A kinase-dead Csf1r mutation associated with adult-onset leukoencephalopathy has a dominant-negative impact on CSF1R signaling

2021 ◽  
Author(s):  
Jennifer Stables ◽  
Emma K. Green ◽  
Anuj Sehgal ◽  
Omkar Patkar ◽  
Sahar Keshvari ◽  
...  
Keyword(s):  
Human Disease ◽  
Bone Marrow Cells ◽  
Negative Impact ◽  
Dominant Negative ◽  
Heterozygous Mutation ◽  
Homozygous Mutation ◽  
Adult Onset ◽  
Tissue Macrophage ◽  
Macrophage Populations

AbstractAmino acid substitutions in the kinase domain of the human CSF1R gene are associated with autosomal dominant adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP). To model the human disease, we created a disease-associated mutation (Glu631Lys; E631K) in the mouse Csf1r locus. Homozygous mutation (Csf1rE631K/E631K) phenocopied the Csf1r knockout; with prenatal mortality or severe postnatal growth retardation and hydrocephalus. Heterozygous mutation delayed the postnatal expansion of tissue macrophage populations in most organs. Bone marrow cells from Csf1rE631K/+ mice were resistant to CSF1 stimulation in vitro, and Csf1rE631K/+ mice were unresponsive to administration of a CSF1-Fc fusion protein which expands tissue macrophage populations in controls. In the brain, microglial cell numbers and dendritic arborization were reduced in the Csf1rE631K/+ mice as in ALSP patients. The microglial phenotype is the opposite of microgliosis observed in Csf1r+/- mice. However, we found no evidence of brain pathology or impacts on motor function in aged Csf1rE631K/+ mice. We conclude that disease-associated CSF1R mutations encode dominant negative repressors of CSF1R signaling. We speculate that leukoencephalopathy associated with human CSF1R mutations requires an environmental trigger and/or epistatic interaction with common neurodegenerative disease-associated alleles.Summary StatementThis study describes the effect of a human disease-associated mutation in the mouse CSF1R gene on postnatal development and growth factor responsiveness of cells of the macrophage lineage.

A Conserved Mechanism of Transcription Factor Competition Controls Hematopoietic Progenitor Self-Renewal.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 91-91
Author(s):  
Shane R. Horman ◽  
Chinamenveni S. Velu ◽  
Tristan Bourdeau ◽  
Avinash Baktula ◽  
Jinfang Zhu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transcription Factor ◽  
Binding Sites ◽  
Bone Marrow Cells ◽  
Dominant Negative ◽  
Wild Type ◽  
Hematopoietic Progenitor ◽  
Self Renewal ◽  
Conditional Deletion

Abstract An intrinsic mechanism of self-renewal is critical for the maintenance of hematopoietic stem cells (HSC), but this HSC function is extinguished during differentiation of progenitors. Here we show that the self-renewal capacity of hematopoietic progenitor cells is regulated through physical competition for occupancy of select DNA binding sites. Initially, we found that conditional deletion of the Growth factor independent-1 (Gfi1) gene results in the accumulation of abnormally persistent myeloid progenitors in vivo. Specifically, while germline Gfi1 deletion induces defective HSC self renewal and a block to granulopoiesis, we find that conditional deletion of Gfi1 induces a severe but transient block to neutrophil development with repopulation of the bone marrow by the remaining wild type HSC within 8 weeks post deletion. However, even though normal levels of granulocyte colony forming units (G-CFU) returned by 8 weeks post deletion, an abnormal Gfi1−/− myeloid progenitor remained in the bone marrow in vivo. Subsequently, we find in vitro that both wild-type bone marrow cells expressing Gfi1-dominant-negative mutants, and Gfi1−/− Lin- bone marrow contain cells that replate indefinitely. We hypothesized that Gfi1 is critical to extinguish self renewal in hematopoietic progenitors. In seemingly unrelated work, we discovered antagonism between the drosophila orthologs of Gfi1 and the Hoxa9/Pbx1/Meis1 transcription factor complex during drosophila embryo segmentation. We extended our drosophila findings to discover that a subset of mammalian DNA regulatory sequences encode DNA binding sites for both Gfi1 and Hoxa9/Pbx1/Meis1. These DNA sequences are able to bind either factor, and function as a molecular switch. Interestingly, composite Gfi1/ Hoxa9/Pbx1/Meis1 binding sites are present in the regulatory regions of the gene encoding Hoxa9. We note that Gfi1 expression is normally induced, while Hoxa9 expression is down-regulated, during the transition from common myeloid progenitor (CMP) to the granulocyte-monocyte progenitor (GMP). CMP have greater self renewal potential than GMP. Conditional deletion of Gfi1 in sorted CMP or GMP both increases Hoxa9 expression and generates progenitors capable of replating indefinitely in vitro. Thus, Gfi1 is critical to limit self renewal in these progenitors. Deregulated Hoxa9 expression or activity appears pivotal to this new Gfi1-null phenotype, because Gfi1 dominant-negative mutants immortalize wild-type (or Hoxa7−/−) but not Hoxa9−/− bone marrow cells in vitro. An abnormal gain of self-renewal can unleash the leukemic potential of progenitor cells. We find that both limiting Gfi1 gene dosage and expression of Gfi1 dominant-negative mutants significantly increases Nup98-Hoxa9-mediated colony formation. In contrast, forced expression of Gfi1 prevents Nup98-Hoxa9 immortalization. Notably, the expression of Hoxa9 (independent of cases with Nup98-Hoxa9 fusions) has been reported to be of significant prognostic value in human acute myeloid leukemia. In conclusion, Gfi1 and the Hoxa9/Pbx1/Meis1 complex compete to control the expression of genes (such as Hoxa9) which are critical to extinguish self renewal and limit the leukemogenic potential of hematopoietic progenitors. The antagonism between these transcription factor complexes is conserved from drosophila segment formation to mammalian hematopoietic progenitor biology.

