Kinome-Wide shRNA Screen Reveals Functional Connections Between FANCA, Mitotic Centrosomes and the Spindle Assembly Checkpoint Tumor Suppressor Pathways

Abstract Fanconi anemia (FA) is a genetic disorder characterized by progressive bone marrow failure, congenital abnormalities and predilection towards development of hematopoietic malignancies, including acute myeloid leukemia (AML). Congenital biallelic disruption of the FA/BRCA signaling network causes Fanconi anemia and somatic mutations within the same genes are increasingly identified in a variety of malignancies in non-FA individuals, consistent with the critical role of this signaling pathway in FA and in the general population. The FA/BRCA tumor suppressor network orchestrates interphase DNA-damage repair (DDR) and DNA replication to maintain genomic stability. Additionally, we and others have demonstrated that the genome housekeeping function of FA/BRCA signaling extends beyond interphase: loss of FA/BRCA signaling perturbs execution of mitosis, including the spindle assembly checkpoint (SAC), centrosome maintenance, cytokinesis and resolution of anaphase DNA bridges. Interphase errors exacerbate mitotic abnormalities and mitotic failure promotes interphase mutagenesis. Consequently, we had demonstrated that primary FA patients' cells accumulate genomic abnormalities consistent with a dual mechanism of impaired interphase DDR/replication and defective mitosis. Previous detailed studies had elucidated multiple mechanisms of interphase DDR-dependent assembly and activation of the FA complex at DNA damage sites to arrest the cell cycle and repair DNA lesions. However, the signaling cross-talk nodes between the FA and mitotic checkpoint pathways remain to be discovered. In this study, we identified functionally relevant mitotic signaling defects resulting from FANCA deficiency via a synthetic lethal kinome-wide pooled shRNA screen in primary patient-derived FANCA -deficient cells compared to isogenic FANCA -corrected cell line. Bioinformatics analysis of our screen results followed by secondary validation of selected hits with alternative shRNAs and small-molecule inhibitors revealed conserved mitotic signal transduction pathways regulating the SAC and centrosome maintenance. Our super-resolution structured illumination (SR-SIM) microscopy coupled with deconvolution imaging revealed that a fraction of FANCA co-localizes with key SAC kinases at mitotic centrosomes and kinetochores, consistent with the role of FANCA in centrosome maintenance and the SAC. Co-immunoprecipitation assays identified the biochemical interaction between FANCA and an essential SAC kinase whose loss is synthetic lethal with FANCA deficiency, providing first insights into the interactions between FA signaling and the canonical SAC network. Together, our study has unraveled functional and biochemical connections between FANCA and the centrosome/SAC kinases, consistent with the essential role of FANCA in cell division. Our ongoing work is aimed at mechanistically dissecting molecular links between these two key tumor suppressor signaling pathways in more detail. We hypothesize that impaired FANCA/SAC cross-talk may contribute to genomic instability in FA-deficient cells and provide opportunities to selectively kill FANCA-/- cells. Disclosures No relevant conflicts of interest to declare.

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Mitotic Errors Promote Genomic Instability and Leukemia in a Novel Mouse Model of Fanconi Anemia

Frontiers in Oncology ◽

10.3389/fonc.2021.752933 ◽

2021 ◽

Vol 11 ◽

Author(s):

Donna M. Edwards ◽

Dana K. Mitchell ◽

Zahi Abdul-Sater ◽

Ka-Kui Chan ◽

Zejin Sun ◽

...

Keyword(s):

