Molecular Basis Of Drug Resistance Research Articles

Old drugs, novel ways out: Drug resistance toward cytotoxic chemotherapeutics.

Efficacy of chemotherapy in the treatment of distinct malignancies is often hampered by drug resistance arising in the tumor. Understanding the molecular basis of drug resistance and translating this knowledge into personalized treatment decisions can enhance therapeutic efficacy and even curative outcome. Over the years, multiple drug resistance mechanisms have been identified that enable tumors to cope with the damage instigated by a specific drug or group of anti-tumor agents. Here we provide an overview of the molecular pathways leading to resistance against conventional anti-cancer drugs, with emphasis on the utility of these pathways for rational selection of treatments for individual cancer patients. We further complement the review by discussing the pitfalls and difficulties in translating these findings into novel treatment strategies for cancer patients.

Mechanism of Adefovir, Tenofovir and Entecavir Resistance: Molecular Modeling Studies of How A Novel Anti-HBV Agent (FMCA) Can Overcome the Drug Resistance

Regardless of significant improvement in the area of anti-HBV therapy, resistance and cross-resistance against available therapeutic agents are the major consideration in drug discovery of new agents. The present study is to obtain the insight of the molecular basis of drug resistance conferred by the B and C domain mutations of HBV-polymerase on the binding affinity of four anti-HBV agents [Adefovir (ADV), Tenofovir (TNF), Entecavir (ETV) & 2'-Fluoro-6'-methylene-carbocyclic adenosine (FMCA)]. In this regard, homology modeled structure of HBV polymerase was used for minimization, conformational search and Glide XP docking followed by binding energy calculation on wild-type as well as on mutant HBV-polymerases (N236T, L180M+M204V+S202G & A194T). Our studies suggest a significant correlation between the fold resistances and the binding affinity of anti-HBV nucleosides. The domain B residue, L180 is indirectly associated with other active-site hydrophobic residues such as A87, F88 and M204, whereas the domain C residue, M204 is closely associated with sugar/pseudosugar ring positioning in the active site. These hydrophobic residues can directly influence the interaction of the incoming nucleoside triphosphates and change the binding efficacy. The carbohydrate ring part of natural substrate dATP, dGTP, FMCA and ETV, are occupied in similar passion in the grooves of HBV polymerase active site. The exocyclic double bond of Entecavir and FMCA occupies in the backside hydrophobic pocket (made by residues A87, F88, L180and M204), which enhances the overall binding affinity. Additional hydrogen bonding interaction of 2'-fluorine of FMCA with R41 residue of polymerase promotes a positive binding in wild-type as well as in ADVr, ETVr and TNFr with respect to that of entecavir.

Alterations in the mitochondrial responses to PENAO as a mechanism of resistance in ovarian cancer cells

ObjectiveThe purpose of this study was to test PENAO, a promising new organoarsenical that is in phase 1 testing in patients with solid tumours, on a range of ovarian cancer cell lines with different histotypes, and to understand the molecular basis of drug resistance exhibited by the endometrioid ovarian cancer cell line, SKOV-3. MethodsProliferation arrest and cell death induced by PENAO in serous (OVCAR-3), endometrioid (SKOV-3, TOV112D), clear cell (TOV21G) and mucinous (EFO27) ovarian cancer cells in culture, and anti-tumour efficacy in a murine model of SKOV-3 and OVCAR-3 tumours, were measured. Cells were analysed for cell cycle arrest, cell death mechanisms, reactive oxygen species production, mitochondrial depolarisation, oxygen consumption and acid production. ResultsPENAO demonstrated promising anti-proliferative activity on the most common (serous, endometrioid) as well as on rare (clear cell, mucinous) subtypes of ovarian cancer cell lines. No cross-resistance with platinum-based drugs was evident. Endometrioid SKOV-3 cells were, however, shown to be resistant to PENAO in vitro and in a xenograft mouse model. This resistance was due to an ability to cope with PENAO-induced oxidative stress, notably through heme oxygenase-1 induction, and a shift in metabolism towards glycolysis. The adaptive glycolytic shift in SKOV-3 was targeted using a mTORC1 inhibitor in combination with PENAO. This strategy was successful with the two drugs acting synergistically to inhibit cell proliferation and to induce cell death via apoptosis and autophagy. ConclusionMitochondria/mTOR dual-targeting therapy may constitute a new approach for the treatment of recurrent/resistant forms of epithelial ovarian cancer.

