Microenvironment Induced Myelophthisis Caused By CYP26 Deficiency

Abstract Hematopoietic stem cells (HSCs) reside in a complex microenvironment that enforces the balance between self-renewal and differentiation. The exact physiologic mechanisms by which the niche controls HSC fate remain elusive. Retinoic acid (RA) is a powerful morphogen that controls stem cell behavior across a variety of systems. In bone marrow (BM), mesenchymal stroma cells (MSCs) express cytochrome P450 (CYP)26 enzymes and can inactivate endogenous and pharmacological retinoids (Ghiaur G et al PNAS 2013, Su M et al. PlosOne 2015, Alonso S et al. 2016). Stromal CYP26 activity is required to maintain human HSCs ex vivo (Ghiaur G et al PNAS 2013). Here we set to study the role of CYP26 in HSC homeostasis in vivo. For this, we induced CYP26 knockout via injection of Tamoxifen in ROSA26CreERT CYP26A1loxP/loxPCYP26B1loxP/loxPmice (CYP26KO) and ROSA26CreERT wildtype mice (CTR). After 5 daily tamoxifen injections, the knockout was confirmed at DNA and RNA level in multiple tissues of CYP26KO mice. Within 4 weeks, CYP26KO mice showed profound leukocytosis (5.97 ±0.37 vs. 21.12 ±3.81 k/mm3, n=4, p<0.01 CTR vs. CYP26KO) with neutrophilia and monocytosis compared to CTR mice. By 6 weeks, they experience profound weight loss, became moribund and had to be sacrificed. At that time, they had massive splenomegaly and lymphadenopathy as well as brittle/pale bones compared to CTR (Figure A). Histological analysis revealed presence of extramedullary hematopoiesis with a predominantly myeloid infiltrate in the spleen (confirmed by flow cytometry analysis) and lymph nodes but also multiple clusters megakaryocytes (Figure B). The mice also displayed decreased BM cellularity (11.25 ±1.01 vs. 5.62 ±0.36 x106/femur, n=5, p<0.01, CTR vs. CYP26KO), increased frequency of CFU-C (44.33 ±3.72 vs. 69.83 ±8.10 CFU/25000 BM mononuclear cells (MNCs), n=10, p=0.02, CTR vs. CYP26KO) with a myeloid bias (26.76±3.89 vs. 44.57 ±3.21 %CFU-GM/G/M, n=10, p<0.01, CTR vs. CYP26KO). When cellularity was taken into account, total CFU-C per femur was comparable between the two groups. The mice also had increased frequency of LSK cells in the BM (0.26 ±0.12 vs 0.73 ±0.18 % LSK of MNCs, n=2, CTR vs. CYP26KO) and an increased frequency of circulating CFU-C in the peripheral blood (37 ±4.04 vs. 179.5 ±43.06 per 200 ml of blood, n=4, p=0.01, CTR vs. CYP26KO). On histological analysis, the BM is dominated by a myeloid infiltrate and shows a striking decrease in radial bone diameter (Figure C,D). MSCs derived from CYP26KO mice have lower levels of CXCL12 and SCF and have impaired osteoblast differentiation potential compared to MSCs derived from control mice (Figure E). These findings were confirmed with ex vivo generated CYP26KO MSCs via retroviral mediated Cre recombination of CYP26A1loxP/loxPCYP26B1loxP/loxPstroma cells. More so, preliminary studies suggest that the hematopoietic phenotype observed depends on cell extrinsic presence of CYP26 activity as transplantation of CYP26A1loxP/loxPCYP26B1loxP/loxP BM cells into wildtype BoyJ recipients did not reproduce the phenotype upon injection with Tamoxifen of the recipient mice in spite of 100% donor derived hematopoiesis. Alternatively, transplant of wildtype cells into CYP26A1loxP/loxPCYP26B1loxP/loxPrecipients, did reproduce the phenotype after injection of Tamoxifen. In conclusion, we show here that dysregulated RA homeostasis in the BM impairs MSCs function and result in egress of hematopoiesis to extramedullary sites. These results come to complement data from RARγKO (Walkley CR et al Cell 2007) and SMRTmRIDmice models (Hong S-H et al PNAS 2013) and suggest a pivotal role of RA signaling in pathogenesis of myeloproliferative neoplasm and myelofibrosis. To what extent these findings correlate with human primary myelofibrosis is currently under investigation. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

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Screen for Small Molecules Capable of Expanding Human Hematopoietic Stem Cell Ex Vivo

Blood ◽

10.1182/blood.v118.21.1919.1919 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1919-1919

Author(s):

Iman Hatem Fares ◽

Jalila Chagraoui ◽

Jana Krosl ◽

Denis-Claude Roy ◽

Sandra Cohen ◽

...

