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PDF file - 25K, Pharmacokinetic parameters of PKC412.

PDF file - 528K, G�6976 inhibits EGFR mutant NSCLC cells independently of PKC inhibition.

PDF file - 142K, EGFR sequencing of the PC-9 ER clone.

PDF file - 21K, Body weight measurement for EGFR T790M xenografts.

XLSX file - 242K, Ambit kinome profiling with 0.5 μM G�6976.

PDF file - 28K, PKC412 is the most selective EGFR T790M inhibitor tested versus EGFR wild-type.

Approximately half of EGFR-mutant non–small cell lung cancer (NSCLC) patients treated with small-molecule EGFR kinase inhibitors develop drug resistance associated with the EGF receptor (EGFR) T790M “gatekeeper” substitution, prompting efforts to develop covalent EGFR inhibitors, which can effectively suppress EGFR T790M in preclinical models. However, these inhibitors have yet to prove clinically efficacious, and their toxicity in skin, reflecting activity against wild-type EGFR, may limit dosing required to effectively suppress EGFR T790M in vivo. While profiling sensitivity to various kinase inhibitors across a large cancer cell line panel, we identified indolocarbazole compounds, including a clinically well-tolerated FLT3 inhibitor, as potent and reversible inhibitors of EGFR T790M that spare wild-type EGFR. These findings show the use of broad cancer cell profiling of kinase inhibitor efficacy to identify unanticipated novel applications, and they identify indolocarbazole compounds as potentially effective EGFR inhibitors in the context of T790M-mediated drug resistance in NSCLC.Significance: EGFR-mutant lung cancer patients who respond to currently used EGFR kinase inhibitors invariably develop drug resistance, which is associated with the EGFR T790M resistance mutation in about half these cases. We unexpectedly identified a class of reversible potent inhibitors of EGFR T790M that do not inhibit wild-type EGFR, revealing a promising therapeutic strategy to overcome T790M-associated drug-resistant lung cancers. Cancer Discov; 3(2); 168–81. ©2012 AACR.See related commentary by Brewer and Pao, p. 138This article is highlighted in the In This Issue feature, p. 125

PDF file - 130K

XLSX file - 109K, Tumor cell line profiling of G�6976 sensitivity.

PDF file - 248K, PKC412 is a reversible EGFR T790M inhibitor.

PDF file - 177K, Comparison of EGFR T790M inhibitors in NR6 cell lines expressing WT EGFR.

PDF file - 78K, Efficacy of PKC412 on tumor growth and signaling in EGFRL858R+T790M transgenic mice.

Supplemental Dataset 1. Expression of AXL, GAS6, Vimentin and E-Cadherin in 644 human cancer cell line using RNAseq technology.

Timelapse movie of HeLa cells treated with R428 (1μM).

Timelapse movie of HeLa cells treated with DMSO.

Timelapse movie of HeLa cells treated with docetaxel (3nM).

Supplemental Dataset 3. Tumor cell line profile of docetaxel in combination with R428

Supplemental Dataset 2. Kinome profile with 1000nM R428

Timelapse movie of HeLa cells treated with docetaxel (3nM) in combination with R428 (1μM).

PBRM1 is an accessory subunit of the PBAF subclass of the SWI/SNF chromatin remodeler and the inactivation of PBRM1 is the second most frequent mutational event in kidney tumorigenesis. However, the impact of PBRM1 loss on chromatin remodeling, especially pertaining to kidney tumorigenesis, has not been examined previously. Here we show that in VHL-deficient ccRCC tumors, PBRM1 deficiency results in altered PBAF complexes that retain the association between SMARCA4 and ARID2 but disengage BRD7 from SMARCA4. The PBRM1-deficient PBAF complexes redistribute from promoter proximal regions to distal enhancer regions containing motifs of pro-tumorigenic factors including NF-κB. Subsequently, PBRM1-deficient cells display heightened NF-κB activity across multiple cell models. The ATPase function of SMARCA4 maintains chromatin occupancy of both pre-existing and newly acquired RELA specific to PBRM1 loss, and activates downstream target gene expression. Proteasome inhibitor bortezomib reverses NF-κB activation by reducing RELA occupancy and delays growth of PBRM1-deficient tumors. In conclusion, PBRM1 safeguards the chromatin by repressing aberrant liberation of pro-tumorigenic NF-κB target genes by residual PBRM1-deficient PBAF complexes. Citation Format: Xiaosai Yao, Jing Han Hong, Amrita Nargund, Michelle Shu Wen Ng, Hong Lee Heng, Zhimei Li, Peiyong Guan, Masahiro Sugiura, Pek Lim Chu, Loo Chien Wang, Xiaofen Ye, Robert Yauch, Kenneth Tou-En Chang, Radoslaw M. Sobota, Patrick Tan, Bin Tean Teh. PBRM1-deficient PBAF complexes target de novo genomic loci to activate NF-κB pathway in clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1494.

