Early Mitosis Research Articles

Phases of cortical actomyosin dynamics coupled to the neuroblast polarity cycle.

The Par complex dynamically polarizes to the apical cortex of asymmetrically dividing Drosophila neuroblasts where it directs fate determinant segregation. Previously, we showed that apically directed cortical movements that polarize the Par complex require F-actin (Oon and Prehoda, 2019). Here, we report the discovery of cortical actomyosin dynamics that begin in interphase when the Par complex is cytoplasmic but ultimately become tightly coupled to cortical Par dynamics. Interphase cortical actomyosin dynamics are unoriented and pulsatile but rapidly become sustained and apically-directed in early mitosis when the Par protein aPKC accumulates on the cortex. Apical actomyosin flows drive the coalescence of aPKC into an apical cap that depolarizes in anaphase when the flow reverses direction. Together with the previously characterized role of anaphase flows in specifying daughter cell size asymmetry, our results indicate that multiple phases of cortical actomyosin dynamics regulate asymmetric cell division.

CBIO-11. HISTONE H3.3 G34R/V MUTATIONS STIMULATE PEDIATRIC HIGH-GRADE GLIOMA FORMATION THROUGH THE INDUCTION OF CHROMOSOMAL INSTABILITY

Abstract H3.3 G34R/V mutations are drivers of high-grade pediatric glioma (pHGG). H3.3 G34R/V mutations are linked to altered H3.3 K36 trimethylation (H3K36me3); implicating epigenetic gene regulation as a possible contributor to pHGG formation. Here we show that H3.3 G34R/V also induces chromosomal instability (CIN); a hallmark of pHGG. If CIN promotes pHGG formation is unknown. We observed that H3.3 G34 mutant pHGG cells have reduced mitotic H3.3 S31 phosphorylation compare to WT H3.3 cell lines. And, H3.3 G34R reduced Chk1 phosphorylation at S31 by >90% in an in vitro kinase assay. Chk1 regulates chromosome segregation through phosphorylation of pericentromeric H3.3 S31 during early mitosis. Overexpression of H3.3 G34R or non-phosphorylatable S31A in H3.3 WT, diploid cells caused a significant increase in CIN. Likewise, H3.3 G34 mutant pHGG cells have significantly elevated rates of CIN as compare to H3.3 WT pHGG cells. During normal cell division, phospho-S31 is lost in anaphase. However, following chromosome missegregation, phospho-S31 spreads and stimulates p53-induced cell cycle arrest. Here we show that WT p53 cells expressing mutant G34 fail to arrest following chromosome mis-segregation. These studies demonstrate that H3.3 G34R/V mutations are sufficient to transform normal, diploid cells into proliferating CIN cells. To determine if this process contributes to tumorigenesis, we used RCAS Nestin-TVA mice to overexpress H3.3 WT, G34R, or S31A – P2A-linked to PDGFB expression in glial precursor cells of newborn mice. Over 100 days, S31A and G34R mice had drastically reduced survival (averaging 77, 81, and 100 days for S31A, G34R, and WT). Furthermore, most G34R and S31A mice developed HGG, while H3.3 WT mice remained tumor-free. Our work implicates CIN as a driver of H3.3 G34 mutant pHGG formation. Our ongoing studies utilize K36M and double mutants to further define the contributions of S31 phosphorylation (CIN) and H3K36me3 (epigenetic gene regulation) to tumorigenesis.

Spatiotemporal coordination of the RSF1-PLK1-Aurora B cascade establishes mitotic signaling platforms

The chromatin remodeler RSF1 enriched at mitotic centromeres is essential for proper chromosome alignment and segregation and underlying mechanisms remain to be disclosed. We here show that PLK1 recruitment by RSF1 at centromeres creates an activating phosphorylation on Thr236 in the activation loop of Aurora B and this is indispensable for the Aurora B activation. In structural modeling the phosphorylated Thr236 enhances the base catalysis by Asp200 nearby, facilitating the Thr232 autophosphorylation. Accordingly, RSF1-PLK1 is central for Aurora B-mediated microtubule destabilization in error correction. However, under full microtubule-kinetochore attachment RSF1-PLK1 positions at kinetochores, halts activating Aurora B and phosphorylates BubR1, regardless of tension. Spatial movement of RSF1-PLK1 to kinetochores is triggered by Aurora B-mediated phosphorylation of centromeric histone H3 on Ser28. We propose a regulatory RSF1-PLK1 axis that spatiotemporally controls on/off switch on Aurora B. This feedback circuit among RSF1-PLK1-Aurora B may coordinate dynamic microtubule-kinetochore attachment in early mitosis when full tension yet to be generated.

