Genomics-based Approach Research Articles

Crossing the streams: Improving data quality and integration across the One Health genomics continuum with data standards and implementation strategies.

The Canadian Genomics Research and Development Initiative for Antimicrobial Resistance (GRDI-AMR) uses a genomics-based approach to understand how health care, food production and the environment contribute to the development of antimicrobial resistance. Integrating genomics contextual data streams across the One Health continuum is challenging because of the diversity in data scope, content and structure. To better enable data harmonization for analyses, a contextual data standard was developed. However, development of standards does not guarantee their use. Implementation strategies are critical for putting standards into practice. This work focuses on the development of implementation strategies to better operationalize data standards across the Canadian federal genomics ecosystem. Results include improved understanding of complex data models that can create challenges for existing systems. Technical implementation strategies included spreadsheet-based solutions, new exchange formats, and direct standards integration into new databases. Data curation exercises highlighted common data collection and sharing issues, which informed improved practices and evaluation procedures. These new practices are contributing to improved data quality and sharing within the GRDI-AMR consortium as evidenced by publicly available datasets. The implementation strategies and lessons learned described in this work are generalizable for other standards and can be applied more broadly within other initiatives.

Genome-wide CRISPR-Cas 9 screens identify BCL family members as modulators of response to regorafenib in experimental glioma.

Registered systemic treatment options for glioblastoma patients are limited. The phase II REGOMA trial suggested an improvement of median overall survival in progressive glioblastoma by the multi-tyrosine kinase inhibitor regorafenib. This has not been confirmed by GBM AGILE. So far, regorafenib has been administered as monotherapy or as an addition to standard of care in newly diagnosed glioblastoma. Rational combination therapies involving regorafenib might be a reasonable strategy. Here, we aimed at identifying functionally-instructed combination therapies involving regorafenib. We applied a genome-wide CRISPR-Cas9-based functional genomics target discovery approach using activation and knockout screens followed by genetic, pharmacological, functional validations. Regorafenib-induced molecular alterations were assessed by RNAsequencing and DigiWest. We investigated selected functionally-instructed combination therapies in three orthotopic glioma mouse models in vivo (syngeneic SMA560/VM/Dk model and two xenograft models) and performed immunohistochemistry of post-treatment brains. We identified potential modifiers of regorafenib response including BCL2, BCL2L1, ITGB3, FOXC1, SERAC1, ARAF, and PLCE1. The combination of regorafenib with Bcl-2/Bcl-xL inhibition was superior to both monotherapies alone in vitro, ex vivo and in vivo. We identified regorafenib-induced regulations of the Bcl-2 downstream target chemokine receptor 1 (CCR1) as one potential underlying molecular mediator. Furthermore, regorafenib led to changes in the myeloid compartment of the glioma-associated microenvironment. This preclinical study uses a functional genomics-based target discovery approach with subsequent validations involving regorafenib. It serves as a biological rationale for clinical translation. Particularly, an investigation of the combination of regorafenib plus navitoclax within a clinical trial is warranted.

Genomics-based timely detection of dengue virus type I genotypes I and V in Uruguay

Genomics-based timely detection of dengue virus type I genotypes I and V in Uruguay

404 (PB392): Genomics-based approach to drug testing in novel patient-derived xenograft models of undifferentiated pleomorphic sarcoma

404 (PB392): Genomics-based approach to drug testing in novel patient-derived xenograft models of undifferentiated pleomorphic sarcoma

