Identification Of Resistance Mechanisms Research Articles

Genomic Epidemiology of Antimicrobial Resistance: Tracking the Global Spread of Resistant Pathogens

The rapid development and spread of antimicrobial resistance have been one of the most prominent health crises globally, which significantly challenges treating infectious diseases. This paper will address the contribution that genomic epidemiology makes to track and understand the global spread of antimicrobial-resistant pathogens. Genomic epidemiology, an area combining pathogen genomics with epidemiological data, allows for an extremely powerful approach in the identification of resistance mechanisms and tracing transmission routes for guiding public health interventions. This review Emphasizes major bacterial pathogens, such as Escherichia coli, Staphylococcus aureus, and Klebsiella pneumoniae, which harbor major resistance genes: blaCTX-M, mecA, and blaKPC. These genes contribute to the worldwide dissemination of resistance, mainly through mechanisms involving horizontal gene transfer and selective antibiotic pressure. Thus, genomic surveillance, phylogenetic analysis, and bioinformatics approaches allow for the pinpointing of transmission hotspots, mapping of resistance gene distribution, and tracking of emerging resistant strains in real time. It also provides information on how genomic data are being used to improve antimicrobial stewardship programs and inform clinical treatment decisions. Public health responses can further be tailored by recognizing regional resistance patterns, hence enhancing interventions. However, several challenges persist—for example, the need for standardized sharing of data, interpretation of genomic data, and integration of these insights into public health systems, especially in low-resource settings. This abstract highlights the transformative impact of genomic epidemiology in the management of the AMR crisis while emphasizing the continued need for global cooperation, enhanced data infrastructures, and policy initiatives to ensure the effective use of genomic data in public health.

PPP3CB overexpression mediates EGFR TKI resistance in lung tumors via calcineurin/MEK/ERK signaling.

Despite initial high response rates to first-line EGFR TKI, all non-small-cell lung cancer (NSCLC) with EGFR-activating mutation will ultimately develop resistance to treatment. Identification of resistance mechanisms is critical to adapt treatment and improve patient outcomes. Here, we show that a PPP3CB transcript that encodes full-length catalytic subunit 2B of calcineurin accumulates in EGFR-mutant NSCLC cells with acquired resistance against different EGFR TKIs and in post-progression biopsies of NSCLC patients treated with EGFR TKIs. Neutralization of PPP3CB by siRNA or inactivation of calcineurin by cyclosporin A induces apoptosis in resistant cells treated with EGFR TKIs. Mechanistically, EGFR TKIs increase the cytosolic level of calcium and trigger activation of a calcineurin/MEK/ERK pathway that prevents apoptosis. Combining EGFR, calcineurin, and MEK inhibitors overcomes resistance to EGFR TKI in both in vitro and in vivo models. Our results identify PPP3CB overexpression as a new mechanism of acquired resistance to EGFR TKIs, and provide a promising therapeutic approach for NSCLC patients that progress under TKI treatment.

Gram-negative bacterial colonization in the gut: Isolation, characterization, and identification of resistance mechanisms

BackgroundThe gut microbiome is made up of a diverse range of bacteria, especially gram-negative bacteria, and is crucial for human health and illness. There is a great deal of interest in the dynamic interactions between gram-negative bacteria and their host environment, especially considering antibiotic resistance. This work aims to isolate gram-negative bacteria that exist in the gut, identify their species, and use resistance-associated gene analysis to define their resistance mechanisms. MethodsSamples were collected from all patients who had a stool culture at a tertiary care center in Lebanon. Each type of bacteria that was identified from the stool samples was subjected to critical evaluations, and all discovered strains underwent antimicrobial susceptibility testing. Polymerase chain reaction was used to profile the genes for Carbapenem-resistant Enterobacteriaceae (CRE), Extended-spectrum beta-lactamase (ESBL), and that of Pseudomonas aeruginosa strains. ResultsEscherichia coli, Klebsiella species, and Pseudomonas aeruginosa turned out to be the predominant microbiota members. Escherichia coli strains had a high frequency of extended-spectrum beta-lactamase genes, with the most discovered gene being bla CTX-M. Additionally, a considerable percentage of isolates had carbapenemase-resistant Enterobacteriaceae genes, suggesting the rise of multidrug-resistant strains. Multidrug resistance genes, such as bla mexR, bla mexB, and bla mexA, were found in strains of Pseudomonas aeruginosa, highlighting the possible difficulties in treating infections brought on by these bacteria. ConclusionThe findings highlight the critical importance of effective surveillance and response measures to maintain the effectiveness of antibiotics considering the introduction of multidrug resistance genes in Pseudomonas aeruginosa and ESBL and CRE genes in Escherichia coli.

