Drug-sensitive Cell Lines Research Articles

The DNA methylation-regulated MCTP1 activates the drug-resistance of esophageal cancer cells.

Accumulating studies have demonstrated that drug-resistance remains a great obstacle for the effective treatment of cancers. Esophageal cancer is still one of the most common cancers worldwide, which also suffers from the drug-resistance during clinical treatment. Here we performed drug-resistance profiling assays and identified several drug-resistant and drug-sensitive esophageal cancer cell lines. The following methylation sequencing showed that the MCTP1 gene is hypermethylated in the drug-resistant esophageal cancer cells. As a result, the expression of MCTP1 is down-regulated in the drug-resistant esophageal cancer cells. Down-regulation of MCTP1 also affects the migration and apoptosis of esophageal cancer cells, as revealed by the wound-healing and apoptosis assays. Further investigations proposed two signaling pathways that might involve in the MCTP1-mediated drug-resistance of esophageal cancer cells. All these results suggested that MCTP1 activates the drug-resistance of esophageal cancer cells, which has implications for further design of new biomarker of esophageal cancer treatment.

Indirubin-3’-Monoxime Acts as a Novel Proteasome Inhibitor: Therapeutic Application in Multiple Myeloma

Multiple myeloma (MM) is still an incurable malignancy of plasma cells. Proteasome inhibitors (PIs) work as the backbone agent and have greatly improved the outcome in majority of newly diagnosed patients with myeloma. However, drug resistance remains the major obstacle causing treatment failure in clinical practice. Indirubin-3’-monoxime (Id-3) is one of the derivatives of Indirubin, a traditional Chinese medicine that has reported to be effective in the treatment of chronic myeloid leukemia and some solid tumors. In this study, we demonstrated the anti-MM activity of Id-3 in both drug sensitive and bortezomib-resistance human MM cell lines and patient-derived cells. Moreover, combination with Id-3 could sensitize MM cells to bortezomib-induced apoptosis. Mechanistically, Id-3 acts as a multifaceted regulator of cell death, which induces DNA damage and cell cycle arrest as well as abrogates NF-κB activation in MM cells. Id-3 efficiently down-regulates USP7 expression, resulting in destabilization of NEK2 and suppression of NF-κB signaling. Importantly, proteasome complex subunits PSME3 (PA28γ) and PSME4 (PA200) were down-regulated in MM cells treated with Id-3. Knockdown of PSME3 or PSME4 by shRNA caused proteasome inhibition and paraprotein accumulation, as well as sensitized MM cells to bortezomib-mediated growth arrest. Clinical data analysis demonstrated that PSME3 and PSME4 are over-expressed in relapsed/refractory MM and associated with inferior overall survival. Taken together, our study indicate that Id-3 is a novel agent triggering proteasome inhibition and represents a promising therapeutic strategy to improve patient outcome in MM. Funding: This work was supported by the Natural Science Foundation of China (8217011925, 81570181, 81920108006), the Non-profit Central Research Institute Fund of the Chinese Academy of Medical Sciences (2018PT31006, 2018RC320012), the Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Sciences CAMS- 2017-I2M-1-005, 2016-I2M-3-023). National Program on Key Basic Research Project 2018ZX09733003. Declaration of Interest: Dr. Kenneth. C. Anderson is the consultant: Pfizer, Amgen, Astrazeneca, Janssen, Precision Biosciences, Mana and Window. Founder/Stock Shareholder: C4 Therapeutics, Oncopep, Raqia and NextRNA. Other authors declared no competing financial interests in relation to the work described. Ethical Approval: The study was approved by the Medical Ethical Committee of the Institute of Hematology & Blood Diseases Hospital, State Key Laboratory of Experimental Hematology, Chinese Academy of Medical Science & Peking Union Medical College, Tianjin, China.

Identification of Tamoxifen-Resistant Breast Cancer Cell Lines and Drug Response Signature.

Breast cancer cell lines are frequently used to elucidate the molecular mechanisms of the disease. However, a large proportion of cell lines are affected by problems such as mislabeling and cross-contamination. Therefore, it is of great clinical significance to select optimal breast cancer cell lines models. Using tamoxifen survival-related genes from breast cancer tissues as the gold standard, we selected the optimal cell line model to represent the characteristics of clinical tissue samples. Moreover, using relative expression orderings of gene pairs, we developed a gene pair signature that could predict tamoxifen therapy outcomes. Based on 235 consistently identified survival-related genes from datasets GSE17705 and GSE6532, we found that only the differentially expressed genes (DEGs) from the cell line dataset GSE26459 were significantly reproducible in tissue samples (binomial test, p = 2.13E-07). Finally, using the consistent DEGs from cell line dataset GSE26459 and tissue samples, we used the transcriptional qualitative feature to develop a two-gene pair (TOP2A, SLC7A5; NMU, PDSS1) for predicting clinical tamoxifen resistance in the training data (logrank p = 1.98E-07); this signature was verified using an independent dataset (logrank p = 0.009909). Our results indicate that the cell line model from dataset GSE26459 provides a good representation of the characteristics of clinical tissue samples; thus, it will be a good choice for the selection of drug-resistant and drug-sensitive breast cancer cell lines in the future. Moreover, our signature could predict tamoxifen treatment outcomes in breast cancer patients.

