FASN Is Critical for Maintenance of NUP98 Fusion-Induced Leukemias

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2377-2377
Author(s):  
Yutaka Shima ◽  
Issay Kitabayashi
Keyword(s):  
Progenitor Cells ◽  
Colony Formation ◽  
Fusion Proteins ◽  
Fatty Acid Synthase ◽  
Fusion Gene ◽  
Mass Spectrometry Analysis ◽  
Transformed Cells ◽  
Nuclear Pore ◽  
Repeat Domain

Abstract NUP98 is a component of the nuclear pore complex (NPC), which plays an important role in molecular traffic between the cytoplasm and the nucleus. The NUP98 gene is rearranged and fused to several partner genes, such as HOXA9 and DDX10, in acute myeloid leukemia and myelodysplastic syndromes and the leukemia with NUP98 rearrangement has a poor prognosis. The role of the NUP98 moiety of NUP98 fusion proteins is not well investigated. NUP98 has two Phe-Gly (FG) repeat domains, which are characteristic of the NPC proteins. To investigate the role of the NUP98 moiety of NUP98 fusion proteins, we purified the protein complexes formed with wild-type NUP98-HOXA9 and its mutants that lack the each FG repeat domain and looked for the proteins that interact with the NUP98 moiety. Mass spectrometry analysis identified that fatty acid synthase (FASN) interacted with the FG repeat domain of NUP98-HOXA9. These data suggest two possibilities. One is that NUP98-HOXA9 may affect the activity of FASN. The other is that FASN may affect activity of NUP98-HOXA9. To test the former possibility, we tested the effect of NUP98-HOXA9 on the activity of FASN in vitro, and we found that NUP98-HOXA9 reduced the FASN activity. FASN is known to be essential for cell growth. In fact, the colony formation of the NUP98-HOXA9-immortalized mouse myeloid stem/progenitor cells was inhibited by shRNA for FASN.　Because NUP98-HOXA9 reduced FASN activity, we hypothesized that FASN activity is low in NUP98 fusion gene-transformed cells and FASN inhibition selectively inhibits the colony formation of these cells. To test this hypothesis, we examined the effect of orlistat, which is the inhibitor of FASN on the colony formation. Orlistat strongly inhibited the colony formation of NUP98-HOXA9- and NUP98-DDX10-transformed cells, but it did not severely affect, the colony-forming activities of other fusion genes-transformed cells and normal progenitor cells. Other FASN inhibitors inhibited the colony formation of NUP98-HOXA9- and NUP98-DDX10-transformed cells. NUP98-HOXA9- and NUP98-DDX10 did not affect the expression level of FASN. The inhibition of FASN did not affect the expression of Hoxa genes and Meis1 gene, which are upregulated by NUP98 fusions. These results suggest that FASN activity is low but is essential for NUP98 fusion-mediated leukemia cells, and that FASN can be a therapeutic target for NUP98 fusion gene-mediated leukemia. Disclosures No relevant conflicts of interest to declare.

scholarly journals MLL Is Essential for NUP98-HOXA9-Induced Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1223-1223
Author(s):  
Yutaka Shima ◽  
Minori Yumoto ◽  
Issay Kitabayashi
Keyword(s):  
Fusion Proteins ◽  
Gene Locus ◽  
Gene Activation ◽  
Full Length ◽  
Mass Spectrometry Analysis ◽  
Nuclear Pore ◽  
Repeat Domain ◽  
Hoxa Gene

Abstract NUP98, a component of the nuclear pore complex (NPC), plays an important role in molecular trafficking between the cytoplasm and the nucleus. NUP98 has two Phe-Gly (FG) repeat domains, which are characteristic of NPC proteins. The NUP98 gene is rearranged and fused to several partner genes, such as HOXA9 and DDX10, in acute myeloid leukemia and myelodysplastic syndromes. Leukemia with NUP98 rearrangement is associated with poor prognosis. However, the molecular mechanism of leukemogenesis induced by NUP98-fusions remains unclear. To investigate the role of NUP98 of NUP98 fusion proteins in leukemogenesis, we generated a set of deletion mutants of NUP98-HOXA9 lacking each FG repeat domain. These mutants were transduced into normal murine progenitor cells and their activity in the immortalization of murine cells was examined by colony-formation assays. Deletion of the second FG domain resulted in a dramatic delay in leukemia induction when cells were transplanted into mice, suggesting that the second FG repeat domain is crucial for immortalization in vitro and leukemogenesis in vivo. qPCR and ChIP analyses showed that the FG repeat domain is necessary for Hoxa gene activation and recruitment to the Hoxa gene locus. To identify interacting proteins of the FG repeat domain of NUP98-HOXA9, protein complexes containing full-length and mutant NUP98-HOXA9 were purified. Mass spectrometry analysis showed that full-length NUP98-HOXA9, but not the mutant lacking the second FG repeat, interacted with MLL. ChIP analysis also showed co-localization of NUP98-HOXA9 and MLL at the Hoxa gene locus in NUP98-HOXA9-expressing cells. Furthermore, analysis of Mll null mice showed that MLL is essential for the recruitment of NUP98-HOXA9 to the Hoxa gene locus and for NUP98-HOXA9-induced immortalization and leukemogenesis. These results suggest that NUP98 fusion proteins interact with MLL to activate HOXA genes during leukemogenesis. Disclosures Kitabayashi: Daiichi Sankyo Co., Ltd.: Research Funding.

