Characterized Resistance Mechanisms Research Articles

Deciphering resistance mechanisms in cancer: final report of MATCH-R study with a focus on molecular drivers and PDX development

BackgroundUnderstanding the resistance mechanisms of tumor is crucial for advancing cancer therapies. The prospective MATCH-R trial (NCT02517892), led by Gustave Roussy, aimed to characterize resistance mechanisms to cancer treatments through molecular analysis of fresh tumor biopsies. This report presents the genomic data analysis of the MATCH-R study conducted from 2015 to 2022 and focuses on targeted therapies.MethodsThe study included resistant metastatic patients (pts) who accepted an image-guided tumor biopsy. After evaluation of tumor content (TC) in frozen tissue biopsies, targeted NGS (10 < TC < 30%) or Whole Exome Sequencing and RNA sequencing (TC > 30%) were performed before and/or after the anticancer therapy.Patient-derived xenografts (PDX) were established by implanting tumor fragments into NOD scid gamma mice and amplified up to five passages.ResultsA total of 1,120 biopsies were collected from 857 pts with the most frequent tumor types being lung (38.8%), digestive (16.3%) and prostate (14.1%) cancer. Molecular targetable driver were identified in 30.9% (n = 265/857) of the patients, with EGFR (41.5%), FGFR2/3 (15.5%), ALK (11.7%), BRAF (6.8%), and KRAS (5.7%) being the most common altered genes. Furthermore, 66.0% (n = 175/265) had a biopsy at progression on targeted therapy. Among resistant cases, 41.1% (n = 72/175) had no identified molecular mechanism, 32.0% (n = 56/175) showed on-target resistance, and 25.1% (n = 44/175) exhibited a by-pass resistance mechanism. Molecular profiling of the 44 patients with by-pass resistance identified 51 variants, with KRAS (13.7%), PIK3CA (11.8%), PTEN (11.8%), NF2 (7.8%), AKT1 (5.9%), and NF1 (5.9%) being the most altered genes. Treatment was tailored for 45% of the patients with a resistance mechanism identified leading to an 11 months median extension of clinical benefit.A total of 341 biopsies were implanted in mice, successfully establishing 136 PDX models achieving a 39.9% success rate. PDX models are available for EGFR (n = 31), FGFR2/3 (n = 26), KRAS (n = 18), ALK (n = 16), BRAF (n = 6) and NTRK (n = 2) driven cancers. These models closely recapitulate the biology of the original tumors in term of molecular alterations and pharmacological status, and served as valuable models to validate overcoming treatment strategies.ConclusionThe MATCH-R study highlights the feasibility of on purpose image guided tumor biopsies and PDX establishment to characterize resistance mechanisms and guide personalized therapies to improve outcomes in pre-treated metastatic patients.Graphical

TRKing down drug resistance in NTRK fusion-positive cancers†.

In a recent issue of The Journal of Pathology, Chen and colleagues established novel patient-derived ex vivo models of NTRK fusion-positive soft tissue sarcoma to characterize resistance mechanisms against targeted therapy with tyrosine kinase inhibitors. Prolonged exposure to escalating concentrations of the tyrosine kinase inhibitor, entrectinib, ultimately led to the occurrence of resistant clones that harbored an inactivating mutation in the NF2 gene, not previously described in this context, accompanied by increased PI3K/AKT/mTOR and Ras/Raf/MEK/ERK signaling. Finally, an inhibitor screen identified, among others, MEK and mTOR inhibitors as potential combination agents. © 2024 The Pathological Society of Great Britain and Ireland.

Rolling the evolutionary dice: Neisseria commensals as proxies for elucidating the underpinnings of antibiotic resistance mechanisms and evolution in human pathogens.

