Hypoxia-activated prodrugs of phenolic olaparib analogues for tumour-selective chemosensitisation.

Poly(ADP-ribose)polymerase inhibitors (PARPi) are used for treatment of tumours with a defect in homologous recombination (HR) repair. Combination with radio- or chemotherapy could broaden their applicability but a major hurdle is enhancement of normal tissue toxicity. Development of hypoxia-activated prodrugs (HAPs) of PARPi has potential to restrict PARP inhibition to tumours thereby avoiding off-target toxicity. We have designed and synthesised phenolic derivatives of olaparib (termed phenolaparibs) and corresponding ether-linked HAPs. Phenolaparib cytotoxicity in HR-proficient and deficient cell lines was consistent with inhibition of PARP-1. Prodrugs were deactivated relative to phenolaparibs in biochemical PARP-1 inhibition assays, and cell culture. Prodrug 7 was selectively converted to phenolaparib 4 under hypoxia and demonstrated hypoxia-selective cytotoxicity, including chemosensitisation of HR-proficient cells in combination with temozolomide. This work demonstrates the feasibility of a HAP approach to PARPi for use in combination therapies.

Radiosensitisation of SCCVII tumours and normal tissues in mice by the DNA-dependent protein kinase inhibitor AZD7648.

BACKGROUND AND PURPOSE: Inhibitors of DNA-dependent protein kinase (DNA-PK) are effective radiation sensitisers in preclinical tumours, but little is known about risks of normal tissue radiosensitisation. Here, we evaluate radiosensitisation of head and neck squamous cell carcinoma (HNSCC) cells by DNA-PK inhibitor AZD7648 under oxia and anoxia in vitro, and tumour (SCCVII), oral mucosa and small intestine in mice. MATERIALS AND METHODS: Radiosensitisation of human (UT-SCC-54C) and murine (SCCVII) HNSCC cells by AZD7648 under oxia and anoxia was evaluated by clonogenic assay. Radiosensitisation of SCCVII tumours in C3H mice by oral AZD7648 (75 mg/kg) was determined by ex vivo clonogenic assay 3.5 days post-irradiation, with evaluation of normal tissue surrogate endpoints using 5-ethynyl-2'-deoxyuridine to facilitate detection of regenerating crypts in the ileum and repopulating S-phase cells in the ileum and oral mucosa of the same animals. RESULTS: AZD7648 potently radiosensitised both cell lines, with similar sensitiser enhancement ratios for 10% survival (SER10) under oxia and anoxia. AZD7648 diffused rapidly through multicellular layers, suggesting rapid equilibration between plasma and hypoxic zones in tumours. SCCVII tumours were radiosensitised by AZD7648 (SER10 2.5). AZD7648 also enhanced radiation-induced body weight loss and suppressed regenerating intestinal crypts and repopulating S-phase cells in the ileum and tongue epithelium with SER values similar to SCCVII tumours. CONCLUSION: AZD7648 is a potent radiation sensitiser of both oxic and anoxic tumour cells, but also markedly radiosensitises stem cells in the small intestine and oral mucosa.

Patient-Derived Xenograft and Organoid Models for Precision Medicine Targeting of the Tumour Microenvironment in Head and Neck Cancer.

Patient survival from head and neck squamous cell carcinoma (HNSCC), the seventh most common cause of cancer, has not markedly improved in recent years despite the approval of targeted therapies and immunotherapy agents. Precision medicine approaches that seek to individualise therapy through the use of predictive biomarkers and stratification strategies offer opportunities to improve therapeutic success in HNSCC. To enable precision medicine of HNSCC, an understanding of the microenvironment that influences tumour growth and response to therapy is required alongside research tools that recapitulate the features of human tumours. In this review, we highlight the importance of the tumour microenvironment in HNSCC, with a focus on tumour hypoxia, and discuss the fidelity of patient-derived xenograft and organoids for modelling human HNSCC and response to therapy. We describe the benefits of patient-derived models over alternative preclinical models and their limitations in clinical relevance and how these impact their utility in precision medicine in HNSCC for the discovery of new therapeutic agents, as well as predictive biomarkers to identify patients' most likely to respond to therapy.