Biomarkers in Immune Checkpoint Inhibitor Cancer Treatment

Immune checkpoint inhibitors (ICIs) have revolutionized cancer therapy by offering durable responses for specific patient populations. The heterogeneity in results among individuals underscores the vital need for reliable predictive biomarkers to guide therapeutic strategies. This review aims to elucidate the current landscape of biomarkers in ICI therapy, focusing on their capability to predict patient responses and guide personalized cancer treatment. We examine both existing and emerging biomarkers—such as programmed death-ligand 1 (PD-L1) expression, tumor mutational burden, circulating tumor DNA, gene expression profiles, and the neutrophil-to-lymphocyte ratio—which provide critical insights into patient classification, therapeutic response monitoring, and identification of resistance mechanisms. Despite their potential, considerable challenges remain, including tumor heterogeneity, variability in biomarker testing, and the complexity and dynamic nature of biomarkers across different tumor types. Progress in multi-biomarker approaches and the use of artificial intelligence demonstrate potential for enhancing prediction precision and facilitating more personalized immunotherapy treatments. Additionally, this review summarizes completed and ongoing clinical trials investigating the effect of predictive biomarkers in directing ICI-based cancer treatments and their ability to tailor therapeutic strategies. In conclusion, the review provides an overview of the role of predictive biomarkers in response to ICI therapy.

The ribosomal protein S6 kinase alpha-1 (RPS6KA1) induces resistance to venetoclax/azacitidine in acute myeloid leukemia

Venetoclax/azacitidine combination therapy is effective in acute myeloid leukemia (AML) and tolerable for older, multimorbid patients. Despite promising response rates, many patients do not achieve sustained remission or are upfront refractory. Identification of resistance mechanisms and additional therapeutic targets represent unmet clinical needs. By using a genome-wide CRISPR/Cas9 library screen targeting 18,053 protein- coding genes in a human AML cell line, various genes conferring resistance to combined venetoclax/azacitidine treatment were identified. The ribosomal protein S6 kinase A1 (RPS6KA1) was among the most significantly depleted sgRNA-genes in venetoclax/azacitidine- treated AML cells. Addition of the RPS6KA1 inhibitor BI-D1870 to venetoclax/azacitidine decreased proliferation and colony forming potential compared to venetoclax/azacitidine alone. Furthermore, BI-D1870 was able to completely restore the sensitivity of OCI-AML2 cells with acquired resistance to venetoclax/azacitidine. Analysis of cell surface markers revealed that RPS6KA1 inhibition efficiently targeted monocytic blast subclones as a potential source of relapse upon venetoclax/azacitidine treatment. Taken together, our results suggest RPS6KA1 as mediator of resistance towards venetoclax/azacitidine and additional RPS6KA1 inhibition as strategy to prevent or overcome resistance.

Activity and rational combinations of a novel, engineered chimeric, TRAIL-based ligand in diffuse large B-cell lymphoma.

