scholarly journals N-Acetylcysteine As a Therapeutic for JAK2V617F Myeloproliferative Neoplasms

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1786-1786
Author(s):  
Brianna Craver ◽  
Gajalakshmi Ramanathan ◽  
Laura Mendez Luque ◽  
Summer Hoang ◽  
Kenza Elalaoui ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Reactive Oxygen Species ◽  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Myeloproliferative Neoplasms ◽  
Oxygen Species ◽  
Wild Type ◽  
Healthy Donors ◽  
Marrow Cells

Abstract Chronic inflammation is common in MPN and drives disease progression and worsens symptom burden. It has been reported that JAK2V617F hematopoietic stem and progenitor cells (HSPCs) exhibit elevated basal oxidative stress compared to wild-type cells. However, JAK2V617F HSCs have a differential response than normal HSPCs upon stimulation with pro-inflammatory cytokines or lipopolysaccharide (LPS) invivo and in vitro. Specifically, JAK2V617F HSPCs do not significantly increase reactive oxygen species, exit quiescence, or increase DNA damage following LPS exposure (Blood 2017 130:4211). Therefore, we hypothesized that oxidative stress is important for differential responses of wild-type and JAK2V617F HSCs to inflammation. To compare cell responses to oxidative stress, we treated bone marrow cells from JAK2V617F knock-in and wild-type mice ex vivo with L-buthionine-S,R-sulfoximine (BSO), which reduces glutathione levels, then measured the impact on myeloid colony formation in methylcellulose. JAK2V617F knock-in bone marrow cells are resistant to oxidative stress-induced reduction in colony formation compared to wild-type bone marrow cells. This data suggests that JAK2V617F myeloid progenitors produce less reactive oxygen species in response to BSO or that these cells are resistant to oxidative stress-induced cell death. Next, we tested the effect of the anti-oxidant n-acetylcysteine (NAC) in a JAK2V617F knock-in mouse model. All hematopoietic cells in this model express JAK2V617F, these mice develop elevated blood counts, splenomegaly, and die suddenly at approximately 2-3 months of age. Surprisingly, addition of NAC (2g/L) in the drinking water extended the lifespan of JAK2V617F-knock in mice (p<0.02, n=6-12). However, aspirin (16mg/L) in drinking water did not extend the lifespan of JAK2V617F-knock in mice. NAC did not alter peripheral blood counts in either JAK2V617F-knock in or wild-type mice, which suggests that NAC's utility in JAK2V617F is not through cytoreduction but is due to its ability to reduce oxidative stress or thrombosis. Additionally, NAC had no effect on thrombin-induced platelet activation, which was assayed by P-selectin expression, phosphatidylserine exposure, and platelet-leukocyte aggregation via flow cytometry. There is a positive correlation between leukocytosis and thrombotic risk in MPN patients. Additionally, MPN patients and mice exhibit elevated neutrophil extracellular trap (NET) formation compared to healthy controls, which contributes to the increased thrombosis in MPN. To test the effect of antioxidants on NET formation in vitro, we treated neutrophils from MPN patients or healthy donors with phorbol myristate acetate concurrently with antioxidants (NAC or ferulic acid) and assayed the presence of extracellular DNA using a SYTOX Green nucleic acid stain. Unstimulated neutrophils from MPN patients exhibited sporadic NET formation while neutrophils from healthy donors did not. Additionally, NAC and ferulic acid reduced DNA release, which is indicative of NET formation. Taken together, these data demonstrate that JAK2V617F progenitors are resistant to oxidative stress-induced cell death. Furthermore, reduction of oxidative stress with n-acetylcysteine in vivo prevented NET-induced thrombosis in JAK2V617F knock-in mice as well as in vitro in normal and MPN neutrophils. These data provide a rationale for investigating the utility of n-acetylcysteine as a therapeutic in myeloproliferative neoplasms. Disclosures No relevant conflicts of interest to declare.

