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The transcription factor p53 (encoded by TP53 ) plays diverse roles in human development and disease. While best known for its role in tumor suppression, p53 signaling also influences mammalian development by triggering cell fate decisions in response to a wide variety of stresses. After over 4 decades of study, a new pathway that triggers p53 activation in response to mitotic delays was recently identified. Termed the mitotic surveillance or mitotic stopwatch pathway, the USP28 and 53BP1 proteins activate p53 in response to delayed mitotic progression to control cell fate and promote genomic stability. In this Minireview, I discuss its identification, potential roles in neurodevelopmental disorders and cancer, as well as explore outstanding questions about its function, regulation and potential use as a biomarker for anti-mitotic therapies.

Centrosomes consist of two centrioles embedded in pericentriolar material and function as the main microtubule organising centres in dividing animal cells. They ensure proper formation and orientation of the mitotic spindle and are therefore essential for the maintenance of genome stability. Centrosome function is crucial during embryonic development, highlighted by the discovery of mutations in genes encoding centrosome or spindle pole proteins that cause autosomal recessive primary microcephaly, including Cep63 and Cep152. In this study we show that Cep63 functions to ensure that centriole duplication occurs reliably in dividing mammalian cells. We show that the interaction between Cep63 and Cep152 can occur independently of centrosome localisation and that the two proteins are dependent on one another for centrosomal localisation. Further, both mouse and human Cep63 and Cep152 cooperate to ensure efficient centriole duplication by promoting the accumulation of essential centriole duplication factors upstream of SAS-6 recruitment and procentriole formation. These observations describe the requirement for Cep63 in maintaining centriole number in dividing mammalian cells and further establish the order of events in centriole formation.

Analysis of cancer mutagenic signatures provides information about the origin of mutations and can inform the use of clinical therapies, including immunotherapy. In particular, APOBEC3A (A3A) has emerged as a major driver of mutagenesis in cancer cells, and its expression results in DNA damage and susceptibility to treatment with inhibitors of the ATR and CHK1 checkpoint kinases. Here, we report the implementation of CRISPR/Cas-9 genetic screening to identify susceptibilities of multiple A3A-expressing lung adenocarcinoma (LUAD) cell lines. We identify HMCES, a protein recently linked to the protection of abasic sites, as a central protein for the tolerance of A3A expression. HMCES depletion results in synthetic lethality with A3A expression preferentially in a TP53-mutant background. Analysis of previous screening data reveals a strong association between A3A mutational signatures and sensitivity to HMCES loss and indicates that HMCES is specialized in protecting against a narrow spectrum of DNA damaging agents in addition to A3A. We experimentally show that both HMCES disruption and A3A expression increase susceptibility of cancer cells to ionizing radiation (IR), oxidative stress, and ATR inhibition, strategies that are often applied in tumor therapies. Overall, our results suggest that HMCES is an attractive target for selective treatment of A3A-expressing tumors.

Adenylosuccinate lyase (ADSL) functions in de novo purine synthesis (DNPS) and the purine nucleotide cycle. ADSL deficiency (ADSLD) causes numerous neurodevelopmental pathologies, including microcephaly and autism spectrum disorder. ADSLD patients have normal serum purine nucleotide levels but exhibit accumulation of dephosphorylated ADSL substrates, S-Ado, and SAICAr, the latter being implicated in neurotoxic effects through unknown mechanisms. We examined the phenotypic effects of ADSL depletion in human cells and their relation to phenotypic outcomes. Using specific interventions to compensate for reduced purine levels or modulate SAICAr accumulation, we found that diminished AMP levels resulted in increased DNA damage signaling and cell cycle delays, while primary ciliogenesis was impaired specifically by loss of ADSL or administration of SAICAr. ADSL-deficient chicken and zebrafish embryos displayed impaired neurogenesis and microcephaly. Neuroprogenitor attrition in zebrafish embryos was rescued by pharmacological inhibition of DNPS, but not increased nucleotide concentration. Zebrafish also displayed phenotypes commonly linked to ciliopathies. Our results suggest that both reduced purine levels and impaired DNPS contribute to neurodevelopmental pathology in ADSLD and that defective ciliogenesis may influence the ADSLD phenotypic spectrum.

