Comparative RNA-seq Research Articles

Comparative RNA-seq analysis reveals a critical role for ethylene in rose (Rosa hybrida) susceptible response to Podosphera pannosa.

Rose is one of the most important ornamental flowers, accounting for approximately one-third of the world’s cut flower market. Powdery mildew caused by Podosphera pannosa is a devastating fungal disease in rose, mainly infecting the young leaves and causing serious economic losses. Therefore, a study on the mechanism of the fungus infecting the rose leaves and the possibility to improve resistance hereby is interesting and meaningful. Accordingly, we conducted transcriptome sequencing of rose leaves infected by P. pannosa at different time points to reveal the molecular mechanism of resistance to powdery mildew. The high-quality reads were aligned to the reference genome of Rosa chinensis, yielding 51,230 transcripts. A total of 1,181 differentially expressed genes (DEGs) were identified in leaves during P. pannosa infection at 12, 24, and 48 hpi. The transcription factors of ERF, MYB, bHLH, WRKY, etc., family were identified among DEGs, and most of them were downregulated during P. pannosa infection. The Kyoto Encyclopedia of Genes and Genomes analysis showed that the hormone signal transduction pathway, especially ethylene signal-related genes, was consistently showing a downregulated expression during powdery mildew infection. More importantly, exogenous 1-MCP (inhibitor of ethylene) treatment could improve the rose leaves’ resistance to P. pannosa. In summary, our transcriptome of rose leaf infected by powdery mildew gives universal insights into the complex gene regulatory networks mediating the rose leaf response to P. pannosa, further demonstrating the positive role of 1-MCP in resistance to biotrophic pathogens.

Genome resequencing and transcriptome profiling reveal molecular evidence of tolerance to water deficit in barley

IntroductionFrequent climate change-induced drought events are detrimental environmental stresses affecting global crop production and ecosystem health. Several efforts have facilitated crop breeding for resilient varieties to counteract stress. However, progress is hampered due to the complexity of drought tolerance; a greater variety of novel genes are required across varying environments. Tibetan annual wild barley is a unique and precious germplasm that is well adapted to abiotic stress and can provide elite genes for crop improvement in drought tolerance. ObjectivesTo identify the genetic basis and unique mechanisms for drought tolerance in Tibetan wild barley. MethodsWhole genome resequencing and comparative RNA-seq approaches were performed to identify candidate genes associated with drought tolerance via investigating the genetic diversity and transcriptional variation between cultivated and Tibetan wild barley. Bioinformatics, population genetics, and gene silencing were conducted to obtain insights into ecological adaptation in barley and functions of key genes. ResultsOver 20 million genetic variants and a total of 15,361 significantly affected genes were identified in our dataset. Combined genomic, transcriptomic, evolutionary, and experimental analyses revealed 26 water deficit resilience-associated genes in the drought-tolerant wild barley XZ5 with unique genetic variants and expression patterns. Functional prediction revealed Tibetan wild barley employs effective regulators to activate various responsive pathways with novel genes, such as Zinc-Induced Facilitator-Like 2 (HvZIFL2) and Peroxidase 11 (HvPOD11), to adapt to water deficit conditions. Gene silencing and drought tolerance evaluation in a natural barley population demonstrated that HvZIFL2 and HvPOD11 positively regulate drought tolerance in barley. ConclusionOur findings reveal functional genes that have been selected across barley’s complex history of domestication to thrive in water deficit environments. This will be useful for molecular breeding and provide new insights into drought-tolerance mechanisms in wild relatives of major cereal crops.

Comparative RNA-Seq analysis reveals insights in Salmonella disease resistance of chicken; and database development as resource for gene expression in poultry

Salmonella, one of the major infectious diseases in poultry, causes considerable economic losses in terms of mortality and morbidity, especially in countries that lack effective vaccination programs. Besides being resistant to diseases, indigenous chicken breeds are also a potential source of animal protein in developing countries. For understanding the disease resistance, an indigenous chicken line Kashmir faverolla, and commercial broiler were selected. RNA-seq was performed after challenging the chicken with Salmonella Typhimurium. Comparative differential expression results showed that following infection, a total of 3153 genes and 1787 genes were differentially expressed in the liver and spleen, respectively. The genes that were differentially expressed included interleukins, cytokines, NOS2, Avβ-defensins, toll-like receptors, and other immune-related gene families. Most of the genes and signaling pathways involved in the innate and adaptive immune responses against bacterial infection were significantly enriched in the Kashmir faverolla. Pathway analysis revealed that most of the enriched pathways were MAPK signaling pathway, NOD-like receptor signaling pathway, TLR signaling pathway, PPAR signaling pathway, endocytosis, etc. Surprisingly some immune-related genes like TLRs were upregulated in the susceptible chicken breed. On postmortem examination, the resistant birds showed small lesions in the liver compared to large necrotic lesions in susceptible birds. The pathological manifestations and RNA sequencing results suggest a balancing link between resistance and infection tolerance in Kashmir faverolla. Here we also developed an online Poultry Infection Database (https://skuastk.org/pif/index.html), the first publicly available gene expression resource for disease resistance in chickens. The available database not only shows the data for gene expression in chicken tissues but also provides quick search, visualization and download capacity.

