Phenotypic Drug Tolerance Research Articles

Parallel reaction monitoring of clinical Mycobacterium tuberculosis lineages reveals pre-existent markers of rifampicin tolerance in the emerging Beijing lineage

The spread of multidrug resistant Mycobacterium tuberculosis is one of the major challenges in tuberculosis control. In Eurasia, the spread of multidrug resistant tuberculosis is driven by the M. tuberculosis Beijing genotype. In this study, we examined whether selective advantages are present in the proteome of Beijing isolates that contribute to the emergence of this genotype. To this end, we compared the proteome of M. tuberculosis Beijing to that of M. tuberculosis H37Rv, both in the presence and absence of the first-line antibiotic rifampicin. During rifampicin exposure, both M. tuberculosis genotypes express proteins belonging to the DosR dormancy regulon, which induces a metabolically hypoactive-, drug tolerant phenotype. However, these markers of rifampicin tolerance were already more abundant in the M. tuberculosis Beijing isolate prior to drug exposure. To determine whether the a priori high abundance of specific proteins contribute to the formation of antibiotic resistance in M. tuberculosis Beijing, we quantified the abundance of 33 selected proteins in 27 clinical isolates from the five most common M. tuberculosis lineages using parallel reaction monitoring. The observed pre-existing high abundance of dormancy proteins in Beijing strains provides an evolutionary advantage that allows these strains to persist for prolonged periods during rifampicin treatment. SignificanceM. tuberculosis is the leading cause of death by a bacterial infection worldwide. Treatment-regimen to eradicate this pathogen make use of the first-line antibiotic rifampicin, which is considered to be the cornerstone of modern day anti-tuberculosis treatment. Despite the potency of rifampicin, there is an increasing occurrence of rifampicin resistant mutants in a specific cluster of M. tuberculosis, the Beijing genotype. Using both a data dependent acquisition and a targeted proteomic approach we identified markers of rifampicin tolerance to be high abundant in members of the M. tuberculosis Beijing genotype, already prior drug exposure. The identification of this M. tuberculosis Beijing specific trait will contribute to improved diagnostics and treatment of M. tuberculosis.

House of cellulose - a new hideout for drug tolerant Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) causes tuberculosis (TB). The treatment of TB requires administration of multiple drugs for long durations because of the unusual drug tolerance of Mtb. The phenotypic drug tolerance of genetically drug-susceptible Mtb in humans can be explained by its ability to form biofilms. Recent studies from different laboratories suggest that Mtb forms biofilms that harbour drug-tolerant bacteria. These findings have created a new area of research in the field of mycobacterial physiology. Recently, my laboratory has reported that Mtb cells organise themselves into biofilms in response to intracellular thiol reductive stress (Trivedi et al. Nature communications. 2016). Bacteria residing in these biofilms are tolerant towards antimycobacterial drugs. Cellulose is a key component of the extracellular polymeric substances that hold mycobacterial cells together in these biofilms. Here, I discuss the implications of these findings and new hypotheses arising from this study on the biology of Mtb biofilms.

Interpreting phenotypic antibiotic tolerance and persister cells as evolution via epigenetic inheritance.

Epigenetic inheritance is the transmission of nongenetic material such as gene expression levels, RNA and other biomolecules from parents to offspring. There is a growing realization that such forms of inheritance can play an important role in evolution. Bacteria represent a prime example of epigenetic inheritance because a large array of cellular components is transmitted to offspring, in addition to genetic material. Interestingly, there is an extensive and growing empirical literature showing that many bacteria can form 'persister' cells that are phenotypically resistant or tolerant to antibiotics, but most of these results are not interpreted within the context of epigenetic inheritance. Instead, persister cells are usually viewed as a genetically encoded bet-hedging strategy that has evolved in response to a fluctuating environment. Here I show, using a relatively simple model, that many of these empirical findings can be more simply understood as arising from a combination of epigenetic inheritance and cellular noise. I therefore suggest that phenotypic drug tolerance in bacteria might represent one of the best-studied examples of evolution under epigenetic inheritance.

Elucidating population-wide mycobacterial replication dynamics at the single-cell level.

