RUNX1-ETO-Dependent Transcriptional Repression of RASSF2 Contributes to t(8;21) Leukemia through Evasion of MST1-Driven Apoptosis Signaling

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1547-1547
Author(s):  
Samuel A. Stoner ◽  
Elizabeth T. Andrews ◽  
Russell Dekelver ◽  
Stephanie Weng ◽  
Miao-Chia Lo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Protein Stability ◽  
Cell Lines ◽  
Transcriptional Repression ◽  
Target Genes ◽  
Mouse Bone Marrow ◽  
Leukemic Transformation ◽  
Self Renewal ◽  
Regulatory Regions ◽  
Primary Mouse

Abstract The t(8;21) chromosomal translocation is among the most frequent recurring cytogenetic abnormalities associated with acute myeloid leukemia (AML), found in 8-12% of de novo AML patients. The t(8;21) results in the stable fusion of the RUNX1 and RUNX1T1 genes, and formation of the oncofusion protein RUNX1-ETO (AML1-ETO). RUNX1-ETO is composed of the N-terminal DNA-binding domain of RUNX1 and nearly the entire ETO protein. RUNX1-ETO promotes leukemia development via the recruitment of transcription factor/transcriptional repression complexes (including NCOR, HDACs, p300, etc.) to regulatory regions of RUNX1 target genes known to be critical for myeloid differentiation and function, such as CEBPA, SPI1 (PU.1), NFE2, and CSF1R. Despite this knowledge, additional RUNX1-ETO target genes remain poorly characterized, and the complete molecular mechanism through which RUNX1-ETO leads to leukemic transformation remains to be elucidated. We propose that a better understanding of additional RUNX1-ETO target genes will lead to the potential for development of novel therapeutics to treat these patients. One such gene that we initially identified as markedly downregulated in RUNX1-ETO leukemia cells using a mouse model of t(8;21) AML is RASSF2 (Lo et al, Blood, 2012). Assessment of publicly available gene expression data revealed that RASSF2 is specifically downregulated in the bone marrow of t(8;21) AML patients compared to patients of different cytogenetic subtypes or to non-t(8;21) FAB subtype M2 AML patients. Additionally, RT-qPCR analysis confirmed that RASSF2 transcript is downregulated 10-100-fold in the t(8;21) AML cell lines, Kasumi-1 and SKNO-1, compared to non-t(8;21) AML cell lines and normal CD34+ hematopoietic cells. Expression of RUNX1-ETO in a non-t(8;21) AML cell line led to a reduction in RASSF2 mRNA expression, while knockdown of RUNX1-ETO in Kasumi-1 cells resulted in a ~5-fold increase in RASSF2 expression. Assessment of published ChIP-seq data showed that RUNX1-ETO directly binds at two regulatory regions within the RASSF2 genomic locus in t(8;21) AML cell lines and patient samples. Re-expression of RASSF2 at physiological levels in t(8;21) AML cell lines resulted in a modest negative growth phenotype, and greatly sensitized these cells to apoptosis following stimulation with various pro-apoptotic agents. Re-expression of RASSF2 in RUNX1-ETO-transduced primary mouse bone marrow caused these cells to lose their long-term self-renewal ability after 3 weeks in a serial replating/colony formation assay. This loss of self-renewal ability in co-transduced cells was accompanied by a marked increase in apoptosis during each of the first three weeks of replating. Mechanistically, re-expression of full-length RASSF2, but not of a deletion mutant lacking the SARAH heterodimerization domain (RASSF2ΔSARAH), in t(8;21) AML cell lines resulted in increased protein amount of the pro-apoptotic kinase, MST1. This suggests that RASSF2 may be a critical regulator of MST1 protein stability in AML cells. Importantly, modest (2-3-fold) overexpression of MST1 in t(8;21) AML cell lines resulted in a significant increase in apoptosis and caused growth arrest. The effects of RASSF2 or MST1 expression in non-t(8;21) AML cell lines were greatly reduced, suggesting that the cellular context of RUNX-ETO-driven leukemias makes them highly susceptible to MST1-dependent apoptosis. Overall, we have identified the importance of a MST1-driven pro-apoptotic signaling axis in t(8;21) leukemia. RUNX1-ETO-dependent transcriptional repression of RASSF2 may be essential for evasion of this apoptosis signaling during leukemic transformation via reduction of MST1 protein stability. MST1, perhaps better known as the mammalian orthologue of the drosophila Hippo kinase, is a critical tumor suppressor in many solid tumor types; and we believe our studies warrant the continued investigation of this pathway in hematological malignancy. Disclosures No relevant conflicts of interest to declare.

