Drug-resistant Lines Research Articles

Urupocidin C: a new marine guanidine alkaloid which selectively kills prostate cancer cells via mitochondria targeting

New bicyclic guanidine alkaloid, urupocidin C (Ur-C) along with the previously known urupocidin A (Ur-A) were isolated from the rare deep-sea marine sponge Monanchora pulchra, harvested in Northwestern Pacific waters. The unique structure of Ur-C was elucidated using 1D and 2D NMR spectroscopy as well as mass spectra. We discovered a promising selectivity of both alkaloids for human prostate cancer (PCa) cells, including highly drug-resistant lines, compared to non-malignant cells. In cancer cells, marine derived compounds were able to induce G1- and S-cell cycle arrest as well as caspase-mediated cell death. For the first time we have identified mitochondrial targeting as a central mechanism of anticancer action for these and similar molecules. Thus, treatment with the isolated alkaloids resulted in mitochondrial membrane permeabilization consequently leading to the release of cytotoxic mitochondrial proteins to cellular cytoplasm, ROS upregulation, consequent activation of caspase-9 and -3, followed by PARP cleavage, DNA fragmentation, and apoptosis. Moreover, synergistic effects were observed when Ur-A and Ur-C were combined with clinically approved PARP inhibitor olaparib. Finally, these alkaloids exhibited additive effects in combination with docetaxel and androgen receptor inhibitor enzalutamide, both applied in PCa therapy. In conclusion, urupocidin-like compounds are promising lead molecules for the development of new drugs for the treatment of advanced PCa.

Molecular mechanism(s) of resistance to vandetanib in medullary thyroid carcinoma.

e15628 Background: Medullary thyroid carcinoma (MTC) is a neuroendocrine tumor arising from parafollicular C cells of the thyroid. Pediatric cases of this cancer are associated with the diagnosis of multiple endocrine neoplasia, which is caused by a mutation in the rearranged during transfection ( RET) gene. Vandetanib, an oral receptor tyrosine kinase inhibitor, is approved for the treatment of patients with progressive MTC. While there is a response rate of 50%, the majority of patients eventually develop resistant disease. The goal of this work is to understand genetic and epigenetic underpinnings of sensitivity and resistance to vandetanib, and develop novel synergistic combination therapies in medullary thyroid carcinoma. Methods: The TT cell line (RET mutation p.C634W) was cultured in increasing concentrations of vandetanib in order to generate a vandetanib resistant cell line. Both vandetanib-sensitive and vandetanib-resistant lines were evaluated. Each line underwent exome sequencing, RNA sequencing, and methylation array analysis. In parallel we performed Genome-wide CRISPR knock-out and CRISPR activation of both cell lines using the TKO Version 3 Library, consisting of 71,090 gRNAs targeting 18,000 genes, and the Calabrese P65-HSF activation Library, using 56,762 guides for 18,000 genes. Results: Both whole exome and RNA sequencing demonstrate increased expression of RET C634W in both cell lines, to a significantly greater extent in the drug-resistant line than in the sensitive line. RNA sequencing demonstrates differential expression of transcripts between the vandetanib-sensitive and vandetanib-resistant cell lines, including multidrug-resistance 1 (which confers drug resistance in other cancers) and autotaxin (promotes cell survival). Genome-wide CRISPR knock-out showed enrichment of genes necessary for growth suppression by vandetanib, including MACROD2, GORASP2, and MAP3K genes. Gene set enrichment analysis of the CRISPR knock-out data showed enrichment of the proteasome pathway as a potential candidate of growth suppression by vandetanib, which was validated via exposure of the cell lines to bortezomib (IC50 = 3.62nM), a molecularly targeted proteasome-inhibitor. Conclusions: DNA mutations and epigenetic modification confer resistance of medullary thyroid carcinoma to tyrosine kinase inhibition. Adding further therapeutic agents to target these genetic alterations is a potential strategy for overcoming resistance.

Genomic stability at the coding regions of the multidrug transporter gene ABCB1: insights into the development of alternative drug resistance mechanisms in human leukemia cells.

