Unraveling a Novel Mechanism of Stat5 Regulation by FAK and Rac1 in Flt3ITD Induced Leukemogenesis

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 858-858
Author(s):  
Anindya Chatterjee ◽  
Joydeep Ghosh ◽  
Baskar Ramdas ◽  
Sasidhar Vemula ◽  
Holly Martin ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Essential Role ◽  
Nuclear Translocation ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Dominant Negative ◽  
Post Transplantation ◽  
Pharmacological Inhibition ◽  
F 14

Abstract Abstract 858 Multiple genetic checks and balances regulate the complex process of hematopoiesis. Despite these measures, mutations in crucial regulatory genes are still known to occur, which in some cases results in abnormal hematopoiesis, including leukemogenesis and/or myeloproliferative neoplasms (MPN). An example of a mutated gene that contributes to leukemogenesis is the FMS- like tyrosine kinase 3 (Flt3) that encodes a receptor tyrosine kinase, which plays an essential role in normal hematopoiesis. Interestingly, Flt3 is one of the most frequently mutated genes (∼30%) in acute myeloid leukemia (AML). Although various pathways downstream of Flt3 activation that lead to leukemic transformation have been extensively studied, effective treatment options for Flt3ITD mediated leukemogenesis is still warranted. In this study we used genetic, pharmacological and biochemical approaches to identify a novel role of Focal adhesion kinase (FAK) in Flt3ITD induced leukemogenesis. We observed hyperactivation of FAK in Flt3ITD expressing human and mouse cell. Treatment with FAK specific small molecule inhibitors F-14 and Y-11, inhibited proliferation and induced cell death of Flt3ITD expressing cells. Similarly, treatment of primary AML patient samples (n=9) expressing Flt3ITD mutations with F-14 inhibited their proliferation. Consistently expression of a dominant negative domain of FAK (FRNK) inhibited hyperproliferation and induced death of Flt3ITD bearing cells. Further, low-density bone marrow (LDBM) cells derived from FAK−/− mice transduced with Flt3ITD showed significantly reduced growth compared to wild-type (WT) LDBM cells transduced with Flt3ITD. We also observed hyperactivation of Rac1 in Flt3ITD expressing cells downstream of FAK, which was downregulated upon treatment with FAK inhibitor F-14 and Y11. Moreover, expression of dominant negative Rac1N17, or treatment with Rac1 inhibitor NSC23766 inhibited hyperproliferation of ITD bearing cells. We next wanted to ascertain the underlying mechanism of FAK mediated activation of Rac1 in Flt3ITD expressing cells. Toward this end, we found RacGEF Tiam1 to be hyperactive in Flt3ITD expressing cells, which was downregulated upon pharmacological inhibition of FAK. A Tiam1-Rac1 complex was also co-immunoprecipitated from Flt3ITD bearing cells, and this association was perturbed upon pharmacological inhibition of FAK. While, Stat5 a key molecule in Flt3ITD mediated leukemic progression, is activated and recruited to the nucleus to express Stat5 responsive genes; however the mechanism of Stat5 translocation to the nucleus is unknown. We observed a novel mechanism involving FAK and Rac1GTPase, in regulating the nuclear translocation of active Stat5. Pharmacological inhibition of FAK and Rac1 resulted in reduced Rac1 and STAT5 translocation into the nucleus, indicating a role of FAK-Rac-STAT5 signaling in Flt3ITD induced leukemogenesis. More importantly, expression of Flt3ITD in Rac1−/− or FAK−/− LDBM cells, showed inhibition of Stat5 activation and its failure to translocate into the nucleus when compared to Flt3ITD expression in WT-LDBM cells. We also observed association between active Rac1 and active Stat5 in the nucleus and in whole cell lysates of Flt3ITD bearing cells, and also in human AML patient samples (n=3), which was attenuated upon pharmacological inhibition of FAK. To determine the effect of FAK inhibition in vivo on Flt3ITD induced MPN, syngeneic transplantation was performed, and mice were treated with vehicle or with FAK inhibitor F-14. While vehicle treated mice developed MPN within 30 days, mice treated with F-14 showed significant overall survival (*p<0.02) and over 50% F-14 treated mice survived till 60 days post transplantation. Inhibition of kinases, and other signaling molecules, that are deregulated in cancer is an exciting new therapeutic strategy. This study indicate an essential role of FAK and Rac1 molecules in Flt3ITD mediated proliferation, survival and leukemogenesis, and demonstrates a novel mechanistic role of FAK/Rac1 in translocating active Stat5 into the nucleus and regulates transformation. To our knowledge, this is also the first time a role of RacGEF Tiam1 is observed in Flt3ITD induced leukemogenesis. Overall, this study demonstrates inhibition of FAK and Rac1 as potentially novel targets, and provides an alternative approach in treating humans suffering from Flt3-ITD induced AML. Disclosures: No relevant conflicts of interest to declare.

