Medulloblastoma (MB) and gliomas are the most frequent high-grade brain tumors (HGBT) in children and adulthood, respectively. The general treatment for these tumors consists in surgery, followed by radiotherapy and chemotherapy. Despite the improvement in patient survival, these therapies are only partially effective, and many patients still die. In the last decades, microtubules have emerged as interesting molecular targets for HGBT, as various microtubule targeting agents (MTAs) have been developed and tested pre-clinically and clinically with encouraging results. Nevertheless, these treatments produce relevant side effects since they target microtubules in normal as well as in cancerous cells. A possible strategy to overcome this toxicity could be to target proteins that control microtubule dynamics but are required by HGBT cells much more than in normal cell types. The genes mutated in primary hereditary microcephaly (MCPH) are ubiquitously expressed in proliferating cells, but under normal conditions are selectively required during brain development, in neural progenitors. There is evidence that MB and glioma cells share molecular profiles with progenitors of cerebellar granules and of cortical radial glia cells, in which MCPH gene functions are fundamental. Moreover, several studies indicate that MCPH genes are required for HGBT expansion. Among the 25 known MCPH genes, we focus this review on KNL1, ASPM, CENPE, CITK and KIF14, which have been found to control microtubule stability during cell division. We summarize the current knowledge about the molecular basis of their interaction with microtubules. Moreover, we will discuss data that suggest these genes are promising candidates as HGBT-specific targets.

Medulloblastoma (MB) is the most common malignant brain tumor in children, and it is classified into four biological subgroups: WNT, Sonic Hedgehog (SHH), Group 3 and Group 4. The current treatment is surgery, followed by irradiation and chemotherapy. Unfortunately, these therapies are only partially effective. Citron kinase protein (CITK) has been proposed as a promising target for SHH MB, whose inactivation leads to DNA damage and apoptosis. D283 and D341 cell lines (Group 3/Group 4 MB) were silenced with established siRNA sequences against CITK, to assess the direct effects of its loss. Next, D283, D341, ONS-76 and DAOY cells were treated with ionizing radiation (IR) or cisplatin in combination with CITK knockdown. CITK depletion impaired proliferation and induced cytokinesis failure and apoptosis of G3/G4 MB cell lines. Furthermore, CITK knockdown produced an accumulation of DNA damage, with reduced RAD51 nuclear levels. Association of IR or cisplatin with CITK depletion strongly impaired the growth potential of all tested MB cells. These results indicate that CITK inactivation could prevent the expansion of G3/G4 MB and increase their sensitivity to DNA-damaging agents, by impairing homologous recombination. We suggest that CITK inhibition could be broadly associated with IR and adjuvant therapy in MB treatment.

Medulloblastoma (MB) is the most frequent brain tumor in children. The standard treatment consists in surgery, followed by radiotherapy and chemotherapy. These therapies are only partially effective since many patients still die and those who survive suffer from neurological and endocrine disorders. Therefore, more effective therapies are needed. Primary microcephaly (MCPH) is a rare disorder caused by mutations in 25 different genes. Centromere-associated protein E (CENPE) heterozygous mutations cause the MCPH13 syndrome. As for other MCPH genes, CENPE is required for normal proliferation and survival of neural progenitors. Since there is evidence that MB shares many molecular features with neural progenitors, we hypothesized that CENPE could be an effective target for MB treatment. In ONS-76 and DAOY cells, CENPE knockdown induced mitotic defects and apoptosis. Moreover, CENPE depletion induced endogenous DNA damage accumulation, activating TP53 or TP73 as well as cell death signaling pathways. To consolidate CENPE as a target for MB treatment, we tested GSK923295, an allosteric inhibitor already in clinical trial for other cancer types. GSK923295, induced effects similar to CENPE depletion with higher penetrance, at low nM levels, suggesting that CENPE’s inhibition could be a therapeutic strategy for MB treatment.

