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Previous studies have shown that sphingosine kinase interacting protein (SKIP) inhibits sphingosine kinase (SK) function in fibroblasts. SK phosphorylates sphingosine producing the potent signaling molecule sphingosine-1-phosphate (S1P). SKIP gene (SPHKAP) expression is silenced by hypermethylation of its promoter in acute myeloid leukemia (AML). However, why SKIP activity is silenced in primary AML cells is unclear. Here, we investigated the consequences of SKIP down-regulation in AML primary cells and the effects of SKIP re-expression in leukemic cell lines. Using targeted ultra-HPLC-tandem MS (UPLC-MS/MS), we measured sphingolipids (including S1P and ceramides) in AML and control cells. Primary AML cells had significantly lower SK activity and intracellular S1P concentrations than control cells, and SKIP-transfected leukemia cell lines exhibited increased SK activity. These findings show that SKIP re-expression enhances SK activity in leukemia cells. Furthermore, other bioactive sphingolipids such as ceramide were also down-regulated in primary AML cells. Of note, SKIP re-expression in leukemia cells increased ceramide levels 2-fold, inactivated the key signaling protein extracellular signal-regulated kinase, and increased apoptosis following serum deprivation or chemotherapy. These results indicate that SKIP down-regulation in AML reduces SK activity and ceramide levels, an effect that ultimately inhibits apoptosis in leukemia cells. The findings of our study contrast with previous results indicating that SKIP inhibits SK function in fibroblasts and therefore challenge the notion that SKIP always inhibits SK activity.

A 3'-protected route toward the synthesis of the diastereomers of clinically active ProTides, NUC-1031 and NUC-3373, is described. The in vitro cytotoxic activities of the individual diastereomers were found to be similar to their diastereomeric mixtures. In the KG1a cell line, NUC-1031 and NUC-3373 have preferential cytotoxic effects on leukemic stem cells (LSCs). These effects were not diastereomer-specific and were not observed with the parental nucleoside analogues gemcitabine and FUDR, respectively. In addition, NUC-1031 preferentially targeted LSCs in primary AML samples and cancer stem cells in the prostate cancer cell line, LNCaP. Although the mechanism for this remains incompletely resolved, NUC-1031-treated cells showed increased levels of triphosphate in both LSC and bulk tumor fractions. As ProTides are not dependent on nucleoside transporters, it seems possible that the LSC targeting observed with ProTides may be caused, at least in part, by preferential accumulation of metabolized nucleos(t)ide analogues.

Purpose: NUC-1031 is a first-in-class ProTide modification of gemcitabine. In PRO-002, NUC-1031 was combined with carboplatin in recurrent ovarian cancer. Patients and Methods: NUC-1031 was administered on days 1 and 8 with carboplatin on day 1 every 3 weeks for up to six cycles. Four dose cohorts of NUC-1031 (500, 625, and 750 mg/m2) with carboplatin (AUC4 or 5) were investigated. Primary endpoint was recommended phase II combination dose (RP2CD). Secondary endpoints included safety, investigator-assessed objective response rate (ORR), clinical benefit rate (CBR), progression-free survival (PFS), and pharmacokinetics. Results: A total of 25 women with recurrent ovarian cancer, a mean of 3.8 prior lines of chemotherapy, and a median platinum-free interval of 5 months (range: 7–451 days) were enrolled; 15 of 25 (60%) were platinum resistant, 9 (36%) were partially platinum sensitive, and 1 (4%) was platinum sensitive. Of the 23 who were response evaluable, there was 1 confirmed complete response (4%), 5 partial responses (17%), and 8 (35%) stable disease. The ORR was 26% and CBR was 74% across all doses and 100% in the RP2CD cohort. Median PFS was 27.1 weeks. NUC-1031 was stable in the plasma and rapidly generated high intracellular dFdCTP levels that were unaffected by carboplatin. Conclusions: NUC-1031 combined with carboplatin is well tolerated in recurrent ovarian cancer. Highest efficacy was observed at the RP2CD of 500 mg/m2 NUC-1031 on days 1 and 8 with AUC5 carboplatin day 1, every 3 weeks for six cycles. The ability to deliver carboplatin at AUC5 and the efficacy of this schedule even in patients with platinum-resistant disease makes this an attractive therapeutic combination.

Mutating the side-chains of amino acids in a peptide ligand, with unnatural amino acids, aiming to mitigate its short half-life is an established approach. However, it is hypothesized that mutating specific backbone peptide bonds with bioisosters can be exploited not only to enhance the proteolytic stability of parent peptides, but also to tune its receptor subtype selectivity. Towards this end, four [Y]6-Angiotensin II analogues are synthesized where amide bonds have been replaced by 1,4-disubstituted 1,2,3-triazole isosteres in four different backbone locations. All the analogues possessed enhanced stability in human plasma in comparison with the parent peptide, whereas only two of them achieved enhanced AT2R/AT1R subtype selectivity. This diversification has been studied through 2D NMR spectroscopy and unveiled a putative more structured microenvironment for the two selective ligands accompanied with increased number of NOE cross-peaks. The most potent analogue, compound 2, has been explored regarding its neurotrophic potential and resulted in an enhanced neurite growth with respect to the established agent C21. © 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. ISSN:0947-6539 ISSN:1521-3765

