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Melanoma is an aggressive and highly metastatic type of skin cancer where the design of new therapies is of utmost importance for the clinical management of the disease. Thus, we have aimed to investigate the mode of action by which a novel methylated analogue of L-Mimosine (e.g., L-SK-4) exerts its therapeutic potency in an in vitro model of malignant melanoma. Cytotoxicity was assessed by the Alamar Blue assay, oxidative stress by commercially available kits, ROS generation, caspase 3/7 activation and mitochondrial membrane depolarisation by flow cytometry, expression of apoptosis-related proteins by western immunoblotting and profiling of lipid biosynthesis by a metabolomic approach. Overall, higher levels of ROS, sphingolipids and apoptosis were induced by L-SK-4 suggesting that the compound’s therapeutic potency is mediated through elevated ROS levels which promote the upregulation of sphingolipid (ceramide) biosynthesis thus leading to the activation of both extrinsic and intrinsic apoptosis, in an experimental model of malignant melanoma.

Cancer cells accumulate iron to supplement their aberrant growth and metabolism. Depleting cells of iron by iron chelators has been shown to be selectively cytotoxic to cancer cells in vitro and in vivo. Iron chelators are effective at combating a range of cancers including those which are difficult to treat such as androgen insensitive prostate cancer and cancer stem cells. This review will evaluate the impact of iron chelation on cancer cell survival and the underlying mechanisms of action. A plethora of studies have shown iron chelators can reverse some of the major hallmarks and enabling characteristics of cancer. Iron chelators inhibit signalling pathways that drive proliferation, migration and metastasis as well as return tumour suppressive signalling. In addition to this, iron chelators stimulate apoptotic and ER stress signalling pathways inducing cell death even in cells lacking a functional p53 gene. Iron chelators can sensitise cancer cells to PARP inhibitors through mimicking BRCAness; a feature of cancers trademark genomic instability. Iron chelators target cancer cell metabolism, attenuating oxidative phosphorylation and glycolysis. Moreover, iron chelators may reverse the major characteristics of oncogenic transformation. Iron chelation therefore represent a promising selective mode of cancer therapy.

An important final year assessment which is common to most UK Undergraduate (UG) degree programmes is the final year project or dissertation. It is considered the capstone module on degree programmes whereby students gain valuable skills valued by employers. Our project engages final year UG students in a collaboration designed with inclusivity and partnership in mind.We ran student-led conferences to communicate the experience of final year students and disseminate their top tips to level 5 students. The project sits across three disciplines in the health and life sciences, the largest faculty in Northumbria University with the aim of developing a widely applicable solution for use across different programmes, departments, faculties and universities. By placing the student experience at its heart, our project seeks to enhance support for student learning through the improvement of graduate attributes including independence of thought and action, curiosity and critical thinking. Thus, employability outcomes are enhanced. The Project conferences aim to respect the diversity of the student body while creating a sense of equality to 1) support supervision quality across the institution. 2) Improve academic experience and 3) enhance graduate characteristics and thus employability.The inclusion of students as partners in pedagogic research in the student-led conferences and in a reflective exercise on the inclusion of students as equal partners in pedagogical research encouraged self- management of their own project and supervisor/student relationship thereby enhancing the student experience whilst developing their key graduate attributes. Student researchers played a major role in data analysis and evaluation. Additionally an eLearning platform organisation site has been constructed continuing the key resources for staff and students alongside video recordings of student presentations. This scalable project supports the development of graduates that are distinguished by their intellectual expertise and employability through meaningful pedagogical research that maximises student satisfaction.

The aim of this study was to understand the experience of Applied science undergraduate (UG) project supervision. The final year UG research project has been coined the pedagogy of the 21st century. The diversity of supervisory styles is a key strength of project modules but is a source of student perceived inequality of experience in a high stakes terminal assessment. This project engaged final year UG students in a collaboration designed with inclusivity and partnership in mind. Student ?researchers? were equal partners and project evolution was led through their involvement in all aspects; study design, methods, resource development and dissemination of the outputs. The overall goal and impact of the project was to provide structure and support to dissertation students and supervisors alike by developing research informed resources that are accessible, engaging and student centred. Three themes were regularly identified; education, practical and emotional support. A key finding was that the student-supervisor relationship strongly influences student experience, satisfaction and success. However, whilst 75 % of students strongly agreed that a good student/supervisor partnership positively affects the success of the final project, only 40 % felt strongly that they had managed to build a partnership with their supervisor. The team used the emergent themes to pull together a ?making supervision work? help guide, a visual model of how the student-supervisor partnership can support the development of skills and progression towards independence. To support this further, a list of discussion points were put together by student researchers aimed at making supervision more effective without making the process a prescriptive one. The answers are personal to each supervisor/student pair but the use of a standard set of questions provides parity, clarity and structure.This project is readily scalable and we strongly believe that this strategic collaboration will see improvements in the student/staff experience of UG supervision.

