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Cell division and organismal development are exquisitely orchestrated and regulated processes. The dysregulation of the molecular mechanisms underlying these processes may cause cancer, a consequence of cell-intrinsic and/or cell-extrinsic events. Cellular DNA can be damaged by spontaneous hydrolysis, reactive oxygen species, aberrant cellular metabolism or other perturbations that cause DNA damage. Moreover, several environmental factors may damage the DNA, alter cellular metabolism or affect the ability of cells to interact with their microenvironment. While some environmental factors are well established as carcinogens, there remains a large knowledge gap of others owing to the difficulty in identifying them because of the typically long interval between carcinogen exposure and cancer diagnosis. DNA damage increases in cells harbouring mutations that impair their ability to correctly repair the DNA. Tumour predisposition syndromes in which cancers arise at an accelerated rate and in different organs - the equivalent of a sensitized background - provide a unique opportunity to examine how gene-environment interactions influence cancer risk when the initiating genetic defect responsible for malignancy is known. Understanding the molecular processes that are altered by specific germline mutations, environmental exposures and related mechanisms that promote cancer will allow the design of novel and effective preventive and therapeutic strategies.

Successful cancer immunotherapy entails activation of innate immune receptors to promote dendritic cell (DC) maturation, antigen presentation, up-regulation of costimulatory molecules, and cytokine secretion, leading to activation of tumor antigen-specific cytotoxic T lymphocytes (CTLs). Here we screened a synthetic library of 100,000 compounds for innate immune activators using TNF production by THP-1 cells as a readout. We identified and optimized a potent human and mouse Toll-like receptor (TLR)1/TLR2 agonist, Diprovocim, which exhibited an EC50 of 110 pM in human THP-1 cells and 1.3 nM in primary mouse peritoneal macrophages. In mice, Diprovocim-adjuvanted ovalbumin immunization promoted antigen-specific humoral and CTL responses and synergized with anti-PD-L1 treatment to inhibit tumor growth, generating long-term antitumor memory, curing or prolonging survival of mice engrafted with the murine melanoma B16-OVA. Diprovocim induced greater frequencies of tumor-infiltrating leukocytes than alum, of which CD8 T cells were necessary for the antitumor effect of immunization plus anti-PD-L1 treatment. [ABSTRACT FROM AUTHOR]

Forward genetics screens with N-ethyl-N-nitrosourea (ENU) provide a powerful way to illuminate gene function and generate mouse models of human disease; however, the identification of causative mutations remains a limiting step. Current strategies depend on conventional mapping, so the propagation of affected mice requires non-lethal screens; accurate tracking of phenotypes through pedigrees is complex and uncertain; out-crossing can introduce unexpected modifiers; and Sanger sequencing of candidate genes is inefficient. Here we show how these problems can be efficiently overcome using whole-genome sequencing (WGS) to detect the ENU mutations and then identify regions that are identical by descent (IBD) in multiple affected mice. In this strategy, we use a modification of the Lander-Green algorithm to isolate causative recessive and dominant mutations, even at low coverage, on a pure strain background. Analysis of the IBD regions also allows us to calculate the ENU mutation rate (1.54 mutations per Mb) and to model future strategies for genetic screens in mice. The introduction of this approach will accelerate the discovery of causal variants, permit broader and more informative lethal screens to be used, reduce animal costs, and herald a new era for ENU mutagenesis. [ABSTRACT FROM AUTHOR]

Here we describe a previously unknown form of inherited immunodeficiency revealed by an N-ethyl-N-nitrosourea–induced mutation called elektra. Mice homozygous for this mutation showed enhanced susceptibility to bacterial and viral infection and diminished numbers of T cells and inflammatory monocytes that failed to proliferate after infection and died via the intrinsic apoptotic pathway in response to diverse proliferative stimuli. They also had a greater proportion of T cells poised to replicate DNA, and their T cells expressed a subset of activation markers, suggestive of a semi-activated state. We positionally ascribe the elektra phenotype to a mutation in the gene encoding Schlafen-2 (Slfn2). Our findings identify a physiological role for Slfn2 in the defense against pathogens through the regulation of quiescence in T cells and monocytes. [ABSTRACT FROM AUTHOR]

Null alleles of the gene encoding NEMO (NF-κB essential modulator) are lethal in hemizygous mice and men, whereas hypomorphic alleles typically cause a syndrome of immune deficiency and ectodermal dysplasia. Here we describe an allele of Ikbkg in mice that impaired Toll-like receptor signaling, lymph node formation, development of memory and regulatory T cells, and Ig production, but did not cause ectodermal dysplasia. Degradation of lwBu, which is considered a primary requirement for NEMO-mediated immune signaling, occurred normally in response to Toll-like receptor stimulation, yet ERK phosphorylation and NF-κB p65 nuclear translocation were severely impaired. This selective loss of function highlights the immunological importance of NEMO-regulated pathways beyond IκBα degradation, and offers a biochemical explanation for rare immune deficiencies in man. [ABSTRACT FROM AUTHOR]