Mycobacterium tuberculosis infections result in a spectrum of clinical outcomes, and frequently the infection persists in a latent, clinically asymptomatic state. The within-host bacterial population is likely to be heterogeneous, and it is thought that persistent mycobacteria arise from a small population of viable, but non-replicating (VBNR) cells. These are likely to be antibiotic tolerant and necessitate prolonged treatment. Little is known about these persistent mycobacteria, since they are very difficult to isolate. To address this, we have successfully developed a replication reporter system for use in M. tuberculosis. This approach, termed fluorescence dilution, exploits two fluorescent reporters; a constitutive reporter allows the tracking of bacteria, while an inducible reporter enables the measurement of bacterial replication. The application of fluorescence single-cell analysis to characterize intracellular M. tuberculosis identified a distinct subpopulation of non-growing mycobacteria in murine macrophages. The presence of VBNR and actively replicating mycobacteria was observed within the same macrophage after 48 h of infection. Furthermore, our results suggest that macrophage uptake resulted in enrichment of non- or slowly replicating bacteria (as revealed by d-cycloserine treatment); this population is likely to be highly enriched for persisters, based on its drug-tolerant phenotype. These results demonstrate the successful application of the novel dual fluorescence reporter system both in vitro and in macrophage infection models to provide a window into mycobacterial population heterogeneity.

Mycobacterium tuberculosis Is Resistant to Isoniazid at a Slow Growth Rate by Single Nucleotide Polymorphisms in katG Codon Ser315.

An important aim for improving TB treatment is to shorten the period of antibiotic therapy without increasing relapse rates or encouraging the development of antibiotic-resistant strains. In any M. tuberculosis population there is a proportion of bacteria that are drug-tolerant; this might be because of pre-existing populations of slow growing/non replicating bacteria that are protected from antibiotic action due to the expression of a phenotype that limits drug activity. We addressed this question by observing populations of either slow growing (constant 69.3h mean generation time) or fast growing bacilli (constant 23.1h mean generation time) in their response to the effects of isoniazid exposure, using controlled and defined growth in chemostats. Phenotypic differences were detected between the populations at the two growth rates including expression of efflux mechanisms and the involvement of antisense RNA/small RNA in the regulation of a drug-tolerant phenotype, which has not been explored previously for M. tuberculosis. Genotypic analyses showed that slow growing bacilli develop resistance to isoniazid through mutations specifically in katG codon Ser315 which are present in approximately 50–90% of all isoniazid-resistant clinical isolates. The fast growing bacilli persisted as a mixed population with katG mutations distributed throughout the gene. Mutations in katG codon Ser315 appear to have a fitness cost in vitro and particularly in fast growing cultures. Our results suggest a requirement for functional katG-encoded catalase-peroxide in the slow growers but not the fast-growing bacteria, which may explain why katG codon Ser315 mutations are favoured in the slow growing cultures.

Confinement-Induced Drug-Tolerance in Mycobacteria Mediated by an Efflux Mechanism.

Tuberculosis (TB) is the world’s deadliest curable disease, responsible for an estimated 1.5 million deaths annually. A considerable challenge in controlling this disease is the prolonged multidrug chemotherapy (6 to 9 months) required to overcome drug-tolerant mycobacteria that persist in human tissues, although the same drugs can sterilize genetically identical mycobacteria growing in axenic culture within days. An essential component of TB infection involves intracellular Mycobacterium tuberculosis bacteria that multiply within macrophages and are significantly more tolerant to antibiotics compared to extracellular mycobacteria. To investigate this aspect of human TB, we created a physical cell culture system that mimics confinement of replicating mycobacteria, such as in a macrophage during infection. Using this system, we uncovered an epigenetic drug-tolerance phenotype that appears when mycobacteria are cultured in space-confined bioreactors and disappears in larger volume growth contexts. Efflux mechanisms that are induced in space-confined growth environments contribute to this drug-tolerance phenotype. Therefore, macrophage-induced drug tolerance by mycobacteria may be an effect of confined growth among other macrophage-specific mechanisms.

Qualitative and quantitative proteomic analysis of Vitamin C induced changes in Mycobacterium smegmatis.

