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Commentary |
1 School of Biomedical Sciences, University of Ulster at Coleraine BT52 1SA, Northern Ireland, 2 Queen Elizabeth University Hospital, Birmingham B15 2TH, UK
The goal of all anti-cancer treatments is to design strategies that are specific for tumours and non-toxic to the patient. Molecular targeting is now becoming a reality with new treatments designed to target processes that are thought to be tumour specific, or where there are quantitative differences in target expression between cancer and normal cells. On 3 March 2005, the Radiation and Cancer Biology committee held a meeting to discuss the targeting of DNA repair pathways, which are often defective in tumours. Prof. Steve Jackson (Cambridge University) started the programme with a discussion of some of the main DNA damage response (DDR) pathways. Repair in normal cells is a hugely efficient process, with individuals requiring repair of an estimated 1018 DNA lesions per day caused by reactive oxygen species alone. Most of the inherited cancer predisposition syndromes involve DDR dysfunction and similar mutations are often found in sporadic cancers. The specificity of DDR targeting agents comes from the need for the faster dividing tumour cells to repair DNA damage more quickly and efficiently than the mostly quiescent, or more slowly cycling normal cells. This may also be compromised by an already defective DDR pathway, which further reduces the ability of the tumour cells to repair efficiently, while being less critical in normal cells. He pointed out the clearly integrated nature of stress response in cells since DNA-PK, ATM and ATR have overlapping roles in DDR, transcriptional regulation, cell cycle control and cell death pathways. These processes are relevant not only to cancer therapy, but also in immune deficiency syndromes, neurodegenerative disorders, infertility, premature ageing and impaired telomere function.
Many laboratory approaches were discussed during the day. Of particular relevance was the talk by Dr Kai Rothkamm (Gray Cancer Institute, Northwood) who discussed the potential uses of the 
H2AX assay to measure double strand breaks (DSB). This relatively new assay allows quantitation of DSBs with more accuracy and at much lower doses than was possible previously. However, as with many assays there are inherent pitfalls as well as advantages of this method, and further work is required to characterize the assay completely. Dr Rothkamm identified possibilities for its use as a low dose exposure assay, using blood lymphocytes, since it is sufficiently sensitive to quantify exposure after diagnostic CT scans. It can also be used in studies of DDR inhibitors to quantitate responses and several speakers during the day reported uses for this assay.
Prof. Jackson proposed that if certain types of cancer possess inherent DNA repair disorders then, in principle, inhibition of the remaining DDR mechanisms should lead to cell death more efficiently that can be achieved in normal cells where the full complement of repair enzymes is available. This theme was exemplified by several of the symposium speakers.
Dr Niall Martin (KuDOS Pharmaceuticals, Cambridge) described two approaches to this strategy. In colorectal tumour cell lines with mis-match repair (MMR) defects, the response to standard cytotoxic agents such as temozolamide is enhanced when combined with inhibitors of PARP-1 (Poly (ADP-ribose) polymerase-1) – an enzyme critical to the early response to single strand breaks (SSB). This combination increases the yield of both SSB and DSB; the latter have been shown using the DSB specific H2AX assay. MMR defects are not found in normal bone marrow cells, so that enhanced acute marrow toxicity is not to be expected. BRCA1/2 are also known to be key proteins involved in the cellular response to DDR. A significant number of breast tumours contain defects in BRAC1/2, including almost all inherited breast tumours. Inhibition of repair with PARP-1 caused a profound sensitization of BRCA1/2 deficient cells affecting G2/M checkpoint arrest, increased chromosome aberrations and tumour regression in the absence of other cytotoxic agents. This offers an exciting opportunity to control this relatively large subset of breast tumours. A similar approach, targeting DNA-PK inhibitors, cause preferential cell kill in ATM -/- cells, again with dramatic effects in vitro.
Prof. Penny Jeggo (Sussex University) described the role in DNA repair of ATM, and a small ATM interacting nuclease, called Artemis. Using quiescent fibroblasts, so that cell cycle differences did not confound the interpretation, she showed that for full restoration of DNA damage caused by some agents, times in the region of 72 h are needed. This is significantly longer than most reported repair studies and interestingly is longer than the time allowed between fractions in conventional radiotherapy; in part this may offer an explanation for the poorer DNA repair capacity in tumour vs normal cells. Although most (
90%) of the DNA repair occurs rapidly, the residual damage is significantly more difficult to deal with. The slower repair process appears to be dependent on the integrity of cell cycle checkpoint control. In addition, more severe lesions, using alpha particles, show a longer time to complete resolution of the damage. She showed evidence that Artemis is required for this process suggesting another potential drug target.
Prof. Hilary Calvert (University of Newcastle) gave a keynote lecture on the current clinical trials involving DNA repair inhibitors. Resistance to methylating agents in many cells is caused by the repair enzyme alkylguanine alkyltransferase (O6AT). This enzyme can be inhibited using 6-benzylguanine (6BG) and 4 bromothenylguanine (Patrin). Unfortunately the clinically tolerated dose of Carmustine must be reduced threefold in combination with 6BG, whereas temozolamide is less affected by combination with Patrin. This suggests that O6AT plays an important role in normal tissue recovery. Phase 2/3 trials are currently determining whether there is an overall therapeutic benefit with this combination. A Phase 1 trial combining a PARP-1 inhibitor with temozolamide is just about to report and further trials are in the planning stages. Prof. Calvert discussed briefly the difficulties of setting up clinical trials in the molecular targeting era, where precise control of sample collection, storage and evaluation must be in place to identify the molecular profile of the tumour and its likely susceptibility to the treatment under investigation.
