Dissertations / Theses on the topic 'Fusion Oncogene Proteins'

The CBFbeta-SMMHC fusion protein is expressed in acute myeloid leukemia (AML) samples with the chromosome inversion inv(16)(p13;q22). This fusion protein binds the transcription factor RUNX with higher affinity than its physiological partner CBFbeta and disrupts the core binding factor (CBF) activity in hematopoietic stem and progenitor cells. Studies in the Castilla laboratory have shown that CBFbeta-SMMHC expression blocks differentiation of hematopoietic progenitors, creating a pre-leukemic progenitor that progresses to AML in cooperation with other mutations. However, the combined function of cumulative cooperating mutations in the pre-leukemic progenitor cells that enhance their expansion to induce leukemia is not known. The standard treatment for inv(16) AML is based on the use of non-selective cytotoxic chemotherapy, resulting in a good initial response, but with limited long-term survival. Therefore, there is a need for developing targeted therapies with improved efficacy in leukemic cells and minimal toxicity for normal cells. Here, we used conditional Nras+/LSL-G12D; Cbfb+/56M; Mx1Cre knock-in mice to show that allelic expression of oncogenic N-RasG12D expanded the multi-potential progenitor (MPP) compartment by 8 fold. Allelic expression of Cbfbeta-SMMHC increased the MPPs and short-term hematopoietic stem cells (ST-HSCs) by 2 to 4 fold both alone and in combination with N-RasG12D expression. In addition, allelic expression of oncogenic N-RasG12D and Cbfbeta-SMMHC increases survival of pre-leukemic stem and progenitor cells. Differential analysis of bone marrow cells determined that Cbfb+/MYH11 and Nras+/G12D; vii Cbfb+/MYH11 cells included increased number of blasts, myeloblasts and promyelocytes and a reduction in immature granulocytes, suggesting that expression of N-RasG12D cannot bypass Cbfbeta-SMMHC driven differentiation block. N-RasG12D and Cbfbeta-SMMHC synergized in leukemia, in which Nras+/G12D; Cbfb+/MYH11 mice have a shorter median latency than Cbfb+/MYH11 mice. In addition, the synergy in leukemogenesis was cell autonomous. Notably, leukemic cells expressing N-RasG12D and Cbfbeta-SMMHC showed higher (over 100 fold) leukemia-initiating cell activity in vivo than leukemic cells expressing Cbfbeta-SMMHC (L-IC activity of 1/4,000 and 1/528,334, respectively). Short term culture and biochemical assays revealed that pre-leukemic and leukemic cells expressing N-RasG12D and Cbfbeta-SMMHC have reduced levels of pro-apoptotic protein Bim compared to control. The Nras+/G12D; CbfbMYH11 pre-leukemic and leukemic cells were sensitive to pharmacologic inhibition of MEK/ERK signaling pathway with increasing apoptosis and Bim protein levels but not sensitive to PI3K inhibitors. In addition, knock-down of Bcl2l11 (Bim) expression in Cbfbeta-SMMHC pre-leukemic progenitors decreased their apoptosis levels. In collaboration with Dr. John Bushweller’s and other research laboratories, we recently developed a CBFbeta-SMMHC inhibitor named AI-10-49, which specifically binds to CBFbeta-SMMHC, prevents its binding to RUNX proteins and restores CBF function. Biochemical analysis in human leukemic cells showed that AI-10-49 has significant specificity in reducing the viability of leukemic cells expressing CBFbeta-SMMHC (IC50= 0.83μM), and negligible toxicity in normal cells. Likewise, mouse Nras+/G12D; viii Cbfb+/MYH11 leukemic cells were sensitive to AI-10-49 (IC50= 0.93μM). By using the NrasLSL-G12D; Cbfb56M mouse model, we also show that AI-10-49 significantly prolongs the survival of mice bearing the leukemic cells. Preliminary mechanistic analysis of AI-10-49 activity has shown that AI-10-49 increased BCL2L11 transcript levels in a dose and time dependent manner in murine and human leukemic cells, suggesting that the viability through BIM-mediated apoptosis may be targeted by both oncogenic signals. My thesis study demonstrates that Cbfbeta-SMMHC and N-RasG12D promote the survival of pre-leukemic myeloid progenitors primed for leukemia by activation of the MEK/ERK/Bim axis, and define NrasLSL-G12D; Cbfb56M mice as a valuable genetic model for the study of inv(16) AML targeted therapies. For instance, the novel CBFbeta-SMMHC inhibitor AI-10-49 shows a significant efficacy in this mouse model. This small molecule will serve as a promising first generation drug for targeted therapy of inv(16) leukemia and also a very useful tool to understand mechanisms of leukemogenesis driving by CBFbeta-SMMHC.

