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

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4095-4095
Author(s):  
Edwin Chen ◽  
Lawrence J Breyfogle ◽  
Rebekka K. Schneider ◽  
Luke Poveromo ◽  
Ross L. Levine ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Myeloproliferative Neoplasm ◽  
Conditional Knockout ◽  
The Other ◽  
Myelogenous Leukemia ◽  
Leukemic Transformation ◽  
The Impact

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

Bone Marrow Stromal Cell Remodeling Is a Common Feature of Diverse Fibrotic Myeloproliferative Neoplasm Models

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 25-25
Author(s):  
Timothy B Campbell ◽  
Si Yi Zhang ◽  
Alexander Valencia ◽  
Emmanuelle Passegue
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Critical Role ◽  
Myeloproliferative Neoplasm ◽  
Myelogenous Leukemia ◽  
Hematopoietic Stem ◽  
Cell Numbers

Abstract Myeloproliferative neoplasms (MPN) are blood cancers initiated by driver mutations that transform hematopoietic stem cells. MPN exhibit gross pathologic bone marrow (BM) stromal remodeling, including damaging myelofibrotic change that leads to dependence on extramedullary hematopoiesis and more severe clinical diseases. Therapies targeting fibrotic change would have broad appeal in the treatment of these diseases. We previously demonstrated a critical role for malignant myeloid cells in remodeling endosteal mesenchymal stromal cells (MSC) into myelofibrotic osteoblast-lineage cells (OBC) in a model of chronic myelogenous leukemia (CML) driven by BCR/ABL (Schepers et al., Cell Stem Cell, 2013). In a separate study in a fibrotic MPN model driven by Jak2V617F, neuropathy and nestin-positive MSC cell death were found critical to disease progression but their involvement in myelofibrosis was not investigated (Arranz et al. Nature. 2014). Our goal is to characterize the type of BM stromal remodeling occurring in non-CML MPN models driven by various mutations and representing a spectrum of disease severity and fibrosis. This includes a minimally fibrotic transgenic Jak2V617F alone model (Jak2V617F model, Xing et al., Blood, 2008) and more advanced fibrotic models driven by MPLW515L expression (MPLW515L model, Pikman et al., PLoS Med, 2006) or combined transgenic Jak2V617F expression with conditional deletion of the polycomb gene EZH2 (Jak2V617F/EZH2-/- model, Sashida et al., JEM, 2016). We found common blood and BM hematopoietic changes in all three models, including thrombocytosis and expansion of myeloid-biased multipotent progenitor BM cells and confirmed the degree of fibrosis using picrosirius red staining of bone sections. Both MPLW515L and Jak2V617F/EZH2-/- heavily fibrotic models demonstrated inhibition of total endosteal MSC, OBC and endothelial cell (EC) numbers during disease development - in most cohorts a greater than 50% decrease in absolute stromal cell numbers was found. In addition, we observed that whole BM cells from Jak2V617F/EZH2-/-mice contained a significantly lower number of totalfibroblast colony forming cells (CFU-F). In co-culture experiments designed to measure direct MSC remodeling induced by malignant cells, both MPLW515L and Jak2V617F/EZH2-/- BM cells inhibited healthy endosteal MSC colony formation over time. In contrast, we found no inhibition of stromal cell numbers or co-culture MSC growth in the minimal fibrotic Jak2V617F model. In initial experiments measuring rare central marrow perivascular MSC, we found reduced LepR+ MSC (Ding et al., Nature, 2012) in both MPLW515L and Jak2V617F/EZH2-/- long bone sections using immunofluorescence. Our results show that fibrotic development in non-CML MPN inhibits stromal cell numbers and function likely via direct effects of malignant hematopoietic cells. This is in contrast to fibrotic CML development where myelofibrotic endosteal stromal cells are expanded. This difference could be partly explained by the type and localization of fibrosis in these various models. The CML model has focal endosteal collagen-I fibrosis which is heavily reliant on osteoblast remodeling, while the MPLW515L and Jak2V617F/EZH2-/- models have more diffuse reticulin central marrow fibrosis which may be produced through a process of stromal cell senescence or differentiation. Overall, this study underscores that a “one size fits all“ approach to understanding myelofibrosis is insufficient. To tease out these differences, we are examining qualitative and quantitative changes in additional central marrow MSC populations, including PDGFR+, Sca-1+ and Gli-1+ MSC, during MPN development as well as assaying the molecular mediators of stromal remodeling. Our long-term goal is to identify therapies that can restore a more normal BM stroma and support healthy hematopoiesis in MPN. Disclosures No relevant conflicts of interest to declare.