Background Gemcitabine is used to treat a wide range of tumours but its efficacy is limited by cancer cell resistance mechanisms. NUC-1031, a phosphoramidate modification of gemcitabine, is the first anti-cancer ProTide to enter the clinic and is designed to overcome these key resistance mechanisms. Methods Sixty-eight patients with advanced solid tumours who had relapsed after treatment with standard therapy were recruited to a dose escalation study to determine the recommended Phase II dose (RP2D) and assess safety of NUC-1031. Pharmacokinetics and anti-tumour activity was also assessed. Results Sixty-eight patients received treatment, 50% of whom had prior exposure to gemcitabine. NUC-1031 was well-tolerated with the most common Grade 3/4 adverse events of neutropaenia, lymphopaenia and fatigue occurring in 13 patients each (19%). In 49 response-evaluable patients, 5 (10%) achieved a partial response and 33 (67%) had stable disease, resulting in a 78% disease control rate. Cmax levels of the active intracellular metabolite, dFdCTP, were 217-times greater than those reported for equimolar doses of gemcitabine, with minimal toxic metabolite accumulation. The RP2D was determined as 825 mg/m2 on Days 1, 8 and 15 of a 28-day cycle. Conclusion NUC-1031 was well-tolerated and demonstrated clinically significant anti-tumour activity, even in patients with prior gemcitabine exposure and in cancers not traditionally perceived as gemcitabine-responsive. Trial registration: clinicaltrials.gov identifier: NCT01621854

Background: NUC-1031 is a ProTide transformation of the nucleoside analog gemcitabine, designed to overcome key cancer resistance mechanisms and generate significantly higher levels of the active metabolite, dFdCTP. Its incorporation into DNA interrupts cell replication and induces DNA damage, leading to cell death. NUC-1031 has shown broad clinical activity across a range of solid tumors as both a single agent and in combination with platinum agents. Increasing evidence in the clinical setting in both ovarian and biliary tract cancer populations suggests potential synergy between NUC-1031 and platinum agents, which may lead to increased anti-tumor activity. The aim of this study was to investigate the dose and time relationships between the incorporation of dFdCTP into DNA, the effect on the cell cycle and DNA damage response in biliary tract cancer cells treated with NUC-1031. Methods: Human intrahepatic cholangiocarcinoma HuCCT1 cells were treated with 500 nM (half-IC50 dose) or 1 µM (IC50 dose) NUC-1031 for 24h and samples were collected every 24h up to 96h. The intracellular conversion of NUC-1031 to its active metabolite dFdCTP was assessed by liquid chromatography mass spectrometry (LC-Q-TOF). This technique was also used to determine the incorporation of dFdCTP into DNA, using the dFdC signature as a surrogate and deoxyguanosine (2dG) for normalization. Flow cytometry was used to measure cell cycle populations and to analyse histone variant H2AX phosphorylation (γH2AX) as a marker for response to DNA damage, where increased signal is related to increase in response. Results: NUC-1031 generated dFdCTP in HuCCT1 cells. The incorporation of this fluorinated deoxycytidine into DNA was dose-dependent over time. At 48h post-treatment, the ratio of dFdC:2dG was 0.70 at half-IC50 dose, with comparable levels at IC50 dose. After 48h, the ratio continued to rise for the 1 µM dose but decreased with 500 nM. The incorporation of the active metabolite was accompanied by an increase in cells in S phase, up to 46% for 500 nM and 64% for 1 µM. This dose-dependent increase coincides with H2AX phosphorylation over time up to 48h. Active metabolite incorporation, S phase population and yH2AX signals reduced towards the 96h time point. Conclusion: NUC-1031 is a potent cytotoxic agent that causes DNA damage through the incorporation of its active metabolite into DNA, in a cell cycle dependant manner. This incorporation induces S phase arrest and evokes the DNA damage response via generation of double-strand breaks. The cytotoxic effect of NUC-1031 is prolonged post-treatment. Ongoing studies are investigating NUC-1031 in combination with cisplatin, where relative levels of dFdCTP incorporated in tumor DNA may act as a pharmacodynamic biomarker to determine synergy in patients who receive the combination treatment. Citation Format: Dillum Patel, Alison L. Dickson, Oliver J. Read, Clarissa M. Czekster, Essam A. Ghazaly, David J. Harrison, Jennifer Bré. NUC-1031 causes incorporation of fluorinated deoxycytidine into DNA, inducing persistent damage in biliary tract cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1003.

Gemcitabine is an anticancer agent acting against several solid tumors. It requires nucleoside transporters for cellular uptake and deoxycytidine kinase for activation into active gemcitabine-triphosphate, which is incorporated into the DNA and RNA. However, it can also be deaminated in the plasma. The intracellular level of gemcitabine-triphosphate is affected by scheduling or by combination with other chemotherapeutic regimens. Moreover, higher concentrations of gemcitabine-triphosphate may affect the toxicity, and possibly the clinical efficacy. As a consequence, different nucleoside analogs have been synthetized with the aim to increase the concentration of gemcitabine-triphosphate into cells. In this review, we summarize currently published evidence on pharmacological factors affecting the intracellular level of gemcitabine-triphosphate to guide future trials on the use of new nucleoside analogs.

Topically applied tetracaine is a local anaesthetic. A novel HPLC method for the rapid and sensitive analysis of tetracaine was developed and compared with a short end direction capillary zone electrophoresis (CZE) method. The method was developed and validated for the separation and quantification of tetracaine in skin samples removed by ‘tape-stripping’. Tetracaine was extracted from tape with 100% methanol, which was then diluted to 50% with water for injection. Tetracaine and the internal standard, procaine, were separated on a reversed-phase Luna PFP(2), 3 µm, 150 × 4.6 mm column at ambient temperature using isocratic elution with KH2PO4 buffer (pH 2.5) and methanol (35:65, v/v). The flow rate was 1 mL/min, with detection at 312 nm. The limit of quantification for tetracaine was 0.03 µg/mL. Calibration lines were linear with r2 values >0.99. The within- and between-assay imprecision and the percentage of inaccuracy for the QC samples including lower and upper limits of quantitation were 92%. Compared with CZE, the mean percentage error and the absolute mean percentage error were 0.62 and 6.29, respectively. The two methods were compared in a number of pharmacokinetic studies. Copyright © 2014 John Wiley & Sons, Ltd.

