On the Path to Seeking Novel Radiosensitizers

Int. J. Radiation Oncology Biol. Phys., Vol. 73, No. 4, pp. 988–996, 2009 Copyright Ó 2009 Elsevier Inc. Printed in the USA. All rights reserved 0360-3016/09/$–see front matter

doi:10.1016/j.ijrobp.2008.12.002

CRITICAL REVIEW

ON THE PATH TO SEEKING NOVEL RADIOSENSITIZERS DAVID KATZ, M.SC.,*y EMMA ITO, M.SC.,*y AND FEI-FEI LIU, M.D., F.R.C.P.C.*yzx * Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada; y Division of Applied Molecular Oncology, University Health Network, Toronto, ON, Canada; z Department of Radiation Oncology, Princess Margaret Hospital, University Health Network, Toronto, ON, Canada; and x Department of Radiation Oncology, University of Toronto, Toronto, ON, Canada Radiation therapy is a highly effective cancer treatment modality, and extensive investigations have been undertaken over the years to augment its efficacy in the clinic. This review summarizes the current understanding of the biologic bases underpinning many of the clinically used radiosensitizers. In addition, this review illustrates how the advent of innovative, high-throughput technologies with integration of different disciplines could be harnessed for an expeditious discovery process for novel radiosensitizers, providing an exciting future for such pursuits in radiation biology and oncology. Ó 2009 Elsevier Inc. Radiation therapy, Sensitizers, Hypoxia, High-throughput screens, In silico analyses.

tagenesis and lethality. Approximately 1 Gy X-ray results in 105 ionization events per cell, producing 1,000 to 2,000 SSBs and 40 DSBs, with the majority of DSB repair occurring within the first 2 h (4). In response to IR-induced injury, a cell must repair the damage, continue to divide despite the damage, or die, activating a complex multitude of DNA damage response (DDR) signaling pathways that can result in cell cycle arrest (5), activation of DNA repair pathways (6), and/or intracellular (7) and extracellular (8) death signals. These responses may function independently; however, there is also considerable cross-talk between pathways.

INTRODUCTION Radiation therapy (RT) is the most common treatment modality for cancer. Its ability to both cure and palliate have underscored its importance in cancer management. In the clinical setting, radiation can be targeted to the area of interest, allowing tumor tissue specificity. Recent advancements in the technology of radiation delivery and treatment planning have improved patient outcomes, particularly in reducing normal tissue toxicity (1). This review will summarize our current understanding of the biology underpinning the agents used to enhance radiation efficacy and will illustrate innovative approaches that could facilitate discovery of novel sensitizers. Biologically, ionizing radiation (IR) is most often delivered as photons of energy in the X-ray wavelength of the electromagnetic spectrum and as particles in the form of electrons (2). The damage caused is indirect as X-rays collide with molecules within the target, ejecting electrons from electron orbitals (called ionization events). These free electrons then either cause direct damage to other molecules (e.g., DNA) or collide with water molecules within the cell, leading to the generation of hydroxyl radicals, subsequently activating a cascade of ionization events responsible for IR damage (3). The lethal effects of IR primarily arise from damage to DNA, exhibited as single-strand breaks (SSB) or doublestrand breaks (DSB). The SSBs are more readily repaired by the cell than the DSBs, which are more likely to cause mu-

COMBINATION OF RT AND CHEMOTHERAPY Although both RT and chemotherapy have been used as single-modality cancer treatments for more than 40 years, the combined chemo-radiotherapy approach has been adopted more recently. The theoretical framework defining the mechanisms by which these two modalities interact was introduced by Steel and Peckham in 1979, with a predominant focus on in vitro cytotoxicity (9). A recent review by Bentzen et al. (10) proposes a modernization of the Steel and Peckham conceptual framework to complement the advent of novel drugs in preclinical/clinical development, along with the recent emergence of molecular oncology therapeutics, defining a more contemporary strategy to best identify and exploit mechanisms of drugradiation interactions. Spatial

Acknowledgments—This work was funded by grants from the OICR and CIHR. David Katz and Emma Ito are recipients of Excellence in Radiation Research for the 21st Century and National Science and Engineering Research Council scholarships, respectively. Received Aug 7, 2008, and in revised form Nov 29, 2008. Accepted for publication Dec 2, 2008.

