Internal tandem duplication in Fms-like tyrosine kinase 3 (FLT3-ITD) is the most frequent mutation observed in acute myeloid leukemia (AML) and correlates with poor prognosis. FLT3 tyrosine kinase ...inhibitors are promising for targeted therapy. Here, we investigated mechanisms dampening the response to the FLT3 inhibitor quizartinib, which is specific to the hematopoietic niche. Using AML primary samples and cell lines, we demonstrate that convergent signals from the hematopoietic microenvironment drive FLT3-ITD cell resistance to quizartinib through the expression and activation of the tyrosine kinase receptor AXL. Indeed, cytokines sustained phosphorylation of the transcription factor STAT5 in quizartinib-treated cells, which enhanced AXL expression by direct binding of a conserved motif in its genomic sequence. Likewise, hypoxia, another well-known hematopoietic niche hallmark, also enhanced AXL expression. Finally, in a xenograft mouse model, inhibition of AXL significantly increased the response of FLT3-ITD cells to quizartinib exclusively within a bone marrow environment. These data highlight a new bypass mechanism specific to the hematopoietic niche that hampers the response to quizartinib through combined upregulation of AXL activity. Targeting this signaling offers the prospect of a new therapy to eradicate resistant FLT3-ITD leukemic cells hidden within their specific microenvironment, thereby preventing relapses from FLT3-ITD clones.
Cytomegalovirus (CMV) is a leading infectious cause of morbidity in immune-compromised patients. γδ T cells have been involved in the response to CMV but their role in protection has not been firmly ...established and their dependency on other lymphocytes has not been addressed. Using C57BL/6 αβ and/or γδ T cell-deficient mice, we here show that γδ T cells are as competent as αβ T cells to protect mice from CMV-induced death. γδ T cell-mediated protection involved control of viral load and prevented organ damage. γδ T cell recovery by bone marrow transplant or adoptive transfer experiments rescued CD3ε-/- mice from CMV-induced death confirming the protective antiviral role of γδ T cells. As observed in humans, different γδ T cell subsets were induced upon CMV challenge, which differentiated into effector memory cells. This response was observed in the liver and lungs and implicated both CD27+ and CD27- γδ T cells. NK cells were the largely preponderant producers of IFNγ and cytotoxic granules throughout the infection, suggesting that the protective role of γδ T cells did not principally rely on either of these two functions. Finally, γδ T cells were strikingly sufficient to fully protect Rag-/-γc-/- mice from death, demonstrating that they can act in the absence of B and NK cells. Altogether our results uncover an autonomous protective antiviral function of γδ T cells, and open new perspectives for the characterization of a non classical mode of action which should foster the design of new γδ T cell based therapies, especially useful in αβ T cell compromised patients.
Objective Regulation of hematopoiesis depends on cytokines, cellular interactions, transcription, and metabolic factors. Among the latter, O2 has been neglected for a long time. Recently, an ...increasing number of publications evidenced the regulatory role of physiological low O2 concentrations (0.1−5%; similar to those in bone marrow) on the in vitro behavior of hematopoietic stem cells. This brief review utilizes the article of Eliasson and colleagues in this Journal to summarize the major results and questions about the relationships between O2 and hematopoiesis. Materials and Methods In order to be concise and interesting for readers unfamiliar with this field, we selected only the most significant data that either reinforce or contradict the conclusions of Eliasson et al., but we also provide references of reviews with a more detailed bibliography. Results A critical analysis of some key publications provides partial answers to three important questions: is the term hypoxia appropriate to describe physiological low O2 concentrations? Is a very low O2 level sufficient to control the quiescence/slow cycling balance of hematopoietic stem cells? Is the O2 concentration able to modify the effect of cytokines on hematopoietic stem cells? Conclusions We propose to use in situ normoxia instead of the confusing term hypoxia when working with normal cells at physiological low O2 concentrations. We suggest that a very low O2 concentration is necessary but not sufficient to induce hematopoietic stem cell quiescence. We review some articles showing that O2 variations modify the effect of cytokines.
Atypical Myeloproliferative Neoplasms (aMPN) share characteristics of MPN and Myelodysplastic Syndromes. Although abnormalities in cytokine signaling are common in MPN, the pathophysiology of ...atypical MPN still remains elusive. Since deregulation of microRNAs is involved in the biology of various cancers, we studied the miRNome of aMPN patients.
