Somatic variants of MAP3K3 are sufficient to cause cerebral and spinal cord cavernous malformations.

Cerebral cavernous malformations (CCMs) and spinal cord cavernous malformations (SCCMs) are common vascular abnormalities of the CNS that can lead to seizure, haemorrhage and other neurological deficits. Approximately 85% of patients present with sporadic (versus congenital) CCMs. Somatic mutations in MAP3K3 and PIK3CA were recently reported in patients with sporadic CCM, yet it remains unknown whether MAP3K3 mutation is sufficient to induce CCMs. Here we analysed whole-exome sequencing data for patients with CCM and found that ∼40% of them have a single, specific MAP3K3 mutation [c.1323C>G (p.Ile441Met)] but not any other known mutations in CCM-related genes. We developed a mouse model of CCM with MAP3K3I441M uniquely expressed in the endothelium of the CNS. We detected pathological phenotypes similar to those found in patients with MAP3K3I441M. The combination of in vivo imaging and genetic labelling revealed that CCMs were initiated with endothelial expansion followed by disruption of the blood-brain barrier. Experiments with our MAP3K3I441M mouse model demonstrated that CCM can be alleviated by treatment with rapamycin, the mTOR inhibitor. CCM pathogenesis has usually been attributed to acquisition of two or three distinct genetic mutations involving the genes CCM1/2/3 and/or PIK3CA. However, our results demonstrate that a single genetic hit is sufficient to cause CCMs.

MLN4924 suppresses the BRCA1 complex and synergizes with PARP inhibition in NSCLC cells.

Like ubiquitination, several studies have demonstrated that neddylation is implicated to be involved in the double strand break repair. BRCA1 is one of the key repair factors in the homologous recombination repair and may play a downstream role of the neddylation. BRCA1 is also a frequently mutated gene in cancers, which serve as the targets for PARP inhibitors. Here we further investigated the correlation between neddylation and BRCA1 complex using neddylation inhibitor MLN4924. MLN4924 efficiently inhibited the recruitment of components of BRCA1 complex to DNA damage sites. Thus MLN4924 may collaborate with PARP inhibitor to suppress tumor. Our results showed that combination MLN4924 and PARP inhibitor Olaparib impaired the DNA repair process in NSCLC cells. Furthermore, MLN4924 and Olaparib significantly inhibited the cancer cell growth. Kaplan-Meier survival analysis from lung cancer patients showed that high expression of NEDD8, BRCA1 and PARPs correlate with worse overall survival. Thus the combination of MLN4924 and PARP inhibitor may serve as a new strategy for NSCLC treatment.

The subcommissural organ regulates brain development via secreted peptides.

The subcommissural organ (SCO) is a gland located at the entrance of the aqueduct of Sylvius in the brain. It exists in species as distantly related as amphioxus and humans, but its function is largely unknown. To explore its function, we compared transcriptomes of SCO and non-SCO brain regions and found three genes, Sspo, Car3, and Spdef, that are highly expressed in the SCO. Mouse strains expressing Cre recombinase from endogenous promoter/enhancer elements of these genes were used to genetically ablate SCO cells during embryonic development, resulting in severe hydrocephalus and defects in neuronal migration and development of neuronal axons and dendrites. Unbiased peptidomic analysis revealed enrichment of three SCO-derived peptides, namely thymosin beta 4, thymosin beta 10, and NP24, and their reintroduction into SCO-ablated brain ventricles substantially rescued developmental defects. Together, these data identify a critical role for the SCO in brain development.

The subcommissural organ regulates brain development via secreted peptides.

The subcommissural organ (SCO) is a gland located at the entrance of the aqueduct of Sylvius in the brain. It exists in species as distantly related as amphioxus and humans, but its function is largely unknown. Here, to explore its function, we compared transcriptomes of SCO and non-SCO brain regions and found three genes, Sspo, Car3 and Spdef, that are highly expressed in the SCO. Mouse strains expressing Cre recombinase from endogenous promoter/enhancer elements of these genes were used to genetically ablate SCO cells during embryonic development, resulting in severe hydrocephalus and defects in neuronal migration and development of neuronal axons and dendrites. Unbiased peptidomic analysis revealed enrichment of three SCO-derived peptides, namely, thymosin beta 4, thymosin beta 10 and NP24, and their reintroduction into SCO-ablated brain ventricles substantially rescued developmental defects. Together, these data identify a critical role for the SCO in brain development.