We have identified a previously unannotated catechol-O-methyl-tranferase (COMT), here designated COMT2, through positional cloning of a chemically induced mutation responsible for a neurobehavioral phenotype. Mice homozygous for a missense mutation in Comt2 show vestibular impairment, profound sensorineuronal deafness, and progressive degeneration of the organ of Corti. Consistent with this phenotype, COMT2 is highly expressed in sensory hair cells of the inner ear. COMT2 enzymatic activity is significantly reduced by the missense mutation, suggesting that a defect in catecholamine catabolism underlies the auditory and vestibular phenotypes. Based on the studies in mice, we have screened DNA from human families and identified a nonsense mutation in the human ortholog of the murine Comt2 gene that causes nonsyndromic deafness. Defects in catecholamine modification by COMT have been previously implicated in the development of schizophrenia. Our studies identify a previously undescribed COMT gene and indicate an unexpected role for catecholamines in the function of auditory and vestibular sense organs. [ABSTRACT FROM AUTHOR]

Abstract: Forward genetic approaches have contributed to our understanding of how the host senses infectious agents such as bacteria, viruses and fungi. Beginning with the initial discovery of Toll-like receptors (TLRs) as primary sensors involved in the recognition of microbial components, our laboratory has taken a forward genetic approach, using N-ethyl-N-nitrosourea (ENU) mutagenesis in mice, to decipher TLR-signaling pathways. This long term effort has helped to elucidate the circuitry of these pathways, identified new molecules, and disclosed new functions for known molecules. Here we review some of the more important insights developed from this approach and discuss its prospects. [Copyright &y& Elsevier]

As machines that reprogramme eukaryotic cells to suit their own purposes, viruses present a difficult problem for multicellular hosts, and indeed, have become one of the central pre-occupations of the immune system. Unable to permanently outpace individual viruses in an evolutionary footrace, higher eukaryotes have evolved broadly active mechanisms with which to sense viruses and suppress their proliferation. These mechanisms have recently been elucidated by a combination of forward and reverse genetic methods. Some of these mechanisms are clearly ancient, whereas others are relatively new. All are remarkably adept at discriminating self from non-self, and allow the host to cope with what might seem an impossible predicament. [ABSTRACT FROM AUTHOR]

The inflammatory response to microbes--and host perception of microbes in general--is largely initiated by a single class of receptors, named for their similarity to the prototypic Toll receptor of Drosophila. The mammalian Toll-like receptors (TLRs) are ultimately responsible for most phenomena associated with infection. This includes both "good" effects of infection (e g., the induction of lasting specific immunity to an infections agent) and "bad" effects of infection (systemic inflammation and shock). Although they are essential for host defense, no other endogenous proteins can match their lethal potential, The TLR complexes transduce the toxicity of lipopolysaccharide (LPS), cysteinyl lipopeptides, and many other molecules of microbial origin. The identification of the TLRs as the key conduit to host awareness of microbial infection was a victory for reductionism, proving that the complexity of infectious inflammation as a phenomenon belies the simplicity if its origins. It was achieved by a classical genetic approach, proceeding from phenotype to gene. Further analysis of the signaling pathways activated by the TLRs has depended on both classical and reverse genetic methods. Additional work will ultimately disclose the extent to which sterile inflammatory diseases are mediated by aberrations in these pathways. [ABSTRACT FROM AUTHOR]

The immunovariant N-ethyl-N-nitrosourea-induced mutations Pococurante (Poc) and Lackadaisical were found to alter MyD88, creating striking receptor-selective effects. Poc, in particular, pre- vented sensing of all MyD88-dependent Toll-like receptor (TLR) ligands except diacyl lipopeptides. Furthermore, Poc-site and classical BB loop mutations caused equivalent phenotypes when en- grafted into any TLR/IL-1 receptor/resistance (TIR) domain. These observations, complemented by data from docking studies and site-directed mutagenesis, revealed that BB loops and Poc sites interact homotypically across the receptor:adapter signaling interface, whereas the C-terminal αE-helices support adapter:adapter and receptor:receptor oligomerization. We have thus defined the TIR domain surface that mediates association between TLR5 and MyD88 and the surface required for MyD88 or TLR oligomerization. Moreover, MyD88 engages individual TLRs differently, suggesting the feasibility of selective pharmacologic TIR domain receptor blockade. [ABSTRACT FROM AUTHOR]

