Impaired binding affinity of YTHDC1 with METTL3/METTL14 results in R-loop accumulation in myelodysplastic neoplasms with DDX41 mutation.

DEAD box helicase 41 (DDX41) mutations are the most prevalent predisposition to familial myelodysplastic syndrome (MDS). However, the precise roles of these variants in the pathogenesis of MDS have yet to be elucidated. Here, we discovered a novel mechanism by which DDX41 contributes to R-loop-induced DNA damage responses (DDR) in cooperation with the m6A-METTL complex (MAC) and YTHDC1 using DDX41 knockout (KO) and DDX41 knock-in (KI, R525H, Y259C) cell lines as well as primary samples from MDS patients. Compared to wild type (WT), DDX41 KO and KI led to increased levels of m6A RNA methylated R-loop. Interestingly, we found that DDX41 regulates m6A/R-loop levels by interacting with MAC components. Further, DDX41 promoted the recruitment of YTHDC1 to R-loops by promoting the binding between METTL3 and YTHDC1, which was dysregulated in DDX41-deficient cells, contributing to genomic instability. Collectively, we demonstrated that DDX41 plays a key role in the physiological control of R-loops in cooperation with MAC and YTHDC1. These findings provide novel insights into how defects in DDX41 influence MDS pathogenesis and suggest potential therapeutic targets for the treatment of MDS.

ZNF212 promotes genomic integrity through direct interaction with TRAIP.

TRAIP is a key factor involved in the DNA damage response (DDR), homologous recombination (HR) and DNA interstrand crosslink (ICL) repair. However, the exact functions of TRAIP in these processes in mammalian cells are not fully understood. Here we identify the zinc finger protein 212, ZNF212, as a novel binding partner for TRAIP and find that ZNF212 colocalizes with sites of DNA damage. The recruitment of TRAIP or ZNF212 to sites of DNA damage is mutually interdependent. We show that depletion of ZNF212 causes defects in the DDR and HR-mediated repair in a manner epistatic to TRAIP. In addition, an epistatic analysis of Zfp212, the mouse homolog of human ZNF212, in mouse embryonic stem cells (mESCs), shows that it appears to act upstream of both the Neil3 and Fanconi anemia (FA) pathways of ICLs repair. We find that human ZNF212 interacted directly with NEIL3 and promotes its recruitment to ICL lesions. Collectively, our findings identify ZNF212 as a new factor involved in the DDR, HR-mediated repair and ICL repair though direct interaction with TRAIP.

PWWP2B promotes DNA end resection and homologous recombination.

Genome instability is one of the leading causes of gastric cancers. However, the mutational landscape of driver genes in gastric cancer is poorly understood. Here, we investigate somatic mutations in 25 Korean gastric adenocarcinoma patients using whole-exome sequencing and show that PWWP2B is one of the most frequently mutated genes. PWWP2B mutation correlates with lower cancer patient survival. We find that PWWP2B has a role in DNA double-strand break repair. As a nuclear protein, PWWP2B moves to sites of DNA damage through its interaction with UHRF1. Depletion of PWWP2B enhances cellular sensitivity to ionizing radiation (IR) and impairs IR-induced foci formation of RAD51. PWWP2B interacts with MRE11 and participates in homologous recombination via promoting DNA end-resection. Taken together, our data show that PWWP2B facilitates the recruitment of DNA repair machinery to sites of DNA damage and promotes HR-mediated DNA double-strand break repair. Impaired PWWP2B function might thus cause genome instability and promote gastric cancer development.

Reciprocal interactions among Cobll1, PACSIN2, and SH3BP1 regulate drug resistance in chronic myeloid leukemia.

Cobll1 affects blast crisis (BC) progression and tyrosine kinase inhibitor (TKI) resistance in chronic myeloid leukemia (CML). PACSIN2, a novel Cobll1 binding protein, activates TKI-induced apoptosis in K562 cells, and this activation is suppressed by Cobll1 through the interaction between PACSIN2 and Cobll1. PACSIN2 also binds and inhibits SH3BP1 which activates the downstream Rac1 pathway and induces TKI resistance. PACSIN2 competitively interacts with Cobll1 or SH3BP1 with a higher affinity for Cobll1. Cobll1 preferentially binds to PACSIN2, releasing SH3BP1 to promote the SH3BP1/Rac1 pathway and suppress TKI-mediated apoptosis and eventually leading to TKI resistance. Similar interactions among Cobll1, PACSIN2, and SH3BP1 control hematopoiesis during vertebrate embryogenesis. Clinical analysis showed that most patients with CML have Cobll1 and SH3BP1 expression at the BC phase and BC patients with Cobll1 and SH3BP1 expression showed severe progression with a higher blast percentage than those without any Cobll1, PACSIN2, or SH3BP1 expression. Our study details the molecular mechanism of the Cobll1/PACSIN2/SH3BP1 pathway in regulating drug resistance and BC progression in CML.

NSMF promotes the replication stress-induced DNA damage response for genome maintenance.

Proper activation of DNA repair pathways in response to DNA replication stress is critical for maintaining genomic integrity. Due to the complex nature of the replication fork (RF), problems at the RF require multiple proteins, some of which remain unidentified, for resolution. In this study, we identified the N-methyl-D-aspartate receptor synaptonuclear signaling and neuronal migration factor (NSMF) as a key replication stress response factor that is important for ataxia telangiectasia and Rad3-related protein (ATR) activation. NSMF localizes rapidly to stalled RFs and acts as a scaffold to modulate replication protein A (RPA) complex formation with cell division cycle 5-like (CDC5L) and ATR/ATR-interacting protein (ATRIP). Depletion of NSMF compromised phosphorylation and ubiquitination of RPA2 and the ATR signaling cascade, resulting in genomic instability at RFs under DNA replication stress. Consistently, NSMF knockout mice exhibited increased genomic instability and hypersensitivity to genotoxic stress. NSMF deficiency in human and mouse cells also caused increased chromosomal instability. Collectively, these findings demonstrate that NSMF regulates the ATR pathway and the replication stress response network for genome maintenance and cell survival.