TRAIL (TNF-related apoptosis inducing ligand) exhibits selective proapoptotic activity in multiple tumor types, while sparing normal cells. This selectivity makes TRAIL an attractive therapeutic candidate. However, despite encouraging activity in preclinical models, clinical trials with TRAIL mimetics/death receptor agonists demonstrated insufficient activity, largely due to emerging resistance to these agents. Herein, we investigated the cytotoxic activity of a novel, TRAIL-based chimeric protein AD-O51.4 combining TRAIL and VEGFA-derived peptide sequences, in hematological malignancies. We characterize key molecular mechanisms leading to resistance and propose rational pharmacological combinations sensitizing cells to AD-O51.4. Sensitivity of DLBCL, classical Hodgkin lymphoma, (cHL), Burkitt lymphoma (BL) and acute myeloid leukemia (AML) to AD-O51.4 was assessed in vitro with MTS assay and apoptosis tests (Annexin V/PI staining). Markers of apoptosis were assessed using immunoblotting, flow cytometry or fluorogenic caspase cleavage assays. Resistant cell lines were obtained by incubation with increasing doses of AD-O51.4. Transcriptomic analyses were performed by RNA sequencing. Sensitizing effects of selected pathway modulators (BCL2, dynamin and HDAC inhibitors) were assessed using MTS/apoptosis assays. AD-O51.4 exhibited low-nanomolar cytotoxic activity in DLBCL cells, but not in other lymphoid or AML cell lines. AD-O51.4 induced death-receptor (DR) mediated, caspase-dependent apoptosis in sensitive DLBCL cells, but not in primary resistant cells. The presence of DRs and caspase 8 in cancer cells was crucial for AD-O51.4-induced apoptosis. To understand the potential mechanisms of resistance in an unbiased way, we engineered AD-O51.4-resistant cells and evaluated resistance-associated transcriptomic changes. Resistant cells exhibited changes in the expression of multiple genes and pathways associated with apoptosis, endocytosis and HDAC-dependent epigenetic reprogramming, suggesting potential therapeutic strategies of sensitization to AD-O51.4. In subsequent analyses, we demonstrated that HDAC inhibitors, BCL2 inhibitors and endocytosis/dynamin inhibitors sensitized primary resistant DLBCL cells to AD-O51.4. Taken together, we identified rational pharmacologic strategies sensitizing cells to AD-O51.4, including BCL2, histone deacetylase inhibitors and dynamin modulators. Since AD-O51.4 exhibits favorable pharmacokinetics and an acceptable safety profile, its further clinical development is warranted. Identification of resistance mechanisms in a clinical setting might indicate a personalized pharmacological approach to override the resistance.

Advances in the research of the mechanism of secondary resistance to imatinib in gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. At present, surgery is the first-line treatment for primary resectable GISTs; however, the recurrence rate is high. Imatinib mesylate (IM) is an effective first-line drug used for the treatment of unresectable or metastatic recurrent GISTs. More than 80% of patients with GISTs show significantly improved 5-year survival after treatment; however, approximately 50% of patients develop drug resistance after 2 years of IM treatment. Therefore, an in-depth research is urgently needed to reveal the mechanisms of secondary resistance to IM in patients with GISTs and to develop new therapeutic targets and regimens to improve their long-term prognoses. In this review, research on the mechanisms of secondary resistance to IM conducted in the last 5 years is discussed and summarized from the aspects of abnormal energy metabolism, gene mutations, non-coding RNA, and key proteins. Studies have shown that different drug-resistance mechanism networks are closely linked and interconnected. However, the influence of these drug-resistance mechanisms has not been compared. The combined inhibition of drug-resistance mechanisms with IM therapy and the combined inhibition of multiple drug-resistance mechanisms are expected to become new therapeutic options in the treatment of GISTs. In addition, implementing individualized therapies based on the identification of resistance mechanisms will provide new adjuvant treatment options for patients with IM-resistant GISTs, thereby delaying the progression of GISTs. Previous studies provide theoretical support for solving the problems of drug-resistance mechanisms. However, most studies on drug-resistance mechanisms are still in the research stage. Further clinical studies are needed to confirm the safety and efficacy of the inhibition of drug-resistance mechanisms as a potential therapeutic target.

995P Effectiveness of combination of osimertinib with another targeted therapy in advanced EGFR mutated non-small cell lung cancer harbouring other oncogenic drivers: The real-world COMPOSIT study