Vitamin C is a critical dietary nutrient in human which has a wide range of regulatory effects on gene expression and physiology of Mycobacterium tuberculosis that leads to a dormant drug-tolerant phenotype. In the presence of iron, vitamin C shows a high bactericidal activity even in the drug resistant phenotype of M. tuberculosis. The regulatory mechanisms underlying vitamin C induced adaptations are largely unknown due to lack of functional genomics data in this field. In this study, we attempt to characterize the direct effect of vitamin C treatment on the physiology of actively growing Mycobacterium smegmatis. The study chose M. smegmatis as it is a fast-growing bacterium and a non-pathogenic model system which shares many physiological features with the pathogenic M. tuberculosis including dormancy and its regulation. The proteomic adaptation of M. smegmatis on vitamin C treatment demonstrates the important changes in cellular and metabolic process such as reversal of tricarboxylic acid cycle, decrease in ATP synthesis, decrease in iron acquisition and storage, and induction of dormancy regulators WhiB3, PhoP, and Lsr2.

Abstract A09: Single-cell RNA sequencing reveals phenotypic plasticity of drug tolerant, clonal populations of cancer cells

Abstract Cancer recurrence and metastatic tumors might arise not only from residual primary tumor cells but even from precancerous cells that are able to spread and seed distant organs. Thus, residual pre-neoplastic and neoplastic cells that persist following treatment constitute an ideal evolutionary niche for cancer evolution. We have investigated the role of the TGF-beta; signaling pathway, which is widely known as a pro-metastatic in late stages of tumor progression, in the context of the cellular response to genotoxic stress induced by chemotherapy drugs. We identified that TGF-beta signaling contribute to drug resistance by repressing the DNA damage response through a coordinate repression of transcription and translation of p53 and the stress response in precancerous and cancer cells. Indeed, TGF-beta/Smad signaling enacts an intrinsic survival pathway in metastatic cancer cells. However, the molecular events accompanying the evolution towards a drug tolerant phenotype are not well understood. Indeed, severe cellular stress might generate or select a subpopulation of reversibly stress-tolerant cells under conditions that are lethal to the majority of the population. Thus, drug tolerance could be attributed to heterogeneity of gene expression within the population to ensure survival of a minority under stress. To address these questions, we developed an experimental paradigm to generate and propagate populations of paclitaxel tolerant precancerous (MCF10A) and metastatic cancer (MDA-MB-231) cells that can resume proliferation after 1 week of consecutive treatment with lethal doses of the drug. We found that drug-tolerant cells reacquired Paclitaxel-sensitivity after expansion. Then we performed whole transcriptome sequencing to identify single nucleotide variants (SNVs) and gene expression profiles at the single-cell level associated with the evolution towards a reversible drug tolerant state in cancer cells. We evaluated untreated, stress-arrested and proliferating drug-tolerant cells from a small (n<64) clonal population. In addition to sequencing the mRNA of single cells, we also performed DNA and RNA sequencing of populations of untreated and drug-tolerant cells to aid in the identification of RNA variants. We performed differential gene expression profiling for single cells and population cells of the three groups to identify the transcriptional stress response and cytotoxic effects of Paclitaxel on gene expression. We find that within untreated, stress-arrested and drug-tolerant cell groups, specific transcriptional programs were enacted while generating high heterogeneity between single cells within and between groups. Drug-tolerant cells contained specific RNA variants encoded by genes involved in microtubule organization and stabilization as well as cell adhesion and cell surface signaling. Unexpectedly, Drug-tolerant cells from a single progenitor cell rapidly reacquired high degree of heterogeneity in gene expression similar to untreated cells, within a few doublings, while uniquely expressing SNVs and genes such as Integrin alpha 6, the histone demethylase KDM5A, and the IGF-1 receptor. Interestingly, despite of the high degree of heterogeneity, the whole gene expression profiles, of each cell rather than small gene sets can identify the response of cells to taxol. Thus, single cell analyses reveal the dynamics of the response to stress in terms of cell-specific SNVs driving heterogeneity, the survival of a minority population through generation of specific genes and RNA variants and the efficient reconversion of drug-tolerant cells back to normalcy, suggesting that such heterogeneity within cancer cell populations might provide evolutionary advantage to increase the pool of drug tolerant cells poising a prospective path towards drug resistance. Citation Format: Fernando Lopez-Diaz, Mei-Chong Wendy Lee, Muhammad Tariq, Shahid Khan, Yelena Dayn, Charlie Vaske, Nader Pourmand, Beverly M. Emerson. Single-cell RNA sequencing reveals phenotypic plasticity of drug tolerant, clonal populations of cancer cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A09.

