Drug-free Medium Research Articles

PO-422 Disagregation in biological medium of promising theranostic agents for cancer

IntroductionWe recently developed a new type of photochemically stable platinum (II) chlorins, which are remarkable photosensitizers that can be used in photodynamic therapy (PDT), due to its therapeutic capacity. Simultaneously, due to its highly luminescence proprieties, in the biological relevant 650–850 nm red and near infrared spectral region, they may be used for biological imaging. In addition, photophysical studies indicate that they may be used as ratiometric oxygen sensors.Material and methodsCompounds with different degrees of hydrophilicity were synthesised and characterised. Photocytotoxicity studies were carried out against two human tumour cell lines, the OE19 line of oesophageal carcinoma and the A375 line of melanocytic melanoma. The formulation of the sensitizers consisted in a 1 mg/mL solution in DMSO and the desired concentrations being achieved by successive dilutions. The sensitizers were administered in several concentrations (from 50 nM to 10 µM) and, 24 hour after incubation, cells were washed with PBS and new drug-free medium was added. Plates were irradiated (<580 nm) with a fluence rate of 7.5 mW/cm2, to reach 10 J. Controls were performed on every test. Evaluation by MTT assay was performed 24 hour after the photodynamic treatment. The IC50 values were calculated.Results and discussionsPreliminary cytotoxicity studies indicate that, in both cell lines, platinum (II) chlorins with more hydrophilic features require lower doses of photosensitizer to induce a significant photocytotoxic effect on tumour cells. Our best results were IC50 value of 165.9 nM (confidence interval at 95%: [77.9; 356.6]) for A375 line and 498.6 nM (confidence interval at 95%: [283.5; 876.5]) for OE19 line. However, and given that the confidence intervals were too large, it was hypothesised that this type of compounds would be aggregating in the biological medium. This hypothesis was corroborated by additional photophysical studies. As such, disaggregation was performed with the use of a non-ionic surfactant since surface active agents (surfactants) has been widely used for enhancing solubilisation of poorly soluble drugs.ConclusionAlthough photocytotoxicity studies reveal that the platinum (II) chlorins tested would be very promising for PDT, further cytotoxicity studies will be carried out using a novel formulation.This work was funded by Fundação para a Ciência e a Tecnologia (FCT): POCI-01–0145-FEDER-PTDC/QEQ-MED/0262/2014;COMPETE 2020 POCI-01–0145-FEDER-007630 and POCI-01–0145-FEDER-007440.

Abstract 3905: Strategic combination of the cyclin-dependent kinase inhibitor CYC065 with venetoclax to target anti-apoptotic proteins in chronic lymphocytic leukemia

Abstract CYC065 is a cyclin-dependent kinase (Cdk) inhibitor that is highly selective towards Cdk2 and Cdk9. In chronic lymphocytic leukemia (CLL), a disease that is addicted to the over-expression of anti-apoptotic proteins for survival, inhibition of Cdk9 by CYC065 reduced phosphorylation of the C-terminal domain of RNA polymerase II and blocked transcription. These actions depleted the intrinsically short-lived anti-apoptotic protein Mcl-1, but not Bcl-2, and induced apoptosis in CLL cells in vitro. The IC50 for CYC065-induced CLL cell death after a 24-hr incubation was 0.8 μM, a concentration that is achievable in the clinic at tolerated doses. CYC065 killed the CLL cells equally efficiently in the presence or absence of the human stromal cell line, StromaNKtert, and with or without a stimulation condition that mimics the lymphoid tissue microenvironment (anti-IgM, anti-CD-40, IL-4). Venetoclax, which specifically inhibits Bcl-2 function, is approved for treatment of CLL with del(17p); however upregulation of Mcl-1 is associated with resistance to venetoclax in the lymph nodes. Therefore, we hypothesized that the combination of CYC065 with venetoclax would target the parallel mechanisms that promote the survival control in CLL cells, and induce synergistic cell death by apoptosis. A time course study of the single agents showed that under conditions that mimic the lymph node microenvironment, cell death induction by venetoclax required 6-8 hr to reach the plateau of cell killing and maximal killing by CYC065 occurred after 24 hr, consistent with the different mechanisms of action of the two compounds. Following the removal of CYC065 or venetoclax after 4, 8, 12, or 24 hr incubations, there was no evidence for additional cell death after an additional 48 hr in drug-free medium regardless of the duration of drug incubation. Immunoblots showed recovery of RNA pol II phosphorylation, and restored Mcl-1 expression upon washout of CYC065. The reversible action of these compounds has potential implications for clinical scheduling combining these compounds. Median effect analysis indicated that CYC065 and venetoclax combined synergistically in CLL samples with or without 17p deletion. A dose reduction analysis confirmed mutual potentiation of each other when combined. Combination of IC50 concentrations of CYC065 and venetoclax for 24 hr was sufficient to decrease the viability of CLL cells by over 90% in the lymph node mimicking microenvironment. Thus, these data provided rationale for clinical combination of CYC065 and venetoclax in CLL. CYC065 is currently in a Phase I clinical trial in patients with advanced solid tumors (NCT02552953) using an intermittent dosing regimen which causes at least 24 hr Mcl-1 downregulation in patient PBMCs at well tolerated dose levels. Citation Format: Rong Chen, Yuling Chen, Sheelagh Frame, David Blake, William G. Wierda, Daniella Zheleva, William Plunkett. Strategic combination of the cyclin-dependent kinase inhibitor CYC065 with venetoclax to target anti-apoptotic proteins in chronic lymphocytic leukemia [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 3905.