BSA-Stabilized Mesoporous Organosilica Nanoparticles Reversed Chemotherapy Resistance of Anaplastic Thyroid Cancer by Increasing Drug Uptake and Reducing Cellular Efflux

Anaplastic thyroid cancer (ATC) is a highly aggressive and the most lethal type of thyroid cancer. The standard-of-care for unresectable ATC is radiotherapy and chemotherapy, usually based on doxorubicin (Dox). However, most patients develop resistance shortly after treatment. To overcome the drug resistance, we synthesized the mesoporous organosilica nanoparticles (MONPs) loaded with Dox and stabilized the nanocomposites by bovine serum albumin (BSA). The surface area and pore volume of MONPs were 612.653 m2/g and 0.589 cm3/g. The loading capacity of Dox-MONPs reached 47.02%. Compared to Dox-MONPs and free Dox, BSA-Dox-MONPs had more durable tumor-killing power on both drug-sensitive cell line HTh74 and drug-resistant cell line HTh74R. The cellular uptake of BSA-Dox-MONPs was 28.14 and 65.53% higher than that of Dox-MONP in HTh74 and HTh74R. Furthermore, the BSA coating decreased the efflux rate of nanocomposites in HTh74 (from 38.95 to 33.05%) and HTh74R (from 43.03 to 32.07%). In summary, BSA-Dox-MONPs reversed the chemotherapy resistance of ATC cells via increased drug uptake and inhibited drug efflux, offering a promising platform for the treatment of chemo-resistant ATC.

Phytochemical characterization and biological activities of green tea (Camellia sinensis) produced in the Azores, Portugal

BackgroundGreen tea is not only one of the most widely consumed beverages worldwide, but is also known for its health promoting and therapeutic effects. Green tea is cultivated in areas with high humidity and acidic soils in China, Indonesia and Japan. Those places have well-marked dry and rainy seasons. In opposite, Azores have a climate with constant average annual rainfall and, unlike eastern regions, relatively constant air humidity throughout the year. While a brand implemented on the Portuguese market, the quality of green tea produced in Azores must be guaranteed. Quality control measures based on phytochemical determination of the chemical composition and biological activities are needed in order to address whenever climate changes interferes significantly with composition and biological effects. PurposeMake the phytochemical characterization of various extracts of green tea leaves coming from Azores and evaluate the anti-cancer activities of the extracts in order to compare the obtained results with those of teas coming from eastern regions. MethodsPhytochemical characterization (catechins, oxyaromatic acids, flavonols, alkaloids and theanine) and total catechins contents (TCC) was performed by using HPLC-DAD analysis, in infusions (5–7 min and 30 min), maceration and methanolic extracts of Camellia sinensis samples coming from Azores, Portugal. The antioxidant activity of extracts was measured by the DPPH assay and the total phenolics contents (TPC) were estimated using the Folin-Ciocalteu colorimetric method. The cytotoxic activity towards drug sensitive and multidrug-resistant leukemia cell lines was determined by the resazurin assay. ResultsThe TCC was higher in methanolic extracts and lower in maceration, as epigallocatechin gallate (EGCG) and epicatechin gallate (ECG) concentrations were significantly higher in methanolic extracts and were only residual in maceration extracts. Maceration extracts showed the highest content of gallic acid, indicating that methanol extracts contained more flavonols of higher molecular weight and/or that maceration may lead to the degalloylation of catechins. The amount of o-caffeoylquinic acid extracted was significantly higher in methanolic samples. Short-term extraction at high temperatures resulted in high amounts of neochlorogenic acid. The contents of glycosylated quercitin-3-d-galactoside and kaempferol-3-glucoside were small in maceration samples and high in methanolic samples. Caffeine was easily extracted by methanol (99%) compared with water, while extraction of the amino-acid l-theanine was unattainable with methanol. TPC values correlated linearly with DPPH•IC50, with infusion samples showing the best antioxidant capacities. The aqueous and the methanol/water extracts were active in multidrug-resistant and drug sensitive cancer cells. ConclusionDespite these climatic differences, the phytochemical characterization revealed that tea coming from the Azores compares well with those from China and Japan. In addition, tea can be produced without fungicides and pesticides to control pests that appear during rainy seasons. This allows the production of high-quality bioorganic tea. Aqueous and methanol/water extracts of the Azorean tea showed to be useful targeting sensitive and drug-resistant tumor cells.