Involvement of Fanconi anemia genes FANCD2 and FANCF in the molecular basis of drug resistance in leukemia.

The Fanconi anemia (FA)‑associated proteins FANCF and FANCD2 are important components of the FA pathway of DNA crosslink repair. FANCF and FANCD2 have been found to be involved in drug‑resistant multiple myeloma, ovarian cancer, non‑small‑cell lung cancer, and head and neck cancer. However, it is unclear whether these two genes participate in adriamycin (ADR)‑resistant leukemia. Therefore, the aim of the current study was to investigate FANCF and FANCD2 expression in drug‑resistant and drug‑sensitive leukemia cells. Western blot analysis revealed enhanced FANCF expression and monoubiquitination of FANCD2 in ADR‑resistant cells. Additionally, it was observed that drug‑resistant cells had reduced DNA damage compared with drug‑sensitive cells. The results of this study indicate that the FA pathway may confer leukemia resistance to ADR via enhanced DNA interstrand crosslink repair.

Genomic diversity of drug-resistant Mycobacterium tuberculosis isolates in Lisbon Portugal: Towards tuberculosis genomic epidemiology

Multidrug- (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) present a challenge to disease control and elimination goals. Lisbon, Portugal, has a high TB incidence rate and unusual and successful XDR-TB strains that have been found in circulation for almost two decades. For the last 20years, a continued circulation of two phylogenetic clades, Lisboa3 and Q1, which are highly associated with MDR and XDR, have been observed. In recent years, these strains have been well characterized regarding the molecular basis of drug resistance and have been inclusively subjected to whole genome sequencing (WGS). Researchers have been studying the genomic diversity of strains circulating in Lisbon and its genomic determinants through cutting-edge next generation sequencing. An enormous amount of whole genome sequence data are now available for the most prevalent and clinically relevant strains circulating in Lisbon.It is the persistence, prevalence and rapid evolution towards drug resistance that has prompted researchers to investigate the properties of these strains at the genomic level and in the future at a global transcriptomic level. Seventy Mycobacterium tuberculosis (MTB) isolates, mostly recovered in Lisbon, were genotyped by 24-loci Mycobacterial Interspersed Repetitive Unit – Variable Number of Tandem Repeats (MIRU-VNTR) and the genomes sequenced using a next generation sequencing platform – Illumina HiSeq 2000.The genotyping data revealed three major clusters associated with MDR-TB (Lisboa3-A, Lisboa3-B and Q1), two of which are associated with XDR-TB (Lisboa3-B and Q1), whilst the genomic data contributed to elucidating the phylogenetic positioning of circulating MDR-TB strains, showing a high predominance of a single SNP cluster group 5. Furthermore, a genome-wide phylogeny analysis from these strains, together with 19 publicly available genomes of MTB clinical isolates, revealed two major clades responsible for MDR/XDR-TB in the region: Lisboa3 and Q1. The data presented by this study contributes to the expanding knowledge of MTB genomic diversity yielding insights on microevolution and identification of novel compensatory mutations. Additionally, the analysis of non-synonymous/synonymous ratios revealed heterogeneities across the chromosome, genotype and Clusters of Orthologous Groups, highlighting possible and different evolution strategies. Overall, the results that are presented support the notion of an increasing genomic diversity that may support both setting and host adaptation.

EpCAM Aptamer-mediated Survivin Silencing Sensitized Cancer Stem Cells to Doxorubicin in a Breast Cancer Model.

