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Page 784 Fabbrizi et al. Cancer Drug Resist 2020;3:775-90 I http://dx.doi.org/10.20517/cdr.2020.49

following ATR inhibition, including the impact on preventing 3D spheroid growth of one of the cell
lines [58,59] . The combination of both ATR and DNA-Pkcs inhibition (KU-0060648) further exacerbated
[59]
the cell killing effects post-irradiation . In relation to ATM as a target for radiosensitisation in HNSCC
cells, the evidence is sparser. A small molecule inhibitor screen identified the compound GSK635416A in
enhancing the radiosensitivity of three HPV-negative HNSCC cell lines, which was then demonstrated to
[60]
act through inhibition of ATM . This study also showed evidence for additive effects of ATM inhibition in
combination with PARP inhibitor (olaparib) in enhancing radiosensitivity. Data recently generated in our lab
has shown a significant impact of the ATM inhibitor KU-55933 in reducing survival of four HPV-negative
HNSCC cell lines (two derived from the oropharynx and one each from the hypopharynx and oral cavity)
[17]
and reducing growth of these 3D spheroid models in response to both photons and protons . Targeting
ATM was also shown to enhance the radiosensitivity of one HPV-positive HNSCC cell line, but not a
second which was the most radiosensitive cell line used.

Several studies have identified that DNA-Pkcs inhibition using different compounds has the ability to
[59]
[61]
increase HNSCC radiosensitivity. The DNA-Pkcs inhibitors KU0060648 and IC87361 have both been
shown to cause reduced survival of HPV-negative HNSCC cell lines (five in total) post-irradiation, the
latter study also suggesting that this approach was more effective than the combination of radiation with
targeting PARP (via olaparib). Evidence using the DNA-Pkcs inhibitor NU7441 has further demonstrated
the effectiveness of targeting DNA-Pkcs in radiosensitising three HPV-negative in addition to three
HPV-positive HNSCC cell lines , suggesting that this is a more general strategy for treatment of HNSCC.
[27]
This study was further progressed in vivo, where a significant delay in growth of a HPV-positive xenograft
model was observed. The slow growth of a HPV-negative xenograft model did not allow for an accurate
assessment of the effectiveness of the combination of the DNA-Pkcs inhibitor with radiation, although this
was shown in two patient-derived xenograft models where tumour growth was significantly suppressed.
Finally, our recent evidence has demonstrated that the DNA-Pkcs inhibitor KU-57788 significantly
enhanced the radiosensitisation of four HPV-negative HNSCC cell lines, including suppression of the
[17]
growth of 3D spheroid models, following both photons and protons . We also observed increased
radiosensitisation of HPV-positive HNSCC cells and spheroids, and in general identified that targeting
DNA-Pkcs was the most effective strategy for radiosensitisation of HNSCC cells in comparison to
inhibitors against ATM and ATR. Cumulatively, these data suggest that targeting the key proteins involved
in the repair of DSBs, particularly ATR and DNA-Pkcs, may be an effective treatment in combination with
RT for HPV-negative HNSCC. Nevertheless, further evidence investigating this strategy in a broader range
of HNSCC cell lines in vitro, as well as utilising in vivo models, are necessary.

Cell cycle
Checkpoint activation allows halting of cell cycle progression and is therefore important in responding to
radiation-induced DNA damage to allow cells to accomplish DNA repair. Notably, due to disruption of
p53/pRb and cell cycle regulation caused by HPV infection, the proteins involved in cell cycle checkpoint
activation, particularly CHK1 and WEE1 in the G2/M checkpoint, are of therapeutic interest in causing
enhanced RT treatment of HPV-positive HNSCC. However, this approach has also been investigated in
pre-clinical studies in HPV-negative models of the disease. Initial evidence using the CHK1 inhibitor
SAR020106 showed enhanced radiosensitivity of two HPV-negative HNSCC cell lines in vitro and
[62]
tumour growth delay of a HPV-negative HNSCC model in vivo . This is supported by a study using the
CHK1 inhibitor CCT244747 in two HPV-negative HNSCC cell lines where increased radiosensitivity
[63]
was observed, along with delayed growth of an HPV-negative HNSCC xenograft model . However, this
study also identified that the triple combination of paclitaxel, CHK1 inhibitor, and radiation was able to
significantly reduce xenograft tumour growth. Subsequent studies using the CHK1 inhibitor PF0477736
demonstrated increased radiosensitisation in two out of three HPV-negative HNSCC cell lines associated
[65]
[64]
with loss of distal chromosome 11q , and only one out of two HPV-negative HNSCC cell lines . The