Determining the role of inflammation in the selection of JAK2 mutant cells in myeloproliferative neoplasms

AbstractMyeloproliferative neoplasm (MPN) is a hematologic malignancy characterized by the clonal outgrowth of hematopoietic cells with a somatically acquired mutation most commonly in JAK2 (JAK2V 617F). This mutation endows upon myeloid progenitors cytokine independent growth and consequently leads to excessive production of myeloid lineage cells. It has been previously suggested that inflammation may play a role in the clonal evolution of JAK2V 617F mutants. In particular, it is possible that one or more cellular kinetic parameters of hematopoietic stem cells (HSCs) are affected by inflammation, such as division or death rates of cells, and the probability of HSC differentiation. This suggests a mechanism that can steer the outcome of the cellular competition in favor of the mutants, initiating the disease. In this paper we create a number of mathematical evolutionary models, from very abstract to more concrete, that describe cellular competition in the context of inflammation. It is possible to build a model axiomatically, where only very general assumptions are imposed on the modeling components and no arbitrary (and generally unknown) functional forms are used, and still generate a set of testable predictions. In particular, we show that, if HSC death is negligible, the evolutionary advantage of mutant cells can only be conferred by an increase in differentiation probability of HSCs in the presence of inflammation, and if death plays a significant role in the dynamics, an additional mechanism may be an increase of HSC’s division-to-death ratio in the presence of inflammation. Further, we show that in the presence of inflammation, the wild type cell population is predicted to shrink under inflammation (even in the absence of mutants). Finally, it turns out that if only the differentiation probability is affected by the inflammation, then the resulting steady state population of wild type cells will contain a relatively smaller percentage of HSCs under inflammation. If the division-to-death rate is also affected, then the percentage of HSCs under inflammation can either decrease or increase, depending on other parameters.

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Cell competition between wild-type and JAK2V617F mutant cells in a murine model of a myeloproliferative neoplasm

10.1101/2020.08.26.267070 ◽

2020 ◽

Author(s):

Melissa Castiglione ◽

Haotian Zhang ◽

Huichun Zhan

Keyword(s):

Stem Cell ◽

Myeloproliferative Neoplasms ◽

Myeloproliferative Neoplasm ◽

List Type ◽

Wild Type ◽

Hematopoietic Stem ◽

Increased Risk ◽

Mutant Cells

AbstractThe myeloproliferative neoplasms (MPNs) are clonal stem cell disorders characterized by overproduction of mature blood cells and increased risk of transformation to frank leukemia. The acquired kinase mutation JAK2V617F plays a central role in a majority of these disorders. The hematopoietic stem cell (HSC) compartment in MPN is heterogeneous with the presence of both JAK2 wild-type and JAK2V617F mutant cells in most patients with MPN. Utilizing in vitro co-culture assays and in vivo competitive transplantation assays, we found that the presence of wild-type cells altered the behavior of co-existing JAK2V617F mutant cells, and a mutant microenvironment (niche) could overcome the competition between wild-type and mutant cells, leading to mutant clonal expansion and overt MPN. We also demonstrated that competition between wild-type and JAK2V617F mutant cells triggered a significant immune response, and there was a dynamic PD-L1 deregulation in the mutant stem/progenitor cells caused by their interactions with the neighboring wild-type cells and the microenvironment. Therefore, while accumulation of oncogenic mutations is unavoidable during aging, our data suggest that, if we could therapeutically enhance normal cells’ ability to compete, we might be better able to control neoplastic cell expansion and prevent the development of a full-blown malignancy.Key PointsThe presence of wild-type cells alters the behavior of co-existing JAK2V617F mutant cellsA mutant microenvironment overcomes the competition between wild-type and JAK2V617F mutant cells, leading to the development of a MPN

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U2AF1(S34F) Mutant Hematopoietic Cells Require Expression of Wild-Type U2af1 for Survival

Blood ◽

10.1182/blood-2018-99-110836 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 941-941

Author(s):

Brian Wadugu ◽

Amanda Heard ◽

Joseph Bradley ◽

Matthew Ndonwi ◽

Jin J Shao ◽

...