Abstract 4836: Targeting transcriptional regulation in multiple myeloma with a covalent CDK7 inhibitor THZ1

Abstract Previous studies have shown that certain tumors such as MYCN-driven neuroblastoma and T-cell ALL may be sensitive to CDK7 inhibition. However, the impact of CDK7 disruption on the proliferation and survival of multiple myeloma (MM) cells, which are regulated, at least in part, by c-Myc, has not as yet been examined. To address this issue, the effects of THZ1, a selective covalent inhibitor of cyclin-dependent kinase CDK7, a component of the general transcription factor TFIIH, was examined in MM cells. Virtually all MM cell lines tested were sensitive to the growth-inhibitory and pro-apoptotic effects of very low (e.g., nM) THZ1 concentrations administered for 24 hr, including H929, OPM2, RPMI8226, and U266 cells, as well as bortezomib- or revlimid- resistant cells. THZ1 induced G2/M arrest and inhibited CDK7-mediated phosphorylation of CDK1/2 at T161 and T160 sites. Notably, THZ1 significantly inhibited general transcription by blocking the phosphorylation of the CTD (carboxy-terminal domain) of RNA Pol II at Ser5, Ser7, and Ser2 sites. Washout studies, in which cells were washed free of THZ1 and resuspended in drug-free medium, demonstrated thatTHZ1 acted irreversibly in blocking CTD phosphorylation. CDK7 inhibition was accompanied by down-regulation of MCL-1, c-Myc (L-myc in U266) and BCL-XL gene expression, all of which exert pro-survival effects. Concordant results were obtained when protein levels were monitored. Moreover, ectopic expression of c-Myc, MCL-1, or BCL-XL in MM cells markedly diminished THZ1 lethality, documenting the functional significance of down-regulation of these proteins in THZ1 activity. CRISPR/Cas9 editing of the CDK7 gene was them utilized to confirm the on-target impact of CDK7 disruption. Cells treated with constructs encoding two independent small guiding RNAs targeting CDK7 (sgCDK7) displayed markedly reduced CDK7 protein levels and sharply diminished H929 and OPM2 cell growth. Furthermore, co-culture with HS-5 cells failed to protect MM cells from THZ1, indicating that THZ1 can circumvent micro-environment forms of drug- resistance. Notably, THZ1 alone showed pronounced antitumor effects (e.g., inhibition of viability), and significantly enhanced proteasome inhibitor (e.g., bortezomib or carfilzomib) lethality in human primary CD138+ MM cells and primitive MM cells (CD138-/19+/20+/27+), but not normal hematopoietic cells (e.g., CD34+ cord blood cells). Finally, administration of THZ1 reduced tumor burden and significantly enhanced survival (P &amp;lt; 0.001) in flank and orthotopic xenograft mouse MM models with minimal toxicity. Collectively, these findings indicate that CDK7 is a potential therapeutic target in MM, and suggest that abrogating CDK7 activity by small molecules like THZ1 warrants further consideration as a therapeutic strategy in MM. Citation Format: Liang Zhou, Yu Zhang, Kanika Sharma, Jacquelyn McCarter, William Craun, Steven Grant. Targeting transcriptional regulation in multiple myeloma with a covalent CDK7 inhibitor THZ1 [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 4836.

PO-423 Novel 4,5,6,7-tetrahydropyrazolo[1,5-a] pyridine fused chlorins as very active photosensitizers against melanoma and bladder cancer cells

Introduction Photodynamic therapy (PDT) is a clinically approved therapeutic procedure, which is entering the mainstream of cancer treatments. Nowadays PDT has been successfully used in the treatment of skin cancers, but the use of PDT against melanoma can be compromised due to the natural resistance mechanism of some melanoma cancer cells. Thus, the search for new photosensitizers is a relevant research goal. Bladder cancer is also an interesting target to PDT, due to easy irradiation accessibility by cystoscopy. Material and methods A375 human melanoma cells and HT1376 human bladder cancer cells were plated. The formulation of the sensitizers consisted in a 1 mg/mL solution in DMSO and the desired concentrations being achieved by successive dilutions. The sensitizers were administered in several concentrations (from 1 nM to 10 mM) and cells were incubated for 24 hour. Controls were performed on every test. Cells were washed with PBS and new drug-free medium was added. Each plate was irradiated with a fluence rate of 7.5 mW/cm2, to reach 10 J. Evaluation by MTT assay was performed 24 hour after the photodynamic treatment in order to evaluate the cytotoxic effect. Results and discussions Our previous in vitro PDT studies demonstrated that the increase of chlorins’ hydrophilicity of leads to higher activity against A375 melanoma cells. Therefore, a series of novel 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-fused chlorins bearing dicarboxylic acid and monocarboxyic moieties were developed showing an interesting biological activity against the A375 and HT1376 cancer cells. Inhibition of the metabolic activity seems to be dependent on the concentration of the sensitizers used. With the experimental metabolic activity values, it was possible to calculate the concentration of the sensitizers that inhibits the proliferation of cultures in 50% (IC50). For this series of compounds, IC50 values ranged from mM to nM concentrations. Nevertheless, a new molecule with an IC50 value of 67,93 nM stood out. Conclusion The compounds tested were active against human melanocytic melanoma A375 cells and human bladder HT1376 cancer cells. MTT assay showed that the metabolic activity was inversely proportional to the concentration of the photosensitizer. Interestingly low IC50 values in the nanomolar range encourage further studies. Funding The Foundation for Science and Technology: POCI-01–0145-FEDER-PTDC/QEQ-MED/0262/2014; (COMPETE 2020). POCI-01–0145-FEDER-007630 and POCI-01–0145-FEDER-007440.

