Transposon Mutagenesis Screen Research Articles

Salmonella Typhimurium screen identifies shifts in mixed-acid fermentation during gut colonization

How enteric pathogens adapt their metabolism to a dynamic gut environment is not yet fully understood. To investigate how Salmonella enterica Typhimurium (S.Tm) colonizes the gut, we conducted an invivo transposon mutagenesis screen in a gnotobiotic mouse model. Our data implicate mixed-acid fermentation in efficient gut-luminal growth and energy conservation throughout infection. During initial growth, the pathogen utilizes acetate fermentation and fumarate respiration. After the onset of gut inflammation, hexoses appear to become limiting, as indicated by carbohydrate analytics and the increased need for gluconeogenesis. In response, S.Tm adapts by ramping up ethanol fermentation for redox balancing and supplying the TCA cycle with α-ketoglutarate for additional energy. Our findings illustrate how S.Tm flexibly adapts mixed fermentation and its use of the TCA cycle to thrive in the changing gut environment. Similar metabolic wiring in other pathogenic Enterobacteriaceae may suggest a broadly conserved mechanism for gut colonization.

Double-Wing Motif Protein is a Novel Biofilm Regulatory Factor of the Plant Disease Biocontrol Agent, Bacillus subtilis.

Transposon mutagenesis screening of Bacillus subtilis YB-1471, a novel rhizosphere biocontrol agent of Fusarium crown rot (FCR) of wheat, resulted in the identification of orf04391, linked to reduced biofilm formation. The gene encodes a protein possessing a putative tertiary structure of a "double-wing" DNA-binding domain. Expression of orf04391 increased during biofilm development in stationary cultures and during rapid growth in shaking cultures. An orf04391 deletion strain showed reduced biofilm production related to lower levels of the extracellular matrix, and the mutant also had reduced sporulation, adhesion, root colonization, and FCR biocontrol efficiency. Transcriptome analysis of YB-1471 and Δorf04391 in stationary culture showed that the loss of orf04391 resulted in altered expression of numerous genes, including sinI, an initiator of biofilm formation. DNA binding was shown with his-tagged Orf04391 binding to the sinIR operon in vivo and in vitro. Orf04391 appears to be a transcriptional regulator of biofilm formation in B. subtilis through the Spo0A-SinI/SinR pathway.

Transposon mutagenesis screen in Klebsiella pneumoniae identifies genetic determinants required for growth in human urine and serum

Klebsiella pneumoniae is a global public health concern due to the rising myriad of hypervirulent and multidrug-resistant clones both alarmingly associated with high mortality. The molecular mechanisms underpinning these recalcitrant K. pneumoniae infection, and how virulence is coupled with the emergence of lineages resistant to nearly all present-day clinically important antimicrobials, are unclear. In this study, we performed a genome-wide screen in K. pneumoniae ECL8, a member of the endemic K2-ST375 pathotype most often reported in Asia, to define genes essential for growth in a nutrient-rich laboratory medium (Luria-Bertani [LB] medium), human urine, and serum. Through transposon directed insertion-site sequencing (TraDIS), a total of 427 genes were identified as essential for growth on LB agar, whereas transposon insertions in 11 and 144 genes decreased fitness for growth in either urine or serum, respectively. These studies not only provide further knowledge on the genetics of this pathogen but also provide a strong impetus for discovering new antimicrobial targets to improve current therapeutic options for K. pneumoniae infections.

Transposon mutagenesis screen in Klebsiella pneumoniae identifies genetic determinants required for growth in human urine and serum.

Klebsiella pneumoniae is a global public health concern due to the rising myriad of hypervirulent and multidrug-resistant clones both alarmingly associated with high mortality. The molecular mechanisms underpinning these recalcitrant K. pneumoniae infection, and how virulence is coupled with the emergence of lineages resistant to nearly all present-day clinically important antimicrobials, are unclear. In this study, we performed a genome-wide screen in K. pneumoniae ECL8, a member of the endemic K2-ST375 pathotype most often reported in Asia, to define genes essential for growth in a nutrient-rich laboratory medium (Luria-Bertani [LB] medium), human urine, and serum. Through transposon directed insertion-site sequencing (TraDIS), a total of 427 genes were identified as essential for growth on LB agar, whereas transposon insertions in 11 and 144 genes decreased fitness for growth in either urine or serum, respectively. These studies not only provide further knowledge on the genetics of this pathogen but also provide a strong impetus for discovering new antimicrobial targets to improve current therapeutic options for K. pneumoniae infections.

