Altered Drug Sensitivity Research Articles

Molecular Dynamics Unveils Multiple-Site Binding of Inhibitors with Reduced Activity on the Surface of Dihydrofolate Reductase.

The sensitivity to protein inhibitors is altered by modifications or protein mutations, as represented by drug resistance. The mode of stable drug binding to the protein pocket has been experimentally clarified. However, the nature of the binding of inhibitors with reduced sensitivity remains unclear at the atomic level. In this study, we analyzed the thermodynamics and kinetics of inhibitor binding to the surface of wild-type and mutant dihydrofolate reductase (DHFR) using molecular dynamics simulations combined with Markov state modeling. A strong inhibitor of methotrexate (MTX) showed a preference for the active site of wild-type DHFR with minimal binding to unrelated (secondary) sites. Deletion of a side-chain fragment in MTX largely destabilized the active site-bound state, with clear evidence of binding to secondary sites. Similarly, the F31V mutation in DHFR diminished the specificity of MTX binding to the active site. These results reveal the presence of multiple-bound states whose stabilities are comparable to or higher than those of the unbound state, suggesting that a reduction in the binding affinity for the active site significantly elevates the fractions of these states. This study presents a theoretical model that more accurately interprets the altered drug sensitivity than the traditional two-state model.

Comprehensive pan-cancer analysis and experiments revealed R3HDM1 as a novel predictive biomarker for prognosis and immune therapy response.

R3HDM1, an RNA binding protein with one R3H domain, remains uncharacterized in terms of its association with tumor progression, malignant cell regulation, and the tumor immune microenvironment. This paper aims to fill this gap by analyzing the potential of R3HDM1 in diagnosis, prognosis, chemotherapy, and immune function across various cancers. Data was collected from the Firehost database (http://gdac.broadinstitute.org) to obtain the TCGA pan-cancer queue containing tumor and normal samples. Additional data on miRNA, TCPA, mutations, and clinical information were gathered from the UCSC Xena database (https://xenabrowser.net/datapages/). The mutation frequency and locus of R3HDM1 in the TCGA database were examined using the cBioPortal. External validation through GEO data was conducted to assess the differential expression of R3HDM1 in different cancers. Protein expression levels were evaluated using the Clinical Proteomics Tumor Analysis Alliance (CPTAC). The differential expression of R3HDM1 was verified in lung adenocarcinoma cell lines and normal lung glandular epithelial cells via RT-qPCR. Cell migration and proliferation experiments were conducted by knocking down the expression of R3HDM1 in two lung adenocarcinoma cell lines using small interfering RNA. The biological role of R3HDM1 in pan-cancer was explored using the GSEA method. Multiple immune infiltration algorithms from the TIMER2.0 database was employed to investigate the correlation between R3HDM1 expression and the tumor immune microenvironment. Validation of transcriptome immune infiltration was based on 140 single-cell datasets from the TISCH database. The study also characterized a pan-cancer survival profile and analyzed the differential expression of R3HDM1 in different molecular subtypes. The relationship between R3HDM1 and drug resistance was investigated using four chemotherapy data sources: CellMiner, GDSC, CTRP and PRISM. The impact of chemicals on the expression of R3HDM1 was explored through the CTD database. The study revealed differential expression of R3HDM1 in various tumors, indicating its potential as an early diagnostic marker. Changes in somatic copy number (SCNA) and DNA methylation were identified as factors contributing to abnormal expression levels. Additionally, the study found that R3HDM1 expression is associated with clinical features, metabolic pathways, and important pathways related to metastasis and the immune system. High expression of R3HDM1 was linked to poor prognosis across different tumors and altered drug sensitivity. Furthermore, the expression of R3HDM1 showed significant correlations with immune modulatory molecules and biomarkers of lymphocyte subpopulation infiltration. Finally, the study highlighted four chemicals that could influence the expression of R3HDM1. Overall, this study proposes that R3HDM1 expression is a promising biomarker for predicting the prognosis of cancer, especially lung adenocarcinoma, and the efficacy of immunotherapy, demonstrating the rationale for further exploration in the development of anti-tumor therapies.

Mycobacterium tuberculosis functional genetic diversity, altered drug sensitivity, and precision medicine.

