Polymerase Beta Research Articles

A database of sequenced genomes of different Streptomyces albus J1074 strains and uses thereof

Streptomyces albus J1074 has recently emerged as a powerful chassis strain for natural product research and discovery, as well as model to investigate various aspects of actinobacterial biology. A number of genetic tools have been developed to facilitate the use of this strain for the aforementioned purposes. One of the promising approaches is to introduce into J1074 genome mutations that would improve the antibiotic-producing capacity of J1074. Particularly, we reported a collection of spontaneous and genomically engineered J1074 mutants carrying mutation in genes for ribosomal protein S12, RNA polymerase beta subunit etc. We took advantage of this collection to build an in-house database which would host (both current and future) the primary and curated sequencing data for these mutants. The database is available at https://biotools.online/media/. The main benefits of the database lie in the known pedigree of the strains, which allows deep interpretation of the data. For example, there is lively – and unresolved – debate on the origins and consequences of the GC composition in actinobacteria. A better understanding of this issue should improve our knowledge of genome evolution in bacteria and will have a number of biotechnological ramifications. We used our Streptomyces albus J1074 dataset as an experimental model to reveal genome-wide spectrum of mutation, which appears to be biased towards elevated GC content. We also included the high-quality genomes of the other streptomycetes into our databse for comparative purposes. The genomic GC content in streptomycetes varies from 75 % to 66.5 %, with median value being 72 %. The GC content of secondary metabolic genes of S. coelicolor is less variable than that of primary metabolic genes, an indicative of different selection pressure on these gene groups. Along with selective constraints, the peculiarities of Streptomyces DNA mismatch repair might contribute to the skewed GC content of their genomes. Further uses of the database may include the development of a more precise knowledge of the mutation rate as well as population genetic processes within this species and genus.

Identification and validation of reference genes for reliable analysis of differential gene expression during antibiotic induced persister formation in Klebsiella pneumoniae using qPCR

The study of differential gene expression in persister cells is compounded by ceasure of conventional cellular metabolic pathways during persistence. There is, hence, a requirement to identify and validate suitable reference genes whose expression remains stable during persistence. We evaluated the suitability of five genes viz. dnaJ, groEL, rpoB, kp751, kp4432 as references to study gene expression using real-time polymerase chain reaction (qPCR) during persister cell formation in Klebsiella pneumoniae. Results obtained showed that while dnaJ and groEL suffered from unstable expression; rpoB, kp751 and kp4432 showed stable expression. Further, it was observed that data normalization using either of the stable genes viz. rpoB, kp751, kp4432 alone, resulted in either too low expression levels or too high variation among replicates. Our study indicates the concurrent use of kp4432 and rpoB as reference genes to be the most suitable for reliable analysis of differential gene expression during antibiotic induced persister formation in K. pneumoniae. kp4432 and rpoB encode NAD-dependant phenylacetaldehyde dehydrogenase and DNA-directed RNA polymerase beta subunit respectively. The outcome of this study will increase the utility of qPCR in studying the temporal changes in gene expression during persistence. The study will also aid in understanding mechanisms underlying persister cell formation particularly in K. pneumoniae.

DNA polymerase β outperforms DNA polymerase γ in key mitochondrial base excision repair activities

DNA polymerase beta (POLβ), well known for its role in nuclear DNA base excision repair (BER), has been shown to be present in the mitochondria of several different cell types. Here we present a side-by-side comparison of BER activities of POLβ and POLγ, the mitochondrial replicative polymerase, previously thought to be the only mitochondrial polymerase. We find that POLβ is significantly more proficient at single-nucleotide gap filling, both in substrates with ends that require polymerase processing, and those that do not. We also show that POLβ has a helicase-independent functional interaction with the mitochondrial helicase, TWINKLE. This interaction stimulates strand-displacement synthesis, but not single-nucleotide gap filling. Importantly, we find that purified mitochondrial extracts from cells lacking POLβ are severely deficient in processing BER intermediates, suggesting that mitochondrially localized DNA POLβ may be critical for cells with high energetic demands that produce greater levels of oxidative stress and therefore depend upon efficient BER for mitochondrial health.

