Abstract
Poly(ADP-ribose)ylation (PARylation) by PAR polymerase 1 (PARP1) and PARylation removal by poly(ADP-ribose) glycohydrolase (PARG) critically regulate DNA damage responses; yet, conflicting reports obscure PARG biology and its impact on cancer cell resistance to PARP1 inhibitors. Here, we found that PARG expression is upregulated in many cancers. We employed chemical library screening to identify and optimize methylxanthine derivatives as selective bioavailable PARG inhibitors. Multiple crystal structures reveal how substituent positions on the methylxanthine core dictate binding modes and inducible-complementarity with a PARG-specific tyrosine clasp and arginine switch, supporting inhibitor specificity and a competitive inhibition mechanism. Cell-based assays show selective PARG inhibition and PARP1 hyperPARylation. Moreover, our PARG inhibitor sensitizes cells to radiation-induced DNA damage, suppresses replication fork progression and impedes cancer cell survival. In PARP inhibitor-resistant A172 glioblastoma cells, our PARG inhibitor shows comparable killing to Nedaplatin, providing further proof-of-concept that selectively inhibiting PARG can impair cancer cell survival.
Highlights
Poly(ADP-ribose)ylation (PARylation) by PAR polymerase 1 (PARP1) and PARylation removal by poly(ADP-ribose) glycohydrolase (PARG) critically regulate DNA damage responses; yet, conflicting reports obscure PARG biology and its impact on cancer cell resistance to PARP1 inhibitors
A strong TR-FRET signal was generated upon assembly of the Tb3+-chelated XRCC1 (FRET donor) with PARylated PARP1, which incorporates fluorescein-labeled ADPribose moieties into PAR chains (FRET acceptor)
As we found that inhibition of PARG increases PARP1 in a PARylated discrete HMW (DHMW) form, we tested whether our PARG inhibitors (PARGi) augments the level of PAR accumulation and of double-stranded DNA breaks in cells treated with ionizing radiation (IR)
Summary
Poly(ADP-ribose)ylation (PARylation) by PAR polymerase 1 (PARP1) and PARylation removal by poly(ADP-ribose) glycohydrolase (PARG) critically regulate DNA damage responses; yet, conflicting reports obscure PARG biology and its impact on cancer cell resistance to PARP1 inhibitors. Our PARG inhibitor sensitizes cells to radiation-induced DNA damage, suppresses replication fork progression and impedes cancer cell survival. Poly(ADP-ribose)ylation (PARylation) by PAR polymerase 1 (PARP1) and its reversal by PAR glycohydrolase (PARG) dictate multiple DNA damage responses[1,2,3]. PARG is recruited to DNA damage sites through interaction with PCNA and PARP126, prevents IR-induced mitotic catastrophe[13] and maintains replication fork stability in a BRCA2dependent manner[27]. PARG genetic knockdown sensitizes various cancer cells to chemotherapeutic agents and radiation[11,13,29,30] and may cause tumor-specific killing in BRCA2-deficient cancer cells[27]. PARG regulates proliferation and differentiation of dendritic cells and T cells via PARP/NF-κB in tumor metastases of colon carcinoma[4]
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