Aim: Despite considerable efforts to reverse clinical multidrug resistance (MDR), targeting the predominant multidrug transporter ABCB1/P-glycoprotein (P-gp) using small molecule inhibitors has been unsuccessful, possibly due to the emergence of alternative drug resistance mechanisms. However, the non-specific P-gp inhibitor cyclosporine (CsA) showed significant clinical benefits in patients with acute myeloid leukemia (AML), which likely represents the only proof-of-principle clinical trial using several generations of MDR inhibitors. Nevertheless, the mutational mechanisms that may underlie unsuccessful MDR modulation by CsA are not elucidated because of the absence of CsA-relevant cellular models. In this study, our aims were to establish CsA-resistant leukemia models and to examine the presence or absence of ABCB1 exonic mutations in these models as well as in diverse types of human cancer samples including AMLs.Methods: Drug-resistant lines were established by stepwise drug co-selection and characterized by drug sensitivity assay, rhodamine-123 accumulation, [3H]-labeled drug export, ABCB1 cDNA sequencing, and RNase protection assay. The genomic stability of the ABCB1 coding regions was evaluated by exome sequencing analysis of variant allele frequencies in human populations. Moreover, the mutational spectrum of ABCB1 was further assessed in diverse types of cancer samples including AMLs in the Cancer Genome Atlas (TCGA) at the National Cancer Institute.Results: We report the development of two erythroleukemia variants, RVC and RDC, which were derived by stepwise co-selection of K562/R7 drug-resistant leukemia cells with the etoposide-CsA and doxorubicin-CsA drug combinations, respectively. Interestingly, both RVC and RDC cell lines, which retained P-gp expression, showed altered multidrug-resistant phenotypes that were resistant to CsA modulation. Strikingly, no mutations were found in the ABCB1 coding regions in these variant cells even under long-term stringent drug selection. Genomically, ABCB1 displayed relatively low variant allele frequencies in human populations when compared with several ABC superfamily members. Moreover, ABCB1 also exhibited a very low mutational frequency in AMLs compared with all types of human cancer. In addition, we found that CsA played a role in undermining the selection of highly drug-resistant cells via induction of low-level and unstable drug resistance.Conclusion: Our data indicate that ABCB1 coding regions are genomically stable and relatively resistant to drug-induced mutations. Non-ABCB1 mutational mechanisms are responsible for the drug-resistant phenotypes in both RVC and RDC cell lines, which are also prevalent in clinical AML patients. Accordingly, we propose several relevant models that account for the development of alternative drug resistance mechanisms in the absence of ABCB1 mutations.

Noninvasive assessment and therapeutic monitoring of drug-resistant colorectal cancer by MR molecular imaging of extradomain-B fibronectin.

Antineoplastic resistance represents a multifaceted challenge for cancer therapy and diagnostics. Extensive molecular heterogeneity, even within neoplasms of the same type, can elicit distinct outcomes of administering therapeutic pressures, frequently leading to the development of drug-resistant populations. Improved success of oncotherapies merits the exploration of precise molecular imaging technologies that can detect not only anatomical but also molecular changes in tumors and their microenvironment, early on in the treatment regimen. To this end, we developed magnetic resonance molecular imaging (MRMI) strategies to target the extracellular matrix oncoprotein, extradomain-B fibronectin (EDB-FN), for non-invasive assessment and therapeutic monitoring of drug-resistant colorectal cancer (CRC).Methods: Two drug-resistant CRC lines generated from parent DLD-1 and RKO cells by long-term treatment with 5ʹ-FU and 5ʹ-FU plus CB-839 respectively, were characterized for functional and gene expression changes using 3D culture, transwell invasion, qRT-PCR, and western blot assays. Contrast-enhanced MRMI of EDB-FN was performed in athymic nu/nu mice bearing subcutaneous tumor xenografts with 40 µmol/kg dose of macrocyclic ZD2-targeted contrast agent MT218 [ZD2-N3-Gd (HP-DO3A)] on a 3T MRS 3000 scanner. Immunohistochemistry was conducted on patient specimens and xenografts using anti-EDB-FN antibody G4.Results: Analyses of TCGA and GTEx databases revealed poor prognosis of colon cancer patients with higher levels of EDB-FN. Similarly, immunohistochemical staining of patient specimens showed increased EDB-FN expression in primary colon adenocarcinoma and hepatic metastases, but none in normal adjacent tissues. Drug-resistant DLD1-DR and RKO-DR cells were also found to demonstrate enhanced invasive potential and significantly elevated EDB-FN expression over their parent counterparts. MRMI of EDB-FN with 40 µmol/kg dose of MT218 (60% lower than the clinical dose) resulted in robust signal enhancement in the drug-resistant CRC xenografts with 84-120% increase in their contrast-to-noise ratios (CNRs) over the non-resistant counterparts. The feasibility of non-invasive therapeutic monitoring using MRMI of EDB-FN was also evaluated in drug-resistant DLD1-DR tumors treated with a pan-AKT inhibitor MK2206-HCl. The treated drug-resistant tumors failed to respond to therapy, which was accurately detected by MRMI with MT218, demonstrating higher signal enhancement and increased CNRs in the 4-week follow-up scans over the pre-treatment scans.Conclusions: EDB-FN is a promising molecular marker for assessing drug resistance. MRMI of EDB-FN with MT218 at a significantly reduced dose can facilitate effective non-invasive assessment and treatment response monitoring of drug-resistant CRC, highlighting its translational potential for active surveillance and management of CRC and other malignancies.