Efflux pumps and membrane permeability contribute to intrinsic antibiotic resistance in Mycobacterium abscessus.

Mycobacterium abscessus is a pulmonary pathogen that exhibits intrinsic resistance to antibiotics, but the factors driving this resistance are incompletely understood. Insufficient intracellular drug accumulation could explain broad-spectrum resistance, but whether antibiotics fail to accumulate in M. abscessus and the mechanisms required for drug exclusion remain poorly understood. We measured antibiotic accumulation in M. abscessus using mass spectrometry and found a wide range of drug accumulation across clinically relevant antibiotics. Of these compounds, linezolid accumulates the least, suggesting that inadequate uptake impacts its efficacy. We utilized transposon mutagenesis screening to identify genes that cause linezolid resistance and found multiple transporters that promote membrane permeability or efflux, including an uncharacterized, M. abscessus-specific protein that effluxes linezolid and several chemically related antibiotics. This demonstrates that membrane permeability and drug efflux are critical mechanisms of antibiotic resistance in M. abscessus and suggests that targeting membrane transporters could potentiate the efficacy of certain antibiotics.

Multiple regulatory inputs including cell envelope stress orchestrate expression of the Escherichia coli rpoN operon.

The rpoN operon, an important regulatory hub in Enterobacteriaceae, includes rpoN encoding sigma factor σ54, hpf involved in ribosome hibernation, rapZ regulating glucosamine-6-phosphate levels, and two genes encoding proteins of the nitrogen-related phosphotransferase system. Little is known about regulatory mechanisms controlling the abundance of these proteins. This study employs transposon mutagenesis and chemical screens to dissect the complex expression of the rpoN operon. We find that envelope stress conditions trigger read-through transcription into the rpoN operon from a promoter located upstream of the preceding lptA-lptB locus. This promoter is controlled by the envelope stress sigma factor E and response regulator PhoP is required for its full response to a subset of stress signals. σE also stimulates ptsN-rapZ-npr expression using an element downstream of rpoN, presumably by interfering with mRNA processing by RNase E. Additionally, we identify a novel promoter in the 3' end of rpoN that directs transcription of the distal genes in response to ethanol. Finally, we show that translation of hpf and ptsN is individually regulated by the RNA chaperone Hfq, perhaps involving small RNAs. Collectively, our work demonstrates that the rpoN operon is subject to complex regulation, integrating signals related to envelope stress and carbon source quality.

Role of Type 4B Secretion System Protein, IcmE, in the Pathogenesis of Coxiella burnetii.

Coxiella burnetii is an obligate intracellular Gram-negative bacterium that causes Q fever, a life-threatening zoonotic disease. C. burnetii replicates within an acidified parasitophorous vacuole derived from the host lysosome. The ability of C. burnetii to replicate and achieve successful intracellular life in the cell cytosol is vastly dependent on the Dot/Icm type 4B secretion system (T4SSB). Although several T4SSB effector proteins have been shown to be important for C. burnetii virulence and intracellular replication, the role of the icmE protein in the host-C. burnetii interaction has not been investigated. In this study, we generated a C. burnetii Nine Mile Phase II (NMII) mutant library and identified 146 transposon mutants with a single transposon insertion. Transposon mutagenesis screening revealed that disruption of icmE gene resulted in the attenuation of C. burnetii NMII virulence in SCID mice. ELISA analysis indicated that the levels of pro-inflammatory cytokines, including interleukin-1β, IFN-γ, TNF-α, and IL-12p70, in serum from Tn::icmE mutant-infected SCID mice were significantly lower than those in serum from wild-type (WT) NMII-infected mice. Additionally, Tn::icmE mutant bacteria were unable to replicate in mouse bone marrow-derived macrophages (MBMDM) and human macrophage-like cells (THP-1). Immunoblotting results showed that the Tn::icmE mutant failed to activate inflammasome components such as IL-1β, caspase 1, and gasdermin-D in THP-1 macrophages. Collectively, these results suggest that the icmE protein may play a vital role in C. burnetii virulence, intracellular replication, and activation of inflammasome mediators during NMII infection.