Structural basis of human separase regulation by securin and CDK1-cyclin B1.

In early mitosis, the duplicated chromosomes are held together by the ring-shaped cohesin complex1. Separation of chromosomes during anaphase is triggered by separase-a large cysteine endopeptidase that cleaves the cohesin subunit SCC1 (also known as RAD212-4). Separase is activated by degradation of its inhibitors, securin5 and cyclin B6, but the molecular mechanisms of separase regulation are not clear. Here we used cryogenic electron microscopy to determine the structures of human separase in complex with either securin or CDK1-cyclin B1-CKS1. In both complexes, separase is inhibited by pseudosubstrate motifs that block substrate binding at the catalyticsite and at nearby docking sites. As in Caenorhabditis elegans7 and yeast8, human securin contains its own pseudosubstrate motifs. By contrast, CDK1-cyclin B1 inhibits separase by deploying pseudosubstrate motifs from intrinsically disordered loops in separase itself. One autoinhibitory loop is oriented by CDK1-cyclin B1 to block the catalyticsites of both separase and CDK19,10. Another autoinhibitory loop blocks substrate docking in a cleft adjacent to the separase catalytic site. A third separase loop contains a phosphoserine6 that promotes complex assembly by binding to a conserved phosphate-binding pocket in cyclin B1. Our study reveals the diverse array of mechanisms by which securin and CDK1-cyclin B1 bind and inhibit separase, providing the molecular basis for the robust control of chromosome segregation.

Feedback control of PLK1 by Apolo1 ensures accurate chromosome segregation.

SUMMARYStable transmission of genetic material during cell division requires accurate chromosome segregation. PLK1 dynamics at kinetochores control establishment of correct kinetochore-microtubule attachments and subsequent silencing of the spindle checkpoint. However, the regulatory mechanism responsible for PLK1 activity in prometaphase has not yet been affirmatively identified. Here we identify Apolo1, which tunes PLK1 activity for accurate kinetochore-microtubule attachments. Apolo1 localizes to kinetochores during early mitosis, and suppression of Apolo1 results in misaligned chromosomes. Using the fluorescence resonance energy transfer (FRET)-based PLK1 activity reporter, we found that Apolo1 sustains PLK1 kinase activity at kinetochores for accurate attachment during prometaphase. Apolo1 is a cognate substrate of PLK1, and the phosphorylation enables PP1γ to inactivate PLK1 by dephosphorylation. Mechanistically, Apolo1 constitutes a bridge between kinase and phosphatase, which governs PLK1 activity in prometaphase. These findings define a previously uncharacterized feedback loop by which Apolo1 provides fine-tuning for PLK1 to guide chromosome segregation in mitosis.

SUMO proteases SENP3 and SENP5 spatiotemporally regulate the kinase activity of Aurora A.

Precise chromosome segregation is mediated by a well-assembled mitotic spindle, which requires balance of the kinase activity of Aurora A (AurA, also known as AURKA). However, how this kinase activity is regulated remains largely unclear. Here, using in vivo and in vitro assays, we report that conjugation of SUMO2 with AurA at K258 in early mitosis promotes the kinase activity of AurA and facilitates the binding with its activator Bora. Knockdown of the SUMO proteases SENP3 and SENP5 disrupts the deSUMOylation of AurA, leading to increased kinase activity and abnormalities in spindle assembly and chromosome segregation, which could be rescued by suppressing the kinase activity of AurA. Collectively, these results demonstrate that SENP3 and SENP5 deSUMOylate AurA to render spatiotemporal control on its kinase activity in mitosis. This article has an associated First Person interview with the first author of the paper.

Quantitative and Site-Specific Chemoproteomic Profiling of Protein O-GlcNAcylation in the Cell Cycle.