Impaired Granulocytic Differentiation In Vitro in Hematopoietic Cells Lacking Retinoic Acid Receptors α1 and γ

Blood ◽  
1998 ◽  
Vol 92 (2) ◽  
pp. 607-615 ◽  
Author(s):  
Jean Labrecque ◽  
Deborah Allan ◽  
Pierre Chambon ◽  
Norman N. Iscove ◽  
David Lohnes ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Retinoic Acid ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Hematopoietic Cell ◽  
Retinoic Acid Receptors ◽  
Wild Type ◽  
Granulocytic Differentiation ◽  
Marrow Cells

Transcripts for the retinoic acid receptors (RARs) α1, α2, γ1, and γ2 were found in the granulocytic lineage (Gr-1+cells) through semiquantitative polymerase chain reaction (PCR) analysis. The screening of single cell cDNA libraries derived from hematopoietic progenitors also showed the presence of RARα and, to a lesser extent, RARγ transcripts in committed granulocyte (colony-forming unit-granulocyte [CFU-G]) or granulocyte-macrophage (CFU-GM) colony-forming cells. The contribution of RARα1 and γ to hematopoietic cell differentiation was therefore investigated in mice bearing targeted disruption of either one or both of these loci. Because RARγ and RARα1γ compound null mutants die shortly after birth, bone marrow cells were collected from fetuses at 18.5 days postcoitum (dpc) and evaluated for growth and differentiation in culture in the presence of Steel factor (SF), interleukin-3 (IL-3), and erythropoietin (Epo). The frequency of colony-forming cells from bone marrow populations derived from RARα1/γ double null mice was not significantly different from that of RARγ or RARα1 single nulls or from wild-type controls. In addition, the distribution of erythroid, granulocyte, and macrophage colonies was comparable between hematopoietic cells from all groups, suggesting that lineage commitment was not affected by the lack of RARα1 and/or RARγ. Colony cells were then harvested individually and evaluated by morphologic criteria. While terminal granulocyte differentiation was evident in wild-type cells and colonies from either single null mutant, colonies derived from RARα1−/−γ−/− bone marrow populations were blocked at the myelocyte and, to a lesser extent, at the metamyelocyte stages, whereas erythroid and macrophage differentiation was not affected. Together, these results indicate that both RARα1 and γ are required for terminal maturation in the granulocytic lineage in vitro, but appear to be dispensable for the early stages of hematopoietic cell development. Our results raise the possibility that in acute promyelocytic leukemia (APL), the different RARα fusion proteins cause differentiation arrest at a stage when further maturation requires not only RARα, but also RARγ. Finally, bone marrow cells appear to differentiate normally in vivo, suggesting an effective compensation mechanism in the RARα1/γ double null mice.

scholarly journals Prostaglandin E2 restrains macrophage maturation via E prostanoid receptor 2/protein kinase A signaling

Blood ◽  
2012 ◽  
Vol 119 (10) ◽  
pp. 2358-2367 ◽  
Author(s):  
Zbigniew Zasłona ◽  
Carlos H. Serezani ◽  
Katsuhide Okunishi ◽  
David M. Aronoff ◽  
Marc Peters-Golden
Keyword(s):  
Bone Marrow ◽  
Protein Kinase ◽  
Prostaglandin E2 ◽  
Protein Kinase A ◽  
Bone Marrow Cells ◽  
Lipid Mediator ◽  
Wild Type ◽  
Kinase A ◽  
Marrow Cells

Abstract Prostaglandin E2 (PGE2) is a lipid mediator that acts by ligating 4 distinct G protein–coupled receptors, E prostanoid (EP) 1 to 4. Previous studies identified the importance of PGE2 in regulating macrophage functions, but little is known about its effect on macrophage maturation. Macrophage maturation was studied in vitro in bone marrow cell cultures, and in vivo in a model of peritonitis. EP2 was the most abundant PGE2 receptor expressed by bone marrow cells, and its expression further increased during macrophage maturation. EP2-deficient (EP2−/−) macrophages exhibited enhanced in vitro maturation compared with wild-type cells, as evidenced by higher F4/80 expression. An EP2 antagonist also increased maturation. In the peritonitis model, EP2−/− mice exhibited a higher percentage of F4/80high/CD11bhigh cells and greater expression of macrophage colony-stimulating factor receptor (M-CSFR) in both the blood and the peritoneal cavity. Subcutaneous injection of the PGE2 analog misoprostol decreased M-CSFR expression in bone marrow cells and reduced the number of peritoneal macrophages in wild-type mice but not EP2−/− mice. The suppressive effect of EP2 ligation on in vitro macrophage maturation was mimicked by a selective protein kinase A agonist. Our findings reveal a novel role for PGE2/EP2/protein kinase A signaling in the suppression of macrophage maturation.

