Modeling and Targeting MLL-PTD/RUNX1 Related MDS/AML In Mouse

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2746-2746
Author(s):  
Yue Zhang ◽  
Xiaomei Yan ◽  
Aili Chen ◽  
Goro Sashida ◽  
Zhijian Xiao ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Median Survival ◽  
Molecular Mechanisms ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Insertion Mutagenesis ◽  
Mouse Bone Marrow ◽  
Hematopoietic Stem ◽  
The Impact

Abstract Myelodysplastic syndromes (MDS) are heterogeneous disorders in which the hematopoietic stem cells (HSCs) in the bone marrow are defective, resulting in insufficient normal blood cells. MDS progress to secondary acute myeloid leukemia (sAML) in about one third of patients, as additional genetic abnormalities are acquired. Because of the similar molecular mechanisms under these two related disease categories, MDS with increased blasts (>5%) and AML with multilineage dysplasia and/or antecedent MDS, are also defined as MDS/AML. MLL and RUNX1/CBFb regulate normal hematopoiesis, and we have shown that they form a regulatory complex to regulate downstream target genes. Mutations of MLL1 (in-frame partial tandem duplication, MLL-PTD, or MLL translocations) or RUNX1 are found in about 28% of MDS, particularly in high-risk MDS or therapy-related MDS. sAML frequently contains both MLL-PTD and RUNX1 mutations, arguing for cooperative leukemogenic synergy between these two molecular lesions. However, Mll-PTD knock-in mice or Runx1Δ/Δ mice do not develop spontaneous MDS or AML. RUNX1 mutations can cause mouse MDS/AML in murine retroviral transduction mediated overexpression and BMT, however, the latency is long (8-14 months) and retroviral vector insertion mutagenesis at Evi1 or Mn1loci seems critical for MDS/AML development in this model. Indeed RUNX1 mutations cooperate with Evi1 upregulation in both murine MDS/AML model and human AML. Thus, we hypothesize that combining RUNX1 mutations with MLL-PTD may facilitate its transformation toward MDS and/or sAML. To understand the impact of RUNX1 mutation cooperativity with MLL-PTD, we first expressed MDS relevant patient-derived RUNX1 mutants (D171N and 291fsX300) in the context of Mll-PTD knock-in mouse bone marrow cells and performed BMT and in vitro CFU replating assay. RUNX1 mutations (D171N and 291fsX300) could not transform WT BM cells. However, they could transform MLL-PTD BM cells and undergo serial replating. Interestingly, D171N and 291fsX300 transformed MLL-PTD cells form different type of clones: MLL-PTD/D171N clones are bigger and diffuse, while MLL-PTD/291fsX300 clones are smaller but denser. In BMT assay, the MLL-PTD/D171N and MLL-PTD/291fsX300 BMT mice developed MDS and MDS/AML (2-10 months) after BMT. The MLL-PTD/D171N BMT mice developed anemia, neutropenia with leukodysplasia and left-shifted differential counts, and a hypo-cellular marrow with excess blasts, while MLL-PTD/291fsX300 BMT mice developed rather similar trilineage dysplasia features but present hyper-cellular marrow with high percent of blasts, some of the mice were diagnosed as MDS/AML. Interestingly, the MLL-PTD/291fsX300 BMT mice also develop myelo-fibrosis (MF) in the BM. We further generated Mll-PTD/Runx1Δ/Δ mice using Mx1-Cre mediated deletion. These mice showed thrombocytopenia one month after pI-pC injection, and developed pancytopenia 2-4 months later. The CBC exhibited increased MCV, RDW and severe anemia. All these Mll-PTD/Runx1Δ/Δ mice died of MDS induced complications within 8 months, and tri-lineages dysplasias (TLD) were found in bone marrow aspiration. Similar but accelerated lethal MDS were found in recipients transplanted with PTD/Runx1Δ/Δ BM cells compared with controls (median survival: 68 days VS undefined). Low dose decitabine (DAC 0.3 mg/kg, twice a week, subcutaneous injection) were used to treat these recipients, and we found significantly longer median survival in DAC treated recipients than controls (median survival: 94.5±6.4 VS 53.5±3.5 days, p<0.001). Neither Mll-PTD nor Runx1Δ/Δ BM cells could replate more than 4 times with M3434 methaltheloase, however, PTD/Runx1Δ/Δ BM cells could be replated more than 6 months in vitro. We also treated these cells in vitro with DAC (0.5 uM). Fewer colony numbers and increased differentiated cells (Gr1+/Mac1+) were found in DAC treated cells than PBS treated controls (CFU numbers/1x105seeded cells: 34±7.7 vs 619±30.5, p<0.001). In conclusion, our study demonstrates that: 1) RUNX1 mutations and complete deletions cause MDS or MDS/AML phenotypes in Mll-PTD background; 2) Decitabine is a promising drug to treat MLL-PTD/RUNX1 related MDS/AML. These exciting new models allow us to identify and analyze MDS/AML-initiating cells (MIC) and major targets that are critical for clonal evolution and pathogenesis of MDS/AML and therapeutic interventions. Disclosures: No relevant conflicts of interest to declare.