Mammalian cell cycle is a central process for tissue growth and maintenance. Protein O-linked β-N-acetylglucosamine (O-GlcNAc) modification has been found to occur on several important cell cycle regulators. However, the O-GlcNAcylated proteome has not been extensively profiled during cell cycle progression. Herein, we report a quantitative profiling of protein O-GlcNAcylation sites in cell proliferation, by using an O-GlcNAc chemoproteomic strategy. In HeLa cells, a total of 902, 439, and 872 high-confidence O-GlcNAcylation sites distributed on 414, 265, and 425 proteins are identified in the interphase, early mitosis, and mitotic exit stages, respectively. The identified O-GlcNAcylation events occur on a variety of important regulators, which are involved in the processes of cell division, DNA repair, and cell death. Furthermore, we show that O-GlcNAcylation is dynamically regulated in a cell cycle stage-dependent manner. Our results provide a valuable resource for investigating the functional roles of O-GlcNAc in the mammalian cell cycle.

HGG-31. HISTONE H3.3 G34R/V MUTATIONS STIMULATE PEDIATRIC HIGH-GRADE GLIOMA FORMATION THROUGH THE INDUCTION OF CHROMOSOMAL INSTABILITY

The histone H3.3 G34R/V mutations are known drivers of high-grade pediatric glioma (pHGG). However, the mechanism(s) for H3.3 G34R/V induced tumor formation are unclear. Chk1 phosphorylates H3.3 S31 at the pericentromere during early mitosis, suggesting a novel mitotic function. We observed that H3.3 G34 mutant pHGG cells have reduced mitotic H3.3 S31 phosphorylation compare to WT H3.3 cell lines. The H3.3 G34R mutation reduced Chk1 phosphorylation at S31 by >90% in an in vitro kinase assay. Overexpression of either H3.3 G34R or non-phosphorylatable S31A in H3.3 WT, diploid cells led to a significant increase in chromosome mis-segregations. Likewise, H3.3 G34 mutant pHGG cells have significantly elevated rates of mis-segregation as compare to H3.3 WT pHGG cells. During normal cell division, phospho-S31 is lost in late anaphase. However, when chromosome missegregation occurs, phospho-S31 spreads and stimulates p53 accumulation in G1 – thus suppressing aneuploid cell proliferation. Here we show that cells expressing mutant G34 fail to arrest following mis-segregation, despite having WT p53. These studies demonstrate that the H3.3 G34R/V mutations are sufficient to transform normal, diploid cells into proliferative, chromosomally instable cells. To determine if this process contributes to tumorigenesis, we used the RCAS/TVA mouse model to overexpress H3.3 WT, G34R, or S31A in the glial precursor cells of mice pups. Over 100 days, S31A and G34R mice had drastically reduced survival (averaging 77, 81, and 100 days for S31A, G34R, and WT mice). Furthermore, most G34R and S31A mice developed HGG, while H3.3 WT mice remained tumor-free and did not develop high-grade tumors. Our work strongly indicates that a major factor in H3.3 G34R pHGG formation is the induction of chromosomal instability – which occurs directly through the suppression of H3.3 S31 phosphorylation.

Actin-dependent membrane polarization reveals the mechanical nature of the neuroblast polarity cycle

SUMMARYThe Par complex directs fate-determinant segregation from the apical membrane of asymmetrically dividing Drosophila neuroblasts. While the physical interactions that recruit the Par complex have been extensively studied, little is known about how the membrane itself behaves during polarization. We examined the membrane dynamics of neuroblasts and surrounding cells using a combination of super-resolution and time-lapse imaging, revealing cellular-scale movements of diverse membrane features during asymmetric division cycles. Membrane domains that are distributed across the neuroblast membrane in interphase become polarized in early mitosis, where they mediate formation of cortical patches of the Par protein atypical protein kinase C (aPKC). Membrane and protein polarity cycles are precisely synchronized and are generated by extensive actin-dependent forces that deform the surrounding tissue. In addition to suggesting a role for the membrane in asymmetric division, our results reveal the mechanical nature of the neuroblast polarity cycle.