scholarly journals Nup98-Homeodomain Fusions Interact with Endogenous Nup98 during Interphase and Localize to Kinetochores and Chromosome Arms during Mitosis

2010 ◽  
Vol 21 (9) ◽  
pp. 1585-1596 ◽  
Author(s):  
Songli Xu ◽  
Maureen A. Powers
Keyword(s):  
Nuclear Pore Complex ◽  
Fusion Proteins ◽  
Cellular Transformation ◽  
Myelogenous Leukemia ◽  
Chromosomal Translocations ◽  
Nuclear Pore ◽  
Dynamic Interactions ◽  
C Terminus ◽  
Repeat Domain ◽  
Acute Myelogenous

Chromosomal translocations involving the Nup98 gene are implicated in leukemias, especially acute myelogenous leukemia. These translocations generate chimeric fusion proteins, all of which have in common the N-terminal half of Nup98, which contains the nucleoporin FG/GLFG repeat motifs. The homeodomain group of Nup98 fusion proteins retain the C-terminus of a homeodomain transcription factor, including the homeobox responsible for DNA binding. Current models for Nup98 leukemogenesis invoke aberrant transcription resulting from recruitment of coregulators by the Nup98 repeat domain. Here we have investigated the behavior of Nup98-homeodomain fusion proteins throughout the cell cycle. At all stages, the fusion proteins exhibit a novel localization distinct from the component proteins or fragments. During interphase, there are dynamic interactions between the Nup98 fusions and endogenous Nup98 that lead to mislocalization of the intranuclear fraction of Nup98, but do not alter the level of Nup98 at the nuclear pore complex. During mitosis, no interaction between the fusion proteins and endogenous Nup98 is observed. However, the fusions are entirely concentrated at kinetochores and on chromosome arms, sites where the APC/C, a target of Nup98 regulation, is also found. Our observations suggest new possibilities for misregulation by which Nup98 translocations may contribute to cellular transformation and leukemogenesis.

scholarly journals Recurrent Non-Coding Mutations in BCL7A May Have Significant Functional Consequence in Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 857-857
Author(s):  
Chandraditya Chakraborty ◽  
Eugenio Morelli ◽  
María Linares ◽  
Kenneth C. Anderson ◽  
Mehmet Kemal Samur ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Viability ◽  
Cell Lines ◽  
Chromatin Remodeling ◽  
Colony Formation ◽  
Ectopic Expression ◽  
Advisory Board ◽  
Mass Spectrometry Analysis ◽  
Rna Seq

