The C-Terminus of the CBFβ-SMMHC Fusion Protein Is Required for the Myeloblastic Transformation of inv16 Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3799-3799
Author(s):  
Yasuhiko Kamikubo ◽  
Lisa Garrett-Beal ◽  
Martha Kirby ◽  
Pu Paul Liu
Keyword(s):  
Fusion Protein ◽  
Myeloid Leukemia ◽  
Fusion Gene ◽  
Homozygous Deletion ◽  
Double Positive ◽  
Developmental Defects ◽  
C Terminus ◽  
Almost All

Abstract Inv(16)(p13q22) is found in almost all human acute myeloid leukemia (AML) subtype M4Eo cases and forms a fusion gene CBFB-MYH11, which encodes a fusion protein CBFβ–SMMHC. CBFβ forms a heterodimeric transcription factor with RUNX1 and both are required for embryonic hematopoiesis, while SMMHC is the smooth muscle myosin heavy chain. Using knock-in mouse strategy, we previously demonstrated that Cbfb+/MYH11 F1 embryos have severe blockage of definitive hematopoiesis and die from CNS hemorrhage. The phenotype was similar to that of embryos with homozygous deletion of Cbfb or Runx1, suggesting that Cbfb-MYH11 dominantly represses Runx1/CBFb function. We further demonstrated that Cbfb-MYH11 is necessary but not sufficient for leukemia to develop, as the Cbfb+/MYH11 mice required additional mutations for leukemogenesis. Several hypotheses have been proposed, based on in vitro studies, to explain how CBFβ-SMMHC dominantly inhibits RUNX1/CBFβ. The C-terminal region of CBFβ-SMMHC is responsible for multimerization and also interacts with corepressors; thus this region might be critical for RUNX1 repression. To determine the importance of this multimerization domain in vivo, we generated knock-in mice expressing CBFβ-SMMHC with a 95 aa C-terminal deletion (CBFβ-SMMHCdC95), which truncates this multimerization/repression domain. CBFβ-SMMHCdC95 expressing F1 heterozygous embryos (Cbfb+/MYH11dC95) developed normally with no hematopoietic defects and no hemorrhage. Hematopoiesis was normal in the adult Cbfb+/MYH11dC95 mice except for mild increase of mature neutrophils and minor T cell developmental defects in the first year. However, the mice proceeded to a lethal myeloproliferative disorder (MPD) during their second year of life. There was a significant increase of Mac1/Gr1 double positive cells in the peripheral blood and the spleen, which were negative for the stem cell/progenitor cell marker, c-kit. Morphologically the erythrocytes and neutrophils were dysplastic, and the mice developed severe splenomegaly. ENU treatment of the Cbfb+/MYH11dC95 mice accelerated the development of the MPD phenotype but could not induce overt, transplantable, myeloid leukemia. These data suggest that the multimerizatin domain of SMMHC is important for both hematopoiesis blockage and leukemogenesis, especially the blastic transformation of inv16 leukemia.

scholarly journals Plag1 and Plagl2 are oncogenes that induce acute myeloid leukemia in cooperation with Cbfb-MYH11

Blood ◽  
2005 ◽  
Vol 105 (7) ◽  
pp. 2900-2907 ◽  
Author(s):  
Sean F. Landrette ◽  
Ya-Huei Kuo ◽  
Karen Hensen ◽  
Sahar Barjesteh van Waalwijk van Doorn-Khosrovani ◽  
Paola N. Perrat ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Fusion Protein ◽  
Myeloid Leukemia ◽  
Fusion Gene ◽  
Cell Renewal ◽  
Chromosome 16 ◽  
Hematopoietic Progenitors ◽  
Acute Myeloid

