Formation of DNA double strand breaks (DSB) at sufficient levels in human and other mammalian genomes, as by IR or certain chemotherapeutic agents, activates a complex signal transduction cascade—the DNA Damage Response (DDR)—that culminates in a range of cellular outcomes including repair of the DNA damage, cell cycle arrest at specific checkpoints, and programmed cell death; the DDR and these various endpoints have been reviewed extensively (Begg et al. 2011; Goodarzi and Jeggo 2013; Ceccaldi et al. 2016; Jeggo and Lobrich 2015; Waters et al. 2014; Jackson 2009) and will be covered here only briefly. Natural and engineered mammalian cells and animals that have gene mutations causing functional defects for components of the DDR are commonly IR hypersensitive to varying degrees, along with displaying “genomic instability” (see later). Human patients and mutant mouse strains with germline DDR gene defects often display increased susceptibility to various malignancies; some also have immunodeficiency related to a failure to repair the programmed DSB formed during the course of immunoglobulin and T-cell receptor gene maturation (Goodarzi and Jeggo 2013; Jackson 2009; Curtin 2012; Weinberg and Hanahan 2011). The finding that DDR gene mutations can cause mammalian cell IR hypersensitivity has given the impetus in recent years for discovery of chemical compounds that could inhibit the functions of various DDR proteins and emulate the effects of such gene defects (sometimes called “pharmacological phenocopy”); these agents would be expected to be candidate preclinical cellular radio- and chemo-sensitizers that might ultimately lead to useful drugs for human cancer medicine (Citrin and Mitchell 2014; Jekimovs et al. 2014; Gavande et al. 2016; Higgins et al. 2015; Raleigh and Haas-Kogan 2013). The finding that inhibition of different facets of the DDR is in fact the molecular mechanism of action for some radiosensitizers now used clinically, as detailed next, serves to validate this strategy.

Following induction of DSB in human nuclear DNA, their presence is sensed by the Mre11/Rad50/NBS1 (MRN) protein complex, which is recruited to the break ends by the human single stranded binding protein 1 (hSSB1); MRN then promotes association of the key damage signaling kinase Ataxia Telangiectasia Mutated (ATM) with the DSB (Begg et al. 2011; Goodarzi and Jeggo 2013; Ceccaldi et al. 2016). ATM kinase is thereby activated toward phosphorylation of itself, of the components of the MRN complex, and of the variant histone H2AX (thereby generating γH2AX) (Begg et al. 2011; Goodarzi and Jeggo 2013; Ceccaldi et al. 2016). γH2AX formation then propagates for many thousands of base pairs from the DSB ends into the DNA strands, and initiates assembly of an array of additional DDR proteins to form a molecular macrostructure, the ionizing radiation induced focus (IRIF) (Goodarzi and Jeggo 2013). ATM signaling is amplified by interactions within the IRIF and then transduced to downstream effectors such as the CHK2 kinase-p53 axis to initiate cell cycle arrest or apoptosis (Begg et al. 2011; Goodarzi and Jeggo 2013; Ceccaldi et al. 2016). Cells with deficient MRN function show severely impaired ATM signaling (Begg et al. 2011; Goodarzi and Jeggo 2013; Ceccaldi et al. 2016). The MRN complex (in particular, the Mre11 nuclease) plays an additional key role in the cellular “choice” between utilization of homologous recombination (HR) versus non-homologous end joining (NHEJ) mechanisms for the repair of DSB in the late-S and G2 phases of the cell cycle ((Begg et al. 2011; Goodarzi and Jeggo 2013; Ceccaldi et al. 2016) and see below). The observed IR sensitivity of MRN function-deficient cells thus makes sense.