Reprint requests to: Fei-Fei Liu, M.D., F.R.C.P.C., Department of Radiation Oncology, Princess Margaret Hospital/Ontario Cancer Institute, 610 University Avenue, Room 7-719, Toronto, ON, Canada M5G 2M9. Tel: (416) 946-2123; Fax: (416) 946-4586; E-mail: [email protected] Conflict of interest: none. 988

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HYPOXIA AND RADIORESPONSIVENESS One of the best-studied biologic processes that modulate tumor response to RT is molecular oxygen. Its importance is illustrated by the observation that higher radiation doses are required under hypoxic conditions. The link between hypoxia and tumor radioresistance can be explained by a physico-chemical phenomenon called the oxygen enhancement effect. The oxygen enhancement ratio (OER) describes the ratio between the RT doses required to achieve equivalent cell killing in the absence vs. presence of oxygen. In vitro, OERs typically range between 2.5 and 3.5 (11), indicating that approximately threefold higher RT doses are required to kill the same number of cells under hypoxic conditions, an effect that can be explained by oxygen’s ability to ‘‘fix’’ free radical–induced damage by IR into a permanent state. It involves oxygen reacting with the radical-induced broken DNA ends, generating stable organic peroxides, which are not readily repaired by the cell. Without this fixation, the free radical damage is transient and hence insufficient to cause genotoxicity. The biologic impact of tumor hypoxia on radioresponsiveness has also been investigated. In 1985, Shrieve and Harris were the first to illustrate that hypoxia is not only a physicochemical effect based on the presence of oxygen but also a biologic response influencing tumor radiosensitivity; demonstrating that sensitivity increases with the duration of hypoxia (12). These results have since been validated by others, establishing that chronically hypoxic cells are more

Cancer

100 C2

Probability (%)

cooperation describes the scenario whereby RT affects locoregional control of the tumor mass, whereas chemotherapy acts on distant metastases, without interaction between the two treatments (i.e., nonoverlapping toxicity profiles). Spatial cooperation is highly theoretical and rarely observed in clinical situations. An alternative interactive scenario is radiation sensitization, describing a state in which chemotherapy cooperates with radiation within the radiation field, resulting in increased killing of cancer cells. The degree of increased cytotoxicity can be either equal to (additive) or greater than (synergistic) the expected sum of killing from each modality. It is also possible for the two treatments to interact in an antagonistic fashion; the killing is less than the expected sum. This scenario can be used to clinical advantage if applied to protect normal tissues, whereby the interaction of the two modalities reduces normal tissue toxicity while maintaining tumor susceptibility to RT. The ability of a drug to induce a radiosensitizing effect can be described by its therapeutic ratio. When delivering concomitant chemo-radiotherapy, the radiation dose–response curves of both the tumor and surrounding normal tissues will shift to the left (Fig. 1). Radiosensitizing compounds, however, will induce a larger shift in the tumor curve compared to that of the normal, ideally keeping the normal tissue curve unchanged. Ultimately, these responses need to be evaluated in clinical trials to determine the true therapeutic ratio of novel radiosensitizers.

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50

ΔDC + Drug

C1

N2

Normal N1

ΔDN + Drug

Favorable therapeutic ratio: ΔDC >ΔDN 0 Radiation Dose (Gy)

Fig. 1. Therapeutic ratio of radiosensitizing compounds. Radiosensitizers with a favorable therapeutic ratio induce a larger change in the radiation dose required for 50% cure probability in cancer tissues (DDC; C1 to C2), than in the dose required for 50% complication probability in normal tissues (DDN; N1 to N2). This is represented by a greater leftward shift in the corresponding dose–response curves. Dotted and solid lines represent cancer and normal tissues, respectively.

