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Background Cisplatin/gemcitabine is standard first-line treatment for patients with advanced biliary tract cancer (ABC). NUC-1031 (phosphoramidate transformation of gemcitabine) is designed to enhance efficacy by maximizing intratumoral active metabolites. Methods Patients with untreated ABC, Eastern Cooperative Oncology Group performance status 0–1 received NUC-1031 (625 or 725 mg/m2) and cisplatin (25 mg/m2) on days 1 and 8, every 21 days. Primary objectives were safety and maximum tolerated dose; secondary objectives were objective response rate (ORR), pharmacokinetics, progression-free survival (PFS), and overall survival (OS). Results Twenty-one patients (median age 61 years, n = 13 male; 17 cholangiocarcinoma, 2 ampullary, and 2 gallbladder cancer) received NUC-1031 625 mg/m2 (n = 8 and expansion n = 7; median six cycles) or 725 mg/m2 (n = 6; median 7.5 cycles). Treatment was well tolerated; most common treatment-emergent grade 3–4 adverse events occurring in more than one patient with 625 mg/m2 NUC-1031 were increased gamma-glutamyl transferase (GGT), 40%; alanine aminotransferase, 20%; bilirubin, 13%; neutropenia, 27%; decreased white cell count, 20%; thrombocytopenia, 13%; nausea, 13%; diarrhea, 13%; fatigue, 13%; and thrombus, 20% and with 725 mg/m2, increased GGT, 67%, and fatigue, 33%. NUC-1031 725 mg/m2 was selected as the recommended dose with cisplatin in ABC. ORR was 33% (one complete response, six partial responses), DCR was 76%, median PFS was 7.2 months (95% confidence interval [CI], 4.3–10.1), and median OS was 9.6 months (95% CI, 6.7–13.1). The median estimates of area under the plasma concentration–time curve from time 0 to last measurable time and maximum concentration were highest for NUC-1031 (218–324 μg•h/mL and 309–889 μg/mL, respectively) and lowest for di-fluoro-deoxycytidine (0.47–1.56 μg•h/mL and 0.284–0.522 μg/mL, respectively). Conclusion This is the first study reporting on the combination of NUC-1031 with cisplatin in ABC and demonstrated a favorable safety profile; 725 mg/m2 NUC-1031 in combination with cisplatin is undergoing phase III trial evaluation in ABC. (ClinicalTrials.gov ID: NCT02351765; EudraCT ID: 2015-000100-26). Implications for Practice The prognosis for patients with advanced biliary tract cancer (ABC) is approximately 1 year, and new treatment options are required. The cisplatin/gemcitabine combination is standard first-line treatment for patients with ABC. NUC-1031 is a phosphoramidate transformation of gemcitabine and is designed to enhance efficacy by maximizing intratumoral active metabolites. This phase Ib study (ABC-08) demonstrated a favorable safety profile of NUC-1031 in combination with cisplatin for the first-line treatment of patients with ABC, and 725 mg/m2 NUC-1031 was recommended in combination with cisplatin for phase III trial evaluation; the NuTide:121 global randomized study is currently enrolling.

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.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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.

AML is a genetically heterogeneous disease and understanding how different co-occurring mutations cooperate to drive leukemogenesis will be crucial for improving diagnostic and therapeutic options for patients. MIR142 mutations have been recurrently detected in IDH-mutated AML samples. Here, we have used a mouse model to investigate the interaction between these two mutations and demonstrate a striking synergy between Mir142 loss-of-function and IDH2R140Q, with only recipients of double mutant cells succumbing to leukemia. Transcriptomic analysis of the non-leukemic single and leukemic double mutant progenitors, isolated from these mice, suggested a novel mechanism of cooperation whereby Mir142 loss-of-function counteracts aberrant silencing of Hoxa cluster genes by IDH2R140Q. Our analysis suggests that IDH2R140Q is an incoherent oncogene, with both positive and negative impacts on leukemogenesis, which requires the action of cooperating mutations to alleviate repression of Hoxa genes in order to advance to leukemia. This model, therefore, provides a compelling rationale for understanding how different mutations cooperate to drive leukemogenesis and the context-dependent effects of oncogenic mutations.

