**3. Strategies for sensitization**

A prediction of the above presented model is that inhibition of any of the proteins required for DDR or DSB repair should lead to radiation sensitization. In general, this prediction has been confirmed 65. However, therapeutic agents that directly inhibit these critical proteins are still years from reaching clinical trial. Encouragingly, several FDA-approved agents have recently been shown to modulate DNA damage response. This property may be explored as therapeutic strategy.

#### **3.1 Molecular rationale for therapeutic window**

Before considering the strategy of radiation sensitization, one must first consider the molecular rationale for therapeutic window. After all, if normal and tumor cells were equally sensitized by the agent, then no therapeutic efficacy is gained.

DNA Damage Response and Repair: Insights into Strategies for Radiation Sensitization 525

EGFR is frequently amplified or mutated in several cancer types, including Non-Small Cell Lung Cancer (NSCLC) and glioblastomas 79-81. As aberrant EGFR signaling is required to sustain tumor survival and proliferation in some cancers, targeted inhibition has led to selective tumor ablation 79. Clinical trial success has led to FDA-approval for treatment of

Several studies have demonstrated that EGFR inhibition sensitized tumor cells to radiation 65. Insights into the mechanism underlying this sensitization have been provided by several recent studies. One series of studies demonstrate that a subset of EGFR travels to the nucleus where it binds to and enhances DNA-PK activity to enhance NHEJ 82, 83}. Indeed, glioblastomas over-expressing an over-active form of EGFR (termed EGFRvIII) exhibit radiation resistance that can be abridged by treatment with DNA-PK inhibitors 84. Another series of studies reveal that EGFR inhibition leads to retention of BRCA1 in the cytoplasm, thereby causing defective HR 85. Finally, other downstream effectors of EGFR, including the Extracellular signal Regulated Kinase (ERK1/2) also modulates HR efficiency 86. It is likely that the radiation sensitization effect of EGFR inhibition represents a culmination of these

There are several other agents that are in mid- to late- clinical trial testing that have also been shown to inhibit DNA damage response. For instance, Histone DeACetylase (HDAC) inhibitors have been shown to down regulate the transcript level of BRCA1 87. These agents have also been shown to disrupt the chromatin re-organization required for ATM activation 41. As another example, Heat Shock Protein 90 (HSP90, the prototypical chaperone protein) inhibitor treatment inhibits ATM autophosphorylation upon DNA damage 88 and destabilizes the MRN complex 89, 90, thereby inhibiting HR. Finally, CDK1 inhibition causes

There are additional modulators of DDR and DNA repair not described here 65. Indeed, the number of pharmacologic inhibitors that either directly or indirectly inhibit DSB repair is being uncovered at a rapid pace. Careful consideration should be given for combination

Radiotherapy is the most effective post-surgical treatment modality in the management of glioblastoma. Adjuvant radiotherapy alone provides a more than doubling of median survival. Incremental gains with additional medical therapy have proven elusive, with most agents showing moderate activity *in vitro* or with encouraging early clinical experience only to demonstrate a lack of benefit in larger trials. Attempts at treatment intensification with radiotherapy have been similarly disappointing. Molecular understanding of DNA damage response and repair, on the other hand, has now afforded novel therapeutic targets. These targets are particularly attractive in the context that oncogenes induce increased DNA damage accumulation and cause tumors to become hyper-dependent on DNA damage response pathways. Encouragingly, several FDA-approved agents modulate critical proteins in DNA damage response/repair, including conventional DNA damaging agents, proteasome inhibitors, and EGFR inhibitors. Clinical trials involving these and other agents

modulating DNA damage response should be designed with this consideration.

the loss of a critical phosphorylation event on BRCA1 required for its HR function 91.

**3.4 Epidermal Growth Factor Receptor (EGFR) inhibitors** 

NSCLC.

individual effects.

**4. Closing remarks** 

**3.5 Other late stage clinical trial agents** 

with radiation therapy in clinical trial design.

A large body has yielded data suggesting that oncogene activation creates a tumor state that increases the accumulation of DNA damage 66-69. This damage, if unrepaired, can be converted into DSBs that eventually lead to cell death. To compensate for this increased DNA damage, the tumor cells require increased utilization of DNA repair processes 69. In this context, the administration of radiation introduces additional DNA damage that further taxes the already over-utilized repair process. This situation, in turn, increases the likelihood of an unrepaired DSB causing cell death. The same effect can be achieved by inhibition of DSB repair. The following sections will review FDA-approved agents with such properties. It is important to note that these agents induce pleiotropic effects beyond DSB repair inhibition.