Identification of resistance mechanisms is crucial to guide therapeutic approaches in patients receiving osimertinib (O). The COMPOSIT study is a French national cohort reporting the effectiveness and tolerance of O in combination with another targeted therapy (TT) in patients with advanced EGFR mutated NSCLC harboring other oncogenic drivers. Patients (pts) treated with O between 01.01.2017 and 30.04.2021 who received a combination of O with another TT were included. Clinical pathological and molecular data were retrospectively collected. The primary endpoint was real-world progression free survival (rwPFS). 55 pts were included from 14 centers. Median age was 59 years (range 29-83); 56.3% were women, 60% never smokers and DelEx19 of EGFR was observed in 69% of cases. Combination therapy was administered to 23 pts who received O as 1st line (41.8%) and to 32 pts in whom O was administrated after receiving chemotherapy and/or 1st or 2nd generation EGFR TT (58.2%). MET pathway was the most targeted mechanism by combination therapies (66.1%). Real world PFS data was available for 54 combinations. Median follow-up was 16.7 months (95% CI; 13.3-19.1). The median rwPFS was 4.1 months (95% CI; 3.2-5.5). Overall response rate (ORR) was 49.0% (95% CI; 35.3-62.7%). The median overall survival (OS) was 11.9 months (95% CI; 8.6-19.6). In MET pathway alterations, rwPFS was 5.3 months (95 CI; 3.9-6.7), ORR 59.4% (95% CI; 42.4-76.4) and OS 14.8 months (95% CI; 7.4-33.2). Adverse events (AE) of grade >2 were reported in 13 patients (23.6%), mainly not related to O (93%). The frequency of drug interruption due to AE was 12.7%.Table: 995PCombination therapies with osimertinib (O) evaluated in COMPOSIT StudyPatients (n=55)Alteration targetedO + Targeted therapy36MET amplificationO + crizotinib (n=19) O + capmatinib (n=7) O + savolitinib (n=4) O + tepotinib (n=5) O + crizotinib then O + capmatinib (n=1)5BRAF fusionO + trametinib5BRAF V600EO + trametinib (n=1) O + dabrafenib (n=2) O + dabrafenib Trametinib (n=2)2EGFR C797S + T790MO + brigatinib1EGFR C797S + T790M cisO + gefitinib1EGFR L861Q + G724S + T790MO + afatinib2RET rearrangementO + praseltinib1ALK rearrangementO + crizotinib1FGFR1 amplificationO + erdafitinib1HER2 amplificationO + trastuzumab Open table in a new tab To our knowledge, this is the largest real-world study of combination therapies in patients with advanced EGFR-mutated NSCLC harboring other oncogenic drivers. Combination approaches with O are already used to overcome resistance by clinicians.

A Variant Carbapenem Inactivation Method (CIM) for Acinetobacter baumannii Group with Shortened Time-to-Result: rCIM-A.

Carbapenem-resistant Acinetobacter baumannii group organisms (CRAB) are challenging because the choice between targeted, new antibiotic drug options and hygiene measures should be guided by a timely identification of resistance mechanisms. In CRAB, acquired class-D carbapenemases (CHDLs) are active against meropenem and imipenem. If PCR methods are not the first choice, phenotypic methods have to be implemented. While promising, the carbapenemase inactivation method (CIM) using meropenem-hydrolysis is, however, hampered by poor performance or overly long time-to-result. We developed a rapid CIM (rCIM-A) with good performance using ertapenem, imipenem, and meropenem disks, 2-h permeabilization and incubation with the test strain in trypticase soy broth, and a read-out of residual carbapenem activity after 6 h, and optionally after 16–18 h. Using clinical isolates and type-strains of Acinetobacter (n = 67) not harboring carbapenemases (n = 28) or harboring acquired carbapenemases (n = 39), the sensitivity of detection was 97.4% with the imipenem disk after 6 h at a specificity of 92.9%. If the inhibition zone around the ertapenem disk at 6 h was 6 or ≤26 mm at 16–18 h, or ≤25.5 mm for meropenem, the specificity was 100%. Because of the high negative predictive value, the rCIM-A seems particularly appropriate in areas of lower CRAB-frequency.

Targeting oncogene and non-oncogene addiction to inflame the tumour microenvironment.

Immune checkpoint inhibitors (ICIs) have revolutionized the clinical management of multiple tumours. However, only a few patients respond to ICIs, which has generated considerable interest in the identification of resistance mechanisms. One such mechanism reflects the ability of various oncogenic pathways, as well as stress response pathways required for the survival of transformed cells (a situation commonly referred to as 'non-oncogene addiction'), to support tumour progression not only by providing malignant cells with survival and/or proliferation advantages, but also by establishing immunologically 'cold' tumour microenvironments (TMEs). Thus, both oncogene and non-oncogene addiction stand out as promising targets to robustly inflame the TME and potentially enable superior responses to ICIs.

An expanded agar-based screening method for azole-resistant Aspergillus fumigatus.