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

Survival in children with relapsed/refractory a cute myeloid leukemia is unsatisfactory. Treatment consists of one course of fludarabine, cytarabine and liposomal daunorubicin, followed by fludarabine and cytarabine and stem-cell transplantation. Study ITCC 020/I-BFM 2009-02 aimed to identify the recommended phase II dose of clofarabine replacing fludarabine in the abovementioned combination regimen (3+3 design). Escalating dose levels of clofarabine (20-40 mg/m²/day x 5 days) and liposomal daunorubicin (40-80 mg/m²/day) were administered with cytarabine (2 g/m²/day x 5 days). Liposomal DNR was given on day 1, 3 and 5 only. The cohort at the recommended phase II dose was expanded to make a preliminary assessment of anti-leukemic activity. Thirty-four children were enrolled: refractory 1st (n=11), early 1st (n=15), ≥2ⁿd relapse (n=8). Dose level 3 (30 mg/m² clofarabine; 60 mg/m² liposomal daunorubicin) appeared to be safe only in patients without subclinical fungal infections. Infectious complications were dose-limiting. The recommended phase II dose was 40 mg/m² clofarabine with 60 mg/m² liposomal daunorubicin. Side-effects mainly consisted of infections. The overall response rate was 68% in 31 response evaluable patients, and 80% at the recommended phase II dose (n=10); 22 patients proceeded to stem cell transplantation. The 2-year probability of event-free survival (pEFS) was 26.5±7.6 and probability of survival (pOS) 32.4±8.0%. In the 21 responding patients, the 2-year pEFS was 42.9±10.8 and pOS 47.6±10.9%. Clofarabine expo-sure in plasma was not significantly different from that in single-agent studies. In conclusion, clofarabine was well tolerated and showed high response rates in relapsed/refractory pediatric acute myeloid leukemia. Patients with (sub)clinical fungal infections should be treated with caution. Clofarabine has been taken forward in the Berlin-Frankfurt-Münster study for newly diagnosed acute myeloid leukemia. The Study ITCC-020 was registered as EUDRA-CT 2009-009457-13; Dutch Trial Registry number 1880. CA extern

Aims Use of the anti-tumour antibiotic actinomycin D is associated with development of hepatotoxicity, particularly in young children. A paucity of actinomycin D pharmacokinetic data make it challenging to develop a sound rationale for defining dosing regimens in younger patients. The study aim was to develop a physiologically based pharmacokinetic (PBPK) model using a combination of data from the literature and generated from experimental analyses. Methods Assays to determine actinomycin D Log P, blood:plasma partition ratio and ABCB1 kinetics were conducted. These data were combined with physiochemical properties sourced from the literature to generate a compound file for use within the modelling-simulation software Simcyp (version 14 release 1). For simulation, information was taken from two datasets, one from 117 patients under the age of 21 and one from 20 patients aged 16-48. Results The final model incorporated clinical renal and biliary clearance data and an additional systemic clearance value. The mean AUC0-26h of simulated subjects was within 1.25-fold of the observed AUC0-26h (84 ng h ml(-1) simulated vs. 93 ng h ml(-1) observed). For the younger age ranges, AUC predictions were within two-fold of observed values, with simulated data from six of the eight age/dose ranges falling within 15% of observed data. Simulated values for actinomycin D AUC0-26h and clearance in infants aged 0-12 months ranged from 104 to 115 ng h ml(-1) and 3.5-3.8 l h(-1) , respectively. Conclusions The model has potential utility for prediction of actinomycin D exposure in younger patients and may help guide future dosing. However, additional independent data from neonates and infants is needed for further validation. Physiological differences between paediatric cancer patients and healthy children also need to be further characterized and incorporated into PBPK models.

The strategy of enzymatic degradation of amino acids to deprive malignant cells of important nutrients is an established component of induction therapy of acute lymphoblastic leukemia. Here we show that acute myeloid leukemia (AML) cells from most patients with AML are deficient in a critical enzyme required for arginine synthesis, argininosuccinate synthetase-1 (ASS1). Thus, these ASS1-deficient AML cells are dependent on importing extracellular arginine. We therefore investigated the effect of plasma arginine deprivation using pegylated arginine deiminase (ADI-PEG 20) against primary AMLs in a xenograft model and in vitro. ADI-PEG 20 alone induced responses in 19 of 38 AMLs in vitro and 3 of 6 AMLs in vivo, leading to caspase activation in sensitive AMLs. ADI-PEG 20-resistant AMLs showed higher relative expression of ASS1 than sensitive AMLs. This suggests that the resistant AMLs survive by producing arginine through this metabolic pathway and ASS1 expression could be used as a biomarker for response. Sensitive AMLs showed more avid uptake of arginine from the extracellular environment consistent with their auxotrophy for arginine. The combination of ADI-PEG 20 and cytarabine chemotherapy was more effective than either treatment alone resulting in responses in 6 of 6 AMLs tested in vivo. Our data show that arginine deprivation is a reasonable strategy in AML that paves the way for clinical trials.

