De Novo Resistance Research Articles

Abstract 2899: Combined inhibition of RAF, MEK and FAK increases PDAC responsiveness to cytotoxic- and immune therapy

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy with poor response rate to therapy. An immune suppressive tumor microenvironment (TME) and oncogenic mutations in KRAS have been implicated as drivers of resistance to both conventional and immune therapies. As such, targeting RAS/MAPK signaling is an attractive strategy. However, RAS/MAPK inhibition has not yet shown clinical efficacy for PDAC, likely due to rapid acquisition of resistance in PDAC cells. Tumor intrinsic mechanisms of resistance to RAS/MAPK have been studied, however, the unique PDAC TME may also be a key driver in resistance. Previous studies have shown that FAK inhibition can reprogram the PDAC TME and delay PDAC progression in animal models. Herein, we found that long-term FAK inhibitor treatment led to hyperactivation of the RAS/MAPK pathway in both genetically engineered mouse models and in post-treatment PDAC tissues from FAK inhibitor clinical trials. Concomitant inhibition of both FAK (VS-4718) and RAF/MEK (avutometinib) signaling dramatically suppressed tumor growth, leading to increased survival across multiple PDAC mouse models. The mechanisms of synergy include both changes in tumor-intrinsic signaling and modulation of tumor/stroma interactions that drive avutometinib resistance. In the TME, we found that cancer associated fibroblasts (CAFs) can impair the downregulation of cMyc by RAF/MEK inhibition in PDAC cells. This resulted in de-novo resistance to RAF/MEK inhibition in fibrotic conditions. By contrast, FAK inhibitors reprogramed CAFs to suppress the production of key growth factors, including FGF1, that drove resistance to RAF/MEK inhibition. While combined FAK and RAF/MEK inhibition only led to disease stasis, the addition of chemotherapy to the combination led to tumor regression and improved long-term survival in PDAC mouse models. Analysis of tumor immunity showed that the combination of FAK and RAF/MEK inhibition improved anti-tumor immunity and improved priming of T cell responses, which was further improved with the addition of chemotherapy. These findings led to testing of FAK (defactinib) plus RAF/MEK (avutometinib) inhibition in combination with gemcitabine and nab-paclitaxel in advanced front-line pancreatic cancer patients (RAMP 205; NCT05669482). Finally, we tested whether addition of immunotherapy could enhance the efficacy of FAKi + RAF/MEKi + chemotherapy and found that addition of either PD-1 or CTLA4/PD-1 blockade led to long term disease control in PDAC animal models. Together, these studies identified FAK inhibition as a novel approach to overcome both tumor intrinsic and stromal-derived resistance to RAS/MAPK inhibition and showed that this combination can be exploited to increase the efficacy of cytotoxic and immunotherapy approaches. Citation Format: Xiuting Liu, John Baer, Brett Knolhoff, Graham Hogg, Faiz Ahmad, Silvia Coma, Jonathan Pachter, Kian Lim, David DeNardo. Combined inhibition of RAF, MEK and FAK increases PDAC responsiveness to cytotoxic- and immune therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2899.

In host evolution of beta lactam resistance during active treatment for Pseudomonas aeruginosa bacteremia.

Multidrug-resistant (MDR) Pseudomonas aeruginosa has been declared a serious threat by the United States Centers for Disease Control and Prevention. Here, we used whole genome sequencing (WGS) to investigate recurrent P. aeruginosa bloodstream infections in a severely immunocompromised patient. The infections demonstrated unusual, progressive increases in resistance to beta lactam antibiotics in the setting of active treatment with appropriate, guideline-directed agents. WGS followed by comparative genomic analysis of isolates collected over 44 days demonstrated in host evolution of a single P. aeruginosa isolate characterized by stepwise acquisition of two de-novo genetic resistance mechanisms over the course of treatment. We found a novel deletion affecting the ampC repressor ampD and neighboring gene ampE, which associated with initial cefepime treatment failure. This was followed by acquisition of a porin nonsense mutation, OprD, associated with resistance to carbapenems. This study highlights the potential for in-host evolution of P. aeruginosa during bloodstream infections in severely immunocompromised patients despite appropriate antimicrobial therapy. In addition, it demonstrates the utility of WGS for understanding unusual resistance patterns in the clinical context.

Pre-extensively drug-resistant tuberculosis among pulmonary multidrug-resistant tuberculosis patients in Eastern Nigeria.