P10.29.A FUNCTIONALLY INSTRUCTED COMBINATION-TREATMENTS INVOLVING REGORAFENIB IN EXPERIMENTAL GLIOMA

Abstract BACKGROUND Glioblastoma are incurable primary tumors of the central nervous system. The standard of care for newly diagnosed glioblastoma outside clinical trials includes a combination of radiation therapy, alkylating chemotherapy and tumor-treating fields. Yet, for progressive glioblastoma, only lomustine is registered in Europe. The REGOMA trial (Lombardi et al., Lancet Oncol 2019) had investigated regorafenib versus lomustine in a phase 2-trial. Furthermore, the use of regorafenib in a compassionate-use-program was recently reported (Tzaridis et al., Neuro Oncol 2019) Yet, the regorafenib arm in the Glioblastoma Adaptive Global Innovative Learning Environment (GBM Agile) master protocol was stopped after an interim analysis that showed a low probability of improvement of overall survival compared with control (press release 21 September 2022). We investigated here a functional genomics-based approach to discover and validate potential modulators of response to regorafenib treatment in experimental glioma in vitro, in vivo and ex vivo. MATERIAL AND METHODS We performed CRISPR/Cas9 activation (Calabrese P65-HSF activation library) and knockout (genome-wide knockout library Brunello) screens during treatment with regorafenib, sequenced and analyzed the genomic DNA of the remaining cells. RESULTS We discovered functionally-instructed molecular hits, designed a drug library for further validation by functional assays in vitro and combination treatments in murine glioma models in vivo. CONCLUSION Taken together, we discovered and validated functionally-instructed combination treatment options involving regorafenib. Our finalized results will be presented.

Development of a transcriptome-based determination of innate immune suppressor (TDIS) assay as an in vitro test for immunotoxicity.

Immunotoxicity has been an important topic in toxicology since inadvertent exposures to xenobiotics were found to alter immune functions in humans. While rodent toxicity tests can reveal some levels of immunotoxicity, alternative methods must be developed to identify the detailed mechanisms. In this study, a method ofin vitroprediction of innate immune suppression by substances was developed using a genomics approach. The primary selection of immune suppressors was based on their ability to downregulate MCP-1, CCL3, TNF, IL-8, and IL-12p40 expression levels in lipopolysaccharide (LPS)-stimulated THP-1 cells. Among 11 substances classified as potent immune suppressors, six including dexamethasone, tacrolimus, tofacitinib, prednisolone, sodium lauryl sulfate, and benzoic acid were used to create a dataset by transcriptomics of chemical-treated THP-1 cells using bulk RNA sequencing. We selected genes that were significantly upregulated by suppressor treatment while filtering out genes also upregulated in LPS-treated THP-1 cells. We identified a 226-gene immunosuppressive gene set (ISG). Innate immune suppressor signature scores were calculated as the median expression of the ISG. In a validation dataset, the signature score predicted acyclovir, cyclosporine, and mercuric chloride as immune suppressors, while selecting genistein as a non-immune suppressor. Although more dataset integration is needed in the future, our results demonstrated the possibility and utility of a novel genomics-based approach, the transcriptome-based determination of innate immune suppressor (TDIS) assay, to evaluate innate immune suppression by different substances. This provides insight into the development of future alternative testing methods because it reflects a comprehensive genetic signature derived from multiple substances rather than one cytokine.

A Subtraction Genomics-Based Approach to Identify and Characterize New Drug Targets in Bordetella pertussis: Whooping Cough.

Bordetella pertussis is a Gram-negative bacterium known to cause pertussis or whooping cough. The disease affects the respiratory system and is contagious. Pertussis causes high mortality among infants aged less than one-year-old, although it can affect anyone of any age. Globally, 16 million cases of pertussis were reported in 2008, 95% of which were in developing nations, and approximately 195,000 children died from the disease. Under a computational subtractive genomics approach, the total proteome of a pathogen is gently trimmed down to a few potential drug targets. First, from NCBI, we obtained the pathogen proteins followed by CD hit for removal of duplicate proteins. The BLAST step was applied to find non-similar proteins, and then, we applied BLAST to these non-similar bacterial proteins with DEG to find essential bacterial proteins. After this, to find the location, these vital proteins were screened via PSORTb; the majority of proteins were in cytoplasm. The KASS server was used to determine the involvement of these proteins in the metabolic pathways of bacteria, and KEGG was applied to find the unique metabolic pathways of the pathogen. Finally, we applied BLAST to these vital, unique, and non-similar proteins with FDA-approved drug targets, and four proteins of the B. pertussis strain B1917 were identified that might be powerful drug targets. A variety of therapeutic molecules could be designed to target these proteins in order to treat infections caused by bacteria.

A Genomics-Based Semirational Approach for Expanding the Postbiotic Potential of Collagen Peptides Using Lactobacillaceae.

