Longitudinal analysis of 25 sequential sample-pairs using a custom multiple myeloma mutation sequencing panel (M(3)P).

Recent advances in genomic sequencing technologies now allow results from deep next-generation sequencing to be obtained within clinically meaningful timeframes, making this an attractive approach to better guide personalized treatment strategies. No multiple myeloma-specific gene panel has been established so far; we therefore designed a 47-gene-targeting gene panel, containing 39 genes known to be mutated in ≥3 % of multiple myeloma cases and eight genes in pathways therapeutically targeted in multiple myeloma (MM). We performed targeted sequencing on tumor/germline DNA of 25 MM patients in which we also had a sequential sample post treatment. Mutation analysis revealed KRAS as the most commonly mutated gene (36 % in each time point), followed by NRAS (20 and 16 %), TP53 (16 and 16 %), DIS3 (16 and 16 %), FAM46C (12 and 16 %), and SP140 (12 and 12 %). We successfully tracked clonal evolution and identified mutation acquisition and/or loss in FAM46C, FAT1, KRAS, NRAS, SPEN, PRDM1, NEB, and TP53 as well as two mutations in XBP1, a gene associated with bortezomib resistance. Thus, we present the first longitudinal analysis of a MM-specific targeted sequencing gene panel that can be used for individual tumor characterization and for tracking clonal evolution over time.

Lessons from next-generation sequencing analysis in hematological malignancies.

Next-generation sequencing has led to a revolution in the study of hematological malignancies with a substantial number of publications and discoveries in the last few years. Significant discoveries associated with disease diagnosis, risk stratification, clonal evolution and therapeutic intervention have been generated by this powerful technology. As part of the post-genomic era, sequencing analysis will likely become part of routine clinical testing and the challenge will ultimately be successfully transitioning from gene discovery to preventive and therapeutic intervention as part of individualized medicine strategies. In this report, we review recent advances in the understanding of hematological malignancies derived through genome-wide sequence analysis.

Genome-wide studies in multiple myeloma identify XPO1/CRM1 as a critical target validated using the selective nuclear export inhibitor KPT-276.

RNA interference screening identified XPO1 (exportin 1) among the 55 most vulnerable targets in multiple myeloma (MM). XPO1 encodes CRM1, a nuclear export protein. XPO1 expression increases with MM disease progression. Patients with MM have a higher expression of XPO1 compared with normal plasma cells (P<0.04) and to patients with monoclonal gammopathy of undetermined significance/smoldering MM (P<0.0001). The highest XPO1 level was found in human MM cell lines (HMCLs). A selective inhibitor of nuclear export compound KPT-276 specifically and irreversibly inhibits the nuclear export function of XPO1. The viability of 12 HMCLs treated with KTP-276 was significantly reduced. KPT-276 also actively induced apoptosis in primary MM patient samples. In gene expression analyses, two genes of probable relevance were dysregulated by KPT-276: cell division cycle 25 homolog A (CDC25A) and bromodomain-containing protein 4 (BRD4), both of which are associated with c-MYC pathway. Western blotting and reverse transcription-PCR confirm that c-MYC, CDC25A and BRD4 are all downregulated after treatment with KPT-276. KPT-276 reduced monoclonal spikes in the Vk*MYC transgenic MM mouse model, and inhibited tumor growth in a xenograft MM mouse model. A phase I clinical trial of an analog of KPT-276 is ongoing in hematological malignancies including MM.

Association between polymorphic variation in VDR and RXRA and circulating levels of vitamin D metabolites.

The vitamin D metabolite 1,25(OH)2D is the bioactive ligand of the vitamin D receptor (VDR). VDR forms a heterodimer with the retinoid X receptors (RXRs) that when bound to ligand influences the transcriptional control of genes that regulate circulating levels of vitamin D metabolites. Whether genetic variation in VDR or RXRA affects circulating levels of 1,25(OH)2D or 25(OH)D has not been established. We used a single nucleotide polymorphism (SNP) tagging approach to evaluate the association between SNPs in VDR and RXRA and serum levels of 1,25(OH)2D and 25(OH)D. A total of 42 tagSNPs in VDR and 32 in RXRA were analyzed in a sample of 415 participants. Principal components analyses revealed a gene-level association between RXRA and serum 1,25(OH)2D concentrations (P=0.01), but not 25(OH)D. No gene-level association was found for VDR with either serum biomarker. At the single-SNP level, a significant positive trend was observed for increasing 1,25(OH)2D levels with each additional copy of the A allele for RXRA SNP rs9409929 (P-trend=0.003). After a multiple comparisons adjustment, no individual SNP in VDR or RXRA was significantly associated with either outcome. These results demonstrate an association between genetic variation in RXRA and 1,25(OH)2D serum concentrations.