Antifungal susceptibility testing is an essential tool for guiding antifungal therapy. Reference methods are complex and usually only available in specialised laboratories. We have designed an expanded agar-based screening method for the detection of azole-resistant Aspergillus fumigatus isolates. Normally, identification of resistance mechanisms is obtained only after sequencing the cyp51A gene and promoter. However, our screening method provides azole resistance detection and presumptive resistance mechanisms identification. A previous agar-based method consisting of four wells containing voriconazole, itraconazole, posaconazole and a growth control, detected azole resistance to clinical azoles. Here, we have modified the concentrations of voriconazole and posaconazole to adapt to the updated EUCAST breakpoints against A.fumigatus. We have also expanded the method to include environmental azoles to assess azole resistance and the azole resistance mechanism involved. We used a collection of A.fumigatus including 54 azole-resistant isolates with Cyp51A modifications (G54, M220, G448S, TR53 , TR34 /L98H, TR46 /Y121F/T289A, TR34 /L98H/S297T/F495I), and 50 azole susceptible isolates with wild-type Cyp51A. The screening method detects azole-resistant A.fumigatus isolates when there is growth in any of the azole-containing wells after 48h. The growth pattern in the seven azoles tested helps determine the underlying azole resistance mechanism. This approach is designed for surveillance screening of A.fumigatus azole-resistant isolates and can be useful for the clinical management of patients prior to antifungal susceptibility testing confirmation.

MA07.03 Paired Liquid and Tissues Biopsies to Guide Treatment for Patients That Progress on 2nd Line Osimertinib Treatment

Despite a high response rate and durability, resistance to osimertinib inevitably occurs in the majority of patients. Identification of resistance mechanisms may guide sequential targeted therapy. We aimed to 1) identify the resistance mechanism using paired liquid and tissue biopsies in patients that progressed on 2nd line osimertinib 2) determine progression free survival (PFS) of post-osimertinib therapy.

Combined FGFR and Akt pathway inhibition abrogates growth of FGFR1 overexpressing EGFR-TKI-resistant NSCLC cells

EGFR tyrosine kinase inhibitor (TKI) resistance in non-small cell lung cancer (NSCLC) patients is inevitable. Identification of resistance mechanisms and corresponding targeting strategies can lead to more successful later-line treatment in many patients. Using spectrometry-based proteomics, we identified increased fibroblast growth factor receptor 1 (FGFR1) expression and Akt activation across erlotinib, gefitinib, and osimertinib EGFR-TKI-resistant cell line models. We show that while combined EGFR-TKI and FGFR inhibition showed some efficacy, simultaneous inhibition of FGFR and Akt or PI3K induced superior synergistic growth inhibition of FGFR1-overexpressing EGFR-TKI-resistant NSCLC cells. This effect was confirmed in vivo. Only dual FGFR and Akt inhibition completely blocked the resistance-mediating signaling pathways downstream of Akt. Further, increased FGFR1 expression was associated with significantly lower PFS in EGFR-TKI-treated NSCLC patients, and increased FGFR1 were demonstrated in a few post- vs. pre-EGFR-TKI treatment clinical biopsies. The superior therapeutic benefit of combining FGFR and Akt inhibitors provide the rationale for clinical trials of this strategy.

Mechanisms of Resistance in Gram-Negative Urinary Pathogens: From Country-Specific Molecular Insights to Global Clinical Relevance.

Urinary tract infections (UTIs) are the most frequent hospital infections and among the most commonly observed community acquired infections. Alongside their clinical importance, they are notorious because the pathogens that cause them are prone to acquiring various resistance determinants, including extended-spectrum beta-lactamases (ESBL); plasmid-encoded AmpC β-lactamases (p-AmpC); carbapenemases belonging to class A, B, and D; qnr genes encoding reduced susceptibility to fluoroquinolones; as well as genes encoding enzymes that hydrolyse aminoglycosides. In Escherichia coli and Klebsiella pneumoniae, the dominant resistance mechanisms are ESBLs belonging to the CTX-M, TEM, and SHV families; p-AmpC; and (more recently) carbapenemases belonging to classes A, B, and D. Urinary Pseudomonas aeruginosa isolates harbour metallo-beta-lactamases (MBLs) and ESBLs belonging to PER and GES families, while carbapenemases of class D are found in urinary Acinetobacter baumannii isolates. The identification of resistance mechanisms in routine diagnostic practice is primarily based on phenotypic tests for the detection of beta-lactamases, such as the double-disk synergy test or Hodge test, while polymerase chain reaction (PCR) for the detection of resistance genes is mostly pursued in reference laboratories for research purposes. As the emergence of drug-resistant bacterial strains poses serious challenges in the management of UTIs, this review aimed to appraise mechanisms of resistance in relevant Gram-negative urinary pathogens, to provide a detailed map of resistance determinants in Croatia and the world, and to discuss the implications of these resistance traits on diagnostic approaches. We summarized a sundry of different resistance mechanisms among urinary isolates and showed how their prevalence highly depends on the local epidemiological context, highlighting the need for tailored interventions in the field of antimicrobial stewardship.