Species within the genus Neisseria are adept at sharing adaptive allelic variation, with commensal species repeatedly transferring resistance to their pathogenic relative Neisseria gonorrhoeae. However, resistance in commensals is infrequently characterized, limiting our ability to predict novel and potentially transferable resistance mechanisms that ultimately may become important clinically. Unique evolutionary starting places of each Neisseria species will have distinct genomic backgrounds, which may ultimately control the fate of evolving populations in response to selection as epistatic and additive interactions coerce lineages along divergent evolutionary trajectories. Alternatively, similar genetic content present across species due to shared ancestry may constrain existing adaptive solutions. Thus, identifying the paths to resistance across commensals may aid in characterizing the Neisseria resistome-or the reservoir of alleles within the genus as well as its depth. Here, we use in vitro evolution of four commensal species to investigate the potential and repeatability of resistance evolution to two antimicrobials, the macrolide azithromycin and the β-lactam penicillin. After 20 days of selection, commensals evolved resistance to penicillin and azithromycin in 11/16 and 12/16 cases, respectively. Almost all cases of resistance emergence converged on mutations within ribosomal components or the mtrRCDE efflux pump for azithromycin-based selection and mtrRCDE, penA, and rpoB for penicillin selection, thus supporting constrained adaptive solutions despite divergent evolutionary starting points across the genus for these particular drugs. Though drug-selected loci were limited, we do identify novel resistance-imparting mutations. Continuing to explore paths to resistance across different experimental conditions and genomic backgrounds, which could shunt evolution down alternative evolutionary trajectories, will ultimately flesh out the full Neisseria resistome.IMPORTANCENeisseria gonorrhoeae is a global threat to public health due to its rapid acquisition of antibiotic resistance to all first-line treatments. Recent work has documented that alleles acquired from close commensal relatives have played a large role in the emergence of resistance to macrolides and beta-lactams within gonococcal populations. However, commensals have been relatively underexplored for the resistance genotypes they may harbor. This leaves a gap in our understanding of resistance that could be rapidly acquired by the gonococcus through a known highway of horizontal gene exchange. Here, we characterize resistance mechanisms that can emerge in commensal Neisseria populations via in vitro selection to multiple antimicrobials and begin to define the number of paths to resistance. This study, and other similar works, may ultimately aid both surveillance efforts and clinical diagnostic development by nominating novel and conserved resistance mechanisms that may be at risk of rapid dissemination to pathogen populations.

Untargeted proteomic differences between clinical strains of methicillin-sensitive and methicillin-resistant Staphylococcusaureus

Staphylococcus aureus is a common disease-causing bacterium that has developed resistances to a wide variety of antibiotics. This increasing antibiotic resistance has made management of these infections difficult. A better understanding of the general differences among clinical S. aureus strains beyond the well characterized resistance mechanisms may help in identifying new anti-microbial targets. This study aimed to identify and compare the general differences in protein profiles among clinical strains of S. aureus sensitive and resistant to methicillin. The proteomic profiles of five methicillin sensitive (MSSA) and five methicillin resistant (MRSA) S. aureus strains were analyzed by ultra-performance liquid chromatography-mass spectrometry. Protein identification was done using Progenesis QI for Proteomics and the UniProt S. aureus database. Proteins that play roles in virulence, metabolism, and protein synthesis were found to be present at different abundances between MSSA and MRSA (Data available via ProteomeXchange with identifier PXD021629). This study shows differences in protein profiles between antibiotic sensitive and antibiotic resistant clinical strains of S. aureus that may affect the resistance mechanism. Further research on these differences may identify new drug targets against methicillin resistant S. aureus strains.

Abstract LB227: Leveraging novel Dato-DXd resistance models to inform biomarker discovery and rational combinations to combat drug resistance

Abstract Datopotamab deruxtecan (Dato-DXd) is an antibody-drug conjugate (ADC) consisting of a humanized anti-TROP2 IgG1 monoclonal antibody covalently linked to a highly potent topoisomerase I (TOP1) inhibitor payload via a stable, tumor-selective, tetrapeptide-based cleavable linker. Despite its promising early clinical signals, drug resistance is a challenge that may emerge with time. The mechanisms of Dato-DXd resistance are currently unknown. Herein, we created and investigated novel models of Dato-DXd resistance to characterize resistance mechanisms and identify novel combinations to overcome Dato-DXd resistance. We used a cyclical dosing method to develop two Dato-DXd acquired resistant NCI-N87 gastric cancer cell line models from in vitro cells (N87-IVR) and in vivo tumors (N87-EVR). The N87-IVR and N87-EVR cells were found to be more than 40-fold resistant to Dato-DXd compared to the parental N87 cells. First, we assessed TROP2 levels, Dato-DXd binding and expression of drug efflux pumps known to have TOP1-inhibitor substrates between the parental and resistant cell populations. Minor changes in each of these were not sufficient to drive Dato-DXd resistance in the drug-tolerant models. Subsequently, we performed a proteogenomic analysis to identify which proteins and pathways were modulated in the resistant phenotype. Both the N87-IVR and N87-EVR cells were divergent from the parental N87 cells and, interestingly, they showed similar yet distinct profiles. Among other changes, our analysis revealed a dramatic loss of SLFN11, a putative DNA/RNA helicase, in both resistant models. Previously published data has shown that a combination with an ATR inhibitor (ATRi) can re-sensitize TOP1 inhibitor-resistant cells in SLFN11-low settings. Consistent with these data, we observed that the ATRi ceralasertib (AZD6738) in combination with Dato-DXd re-sensitizes the resistant NCI-N87 cells in vitro and in vivo. In addition to SLFN11 loss, we also identified a number of other modulations with the potential to drive resistance, e.g. downregulation of the caveolin internalization machinery and interferon response genes. These findings help us refine the mechanism of action of Dato-DXd and shed light on the potential mechanisms of Dato-DXd resistance that may emerge clinically. These data have been used to uncover new biomarkers of Dato-DXd sensitivity and can lead to the development of new clinical strategies to combat Dato-DXd resistance, e.g. the combination with ATRi. Citation Format: Anna Przybyla, Chetan Rane, Gopi Patel, Deepa Bhavsar, Shenghua Wen, David Clark, Yeoun Jin Kim, Alan Lau, Yann Wallez, Jay Mettetal, Wenyan Zhong, Matthew Sung. Leveraging novel Dato-DXd resistance models to inform biomarker discovery and rational combinations to combat drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB227.