Abstract 12726: Crucial Role of Jak2 V617f-positive Myeloproliferative Neoplasm in the Development of Aortic Aneurysm

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Tetsuro Yokokawa ◽  
Tomofumi Misaka ◽  
Yusuke KIMISHIMA ◽  
Kento Wada ◽  
Keiji Minakawa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Angiotensin Ii ◽  
Aortic Aneurysm ◽  
Mrna Expression ◽  
Abdominal Aorta ◽  
Myeloproliferative Neoplasms ◽  
Myeloproliferative Neoplasm ◽  
Jak2 V617f ◽  
Vascular Complication ◽  
The Impact

Objective: To investigate the impact of hematopoietic JAK2V617F, which causes myeloproliferative neoplasms (MPNs), on the development of aortic aneurysm (AA). Approach and Results: We applied a bone marrow transplantation (BMT) strategy using the donor cells from Jak2 V617F transgenic (JAK2 V617F ) mice into the lethally irradiated apolipoprotein E-deficient mice. To induce the AA formation and progression, the recipient mice (BMT mice) were subjected to continuous angiotensin II infusion. Abdominal aortic diameter in JAK2 V617F -BMT mice was significantly enlarged compared to the control wild-type (WT)-BMT mice in response to angiotensin II. The incidence of abdominal AA was significantly higher in JAK2 V617F -BMT mice than in WT-BMT mice. Hematopoietic JAK2V617F accelerated aortic elastic lamina degradation as well as activation of matrix metalloproteinase (MMP)-2 and MMP-9 in the abdominal aorta. The numbers of CD68 + macrophages and Ly6B.2 + neutrophils and cytokine expressions such as Ccl6 and Tgfb1 were significantly increased in the abdominal aorta of JAK2 V617F -BMT mice accompanied by STAT3 activation. Bone marrow-derived macrophages carrying JAK2V617F showed elevations of both Mmp2 and Mmp9 mRNA expression. Finally, we found that 23% of MPN patients with JAK2 V617F mutation showed the presence of AA and increases in TGFB3 and IL-8 mRNA expression of the peripheral leukocytes. Conclusions: Hematopoietic JAK2V617F was involved in the development of AA through increases in the infiltration of inflammatory cells and MMP activation. Our findings provide a novel feature of vascular complication of AA in MPNs due to constitutive activation of the hematopoietic JAK-STAT pathway.

scholarly journals Incorporation of Clinical Information to Decipher Driver Mutations in Myeloproliferative Neoplasms

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5489-5489
Author(s):  
Murtadha K. Al-Khabori ◽  
Shoaib Al-Zadjali ◽  
Mohamed Al-Rawahi ◽  
Iman Al Noumani ◽  
Khalil Al Farsi ◽  
...  
Keyword(s):  
Platelet Count ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Myeloproliferative Neoplasm ◽  
Clinical Information ◽  
Primary Myelofibrosis ◽  
Mutation Load ◽  
Female Ratio ◽  
Epigenetic Regulators ◽  
The Impact

Abstract Introduction: Mutations in the epigenetic regulators are commonly found in myeloid disorders including Myeloproliferative Neoplasm (MPN). Primary myelofibrosis, dysplastic changes and severity of the disease were associated with the mutation load. Most of the studies had a limited number of targeted genes and included a mixture of JAK2 positive and negative disease. The objective of this study is to assess the impact of mutations in the epigenetic regulators on the presentation of patients with JAK2 V617F positive MPN. Methods: We retrieved the clinical and laboratory information on 61 consecutive eligible patients. Mutation analysis of the entire coding regions of ASXL1, ASXL2, CBL, CEBPA, CSF3R, DNMT3A, EZH2, IDH2, TET1 and TET2 genes was performed using next generation sequencing (NGS; Ion PGM Sequencer; Thermo Fisher ScientificÒ). The library was constructed and the templates were prepared using the PGM tool. The variants were annotated using the ClinVar database and the prediction from the Scale-Invariant Feature Transform (SIFT) and or Polymorphism Phenotyping (Polyphen) algorithms. Alignment, variant filtering and annotation were performed using Ion Torrent Suite. Standard descriptive and analytical statistics were used as appropriate to describe and compare different groups. The MPN subtype, bleeding, thrombosis, hemoglobin, platelet count, White Cell Count (WBC), Lactate Dehydrogenase (LDH) and erythropoietin level were compared for each candidate variant. An alpha threshold of 0.05 was used with no adjustment for multiple comparisons as the analyses were considered exploratory. All statistics were performed using R program. Variants were selected for further experimentation based on their frequency and association with the clinical information at diagnosis. Results: Sixty-one patients were included (Polycythemia Vera: 29, Essential Thrombocythemia: 21, Primary Myelofibrosis: 9, MPN unclassifiable: 2) with a median age of 62 years (Interquartile Range [IQR]: 44 - 70). Male to female ratio was 35:26. The median hemoglobin, WBC, platelet count, LDH and erythropoietin were 14.6 g/dL (IQR: 12.8 - 16.8), 11.5 *109/L (IQR: 11.5 - 14.4), 507 *109/L (IQR: 391 - 779), 265 mU/mL (IQR: 231 - 409) and 1.2 mU/mL (IQR: 1.0 - 4.8) respectively. At presentation, 54% had splenomegaly, 23% had an arterial or venous thrombosis, and 5% had bleeding. Sixty-three variants were found in the samples tested. The median mutation load was 13 variants (Range: 11-14). Patients with higher than the median mutation load had higher mean erythropoietin (7.8 vs. 0.9 g/dL; p = 0.02016). ASXL1 p.Leu815Pro variant was found in all patients. Only three variants were found in the ClinVar database. Seven variants were predicted to be pathogenic (ASXL1: 1, EZH2: 1, IHD1: 1, TET1: 1 and TET2: 3). Patients with TET2: p.Leu1721Trp variant had 6.4 higher odds of bleeding (p = 0.04345). Patients with TET2: p.His1778Arg variant had a lower WBC (9.1 vs. 13.9 *109/L; p = 0.01699) and LDH (213 vs. 348 mU/mL; p = 0.0006528) while those with TET1: p.Lle1123Met variant had a higher WBC (13.5 vs. 8.3 *109/L; p = 0.02756). None of the remaining comparisons were statistically significant. Conclusions: Incorporation of clinical information facilitates the prioritization of variants from DNA sequencing experiments. In MPN, we recommend ASXL1: p.Leu815Pro (expressed in all patients), TET2: p.Leu1721Trp (associated with bleeding), TET2: p.His1778Arg (correlated with the WBC and LDH) and TET1: p.Lle1123Met (correlated with LDH) for further functional experimentation. Disclosures No relevant conflicts of interest to declare.