Extracellular organic disulfide reduction by Shewanella oneidensis MR-1.

Microbial reduction of organic disulfides affects the macromolecular structure and chemical reactivity of natural organic matter. Currently, the enzymatic pathways that mediate disulfide bond reduction in soil and sedimentary organic matter are poorly understood. In this study, we examined the extracellular reduction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) by Shewanella oneidensis strain MR-1. A transposon mutagenesis screen performed with S. oneidensis resulted in the isolation of a mutant that lost ~90% of its DTNB reduction activity. Genome sequencing of the mutant strain revealed that the transposon was inserted into the dsbD gene, which encodes for an oxidoreductase involved in cytochrome c maturation. Complementation of the mutant strain with the wild-type dsbD partially restored DTNB reduction activity. Because DsbD catalyzes a critical step in the assembly of multi-heme c-type cytochromes, we further investigated the role of extracellular electron transfer cytochromes in organic disulfide reduction. The results indicated that mutants lacking proteins in the Mtr system were severely impaired in their ability to reduce DTNB. These findings provide new insights into extracellular organic disulfide reduction and the enzymatic pathways of organic sulfur redox cycling.IMPORTANCEOrganic sulfur compounds in soils and sediments are held together by disulfide bonds. This study investigates how Shewanella oneidensis breaks apart extracellular organic sulfur compounds. The results show that an enzyme involved in the assembly of c-type cytochromes as well as proteins in the Mtr respiratory pathway is needed for S. oneidensis to transfer electrons from the cell surface to extracellular organic disulfides. These findings have important implications for understanding how organic sulfur decomposes in terrestrial ecosystems.

MBARq: a versatile and user-friendly framework for the analysis of DNA barcodes from transposon insertion libraries, knockout mutants, and isogenic strain populations.

DNA barcoding has become a powerful tool for assessing the fitness of strains in a variety of studies, including random transposon mutagenesis screens, attenuation of site-directed mutants, and population dynamics of isogenic strain pools. However, the statistical analysis, visualization, and contextualization of the data resulting from such experiments can be complex and require bioinformatic skills. Here, we developed mBARq, a user-friendly tool designed to simplify these steps for diverse experimental setups. The tool is seamlessly integrated with an intuitive web app for interactive data exploration via the STRING and KEGG databases to accelerate scientific discovery. The tool is implemented in Python. The source code is freely available (https://github.com/MicrobiologyETHZ/mbarq) and the web app can be accessed at: https://microbiomics.io/tools/mbarq-app.

Inhibition of Pseudomonas aeruginosa quorum sensing by chemical induction of the MexEF-oprN efflux pump.

The cell-to-cell communication system quorum sensing (QS), used by various pathogenic bacteria to synchronize gene expression and increase host invasion potentials, is studied as a potential target for persistent infection control. To search for novel molecules targeting the QS system in the Gram-negative opportunistic pathogen Pseudomonas aeruginosa, a chemical library consisting of 3,280 small compounds from LifeArc was screened. A series of 10 conjugated phenones that have not previously been reported to target bacteria were identified as inhibitors of QS in P. aeruginosa. Two lead compounds (ethylthio enynone and propylthio enynone) were re-synthesized for verification of activity and further elucidation of the mode of action. The isomeric pure Z-ethylthio enynone was used for RNA sequencing, revealing a strong inhibitor of QS-regulated genes, and the QS-regulated virulence factors rhamnolipid and pyocyanin were significantly decreased by treatment with the compounds. A transposon mutagenesis screen performed in a newly constructed lasB-gfp monitor strain identified the target of Z-ethylthio enynone in P. aeruginosa to be the MexEF-OprN efflux pump, which was further established using defined mex knockout mutants. Our data indicate that the QS inhibitory capabilities of Z-ethylthio enynone were caused by the drainage of intracellular signal molecules as a response to chemical-induced stimulation of the MexEF-oprN efflux pump, thereby inhibiting the autogenerated positive feedback and its enhanced signal-molecule synthesis.

A putative lipase affects Pseudomonas aeruginosa biofilm matrix production.