Genetic diversity of Bartonella species in small mammals in the Qaidam Basin, western China

Investigation of the prevalence and diversity of Bartonella infections in small mammals in the Qaidam Basin, western China, could provide a scientific basis for the control and prevention of Bartonella infections in humans. Accordingly, in this study, small mammals were captured using snap traps in Wulan County and Ge’ermu City, Qaidam Basin, China. Spleen and brain tissues were collected and cultured to isolate Bartonella strains. The suspected positive colonies were detected with polymerase chain reaction amplification and sequencing of gltA, ftsZ, RNA polymerase beta subunit (rpoB) and ribC genes. Among 101 small mammals, 39 were positive for Bartonella, with the infection rate of 38.61%. The infection rate in different tissues (spleens and brains) (χ2 = 0.112, P = 0.738) and gender (χ2 = 1.927, P = 0.165) of small mammals did not have statistical difference, but that in different habitats had statistical difference (χ2 = 10.361, P = 0.016). Through genetic evolution analysis, 40 Bartonella strains were identified (two different Bartonella species were detected in one small mammal), including B. grahamii (30), B. jaculi (3), B. krasnovii (3) and Candidatus B. gerbillinarum (4), which showed rodent-specific characteristics. B. grahamii was the dominant epidemic strain (accounted for 75.0%). Furthermore, phylogenetic analysis showed that B. grahamii in the Qaidam Basin, might be close to the strains isolated from Japan and China. Overall, we observed a high prevalence of Bartonella infection in small mammals in the Qaidam Basin. B. grahamii may cause human disease, and the pathogenicity of the others Bartonella species needs further study, the corresponding prevention and control measures should be taken into consideration.

Lincomycin-Induced Secondary Metabolism in Streptomyces lividans 66 with a Mutation in the Gene Encoding the RNA Polymerase Beta Subunit.

Activating the genetic potential of Streptomyces strains to produce secondary metabolites can improve the production of useful biologically active compounds and facilitate the discovery of novel biologically active compounds. In this study, we found that Streptomyces lividans carrying the R440H mutation in rpoB, encoding the RNA polymerase beta subunit, grown in the presence of lincomycin at concentrations below the minimum inhibitory concentration (MIC) produced abundant amounts of actinorhodin and certain cryptic secondary metabolites despite culture conditions that restrict their production by the wild-type strain. The results indicate that lincomycin at concentrations below the MIC may strongly potentiate secondary metabolite production by Streptomyces strains carrying a specific rpoB mutation. In this study, we report an interesting phenomenon induced by combining the positive effects of certain rpoB mutations and concentration-dependent responses to lincomycin on secondary metabolism in S. lividans 66 and discuss the mechanisms and their applicability in exploring cryptic secondary metabolite production in streptomycetes.

Lymphocyte predominant cells detect Moraxella catarrhalis-derived antigens in nodular lymphocyte-predominant Hodgkin lymphoma

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is a rare lymphoma of B-cell origin with frequent expression of functional B-cell receptors (BCRs). Here we report that expression cloning followed by antigen screening identifies DNA-directed RNA polymerase beta’ (RpoC) from Moraxella catarrhalis as frequent antigen of BCRs of IgD+ LP cells. Patients show predominance of HLA-DRB1*04/07 and the IgVH genes encode extraordinarily long CDR3s. High-titer, light-chain-restricted anti-RpoC IgG1/κ-type serum-antibodies are additionally found in these patients. RpoC and MID/hag, a superantigen co-expressed by Moraxella catarrhalis that is known to activate IgD+ B cells by binding to the Fc domain of IgD, have additive activation effects on the BCR, the NF-κB pathway and the proliferation of IgD+ DEV cells expressing RpoC-specific BCRs. This suggests an additive antigenic and superantigenic stimulation of B cells with RpoC-specific IgD+ BCRs under conditions of a permissive MHC-II haplotype as a model of NLPHL lymphomagenesis, implying future treatment strategies.

