G2 checkpoint targeting via Wee1 inhibition radiosensitizes EGFRvIII-positive glioblastoma cells.

BACKGROUND: The gene of the Epidermal growth factor receptor (EGFR) is one of the most frequently altered genes in glioblastoma (GBM), with deletions of exons 2-7 (EGFRvIII) being amongst the most common genomic mutations. EGFRvIII is heterogeneously expressed in GBM. We already showed that EGFRvIII expression has an impact on chemosensitivity, replication stress, and the DNA damage response. Wee1 kinase is a major regulator of the DNA damage induced G2 checkpoint. It is highly expressed in GBM and its overexpression is associated with poor prognosis. Since Wee1 inhibition can lead to radiosensitization of EGFRvIII-negative (EGFRvIII-) GBM cells, we asked, if Wee1 inhibition is sufficient to radiosensitize also EGFRvIII-positive (EGFRvIII+) GBM cells. METHODS: We used the clinically relevant Wee1 inhibitor adavosertib and two pairs of isogenetic GBM cell lines with and without endogenous EGFRvIII expression exhibiting different TP53 status. Moreover, human GBM samples displaying heterogenous EGFRvIII expression were analyzed. Expression of Wee1 was assessed by Western blot and respectively immunohistochemistry. The impact of Wee1 inhibition in combination with irradiation on cell cycle and cell survival was analyzed by flow cytometry and colony formation assay. RESULTS: Analysis of GBM cells and patient samples revealed a higher expression of Wee1 in EGFRvIII+ cells compared to their EGFRvIII- counterparts. Downregulation of EGFRvIII expression by siRNA resulted in a strong decrease in Wee1 expression. Wee1 inhibition efficiently abrogated radiation-induced G2-arrest and caused radiosensitization, without obvious differences between EGFRvIII- and EGFRvIII+ GBM cells. CONCLUSION: We conclude that the inhibition of Wee1 is an effective targeting approach for the radiosensitization of both EGFRvIII- and EGFRvIII+ GBM cells and may therefore represent a promising new therapeutic option to increase response to radiotherapy.

Impaired DNA double-strand break repair and effective radiosensitization of HPV-negative HNSCC cell lines through combined inhibition of PARP and Wee1.

Objectives: In head and neck squamous cell carcinoma (HNSCC), tumors negative for Human Papillomavirus (HPV) remain a difficult to treat entity and the morbidity of current multimodal treatment is high. Radiotherapy in combination with molecular targeting could represent suitable, less toxic treatment options especially for cisplatin ineligible patients. Therefore, we tested dual targeting of PARP and the intra-S/G2 checkpoint through Wee1 inhibition for its radiosensitizing capacity in radioresistant HPV-negative HNSCC cells. Materials and methods: Three radioresistant HPV-negative cell lines (HSC4, SAS, UT-SCC-60a) were treated with olaparib, adavosertib and ionizing irradiation. The impact on cell cycle, G2 arrest and replication stress was assessed through flow cytometry after DAPI, phospho-histone H3 and γH2AX staining. Long term cell survival after treatment was determined through colony formation assay and DNA double-strand break (DSB) levels were assessed through quantification of nuclear 53BP1 foci in cell lines and patient-derived HPV± tumor slice cultures. Results: Wee1 and dual targeting induced replication stress but failed to effectively inhibit radiation-induced G2 cell cycle arrest. Single as well as combined inhibition increased radiation sensitivity and residual DSB levels, with the largest effects induced through dual targeting. Dual targeting also enhanced residual DSB levels in patient-derived slice cultures from HPV-negative but not HPV+ HNSCC (5/7 vs. 1/6). Conclusion: We conclude that the combined inhibition of PARP and Wee1 results in enhanced residual DNA damage levels after irradiation and effectively sensitizes radioresistant HPV-negative HNSCC cells. Ex vivo tumor slice cultures may predict the response of individual patients with HPV-negative HNSCC to this dual targeting approach.

A Lack of Effectiveness in the ATM-Orchestrated DNA Damage Response Contributes to the DNA Repair Defect of HPV-Positive Head and Neck Cancer Cells.