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.

3030 Background: The inhibition of cellular nucleotide metabolism to promote apoptosis is a key principle of cancer therapy. This, in combination with platinum-induced DNA-damage, is key to promoting anti-cancer activity in a variety of tumors, including ovarian, biliary tract, lung, breast and bladder. NUC-1031, a phosphoramidate transformation of gemcitabine is designed to overcome resistance mechanisms that limit the efficacy of this nucleoside analog. NUC-1031 has shown broad clinical activity across multiple solid tumors as both a single agent and in combination with platinum agents. We show potential synergism between NUC-1031 and a platinum agent in advanced ovarian (OC) and biliary tract (BTC) cancers. Methods: PRO-002 was a phase Ib study; 25 patients (pts) with recurrent OC who had exhausted all other therapy options received NUC-1031 + carboplatin. 17 pts were considered platinum resistant (10) or platinum refractory (7). ABC-08 is a phase Ib study, 14 pts with advanced BTC treated in the first-line setting with NUC-1031 + cisplatin. Results: In PRO-002, strong efficacy signals were observed in non-platinum-responsive patients. Of the 17 response-evaluable platinum-resistant or refractory pts, 5 partial responses (PRs) and 11 stable diseases (SDs) were achieved, resulting in an ORR of 29% and a DCR of 94%. NUC-1031 + carboplatin was well-tolerated with no unexpected AEs; DLTs were myelosuppression and fatigue. Encouraging response rates were also observed in ABC-08 compared to historical standard of care (ABC-02). One CR (7%), 6 PRs (43%) and 1 SD (7%) were observed, resulting in an ORR of 50%. NUC-1031 + cisplatin was well-tolerated, with no unexpected AEs or DLTs. Complementary in vitro evidence suggests that the beneficial interaction occurs whereby platinum treatment sensitizes cells to NUC-1031. Conclusions: Increasing evidence suggests that NUC-1031 in combination with a platinum agent may have synergistic properties, leading to enhanced anti-cancer activity. In both OC and BTC, durable tumor shrinkage was observed. This was particularly encouraging in a platinum resistant/refractory OC population. Future studies utilizing both NUC-1031 plus a platinum agent will further elucidate the potential of this therapeutic combination.

Ciclosporin is used as an immunosuppressant in post-organ transplantation. Recently, many questions have been raised about using generic substitutes, especially with narrow therapeutic index drugs (NTIDs). In this study, a simple high-performance liquid chromatography (HPLC) method was developed, validated and applied to detection ciclosporin in dissolution testing. Seven ciclosporin products (gelatin capsules) were included in this study, obtained from Columbia (C), Egypt (E), India (I), Jordan (J), Pakistan (P), Saudi Arabia (S), and Turkey (T). The dissolution test was done for all capsules. The Conditions were as follows: 500 ml deionized water as the medium in apparatus 2 (Pharmatest, Germany), temperature 37.5 ± 0.5°C; 50 rev/min, sampling times were 5, 10, 15, 30, 60 and 90 min with 5 ml for each sample. HPLC separation was done by a C18 column, 5 μm, (4.6 × 250 mm, ACE 5) held at 50 ± 0.3°C. Analytes were isocratically eluted at 0.7 ml/min with acetonitrile and water (70+30%) and 0.03% trifluoroacetic acid, over the 25-min run time. The intra-day and inter-day imprecision for ciclosporin across the standard range was

TPS3617Background: 5-FU and its other forms, floxuridine and capecitabine, exert their anti-cancer activity mainly due to the active metabolite, fluorodeoxyuridine-monophosphate (FUDR-MP), which in...