Keyword(s):

Bone Marrow ◽

Rna Splicing ◽

Bone Marrow Cells ◽

Hematopoietic Cells ◽

Poly I:C ◽

Wild Type ◽

Hematopoietic Stem ◽

Mutant Cells ◽

Marrow Cells

Abstract Somatic mutations in U2AF1, a spliceosome gene involved in pre-mRNA splicing, occur in up to 11% of MDS patients. While we reported that mice expressing mutant U2AF1(S34F) have altered hematopoiesis and RNA splicing, similar to mutant MDS patients, the role of wild-type U2AF1 in normal hematopoiesis has not been studied. U2AF1mutations are always heterozygous and the wild-type allele is expressed, suggesting that mutant cells require the residual wild-type (WT) allele for survival. A complete understanding of the role of wild-type U2AF1 on hematopoiesis and RNA splicing will enhance our understanding of how mutant U2AF1 contributes to abnormal hematopoiesis and splicing in MDS. In order to understand the role of wild-type U2af1 in normal hematopoiesis, we created a conditional U2af1 knock-out (KO) mouse (U2af1flox/flox). Homozygous embryonic deletion of U2af1using Vav1-Cre was embryonic lethal and led to reduction in fetal liver hematopoietic stem and progenitor cells (KLS and KLS-SLAM, p ≤ 0.05) at embryonic day 15, suggesting that U2af1 is essential for hematopoiesis during embryonic development. To study the hematopoietic cell-intrinsic effects of U2af1 deletion in adult mice, we performed a non-competitive bone marrow transplant of bone marrow cells from Mx1-Cre/U2af1flox/flox, Mx1-Cre/U2af1flox/wtor Mx1-Cre/U2af1wt/wtmice into lethally irradiated congenic recipient mice. Following poly I:C-induced U2af1deletion, homozygous U2af1 KOmice, but not other genotypes (including heterozygous KO mice), became moribund. Analysis of peripheral blood up to 11 days post poly I:C treatment revealed anemia (hemoglobin decrease >1.7 fold) and multilineage cytopenias in homozygous U2af1 KOmice compared to all other genotypes(p ≤ 0.001, n=5 each).Deletion of U2af1 alsoled to rapid bone marrow failure and a reduction in the absolute number of bone marrow neutrophils (p ≤ 0.001), monocytes (p ≤ 0.001), and B-cells (p ≤ 0.05), as well as a depletion of hematopoietic progenitor cells (KL, and KLS cells, p ≤ 0.001, n=5 each). Next, we created mixed bone marrow chimeras (i.e., we mixed equal numbers of homozygous KO and wild-type congenic competitor bone marrow cells and transplanted them into lethally irradiated congenic recipient mice) to study the effects of U2af1 deletion on hematopoietic stem cell (HSC) function. As early as 10 days following Mx1-Cre-induction, we observed a complete loss of peripheral blood neutrophil and monocyte chimerism of the U2af1 KOcells, but not U2af1 heterozygous KO cells, and at 10 months there was a complete loss of homozygous U2af1 KObone marrow hematopoietic stem cells (SLAM, ST-HSCs, and LT-HSCs), neutrophils, and monocytes, as well as a severe reduction in B-cells and T-cells (p ≤ 0.001, n=3-4 for HSCs. p ≤ 0.001, n=9-10 for all other comparisons). The data indicate that normal hematopoiesis is dependent on wild-type U2af1expression, and that U2af1 heterozygous KO cells that retain one U2af1 allele are normal. Next, we tested whether mutant U2AF1(S34F) hematopoietic cells require expression of wild-type U2AF1 for survival. To test this, we used doxycycline-inducible U2AF1(S34F) or U2AF1(WT) transgenic mice. We generated ERT2-Cre/U2af1flox/flox/TgU2AF1-S34F/rtTA(S34F/KO), and ERT2-Cre/U2af1flox/flox/TgU2AF1-WT/rtTA,(WT/KO) mice, as well as all other single genotype control mice. We then created 1:1 mixed bone marrow chimeras with S34F/KO or WT/KO test bone marrow cells and wild-type competitor congenic bone marrow cells and transplanted them into lethally irradiated congenic recipient mice. Following stable engraftment, we induced U2AF1(S34F) (or WT) transgene expression with doxycycline followed by deletion of endogenous mouse U2af1 using tamoxifen. As early as 2 weeks post-deletion of U2af1, S34F/KO neutrophil chimerism dropped to 5.4% indicating loss of mutant cells, while WT/KO neutrophil chimerism remained elevated at 31.6% (p = 0.01, n=6-8). The data suggest that mutant U2AF1(S34F) hematopoietic cells are dependent on expression of wild-type U2af1 for survival. Since U2AF1mutant cells are vulnerable to loss of the residual wild-type U2AF1allele, and heterozygous U2af1KO cells are viable, selectively targeting the wild-type U2AF1allele in heterozygous mutant cells could be a novel therapeutic strategy. Disclosures No relevant conflicts of interest to declare.