Pre-extensively drug-resistant tuberculosis (Pre-XDR-TB), an emerging form of drug-resistant tuberculosis, is challenging efforts at tuberculosis control, leading to treatment failure among multidrug-resistant tuberculosis (MDR-TB) patients and progression to extensively drug-resistant tuberculosis (XDR-TB). We determined the rate of Pre-XDR-TB among multidrug-resistant patients in Southeast, Nigeria. A prospective laboratory-based study was carried out at the South East Zonal Tuberculosis Reference Laboratory from January 2021 to December 2021. Second-line drug (SLD) resistance was performed on 225 sputum samples of multidrug-resistant patients prior to treatment initiation using GenoType MTBDRsl genotypic drug susceptibility testing (DST) method. The rate of Pre-XDR-TB among 225 MDR-TB cases was 3.1%. Fluoroquinolone-resistant Pre-XDR-TB was observed (100%) in previously treated tuberculosis cases. Only one (0.4%) case showed resistance to both fluoroquinolone (FQ) and one second-line injectable drug (XDR-TB). The extensively drug-resistant case observed was a de-novo resistance. Exactly 0.9% of the multidrug-resistant cases showed resistance to second-line injectables. The prevalence of Pre-XDR-TB among MDR-TB cases was high. There is need for rapid detection of Pre-XDR-TB among MDR-TB cases before treatment initiation.

Epigenetic derepression converts PPAR\u03b3 into a druggable target in triple-negative and endocrine-resistant breast cancers

Clinical trials repurposing peroxisome proliferator-activated receptor-gamma (PPARγ) agonists as anticancer agents have exhibited lackluster efficacy across a variety of tumor types. Here, we report that increased PPARG expression is associated with a better prognosis but is anticorrelated with histone deacetylase (HDAC) 1 and 2 expressions. We show that HDAC overexpression blunts anti-proliferative and anti-angiogenic responses to PPARγ agonists via transcriptional and post-translational mechanisms, however, these can be neutralized with clinically approved and experimental HDAC inhibitors. Supporting this notion, concomitant treatment with HDAC inhibitors was required to license the tumor-suppressive effects of PPARγ agonists in triple-negative and endocrine-refractory breast cancer cells, and combination therapy also restrained angiogenesis in a tube formation assay. This combination was also synergistic in estrogen receptor-alpha (ERα)–positive cells because HDAC blockade abrogated ERα interference with PPARγ-regulated transcription. Following a pharmacokinetics optimization study, the combination of rosiglitazone and a potent pan-HDAC inhibitor, LBH589, stalled disease progression in a mouse model of triple-negative breast cancer greater than either of the monotherapies, while exhibiting a favorable safety profile. Our findings account for historical observations of de-novo resistance to PPARγ agonist monotherapy and propound a therapeutically cogent intervention against two aggressive breast cancer subtypes.

Comparative metagenomics analysis of the rhizosphere microbiota influence on Radix Angelica sinensis in different growth soil environments in China

Abstract We studied the correlation between the soil microbial status and the crop yield or medicinal properties of Angelica s.. Soil from different counties of China were collected and used in this study. Readfq, SOAP denovo, MetaGeneMark, and DIAMOND Resistance Gene Identifier (RGI) software were used in the analysis. A total of 131,345 genes of high quality were obtained, with the number of genes in all samples ranging from 483-42,545 and the average length ranging from 354.88-523.47. The acidobacteria in the non-sterilized group were significantly increased by comparing with the sterilized group, while actinobacteria in the non-sterilized group were significantly reduced by comparing with the sterilized group. There was a strong correlation between the two groups of the planting group and the two groups of the non-planting group, while the correlation between the planting and non-planting group was very weak. There were significant differences in the soil microbial community structure between Min county and Yunnan, as well as significant differences between the dominant genera of Min county and Yunnan. In summary, our study showed that the soil microbial flora in different areas affected the characteristics of Angelica s. roots, while sterilized soil was not conducive to the growth of Angelica s.

De Novo Resistance to Arg10-Teixobactin Occurs Slowly and Is Costly.