Dissecting Proto-Oncogenic PIM Serine/Threonine Kinases in FLT3-ITDInduced Leukemogenesis: PIM1 Regulates CXCL12/CXCR4-Mediated Homing and Migration

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3796-3796
Author(s):  
Christelle Gasser ◽  
Rebekka Grundler ◽  
Laurent Brault ◽  
Alec Bullock ◽  
Tobias Dechow ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Small Molecule ◽  
Bone Marrow Cells ◽  
Dominant Negative ◽  
Cell Homing ◽  
Threonine Kinases ◽  
And Migration ◽  
Marrow Cells

Abstract Previous work has shown that FLT3-ITD mediated leukemogenesis is associated with increased expression of PIM1 and PIM2 serine/threonine kinases. Here we show that retroviral expression of FLT3-ITD could not compensate impaired clonogenic in vitro growth of PIM1−/− bone marrow cells. Induction of a lethal myelo- and lymphoproliferative disorder by FLT3-ITD in vivo was independent of PIM2, but rather unexpectedly, lethally irradiated recipients could not be reconstituted with FLT3-ITD expressing bone marrow cells lacking PIM1. Transplants of CSFE-labeled PIM1−/− cells revealed an impaired homing capacity to bone marrow and spleen. Expression of lower surface CXCR4 levels (while maintaining normal total CXCR4 levels) in PIM1−/− bone marrow cells was associated with significantly reduced migration towards a CXCL12 gradient and impaired CXCL12-mediated intracellular Ca2+ release. Using siRNA-mediated knockdown, a small molecule PIM inhibitor, expression of a dominant-negative acting PIM1 mutant or re-expression of PIM1 in knockout cells, we observed that PIM1 activity was critical for CXCR4 surface expression. In vitro kinase assays and masspectrometric analysis further revealed that PIM1 directly phosphorylated serine 339 located in the CXCR4 intracellular domain known to be essential for proper receptor recycling. Interestingly, in leukemic blasts from acute myeloid leukemia (AML) patients, we found an association of increased PIM1 expression and high-level of surface CXCR4. In addition, treatment of the cells with a small molecule PIM inhibitor resulted in decreased surface CXCR4 expression in some patients. Our work suggests that PIM1 exerts its oncogenic activity not only by supporting proliferation and survival but also by regulation of cell homing and migration through direct modification of the CXCL12/CXCR4 axis. As CXCR4 is a key mediator of cancer stem cell homing and metastasis, targeting of PIM1 may offer new therapeutic avenues against tumor progression and relapse.

scholarly journals Metabolism of tissue macrophages in homeostasis and pathology

2021 ◽  
Author(s):  
Stefanie K. Wculek ◽  
Gillian Dunphy ◽  
Ignacio Heras-Murillo ◽  
Annalaura Mastrangelo ◽  
David Sancho
Keyword(s):  
Immune Cells ◽  
Current Knowledge ◽  
Cellular Metabolism ◽  
Structural Components ◽  
Tissue Macrophage ◽  
Cellular Energy ◽  
Pathological Conditions ◽  
Macrophage Populations ◽  
Adult Bone

