Off-target Resistance Research Articles

Bystander Selection for Antimicrobial Resistance: Implications for Patient Health.

Antimicrobial therapy promotes resistance emergence in target infections and in off-target microbiota. Off-target resistance emergence threatens patient health when off-target populations are a source of future infections, as they are for many important drug-resistant pathogens. However, the health risks of antimicrobial exposure in off-target populations remain largely unquantified, making rational antibiotic stewardship challenging. Here, we discuss the contribution of bystander antimicrobial exposure to the resistance crisis, the implications for antimicrobial stewardship, and some novel opportunities to limit resistance evolution while treating target pathogens.

Abstract 330: High-throughput drug screens in osimertinib-resistant NSCLC cell lines, using continuous high-content imaging, identify potential co-targets to overcome acquired resistance

Abstract INTRODUCTION: Oncogenic mutations in EGFR are found in 15-30% of non-small cell lung cancers (NSCLC). Patients with EGFR-mutant NSCLC derive clinical benefit from EGFR tyrosine kinase inhibitors (TKIs), such as erlotinib and afatinib. Recently, a 3rd-generation EGFR TKI, osimertinib, was approved for first-line treatment of metastatic EGFR-mutant NSCLC based on improved progression-free survival compared to earlier EGFR TKIs. Unfortunately, resistance to osimertinib and other EGFR TKIs invariably develops. While secondary mutations in EGFR are a common resistance mechanism, resistance also frequently arises independent of EGFR alterations. We hypothesize that “bypass” signaling pathways are responsible for much of this off-target drug resistance. Moreover, we hypothesize that these bypass signaling pathways are pharmacologically targetable. DESIGN: Osimertinib-resistant cell lines were developed for 6 months and characterized via Western blot, CellTiter Blue, and RNA-seq. Drug screens were conducted with nine matched isogenic osimertinib-sensitive and -resistant cell lines, using continual imaging and cell counting on an automated high-content imaging microscope, in the presence and absence of osimertinib. The drug-induced proliferation (DIP) rate metric was used to generate time-resolved analyses of drug effect while minimizing effect of deviations in plating density. A library of 1176 compounds was selected based on: (1) published bioactivity/safety data; (2) prior clinical investigation; (3) target diversity; (4) minimized chemical redundancy. RESULTS: No de novo secondary EGFR alterations were found in any osimertinib-resistant cell line. Clear evidence of epithelial-to-mesenchymal transition was apparent in 4 of 9 resistant lines, which correlated with decreased EGFR and ErbB-family protein expression. FGFR1 mRNA expression was elevated in 8 of 9 resistant lines, and protein expression increased in 4 of 9 osimertinib-resistant lines. Initial drug screens have identified candidate “hits” which are more efficacious in an osimertinib-resistant cell line than its matched parental line. CONCLUSION: These results suggest that acquired osimertinib resistance, relying on certain “bypass” signaling pathways, arise independently in multiple cell culture models. Secondly, these results suggest that such “bypass”-mediated resistance can be identified and is pharmacologically targetable. Lastly, screening data demonstrates the utility and robustness of the DIP rate metric in image-based high-throughput drug screens. Citation Format: David Westover, Yun-Kai Zhang, Yingjun Yan, Stephen Himmelberg, Darren R. Tyson, Joshua A. Bauer, Christine M. Lovly. High-throughput drug screens in osimertinib-resistant NSCLC cell lines, using continuous high-content imaging, identify potential co-targets to overcome acquired resistance [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 330.

ATP-competitive DNA gyrase and topoisomerase IV inhibitors as antibacterial agents