Comparative transcriptome analysis of compatible and incompatible Brassica napus-Xanthomonas campestris interactions.

Black rot caused by the vascular pathogenic bacterium Xanthomonas campestris pv. campestris (Xcc) is widespread in Brassicaceae plants and an infectious disease that causes large yield losses in oil seed rape (Brassica napus L.). Improvement of resistance through breeding is a crucial strategy to prevent black rot disease in B. napus, but presently hampered by insufficient understanding of Xcc-Brassica interactions. This study compares two EMS-mutagenized B. napus lines that show contrasting resistance levels to their susceptible progenitor. Patterns of differential gene expression between these B. napus lines were evaluated at three time points post inoculation by comparative RNA-seq analysis. In line with the observed disease phenotypes, the susceptible line ZS9mXccS-1 displayed a steady amount of differentially expressed genes (DEGs) at different time points of infection, whereas the resistant line ZS9mXccR-1 displayed a gradual increase in DEGs throughout the course of infection. Weighted gene co-expression network analysis (WGCNA) pinpointed multiple defense-related hub genes with potential central roles in immunity, including the cell surface receptor genes CRK11 and BIR1, and the associated downstream regulatory genes WRKY11 and PBL30. KEGG analysis of DEGs belonging to two distinct co-expression modules revealed enriched pathways associated with defense, including Ca2+-signaling, receptor-mediated immunity, and phytohormone balance. Taken together, our comparative transcriptome analysis provides new avenues to unravel the mechanisms underlying black rot resistance in B. napus.

Comparative RNA-seq analysis and ceRNA network of genistein-treated GT1-7 neurons

Comparative RNA-seq analysis and ceRNA network of genistein-treated GT1-7 neurons

Comparative RNA-Seq analysis unravels molecular mechanisms regulating therapeutic properties in the grains of traditional rice Kavuni

Developing rice varieties with enhanced levels of functional bioactives is an important intervention for achieving food and nutritional security in Asia where rice is the staple food and Type II diabetes incidences are higher. The present study was aimed at dissecting out the molecular events underlying the accumulation of bio active compounds in pigmented traditional rice Kavuni. Comparative transcriptome profiling in the developing grains of Kavuni and a white rice variety ASD 16 generated 37.7 and 29.8 million reads respectively. Statistical analysis identified a total of 9177 exhibiting significant differential expression (DEGs) between the grains of Kavuni and ASD 16. Pathway mapping of DEGs revealed the preferential up-regulation of genes involved in the biosynthesis of amylose and dietary fibres in Kavuni accounting for its low glycemic index (GI). Transcripts involved in the biosynthesis of carotenoids, flavonoids, anthocyanins, phenolic acids and phenylpropanoids were also found to be up-regulated in the grains of Kavuni. This study identified up-regulation of key transcripts involved in the accumulation of phenolic acids having potential for inhibiting major hydrolytic enzymes α-amylase and α-glucosidase and thus accounting for the slow digestibility leading to low GI. Overall, this study has identified molecular targets for the genetic manipulation of anti-diabetic and anti-oxidant traits in rice.

Screening and Verification of Photosynthesis and Chloroplast-Related Genes in Mulberry by Comparative RNA-Seq and Virus-Induced Gene Silencing.

Photosynthesis is one of the most important factors in mulberry growth and production. To study the photosynthetic regulatory network of mulberry we sequenced the transcriptomes of two high-yielding (E1 and E2) and one low-yielding (H32) mulberry genotypes at two-time points (10:00 and 12:00). Re-annotation of the mulberry genome based on the transcriptome sequencing data identified 22,664 high-quality protein-coding genes with a BUSCO-assessed completeness of 93.4%. A total of 6587 differentially expressed genes (DEGs) were obtained in the transcriptome analysis. Functional annotation and enrichment revealed 142 out of 6587 genes involved in the photosynthetic pathway and chloroplast development. Moreover, 3 out of 142 genes were further examined using the VIGS technique; the leaves of MaCLA1- and MaTHIC-silenced plants were markedly yellowed or even white, and the leaves of MaPKP2-silenced plants showed a wrinkled appearance. The expression levels of the ensiled plants were reduced, and the levels of chlorophyll b and total chlorophyll were lower than those of the control plants. Co-expression analysis showed that MaCLA1 was co-expressed with CHUP1 and YSL3; MaTHIC was co-expressed with MaHSP70, MaFLN1, and MaEMB2794; MaPKP2 was mainly co-expressed with GH9B7, GH3.1, and EDA9. Protein interaction network prediction revealed that MaCLA1 was associated with RPE, TRA2, GPS1, and DXR proteins; MaTHIC was associated with TH1, PUR5, BIO2, and THI1; MaPKP2 was associated with ENOC, LOS2, and PGI1. This study offers a useful resource for further investigation of the molecular mechanisms involved in mulberry photosynthesis and preliminary insight into the regulatory network of photosynthesis.

