Aurora A Kinase Is Required For Hematopoiesis and Couples Polyploidization With Terminal Differentiation In Megakaryocytes Through Phosphorylation Of NF-E2

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 219-219
Author(s):  
Benjamin Goldenson ◽  
Qiang Jeremy Wen ◽  
John D Crispino
Keyword(s):  
Bone Marrow ◽  
Transcription Factors ◽  
Primary Cultures ◽  
Terminal Differentiation ◽  
Lymphoid Cells ◽  
Conditional Knockout ◽  
Kinase Assay ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Megakaryocyte Differentiation

Abstract We have recently shown that small molecule inhibitors of Aurora A kinase (AURKA) including dimethyfasudil and MLN827 induce polyploidization and differentiation of normal and malignant megakaryocytes, as evidenced by increased DNA content and upregulation of the cell surface markers CD41 and CD42. Furthermore, our pre-clinical studies demonstrate that MLN8237 shows potent anti-leukemia activity and anti-myelofibrotic activity in MPNs by promoting polyploidization and terminal differentiation of abnormal megakaryocytes. To determine the mechanism by which AURKA inhibitors ameliorate the leukemic and myelofibrotic phenotypes, we have examined the functional requirement for Aurka in adult hematopoiesis. To circumvent the embryonic lethality of the germline knockout and study the requirement for AURKA in adult hematopoiesis, we induced deletion in Aurka floxed mice with Mx1-Cre. Complete loss of AURKA caused a rapid and profound defect in hematopoiesis, with the mice developing pancytopenia and marked hypocellularity of the bone marrow. Notably, we observe an increase in the proportion of CD41 and CD42-positive megakaryocytes in contrast to the deficiency of myeloid and lymphoid cells in the bone marrow. To determine whether the observed defects are cell autonomous, we transplanted Aurkafl/fl, Mx1-Cre bone marrow cells to lethally irradiated recipients, confirmed engraftment, and induced deletion with pIpC. Upon AURKA deletion, the transplanted mice develop an identical phenotype to the one observed in the conditional knockout mice, demonstrating a cell autonomous requirement for AURKA during hematopoiesis. Moreover, in competitive transplantation experiments, Aurka-/- cells are selectively lost and fail to contribute to hematopoiesis. Mechanistically, loss of AURKA led to a significant degree of apoptosis in hematopoietic cells, likely due to mitotic catastrophe resulting from impaired chromosome segregation. To bypass the requirement for AURKA in progenitor cells and examine AURKA function in the megakaryocyte lineage, we deleted AURKA in megakaryocytes ex vivo by infecting Aurka floxed bone marrow with MSCV-Cre. We found that deletion of AURKA resulted in increased CD41 and CD42 expression as well as increased ploidy of the megakaryocyte fraction. Together these results are consistent with a selective differentiation effect of AURKA deficiency on megakaryocytes. To investigate whether AURKA modulates differentiation of megakaryocytes through interactions with lineage specific transcription factors, we performed co-immunoprecipitation experiments between AURKA and megakaryocyte transcription factors containing consensus AURKA phosphorylation sites in primary megakaryocytes. Results confirmed a robust interaction between AURKA and p45 NF-E2. Additionally, we found by an in vitro kinase assay that AURKA phosphorylates p45 NF-E2 on the S170 residue, as the S170A mutant of NF-E2 cannot be phosphorylated by AURKA. To explore the functional importance of NF-E2 phosphorylation at S170, we created a phosphomimetic mutant, S170D, and assayed its ability to induce megakaryocyte differentiation in primary cultures. While overexpression of wild-type NF-E2 significantly increased the expression of the megakaryocyte differentiation markers CD41 and CD42, overexpression of the S170D mutant was markedly less effective in promoting megakaryocyte differentiation, indicating that the loss of phosphorylation promotes NF-E2 activity and that NF-E2 activity may be suppressed by AURKA phosphorylation. Finally, to determine if AURKA inhibitors promote megakaryocyte differentiation by allowing activation of NF-E2, we knocked-down down NF-E2 in megakaryocytic cell lines and subsequently treated with diMF or MLN8237. Strikingly, cells with NF-E2 knocked-down displayed significantly less CD41 and CD42 expression in response to AURKA inhibitor treatment compared to control knockdowns. This experiment demonstrates that NF-E2 is required for the differentiation effect caused by AURKA inhibition. Taken together, our data show that Aurora A kinase is required for adult hematopoiesis and that Aurora A regulates NF-E2 function during megakaryocyte differentiation. Our results also reveal that inhibition of AURKA kinase activity couples polyploidization and terminal differentiation of megakaryocytes. Disclosures: Crispino: Sanofi: Research Funding.