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Fold Increase ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Abstract 1919 Hematopoietic stem cell (HSC) transplantation is a life saving procedure whose applicability is restricted by the lack of suitable donors, by poor responsiveness to mobilization regimens in preparation of autologous transplantations, by insufficient HSC numbers in individual cord blood units, and by the inability to sufficiently amplify HSCs ex vivo. Characterization of Stemregenin (SR1), an aryl hydrocarbon receptor (AHR) antagonist that promotes HSC expansion, provided a proof of principle that low molecular weight (LMW) compounds have the ability to promote HSC expansion. To identify novel putative agonists of HSC self-renewal, we initiated a high throughput screen (HTS) of a library comprising more than 5,000 LMW molecules using the in vitro maintenance of the CD34+CD45RA- phenotype as a model system. Our study was based on the fact that mobilized peripheral blood-derived CD34+CD45RA- cells cultured in media supplemented with: stem cell factor, thrombopoietin, FLT3 ligand and interleukin 6, would promote the expansion of mononuclear cells (MNC) concomitant with a decrease in CD34+CD45RA- population and HSC depletion. LMW compounds preventing this loss could therefore act as agonists of HSC expansion. In a 384-well plate, 2000 CD34+cells were initially cultured/well in 50μl medium comprising 1μM test compounds or 0.1% DMSO (vehicle). The proportions of CD34+CD45RA− cells were determined at the initiation of experiment and after a 7-day incubation. Six of 5,280 LMW compounds (0.11%) promoted CD34+CD45RA− cell expansion, and seventeen (0.32%) enhanced differentiation as determined by the increase in proportions of CD34−CD45RA+ cells compared to control (DMSO). The 6 LMW compounds promoting expansion of the CD34+CD45RA− cell population were re-analyzed in a secondary screen. Four out of these 6 molecules suppressed the transcriptional activity of AHR, suggesting that these compounds share the same molecular pathway as SR1 in stimulating HSC expansion, thus they were not further characterized. The remaining 2 compounds promoted, similar to SR1 or better, a 10-fold and 35-fold expansion of MNC during 7 and 12-day incubations, respectively. The expanded cell populations comprised 65–75% of CD34+ cells compared to 12–30% determined for DMSO controls. During 12-day incubation with these compounds, the numbers of CD34+ cells increased ∼25-fold over their input values, or ∼ 6-fold above the values determined for controls. This expansion of CD34+ cells was associated with a ∼5-fold increase in the numbers of multilineage CFC (granulocyte, erythroid, monocyte, and megakaryocyte, or CFU-GEMM) compared to that found in DMSO control cultures. The ability of the 2 newly identified compounds to expand functional HSCs is currently being evaluated in vivo usingimmunocompromised mice. In conclusion, results of our initial screen suggest that other mechanism, besides inhibition of AhR, are at play for expansion of human HSC. Disclosures: No relevant conflicts of interest to declare.

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Absence of the Rho GTPase Activating Protein p190-B Enhances Long Term Hematopoietic Stem Cell Engraftment

Blood ◽

10.1182/blood.v112.11.614.614 ◽

2008 ◽

Vol 112 (11) ◽

pp. 614-614 ◽

Cited By ~ 1

Author(s):

Haiming Xu ◽

Hartmut Geiger ◽

Kathleen Szczur ◽

Deidra Deira ◽

Yi Zheng ◽

...

Keyword(s):