Dna Damage ◽

Genomic Instability ◽

Fanconi Anemia ◽

Spindle Assembly Checkpoint ◽

Dna Damage Repair ◽

Spindle Assembly ◽

Dependent Manner ◽

Damage Repair

Fanconi anemia (FA) is a disease of genomic instability and cancer. In addition to DNA damage repair, FA pathway proteins are now known to be critical for maintaining faithful chromosome segregation during mitosis. While impaired DNA damage repair has been studied extensively in FA-associated carcinogenesis in vivo, the oncogenic contribution of mitotic abnormalities secondary to FA pathway deficiency remains incompletely understood. To examine the role of mitotic dysregulation in FA pathway deficient malignancies, we genetically exacerbated the baseline mitotic defect in Fancc-/- mice by introducing heterozygosity of the key spindle assembly checkpoint regulator Mad2. Fancc-/-;Mad2+/- mice were viable, but died from acute myeloid leukemia (AML), thus recapitulating the high risk of myeloid malignancies in FA patients better than Fancc-/-mice. We utilized hematopoietic stem cell transplantation to propagate Fancc-/-; Mad2+/- AML in irradiated healthy mice to model FANCC-deficient AMLs arising in the non-FA population. Compared to cells from Fancc-/- mice, those from Fancc-/-;Mad2+/- mice demonstrated an increase in mitotic errors but equivalent DNA cross-linker hypersensitivity, indicating that the cancer phenotype of Fancc-/-;Mad2+/- mice results from error-prone cell division and not exacerbation of the DNA damage repair defect. We found that FANCC enhances targeting of endogenous MAD2 to prometaphase kinetochores, suggesting a mechanism for how FANCC-dependent regulation of the spindle assembly checkpoint prevents chromosome mis-segregation. Whole-exome sequencing revealed similarities between human FA-associated myelodysplastic syndrome (MDS)/AML and the AML that developed in Fancc-/-; Mad2+/- mice. Together, these data illuminate the role of mitotic dysregulation in FA-pathway deficient malignancies in vivo, show how FANCC adjusts the spindle assembly checkpoint rheostat by regulating MAD2 kinetochore targeting in cell cycle-dependent manner, and establish two new mouse models for preclinical studies of AML.

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A tumor suppressor role of the Bub3 spindle checkpoint protein after apoptosis inhibition

The Journal of Cell Biology ◽

10.1083/jcb.201210018 ◽

2013 ◽

Vol 201 (3) ◽

pp. 385-393 ◽

Cited By ~ 40

Author(s):

Sara Morais da Silva ◽

Tatiana Moutinho-Santos ◽

Claudio E. Sunkel

Keyword(s):

Tumor Suppressor ◽

Spindle Assembly Checkpoint ◽

Mitotic Checkpoint ◽

Proliferative Potential ◽

Altered Expression ◽

Apoptosis Inhibition ◽

Wing Imaginal Discs ◽

A Minor ◽

Checkpoint Genes

Most solid tumors contain aneuploid cells, indicating that the mitotic checkpoint is permissive to the proliferation of chromosomally aberrant cells. However, mutated or altered expression of mitotic checkpoint genes accounts for a minor proportion of human tumors. We describe a Drosophila melanogaster tumorigenesis model derived from knocking down spindle assembly checkpoint (SAC) genes and preventing apoptosis in wing imaginal discs. Bub3-deficient tumors that were also deficient in apoptosis displayed neoplastic growth, chromosomal aneuploidy, and high proliferative potential after transplantation into adult flies. Inducing aneuploidy by knocking down CENP-E and preventing apoptosis does not induce tumorigenesis, indicating that aneuploidy is not sufficient for hyperplasia. In this system, the aneuploidy caused by a deficient SAC is not driving tumorigenesis because preventing Bub3 from binding to the kinetochore does not cause hyperproliferation. Our data suggest that Bub3 has a nonkinetochore-dependent function that is consistent with its role as a tumor suppressor.

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Coupling of Cdc20 inhibition and activation by BubR1

10.1101/2020.12.18.423461 ◽

2020 ◽

Author(s):

Jamin Hein ◽

Dimitriya H Garvanska ◽

Isha Nasa ◽

Arminja Kettenbach ◽

Jakob Nilsson

Keyword(s):

