The cellular DNA damage response (DDR) is a key factor in tumor suppression and tumor responses to genotoxic chemo- and radiotherapy. Master DDR regulators include three phosphatidyl inositol 3′ kinase–related kinases (PIKK) called ATM, ATR, and the catalytic subunit of DNA-dependent protein kinase, DNA-PKcs. Among their many functions, PIKKs regulate repair of DNA double-strand breaks (DSB) by homologous recombination (HR) and nonhomologous end-joining (NHEJ). Ionizing radiation induces DSBs that are either widely dispersed and efficiently repaired, or clustered and poorly repaired by the dominant NHEJ pathway. The inefficient repair of clustered DSBs by NHEJ shifts repair toward the competing HR pathway. In this issue of Cancer Research, Zhou and colleagues revealed a novel synthetic lethal approach in which the greater dependency on HR to repair clustered DSBs induced by protons is exploited to enhance killing of tumor cells and tumor xenografts by suppressing HR with an ATM inhibitor or mutant BRCA1. This is an important step toward precision cancer radiotherapy.

See related article by Zhou et al., p. 3333

Genotoxic chemo- and radiotherapy kill tumor cells by inducing cytotoxic DNA damage, but these agents also damage exposed normal tissue, limiting doses and causing severe side effects. Intrinsic and acquired tumor resistance to these agents also limit treatment efficacy. Despite these limitations, genotoxic drugs and ionizing radiation are widely used to fight cancer. This is because no cell, cancer or otherwise, can proliferate unless its DNA is (relatively) accurately transmitted to daughter cells, and DNA damage can block or reduce the fidelity of DNA replication and chromosome segregation. Thus, an important goal is to improve the cancer specificity, or precision, of genotoxic therapeutics. The study by Zhou and colleagues (1) is an important step toward achieving this goal.

Cells respond to DNA damage by activating a complex DNA damage response (DDR) network comprising DNA repair systems, and DNA damage checkpoints that slow or arrest cell-cycle progression, stimulate DNA repair, and trigger apoptosis if damage is excessive (2). The DDR has received considerable attention because DDR defects are important cancer drivers that increase genome instability and because these systems regulate tumor responses to genotoxic therapeutics (3, 4). An effective way to exploit the DDR for therapeutic gain is to identify synthetic lethal relationships. For example, tumors with homologous recombination (HR) defects, such as those harboring mutant BRCA1 or BRCA2, are dependent on PARP1-dependent repair of ssDNA damage and are thus hypersensitive to PARP1 inhibitors (5). A related approach uses drugs to transiently inhibit DDR factors to sensitize tumor cells to genotoxic agents. This second approach is more broadly applicable because it does not require mutations in specific DDR genes, but therapeutic gain can only be achieved if tumor cells are sensitized more than normal cells, or if sensitization and/or DNA damage are largely focused to the tumor volume, as is routine in radiotherapy.

Ionizing radiation induces >100 different types of DNA lesions, including base damage, single-strand breaks, and highly cytotoxic double-strand breaks (DSB). DSBs are repaired by the dominant nonhomologous end-joining (NHEJ) pathway and by the competing HR pathway. NHEJ is error-prone, while HR is generally accurate because it employs a homologous repair template, usually the sister chromatid. The balance between NHEJ and HR is principally regulated by resection of DSB ends, which creates long, 3′ single-strand tails that are coated by RAD51 and catalyze invasion of a homologous repair template to effect DSB repair by HR (6). NHEJ, in contrast, does not require end resection. ATM, ATR, and DNA-dependent protein kinase (DNA-PKcs) play central roles in the DDR. Although these phosphatidyl inositol 3′ kinase–related kinases show significant cross-talk, the current consensus is that ATM is critical for HR-mediated repair of frank DSBs (such as those induced by radiation or nucleases), ATR is critical for HR-mediated repair of DSBs at collapsed replication forks, and DNA-PKcs plays a central role in NHEJ.