Rapid screening drug susceptibility test in tuberculosis using sandwich electrochemical immunosensor

Drug susceptibility testing (DST) for Mycobacterium tuberculosis currently faces multiple challenges, including lengthy ineffective standard methods, the expensive cost of molecular tests, and large bulky diagnostic machines. In this work, a disposable MPT64 sensor was developed to rapidly determine drug susceptibility (DS-TB) and multidrug resistant tuberculosis (MDR-TB) using an electrochemical sandwich-immunosensor for the detection of MPT64 as an indicator of Mycobacterium tuberculosis growth. Anti-MPT64 (as capture antibody; cAb) was immobilised on screen printed carbon electrodes to specifically bind with MPT64 target protein. A reporter probe of anti-MPT64 conjugated with horseradish peroxidase (HRP labeled rAb) then completed the sandwich immunocomplex. The current signals were received from the catalytic reaction between 3,3′-5,5′-tetramethylbenzidine (TMB), H2O2 and HRP labeled rAb using the chronoamperometric mode on a portable potentiostat. Increasing MPT64 concentration was taken as an indicator of growth, which was measured by the disposable MPT64 sensor. This sensor shows the possibility of determining DST by comparing the signals of DS-TB and MDR-TB growth in drug-free and drug-containing liquid medium. The time required to identify DS-TB and MDR-TB in pure culture MTB and leftover sputum sediments from patients are 3 days and 4–6 days, respectively. Therefore, using this sensor is significantly rapid and inexpensive and has the potential to be used for drug susceptibility testing of tuberculosis in middle to low income countries.

Stress-Adaptive Responses Associated with High-Level Carbapenem Resistance in KPC-Producing Klebsiella pneumoniae.

Carbapenem-resistant Enterobacteriaceae (CRE) organisms have emerged to become a major global public health threat among antimicrobial resistant bacterial human pathogens. Little is known about how CREs emerge. One characteristic phenotype of CREs is heteroresistance, which is clinically associated with treatment failure in patients given a carbapenem. Through in vitro whole-transcriptome analysis we tracked gene expression over time in two different strains (BR7, BR21) of heteroresistant KPC-producing Klebsiella pneumoniae, first exposed to a bactericidal concentration of imipenem followed by growth in drug-free medium. In both strains, the immediate response was dominated by a shift in expression of genes involved in glycolysis toward those involved in catabolic pathways. This response was followed by global dampening of transcriptional changes involving protein translation, folding and transport, and decreased expression of genes encoding critical junctures of lipopolysaccharide biosynthesis. The emerged high-level carbapenem-resistant BR21 subpopulation had a prophage (IS1) disrupting ompK36 associated with irreversible OmpK36 porin loss. On the other hand, OmpK36 loss in BR7 was reversible. The acquisition of high-level carbapenem resistance by the two heteroresistant strains was associated with distinct and shared stepwise transcriptional programs. Carbapenem heteroresistance may emerge from the most adaptive subpopulation among a population of cells undergoing a complex set of stress-adaptive responses.

Effects of Tacrolimus or Sirolimus on the adhesion of vascular wall cells: Controlled in-vitro comparison study.

In drug eluting stents the cytostatic drugs Sirolimus or Tacrolimus are used to inhibit blood vessel restenosis by limiting the proliferation of smooth muscle cells. However, the cytostatic activity of both drugs was shown to be not cell specific and could also affect the stent endothelialisation, respectively. Currently, only limited in vitro data are available about the impact of Sirolimus and Tacrolimus on endothelial cell proliferation over a broad concentration range. To answer this question the following study was performed.Commercially obtained HUVEC were expanded with DMEM cell culture medium (GIBCO, Germany) supplemented with 5 vol% fetal calf serum on non-coated regular polystyrene-based 24-multiwell plates. For drug testings 2×104 cells/cm2 were seeded and grown for 24 h until 30-40% of the multiwell surfaces were covered and then exposed to Sirolimus (1.0×10-11 - 1.0×10-5 mol/l) or Tacrolimus (2.0×10-8 - 6.2×10-5 mol/l), both dissolved in DMSO. 12, 24 and 48 h after adding the drugs cell numbers per area were quantified by counting the cells in six wells with four fields of view per well, representing 0.6 mm2, using a confocal laser microscope.After 48 h of cell growth in the drug-free cell culture medium, the HUVEC number increased from 2.0×104 to 3.55×104 cells/cm2 (mean cell doubling time: 53.6 h, n = 6). At lower concentrations (≤2.0×10-6 mol/l) Tacrolimus reduced the number of adherent HUVEC significantly less than Sirolimus (p < 0.05). However, at higher concentrations (≥2.07×10-5 mol/l) the effect of Tacrolimus on the number of adherent endothelial cells was significantly greater than that of Sirolimus (p < 0.05). At the highest concentration applied (6.22×10-5 mol/l), Tacrolimus induced detachment of all HUVECs within 12 h after drug application. The number of adherent HUVEC decreased only slightly (about 9%) after Sirolimus application at the highest concentration (1.09×10-5 mol/l).These data show that in a non-flow model the cytostatic drug Tacrolimus reduced the number of adherent endothelial cells less than Sirolimus, as long as the drug concentration did not surpass 10-6 mol/l. At the limits of solubility, Sirolimus (1×10-5 mol/l) reduced the number of adherent endothelial cells less than Tacrolimus (6×10-5 mol/l), which induced detachment of endothelial cells.