ABSTRACTIntroduction: The bacterial topoisomerases DNA gyrase and topoisomerase IV are validated targets for development of novel antibacterial agents. Fluoroquinolones inhibit the catalytic GyrA and/or ParC(GrlA) subunit and have been commonly used, although these have toxicity liabilities that restrict their use. The ATPase GyrB and ParE(GrlB) subunits have been much less explored and after withdrawal of novobiocin, there are no further marketed inhibitors . ATP-competitive inhibitors of GyrB and/or ParE(GrlB) are of special interest, as this target has been validated, and it is expected that many of the problems associated with fluoroquinolones can be avoided.Areas covered: This review summarises the development of ATP-competitive inhibitors of GyrB and/or ParE(GrlB) as novel antibacterial agents over the last 10 years. Structural features of the new inhibitors and their optimisation strategies are highlighted.Expert opinion: The development of novel ATP-competitive inhibitors of GyrB and/or ParE(GrlB) is ongoing in industrial and academical research. Development of resistance is one of the most problematic issues, but GyrB/ParE(GrlB) inhibitors do not show cross-resistance with fluoroquinolones. Other common issues, such as low solubility, high protein binding, development of off-target resistance, are related to the structures of the inhibitors themselves, which is thus a main focus of design strategies. With some now in early clinical development, there is reasonable expectation that novel ATP-competitive inhibitors of GyrB/ParE(GrlB) will reach the market in the near future.

1771. Gut Resistome Changes in Response to Prophylactic Antibiotic Administration During Chemotherapy in Children With Acute Lymphoblastic Leukemia

BackgroundAntibiotic resistance harbored in gut microbiome contributes to the emergence of multi–drug-resistant organisms (MDRO). Pediatric leukemia patients typically receive extensive antibiotics and are at higher risk for infection due to MDRO.MethodsA prospective cohort of children (n = 242) with acute lymphoblastic leukemia self-collected stool samples at diagnosis and after induction chemothearpy. A third of patients (n = 69) underwent protocol-driven antibiotic prophylaxis: Levofloxacin (LV) given once neutropenia develops. With neutropenic fever patients on prophylaxis stopped LV and all patients received cefepime. Using metagenomic sequencing, we identified bacterial community composition and after alignment to the Comprehensive Antibiotic Resistance Database were able to determine the presence of bacterial resistance genes in 168 stool samples from 49 patients.ResultsExpected changes in the community composition were discovered with LV prophylaxis, including the loss of many Enterobacteriaceae and Enterococcaceae species, offset by increases in Bacteroides species. Unexpectedly, LV prophylaxis reduced the acquisition of VanA cluster of vancomycin resistance genes and did not increase acquisition of β-lactamase or fluoroquinolone (FQ) resistance gene families.ConclusionLV prophylaxis during leukemia treatment imparts predictable changes in gut bacterial communities but counter intuitively decreases antibiotic resistance in the gut microbiome reservoir. The reduction in VanA cluster of genes is likely due to depletion of Enterococcaceae species via direct killing or loss of synergistic partners. The lack of increase in target (FQ) or off-target resistance suggests that prophylaxis altered community selective pressures or prophylaxis drug concentrations were sufficient to limit the outgrowth of resistant mutants.Disclosures J. Wolf, Karius Inc.: Investigator, Research support.

Single-Molecule Sequencing Reveals Patterns of Preexisting Drug Resistance That Suggest Treatment Strategies in Philadelphia-Positive Leukemias

Purpose: Sequential treatment with targeted therapies can result in complex combinations of resistance mutations in drug targets. This mutational complexity has spurred the development of pan-target inhibitors, i.e., therapies for which no single target mutation can cause resistance. Because the propensity for on- versus off-target resistance varies across cancer types, a deeper understanding of the mutational burden in drug targets could rationalize treatment outcomes and prioritize pan-target inhibitors for indications where on-target mutations are most likely.Experimental Design: To measure and model the mutational landscape of a drug target at high resolution, we integrated single-molecule Duplex Sequencing of the ABL1 gene in Philadelphia-positive (Ph+) leukemias with computational simulations.Results: A combination of drug target mutational burden and tumor-initiating cell fraction is sufficient to predict that most patients with chronic myeloid leukemia are unlikely to harbor ABL1 resistance mutations at the time of diagnosis, rationalizing the exceptional success of targeted therapy in this setting. In contrast, our analysis predicts that many patients with Ph+ acute lymphoblastic leukemia (Ph+ ALL) harbor multiple preexisting resistant cells with single mutants. The emergence of compound mutations can be traced to initial use of an ABL1 inhibitor that is susceptible to resistance from single point mutations.Conclusions: These results argue that early use of therapies that achieve pan-inhibition of ABL1 resistance mutants might improve outcomes in Ph+ ALL. Our findings show how a deep understanding of the mutational burden in drug targets can be quantitatively coupled to phenotypic heterogeneity to rationalize clinical phenomena. Clin Cancer Res; 24(21); 5321-34. ©2018 AACR.

