Identification of IRF8 As a Potent Tumor Suppressor in Murine Acute Promyelocytic Leukemia

Abstract The classical paradigm suggests that PML/RARA fusion protein is the main driver of pathogenesis in APL. It is believed that the fusion oncogenic protein mediates this effect by potentially repressing key myeloid maturation genes involved in differentiation processes. However, the underlying mechanism is not completely understood. We recently challenged this model re-opening fundamental questions as to understand the precise contribution of the fusion protein to leukemic transformation. This knowledge on the mechanistic pathways can lead to better tailored combinatorial therapeutics. To understand the role of the PML/RARA fusion protein in leukemogenesis, we initially did a transcriptome analysis in our murine MRP8-PML/RARA APL model. Interestingly, we observed only moderate alterations in gene expression pattern of the key myeloid genes that we thought to be actively involved in differentiation processes. Of particular note, we found significant downregulation of the Irf8 in our promyelocyte compartments. IRF8 is a known regulator of hematopoiesis. The IRF8 myeloid transcription factor (TF) is expressed in several lineages of the hematopoietic tree and plays an important role in orchestrating specification and differentiation of B cells, dendritic cells and monocytes. Herein, we speculate lower levels of IRF8 could potentially impact tumorigenesis in the context of PML/RARA. In order to address this question, technically, we employed stringent staining and sorting strategy to distinctly differentiate early and late promyelocytes and looked at the expression pattern of Irf8 gene both at the transcript and protein levels. Results from qRT-PCR demonstrated 4.8 fold decrease in Irf8 expression compared to wildtype controls both in preleukemic promyelocytes and fully differentiated leukemic cells suggesting PML/RARA could be a target of IRF8 and this association could potentially be involved in the emergence and maintenance of leukemia. We next asked whether these changes are reflective at the protein levels and performed a Western blot analysis in our highly purified promyelocyte population and found a dramatic decrease in IRF8 levels in comparison to wild type controls again suggesting a possible protein-protein interaction under normal conditions that may provide an advantage for the cells from turning oncogenic. In order to study how low levels of IRF8 impact promyelocyte expansion, we generated double knock-outs of mice harboring both PML/RARA Irf8-/- mutations and compared their phenotype with mice harboring single mutations in either PML/RARA or Irf8 gene. As previously observed, young PML/RARA mice had a substantially increased number of marrow promyelocytes in comparison to wild-type mice. Fascinatingly, loss of Irf8 alone resulted in an essentially identical expansion of promyelocytes (as well as a loss of earlier myeloid progenitors in the bone marrow, not seen in PML/RARA mice) and a combination of PML/RARA expression and IRF8 loss did not result in a statistically significant further expansion of promyelocytes. These results suggest an epistatic relationship between PML/RARA and IRF8, compatible with downregulation of IRF8 by PML/RARA as being a key mechanism by which t(15;17) expands promyelocytes in the initiation of APL. Furthermore, in order to assess the impact of single/double genetic alterations on the overall and leukemia free survival we transplanted lethally irradiated mice with bone marrow cells derived from PML/RARA, Irf8-/- and PML/RARA Irf8-/-double knock outs and followed these mice over a period of one year. We observed there is no difference in their overall survival rate among the different groups of mice. However, looking specifically at the acute leukemic deaths, we observed a reduced latency in our PML/RARA Irf8-/- cohorts compared to mice carrying single mutation at PML/RARA loci. We also noticed that all the acute leukemias in the PML/RARA Irf8-/- cohort occurred prior to the first appearance of acute leukemia in the PML/RARA cohorts. Altogether, these data support a model of APL leukemogenesis in which the translocation of chromosomes 15 and 17 initiates leukemia development, in part by downregulating IRF8, and in which the resulting expansion of the promyelocyte compartment contributes to acquisition of additional cooperating events (e.g. trisomy of chromosome 8, mutation of FLT3) that complete leukemic transformation. Disclosures No relevant conflicts of interest to declare.

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Leukemic transformation by the APL fusion protein PRKAR1A-RARα critically depends on recruitment of RXRα

Blood ◽

10.1182/blood-2009-07-232652 ◽

2010 ◽

Vol 115 (3) ◽

pp. 643-652 ◽

Cited By ~ 18

Author(s):

Jihui J. Qiu ◽

Xiaoxi Lu ◽

Bernd B. Zeisig ◽

Zhigui Ma ◽

Xun Cai ◽

...