Bone Marrow ◽

Ex Vivo ◽

Gtpase Activating Protein ◽

Self Renewal ◽

Hematopoietic Stem ◽

Proliferation And Differentiation ◽

Serial Transplantation

Abstract Hematopoietic stem cell (HSC) engraftment is a multistep process involving HSC homing to bone marrow (BM), self-renewal, proliferation and differentiation to mature blood cells. However, the molecular regulation of HSC engraftment is still poorly defined. Small Rho GTPases are critical regulator of cell migration, proliferation and differentiation in multiple cell types. While their role in HSC functions has begun to be understood, the role of their regulator in vivo has been understudied. P190-B GTPase Activating Protein (GAP), a negative regulator of Rho activity, has been implicated in regulating cell size and adipogenesis-myogenesis cell fate determination during fetal development (Sordella, Dev Cell, 2002; Cell 2003). Here, we investigated the role of p190-B in HSC/P engraftment. Since mice lacking p190-B die before birth, serial competitive repopulation assay was performed using fetal liver (FL) tissues from day E14.5 WT and p190-B−/− embryos. WT and p190-B−/− FL cells exhibited similar levels of engraftment in primary recipients. However, the level of contribution of p190-B−/− cells to peripheral blood and bone marrow was maintained between the primary and secondary recipients and still easily detectable in tertiary recipients, while the level of contribution of FL WT cells dramatically decreased with successive serial transplantion and was barely detectable in tertiary recipients. The contribution to T cell, B cell and myeloid cell reconstitution was similar between the genotypes. A pool of HSC was maintained in serially transplanted p190-B−/− animals, since LinnegScaposKitpos (LSK) cells were still present in the BM of p190-B−/− secondary engrafted mice while this population disappeared in WT controls. Importantly, this enhanced long term engraftment was due to a difference in the functional capacity of p190-B−/− HSC compared to WT HSC since highly enriched p190-B−/− HSC (LSK) demonstrated similar enhanced serial transplantation potential. Because previous studies have suggested that the loss of long term function of HSC during serial transplantation can depend, at least in part, on the upregulation of the cyclin dependent kinase inhibitor p16Ink4a (Ito et al, Nat Med 2006), the expression of p16Ink4a was examined during serial transplantation. While expression of p16Ink4a increased in WT HSC in primary and secondary recipients, p16Ink4a remained low in p190-B−/− HSC, which indicated that p190-B-deficiency represses the upregulation of p16Ink4a in HSC in primary and secondary transplant recipients. This provides a possible mechanism of p190-B-mediated HSC functions. We next examined whether p190-B-deficiency may preserve the repopulating capacity of HSC/P during ex vivo cytokine-induced culture. While freshly isolated LSK cells from WT and p190-B−/− mice exhibited comparable intrinsic clonogenic capacity, the frequency of colony-forming unit after 7 days in culture was 2 fold-higher in p190-B−/− compared with WT cultures, resulting in a net CFU expansion. Furthermore, competitive repopulation assays showed significantly higher repopulating activity in mice that received p190-B−/− cultured cells compared with WT cells equivalent to a 4.4-fold increase in the estimated frequency of repopulating units. Interestingly, p190-deficiency did not alter cell cycling rate or survival both in vivo and in vitro. Therefore, p190-B-deficiency maintains key HSC functions either in vivo or in ex vivo culture without altering cycling rate and survival of these cells. These findings define p190-B as a critical regulator of HSC functions regulating self renewal activity while maintaining a balance between proliferation and differentiation.

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Macrophages Prevent Human Red Blood Cell Reconstitution In NOD/SCID and NOD/SCID/γc−/− Mice.

Blood ◽

10.1182/blood.v116.21.3723.3723 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3723-3723

Author(s):

Zheng Hu ◽

Yong-Guang Yang

Keyword(s):

Mononuclear Cells ◽

Conflicts Of Interest ◽

Embryonic Stem ◽

Humanized Mice ◽

In Vivo Model ◽

Recipient Mouse ◽

Hematopoietic Stem ◽

Immunodeficient Mice ◽

Human Rbcs

Abstract Abstract 3723 An animal model supporting human erythropoiesis will be highly valuable for assessing the biological function of human RBCs under physiological and disease settings, and for evaluating protocols of in vitro RBC differentiation from human embryonic stem cells. Although immunodeficient mice on the NOD background have been widely used to study human hematopoietic stem cell function in vivo, the successful use of these mice in the study of human erythropoiesis and RBC function has not been reported. We have previously shown that co-transplantation of human fetal thymic tissue (under renal capsule) and CD34+ fetal liver cells (FLCs; i.v.) in NOD/SCID or NOD/SCID/γc−/− mice results in the development of multilineage human hematopoietic cells. Here, we analyzed human RBC reconstitution in these humanized mice. Although a large number of human erythrocytes, which consisted predominantly of immature nucleated erythrocytes, were detected in the bone marrow of human fetal thymus/CD34+ FLC-grafted mice, human RBCs were undetectable in blood of these mice, even in those with nearly full human chimerism in peripheral blood mononuclear cells (PBMCs). Recipient mouse macrophage-mediated rejection is, at least, one of the major mechanisms responsible for the lack of human RBCs in these mice, as human RBCs became detectable in blood following macrophage depletion and disappeared again after withdrawal of treatment. Furthermore, treatment with human erythropoietin (EPO) and human IL-3 significantly increased human RBC reconstitution in mice that were depleted of macrophages. Like the human RBCs developed in the humanized mice, exogenously injected normal human RBCs were also rapidly rejected by macrophages in NOD/SCID mice. Taken together, our data demonstrate that human RBCs are highly susceptible to rejection by macrophages in immunodeficient mice. Thus, strategies for preventing human RBC rejection by macrophages are required for using immunodeficient mice as an in vivo model to study human erythropoiesis and RBC function. Disclosures: No relevant conflicts of interest to declare.

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A Critical Role of Mir-9 in Myelopoesis and EVI1-Induced Leukemogenesis.

