New rationale for cancer drug discovery


29 Aug 2016

New rationale for cancer drug discovery

New rationale for cancer drug discovery involving cellular DNA repair machinery

Dr Benu Brata Das, Intermediate Fellow

Indian Association for the Cultivation of Science, Kolkata

“Synthetic lethality” or “Cancer specific cell killing” is the most exciting change in cancer treatment strategy since the invention of recent personalized chemotherapy. In this connection, Poly (ADP-ribose) polymerase (PARP) inhibitors have gained an immense interest in the clinical trial as a single agent for the treatment of breast and ovarian cancer due to mutations in the DNA repair genes like BRCA1 and BRCA2 or in combination with DNA topoisomerase 1 (Top1) inhibitors. FDA recently approved PARP inhibitors for ovarian cancer.

PARP is a family of 17 nuclear proteins involved in DNA repair and programmed cell death. One of the family members, PARP1, has been proposed to play a critical role in the early detection and repair of Top1cc-induced DNA breaks, a process known as PARylation. DNA topoisomerase 1, Top1, regulates DNA supercoiling both in the nucleus and mitochondria to enable faithful transmission of our genetic information to the offspring. However, Top1 is toxic when trapped on the DNA (Top1 cleavage complexes; Top1cc) in the presence of anticancer drug camptothecin (CPT) or endogenous DNA damage generated by reactive oxygen species (ROS). PARP1 catalyzes the addition of ADP-ribose polymers (PAR polymers) onto itself and Top1 and PARP inhibitors enhance the cytotoxicity of CPT in the clinical trials.  Until the current study, PARP inhibitors were assumed to kill cancer cells by inhibiting the repair of Top1-induced DNA lesions. However, the molecular mechanism by which PARylation regulates Top1 nuclear dynamics is not fully understood.

In our recently published study we describe a novel role for PAR polymers on Top1 nuclear dynamics, which is independent from Top1-PARP1 interaction but dependent on PARP catalytic activity. We examine the effects of PARP inhibitors and/or in combination with CPT on fluorescently tagged-human Top1 subnuclear dynamics in living cells. Using a combination of live cell microscopy and fluorescence recovery after photobleaching (FRAP) experiments, we establish that orally bioactive PARP inhibitors (Veliparib, ABT-888) efflux Top1 from the nucleolus to the nucleoplasm. FRAP data reveals that combination of ABT-888 with CPT markedly increased the bound/immobile fraction of Top1 (Top1cc) across the nuclear genome compared to CPT alone, which is associated with increased cytotoxicity in the proliferating cells exposed to the combination of PARP inhibitor with Top1 inhibitor. In an unprecedented finding, we conclusively demonstrate that mutant Top1 (Top1N722S) is restricted to the nucleolus due to its deficiency in accumulation of CPT-induced Top1-PARylation and Top1cc formation.

The present study provides new mechanistic insights into the action of PARP inhibitors in combination with Top1 inhibitors relevant for cancer chemotherapy. PARylation of Top1 counteracts CPT-induced stabilization of Top1cc. Therefore ABT-888 markedly increased CPT-induced trapping of Top1 across the nuclear genome, which is coupled with increased cytotoxicity in cancer cells.

Poly(ADP-ribose) polymers regulate DNA topoisomerase I (Top1) nuclear dynamics and camptothecin sensitivity in living cells.Das, S.K., Rehman, I., Ghosh, A., Sengupta, S., Majumder, P., Jana, B and Das BB*. Nucleic. Acids Res., 2016 July