In the face of the unrelenting global burden of tuberculosis (TB), antibiotics remain our most effective tools to save lives and control the spread of Mycobacterium tuberculosis (Mtb). However, we confront a dual challenge in our use of antibiotics: simplifying and shortening the TB drug regimen while also limiting the emergence and propagation of antibiotic resistance. This task is now more feasible due to the increasing availability of bacterial genomic data at or near the point of care. These resources create an opportunity to envision how integration of bacterial genetic determinants of antibiotic response into treatment algorithms might transform TB care. Historically, Mtb drug resistance studies focused on mutations in genes encoding antibiotic targets and the resulting increases in the minimal inhibitory concentrations (MICs) above a breakpoint value. But recent progress in elucidating the effects of functional genetic diversity in Mtb has revealed various genetic loci that are associated with drug phenotypes such as low-level MIC increases and tolerance which predict the development of resistance and treatment failure. As a result, we are now poised to advance precision medicine approaches in TB treatment. By incorporating information regarding Mtb genetic characteristics into the development of drug regimens, clinical care which tailors antibiotic treatment to maximize the likelihood of success has come into reach.

Development of Human Cell-Based In Vitro Infection Models to Determine the Intracellular Survival of Mycobacterium avium.

The Mycobacterium avium (Mav) complex accounts for more than 80% of all pulmonary diseases caused by non-tuberculous mycobacteria (NTM) infections, which have an alarming increase in prevalence and vary in different regions, currently reaching 0.3–9.8 per 100,000 individuals. Poor clinical outcomes, as a result of increasing microbial drug resistance and low treatment adherence due to drug-toxicities, emphasize the need for more effective treatments. Identification of more effective treatments, however, appears to be difficult, which may be due to the intracellular life of NTM and concomitant altered drug sensitivity that is not taken into account using traditional drug susceptibility testing screenings. We therefore developed human cell-based in vitro Mav infection models using the human MelJuSo cell line as well as primary human macrophages and a fluorescently labeled Mav strain. By testing a range of multiplicity of infection (MOI) and using flow cytometry and colony-forming unit (CFU) analysis, we found that an MOI of 10 was the most suitable for Mav infection in primary human macrophages, whereas an MOI of 50 was required to achieve similar results in MelJuSo cells. Moreover, by monitoring intracellular bacterial loads over time, the macrophages were shown to be capable of controlling the infection, while MelJuSo cells failed to do so. When comparing the MGIT system with the classical CFU counting assay to determine intracellular bacterial loads, MGIT appeared as a less labor-intensive, more precise, and more objective alternative. Next, using our macrophage Mav infection models, the drug efficacy of the first-line drug rifampicin and the more recently discovered bedaquiline on intracellular bacteria was compared to the activity on extracellular bacteria. The efficacy of the antibiotics inhibiting bacterial growth was significantly lower against intracellular bacteria compared to extracellular bacteria. This finding emphasizes the crucial role of the host cell during infection and drug susceptibility and highlights the usefulness of the models. Taken together, the human cell-based Mav infection models are reliable tools to determine the intracellular loads of Mav, which will enable researchers to investigate host–pathogen interactions and to evaluate the efficacy of (host-directed) therapeutic strategies against Mav.

Immunosuppression broadens evolutionary pathways to drug resistance and treatment failure during Acinetobacter baumannii pneumonia in mice

Acinetobacter baumannii is increasingly refractory to antibiotic treatment in healthcare settings. As is true of most human pathogens, the genetic path to antimicrobial resistance (AMR) and the role that the immune system plays in modulating AMR during disease are poorly understood. Here we reproduced several routes to fluoroquinolone resistance, performing evolution experiments using sequential lung infections in mice that are replete with or depleted of neutrophils, providing two key insights into the evolution of drug resistance. First, neutropenic hosts acted as reservoirs for the accumulation of drug resistance during drug treatment. Selection for variants with altered drug sensitivity profiles arose readily in the absence of neutrophils, while immunocompetent animals restricted the appearance of these variants. Secondly, antibiotic treatment failure in the immunocompromised host was shown to occur without clinically defined resistance, an unexpected result that provides a model for how antibiotic failure occurs clinically in the absence of AMR. The genetic mechanism underlying both these results is initiated by mutations activating the drug egress pump regulator AdeL, which drives persistence in the presence of antibiotic. Therefore, antibiotic persistence mutations present a two-pronged risk during disease, causing drug treatment failure in the immunocompromised host while simultaneously increasing the emergence of high-level AMR.

Construction of a Co-Expression Network for lncRNAs and mRNAs Related to Urothelial Carcinoma of the Bladder Progression.