Gemcitabine is a nucleoside analogue commonly used in cancer therapy but with limited efficacy due to a high susceptibility to cancer cell resistance. The addition of a phosphoramidate motif to the gemcitabine can protect it against many of the key cancer resistance mechanisms. We have synthesized a series of gemcitabine phosphoramidate prodrugs and screened for cytostatic activity in a range of different tumor cell lines. Among the synthesized compounds, one in particular (NUC-1031, 6f) was shown to be potent in vitro. Importantly, compared with gemcitabine, 6f activation was significantly less dependent on deoxycytidine kinase and on nucleoside transporters, and it was resistant to cytidine deaminase-mediated degradation. Moreover, 6f showed a significant reduction in tumor volumes in vivo in pancreatic cancer xenografts. The ProTide 6f is now in clinical development with encouraging efficacy signals in a Phase I/II study, which strongly supports the ProTide approach to generate promising new anticancer agents.

Targeted therapies have yet to have significant impact on the survival of patients with bladder cancer. In this study, we focused on the urea cycle enzyme argininosuccinate synthetase 1 (ASS1) as a therapeutic target in bladder cancer, based on our discovery of the prognostic and functional import of ASS1 in this setting. ASS1 expression status in bladder tumors from 183 Caucasian and 295 Asian patients was analyzed, along with its hypothesized prognostic impact and association with clinicopathologic features, including tumor size and invasion. Furthermore, the genetics, biology, and therapeutic implications of ASS1 loss were investigated in urothelial cancer cells. We detected ASS1 negativity in 40% of bladder cancers, in which multivariate analysis indicated worse disease-specific and metastasis-free survival. ASS1 loss secondary to epigenetic silencing was accompanied by increased tumor cell proliferation and invasion, consistent with a tumor-suppressor role for ASS1. In developing a treatment approach, we identified a novel targeted antimetabolite strategy to exploit arginine deprivation with pegylated arginine deiminase (ADI-PEG20) as a therapeutic. ADI-PEG20 was synthetically lethal in ASS1-methylated bladder cells and its exposure was associated with a marked reduction in intracellular levels of thymidine, due to suppression of both uptake and de novo synthesis. We found that thymidine uptake correlated with thymidine kinase-1 protein levels and that thymidine levels were imageable with [18F]-fluoro-L-thymidine (FLT)–positron emission tomography (PET). In contrast, inhibition of de novo synthesis was linked to decreased expression of thymidylate synthase and dihydrofolate reductase. Notably, inhibition of de novo synthesis was associated with potentiation of ADI-PEG20 activity by the antifolate drug pemetrexed. Taken together, our findings argue that arginine deprivation combined with antifolates warrants clinical investigation in ASS1-negative urothelial and related cancers, using FLT-PET as an early surrogate marker of response. Cancer Res; 74(3); 896–907. ©2013 AACR.

Background: NUC-3373 is the first nucleotide analogue to bypass the key cancer resistance mechanisms associated with 5-fluorouracil (5-FU) and 5-fluorodeoxyuridine (FUDR) that significantly impede their clinical activity. In EC50 assays, NUC-3373 demonstrated up to 330x greater activity than 5-FU across multiple cancer cell lines and retained its activity in conditions mimicking drug resistance: thymidine kinase inhibition, thymidine phosphorylase over-expression and membrane transporter inhibition. In addition, whilst dihydropyrimidine dehydrogenase (DPD) significantly degraded 5-FU in cell lysates (p = 0.039), levels of NUC-3373 remained unaffected. Importantly, NUC-3373 generated 363x higher intracellular levels of the anti-cancer agent 5-fluorodeoxyuridine monophosphate, the active metabolite of 5-FU and FUDR. In colorectal cancer xenografts, NUC-3373 administered at 30 mg/kg 5-FU equimolar dose, demonstrated significantly greater tumour growth inhibition (47%) than 5-FU (25%). Finally, in formal toxicology studies NUC-3373 was significantly better tolerated than 5-FU, showing low conversion into the metabolite dihydrofluorouracil, dhFU (AUC plasma ratio dhFU:NUC-3373 = 0.03). NUC-3373 warrants clinical evaluation. Study Design: A two-part, Phase I open label, dose escalation and expansion study will be conducted to assess safety, pharmacokinetics and clinical activity of single agent NUC-3373 in participants with advanced solid tumours. A standard 3+3 dose escalation scheme will be used to identify the recommended Phase 2 dose (RP2D) of NUC-3373 comparing, in Part 1, a weekly schedule of administrationon days 1, 8, 15 and 22 of a 28-day cycle; and in Part 2 a fortnightly schedule of administration on days 1 and 15 of a 28-day cycle. Objectives: The primary objective is to establish the RP2D of NUC-3373 administered weekly and fortnightly. The secondary objectives are to assess the safety, pharmacokinetics, pharmacodynamics and anti-tumour activity of the agent. Tertiary objectives are to identify biomarkers that may help select those patients more likely to benefit from NUC-3373 than 5-FU. Major eligibility criteria: Histologically confirmed diagnosis of solid tumour, which is not amenable to, or refractory to standard chemotherapy, or for which no standard chemotherapy exists; measurable/evaluable disease; ECOG score 0-2; adequate bone marrow, liver and renal functions. Patients with a history of allergic reactions attributed to previous 5-FU or capecitabine treatment are excluded. Conclusions: NUC-3373 overcomes key cancer resistance mechanisms associated with 5-FU and a Phase I clinical study has been initiated. Citation Format: Sarah P. Blagden, Magdalena Slusarczyk, Michaela Serpi, Christopher McGuigan, Essam A. Ghazaly. First-in-human phase I study of the nucleotide analogue NUC-3373 designed to overcome fluoropyrimidine drug resistance mechanisms. [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 CT028.