Drug-Resistant Tuberculosis and Lines of Mycobacterium Tuberculosis Circulating in Kinshasa

Drug-Resistant Tuberculosis and Lines of Mycobacterium Tuberculosis Circulating in Kinshasa

Abstract 2535: Enhanced gene expression of replication fork and other E2F targets genes is associated with sensitivity and, paradoxically, also with acquired drug resistance, to the Chk1 inhibitor prexasertib

Abstract Many cancers are under replicative stress (RS) arising from the combined influence of oncogenic drivers (i.e. Cyclin E, E2F, Myc), genomic instability and/or deficiencies in DNA Damage Repair (DDR). Tumors survive RS by upregulating checkpoints such as those driven by the ATR/CHK1 pathway. Dependence of tumors under high RS on the Chk1 pathway is currently being explored in the clinic with the Chk1 kinase inhibitor prexasertib. Preclinical and clinical studies have demonstrated evidence of efficacy in various cancers supporting the notion that this drug has the potential to improve the standard of care for patients with these malignancies. Two pivotal steps to ultimately fulfill the promise of a novel therapeutic in the clinic are, 1) to enable a patient selection strategy via predictive biomarkers of drug response or de novo resistance; 2) to gain insight into mechanisms underlying the development of acquired resistance, a common phenomenon of cancer therapeutics that contributes to non-durable response. Two experimental objectives to address these challenges for prexasertib were set, 1) to profile a large pan-cancer tumor cell line panel for prexasertib response and explore the association between drug response and baseline molecular profile via transcriptomic profiling, (2) to establish an in vitro tumor line with acquired resistance to prexasertib and characterize it via transcriptomic and targeted proteomic profiling to probe the mechanism of acquired resistance to prexasertib. Key observations from this study: A) pathways corresponding to E2F targets, G2M checkpoint and the Spindle Assembly Checkpoint (SAC), with a distinct enrichment for replication fork genes emerged across three different studies, (a) across a pan-cancer tumor line panel associated with prexasertib sensitivity, (b) associated with resistance in a tumor cell line with acquired resistance to prexasertib and (c) as top hits in an unbiased functional shRNA screen aimed at identifying genes that upon knockdown sensitize the resistant tumor cell line to prexasertib. B)knockdown of specific replication fork components in the drug-resistant tumor line was associated with marked sensitization to prexasertib and concomitant evidence of RS and DNA damage.To explain these paradoxical observations, we postulate that tumors may upregulate the expression of E2F target/G2M/SAC genes to resist oncogene-induced RS and that they may rely on similar genes to survive prexasertib-induced RS. Based on this data, transcriptomic profiling may prove of utility in the identification of patient tailoring biomarkers for prexasertib and as tool to unravel mechanisms of acquired resistance. Citation Format: Wayne D. Blosser, Jack A. Dempsey, Ann M. McNulty, Wenjuan Wu, Philip J. Ebert, Jason C. Ting, Yue W. Webster, Trent R. Stewart, Xueqian Gong, Farhana F. Merzoug, Sean Buchanan, Ricardo Martinez. Enhanced gene expression of replication fork and other E2F targets genes is associated with sensitivity and, paradoxically, also with acquired drug resistance, to the Chk1 inhibitor prexasertib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2535.

High-throughput RNA sequencing for screening the circular RNA expression profiling associated with chemo-resistance inosteosarcoma

Objective To screen the expression profile of circular RNA associated with chemo-resistance in osteosarcoma, and to analyze and identify its possible molecular functions. Methods Three pairs of matched drug-resistant and sensitive human osteosarcoma cell lines (MG63/DXR vs MG63, U2OS/DXR vs U2OS, KHOS/DXR vs KHOS) were first tested by CCK-8 assay for three commonly used osteosarcoma chemotherapy drugs (doxorubicin, cisplatin, and methotrexate); then, using next-generation high-throughput RNA sequencing technology, comparative analysis of circRNA expression was performed in three pairs of matched multidrug-resistant and sensitive human osteosarcoma cell lines; followed by real-time quantitative PCR (qRT-PCR) to confirm the reliability and accuracy of sequencing data in chemotherapy-resistant osteosarcoma cell lines and tissues. In addition, a variety of bioinformatics analyses, including GO, KEGG pathway analysis and the construction of circRNA-miRNA networks, were performed to predict potential molecular functions of differentially expressed circRNAs and to construct relevant regulatory pathways or networks. Results Three osteosarcoma-resistant cells (MG63/DXR, U2OS/DXR, KHOS/DXR) were significantly resistant to the three common osteosarcoma chemotherapy drugs compared with control cells (MG63, U2OS, KHOS), which laid a solid foundation for the further experiments. RNA sequencing revealed a total of 80 circRNAs differentially expressed between the two groups. Compared with drug-sensitive osteosarcoma cells, 57 circRNAs were upregulated and 23 circRNAs weredownregulated in the drug-resistant osteosarcoma cell lines (fold change≥ 2 or ≤0.5, P<0.05). Tenrandomly selected circRNAs were verified by qRT-PCR and the results showed that 9 of the 10 circRNAswere consistent with the sequencing results. In addition, KEGG pathway analysis showed that 56 pathways were significantly enriched in differentially expressed circRNAs, including Glycolysis/gluconeogenesis, ABC transporter, VEGF signaling pathway, and so on. Moreover, the differently expressedcircRNA-hsa_circ_0004674 with the highest fold change was highly expressedin the chemotherapy-resistant osteosarcoma cells and tissues, associated with poor prognosis in osteosarcoma patients. Some potential endogenous competitive RNA (ceRNA) regulatory pathways associated with hsa_circ_0004674, such as hsa_circ_0004674-miR-490-3p-ABCC2 and hsa_circ_0004674- miR-1254- EGFR, were constructed by use of the authoritative databases (target scan and miRanda) and literature searching and the miRNA response element sequences (MREs) between miRNAs that have a potential ceRNA regulatory relationship with hsa_circ_0004674 were alsopredicted. Conclusion CircRNA is closely related to tumor progression and may play a role in osteosarcoma chemo-resistance. Besides, hsa_circ_0004674 may be a potential candidate for reversing drug resistance of osteosarcoma. Key words: Sarcoma; RNA; Drug resistance, neoplasm; MicroRNAs; High-throughput screening assays

