Targeted next-generation sequencing of DNA regions proximal to a conserved GXGXXG signaling motif enables systematic discovery of tyrosine kinase fusions in cancer

Tyrosine kinase (TK) fusions are attractive drug targets in cancers. However, rapid identification of these lesions has been hampered by experimental limitations. Our in silico analysis of known cancer-derived TK fusions revealed that most breakpoints occur within a defined region upstream of a conserved GXGXXG kinase motif. We therefore designed a novel DNA-based targeted sequencing approach to screen systematically for fusions within the 90 human TKs; it should detect 92% of known TK fusions. We deliberately paired ‘in-solution’ DNA capture with 454 sequencing to minimize starting material requirements, take advantage of long sequence reads, and facilitate mapping of fusions. To validate this platform, we analyzed genomic DNA from thyroid cancer cells (TPC-1) and leukemia cells (KG-1) with fusions known only at the mRNA level. We readily identified for the first time the genomic fusion sequences of CCDC6-RET in TPC-1 cells and FGFR1OP2-FGFR1 in KG-1 cells. These data demonstrate the feasibility of this approach to identify TK fusions across multiple human cancers in a high-throughput, unbiased manner. This method is distinct from other similar efforts, because it focuses specifically on targets with therapeutic potential, uses only 1.5 µg of DNA, and circumvents the need for complex computational sequence analysis.     

Y Fuse? Sex Chromosome Fusions in Fishes and Reptiles

Chromosomal fusion plays a recurring role in the evolution of adaptations and reproductive isolation among species, yet little is known of the evolutionary drivers of chromosomal fusions. Because sex chromosomes (X and Y in male heterogametic systems, Z and W in female heterogametic systems) differ in their selective, mutational, and demographic environments, those differences provide a unique opportunity to dissect the evolutionary forces that drive chromosomal fusions. We estimate the rate at which fusions between sex chromosomes and autosomes become established across the phylogenies of both fishes and squamate reptiles. Both the incidence among extant species and the establishment rate of Y-autosome fusions is much higher than for X-autosome, Z-autosome, or W-autosome fusions. Using population genetic models, we show that this pattern cannot be reconciled with many standard explanations for the spread of fusions. In particular, direct selection acting on fusions or sexually antagonistic selection cannot, on their own, account for the predominance of Y-autosome fusions. The most plausible explanation for the observed data seems to be (a) that fusions are slightly deleterious, and (b) that the mutation rate is male-biased or the reproductive sex ratio is female-biased. We identify other combinations of evolutionary forces that might in principle account for the data although they appear less likely. Our results shed light on the processes that drive structural changes throughout the genome.     

Kinase gene fusions in defined subsets of melanoma

Genomic rearrangements resulting in activating kinase fusions have been increasingly described in a number of cancers including malignant melanoma, but their frequency in specific melanoma subtypes has not been reported. We used break‐apart fluorescence in situ hybridization (FISH) to identify genomic rearrangements in tissues from 59 patients with various types of malignant melanoma including acral lentiginous, mucosal, superficial spreading, and nodular. We identified four genomic rearrangements involving the genes BRAF, RET, and ROS1. Of these, three were confirmed by Immunohistochemistry (IHC) or sequencing and one was found to be an ARMC10‐BRAF fusion that has not been previously reported in melanoma. These fusions occurred in different subtypes of melanoma but all in tumors lacking known driver mutations. Our data suggest gene fusions are more common than previously thought and should be further explored particularly in melanomas lacking known driver mutations.     

Characterization of a group of anaerobically induced, fnr-dependent genes of Salmonella typhimurium.