WT1 Promotes Myeloid Development and Inhibits Lymphoid Development.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1539-1539
Author(s):  
Aschwin L. Menke ◽  
G.H. J. Knops ◽  
P.C. M. Linssen ◽  
G. Nikoloski ◽  
A. Pennings ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Target Genes ◽  
Bone Marrow Cells ◽  
Biological Activities ◽  
Mouse Bone Marrow ◽  
Mutant Proteins ◽  
Empty Vector ◽  
Primary Mouse ◽  
Marrow Cells

Abstract The Wilms’ Tumor 1 (WT1) gene is highly expressed in bone marrow progenitor cells, and is downregulated during the differentiation towards mature blood cells. Several lines of evidence suggest that WT1 plays an important role in leukemia development. WT1 overexpression can be detected in more than 80% of acute leukemia’s and an inverse correlation has been found between the expression levels of WT1 and the overall survival of patients. In addition, in about 9% of AML cases and 3% of ALL cases, WT1 is mutated and the presence of these mutations may have an adverse effect on the survival of the patients. So far, little is known about the biological activities of the wildtype and the WT1-mutant proteins during hematopoiesis and the presence of different isoforms with different biological activities has hampered a clear interpretation of the results so far. In a comprehensive study, we have investigated the function of all four major WT1 isoforms in primary mouse bone marrow cells using in-vitro and in-vivo assays. In addition, we have studied for each isoform the effect of mutations on the biological activity. In-vitro studies: 4 wildtype WT1 isoforms, 6 mutants and an empty vector control were retrovirally transduced into primary murine bone marrow cells. Subsequently, the transduced cells were FACS-sorted and used for various assays. WT1 inhibited the in-vitro colony formation (CFU-GM) by 60–95%, depending on the expressed isoform. In contrast, expression of the corresponding WT1 mutant proteins had no effect on colony formation. To study the underlying mechanism, we cultured the WT1-transduced bone marrow cells and analyzed the cells each day for proliferation (Cell count & DNA histograms), differentiation (Mac1, Gr1) and apoptosis (Annexin V) using FACS analysis. In agreement with the colony assays, the expression of all 4 wildtype WT1 isoforms induced growth arrest and resulted in accelerated differentiation. Target genes: To investigate which genes may be involved in the observed phenotypes, we quantitatively analyzed the expression levels of 34 putative WT1-target genes in the transduced murine bone marrow cells. Briefly, primary mouse bone marrow cells were retrovirally transduced with 4 different wildtype WT1 isoforms, 4 different mutant isoforms or an empty vector control. Sixteen hours after transduction, the transduced cells were FACS-sorted and RNA was extracted for quantitative real-time RT-PCR analysis. We have identified a number of putative WT1-target genes that are differentially regulated by the 4 wildtype WT1 isoforms but not by the WT1 mutant proteins: E-cadherin, syndecan, NGF-receptor, Egr-1, TGF-b, c-Myc, Vitamin D-receptor, insulin-receptor thrombospondin and the taurine transporter. In-vivo studies: To study the effect of WT1 on more immature bone marrow stem cells, we have transplanted WT1-transduced primary mouse CD45.2 bone marrow cells together with empty-vector-transduced primary mouse CD45.1 bone marrow cells into ablatively irradiated syngenic CD45.1/CD45.2 heterozygous mice. Six weeks after transplantation, 5-colour FACS analysis of peripheral blood indicated that the expression of WT1 promotes myeloid differentiation (Mac1 & Gr1) and inhibits the formation of B- (IgM/B220) and T-cells (CD4 & CD8).

Identification of Meis1 as a Target of CREB in Myeloid Leukemogenesis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1945-1945
Author(s):  
Samuel D. Esparza ◽  
Deepa Shankar ◽  
Stanley Nelson ◽  
Kathleen Sakamoto
Keyword(s):  
Bone Marrow ◽  
Transcription Factor ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leucine Zipper ◽  
Target Genes ◽  
Bone Marrow Cells ◽  
Mrna Level ◽  
Leukemia Cell ◽  
Self Renewal