Blood ◽

10.1182/blood.v120.21.2332.2332 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2332-2332

Author(s):

Vitalyi Senyuk ◽

Yunyuan Zhang ◽

Yang Liu ◽

Ming Ming ◽

Jianjun Chen ◽

...

Keyword(s):

Cell Transformation ◽

Conflicts Of Interest ◽

Critical Role ◽

Ectopic Expression ◽

Myeloid Differentiation ◽

Dna Hypermethylation ◽

Hematopoietic Stem

Abstract Abstract 2332 MicroRNA-9 (miR-9) is required for normal neurogenesis and organ development. The expression of miR-9 is altered in several types of solid tumors suggesting that it may have a function in cell transformation. However the role of this miR in normal hematopoiesis and leukemogenesis is unknown. Here we show that miR-9 is expressed at low levels in hematopoietic stem/progenitor cells (HSCs/HPCs), and that it is upregulated during hematopoietic differentiation. Ectopic expression of miR-9 strongly accelerates terminal myelopoiesis, while promoting apoptosis in vitro and in vivo. In addition, the inhibition of miR-9 in HPC with a miRNA sponge blocks myelopoiesis. EVI1, required for normal embryogenesis, and is considered an oncogene because inappropriate upregulation induces malignant transformation in solid and hematopoietic cancers. In vitro, EVI1 severely affects myeloid differentiation. Here we show that EVI1 binds to the promoter of miR-9–3 leading to DNA hypermethylation of the promoter as well as repression of miR-9. We also show that ectopic miR-9 reverses the myeloid differentiation block that is induced by EVI1. Our findings suggest that inappropriately expressed EVI1 delays or blocks myeloid differentiation, at least in part by DNA hypermethylation and downregulation of miR-9. It was previously reported that FoxOs genes inhibit myeloid differentiation and prevent differentiation of leukemia initiating cells. Here we identify FoxO3 and FoxO1 as new direct targets of miR-9 in hematopoietic cells, and we find that upregulation of FoxO3 in miR-9-positive cells reduces the acceleration of myelopoiesis. These results reveal a novel role of miR-9 in myelopoiesis and in the pathogenesis of EVI1-induced myeloid neoplasms. They also provide new insights on the potential chromatin-modifying role of oncogenes in epigenetic changes in cancer cells. Disclosures: No relevant conflicts of interest to declare.

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hnRNP K: A Tumor Suppressor or Oncogene?

Blood ◽

10.1182/blood.v124.21.259.259 ◽

2014 ◽

Vol 124 (21) ◽

pp. 259-259

Author(s):

Miguel Gallardo ◽

Hun Ju Lee ◽

Carlos E. Bueso-Ramos ◽

Xiaorui Zhang ◽

Laura R. Pageon ◽

...

Keyword(s):