STAT3 signaling pathway in drug-resistant bladder cancer cell line.

STAT3 signaling pathway is related to the proliferation, apoptosis and metastasis of tumor cells. The relationship between STAT3 and drug resistance is still unknown. We studied the inhibitors in STAT3 pathway and its downstream molecules to analyze the unique effects in drug-resistant bladder cancer cells. qRT-PCR and Western blot were implemented to study the expression level of JAK2, STAT3, p-STAT3, MMP2 and Cyclin D1 in Pumc-91 and Pumc-91/ADM cell lines, respectively. The effects of AG490 on the expression of STAT3, p-STAT3, MMP2 and Cyclin D1 in Pumc-91 were evaluated using qRT-PCR and Western blot. Pumc-91/ADM cells were treated with AG490. CCK-8 and wound healing assay were used to detect the cell proliferation and metastasis. Compared to Pumc-91, an obvious decrease of JAK2, p-STAT3 and increase of MMP2 were shown in Pumc-91/ADM cell line. After inhibition of STAT3 signaling pathway, the mRNA and protein levels of STAT3, p-STAT3, MMP2 and Cyclin D1 obviously decreased in the test group. The proliferation and migration of Pumc-91/ADM were suppressed by inhibiting of STAT3. STAT3 pathway regulated the proliferation and migration of bladder cancer drug-resistant cells by modulating the expression of Cyclin D1 and MMP2.

Resistance of Leishmania (Viannia) Panamensis to Meglumine Antimoniate or Miltefosine Modulates Neutrophil Effector Functions.

Leishmania (Viannia) panamensis (L. (V.) p.) is the main causative agent of cutaneous leishmaniasis in Colombia and is usually treated with either meglumine antimoniate (MA) or miltefosine (MIL). In recent years, there has been increasing evidence of the emergence of drug-resistance against these compounds. Neutrophils are known to play an important role in immunity against Leishmania. These cells are rapidly recruited upon infection and are also present in chronic lesions. However, their involvement in the outcome of infection with drug-resistant Leishmania has not been examined. In this study, human and murine neutrophils were infected in vitro with MA or MIL drug-resistant L. (V.) p. lines derived from a parental L. (V.) p. drug-susceptible strain. Neutrophil effector functions were assessed analyzing the production of reactive oxygen species (ROS), the formation of neutrophil extracellular trap (NET) and the expression of cell surface activation markers. Parasite killing by neutrophils was assessed using L. (V.) p. transfected with a luciferase reporter. We show here that MA and MIL-resistant L. (V.) p. lines elicited significantly increased NET formation and MA-resistant L. (V.) p. induced significantly increased ROS production in both murine and human neutrophils, compared to infections with the parental MIL and MA susceptible strain. Furthermore, neutrophils exposed to drug-resistant lines showed increased activation, as revealed by decreased expression of CD62L and increased expression of CD66b in human neutrophils yet presented higher survival within neutrophils than the drug-susceptible strain. These results provide evidence that parasite drug-susceptibility may influences neutrophil activation and function as well as parasite survival within neutrophils. Further investigaton of the inter-relationship of drug susceptibility and neutrophil effector function should contribute to better understanding of the factors involved in susceptibility to anti-Leishmania drugs.

Ewing sarcoma resistance to SP-2509 is not mediated through KDM1A/LSD1 mutation.