Chemotherapy medication of Vincristine and Vinblastine

Cancers treated with Vincristine and vinblastine include: acute leukemia, Hodgkin's and non- Hodgkin's lymphoma, neuroblastoma, rhabdomyosarcoma, Ewing's sarcoma, Wilms' tumor, multiple myeloma, chronic leukemias, thyroid cancer, brain tumors, non-small cell lung cancer, bladder cancer, melanoma, and testicular cancer andIt is also used to treat some blood disorders. It is given by injection into a vein. Vincristine and vinblastine exhibit differential activity against tumors and normal tissues. In this work, a number of cultured cell lines were assayed for their sensitivity to the antiproliferative and cytotoxic effects of the two drugs following short-term (4 hr) or during continuous exposures. Differential activity was not seen when cells were subjected to continuous exposures. The concentrations of Vincristine and vinblastine, respectively, that inhibited growth rates by 50% were: mouse leukemia L1210 cells, 4.4 and 4.0 nw; mouse lymphoma S49 cells, 5 and 3.5 nM; mouse neuroblastoma cells, 33 and 15 nw; HeLa cells, 1.4 and 2.6 nw; and human leukemia HL-60 cells, 4.1 and 5.3 nM. In contrast, differential toxicity was seen when cells were subjected to 4-hr exposures and transferred to drug-free medium: the 50% growth-inhibitory concentrations for Vincristine and vinblastine, respectively, for inhibition (a) of proliferation of L1210 cells were 100 and 380 nM and of HL-60 cells were 23 and 900 nM and (b) of colony formation of L1210 cells were 6 and &gt;600 nM and of HeLa cells were 33 and 62 nM. Uptake and release of [3H]- vincristine and [3H]vinblastine were examined in L1210 cells under the conditions of growth experiments. Uptake of both drugs was dependent on the pH of culture media, and signifi cantly greater amounts of [3H]vinblastine than of [3H]vincristine were associated with cells after 4-hr exposures to equal concen trations of either drug. When cells were transferred to drug-free medium after 4-hr exposures, vinblastine was released much more rapidly from cells than was Vincristine, and by 0.5 hr after resuspension of cells, the amount of Vincristine associated with the cells was greater than the amount of vinblastine and remained so for up to at least 6 hr.

Daptomycin Resistance in Clinical MRSA Strains Is Associated with a High Biological Fitness Cost.

Daptomycin remains as one of the main treatment options for Methicillin-Resistant Staphylococcus aureus (MRSA). Sporadic resistance cases reported in patients treated with either daptomycin or glycopeptides are a growing concern. In a previous study, we described a clinical case of a patient with a community-acquired MRSA infection resistant to daptomycin and with intermediate resistance to vancomycin who developed a recurrent infection with a susceptible isogenic strain. In the present work, we further investigated the sequential events to determine whether the switch from a daptomycin resistance to a susceptible phenotype was due to a phenomenon of resistance reversion or recurrent infection with a susceptible strain. Pairwise competition experiments showed that the susceptible clinical recurrent SA6850 strain had increased fitness when compared to the resistant counterpart SA6820 strain. In fact, although we have demonstrated that reversion of daptomycin resistance to daptomycin susceptible can occur in vitro after serial passages in drug-free media, phylogenetic analysis suggested that the in vivo process was the result of a recurrent infection with a previous susceptible isolate carried by the patient rather than a resistance reversion of the strain. Whole genome sequence of evolved strains showed that daptomycin resistance in MRSA is associated with a high fitness cost mediated by mutations in mprF gene, revealed as a key element of the biological cost. Moreover, we determined that daptomycin resistance-associated fitness cost was independent of vancomycin intermediate resistance phenotype, as demonstrated in additional clinical MRSA vancomycin susceptible strains. This study highlights important observations as, despite daptomycin offers a useful treatment option for the patients with persistent infections, it has to be carefully monitored. The high fitness cost associated to daptomycin resistance may explain the reduced dissemination of daptomycin resistance and the absence of daptomycin reported outbreaks.

Impact of Morphological Sectors on Antifungal Susceptibility Testing and Virulence Studies.

Morphological heterogeneity of Aspergillus terreus cultures was observed during continued cultivation of amphotericin B (AMB)-resistant isolates on drug-free medium. Outgrowth leads to the emergence of multiple sectors that might result from increased growth rates at drug-free conditions. We evaluated the differences in AMB susceptibility and virulence between sector subcultures (ATSec), AMB-resistant (ATR) strains, and AMB-susceptible (ATS) strains. By comparing A. terreus AMB-resistant (ATR) strains and A. terreus sector (ATSec) cultures we observed a highly significant reduction of AMB MICs in ATSec (ATR MIC, 2 to 32 μg/ml; ATSec MIC, 0.12 to 5 μg/ml). Furthermore, Galleria mellonella survival studies revealed an enhanced virulence of ATSec, which was comparable with that of AMB-sensitive Aspergillus terreus strains (median survival rates for ATS isolates, 72 h; for ATSec isolate ATSecG1, 84 h; for ATR isolates, 144 h). Our findings clearly demonstrate that spontaneous culture degeneration occurs in A. terreus and, most importantly, crucially impacts drug efficacy and virulence.

Abstract PR17: Single-cell analysis reveals an adaptive, slowly-dividing, de-differentiated, drug-resistant cell state selectively inhibitable by drug combinations