Heat treatment of sufficient temperature and duration very potently sensitizes malignant human cells to killing by IR, a phenomenon termed hyperthermic radiosensitization (HtRs) (Dewey 2009). Given the magnitude of this effect, clinical application of HtRs has been of interest for decades, but implementation of this has been hindered previously by the engineering challenges connected with heating deep tumors in situ; this obstacle might now be overcome, however, with the development of MR-guided focused ultrasound technology (see Chapter XX of this text). The molecular basis of HtRs has been elucidated using a genetic approach termed epistasis analysis; this strategy is predicated on the fact that two separate functional defects (“hits”) in the same mechanistic pathway should have no more consequence than either single hit alone does. In contrast, hits to separate pathways that function in parallel in a complementary fashion (for example, the NHEJ and HR mechanisms for DSB repair; see below) typically have a greater impact on cell physiology than either hit alone. Previous work had implicated one or more of the MRN complex protein components in HtRs, possibly via active export of these proteins out of the nucleus in response to heat treatment (Seno and Dynlacht 2004). The effect heating on clonogenic inactivation by IR was next investigated using cells having natural (cells derived from Mre11- or Nbs1-defective patients) or engineered (siRNA knockdown) deficiencies of Mre11, Nbs1, or Rad50 function (Dynlacht et al. 2011). For Nbs1 and Rad50, unheated cells were hypersensitive to killing by IR compared to normal cells, but that sensitivity was increased still further by the heat treatment. In contrast, heating of the Mre11 cells did not alter their already marked sensitivity to IR (Dynlacht et al. 2011). Purified Mre11 protein was also found to be unusually sensitive to heat denaturation in vitro (Dynlacht et al. 2011). The interpretation of these findings is that the Mre11-mutant cells are already maximally deficient for MRN-ATM signaling after DNA damage so that heat denaturation of any residual Mre11 protein has no consequence. In contrast, low levels of Nbs1 or Rad50 activity in the respective mutant cells did support some MRN-ATM signaling after IR, and this was abolished by total heat inactivation of cellular Mre11. In keeping with these findings, recently developed small molecule inhibitors of the Mre11 endo- and exonuclease activities are radiosensitizing agents in vitro (Shibata et al. 2014).

Repair of DNA DSB in eukaryotic cell relies on two fundamentally distinct mechanisms, nonhomologous end joining (NHEJ) and homologous recombination (HR) repair (sometimes called homology directed repair) (Begg et al. 2011; Goodarzi and Jeggo 2013; Ceccaldi et al. 2016; Waters et al. 2014; Pannunzio et al. 2014; Moynahan and Jasin 2010). NHEJ entails the nucleolytic processing and direct ligation of DSB ends that may approximately restore the original configuration of a stretch of DNA (typically with loss of some of the nucleotides adjacent to the strand breaks). Alternatively, DNA ends from remote parts of the genome might be brought together by NHEJ repair, forming a chromosomal translocation or inversion (Begg et al. 2011; Goodarzi and Jeggo 2013; Ceccaldi et al. 2016). The kinetics of end joining by this mechanism is determined by the chemistry of the DNA termini and by the chromatin configuration of the DNA within which the DSB occurs; chemically complex ends produced by relatively high LET radiations, and the compacted chromatin associated with heterochromatic chromosomal regions lead to slower end rejoining (Begg et al. 2011; Goodarzi and Jeggo 2013; Ceccaldi et al. 2016). Quite recently, it has been possible to distinguish between so-called canonical NHEJ (the mechanism identified first) and one or more “alternative” NHEJ pathways (Begg et al. 2011; Goodarzi and Jeggo 2013; Ceccaldi et al. 2016; Pannunzio et al. 2014); these differ with respect to the specific proteins involved and the precise molecular details of DNA end synapsis during DSB end rejoining (Begg et al. 2011; Goodarzi and Jeggo 2013; Ceccaldi et al. 2016; Pannunzio et al. 2014). How these various NHEJ processes might be differently targeted for achieving radiosensitization remains to be seen.

HR repair of DSB is initiated by quite extensive exonucleolytic degradation of DNA, starting at the break ends and proceeding in the 5′ → 3′ direction, thereby forming long single strands having 3′ hydroxyl termini (Begg et al. 2011; Goodarzi and Jeggo 2013; Ceccaldi et al. 2016; Moynahan and Jasin 2010). Those strands invade nearby intact homologous DNA (this is typically afforded by the adjacent sister chromatid following replication) and prime the synthesis of new DNA corresponding to the regions surrounding the DSB, using the intact complementary strands as templates. The new DNA strands are then extracted from the sister chromatid and reannealed; ligation of the new DNA 3′ ends to 5′ ends of the broken chromatid completes repair of the DSB (Begg et al. 2011; Goodarzi and Jeggo 2013; Ceccaldi et al. 2016; Moynahan and Jasin 2010).