Abstract The cyclic AMP response element binding protein, CREB, is a leucine zipper transcription factor that induces the expression of genes that regulate proliferation and survival. We previously demonstrated that CREB expression was increased in bone marrow cells from patients with acute leukemia. Transgenic mice that overexpress CREB in myeloid cells develop a myeloproliferative/myelodysplastic syndrome after one year. Although CREB has been implicated in the development of leukemia, the mechanisms by which CREB overexpression leads to a malignant phenotype remain elusive. To identify CREB target genes, we performed microarray analysis with RNA from two CREB overexpressing cell lines, K562 and NFS60. Both murine (NFS60) and human (K562) myeloid leukemia cell lines were stably transfected with CREB under the control of the myeloid-specific HMRP-8 promoter. Meis1, an important transcription factor in hematopoiesis, was found to be upregulated 40-fold in CREB overexpressing cells compared to controls. In the development of granulocytes, Meis1 expression decreases as cells differentiate. Similar to CREB, Meis1 is expressed in CD34+ hematopoietic cells but not in CD34- cells. Meis1 expression has been reported to promote self-renewal of stem cells and inhibit G-CSF-dependent differentiation into granulocytes. We hypothesized that CREB overexpression may lead to persistent expression of Meis1, increased self-renewal, and a block in differentiation of primitive hematopoietic progenitor cells, thereby contributing to leukemogenesis. We first examined Meis1 expression in CREB overexpressing K562 and NFS60 cells. Significantly higher levels of Meis1 were detected in Western blot analysis with lysates from CREB overexpressing K562 cells compared to parental cells. In addition, the degree of CREB expression correlated with the degree of Meis1 expression when evaluated at the protein level. Experiments performed using the NFS60 cell line showed similar results. To investigate Meis1 expression in primary myeloid leukemia cells, bone marrow from patients with acute myeloid leukemia (AML) were analyzed for CREB and Meis1 expression. Samples from three of the four patients had elevated CREB expression. Patients whose cells had the highest levels of CREB also had highest expression of Meis1. The one patient with minimal CREB expression showed no detectible levels of Meis1. Evaluation of cell lines and primary cells using quantitative real time PCR is underway to determine potential differences in CREB and Meis1 expression at the mRNA level. Chromatin immunoprecipitation will also be performed to determine whether Meis1 is a direct target of CREB. Our data demonstrate that CREB overexpression correlates with overexpression of Meis1 in both cell lines and primary AML cells. These results suggest that CREB and Meis1 are co-regulated in AML cells and may contribute to the leukemia phenotype.

A Potent Stimulator of Self-Renewal in Combination with MEIS1 Overexpression Allows the Transformation of Late Committed Myeloid Progenitors.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1434-1434
Author(s):  
Tobias Berg ◽  
Florian Kuchenbauer ◽  
Anisa Salmi ◽  
David Lai ◽  
Michael Heuser ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Rapid Onset ◽  
Incomplete Penetrance ◽  
Mouse Bone Marrow ◽  
Leukemic Transformation ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Myeloid Progenitors

