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.

Potent Cooperation Between NUP98-NSD1 and FLT3-ITD In AML Induction

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3797-3797
Author(s):  
Angeliki Thanasopoulou ◽  
Alexandar Tzankov ◽  
Juerg Schwaller
Keyword(s):  
Bone Marrow ◽  
Fusion Protein ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Latency Period ◽  
Histopathological Analysis ◽  
Murine Bone Marrow ◽  
Marrow Cells

Abstract The NUP98-NSD1 fusion protein, product of the t(5;11)(q35;p15.5) chromosomal translocation, is an AML-associated cytogenetically silent genetic aberration, recently identified as the most frequent fusion in pediatric AML, generally associated with aggressive disease and poor prognosis. Interestingly, the vast majority (>70%) of the reported NUP98-NSD1-positive cases also carried an activating FLT3-ITD mutation suggesting functional cooperation. The purpose of this study was to search for experimental evidence of a functional cooperation between NUP98-NSD1 and FLT3-ITD in the transformation of murine hematopoietic cells in vitro and in vivo. Lineage surface marker-depleted murine bone marrow cells were transduced with either pMSCV-NUP98-NSD1-neo or pMSCV-FLT3-ITD-GFP or both expression constructs on fibronectin-coated plates. Serial colony formation assays in myeloid favoring medium and immunophenotypic analysis by flow cytometry indicated that retroviral expression of NUP98-NSD1 provided increased self-renewal capacity and impaired differentiation of murine bone marrow stem and progenitor cells. NUP98–NSD1 expressing cells displayed a typical myeloblastic morphology and co-expressed myeloid and early stem cell surface markers (CD34low/c-kit+/FcgR+/Gr-1+/ Mac-I+/B220-). Co-expression of FLT3-ITD resulted in high rates of cell proliferation, showed a more differentiated phenotype and concomitantly impaired the in vitro clonogenic capacity in methylcellulose cultures. Bone marrow cells expressing NUP98-NSD1 with or without FLT3-ITD were harvested from methylcellulose cultures and transplanted into sub-lethally irradiated syngeneic mice. All mice receiving cells co-expressing NUP98-NSD1 and FLT3-ITD developed AML that was transplantable into all secondary recipients. Myeloid leukemic blasts that co-expressed NUP98-NSD1 and FLT3-ITD were present in abundance both in BM preparations and in blood smears, and histopathological analysis showed widespread infiltration into solid organs. By contrast, no AML ever developed in mice receiving cells expressing only NUP98-NSD1. These mice, similar to mice receiving cells expressing FLT3-ITD only, developed signs of a chronic myeloproliferative disorder, characterized by expansion of Mac-1+/Gr-1+ BM cells with granulocytic/monocytic differentiation that in some cases caused severe distress after a latency period of more than one year. Intriguingly, upon injection with double transduced NUP98-NSD1 and FLT3-ITD progenitors rather different latency periods of the AML development were observed between different experiments. Interestingly, the latency periods could be correlated to the ratio of expression levels of FLT3-ITD to wildtype FLT3, with higher FLT3-ITD levels associated with a shorter latency. To further investigate the significance of aberrant FLT3 signaling, in vitro and in vivo transformed NUP98-NSD1 and NUP98-NSD1/FLT3-ITD cells were treated with a selective FLT3 tyrosine kinase inhibitor (PKC412). The higher sensitivity of cells co-expressing NUP98-NSD1 and FLT3-ITD to PKC412, compared to cells expressing NUP98-NSD1 only, indicated that proliferation and survival were dependent on FLT3-derived signals. Taken together, these observations demonstrate a potent cooperation between NUP98-NSD1 fusion and FLT3-ITD in leukemic transformation. However, neither the NUP98-NSD1 fusion protein nor the FLT3-ITD mutation alone was sufficient to induce AML. Moreover, the high sensitivity of NUP98-NSD1 and FLT3-ITD co-expressing leukemic blasts to FLT3 signaling inhibition suggests a possible therapeutic strategy to be further explored in this AML subgroup. Disclosures: No relevant conflicts of interest to declare.