Ewing sarcoma is the second most common solid bone malignancy diagnosed in pediatric and young adolescent populations. Despite global co-operative efforts, outcomes for patients with relapsed and refractory disease remains obstinately poor. It has become increasingly clear that disruption of the epigenome as a result of alterations in epigenetic regulators, plays a pivotal role in tumorigenesis. As such, this study investigated Ewing sarcoma mechanisms of acquired resistance to the small molecule reversible lysine specific demethylase (LSD1/KDM1A) inhibitor SP-2509. Surprisingly, whole exome sequencing analysis of our generated A673 SP-2509 drug resistant cell line revealed an absence of mutations in KDM1A. Compared to parental counterparts, SP-2509 drug resistant cells demonstrated decreased anchorage independent growth capacity, enhanced sensitivity to the HDAC inhibitors vorinostat/entinostat and a distinct transcriptional profile that was enriched for extracellular matrix proteins. SP-2509 drug resistant cells also exhibited elevated expression levels of the multi-drug resistance genes ABCB1, ABCC3, and ABBC5 and decreased expression of the transcriptional repressor RCOR1/CoREST. Following several months of SP-2509 withdrawal, low level SP-2509 resistance was still apparent (6.3 fold increase in IC50), with drug resistant cell populations maintaining their distinct transcriptional profile. Furthermore, compared to parental cells, washout drug resistant lines displayed equal sensitivity to the standard Ewing sarcoma chemotherapeutic agent's vincristine and doxorubicin. Together these findings indicate that resistance to SP-2509 is not fully reversible or driven by direct mutation in KDM1A.

Cytotoxicity of abietane diterpenoids from Salvia multicaulis towards multidrug-resistant cancer cells

Diterpenoids salvimulticanol (1) and salvimulticaoic acid (2) together with known diterpenoid (3–6) were isolated from Salvia multicaulis. Structures were elucidated by spectroscopic techniques including HRESIMS as well as 1D-, and 2D-NMR. In-vitro cytotoxicity was assayed against human cancer cell lines. As several metabolites exhibited activity against drug-resistance lines, compounds were screened against a panel of human drug-sensitive and multidrug-resistant cancer lines. A proposed biosynthetic pathway for these new diterpenoids (1–2) as well as the cytotoxic structure-activity relationship of all identified compounds were discussed. Compound 1 and 6 showed the most potent cytotoxicity with IC50 11.58 and 4.13 towards leukemia cell lines CCRF-CEM and CEM-ADR5000, respectively.

PO-492 Establishment of new drug-resistant gastric cancer cell lines

IntroductionGastric cancer (GC) is an important public health problem in Chile, because constitutes the first cause of death for cancer in man and the third in women. Most GC patients are diagnosed at advanced stages where surgery is not effective. In these cases, the GC treatment is mainly based in chemotherapy with Cisplatin (CDDP) or 5-fluorouracil (5-FU). However, tumour cells usually develop chemotherapy resistance, increasing the rate of recurrence. The establishment of drug resistant cell lines could serve as an initial screen for agents that might modulate drug resistance in gastric cancer.Material and methodsTwo gastric cancer cell lines AGS and MKN-28 were treated with incremental doses of cisplatin or 5-FU to generate resistant variants (continuous growth method with increasing dose). The drug sensitivity of parental and resistant cells was determined by dose-dependent cytotoxicity curve using standard MTT assay. The inhibitory concentration 50% (IC50) values were calculated by non-linear regression test using GraphPad PRISM 5.0 software. The resistance index (RI) was determined as the ratio of the IC50 of the resistant cell line to the IC50 of parental cell line. Relative expression of resistance marker genes to CDDP or 5-FU was determined by qRT-PCR using 2-(ddCt) method. U-Mann Whitney test was used to compare groups (at statistical significance level of p<0.05). Changes in cell morphology were monitored continuously during the development of resistance clones using an inverted phase contrast microscope. The stabilisation of drug resistance of cell lines was tested after two months in drug-free medium.Results and discussionsAfter 10 months of treatment, AGS resistant to CDDP, MKN-28 resistant to CDDP and AGS resistant to 5-FU exhibited an increase of 3.9, 2.6 and 3.4 fold of resistance, respectively. Resistance marker genes for CDDP (ABCC2 and CTR1) and 5-FU (TYMS) were differentially expressed in resistant cells compared to their parental cells. Changes in cell morphology were observed in resistant cells compared to their parental cells. The resistant phenotype was very stable and the values of IC50 and RI had no significant change after 2 months in drug-free medium.ConclusionThe three drug-resistant lines selected by continuous growth method with increasing dose may serve as appropriate models for the study of mechanisms of drug resistance in GC. Further studies are necessary in order to identify the genes involved in the resistant-phenotype which could help to find new targets for GC therapy.

Differential protein expression and post-translational modifications in metronidazole-resistant Giardia duodenalis.