AbstractRecurrent chromosomal rearrangements are associated with the development of acute myeloid leukemia (AML). The frequent inversion of chromosome 16 creates the CBFB-MYH11 fusion gene that encodes the fusion protein CBFβ-SMMHC. This fusion protein inhibits the core-binding factor (CBF), resulting in a block of hematopoietic differentiation, and induces leukemia upon the acquisition of additional mutations. A recent genetic screen identified Plag1 and Plagl2 as CBFβ-SMMHC candidate cooperating proteins. In this study, we demonstrate that Plag1 and Plagl2 independently cooperate with CBFβ-SMMHC in vivo to efficiently trigger leukemia with short latency in the mouse. In addition, Plag1 and Plagl2 increased proliferation by inducing G1 to S transition that resulted in the expansion of hematopoietic progenitors and increased cell renewal in vitro. Finally, PLAG1 and PLAGL2 expression was increased in 20% of human AML samples. Interestingly, PLAGL2 was preferentially increased in samples with chromosome 16 inversion, suggesting that PLAG1 and PLAGL2 may also contribute to human AML. Overall, this study shows that Plag1 and Plagl2 are novel leukemia oncogenes that act by expanding hematopoietic progenitors expressing CbFβ-SMMHC.

scholarly journals Gp37 Regulates the Pathogenesis of Avian Leukosis Virus Subgroup J via Its C Terminus

2020 ◽  
Vol 94 (11) ◽  
Author(s):  
Tuofan Li ◽  
Xiaohui Yao ◽  
Chunping Li ◽  
Jun Zhang ◽  
Quan Xie ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Antiviral Drug ◽  
Underlying Mechanism ◽  
Vital Role ◽  
Significant Loss ◽  
Viral Shedding ◽  
C Terminus ◽  
New Perspective

ABSTRACT Different from other subgroups of avian leukosis viruses (ALVs), ALV-J is highly pathogenic. It is the main culprit causing myeloid leukemia and hemangioma in chickens. The distinctiveness of the env gene of ALV-J, with low homology to those of other ALVs, is linked to its unique pathogenesis, but the underlying mechanism remains unclear. Previous studies show that env of ALV-J can be grouped into three species based on the tyrosine motifs in the cytoplasmic domain (CTD) of Gp37, i.e., the inhibitory, bifunctional, and active groups. To explore whether the C terminus or the tyrosine motifs in the CTD of Gp37 affect the pathogenicity of ALV-J, a set of ALV-J infectious clones containing different C termini of Gp37 or the mutants at the tyrosine sites were tested in vitro and in vivo. Viral growth kinetics indicated not only that ALV-J with active env is the fastest in replication and ALV-J with inhibitory env is the lowest but also that the tyrosine sites essentially affected the replication of ALV-J. Moreover, in vivo studies demonstrated that chickens infected by ALV-J with active or bifunctional env showed higher viremia, cloacal viral shedding, and viral tissue load than those infected by ALV-J with inhibitory env. Notably, the chickens infected by ALV-J with active or bifunctional env showed significant loss of body weight compared with the control chickens. Taken together, these findings reveal that the C terminus of Gp37 plays a vital role in ALV-J pathogenesis, and change from inhibitory env to bifunctional or active env increases the pathogenesis of ALV-J. IMPORTANCE ALV-J can cause severe immunosuppression and myeloid leukemia in infected chickens. However, no vaccine or antiviral drug is available against ALV-J, and the mechanism for ALV-J pathogenesis needs to be elucidated. It is generally believed that gp85 and LTR of ALV contribute to its pathogenesis. Here, we found that the C terminus and the tyrosine motifs (YxxM, ITIM, and ITAM-like) in the CTD of Gp37 of ALV-J could affect the pathogenicity of ALV-J in vitro and in vivo. The pathogenicity of ALV-J with Gp37 containing ITIM only was significantly less than ALV-J with Gp37 containing both YxxM and ITIM and ALV-J with Gp37 containing both YxxM and ITAM-like. This study highlights the vital role of the C terminus of Gp37 in the pathogenesis of ALV-J and thus provides a new perspective to elucidate the interaction between ALV-J and its host and a molecular basis to develop efficient strategies against ALV-J.