Bacterial ecology and antibiotic resistance mechanisms of isolated resistant strains from diabetic foot infections in the north west of Algeria.

In front of the polymorphic bacterial ecology and antibiotic resistance in diabetic patients with foot infections and good patient care, collaboration between clinicians and microbiologists is needed to improve assessment and management of patients with this pathology. This study was designed to characterize the bacterial ecology of diabetic foot infection (DFIs) and to determine the different mechanisms of resistance involved. In this study bacterial strains and antibiotic resistance profiles were determined from diabetic foot infections patients (n = 117). The identification of resistance mechanisms, such as penicillinase and/or extended-spectrum β-lactamase production (ESBL), methicillin-resistant Staphylococcus aureus (MRSA) and efflux pump over-expression were performed. A high prevalence of Gram-negative bacteria (61%) with Escherichia coli, and other Enterobacteriaceae and Pseudomonas aeruginosa being the predominant isolates. Gram positive bacteria mainly represented by Staphylococcus aureus accounted for 39% of the isolates. 93.5% of the Enterobacteriaceae were resistant to, at least, one molecule in the β-lactam family, while the majority of the Staphylococci were resistant to penicillin G and tetracycline (93.3% and 71.7%). The majority of non-fermenting Gram negative bacteria were also resistant to fluoroquinolones. β-lactamase detection tests revealed the presence of extended-spectrum β-lactamase in 43.5% of the Enterobacteriaceae, while methicillin-resistant Staphylococcus aureus represented 18.2% of the isolates. Additionally, 50.9% of non-fermenting Gram negative bacteria were overproducing efflux pumps. All Acinetobacter Baumannii were Multidrug-Resistant (MDR), as the majority of Staphylococci, and Enterobacteriaceae. These results should be taken into account by the clinician in the prescription of probabilistic antibiotic therapy in this context.

Abstract B40: Analytical validation and initial clinical utility of multianalyte transcriptomic biomarker profiling of circulating tumor cells using automated exclusion-based sample preparation technology

Abstract The efficacy of androgen receptor (AR) signaling inhibitors (ARSIs) in prostate cancer is limited by both de novo and acquired resistance. Two well-described mechanisms of resistance to ARSIs are the development of constitutively activated AR splice variants (AR-Vs) or neuroendocrine prostate cancer (NEPC). Expression of NEPC markers is traditionally evaluated in solid tumor biopsies while AR-Vs can be detected in circulating tumor cells (CTCs) and have prognostic relevance in prostate cancer. Unlike solid tumor biopsy-based detection strategies, liquid biopsies can be performed periodically over the course of treatments, enabling early identification of resistance mechanisms. To evaluate for ARSI resistance, we developed a multiplex gene expression panel of AR-Vs and NEPC markers. We evaluated potential clinical utility of this assay in CTCs captured using an exclusion-based sample preparation technology. This test was evaluated with a cohort of 46 patients, showing 39% sensitivity and 94% specificity at detecting ARSI resistance. To develop a clinical-grade liquid biopsy assay with this multiplexed gene expression panel, we utilized a high-throughput automated exclusion-based sample preparation (ESP) technology for CTC capture, mRNA extraction, and gene expression analysis using RT-qPCR. Cell lines were used to evaluate the analytical performance of the methodology, and spike-in control materials were designed to monitor the efficiency of mRNA extraction for each individual patient sample. Serial dilutions of cell lines demonstrated the sensitivity to extract and quantify mRNA at the single-cell level with high precision between evaluations performed on three different days. Increasing numbers of cells resulted in the quantification of proportionally higher amounts of both AR-Vs and NEPC markers, demonstrating the analytical accuracy of the methodology (R2 values of 0.8 and above). To monitor for RNA degradation, a grape mRNA amplicon material was designed as an internal spike-in control for daily use in testing patient samples. Healthy donor PBMCs were used to confirm primers were specific for cells of cancer origin. ESP-mRNA extraction followed by RT-qPCR demonstrates single-cell resolution for the detection of AR-V/NEPC transcriptomic expression, with high precision in evaluating actual patient samples. The high analytical performance, and the promising preliminary correlation with ARSI resistance, warrant further efforts towards implementation into clinical laboratory testing environments and testing in prospective clinical trials. Citation Format: Zachery D. Schultz, Jennifer L. Schehr, Rory M. Bade, Molly M. Morgan, Madalyn S. Gill, Hannah M. Pezzi, Jaime M. Sperger, Charlotte N. Stahlfeld, Anupama Singh, Jay W. Warrick, David J. Beebe, Joshua M. Lang. Analytical validation and initial clinical utility of multianalyte transcriptomic biomarker profiling of circulating tumor cells using automated exclusion-based sample preparation technology [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B40.

