New Mechanism Of Drug Resistance Research Articles

Coregulation of extracellular vesicle production and fluconazole susceptibility in Cryptococcus neoformans.

Resistance to fluconazole (FLC), the most widely used antifungal drug, is typically achieved by altering the azole drug target and/or drug efflux pumps. Recent reports have suggested a link between vesicular trafficking and antifungal resistance. Here, we identified novel Cryptococcus neoformans regulators of extracellular vesicle (EV) biogenesis that impact FLC resistance. In particular, the transcription factor Hap2 does not affect the expression of the drug target or efflux pumps, yet it impacts the cellular sterol profile. Subinhibitory FLC concentrations also downregulate EV production. Moreover, in vitro spontaneous FLC-resistant colonies showed altered EV production, and the acquisition of FLC resistance was associated with decreased EV production in clinical isolates. Finally, the reversion of FLC resistance was associated with increased EV production. These data suggest a model in which fungal cells can regulate EV production in place of regulating the drug target gene expression as a first line of defense against antifungal assault in this fungal pathogen. IMPORTANCE Extracellular vesicles (EVs) are membrane-enveloped particles that are released by cells into the extracellular space. Fungal EVs can mediate community interactions and biofilm formation, but their functions remain poorly understood. Here, we report the identification of the first regulators of EV production in the major fungal pathogen Cryptococcus neoformans. Surprisingly, we uncover a novel role of EVs in modulating antifungal drug resistance. Disruption of EV production was associated with altered lipid composition and changes in fluconazole susceptibility. Spontaneous azole-resistant mutants were deficient in EV production, while loss of resistance restored initial EV production levels. These findings were recapitulated in C. neoformans clinical isolates, indicating that azole resistance and EV production are coregulated in diverse strains. Our study reveals a new mechanism of drug resistance in which cells adapt to azole stress by modulating EV production.

The Identification of a Group of Self-Assembling, Trimetallic Cryptands with Multi-Kinase Inhibitor Activity Using a Phenotypic Approach to Drug Discovery

Despite significant advances in the development of targeted anti-cancer drugs, the much hoped for paradigm shift in cancer survival has been blunted by the emergence of new mechanisms of drug resistance. The development of drugs with multiple mechanisms of action is one approach that has been adopted to circumvent this problem and in this talk, the use of a phenotypic approach to drug discovery based on potency and selectivity in vitro has been used to identify compounds that have multiple mechanisms of action. This talk will specifically focus on the discovery, preclinical activity and mechanism of action of self-assembling trimetallic cryptands that have potent but selective activity against cancer cells in vitro and in vivo.

Cancer-type organic anion transporting polypeptide 1B3 (Ct-OATP1B3) is localized in lysosomes and mediates resistance against kinase inhibitors.

Cancer-type organic anion transporting polypeptide 1B3 (Ct-OATP1B3), a splice variant of the hepatic uptake transporter OATP1B3 (liver-type; Lt-OATP1B3), is expressed in several tumor entities including colorectal carcinoma (CRC) and breast cancer. In CRC, high OATP1B3 expression has been associated with reduced progression-free and overall survival. Several kinase inhibitors used for antitumor treatment are substrates and/or inhibitors of OATP1B3 (e.g. encorafenib, vemurafenib). The functional importance of Ct-OATP1B3 has not been elucidated so far. HEK293 cells stably overexpressing Ct-OATP1B3 protein were established and compared with control cells. Confocal laser scanning microscopy, immunoblot, and proteomics-based expression analysis demonstrated that Ct-OATP1B3 protein is intracellularly localized in lysosomes of stably-transfetced cells. Cytotoxicity experiments showed that cells recombinantly expressing the Ct-OATP1B3 protein were more resistant against the kinase inhibitor encorafenib compared to control cells [e.g. encorafenib (100 µM) survival rates: 89.5% vs. 52.8%]. In line with these findings, colorectal cancer DLD1 cells endogenously expressing Ct-OATP1B3 protein had poorer survival rates when the OATP1B3 substrate bromosulfophthalein (BSP) was coincubated with encorafenib or vemurafenib compared to the incubation with the kinase inhibitor alone. This indicates a competitive inhibition of Ct-OATP1B3-mediated uptake into lysosomes by BSP. Accordingly, mass spectrometry-based drug analysis of lysosomes showed a reduced lysosomal accumulation of encorafenib in DLD1 cells additionally exposed to BSP. These results demonstrate that Ct-OATP1B3 protein is localized in the lysosomal membrane and can mediate transport of certain kinase inhibitors into lysosomes revealing a new mechanism of resistance. Significance Statement We describe the characterization of a splice variant of the liver-type uptake transporter OATP1B3 expressed in several tumor entities. This variant is localized in lysosomes mediating resistance against kinase inhibitors which are substrates of this transport protein by transporting them into lysosomes and thereby reducing the cytoplasmic concentration of these antitumor agents. Therefore, the expression of the Ct-OATP1B3 protein is associated with a better survival of cells revealing a new mechanism of drug resistance.