We have previously reported the isolation of a group of anaerobically regulated, fnr-dependent lac fusions in Salmonella typhimurium and have grouped these oxd genes into classes based on map position. In order to identify these genes, we have replaced the original Mud-lac fusion in a member of each oxd class with the much smaller Mud-cam element, cloned the fusion, and determined DNA sequence sufficient to define the oxd gene. Several of the fusions correspond to previously known genes from S. typhimurium or Escherichia coli: oxd-4 = cbiA and oxd-11 = cbiK, oxd-5 = hybB, oxd-7 = dcuB, oxd-8 = moaB, oxd-12 = dmsA, and oxd-14 = napB (aeg-46. 5). Two other fusions correspond to previously unknown loci: oxd-2 encodes an acetate/propionate kinase, and oxd-6 encodes a putative ABC transporter present in S. typhimurium but not in E. coli.     

Subcellular localization of avian sarcoma virus and human immunodeficiency virus type 1 integrases.

The composition and subcellular trafficking of subviral preintegration complexes are reported to vary among the different retroviruses. The process by which the avian sarcoma virus (ASV) preintegration complex gains access to target chromatin remains unknown. Here we report that ASV integrase (IN) expressed as a fusion to beta-galactosidase accumulates in the nuclei of transfected COS-1 cells. In contrast, human immunodeficiency type 1 (HIV-1) IN-beta-galactosidase fusions expressed similarly are predominantly cytoplasmic. To identify the region of ASV IN that specifies nuclear localization, various subdomains of the protein were expressed as beta-galactosidase fusions and their subcellular locations were assessed cytochemically and by indirect immunofluorescence. These analyses showed that the ASV IN protein possesses a functional nuclear localization signal that spans amino acids 206 to 235 and displays limited homology with known nuclear transport signals.     

Multiple independent fusions of glucose-6-phosphate dehydrogenase with enzymes in the pentose phosphate pathway

Fusions of the first two enzymes in the pentose phosphate pathway, glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconolactonase (6PGL), have been previously described in two distant clades, chordates and species of the malarial parasite Plasmodium. We have analyzed genome and expressed sequence data from a variety of organisms to identify the origins of these gene fusion events. Based on the orientation of the domains and range of species in which homologs can be found, the fusions appear to have occurred independently, near the base of the metazoan and apicomplexan lineages. Only one of the two metazoan paralogs of G6PD is fused, showing that the fusion occurred after a duplication event, which we have traced back to an ancestor of choanoflagellates and metazoans. The Plasmodium genes are known to contain a functionally important insertion that is not seen in the other apicomplexan fusions, highlighting this as a unique characteristic of this group. Surprisingly, our search revealed two additional fusion events, one that combined 6PGL and G6PD in an ancestor of the protozoan parasites Trichomonas and Giardia, and another fusing G6PD with phosphogluconate dehydrogenase (6PGD) in a species of diatoms. This study extends the range of species known to contain fusions in the pentose phosphate pathway to many new medically and economically important organisms.     

Oncogenic Neuregulin 1 gene (NRG1) fusions in cancer: A potential new therapeutic opportunities

It is widely established that chromosomal rearrangements induce oncogenesis in solid tumors. However, discovering chromosomal rearrangements that are targetable and actionable remains a difficulty. Targeting gene fusion or chromosomal rearrangement seems to be a powerful strategy to address malignancies characterized by gene rearrangement. Oncogenic NRG1 fusions are relatively rare drivers that infrequently occur across most tumor types. NRG1 fusions exhibit unique biological properties and are difficult to identify owing to their large intronic regions. NRG1 fusions can be detected using a variety of techniques, including fluorescence in situ hybridization, immunohistochemistry, or next-generation sequencing (NGS), with NGS-based RNA sequencing being the most sensitive. Previous studies have shown that NRG1 fusion protein induces tumorigenesis, and numerous therapies targeting the ErbB signaling pathway, such as ErbB kinase inhibitors and monoclonal antibodies, have initially demonstrated encouraging anticancer efficacy in malignant tumors carrying NRG1 fusions. In this review, we present the characteristics and prevalence of NRG1 fusions in solid tumors. Additionally, we discuss the laboratory approaches for diagnosing NRG1 gene fusions. More importantly, we outline promising strategies for treating malignancies with NRG1 fusion.     