Bone Marrow ◽

Tumor Suppressor ◽

Conflicts Of Interest ◽

Chromosome 9 ◽

Wild Type ◽

Differentiation Potential ◽

Hematopoietic Stem ◽

Hnrnp K ◽

Differentiation And Proliferation

Abstract Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is an RNA and DNA binding protein that regulates critical pathways controlling differentiation and proliferation programs. While alterations in hnRNP K expression are associated with neoplastic malignancies, we currently do not understand how changes in hnRNP K expression contribute to tumor phenotypes in vivo. Previous biochemical and cell line studies demonstrate that hnRNP K transcriptionally regulates p53-dependent activities, suggesting it functions as a potential tumor suppressor. However, hnRNP K has also been shown to positively regulate c-Myc expression, indicating it may behave as an oncogene. The HNRNP K gene maps to a region of chromosome 9 (9q21.32), which is lost in a subset of patients with acute myeloid leukemia (AML). RNA expression analyses of patient samples with AML that harbor 9q21.32 deletions revealed a significant reduction in HNRNP K expression compared to wild type control samples, supporting the notion that hnRNP K acts as a tumor suppressor (Figure 1A). However, patients with AML who do not harbor a 9q21.32 deletion displayed a significant increase in hnRNP K expression (Figure 1A). Thus, to examine the association between altered hnRNP K expression and disease status in patients with AML, we performed reverse phase protein array (RPPA) analysis on CD34+ bone marrow cells from 415 de novo AML patient as well as healthy donor controls. Interestingly, we observed a significant correlation between elevated hnRNP K levels and poor outcomes, which supports the idea that hnRNP K has oncogenic potential (Figure 1A). Together, these observations indicate that any change in hnRNP K expression may contribute to the etiology of AML and supports the idea that hnRNP K may potentially act as either a haploinsufficient tumor suppressor or oncogene in AML. To directly interrogate these possibilities in vivo, we generated mouse models that either harbor a deletion of one hnRNP K allele (hnRNP K+/-) or overexpressed hnRNP K (hnRNP KTg) in the hematological compartment. Western blot analyses demonstrated that hnRNP K haploinsufficiency results in a significant reduction in hnRNP K expression while tissue-specific activation of hnRNP K resulted in overexpression of hnRNP K. Similar to our observation in AML patients, either hnRNP K haploinsufficiency or overexpression resulted in similar phenotypes in vitro and in vivo. Lin-CD117+ hematopoietic stem cells (HSCs) from hnRNP K+/- and hnRNP KTg mice had significant increases in differentiation and proliferation potential as determined by colony formation assays. In these experiments, we observed a significant increase in the number of total colonies and number of cells per colony in both hnRNP K+/- and hnRNP KTg HSCs as compared to wild type HSCs (Figure 1B). In vivo analyses of the hnRNP K+/- and hnRNP KTg mice revealed a significant increase in myeloid hyperplasia in the peripheral blood and bone marrow, increased tumor formation, genomic instability, and decreased survival compared to wild type mice (Figure 1C). Interestingly, both increased and decreased hnRNP K expression resulted in alterations in similar pathways that regulate differentiation and proliferations potential (e.g.; p53 and c-Myc pathways and alterations in C/EBP expression). Together, these clinical and animal model studies illustrate that either over-expression or under-expression of hnRNP K lead to strikingly similar phenotypes that directly impact the etiology of AML. Furthermore, these data not only implicate that hnRNP K behaves as both a tumor suppressor and oncogene, but also suggest that it functions as a master toggle that dictates the proliferation and differentiation potential of HSCs. We are currently using Whole Transcriptome Shotgun Sequencing (RNA-Seq) and ChIP-Seq to evaluate the mechanisms by which increased and decreased hnRNP K expression impact hematologic malignancies. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Low Density Granulocytes in Patients after Allogeneic Hematopoietic Stem Cells Transplantation (allo-HSCT): A Distinct Class of Neutrophils in Systemic Alloimmunity

Blood ◽

10.1182/blood.v126.23.4614.4614 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4614-4614

Author(s):

Ekaterina Mikhaltsova ◽

Valeri G. Savchenko ◽

Larisa A. Kuzmina ◽

Mikhail Drokov ◽

Vera Vasilyeva ◽

...

Keyword(s):

Lupus Erythematosus ◽

Peripheral Blood ◽

Hematological Malignancies ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Human Peripheral Blood ◽

Unrelated Donor ◽

Low Density ◽

Hematopoietic Stem

Abstract Introduction It's generally considered that all alloimmune process such as acute graft-versus host disease (aGVHD) after allo-HSCT are mostly controlled by lymphocytes. The role of neutrophils in systemic alloimmunity after allo-HSCT is still illusive. In 1987 a distinct subset of proinflammatory, low-density granulocytes (LDGs) isolated from the peripheral blood mononuclear cell fractions of patients with system lupus erythematosus has been described. There is no LDG's in healthy donors. While the origin and role of LDGs still needs to be fully characterized, we try to describe this population in patients with hematological malignancies after allo-HSCT Patients and methods. Peripheral blood samples were collected in EDTA-tubes before allo-HSCT, on day +30,+60,+90 after allo-HSCT and at day of aGVHD from 47 patients with hematological malignancies (AML=22, ALL n=17, LPD=3, MDS =2; CML=2; 17 with active disease, 30 - in CR) after allo-HSCT (from matched unrelated donor n=34, from matched related donor n=13; MAC = 13, RIC=34). Isolation of mononuclear cells from human peripheral blood was made by standard protocol using Lympholyte®-M Cell Separation Media (Cedarlane Labs). The anti-CD66b-PE (Biolegend, USA) antibodies and FSC/SSC were used to determine LDGs cells as FSChigh \SSChigh \CD66b+. 100000 of cells were analyzed on a BD FACSCanto II (Becton Dickinson, USA). Results. Results of blood evaluation of 47 patients with hematological malignancies, whose blood was examined after allo-HSCT presented in table 1. Conclusion Despite the fact that we don't get significant differences. LDG's detection in allo-HSCT patients need further investigation. Table 1. Incidence of LDG after allo-HSCT in patients with and without aGVHD Table 1. Incidence of LDG after allo-HSCT in patients with and without aGVHD Disclosures No relevant conflicts of interest to declare.

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The Role of STAT5 in HOXA9-Associated Leukemia

Blood ◽

10.1182/blood.v128.22.3919.3919 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3919-3919

Author(s):

Peilin Ma ◽

Yuqing Sun ◽

Jingya Wang ◽

Weihua Song ◽

Tao Xu ◽

...