Understanding the molecular basis of drug resistance and utilising this information to overcome chemoresistance remains a key challenge in oncology. Here we report that survivin, a key protein implicated in drug resistance, is overexpressed in cancer stem cell pool of doxorubicin-resistant breast cancer cells. Moreover, by utilising an active targeting system consisting of an RNA aptamer targeted against the epithelial cell adhesion molecule and a Dicer substrate survivin siRNA, we could deliver a high dose of the siRNA to cancer stem cells in xenograft tumours. Importantly, silencing of survivin with this aptamer-siRNA chimera in cancer stem cell population led to the reversal of chemoresistance, such that combined treatment with low dose of doxorubicin inhibited stemness, eliminated cancer stem cells via apoptosis, suppressed tumour growth, and prolonged survival in mice bearing chemoresistant tumours. This strategy for in vivo cancer stem cell targeting has wide application for future effective silencing of anti-death genes and in fact any dysregulated genes involved in chemoresistance and tumour relapse.

Abstract 897: Elucidating the molecular basis of intrinsic taxane resistance in gastric cancer

Abstract Gastric cancer (GC) is histologically divided into intestinal (INT) and diffuse (DIF) clinical subtypes. Even though these two GC groups are structurally and biologically different, this classification is not used to inform choice of treatment. The taxanes (i.e., docetaxel and cabazitaxel) are widely used for the treatment of cancer and for GC specifically the TAX-325 study revealed a significantly improved survival when docetaxel (DTX) was added to cisplatin and fluorouracil. Despite this advancement, patients still exhibit intrinsic or acquired resistance to DTX. Yet, the molecular basis of drug resistance remains unknown. We performed a post-hoc analysis of the TAX-325 study to examine the potential influence of GC subtypes in clinical response to DTX. We classified randomized patients as diffuse or non-diffuse histology and correlated histology with clinical outcomes using a Cox proportional hazards model. Non-diffuse GC showed a significant improvement in overall survival with the addition of DTX (12.1 v 8.8 mo, p=0.002), whereas diffuse histology was not associated with an improvement in survival (8.3 v 8.5 mo, p=0.66). To investigate the molecular mechanism of GC DIF subtype resistance to taxanes, we used a panel of 12 GC cell lines representative of both subtypes (4 intestinal subtype, 8 diffuse subtype). DTX cytotoxicity assays revealed that similarly to what we observed clinically, 5/8 (63%) DIF GC cell lines were resistant (IC50 > 600 nM) to DTX compared to 1/4 (25%) INT GC cell lines. Further functional studies revealed that there was minimal DTX drug-target engagement in the DIF GC cells, as, assessed by confocal microscopy of the microtubule network and tubulin polymerization assays. These results suggested that DTX interaction with its target, microtubule, was impaired in the DIF GC cell lines. To rule out multi-drug resistance (MDR) as potential cause of intrinsic DTX resistance in these cells we performed flow cytometric evaluation of P-glycoprotein and found that all of the DIF GC cell lines were negative. Additionally, drug accumulation studies with C-14 radiolabeled DTX revealed that the drug accumulated intracellularly in all of GC cell lines in our panel. Next generation sequencing of our panel of untreated or DTX-treated GC cell lines revealed a number of genes/pathways that are differentially expressed in the drug-sensitive versus the resistant cell lines. Gene set enrichment analysis is currently ongoing in order to identify a subset of genes/pathways implicated in intrinsic DTX resistance in GC. These studies will provide novel insights into mechanism of intrinsic drug resistance and will ultimately allow us to design more effective targeted therapies to overcome chemo-resistance and eventually prolong patient survival. Citation Format: Giuseppe Galletti, Kyle Cleveland, Chao Zhang, Ada Gjyrezi, Alexandre Matov, Doron Betel, Manish A. Shah, Paraskevi Giannakakou. Elucidating the molecular basis of intrinsic taxane resistance in gastric cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 897. doi:10.1158/1538-7445.AM2014-897

A report of rifampin-resistant leprosy from northern and eastern India: identification and in silico analysis of molecular interactions

