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Nickoloff et al. Cancer Drug Resist 2021;4:244-63 I http://dx.doi.org/10.20517/cdr.2020.89 Page 252

Mutant forms of EGFR (D746-750, L858R, and the targeted-therapy resistant T790M mutant) confer
radiosensitivity to hypoxic lung cancer cells, at least in part due to downregulation of RAD50, a member of
the MRE11/RAD50/NBS1 complex that plays early end-processing and signaling roles in NHEJ and HR [190] .
These results suggest that tumor EGFR status can be used to personalize radiotherapy treatment plans
and augmentation with NHEJ inhibitors. The link between EGFR and DSB repair suggests strategies to
modulate tumor radiosensitivity by inhibiting NHEJ indirectly with available drugs that target EGFR and
AKT1/3 pathways [148,160] .

TARGETING HR
A key step in HR is formation of RAD51 nucleoprotein filaments that seek and invade homologous duplex
DNA repair template [Figure 2]. RAD51 sub-nuclear foci are observed ~1 h after irradiation and are often
interpreted as evidence of “HR activity”. However, RAD51 nucleoprotein filament formation marks only the
initial phase of HR; once the filament invades a donor duplex, RAD51 must dissociate to allow extension
of the invading strand by repair-associated DNA polymerases [191] . Thus, RAD51 foci are markers of HR
initiation, but persistent RAD51 foci may reflect failure to complete HR due to downstream HR defects [192] .
Functional HR, therefore, is best assayed by directly detecting HR products. There are several types of HR
assay systems, including plasmid transfection systems, integrated HR repeat substrates, and HR-mediated
gene editing [193,194] . When assaying RAD51-dependent HR using linked (direct or inverted) repeats, it is
important that the design detects RAD51-dependent gene conversion but not RAD51-independent single-
[62]
strand annealing . Plasmid transfection assays are convenient, but substrates may not be chromatinized
before or during HR, and therefore may not accurately reflect the full constellation of HR functions in
chromatin [195] . Similarly, gene editing involves transfection of a non-chromatinized, homologous donor
DNA sequence. Plasmid and gene editing assays are useful in rapid HR screens that can be complemented
by analysis of HR products in a chromosomal context.

HR is important for repair of frank DSBs, but its other critical role is repairing single-ended DSBs that
arise when replication stress causes fork collapse [Figure 3] [196] . A 1-Gy dose of ionizing radiation induces
~40 frank DSBs, but hundreds of single-strand lesions that can cause “secondary DSBs” due to fork
collapse [197,198] . HR is critical for repair of these one-ended DSBs because mis-repair by NHEJ necessarily
involves a distant DSB end (from a different broken replication fork or a frank DSB), causing large-scale
genome rearrangements including deletions, translocations, and dicentric chromosomes that can trigger
cell death or genome instability from persistent bridge-breakage-fusion cycles [199] . Thus, care must be taken
when interfering with HR to enhance radiotherapy, as HR is critical for maintaining genome stability in
normal tissues to prevent induction of secondary cancers.

Because RAD51 plays a central role in HR, it is an attractive target for radiosensitization. The Bishop and
Connell labs developed a small molecule RAD51 inhibitor, RI-1, that blocks RAD51 binding to ssDNA [200]
and radiosensitizes glioma and glioblastoma cells [161,162] . New RAD51 inhibitors have been developed,
including one that blocks D-loop formation (strand invasion) and HR but does not affect RAD51 binding
to ssDNA or formation of radiation-induced RAD51 foci [201,202] . A recently developed antibody fragment
linked to a cell-penetrating peptide blocks RAD51 DNA binding, sensitizes cells to radiation, and is
synthetically lethal with PTEN defects in glioma and melanoma cells [163-165] . Another small molecule RAD51
inhibitor, CYT-0851, is currently in a clinical trial as monotherapy against several types of cancer [166] .

HR defects pre-dispose to cancer, including breast, ovarian, and other cancers with defects in BRCA1,
BRCA2, PALB2, MRE11, and RAD51, as well as DDR factors that regulate HR, such as ATM [203-206] . HR
proteins function as tumor suppressors by maintaining genome stability by promoting accurate DSB repair,
stabilizing stressed replication forks, and repairing and restarting collapsed replication forks [207] . PARP1
inhibitors cause replication stress by inhibiting PARP1-dependent repair of single-strand damage and