Keyword(s):

Binding Sites ◽

Myeloid Leukemia ◽

Tandem Duplication ◽

Conflicts Of Interest ◽

Hematopoietic Stem ◽

Constitutive Activation ◽

Oncogenic Mutation

Abstract Homeobox A9 (HOXA9) is a homeodomain-containing transcription factor that is essential for hematopoietic stem cell expansion and differentiation. Deregulation of HOXA9 is commonly observed in human acute myeloid leukemia (AML). About half of AML patients overexpress HOXA9 as a result of MLL rearrangements, NUP98 translocations, NPM1 mutations or CDX2/CDX4 overexpression. Despite its central importance in leukemia, the mechanism of transcriptional regulation by HOXA9 and its downstream effectors are poorly understood. HOXA9 physically interacts with MEIS1, a cofactor that greatly accelerates leukemia development in transplanted animals. Our group recently identified a number of transcription factors as HOXA9 potential collaborators by genomic profiling of HOXA9 binding sites and mass spectroscopy. One of these putative collaborators is signal transducer and activator of transcription 5 (STAT5), which coimmunoprecipitates with HOXA9. Furthermore STAT motifs extensively overlap with HOXA9 binding sites. STAT5 is important for survival, proliferation and differentiation of hematopoietic cells and constitutive activation of STAT5 has also been observed in human leukemias bearing oncogenic mutation of Jak2, Bcr-Abl, c-Kit and Flt3. FLT3 internal tandem duplication (FLT3-ITD) is observed in 25% of patients with MLL-partial tandem duplication (MLL-PTD) and is associated with HOXA9 upregulation and unfavorable prognosis. Therefore, we hypothesized that the interaction of HOXA9 and STAT5 may play a role in HOXA9-associated leukemogenesis. Treatment of human cell lines bearing MLL-AF9 and FLT3-ITD with specific FLT3 and STAT5 inhibitors showed that suppression of the constitutive activation of STAT5 significantly inhibits the hyper-proliferation of these cells. We then overexpressed FLT3-ITD or active mutation of STAT5 (STAT5 1*6) in mouse hematopoietic stem cells /progenitor cells (HSC/PCs) transduced with MLL-AF9 or HOXA9 and found that constitutively active STAT5 enhances cell proliferation in vitro. We next transduced HOXA9 into HSC/Pcs from wild type (WT) or FLT3-ITD transgenic mice and transplanted these cells into sublethally irradiated WT mice. All of these recipients developed myeloid leukemia, with recipients transplanted with FLT3-ITD (n=4) developing leukemia significantly earlier than WT controls (n=5, p<0.05), suggesting that FLT3-ITD mediated STAT5 activation enhanced HOXA9-induced leukemogenesis in vivo. To further assess the role of STAT5 in HOXA9-mediated transformation, we performed ChIP-Seq assay with HOXA9-transformed cells and identified nearly half of STAT5 binding sites (228 out of 596) colocalized with HOXA9. Most of these cobound sites are located in distal intergenic (61.0%) and intron (35.1%) regions. Five cobound regions (Il2rα, Fgf1, Pdlim5, Pim1, Fabp5) were selected and confirmed by ChIP-qPCR. To further characterize the interaction between HOXA9 and STAT5, GST pull-down assays were performed that showed that the c-terminal of HOXA9 is critical for interaction with STAT5. Overall, the findings suggest that STAT5 promotes HOXA9-induced transformation by functionally interacting with HOXA9 at HOXA9-regulated enhancers. Disclosures No relevant conflicts of interest to declare.

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Thalassemia Gene Therapy By In Vivo Transduction of Mobilized Hematopoietic Stem Cells (HSCs) with an Integrating Hybrid Adenovirus Vector System

Blood ◽

10.1182/blood-2018-99-116787 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2193-2193

Author(s):

Afrodite Georgakopoulou ◽

Hongjie Wang ◽

Chrysi Kapsali ◽

Nikoleta Psatha ◽

Angeliki Koufali ◽

...

Keyword(s):

Gene Therapy ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Hematological Parameters ◽

