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.

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

Direct Binding of Grb2 Is Required for Efficient Induction of Myeloproliferative Disease in Mice by ETV6/FLT3.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2903-2903
Author(s):  
Kazuhisa Chonabayashi ◽  
Masakatsu Hishizawa ◽  
Shin Kawamata ◽  
Masashi Matsui ◽  
Tatsuharu Ohno ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tyrosine Kinase ◽  
Binding Sites ◽  
Bone Marrow Cells ◽  
Myeloproliferative Disease ◽  
Juxtamembrane Domain ◽  
Murine Bone Marrow ◽  
Direct Binding ◽  
Marrow Cells

Abstract Abstract 2903 Poster Board II-879 FMS-like tyrosine kinase 3 (FLT3), a class III receptor tyrosine kinase, is one of the most frequently mutated genes in hematological malignancies. The most common mutations of FLT3 are internal tandem duplications (ITDs) within the juxtamembrane domain: these mutations occur in 20% to 30% of patients with AML and are closely associated with a poor prognosis. In a small number of patients with myeloproliferative neoplasms (MPNs), FLT3 has been reported to fuse to ETV6 (TEL) and contribute to leukemogenesis, but the leukemogenic mechanism of ETV6/FLT3 remains unclear. We encountered a case of ETV6/FLT3 fusion in a patient with MPN complicated with T-cell lymphoblastic lymphoma. In this case, both myeloid and lymphoma cells shared the same chromosomal translocation, t(12;13)(p13;q12), and allogeneic hematopoietic stem cell transplantation led to complete remission for 3 years. Full-length ETV6/FLT3 fusion cDNA was cloned from the patient's bone marrow cells. Sequence analysis of the PCR product revealed that, in contrast to the finding of previously reported two cases of ETV6/FLT3-positive MPN, ETV6 exon 6 was fused to FLT3 exon 14 and that the fused portion of ETV6 contained 2 potential Grb2-binding sites (Vu et al., Leukemia 2006; Walz et al., Blood 2007a). The ETV6/FLT3 conferred IL-3-independent growth to Ba/F3 and 32Dcl3 cells. Using a dominant negative approach, we showed that both STAT5 and Ras played important roles in ETV6/FLT3-mediated transformation of the hematopoietic cell lines. To investigate the role of the ETV6/FLT3 fusion protein in vivo, we used a murine bone marrow transplant model. Retroviral transduction of the ETV6/FLT3 into primary murine bone marrow cells resulted in a CML-like myeloproliferative disease (MPD) with complete penetrance in the transplanted mice. The disease progressed to cause death at a median of 18 days after transplantation (n = 16). The transplanted mice developed severe leukocytosis (159 × 103 /μl to 417 × 103 /μl), splenomegaly, and extensive infiltration of myeloid cells in the bone marrow, spleen, liver, and peripheral blood. ETV6/FLT3-induced MPD was oligoclonal and only 2 of the 9 secondary transplant recipients developed similar MPD when 5 × 106 spleen cells from 3 independent diseased mice were used as donors. We assayed the mutant forms of the ETV6/FLT3 to test their ability to transform hematopoietic cells. Induction of MPD required the oligomerization domain of ETV6 and the tyrosine kinase activity of FLT3. Mice that received the double tyrosine-to-phenylalanine mutant of ETV6/FLT3 at sites 589 and 591 (Y589/591F) in the juxtamembrane domain of FLT3, which are critical for FLT3-ITD-induced MPD, also developed a similar MPD phenotype. Unlike FLT3-ITDs, Y589/591F mutation did not abrogate STAT5 activation in Ba/F3 and 32Dcl3 cells transformed by ETV6/FLT3. A recent study has shown that direct binding of Grb2 to tyrosine 768, 955, and 969 of FLT3 is important for FLT3-ITD-mediated proliferation and survival of hematopoietic cells. Tyrosine 314 in exon 5 of ETV6 has also been reported as the principal Grb2-binding site that contributes to leukemogenesis via oncogenic ETV6 fusion proteins such as ETV6/ABL. Thus, we next investigated the role of Grb2 binding in ETV6/FLT3-mediated leukemogenesis. Using coimmunoprecipitation assays, we demonstrated that Grb2 also binds to the tyrosine 314 and 354 of ETV6 of the ETV6/FLT3, in addition to the tyrosine 768, 955, and 969 of FLT3. Both ETV6/FLT3-Y314/354F and ETV6/FLT3-Y768/955/969F retained their interaction with Grb2 and induced rapidly fatal MPD when they were transduced into primary murine bone marrow cells. On the other hand, the ETV6/FLT3 mutant at all the binding sites of Grb2 (Y314/354/768/955/969F) significantly attenuated MPD development in mice. Simultaneous mutation of these 5 tyrosine residues completely abolished the binding of Grb2 and resulted in a marked decrease in the binding and phosphorylation of Gab2 and impaired activation of STAT5 and Akt in Ba/F3 cells. These results indicate that tyrosine 589 and 591 of FLT3 are dispensable for the ETV6/FLT3-induced MPD phenotype, and suggest that both ETV6 and FLT3 portions contribute to the ETV6/FLT3-mediated leukemogenesis by binding directly to Grb2. Our observations provide deep insights into the oncogenic signaling induced by active FLT3 mutants as well as provide a potential target for therapies. Disclosures: No relevant conflicts of interest to declare.