MiRNome and mutations in epigenetic regulator genes ASXL1, TET2, DNMT3A, EZH2 and IDH1/2 were explored in aMPN patients. Epigenetic regulation of miR-10a and HOXB4 expression was investigated by treating hematopoietic cell lines with 5-aza-2'deoxycytidine, valproic acid and retinoic acid. Functional effects of miR-10a overexpression on cell proliferation, differentiation and self-renewal were studied by transducing CD34
cells with lentiviral vectors encoding the pri-miR-10a precursor.
MiR-10a was identified as the most significantly up-regulated microRNA in aMPN. MiR-10a expression correlated with that of HOXB4, sitting in the same genomic locus. The transcription of these two genes was increased by DNA demethylation and histone acetylation, both necessary for optimal expression induction by retinoic acid. Moreover, miR-10a and HOXB4 overexpression seemed associated with DNMT3A mutation in hematological malignancies. However, overexpression of miR-10a had no effect on proliferation, differentiation or self-renewal of normal hematopoietic progenitors.
MiR-10a and HOXB4 are overexpressed in aMPN. This overexpression seems to be the result of abnormalities in epigenetic regulation mechanisms. Our data suggest that miR-10a could represent a simple marker of transcription at this genomic locus including HOXB4, widely recognized as involved in stem cell expansion.
Xenotransplantation models allowing the identification and quantification of human Hematopoietic stem cells (HSC) in immunodeficient mice remain the only way to appropriately address human HSC ...function despite the recent progress in phenotypic characterization. However, these in vivo experiments are technically demanding, time consuming and expensive. Indeed, HSCs engraftment in mouse requires pre-conditioning of animals either by irradiation or cytotoxic drugs to allow homing of injected cells in specific stem cell niches and their subsequent expansion and differentiation in bone marrow. Recently, the development of busulfan pre-conditioning of animals improved the flexibility of experimentation in comparison with irradiation.
In order to further facilitate the organization of these complex experiments we investigated the effect of extending the period between mice pre-conditioning and cell injection on the engraftment efficiency. In the meantime, we also explored the role of busulfan doses, mouse gender and intravenous injection route (caudal or retro orbital) on engraftment efficiency.
We showed that a period of up to 7 days did not modify engraftment efficiency of human HSCs in NSG model. Moreover, retro orbital cell injection to female mice pre-conditioned with 2x25 mg/kg of busulfan seems to be the best adapted schema to detect the human HSC in xenotransplantation experiments.
Physiological bone marrow oxygen concentrations are everywhere lower than 4% and almost null in some areas. We compared the effects of 20%, 3%, and 0.1% O2 concentrations on cord blood CD34+ cell ...survival, cycle, and functionality in serum-free cultures for 72 hours with or without interleukin-3 (IL-3). As from 24 hours, IL-3 improved cell survival and proliferation in all conditions. After 72 hours, cells were 1.5 and 2.5 times more in quiescence (G0) at 3% and 0.1% O2, respectively, than at 20%; transforming growth factor-beta signaling seemed not to be involved. To explore cell cycle further, fresh CD34+ cells were stained with PKH26 and cultured for 72 hours, and then undivided and divided cells were sorted. At 0.1% O2, 46.5%+/-19.1% of divided cells returned to G0 compared with 7.9%+/-0.3% at 20%. Colony formation and nonobese diabetic/severe combined immunodeficient mice engraftment efficiency were similar after 3 days at 20% and 0.1% O2 concentrations but lower than at T0. In conclusion, a low O2 concentration, close to those found in bone marrow stem cell niches, induces the G0 return of CD34+ cells without impairing their functional capacity.
Culture conditions used for the expansion of hematopoietic stem and progenitor cells (HSCs and HPCs, collectively HSPCs) should ideally favor the self renewal of long-term HSCs. At 20% O2, the ...synthesis of HIF-1α is balanced by its hydroxylation and proteasomal degradation. This favors HSPC differentiation, but can be prevented by culturing CD34+ cord blood cells in the presence of dimethyloxaloylglycine (DMOG). This differentiation may also be reduced by culturing the cells in the presence of Stemregenin 1, an antagonist of the aryl hydrocarbon receptor (AhR). The objective of this study was to investigate how hypoxia, DMOG and Stemregenin 1 might affect the expansion of HSPCs with the aim of identifying optimal conditions for expansion in culture. It was found that DMOG decreased proliferation but was effective in preserving the number of cells in the primitive hematopoietic sub-populations in vitro. The effect of DMOG was similar to hypoxia, although differences were observed with regard to the side population and CD34+ sub-populations. Stemregenin 1 on the other hand increased the size of the primitive as well as the other HSC sub-populations. The use of Stemregenin 1 with DMOG increased the proportion of primitive HSCs to 3.54% compared to 2.61% for Stemregenin 1 alone. In vivo engraftment studies confirmed these findings and showed that fewer cells (3710) are required for long-term engraftment when HSCs are grown in Stemregenin 1 together with hypoxia than in Stemregenin 1 under conditions of normoxia (13430).