Argininosuccinate synthetase 1 (ASS1) is the rate-limiting enzyme for arginine biosynthesis. ASS1 expression is lost in a range of different tumour types, including 50% of malignant pleural mesotheliomas. Starving ASS1-deficient cells of arginine with arginine blockers such as ADI-PEG20 can induce selective lethality and has shown great promise in the clinic. However, recent data has shown that ASS1-deficient tumours can become resistant to this therapy although the mechanisms behind this resistance remain unclear. We have generated the first model of ADI-PEG20 resistance in mesothelioma cells whereby we observe re-expression of ASS1, via demethylation of the ASS1 promoter. Through coordinated transcriptomic and metabolomic profiling, we have shown that ASS1-deficient cells have decreased levels of acetylated polyamines, resulting in an increased activation of the polyamine synthesis pathway. Upon arginine deprivation, we observe a decrease in polyamine metabolites in the ASS1-deficient cells only, suggesting that exogenous arginine is required to maintain polyamine biosynthesis in the absence of ASS1. We identify for the first time a compensatory increase in polyamine synthesis gene expression upon ASS1 loss and highlight a synthetic lethal dependence between ASS1-deficiency and polyamine metabolism, which could potentially be exploited for the treatment of ASS1-negative cancers. Citation Format: Peter W. Szlosarek, Matthew Locke, Essam Ghazaly, Laura Lattanzio, Cristiana Lo Nigro, Sarah A. Martin. Inhibition of the polyamine synthesis pathway is synthetically lethal with loss of argininosuccinate synthase 1 in cancer. [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 1050.

5565Background: Acelarin is a first-in-class nucleotide analogue utilising phosphoramidate chemistry to overcome the key gemcitabine resistance mechanisms and generate high intracellular levels of ...

Lapatinib is a specific HER1 and 2 targeted tyrosine kinase inhibitor now widely used in combination with chemotherapy in the clinical setting. In this work, we investigated the interactions between lapatinib and specific chemotherapy agents (cisplatin, SN-38, topotecan) in a panel of cell lines [breast (n = 2), lung (n = 2), testis (n = 4)]. A high-sensitivity cell proliferation/cytotoxicity ATP assay and flow cytometry were used to determine cell viability, apoptosis, and the effect of the drugs on cell-cycle distribution. CalcuSyn analysis was employed to formally identify synergistic interactions between drugs. Intracellular concentrations of SN-38 were measured using a novel high-performance liquid chromatography (HPLC) technique. Flow cytometry and HPLC techniques were used to identify the effect of lapatinib on drug influx and efflux pumps, using specific substrates and inhibitors of these pumps. Results showed significant synergy between SN-38, and lapatinib in the majority of cell lines (combination index < 0.75), associated with increased apoptosis. This synergy was not universal but, when observed (Susa S/R, H1975, H358, and MDA-MB-231 cell lines), was related to SN-38 intracellular accumulation (2.2- to 4.8-fold increase, P < 0.05 for each), attributable to the inhibition of the breast cancer–related protein (BCRP) efflux pump by lapatinib. Flow cytometry analysis showed that lapatinib (10 μmol/L) inhibited the efflux of mitoxantrone, a specific substrate of the BCRP pump, in a manner similar to fumitremorgin C, a known BCRP inhibitor, confirming lapatinib as a BCRP inhibitor. This work shows that lapatinib has a direct inhibitory effect on BCRP accounting for the synergistic findings. The synergy is cell line dependent and related to the activity of specific efflux pumps. Mol Cancer Ther; 9(12); 3322–9. ©2010 AACR.