Abstract 405: A new mechanism of drug resistance in cancer: extracellular ATP-induced resistance by macropinocytosis-mediated internalization and redox changes

Abstract Drug resistance are responsible for most relapses and up to 90% of cancer related death. The opportunistic uptake of extracellular molecules has been named as a key emerging hallmark of cancer metabolism. In tumor microenvironment, intratumoral extracellular ATP (eATP) levels are 1,000 times or more higher than those in corresponding normal tissues. Our previous studies have shown that eATP can be taken up by cancer cell via macropinocytosis, leading to substantial increase in intracellular ATP levels. We found that this high intracellular ATP levels contribute to drug resistance in several ways: first, it directly fueled and enhanced drug-pumping activity of major ATP-binding cassette (ABC) multidrug transporters, which are located in plasma membrane and can pump out a wide range of substrates including anticancer drugs, leading to reduced intracellular drug retention. Secondly, ATP treatment upregulated gene expression of the major ABC transporters, and this gene regulatory effect of ATP was comparable to TGF-beta, which is a well-established epithelial-mesenchymal transition (EMT) inducer, and EMT is known to induce ABC transporter expression and drug resistance. Moreover, eATP treatment led to an increase in intracellular glutathione (GSH) levels, probably by enhancing GSH synthesis. Increased GSH levels have been described to significantly contribute to drug resistance. We also found eATP altered cellular redox state, which potentially stimulates EMT. Furthermore, we performed RNA-sequencing and identified several genes that may serve as downstream factors of eATP in promoting EMT and drug resistance. We are currently investigating the roles of these genes by knocking down gene expression or knocking out these genes. Citation Format: Haiyun Zhang, Xiaozhuo Chen. A new mechanism of drug resistance in cancer: extracellular ATP-induced resistance by macropinocytosis-mediated internalization and redox changes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 405.

FLT3 Inhibitors Induce p53 Instability Due to Increased Interaction between p53 and MDM2 By Inhibiting STAT5 Phosphorylation in Acute Myeloid Leukemia

Frequently mutated in Acute myeloid leukemia (AML), FLT3 is considered as one of the favorable targets for treatment. The FLT3 internal tandem duplication (ITD) mutation enhances kinase activity and causes hyperactivation of downstream signal transduction. Several small molecule FLT3 inhibitors have developed, but their clinical efficacy is limited due to generation of drug resistance. In this study, we define a new mechanism of drug resistance toward tyrosine kinase inhibitors (TKIs). Initially, we found a rapid decrease in the protein level of tumor suppressor p53 in FLT3-ITD-positive MV4-11 and MOLM13 cells and peripheral blood mononuclear cells (PBMCs) from FLT3-ITD AML patients upon treatment with TKIs including sorafenib, sunitinib and quizartinib. The decrease is not caused by changes in mRNA expression as revealed by qPCR analyses but rather by accelerated protease degradation because the p53 protein was stabilized by proteasome inhibitor MG132. Furthermore, treatment of cells with RG7388, a potent disruptor of p53 and MDM2 interaction, prevented the TKI-induced p53 loss. Since MDM2 is the most important E3 ligase responsible for ubiquitination of p53, the data suggest that TKIs may lead to the degradation of p53 by promoting ubiquitination. Indeed, ubiquitination assays verified that TKIs promoted K48 poly-ubiquitination of p53. Previous studies have demonstrated that activations of FLT3 downstream signaling components such as ERKs and Akt reduce p53 protein stability through ubiquitination by activating MDM2. It is somewhat unexpected that inhibition of FLT3-ITD and its downstream signaling pathways also resulted in decreased p53 stability due to increased ubiquitination. We treated FLT3-ITD-containing cells with specific ERK, AKT and STAT5 inhibitors. Interestingly, while inhibition of ERKs and AKT had no significant effect on the stability of p53, STAT5 inhibition resulted in a reduced level of p53 accompanied by increased K48 poly-ubiquitination. We further analyzed the interaction of p53 with MDM2 in AML cells by using immunoprecipitation. The results showed that the p53-MDM2 interaction was significantly enhanced after treatment with TKIs and STAT5 inhibitors, which was diminished in the presence of RG7388. Subcellular fractionation revealed the presence of p53 and STAT5 in both nucleus and cytoplasm. Treatment of cells with TKIs resulted in a decreased level of p53 and STAT5 in the nucleus, and immunoprecipitation of nuclear proteins with a p53 antibody revealed a reduced association of p53 with STAT5. Taken together, the data suggest that FLT3 inhibitors inhibited nuclear translocation of STAT5 and reduced its interaction of p53 thereby facilitating p53/MDM2 interaction and subsequent ubiquitination and degradation of p53. This study reveals a novel mechanism by which drug resistance to TKIs may occur and further support the use of MDM2/p53 interaction inhibitors in combination with TKIs for treatment of AML. DisclosuresNo relevant conflicts of interest to declare.