The mouse cytomegalovirus (MCMV) resistome is the set of host genes with nonredundant functions in resistance to MCMV infection. By screening 3500 G3 germline mutant mice (∼1750 gamete equivalents), we have identified eight transmissible mutations that create MCMV susceptibility in C57BL/6 mice. Among these, a mutation called Domino was noted to cause macrophage susceptibility to vesicular stomatitis virus (VSV) in vitro. This accessory phenotype was not corrected by type I interferon (IFN), which suggested a defect of the type I IFN pathway. Domino corresponds to a point mutation that alters the DNA binding domain of STAT1, leading to a defect of STAT1 activation. Identification of the Domino mutation demonstrates that an in vivo MCMV susceptibility screen is feasible and illustrates how it can provide insight into the resistome. Moreover, some mutations are far more deleterious than Domino in MCMV-infected mice, consistent with the interpretation that certain protein(s) unrelated to IFN production or signaling are more important than IFNs with regard to their net antiviral effects. [ABSTRACT FROM AUTHOR]

Many genes, and conceivably most genes, are constitutively expressed yet have conditional functions. Their products are utilized only under special circumstances, and enforce homeostatic regulation. Mutations do not disclose the function of such genes unless the proper conditions are applied. The genes that encode the Toll-like receptors (TLRs) fall into this category. The TLRs represent the principal sensors of infection in mammals. Absent infection, mammals have little need for the TLRs; they are essential only when microbes gain access to the interior milieu of the host. The function of the TLRs in mammals was first disclosed by a spontaneous mutation in a locus called Lps, when it was shown by positional cloning to be identical to Tlr4. Random germline mutagenesis has since permitted an estimate of the total number of proteins required for TLR signaling to the level of tumor necrosis factor (TNF) synthesis and activity, and has also shown that these sensors are extremely broad in their ability to detect microbes. Ultimately, the TLRs are responsible for most infection-related phenomena, both good and bad. These include the development of fever, shock, and tissue injury, but also the activation of innate and adaptive effector mechanisms that lead to the elimination of microbes. [ABSTRACT FROM AUTHOR]

Resistance to infection is largely inherited rather than acquired, and is encoded by a definable set of host genes designated the ‘resistome’. Logically speaking, piecemeal disruption of the resistome gives us the best chance to define it, and the most spectacular advances in understanding innate immunity have grown from spontaneous or induced germline mutations of the resistome. Mutations induced by random germline mutagenesis have now become so numerous that we are nearly in a position to define the size of the resistome, and both random and targeted mutations give us a fairly nice sketch of its components and how they interact. Our own N-ethyl-N-nitrosourea mutagenesis effort, which recently showed that components of Toll-like receptor signaling are essential constituents of the arsenal against MCMV infections, validated the forward genetic approach as a powerful tool to define the resistome. [Copyright &y& Elsevier]

Though sometimes portrayed as “new,” the science of innate immunity made its start more than 100 years ago. Recent progress has reflected the application of new methods to old problems. In particular, genetic dissection of innate immune pathways has been pursued with great success in model organisms. This has opened the way to an understanding of innate immune sensing. The effector arm of innate immunity has also been tackled, largely though the use of biochemical methods. [Copyright &y& Elsevier]

Both forward and reverse genetic techniques have been used to define components of the mammalian lipopolysaccharide (LPS) receptor. TLR4, identified by a forward genetic approach as the product of the classical Lps locus, is the only known transmembrane component of the mammalian LPS receptor. Gene knockout work has also established that LPS signal transduction requires the integrity of CD14, MD-2, and, in part, MyD88, IRAK4, and TRAF-6. However, there is no reason to believe that these are the only proteins that make up the receptor/transducer apparatus. To examine the possibility that other proteins may be involved, we initiated a mutagenesis program, in which germline mutations are induced in mice using N-ethyl-N-nitrosourea (ENU), and macrophages from individual animals are screened for their competence to respond to LPS. We now report the existence of a new locus, Lps2, which is required for TNF production in response to LPS. The Lps2 mutation that we have identified is co-dominant, is similar in phenotypic effect to Lps[sup d] , and does not represent a novel allele of any of the genes that are known to encode the 'core' LPS signaling apparatus. The Lps2 mutation does not preclude signaling initiated by peptidoglycan or unmethylated DNA. Hence, genetic data suggest that there is at least one 'missing' component of the LPS receptor complex that has yet to be found. [ABSTRACT FROM AUTHOR]

Genetic tools (especially classical tools) have enlightened our understanding of biology as no other approach could. Mendel, Morgan, Bridges, and their heirs began with phenotypic traits and ended with genes. At first, the chemical identity of genes was not known, and even after years of methodologic refinement, more years of effort were needed to find the gene and the mutational difference that caused a particular phenotype. Chemistry has now outraced phenotypic analysis; all the genes are suddenly known, but most of their functions are not. The greatest challenge confronting all fields in biology is to establish correspondence between genes and discrete biologic functions. Sepsis is a devastating problem that has eluded a solution, despite the introduction of highly effective antimicrobial agents. Sepsis is an orchestrated process, understood in broad outline, but not in all details. Which genes are involved in the pathogenesis of sepsis? As in the golden age of genetics, the answer requires the solution of phenotypic puzzles, which, in turn, requires the creation of phenotypes. [ABSTRACT FROM AUTHOR]