Background: NUC-3373 is the first Nucleotide Analogue able to bypass the key drug resistance mechanisms associated with 5-fluorouracil (5-FU) and 5-fluorodeoxyuridine (FUDR) that severely hinder their clinical activity. The key cellular mechanisms causing drug resistance in cancer cells are the limited expression of activating kinases and nucleoside transporters, and overexpression of catabolic enzymes. The anti-neoplastic activity of 5-FU is largely attributed to its active metabolite, 5-fluorodeoxyuridine monophosphate (FdUMP), which inhibits the enzyme thymidylate synthase. As it already bears the monophosphate moiety, NUC-3373 is a pre-activated form of the active anti-cancer agent FdUMP. Here, we report potent in vitro and in vivobiological activity of NUC-3373. Methods: The cytotoxicity of 5-FU and the ProTide NUC-3373 was monitored with EC50 in vitro viability assays using 4 colorectal (Colo-205-luc; HT-29; HCT-116 and SW620); 3 lung (H1975; H1703 and SK-MES-1); 2 ovarian (OVCAR3 and A2780); 1 acute lymphoblastic leukemia (CCRF-CEM) and 1 cervix (HeLa) human tumour cell lines. These assays were also performed in conditions mimicking cancer resistance, where the activating enzyme thymidine kinase (TK) and the nucleoside transporter, hENT1, were mutated. Sensitivity of NUC-3373 and 5-FU to dihydropyrimidine dehydrogenase (DPD) degradation was assessed by absorption spectroscopy and UPLC-MS/MS. NUC-3373 anti-cancer activity was further evaluated in HT-29 nude mouse xenograft models. Results: NUC-3373 was more cytotoxic than 5-FU, achieving 2 to 333 fold lower EC50 values in the majority of cancer cell lines tested. TK inhibition reduced the cytotoxicity of NUC-3373 and FUDR by 4- and 136-fold respectively, suggesting NUC-3373 is more independent of TK. Inhibition of nucleoside transport decreased cytotoxic activity of NUC-3373 only mildly compared to a 63-fold reduction in FUDR cytotoxicity. NUC-3373 and 5-FU sensitivity to DPD degradation was assessed in cell lysates with or without the DPD inhibitor, gimeracil. NUC-3373 concentrations remained unaffected in the cell lysate irrespective of gimeracil treatment. In contrast, when the experiment was conducted with 5-FU, gimeracil treatment significantly increased the 5-FU concentration (p = 0.039) suggesting that NUC-3373, unlike 5-FU, is not a substrate for DPD metabolism. In colorectal cancer xenografts, NUC-3373 demonstrated greater tumour growth inhibition (47%) than 5-FU (25%). A toxicology study in which NUC-3373 was administered daily for 5 days/week for 4 consecutive weeks, at doses greater than 4 mg/kg/day in beagle dogs, compared favourably with that described following a single bolus intravenous administration of 5-FU. Conclusions: The novel ProTide, NUC-3373, overcomes the key cancer resistance mechanisms associated with 5-FU. NUC-3373 has efficacy in vitro and in vivo, and is resistant to DPD-mediated degradation. Results from formal toxicology assessments support the initiation of human clinical studies. A Phase I/II clinical study at Oxford University has been initiated to explore the safety, pharmacokinetic and clinical activity of NUC-3373 in participants with advanced solid tumours. Citation Format: Essam A. Ghazaly, Magdalena Slusarczyk, Christopher McGuigan, David Harrison, Sarah P. Blagden. NUC-3373: A novel pyrimidine nucleotide analogue that overcomes key cancer drug resistance limiting patient survival. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B46.

Introduction: Chemotherapeutic resistance is a common issue that severely limits the clinical utility of all nucleoside analogues. The key cellular mechanisms causing resistance are limited expression of activating kinases and nucleoside transporters, and overexpression of catabolising enzymes. Phosphoramidate chemistry has been applied to nucleoside analogues to generate a new class of anti-cancer agents, ProTides, which overcome all these resistance mechanisms. Here, we report potent in vitro and in vivo biological activity of NUC-1031, a gemcitabine-derived ProTide. Methods: The cytotoxicity of the ProTide NUC-1031 and gemcitabine was monitored through in vitro viability assays. Both compounds were tested against a broad range of cancer cell lines including the pancreatic lines MiaPaCa-2 and BxPC-3 (partially or fully resistant to gemcitabine). Mimicking cancer resistance conditions, the activating enzyme, deoxycytidine kinase (dCK) was inhibited with deoxycytidine, and the nucleoside transporter, hENT1, was blocked with dipyridamole. The resulting intracellular concentrations of the active moiety, dFdCTP, were then measured. The ability of gemcitabine and NUC-1031 to resist degradation by cytidine deaminase (CDA) was assessed using absorption spectroscopy. Activity of NUC-1031 and gemcitabine were further evaluated in MiaPaCa-2 and BxPC-3 nude mouse xenograft models. Results: NUC-1031 was more cytotoxic than gemcitabine in vitro, with a lower EC50. NUC-1031 activity was not significantly affected by dCK inhibition (EC50= 0.2nM to 0.7nM in the presence of deoxycytidine) whereas gemcitabine cytotoxicity was reduced by 75 fold (EC50= 1.4nM to 104.9nM). Inhibition of nucleoside transport with dipyridamole significantly decreased gemcitabine cytotoxicity (EC50= 611μM rose to >2000μM). In contrast, NUC-1031 cytotoxic activity remained unchanged and significantly lower than gemcitabine (EC50= 190μM to 162μM). Measured intracellular concentrations of dFdCTP were consistent with these results. Additionally, gemcitabine was catabolized within one minute of exposure to CDA, while NUC-1031 was impervious to deamination. Finally, in pancreatic cancer xenografts, at 11, 14, 21 & 25 days, NUC-1031 showed greater reduction in tumor volumes compared to gemcitabine and control. Conclusions: The novel ProTide, NUC-1031, overcomes the key cancer resistance mechanisms associated with gemcitabine. Notably, NUC-1031 reaches up to 20 fold higher intracellular levels of dFdCTP, even in resistant cells. A Phase I clinical study of NUC-1031 is nearing completion and has provided significant disease control in both patients refractory to, or deemed unsuitable for, gemcitabine therapy. Citation Format: Essam A. Ghazaly, Magdalena Slusarczyk, Malcolm Mason, John Gribben, Christopher McGuigan, Sarah Blagden. NUC-1031: A novel ProTide that overcomes the key cancer resistance mechanisms associated with poor survival. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT401. doi:10.1158/1538-7445.AM2014-CT401