Aurora a kinase (AURKA) is required for male germline maintenance and regulates sperm motility in the mouse

2021 ◽  
Author(s):  
William C Lester ◽  
Taylor Johnson ◽  
Ben Hale ◽  
Nicholas Serra ◽  
Brian Elgart ◽  
...  
Keyword(s):  
Cell Division ◽  
Sperm Count ◽  
Mitotic Cell ◽  
Vital Role ◽  
Conditional Knockout ◽  
Testis Size ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Mitotic Kinase ◽  
Knockout Animals

Abstract Aurora A kinase (AURKA) is an important regulator of cell division and is required for assembly of the mitotic spindle. We recently reported the unusual finding that this mitotic kinase is also found on the sperm flagellum. To determine its requirement in spermatogenesis, we generated conditional knockout animals with deletion of the Aurka gene in either spermatogonia or spermatocytes to assess its role in mitotic and postmitotic cells, respectively. Deletion of Aurka in spermatogonia resulted in disappearance of all developing germ cells in the testis, as expected given its vital role in mitotic cell division. Deletion of Aurka in spermatocytes reduced testis size, sperm count, and fertility, indicating disruption of meiosis or an effect on spermiogenesis in developing mice. Interestingly, deletion of Aurka in spermatocytes increased apoptosis in spermatocytes along with an increase in the percentage of sperm with abnormal morphology. Despite the increase in abnormal sperm, sperm from spermatocyte Aurka knockout mice displayed increased progressive motility. In addition, sperm lysate prepared from Aurka knockout animals had decreased protein phosphatase 1 (PP1) activity. Together, our results show that AURKA plays multiple roles in spermatogenesis, from mitotic divisions of spermatogonia to sperm morphology and motility.

Hhex Is a Critical Gene In The Development Of Normal and Malignant Lymphoid Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3788-3788
Author(s):  
Charnise Goodings ◽  
Stephen B. Smith ◽  
Elizabeth Mathias ◽  
Elizabeth Smith ◽  
Rati Tripathi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Transgenic Mice ◽  
Conflicts Of Interest ◽  
Lymphoid Cells ◽  
Conditional Knockout ◽  
Hematopoietic Stem ◽  
Direct Transcriptional Target ◽  
Cell Counts ◽  
Transcriptional Target

Abstract Hematopoietically expressed homeobox (Hhex) is a T-cell oncogene. It is frequently deregulated in murine retroviral insertional mutagenesis screens and its enforced expression induces T-cell leukemia in bone marrow transduction and transplantation experiments. We discovered that HHEX is a direct transcriptional target of an LIM domain Only-2 (LMO2)-associated protein complex. HHEX clusters with LMO2-overexpressing T-ALLs and is especially overexpressed in Early T-cell Precursor (ETP) – ALL where it is a direct transcriptional target of LMO2. To further understand Hhex's function, we induced a conditional knockout in floxed Hhex mice with the Vav-iCre transgene. Mice were viable and showed normal blood cell counts with highly efficient deletion of Hhex in all hematopoietic tissues. Thymocytes from conditional knockouts showed a normal pattern of development. Most impressively, Hhex conditional knockout markedly prolonged the latency of T-ALL onset in CD2-Lmo2 transgenic mice (figure 1). Hhex conditional knockouts (Hhex cKOs) also had a significant decrease in mature B cells in the spleen and bone marrow. Interestingly, hematopoietic stem and progenitor cells plated on OP9-GFP or OP9-DL1 stromal cells showed proliferative defects and incomplete differentiation towards both B and T lineage. Also under stress conditions such as sublethal irradiation and competitive bone marrow transplants, Hhex conditional knockouts show a marked defect in both B and T lineages but an increase in early progenitor populations. Our experiments show that Hhex is a critical transcription factor in lymphoid development and in LMO2-induced T-ALL.Figure 1Hhex conditional knockout markedly prolonged the latency of T-ALL onset in CD2-Lmo2 transgenic miceFigure 1. Hhex conditional knockout markedly prolonged the latency of T-ALL onset in CD2-Lmo2 transgenic mice Disclosures: No relevant conflicts of interest to declare.