Inhibition of HDAC8 Reactivates p53 and Abrogates Leukemia Stem Cell Activity in CBFβ-SMMHC Associated Acute Myeloid Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 363-363
Author(s):  
Jing Qi ◽  
Qi Cai ◽  
Sandeep Singh ◽  
Ling Li ◽  
Hongjun Liu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Cell Activity ◽  
Disease Free Survival ◽  
Hematopoietic Stem ◽  
The Impact ◽  
Acute Myeloid

Abstract The inv(16)-created CBFβ-SMMHC fusion protein inhibits differentiation of hematopoietic stem and progenitor cells (HSPCs) and creates pre-leukemic populations predisposed to acute myeloid leukemia (AML) transformation. However, the molecular mechanism underlying the leukemogenic function of CBFβ-SMMHC has been elusive. Given the low TP53 mutation rate in AML, alternative mechanisms disrupting p53 function are expected. We showed thatCBFβ-SMMHC impairs p53 acetylation and p53 target gene activation through formation of an aberrant protein complex with p53 and HDAC8 (Blood, 120: A772; 122(21): 224). We now show that CBFβ-SMMHC binds to p53 and HDAC8 independently through distinct regions and that HDAC8 mediates the deacetylation of p53 associated with CBFβ-SMMHC. In addition, we generated mice carrying a floxed Hdac8 (Hdac8f) allele and crossed with Cbfb56M/+/Mx1-Cre (Kuo YH et al, Cancer Cell 2006). Deletion of Hdac8 signifiacntly (p<0.0001) reduced the incidence of AML and prolonged disease-free survival. Pharmacologic inhibition of HDAC8 activity with HDAC8-selective inhibitors (HDAC8i) reactivates p53 and selectively induces apoptosis of inv(16)+ AML CD34+ cells while sparing normal HSPCs. To test the effect of HDAC8i on LSC engraftment and leukemia-initiating capacity, we generated Cbfb56M/+/Mx1-Cre mice with a Cre-reporter line expressing tdTomato fluorescence protein following Cre-mediated recombination. AML cells (dTomato+/cKit+) treated with HDAC8i (22d) ex vivo showed reduced engraftment (p=0.025) and enhanced survival (p=0.025) in transplanted mice. To examine whether HDAC8i 22d treatment affects the engraftment capacity on surviving cells, we transplanted equal number (2 x 106) of AML cells treated with either 22d or vehicle in another cohort of mice (n=4). We show that HDAC8i 22d treatment reduced the engraftment of dTomato+/cKit+ AML cells and enhanced survival, suggesting that the engraftment capacity is altered in addition to reducing AML cell survival. We next performed preclinical studies to determine the efficacy of in vivo administration of HDAC8i 22d. AML transplanted mice were randomized into two groups, one group treated with vehicle and the other treated with HDAC8i 22d for 2 weeks. Flow cytometry analysis revealed significantly reduced frequency (p=0.0097) and number (p=0.0101) of dTomato+/cKit+ AML cells in the bone marrow and spleen of 22d treated mice compared to vehicle treated group. To further assess the impact on LSC activity, we transplanted bone marrow cells from these treated mice into secondary recipients and analyzed for AML engraftment. Significant reduction in the frequency (p<0.0001) and the number (p=0.0006) of dTomato+/cKit+ AML cells was observed in the bone marrow and spleen. Furthermore, HDAC8i 22d treated transplants showed no signs of leukemia while vehicle treated transplants are moribund with aggressive AML. These results indicate that HDAC8 inhibition by 22d treatment effectively eliminates engraftment and leukemia-initiating capacity of AML LSCs. In conclusion, our studies identify a novel post-translational p53-inactivating mechanism and demonstrate selective HDAC8 inhibition as a promising approach to target inv(16)+ AML LSCs. Disclosures No relevant conflicts of interest to declare.

Autophagy Regulation and Dysfunction in Bone Marrow Malignancies

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4623-4623
Author(s):  
Fernando V Pericole ◽  
Mariana Lazarini ◽  
Adriana S. S. Duarte ◽  
João Machado-Neto ◽  
Sara T. Olalla Saad
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Low Risk ◽  
Control Group ◽  
Hematopoietic Stem ◽  
Combination Treatments ◽  
Molecular Machinery ◽  
Total Bone Marrow

