Three mechanisms have been proposed to define CPT drug resistance mechanisms: the lack of accumulation of the drug due to ABC transporters/multi-drug resistance (MDR) genes, mutations in topoI that alter the topoI-DNA-CPT interaction, and the rate of ubiquitin proteasomal pathway (UPP) dependent degradation of topoI. However, now it has been established that CPTs are not MDR substrates and topoI mutations are very rare in the patient population, making UPP-mediated degradation of topoI a key regulator of CPT response. One of the most remarkable phenomena observed in the cellular response to CPTs is the degradation of topoI. Importantly, the cells that degrade topoI rapidly are resistant to irinotecan. The seminal work carried out at Dr. Bharti’s lab defined the precise mechanism of topoI degradation by UPP and identified the molecular determinants of this pathway. Briefly, a decade of work has demonstrated that: i) topoI associates with DNA-PK, and DNA-PKcs phosphorylates topoI at Serine 10; ii) Phosphorylated topoI is ubiquitinated by BRCA1; iii) cells with higher basal levels of topoI-pS10 degrade topoI rapidly and are resistant to irinotecan; iv) the higher basal level of topoI-pS10 is maintained by phosphatases that deregulate kinase cascade and maintains steady state DNA-PKcs activation. The pathway has since been validated and published at several levels, including demonstration that CPT-resistant cells have a higher basal level of phosphorylated topoI-S10.
Fig.1. Schematic representation of deregulated kinase cascade effecting the rate of topoI degradation and visualization of topoI-pS10 in patient FFPE tissue