Keyword(s):

Bone Marrow ◽

Retinoic Acid ◽

Dna Binding ◽

Fusion Protein ◽

Point Mutations ◽

Wild Type ◽

Leukemic Transformation ◽

Interaction Domain ◽

Binding Characteristics ◽

Retinoic Acid Response Element

Abstract PRKAR1A (R1A)–retinoic acid receptor-α (R1A-RARα) is the sixth RARα–containing fusion protein in acute promyelocytic leukemia (APL). Using the murine bone-marrow retroviral transduction/transformation assay, we showed that R1A-RARα fusion protein could transform bone-marrow progenitor/stem cells. In gel-shift assays, R1A-RARα was able to bind to a panel of retinoic acid response elements both as a homodimer and as a heterodimer with RXRα, and demonstrated distinct DNA-binding characteristics compared with wild-type RARα/RXRα or other X-RARα chimeric proteins. The ratio of R1A-RARα to RXRα proteins affected the retinoic acid response element interaction pattern of R1A-RARα/RXRα complexes. Studies comparing R1A-RARα with R1A-RARα(ΔRIIa) demonstrated that the RIIa protein interaction domain located within R1A was responsible for R1A-RARα homodimeric DNA binding and interaction with wild-type R1A protein. However, the RIIa domain was not required for R1A-RARα–mediated transformation because its deletion in R1A-RARα(ΔRIIa) did not compromise its transformation capability. In contrast, introduction of point mutations within the RARα portion of either R1A-RARα or R1A-RARα(ΔRIIa), previously demonstrated to eliminate RXRα interaction or treatment of transduced cells with RXRα shRNA or a RXRα agonist, reduced transformation capability. Thus, leukemic transformation by APL fusion protein PRKAR1A-RARα is critically dependent on RXRα, which suggests RXRα is a promising target for APL.

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Titanium Dioxide Presents a Different Profile in Dextran Sodium Sulphate-Induced Experimental Colitis in Mice Lacking the IBD Risk Gene Ptpn2 in Myeloid Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22020772 ◽

2021 ◽

Vol 22 (2) ◽

pp. 772

Author(s):

Javier Conde ◽

Marlene Schwarzfischer ◽

Egle Katkeviciute ◽

Janine Häfliger ◽

Anna Niechcial ◽

...

Keyword(s):

Bone Marrow ◽

Titanium Dioxide ◽

Genetic Risk ◽

Intestinal Inflammation ◽

Sodium Sulphate ◽

Food Additive ◽

Wild Type ◽

Dextran Sodium Sulphate ◽

Risk Gene ◽

The Impact

Environmental and genetic factors have been demonstrated to contribute to the development of inflammatory bowel disease (IBD). Recent studies suggested that the food additive; titanium dioxide (TiO2) might play a causative role in the disease. Therefore, in the present study we aimed to explore the interaction between the food additive TiO2 and the well-characterized IBD risk gene protein tyrosine phosphatase non-receptor type 2 (Ptpn2) and their role in the development of intestinal inflammation. Dextran sodium sulphate (DSS)-induced acute colitis was performed in mice lacking the expression of Ptpn2 in myeloid cells (Ptpn2LysMCre) or their wild type littermates (Ptpn2fl/fl) and exposed to the microparticle TiO2. The impact of Ptpn2 on TiO2 signalling pathways and TiO2-induced IL-1β and IL-10 levels were studied using bone marrow-derived macrophages (BMDMs). Ptpn2LysMCre exposed to TiO2 exhibited more severe intestinal inflammation than their wild type counterparts. This effect was likely due to the impact of TiO2 on the differentiation of intestinal macrophages, suppressing the number of anti-inflammatory macrophages in Ptpn2 deficient mice. Moreover, we also found that TiO2 was able to induce the secretion of IL-1β via mitogen-activated proteins kinases (MAPKs) and to repress the expression of IL-10 in bone marrow-derived macrophages via MAPK-independent pathways. This is the first evidence of the cooperation between the genetic risk factor Ptpn2 and the environmental factor TiO2 in the regulation of intestinal inflammation. The results presented here suggest that the ingestion of certain industrial compounds should be taken into account, especially in individuals with increased genetic risk

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Elimination of 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid 9-phosphate synthase activity from human N-acetylneuraminic acid 9-phosphate synthase by a single mutation

Biochemical Journal ◽

10.1042/bj20052034 ◽

2006 ◽

Vol 397 (1) ◽

pp. 195-201 ◽

Cited By ~ 9

Author(s):

Jijun Hao ◽

Willie F. Vann ◽

Stephan Hinderlich ◽

Munirathinam Sundaramoorthy

Keyword(s):