Abstract 3767: Transcriptional and proteomic profiling of drug-tolerant cancer cell subpopulations using colonies that emerge in the presence of anticancer agents

Abstract Background: Cancer relapse after curative treatment is thought to originate from drug-tolerant cancer cell subpopulations. To isolate and characterize the drug-tolerant cell subpopulations, we collected individual colonies that emerged in the presence of anticancer drugs and performed proteomic profiling using reverse-phase protein arrays (RPPAs). Materials and Methods: Cancer cells (HCT116, HeLa, HT29, MCF7, and MKN45) were sparsely disseminated (10-100 cells/cm2) into the presence of anticancer drugs (cisplatin, docetaxel, gefitinib, and sorafenib) of six serially diluted concentrations on the basis of 50% colony inhibitory concentration (CI50) for each drug. A total of 2,400 individual colony lysates (20 colony lysates per drug concentration) were printed on glass slides using a dedicated microarrayer followed by immunochemical detection of 44 functionally-diverse proteins. A transcriptional profiling followed by gene ontology (GO) analysis was also performed. Results: CI50 of cisplatin and docetaxel were >10 times lower than the 50% growth inhibitory concentration (GI50) in all cell lines. This indicates that colony formation is functionally different from exponential cell growth, and drug-tolerant colony initiation can be suppressed by traditional anticancer drugs with very low doses. Colony lysate arrays revealed that the colonies expressing stemness, pluripotent, and mesenchymal markers tended to show low levels of epithelial proteins. However, individual protein expression levels were not well associated with drug concentrations, suggesting that these protein phenotypes of colonies were not necessarily induced by anticancer drugs. Instead, they may have emerged spontaneously. To identify molecular fractions important to drug-tolerant phenotypes, we screened compounds inhibiting chromatin formation, transcription, and protein biosynthesis. A transcriptional inhibitor eliminated drug-tolerant colonies within a short (four-hour) temporal treatment, suggesting that putative drug-tolerance inducing factors were controlled at the transcriptional level. Analysis of cDNA microarray using drug-free and cisplatin-resistant colonies followed by GO analysis revealed that most expressed genes in cisplatin-resistant colonies were specifically associated with embryonic development. Conclusion: These findings suggest that the regulatory system at the protein level is unlikely to be directly associated with drug-tolerant colony formation. Instead, transcriptional regulation appears to play a crucial role in forming drug-tolerant colonies. Citation Format: Kohei Kume, Satoshi Nishizuka, Miyuki Ikeda, Sawako Miura, Fumitaka Endo, Katagiri Hirokatsu, Kaoru Ishida, Kei Sato, Chihaya Maesawa, Go Wakabayashi. Transcriptional and proteomic profiling of drug-tolerant cancer cell subpopulations using colonies that emerge in the presence of anticancer agents. [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 3767. doi:10.1158/1538-7445.AM2014-3767

Abstract 1981: Drug-tolerant gastric cancer cell subpopulation enriched by 5-fluorouracil acquires malignant phenotype

