Trastuzumab deruxtecan in patients with HER2-positive advanced colorectal cancer (DESTINY-CRC02): primary results from a multicentre, randomised, phase 2 trial.

BACKGROUND: Trastuzumab deruxtecan has shown encouraging activity in patients with treatment-refractory HER2-positive, RAS wild-type and BRAF wild-type metastatic colorectal cancer. Dose optimisation and further antitumour assessments in patients with RAS mutations and those with previous anti-HER2 therapy are warranted. We aimed to evaluate two doses of trastuzumab deruxtecan (5·4 mg/kg and 6·4 mg/kg) to establish the recommended dose in patients with pretreated HER2-positive, RAS wild-type or mutant metastatic colorectal cancer. METHODS: DESTINY-CRC02 was a multicentre, randomised, two-stage, two-arm, phase 2 study done in 53 research hospitals and medical centres in Australia, Belgium, France, Italy, Japan, South Korea, Spain, Taiwan, the UK, and the USA. Eligible patients were aged 18 years and older or 20 years and older (depending on region) with pretreated pathologically documented, unresectable, recurrent, or metastatic HER2-positive, and RAS wild-type or mutant colorectal cancer. Patients were required to have an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1 and have received previous chemotherapy, and anti-EGFR, anti-VEGF, or anti-PD-L1 therapy, if clinically indicated. In stage 1, patients were randomly assigned (1:1), via a secure interactive response technology system, to receive 5·4 mg/kg or 6·4 mg/kg trastuzumab deruxtecan administered intravenously every 21 days. Stratification factors were ECOG performance status, HER2 status, and RAS status. In stage 2, patients were assigned into the 5·4 mg/kg treatment group only. The primary endpoint was confirmed objective response rate by blinded independent central review, assessed in all patients for whom treatment was assigned (full analysis set). Safety was assessed in all patients who received at least one dose of study drug. This trial is registered with ClinicalTrials.gov, NCT04744831, and is ongoing (not recruiting). FINDINGS: Between March 5, 2021, and March 29, 2022, 135 patients were centrally screened, 122 of whom were enrolled. In stage 1, 40 patients each were randomly assigned to receive trastuzumab deruxtecan 5·4 mg/kg and 6·4 mg/kg. In stage 2, an additional 42 patients were enrolled in the 5·4 mg/kg group. 64 (52%) participants were male and 58 (48%) were female. The median duration of follow-up was 8·9 months (IQR 6·7-10·5) in the 5·4 mg/kg group and 10·3 months (5·9-12·7) in the 6·4 mg/kg group. The confirmed objective response rate by blinded independent central review was 37·8% (31/82 [95% CI 27·3-49·2]) in the 5·4 mg/kg group and 27·5% (11/40 [14·6-43·9]) in the 6·4 mg/kg group. 34 (41%) of 83 patients in the 5·4 mg/kg group and 19 (49%) of 39 in the 6·4 mg/kg group had grade 3 or worse drug-related treatment-emergent adverse events. The most common grade 3 or worse drug-related treatment-emergent adverse events were neutrophil count decreased (13 [16%] of 83 patients), anaemia (six [7%]), nausea (six [7%]), and white blood cell count decreased (five [6%]) in the 5·4 mg/kg group; and were neutrophil count decreased (10 [26%] of 39 patients), anaemia (eight [21%]), platelet count decreased (four [10%]), and white blood cell count decreased (four [10%]) in the 6·4 mg/kg group. Drug-related serious adverse events occurred in 11 (13%) of 83 patients in the 5·4 mg/kg group and six (15%) of 39 patients in the 6·4 mg/kg group; the most common in the 5·4 mg/kg group was nausea (three [4%] patients) and the most common in the 6·4 mg/kg group were fatigue (two [5%] patients), neutropenia (two [5%]), and thrombocytopenia (two [5%]). A drug-related treatment-emergent adverse event related to death occurred in one (1%) patient in the 5·4 mg/kg group (due to hepatic failure). Adjudicated drug-related interstitial lung disease or pneumonitis events were observed in seven (8%) patients in the 5·4 mg/kg group (all grade 1 or 2) and in five (13%) patients in the 6·4 mg/kg group (four grade 1 or 2; one grade 5). INTERPRETATION: The promising antitumour activity and favourable safety profile support trastuzumab deruxtecan 5·4 mg/kg as the optimal single-agent dose for patients with pretreated HER2-positive metastatic colorectal cancer, including those with RAS mutations, previous anti-HER2 therapy, or both. FUNDING: Daiichi Sankyo and AstraZeneca.

