In tumor cells, DNA replication is constantly challenged by endogenous and exogenous sources, referred to as replication stress, and various pathways have evolved to mitigate this stress in cancer. We recently identified an extracellular matrix (ECM)-induced DNA repair pathway involving N-myc downstream regulated gene 1 (NDRG1). Matrix-induced signaling results in NDRG1-dependent protection from chemotherapy-induced replication stress. To uncover further mechanistic details of NDRG1-mediated effects on DNA replication, we identified transglutaminase 2 (TGM2) as a novel NDRG1-binding partner. TGM2 is an acyltransferase that catalyzes Ca(2+)-dependent protein modifications. This interaction was enriched upon chemotherapy-induced replication stress and also upon ECM-induced signaling. Our data show that TGM2 depletion significantly slows replication fork progression, and this phenotype is dependent on TGM2 catalytic activity and its nuclear localization. Our study further identifies a putative NDRG1–TGM2 binding site and shows that the physical interaction between NDRG1 and TGM2 is required for efficient DNA replication. Implications: This study reveals a previously unrecognized nuclear function for NDRG1 and TGM2 in regulating DNA replication fork stability and recovery, and uncovers a stress-responsive mechanism that supports replication homeostasis in cancer cells and advances our understanding of how extracellular signals are integrated with replication and repair pathways.

Background/Objectives: Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with limited treatment options. Patients are treated with DNA damaging chemotherapies which act by inducing DNA damage in rapidly dividing tumor cells. Unfortunately, these tumors frequently develop treatment resistance, underscoring the need to understand resistance mechanisms in order to develop better treatment strategies. DNA damage response (DDR) detects and repairs DNA damage, and the DDR pathway has been shown to contribute to chemoresistance. Another factor known to drive chemoresistance in PDAC is the dense stroma, composed of extracellular matrix proteins secreted by cancer-associated fibroblasts (CAFs). Our recent work identified a CAF-induced resistance mechanism involving N-myc downstream regulated gene 1 (NDRG1). CAF-induced signaling resulted in the phosphorylation of NDRG1 and NDRG1-dependent DNA repair and protection from chemotherapies. Loss of NDRG1 resulted in increased chemotherapy-induced DNA damage and decreased replication fork speed and recovery. Methods: To gain insight into the molecular mechanism of NDRG1-mediated DNA repair and replication, we performed a BioID screen to identify binding partners of NDRG1. We further assessed the mechanistic roles of the identified interaction partners on DNA repair using DNA replication and repair assays such as the Comet assay and DNA fiber assays. Results: Our BioID screen identified meiotic recombination 11 (MRE11) protein, a nuclease involved in DDR, as a putative NDRG1 interacting protein. Interaction between MRE11 and NDRG1 was enriched during the late S/early G2 cell cycle phases and under replication stress. However, this interaction is likely indirect as the interaction only occurred in a cellular context and not with in vitro purified proteins. Blocking NDRG1 phosphorylation or blocking MRE11 exonuclease activity both resulted in protection of newly synthesized DNA at stalled replication forks. In NDRG1 knockout cells, blocking MRE11 led to decreased protection of nascent DNA, suggesting that NDRG1 and MRE11 may be acting in the same pathway and that NDRG1 is required for MRE11’s activity at stalled forks. Conclusions: In summary, our work has uncovered a protein complex between NDRG1 and MRE11 that may play a key role in chemoresistance due to its role in the processing of stalled replication forks.

In tumor cells, DNA replication is constantly challenged by endogenous and exogenous sources, referred to as replication stress, and various pathways have evolved to mitigate this stress in cancer. We recently identified an extracellular matrix (ECM)-induced DNA repair pathway involving N-myc downstream regulated gene 1 (NDRG1). Matrix-induced signaling results in NDRG1-dependent protection from chemotherapy-induced replication stress. To uncover further mechanistic details of NDRG1-mediated effects on DNA replication, we identified transglutaminase 2 (TGM2) as a novel NDRG1-binding partner. TGM2 is an acyltransferase that catalyzes Ca(2+)-dependent protein modifications. This interaction was enriched upon chemotherapy-induced replication stress and also upon ECM-induced signaling. Our data show that TGM2 depletion significantly slows replication fork progression, and this phenotype is dependent on TGM2 catalytic activity and its nuclear localization. Our study further identifies a putative NDRG1–TGM2 binding site and shows that the physical interaction between NDRG1 and TGM2 is required for efficient DNA replication.Implications:This study reveals a previously unrecognized nuclear function for NDRG1 and TGM2 in regulating DNA replication fork stability and recovery, and uncovers a stress-responsive mechanism that supports replication homeostasis in cancer cells and advances our understanding of how extracellular signals are integrated with replication and repair pathways.

Supplementary Figure S4. Inhibition of TGM2 catalytic activity does not sensitize cells to fork stalling drugs.

Figure S2. Regulation of NDRG1-TGM2 interaction by SGK-1-mediated phosphorylation is cell type-specific.