Abstract Abstract 1434 Poster Board I-457 Which cells are susceptible to leukemic transformation and which cellular properties need to be altered to cause transformation are critical issues in the understanding of leukemogenesis. We have identified that the engineered fusion gene between NUP98 and the homeodomain of HOXA10 (NA10hd) exhibits an extraordinary capacity to induce stem cell self-renewal in vitro without blocking differentiation in vivo and without having any detectable leukemogenic activity on its own. However, NA10hd induces rapid-onset leukemia in mice when it is co-expressed with Meis1. We have exploited this unique combination of defined factors to investigate the role of reactivation/intensification of the self-renewal program in leukemic transformation by assessing whether committed myeloid progenitors with essentially no intrinsic self-renewal activity can be transformed into cells with “stem-cell” like characteristics that drive leukemia. Populations enriched for hematopoietic stem cells (lin-Sca1+ckit+), common myeloid progenitors (lin-Sca1-ckit+CD34+CD16/32lo, CMP) and granulocyte-monocyte progenitors (lin-Sca1-ckit+CD34+CD16/32hi, GMP) were sorted from mouse bone marrow. Expression of NA10hd and Meis1 was achieved by retroviral transduction with MSCV based vectors carrying cassettes for NA10hd-IRES-GFP or Meis1-IRES-YFP. Transduced cells were assessed for colony formation (CFC assay) and transplanted into irradiated recipients to assess their potential to give rise to leukemia. We first temporally dissociated the potential stimulation of self-renewal by NA10hd as a “first hit” from Meis1 overexpression as a “second hit” by using bulk bone marrow cells or highly purified CMP and GMP from mice previously reconstituted with NA10hd-transduced stem cells. At the time of harvest, mice were healthy and progenitor populations from these mice showed a regular distribution and a limited replating potential. However, upon infection with Meis1 a sustained replating potential over multiple CFC rounds was induced. NA10hd-expressing CMP and GMP transduced with Meis1 also gave rise to rapid onset and aggressive AML (median latency of 46.5 days for bulk, 54 days for CMP and 92 days for GMP respectively) when transplanted into irradiated recipients. To further assess whether NA10hd plus Meis1 were sufficient for transformation of later progenitors, CMP and GMP were isolated from wildtype, normal mice and cotransduced with NA10hd and Meis. To minimize the chance of contamination of fractions, infections were also carried out with purified cells at limiting number (50 cells per culture). Neither CMP nor GMP transduced with NA10hd alone gave rise to significant engraftment and both were non-leukemogenic. In contrast, both CMP and GMP co-transduced with NA10hd and Meis1 gave rapid onset leukemia. Leukemias derived from CMP and GMP showed no obvious differences with respect to morphology, immunophenotype or capability to give rise to secondary transplants. However, there was an apparent small diminution in the potency of GMP to give rise to leukemias as indicated by slightly longer latency and incomplete penetrance (3/7 mice surviving with transduced bulk GMP). We conclude that late committed myeloid progenitor cells can give rise to leukemia in our model system and that reactivation of self-renewal potential appears to be a critical step in this transformation process. This novel model of leukemia induction now opens up possibilities to dissect the molecular mechanism by which transformation of differentiated progenitors can occur. Disclosures No relevant conflicts of interest to declare.

scholarly journals Establishment of Cell Lines from Both Myeloma Bone Marrow and Plasmacytoma: SNU_MM1393_BM and SNU_MM1393_SC from a Single Patient

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Youngil Koh ◽  
Woo-June Jung ◽  
Kwang-Sung Ahn ◽  
Sung-Soo Yoon
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Cultured Cells ◽  
Clonal Evolution ◽  
Myeloma Cell ◽  
Mouse Bone Marrow ◽  
Single Patient ◽  
U266 Cell

Purpose.We tried to establish clinically relevant human myeloma cell lines that can contribute to the understanding of multiple myeloma (MM).Materials and Methods.Mononuclear cells obtained from MM patient’s bone marrow were injected via tail vein in an NRG/SCID mouse. Fourteen weeks after the injection, tumor developed at subcutis of the mouse. The engraftment of MM cells into mouse bone marrow (BM) was also observed. We separated and cultured cells from subcutis and BM.Results.After the separation and culture of cells from subcutis and BM, we established two cell lines originating from a single patient (SNU_MM1393_BM and SNU_MM1393_SC). Karyotype of the two newly established MM cell lines showed tetraploidy which is different from the karyotype of the patient (diploidy) indicating clonal evolution. In contrast to SNU_MM1393_BM, cell proliferation of SNU_MM1393_SC was IL-6 independent. SNU_MM1393_BM and SNU_MM1393_SC showed high degree of resistance against bortezomib compared to U266 cell line. SNU_MM1393_BM had the greater lethality compared to SNU_MM1393_SC.Conclusion.Two cell lines harboring different site tropisms established from a single patient showed differences in cytokine response and lethality. Our newly established cell lines could be used as a tool to understand the biology of multiple myeloma.

Abstract 454: Myeloid CD98hc Deficiency Reduces Atherosclerotic Plaque Development via Impaired Proliferation of Macrophages

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Sara McCurdy ◽  
William A Boisvert
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Atherosclerotic Plaque ◽  
Bone Marrow Cells ◽  
Transmembrane Protein ◽  
Mouse Bone Marrow ◽  
Plaque Development ◽  
Primary Mouse