radiosensitive than acutely hypoxic cells, as well as normoxic cells when both are irradiated under oxygenated conditions due to accumulation in the IR-sensitive late-G1 phase (13, 14), indicating that hypoxia can exert biologic effects on tumors that influence radioresponsiveness, with not all effects being radioprotective. The best-characterized biologic pathway related to hypoxia is regulated via hypoxia inducible factor–1 (HIF-1), a heterodimeric transcription factor. The HIF-1 molecule is stabilized under hypoxic conditions and is capable of regulating hundreds of genes that affect radioresponsiveness (15), increasing its expression approximately twofold 24 to 48 h post-IR (16). Inhibition of HIF-1 has also been reported to increase tumor radiosensitivity (17). However, just as the relationship between radioresponsiveness and hypoxia is complex, so is the interplay between radiosensitivity and HIF-1; not all HIF-1–mediated effects are radioprotective for tumors, such as promoting adenosine triphosphate (ATP) metabolism, proliferation, and p53 activation (14). It appears that the strongest evidence for HIF-1–induced radioresistance exists in relation to its interplay with the tumor microenvironment, specifically the tumor vasculature, as discussed in a recent review by Dewhirst et al. (18). Animals lacking HIF-1 demonstrate a defect in the expression of vascular endothelial growth factor (VEGF) (19). The VEGF receptor is a cell surface receptor that signals to the antiapoptotic machinery within the vascular endothelium, rendering such cells radioresistant. Accordingly, blocking VEGF signaling has been reported to enhance the radiosensitivity of tumor vasculature (20). As a major regulator of VEGF expression, HIF-1 inhibition has also been shown to recapitulate this effect, inducing significant radiosensitization of the tumor vasculature, translating into improved overall tumor control by IR (16). These data collectively suggest that the microenvironmental effects of HIF-1 activation by hypoxia may outweigh some of the apparently counteracting cellular effects of HIF-1 activation.

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DRUG-INDUCED CHEMICAL VS. BIOLOGIC MODULATION OF RADIATION RESPONSE

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(A)

CHEMICALLY TARGETED RADIOSENSITIZERS

Because of the two phases of RT, the generation and subsequent repair of DSBs, a biphasic mode of modulating RT response exists. Chemically mediated interventions exploit the physico-chemical properties of the irradiated cell to enhance DNA damage. For example, a chemical compound might prolong the stability or amount of free radicals present in the nucleus, thereby augmenting the number of DSBs per unit radiation (Fig. 2A). Biologically mediated interventions, in contrast, involve targeting pathways that modulate tumor RT response; for example, a compound that abrogates the ability of a cell to repair DSBs would increase cell death per unit of radiation (Fig. 2B). Currently available radiosensitizers and those agents undergoing preclinical/clinical development will be reviewed in the subsequent sections. BIOREDUCTIVE RADIOSENSITIZERS Bioreductive agents must undergo reduction to form active cytotoxic species. Nitroimidazoles, the first-generation radiosensitizers, become active under hypoxia. They are similar to oxygen in their ability to fix free radical damage on DNA; thus, in the absence of oxygen, they will cause increased DSBs when delivered with IR. The tumor selectivity of these compounds is based on a bioreductive process that exists only under hypoxic conditions; hence, there are minimal effects on normoxic cells. This therapeutic advantage has been demonstrated in both preclinical (21) and clinical systems (22), and has been recently reviewed (23). Another bioreductive agent currently being assessed in the clinic is tirapazamine (TPZ). This substance induces a greater than 100-fold increase in cytotoxicity under anoxic conditions because of its electron-donating ability. It donates electrons to surrounding molecules, generating transient oxidizing radicals; under normoxic states, surrounding molecular oxygen quenches these radicals. These transient oxidizing radicals form DNA radicals by abstracting a proton from the C4 of the deoxyribose ring (24). Combination therapy of TPZ with RT has shown promising results in head and neck cancer, and is being evaluated in patients with cervical cancer (25, 26). RADIOSENSITIZERS AFFECTING DNA/RNA PROCESSES Platinum analogs Cisplatin is the most commonly used agent in combination with RT. It interacts with DNA to form inter-/intrastrand cross-links, as well as DNAprotein cross-links, inhibiting DNA replication and RNA transcription and ultimately inducing mutagenesis or apotosis (27). The precise mechanism underlying the radiosensitization of cisplatin remains a subject of debate; both chemically and biologically mediated processes have been suggested. When cisplatin is delivered concurrently with RT, there is an increase in the number of toxic platinum intermediates