Abstract 1108: A new mechanism of drug resistance in cancer: Extracellular ATP-induced resistance through ATP internalization and upregulation of ATP-binding cassette multidrug transporters

Abstract Cancer is the second leading cause of death in the US. Multidrug resistance (MDR) and the resulting ineffectiveness of the drug treatment are responsible for most relapses of cancer and cancer-related death. One major mechanism of MDR is the efflux of multiple drugs mediated by ATP-binding Cassette (ABC) multidrug transporters. ABC transporters pump drugs with different structures and mechanisms of action out of cancer cells using ATP-provided energy. The opportunistic uptake of extracellular molecules has been named as a key emerging hallmark of cancer metabolism1. Intratumoral extracellular ATP levels of various cancer types are 103 to 104 times higher than those in their corresponding normal tissues2. Our previous studies have shown that extracellular ATP is internalized by cancer cell via macropinocytosis, and promotes cancer drug resistance by fueling ABC transporters and increase its efflux activities3. However, whether extracellular ATP can indeed enhance efflux of drugs, and the underlying mechanism besides providing energy remain unclear. Our current study results showed extracellular ATP decreased intracellular accumulation and thereby enhanced efflux of 3H-labeled chemotherapeutic drug and targeted drug in human cancer cells. The results of quantitative reverse transcription PCR, western blot and immunofluorescence microscopy demonstrated that extracellular ATP can also significantly upregulate ABC transporters at mRNA and protein levels, and change the intracellular localization of the latter. These results suggest that extracellular ATP has profound regulatory effects on ABC transporters and thus promotes drug resistance, linking the ATP-rich tumor microenvironment with cancer drug resistance. The completion of the study will expand our understanding of the roles of extracellular ATP in cancer, and reveal new therapeutic targets for reducing drug resistance and enhance anticancer efficacy of chemotherapeutics and targeted drugs. 1. Pavlova N, Thompson C. Cell Metab. (2016) 23:27-47 2. Falzoni S, et al., Interface Focus. (2013) 3: 20120101 3. Wang X, et al., Oncotarget. (2017) 8(50):87860-87877 Citation Format: Haiyun Zhang, Xuan Wang, Xiaozhuo Chen. A new mechanism of drug resistance in cancer: Extracellular ATP-induced resistance through ATP internalization and upregulation of ATP-binding cassette multidrug transporters [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1108.

Epigenetic Drugs Selectively Target a Population of AML Cells Which Are Positive for CD123 Cell Surface Marker and Are Chemoresistant

Acute Myeloid leukemia (AML) is the most common acute leukemia in adults. While Cytarabine (Ara-C) based chemotherapy has been a standard for initial treatment, over 50% of patients will eventually relapse within a period of time. Chemoresistance remains a major burden for treatment of relapse patient. Recent studies suggest that AML relapse is caused by the survival of drug resistant leukemia stem cell (LSC) population. Since LSCs are in low frequency and remain quiescent in patients, it is difficult to study the chemoresistant mechanism on this population of cells. It has been previously shown that cell lines that derived from patient sample might be considered as attractive models aimed to better characterize LSC biological properties and their chemotherapy resistance mechanisms. Multiple LSC cell surface markers has been identified in patient sample and cell lines. However, it remains to be determined whether subpopulations with different LSC markers has similar level of chemoresistance.To better understand the mechanism of drug resistance and whether it is correlated to the expression of certain group of LSC surface markers, we collected various human AML cell lines and examined correlations of their IC50 of Ara-C and percentage of individual LSC surface markers in these cell lines. We found that CD34 surface marker is not correlated to chemoresistance. Other LSC surface markers, such as CD13, CD44, CD117, CD96 and CD45RA are also not correlated to chemoresistance. Interestingly, CD47 (R square = 0.6660, P value = 0.0476) and CD 123 (R square = 0.7421, P value = 0.0275) are positively correlated to chemoresistance. In order to rule out heterogeneous genetic background contribute to chemoresistance of cell lines, we generated a drug resistant cell line from an drug sensitive AML cell line, OCI-AML2, by gradually increased the Ara-C concentration in culture media. During the selection, the percentage of cells with CD47 and CD123 gradually increased. When the resistant cell line is established, the percent of CD123 positive cells are over 90 percent. Through cell tracking experiments, we found that resistant cells grow at least two cycles slower than sensitive cells in a 5 day tracking assay. RNAseq analysis shows that genes related to cell cycle regulator and resistance of apoptosis changed significantly. However, genes that related to stemness, such as Oct4, Nanog, KLF4, Myc, FLT3, are no significant changed in drug resistant cells compare with the sensitive line, although Sox2 increased 50% and ALDH1A3 increased 3 folds. Therefore, chemoresistant cells may represent cells in a slow growing, de-differentiate stage, however, it may not bare all characteristics of a LSC.We further investigate the role of epigenetic factors in regulating the survival of chemoresistant leukemia cells. Our data shows epigenetic drugs that targeting histone deacetylases, bromodomain proteins and PRC1 complex are effective to induce apoptosis and cell death of chemoresistant cells, therefore they may be potential second line of chemotherapy drug for chemoresistant patients. Histone deacetylase inhibitor Romidepsin efficiently cleared chemoresistant AML blast in xenographed NSG mice. Furthermore, we found Romidepsin has synergetic effect when combine with Ara-C. Interestingly, Romedepsin preferentially target CD123 positive, slow growing cells while Ara-C mainly target fast growing, CD123 negative cells. Therefore, combine treatment with Romedepsin and Ara-C may prevent the development of chemoresistance in AML cells. In summary, we identify CD123 and CD47cell surface markers may potentially serve as biomarker for chemresistance AML cells. Our study also sheds light on a new mechanism of drug resistance in leukemia, and provide a rationale to develop and test epigenetic-targeted therapies in leukemia, especially in drug resistant relapse patients. DisclosuresNo relevant conflicts of interest to declare.

