Supplementary Table 1 from Chemosensitization of B-Cell Lymphomas by Methylseleninic Acid Involves Nuclear Factor-κB Inhibition and the Rapid Generation of Other Selenium Species

Supplementary Data from A Synergistic Interaction between Lapatinib and Chemotherapy Agents in a Panel of Cell Lines Is Due to the Inhibition of the Efflux Pump BCRP

Supplementary Figures 1-3, Table 1 from AZD1152 Rapidly and Negatively Affects the Growth and Survival of Human Acute Myeloid Leukemia Cells In vitro and In vivo

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.

Background Despite the high rate of sudden death after myocardial infarction among patients with a low ejection fraction, implantable cardioverter–defibrillators are contraindicated until 40 to 90 days after myocardial infarction. Whether a wearable cardioverter–defibrillator would reduce the incidence of sudden death during this high‐risk period is unclear. Methods We randomly assigned (in a 2:1 ratio) patients with acute myocardial infarction and an ejection fraction of 35% or less to receive a wearable cardioverter–defibrillator plus guideline‐directed therapy (the device group) or to receive only guideline‐directed therapy (the control group). The primary outcome was the composite of sudden death or death from ventricular tachyarrhythmia at 90 days (arrhythmic death). Secondary outcomes included death from any cause and nonarrhythmic death. Results Of 2302 participants, 1524 were randomly assigned to the device group and 778 to the control group. Participants in the device group wore the device for a median of 18.0 hours per day (interquartile range, 3.8 to 22.7). Arrhythmic death occurred in 1.6% of the participants in the device group and in 2.4% of those in the control group (relative risk, 0.67; 95% confidence interval [CI], 0.37 to 1.21; P=0.18). Death from any cause occurred in 3.1% of the participants in the device group and in 4.9% of those in the control group (relative risk, 0.64; 95% CI, 0.43 to 0.98; uncorrected P=0.04), and nonarrhythmic death in 1.4% and 2.2%, respectively (relative risk, 0.63; 95% CI, 0.33 to 1.19; uncorrected P=0.15). Of the 48 participants in the device group who died, 12 were wearing the device at the time of death. A total of 20 participants in the device group (1.3%) received an appropriate shock, and 9 (0.6%) received an inappropriate shock. Conclusions Among patients with a recent myocardial infarction and an ejection fraction of 35% or less, the wearable cardioverter–defibrillator did not lead to a significantly lower rate of the primary outcome of arrhythmic death than control. (Funded by the National Institutes of Health and Zoll Medical; VEST ClinicalTrials.gov number, NCT01446965.)

Left bundle branch block (LBBB) has been classically depicted as an electrical delay of conduction manifested by a mechanical dysynchrony of left ventricular contraction with a decrease in systolic function. Until recently, direct correction of LBBB has not been able to be demonstrated. With

The purpose of this study was to determine whether hypnosis would be more effective than standard behavioral counseling in helping smokers to remain abstinent. A total of 140 current smokers were enrolled in a randomized controlled smoking cessation trial at an urban Veterans Affairs medical center. Participants (n=102) who were able to quit for at least 3 days received either a hypnosis or behavioral relapse prevention intervention. Both relapse prevention interventions consisted of two 60 min face-to-face sessions and four 20 min follow-up phone calls (two phone calls per week). At 26weeks, the validate\d point-prevalence quit rate was 35% for the hypnosis group and 42% for the behavioral counseling group (relative risk=0.85; 95% confidence interval: 0.52-1.40). At 52weeks, the validated quit rate was 29% for the hypnosis group and 28% for the behavioral group (relative risk =1.03; 95% confidence interval: 0.56-1.91). It was concluded that hypnosis warrants further investigation as an intervention for facilitating maintenance of quitting.

