The activating mutation JAK2 V617F plays a central role in the pathogenesis of polycythemia vera, essential thrombocythemia, and primary myelofibrosis. Inhibition of JAK2 activity leads to growth ...inhibition and apoptosis in cells with mutated JAK2. However, the proapoptotic proteins involved in JAK2 inhibition-induced apoptosis remain unclear. In this study, we show that JAK2 inhibition-induced apoptosis correlated with up-regulation of the nonphosphorylated form of the BH3-only protein Bim in hematopoietic cell lines bearing JAK2 mutations. Knockdown of Bim dramatically inhibited apoptosis induced by JAK2 inhibition, which was reversed by the BH3 mimetic agent ABT-737. In addition, ABT-737 enhanced the apoptosis induced by JAK2 inhibition in JAK2 V617F+ HEL and SET-2 cells. The combination of JAK inhibitor I and ABT-737 reduced the number of erythroid colonies derived from CD34+ cells isolated from JAK2 V617F+ polycythemia vera patients more efficiently than either drug alone. These data suggest that Bim is a key effector molecule in JAK2 inhibition-induced apoptosis and that targeting this apoptotic pathway could be a novel therapeutic strategy for patients with activating JAK2 mutations.
Cb2, the gene encoding the peripheral cannabinoid receptor, is located in a common virus integration site and is overex-pressed in retrovirally induced murine myeloid leukemias. Here we show that ...this G protein-coupled receptor (GPCR) is also aberrantly expressed in a high percentage of human acute myeloid leukemias. We investigated the mechanism of transformation by Cb2 and demonstrate that aberrant expression of this receptor on hematopoietic precursor cells results in distinct effects depending on the ligand used. Cb2-expressing myeloid precursors migrate upon stimulation by the endocannabinoid 2-arachidonoylglycerol and are blocked in neutrophilic differentiation upon exposure to another ligand, CP55940. Both effects depend on the activation of Gαi proteins and require the mitogen-induced extracellular kinase/extracellular signal-regulated kinase (MEK/ERK) pathway. Down-regulation of cyclic adenosine monophosphate (cAMP) levels upon Gαi activation is important for migration induction but is irrelevant for the maturation arrest. Moreover, the highly conserved G protein-interacting DRY motif, present in the second intracellular loop of GPCRs, is critical for migration but unimportant for the differentiation block. This suggests that the Cb2-mediated differentiation block requires interaction of Gαi proteins with other currently unknown motifs. This indicates a unique mechanism by which a transforming GPCR, in a ligand-dependent manner, causes 2 distinct oncogenic effects: altered migration and block of neutrophilic development. (Blood. 2004;104:526-534)
Chronic inflammation is a hallmark of autoinflammatory disorders such as rheumatoid arthritis and inflammatory bowel disease. It is characterized by excessive production of cytokines and chemokines. ...Recent evidence showed that hematopoietic stem cells (HSCs) directly respond to these signaling molecules. In order to investigate how chronic inflammation may affect HSCs, we employed a mouse model suffering from chronic multifocal osteomyelitis (CMO). CMO mice exhibit a progressive autoinflammatory disorder that resembles human chronic recurrent multifocal osteomyelitis. CMO mice develop swollen paws, tail kinks, increased bone marrow (BM) cellularity, number of granulocytes, and production of cytokines such as IL-1b, IL-6, and MIP-1a. Remarkably, we observed expansion of HSCs already in asymptomatic mice. To assess whether the phenotypical expansion affects their functionality, we performed HSC limiting dilution assays and observed that CMO HSCs have significantly impaired engraftment ability compared to WT HSCs. Interestingly, by crossing MyD88-deficient mice with CMO mice, we observed that most of the CMO phenotype could be rescued, however, the HSC expansion was preserved. Furthermore, we generated chimeras to investigate the effect of the immune compartment and the BM niche on HSCs. Our results showed that both CMO compartments reduce HSC fitness. RNAseq data suggested that the loss of HSC function is mediated via upregulation of the IL-6/Jak/STAT3 signaling pathway. Accordingly, treatment of CMO mice with STAT3 inhibitor demonstrated a significant reduction of the HSC expansion. Altogether, our data indicate that chronic autoinflammatory conditions have a detrimental effect on HSCs, and highlight the possibility of adding clinically available STAT3 inhibitors to the current treatment to preserve stem cell functions.