Pan-active imidazolopiperazine antimalarials target the Plasmodium falciparum intracellular secretory pathway

A promising new compound class for treating human malaria is the imidazolopiperazines (IZP) class. IZP compounds KAF156 (Ganaplacide) and GNF179 are effective against Plasmodium symptomatic asexual blood-stage infections, and are able to prevent transmission and block infection in animal models. But despite the identification of resistance mechanisms in P. falciparum, the mode of action of IZPs remains unknown. To investigate, we here combine in vitro evolution and genome analysis in Saccharomyces cerevisiae with molecular, metabolomic, and chemogenomic methods in P. falciparum. Our findings reveal that IZP-resistant S. cerevisiae clones carry mutations in genes involved in Endoplasmic Reticulum (ER)-based lipid homeostasis and autophagy. In Plasmodium, IZPs inhibit protein trafficking, block the establishment of new permeation pathways, and cause ER expansion. Our data highlight a mechanism for blocking parasite development that is distinct from those of standard compounds used to treat malaria, and demonstrate the potential of IZPs for studying ER-dependent protein processing.

Abstract A118: Identification of resistance mechanisms to FGFR4 targeted therapy in hepatocellular carcinoma

Abstract When on-target resistance mutations occur in response to potent and selective therapeutics, the target is often considered a validated driver of disease. Hepatocellular carcinoma (HCC) is a leading cause of cancer mortality worldwide whose drivers remain unvalidated because approved therapies utilize multi-kinase inhibitors or immune modulation. In this study, we validate for the first time a driver of disease in the FGF19 expressing subset of HCC through the identification of Fisogatinib resistance mutations in FGFR4. Fisogatinib (formerly known as BLU-554), an investigational agent, was developed to specifically inhibit the tyrosine kinase activity of FGFR4. Approximately one third of HCC patients harbor aberrant expression of fibroblast growth factor 19 (FGF19), a ligand hypothesized to drive paracrine FGFR4 activation and enhance mitogenic signaling in HCC cells. The identification of FGF19 amplifications in a subset of HCC patients through genomic profiling is further suggestive of the involvement of the FGF19/FGFR4 signaling axis in oncogenesis. The first-in-human, phase I study of Fisogatinib in patients with advanced HCC (NCT02508467) suggests that expression of FGF19 as assessed by immunohistochemistry is responsible for oncogenesis and predictive of response to treatment. We identified FGFR4 gatekeeper (V550) and hinge-1 (C552) mutations upon disease progression in two patients treated with Fisogatinib. These resistance mutations had been predicted by our structure-based studies and were also identified through in vitro and in vivo Fisogatinib resistance screens. We confirm by liquid chromatography tandem mass-spectrometry that Fisogatinib binding to FGFR4 is prevented when either the gatekeeper or the hinge-1 mutation is present and demonstrate the capability of these mutations to mediate resistance to Fisogatinib through genetic engineering. Using a gatekeeper-agnostic pan-FGFR inhibitor we show, in preclinical models, continued FGF19/FGFR4 pathway dependence. These results validate FGF19/FGFR4 as an oncogenic driver and inform the profile of potential next-generation inhibitors designed to facilitate improved patient outcomes. Citation Format: Megan A Hatlen, Oleg Schmidt-Kittler, Cori-Ann Sherwin, Emily Rozsahegyi, Nooreen Rubin, Michael Sheets, Joseph L Kim, Chandra Miduturu, Neil Bifulco, Natasja Brooijmans, Hongliang Shi, Timothy Guzi, Andy Boral, Christoph Lengauer, Marion Dorsch, Richard D Kim, Yoon-Koo Kang, Beni B Wolf, Klaus P Hoeflich. Identification of resistance mechanisms to FGFR4 targeted therapy in hepatocellular carcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A118. doi:10.1158/1535-7163.TARG-19-A118