#### **3.2 DNA damaging agents**

Conventional chemotherapy involves DNA damaging agents that are often used in conjunction with radiation. In this context, these FDA-approved agents often sensitize the tumoricidal effects of radiation. The mechanism of this sensitization is thought to be related to the generation of DNA damages that sequester critical DNA repair proteins. For instance, lesions generated by cisplatin bind to and sequester the Ku70/80 heterodimer and thereby compromise the efficiency of NHEJ 70. Further, most DNA damages induced by conventional chemotherapy are ultimately converted to DSBs 71. These DSBs will titrate away the repair proteins available to repair the DSBs induced by subsequent radiation. These types of mechanisms likely account for the increased glioblastoma patient survival observed in the context of concurrent radiation/ temozolomide treatment relative to radiation treatment alone 3, 4.

#### **3.3 Proteasome inhibitors**

As a result of extreme aneuploidy, copy-number variation, and transcriptional alteration that are present in many cancer cells, there is increased stress on the chaperone pathways (such as heat shock proteins) to maintain folding of over-expressed proteins. When the capacity of these chaperone proteins becomes saturated, the unfolded proteins require degradation by the proteasome complex 72. Thus, tumor cells exhibit increased dependency on proteasome function. Indeed, proteasome inhibition has been demonstrated to selectively ablate cancer cells both *in vitro* and *in vivo* 73. The proteasome inhibitor bortezomib has attained FDA-approval as a treatment for multiple myeloma and mantle cell lymphoma.

Recent studies implicate proteasome function in DSB repair. The yeast Sem1 protein is a subunit of the 19S proteasome that is required for efficient HR 74. The human Sem1 homologue, DSS1, physically interacts with the HR protein, BRCA2, and is required for its stability and function 75-77. Using the DR-GFP assay to directly assess HR efficiency, Murakawa et al. demonstrated that HR efficiency is significantly reduced by proteasome inhibition 78. As a whole, these studies suggest proteasome inhibition as a means to target HR in cancer therapy.

The mechanism by which proteasome inhibition modulates HR remains an area of investigation. One hypothesis frequently put forth is the following. The proteins destined for proteasome degradation are typically modified by attachment of multiple ubiquitin moieties 74. Processing of the tagged protein releases the tagged ubiquitin to replete the intracellular pool. Proteasome inhibition, thus, leads to accumulation of ubiquitinated proteins. This accumulation, in turn, depletes the intracellular ubiquitin pool. Since free ubiquitins are required to activate HR, the repair process is compromised by proteasome inhibition.

### **3.4 Epidermal Growth Factor Receptor (EGFR) inhibitors**

EGFR is frequently amplified or mutated in several cancer types, including Non-Small Cell Lung Cancer (NSCLC) and glioblastomas 79-81. As aberrant EGFR signaling is required to sustain tumor survival and proliferation in some cancers, targeted inhibition has led to selective tumor ablation 79. Clinical trial success has led to FDA-approval for treatment of NSCLC.

Several studies have demonstrated that EGFR inhibition sensitized tumor cells to radiation 65. Insights into the mechanism underlying this sensitization have been provided by several recent studies. One series of studies demonstrate that a subset of EGFR travels to the nucleus where it binds to and enhances DNA-PK activity to enhance NHEJ 82, 83}. Indeed, glioblastomas over-expressing an over-active form of EGFR (termed EGFRvIII) exhibit radiation resistance that can be abridged by treatment with DNA-PK inhibitors 84. Another series of studies reveal that EGFR inhibition leads to retention of BRCA1 in the cytoplasm, thereby causing defective HR 85. Finally, other downstream effectors of EGFR, including the Extracellular signal Regulated Kinase (ERK1/2) also modulates HR efficiency 86. It is likely that the radiation sensitization effect of EGFR inhibition represents a culmination of these individual effects.

#### **3.5 Other late stage clinical trial agents**

There are several other agents that are in mid- to late- clinical trial testing that have also been shown to inhibit DNA damage response. For instance, Histone DeACetylase (HDAC) inhibitors have been shown to down regulate the transcript level of BRCA1 87. These agents have also been shown to disrupt the chromatin re-organization required for ATM activation 41. As another example, Heat Shock Protein 90 (HSP90, the prototypical chaperone protein) inhibitor treatment inhibits ATM autophosphorylation upon DNA damage 88 and destabilizes the MRN complex 89, 90, thereby inhibiting HR. Finally, CDK1 inhibition causes the loss of a critical phosphorylation event on BRCA1 required for its HR function 91.

There are additional modulators of DDR and DNA repair not described here 65. Indeed, the number of pharmacologic inhibitors that either directly or indirectly inhibit DSB repair is being uncovered at a rapid pace. Careful consideration should be given for combination with radiation therapy in clinical trial design.