Nrf2, a Critical Regulator of Oxidative Stress, Is Required for HSC Function and Cytokine Response.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1492-1492
Author(s):  
Akil Merchant ◽  
Anju Singh ◽  
Giselle Joseph ◽  
Qiuju Wang ◽  
Ping Zhang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Stem Cells ◽  
Bone Marrow ◽  
Transcription Factor ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
Cytokine Signaling ◽  
Wild Type ◽  
Increased Sensitivity ◽  
Marrow Cells

Abstract Abstract 1492 Poster Board I-515 Previous studies have established an important role for reactive oxygen species (ROS) in regulating the function and life-span of hematopoietic stem cells (HSC). Nuclear factor erythroid-2–related factor 2 (Nrf2) is a redox-sensitive transcription factor that regulates cellular responses to ROS and detoxification pathways implicated in chemoresistance, however, its role in normal stem cells is unknown. We analyzed Nrf2null mice and found increased total bone marrow cellularity, cKit+Sca1+Lin− (KSL) stem-progenitor cells, and long-term quiescent HSC (CD34−KSL) compared to wild type mice (p<0.05). Transplantation of equal numbers of KSL cells from Nrf2wt and Nrf2null resulted in a five-fold decrease in peripheral blood chimerism from Nrf2null derived cells at 16 weeks (15% wild type vs. 3% null, p<0.05). Unlike other models of deficiencies in genes associated with ROS handling, such as ATM or the FoxO family of transcription factors, basal ROS levels were not elevated in Nrf2null HSC. However, Nrf2null bone marrow cells demonstrated increased sensitivity to induced oxidative stress and in vitro treatment with H2O2 resulted in a 2 fold decrease in colony formation in methylcellulose. We also examined the in vivo sensitivity of Nrf2null cells to oxidative stress by irradiating (400 rads) stably chimeric mice 20 weeks following transplantation with either Nrf2wt or Nrf2null HSC. Mice receiving Nrf2null HSC demonstrated a 50% decrease in peripheral blood chimerism at 4 months following radiation compared to no change in Nrf2wt recipients (p<0.05) confirming that loss of Nrf2 leads to increased sensitivity to oxidative stress. Microarray gene expression analysis from Nrf2wt and Nrf2null mice revealed down regulation of the G-CSF cytokine receptor in Nrf2null HSC and suggested that defective cytokine signaling may contribute to the HSC dysfunction seen in Nrf2null bone marrow cells. To test this hypothesis, we attempted to rescue the function of Nrf2null HSC by treating mice with exogenous G-CSF. Nrf2wt and Nrf2null mice were treated with one week of daily G-CSF and then HSC were harvested and transplanted. In contrast to the defects in engraftment of untreated Nrf2null HSC, there was no significant difference in peripheral blood chimerism following transplantation of G-CSF treated Nrf2wt or Nrf2null HSC, thus demonstrating that G-CSF treatment could rescue the HSC defect in mutant mice. In conclusion, the Nrf2 transcription factor appears to be a novel and essential regulator of normal HSC function through the modulation of oxidative stress response and cytokine signaling. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A limited role for p16Ink4a and p19Arf in the loss of hematopoietic stem cells during proliferative stress

Blood ◽  
2005 ◽  
Vol 106 (3) ◽  
pp. 827-832 ◽  
Author(s):  
Lilia Stepanova ◽  
Brian P. Sorrentino
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Dominant Role ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Serial Transplantation ◽  
Marrow Cells

Abstract It has long been known that prolonged culture or serial transplantation leads to the loss of hematopoietic stem cells (HSCs); however, the mechanisms for this loss are not well understood. We hypothesized that expression of p16Ink4a or p19Arf or both may play a role in the loss of HSCs during conditions of enhanced proliferation, either in vitro or in vivo. Arf was not expressed in freshly isolated HSCs from adult mice but was induced in phenotypically primitive cells after 10 to 12 days in culture. When cultured bone marrow cells from either Arf–/– or Ink4a-Arf–/– mice were compared to wild-type cells in a competitive repopulation assay, no significant differences in HSC activity were seen. We then evaluated the role of p19Arf and p16Ink4a in the loss of HSCs during serial transplantation. Bone marrow cells from Ink4a-Arf–/–, but not Arf–/–, mice had a modestly extended life span and, on average, supported reconstitution of one additional recipient compared to wild-type cells. Mice given transplants of Ink4a-Arf–/–cells eventually did die of hematopoietic failure in the next round of transplantation. We conclude that mechanisms independent of the Ink4a-Arf gene locus play a dominant role in HSC loss during conditions of proliferative stress.