Abstract Background: Post-operative adjuvant chemotherapy for gastric cancer is a treatment for suppressing the growth of invisible cancer cells, but substantial numbers of patients experience recurrence despite of this therapy. We attempted to clarify the mechanism of cancer relapse after chemotherapy using 5-FU tolerant human gastric cancer cell line, MKN45. Methods: Through a stepwise dose escalation of 5-fluorouracil (5-FU) for one year, a 5-FU-tolerant cell subpopulation, MKN45T, was established from a human gastric cancer cell line, MKN45. Nucleotide variation was screened with a panel of 46 cancer-associated genes, and western blot analyses were performed to examine whether known proteins were involved in the acquisition of the drug-tolerant phenotype. For molecular profiling, cancer cell subpopulations emerging as colonies in the presence of anticancer drugs including 5-FU, cisplatin, and docetaxel, “reverse-phase” protein arrays (RPPAs) were produced with the drug-tolerant 480 colonies. Subcutaneous or orthotopic xenografts of MKN45 and MKN45T to immunodeficient mice were performed to examine tumorigenicity. Results: Western blot analysis revealed that most of protein expression levels were not visibly different between MKN45 and MKN45T, except that p53 showed slight reduction in MKN45T. Analysis of 46 cancer-associated genes revealed that all gene variations were identical in both MKN45 and MKN45T, suggesting that gene mutations had a limited effect on the acquisition of the drug-tolerant phenotype. RPPA analysis revealed that colonies from MKN45T exhibited a protein level increase in a 5-FU concentration-dependent manner in Atg5, Atg7, and PI3K, suggesting a functional association between drug-tolerance and autophagy/glucose metabolism. An orthotopic xenograft to the stomach demonstrated tumors in the stomach (9 out of 9, 100%), and lymph node and liver metastases only in MKN45T at a high frequency (6 out of 9, 67%). No difference in tumorigenicity was observed when MKN45 and MKN45T were transplanted subcutaneously. The orthotopic xenograft of MKN45T followed by administration of 5-FU (30mg/kg/day) for five post-xenograft days suppressed metastasis in 4 out of 5 mice (80%) at 42 post-xenograft days, indicating that chemotherapy is particularly effective if it is performed before initiating tumor formation. Conclusion: Chemotherapy does not only reduce tumor size, but may also be selecting for tumors that are resistant to the chemotherapy. Our clinical experiences are well-explained by this observation, as relapse after chemotherapy is often chemotherapy resistant. Citation Format: Kaoru Ishida, Satoshi S. Nishizuka, Kohei Kume, Mamoru Nukatsuka, Kei Sato, Fumitaka Endo, Hirokatsu Katagiri, Takashi Kobunai, Teiji Takechi, Keisuke Koeda, Go Wakabayashi. Drug-tolerant gastric cancer cell subpopulation enriched by 5-fluorouracil acquires malignant phenotype. [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 1981. doi:10.1158/1538-7445.AM2014-1981

The Spectrum of Drug Susceptibility in Mycobacteria.

A major factor complicating efforts to control the tuberculosis epidemic is the long duration of treatment required to successfully clear the infection. One reason that long courses of treatment are required may be the fact that mycobacterial cells arise during the course of infection that are less susceptible to antibiotics. Here we describe the paradigms of phenotypic drug tolerance and resistance as they apply to mycobacteria. We then discuss the mechanisms by which phenotypically drug-tolerant and -resistant cells arise both at a population level and in specialized subpopulations of cells that may be especially important in allowing the bacterium to survive in the face of treatment. These include general mechanisms that have been shown to alter the susceptibility of mycobacteria to antibiotics including growth arrest, efflux pump induction, and biofilm formation. In addition, we discuss emerging data from single-cell studies of mycobacteria that have identified unique ways in which specialized subpopulations of cells arise that vary in their frequency, in their susceptibility to drug, and in their stability over time.

Global adaptation to a lipid environment triggers the dormancy-related phenotype of Mycobacterium tuberculosis.

ABSTRACTStrong evidence supports the idea that fatty acids rather than carbohydrates are the main energy source of Mycobacterium tuberculosis during infection and latency. Despite that important role, a complete scenario of the bacterium’s metabolism when lipids are the main energy source is still lacking. Here we report the development of an in vitro model to analyze adaptation of M. tuberculosis during assimilation of long-chain fatty acids as sole carbon sources. The global lipid transcriptome revealed a shift toward the glyoxylate cycle, the overexpression of main regulators whiB3, dosR, and Rv0081, and the increased expression of several genes related to reductive stress. Our evidence showed that lipid storage seems to be the selected mechanism used by M. tuberculosis to ameliorate the assumed damage of reductive stress and that concomitantly the bacilli acquired a slowed-growth and drug-tolerant phenotype, all characteristics previously associated with the dormant stage. Additionally, intergenic regions were also detected, including the unexpected upregulation of tRNAs that suggest a new role for these molecules in the acquisition of a drug-tolerant phenotype by dormant bacilli. Finally, a set of lipid signature genes for the adaptation process was also identified. This in vitro model represents a suitable condition to illustrate the participation of reductive stress in drugs’ activity against dormant bacilli, an aspect scarcely investigated to date. This approach provides a new perspective to the understanding of latent infection and suggests the participation of previously undetected molecules.