Abstract Partial responsiveness of tumor cells due to drug adaptation (or tolerance) during the early phase of treatment with targeted therapeutics seems to be essential for creating a population of viable tumor cells from which resistant clones eventually arise. Thus, understanding transient drug adaptation is likely to be important for both improving the initial effectiveness of treatment and delaying or controlling acquired resistance. Despite the wealth of information available about the molecular events and feedback mechanisms leading to drug tolerance or adaptation, most of our knowledge in this area comes from studying drug response in bulk tumor cell populations. Furthermore, the phenotypic consequences of drug adaptation have been studied most frequently at a few fixed time-points, when drug-adapted cells exhibit a high population-average activity in multiple pro-growth or pro-survival signaling cascades. Therefore, it remains unclear how uniform or heterogeneous the early drug adaptation is across individual cells within a tumor cell population, and how the fate of drug-adapted cells is determined by a diversity of early drug-induced adaptive signaling responses. Uncovering the evolution of biochemical and phenotypic heterogeneity in drug-adapted cell populations is key to designing optimal combinations of drugs to overcome resistance and to achieve durable therapy. In this study, we monitor the responses of BRAFV600E melanoma cells to RAF/MEK inhibitors at the single-cell level in real time using time-lapse live-cell imaging, and then analyze the resulting cell states using transcriptional, biochemical and phenotypic profiling. We found that exposure of tumor cells to RAF/MEK inhibitors elicits heterogeneous and time-variable responses in which some cells die, some arrest and a fraction of slowly-cycling cells adapts to drug, adopting a reversible drug-resistance phenotype characterized by up-regulation of markers of neural crest, a melanocyte precursor, including NGFR (the low affinity nerve growth factor receptor, also known as p75NTR or CD271). The slowly-cycling NFGRHigh state induced by RAF/MEK inhibitors is only transiently stable: after 1-2 weeks of outgrowth in drug-free medium, such cells reset to their initial state as measured by the restoration of RAF/MEK inhibitor sensitivity, accelerated rate of cell division and reduced expression of NGFR. Transcriptional and biochemical profiling of cell lines and human tumors implicates a role for the c-Jun/ECM/FAK/Src cascade in driving the de-differentiated (NGFRHigh) resistance program. We identify multiple drugs targeting this cascade as well as BET bromodomain inhibitors that block the slowly-cycling NGFRHigh state in cell lines and in a BRAFV600E melanoma xenograft model and increase sensitivity to RAF/MEK inhibitors. Our study reveals directly how drug adaptation happens in individual tumor cells leading to emergence of heterogeneous cell sub-populations with reduced drug-sensitivity that may be selectively targeted by drug combinations. Citation Format: Mohammad Fallahi-Sichani, Verena Becker, Benjamin Izar, Gregory J. Baker, Jia-Ren Lin, Sarah A. Boswell, Levi A. Garraway, Peter K. Sorger. Single-cell analysis reveals an adaptive, slowly-dividing, de-differentiated, drug-resistant cell state selectively inhibitable by drug combinations [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr PR17.

Methyl pyruvate protects a normal lung fibroblast cell line from irinotecan-induced cell death: Potential use as adjunctive to chemotherapy.

The Warburg Effect, characterized by increased rate of glycolysis even under normoxic conditions, is one of the hallmarks of cancer. Relatively lower oxidative phosphorylation (OXPHOS) is also a characteristic feature in cancer cells. We hypothesized that interference with this phenomenon, by introducing exogenous pyruvate, would upset this cancer phenotype and boost the energy requirements of normal cells. We find that methyl pyruvate protects irinotecan-treated normal lung fibroblast cell line (MRC-5) probably by turning off the p53/p21 axis of the apoptotic pathways. When the MRC-5 fibroblasts recover in drug-free medium, the intrinsic apoptotic pathway is also turned off and the cells survive with no discernible exponential growth during the observation period. In contrast, the mere introduction of exogenous pyruvate kills the lung cancer cell line (A549). Although, functional p53 is important in the drug-induced cancer cell death, it is probably not essential because cancer cell lines with mutated p53 also die albeit less efficiently. We conclude that methyl pyruvate may preferentially kill cancer cells and protect normal cells during chemotherapy.

Abstract 4103: IL-6 dynamics regulate neuroendocrine transformation in gefitinib acquired resistance EGFR mutant lung cancer cells

Abstract Transformation to small-cell lung cancer (SCLC, one of aggressive neuroendocrine [NE] tumor) is reported when activating epidermal growth factor receptor (EGFR) mutant non-small-cell lung cancer acquired resistance to tyrosine kinase inhibitors (TKI, such as gefitinib). IL-6 activation confers to acquire TKI resistance and associates with p53 and RB inactivation those are SCLC hallmark changes. Whether NE transformation could phenocopy in isogenic acquired resistance cell line and the role of IL-6 in this process remain unknown. We established 827GRs (including 827GR, 827GR+ and 827GR.M6) acquired resistance to gefitinib from HCC827 cells by long term stepwise treated with gefitinib and they still had EGFR exon 19 deletion without acquired T790M. 827GR was parental resistance line with unstable gefitinib resistance in drug-free medium by passage. We maintained 827GR in medium with or without 1μM gefitinib over 6 months to generate stable clones: 827GR+ and 827GR.M6. 827GRs had SCLC hallmark changes, i.e., inactivation of p53, RB and Notch by western blot and gene set enrichment analysis. Compared to HCC827, 827GRs were more sensitive to cisplatin and etoposide but not paclitaxel. IL-6 level was positive correlated with gefitinib resistance among 827GRs by cytokine array and ELISA. Interestingly, among 827GRs, 827GR.M6 harbored low IL-6 secretion had obviously NOTCH-ASCL1-DLL3 alteration, high NE marker expression and significant inter-rater agreement with selected Byers’ SCLC gene signature than high IL-6 secretion 827GR+, suggesting IL-6 dynamics might regulate NE marker expression. IL-6 genetic manipulation in HCC827 and 827GR+ also demonstrated this phenomenon. Moreover, IL-6 dynamics correlate with NE expression also showed in patient derived lung cancer cell line in published microarray dataset (GSE64322). In conclusion, our work demonstrated activating EGFR mutant lung cancer acquired resistance to TKI with NE transformation could phenocopy in isogenic cell line model and IL-6 dynamics might regulate this process. Citation Format: Shang-Yin Wu, Hsuan-Heng Yeh, Chun-Hua Hung, Chien-Chung Lin, Wen-Pin Su, Wu-Chou Su. IL-6 dynamics regulate neuroendocrine transformation in gefitinib acquired resistance EGFR mutant lung cancer cells [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 4103. doi:10.1158/1538-7445.AM2017-4103