Background CLL-induced severe T-cell dysfunction and ineffective anti-tumor immune-responses are hallmarks of the disease, but the specific interactions remain poorly understood. The PD-1/PD-L1 axis is an important mediator of T-cell dysfunction in solid tumors, and we have previously demonstrated that T cells from CLL patients exhibit impaired immunological synapse (IS) formation, predominantly mediated by PD-L1 (CD274) expression on CLL cells. We have also shown that the corresponding T-cell ligand PD-1 (CD279) is also upregulated, probably as a result of chronic antigenic stimulation, and that T cells have similarities to exhausted T cells observed in the context of chronic viral infection. Recent studies demonstrated that ibrutinib has impressive clinical activity in CLL, and mechanisms of action include irreversible binding of essential components of both B-cell- and T-cell-receptor signaling and interactions with the tumor microenvironment. Modulation of PD-1/PD-L1 interactions might therefore be an additional potential mode of action of this drug. Using the well-established Eμ-TCL1 (TCL1) mouse model of CLL, our aims were to demonstrate that (1) altered expression of PD-L1 on CLL cells and PD-1 on T cells and CLL are causally related, (2) the second ligand of PD-1, PD-L2, is also involved in mediating T-cell dysfunction, (3) T-cell effector function and IS formation are directly linked to PD-1 expression and (4) PD-1 associated in vivo T-cell responses can be modulated by treatment with ibrutinib. Methods As we have previously demonstrated that the spleen is the major organ of disease and representative of T-cell changes in peripheral blood and lymph nodes, experiments were performed on spleens from young TCL1 and wild-type (WT) C57Bl/6 mice with established CLL after adoptive transfer (AT) of syngeneic CLL cells (n=10), and on matched litter-mates after AT of healthy mouse B cells (n=10). An additional 12 mice were randomized to treatment with 25 mg/kg/d ibrutinib in 10% HP-β-CD, vehicle control, or sterile water, all administered by gavage, three weeks after AT of syngeneic CLL cells, and sacrificed 20 days later at a pre-defined endpoint. Multicolor flow cytometry was used to characterize T-cell subsets, expression of PD-1, PD-L1 and PD-L2 and T-cell effector function. Entire population CD8 T cells, PD-1+ve and PD-1-ve CD8 T cells were flow-sorted and used in IS formation assays with healthy murine B cells as antigen-presenting cells. Results Our previous studies using aged TCL1 mice and age-matched WT controls indicated that CLL-related PD-1 upregulation on antigen-experienced CD44+ CD8 T cells is masked by aging. However, PD-1 expression could also be induced in young TCL1 and WT mice by AT of CLL cells but not healthy B cells, suggesting a causal relationship with disease. Both PD-L1 and PD-L2 surface expression on CLL B cells were significantly increased compared to healthy B cells. Using TCL1 mice at early stages of CLL development when a healthy CD19+ B-cell population is still present, we were able to confirm that PD-L2 expression is a unique feature of CLL cells, with PD-L2 being virtually absent on healthy B cells. We next compared effector function and the ability to form IS of PD-1+ve and PD-1-ve antigen-experienced CD44+ T-cell subsets in mice with CLL. While proliferation was equally impaired in these subsets, they were both able to degranulate but generally failed to localize granzyme B to the IS. Although subsets produced some IL2/TNFα/IFNγ cytokine responses, PD-1+ve cells had significantly impaired TNFα and slightly impaired IL2 and IFNγ production, and a highly significant impaired ability to form IS compared to PD-1-ve cells. Treatment with ibrutinib reduced PD-1 expression on antigen-experienced CD44+ CD8 T cells and promoted stronger IFNγ production of entire population CD8 T cells, but failed to restore proliferation and granzyme B relocation to the IS. Conclusion Our in vivo data suggests that CLL and PD-1/PD-L1-mediated T-cell dysfunction are causally related, but that phenotypic and functional T-cell changes are not absolute and might be at least partly reversible by ibrutinib treatment. We also show that the second ligand of PD-1, PD-L2, is also a critical mediator of PD-1 associated T-cell dysfunction in CLL. Disclosures: Riches: Celgene: Research Funding. Gribben:Celgene: Research Funding; Pharmacyclics: Honoraria; Roche: Honoraria.

Background Arginine is an important amino acid for tumor cell growth and development. Argininosuccinate synthetase 1 (ASS1) is a key enzyme required for biosynthesis of arginine. Preclinically, ASS1-deficient tumor cells are particularly sensitive to arginine depletion, and randomised trials exploring this strategy are in progress in hepatocellular carcinoma and mesothelioma using the drug pegylated arginine deiminase (ADI-PEG20). In this study, we determined the metabolic changes induced by ADI-PEG20 treatment in a panel of bladder cancer and mesothelioma cell lines with promoter methylation-dependent silencing of ASS1. We used two cancer cell lines expressing ASS1 as a control. Methods Malignant mesothelioma, ASS1-negative (H2591, MSTO and JU77) and ASS1- positive (H28) cell lines were used. For bladder cancer, ASS1-negative (T24, 253J, UMUC-3) and ASS1-positive (RT112) cell lines were also used. All cells were treated with 750 ng/ml of ADI-PEG20 for 24 hours which induces up to 90% killing of arginine-dependent cell lines. Then, ultra performance liquid chromatography-mass spectrometry (UPLC-MS) technique was employed for untargeted quantitation of the metabolomic changes induced by ADI-PEG20. Results: All cell lines treated with ADI-PEG20 could be clearly discriminated from their untreated control pair when using PCA multivariate analysis. Arginine depletion was noted in all treated cell lines irrespective of ASS1 expression, however the reduction was at least one-log-fold greater in the ASS1-negative tumor cells. Citrulline, n-a-acetylcitrulline, and glutamine were upregulated specifically in ASS1-negative tumor cell lines. The main impact of ADI-PEG20 treatment was on pyrimidine metabolism in the ASS1-deficient tumor cells with upregulation of thymine and downregulation of thymidine, ureidosuccinic acid, uridine monophosphate and 5-hydroxymethyluracil. Notably, we identified that the reduction of the thymidine nucleotide pool was linked to suppression of thymidylate synthetase and dihydrofolate reductase, and paradoxically, reduced uptake of 3H-FLT. Finally, ADI-PEG20 caused variable effects on cytidine, uridine and ornithine levels in different cancer cell lines. Conclusion This study provides an insight into possible metabolic pathways affected by ADI-PEG20. The impact of ADI-PEG20 on thymidine metabolism, in particular, may be employed as a potential biomarker for optimizing the efficacy of ADI-PEG20 in the treatment of arginine auxotophic cancers. Citation Format: Essam A. Ghazaly, Phuong Luong, Malgorzata Chmielewska-Kassasir, Chantelle Hudson, John S. Bomalaski, L Wozniak, Norbert E. Avril, Simon P. Joel, Peter W. Szlosarek. A comprehensive untargeted UPLC-MS based metabolomic analysis of ASS1-deficient solid tumor cell lines treated with arginine deiminase. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1885. doi:10.1158/1538-7445.AM2013-1885