Aldol Condensation ◽

Saturation Mutagenesis ◽

Single Mutation ◽

Wild Type ◽

Wild Type Enzyme ◽

High Sequence Identity ◽

Acetylneuraminic Acid ◽

Nonulosonic Acid ◽

The Impact ◽

Phosphate Synthase

The most commonly occurring sialic acid Neu5Ac (N-acetylneuraminic acid) and its deaminated form, KDN (2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid), participate in many biological functions. The human Neu5Ac-9-P (Neu5Ac 9-phosphate) synthase has the unique ability to catalyse the synthesis of not only Neu5Ac-9-P but also KDN-9-P (KDN 9-phosphate). Both reactions are catalysed by the mechanism of aldol condensation of PEP (phosphoenolpyruvate) with sugar substrates, ManNAc-6-P (N-acetylmannosamine 6-phosphate) or Man-6-P (mannose 6-phosphate). Mouse and putative rat Neu5Ac-9-P synthases, however, do not show KDN-9-P synthase activity, despite sharing high sequence identity (>95%) with the human enzyme. Here, we demonstrate that a single mutation, M42T, in human Neu5Ac-9-P synthase can abolish the KDN-9-P synthase activity completely without compromising the Neu5Ac-9-P synthase activity. Saturation mutagenesis of Met42 of the human Neu5Ac-9-P synthase showed that the substitution with all amino acids except leucine retains only the Neu5Ac-9-P synthase activity at levels comparable with the wild-type enzyme. The M42L mutant, like the wild-type enzyme, showed the additional KDN-9-P synthase activity. In the homology model of human Neu5Ac-9-P synthase, Met42 is located 22 Å (1 Å=0.1 nm) away from the substrate-binding site and the impact of this distant residue on the enzyme functions is discussed.

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Understanding the Impact of Inflammation on Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v116.21.2629.2629 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2629-2629

Author(s):

Ying Zhao ◽

Flora Ling ◽

Hong-Cheng Wang ◽

Xiao-Hong Sun

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Chronic Inflammation ◽

Bone Marrow Cells ◽

Wild Type ◽

Hematopoietic Stem ◽

Inflammatory Conditions ◽

The Impact ◽

Lps Treatment ◽

Marrow Cells

Abstract Abstract 2629 The overall objectives of this study are to investigate the impact of inflammatory conditions on hematopoietic stem cell (HSC) maintenance and to elucidate the underlying mechanisms. HSCs are exposed to a variety of inflammatory conditions through life. How these conditions influence the integrity of HSCs is a fundamental issue of clinical importance but it is poorly understood. Equally unknown is the molecular regulation of HSC maintenance during inflammatory. In this context, our focus is on the role of basic helix-loop-helix (bHLH) proteins, which include transcription activators such as E2A proteins and their inhibitors including Id proteins. We and others have shown that these regulators are involved in normal hematopoiesis such as stem cell function and lineage specific differentiation. Recently, we have obtained evidence to suggest that signaling through Toll-like receptors (TLRs), which is closely linked to inflammation, causes down-regulation of E2A function by stimulating Id1 expression. Therefore, we hypothesize that inflammatory conditions causes down-regulation of E protein function, which disturbs the quiescence of long-term (LT)-HSC, leading to stem cell exhaustion over time. To test this hypothesis, we induced chronic inflammation in wild type and Id1-/- mice by daily injection of 1 mg of LPS, i.p. for 30 days. Peripheral blood was collected on days 15 and 30 and levels of a panel of inflammatory cytokines were assayed using a Luminex multiplex kit. On day 15, dramatic increases were found in the levels of IL-10, IL-6, KC and TNFα but not IFN-γ, IL12-p70 and IL-1β. Interestingly, levels of IL-6 and TNFα were significantly lower in Id1-/- mice compared to wild type mice. By day 30 of LPS treatment, levels of these cytokines returned to the levels in animals without LPS injection. These results suggest that this chronic LPS treatment indeed elicited an inflammatory response that included transient elevation of inflammatory cytokines. Whether secretion of these cytokines has any direct effects on HSCs remains to be determined. To measure HSC activity in these LPS-treated mice, we performed serial bone marrow transplant assays. Lin−Sca-1+c-kit+ (LSK) stem/progenitor cells were isolated from wild type or Id1-/- mice treated with or without LPS. These cells were transplanted into lethally irradiated CD45.1+ recipients along with equal numbers of YFP-expressing LSK as competitors. Six weeks later, cohorts of mice were sacrificed and bone marrow cells were collected. Pooled whole bone marrow cells within each cohort were injected into lethally irradiated secondary recipients. Secondary recipients were sacrificed 8 and 16 weeks post transplant. For assessment of primary and secondary engraftment, bone marrow cells were examined for expression of donor and lineage specific markers. Robust engraftment was observed in primary or secondary recipients. Donor derived cells were then gated for YFP− and YFP+ cells, which separate cells originated from tester and competitor LSK, respectively. While YFP− and YFP+ cells engrafted equivalently in primary recipients transplanted with cells treated with or without LPS, LPS treatment of wild type mice caused a great disparity in secondary recipients. In contrast, HSC in Id1-/- mice did not appear to be affected by the same treatment even though HSCs in Id1 deficient mice are normally lower in numbers and activities as we previously reported. These results suggest that chronic inflammation diminishes the LT-stem cell activity and this may involve the up-regulation of Id1 expression. To investigate the underlying mechanism, we performed label retaining assays to examine the quiescence of LT-HSCs. We found that BrdU-labeling in HSCs was 2-fold lower in mice treated with LPS compared to the untreated controls, suggesting that treatment with LPS promoted the cycling of HSCs, thus impairing their stem cell function. Taken together, our study illustrates that chronic inflammation has a detrimental effect on LT-stem cell activity. Although HSCs have an enormous capability to repopulate the bone marrow by compensatory proliferation, pro-longed inflammation could eventually lead to stem cell exhaustion and seriously compromise hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