Sleeping Beauty ◽

Vector System ◽

Good Tolerability ◽

Hematopoietic Stem

Abstract To overcome the cost and complexity of current thalassemia ex vivogene therapy protocols, we developed a minimally invasive and readily translatable approach for in vivo HSC gene delivery which abrogates the need for HSC leukapheresis, CD34+ cell selection, ex vivo HSC culture, myeloablation and ultimately, transplantation. Our approach involves HSC mobilization with G-CSF/AMD3100 andintravenous injections of a hybrid vector system consisting ofa CD46-targeting, helper-dependent adenoviral vector and the hyperactive Sleeping Beauty transposase (SB100x) that mediates integration of thevector-encoded γ-globin and mgmtP140K genes. Pretreatment with glucocorticoids before virus injectionsis used to blockthe release of pro-inflammatory cytokines andimmunosuppression is applied in order to avoid responses against human g-globin- and MGMT protein-expressing cells. We tested our approach in a mouse model recapitulating the phenotypeof human β-thalassemia intermedia (Hbbth-3/hCD46++ mice). At week 8 post transduction, hCD46+/+/Hbbth-3 mice expressed HbF in 31.2±2.7% of circulating erythrocytes. Due to a significant drop in HbF expression by week 16 (11.9±3.0%), a 4-dose O6BG/BCNU treatment was administeredin order to in vivo select forgene corrected hematopoietic progenitors, thus recovering the HbF expression in76.0±5.7% of the circulating erythrocytes, by week 29 post in vivo transduction. With an average vector copy number of 1.4/cell, the human γ-globin to mouse α-globin expression was ~10% by HPLC and the human γ-globin to mouse β-globin mRNA ratio ~10%, by qRT-PCR. Hematological parameters (RBCs, Ht, Hb, MCV, RDW, Reticulocytes) at week 29 post in vivo transduction, were significantly improved over baseline or were indistinguishable from normal values, suggesting near to complete phenotypic correction. Treated mice showed significant reduction of spleen size, extramedullary erythropoiesis and parenchymal hemosiderosis. After secondary transplantation and without in vivo selection, more than 90% of donor-derived erythrocytes (CD46+) were g-globin-positive, up to 20 weeks post-transplant. Safety was demonstrated by the good tolerability of treatment, the absence of alterations in hematopoiesis, the normal colony-forming potential of bone marrow cells and the random integration pattern of our vector system. Overall, we present a simplified platform for gene therapy of thalassemia, which can serve as a cost-efficient and "portable" approach to make gene therapy accessible even to resource-poor regions where thalassemia major is endemic but only minimally complex strategies could be adopted. Disclosures No relevant conflicts of interest to declare.

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Plasma EDA Fibronectin in Primary Myelofibrosis Correlates with Bone Marrow Fibrosis and Predicts Splenomegaly

Blood ◽

10.1182/blood-2019-130735 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1688-1688

Author(s):

Alessandro Malara ◽

Cristian Gruppi ◽

Margherita Massa ◽

Vittorio Rosti ◽

Giovanni Barosi ◽

...

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Philadelphia Chromosome ◽

Myeloproliferative Neoplasm ◽

Primary Myelofibrosis ◽

Enzyme Linked Immunosorbent Assay ◽

Costal Margin ◽

Cell Transplant ◽

Healthy Controls ◽

Hematopoietic Stem

Introduction: Primary myelofibrosis (PMF) is a Philadelphia chromosome negative myeloproliferative neoplasm with adverse prognosis characterized by bone marrow (BM) fibrosis and extramedullary hematopoiesis. Fibronectin (FN) is an extracellular matrix glycoprotein that plays vital roles during tissue repair and regeneration. It exists in different forms. Plasma FN is synthesized by hepatocytes and secreted into the blood plasma, where circulates at a concentration of 300-600 μg/ml in a soluble, compact form. Differently, cellular FN is synthesized by several cell types, such as fibroblasts, endothelial cells, chondrocytes and myocytes. The alternative splicing of EDA and EDB and more complex splicing of the V domain, during transcription of FN1 gene, allows different isoforms of FN to be expressed in a tissue-dependent and temporally regulated manner. Very low levels (1.3-3 μg/ml) of FN containing EDA and/or EDB are present in plasma. Although its function is not well understood, EDA containing FN (EDA-FN) is known to agonize Toll like receptor 4 (TLR4), resulting in NF-κβ-dependent cytokine release; to induce myofibroblast differentiation during wound healing; and to increase agonist-induced platelet aggregation and thrombus formation in vivo. We previously showed that EDA-FN levels are increased in plasma and BM biopsies of PMF patients. Mechanistically, BM EDA-FN sustains megakaryocyte proliferation through TLR4 binding and confer a pro-inflammatory phenotype to cell niches promoting fibrosis progression in Romiplostim-treated mice. In this work we measured the plasma levels of EDA-FN in 104 well characterized patients with PMF to determine whether elevated levels of EDA-FN predict the occurrence of disease-related events. Methods: Plasma circulating EDA FN was measured with an enzyme linked immunosorbent assay developed at the University of Pavia, by our group. We obtained plasma EDA-FN concentration values and health care data of persons with PMF from the data-base of the Centre for the Study of Myelofibrosis at the IRCCS Policlinico S. Matteo Foundation in Pavia. We sequentially excluded persons treated with disease-modifying drugs at any time before or on the date of base-cohort entry, and those who had been splenectomized or had received a stem cell transplant. We also excluded persons with acute inflammatory diseases, autoimmune diseases, other neoplasms, and severe liver or renal dysfunction. For this study we selected everyone giving written informed consent and the study was approved by the local Ethic Committee. Immunofluorescence was performed on spleen sections from PMF patients who underwent splenectomy either because of anemia or symptomatic splenomegaly, or both; and healthy controls that were splenectomized following traumatic lesion of the spleen. Data were analyzed using STATISTICA software. Results: A homozygous JAK2V617F genotype was the major determinant of elevated plasma EDA-FN. Elevated EDA-FN levels were associated with anemia, increased levels of high-sensitivity C-reactive protein, BM fibrosis and splanchnic vein thrombosis at diagnosis. We interpreted these associations as reflecting the role EDA-FN plays in tissue remodeling, inflammation and vascular injury. Interestingly, EDA-FN levels resulted also associated with spleen size, and elevated levels of EDA-FN at diagnosis predicted large splenomegaly (more than 10 cm from the left costal margin) outcome. The evidence that plasma EDA-FN levels were not associated with the CD34+ hematopoietic stem cells mobilization, drove us to hypothesize that EDA-FN could reflect spleen endothelial cell activation and/or neoangiogenesis. Immunofluorescence analysis of spleen specimens from PMF patients and healthy controls revealed that high levels of EDA-FN were present in pathological spleens in strong association with endothelial neoangiogenesis. Conclusions: Quantification of EDA-FN level in PMF strongly correlates with BM fibrosis and may be the first marker of an altered spleen microvasculature that contributes to splenomegaly. Understanding the role of this FN isoform in PMF would be useful for testing new mechanisms of disease progression and new hypotheses about the treatment of splenomegaly in PMF. Disclosures No relevant conflicts of interest to declare.