Bacterial pathogens are rapidly evolving resistance to all clinically available antibiotics. One part of the solution to this complex issue is to better understand the resistance mechanisms to new and existing antibiotics. Here, we focus on two antibiotics. Teixobactin is a recently discovered promising antibiotic that is claimed to "kill pathogens without detectable resistance" (L. L. Ling, T. Schneider, A. J. Peoples, A. L. Spoering, et al., Nature 517:455-459, 2015, https://doi.org/10.1038/nature14098). Moenomycin A has been extensively used in animal husbandry for over 50 years with no meaningful antibiotic resistance arising. However, the nature, mechanisms, and consequences of the evolution of resistance to these "resistance-proof" compounds have not been investigated. Through a fusion of experimental evolution, whole-genome sequencing, and structural biology, we show that Staphylococcus aureus can develop significant resistance to both antibiotics in clinically meaningful timescales. The magnitude of evolved resistance to Arg10-teixobactin is 300-fold less than to moenomycin A over 45 days, and these are 2,500-fold and 8-fold less than evolved resistance to rifampicin (control), respectively. We have identified a core suite of key mutations, which correlate with the evolution of resistance, that are in genes involved in cell wall modulation, lipid synthesis, and energy metabolism. We show the evolution of resistance to these antimicrobials translates into significant cross-resistance against other clinically relevant antibiotics for moenomycin A but not Arg10-teixobactin. Lastly, we show that resistance is rapidly lost in the absence of antibiotic selection, especially for Arg10-teixobactin. These findings indicate that teixobactin is worth pursuing for clinical applications and provide evidence to inform strategies for future compound development and clinical management.

Abstract P4-04-07: A DNA-methylation signature to predict resistance to the CDK4/6 inhibitor palbociclib

Abstract Introduction: CDK4/6 inhibitors, such as palbociclib, target the cyclin-dependent kinases 4 and 6, essential for cell cycle regulation. Preclinical studies have suggested potential determinants of palbociclib sensitivity and clinical data have recently indicated loss-of function mutations in RB1 and increased expression of CCNE1 as potential biomarkers of resistance in a fraction of breast cancer (BC) patients. In line with these studies, using a panel of resistant luminal BC cell lines made resistant to palbociclib we recently demonstrated a common transcriptional signature of resistance to this agent, including the over-expression of CCNE1 and under-expression of RB1. Based on these data, we developed a minimal signature of de-novo resistance to palbociclib (ratio between CCNE1 and RB1, CCNE1/RB1) and demonstrated its prognostic role in both preclinical and clinical datasets. Gene expression signatures, such as CCNE1/RB1, are limited to the analysis of tissue samples because they are not easily detectable in liquid biopsies. Based on the key role DNA-methylation (DNAm) plays in regulating gene expression in human cells, we hypothesized that DNAm could be used as surrogate of our gene expression signature, with the further advantage of being easily adapted to non-invasive screenings. Exploiting this concept, here we report on a DNAm-based signature mirroring CCNE1/RB1 and its potential role in predicting resistance to palbociclib in BC patients. Materials and methods: Seven estrogen receptor positive breast cancer cell lines with acquired in vitro resistance to palbociclib were used. DNAm profiles of untreated parental cells and of their palbociclib-resistant counterparts were generated through Illumina Infinium MethylationEPIC BeadChip. For each cell line, candidate DNAm sites associated with resistance to palbociclib were selected by differential DNAm analysis in resistant versus sensitive counterparts. To identify DNAm sites/regions distinguishing CCNE1/RB1 high vs low patients, RNA-seq data from ER+/HER2- patients in the TCGA-BRCA dataset were considered and patients were classified as CCNE1/RB1 high (≥90% quantile) or low (≤10% quantile). Matched DNAm data were exploited to perform differential DNAm analysis between CCNE1/RB1 high and low samples by Rocker-meth, a tool recently developed by our group to perform differential DNAm analysis. The pharmacogenomic cancerrxgene dataset was used to test the association between the CCNE1/RB1 DNAm signature and the measure of effectiveness of palbociclib across multiple BC cell lines. Results: From the differential methylation analysis of CCNE1/RB1 high versus low ER+/HER2- BC patients from the TCGA-BRCA dataset we identified the set of sites and regions that compose our DNAm-based signature. In-silico functional analysis of differentially methylated sites and regions showed significant enrichment of regulatory-related terms. Both hyper- and hypo- DNAm sites in resistant versus untreated sensitive cell lines were significantly enriched in the set of differentially methylated sites in CCNE1/RB1 high versus low TCGA samples. In the cancerrxgene dataset, the CCNE1/RB1 DNAm signature correlated with higher IC50 in breast cancer cell lines. Conclusions: This study shows that DNAm is a potential predictor of resistance to palbociclib in ER+/HER2- patients. The DNAm-based signature mirroring CCNE1/RB1 developed using TCGA-BRCA dataset was validated in our preclinical BC cell lines and associated with effectiveness of palbociclib in an independent BC cell line dataset. Ongoing studies on the analysis of DNAm in liquid biopsies of metastatic luminal BC patients will evaluate the reliability of this DNAm signature in predicting resistance to palbociclib. Citation Format: Matteo Benelli, Martina Bonechi, Dario Romagnoli, Chiara Biagioni, Ilenia Migliaccio, Francesca De Luca, Francesca Galardi, Angelo Di Leo, Luca Malorni. A DNA-methylation signature to predict resistance to the CDK4/6 inhibitor palbociclib [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-04-07.