Octamerization of lambda CI repressor is needed for effective repression of P(RM) and efficient switching from lysogeny.

The CI repressor of bacteriophage lambda is a model for the role of cooperativity in the efficient functioning of genetic switches. Pairs of CI dimers interact to cooperatively occupy adjacent operator sites at O(R) and at O(L). These CI tetramers repress the lytic promoters and activate transcription of the cI gene from P(RM). CI is also able to octamerize, forming a large DNA loop between O(R) and O(L), but the physiological role of this is unclear. Another puzzle is that, although a dimer of CI is able to repress P(RM) by binding to the third operator at O(R), O(R)3, this binding seems too weak to affect CI production in the lysogenic state. Here we show that repression of P(RM) at lysogenic CI concentrations is absolutely dependent on O(L), in this case 3.8 kb away. A mutant defective in this CI negative autoregulation forms a lysogen with elevated CI levels that cannot efficiently switch from lysogeny to lytic development. Our results invalidate previous evidence that Cro binding to O(R)3 is important in prophage induction. We propose the octameric CI:O(R)-O(L) complex increases the affinity of CI for O(R)3 by allowing a CI tetramer to link O(R)3 and the third operator at O(L), O(L)3.

Establishing lysogenic transcription in the temperate coliphage 186.

A single-copy chromosomal reporter system was used to measure the intrinsic strengths and interactions between the three promoters involved in the establishment of lysogeny by coliphage 186. The maintenance lysogenic promoter p(L) for the immunity repressor gene cI is intrinsically approximately 20-fold weaker than the lytic promoter p(R). These promoters are arranged face-to-face, and transcription from p(L) is further weakened some 14-fold by the activity of p(R). Efficient establishment of lysogeny requires the p(E) promoter, which lies upstream of p(L) and is activated by the phage CII protein to a level comparable to that of p(R). Transcription of p(E) is less sensitive to converging p(R) transcription and raises cI transcription at least 55-fold. The p(E) promoter does not occlude p(L) but inhibits lytic transcription by 50%. This interference is not due to bound CII preventing elongation of the lytic transcript. The p(E) RNA is antisense to the anti-immune repressor gene apl, but any role of this in the establishment of lysogeny appears to be minimal.

Establishment of lysogeny in bacteriophage 186. DNA binding and transcriptional activation by the CII protein.

The CII protein of bacteriophage 186 is a transcriptional activator of the helix-turn helix family required for establishment of the lysogenic state. DNA binding by 186 CII is unusual in that the invertedly repeated half sites are separated by 20 base pairs, or two turns of the DNA helix, rather than the one turn usually associated with this class of proteins. Here, we investigate quantitatively the DNA binding properties of CII and its interaction with RNA polymerase at the establishment promoter, p(E). The stoichiometry of CII binding was determined by sedimentation equilibrium experiments using a fluorescein-labeled oligonucleotide and purified CII. These experiments indicate that the CII species bound to DNA is a dimer, with additional weak binding of a tetrameric species at high concentrations. Examination of the thermodynamic linkages between CII self-association and DNA binding shows that CII binds to the DNA as a preformed dimer (binding free energy, 9.9 kcal/mol at 4 degrees C) rather than by association of monomers on the DNA. CII binding induces in the DNA a bend of 41 (+/- 5) degrees. The spacing between the binding half sites was shown to be important for CII binding, insertion or removal of just 1 base pair significantly reducing the affinity for CII. Removal of 5 or 10 base pairs between binding half sites eliminated binding, as did insertion of an additional 10 base pairs. CII binding at p(E) was improved marginally by the presence of RNA polymerase (DeltaDeltaG = -0.5 (+/- 0.3) kcal/mol). In contrast, the binding of RNA polymerase at p(E) was undetectable in the absence of CII but was improved markedly by the presence of CII. Thus, CII appears to recruit RNA polymerase to the promoter. The nature of the base pair changes in mutant phage, selected by their inability to establish lysogeny, are consistent with this mechanism of CII action.