Pseudomonas aeruginosa is an opportunistic pathogen that is widely known for infecting patients with underlying conditions. This species often survives antibiotic therapy by forming biofilms, in which the cells produce a protective extracellular matrix. P. aeruginosa also produces virulence factors that enhance its ability to cause disease. One signaling pathway that influences virulence is the nitrogen-related phosphotransferase system (Nitro-PTS), which consists of an initial phosphotransferase, PtsP, a phosphocarrier, PtsO, and a terminal phosphate receptor, PtsN. The physiological role of the Nitro-PTS in P. aeruginosa is poorly understood. However, PtsN, when deprived of its upstream phosphotransfer proteins, has an antagonistic effect on biofilm formation. We thus conducted a transposon mutagenesis screen in an unphosphorylated-PtsN (i.e., ∆ptsP) background to identify downstream proteins with unacknowledged roles in PtsN-mediated biofilm suppression. We found an unstudied gene, PA14_04030, whose disruption restored biofilm production. This gene encodes a predicted phospholipase with signature alpha/beta hydrolase folds and a lipase signature motif with an active-site Ser residue. Hence, we renamed the gene bipL, for biofilm-impacting phospholipase. Deletion of bipL in a ∆ptsP background increased biofilm formation, supporting the idea that BipL is responsible for reducing biofilm formation in strains with unphosphorylated PtsN. Moreover, substituting the putative catalytic Ser for Ala phenocopied bipL deletion, indicating that this residue is important for the biofilm-suppressive activity of BipL in vivo. As our preliminary data suggest that BipL is a lipase, we performed lipidomics to detect changes in the lipid profile due to bipL deletion and found changes in some lipid species. IMPORTANCE Biofilm formation by bacteria occurs when cells secrete an extracellular matrix that holds them together and shields them from environmental insults. Biofilms of bacterial opportunistic human pathogens such as Pseudomonas aeruginosa pose a substantial challenge to clinical antimicrobial therapy. Hence, a more complete knowledge about the bacterial factors that influence and regulate production of the biofilm matrix is one key to formulate more effective therapeutic strategies. In this study, we screen for factors that are important for reducing biofilm matrix production in certain genetic backgrounds. We unexpectedly found a gene encoding a putative lipase enzyme and showed that its predicted catalytic site is important for its ability to reduce biofilm formation. Our findings suggest that lipase enzymes have previously uncharacterized functions in biofilm matrix regulation.

A genome-wide transposon mutagenesis screening identifies LppB as a key factor associated with Mycoplasma bovis colonization and invasion into host cells.

Mycoplasma spp., the smallest self-replicating and genome-reduced organisms, have raised a great concern in both the medical and veterinary fields due to their pathogenicity. The molecular determinants of these wall-less bacterium efficiently use their limited genes to ensure successful infection of the host remain unclear. In the present study, we used the ruminant pathogen Mycoplasma bovis as a model to identify the key factors for colonization and invasion into host cells. We constructed a nonredundant fluorescent transposon mutant library of M. bovis using a modified transposon plasmid, and identified 34 novel adhesion-related genes based on a high-throughput screening approach. Among them, the ΔLppB mutant exhibited the most apparent decrease in adhesion to embryonic bovine lung (EBL) cells. The surface-localized lipoprotein LppB, which is highly conserved in Mycoplasma species, was then confirmed as a key factor for M. bovis adhesion with great immunogenicity. LppB interacted with various components (fibronectin, vitronectin, collagen IV, and laminin) of host extracellular matrix (ECM) and promoted plasminogen activation through tPA to degrade ECM. The 439-502 amino acid region of LppB is a critical domain, and F465 and Y493 are important residues for the plasminogen activation activity. We further revealed LppB as a key factor facilitating internalization through clathrin- and lipid raft-mediated endocytosis, which helps the Mycoplasma invade the host cells. Our study indicates that LppB plays a key role in Mycoplasma infection and is a potential new therapeutic and vaccine target for Mycoplasma species.