In Vitro Generation Of Osteoclasts From Interleukin-3 (IL-3)-Dependent Mouse Hematopoietic Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4842-4842
Author(s):  
Shunqing Wang ◽  
Huixian Hong
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Control Culture ◽  
Significant Loss ◽  
Murine Bone Marrow ◽  
New Strategy ◽  
Differentiation And Proliferation ◽  
Marrow Cells

Abstract Objective This work aims to develop a new strategy to generate murine osteoclasts in vitro using IL-3-dependent cells prepared by 6-day IL-3 treatment of murine bone marrow cells Methods 1. Here, we describe an alternative method for in vitro generation of osteoclasts, which involves the use of interleukin (IL)-3-dependent murine bone marrow cells. Bone marrow cells, isolated from 6- to 8-week old C57BL/6 were cultured in α-MEM containing 10% FBS in tissue culture dishes overnight to remove stromal cells. Then, non-adherent bone marrow cells were harvested and continued in α-MEM containing 10% FBS without (control) or with IL-3 (1 ng/ml) for 6 days. While no cells survived in the control culture after the 6-day culturing, the IL-3-treated culture gave rise to a significant number of surviving cells. These IL-3-depedent cells were capable of differentiating to osteoclasts in response to M-CSF and RANKL stimulation. Moreover, these IL-3-dependent cells can be further expanded by plating them in non-treated plastic dishes followed with M-CSF treatment; they continued to survive and proliferate in non-treated plastic dishes in the presence of M-CSF for up to 4 days. After 4-day M-CSF treatment, these cells can be lifted by EDTA, and they were still able to differentiate into osteoclasts upon subsequent stimulation of M-CSF and RANKL. 2. We performed the in vitro bone resorption assay, Semiquantitative Reverse Transcription (RT)-PCR, Western Analysis, Infection of Murine Bone Marrow Cells (BMCs) to test whether the osteoclasts generated from IL-3-dependent murine bone marrow cells are different from the osteoclasts generated from traditional method. Results 1. IL-3 can maintain the survival of murine bone marrow cells for up to 6 days and these cells still keep their capacity to generate osteoclasts. The capacity of IL-3-dependent cells to form osteoclasts decreases with time of IL-3 treatment and IL-3 dependent cells can be further expanded by M-CSF without significant loss of the osteoclastogenic potential. 2. IL-3-dependent cells can form functional osteoclasts. RANKL induces the expression of osteoclast genes in IL-3-dependent cells. RANKL activates some of RANK signaling pathways in IL-3-dependent cells. Importantly, we found that IL-3 dependent murine bone marrow cells can be infected by retrovirus encoding GFP. Conclusions 1) We have developed a new strategy to generate murine osteoclasts in vitro using IL-3-dependent cells prepared by 6-day IL-3 treatment of murine bone marrow cells. 2) IL-3-dependent cells can be infected by retrovirus, permitting further experimental manipulations to express or knock down genes in IL-3-dependent cells for studying the molecular mechanism controlling differentiation and proliferation of osteoclast precursors or delineating molecular events in early osteoclastogenesis. Disclosures: No relevant conflicts of interest to declare.

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.

Prospective Isolation Of Human Erythroid Lineage-Committed Progenitors

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3418-3418
Author(s):  
Yasuo Mori ◽  
Jun Seita ◽  
John V. Pluvinage ◽  
James Y. Chen ◽  
Irving L. Weissman
Keyword(s):  
Bone Marrow ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Marrow Cell ◽  
Significant Loss ◽  
Refractory Anemia ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow

Abstract Anemia is one of the findings most frequently seen in patients with myelodysplastic syndrome (MDS); however, in our hands, significant loss of megakaryocyte/erythrocyte progenitors (MEP) is not observed among these patients (PNAS, 2013, 110(8):3011-6). Unipotent erythroid-committed progenitors (EPs), estimated to exist downstream of bipotent MEPs, may be involved in the pathogenesis of refractory anemia among MDS patients. Previously, we identified mouse (m) EPs (mEPs) in bone marrow. The expression of endoglin (CD105) was a key marker to isolate mEPs: mEPs were the Lineage(lin)-Sca-1-c-kit+CD16/32-CD150+CD105+CD41- population in murine bone marrow (Cell Stem Cell, 2007, 1(4):428-42). Here we show that the human (h) counterpart (hEPs) is prospectively isolatable in human bone marrow. We analyzed the expression of hCD105 in addition to hCD71 (known to be an early erythroid marker) in human stem and progenitor populations, and found a fraction (32.9±13.6%, n = 5) of common myeloid progenitors (CMPs; lin-CD34+CD38+CD45RA-IL-3Ra+) and a major part (85.1±5.5%) of MEPs (lin-CD34+CD38+CD45RA-IL-3Ra-) expressed hCD71. A portion of CD71+ MEPs co-expressed CD105 (CD105+MEPs; 32.3±6.5% of MEPs). Neither hCD105 nor hCD71 was detectable in hematopoietic stem cells (HSCs; lin-CD34+CD38-CD45RA-), common lymphoid progenitors (CLPs; lin-CD34+CD38+CD10+), or granulocyte/monocyte progenitors (GMPs; lin-CD34+CD38+CD45RA+IL-3Ra+) by FACS. In vitro, CD71+ cells within the CMP fraction showed differentiation potential skewed toward MegE lineage, representing the transitional stage to MEPs. CD71+MEPs mostly generated BFU-E, although they still retained some MegK potential, while output of CD105+MEPs was completely restricted to erythroid lineage, and colonies from this fraction contained a small number of mature (enucleated) erythrocytes (CFU-E type). Thus we termed CD71+MEPs and CD105+MEPs as human erythroid-biased MEPs (E-MEPs) and hEPs, respectively. In short-term liquid culture, hE-MEPs gave rise to hEPs in the presence of SCF, TPO and EPO, whereas hEPs could not generate hE-MEPs, suggesting a hierarchical progression from E-MEPs to EPs. These newly classified populations might be a very useful tool for understanding the molecular mechanisms of human erythroid development, and should be analyzed in patients with erythroid-related disorders (e.g., MDS, polycythemia vera, or aplastic anemia). Disclosures: No relevant conflicts of interest to declare.

scholarly journals Differential Contribution of N- and C-Terminal Regions of HIF1α and HIF2α to Their Target Gene Selectivity

2020 ◽  
Vol 21 (24) ◽  
pp. 9401
Author(s):  
Antonio Bouthelier ◽  
Florinda Meléndez-Rodríguez ◽  
Andrés A. Urrutia ◽  
Julián Aragonés
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Cellular Response ◽  
Target Gene ◽  
Target Genes ◽  
Transactivation Domain ◽  
Transactivation Domains ◽  
Relative Contribution

Cellular response to hypoxia is controlled by the hypoxia-inducible transcription factors HIF1α and HIF2α. Some genes are preferentially induced by HIF1α or HIF2α, as has been explored in some cell models and for particular sets of genes. Here we have extended this analysis to other HIF-dependent genes using in vitro WT8 renal carcinoma cells and in vivo conditional Vhl-deficient mice models. Moreover, we generated chimeric HIF1/2 transcription factors to study the contribution of the HIF1α and HIF2α DNA binding/heterodimerization and transactivation domains to HIF target specificity. We show that the induction of HIF1α-dependent genes in WT8 cells, such as CAIX (CAR9) and BNIP3, requires both halves of HIF, whereas the HIF2α transactivation domain is more relevant for the induction of HIF2 target genes like the amino acid carrier SLC7A5. The HIF selectivity for some genes in WT8 cells is conserved in Vhl-deficient lung and liver tissue, whereas other genes like Glut1 (Slc2a1) behave distinctly in these tissues. Therefore the relative contribution of the DNA binding/heterodimerization and transactivation domains for HIF target selectivity can be different when comparing HIF1α or HIF2α isoforms, and that HIF target gene specificity is conserved in human and mouse cells for some of the genes analyzed.

