Anthony G. McCluskey

[131I]meta-iodobenzylguanidine and topotecan combination treatment of tumors expressing the noradrenaline transporter.

Purpose: Both [131I]meta-iodobenzylguanidine ([131I]MIBG) and the topoisomerase I inhibitor topotecan are effective as single-agent treatments of neuroblastoma. The aim of this study was to investigate the efficacy of [131I]MIBG in combination with topotecan in vitro and in vivo. Experimental Design: The cell lines used were SK-N-BE(2c) (human neuroblastoma) and UVW/NAT (glioma cell line transfected with the noradrenaline transporter gene). Three different treatment schedules were assessed: topotecan given before (schedule 1), after (schedule 2), or simultaneously (schedule 3) with [131I]MIBG. DNA strand breakage was evaluated by comet assay, and cytotoxicity was determined by clonogenic survival. Efficacy was also measured by growth delay of tumor xenografts in nude mice. Results: Combination schedules 2 and 3 caused more cytotoxicity than schedule 1. Similarly, significant DNA damage was observed following treatment schedules 2 and 3 (P < 0.005) but not schedule 1. The mean number of days for a doubling in volume of SK-N-BE(2c) tumors and a 10-fold increase in volume of UVW/NAT tumors were 10.4 and 18.6 (untreated), 19.7 and 25.3 (topotecan alone), 22.8 and 31.9 ([131I]MIBG alone), 26.3 and 37.1 (combination schedule 1), 34.3 and 49.7 (combination schedule 2), and 53.2 and >71 (combination schedule 3), respectively. The highest rate of cure of both xenografts was observed following treatment with combination schedule 3. Conclusions: The combination of topotecan and [131I]MIBG compared with either treatment alone gave rise to greater than additive DNA damage, clonogenic cell kill, and tumor growth delay. These effects were dependent on the scheduling of the two agents.

Deletion of the Dual Specific Phosphatase-4 (DUSP-4) Gene Reveals an Essential Non-redundant Role for MAP Kinase Phosphatase-2 (MKP-2) in Proliferation and Cell Survival

Mitogen-activated protein kinase phosphatase-2 (MKP-2) is a type 1 nuclear dual specific phosphatase (DUSP) implicated in a number of cancers. We examined the role of MKP-2 in the regulation of MAP kinase phosphorylation, cell proliferation, and survival responses in mouse embryonic fibroblasts (MEFs) derived from a novel MKP-2 (DUSP-4) deletion mouse. We show that serum and PDGF induced ERK-dependent MKP-2 expression in wild type MEFs but not in MKP-2−/− MEFs. PDGF stimulation of sustained ERK phosphorylation was enhanced in MKP-2−/− MEFs, whereas anisomycin-induced JNK was only marginally increased. However, marked effects upon cell growth parameters were observed. Cellular proliferation rates were significantly reduced in MKP-2−/− MEFs and associated with a significant increase in cell doubling time. Infection with adenoviral MKP-2 reversed the decrease in proliferation. Cell cycle analysis revealed a block in G2/M phase transition associated with cyclin B accumulation and enhanced cdc2 phosphorylation. MEFs from MKP-2−/− mice also showed enhanced apoptosis when stimulated with anisomycin correlated with increased caspase-3 cleavage and γH2AX phosphorylation. Increased apoptosis was reversed by adenoviral MKP-2 infection and correlated with selective inhibition of JNK signaling. Collectively, these data demonstrate for the first time a critical non-redundant role for MKP-2 in regulating cell cycle progression and apoptosis.

Radiation quality-dependent bystander effects elicited by targeted radionuclides

The efficacy of radiotherapy may be partly dependent on indirect effects, which can sterilise malignant cells that are not directly irradiated. However, little is known of the influence of these effects in targeted radionuclide treatment of cancer. We determined bystander responses generated by the uptake of radioiodinated iododeoxyuridine ([*I]IUdR) and radiohaloanalogues of meta‐iodobenzyl‐guanidine ([*I]MIBG) by noradrenaline transporter (NAT) gene‐transfected tumour cells. NAT specifically accumulates MIBG. Multicellular spheroids that consisted of 5% of NAT‐expressing cells, capable of the active uptake of radiopharmaceutical, were sterilised by treatment with 20 kBqmL−1 of the α‐emitter meta‐[211At]astatobenzylguanidine ([211At]MABG). Similarly, in nude mice, retardation of the growth of tumour xenografts containing 5% NAT‐positivity was observed after treatment with [131I]MIBG. To determine the effect of subcellular localisation of radiolabelled drugs, we compared the bystander effects resulting from the intracellular concentration of [131I]MIBG and [131I]IUdR (low linear energy transfer (LET) β‐emitters) as well as [123I]MIBG and [123I]IUdR (high LET Auger electron emitters). [*I]IUdR is incorporated in DNA whereas [*I]MIBG accumulates in extranuclear sites. Cells exposed to media from [131I]MIBG‐ or [131I]IUdR‐treated cells demonstrated a dose‐response relationship with respect to clonogenic cell death. In contrast, cells receiving media from cultures treated with [123I]MIBG or [123I]IUdR exhibited dose‐dependent toxicity at low dose but elimination of cytotoxicity with increasing radiation dose (i.e. U‐shaped survival curves). Therefore radionuclides emitting high LET radiation may elicit toxic or protective effects on neighbouring untargeted cells at low and high dose respectively. It is concluded that radiopharmaceutical‐induced bystander effects may depend on LET of the decay particles but are independent of site of intracellular concentration of radionuclide.

