Elucidating the mechanistic role of NDRG1 in DNA repair, replication, and chemoresistance through characterization of novel protein-protein interactions

Abstract

Faithful DNA replication is critical for maintaining genomic stability but is constantly challenged by endogenous and exogenous replication stress, a hallmark of cancer. While many chemotherapies used to treat cancer induce replication stress, cancer cells frequently develop resistance, underscoring the need to understand mechanisms by which tumor cells overcome this stress and enhance their DNA repair efficiency. We have identified a novel ECM-induced resistance pathway involving N-myc downstream regulated gene 1 (NDRG1) that results in NDRG1-dependent protection from chemotherapy-induced replication stress in pancreatic cancer cells. To uncover the molecular basis of NDRG1-mediated DNA repair, we utilized a BioID proximity labeling screen and identified Transglutaminase 2 (TGM2) and Meiotic Recombination 11 (MRE11) as two novel NDRG1 binding partners. We characterized the physical protein-protein interactions and functional interactions of these complexes using biochemical, co-immunoprecipitation, and DNA fiber assays. We found that the NDRG1-TGM2 interaction is enriched upon hydroxyurea and gemcitabine-induced replication stress and is regulated by ECM-induced signaling, and serum- and glucocorticoid-induced kinase 1 (SGK1)-mediated NDRG1 phosphorylation. Importantly, we discovered that TGM2 modulates DNA replication fork homeostasis, and this function was dependent on both the nuclear localization and transamidase catalytic activity of TGM2. Furthermore, we identified a putative NDRG1-TGM2 binding site and demonstrated that this physical interaction contributes to efficient DNA replication fork progression and stalled fork recovery. This work establishes TGM2, an enzyme traditionally regarded to function in the cytoplasm and extracellular space, as a novel regulator of nuclear DNA replication. The NDRG1-MRE11 interaction was found to be enriched during late S/early G2 phases and upon treatment with fork stalling agents. The lack of binding between purified MRE11 and NDRG1 suggests the interaction is indirect, implying the existence of a regulatory scaffold or post translational modification necessary to mediate the interaction. Functionally, we demonstrated that NDRG1 and MRE11 cooperate to promote the degradation of nascent DNA at stalled forks. Taken together, our work identified novel mechanistic functions of NDRG1 in DNA replication and repair, through its interactions with TGM2 and MRE11, and provided fundamental new insights into the functions and interplay between these proteins. Furthermore, our work positions NDRG1 as a promising therapeutic target to suppress PDAC chemoresistance.