Translating Provider and Staff Engagement Results to Actionable Planning and Outcomes.

Staff and provider engagement leads to better quality and experience of care and less turnover and burnout. In this program, we describe an approach to better understand underlying factors that lead to low staff and provider engagement and address such factors by creating actionable plans that drive improved engagement measures. Focus groups were conducted with staff, advance practice providers, and faculty to better understand low scored areas in an annual third-party engagement survey. Focus group results were analyzed, and thematic action plans were then developed by a leadership team. These plans and the status of addressing the identified issues were published and disseminated back to all staff and providers using a "stoplight report." The leadership team met every 2 to 4 weeks until all issues were addressed and communicated back to the department. The subsequent year's engagement scores statistically increased across all engagement score domains for both staff and faculty. We conclude that using a qualitative approach to understanding low-scored engagement domains will allow a deeper and authentic understanding of the root factors that drive low engagement scores. This approach allows teams to develop responsive action plans, resulting in higher engagement scores, which will eventually lead to better service and care to patients.

Corrigendum: Insulin-Like Growth Factor Binding Protein-5 in Physiology and Disease.

[This corrects the article DOI: 10.3389/fendo.2020.00100.].

Insulin-Like Growth Factor Binding Protein-5 in Physiology and Disease.

Insulin-like growth factor (IGF) signaling is regulated by a conserved family of IGF binding proteins (IGFBPs) in vertebrates. Among the six distinct types of IGFBPs, IGFBP-5 is the most highly conserved across species and has the broadest range of biological activities. IGFBP-5 is expressed in diverse cell types, and its expression level is regulated by a variety of signaling pathways in different contexts. IGFBP-5 can exert a range of biological actions including prolonging the half-life of IGFs in the circulation, inhibition of IGF signaling by competing with the IGF-1 receptor for ligand binding, concentrating IGFs in certain cells and tissues, and potentiation of IGF signaling by delivery of IGFs to the IGF-1 receptor. IGFBP-5 also has IGF-independent activities and is even detected in the nucleus. Its broad biological activities make IGFBP-5 an excellent representative for understanding IGFBP functions. Despite its evolutionary conservation and numerous biological activities, knockout of IGFBP-5 in mice produced only a negligible phenotype. Recent research has begun to explain this paradox by demonstrating cell type-specific and physiological/pathological context-dependent roles for IGFBP-5. In this review, we survey and discuss what is currently known about IGFBP-5 in normal physiology and human disease. Based on recent in vivo genetic evidence, we suggest that IGFBP-5 is a multifunctional protein with the ability to act as a molecular switch to conditionally regulate IGF signaling.

Gonadotropin-releasing hormone neuron development in vertebrates.

Gonadotropin-releasing hormone (GnRH) neurons are master regulators of the reproductive axis in vertebrates. During early mammalian embryogenesis, GnRH1 neurons emerge in the nasal/olfactory placode. These neurons undertake a long-distance migration, moving from the nose to the preoptic area and hypothalamus. While significant advances have been made in understanding the functional importance of the GnRH1 neurons in reproduction, where GnRH1 neurons come from and how are they specified during early development is still under debate. In addition to the GnRH1 gene, most vertebrate species including humans have one or two additional GnRH genes. Compared to the GnRH1 neurons, much less is known about the development and regulation of GnRH2 neuron and GnRH3 neurons. The objective of this article is to review what is currently known about GnRH neuron development. We will survey various cell autonomous and non-autonomous factors implicated in the regulation of GnRH neuron development. Finally, we will discuss emerging tools and new approaches to resolve open questions pertaining to GnRH neuron development.

IGF-Binding Proteins: Why Do They Exist and Why Are There So Many?