DSB Creation

(B)

BIOLOGICALY TARGETED RADIOSENSITIZERS Biological Molecules

DSB Repair

Fig. 2. Chemical and biologic targeting of radiosensitizers. (A) Radiosensitizers that are chemically mediated augment the chemical reactions, or extend the half-life of radical species involved in the creation of double-strand breaks (DSBs). (B) Biologically targeted radiosensitizers affect pathways involved in the response to DSBs, resulting in prolonged presence of DSBs, and increased cytotoxicity.

in the presence of oxygen free radicals generated by IR (28). As well, increased uptake of cisplatin through the cell membrane (29), and inhibition of DNA repair (30), have been reported. Nonetheless, cisplatin has been shown to improve overall outcome in numerous Phase III clinical trials for the treatment of non–small-cell lung carcinoma (NSCLC) (31), head-and-neck squamous cell carcinoma (HNSCC) (32), and cancers of the cervix (33). A third-generation cisplatin analog, oxaliplatin, has also demonstrated potential radiosensitization (34) and cytotoxicity in cisplatin-resistant systems (35). This agent reacts with DNA to form intra-strand linkages with adjacent guanine or guanine and adenine residues (36). Oxaliplatin is currently being investigated in clinical trials for the treatment of rectal cancer in combination with RT (37). Temozolomide Temozolomide (TMZ) is a DNA alkylating agent that methylates guanine on the O6 position (38). These alkylated lesions are usually processed by the DNA repair enzyme, O6-methylguanine DNA-methyltransferase (MGMT) (38); hence, tumors with MGMT mutations are preferentially radiosensitized (39). The agent TMZ does not act directly with IR, which contributes to its favorable toxicity profile, rendering it a highly efficacious and well-tolerated therapeutic regimen combined with RT for patients with glioblastoma (40). 5-Fluorouracil 5-Fluorouracil (5-FU) is a halogenated pyrimidine nucleososide analog commonly used with RT in the clinic. The analog 5-FU is phosphorylated by cellular thymidine kinase to form 5-fluoro-20 -deoxy-uridine-monophosphate (FdUMP),

On the path to seeking novel radiosensitizers d D. KATZ et al.

which inhibits thymidylate synthase (TS). The intracellular pools of thymidine 50 -monophosphate and thymidine 50 -triphosphate subsequently become depleted, leading to the inhibition of DNA synthesis and interference of DNA repair (41). Alternatively, 5-FU can be metabolized to 5-FU triphosphate, a substrate for RNA polymerase, which, upon incorporation into RNA, inhibits mRNA polyadenylation with subsequent modification of its secondary structure (41). The underlying mechanism of 5-FU–mediated radiosensitization is not clearly understood. Noncytotoxic concentrations of 5-FU can be radiosensitizing, but must be administered before IR (42). Furthermore, these effects appear to be dependent on inappropriate progression through the S-phase (43). Phase III trials of 5-FU and RT have demonstrated benefit in cancers of the esophagus, cervix, and anal canal (44–46). Capecitabine, an oral pro-drug of 5-FU, has also been evaluated in clinical trials. It is metabolized by three enzymes to form the active state; the final enzyme being thymidine phosphorylase (TP), whose activity is upregulated by IR, which in turn enhances the efficacy of capecitabine (47). Gemcitabine Gemcitabine is a pyrimidine analog with radiosensitizing effects against several different cancer cell lines. Clinical trials have also shown promising results for the treatment of NSCLC and pancreatic cancer (48, 49). Radiosensitization is observed when gemcitabine is administered at low concentrations at least 24 h before RT (50). It depletes the intracellular pool of deoxynucleoside triphosphate (dNTP) via ribonucleotide reductase inhibition, resulting in misincorporation of gemcitabine into DNA and, ultimately, in radiosensitization (51). Texaphyrins Texaphyrins are porphyrin-like macrocycles that form stable complexes with large metal cations. When motexafin gadolinium (MGd) is delivered to tumor cells with IR, an increase in oxidative stress and DSBs are observed. Under basal conditions, radical species created by IR are neutralized by endogenous antioxidants, such as ascorbate and glutathione (52). In vitro studies have suggested that MGd can bind to these reducing metabolites, preventing them from scavenging cellular free radicals (53), leading to increased DSBs and toxicity because of oxidative stress. Phase III trials have demonstrated NSCLC patients with brain metastases to show a delay in neurologic progression when treated with whole-brain RT and MGd vs. RT alone (54). RADIOSENSITIZERS TARGETING SIGNALING PATHWAYS Epidermal growth factor receptor–targeted therapies Members of the epidermal growth factor receptor (EGFR) family, specifically EGFR, are overexpressed in many epithelial tumors (55). Cancer cells have been shown to upregulate the tyrosine kinase activity of EGFR in response to RT via reactive oxygen species–mediated inhibition of protein