Clinical and Molecular Cytogenetic Characteristics of Five Cases with Isodicentric Y Chromosome

Isodicentric Y chromosome [idic(Y)] is one of the most common structural abnormalities of the Y chromosome and has been observed in patients with reproductive disorders and in patients with disorders of sexual development. Most idic(Y) chromosomes are found in mosaic form with a 45,X cell line. These chromosomes are highly unstable during mitosis due to the presence of 2 centromers, which explains their probable loss in early mitosis or mitosis of the embryo and therefore the presence of the 45,X line. It has been hypothesized that the proportion of 45,X cells in various tissues probably influences the phenotypic sex of individuals carrying an idic(Y) chromosome, ranging from infertile men, hypospadias, ambiguous genitalia, and Turner syndrome to sex reversal. In this article we present 5 cases of patients with idic(Y) referred for suspected disorder of sex development (DSD), 3 with a male assignment and 2 with a female assignment. All cases have variable clinical characteristics, which were assessed by the transdisciplinary group of Disorders of Sex Development of the Hospital Universitario San Ignacio, Bogotá, Colombia. Patients were analyzed by conventional and molecular cytogenetics using high-resolution G-band and FISH techniques. Our findings highlight the importance of cytogenetic studies in the diagnosis of DSD patients.

Reduced USP22 Expression Impairs Mitotic Removal of H2B Monoubiquitination, Alters Chromatin Compaction and Induces Chromosome Instability That May Promote Oncogenesis.

Simple SummaryChromosome instability (CIN) promotes cancer initiation and progression, but its genetic origins remain largely unknown. As aberrant mitotic processes such as chromatin compaction defects can induce CIN, characterizing the molecular determinants of mitotic fidelity may reveal novel CIN genes. In vitro, monoubiquitination of histone H2B at lysine 120 (H2Bub1) impairs chromatin compaction, while in vivo H2Bub1 is rapidly depleted in early mitosis. USP22 is a major deubiquitinating enzyme catalyzing H2Bub1 removal in interphase and may also be responsible for H2Bub1 depletion in mitosis. To assess whether mitotic removal of H2Bub1 is required for mitotic fidelity, we employ complementary genetic and single-cell imaging microscopy approaches to assess the impact reduced USP22 expression has on H2Bub1 abundance, chromatin compaction and chromosome stability. We show that USP22 deficiency impairs H2Bub1 removal and induces chromatin compaction defects. Further, we identify USP22 as a novel CIN gene, indicating that USP22 deletions in tumors may drive CIN and contribute to oncogenesis.Chromosome instability (CIN) is an enabling feature of oncogenesis associated with poor patient outcomes, whose genetic determinants remain largely unknown. As mitotic chromatin compaction defects can compromise the accuracy of chromosome segregation into daughter cells and drive CIN, characterizing the molecular mechanisms ensuring accurate chromatin compaction may identify novel CIN genes. In vitro, histone H2B monoubiquitination at lysine 120 (H2Bub1) impairs chromatin compaction, while in vivo H2Bub1 is rapidly depleted from chromatin upon entry into mitosis, suggesting that H2Bub1 removal may be a pre-requisite for mitotic fidelity. The deubiquitinating enzyme USP22 catalyzes H2Bub1 removal in interphase and may also be required for H2Bub1 removal in early mitosis to maintain chromosome stability. In this study, we demonstrate that siRNA-mediated USP22 depletion increases H2Bub1 levels in early mitosis and induces CIN phenotypes associated with mitotic chromatin compaction defects revealed by super-resolution microscopy. Moreover, USP22-knockout models exhibit continuously changing chromosome complements over time. These data identify mitotic removal of H2Bub1 as a critical determinant of chromatin compaction and faithful chromosome segregation. We further demonstrate that USP22 is a CIN gene, indicating that USP22 deletions, which are frequent in many tumor types, may drive genetic heterogeneity and contribute to cancer pathogenesis.

The myosin regulatory light chain Myl5 localizes to mitotic spindle poles and is required for proper cell division.

Myosins are ATP-dependent actin-based molecular motors critical for diverse cellular processes like intracellular trafficking, cell motility, and cell invasion. During cell division, myosin MYO10 is important for proper mitotic spindle assembly, the anchoring of the spindle to the cortex, and positioning of the spindle to the cell mid-plane. However, myosins are regulated by myosin regulatory light chains (RLCs), and whether RLCs are important for cell division has remained unexplored. Here, we have determined that the previously uncharacterized myosin RLC Myl5 associates with the mitotic spindle and is required for cell division. We show that Myl5 localizes to the leading edge and filopodia during interphase and to mitotic spindle poles and spindle microtubules during early mitosis. Importantly, depletion of Myl5 led to defects in mitotic spindle assembly, chromosome congression, and chromosome segregation and to a slower transition through mitosis. Furthermore, Myl5 bound to MYO10 in vitro and co-localized with MYO10 at the spindle poles. These results suggest that Myl5 is important for cell division and that it may be performing its function through MYO10.