The CBFbeta-SMMHC fusion protein is expressed in acute myeloid leukemia (AML) samples with the chromosome inversion inv(16)(p13;q22). This fusion protein binds the transcription factor RUNX with higher affinity than its physiological partner CBFbeta and disrupts the core binding factor (CBF) activity in hematopoietic stem and progenitor cells. Studies in the Castilla laboratory have shown that CBFbeta-SMMHC expression blocks differentiation of hematopoietic progenitors, creating a pre-leukemic progenitor that progresses to AML in cooperation with other mutations. However, the combined function of cumulative cooperating mutations in the pre-leukemic progenitor cells that enhance their expansion to induce leukemia is not known. The standard treatment for inv(16) AML is based on the use of non-selective cytotoxic chemotherapy, resulting in a good initial response, but with limited long-term survival. Therefore, there is a need for developing targeted therapies with improved efficacy in leukemic cells and minimal toxicity for normal cells. Here, we used conditional Nras+/LSL-G12D; Cbfb+/56M; Mx1Cre knock-in mice to show that allelic expression of oncogenic N-RasG12D expanded the multi-potential progenitor (MPP) compartment by 8 fold. Allelic expression of Cbfbeta-SMMHC increased the MPPs and short-term hematopoietic stem cells (ST-HSCs) by 2 to 4 fold both alone and in combination with N-RasG12D expression. In addition, allelic expression of oncogenic N-RasG12D and Cbfbeta-SMMHC increases survival of pre-leukemic stem and progenitor cells. Differential analysis of bone marrow cells determined that Cbfb+/MYH11 and Nras+/G12D; vii Cbfb+/MYH11 cells included increased number of blasts, myeloblasts and promyelocytes and a reduction in immature granulocytes, suggesting that expression of N-RasG12D cannot bypass Cbfbeta-SMMHC driven differentiation block. N-RasG12D and Cbfbeta-SMMHC synergized in leukemia, in which Nras+/G12D; Cbfb+/MYH11 mice have a shorter median latency than Cbfb+/MYH11 mice. In addition, the synergy in leukemogenesis was cell autonomous. Notably, leukemic cells expressing N-RasG12D and Cbfbeta-SMMHC showed higher (over 100 fold) leukemia-initiating cell activity in vivo than leukemic cells expressing Cbfbeta-SMMHC (L-IC activity of 1/4,000 and 1/528,334, respectively). Short term culture and biochemical assays revealed that pre-leukemic and leukemic cells expressing N-RasG12D and Cbfbeta-SMMHC have reduced levels of pro-apoptotic protein Bim compared to control. The Nras+/G12D; CbfbMYH11 pre-leukemic and leukemic cells were sensitive to pharmacologic inhibition of MEK/ERK signaling pathway with increasing apoptosis and Bim protein levels but not sensitive to PI3K inhibitors. In addition, knock-down of Bcl2l11 (Bim) expression in Cbfbeta-SMMHC pre-leukemic progenitors decreased their apoptosis levels. In collaboration with Dr. John Bushweller’s and other research laboratories, we recently developed a CBFbeta-SMMHC inhibitor named AI-10-49, which specifically binds to CBFbeta-SMMHC, prevents its binding to RUNX proteins and restores CBF function. Biochemical analysis in human leukemic cells showed that AI-10-49 has significant specificity in reducing the viability of leukemic cells expressing CBFbeta-SMMHC (IC50= 0.83μM), and negligible toxicity in normal cells. Likewise, mouse Nras+/G12D; viii Cbfb+/MYH11 leukemic cells were sensitive to AI-10-49 (IC50= 0.93μM). By using the NrasLSL-G12D; Cbfb56M mouse model, we also show that AI-10-49 significantly prolongs the survival of mice bearing the leukemic cells. Preliminary mechanistic analysis of AI-10-49 activity has shown that AI-10-49 increased BCL2L11 transcript levels in a dose and time dependent manner in murine and human leukemic cells, suggesting that the viability through BIM-mediated apoptosis may be targeted by both oncogenic signals. My thesis study demonstrates that Cbfbeta-SMMHC and N-RasG12D promote the survival of pre-leukemic myeloid progenitors primed for leukemia by activation of the MEK/ERK/Bim axis, and define NrasLSL-G12D; Cbfb56M mice as a valuable genetic model for the study of inv(16) AML targeted therapies. For instance, the novel CBFbeta-SMMHC inhibitor AI-10-49 shows a significant efficacy in this mouse model. This small molecule will serve as a promising first generation drug for targeted therapy of inv(16) leukemia and also a very useful tool to understand mechanisms of leukemogenesis driving by CBFbeta-SMMHC.

