Dr Hannah Bolland

Platinum(IV) anticancer agents have demonstrated the potential to overcome the limitations associated with the widely used Pt(II) chemotherapeutics, cisplatin, carboplatin and oxaliplatin. In order to identify therapeutic scenarios where this type of chemotherapy can be applied, an improved understanding on the intracellular reduction of Pt(IV) complexes is needed. Here, we report the synthesis of two fluorescence responsive oxaliplatin(IV)(OxPt) complexes, OxaliRes and OxaliNap. Sodium ascorbate (NaAsc) was shown to reduce each OxPt(IV) complex resulting in increases in their respective fluorescence emission intensities at 585 nm and 545 nm. The incubation of each OxPt(IV) complex with a colorectal cancer cell line resulted in minimal changes to the respective fluorescence emission intensities. In contrast, the treatment of cells with NaAsc showed a dose-dependent increase in fluorescence emission intensity. With this knowledge in hand, we tested the reducing potential of tumor hypoxia, where an oxygen-dependent bioreduction was observed for each OxPt(IV) complex with <0.1 % O2 providing the greatest fluorescence signal. Clonogenic cell survival assays correlated with these observations demonstrating significant differences in toxicity between hypoxia (<0.1 % O2) and normoxia (21 % O2). To the best of our knowledge, this is the first report showing carbamate-functionalized OxPt(IV) complexes as potential hypoxia-activated prodrugs .   

Purpose: DNA polymerase theta (Polθ, encoded by the POLQ gene) is a DNA repair enzyme critical for microhomology mediated end joining (MMEJ). Polθ has limited expression in normal tissues but is frequently overexpressed in cancer cells and, therefore, represents an ideal target for tumor-specific radiosensitization. In this study we evaluate whether targeting Polθ with novel small-molecule inhibitors is a feasible strategy to improve the efficacy of radiotherapy.

Experimental design: We characterized the response to Polθ inhibition in combination with ionizing radiation in different cancer cell models in vitro and in vivo.

Results: Here, we show that ART558 and ART899, two novel and specific allosteric inhibitors of the Polθ DNA polymerase domain, potently radiosensitize tumor cells, particularly when combined with fractionated radiation. Importantly, noncancerous cells were not radiosensitized by Polθ inhibition. Mechanistically, we show that the radiosensitization caused by Polθ inhibition is most effective in replicating cells and is due to impaired DNA damage repair. We also show that radiosensitization is still effective under hypoxia, suggesting that these inhibitors may help overcome hypoxia-induced radioresistance. In addition, we describe for the first time ART899 and characterize it as a potent and specific Polθ inhibitor with improved metabolic stability. In vivo, the combination of Polθ inhibition using ART899 with fractionated radiation is well tolerated and results in a significant reduction in tumor growth compared with radiation alone.

Conclusions: These results pave the way for future clinical trials of Polθ inhibitors in combination with radiotherapy.

Hypoxia (low oxygen levels) occurs in a range of biological contexts, including plants, bacterial biofilms, and solid tumors; it elicits responses from these biological systems that impact their survival. For example, conditions of low oxygen make treating tumors more difficult and have a negative impact on patient prognosis. Therefore, chemical probes that enable the study of biological hypoxia are valuable tools to increase the understanding of disease-related conditions that involve low oxygen levels, ultimately leading to improved diagnosis and treatment. While small-molecule hypoxia-sensing probes exist, the majority of these image only very severe hypoxia (<1% O2) and therefore do not give a full picture of heterogeneous biological hypoxia. Commonly used antibody-based imaging tools for hypoxia are less convenient than small molecules, as secondary detection steps involving immunostaining are required. Here, we report the synthesis, electrochemical properties, photophysical analysis, and biological validation of a range of indolequinone-based bioreductive fluorescent probes. We show that these compounds image different levels of hypoxia in 2D and 3D cell cultures. The resorufin-based probe 2 was activated in conditions of 4% O2 and lower, while the Me-Tokyo Green-based probe 4 was only activated in severe hypoxia—0.5% O2 and less. Simultaneous application of these compounds in spheroids revealed that compound 2 images similar levels of hypoxia to pimonidazole, while compound 4 images more extreme hypoxia in a manner analogous to EF5. Compounds 2 and 4 are therefore useful tools to study hypoxia in a cellular setting and represent convenient alternatives to antibody-based imaging approaches.

Aryl boronate fluorescent probes allow the non-invasive study of dynamic cellular processes involving the reactive species, hydrogen peroxide (H2O2) and peroxynitrite (ONOO−). However, the ability of these probes to differentiate between these two species remains unclear. Here, we report a boronate-functionalised hemicyanine dye (HD-BPin) as a potential strategy to distinguish between H2O2 at 704 nm (red channel) and ONOO− at 460 nm (blue channel) in solution and in cells. This work also highlights the choice of fluorophore before boronate functionalization can dictate the observed selectivity between these two species.