Gfi1-N382S Mutants from Human Severe Congenital Neutropenia Patients Function through a Transcriptional Dominant Negative Mechanism.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1185-1185
Author(s):  
Adrian P. Zarebski ◽  
Avinash M. Baktula ◽  
Sudeep Basu ◽  
John O. Trent ◽  
H. Leighton Grimes
Keyword(s):  
Bone Marrow ◽  
Dna Binding ◽  
Bone Marrow Cells ◽  
Severe Congenital Neutropenia ◽  
Wild Type ◽  
Congenital Neutropenia ◽  
Murine Bone Marrow ◽  
Amino Terminal ◽  
Marrow Cells

Abstract The Growth factor independence 1 (Gfi1) zinc finger transcriptional repressor is a T cell leukemia oncoprotein that also plays a crucial role in granulopoiesis in both mice and humans. A single point mutation in the amino terminal SNAG repressor domain (P2A) is able to ablate both Gfi1 transcriptional repression activity and linked oncogenic activity in T lymphoctyes. Mice deleted for Gfi1 are lymphopenic, but also lack mature neutrophils. Gfi1−/− mice display a profound block to myeloid differentiation and abnormal promyelocytes accumulate in the blood. Humans with Severe Congenital Neutropenia (SCN) with heterozygous mutations in Gfi1 have similar abnormal promyelocytes. We introduced the SCN patient Gfi1N382S DNA-binding-deficient mutation into murine Gfi1 and overexpressed it in primary murine bone marrow cells. While expression of the wild type Gfi1 resulted almost exclusively in mature granulocyte differentiation, forced expression of the N382S mutant resulted almost exclusively in monocytic differentiation. Flow cytometric analysis revealed a population of N382S-expressing cells with markers of both monocytes and neutrophils resembling the atypical Gfi1−/− promyelocytes. To determine if mutation of the N382 residue is uniquely able to block Gfi1 function, we constructed a virtual model of Gfi1 zinc fingers 3, 4 and 5 interacting with DNA. The model revealed several possible protein-DNA interactions. In order to validate the model we mutated those residues to alanine and performed EMSA with in vitro transcribed/translated proteins. The same alanine substitution mutants were expressed in primary murine bone marrow and tested for their ability to control myelopoiesis. Lack of DNA binding in EMSA tightly correlated with impaired granulopoesis in our in vitro model, suggesting the necessity of intact DNA binding for proper Gfi1 function. These data suggested that the non-DNA binding mutants were able to inhibit repression by wild type endogenous Gfi1, perhaps through the sequestration of limiting corepressor proteins. The Gfi1P2A mutant is unable to repress transcription. We therefore tested the effect of Gfi1P2A expression on myelopoiesis and found that it blocked granulopoiesis equivalently to Gfi1N382S. To rigorously determine whether the titration of limiting corepressors was the cause of N382S neutropenia, we constructed a compound mutant containing both N382S and P2A and expressed it in primary murine bone marrow cells. Expression of Gfi1P2A-N382S had little effect on myelopoiesis. We conclude that SCN patients with heterozygous Gfi1 mutations have blocked granulopoiesis because the non-DNA binding mutant protein competes with the wild type allele for titratable associated cofactors.