The International University of Andalucía (UNIA) Current Trends in Biomedicine Workshop on Molecular Causes of Primary Microcephaly and Related Diseases took place in Baeza, Spain, November 18–20, 2019. This meeting brought together scientists from Europe, the USA and China to discuss recent advances in our molecular and genetic understanding of a group of rare neurodevelopmental diseases characterised by primary microcephaly, a condition in which head circumference is smaller than normal at birth. Microcephaly can be caused by inherited mutations that affect key cellular processes, or environmental exposure to radiation or other toxins. It can also result from viral infection, as exemplified by the recent Zika virus outbreak in South America. Here we summarise a number of the scientific advances presented and topics discussed at the meeting.

Ataxia-telangiectasia mutated (ATM) protein kinase regulates the DNA damage response (DDR) and is associated with cancer suppression. Here we report a cancer-promoting role for ATM. ATM depletion in metastatic cancer cells reduced cell migration and invasion. Transcription analyses identified a gene network, including the chemokine IL-8, regulated by ATM. IL-8 expression required ATM and was regulated by oxidative stress. IL-8 was validated as an ATM target by its ability to rescue cell migration and invasion defects in ATM-depleted cells. Finally, ATM-depletion in human breast cancer cells reduced lung tumors in a mouse xenograft model and clinical data validated IL-8 in lung metastasis. These findings provide insights into how ATM activation by oxidative stress regulates IL-8 to sustain cell migration and invasion in cancer cells to promote metastatic potential. Thus, in addition to well-established roles in tumor suppression, these findings identify a role for ATM in tumor progression. Damaged DNA threatens the normal activity of living cells, so cells use a number of mechanisms to ensure that this damage is repaired. When DNA is damaged, an enzyme called ATM activates several other proteins that ultimately lead to the DNA being repaired. Problems with detecting and repairing damaged DNA have been linked to cancer. Thus, these pathways, including the activity of ATM, were previously thought to only be involved in cancer suppression. Now, Chen et al. report a new cancer-promoting role for ATM. The experiments reveal that reducing the amount of ATM in cancer cells actually made them less able to migrate and less invasive. Likewise, human breast cancer cells in which the levels of ATM had been depleted formed fewer lung tumors than normal breast cancer cells when they were transplated into mice. Oxidative stress—a build-up of harmful chemicals inside cells—is a signature feature of cancer cells and is known to be another signal that activates ATM. Chen et al. found that activating ATM through oxidative stress, but not by DNA damage, encouraged the cancer cells to migrate and become invasive. Further analysis of cellular responses following ATM activation by oxidative stress revealed that this enzyme regulates the production of a small protein called interleukin-8. This protein is an important pro-inflammatory molecule that has been implicated in cancer, in particular, in helping cancer cells to migrate to other tissues. When interleukin-8 was added to ATM-depleted cancer cells, it rescued their defects in spreading and invasiveness, thereby providing strong evidence that interleukin-8 is a biologically important target of ATM. Clinical data confirmed that breast cancer cells that had also spread to the patient's lungs often produced high levels of interleukin-8. Together, these findings suggest that ATM could be a potential target for anti-cancer therapies, as inhibiting this enzyme would inhibit interleukin-8, and in turn slow the progression and spread of cancer.

Metabolomics experiments identify metabolites whose abundance varies as the conditions under study change. Pathway enrichment tools help in the identification of key metabolic processes and in building a plausible biological explanation for these variations. Although several methods are available for pathway enrichment using experimental evidence, metabolomics does not yet have a comprehensive overview in a network layout at multiple molecular levels. We propose a novel pathway enrichment procedure for analysing summary metabolomics data based on sub-network analysis in a graph representation of a reference database. Relevant entries are extracted from the database according to statistical measures over a null diffusive process that accounts for network topology and pathway crosstalk. Entries are reported as a sub-pathway network, including not only pathways, but also modules, enzymes, reactions and possibly other compound candidates for further analyses. This provides a richer biological context, suitable for generating new study hypotheses and potential enzymatic targets. Using this method, we report results from cells depleted for an uncharacterised mitochondrial gene using GC and LC-MS data and employing KEGG as a knowledge base. Partial validation is provided with NMR-based tracking of 13C glucose labelling of these cells.