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tyrosine phosphatases, the enzymes responsible for dephosphorylating activated tyrosine kinases (56). Signaling by EGFR can also be upregulated in a delayed manner through the induction of paracrine growth factors, such as transforming growth factor–a, a ligand for EGFR (57). Accordingly, EGFR overexpression has been linked to poor clinical outcomes with RT (58) and is targeted as a strategy for radiosensitization. Cetuximab, a chimeric monoclonal antibody targeting EGFR, has been shown to enhance tumor radiocurability in HNSCC xenograft models (59). Furthermore, a landmark Phase III randomized trial has reported diseasefree and overall survival advantages for HNSCC patients treated with both cetuximab and RT (60). Because of the pleiotropic effects of EGFR signaling, the precise mechanism of radiosensitization by EGFR blockade has not been fully elucidated; however, evidence favors inhibition of cell proliferation and induction of apoptosis (61). Farnesyl transferase–targeted therapies The Ras proto-oncogene is mutated in 30% of all cancers and has been implicated in radioresistant phenotypes. Ras acts downstream of many receptor tyrosine kinases, including EGFR, linking it to Raf and mitogen-activated protein kinases pathways (62). Ras activation is dependent upon posttranslational modification by farnesyl transferase (FT), which prenylates Ras, allowing it to dock to the inner leaflet of the plasma membrane (63). Ras can subsequently act as a molecular switch that flips between the active (GDP-bound) and inactive (GTP-bound) configurations (63). When Ras is mutated, it is permanently fixed in the active state, constitutively delivering proliferative signals despite the absence of any ligands. To reverse the radioresistant effects of activated Ras, many FT inhibitors have been developed, including FTI-277, L-744,832, and L-778,123; all of which have demonstrated success in preclinical models (64, 65). L-778,123 is the only FT inhibitor to have proceeded to clinical testing with RT, showing promising outcomes for HNSCC and NSCLC patients (66). Antiangiogenic therapies Angiogenesis is regulated by many pro- and antiangiogenic factors, with VEGF playing a central role. Angiogenesis is essential for tumor growth and progression and has therefore been targeted for many cancer treatments (67). Signaling by VEGF can be disrupted by targeting either the VEGF ligand (e.g., bevacizumab) or its receptor (e.g., PTK787, SU5416). The effect of combining anti-VEGF therapy with RT remains controversial. It has been predicted that destruction of tumor vessels by antiangiogenic agents will lead to increased tumor hypoxia, resulting in radioresistance (68). However, preclinical evaluations have demonstrated anti-VEGF treatments to radiosensitize tumors, crediting this effect to transient ‘‘normalization’’ of the tumor vasculature, leading to increased tumor oxygenation and thereby radiosensitization (67). Use of SU5416 plus RT has shown promising results in both glioblastoma and melanoma