AbstractCellular metabolism orchestrates the intricate use of tissue fuels for catabolism and anabolism to generate cellular energy and structural components. The emerging field of immunometabolism highlights the importance of cellular metabolism for the maintenance and activities of immune cells. Macrophages are embryo- or adult bone marrow-derived leukocytes that are key for healthy tissue homeostasis but can also contribute to pathologies such as metabolic syndrome, atherosclerosis, fibrosis or cancer. Macrophage metabolism has largely been studied in vitro. However, different organs contain diverse macrophage populations that specialize in distinct and often tissue-specific functions. This context specificity creates diverging metabolic challenges for tissue macrophage populations to fulfill their homeostatic roles in their particular microenvironment and conditions their response in pathological conditions. Here, we outline current knowledge on the metabolic requirements and adaptations of macrophages located in tissues during homeostasis and selected diseases.

scholarly journals Disease-Associated Human Telomerase RNA Variants Show Loss of Function for Telomere Synthesis without Dominant-Negative Interference

2008 ◽  
Vol 28 (20) ◽  
pp. 6510-6520 ◽  
Author(s):  
Timothy M. Errington ◽  
Dragony Fu ◽  
Judy M. Y. Wong ◽  
Kathleen Collins
Keyword(s):  
Negative Impact ◽  
Dominant Negative ◽  
Telomere Maintenance ◽  
General Mechanism ◽  
Genome Replication ◽  
Telomerase Rna ◽  
Loss Of Function ◽  
Wild Type ◽  
Human Telomerase

ABSTRACT Telomerase adds simple-sequence repeats to chromosome ends to offset the terminal sequence loss inherent in each cycle of genome replication. Inherited mutations in genes encoding subunits of the human telomerase holoenzyme give rise to disease phenotypes including hematopoietic failure and pulmonary fibrosis. Disease-associated variants of the human telomerase RNA are expressed in heterozygous combination with wild-type telomerase RNA. Here, we exploit a sensitized human primary cell assay system to investigate the biological function of disease-linked telomerase RNA variants and their impact on the function of coexpressed wild-type telomerase RNA. We find that telomerase RNA variants discovered in patients with dyskeratosis congenita or aplastic anemia show loss of function without any indication of dominant-negative impact on telomere maintenance by the coexpressed wild-type RNA. To reconcile this result with contradictory findings from reconstitution assays in vitro, we demonstrate that the lack of dominant-negative impact on telomere maintenance correlates with physiological assembly of active human telomerase holoenzyme ribonucleoproteins harboring monomers rather than higher-order multimers of telomerase RNA and telomerase reverse transcriptase. These findings support loss of function of telomerase RNA as a general mechanism of human disease.

Structural and Functional Characterization of the NHR2 and Runt Domains of AML1/ETO.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 482-482
Author(s):  
Matthew D. Cheney ◽  
Yizhou Liu ◽  
Yunpeng Zhou ◽  
Maksymilian Chruszcz ◽  
Thomas M. Laue ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Dominant Negative ◽  
Heptad Repeat ◽  
Mouse Bone Marrow ◽  
Runt Domain ◽  
Primary Mouse ◽  
Dominant Negative Activity ◽  
Marrow Cells