Carcinoma of urinary bladder is the most familiar cancer of the urinary tract, with the highest incidence in men. However, its prognosis and treatment have not improved significantly in the last 30 years. The main reason for this may be related to the alteration and regulation of genes. These alterations in genes that play a crucial role in cell cycle regulation may result in high-grade tumors and may alter drug sensitivity. Notably, the role of lncRNA in bladder cancer, especially the lncRNA-mRNA regulatory network, has not been fully elucidated. In this manuscript, we compared RNA sequencing (RNA-seq) data from 19 normal bladder tissues and 411 primary bladder tumor tissues using The Cancer Genome Atlas (TCGA) data bank, subjected differentially expressed mRNAs and lncRNAs to weighted gene co-expression network analysis, and screened out modules highly correlated with tumor progression. Subsequently, a lncRNA-mRNA co-expression network was built, and two key mRNAs were identified via COX regression analysis. Kaplan-Meier curve analysis revealed that the overall survival of sick people in the high-risk section was significantly shorter than those in the low-risk section. Therefore, this lncRNA-mRNA-based co-expression pattern may be used clinically to predict the prognosis of carcinoma of urinary bladder people. Our study not only provides a genetic target for carcinoma of urinary bladder therapy but also provides new ideas for people in the medical profession to discover the treatment of various tumors.

Phenotypic Consequences of SLC25A40-ABCB1 Fusions beyond Drug Resistance in High-Grade Serous Ovarian Cancer.

Simple SummaryAmong the plethora of malignancies affecting the female reproductive tract, those concerning the ovary are the most frequently fatal. In particular, chemotherapy-resistant High-Grade Serous Ovarian Cancer (HGSOC) remains a clinically intractable disease with a high rate of mortality. We previously identified SLC25A40-ABCB1 transcriptional fusions as the driving force behind drug resistance in HGSOC. As success in the clinical arena will only be achieved by enhancing our fundamental understanding of the drivers that mediate cellular drug resistance, this report sought to elucidate the phenotypic, metabolomic and transcriptional consequences of SLC25A40-ABCB1 fusions beyond drug resistance. High-throughput FDA drug screening was also undertaken to identify new therapeutic avenues against drug-resistant cellular populations.Despite high response rates to initial chemotherapy, the majority of women diagnosed with High-Grade Serous Ovarian Cancer (HGSOC) ultimately develop drug resistance within 1–2 years of treatment. We previously identified the most common mechanism of acquired resistance in HGSOC to date, transcriptional fusions involving the ATP-binding cassette (ABC) transporter ABCB1, which has well established roles in multidrug resistance. However, the underlying biology of fusion-positive cells, as well as how clonal interactions between fusion-negative and positive populations influences proliferative fitness and therapeutic response remains unknown. Using a panel of fusion-negative and positive HGSOC single-cell clones, we demonstrate that in addition to mediating drug resistance, ABCB1 fusion-positive cells display impaired proliferative capacity, elevated oxidative metabolism, altered actin cellular morphology and an extracellular matrix/inflammatory enriched transcriptional profile. The co-culture of fusion-negative and positive populations had no effect on cellular proliferation but markedly altered drug sensitivity to doxorubicin, paclitaxel and cisplatin. Finally, high-throughput screening of 2907 FDA-approved compounds revealed 36 agents that induce equal cytotoxicity in both pure and mixed ABCB1 fusion populations. Collectively, our findings have unraveled the underlying biology of ABCB1 fusion-positive cells beyond drug resistance and identified novel therapeutic agents that may significantly improve the prognosis of relapsed HGSOC patients.

Effect of cell microenvironment on the drug sensitivity of hepatocellular cancer cells.

The native hepatocellular cancer (HCC) microenvironment is characterized by more hypoxic, hypoglycemic, and acidic conditions than those used in standard cell culture. This study aimed to investigate whether HCC cells cultured in more native conditions have an altered phenotype and drug sensitivity compared to those cultured in standard conditions. Six HCC cell lines were cultured in “standard” (21% O2, 25 mM glucose) or more “native” (1% O2, 5 mM glucose, 10 mM lactate) conditions. Cells were assessed for growth rates, cell cycle distribution, relevant metabolite and protein levels, genome-wide gene expression, mitochondrial DNA sequence and sensitivity to relevant drugs. Many differences in cellular and molecular phenotypes and drug sensitivity were observed between the cells. HCC cells cultured in native conditions had slower doubling times, increased HK2 and GLUT, lower PHDA and ATP levels, and mutations in mitochondrial DNA. Thirty-one genes, including the hypoxia-associated NDRG1, were differentially expressed between the cells. HCC patients in The Cancer Genome Atlas (TCGA) with tumors with a high score based on these 31 genes had a poorer prognosis than those with a low score (p = 0.002). From 90 comparisons of drug sensitivity, increased resistance and sensitivity for cells cultured in native conditions was observed in 14 (16%) and 8 (9%) comparisons respectively. In conclusion, cells cultured in more native conditions can have a more glycolytic and aggressive phenotype and varied drug sensitivity to those cultured in standard conditions, and may provide new insights to understanding tumor biology and drug development.