Mantle cell lymphoma (MCL) is a clinically heterogeneous, but often aggressive lymphoma characterized by the IGH:CCND1 translocation and cyclin D1 (CCND1) over-expression. Chromosomal instability, due to disrupted DNA damage response, in conjunction with abnormal activation of cell survival mechanisms underlies the aggressive clinical course in MCL (Jares et al, 2012). In recent years, improved understanding of lymphoma biology has led to the development of a number of small molecule inhibitors. However, the relative rarity of MCL (incidence 0·55 per 100 000) (Smedby & Hjalgrim, 2011) poses a challenge in effectively evaluating these drugs in patients. In vitro studies have been limited by the difficulty of culturing primary MCL cells. Murine models of MCL cell lines are relatively easy to establish in SCID or NOD/SCID/IL2Rγ null (NSG) mice (Wang et al, 2007, 2008a; Weston et al, 2010) but have their limitations. Until a couple of years ago, the only primary mouse model of human MCL described in the literature was established by injection of primary MCL cells into subcutaneous human bone grafts implanted in SCID mice (SCID-Hu model) (Wang et al, 2008b). Recently, however, disseminated models of human primary MCL have been established in NSG mice (Iyengar et al, 2012; Klanova et al, 2014). We report our experience here in further detail, focussing on the characteristics of MCL engraftment in this model. We used 8- to 12-week-old NSG mice that were sub-lethally irradiated (3·75 Gy) 24 h prior to transplantation. Before undertaking xenograft studies with primary cells, we used the MCL cell line JEKO-1 to assess kinetics, disease burden and distribution of MCL cells in NSG mice. JEKO-1 cells were transduced with firefly luciferase and injected intravenously into irradiated mice at two doses – 0·5 × 106 and 2 × 106 cells. Bioluminescent imaging was performed at weekly intervals following injection of D-luciferin. All mice became ill with marked weight loss and had to be sacrificed by day 29. Bioluminescence was observed in the bone marrow and spleen in all mice. Mice injected with the higher cell dose had more rapid disease progression, developed hind leg weakness and had bioluminescence in the central nervous system (CNS) on imaging, indicating involvement (Fig 1). Following this, seven cryopreserved primary MCL samples were identified from the Barts Cancer Institute tissue bank. An additional fresh primary sample derived from a splenectomy was included in the cohort. Ethical approval was obtained from East London and the City Local Research Ethics Committee. Written informed consent was obtained from patients according to the Declaration of Helsinki. All samples had a classical MCL phenotype with CD5/CD20 positivity and were confirmed to have the IGH:CCND1 translocation by fluorescence in situ hybridization (FISH). Irradiated NSG mice were injected intravenously with a dose of 107 unselected MCL cells each. Flow cytometry was performed for mouse CD45 and human CD45, CD3, CD5 and CD20 on peripheral blood samples taken from mice at 3, 6 and 12 weeks. Mice were sacrificed at 20 weeks, or earlier if they met Home Office guidelines, and tissue was harvested for immunohistochemistry (IHC). Cells were flushed from mouse femur for flow cytometry. At 20 weeks, MCL cells were found in the bone marrow and spleen of mice injected with 2 out of the 7 cryopreserved primary samples. Both samples that engrafted had blastoid morphology and one was obtained from a patient with relapsed disease. FISH for IGH:CCND1 on cell suspensions prepared from spleen of NSG mice further confirmed engraftment. None of the mice that engrafted appeared to have bowel involvement as assessed by IHC. Lymphadenopathy was not found at sacrifice. Scattered human CD20-positive cells were seen in the liver but this was not a consistent feature. Mice remained relatively well until sacrifice (Fig 2A–F). As a next step, secondary transplantation of MCL cells isolated from NSG spleen (107 cells per mouse) was undertaken. Once again, engraftment was seen in mouse spleen and bone marrow on sacrifice at 20 weeks (Fig 2G). In addition to the two cryopreserved samples, evidence of engraftment was also seen in the spleen of NSG mice injected with the fresh primary sample (non-blastoid). Interestingly, there appeared to be co-existence of MCL cells and T-cells in the spleen of mice injected with fresh MCL cells, with tumour cells concentrated around blood vessels. However, these mice had to be sacrificed at 7 weeks due to illness and T-cell infiltration was found in the liver and bone marrow, without evidence of MCL. We found a similar proliferation of T cells but without evidence of MCL in one of the seven cryopreserved samples that had high T-cell content (>10%), indicating T-cell depletion may be important in this scenario. Therefore, similar to the recent report by Klanova et al (2014), we demonstrate human primary MCL engraftment in NSG mice. In contrast to their study where mice were injected with a variable cell dose (1–8 × 107 cells), we injected all mice with a fixed dose of 107 cells. This may explain the lower rate of engraftment in our study. Both cryopreserved samples that engrafted in our study had blastoid morphology, suggesting that a higher cell dose may be required for engraftment of non-blastoid MCL in this model. In our experiments, mice with primary MCL engraftment were not visibly ill at 20 weeks and disease burden was heaviest in the spleen. In contrast, disease progression was rapid in the JEKO-1 xenograft, with CNS involvement and hind leg weakness developing by 4 weeks. These findings mirror those of Klanova et al (2014), and are important considerations when designing pre-clinical experiments involving these models. The longer overall survival of NSG mouse models of primary human MCL could be an advantage for pre-clinical testing of newer agents, which often require longer periods of administration for efficacy. Finally, our study demonstrates, similar to the findings of Klanova et al (2014), that secondary transplantation can be successfully carried out in this model, highlighting the self-renewal and tumour-initiating capacity of primary MCL cells. In summary, this NSG model of human primary MCL is a promising in vivo model for both pre-clinical drug testing and further understanding MCL biology. Our research provides further insight into the advantages and limitations of this model, which will be crucial for its effective use in pre-clinical research. This work was funded by Cancer Research UK (D.B.) and a Roger Counter Foundation award from the British Society of Haematology (S.I.). We are indebted to patients who gave samples. We thank the Animal Care and Flow Cytometry core facilities staff at the London Research Institute for valuable technical help. S.I.: design and performance of experiments, data analysis and interpretation, manuscript writing; L.A.M.: design and performance of experiments, data analysis and interpretation; A.C. and S.I.: help in sample processing, research and data analysis; R.A., D.T. and J.G.: provision of vital patient samples, materials and data; A.R. and D.L. performance of FISH analysis; D.B.: design of experiments, data analysis, manuscript writing. The authors declare no potential conflicts of interest.