Annotation and characterization of Babesia gibsoni apicoplast genome

BackgroundBabesia gibsoni is an apicomplexan parasite transmitted by ticks, which can infect canine species and cause babesiosis. The apicoplast is an organelle associated with isoprenoids metabolism, is widely present in apicomplexan parasites, except for Cryptosporidium. Available data indicate that the apicoplast is essential for the survival of apicomplexan parasites.MethodsHere, the apicoplast genome of B. gibsoni was investigated by high-throughput genome sequencing, bioinformatics analysis, and conventional PCR.ResultsThe apicoplast genome of B. gibsoni-Wuhan strain (B. gibsoni-WH) consists of a 28.4 kb circular molecule, with A + T content of 86.33%, similar to that of B. microti. Specifically, this genome encodes genes involved in maintenance of the apicoplast DNA, transcription, translation and maturation of organellar proteins, which contains 2 subunits of ribosomal RNAs, 17 ribosomal proteins, 1 EF-Tu elongation factor (tufA), 5 DNA-dependent RNA polymerase beta subunits, 2 Clp protease chaperones, 23 tRNA genes and 5 unknown open reading frames (hypothetical proteins). Phylogenetic analysis revealed high similarity of B. gibsoni apicoplast genome to that of B. orientalis and B. bovis.ConclusionsTo our knowledge, this is the first report of annotation and characterization of B. gibsoni-WH apicoplast genome. The results will facilitate the development of new anti-Babesia drug targets.

Salt‐dependent protein splicing of an intein from the halophile Haloferax volcanii

Protein splicing is a post‐translational process facilitated by an intervening polypeptide called an intein, or internal protein. The intein is located between two flanking polypeptides, called exteins. The intein catalyzes its excision from the extein concomitant with the ligation of the exteins. The extremophile Haloferax volcanii grows in high salt environments. We are interested in the salt‐dependence of the protein splicing and nuclease activity of the intein that has invaded its DNA Polymerase beta gene. We have shown that we can induce protein splicing of the intein in vitro as a function of salt concentration and time. The intein has an intervening homing endonuclease domain and we are interested to learn if mutations within this domain would influence splicing. Characterization of a conditionally‐active nuclease may have utility in biotechnology applications.Support or Funding InformationThis work was supported by the National Science Foundation (grant MCB‐1517138), National Institutes of Health (grant 1R15GM132817‐01), and by the Camille & Henry Dreyfus Foundation

"Catch and Release": A Variation of the Archetypal Nucleotidyl Transfer Reaction.

Nucleotidyl transfer is an archetypal enzyme reaction central to DNA replication and repair. Here we describe a variation of the nucleotidylation reaction termed "catch and release" that is used by an antibiotic modifying enzyme. The aminoglycoside nucleotidyl transferase 4' (ANT4') inactivates antibiotics such as kanamycin and neomycin through nucleotidylation within an active site that shares significant structural, and inferred underlying catalytic similarity, with human DNA polymerase beta. Here we follow the entire nucleotidyl transfer reaction coordinate of ANT4' covalently inactivating neomycin using X-ray crystallography. These studies show that although the underlying reaction mechanism is conserved with polymerases, a short 2.35 A hydrogen bond is initially formed to facilitate tight binding of the aminoglycoside substrate and is subsequently disrupted by the assembly of the catalytically active ternary complex. This enables the release of products post catalysis due to a lower free energy of the product state compared to the starting substrate complex. We propose that this "catch and release" mechanism of antibiotic turnover observed in ANT4' is a variation of nucleotidyl transfer that has been adapted for the inactivation of antibiotics.