Food-derived bioactive peptides (BPs) have received considerable attention as postbiotics for human gut health. Here we used a genomics-based semirational approach to expand the postbiotic potential of collagen peptides (CPs) produced from probiotic fermentation. In silico digestion revealed distinct BPs embedded in fish collagen in a protease-dependent manner. Anaerobic digestion of collagen by representative Lactobacillaceae species revealed differential substrate utilization and collagen degradation patterns. Nanoliquid chromatography-mass spectrometry analysis of CPs showed that each species exhibited different cleavage patterns and unique peptide profiles. Remarkably, the 1-10 kDa CPs produced by Lacticaseibacillus paracasei showed agonistic activities toward G protein-coupled receptor 35 (GPR35). These CPs could repair intestinal epithelium through the GPR35-mediated extracellular signal-regulated protein kinase (ERK) 1/2 signaling pathway, suggesting that probiotic-aided collagen hydrolysates can serve as postbiotics for host-microbe interactions. Therefore, this study provides an effective strategy for the rapid screening of CPs for gut health in the gastrointestinal tract.

Systematic genomics-based approach for developing new therapeutics and diagnostics for esophageal cancer.

e16034 Background: As the prognosis of advanced esophageal cancer patients is still poor after standard therapies, development of new molecular therapies with minimum risk of adverse events and cancer biomarkers for companion diagnostics is eagerly awaited. Methods: We have been screening new therapeutic target molecules and biomarkers for esophageal cancers as follows; i) To identify highly expressed genes in esophageal cancers by gene expression microarray analysis, ii) To verify the target molecules for their low expression in normal tissues, iii) To validate the clinicopathological significance of their protein expression by tissue microarray covering 265 esophageal cancers, and iv) To confirm their function for the growth and/or invasive ability of esophageal cancer cells by siRNAs and gene transfection assays. Results: We identified dozens of candidate oncoproteins and selected ESOC1 (esophageal cancer-associated oncoprotein 1) that has a methyltransferase domain. We first confirmed its in vitro methyltransferase activity. Immunohistochemical analysis showed that ESOC1 positivity was detected in 68.5% of esophageal cancers and associated with tumor size. Moreover ESOC1 positivity was an independent prognostic factor for patients with esophageal cancer. Knockdown of ESOC1 expression by siRNAs suppressed growth of esophageal cancer cells. Exogenous expression of ESOC1 increased the growth of mammalian cells. Therefore, ESOC1 is likely to be a prognostic biomarker and therapeutic target for esophageal cancers. Conclusions: Systematic genomics-based approach could be useful for the selection of novel candidate cancer biomarkers and therapeutic targets for small molecules, antibodies, nucleic acid drugs, and immunotherapies.

A molecular diagnostic for Moniliophthora perniciosa, the causal agent of witches' broom disease of cacao, that differentiates it from its sister taxon Moniliophthora roreri

Moniliophthora perniciosa is a hemibiotrophic plant pathogen that causes witches' broom disease (WBD) in Theobroma , Herrania, and other plant species. This disease is one of the primary threats that reduce the yield of cacao and cupuaçu plantations in Tropical America. Thereby, there is a growing concern that the pathogen may spread to other areas. The fungus can infect flowers, pods, and stems, causing various symptoms, including stem swellings and dark brown necrotic pods. In some cases, these symptomatologies are very similar to other cacao diseases, such as the black pod ( Phytophthora spp.) and frosty pod rot ( M. roreri ) of cacao. Pathogens isolation and identification from diseased samples need expertise. Thus, the objective of this work was to develop a diagnostic guide with standard procedures with the aid of a molecular marker for M. perniciosa identification that is specific, sensitive, and cost-effective. A genomics-based approach was used to identify unique primer pairs to M. perniciosa . The specificity of 24 primer pairs in identifying M. perniciosa from pure culture gDNA was evaluated by conventional polymerase chain reaction (PCR) with approximately 400 isolates from ten fungal species of common occurrence in cacao plantations, including M. roreri . No amplicon was detected from other fungal species, confirming the high specificity and sensibility of the M. perniciosa specific markers Mp44 and Mp47 for molecular identification of M. perniciosa from pure culture. Finally, the present study provides a set of standardized WBD diagnosis procedures that associate a classical with a molecular approach by PCR-based identification of M. perniciosa . • Bioinformatics tools were used to find species-specific sequences to M. perniciosa. • Primer sets did not cross amplify other cacao plant pathogens such as M. roreri and P. palmivora. • Molecular detection of M. perniciosa by convetional PCR. • The two validated markers differentiated M. perniciosa and M. roreri .