The mammalian SWI/SNF helicase SMARCA4 is frequently mutated in cancer and inactivation results in a cellular dependence on its paralog, SMARCA2, thus making SMARCA2 an attractive synthetic lethal target. However, published data indicates that achieving a high degree of selective SMARCA2 inhibition is likely essential to afford an acceptable therapeutic index, and realizing this objective has been challenging due to the homology with the SMARCA4 paralog. Herein we report the discovery of the first potent and selective SMARCA2 proteolysis-targeting chimera (PROTAC) molecule (A947). Selective SMARCA2 degradation was achieved in the absence of selective SMARCA2/4 PROTAC binding and translated to potent in vitro growth inhibition and in vivo efficacy in SMARCA4 mutant models, compared to wild type models. Global ubiquitin mapping and proteome profiling revealed no unexpected off-target degradation related to A947 treatment. Our study thus highlights the ability to transform a non-selective SMARCA2/4-binding ligand into a selective and efficacious in vivo SMARCA2-targeting PROTAC, and thereby provides a potential new therapeutic opportunity for patients whose tumors contain SMARCA4 mutations.

Background: To know the knowledge on inhaler devices in medical staff, this study surveyed knowledge of inhaled therapy and the use of inhaler devices in nurses in China.Methods: 1831 nurses were selected to survey on a new self-designed questionnaire online. The questionnaire has 11 questions, including the storage location of the inhaler devices, steps of using the inhaler device, and the common errors when using various devices, etc. Results: Among the 1831 participants, 816(44.57%), 122(6.66%) and 893(48.77%) nurses worked in community hospital, secondary hospital and tertiary hospital respectively. Adequate knowledge of inhaler devices was demonstrated by nurses working in community hospital(20.10%)、Secondary hospital(8.2%) and Tertiary hospital(13.1%). 27.70% nurses in community hospital knew operational key points in using inhaler compared to in secondary hospital (15.57%) and tertiary hospital (23.18%) PConclusion: The focus on community hospitals has some results in China. However, knowledge on inhaler devices should be continued to strengthen among nurses in hospital. It is necessary to create training opportunities for nurses to increase their awareness and knowledge of chronic respiratory diseases management.

Objective: The main objective of the present study is to evaluate the psychometric properties of the Chinese version of Beliefs about Medicines Questionnaire-specific among asthma patients; and to assess the association between patients’ belief and adherence to inhaled corticosteroid therapy. Methods: A cross-sectional survey was carried out in the asthma clinic of Zhongshan Hospital, to Fudan University (Shanghai, China) between April 2016 and March 2018. The Beliefs about Medicines Questionnaire-specific was translated into Chinese according to international guidelines. Internal consistency, test–retest reliability, and confirmatory factor analysis were calculated to validate the Beliefs about Medicines Questionnaire-specific. The relationship between the adherence and the belief subscale were assessed using Kruskal–Wallis test. Results: Two hundred and seventeen patients were recruited in this study. The Beliefs about Medicines Questionnaire-specific was deemed reliable based on the results of Cronbach’s alpha coefficient and test–retest intraclass correlation coefficient (ICC, ICC= 0.759). Confirmatory factor analysis showed acceptable model fit for the two-factor model. Patients’ compliance was closely related to their belief about inhaled corticosteroid. The adherence rates were highest for the accepting groups, and lowest for the skeptical groups. Higher adherence was significantly associated with higher necessity-concerns differential (p = .001) and lower concern (p = .004). Conclusions: The Chinese version of the Beliefs about Medicines Questionnaire-specific can be used as a reliable tool by the clinicians to identify beliefs and behaviors of individual to improve adherence in Chinese patients.