Non-Canonical NF-Kb Signaling Regulates Hematopoietic Stem Cell Self-Renewal and Microenvironment Interactions

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 859-859 ◽  
Author(s):  
Chen Zhao ◽  
Yan Xiu ◽  
John M Ashton ◽  
Lianping Xing ◽  
Yoshikazu Morita ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Wild Type ◽  
Double Knockout ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Physiological Processes ◽  
Marrow Cells

Abstract Abstract 859 RelB and NF-kB2 are the main effectors of NF-kB non-canonical signaling and play critical roles in many physiological processes. However, their role in hematopoietic stem/progenitor cell (HSPC) maintenance has not been characterized. To investigate this, we generated RelB/NF-kB2 double-knockout (dKO) mice and found that dKO HSPCs have profoundly impaired engraftment and self-renewal activity after transplantation into wild-type recipients. Transplantation of wild-type bone marrow cells into dKO mice to assess the role of the dKO microenvironment showed that wild-type HSPCs cycled more rapidly, were more abundant, and had developmental aberrancies: increased myeloid and decreased lymphoid lineages, similar to dKO HSPCs. Notably, when these wild-type cells were returned to normal hosts, these phenotypic changes were reversed, indicating a potent but transient phenotype conferred by the dKO microenvironment. However, dKO bone marrow stromal cell numbers were reduced, and bone-lining niche cells supported less HSPC expansion than controls. Further, increased dKO HSPC proliferation was associated with impaired expression of niche adhesion molecules by bone-lining cells and increased inflammatory cytokine expression by bone marrow cells. Thus, RelB/NF-kB2 signaling positively and intrinsically regulates HSPC self-renewal and maintains stromal/osteoblastic niches and negatively and extrinsically regulates HSPC expansion and lineage commitment through the marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The expansion of murine bone marrow cells preincubated in hypoxia as an in vitro indicator of their marrow-repopulating ability

Leukemia ◽  
2000 ◽  
Vol 14 (4) ◽  
pp. 735-739 ◽  
Author(s):  
MG Cipolleschi ◽  
E Rovida ◽  
Z Ivanovic ◽  
V Praloran ◽  
M Olivotto ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Murine Bone Marrow ◽  
Marrow Repopulating Ability ◽  
Marrow Cells

Characterization of cytotoxic cells generated from in vitro cultures of murine bone marrow cells

1985 ◽  
Vol 92 (1) ◽  
pp. 1-13 ◽  
Author(s):  
Gary R. Klimpel ◽  
Marcella Sarzotti ◽  
Victor E. Reyes ◽  
Kathleen D. Klimpel
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
In Vitro Cultures ◽  
Murine Bone Marrow ◽  
Cytotoxic Cells ◽  
Marrow Cells