Brain size and cellular heterogeneity are tightly regulated by species-specific proliferation and differentiation of multipotent neural progenitor cells (NPCs). Errors in this process are among the mechanisms of primary hereditary microcephaly (MCPH), a group of disorders characterized by reduced brain size and intellectual disability. Biallelic citron rho-interacting serine/threonine kinase ( CIT ) missense variants that disrupt kinase function ( CIT ^KI/KI ) and frameshift loss-of-function variants ( CIT ^FS/FS ) are the genetic basis for MCPH17; however, the function of CIT catalytic activity in brain development and NPC cytokinesis is unknown. Therefore, we created the Cit ^KI/KI mouse model and found that it did not phenocopy human microcephaly, unlike biallelic Cit ^FS/FS animals. Nevertheless, both Cit models exhibited binucleation, DNA damage, and apoptosis. To investigate human-specific mechanisms of CIT microcephaly, we generated CIT ^KI/KI and CIT ^FS/FS human forebrain organoids. We found that CIT ^KI/KI and CIT ^FS/FS organoids lost cytoarchitectural complexity, transitioning from pseudostratified to simple neuroepithelium. This change was associated with defects that disrupted the polarity of NPC cytokinesis, in addition to elevating apoptosis. Together, our results indicate that both CIT catalytic and scaffolding functions in NPC cytokinesis are critical for human corticogenesis. Species differences in corticogenesis and the dynamic 3D features of NPC mitosis underscore the utility of human forebrain organoid models for understanding human microcephaly.

Goldberg–Shprintzen disease (GOSHS) is a rare microcephaly syndrome accompanied by intellectual disability, dysmorphic facial features, peripheral neuropathy and Hirschsprung disease. It is associated with recessive mutations in the gene encoding kinesin family member 1-binding protein (KIF1BP, also known as KIFBP). The encoded protein regulates axon microtubules dynamics, kinesin attachment and mitochondrial biogenesis, but it is not clear how its loss could lead to microcephaly. We identified KIF1BP in the interactome of citron kinase (CITK, also known as CIT), a protein produced by the primary hereditary microcephaly 17 (MCPH17) gene. KIF1BP and CITK interact under physiological conditions in mitotic cells. Similar to CITK, KIF1BP is enriched at the midbody ring and is required for cytokinesis. The association between KIF1BP and CITK can be influenced by CITK activity, and the two proteins may antagonize each other for their midbody localization. KIF1BP knockdown decreases microtubule stability, increases KIF23 midbody levels and impairs midbody localization of KIF14, as well as of chromosome passenger complex. These data indicate that KIF1BP is a CITK interactor involved in midbody maturation and abscission, and suggest that cytokinesis failure may contribute to the microcephaly phenotype observed in GOSHS.

Citron Kinase (CITK) is a protein encoded by the CIT gene, whose pathogenic variants underlie microcephalic phenotypes that characterize MCPH17 syndrome. In neural progenitors, CITK loss leads to microtubule instability, resulting in mitotic spindle positioning defects, cytokinesis failure, and accumulation of DNA double strand breaks (DSBs), ultimately resulting in TP53-dependent senescence and apoptosis. Although DNA damage accumulation has been associated with impaired homologous recombination (HR), the role of CITK in this process and whether microtubule dynamics are involved is still unknown. In this report we show that CITK is required for proper BRCA1 localization at sites of DNA DSBs. We found that CITK’s scaffolding, rather than its catalytic activity, is necessary for maintaining BRCA1 interphase levels in progenitor cells during neurodevelopment. CITK regulates the nuclear levels of HDAC6, a modulator of both microtubule stability and DNA damage repair. Targeting HDAC6 in CITK-deficient cells increases microtubule stability and recovers BRCA1 localization defects and DNA damage levels to that detected in controls. In addition, the CIT-HDAC6 axis is functionally relevant in a MCPH17 zebrafish model, as HDAC6 targeting recovers the head size phenotype produced by interfering with the CIT orthologue gene. These data provide novel insights into the functional interplay between HR and microtubule dynamics and into the pathogenesis of CITK based MCPH17, which may be relevant for development of therapeutic strategies.