Leeomic: A comprehensive proteomic analysis towards discovery of predictive patterns of protein expression to ribociclib sensitivity and resistance—A compLEEment-1 Canadian correlative sub-study.

TPS3170 Background: Despite developments in the treatment of advanced hormone receptor positive (HR+), human epidermal growth factor receptor 2 negative (HER2-) breast cancer, primary or acquired resistance eventually occurs in all cases and there is still very limited understanding of the mechanisms of resistance to therapy. LEEOMIC is a sub-study of the main CompLEEment-1 ( N = 3255 patients enrolled, CLEE011A2404 v03) trial, an open-label, phase 3b study evaluating ribociclib + letrozole as first-line therapy in an expanded advanced breast cancer patient population which recruited over 250 Canadian patients. The purpose of this Canadian correlative sample collection study is to explore the mechanisms of response and resistance to ribociclib in combination with letrozole through proteomic and ctDNA analysis. Methods: The British Columbia Cancer Research Centre team developed a novel and optimized MS/MS platform called SP3-Clinical Tissue Proteomics (SP3-CTP) to perform in-depth proteome profiling ( > 8,000 proteins) from formalin fixed paraffin embedded (FFPE) material (10-micron section). SP3-CTP analysis of the proteome of the study patients who did not achieve clinical benefit (primary resistance: progression within 3 months of treatment) will be compared to the proteome of the sub-group of prolonged responders (time to progression of 22 months or more) in order to identify biomarkers that can predict response or de-novo resistance to therapy. Archival tumor biopsies (primary or metastatic) collected from the study will be submitted for proteomic analysis to identify proteomic expression levels that may serve as predictor of response. It is anticipated that over 150 samples will be collected. If available, blood samples taken at time of progression or end of treatment will also be analyzed for ctDNA for genetic profiling and to study if there is any correlation between genetic mutations and response or resistance to therapy. Currently, both tissue and blood samples are being collected and no analysis has been conducted thus far. Clinical trial information: NCT03613220.

MA15.01 Strong PD-L1 Expression Predicts Poor Response and de Novo Resistance to EGFR TKIs Among Non-Small Cell Lung Cancer Patients with EGFR Mutation

MA15.01 Strong PD-L1 Expression Predicts Poor Response and de Novo Resistance to EGFR TKIs Among Non-Small Cell Lung Cancer Patients with EGFR Mutation

Strong Programmed Death Ligand 1 Expression Predicts Poor Response and De Novo Resistance to EGFR Tyrosine Kinase Inhibitors Among NSCLC Patients With EGFR Mutation

IntroductionThis study evaluated whether tumor expression of programmed death ligand 1 (PD-L1) could predict the response of EGFR-mutated NSCLC to EGFR tyrosine kinase inhibitor (TKI) therapy. MethodsWe retrospectively evaluated patients who received EGFR-TKIs for advanced NSCLC at the Guangdong Lung Cancer Institute between April 2016 and September 2017 and were not enrolled in clinical studies. The patients' EGFR and PD-L1 statuses were simultaneously evaluated. ResultsAmong the 101 eligible patients, strong PD-L1 expression significantly decreased objective response rate, compared with weak or negative PD-L1 expression (35.7% versus 63.2% versus 67.3%, p = 0.002), and shortened progression-free survival (3.8 versus 6.0 versus 9.5 months, p < 0.001), regardless of EGFR mutation type (19del or L858R). Furthermore, positive PD-L1 expression was predominantly observed among patients with de novo resistance rather than acquired resistance to EGFR-TKIs (66.7% versus 30.2%, p = 0.009). Notably, we found a high proportion of PD-L1 and cluster of differentiation 8 (CD8) dual-positive cases among patients with de novo resistance (46.7%, 7 of 15). Finally, one patient with de novo resistance to EGFR-TKIs and PD-L1 and CD8 dual positivity experienced a favorable response to anti–programmed death 1 therapy. ConclusionsThis study revealed the adverse effects of PD-L1 expression on EGFR-TKI efficacy, especially in NSCLC patients with de novo resistance. The findings indicate the reshaping of an inflamed immune phenotype characterized by PD-L1 and CD8 dual positivity and suggest potential therapeutic sensitivity to programmed death 1 blockade.