Meis1-mediated apoptosis is caspase dependent and can be suppressed by coexpression of HoxA9 in murine and human cell lines

Blood ◽  
2005 ◽  
Vol 105 (3) ◽  
pp. 1222-1230 ◽  
Author(s):  
Peter J. Wermuth ◽  
Arthur M. Buchberg
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Cell Types ◽  
Selective Advantage ◽  
Myeloid Differentiation ◽  
Homeodomain Protein ◽  
Murine Bone Marrow ◽  
Differentiation Pathways

AbstractCoexpression of the homeodomain protein Meis1 and either HoxA7 or HoxA9 is characteristic of many acute myelogenous leukemias. Although Meis1 can be overexpressed in bone marrow long-term repopulating cells, it is incapable of mediating their transformation. Although overexpressing HoxA9 alone transforms murine bone marrow cells, concurrent Meis1 overexpression greatly accelerates oncogenesis. Meis1-HoxA9 cooperation suppresses several myeloid differentiation pathways. We now report that Meis1 overexpression strongly induces apoptosis in a variety of cell types in vitro through a caspase-dependent process. Meis1 requires a functional homeodomain and Pbx-interaction motif to induce apoptosis. Coexpressing HoxA9 with Meis1 suppresses this apoptosis and provides protection from several apoptosis inducers. Pbx1, another Meis1 cofactor, also induces apoptosis; however, coexpressing HoxA9 is incapable of rescuing Pbx-mediated apoptosis. This resistance to apoptotic stimuli, coupled with the previously reported ability to suppress multiple myeloid differentiation pathways, would provide a strong selective advantage to Meis1-HoxA9 coexpressing cells in vivo, leading to leukemogenesis.

MN1 Inhibits Myeloid Differentiation by Transcriptional Repression of EGR2

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 229-229
Author(s):  
Michael Heuser ◽  
Eric Yung ◽  
Courteney Lai ◽  
Bob Argiropoulos ◽  
Florian Kuchenbauer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Transcriptional Activation ◽  
Transcriptional Repression ◽  
Target Genes ◽  
Bone Marrow Cells ◽  
Function Analysis ◽  
Myeloid Differentiation ◽  
Marrow Cells

