Evaluating the Therapeutic Potential of a Small Molecule ATR Inhibitor Combined with Radiotherapy for Head and Neck Squamous Cell Carcinoma

Abstract

<h3>Purpose/Objective(s)</h3> Radiotherapy (RT) plays a major role in HNSCC treatment. Unfortunately, recurrence rates approach 50% for locally advanced, HPV-negative HNSCC, and treatment options for recurrent HNSCC are limited. New therapies are needed to enhance RT response and improve local tumor control. Ataxia telangiectasia and Rad 3-related (ATR) is a DNA damage response protein activated by replication stress (common in HNSCC) and single strand DNA breaks, such as those induced by RT. We evaluated the effect of ATR inhibitor BAY1895344 (BAY) +/- RT in preclinical HNSCC models, including the radioresistant MOC2 model. <h3>Materials/Methods</h3> HNSCC cells were treated with vehicle or serial dilutions of BAY +/- 4 Gy, and IC50 was determined by MTT assay at 72h. Sensitivity of immortalized oral keratinocyte (IMOK) cells to BAY was evaluated as a normal tissue control. To evaluate inhibition of phosphorylation of ATR downstream effector CHK1, cell lysates were harvested after treatment with BAY +/- 4 Gy (1 hr post-RT) and immunoblot performed. Radiosensitization was evaluated by clonogenic assay and quantification of yH2AX foci at 1, 3, 8, and 24 hr after 4 Gy +/- 100nM BAY. Heterotopic MOC2 tumors were induced in the hind limb of C57BL/6 mice. When tumors reached 60-110 mm³, mice were randomized to vehicle, BAY, 10 Gy, or 10 Gy + BAY (n=7-12/group). BAY was given PO (30 mg/kg BID x 3d) with RT given after the 2nd dose. Tumors were measured 3x/week, and times to tumor quintupling (TTQ) compared by ANOVA with Tukey's multiple comparisons post-test. In an ongoing experiment, mice were randomized to vehicle, BAY (50 mg/kg BID 3 days on/4 days off, continued weekly), 10 Gy, or 10 Gy+BAY (n=9/group). <h3>Results</h3> BAY inhibited CHK1 phosphorylation in a dose-dependent manner and led to persistent yH2AX foci at 24h post-RT. MTT assay showed cooperative effect between BAY and RT (IC50=63 nM for BAY vs 15nM for BAY + 4Gy) in MOC2 cells, with similar results across several primary murine HNSCC cell lines and the human FaDu cell line. IMOK cells were relatively resistant to BAY (IC50=3 uM). Clonogenic assay showed radiosensitization with 100 nM BAY (RT dose enhancement ratio = 3.7 at 4 Gy for MOC2). In the MOC2 model, BAY increased <i>in vivo</i> mean TTQ by 7.4 d vs vehicle (p=0.002), and 10 Gy + BAY increased TTQ by 7.3 d vs 10 Gy alone (p<0.001). A trend towards longer growth delay was observed 10 Gy + BAY vs BAY (mean TTQ increased by 4.3 d; p=0.08). With continued BAY dosing (3d on/4d off), median TTQ was 9.3 d with vehicle, 16.8 d with RT, and not yet reached for BAY or 10 Gy + BAY. <h3>Conclusion</h3> BAY 1895344 radiosensitizes HNSCC <i>in vitro</i> and increases tumor growth delay <i>in vivo</i> as a monotherapy or in combination with RT. Given these promising preclinical results, safety and efficacy of BAY 1895344 with stereotactic body radiation therapy (SBRT) reirradiation and pembrolizumab for recurrent HNSCC will be evaluated in an ongoing phase I CTEP trial (NCT04576091).