The Arabidopsis GRAS-type SCL28 transcription factor controls the mitotic cell cycle and division plane orientation

Gene expression is reconfigured rapidly during the cell cycle to execute the cellular functions specific to each phase. Studies conducted with synchronized plant cell suspension cultures have identified hundreds of genes with periodic expression patterns across the phases of the cell cycle, but these results may differ from expression occurring in the context of intact organs. Here, we describe the use of fluorescence-activated cell sorting to analyze the gene expression profile of G2/M cells in the growing root. To this end, we isolated cells expressing the early mitosis cell cycle marker CYCLINB1;1-GFP from Arabidopsis root tips. Transcriptome analysis of these cells allowed identification of hundreds of genes whose expression is reduced or enriched in G2/M cells, including many not previously reported from cell suspension cultures. From this dataset, we identified SCL28, a transcription factor belonging to the GRAS family, whose messenger RNA accumulates to the highest levels in G2/M and is regulated by MYB3R transcription factors. Functional analysis indicates that SCL28 promotes progression through G2/M and modulates the selection of cell division planes.

Ongoing replication forks delay the nuclear envelope breakdown upon mitotic entry

DNA replication is a major contributor to genomic instability, and protection against DNA replication perturbation is essential for normal cell division. Certain types of replication stress agents, such as aphidicolin and hydroxyurea, have been shown to cause reversible replication fork stalling, wherein replisome complexes are stably maintained with competence to restart in the S phase of the cell cycle. If these stalled forks persist into the M phase without a replication restart, replisomes are disassembled in a p97-dependent pathway and under-replicated DNA is subjected to mitotic DNA repair synthesis. Here, using Xenopus egg extracts, we investigated the consequences that arise when stalled forks are released simultaneously with the induction of mitosis. Ara-cytidine-5′-triphosphate–induced stalled forks were able to restart with the addition of excess dCTP during early mitosis before the nuclear envelope breakdown (NEB). However, stalled forks could no longer restart efficiently after the NEB. Although replisome complexes were finally disassembled in a p97-dependent manner during mitotic progression whether or not fork stalling was relieved, the timing of the NEB was delayed with the ongoing forks, rather than the stalled forks, and the delay was dependent on Wee1/Myt1 kinase activities. Thus, ongoing DNA replication was found to be directly linked to the regulation of Wee1/Myt1 kinases to modulate cyclin-dependent kinase activities because of which DNA replication and mitosis occur in a mutually exclusive and sequential manner.

Destabilization of Long Astral Microtubules via Cdk1-Dependent Removal of GTSE1 from Their Plus Ends Facilitates Prometaphase Spindle Orientation

The precise regulation of microtubule dynamics over time and space in dividing cells is critical for several mitotic mechanisms that ultimately enable cell proliferation, tissue organization, and development. Astral microtubules, which extend from the centrosome toward the cell cortex, must be present for the mitotic spindle to properly orient, as well as for the faithful execution of anaphase and cytokinesis. However, little is understood about how the dynamic properties of astral microtubules are regulated spatiotemporally, or the contribution of astral microtubule dynamics to spindle positioning. The mitotic regulator Cdk1-CyclinB promotes destabilization of centrosomal microtubules and increased microtubule dynamics as cells enter mitosis, but how Cdk1 activity modulates astral microtubule stability, and whether it impacts spindle positioning, is unknown. Here, we uncover a mechanism revealing that Cdk1 destabilizes astral microtubules in prometaphase and thereby influences spindle reorientation. Phosphorylation of the EB1-dependent microtubule plus-end tracking protein GTSE1 by Cdk1 in early mitosis abolishes its interaction with EB1 and recruitment to microtubule plus ends. Loss of Cdk1 activity, or mutation of phosphorylation sites in GTSE1, induces recruitment of GTSE1 to growing microtubule plus ends in mitosis. This decreases the catastrophe frequency of astral microtubules and causes an increase in the number of long astral microtubules reaching the cell cortex, which restrains the ability of cells to reorient spindles along the long cellular axis in early mitosis. Astral microtubules thus must not only be present but also dynamic to allow the spindle to reorient, a state assisted by selective destabilization of long astral microtubules via Cdk1.