Use of CALM-AF10 and NUP98-HOXD13 fusions to Identify Oncogenic Pathways That Collaborate with HOXA9 Overexpression

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2499-2499
Author(s):  
Rachel L Novak ◽  
David P Harper ◽  
David L. Caudell ◽  
Sarah H Beachy ◽  
Yang Jo Chung ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Insertional Mutagenesis ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Fusion Gene ◽  
Insertion Site ◽  
Cell Number ◽  
Incomplete Penetrance ◽  
Leukemic Transformation

Abstract Abstract 2499 Acute myeloid leukemias (AMLs) are thought to result from a series of complementary mutations that affect several vital pathways, including differentiation, self-renewal, apoptosis, and proliferation. Several mutations associated with AML (MLL fusions, NUP98 fusions, CALM-AF10 fusion, and NPM1 mutations) are thought to impair hematopoietic differentiation by dysregulation of target genes; one of the most consistently dysregulated target genes is HOXA9. Mice that express a CALM-AF10 (CA10) or NUP98-HOXD13 (NHD13) fusion gene overexpress HOXA9 and develop AML with a delayed onset and incomplete penetrance, suggesting the need for complementary mutations. We have used three techniques including candidate gene resequencing, retroviral insertional mutagenesis (RIM), and gene expression profiling to compare and contrast the complementary mutations that occur in these mice. The frequency of Ras pathway mutations is similar for both CA10 (20%) and NHD13 (32%) leukemic mice, and previously reported RIM studies identified common integration sites near Mn1 and Evi1 in both models, suggesting that dysregulation of similar collaborative pathways can lead to leukemia in both models. However, there were significant differences between these models. The aforementioned RIM studies identified Zeb2 as the most frequent insertion site for CA10 leukemias, suggesting that Zeb2 collaborated with CA10, whereas no Zeb2 insertions were found in the NHD13 RIM study. Another distinction between the NHD13 and CA10 leukemias is the expression of the HOXA9 co-regulatory factor Meis1. Meis1 is markedly upregulated in bone marrow from clinically healthy, pre-leukemic CA10 mice and CA10 leukemic tissues compared to wildtype bone marrow. Conversely, Meis1 expression was decreased in bone marrow from clinically healthy NHD13 mice compared to wildtype, and only occasionally overexpressed in NHD13 leukemias. Interestingly, RIM analysis of NHD13 mice identified Meis1 as the most frequent insertion site, suggesting that Meis1 overexpression can collaborate with NHD13 during leukemic transformation. Flt3, which is frequently mutated in patients with AML, has been shown to be transcriptionally activated by Meis1. Of note, Flt3 is consistently overexpressed in CA10 leukemias but is overexpressed only in those NHD13 leukemias that have upregulated Meis1. Intriguingly, the frequency of Flt3 activating mutations in CA10 leukemias (which overexpress Flt3) was 25%, but none were found in the NHD13 leukemias (which do not overexpress Flt3). Lastly, using gene expression arrays, N-myc was identified as one of the most differentially overexpressed mRNAs, suggesting that it may cooperate with CA10 during leukemic transformation. Furthermore, N-myc has recently been shown to promote AML in mice, and our preliminary experiments suggest that N-myc and CA10 collaborate and lead to increased cell number and replating potential in bone marrow colony formation assays. In conclusion, we have taken several genetic approaches to identify key similarities and differences in the biology of two leukemic models (CA10 and NHD13), both of which overexpress HOXA9. Disclosures: No relevant conflicts of interest to declare.