Presence of point mutations within the drug resistance determining regions of Mycobacterium leprae (M. leprae) genome confers molecular basis of drug resistance to dapsone, rifampin and ofloxacin in leprosy. This study is focused on the identification of mutations within the rpoB gene region of M. leprae that are specific for rifampin interaction, and further in silico analysis was carried out to determine the variations in the interactions. DNA and RNA were isolated from slit skin scrapings of 60 relapsed leprosy patients. PCR targeting rpoB gene region and amplicon sequencing was performed to determine point mutations. mRNA expression levels of rpoB and high-resolution melt analysis of mutants were performed using Rotor Gene Q Realtime PCR. Molecular docking was performed using LigandFit Software. Ten cases having point mutations within the rpoB gene region were identified and were clinically confirmed to be resistant to rifampin. A new mutation at codon position Gln442His has been identified. There is a 9.44-fold upregulation in the mRNA expression of rpoB gene in mutant/resistant samples when compared with the wild/sensitive samples. In silico docking analysis of rifampin with wild-type and Gln442His mutant RpoB proteins revealed a variation in the hydrogen-bonding pattern leading to a difference in the total interaction energy and conformational change at position Asp441. These preliminary downstream functional observations revealed that the presence of point mutations within the rifampin resistance determining regions of rpoB gene plays a vital role in conferring genetic and molecular basis of resistance to rifampin in leprosy.

An upstream truncation of the furA-katG operon confers high-level isoniazid resistance in a Mycobacterium tuberculosis clinical isolate with no known resistance-associated mutations.

Although the major causes of isoniazid (INH) resistance in Mycobacterium tuberculosis are confined to structural mutations in katG and promoter mutations in the mabA-inhA operon, a significant proportion of INH-resistant strains have unknown resistance mechanisms. Recently, we identified a high-level INH-resistant M. tuberculosis clinical isolate, GB005, with no known resistance-associated mutations. A comprehensive study was performed to investigate the molecular basis of drug resistance in this strain. Although no mutations were found throughout the katG and furA-katG intergenic region, the katG expression and the catalase activity were greatly diminished compared to those in H37Rv (P < 0.01). Northern blotting revealed that the katG transcript from the isolate was smaller than that of H37Rv. Sequencing analysis of furA and upstream genes discovered a 7.2-kb truncation extended from the 96th base preceding the initiation codon of katG. Complementation of the M. tuberculosis Δ(furA-katG) strain with katG and different portions of the truncated region identified a 134-bp upstream fragment of furA that was essential for full catalase activity and INH susceptibility in M. tuberculosis. The promoter activity of this fragment was also shown to be stronger than that of the furA-katG intergenic region (P < 0.01). Collectively, these findings demonstrate that deletion of the 134-bp furA upstream fragment is responsible for the reduction in katG expression, resulting in INH resistance in GB005. To our knowledge, this is the first report showing that deletion of the upstream region preceding the furA-katG operon causes high-level INH resistance in a clinical isolate of M. tuberculosis.

Inducible drug modification: A new form of resistance

Inducible drug modification: A new form of resistance

Drug Resistance Mechanisms in Mycobacterium tuberculosis

Tuberculosis (TB) is a serious public health problem worldwide. Its situation is worsened by the presence of multidrug resistant (MDR) strains of Mycobacterium tuberculosis, the causative agent of the disease. In recent years, even more serious forms of drug resistance have been reported. A better knowledge of the mechanisms of drug resistance of M. tuberculosis and the relevant molecular mechanisms involved will improve the available techniques for rapid drug resistance detection and will help to explore new targets for drug activity and development. This review article discusses the mechanisms of action of anti-tuberculosis drugs and the molecular basis of drug resistance in M. tuberculosis.

Pharmacogenetics research on chemotherapy resistance in colorectal cancer over the last 20 years.

During the past two decades the first sequencing of the human genome was performed showing its high degree of inter-individual differentiation, as a result of large international research projects (Human Genome Project, the 1000 Genomes Project International HapMap Project, and Programs for Genomic Applications NHLBI-PGA). This period was also a time of intensive development of molecular biology techniques and enormous knowledge growth in the biology of cancer. For clinical use in the treatment of patients with colorectal cancer (CRC), in addition to fluoropyrimidines, another two new cytostatic drugs were allowed: irinotecan and oxaliplatin. Intensive research into new treatment regimens and a new generation of drugs used in targeted therapy has also been conducted. The last 20 years was a time of numerous in vitro and in vivo studies on the molecular basis of drug resistance. One of the most important factors limiting the effectiveness of chemotherapy is the primary and secondary resistance of cancer cells. Understanding the genetic factors and mechanisms that contribute to the lack of or low sensitivity of tumour tissue to cytostatics is a key element in the currently developing trend of personalized medicine. Scientists hope to increase the percentage of positive treatment response in CRC patients due to practical applications of pharmacogenetics/pharmacogenomics. Over the past 20 years the clinical usability of different predictive markers has been tested among which only a few have been confirmed to have high application potential. This review is a synthetic presentation of drug resistance in the context of CRC patient chemotherapy. The multifactorial nature and volume of the issues involved do not allow the author to present a comprehensive study on this subject in one review.