Impaired condensin complex and Aurora B kinase underlie mitotic and chromosomal defects in hyperdiploid B-cell ALL

AbstractB-cell acute lymphoblastic leukemia (ALL; B-ALL) is the most common pediatric cancer, and high hyperdiploidy (HyperD) identifies the most common subtype of pediatric B-ALL. Despite HyperD being an initiating oncogenic event affiliated with childhood B-ALL, the mitotic and chromosomal defects associated with HyperD B-ALL (HyperD-ALL) remain poorly characterized. Here, we have used 54 primary pediatric B-ALL samples to characterize the cellular-molecular mechanisms underlying the mitotic/chromosome defects predicated to be early pathogenic contributors in HyperD-ALL. We report that HyperD-ALL blasts are low proliferative and show a delay in early mitosis at prometaphase, associated with chromosome-alignment defects at the metaphase plate leading to robust chromosome-segregation defects and nonmodal karyotypes. Mechanistically, biochemical, functional, and mass-spectrometry assays revealed that condensin complex is impaired in HyperD-ALL cells, leading to chromosome hypocondensation, loss of centromere stiffness, and mislocalization of the chromosome passenger complex proteins Aurora B kinase (AURKB) and Survivin in early mitosis. HyperD-ALL cells show chromatid cohesion defects and an impaired spindle assembly checkpoint (SAC), thus undergoing mitotic slippage due to defective AURKB and impaired SAC activity, downstream of condensin complex defects. Chromosome structure/condensation defects and hyperdiploidy were reproduced in healthy CD34+ stem/progenitor cells upon inhibition of AURKB and/or SAC. Collectively, hyperdiploid B-ALL is associated with a defective condensin complex, AURKB, and SAC.

Drosophila Tubulin-Specific Chaperone E Recruits Tubulin around Chromatin to Promote Mitotic Spindle Assembly

Proper assembly of mitotic spindles requires microtubule nucleation not only at the centrosomes but also around chromatin. In this study, we found that the Drosophila tubulin-specific chaperone dTBCE is required for the enrichment of tubulin in the nuclear space after nuclear envelope breakdown and for subsequent promotion of spindle microtubule nucleation. These events depend on the CAP-Gly motif found in dTBCE and are regulated by Ran and lamin proteins. Our data suggest that during early mitosis, dTBCE and nuclear pore proteins become enriched in the nucleus, where they interact with the Ran GTPase to promote dynamic tubulin enrichment. We propose that this novel mechanism enhances microtubule nucleation around chromatin, thereby facilitating mitotic spindle assembly.

CBIO-06. MKlp2 INHIBITION AS A NOVEL ANTIMITOTIC FOR GLIOBLASTOMA

Abstract Despite innovations in therapy and surgery, glioblastoma remains one of the deadliest tumor types with a 5-year survival rate of approximately 5%. There is a desperate need for new therapeutic strategies to block the growth and survival of glioblastoma. To circumvent toxicities associated with traditional antimitotics, there has been a growing interest in developing therapies that impede mitosis-specific microtubule functions, such as the inhibition of kinesins, mitotic motor proteins that travel along microtubules. Our work with motor kinesin-like protein 2 (MKlp2), in combination with reports from published literature, suggest a previously undescribed role for MKlp2 in early mitosis that would make its inhibition effective at impeding glioblastoma cell growth without neurologic side effects. The objective of this study was to further define this novel function of MKlp2 and to determine the efficacy of MKlp2 inhibition in glioblastoma. Our data indicate that MKlp2 inhibition induced a spindle assembly checkpoint-mediated arrest, suggesting that function of MKlp2 is important for recovery progression to anaphase in addition to its well-known function in late mitosis. In addition, MKlp2 inhibition with a small molecule MKlp2 inhibitor significantly decreased short-term and long-term survival of human glioblastoma patient-derived lines. Furthermore, our data indicate that it is loss of this early mitotic function that is responsible for the anti-proliferative effects caused by MKlp2 inhibition. Ongoing work will continue to delineate the mechanism of MKlp2 in early mitosis and determine the effect of small molecule MKlp2 inhibition in vivo, all the while exploring a new therapeutic strategy for glioblastoma