Insulin-like growth factors (IGFs) are key growth-promoting peptides that act as both endocrine hormones and autocrine/paracrine growth factors. In the bloodstream and in local tissues, most IGF molecules are bound by one of the members of the IGF-binding protein (IGFBP) family, of which six distinct types exist. These proteins bind to IGF with an equal or greater affinity than the IGF1 receptor and are thus in a key position to regulate IGF signaling globally and locally. Binding to an IGFBP increases the half-life of IGF in the circulation and blocks its potential binding to the insulin receptor. In addition to these classical roles, IGFBPs have been shown to modulate IGF signaling locally under various conditions. Although members of the IGFBP family share significant sequence homology, they each have unique structural features and play distinct roles. These IGFBP genes also have different modes of regulation and distinct expression patterns. Some IGFBPs have been found to bind to their own receptors or to translocate into the interior compartments of cells where they may execute IGF-independent actions. In spite of this functional and regulatory diversity, it has been puzzling that loss-of-function studies have yielded relatively little information about the physiological functions of IGFBPs. In this review, we suggest that evolution has tended to retain an array of IGFBPs in order to facilitate fine-tuning of IGF signaling. We explore the emerging explanation that many IGFBP functions have evolved to allow the targeted adjustment of IGF signaling under stressful or irregular conditions, which would likely not be revealed in a standard laboratory setting.

Thymic stromal lymphopoietin receptor blockade reduces allergic inflammation in a cynomolgus monkey model of asthma.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) pathway blockade is a potential strategy for asthma treatment because the main activities of TSLP are activation of myeloid dendritic cells (mDCs) and modulation of cytokine production by mast cells. TSLP-activated mDCs prime the differentiation of naive T cells into inflammatory TH2 cells. OBJECTIVE: We sought to investigate mechanisms underlying the development of allergic lung inflammation in cynomolgus monkeys using gene expression profiling and to assess the effect of thymic stromal lymphopoietin receptor (TSLPR) blockade in this model. METHODS: An mAb against human TSLPR was generated and confirmed to be cross-reactive to cynomolgus monkey. Animals were dosed weekly with either vehicle or anti-TSLPR mAb for 6 weeks, and their responses to allergen challenge at baseline, week 2, and week 6 were assessed. RESULTS: After 6 weeks of treatment, anti-TSLPR mAb-treated animals showed reduced bronchoalveolar lavage (BAL) fluid eosinophil counts, reduced airway resistance in response to allergen challenge, and reduced IL-13 cytokine levels in BAL fluid compared with values seen in vehicle-treated animals. Expression profiling of BAL fluid cells collected before and after challenge showed a group of genes upregulated by allergen challenge that strongly overlapped with 11 genes upregulated in dendritic cells (DCs) when in vitro stimulated by TSLP (TSLP-DC gene signature). The number of genes differentially expressed in response to challenge was reduced in antibody-treated animals after 6 weeks relative to vehicle-treated animals. Expression of the TSLP-DC gene signature was also significantly reduced in antibody-treated animals. CONCLUSION: These results demonstrate promising efficacy for TSLPR blockade in an allergic lung inflammation model in which TSLP activation of mDCs might play a key role.

Natural regulatory T cells are resistant to calcium release-activated calcium (CRAC/ORAI) channel inhibition.

Organ transplant patients are often treated with immunosuppressants, such as the calcineurin phosphatase inhibitor, cyclosporin A, to block T cell-mediated graft rejection. The calcium release-activated calcium (CRAC/ORAI) channels, which act upstream of calcineurin, are essential for calcium entry and CD4(+) T-cell activation. Although cyclosporine A has also been shown to inhibit FoxP3(+) Tregs both in vitro and in vivo, the role of ORAI channel inhibition in natural Tregs (nTregs) or inducible Tregs (iTregs) has not been investigated. We found that, despite inhibition of calcium influx through the ORAI channels, ORAI channel inhibitors were unable to repress FoxP3 expression in mouse and human nTregs, whereas FoxP3 expression was inhibited in iTregs. In contrast, cyclosporin A inhibited FoxP3 expression in both nTregs and iTregs. We also generated mice with a T cell-specific, conditional knockout of ORAI1 and found that the mice have normal nTreg development and suppressive activity. Moreover, iTregs derived from ORAI1 conditional knockout mice develop normally and are still susceptible to ORAI channel inhibition. Our data indicate that unlike CD4(+) T cells and iTregs, nTregs are resistant to ORAI-mediated inhibition. Targeting ORAI channels potentially offers a novel way to inhibit pathologic T cells, while sparing nTreg-mediated tolerance.