Oxidative DNA-protein crosslinks formed in mammalian cells by abasic site lyases involved in DNA repair

Free radical attack on C1′ of deoxyribose forms the oxidized abasic (AP) site 2-deoxyribonolactone (dL). In vitro, dL traps the major base excision DNA repair enzyme DNA polymerase beta (Polβ) in covalent DNA-protein crosslinks (DPC) via the enzyme’s N-terminal lyase activity acting on 5′-deoxyribose-5-phosphate residues. We previously demonstrated formation of Polβ-DPC in cells challenged with oxidants generating significant levels of dL. Proteasome inhibition under 1,10-copper-ortho-phenanthroline (CuOP) treatment significantly increased Polβ-DPC accumulation and trapped ubiquitin in the DPC, with Polβ accounting for 60–70 % of the total ubiquitin signal. However, the identity of the remaining oxidative ubiquityl-DPC remained unknown. In this report, we surveyed whether additional AP lyases are trapped in oxidative DPC in mammalian cells in culture. Poly(ADP-ribose) polymerase 1 (PARP1), Ku proteins, DNA polymerase λ (Polλ), and the bifunctional 8-oxoguanine DNA glycosylase 1 (OGG1), were all trapped in oxidative DPC in mammalian cells. We also observed significant trapping of Polλ, PARP1, and OGG1 in cells treated with the alkylating agent methylmethane sulfonate (MMS), in addition to dL-inducing agents. Ku proteins, in contrast, followed a pattern of trapping similar to that for Polβ: MMS failed to produce Ku-DPC, while treatment with CuOP or (less effectively) H2O2 gave rise to significant Ku-DPC. Unexpectedly, NEIL1 and NEIL3 were trapped following H2O2 treatment, but not detectably in cells exposed to CuOP. The half-life of all the AP lyase-DPC ranged from 15−60 min, consistent with their active repair. Accordingly, CuOP treatment under proteasome inhibition significantly increased the observed levels of DPC in cultured mammalian cells containing PARP1, Ku protein, Polλ, and OGG1 proteins. As seen for Polβ, blocking the proteasome led to the accumulation of DPC containing ubiquitin. Thus, the ubiquitin-dependent proteolytic mechanisms that control Polβ-DPC removal may also apply to a broad array of oxidative AP lyase-DPC, preventing their toxic accumulation in cells.

Detection of Mutations in 81-bpRifampin Resistance Determining Region (RRDR) of rpoB gene in Mycobacterium tuberculosis using GeneXpert MTB/RIF in Clinical Specimens from Quetta, Pakistan

GeneXpert MTB/RIF has revolutionized the tuberculosis diagnosis by simultaneous detection of Mycobacterium tuberculosis and resistance to RIF (rifampicin), a surrogate marker for multidrug-resistant TB in less than two hours. The RIF-resistance pattern in Balochistan, Pakistan, is not documented. This study was aimed to detect RIF-resistant TB and mutations in RNA polymerase beta (rpoB) gene of M. tuberculosis within 81-bp RRDR in Quetta, Pakistan using GeneXpert® MTB/RIF assay. In total, 2300 clinical specimens were collected from suspected TB patients at Fatima Jinnah General and Chest Hospital Quetta, Pakistan between January and August 2017. These specimens were analyzed by GeneXpert® MTB/RIF assay. The data was statistically analyzed using SPSS software. Out of 2300 clinical specimens, M. tuberculosis was positive in in 899 (39.1%) cases by GeneXpert® MTB/RIF assay [positive respiratory cases 42.9% (871/2032) and non-respiratory 10.4% (28/268) with statistically significant difference (χ2= 104.5, p<0.001)]. Among 899 MTB positive cases, 46 (5.1%) were RIF-resistance caused by various rpoB gene mutations within 81-bp RRDR. Most of the RIF-resistant isolates were observed to harbor mutationsin Probe E 78.3% (n=36) whereas mutations in Probe A, B, D were observed 2.2% (n=1), 4.3% (n=2), and 6.5% (n=3), respectively. However, none of cases had RIF-resistance associated with Probe C. Out of 46 RRD cases, 21 (45.7 %) were males and 25 (54.3 %) were females. Additionally, Xpert® test showed higher detection rate than fluorescent microscopy (39.1% vs 31.2%, P<0.05) and detected MTB in 186 (11.8%) smear-negative specimens. Among 42 confirmed TB patients had MDR contact and eight patients were co-infected with HIV. In conclusion, 5.1% of the TB patients showed rifampicin resistance. The most frequent rpoB genetic mutations were observed in codons 531/533 (Probe E, 78.3%) whereas the least within the sequence 511 (Probe A, 2.2%).