Effects of Cryopreservation on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes for Assessing Drug Safety Response Profiles.

SummaryBurgeoning applications of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in disease modeling, regenerative medicine, and drug screening have broadened the usage of hiPSC-CMs and entailed their long-term storage. Cryopreservation is the most common approach to store hiPSC-CMs. However, the effects of cryopreservation and recovery on hiPSC-CMs remain poorly understood. Here, we characterized the transcriptome, electro-mechanical function, and drug response of fresh hiPSC-CMs without cryopreservation and recovered hiPSC-CMs from cryopreservation. We found that recovered hiPSC-CMs showed upregulation of cell cycle genes, similar or reduced contractility, Ca2+ transients, and field potential duration. When subjected to treatment of drugs that affect electrophysiological properties, recovered hiPSC-CMs showed an altered drug response and enhanced propensity for drug-induced cardiac arrhythmic events. In conclusion, fresh and recovered hiPSC-CMs do not always show comparable molecular and physiological properties. When cryopreserved hiPSC-CMs are used for assessing drug-induced cardiac liabilities, the altered drug sensitivity needs to be considered.

TP53 in Acute Myeloid Leukemia: Evolving from Just Another Prognostic Marker to an Actionable Biomarker Informing Treatment

Introduction Acute Myeloid Leukemia (AML) is a devastating disease with poor overall survival. Access to Precision Medicines is revolutionizing AML care and is driving an increase in Next Generation Sequencing (NGS) utilization to determine the genomic profile of patients with AML. Collaborative efforts to identify and promote emerging and established actionable biomarkers benefits all stakeholders in the patient diagnostic pathway and, ultimately, leads to more patients getting the right test for the right treatment. TP53 plays a central role in key cellular processes such as oncogenesis, cellular proliferation, and DNA repair.1 TP53 is commonly mutated in most cancers. However, a vast majority of AML patients harbor intact, wild-type TP53 in which TP53 pathway dysregulation is promoted by mechanisms such as FLT3 mutations and chromosomal translocations. TP53 mutations lead to clonal propagation and promotes leukemogenesis, and are associated with altered drug sensitivity, chemoresistance, and high risk of relapse.Thus, mutated TP53 is a powerful independent prognostic indicator of poor outcome in AML. TP53 mutations are common in AML cases characterized by increased genomic instability, relapse, and the elderly. Therefore, treating AML patients by directly targeting TP53 mutant AMLs, or leveraging wild-type TP53 AMLs with therapeutic strategies in combination with standard AML chemotherapy or other targeted therapies requires that all AML patients have their TP53 status tested.1 Methods We assessed the testing landscape for determining TP53 status in AML cancer using a data set of 7758 newly diagnosed and relapsed/refractory (R/R) AML patients in the US from Q1-2017 through Q2-2019 identified from Diaceutics' proprietary Global Diagnostic Index (GDI). An analysis of real-world TP53 and Next Generation Sequencing (NGS)-associated testing data and a laboratory profile mapping exercise of 806 US labs was carried out using Diaceutics' proprietary methods and data sources to evaluate TP53 testing rates, test availability, and testing methodology. AML biomarker testing rates were determined from 1176 patients and results available for 437 of them were used to obtain positivity rates. Results Overall, 95 labs performed in-house AML molecular biomarker testing. Of these, 50 performed TP53 testing, 65 performed IDH testing, and 84 performed FLT3 testing. Testing rates on bone marrow aspirates for TP53, IDH1/2, and FLT3 were 59%, 63% and 68%, respectively, with corresponding positivity rates of 15%, 9% (IDH1), 13% (IDH2), 17%, respectively. Simultaneous testing with cytogenetic markers occurred 77% of the time vs 23% of the time following the results from cytogenetic testing. A total of 43 labs included TP53 as part of comprehensive NGS panels, with 3 labs exclusively using Sanger Sequencing and 4 labs testing TP53 via single gene (Sanger and/or NGS) and comprehensive NGS panels. Co-mutation rates between mutated TP53 and FLT3 or IDH1 were 1.3% and 4.2%, respectively. Co-mutation rates between unmutated TP53 and FLT3 or IDH1 were 17.5% and 9.3%, respectively. Conclusion TP53 status is evolving from simply a poor prognostic indicator to an attractive actionable biomarker in newly diagnosed and R/R AML patients. Thus, TP53 status should always be properly tested in parallel with other actionable AML biomarkers. Stakeholder awareness should be increased and recommendations for TP53 clinical utility, preferred testing methods, gene regions to test, and results reporting should be updated, to advance standardization and harmonization that ultimately improves treatment outcomes for AML patients. Reference Barbosa K, Li S, Adams PD, Deshpande AJ. The role of TP53 in acute myeloid leukemia: Challenges and opportunities. Genes Chromosomes Cancer. 2019;58:875-888. https://doi.org/10.1002/gcc.2279 Disclosures Riccelli: Diaceutics: Current Employment, Current equity holder in publicly-traded company. Hoefsmit:Diaceutics: Current Employment, Current equity holder in publicly-traded company. Moore:Diaceutics: Current Employment. Idica:Diaceutics: Current Employment, Current equity holder in publicly-traded company. Sornkom:Diaceutics: Current Employment. Slifko:Diaceutics: Current Employment, Current equity holder in publicly-traded company. Delic:Diaceutics: Current Employment, Current equity holder in publicly-traded company. Entwistle:Diaceutics: Current Employment, Current equity holder in publicly-traded company. Munksted:Diaceutics: Current Employment, Current equity holder in publicly-traded company. Clark:Diaceutics: Current Employment, Current equity holder in publicly-traded company.