A Novel Aryl-Guanidinium Based Compound Targets the STAT3 Signalling Pathway, Downregulates MCL-1 Expression and Induces Anti-Myeloma Activity

Multiple myeloma (MM) is a hematological malignancy characterized by the proliferation of malignant plasma cells in the bone marrow microenvironment. Increased understanding of the pathophysiology of MM and advances in therapeutics have led to an increased survival of patients in the past decade. However, despite the effectiveness of the first-line treatments, patients invariably relapse and become drug refractory due to genetic heterogeneity and induction of prosurvival signalling cascades in the bone marrow microenvironment. There is, therefore, a need for novel therapeutics which target key prosurvival signalling pathways in MM. The Janus kinase (JAK)/Signal transducer and activator of transcription 3 (STAT3) pathway in MM can be both constitutively active due to autonomous mutations or activated by growth and survival factors, such as interleukin-6 (IL-6), secreted by bone marrow stromal cells and other cells within the bone marrow microenvironment. STAT3 is upregulated in MM and increases the expression of genes involved proliferation, angiogenesis and evasion of apoptosis. STAT3 target genes include prosurvival BCL2 family members, such as MCL-1 which is overexpressed in MM, leading to apoptosis resistance and is associated with shorter patient survival. Several studies have shown promise in targeting prosurvival BCL2 family members with BCL2 homology domain 3 (BH3) mimetics in MM. We have previously described a member of a series of guanidinium based compounds which inhibits BRAF, a serine/threonine kinase component of the RAS/ERK signalling pathway, through a putative type-III allosteric mechanism [Diez-Cecilia et al, (2015) Bioorg. Med. Chem. Lett. 25, 4287-4292]. Here, we describe a new derivative of this series, VP79s, which exhibits cytotoxicity in MM cell lines with IC50 values in the low micromolar range; however, VP79s does not inhibit RAS/ERK signalling suggesting a novel mechanism of action. VP79s was found to induce apoptosis in NCI-H929 and U266B1 MM cell lines in a dose- and time-dependent manner. VP79s was more potent in inducing cell death in a panel of drug sensitive and drug resistant MM cell lines compared to standard and emerging therapeutic agents for MM. VP79s rapidly inhibited both constitutively active and IL-6-induced STAT3 signalling in MM cells. Examination of upstream mediators of STAT3 signalling suggested a mechanism for VP79s inhibition of STAT3 activation via inhibition of JAK2 phosphorylation and downregulation of the IL-6 receptors, CD126 and CD130. Analysis of downstream STAT3 targets demonstrated that treatment with VP79s resulted in a rapid time-dependent decrease in expression of MCL-1. VP79s was shown to overcome adhesion mediated drug resistance in MM cells in a co-culture model system with bone marrow stromal cells which induces MM cell resistance to treatment with the proteasome inhibitor bortezomib. Combining VP79s with bortezomib synergistically enhanced apoptosis in MM cells compared with single agent treatment. VP79s showed selective cytotoxicity in patient derived MM cells compared to normal donor lymphocytes and reduced the viability of MM patient samples in a dose-responsive manner suggesting its potential as an anti-MM therapeutic. In conclusion, the novel agent VP79s inhibits the JAK/STAT3 signalling pathway resulting in rapid downregulation of MCL-1 expression and inducing potent anti-MM activity. This mechanism may suggest a novel strategy for targeting MCL-1, an emerging target in MM. Disclosures No relevant conflicts of interest to declare.

Steroidal saponins from Raphia vinifera and their cytotoxic activity

Phytochemical analysis of the fruits of Raphia vinifera led to the isolation of four new steroidal saponins (1–4), along with six known secondary metabolites (6–10). The structures of the isolated compounds were determined based on the analyses of NMR and mass spectrometric data, and chemical degradation reactions. Among the compounds tested, 1 and 4 showed the most promising cytotoxic activity against the drug-sensitive CCRF-CEM leukemia cell lines, with IC50 values of 3.55 µM and 7.14 µM, respectively.

Fucoxanthin extracted from Laminaria Japonica inhibits metastasis and enhances the sensitivity of lung cancer to Gefitinib

Ethnopharmacological relevanceLaminaria japonica, a brown seaweed, has been used in Traditional Chinese Medicine (TCM) to treat a variety of diseases including lung cancer. Aim of the studyTo demonstrate the effects of Fucoxanthin (FX), a major active component extracted from Laminaria japonica on metastasis and Gefitinib (Gef) sensitivity in human lung cancer cells both in vitro and in vivo. Materials and methodsInvasion and migration of lung cancer cells were detected using the wound healing assay and transwell assay. Epithelial-to-mesenchymal transition (EMT) factors and PI3K/AKT/NF-κB pathways were analyzed by western blotting. RNA interference (RNAi) technology was used to silence TIMP-2 gene expression in A549 cells. The anti-metastatic effect of FX was evaluated in vivo in an experimental lung metastatic tumor model. On the other hand, cell counting kit-8 assay was used to study the cell viability of human lung cancer PC9 cells and Gef resistant PC9 cells (PC9/G) after Gef, FX or FX combined with Gef treatment. PC9 xenograft model was established to explore the anti-tumor effect of FX or combined with Gef. Immunohistochemistry staining assay and immunofluorescence staining assay were used to reveal the effects of FX on lung cancer cell proliferation and apoptosis. ResultsFX was able to significantly inhibit lung cancer cells migration and invasion in vitro. FX suppressed the expressions of Snail, Twist, Fibronectin, N-cadherin, MMP-2, PI3K, p-AKT and NF-κB, and increased the expression of TIMP-2. Furthermore, knockdown of TIMP-2 attenuated FX-mediated invasion inhibition. Additionally, we demonstrated that FX inhibited lung cancer cells metastasis in vivo. The anti-metastatic effects of FX on lung cancer cells might be attributed to inhibition of EMT and PI3K/AKT/NF-κB pathway. We further demonstrated that the anti-tumor activity of FX was not only limited to the drug sensitive cell lines, but also prominent on lung cancer cells with Gef resistant phenotype. Furthermore, in vivo xenograft assay confirmed that FX inhibited tumor growth and enhanced the sensitivity of lung cancer cells to Gef and this effect may be due to inhibition of tumor cell proliferation and activation of apoptosis. ConclusionCollectively, our findings suggested that FX suppresses metastasis of lung cancer cells and overcomes EGFR TKIs resistance. Thus, FX is worthy of further investigation as a drug candidate for the treatment of lung cancer.