Multiple myeloma (MM) is a complex hematological malignancy characterized by gene pathway deregulations. Initial sequencing approaches have failed to identify any single frequent (>25%) mutation in the coding genome. We, therefore performed a deep (average coverage > 80X) whole genome sequencing (WGS) on 260 MM samples (208 newly diagnosed and 52 first relapse after uniform treatment) to comprehensively identify recurrent somatic alterations in non-coding regions. We have identified the most frequently involved genes affected by perturbation in neighboring non-coding region and integrate their expression using our matching deep RNA-seq data from the same patients. One of the most prominent examples is mutations in the 5' untranslated region and intron 1 of the BCL7A gene in 76% of myeloma patients. Integration of WGS with RNA-seq data confirmed significant downregulation of its expression (p values < 1e-5) in the MM cells as compared to normal plasma cells (PC). This led us to investigate the consequences of BCL-7A loss in MM. To evaluate the role of BCL7A in MM, using gain of- (GOF) and loss-of-function (LOF) approaches, we have utilized a large panel of MM cell lines with differential expression of BCL7A at the RNA and protein levels. Ectopic expression of BCL7A in a panel of 3 MM cell lines with low basal levels of BCL7a significantly reduced cell viability and colony formation over time. Inhibition of cell viability was associated with induction of apoptotic cell death in the BCL7A overexpressing cells compared to control cells. LOF studies in 3 MM cell lines with relatively higher expression of BCL7a using 3 BCL7A-specific shRNA constructs showed a more proliferative phenotype, with increased growth and viability and enhanced colony formation. The effects of BCL7A loss in MM cells were further confirmed using CRISPR-Cas9 system. BCL7a-KO cells had higher proliferative rate compared to WT cells and add back of lentiviral BCL7a plasmid reversed this effect. BCL7A is part of the SWI/SNF chromatin remodeling complex. Mutations in the genes encoding m-SWI/SNF subunits are found in more than 20% of human cancers, with subunit- and complex-specific functions. We confirmed that when expressed, BCL7A interacts with BCL11A into the SWI/SNF complex in MM cells. Comparative, mass spectrometry analysis in fact revealed SMARCC2 (BAF170), an integral subunit of SWI/SNF complex, to bind with BCL7A-BCL11A complex. However, BCL7A loss causes decreased SMARCC2 incorporation into SWI/SNF, thus suggesting that presence of BCL7A is crucial in the formation of SWI/SNF complex in MM cells and might play an important role in chromatin remodeling. Interestingly, oncogenes DEK (DNA binding oncogene) and TPD52 (tumor protein D52) involved in cancer cell proliferation and chromatin remodeling formed complex with BCL11A in BCL7A KO MM cells. Additionally, several anti-apoptotic proteins such as ANXA-1 and BCL2 are in complex with BCL11A when BCL7A is lost, suggesting the formation of an anti-apoptotic complex with consequences on MM cell survival. Currently ongoing studies are investigating the molecular mechanism of non-coding mutations impacting BCL7A expression and pathways affected by its downregulation with impact on MM cell growth and survival. In conclusion, we report biological consequences of a frequent (>75% patients) non-coding mutation in MM with cellular and molecular effects of BCL7A loss in which implicates a functional role of the m-SWI/SNF complex in driving a MM cell proliferative phenotype. Disclosures Anderson: Gilead Sciences: Other: Advisory Board; Janssen: Other: Advisory Board; Sanofi-Aventis: Other: Advisory Board; C4 Therapeutics: Other: Scientific founder ; OncoPep: Other: Scientific founder . Munshi:Abbvie: Consultancy; Abbvie: Consultancy; Amgen: Consultancy; Amgen: Consultancy; Adaptive: Consultancy; Adaptive: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Takeda: Consultancy; Oncopep: Consultancy; Oncopep: Consultancy; Celgene: Consultancy.

scholarly journals MBNL1 As a New Therapeutic Target in MLL-Fusion Gene Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 462-462 ◽  
Author(s):  
Svetlana S Itskovich ◽  
Jason Clark ◽  
James C. Mulloy ◽  
Matthew D Disney ◽  
Ashish R Kumar
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Peripheral Blood ◽  
Fusion Gene ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Transformed Cells ◽  
Wild Type

Abstract Translocations of the Mixed Lineage Leukemia (MLL) gene located on chromosome 11 are commonly found in infants with AML or ALL and in secondary leukemia at all ages. A majority of patients with these translocations have a poor prognosis. Gene expression profiling studies demonstrate that one of the most consistently overexpressed genes in these leukemias (compared to all other leukemias) is muscleblind-like 1 (MBNL1). Further, MBNL1 was also identified as a direct transcriptional target of MLL-fusion proteins. An RNA-binding protein, MBNL1 is known to be a key factor in the pathophysiology of Myotonic Dystrophy Type I (DM), where sequestration of MBNL1 leads to splicing defects in muscle and neuronal cells. However, the role of MBNL1 in hematopoiesis and leukemogenesis is unknown. To determine the role of MBNL1 in normal hematopoiesis we studied MBNL1-/- mice. Compared to littermate controls, MBNL1-/- mice showed no differences in peripheral blood counts or bone marrow cellularity. When challenged with 5-FU, both MBNL1-/- and wild type mice displayed similar kinetics of peripheral blood cytopenia and recovery. Next we examined the role of MBNL1 in hematopoietic stem cell function using a competitive transplantation assay. Lethally irradiated mice were transplanted with a 1:1 mix of CD45.1 and CD45.2 bone marrow, with the latter being wild-type or MBNL1-/-. Flow cytometry analysis of peripheral blood at 4 weeks post-transplant showed donor chimerism being 53±4.14% in recipients of wild type marrow and 25±5.41 % in the MBNL1-/- recipients. Successive analyses every 4 weeks showed the chimerism to be stable over the next 16 weeks. To determine the role of MBNL1 in leukemia, we transformed MBNL1-/- or wild type bone marrow cells with various oncogenes delivered via retroviral transduction and compared them in methylcellulose colony replating assays. Absence of MBNL1 significantly reduced colony formation in MLL-AF9 and E2A-HLF transformed cells by 59.5% (± 27.1) and 50.7% (± 23) respectively, compared to controls. To assess the role of MBNL1 in leukemia in vivo, we transplanted MLL-AF9-transformed wild type or MBNL1-/- cells into irradiated mice. All recipients injected with wild-type MLL-AF9-transformed cells succumbed to leukemia with a median time of 106 days. In contrast, the majority of recipients of MBNL1-/- cells survived leukemia-free for at least 140 days post-transplantation (p=0.0017, log rank test). We next assessed the role of MBNL1 in human leukemia cells. Lentiviral-shRNA knockdown of MBNL1 in leukemia cell lines (MV4;11, THP-1) significantly inhibited cell growth, both in liquid culture and methylcellulose colony forming assays. To determine the requirement of MBNL1 for leukemia propagation in vivo, we used cord blood-derived leukemia cells bearing the MLL-AF9 fusion gene and mutant NRAS (MA9NRAS). MA9NRAS cells transduced with MBNL1-specific or control (non-targeting, NT) shRNA were transplanted into immunodeficient mice. Six weeks after transplant, bone marrow aspirates showed persistence of lentiviral-transduced cells in 85% of the NT-group. On the other hand, MBNL1-shRNA transduced cells were not detected in any of the recipient mice. These results suggest that MBNL1 is essential for leukemia cell propagation in vivo. Finally, we tested therapeutic targeting of MBNL1 in MLL-fusion gene leukemia. A lead inhibitor that prevents binding of MBNL1 to its targets was recently identified. Treatment of MA9NRAS cells with the inhibitor for 48 hours led to significant apoptosis whereas normal cord blood CD34+ cells were relatively less sensitive. Blockade of MBNL1 in leukemia cells either by shRNA-knockdown or by the inhibitor showed identical changes in splicing patterns of known MBNL1 target genes. Collectively, our data suggest that MBNL1 is required for the initiation and propagation of MLL-fusion gene leukemia while it appears relatively dispensable for normal hematopoiesis. Further, we have identified a promising lead inhibitor that could be developed for novel treatments for therapy-resistant leukemias. Disclosures No relevant conflicts of interest to declare.