Calr Mutants Retroviral Mouse Models Lead to a Myeloproliferative Neoplasm Mimicking an Essential Thrombocythemia Progressing to a Myelofibrosis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 157-157 ◽  
Author(s):  
Caroline Marty ◽  
Nivarthi Harini ◽  
Christian Pecquet ◽  
Ilyas Chachoua ◽  
Vitalina Gryshkova ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Myeloproliferative Neoplasm ◽  
In Vivo Model ◽  
Common Mechanism ◽  
Loss Of Function ◽  
Hematopoietic Stem ◽  
Platelet Counts

Abstract Classical BCR-ABL-negative myeloproliferative neoplasms (MPN) include Polycythemia Vera (PV), Essential Thrombocytemia (ET) and Primary Myelofibrosis (PMF). They are malignant homeopathies resulting from the transformation of a multipotent hematopoietic stem cell (HSC). The common mechanism of transformation is the constitutive activation of the cytokine receptor/JAK2 pathway that leads to the myeloproliferation. The acquired point mutation JAK2V617F is the most prevalent (95% of PV and 60% of ET or PMF). In addition, other mutations affecting the same signaling pathway have been described such as JAK2 exon 12 mutations, mutations of MPL affecting W515, and loss-of-function mutations of LNK and also mutations of c-Cbl in 3% of PMF. Recently, whole exome sequencing allowed identifying a new recurrent genetic abnormalities in the exon 9 of the calreticulin gene (CALR) in about 30% of ET and PMF patients. All CALR mutants induce a frameshift of the same alternative reading frame and generate a novel C-terminus tail. To address the role of these new mutants in the pathophysiology of MPN, the goal of this study was to investigate the effect of the CALR mutant (del52 and ins5) expression by a retroviral mouse modeling. For that purpose, we transduced bone marrow cells with retrovirus expressing either CALRdel52, CALRins5, CALRWT or CALRDexon9 and performed a transplantation in lethally irradiated recipient mice (10 mice / group), which were then followed over one year. CALRdel52 expressing mice showed a rapid and strong increased in platelet counts (over 5 x106/mL) without any other changes in blood parameters during 6 months. In contrast, CALRins5 expressing mice presented platelet counts much lower than CALRdel52 but significantly higher than CALRWT or CALRDexon9 expressing mice. After 6 months, CALRdel52 expressing mice showed a decreased in platelets count associated with anemia and development of splenomegaly suggesting the progression to a myelofibrosis. Importantly, the disease was transplantable to secondary recipient for both CALRdel52 and CALRins5 mutants. The bone marrow and spleen were also analyzed over time. We observed a progressive increased in immature progenitors (SLAM cells) as well as a hypersensitivity of the megakaryocytic progenitors (CFU-MK) to thrombopoietin. Altogether, these results demonstrate that CALR mutants are able and sufficient to induce a thrombocytosis progressing to myelofibrosis in retroviral mouse model, thus mimicking the natural history of MPN patients. It will offer a good in vivo model to investigate therapeutic approaches for CALR-positive MPN. Disclosures No relevant conflicts of interest to declare.

scholarly journals Depletion of Neoplastic CD11b Positive Cells in Jak2V617F Mutant Mice Reduced Fibrocytes in Bone Marrow and Improved Myelofibrosis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 310-310
Author(s):  
Yoshinori Ozono ◽  
Kotaro Shide ◽  
Takuro Kameda ◽  
Ayako Kamiunten ◽  
Yuki Tahira ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Blood Cells ◽  
Diphtheria Toxin ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
The Other ◽  
Immunofluorescent Staining ◽  
Control Group ◽  
Other Hand