Development of a Genomics-Based Approach To Identify Putative Hypervirulent Nontyphoidal Salmonella Isolates: Salmonella enterica Serovar Saintpaul as a Model.

ABSTRACTWhile differences in human virulence have been reported across nontyphoidal Salmonella (NTS) serovars and associated subtypes, a rational and scalable approach to identify Salmonella subtypes with differential ability to cause human diseases is not available. Here, we used NTS serovar Saintpaul (S. Saintpaul) as a model to determine if metadata and associated whole-genome sequence (WGS) data in the NCBI Pathogen Detection (PD) database can be used to identify (i) subtypes with differential likelihoods of causing human diseases and (ii) genes and single nucleotide polymorphisms (SNPs) potentially responsible for such differences. S. Saintpaul SNP clusters (n = 211) were assigned different epidemiology types (epi-types) based on statistically significant over- or underrepresentation of human clinical isolates, including human associated (HA; n = 29), non-human associated (NHA; n = 23), and other (n = 159). Comparative genomic analyses identified 384 and 619 genes overrepresented among isolates in 5 HA and 4 NHA SNP clusters most significantly associated with the respective isolation source. These genes included 5 HA-associated virulence genes previously reported to be present on Gifsy-1/Gifsy-2 prophages. Additionally, premature stop codons in 3 and 7 genes were overrepresented among the selected HA and NHA SNP clusters, respectively. Tissue culture experiments with strains representing 4 HA and 3 NHA SNP clusters did not reveal evidence for enhanced invasion or intracellular survival for HA strains. However, the presence of sodCI (encoding a superoxide dismutase), found in 4 HA and 1 NHA SNP clusters, was positively correlated with intracellular survival in macrophage-like cells. Post hoc analyses also suggested a possible difference in intracellular survival among S. Saintpaul lineages.IMPORTANCE Not all Salmonella isolates are equally likely to cause human disease, and Salmonella control strategies may unintentionally focus on serovars and subtypes with high prevalence in source populations but are rarely associated with human clinical illness. We describe a framework leveraging WGS data in the NCBI PD database to identify Salmonella subtypes over- and underrepresented among human clinical cases. While we identified genomic signatures associated with HA/NHA SNP clusters, tissue culture experiments failed to identify consistent phenotypic characteristics indicative of enhanced human virulence of HA strains. Our findings illustrate the challenges of defining hypo- and hypervirulent S. Saintpaul and potential limitations of phenotypic assays when evaluating human virulence, for which in vivo experiments are essential. Identification of sodCI, an HA-associated virulence gene associated with enhanced intracellular survival, however, illustrates the potential of the framework and is consistent with prior work identifying specific genomic features responsible for enhanced or reduced virulence of nontyphoidal Salmonella.

Very Elevated IgE, Atopy, and Severe Infection: A Genomics-Based Diagnostic Approach to a Spectrum of Diseases

Elevated IgE has been long recognized as an important clinical marker of atopy but can be seen in a myriad of conditions. The discovery of autosomal dominant STAT3 deficiency marked the first recognition of hyper-IgE syndrome (HIES) and the first primary immunodeficiency linked to elevated IgE. Since then, genomic testing has increased the number of defects with associated mutations causing hyper-IgE syndrome and atopic diseases with FLG, DOCK8, SPINK5, and CARD11, among others. A spectrum of recurrent infections and atopy are hallmarks of elevated IgE with significant phenotypic overlap between each underlying condition. As treatment is predicated on early diagnosis, genomic testing is becoming a more commonly used diagnostic tool. We present a 6-year-old male patient with markedly elevated IgE and severe atopic dermatitis presenting with staphylococcal bacteremia found to have a heterozygous variant in FLG (p.S3247X) and multiple variants of unknown significance in BCL11B, ZAP70, LYST, and PTPRC. We review the genetic defects underpinning elevated IgE and highlight the spectrum of atopy and immunodeficiency seen in patients with underlying mutations. Although no one mutation is completely causative of the constellation of symptoms in this patient, we suggest the synergism of these variants is an impetus of disease.