A simple and rapid ultra-high performance liquid chromatography-mass spectrometry/mass spectrometry (UPLC-MS/MS) method has been developed for measuring intracellular concentrations of the anticancer agent 7-ethyl-10-hydroxycamptothecin (SN-38) in tumour cells using camptothecin (CPT) as internal standard. SN-38 extraction was carried out using acidified acetonitrile. SN-38 and CPT were separated on a PFP column using gradient elution with acidified water and acetonitrile. SN-38 and CPT were quantified using a triple quadrupole mass spectrometry system. Least square regression calibration lines were obtained with average correlation coefficients of R(2)=0.9993±0.0016. The lower limit of detection (LOD) and lower limit of quantification (LOQ) for SN-38 were 0.1 and 0.3ng/ml, respectively. CPT recovery was 98.5±13% and SN-38 recoveries at low quality control (LQC, 5ng/ml) and high quality control (HQC, 500ng/ml) were 89±6% and 95±8%, respectively. The intra- and inter-day imprecision for LQC was 5.8 and 8.5%, and for HQC was 6.3 and 4.4%, respectively. The method was compared to a validated high performance liquid chromatography-fluorescent method. In addition, the method has been successfully applied to determine the intracellular accumulation of SN-38 investigating the transport through ABCB1 (P-gp) and ABCG2 (BCRP) efflux pumps in colorectal cancer cell lines.

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.

Phosphoinositide-3 kinase (PI3K) pathway activation contributes to mantle cell lymphoma (MCL) pathogenesis, but early-phase studies of the PI3K p110δ inhibitor GS-1101 have reported inferior responses in MCL compared with other non-Hodgkin lymphomas. Because the relative importance of the class IA PI3K isoforms p110α, p110β, and p110δ in MCL is not clear, we studied expression of these isoforms and assessed their contribution to PI3K signaling in this disease. We found that although p110δ was highly expressed in MCL, p110α showed wide variation and expression increased significantly with relapse. Loss of phosphatase and tensin homolog expression was found in 16% (22/138) of cases, whereas PIK3CA and PIK3R1 mutations were absent. Although p110δ inhibition was sufficient to block B-cell receptor-mediated PI3K activation, combined p110α and p110δ inhibition was necessary to abolish constitutive PI3K activation. In addition, GDC-0941, a predominantly p110α/δ inhibitor, was significantly more active compared with GS-1101 against MCL cell lines and primary samples. We found that a high PIK3CA/PIK3CD ratio identified a subset of primary MCLs resistant to GS-1101 and this ratio increased significantly with relapse. These findings support the use of dual p110α/p110δ inhibitors in MCL and suggest a role for p110α in disease progression.