Abstract Abstract 229 Overexpression of MN1 (meningioma 1) is a negative prognostic factor in acute myeloid leukemia (AML) patients with normal cytogenetics, and induces a rapidly lethal AML in mice. We have shown previously that MN1, a transcription cofactor of retinoic acid receptor alpha (RARA), increases resistance to all-trans retinoic acid (ATRA) by greater than 3000-fold in an in-vitro differentiation model. We investigated the molecular mechanisms involved in the MN1-induced myeloid differentiation block by fusing potent transcriptional activation or repression domains to MN1, conducting a structure-function analysis of MN1, gene expression profiling, ChIP-on chip experiments, and functional validation of MN1 target genes. We found that (1) MN1 inhibits myeloid differentiation through transcriptional repression; (2) the C-terminal domain of MN1 is critical for induction of resistance to ATRA; (3) EGR2 is a putative direct target of MN1 and RARA that is repressed in MN1 leukemias; and (4) that constitutive upregulation of EGR2 in MN1 leukemias permits differentiation and prevents engraftment of transplanted cells. To investigate whether MN1 impacts on myeloid differentiation through transcriptional activation or repression we fused a strong transcriptional activation domain (VP16) or repression domain (M33) to MN1. MN1VP16 immortalized murine bone marrow cells, however, these cells could differentiate to mature granulocytes, and succumbed to cell cycle arrest upon treatment with ATRA. Mice receiving transplants of MN1VP16 cells had a median survival of 143 days (n=16) compared to 35 days in mice receiving MN1-transduced cells (n=18; p<.001). Morphologic analysis of bone marrow mostly showed mature granulocytes with less than 20 percent immature forms consistent with a diagnosis of myeloproliferative-like disease. Conversely, mice receiving transplants with cells transduced with the fusion of MN1 to the transcriptional repression domain of M33 (n=7) developed leukemia with a similar latency and phenotype as mice receiving transplants from MN1-transduced cells (survival, P=.6). These data suggest that MN1 inhibits myeloid differentiation by transcriptional repression rather than activation of its target genes. A structure-function analysis was performed to identify the domain(s) of MN1 required to inhibit myeloid differentiation. Consecutive stretches of 200 amino acids of MN1 were interrogated The deletion constructs were subsequently transduced into bone marrow cells immortalized by NUP98-HOXD13 (ND13). ND13 cells are very sensitive to ATRA-induced differentiation and cell cycle arrest with an IC50 of 0.1 μ M, whereas overexpression of MN1 increases resistance greater than 3000-fold. Interestingly, deletion of the 200 C-terminal amino acids of MN1 restored ATRA sensitivity of ND13 cells compared to full-length MN1, suggesting that the C-terminus of MN1 is required for inhibition of myeloid differentiation. To identify MN1-regulated genes important for the myeloid differentiation block we performed gene expression profiling of MN1- and MN1VP16-transduced bone marrow cells. To further identify genes that might be directly regulated by MN1 we performed ChIP-on-chip using anti-MN1 and anti-RARA antibodies. EGR2, CCL5, CMAH, among others, were identified as targets of both MN1 and RARA whose gene expression was low in MN1 but high in MN1VP16 cells. Overexpression of these genes in MN1-transduced leukemic cells was used to validate their function. Blast percentage of in vitro cultured bone marrow cells was 93, 58, 83, and 41 percent in MN1+CTL cells, MN1+EGR2, MN1+CCL5, and MN1+CMAH cells, respectively. MN1+EGR2 cell engraftment in peripheral blood of mice declined from 2.2 percent at 4 weeks to undetectable levels at 8 weeks (n=4), whereas MN1+CCL5 and MN1+CMAH cell engraftment was 23 (n=4) and 26 (n=4) percent at 4 weeks, and 14 and 30 percent at 8 weeks, respectively. At time of death, EGR2 was not detectable in mice whereas leukemias of mice receiving MN1+CCL5 or MN1+CMAH- transduced cells were positive for CCL5 or CMAH, respectively. In conclusion, our data suggest that MN1 inhibits myeloid differentiation by transcriptional repression of a subset of its target genes, and that re-expression of EGR2, a zinc-finger transcription factor, may prevent outgrowth of MN1 leukemias in mice. Pharmacologic activation of EGR2 may become a novel antileukemic strategy. Disclosures: No relevant conflicts of interest to declare.

ATRA and the RARα Specific Agonist, NRX195183, Have Opposing Effects on the In Vitro Clonogenicity of Pre-Leukemic Murine AML1-ETO Bone Marrow Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 999-999
Author(s):  
Lynette C.Y. Chee ◽  
Jean Hendy ◽  
Louise Purton ◽  
Grant A. McArthur
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Myeloid Cells ◽  
Myeloid Cell ◽  
Sustained Remission ◽  
Self Renewal ◽  
Cells Cultured ◽  
Marrow Cells