RNAi-Mediated Inhibition of Mcl-1 Expression Enhances Apoptosis in Imatinib-Treated CML Progenitors

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1669-1669
Author(s):  
Su Chu ◽  
Ravi Bhatia
Keyword(s):  
Flow Cytometry ◽  
Progenitor Cells ◽  
Colony Formation ◽  
Cell Expansion ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Family Members ◽  
Control Shrna ◽  
Cd34 Cells

Abstract Abstract 1669 Tyrosine kinase inhibitor (TKI) treatment inhibits proliferation in CML stem/progenitor cells, but only modestly increases apoptosis. Residual leukemia stem cells remain a potential source of disease relapse in IM-treated patients. The Bcl-2 family of anti-apoptotic proteins plays a central role in the regulation of apoptosis. Several Bcl-2 inhibitors are being evaluated in preclinical and clinical studies and there is considerable interest in evaluating their ability to induce apoptosis in CML stem and progenitor cells. However these agents have considerable toxicity possibly related to lack of selectivity for individual family members. We performed a functional siRNA screen to determine the role of individual Bcl-2 family members in maintaining survival of in CML and normal CD34+ cells. CML and normal CD34+ cells were transfected with siRNAs targeting Bcl-2, Bcl-2L1, Bcl-2L2, Bcl-2L10, Mcl-1 and Bcl2A1. In this screen Mcl-1 knockdown resulted in significant reduction in viability of CML CD34+ cells, with or without co-treatment with IM (1uM). Significant reduction in normal CD34+ viability was not seen. These results were validated using different siRNA sequences to knockdown Mcl1 expression. Increased apoptosis of CML but not normal CD34+ cells was seen (23±8% for CML vs. 4.2±1.5% for normal CD34+ cells, n=3, p<0.5). CML CD34+ cell apoptosis was further enhanced by combination of Mcl-1 inhibition with IM treatment (48±15% for CML vs. 7.2±3% for CB progenitors, p<0.1). To further evaluate the role of Mcl-1 in regulating CML CD34+ cell growth, an anti-Mcl-1 shRNA construct was cloned into the pHIV7-SF-RFP lentivirus vector. Cord blood and CML CD34+ cells were transduced with Mcl-1 specific or control, non-specific shRNA expressing vectors. Western blotting demonstrated effective knockdown of Mcl-1 protein levels in Mcl-1 shRNA transduced CD34+cells (82% reduction in CML and 78% in normal CD34+ cells). CD34+ RFP+ cells were selected by flow cytometry and cultured in presence and absence of IM. A significant increase in apoptosis was seen in Mcl-1 knockdown CML CD34+ cells compared with control shRNA-transduced cells, and further increase in apoptosis was seen following IM treatment (4.7±0.5 for control shRNA-transduced cells VS 25.7±2.1 for Mcl-1 knockdown cells). Mcl-1 knockdown CML CD34+ cells generated fewer colonies in methylcellulose progenitor culture (93 colonies for control siRNA transduced cells vs. 31 colonies for Mcl-1 knockdown cells) and demonstrated reduced cell expansion following culture with growth factor (SCF; IL3; GM-CSF and G-CSF) compared with control shRNA transduced cells (383,750± 172,476 for control shRNA-transduced cells 224,250± 87,044 for Mcl-1 knockdown cells). Cell expansion was further reduced with IM treatment. Mcl-1 knockdown resulted in complete loss of erythroid colony formation. Analysis of cell differentiation by flow cytometry after culture for 4 or 7 days revealed that Mcl-1 knockdown resulted in reduced generation of both erythroid (GPA+) and myeloid (CD33+ and CD14+) cells. In contrast to the results of the initial siRNA studies, shRNA-mediated Mcl-1 knockdown also resulted in significantly increased apoptosis of normal CD34+ cells (12.6± 1.6% for control shRNA-transduced cells and 24.5± 0.9% for Mcl-1 knockdown cells) associated with reduced colony formation and reduced growth in culture (1.265e+006± 273,892 for control shRNA-transduced cells 589,000 ± 188,082 for Mcl-1 knockdown cells). We conclude that RNAi-mediated Mcl-1 knockdown inhibits CML CD34+ cell survival and proliferation and enhances apoptosis after IM treatment, but also reduces viability of normal CD34+ cells. Since Mcl-1 protein expression is subject to multiple levels of regulation, our results suggest that strategies to selectively target Mcl-1 regulatory mechanisms active in CML but not normal progenitors may be less toxic and have greater clinical utility than direct targeting of the protein. Disclosures: No relevant conflicts of interest to declare.