Progressive multiple sclerosis is associated with metabolic failure of the axon and excitotoxicity that leads to chronic neurodegeneration. Global sodium-channel blockade causes side effects that can limit its use for neuroprotection in multiple sclerosis. Through selective targeting of drugs to lesions we aimed to improve the potential therapeutic window for treatment. This was assessed in the relapsing-progressive experimental autoimmune encephalomyelitis ABH mouse model of multiple sclerosis using conventional sodium channel blockers and a novel central nervous system-excluded sodium channel blocker (CFM6104) that was synthesized with properties that selectively target the inflammatory penumbra in experimental autoimmune encephalomyelitis lesions. Carbamazepine and oxcarbazepine were not immunosuppressive in lymphocyte-driven autoimmunity, but slowed the accumulation of disability in experimental autoimmune encephalomyelitis when administered during periods of the inflammatory penumbra after active lesion formation, and was shown to limit the development of neurodegeneration during optic neuritis in myelin-specific T cell receptor transgenic mice. CFM6104 was shown to be a state-selective, sodium channel blocker and a fluorescent p-glycoprotein substrate that was traceable. This compound was >90% excluded from the central nervous system in normal mice, but entered the central nervous system during the inflammatory phase in experimental autoimmune encephalomyelitis mice. This occurs after the focal and selective downregulation of endothelial p-glycoprotein at the blood-brain barrier that occurs in both experimental autoimmune encephalomyelitis and multiple sclerosis lesions. CFM6104 significantly slowed down the accumulation of disability and nerve loss in experimental autoimmune encephalomyelitis. Therapeutic-targeting of drugs to lesions may reduce the potential side effect profile of neuroprotective agents that can influence neurotransmission. This class of agents inhibit microglial activity and neural sodium loading, which are both thought to contribute to progressive neurodegeneration in multiple sclerosis and possibly other neurodegenerative diseases.

2525 Background: GSK1070916A is a potent and selective inhibitor of Aurora B and C. This phase I study in collaboration with GlaxoSmithKline was part of the Cancer Research UK Clinical Development Programme. Methods: Patients (pts) with advanced/metastatic solid cancers for whom there was no standard therapy, with adequate performance status and organ function were eligible for GSK1070916A (1 hour i.v. infusion days 1 – 5, every 21 days). The primary objectives were to determine the safety profile, dose limiting toxicity (DLT) and maximum tolerated dose (MTD) of GSK1070916A. The starting dose was 5mg/m2/day, with initial single pt cohorts, followed by “3 + 3” cohorts and expansion at the MTD. DLTs included prolonged (> 5 days) or complicated grade 4 neutropenia; the MTD was the highest dose at which < 1 of 3 - 6 pts experienced DLT. Cycle 1 blood and healthy skin biopsies were obtained for PK and PD assays. The expanded cohort included 6 pts having pre- and post-treatment functional imaging studies (FDG PET-CT and MRI), and a further 6 having paired tumour biopsies for PD studies. Results: Nine single pt cohorts received up to 73mg/m2/day of GSK1070916A with no grade 3 or 4 related adverse events. At 102.2mg/m2/day, 1 pt had a DLT (febrile neutropenia) and 2 pts non-DLT grade 4 neutropenia; this dose was considered unacceptably toxic and 23 pts received a lower dose of 85mg/m2/day; 7/23 pts had prolonged/complicated grade 4 neutropenia, 5 of whom continued GSK1070916A with dose reduction +/- delay. There were no treatment related deaths. A pt with ovarian cancer (102.2mg/m2/day) had a RECIST PR; 19 pts had stable disease for < 223 days. GSK1070916A PK were linear with a strong correlation between exposure (AUC) and reduction in neutrophils (r2 0.91). At the 85 mg/m2 dose, mean day 1 t1/2 was 8.98 hours and Cl 9.2 l/h; AUCinf was 10% higher on day 5 than day 1. PD results in healthy skin (phosphoHistone-H3, Ki 67 and cleaved caspase-3) were inconsistent. Conclusions: The MTD of GSK1070916A as a 1 hour i.v. infusion on days 1 – 5, every 21 days is 85mg/m2/day, with predictable and manageable neutropenia as the DLT and evidence of clinical activity. Serum levels of cytokeratin-18, tumour PD and functional imaging data will be presented. Clinical trial information: NCT01118611.