Cul4A Is Required for Cell Viability and Its Deficiency in Hematopoietic Cells Causes Apoptosis and Is Fatal.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 639-639
Author(s):  
Kristin T. Chun ◽  
David L. Waning ◽  
Binghui Li ◽  
Nan Jia ◽  
Yahaira M. Naaldijk ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Small Intestine ◽  
Peripheral Blood ◽  
Hematopoietic Cells ◽  
Lymphoid Cells ◽  
Conditional Knockout ◽  
Hematopoietic Stem ◽  
Mutant Cells ◽  
Total Cellularity

Abstract Critical regulators of hematopoiesis are controlled by ubiquitin-mediated proteolysis. Cul4A encodes a core subunit of one ubiquitin ligase, and previous results with hematopoietic cell lines and with Cul4A haploinsufficient mice indicate that Cul4A is required for hematopoietic stem cell function and to maintain the homeostasis of progenitors, precursors, and mature hematopoietic cells. Because Cul4A-deficiency is embryonic lethal, we generated Cul4A conditional knockout mice to examine the requirement of Cul4A for hematopoiesis in adult mice. A mutant Cul4A allele (Cul4Aflox) was constructed where its first coding exon was flanked by loxP sites. Transgenic mice with this mutant allele and the interferon-inducible Cre transgene, Mx1-Cre, were derived. When deletion of Cul4A was induced in Cul4Aflox/flox Mx1-Cre mice, the animals died within 3–10 days of the beginning of induction. Necropsies performed four days after the beginning of induction showed that all of the tissues where Mx1-Cre was reported to be expressed appeared normal, except the bone marrow, spleen, and small intestine. The red pulp in the spleen was diminished, there were many fewer nucleated cells in the bone marrow, and the microvilli of the small intestine (duodenum) were dramatically shortened. The mass and total cellularity of mutant spleens were half of controls (Cul4Aflox/flox mice without Mx1-Cre), and bone marrow total cellularity was one-tenth of controls. The frequency of mutant hematopoietic progenitors was reduced 3800-fold in the bone marrow and 80-fold in the spleen. Peripheral blood counts of mature myeloid and lymphoid cells were also dramatically reduced. To separate the in vivo effects of Cul4A-deficiency in hematopoietic cells from those in other cell types, conditional mutant bone marrow was transplanted into wild type recipients, these cells were allowed to engraft for 2 months, and then Cul4A deletion was induced. Mutant animals died within 9–11 days of the beginning of induction with bone marrow nearly empty of cells, spleens only 29% the mass of controls, myeloid and lymphoid counts in the peripheral blood reduced to nearly zero, hematocrits at only 21% of controls, and platelet counts at only 10% of controls. The small intestine, however appeared normal, indicating that Cul4A-deficiency in hematopoietic cells is sufficient to cause death. To examine the fate of Cul4A-deficient hematopoietic cells, deletion was induced in vivo in Cul4Aflox/flox Mx1-Cre and control mice, and then bone marrow was harvested and cultured in vitro. Apoptotic cells were detected (either Annexin V positive, 7-AAD negative or TUNEL positive cells) 2–5 days after induction. At 4 and 5 days after induction, the frequency of apoptotic mutant cells was significantly greater than controls (P=0.01 and 0.03, respectively), and at 5 days the frequency of TUNEL positive cells was 4.5-fold greater in the mutant cells. Together, these results indicate that Cul4A-deficiency in hematopoietic cells results in apoptosis, a failure of the hematopoietic system, and death. Analyses of how the expression levels of Cul4A target proteins are altered by Cul4A-deficiency will be presented.

Ontogeny of Hematopoiesis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. SCI-4-SCI-4
Author(s):  
Elaine Dzierzak
Keyword(s):  
Bone Marrow ◽  
Endothelial Cells ◽  
Transcription Factors ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Developmental Time ◽  
Hematopoietic Cell ◽  
Conditional Knockout ◽  
Hematopoietic Stem ◽  
And Function

Abstract The current challenge in hematopoietic transplantation and regeneration therapies is acquiring and/or producing a reliable and plentiful source of hematopoietic stem cells (HSCs). Given that HSCs from bone marrow, peripheral, or umbilical cord blood undergo only limited/no expansion ex vivo, there is a high interest in understanding how the adult cohort of multipotent self-renewing HSCs are generated and expanded during embryonic development. The development of HSCs in vertebrate embryos begins in the major vasculature. HSCs are generated in a short window of developmental time starting at embryonic day E10.5 until E12 in the mouse embryo, and from gestational weeks four to six in the human embryo. The first HSCs, which are as potent as bone marrow HSCs in transplantation procedures, are generated in the aorta-gonad-mesonephros (AGM) region. HSCs are found in the major vasculature – aorta, vitelline artery, and umbilical artery – subsequent to the appearance of hematopoietic cell clusters closely associated with the lumenal walls of these vessels. The relationship of HSCs to these clusters and the identification of the precursors to HSCs have been recently established through genetic, phenotypic, and real-time imaging studies. Remarkably, HSCs and hematopoietic progenitors arise directly from a subset of endothelial cells (hemogenic endothelial cells) in a natural transdifferentiation event. They are made through a process called endothelial to hematopoietic cell transition (EHT). EHT and HSC generation is in part regulated through ventral-derived developmental signals and a group of pivotal (core) transcription factors, including Runx1 and Gata2. Conditional knockout strategies show that these transcription factors are required for the generation of vascular hematopoietic clusters and HSCs, suggesting a role in hematopoietic fate induction and/or cell expansion. Interestingly, whereas both Runx1 and Gata2 are required for HSC generation, only Gata2 remains essential in HSCs after their production. We are profiling hemogenic endothelial and HSCs by RNA sequencing so as to understand the complete genetic program that leads to generation of HSCs. These results will be discussed in the context of developmental signaling pathways (BMP4, Hedgehog, etc.) that appear to impact HSC generation and expansion, and the localized dynamic expression and function of Gata2 and Runx1 in vascular endothelial and hematopoietic cluster cells. Disclosures: No relevant conflicts of interest to declare.