Amphotericin B and Other Polyenes-Discovery, Clinical Use, Mode of Action and Drug Resistance.

Although polyenes were the first broad spectrum antifungal drugs on the market, after 70 years they are still the gold standard to treat a variety of fungal infections. Polyenes such as amphotericin B have a controversial image. They are the antifungal drug class with the broadest spectrum, resistance development is still relatively rare and fungicidal properties are extensive. Yet, they come with a significant host toxicity that limits their use. Relatively recently, the mode of action of polyenes has been revised, new mechanisms of drug resistance were discovered and emergent polyene resistant species such as Candida auris entered the picture. This review provides a short description of the history and clinical use of polyenes, and focusses on the ongoing debate concerning their mode of action, the diversity of resistance mechanisms discovered to date and the most recent trends in polyene resistance development.

Abstract 3008: A new mechanism of drug resistance in cancer: Extracellular ATP-induced resistance through ATP internalization and upregulation of ATP-binding cassette multidrug transporters

Abstract Cancer is the second leading cause of death in the US. Multidrug resistance (MDR) and the resulting ineffectiveness of the drug treatment are responsible for most relapses of cancer and cancer-related death. One major mechanism of MDR is the efflux of multiple drugs mediated by ATP-binding Cassette (ABC) multidrug transporters. ABC transporters pump drugs with different structures and mechanisms of action out of cancer cells using ATP-provided energy. And ABC activities is also intimately linked to epithelial mesenchymal transition and metastasis. The opportunistic uptake of extracellular molecules has been named as a key emerging hallmark of cancer metabolism. Intratumoral extracellular ATP levels of various cancer types are 103 to 104 times higher than those in their corresponding normal tissues. Our previous studies have shown that extracellular ATP is internalized by cancer cell via macropinocytosis, and promotes cancer drug resistance by directly fueling ABC transporters and increase its drug efflux activities. However, whether extracellular ATP can induce change in the expression of ABC transporters and the underlying mechanisms remain unclear. Our results from quantitative reverse transcription PCR and flow cytometry show that extracellular ATP significantly alters mRNA levels and cell surface expression of ABC transporters. These results suggest that extracellular ATP has profound regulatory effects on ABC transporters and thus promotes drug resistance, linking the ATP-rich tumor microenvironment with cancer drug resistance. On-going studies include identification of mechanisms for the extracellular ATP-induced drug resistance via ABC transporters. The completion of the study will expand our understanding of the roles of extracellular ATP in cancer, and reveal new therapeutic targets for reducing drug resistance and enhancing anticancer efficacy of chemotherapeutics and targeted drugs. Citation Format: Haiyun Zhang, Xuan Wang, Xiaozhuo Chen. A new mechanism of drug resistance in cancer: Extracellular ATP-induced resistance through ATP internalization and upregulation of ATP-binding cassette multidrug transporters [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3008.