Hypoxia-selective radiosensitisation by SN38023, a bioreductive prodrug of DNA-dependent protein kinase inhibitor IC87361.

DNA-dependent protein kinase (DNA-PK) plays a key role in repair of radiation-induced DNA double strand breaks (DSB) by non-homologous end-joining. DNA-PK inhibitors (DNA-PKi) are therefore efficient radiosensitisers, but normal tissue radiosensitisation represents a risk for their use in radiation oncology. Here we describe a novel prodrug, SN38023, that is metabolised to a potent DNA-PKi (IC87361) selectively in radioresistant hypoxic cells. DNA-PK inhibitory potency of SN38023 was 24-fold lower than IC87361 in cell-free assays, consistent with molecular modelling studies suggesting that SN38023 is unable to occupy one of the predicted DNA-PK binding modes of IC87361. One-electron reduction of the prodrug by radiolysis of anoxic formate solutions, and by metabolic reduction in anoxic HCT116/POR cells that overexpress cytochrome P450 oxidoreductase (POR), generated IC87361 efficiently as assessed by LC-MS. SN38023 inhibited radiation-induced Ser2056 autophosphorylation of DNA-PK catalytic subunit and radiosensitised HCT116/POR and UT-SCC-54C cells selectively under anoxia. SN38023 was an effective radiosensitiser in anoxic HCT116 spheroids, demonstrating potential for penetration into hypoxic tumour tissue, but in spheroid co-cultures of high-POR and POR-null cells it showed no evidence of bystander effects resulting from local diffusion of IC87361. Pharmacokinetics of IC87361 and SN38023 at maximum achievable doses in NIH-III mice demonstrated sub-optimal exposure of UT-SCC-54C tumour xenografts and did not provide significant tumour radiosensitisation. In conclusion, SN38023 has potential for exploiting hypoxia for selective delivery of a potent DNA-PKi to the most radioresistant subpopulation of cells in tumours. However, prodrugs providing improved systemic pharmacokinetics and that release DNA-PKi that elicit bystander effects are needed to maximise therapeutic utility.

Radiosensitization of head and neck squamous cell carcinoma lines by DNA-PK inhibitors is more effective than PARP-1 inhibition and is enhanced by SLFN11 and hypoxia.

Background and purpose: Poly(ADP-ribose)polymerase-1 (PARP1) and DNA-dependent protein kinase (DNA-PK) play key roles in the repair of radiation-induced DNA double strand breaks, but it is unclear which is the preferred therapeutic target in radiotherapy. Here we compare small molecule inhibitors of both as radiosensitizers of head and neck squamous cell carcinoma (HNSCC) cell lines.Methods: Two PARP1 inhibitors (olaparib, veliparib) and two DNA-PK inhibitors (KU57788, IC87361) were tested in 14 HNSCC cell lines and two non-tumorigenic lines (HEK-293 and WI-38/Va-13), with drug exposure for 6 or 24 h post-irradiation, using regrowth assays. For three lines (UT-SCC-54C, -74B, -76B), radiosensitization was also assessed by clonogenic assay under oxia and acute (6 h) anoxia, and for 54C cells under chronic hypoxia (0.2% O2 for 48 h). Relationships between sensitizer enhancement ratios (SER) and gene expression, assessed by RNA sequencing, were evaluated.Results: The inhibitors were minimally cytotoxic in the absence of radiation, with 74B and 54C cells the most sensitive to both olaparib and KU57788. Median SER values for each inhibitor at 1.1 µM were 1.12 (range 1.02-1.24) for olaparib, 1.08 (1.04-1.13) for veliparib, 1.35 (1.10-1.64) for IC87361 and 1.77 (1.41-2.38) for KU57788. The higher SER values for the DNA-PK inhibitors were observed with all cell lines (except HEK-293) and all concentrations tested and were confirmed by clonogenic assay. Radiosensitization by the DNA-PK inhibitors correlated with expression of SLFN11 mRNA. Radiosensitization by IC87361 and olaparib was significantly enhanced under acute anoxia and chronic hypoxia.Conclusions: The DNA-PK inhibitors KU57788 and IC87361 are more effective radiosensitizers than the PARP-1 inhibitors olaparib and veliparib at non-cytotoxic concentrations in HNSCC cell cultures and their activity is enhanced by SLFN11 and hypoxia.