Abstract Abstract 4623 Introduction: Autophagy is a catabolic pathway by which cytoplasmic materials are degraded into the lysosome and it is also a quality control system for proteins and organelles. Autophagy plays an important role in cell adaptation to starvation, hypoxia, cell survival and cancer. Its core molecular machinery is tightly linked to metabolic pathways, such as LKB1/AMPK and mTORC1. Autophagy has been shown to play several important roles in cancer. Indeed, multiple autophagy genes have been characterized as tumor suppressor genes. In hematopoietic system, autophagy is required during myeloid and lymphoid differentiation, terminal erythroid mitochondrial clearance, production of proplatelets and also differentiation of monocytes into macrophages. Interestingly, autophagy seems disturbed in most bone marrow malignancies. Evidence in mice suggests that autophagy suppression (ATG7 or ATG5 knockdown models) in hematopoietic stem cells may be implicated in Acute Myeloid Leukemia (AML) pathogenesis. In Multiple Myeloma (MM), in vitro studies using cell lines showed autophagy activation and lysosome inhibitors (such as chloroquine) are currently been used in various combination treatments in clinical trials. Aim: The aim was to characterize the expression of autophagy machinery key genes (BECN1, MAP1LC3A, SQSTM1), as well as hypoxia master regulator (HIF1A) in total bone marrow cells from bone marrow malignancies: myelodysplasia (MDS), MM and AML patients, excluding acute promyelocytic leukemia. Methods: BECN1, MAP1LC3A, SQSTM1 and HIF1A levels were verified, by q-PCR, in diagnostic (or without any treatment) BM aspirates from 22 normal donors, 30 MDS (17 low-risk and 13 high-risk, according 2008 WHO classification), 43 AML and 11 MM patients. Results: BECN1 gene expression was increased in MM, compared with control group. All other groups did not differ from the control group. Comparing diseases amongst each other, AML had a lower BECN1 expression, compared with low-risk MDS and with MM (Figure 1A). Disclosures: No relevant conflicts of interest to declare.

scholarly journals Human Cytomegalovirus Selectively Suppresses the Megakaryo/Thrombopoiesis of PDGFR+ and αvβ3+ Megakaryocytes Via the TPO/c-Mpl Pathway after Allo-HSCT

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 25-25
Author(s):  
Feng-qi Liu ◽  
Fei-er Feng ◽  
Gao-chao Zhang ◽  
Yan Su ◽  
Xue-yan Sun ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Human Cytomegalovirus ◽  
Hcmv Infection ◽  
Conflicts Of Interest ◽  
Antiviral Treatment ◽  
Infection Model ◽  
Hematopoietic Stem ◽  
The Impact

Introduction Virus-induced thrombocytopenia is a severe complication in immunocompromised hosts. Among patients following allogeneic hematopoietic stem cell transplantation (allo-HSCT), human cytomegalovirus (HCMV) infection contributes to a variety of end-organ diseases and hematological complications, leading to increased mortality. Even with antiviral treatment, HCMV remains a potentially lethal infection due to the lack of understanding of the underlying mechanisms of host-virus interactions. The key to solving this problem is to identify the factors that predispose patients to HCMV infection and carry out targeted therapy. Here, we investigated the megakaryo/thrombopoiesis process, including the thrombopoietin (TPO)/c-Mpl pathway, after HCMV infection in vivo and in vitro, screened for susceptible subsets of megakaryocytes (MKs) and explored novel therapeutic targets for HCMV infection. Methods To test whether thrombocytopenia induced by HCMV results from an impaired megakaryo/thrombopoiesis process, we studied the impact of HCMV in an in vivo model of HCMV DNAemia patients following allo-HSCT and an in vitro model of bone marrow CD34+-derived MKs infected with serum from HCMV DNAemia patients. Forty patients who had received allo-HSCT were enrolled in this study, among whom 18 recipients had HCMV DNAemia and 22 were HCMV negative, and bone marrow-derived mononuclear cells (MNCs) from patients were tested for CD41, vWF, pp65, c-Mpl, PDGFR, αvβ3 and TLR2 using flow cytometry (FCM). Transmission electron microscopy (TEM) was used to detect HCMV capsids inside MKs. Cell apoptosis was measured by Annexin V. MK ploidy was determined by FCM for propidium iodide (PI) staining. Finally, inhibitors of PDGFR (IMC-3G3 and Gleevec), αvβ3 and TLR2 were cocultured with MKs. Results Our data showed that pp65+ cells accounted for 40.59±6.12% of total CD41+vWF+ MKs from HCMV DNAemia patients, and there was a significant increase in the expression of αvβ3, PDGFR and TLR2 in pp65+ MKs compared with that in control patients. Furthermore, the percentage of PDGFR+αvβ3+ MKs emerged as an independent factor associated with HCMV infection in multivariate analysis (p = 0.008). MKs in HCMV-infected patients showed increased apoptosis and necrosis and different patterns of MK ploidy distribution compared with those in HCMV-negative patients, with a decreased proportion from 16N to 64N and a peak at 8N. Meanwhile, the expression of TPO receptor c-Mpl was lower in pp65+ MKs from HCMV DNAemia patients (0.77±0.38% in pp65+ MKs from HCMV DNAemia patients, 1.75±0.40% in pp65- MKs from HCMV DNAemia patients, 1.97±0.67% in MKs from HCMV-negative patients, and 2.06±0.29% in MKs from healthy controls, p&lt;0.01) while the TPO level in serum was increased compared with that in controls. Next, we established an in vitro HCMV infection model of CD34+-derived MKs with serum from HCMV DNAemia patients, and the laboratory HCMV strain Towne was used as a positive control. After 9 days of coculturing, the viral capsids of HCMV were observed in the nuclei of MKs (Figure 1A), and HCMV infection increased the apoptosis of MKs and shifted them to low ploidy, with a significant decrease in platelet release. As with the in vivo results, c-Mpl was downregulated in HCMV-infected MKs. The expression levels of PDGFR, TLR2 and αvβ3 on MKs were increased in coculture with HCMV DNAemia serum, and pp65-positive MKs were decreased compared with the control after treatment with inhibitors of PDGFR and αvβ3 (Figure 1B). However, neither Gleevec nor anti-TLR2 altered the HCMV infection rate. Conclusions Our study showed that HCMV could impair megakaryopoiesis throughout maturation, apoptosis, and platelet generation via the TPO/c-Mpl pathway both in vivo and in vitro. MKs with PDGFR+ and αvβ3+ phenotypes are susceptible to HCMV infection and we proposed PDGFR and αvβ3 inhibitors as potential therapeutic alternatives for allo-HSCT patients with HCMV infection. Disclosures No relevant conflicts of interest to declare.