MLL-AF9 and MLL-ENL Induce Deregulation of Similar Downstream Candidate Genes: An Analysis across Experimental Protocols and Laboratories.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3372-3372
Author(s):  
Ashish R. Kumar ◽  
Robert K. Slany ◽  
Jay L. Hess ◽  
John H. Kersey
Keyword(s):  
Gene Expression ◽  
Fusion Protein ◽  
Fusion Gene ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Expression Systems ◽  
Rt Pcr ◽  
Conditional Expression

Expression profiling has become an important tool for understanding gene deregulation in MLL-fusion leukemias. However, the results of gene profiling experiments are difficult to interpret when applied to leukemia cells because (i) leukemias arise in cells that differ greatly in their gene expression profiles, and (ii) leukemias most often require secondary genetic events in addition to the MLL fusion gene. Two principal model systems have been used to understand the direct effects of MLL-fusion genes. Knock-in models have the advantage of the fusion gene being under control of the physiologic promoter. On the other hand, conditional expression systems offer the ability to conduct short term experiments, permitting the analysis of direct effects on downstream genes. In the present combined-analysis, we used the Affymetrix U74Av2 oligonucleotide microarray to evaluate the effects of the MLL-fusion gene in vivo and in vitro respectively using two closely related MLL fusion genes - MLL-AF9 for knock-in and MLL-ENL for conditional expression. In the MLL-AF9 study, we compared gene expression profiles of bone marrow cells from MLL-AF9 knock-in mice (C57Bl/6, MLL-AF9+/−) to those of age-matched wild type mice (Kumar et. al. 2004, Blood). We used a t-test (p<0.05) to selected genes that showed significant changes in expression levels. In the MLL-ENL study, we transformed murine primary hematopoietic cells with a conditional MLL-ENL vector (MLL-ENL fused to the modified ligand-binding domain of the estrogen receptor) such that the fusion protein was active only in the presence of tamoxifen. We then studied the downstream effects of the fusion protein by comparing gene expression profiles of the cells in the presence and absence of tamoxifen. We used a pair-wise comparison analysis to select genes that showed a change in expression level of 1.5 fold or greater in at least two of three experiments (Zeisig et. al. 2004, Mol. Cell Biol.). Those genes that were up-regulated in both datasets were then compiled together. This list included Hoxa7, Hoxa9 and Meis1. The results for these 3 genes were confirmed by quantitative RT-PCR in both the MLL-AF9-knock-in and the MLL-ENL-conditional-expression systems. The remaining candidate genes in the common up-regulated gene set (not yet tested by quantitative RT-PCR) include protein kinases (Bmx, Mapk3, Prkcabp, Acvrl1, Cask), RAS-associated proteins (Rab7, Rab3b), signal transduction proteins (Notch1, Eat2, Shd, Fpr1), cell membrane proteins (Igsf4), chaperones (Hsp70.2), transcription factors (Isgf3g), proteins with unknown functions (Olfm1, Flot1), and hypothetical proteins. The results of the combined analysis demonstrate that these over-expressions are (i) a direct and sustained effect of the MLL-fusion protein, (ii) are independent of secondary events that might be involved in leukemogensis, and (iii) are independent of the two partner genes that participate in these fusions. The over-expression of a few genes in both the -in vitro and in vivo experimental systems makes these molecules very interesting for further studies, to understand the biology of MLL-fusion leukemias and for development of new therapeutic strategies.

scholarly journals Ezh2 augments leukemogenicity by reinforcing differentiation blockage in acute myeloid leukemia

Blood ◽  
2012 ◽  
Vol 120 (5) ◽  
pp. 1107-1117 ◽  
Author(s):  
Satomi Tanaka ◽  
Satoru Miyagi ◽  
Goro Sashida ◽  
Tetsuhiro Chiba ◽  
Jin Yuan ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Target Genes ◽  
Fusion Gene ◽  
Hematologic Malignancies ◽  
Leukemic Cells ◽  
Polycomb Repressive Complex 2 ◽  
Acute Myeloid