The Cell Wall, Cell Membrane and Virulence Factors of Staphylococcus aureus and Their Role in Antibiotic Resistance.

Antibiotic resistant strains of bacteria are a serious threat to human health. With increasing antibiotic resistance in common human pathogens, fewer antibiotics remain effective against infectious diseases. Staphylococcus aureus is a pathogenic bacterium of particular concern to human health as it has developed resistance to many of the currently used antibiotics leaving very few remaining as effective treatment. Alternatives to conventional antibiotics are needed for treating resistant bacterial infections. A deeper understanding of the cellular characteristics of resistant bacteria beyond well characterized resistance mechanisms can allow for increased ability to properly treat them and to potentially identify targetable changes. This review looks at antibiotic resistance in S aureus in relation to its cellular components, the cell wall, cell membrane and virulence factors. Methicillin resistant S aureus bacteria are resistant to most antibiotics and some strains have even developed resistance to the last resort antibiotics vancomycin and daptomycin. Modifications in cell wall peptidoglycan and teichoic acids are noted in antibiotic resistant bacteria. Alterations in cell membrane lipids affect susceptibility to antibiotics through surface charge, permeability, fluidity, and stability of the bacterial membrane. Virulence factors such as adhesins, toxins and immunomodulators serve versatile pathogenic functions in S aureus. New antimicrobial strategies can target cell membrane lipids and virulence factors including anti-virulence treatment as an adjuvant to traditional antibiotic therapy.

641. Activity of Gepotidacin Tested Against Molecularly Characterized Escherichia coli Isolates Resistant to Commonly Used Oral Therapies for UTI in the US (2019-2020)

Abstract Background Gepotidacin is a novel first in class triazaacenaphthylene antibiotic in Phase 3 clinical trials for the treatment of gonorrhea and uncomplicated urinary tract infection (UTI). This study evaluates the epidemiology of E. coli (EC) causing UTI in US patients and the activity of gepotidacin and comparators against various subsets, including those with characterized resistance mechanisms. Methods 1,993 EC collected from 45 US sites were included as part of the Gepotidacin UTI Global Surveillance Study as part of the SENTRY Antimicrobial Surveillance Program (2019-2020). Isolates were tested for susceptibility (S) by CLSI methods and CLSI interpretations were applied. Isolates that met MIC criteria for screening of extended-spectrum β-lactamase (ESBL) genes were subjected to genome sequencing followed by ESBL gene screening and epidemiology typing (MLST, O:H, and fimH). Results A total of 84.4% (1,682/1,993) EC were ESBL negative (Table). Among these isolates, 16.5% (278/1,682) were not S to the fluoroquinolones (FQ) ciprofloxacin and/or levofloxacin, whereas 26.2% (440/1,682) were not S to trimethoprim-sulfamethoxazole (SXT). An ESBL phenotype was noted in 15.6% (311/1,993) of EC, which tended to be mostly not S to the FQs (79.7%), SXT (61.7%), amoxicillin-clavulanate (52.9%), and oral cephalosporins (99.7%). Most ESBL isolates carried CTX-M alleles alone (81.4%; 253/311), whereas 9.6% (30/311) had plasmid AmpC genes. Approximately half (56.3%; 175/311) of ESBL isolates belonged to clonal complex (CC) 131, of which 70.9% (124/175) were O25b:H4 and carried fimH30. Overall, gepotidacin had MIC90 of 2-4 mg/L against various phenotypic/genotypic subsets. Conclusion High rates of EC not S to commonly used oral agents (FQs and SXT) were observed. ESBL phenotype further compromised the activity of oral agents, including oral cephalosporins. Gepotidacin had potent and stable in vitro activity against various subsets, including the resistant CC131 O25b:H4 clone. These data support the further clinical development of gepotidacin as a treatment option for UTI caused by EC, including resistant isolates against which other oral treatment options are limited. Disclosures Rodrigo E. Mendes, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Nabriva Therapeutics: Grant/Research Support|Office for Assistant Secretary of Defense for Health Affairs: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support|Spero Therapeutics: Grant/Research Support SJ Ryan Arends, PhD, AbbVie: Grant/Research Support|GSK: Grant/Research Support|Nabriva Therapeutics: Grant/Research Support|Shionogi: Grant/Research Support John H. Kimbrough, PhD, AbbVie: Grant/Research Support|GSK: Grant/Research Support Valerie Kantro, BA, AbbVie: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Shionogi: Grant/Research Support Nicole E. Scangarella-Oman, MS, GlaxoSmithKline plc.: Employee and shareholder Jennifer M. Streit, BS, MT(ASCP), Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Shionogi: Grant/Research Support Mariana Castanheira, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support.