Abstract AML1/ETO is the chimeric fusion protein resulting from the t(8;21) found in AML of the M2 subtype. It contains the N-terminal 177 amino acids of RUNX1 and virtually all (575aa) of ETO. The RUNX1 component includes the Runt domain, which mediates both DNA binding and heterodimerization with CBFβ, but lacks the more C-terminal sequences required for transactivation. AML1/ETO occupies RUNX target genes in vivo and is associated with a repressive chromatin structure characterized by reduced levels of acetylated histone H3. AML1/ETO is thought to repress transcription by recruiting a SMRT (N-CoR)/Sin3A/HDAC complex to chromatin via sequences in ETO. ETO is the human homologue of the Drosophila Nervy protein and shares 4 regions of homology with Nervy called Nervy Homology Regions (NHR) 1–4. Deletion studies have shown that three of the AML1/ETO domains essential for its repressive function are the Runt domain, NHR2, and NHR4. The NHR2 domain is a hydrophobic heptad repeat that mediates oligomerization of AML1/ETO, interaction with ETO family members, and also with mSin3A and HDACs. We recently solved an x-ray structure of the NHR2 domain and found it to be an alpha-helical tetramer. Based on this structure we have introduced amino acid substitutions into the NHR2 domain that disrupt tetramer formation but not AML1/ETO stability. These mutations impair the ability of AML1/ETO to inhibit the differentiation of GR-1+/Mac-1+ cells following retroviral transduction into primary mouse bone marrow cells, and also inhibit the serial replating ability of AML1/ETO expressing bone marrow cells in vitro. We additionally show that mutations reported by Amann et al. (Mol Cell Biol. 21, 6470, 2001) to disrupt mSin3A binding to NHR2 do not affect the biological activity of AML1/ETO in vitro. We also introduced mutations in the Runt domain of AML1/ETO that disrupt CBFβ binding by defined amounts (40-fold, 200-fold, 500-fold), and demonstrated that CBFβ binding by AML1/ETO is essential for its dominant negative activity. The latter results suggest that small molecules designed to selectively impair heterodimerization of AML1/ETO with CBFβ could potentially block AML1/ETO’s dominant negative activity.

scholarly journals CSF1R-dependent macrophages control postnatal somatic growth and organ maturation

2020 ◽  
Author(s):  
Sahar Keshvari ◽  
Melanie Caruso ◽  
Lena Batoon ◽  
Anuj Sehgal ◽  
Ngari Teakle ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Somatic Growth ◽  
Mononuclear Phagocyte ◽  
Homozygous Mutation ◽  
Blood Monocytes ◽  
Term Survival ◽  
Tissue Macrophage ◽  
Organ Systems ◽  
Macrophage Populations ◽  
Mutant Phenotypes

AbstractThe development of the mononuclear phagocyte system (MPS) is controlled by signals from the CSF1 receptor (CSF1R). Homozygous mutation of the Csf1r locus (Csf1rko) in inbred rats led to the loss of non-classical monocytes and tissue macrophage populations, reduced postnatal somatic growth, severe developmental delay impacting all major organ systems and early mortality. The developmental impacts overlap with growth hormone/insulin-like growth factor (GH/IGF1) deficiency and Csf1rko rats lacked circulating IGF1. The liver is the main source of circulating IGF1. Expression profiling of juvenile wild-type and Csf1rko livers identified 2760 differentially expressed genes associated with the loss of macrophages, severe hypoplasia, delayed hepatocyte maturation, disrupted lipid metabolism and dysregulation of the GH/IGF1 system. Transfer of WT bone marrow at weaning restored circulating IGF1 and reversed the mutant phenotypes enabling long term survival and fertility. Phenotypic rescue was achieved without reconstituting blood monocytes or CSF1-responsive bone marrow progenitors. The results demonstrate that CSF1R-dependent macrophages control the growth and maturation of the liver and regulate the GH/IGF1 axis.

scholarly journals Role of the novel endoribonuclease SLFN14 and its disease causing mutations in ribosomal degradation

2018 ◽  
Author(s):  
Sarah J. Fletcher ◽  
Vera P. Pisareva ◽  
Abdullah Khan ◽  
Andrew Tcherepanov ◽  
Neil V. Morgan ◽  
...  
Keyword(s):  
In Vitro Models ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Heterozygous Mutation ◽  
Missense Mutations ◽  
The Novel ◽  
Excessive Bleeding ◽  
Negative Effect ◽  
Inherited Thrombocytopenia

ABSTRACTPlatelets are anucleate and mostly ribosome-free cells within the bloodstream, derived from megakaryocytes within bone marrow and crucial for cessation of bleeding at sites of injury. Inherited thrombocytopenias are a group of disorders characterized by alow platelet count and are frequently associated with excessive bleeding. SLFN14 is one of the most recently discovered genes linked to inherited thrombocytopenia where several heterozygous missense mutations in SLFN14 were identified to cause defective megakaryocyte maturation and platelet dysfunction. Yet, SLFN14 was recently described as a ribosome-associated protein resulting in rRNA and ribosome-bound mRNA degradation in rabbit reticulocytes. To unveil the cellular function of SLFN14 and the link between SLFN14 and thrombocytopenia, we examined SLFN14 (WT/mutants) in in vitro models. Here, we show that all SLFN14 variants co-localize with ribosomes and mediate rRNA endonucleolytic degradation and ribosome clearance. Compare dto SLFN14 WT, expression of mutants is dramatically reduced as a result of post-translational degradation due to partial misfolding of the protein. Moreover, all SLFN14 variants tend to form oligomers. These findings could explain the dominant negative effect of heterozygous mutation on SLFN14 expression in patients’ platelets. Overall we suggest that SLFN14 could be involved in ribosome degradation during platelet formation and maturation.