Transcripts 202 and 205 of IL-6 confer resistance to Vemurafenib by reactivating the MAPK pathway in BRAF(V600E) mutant melanoma cells

BRAF mutations occur in approximately 50% of melanoma patients. The mutated BRAF kinase continuously activates the mitogen-activated protein kinase (MAPK) pathway to promote cell growth and proliferation. Vemurafenib as a specific BRAF inhibitor can significantly prolong progression-free survival in melanoma patients. However, most patients developed resistance to Vemurafenib after 6 months. The mechanism of drug resistance is not yet fully understood. In this study, we found that proteins secreted by drug-resistant cells protect sensitive cells from Vemurafenib. By RNA-seq, we compared differentially expressed genes between resistant and sensitive cells. We demonstrated that drug-resistant cells secrete more IL-6 protein than sensitive cells. For the first time, we found that IL-6 expressed by drug-resistant cells consists of the following transcripts: IL6-201, IL6-202 and IL6-205. We confirmed that it is the IL6-202 and IL6-205 transcripts that confer drug resistance to Vemurafenib by reactivating the MAPK pathway while IL6-201 is not responsible for the resistance in A375 melanoma cells. Neutralizing IL-6 significantly increased the sensitivity of drug-resistant cells to Vemurafenib. Overall, these results reveal a new mechanism of drug resistance in melanoma.

Mycobacterium abscessus, an Emerging and Worrisome Pathogen among Cystic Fibrosis Patients.

Nontuberculous mycobacteria (NTM) have recently emerged as important pathogens among cystic fibrosis (CF) patients worldwide. Mycobacterium abscessus is becoming the most worrisome NTM in this cohort of patients and recent findings clarified why this pathogen is so prone to this disease. M. abscessus drug therapy takes up to 2 years and its failure causes an accelerated lung function decline. The M. abscessus colonization of lung alveoli begins with smooth strains producing glycopeptidolipids and biofilm, whilst in the invasive infection, “rough” mutants are responsible for the production of trehalose dimycolate, and consequently, cording formation. Human-to-human M. abscessus transmission was demonstrated among geographically separated CF patients by whole-genome sequencing of clinical isolates worldwide. Using a M. abscessus infected CF zebrafish model, it was demonstrated that CFTR (cystic fibrosis transmembrane conductance regulator) dysfunction seems to have a specific role in the immune control of M. abscessus infections only. This pathogen is also intrinsically resistant to many drugs, thanks to its physiology and to the acquisition of new mechanisms of drug resistance. Few new compounds or drug formulations active against M. abscessus are present in preclinical and clinical development, but recently alternative strategies have been investigated, such as phage therapy and the use of β-lactamase inhibitors.

Abstract 5848: Histone deacetylase inhibitor selectively targets CD123 positive chemoresistant AML cells

Abstract Acute Myeloid leukemia (AML) is the most common acute leukemia in adults. Chemoresistance remains a major burden for treatment of relapse patient. Studies suggest that AML relapse related to the survival of drug resistant leukemia stem cell (LSC) population. To better understand the mechanism of drug resistance and whether it is correlated to the expression of certain group of LSC surface markers, we collected various human AML cell lines and examined correlations of their IC50 of Ara-C and percentage of individual LSC surface markers in these cell lines. We found that only CD47 (R square = 0.6660, P value = 0.0476) and CD 123 (R square = 0.7421, P value = 0.0275) are positively correlated to chemoresistance. In order to rule out heterogeneous genetics background contribute to chemoresistance of cell lines, we generated a resistant cell line from a drug sensitive AML cell line, OCI-AML2, by gradually increased the Ara-C concentration in culture media. During the selection, the percentage of cells with CD47 and CD123 gradually increased. When the resistant cell line is established, the percent of CD123 positive cells are over 90 percent. RNA-seq analysis shows that genes related to cell cycle regulator and resistance of apoptosis changed significantly. However, signature genes that related to stemness, such as Oct4, Nanog, KLF4, Myc, FLT3, are no significant changed in resistant cells compare with the sensitive line, although Sox2 increased 50% and ALDH1A3 increased 3 folds. Therefore, chemoresistant cells may represent cells in a slow growing, de-differentiate stage, however, they may not bare all characteristics of LSCs. We further investigate the role of epigenetic factors in regulating the survival of chemoresistant leukemia cells. Our data shows epigenetic drugs that targeting histone deacetylases, bromodomain proteins and PRC1 complex are effective to induce apoptosis and cell death of chemoresistant cells, therefore they may be potential second line of chemotherapy drug for chemoresistant patients. Histone deacetylase inhibitor Romidepsin efficiently cleared chemoresistant AML blast in xenographed NSG mice. Furthermore, we found Romidepsin has synergetic effect when combine with Ara-C. Interestingly, Romedepsin preferentially target CD123 positive, slow growing cells while Ara-C mainly target fast growing, CD123 negative cells. Therefore, combine treatment with Romedepsin and Ara-C may prevent the development of chemoresistance in AML cells. In summary, we identify CD123 and CD47 cell surface markers may potentially serve as biomarker for chemresistance AML cells. Our study also sheds light on a new mechanism of drug resistance in leukemia, and provide a rationale to develop and test epigenetic-targeted therapies in leukemia, especially in drug resistant relapse patients. Citation Format: Bowen Yan, Suming Huang, Yi Qiu. Histone deacetylase inhibitor selectively targets CD123 positive chemoresistant AML cells [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 5848.