Inhibition of root growth by alkaline salts due to disturbed ion transport and accumulation in Leymus chinensis

Soil alkalization severely affects plant growth and agricultural production. Compared with neutral salts, alkaline salts cause a combined stress of ion toxicity and high pH in plants. The alkali-resistant halophyte Leymus chinensis is a dominant grass growing on soda (NaHCO3 and Na2CO3, pH > 9) saline-alkali soil in northeast China. However, the molecular mechanism of alkaline salt tolerance in L. chinensis roots remains unclear. In this study, the seedling phenotype, ion accumulation, root ion dynamics, and gene expression profiles in L. chinensis under neutral salt (100 mM NaCl), alkaline salt (100 mM NaHCO3 and 50 mM Na2CO3), and high pH (pH = 10) stresses were investigated and compared. Phenotypic analysis showed that alkaline salt more severely inhibited the growth of seedling roots compared to neutral salt and high pH. Ion content detection revealed that alkaline salt-treated seedlings had higher Na+ accumulation and lower nutrient content compared to neutral salt and high pH-treated seedlings. Furthermore, the non-invasive micro-test showed that alkaline salt-induced high pH conditions perturb ion flux in seedling roots, which may be associated with impaired nutrient ion uptake and aggravated Na+ accumulation. Moreover, using full-length transcriptome combined with comparative RNA-seq, the identified alkaline salt-specific response genes were most significantly enriched to the transporter activity term, which contained a large number of genes encoding transporters of nutrient ions. Our results provide new insights into the unique response mechanism of L. chinensis roots to alkaline salt.

A novel aldo-keto reductase gene is involved in 6'-deoxychalcone biosynthesis in dahlia (Dahlia variabilis).

A novel gene belonging to the aldo-keto reductase 13 family is involved in isoliquiritigenin biosynthesis in dahlia. The yellow pigments of dahlia flowers are derived from 6'-deoxychalcones, which are synthesized via a two-step process, involving the conversion of 3-malonyl-CoA and 4-coumaloyl-CoA into isoliquiritigenin in the first step, and the subsequent generation of butein from isoliquiritigenin. The first step reaction is catalyzed by chalcone synthase (CHS) and aldo-keto reductase (AKR). AKR has been implicated in the isoflavone biosynthesis in legumes, however, isolation of butein biosynthesis related AKR members are yet to be reported. A comparative RNA-seq analysis between two dahlia cultivars, 'Shukuhai' and its butein-deficient lateral mutant 'Rinka', was used in this study to identify a novel AKR gene involved in 6'-deoxychalcone biosynthesis. DvAKR1 encoded a AKR 13 sub-family protein with significant differential expression levels, and was phylogenetically distinct from the chalcone reductases, which belongs to the AKR 4A sub-family in legumes. DNA sequence variation and expression profiles of DvAKR1 gene were correlated with 6'-deoxychalcone accumulation in the tested dahlia cultivars. A single over-expression analysis of DvAKR1 was not sufficient to initiate the accumulation of isoliquiritigenin in tobacco, in contrast, its co-overexpression with a chalcone 4'-O-glucosyltransferase (Am4'CGT) from Antirrhinum majus and a MYB transcription factor, CaMYBA from Capsicum annuum successfully induced isoliquiritigenin accumulation. In addition, DvAKR1 homologous gene expression was detected in Coreopsideae species accumulating 6'-deoxychalcone, but not in Asteraceae species lacking 6'-deoxychalcone production. These results not only demonstrate the involvement of DvAKR1 in the biosynthesis of 6'-deoxychalcone in dahlia, but also show that 6'-deoxychalcone occurrence in Coreopsideae species developed evolutionarily independent from legume species.

The Functional Role and Regulatory Mechanism of Bromodomain-Containing Protein 9 in Human Uterine Leiomyosarcoma.

