Pharmacological inhibition of BAP1 recruits HERC2 to competitively dissociate BRCA1-BARD1, suppresses DNA repair and sensitizes CRC to radiotherapy

Radiotherapy is widely used in the management of advanced colorectal cancer (CRC). However, the clinical efficacy is limited by the safe irradiated dose. Sensitizing tumor cells to radiotherapy via interrupting DNA repair is a promising approach to conquering the limitation. The BRCA1-BARD1 complex has been demonstrated to play a critical role in homologous recombination (HR) DSB repair, and its functions may be affected by HERC2 or BAP1. Accumulated evidence illustrates that the ubiquitination-deubiquitination balance is involved in these processes; however, the precise mechanism for the cross-talk among these proteins in HR repair following radiation hasn't been defined. Through activity-based profiling, we identified PT33 as an active entity for HR repair suppression. Subsequently, we revealed that BAP1 serves as a novel molecular target of PT33 via a CRISPR-based deubiquitinase screen. Mechanistically, pharmacological covalent inhibition of BAP1 with PT33 recruits HERC2 to compete with BARD1 for BRCA1 interaction, interrupting HR repair. Consequently, PT33 treatment can substantially enhance the sensitivity of CRC cells to radiotherapy in vitro and in vivo. Overall, these findings provide a mechanistic basis for PT33-induced HR suppression and may guide an effective strategy to improve therapeutic gain.

NAD+ supplementation reverses the oxidative stress induced PARP1 signalling in D. Discoideum

Increased oxidative stress leads to cell death by inducing DNA damage, PARP activation and energy depletion in age related disorders which are a growing concern due to increased life expectancy. Indeed, cellular NAD+ levels, depletion of which is one of the consequences of overactive PARP, also decline with age. We previously showed rescue in oxidative stress induced paraptotic and necrotic cell death by PARP1 inhibition in D. discoideum. Inhibition of PARP1 activity prevented cellular depletion of its substrate NAD+. To understand the significance of NAD+ depletion in PARP1 mediated oxidative stress induced cell death, exogenous addition of NAD+ was done. Addition of NAD+ prevented PARP1 mediated oxidative stress induced cell death at low doses upto 10 mM NAD+, nevertheless led to an anticipated increase in PARP1 activity. NAD+ significantly prevented oxidative stress induced cell death in D. discoideum. Exogenous NAD+ averted depletion of cellular NAD+ and mitochondrial membrane potential changes that were triggered by oxidative stress, without getting affected by the elevated ROS levels. Altogether, this study ascertains that NAD+ replenishment overcomes cadmium or H2O2 induced cell death by preventing cellular energy collapse incited by PARP1 activation. Thus, our results explicitly demonstrate that PARP1 overactivation led NAD+ depletion but not PARP1 activity per se is of consequential significance in causing oxidative stress induced D. discoideum cell death. Moreover, NAD+ supplementation could be a beneficial approach in aging and age-related disorders mediated by PARP1