Inhibition of Poly(ADP-Ribose) Polymerase Enhances the Toxicity of 131I-Metaiodobenzylguanidine/Topotecan Combination Therapy to Cells and Xenografts That Express the Noradrenaline Transporter

Targeted radiotherapy using 131I-metaiodobenzylguanidine (131I-MIBG) has produced remissions in some neuroblastoma patients. We previously reported that combining 131I-MIBG with the topoisomerase I inhibitor topotecan induced long-term DNA damage and supraadditive toxicity to noradrenaline transporter (NAT)–expressing cells and xenografts. This combination treatment is undergoing clinical evaluation. This present study investigated the potential of poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP-1) inhibition, in vitro and in vivo, to further enhance 131I-MIBG/topotecan efficacy. Methods: Combinations of topotecan and the PARP-1 inhibitor PJ34 were assessed for synergism in vitro by combination-index analysis in SK-N-BE(2c) (neuroblastoma) and UVW/NAT (NAT-transfected glioma) cells. Three treatment schedules were evaluated: topotecan administered 24 h before, 24 h after, or simultaneously with PJ34. Combinations of PJ34 and 131I-MIBG and of PJ34 and 131I-MIBG/topotecan were also assessed using similar scheduling. In vivo efficacy was measured by growth delay of tumor xenografts. We also assessed DNA damage by γH2A.X assay, cell cycle progression by fluorescence-activated cell sorting analysis, and PARP-1 activity in treated cells. Results: In vitro, only simultaneous administration of topotecan and PJ34 or PJ34 and 131I-MIBG induced supraadditive toxicity in both cell lines. All scheduled combinations of PJ34 and 131I-MIBG/topotecan induced supraadditive toxicity and increased DNA damage in SK-N-BE(2c) cells, but only simultaneous administration induced enhanced efficacy in UVW/NAT cells. The PJ34 and 131I-MIBG/topotecan combination treatment induced G2 arrest in all cell lines, regardless of the schedule of delivery. In vivo, simultaneous administration of PJ34 and 131I-MIBG/topotecan significantly delayed the growth of SK-N-BE(2c) and UVW/NAT xenografts, compared with 131I-MIBG/topotecan therapy. Conclusion: The antitumor efficacy of topotecan, 131I-MIBG, and 131I-MIBG/topotecan combination treatment was increased by PARP-1 inhibition in vitro and in vivo.

Experimental treatment of neuroblastoma using [131I]meta-iodobenzylguanidine and topotecan in combination

The radiopharmaceutical [(131)I]meta-iodobenzylguanidine ([(131)I]MIBG) and the topoisomerase I inhibitor topotecan are both effective as single-agent treatments of neuroblastoma. Our purpose was to assess the therapeutic potential of [(131)I]MIBG and topotecan in combination using SK-N-BE(2c) neuroblastoma cells and UVW/NAT glioma cells expressing the noradrenaline transporter transgene. Topotecan treatment was given (i) before, (ii) after or (iii) simultaneously with [(131)I]MIBG. DNA fragmentation was evaluated by comet assay and cell cycle redistribution was determined by fluorescence-activated cell sorting. Combination index analysis indicated that delivery schedules (ii) and (iii) were more effective than schedule (i) with respect to clonogenic cell kill. Similarly, significant DNA damage was observed following treatment schedules (ii) and (iii) (p <0.005), but not (i). Prior exposure to topotecan did not significantly enhance [(131)I]MIBG uptake in athymic mice bearing tumour xenografts. We conclude that the enhancement of the efficacy of [(131)I]MIBG by combining it with topotecan was the result of inhibition of DNA damage repair rather than an increase in expression of the noradrenaline transporter by tumour.

Lysosomotropism depends on glucose : a chloroquine resistance mechanism

There has been long-standing interest in targeting pro-survival autophagy as a combinational cancer therapeutic strategy. Clinical trials are in progress testing chloroquine (CQ) or its derivatives in combination with chemo- or radiotherapy for solid and haematological cancers. Although CQ has shown efficacy in preclinical models, its mechanism of action remains equivocal. Here, we tested how effectively CQ sensitises metastatic breast cancer cells to further stress conditions such as ionising irradiation, doxorubicin, PI3K-Akt inhibition and serum withdrawal. Contrary to the conventional model, the cytotoxic effects of CQ were found to be autophagy-independent, as genetic targeting of ATG7 or the ULK1/2 complex could not sensitise cells, like CQ, to serum depletion. Interestingly, although CQ combined with serum starvation was robustly cytotoxic, further glucose starvation under these conditions led to a full rescue of cell viability. Inhibition of hexokinase using 2-deoxyglucose (2DG) similarly led to CQ resistance. As this form of cell death did not resemble classical caspase-dependent apoptosis, we hypothesised that CQ-mediated cytotoxicity was primarily via a lysosome-dependent mechanism. Indeed, CQ treatment led to marked lysosomal swelling and recruitment of Galectin3 to sites of membrane damage. Strikingly, glucose starvation or 2DG prevented CQ from inducing lysosomal damage and subsequent cell death. Importantly, we found that the related compound, amodiaquine, was more potent than CQ for cell killing and not susceptible to interference from glucose starvation. Taken together, our data indicate that CQ effectively targets the lysosome to sensitise towards cell death but is prone to a glucose-dependent resistance mechanism, thus providing rationale for the related compound amodiaquine (currently used in humans) as a better therapeutic option for cancer.