Uterine leiomyosarcoma (uLMS) is the most common type of uterine sarcoma associated with poor prognosis, high rates of recurrence, and metastasis. There is currently limited information about uLMS molecular mechanisms of origin and development. Bromodomain (BRD)-containing proteins are involved in many biological processes, most notably epigenetic regulation of transcription, and BRD protein dysfunction has been linked to many diseases including tumorigenesis. However, the role of BRD proteins in the pathogenesis of uLMS is unknown. Here, we show for the first time that BRD9 is aberrantly overexpressed in uLMS tissues compared to adjacent myometrium. BRD9 expression is also upregulated in uLMS cell lines compared to benign uterine fibroid and myometrium cell lines. Inhibition of BRD9 using the specific inhibitor (TP-472) suppressed uLMS cell proliferation via inducing apoptosis and cell cycle arrest. To further characterize the mechanistic basis for TP-472 inhibition of uLMS cell growth, we performed a comparative RNA-seq analysis of vehicle-treated and TP-472-treated uLMS cells (n = 4 each). Bioinformatics analysis revealed that TP-472 treatment distinctly altered the uLMS cell transcriptome. Gene set enrichment analysis identified critical pathways altered by BRD9 inhibition, including interferon-alpha response, KRAS signaling, MYC targets, TNF-a signaling via NFkB, and MTORC1 signaling. Parsimonious gene correlation network analysis identified nine enriched modules, including cell cycle and apoptosis modules. Moreover, the ENCODE Histone Modifications gene set and TargetScan microRNA analysis in Enrichr suggested that TP-472-induced BRD9 inhibition may alter the uLMS cell transcriptome by reprograming the oncogenic epigenome and inducing miRNA-mediated gene regulation. Therefore, BRD9 constitutes a specific vulnerability in malignant uLMS, and targeting non-BET BRD proteins in uLMS may provide a promising and novel strategy for treating patients with this aggressive uterine cancer.

Rye B chromosomes differently influence the expression of A chromosome–encoded genes depending on the host species

The B chromosome (B) is a dispensable component of the genome in many species. To evaluate the impact of Bs on the transcriptome of the standard A chromosomes (A), comparative RNA-seq analyses of rye and wheat anthers with and without additional rye Bs were conducted. In both species, 5–6% of the A-derived transcripts across the entire genomes were differentially expressed in the presence of 2Bs. The GO term enrichment analysis revealed that Bs influence A chromosome encoded processes like “gene silencing”; “DNA methylation or demethylation”; “chromatin silencing”; “negative regulation of gene expression, epigenetic”; “post-embryonic development”; and “chromosome organization.” 244 B chromosome responsive A-located genes in + 2B rye and + B wheat shared the same biological function. Positively correlated with the number of Bs, 939 and 1391 B-specific transcripts were identified in + 2B and + 4B wheat samples, respectively. 85% of B-transcripts in + 2B were also found in + 4B transcriptomes. 297 B-specific transcripts were identified in + 2B rye, and 27% were common to the B-derived transcripts identified in + B wheat. Bs encode mobile elements and housekeeping genes, but most B-transcripts were without detectable similarity to known genes. Some of these genes are involved in cell division-related functions like Nuf2 and might indicate their importance in maintaining Bs. The transcriptome analysis provides new insights into the complex interrelationship between standard A chromosomes and supernumerary B chromosomes.

Poster 101: Human pluripotent stem cell-derived chondroprogenitors committed to form distinct types of chondrocytes for the future cartilage tissue engineering