scholarly journals TRIM31 facilitates K27-linked polyubiquitination of SYK to regulate antifungal immunity

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Xueer Wang ◽  
Honghai Zhang ◽  
Zhugui Shao ◽  
Wanxin Zhuang ◽  
Chao Sui ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Receptor Tyrosine Kinase ◽  
Fungal Pathogen ◽  
Essential Role ◽  
Molecular Mechanisms ◽  
Systemic Infection ◽  
Lectin Receptors ◽  
Innate And Adaptive Immunity ◽  
Cytokines And Chemokines

AbstractSpleen tyrosine kinase (SYK) is a non-receptor tyrosine kinase, which plays an essential role in both innate and adaptive immunity. However, the key molecular mechanisms that regulate SYK activity are poorly understood. Here we identified the E3 ligase TRIM31 as a crucial regulator of SYK activation. We found that TRIM31 interacted with SYK and catalyzed K27-linked polyubiquitination at Lys375 and Lys517 of SYK. This K27-linked polyubiquitination of SYK promoted its plasma membrane translocation and binding with the C-type lectin receptors (CLRs), and also prevented the interaction with the phosphatase SHP-1. Therefore, deficiency of Trim31 in bone marrow-derived dendritic cells (BMDCs) and macrophages (BMDMs) dampened SYK-mediated signaling and inhibited the secretion of proinflammatory cytokines and chemokines against the fungal pathogen Candida albicans infection. Trim31−/− mice were also more sensitive to C. albicans systemic infection than Trim31+/+ mice and exhibited reduced Th1 and Th17 responses. Overall, our study uncovered the pivotal role of TRIM31-mediated K27-linked polyubiquitination on SYK activation and highlighted the significance of TRIM31 in anti-C. albicans immunity.

scholarly journals Depletion of Centromeric MCAK Leads to Chromosome Congression and Segregation Defects Due to Improper Kinetochore Attachments

2004 ◽  
Vol 15 (3) ◽  
pp. 1146-1159 ◽  
Author(s):  
Susan L. Kline-Smith ◽  
Alexey Khodjakov ◽  
Polla Hergert ◽  
Claire E. Walczak
Keyword(s):  
Essential Role ◽  
Dominant Negative ◽  
Primary Role ◽  
Complex Behavior ◽  
Chromosome Movement ◽  
Immunofluorescence Localization ◽  
Microtubule Attachment ◽  
Chromosome Congression

The complex behavior of chromosomes during mitosis is accomplished by precise binding and highly regulated polymerization dynamics of kinetochore microtubules. Previous studies have implicated Kin Is, unique kinesins that depolymerize microtubules, in regulating chromosome positioning. We have characterized the immunofluorescence localization of centromere-bound MCAK and found that MCAK localized to inner kinetochores during prophase but was predominantly centromeric by metaphase. Interestingly, MCAK accumulated at leading kinetochores during congression but not during segregation. We tested the consequences of MCAK disruption by injecting a centromere dominant-negative protein into prophase cells. Depletion of centromeric MCAK led to reduced centromere stretch, delayed chromosome congression, alignment defects, and severe missegregation of chromosomes. Rates of chromosome movement were unchanged, suggesting that the primary role of MCAK is not to move chromosomes. Furthermore, we found that disruption of MCAK leads to multiple kinetochore–microtubule attachment defects, including merotelic, syntelic, and combined merotelic-syntelic attachments. These findings reveal an essential role for Kin Is in prevention and/or correction of improper kinetochore–microtubule attachments.

