54P - Inhibition of ATM vs ATR in combination with radiotherapy affects cellular toxicity and expression of immune checkpoint molecules differently in HNSCC

Background

Treatment of human papilloma virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) is still challenging, regarding its radio resistance and sparing of healthy surrounding tissue when applying radiotherapy (RT). Small molecules kinase inhibitors (smKI), targeting components of the DNA damage repair (DDR) pathway such as ATM and ATR, in combination with RT are promising to overcome these challenges. We hypothesized that inhibition von ATM vs. ATR concomitant to RT increases cellular toxicity and leads to diverse immune surface marker expression on HNSCC cells.

Methods

The effect of smKIs AZD0156 (ATMi) and VE-822 (ATRi) in combination with RT on HPV-pos. and HPV-neg. human HNSCC was analyzed. Colony formation (Co, smKI, RT, smKI+RT), immunogenic and non-immunogenic cell death (necrosis, apoptosis) were measured using Annexin/PI staining (flow cytometry). Immune-stimulating (ICOS-L, OX40-L, TNFSFR9, CD70) and immune-suppressive (PD-L1, PD-L2, HVEM) surface-marker were measured after 48h of treatment of HNSCC cells (HSC4, Cal33, UM-SCC-47, UD-SCC-2).

Results

Colony forming was significantly inhibited by smKI+RT in cancer cells, while sparing toxicity in healthy fibroblasts. Effects were more prominent in HPV-pos. compared to HPV-neg. HNSCC cells. Furthermore, ATRi demonstrated stronger cellular toxicity by inducing cell death at 0.1 μM compared to 1 μM ATMi. In contrast, ATMi only in combination with RT led to significant increase of apoptosis. After treatment with ATRi, upregulation of immune-suppressive checkpoint molecules on the cell surfaces was observed predominantly, but less influence on immune-stimulatory surface marker was found. Of note, ATMi treatment w/o RT led to even increased expression of both suppressive and stimulatory immune checkpoint molecules.

Conclusions

Inhibition of ATR shows greater toxicity, but ATM inhibition has stronger influence on the expression of immune checkpoint molecules. Taken together, combined treatment has the potential to be a therapeutic option that could improve tumor control without increasing toxicity in HNSCC.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

Strahlenklinik Erlangen.

Funding

Bundesministerium für Bildung und Forschung (GREWIS-alpha, 02NUK050E).

Disclosure

The author has declared no conflicts of interest.