Objectives:Chondrogenic mesenchymal cells have been developed from human pluripotent stem cells (hPSCs) as a human model of embryonic chondrogenesis. These cells are expandable in the presence of fibroblast growth factor (FGF), and the types of cartilage they tend to generate have been characterized in vitro and in vivo. Resistance to endochondral ossification is one of the most important characteristics of the tissue engineered cartilage for cartilage regenerative therapy. Despite some reports on directed derivation of endochondral ossification resistant chondrocytes from hPSCs, their precursors (chondrogenic mesenchymal cells) have been poorly characterized. We have developed methods to generate two types of chondrogenic mesenchymal cells that lead to cartilage pellets expressing high or very low levels of hypertrophic chondrocyte markers in vitro. These cartilage pellets, when transplanted, became either fully mineralized bony tissue or remained unmineralized, respectively. Here, we present results from cell-type analyses of the two types of mesenchymal cells using the genome-wide RNA-seq technology, aiming to provide mechanistical insights into how the two types of mesenchymal cells prefer to form different types of cartilage.Methods:Human PSC lines were differentiated toward paraxial mesodermal progeny in a chemically-defined medium (CDM) as previously described (1, 2), and mesodermal cells were isolated by cell sorting (2, 3). The isolated cells were then cultured in CDM containing FGF2, platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-beta) inhibitor and glycogen synthase kinase (GSK) inhibitor (1), which were then subjected to CDM containing FGF2, TGFbeta inhibitor and GSK inhibitor (FSbC medium) as well as to CDM containing PDGF and BMP inhibitor (PN medium) to generate mesenchymal cells of distinct size and shape. The resulting cells were subjected to RT-PCR and bulk RNA-seq analyses for cell-type determination. Chondrogenesis was done by pellet culture using PDGF, TGF-beta3 and bone morphogenetic proteins, as described previously (1, 2). Some cartilage pellets were fixed, sectioned, and stained with Toluidine Blue and von Kossa, or immunostained with anti-collagen type I, II and X (COL1, 2, 10) antibodies. DNA, RNA, and sulfated glycosaminoglycan were isolated and quantified from unfixed cartilage pellets. Some cartilage pellets were also subcutaneously transplanted into NSG (severely immunocompromised) mice and the recovered cartilage pellets were similarly analyzed. All the methods involved were previously described in detail (1, 2).Results:The PN cultured mesenchymal cells (Fig. 1) developed COL2+ cartilage pellets that expressed COL10A1 at 10 to 100-fold lesser levels and PRG4 (Lubricin gene) at higher levels than those derived from the FSbC cultured mesenchymal cells, and were resistant to endochondral ossification after subcutaneous transplantation for 8 weeks (Fig. 2) (1, 2). In contrast, the FSbC cultured mesenchymal cells (Fig. 1) developed cartilage pellets that expressed COL10A1 at higher levels than those derived from the PN cultured mesenchymal cells, and were readily mineralized after subcutaneous transplantation (Fig. 2) (1, 2). The comparative RNA-seq analyses of the two mesenchymal cell populations showed that the FSbC cells resembled ectomesenchymal cells derived from neural crest progeny of hPSCs (1, 2) and the PN cells consisted of cells resembling mesenchymal stromal cells and ligament/tendon progenitors (Fig. 3).Conclusions:From hPSCs, chondrogenic mesenchymal cells of distinct chondrogenesis activity can be generated in vitro in a controlled fashion. One type leads to chondrocytes prone to endochondral ossification, and the other type leads to rather stable cartilage that seems to resist endochondral ossification. Our results suggest that the fate of hPSC-derived chondrocytes can be controlled at the mesenchymal cell (i.e., chondrocyte precursor)-stage, which seems to be dependent on how the mesenchymal cells are generated from hPSCs and maintained in culture. Further biological studies on these cells will not only enable hPSC-derived chondrocytes/chondroprogenitors to be used directly for cartilage regenerative therapy, but also may lead to a critical mechanism that allows therapeutically relevant adult chondrogenic cells such as skeletal stem cells to reproducibly regenerate hyaline permanent cartilage during cartilage repair.Fig. 1Fig. 2Fig. 3

Transcriptomic and proteomic profiling of peptidase expression in Fasciola hepatica eggs developing at host’s body temperature

Fasciola hepatica is a global parasite of livestock which also causes a neglected zoonosis in humans. The parasite’s communication with the host during its complicated lifecycle is based on an ingenious enzymatic apparatus which includes a variety of peptidases. These enzymes are implicated in parasite migration, pathogenesis of the disease, and modification of host immune response. Although the dynamics of proteolytic machinery produced by intra-mammalian F. hepatica life stages has been previously investigated in great detail, peptidases of the eggs so far received little scientific attention. In this study, we performed a comparative RNA-seq analysis aimed at identification of peptidases expressed in F. hepatica eggs, cultured at 37 °C to represent gall bladder retained eggs, for different time periods and employed mass spectrometry in order to identify and quantify peptidases translated in F. hepatica egg lysates. We demonstrated that F. hepatica eggs undergo significant molecular changes when cultured at the physiological temperature of the definitive host. Egg transcriptome is subject to numerous subtle changes while their proteome is even more variable. The peptidase profile is considerably modified on both transcriptome and proteome level. Finally, we measured and classified proteolytic activities in extracts from F. hepatica eggs using a library of fluorogenic substrates and peptidase class-selective inhibitors. Activities of threonine peptidases were detected constantly, while the cysteine peptidases prevailing in freshly laid eggs are substituted by aspartic peptidase and metallopeptidase activities in the later stages of egg development.