Distinct roles of XRCC1 in genome integrity in Xenopus egg extracts.

Oxidative DNA damage represents one of the most abundant DNA lesions. It remains unclear how DNA repair and DNA damage response (DDR) pathways are co-ordinated and regulated following oxidative stress. While XRCC1 has been implicated in DNA repair, it remains unknown how exactly oxidative DNA damage is repaired and sensed by XRCC1. In this communication, we have demonstrated evidence that XRCC1 is dispensable for ATR-Chk1 DDR pathway following oxidative stress in Xenopus egg extracts. Whereas APE2 is essential for SSB repair, XRCC1 is not required for the repair of defined SSB and gapped plasmids with a 5'-OH or 5'-P terminus, suggesting that XRCC1 and APE2 may contribute to SSB repair via different mechanisms. Neither Polymerase beta nor Polymerase alpha is important for the repair of defined SSB structure. Nonetheless, XRCC1 is important for the repair of DNA damage following oxidative stress. Our observations suggest distinct roles of XRCC1 for genome integrity in oxidative stress in Xenopus egg extracts.

Construction and application of a “superplasmid” for enhanced production of antibiotics

More than two-third of known antibiotics are produced by actinomycetes of the genus Streptomyces. Unfortunately, the production rate from Streptomyces natural antibiotic is extremely slow and thus cannot satisfy industrial demand. In this study, the production of antibiotics by Streptomyces is enhanced by a "superplasmid" which including global regulatory factors afsR, cyclic adenosine receptor protein (CRP), RNA polymerase beta subunits (rpoB) with point mutation and acetyl coenzyme A carboxylase gene (accA2BE), these elements are controlled by the PermE* promoter and then transfer into Streptomyces coelicolor M145, Streptomyces mutabilis TRM45540, Streptomyces hygroscopicus XM201, and Streptomyces hygroscopicus ATCC29253 by conjugation to generate exconjugants. NMR, HPLC, and LC-MS analyses revealed that the superplasmid led to the overproduction of actinorhodin (101.90%), undecylprodigiosin (181.60%) in S. coelicolor M145:: pLQ003, of rapamycin (110%), hygrocin A (163.4%) in S. hygroscopicus ATCC29253:: pLQ003, and of actinomycin D (11.78%) in S. mutabilis TRM45540:: pLQ003, and also to the downregulation of geldanamycin in S. hygroscopicus XM201, but we found that mutant strains in mutant strains of S. hygroscopicus XM201 with regulatory factors inserted showed several peaks that were not found in wild-type strains. The results of the present work indicated that the regulator net working in Streptomyces was not uniform, the superplasmid we constructed possibly caused this overproduction and downregulation in different Streptomyces.

Clostridium perfringens type A Causing Necrotic Enteritis Outbreaks among Chickens in Egypt

Undoubtedly, necrotic enteritis is identified as one of the most threatening diseases which face poultry industry in Egypt and need radical solution to avoid huge economic losses. The present work was designed for typing Clostridium perfringens isolated from diseased chickens suspected to have necrotic enteritis from different outbreaks in six Governorates in Egypt (Gharbia, Dakhalia, Sharkia, Ismailia, North Sinai and Kafr El Sheikh) during the period from August to December 2018. Intestine and liver samples were obtained from eighty six diseased broiler chickens representing 60 flocks. C. perfringens was isolated and toxigenicity of the recovered isolates was determined by Nagler's and dermonecrotic reactions. Furthermore, multiplex polymerase chain reaction (PCR) targeting alpha, beta, epsilon and iota toxins' genes was performed for result confirmation and typing of the toxigenic isolates. Sixty-six C.perfringens isolates (38.37%) were recovered from 172 intestine and liver samples and twenty isolates (30.3%) were toxigenic and typed as C. perfringens type A producing alpha toxin only. These findings established the fact that alpha toxin is the only main toxin of C. perfringens type A which is basically responsible for its pathogenicity and virulence. In addition, most of the positively toxigenic isolates were isolated from hepatic lesions (15 isolates) rather than intestinal lesion (5 isolates). In conclusion, alpha toxin is a major toxin for NE development in chickens. Genotyping of Clostridium perfringens by multiplex PCR is a useful adjunct to diagnosis of necrotic enteritis in chickens.