Exploration of the Plasmodium falciparum Resistome and Druggable Genome Reveals New Mechanisms of Drug Resistance and Antimalarial Targets.

Plasmodium parasites, the causative agent of malaria infections, rapidly evolve drug resistance and escape detection by the human immune response via the incredible mutability of its genome. Understanding the genetic mechanisms by which Plasmodium parasites develop antimalarial resistance is essential to understanding why most drugs fail in the clinic and designing the next generation of therapies. A systematic genomic analysis of 262 Plasmodium falciparum clones with stable in vitro resistance to 37 diverse compounds with potent antimalarial activity was undertaken with the main goal of identifying new drug targets. Despite several challenges inherent to this method of in vitro drug resistance generation followed by whole genome sequencing, the study was able to identify a likely drug target or resistance gene for every compound for which resistant parasites could be generated. Known and novel P falciparum resistance mediators were discovered along with several new promising antimalarial drug targets. Surprisingly, gene amplification events contributed to one-third of the drug resistance acquisition events. The study can serve as a model for drug discovery and resistance analyses in other similar microbial pathogens amenable to in vitro culture.

Abstract 3194: A new mechanism of drug resistance in cancer: extracellular ATP-induced resistance through ATP internalization and upregulation of protein phosphorylation in Akt and ERK pathways

Abstract The opportunistic uptake of extracellular molecules has been named as a key emerging hallmark of cancer metabolism[1]. Extracellular ATP (eATP) levels of various cancer types are 103 to 104 times higher than those in their corresponding normal tissues[2]. However, the biological significance of the high ATP concentrations is not clear. We recently reported that cancer cells uptake extracellular ATP via different endocytoses to enhance growth, survival, and drug resistance to tyrosine kinase inhibitors (TKIs)[3 ,4]. We hypothesized that eATP induces resistance to TKIs by endocytoses-mediated internalization of eATP, which competes with TKIs for the ATP-binding site of receptor TKs (RTKs), phosphorylating and activating RTKs and RTK-mediated signaling pathways. In contrast to previous reports that synthesized intracellular ATP (iATP) elevation contributed acquired drug resistance, here we report a novel intrinsic drug resistance in which eATP was internalized and substantially increased iATP levels and promoted cancer cell drug resistance to the TKI sunitinib in human NSCLC A549 cells. The iATP level elevation and drug resistance were mediated primarily by macropinocytosis. The resistance was reduced when macropinocytosis was suppressed by inhibitors or an siRNA knockdown of a key micropinocytosis protein PAK1. Intracellularly, the elevated iATP upregulated phosphorylation of PDGFR and proteins/enzymes in the PDGFR-mediated Akt-mTOR and Raf-ERK signaling pathways, resulting in reduced apoptosis triggered by sunitinib. Furthermore, both in vitro and in A549 tumors, eATP partially restored phosphorylation levels of PDGFR and PDGFR-mediated proteins/enzymes suppressed by sunitinib. The resistance cannot be accounted for by the overall purinergic receptor-mediated signaling, glycolysis, or mitochondrial OXPHOS. These results strongly suggest that the eATP-elevated intracellular ATP levels reversed the inhibition of TKIs by using the mechanism we hypothesized, linking for the first time the ATP-rich tumor microenvironment with cancer drug resistance. All these findings significantly expand our understanding of the roles of extracellular ATP in cancer, and offer new anti-drug resistance targets. 1. Pavlova N, Thompson C. Cell Metab. (2016) 23:27-47. 2. Falzoni et al., Interface Focus.(2013) 3: 20120101. 3. Qian Y, Wang X, et al., Cancer Lett. (2014)351: 242-5. 4. Qian Y, Wang X et al., Mol Cancer Res (2016) 14:1087-96 Citation Format: Xuan Wang, Yunsheng Li, Yanrong Qian, Yanyang Cao, Xiaozhuo Chen. A new mechanism of drug resistance in cancer: extracellular ATP-induced resistance through ATP internalization and upregulation of protein phosphorylation in Akt and ERK pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3194. doi:10.1158/1538-7445.AM2017-3194

Regulators of Stem Cells Proliferation in Tissue Regeneration

This review summarizes recent breakthroughs in studies regarding proliferation of adult stem cells which complement and extend our knowledge to various aspects of cell biology and signal pathways. In recent years, many interesting results and discoveries were achieved regarding the proliferative properties of stem cells. In vitro expansion of stem cells may benefit from high proliferation rates, which can produce a large amount of cells to regenerate tissue defects. Meanwhile, optimizing the culture conditions for stem cell propagation is helpful in maintaining the pluripotency and differentiation potential of stem cells, which are critical for tissue engineering and regenerative medicine. Additionally, understanding the proliferation of stem cells may help us to uncover new mechanisms of drug resistance, since stem cells are believed to play an important role in drug resistance. In this review, we focus on signaling pathways regulating the proliferation of various stem cells isolated from adult tissues. We also emphasize the significance of stem cells proliferation in cell research, as well as rapid propagation of adult stem cells for regenerative medicine.