Abstract EZH2, a catalytic component of the polycomb repressive complex 2, trimethylates histone H3 at lysine 27 (H3K27) to repress the transcription of target genes. Although EZH2 is overexpressed in various cancers, including some hematologic malignancies, the role of EZH2 in acute myeloid leukemia (AML) has yet to be examined in vivo. In the present study, we transformed granulocyte macrophage progenitors from Cre-ERT;Ezh2flox/flox mice with the MLL-AF9 leukemic fusion gene to analyze the function of Ezh2 in AML. Deletion of Ezh2 in transformed granulocyte macrophage progenitors compromised growth severely in vitro and attenuated the progression of AML significantly in vivo. Ezh2-deficient leukemic cells developed into a chronic myelomonocytic leukemia–like disease with a lower frequency of leukemia-initiating cells compared with the control. Chromatin immunoprecipitation followed by sequencing revealed a significant reduction in the levels of trimethylation at H3K27 in Ezh2-deficient leukemic cells, not only at Cdkn2a, a known major target of Ezh2, but also at a cohort of genes relevant to the developmental and differentiation processes. Overexpression of Egr1, one of the derepressed genes in Ezh2-deficient leukemic cells, promoted the differentiation of AML cells profoundly. Our findings suggest that Ezh2 inhibits differentiation programs in leukemic stem cells, thereby augmenting their leukemogenic activity.

SRPK1 Is a Therapeutic Vulnerability in Acute Myeloid Leukemia through Its Effects on Alternative Isoforms of Epigenetic Regulators Including BRD4

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 781-781
Author(s):  
Konstantinos Tzelepis ◽  
Etienne De Braekeleer ◽  
Isaia Barbieri ◽  
Vijay Baskar ◽  
Demetrios Aspris ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Fusion Gene ◽  
Ectopic Expression ◽  
Pharmacological Inhibition ◽  
Epigenetic Regulators ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is an aggressive cancer with a poor prognosis, for which the therapeutic landscape has changed little for decades. Aberrant mRNA splicing plays an important role in cancer development and genes coding for several of the major components of the spliceosome are targeted by somatic mutations in several cancers including myelodysplastic syndromes and AML. Recently, myeloid neoplasms harbouring spliceosome gene mutations were shown to be preferentially susceptible to pharmacological disruption of the spliceosome. Here we report that targeting particular pathways of the spliceosome machinery can also be an effective therapeutic strategy in other types of AML. Recently, we generated a comprehensive catalogue of genetic vulnerabilities in AML using CRISPR-Cas9 genome-wide recessive screens and reported several novel intuitive and non-intuitive therapeutic candidates. Amongst these we identified SRPK1, the gene coding for a serine-threonine kinase that phosphorylates the major spliceosome protein SRSF1. Here, we demonstrate that targeted genetic disruption of SRPK1 in MLL-rearranged AMLs leads to differentiation and apoptosis (Fig. 1A). Additionally, the survival of immunocompromised mice transplanted with human AML cell lines carrying the MLL-AF9 fusion gene, namely MOLM-13 and THP-1, was significantly prolonged by genetic disruption of SRPK1 with CRISPR-Cas9. Similar effects were seen with pharmacological inhibition of SRPK1 in vitro and in vivo, using the novel SRPK1-specific kinase inhibitor SPHINX31 (Fig. 1B-C). Importantly, we go on to demonstrate that, while the SRPK1 kinase activity is required for AML cell survival, it is dispensable for normal hematopoiesis. At the molecular level, we show that genetic or pharmacological inhibition of SRPK1 was associated with widespread changes in the splicing of multiple genes including several with roles in leukemogenesis such as MYB, BRD4 and MED24 . We focused on BRD4 as its splicing isoforms have distinct molecular properties and found that SRPK1 inhibition led to a substantial switch from the short (BRD4S) to the long (BRD4L) isoform at the mRNA and protein levels (Fig. 1D-E). This was associated with BRD4 eviction from genomic loci involved in myeloid leukemogenesis including BCL2 and MYC. Notably, ectopic expression of the short (BRD4S) isoform rescued the phenotype of SRPK1 inhibition suggesting that the observed BRD4 splicing switch mediates at least part of the anti-leukemic effects of SRPK1 inhibition. Furthermore, we show that the BRD inhibitor iBET-151 synergizes with SRPK1 inhibition to kill human MLL-AF9 -driven AMLs in vitro and in vivo. Collectively our findings reveal that SRPK1 is required for normal splicing of key epigenetic regulators including BRD4 and represents a novel therapeutic vulnerability in AML that can be used alone or in combination with clinically relevant epigenetic drugs to enhance their anti-leukemic effects. Disclosures No relevant conflicts of interest to declare.