Use of bacteriophage to target bacterial surface structures required for virulence: a systematic search for antibiotic alternatives.

Bacteriophages (phage) that infect pathogenic bacteria often attach to surface receptors that are coincidentally required for virulence. Receptor loss or modification through mutation renders mutants both attenuated and phage resistant. Such attenuated mutants frequently have no apparent laboratory growth defects, but in the host, they fail to exhibit properties needed to produce disease such as mucosal colonization or survival within professional phagocytic cells. The connection between attenuation and phage resistance has been exploited in experimental demonstrations of phage therapy. In such experiments, phage resistant mutants that arise naturally during therapy are inconsequential because of their attenuated status. A more contemporary approach to exploiting this connection involves identifying small effector molecules, identified in high-throughput screens, that inhibit one or more of the steps needed to produce a functioning phage receptor. Since such biosynthetic steps are unique to bacteria, inhibitors can be utilized therapeutically, in lieu of antibiotics. Also, since the inhibitor is specific to a particular bacterium or group of bacteria, no off-target resistance is generated in the host's commensal bacterial population. This brief review covers examples of how mutations that confer phage resistance produce attenuation, and how this coincidental relationship can be exploited in the search for the next generation of therapeutic agents for bacterial diseases.

Abstract NG02: Polyclonal and heterogeneous resistance to targeted therapy in leukemia

Abstract NG02: Polyclonal and heterogeneous resistance to targeted therapy in leukemia

Abstract 1893: Activating NRAS mutations are associated with FLT3-independent resistance to the clinically active FLT3 inhibitor AC220 in vitro

Abstract Background: Clinical outcomes associated with acute myeloid leukemia (AML) have not improved substantially in decades. Activating FMS-like tyrosine kinase-3 (FLT3) mutations are detected in ∼30% of AML cases with internal tandem duplication (ITD) mutations conferring worse prognosis. Recently, the potent FLT3 inhibitor AC220 has achieved composite complete remission in 45% of patients with relapsed/refractory FLT3-mutant AML (Cortes et al, EHA 2011). Translational studies from our laboratory implicate reactivation of FLT3-ITD through kinase domain mutations in the majority AC220 relapse cases (Smith et al, ASH 2011). Given that less than half of treated patients initially achieve deep remissions on AC220, we hypothesize that off-target (non-FLT3 mediated) resistance is responsible for primary resistance to AC220, and may mediate loss of response in a subset of patients. Results: To model and identify putative mechanisms of off-target resistance to AC220 in vitro, we cultured the FLT3-ITD+ patient derived cell lines MV4;11 and Molm14 in escalating concentrations of AC220. To date, we have isolated 8 Molm14 subclones that are resistant to at least 20nM AC220, which is >60-fold the IC50 of the parental line (0.3nM), while efforts to generate resistant MV4;11 cells have repeatedly failed. Analysis of FLT3 signaling revealed persistent FLT3 phosphorylation in 5 clones in the presence of 10nM AC220. Sequencing of the FLT3 kinase domain identified acquired secondary activation loop mutations suggesting on-target acquired resistance. The remaining 3 clones demonstrated off-target resistance as evidenced by the absence of a FLT3 mutation and loss of FLT3 phosphorylation with 10nM AC220 treatment. Despite FLT3 inhibition, the clones failed to undergo apoptosis and maintained ERK phosphorylation when treated with AC220. Sequencing of K- and NRAS revealed an NRAS G12C mutation in 2 clones and an NRAS Q61K mutation in the third clone, both of which are known activating RAS mutations. The AC220 IC50 values for the G12C mutant clones were determined to be 134nM and 329nM AC220 and the Q61K mutant was one of the most resistant of all clones with an IC50 of >1000nM AC220. Further, all 3 NRAS mutant clones showed increased sensitivity to the MEK inhibitor PD0325901 compared to the parental cells and on-target resistant clones. Conclusions: Generation of 8 independent AC220-resistant AML cell lines revealed evidence for on- or off-target resistance mechanisms. We have identified oncogenic RAS as a putative mechanism of acquired AC220 off-target resistance in vitro, suggesting that pathologic activation of the RAS/ERK pathway may result in failure of clinically active FLT3 inhibitor therapy in AML patients. Assessment for activating RAS mutations in patient samples with primary and acquired resistance to AC220 and other clinically effective FLT3 inhibitors is currently ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1893. doi:1538-7445.AM2012-1893