Actions of sesquiterpene lactones isolated from Moquiniastrum polymorphum subsp. floccosum in MCF7 cell line and their potentiating action on doxorubicin

BackgroundIn order to obtain better clinical results in anticancer therapies, polychemotherapy or combination therapies are used. For this, the combinations are required to increase the efficacy and reduce the adverse reactions of the associated chemotherapies. The aim of this study was to evaluate the cytotoxic, apoptotic and (anti)proliferative potential of two sesquiterpene lactones isolated from Moquiniastrum polymorphum, 11,13-diidrozaluzanin C (1) and gochnatiolide C (2), and their associations with chemotherapeutic agents irinotecan, tamoxifen, cisplatin, 5-fluouracyl and doxorubicin in the tumoral lineage of MCF-7 breast adenocarcinoma.MethodsThe analyses were performed by MTT cytotoxicity assays, drug combination index (CI), apoptosis morphological assay and cell proliferation assay. Treatments were evaluated with short exposure times (4 h), followed or not by recovery in drug-free medium for 24 h. For the cell viability assay the statistical analysis was performed using software INSTAT, and the ANOVA/Tukey test was applied. Combination Indices (CI) was made using CompuSyn software and demonstrated through isoboles. The assays that evaluated cell death and proliferation used statistical analysis SAS 9.4 (Statistical Analysis System), and the procedure adopted was PROC NPAR1WAY. The Wilcoxon test at 5% level was applied for comparing statistical differences.ResultsThe results demonstrated that the compounds decrease cell viability and increase their action when associated with irinotecan, tamoxifen and doxorubicin (CI < 1 and CI = 1). In periods of 4 h-exposure, the compounds cause cell death by apoptosis and after 24 h, they increase the mean number of cells in programmed cell death, especially when treated with 2. In addition, the association with doxorubicin increases the apoptotic potential induced by tested compounds. Both isolates had effect on the reduction of the number of mitoses, especially when 2 at its highest concentration is associated with doxorubicin.ConclusionsFinally, these compounds are presented as potential agents in chemotherapy combined with doxorubicin, since they trigger the mechanism of apoptosis, which, through the mechanism of action of sesquiterpene lactones, leads to a reduction in toxicity. In addition, the tested compounds have the ability to exert a synergistic action with doxorubicin, possibly by down-regulating the drug resistance mechanisms.

Abstract P3-07-15: Treatment with a Jumonji demethylase inhibitor JIB-04 sensitizes resistant breast cancer cells to chemotherapeutic drugs in an in vitro model of intrinsic resistance

Abstract We have recently developed a usable model of panresistance in triple-negative breast cancer. We have shown that only 0.01% cells survive a metabolic challenge involving lack of glutamine in culture medium of SUM149 triple-negative Inflammatory Breast Cancer cell line. These cells, designated as SUM149-MA for metabolic adaptability, are resistant to chemotherapeutic drugs, and they efficiently metastasize to multiple organs in nude mice. The MA cells can survive a variety of challenges in the metastasis process because of their embryo-like nature and high adaptability. Epigenetic state is a key player in cancer evolution. Therefore, a variety of compounds are being developed that would target epigenome for anticancer therapy. We believe that the identification of potentially effective epigenetic drugs can benefit from their evaluation in a usable system of panresistant cancer cells such as our model system of MA cells. This is particularly true for the compounds that may affect cellular adaptability upon a long treatment rather than affecting cell proliferation or apoptosis in a short period. In this study we evaluated JIB-04, a pan-inhibitor of Jumonji family of histone demethylases, for its ability to affect intrinsic resistance in MA cells. First, we found that MA cells are more resistant to JIB-04 treatment in a comparative evaluation with the parental SUM149 cells line. A treatment with 125-250 nM JIB-04 for 18-20 days killed all parental cells but not all MA cells; at least 20 MA cells survived and formed colonies after a 14-days recovery. Next, we asked whether JIB-04 treatment would affect the intrinsic resistance of MA cells, e.g., sensitize them to treatment with chemotherapeutic drugs. In order to assess the most resistant cells in population, these experiments involved treatment of MA cells with 125 nM JIB-04 for 10 days followed by a recovery in drug-free medium for 7 days; then we treated the cells with 5 nM paclitaxel or 100 nM doxorubicin for 6-8 days followed by a recovery for 1-12 days. We found that JIB-04 pre-treatment sensitized MA cells and the parental SUM149-Luc cell line to both these chemotherapeutic drugs as assessed by a dramatic drop in the number of colonies as compared to those obtained after treatment of control cells with the same chemotherapeutic drugs. To consider how our results may apply in the context of evolving disease in patients who are not taking chemotherapeutic drugs, we asked whether JIB-04 treatment would alter expression of surface molecules on resistant breast cancer cells such that they can be destroyed by immune system. In this regard, we specifically found that JIB-04 treatment increased the expression of PD-L1 in MA cells. Published results from a clinical study suggest that PD-L1 expression on breast cancer cells renders them responsive to various therapies, not just immune checkpoint blockade targeting PD-L1. In conclusion, our results suggest a novel approach for evaluating potential anticancer agents such as JIB-04 that would halt cancer evolution and prevent development of resistance to currently offered therapies. Supported by a State of Texas Grant for Rare and Aggressive Cancers. Citation Format: Singh B, Washburn LJ, Kinne HE, Milligan RD, Lucci A. Treatment with a Jumonji demethylase inhibitor JIB-04 sensitizes resistant breast cancer cells to chemotherapeutic drugs in an in vitro model of intrinsic resistance [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-07-15.

Establishment of Proliferative Tetraploid Cells from Nontransformed Human Fibroblasts.