Discovery of daspyromycins A and B, 2-aminovinyl-cysteine containing lanthipeptides, through a genomics-based approach

Lanthipeptides are one of the largest groups of ribosomally synthesized and post-translationally modified peptides (RiPPs) and are characterized by the presence of lanthionine (Lan) or methyllanthionine residues (MeLan). Only very few lanthipeptides contain a C-terminal 2-aminovinyl-cysteine (AviCys) motif, but all of them show potent antibacterial activities. Recent advances of genome sequencing led to the rapid accumulation of new biosynthetic gene clusters (BGCs) for lanthipeptides. In this study, through our genome mining strategy, we found the AviCys containing lanthipeptides are widespread in the bacterial kingdom. A lanthipeptide-type biosynthetic gene cluster was identified from public bacterial genome database. Two new lanthipeptides, daspyromycins A and B (1 and 2) containing AviCys motif, along with two degraded products, daspyromycins C and D (3 and 4), were obtained after heterologous expression of the gene cluster in Streptomyces albus J1074. Daspyromycins A and B showed potent antimicrobial activity against a spectrum of Gram-positive and -negative bacteria including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE).

PseudoChecker: an integrated online platform for gene inactivation inference.

The rapid expansion of high-quality genome assemblies, exemplified by ongoing initiatives such as the Genome-10K and i5k, demands novel automated methods to approach comparative genomics. Of these, the study of inactivating mutations in the coding region of genes, or pseudogenization, as a source of evolutionary novelty is mostly overlooked. Thus, to address such evolutionary/genomic events, a systematic, accurate and computationally automated approach is required. Here, we present PseudoChecker, the first integrated online platform for gene inactivation inference. Unlike the few existing methods, our comparative genomics-based approach displays full automation, a built-in graphical user interface and a novel index, PseudoIndex, for an empirical evaluation of the gene coding status. As a multi-platform online service, PseudoChecker simplifies access and usability, allowing a fast identification of disruptive mutations. An analysis of 30 genes previously reported to be eroded in mammals, and 30 viable genes from the same lineages, demonstrated that PseudoChecker was able to correctly infer 97% of loss events and 95% of functional genes, confirming its reliability. PseudoChecker is freely available, without login required, at http://pseudochecker.ciimar.up.pt.

Cancer genomics-based screening of new therapeutic targets and biomarkers for esophageal cancer.

e16514 Background: Since the number of esophageal cancer patients who show objective response to standard therapies is still small, development of new anti-cancer agents with minimum risk of adverse events and highly precise molecular biomarkers is eagerly awaited. Methods: We have been screening novel therapeutic targets and their companion diagnostics for esophageal cancers as follows; i) To identify up-regulated genes in esophageal cancers by the gene expression profile analysis, ii) To verify the candidate genes for their low expression in normal tissues, iii) To validate the clinicopathological significance of their protein expression by tissue microarray covering 265 esophageal cancers, and iv) To verify their function for the growth of the esophageal cancer cells by siRNAs and gene transfection assays. Results: We identified dozens of candidate oncoproteins and selected a metyltransferase ESOC1 (esophageal cancer-associated oncoprotein 1). Immunohistochemical analysis revealed that ESOC1 positivity was observed in 68.5% of esophageal cancers and associated with tumor size. Moreover ESOC1 expression was an independent prognostic factor for esophageal cancer patients. Suppression of ESOC1 expression by its siRNAs inhibited growth of esophageal cancer cell lines. Introduction of ESOC1 increased the growth activity of mammalian cells, suggesting that ESOC1 is likely to be a prognostic biomarker and therapeutic target for esophageal cancers. Conclusions: Cancer genomics-based approach could be useful for the development of new cancer biomarkers as well as therapeutic targets for small molecules, antibodies, nucleic acid drugs, and immunotherapies.