The sequence of the human genome is providing researchers with the scaffold upon which genes are built. The definition of the boundaries of the genes themselves and of their complex architecture requires a mapping of the transcriptome to the genome. A methodology was developed for generating a detailed transcriptome map and for reconstituting transcripts by using the genome as a template. As a demonstration of the potential of this method, the structure of the human Toll-like receptor (TLR) genes was reevaluated. For all TLR genes for which a genomic sequence was available (i.e., all except TLR10), novel features of the gene structure were discovered. These features include multiple alternative polyadenylation sites, additional exons or splice variants, and overlaps with other genes. These findings have implications for the analysis of TLR gene expression and for the diversity of the proteins encoded by these genes. [ABSTRACT FROM AUTHOR]

The immune system provides protection from a wide range of pathogens. One component of immunity, the phylogenetically ancient innate immune response, fights infections from the moment of first contact and is the fundamental defensive weapon of multicellular organisms. The Toll family of receptors has a crucial role in immune defence. Studies in fruitflies and in mammals reveal that the defensive strategies of invertebrates and vertebrates are highly conserved at the molecular level, which raises the exciting prospects of an increased understanding of innate immunity. [ABSTRACT FROM AUTHOR]

In the course of a large-scale program of ENU mutagenesis, we isolated a dominant mutation, called Velvet. The mutation was found to be uniformly lethal to homozygotes, which do not survive E13.5. Mice heterozygous for the Velvet mutation are born with eyelids open and demonstrate a wavy coat and curly vibrissae. The mutation was mapped to the proximal end of chromosome 11 by genome-wide linkage analysis. On 249 meioses, the locus was confined to a 2.7-Mb region, which included the epidermal growth factor receptor gene (Egfr). An A → G transition in the Egfr coding region of Velvet mice was identified, causing the amino acid substitution D833G. This substitution alters an essential triad of amino acids (DFG → GFG) that is normally required for coordination of the ATP substrate. As such, kinase activity is at least mostly abolished, but quaternary structure of the receptor is presumably maintained, accounting for the dominant effect. Velvet is the first known dominant representative of the Egfr allelic series that is fully viable, a fact that makes it particularly useful for developmental studies. [ABSTRACT FROM AUTHOR]

Introduction: Human studies show congenital heart disease (CHD) has a complex non-Mendelian genetic etiology. We previously conducted a recessive N-ethyl-N-nitrosourea (ENU) mouse mutagenesis screen using fetal ultrasound and recovered over 150 recessive mutations causing CHD. In the present study, we conducted a sensitized screen with driver mutations to recover dominant mutations causing CHD. We hypothesize this dominant screen can help to elucidate the complex genetic architecture of CHD. Methods: G1 males with heterozygous ENU induced mutations were mated to females harboring heterozygous "driver" mutation in Kif7, Anks6, or Sap130 that can cause CHD when homozygous. The resulting G2 offspring were screened for CHD by fetal ultrasound. Mutations in G1 males were identified by whole exome sequencing and the G2 offspring were genotyped using Ion Torrent sequencing. Linkage analysis was conducted to identify the CHD causing mutation. Results: We found 9.3% of the G1 pedigrees (n=551) yielded CHD mutants, similar to the 8.2% observed in the previous recessive screen. However, only 2.5 mutants were recovered per line vs. 4.5 in the recessive screen despite having screened similar number of embryos, supporting non-Mendelian genetics in the dominant screen. Genotype-phenotype analysis revealed only 32%, 45%, and 55% of the CHD mutants recovered carried the Kif7, Anks6, or Sap130 driver mutations, respectively, suggesting the driver mutations are not required for CHD pathogenesis. For mouse line 4142 with DORV/OA, we identified a heterozygous p21/Rac-activated kinase 2 (Pak2) mutation as disease causing (p=4.8e-12). CHD penetrance was 83% among Pak2 +/m embryos. Among surviving 4142 G2 offspring, only 14% have the Pak2 mutation vs. 50% expected. We observed 75% of the surviving Pak2 mutant mice also carry a mutation in Zbtb2, a gene identified to regulate p21, suggesting this may act as a protective modifier. Conclusion: We showed the feasibility of using mouse mutagenesis to recover dominant mutations causing CHD, and showed for the first time a role for Pak2 in CHD. These findings demonstrate that the genetic diversity created by ENU mutagenesis can provide the genomic context to investigate the complex genetics and genetic architecture of CHD. [ABSTRACT FROM AUTHOR]