Identifying plant genes shaping microbiota composition in the barley rhizosphere

A prerequisite to exploiting soil microbes for sustainable crop production is the identification of the plant genes shaping microbiota composition in the rhizosphere, the interface between roots and soil. Here, we use metagenomics information as an external quantitative phenotype to map the host genetic determinants of the rhizosphere microbiota in wild and domesticated genotypes of barley, the fourth most cultivated cereal globally. We identify a small number of loci with a major effect on the composition of rhizosphere communities. One of those, designated the QRMC-3HS, emerges as a major determinant of microbiota composition. We subject soil-grown sibling lines harbouring contrasting alleles at QRMC-3HS and hosting contrasting microbiotas to comparative root RNA-seq profiling. This allows us to identify three primary candidate genes, including a Nucleotide-Binding-Leucine-Rich-Repeat (NLR) gene in a region of structural variation of the barley genome. Our results provide insights into the footprint of crop improvement on the plant’s capacity of shaping rhizosphere microbes.

Abstract 2336: Pan-cancer analysis of NAMPT inhibitors reveals unique sensitivities to multiple NAMPT inhibitors in several cancer types

Abstract Nicotinamide phosphoribosyltransferase (NAMPT) plays a critical role in cellular metabolism and as such has been an active target for cancer drug therapy. NAMPT inhibition, via small molecule NAMPT inhibitors, disrupts cellular energy metabolism as well as other NAD+-dependent processes and has been shown to suppress cancer cell proliferation. The early clinical translation of NAMPT inhibitors has been hindered by toxicity, therefore the successful development of newer classes of NAMPT inhibitors will depend on identifying cancers with unique sensitivity to, and molecular biomarkers of susceptibility to NAMPT inhibition. Through our pan-cancer screens using several different NAMPT inhibitors on molecularly characterized cancer cell line panels, we have identified specific cancer types and subtypes that are consistently sensitive to NAMPT inhibition. In order to determine the efficacy of NAMPT inhibitors in different cancer cells, we performed proliferation assays on a large and fully characterized panel of human cancer cell lines (n=496) for response to several NAMPT inhibitors including FK866, KPT9274 and LSN3154567. Briefly, cells were treated with six dilutions of these inhibitors in duplicate, counted following a 6-day treatment window and IC50 values were determined for each cell line. Reverse-phase protein array (RPPA) analysis, comparative genomic hybridization (CGH), point mutation and RNAseq data for each cell line were then queried for correlation with NAMPT sensitivity/resistance. Proliferation assays indicate a strong correlation between sensitive and resistant cancer cell types to all three compounds despite their different mechanisms of action. Most notably, acute myeloid leukemia (AML), ovarian cancer, Ewing’s sarcoma and small cell lung cancer cells (SCLC) consistently demonstrated sensitivity to treatment with NAMPT inhibitors. Additional analysis revealed several baseline genomic and proteomic markers that were strongly associated with response to NAMPT inhibition. Our screening process provides a comprehensive overview of the cancer types and subtypes that demonstrate sensitivity to NAMPT inhibition. Furthermore, the molecular predictors of response identified in our screen may ultimately be useful in developing diagnostic tools for enrollment in biomarker-enriched clinical trials aimed at determining the potential use of NAMPT inhibitors. These results suggest the inhibitors’ therapeutic potential in patient populations presenting with AML, ovarian cancer, Ewing’s sarcoma and SCLC all of which currently have high unmet needs. Citation Format: Jenny J. Hong, Martina S. McDermott, Jewel K. Ng, Prita Pandya, Dennis J. Slamon. Pan-cancer analysis of NAMPT inhibitors reveals unique sensitivities to multiple NAMPT inhibitors in several cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2336.

Abstract 3135: Postmenopausal obese mammary adipocytes promote breast tumorigenesis via the LCN2/IL-6/STAT3 signaling axis