Bleomycin induces molecular changes directly relevant to idiopathic pulmonary fibrosis: a model for "active" disease.

The preclinical model of bleomycin-induced lung fibrosis, used to investigate mechanisms related to idiopathic pulmonary fibrosis (IPF), has incorrectly predicted efficacy for several candidate compounds suggesting that it may be of limited value. As an attempt to improve the predictive nature of this model, integrative bioinformatic approaches were used to compare molecular alterations in the lungs of bleomycin-treated mice and patients with IPF. Using gene set enrichment analysis we show for the first time that genes differentially expressed during the fibrotic phase of the single challenge bleomycin model were significantly enriched in the expression profiles of IPF patients. The genes that contributed most to the enrichment were largely involved in mitosis, growth factor, and matrix signaling. Interestingly, these same mitotic processes were increased in the expression profiles of fibroblasts isolated from rapidly progressing, but not slowly progressing, IPF patients relative to control subjects. The data also indicated that TGFß was not the sole mediator responsible for the changes observed in this model since the ALK-5 inhibitor SB525334 effectively attenuated some but not all of the fibrosis associated with this model. Although some would suggest that repetitive bleomycin injuries may more effectively model IPF-like changes, our data do not support this conclusion. Together, these data highlight that a single bleomycin instillation effectively replicates several of the specific pathogenic molecular changes associated with IPF, and may be best used as a model for patients with active disease.

Characterization of a novel CRAC inhibitor that potently blocks human T cell activation and effector functions.

Store operated calcium entry (SOCE) downstream of T cell receptor (TCR) activation in T lymphocytes has been shown to be mediated mainly through the Calcium Release Activated Calcium (CRAC) channel. Here, we compared the effects of a novel, potent and selective CRAC current inhibitor, 2,6-Difluoro-N-{5-[4-methyl-1-(5-methyl-thiazol-2-yl)-1,2,5,6-tetrahydro-pyridin-3-yl]-pyrazin-2-yl}-benzamide (RO2959), on T cell effector functions with that of a previously reported CRAC channel inhibitor, YM-58483, and a calcineurin inhibitor Cyclosporin A (CsA). Using both electrophysiological and calcium-based fluorescence measurements, we showed that RO2959 is a potent SOCE inhibitor that blocked an IP3-dependent current in CRAC-expressing RBL-2H3 cells and CHO cells stably expressing human Orai1 and Stim1, as well as SOCE in human primary CD4(+) T cells triggered by either TCR stimulation or thapsigargin treatment. Furthermore, we demonstrated that RO2959 completely inhibited cytokine production as well as T cell proliferation mediated by TCR stimulation or MLR (mixed lymphocyte reaction). Lastly, we showed by gene expression array analysis that RO2959 potently blocked TCR triggered gene expression and T cell functional pathways similar to CsA and another calcineurin inhibitor FK506. Thus, both from a functional and transcriptional level, our data provide evidence that RO2959 is a novel and selective CRAC current inhibitor that potently inhibits human T cell functions.

Comparing the differential effects of LPA on the barrier function of human pulmonary endothelial cells.

Lysophosphatidic acid (LPA) is a class of bioactive lyso-phospholipid that mediates most of its biological effects through a family of G protein-coupled receptors of which six have been identified. The role of the LPA pathway in driving chronic lung diseases such as idiopathic pulmonary fibrosis (IPF) has gained considerable academic and industry attention. Modulation of the pulmonary artery endothelial barrier function by the LPA1 receptor has been shown to drive pulmonary fibrosis in murine models of disease. The purpose of this study was (i) to assess the effect of LPA on the barrier function of human pulmonary arterial (HPAEC) and microvascular (HMVEC) endothelial cells and (ii) to identify the LPA receptor subtype(s) responsible for changes in human pulmonary endothelial cell permeability using LPA receptor antagonists and siRNA technology. Analysis of the LPA receptor subtype expression demonstrated predominant expression of LPA2 and LPA6 receptor subtypes in both HPAECs and HMVECs. HPAECs also exhibit low expression of LPA1, LPA3, and LPA4 receptor subtypes. Treatment of cells with increasing concentrations of LPA caused loss of barrier function in HPAECs but not HMVECs, despite both cell types exhibiting very similar LPA receptor expression profiles. The LPA-mediated loss of barrier function in HPAECs appears to be independent of the LPA1 receptor and likely to be mediated via the LPA6 receptor although we cannot exclude an additional role for the LPA2 and LPA4 receptors in mediating these effects. These results suggest cell-specific mechanisms exist in human pulmonary endothelial cells to permit regulation of barrier function downstream of LPA receptors. More importantly, our data indicate that selective LPA1 receptor antagonism may be insufficient for therapeutic use in pulmonary diseases where impaired endothelial barrier function is related to disease initiation and progression.