Aurora A Kinase Is Required for Hematopoiesis

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1201-1201
Author(s):  
Benjamin Goldenson ◽  
Jeremy Q Wen ◽  
John Crispino
Keyword(s):  
Bone Marrow ◽  
Steady State ◽  
Small Molecule ◽  
Dna Content ◽  
Colony Formation ◽  
Conflicts Of Interest ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Wild Type ◽  
Significant Difference

Abstract Abstract 1201 In acute megakaryocytic leukemia (AMKL), there is a failure of megakaryocytes to differentiate, become polyploid and stop dividing. We used an integrated screening approach that included chemical, proteomic and genetic screens to identify small molecules and their targets that control polyploidization and differentiation of normal and malignant megakaryocytes. We identified several small molecule inducers of polyploidy and used siRNA and proteomic target ID approaches to determine the cellular targets of the lead small molecule dimethylfasudil (diMF). Aurora Kinase A (AURKA) was identified as one of the top targets of diMF. AURKA is an attractive target in AMKL for several reasons. First, AURKA is overexpressed in AMKL cells. Second, at the biologically effective doses used in our cell-based assays, AURKA inhibition was selective for the megakaryocyte lineage. Third, AURKA inhibition by diMF or the selective AURKA inhibitor MLN8237 increased MK polyploidy, induced features of differentiation, blocked proliferation of AMKL blasts, and improved survival in an AMKL mouse model. AURKA is important in mitotic spindle assembly, mitosis, chromosomal alignment and segregation. Moreover, it is required for embryonic development, as Aurka−/− embryos fail to grow beyond the blastocyst stage. However, the extent to which AURKA is necessary for steady state hematopoiesis in adults is unknown. To investigate the necessity of AURKA in hematopoiesis, we utilized a conditionally targeted strain of mice (Aurkaflox/flox). To delete AURKA in megakaryocytes ex vivo, Aurkaflox/flox bone marrow cells were expanded, transduced with a retrovirus expressing Cre and GFP, and then cultured in the presence of THPO for 72 hours. We found that deletion of AURKA resulted in increased CD41 and CD42 expression as well as increased DNA content. Assays for apoptosis by Annexin V staining of Aurkaflox/flox cells infected with Cre also showed increased apoptosis in AURKA-deleted cells at 24 and 48 hours. To delete AURKA in vivo, we crossed Aurkaflox/flox mice to MX1-Cre mice and injected wild-type, heterozygous and homozygous floxed mice expressing MX1-Cre with pIpC every other day for six days. We found that deletion of AURKA in hematopoietic progenitors leads to pancytopenia, profound bone marrow defects and death within two weeks. Colony formation assays showed significantly decreased myeloid, erythroid and megakaryocyte colony formation with AURKA deficiency. Bone marrow histology displayed markedly hypocellular marrow, but curiously, flow cytometry revealed a significant increase in the percentage of CD41 and CD42 positive cells. This observation suggests that AURKA normally acts to restrain terminal differentiation of megakaryocytes and is consistent with the CD41 and CD42 inducing ability of AURKA inhibitors. To confirm that AURKA is the key target of our recently identified polyploidy inducers, we assayed the effects of diMF and MLN8237 on Aurka+/+, Aurka+/− and Aurka−/− megakaryocytes. 300 nM diMF and 100 nM MLN8237, concentrations that strongly induce polyploidy, did not increase MK polyploidization in Aurka−/− MKs. diMF and MLN8237 treatment increased polyploidy in Aurka+/− MKs with no significant difference in comparison to Aurka+/+ MKs. We also assayed the ability of wild-type or the T217D mutant of AURKA, which is resistant to inhibition by MLN8237, to reduce the induction of polyploidy caused by diMF and MLN8237 upon overexpression. CMK cells were infected with viruses harboring wild-type or T217D AURKA, treated with DMSO, 3 μM diMF or 30 nM MLN8237 for 72 hours, and then evaluated for DNA content. The increase in polyploidization induced by both compounds was significantly decreased in cells overexpressing the T217D mutant of AURKA. With overexpression of the wild-type AURKA, there was a trend towards reduction in polyploidy, but more variable effects and no significant difference. Thus, AURKA T217D overexpression reduced the ability of diMF and MLN8237 to induce polyploidization, consistent with our conclusion that diMF targets AURKA. Together, our data support a role of AURKA in megakaryocyte polyploidization and differentiation and show that AURKA is required for steady state hematopoiesis. The results also show that AURKA is the key target of diMF in the induction of polyploidization of megakaryocytes and support the development of Aurora A kinase inhibitors in clinical trials for AMKL. Disclosures: No relevant conflicts of interest to declare.