PML-RARα Deregulates an Unexpectedly Small Number of Genes in Pre-Leukemic Promyelocytes

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3526-3526
Author(s):  
Coline M Gaillard ◽  
Taku A Tokuyasu ◽  
Emmanuelle Passegué ◽  
Scott C. Kogan
Keyword(s):  
Bone Marrow ◽  
Fusion Protein ◽  
Conflicts Of Interest ◽  
Fusion Gene ◽  
Leukemic Cells ◽  
Cytokine Signaling ◽  
Proliferation Capacity ◽  
Number Of Genes

Abstract Abstract 3526 Background: Acute Promyelocytic Leukemia (APL) is characterized by the accumulation in the blood and bone marrow of abnormal promyelocytes, which have the ability to transfer the disease to secondary recipients in animal models. The PML-RARα fusion protein is thought to be the primary abnormality implicated in the pathology, and is believed to prevent transcription of genes necessary for normal myeloid development and differentiation. Identifying PML-RARα targets is critical for understanding the road to leukemic transformation. However, such targets have so far been identified using cell line assays in vitro, murine cells differentiated into promyelocytes in vitro, or fully transformed murine or human leukemic cells. Focusing on the cell population in which the transforming potential is acquired, we describe here a novel strategy to identify the transcriptomic dysregulation induced by PML-RARα expression in maturing myeloid populations in vivo. Methods: We utilize a murine model of human APL in which the human PML-RARα fusion gene is expressed under the control of the MRP8 promoter, driving its expression in maturing myeloid populations. Those animals can be described as pre-leukemic since they eventually develop leukemia when additional mutations occur. Fresh bone marrows from normal (Fvb/n) or pre-leukemic (PML-RARα) animals were harvested. Using an improved cell surface antigen staining strategy and fluorescence-activated cell sorting, three populations of increasingly differentiated myeloid populations have been sorted (Granulocyte Macrophage Progenitor, Early promyelocyte and Late promyelocyte). RNA was extracted and submitted for whole-genome microarray analysis. In addition, we are using a variety of bioinformatics approaches to decipher the network of novel interactions driven by PML-RARα expression. Results: Markers used in our sorting strategy were validated in the dataset, including CD34 and Gr1. In the normal samples, markers of neutrophil maturation increased, largely as expected, and a number of early transcription factors decreased in an expected manner including Hoxa9 and Meis1. One remarkable finding was that despite the previously described ability of PML-RARα to regulate transcription from multiple sites in the genome, only a small number of genes were differentially impacted by the expression of this protein. Surprisingly, well-known regulators of myeloid differentiation that have been implicated in the retinoic acid responsiveness of APL including Sfpi1 (PU.1) and Cebpa were not differentially expressed. However, in pre-leukemic samples PML-RARα did cause decreased expression of multiple neutrophilic granule genes including Ltf, Mmp9 and Ngp. The gene most upregulated in the pre-leukemic samples was Spp1 which encodes the osteopontin phosphoprotein. Of interest, we identified the myeloid tumor suppressor Irf8 to be downregulated 5 fold in the presence of PML-RARα. To investigate the importance of IRF8 levels in APL initiation, we transplanted Irf8+/+ PML-RARα or Irf8+/− PML-RARα bone marrow into irradiated recipients. Despite the potential for decreased expression of IRF8 to contribute to APL, we observed no difference. This result does not confirm a role for IRF8 in APL pathogenesis, but further investigations are needed to exclude such a role. Bioinformatics studies highlighted enrichment in cell cycle-related genes upon PML-RARα expression, suggesting a possible difference in the proliferation capacity of the pre-leukemic cells, which is currently under investigation. Conclusions: We found that in vivo the transcriptome was only modestly dysregulated by the presence of PML-RARα. These observations open up new questions on the role of the fusion protein in pathogenesis: How does PML-RARα prime pre-leukemic cells for full transformation? How do secondary events allow an initiated cell to advance to a fully transformed state? Such questions are currently being investigated, with a special interest on looking at the cooperation between PML-RARα and activated cytokine signaling in leukemia initiation. Disclosures: No relevant conflicts of interest to declare.