This study aimed to investigate the relationship between illness perceptions, medication beliefs, and self-reported adherence to inhaled corticosteroid (ICS) therapy in adult Chinese patients with asthma.A cross-sectional survey was conducted in the asthma outpatient clinic of Zhongshan Hospital, Fudan University (Shanghai, China) between October 2018 and September 2019. Illness perceptions, medication beliefs, and medication adherence were assessed using validated scales, specifically the Medication Adherence Report Scale for Asthma, Beliefs about Medicines Questionnaire -Specific, and the Brief Illness Perception Questionnaire. Spearman correlation and multiple logistic regression were used to determine the relationship among these factors.Beliefs about the necessity of medication and emotional response to the illness have a strong influence on self-reported medication adherence in adult patients with asthma in China. Interventions targeted adherence improvement among patients with asthma may be tailored to the individual's baseline perceptions and medication beliefs, and focus on modifying inaccurate illness perceptions and medication beliefs as the main targets.

This paper chooses 14 listed banks in China June 2013 to March 2016 quarter data as sample, exploding the effect of “Bao bao” Internet monetary funds on deposits in commercial banks based on the econometric model. The empirical results show that “Bao bao” Internet monetary funds have not been diverted the deposits balance of commercial banks, but it increases the cost of deposits of commercial banks, and brings a certain amount of pressure on interest payments of Chinese commercial banks.

Colorectal cancer remains a major unmet medical need, prompting large-scale genomics efforts in the field to identify molecular drivers for which targeted therapies might be developed. We previously reported the identification of recurrent translocations in R-spondin genes present in a subset of colorectal tumours. Here we show that targeting RSPO3 in PTPRK-RSPO3-fusion-positive human tumour xenografts inhibits tumour growth and promotes differentiation. Notably, genes expressed in the stem-cell compartment of the intestine were among those most sensitive to anti-RSPO3 treatment. This observation, combined with functional assays, suggests that a stem-cell compartment drives PTPRK-RSPO3 colorectal tumour growth and indicates that the therapeutic targeting of stem-cell properties within tumours may be a clinically relevant approach for the treatment of colorectal tumours.

The mammalian SWI/SNF complex catalyzes the remodeling of chromatin through the helicase activity of two mutually-exclusive, paralogous subunits, BRG1 and BRM. BRG1 is frequently mutated in cancer and its inactivation results in a cellular dependence on BRM. Despite the attractiveness of BRM as a synthetic lethal therapeutic target, the selective inhibition of BRM represents a considerable challenge due to the high degree of homology between BRM and BRG1. Furthermore, published data indicate that achieving such selectivity is likely essential to afford an acceptable therapeutic index. We sought to mimic the synthetic lethality observed in BRG1-mutant cancers by identifying proteolysis-targeting chimera (PROTAC®) molecules capable of selectively degrading BRM via trimeric complex formation with the von Hippel-Lindau (VHL) E3 ligase. In this disclosure, we report our initial discovery of potent and selective chimeric BRM-degrader molecules which exhibit BRM DC50 values 25. Importantly, selective BRM degradation can be achieved in the absence of selective PROTAC® binding (BRG1/BRM Kd ratios Citation Format: Michael Berlin, Jennifer Cantley, Jing Wang, Mark Bookbinder, Gregory Cadelina, Emily Chan, Huifen Chen, Xin Chen, Kim Davenport, Tharu Fernando, Debbie Gordon, Brian Hamman, Roy Haskell, Alexey Ishchenko, Donald S. Kirkpatrick, Jonathan Maher, Mark Merchant, John Moffat, Alicia Morgan, An Nguyen, Jennifer Pizzano, Connor Quinn, Christopher M. Rose, Emma Rousseau, Vijay Sethuraman, Leanna Staben, Catherine Wilson, Xiaofen Ye, Fabio Broccatelli, Robert Yauch, Peter S. Dragovich. Selective, chemically-induced degradation of BRM (SMARCA2) enables in vivo efficacy in BRG1 (SMARCA4)-deficient xenograft tumor models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5687.

In this paper, an exact analytical solution is presented for a transversely isotropic functionally graded magneto-electro-elastic (FGMEE) cantilever beam, which is subjected to a uniform load on its upper surface, as well as the concentrated force and moment at the free end. This solution can be applied for any form of gradient distribution. For the basic equations of plane problem, all the partial differential equations governing the stress field, electric, and magnetic potentials are derived. Then, the expressions of Airy stress, electric, and magnetic potential functions are assumed as quadratic polynomials of the longitudinal coordinate. Based on all the boundary conditions, the exact expressions of the three functions can be determined. As numerical examples, the material parameters are set as exponential and linear distributions in the thickness direction. The effects of the material parameters on the mechanical, electric, and magnetic fields of the cantilever beam are analyzed in detail.