Genomic alterations (GA) predicted to confer lack of benefit from trastuzumab in advanced esophagogastric cancers (EGC): Analysis of 527 HER2-amplified (HER2amp) cases.

44 Background: The addition of trastuzumab to chemotherapy improves survival in first line treatment of advanced HER2 overexpressing EGC. However, the response rate is under 50% in this molecularly heterogeneous group of cancers. Genomic alterations (GA) associated with lack of benefit from trastuzumab are understudied. Methods: We prospectively analyzed clinical samples from patients with EGC using hybrid-capture based comprehensive genomic profiling (CGP). Pre-specified literature review was used to determine GA associated with de-novo trastuzumab resistance. Results: From 2,245 gastroesophageal junction (GEJ) adenocarcinomas and 1,883 gastric adenocarcinomas (GC) we identified 395 HER 2-amplified GEJ (18%) and 132 HER 2-amplified (HER2amp) GC (7.0%) cases. Median HER 2 copy number was 19 in GEJ and 16 in GC samples. PIK3CA GA and METamp were observed in ~9% and ~5% of both HER 2amp and non- HER2amp EGC cases; however, co-amplification of cell-cycle mediators CDK6 and CCCNE1 were each significantly enriched in HER 2amp cases (Table). MYCamp and deleterious SMAD4 GA were also significantly enriched in cases with Her2amp. CDKN2A GA were common regardless of HER 2amp, particularly in GEJ . In cases with &gt;100 estimated HER 2 copies, only PIK3CA GA were significantly less frequent than in those with 6-99 copies (2.3% vs. 9.8%, P =0.04). All HER 2amp cases were microsatellite stable. A clinically annotated HER 2amp cohort will be presented. Conclusions: GA predicted to decrease trastuzumab sensitivity exist in a significant portion of HER 2amp EGC, although not all are enriched relative to non- HER 2amp cases. CGP may provide additive information to traditional HER2 testing and identify patients less likely to benefit from therapy. Larger clinical datasets are needed to validate our observations. [Table: see text]

Parasex Generates Phenotypic Diversity de Novo and Impacts Drug Resistance and Virulence in Candida albicans.

Candida albicans is a diploid fungus that is a frequent cause of mucosal and systemic infections in humans. This species exhibits an unusual parasexual cycle in which mating produces tetraploid cells that undergo a nonmeiotic program of concerted chromosome loss to return to a diploid or aneuploid state. In this work, we used a multipronged approach to examine the capacity of parasex to generate diversity in C. albicans First, we compared the phenotypic properties of 32 genotyped progeny and observed wide-ranging differences in fitness, filamentation, biofilm formation, and virulence. Strikingly, one parasexual isolate displayed increased virulence relative to parental strains using a Galleria mellonella model of infection, establishing that parasex has the potential to enhance pathogenic traits. Next, we examined parasexual progeny derived from homothallic, same-sex mating events, and reveal that parasex can generate diversity de novo from identical parental strains. Finally, we generated pools of parasexual progeny and examined resistance of these pools to environmental stresses. Parasexual progeny were generally less fit than control strains across most test conditions, but showed an increased ability to grow in the presence of the antifungal drug fluconazole (FL). FL-resistant progeny were aneuploid isolates, often being diploid strains trisomic for both Chr3 and Chr6. Passaging of these aneuploid strains frequently led to loss of the supernumerary chromosomes and a concomitant decrease in drug resistance. These experiments establish that parasex generates extensive phenotypic diversity de novo, and that this process has important consequences for both virulence and drug resistance in C. albicans populations.

Antiviral Activity and Resistance Analysis of NS3/4A Protease Inhibitor Grazoprevir and NS5A Inhibitor Elbasvir in Hepatitis C Virus GT4 Replicons.