Maspin is a non-inhibitory member of the serpin family that affects cell behaviours related to migration and survival. We have previously shown that peptides of the isolated G α-helix (G-helix) domain of maspin show bioactivity. Migration, invasion, adhesion and proliferation of vascular smooth muscle cells (VSMC) are important processes that contribute to the build-up of atherosclerotic plaques. Here we report the use of functional assays of these behaviours to investigate whether other maspin-derived peptides impact directly on VSMC; focusing on potential anti-atherogenic properties. We designed 18 new peptides from the structural moieties of maspin above ten amino acid residues in length and considered them beside the existing G-helix peptides. Of the novel peptides screened those with the sequences of maspin strand 4 and 5 of beta sheet B (S4B and S5B) reduced VSMC migration, invasion and proliferation, as well as increasing cell adhesion. A longer peptide combining these consecutive sequences showed a potentiation of responses, and a 7-mer contained all essential elements for functionality. This is the first time that these parts of maspin have been highlighted as having key roles affecting cell function. We present evidence for a mechanism whereby S4B and S5B act through ERK1/2 and AMP-activated protein kinase (AMPK) to influence VSMC responses.

The prognostic value of cancer stem cell markers in various cancer subtypes is a well documented research area. Our findings show that the stem cell marker Lgr5 is associated with an aggressive phenotype in neuroblastoma. Here, we discuss these findings within the context of recent studies in several cancers such as lung, colorectal and intestinal cancer, glioblastoma and ewing's sarcoma. Neuroblastoma continues to be an elusive disease, due to its heterogeneous presentation ranging from spontaneous regression to aggressive metastatic disease and intertwined genetic variability. Currently, the most significant prognostic marker of high risk disease and poor prognosis is amplification of the MYCN oncogene, which is found in approximately 25% of cases (Huang and Weiss, 2013). With this in mind, there is still much to learn about the driving mechanisms of this aggressive pediatric tumor. Neuroblastoma development is thought to be the result of aberrant differentiation of the cell of origin, embryonic neural crest cells which then migrate and invade during the developmental stage (Joshi et al., 2007). Aberrant cells are those which would, under normal conditions form the mature tissues of the sympathetic ganglia and adrenal medulla. Tumors are known to develop indiscriminately along the radius of the sympathetic ganglia, although it is well established that the adrenal glands are fundamentally the most common primary site (Jessen and Mirsky, 2005).

The stress-inducible transcription complex NF-κB induces the transcription of genes that regulate proliferation and apoptosis. Constitutively activated NF-κB is common in breast cancers, and contributes to malignant progression and therapeutic resistance. Ataxia telangiectasia mutated (ATM) is a key regulator of the cellular response to DNA double strand breaks (DSBs), and recent reports have demonstrated that ATM is required for the activation of NF-κB following DNA damage. We investigated the role of ATM in the NF-κB signalling cascade induced by ionising radiation (IR) in breast cancer cell lines using KU55933, a novel and specific inhibitor of ATM. KU55933 suppressed IR-induced IκBα degradation, p50/p65 nuclear translocation and binding to kB consensus sequences. KU55933 also suppressed transcription of an NF-κB dependent reporter gene and inhibited IR-induced DSB repair as assessed by the neutral Comet assay. KU55933 sensitised cells to IR, with a concurrent increase in caspase 3 activity. Importantly, KU55933 sensitised IKKβ(+/+) and p65(+/+), but not IKKβ(-/-) or p65(-/-), mouse embryonic fibroblasts to IR, despite the equivalent inhibitory effects of KU55933 on DSB repair in both the proficient and the deficient cell lines. P65 siRNA had no effect on DSB repair in either breast cancer cell line. When combined with KU55933, DSB repair was inhibited to the same extent as KU55933 alone in both breast cancer cell lines. P65 siRNA alone sensitised both cell lines to IR. A combination of p65 siRNA and KU55933 resulted in no further sensitisation compared to either one alone. Taken together these data support the hypothesis that KU55933-mediated radio-sensitisation is solely a consequence of its inhibition of NF-κB activation. We conclude that radiotherapy deploying ATM inhibitors may be particularly advantageous in tumours where NF-κB is constitutively activated.