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Alterations In the Bone Marrow Microenvironment Contribute to Oxidative Stress and DNA Damage In Hematopoietic Stem/Progenitors Carrying a Csf3r Truncation Mutation

Blood ◽

10.1182/blood.v116.21.387.387 ◽

2010 ◽

Vol 116 (21) ◽

pp. 387-387

Author(s):

Ghada M Kunter ◽

Jill Woloszynek ◽

Daniel C. Link

Keyword(s):

Oxidative Stress ◽

Bone Marrow ◽

Dna Damage ◽

Single Dose ◽

Bone Marrow Failure ◽

Bone Marrow Microenvironment ◽

Wild Type ◽

Leukemic Transformation ◽

Hematopoietic Stem ◽

Increased Risk

Abstract Abstract 387 A shared feature of many bone marrow failure syndromes is their propensity to develop myelodysplasia (MDS) or acute myeloid leukemia (AML). The molecular mechanisms that underlie this susceptibility are largely unknown. Severe congenital neutropenia (SCN) is an inherited disorder of granulopoiesis that is associated with a marked increased risk of developing MDS/AML. Somatic mutations of CSF3R, encoding the G-CSF receptor (G-CSFR), that truncate the carboxy-terminal tail are associated with the development of MDS/AML in SCN. Transgenic mice carrying a ‘knock-in’ mutation of their Csf3r (termed d715 G-CSFR) reproducing a mutation found in a patient with SCN have normal basal granulopoiesis but an exaggerated neutrophil response to G-CSF treatment. We previously reported that the d715 G-CSFR is able to cooperate with the PML-RARƒÑ oncogene to induce AML in mice. Herein, we summarize data supporting the hypothesis that alterations in the bone marrow microenvironment induced by G-CSF contribute to oxidative DNA damage in hematopoietic stem/progenitors cells (HSPCs) and possibly leukemic transformation. We previously showed that G-CSF treatment is associated with a marked loss of osteoblasts in the bone marrow, thereby potentially disrupting the osteoblast stem cell niche (Semerad, Blood 2005). Of note, patients with SCN chronically treated with G-CSF are prone to develop osteopenia, suggesting that osteoblast suppression by G-CSF also may occur in humans. We first asked whether the d715 G-CSFR was able to mediate this response. Wild-type or d715 G-CSFR were treated with G-CSF for 1–7 days and osteoblast activity in the bone marrow measured by expression of CXCL12 and osteocalcin. Consistent with previous reports, a decrease in osteocalcin and CXCL12 was not apparent until after 3 days of G-CSF treatment and reached a maximum after 7 days. Surprisingly, the magnitude of osteoblast suppression was greater in d715 G-CSFR compared with wild-type mice. The fold-decrease in osteocalcin mRNA from baseline in wild-type mice was 147 ± 70.1 versus 1,513 ± 1091 in d715 G-CSFR mice (p < 0.001). Likewise, a greater fold-decrease in CXCL12 mRNA was observed. We next assessed oxidative stress in c-KIT+ Sca+ lineage− (KSL) progenitors after G-CSF treatment. In both wild-type and d715 G-CSFR KSL cells no increase in reactive oxygen species (ROS) was observed at baseline or 12 hours after a single dose of G-CSF. However, after 7 days of G-CSF, a significant increase (3.4 ± 0.1 fold; p = 0.009) in ROS was observed in d715 G-CSFR but not wild-type KSL cells. To determine whether oxidative stress contributed to DNA damage, histone H2AX phosphorylation (pH2AX) was measured by flow cytometry. No increase in pH2AX was observed after short-term (less than 24 hour) G-CSF treatment. However, a modest but significant (1.9 ± 0.1 fold; p = 0.0007) increase in pH2AX was observed in d715 G-CSFR but not wild-type KSL cells after 7 days of G-CSF. To determine whether increased oxidative stress was casually linked to DNA damage, we co-administered the antioxidant N-acetyl cysteine (NAC) during G-CSF treatment. As expected, induction of ROS in KSL cells was markedly suppressed by NAC administration. Importantly, the increase in pH2AX levels in d715 G-CSFR KSL cells induced by G-CSF was completely blocked by NAC administration. Finally, to determine whether alterations in the bone marrow microenvironment, specifically decreased CXCL12 expression, contributed to DNA damage, we treated mice with AMD3100, a specific antagonist of CXCR4 (the major receptor for CXCL12). Treatment of wild-type or d715 G-CSFR mice with a single dose of G-CSF (3 hour time point) or with AMD3100 alone did not induce H2AXp. However, co-administration of AMD3100 with a single dose of G-CSF induced modest but significant H2AXp in d715 G-CSFR KSL cells (5.74 ± 1.06 fold; P<0.001). Collectively, these data suggest a model in which alterations in the bone marrow microenvironment induced by G-CSF may contribute to genetic instability in HSPCs and ultimately leukemic transformation. The mutant CSF3R may contribute to leukemogenesis through both increased ROS production in HSPCs and increased suppression of osteoblasts. Disclosures: No relevant conflicts of interest to declare.