Abstract Obesity is associated with a substantially increased risk (~50%) and a poor prognosis of breast cancer (BC) in postmenopausal (PM) women. The mechanism(s) underlying obesity-related BC are not clearly understood and, to date, most studies focus on the systemic effects of subcutaneous or visceral adipocytes on BC. We hypothesized that the increased local presence of ‘obese’ mammary adipocytes within the breast microenvironment promotes the acquisition of an invasive and vascular breast tumor cell phenotype and thereby markedly accelerates tumor proliferation and progression. We first asked whether local interactions between mammary adipocytes and BC cells might promote tumor growth and if so, what the underlying mechanism(s) might be. BC cells (ER- and ER+), when treated with the secretome of mammary adipocytes from obese women (ObAdCM), upregulated potent angiogenic/inflammatory factors, including VEGF, bFGF, lipocalin-2 (LCN2), IL-6 and significantly suppressed the angiogenesis inhibitor Tsp-1. ObAdCM stimulated endothelial cell (EC) recruitment by BC cells and increased EC proliferation, indispensable steps in tumor neovascularization. PM ObAdCM significantly stimulated BC cell proliferation, migration and invasion. Both IL-6 and LCN2-neutralization resulted in significant reduction of the ObAdCM-stimulated BC cell migration and proliferation, suggesting that these factors play a role in obesity-mediated BC progression. ObAdCM induced pSTAT3/pAkt signaling in BC cells and IL-6 and LCN2 could independently stimulate pSTAT3 in BC cells. STAT3 inhibition abrogated the ObACDM-mediated effects on BC cell proliferation and migration, suggesting that the pro-tumorigenic effects of PM obese mammary adipocytes are mediated by LCN2/IL-6/STAT3 signaling. Comparative RNAseq analyses identified >30 differentially expressed genes (DEGs) in PM compared to pre-menopausal (Pre-M) obese mammary adipocytes. Furthermore, GO and KEGG enrichment analysis of DEGs suggested an upregulation of PI3K/Akt signaling, ECM-receptor interactions, focal adhesions and lipid/fatty acid metabolism in PM mammary adipocytes. Taken together, our results indicate that obese mammary adipocytes in the breast tumor microenvironment can promote migration, proliferation, invasion and the vascular phenotype of BC cells and suggest that targeting the LCN2/IL-6/STAT3 signaling axis may be a useful strategy in the treatment of obesity-driven breast tumorigenesis. [Supported by NIH RO1CA185530, the Breast Cancer Research Foundation, the Karp Family Foundation and the Nile Albright Research Foundation] Citation Format: Roopali Roy, Emily Man, Katherine Gonzalez, Lauren Merritt, Cassandra Daisy, Rama Aldakhallah, Liang Sun, Biju Isaac, Shira Rockowitz, Margaret Lotz-Bousvaros, Susan Pories, Marsha A. Moses. Postmenopausal obese mammary adipocytes promote breast tumorigenesis via the LCN2/IL-6/STAT3 signaling axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3135.

Abstract 2981: Loss of the small GTPase RHOA enhances lymphomagenesis and impairs the activity of lenalidomide in mantle cell lymphoma

Abstract The small GTPase Ras homolog family member A (RHOA) is one of the most extensively investigated members of the Rho GTPase family, that acts as a molecular switch controlling a wide variety of signal transduction pathways. Although RHOA has long been implicated in malignant transformation in solid tumors, recent evidences have demonstrated its tumor suppressor activity in different subgroups of B-cell non-Hodgkin lymphoma (B-NHL). Here, using a panel of 11 cell lines covering the most common and/or aggressive B-NHL subtypes, we observed that the lowest mRNA and protein levels of RHOA are found in mantle cell lymphoma (MCL), the most aggressive entity with a median overall survival of 5-7 years. Depletion of RHOA expression and activity was carried out in the two RHOA+ MCL cell lines, REC-1 and Z-138, by CRISPR/Cas9-mediated gene edition followed by RHOA pulldown activation assay. In RHOA knockout (KO) subclones, cell proliferation was increased by 40%, in association with a 10-fold increase in mitotic index and a 3-fold potentiation of cell migration, as assessed respectively by CellTitter-Glo assay, histone H3-pSer10 labeling and Transwell assay, thus suggesting a tumor suppressor role of RHOA in MCL malignancy. Comparative RNA-seq analysis of RHOAWT and RHOAKO MCL subclones then highlighted a crucial role of RHOA and its downstream signaling in the control of MCL cell proliferation, cell cycle and cell migration. Interestingly, the immunomodulatory drug lenalidomide has previously shown to regulate such processes and to be clinically active in patients with relapsed/refractory MCL. In a co-culture system associating RHOAWT MCL cells with peripheral blood-derived effector cells, we observed that lenalidomide treatment facilitated the formation of immunological synapse, as revealed by simultaneous immunodetection of effector T cells, target B cells and perforin, and by quantification of lactate dehydrogenase (LDH) release. Conversely, lenalidomide exhibited minimal effect in the RHOAKO MCL co-cultures, as shown by a 30-40% decrease in the formation of immune synapse and a 4-fold decrease in LDH secretion. Taken together, these data provide the first insight into the tumor suppressive activity of RHOA in MCL and into its requirement for the immune-mediated mechanism of action of lenalidomide in this disease. Ongoing in vivo validation studies will be presented. Citation Format: Juliana C. Santos, Marcelo L. Ribeiro, Gael Roue. Loss of the small GTPase RHOA enhances lymphomagenesis and impairs the activity of lenalidomide in mantle cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2981.

Metastasis Related Epithelial-Mesenchymal Transition Signature Predicts Prognosis and Response to Immunotherapy in Gastric Cancer.

