5-FU in combination with PARP inhibitor ABT-888 deregulates MGMT-dependent mismatch repair (MMR) pathway in MMR-proficient colorectal cancer stem cells by modulating MGMT/PARP1/MSH6 complex

BACKGROUND Previous study showed the PARP inhibitor ABT-888 potentiates the cytotoxicity of 5-fluorouracil (5-FU) by inhibiting PARP1-mediated mismatch repair (MMR) pathway via MSH6 deregulation in MMR-proficient colorectal cancer stem cells (CRC-CSCs). Emerging evidence indicates 5-FU regulates O6 -methylguanine-DNA-methyltransferase (MGMT) activity, but the mechanistic basis of MGMT involvement in PARP1-mediated MMR pathway following 5-FU treatment remains complex and poorly defined.RESEARCH DESIGN AND METHODS This study delineates the role of MGMT in 5-FU-induced MMR pathway activation and evaluates 5-FU+ABT-888 combination effects on MGMT modulation in CRC-CSCs. The molecular mechanism has been studied by using colocalization, western blot, co-immunoprecipitation, MGMT gene-knockdown, and molecular docking in in vitro, in silico, and ex vivo preclinical models.RESULTS 5-FU treatment induced PARylated-PARP1 in CRC-CSCs, promoting PARP1-MGMT-MSH6 interactions that activated MMR. ABT-888 inhibited PARylation in 5-FU-pre-exposed CSCs. Therefore, PARP1 could not physically interact with both MGMT and MSH6, and complete abolishment of MGMT and MSH6, and MMR protein downregulation were observed in combination treatment. MGMT silencing confirmed its critical role in PARP1-mediated MMR activation. Similar findings were obtained in in silico and ex vivo models.CONCLUSIONS 5-FU+ABT-888 enhanced CRC-CSCs death by inhibiting the PARP1-MGMT-MSH6 interaction and simultaneously inhibiting the MGMT-dependent PARP1-mediated MMR pathway in MMR-proficient CRC-CSCs.Schematic model illustrating 5-FU and ABT-888 combination treatment induces apoptosis by inhibiting the MGMT-dependent MMR pathway in CRC-CSCs. The diagram proposes a mechanistic link between PARP1, MGMT, and MMR signaling in regulating 5-FU response and PARP-inhibitor-mediated cytotoxicity in MMR-proficient CRC-CSCs. Left panel (Cancer survival): (1) 5-FU is incorporated into DNA, generating 5-FU-induced DNA adducts in MMR-proficient CRC-CSCs. (2) PARP1 is recruited to damaged DNA and (3) becomes activated, leading to PARylation. (4) MGMT acts as a PAR acceptor and binds activated PARP1 at sites of damage. (5) MSH6, together with MSH2 (MutSα), serves as an additional PAR acceptor and associates with PARylated PARP1 and MGMT, promoting recruitment of downstream MMR heterodimers. (6) The MutL complex (MLH1-PMS2) is subsequently recruited to the mismatch site, forming a multi-protein repair complex with PARP1, MGMT, MSH2, and MSH6. (7) This cascade enables efficient MMR processing and pathway activation, thereby sustaining survival of MMR-proficient CRC-CSCs. Right panel (Cancer reduction): (8) Upon treatment with the PARP inhibitor ABT-888, PARP1-PARylation is blocked. (9) Consequently, MGMT and MSH6 fail to interact with PARP1, preventing further recruitment of MMR components and leading to defective MGMT-dependent MMR signaling in 5-FU pre-exposed CRC-CSCs, ultimately triggering apoptosis and reducing the cancer stem cell population. Created in BioRender (https://biorender.com/i29644a).