TPS544 Background: The UK ABC-02 study established cisplatin and gemcitabine as the reference regimen for first-line treatment of patients (pts) with advanced biliary tract cancers (BTCs) (median overall survival (OS): 11.7 months). No clinical studies since ABC-02 have reported an extension in OS, and therefore effective new agents/combinations are required. NUC-1031 was designed to improve on gemcitabine’s relatively poor efficacy by overcoming its associated key cancer resistance mechanisms, through cellular uptake independent of nucleoside transporters, activation independent of deoxycytidine kinase and protection from cytidine deaminase inactivation, resulting in over 200x the intracellular levels of the anti-cancer metabolite, dFdCTP, greater stability and reduction in the generation of toxic metabolites. NUC-1031 showed activity as monotherapy in a phase I/II study in 7 pts with BTC, refractory to all standard treatments (Blagden et al ASCO 2015; abstract 2514). Methods: ABC-08 is a multi-centre phase Ib study of NUC-1031 combined with cisplatin in pts with non-resectable or recurrent/metastatic cholangiocarcinoma, gallbladder or ampullary carcinoma, aged ≥18 years with an ECOG performance status of 0-1, who have received no prior systemic therapy. The starting dose for NUC-1031 is 625 mg/m2 administered IV on days 1 and 8 in combination with cisplatin (standard dose of 25 mg/m2) (21 day schedule). The dose will be escalated sequentially in cohorts of 3-6 pts using an accelerated titration procedure (725mg/m2, 825mg/m2, 925mg/m2). Treatment will continue until intolerable toxicity/progressive disease. The primary endpoints are safety and RP2D. Secondary endpoints are progression-free survival, OS, response rate and pharmacokinetic endpoints, including assessments of multiple plasma and intracellular analytes: NUC-1031, cisplatin, dFdC, dFdCMP, dFdCDP, dFdCTP and dFdU, and will be correlated with safety profile and clinical activity. Planned accrual is 15-24 pts over 2 years. Cohort 1 has been completed without dose-limiting toxicities. Enrolment to cohort 2 is on-going. Clinical trial information: NCT02351765.

Ovarian cancer is the most lethal of gynecological cancers killing 60% of women diagnosed with the disease within 5 years. The major contributor to this high mortality is the emergence of chemotherapy resistance; the tumor is initially sensitive to chemotherapy (especially cisplatin, the mainstay of treatment) but recurs with increasingly resistant disease. Effective methods of overcoming treatment resistance are a major unmet medical need and would prolong survival and improve quality of life for women with this disease. We have shown previously that the RNA binding protein La-related protein 1 (LARP1) binds and post-transcriptionally regulates the stability of mRNAs encoding cell survival and stress response proteins including mTOR, BCL2 and BIK. In ovarian cancer tissue, elevated levels of LARP1 protein correlate with adverse survival outcome and chemotherapy resistance. In vivo inhibition of LARP1 using therapeutic RNA interference (packaged in DOPC nanoliposomes) restores cisplatin sensitivity in resistant ovarian cancer xenograft models. In concurrent studies, using a novel ultra-high performance liquid chromatography tandem mass spectrometry method, we have quantified LARP1 in the circulation of ovarian cancer patients and found that high levels correspond with poor prognosis. Circulating LARP1 has prognostic significance and may act as a companion biomarker to a LARP1 inhibitor. We conclude that LARP1, through its regulation of multiple mRNAs within critical pathophysiological pathways, is an important cancer therapeutic target and that RNA-based drugs designed to target LARP1 restore chemotherapy sensitivity in xenograft models. Citation Format: Essam A. Ghazaly, John Le Quesne, Dahai Jiang, Selanere L. Mangala, James Chettle, Cristian Rodriguez-Aguayo, Gabriel Lopez-Berestein, Chathunissa Gnanaranjan, Manuela Mura, Chara Stavraka, Anil K. Sood, Sarah P. Blagden. The RNA-binding protein LARP1 is a cancer therapeutic target. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr B30.

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.

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.