Regions of low oxygen (hypoxia) are found in >50% of breast tumours, most frequently in the more aggressive triple negative breast cancer subtype (TNBC). Metastasis is the cause of 90% of breast cancer patient deaths. Regions of tumour hypoxia tend to be more acidic and both hypoxia and acidosis increase tumour metastasis. In line with this the metastatic process is dependent on pH regulatory mechanisms. We and others have previously identified increased hypoxic expression of Na+ driven bicarbonate transporters (NDBTs) as a major mechanism of tumour pH regulation. Hypoxia induced the expression of NDBTs in TNBC, most frequently SLC4A4 and SLC4A5. NDBT inhibition (S0859) and shRNA knockdown suppressed migration (40% reduction) and invasion (70% reduction) in vitro. Tumour xenograft metastasis in vivo was significantly reduced by NDBT knockdown. To investigate the mechanism by which NDBTs support metastasis, we investigated their role in regulation of phospho-signalling, epithelial-to-mesenchymal transition (EMT) and metabolism. NDBT knockdown resulted in an attenuation in hypoxic phospho-signalling activation; most notably LYN (Y397) reduced by 75%, and LCK (Y394) by 72%. The metastatic process is associated with EMT. We showed that NDBT knockdown inhibited EMT, modulating the expression of key EMT transcription factors and ablating the expression of vimentin whilst increasing the expression of E-cadherin. NDBT knockdown also altered metabolic activity reducing overall ATP and extracellular lactate levels. These results demonstrate that targeting hypoxia-induced NDBT can be used as an approach to modulate phospho-signalling, EMT, and metabolic activity and reduce tumour migration, invasion, and metastasis in vivo.

Hypoxia is a feature of most solid tumours and predicts for poor prognosis. In radiobiological hypoxia (<0.1% O2) cells become up to three times more resistant to radiation. The biological response to radiobiological hypoxia is one of few physiologically relevant stresses that activates both the unfolded protein and DNA damage responses (UPR and DDR). Links between these pathways have been identified in studies carried out in normoxia. Based in part on these previous studies and recent work from our laboratory, we hypothesised that the biological response to hypoxia likely includes overlap between the DDR and UPR. While inhibition of the DDR is a recognised strategy for improving radiation response, the possibility of achieving this through targeting the UPR has not been realised. We carried out a systematic review to identify links between the DDR and UPR, in human cell lines exposed to <2% O2. Following PRISMA guidance, literature from January 2010 to October 2020 were retrieved via Ovid MEDLINE and evaluated. A total of 202 studies were included. LAMP3, ULK1, TRIB3, CHOP, NOXA, NORAD, SIAH1/2, DYRK2, HIPK2, CREB, NUPR1, JMJD2B, NRF2, GSK-3B, GADD45a, GADD45b, STAU1, C-SRC, HK2, CAV1, CypB, CLU, IGFBP-3 and SP1 were highlighted as potential links between the hypoxic DDR and UPR. Overall, we identified very few studies which demonstrate a molecular link between the DDR and UPR in hypoxia, however, it is clear that many of the molecules highlighted warrant further investigation under radiobiological hypoxia as these may include novel therapeutic targets to improve radiotherapy response.

CREBBP (CBP/KAT3A) and its paralogue EP300 (KAT3B) are lysine acetyltransferases (KATs) that are essential for human development. They each comprise 10 domains through which they interact with >400 proteins, making them important transcriptional co-activators and key nodes in the human protein–protein interactome. The bromodomains of CREBBP and EP300 enable the binding of acetylated lysine residues from histones and a number of other important proteins, including p53, p73, E2F, and GATA1. Here, we report a work to develop a high-affinity, small-molecule ligand for the CREBBP and EP300 bromodomains [(−)-OXFBD05] that shows >100-fold selectivity over a representative member of the BET bromodomains, BRD4(1). Cellular studies using this ligand demonstrate that the inhibition of the CREBBP/EP300 bromodomain in HCT116 colon cancer cells results in lowered levels of c-Myc and a reduction in H3K18 and H3K27 acetylation. In hypoxia (<0.1% O2), the inhibition of the CREBBP/EP300 bromodomain results in the enhanced stabilization of HIF-1α.

Tumor hypoxia is associated with therapy resistance and poor patient prognosis. Hypoxia-activated prodrugs, designed to selectively target hypoxic cells while sparing normal tissue, represent a promising treatment strategy. We report the pre-clinical efficacy of 1-methyl-2-nitroimidazole panobinostat (NI-Pano, CH-03), a novel bioreductive version of the clinically used lysine deacetylase inhibitor, panobinostat. NI-Pano was stable in normoxic (21% O2) conditions and underwent NADPH-CYP-mediated enzymatic bioreduction to release panobinostat in hypoxia (<0.1% O2). Treatment of cells grown in both 2D and 3D with NI-Pano increased acetylation of histone H3 at lysine 9, induced apoptosis, and decreased clonogenic survival. Importantly, NI-Pano exhibited growth delay effects as a single agent in tumor xenografts. Pharmacokinetic analysis confirmed the presence of sub-micromolar concentrations of panobinostat in hypoxic mouse xenografts, but not in circulating plasma or kidneys. Together, our pre-clinical results provide a strong mechanistic rationale for the clinical development of NI-Pano for selective targeting of hypoxic tumors.

Inhibition of the ATR kinase has emerged as a therapeutically attractive means to target cancer since the development of potent inhibitors, which are now in clinical testing. We investigated a potential link between ATR inhibition and the autophagy process in esophageal cancer cells using four ATR inhibitors including two in clinical testing. The response to pharmacological ATR inhibitors was compared with genetic systems to investigate the ATR dependence of the effects observed. The ATR inhibitor, VX-970, was found to lead to an accumulation of p62 and LC3-II indicative of a blocked autophagy. This increase in p62 occurred post-transcriptionally and in all the cell lines tested. However, our data indicate that the accumulation of p62 occurred in an ATR-independent manner and was instead an off-target response to the ATR inhibitor. This study has important implications for the clinical response to pharmacological ATR inhibition, which in some cases includes the blockage of autophagy.