MLL Fusion Protein Driven AML Is Selectively Inhibited by Targeted Disruption of the MLL-PAFc Interaction

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 56-56 ◽  
Author(s):  
Andrew G. Muntean ◽  
Eric M Granowicz ◽  
Jay L. Hess
Keyword(s):  
Fusion Protein ◽  
Colony Formation ◽  
Fusion Proteins ◽  
Target Genes ◽  
Hox Genes ◽  
Conflicts Of Interest ◽  
Dominant Negative ◽  
Conditional Knockout ◽  
Transformed Cells ◽  
Mouse Bone Marrow

Abstract Abstract 56 Balanced chromosomal translocations of the MLL gene located on chromosome 11q23 result in the expression of a chimeric fusion proteins with enhanced transcriptional activity. The HOX genes and their co-factors, such as MEIS1 and PBX2, are critical downstream targets of MLL fusion proteins and essential for transformation. Previously we showed MLL fusion proteins are critically dependent on a direct interaction with the RNA Pol II Associated Factor complex (PAFc). PAFc is a protein complex important for the initiation, elongation and termination of transcription. It is also necessary for histone H2B K120 mono-ubiquitination through the direct recruitment of the BRE1/RAD6 E3 ubiquitin ligase complex. MLL fusion proteins make two direct contacts with the PAF1 and CTR9 subunits of the PAFc that are crucial for MLL fusion protein mediated transformation. Deletion of regions of MLL that interact with PAFc abrogates AML in mouse bone marrow transplantation assays. Here we tested the general requirement for PAFc in AML using a conditional knockout mouse model of one component of PAFc, Cdc73. These studies show that PAFc is necessary for growth of both E2A-HLF and MLL-AF9 transformed cells. Excision of Cdc73 leads to decreased expression of the MLL target genes Hoxa9 and Meis1, decreased colony formation and decreased proliferation of leukemic blasts and ultimately apoptosis. We then performed chromatin immunoprecipitation assays to assess the binding of PAFc and MLL to target loci with and without Cdc73. Excision of Cdc73 leads to a rapid decrease in association of PAFc as well as MLL fusion proteins and wild type MLL at target loci confirming that proper targeting of MLL fusion proteins requires PAFc. A decrease in H3K4me3 and H2Bub is also observed and consistent with a role of PAFc in the deposition of these epigenetic marks. We then sought to disrupt the MLL-PAFc interaction through expression of a small 40 amino acid fragment of MLL that interacts with the PAF1 subunit of PAFc. As the MLL-PAFc interaction involves interactions between MLL and both CTR9 and PAF1, it was unknown whether targeting one interaction site would be sufficient to disrupt transformation. Indeed, expression of the short fragment encompassing the pre-CxxC region of MLL acts as a dominant negative and disrupts the MLL-PAFc interaction, significantly decreasing Hox gene expression, colony formation and cell proliferation of MLL-AF9 transformed cells. Importantly, expression of the MLL fragment selectively inhibited MLL fusion mediated leukemic transformation and cell growth while the growth and proliferation of E2A-HLF cells is unaffected. Together these data show that targeting the MLL-PAFc interaction with a small MLL fragment can act as a dominant negative and selectively inhibit the growth of AML cells transformed with MLL fusion proteins. These data also suggest the MLL-PAF1 interaction surface is a promising region for therapeutic targeting. Disclosures: No relevant conflicts of interest to declare.