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to be responsible for carcinogenesis in such tissues. Although proliferative polyploid cells are requisite for analyzing chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is rather challenging. Induction of tetraploidy by chemical agents usually results in a mixture of diploid and tetraploid populations, and most studies employed fluorescence-activated cell sorting or cloning by limiting dilution to separate tetraploid from diploid cells. However, these procedures are time-consuming and laborious. The present report describes a relatively simple protocol to induce proliferative tetraploid cells from normal human fibroblasts with minimum contamination by diploid cells. Briefly, the protocol is comprised of the following steps: arresting cells in mitosis by demecolcine (DC), collecting mitotic cells after shaking off, incubating collected cells with DC for an additional 3 days, and incubating cells in drug-free medium (They resume proliferation as tetraploid cells within several days). Depending on cell type, the collection of mitotic cells by shaking off might be omitted. This protocol provides a simple and feasible method to establish proliferative tetraploid cells from normal human fibroblasts. Tetraploid cells established by this method could be a useful model for studying chromosome instability and the oncogenic potential of polyploid human cells.

Mechanisms of Resistance to Apto-253

APTO-253 is a small molecule indolyl-phenanthroline-imidazole that exerts potent cytotoxicity against human acute myeloid leukemia and lymphoma cells but not normal PBMC or bone marrow cells. The goal of this project was to provide fundamental information on the mechanism of resistance of APTO-253 so as to identify synthetic lethal interactions that can guide combination drug studies.The human Burkitt’s lymphoma cell line Raji was exposed to progressively increasing concentrations of APTO-253 over a period of 6 months to generate the resistant subline Raji/253R. Raji/253R cells were 16.7 ± 3.9-fold resistant to a 120 h exposure to APTO-253 (IC50: Raji IC50 91.9 ± 22.3 nm; Raji /253R IC50 1387.7 ± 98.5 nm). The level of resistance remained stable for at least 3 month during culture in drug-free media. Exposure of the parental Raji cells to 500 nM APTO-253 for 24 h produced: a) G1 arrest accompanied by up-regulation of proapoptotic proteins BIK and BAD and down-regulation of anti-apoptotic protein MCL-1; b) cleavage of PARP; and, c) time-dependent up-regulation of γH2AX. None of these changes were detected in the Raji/253R cells subjected to the same exposure. RNA-seq analysis of the parental and resistant cells in the absence of drug exposure demonstrated marked up-regulation of ATP-binding cassette sub-family G member 2 (ABCG2) in the Raji/253R cells that was confirmed by qRT-PCR and Western blot analysis. Ko143 is a specific inhibitor of ABCG2 that was not toxic to either Raji or Raji/253R when used as a single agent. When Raji/253R cells were concurrently treated with either 5 nM or 50 nM Ko143 and APTO-253, resistance to APTO-253 was reversed by 1.6- and 6.8-fold, respectively, confirming that overexpression of ABCG2 is one mechanism of resistance to APTO-253. The overexpression of ABCG2 in Raji/253R resulted in 2.2-fold less intracellular APTO-253 in Raji/253R cells relative to the Raji cells after a 6 h exposure to 500 nM APTO-253. Raji/253R was found to be cross-resistant to topotecan, a known ABCG2 substrate, and Ko143 also reverses topotecan resistance in Raji/253R. Etoposide is also an ABCG2 substrate but, surprisingly, Raji/253R cells were found to be hypersensitive to etoposide. Etoposide is a non-intercalating topoisomerase II inhibitor and a potent inducer double strand breaks. The hypersensitivity to etoposide in Raji/253R suggests that DNA repair pathways are aberrant in the Raji/253R cells. The second major finding in the Raji/253R cells is that they express only a truncated 45 kDa form of c-Myc in which 159 nucleotides (53 amino acids) are missing from exon 2. Sanger sequencing disclosed microhomologies at each end of the deletion suggesting that microhomology mediated end-joining (MMEJ) repair of a double strand break produced as a result of APTO-253 exposure was responsible for the deletion. BRCA1 plays crucial roles in both homologous recombination (HR) and non-homologous end joining (NHEJ) repair. The RNA-seq RPKM (reads per kilobase exon per million) data revealed that there was less BRCA1 in Raji/253R suggesting that the two classic DNA repair mechanisms (HR and NHEJ) may be impaired in the Raji/253R cells. We conclude that high level resistance to APTO-253 is mediated in part by ABCG2, and that adaption to the drug has resulted in expression of an altered form of c-Myc whose link to the resistant phenotype is not yet established. DisclosuresNo relevant conflicts of interest to declare.

Abstract 2140: Cabazitaxel is more active than first generation taxanes in ABCB1(+) cell lines due to its higher intracellular accumulation and reduction in ATPase stimulation