Systematic identification of signaling pathways with potential to confer anticancer drug resistance.

Cancer cells can activate diverse signaling pathways to evade the cytotoxic action of drugs. We created and screened a library of barcoded pathway-activating mutant complementary DNAs to identify those that enhanced the survival of cancer cells in the presence of 13 clinically relevant, targeted therapies. We found that activation of the RAS-MAPK (mitogen-activated protein kinase), Notch1, PI3K (phosphoinositide 3-kinase)-mTOR (mechanistic target of rapamycin), and ER (estrogen receptor) signaling pathways often conferred resistance to this selection of drugs. Activation of the Notch1 pathway promoted acquired resistance to tamoxifen (an ER-targeted therapy) in serially passaged breast cancer xenografts in mice, and treating mice with a γ-secretase inhibitor to inhibit Notch signaling restored tamoxifen sensitivity. Markers of Notch1 activity in tumor tissue correlated with resistance to tamoxifen in breast cancer patients. Similarly, activation of Notch1 signaling promoted acquired resistance to MAPK inhibitors in BRAF(V600E) melanoma cells in culture, and the abundance of Notch1 pathway markers was increased in tumors from a subset of melanoma patients. Thus, Notch1 signaling may be a therapeutic target in some drug-resistant breast cancers and melanomas. Additionally, multiple resistance pathways were activated in melanoma cell lines with intrinsic resistance to MAPK inhibitors, and simultaneous inhibition of these pathways synergistically induced drug sensitivity. These data illustrate the potential for systematic identification of the signaling pathways controlling drug resistance that could inform clinical strategies and drug development for multiple types of cancer. This approach may also be used to advance clinical options in other disease contexts.

A Gripping New Mechanism of Drug Resistance in HIV-1 Reverse Transcriptase

HIV-1 Reverse Transcriptase (RT) converts viral RNA into dsDNA that is integrated into the human genome. Nonnucleoside RT inhibitors (NNRTIs) are highly effective in the treatment and prevention of HIV. NNRTI resistance mutations arise from therapy, yet the mechanism(s) of how these mutations inhibit polymerization by RT is unclear. We examine the role of NNRTI on the dynamics between RT and its Template/Primer (T/P) substrate using a combination of molecular modeling, single-molecule and bulk fluorescence techniques and provide an unprecedented glimpse into the dynamics of RT-T/P interaction as well as the intramolecular conformation of RT itself while bound to its substrate. The data suggests a unique mechanism of resistance that is mediated by interplay between intramolecular conformational changes in RT and intermolecular dynamics of the RT-template/primer-dNTP complex.

Establishment of a 5-fluorouracil-resistant triple-negative breast cancer cell line

Triple-negative breast cancers (TNBCs) are defined as tumors that lack expression of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2. Clinically, TNBC patients are treated with cytotoxic drugs including 5-fluorouracil (5-FU). However, TNBCs develop resistance to such drugs after a series of treatments. To elucidate the mechanisms of drug resistance, establishment of drug-resistant cancer cell lines should be one of the most useful model systems. However, 5-FU-resistant TNBC cell lines have not been previously reported. In this study, we established a 5-FU-resistant cell line, MDA-MB-231/5-FU, from the human TNBC cell line MDA-MB-231, by repeated exposure to stepwise increases in the concentration of 5-FU. The IC₅₀ value of 5-FU for MDA-MB-231/5-FU was 5.5-fold that for the parental cells. The MDA-MB-231/5-FU cell line acquired resistance to not only 5-FU, but also vinorelbine, paclitaxel and gemcitabine. Additionally, we performed iTRAQ-based quantitative proteomics in MDA-MB-231/5-FU cells and the parental cells in order to characterize MDA-MB-231/5-FU. The proteins upregulated in the newly established cells were mainly classified into the categories of 'DNA recombination', 'cell cycle', 'complex assembly', 'cytoskeleton organization', 'transport' and 'negative regulation of cell death'. These proteins may be related to mechanisms of drug resistance in TNBCs. Our established MDA-MB-231/5-FU cell line should be a useful tool for identifying new mechanisms of drug resistance and new drug targets in TNBCs.

Abstract 2105: Advanced glycation end products of type I collagen promote cell proliferation and resistance against doxorubicin in HT-1080 cancer cells.