Myelofibrosis (MF) associated with myeloproliferative neoplasms (MPN) has been considered to be a reactive phenomenon caused by mesenchymal stromal cells (MSCs) stimulated by cytokines such as TGFb-1 overproduced by neoplastic megakaryocytes (MKs) and platelets. TGFb-1 stimulates non-neoplastic mesenchymal cells to produce collagen and fibronectin and to induces bone marrow (BM) fibrosis. However, the involvement of neoplastic fibrocyte in MF has recently been reported (Verstovsek et al. JEM 2016), and among blood cells, monocytes in particular are considered to be the main source of neoplastic fibrocytes. In this study, we assesed the role of neoplastic fibrocytes using a mouse model of MPN induced by Jak2V617F (Shide et al. Leukemia 2008). First, the distribution of neoplastic fibrocyte in the BM of Jak2V617F transgenic (TG) mice was examined. We transplanted wild-type (WT) or Jak2V617F TG cells (B6-CD45.2), together with WT BM cells (B6-CD45.1) into irradiated WT recipient mice (B6-CD45.1). Only recipient mice transplanted with a mixture of Jak2V617F cells and WT cells developed BM fibrosis. In immunofluorescent staining of fibrotic BM, cells expressing the fibrocyte marker CD45/Collagen-1(Col-1) were observed much more than cells expressing the fibroblast marker CD90(usually positive for MSCs)/Col-1. As for CD45/Col-1 positive cells, cells expressing CD45.2/Col-1 were much more than cells expressing CD45.1/Col-1, clearly indicating that these cells were derived from Jak2V617F mutant blood cells. On the other hand, in the BM of recipient mice transplanted with control WT cells, few cells expressing CD45/Col-1 or CD90/Col-1 were present. To examine the differentiation ability of Jak2V617F blood cells to fibrocytes directly, peripheral blood (PB) mononuclear cells (MNC) of Jak2V617F mice or WT mice were cultured in vitro. After 5 days of culture, PB MNCs from Jak2V617F mice differentiated into mature fibrocytes exhibiting a long spindle shape with Col-1 expression. On the other hand, there were very few fibrocytes differentiated from PB MNC from WT mice. Next, we depleted monocytes, the main source of fibrocytes, and observed its effects on BM fibrosis in vivo. Jak2V617F TG mice were mated with CD11b-diphtheria toxin receptor (DTR) TG mice (Duffield et al. JCI 2005) to obtain Jak2V617F/CD11b-DTR double TG mice. Mice transplanted with BM cells from Jak2V617F/CD11b-DTR double TG mice (hereinafter called Jak2V617F/CD11b-DTR mice) exhibit leukocytosis, thrombocytosis, anemia, splenomegaly, and BM fibrosis with increased megakaryocytes. Jak2V617F/CD11b-DTR mice was administered diphtheria toxin (DT) intraperitoneally to deplete monocytes. One day after DT administration, the number of PB monocytes (CD11b+/F4/80+) drastically decreased in Jak2V617F/CD11b-DTR mice, and the reduction of monocyte was maintained by every-other-day DT administration. After 8 weeks DT treatment, mice were sacrificed and analyzed. As a control group, Jak2V617F/CD11b-DTR mice treated with PBS were examined. DT treatment drastically decreased the number of neoplastic fibrocytes expressing CD45.2/Col-1 in BM and spleen of Jak2V617F/CD11b-DTR mice compared with control mice treated with PBS. Consistently, reticulin fibers were eliminated almost completely and collagen fibers almost fully disappeared in BM, which led to a reversal of the decrease in BM cellularity, although the number of MKs was not affected. Similar findings were observed in the spleen, although not completely. Plasma TGF-b1 level were about 2-fold higher in Jak2V617F/CD11b-DTR mice than in WT mice. Neoplastic monocyte depletion significantly decreased TGF-b1 level. Since MK numbers did not change, this indicates that fibrocytes are one of the main sources of TGF-b1. In other features of MF in Jak2V617F/CD11b-DTR mice, splenomegaly was ameliorated by DT treatment. Microscopic analysis revealed an improvement in the damaged spleen architecture and the disappearance of splenic fibrosis. In summary, most collagen-producing cells in BM were neoplastic fibrocytes in Jak2V617F-induced MPN, indicating that neoplastic fibrocytes played an essential role and mesenchymal fibroblasts had a minor contribution in fibrosis in MPN. Depletion of neoplastic monocytes also improved splenomegaly as well as BM fibrosis in mice, and this cell fraction could be a promising therapeutic target. Disclosures No relevant conflicts of interest to declare.

Mesenchymal Stromal Cells From AML Bone Marrow Are Abnormal by Gene Expression Profiling.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1055-1055
Author(s):  
Wencai Ma ◽  
R. Eric Davis ◽  
Rodrigo Jacamo ◽  
Marina Konopleva ◽  
Ramiro Garzon ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Extracellular Matrix ◽  
Conflicts Of Interest ◽  
Single Gene ◽  
Gene Set Enrichment Analysis ◽  
Myelogenous Leukemia ◽  
Downregulated Gene ◽  
Gene Set ◽  
Gene Sets