BACKGROUND:Tumor classification based on their predicted responses to kinase inhibitors is a major goal for advancing targeted personalized therapies. Here, we used a phosphoproteomic approach to investigate biological heterogeneity across hematological cancer cell lines including acute myeloid leukemia, lymphoma, and multiple myeloma.RESULTS:Mass spectrometry was used to quantify 2,000 phosphorylation sites across three acute myeloid leukemia, three lymphoma, and three multiple myeloma cell lines in six biological replicates. The intensities of the phosphorylation sites grouped these cancer cell lines according to their tumor type. In addition, a phosphoproteomic analysis of seven acute myeloid leukemia cell lines revealed a battery of phosphorylation sites whose combined intensities correlated with the growth-inhibitory responses to three kinase inhibitors with remarkable correlation coefficients and fold changes (> 100 between the most resistant and sensitive cells). Modeling based on regression analysis indicated that a subset of phosphorylation sites could be used to predict response to the tested drugs. Quantitative analysis of phosphorylation motifs indicated that resistant and sensitive cells differed in their patterns of kinase activities, but, interestingly, phosphorylations correlating with responses were not on members of the pathway being targeted; instead, these mainly were on parallel kinase pathways.CONCLUSION:This study reveals that the information on kinase activation encoded in phosphoproteomics data correlates remarkably well with the phenotypic responses of cancer cells to compounds that target kinase signaling and could be useful for the identification of novel markers of resistance or sensitivity to drugs that target the signaling network.