Recent reports implicate poly(ADP-ribose) polymerase-1 (PARP-1) in the activation of nuclear factor kappaB (NF-kappaB). We investigated the role of PARP-1 in the NF-kappaB signalling cascade induced by ionizing radiation (IR). AG14361, a potent PARP-1 inhibitor, was used in two breast cancer cell lines expressing different levels of constitutively activated NF-kappaB, as well as mouse embryonic fibroblasts (MEFs) proficient or deficient for PARP-1 or NF-kappaB p65. In the breast cancer cell lines, AG14361 had no effect on IR-induced degradation of IkappaBalpha or nuclear translocation of p50 or p65. However, AG14361 inhibited IR-induced NF-kappaB-dependent transcription of a luciferase reporter gene. Similarly, in PARP-1(-/-) MEFs, IR-induced nuclear translocation of p50 and p65 was normal, but kappaB binding and transcriptional activation did not occur. AG14361 sensitized both breast cancer cell lines to IR-induced cell killing, inhibited IR-induced XIAP expression and increased caspase-3 activity. However, AG14361 failed to increase IR-induced caspase activity when p65 was knocked down by siRNA. Consistent with this, AG14361 sensitized p65(+/+) but not p65(-/-) MEFs to IR. We conclude that PARP-1 activity is essential in the upstream regulation of IR-induced NF-kappaB activation. These data indicate that potentiation of IR-induced cytotoxicity by AG14361 is mediated solely by inhibition of NF-kappaB activation.

DNA-dependent protein kinase (DNA-PK) and poly (ADP-ribose) polymerase-1 (PARP-1) participate in nonhomologous end joining and base excision repair, respectively, and are key determinants of radio- and chemo-resistance. Both PARP-1 and DNA-PK have been identified as therapeutic targets for anticancer drug development. Here we investigate the effects of specific inhibitors on enzyme activities and DNA double-strand break (DSB) repair. The enzyme activities were investigated using purified enzymes and in permeabilized cells. Inhibition, or loss of activity, was compared using potent inhibitors of DNA-PK (NU7026) and PARP-1 (AG14361), and cell lines proficient or deficient for DNA-PK or PARP-1. Inactive DNA-PK suppressed the activity of PARP-1 and vice versa. This was not the consequence of simple substrate competition, since DNA ends were provided in excess. The inhibitory effect of DNA-PK on PARP activity was confirmed in permeabilized cells. Both inhibitors prevented ionizing radiation-induced DSB repair, but only AG14361 prevented single-strand break repair. An increase in DSB levels caused by inhibition of PARP-1 was shown to be caused by a decrease in DSB repair, and not by the formation of additional DSBs. These data point to combined inhibition of PARP-1 and DNA-PK as a powerful strategy for tumor radiosensitization.

This chapter discusses the inhibition of DNA repair as a therapeutic target. Inhibition of DNA repair represents an excellent target for cancer therapy. DNA repair inhibitors can increase the specificity and toxicity of radio- and chemotherapy, or be stand-alone treatments for cancers that are defective in other DNA repair pathways by amplifying endogenous lesions present in the cancer cells. DNA repair can affect the response to cancer therapy in two ways. First, as radiotherapy and most chemotherapy drugs cause DNA damage, DNA repair is an important determinant for treatment efficiency. High levels of DNA repair contribute to removal of the DNA lesions, resulting in resistance and failed cancer treatments. Secondly, the defects in DNA repair in cancers influence the chemotherapy outcome. For instance, mismatch repair defective tumors are resistant to a variety of drugs, while homologous recombination defective tumors are highly sensitive to cross-linkers. There is a major therapeutic potential to enhance the persistence of toxic lesions in cancer cells through inhibition of DNA repair enzymes. The success following preclinical and clinical trials using inhibition of O6-alkylguanine DNA alkyltransferase, poly (ADP-ribose) polymerase, and DNA dependent protein kinase provides a strong rationale for further development of inhibitors for DNA repair as future sensitizers to current anticancer treatments.