Effects of the Homeodomain Gene Pitx2 on Self-Renewal of Hematopoietic Stem Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 95-95 ◽  
Author(s):  
Hui Z. Zhang ◽  
Svetlana Rogulina ◽  
Wendy Chen ◽  
Barbara A. Degar ◽  
Bernard G. Forget
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Leukemic Cells ◽  
Wild Type ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Colony Forming Units ◽  
Marrow Cells

Abstract Pitx2, a homeodomain gene preferentially expressed in murine hematopoietic stem/progenitor cells, is also a downstream target of genes important for hematopoiesis such as MLL and Wnt/Dvl/β-Catenin. We have previously reported that Pitx2 null hematopoietic stem cells (HSCs) can contribute to multi-lineage hematopoiesis under physiologic conditions. We have now carried out serial bone marrow transplantation experiments and demonstrated that after the 3rd round of serial transplantation, Pitx2 null cells reconstituted only 28.6% of the recipient hematopoietic cells as compared to 60% in the case of wild type cells (P<0.001). There were no Pitx2 null donor-derived cells in recipient mice after the 4th round of transplantation, whereas donor-derived chimerism was 57% with wild type cells (P<0.001), and 26% with Pitx2 +/− cells (P<0.001). Therefore, Pitx2 null HSCs have decreased self renewal capacity. To further study the function of Pitx2 in HSC, we constitutively overexpressed the Pitx2 gene in murine bone marrow cells following transduction using a MSCV/IRES/GFP retroviral vector, and analyzed the effects on hematopoiesis in vitro and in vivo. Bone marrow cells overexpressing Pitx2 were isolated on the basis of their GFP expression and analyzed for their colony forming ability in vitro. Retrovirally transduced bone marrow cells were also transplanted into lethally irradiated mice, and the transplanted mice were observed for long-term reconstitution. Colony-forming unit assays showed that Pitx2 overexpressing bone marrow cells, compared to control cells transduced with vector only, had increased numbers of GM colony forming units and reduced numbers of megakaryocytic colony forming units. Pitx2-overexpressing cells continued to form GM colonies after more than eight serial replatings. When these cells were cultured in liquid medium containing SCF, IL-3 and IL-6, they gave rise to cells that stained positively either for alpha naphthyl butyrate, indicating monocytic differentiation, or for peroxidase, indicating neutrophilic differentiation. The ability of these GM-colony forming cells to cause leukemia is currently under investigation. Long-term reconstitution of hematopoiesis in mice by Pitx2 over-expressing HSCs was demonstrated by identifying GFP positive multi-lineage peripheral blood cells four months following transplantation. One of these mice manifested leukemia at this time, as evidenced by a markedly elevated WBC count and other hematologic abnormalities. The leukemic WBCs had very high levels of GFP and Pitx2 expression and were shown to contain two retroviral integration sites, neither of which involved a known oncogene or overexpression of the gene at the integration site. Immunophenotyping by flow cytometry demonstrated that the majority of the leukemic cells were c-kit positive and expressed the megakaryocytic marker CD41, as well as the common myeloid progenitor marker, CD16/32. Some of the cells expressed the erythroid marker Ter119. The leukemic cells did not express any lymphoid markers, including CD3ε, B220, CD19, and IL7R3. This Pitx2-overexpression-associated leukemia was transplantable. Experiments are under way to characterize the leukemia initiating cells. Taken together, our results provide evidence that the homeodomain gene Pitx2 plays a role in the self-renewal of hematopoietic stem/progenitor cells.

Inhibition of AKT Signaling Potently Inhibits the Growth of JAK and MPL-Mutant Cells in the Myeloproliferative Neoplasms In Vitro and In Vivo,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3862-3862
Author(s):  
Irum Khan ◽  
Zan Huang ◽  
Qiang Jeremy Wen ◽  
Priya Koppikar ◽  
Ross L Levine ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Signaling Pathways ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Akt Signaling ◽  
Murine Bone Marrow ◽  
Marrow Cells