Reversal ofMycobacterium tuberculosisphenotypic drug resistance by 2-aminoimidazole-based small molecules

The expression of phenotypic drug resistance or drug tolerance serves as a strategy for Mycobacterium tuberculosis to survive in vivo antimicrobial drug treatment; however, the mechanisms are poorly understood. Progress toward a more in depth understanding of in vivo drug tolerance and the discovery of new therapeutic strategies designed specifically to treat drug-tolerant M. tuberculosis are hampered by the lack of appropriate in vitro assays. A library of 2-aminoimidazole-based small molecules combined with the antituberculosis drug isoniazid was screened against M. tuberculosis expressing in vitro drug tolerance as microbial communities attached to an extracellular matrix derived from lysed leukocytes. Based on the ability of nine of ten 2-aminoimidazole compounds to inhibit Mycobacterium smegmatis biofilm formation and three of ten molecules capable of dispersing established biofilms, two active candidates and one inactive control were tested against drug-tolerant M. tuberculosis. The two active compounds restored isoniazid susceptibility as well as reduced the in vitro minimum inhibitory concentrations of isoniazid in a dose-dependent manner. The dispersion of drug-tolerant M. tuberculosis with 2-aminoimidazole-based small molecules as an adjunct to antimicrobial treatment has the potential to be an effective antituberculosis treatment strategy designed specifically to eradicate drug-tolerant M. tuberculosis.

Alternative explanation for indole-induced antibiotic tolerance in Salmonella

We were interested to read the recent publication by Vega et al. (1), which suggests that indole is an interspecies signal that causes Salmonella to become less susceptible to antibiotics due to activation of the oxidative stress response. However, we believe that there is an alternative explanation for their data: the drug tolerance phenotype is due to increased production of one or more multidrug efflux pumps. The drugs tested by Vega et al., ciprofloxacin and carbenicillin, are known substrates of the AcrB transporter, and indole has been shown to induce the production of efflux pumps in both Escherichia coli and Salmonella (2). In Salmonella, induction is mediated by increased expression of the transcriptional activator RamA, which regulates expression of acrAB (2). Therefore, the indole-induced drug tolerance seen by Vega et al. could result from induction of the multidrug resistance (MDR) AcrAB-TolC efflux system, leading to increased tolerance to these two drugs. This hypothesis is noted by the authors but dismissed after their RT-PCR experiments showed that expression of the ramA gene was reduced in the presence of indole. We are perplexed by this observation because it conflicts with all previous studies including measurements using gene reporter constructs, RT-PCR, microarray, and Western blotting (2), which reveal that ramA/RamA is induced by indole in numerous different Salmonella strains including strain LT2 used by Vega et al. in their experiments. Furthermore, the authors used these RT-PCR data as evidence that efflux is not involved in the phenotype. We believe that this is a mistaken assumption for two reasons. First, efflux was not measured. This is surprising because efflux of ciprofloxacin and other compounds can be measured easily using one of several published methods to quantify accumulation or efflux of fluorescent substrates (3, 4). Second, regulation of MDR efflux pumps in Gram-negative bacteria is complex and multifactorial. For instance, other transcription factors, including MarA, SoxS, and Rob, can also regulate expression of MDR efflux pumps, and indole induces expression of soxS in E. coli (5).

Abstract PR05: Single-cell RNA sequencing reveals phenotypic plasticity of drug tolerant clonal populations of cancer cells