Kinase Activity of RTKs Is Dispensable for Factor Independent Growth and Transformation of a Myeloid Cell Line 32D in the Presence of Cbl Mutants.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3970-3970
Author(s):  
◽  
Srinivasa Rao Bandi ◽  
Marion Rensinghoff ◽  
Rebekka Grundler ◽  
Lara Tickenbrock ◽  
...  
Keyword(s):  
Cell Line ◽  
Tyrosine Kinases ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Conflicts Of Interest ◽  
Myeloid Cell ◽  
Dominant Negative ◽  
Class Iii ◽  
Primary Resistance

Abstract Abstract 3970 Poster Board III-906 Purpose The Cbl proto-oncogene products have emerged as important components of the signal transduction cascades downstream of both non-receptor and receptor tyrosine kinases (RTKs). By regulation of receptor trafficking and degradation, they have been shown to tightly regulate the intensity and amplitude of RTK activation. c-Kit belongs to the family of the class-III RTKs and plays an important role in the pathogenesis of acute myeloid leukemia (AML). So far, very little is known about the role of c-Cbl mutants in the role of c-Kit signaling. Results We analyzed the interaction of c-Cbl with c-Kit and the functional relevance of this interaction in the IL-3-dependent murine myeloid progenitor cell line 32Dcl3. We recently identified the first c-Cbl mutation in human disease in an AML patient, named Cbl-R420Q. Co-expression of two different dominant negative mutants of c-Cbl (Cbl-R420Q or Cbl-70Z) with Kit induced cytokine-independent proliferation, survival and clonogenic growth. Importantly, transformation was observed also with kinase-dead forms of Kit and Flt3 in the presence of Cbl-70Z, but not in the absence of Kit or Flt3, suggesting a mechanism dependent on RTKs, but independent of their kinase activity. Instead, transformation appeared to depend on Src family kinases (SFKs), as c-Cbl co-immunoprecipitated with SFKs and SFK inhibition abolished transformation. Conclusion Our results indicate that c-Cbl has an important role in c-Kit signal mitigation. They demonstrate that disturbed mechanisms of c-Kit internalization have important implications for its transforming potential, possibly in the development of AML. Furthermore, these findings may explain primary resistance to tyrosine kinase inhibitors targeted at RTKs. Disclosures: No relevant conflicts of interest to declare.

Frequency and Determinants of Thrombotic and Bleeding Complications in Ph Negative Myeloproliferative Neoplasms (MPN): The Role of Adamts-13 and VWF Activities in an Italian Cohort 0f 88 Well Characterized Patients

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3369-3369
Author(s):  
Augusto B. Federici ◽  
Maria C Carraro ◽  
Antonella Lattuada ◽  
Chiara Vanelli ◽  
Veronica Sciumbata ◽  
...  
Keyword(s):  
Risk Factors ◽  
Multivariate Analysis ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Thrombotic Event ◽  
Previous History ◽  
Bleeding Complications ◽  
Post Surgery ◽  
Bleeding Episodes