Patients with human papillomavirus-positive squamous cell carcinoma of the head and neck (HPV+ HNSCC) have a favorable prognosis compared to those with HPV-negative (HPV-) ones. We have shown previously that HPV+ HNSCC cell lines are characterized by enhanced radiation sensitivity and impaired DNA double-strand break (DSB) repair. Since then, various publications have suggested a defect in homologous recombination (HR) and dysregulated expression of DSB repair proteins as underlying mechanisms, but conclusions were often based on very few cell lines. When comparing the expression levels of suggested proteins and other key repair factors in 6 HPV+ vs. 5 HPV- HNSCC strains, we could not confirm most of the published differences. Furthermore, HPV+ HNSCC strains did not demonstrate enhanced sensitivity towards PARP inhibition, questioning a general HR defect. Interestingly, our expression screen revealed minimal levels of the central DNA damage response kinase ATM in the two most radiosensitive HPV+ strains. We therefore tested whether insufficient ATM activity may contribute to the enhanced cellular radiosensitivity. Irrespective of their ATM expression level, radiosensitive HPV+ HNSCC cells displayed DSB repair kinetics similar to ATM-deficient cells. Upon ATM inhibition, HPV+ cell lines showed only a marginal increase in residual radiation-induced γH2AX foci and induction of G2 cell cycle arrest as compared to HPV- ones. In line with these observations, ATM inhibition sensitized HPV+ HNSCC strains less towards radiation than HPV- strains, resulting in similar levels of sensitivity. Unexpectedly, assessment of the phosphorylation kinetics of the ATM targets KAP-1 and Chk2 as well as ATM autophosphorylation after radiation did not indicate directly compromised ATM activity in HPV-positive cells. Furthermore, ATM inhibition delayed radiation induced DNA end resection in both HPV+ and HPV- cells to a similar extent, further suggesting comparable functionality. In conclusion, DNA repair kinetics and a reduced effectiveness of ATM inhibition clearly point to an impaired ATM-orchestrated DNA damage response in HPV+ HNSCC cells, but since ATM itself is apparently functional, the molecular mechanisms need to be further explored.

Patient derived ex vivo tissue slice cultures demonstrate a profound DNA double-strand break repair defect in HPV-positive oropharyngeal head and neck cancer.

BACKGROUND: HPV-positive head and neck squamous cell carcinoma of the oropharynx (OPSCC) are more sensitive towards radiation than HPV-negative OPSCC. Two main theories exist regarding the underlying mechanism. Stronger lymphocyte infiltration points to an enhanced immunogenicity, whereas data from HPV-positive HNSCC cell lines suggest an enhanced cellular radiosensitivity based on a defect in DNA double-strand break (DSB) repair. The critical limitation of the latter theory is that the evidence was largely derived from a small number of established HPV-positive HNSCC cell lines. METHODS AND MATERIALS: Fresh patient-derived OPSCC samples were cut in 400 µm sections and cultured on cell culture inserts. Slice cultures were irradiated, in part combined with ATM inhibition, and fixed and frozen after 2 and 24 h. DSBs were analyzed by quantification of 53BP1 foci in nuclei co-stained with the SCC marker p63 via immunofluorescence microscopy. RESULTS: Ex vivo OPSCC tumor slice cultures maintained stable oxygenation and proliferation characteristics for at least 3 days. Areas of p63-positivity in immunofluorescence microscopy matched histologically confirmed tumor cell areas in serial sections, indicating the suitability of p63 as a tumor cell marker. p63-positive nuclei in HPV-positive OPSCC tissues (n = 14) showed profoundly elevated numbers of residual radiation-induced DSBs as compared to those from HPV-negative OPSCC (n = 12) (3 Gy: on average 4.9 vs. 1.2 foci per nucleus; p < 0.0001). Within the HPV-positive subgroup, samples derived from patients with a smoking history of less than 10 pack years demonstrated higher residual DSBs as compared to those derived from patients with 10 or more pack years (3 Gy: on average 6.5 vs. 3.2 foci per nucleus; p = 0.0105). Additional ATM inhibition resulted in a substantial increase in residual foci in all 4 HPV-negative samples tested but strikingly only in 2 out of 11 HPV-positive samples. CONCLUSIONS: In summary, our data provide robust, cell line-independent experimental evidence for an intrinsic DSB repair deficiency in HPV-positive OPSCC, strongly suggesting a meaningful contribution to the enhanced clinical radiosensitivity. The reduced effectiveness of ATM inhibition indicates a defect in the ATM-orchestrated DNA damage response. Lower numbers of residual 53BP1 nuclear foci in the ex vivo assay may identify HPV-positive patients with effective DSB repair who should potentially be excluded from de-intensification approaches.

Dual Inhibition of PARP and the Intra-S/G2 Cell Cycle Checkpoints Results in Highly Effective Radiosensitization of HPV-Positive HNSCC Cells.