CBIO-21. H3F3A G34R/V MUTATIONS INDUCE CHROMOSOMAL INSTABILITY AND PROMOTE PEDIATRIC HIGH-GRADE GLIOMA FORMATION

Abstract Histone H3.3 G34R/V mutations are drivers of pediatric high-grade glioma (pHGG). However, the mechanism(s) responsible for G34R/V induced tumorigenesis are unclear. We observed that pHGG cells with H3.3 G34 mutations have significantly reduced phosphorylation at H3.3 S31 as compared to H3.3 WT cell lines. And, in vitro, G34 mutation completely blocks H3.3 S31 phosphorylation by Chk1 – the kinase responsible for S31 phosphorylation. During early mitosis, phospho-S31 is enriched in the chromosomal pericentromere; which plays a vital structural role in chromosome segregation. Over-expression of GFP-H3.3 G34R or non-phosphorylatable GFP-H3.3 S31A in chromosomally stable cell lines results in a significant increase in chromosome mis-segregations. Likewise, H3.3 G34V pHGG derived cells showed a higher frequency of mis-segregation than H3.3 WT cells. During cell division, phospho-S31 is lost in late anaphase. However, when chromosome missegregation occurs, phospho-S31 spreads to the chromosome arms where it stimulates p53 accumulation in G1 – thus acting as a sensor to suppress the proliferation of aneuploid cells. Here we show that cells expressing mutant G34 fail to arrest following mis-segregation, despite having WT p53. Taken together this work demonstrates that the H3.3 G34R/V mutations are sufficient to transform normal, diploid cells into proliferative, chromosomally instable cells. To determine if this process contributes to tumorigenesis, we expressed WT H3.3, H3.3-G34R or H3.3-S31A in a mouse model of pHGG. H3.3 WT controls developed low-grade tumors and all survived 80 days. Mice with H3.3-G34R or H3.3-S31A developed low- and high-grade tumors. And 78% and 50% of H3.3-G34R and H3.3-S31A mice, respectively, survived to 80 days. Our work suggests that H3.3 G34R/V drives chromosomal instability through the suppression of H3.3 S31 phosphorylation AND that chromosomal instability is a contributing driver of glioma formation in G34 mutant tumors.

Wip1 controls the translocation of the chromosomal passenger complex to the central spindle for faithful mitotic exit.

Dramatic cellular reorganization in mitosis critically depends on the timely and temporal phosphorylation of a broad range of proteins, which is mediated by the activation of the mitotic kinases and repression of counteracting phosphatases. The mitosis-to-interphase transition, which is termed mitotic exit, involves the removal of mitotic phosphorylation by protein phosphatases. Although protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) drive this reversal in animal cells, the phosphatase network associated with ordered bulk dephosphorylation in mitotic exit is not fully understood. Here, we describe a new mitotic phosphatase relay in which Wip1/PPM1D phosphatase activity is essential for chromosomal passenger complex (CPC) translocation to the anaphase central spindle after release from the chromosome via PP1-mediated dephosphorylation of histone H3T3. Depletion of endogenous Wip1 and overexpression of the phosphatase-dead mutant disturbed CPC translocation to the central spindle, leading to failure of cytokinesis. While Wip1 was degraded in early mitosis, its levels recovered in anaphase and the protein functioned as a Cdk1-counteracting phosphatase at the anaphase central spindle and midbody. Mechanistically, Wip1 dephosphorylated Thr-59 in inner centromere protein (INCENP), which, subsequently bound to MKLP2 and recruited other components to the central spindle. Furthermore, Wip1 overexpression is associated with the overall survival rate of patients with breast cancer, suggesting that Wip1 not only functions as a weak oncogene in the DNA damage network but also as a tumor suppressor in mitotic exit. Altogether, our findings reveal that sequential dephosphorylation of mitotic phosphatases provides spatiotemporal regulation of mitotic exit to prevent tumor initiation and progression.