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Role of the Polycomb Methyltransferase Ezh1 in Myelodysplastic Syndrome Induced By Ezh2 Insufficiency

Blood ◽

10.1182/blood.v128.22.1968.1968 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1968-1968

Author(s):

Kazumasa Aoyama ◽

Makiko Mochizuki-Kashio ◽

Motohiko Oshima ◽

Shuhei Koide ◽

Yaeko Nakajima-Takagi ◽

...

Keyword(s):

Myelodysplastic Syndrome ◽

Intraperitoneal Injection ◽

Target Genes ◽

Myeloproliferative Neoplasms ◽

Hematologic Malignancies ◽

Gene Set Enrichment Analysis ◽

Wild Type ◽

Hematopoietic Stem ◽

The Impact

Abstract Ezh1 and Ezh2, the catalytic components of polycomb-repressive complex 2 (PRC2), negatively control gene expression by catalyzing mono, di, and tri-methylation of histone H3 at lysine 27 (H3K27me1/me2/me3). Loss-of-function mutations of EZH2, but not those of EZH1, have been found in patients with hematologic malignancies such as myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPNs), and MDS/MPN overlap disorders. We previously demonstrated that hematopoietic cell-specific Ezh2 knockout mice (Ezh2Δ/Δ) developed hematologic malignancies including MDS and MDS/MPN. Although deletion of Ezh1, another enzymatic component of PRC2, (Ezh1-/-) did not significantly affect global H3K27me3 levels or hematopoiesis, deletion of both Ezh1 and Ezh2 in mice (Ezh1-/-Ezh2Δ/Δ) caused rapid exhaustion of hematopoietic stem cells (HSCs). Given that only Ezh1 and Ezh2 are known as enzymatic components of PRC2, we concluded that residual PRC2 enzymatic activity is required for HSC maintenance and development of hematologic malignancies in the setting of EZH2 insufficiency frequently observed in MDS. However, the role of Ezh1 in Ezh2-insufficient hematologic malignancies is still not fully understood since hematopoiesis could not be maintained in Ezh1-/-Ezh2Δ/Δ mice. Here we analyzed the impact of Ezh1 heterozygosity on Ezh2-null hematopoiesis (Ezh1+/-Ezh2Δ/Δ), in which PRC2 activity is mediated by a single allele of Ezh1, for better understanding of Ezh2-deficient hematologic malignancies. We first transplanted BM cells from Ezh1+/-Ezh2flox/flox CD45.2 mice with CD45.1 wild-type competitor cells into lethally irradiated CD45.1 recipient mice and deleted Ezh2 by intraperitoneal injection of tamoxifen. Ezh1+/-Ezh2Δ/Δ cells exhibited a lower repopulation capacity than Ezh2Δ/Δ but established persistent repopulation for at least 6 months after the deletion of Ezh2 while double knockout cells (Ezh1-/-Ezh2Δ/Δ) were outcompeted by competitor cells immediately. We next transplanted BM cells from Ezh1+/-Ezh2flox/flox CD45.2 mice without CD45.1 wild-type competitor cells into lethally irradiated CD45.1 recipient mice and deleted Ezh2 by intraperitoneal injection of tamoxifen. Importantly, recipient mice reconstituted with Ezh1+/-Ezh2Δ/Δ cells exhibited MDS-like phenotypes including anemia and morphological myelodysplasia, which were more pronounced than those of Ezh2Δ/Δ mice. Ezh1+/-Ezh2Δ/Δ mice also showed more advanced hematological abnormalities such as erythroid differentiation block, increased apoptosis of erythroid cells, and extramedullary hematopoiesis in the spleen than Ezh2Δ/Δ mice did. These results suggest that Ezh1 heterozygosity promotes the development of myelodysplasia in the setting of Ezh2insufficiency. Next we examined the molecular mechanism by which the loss of Ezh1 promotes myelodysplasia. Western blot and ChIP-sequence analyses revealed that global levels of H3K27me3 were not significantly changed but H3K27me3 levels at promoter regions of the PRC2 target genes were obviously reduced by Ezh1 heterozygosity in Ezh2Δ/Δ HSPCs. As a consequence, PRC2 target genes were highly de-repressed in Ezh1+/-Ezh2Δ/Δ LSK HSPCs compared with Ezh2Δ/Δ HSPCs. Among these, several genes appeared to be associated with MDS such as S100A9, encoding an inflammatory protein implicated in dyserythropoiesis in MDS. Furthermore, gene set enrichment analysis showed that the genes highly expressed in myeloid cells were positively enriched by Ezh1 heterozygosity in Ezh2Δ/ΔHSPCs. These findings indicate that dosage of Ezh1 is critical in the maintenance of Ezh2-insufficient hematopoiesis as well as the progression of MDS with Ezh2 insufficiency. Disclosures No relevant conflicts of interest to declare.

