Predictive biomarkers correlate with the impact of certain cancer treatments and thus, patient outcome. Accurate predictive biomarkers are difficult to obtain as many variables exist that may affect the validity of a marker, including but not limited to, downstream or upstream effectors of the biomarker, transcriptional regulators, and/or DNA modification.
The supercoiled nature of DNA requires topological modification during transcription, replication and DNA repair. (See, Gilbert, D C., et al., Cancer Research UK (2012) 106:18-24). These modifications are commonly carried out by DNA topoisomerases that that cleave the sugar-phosphate backbone of DNA without altering its chemical composition. Top1 is ubiquitously expressed and plays key roles in DNA replication, transcription, recombination and repair, primarily functioning to break a single strand of double-stranded, supercoiled DNA which enables the DNA to relax before it is re-ligated. (See, Wang, J C. J. Mol Biol (1971) 55:523-533). Top1 is overexpressed in many types of tumors making it the target of many anti-tumor, chemotherapeutic agents (See, Gilbert, D C., et al. (2012)).
The antitumor activity of camptothecin(s) (CPT), a known DNA topoisomerase 1-directed anti-cancer agent, is attributed to CPTs ability to bind TOP1 and inhibit the re-ligation step of the breakage/re-annealing reaction of DNA during the cell-cycle. CPTs covalently trap TOP1-DNA intermediates, resulting in accumulation of a reversible Top1-CPT-DNA covalent complex, often referred to as the cleavable complex (TOP1 ccs) (See, e.g., Zhang, H-F, et al., Cancer Research (2004) 64:1114-1121). The Top1 cleavable complex acts as a road block, colliding with moving replication forks or the RNA polymerase elongation complexes, thereby facilitating the conversion of single strand DNA breaks into the lethal DNA double stranded breaks (See, Chen, A Y., Annu. Rev. Pharmacol. Toxicol. (1994) 34:191-218). Following CPT treatment, Top1 is rapidly degraded by the ubiquitin-proteasome pathway, which is believed to be a critical mechanism to control the cellular response to the repair of CPT-induced DNA lesions and promote resistance to CPT cytotoxicity.
Ubiquitin-dependent proteolysis is carried out by the sequential action of three enzymes, the E1 ubiquitin-activating enzyme, the E2 ubiquitin-conjugating enzyme, and the E3 ubiquitin ligase among which the E3 ubiquitin ligase binds directly to the target protein and thus is exclusively responsible for conferring substrate specificity (See, Hershko, A., J. Biol. Chem. (1983) 258:8206-8214). The cullin family of proteins are the major components of the E3 ubiquitin ligase complex, generally functioning as scaffold proteins binding RING-finger proteins (See, Zhang, H-F, et al., (2004)). In mammalian cells there are two distinct Cullin 4 genes, CUL4A and CUL4B that share 82% sequence identity and overlapping functions. Distinct activities of the two CUL4s have also been reported, resulting from their differential expression during male meiosis and from their preferential subcellular distribution (predominantly cytoplasm for CUL4A and nuclear for CUL4B). CUL4B has a unique N-terminal extension that enables the protein's ability to bind unique adaptors (e.g. Aryl hydrocarbon receptor) and assemble a CUL4B-specific E3 ligase complex. To date, the degree of functional redundancy and difference between CUL4A and CUL4B, and the specific targets of CUL4A or CUL4B are not fully understood.