Induction of acute myeloid leukemia in mice by the human leukemia-specific fusion gene NUP98-HOXD13 in concert with Meis1

Blood ◽  
2003 ◽  
Vol 101 (11) ◽  
pp. 4529-4538 ◽  
Author(s):  
Nicolas Pineault ◽  
Christian Buske ◽  
Michaela Feuring-Buske ◽  
Carolina Abramovich ◽  
Patty Rosten ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Hox Genes ◽  
Fusion Gene ◽  
Disease Onset ◽  
Human Leukemia ◽  
Hoxa Cluster ◽  
Acute Myeloid

Abstract HOX genes, notably members of the HOXA cluster, and HOX cofactors have increasingly been linked to human leukemia. Intriguingly, HOXD13, a member of the HOXD cluster not normally expressed in hematopoietic cells, was recently identified as a partner of NUP98 in a t(2;11) translocation associated with t-AML/MDS. We have now tested directly the leukemogenic potential of the NUP98-HOXD13 t(2; 11) fusion gene in the murine hematopoietic model. NUP98-HOXD13 strongly promoted growth and impaired differentiation of early hematopoietic progenitor cells in vitro; this effect was dependent on the NUP98 portion and an intact HOXD13 homeodomain. Expression of the NUP98-HOXD13 fusion gene in vivo resulted in a partial impairment of lymphopoiesis but did not induce evident hematologic disease until late after transplantation (more than 5 months), when some mice developed a myeloproliferative-like disease. In contrast, mice transplanted with bone marrow (BM) cells cotransduced with NUP98-HOXD13 and the HOX cofactor Meis1 rapidly developed lethal and transplantable acute myeloid leukemia (AML), with a median disease onset of 75 days. In summary, this study demonstrates that NUP98-HOXD13 can be directly implicated in the molecular process leading to leukemic transformation, and it supports a model in which the transforming properties of NUP98-HOXD13 are mediated through HOX-dependent pathways.

scholarly journals C-Terminal Domain of ABL Family Kinases, ABL and ARG, Defines Their Distinct Leukemogenic Activities in Vivo

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2368-2368 ◽  
Author(s):  
Asumi Yokota ◽  
Hideyo Hirai ◽  
Tsukimi Shoji ◽  
Taira Maekawa ◽  
Keiko Okuda
Keyword(s):  
Bone Marrow ◽  
Mast Cells ◽  
Tyrosine Kinase ◽  
Fusion Proteins ◽  
Myeloid Leukemia ◽  
Bone Marrow Cells ◽  
Terminal Domain ◽  
C Terminus