Comparison of the transport of chlorozotocin and CCNU in L1210 leukemia and murine bone marrow cells in vitro

1983 ◽  
Vol 11 (3) ◽  
Author(s):  
Philip Lazarus ◽  
JudithSt Germina ◽  
Maurice Dufour ◽  
Greg Palmer ◽  
Deborah Wallace ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
L1210 Leukemia ◽  
Murine Bone Marrow ◽  
Marrow Cells

Myeloproliferative Disease or Myeloid Leukemia Caused by Cbl Mutants in a Mouse Transplantation Model.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3973-3973
Author(s):  
◽  
Srinivasa Rao Bandi ◽  
Marion Rensinghoff ◽  
Rebekka Grundler ◽  
Lara Tickenbrock ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myeloid Leukemia ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Myeloid Cells ◽  
Myeloproliferative Disease ◽  
Murine Bone Marrow ◽  
Hematologic Disorder ◽  
Almost All ◽  
Marrow Cells

Abstract Abstract 3973 Poster Board III-909 Purpose Somatic mutations of Kit have been found in leukemias and gastrointestinal stromal tumors. The proto-oncogene c-Cbl negatively regulates Kit and Flt3 by its E3 ligase activity and acts as a scaffold for several signaling adaptor molecules. We recently identified the first c-Cbl mutation in human disease in an AML patient, called Cbl-R420Q. Results We transduced primary murine bone marrow retrovirally with c-Cbl mutants and transplanted it into lethally irradiated mice. Almost all recipients of bone marrow cells transduced with Cbl mutants developed a lethal hematologic disorder with a mean latency of 341 days in the Cbl-R420Q group and 395 days in the Cbl-70Z group. Eleven out of 13 mice and 8 out of 11 mice died in the Cbl-R420Q group and Cbl-70Z group, respectively. Two animals succumbed to a myeloid leukemia, the other mice developed a myeloproliferative disease. The leukemic mice showed a leukocytosis of up to 140.000/μL. They developed a splenomegaly with massive expansion of myeloid cells in liver and spleen. Histology sections of spleen, liver and bone marrow and FACS analyses of spleen, bone marrow and peripheral blood showed extensive infiltration of myeloid cells. Conclusion Thus, transplantation of bone marrow cells expressing Cbl mutants leads to a myeloid leukemia or to a myeloproliferative disease with long latency and high penetrance. Disclosures: No relevant conflicts of interest to declare.

Functional Role of BAALC In Leukemogenesis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4194-4194
Author(s):  
Tobias Berg ◽  
Michael Heuser ◽  
Florian Kuchenbauer ◽  
Gyeongsin Park ◽  
Stephen Fung ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Myeloid Differentiation ◽  
Self Renewal ◽  
Murine Bone Marrow ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Marrow Cells