Flammer Syndrome & Glaucoma

The need of blood flow to different organs varies rapidly over time which is why there is a need for sophisticated local regulation of blood flow. The term “dysregulation” just simply means that blood flow is not properly adapted to this need. Dysregulative mechanisms can therefore lead to an over- or under-perfusion. A constant over- or under-perfusion does not normally induce long-term damage. A repeated under-perfusion, such as a repeated mild reperfusion injury, however, leads to damage. Systemic dysregulation can be primary or secondary of nature. A secondary dysregulation (SVD) is due to other diseases such as autoimmune diseases. The term Flammer Syndrome (FS) named after the famous physician J. Flammer refers to a clinical entity comprising a complex of clinical features caused mainly by dysregulation of the blood supply which has previously been called primary vascular dysregulation. People with FS tend to have cold extremities, prolonged sleep-onset time, altered drug sensitivity, low blood pressure and higher smell score, and increased retinal venous pressures as measured by means of ophthalmodynamometry. In the eye, the spatial irregularity of the retinal arteries is increased, and optic nerve head blood flow is correlated with finger blood flow indirectly indicating that the local regulation is disturbed. Blood flow is, on average, reduced in glaucoma patients, particularly in patients with normal-tension glaucoma suffering from FS, and in patients with high-tension glaucoma, which progress despite a normalized intraocular pressure (IOP). A constant reduction of blood flow (as we see in SVD) can lead to atrophy but does not contribute to glaucomatous atrophy. An increased variation of microcirculation as commonly seen in glaucoma patients with FS, however, is clearly linked to occurrence and progression of glaucomatous optic neuropathy (GON). Oxygen supply to the eye fluctuates, either if IOP fluctuates on a high level or blood pressure on a low level or if autoregulation is disturbed. Autoregulation is disturbed in patients with primary vascular dysregulation (PVD). Unstable oxygen supply to the optic nerve head leads to oxidative stress, which in turn, leads to the production of peroxynitrite (ONOO-) which finally kills the cells. In this review, we are talking about pathogenesis of the FS and some suggested therapeutic options for it.