scholarly journals The MEK/ERK Inhibitor Trametinib Reduces Fibrosis in a Transduction-Transplantation Model of Mutated Calreticulin

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 635-635 ◽  
Author(s):  
Thanh Kim Nguyen ◽  
Prasanthi Tata ◽  
Stefan Brooks ◽  
Nilamani Jena ◽  
Sarah J Morse ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Colony Formation ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Average Score ◽  
Spleen Weight ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
The Impact ◽  
Transplantation Model

Abstract Insertion or deletion mutations in calreticulin (CALR) are present in the majority of JAK2V617F-negative MPN patients. We utilized a murine retroviral transduction-transplantation model to express the 52bp CALR deletion mutation (CALRDEL) in both BALB/c and C57B/6 backgrounds. As described previously (Marty et al., Blood 2016;127:1317), recipients of CALRDEL-transduced marrow developed persistent thrombocytosis without leukocytosis or erythrocytosis by two months post-transplant. Mice were euthanized at six and nine months post-transplant to evaluate the tempo of disease progression. At six months CALRDEL mice had impressive expansion of megakaryocytes expressing the CALRDEL mutant protein in the bone marrow (BM) without fibrosis or significant splenomegaly. By nine months BM fibrosis and splenomegaly were present. Both whole BM and spleen cells were able to serially transplant the MPN phenotype into secondary recipients. When cultured in collagen-based media supplemented with thrombopoietin, CALRDEL BM cells produced an increased number of megakaryocyte colonies as compared to empty vector. The increased colony formation potential of CALRDEL bone marrow cells was limited to megakaryocytes, we found no increase in colony formation from CALRDEL hematopoietic stem and progenitor cells in methylcellulose with cytokines supporting erythroid and GM colony formation. However, CALRDEL enhanced the serial replating ability of LKS (lineageneg, c-kit+ Sca-1+) cells. Both pSTAT5 and pERK were increased in whole spleen lysates from CALRDEL mice as compared to wild-type BALB/c mice. Therefore, we tested the impact of ruxolitinib, a JAK1/2 inhibitor, and trametinib, a MAPK/ERK inhibitor, on the MPN phenotype of CALRDEL mice. At six months post-transplant mice were treated with either ruxolitinib (90mg/kg PO BID), trametinib (3mg/kg PO daily), or vehicle for 40 days. Ruxolitinib reduced pSTAT5 but caused a paradoxical increase in pERK in whole spleen lysates, while trametinib reduced pERK but not pSTAT5. Trametinib caused a transient increase in platelets and white cells. In spite of pharmacodynamic evidence of effective dosing, ruxolitinib had no significant effect on platelet or leukocyte count but did reduce hemoglobin slightly. Both ruxolitinib and trametinib reduced spleen weight. Ruxolitinib reduced the fraction of the mutant CALRDEL allele (inferred from percentage of GFP+ cells) in the spleen but not the bone marrow, while trametinib had no impact on disease allele burden in any organ. Neither ruxolitinib nor trametinib reduced the expansion of megakaryocytes in the bone marrow but trametinib significantly reduced marrow fibrosis (average score MF-2.5 for vehicle, MF-1.75 for ruxolitinib, MF-1 for trametinib). To assess the role of STAT5 in the pathogenesis of the ET-like MPN induced by the CALRDEL mutant, we transduced BM from syngeneic Balb/c donors carrying a floxed Stat5ab allele in combination with a Stat5ab null allele (Mx-Cre;Stat5abfl/-; Walz et al., Blood 2012;119:3550). Haploinsufficiency for Stat5ab significantly delayed the development of ET-like MPN and attenuated thrombocytosis, implicating JAK2-STAT5 signaling directly in the pathogenesis of this disease. In summary, this CALRDELmouse model results in an MPN phenotype resembling essential thrombocythemia followed by myelofibrosis. CALRDELresults in expansion of megakaryocytes and platelets without expansion of other myeloid cell types. Both pSTAT5 and pERK are increased in our CALRDEL model and pharmacologic inhibition of pERK results in reduction of fibrosis without reducing megakaryocytes. These studies implicate pERK as a potential anti-fibrosis therapeutic target in MPN. Disclosures No relevant conflicts of interest to declare.