Abstract Abstract 3369 Background: Patients with Ph-negative Myeloproliferative Neoplasms (MPN) such as Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) can be exposed during the course of these MPN to thrombotic and bleeding complications, with increased morbidity and mortality. Age, previous history of thrombosis, increased White Blood Cell (WBC) and Jak2 allele burden have been proposed as risk factors for Venous (VTE) and Arterial (ATE) thromboses while bleeding has been previously associated with abnormalities of the von Willebrand factor (VWF). Aims: To investigate any significant role of ADAMTS-13 and VWF activities in the thrombotic and bleeding complications observed in a small but well characterized cohort of MPN patients. Patients and Methods: 88 consecutive patients were diagnosed at the Hematology and Transfusion Medicine Division, L.SACCO University Hospital of Milan, according to WHO criteria. Patients signed an informed consent to participate in this clinical study with a protocol approved by local IRB and they showed MPN type (%), mean age (range), gender M/F and Jak2 positivity (%) as follows: PV[n=42 (48%), 68 (36–86), 18/24; 85.7%]; ET [n=34 (38%), 66 (30–93), 10/24, 61.7%]; PMF [n=12 (14%), 67 (37–88), 7/5, 58%]. Thrombotic and bleeding episodes were recorded and managed from the time of diagnosis and associated with the use of aspirin (ASA) and of other MPN therapies. Among additional lab parameters, plasmatic ADAMTS-13 and VWF activities were also measured at enrolment as endothelial/platelet marker. These activities were assayed with Technozym ADAMTS-13 activity (Technoclone GmbH, Austria), Innovance VWF-GPIb activity (Siemens AG, Germany) and HemosIL-VWF antigen (Instrumentation Laboratory, USA). Multimeric analyses were also tested using very sensitive intermediate SDS-agarose gel electrophoresis. Statistical analyses were performed by SPSS-17.2. Results: 59/88 (67%) patients did not show any thrombotic or bleeding complications during the 6-year follow-up. In these cases mean (range) values of VWF:GPIb and VWF:Ag were 104 (29–202) and 133 (52–288) U/dL while ADAMTS-13 was 102 (63–143). 20/88 (23%) cases showed at least one thrombotic event (13ATE/7VTE): AMI (6), STROKE (6), TIA (2), PE (1), DVT (7). Patients with thromboses showed relatively higher values VWF:GPIb and lower ADAMTS-13 and this was confirmed in multivariate analysis especially for ET [VWF:GPIb=135 (61–237) U/dL, p=0.004 and ADAMTS-13=89(62–134), p=0.009]. Major bleeding episodes mainly mucosal (5 gastrointestinal, 3 post-surgery, 1 severe menorrhagia) requiring blood transfusions or hysterectomy were observed in 9/88 (10%) patients. At the multivariate analysis, major bleedings were significantly associated with lower VWF:GPIb [68 (25–111) U/dL, p=0.022), lower VWF:Ag [93 (35–146) U/dL, p=0.016] and to the ASA intake (p=0.006). Most of these bleeders showed also a relative loss of the highest molecular weight multimers. Conclusions: Based on these observations, we confirm that thrombotic events in MPN may certainly have multiple risk factors: however, lower ADAMTS-13 and higher VWF activities might play a role as additional risk factors especially in ET. Conversely, lower levels of VWF with loss of the largest multimers are important risk factors for bleeding in MPN especially in patients treated with ASA. Disclosures: No relevant conflicts of interest to declare.

The Role Of Deregulated HMGA2 Expression With Promoter Methylation Of p16 In Myeloproliferative Neoplasms

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1606-1606
Author(s):  
Kayo Shirado Harada ◽  
Kazuhiko Ikeda ◽  
Kazuei Ogawa ◽  
Hideyoshi Noji ◽  
Hideo Kimura ◽  
...  
Keyword(s):  
Promoter Methylation ◽  
Blood Cells ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Expression Levels ◽  
Allele Burden ◽  
Polycomb Group Genes ◽  
Micro Rnas ◽  
Jak2v617f Allele Burden