Introduction: Acelarin® is the first anti-cancer ProTide to enter the clinic and has achieved a very high rate of disease control in a Phase I study (ProGem1) of patients with advanced progressive solid tumours. It is a first-in-class nucleotide analogue specifically designed to bypass key cancer resistance pathways associated with gemcitabine, including altered expression of human equilibrative nucleoside transporter 1 (hENT1), deoxycytidine kinase (dCK) and cytidine deaminase (CDA). We have used a cytological RNA-based assay, RNAscope® and quantitative Reverse Transcriptase Polymerase Chain Reaction (qPCR) analysis to quantify these three biomarkers in seven cancer cell lines, and reviewed and tested a panel of commercially available antibodies against each protein. The aim was to select and validate a method suitable for stratifying tumours according to their molecular signature and allow the identification of patients unlikely to derive clinical benefit from gemcitabine. Methods: Cells from seven cancer lines (BxPC-3, MiaPaCa-2, PANC-1, SK-MES-1, NCI-H1703, OVCAR-3 and OVCAR-8) were prepared in formalin-fixed paraffin-embedded blocks. In addition, transiently transfected MiaPaCa-2 cells overexpressing hENT1, dCK and CDA were prepared. Expression of mRNA was detected using RNAscope and quantified by SpotStudio as well as by qPCR. Antibodies were examined by Western-blot analysis, reverse phase protein arrays (RPPA) and immunohistochemistry. Protein expression by immunofluorescence was quantified using Aqua HistoRx technology. Results: RNAscope detected mRNA for CDA at low levels in all cell lines. dCK and hENT1 were detected at low (in BxPC-3) to high (in NCI-H1703) expression levels across the cell lines. The qPCR method quantified precisely the amount of RNA, which demonstrated a positive and significant correlation with RNAscope data (Spearman correlation rho = 0.76, p = 0.037 for CDA, rho = 0.67, p = 0.08 for dCK, rho = 0.67, p = 0.08). Most antibodies were rejected because of low specificity as observed by Westerns, RPPA and immunohistochemistry. In addition, both techniques identified the gene over-expression in the transfected cell lines where the expression was up to 100 fold higher in transfected cells compared to their wild-type pairs. Conclusions: Selection of antibodies against hENT1, CDA and DCK for clinical use remains challenging. The RNAscope technology was validated, allowing reliable detection of biomarkers thought to be associated with cancer cell resistance to gemcitabine. Citation Format: Essam A. Ghazaly, Chathunissa Gnanaranjan, Bill Greenhalf, Sarah P. Blagden, Peter Mullen, David Harrison, John G. Gribben. Validating the RNAscope for molecular profiling of key biomarkers associated with gemcitabine resistance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3390. doi:10.1158/1538-7445.AM2015-3390

Background Approximately 50% of all malignant pleural mesotheliomas (MPM) are deficient in argininosuccinate synthetase (ASS1), the rate-limiting enzyme in arginine biosynthesis, and are therefore sensitive to arginine deprivation. This discovery in MPM has been translated into the clinic using the arginine depletor pegylated arginine deiminase (ADI-PEG20) which showed a halving in the risk of disease progression in a randomized phase II study (Szlosarek et al, ASCO 2014). However, unstudied to date, stromal resistance to ADI-PEG20 may reduce its efficacy. Here, we studied the effect of macrophages, which are abundant in mesothelioma, on the tumor cytotoxicity of ADI-PEG20. Methods ASS1 negative MPM cell lines treated with ADI-PEG20 were analysed using the Affymetrix Human Genome U133 Plus 2.0 array, with validation of genes involved in stromal-tumor cell communication. Additional studies involved using tumor-macrophage co-culture experiments, mass spectrometry and an MPM xenograft model. Results A distinct pro-inflammatory cytokine gene expression signature involved in macrophage recruitment and activation was identified in the ADI-PEG20-treated ASS1 negative MPM cell lines. Notably, a significant increase in ASS1 negative MPM cell viability was seen upon co-culture with macrophages in the presence of ADI-PEG20. This was accompanied by a significant increase in ASS1 expression in co-cultured macrophages, with a corresponding increase in argininosuccinate lyase (ASL) expression in co-cultured tumor cells and a doubling in levels of the arginine precursor, argininosuccinate, in cell supernatant. The addition of argininosuccinate to tumor cell media rescued ASS1 negative MPM cells from ADI-PEG20 cytotoxicity, while the macrophage-mediated resistance to ADI-PEG20 was abrogated following ASL knockdown in MPM cells. Finally, xenograft studies demonstrated a significant reduction in tumor volume in mice treated with ADI-PEG20 in combination with macrophage depletion, compared with ADI-PEG20 monotherapy. Conclusion Collectively, our data indicate that as a result of metabolic ‘cross-talk’ between macrophages and ASS1 negative MPM cells, macrophages mediate MPM resistance to ADI-PEG20 via the provision of argininosuccinate. Our studies provide a rationale for combining ADI-PEG20 with an inhibitor of macrophage recruitment in the treatment of ASS1-deficient mesothelioma. Citation Format: Melissa M. Phillips, Ramsay Khadeir, Laura Tookman, Fiona McCarthy, Jeremy Steele, John Bomalaski, Essam Ghazaly, Peter W. Szlosarek. Macrophages promote resistance to pegylated arginine deiminase in malignant pleural mesothelioma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3360. doi:10.1158/1538-7445.AM2015-3360

Blagden, Sarah Patricia, et al. "A first in human Phase I/II study of NUC-1031 in patients with advanced gynecological cancers." ASCO Annual Meeting Proceedings. Vol. 33. No. 15_suppl. 2015.

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.