BackgroundMetronidazole (Mtz) is the frontline drug treatment for multiple anaerobic pathogens, including the gastrointestinal protist, Giardia duodenalis. However, treatment failure is common and linked to in vivo drug resistance. In Giardia, in vitro drug-resistant lines allow controlled experimental interrogation of resistance mechanisms in isogenic cultures. However, resistance-associated changes are inconsistent between lines, phenotypic data are incomplete, and resistance is rarely genetically fixed, highlighted by reversion to sensitivity after drug selection ceases or via passage through the life cycle. Comprehensive quantitative approaches are required to resolve isolate variability, fully define Mtz resistance phenotypes, and explore the role of post-translational modifications therein.FindingsWe performed quantitative proteomics to describe differentially expressed proteins in 3 seminal Mtz-resistant lines compared to their isogenic, Mtz-susceptible, parental line. We also probed changes in post-translational modifications including protein acetylation, methylation, ubiquitination, and phosphorylation via immunoblotting. We quantified more than 1,000 proteins in each genotype, recording substantial genotypic variation in differentially expressed proteins between isotypes. Our data confirm substantial changes in the antioxidant network, glycolysis, and electron transport and indicate links between protein acetylation and Mtz resistance, including cross-resistance to deacetylase inhibitor trichostatin A in Mtz-resistant lines. Finally, we performed the first controlled, longitudinal study of Mtz resistance stability, monitoring lines after cessation of drug selection, revealing isolate-dependent phenotypic plasticity.ConclusionsOur data demonstrate understanding that Mtz resistance must be broadened to post-transcriptional and post-translational responses and that Mtz resistance is polygenic, driven by isolate-dependent variation, and is correlated with changes in protein acetylation networks.

Racemic vs. enantiopure inert Ti(iv) complex of a single diaminotetrakis(phenolato) ligand in anticancer activity toward human drug-sensitive and -resistant cancer cell lines.

A tetrakis(phenolato) Ti(iv) complex was synthesized in racemic and optically pure form, exhibiting high hydrolytic stability, and similar cytotoxicity for all stereochemical forms on HT-29 and A2780 cancer cells. Higher activity of the racemate on drug-resistant A2780cp and A2780adr lines implies a beneficial activity of both enantiomers rendering enantiomeric resolution unnecessary.

Streamlined Total Synthesis of Trioxacarcins and Its Application to the Design, Synthesis, and Biological Evaluation of Analogues Thereof. Discovery of Simpler Designed and Potent Trioxacarcin Analogues.

A streamlined total synthesis of the naturally occurring antitumor agents trioxacarcins is described, along with its application to the construction of a series of designed analogues of these complex natural products. Biological evaluation of the synthesized compounds revealed a number of highly potent, and yet structurally simpler, compounds that are effective against certain cancer cell lines, including a drug-resistant line. A novel one-step synthesis of anthraquinones and chloro anthraquinones from simple ketone precursors and phenylselenyl chloride is also described. The reported work, featuring novel chemistry and cascade reactions, has potential applications in cancer therapy, including targeted approaches as in antibody-drug conjugates.

Transcriptome profiling identifies genes/pathways associated with experimental resistance to paromomycin in Leishmania donovani

Widespread resistance towards antimony and reports of relapses following miltefosine treatment has severely affected the management of visceral leishmaniasis (VL) in the Indian subcontinent. Paromomycin (PMM), an aminoglycoside antibiotic, has been licensed for VL treatment in India in 2007. Although its use is still restricted in the field, unraveling the molecular mechanism of resistance towards PMM is the key to preserve the drug. In this study, PMM resistant lines were selected up to 100 μM of PMM in three distinct field isolates of Leishmania donovani at promastigote stage. The resistance induced at promastigote level was also evident in amastigotes which showed 6 fold decreases in PMM susceptibility. Comparative transcriptome profiling of PMM resistant (PMM-R) and the corresponding PMM sensitive (PMM-S) parasites revealed modulated expression of 500 genes (1.5 fold cut off) in PMM-R parasites. Selected genes were validated for their modulated expression by quantitative real-time PCR. Functional classification and pathway analysis of modulated genes indicated probable adaptations in drug resistant lines which included a) reduced oxidative phosphorylation; b) increased glycosomal succinate fermentation and substrate level phosphorylation; c) dependency on lipids and amino acids for energy generation; d) reduced DNA synthesis and increased DNA damage repair and e) decreased protein synthesis and degradation. Interestingly, PMM-R parasites showed a marked increase in PMM susceptibility in presence of verapamil and amlodipine, antagonists of Ca2+ channel that are also modulators of ABC transporters. Moreover, infection of macrophages by PMM-R parasites led to modulated nitric oxide (NO) levels while reactive oxygen species (ROS) level remained unaltered. The present study highlights the putative mechanisms of PMM resistance in Leishmania.