Abstract 1873: Drivers of secondary resistance to anti-EGFR therapy in metastatic colorectal cancer

Abstract Secondary Resistance (SR) evolves in virtually all metastatic colorectal cancers (mCRC) treated with anti-EGFR targeting drugs such as Cetuximab (Cmab). Yet, SR patient-derived xenograft (pdx) models are largely lacking. Here, we established eight SR pdx models through chronic Cmab treatment of drug-sensitive mCRC pdx. All pdx models were initially wild-type for KRAS, NRAS, PI3K and BRAF, as required for Cmab treatment. Post SR establishment, two models showed KRAS mutations and were excluded from further studies. Whole exome sequencing of the remaining six SR pdx models did not reveal any known driver mutations which could confer SR. However, global gene expression analyses identified widespread transcriptional reprograming with many genes able to activate KRAS and/or PI3K signaling pathways. Among the highly dysregulated genes, fibroblast growth factors FGF13, FGF19, FGF20, and insulin-like growth factor IGF2 stood out as potential drivers of SR through upregulation of RTK signaling. Functional molecular analyses of these factors were performed using lentiviral vector mediating gene transduction. Cell viability, proliferation assays and western blot (WB) analyses were applied to assess the molecular functionality of FGFs and IGF2-ligands in vitro. Finally, transgenic cell lines were injected into nude mice to test their therapeutic response towards Cmab treatment following upregulation or downregulation of the chosen candidates. Stable lentiviral overexpression of FGF13 and IGF2 in drug-sensitive cell lines and pdx models triggered SR both in vitro and in vivo. WB analyses showed that FGF13 and IGF2 overexpression induced phosphorylation of pAKT, pS6, and pERK. Conversely, knockdown of IGF2 in a cell line with high endogenous IGF2 expression improved Cmab sensitivity. Lastly, when subjecting a Cmab SR pdx model with high endogenous overexpression of FGF13 and FGF19 to a Pan-FGF inhibitor (LY2874455) in combination with Cmab, we observed a conversion of the SR into partial response. Taken together, these results support a role of IGF2 and FGFs as candidate proteins conferring SR in an autocrine fashion in the setting of KRAS wild-type mCRC under chronic anti-EGFR treatment. Importantly, these results also suggest that potential combination therapies of Cmab and an IGF2 or FGF inhibitor could prevent establishment of SR through these pathways and offer new treatment opportunities for patients with SR mCRC. Citation Format: Soha Hussein Noseir, Stephan Hahn, Abdelouahid Maghnouj, Swetlana Ladigan. Drivers of secondary resistance to anti-EGFR therapy in metastatic colorectal cancer [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 1873.

Abstract 5264: Selective toxicity of MX-106-4C, a survivin inhibitor, in P-glycoprotein-mediated multidrug resistant colon cancer

Abstract One of the major challenges in colon cancer chemotherapy is multidrug resistance (MDR), which is typically mediated by the overexpression of ATP-binding cassette (ABC) transporters, particularly P-glycoprotein (P-gp, ABCB1, MDR1). A number of P-gp inhibitors have been developed, however, none of these compounds have improved chemotherapeutic efficacy due to undesirable pharmacokinetic profiles or adverse effects, resulting in limited clinical success. Therefore, alternative approaches are urgently needed to circumvent MDR cancer. In previous study, a series of synthesized analogs of MX-106, as anti-cancer drugs targeting survivin, exhibited collateral sensitivity (CS) effect to P-gp overexpressing MDR colon cancer cells as well as ABCB1 gene transfected cells, reflected by more than 10-fold cytotoxic effect in P-gp positive MDR cell lines compared to drug sensitive cell lines. Among the analogs, MX-106-4C was identified as the leading compound with the most potent selective toxicity to P-gp overexpressing cells. MX-106-4C-induced CS effect was observed in both intrinsic and acquired P-gp overexpressing colon cancer cells, which was only partially reversed with the presence of a P-gp inhibitor. Nevertheless, this CS effect was abolished in ABCB1-knockout cells, indicating that the selective cytotoxicity was P-gp expression dependent, but only partially related to P-gp function. Furthermore, we found that MX-106-4C did not significantly affect P-gp ATPase activity or drug accumulation and efflux in P-gp-overexpressing cells. In P-gp overexpressing colon cancer cells, short-term (up to 72 h) incubation of MX-106-4C significantly down regulated P-gp expression at transcriptional level but not protein level, whereas long-term (14 d) incubation of MX-106-4C significantly down regulated P-gp protein expression and re-sensitized MDR colon cancer cells to doxorubicin. These findings suggested an indirect interaction and regulation between MX-106-4C and P-gp. Further study revealed that the selective cytotoxic effects of MX-106-4C were associated with cell cycle arrest at G1 phase and apoptosis through the downregulation of CDK4. Overall, this study demonstrates that MX-106-4C selectively kills P-gp positive MDR colon cancer cells and indirectly regulates P-gp, which provides a clue for CS compound design and a novel strategy to obviate P-gp-mediated colon cancer MDR by re-sensitizing heterogeneous tumors with CS agents. Citation Format: Zi-Ning Lei, Zhongzhi Wu, Qiu-Xu Teng, Min Xiao, Wei Li, John N. Wurpel, Zhe-Sheng Chen. Selective toxicity of MX-106-4C, a survivin inhibitor, in P-glycoprotein-mediated multidrug resistant colon cancer [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 5264.