Three types of radiation are currently used in external beam radiotherapy: X-rays, protons, and carbon ions. X-rays are high energy photons that interact weakly with tissue and pass completely through the body. To achieve high doses in tumors while sparing adjacent normal tissue, X-rays are focused with multi-leaf beam collimators, “intensity modulated,” and delivered from multiple angles. Protons and carbon ions are charged particles that strongly interact with tissue, and the majority of the radiation dose is delivered when particles slow and stop at a defined depth in tissue (i.e., in the tumor volume), determined by initial particle energy. With charged particle radiation, distal (normal) tissue is protected because it receives essentially no dose. The sharp dose escalation at the end of a charged particle track is termed the Bragg peak. X-rays are sparsely ionizing, described as low linear energy transfer (LET) radiation. Protons and carbon ions are relatively sparsely ionizing (low LET) in the “entrance” region (proximal to the Bragg peak), similar to X-rays, but LET increases dramatically in the Bragg peak.

All types of ionizing radiation cause DNA damage by the same mechanisms, predominantly due to attack by reactive oxygen species created by radiolysis of water, and by direct energy absorption. Low LET X-rays largely induce dispersed DNA lesions, whereas high LET Bragg peak protons and carbon ions induce dispersed and clustered lesions, including clustered DSBs, defined as two or more DSBs separated by <200 bp. Importantly, clustered DSBs are repaired less efficiently than dispersed DSBs and are hence more cytotoxic (7). Thus, high LET Bragg peak protons and carbon ions are more cytotoxic than an equivalent dose of low LET X-rays because high LET radiation induces poorly repaired clustered DSBs more efficiently than low LET radiation, characterized as “relative biological effect” (RBE). The RBE of X-rays is defined as 1.0, the measured Bragg peak proton RBE is 1.5–1.7, and the carbon ion RBE is 2.5–3 (7, 8). In other words, Bragg peak protons and carbon ions are 1.5–3 times more cytotoxic than an equivalent dose of X-rays. From this discussion, it is clear that RBE increases with LET, reflecting the greater efficiency of clustered DSB induction with high LET radiation and the inefficient repair (greater cytotoxicity) of clustered DSBs. The inefficient repair of clustered DSBs is due, at least, in part, to the inefficient activation of NHEJ by short DNA fragments (9). For this reason, HR plays a more important role in the repair of clustered DSBs induced by high LET radiation, than dispersed DSBs induced by low LET radiation (7).

Zhou and colleagues (1) exploited this shift toward HR repair of clustered DSBs to selectively sensitize tumor cells to high LET Bragg peak protons. This group showed that DSBs induced by high LET Bragg peak protons are repaired more slowly than DSBs induced by low LET X-rays or entrance protons and that this slow repair correlated with a higher RBE. They then showed greater resection of DSBs induced by Bragg peak protons as well as more RAD51 foci, relative to low LET X-rays and entrance protons, which is consistent with prior evidence for increased dependence on HR with high LET carbon ions (7). The next key findings were that suppression of HR-mediated repair of frank DSBs with an ATM inhibitor (ATMi) selectively increased RBE when cells were treated with high LET Bragg peak protons, but not low LET entrance protons or X-rays. Importantly, the RBE increase with ATMi was mimicked by a genetic HR defect (mutant BRCA1), but not by inhibition of ATR or DNA-PKcs. This indicates that ATM-dependent HR is the key repair pathway that confers resistance to high LET radiation–induced (clustered) DSBs, rather than ATR-dependent HR repair of DSBs at collapsed replication forks, or DNA-PKcs–dependent DSB repair by NHEJ. Thus, despite the cross-talk among ATM, ATR, and DNA-PKcs (2–4), high LET particle radiation reveals a clear separation of function of these DDR kinases. Interestingly, the marked RBE increase seen with Bragg peak protons and ATMi depends on functional NHEJ, suggesting that failure to repair clustered DSBs by HR shunts DSBs toward error-prone, “toxic” NHEJ. These findings were then extended to preclinical mouse xenograft studies, in which tumors were treated with ATMi and irradiated with Bragg peak protons, entrance protons, or X-rays. Although ATMi suppressed tumor growth with all three radiation types, the greatest effect was seen with high LET Bragg peak protons.