Screening transposon-mutant library of methicillin-resistant Staphylococcus aureus for mutants with altered sensitivity toward three antimicrobial agents

Background and Hypothesis: Methicillin-resistant Staphylococcus aureus (MRSA) is resistant to almost all beta-lactam antibiotics, including oxacillin. To combat MRSA infections, recently, new classes of drugs are developed, such as oxadiazole DR1 and ESP404. We hypothesize that, if we identify MRSA mutants with altered sensitivity toward the drugs, those mutants will help us understand the antimicrobial mechanisms of the drugs.
 Experimental Design or Project Methods: The screening was carried out on the Nebraska library consisting of 1,920 non-redundant transposon mutants of USA300 strain in 96 well plates. First, the minimum inhibitory concentration (MIC) of each drug was determined. Then the transposon mutants were grown in tryptic soy broth (TSB) containing either 1/4× MIC or 4× MIC of the drugs for sensitive mutants and resistant mutants respectively. Due to the limited availability of ESP404, the drug was used to screen for sensitive mutants only.
 Results: The first screening identified 81 mutants with altered drug sensitivity: 23 for oxadiazole DR1 (5 sensitive, 18 resistant); 20 for ESP404 (all sensitive); 38 for oxacillin (18 sensitive, 5 resistant).
 Conclusion and Potential Impact: Screening of the transposon-mutant library identified multiple mutants that have altered sensitivity toward three drugs against S. aureus. Further verification of the phenotype and characterization of those mutants are expected to shed light on the antimicrobial mechanism of the drugs and open a door for the development of drug-potentiators.

2003P - Expression of mutant p53 affects cancer cell sensitivity to topotecan

BackgroundInactivation of tumor suppressor p53 is a common event in tumor progression. In almost 50% of cases p53 inactivation is caused by mutations that primarily affect DNA-binding domain. Oncogenic missense mutation Y220C is the ninth most common for p53 and is annually observed in ∼100,000 new diagnosed cancer cases worldwide. Presence of this mutation disturbs tertiary structure of the p53 DNA-binding domain that further leads to destabilization of the whole protein, its partial denaturation and loss of transcriptional activity. In some cases, p53 mutations result in more aggressive cancer cells and alter drug sensitivity. MethodsWe employ CRISPR-Cas9 gene editing technology to generate p53 knock-out and Y220C mutant MCF-7 cell lines. Quantitative analysis of alterations in intracellular protein levels were performed by immunoblotting, analysis of p53-dependent gene expression by quantitative real-time reverse transcription PCR; cell proliferation and chemotherapy cytotoxicity by MTS test. Statistical analysis was performed using ANOVA and Holm-Sidak method for multiple comparison, p≤0.05 was considered to be statistically significant. ResultsWe have confirmed that p53 mutation Y220C leads to p53 inactivation. Proliferation rate of p53 knockout and mutant MCF-7 cell lines was 15% higher than wild type. We have shown statistically significant decrease in topotecan cytotoxicity towards knockout and mutant cells compared to wild-type – 14% and 26%, respectively. ConclusionsDecline of topotecan cytotoxicity in mutant and knockout cells can be explained by topotecan-mediated induction of p53 that leads to higher levels of cell death in wild-type MCF-7. References Bulatov, E., Zagidullin, A., Valiullina, A., Sayarova, R., Rizvanov, A. (2018). Small molecule modulators of RING-type E3 ligases: MDM and Cullin families as targets. Frontiers in pharmacology, 9, 450. Legal entity responsible for the studyAlbert Rizvanov. FundingGrant of the President of the Russian Federation (MK-4253.2018.4). DisclosureAll authors have declared no conflicts of interest.

Extensive transcriptional responses are co-ordinated by microRNAs as revealed by Exon-Intron Split Analysis (EISA).

Epithelial–mesenchymal transition (EMT) has been a subject of intense scrutiny as it facilitates metastasis and alters drug sensitivity. Although EMT-regulatory roles for numerous miRNAs and transcription factors are known, their functions can be difficult to disentangle, in part due to the difficulty in identifying direct miRNA targets from complex datasets and in deciding how to incorporate ‘indirect’ miRNA effects that may, or may not, represent biologically relevant information. To better understand how miRNAs exert effects throughout the transcriptome during EMT, we employed Exon–Intron Split Analysis (EISA), a bioinformatic technique that separates transcriptional and post-transcriptional effects through the separate analysis of RNA-Seq reads mapping to exons and introns. We find that in response to the manipulation of miRNAs, a major effect on gene expression is transcriptional. We also find extensive co-ordination of transcriptional and post-transcriptional regulatory mechanisms during both EMT and mesenchymal to epithelial transition (MET) in response to TGF-β or miR-200c respectively. The prominent transcriptional influence of miRNAs was also observed in other datasets where miRNA levels were perturbed. This work cautions against a narrow approach that is limited to the analysis of direct targets, and demonstrates the utility of EISA to examine complex regulatory networks involving both transcriptional and post-transcriptional mechanisms.