Recent Advances in β-lactam Derivatives as Potential Anticancer Agents.

Cancer, accounts for around 10 million deaths annually, is the second leading cause of death globally. The continuous emergency of drug-resistant cancers and the low specificity of anticancer agents are the main challenges in the control and eradication of cancers, so it is imperative to develop novel anticancer agents. Immense efforts have been made in developing new lead compounds and novel chemotherapeutic strategies for the treatment of various forms of cancers in recent years. β-Lactam derivatives constitute versatile and attractive scaffolds for the drug discovery since these kinds of compounds possess a variety of pharmacological properties, and some of them exhibited promising potency against both drug-sensitive and drug-resistant cancer cell lines. Thus, β-lactam moiety is a useful template for the development of novel anticancer agents. This review will provide an overview of β-lactam derivatives with the potential therapeutic application for the treatment of cancers covering articles published between 2000 and 2020. The mechanisms of action, the critical aspects of design and structureactivity relationships are also discussed.

Abstract A56: Novel L-sugar linked glycosylated antitumor ether lipids for killing platinum-resistant human epithelial ovarian cancer cells

Abstract Glycosylated antitumor ether lipids (GAELs) are a promising class of investigational anticancer agents with potent antitumor activity against a range of cancers, including platinum-resistant high-grade serous ovarian cancer (HGSOC). We previously demonstrated that epithelial ovarian cancer (EOC) cell lines (HGSOC and endometrioid) and primary cells derived from patient ascites (HGSOC and clear cell) were sensitive to the cell-killing effects of D-glucosamine-derived GAELs via an apoptosis-independent mechanism. However, D-linked carbohydrates are metabolized by endogenous glucosidases in vivo, rendering the GAEL inactive. Thus, the aim of the current study was to synthesize a novel class of GAELs with L-linked carbohydrates and to evaluate their efficacy in EOC cell lines and patient samples. Out of seven novel compounds tested, L-rhamnose-GAEL was identified as the compound with the greatest efficacy for killing EOC cell lines and patient cells that were grown under adherent or nonadherent (3D) conditions. The drug-sensitive and drug-resistant syngeneic endometrioid EOC cell lines, A2780s and A2780cp, respectively, as well as the NIH:OVCAR-3 and COV362 HGSOC lines, were incubated with L-rhamnose-GAEL for 48 hours and the CC50 determined as 15 μM for A2780s, 22.5 μM for A2780cp, and 5 μM for NIH:OVCAR-3 and COV362 cells. Similar experiments were conducted with primary EOC cells, chemo-naïve EOC126 (clear-cell adenocarcinoma), and platinum-resistant EOC 183I (HGSOC). Cell viability decreased in a dose-dependent manner with CC50 of 12 μM for EOC126 and 22.5 μM for EOC183l. For comparison, when EOC126 and EOC183I were grown as 3D cultures the CC50for cisplatin was 20 μM and 40 μM, respectively. To evaluate the effect of using low-dose treatment of L-rhamnose-GAEL on primary EOC cells (chemo-naive and chemoresistant), cells were exposed to L-rhamnose-GAEL for 96 hours. These studies revealed that prolonged incubation led to low CC50 of 1-5 μM. These results showed that primary EOC cells derived from chemo-naïve and platinum-resistant patients were sensitive to the L-rhamnose-GAEL and offer a novel drug class capable of killing chemotherapy-resistant EOC cells. Citation Format: Mark W. Nachtigal, Frank Schweizer, Gilbert Arthur. Novel L-sugar linked glycosylated antitumor ether lipids for killing platinum-resistant human epithelial ovarian cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A56.

Drug mechanism-of-action discovery through the integration of pharmacological and CRISPR screens.