Abstract The notion that most cancers are ecosystems of evolving clones is now widely accepted. Hence, residual neoplastic cells that persist following treatment constitute an ideal evolutionary niche from which recurrent cancers arise and are a major obstacle to the successful treatment of human cancers. We have identified that TGFβ signaling contribute to drug resistance by coordinately repressing transcription and translation of p53 in precancerous and cancer cells. However, the molecular events accompanying the evolution towards a drug tolerant phenotype are not well understood. Indeed, severe cellular stress might generate a subpopulation of reversibly stress-tolerant cells under conditions that are lethal to the majority of the population. Drug tolerance attributed to heterogeneity of gene expression within the population to ensure survival of a minority under stress. To address these questions, we developed an experimental paradigm to generate and propagate populations of paclitaxel tolerant precancerous (MCF10A) and metastatic cancer (MDA-MB-231) cells that can resume proliferation after 1 week of consecutive treatment with lethal doses of the drug . We found that drug-tolerant cells reacquired Paclitaxel-sensitivity after expansion. Then we performed whole transcriptome sequencing to identify single nucleotide variants (SNVs) and gene expression profiles at the single-cell level associated with the evolution towards a reversible drug tolerant state in cancer cells. We evaluated untreated, stress-arrested and proliferating drug-tolerant cells from a small (n<64) clonal population. In addition to sequencing the mRNA of single cells, we also performed DNA and RNA sequencing of populations of untreated and drug-tolerant cells to aid in the identification of RNA variants. We performed differential gene expression profiling for single cells and population cells of the three groups to identify the transcriptional stress response and cytotoxic effects of Paclitaxel on gene expression. We find that within untreated, stress-arrested and drug-tolerant cell groups, specific transcriptional programs were enacted while generating high heterogeneity between single cells within and between groups. Drug-tolerant cells contained specific RNA variants encoded by genes involved in microtubule organization and stabilization as well as cell adhesion and cell surface signaling. Unexpectedly, Drug-tolerant cells from a single progenitor cell rapidly reacquired high degree of heterogeneity in gene expression similar to untreated cells, within a few doublings, while uniquely expressing SNVs and genes such as Integrin alpha 6, the histone demethylase KDM5A, and the IGF-1 receptor. Thus, single cell analyses reveal the dynamics of the response to stress in terms of cell-specific SNVs driving heterogeneity, the survival of a minority population through generation of specific genes and RNA variants and the efficient reconversion of drug-tolerant cells back to normalcy, suggesting that such heterogeneity within cancer cell populations might provide evolutionary advantage to increase the pool of drug tolerant cells poising a prospective path towards drug resistance. This abstract is also presented as poster B68. Citation Format: Fernando J. Lopez-Diaz, Mei-Chong Wendy Lee, Muhammad A. Tariq, Yelena Dayn, Shahid Y. Khan, Charlie Vaske, Nader Pourmand, Beverly M. Emerson. Single-cell RNA sequencing reveals phenotypic plasticity of drug tolerant clonal populations of cancer cells. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr PR05.

Abstract 4060: Analysis of cancer cell populations by isolated single colonies for the identification of drug-tolerance inducing factors.

Abstract [Background] Functional heterogeneity within cancer cell populations plays an important role in responses to genotoxic stresses caused by anticancer agents. We have characterized 2,400 colonies derived in the presence of anticancer drugs using human cancer cell lines by newly introduced colony lysate array technique (CoLA). In the present study, we attempted to identify molecular components that could induce anticancer drug-tolerance phenotypes. [Materials and Methods] Using five cancer cell lines and four anticancer agents, CoLA was produced using 2,400 drug-tolerant colonies, followed by protein expression analysis employing specific immunodetection and using a panel of primary antibodies. Transcriptional products that may be important to the formation of colonies in the presence of anticancer drugs were identified by DNA microarray. Candidate genes were examined to determine whether they were associated with the suppression of colony formation, using gene knockdown by siRNA. [Results] This study revealed that CoLA is capable of analyzing lysates from individual colonies in a quantitative manner, by a panel of protein markers. Colonies that expressed pluripotent and CSC markers tended to express low levels of epithelial proteins. However, individual protein expression levels were not well-associated with drug concentrations, suggesting that the phenotypes of drug-tolerant colonies were not necessarily induced by drugs, and emerged spontaneously. Subsequent analysis identifying molecular fractions important to drug-tolerant phenotypes revealed that transcriptional products play an important role in developing these phenotypes. DNA microarray analysis allowed for the narrowing down of 12 genes that are potentially associated with these phenotypes. A gene knockdown by siRNA identified two out of 12 candidate genes as involved in the suppressed colony formation of MCF7 human breast cancer cell line in the presence of cisplatin. [Discussion] CoLA assay revealed that a number of CSC marker-negative colonies exist in the presence of anticancer drugs, suggesting that drug-tolerant cells may not always be associated with CSCs. In the context of drug-tolerant phenotype acquisition, protein expression may merely be a handy marker, whereas transcriptional control may be more critical. Further functional analysis of candidate genes for colony suppression in the presence of anticancer drugs is necessary. Together, de novo drug-tolerance inducing factors are possibly involved in the heterogeneity of cancer cell populations. Citation Format: Kohei Kume, Satoshi Nishizuka, Miyuki Ikeda, Sawako Miura, Yuriko Wada, Shuji Fujikawa, Chihaya Maesawa, Go Wakabayashi. Analysis of cancer cell populations by isolated single colonies for the identification of drug-tolerance inducing factors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4060. doi:10.1158/1538-7445.AM2013-4060