Bacterial cell cycle and growth phase switch by the essential transcriptional regulator CtrA.

Many bacteria acquire dissemination and virulence traits in G1-phase. CtrA, an essential and conserved cell cycle transcriptional regulator identified in the dimorphic alpha-proteobacterium Caulobacter crescentus, first activates promoters in late S-phase and then mysteriously switches to different target promoters in G1-phase. We uncovered a highly conserved determinant in the DNA-binding domain (DBD) of CtrA uncoupling this promoter switch. We also show that it reprograms CtrA occupancy in stationary cells inducing a (p)ppGpp alarmone signal perceived by the RNA polymerase beta subunit. A simple side chain modification in a critical residue within the core DBD imposes opposing developmental phenotypes and transcriptional activities of CtrA and a proximal residue can direct CtrA towards activation of the dispersal (G1-phase) program. Hence, we propose that this conserved determinant in the CtrA primary structure dictates promoter reprogramming during the growth transition in other alpha-proteobacteria that differentiate from replicative cells into dispersal cells.

Mutation in DNA Polymerase Beta Causes Spontaneous Chromosomal Instability and Inflammation-Associated Carcinogenesis in Mice.

DNA polymerase beta (Pol β) is a key enzyme in the base excision repair (BER) pathway. Pol β is mutated in approximately 40% of human tumors in small-scale studies. The 5´-deoxyribose-5-phosphate (dRP) lyase domain of Pol β is responsible for DNA end tailoring to remove the 5’ phosphate group. We previously reported that the dRP lyase activity of Pol β is critical to maintain DNA replication fork stability and prevent cellular transformation. In this study, we tested the hypothesis that the human gastric cancer associated variant of Pol β (L22P) has the ability to promote spontaneous chromosomal instability and carcinogenesis in mice. We constructed a Pol β L22P conditional knock-in mouse model and found that L22P enhances hyperproliferation and DNA double strand breaks (DSBs) in stomach cells. Moreover, mouse embryonic fibroblasts (MEFs) derived from L22P mice frequently induce abnormal numbers of chromosomes and centrosome amplification, leading to chromosome segregation errors. Importantly, L22P mice exhibit chronic inflammation accompanied by stomach tumors. These data demonstrate that the human cancer-associated variant of Pol β can contribute to chromosomal instability and cancer development.

Bartonella Species Detected in Rodents from Eastern China.

Bartonella are vector borne gram-negative facultative intracellular bacteria. Bartonella species are associated with rodents and their flea parasites worldwide. The genetic variation and distribution of Bartonella species in rodents are not clear in China. We investigated the presence and genetic diversity of Bartonella species in rodents from eastern China. We captured rodents from 2015 to 2016 in Jiaonan County, Shandong Province, and detected Bartonella species in the spleen of rodents by PCR amplification of the citrate synthase (gltA) gene and RNA polymerase beta subunit (rpoB) gene. We found that 8.38% (16/191) of the rodents were Bartonella positive by PCR for both gltA and rpoB genes; that Bartonella sequences from the rodents were phylogenetically divided into five clades, which were closely related to B. tribocorum, B. rattimassiliensis, B. grahamii, B. fuyuanensis, and B. queenslandensis, respectively; and that each Bartonella species is rodent species-specific with B. rattimassiliensis and B. tribocorum for Rattus norvegicus, B. grahamii for Tscherskia triton, B. fuyuanensis for Apodemus agrarius, and B. queenslandensis for Niviventer confucianus. This study indicated that Bartonella organisms have a broad distribution and a variety of genotypes in rodents in eastern China and the threats to public health by these Bartonella species should be monitored in China.

Isoforms of Base Excision Repair Enzymes Produced by Alternative Splicing.