Low success rates during drug development are due, in part, to the difficulty of defining drug mechanism‐of‐action and molecular markers of therapeutic activity. Here, we integrated 199,219 drug sensitivity measurements for 397 unique anti‐cancer drugs with genome‐wide CRISPR loss‐of‐function screens in 484 cell lines to systematically investigate cellular drug mechanism‐of‐action. We observed an enrichment for positive associations between the profile of drug sensitivity and knockout of a drug's nominal target, and by leveraging protein–protein networks, we identified pathways underpinning drug sensitivity. This revealed an unappreciated positive association between mitochondrial E3 ubiquitin–protein ligase MARCH5 dependency and sensitivity to MCL1 inhibitors in breast cancer cell lines. We also estimated drug on‐target and off‐target activity, informing on specificity, potency and toxicity. Linking drug and gene dependency together with genomic data sets uncovered contexts in which molecular networks when perturbed mediate cancer cell loss‐of‐fitness and thereby provide independent and orthogonal evidence of biomarkers for drug development. This study illustrates how integrating cell line drug sensitivity with CRISPR loss‐of‐function screens can elucidate mechanism‐of‐action to advance drug development.

Personalized Network Modeling of the Pan-Cancer Patient and Cell Line Interactome.

PURPOSEPersonalized network inference on diverse clinical and in vitro model systems across cancer types can be used to delineate specific regulatory mechanisms, uncover drug targets and pathways, and develop individualized predictive models in cancer.METHODSWe developed TransPRECISE (personalized cancer-specific integrated network estimation model), a multiscale Bayesian network modeling framework, to analyze the pan-cancer patient and cell line interactome to identify differential and conserved intrapathway activities, to globally assess cell lines as representative models for patients, and to develop drug sensitivity prediction models. We assessed pan-cancer pathway activities for a large cohort of patient samples (> 7,700) from the Cancer Proteome Atlas across ≥ 30 tumor types, a set of 640 cancer cell lines from the MD Anderson Cell Lines Project spanning 16 lineages, and ≥ 250 cell lines’ response to > 400 drugs.RESULTSTransPRECISE captured differential and conserved proteomic network topologies and pathway circuitry between multiple patient and cell line lineages: ovarian and kidney cancers shared high levels of connectivity in the hormone receptor and receptor tyrosine kinase pathways, respectively, between the two model systems. Our tumor stratification approach found distinct clinical subtypes of the patients represented by different sets of cell lines: patients with head and neck tumors were classified into two different subtypes that are represented by head and neck and esophagus cell lines and had different prognostic patterns (456 v 654 days of median overall survival; P = .02). High predictive accuracy was observed for drug sensitivities in cell lines across multiple drugs (median area under the receiver operating characteristic curve > 0.8) using Bayesian additive regression tree models with TransPRECISE pathway scores.CONCLUSIONOur study provides a generalizable analytic framework to assess the translational potential of preclinical model systems and to guide pathway-based personalized medical decision making, integrating genomic and molecular data across model systems.

Reversal of ABCB1-related multidrug resistance by ERK5-IN-1

BackgroundInhibition of ABC transporters is considered the most effective way to circumvent multidrug resistance (MDR). In the present study, we evaluated the MDR modulatory potential of ERK5-IN-1, a potent extracelluar signal regulated kinase 5 (ERK5) inhibitor.MethodsThe cytotoxicity and MDR reversal effect of ERK5-IN-1 were assessed by MTT assay. The KBv200-inoculated nude mice xenograft model was used for the in vivo study. Doxorubicin efflux and accumulation were measured by flow cytometry. The modulation of ABCB1 activity was measured by colorimetric ATPase assay and [125I]-iodoarylazidoprazosin (IAAP) photolabeling assay. Effect of ERK5-IN-1 on expression of ABCB1 and its downstream markers was measured by PCR and/or Western blot. Cell surface expression and subcellular localization of ABCB1 were tested by flow cytometry and immunofluorescence.ResultsOur results showed that ERK5-IN-1 significantly increased the sensitivity of vincristine, paclitaxel and doxorubicin in KBv200, MCF7/adr and HEK293/ABCB1 cells, respectively. This effect was not found in respective drug sensitive parental cell lines. Moreover, in vivo combination studies showed that ERK5-IN-1 effectively enhanced the antitumor activity of paclitaxel in KBv200 xenografts without causing addition toxicity. Mechanistically, ERK5-IN-1 increased intracellular accumulation of doxorubicin dose dependently by directly inhibiting the efflux function of ABCB1. ERK5-IN-1 stimulated the ABCB1 ATPase activity and inhibited the incorporation of [125I]-iodoarylazidoprazosin (IAAP) into ABCB1 in a concentration-dependent manner. In addition, ERK5-IN-1 treatment neither altered the expression level of ABCB1 nor blocked the phosphorylation of downstream Akt or Erk1/2. No significant reversal effect was observed on ABCG2-, ABCC1-, MRP7- and LRP-mediated drug resistance.ConclusionsCollectively, these results indicated that ERK5-IN-1 efficiently reversed ABCB1-mediated MDR by competitively inhibiting the ABCB1 drug efflux function. The use of ERK5-IN-1 to restore sensitivity to chemotherapy or to prevent resistance could be a potential treatment strategy for cancer patients.

