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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 785

latter study in fact showed that four out of five HPV-positive HNSCC cell lines tested were radiosensitised
with CHK1 inhibition, which was supported by a follow-up study performed by the same research
[54]
group in two HPV-positive HNSCC cell lines . Additional evidence acquired in three HPV-positive
HNSCC cell lines showed the impact of the CHK1 inhibitors LY2603618 and MK8776 in increasing
cellular radiosensitivity, but also that the additive effect of the WEE1 inhibitor AZD1775 was relatively
[66]
mild . Interestingly, the WEE1 inhibitor alone in combination with radiation only appeared effective in
radiosensitising one of the three HPV-positive HNSCC cell lines.

Studies have also examined combinatorial strategies using inhibitors targeting both cell cycle checkpoints,
and DNA repair (via PARP). Here, the additive effect of PARP inhibition (using olaparib) in combination
with CHK1 inhibitor PF00477736 in reducing radiation-induced survival of two HPV-positive HNSCC
cell lines has been shown . This is supported by a more recent study demonstrating the added benefit
[54]
of CHK1 (MK8776) and PARP (niraparib) inhibition in enhancing radiosensitivity of one HPV-positive
[56]
HNSCC cell line, but which was not effective in one HPV-negative HNSCC cell line . In contrast, it was
additionally observed that the HPV-negative HNSCC cell line was more susceptible to the combination
of PARP and WEE1 (MK1775) inhibition, which was suggested to be due to more potent activation of the
G2/M checkpoint caused by WEE1 inhibition that is downstream of CHK1. Noteworthy, a phase I dose-
finding multicenter trial has begun this year with the aim of determining a safe dose of the WEE1 inhibitor
AZD1775, both for preoperative patients treated with cisplatin and for post-operative patients treated with
cisplatin-based chemoradiation [102] .

Another promising therapeutic target for radiosensitising HNSCC is CDK4/6, utilising the inhibitors
palbociclib and ribociclib. Palbociclib has been demonstrated to selectively increase the radiosensitisation
of HPV-negative, but not HPV-positive HNSCC cell lines both in normoxic and in hypoxic (1%)
[67]
conditions . This treatment was shown to cause a reduction in HR efficiency post-irradiation, ultimately
leading to increased chromosomal damage and cell death. Palbociclib is currently under testing in the
UPSTREAM trial in patients with recurrent or metastatic HNSCC who show cyclin D1 amplification
and p16 negativity. Ribociclib is a small inhibitor of CDK4/6, which has showed promising safety and
efficacy results in a phase I study in patients with local or metastatic recurrence of HNSCC together with
cetuximab [103] . However, the impact of ribociclib in combination with radiation on HNSCC cell models
has yet to be investigated. Given the promising evidence that targeting the cell cycle can enhance the
radiosensitivity of HNSCC cell lines, particularly relatively radioresistant HPV-negative HNSCC, more
comprehensive studies are warranted to accumulate this important preclinical data.

CONCLUSION
Promising data has been reported demonstrating that the radiosensitivity of HNSCC, particularlyrelatively
radioresistant HPV-negative HNSCC, can be increased by inhibitors targeting proteins involved in DNA
damage repair (PARP, ATR, and DNA-Pkcs) and in DNA damage checkpoint activation (CHK1). These
preclinical studies have largely been performed in vitro using the large numbers of established HNSCC cell
lines that exist [104] , but that variability in the degree of radiosensitisation is observed which is dependent on
the specific cell line used. These studies also differ in the precise inhibitors utilised that harbour different
selectivity and potency. Therefore, the challenge now is to accumulate preclinical evidence using the most
selective and up to date inhibitors in a larger cohort of HNSCC models, to convincingly demonstrate that
such approaches can enhance the radiosensitivity of HNSCC. These models should include HPV-positive
and HPV-negative cell lines from the oropharynx, but also those originating from different tumour origins,
including the larynx, hypopharynx, and oral cavity which are largely HPV-negative, to understand whether
or not the strategies investigated can be utilised more generally in treating HNSCC. It is also apparent
that more advanced HNSCC models should be employed in this research, including 3D spheroids and
well characterized patient-derived organoids, that mimic more precisely the structure and environment