Abstract ARG (ABL2) is a member of ABL family kinases and highly homologous to ABL (ABL1) except the C-terminal domain adjacent to the kinase domain. TEL/ARG that consists of ARG fused to TEL (ETV6) has been found in AML M3, M4 or T-ALL patients, with additional chromosomal abnormalities of t(15;17)(q12;q21), inv(16)(p13;q12) or t(1;10;12)(q25;q23;p13) translocation, respectively. The structure of TEL/ARG is similar to that of TEL/ABL, which has been found in patients with T-ALL, B-ALL, AML and CML. TEL mediates homo-oligomerization of these fusion proteins, TEL/ABL and TEL/ARG, resulting in constitutive activation of the tyrosine kinases. Although ABL fusion proteins such as BCR/ABL and TEL/ABL have been intensively investigated, the involvement of TEL/ARG in leukemogenesis is not fully elucidated yet. We have recently reported that in vitro transforming activity of TEL/ARG was significantly lower than that of TEL/ABL although their kinase activities were almost identical. Interestingly, the in vitro transforming activities of C-terminus-swapped mutants, TEL/ABL with C-terminal domain of ARG [TEL-ABL (ARG-C)] or TEL/ARG with C-terminal domain of ABL [TEL/ARG (ABL-C)], were comparable to those of TEL/ARG or TEL/ABL, respectively, while kinase activities in the swapped mutants were not altered. These results suggest that C-termini of ABL family kinases contain some functional domain that defines their distinct transforming activities. The purpose of this study is to compare the in vivo leukemogenic activities of TEL/ABL and TEL/ARG, and evaluate the impact of the C-terminal domains. First, we investigated whether TEL/ABL or TEL/ARG caused leukemia in mice. Each fusion gene together with GFP gene was retrovirally transduced into the bone marrow cells harvested from C57BL/6 mice treated with 5-fluorouracil, and the transduced cells were transplanted into lethally irradiated mice. Similar to BCR/ABL, transplantation of TEL/ABL-transduced cells induced rapid myeloproliferative status accompanied by hepatomegaly and/or splenomegaly, and all the recipient mice died within 33 days after transplantation, indicating the development of myeloid leukemia. In contrast, the recipient mice transplanted with TEL/ARG-transduced cells did not develop myeloid leukemia but infiltrative mastocytosis, and died around 200 days after transplantation (Figure 1). Hemophagocytic mast cells accumulating in the bone marrow, and mast cells circulating in the peripheral blood were also observed in these mice. Next we investigated the roles of C-terminal domains of ABL and ARG in their in vivo leukemogenic activities. C-terminus-swapped mutants, TEL/ABL (ARG-C) and TEL/ARG (ABL-C) were retrovirally transduced into bone marrow cells and the transduced cells were transplanted as described above. Intriguingly, TEL/ABL (ARG-C) mutant failed to cause myeloproliferative status or leukemia at day 153 (Figure 2A). On the other hand, TEL/ARG (ABL-C) induced lethal myeloid leukemia in 4 out of 13 mice (30.8%) within 111 days after transplantation (Figure 2B). Collectively, the in vivo phenotypes induced by TEL/ABL (ARG-C) or TEL/ARG (ABL-C) resembled those induced by TEL/ARG or TEL/ABL, respectively. Mastocytosis, a characteristic of TEL-ARG-induced phenotype, has not been observed so far in any of the recipients of TEL/ABL (ARG-C) or TEL/ARG (ABL-C). In conclusion, these results indicate that C-terminal domain of ABL family kinases defines their distinct leukemogenic activities in vivo through modulating both proliferation and differentiation. Notably, C-terminus of ARG strongly suppressed the in vivo leukemogenic activity of TEL/ABL without impairing the tyrosine kinase activity. Further clarification of the molecular mechanisms underlying the suppressive activity of C-terminus of ARG will lead to development of a novel therapeutic strategy, especially for patients with CML harboring mutations, which are resistant to tyrosine kinase inhibitors. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

scholarly journals Intracellular delivery of anti-BCR/ABL antibody by PLGA nanoparticles suppresses the oncogenesis of chronic myeloid leukemia cells