The use of oriC-dependent phage infection to characterize the ultra violet (UV)-induced inhibition of initiation of DNA replication in Escherichia coli.

The oriC transducing phage lambda poriCc is a pseudovirulent phage capable of forming plaques on a lambda lysogen. This phenotype is dependent upon the presence of the oriC insert. The ability of lambda poriCc to form plaques on a lambda lysogen represents a potential phage assay system for studying aspects of oriC function. In the present study we establish that lambda poriCc infection of a lambda lysogen is a legitimate assay for oriC function. We use this assay to confirm the previously reported observation that initiation of DNA replication from oriC is transiently inhibited in a ultra violet (UV) irradiated cell at doses greater than 60 J/m2. We further demonstrate using this assay that the UV induced inhibition of initiation of DNA replication from oriC is not a SOS function nor a heat shock function. In the course of these studies, we found that lambda poriCc infection of a non-lysogenic cell is extremely sensitive to pre-irradiation of the Escherichia coli host. We postulate that the sensitivity of lambda poriCc replication to host cell pre-irradiation reflects in some way the transient inhibition of initiation of DNA replication from oriC following UV irradiation.

The late-expressed region of the temperate coliphage 186 genome.

The late-lytic region of the genome of bacteriophage 186 encodes the phage proteins that synthesize the complex viral particle and lyse the bacterial host. We report the completion of the DNA sequence of the late region and the assignment of 18 previously identified genes to open reading frames in the sequence. The 186 late region is similar to the late region of phage P2, sharing 26 genes of known function: the single gene for activation of late gene transcription, 6 genes for construction of DNA-containing heads, 16 for tail morphogenesis, and 3 for cell lysis. We identified two 186 late genes with unknown function; one is homologous to previously unrecognised genes in P2, HP1, and phiCTX, and the other may modulate DNA packaging. The 186 late region, like the rest of the genome, lacks the lysogenic conversion genes that are carried by P2, allowing the 186 late region to be transcribed from only three late promoters rather than four. The relative absence of lysogenic conversion genes in 186 suggests that the two phages have evolved to use the lytic and lysogenic reproductive modes to different extents.

The Tum protein of coliphage 186 is an antirepressor.

The tum gene of coliphage 186, encoded on a LexA controlled operon, is essential for UV induction of a 186 prophage. Primer extension analysis is used to confirm that Tum is the sole phage function required for prophage induction and that it acts against the maintenance repressor, CI, to relieve repression of the lytic promoters, pR and pB, and thereby bring about lytic development. In vitro experiments with purified proteins demonstrate that Tum prevents CI binding to its operator sites. Tum does not compete with CI for binding sites on DNA, and unlike RecA mediated induction of lambda prophage, the action of Tum on CI is reversible. Mechanisms by which Tum may act against CI are discussed.

Metal- and DNA-binding properties and mutational analysis of the transcription activating factor, B, of coliphage 186: a prokaryotic C4 zinc-finger protein.