Antibiotic Susceptibility Profiles and Resistance Mechanisms to β-Lactams and Polymyxins of Escherichia coli from Broilers Raised under Intensive and Extensive Production Systems.

The intensive and extensive broiler production systems imply different veterinary interventions, including the use of antimicrobials. This study aimed to compare the antimicrobial susceptibility profiles of Escherichia coli isolated from both systems, characterize resistance mechanisms to β-lactams and polymyxins, and identify genetic elements such as integrons. E. coli isolates recovered from broiler cecal samples were assayed for antimicrobial susceptibility through the broth microdilution technique. The molecular characterization of acquired resistance mechanisms to β-lactams and colistin and the detection of integrons was performed by a multiplex PCR. For most antibiotics tested, the prevalence of reduced susceptibility is higher in commensal and extended-spectrum β-lactamases (ESBL)/AmpC producers from broilers raised in the intensive system, compared with those raised under extensive conditions. SHV-12 was the most common ESBL enzyme found in both production systems. Other ESBL variants such as CTX-M-1, CTX-M-55, CTX-M-14, CTX-M-32, CTX-M-9, TEM-52, and plasmid-encoded AmpC enzyme CMY-2 were also present. MCR-1 was identified in a colistin-resistant isolate from broilers raised under the intensive system. This study highlights the differences in E. coli antibiotic susceptibility from both production types and emphasizes that a great deal of work remains to decrease consumption and antimicrobial resistance levels.

Genetic correlation of crizotinib efficacy and resistance in ALK- rearranged non-small-cell lung cancer

ObjectivesCrizotinib remains one of the most commonly used targeted therapies for ALK fusion-positive patients. However, the mutational profiles and mechanisms of resistance to first-line crizotinib treatment remain to be thoroughly examined. Materials and MethodsWe retrospectively reviewed 125 ALK-positive patients with histological and/or cytological diagnosis of NSCLC. Of these, baseline samples were available from 62 patients and 63 had resistance samples following first-line crizotinib treatment, with 18 patients having paired baseline and resistance samples. All patients were genetically profiled by NGS using a 139 lung cancer gene panel (Pulmocan®, Nanjing Geneseeq Technology Inc.). Survival associations of progression-free survival (PFS) and resistance mechanisms were evaluated in relation to ALK fusion variants and background genetic alterations. ResultsThe median age of the cohort was 53 years old (range 26–78; 46.4 % females). Three novel ALK fusion partners were identified, including PSME4, cullin3 (CUL3) and coiled-coil domain containing 85A (CCDC85A). Among the different ALK fusion genes, patients carrying the v3 variant experienced worse PFS outcome compared with other non-v3 fusions (P = 0.01) in response to first-line crizotinib. Profiling of the genetic landscape revealed TP53 as the most frequently co-mutated gene, alterations of which were associated with unfavorable outcome (P = 0.024) and were among the secondary acquired mutations in the resistance samples. Examinations of the resistance mechanisms showed that the v3 variant was more likely to acquire ALK activating mutations (P = 0.04). Off-target resistance mechanisms included mutations in genes in the RAS/MAPK and its parallel pathway genes, such as ERBB2, BRAF, KRAS, FGFR3, NF1 and CREBBP. ConclusionIn this study, through profiling of the mutational landscape of ALK-positive advanced NSCLCs both at baseline and disease progression, we characterized resistance mechanisms and molecular correlations of PFS in response to first-line crizotinib. Our findings may facilitate rational selection of subsequent ALK TKIs in the clinic.