scholarly journals Busulfan-Cyclophosphamide Worsens Peripheral Gvhd While Fludarabine-Busulfan Results in Wide Bone Marrow Involvement in a Murine Model of Acute Gvhd

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5791-5791
Author(s):  
Xin He ◽  
Xiaojun Xu ◽  
Yongbin Ye ◽  
Qifa Liu
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Bone Marrow Involvement ◽  
Conditioning Regimen ◽  
Clinical Manifestations ◽  
Major Complication ◽  
Clinical Situation ◽  
Hematopoietic Stem ◽  
The Impact

Abstract Acute graft-versus-host disease (aGVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT) and A proper conditioning regimen is vital to stop the development of aGVHD. To provide a platform for the study of aGVHD and evaluate the impact of different conditioning regimens, we established a murine aGVHD model that simulates the clinical situation and can be conditioned with Busulfan-Cyclophosphamide (BU-Cy) and Fludarabine-BU (Flu-BU). In our study, BALB/c mice were conditioned with BU-Cy or Flu-BU and transplanted with 2×107 bone marrow cells and 2×107splenocytes from either allogeneic (C57BL/6) or syngeneic (BALB/c) donors. The allogeneic recipients conditioned with BU-Cy had shorter survival (P<0.05) and more severe hepatic and intestinal clinical manifestations and pathological changes associated with increased INF-γ expression and diminished IL-4 expression in serum compared to allogeneic recipients conditioned with Flu-BU. Meanwhile increased donor-derived T-cell infiltration and impaired bone marrow B-cell development could be seen in the aGVHD mice conditioned with Flu-BU. Our study showed that the conditioning regimen with BU-Cy resulted in more severe peripheral aGVHD, while the Flu-BU regimen was associated with aGVHD with wide bone marrow involvement. Disclosures No relevant conflicts of interest to declare.

Decitabine Increase Platelet Count by Two Mechanisms.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3507-3507
Author(s):  
Jianhui Wang ◽  
Zongdong Li
Keyword(s):  
Bone Marrow ◽  
Platelet Count ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Current Data ◽  
Dna Demethylation ◽  
Mouse Bone Marrow ◽  
Platelet Release ◽  
Marrow Cells

Abstract Abstract 3507 Poster Board III-444 Thrombocytopenia is frequently associated with the myelodysplastic syndromes (MDS). 5-aza-2'-deoxycytidine (Decitabine) has been used to treat MDS with an approximately 20% response rate in thrombocytopenia. In this study, we have investigated the effect of Decitabine on platelet count in mouse. We report here that enhanced platelet release and maturation of megakaryocyte are two mechanisms involved in Decitabine induced elevation of platelet count. We first noted that a 30% of platelet count increase was found in the Balb/c mice 12 hours after the injection of Dectiabine at a clinically relevant dose (15 mg/m2) suggesting an instant platelet release from spleen or from megkaryocyte of bone marrow. The effect of Decitabine on megakaryocyte maturation was studied in in vitro differentiation of mouse bone marrow cells and megakaryoblastic cell line L8057. Decitabine (2.5 mm) is able to induce L8057 cells to differentiate into a megakaryocyte like polyploidy cells with positive marker of acetylcholinesterase and αIIb integrin. High expression of αIIb integrin was also found in the primary bone marrow cells cultured with both thrombopoietin and Decitabine as compared to thrombopoietin alone. The demethylation-induced transcription of GP6 has been reported in thrombopoietin induced megakaryocyte differentiation. Since Decitabine is a DNA demethylation reagent, we have investigated the GP6 expression in Decitabine treated L8057 cells and have found upregualtion of GP6 expression. Although the role of DNA demethylation in megkaryoctye differentiation still needs to be verified, our current data support that Decitabine is able to drive magakaryocyte maturation. Disclosures: No relevant conflicts of interest to declare.

Diamond-Blackfan Anemia: Erythropoiesis Lost in Ribosome Biosynthesis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. SCI-2-SCI-2
Author(s):  
Stefan Karlsson ◽  
Johan Flygare ◽  
Pekka Jaako ◽  
David Bryder
Keyword(s):  
Bone Marrow ◽  
Platelet Count ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Macrocytic Anemia ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem ◽  
Diamond Blackfan Anemia