Introduction Malignant cells require amino acids for a wide range of core functions. Amino acid deprivation using enzymatic degradation has been used to induce remission in acute lymphoblastic leukemia for decades. Amino acid deprivation may also benefit patients with acute myeloid leukemia (AML). We have previously shown that AML cells lack of argininosuccinate synthetase 1(ASS1), a key enzyme in the pathway that produces arginine (1). Here we tested the effect of an arginine depleting agent, pegylated arginine deiminase (ADI-PEG 20) on primary AML cells in a xenograft model of AML. Methods NOD/SCID/interleukin 2 gamma chain null (NSG) mice were transplanted with 6 primary AML samples. 12 weeks after transplantation of AML mice received either ADI-PEG 20, cytarabine, ADI-PEG 20 plus cytarabine or vehicle. ADI was administered weekly for 4 doses and cytarabine was given for 10 consecutive days (0.2mg per day, roughly equivalent to 40mg in humans). Five weeks after treatment started, mice were killed and the percentage of AML in the bone marrow was determined by flow cytometry. Blood was collected to quantify plasma arginine using liquid chromatography-mass spectrometry/mass spectrometry. Results Plasma arginine levels were depressed following ADI-PEG 20 administration confirming that arginine depletion was achieved in vivo. In all six experiments the combination of ADI-PEG 20 and cytarabine induced a significant reduction in levels of AML compared to control (Figure 1). Critically the combination of ADI-PEG 20 and cytarabine was significantly better than cytarabine alone in three of six experiments. Conclusion Our experiments show that arginine deprivation by ADI-PEG 20 can decrease the leukemic burden in mice transplanted with primary AML cells. The combination of ADI with cytarabine had a greater effect than cytarabine alone in half the experiments. These results provide the rationale to test ADI-PEG 20 with cytarabine in clinical trials. 1. Peter W. Szlosarek, Fiona Luong, Andrew Clear, David Taussig, Simon Joel, Maria Calaminici, Silvana Debernardi, Jude Fitzgibbon, John S. Bomalaski, Arthur E. Frankel, and Dominique Bonnet. Pegylated arginine deiminase (ADI-PEG 20) as a potential novel therapy for argininosuccinate synthetase-deficient acute myeloid leukemia. Cancer Research: April 15, 2011. AACR 102nd Annual Meeting 2011, (AACR Abstract # 467) F. M-M and L. A-M contributed equally D.B., P.W.S. and D.C.T contributed equally Disclosures: Bomalaski: Polaris Group: Employment, Equity Ownership. Szlosarek:Polaris Group: Research Funding.

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

Abstract 1654 Background: Mantle cell lymphoma (MCL) is an incurable, aggressive subtype of non-Hodgkin lymphoma in which there is a need for novel targeted therapies. Activation of the PI3K-Akt pathway and its role in the pathogenesis of MCL has been highlighted in a number of studies. Constitutive activation of the PI3K pathway inactivates GSK-3β, a downstream target of Akt, that can phosphorylate cyclin D1 resulting in its nuclear export. There is also evidence that cyclin D1 mRNA stability and translation is enhanced by this pathway. The class Ia PI3K p110 catalytic subunit isoforms α, β and δ are primarily implicated in oncogenesis. While the PI3K p110δ isoform is known to be enriched in lymphocytes, a gain of PIK3CA (the gene encoding PI3K p110α) copy number has been shown to be a frequent alteration in MCL. The expression and relative importance of the individual Class Ia PI3K isoforms has not been documented in this disease. With the development of isoform selective inhibitors, this is an important issue that needs to be addressed. Aims: We studied the expression of class Ia PI3K isoforms in primary MCL with relation to morphological variants and disease status. We also compared the efficacy of PI3K inhibition in MCL cell lines and primary samples using two novel inhibitors, GDC-0941(predominantly p110α/δ-selective) and CAL-101 (δ-selective), both of which are in early phase clinical trials. Methods: Tissue microarrays were constructed from triplicate 1mm cores from 144 MCL biopsies and 16 tonsil controls. The levels of p110α, p110β and p110δ isoforms were then determined by immunohistochemistry using isoform-specific antibodies. The in vitro effect of PI3K inhibitors on cell viability and apoptosis was studied in 4 MCL cell lines, (Jeko-1, Granta519, REC-1 and JVM-2), and 15 primary MCL samples. Expression of the class Ia PI3K isoforms and changes in downstream targets of PI3K were determined by western blotting. Results: P110δ was expressed at a consistently higher level in MCL samples and normal tonsil controls compared to the α and β isoforms, while p110β expression was weak and significantly lower than p110α expression. On comparing expression of isoforms at diagnosis and relapse, p110α expression was significantly increased beyond 1st relapse compared to diagnostic biopsies (p=0.04) and tonsil controls (p=0.02), an observation that was even more apparent in 6 paired samples [p=0.008, median IHC score 19.6 (5.0−53.2) at diagnosis vs. 91.5 (38.6 − 129) beyond 1st relapse]. No significant change was found in the expression of p110β or p110δ between diagnostic and relapse samples. There was no significant difference in expression levels of the 3 isoforms between blastoid and non-blastoid morphological variants. Expression of both the p110α and δ isoforms was detected by western blotting in 4 MCL cell lines, but only Jeko-1 cells were sensitive to inhibition with GDC-0941. CAL-101 produced little or no apoptosis in all 4 cell lines. In primary MCL samples, GDC-0941 was consistently more potent than CAL-101, with decrease in cell viability of 32 vs. 20% at 1μM (p=0.15), 51 vs. 25% at 5μM (p=0.02) and 67 vs. 35% at 10μM (p Conclusion: Our studies demonstrate that although p110δ is the most consistently expressed isoform, the expression of the p110α subunit increases significantly in multiply relapsed MCL. This observation, in combination with significantly greater in vitro sensitivity of MCL primary samples to GDC-0941, compared to the p110δ-selective inhibitor CAL-101, provides strong evidence for further evaluation of GDC-0941 in this disease. Disclosures: Gribben: Roche: Honoraria; Celgene: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; Pharmacyclics: Honoraria. Joel:Astra Zeneca: Research Funding; Intellikine: Research Funding.