Studies Towards Hypoxia-Activated Prodrugs of PARP Inhibitors.

Poly(ADP-ribose)polymerase (PARP) inhibitors (PARPi) have recently been approved for the treatment of breast and ovarian tumors with defects in homologous recombination repair (HRR). Although it has been demonstrated that PARPi also sensitize HRR competent tumors to cytotoxic chemotherapies or radiotherapy, normal cell toxicity has remained an obstacle to their use in this context. Hypoxia-activated prodrugs (HAPs) provide a means to limit exposure of normal cells to active drug, thus adding a layer of tumor selectivity. We have investigated potential HAPs of model PARPi in which we attach a bioreducible "trigger" to the amide nitrogen, thereby blocking key binding interactions. A representative example showed promise in abrogating PARPi enzymatic activity in a biochemical assay, with a ca. 160-fold higher potency of benzyl phthalazinone 4 than the corresponding model HAP 5, but these N-alkylated compounds did not release the PARPi upon one-electron reduction by radiolysis. Therefore, we extended our investigation to include NU1025, a PARPi that contains a phenol distal to the core binding motif. The resulting 2-nitroimidazolyl ether provided modest abrogation of PARPi activity with a ca. seven-fold decrease in potency, but released the PARPi efficiently upon reduction. This investigation of potential prodrug approaches for PARPi has identified a useful prodrug strategy for future exploration.

Cellular pharmacology of evofosfamide (TH-302): A critical re-evaluation of its bystander effects.

Evofosfamide (TH-302) is a clinical-stage hypoxia-activated prodrug with proven efficacy against hypoxic cells in preclinical tumour models. TH-302 is designed to release the DNA crosslinking agent bromo-isophosphoramide mustard (Br-IPM) when reduced in hypoxic tissue. Br-IPM is considered to diffuse locally from hypoxic regions, eliciting additional tumour cell killing, but the latter 'bystander effect' has not been demonstrated directly. Previous studies with multicellular co-cultures that included cells expressing the E. coli nitroreductase NfsA as a model TH-302 reductase have provided clear evidence of a bystander effect (which we confirm in the present study). However, NfsA is an oxygen-insensitive two-electron reductase that is not expected to generate the nitro radical intermediate that has been demonstrated to fragment to release Br-IPM. Here, we use mass spectrometry methods to characterise TH-302 metabolites generated by one-electron reduction (steady-state radiolysis by ionising radiation and cellular metabolism under hypoxia, including HCT116 cells that overexpress P450 oxidoreductase, POR) or by NfsA expressed in HCT116 cells under oxic conditions, and investigate the stability and cytotoxicity of these products. Br-IPM is shown to have very low cytotoxic potency when added to extracellular culture medium and to be rapidly converted to other hydrophilic products including dichloro-isophosphoramide mustard (IPM). Only traces of Br-IPM or IPM were detected in the extracellular medium when generated by cellular metabolism of TH-302. We identify, in NfsA-expressing cells, the hydroxylamine metabolite of TH-302, and downstream products resulting from rearrangement or hydration of the imidazole ring, and demonstrate that formation of these candidate bystander effect mediators is suppressed by hypoxia. This characterisation of the cellular pharmacology of TH-302 implies that bystander effects from hypoxic activation of TH-302 are unlikely to contribute to its anticancer activity.