Distinct shared and compartment-enriched oncogenic networks drive primary versus metastatic breast cancer

Metastatic breast-cancer is a major cause of death in women worldwide, yet the relationship between oncogenic drivers that promote metastatic versus primary cancer is still contentious. To elucidate this relationship in treatment-naive animals, we hereby describe mammary-specific transposon-mutagenesis screens in female mice together with loss-of-function Rb, which is frequently inactivated in breast-cancer. We report gene-centric common insertion-sites (gCIS) that are enriched in primary-tumors, in metastases or shared by both compartments. Shared-gCIS comprise a major MET-RAS network, whereas metastasis-gCIS form three additional hubs: Rho-signaling, Ubiquitination and RNA-processing. Pathway analysis of four clinical cohorts with paired primary-tumors and metastases reveals similar organization in human breast-cancer with subtype-specific shared-drivers (e.g. RB1-loss, TP53-loss, high MET, RAS, ER), primary-enriched (EGFR, TGFβ and STAT3) and metastasis-enriched (RHO, PI3K) oncogenic signaling. Inhibitors of RB1-deficiency or MET plus RHO-signaling cooperate to block cell migration and drive tumor cell-death. Thus, targeting shared- and metastasis- but not primary-enriched derivers offers a rational avenue to prevent metastatic breast-cancer.

Abstract 4607: TRPS1 inhibits angiogenesis in triple negative breast cancer down regulating JAG1 and TYMP genes involved in angiogenesis pathways

Abstract Background. Triple-negative breast cancer (TNBC), the most difficult subtype to treat, is defined as estrogen receptor, HER2, and progesterone receptor negative. TNBC constitutes 10-20% of all breast cancer and has a higher rate of distal recurrence and a poorer prognosis than other breast cancer subtypes. Less than 30% of women with metastatic TNBC survive 5 years and almost all die from their disease despite adjuvant chemotherapy. Angiogenesis is a critical factor in the development of tumors and metastases in numerous cancers. Clinical trials with anti-angiogenic drugs showed to be inadequate, with moderate response rates and insignificant survival gains for patients. To understand the genetic forces involved in TNBC, we performed a transposon mutagenesis screen in Pten mutant mice that identified several candidate trunk drivers and progression genes. A major finding of our screen was the discovery and functional validation of, Trichorhinophalangeal syndrome type 1 (TRPS1), a GATA-like transcription factor, which functions as a transcriptional repressor or activator, depending on cell type, stage of development, or pathological conditions. Based on this data, we explored the role of TRPS1 in angiogenesis. Methods and Results. Tube formation and sprouting assays were performed using overexpression and inactivation of TRPS1 in MDA-MD-231 and HCC70 cells, respectively. Interestingly, inactivation of TRPS1 expression accelerates tube formation structures compared to the vector control as well as cell branching in the sprouting assay. Overexpression of TRPS1 prevents tubing and branching formation in vitro assays. Moreover, immunohistochemistry staining of CD31 detected a reduced number of blood vessels in MDA-MB-231 tumor xenografts overexpressing TRPS1, and an increase of angiogenic vasculature in HCC70 TRPS1-shRNA tumor xenografts. In vitro and in vivo assays demonstrate the role of TRPS1 in tumor angiogenesis. Furthermore, human ChIP qPCR angiogenesis array identified 10 top candidate genes potentially regulated by TRPS1 transcription factor. Of these candidates, JAG1 and TYMP showed to have a higher fold enrichment compared to other angiogenic related genes. Finally, we validated its direct functional binding by using luciferase reporter assay, such demonstrated that TRPS1 is a direct transcriptional regulator of JAG1 and TYMP. Conclusion. TRPS1 is a tumor suppressor involved in the mechanisms regulating tumor angiogenesis by repressing the expression of JAG1 and TYMP genes in TNBC. Citation Format: Liliana Guzman. TRPS1 inhibits angiogenesis in triple negative breast cancer down regulating JAG1 and TYMP genes involved in angiogenesis pathways. [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 4607.

Biotin-dependent cell envelope remodelling is required for Mycobacterium abscessus survival in lung infection.

Mycobacterium abscessus is an emerging pathogen causing lung infection predominantly in patients with underlying structural abnormalities or lung disease and is resistant to most frontline antibiotics. As the pathogenic mechanisms of M. abscessus in the context of the lung are not well-understood, we developed an infection model using air-liquid interface culture and performed a transposon mutagenesis and sequencing screen to identify genes differentially required for bacterial survival in the lung. Biotin cofactor synthesis was required for M. abscessus growth due to increased intracellular biotin demand, while pharmacological inhibition of biotin synthesis prevented bacterial proliferation. Biotin was required for fatty acid remodelling, which increased cell envelope fluidity and promoted M. abscessus survival in the alkaline lung environment. Together, these results indicate that biotin-dependent fatty acid remodelling plays a critical role in pathogenic adaptation to the lung niche, suggesting that biotin synthesis and fatty acid metabolism might provide therapeutic targets for treatment of M. abscessus infection.