In summary, the study by Zhao and colleagues represents an important extension of the synthetic lethal concept. Specifically, synthetic lethality is reflected in increased RBE when clustered DSBs induced by high LET Bragg peak protons, which require HR for efficient repair, are “synthesized” (i.e., combined) with HR suppression by ATMi or defective BRCA1. This approach is also likely to be effective against tumors with BRCA2 defects and other HR defects, and with high LET carbon ions. Although high LET radiation shifts DSB repair toward HR, NHEJ appears to remain the dominant DSB repair pathway (10). Thus, Zhao and colleagues revealed that synthetic lethal vulnerabilities can be realized with subtle shifts in the HR-NHEJ balance. Their study also illustrates how an understanding of the DDR can be exploited to enhance cancer therapy, giving us a glimpse of the future of precision cancer radiotherapy.

No disclosures were reported.

1.
Zhou
Q
,
Howard
ME
,
Tu
X
,
Zhu
Q
,
Denbeigh
JM
,
Remmes
NB
, et al
Inhibition of ATM induces hypersensitivity to proton irradiation by upregulating toxic end joining
.
Cancer Res
2021
;
81
:
3333
46
.
2.
Blackford
AN
,
Jackson
SP
. 
ATM, ATR, and DNA-PK: the trinity at the heart of the DNA damage response
.
Mol Cell
2017
;
66
:
801
17
.
3.
Pilie
PG
,
Tang
C
,
Mills
GB
,
Yap
TA
. 
State-of-the-art strategies for targeting the DNA damage response in cancer
.
Nat Rev Clin Oncol
2019
;
16
:
81
104
.
4.
Nickoloff
JA
,
Taylor
L
,
Sharma
N
,
Kato
TA
. 
Exploiting DNA repair pathways for tumor sensitization, mitigation of resistance, and normal tissue protection in radiotherapy
.
Cancer Drug Res
2020
;
3
.
DOI: 10.20517/cdr.2020.89
.
5.
Pommier
Y
,
O'Connor
MJ
,
de Bono
J
. 
Laying a trap to kill cancer cells: PARP inhibitors and their mechanisms of action
.
Sci Transl Med
2016
;
8
:
362ps17
.
6.
Wright
WD
,
Shah
SS
,
Heyer
WD
. 
Homologous recombination and the repair of DNA double-strand breaks
.
J Biol Chem
2018
;
293
:
10524
35
.
7.
Nickoloff
JA
,
Sharma
N
,
Taylor
L
. 
Clustered DNA double-strand breaks: biological effects and relevance to cancer radiotherapy
.
Genes
2020
;
11
:
99
116
.
8.
Paganetti
H
. 
Relative biological effectiveness (RBE) values for proton beam therapy. Variations as a function of biological endpoint, dose, and linear energy transfer
.
Phys Med Biol
2014
;
59
:
R419
72
.
9.
Pang
D
,
Winters
TA
,
Jung
M
,
Purkayastha
S
,
Cavalli
LR
,
Chasovkikh
S
, et al
Radiation-generated short DNA fragments may perturb non-homologous end-joining and induce genomic instability
.
J Radiat Res
2011
;
52
:
309
19
.
10.
Takahashi
A
,
Kubo
M
,
Ma
H
,
Nakagawa
A
,
Yoshida
Y
,
Isono
M
, et al
Nonhomologous end-joining repair plays a more important role than homologous recombination repair in defining radiosensitivity after exposure to high-LET radiation
.
Radiat Res
2014
;
182
:
338
44
.