Zejin Sun

Normal hematopoiesis and neurofibromin-deficient myeloproliferative disease require Erk

Neurofibromatosis type 1 (NF1) predisposes individuals to the development of juvenile myelomonocytic leukemia (JMML), a fatal myeloproliferative disease (MPD). In genetically engineered murine models, nullizygosity of Nf1, a tumor suppressor gene that encodes a Ras-GTPase-activating protein, results in hyperactivity of Raf/Mek/Erk in hematopoietic stem and progenitor cells (HSPCs). Activated Erk1/2 phosphorylate kinases and transcription factors with myriad mitogenic roles in diverse cell types. However, genetic studies examining Erk1/2s differential and/or combined control of normal and Nf1-deficient myelopoiesis are lacking. Moreover, prior studies relying on chemical Mek/Erk inhibitors have reached conflicting conclusions in normal and Nf1-deficient mice. Here, we show that while single Erk1 or Erk2 disruption did not grossly compromise myelopoiesis, dual Erk1/2 disruption rapidly ablated granulocyte and monocyte production in vivo, diminished progenitor cell number, and prevented HSPC proliferation in vitro. Genetic disruption of Erk1/2 in the context of Nf1 nullizygosity (Mx1Cre(+)Nf1(flox/flox)Erk1(-/-)Erk2(flox/flox)) fully protects against the development of MPD. Collectively, we identified a fundamental requirement for Erk1/2 signaling in normal and Nf1-deficient hematopoiesis, elucidating a critical hematopoietic function for Erk1/2 while genetically validating highly selective Mek/Erk inhibitors in a leukemia that is otherwise resistant to traditional therapy.

Fanconi anemia signaling network regulates the spindle assembly checkpoint

Fanconi anemia (FA) is a heterogenous genetic disease 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, leading to cancer. Here, we systematically evaluated the role of the FA proteins in chromosome segregation through functional RNAi screens and analysis of primary cells from patients with FA. We found that FA signaling is essential for the spindle assembly checkpoint and is therefore required for high-fidelity chromosome segregation and prevention of aneuploidy. Furthermore, we discovered that FA proteins differentially localize to key structures of the mitotic apparatus in a cell cycle-dependent manner. The essential role of the FA pathway in mitosis offers a mechanistic explanation for the aneuploidy and malignant transformation known to occur after disruption of FA signaling. Collectively, our findings provide insight into the genetically unstable cancers resulting from inactivation of the FA/BRCA pathway.

Pak2 regulates hematopoietic progenitor cell proliferation, survival, and differentiation.

p21‐Activated kinase 2 (Pak2), a serine/threonine kinase, has been previously shown to be essential for hematopoietic stem cell (HSC) engraftment. However, Pak2 modulation of long‐term hematopoiesis and lineage commitment remain unreported. Using a conditional Pak2 knockout mouse model, we found that disruption of Pak2 in HSCs induced profound leukopenia and a mild macrocytic anemia. Although loss of Pak2 in HSCs leads to less efficient short‐ and long‐term competitive hematopoiesis than wild‐type cells, it does not affect HSC self‐renewal per se. Pak2 disruption decreased the survival and proliferation of multicytokine stimulated immature progenitors. Loss of Pak2 skewed lineage differentiation toward granulocytopoiesis and monocytopoiesis in mice as evidenced by (a) a three‐ to sixfold increase in the percentage of peripheral blood granulocytes and a significant increase in the percentage of granulocyte‐monocyte progenitors in mice transplanted with Pak2‐disrupted bone marrow (BM); (b)Pak2‐disrupted BM and c‐kit+ cells yielded higher numbers of more mature subsets of granulocyte‐monocyte colonies and polymorphonuclear neutrophils, respectively, when cultured in the presence of granulocyte‐macrophage colony‐stimulating factor. Pak2 disruption resulted, respectively, in decreased and increased gene expression of transcription factors JunB and c‐Myc, which may suggest underlying mechanisms by which Pak2 regulates granulocyte‐monocyte lineage commitment. Furthermore, Pak2 disruption led to (a) higher percentage of CD4+CD8+ double positive T cells and lower percentages of CD4+CD8− or CD4−CD8+ single positive T cells in thymus and (b) decreased numbers of mature B cells and increased numbers of Pre‐Pro B cells in BM, suggesting defects in lymphopoiesis. Stem Cells 2015;33:1630–1641

Human FANCC is hypomorphic in murine Fancc-deficient cells

Fancc suppresses cross-linker-induced genotoxicity, modulates growth-inhibitory cytokine responses, and modulates endotoxin responses. Although loss of the latter function is known to account for endotoxin-induced marrow failure in murine Fancc (mFancc)-deficient mice, some argue that cytokine and endotoxin hypersensitivities devolve simply from genomic instability. Seeking to resolve this question, we planned to ectopically express instructive human FANCC (hFANCC) mutants in murine Fancc-deficient hematopoietic stem cells. To first assure that hFANCC cDNA was competent in murine cells, we compared hFANCC and mFancc in complementation assays for cross-linking agent hypersensitivity and endotoxin hypersensitivity. We found that mFancc complemented murine Fancc-deficient cells in both assays, but that hFANCC fully suppressed only endotoxin hypersensitivity, not cross-linking agent hypersensitivity. These results support the notions that Fancc is multifunctional and that structural prerequisites for its genoprotective functions differ from those required to constrain endotoxin responses known to lead to marrow failure in Fancc-deficient mice.

Leukemia and chromosomal instability in aged Fancc / mice

Fanconi anemia (FA) is an inherited disorder of genomic instability associated with high risk of myelodysplasia and acute myeloid leukemia (AML). Young mice deficient in FA core complex genes do not naturally develop cancer, hampering preclinical studies on malignant hematopoiesis in FA. Here we describe that aging Fancc(-/-) mice are prone to genomically unstable AML and other hematologic neoplasms. We report that aneuploidy precedes malignant transformation during Fancc(-/-) hematopoiesis. Our observations reveal that Fancc(-/-) mice develop hematopoietic chromosomal instability followed by leukemia in an age-dependent manner, recapitulating the clinical phenotype of human FA and providing a proof of concept for future development of preclinical models of FA-associated leukemogenesis.

Correction: INPP5E preserves genomic stability through regulation of mitosis [Molecular and Cellular Biology, 37, 6, (2017) (e00500-16)] DOI: 10.1128/MCB.00500-16

Elizabeth A. Sierra Potchanant,a Donna Cerabona,a,b Zahi Abdul Sater,a Ying He,a Zejin Sun,a Jeff Gehlhausen,a,b Stéphane Schurmans,e Stéphanie Gayral,f Grzegorz Nalepaa,b,c,d Department of Pediatrics, Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, Indiana, USAa; Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, USAb; Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, Indiana, USAc; Division of Pediatric Hematology-Oncology, Riley Hospital for Children, Indianapolis, Indiana, USAd; Laboratory of Functional Genetics, GIGA Research Center, University of Liège, Liège, Belgiume; Institut National de la Santé et de la Recherche Médicale (INSERM), UMR 1048, Institute of Metabolic and Cardiovascular Diseases, Toulouse, Francef

INPP5E Preserves Genomic Stability through Regulation of Mitosis

ABSTRACT 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.