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...

2514 Background: NUC-1031 is a first-in-class nucleotide analogue utilising phosphoramidate chemistry to enhance anti-cancer efficacy and safety. In preclinical studies NUC-1031 demonstrated potent...

Immunomodulatory drugs (IMiDs), such as lenalidomide provide a tool to enhance both direct anti-tumor and graft-versus-tumor effects after allogeneic haematopoietic stem-cell transplantation (AHCT). However, early clinical experience with IMiDs after AHCT using adult peripheral blood (APB) as a stem cell source has been limited by induction of graft-versus-host disease. Characterization of the mechanisms by which IMIDs can modulate alloresponses of T-cells and identification of differential effects on T-cells from disparate cell sources could facilitate more effective use of these drugs in the setting of AHCT. We therefore used in vitro modelling, multi-parameter flow cytometry and gene expression analysis to delineate the impact of the widely used IMiD lenalidomide on quantitative and qualitative alloresponses mediated by T-cells derived from APB and umbilical cord blood (UCB). We co-cultured carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled peripheral blood mononuclear cells (PBMC) from healthy adult donors or donated UCB units with irradiated allogeneic PBMC in the presence of 1μM lenalidomide or vehicle control. In this model, cellular and supernatant concentrations of lenalidomide (determined by mass spectrometry) were similar to in vivo levels achieved at doses used as maintenance after AHSCT in published studies. Functional alloresponses were quantified after 7-9 days of allogeneic co-culture by flow cytometry. In addition, co-culture responders were flow-sorted into alloproliferative or non-proliferative fractions and extracted RNA used for gene expression profiling. We demonstrate that lenalidomide potentiates net alloproliferation of APB derived T-cells cells by selectively enhancing allospecific proliferation of CD8+ T-cells (median 58% (lenalidomide-treated) versus 43% (untreated), n=40, p In common with our findings in APB T-cells, lenalidomide also potentiated proliferation of alloreactive CD8+ T-cells from UCB (median 43% (lenalidomide-treated) vs 24% (untreated), n=17, p=0.0005) with similar polyfunctional effector memory differentiation, immunoregulatory gene expression changes and cellular ikaros depletion. Importantly, lenalidomide reduced allospecific proliferation of UCB CD4+ T-cells (median 58% (untreated) versus 41% (lenalidomide-treated), n=17, p Our findings show that lenalidomide has a qualitatively different impact on alloresponses of T-cells from different cell sources; alloresponses of APB T-cells are increased by lenalidomide via selective expansion of polyfunctional CD8+ effectors while lenalidomide limits alloresponses of UCB T-cells by reducing CD4+ effector expansion and increasing tolerogenic Treg. These findings have important implications for the future use of IMiDs in the setting of AHCT. Disclosures No relevant conflicts of interest to declare.