Abstract 16: Evaluation of the efficacy of clotrimazole treatment in human melanoma cell lines that overexpress the multidrug resistance-associated protein (MRP) and the lung resistance-related protein (LRP)

Abstract Drug resistance to chemotherapy, mediated in part by overexpression of P-glycoprotein (MDR-1), multidrug resistance-associated protein (MRP), or lung resistance-realted protein (LRP), continues to be a major problem in the treatment of melanoma. Because we previously demonstrated the ability of clotrimazole to enhance cytotoxicity in human metastatic melanoma cells, here we evaluated the ability of clotrimazole to treat drug-resistant human melanoma cells that overexpress MRP and/or LRP. We utilized human melanoma cell lines that overexpress either MDR or LRP, and human melanoma cell lines that overexpress both. Cell growth and cell death were analyzed by flow cytometry, proliferation assays, and immunoblotting after drug treatments and RNA interference. Clotrimazole caused significant decreases in proliferation and increases in cell death in both drug-sensitive melanoma lines and those overexpressing MRP and LRP. No extensive cell death was induced by clotrimazole treatment in noncancerous human skin cell lines. These results indicate that clotrimazole selectively induces cytotoxicity in both drug-sensitive and drug-resistant melanoma lines. As clotrimazole is known to inhibit various transient receptor potential (TRP) ion channels, we pretreated these melanoma lines with TRP inhibitors or RNAi. Inhibitors of the TRP melastatin-8 channel (TRPM8) or TRP vanilloid-1 (TRPV1) failed to produce comparable levels of cell death caused by clotrimazole. However, RNAi silencing of the TRPM2 cation channel caused significant levels of cell death in both drug-sensitive and drug-resistant melanoma lines. These results indicated that inhibition of TRPM2 channels may have a primary role in the ability of clotrimazole to treat drug-sensitive and drug-resistant melanoma lines. In conclusion, this study demonstrated that clotrimazole selectively increases cell death in drug-resistant melanoma lines, with minimal deleterious effects in normal skin cells. Taken together, we conclude that clotrimazole has in vitro efficacy toward the treatment of drug-resistant human melanoma cells. Thus, these preliminary studies indicate that clotrimazole has the potential to successfully treat drug resistant melanoma in the future. Citation Format: Steven D. Blake, Shelby G. McKamey, Christopher M. Tweed, David W. Koh. Evaluation of the efficacy of clotrimazole treatment in human melanoma cell lines that overexpress the multidrug resistance-associated protein (MRP) and the lung resistance-related protein (LRP) [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 16. doi:10.1158/1538-7445.AM2017-16

Determining the LC50 of a Novel MetAP Inhibitor in Lung Carcinoma Cells

The rise of multi‐drug resistant bacterial infections grows at an alarming rate, thus necessitating the discovery of new antibiotics and antimicrobials. Dr. Omonike Olaleye, a colleague from Texas State University, discovered 75 compound inhibitors against Methionine aminopeptidase (MetAP) after a high‐throughput screen. MetAP is a universally conserved, essential protein that removes the initial methionine from a newly synthesized polypeptide, which allows for further modification to form a completed protein; studies have indicated that MetAP deletion is lethal. Interestingly, MetAP is conserved across species, and prokaryotic and eukaryotic MetAP are dissimilar enough to make MetAP an attractive potential therapeutic target. This project is currently focused on a compound (UST‐001) efficacious in inhibiting Mycobacterium tuberculosis; however, the effect on mammalian cells is unknown. Relative toxicities must be determined before further tests can be conducted. We determined the in‐vitro LC50 of UST‐001 on a human lung carcinoma (H1299) line to be 3.2μM by using a combination of an MTT assay and flow cytometry. However, because the H1299s are a cancerous and thus drug resistant line, the experimental calculated value may not be representative of the MetAP inhibitor's toxicity in a non‐cancerous cell line. We plan to overcome this limitation in the future by using a primary lung cell line, and to compare the relative toxicities between both cell lines. The data gathered from this project will facilitate further research in determining whether or not this UST‐001 will be a viable clinical candidate.

Zoledronic acid reverses cisplatin resistance in nasopharyngeal carcinoma cells by activating the mitochondrial apoptotic pathway.

Despite improvements to radiotherapeutic strategies, resistance to adjuvant chemotherapy remains the main problem underlying the low 5-year survival rate in patients with nasopharyngeal carcinoma (NPC). In the present study, the human NPC cell line HNE1 was exposed to gradually increasing concentrations of cisplatin (CDDP) in order to establish a drug-resistant sub-cell line, HNE1/CDDP. HNE1/CDDP cells exhibited multidrug resistance and a prolonged doubling time, as compared with the parent HNE1 cells. Furthermore, pretreatment with zoledronic acid (ZOL) appeared to resensitize the CDDP-resistant cells by inducing S-phase cell cycle arrest and the mitochondrial apoptotic pathway by upregulating the expression of B-cell lymphoma-2 (BCL-2)-associated X protein and caspase-9 and downregulating the expression of BCL-2. The results of the present study suggested that HNE1/CDDP cells are a stable, multidrug-resistant NPC cell line that may serve as an important tool for research in drug resistance. In addition, the application of ZOL may hold clinical therapeutic potential for the treatment of drug resistance in NPC.