Abstract Abstract 1055 Cytogenetic and other evidence suggests that the mesenchymal stromal cell (MSC) is abnormal in bone marrow (BM) affected by acute myelogenous leukemia (AML). To gain further insight into molecular and physiologic abnormalities, we used Affymetrix HG-U133 Plus 2 microarrays to compare gene expression between BM-MSCs from 12 AML patients and BM-MSCs from 4 normal donors (ND). BM-MSCs were purified by in vitro culture as adherent cells with a purity of over 95%. Comparison at the single-gene level between AML and ND samples found only one differentially-expressed probe by t tests at a false-discovery rate (FDR) of 0.1. Comparison by the gene set enrichment analysis (GSEA) method of Subramanian et al., which is a more powerful way to find small differences that are significantly enriched within sets of biologically-related genes, first found that many enriched gene sets were predominantly the result of data from one AML sample. After excluding this sample, GSEA at an FDR of 0.25 found 115 downregulated gene sets for AML BM-MSCs from the Gene Ontology-based “C5” category of the mSigDB collection of gene sets. 19 of the 20 most significantly enriched downregulated gene sets were related to cell cycle progression, indicating that BM-MSCs are less proliferative in AML than in normal BM. An upregulated enriched gene set in AML BM-MSCs, from the “C2” category of curated gene sets, was composed of extracellular matrix genes for keratins, collagen, and laminin; while surprising, this is consistent with reports of BM-derived MSCs differentiating into epithelial cells after autografting, and suggest that BM-MSCs in AML may remodel the extracellular matrix. Overall, these results indicate that BM-MSCs in AML patients are substantially different from normal BM-MSCs. These and other differences could have substantial effects on the BM microenvironment and therapy response in AML, and should be studied further. Disclosures: No relevant conflicts of interest to declare.

SETD2, a H3K36 Lysine Methyltransferase, Is Essential for Adult Normal Hematopoiesis and Leukemia Stem Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1055-1055
Author(s):  
Yile Zhou ◽  
Yunzhu Dong ◽  
Jiachen Bu ◽  
Xiaomei Yan ◽  
Yoshihiro Hayashi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Conditional Knockout ◽  
The Other ◽  
Leukemia Stem Cells ◽  
Transcriptional Networks ◽  
Loss Of Function ◽  
Hematopoietic Stem

Abstract Hematopoietic stem cells (HSCs) are characterized by their capability for self-renewal and multi-potency. Hematopoiesis is dynamically controlled by the interplay between epigenetic and transcriptional networks. Dysregulation of these networks can lead to unfitness of hematopoiesis, cell transformation, and hematological diseases. The human SETD2 gene was originally isolated from HSCs and progenitors. SETD2 is a histone methyltransferase, which specifically catalyzes tri-methylation of histone 3 lysine 36 (H3K36me3). SETD2 functions as a tumor suppressor, as loss-of-function mutations have been identified in many cancers. However, the role of SETD2 in hematopoiesis has not been fully understood. To assess the function of Setd2 in hematopoiesis, we generated three Setd2 mouse alleles with Crispr/CAS9 technology; Setd2F2478/WT knock-in, Setd2Exon6-Δ/WT, and Setd2-Exon6flox/flox/Mx1-Cre conditional knockout alleles, as homozygous Setd2 mutation showed embryonic lethality. Setd2-F2478 point mutation, which is located in the SRI domain, can express SETD2 mutant protein but completely lose the interaction with RNA pol II. Setd2Exon6-Δ/WT allele results in a frame shift and nonsense mediated decay of Setd2 mRNA and protein. After induction of excision with pIpC injection, Setd2-exon6flox/flox/Mx1-Cre+ (Setd2Exon6-Δ/Δ) mice showed severe anemia, increased platelet count, and a reduction in bone marrow (BM) cellularity compared to wild-type (WT) mice, while Setd2F2478/WT and Setd2Exon6-Δ/WT mice did not show any obvious hematological changes. The Lin- Sca-1+ c-Kit+ (LSK) population in Setd2Exon6-Δ/Δ mice was 2.5-fold decreased compared to those in WT, while the LSK populations in Setd2F2478/WT and Setd2Exon6-Δ/WT mice were comparable with those in WT. Interestingly, all three of these Setd2 mutant alleles showed a higher frequency of Lin- Sca-1- c-Kit+ (LK) cells in the BM. In the LK populations, we found an increased CMP population in Setd2F2478/WT and Setd2Exon6-Δ/WT mice; of note, the CMP population in the Setd2Exon6-Δ/Δ mice had disappeared while the MEP population expanded with higher expression of CD16/32. Next, to assess the function of the HSPCs, we performed CFU assays and competitive bone marrow transplantations (CBMT). Consistent with our phenotypic findings, the number of colonies derived from Setd2F2478/WT and Setd2Exon6-Δ/WT BM cells was increased in the first two passages, while the number of colonies derived from Setd2Exon6-Δ/Δ mice was significantly decreased. In CBMT, we found that mice transplanted with Setd2Exon6-Δ/Δ BM cells showed anemia and an impaired BM reconstitution, compared to the control (p = 0.0002). On the other hand, the Setd2F2478/WT and Setd2Exon6-Δ/WT models showed comparable capabilities of BM reconstitution. Taken together, these results suggest that Setd2 has an essential role in the maintenance of adult hematopoiesis. SETD2 mutations (mainly one allele mutation) have been frequently identified in acute leukemia, especially in about 22% of MLL leukemia. To understand the role of SETD2 in leukemic stem cells, Setd2 mutant mice were bred with the Mll-AF9 knock-in mouse. The Mll-AF9/ Setd2F2478/WT and Mll-AF9/ Setd2Exon6-Δ/WT mice showed higher frequencies of LK and LSK populations compared to Mll-AF9 mice, indicating that Setd2 mutations may increase the stemness of leukemia stem cells (LSCs). The cells derived from Mll-AF9/ Setd2F2478/WT and Mll-AF9/ Setd2Exon6-Δ/WT mice resulted in a significantly higher yield of colonies and growth advantage in serial replating CFU assay compared to the cells derived from Mll-AF9 mice. After BMT of equal numbers of cells from Mll-Af9 or Mll-AF9/ Setd2F2478/WT mice into recipient mice, the Mll-AF9/ Setd2F2478/WTBMT mice developed leukemia with significantly shortened latencies compared with MLL-Af9 BMT mice. In conclusion, our data suggests that Setd2 plays an important role in maintaining normal HSPCs. Half the doses of Setd2 can still maintain the normal hematopoiesis while a total loss of Setd2 leads to a failure of hematopoiesis. In leukemia, heterozygous mutants of Setd2 can accelerate leukemogenesis by expanding LSCs. Whether the remaining WT allele is required for leukemia maintenance is unclear. Further reduction of Setd2 levels, or complete deletion of the other WT allele, may diminish SETD2-mutated leukemia. Such tumor vulnerability can be explored as a therapeutic strategy. Disclosures No relevant conflicts of interest to declare.