In head and neck squamous cell carcinoma (HNSCC), tumors positive for human papillomavirus (HPV) represent a distinct biological entity with favorable prognosis. An enhanced radiation sensitivity of these tumors is evident in the clinic and on the cellular level when comparing HPV-positive and HPV-negative HNSCC cell lines. We could show that the underlying mechanism is a defect in DNA double-strand break repair associated with a profound and sustained G2 arrest. This defect can be exploited by molecular targeting approaches additionally compromising the DNA damage response to further enhance their radiation sensitivity, which may offer new opportunities in the setting of future de-intensified regimes. Against this background, we tested combined targeting of PARP and the DNA damage-induced intra-S/G2 cell cycle checkpoints to achieve effective radiosensitization. Enhancing CDK1/2 activity through the Wee1 inhibitor adavosertib or a combination of Wee1 and Chk1 inhibition resulted in an abrogation of the radiation-induced G2 cell cycle arrest and induction of replication stress as assessed by γH2AX and chromatin-bound RPA levels in S phase cells. Addition of the PARP inhibitor olaparib had little influence on these endpoints, irrespective of checkpoint inhibition. Combined PARP/Wee1 targeting did not result in an enhancement in the absolute number of residual, radiation induced 53BP1 foci as markers of DNA double-strand breaks but it induced a shift in foci numbers from S/G2 to G1 phase cells. Most importantly, while sole checkpoint or PARP inhibition induced moderate radiosensitization, their combination was clearly more effective, while exerting little effect in p53/G1 arrest proficient normal human fibroblasts, thus indicating tumor specificity. We conclude that the combined inhibition of PARP and the intra-S/G2 checkpoint is a highly effective approach for the radiosensitization of HPV-positive HNSCC cells and may represent a viable alternative for the current standard of concomitant cisplatin-based chemotherapy. In vivo studies to further evaluate the translational potential are highly warranted.

Radiosensitisation and enhanced tumour growth delay of colorectal cancer cells by sustained treatment with trifluridine/tipiracil and X-rays.

Trifluridine/tipiracil (FTD/TPI; marketed as Lonsurf®) has shown clinically relevant activity after fluoropyrimidine failure in colorectal cancer and may thus be of increased efficacy compared with current standard capecitabine chemoradiation. Here we investigated the colorectal cancer cell lines HT29, HCT116, SW48 and Caco-2 to provide a preclinical rationale for FTD/TPI-based chemoradiation treatment. All lines incorporated similar amounts of FTD, irrespective of treatment concentration and duration, then arrested in S phase, showed persistent γH2AX induction and eventually underwent endoreplication, resulting in polyploidy. Clonogenic assays performed for four combined treatment schedules demonstrated additivity for treatments given within 6 h of each other. However, 24 h FTD/TPI treatment prior to irradiation caused 1.6-2.4 fold radiosensitisation. Combined in vivo treatment was well tolerated and caused a marked tumour growth delay, similar to capecitabine radiochemotherapy regimes. Prolonged S phase arrest, persistent γH2AX signalling, endoreplication and polyploidy may contribute to the cytotoxicity of FTD/TPI. The strong radiosensitising effect observed in vitro after prolonged treatment with FTD/TPI and equivalence with capecitabine-based chemoradiation in vivo support a daily fractionated combined regime of FTD/TPI and radiation in rectal cancer treatment. This is now being tested in a phase I/II clinical trial (NCT04177602).

The inflammation-reducing compatible solute ectoine does not impair the cytotoxic effect of ionizing radiation on head and neck cancer cells.

Ectoine is a natural protectant expressed by halophile bacteria to resist challenges of their natural environments, such as drought, heat or high salt concentrations. As a compatible solute, ectoine does not interfere with the cell's metabolism even at high molar concentrations. External application of ectoine results in surface hydration and membrane stabilization. It can reduce inflammation processes and was recently tested in a pilot study for the prevention and treatment of chemotherapy-induced oral mucositis. Oral mucositis is especially frequent and severe in patients with head and neck squamous cell carcinoma (HNSCC), who receive radiotherapy or chemoradiation. It is extremely painful, can limit nutritional intake and may necessitate treatment interruptions, which can critically compromise outcome. As it was recently reported that in vitro ectoine has the ability to protect DNA against ionizing irradiation, it was the aim of this study to test whether ectoine may protect HNSCC cells from radiotherapy. Using HNSCC cell lines and primary human fibroblasts, we can show that in living cells ectoine does not impair DNA damage induction and cytotoxicity through ionizing radiation. We therefore conclude that testing the ectopic application of ectoine for its ability to alleviate early radiotherapy/chemoradiation-induced side effects is safe and feasible.