Macrophage accumulation is a key process affecting all stages of atherosclerosis. Whether these cells accumulate in plaque solely by recruitment of monocytes from circulation or by proliferation within the plaque is an important question that has garnered much interest in recent years. Originally identified as a lymphocyte activation marker, CD98hc (SLC3A2) is a transmembrane protein involved in cell proliferation and survival via integrin signaling and MAP kinase activation. We hypothesized that CD98hc deficiency in myeloid cells would have a protective effect on atherosclerosis development and plaque composition by limiting macrophage proliferation. For the studies described, we utilized mice with myeloid-specific deletion of the CD98hc ( CD98hc fl/fl LysMCre + ) to determine the effects of CD98hc deficiency on macrophage function in the context of atherosclerosis . We performed in vitro assays to investigate the role of CD98hc in the proliferation and survival of primary mouse bone marrow derived macrophages. Although we found no differences in the number of bone marrow cells isolated from control or CD98hc -/- animals, after differentiation with MCS-F for 7 days, the number of macrophages obtained from CD98hc -/- mice was approximately 80% lower (7.2 ± 2.2 x 10 6 vs. 42.4 ± 4.6 x 10 6 per mouse) compared to control mice. Proliferation assays in vitro using EdU revealed approximately 50% (15.4 ± 2.5% vs. 7.5±1.8%) reduced cell proliferation in CD98hc -/- macrophages compared to control cells that could not be rescued with the addition M-CSF. In a 6-week atherosclerosis study using Ldlr -/- CD98hc fl/fl LysMCre + mice, Oil-Red O staining of whole aortae as well as aortic sinus sections showed that atherosclerotic plaque development was reduced compared to Ldlr -/- CD98hc fl/fl LysMCre - control mice. Additionally, immunohistochemical staining of atherosclerotic tissues revealed a reduction in macrophage abundance and proliferation within the plaque of Ldlr -/- CD98hc fl/fl LysMCre + mice compared to control mice. These findings support an important role of CD98hc in macrophage proliferation within the plaque environment, and provide a novel target for reducing atherosclerosis.

scholarly journals Expression of the c-kitligand and interleukin 6 genes in mouse bone marrow stromal cell lines

Stem Cells ◽  
1994 ◽  
Vol 12 (4) ◽  
pp. 409-415 ◽  
Author(s):  
Takeshi Otsuka ◽  
Tomonori Ogo ◽  
Teruaki Nakano ◽  
Hiroaki Niiro ◽  
Seiji Kuga ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Interleukin 6 ◽  
Stromal Cell ◽  
Bone Marrow Stromal Cell ◽  
Mouse Bone Marrow ◽  
Marrow Stromal Cell

scholarly journals Calcium ionophore but not phorbol ester promotes eicosanoids release by proliferating interleukin-3-dependent bone marrow cells

Blood ◽  
1990 ◽  
Vol 76 (8) ◽  
pp. 1586-1592 ◽  
Author(s):  
Y Shibata ◽  
PG McCaffrey ◽  
H Sato ◽  
Y Oghiso
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Phorbol Ester ◽  
Bone Marrow Cells ◽  
Marrow Cell ◽  
Calcium Ionophore ◽  
Mouse Bone Marrow ◽  
Ionophore A23187 ◽  
Interleukin 3 ◽  
Marrow Cells

Abstract Eicosanoid release during multilineage hematopoiesis was assessed using freshly isolated mouse bone marrow cells cultured in the presence of interleukin-3 (IL-3) (10% WEHI-3 culture-conditioned medium). Cells that could release prostaglandin E2 (PGE2) when stimulated with calcium ionophore A23187, but not with phorbol ester (PMA), appeared within 4 days. The cells harvested on day 10 released 42 ng of PGE2/10(6) cells/mL after A23187 stimulation. Leukotriene B4 (LTB4) (4 ng/mL) was also detected after A23187 stimulation, but there was no detectable LTC4 (less than 0.5 ng/mL). Nonadherent bone marrow cells were isolated from 28-day cultures and cloned. All clones were strongly IL-3- dependent. Although other growth factors such as granulocyte colony- stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), and CSF-1 failed to promote survival or support proliferation of the cells, three clones (11–1-A6, 3–2-D5, and 11–1-A1) showed significant increases in 3H-thymidine incorporation, respectively, after PMA treatment for 24 hours. Surviving cells displayed dominantly myeloid type morphology and phenotypic characteristics. The data suggest that IL-3 is important in the formation of PGE2-producing cells. In contrast to many macrophages (MO), neither the IL-3-dependent cell lines nor the IL-3-cultured bone marrow cells released significant amounts of PGE2 when stimulated with PMA or IL-3, although PMA and IL-3 both induced translocation of protein kinase C (PKC) to the membrane fraction. The lack of production of PGE2 and other eicosanoids by the PMA- and IL-3- stimulated cell lines was confirmed by measuring the release of 3H- arachidonic acid. The data suggest that in IL-3-dependent bone marrow cell lines the activation of eicosanoid metabolism requires elevated cellular Ca2+; PKC activation alone does not appear to be a sufficient stimulus.

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