Abstract 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

Epigenetic therapy has an increasing role in the treatment of cancers, particularly haematological malignancies. Histone deacetylase (HDAC) inhibitors are a promising new class of anti-cancer agents (Marson, 2009) and have widespread effects both within and beyond the genome. The latter includes cytoskeletal proteins, molecular chaperones and transcription factors. Such proteins are acetylated by a post-translational modification event that regulates their function. Four main classes of HDAC have been described to date, all sharing a zinc-dependent catalytic domain. HDAC6 (class 2) is unique in its two active HDAC domains and ubiquitin binding zinc finger that participates in the aggresomal pathway of protein degradation (Kawaguchi et al, 2003). Vorinostat and panabinostat are the most clinically advanced HDAC inhibitors (HDACis) and are currently under evaluation in clinical trials for lymphoid malignancies (Dimopoulos et al, 2011; Wolf et al, 2012). As part of our programme of the development of novel HDAC inhibitors (Marson et al, 2007), we designed, synthesized and evaluated a set of compounds containing bifurcation predicted to increase binding across the protein periphery (‘cap’ region) of HDACs. One compound, UCL67022 (2-(N-phenylcarbamoyl)suberoylanilide-8-hydroxamic acid) was identified as a potent hydroxamic acid inhibitor of HDACs (half maximal effective concentration [EC50] = 40 nmol/l vs. 260 nmol/l for vorinostat in a cell-free HDAC assay) that, at 10 mg/kg, produced near-cytostatic tumour growth in a mouse xenograft model of breast cancer (Joel & Marson, 2008). The increased potency of UCL67022 is attributable to the additional side-chain, which is absent in vorinostat, and gives greater interactions with the HDAC periphery than does vorinostat (Figure S1). Furthermore, this may also enhance HDAC isoform selectivity. On this basis we investigated the activity of UCL67022 in B cell malignancies. UCL67022 demonstrated potent activity in both multiple myeloma (MM) and a variety of non-Hodgkin lymphoma (NHL) cell lines and patient-derived samples (isolated and cultured as previously described (Maharaj et al, 2012; Popat et al, 2013). EC50 values for cytotoxicity assays were generally in the nanomolar range and up to 20 times lower than that obtained with vorinostat (supplementary tables 1 & 2). Reassuringly, minimal cytotoxicity was demonstrated when cultured with peripheral blood mononuclear cells, and concentrations over 10 times the EC50 were required to significantly reduce viability. Similarly, no cytotoxicity to patient bone marrow stromal cell (BMSC) viability was observed with concentrations lethal to myeloma cells (data not shown). The impact of the MM bone marrow microenvironment upon drug resistance was simulated using interleukin 6 (IL6) and insulin-like growth factor (IGF1; R&D systems, Abingdon, UK) supplementation and co-culture with BMSCs (Fig 1A). Under both conditions, UCL67022 retained activity in both IL6-dependent (U266) and independent (MM1S) cell lines despite an impairment in dexamethasone mediated cytotoxicity (Figure S2). To evaluate the role of BMSC humoral growth factors, the levels of cytokines were analysed from supernatants of 48-h co-cultures of HS-5/patient-derived BMSCs and primary CD138+ve MM cells. Negligible levels were detected from MM cells cultured alone, whereas significant levels were found in the supernatants from BMSCs. MM-BMSC co-cultures demonstrated increases in vascular endothelial growth factor (VEGF), IL6 and IL8 levels comparatively, in keeping with paracrine loops. The addition of UCL67022 down-regulated such secretion in a dose-dependent manner and more so than bortezomib (Fig 1B, C). Drug mechanism of action was confirmed by acetylation of histone H3, in keeping with HDAC inhibitory activity and acetylation of α-tubulin confirming HDAC6 inhibition. In comparison to vorinostat, a 10-fold lower concentration was required (histone H3: 0·3 vs 3 μmol/l and α-tubulin 0·1 vs. 1 μmol/l). Furthermore, UCL67022 induced reduction (suggesting cleavage) of 8 but not caspase 9 at 0·3 μmol/l. Reduction of caspase 8 was associated with a reduction in full length poly ADP ribose polymerase (PARP), indicating apoptosis (Fig 1D, E). Immunofluorescence of MM1S cells cultured with UCL67022 for 24 h showed an increase in thick acetylated α-tubulin bundles throughout the cytoskeleton, whereas bortezomib induced the formation of discrete perinuclear structures (Fig 1F). UCL67022 caused a diffuse increase in ubiquitin staining throughout the cytoplasm whereas bortezomib induced localization of ubiquitinated proteins to a perinuclear structure similar to that with α-tubulin (Fig 2A). The combination of UCL67022 and bortezomib caused the dispersal of aggregated ubiquitin into smaller widespread aggregates throughout the cytoplasm (Fig 2B). Consequently, the cytotoxicity of this combination was assessed in a variety of exposure schedules for three different MM cell lines. Using calcusyn software, combination indices (.I) ranging between 0·5 and 1·5 were obtained for all schedules of culture, suggesting additive cytotoxicity (representative plot in Fig 2). Primary NHL samples cultured in a CD40 system for 24 h prior to the addition of vorinostat or UCL67022 and bortezomib at 3 different concentrations demonstrated synergistic cytotoxicity with sub-lethal concentrations of both HDACi; however a more pronounced interaction was observed with UCL67022 (median CI values range: UCL67022: 0·19–0·90 vs. vorinostat: 0·52–1·41; Fig 2C, D, E). Of note, synergism was demonstrated in 3 different NHL sub-types (mantle cell, follicular and diffuse large B cell). In summary, we have characterized a novel hydroxamic acid-based HDACi, UCL67022, with potent in vitro activity in MM and NHL. Notably, EC50 values were 10-fold lower than that demonstrated with vorinostat. Additionally, UCL67022 surmounted the protective effects of the key bone marrow microenvironment cytokines IL6 and IGF1, retained significant activity in BMSC co-cultures and modulated cytokine secretion from BMSCs. UCL67022 was able to disrupt proteasome inhibition-induced ubiquitin aggregates, which have previously been demonstrated to represent a second protein degradation pathway known as the aggresome (Kawaguchi et al, 2003). This pathway serves as a cytoprotective response to proteasome inhibition and is mediated by HDAC 6. Consequently, UCL67022 was found to enhance the cytotoxicity of bortezomib in MM and NHL models. A number of HDACis have been described as having either additive (Maiso et al, 2006) or potentially synergistic responses (Catley et al, 2006) with bortezomib. However the phase 3 clinical trial of vorinostat and bortezomib in MM demonstrated a minimal progression-free survival benefit to bortezomib alone (Dimopoulos et al, 2011). Given the potency of UCL67022 and its pre-clinical superiority to vorinostat, we recommend further evaluation into clinical trials. RP was funded by a Clinical Training Fellowship from Barts and the London Charity. The award of a BBSRC studentship (to BJM) and support from the MandevilleTrust are gratefully acknowledged. RP, LM, SJ designed the research study, performed the research, analysed the data and wrote the paper; CM, BM, AR formulated and synthesized UCL67022; HO, JC contributed essential patient samples and clinical data. JP performed the research and anlysed data. All authors were involved in drafting and approving the manuscript. The authors declare no conflict of interest. Fig S1. Chemical structure of vorinostat and UCL67022. Note the additional side-chain in UCL67022 that enhances interactions of its cap region the HDAC. Fig S2. UCL67022 retains activity in (A) MM1S (B) U266 HMCLs supplemented with IL-6 and IGF-1 whereas (C) Dexamethasone reduces its cytotoxicity with IL-6 and IGF-1. Table SI. EC50 values for UCL67022 and vorinostat for myeloma cell lines and 2 patient derived myeloma samples. Table SII. (A) Comparison of EC50s of vorinostat and UCL67022 for DLBCL and MCL cell lines (B) Comparison of EC50s of vorinostat and UCL67022 for primary lymphoma samples. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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]