H5N1 influenza virus pathogenesis in genetically diverse mice is mediated at the level of viral load.

UNLABELLED: The genotype of the host is one of several factors involved in the pathogenesis of an infectious disease and may be a key parameter in the epidemiology of highly pathogenic H5N1 influenza virus infection in humans. Gene polymorphisms may affect the viral replication rate or alter the host's immune response to the virus. In humans, it is unclear which aspect dictates the severity of H5N1 virus disease. To identify the mechanism underlying differential responses to H5N1 virus infection in a genetically diverse population, we assessed the host responses and lung viral loads in 21 inbred mouse strains upon intranasal inoculation with A/Hong Kong/213/03 (H5N1). Resistant mouse strains survived large inocula while susceptible strains succumbed to infection with 1,000- to 10,000-fold-lower doses. Quantitative analysis of the viral load after inoculation with an intermediate dose found significant associations with lethality as early as 2 days postinoculation, earlier than any other disease indicator. The increased viral titers in the highly susceptible strains mediated a hyperinflamed environment, indicated by the distinct expression profiles and increased production of inflammatory mediators on day 3. Supporting the hypothesis that viral load rather than an inappropriate response to the virus was the key severity-determining factor, we performed quantitative real-time PCR measuring the cytokine/viral RNA ratio. No significant differences between susceptible and resistant mouse strains were detected, confirming that it is the host genetic component controlling viral load, and therefore replication dynamics, that is primarily responsible for a host's susceptibility to a given H5N1 virus. IMPORTANCE: Highly pathogenic H5N1 influenza virus has circulated in Southeast Asia since 2003 but has been confirmed in relatively few individuals. It has been postulated that host genetic polymorphisms increase the susceptibility to infection and severe disease. The mechanisms and host proteins affected during severe disease are unknown. Inbred mouse strains vary considerably in their ability to resist H5N1 virus and were used to identify the primary mechanism determining disease severity. After inoculation with H5N1, resistant mouse strains had reduced amounts of virus in their lungs, which subsequently resulted in lower production of proinflammatory mediators and less pathology. We therefore conclude that the host genetic component controlling disease severity is primarily influencing viral replication. This is an important concept, as it emphasizes the need to limit virus replication through antiviral therapies and it shows that the hyperinflammatory environment is simply a reflection of more viral genetic material inducing a response.

Comparative endocrinology of aging and longevity regulation.

Hormones regulate growth, development, metabolism, and other complex processes in multicellular animals. For many years it has been suggested that hormones may also influence the rate of the aging process. Aging is a multifactorial process that causes biological systems to break down and cease to function in adult organisms as time passes, eventually leading to death. The exact underlying causes of the aging process remain a topic for debate, and clues that may shed light on these causes are eagerly sought after. In the last two decades, gene mutations that result in delayed aging and extended longevity have been discovered, and many of the affected genes have been components of endocrine signaling pathways. In this review we summarize the current knowledge on the roles of endocrine signaling in the regulation of aging and longevity in various animals. We begin by discussing the notion that conserved systems, including endocrine signaling pathways, "regulate" the aging process. Findings from the major model organisms: worms, flies, and rodents, are then outlined. Unique lessons from studies of non-traditional models: bees, salmon, and naked mole rats, are also discussed. Finally, we summarize the endocrinology of aging in humans, including changes in hormone levels with age, and the involvement of hormones in aging-related diseases. The most well studied and widely conserved endocrine pathway that affects aging is the insulin/insulin-like growth factor system. Mutations in genes of this pathway increase the lifespan of worms, flies, and mice. Population genetic evidence also suggests this pathway's involvement in human aging. Other hormones including steroids have been linked to aging only in a subset of the models studied. Because of the value of comparative studies, it is suggested that the aging field could benefit from adoption of additional model organisms.