A monoclonal antibody, mAb 203.37, raised against purified nuclear matrix proteins identified a single ~270 kDa protein that localized to the nuclear envelope. Double-label immunofluorescent microscopy using differential permeabilization protocols showed that this protein was present exclusively on the nucleoplasmic side of the nuclear envelope and that it co-localized with components of the nuclear pore complex. The nucleotide sequence of clones isolated using mAb 203.37 identified this protein as Tpr, a protein previously shown to be involved in oncogenic fusions with a number of protein kinases. Sequence analysis showed Tpr to be a 2348 amino acid protein with a predicted molecular weight of 265 kDa protein and a bipartite structure consisting of an ~1600 amino acid N-terminal domain that is almost entirely an α-helical coiled-coil followed by a highly acidic non-coiled carboxy-terminus. Ectopic expression of epitope-tagged Tpr constructs revealed two functional domains for Tpr: a nuclear pore complex binding domain and a nuclear localization sequence. The amino-terminus of Tpr, the portion of the protein shown to activate protein kinase oncogenes, did not localize to the nuclear pore complex indicating that the transforming activity of Tpr-protein kinase chimeras did not involve interactions with the nuclear pore complex. Ectopic expression of Tpr and a number of Tpr constructs resulted in the accumulation of poly (A)+ RNA in the nuclear interior but did not effect the import of a reporter protein into the nucleus indicating a role for Tpr in the export of mRNA from the nucleus.

A common phenotype observed in most cancers is chromosomal instability. This includes both structural and numerical chromosomal aberrations, which can promote carcinogenesis. The fusion gene CBFB/MYH11 is created by the structural chromosomal inversion(16)(p13.1q22), resulting in the fusion protein CBFβ-SMMHC, which blocks differentiation in hematopoietic progenitor cells. This mutation alone, however, is not sufficient for transformation, and at least one additional cooperating mutation is necessary. The role of wildtype Cbfb in modulating the oncogenic function of the fusion protein Cbfβ-SMMHC in mice was examined. Transgenic mice expressing the fusion protein, but lacking a wild-type copy of Cbfb, were created to model the effects of these combined mutations. It was found that wild-type Cbfb is necessary for maintaining normal hematopoietic differentiation. Consequently, complete loss of wild-type Cbfb accelerates leukemogenesis in Cbfb/MYH11 mice compared to mice expressing both the fusion and wild-type proteins. While there is no evidence in human patient samples that loss of wild-type Cbfb expression cooperates with the fusion protein to cause transformation, it is apparent from these experiments that wild-type Cbfβ does play a role in maintaining genomic integrity in the presence of Cbfβ-SMMHC. Experiments have also shown that loss of Cbfb leads to accumulation of hematopoietic progenitor cells, which may acquire additional cooperating mutations. Not unlike CBFB/MYH11, the human papillomavirus (HPV) E6 and E7 proteins are not sufficient for cellular transformation. Instead, high risk HPV E7 causes numerical chromosomal aberrations, which can lead to accumulation of additional cooperating mutations. Expression of HPV-16 E7 and subsequent downregulation of the retinoblastoma protein (Rb) has been shown to induce polyploidy in human keratinocytes. Polyploidy predisposes cells to aneuploidy and can eventually lead to transformation in HPV positive cells. There are several possible mechanisms through which E7 may lead to polyploidization, including abrogation of the spindle assembly checkpoint, cleavage failure, abrogation of the postmitotic checkpoint, and re-replication. Rb-defective mouse and human cells were found to undergo normal mitosis and complete cytokinesis. Furthermore, DNA re-replication was not found to be a major mechanism to polyploidization in HPV-E7 cells upon microtubule disruption. Interestingly, upon prolonged mitotic arrest, cells were found to adapt to the spindle assembly checkpoint and halt in a G1-like state with 4C DNA content. This post-mitotic checkpoint is abrogated by E7-induced Rb-downregulation leading to S-phase induction and polyploidy. This dissertation explores two examples of the multi-step pathway in human cancers. While certain genes or genetic mutations are often characteristic of specific cancers, those mutations are often not sufficient for transformation. The genetic or chromosomal abnormalities that they produce often stimulate the additional mutations necessary for oncogenesis. The studies with Cbfb/MYH11 and HPV E7 further exemplify the significance of numerical and structural chromosomal aberrations in multi-step carcinogenesis.