Genetic Models Demonstrate a Requirement of MEIS1 in Survival of MLL-AF9 Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 120-120 ◽  
Author(s):  
Jayeeta Roychoudhury ◽  
Jason P Clark ◽  
Kevin A. Link ◽  
Gang Huang ◽  
James C. Mulloy ◽  
...  
Keyword(s):  
Median Time ◽  
Colony Formation ◽  
Leukemia Cell ◽  
The Other ◽  
Tamoxifen Treatment ◽  
Leukemia Cells ◽  
Transformed Cells ◽  
Other Hand ◽  
Leukemia Development

Abstract Abstract 120 MEIS1 is highly expressed in several leukemias, particularly those associated with MLL-translocations. Over-expression of MEIS1 accelerates leukemia development in experimental models of leukemia while shRNA mediated knock-down of MEIS1 inhibits leukemia cell growth, suggesting that MEIS1 may be a suitable target for therapy. These previous studies are however marred by potential off-target effects and were carried out on cultured cell lines. We thus studied the role of MEIS1 using a more robust inducible-deletion system. Mice engineered to carry loxp sites flanking the homeodomain of MEIS1 (MEIS1-flox) were crossed with Rosa26CreER mice. Tamoxifen treatment of the resulting MEIS1-flox/CreER mice resulted in efficient recombination and deletion of MEIS1. We transformed MEIS1-flox/CreER or CreER bone marrow cells with various oncogenes delivered via retroviral transduction and then evaluated the effect of MEIS1-deletion. Transformed cells were cultured in methylcellulose supplemented with 4-hydroxy-tamoxifen (4-OHT) or vehicle (ethanol) and colony numbers were counted after 7 days of culture. Loss of MEIS1 significantly reduced colony formation in MLL-AF9 and MLL-GAS7 transformed cells by 74.7 ± 3.2%, 43.9 ± 2.5%, respectively compared to controls. To validate the specificity of this effect, we studied the effect of MEIS1-deletion on cells transformed by the AML1-ETO fusion oncogene. These leukemias display normal or low levels of MEIS1, suggesting that MEIS1 is unlikely to play a role in transformation by AML1-ETO. As expected, MEIS1 deletion had no effect on colony formation by AML1-ETO transformed cells. To substantiate the role of MEIS1 in a more physiologic model of leukemia, we crossed the MEIS1-flox/CreER mice with MLL-AF9 knock-in mice. The knock-in mice carry a single copy of the MLL-AF9 fusion gene in every cell and develop AML with a medial latency of ∼6 months. In methylcellulose culture assays, MEIS1 deletion decreased colony formation of the MLL-AF9 knock-in cells by 81.5 ± 2.8% compared to controls (1.9 ± 0.1%). Further analysis of leukemia cells showed that MEIS1 deletion led to significant increase in apoptosis (as measured by Annexin V staining) to 24.6 ± 4.0% compared to that in controls (0.4 ± 0.33%). To assess the role of MEIS1 in leukemia in vivo, we transplanted MLL-AF9/MEIS1-flox/CreER cells into irradiated mice. Two weeks after transplantation, recipient mice were treated with either Tamoxifen or vehicle (corn oil). Deletion of MEIS1 was confirmed by PCR analysis on peripheral blood. Survival analysis showed that the two control groups - vehicle treated MLL-AF9/MEIS1-flox/CreER mice and Tamoxifen-treated MLL-AF9/CreER mice - succumbed to leukemia with a median time of 130 and 147 days, respectively. On the other hand, MEIS1-deletion led to a significantly prolonged survival with median time of 250 days (p < 0.003, long rank test). Re-expression of non-deletable MEIS1 by retrovirus rescued leukemia-development in MEIS1-deleted mice, confirming that the effect was MEIS1-mediated. On the other hand, re-expression of MEIS1 lacking the homeodomain, carboxyl-terminal domain or the PBX1-interacting domain did not rescue leukemia development, indicating that these domains and interactions of MEIS1 play important roles in the maintenance of leukemia. Finally, to determine the molecular pathways regulated by MEIS1 in leukemia, we performed gene expression profiling of control and MEIS1-deleted MLL-AF9 knock-in leukemia cells using RNA-sequencing. Preliminary analysis of the data revealed that MEIS1-deletion led to decreased expression of genes associated with the hypoxia-response pathway. Collectively, these results demonstrate that MEIS1 is plays an essential role in MLL-AF9 leukemia wherein it promotes survival. Thus, MEIS1-targeting could be a useful therapeutic strategy. Our data will also provide novel information on the downstream targets of MEIS1 in leukemia, revealing pathways that may be more readily amenable to suppression by available or novel inhibitory-agents. Disclosures: No relevant conflicts of interest to declare.