Multiciliated cells (MCCs) in the brain reside in the ependyma and the choroid plexus (CP) epithelia. The CP secretes cerebrospinal fluid that circulates within the ventricular system, driven by ependymal cilia movement. Tumors of the CP are rare primary brain neoplasms mostly found in children. CP tumors exist in three forms: CP papilloma (CPP), atypical CPP, and CP carcinoma (CPC). Though CPP and atypical CPP are generally benign and can be resolved by surgery, CPC is a particularly aggressive and little understood cancer with a poor survival rate and a tendency for recurrence and metastasis. In contrast to MCCs in the CP epithelia, CPCs in humans are characterized by solitary cilia, frequent TP53 mutations, and disturbances to multiciliogenesis program directed by the GMNC-MCIDAS transcriptional network. GMNC and MCIDAS are early transcriptional regulators of MCC fate differentiation in diverse tissues. Consistently, components of the GMNC-MCIDAS transcriptional program are expressed during CP development and required for multiciliation in the CP, while CPC driven by deletion of Trp53 and Rb1 in mice exhibits multiciliation defects consequent to deficiencies in the GMNC-MCIDAS program. Previous studies revealed that abnormal NOTCH pathway activation leads to CPP. Here we show that combined defects in NOTCH and Sonic Hedgehog signaling in mice generates tumors that are similar to CPC in humans. NOTCH-driven CP tumors are monociliated, and disruption of the NOTCH complex restores multiciliation and decreases tumor growth. NOTCH suppresses multiciliation in tumor cells by inhibiting the expression of GMNC and MCIDAS, while Gmnc-Mcidas overexpression rescues multiciliation defects and suppresses tumor cell proliferation. Taken together, these findings indicate that reactivation of the GMNC-MCIDAS multiciliogenesis program is critical for inhibiting tumorigenesis in the CP, and it may have therapeutic implications for the treatment of CPC.

The Tousled-like kinases (TLKs) are an evolutionarily conserved family of serine–threonine kinases that have been implicated in DNA replication, DNA repair, transcription, chromatin structure, viral latency, cell cycle checkpoint control and chromosomal stability in various organisms. The functions of the TLKs appear to depend largely on their ability to regulate the H3/H4 histone chaperone ASF1, although numerous TLK substrates have been proposed. Over the last few years, a clearer picture of TLK function has emerged through the identification of new partners, the definition of specific roles in development and the elucidation of their structural and biochemical properties. In addition, the TLKs have been clearly linked to human disease; both TLK1 and TLK2 are frequently amplified in human cancers and TLK2 mutations have been identified in patients with neurodevelopmental disorders characterized by intellectual disability (ID), autism spectrum disorder (ASD) and microcephaly. A better understanding of the substrates, regulation and diverse roles of the TLKs is needed to understand their functions in neurodevelopment and determine if they are viable targets for cancer therapy. In this review, we will summarize current knowledge of TLK biology and its potential implications in development and disease.

Tousled-like kinases (TLKs) are required for genome stability and normal development in numerous organisms and have been implicated in breast cancer and intellectual disability. In humans, the similar TLK1 and TLK2 interact with each other and TLK activity enhances ASF1 histone binding and is inhibited by the DNA damage response, although the molecular mechanisms of TLK regulation remain unclear. Here we describe the crystal structure of the TLK2 kinase domain. We show that the coiled-coil domains mediate dimerization and are essential for activation through ordered autophosphorylation that promotes higher order oligomers that locally increase TLK2 activity. We show that TLK2 mutations involved in intellectual disability impair kinase activity, and the docking of several small-molecule inhibitors of TLK activity suggest that the crystal structure will be useful for guiding the rationale design of new inhibition strategies. Together our results provide insights into the structure and molecular regulation of the TLKs. The Tousled-like kinase (TLKs) family belongs to a distinct branch of Ser/Thr kinases that exhibit the highest levels of activity during DNA replication. Here the authors present the crystal structure of the kinase domain from human TLK2 and propose an activation model for TLK2 based on biochemical and phosphoproteomics experiments.