Abstract Resistance to apoptosis and chemotherapy is a critical factor in cancer recurrence and patient relapse. Extracellular matrix via its receptors, the integrins, has emerged as a major pathway contributing to cancer cell survival and resistance to chemotherapy (Aoudjit and Vuori, Chemother Res Pract. 2012;2012:283181). However, the effects of extracellular matrix advanced glycation end products (AGE) on cell proliferation and sensitivity to chemotherapy is not well established. In this study, we investigated whether type I collagen and particularly its AGE are involved in cell proliferation and resistance to doxorubicin in human fibrosarcoma HT-1080 cancer cells. Type I collagen was extracted from rats tail tendons (10 days, 8 and 96 weeks-old) and prepared as already described (Millerot-Serrurot et al., 2010, Cancer Cell Int, 13: 10-26). First, we characterized the glycation pattern of the different collagens using SDS-PAGE electrophoresis and fluorescence spectroscopy. A significant increase in AGE level has been observed in collagen extracted from 8 and 96 weeks-old rats. AGE level was significantly higher in collagen extracted from 96 weeks-old rats when compared to 8 weeks-old ones. Only collagen extracted from 96 weeks-old rats was able to induce 3-fold increase in proliferation of HT-1080 cells. Western-blot analysis showed that activation of MAPK/ERK, but not PI3K/AKT, was required for collagen AGE mediated cell proliferation. The MAPK/ERK inhibitor U0126 abolished this effect. Both collagens were able to inhibit doxorubicin-induced apoptosis in HT-1080 cells. Moreover, protection against apoptosis was significantly higher in the presence of collagen extracted from 96 weeks-old rats. Our data suggest extracellular matrix AGE signalling as an important promoter of cancer cell proliferation and that this pathway may contribute to a new mechanism of drug resistance. Citation Format: Charles Saby, Hassan EL Btaouri, Laurence Van Gulick, Marie Guilbert, Pierre Jeannesson, Hamid Morjani. Advanced glycation end products of type I collagen promote cell proliferation and resistance against doxorubicin in HT-1080 cancer cells. [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 2105. doi:10.1158/1538-7445.AM2013-2105

Targeting Mcl-1 for Multiple Myeloma (MM) Therapy: Drug-Induced Generation of Mcl-1 Fragment Mcl-1128–350 Triggers MM Cell Death Via c- Jun Upregulation

AbstractAbstract 3959Myeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic protein of the Bcl-2 family located on chromosome 1q21. Gains of 1q21 are unfavorable genetic prognostic factors for Multiple Myeloma (MM) patients. Our present data confirm significant overexpression of Mcl-1 gene in tumor versus plasma cells derived from healthy donors. Moreover, a significant link between Mcl-1 overexpression and poor prognosis was found in a data set of 590 MM patients, supporting a key role for Mcl-1 in MM survival and drug resistance. Mcl-1 is therefore a potential therapeutic target in MM. Strategies to inhibit Mcl-1 activity in MM and other malignancies may not only include induction of proteasome-dependent Mcl-1 degradation and modulation of Mcl-1 interaction with BH3-only proteins, but also induction of proteasome- independent Mcl-1 degradation. Indeed, our own and other previous data demonstrate that in contrast to full length Mcl-1 a caspase- induced 28kD Mcl-1 fragment, Mcl-1D128–350, inhibits MM cell proliferation and triggers apoptosis. We therefore performed a large-scale drug screen for agents which induce generation of Mcl-1D128–350 in MM cells. Our results show that bortezomib and staurosporine (STS) but not melphalan, doxorubicin or thalidomide induce generation of Mcl-1D128–350. We next sought to identify molecular sequelae downstream of Mcl-1D128–350 which mediate its anti-proliferative and pro-apoptotic effect in MM cells. Surprisingly, we observed nuclear accumulation of drug- induced Mcl-1D128–350, enhanced AP-1 reporter activity as well as elevated protein levels of c-Jun. Conversely, drug- induced c-Jun upregulation was blocked after introduction of point mutations (A127D, A157D) into the two highly conserved Mcl-1 caspase-cleavage sites Asp127 and Asp157. Gene profiles on MM cells transfected with either Mcl-1wt or Mcl-1D128–350 identified a group of differentially expressed genes associated with MM cell proliferation, survival and drug resistance. Consistent with these data, treatment with bortezomib or STS triggered c-Jun upregulation followed by apoptosis in Mcl-1wt/wt but not Mcl-1d/null murine embryonic fibroblasts (MEFs). Importantly, STS-induced c-Jun upregulation and cell death was restored in Mcl-1d/null MEFs transfected with a plasmid carrying Mcl-1wt. In ongoing studies we are analyzing genome sequencing data in MM cells for the existence of point mutations in the Mcl-1 caspase cleavage sites. The presence of such mutations could represent a potential new mechanism of drug resistance. Taken together, our data add another facet to the complex cellular functions of Mcl-1 in MM and derive a promising therapeutic and potential prognostic role for Mcl-1D128–350 in MM. Disclosures:No relevant conflicts of interest to declare.