Abstract Myeloproliferative Neoplasms (MPNs) are characterized by clonal proliferative hematopoiesis with increased mature blood cells. The signal-activating mutations such as JAK2V617F increase blood cells, but it remains uncertain how an abnormal hematopoietic cell clone expands in MPNs. We have recently showed that overexpression of the high mobility group AT-hook 2 (HMGA2) causes proliferative hematopoiesis with providing a clonal growth advantage to hematopoietic cells in mice (Ikeda et al, Blood, 2011), suggesting the possibility that HMGA2 contributes to the pathogenesis of MPNs. However, since only a few studies have evaluated expression of HMGA2 mRNA in patients with MPNs, the role of HMGA2 in the pathogenesis of MPNs is yet unclear. MPNs also show mutations in epigenetic modifiers involving DNA methylation such as polycomb group genes (PcG) and aberrant expressions of micro RNAs (miRNA) that negatively regulate expressions of targeted genes. Interestingly, deficiency in either PcG-related BMI1 (Oguro et al, J Exp Med, 2012) or let-7-family miRNA (Mayr et al, Science, 2007) causes deregulation of HMGA2 expression, leading to its oncogenic activity in part by negatively regulating tumor suppressor p16. Thus, in this study, to clarify the role of HMGA2 in MPNs, we investigated expression of HMGA2 mRNA in peripheral granulocytes of 56 patients with MPNs including 23 polycythemia vera (PV), 26 essential thrombocythemia (ET) and 7 primary myelofibrosis (PMF) along with clinical findings, JAK2V617F allele burden, expressions of BMI1 mRNA and let-7-family miRNAs, and promoter methylation of p16. Quantitative RT-PCR (qPCR) showed significantly higher expression of HMGA2 mRNA relative to internal control HPRT1 mRNA in PMF (mean ± SD; 31.7 ± 42.8, p<0.01), but not PV (15.7 ± 53.2) or ET (2.14 ± 7.70), compared with 12 healthy volunteers (HV; 0.431 ± 0.366). In addition, deregulated HMGA2 expression (>1.2), which was determined as relative expression level above mean + 2SD of HMGA2 mRNA in 12 HV, was most frequently detected in patients with PMF [7/7 (100%)] (p<0.01), compared with PV [5/23 (21.7%)] and ET [6/26 (23.1%)]. We also found a significant positive correlation in expression levels of HMGA2 mRNA with serum LDH values (r=0.531, p<0.01) rather than JAK2V617F allele burden (r=0.25, p=0.08). These data suggested that expression of HMGA2 mRNA independently correlated with disease phenotype and status in MPNs. We next explored the cause of deregulated expression of HMGA2 mRNA and found lower expression of let-7a (0.19 ± 0.13 vs. 0.42 ± 0.39, p=0.04) and -7c (0.57 ± 0.60 vs. 1.14 ± 0.94, p=0.06) rather than -7b (p=0.2) by qPCR, in patients with deregulated expression of HMGA2 mRNA compared with other patients. However, HMGA2-involved chromosomal abnormality in 12q13-15 was not detected in any patient, and there was no difference in expression of BMI1 mRNA between patients with deregulated expression of HMGA2 mRNA and other patients. Thus, decreased expression of let-7 miRNAs might contribute to deregulated expression of HMGA2 mRNA in MPNs. Finally, we investigated correlation of deregulated expression of HMGA2 mRNA with promoter methylation of p16. Methylation-specific PCR assay detected promoter methylation of p16 in 17/56 (30.4%) patients with MPNs. Strikingly, patients with deregulated expression of HMGA2 mRNA significantly more often showed promoter methylation of p16 compared with other patients [10/18 (55.6%) vs. 7/38 (18.4%), p<0.01]. Furthermore, patients with promoter methylation of p16 showed higher expression levels of HMGA2 mRNA than patients without the methylation, especially in patients with PMF (2.33 ± 0.90 vs. 70.9 ± 38.3, p=0.01). In conclusion, deregulated expression of HMGA2 in association with decreased expression of let-7 miRNAs may play a crucial role in the pathogenesis of MPNs possibly through p16. Disclosures: No relevant conflicts of interest to declare.

Flt3/ITD Blocks Myeloid Differentiation Of Hematopoietic Cells By Up-Regulating Runx1

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3808-3808
Author(s):  
Tomohiro Hirade ◽  
Mariko Abe ◽  
Chie Onishi ◽  
Seiji Yamaguchi ◽  
Seiji Fukuda
Keyword(s):  
Conflicts Of Interest ◽  
Hematopoietic Cells ◽  
Cell Number ◽  
Dominant Negative ◽  
Leukemia Cells ◽  
Myeloid Differentiation ◽  
Differentiation And Proliferation ◽  
Runx1 Expression ◽  
Novel Therapeutic