Genetic and molecular basis of drug resistance and species-specific drug action in schistosome parasites.

Oxamniquine resistance evolved in the human blood fluke (Schistosoma mansoni) in Brazil in the 1970s. We crossed parental parasites differing ~500-fold in drug response, determined drug sensitivity and marker segregation in clonally derived second-generation progeny, and identified a single quantitative trait locus (logarithm of odds = 31) on chromosome 6. A sulfotransferase was identified as the causative gene by using RNA interference knockdown and biochemical complementation assays, and we subsequently demonstrated independent origins of loss-of-function mutations in field-derived and laboratory-selected resistant parasites. These results demonstrate the utility of linkage mapping in a human helminth parasite, while crystallographic analyses of protein-drug interactions illuminate the mode of drug action and provide a framework for rational design of oxamniquine derivatives that kill both S. mansoni and S. haematobium, the two species responsible for >99% of schistosomiasis cases worldwide.

Designing personalised cancer treatments

The concept of personalised medicine for cancer is not new. It arguably began with the attempts by Salmon and Hamburger to produce a viable cellular chemosensitivity assay in the 1970s, and continues to this day. While clonogenic assays soon fell out of favour due to their high failure rate, other cellular assays fared better and although they have not entered widespread clinical practice, they have proved to be very useful research tools. For instance, the ATP-based chemosensitivity assay was developed in the early 1990s and is highly standardised. It has proved useful for evaluating new drugs and combinations, and in recent years has been used to understand the molecular basis of drug resistance and sensitivity to anti-cancer drugs.Recent developments allow unparalleled genotyping and phenotyping of tumours, providing a plethora of targets for the development of new cancer treatments. However, validation of such targets and new agents to permit translation to the clinic remains difficult. There has been one major disappointment in that cell lines, though useful, do not often reflect the behaviour of their parent cancers with sufficient fidelity to be useful. Low passage cell lines — either in culture or xenografts are being used to overcome some of these issues, but have several problems of their own. Primary cell culture remains useful, but large tumours are likely to receive neo-adjuvant treatment before removal and that limits the tumour types that can be studied. The development of new treatments remains difficult and prediction of the clinical efficacy of new treatments from pre-clinical data is as hard as ever. One lesson has certainly been that one cannot buck the biology — and that understanding the genome alone is not sufficient to guarantee success. Nowhere has this been more evident than in the development of EGFR inhibitors. Despite overexpression of EGFR by many tumour types, only those with activating EGFR mutations and an inability to circumvent EGFR blockade have proved susceptible to treatment.The challenge is how to use advanced molecular understanding with limited cellular assay information to improve both drug development and the design of companion diagnostics to guide their use. This has the capacity to remove much of the guesswork from the process and should improve success rates.

Interview with Celia Schiffer

Celia Schiffer, a Professor in Biochemistry and Molecular Pharmacology; a former Director of UMass Center for AIDS Research; and a Founder and Co-Director for the Institute for Drug Resistance (University of Massachusetts Medical School, MA, USA). Schiffer has an undergraduate degree in physics from the University of Chicago, with a PhD in biophysics from University of California, San Francisco (CA, USA). She was a postdoctoral associate first at the ETH in Zurich and then at Genentech in San Francisco. Schiffer has published more than 100 peer reviewed journal articles. Her laboratory primarily uses structural biology, biophysical and chemistry techniques to study the molecular basis for drug resistance in anti-virals including: HIV, Hepatitis C, Influenza and Dengue. Her laboratory designs, synthesizes and tests new antiviral inhibitors that should be more robust to resistance. She has overseen more than US$30 million in research funding. She is also an outstanding mentor and educator, receiving a faculty mentoring award, a highly sought after PhD advisor. Interview conducted by Hannah Coaker, Assistant Commissioning Editor.