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Tet2 Loss Accelerates The Myeloproliferative Neoplasm (MPN) Phenotype Of Jak2V617F Knockin Mice But Is Insufficient To Cause Leukemic Transformation

Blood ◽

10.1182/blood.v122.21.4095.4095 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4095-4095

Author(s):

Edwin Chen ◽

Lawrence J Breyfogle ◽

Rebekka K. Schneider ◽

Luke Poveromo ◽

Ross L. Levine ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Myeloproliferative Neoplasm ◽

Conditional Knockout ◽

The Other ◽

Myelogenous Leukemia ◽

Leukemic Transformation ◽

The Impact

Abstract TET2 mutations are early somatic events in the pathogenesis of acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and myeloproliferative neoplasms (MPN) and are one of the most common genetic lesions found in these diseases. In MPN, TET2 mutations are enriched within more advanced disease phenotypes such as myelofibrosis and leukemic transformation and often co-occur with the JAK2V617F mutation, which is present in the majority of MPN patients. We have developed and characterized a Jak2V617F conditional knockin mouse (Jak2VF/+), the phenotype of which closely recapitulates the features of human MPN. To determine the impact of Tet2 loss on Jak2V617F-mediated MPN, we crossed Tet2 conditional knockout mice with Jak2VF/+ knockin and Vav-Cre transgenic mice and backcrossed the compound mutant animals. We then characterized the effects of heterozygous and homozygous loss of Tet2 on the phenotype of Jak2VF/+ mice. We assessed peripheral blood counts, histopathology, hematopoietic differentiation using flow cytometry, colony formation and re-plating capacity. We also evaluated the effects of Tet2 loss on the transcriptome of the HSC compartment using gene expression microarrays and on HSC function using competitive bone marrow transplantation assays. Similar to Jak2VF/+/VavCre+ mice, Tet2+/-/Jak2VF/+/VavCre+ and Tet2-/-/Jak2VF/+/VavCre+ mice develop leukocytosis, elevated hematocrits (HCT) and thrombocytosis. Tet2-/-/Jak2VF/+/VavCre+ mice demonstrate enhanced leukocytosis and splenomegaly compared to the other groups. All groups demonstrate myeloid expansion, erythroid hyperplasia and megakaryocytic abnormalities consistent with MPN in the bone marrow and spleen, while more prominent myeloid expansion and megakaryocytic morphological abnormalities are observed in Tet2-/-/Jak2VF/+/VavCre+ mice as compared to the other groups. Notably, we do not see the development of acute myelogenous leukemia (AML) in Tet2-/-/Jak2VF/+/VavCre+ mice at 6 months. We see enhanced expansion of lineagelowSca1+cKithigh (LSK) cells (enriched for HSC) most prominently in the spleens of Tet2+/-/Jak2VF/+/VavCre+ and Tet2-/-/Jak2VF/+/VavCre+ mice as compared to Jak2VF/+/VavCre+ mice. In colony forming assays, we find that Tet2-/-/Jak2VF/+/VavCre+ LSK cells have enhanced re-plating activity compared to Jak2VF/+/VavCre+ LSK cells and that Tet2-/-/Jak2VF/+/VavCre+ LSK cells form more colonies that Tet2-/-/Jak2+/+/VavCre+ cells. Gene expression analysis demonstrates enrichment of a HSC self-renewal signature inTet2-/-/Jak2VF/+/VavCre+ LSK cells. Concordant with this, we find that Tet2-/-/Jak2VF/+/VavCre+ LSK cells have enhanced competitive repopulation at 16 weeks as compared to Jak2VF/+/VavCre+ and Tet2+/-/Jak2VF/+/VavCre+ LSK cells. In aggregate these findings demonstrate that Tet2 loss promotes disease progression in MPN but is insufficient to drive full leukemic transformation. Disclosures: No relevant conflicts of interest to declare.