Suppression of CML Progenitor but Not Stem Cells Requires Simultaneous Inhibition of KIT and BCR-ABL1.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2778-2778
Author(s):  
Zhimin Gu ◽  
Amie S. Corbin ◽  
Thomas O'Hare ◽  
Anna M. Eiring ◽  
Tian yi Zhang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Colony Formation ◽  
Kinase Activity ◽  
Colony Growth ◽  
Newly Diagnosed ◽  
Cd34 Cells ◽  
Simultaneous Inhibition

Abstract Abstract 2778 In chronic myeloid leukemia (CML), imatinib and other tyrosine kinase inhibitors (TKIs) inhibit BCR-ABL1 tyrosine kinase activity but also target additional kinases including KIT. The role of KIT inhibition in the therapeutic efficacy of TKIs is controversial. We used TKIs with selective activity against ABL (PPY-A) or KIT (BAW667) and genetic tools to assess the role of KIT signaling for growth of CML cell lines and primary CML progenitor and stem cells. In Mo7eBCR-ABL1 or newly diagnosed CML CD34+ progenitor cells, immunoblotting confirmed that PPY-A (1 μM) suppresses BCR-ABL1 phosphorylation but not KIT tyrosine phosphorylation. In contrast, treatment of cells with a KIT-blocking antibody (K44.2, 200ng/mL), shRNA targeting KIT (shKIT), or the KIT selective inhibitor BAW667 (1 μM), suppressed KIT activity without affecting BCR-ABL1 kinase activity. Therefore, these systems are suitable to isolate the role of BCR-ABL1 vs. KIT inhibition. Treatment of Mo7eBCR-ABL1 cells with PPY-A resulted in suppression of growth by 91.7% (p<0.003). When PPY-A was combined with KIT activation by SCF, proliferation was restored, indicating KIT signaling must be inactivated to induce cell death by BCR-ABL1 inhibition. Immunoblot analysis of Mo7eBCR-ABL1 cells revealed that culture in SCF rapidly activated AKT and ERK1/2 in the presence but not absence of PPY-A. Simultaneous inhibition of AKT with LY294002 abolished SCF-mediated rescue of cell proliferation, whereas ERK1/2 inhibition with PD98059 only partially abrogated SCF rescue. These data indicate that SCF rescue of Mo7eBCR-ABL1 cells upon BCR-ABL1 inhibition critically depends on AKT. To assess BCR-ABL1 vs. KIT inhibition in primary cells, CD34+ cells from newly diagnosed CML patients (n=4) and normal controls (n=3) were cultured in semisolid medium supplied with IL-3 and GM-CSF (no SCF), in the presence of 1 μM PPY-A combined with shKIT or 1 μM BAW667. KIT inhibition by shKIT or 1 μM BAW667 reduced CFU-GM formation by 40% compared to controls (p<0.04) even in the absence of SCF, with no effects were seen in normal CD34+ cells, indicating that BCR-ABL1-dependent KIT activation occurs in the absence of SCF stimulation. PPY-A reduced colony formation by 54.7%, while PPY-A plus shKIT and PPY-A plus BAW667 suppressed CFU-GM colony formation by 79.7% and 72.1%, comparable to the effects of imatinib (71.9%). Addition of SCF partially rescued colony growth from the effects of PPY-A, consistent with results on Mo7eBCR-ABL1 cells. In a separate set of experiments lineage-negative (Lin−) cells from newly diagnosed patients (n=4) were cultured on HS-5 stromal cells containing K44.2, PPY-A, K44.2 plus PPY-A or 2 mM imatinib, followed by clonogenic assays. Only the PPY-A / K44.2 combination suppressed CFU-GM; isolated BCR-ABL1 or KIT block did not. These data demonstrate that both BCR-ABL1 and KIT contribute to CML progenitor cell survival under physiologically relevant conditions, and that inhibition of both pathways is required for imatinib-mediated suppression of CML progenitor cells. To assess the role of KIT vs. BCR-ABL1 inhibition on primitive CML cells we performed long-term culture-initiating cell (LTC-IC) assays on M2–10B4 murine stromal cells, using Lin− cells from newly diagnosed patients (n=3). Cultures were performed with K44.2, PPY-A, K44.2 plus PPY-A or 2 mM imatinib, with colonies plated at 1, 3, and 6 weeks. At 1 week colonies were reduced by 30% with K44.2 and 70% with PPY-A, but by 90% with the PPY-A / K44.2 combination or with imatinib. In contrast, at 6 weeks colony formation was unaffected by K44.2 but reduced by >95% with PPY-A, the PPY-A / K44.2 combination or imatinib. Week 3 colony growth was intermediate. Consistent with the LTC-IC assay, KIT inhibition with BAW667 enhanced PPY-A suppression of colony formation in Lin−CD34+CD38+ progenitor cells from newly diagnosed patients (n=3) by 18.7% (p<0.05), with no significant effect on primitive Lin−CD34+CD38− cells (7.7%, p=ns). Our findings suggest KIT inhibition is much more critical for suppression of mature progenitors compared to primitive CML cells. Since AKT is active in CML progenitors but suppressed by TGFβ in stem cells (Nature, 2010;463(7281):676; JCI, 2011;121(1):396), we speculate that upon BCR-ABL1 inhibition CML progenitors but not stem cells switch to an SCF-dependent mode of AKT activation, which renders these cells uniquely sensitive to dual inhibition of BCR-ABL1 and KIT signaling. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The scaffolding function of the RLTPR protein explains its essential role for CD28 co-stimulation in mouse and human T cells