Sentinel Surveillance Reveals Emerging Daptomycin-Resistant ST736 Enterococcus faecium and Multiple Mechanisms of Linezolid Resistance in Enterococci in the United States.

Enterococcus faecalis and faecium with resistance to daptomycin and/or linezolid are emerging globally. We present the genomic characterization of daptomycin- and linezolid-resistant E. faecalis and E. faecium surveillance isolates from the United States, 2013–2016. Daptomycin resistance was low among E. faecalis (2/364, 0.5%) and E. faecium (17/344, 5%). The majority (71%, 12/17) of daptomycin-resistant E. faecium isolates belonged to the emerging ST736 clone and contained mutations in liaFSR and cls previously associated with resistance. However, 1/2 E. faecalis and 3/17 E. faecium did not contain these mutations previously associated with daptomycin resistance. Linezolid resistance was rare among E. faecalis (1/364, 0.3%) and E. faecium (2/344, 0.6%). These two E. faecium isolates, one of which was also resistant to daptomycin and vancomycin, contained the 23S rRNA nucleotide mutation (G2576T) associated with linezolid resistance. Long-read sequencing revealed the linezolid-resistant E. faecalis isolate contained chromosomal- and plasmid-encoded copies of optrA. The chromosomal optrA was located on the recently described Tn6674 multiresistance transposon. The second copy of optrA was encoded on an ∼65 kb mosaic plasmid, with component regions sharing high sequence identity to optrA-encoding multiresistance plasmids of animal origin. The optrA-encoding plasmid contained open reading frames predicted to encode proteins associated with a pheromone-responsive plasmid transfer system, and filter mating experiments confirmed the plasmid was conjugative. Continued surveillance of enterococci is necessary to assess the prevalence and trends of daptomycin and linezolid resistance in the United States, characterize resistance mechanisms and how they transfer, and monitor for emerging sequence types associated with resistance.

The MK2/Hsp27 axis is a major survival mechanism for pancreatic ductal adenocarcinoma under genotoxic stress.

Combination chemotherapies remain the cornerstone treatment for pancreatic ductal adenocarcinoma (PDAC), but de novo and acquired resistance is common. In this study, we aimed to identify and characterize resistance mechanisms to a FIRINOX chemotherapy regimen (a combination of 5-fluorouracil, irinotecan, and oxaliplatin) because it is the most aggressive regimen currently used clinically for patients with PDAC. Using an unbiased reverse-phase protein array, we detected phospho-activation of heat shock protein 27 (Hsp27) as the most up-regulated event after FIRINOX treatment in PDAC cells. Silencing HSP27 by RNA interference or by a small-molecule inhibitor enhanced apoptosis caused by FIRINOX in vitro. Mechanistically, FIRINOX up-regulated tumor necrosis factor–α (TNFα), causing autocrine phosphorylation and activation of transforming growth factor–β–activated kinase 1 (TAK1), MAPK activated protein kinase 2 (MAPKAPK2 or MK2), and, ultimately, Hsp27. Targeting MK2, the kinase that directly phosphorylates Hsp27, abrogated Hsp27 activation, sensitized PDAC cells to apoptosis, and suppressed SN-38–induced protective autophagy in vitro, in part by blocking phospho-activation of Beclin1. In an autochthonous PDAC mouse model, the MK2 inhibitor ATI-450 decreased PDAC development and progression. When combined with FIRINOX, ATI-450 eliminated most PDAC foci and marked prolonged mouse survival without causing additional toxicity. Last, we found that high phospho-MK2 expression in tumors was associated with poorer survival of patients with PDAC. Our study identified MK2 as a mediator of genotoxic stress–induced activation of prosurvival pathways and provides preclinical support for combining an MK2 inhibitor with FIRINOX-based chemotherapies to treat PDAC.

P76.27 ORCHARD: A Biomarker-Directed Phase 2 Platform Study in pts with Advanced EGFRm NSCLC Progressing on First-Line Osimertinib

