Browsing by Subject "Spindle assembly checkpoint"

Now showing 1 - 4 of 4
  • Thumbnail Image
    Item
    FANCA maintains genomic stability through regulating BUBR1 acetylation
    (2017-08) Abdul Sater, Zahi Abass; Nalepa, Grzegorz; Clapp, Wade; Goebl, Mark; Wek, Ronald
    Fanconi Anemia (FA), a chromosomal instability syndrome, is characterized by bone marrow failure, genetic malformations, and predisposition to malignancies like acute myeloid leukemia (AML) and solid tumors. FA is caused by germline bi-allelic mutations in one of 21 known FA pathway genes and somatic mutations in FA genes are also found in a variety of sporadic cancers. Recently, numerous reports have discovered that the protective function of the FA pathway extends beyond its canonical role in regulation of DNA repair in interphase. In particular, the FA pathway has been shown to function in essential mitotic processes including spindle assembly checkpoint (SAC), cytokinesis, and centrosome maintenance. Understanding of the mechanistic origins of genomic instability leading to carcinogenesis and bone marrow failure has important scientific and clinical implications. To this end, using a micronucleus assay, we showed that both interphase DNA damage and mitotic errors contribute to genomic instability in FA ex vivo and in vivo. Functional studies of primary FA patient cells coupled with super-resolution microscopy revealed that FANCA is important for centrosome dependent spindle assembly supporting the protective role of FA pathway in mitotic processes. Furthermore, we dissected the interactions between the FA pathway and cellular kinase networks by employing a synthetic lethality sh-RNA screen targeting all human kinases. We mapped kinases that were synthetically lethal upon loss of FANCA, particularly those involved in highly conserved signal transduction pathways governing proliferation and cell cycle homeostasis. We mechanistically show that loss of FANCA, the most abundant FA subtype, results in in premature degradation of the mitotic kinase BUBR1 and faster mitotic exit. We further demonstrate that FANCA is important for PCAF-dependent acetylation of BUBR1 to prevent its premature degradation. Our results deepen our understanding of the molecular functions of the FA pathway in mitosis and uncover a mechanistic connection between FANCA and SAC phosphosignaling networks. These findings support the notion that further weakening the SAC through targeting kinases like BUBR1 in FA-deficient cancers may prove to be a rational therapeutic strategy.
  • Thumbnail Image
    Item
    Fancc regulates the spindle assembly checkpoint to prevent tumorigenesis in vivo
    (2017-06) Edwards, Donna Marie; Clapp, D. Wade; Nalepa, Grzegorz; Harrington, Maureen A.; Goebl, Mark G.
    The Fanconi anemia (FA) pathway consists of 21 genes that maintain genomic stability and prevent cancer. Biallelic mutations within this network cause Fanconi anemia, an inherited bone marrow failure and cancer predisposition syndrome. Heterozygous inborn mutations in FA genes increase risk of breast/ovarian cancers, and somatic mutations occur in malignancies in non-Fanconi patients. Understanding the tumor suppressive functions of FA signaling is important for the study of Fanconi anemia, inherited cancers, and sporadic cancers. The FA network functions as a genome guardian throughout the cell cycle. In addition to the well-established roles of FA proteins in interphase DNA replication/repair, the FA pathway controls mitosis by regulating the spindle assembly checkpoint (SAC) to ensure proper chromosome segregation. The SAC consists of several tumor suppressors, including Mad2, and SAC impairment predisposes to aneuploidy and cancer. However, the in vivo contribution of SAC dysfunction to malignant transformation of FA-deficient cells remains unknown. Furthermore, the mechanisms by which FA proteins regulate the SAC are unclear. To test whether SAC dysfunction drives genomic instability and tumorigenesis in FA, we generated a novel FA-SAC model by intercrossing Fancc-/- and Mad2+/- mice. The intercrossed mice displayed heightened aneuploidy secondary to exacerbated SAC dysfunction. Importantly, these mice were prone to developing hematologic malignancies, particularly leukemia, faithfully recapitulating the clinical phenotype of Fanconi anemia. Upon establishing SAC dysfunction as a driver of tumorigenesis in FA, we next explored the mechanism by which FANCC regulates the SAC. We demonstrated that the mitotic kinase CDK1 phosphorylates FANCC to regulate subcellular localization and SAC function of FANCC during mitosis. Our study highlights the essential role of compromised chromosome segregation in the development of leukemia due to impaired FA signaling. This work furthers our knowledge of FANCC signaling at the SAC, and has implications for future use of mitotic-centered therapies for FA-associated tumors.
  • Thumbnail Image
    Item
    INPP5E Preserves Genomic Stability through Regulation of Mitosis
    (American Society for Microbiology, 2017-03-15) Sierra Potchanant, Elizabeth A.; Cerabona, Donna; Sater, Zahi Abdul; He, Ying; Sun, Zejin; Gehlhausen, Jeff; Nalepa, Grzegorz; Department of Pediatrics, IU School of Medicine
    The partially understood phosphoinositide signaling cascade regulates multiple aspects of cellular metabolism. Previous studies revealed that INPP5E, the inositol polyphosphate-5-phosphatase that is mutated in the developmental disorders Joubert and MORM syndromes, is essential for the function of the primary cilium and maintenance of phosphoinositide balance in nondividing cells. Here, we report that INPP5E further contributes to cellular homeostasis by regulating cell division. We found that silencing or genetic knockout of INPP5E in human and murine cells impairs the spindle assembly checkpoint, centrosome and spindle function, and maintenance of chromosomal integrity. Consistent with a cell cycle regulatory role, we found that INPP5E expression is cell cycle dependent, peaking at mitotic entry. INPP5E localizes to centrosomes, chromosomes, and kinetochores in early mitosis and shuttles to the midzone spindle at mitotic exit. Our findings identify the previously unknown, essential role of INPP5E in mitosis and prevention of aneuploidy, providing a new perspective on the function of this phosphoinositide phosphatase in health and development.
  • Thumbnail Image
    Item
    Mitotic Errors Promote Genomic Instability and Leukemia in a Novel Mouse Model of Fanconi Anemia
    (Frontiers Media, 2021-11-05) Edwards, Donna M.; Mitchell, Dana K.; Abdul-Sater, Zahi; Chan, Ka-Kui; Sun, Zejin; Sheth, Aditya; He, Ying; Jiang, Li; Yuan, Jin; Sharma, Richa; Czader, Magdalena; Chin, Pei-Ju; Liu, Yie; de Cárcer, Guillermo; Nalepa, Grzegorz; Broxmeyer, Hal E.; Clapp, D. Wade; Potchanant, Elizabeth A. Sierra; Pediatrics, School of Medicine
    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.