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Transplantation for bone marrow failure: current issues

Hematology ◽

10.1182/asheducation-2016.1.90 ◽

2016 ◽

Vol 2016 (1) ◽

pp. 90-98 ◽

Cited By ~ 13

Author(s):

Régis Peffault de Latour

Keyword(s):

Bone Marrow Failure ◽

Clonal Evolution ◽

Human Leukocyte ◽

Unrelated Donor ◽

Hematopoietic Stem ◽

Leukocyte Antigen ◽

Sibling Donor ◽

Alternative Donor

Abstract The preferred treatment of idiopathic aplastic anemia (AA) is allogeneic hematopoietic stem cell transplantation (HSCT) from a human leukocyte antigen (HLA)–identical sibling donor. Transplantation from a well-matched unrelated donor (MUD) may be considered for patients without a sibling donor after failure of immunosuppressive therapy, as may alternative transplantation (mismatched, cord blood or haplo-identical HSCT) for patients without a MUD. HSCT may also be contemplated for congenital disorders in cases of pancytopenia or severe isolated cytopenia. Currently, HSCT aims are not only to cure patients but also to avoid long-term complications, notably chronic graft-versus-host disease (GVHD), essential for a good quality of life long term. This paper summarizes recent advances in HSCT for idiopathic and inherited AA disorders. The effect of age on current transplantation outcomes, the role of transplantation in paroxysmal nocturnal hemoglobinuria, and the prevention of GVHD are also discussed. Emerging strategies regarding the role of up-front unrelated donor and alternative donor HSCT in idiopathic AA, along with advances in the treatment of clonal evolution in Fanconi anemia, are also examined.

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A Case of Myeloproliferative Neoplasm with BCR-FGFR1 Rearrangement: Favorable Outcome after Haploidentical Allogeneic Transplantation

Case Reports in Hematology ◽

10.1155/2018/5724960 ◽

2018 ◽

Vol 2018 ◽

pp. 1-4 ◽

Cited By ~ 1

Author(s):

Paola Villafuerte-Gutiérrez ◽

Montserrat López Rubio ◽

Pilar Herrera ◽

Eva Arranz

Keyword(s):

Kinase Inhibitors ◽

Fusion Gene ◽

Myeloproliferative Neoplasms ◽

Therapeutic Option ◽

Myeloproliferative Neoplasm ◽

Allogeneic Transplantation ◽

Term Survival ◽

Hematopoietic Stem ◽

Health Organization

Hematopoietic myeloproliferative neoplasms with FGFR1 rearrangement result in the 8p11 myeloproliferative syndrome that in the current Word Health Organization classification is designated as “myeloid and lymphoid neoplasm with FGFR1 abnormalities.” We report the case of a 66-year-old man who had clinical features that resembled chronic myeloid leukaemia (CML), but bone marrow cytogenetic and fluorescent in situ hybridization (FISH) studies showed t(8;22)(p11;q11) and BCR-FGFR1 fusion gene. He was initially managed with hydroxyurea, and given the aggressive nature of this disease, four months later, the patient underwent an allogeneic hematopoietic stem-cell transplantation (HSCT) from an HLA-haploidentical relative. Currently, HSCT may be the only therapeutic option for long-term survival at least until more efficacious tyrosine kinase inhibitors (TKIs) become available.