Osimertinib is a third-generation, irreversible, oral epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) that potently and selectively inhibits both EGFR-TKI sensitizing (EGFRm) and EGFR T790M resistance mutations, and has demonstrated efficacy in EGFRm advanced NSCLC, including central nervous system metastases. Most patients’ (pts) tumors develop resistance to osimertinib through secondary EGFR mutations (including C797S, G724S and L718V/Q) and off-target genomic alterations (including MET amplification and gene fusions). Data suggest that off-target alterations such as ALK and RET fusions may be resistance mechanisms to first-line osimertinib in some pts (each <5%). The ORCHARD study (NCT03944772) aims to better characterize resistance mechanisms to first-line osimertinib, and evaluate post-progression treatments. The flexible, platform-based design allows for new cohorts and treatment arms, for broader exploration of targeting resistance mechanisms. Previously presented: WCLC 2019 (Yu H, et al. J Thorac Oncol;14[10 suppl]:S647). In this open-label, multicenter, multidrug, biomarker-directed Phase 2 platform-based study (Figure), adult pts with locally advanced/metastatic EGFRm NSCLC whose disease progressed on first-line osimertinib are eligible. Treatment is assigned per molecular characterization of a mandatory tissue biopsy from a progressing lesion, analyzed by next-generation sequencing (Foundation Medicine Inc.). The protocol has recently been amended: Group A now includes treatment arms for pts with ALK/RET rearrangements, who will receive osimertinib plus alectinib/selpercatinib, respectively. The ‘MET alterations’ arm has been expanded from MET-amplification to also include MET exon 14 skipping, and the ‘EGFR alterations’ arm has been expanded from EGFR-amplification to also include mutations at the EGFR L718/G724 residue, or insertion in EGFR exon 20. Group B encompasses pts with no identifiable resistance mechanism; Group C is observational, for pts whose tumors harbor resistance mechanisms not currently addressed within the study. Tumor assessments (RECIST 1.1) will be performed every 6 weeks for 24 weeks, and every 9 weeks thereafter until disease progression or treatment discontinuation. Interim analyses will be performed on the first 16 pts in each cohort who have had the opportunity for two post-baseline RECIST assessments; individual cohorts may be stopped (if there is <10% probability for objective response rate [ORR] to be above target value [45%, equaling ≤4 of the 16 pts with confirmed responses]), or expanded to 30–40 pts for further evaluation. Primary endpoint: confirmed ORR (investigator assessed); secondary endpoints include: progression-free survival, duration of response, overall survival, and pharmacokinetics. Safety will also be reported. A summary of the observed genomic landscape during enrolment will be presented. osimertinib, non-small cell lung cancer, resistance

Molecular characterization of western corn rootworm pyrethroid resistance.

Western corn rootworm (WCR) pyrethroid resistance has been confirmed in the western US Corn Belt. Toxicological and biochemical studies indicated that multiple mechanisms of resistance might be involved in the resistance trait, such as enhanced metabolism and/or kdr target-site mutation(s) in the voltage-gated sodium channels. To characterize the mechanisms of WCR pyrethroid resistance at the molecular level, pairwise comparisons were made between RNA-Seq data collected from pyrethroid-resistant and -susceptible WCR populations. Gene expression levels and sodium channel sequences were evaluated. Seven transcripts exhibited significantly different expression (q ≤ 0.05) when comparing field-collected pyrethroid-resistant (R-Field) and -susceptible (S-Field) WCR populations. Three of the differentially expressed transcripts were P450s overexpressed in R-Field (9.2-26.2-fold). A higher number (99) of differentially expressed transcripts was found when comparing laboratory-derived pyrethroid-resistant (R-Lab) and -susceptible (S-Lab) WCR populations. Eight of the significant transcripts were P450s overexpressed in R-Lab (2.7-39.8-fold). This study did not detect kdr mutations in pyrethroid-resistant WCR populations. Other differentially expressed transcripts that may play a role in WCR pyrethroid resistance are discussed. This study revealed that P450-mediated metabolism is likely to be a major mechanism of WCR pyrethroid resistance, which could affect the efficacy of other insecticides sharing similar metabolic pathways. Additionally, results suggested that although laboratory selection of a pyrethroid-resistant WCR population may help to characterize resistance mechanisms, a field-selected population provided rare and perhaps major variants corresponding to the resistance trait.

Activity of cefepime/zidebactam (WCK 5222) against Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii endemic to New York City medical centres.

The combination of cefepime and zidebactam (WCK5222), a novel β-lactam enhancer, has demonstrated activity against a wide variety of Gram-negative pathogens and is currently under clinical evaluation. To examine the activity of cefepime/zidebactam against: (i) a contemporary collection of Gram-negative isolates from New York City; (ii) a collection of carbapenem-resistant clinical isolates; and (iii) a collection of isolates with characterized resistance mechanisms. Susceptibility tests were performed using broth microdilution for cefepime, zidebactam and cefepime/zidebactam (1:1). More than 99% of a contemporary collection of Escherichia coli, Klebsiella pneumoniae and Enterobacter spp. had cefepime/zidebactam MICs ≤2 mg/L, the susceptibility breakpoint for cefepime. For K. pneumoniae, the acquisition of blaKPC resulted in increased MICs, although MICs remained ≤2 mg/L for 90% of KPC-possessing isolates. Overall for Pseudomonas aeruginosa, 98% of isolates had MICs ≤8 mg/L and MICs were affected by increased expression of ampC. For carbapenem-resistant P. aeruginosa, 78% of isolates had cefepime/zidebactam MICs ≤8 mg/L. The activity of cefepime/zidebactam against Acinetobacter baumannii was lower, with 85% of all isolates and 34% of carbapenem-resistant isolates with MICs ≤8 mg/L (cefepime interpretative criteria). Cefepime/zidebactam demonstrated excellent activity against Enterobacteriaceae and P. aeruginosa, although activity was reduced in carbapenem-non-susceptible isolates. The activity against A. baumannii was reduced and studies examining the therapeutic efficacy in strains with high cefepime/zidebactam MICs are warranted.

