Tumour hypoxia renders cancer cells resistant to cancer therapy, resulting in markedly worse clinical outcomes. To find clinical candidate compounds that reduce hypoxia in tumours, we conduct a high-throughput screen for oxygen consumption rate (OCR) reduction and identify a number of drugs with this property. For this study we focus on the anti-malarial, atovaquone. Atovaquone rapidly decreases the OCR by more than 80% in a wide range of cancer cell lines at pharmacological concentrations. In addition, atovaquone eradicates hypoxia in FaDu, HCT116 and H1299 spheroids. Similarly, it reduces hypoxia in FaDu and HCT116 xenografts in nude mice, and causes a significant tumour growth delay when combined with radiation. Atovaquone is a ubiquinone analogue, and decreases the OCR by inhibiting mitochondrial complex III. We are now undertaking clinical studies to assess whether atovaquone reduces tumour hypoxia in patients, thereby increasing the efficacy of radiotherapy.

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Autophagy is a conserved constitutive cellular process, responsible for the degradation of dysfunctional proteins and organelles. Autophagy plays a role in many diseases such as neurodegeneration a...

Restoration of hypoxia-induced apoptosis in tumors harboring p53 mutations has been proposed as a potential therapeutic strategy; however, the transcriptional targets that mediate hypoxia-induced p53-dependent apoptosis remain elusive. Here, we demonstrated that hypoxia-induced p53-dependent apoptosis is reliant on the DNA-binding and transactivation domains of p53 but not on the acetylation sites K120 and K164, which, in contrast, are essential for DNA damage–induced, p53-dependent apoptosis. Evaluation of hypoxia-induced transcripts in multiple cell lines identified a group of genes that are hypoxia-inducible proapoptotic targets of p53, including inositol polyphosphate-5-phosphatase (INPP5D), pleckstrin domain–containing A3 (PHLDA3), sulfatase 2 (SULF2), B cell translocation gene 2 (BTG2), cytoplasmic FMR1-interacting protein 2 (CYFIP2), and KN motif and ankyrin repeat domains 3 (KANK3). These targets were also regulated by p53 in human cancers, including breast, brain, colorectal, kidney, bladder, and melanoma cancers. Downregulation of these hypoxia-inducible targets associated with poor prognosis, suggesting that hypoxia-induced apoptosis contributes to p53-mediated tumor suppression and treatment response. Induction of p53 targets, PHLDA3, and a specific INPP5D transcript mediated apoptosis in response to hypoxia through AKT inhibition. Moreover, pharmacological inhibition of AKT led to apoptosis in the hypoxic regions of p53-deficient tumors and consequently increased radiosensitivity. Together, these results identify mediators of hypoxia-induced p53-dependent apoptosis and suggest AKT inhibition may improve radiotherapy response in p53-deficient tumors.

ATM-mediated signaling in response to DNA damage is a barrier to tumorigenesis. Here we asked whether replication stress could also contribute to ATM signaling. We demonstrate that, in the absence of DNA damage, ATM responds to replication stress in a hypoxia-induced heterochromatin-like context. In certain hypoxic conditions, replication stress occurs in the absence of detectable DNA damage. Hypoxia also induces H3K9me3, a histone modification associated with gene repression and heterochromatin. Hypoxia-induced replication stress together with increased H3K9me3 leads to ATM activation. Importantly, ATM prevents the accumulation of DNA damage in hypoxia. Most significantly, we describe a stress-specific role for ATM in maintaining DNA replication rates in a background of increased H3K9me3. Furthermore, the ATM-mediated response to oncogene-induced replication stress is enhanced in hypoxic conditions. Together, these data indicate that hypoxia plays a critical role in the activation of the DNA damage response, therefore contributing to this barrier to tumorigenesis.

Hydrogen peroxide (H2O2) plays a vital role in normal cellular processes but at supraphysiological concentrations causes oxidative stress and cytotoxicity, a property that is potentially exploitable for the treatment of cancer in combination with radiotherapy (RT). We report the first Phase I trial testing the safety and tolerability of intratumoural H2O2 + external beam RT as a novel combination in patients with breast cancer, and exploratory plasma marker analyses investigating possible mechanisms of action. 12 patients with breast tumours ≥3 cm (surgically or medically inoperable) received intratumoural H2O2 with either 36 Gy in 6 twice-weekly fractions (n=6) or 49.5 Gy in 18 daily fractions (n=6) to the whole breast +/- loco-regional lymph nodes in a single-centre, non-randomised study. H2O2 was mixed in 1% sodium hyaluronate gel (final H2O2 concentration 0.5%) before administration to slow drug release and minimise local discomfort. The mixture was injected intratumourally under ultrasound guidance twice-weekly 1h prior to RT. The primary endpoint was patient-reported maximum intratumoural pain intensity before and 24h post-injection. Secondary endpoints included ≥grade 3 skin toxicity and tumour response by ultrasound. Blood samples were collected before, during and at the end of treatment for cell-death and immune marker analysis. Compliance with H2O2 and RT was 100%. 5/12 patients reported moderate pain following injection (grade 2 CTCAE v4.02) with median duration 60min (interquartile range 20-120min). Skin toxicity was comparable to RT alone, with maintained partial/complete tumour response relative to baseline in 11/12 patients at last follow-up (median 12 months). Blood marker analysis highlighted significant associations of TRAIL, IL-1β, IL-4 and MIP-1α with tumour response. Intratumoural H2O2 with RT is well-tolerated with no additional toxicity compared to RT alone. If efficacy is confirmed in a randomised Phase II trial, the approach has potential as a cost-effective radiation response enhancer in multiple cancer types where locoregional control after RT alone remains poor.