Functional Coupling of K+-Cl- Cotransporter (KCC) to GABA-Gated Cl- Channels in the Central Nervous System of Drosophila melanogaster Leads to Altered Drug Sensitivities.

GABAergic signaling is the cornerstone for fast synaptic inhibition of neural signaling in arthropods and mammals and is the molecular target for insecticides and pharmaceuticals, respectively. The K+-Cl- cotransporter (KCC) is the primary mechanism by which mature neurons maintain low intracellular Cl- concentration, yet the fundamental physiology, comparative physiology, and toxicological relevance of insect KCC is understudied. Considering this, we employed electrophysiological, genetic, and pharmacological methods to characterize the physiological underpinnings of KCC function to the Drosophila CNS. Our data show that genetic ablation or pharmacological inhibition of KCC results in an increased spike discharge frequency and significantly ( P < 0.05) reduces the CNS sensitivity to γ-aminobutyric acid (GABA). Further, simultaneous inhibition of KCC and ligand-gated chloride channel (LGCC) complex results in a significant ( P < 0.001) increase in CNS spontaneous activity over baseline firing rates that supports functional coupling of KCC to LGCC function. Interestingly, 75% reduction in KCC mRNA did not alter basal neurotransmission levels indicating that only a fraction of the KCC population is required to maintain the Cl- ionic gradient when at rest, but prolonged synaptic activity increases the threshold for GABA-mediated inhibition and reduces nerve sensitivity to GABA. These data expand current knowledge regarding the physiological role of KCC in a model insect and provides the necessary foundation to develop KCC as a novel biochemical target of insecticides, as well as complements existing research to provide a holistic understanding of the plasticity in mammalian health and disease.

Genome-scale network model of metabolism and histone acetylation reveals metabolic dependencies of histone deacetylase inhibitors

Histone acetylation plays a central role in gene regulation and is sensitive to the levels of metabolic intermediates. However, predicting the impact of metabolic alterations on acetylation in pathological conditions is a significant challenge. Here, we present a genome-scale network model that predicts the impact of nutritional environment and genetic alterations on histone acetylation. It identifies cell types that are sensitive to histone deacetylase inhibitors based on their metabolic state, and we validate metabolites that alter drug sensitivity. Our model provides a mechanistic framework for predicting how metabolic perturbations contribute to epigenetic changes and sensitivity to deacetylase inhibitors.

Cellular polarity modulates drug resistance in primary colorectal cancers via orientation of the multidrug resistance protein ABCB1.

Colonic epithelial cells are highly polarised with a lumen‐facing apical membrane, termed the brush border, and a basal membrane in contact with the underlying extracellular matrix (ECM). This polarity is often maintained in cancer tissue in the form of neoplastic glands and has prognostic value. We compared the cellular polarity of several ex vivo spheroid colonic cancer cultures with their parental tumours and found that those grown as non‐attached colonies exhibited apical brush border proteins on their outer cellular membranes. Transfer of these cultures to an ECM, such as collagen, re‐established the centralised apical polarity observed in vivo. The multidrug resistance protein ABCB1 also became aberrantly polarised to outer colony membranes in suspension cultures, unlike cultures grown in collagen, where it was polarised to central lumens. This polarity switch was dependent on the presence of serum or selected serum components, including epidermal growth factor (EGF), transforming growth factor‐β1 (TGF‐β1) and insulin‐like growth factor‐1 (IGF‐1). The apical/basal orientation of primary cancer colon cultures cultured in collagen/serum was modulated by α2β1 integrin signalling. The polarisation of ABCB1 in colonies significantly altered drug uptake and sensitivity, as the outward polarisation of ABCB1 in suspension colonies effluxed substrates more effectively than ECM‐grown colonies with ABCB1 polarised to central lumens. Thus, serum‐free suspension colonies were more resistant to a variety of anti‐cancer drugs than ECM‐grown colonies. In conclusion, the local stroma, or absence thereof, can have profound effects on the sensitivity of colorectal cultures to drugs that are ABCB1 substrates. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Tumor‐stroma interactions differentially alter drug sensitivity based on the origin of stromal cells

