NEK2 inhibition triggers anti-pancreatic cancer immunity by targeting PD-L1.

Despite the substantial impact of post-translational modifications on programmed cell death 1 ligand 1 (PD-L1), its importance in therapeutic resistance in pancreatic cancer remains poorly defined. Here, we demonstrate that never in mitosis gene A-related kinase 2 (NEK2) phosphorylates PD-L1 to maintain its stability, causing PD-L1-targeted pancreatic cancer immunotherapy to have poor efficacy. We identify NEK2 as a prognostic factor in immunologically "hot" pancreatic cancer, involved in the onset and development of pancreatic tumors in an immune-dependent manner. NEK2 deficiency results in the suppression of PD-L1 expression and enhancement of lymphocyte infiltration. A NEK binding motif (F/LXXS/T) is identified in the glycosylation-rich region of PD-L1. NEK2 interacts with PD-L1, phosphorylating the T194/T210 residues and preventing ubiquitin-proteasome pathway-mediated degradation of PD-L1 in ER lumen. NEK2 inhibition thereby sensitizes PD-L1 blockade, synergically enhancing the anti-pancreatic cancer immune response. Together, the present study proposes a promising strategy for improving the effectiveness of pancreatic cancer immunotherapy.

NEK9 regulates primary cilia formation by acting as a selective autophagy adaptor for MYH9/myosin IIA.

Autophagy regulates primary cilia formation, but the underlying mechanism is not fully understood. In this study, we identify NIMA-related kinase 9 (NEK9) as a GABARAPs-interacting protein and find that NEK9 and its LC3-interacting region (LIR) are required for primary cilia formation. Mutation in the LIR of NEK9 in mice also impairs in vivo cilia formation in the kidneys. Mechanistically, NEK9 interacts with MYH9 (also known as myosin IIA), which has been implicated in inhibiting ciliogenesis through stabilization of the actin network. MYH9 accumulates in NEK9 LIR mutant cells and mice, and depletion of MYH9 restores ciliogenesis in NEK9 LIR mutant cells. These results suggest that NEK9 regulates ciliogenesis by acting as an autophagy adaptor for MYH9. Given that the LIR in NEK9 is conserved only in land vertebrates, the acquisition of the autophagic regulation of the NEK9-MYH9 axis in ciliogenesis may have possible adaptive implications for terrestrial life.

NEK10 tyrosine phosphorylates p53 and controls its transcriptional activity.

In response to genotoxic stress, multiple kinase signaling cascades are activated, many of them directed towards the tumor suppressor p53, which coordinates the DNA damage response (DDR). Defects in DDR pathways lead to an accumulation of mutations that can promote tumorigenesis. Emerging evidence implicates multiple members of the NimA-related kinase (NEK) family (NEK1, NEK10, and NEK11) in the DDR. Here, we describe a function for NEK10 in the regulation of p53 transcriptional activity through tyrosine phosphorylation. NEK10 loss increases cellular proliferation by modulating the p53-dependent transcriptional output. NEK10 directly phosphorylates p53 on Y327, revealing NEK10's unexpected substrate specificity. A p53 mutant at this site (Y327F) acts as a hypomorph, causing an attenuated p53-mediated transcriptional response. Consistently, NEK10-deficient cells display heightened sensitivity to DNA-damaging agents. Further, a combinatorial score of NEK10 and TP53-target gene expression is an independent predictor of a favorable outcome in breast cancers.

Structural mechanism for NEK7-licensed activation of NLRP3 inflammasome.

The NLRP3 inflammasome can be activated by stimuli that include nigericin, uric acid crystals, amyloid-ß fibrils and extracellular ATP. The mitotic kinase NEK7 licenses the assembly and activation of the NLRP3 inflammasome in interphase. Here we report a cryo-electron microscopy structure of inactive human NLRP3 in complex with NEK7, at a resolution of 3.8 Å. The earring-shaped NLRP3 consists of curved leucine-rich-repeat and globular NACHT domains, and the C-terminal lobe of NEK7 nestles against both NLRP3 domains. Structural recognition between NLRP3 and NEK7 is confirmed by mutagenesis both in vitro and in cells. Modelling of an active NLRP3-NEK7 conformation based on the NLRC4 inflammasome predicts an additional contact between an NLRP3-bound NEK7 and a neighbouring NLRP3. Mutations to this interface abolish the ability of NEK7 or NLRP3 to rescue NLRP3 activation in NEK7-knockout or NLRP3-knockout cells. These data suggest that NEK7 bridges adjacent NLRP3 subunits with bipartite interactions to mediate the activation of the NLRP3 inflammasome.

NEK7 regulates dendrite morphogenesis in neurons via Eg5-dependent microtubule stabilization.

Organization of microtubules into ordered arrays is best understood in mitotic systems, but remains poorly characterized in postmitotic cells such as neurons. By analyzing the cycling cell microtubule cytoskeleton proteome through expression profiling and targeted RNAi screening for candidates with roles in neurons, we have identified the mitotic kinase NEK7. We show that NEK7 regulates dendrite morphogenesis in vitro and in vivo. NEK7 kinase activity is required for dendrite growth and branching, as well as spine formation and morphology. NEK7 regulates these processes in part through phosphorylation of the kinesin Eg5/KIF11, promoting its accumulation on microtubules in distal dendrites. Here, Eg5 limits retrograde microtubule polymerization, which is inhibitory to dendrite growth and branching. Eg5 exerts this effect through microtubule stabilization, independent of its motor activity. This work establishes NEK7 as a general regulator of the microtubule cytoskeleton, controlling essential processes in both mitotic cells and postmitotic neurons.

NEK8 regulates DNA damage-induced RAD51 foci formation and replication fork protection.

Proteins essential for homologous recombination play a pivotal role in the repair of DNA double strand breaks, DNA inter-strand crosslinks and replication fork stability. Defects in homologous recombination also play a critical role in the development of cancer and the sensitivity of these cancers to chemotherapy. RAD51, an essential factor for homologous recombination and replication fork protection, accumulates and forms immunocytochemically detectable nuclear foci at sites of DNA damage. To identify kinases that may regulate RAD51 localization to sites of DNA damage, we performed a human kinome siRNA library screen, using DNA damage-induced RAD51 foci formation as readout. We found that NEK8, a NIMA family kinase member, is required for efficient DNA damage-induced RAD51 foci formation. Interestingly, knockout of Nek8 in murine embryonic fibroblasts led to cellular sensitivity to the replication inhibitor, hydroxyurea, and inhibition of the ATR kinase. Furthermore, NEK8 was required for proper replication fork protection following replication stall with hydroxyurea. Loading of RAD51 to chromatin was decreased in NEK8-depleted cells and Nek8-knockout cells. Single-molecule DNA fiber analyses revealed that nascent DNA tracts were degraded in the absence of NEK8 following treatment with hydroxyurea. Consistent with this, Nek8-knockout cells showed increased chromosome breaks following treatment with hydroxyurea. Thus, NEK8 plays a critical role in replication fork stability through its regulation of the DNA repair and replication fork protection protein RAD51.