Abstract Abstract 3862 Somatic mutations in JAK2 and MPL are associated with the BCR-ABL negative myeloproliferative neoplasms (MPNs) including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). While oral JAK2 inhibitors improve peripheral blood counts and splenomegaly, these drugs show varying effects on JAK2 mutant allele burden and do not eliminate the malignant clone in humans or in animal models of MPN. Although much of the research to date has focused on JAK/STAT signaling, other pathways downstream of the class I cytokine receptors, including PI3K/AKT and ERK are also activated in MPNs. Our hypothesis is that persistent activation of these signaling pathways contributes to the progression of myeloproliferative neoplasms (MPNs). Multiple studies have shown that overexpression of activated JAK2 and MPL mutants (e.g. JAK2 V617F or MPL W515L) in primary murine bone marrow cells causes robust AKT, STAT3, and STAT5, JNK, ERK phosphorylation. To determine the extent to which these signaling pathways are involved in the disease, we cultured JAK2 V617F and MPL W515L expressing clones of the G1ME and 6133 megakaryocytic cell lines with a panel of small molecule kinase inhibitors. We discovered that inhibition of the PI3K/AKT and MAPK/JNK signaling pathways with triciribine and SP600125 respectively, potently suppressed growth of these cells by inducing G1 arrest and apoptosis. In contrast, inhibitors of Ras, ERK, Src family tyrosine kinase, and PKC failed to significantly inhibit proliferation of the JAK2 or MPL mutant expressing cells. Murine bone marrow cells transduced with MPL W515L show a dramatic expansion of megakaryocytes (CFU-MKs) and this expansion was abrogated in the presence of PI3K/AKT inhibitors suggesting a requirement for this pathway in aberrant megakaryocyte expansion. Since AKT inhibition showed the strongest effect, we assayed the activity of MK-2206, a potent and selective allosteric AKT inhibitor, on multiple models of MPNs. We discovered that MK-2206 induced proliferative arrest and apoptosis accompanied by suppression of PI3K/AKT signaling in G1ME and 6133 cells expressing MPLW515L. MK-2206 also potently inhibited liquid culture growth and colony formation of PMF patient CD34+ cells in vitro. Finally, in preliminary murine transplants experiments with MPLW515L expressing bone marrow progenitors, treatment with MK-2206 led to significant reductions in peripheral blood leukocytosis and extramedullary hematopoiesis. Together, these findings demonstrate that the PI3K/AKT axis represents a rational target for therapy in human myeloproliferative neoplasms. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Estrogen responsiveness of bone formation in vitro and altered bone phenotype in aged estrogen receptor-α-deficient male and female mice

2005 ◽  
Vol 152 (2) ◽  
pp. 301-314 ◽  
Author(s):  
Vilhelmiina Parikka ◽  
ZhiQi Peng ◽  
Teuvo Hentunen ◽  
Juha Risteli ◽  
Teresa Elo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Formation ◽  
Bone Marrow Cells ◽  
Type I ◽  
Wild Type ◽  
Male And Female ◽  
Female Mice ◽  
Bone Phenotype ◽  
Marrow Cells

Objective: Although the beneficial effects of estrogen on bone are well known, the roles of estrogen receptors (ERs) in mediating these effects are not fully understood. Methods: To study the effects of long-term ERα deficiency, bone phenotype was studied in aged ERα knockout (ERKO) mice. In addition, ERKO osteoclasts and osteoblasts were cultured in vitro. Design and results: Histomorphometric analysis showed that the trabecular bone volume and thickness were significantly increased and the rate of bone formation enhanced in both male and female ERKO mice in comparison to the wild-type animals. In ERKO males, however, the bones were thinner and their maximal bending strengths decreased. Consistent with previous reports, the bones of knockout mice, especially of female mice, were shorter than those of wild-type mice. In addition, the growth plates were totally absent in the tibiae of aged ERKO females, whereas the growth plate cartilages were detectable in wild-type females as well as in all the males. Analysis of cultured bone marrow cells from 10- to 12-week-old mice demonstrated that 17β-estradiol could stimulate osteoblastic differentiation of bone marrow cells derived from ERKO mice relatively to the same extent as those derived from wild-type mice. This was demonstrated by increases in synthesis of type I collagen, activity of alkaline phosphatase and accumulation of calcium in cultures. Total protein content was, however, reduced in ERKO osteoblast cultures. Conclusions: These results show altered bone phenotype in ERKO mice and demonstrate the stimulatory effect of estrogen on osteoblasts even in the absence of full-length ERα.

scholarly journals IL13 Contributes to Fibrotic Progression of the Myeloproliferative Neoplasms

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 60-60
Author(s):  
Johanna Melo-Cardenas ◽  
Lavanya Bezavada ◽  
Jeremy Chase Crawford ◽  
Sandeep Gurbuxani ◽  
Anitria Cotton ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mast Cells ◽  
Mouse Model ◽  
Single Cell ◽  
Research Funding ◽  
Bone Marrow Cells ◽  
Myeloproliferative Neoplasms ◽  
Disease Process ◽  
Wild Type ◽  
Marrow Cells