Coliphage 186 B is a 72-amino acid protein belonging to the Ogr family of analogous transcription factors present in P2-like phage, which contain a Cys-X2-Cys-X22-Cys-X4-Cys presumptive zinc-finger motif. The molecular characterization of these proteins has been hampered by their insolubility, a difficulty overcome in the present study by obtaining B as a soluble cadmium-containing derivative (CdB). Atomic absorption spectroscopy showed the presence of one atom of cadmium per molecule of purified CdB. The UV absorption spectrum revealed a shoulder at 250 nm, characteristic of CysS-Cd(II) ligand-to-metal charge-transfer transitions, and the difference absorption coefficient after acidification (delta epsilon 248, 24 mM-1 cm-1) indicated the presence of a Cd(Cys-S)4 center. Gel mobility shift analysis of CdB with a 186 late promoter demonstrated specific DNA-binding (KD, app 3-4 microM) and the protein was shown to activate transcription in vitro from a promoter-reporter plasmid construct. The B DNA-binding site was mapped by gel shift and DNAase I cleavage protection experiments to an area between-70 and -43 relative to the transcription start site, coincident with the consensus sequence, GTTGT-N8-TNANCCA, from -66 to -47 of the 186 and P2 late promoters. Inactive B point mutants were obtained in the putative DNA-binding loop of the N-terminal zinc-finger motif and in a central region thought to interact with the Escherichia coli RNA polymerase alpha-subunit. A truncated B mutant comprising the first 53 amino acids (B1-53) exhibited close to wild-type activity, showed a DNA-binding affinity similar to that of the full-length protein, and could be reconstituted with either Cd or Zn. Gel permeation analysis revealed that B1-53 was a majority dimeric species whereas wild-type B showed larger oligomers. 186 B therefore exhibits a potentially linear organization of functional regions comprising an N-terminal C4 zinc-finger DNA-binding region, a dispensable C-terminal region involved in protein self-association, and a central region that interacts with RNA polymerase.

The dual role of Apl in prophage induction of coliphage 186.

In the present study we show that the Apl protein of the temperate coliphage 186 combines, in one protein, the activities of the coliphage lambda proteins Cro and Xis. We have shown previously that Apl represses both the lysogenic promoter, pL, and the major lytic promoter, pR, and is required for excision of the prophage. Apl binds at two locations on the phage chromosome, i.e. between pR and pL and at the phage-attachment site. Using an in vivo recombination assay, we now show that the role of Apl in excision is in the process itself and is not simply a consequence of repression of pR or pL. To study the repressive role of Apl at the switch promoters we isolated Apl-resistant operator mutants and used them to demonstrate a requirement for Apl in the efficient derepression of the lysogenic promoter during prophage induction. We conclude that Apl is both an excisionase and transcriptional repressor.

The CII protein of bacteriophage 186 establishes lysogeny by activating a promoter upstream of the lysogenic promoter.

We have shown previously that the cII gene product of the non-lambdoid temperate bacteriophage 186 is required for the establishment of lysogeny. We show here that CII, a potential helix-turn-helix DNA-binding protein, establishes lysogeny by activating a promoter (PE) which spans the apl/cII intergenic region, upstream of the lysogenic promoter, PL. The start site of the PE transcript (+1) has been mapped by primer extension and we have identified the CII binding determinants at PE by DNase I footprinting. CII binds to inverted repeat sequences separated by two turns of the helix, with binding half-sites centred at the 38 and -58 positions of PE. Oligomerisation studies with purified CII protein indicate that a CII tetramer may be the species that binds to this site. We also show that PE is subject to direct negative feedback by the CI repressor.

Purification and self-association equilibria of the lysis-lysogeny switch proteins of coliphage 186.

The CI repressor protein, responsible for maintenance of the lysogenic state, and the Apl protein, required for efficient prophage induction, are the two control proteins of the lysis-lysogeny transcriptional switch of coliphage 186. These proteins have been overexpressed, purified, and their self-association behavior examined by sedimentation equilibrium. Phage 186 CI dimers self-associate in solution through tetramers to octamers in a concerted process. The Apl protein of 186 is an unusual example of a helix-turn-helix protein which is monomeric in solution.

DNA binding by the coliphage 186 repressor protein CI.

The cI gene of coliphage 186 maintains lysogeny and confers immunity to 186 infection by repressing the major early promoter, p(R), and the promoter for the late transcription activator gene, p(B). Gel mobility shirt and DNase I footprinting show that CI protein binds to the DNA at p(R) and p(B) and also to sites approximately 300 base pairs upstream and downstream of p(R), called FL and FR. Mutations which cause virulence reduce CI binding to p(R). The biochemical and genetic data identify three CI operators at p(R), two at p(B), and single operators at FL and FR. The operators at the p(B), FL, FR, and central p(R) sites are inverted repeat sequences, separated by 5 base pairs (Type A) or, in the case of p(R), by 4 base pairs (Type A'). A different inverted repeat operator sequence (Type B) is proposed for the binding sites on each side of the central site at p(R). Thus, CI appears to recognize two distinct DNA sequences. CI binds cooperatively to adjacent operators, and binding at p(R) is strongly dependent on these cooperative interactions. A high order CI multimer appears to be the active DNA binding species, even at single operators.