Ubiquitin Ligase ◽

Cross Talk ◽

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Cyclin B1 ◽

Mitotic Checkpoint ◽

Anaphase Onset ◽

Mitotic Checkpoint Complex

Tight regulation of the APC/C-Cdc20 ubiquitin ligase that targets Cyclin B1 for degradation is important for mitotic fidelity. The spindle assembly checkpoint (SAC) inhibits Cdc20 through the mitotic checkpoint complex (MCC). In addition, phosphorylation of Cdc20 by Cyclin B1-Cdk1 independently inhibits APC/C-Cdc20 activation. This creates a conundrum for how Cdc20 gets activated prior to Cyclin B1 degradation. Here we show that the MCC component BubR1 harbours both Cdc20 inhibition and activation activities, allowing for cross-talk between the two Cdc20 inhibition pathways. Specifically BubR1 acts as a substrate specifier for PP2A-B56 to enable efficient Cdc20 dephosphorylation in the MCC. A mutant Cdc20 mimicking the dephosphorylated state escapes a mitotic checkpoint arrest arguing that restricting Cdc20 dephosphorylation to the MCC is important. Collectively our work reveals how Cdc20 can be dephosphorylated in the presence of Cyclin B1-Cdk1 activity without causing premature anaphase onset.

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The role of MAD2L1BP in the silencing of the spindle-assembly checkpoint and the DNA damage checkpoint

10.14711/thesis-b1070168 ◽

2009 ◽

Author(s):

Kin Fan On

Keyword(s):

Dna Damage ◽

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Dna Damage Checkpoint

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Coupling of Cdc20 inhibition and activation by BubR1

The Journal of Cell Biology ◽

10.1083/jcb.202012081 ◽

2021 ◽

Vol 220 (5) ◽

Author(s):

Jamin B. Hein ◽

Dimitriya H. Garvanska ◽

Isha Nasa ◽

Arminja N. Kettenbach ◽

Jakob Nilsson

Keyword(s):

Ubiquitin Ligase ◽

Cross Talk ◽

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Cyclin B1 ◽

Mitotic Checkpoint ◽

Anaphase Onset ◽

Mitotic Checkpoint Complex

Tight regulation of the APC/C-Cdc20 ubiquitin ligase that targets cyclin B1 for degradation is important for mitotic fidelity. The spindle assembly checkpoint (SAC) inhibits Cdc20 through the mitotic checkpoint complex (MCC). In addition, phosphorylation of Cdc20 by cyclin B1–Cdk1 independently inhibits APC/C–Cdc20 activation. This creates a conundrum for how Cdc20 is activated before cyclin B1 degradation. Here, we show that the MCC component BubR1 harbors both Cdc20 inhibition and activation activities, allowing for cross-talk between the two Cdc20 inhibition pathways. Specifically, BubR1 acts as a substrate specifier for PP2A-B56 to enable efficient Cdc20 dephosphorylation in the MCC. A mutant Cdc20 mimicking the dephosphorylated state escapes a mitotic checkpoint arrest, arguing that restricting Cdc20 dephosphorylation to the MCC is important. Collectively, our work reveals how Cdc20 can be dephosphorylated in the presence of cyclin B1-Cdk1 activity without causing premature anaphase onset.

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FANCA Controls Mitotic Phosphosignaling Networks To Ensure Genome Stability During Cell Division

Blood ◽

10.1182/blood.v122.21.801.801 ◽

2013 ◽

Vol 122 (21) ◽

pp. 801-801

Author(s):

Rikki Enzor ◽

Zahi Abdul Sater ◽

Donna Cerabona ◽

Zejin Sun ◽

Su-jung Park ◽

...

Keyword(s):