BackgroundIncreasing evidence has revealed the effect of epithelial-mesenchymal transition (EMT) on tumor microenvironment and cancer treatment. However, an EMT-based signature to predict the prognosis and therapeutic effect in gastric cancer (GC) has rarely been established.MethodsDifferentially expressed genes (DEGs) between paired primary gastric and ovarian metastatic tumors were identified through comparative RNA-seq analysis, followed by the construction of metastasis-related EMT signature (MEMTS) based on DEGs and EMT gene set. Then, both The Cancer Genome Atlas (TCGA) cohort and the Asian Cancer Research Group (ACRG) cohort were analyzed to explore the potential association between MEMTS and prognosis in GC. Samsung Medical Center (SMC) cohort and two individual immunotherapy treatment cohorts, including Kim cohort and Hugo cohort, were utilized to evaluate the predictive value of MEMTS on the response to adjuvant therapy and immunotherapy, respectively. Finally, the potential association of MEMTS with tumor environment and immune escape mechanisms was investigated.ResultsHigh MEMTS predicted a poor prognosis in patients with GC. Patients with low MEMTS potentially gained more benefits from adjuvant chemoradiotherapy than those with high MEMTS. MEMTS reliably predicted the response to immunotherapy in GC (area under the curve = 0.896). MEMTS was significantly associated with cancer-associated fibroblasts and stromal score in the aspect of the tumor microenvironment.ConclusionMEMTS serves as a potential biomarker to predict the prognosis and response to adjuvant therapy and immunotherapy in GC. MEMTS-based evaluation of individual tumors enables personalized treatment for GC patients in the future.

An Aux/IAA Family Member, RhIAA14, Involved in Ethylene-Inhibited Petal Expansion in Rose (Rosa hybrida).

Flower size, a primary agronomic trait in breeding of ornamental plants, is largely determined by petal expansion. Generally, ethylene acts as an inhibitor of petal expansion, but its effect is restricted by unknown developmental cues. In this study, we found that the critical node of ethylene-inhibited petal expansion is between stages 1 and 2 of rose flower opening. To uncover the underlying regulatory mechanism, we carried out a comparative RNA-seq analysis. Differentially expressed genes (DEGs) involved in auxin-signaling pathways were enriched. Therefore, we identified an auxin/indole-3-acetic acid (Aux/IAA) family gene, RhIAA14, whose expression was development-specifically repressed by ethylene. The silencing of RhIAA14 reduced cell expansion, resulting in diminished petal expansion and flower size. In addition, the expressions of cell-expansion-related genes, including RhXTH6, RhCesA2, RhPIP2;1, and RhEXPA8, were significantly downregulated following RhIAA14 silencing. Our results reveal an Aux/IAA that serves as a key player in orchestrating petal expansion and ultimately contributes to flower size, which provides new insights into ethylene-modulated flower opening and the function of the Aux/IAA transcription regulator.

Staphylococcal saoABC Operon Codes for a DNA-Binding Protein SaoC Implicated in the Response to Nutrient Deficit.

Whilst a large number of regulatory mechanisms for gene expression have been characterised to date, transcription regulation in bacteria still remains an open subject. In clinically relevant and opportunistic pathogens, such as Staphylococcus aureus, transcription regulation is of great importance for host-pathogen interactions. In our study we investigated an operon, exclusive to staphylococci, that we name saoABC. We showed that SaoC binds to a conserved sequence motif present upstream of the saoC gene, which likely provides a negative feedback loop. We have also demonstrated that S. aureus ΔsaoB and ΔsaoC mutants display altered growth dynamics in non-optimal media; ΔsaoC exhibits decreased intracellular survival in human dermal fibroblasts, whereas ΔsaoB produces an elevated number of persisters, which is also elicited by inducible production of SaoC in ΔsaoBΔsaoC double mutant. Moreover, we have observed changes in the expression of saoABC operon genes during either depletion of the preferential carbon or the amino acid source as well as during acidification. Comparative RNA-Seq of the wild type and ΔsaoC mutant demonstrated that SaoC influences transcription of genes involved in amino acid transport and metabolism, and notably of those coding for virulence factors. Our results suggest compellingly that saoABC operon codes for a DNA-binding protein SaoC, a novel staphylococcal transcription factor, and its antagonist SaoB. We linked SaoC to the response to nutrient deficiency, a stress that has a great impact on host-pathogen interactions. That impact manifests in SaoC influence on persister formation and survival during internalisation to host cells, as well as on the expression of genes of virulence factors that may potentially result in profound alternations in the pathogenic phenotype. Investigation of such novel regulatory mechanisms is crucial for our understanding of the dynamics of interactions between pathogenic bacteria and host cells, particularly in the case of clinically relevant, opportunistic pathogens such as Staphylococcus aureus.