Resistance to MAPK Inhibitors in Melanoma Involves Activation of the IGF1R-MEK5-Erk5 Pathway.

Combined treatment of metastatic melanoma with BRAF and MEK inhibitors has improved survival, but the emergence of resistance represents an important clinical challenge. Targeting ERK is a suitable strategy currently being investigated in melanoma and other cancers. To anticipate possible resistance to ERK inhibitors (ERKi), we used SCH772984 (SCH) as a model ERKi to characterize resistance mechanisms in two BRAF V600E melanoma cell lines. The ERKi-resistant cells were also resistant to vemurafenib (VMF), trametinib (TMT), and combined treatment with either VMF and SCH or TMT and SCH. Resistance to SCH involved stimulation of the IGF1R-MEK5-Erk5 signaling pathway, which counteracted inhibition of Erk1/2 activation and cell growth. Inhibition of IGF1R with linsitinib blocked Erk5 activation in SCH-resistant cells and decreased their growth in 3D spheroid growth assays as well as in NOD scid gamma (NSG) mice. Cells doubly resistant to VMF and TMT or to VMF and SCH also exhibited downregulated Erk1/2 activation linked to stimulation of the IGF1R-MEK5-Erk5 pathway, which accounted for resistance. In addition, we found that the decreased Erk1/2 activation in SCH-resistant cells involved reduced expression and function of TGFα. These data reveal an escape signaling route that melanoma cells use to bypass Erk1/2 blockade during targeted melanoma treatment and offer several possible targets whose disruption may circumvent resistance. SIGNIFICANCE: Activation of the IGF1R-MEK5-Erk5 signaling pathway opposes pharmacologic inhibition of Erk1/2 in melanoma, leading to the reactivation of cell proliferation and acquired resistance.

Kernel volatiles of some pigmented wheats do not elicit a preferential orientation in Sitophilus granarius adults

The granary weevil, Sitophilus granarius (L.) (Coleoptera, Dryophthoridae), is a primary pest of stored cereals worldwide. To identify possible sources of wheat resistance toward this pest, two commercial durum and bread wheat varieties with yellow pericarp were compared with anthocyanin-pigmented durum and bread wheat genotypes that have never entered commercial production. The composition of the kernel head-space volatile organic compounds (VOCs) and the olfactory responses of granary weevil adults to these kernel VOCs were investigated. Head-space solid-phase micro-extraction and gas chromatography–mass spectrometry analysis highlighted 17 and 13 kernel VOCs from durum and bread wheats, respectively. These compounds mainly included aldehydes and alcohols, and to a lesser extent, terpenes and benzene derivatives. Quantitative and qualitative differences were seen between the odor profiles of yellow and pigmented wheat kernels. In two-choice behavioral bioassays, granary weevil adults were significantly attracted by the kernel odors from the yellow commercial wheat varieties and their hexane extracts, but not by those of the pigmented wheat genotypes and their hexane extracts. Electroantennography confirmed the presence of VOCs in all of the hexane extracts that stimulated the olfactory system of both sexes of the granary weevil in a dose-dependent manner. Thus, differences among the odor blends were responsible for the different olfactory responses of granary weevils to the yellow and pigmented wheat kernels. These differences in VOC emissions and olfactory responses induced in granary weevils by the yellow and pigmented wheat kernels can be exploited to characterize resistance mechanisms associated with different genotypes and to incorporate resistance into improved varieties.