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CXCR4-SDF-1 Signaling in Nestin+ Mesenchymal Stem Cell Is Required for HSC Maintenance during Homeostasis and Regeneration after Irradiation

Blood ◽

10.1182/blood.v128.22.3883.3883 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3883-3883 ◽

Cited By ~ 1

Author(s):

Pratibha Singh ◽

Louis M. Pelus

Keyword(s):

Stem Cells ◽

Stromal Cell ◽

Cxcr4 Expression ◽

Wild Type ◽

Post Transplantation ◽

Hematopoietic Stem ◽

Cxcr4 Signaling ◽

Hematopoietic Recovery ◽

Hematopoietic Reconstitution

Hematopoietic stem cells (HSC) reside in a complex microenvironment (niche) within the bone marrow (BM), where multiple populations of microenvironmental stromal cells regulate and finely tune their proliferation, differentiation and trafficking. Recent studies have shown that mesenchymal stem cells (MSC) are an essential component of the HSC niche. Intrinsic HSC CXCR4-SDF-1 signaling has been implicated in self-renewal and quiescence; however, the role of microenvironment CXCR4-SDF-1 signaling in supporting HSC function remains unclear. We previously demonstrated that microenvironmental stromal cell-derived CXCR4 is important for HSC recovery, as transplantation of wild-type HSC into CXCR4 deficient recipients showed reduced HSC engraftment. In this study, we now show that CXCR4-SDF-1 signaling in nestin+ MSC regulates HSC maintenance under normal homeostatic conditions and promotes hematopoietic regeneration after irradiation. Multivariate flow cytometry analysis of marrow stroma cells revealed that mouse BM MSCs identified as CD45-Ter119-CD31-Nestin+PDGFR+CD51+ express the CXCR4 receptor, which was confirmed by RT-PCR analysis. To investigate the role of MSC CXCR4 signaling in niche maintenance and support of HSC function, we utilized genetic mouse models, in which CXCR4 could be deleted in specific stromal cell types. Selective deletion of CXCR4 from nestin+ MSC in adult tamoxifen inducible nestin-cre CXCR4flox/flox mice resulted in reduced total MSC in BM (Control vs. Deleted: 647±128 vs. 209±51/femur, respectively, n=5, p<0.05), which was associated with a significant reduction in Lineage-Sca-1+c-Kit+ (LSK) cells (Control vs. Deleted: 18,033±439 vs. 4523±358/femur, respectively n=5, p<0.05). Selective CXCR4 deletion in nestin+ MSC also resulted in enhanced LSK cell egress to the peripheral circulation (Control vs. Deleted: 1022±106 vs. 2690±757/ml blood, respectively n=5, p<0.05), with no detectable difference in HSC cell cycle or apoptosis. However, the repopulation ability of HSC obtained from mice where CXCR4 was deleted in nestin+ MSC was reduced by >2 fold. In contrast, deletion of CXCR4 from osteoblasts using osteocalcin cre CXCR4flox/flox mice had no effect on HSC numbers in BM and blood.To investigate the role of nestin+ MSC CXCR4 signaling in BM niche reconstruction and hematopoietic recovery, we transplanted BM cells from wild-type mice into syngeneic wild-type or nestin+ MSC CXCR4 deleted recipients after lethal irradiation (950 rad) and analyzed HSC homing, niche recovery and hematopoietic reconstitution. Deletion of CXCR4 from nestin expressing MSC resulted in significantly reduced LSK cell homing at 16 hrs post transplantation (Control vs. Deleted: 8643±1371 vs. 3004±1044/ mouse, respectively, n=5, p<0.05). Robust apoptosis and senescence after total body irradiation was observed in nestin expressing MSCs lacking CXCR4 expression. At 15 days post-transplantation, chimeric mice with nestin+ MSC lacking CXCR4 expression displayed attenuated niche recovery and hematopoietic reconstitution compared to mice with wild-type stroma. In conclusion, our study suggests that CXCR4-SDF-1 signaling in nestin+ MSC is critical for the maintenance and retention of HSC in BM during homeostasis and promotes niche regeneration and hematopoietic recovery after transplantation. Furthermore, our data suggest the modulating CXCR4 signaling in the hematopoietic niche could be beneficial as a means to enhance HSC recovery following clinical hematopoietic transplantation or radiation/chemotherapy injury. Disclosures No relevant conflicts of interest to declare.