An integrative genomic analysis identifies Bhmt2 as a diet-dependent genetic factor protecting against acetaminophen-induced liver toxicity.

Acetaminophen-induced liver toxicity is the most frequent precipitating cause of acute liver failure and liver transplant, but contemporary medical practice has mainly focused on patient management after a liver injury has been induced. An integrative genetic, transcriptional, and two-dimensional NMR-based metabolomic analysis performed using multiple inbred mouse strains, along with knowledge-based filtering of these data, identified betaine-homocysteine methyltransferase 2 (Bhmt2) as a diet-dependent genetic factor that affected susceptibility to acetaminophen-induced liver toxicity in mice. Through an effect on methionine and glutathione biosynthesis, Bhmt2 could utilize its substrate (S-methylmethionine [SMM]) to confer protection against acetaminophen-induced injury in vivo. Since SMM is only synthesized in plants, Bhmt2 exerts its beneficial effect in a diet-dependent manner. Identification of Bhmt2 and the affected biosynthetic pathway demonstrates how a novel method of integrative genomic analysis in mice can provide a unique and clinically applicable approach to a major public health problem.

Testicular gene expression profiling following 2-methoxyethanol and 2-ethoxyethanol exposure in male rats reveals abnormal expression of the actin binding protein cortactin in degenerating spermatocytes.

The glycol ether solvents 2-methoxyethanol (2-ME) and 2-ethoxyethanol (2-EE) produce testicular toxicity characterized by spermatocyte degeneration, while a similar glycol ether, 2-butoxyethanol (2-BE), has no testicular effects. The goal of the current study was to better understand the mechanism of glycol ether testicular toxicity through gene expression profiling and functional classification of differentially expressed genes. Male rats were administered 2-ME (150 and 50mg/kg/day), 2-EE (500 mg/kg/day), 2-BE (125 mg/kg/day), or vehicle for 3 days, and testes were collected for histopathological and gene expression analysis. Histopathological changes in the testes were observed only in animals given 150 mg/kg/day 2-ME, consisting of degeneration and necrosis of spermatocytes and reductions in spermatocyte numbers. Microarray analysis of testicular samples from these animals revealed a large number of differentially expressed genes from animals exposed to 2-EE or to 50mg/kg or 150 mg/kg 2-ME (>900 each at >1.5-fold changed), compared to 28 genes from 2-BE treated animals. Expression Analysis Systematic Explorer (EASE) analysis of these genes demonstrated statistical enrichment in genes in categories including protein transport, endocytosis, protein kinase activity, cell cycle, and meiosis. Quantitative PCR confirmation of select genes confirmed increased expression of the actin binding protein cortactin and the transcription factor Wilm's tumor 1 (Wt1) following 2-ME exposure. Increased localization of cortactin in abnormal spermatocytes was also observed by immunohistochemistry, consistent with a possible role for this protein in the mechanism of toxicity.

Persistent activation of an innate immune response translates respiratory viral infection into chronic lung disease.

To understand the pathogenesis of chronic inflammatory disease, we analyzed an experimental mouse model of chronic lung disease with pathology that resembles asthma and chronic obstructive pulmonary disease (COPD) in humans. In this model, chronic lung disease develops after an infection with a common type of respiratory virus is cleared to only trace levels of noninfectious virus. Chronic inflammatory disease is generally thought to depend on an altered adaptive immune response. However, here we find that this type of disease arises independently of an adaptive immune response and is driven instead by interleukin-13 produced by macrophages that have been stimulated by CD1d-dependent T cell receptor-invariant natural killer T (NKT) cells. This innate immune axis is also activated in the lungs of humans with chronic airway disease due to asthma or COPD. These findings provide new insight into the pathogenesis of chronic inflammatory disease with the discovery that the transition from respiratory viral infection into chronic lung disease requires persistent activation of a previously undescribed NKT cell-macrophage innate immune axis.

In silico and in vitro pharmacogenetic analysis in mice.