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Enforced Differentiation of Dnmt3a-Null Bone Marrow Leads to Failure with c-Kit Mutations Driving Leukemic Transformation

Blood ◽

10.1182/blood.v124.21.837.837 ◽

2014 ◽

Vol 124 (21) ◽

pp. 837-837

Author(s):

Hamza Celik ◽

Cates Mallaney ◽

Alok Kothari ◽

Christopher A Miller ◽

Jasreet Hundal ◽

...

Keyword(s):

Bone Marrow ◽

Survival Curve ◽

Lymphoblastic Leukemia ◽

Loss Of Function ◽

Wild Type ◽

Leukemic Transformation ◽

Kaplan Meier ◽

Cell Acute Lymphoblastic Leukemia ◽

Meier Survival Curve

Abstract Genome sequencing studies of patient samples have implicated the involvement of various components of the epigenetic machinery in myeloid diseases, including the de novo DNA methyltransferase DNMT3A (Cancer Genome Atlas Research, N Engl J Med, 2013). We have recently shown that Dnmt3a is essential for normal hematopoietic stem cell (HSC) differentiation. Genetic ablation of Dnmt3a resulted in HSCs that showed diminished capacity for peripheral blood generation after serial transplantation (on a per-HSC basis), while phenotypically-defined HSCs accumulated in the bone marrow (Challen et al., Nature Genetics, 2012). Although this differentiation arrest was insufficient to cause overt disease, in these competitive transplants the presence of wild-type whole bone marrow may have suppressed malignant transformation of the mutant HSCs. Dnmt3a-null HSCs were less proliferative than counterpart control HSCs in this transplantation setting, suggesting that the cellular turnover threshold necessary to generate additional genetic and/or epigenetic lesions required for leukemogenesis was not achieved. To further understand the contribution of Dnmt3a loss-of-function in hematopoiesis, we performed non-competitive transplantation of Dnmt3a-null bone marrow. This forces the mutant HSCs to divide in vivoto regenerate the hematopoietic system following lethal irradiation, and should uncover any predispositions to transformation. Mice transplanted with Dnmt3a-null bone marrow in the absence of wild-type support cells succumbed principally to bone marrow failure (median survival 328 days) characteristic of myelodysplastic syndromes (MDS) with symptoms including anemia, neutropenia, bone marrow hypercellularity and splenomegaly with myeloid infiltration. 2/25 mice developed myeloid leukemia with >20% blasts in the blood and bone marrow. 4/25 primary mice succumbed to myeloproliferative disorders, some of which progressed to secondary leukemia after long latency. Exome sequencing was performed to identify co-operating mutations that drove leukemic transformation, and revealed c-Kit mutations found only in the Dnmt3a-null AML samples. As DNMT3A and KIT mutations can co-occur in AML and mastocytosis, we tested whether these two pathways could co-operate in vivo by ectopic introduction of c-Kit variants into hematopoietic progenitors followed by bone marrow transplantation (Figure 1). As previously reported, expression of c-KitD814V in wild-type cells lead to development of B-cell acute lymphoblastic leukemia (B-ALL). However, expression of c-KitD814V in a Dnmt3a-null background lead to acute leukemia with a much shorter latency (median survival 67 days), implicating a synergism between these pathways in vivo. Moreover, the absence of Dnmt3a also distorted the spectrum of leukemia resulting from enforced c-Kit signaling. While some of the mice transplanted with Dnmt3a-null c-KitD814V cells also succumbed to a B-ALL, 4/13 (31%) developed mastocytosis with involvement of myeloid blasts, and 4/13 (31%) mice developed a T-cell acute lymphoblastic leukemia (T-ALL). We show for the first time that these pathways can co-operate to accelerate transformation in vivo. This Dnmt3a/c-Kit disease model resembles the classical “two-hit” model of leukemogenesis in which one mutation in a hematopoietic progenitor cell inhibits differentiation (Dnmt3a loss-of-function), whilst another drives proliferation (c-Kit gain-of-function). Such mouse models present a unique opportunity to study the sequence of early events leading to HSC transformation following Dnmt3a-inactivation. Figure 1 Kaplan-Meier survival curve of mice transplanted with control or Dnmt3a-KO bone marrow progenitor cells transduced with a lentivirus expressing c-KitD814V. *** p <0.001. Figure 1. Kaplan-Meier survival curve of mice transplanted with control or Dnmt3a-KO bone marrow progenitor cells transduced with a lentivirus expressing c-KitD814V. *** p <0.001. Disclosures No relevant conflicts of interest to declare.