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The Power of Extracellular Vesicles in Myeloproliferative Neoplasms: “Crafting” a Microenvironment That Matters

Cells ◽

10.3390/cells10092316 ◽

2021 ◽

Vol 10 (9) ◽

pp. 2316

Author(s):

Lucia Catani ◽

Michele Cavo ◽

Francesca Palandri

Keyword(s):

Extracellular Vesicles ◽

Immune Escape ◽

Myeloproliferative Neoplasms ◽

Cell Communication ◽

Bioactive Molecules ◽

Driver Genes ◽

Hematopoietic Stem ◽

Increased Risk ◽

Patients Experience

Myeloproliferative Neoplasms (MPN) are acquired clonal disorders of the hematopoietic stem cells and include Essential Thrombocythemia, Polycythemia Vera and Myelofibrosis. MPN are characterized by mutations in three driver genes (JAK2, CALR and MPL) and by a state of chronic inflammation. Notably, MPN patients experience increased risk of thrombosis, disease progression, second neoplasia and evolution to acute leukemia. Extracellular vesicles (EVs) are a heterogeneous population of microparticles with a role in cell-cell communication. The EV-mediated cross-talk occurs via the trafficking of bioactive molecules such as nucleic acids, proteins, metabolites and lipids. Growing interest is focused on EVs and their potential impact on the regulation of blood cancers. Overall, EVs have been suggested to orchestrate the complex interplay between tumor cells and the microenvironment with a pivotal role in “education” and “crafting” of the microenvironment by regulating angiogenesis, coagulation, immune escape and drug resistance of tumors. This review is focused on the role of EVs in MPN. Specifically, we will provide an overview of recent findings on the involvement of EVs in MPN pathogenesis and discuss opportunities for their potential application as diagnostic and prognostic biomarkers.

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Synergic Crosstalk between Inflammation, Oxidative Stress, and Genomic Alterations in BCR–ABL-Negative Myeloproliferative Neoplasm

Antioxidants ◽

10.3390/antiox9111037 ◽

2020 ◽

Vol 9 (11) ◽

pp. 1037

Author(s):

Alessandro Allegra ◽

Giovanni Pioggia ◽

Alessandro Tonacci ◽

Marco Casciaro ◽

Caterina Musolino ◽

...

Keyword(s):

Oxidative Stress ◽

Stem Cell ◽

Chronic Inflammation ◽

Myeloproliferative Neoplasms ◽

Myeloproliferative Neoplasm ◽

Tumor Stage ◽

Driver Mutations ◽

Hematopoietic Stem ◽

Chronic Myeloproliferative Neoplasms ◽

Inflammatory State

Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) have recently been revealed to be related to chronic inflammation, oxidative stress, and the accumulation of reactive oxygen species. It has been proposed that MPNs represent a human inflammation model for tumor advancement, in which long-lasting inflammation serves as the driving element from early tumor stage (over polycythemia vera) to the later myelofibrotic cancer stage. It has been theorized that the starting event for acquired stem cell alteration may occur after a chronic inflammation stimulus with consequent myelopoietic drive, producing a genetic stem cell insult. When this occurs, the clone itself constantly produces inflammatory components in the bone marrow; these elements further cause clonal expansion. In BCR–ABL1-negative MPNs, the driver mutations include JAK 2, MPL, and CALR. Transcriptomic studies of hematopoietic stem cells from subjects with driver mutations have demonstrated the upregulation of inflammation-related genes capable of provoking the development of an inflammatory state. The possibility of acting on the inflammatory state as a therapeutic approach in MPNs appears promising, in which an intervention operating on the pathways that control the synthesis of cytokines and oxidative stress could be effective in reducing the possibility of leukemic progression and onset of complications.