Aurora A Kinase Is a Novel Therapeutic Target In The Myeloproliferative Neoplasms

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 109-109 ◽  
Author(s):  
Qiang Jeremy Wen ◽  
Benjamin Goldenson ◽  
Sebastien Malinge ◽  
Brady L Stein ◽  
Terra L Lasho ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mouse Models ◽  
Disease Burden ◽  
Jak Inhibitor ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Heterozygous Deletion ◽  
Jak Inhibitors

Abstract We recently reported that the induction of polyploidization of malignant megakaryocytes shows great promise as a new therapy for acute leukemia. Polyploidization inducers such as dimethylfasudil (diMF) and MLN8237, both of which target Aurora A kinase (AURKA), induce proliferation arrest, polyploidization, expression of megakaryocyte differentiation markers and apoptosis of leukemic megakaryocytes in vitro and in vivo. Since megakaryocytes in primary myelofibrosis (PMF) show impaired polyploidization and maturation, and likely directly contribute to the disease, we predicted that polyploidization inducers would provide a new therapeutic strategy. To determine the effect of these compounds on the growth of MPN cells, we first treated the JAK2 V617F mutant megakaryocytic SET2 cell line with varying doses of MLN8237 and diMF. Both compounds effectively and dose dependently inhibited proliferation, induced polyploidization and upregulation of lineage specific markers CD41 and CD42, and increased apoptosis. Furthermore, MLN8237 synergized with ruxolitinib to induce apoptosis of the SET2 cells and also potently induced growth arrest of JAK2 inhibitor persistent SET2 cells. We observed a similar polyploidization and differentiating activity of MLN8237 and diMF on megakaryocytes derived from primary human PMF progenitors. The ability of these agents to induce polyploidization was specific, as the non-megakaryocyte fractions of the cultures were not affected. Next, we assayed the activity of polyploidization inducers on progression of MPNs in two mouse models: JAK2V617F conditional knockin mice and mice engrafted with MPLW515L expressing bone marrow progenitors. Of note, spleens from both mouse models displayed a robust increase in both total and phosphorylated forms of AURKA relative to control animals, further suggesting that AURKA is a rational target in this disease. We first assayed the activities of MLN8237 and diMF in the MPLW515L bone marrow transplantation model. Recipient mice develop a rapid MPN characterized by leukocytosis, thrombocytosis and bone marrow fibrosis. Both MLN8237 and diMF reduced the disease burden, as evidenced by significant reductions in the liver and spleen weights, white cell counts and platelet counts. Both compounds also led to a significant decrease of fibrosis in the bone marrow, diminished infiltration of megakaryocytes and granulocytes in the liver, and a profound reduction in the numbers of megakaryocytes within the spleen. Moreover, plasma levels of TGF-β a known myelofibrogenic cytokine, were decreased by more than 3-fold by the drug treatment. Both diMF and MLN8237 led to selective polyploidization of megakaryocytes in the spleen as well as marked reductions in the levels of p-AURKA. Of note, neither agent affected the extent of phosphorylation of STAT3 or STAT5. Therefore, we tested whether the combined use of a JAK inhibitor and a polyploidy inducer would show enhanced activity in vivo. Indeed, the combination of MLN8237 and ruxolitinib led to greater reductions in tumor burden in the MPLW515L mouse model than either agent alone. Similar results were obtained using the JAK2V617F knock-in model. To further validate our conclusion that AURKA is a target in PMF, we infected Aurkafl/fl floxed bone marrow progenitors with MPLW515L and transplanted the cells to irradiated recipients. Excision of both alleles of Aurka by Cre mediated recombination completely resolved the disease, while heterozygous deletion of Aurka significantly reduced the disease burden, in a manner similar to treatment with MLN8237. Given that heterozygous deletion of Aurka does not alter normal hematopoiesis in mice, the fact that a 50% reduction in kinase expression was associated with a significant decrease in disease burden suggests that there is an effective therapeutic window in which AURKA inhibitors will be effective against MPN while sparing normal hematopoiesis. Although JAK inhibitors provide symptomatic relief, it is becoming clear that they are not curative. Thus, there is an urgent need to develop new agents to use in combination with JAK inhibitors. Our data reveal that inducing polyploidization and differentiation of dysplastic megakaryocytes in PMF ameliorates features of the disease both in vitro and in vivo. These results support the initiation of clinical studies that combine a JAK inhibitor with an AURKA inhibitor. Disclosures: Crispino: Sanofi: Research Funding.