HR contrasts with NHEJ in that the latter has no mechanistic requirement for the presence intact homologous DNA nearby (Begg et al. 2011; Goodarzi and Jeggo 2013; Ceccaldi et al. 2016). For this reason, NHEJ is practicable throughout the cell cycle, while HR is confined to the G2 phase and to those genomic regions that have already undergone replication during S phase (Begg et al. 2011; Goodarzi and Jeggo 2013; Ceccaldi et al. 2016). NHEJ appears to be responsible for the majority of DSB repair throughout the cell cycle in mammalian cells, this despite it being much more “error prone” than HR, given the likelihood of small DNA sequence changes in the vicinity of the DSB and the potential to form gross chromosomal aberrations (Begg et al. 2011; Goodarzi and Jeggo 2013; Ceccaldi et al. 2016; Pannunzio et al. 2014). The mechanistic basis for the cellular “decision” between use of NHEJ versus HR for the repair of a given DSB during G2 or S phase is at present poorly understood and is the subject of much research activity, although the MRN complex appears to be intimately involved (Begg et al. 2011; Goodarzi and Jeggo 2013; Ceccaldi et al. 2016; Shibata et al. 2014).

Following DNA damage and several other cellular stresses, many normal cells and certain malignant cells undergo a process of programmed cell death termed apoptosis (Balcer-Kubiczek 2012). For cells exposed to IR, DDR signaling through ATM ultimately impinges upon the tumor suppressor protein p53 and activates it as a transcription factor (Begg et al. 2011; Goodarzi and Jeggo 2013; Ceccaldi et al. 2016; Balcer-Kubiczek 2012). Depending upon cellular context, p53 activation promotes either apoptosis or cell cycle arrest in G1 phase by transcriptional regulation of specific genes (Balcer-Kubiczek 2012). In the checks and balances of cellular governance, pro-apoptotic influences are countered by pro-survival signaling; the Akt serine/threonine kinases are key mediators of the latter process (Balcer-Kubiczek 2012; Toulany and Roderman 2015). Loss of proper apoptotic response to cellular stress appears to be an essential component of carcinogenesis for many cell types (Weinberg and Hanahan 2011); enhanced Akt signaling by several different mechanisms has been found to be one means by which malignant cells can achieve this abrogation of apoptosis (Cengel et al. 2007; Mckenna et al. 2003; Cheung and Testa 2013). Akt activation in tumors is associated with chemotherapy and radiotherapy treatment resistance (Cengel et al. 2007; Mckenna et al. 2003; Cheung and Testa 2013; Sekhar et al. 2011; Kao et al. 2007; Misale et al. 2012; Garrido-Laguna et al. 2012), and down regulation of Akt signaling by dominant negative inhibition or drug targeting of its upstream signaling partner PI3 kinase (PI3K) has been shown in some cases to cause radiosensitization (Tanno et al. 2004). These finding are believed to reflect, at least in some cells, mitigation of the killing effects of IR by pro-survival Akt signaling by these inhibitory interventions (Toulany and Roderman 2015; Tanno et al. 2004; Brognard et al. 2001). Importantly, however, more recent results indicate that activated Akt also plays significant, directs role in cellular responses to IR including DNA DSB sensing/signaling and regulation of NHEJ; targeting activated Akt may thus instead (or in addition) radiosensitize some cells by inhibiting DSB repair (Toulany et al. 2008, 2012; Park et al. 2009).

Akt is a downstream effector of several mitogenic (pro-growth) signaling cascades that are frequently found to be corrupted in tumor cells (Toulany and Roderman 2015; Cheung and Testa 2013); prominent among these are activating mutations and amplification of genes encoding cell surface receptor tyrosine kinases (RTKs) such as EGFR (Weinberg and Hanahan 2011; Toulany and Roderman 2015; Cheung and Testa 2013). One target of deregulated EGFR signaling in tumors, via PI3K, is Akt (Toulany and Roderman 2015; Cheung and Testa 2013). Significant correlations have been found for many tumors between EGFR activation and Akt activation (Cheung and Testa 2013; Nijkamp et al. 2011), and between EGFR activation and chemo/radiation resistance (Ang et al. 2002; Nakamura 2007); findings such as these have made EGFR signaling an attractive target for cancer therapy (Seshacharyulu et al. 2012; Dassonville et al. 2007). Cetuximab, a recombinant monoclonal antibody therapeutic that down regulates EGFR signaling has been investigated in combination with XRT patients with colon cancer, non-small cell lung cancer, and head and neck cancers (Seshacharyulu et al. 2012), and is now in routine use for the latter given its efficacy as a radiosensitizer (Bobber et al. 2010). Erlotinib, a small molecule EGFR TK inhibitor also shows radiosensitizing activity for some malignant cells, and has shown this activity in the clinic in a promising way for patients with brain metastases of non-small cell lung cancer (Zheng et al. 2016).