Abstract Abstract SCI-2 Diamond-Blackfan anemia (DBA) is a rare congenital erythroid hypoplasia that presents early in infancy. The classic hematologic profile of DBA consists of macrocytic anemia with selective absence of erythroid precursors in a normocellular bone marrow, normal or slightly decreased neutrophil, and variable platelet count. During the course of the disease some patients show decreased bone marrow cellularity that often correlates with neutropenia and thrombocytopenia. DBA is a developmental disease since almost 50% of the patients show a broad spectrum of physical abnormalities. All known DBA disease genes encode for ribosomal proteins that collectively explain the genetic basis for approximately 55% of DBA cases. Twenty-five percent of the patients have mutations in a gene encoding for ribosomal protein S19 (RPS19). All patients are heterozygous with respect to RPS19 mutations suggesting a functional haploinsufficiency of RPS19 as basis for disease pathology. Despite the recent advances in DBA genetics, the pathophysiology of the disease remains elusive. Cellular studies on patients together with successful marrow transplantation have demonstrated the intrinsic nature of the hematopoietic defect. DBA patients have a variable deficit in burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) progenitors. The frequency of immature hematopoietic progenitors in DBA patients is normal but their proliferation is impaired in vitro. Generation of animal models for RPS19-deficient DBA is pivotal to understand the disease mechanisms and to evaluate novel therapies. Several DBA models have been generated in mice or zebrafish. Although these models have provided important insights on DBA, they are limited in a sense that the hematopoietic phenotype and molecular mechanisms are likely to be influenced by the level of RPS19 downregulation. We have generated mouse models for RPS19-deficient DBA by taking advantage of transgenic RNAi. These models are engineered to contain a doxycycline-regulatable RPS19-targeting shRNA, allowing a reversible and dose-dependent downregulation of RPS19 expression. We demonstrate that the RPS19-deficient mice develop a macrocytic anemia together with leukocytopenia and variable platelet count and the severity of the phenotype depends on the level of RPS19 downregulation. We show further that a chronic RPS19 deficiency leads to irreversible exhaustion of hematopoietic stem cells and subsequent bone marrow failure. Overexpression of RPS19 following gene transfer rescues the proliferative and apoptotic phenotype of RPS19-deficient hematopoietic progenitors in vitro, demonstrating that the phenotype is specifically caused by the RPS19 deficiency. Expression analysis of RPS19-deficient hematopoietic progenitors reveals an activation of the p53 pathway. By intercrossing the DBA mice with p53 null mice we demonstrate that inactivation of p53 in vivo results in a variable rescue of the hematopoietic phenotype depending on the level of RPS19 downregulation. Therefore, we conclude that increased activity of p53 plays a major role in causing the DBA phenotype but that other hitherto unidentified pathways also play a role, specifically in patients that have low levels of functional RPS19. Disclosures: No relevant conflicts of interest to declare.

The Ryk Receptor Regulates Hematopoietic Stem and Progenitor Cell Proliferation and Their Sensitivity To Myeloablative Agents

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 796-796
Author(s):  
Benjamin Povinelli ◽  
Michael Nemeth
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Molecular Mechanisms ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Peripheral Blood Cells ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract The molecular mechanisms that control the balance between quiescence and proliferation of hematopoietic stem and progenitor cells (HSPCs) are critical for maintaining life-long hematopoiesis. In a recent study (Povinelli, et al. Stem Cells, In Press, 2013) we demonstrated that the Wnt5a ligand inhibits HSPC proliferation through a functional interaction with a non-canonical Wnt ligand receptor termed Related to Receptor Tyrosine Kinase (Ryk). Expression of Ryk on HSPCs in vivo was associated with a decreased rate of proliferation. Following treatment with fluorouracil (5-FU), the percentage of Ryk+ HSPCs increased at the expense of Ryk-/low HSPCs. Based on these data, we hypothesized that one function of the Ryk receptor is to protect HSPCs from the effects of myeloablative agents. To test this hypothesis, we injected 6-8 week old C57BL/6 mice with 150 mg/kg of 5-FU and analyzed bone marrow 48 hours later for the presence of apoptotic HSPCs, defined as lineage negative (Lin-), Sca-1+, CD48- cells positive for active caspase-3. There was a 2.5-fold decrease in the percentage of apoptotic Ryk+ HSPCs (12.9 ± 1.7%) compared to Ryk-/low HSPCs (32.4 ± 5.3%, p < 0.001, n = 3). To test whether this effect was limited to 5-FU, we performed a similar study in which we irradiated C57BL/6 mice with 3 cGy of total body irradiation (TBI) and analyzed bone marrow 72 hours later for apoptotic HSPCs (for this experiment, defined by a Lin-, c-kit+, Sca-1+, CD150+, CD48- immunophenotype or LSK, SLAM). Comparable to the effects of 5-FU, there was a significant 3.0-fold reduction in the percentage of apoptotic Ryk+ HSPCs (3.1 ± 0.2%) compared to Ryk-/low HSPCs (9.2 ± 1.5%, p < 0. 001, n = 3) in mice receiving 3 cGy TBI. These results demonstrated an association between Ryk expression and survival of HSPCs following myeloablative injury. To determine whether in vivo targeting of the Ryk receptor would increase the sensitivity of HSPCs to myeloablative injury, we utilized a neutralizing rabbit anti-Ryk antibody (α-Ryk). We injected C57BL/6 mice with 5 mg/kg α-Ryk or rabbit IgG isotype for 2 consecutive days. Twenty-four hours after the second dose, we determined the frequency and cell cycle status of LSK SLAM cells. Treatment with α-Ryk significantly increased the percentage of LSK SLAM cells in the S/G2/M phases compared to control (α-Ryk: 17.8 ± 2.2%; isotype IgG: 11.6 ± 2.7%, p < 0.05, n = 3). This was associated with a decrease in the percentage of LSK, SLAM cells in G1 following treatment with α-Ryk (α-Ryk: 40.5 ± 3.2%, isotype IgG: 51.3 ± 2.2; p < 0.01, n = 3). The percentage of G0 LSK SLAM cells was unchanged (α-Ryk: 37.9 ± 2.6, isotype IgG: 35.7 ± 3.1% n = 3) indicating that inhibiting Ryk promoted the exit of LSK SLAM cells from G1. Treatment with α-Ryk also increased the percentage of whole bone marrow cells expressing the LSK SLAM phenotype by 1.4-fold compared to controls (p < 0.05, n = 3). To determine if α-Ryk treatment altered HSPC function, we transplanted whole bone marrow cells from C57BL/6 mice treated with two days of α-Ryk or isotype IgG at a 1:1 ratio with whole bone marrow from untreated Ubc-GFP transgenic mice into lethally irradiated B6.SJL mice. Four weeks after transplant, we analyzed peripheral blood cells for the percentage of CD45.2+ GFP- cells. There was no difference in engraftment by transplanted bone marrow cells from mice treated with α-Ryk or isotype IgG (α-Ryk: 61.6 ± 6.1% n = 4, isotype IgG: 52.8 ± 13.6%, n = 5), indicating that the neutralizing antibody does not inhibit short-term HSPC function on its own. We then tested whether blocking Ryk function resulted in greater sensitivity of HSPCs to 5-FU. We treated B6.SJL mice with 5 mg/kg α-Ryk or isotype IgG for 2 consecutive days, followed by 150 mg/kg of 5-FU. Forty-eight hours after 5-FU treatment, we transplanted 2x106 C57BL/6 whole bone marrow cells into treated B6.SJL mice without additional conditioning. Four weeks after transplant, we determined the percentage of donor-derived CD45.2+ peripheral blood cells. Treatment of recipient mice with α-Ryk prior to 5-FU treatment resulted in increased engraftment of donor bone marrow by 3.6-fold compared to isotype (p < 0.05, n = 5), suggesting that inhibition of Ryk resulted in increased elimination of host HSPCs by 5-FU. Collectively, these data suggest a model in which inhibition of the Ryk receptor results in increased proliferation of HSPCs, rendering them more sensitive to the effects of myeloablative agents such as chemotherapy or TBI. Disclosures: No relevant conflicts of interest to declare.