Acute myeloid leukemia (AML) is a malignant neoplasia of the hematopoietic system characterized by the accumulation of immature and non-functional leukemic blasts in the bone marrow and peripheral ...tissues. Mechanistically, the development of AML is explained by the "two-hit" theory, based on the accumulation of driver mutations that will cooperate to induce transformation. However, a significant percentage of AML patients exhibit only one driver mutation, and thus, how leukemic transformation occurs in these cases is unclear. Accumulating evidence suggests that non-genetic factors, such as chronic inflammation, might influence AML development, and accordingly, clinical data reported that patients with chronic inflammatory disorders have an increased risk of developing hematological malignancies. Here, using a mouse model of chronic inflammation, we demonstrate that systemic elevated levels of cytokines and chemokines, and hyper-activation of the Jak/Stat3 signaling pathway, might substitute "second hit" mutations and accelerate tumorigenesis. Altogether, our data highlights chronic inflammation as an additional factor in the development of AML, providing additional understanding of the mechanisms of transformation, and opening new venues for the treatment of this disease.Acute myeloid leukemia (AML) is a malignant neoplasia of the hematopoietic system characterized by the accumulation of immature and non-functional leukemic blasts in the bone marrow and peripheral tissues. Mechanistically, the development of AML is explained by the "two-hit" theory, based on the accumulation of driver mutations that will cooperate to induce transformation. However, a significant percentage of AML patients exhibit only one driver mutation, and thus, how leukemic transformation occurs in these cases is unclear. Accumulating evidence suggests that non-genetic factors, such as chronic inflammation, might influence AML development, and accordingly, clinical data reported that patients with chronic inflammatory disorders have an increased risk of developing hematological malignancies. Here, using a mouse model of chronic inflammation, we demonstrate that systemic elevated levels of cytokines and chemokines, and hyper-activation of the Jak/Stat3 signaling pathway, might substitute "second hit" mutations and accelerate tumorigenesis. Altogether, our data highlights chronic inflammation as an additional factor in the development of AML, providing additional understanding of the mechanisms of transformation, and opening new venues for the treatment of this disease.
Mutations in the adaptor protein PSTPIP2 are the cause of the autoinflammatory disease chronic multifocal osteomyelitis in mice. This disease closely resembles the human disorder chronic recurrent ...multifocal osteomyelitis, characterized by sterile inflammation of the bones and often associated with inflammation in other organs, such as the skin. The most critical process in the disease's development is the enhanced production of IL-1β. This excessive IL-1β is likely produced by neutrophils. In addition, the increased activity of macrophages, osteoclasts, and megakaryocytes has also been described. However, the molecular mechanism of how PSTPIP2 deficiency results in this phenotype is poorly understood. Part of the PSTPIP2 inhibitory function is mediated by protein tyrosine phosphatases from the proline-, glutamic acid-, serine- and threonine-rich (PEST) family, which are known to interact with the central part of this protein, but other regions of PSTPIP2 not required for PEST-family phosphatase binding were also shown to be indispensable for PSTPIP2 function. In this article, we show that PSTPIP2 binds the inhibitory enzymes Csk and SHIP1. The interaction with SHIP1 is of particular importance because it binds to the critical tyrosine residues at the C terminus of PSTPIP2, which is known to be crucial for its PEST-phosphatase-independent inhibitory effects in different cellular systems. We demonstrate that in neutrophils this region is important for the PSTPIP2-mediated suppression of IL-1β processing and that SHIP1 inhibition results in the enhancement of this processing. We also describe deregulated neutrophil response to multiple activators, including silica, Ab aggregates, and LPS, which is suggestive of a rather generalized hypersensitivity of these cells to various external stimulants.