Genome-based analysis for the bioactive potential of Streptomyces yeochonensis CN732, an acidophilic filamentous soil actinobacterium

BackgroundAcidophilic members of the genus Streptomyces can be a good source for novel secondary metabolites and degradative enzymes of biopolymers. In this study, a genome-based approach on Streptomyces yeochonensis CN732, a representative neutrotolerant acidophilic streptomycete, was employed to examine the biosynthetic as well as enzymatic potential, and also presence of any genetic tools for adaptation in acidic environment.ResultsA high quality draft genome (7.8 Mb) of S. yeochonensis CN732 was obtained with a G + C content of 73.53% and 6549 protein coding genes. The in silico analysis predicted presence of multiple biosynthetic gene clusters (BGCs), which showed similarity with those for antimicrobial, anticancer or antiparasitic compounds. However, the low levels of similarity with known BGCs for most cases suggested novelty of the metabolites from those predicted gene clusters. The production of various novel metabolites was also confirmed from the combined high performance liquid chromatography-mass spectrometry analysis. Through comparative genome analysis with related Streptomyces species, genes specific to strain CN732 and also those specific to neutrotolerant acidophilic species could be identified, which showed that genes for metabolism in diverse environment were enriched among acidophilic species. In addition, the presence of strain specific genes for carbohydrate active enzymes (CAZyme) along with many other singletons indicated uniqueness of the genetic makeup of strain CN732. The presence of cysteine transpeptidases (sortases) among the BGCs was also observed from this study, which implies their putative roles in the biosynthesis of secondary metabolites.ConclusionsThis study highlights the bioactive potential of strain CN732, an acidophilic streptomycete with regard to secondary metabolite production and biodegradation potential using genomics based approach. The comparative genome analysis revealed genes specific to CN732 and also those among acidophilic species, which could give some insights into the adaptation of microbial life in acidic environment.

TARGETED RESEQUENCING OF WETLAND SEDIMENT AS A TOOL FOR AVIAN INFLUENZA VIRUS SURVEILLANCE

Surveillance methods for avian influenza virus (AIV) based upon collecting and testing samples from individual wild birds have several significant limitations primarily related to the difficulties associated with obtaining samples. Because AIVs are shed in waterfowl feces, the use of environmental substrates where waterfowl feces accumulate may overcome some of these limitations. However, these substrates are difficult to analyze using traditional diagnostic techniques, such as virus culture and PCR, because of virus inactivation, RNA degradation, low concentration of target RNA, microbial complexity, presence of inhibitory substances, and other factors. We investigated the use of a genomics-based approach called targeted resequencing to detect and characterize AIVs in wetland sediments during the 2014-15 North American highly pathogenic avian influenza outbreak. We identified AIV in 20.6% (71/345) sediment samples obtained from wetlands (n=15) and outdoor waterbodies on AIV-infected poultry farms (n=10) in British Columbia, Canada (the first area affected during the outbreak). Thirteen hemagglutinin (HA) and nine neuraminidase (NA) subtypes were detected, including H5, N1, and N8 sequences that clustered with other sequences associated with the North American outbreak. Additionally, as many as eight HA and eight NA subtypes could be detected in a single sediment sample. This proof-of-concept study shows the potential utility of sediment sampling coupled with genomics-based analysis as a tool for AIV surveillance.

A Toxicity Screening Approach to Identify Bacteriophage-Encoded Anti-Microbial Proteins.

The rapid emergence of antibiotic resistance among many pathogenic bacteria has created a profound need to discover new alternatives to antibiotics. Bacteriophages, the viruses of microbes, express special proteins to overtake the metabolism of the bacterial host they infect, the best known of which are involved in bacterial lysis. However, the functions of majority of bacteriophage encoded gene products are not known, i.e., they represent the hypothetical proteins of unknown function (HPUFs). In the current study we present a phage genomics-based screening approach to identify phage HPUFs with antibacterial activity with a long-term goal to use them as leads to find unknown targets to develop novel antibacterial compounds. The screening assay is based on the inhibition of bacterial growth when a toxic gene is expression-cloned into a plasmid vector. It utilizes an optimized plating assay producing a significant difference in the number of transformants after ligation of the toxic and non-toxic genes into a cloning vector. The screening assay was first tested and optimized using several known toxic and non-toxic genes. Then, it was applied to screen 94 HPUFs of bacteriophage φR1-RT, and identified four HPUFs that were toxic to Escherichia coli. This optimized assay is in principle useful in the search for bactericidal proteins of any phage, and also opens new possibilities to understanding the strategies bacteriophages use to overtake bacterial hosts.