Drug combinations as effective anti-leishmanials against drug resistant Leishmania mexicana

Leishmania is a parasite that causes the disease leishmaniasis, and 700 000 to 1 million new cases occur each year. There are few drugs that treat the disease and drug resistance in the parasite limits the clinical utility of existing drugs. One way to combat drug resistance is to use combination therapy rather than monotherapy. In this study we have compared the effect of single and combination treatments with four different compounds, i.e. alkylphosphocholine analogues APC12 and APC14, miltefosine (MIL), ketoconazole (KTZ), and amphotericin B (AmpB), on the survival of Leishmania mexicana wild-type promastigotes and a cell line derived from the WT with induced resistance to APC12 (C12Rx). The combination treatment with APC14 and APC16 had a synergistic effect in killing the WT while the combination treatment with KTZ and APC12 or APC14 or APC12 and APC14 had a synergistic effect against C12Rx. More than 90% killing efficiency was obtained using APC12 alone at >1 mg ml-1 against the C12Rx strain; however, combinations with APC14 produced a similar killing efficiency using APC12 at 0.063-0.25 mg ml-1 and APC14 at 0.003-0.5 mg ml-1. These results show that combination therapy can negate induced drug resistance in L. mexicana and that the use of this type of screening system could accelerate the development of drug combinations for clinical use.

Design, synthesis and SAR study of 3rd-generation taxoids bearing 3-CH3, 3-CF3O and 3-CHF2O groups at the C2-benzoate position.

It has been shown that inclusion of CF3O and CHF2O groups to drug candidates often improve their pharmacological properties, especially metabolic stability, membrane permeability and PK profile. Moreover, the unique non-spherical structure of the OCHF2 group can provide interesting and beneficial characteristics. Accordingly, new 3rd-generation taxoids, bearing 3-OCF3 or 3-OCF2H (and 3-CH3 for comparison) at the C2 benzoate moiety, were synthesized and their potencies against drug-sensitive and drug-resistant cancer cell lines examined. In this study, our previous SAR studies on 3rd-generation taxoids were expanded to disclose that CH3, CF3O and CHF2O groups are well tolerated at this position and enhance potency, especially against MDR-cancer cell lines so that these taxoids can virtually overcome MDR. These new taxoids exhibit up to 7 times higher cytotoxicity (IC50) than paclitaxel against drug-sensitive cancer cell lines (MCF7 and LCC6-WT) and 2-3 orders of magnitude higher potency than paclitaxel against drug-resistant ovarian, breast and colon cancer cell lines with MDR-phenotype (NCI/ADR, LCC6-MDR and LDL-1), as well as pancreatic cancer cell line, CFPAC-1. Since it has been shown that a bulky group at this position reduces potency, it is noteworthy that rather bulky CF3O and CHF2O groups are well tolerated. Molecular modeling analysis indicated the favorable van der Waals interactions of CF3O and CHF2O groups in the binding site. It is also worthy of note that new taxoids, bearing a CHF2O group at the C2 benzoate position (1-06 series), exhibited the highest potencies against MDR-cancer cell lines and cancer stem cell (CSC)-enriched cancer cell lines. These new 3rd-generation taxoids are promising candidates for highly potent chemotherapeutic agents, as well as payloads for tumor-targeting drug conjugates such as antibody-drug conjugates (ADCs).

Antiproliferative properties of labdane diterpenoids from Croton sylvaticus Hochst against drug sensitive and resistant leukemia cell lines

Antiproliferative properties of labdane diterpenoids from Croton sylvaticus Hochst against drug sensitive and resistant leukemia cell lines

Anticancer Activity of a Highly Potent Small Molecule Tubulin Polymerization Inhibitor, AB8939