Subunits of the SWI/SNF chromatin remodeling complex are frequently mutated in human cancers leading to epigenetic dependencies that are therapeutically targetable. The dependency on the polycomb repressive complex (PRC2) and EZH2 represents one such vulnerability in tumors with mutations in the SWI/SNF complex subunit, SNF5; however, whether this vulnerability extends to other SWI/SNF subunit mutations is not well understood. Here we show that a subset of cancers harboring mutations in the SWI/SNF ATPase, SMARCA4, is sensitive to EZH2 inhibition. EZH2 inhibition results in a heterogenous phenotypic response characterized by senescence and/or apoptosis in different models, and also leads to tumor growth inhibition in vivo. Lower expression of the SMARCA2 paralog was associated with cellular sensitivity to EZH2 inhibition in SMARCA4 mutant cancer models, independent of tissue derivation. SMARCA2 is suppressed by PRC2 in sensitive models, and induced SMARCA2 expression can compensate for SMARCA4 and antagonize PRC2 targets. The induction of SMARCA2 in response to EZH2 inhibition is required for apoptosis, but not for growth arrest, through a mechanism involving the derepression of the lysomal protease cathepsin B. Expression of SMARCA2 also delineates EZH2 inhibitor sensitivity for other SWI/SNF complex subunit mutant tumors, including SNF5 and ARID1A mutant cancers. Our data support monitoring SMARCA2 expression as a predictive biomarker for EZH2-targeted therapies in the context of SWI/SNF mutant cancers.

Molecularly targeted drug therapies have revolutionized cancer treatment; however, resistance remains a major limitation to their overall efficacy. Epithelial-to-mesenchymal transition (EMT) has been linked to acquired resistance to tyrosine kinase inhibitors (TKI), independent of mutational resistance mechanisms. AXL is a receptor tyrosine kinase associated with EMT that has been implicated in drug resistance and has emerged as a candidate therapeutic target. Across 643 human cancer cell lines that were analyzed, elevated AXL was strongly associated with a mesenchymal phenotype, particularly in triple-negative breast cancer and non–small cell lung cancer. In an unbiased screen of small-molecule inhibitors of cancer-relevant processes, we discovered that AXL inhibition was specifically synergistic with antimitotic agents in killing cancer cells that had undergone EMT and demonstrated associated TKI resistance. However, we did not find that AXL inhibition alone could overcome acquired resistance to EGFR TKIs in the EMT setting, as previously reported. These findings reveal a novel cotreatment strategy for tumors displaying mesenchymal features that otherwise render them treatment refractory. Cancer Res; 74(20); 5878–90. ©2014 AACR.

Cellular senescence is an irreversible proliferation arrest of primary cells and an important tumor suppression process. Senescence is often characterized by domains of facultative heterochromatin, called senescence-associated heterochromatin foci (SAHF), which repress expression of proliferation-promoting genes. Formation of SAHF is driven by a complex of histone chaperones, HIRA and ASF1a, and depends upon prior localization of HIRA to PML nuclear bodies. However, how the SAHF assembly pathway is activated in senescent cells is not known. Here we show that expression of the canonical Wnt2 ligand and downstream canonical Wnt signals are repressed in senescent human cells. Repression of Wnt2 occurs early in senescence and independently of the pRB and p53 tumor suppressor proteins and drives relocalization of HIRA to PML bodies, formation of SAHF and senescence, likely through GSK3beta-mediated phosphorylation of HIRA. These results have major implications for our understanding of both Wnt signaling and senescence in tissue homeostasis and cancer progression.