Abstract Internal-Tandem-Duplication mutations in the FLT3 (FLT3/ITD) gene are detected in 30% of patients with acute myeloid leukemia (AML) and are associated with extremely poor prognoses. The lack of significant efficacy of FLT3/ITD inhibitors underscores the need to identify FLT3/ITD-specific signaling pathways that are distinct from those that occur in normal hematopoietic cells to develop novel therapeutic approaches. FLT3/ITD is classified as a “class I mutation” that drives the proliferation of leukemia cells. In addition to mutation of FLT3/ITD, a “class II mutation” that blocks differentiation of the pre-leukemic clone is generally required for the development of AML. For instance, dominant negative mutations of RUNX1 are occasionally found in patients with AML. These mutations of RUNX1 cause AML by blocking the differentiation of leukemia cells in combination with the mutation of FLT3/ITD. RUNX1 is a core-binding transcription factor and plays an important role in hematopoietic homeostasis, particularly differentiation and proliferation. Loss of RUNX1 blocks hematopoietic differentiation and is associated with the emergence of a primitive hematopoietic compartment, suggesting that RUNX1 generally induces differentiation of hematopoietic cells. However, the functional role of RUNX1 as a down-stream effector of FLT3/ITD has not been characterized. Herein, we investigated the role of Runx1 in aberrant proliferation and differentiation of hematopoietic cells induced by Flt3 /ITD. A comparison of RUNX1 expression levels in AML patients for whom information has been deposited in the public gene expression profile database (GSE1159) revealed that RUNX1 mRNA expression was significantly higher in FLT3/ITD+AML cells (N=78) than in FLT3/ITD-AML cells (N=190, P<0.05). The mRNA microarray analysis consistently demonstrated that Runx1 is up-regulated by Flt3/ITD in Ba/F3 cells. Up-regulation of Runx1 by Flt3/ITD was validated in Ba/F3 cells and 32D cells by quantitative RT-PCR. Incubation of control 32D cells with 20 ng/ml of G-CSF increased the number of Gr-1+/Mac-1+cells, whereas the induction of myeloid differentiation by G-CSF was abrogated by the overexpression of Flt3/ITD in 32D cells. By contrast, transduction of shRNA specific for Runx1 into Flt3/ITD+32D cells inhibited the expression of Runx1 mRNA by 60 % but increased the number and the proportion of Gr-1+/Mac-1+cells ; these effects were enhanced by incubation with G-CSF. These data indicate that Runx1 mediates the block of differentiation toward the myeloid lineage that is induced by Flt3/ITD. Moreover, the number of colony-forming units (CFU) over-expressing Flt3/ITD cultured in the absence of growth factors was reduced by Runx1-shRNA without affecting the total cell number in the suspension culture, as compared to Flt3/ITD+32D cells transduced with control-shRNA. This implies that antagonizing Runx1 facilitates the production of terminally differentiated cells that have lost colony-forming ability, thereby reducing the CFU number without altering the total number of cells. Finally, Runx1-shRNA inhibited the formation of secondary CFU colonies derived from the primary Flt3-ITD-over-expressing CFU colonies. Our results suggest that Flt3/ITD blocks myeloid differentiation of Flt3/ITD+cells by up-regulating Runx1 expression. The blocking of differentiation mediated by Runx1 in Flt3/ITD+cells is in contrast to the cell differentiation-inducing role of Runx1 in normal hematopoiesis, suggesting that the function of Runx1 in Flt3/ITD+cells may be distinct from that in normal cells. The reduction of secondary CFU colonies by Runx1-shRNA suggests that Runx1 may mediate self-renewal of Flt3/ITD+hematopoietic progenitor cells. These findings suggest that antagonizing RUNX1 may represent a novel therapeutic strategy to induce terminal differentiation of FLT3/ITD+AML cells in AML patients, in addition to inhibiting their aberrant proliferation. Disclosures: No relevant conflicts of interest to declare.

