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Fabbrizi et al. Cancer Drug Resist 2020;3:775-90 I http://dx.doi.org/10.20517/cdr.2020.49 Page 783

survival of one HPV-negative HNSCC cell line post-irradiation, but had a ~1.5-fold enhanced effect on two
[26]
HPV-positive HNSCC cell lines due to their reported DSB repair defect . This is supported by enhanced
radiosensitivity of three HPV-positive HNSCC cell lines using high doses of veliparib (10 and 20 µmol/L),
[27]
although one out of three HPV-negative HNSCC cell lines also displayed marked radiosensitisation .
Interestingly though, veliparib only had a relatively mild effect on suppressing growth of HPV-positive
HNSCC xenografts.

Several studies have utilised the PARP inhibitor olaparib as a radiosensitizer, although these have shown
mixed results particularly regarding degree of radiosensitisation of HPV-negative HNSCC cell lines, which
are deemed DSB repair proficient. Indeed, it has been suggested that HPV-negative HNSCC cell lines are
[52]
more responsive to olaparib (1 µmol/L) radiosensitisation when the cells are specifically HR-deficient ,
although individual variability in cellular responses was observed. A recent study has shown that olaparib
(at 1 and 5 µmol/L) is effective in combination with radiation in decreasing the survival of HPV-negative
[53]
HNSCC cells, but only those that are SMAD4 deficient . Despite this, only a modest effect of olaparib in
combination with radiation, versus radiation alone, was observed on growth of SMAD4-deficient HNSCC
xenografts. Nevertheless, we have reported that two HPV-negative HNSCC cell lines were significantly
[25]
radiosensitised with olaparib (0.1 µmol/L) with dose enhancement ratios (DER) of 1.79 and 3.34 . This
was in comparison to two HPV-positive HNSCC cell lines, whereby only one of these showed increased
radiosensitisation in the presence of olaparib (DER = 1.51). A separate study using five HPV-positive
HNSCC cell lines displayed increased radiosensitivity after olaparib (1 µmol/L) treatment, although
[54]
markedly only in four cell lines following a relatively high radiation dose (6 Gy) , suggesting that the
radiation dose plays a fundamental role together with the DNA repair capacity of the cells. Using the PARP
inhibitor niraparib (1 µmol/L), this was recently shown to equally increase the sensitivity of two HPV-positive
and two HPV-negative HNSCC cell lines to conventional radiation (DER = 1.06-1.21) and proton
[55]
irradiation (DER = 1.12-1.31) . Both sets of cell lines displayed higher numbers of residual 53BP1 foci
present when niraparib was used in combination with either form of radiation. In contrast, another recent
study has demonstrated that niraparib (1 µmol/L) had a greater impact of the radiosensitisation of two
HPV-negative (DER = 1.94-2.13) compared to two HPV-positive (DER = 1.36-1.43) HNSCC cell lines .
[56]
Cumulatively, although positive results are observed when HNSCC cells are treated with PARP inhibitors
prior to irradiation, further studies are required to investigate in depth the potential selectivity towards
HPV-positive and HPV-negative HNSCC, and therefore provide evidence for PARP as a potentially
promising therapeutic target in the clinical setting.

DSB repair
Targeting the proteins involved in the repair of DSBs, that are potentially toxic lesions induced by RT, is
considered a promising strategy in HNSCC treatment. As detailed previously, HPV-positive HNSCC in
general have been found to be DSB repair deficient which contributes to their increased radiosensitivity
in vitro and in vivo, and so strategies have mostly focused on enhancing radiosensitivity of relatively
radioresistant HPV-negative HNSCC. Given that DSBs are sensed by the protein kinases ATM, ATR,
and DNA-Pkcs, these enzymes have been investigated as targets for inhibitors to increase HNSCC
radiosensitisation. A number of studies have focused on ATR and found this to be a successful approach
for increasing radiosensitivity in HPV-negative HNSCC cell lines. In particular, the ATR inhibitor VE821
was shown to enhance the radiosensitivity of a single HPV-negative HNSCC cell line in both normoxia
and hypoxia (2% and < 0.02% oxygen) . Similarly using the same inhibitor, we have also recently observed
[57]
radiosensitisation of two oropharyngeal HPV-negative HNSCC cell lines grown as colonies but also as
3D spheroids following both photons and (low-LET) protons . However, our study also demonstrated
[17]
that a hypopharyngeal HPV-negative HNSCC spheroid model was not radiosensitized in the presence of
VE821, suggesting cell and tumour-specific radiosensitisation. Using the ATR inhibitor AZD6738, two
studies have demonstrated that HPV-negative HNSCC cell lines (four in total) can be radiosensitised