A synthetic lethality caused by EZH2 inhibition in the context of SNF5 mutations is supported by both preclinical and recent clinical data, however the extent of the synthetic lethal relationship in the context of other SWI/SNF subunit mutations is not well understood. We determined that a subset of SMARCA4 mutant cancer models are sensitive to EZH2 inhibition. EZH2 inhibition resulted in a heterogenous phenotypic response characterized by senescence and/or apoptosis amongst models, and further lead to tumor growth inhibition in vivo. The differential sensitivity to EZH2 inhibition was not caused by a differential pharmacodynamic effect of the drug, nor differences in basal histone methylation or PRC2 subunit levels. However, expression of the SWI/SNF subunit, SMARCA2, delineated sensitivity amongst SMARCA4 mutant models tested. Expression of SMARCA2 further delineated sensitivity amongst other SWI/SNF mutant models tested, including SNF5 and ARID1A mutants. We determined that SMARCA2 is under PRC2 mediated suppression and the derepression of SMARCA2 was necessary for apoptosis, but not senescence, in response to EZH2 inhibition. SMARCA2 has been shown to be concurrently lost in a high percentage of SNF5 mutant malignant rhabdoid tumors and SMARCA4 mutant SCCOHT tumors, however we determined that ≈15% of SMARCA4 mutant NSCLCs concurrently lose SMARCA2. Our data supports monitoring SMARCA2 expression as a predictive biomarker for EZH2-targeted therapies that are currently being developed in the context of SWI/SNF mutant cancers. Citation Format: Thomas Januario, Xiaofen Ye, Russell Bainer, Bruno Alicke, Margaret Solon, Benjamin Haley, Zora Modrusan, Stephen Gould, Hartmut Koeppen, Robert L. Yauch. PRC2 mediated repression of SMARCA2 predicts for EZH2 inhibitor activity in tumors with SWI/SNF mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2790. doi:10.1158/1538-7445.AM2017-2790

Plasma protein C functions as an anticoagulant when it is converted to the active form of serine protease. Protein C activation has been found to be mediated by the endothelial cell surface thrombin/thrombomodulin (TM) complex. In addition, we recently identified the endothelial cell protein C/activated protein C receptor (EPCR) which is capable of high-affinity binding for protein C. In this study, we established monoclonal antibodies (mAbs) against EPCR including several function blocking antibodies. Immunohistochemical analysis using these mAbs demonstrated that EPCR is widely expressed in the endothelial cells of arteries, veins, and capillaries in the lung, heart, and skin. Function blocking anti-EPCR mAbs strongly inhibited protein C activation mediated by primary cultured arterial endothelial cells which express abundant EPCR. Anti-EPCR mAbs also prevent protein C activation mediated by microvascular endothelial cells. These results indicate that EPCR functions as an important regulator for the protein C pathway in various types of vessels.

Epithelial-to-mesenchymal transition (EMT) is a cellular process essential to the development and maintenance of solid tissues. In cancer, EMT suppresses apoptosis, but the mechanisms remain unclear. EMT selectively attenuated apoptosis signaling via the death receptors DR4 and DR5. Loss of the epithelial cell adhesion protein E-cadherin recapitulated this outcome, whereas homotypic E-cadherin engagement promoted apoptotic signaling via DR4/DR5, but not Fas. Depletion of α-catenin, which couples E-cadherin to the actin cytoskeleton, or actin polymerization inhibitors similarly attenuated DR4/DR5-induced apoptosis. E-cadherin bound specifically to ligated DR4/DR5, requiring extracellular cadherin domain 1 and calcium. E-cadherin augmented DR4/DR5 clustering and assembly of the death-inducing signaling complex (DISC), increasing caspase-8 activation in high molecular weight cell fractions. Conversely, EMT attenuated DR4/DR5-mediated DISC formation and caspase-8 stimulation. Consistent with these findings, epithelial cancer cell lines expressing higher E-cadherin levels displayed greater sensitivity to DR4/DR5-mediated apoptosis. These results have potential implications for tissue homeostasis as well as cancer therapy.

Approximately half of EGFR-mutant non–small cell lung cancer (NSCLC) patients treated with small-molecule EGFR kinase inhibitors develop drug resistance associated with the EGF receptor (EGFR) T790M “gatekeeper” substitution, prompting efforts to develop covalent EGFR inhibitors, which can effectively suppress EGFR T790M in preclinical models. However, these inhibitors have yet to prove clinically efficacious, and their toxicity in skin, reflecting activity against wild-type EGFR, may limit dosing required to effectively suppress EGFR T790M in vivo. While profiling sensitivity to various kinase inhibitors across a large cancer cell line panel, we identified indolocarbazole compounds, including a clinically well-tolerated FLT3 inhibitor, as potent and reversible inhibitors of EGFR T790M that spare wild-type EGFR. These findings show the use of broad cancer cell profiling of kinase inhibitor efficacy to identify unanticipated novel applications, and they identify indolocarbazole compounds as potentially effective EGFR inhibitors in the context of T790M-mediated drug resistance in NSCLC. Significance: EGFR-mutant lung cancer patients who respond to currently used EGFR kinase inhibitors invariably develop drug resistance, which is associated with the EGFR T790M resistance mutation in about half these cases. We unexpectedly identified a class of reversible potent inhibitors of EGFR T790M that do not inhibit wild-type EGFR, revealing a promising therapeutic strategy to overcome T790M-associated drug-resistant lung cancers. Cancer Discov; 3(2); 168–81. ©2012 AACR. See related commentary by Brewer and Pao, p. 138 This article is highlighted in the In This Issue feature, p. 125