The stress-inducible transcription factor, nuclear factor (NF)-κB induces genes involved in proliferation and apoptosis. Aberrant NF-κB activity is common in cancer and contributes to therapeutic-resistance. Poly(ADP-ribose) polymerase-1 (PARP-1) is activated during DNA strand break repair and is a known transcriptional co-regulator. Here, we investigated the role of PARP-1 function during NF-κB activation using p65 small interfering RNA (siRNA), PARP siRNA or the potent PARP-1 inhibitor, AG-014699. Survival and apoptosis assays showed that NF-κB p65(-/-) cells were more sensitive to ionizing radiation (IR) than p65(+/+) cells. Co-incubation with p65 siRNA, PARP siRNA or AG-014699 radio-sensitized p65(+/+), but not p65(-/-) cells, demonstrating that PARP-1 mediates its effects on survival via NF-κB. Single-strand break (SSB) repair kinetics, and the effect SSB repair inhibition by AG-014699 were similar in p65(+/+) and p65(-/-) cells. As preventing SSB repair did not radio-sensitize p65(-/-) cells, we conclude that radio-sensitization by AG-014699 is due to downstream inhibition of NF-κB activation, and independent of SSB repair inhibition. PARP-1 catalytic activity was essential for IR-induced p65 DNA binding and NF-κB-dependent gene transcription, whereas for tumor necrosis factor (TNF)-α-treated cells, PARP-1 protein alone was sufficient. We hypothesize that this stimulus-dependent differential is mediated via stimulation of the poly(ADP-ribose) polymer, which was induced following IR, not TNF-α. Targeting DNA damage-activated NF-κB using AG-014699 may therefore overcome toxicity observed with classical NF-κB inhibitors without compromising other vital inflammatory functions. These data highlight the potential of PARP-1 inhibitors to overcome NF-κB-mediated therapeutic resistance and widens the spectrum of cancers in which these agents may be utilized.

namic gene delivery technique; this permitted hepatocytes to produce and secrete the soluble protein. This technique uses hydrodynamic force, generated by an injection of a large volume (10% of body weight) of DNA solution (30 ig/mL) over 5–7 seconds through the tail vein, to permeabilize the capillary endothelium of the liver and generate ‘pores’ in the plasma membrane of the surrounding parenchymal cells, thereby allowing DNA to reach the cell interior. With time, the membrane pores close, trapping these molecules inside. At that point, transfected cells transiently produce the molecule encoded by the vector. We generated a CD40L construct consisting of the soluble part of human CD40L and the trimerization domain of adiponectin (ADPN-CD40L). We also created a similar construct for human interleukin (IL)-4. The adiponectin domain creates a trimer that can also dimerize to a hexamer, creating multimeric forms with greater biological activity. Furthermore, glycosylation of adiponectin increases the stability of these molecules. After hydrodynamic gene delivery of the ADPN-CD40L or the ADPN-IL-4 expression vector, high levels of ADPN-CD40L ( 5 ng/mL) and ADPN-IL-4 ( 3 ng/mL) were found in the sera of treated mice for more than 6 months. Similar data were obtained when both constructs were injected simultaneously. Next, CFSE-labeled CLL peripheral blood mononuclear cells, followed by anti-CD3 monoclonal antibodies, were injected in these mice. Untreated mice and mice that were co-injected with allogeneic monocytes served as negative and positive controls. Ten days after CLL cell injection into mice with circulating ADPN-CD40L, we documented leukemia cell proliferation in the blood and spleen by CFSE dilution for every CLL sample studied, although the degree of proliferation varied among samples. Nevertheless, proliferation could be extensive; in some cases, leukemic cells underwent more than 6 rounds of division. Mice that received only ADPN-IL-4 did not show enhanced CLL cell proliferation but had increased viability over control animals. Finally, mice that received ADPN-CD40L plus ADPN-IL-4 had the greatest degree of CLL cell proliferation and viability, exceeding that in mice receiving ADPN-CD40L or ADPN-IL-4 alone. At the same time point, no proliferation in untreated mice and negligible or minimal CLL cell proliferation was found in mice receiving monocytes. Thus, this approach reduces or may eliminate a downside of our initial model, in which necessary but uncontrolled T-cell expansion to allo-antigens generated graft-versus-host disease and a graftversus-leukemia effect, making the model short term. This new approach may lead to a long-term model that would allow more complex preclinical studies or would be a better tool to study the basic biology of CLL cells in vivo. Experiments testing this are currently in progress.