Population-based inference of aminoglycoside resistance mechanisms in Escherichia coli

BackgroundInterpretative reading of antimicrobial susceptibility test (AST) results allows inferring biochemical resistance mechanisms from resistance phenotypes. For aminoglycosides, however, correlations between resistance pathways inferred on the basis of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breakpoints and expert rules versus genotypes are generally poor. This study aimed at developing and validating a decision tree based on resistance phenotypes determined by disc diffusion and based on epidemiological cut-offs (ECOFFs) to infer the corresponding resistance mechanisms in Escherichia coli. MethodsPhenotypic antibiotic susceptibility of thirty wild-type and 458 aminoglycoside-resistant E. coli clinical isolates was determined by disc diffusion and the genomes were sequenced. Based on well-defined cut-offs, we developed a phenotype-based algorithm (Aminoglycoside Resistance Mechanism Inference Algorithm - ARMIA) to infer the biochemical mechanisms responsible for the corresponding aminoglycoside resistance phenotypes. The mechanisms inferred from susceptibility to kanamycin, tobramycin and gentamicin were analysed using ARMIA- or EUCAST-based AST interpretation and validated by whole genome sequencing (WGS) of the host bacteria. FindingsARMIA-based inference of resistance mechanisms and WGS data were congruent in 441/458 isolates (96·3%). In contrast, there was a poor correlation between resistance mechanisms inferred using EUCAST CBPs/expert rules and WGS data (418/488, 85·6%). Based on the assumption that resistance mechanisms can result in therapeutic failure, EUCAST produced 63 (12·9%) very major errors (vME), compared to only 2 (0·4%) vME with ARMIA. When used for detection and identification of resistance mechanisms, ARMIA resolved >95% vMEs generated by EUCAST-based AST interpretation. InterpretationThis study demonstrates that ECOFF-based analysis of AST data of only four aminoglycosides provides accurate information on the resistance mechanisms in E. coli. Since aminoglycoside resistance mechanisms, despite having in certain cases a minimal effect on the minimal inhibitory concentration, may compromise the bactericidal activity of aminoglycosides, prompt detection of resistance mechanisms is crucial for therapy. Using ARMIA as an interpretative rule set for editing AST results allows for better predictions of in vivo activity of this drug class.

Abstract 3819: Amphiregulin in damaged tumor microenvironment potentiates therapeutic resistance and enables PD-L1-mediated immunosuppression

Abstract Therapeutic resistance is the major barrier to conquer human cancer, the most lethal pathology that claims numerous lives per year. Although identification of resistance mechanisms is the key to develop optimal strategies, progress has been limited due to long term failure to reveal the most critical targets. Cancer cells exhibit high plasticity even in clinical settings, a phenomenon that contributes to disease refraction by developing intrinsic or acquired resistance. Here we highlight the multifaceted and genetically conserved senescence-associated secretory phenotype (SASP), which encodes a large array of soluble factors including amphiregulin (AREG). Expression of AREG is markedly inducible by DNA damage to stromal cells in the tumor microenvironment (TME), a process mediated predominantly by the NF-κB complex and C/EBP family. Stromal AREG remarkably enhances the malignancy of cancer cells including acquired resistance, mainly by activating the EGFR pathway and causing a distinct epithelial to endothelial transition (EET). Paracrine AREG induces PD-L1 expression in recipient cancer cells, thereby creating an immunosuppressive TME via immune checkpoint activation. Therapeutic targeting AREG by a monoclonal antibody not only remarkably deprived cancer cells of chemoresistance, but achieved prominent efficiency when combined with classical chemotherapy in humanized animals with restored immunocompetency. Stromal AREG enters circulation and is readily detectable in the peripheral blood of post-treatment patients, representing a novel liquid biopsy biomarker for therapeutic evaluation and disease prognosis. Our study substantiates the potential of in vivo SASP in driving the TME-mediated drug resistance and shaping an immunosuppressive niche, and provides the proof-of-principle of targeting major SASP factors to effectively improve therapeutic index with reduced mortality. Citation Format: Qixia Xu, Jean-Philippe Coppé, Judith Campisi, Eric Lam, Yu Sun. Amphiregulin in damaged tumor microenvironment potentiates therapeutic resistance and enables PD-L1-mediated immunosuppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3819.