Topological characterization of the essential Escherichia coli cell division protein FtsW

The membrane topology of Escherichia coli FtsW, a 46-kDa essential protein, was analyzed using a set of 28 ftsW-alkaline phosphatase (ftsW-phoA) and nine ftsW-beta-lactamase (ftsW-bla) gene fusions obtained by in vivo and in vitro methods. The alkaline phosphatase activities or resistance pattern of cells expressing the FtsW-PhoA or FtsW-Bla fusions confirmed only eight out of 10 transmembrane segments predicted by computational methods. After comparison with the recent topology of Streptococcus pneumoniae FtsW, we could identify all the fusions in absolute agreement with the predicted model: N-terminal and C-terminal ends in the cytoplasm, 10 transmembrane segments and one large loop of 67 amino acids (E240-E306) located in the periplasm.     

KCl stimulation and ultrastructural responses of tannic acid-stained cholinergic synaptic terminals

The quantal-vesicular hypothesis equates miniature end-plate potentials (MEPPs) with fusions of synaptic vesicles. MEPP production thus predicts vesicle losses, increases in vesicle fusions and increases in terminal plasma membrane. MEPP production and these ultrastructural parameters have been evaluated in the cholinergic presynaptic terminals of skate electric organ following tannic acid saline incubation, known to promote capture and selective staining of dense-core granule fusions, and KCl stimulation, known to elevate MEPP production dramatically in these cholinergic terminals. After pretreatment in tannic acid-elasmobranch saline, KCl stimulation produced MEPPs at 40/s/microm(2)of terminal surface for several minutes with gradual reduction to spontaneous levels by 25-30 min. No loss of vesicles, no vesicle fusions, no expansions of plasma membrane and no tannic acid enhanced staining of vesicles or vacuoles accompanied the generation of 800 MEPPs/microm(3)of terminals having densities of 567 vesicles/microm(3). No ultrastructural footprints were found to support the notion that unnaturally high rates of vesicular exocytosis had occurred.     

A visual screen of a GFP-fusion library identifies a new type of nuclear envelope membrane protein.

The nuclear envelope (NE) is a distinct subdomain of the ER, but few membrane components have been described that are specific to it. We performed a visual screen in tissue culture cells to identify proteins targeted to the NE. This approach does not require assumptions about the nature of the association with the NE or the physical separation of NE and ER. We confirmed that screening a library of fusions to the green fluorescent protein can be used to identify proteins targeted to various subcompartments of mammalian cells, including the NE. With this approach, we identified a new NE membrane protein, named nurim. Nurim is a multispanning membrane protein without large hydrophilic domains that is very tightly associated with the nucleus. Unlike the known NE membrane proteins, it is neither associated with nuclear pores, nor targeted like lamin-associated membrane proteins. Thus, nurim is a new type of NE membrane protein that is localized to the NE by a distinct mechanism.     

RpoS-regulated genes of Escherichia coli identified by random lacZ fusion mutagenesis

RpoS is a conserved alternative sigma factor that regulates the expression of many stress response genes in Escherichia coli. The RpoS regulon is large but has not yet been completely characterized. In this study, we report the identification of over 100 RpoS-dependent fusions in a genetic screen based on the differential expression of an operon-lacZ fusion bank in rpoS mutant and wild-type backgrounds. Forty-eight independent gene fusions were identified, including several in well-characterized RpoS-regulated genes, such as osmY, katE, and otsA. Many of the other fusions mapped to genes of unknown function or to genes that were not previously known to be under RpoS control. Based on the homology to other known bacterial genes, some of the RpoS-regulated genes of unknown functions are likely important in nutrient scavenging.     

Identification of new genes regulated by the marRAB operon in Escherichia coli.