Abstract The taxanes paclitaxel (Taxol) and docetaxel (Taxotere) have substantial clinical activity in breast, ovarian, lung, and other cancers, but their clinical efficacy is limited by preexisting or acquired drug resistance, such as the expression of the ATP-dependent multidrug resistance (MDR) transporter P-glycoprotein (P-gp). Cabazitaxel (Jevtana), the dimethoxy derivative of docetaxel, was identified because of its activity in taxane-resistant models both in vitro and in vivo. Our studies confirm that cabazitaxel is more active in cell variants that express P-gp, and the objective of this study was to investigate cabazitaxel's affinity for the transporter. Taxane accumulation patterns were studied in ABCB1(+) variants using [14C]-radiolabeled docetaxel and cabazitaxel over a time course up to 1 h. Time points were collected and spun (10,000 x g, 1 min) through Nyosil M20 oil thereby terminating uptake, cell pellets lysed with 2% (w/v) SDS, and counts determined by liquid scintillation, with all measurements normalized to protein content. The kinetics of drug accumulation between the two taxanes is different, with the maximum intracellular drug concentration achieved faster with cabazitaxel (5 min) than docetaxel (15-30 min) in all cell lines. ABCB1(+) variants accumulated less taxane, and these levels could be restored to parental levels in the presence of known P-gp inhibitors (2 μM PSC-833, valspodar). However, the MDR cell models tested accumulated twice as much cabazitaxel than docetaxel under identical experimental conditions. Efflux in drug-free medium confirmed that ABCB1(+) variants retained 2.2x more cabazitaxel than docetaxel. We observed a strong association (r2 = 0.91) between the degree of taxane resistance conferred by P-gp expression and the accumulation differences observed with the two taxanes. Several low P-gp-expressing cell models were not cross-resistant to cabazitaxel while demonstrating resistance to docetaxel. Furthermore, we determined ATPase stimulation in membranes isolated from our MDR cell models as an indirect measure of P-gp activity following taxane treatment. We observed a 1.9x reduction in sodium orthovanadate-sensitive ATPase stimulation resulting from treatment with cabazitaxel than with docetaxel (1 μM for 30 min). Future experiments will include direct photoaffinity labeling of P-gp with [3H]-azido-cabazitaxel and docetaxel, and competition assays with known P-gp substrates. Our studies indicate that the improved activity of cabazitaxel in MDR models might be due to its reduced affinity for P-gp compared to docetaxel. Citation Format: George E. Duran, Dietmar Weitz, Dorothée Sémiond, Diego Gianolio, Sandrine Macé, Branimir I. Sikic. Cabazitaxel is more active than first generation taxanes in ABCB1(+) cell lines due to its higher intracellular accumulation and reduction in ATPase stimulation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2140.

Effect of a tretinoin derivative ECPIRM on retinoic acid receptors and skin irritation responses to it in mice

Objective To estimate the effect of a tretinoin derivative ECPIRM on retinoic acid receptors(RARs), and to observe skin irritation responses to it in mice. Methods Cultured SCL-1 cells were divided into 2 groups to be treated with culture medium containing 10 μmol/L ECPIRM(ECPIRM group)or 10 μmol/L all-trans retinoic acid(ATRA)(ATRA group)for 24 hours, and those treated with drug-free culture medium served as the control group. Western blot analysis and real-time fluorescence-based quantitative PCR were performed to quantify the protein and mRNA expressions of RARs(RARα, RARβ, RARγ and RXRα)respectively. In addition, real-time fluorescence-based quantitative PCR was conducted to measure the mRNA expressions of two target genes of the activated RAR signaling pathway, i.e., cytochrome P450 26A1(CYP26A1)and tazarotene-induced gene 1(TIG1). Eight BALB/c mice were equally divided into 2 groups to be topically treated with 0.075% ECPIRM gel or 0.05% ATRA cream at equal molar concentrations on the shaved skin once daily for 21 successive days. Skin irritation reactions were assessed in these mice. Results Compared with the control group, the ATRA group showed significantly increased protein and mRNA expressions of RARα, RARβ and RARγ(all P 0.05). Obvious Skin irritation reactions such as erythema and desquamation were observed in BALB/c mice after 2-day topical treatment with ATRA cream, and their degree peaked after 5-day treatment. However, neither erythema nor desquamation was observed in BALB/c mice during 21-day treatment with 0.075% ECPIRM gel. Conclusion Unlike ATRA, ECPIRM cannot activate the canonical RAR signaling pathway or cause skin irritation reactions. Key words: Tretinoin; Skin irritancy tests; Mice; ECPIRM; Retinoic acid receptor

Alternative methods for the Plasmodium falciparum artemisinin ring-stage survival assay with increased simplicity and parasite stage-specificity.

BackgroundArtemisinin-based combination therapy is recommended to treat Plasmodium falciparum worldwide, but observations of longer artemisinin (ART) parasite clearance times (PCTs) in Southeast Asia are widely interpreted as a sign of potential ART resistance. In search of an in vitro correlate of in vivo PCT after ART treatment, a ring-stage survival assay (RSA) of 0–3 h parasites was developed and linked to polymorphisms in the Kelch propeller protein (K13). However, RSA remains a laborious process, involving heparin, Percoll gradient, and sorbitol treatments to obtain rings in the 0–3 h window. Here two alternative RSA protocols are presented and compared to the standard Percoll-based method, one highly stage-specific and one streamlined for laboratory application.MethodsFor all protocols, P. falciparum cultures were synchronized with 5 % sorbitol treatment twice over two intra-erythrocytic cycles. For a filtration-based RSA, late-stage schizonts were passed through a 1.2 μm filter to isolate merozoites, which were incubated with uninfected erythrocytes for 45 min. The erythrocytes were then washed to remove lysis products and further incubated until 3 h post-filtration. Parasites were pulsed with either 0.1 % dimethyl sulfoxide (DMSO) or 700 nM dihydroartemisinin in 0.1 % DMSO for 6 h, washed twice in drug-free media, and incubated for 66–90 h, when survival was assessed by microscopy. For a sorbitol-only RSA, synchronized young (0–3 h) rings were treated with 5 % sorbitol once more prior to the assay and adjusted to 1 % parasitaemia. The drug pulse, incubation, and survival assessment were as described above.ResultsRing-stage survival of P. falciparum parasites containing either the K13 C580 or C580Y polymorphism (associated with low and high RSA survival, respectively) were assessed by the described filtration and sorbitol-only methods and produced comparable results to the reported Percoll gradient RSA. Advantages of both new methods include: fewer reagents, decreased time investment, and fewer procedural steps, with enhanced stage-specificity conferred by the filtration method.ConclusionsAssessing P. falciparum ART sensitivity in vitro via RSA can be streamlined and accurately evaluated in the laboratory by filtration or sorbitol synchronization methods, thus increasing the accessibility of the assay to research groups.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-016-1148-2) contains supplementary material, which is available to authorized users.