A virulence activator of a surface attachment protein in Burkholderia pseudomallei acts as a global regulator of other membrane-associated virulence factors.

Burkholderia pseudomallei (Bp), causing a highly fatal disease called melioidosis, is a facultative intracellular pathogen that attaches and invades a variety of cell types. We previously identified BP1026B_I0091 as a surface attachment protein (Sap1) and an essential virulence factor, contributing to Bp pathogenesis in vitro and in vivo. The expression of sap1 is regulated at different stages of Bp intracellular lifecycle by unidentified regulator(s). Here, we identified SapR (BP1026B_II1046) as a transcriptional regulator that activates sap1, using a high-throughput transposon mutagenesis screen in combination with Tn-Seq. Consistent with phenotypes of the Δsap1 mutant, the ΔsapR activator mutant exhibited a significant reduction in Bp attachment to the host cell, leading to subsequent decreased intracellular replication. RNA-Seq analysis further revealed that SapR regulates sap1. The regulation of sap1 by SapR was confirmed quantitatively by qRT-PCR, which also validated the RNA-Seq data. SapR globally regulates genes associated with the bacterial membrane in response to diverse environments, and some of the genes regulated by SapR are virulence factors that are required for Bp intracellular infection (e.g., type III and type VI secretion systems). This study has identified the complex SapR regulatory network and its importance as an activator of an essential Sap1 attachment factor.

Transmicron: accurate prediction of insertion probabilities improves detection of cancer driver genes from transposon mutagenesis screens.

Transposon screens are powerful in vivo assays used to identify loci driving carcinogenesis. These loci are identified as Common Insertion Sites (CISs), i.e. regions with more transposon insertions than expected by chance. However, the identification of CISs is affected by biases in the insertion behaviour of transposon systems. Here, we introduce Transmicron, a novel method that differs from previous methods by (i) modelling neutral insertion rates based on chromatin accessibility, transcriptional activityand sequence context and (ii) estimating oncogenic selection for each genomic region using Poisson regression to model insertion counts while controlling for neutral insertion rates. To assess the benefits of our approach, we generated a dataset applying two different transposon systems under comparable conditions. Benchmarking for enrichment of known cancer genes showed improved performance of Transmicron against state-of-the-art methods. Modelling neutral insertion rates allowed for better control of false positives and stronger agreement of the results between transposon systems. Moreover, using Poisson regression to consider intra-sample and inter-sample information proved beneficial in small and moderately-sized datasets. Transmicron is open-source and freely available. Overall, this study contributes to the understanding of transposon biology and introduces a novel approach to use this knowledge for discovering cancer driver genes.

Ring Finger Protein 125 Is an Anti-Proliferative Tumor Suppressor in Hepatocellular Carcinoma.