The Escherichia coli retrons Ec67 and Ec86 replace DNA between the cos site and a transcription terminator of a 186-related prophage.

Retrons are unusual, reverse transcriptase-encoding elements found in bacteria. Although there are a number of indications that retrons are mobile elements, their transposition has not been observed. The Escherichia coli retrons Ec67 and Ec86 are different retrons inserted at the same site and we have further characterized this site in search of clues to the mechanism of retron transposition. We confirm, by extending previous sequence analysis, that Ec67 and Ec86 are inserted into prophages related to coliphage 186. Comparison with the recently published sequence of the 186 96-2% region indicates that the retrons have replaced approximately 180 bp of DNA between the phage cohesive end site (cos) and the transcription terminator of a phage DNA-packaging gene. These features--DNA replacement at the insertion site and the location of retron junctions near transcription terminators or DNA cleavage sites--are shared with other retrons and suggest ways in which retron transposition might have occurred.

Defining the SOS operon of coliphage 186.

We have sequenced the LexA-controlled operon of coliphage 186 that carries the tum gene, whose product is necessary for UV induction of the 186 prophage. The operon consists of orf95 and orf97, and we have identified orf95 as the tum gene. The major translation products from orf95 result from internal initiations and modulate Tum activity. Tum is the product of the full-length Orf95 protein. The second gene of the operon, orf97, is of unknown function but, while it has little effect on prophage induction, its presence in the cell totally blocks infection of that cell by 186.

DNA sequence of tail fiber genes of coliphage 186 and evidence for a common ancestor shared by dsDNA phage fiber genes.

We present here the nucleotide sequence of the tail fiber genes of phage 186. Marker rescue was used to associate an open reading frame (ORF) of 462 codons with the previously known tail gene K. A downstream ORF, encoding a 166-amino-acid product, was designated orf 45. Comparative studies suggested that K encodes the tail fiber protein and that orf 45 encodes an assembly protein. K protein contains a succession of short amino acid sequences (motifs) that are homologous with sequences from the tail fiber proteins of unrelated bacteriophages. The fact that these sequence motifs are variously present in the tail fiber proteins of unrelated bacteriophages has been advanced as evidence for horizontal transfer in the evolution of the associated tail fiber genes. However, the fact that the order of the various motifs in the proteins is invariant emphasizes the probability that independent divergence from a common ancestor also played a major role in the evolution of the tail fiber genes.

Tail sheath and tail tube genes of the temperate coliphage 186.

The nucleotide sequence of the phage 186 genome from 46.3-52.5% was determined and was found to contain two open reading frames highly similar to the tail sheath gene FI and tail tube gene FII of phage P2. The reading frames were identified as genes J and I by marker rescue experiments. A late promoter pJ was identified by galK reporter and primer extension studies. Northern analysis suggested that the pJ transcript predominantly terminated immediately after gene I, although some transcripts could extend to the terminator tB at 67.3%.

DNA replication studies with coliphage 186: the involvement of the Escherichia coli DnaA protein in 186 replication is indirect.

The inability of coliphage 186 to infect productively a dnaA(Ts) mutant at a restrictive temperature was confirmed. However, the requirement by 186 for DnaA is indirect, since 186 can successfully infect suppressed dnaA (null) strains. The block to 186 infection of a dnaA(Ts) strain at a restrictive temperature is at the level of replication but incompletely so, since some 20% of the phage specific replication seen with infection of a dnaA+ host does occur. A mutant screen, to isolate host mutants blocked in 186-specific replication but not in the replication of the close relative coliphage P2, which has no DnaA requirement, yielded a mutant whose locus we mapped to the rep gene. A 186 mutant able to infect this rep mutant was isolated, and the mutation was located in the phage replication initiation endonuclease gene A, suggesting direct interaction between the Rep helicase and phage endonuclease during replication. DNA sequencing indicated a glutamic acid-to-valine change at residue 155 of the 694-residue product of gene A. In the discussion, we speculate that the indirect need of DnaA function is at the level of lagging-strand synthesis in the rolling circle replication of 186.