Abstract 526: A Transcriptomic Analysis of Cardiac Myofibroblasts Reveals Novel Anti-fibrotic and Immunomodulatory Genes Modulated by CDC-Derived Exosomes

Background: Cardiac fibroblasts are the most prevalent cell in the heart and play an important role in both the healthy and diseased myocardium. Following myocardial infarction, TGF-β secretion promotes a phenotype transition of fibroblasts to activated myofibroblasts. We have previously shown that CDC-derived exosomes promote cardiomyocyte proliferation and survival, stimulate angiogenesis, and polarize cardiac macrophages to a unique Arg1-high phenotype. We hypothesized that CDC-derived exosomes modulate the gene expression of myofibroblasts to attenuate fibrosis in the remodeling heart. Methods: We took a genomics-based approach to investigate CDC-exosome mediated differential gene expression and regulation of operant signaling pathways in TGF-β induced ventricular myofibroblasts. Cardiac fibroblasts were isolated from C57BL/6 mice (n=3) using a Langendorff-free method of in situ enzymatic dissociation. Cells were plated at 1x10 5 cells/mL, serum depleted to induce cell cycle arrest and exposed to 4 different conditions: media alone, TGF-β (4 ng/ml) “myofibroblasts”, CDC-exosomes (20 μg/ml), or TGF-β and CDC-exosomes. After 24 hours, RNA was extracted and analyzed by RNA-seq. Results/Conclusions: Gene ontology analysis showed attenuation of a pro-fibrotic gene expression profile in the TGF-β + CDC-exo group relative to TGF-β treatment alone. Genes involved in blunting of the pro-fibrotic response ( Mmp14 and Qsox1) , as well those implicated in attenuating the transition of fibroblasts to myofibroblasts ( Gsto1 ) were also upregulated in CDC-exosome treated myofibroblasts (edgeR; FDR-corrected p<0.05). Interesting, we found a highly significant enrichment of genes involved in macrophage migration ( Kars , Mmp14 , and Tnfsf18 ) in the CDC-exosome treated myofibroblasts relative to the TGF-β group (GO:1905523, p=8.42x10 -6 ). These data suggest active cross talk between myofibroblasts and macrophages and highlight a novel immunomodulatory role of CDC-derived exosomes on myofibroblasts post MI.

Integrated genomics-based approach to identify new therapeutic targets and cancer biomarkers for lung cancer.

e13019 Background: Because the number of lung cancer patients who show good response to standard therapies is still limited, development of new anti-cancer agents with minimum risk of adverse effects and highly sensitive molecular biomarkers is urgently required. Methods: We have been screening novel therapeutic targets and their companion biomarkers for lung cancer as follows; i) To identify up-regulated genes in lung cancers by the gene microarray analysis, ii) To verify the candidate genes for their low expression in normal organs, iii) To validate the clinicopathological significance of their protein expression by tissue microarray covering hundreds of lung cancers, and iv) To verify their function for the growth of lung cancer cells by siRNAs. Results: We identified dozens of candidate oncoproteins and selected a serine/threonine kinase LASK2 (lung cancer-associated kinase 2). Immunohistochemical analysis showed that strong LASK2 positivity was an independent prognostic factor for non-small cell lung cancer patients (P < 0.0001). Suppression of LASK2 expression by its siRNAs inhibited proliferation of lung cancer cells. Introduction of LASK2 in mammalian cells also enhanced cellular growth in vitro and in mice model. Induction of LASK2 appeared to increase the levels of phosphorylation of oncogenic signal proteins for lung cancer. The data indicate that LASK2 is a prognostic biomarker and therapeutic target for lung cancers. Conclusions: Integrated genomics-based approach could facilitate the development of new cancer biomarkers as well as therapeutic targets for small molecules, monoclonal antibodies, nucleic acid drugs, and immunotherapies.