Thesis (Ph.D. in Pharmacology) -- University of Colorado Denver, 2008.
Typescript. Includes bibliographical references (leaves 112-125). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Interactions between the retinoic acid receptor alpha (RARalpha) and coregulators play a key role in coordinating gene transcription and myeloid differentiation. In acute promyelocytic leukemia (APL), RARalpha is fused with the promyelocytic leukemia (PML) gene, resulting in the expression of the fusion protein PML/RARalpha. Here, I report that topoisomerase II beta (topoIIbeta) associates with and negatively modulates PML/RARalpha and RARalpha transcriptional activity, and increased levels and association of topoIIbeta cause resistance to retinoic acid (RA) in APL cell lines. Knock down of topoIIbeta was able to overcome resistance by permitting RA-induced differentiation and increased RA-gene expression. Overexpression of topoIIbeta, in clones from an RA-sensitive cell line, conferred resistance by a reduction in RA-induced expression of target genes and differentiation. Chromatin immunoprecipitation assays indicate that topoIIbeta is bound to an RA-response element, and inhibition of topoIIbeta causes hyper-acetylation of histone 3 at lysine 9 and activation of transcription. These results identify a novel mechanism of resistance in APL and provide further insights to the role of topoIIbeta in gene regulation and differentiation.
Studies to determine the mechanism by which topoIIbeta protein is regulated found that levels of protein kinase C delta (PKCdelta) correlated with topoIIbeta protein expression. Moreover, activation of PKCdelta, by RA or PMA, led to an increase of topoIIbeta protein levels. Most notably, in NB4-MR2 cells, we observed increased phosphorylation levels of threonine 505 on PKCdelta, a marker of activation. Inhibition of PKCdelta was able to overcome the topoIIbeta repressive effects on RA-target genes. In addition, the combination of RA and PKCdelta inhibition led to increased expression of the granulocytic marker, CD11c, in NB4 and NB4-MR2 cells. These results suggest that PKCdelta regulates topoIIbeta expression, and a constitutively active PKCdelta in the NB4-MR2 cell line leads to overexpression of topoIIbeta.
In conclusion, these studies demonstrate that topoIIbeta associates with RARalpha, binds to RAREs and plays a critical role in RA dependent transcriptional regulation and granulocytic differentiation. In addition, I show that topoIIbeta overexpression leads to RA resistance and provide evidence that topoIIbeta protein levels are regulated via a mechanism involving the PKCdelta pathway. This work has contributed to an enhanced understanding of the role of topoIIbeta in gene regulation and brings novel perspectives in the treatment of RA-resistance in APL.