Axl is expressed in various types of cancer and is involved in multiple processes of tumorigenesis, including promoting tumor cell growth, migration, invasion, metastasis as well as angiogenesis. To evaluate further the mechanisms involved in the expression/activation of Axl in various aspects of tumorigenesis, especially its roles in modulating tumor stromal functions, we have developed a phage-derived mAb (YW327.6S2) that recognizes both human and murine Axl. YW327.6S2 binds to both human and murine Axl with high affinity. It blocks the ligand Gas6 binding to the receptor, downregulates receptor expression, inhibits receptor activation and downstream signaling. In A549 non-small-cell lung cancer (NSCLC) and MDA-MB-231 breast cancer models, YW327.6S2 attenuates xenograft tumor growth and potentiates the effect of anti-VEGF treatment. In NSCLC models, YW327.6S2 also enhances the effect of erlotinib and chemotherapy in reducing tumor growth. Furthermore, YW327.6S2 reduces the metastasis of MDA-MB-231 breast cancer cells to distant organs. YW327.6S2 induces tumor cell apoptosis in NSCLC, reduces tumor-associated vascular density and inhibits the secretion of inflammatory cytokines and chemokines from tumor-associated macrophages in the breast cancer model. In conclusion, anti-Axl mAb can enhance the therapeutic efficacy of anti-VEGF, EGFR small-molecule inhibitors as well as chemotherapy. Axl mAb affects not only tumor cells but also tumor stroma through its modulation of tumor-associated vasculature and immune cell functions.

To perform structure/function analyses of a protein in vivo, ideally one should be able to simultaneously abolish expression of the endogenous wild-type protein, substitute it with a form of the protein containing a targeted mutation, and analyze the functional consequences. Until recently, this was a highly challenging and/or laborious approach in mammalian systems, requiring a targeted gene knockin in a human cell line or mouse. Herein is described a RNA interference (RNAi)-based approach to achieve this much more simply in mammalian cells. A single retrovirus has been constructed, which directs expression of a short hairpin RNA (shRNA) to knockdown expression of the endogenous protein of interest; a cDNA coding for a wild-type or mutant version of the same protein that also contains "silent mutations" that do not affect the protein sequence, but do make the mRNA resistant to the shRNA; and a puromycin-resistance gene to allow rapid drug selection of the virus-infected cells. Using this virus, expression of the endogenous Anti-Silencing Function 1a (ASF1a) histone chaperone has been efficiently replaced in primary human cells, by an ectopically expressed epitope-tagged version. Moreover, the virus is designed so that other shRNA and shRNA-resistant cDNA cassettes can easily be substituted, making the approach readily applicable to other protein targets.

Toxigenic serogroups O1 and O139 of Vibrio cholerae may cause cholera epidemics or pandemics. Nontoxigenic strains within these serogroups also exist in the environment, and also some may cause sporadic cases of disease. Herein, we investigate the genomic diversity among toxigenic and nontoxigenic O1 and O139 strains by comparative genomic microarray hybridization with the genome of El Tor strain N16961 as a base. Conservation of the toxigenic O1 El Tor and O139 strains is found as previously reported, whereas accumulation of genome changes was documented in toxigenic El Tor strains isolated within the 40 years of the seventh pandemic. High phylogenetic diversity in nontoxigenic O1 and O139 strains is observed, and most of the genes absent from nontoxigenic strains are clustered together in the N16961 genome. By comparing these toxigenic and nontoxigenic strains, we observed that the small chromosome of V. cholerae is quite conservative and stable, outside of the superintegron region. In contrast to the general stability of the genome, the superintegron demonstrates pronounced divergence among toxigenic and nontoxigenic strains. Additionally, sequence variation in virulence-related genes is found in nontoxigenic El Tor strains, and we speculate that these intermediate strains may have pathogenic potential should they acquire CTX prophage alleles and other gene clusters. This genome-wide comparison of toxigenic and nontoxigenic V. cholerae strains may promote understanding of clonal differentiation of V. cholerae and contribute to an understanding of the origins and clonal selection of epidemic strains.