Dr Stephany Veuger (Newcastle University) presented further work on PARP-1 inhibition. NF-kB is a stress inducible transcription complex that induces genes that control proliferation responses and suppress apoptotic cascades. Aberrant activation of NF-kB is common in tumours and recently it has been noticed that its activation in PARP-1 deficient cells is reduced. The involvement of these two proteins in the presence or absence of a potent PARP-1 inhibitor (AG14361) was investigated when cells were also exposed to 20 Gy ionizing radiation (IR). The data provided evidence that PARP-1 function is required for NF-kB activity following exposure to IR. The results suggested that potentiation of IR-induced radiosensitivity may be through inhibition of NF-kB rather than as a direct consequence of PARP-1 mediated inhibition of DNA repair. This result clearly has implications for rationale design of PARP-1 inhibitors in the treatment of cancer.
Dr Paul Mullan (Queen's University, Belfast) showed the power of an initial microarray screen to identify differences in BRCA1 competent and deficient cells. Dr Mullan and colleagues have identified a family of calcium binding proteins that are novel BRCA-1 repressed targets. S100A7 (psoriasin) is dependent on functional c-Myc and is also inducible by DNA damage in a BRCA-1 dependent manner. They linked this to a novel pathway of p27kip1 down-regulation that has previously been seen to be consistently down-regulated in BRCA1 mutated cells. The data have allowed identification of a novel pathway that could provide a target for molecular targeting agents.
Targeting of DNA base excision repair was discussed by Dr Srinivasan Madhusudan (CRUK, Weatherall Institute of Molecular Medicine, Oxford). The multifunctional protein endonuclease HAP-1/APE-1/Ref-1 is involved in base excision repair and is implicated in the pathogenesis of several human tumours. Its over-expression is linked to both chemoresistance and radioresistance. Using a high throughput chemical screen, the Oxford group has identified KM09181 as a lead inhibitor of HAP-1 with an IC50 value of 3.5 mM. At non-toxic concentrations it causes significant potentiation of the cytotoxicity of a number of agents. This report is the first biological evidence for the direct targeting of this DNA repair enzyme.
A series of novel PARP-1 inhibitors were described by Dr Esther Woon (University of Bath). Previously, they had identified 5-aminoisoquinolin-1-one (5-AIQ), which shows a wide range of therapeutic activity in vivo. Using the PARP-1 crystal structure, they designed a series of compounds similar to 5-AIQ, with the aim of identifying novel compounds with more potent PARP-1 inhibitory activity while retaining the excellent biopharmaceutical properties of 5-AIQ. A compound, 5-amino-3-methylisoquinolin-1-one (3-Me-5AIQ) was identified, which was 7 times more potent than 5-AIQ.
A rather surprising result was reported by Dr S C Sak (CRUK, Leeds) who used immunohistochemistry to assess expression of two DDR proteins, APE-1 and XRCC1, in biopsy samples from 90 muscle invasive bladder tumours. High levels of these proteins correlated with survival after radical radiotherapy. On first reflection, high expression should protect tumours from IR. However, others factors may be invoked to explain this. Since the median patient age was 75 years, it is possible that depletion of natural radioprotectors, e.g. glutathione and other sulphydryl compounds, might allow more DSB damage to occur per unit dose with enhanced repair responses in patients who are cured. Another potential explanation could be the occurrence of enhanced mis-repair in these patients. This enigmatic finding needs further investigation.
The response of DDR pathways following exposure to low dose radiation (0–2 Gy) was discussed by Dr Susan Short (Gray Cancer Institute, Northwood). She reported the response of a number of genes in two cell lines, +/- for low dose hypersensitivity (HRS). ATM signalling to downstream targets such as P53, CHK1 and CHK2 is functional at doses as low as 0.2 Gy. The induction of DSB, measured using H2AX, appear to be linear with dose, but inhibition of DNA repair produces an exaggerated effect when using ATM inhibitors. DNA-PK inhibitors have a lesser effect on low dose responses, but Rad51/BRCA2 mediated repair events may increase at doses below 1 Gy, which may be applicable to normal tissue responses during radiotherapy.
Overall, the workshop provided an excellent update on the progress of molecular targeting of DNA repair as a strategy for enhancing anti-cancer treatments. Significant progress has been made in recent years. The processes are better understood and the development of the H2AX assay has allowed the interrogation of effects in the low clinically relevant range. New and better drugs are currently being tested and there is an expectation that these strategies will be successful in controlling at least a subset of solid tumour treatment responses where DDR pathways are already significantly compromised. There are some important caveats and implications to radiotherapy, including a theoretical risk of enhanced carcinogenesis in normal tissues; malignant transformation assays should be performed to investigate the potential magnitude of this risk and whether there is a synergy with concomitant radiation and/or chemotherapy. The use of proton beam radiotherapy might allow these agents to be used more safely due to the reduced collateral radiation of normal tissues; intensity-modulated radiotherapy (IMRT), associated with a dose bath effect of low to medium dose in surrounding normal tissues would need very careful assessment, although dose escalation may not be so necessary in the presence of DNA repair inhibitors. It is also self-evident that the extant mathematical models of repair used in radiotherapy might require specific changes to accommodate the mechanisms described in this paper. Changes in radiotherapy fractionation (dose per fraction and interfraction interval) might follow the determination of precise repair capacity in tumours relative to normal tissues. Robust laboratory, clinical and analytical methodology is necessary in order to determine whether enhanced cure rates and an improved therapeutic index can be achieved by exploitation of altered repair systems in some types of cancer. Based on the content of this meeting, the prospects seem good.
Received for publication March 24, 2005. Revision received June 8, 2005. Accepted for publication July 13, 2005.
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