Aurora-A Kinase Expression In Untreated Patients With Chronic Lymphocytic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4896-4896
Author(s):  
Gurhan Kadikoylu ◽  
Deniz Cetin ◽  
Gokay Bozkurt ◽  
Firuzan Kacar ◽  
Irfan Yavasoglu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Immunohistochemical Staining ◽  
Conflicts Of Interest ◽  
Deficiency Anemia ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Rt Pcr ◽  
Gapdh Mrna ◽  
Roche Diagnostics ◽  
Kinase Expression

Abstract Aurora-A kinase is a cell-cyle regulating kinase required for chromosomal segregation. Overexpression of Aurora-A kinase has been detected some solid tumors and hematological malignancies such as multiple myelomai non-Hodgkin's lymhoma, and acute leukemia. But there are only two studies in chronic lymphocyic leukemia (CLL). This prospective study was approved by Ethical Committee of University. We investigated Aurora-A kinase in bone marrow of 41 untreated patients (22 male and 19 female with mean age of 70±10 years) with CLL and 19 patients (8 male and 11 female with mean age of 54±20 years) with anemia such as megaloblastic, autoimmune hemolytic, and iron deficiency anemia using a quantitative reverse transcriptase-PCR (RT-PCR) method. β-Actin and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) mRNA was used as the internal controls. Total RNA was extracted from bone marrow cells using the Trizol method (High Pure Isolation Kit, Roche Diagnostics). cDNA was prepared with the Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics). Aurora-A cDNA was quantified using TaqMan Universal PCR Mastermix (Applied Biosystems) and the Aurora-A TaqMan Gene Expression Assay. β-Actin was assayed using TaqMan Universal PCR Mastermix with forward (CCCTGGCACCCAGCAC) and reverse (GCCGATCCACACGGAGTAC) primers at 400nM each and probe (fam-ATCAAGATCATTGCTCCTCCTGAGCGC-bhq) at 100nM concentrations. Real-time quantitative RT-PCR was performed in LightCycler 480II (Roche Diagnostics). Relative RNA level was reported via standard delta delta Ct (dd Ct). Immunhistochemical analysis was performed using formalin-fixed, parafin-embedded sections of bone marrow biopsy specimens from patients and controls. Tissue sections were incubated for 60 minutes with Aurora-A (Novus Biologicals Inc. Littleton CO, 1:100 dilution). Aurora-A kinase is establish as positive if exist >10% in the cytoplasms and nuclei of the neoplastic cells in all cases. If this staining is 0-10% of cells, it is accepted as slight positive. If these cells is never (0%) stained, it is negative. For the comparison of values, Mann-Whitney-U, Chi-square, and One-Way ANOVA tests were used by SPSS 15.0 for Windows. With FISH method, 17p and 13q deletions were detected in 10% and 37% of the patients CLL, respectively. There was trisomy 12 in 7% of teh patients. 68% of the patients with CLL were in Binet-A stage. By immunohistochemical analysis, while Aurora-A kinase was positive in 61% of the patients, it was negative in 72% and slight positive 28% of controls. These positivity was statistically significant (p<0.001). By RT-PCR, β-Actin and GAPDH mRNA values were 3.85±2.61 and 3.49±2.32 in the patients with CLL, respectively. These values were 3.80±2.73 and 4.34±2.36 in controls, respectively. There was no difference for both β-Actin and GAPDH mRNA values between two groups (p>0.05). According to Binet classification, there was no difference for Aurora-A kinase expression with RT-PCR and immunohistochemical staining (p>0.05). Moreover there was no difference for both expression of Aurora-A kinase and immunhistochemical staining in between the patients with chromosmal ambnormalities and without (p>0.05). Although overexpression of Aurora-A kinase expression was not detected, significant immunohistochemical staining represented that Aurora-A kinase can be potential therapeutic target in CLL. Disclosures: No relevant conflicts of interest to declare.