Random TnphoA and TnlacZ translational fusions were introduced into an Escherichia coli strain with a deletion of the multiple antibiotic resistance (mar) locus, complemented in trans by a temperature-sensitive plasmid bearing the mar locus with a constitutively expressed mar operon. Five gene fusions (two with lacZ and three with phoA) regulated by the mar operon were identified by increased or decreased marker enzyme activity following loss of the complementary plasmid at the restrictive temperature. Expression of LacZ from both lacZ fusions increased in the presence of the mar operon; expression from the three phoA fusions was represented by the mar operon. The lacZ fusions were mapped at 31.5 and 14 min on the Escherichia coli chromosome. One of the phoA fusions was located at 51.6 min while the two others mapped at 77 min. Cloning and sequencing of a portion of the fused genes showed all of them to be different. The phoA fusions at 77 min were located in a recently identified gene, slp, a lipoprotein of unknown function (D.M. Alexander and A. C. St. John, Mol. Microb. 11:1059-1071, 1994). The others showed no homology with any known genes of E. coli. The insertions caused small but reproducible changes in the antibiotic susceptibility profile. This approach has enabled the identification of new genes in E. coli which are regulated by the marRAB operon and involved in the Mar phenotype.     

The use of gene fusions to examine the membrane topology of the L-subunit of the photosynthetic reaction center and of the cytochrome b subunit of the bc1 complex from Rhodobacter sphaeroides.

The topology of the cytochrome b subunit of the bc1 complex from Rhodobacter sphaeroides has been examined by generating gene fusions with alkaline phosphatase. Gene fusions were generated at random locations within the fbcB gene encoding the cytochrome b subunit. These fusion products were expressed in Escherichia coli and were screened for alkaline phosphatase activity on chromogenic plates. 33 in-frame fusions which showed activity were further characterized. The fusion junctions of all those fusions which had a high specific activity were clustered in three regions of the cytochrome b polypeptide, and thus these regions were tentatively assigned as being near the periplasmic surface. The data are consistent with a model containing eight transmembrane helices. In order to explore the validity of the gene fusion approach for a protein not normally expressed in E. coli, the topology of the L-subunit of the photosynthetic reaction center from R. sphaeroides was also explored using phoA gene fusions. A similar protocol was used as with the cytochrome b subunit. The gene fusions with high specific activity were shown to be in regions of the L-subunit polypeptide known to be at or near the periplasmic surface, as defined by the high resolution structure determined by X-ray crystallography. These data demonstrate the utility of this approach for determining membrane protein topology and extend potential applications to include at least some proteins not normally expressed in E. coli.     

Evidence that modulation requires sequences downstream of the promoters of two vir-repressed genes of Bordetella pertussis.

Gene expression in Bordetella pertussis is altered by environmental signals in a process called antigenic modulation. In the presence of modulating signals, expression of several known virulence factors and outer membrane proteins is coordinately reduced. From a bank of TnphoA fusions, we have identified five genes whose expression profiles are reciprocal of those of the major virulence determinants; that is, alkaline phosphatase activity is maximal during growth in the presence of the modulators nicotinic acid and MgSO4 (S. Knapp and J. J. Mekalanos, J. Bacteriol. 170:5059-5066, 1988). We have called these loci vir-repressed genes (vrg). Two of these gene fusions (vrg-6 and vrg-18) have been cloned in Escherichia coli, returned on low-copy-number plasmids to several strains of B. pertussis, and found to be regulated similarly to the fusions harbored on the chromosome. Deletions of the two vrg promoters were constructed and returned to B. pertussis. Regulation was maintained even when all but 24 nucleotides upstream of the vrg-18 initiation codon and 60 nucleotides upstream of the vrg-6 initiation codon were deleted, suggesting that cis-acting regulatory elements of these genes lie very near or within the coding region. We observed a 21-base palindromic sequence overlapping an 8-base direct repeat within the signal sequence coding region of vrg-6; insertion of a 6-bp linker in this region abolished regulation. These repetitive sequences are also at the site of greatest primary sequence identify between vrg-6 and vrg-18 and correspond to the signal sequence coding region. We propose models that involve recognition of this region by a vir-regulated gene product.