Isolation of human mAbs that directly modulate FMS-related tyrosine kinase 3 signaling

FMS-related tyrosine kinase 3 (FLT3) is a class III receptor tyrosine kinase that plays important roles in hematopoiesis, including early progenitors and dendritic cell development. FLT3 is expressed at high levels in 70-100% of cases of AML and in virtually all cases of B-lineage acute lymphoblastic leukemia. FLT3 is regarded as a molecular target in the development of novel therapies for acute leukemia patients. Currently, many small-molecule FLT3 inhibitors have been developed, but clinical trials have resulted in limited antileukemia effects because of off-target toxicities and drug resistance. The development of anti-FLT3 Abs might overcome these difficulties and enhance the antileukemia efficacy of FLT3 inhibitors. In the present study, we demonstrate the isolation of novel human mAbs against FLT3 with antagonistic or agonistic activities. An antagonistic Ab, designated A2, continuously inhibits FLT3 ligand (FL)-induced phosphorylation of FLT3 and MAPK. A2 cooperatively induces apoptosis with daunorubicin, even in the presence of FL. An agonistic Ab, designated 3E6, surprisingly induces the phosphorylation of FLT3 and MAPK, and supports the growth of a factor-dependent cell line independently of FL addition. In addition, A2 showed complement-dependent cytotoxicity activity, but was devoid of Ab-dependent cell mediated cytotoxicity. Finally, we evaluated Ab internalization in a cell line. Immunofluorescence and flow cytometry analyses revealed that A2 is efficiently internalized. Collectively, these data demonstrate that A2 is a potent human Ab that might be capable of delivering cytotoxic reagents and that has antagonistic effects on FLT3 signaling. In addition, 3E6 might be a potential scaffold for novel dendritic cell-based immunotherapies.

Molecular mechanisms of cisplatin resistance

Platinum-based drugs, and in particular cis-diamminedichloroplatinum(II) (best known as cisplatin), are employed for the treatment of a wide array of solid malignancies, including testicular, ovarian, head and neck, colorectal, bladder and lung cancers. Cisplatin exerts anticancer effects via multiple mechanisms, yet its most prominent (and best understood) mode of action involves the generation of DNA lesions followed by the activation of the DNA damage response and the induction of mitochondrial apoptosis. Despite a consistent rate of initial responses, cisplatin treatment often results in the development of chemoresistance, leading to therapeutic failure. An intense research has been conducted during the past 30 years and several mechanisms that account for the cisplatin-resistant phenotype of tumor cells have been described. Here, we provide a systematic discussion of these mechanism by classifying them in alterations (1) that involve steps preceding the binding of cisplatin to DNA (pre-target resistance), (2) that directly relate to DNA-cisplatin adducts (on-target resistance), (3) concerning the lethal signaling pathway(s) elicited by cisplatin-mediated DNA damage (post-target resistance) and (4) affecting molecular circuitries that do not present obvious links with cisplatin-elicited signals (off-target resistance). As in some clinical settings cisplatin constitutes the major therapeutic option, the development of chemosensitization strategies constitute a goal with important clinical implications.

High-frequency transposition for determining antibacterial mode of action

Connecting bacterial growth inhibitors to molecular targets at the whole-cell level is a major impediment to antibacterial development. Herein we report the design of a highly efficient and versatile bacteriophage-based mariner transposon delivery system in Staphylococcus aureus for determining inhibitor mode of action. Using bacteriophage-mediated delivery of concatameric minitransposon cassettes, we generated nonclonal transposant libraries with genome-wide insertion-site coverage in either laboratory or methicillin-resistant strain backgrounds and screened for drug resistance in situ on a single agar plate in one step. A gradient of gene-target expression levels, along with a correspondingly diverse assortment of drug-resistant phenotypes, was achieved by fitting the transposon cassette with a suite of outward-facing promoters. Using a panel of antibiotics, we demonstrate the ability to unveil not only an inhibitor's molecular target but also its route of cellular entry, efflux susceptibility and other off-target resistance mechanisms.