Combining the experimental efficiency of a murine hepatic in vitro drug biotransformation system with in silico genetic analysis produces a model system that can rapidly analyze interindividual differences in drug metabolism. This model system was tested by using two clinically important drugs, testosterone and irinotecan, whose metabolism was previously well characterized. The metabolites produced after these drugs were incubated with hepatic in vitro biotransformation systems prepared from the 15 inbred mouse strains were measured. Strain-specific differences in the rate of 16 alpha-hydroxytestosterone generation and irinotecan glucuronidation correlated with the pattern of genetic variation within Cyp2b9 and Ugt1a loci, respectively. These computational predictions were experimentally confirmed using expressed recombinant enzymes. The genetic changes affecting irinotecan metabolism in mice mirrored those in humans that are known to affect the pharmacokinetics and incidence of adverse responses to this medication.

Genetic mapping in mice identifies DMBT1 as a candidate modifier of mammary tumors and breast cancer risk.

Low-penetrance breast cancer susceptibility alleles seem to play a significant role in breast cancer risk but are difficult to identify in human cohorts. A genetic screen of 176 N2 backcross progeny of two Trp53(+/-) strains, BALB/c and C57BL/6, which differ in their susceptibility to mammary tumors, identified a modifier of mammary tumor susceptibility in an approximately 25-Mb interval on mouse chromosome 7 (designated SuprMam1). Relative to heterozygotes, homozygosity for BALB/c alleles of SuprMam1 significantly decreased mammary tumor latency from 70.7 to 61.1 weeks and increased risk twofold (P = 0.002). Dmbt1 (deleted in malignant brain tumors 1) was identified as a candidate modifier gene within the SuprMam1 interval because it was differentially expressed in mammary tissues from BALB/c-Trp53(+/-) and C57BL/6-Trp53(+/-) mice. Dmbt1 mRNA and protein was reduced in mammary glands of the susceptible BALB/c mice. Immunohistochemical staining demonstrated that DMBT1 protein expression was also significantly reduced in normal breast tissue from women with breast cancer (staining score, 1.8; n = 46) compared with cancer-free controls (staining score, 3.9; n = 53; P < 0.0001). These experiments demonstrate the use of Trp53(+/-) mice as a sensitized background to screen for low-penetrance modifiers of cancer. The results identify a novel mammary tumor susceptibility locus in mice and support a role for DMBT1 in suppression of mammary tumors in both mice and women.

Understanding our drugs and our diseases.

Analysis of mouse genetic models of human disease-associated traits has provided important insight into the pathogenesis of human disease. As one example, analysis of a murine genetic model of osteoporosis demonstrated that genetic variation within the 15-lipoxygenase (Alox15) gene affected peak bone mass, and that treatment with inhibitors of this enzyme improved bone mass and quality in rodent models. However, the method that has been used to analyze mouse genetic models is very time consuming, inefficient, and costly. To overcome these limitations, a computational method for analysis of mouse genetic models was developed that markedly accelerates the pace of genetic discovery. It was used to identify a genetic factor affecting the rate of metabolism of warfarin, an anticoagulant that is commonly used to treat clotting disorders. Computational analysis of a murine genetic model of narcotic drug withdrawal suggested a potential new approach for treatment of narcotic drug addiction. Thus, the results derived from computational mouse genetic analysis can suggest new treatment strategies, and can provide new information about currently available medicines.

In silico pharmacogenetics of warfarin metabolism.

Pharmacogenetic approaches can be instrumental for predicting individual differences in response to a therapeutic intervention. Here we used a recently developed murine haplotype-based computational method to identify a genetic factor regulating the metabolism of warfarin, a commonly prescribed anticoagulant with a narrow therapeutic index and a large variation in individual dosing. After quantification of warfarin and nine of its metabolites in plasma from 13 inbred mouse strains, we correlated strain-specific differences in 7-hydroxywarfarin accumulation with genetic variation within a chromosomal region encoding cytochrome P450 2C (Cyp2c) enzymes. This computational prediction was experimentally confirmed by showing that the rate-limiting step in biotransformation of warfarin to its 7-hydroxylated metabolite was inhibited by tolbutamide, a Cyp2c isoform-specific substrate, and that this transformation was mediated by expressed recombinant Cyp2c29. We show that genetic variants responsible for interindividual pharmacokinetic differences in drug metabolism can be identified by computational genetic analysis in mice.