Extrinsic Factors in the In Vivo Macroenvironment Generate Phenotypic Resistance to BTK/Bcl-2 Targeted Therapies in Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma

Ibrutinib (IBR), an inhibitor of Bruton's Tyrosine Kinase (BTK), has been FDA approved for Chronic Lymphocytic Leukemia (CLL) and Mantle Cell Lymphoma (MCL). However, IBR responses are incomplete in most cases, and of short duration for MCL and higher-risk CLL subtypes. We hypothesize that intrinsic resistance, incomplete responses, and rapid recurrence can be due to adaptive signaling that should be co-targeted with BTK inhibition to achieve deeper and more durable responses. We previously showed that venetoclax (VEN), an inhibitor of Bcl-2, generated synergistic cytotoxicity with IBR in MCL lines as well as circulating leukemic B cells in 13/19 CLL and 4/5 MCL patient samples treated ex vivo (Jayappa KD et al. ASH 2015; Portell CA et al. ASH 2014). However, the sensitivity to VEN or IBR+VEN was highly variable among patient samples, and did not correlate with the diagnostic genetic lesions in the CLL/MCL cells or clinical characteristics of the patients. Here, we show that resistance to IBR+VEN can be induced by interaction with soluble factors found in the in vivo "macroenvironment" of the circulating cancer cells.To gain insight into changes in signaling pathways that might underlie mechanisms of drug synergy and resistance, we analyzed drug resistant MCL lines treated with IBR, VEN, and the combination by Reverse Phase Protein Arrays. We observed downregulation of PI3K-Akt, MAPK, JAK-STAT, and NOTCH signaling after IBR and IBR+VEN treatment, cleavage of caspases and PARP after VEN-treatment, and greatly enhanced cleavage of caspases and PARP after IBR+VEN treatment. A notable exception was significantly increased phosphorylation on p65 S536 of the NF-kB pathway at longer times after IBR+VEN treatment, indicating a possible role of NF-kB signalling in generating resistance to IBR and VEN in cells that survived treatment.To determine whether extrinsic factors in the cancer cell environment might be able to induce a drug-resistant phenotype, we co-cultured or pre-incubated PBMC with a diverse panel of stromal cells, cytokines, and other agonists. We found that the combination of soluble CD40L, IL-10, and CpG DNA (agonist mix) generated nearly complete resistance to IBR+VEN in CLL and MCL patient samples, with CpG DNA being the most effective single agent. Every sample treated with agonist mix displayed increased resistance to IBR+VEN drug combination, suggesting that responsiveness transcends genetic heterogeneity and reflects the developmental lineage of the cancer cells.We investigated whether the extrinsic agonists induce drug resistance by activating the NF-κB pathway, by analyzing nuclear localization of NF-kB transcription factors RelA and RelB using ImageStream and cell fractionation. We observed robust activation of alternative-NF-kB signaling in primary CLL and MCL cells treated with agonist mix, with little effect on canonical NF-κB. PKC, MAPK and PI3K-Akt signaling also showed evidence of activation by agonist mix. Agonist mix treatment also caused significant overexpression of anti-apoptotic proteins Mcl-1, Bcl-xL, and survivin, but not Bcl-2. Inhibitors of NF-κB blocked RelB translocation and overexpression of Mcl-1, Bcl-xL and survivin. Inhibitors of NF-kB or of upregulated anti-apoptotic proteins overcame drug resistance induced by agonist mix. Inhibitors of the other activated pathways (MAPK, PI3K-Akt, PKC) did not block agonist-induced drug resistance at pharmacologically relevant concentrations.To determine whether extra-nodal agonists in the blood of patients could generate resistance to IBR and VEN, we analyzed drug cytotoxicity in CLL patient PBMCs cultured with autologous plasma. We found that autologous plasma was able to induce increased resistance to IBR+VEN in 3/7 CLL samples and this resistance was blocked by treatment with an NF-kB inhibitor.In conclusion, soluble agonists in the patient macroenvironment of circulating CLL/MCL cells can generate resistance to IBR+VEN by activating alternative-NF-kB signaling and over-expression of multiple anti-apoptotic proteins. Inhibitors of NF-kB or of the upregulated anti-apoptotic proteins overcame IBR+VEN resistance generated by these extrinsic factors. We suggest that survival signals generated by extra-nodal agonists in the patient macroenvironment generate drug resistance in CLL and MCL, and that improved responses could occur with interventions that block these survival signals. DisclosuresPortell:AbbVie: Research Funding; Roche/Genentech: Research Funding; Infinity: Research Funding; Acerta: Research Funding. Wulfkuhle:Theranostics Health, LLC: Equity Ownership. Petricoin:Perthera, Inc.: Consultancy, Equity Ownership; Ceres Nanosciences, Inc.: Consultancy, Equity Ownership, Patents & Royalties; Avant Diagnostics, Inc.: Equity Ownership. Williams:University of Virginia: Employment; Janssen and Pharmacyclics: Research Funding.