We have identified the small chemical molecule AB8939 as being a structurally novel, synthesized tubulin inhibitor that can circumvent resistance mechanisms known to limit the effectiveness of existing tubulin inhibitors; e.g., P-glycoprotein (Pgp) and myeloperoxidase (MPO) mediated resistance. A series of in vitro preclinical studies provide proof-of-concept that AB8939 has broad applicability as a potent anticancer drug, particularly in tumors of hematopoietic and lymphoid tissues, including acute myeloid leukemia (AML). Regarding mechanism of action, x-ray crystallography demonstrated that AB8939 binds to the colchicine-binding site on the beta-subunit of tubulin. Cell cycle arrest in the G2/M phase was evaluated using HCT116 cells (a human colorectal tumor), treated at various concentrations of AB8939 for 24 hours. It was seen that AB8939 produced a strong mitotic arrest at the sub-micromolar concentration range (90% of cells in G2/M phase at 10 nM), which was of comparable strength to that of established microtubule targeting agents, each at a concentration of 100 nM. Additional assays using cytarabine (Ara-C) resistant MOLM14 AML cells confirmed this activity, also demonstrating dose dependent (2 to 20 nM) G2/M phase cell cycle arrest in patient-derived AML blasts and that G2/M cell cycle arrest lead to cellular death by apoptosis at nanomolar concentrations. The effect of AB8939 (100 nM) on the integrity of the microtubule and actin networks was tested in 3T3NIH cells (murine embryonic fibroblast cell line). AB8939 induced a rapid (within 1 hour) and radical destabilization of the microtubule network but did not affect the actin network. Similarly, destabilization of the microtubule network was observed in human primary cardiomyocytes and primary human lung fibroblast cells treated for 24 hours at 10 to 1000 nM AB8939. Further in vitro analysis showed that AB8939 produces a direct and potent, dose-dependent depolymerization effect (50% inhibition of in vitro microtubule polymerization at around 1 µM, with 100% inhibition at >5 µM). The potential of AB8939 to overcome resistance to chemotherapeutic agents in Pgp-dependent multidrug-resistant cell lines was assessed using the drug-sensitive human sarcoma cell line MES-SA (parental) and its multidrug-resistant counterparts MES-SA/MX2 and MES-SA/Dx5 in a 6-day proliferation/survival assay. AB8939 efficiently inhibited each of these cells with an IC50 ≤10 nM. By comparison, the MES-SA/MX2 and MES-SA/Dx5 cell lines were highly resistant to the chemotherapeutic agents of doxorubicin and vincristine, as compared with the effect on parental cells (IC50 <1.5 - 2.0 µM versus 20 nM, respectively). Additional tests showed that AB8939 is a very poor substrate of Pgp efflux pump, comparable with combretastatin-4, and therefore has the potential to overcome multidrug resistance in cancer patients. The anti-proliferative activity of AB8939 in various hematopoietic tumors and solid tumors was evaluated using a colorimetric cell proliferation and viability assay. AB8939 produced good anti-tumor activity after 72 hours (IC50 of ≤50 nM) in 19 hematopoietic tumor cell lines tested, including AML (3 cell lines), B cell lymphoma (8 cell lines), T cell lymphoma (6 cell lines), and multiple myeloma (2 cell lines). AB8939 also showed good anti-tumor activity after 6 days (IC50 of ≤10 nM) in several solid tumor cell lines, including breast, colon, glioblastoma, head and neck, lung, kidney, melanoma neuroblastoma, ovary, pancreas and prostate cell lines. The therapeutic potential of AB8939 in refractory/resistant AML was investigated further on doxorubicin-resistant AML cell lines (HL60 and U937), doxorubicin being a commonly used AML induction drug and Pgp substrate. AB8939 produced a strong anti-proliferative effect in both cell lines whereas both were resistant to doxorubicin, thus demonstrating AB8939's potential to overcome refractory/resistant AML. Notably, HL60 and U937 are respectively MPO-positive and MPO-negative, indicating that unlike vinca alkaloids (e.g. vincristine or vinblastine) AB8939 it is not deactivated by this myeloid enzyme. These data show that AB8939 is a prolific and highly potent (nanomolar concentrations) Pgp-independent, next-generation microtubule-destabilizer drug for cancer therapy; in particular, difficult to treat hematopoietic tumors such as relapsed/refractory AML. Disclosures Humbert: AB Science: Employment. Goubard:AB Science: Employment. Mansfield:AB Science: Employment, Patents & Royalties. Hermine:AB Science: Membership on an entity's Board of Directors or advisory committees. Dubreuil:AB Science: Employment, Membership on an entity's Board of Directors or advisory committees, Research Funding. AB8939 Study Group:AB Science: Consultancy, Employment.

Leaf Extracts from Dillenia philippinensis Rolfe Exhibit Cytotoxic Activity to both Drug-Sensitive and Multidrug-Resistant Cancer Cells.

Background:Cancer is one of the leading causes of illness and death worldwide. Only palliative therapeutic options are available for many types of cancers, and most anticancer drugs in clinical use exhibit significant side effects. It is therefore important to develop new anticancer drugs that are more effective and less toxic. In this study, we evaluate the bioactivity of a Philippine endemic plant, “katmon” or Dillenia philippinensis, and its potential use in cancer therapy. Methods:The cytotoxicity of the crude leaf extract, partitions, and isocratic column chromatography fractions of Dillenia philippinensis was determined in vitro by MTT assay against drug-sensitive cancer cell lines MCF7 (human breast adenocarcinoma) and HCT 116 (human colorectal carcinoma), as well as against moderately multidrug resistant (MDR) cancer cell line HCT-15 (human colorectal carcinoma) and its highly MDR subline HCT-15/Dox. The selectivity of the extract to cancer cells was determined by computing for the selectivity index (SI) with respect to normal mouse embryonic fibroblasts (NIH/3T3) cell line. To check for a possible mechanism for overcoming cancer multiple drug resistance, Calcein-AM assay was performed to assess the activity of the extract against P-glycoprotein-activated efflux pump. Results: Dillenia philippinensis (DP1) fraction from the hexane partition exhibited cytotoxicity (IC50< 30 µg/ml) against MCF7, HCT 116, HCT-15, and HCT-15/Dox cells. DP1 also exhibited a moderate level of selectivity against cancer cells over normal cells as supported by the SI computed from the IC50 value obtained for the normal cell line. DP1 was able to inhibit P-glycoprotein (P-gp) activity in a dose-dependent manner, suggesting its possible role in targeting cancer cells with overexpressed P-gp. Conclusion:The present findings thus demonstrate the potential chemotherapeutic properties of D. philippinensis which can be promising for future drug development against cancer.