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Mycobacterium tuberculosis Requires Cholesterol Oxidase to Disrupt TLR2 Signalling in Human Macrophages

Mediators of Inflammation ◽

10.1155/2019/2373791 ◽

2019 ◽

Vol 2019 ◽

pp. 1-17

Author(s):

Izabela Szulc-Kielbik ◽

Michal Kielbik ◽

Patrycja Przygodzka ◽

Anna Brzostek ◽

Jaroslaw Dziadek ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Cholesterol Oxidase ◽

Interleukin 10 ◽

Signalling Pathway ◽

Mrna Levels ◽

Dependent Manner ◽

Wild Type ◽

Human Macrophages ◽

Protein Levels ◽

The Impact

This study tested the hypothesis that Mycobacterium tuberculosis (Mtb) uses a cholesterol oxidase enzyme (ChoD) to suppress a toll-like receptor type 2- (TLR2-) dependent signalling pathway to modulate macrophages’ immune response. We investigated the impact of Mtb possessing or lacking ChoD as well as TBChoD recombinant protein obtained from Mtb on the expression and activation of two key intracellular proteins involved in TLR2 signalling in human macrophages. Finally, the involvement of TLR2-related signalling proteins in an inflammatory/immunosuppressive response of macrophages to Mtb was evaluated. We demonstrate that wild-type Mtb but not the ∆choD mutant decreased the cytosolic IRAK4 and TRAF6 protein levels while strongly enhancing IRAK4 and TRAF6 mRNA levels in macrophages. Our data show that the TLR2 present on the surface of macrophages are involved in disturbing the signalling pathway by wild-type Mtb. Moreover, recombinant TBChoD effectively decreased the cytosolic level of TRAF6 and lowered the phosphorylation of IRAK4, which strongly confirm an involvement of cholesterol oxidase in affecting the TLR2-related pathway by Mtb. Wild-type Mtb induced an immunosuppressive response of macrophages in an IRAK4- and TRAF6-dependent manner as measured by interleukin 10 production. In conclusion, ChoD is a virulence factor that enables Mtb to disturb the TLR2-related signalling pathway in macrophages and modulate their response.

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Cytotoxic Injury to Thymic Stroma and Impaired Reconstitution.

Blood ◽

10.1182/blood.v104.11.1232.1232 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1232-1232

Author(s):

Susan E. Prockop ◽

Richard J. O’Reilly ◽

Howard Petrie

Keyword(s):

Bone Marrow ◽

T Cell ◽

De Novo ◽

Immunosuppressive Treatment ◽

Lymphoid Cells ◽

Cytotoxic Agents ◽

Cell Transplant ◽

Wild Type ◽

Long Term Outcome ◽

The Impact

Abstract A key component of long-term outcome after stem cell transplant (SCT) is successful reconstitution of the immune system. Effective reconstitution of antigen-specific T-cell immunity requires de novo T cell generation. Bone marrow derived progenitors seed the thymus and undergo a complex process involving lineage commitment, proliferation and selection. Coordinated interaction of marrow-derived lymphoid progenitors with thymic stromal cells is required for successful T lymphopoiesis in the post-natal thymus. Disruption of the microenvironment can result in disrupted T cell lymphopoiesis. One cause of prolonged defects in generating functional T lymphocytes after BMT is damage to the thymic microenvironment induced by radiation or cytotoxic therapy. However, the impact of individual agents, administered at myeloablative or non-myeloablative doses, on the thymic microenvironment has not been fully evaluated. In addition, mechanisms by which stromal injury modifies T cell production and maturation have only begun to be understood. We have developed a model system using immunodeficient mice as a platform on which to assess thymic reconstitution. The thymus of mice deficient for the alpha chain of the IL-7 receptor (IL7R−/−) is relatively depleted of lymphoid cells and can be reconstituted following transplant of wild type marrow administered without myeloablative or immunosuppressive treatment. Injection of low doses of wild type bone marrow into these mice results in low levels of marrow chimerism and a normally cellular thymus repopulated with donor-derived lymphocytes. The ability to achieve this reconstitution appears to depend on absolute numbers of early intra-thymic precursors, rather than on total thymic cellularity. We have exploited this model to differentially assess the effects of cytotoxic agents including radiation and immunosuppressive drugs, on the capacity of the thymic microenvironment to support the maturation of normal lympoid progenitors (Figure 1). We demonstrate that some agents do not affect the ability of the thymic microenvironment to support reconstitution (eg fludarabine), others nearly ablate it (cyclophosphamide). We are also able to show dose, schedule, and synergistic effects on the ability of the thymic microenvironment to support de novo T cell lymphopoeisis. Distinct morphologic and phenotypic effects can be demonstrated by different agents (eg busulfan versus thiotepa) with preliminary data suggesting that the effects are mediated by injury to different stromal subsets. It is anticipated that this information will lead to strategies to both minimize delayed immune reconstitution and to augment T cell lymphopoiesis post-transplant. In addition, further evaluation of impaired thymic reconstitution will augment the understanding of lymphostromal interactions crucial to normal T cell lymphopoiesis.