Gtpase Immunity-Associated Protein Family Member 4 Contributes to the Enhanced Expansion of HSPCs in RUNX1 Deficient Mice

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4344-4344
Author(s):  
Amanda Scholl ◽  
Kentson Lam ◽  
Alex Muselman ◽  
Tingdong Tang ◽  
Shinobu Matsuura ◽  
...  
Keyword(s):  
Family Member ◽  
Conflicts Of Interest ◽  
Protein Family ◽  
Dominant Negative ◽  
Negative Regulator ◽  
Wild Type ◽  
Double Knockout ◽  
Hematopoietic Stem ◽  
Myeloid Progenitor

Abstract RUNX1 is the transcription factor described as the master regulator of hematopoiesis. Due to its central role during blood development, numerous RUNX1 mutations have been reported in hematologic abnormalities. Mice null for Runx1 die during embryogenesis, lacking definitive HSCs. Conditional Runx1Δ/Δ mice are viable, but exhibit a variety of blood abnormalities. The most salient defect in these Runx1Δ/Δ mice is expansion of the hematopoietic stem and progenitor cell (HSPC) population, measured as an increase in number of lineage negative, Sca1 positive, cKit positive (LSK) cells. A shortened form of RUNX1 (RUNX1SF) lacking the C-terminal and part of the N-terminal domain (41-214) acts as a dominant negative regulator of RUNX1 and hence also models RUNX1 loss-of-function. A differential gene expression analysis of HSPCs derived from Runx1Δ/Δ compared to wild type mice uncovered GTPase immunity-associated protein family member 4 (GIMAP4) as one of the genes most highly upregulated. Previous studies have focused almost exclusively on the role of GIMAP4 as a pro-apoptotic protein during T-cell development. This study illuminates a novel non-apoptotic role of GIMAP4 in a formerly unstudied HSPC context. Runx1Δ/Δ mice were crossed with Gimap4-/- mice to generate a double knockout (dKO) mouse line. These dKO mice exhibited attenuated HSPC proliferation in comparison to Runx1Δ/Δ mice, suggesting that GIMAP4 functions in this HSPC expansion phenotype. BMT experiments using lethally irradiated C57 mice and RUNX1SF transduced wild type versus Gimap4-/-bone marrow confirmed this result. GIMAP4 also worked independently and coordinately with RUNX1 to influence individual progenitor populations. Common lymphoid progenitors (CLP) were affected only by GIMAP4. Gimap4-/- mice exhibited an expansion of the CLP population, consistent with its pro-apoptotic role in lymphoid populations. Conversely, both RUNX1 and GIMAP4 coordinately exerted an effect on myeloid progenitor populations. Runx1Δ/Δ mice harbored expanded granulocyte-macrophage progenitor (GMP) and common myeloid progenitor (CMP) populations. This expansion was not observed when GIMAP4 was also ablated. This suggests a pro-proliferative role of GIMAP4 specifically in myeloid populations. These opposing roles of GIMAP4 in lymphoid versus myeloid cells suggest a more contextual, cell-specific role of this GTPase protein. Ultimately, this study provides insight into how RUNX1 and GIMAP4 may coordinate to maintain HSPC homeostasis. Disclosures No relevant conflicts of interest to declare.

scholarly journals p130Cas mediates the transforming properties of the anaplastic lymphoma kinase

Blood ◽  
2005 ◽  
Vol 106 (12) ◽  
pp. 3907-3916 ◽  
Author(s):  
Chiara Ambrogio ◽  
Claudia Voena ◽  
Andrea D. Manazza ◽  
Roberto Piva ◽  
Ludovica Riera ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Anaplastic Lymphoma Kinase ◽  
Kinase Activity ◽  
Adaptor Protein ◽  
Dominant Negative ◽  
Lymphoid Cells ◽  
Src Tyrosine Kinase ◽  
Anaplastic Lymphoma ◽  
Proteomic Approach

Translocations of the anaplastic lymphoma kinase (ALK) gene have been described in anaplastic large-cell lymphomas (ALCLs) and in stromal tumors. The most frequent translocation, t(2;5), generates the fusion protein nucleophosmin (NPM)–ALK with intrinsic tyrosine kinase activity. Along with transformation, NPM-ALK induces morphologic changes in fibroblasts and lymphoid cells, suggesting a direct role of ALK in cell shaping. In this study, we used a mass-spectrometry–based proteomic approach to search for proteins involved in cytoskeleton remodeling and identified p130Cas (p130 Crk-associated substrate) as a novel interactor of NPM-ALK. In 293 cells and in fibroblasts as well as in human ALK-positive lymphoma cell lines, NPM-ALK was able to bind p130Cas and to induce its phosphorylation. Both of the effects were dependent on ALK kinase activity and on the adaptor protein growth factor receptor–bound protein 2 (Grb2), since no binding or phosphorylation was found with the kinase-dead mutant NPM-ALKK210R or in the presence of a Grb2 dominant-negative protein. Phosphorylation of p130Cas by NPM-ALK was partially independent from Src (tyrosine kinase pp60c-src) kinase activity, as it was still detectable in Syf-/- cells. Finally, p130Cas-/- (also known as Bcar1-/-) fibroblasts expressing NPM-ALK showed impaired actin filament depolymerization and were no longer transformed compared with wild-type cells, indicating an essential role of p130Cas activation in ALK-mediated transformation.