Simple SummaryHCC is a leading cause of cancer-related deaths worldwide. However, its tremendous inter- and intra-tumor heterogeneity has made it difficult to identify driver genes for HCC. Transposon mutagenesis is a versatile in vivo tool to identify cancer genes in tissues of interest. Herein, we analyzed transposon mutagenesis screening data in the liver and discovered a novel tumor suppressor gene, RNF125. This gene functions as a negative regulator of cell proliferation through transcriptional suppression of multiple genes important for cell proliferation and liver regeneration. This gene is frequently inactivated in human HCC and has a significant impact on patient prognosis. Our findings identify a new regulatory network of cell proliferation mediated by RNF125 and its contribution to HCC development and progression.Hepatocellular carcinoma (HCC) is one of the deadliest cancers worldwide and the only cancer with an increasing incidence in the United States. Recent advances in sequencing technology have enabled detailed profiling of liver cancer genomes and revealed extensive inter- and intra-tumor heterogeneity, making it difficult to identify driver genes for HCC. To identify HCC driver genes, we performed transposon mutagenesis screens in a mouse HBV model of HCC and discovered many candidate cancer genes (SB/HBV-CCGs). Here, we show that one of these genes, RNF125 is a potent anti-proliferative tumor suppressor gene in HCC. RNF125 is one of nine CCGs whose expression was >3-fold downregulated in human HCC. Depletion of RNF125 in immortalized mouse liver cells led to tumor formation in transplanted mice and accelerated growth of human liver cancer cell lines, while its overexpression inhibited their growth, demonstrating the tumor-suppressive function of RNF125 in mouse and human liver. Whole-transcriptome analysis revealed that RNF125 transcriptionally suppresses multiple genes involved in cell proliferation and/or liver regeneration, including Egfr, Met, and Il6r. Blocking Egfr or Met pathway expression inhibited the increased cell proliferation observed in RNF125 knockdown cells. In HCC patients, low expression levels of RNF125 were correlated with poor prognosis demonstrating an important role for RNF125 in HCC. Collectively, our results identify RNF125 as a novel anti-proliferative tumor suppressor in HCC.

Fluoropyrimidine Bioactivation and Metabolism by the Gut Microbiome

Despite decades of research into why populations of cancer patients differentially respond to the same treatment, variation in response to therapy regimens remains a significant challenge in the oncology field. However, our understanding of the causal mechanisms of interpatient variability remains lacking; up to 35% of patients require dose adjustments and 10% of patients are removed from the fluoropyrimidine capecitabine altogether. This research seeks to further our understanding of the microbiome’s role in fluoropyrimidine treatment by identifying gut bacterial strains responsible for capecitabine activation as well as biochemically characterizing drug inactivating enzymes from the human gut microbiome. Growth curve data show that knockouts of bacterial enzymes homologous to mammalian enzymes known to be involved in capecitabine metabolism increase bacterial resistance to capecitabine. Additionally, an unbiased transposon mutagenesis screen identifies enzymes known to increase resistance as well as enzymes both within and outside of nucleotide metabolism. These two pieces of data suggest that bacteria have the capacity to metabolize capecitabine, upending the paradigm that activation of this prodrug happens exclusively in the liver and tumor. Confirmation of these metabolites with high resolution triple quadrupole linear ion trap mass spectrometry is ongoing. Separately, previous studies indicate that gut bacteria are capable of degrading 5‐fluorouracil, however it is not clear how efficient these pathways are or to what extent they occur in the host. Enzyme kinetics of homologues of this enzyme within the microbiome show that degradative reaction for uracil is essentially irreversible. Additionally, this enzymatic characterization has identified the non‐enzymatic enantiomer of the degraded fluoropyrimidine as a potential inhibitor for this enzyme, which might allow for co‐dosing and inhibition of bacterial metabolism. Metagenomic sequencing of the degradative enzymes within patient samples is ongoing and will potentially reveal the main drivers of this activity in patients. These avenues of research will uncover new bacterial metabolism pathways of xenobiotics, compare these metabolic pathways between microbiota and host, and have the potential to lead to new advances in disrupting microbial metabolism of chemotherapeutics.

Genetic alterations of the SUMO isopeptidase SENP6 drive lymphomagenesis and genetic instability in diffuse large B-cell lymphoma

SUMOylation is a post-translational modification of proteins that regulates these proteins’ localization, turnover or function. Aberrant SUMOylation is frequently found in cancers but its origin remains elusive. Using a genome-wide transposon mutagenesis screen in a MYC-driven B-cell lymphoma model, we here identify the SUMO isopeptidase (or deconjugase) SENP6 as a tumor suppressor that links unrestricted SUMOylation to tumor development and progression. Notably, SENP6 is recurrently deleted in human lymphomas and SENP6 deficiency results in unrestricted SUMOylation. Mechanistically, SENP6 loss triggers release of DNA repair- and genome maintenance-associated protein complexes from chromatin thereby impairing DNA repair in response to DNA damages and ultimately promoting genomic instability. In line with this hypothesis, SENP6 deficiency drives synthetic lethality to Poly-ADP-Ribose-Polymerase (PARP) inhibition. Together, our results link SENP6 loss to defective genome maintenance and reveal the potential therapeutic application of PARP inhibitors in B-cell lymphoma.