Introduction Relapsed/refractory pediatric AML has a poor prognosis despite salvage therapy including stem-cell transplantation. Chemotherapy using FLAG plus liposomal daunorubicine (FLAG-DNX) is currently considered the standard in 1strelapse in Europe (Kaspers et al, JCO 2013). FLAG is based on potentiation of cytarabine (Ara-C) by fludarabine (Flu) by increasing Ara-CTP levels. Clofarabine (CLO) is a novel purine nucleoside analog, designed to have improved efficacy. Methods We initiated an ongoing phase 1B dose-escalation study using a ‘3x3 design’ to define the optimal dose of CLO, replacing FLU in FLAG-DNX. Dosages consisted of Ara-C 2gr/m2/day (day 1-5), with escalation of DNX from 40, 60 to 80 mg/m2/day (day 1, 3 and 5), and CLO from 20, 30 to 40 mg/m2/day (day 1-5) in 5 dose-levels, without GCSF priming. At day 6 intrathecal Ara-C was administered. Dose-level 1-4 recruited early 1st relapse, refractory 1st relapse and 2nd relapse of AML, and escalated CLO to 40 mg/m2 with DNX 60 mg/m2. Dose level 5 is open to 1st relapse pts and escalates DNX to 80 mg/m2. Responding patients received a 2ndconsolidation with CLARA-DNX or treated by investigator discretion. Other inclusion criteria consisted of performance status ≥60%, and normal liver and renal function. Maximum tolerated dose was the primary endpoint. Hematologic DLTs were defined as count recovery in responding pts >42 days. Serum and CSF were collected for pharmacokinetics (PK), taken at day 1: predose, T=2hrs and T=5 hrs post-infusion, and repeated on day 5, including a T=24 sample on day 6, together with a CSF sample just prior to intrathecal medication. CLO plasma and CSF concentrations were analyzed using LC-MS/MS. Efficacy data are awaiting central review and not released until the end of the study. The study is registered in the Dutch Trial Registry (nr. 1880). Results We here report safety and PK data on the first 4 dose-levels after accrual of 22 AML patients (≥2nd relapse, n=7; refractory 1st relapse, n=9; early 1strelapse, n=6). Patients were treated at dose-level (DL) 1, n=4; 2, n=3; 3, n=12; 4, n=3. Eleven patients had been transplanted prior to enrollment. In total 27 CLARA-DNX courses were administered. Median age was 6.4 years, 14 patients were male. 20 SAEs were reported, consisting of febrile neutropenia (n=11), typhlitis (n=1), lung-infection fungal (n=3), lung-infection bacterial (n=2), candida sepsis (n=1), rash (hand-foot skin reaction) (n=1), and tumor lysis/capillary leak syndrome (n=1). Other grade 3-4 AEs reported in at least 2 patients consisted of abdominal pain, nausea and vomiting, diarrhea, anorexia and allergic reaction. Two patients had bilirubin elevation (max 61 umol/l); six patients had mild transaminase elevation > 100 U/l (max. 330U/l), which was reversible. At dose-level 3, two patients experienced DLTs (fungal infection) halting dose-escalation. Detailed analysis showed that most patients had evidence of subclinical fungal infections prior to enrollment. Therefore a protocol amendment was issued that required screening patients with CT-scan and serum-galactomannan prior to enrollment. After enrolling another cohort of 6 patients at DL3 this appeared to be safe (1 DLT of Candida sepsis out of 6 patients). No DLTs occurred at dose-level 4 in 3 patients. No hematological DLTs occurred. PK samples were available from 19 patients; in 2 patients sampling was repeated at course 2. At day 1 the median AUC was 28 ng/ml.mg.hr (range 6-401), with a mean T1/2 of 1.5 hrs. Day 1 and day 5 results were similar. CSF levels were not measurable in most patients and were 0.1-0.2 ng/ml.mg in the 3 patients with detectable levels. Our PK-data in combination are not significantly different from our data with single-agent CLO in relapsed ALL patients (Joel et al, AACR, 2007), and fit the CLO single agent PK model of Bonate et al (J Clin Pharmacol 2004, 44: 1309-32) . Conclusions The RP2D of CLO in a CLARA/DNX course in relapsed/refractory pediatric AML is 40 mg/m2, excluding patients with evidence of prior subclinical aspergillus. There is no evidence for PK interaction between CLO and the other drugs. We are currently testing the safety of an augmented dose of DNX (80 mg/m2) in 1strelapse AML patients. When tolerable this dose may be randomized against other induction regimens in front-line therapy in pediatric AML. Acknowledgment This study was financially supported by Sanofi and by the KiKa-foundation grant nr. 26. Disclosures: Off Label Use: Clofarabine for pediatric AML.

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]