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Aberrant epigenetic regulators control expansion of human CD34+ hematopoietic stem/progenitor cells
(2013)
Transcription is a tightly regulated process ensuring the proper expression of numerous genes regulating all aspects of cellular behavior. Transcription factors regulate multiple genes including other transcription factors that together control a highly complex gene network. The transcriptional machinery can be “hijacked” by oncogenic transcription factors, thereby leading to malignant cell transformation. Oncogenic transcription factors manipulate a variety of epigenetic control mechanisms to fulfill gene regulatory and cell transforming functions. These factors assemble epigenetic regulators at target gene promoter sequences, thereby disturbing physiological gene expression patterns. Retroviral vector technology and the availability of “healthy” human hematopoietic CD34+ progenitor cells enable the generation of pre-leukemic cell models for the analysis of aberrant human hematopoietic progenitor cell expansion mediated by leukemogenic transcription factors. This review summarizes recent findings regarding the mechanism by which leukemogenic gene products control human hematopoietic CD34+ progenitor cell expansion by disrupting the normal epigenetic program.
Acute myeloid leukemia (AML) is characterized by an aberrant self-renewal of hematopoietic stem cells (HSC) and a block in differentiation. The major therapeutic challenge is the characterization of the leukemic stem cell as a target for the eradication of the disease. Until now the biology of AML-associated fusion proteins (AAFPs), such as the t(15;17)-PML/RARα, t(8;21)-RUNX1/RUNX1T1 and t(6;9)-DEK/NUP214, all able to induce AML in mice, was investigated in different models and genetic backgrounds, not directly comparable to each other. To avoid the bias of different techniques and models we expressed these three AML-inducing oncogenes in an identical genetic background and compared their influence on the HSC compartment in vitro and in vivo.
These AAFPs exerted differential effects on HSCs and PML/RARα, similar to DEK/NUP214, induced a leukemic phenotype from a small subpopulation of HSCs with a surface marker pattern of long-term HSC and characterized by activated STAT3 and 5. In contrast the established AML occurred from mature populations in the bone marrow. The activation of STAT5 by PML/RARα and DEK/NUP214 was confirmed in t(15;17)(PML/RARα) and t(6;9)(DEK/NUP214)-positive patients as compared to normal CD34+ cells. The activation of STAT5 was reduced upon the exposure to Arsenic which was accompanied by apoptosis in both PML/RARα- and DEK/NUP214-positive leukemic cells. These findings indicate that in AML the activation of STATs plays a decisive role in the biology of the leukemic stem cell. Furthermore we establish exposure to arsenic as a novel concept for the treatment of this high risk t(6;9)-positive AML.
RP1 (synonym: MAPRE2, EB2) is a member of the microtubule binding EB1 protein family, which interacts with APC, a key regulatory molecule in the Wnt signalling pathway. While the other EB1 proteins are well characterized the cellular function and regulation of RP1 remain speculative to date. However, recently RP1 has been implicated in pancreatic cancerogenesis. CK2 is a pleiotropic kinase involved in adhesion, proliferation and anti-apoptosis. Overexpression of protein kinase CK2 is a hallmark of many cancers and supports the malignant phenotype of tumor cells. In this study we investigate the interaction of protein kinase CK2 with RP1 and demonstrate that CK2 phosphorylates RP1 at Ser236 in vitro. Stable RP1 expression in cell lines leads to a significant cleavage and down-regulation of N-cadherin and impaired adhesion. Cells expressing a Phospho-mimicking point mutant RP1-ASP236 show a marked decrease of adhesion to endothelial cells under shear stress. Inversely, we found that the cells under shear stress downregulate endogenous RP1, most likely to improve cellular adhesion. Accordingly, when RP1 expression is suppressed by shRNA, cells lacking RP1 display significantly increased cell adherence to surfaces. In summary, RP1 phosphorylation at Ser236 by CK2 seems to play a significant role in cell adhesion and might initiate new insights in the CK2 and EB1 family protein association.
Rho GTPases are involved in homing and mobilization of hematopoietic stem and progenitor cells due to their impact on cytoskeleton remodeling. We have previously shown that inhibition of Rho, Rac and Cdc42 clearly impairs adhesion of normal and leukemic hematopoietic progenitor cells (HPC) to fibronectin and migration in a three-dimensional stromal cell model. Here, we identified the Ras GTPase-Activating Protein SH3 Domain-Binding Protein (G3BP) as a target gene of Rho GTPases and analysed its role in regulating HPC motility. Overexpression of G3BP significantly enhanced adhesion of murine 32D HPC to fibronectin and human umbilical vein endothelial cells, increased the proportion of adherent cells in a flow chamber assay and promoted cell migration in a transwell assay and a three-dimensional stromal cell model suggesting a strong impact on the cytoskeleton. Immunofluorescent staining of G3BP-overexpressing fibroblasts revealed a Rho-like phenotype characterized by formation of actin stress fibers in contrast to the Rac-like phenotype of control fibroblasts. This is the first report implicating a role for G3BP in Rho GTPase-mediated signalling towards adhesion and migration of HPC. Our results may be of clinical importance, since G3BP was found overexpressed in human cancers.
Reciprocal t(9;22) ABL/BCR fusion proteins: leukemogenic potential and effects on B cell commitment
(2009)
Background: t(9;22) is a balanced translocation, and the chromosome 22 breakpoints (Philadelphia chromosome – Ph+) determine formation of different fusion genes that are associated with either Ph+ acute lymphatic leukemia (Ph+ ALL) or chronic myeloid leukemia (CML). The "minor" breakpoint in Ph+ ALL encodes p185BCR/ABL from der22 and p96ABL/BCR from der9. The "major" breakpoint in CML encodes p210BCR/ABL and p40ABL/BCR. Herein, we investigated the leukemogenic potential of the der9-associated p96ABL/BCR and p40ABL/BCR fusion proteins and their roles in the lineage commitment of hematopoietic stem cells in comparison to BCR/ABL. Methodology: All t(9;22) derived proteins were retrovirally expressed in murine hematopoietic stem cells (SL cells) and human umbilical cord blood cells (UCBC). Stem cell potential was determined by replating efficiency, colony forming - spleen and competitive repopulating assays. The leukemic potential of the ABL/BCR fusion proteins was assessed by in a transduction/transplantation model. Effects on the lineage commitment and differentiation were investigated by culturing the cells under conditions driving either myeloid or lymphoid commitment. Expression of key factors of the B-cell differentiation and components of the preB-cell receptor were determined by qRT-PCR. Principal Findings: Both p96ABL/BCR and p40ABL/BCR increased proliferation of early progenitors and the short term stem cell capacity of SL-cells and exhibited own leukemogenic potential. Interestingly, BCR/ABL gave origin exclusively to a myeloid phenotype independently from the culture conditions whereas p96ABL/BCR and to a minor extent p40ABL/BCR forced the B-cell commitment of SL-cells and UCBC. Conclusions/Significance: Our here presented data establish the reciprocal ABL/BCR fusion proteins as second oncogenes encoded by the t(9;22) in addition to BCR/ABL and suggest that ABL/BCR contribute to the determination of the leukemic phenotype through their influence on the lineage commitment.