Ang1 Functions as An Autocrine Activating Factor of Tie2 Signaling In Hematopoietic Stem Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1548-1548
Author(s):  
Haruka Momose ◽  
Kazuya Takizawa ◽  
Madoka Kuramitsu ◽  
Takuo Mizukami ◽  
Atsuko Masumi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Molecular Mechanisms ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Lentiviral Vector ◽  
Down Regulation ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Abstract 1548 Hematopoietic stem cells (HSCs) are clonogenic cells that possess the self-renewal capacity to produce more HSCs, as well as the multilineage potential that gives rise to a defined set of mature differentiated progeny for maintenance or repair of the whole blood system. HSCs lie in the hematopoietic niches located along the inner surface of the bone or the sinusoidal endothelium, and are in contact with niche cells. The cell-cell interactions with niche cells are believed to be an important prerequisite to trigger signaling events in HSCs, thereby controlling the balance between HSC self-renewal and differentiation. However, the precise molecular mechanisms regulating niche cell-HSC interactions are not well understood. One of the key molecules for those interactions is Angiopoietin-1 (Ang1). Ang1 is expressed by the niche cells and has been identified as an activating ligand for Tie2 (tyrosine kinase with Ig-like loops and epidermal growth factor homology domains 2). The expression of Tie2 is dominant in HSCs, and Tie2 in HSCs is supposed to be stimulated by Ang1 derived from niche cells. However, Ang1 is also expressed in HSCs. Detailed analysis has shown that Ang1 expression was found to be restricted in long-term HSCs (CD34-lineage-Sca-1+c-Kit+), indicating that Ang1 derived from HSCs plays a role in regulating HSCs. We attempted to elucidate a novel regulating system for HSCs through Ang1-Tie2 signaling by utilizing a hematopoietic cell line in which Tie2 was stably expressed (Ba/F3-Tie2). In Ba/F3-Tie2 cells, Tie2 was found to be phosphorylated on tyrosine residues, even without exogenous addition of Ang1. In the same cells, the expression level of endogenous Ang1 was increased four-fold. When Ang1 expression was down-regulated by transduction with a lentiviral vector expressing short hairpin RNA (shRNA) for Ang1 (shAng1), the phosphorylation of Tie2 was suppressed, suggesting that Tie2 expressed in Ba/F3-Tie2 cells could be stimulated by endogenous Ang1. To mimic the physiological circumstances of the bone marrow, Ba/F3-Tie2 cells were cultured on OP9 stromal cells. Under these culture conditions, the effect of endogenous Ang1 was investigated. Down-regulation of Ang1 by shAng1 demonstrated an approximate 50% reduction in the proliferation of Ba/F3-Tie2 cells on the OP9 cell layer. A HSC-rich population of cells prepared from bone marrow (lineage-Sca-1+c-Kit+; LSK) was also analyzed on OP9 cell layers. Similar to the results obtained from the analysis of Ba/F3-Tie2 cells, down-regulation of Ang1 by shAng1 resulted in an approximately 70% decrease in the proliferation of LSK cells cultured on OP9 monolayers. We confirmed that the suppressive effect on HSC proliferation was due to the lack of Ang1 from HSCs by culturing on Ang1-defective OP9 cells. Finally, we performed in vivo analysis to confirm the importance of endogenous Ang1 to HSCs. Ly5.2 LSK cells transduced with the shAng1 expressing vector were transplanted along with Ly5.1xLy5.2 bone marrow cells into lethally irradiated Ly5.1 mice. The Ly5.2 donor-derived cells in the recipient's peripheral blood were monitored every 2 weeks. As expected, shAng1-introduced donor cells were at decreased ratios at week four (mean ratios, 31.5% for control vs. 17.5% for shAng1), and were reduced to an even lower level at week 12 (mean ratios, 27.1% for control vs. 6.79% for shAng1). This phenomenon was also confirmed by histochemical results, where statistically fewer HSCs existed in the bone marrow of recipient mice in which shAng1-introduced HSCs were transplanted, as compared to the control. Altogether, our data suggested that Tie2 in HSCs could be stimulated by the Ang1 produced by the surrounding HSCs, and this possible autocrine regulation might control the functions of HSCs. Disclosures: No relevant conflicts of interest to declare.