Clinical and Haematological Features of Genetic Mutations in MPN Patients with Diagnosis of Prefibrotic Idiopathic Myelofibrosis (MF0)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5176-5176
Author(s):  
Bruno Martino ◽  
Claudia Labate ◽  
Bruna Greve ◽  
Domenica Ielo ◽  
Domenico Rotilio ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Triple Negative ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Interquartile Range ◽  
The Other ◽  
Prognostic Impact ◽  
Idiopathic Myelofibrosis ◽  
Thrombotic Risk ◽  
Exon 9

Abstract Background: Diagnosis of a prefibrotic state (MF0) presents histological diagnostic difficulties. MF0 has a worst prognostic impact than Essential Thrombocythemia (TE) as regard the thrombotic risk and a higher risk towards Idiopathic Myelofibrosis (MI) and acute leukemia evolution. For this reason it is very useful to identify this group of patients. Aims: The aim of this study was to evaluate clinical and hematological impact of the mutational status in patients with an MF0 diagnosis. Methods: A retrospective chart review was performed from January 2010 to July 2015 in a single center in 317 patients affected by Ph negative chronic myeloproliferative neoplasms. Polycythemia vera cases were excluded. Thirty-three patients have been classified as MF0 on the base of the bone marrow histological examination. Onset features include cytogenetics, blood counts, peripheral CD34-positive cells, spleen and liver size, thrombotic events (prior to and post-diagnosis) and thrombotic risk. Fragment analysis was performed to study exon 9 CALR mutation, Real-time PCR was applied for exon 14 JAK2 V617F mutation and Sanger sequencing was used to identify exon 10 MPL mutations for 505 and 515 codons. According to genetic results, patients were identified into four groups according to a positivity for JAK2, MPL, CALR and for triple negativity. Results: The 33 patients with MF0 were: 11 (33%) JAK2 positive, 10 (30%) CALR positive, (10) 30% triple negative and 2 (7%) MPL positive. The latter group was not further considered for analysis due to the low number of cases. Eighty percent of CALR positive patients had a deletion on the exon 9 of the gene (8 del52bp and 2 del46bp), while 20% had the type 2 mutation (ins5bp). The average of JAK2 allelic burden was 20%. The 3 groups of patients were comparable for age, white blood counts and hemoglobin values. There was a female prevalence (23 vs 10 males). Platelet count was higher (median 875.500 103 µl, interquartile range 303.000 103 µl , p = 0.008) in CALR positive patients compared to JAK2 positive (median 569.000 103 µl, interquartile range 433.000 103 µl) and to triple negative patients (median PLT count 629.500 103 µl, interquartile range 378.250 103 µl). Noteworthy, bone marrow cytogenetic exam was normal in all patients. JAK2 positive patients had a larger spleen compared to the other two groups. Pre-diagnosis thrombotic events were exclusive for JAK2 positive patients and absent in the other groups. Triple negative patients do not have a negative prognostic impact for the thrombotic risk. Conclusions: CALR deletion could be considered as an MF0 marker and should be included in diagnostic work-up. JAK2 positivity in MF0 is associated with a high thrombotic risk. Disclosures No relevant conflicts of interest to declare.