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Unbalanced X Chromosome Inactivation Independent of Telomere Shortening in Mice Heterozygous for a Mutant Dyskerin Allele.

Blood ◽

10.1182/blood.v108.11.184.184 ◽

2006 ◽

Vol 108 (11) ◽

pp. 184-184

Author(s):

Baiwei Gu ◽

Jun He ◽

Monica Bessler ◽

Philip J. Mason

Keyword(s):

Bone Marrow ◽

Ribosome Biogenesis ◽

Es Cells ◽

X Inactivation ◽

Telomere Maintenance ◽

Male Mice ◽

Wild Type ◽

Telomere Shortening ◽

Ribonucleoprotein Complexes ◽

The Impact

Abstract X-linked Dyskeratosis Congenita (DC) is a rare recessive disorder caused by mutations in the DKC1 gene that encodes dyskerin. Dyskerin is part of ribonucleoprotein complexes that participate in two different pathways: ribosome biogenesis and telomere maintenance. It is the subject of intense debate whether disease manifestations in DC are due to dysfunctional telomere maintenances or are caused by a defect in ribosome biogenesis. Pathogenic mutations in dyskerin cause telomere shortening and patients with X-linked DC have critically short telomeres, However, whether there is an additional defect in ribosome biogenesis is difficult to investigate. To dissect the impact of a pathogenic dyskerin mutation on telomeres from the possible additional impact on ribosome biogenesis in an in vivo model, we generated mice expressing a mutant dyskerin protein. Because laboratory mice have very long telomeres a short telomere phenotype requires several generations of inbreeding, whereas a phenotype seen in the first generation is likely to be caused by the defect in ribosome biogenesis. To delete the last 21 amino acids of dyskerin (Del15) we used homologous recombination followed by conditional gene deletion in murine embryonic stem (ES) cells and in mice. Six independent ES cell clones with the deleted Dkc1 gene were obtained. In vitro analysis of the ES cells showed that the Del15 mutation led to dramatically decreased expression of a truncated dyskerin protein with decreased accumulation of the telomerase RNA. In addition, both reduction in telomerase activity and significant telomere shortening after 65 passages were observed. These findings indicate that the Del15 mutation impairs the telomerase maintenance pathway. After testing the accumulation of a series of mouse H/ACA snoRNA in Del15 ES cells, we found a decrease of the mU68 and mE1 snoRNAs suggesting the mutation may also confer effects which are outside the telomerase pathway. We therefore went on to produce a line of mice expressing the truncated Dkc1 protein and were able to obtain male mice hemizygous for the mutant Dkc1 gene as well as female heterozgotes. The male mice express the truncated dyskerin protein and show no gross abnormality up to 6 months of age. Interestingly, heterozygous female mice were healthy as well but the truncated dyskerin protein was dramatically decreased in expression compared to the wild type dyskerin in spleen, thymus, and bone marrow, but not in liver and brain. This result must derive from preferential proliferation of cells expressing wild type dyskerin after random X-inactivation in early embryogenesis. Our analysis indicates that the mutant dyskerin impairs the proliferation in hematopoietic tissues while it does not affect cells which are not rapidly proliferating such as those in liver and brain. Because of the early appearance of the skewed X-inactivation phenotype we conclude that skewing in these mice is caused by a telomere independent mechanism. Interestingly, the lack of overt DC-like abnormalities in the male hemizygous mice indicates that this proliferative disadvantage is insufficient to cause bone marrow failure but in combination with impaired telomere maintenance may accelerate the onset and severity of disease and thus explain the earlier and more severe manifestation in X-linked DC compared to autosomal dominant DC which only affects the telomerase pathway.

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