bacterial meningitis (OR=1.30; P=0.0024).

Robust isolation of malignant plasma cells in multiple myeloma.

Molecular characterization of malignant plasma cells is increasingly important for diagnostic and therapeutic stratification in multiple myeloma. However, the malignant plasma cells represent a relatively small subset of bone marrow cells, and need to be enriched prior to analysis. Currently, the cell surface marker CD138 (SDC1) is used for this enrichment, but has an important limitation in that its expression decreases rapidly after sampling. Seeking alternatives to CD138, we performed a computational screen for myeloma plasma cell markers and systematically evaluated 7 candidates. Our results conclusively show that the markers CD319 (SLAMF7/CS1) and CD269 (TNFRSF17/BCMA) are considerably more robust than CD138 and enable isolation of myeloma plasma cells under more diverse conditions, including the samples that have been delayed or frozen. Our results form the basis of improved procedures for characterizing cases of multiple myeloma in clinical practice.

The retinoblastoma gene undergoes rearrangements in BRCA1-deficient basal-like breast cancer.

Breast tumors from BRCA1 germ line mutation carriers typically exhibit features of the basal-like molecular subtype. However, the specific genes recurrently mutated as a consequence of BRCA1 dysfunction have not been fully elucidated. In this study, we used gene expression profiling to molecularly subtype 577 breast tumors, including 73 breast tumors from BRCA1/2 mutation carriers. Focusing on the RB1 locus, we analyzed 33 BRCA1-mutated, 36 BRCA2-mutated, and 48 non-BRCA1/2-mutated breast tumors using a custom-designed high-density oligomicroarray covering the RB1 gene. We found a strong association between the basal-like subtype and BRCA1-mutated breast tumors and the luminal B subtype and BRCA2-mutated breast tumors. RB1 was identified as a major target for genomic disruption in tumors arising in BRCA1 mutation carriers and in sporadic tumors with BRCA1 promoter methylation but rarely in other breast cancers. Homozygous deletions, intragenic breaks, or microdeletions were found in 33% of BRCA1-mutant tumors, 36% of BRCA1 promoter-methylated basal-like tumors, 13% of non-BRCA1-deficient basal-like tumors, and 3% of BRCA2-mutated tumors. In conclusion, RB1 was frequently inactivated by gross gene disruption in BRCA1 hereditary breast cancer and BRCA1-methylated sporadic basal-like breast cancer but rarely in BRCA2 hereditary breast cancer and non-BRCA1-deficient sporadic breast cancers. Together, our findings show the existence of genetic heterogeneity within the basal-like breast cancer subtype that is based upon BRCA1 status.

Neutrophil elastase sorting involves plasma membrane trafficking requiring the C-terminal propeptide.

The primary granules/secretory lysosomes of neutrophils store mature neutrophil elastase (NE) as a luminal protein after proteolytic removal of N-terminal and C-terminal pro-peptides from a proform of NE. The N-terminal pro-peptide prevents premature activation that might be toxic to the cell, but the C-terminal pro-peptide has no defined function. In this study, we investigated the role of the C-terminal pro-peptide in trafficking of NE by expressing, in rat basophilic leukemia (RBL) cells, both wild-type NE and the mutant NE/Delta248-267, which lacks the C-terminal pro-peptide. Both transfected proteins were found to be targeted to secretory lysosomes. In addition, results from antibody ligation and cell-surface biotinylation indicated that proform of NE was targeted to the plasma membrane, and then subjected to endocytosis. The results were supported by the detection of targeting of the proform to the plasma membrane followed by internalization both in RBL cells and normal granulopoietic precursor cells. Targeting of NE to the plasma membrane required the C-terminal pro-peptide as NE/Delta248-267 expressed in RBL cells bypassed plasma membrane trafficking. Our results indicate targeting of a population of NE to the plasma membrane and internalization dependent on the C-terminal NE pro-peptide.

Artificially controlled aggregation of proteins and targeting in hematopoietic cells.

The targeting mechanisms for granule proteins in hematopoietic cells are largely unknown. Aggregation is believed to be important for protein sorting-for-entry and sorting-by-retention in endocrine and neuroendocrine cells. We asked whether artificially induced multimerization/aggregation of chimeric proteins could affect their sorting in hematopoietic cells. A system was used that permits ligand-controlled intracellular oligomerization of hybrid proteins containing the FK506-binding protein (FKBP). The hybrid proteins ELA-(FKBP)3 with neutrophil elastase (ELA) and (FKBP*)4-FCS-hGH with a furin cleavage site (FCS) and human growth hormone (hGH) were expressed in the myeloblastic 32D and the rat basophilic leukemia (RBL-1) hematopoietic cell lines. ELA alone is normally targeted to secretory lysosomes. However, the hybrid proteins and ligand-induced aggregates of them were constitutively secreted and not targeted. The hGH that was released at the FCS in (FKBP*)4-FCS-hGH was also constitutively secreted. We conclude that protein multimerization/aggregation per se is not enough to facilitate sorting-for-entry to secretory lysosomes in hematopoietic cells and that improperly folded proteins may be eliminated from sorting by constitutive secretion.

Sorting of soluble TNF-receptor for granule storage in hematopoietic cells as a principle for targeting of selected proteins to inflamed sites.

Hematopoietic cells have secretory lysosomes that degranulate at the inflammatory site upon stimulation. We asked whether one could target exogenous proteins with a therapeutic potential to secretory lysosomes in hematopoietic cells. For this purpose, we expressed a soluble tumor necrosis factor (TNF) receptor form (sTNFR1) in hematopoietic cell lines. In order to accomplish targeting to secretory lysosomes, both endoplasmic reticulum (ER) retention and constitutive secretion have to be prevented. ER export was facilitated by addition of a transmembrane (tm) sequence, and constitutive secretion was overcome by incorporating a cytosolic sorting signal (Y) from CD63. This signal directed the resulting sTNFR1-tm-Y to secretory lysosomes. Confirmation of these results was provided by biosynthetic radiolabeling, subcellular fractionation, immunofluorescence microscopy, and immunoelectron microscopy. The tm-Y fragment was cleaved by proteolysis, resulting in generation of the membrane-free sTNFR1 in secretory lysosomes. Our results suggest a potential for using the storage organelles of hematopoietic cells as vehicles for targeting sites of inflammation with therapeutically active agents.