A novel serine/threonine protein phosphatase type 5 from second-generation merozoite of Eimeria tenella is associated with diclazuril-induced apoptosis

Screening the anticoccidial drug targets is very important for developing novel drugs and revealing the molecular basis of drug resistance in coccidia. Due to high effectivity and safety, diclazuril was used widely in the poultry industry. To assess the roles of the serine/threonine protein phosphatase type 5 of second-generation merozoites in Eimeria tenella (EtPP5) in the anticoccidial activity of diclazuril against chicken coccidiosis, EtPP5 was cloned using reverse transcriptase polymerase chain reaction and rapid amplification of cDNA ends. Ultrastructural changes in second-generation merozoites and mRNA expression level of EtPP5 were monitored by transmission electron microscopy (TEM) and quantitative real-time PCR, respectively. The results showed that the full length of the cloned EtPP5 cDNA (2,495 bp) encompassed a 1,647-bp open reading frame encoding a polypeptide of 548 residues with an estimated molecular mass of 60.82 kDa and a theoretical isoelectric point of 5.89. Molecular analysis of EtPP5 reveals the presence of a C-terminal phosphatase domain and an extended N-terminal tetratricopeptide repeat motif, a typical feature of protein phosphatases. The cDNA sequence has been submitted to the GenBank database with accession number JX987508. EtPP5 shared 89% homology with the published sequence of a PP5 ortholog of Toxoplasma gondii at the amino acid level (GenBank XP_002364442.1). TEM observed that diclazuril induced ultrastructural changes in second-generation merozoites. Quantitative real-time PCR analysis showed that compared with the control group, the level of EtPP5 mRNA expression was significantly downregulated by 51.4% by diclazuril treatment. The high similarity of EtPP5 to previously described PP5 of other organisms, as well as its downregulated expression and connection with apoptosis in the second-generation merozoites induced by diclazuril, suggests that it could act an important role in understanding the signaling mechanism underlining the diclazuril-induced merozoites apoptosis.

Aromatic interaction profile to understand the molecular basis of raltegravir resistance

Integrase (IN) is the enzyme of human immunodeficiency virus (HIV) which inserts the viral DNA (vDNA) into the host genome for successful viral replication leading to the infection. However, the chemical basis of HIV IN catalysis is speculative due to lack of complete co-crystal structure. Using the recently published prototype foamy virus IN crystal structure, we developed a model structure of HIV IN showing interaction of vDNA, the metal (Mg2+) cofactor, and raltegravir (RLT) in the active site. Molecular docking and dynamics simulations studies showed that RLT uses it core central ring with diketo motif for Mg2+ chelation and bridge interaction with DDE motif. The triple arene interactions mediated by RLT with neighboring molecular motifs (Y143, cytosine, and adenine) is maintained during long simulation in wild type (WT). The fluorobenzyl and oxadiazole moieties of RLT forms aromatic stacking with cytosine base (head stacking) aromatic side chain of Y143 (tail stacking), respectively, while central ring further establishes aromatic stacking with distorted adenine base of vDNA (central stacking). The novel triple stacking systems were further explored to understand the molecular basis of drug resistance by molecular simulation. The in silico mutation (N155H, Q148H, and Q148H + G140S) and simulation studies elucidated the structural mechanism of resistance to RLT. The simulation studies provided the molecular basis for interdependency observed for the primary and secondary (Q148H and G140S) mutations and also explained the mechanism of viral fitness regain. Our study reveals that triple stacking and its consequence in terms of VdW energetic profile acts as a critical point to understand the drug-resistance. Here, we demonstrate that the root mean square deviation of centroid system (aromatic stacking) can be used as a major determinant of RLT binding toward the fold resistance. This is first kind of report, which discloses a strategy to explore the molecular level of drug resistance profile using aromatic interactions.