Impact of RAS Mutations In Myelodysplastic Syndrome (MDS)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2926-2926
Author(s):  
Aref Al-Kali ◽  
Rajyalakshmi Luthra ◽  
Carlos E. Bueso-Ramos ◽  
Sherry Pierce ◽  
Tapan Kadia ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Overall Survival ◽  
Conflicts Of Interest ◽  
Prognostic Significance ◽  
Prognostic Impact ◽  
Leukemic Transformation ◽  
Ras Mutation ◽  
Ras Mutations ◽  
Mutational Status ◽  
The Impact

Abstract Abstract 2926 Background: RAS proteins are encoded by 3 proto-oncogenes (K-RAS, N-RAS, and H-RAS) and regulate the growth and differentiation of many cellular types, including the myeloid compartment. Codons 12, 13, and 61 (in N-RAS and K-RAS) have been reported to be common targets of mutations. These mutations result in constitutive activation of the RAS pathway by increasing intracellular RAS GTP levels, which result in activation of the RAS/MEK and the RAS/PI3K pathways. Its prognostic significance in MDS is yet to be clarified. Because the potential prognostic and therapeutic implications of RAS mutations in MDS, we decided to study the impact of RAS mutations in patients (pts) with MDS. Methods: Mutation analysis of KRAS and NRAS codons 12, 13, and 61 was performed by Pyrosequencing using biotinylated reverse primers. All pts referred to MD Anderson between 2000–2009 were reviewed. In addition to standard clinical characteristics, including karyotyping, mutational status of Flt3, c-Kit and JAK2 was also analyzed. Overall survival was calculated based on Kaplan-Meier. Results: Of the 1067 pts with MDS of age above 17 evaluated for RAS mutation, 43 pts (4%) were positive. Seventy nine percent of RAS mutations were of N-RAS type (codon12 (55%), codon 13 (12%), codon 61(12%)) with K-RAS 12 comprising the rest (21%). Median age was 68 years (range, 18–94) with 66% being males (703 pts). Their median white blood cell (WBC) was 3.3×109/L (range, 0.3–72.2), hemoglobin 9.8 (range, 3–17.5), and platelets 73×109/liter (range, 1–1040). Median bone marrow blast was 5% and 77 pts (7%) had leukemic transformation. More than half of the pts had RAEB or RAEB-t (44% and 9 % respectively) which translated into fewer patients in the low IPSS category (19%). The median age was 66 years (range 26–84) in the RAS mutated (muRAS) group compared to 68 in the wild type (wtRAS). WBC was higher in the muRAS group compared to wtRAS (6.8 ×109/L vs. 3.2 ×109/L, p<0.001 by Mann-Whitney median test (M-W-T)). Percent of bone marrow blasts was higher (9%) in muRAS group compared to 5% in wtRAS (p<0.001 by M-W-T). High (Int-2 and high) risk by IPSS was more frequent in muRAS 51% compared to 40% in wtRAS group (p=0.11). Leukemic transformation was 9% and 7% (p=0.61) in both muRAS and wtRAS groups respectively. RAS mutation was detected more frequently in the CMML subgroup (12/80 (15%) pts). There were no differences in terms of response to DNA methylating inhibitors (CR in 41% in muRAS versus 40% in wtRAS). ARA-C-based regimens resulted in better CR in wtRAS than muRAS (59% vs 25%); however, the size of muRAS group was too small (1 out 4 pts).There was a trend towards shorter overall survival (OS) in the muRAS group compared to wtRAS (395 vs. 500 days, p value=0.057). MuRAS RAEB (325 vs. 450 days, p= 0.13) and RAEB-t (125 vs. 375 days, p= 0.27) subgroups showed a trend towards worse OS compared to wtRAS group. There was a significant OS decrease in RCMD (150 vs. 700 day, p= 0.015). Surprisingly, muRAS CMML group showed a trend towards better OS compared to wtRAS (728 vs. 687 days, p=0.44). Additionally we analyzed patients for presence of Flt3 mutation. These were present in only 18 of the pts (4%) but none of them had also a RAS mutation. There was no c-Kit mutations found in this cohort of pts, compared to 9/37 (24%) pts tested positive for JAK2V617F. Conclusion: MuRAS occurs in 4% of pts with MDS. In the muRAS group, pts tended to have higher risk disease and risk of transformation to acute leukemia compared with wtRAS. OS tended to be worse in some subgroups in muRAS. Although the prognostic impact of RAS mutations in MDS appears to be limited, mutations could be used to develop targeted therapeutic interventions. Disclosures: No relevant conflicts of interest to declare.

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