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.

The prognostic impact of B7-H3 and B7-H4 in head and neck squamous cell carcinoma.

PURPOSE: Immune checkpoint inhibition is a therapeutic option in many cancer entities. In head and neck squamous cell carcinoma (HNSCC) targeting of the PD-1/PD-L1 (B7-H1) axis is approved in recurrent/metastatic disease and is being explored in the curative setting. Here, we evaluated two related members of the B7 family, B7-H3 & B7-H4, for their prognostic impact under standard treatment. METHODS: A tissue microarray (TMA) of a single center HNSCC cohort was stained for B7-H3 and B7-H4. Staining intensity and the number of tumor cells stained were assessed, and the expression was scored according to an established algorithm. Staining scores were correlated with clinicopathological parameters and associated with patient survival. mRNA levels of both proteins were associated with patient outcome using the TCGA dataset. RESULTS: mRNA levels of B7-H3 and B7-H4 were not significantly associated with patient survival. TMA analysis revealed interpretable protein staining in 408 samples. Strong staining was the most frequent category for B7-H3 and no staining for B7-H4. In patients with p16-negative oropharyngeal SCC (OPSCC) and in a pooled cohort consisting of p16-negative OPSCC, laryngeal, hypopharyngeal and oral cavity SCC, strong B7-H3 expression was associated with better overall survival. For the latter cohort, this was in part due to reduced lymph node involvement. B7-H3 expression in p16-positive OPSCC and B7-H4 expression were not associated with outcome. CONCLUSION: Despite a possible role in tumor immune escape, B7-H3 was associated with favorable prognosis in HPV-negative HNSCC in our cohort. The underlying mechanisms and a potential impact for B7-H3 targeting remain to be elucidated.

Tissue Microarray Analyses Suggest Axl as a Predictive Biomarker in HPV-Negative Head and Neck Cancer.

The receptor tyrosine kinase Axl is described to promote migration, metastasis and resistance against molecular targeting, radiotherapy, and chemotherapy in various tumor entities, including head and neck squamous cell carcinoma (HNSCC). Since clinical data on Axl and its ligand Gas6 in HNSCC are sparse, we assessed the association of Axl and Gas6 expression with patient survival in a single center retrospective cohort in a tissue microarray format. Expression was evaluated manually using an established algorithm and correlated with clinicopathological parameters and patient survival. A number of 362 samples yielded interpretable staining, which did not correlate with T- and N-stage. Protein expression levels were not associated with the survival of patients with p16-positive oropharyngeal SCC. In HPV-negative tumors, Axl expression did not impact patients treated with primary or adjuvant radio(chemo)therapy, but was significantly associated with inferior overall and recurrence-free survival in patients treated with surgery alone. Gas6 was a positive predictor of survival in patients whose treatment included radiotherapy. Associations remained significant in multivariable analysis. Our data question a meaningful contribution of the Axl/Gas6 pathway to radio-resistance in HNSCC and instead suggest that strong Axl expression identifies tumors requiring adjuvant radio(chemo)therapy after surgery.

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.

Analyzing tyrosine kinase activity in head and neck cancer by functional kinomics: Identification of hyperactivated Src family kinases as prognostic markers and potential targets.

Signal transduction via protein kinases is of central importance in cancer biology and treatment. However, the clinical success of kinase inhibitors is often hampered by a lack of robust predictive biomarkers, which is also caused by the discrepancy between kinase expression and activity. Therefore, there is a need for functional tests to identify aberrantly activated kinases in individual patients. Here we present a systematic analysis of the tyrosine kinases in head and neck cancer using such a test-functional kinome profiling. We detected increased tyrosine kinase activity in tumors compared with their corresponding normal tissue. Moreover, we identified members of the family of Src kinases (Src family kinases [SFK]) to be aberrantly activated in the majority of the tumors, which was confirmed by additional methods. We could also show that SFK hyperphosphorylation is associated with poor prognosis, while inhibition of SFK impaired cell proliferation, especially in cells with hyperactive SFK. In summary, functional kinome profiling identified SFK to be frequently hyperactivated in head and neck squamous cell carcinoma. SFK may therefore be potential therapeutic targets. These results furthermore demonstrate how functional tests help to increase our understanding of cancer biology and support the expansion of precision oncology.

Mass Spectrometric Comparison of HPV-Positive and HPV-Negative Oropharyngeal Cancer.

Squamous cell carcinoma of the head and neck (HNSCC) consist of two distinct biological entities. While the numbers of classical, tobacco-induced HNSCC are declining, tumors caused by human papillomavirus (HPV) infection are increasing in many countries. HPV-positive HNSCC mostly arise in the oropharynx and are characterized by an enhanced sensitivity towards radiotherapy and a favorable prognosis. To identify molecular differences between both entities on the protein level, we conducted a mass spectrometric comparison of eight HPV-positive and nine HPV-negative oropharyngeal tumors (OPSCC). Overall, we identified 2051 proteins, of which 31 were found to be differentially expressed. Seventeen of these can be assorted to three functional groups, namely DNA replication, nuclear architecture and cytoskeleton regulation, with the differences in the last group potentially reflecting an enhanced migratory and invasive capacity. Furthermore, a number of identified proteins have been described to directly impact on DNA double-strand break repair or radiation sensitivity (e.g., SLC3A2, cortactin, RBBP4, Numa1), offering explanations for the differential prognosis. The unequal expression of three proteins (SLC3A2, MCM2 and lamin B1) was confirmed by immunohistochemical staining using a tissue microarray containing 205 OPSCC samples. The expression levels of SLC3A2 and lamin B1 were found be of prognostic relevance in patients with HPV-positive and HPV-negative OPSCC, respectively.