3P258 On phenotypic drug tolerance based on expression noise of antibiotic resistant gene(20. Origin of life & Evolution,Poster)

3P258 On phenotypic drug tolerance based on expression noise of antibiotic resistant gene(20. Origin of life & Evolution,Poster)

The Role of Transport Mechanisms in Mycobacterium Tuberculosis Drug Resistance and Tolerance

In the fight against tuberculosis, cell wall permeation of chemotherapeutic agents remains a critical but largely unsolved question. Here we review the major mechanisms of small molecule penetration into and efflux from Mycobacterium tuberculosis and other mycobacteria, and outline how these mechanisms may contribute to the development of phenotypic drug tolerance and induction of drug resistance. M. tuberculosis is intrinsically recalcitrant to small molecule permeation thanks to its thick lipid-rich cell wall. Passive diffusion appears to account for only a fraction of total drug permeation. As in other bacterial species, influx of hydrophilic compounds is facilitated by water-filled open channels, or porins, spanning the cell wall. However, the diversity and density of M. tuberculosis porins appears lower than in enterobacteria. Besides, physiological adaptations brought about by unfavorable conditions are thought to reduce the efficacy of porins. While intracellular accumulation of selected drug classes supports the existence of hypothesized active drug influx transporters, efflux pumps contribute to the drug resistant phenotype through their natural abundance and diversity, as well as their highly inducible expression. Modulation of efflux transporter expression has been observed in phagocytosed, non-replicating persistent and multi-drug resistant bacilli. Altogether, M. tuberculosis has evolved both intrinsic properties and acquired mechanisms to increase its level of tolerance towards xenobiotic substances, by preventing or minimizing their entry. Understanding these adaptation mechanisms is critical to counteract the natural mechanisms of defense against toxic compounds and develop new classes of chemotherapeutic agents that positively exploit the influx and efflux pathways of mycobacteria.

Comparative analysis of mycobacterial NADH pyrophosphatase isoforms reveals a novel mechanism for isoniazid and ethionamide inactivation

NADH pyrophosphatase (NudC) catalyses the hydrolysis of NAD(H) to AMP and NMN(H) [nicotinamide mononucleotide (reduced form)]. NudC multiple sequence alignment reveals that homologues from most Mycobacterium tuberculosis isolates, but not other mycobacterial species, have a polymorphism at the highly conserved residue 237. To elucidate the functional significance of this polymorphism, comparative analyses were performed using representative NudC isoforms from M. tuberculosis H37Rv (NudCRv) and M. bovis BCG (NudCBCG). Biochemical analysis showed that the P237Q polymorphism prevents dimer formation, and results in a loss of enzymatic activity. Importantly, NudCBCG was found to degrade the active forms of isoniazid (INH), INH-NAD and ethionamide (ETH), ETH-NAD. Consequently, overexpression of NudCBCG in Mycobacterium smegmatis mc2155 and M. bovis BCG resulted in a high level of resistance to both INH and ETH. Further genetic studies showed that deletion of the nudC gene in M. smegmatis mc2155 and M. bovis BCG resulted in increased susceptibility to INH and ETH. Moreover, inactivation of NudC in both strains caused a defect in drug tolerance phenotype for both drugs in exposure assays. Taken together, these data suggest that mycobacterial NudC plays an important role in the inactivation of INH and ETH.

Tumor heterogeneity and strategies to overcome kinase inhibitor resistance in cancer: lessons from melanoma

Tumor heterogeneity and strategies to overcome kinase inhibitor resistance in cancer: lessons from melanoma