Gamma irradiation and targeted radionuclides enhance the expression of the noradrenaline transporter transgene controlled by the radio-inducible p21(WAF1/CIP1) promoter.

The use of radiation-inducible promoters to drive transgene expression offers the possibility of temporal and spatial regulation of gene activation. This study assessed the potential of one such promoter element, p21WAF1/CIP1 (WAF1), to drive expression of the noradrenaline transporter (NAT) gene, which conveys sensitivity to radioiodinated meta-iodobenzylguanidine (MIBG). An expression vector containing NAT under the control of the radiation-inducible WAF1 promoter (pWAF/NAT) was produced. The non-NAT expressing cell lines UVW (glioma) and HCT116 (colorectal cancer) were transfected with this construct to assess radiation-controlled WAF1 activation of the NAT gene. Transfection of UVW and HCT cells with pWAF/NAT conferred upon them the ability to accumulate [131I]MIBG, which led to increased sensitivity to the radiopharmaceutical. Pretreatment of transfected cells with γ radiation or the radiopharmaceuticals [123I]MIBG or [131I]MIBG induced dose- and time-dependent increases in subsequent [131I]MIBG uptake and led to enhanced efficacy of [131I]MIBG-mediated cell kill. Gene therapy using WAF1-driven expression of NAT has the potential to expand the use of this therapeutic modality to tumors that lack a radio-targetable feature.

Activation of the noradrenaline transporter gene using the radiation-inducible WAF1-1 promoter sensitizes tumour cells to [I-131] MIBG targeted radiotherapy

The Syrian hamster embryo (SHE) assay (pH6.7) is being touted as a ‘‘3R’s’’ alternative in animal laboratory studies. In the SHE assay, traditionally, colonies are counted and scored by eye to determine the transforming potential of test chemicals. Application of infrared (IR) spectroscopy opens up the possibility of comparing test chemicals with negative and positive controls in a high-throughput fashion (1) through objective pattern recognition methods. Such methods are under development under the 1) ‘‘openness’’, and 2) multiple-class requirements: 1) computer systems need to be ‘‘open’’ to new data to refine existing classifiers; 2) furthermore, the existence of multiple classes (i.e., chemical treatment conditions) calls for composite architectures containing many simple classifiers instead a single complicated one. In this study we present two classification strategies contemplating these two principles. The proposed strategies are compared to well established ‘‘closed’’, single-model classifiers. The dataset used in the study was derived from a SHE assay where eight treatment conditions were present [vehicle control (DMSO), D-M, B[a]P, 3-MCA, Anthracene, o-T, 2,4-dT, and MNNG] (2). From the assay, IR spectra (n¼14,000) were obtained using attenuated total reflection Fourier-transform IR spectroscopy. Gradual feeding of the proposed models with training data is shown to gradually improve the classification of test data. Segregation of data along chemical mode of action was observed. Overall, the results strengthen arguments towards using the SHE assay in toxicological assessments and point to IR spectroscopy as a possible alternative to visual scoring.DNA is not chemically inert but faces constant challenges to its stability. One of these is the fusion of adjacent pyrimidine bases by ultra violet (UV) radiation to create cyclobutane pyrimidine dimers (CPDs). Numerous methods of DNA repair have evolved within cells, of which nucleotide excision repair (NER) is responsible for the removal of CPDs and other bulky adducts. To investigate this and other repair pathways various techniques have been developed to detect DNA damage at low resolutions in whole genomes or high resolutions over small sections of a genome. We have developed a novel microarray based method for the genome wide high resolution analysis of DNA damage in yeast which combines the advantages of these, allowing detailed measurement of repair across entire genomes. A program has been written to predict the expected CPD formation based on sequence; this has shown that the genome wide damage detection method is accurate. Additionally, ChIPchip has been used to determine the binding positions of proteins involved in NER and analyse histone modifications after damage induction. Combining these datasets allows protein binding and acetylation levels to be correlated with repair rates. These datasets require bioinformatic tools to analyse and extract results. I have developed a suite of novel tools to process, normalise, display and interrogate these datasets including a new normalisation method which allows accurate comparisons to be made between different factors, revealing changes in acetylation profiles following UV and between different mutant strains, a peak detection method to distinguish protein binding peaks from a background of nonbound regions, revealing many novel binding sites for proteins such as Abf1 and Rad16, and graphical displays to determine patterns that occur at multiple positions throughout genomes, revealing patterns of varying repair rates at regions such as centromeres and telomeres.