Understanding of the hematopoietic stem and progenitor cell biology has important implications for regenerative medicine and the treatment of hematological pathologies. Despite the most relevant data ...that can be acquired using in vivo models or primary cultures, the low abundance of hematopoietic stem and progenitor cells considerably restricts the pool of suitable techniques for their investigation. Therefore, the use of cell lines allows sufficient production of biological material for the performance of screenings or assays that require large cell numbers. Here we present a detailed description, readout, and interpretation of proliferation and differentiation assays which are used for the investigation of processes involved in myelopoiesis and neutrophilic differentiation. These experiments employ the 32D/G-CSF-R cytokine dependent murine myeloid cell line, which possesses the ability to proliferate in the presence of IL-3 and differentiate in G-CSF. We provide optimized protocols for handling 32D/G-CSF-R cells and discuss major pitfalls and drawbacks that might compromise the described assays and expected results. Additionally, this article contains protocols for lentiviral and retroviral production, titration, and transduction of 32D/G-CSF-R cells. We demonstrate that genetic manipulation of these cells can be employed to successfully perform functional and molecular studies, which can complement results obtained with primary hematopoietic stem and progenitor cells or in vivo models.
The DLX homeodomain genes are part of the Drosophila distal-less family, originally identified in the forebrain of the developing mouse embryo. DLX1 gene is expressed also in the hematopoietic cells. ...Our previous data showed that patients with FLT3/ITD (internal tandem duplication) mutation representing about 35% of all acute myeloid leukemia (AML) cases have higher expression of DLX1 compared to non-FLT3/ITD AML patients. Further, FLT3 signaling was described to regulate DLX1 gene expression.
In the current study we found an association between DLX1 expression level and overall survival using GEP data from TCGA database within the group of FLT3/ITD-positive patients. Worse overall survival was linked to lower expression of DLX1 (p=0.003, n=46) even though generally this group of patients is characterized by their higher DLX1 level. This association was also observed when all AML patients were analyzed (p=0.01, n=197). Since the role of DLX1 in the leukemogenic process is not yet described we aimed to characterize the phenotype of leukemic cells with different expression levels of DLX1 gene.
We designed two functional shRNAs (pGhU6-sh1_DLX1 and pGhU6-sh2_DLX1, referred as sh1 and sh2) to downregulate DLX1 expression in MV4;11 (FLT3/ITD positive) leukemic cells, which present high endogenous level of DLX1, and a non-silenced control (pGhU6-NSC). In vitro studies showed that cells with silenced DLX1 were arrested in G0 phase of cell cycle (35%±4.8 (NSC) to 67.5%±2.2 (sh1; p <0.01) and 65.5%±2.5 (sh2; p <0.01); flow cytometry - Pyronin Y/ Hoechst 33342 staining) and had lower proliferative activity (trypan blue over the period of 10 passages). Moreover, cells with silenced DLX1 were less apoptotic (Annexin V/PI staining).
Next, we studied the impact of DLX1 downregulation on leukemic cell infiltration in vivo using sub-lethally irradiated NSG (NOD SCID gamma) mice. We injected 1x106 silenced or non-silenced MV4;11 cells via tail vein (n=20, 6 mice/group, 2 control mice). After two weeks we measured the absolute number of MV4;11 cells by flow cytometry (CD33+/GFP+/DAPI-) in bone marrow (BM) and spleen (SP) of recipient mice. Absolute cell counts of leukemic cells with silenced DLX1 were 1.26x106 (sh1; p=0.015) and 2.89x106 (sh2; p<0.0001) vs NSC cells 0.52x106 in BM and 1.59x106 (p=0.036) and 14.2x106 (p=0.025) vs 0.34x106 in SP. To ensure that the increased cell numbers were not the result of enhanced homing of DLX1 silenced cells, we performed homing experiments. Control and DLX1 silenced cells were transplanted and the number of cells in BM and SP was determined 16 hours after transplantation. We observed no differences between the studied groups, indicating that DLX1 silencing does not affect the homing ability of MV4;11 cells. Moreover, we analyzed cell cycle in leukemic cells isolated from recipient mice three weeks after transplantation. In agreement with our in vitro results, leukemic cells with silenced DLX1 had higher percentage of cells arrested in G0 phase (48.6%±6.3 (sh1; p <0.001) and 80.9%±9.3 (sh2; p <0.001) vs 26.4%±4.8 (NSC) in SP). Overall, mice with silenced DLX1 presented worse fitness and bigger splenomegaly.