Chromosomal alterations involving the RUNXI or CBFB genes are specifically and recurrently associated with human acute myeloid leukemia (AML). One such chromosomal alteration, a pericentric inversion of chromosome 16, is present in the majority of cases of the AML subtype M4Eo. This inversion joins CBFB with the smooth muscle myosin gene MYH11 creating the fusion CBFB-MYH11. Knock-in studies in the mouse have demonstrated that expression of the protein product of the Cbfb-MYH11fusion, Cbfβ-SMMHC, predisposes mice to AML and that chemical mutagenesis both accelerates and increases the penetrance of the disease (Castilla et al., 1999). However, the mechanism of transformation and the associated collaborating genetic events remain to be resolved. As detailed in Chapter 2, we used retroviral insertional mutagenesis (RIM) to identify mutations in Cbfb-MYH11 chimeric mice that contribute to AML. The genetic screen identified 54 independent candidate cooperating genes including 6 common insertion sites: Plag1, Plagl2, Runx2, H2T23, Pstpip2, and Dok1. Focusing on the 2 members of the Plag family of transcription factors, Chapter 3 presents experiments demonstrating 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 and in vitro cell renewal in Cbfβ-SMMHC hematopoietic progenitors. Furthemore, PLAG1 and PLAGL2 expression was increased in 20% of human AML samples suggesting that PLAG1 and PLAGL2 may also contribute to human AML. Interestingly, PLAGL2was preferentially increased in samples with chromosome 16 inversion, t(8;21), and t(15;17). To define the mechanism by which PLAGL2 contributes to leukemogenesis, Chapter 4 presents studies assessing the role of the Mp1 signaling cascade as a Plagl2 downstream pathway in leukemia development. Using microarray analysis we discovered that PLAGL2 induces the expression of Mp1 transcript in primary bone marrow cells that express Cbfβ-SMMHC and that this induction is maintained in leukemogenesis. We have also performed luciferase assays to confirm that the Mp1 proximal promoter can be directly bound and activated by PLAGL2. Furthermore, we demonstrate increased Mp1 expression leads to hypersensitivity to the Mp1 ligand thrombopoietin (TPO) in PLAGL2/Cbfβ-SMMHC leukemic cells. To test the functional relevance in leukemia formation, we performed a bone-marrow transplantation assay and demonstrate that overexpression of Mp1 is indeed sufficient to cooperate with Cbfβ-SMMHC in leukemia induction. This data reveals that PLAGL2 cooperates with Cbfβ-SMMHC at least in part by inducing the expression of the cytokine receptor Mp1. Thus, we have identified the Mp1 signal transduction pathway as a novel target for therapeutic intervention in AML.

Leukemia is a hematopoietic cancer that is characterized by the abnormal differentiation and proliferation of hematopoietic cells. It is ranked 7th by death rate among cancer types in USA, even though it is not one of the top 10 cancers by incidence (USCS, 2010). This indicates an urgent need for more effective treatment strategies. In order to design the new ways of prevention and treatment of leukemia, it is important to understand the molecular mechanisms involved in development of the disease. In this study, we investigated mechanisms involved in the development of acute myeloid leukemia (AML) that is associated with CBF fusion genes. The RUNX1 and CBFB genes that encode subunits of a transcriptional regulator complex CBF, are mutated in a subset (20 – 25%) of AML cases. As a result of these mutations, fusion genes called CBFB-MYH11 and RUNX1-ETO arise. The chimeric proteins encoded by the fusion genes provide block in proliferation for myeloid progenitors, but are not sufficient for AML development. Genetic studies have indicated that activation of cytokine receptor signaling is a major oncogenic pathway that cooperates in leukemia development. The main goal of my work was to determine a role of two factors that regulate cytokine signaling activity, the microRNA cluster miR-17-92 and the thrombopoietin receptor MPL, in their potential cooperation with the CBF fusions in AML development. We determined that the miR-17-92 miRNA cluster cooperates with Cbfb-MYH11 in AML development in a mouse model of human CBFB-MYH11 AML. We found that the miR-17-92 cluster downregulates Pten and activates the PI3K/Akt pathway in the leukemic blasts. We also demonstrated that miR-17-92 provides an anti-apoptotic effect in the leukemic cells, but does not seem to affect proliferation. The anti-apoptotic effect was mainly due to activity of miR-17 and miR-20a, but not miR-19a and miR-19b. Our second study demonstrated that wild type Mpl cooperated with RUNX1-ETO fusion in development of AML in mice. Mpl induced PI3K/Akt, Ras/Raf/Erk and Jak2/Stat5 signaling pathways in the AML cells. We showed that PIK3/Akt pathway plays a role in AML development both in vitro and in vivo by increasing survival of leukemic cells. The levels of MPL transcript in the AML samples correlated with their response to thrombopoietin (THPO). Moreover, we demonstrated that MPL provides pro-proliferative effect for the leukemic cells, and that the effect can be abrogated with inhibitors of PI3K/AKT and MEK/ERK pathways. Taken together, these data confirm important roles for the PI3K/AKT and RAS/RAF/MEK pathways in the pathogenesis of AML, identifies two novel genes that can serve as secondary mutations in CBF fusions-associated AML, and in general expands our knowledge of mechanisms of leukemogenesis.