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Guoyun Jiang ◽  
Zhenglan Huang ◽  
Ying Yuan ◽  
Kun Tao ◽  
Wenli Feng
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Myeloid Leukemia ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Fusion Gene ◽  
Intracellular Delivery ◽  
Tyrosine Kinase Activity

Abstract Background The pathogenesis of chronic myeloid leukemia (CML) is the formation of the BCR/ABL protein, which is encoded by the bcr/abl fusion gene, possessing abnormal tyrosine kinase activity. Despite the wide application of tyrosine kinase inhibitors (TKIs) in CML treatment, TKIs drug resistance or intolerance limits their further usage in a subset of patients. Furthermore, TKIs inhibit the tyrosine kinase activity of the BCR/ABL oncoprotein while failing to eliminate the pathologenic oncoprotein. To develop alternative strategies for CML treatment using therapeutic antibodies, and to address the issue that antibodies cannot pass through cell membranes, we have established a novel intracellular delivery of anti-BCR/ABL antibodies, which serves as a prerequisite for CML therapy. Methods Anti-BCR/ABL antibodies were encapsulated in poly(d, l-lactide-co-glycolide) nanoparticles (PLGA NPs) by a double emulsion method, and transferrin was labeled on the surface of the nanoparticles (Ab@Tf-Cou6-PLGA NPs). The characteristics of nanoparticles were measured by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Cellular uptake of nanoparticles was measured by flow cytometry (FCM). The effect of nanoparticles on the apoptosis and proliferation of CML cells was testified by FCM and CCK-8 assay. In addition, the anti-cancer impact of nanoparticles was evaluated in mouse models of CML. Results The results demonstrated that the Ab@Tf-Cou6-PLGA NPs functioned as an intracellular deliverer of antibodies, and exhibited an excellent effect on degrading BCR/ABL oncoprotein in CML cells via the Trim-Away pathway. Treatment with Ab@Tf-Cou6-PLGA NPs inhibited the proliferation and induced the apoptosis of CML cells in vitro as well as impaired the oncogenesis ability of CML cells in vivo. Conclusions In conclusion, our study indicated that this approach achieved safe and efficient intracellular delivery of antibodies and degraded BCR/ABL oncoprotein via the Trim-Away pathway, which provides a promising therapeutic strategy for CML patients, particularly those with TKI resistance.

scholarly journals The UreEF Fusion Protein Provides a Soluble and Functional Form of the UreF Urease Accessory Protein

2006 ◽  
Vol 188 (24) ◽  
pp. 8413-8420 ◽  
Author(s):  
Jong Kyong Kim ◽  
Scott B. Mulrooney ◽  
Robert P. Hausinger
Keyword(s):  
Fusion Protein ◽  
Metal Binding ◽  
Accessory Proteins ◽  
Wild Type ◽  
Binding Properties ◽  
C Terminus ◽  
Urease Accessory Protein

ABSTRACT Four accessory proteins (UreD, UreE, UreF, and UreG) are typically required to form the nickel-containing active site in the urease apoprotein (UreABC). Among the accessory proteins, UreD and UreF have been elusive targets for biochemical and structural characterization because they are not overproduced as soluble proteins. Using the best-studied urease system, in which the Klebsiella aerogenes genes are expressed in Escherichia coli, a translational fusion of ureE and ureF was generated. The UreEF fusion protein was overproduced as a soluble protein with a convenient tag involving the His-rich region of UreE. The fusion protein was able to form a UreD(EF)G-UreABC complex and to activate urease in vivo, and it interacted with UreD-UreABC in vitro to form a UreD(EF)-UreABC complex. While the UreF portion of UreEF is fully functional, the fusion significantly affected the role of the UreE portion by interrupting its dimerization and altering its metal binding properties compared to those of the wild-type UreE. Analysis of a series of UreEF deletion mutants revealed that the C terminus of UreF is required to form the UreD(EF)G-UreABC complex, while the N terminus of UreF is essential for activation of urease.

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