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JAK2 V617F impairs hematopoietic stem cell function in a conditional knock-in mouse model of JAK2 V617F–positive essential thrombocythemia

Blood ◽

10.1182/blood-2009-12-259747 ◽

2010 ◽

Vol 116 (9) ◽

pp. 1528-1538 ◽

Cited By ~ 136

Author(s):

Juan Li ◽

Dominik Spensberger ◽

Jong Sook Ahn ◽

Shubha Anand ◽

Philip A. Beer ◽

...

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell ◽

Essential Thrombocythemia ◽

Cell Function ◽

Myeloproliferative Neoplasms ◽

Myeloproliferative Neoplasm ◽

Jak2 V617f ◽

Transgenic Models ◽

Disease Phenotype ◽

Hematopoietic Stem

The JAK2 V617F mutation is found in most patients with a myeloproliferative neoplasm and is sufficient to produce a myeloproliferative phenotype in murine retroviral transplantation or transgenic models. However, several lines of evidence suggest that disease phenotype is influenced by the level of mutant JAK2 signaling, and we have therefore generated a conditional knock-in mouse in which a human JAK2 V617F is expressed under the control of the mouse Jak2 locus. Human and murine Jak2 transcripts are expressed at similar levels, and mice develop modest increases in hemoglobin and platelet levels reminiscent of human JAK2 V617F–positive essential thrombocythemia. The phenotype is transplantable and accompanied by increased terminal erythroid and megakaryocyte differentiation together with increased numbers of clonogenic progenitors, including erythropoietin-independent erythroid colonies. Unexpectedly, JAK2V617F mice develop reduced numbers of lineage−Sca-1+c-Kit+ cells, which exhibit increased DNA damage, reduced apoptosis, and reduced cell cycling. Moreover, competitive bone marrow transplantation studies demonstrated impaired hematopoietic stem cell function in JAK2V617F mice. These results suggest that the chronicity of human myeloproliferative neoplasms may reflect a balance between impaired hematopoietic stem cell function and the accumulation of additional mutations.

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Cse1p Is Involved in Export of Yeast Importin α from the Nucleus

Molecular and Cellular Biology ◽

10.1128/mcb.18.11.6805 ◽

1998 ◽

Vol 18 (11) ◽

pp. 6805-6815 ◽

Cited By ~ 88

Author(s):

Jens Solsbacher ◽

Patrick Maurer ◽

F. Ralf Bischoff ◽

Gabriel Schlenstedt

Keyword(s):

Nuclear Export ◽

Fluorescent Protein ◽

Nuclear Pore ◽

Wild Type ◽

Importin Α ◽

Localization Signal ◽

Green Fluorescent ◽

Mutant Cells

ABSTRACT Proteins bearing a nuclear localization signal (NLS) are targeted to the nucleus by the heterodimeric transporter importin. Importin α binds to the NLS and to importin β, which carries it through the nuclear pore complex (NPC). Importin disassembles in the nucleus, evidently by binding of RanGTP to importin β. The importin subunits are exported separately. We investigated the role of Cse1p, theSaccharomyces cerevisiae homologue of human CAS, in nuclear export of Srp1p (yeast importin α). Cse1p is located predominantly in the nucleus but also is present in the cytoplasm and at the NPC. We analyzed the in vivo localization of the importin subunits fused to the green fluorescent protein in wild-type and cse1-1 mutant cells. Srp1p but not importin β accumulated in nuclei ofcse1-1 mutants, which are defective in NLS import but not defective in NLS-independent import pathways. Purified Cse1p binds with high affinity to Srp1p only in the presence of RanGTP. The complex is dissociated by the cytoplasmic RanGTP-binding protein Yrb1p. Combined with the in vivo results, this suggests that a complex containing Srp1p, Cse1p, and RanGTP is exported from the nucleus and is subsequently disassembled in the cytoplasm by Yrb1p. The formation of the trimeric Srp1p-Cse1p-RanGTP complex is inhibited by NLS peptides, indicating that only NLS-free Srp1p will be exported to the cytoplasm.

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