Due to tumor heterogeneity, most believe that effective treatments should be tailored to the features of an individual tumor or tumor subclass. It is still unclear, however, what information should be considered for optimal disease stratification, and most prior work focuses on tumor genomics. Here, we focus on the tumor microenvironment. Using a large‐scale coculture assay optimized to measure drug‐induced cell death, we identify tumor–stroma interactions that modulate drug sensitivity. Our data show that the chemo‐insensitivity typically associated with aggressive subtypes of breast cancer is not observed if these cells are grown in 2D or 3D monoculture, but is manifested when these cells are cocultured with stromal cells, such as fibroblasts. Furthermore, we find that fibroblasts influence drug responses in two distinct and divergent manners, associated with the tissue from which the fibroblasts were harvested. These divergent phenotypes occur regardless of the drug tested and result from modulation of apoptotic priming within tumor cells. Our study highlights unexpected diversity in tumor–stroma interactions, and we reveal new principles that dictate how fibroblasts alter tumor drug responses.

Abstract LB-056: Novel molecular mechanism underlying cancer drug resistance: Involvement of p58IPKchaperone pathway

Abstract Rhabdomyosarcoma (RMS) is an aggressive and highly malignant form of pediatric cancer that develops from skeletal muscle cells. A third of these cancers become drug resistant, metastatic and fatal. Current therapeutic approaches have not resulted in significant improvement in outcome for patients with recurrent and/or metastatic disease. Here, employing two RMS-derived cell lines (RH30, a drug sensitive and RD, a drug resistant), we demonstrate a novel underlying molecular mechanism of drug resistance in these tumor subtypes. We show that in response to chemotherapeutics drugs [actinomysin D, torisol, etoposide and arsenic trioxide (ATO)], endoplasmic reticulum (ER) stress associated unfolded protein response (UPR) signaling pathway could be induced in RH30 cell only, while drug resistance RD cells largely lack this ability. The mechanism underlying UPR induction involves activation of multifunctional molecular chaperone P58IPK that besides regulating protein homeostasis is an inhibitor of the interferon-induced double-stranded RNA-activated protein kinase (PKR). We observed that the expression of p58IPK is high in drug resistance RD cells as compared to drug sensitive RH30 cells. To confirm its role in drug resistance, we ablated or overexpressed p58IPK respectively in drug resistant RD and drug sensitive RH30 cells. The p58IPK depleted RD cells became sensitive to chemotherapeutic agents while overexpressing p58IPK RH30 cells manifested drug resistance. To further demonstrate the known functional consequences of manipulation in the expression of p58IPK we found that P58IPK over-expression in RH30 cells inhibited eIF2α phosphorylation and diminished apoptosis by downregulating ATF4/CHOP/Cleaved caspase-3, while silencing of p58IPK in RD cells enhanced eIF2α phosphorylation and increased ATF4/CHOP/Cleaved caspase-3-dependent apoptosis in response to chemotherapeutic drug treatment. The underlying mechanism by which P58IPK alters drug sensitivity in RMS cells involves its chaperone function by its binding to drug transporters ABCB-1 and ABCC-1. At baseline, in RD cells with high p58IPK manifest significantly higher dye efflux as compared to RH30 with low p58IPK. This baseline drug exclusion profile correlated with the ATPase activity of these ABC transporters. However, ablation or overexpression of p58IPK respectively, in RH30/RD cells reversed their drug resistance phenotype. Thus, we have identified a novel drug resistance mechanism in RMS. These results may have translational implications in overcoming drug-resistance in deadly neoplasm. These studies are in progress in our laboratory. Citation Format: Mohammad Athar, Ritesh Srivastava. Novel molecular mechanism underlying cancer drug resistance: Involvement of p58IPKchaperone pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-056.

Rationalizing Drug Response in Cancer Cell Lines

Cancer cell lines (CCLs) play an important role in the initial stages of drug discovery allowing, among others, for the screening of drug candidates. As CCL panels continue to grow in size and diversity, many polymorphisms in genes encoding drug-metabolizing enzymes, transporters and drug targets, as well as disease-related genes have been linked to altered drug sensitivity. However, identifying the correlation between this variability and pharmacological responses remains challenging due to the heterogeneity of cancer biology and the intricate interplay between cell lines and drug molecules. Here, we propose a network-based strategy that exploits information on gene expression and somatic mutations of CCLs to group cells according to their molecular similarity. We then identify genes that are characteristic of each cluster and correlate their status with drug response. We find that CCLs with similar characteristic active network regions present specific responses to certain drugs, and identify a limited set of genes that might be directly involved in drug sensitivity or resistance.