HUWE1-dependent DNA-PKcs neddylation modulates its autophosphorylation in DNA damage response.

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is the core component of DNA-PK complex in the non-homologous end-joining (NHEJ) repair of DNA double-strand breaks, and its activity is strictly controlled by DNA-PKcs phosphorylation. The ubiquitin-like protein, NEDD8 is involved in regulation of DNA damage response, but it remains mysterious whether and how NEDD8-related neddylation affects DNA-PKcs and the NHEJ process. Here, we show that DNA-PKcs is poly-neddylated at its kinase domain. The neddylation E2-conjugating enzyme UBE2M and E3 ligase HUWE1 (HECT, UBA, and WWE domain containing E3 ubiquitin protein ligase 1) are responsible for the DNA-PKcs neddylation. Moreover, inhibition of HUWE1-dependent DNA-PKcs neddylation impairs DNA-PKcs autophosphorylation at Ser2056. Finally, depletion of HUWE1-dependent DNA-PKcs neddylation reduces the efficiency of NHEJ. These studies provide insights how neddylation modulates the activity of NHEJ core complex.

RBX1 prompts degradation of EXO1 to limit the homologous recombination pathway of DNA double-strand break repair in G1 phase.

End resection of DNA double-strand breaks (DSBs) to form 3' single-strand DNA (ssDNA) is critical to initiate the homologous recombination (HR) pathway of DSB repair. HR pathway is strictly limited in the G1-phase cells because of lack of homologous DNA as the templates. Exonuclease 1 (EXO1) is the key molecule responsible for 3' ssDNA formation of DSB end resection. We revealed that EXO1 is inactivated in G1-phase cells via ubiquitination-mediated degradation, resulting from an elevated expression level of RING-box protein 1 (RBX1) in G1 phase. The increased RBX1 significantly prompted the neddylation of Cullin1 and contributed to the G1 phase-specific degradation of EXO1. Knockdown of RBX1 remarkedly attenuated the degradation of EXO1 and increased the end resection and HR activity in γ-irradiated G1-phase cells, as demonstrated by the increased formation of RPA32, BrdU, and RAD51 foci. And EXO1 depletion mitigated DNA repair defects due to RBX1 reduction. Moreover, increased autophosphorylation of DNA-PKcs at S2056 was found to be responsible for the higher expression level of the RBX1 in the G1 phase. Inactivation of DNA-PKcs decreased RBX1 expression, and simultaneously increased EXO1 expression and DSB end resection in G1-phase cells. This study demonstrates a new mechanism for restraining the HR pathway of DNA DSB repair in G1 phase via RBX1-prompted inactivation of EXO1.

TIP60 K430 SUMOylation attenuates its interaction with DNA-PKcs in S-phase cells: Facilitating homologous recombination and emerging target for cancer therapy.

Nonhomologous end joining (NHEJ) and homologous recombination (HR) are major repair pathways of DNA double-strand breaks (DSBs). The pathway choice of HR and NHEJ is tightly regulated in cellular response to DNA damage. Here, we demonstrate that the interaction of TIP60 with DNA-PKcs is attenuated specifically in S phase, which facilitates HR pathway activation. SUMO2 modification of TIP60 K430 mediated by PISA4 E3 ligase blocks its interaction with DNA-PKcs, whereas TIP60 K430R mutation recovers its interaction with DNA-PKcs, which results in abnormally increased phosphorylation of DNA-PKcs S2056 in S phase and marked inhibition of HR efficiency, but barely affects NHEJ activity. TIP60 K430R mutant cancer cells are more sensitive to radiation and PARP inhibitors in cancer cell killing and tumor growth inhibition. Collectively, coordinated regulation of TIP60 and DNA-PKcs facilitates HR pathway choice in S-phase cells. TIP60 K430R mutant is a potential target of radiation and PARPi cancer therapy.