Further, we investigated the signaling pathways which could lead to G0 arrest. Since DLX1 inhibits TGF-β pathway through direct interaction with SMAD4, a key downstream effector of TGF-β/BMP signaling, we studied the changes in target genes in our model cell lines. Expression of CUTL-1 (sh1 - 1.5-fold, sh2 - 2-fold change to NSC), PAI-1 (3.5-fold, 4.2-fold) and CDKN1C (1.3-fold, 2.3-fold) were significantly increased in DLX1 silenced cells. PAI-1 was shown to induce replicative senescence and CDKN1C is an inhibitor of cell cycle progression. While both targets disturb cell cycle and could be responsible for the phenotype we have observed, this hypothesis needs to be elucidated in future experiments.
Altogether, our data demonstrate that dysregulation of DLX1 gene in leukemic cells changes the cell phenotype. Lower level of DLX1 gene leads to arrest in G0 phase which in vitro slows down the proliferation whereas in vivo it allows the cells to persist in spleen and BM. We hypothesize that the DLX1 silenced cells become more resistant to external effects which could then be reflected as a reduced survival observed in patients with low levels of DLX1.
Supported by P304/12/2214.
No relevant conflicts of interest to declare.
Abstract The knowledge about the contribution of the innate immune system to health and disease is expanding. However, to obtain reliable results, it is critical to select appropriate mouse models ...for in vivo studies. Data on genetic and phenotypic changes associated with different mouse strains can assist in this task. Such data can also facilitate our understanding of how specific polymorphisms and genetic alterations affect gene function, phenotypes, and disease outcomes. Extensive information is available on genetic changes in all major mouse strains. However, comparatively little is known about their impact on immune response and in particular on innate immunity. Here, we analyzed a mouse model of chronic multifocal osteomyelitis (CMO), an autoinflammatory disease driven exclusively by the innate immune system, which is caused by an inactivating mutation in the Pstpip2 gene. We investigated how the genetic background of BALB/c, C57BL/6J, and C57BL/6NCrl strains alters the molecular mechanisms controlling disease progression. While all mice developed the disease, symptoms were significantly milder in BALB/c and partially also in C57BL/6J when compared to C57BL/6NCrl. Disease severity correlated with the number of infiltrating neutrophils and monocytes and with the production of chemokines attracting these cells to the site of inflammation. It also correlated with increased expression of genes associated with autoinflammation, rheumatoid arthritis, neutrophil activation, and degranulation, resulting in altered neutrophil activation in vivo. Together, our data demonstrate striking effects of genetic background on multiple parameters of neutrophil function and activity influencing the onset and course of the CMO disease.
We previously identified a novel common virus integration site, Evi11, by means of retroviral insertional mutagenesis. We demonstrated that the gene encoding the peripheral cannabinoid receptor (Cb2) ...is the potential target, suggesting that Cb2 is a proto-oncogene. To elucidate a role for this G protein–coupled receptor (GPCR) in leukemic transformation we generated a Cb2-EGFP cDNA construct that was introduced into 32D/G-CSF-R cells. These cells require interleukin 3 (IL-3) to proliferate in vitro, whereas in the presence of granulocyte–colony-stimulating factor (G-CSF) they differentiate toward mature neutrophils. We demonstrate that 32D/G-CSF-R/Cb2-EGFP cells migrate in a transwell assay in reponse to the Cb2 ligand 2-arachidonoylglycerol (2-AG), indicating that the fusion protein was functional. When cultured in the presence of G-CSF neutrophilic differentiation of Cb2-EGFP–expressing 32D/G-CSF-R cells was completely blocked. Moreover, a Cb2-specific antagonist fully recovered the G-CSF–induced neutrophilic differentiation of 32D/G-CSF-R/Cb2-EGFP cells. To investigate which signal transduction pathway(s) may be involved in the block of neutrophilic maturation, differentiation experiments were carried out using specific inhibitors of signaling routes. Interestingly, full rescue of G-CSF–induced neutrophilic differentiation was observed when cells were cultured with the mitogen-induced extracellular kinase (MEK) inhibitors, PD98059 or U0126, and partial recovery was detected with the phosphoinositide 3-kinase (PI3-K) inhibitor LY-294002. These studies demonstrate that the Cb2 receptor is an oncoprotein that blocks neutrophilic differentiation when overexpressed in myeloid precursor cells. Cb2 appears to mediate its activity through MEK/extracellular signal-related kinase (ERK) and PI3-K pathways.
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