Transcripts of many enzymes involved in base excision repair (BER) undergo extensive alternative splicing, but functions of the corresponding alternative splice variants remain largely unexplored. In this review, we cover the studies describing the common alternatively spliced isoforms and disease-associated variants of DNA glycosylases, AP-endonuclease 1, and DNA polymerase beta. We also discuss the roles of alternative splicing in the regulation of their expression, catalytic activities, and intracellular transport.

Aberrant DNA Polymerase Beta Enhances H. pylori Infection Induced Genomic Instability and Gastric Carcinogenesis in Mice.

H. pylori is a significant risk factor of gastric cancer that induces chronic inflammation and oxidative DNA damage to promote gastric carcinoma. Base excision repair (BER) is required to maintain the genome integrity and prevent oxidative DNA damage. Mutation in DNA polymerase beta (Pol β) impacts BER efficiency and has been reported in approximately 30–40% of gastric carcinoma tumors. In this study, we examined whether reduced BER capacity associated with mutation in the POLB gene, along with increased DNA damage generated by H. pylori infection, accelerates gastric cancer development. By infecting a Pol β mutant mouse model that lacks dRP lyase with H. pylori, we show that reactive oxygen and nitrogen species (RONS) mediated DNA damage is accumulated in Pol β mutant mice (L22P). In addition, H. pylori infection in Leu22Pro (L22P) mice significantly increases inducible nitric oxide synthesis (iNOS) mediated chronic inflammation. Our data show that L22P mice exhibited accelerated H. pylori induced carcinogenesis and increased tumor incidence. This work shows that Pol β mediated DNA repair under chronic inflammation conditions is an important suppressor of H. pylori induced stomach carcinogenesis.

Phase 1/2 trial of FF-10502-01, a pyrimidine antimetabolite, in patients with advanced cholangiocarcinoma and solid tumors.

3008 Background: FF10502 is a synthetic pyrimidine nucleoside similar to gemcitabine (gem) with a sulfur in the pentose ring. FF10502 is a more potent inhibitor of DNA polymerase Beta than gem with activity in gem resistant patient (pt) derived xenograft models. FF10502 is avidly taken up into DNA and has greater activity against quiescent cells than gem. Methods: Pts > 18 years old with advanced disease who had progressed on standard of care were enrolled into 9 dose levels to determine maximum tolerated dose (MTD) and dose limiting toxicities (DLTs) and subsequently into two expansion cohorts: biliary or solid tumors (ST). FF10502 at doses of 8 to 135 mg/m2 was administered iv on days 1, 8, 15 of a 28-day cycle until progressive disease or toxicity. PK/PD evaluations were performed on all pts. Response was assessed by RECIST 1.1. Results: 76 pts were treated; 35 pts in dose escalation, including 7 cholangiocarcinoma pts. MTD was 90 mg/m2. DLTs included 2 pts with hypotension at 135mg/m2 (G3 and G4) and 1 pt each with G3 fatigue and G2 rash at 100mg/m2. In expansion, 19 cholangiocarcinoma, 3 gallbladder and 19 other pts (13 pancreatic, 2 urothelial, and 1 each ovarian, prostate, NSCLC, SCCHN each) were treated. 1 pt with prior rituximab for ITP developed PML. G3 treatment-related low platelets occurred in 3 pts at 90mg/m2 after cycle 1. There were 5 partial responses (PRs), including 4 pts who had progressed on prior gemcitabine: 3 of 26 pts with cholangiocarcinoma, 1 urothelial carcinoma and 1 chondroblastic osteosarcoma. 7 cholangiocarcinoma pts stayed on therapy for ≥6 months. FF10502 incorporation intoperipheral blood cellular DNA was seen, andbiomarkeranalysisdata to identify pts with higher potential for clinical response will be presented. Conclusions: FF10502 is well tolerated in pts with advanced cancers refractory to standard therapies. Early signals of efficacy warranting further exploration were seen in heavily pretreated cholangiocarcinoma pts (median: 4 prior therapies). Patient selection based on differential effects of FF10502 on DNA polymerases will be explored. Clinical trial information: NCT02661542.