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xenograft models (20), and is currently being evaluated in clinical trials (http://clinicaltrials.gov). Novel radiosensitizer discovery Although a long list of potential radiosensitizers exists, many of which have shown promise in clinical trials, there remain significant opportunities for improvement, an example being in the management of head-and-neck cancer (HNC). Standard therapy for early stage HNC is either curative RT or surgery, with both achieving similar results (69). However, for patients with locally advanced disease, the outcome is significantly worse, primarily because of local recurrences. Hence, many clinical trials have been conducted using combined RT and chemotherapy, with the majority of successful trials using platinum analogs. Meta-analyses of concomitant chemotherapy with standard RT fractionations have demonstrated a modest survival benefit of only 8% (70), which unfortunately is associated with significant normal tissue toxicity (71). Such challenges have prompted investigators to explore novel avenues for the discovery of more potent and, hopefully, more clinically relevant radiosensitizing compounds. Two promising strategies, high-throughput screening (HTS) and in silico data mining, will be discussed. High-throughput screening High-throughput screening–based drug discovery programs have recently been developed in academic institutions, and have emerged as a fruitful enterprise by integrating the disciplines of biology and chemistry for the discovery of novel biologic pathways and therapeutics. In the domain of chemical genetics, HTS can be divided into two distinct approaches (72) (Fig. 3). Forward chemical genetics, or phenotype-based screens, assay the response of an experimental system to chemical perturbations via phenotypic read-outs. Genetic markers measure alterations in the expression of specific genes as indicators of broad cellular responses; for example, extracellular signal–regulated kinase (ERK) activation served as a read-out in a recent functional genomic HTS for novel regulators of the receptor tyrosine kinase (RTK)/ERK signaling pathway (73). Functional assays directly monitor cellular processes, such as cell viability (74). Microscopic imaging assays detect visual differences within the cell (75). Reverse chemical genetics, or target-based screens in contrast, target a specific protein or gene within the cell, and thereafter elucidate the cellular phenotype (76). Use of HTS has been fruitful in identifying drug candidates in many domains; however, technical obstacles have impeded the development of HTS for radiosensitizers, mainly with respect to read-outs. The gold standard for measuring radiosensitization is the colony formation assay (CFA), whereby tumor cells are treated with radiation and/or anticancer agents, plated in multi-well plates, then assessed for clonogenicity via manual colony counting. Our laboratory has successfully developed an automated CFA in high-throughput format, demonstrating in a proof-of-principle study that it can potentially be useful in the discovery of potent anticancer compounds (77). Although CFA is the ideal assay, radio-

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FORWARD

REVERSE Identify protein of interest

Plate cells

Screen for ligand

Identify compound from phenotype-based screen

Test on in vitro systems

Test on in vitro systems

Determine cellular phenotype

Identify protein target

Fig. 3. High-throughput screening approaches. Forward chemical– biology screening uses a phenotype-based approach to identify a chemical of interest, the target for which is later identified. The reverse chemical–biology approach begins with a protein of interest and a screen to identify its ligand; the phenotypic outcome is assayed subsequently.

sensitizers have been identified through forward screens using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) assay, a read-out for cell viability; identifying NS-123 as a radiosensitizing molecule that inhibits DSB repair (78). Several reverse screens have also used known molecular targets in the DSB repair pathway as read-outs, identifying KU-55933 (ATM inhibitor) (79) and NU7441 (DNAPKCS inhibitor) (80) as potential radiosensitizing agents from libraries of small-molecule compounds. Short-interfering RNAs (siRNAs), which are 21- to 23-nucleotide-long RNA molecules that silence gene expression, have recently emerged as powerful tools for discovering novel genes involved in radiation response (75). Kolas et al. combined siRNA-mediated HTS with high-content microscopy to examine p53-binding protein–1 foci formation in response to IR, identifying ubiquitin ligase RNF8 as a key player in DDR (75). Thus, such HTS prove to be valuable resources for future delineation of radiation response pathways, with subsequent development of drugs targeting these cascades.

In silico data mining Traditional HTS methods use in vitro assays to evaluate cellular processes of interest, leading to the generation of lead compounds for drug development. However, with the vast amount of data generated by gene expression profiles in the recent years, novel in silico approaches of

On the path to seeking novel radiosensitizers d D. KATZ et al.