Although genotype 4 (GT4)-infected patients represent a minor overall percentage of the global hepatitis C virus (HCV)-infected population, the high prevalence of the genotype in specific geographic regions coupled with substantial sequence diversity makes it an important genotype to study for antiviral drug discovery and development. We evaluated two direct-acting antiviral agents-grazoprevir, an HCV NS3/4A protease inhibitor, and elbasvir, an HCV NS5A inhibitor-in GT4 replicons prior to clinical studies in this genotype. Following a bioinformatics analysis of available GT4 sequences, a set of replicons bearing representative GT4 clinical isolates was generated. For grazoprevir, the 50% effective concentration (EC50) against the replicon bearing the reference GT4a (ED43) NS3 protease and NS4A was 0.7 nM. The median EC50 for grazoprevir against chimeric replicons encoding NS3/4A sequences from GT4 clinical isolates was 0.2 nM (range, 0.11 to 0.33 nM; n = 5). The difficulty in establishing replicons bearing NS3/4A resistance-associated substitutions was substantially overcome with the identification of a G162R adaptive substitution in NS3. Single NS3 substitutions D168A/V identified from de novo resistance selection studies reduced grazoprevir antiviral activity by 137- and 47-fold, respectively, in the background of the G162R replicon. For elbasvir, the EC50 against the replicon bearing the reference full-length GT4a (ED43) NS5A gene was 0.0002 nM. The median EC50 for elbasvir against chimeric replicons bearing clinical isolates from GT4 was 0.0007 nM (range, 0.0002 to 34 nM; n = 14). De novo resistance selection studies in GT4 demonstrated a high propensity to suppress the emergence of amino acid substitutions that confer high-potency reductions to elbasvir. Phenotypic characterization of the NS5A amino acid substitutions identified (L30F, L30S, M31V, and Y93H) indicated that they conferred 15-, 4-, 2.5-, and 7.5-fold potency losses, respectively, to elbasvir. The activity profiles of grazoprevir and elbasvir supported the testing of the direct-acting antivirals in clinical studies.

MA16.07 Drug Repurposing to Overcome De Novo Resistance of Non-Traditional EGFR Mutations

Approximately 10% of EGFR mutant NSCLCs have an in-frame insertion in exon 20 of EGFR resulting in innate resistance to 1st generation TKIs. The reported response rate of patients with EGFR exon 20 insertions to gefitinib and erlotinib is 5% with a median progression free survival of 1.5 months. It has been shown that exon 20 insertions stabilize the active conformation of EGFR and increase affinity to ATP over TKIs. We hypothesize that exon 20 insertions induce conformational changes to the drug binding pocket resulting in EGFR TKI resistance which can be overcome by covalently binding TKIs. Ba/F3 cells expressing 7 different clinically observed EGFR exon 20 insertion mutations spanning the helix (residues 763-766) and loop (residues 767-773) regions were generated and screened against 1st, 2nd, and 3rd generation EGFR inhibitors including erlotinib, gefitinib, afatinib, dacomitinib, neratinib, poziotinib, ibrutinib rocilentinib, EGF816, and osimertinib. Computational modeling was conducted to analyze the conformational changes and drug binding affinity. In Ba/F3 cells with EGFR exon 20 insertions, most 1st and 3rd generation TKIs failed to inhibit growth of EGFR exon 20 insertions after residue 767 with IC50 values above 100nM. However, poziotinib significantly inhibited cell growth of all EGFR exon 20 insertions tested across the helix and loop regions with an average IC50 value of 2.9nM, as compared to osimertinib and rocilentinib (IC50 values =103nM and 850nM, respectively). Further characterization using three-dimensional modeling revealed that exon 20 insertions induce conformational changes which cause a decreased affinity for 1st generation TKIs and steric hindrance of C797 reducing the ability of 3rd generation TKIs to covalently bind. A significant shift of the c-helix and p-loop result in a sterically hindered binding pocket. Therefore, the smaller, more flexible 1,2-dichloro-3-fluorobenzene terminal group of poziotinib can overcome the structural changes induced by the exon 20 insertions, whereas the ridged 1-methylindole terminal group of osimertinib cannot. EGFR exon 20 insertions induce a shift of the p-loop and c-helix resulting in steric hindrance of the binding pocket thereby preventing binding of 1st generation and 3rd generation EGFR inhibitors including rocilentinib and osimertinib. A smaller, more flexible inhibitor such as poziotinib can overcome the steric hindrance of the drug binding pocket. Currently, in vivo studies of the EGFR D770insNPG GEMM, and EGFR H773insNPH PDX model with poziotinib are underway, and a clinical trial testing poziotinib in EGFR exon 20 mutant NSCLC patients will begin enrollment this year.

PTC725, an NS4B-Targeting Compound, Inhibits a Hepatitis C Virus Genotype 3 Replicon, as Predicted by Genome Sequence Analysis and Determined Experimentally.