Abstract 3588 Poor prognosis patients with chronic lymphocytic leukaemia (CLL) can be identified by cytogenetic abnormalities such as del(17p), and del(11q) and corresponding mutations in TP53 and ATM (ataxia telangiectasia mutated kinase) respectively, which are associated with chemoresistance by virtue of defects in the DNA damage response pathway. We have demonstrated that overexpression of DNA-dependent protein kinase (DNA-PK), which mediates non-homologous end joining, is also associated with poor prognosis CLL1. Recent data show that constitutive activation of the p65 subunit of the transcription factor, NF-κB, confers poor survival in CLL. Since the repertoire of genes activated by NF-κB includes anti-apoptotic and pro-survival genes, NF-κB therefore represents an attractive target for therapeutic intervention. The parthenolide analogue, LC-1, has been shown to be synergisitic with fludarabine in ex vivo studies on CLL cells2, and has validated the concept of targeting NF-κB and prompted clinical trials in CLL patients. Previous data from our laboratory using cell line models demonstrated that DNA damage-induced NF-κB activation requires the base excision repair protein, poly(ADP-ribose) polymerase (PARP), to confer radioresistance3. The DNA damage sensor, ATM, also mediates the response to DNA damage, via phosphorylation of IKK-γ (Inhibitory κ-Kinase/NEMO). In fact, we demonstrated that radio-sensitization by an ATM inhibitor is mediated via NF-κB, rather than by the inhibition of double strand break repair4. Since these observations identify key DNA damage response proteins as regulators of NF-κB activity, we hypothesized that inhibitors of PARP, ATM and DNA-PK, could chemosensitize CLL cells via inhibition of NF-κB. Here, we analyzed NF-κB activity by quantifying NF-κB subunit DNA binding (measured by ELISA) in an unselected CLL cohort. Constitutive activation of the p65 and p50 subunits correlated closely (P= 0.001, n= 57), and predicted shorter time to first treatment (TTFT) and overall survival (OS). Importantly, higher activation occurred in del(17p) (e.g. p50; P= 0.05, n= 49) cases and in those cases that had received treatment. p52 and c-Rel activation correlated with p65 and p50 activation. However, although high p65 and p50 activation predicted shorter OS and TTFT, increased p52 and c-Rel activation were associated with a longer TTFT (up to 40 months) demonstrating the complex crosstalk between the NF-κB subunits in CLL. We were able to correlate p50 and p65 subunit activation with ex vivo resistance to fludarabine and chlorambucil in 12 cases: the most chemoresistant cases had higher p50 and p65 DNA binding activity. Preliminary data indicates that levels of activated NF-κB binding seen in the ELISA correlates with protein expression in nuclear extracts isolated from CLL cells (P= 0.0013, n=26). Here we make the novel observation that the increase in DNA damage-induced NF-κB activation is linked to increased DNA-PK activation. DNA-PK catalytic subunit levels were significantly higher in patients with high p65 activation (P= 0.02, n= 30), regardless of treatment status. Furthermore, a selective inhibitor of DNA-PK, NU7441, increased mitoxantrone-induced cytotoxicity in CLL cells (up to 50-fold) and reduced p65 and p50 DNA binding, indicating a direct link between DNA-PK and NF-κB activation. Ongoing studies are investigating this mechanistic link further, using chromatin immunoprecipitation (ChIP) and using siRNA knockdown. CLL cells were radiosensitized by either the DNA-PK inhibitor, NU7441 or the pan IKK inhibitor, BAY 11–7082. Strikingly, a combination of these two inhibitors had no further effect on radiosensitization, suggesting that DNA-PK and NF-κB act in a common pathway. We have demonstrated that inhibition of ATM sensitizes CLL cells to DNA damage, and future work will assess the impact of ATM and PARP inhibition on NF-κB activity in CLL cells. These data present a novel role for DNA-PK in the regulation of NF-κB and highlight important new therapeutic avenues for the use of DNA-PK inhibitors, which may prove useful in overcoming NF-κB mediated therapeutic resistance in CLL. Willmore E, et al, Clin Cancer Res. 2008. Hewamana S, et al, Clin Cancer Res. 2008. Hunter JE, et al, Proceedings of the AACR. 2009. Veuger SJ et al, Under review, DNA repair. 2010. Disclosures: No relevant conflicts of interest to declare.