Abstract Abstract 4194 Cytogenetically normal acute myeloid leukemia (CN-AML) patients with high BAALC or MN1 expression have a poor prognosis. Whereas the oncogenic function of MN1 is well established, the functional role of BAALC in hematopoiesis is not known. We therefore compared the expression of BAALC and MN1 in 140 CN-AML patients by quantitative PCR. To further assess the impact of BAALC on leukemogenesis we used retroviral gene transfer into primary murine bone marrow cells and cells immortalized with NUP98-HOXD13 (ND13) and HOXA9. Transduced cells were assessed in vitro by colony forming assays and for their sensitivity to treatment with all-trans retinoic acid (ATRA). They were also evaluated by in vivo transplantation into lethally-irradiated mice. In the 140 CN-AML patients analyzed, the expression of BAALC and MN1 was highly correlated (R=0.71). Retroviral overexpression of MN1 or BAALC in the Hox gene-immortalized bone marrow cells did not cause upregulation of the other gene, suggesting that these genes do not regulate each other. In murine bone marrow cells BAALC did not immortalize the cells in vitro as assessed by serial replating of transduced cells in methylcellulose assays. Transplantation of transduced cells resulted in negligible engraftment of approximately 1 percent at 4 weeks after transplantation. However, co-transduction of BAALC into NUP98-HOXD13 cells (which are very sensitive to the treatment with all-trans retinoic acid) increased the 50 percent inhibitory concentration (IC50) of ATRA by 4.3-fold, suggesting a negative impact of BAALC on myeloid differentiation. We next evaluated whether the differentiation inhibiting effects of BAALC may cooperate with the self renewal-promoting effects of HOXA9 to induce leukemia in mice. Mice receiving transplants of murine bone marrow cells transduced with BAALC and HOXA9 developed myeloid leukemias with a median latency of 139.5 days that were characterized by leukocytosis, massively enlarged spleens (up to 1.02 g), anemia and thrombocytopenia. Infiltrations of myeloid cells were also found in liver, spleen, and kidney. The disease was transplantable into secondary animals. By Southern blot analysis we found one to two BAALC viral integrations per mouse, suggesting that clonal disease had developed from BAALC-transduced cells. We demonstrate for the first time that BAALC blocks myeloid differentiation and promotes leukemogenesis when combined with the self-renewal promoting oncogene HOXA9. Due to its prognostic and functional effects BAALC may become a valuable therapeutic target in leukemia patients. Disclosures: No relevant conflicts of interest to declare.

The bHLH Transcription Factors SCL and LYL1 Awaken Hematopoietic Stem Cells by Repression of p21

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 214-214
Author(s):  
David J. Curtis ◽  
Nhu-Y Nguyen ◽  
Jessica Salmon
Keyword(s):  
Cell Cycle ◽  
Transcription Factors ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Short Term ◽  
Hematopoietic Stem ◽  
Bhlh Factors ◽  
Mild Defect ◽  
Bhlh Transcription Factors

Abstract Abstract 214 The basic helix-loop-helix (bHLH) transcription factors SCL (TAL1) and LYL1 are regulators of adult hematopoietic stem cell (HSC) activity with significant functional redundancy: HSCs lacking SCL (SCLδ/δ) have a mild defect in short-term repopulating activity whilst HSCs lacking LYL1 (LYL1−/−) have normal repopulating activity. In contrast, we have shown previously that HSCs lacking both SCL and LYL1 (DKO) are unable to grow in vitro and have no in vivo repopulating activity. Phenotypic and expression analyses of SCLδ/δ, LYL1−/− and DKO mice were performed to determine how bHLH factors regulate HSC activity. Consistent with the short-term repopulating defects of SCLδ/δ HSC, Lineage negative Sca-1+ c-Kit+ (LSK) bone marrow cells from SCLδ/δ mice had reduced in vitro replating activity associated with increased quiescence – 90% in G0 compared with 70% in normal LSK. Increased quiescence was associated with delayed hematopoietic recovery following treatment of mice with 5-Fluorouracil. Consistent with the increased quiescence, expression of the cell cycle inhibitor, Cdkn1a (p21) was increased three-fold in SCLδ/δ and LYL1−/− LSK. Moreover, p21 levels in LSK isolated from DKO mice were increased 50-fold. To determine the functional relevance of the elevated levels of p21 in DKO HSCs, we generated DKO mice on a p21-deficient (p21−/−) background. Remarkably, loss of p21 rescued in vitro cell growth of DKO progenitors. More importantly, primary and secondary competitive repopulation assays demonstrated multi-lineage repopulating activity of p21−/− DKO HSCs. These results suggest the bHLH factors SCL and LYL1 function as repressors of p21, allowing HSCs to enter cell cycle during stress hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

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