Background: Sphingolipids are important in cancer cell signalling. Sphingosine 1 phosphate (S1P) promotes cell survival and resistance to apoptosis, while S1P precursors ceramide (CER) and sphingosine (SPH), mediate antiproliferative and apoptotic responses. S1P is generated from SPH by sphingosine kinase (SK) enzymes (SK1 and SK2), with SK activity and localisation regulated by other proteins, including PKC, PKA and a SK anchoring protein (SKAP) that has been reported to negatively regulate SK1 activity in fibroblasts. S1P localisation is thought to play an important role in its function. Based on our preliminary observation in primary AML cells that SKAP expression resulted in an increase in S1P, we have investigated the effect of SKAP transfection on S1P production and localisation. Methods: K562, (and for some confirmatory experiments MCF-7), cells were transfected with the SKAP gene using standard techniques. SKAP is normally silenced in both cell lines. Transfection was confirmed by RNA expression. Intracellular and extracellular S1P and SPH, and intracellular SK activity (based on the production of C17 S1P from C17 SPH, an unnatural SPH that is a SK substrate) in intact cells were measured by LC-MS/MS. Phorbol 12-myristate 13-acetate (PMA) was used to induce membrane associated SK function, and MK-571 and fumitremorgen C (FTC) were used to block S1P efflux through ABCC1 and ABCG2 efflux pumps, respectively. Chemosensitivity to doxorubicin and imatinib in transfected cells was also studied. Results: K562 cells transfected with the SKAP gene showed a 2.5 fold increase in intracellular and extracellular levels of basal S1P compared to vector alone control. (In MCF-7 cells SKAP transfection resulted in an almost 10-fold increase in S1P). Further studies in K562 cells confirmed a significant increase in intracellular SK activity in SKAP transfected compared to vector alone cells, based on C17 S1P production (8.8 ± 2.6 vs 1.4 ± 0.4 ng/106 cells respectively after 24 hrs, p< 0.05). This increase was also observed, though to a lesser extent, in extracellular C17 S1P (678 ± 50 in SKAP vs 462 ± 47 pg/ml in vector alone, p< 0.05). In a proliferation assay this increase in SK activity was associated with a 25% increase in viable cell number (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3256. doi:1538-7445.AM2012-3256

Tumors deficient in argininosuccinate synthetase-1 (ASS1), a key enzyme involved in arginine synthesis, are auxotrophic for arginine and sensitive to amino acid deprivation. Currently, several trials are testing the arginine-depleting agent, pegylated arginine deiminase (ADI-PEG20) in patients with cancer, including a randomised phase II multicenter UK study in patients with ASS1-deficient mesothelioma (NCT01279967). Here, we sought to identify key pathways involved in the mechanism of action of ADI-PEG20 using a panel of ASS1-deficient malignant mesothelioma (MPM) cell lines as our model. Epithelioid (2591), biphasic (MSTO) and sarcomatoid (JU77) mesothelioma cells were exposed to ADI-PEG20 and analysed using Affymetrix gene expression profiling with validation of candidate genes by qPCR and western blotting, and metabolic studies by LC-MS and 1H-NMR. ADI-PEG20 modulated several metabolic genes involved in nucleotide synthesis in the MPM cell lines. We identified suppression of ribonucleotide reductase (M1 and M2 isoforms), thymidylate synthase and dihydrofolate reductase by 24hrs, the latter enzymes being known targets of pemetrexed, a multitargeted antifolate used in the first-line therapy of mesothelioma. Moreover, metabolic profiling revealed upregulation of several modified nucleosides, including ribothymidine and downregulation of the nucleotide, guanosine monophosphate. Lastly, in vitro studies confirmed potentiation between ADI-PEG20 and pemetrexed and sequential therapy led to reduced tumor growth in vivo. In conclusion, arginine depletion modulates several key enzymes involved in nucleotide synthesis, providing a rationale for combining ADI-PEG20 with pemetrexed-containing regimens in the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B139.