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Endogenous TGF-beta1 Mediated Osteogenic Impairment of Medullar Mesenchymal Stromal Cells in Primary Myelofibrosis

Blood ◽

10.1182/blood.v124.21.1873.1873 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1873-1873

Author(s):

Christophe Martinaud ◽

Christophe Desterke ◽

Johanna Konopacki ◽

Lisa Pieri ◽

Rachel Golub ◽

...

Keyword(s):

Bone Marrow ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasm ◽

Hematopoietic Cells ◽

Primary Myelofibrosis ◽

Osteogenic Potential ◽

Healthy Donors

Abstract Primary myelofibrosis (PMF) is myeloproliferative neoplasm characterized by clonal myeloproliferation, dysmegakaryopoiesis, extramedullary hematopoiesis associated with myelofibrosis and altered stroma in bone marrow and spleen. Mesenchymal stromal cells (MSCs) are reported to play a pivotal role in fibrosis and stromal changes are considered as a reactive counterpart of the cytokine production by clonal hematopoietic cells. The present study shows that MSCs from patients demonstrate functional abnormalities that are unexpectedly maintained ex-vivo, in culture. Material and Methods: we studied MSCs and bone marrow sections from PMF patients (n=12) as compared to healthy donors (HDs) (n=6). We tested their proliferation, immunophenotype, hematopoiesis supporting capacities, differentiation abilities, in-vivo osteogenic assays, and performed secretome and transcriptome analysis. Results: We found that PMF-MSCs exhibit similar proliferative capacity and long-term hematopoiesis supporting abilities as compare to healthy donors. They overproduce interleukin 6, VEGF, RANTES, PDGF, BMP-2 and surprisingly TGF-beta1. MSCs from fibrotic PMF patients express high levels of glycosaminoglycans. Adipocytes and chondrocytes differentiation abilities were not different as compared to HDs but PMF-MSCs exhibit an increased in vitro potential. Implementation on scaffold in nude mice confirmed, in vivo, this increased osteogenic potential. We then looked into gene expression and discovered that PMF-MSCs show an original transcriptome signature related to osteogenic lineage and TGF-beta1. Indeed, osteogenic genes such as Runx2, Dlx5, Twist1, Noggin, Sclerostin, GDF5 and Serpine1 are deregulated and suggest a potential osteoprogenitor priming of PMF-MSCs. These molecular results also advocated for a TGF-beta1 impregnation that prompted us to study its impact on PMF-MSCs osteogenic differentiation. First, we then showed that Smad2 is intrinsically over-activated in PMF-MSC and that stimulation by TGF-beta1 is associated with an increase phospho-Smad2 level and an enhancement of bone master gene regulator Runx2 expression. Then, we inhibited TGF-beta1 pathway by by SB-431542 and evidenced a specific behavior of osteogenic MSCs differentiation in patients, suggesting involvement of TGF-beta1 in osteogenic impairment. Conclusion: Altogether, our results identify a signature of PMF-MSCs and suggest that they participate in PMF osteogenic dysregulation independently from in vivo local stimulation by clonal hematopoietic cells Disclosures No relevant conflicts of interest to declare.

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