Hunter 2014 PhD Thesis

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Hunter FW (2014) Old target; new paradigm: Determinants of sensitivity to hypoxia-directed anticancer prodrugs. PhD Thesis:1-353.

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Hunter FW (2014) PhD Thesis

Abstract: Targeting tumour hypoxia is a long-standing goal in cancer drug development. An approach developed over the last three decades involves design of hypoxia-activated prodrugs (HAPs) of DNA replication fork poisons. These prodrugs are reductively activated in the absence of oxygen to release cytotoxic effectors, thus potentially eliminating this relatively radioresistant and chemoresistant subpopulation of tumour cells. However, experience with early HAPs has established that biomarkers for tumour hypoxia, expression of HAP reductases and drug sensitivity are urgently needed to stratify patients in clinical development. Thus this thesis aimed to discover and characterise genes that determine sensitivity to investigational HAPs in preclinical cancer models.

We examined benzotriazine di-N-oxide (BTO) HAPs tirapazamine and SN30000, nitroaromatic mustards PR-104 and TH-302, nitro-chloromethylbenzindolines (nitroCBI) SN29428 and SN30548 in addition to cytotoxic effectors and reference chemotherapeutic agents. Candidate determinants relating to specific aspects of HAP pharmacology (enzymatic activation, DNA repair) were modulated using gain- and loss-of-function genetic techniques in cell lines and xenografts. Unbiased screens for further sensitivity genes were performed in tumour cell lines by correlating Affymetrix microarray data with phenotypic measures and by interrogating genome-wide shRNA libraries.


O2k-Network Lab: NZ Auckland Hickey AJ


Labels: MiParea: Respiration, Pharmacology;toxicology  Pathology: Cancer 

Organism: Other mammals  Tissue;cell: CHO  Preparation: Intact cells 


Pathway: S, ROX  HRR: Oxygraph-2k, O2k-Fluorometer 

Amplex UltraRed 

Abstract continued

The DNA crosslinking HAPs PR-104 and TH-302 were highly cytotoxic to cells with dysfunctional homologous recombination (HR) repair (BRCA1, PALB2 knockdown, Rad51d, BRCA2 knockout). These prodrugs showed potent single-agent activity and were superior to platinum chemotherapy in a xenograft model of HR-deficient cancer, while BTO and nitroCBI HAPs were less active in this context. Transcriptomic analysis of 23 cancer cell lines identified iv FOXRED2 as a novel one-electron flavoreductase capable of activating multiple HAPs under hypoxia. High-throughput shRNA screening identified P450 (cytochrome) oxidoreductases (POR) to be the predominant determinant of sensitivity to SN30000 under hypoxia in multiple cell lines. POR suppression abrogated reductive activation of SN30000 and enhanced clonogenic survival.

Although all HAPs share the same fundamental design concept, this thesis reveals important differences in their molecular targets. Clinical evaluation of a DNA crosslinking HAP in HR-deficient malignancies is warranted. POR shows promise as companion diagnostic predictive of SN30000 sensitivity. High-throughput genomic screens can be leveraged to resolve variability observed in HAP sensitivity between tumours.

Correction

An Oroboros Oxygraph-2k was used in this thesis, whereas the Anton Paar/Oroboros Oxygraph was the first-generation instrument for high-resolution respirometry, which was replaced by the Oxygraph-2k in 2002.