A Quadratic Phenotypic Optimization Drug Screening Platform (QPOP) Identified Chromatin Modification as a Potential Strategy to Target Radioresistant (RR) Head and Neck Cancers (HNC)

Purpose/Objective(s)

Radiotherapy (RT) is a primary treatment modality for most HNC. However, locoregional recurrence post-RT remains a pertinent clinical problem, given that salvage therapeutic options are often limited and ineffective. Resistance to RT is thus an important unmet clinical need in HNC, but therapeutic strategies against radioresistance remain suboptimal, partly owing to the divergent tumor and microenvironmental factors underpinning radioresistance. We had previously developed a high-throughput drug screening method – QPOP that was applied successfully in drug-resistant multiple myeloma (Rashid M. et al., 2018). Here, we investigated the utility of QPOP for the discovery of novel drug combinations in RR-HNC models, and hypothesize that QPOP can accurately identify optimal drug combinations that resensitize RR cells.

Materials/Methods

In vitro isogenic models of RR-HNC were generated by exposing two wild-type (WT) HNC cell lines, FaDu and HK-1, to 90 Gy of 2 Gy irradiation (IR) (45 fr x 2 Gy/fr), and validated using clonogenic assays. QPOP is a complex system analytics platform that rapidly and efficiently ranks and compares all possible system-specific drug combinations from a large drug search set. Pre-experimental selection of drug candidates consisted of standard chemotherapy (cisplatin, carboplatin, and docetaxel), as well as small molecule inhibitors and targeted therapies specific to HNC. Validation of top and lowest QPOP-ranked drug combinations include synergy scoring (Bliss model), combination index theorem of Chou-Talalay, and apoptotic response (Annexin V assay). Drug targeting was validated by western blot analyses, and the Comet assay was used to quantify DNA damage.

Results

A preliminary QPOP-based combinatorial drug screen identified distinct drug sensitivities between WT- and RR-FaDu cells. Validation of the top 5 QPOP-ranked drug combinations revealed that co-treatment of panobinostat (HDAC inhibitor, Pano) and AZD7762 (CHK1 inhibitor) exhibited the most distinct and contrasting response between WT and RR cells. Response surface mapping highlighted Pano and AZD7762 synergy in RR-FaDu, further supported by Bliss synergy scoring that showed synergistic growth inhibition in RR (20.1, synergistic), but not in WT (8.3, additive) cells. These results were corroborated by H3K27 hyperacetylation, and increased downstream caspase-dependent apoptosis and DNA damage in RR-FaDu following combinatorial treatment, compared with single drug treatments. Next, we interrogated if Pano can re-sensitize RR-FaDu and RR-HK-1 to IR. Interestingly, exposure to Pano before 4 Gy IR led to radiosensitization of RR-HNC cells (radiosensitization ratio: FaDu=1.77 [P=0.06]; HK-1=1.81 [P=0.041]), but not WT-HNC cells (FaDu=0.90 [P=0.60]; HK-1=1.26 [P=0.29]).

Conclusion

Herein, QPOP identified the potential of chromatin modification using Pano, in combination with CHK1 blockade or re-IR for targeting RR-HNC.