The Role of Forkhead Transcription Factors FoxO1, FoxO3 and FoxO4 in Hematopoiesis and Leukemogenesis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1350-1350
Author(s):  
Zuzana Tothova ◽  
Ramya Kollipara ◽  
Ronald A. DePinho ◽  
D. Gary Gilliland
Keyword(s):  
Bone Marrow ◽  
Transcription Factors ◽  
Family Members ◽  
Hematopoietic Cells ◽  
Conditional Knockout ◽  
Forkhead Transcription Factors ◽  
Anaplastic Lymphoma ◽  
Efficient Transformation ◽  
Survival Signals

Abstract FoxO is a family of forkhead transcription factors that negatively regulate proliferation and survival signals in hematopoietic cells. We and others have previously shown that inhibition of the three members of this family (FoxO1, FoxO3 and FoxO4) by leukemogenic tyrosine kinase fusion genes results in enhanced proliferative and survival signaling in leukemic cells. For example, the transforming activities of the lymphoma associated NPM-ALK (nucleophosmin-anaplastic lymphoma kinase) fusion, BCR-ABL, or FLT3-ITD, are mediated in part by inactivation of FoxO through phosphorylation and ubiquitin mediated degradation by constitutively active Akt (Gu TL, et al. Blood 2004), with subsequent induction of proliferative and survival signals. Furthermore, inhibition of FoxO is required for efficient transformation of hematopoietic cells. However, the roles of FoxO in adult hematopoiesis are unknown. We have initiated studies to examine the role of FoxO in the context of normal hematopoiesis and leukemogenesis using a triple conditional knockout mouse for each of the FoxO1, FoxO3 and FoxO4 alleles. The FoxO alleles are flanked by lox-P sites and conditional excision is mediated by Cre expression under the control of the interferon inducible Mx1 promoter. Based on the normal function of FoxO family members to repress proliferative and survival signals, we hypothesized that the deletion of FoxO subfamily members would lead to an enhanced proliferation and survival in the hematopoietic compartment, and might contribute to the development of a myeloproliferative and/or lymphoproliferative phenotype in vivo. Triple homozygous conditional FoxO knockout mice were generated in an Mx1-Cre background to allow for excision of the FoxO alleles in the hematopoietic stem cell compartment after treatment with pIpC. Complete excision of each of the three alleles in the hematopoietic compartment was confirmed. However, in contrast with our working hypothesis, we observed that loss of function of FoxO family members was associated with a relatively subtle hematopoietic phenotype with 12 months of follow-up. The phenotype includes a non-fatal mild myeloproliferative phenotype that is progressive over time and characterized by modest splenomegaly, extramedullary hematopoiesis and increased mature myeloid populations in bone marrow and spleen. In addition, there are subtle alterations in both B and T lymphoid cell populations, including a decrease in both immature and mature B cells in the spleen and bone marrow; and abnormalities of CD4+CD8+ double positive and CD4+ and CD8+ T cells in the thymus. Examination of stem and progenitor populations also revealed subtle differences in the HSC and CLP progenitor populations at 4 weeks post pIpC. Thus, these data indicate that complete loss of FoxO function in the adult hematopoietic compartment results in a relatively subtle hematopoietic phenotype. They further demonstrate that although inhibition of FoxO family members is required for efficient transformation of hematopoietic cells by leukemogenic fusion tyrosine kinases, loss of FoxO function alone is not sufficient to induce a leukemic phenotype.

Satb1 Determines Hematopoietic Stem Cell Differentiation Toward the Lymphoid Lineages

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 388-388
Author(s):  
Yusuke Satoh ◽  
Takafumi Yokota ◽  
Motonari Kondo ◽  
Paul W. Kincade ◽  
Taku Kouro ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transcription Factors ◽  
Es Cells ◽  
Lymphoid Cells ◽  
The Other ◽  
Hematopoietic Stem ◽  
Over Expression ◽  
Lymphoid Lineage ◽  
Other Hand ◽  
B Lineage