SummaryIncreasing chemotherapy delivery to tumors, while enhancing drug uptake and reducing side effects, is a primary goal of cancer research. In mouse and human cancer models in vivo, we show that coadministration of low-dose Cilengitide and Verapamil increases tumor angiogenesis, leakiness, blood flow, and Gemcitabine delivery. This approach reduces tumor growth, metastasis, and minimizes side effects while extending survival. At a molecular level, this strategy alters Gemcitabine transporter and metabolizing enzyme expression levels, enhancing the potency of Gemcitabine within tumor cells in vivo and in vitro. Thus, the dual action of low-dose Cilengitide, in vessels and tumor cells, improves chemotherapy efficacy. Overall, our data demonstrate that vascular promotion therapy is a means to improve cancer treatment.

7587 Background: Arginine is a key amino acid for tumorigenesis modulating a diverse array of metabolic pathways, from synthesis of proteins, NO and polyamines to nucleotide turnover and mTOR signa...

2531 Background: Acelarin (NUC-1031) is a first-in-class nucleotide designed to overcome key cancer cell resistance by having nucleoside transporter-independent cellular uptake, activity independen...

Background: Sphingosine kinase interacting protein (SKIP) has been shown to be mostly silenced by hypermethylation in AML [1]. SKIP interacts with and regulates the function of sphingosine kinase (SK) enzyme. SK activity results in phosphorylation of sphingosine (SPH) to form sphingosine 1 phosphate (S1P), which promotes cell survival and resistance to apoptosis. On the other hand, S1P precursors ceramide (CER) and SPH mediate antiproliferative and apoptotic responses. SKIP has been reported to negatively regulate SK1 activity in fibroblasts. Therefore, we investigated the consequences of SKIP silencing in primary AML cells. In addition, we studied the effects of SKIP re-expression in leukemic cell lines. Methods: CTS and K562 cells were transfected with SKIP gene using standard techniques. SKIP is normally silenced in both cell lines. Intracellular and extracellular S1P, SPH and CER were measured by UPLC-MS/MS. In addition, intracellular SK activity was determined based on C17 S1P production from C17 SPH substrate. Chemosensitivity to doxorubicin, Imatinib and Ara-C in transfected cells was also studied. Results: In Primary AML cells, intracellular S1P and CER concentrations were reduced compared to G-CSF-mobilized peripheral blood mononuclear cells. In addition, SK activity was found to be downregulated in AML primary cells. When we transfected leukemic cell lines with SKIP gene, S1P and CER showed at least 2 fold increase in intracellular and extracellular basal levels compared to vector alone control (Figure 1). Further studies confirmed a significant increase in intracellular SK activity in SKIP transfected compared to vector alone cells, based on C17 S1P production (8.8 ± 2.6 vs 1.4 ± 0.4 ng/106 cells respectively after 24 hrs, p< 0.05). This increase in S1P and CER was associated with increasing apoptotic signals as evidenced by Annexin V staining and cleaved PARP expression. Moreover, chemosensitivity to Imatinib and AraC was significantly increased in SKIP transfected cell lines. These experiments confirm the regulation of SK1 function by SKIP. Conclusion: These data indicate that SKIP is downregulated in AML leading to reduced SK activity, which ultimately inhibits the apoptosis response. Figure 1. Effect of SKIP transfection on intracellular concentrations of S1P. Figure 1. Effect of SKIP transfection on intracellular concentrations of S1P. 1. Saied, M.H., et al., Genome wide analysis of acute myeloid leukemia reveal leukemia specific methylome and subtype specific hypomethylation of repeats. PLoS One, 2012. 7(3): p. e33213. EAG and PS contributed equally JG and DT contributed equally Disclosures No relevant conflicts of interest to declare.

2576 Background: NUC-1031 is a novel nucleotide (ProTide) that evades all three key cellular resistance mechanisms associated with gemcitabine (dFdC). NUC-1031 bypasses nucleoside transporters, is activated independent of deoxycytidine kinase and is resistant to cytidine deaminase-mediated degradation. NUC-1031 has demonstrated broad antiproliferative activity in vitro and in vivo. Methods: Patients with relapsed/refractory advanced solid tumors entered in sequential cohorts of up to 6 patients, with escalating doses of NUC-1031 administered as a 5-10 minute IV injection weekly or twice-weekly. Ongoing objectives are to determine recommended phase II dose, safety profile, pharmacokinetics (PK) and preliminary anti-tumor activity. Results: 8 patients (5 female, 3 male) with pancreatic (2), colorectal (2), breast (1), and ovarian (1) cancers; cholangiocarcinoma (1) and unknown primary (1) have been enrolled. Two dose levels - 500mg/m2 (4) and 1000mg/m2 (1) weekly and one dose level - 375 mg/m2(3) twice-weekly. No DLTs have been observed. Mean AUC (0 - 24 h) for NUC-1031 was 150.3 ± 84.8 µM/h (n=5). dFdC and dFdU were detected in plasma up to 24 h (range of 0 - 5.8 µM for dFdC and 0 - 14.9 µM for dFdU). NUC-1031 excreted in urine mainly as dFdU. The Table shows rapid elimination of NUC-1031 from plasma and high intracellular levels of the active gemcitabine triphosphate at 2 and 24 h. Stable disease achieved in 1 patient with rapidly progressing breast cancer. Two further patients had symptomatic relief and improved QOL, including a dramatic reduction in ascites and pain. Conclusions: PK data show NUC-1031 has ≥ 10x higher intracellular levels of the active compound, dFdCTP, and significantly lower plasma Cmax levels of the toxic metabolite, dFdU, compared to equivalent levels of gemcitabine. NUC-1031 has shown better intracellular delivery and toxicity profile than gemcitabine with some promising early indicators of clinical efficacy. Clinical trial information: NCT01621854. [Table: see text]