Abstract Pre-fibrotic-primary myelofibrosis PMF (Pre-PMF) is an indolent form of PMF that frequently progresses to overt-PMF. While both stages of the disease are characterized by the presence of dysplastic megakaryocytes, progression to fulminant disease is associated with significantly increased fibrosis in the bone marrow. We have previously shown that megakaryocyte maturation in overt-PMF is impaired due to a GATA1 deficiency. However, in Pre-PMF patients most megakaryocytes express GATA1. This raises the possibility that alterations in megakaryocyte development occur during the progression of the disease and may contribute to fibrosis. This progression in megakaryocyte defects is likely driven not only by the aberrant JAK/STAT signaling but also microenvironmental factors. To identify such factors, we performed studies in two mouse models of PMF (driven by JAK2V617 and MPLW515L mutations) before and after development of fibrosis. Using an unbiased approach, we measured the levels of different cytokines in the bone marrow, plasma, and spleen. In addition, we performed single cell RNAseq in bone marrow populations. We observed extensive changes in the level of cytokines in the bone marrow of the MPLW515L mouse model compared to the JAK2V617F model. We initially focused on those cytokines that are elevated in the bone marrow of both murine models, including IL13 (Figure 1A), because previous studies have shown that IL13 is elevated in PMF patients and that JAK2 inhibitors do not decrease IL13 (1-3). Moreover, elevated IL13 has been identified in patients who progress to secondary AML (2). How IL13 may contribute to the progression of the disease has not been investigated. We assayed the effect of IL13 on megakaryocytes in vitro and discovered that it promoted megakaryocyte differentiation in the absence of thrombopoietin (TPO) and potentiated the effect of TPO. This effect was observed in cultures of both wild-type and MPLW515L megakaryocytes. Next, we assayed for expression of the IL13 receptor (Il13ra1) in the bone marrow of JAK2V617F and MPLW515L mutant mice and found that it was highly upregulated compared to wild-type animals. IL13ra1 expression was particularly intense in the megakaryocyte lineage, and its expression increased with disease progression (Figure 1B). Next, we asked whether IL13 is essential for myeloproliferative neoplasm (MPN) development in vivo. To study this, we transplanted bone marrow cells from Il4/13 f/f Mx1-Cre mice expressing MPLW515L to irradiated recipients, waited until MPN developed, and then excised by pIpC injection. This experiment revealed that loss of IL13 and IL4 led to a profound reduction in disease burden (Figure 1C), decreased splenomegaly, and diminished degree of bone marrow fibrosis. Moreover, loss of IL13 and IL4 decreased the levels of pro-inflammatory cytokines in the bone marrow and spleen (Figure 1D). We attribute this effect to deletion of IL13 because IL4 was only moderately increased in the bone marrow of the MPLW515L mouse model, and because IL4 has been reported to not be altered in the MPNs. Finally, we performed single cell RNA-seq on bone marrow cells from mice transplanted with JAK2V617F or control progenitor cells early and late in the disease process (Figure 1E). Our results revealed that there was decreased myeloid progenitors but an enhancement in the mast cell lineage that tracked with the degree of fibrosis. We confirmed the presence of elevated numbers of mast cells in the bone marrow by immunohistochemistry (Figure 1F). Mast cells produce IL13, and therefore they are the likely source for the increased IL13. Finally, consistent with the observation that IL13 signaling is primarily mediated through STAT6, we found enrichment of STAT6 target genes in megakaryocyte progenitors from the late timepoint in our scRNAseq data (Figure 1G). In summary, our data demonstrate that IL13 is involved in the progression of PMF and that inhibition of the IL13 signaling pathway should be investigated as a therapeutic option in PMF. 1. Tefferi A, et al. J Clin Oncol (2011) 2. Fisher DAC, et al. Leukemia (2019) 3. Chen P, et al. Front Med (2021) Figure 1 Figure 1. Disclosures Crispino: Forma Therapeutics: Research Funding; Scholar Rock: Research Funding; MPN Research Foundation: Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Consultancy.

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