Pisatin involvement in the variation of inhibition of Fusarium oxysporum f. sp. pisi spore germination by root exudates of Pisum spp. germplasm

Fusarium wilt caused by Fusarium oxysporum f. sp. pisi (Fop) is one of the major constraints of pea worldwide. Its control is difficult and is mainly based on the use of resistant cultivars. This study aimed to identify and characterize resistance mechanisms interfering with Fop spore germination, as an additional pre‐penetration resistance mechanism little explored so far. For this, root exudates were collected from 12 pea accessions with differential responses to the disease, from resistant to susceptible, and their effects on Fop germination and growth were determined. While root exudates from most accessions stimulated Fop germination, the root exudates of three accessions, JI 1412, JI 2480 and P42, did not stimulate, or even inhibited, Fop germination. Although some additional compounds might be involved, the analysis showed that the most active metabolite was the pea phytoalexin pisatin. Pisatin was identified in the active fraction of pea root exudate extracts and its amount in the root exudates was negatively correlated with the extent of Fop germination. This suggests an important role of pisatin in the constitutive defence of pea against F. oxysporum.

Mechanisms of resistance against NITD-916, a direct inhibitor of Mycobacterium tuberculosis InhA

Isoniazid inhibits Mycobacterium tuberculosis InhA and is a key component of drug regimens that treat tuberculosis. However, the high rate of resistance against isoniazid is a contributing factor to the emergence of multi-drug resistance strains of M. tuberculosis. The 4-hydroxy-2-pyridine NITD-916 is a direct inhibitor of M. tuberculosis InhA that has comparable efficacy to isoniazid in mouse models of TB infection but a lower frequency of resistance. To characterize resistance mechanisms against NITD-916 we isolated resistant mutants in H37Rv (Euro-American lineage) and HN878 (East-Asian lineage) strains of M. tuberculosis. The resistance frequency was similar in both strains. Mutations were identified in residues within or near to the active of InhA or in the fabG1inhA promoter region. All mutants were resistant to NITD-916 but were not cross resistant to isoniazid, despite homology to SNPs identified in isoniazid resistant clinical isolates.

Distribution of resistance genetic determinants among Vibrio cholerae isolates of 2012 and 2013 outbreaks in IR Iran.

The objective of this study was to characterize antimicrobial resistance determinants in relation to antimicrobial susceptibility and genotyping profile in 20 clinical isolates of Vibrio cholerae. All of the isolates were resistant to streptomycin. The second most prevalent resistance was observed to trimethoprim (75%), co-trimoxazole (60%), tetracycline (50%), and minocycline (45%). About 50% of the isolates fulfilled the criteria of Multi Drug Resistance (MDR) phenotype. None of the isolates carried tet A, B, C, and, D determinants. This finding shows that tetracycline resistance determinants recognized so far, does not satisfactorily describe the 50% tetracycline resistance phenotype in this study, suggesting the possible contribution of other not yet characterized resistance mechanisms involved. Class 1 integron, widely distributed among enteric bacteria, was not detected among V.cholerae strains under study. Conversely, 100% of the isolates harbored SXT constin(int), among which 70% were positive for dfrA1, strA, and strB genes. The sul1gene was present in 60% of the isolates while none of them contained floR gene. All the isolates uniformly appeared to be identical in fingerprinting profiles expected from outbreak strains. In conclusion, SXT element with its mosaic structure was the exclusive antimicrobial resistance determinant of clonal V.cholerae isolates taken from outbreaks of 2012 and 2013 in Iran.

Inhibition of oxidative phosphorylation suppresses the development of osimertinib resistance in a preclinical model of EGFR-driven lung adenocarcinoma.

Metabolic plasticity is an emerging hallmark of cancer, and increased glycolysis is often observed in transformed cells. Small molecule inhibitors that target driver oncogenes can potentially inhibit the glycolytic pathway. Osimertinib (AZD9291) is a novel EGFR tyrosine kinase inhibitor (TKI) that is potent and selective for sensitising (EGFRm) and T790M resistance mutations. Clinical studies have shown osimertinib to be efficacious in patients with EGFRm/ T790M advanced NSCLC who have progressed after EGFR-TKI treatment. However experience with targeted therapies suggests that acquired resistance may emerge. Thus there is a need to characterize resistance mechanisms and to devise ways to prevent, delay or overcome osimertinib resistance. We show here that osimertinib suppresses glycolysis in parental EGFR-mutant lung adenocarcinoma lines, but has not in osimertinib-resistant cell lines. Critically, we show osimertinib treatment induces a strict dependence on mitochondrial oxidative phosphorylation (OxPhos), as OxPhos inhibitors significantly delay the long-term development of osimertinib resistance in osimertinib-sensitive lines. Accordingly, growth conditions which promote a less glycolytic phenotype confer a degree of osimertinib resistance. Our data support a model in which the combination of osimertinib and OxPhos inhibitors can delay or prevent resistance in osimertinib-naïve tumour cells, and represents a novel strategy that warrants further pre-clinical investigation.