2016 ◽  
Vol 213 (11) ◽  
pp. 2437-2457 ◽  
Author(s):  
Romain Roncagalli ◽  
Margot Cucchetti ◽  
Nicolas Jarmuzynski ◽  
Claude Grégoire ◽  
Elise Bergot ◽  
...  
Keyword(s):  
T Cells ◽  
Signaling Pathway ◽  
Affinity Purification ◽  
Mass Spectrometry Analysis ◽  
Pleckstrin Homology Domain ◽  
Spectrometry Analysis ◽  
Human T Cells ◽  
Repeat Domain ◽  
Human And Mouse

The RLTPR cytosolic protein, also known as CARMIL2, is essential for CD28 co-stimulation in mice, but its importance in human T cells and mode of action remain elusive. Here, using affinity purification followed by mass spectrometry analysis, we showed that RLTPR acts as a scaffold, bridging CD28 to the CARD11/CARMA1 cytosolic adaptor and to the NF-κB signaling pathway, and identified proteins not found before within the CD28 signaling pathway. We further demonstrated that RLTPR is essential for CD28 co-stimulation in human T cells and that its noncanonical pleckstrin-homology domain, leucine-rich repeat domain, and proline-rich region were mandatory for that task. Although RLTPR is thought to function as an actin-uncapping protein, this property was dispensable for CD28 co-stimulation in both mouse and human. Our findings suggest that the scaffolding role of RLTPR predominates during CD28 co-stimulation and underpins the similar function of RLTPR in human and mouse T cells. Along that line, the lack of functional RLTPR molecules impeded the differentiation toward Th1 and Th17 fates of both human and mouse CD4+ T cells. RLTPR was also expressed in both human and mouse B cells. In the mouse, RLTPR did not play, however, any detectable role in BCR-mediated signaling and T cell-independent B cell responses.

scholarly journals Interleukin 13: novel role in direct regulation of proliferation and differentiation of primitive hematopoietic progenitor cells.

1994 ◽  
Vol 180 (1) ◽  
pp. 75-82 ◽  
Author(s):  
S E Jacobsen ◽  
C Okkenhaug ◽  
O P Veiby ◽  
D Caput ◽  
P Ferrara ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Colony Formation ◽  
Biological Activities ◽  
Hematopoietic Progenitor Cells ◽  
Synergistic Activity ◽  
Hematopoietic Progenitor ◽  
Interleukin 13 ◽  
Gm Csf ◽  
Macrophage Colony

The recently cloned interleukin 13 (IL-13) shares most investigated biological activities on B lymphocytes and monocytes with IL-4. In this study we investigated for the first time the potential role of IL-13 in the regulation of the growth of hematopoietic progenitor cells. IL-13 enhanced stem cell factor (SCF)-induced proliferation of Lin-Sca-1+ bone marrow progenitor cells more potently than IL-4. The effect of IL-13 was purely synergistic, since IL-13 alone stimulated no colony formation. Single cell experiments suggested that the synergistic effect of IL-13 on Lin-Sca-1+ progenitors was directly mediated. In contrast, IL-13 had no synergistic activity on SCF-induced proliferation of the more mature Lin-Sca-1- progenitor cells. Thus, the cloning frequency in response to SCF + IL-13 was at least 20-fold higher in the Lin-Sca-1+ than the Lin-Sca-1- progenitor cell population. Furthermore, IL-13 but not IL-4 synergistically enhanced colony formation of Lin-Sca-1+ progenitors in response to granulocyte/macrophage colony-stimulating factor (GM-CSF) (threefold), whereas both IL-4 and IL-13 enhanced G-CSF-induced colony formation (threefold), and neither of the two significantly affected CSF-1 and IL-3-induced proliferation. Finally, whereas stimulation of Lin-Sca-1+ progenitors by SCF + G-CSF resulted in the formation of 90% granulocytes, the addition of IL-13 resulted in the production of macrophages exclusively. This novel effect on differentiation was directly mediated, shared with IL-4, and could not be observed on Lin-Sca-1- progenitor cells. Collectively, these findings indicate a novel role of IL-13 in early myelopoiesis, partially overlapping but also different from that of IL-4.

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