•G-CSF provided a 3.2 fold increase in cell proliferation and actual number of cells with the side population characteristics.•DMOG provided similar, but not the same observations compared to hypoxia.•The use of Stemregenin 1 provided significant increase in the side population as well as secondary engraftment.•Stemregenin 1 with DMOG significantly inhibited cell proliferation and provided the highest proportion of side population.•The use of Stemregenin 1 with hypoxia provided less sells but similar engraftment levels compared to the use of Stemregenin 1.
Mutations in
observed in myeloproliferative neoplasms (MPN) were recently shown to be pathogenic via their interaction with MPL and the subsequent activation of the Janus Kinase - Signal Transducer ...and Activator of Transcription (JAK-STAT) pathway. However, little is known on the impact of those variant CALR proteins on endoplasmic reticulum (ER) homeostasis.
The impact of the expression of Wild Type (WT) or mutant CALR on ER homeostasis was assessed by quantifying the expression level of Unfolded Protein Response (UPR) target genes, splicing of X-box Binding Protein 1 (XBP1), and the expression level of endogenous lectins. Pharmacological and molecular (siRNA) screens were used to identify mechanisms involved in CALR mutant proteins degradation. Coimmunoprecipitations were performed to define more precisely actors involved in CALR proteins disposal.
We showed that the expression of CALR mutants alters neither ER homeostasis nor the sensitivity of hematopoietic cells towards ER stress-induced apoptosis. In contrast, the expression of CALR variants is generally low because of a combination of secretion and protein degradation mechanisms mostly mediated through the ER-Associated Degradation (ERAD)-proteasome pathway. Moreover, we identified a specific ERAD network involved in the degradation of CALR variants.
We propose that this ERAD network could be considered as a potential therapeutic target for selectively inhibiting CALR mutant-dependent proliferation associated with MPN, and therefore attenuate the associated pathogenic outcomes.
BACKGROUND: The liquid culture of murine bone marrow cells at 1‐percent oxygen maintains the balance between primative progenitor cell renewal and clonogenic progenitor expansion better than that at ...20‐percent oxygen. These results are of potential interest for the ex vivo expansion of human progenitor cells, as low O2 tension could preserve the engraftment potential of cultured apheresis products.
STUDY DESIGN AND METHODS: G–CSF‐mobilized blood cells collected by apheresis, now the main source of progenitor cells for autologous transplantation, were cultured at 1‐percent and 20‐percent O2 for 7 days in serum‐free liquid cultures in the presence of IL‐3 and SCF (5 ng/mL). The growth of the clonogenic progenitors (CFU–GM, BFU–E, CFU–Mix) and of the more primitive human HPCs that are capable of generating clongenic progenitors in secondary liquid culture, as well as the proliferation and differentiation of total and CD34+ cells, was analyzed.
RESULTS: The expansion of CD34+ cells and of clonogenic progenitors was significantly lower in liquid cultures at 1‐percent O2 than at 20‐percent O2. On the contrary, the primitive human HPCs were better maintained and expanded at 1‐percent O2, although the number of CD34+ cells remaining quiescent was lower. After 7 days of liquid culture at 1‐percent or 20‐percent O2 the percentage of CD34+ cells was similar. However, the CD34+ cells that divided more than four times (PKH2 staining) were more numerous in liquid cultures incubated at 1‐percent O2.
CONCLUSION: When cultured at 1‐percent O2 for 7 days in presence of IL‐3 and SCF, the CD34+ cells present in apheresis components underwent more cell divisions and better maintained their primitive progenitor cell potential. As suggested by previous results in mice, our data on human cells emphasize the potential interest of cultures at low O2 tension (1%) for cell therapy protocols aimed at expanding primitive HPCs in autografts.
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