Cell Cycle ◽

Cell Division ◽

Chromosome Segregation ◽

Fanconi Anemia ◽

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Signaling Network ◽

Mitotic Apparatus ◽

Risk Of Cancer

Abstract Fanconi anemia (FA) is a heterogenous genome instability syndrome with a high risk of cancer. The FA proteins are essential for interphase DNA damage repair. However, it is incompletely understood why FA-deficient cells also develop gross aneuploidy and multinucleation, which are symptoms of error-prone chromosome segregation. Emerging evidence indicates that the FA signaling network functions as a guardian of the genome throughout the cell cycle, including chromosome segregation during mitosis. However, the mechanistic aspects of the critical role of the FA signaling in mitosis remain poorly understood. We have recently shown that the FA signaling network localizes to the mitotic apparatus to control the spindle assembly checkpoint and centrosome maintenance (J Clin Invest 2013, in press). The spindle assembly checkpoint (SAC) is a complex tumor suppressor signaling network that prevents premature separation of sister chromatids by delaying the metaphase-to-anaphase transition until all the kinetochores are properly attached to the mitotic spindle. Since weakened SAC promotes stochastic chromosome segregation, mutagenesis and cancer, these findings shed new light on the role of FA signaling in maintenance of genomic stability. We found the subcellular localization of FA proteins to the mitotic apparatus is spatiotemporally regulated as cells divide. Our new data revealed the pathways connecting the FANCA protein with canonical mitotic phosphosignaling networks. We have employed unbiased kinome-wide phospho-mass spectrometry to compare the landscape of abnormalities of mitotic signaling pathways in primary FANCA-/- patient cells and gene-corrected isogenic cells. These experiments led us to identify and quantify a wide range of phosphorylation abnormalities of multiple FANCA-dependent centrosome-, kinetochore- and chromosome-associated regulators of mitosis. Our data illuminated the role for FA signaling in three critical stages of cell division: (1) the spindle assembly checkpoint, (2) anaphase and (3) cytokinesis. Thus, we employed live phase-contrast imaging of primary FANCA-/- patient cells in comparison to gene-corrected cells to separately quantify aberrations in (1) chromosome congression and metaphase-anaphase transition (SAC malfunction), (2) execution of anaphase and (3) completion of cytokinesis. Our findings further our understanding of human cell cycle control and provide new insights into the origins of genomic instability in Fanconi anemia by establishing mechanistic connection between the FANCA protein and key mitotic signaling networks. The identification of cell division pathways regulated by FANCA has implications for future targeted drug development in Fanconi anemia and FA-deficient malignancies in the general population. Disclosures: No relevant conflicts of interest to declare.

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Faculty Opinions recommendation of DNA damage during mitosis in human cells delays the metaphase/anaphase transition via the spindle-assembly checkpoint.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1010362.151807 ◽

2002 ◽

Author(s):

Orna Cohen-Fix

Keyword(s):

Dna Damage ◽

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Human Cells

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A Screen for Dynein Synthetic Lethals in Aspergillus nidulans Identifies Spindle Assembly Checkpoint Genes and Other Genes Involved in Mitosis

Genetics ◽

10.1093/genetics/149.1.101 ◽

1998 ◽

Vol 149 (1) ◽

pp. 101-116

Author(s):

Vladimir P Efimov ◽

N Ronald Morris

Keyword(s):

Aspergillus Nidulans ◽

Spindle Assembly Checkpoint ◽

Genetic Interaction ◽

Synthetic Lethality ◽

Spindle Assembly ◽

Nuclear Migration ◽

Cytoplasmic Dynein ◽

Vesicle Transport ◽

Synthetic Lethal ◽

Checkpoint Genes

Abstract Cytoplasmic dynein is a ubiquitously expressed microtubule motor involved in vesicle transport, mitosis, nuclear migration, and spindle orientation. In the filamentous fungus Aspergillus nidulans, inactivation of cytoplasmic dynein, although not lethal, severely impairs nuclear migration. The role of dynein in mitosis and vesicle transport in this organism is unclear. To investigate the complete range of dynein function in A. nidulans, we searched for synthetic lethal mutations that significantly reduced growth in the absence of dynein but had little effect on their own. We isolated 19 sld (synthetic lethality without dynein) mutations in nine different genes. Mutations in two genes exacerbate the nuclear migration defect seen in the absence of dynein. Mutations in six other genes, including sldA and sldB, show a strong synthetic lethal interaction with a mutation in the mitotic kinesin bimC and, thus, are likely to play a role in mitosis. Mutations in sldA and sldB also confer hypersensitivity to the microtubule-destabilizing drug benomyl. sldA and sldB were cloned by complementation of their mutant phenotypes using an A. nidulans autonomously replicating vector. Sequencing revealed homology to the spindle assembly checkpoint genes BUB1 and BUB3 from Saccharomyces cerevisiae. Genetic interaction between dynein and spindle assembly checkpoint genes, as well as other mitotic genes, indicates that A. nidulans dynein plays a role in mitosis. We suggest a model for dynein motor action in A. nidulans that can explain dynein involvement in both mitosis and nuclear distribution.

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