Regulation of Myelopoiesis by Differentiation Stage-Specific Activities of HOXA9 and HOXA10

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. SCI-29-SCI-29
Author(s):  
Elizabeth A. Eklund
Keyword(s):  
Bone Marrow ◽  
Transcription Factors ◽  
Gene Networks ◽  
Target Gene ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Murine Bone Marrow

Abstract Abstract SCI-29 HOXA9 and HOXA10 are homeodomain (HD) transcription factors that are implicated in control of myelopoiesis and contribute to myeloid leukemogenesis. These proteins are expressed coordinately during hematopoiesis, with maximal expression in granulocyte/monocyte progenitor (GMP) cells. Engineered overexpression of Hoxa9 or Hoxa10 in primary bone marrow cells expands the GMP population in vitro, and results in myeloproliferation in murine bone marrow transplant experiments. Mice transplanted with Hoxa9- or Hoxa10-overexpressing bone marrow develop acute myeloid leukemia (AML) over time. Consistent with this, increased and sustained expression of a set of HD proteins, including HOXA9 and HOXA10, is found in a subset of human AML, including AML with MLL gene translocations (11q23-AML). Since the DNA-binding HDs of HOXA9 and HOXA10 are highly conserved, we hypothesize that they recognize a common set of target genes. However, since HOXA9 and HOXA10 diverge outside the HD, we considered the unexplored possibility that they perform different functions in regulating such genes. To identify molecular mechanisms for HOX-induced GMP expansion and leukemogenesis, we performed a chromatin immunoprecipitation-based screen for HOXA10 target genes. Gene ontology studies determined that the identified set is enriched for genes encoding growth factors and receptors, including fibroblast growth factor 2 (FGF2). We found that production of FGF2 by Hoxa10-overexpressing GMP stabilizes β-catenin and induces proliferation in an autocrine manner. We also found that HOXA9 and HOXA10 activate common FGF2 cis elements. The Hoxa10-target-gene set is also enriched for HD-transcription factors, including CDX4. We determined that Cdx4 transcription is activated by HOXA10 in GMP, but repressed by HOXA9 in differentiating myeloid cells. CDX4 activates transcription of both Hoxa9 and Hoxa10, identifying a HOX-CDX cross-regulatory mechanism. This mechanism may be influenced by Fgf2, since Hoxa10 and Cdx4 are β-catenin target genes, but β-catenin activity decreases Hoxa9 expression. Gene expression profiling studies indicate that HOXA9, HOXA10, CDX4, and FGF2 are increased in 11q23-AML, suggesting clinical relevance. Arih2 (encoding the E3 ligase Triad1) is another common HOXA9 and HOXA10 target gene that may influence Fgf2 activity. We found that Arih2 transcription is repressed by HOXA9 in myeloid progenitors, but activated by HOXA10 in differentiating phagocytes. FGF receptors are destabilized by ubiquitination, and we found increased FGF-R ubiquitination in Hoxa10-overexpressing cells. Therefore, Triad1-dependent regulation of FGF-R stability is another mechanism for control of FGF2 activity and myeloproliferation by HOXA9 and HOXA10. Therefore, HOXA9 and HOXA10 regulate a common set of target genes that control GMP expansion in a manner that is antagonistic for some genes and cooperative for others. Clinical correlative studies suggest that coordinate control of these genes by HOXA9 and HOXA10 is dysregulated in HOX-overexpressing leukemia. Understanding HOX-regulated gene networks may identify therapeutic targets for HOX-overexpressing leukemias. For example, blocking FGF-related signaling pathways may ameliorate cytokine hypersensitivity in such leukemias, and would be a topic of interest for additional studies. Disclosures: No relevant conflicts of interest to declare.

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