PTC725 is a small molecule NS4B-targeting inhibitor of hepatitis C virus (HCV) genotype (gt) 1 RNA replication that lacks activity against HCV gt2. We analyzed the Los Alamos HCV sequence database to predict susceptible/resistant HCV gt's according to the prevalence of known resistance-conferring amino acids in the NS4B protein. Our analysis predicted that HCV gt3 would be highly susceptible to the activity of PTC725. Indeed, PTC725 was shown to be active against a gt3 subgenomic replicon with a 50% effective concentration of ∼5 nM. De novo resistance selection identified mutations encoding amino acid substitutions mapping to the first predicted transmembrane region of NS4B, a finding consistent with results for PTC725 and other NS4B-targeting compounds against HCV gt1. This is the first report of the activity of an NS4B targeting compound against HCV gt3. In addition, we have identified previously unreported amino acid substitutions selected by PTC725 treatment which further demonstrate that these compounds target the NS4B first transmembrane region.

ISY-4-2 - Overcoming endocrine therapy resistance in breast cancer, a cat and mouse game?

Overcoming resistance to endocrine therapy is the most intensive research area in estrogen receptor (ER)- positive breast cancer. Both de-novo resistance and acquired resistance are experienced in the treatment of advanced breast cancer patients, however, it is unclear whether underlying mechanism of this drug resistance could be common for both situation or specific. We know that patient who progress to one endocrine drug still has a chance to response to the other endocrine drug, and in some cases, drug in same category shows benefit when it used later part of her treatment history. At least from clinical experience, resistance mechanism has plasticity in some degree and seems not absolutely be settled until very late stage. In this session, we would discuss about plasticity of endocrine therapy resistance and would like to ask whether Tom can catch Jerry.

Abstract C65: Defining the implications of sugar derived metabolites (AGEs) to tamoxifen resistance and breast cancer disparity: Is it a question of lifestyle?

Abstract Tamoxifen is the most widely prescribed adjuvant therapy for estrogen receptor positive (ER+) breast cancer which comprises around 70% of all breast cancer cases. While many patients respond positively to tamoxifen treatment around 50% have de-novo resistance and approximately 30% of responsive tumors recur due to acquired drug resistance. Compared to European American women, African-American women with ER+ breast cancer, have worse progression-free and overall survival which coincides with increased resistance to anti-cancer therapies such as tamoxifen. Poor diet, low income, obesity and a lack of exercise are established lifestyle factors that are known to increase cancer burden and are often more prevalent in African American communities. Our research has identified a potential mechanistic link between sugar derived metabolites and estrogen receptor (ER) phosphorylation which provides a biological consequence of these established lifestyle factors which may directly impact tamoxifen therapy and minority health. Glycation is the non-enzymatic glycosylation of sugar moieties to biological macromolecules such as protein and DNA which produces reactive metabolites known as advanced glycation end products (AGE's). AGE's accumulate in our tissues as we grow older and drive many of the complications associated with diseases displaying health disparity including diabetes, metabolic syndrome, Alzheimer's and heart disease. Significantly, low income, obesity and an inactive lifestyle are established factors driving health disparity that also contribute to increased AGE accumulation levels in our bodies. In particular, AGE content in the Western Diet has consistently increased over the last 50 years due to increased consumption of sugar laden and cheap processed/manufactured foods which are high in reactive AGE metabolites and promote obesity. Due to the established links with lifestyle and cancer disparity we examined the biological effects of AGEs on tamoxifen therapy and found that elevated AGE levels can directly affect the response to tamoxifen treatment and promote tamoxifen resistance. AGE treatment in ER+ breast cancer cell models promoted tamoxifen resistance via the activation of the MAPK and AKT pathways leading to resistance associated changes in ERα phosphorylation. We also observed greater levels of AGE and its cognate receptor for AGE (RAGE) within breast cancer tumor and serum samples and showed a correlation between tumor progression and intra-tumoral AGE concentration. Strategies to eliminate or delay the occurrence of tamoxifen resistance would contribute to increasing overall survival in minority populations. By associating lifestyle-derived AGEs with tamoxifen resistance, opportunities exist for impacting cancer treatment initiatives arising through health and nutritional education and community outreach programs driven by basic, translational, epidemiological and cancer prevention research initiatives. Citation Format: Katherine R. Walter, Danzell M. Smith, Van Phan, Laura spruill, Marvella E. Ford, Victoria J. Findlay, David P. Turner. Defining the implications of sugar derived metabolites (AGEs) to tamoxifen resistance and breast cancer disparity: Is it a question of lifestyle? [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr C65.

Hit me with your best shot: dolutegravir - a space in the next WHO guidelines?

Hit me with your best shot: dolutegravir - a space in the next WHO guidelines?