La Leucémie Aiguë Promyélocytaire (LAP) est due à une translocation chromosomique fusionnant le gène du récepteur alpha à l'acide rétinoïque (RARA) au gène promyelocytic leukemia (PML). L'expression de la protéine de fusion PML-RARA permet l'expansion d'un clone leucémique présentant des propriétés d'auto-renouvellement ainsi qu'un blocage caractéristique de la différenciation au stade promyélocyte. Le traitement par l'acide rétinoïque (AR) provoque une différenciation massive des cellules leucémiques et une régression tumorale qui se traduisent par l'obtention de rémissions complètes chez les patients atteints de LAP. Au niveau moléculaire, l'AR réactive la transcription des gènes réprimés par PML-RARA et déclenche la dégradation de la protéine de fusion. Par des approches de thérapie expérimentale sur des modèles murins de LAP, j'ai apporté de nouvelles preuves d'un découplage entre différenciation des blastes et éradication de la maladie, et d'une contribution majeure de la dégradation de PML-RARA à la guérison. J'ai également pu montrer que la perte d'auto-renouvellement des cellules leucémiques en réponse au traitement est sous-tendue par l'activation d'une voie atypique de P53, dont j'ai vérifié fonctionnellement l'implication. Enfin, j'ai mis en évidence le rôle central de PML, dont la reformation des corps nucléaires suit la dégradation de PML-RARA, dans cette activation de P53. Par conséquent, nos données soutiennent un modèle dans lequel la dégradation de PML-RARA induit la reformation des corps PML qui permet l'activation d'une réponse P53 menant à la perte de l'auto-renouvellement des blastes leucémiques et à l'éradication de la maladie
Acute promyelocytic leukemia (APL) arises from a chromosomic translocation fusing a part of the promyelocytic leukemia (PML) gene to a part of the retinoic acid receptor alpha (RARA) gene. The expression of the PML-RARA fusion protein allows the expansion of a leukemic clone presenting hyper-proliferation and self-renewing capacities, as well as a characteristic block at the promyelocyte stage of myeloid differentiation. Treatment with therapeutic doses of retinoic acid (RA) induces the massive differentiation of APL cells and a dramatic tumor regression, eventually achieving complete remissions in APL patients. At the molecular level, RA both reverses the transcriptional repression imposed by PML-RARA and triggers the degradation of the fusion protein. Through experimental therapeutics in mouse models of APL, I could produce new evidence of a complete uncoupling between blast differentiation and disease eradication, as well as of a major contribution of PML-RARA degradation to APL cure. I further demonstrated that the activation of an atypical P53 response underlies the loss of self-renewal of leukemia cells following PML-RARA degradation by RA. Finally, I was able to uncover the central role of the PML protein, which reforms nuclear bodies upon treatment, in this mechanism, upstream of P53 activation. Our data thus tend to support a sequential model in which therapy-triggered degradation of PML-RARA allows the reformation of PML nuclear bodies, which in tum activate a P53 response, resulting in the loss of leukemia cell self-renewal and ultimately leading to APL cure

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, South Africa, in fulfillment of the requirements for the degree of Doctor of Philosophy. Johannesburg, 1996
The NPM-ALK fusion gene, formed by the t(2;5)(p23;q35) in non-Hodgkin's lymphoma encodes a 75kDa hybrid protein that contains the amino-terminal 118 amino acid residues of the nucleolar phosphoprotein nucleophosmin (NPM) joined to the entire cytoptasmic portion of the receptor tyrosine kinase, anaplastic lymphoma kinase (ALK). The transforming ability of NPM-ALK is demonstrated and it is shown that oncogenesis by the chimaeric protein requires the activation of its kinase function as a result of oligomerisation mediated by the NPM segment. [Abbreviated Abstract. Open document to view full version]