Abstract Abstract 388 There is accumulating evidence that combinations of transcription factors coordinately and sequentially regulate lymphopoiesis. Five transcription factors, PU.1, Ikaros, E2A, EBF, and Pax5 are known to be hierarchically involved in early steps in B-lineage differentiation. However, it remains unclear whether the initiation of lymphoid differentiation is regulated entirely by transcription factors in a hierarchical manner. A major goal of our study was to find key genes involved in specification of lymphoid fates. For this purpose, we compared gene expression profiles between hematopoietic stem cells (HSC) and early lymphoid progenitors (ELP), which were sorted from E14.5 fetal liver of Rag1/GFP knock-in heterozygous embryos. As a result, we found that the expression of Satb1, a global chromatin regulator, was significantly increased along the differentiation of HSC to ELP. To explore roles of Satb1 in early lymphoid specification, we performed transplantation experiments, injecting HSC isolated from bone marrow of 2 weeks old Satb1-null mice into wild-type (WT) mice. We observed that Satb1-null HSC could not reconstitute CD3+ T cells in lethally irradiated WT recipients. Indeed, CD3+ T lineage recoveries from Satb1-null HSC were decreased approximately 10-fold compared with WT HSC. On the other hand, we observed varied levels of reconstitution of the B lineage and no reduction in reconstitution of the myeloid lineage resulted from Satb1 ablation. These results demonstrate that expression of Satb1 in HSC is indispensable for lymphopoiesis, but not for myelopoiesis. Furthermore, our data indicate that abnormalities of lymphoid development observed in Satb1-null mice are intrinsic to Satb1-deficient HSC. Next we conducted over-expression experiments to define the role of Satb1 in lineage fate decisions of HSC. Limiting dilution assays in MS5 co-culture condition revealed 1 in 41 Satb1-transduced Flt3−Lineage−Sca1+c-Kit+ (LSK) cells produced B cells. However, only 1 in 143 control ones were lymphopoietic under these conditions. These results suggest that Satb1 affects the lineage fate of HSC and promotes their commitment to lymphoid cells. Next we examined if the exogenous expression of Satb1 promotes B lymphocyte growth from ES cells in the OP9 co-culture system. We established ES cell clones, which can be induced to express Satb1/GFP on removal of tetracycline (Tet) from the culture medium. Eight days after Tet deprivation, 22 % of GFP+ cells expressed CD45 and CD19. On the other hand, only 1% of GFP− cells expressed same cell-surface markers. A majority of the CD19+ cells in Satb1/GFP+ ES-derived cells were positive for CD11b/Mac1 and/or CD5, suggesting that B1-B lineage cells were produced. In addition, Igh rearrangement assay revealed that DH-JH recombination occurred in the Satb1/GFP+ ES-derived cells. These results indicate that Satb1 over-expression directs even ES cells to differentiate toward the lymphoid lineage. Lymphopoietic activity becomes compromised during aging. Accumulating evidence suggests that the earliest lymphoid progenitor pools proximal to HSC are deficient in aged bone marrow. It is likely that the down-regulation of genes mediating lymphoid specification and function is involved as a major cause. Satb1 has been listed in microarray panels as one of the genes that are down-regulated in aged HSC (Rossi et al., 2005). To confirm this observation, we sorted CD150+ LSK cells from bone marrow of 6-weeks or 2-years old mice and examined expressions of Satb1. As a result, the aged CD150+LSK cells showed reduced expression (about 6-fold) of Satb1 compared with these cells from young mice. We then examined whether Satb1 expression could restore the lymphopoietic activity of progenitors derived from aged mice. Satb1-transduced Rag1/GFP− LSK cells produced significantly higher percentages of B220+ cells than control cells. With respect to the recovered B-lineage cell counts, about 3-fold more B220+ Rag1/GFP+ Mac1−cells were obtained by Satb1transduction than mock transduction. These results demonstrate that Satb1 can at least partially restore the lymphopoietic activity of aged hematopoietic progenitors. In conclusion, our results indicate that Satb1 plays critical roles in producing lymphoid lineage from primitive stem/progenitor cells. Such activity in generating lymphoid cells may be of clinical significance and useful to overcome immuno-senescence. Disclosures: No relevant conflicts of interest to declare.

Centrosome Aurora A gradient ensures single polarity axis in C. elegans embryos

2020 ◽  
Vol 48 (3) ◽  
pp. 1243-1253 ◽  
Author(s):  
Sukriti Kapoor ◽  
Sachin Kotak
Keyword(s):  
Symmetry Breaking ◽  
Cell Fate ◽  
Cell Cortex ◽  
Axis Formation ◽  
Aurora A Kinase ◽  
Aurora A ◽  
C Elegans ◽  
Cell Embryo ◽  
Polarity Establishment

Cellular asymmetries are vital for generating cell fate diversity during development and in stem cells. In the newly fertilized Caenorhabditis elegans embryo, centrosomes are responsible for polarity establishment, i.e. anterior–posterior body axis formation. The signal for polarity originates from the centrosomes and is transmitted to the cell cortex, where it disassembles the actomyosin network. This event leads to symmetry breaking and the establishment of distinct domains of evolutionarily conserved PAR proteins. However, the identity of an essential component that localizes to the centrosomes and promotes symmetry breaking was unknown. Recent work has uncovered that the loss of Aurora A kinase (AIR-1 in C. elegans and hereafter referred to as Aurora A) in the one-cell embryo disrupts stereotypical actomyosin-based cortical flows that occur at the time of polarity establishment. This misregulation of actomyosin flow dynamics results in the occurrence of two polarity axes. Notably, the role of Aurora A in ensuring a single polarity axis is independent of its well-established function in centrosome maturation. The mechanism by which Aurora A directs symmetry breaking is likely through direct regulation of Rho-dependent contractility. In this mini-review, we will discuss the unconventional role of Aurora A kinase in polarity establishment in C. elegans embryos and propose a refined model of centrosome-dependent symmetry breaking.

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