Abstract Abstract 999 All-trans retinoic acid (ATRA) is used successfully to treat acute promyelocytic leukemia (APML), however, to date it has not shown promise in treating other AML subtypes. ATRA has been shown to enhance hematopoietic stem cell (HSC) self-renewal (requiring RARγ activation) but promotes differentiation of myeloid progenitors likely through RARα activation. We hypothesized that (1) the lack of success of ATRA in treating other AML subtypes may be due to the potential ability of ATRA to enhance self-renewal of the leukemic stem cell and (2) the use of a specific RARα agonist may have more promise in enhancing AML differentiation. We therefore compared the effects of pharmacological levels (1μM) of ATRA and an RARα-specific agonist, NRX195183, on bone marrow cells harvested from a Cre-inducible conditional AML1-ETO (AE) knock-in murine model. AE cells cultured for 2 weeks with ATRA showed significant reductions in the proportions of mature myeloid cells (Gr1brightCD11b+) by fluorescence activated cell sorting (FACS) (DMSO: 14.2±4.3%, ATRA: 4.0±1.6%, p=0.04, n=4). By 4 weeks of culture, ATRA-treated AE cells had increased blast and reduced maturing myeloid cell proportions (Blasts %: DMSO 70.2 ± 3.0, ATRA 95.3 ± 1.2, p=0.08; Intermediate %: DMSO 14.3 ± 2.6, ATRA 3.8 ± 1.0, p=0.01; Neutrophils %: DMSO 2.3± 1.0, ATRA 0.3 ± 0.2, p=0.07, n=6). Furthermore, ATRA potentiated the clonogenicity of the AE cells after 5 weeks of treatment in vitro (Mean±SEM for colony #/ 5×104 cells: DMSO 505.8±337.0, ATRA 4394±388.9, p=0.001; n=6). In contrast, AE cells cultured for 2 weeks with NRX195183 showed significant increases in the proportions of mature myeloid cells by FACS (DMSO: 15.8±3.5%, NRX195183 26.7±3.0%, p=0.03; n=5). By 4 weeks of culture, NRX195183-treated AE cells had decreased blast and increased maturing myeloid cell proportions (Blasts %: DMSO 82.4±3.0, NRX195183 58.8±9.1, p=0.03; Intermediate %: DMSO 14.5±2.5, NRX195183 29.0±6.8, p=0.07; Neutrophils %: DMSO 1.6±0.8, NRX195183 8.2±4.7 p=ns; DMSO n=8, NRX195183 n=5). Moreover, NRX195183 reduced the clonogenicity of the AE cells after 5 weeks of treatment in vitro (Mean±SEM for colony #/ 5×104 cells DMSO 554.8±252.6, NRX195183 82.6±61.6, p=0.05; n=8). Short-term in vivo transplants of fetal liver cells overexpressing the truncated AE gene, AE9a, into sublethally irradiated recipients revealed similar findings in the NRX195183-treated mice with a decrease in blasts and an increase in mature neutrophils in the peripheral blood on morphological analysis after 4 weeks of treatment (Blasts x106/ml: DMSO 3.1±1.0, NRX195183 0.9±0.3, p=0.08; Neutrophils x106/ml: DMSO 0.5±0.1, NRX195183 0.8±0.1, p=0.04; DMSO n=16, NRX195183 n=11). Taken together, these findings support a model whereby ATRA promotes self-renewal of leukemic blasts whilst NRX195183 has the opposing effect. To understand the mechanism by which ATRA promotes self-renewal in AE cells, we performed genome-wide gene expression analyses on the ATRA- versus control-treated AE cells. This revealed 16 differentially upregulated genes after 24 hours of treatment. Using Ingenuity Pathway Analysis, the top scoring network in the ATRA-treated AE cells was cell-to-cell signalling and interaction (p=1.1E-7-2.4E-3), lipid metabolism (p=2.3E-7-2.0E-3) and small molecule biochemistry (p=2.3E-7-2.1E-3); SERPINE1 and BMP2 were the genes with the highest connectivity within the network interacting with molecules known for their roles in tumorigenesis, including AKT, NF-kβ complex and TGFβ1. SERPINE1 upregulation has been shown to be RARα-mediated whilst BMP2 has been shown to be a RARγ-regulated gene. Interestingly, the specific RARγ agonist, NRX204723, had no effect on the clonogenic potential of these AE progenitors thus raising the hypothesis that both RARα and RARγ activation are required to promote self-renewal of the AE progenitors. Further studies using both RARα/RARγ agonists are warranted to assess if the ATRA effects on AE cells are phenocopied. Collectively, these findings reveal the contrasting roles of specific RARα activation in promoting loss of self-renewal ability and enhancing differentiation in the AE cells whilst ATRA promotes clonogenicity of these cells. This has potential significant implications in AML treatment as specific RARα agonists may be beneficial in improving the efficacy of current treatment modalities to achieve sustained remission in other AML subtypes. Disclosures: No relevant conflicts of interest to declare.

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