high-throughput discovery have emerged; one of which is the Connectivity Map (CMap) (81), as shown in Fig. 4. CMap is a powerful Web-based tool (www.broad.mit.edu/ cmap) that attempts to bridge the domains of clinical medicine, molecular genetics, and chemistry, using the common language of gene expression profiling, a DNA microarraybased technology measuring the expression of thousands of genes simultaneously to create a global picture of cellular function. CMap comprises a large gene expression database generated from human cancer cell lines treated with different chemicals, which is then used to compare various disease states. In its first release, CMap contained data for 164 distinct chemicals at varying concentrations, on different cell lines, generating a library of 564 searchable gene expression profiles (82). A recently released version contained datasets for 1,309 chemicals, generating a library of 7,056 expression profiles. Volumes of gene expression profiles exist in the current literature for various types of interactions involving IR. The largest repository of such data belongs to the National Center for Biotechnology Information in Gene Expression Omnibus (GEO), which contains datasets from more than 235,000 samples, organized into more than 9,000 series (http://www.ncbi. nlm.nih.gov/geo/) (83). A simple search of the terms ‘‘ionizing’’ and ‘‘radiation’’ yields 61 individual GEO datasets, each containing multiple expression profiling results that can potentially be analyzed with CMap to identify novel compounds that affect radiation response in a similar manner. Hassane et al. recently exploited this approach to successfully identify agents that eradicated leukemia stem cells (84). Therefore, culling these profiles via CMap may prove to be a cost-effective method in developing novel hypotheses for the evaluation of radiation response, with the ultimate goal of discovering novel radiosensitizing compounds. Another potentially fruitful method for discovering novel radiosensitizers is to generate new gene expression profiles for a disease state of interest, then subjecting them to CMap analysis. For example, if new expression profiles for radioresistance in locally recurrent HNC were generated and analyzed with CMap, novel radiosensitizers specific for recurrent HNC could be discovered. The utility of this approach is supported by the recent identification of rapamycin

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as a modulator of glucocorticoid resistance in acute lymphoblastic leukemia (85). CMap can also assist in the elucidation of mechanisms of action for known radiosensitizers. For example, cisplatin has long been used clinically, but its precise mechanism of sensitization remains unclear. If an expression profile of cisplatintreated cells were generated and then analyzed with CMap, drugs that induce a similar profile could hence be identified. If one or more of these ‘‘hits’’ already has a defined mechanism of action, then this process might also explain the radiosensitizing actions of cisplatin. This approach has indeed been undertaken, successfully identifying gedunin as a heat shock protein 90 inhibitor (86). The integration of gene expression profiles for various disease states with those of drug-treated states has transpired into a powerful tool. By exploiting large data repositories, such as GEO and CMap, it is conceivable that novel lead radiosensitizing compounds could be rationally selected without ever performing experiments at the bench. Realistically, such tools will never replace the subsequent unraveling of mechanisms of interactions, which will always be experimentally based with clearly defined scientific hypotheses. However, the use of CMap as a preliminary hypothesis-generating tool could translate into significant cost savings, leaving more resources to be dedicated to the subsequent understanding and optimization of lead compounds. CONCLUSION AND FUTURE DIRECTIONS A vast array of anticancer compounds used in combination with RT exists in the clinic today. As our understanding of the mechanisms underlying radiosensitization continues to expand, so does the number of potential therapeutic targets, although there remains the challenge of translating these targets into clinical drugs. To continue to advance drug discovery, it is therefore necessary to exploit cutting-edge technology, with HTS and in silico data mining being two approaches recently undertaken by academia. With the flurry of promising new drugs and the requirement for larger, more complex trials, the biggest challenge will be to bring the drug development pipeline up to parity; the clinical trial stage being the major bottleneck prolonging the discovery-to-

Fig. 4. In silico hypothesis generation with Connectivity Map (CMap). One starts with a disease state of interest (e.g., radiosensitive, radioresistant), followed by in silico analysis with CMap datasets, which are then validated using in vitro/in vivo approaches and ultimately in clinical trials.

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market timeline. With the increasing use of innovative technologies to advance drug discovery, it is only fitting that the Internet is emerging as a promising approach to expedite clinical trials (87). E-clinical trials are proving to be cost-effective solutions for streamlining areas such as patient recruitment, randomization, real-time data entry/analysis,

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communications, and project management (88, 89), rapidly changing the current paradigms of clinical research. The integration of such tools in the field of radiation oncology promises to be an exciting future for the discovery and development of novel radiosensitizers, with the ultimate goal of improving clinical outcomes for cancer patients.

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