Impact of Hypoxia As Novel Microenvironmental Factor On Drug Susceptibility of Primary CLL Cells

AbstractAbstract 3918 Background:CLL cells circulating in the peripheral blood are sensitive to therapy while malignant cells residing in the microenvironment survive and are the source of relapse. One of the strongest microenvironmental stimuli is CD40 ligand (CD40L)-CD40 interaction, which induces proliferative/anti-apoptotic genes in CLL cells, protecting them from apoptosis and many cytotoxic drugs. Despite the evident importance of CD40 activation further stimuli have to be considered, especially hypoxia. Lymph nodes, particularly those being infiltrated by malignant cells, show a low oxygen tension (<1%). Prior CLL investigations never took this important factor into account, hence the impact of hypoxia on cell survival and drug-resistance is still unrevealed. Methods:We have established an in vitro model, which mimics hypoxic conditions and CD40L-CD40 interaction, in order to understand the molecular basis of drug resistance of CLL cells resident in the microenvironment. CLL cells were cultured on CD40L feeder cells and kept up to 96 hours in hypoxia (1% O2) or normoxia (21% O2). We determined how proliferation rates in CLL are affected by these conditions and subsequently applied several drugs to investigate differences in drug efficacy between normoxia and hypoxia. Apoptosis was determined by AnnexinV/7AAD-staining and subsequent flow cytometry. Expression of potential target molecules was determined by qRT-PCR and Western Blotting. Results:Hypoxia is known to protect malignant cells in solid cancers from chemotherapy. We made similar observations, since classical DNA-targeting drugs were inefficient to kill CLL cells under hypoxic conditions. However, we identified ABT-737, which affects mitochondrial integrity, to be even more efficient under hypoxic conditions compared to normoxia. In order to explain this discrepancy we investigated the expression of several mitochondrial localized anti-/proapoptotic genes on RNA and protein level. We show that the de-regulation of BclXL and Mcl-1 under hypoxic conditions is essential for ABT-737 sensitivity. BclXL deregulation depends on a general reduction in protein translation in hypoxic cells. Mcl-1 protein expression differs from its mRNA expression, hence we expected regulation subsequent to protein synthesis. Indeed we could identify an increased activity of the proteasome in hypoxia, as Mcl-1 is a short-lived protein with a rapid proteasomal turnover this is a feasible explanation for the observed downregulation. Interestingly, hypoxia has a great impact on proliferation of primary CLL cells under different stimuli in vitro. Conclusion:These are the first experiments investigating the impact of oxygen tension on survival and response to chemotherapy of CLL cells. We show that hypoxia renders CLL cells resistant to classical DNA-targeting agent Fludarabine and Bendamustine. Furthermore we point out that small molecules like ABT-737, which specifically target mitochondria, might be efficient in targeting CLL cells protected by hypoxia and CD40L-CD40 interaction within the microenvironment. Development of novel in vitro models like ours will help us understand the specific molecular changes induced by microenvironmental stimuli and their impact on drug efficacy. These findings will allow us to identify novel therapeutic targets.M.Hu. and L.P.F. contributed equally to this work Disclosures:No relevant conflicts of interest to declare.

MicroScale: miniaturizing cell arrays for macrogenetic screens

MicroScale: miniaturizing cell arrays for macrogenetic screens

Understanding the basis of drug resistance of the mutants of αβ-tubulin dimer via molecular dynamics simulations.

The vital role of tubulin dimer in cell division makes it an attractive drug target. Drugs that target tubulin showed significant clinical success in treating various cancers. However, the efficacy of these drugs is attenuated by the emergence of tubulin mutants that are unsusceptible to several classes of tubulin binding drugs. The molecular basis of drug resistance of the tubulin mutants is yet to be unraveled. Here, we employ molecular dynamics simulations, protein-ligand docking, and MMPB(GB)SA analyses to examine the binding of anticancer drugs, taxol and epothilone to the reported point mutants of tubulin - T274I, R282Q, and Q292E. Results suggest that the mutations significantly alter the tubulin structure and dynamics, thereby weaken the interactions and binding of the drugs, primarily by modifying the M loop conformation and enlarging the pocket volume. Interestingly, these mutations also affect the tubulin distal sites that are associated with microtubule building processes.