scholarly journals Role of Iron-Responsive Mitochondrial Metabolism and ROS in Anemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 216-216
Author(s):  
Chanté L Richardson ◽  
Valerie M Schrott ◽  
Claudette M St. Croix ◽  
Yinna Wang ◽  
Catherine G Corey ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Iron Deficiency ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Mitochondrial Metabolism ◽  
Superoxide Production ◽  
Deficiency Anemia ◽  
Erythroid Progenitor ◽  
Iron Restriction

Abstract Iron and erythropoietin (Epo) are intimately linked regulators of erythropoiesis. Moderate iron restriction suppresses erythropoiesis at the Epo-dependent, CFU-E stage, without induction of apoptosis and without suppression of other hematopoietic cell lineages. Iron modulates Epo bioactivity in patients with iron deficiency anemia (IDA) and patients with anemia of chronic disease and inflammation (ACDI). To conserve iron when supplies are low, this erythroid iron restriction response reduces iron consumption by suppressing erythropoiesis. The erythroid iron sensor is unknown. Aconitases are multifunctional iron-sulfur cluster proteins localized in the cytosol (Aco1) and mitochondria (Aco2) that convert citrate into isocitrate. We have shown that iron restriction inhibits Aco2 enzymatic activity leading to suppression of erythropoiesis in vitro, and these effects are reversed by isocitrate. Isocitrate corrects IDA in mice and ACDI in rats (Bullock GC, et al. Blood. 2010;116:97-108; Richardson CL, et al. J Clin Invest. 2013 Aug 1;123(8):3614-3623). Iron restriction also alters the cross-talk between transferrin receptor and Epo receptor signaling pathways. These results suggest that Aco2 is an iron-responsive regulator of erythropoiesis. We are investigating the downstream molecular signaling mechanisms by which iron restriction induced-inactivation of Aco2 suppresses erythropoiesis. Our novel preliminary data show that mitochondrial oxidative metabolism rates change over time during erythropoiesis and that iron restriction reduces erythroid mitochondrial metabolism 4 to 7-fold compared to iron replete controls. This iron restriction induced change in respiration is associated with a significant, 1.5 to 3-fold, increase in mitochondrial superoxide production without a corresponding increase in hydrogen peroxide. Importantly, these mitochondrial alterations are reproduced by direct inhibition of aconitase with fluoroacetate (FA) and are not due to changes in mitochondrial number. Further, isocitrate reverses the effects of iron restriction or aconitase inhibition on mitochondrial metabolism and attenuates superoxide production. Based on these data and the known role of reactive oxygen species (superoxide/hydrogen peroxide) in Epo signaling, we propose the overarching hypothesis that iron restriction inhibits mitochondrial aconitase which, in turn, alters erythroid mitochondrial metabolism and ROS signaling resulting in suppression of erythropoiesis (Figure 1). We show for the first time bioenergetics profiles from iron restricted and iron replete primary human erythroid progenitor cells undergoing erythropoiesis. We also show that moderate levels of iron restriction cause mitochondrial dysfunction and alterations in mitochondrial ROS in differentiating erythroid progenitors. The clinical relevance of this project lies in its potential for the development of new iron-free agonists and antagonists of red blood cell production. Agonists may benefit patients with anemia due to iron deficiency or chronic inflammation and antagonists may benefit patients with myeloproliferative neoplasms. Figure 1: Proposed mechanism of iron-dependent regulation of erythropoiesis Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document