237P - Characteristics of De Novo Resistance to Androgen Targeting Therapeutics (Ar Tx) Through Circulating Tumor Cells (Ctcs) Analysis in Metastatic Castration Resistant Prostate Cancer (Mcrpc) Patients

ABSTRACT Aim: The prospective identification of patients (pts) with tumors that are resistant to AR tx de novo (DN) reduces pt morbidity and enables the early exploration of alternative approaches. We examined CTCs, the incidence of specific CTC subpopulations, AR protein expression and AR localization from blood samples taken prior to AR Tx, inclusive of abiraterone acetate + prednisone (AA + P), enzalutamide (E) or taxanes (T) to determine if a prediction model could be developed for DN resistance. Methods: 40 blood samples were collected from mCRPC pts immediately prior to treatment with AR tx (15 AA + P, 9 E, & 16 T). Pts were treated with AR Tx and followed with outcomes classified as true response (TR) (n = 10), acquired resistance (AcR, initial response followed by progression within the first 6 months of treatment) (n = 13), or DN resistance (n = 17). Samples were processed utilizing the Epic Sciences platform. CTCs, defined as traditional (CK + , CD45-, intact nucleus, distinct morphology), cluster (≥ 2 attached CTCs), CK- (CK-, CD45-, distinct morphology, intact nucleus), and small (CK + , CD45-, small intact nucleus) were examined for AR. PSA, CellSearch®, and clinical history were recorded. Results: DN pt blood draws had higher numbers of all cell events and AR+ CTCs than AcR or TR. CTC Events per 7.5mL of Blood True Responders (n = 10) Acquired Resistors (n = 13) de novo Resistance (n = 17) Mean PSA (range) 13.36 (0.51–24.98) 307.76 (7.94–1774.5) 435.08 (0.71–2589.9) CellSearch® ≥ 5 CTC CellSearch® (Unfavorable) 20% 69% 65% Epic CTCs ≥ 5 Traditional CTCs 90% 100% 94% Mean total CTC events per patient 130 193 629 Mean AR+ CTCs per patient 20 51 266 Mean CK- CTCs per patient 22 29 52 Conclusions: Patients with DN resistance to AR tx have higher baseline CTC event frequency than responders (including traditional, CK-ve and AR expressing CTCs). Higher CTC event frequency was also observed in AcR as compared with TR responders. By analysing overall CTC counts (including subpopulations), DN resistant patients may be identified through analysis of a baseline blood draw. Analysis of baseline CTC heterogeneity in AcR may also provide insights to minor resistant subclonal populations. Further CTC analysis of AcR and DN baseline samples is warranted. Disclosure: J. Louw, R. Krupa, A. Jendrisak, D. Marrinucci and R. Dittamore: I am an employee and stockholder of Epic Sciences; H.I. Scher: Research funding from Janssen, Janssen Research, and Medivation Uncompensated advisor for Janssen, Janssen Research, and Medivation. All other authors have declared no conflicts of interest.

Rapid response to selection, competitive release and increased transmission potential of artesunate-selected Plasmodium chabaudi malaria parasites.

The evolution of drug resistance, a key challenge for our ability to treat and control infections, depends on two processes: de-novo resistance mutations, and the selection for and spread of resistant mutants within a population. Understanding the factors influencing the rates of these two processes is essential for maximizing the useful lifespan of drugs and, therefore, effective disease control. For malaria parasites, artemisinin-based drugs are the frontline weapons in the fight against disease, but reports from the field of slower parasite clearance rates during drug treatment are generating concern that the useful lifespan of these drugs may be limited. Whether slower clearance rates represent true resistance, and how this provides a selective advantage for parasites is uncertain. Here, we show that Plasmodium chabaudi malaria parasites selected for resistance to artesunate (an artemisinin derivative) through a step-wise increase in drug dose evolved slower clearance rates extremely rapidly. In single infections, these slower clearance rates, similar to those seen in the field, provided fitness advantages to the parasite through increased overall density, recrudescence after treatment and increased transmission potential. In mixed infections, removal of susceptible parasites by drug treatment led to substantial increases in the densities and transmission potential of resistant parasites (competitive release). Our results demonstrate the double-edged sword for resistance management: in our initial selection experiments, no parasites survived aggressive chemotherapy, but after selection, the fitness advantage for resistant parasites was greatest at high drug doses. Aggressive treatment of mixed infections resulted in resistant parasites dominating the pool of gametocytes, without providing additional health benefits to hosts. Slower clearance rates can evolve rapidly and can provide a strong fitness advantage during drug treatment in both single and mixed strain infections.