Oridonin-Generated Cleavage Fragment of AML1-ETO Inhibits Its Oligomerization and Oncogenic Function Leading to Differentiation and Apoptosis of Leukemic Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1051-1051
Author(s):  
Chuanfeng Wu ◽  
Tao Zhen ◽  
Guangbiao Zhou ◽  
Ping Liu ◽  
Zhu Chen ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Target Genes ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Dominant Negative ◽  
Leukemic Cells ◽  
Granulocytic Differentiation ◽  
Regulatory Pathways ◽  
Cleavage Fragment

Abstract Abstract 1051 Poster Board I-73 Oligomerization through the NHR2 domain is essential for AML1-ETO's inhibition of granulocytic differentiation and enhanced clonogenic potential of primary bone marrow cells. We show here that Oridonin interferes with AML1-ETO oligomerization through its cleavage fragment DAML1-ETO, which consists of the amino acids (aa) 188-752 of the parental oncoprotein or aa 40-604s of the wild-type ETO. DAML1-ETO interacts with the parental AML1-ETO through NHR2 and exerts dominant negative effects on AML1-ETO with regard to DNA binding, transregulatory activity on target genes and regulation of leukemic cell survival, differentiation and proliferation both in vitro and in vivo. Moreover, Oridonin can activate retinoic acid and cAMP/PKA pathways, and potentiate differentiation induced by all-trans retinoic acid (ATRA) and G-CSF. Consistently, combined use of Oridonin, ATRA and G-CSF significantly prolongs lifespan of t (8;21) leukemic mice and, interestingly, we find that this treatment targets the Lin-/Sca-1+/C-KIT+ and Lin-/Sca-1-/C-KIT+ leukemia initiating cells. These data suggest that Oridonin, and potentially other small molecules, can inhibit AML1-ETO oligomerization and leukemogenic function, thus providing a targeted therapy that activates key regulatory pathways for myelomonocytic cell differentiation and apoptosis. Disclosures: No relevant conflicts of interest to declare.

SHP2 drives inflammation-triggered insulin resistance by reshaping tissue macrophage populations

2021 ◽  
Vol 13 (591) ◽  
pp. eabe2587
Author(s):  
Romain Paccoud ◽  
Céline Saint-Laurent ◽  
Enzo Piccolo ◽  
Mylène Tajan ◽  
Alizée Dortignac ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Bone Marrow ◽  
Tyrosine Phosphatase ◽  
Lipid Management ◽  
Tissue Macrophage ◽  
Depth Analysis ◽  
Macrophage Populations

Insulin resistance is a key event in type 2 diabetes onset and a major comorbidity of obesity. It results from a combination of fat excess–triggered defects, including lipotoxicity and metaflammation, but the causal mechanisms remain difficult to identify. Here, we report that hyperactivation of the tyrosine phosphatase SHP2 found in Noonan syndrome (NS) led to an unsuspected insulin resistance profile uncoupled from altered lipid management (for example, obesity or ectopic lipid deposits) in both patients and mice. Functional exploration of an NS mouse model revealed this insulin resistance phenotype correlated with constitutive inflammation of tissues involved in the regulation of glucose metabolism. Bone marrow transplantation and macrophage depletion improved glucose homeostasis and decreased metaflammation in the mice, highlighting a key role of macrophages. In-depth analysis of bone marrow–derived macrophages in vitro and liver macrophages showed that hyperactive SHP2 promoted a proinflammatory phenotype, modified resident macrophage homeostasis, and triggered monocyte infiltration. Consistent with a role of SHP2 in promoting inflammation-driven insulin resistance, pharmaceutical SHP2 inhibition in obese diabetic mice improved insulin sensitivity even better than conventional antidiabetic molecules by specifically reducing metaflammation and alleviating macrophage activation. Together, these results reveal that SHP2 hyperactivation leads to inflammation-triggered metabolic impairments and highlight the therapeutical potential of SHP2 inhibition to ameliorate insulin resistance.

Sign in / Sign up

Export Citation Format

Share Document