RAR and AML1 transcription factors are found in leukemias as fusion proteins with PML and ETO, respectively. Association of PML-RAR and AML1-ETO with the nuclear corepressor (N-CoR)/histone ...deacetylase (HDAC) complex is required to block hematopoietic differentiation. We show that PML-RAR and AML1-ETO exist in vivo within high molecular weight (HMW) nuclear complexes, reflecting their oligomeric state. Oligomerization requires PML or ETO coiled-coil regions and is responsible for abnormal recruitment of N-CoR, transcriptional repression, and impaired differentiation of primary hematopoietic precursors. Fusion of RAR to a heterologous oligomerization domain recapitulated the properties of PML-RAR, indicating that oligomerization per se is sufficient to achieve transforming potential. These results show that oligomerization of a transcription factor, imposing an altered interaction with transcriptional coregulators, represents a novel mechanism of oncogenic activation.
Acute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia characterized by a block of differentiation at the promyelocytic stage. APL patients respond to pharmacological ...concentrations of all-trans retinoic acid (RA) and disease remission correlates with terminal differentiation of leukemic blasts. The PML/RAR oncogenic transcription factor is responsible for both the pathogenesis of APL and for its sensitivity to RA. In order to identify physiological targets of RA therapy, we analysed gene expression profiles of RA-treated APL blasts and found 1056 common target genes. Comparing these results to those obtained in RA-treated U937 cell lines revealed that transcriptional response to RA is largely dependent on the expression of PML/RAR. Several genes involved in the control of differentiation and stem cell renewal are early targets of RA regulation, and may be important effectors of RA response. Modulation of chromatin modifying genes was also observed, suggesting that specific structural changes in local chromatin domains may be required to promote RA-mediated differentiation. Computational analysis of upstream genomic regions in RA target genes revealed nonrandom distribution of transcription factor binding sites, indicating that specific transcriptional regulatory complexes may be involved in determining RA response.
We have analyzed the differentiation program of growth factor-dependent TF-1 erythroleukemia cells as well as clones with inducible expression of the APL-specific PML/RARα protein. We have shown that ...TF-1 cells may be induced to megakaryocytic differentiation by phorbol ester (phorbol dibutyrate, PDB) addition, particularly when combined with thrombopoietin (Tpo). RT-PCR studies showed that Tpo induces Tpo receptor (TpoR or c-mpl), whose expression was further potentiated by PDB addition. When the cells are induced with both PDB and Tpo erythropoietin receptor (EpoR) expression was inhibited. In the absence of Zn2+-induced PML/RARα expression, PDB and Tpo induced megakaryocytic differentiation of TF-1 MTPR clones as observed in ‘wild-type’ TF-1 cells. Conversely, when PML/RARα expression was induced by Zn2+, PDB and Tpo treatment of these clones caused only a reduced level of megakaryocytic differentiation. These observations indicate that: (1) TF-1 cells as well as other erythroleukemic cells, possess the capacity to differentiate to megakaryocytic cells when grown in the presence of protein kinase (PKC) activators and more efficiently when combined with Tpo; (2) the PML/RARα gene has a wide capacity to interfere with the program of hematopoietic differentiation, including megakaryocytic differentiation. Finally, we also observed that PML/RARα expression in TF-1 cells induces an up-modulation of interleukin-3 receptor, c-kit and c-mpl, a phenomenon which may offer these cells a growth advantage.
The pathogenesis of acute myeloid leukemia is associated with the appearance of oncogenic fusion proteins generated as a consequence of specific chromosome translocations. Of the two components of ...each fusion protein, one is generally a transcription factor, whereas the other partner is more variable in function, but often involved in the control of cell survival and apoptosis. As a consequence, AML-associated fusion proteins function as aberrant transcriptional regulators that interfere with the process of myeloid differentiation, determine a stage-specific arrest of maturation and enhance cell survival in a cell-type specific manner. The abnormal regulation of transcriptional networks occurs through common mechanisms that include recruitment of aberrant co-repressor complexes, alterations in chromatin remodeling, and disruption of specific subnuclear compartments. The identification and analysis of common and specific target genes regulated by AML fusion proteins will be of fundamental importance for the full understanding of acute myeloid leukemogenesis and for the implementation of disease-specific drug design.
The block of terminal differentiation is a prominent feature of acute promyelocytic leukemia (APL) and its release by retinoic acid correlates with disease remission. Expression of the APL-specific ...PML/RAR alpha fusion protein in hematopoietic precursor cell lines blocks terminal differentiation, suggesting that PML/RAR alpha may have the same activity in APL blasts. We expressed different PML/RAR alpha mutants in U937 and TF-1 cells and demonstrated that the integrity of the PML protein dimerization and RAR alpha DNA binding domains is crucial for the differentiation block induced by PML/RAR alpha , and that these domains exert their functions only within the context of the fusion protein. Analysis of the in vivo dimerization and cell localization properties of the PML/RAR alpha mutants revealed that PML/RAR alpha -PML and PML/RAR alpha -RXR heterodimers are not necessary for PML/RAR alpha activity on differentiation. We propose that a crucial mechanism underlying PML/RAR alpha oncogenic activity is the deregulation of a transcription factor, RAR alpha , through its fusion with the dimerization interface of another nuclear protein, PML.
The block of terminal differentiation is a prominent feature of acute promyelocytic leukemia (APL) and its release by retinoic acid correlates with disease remission. Expression of the APL‐specific ...PML/RARalpha fusion protein in hematopoietic precursor cell lines blocks terminal differentiation, suggesting that PML/ RARalpha may have the same activity in APL blasts. We expressed different PML/RARalpha mutants in U937 and TF‐1 cells and demonstrated that the integrity of the PML protein dimerization and RARalpha DNA binding domains is crucial for the differentiation block induced by PML/RARalpha, and that these domains exert their functions only within the context of the fusion protein. Analysis of the in vivo dimerization and cell localization properties of the PML/RARalpha mutants revealed that PML/RARalpha–PML and PML/RARalpha–RXR heterodimers are not necessary for PML/RARalpha activity on differentiation. We propose that a crucial mechanism underlying PML/RARalpha oncogenic activity is the deregulation of a transcription factor, RARalpha, through its fusion with the dimerization interface of another nuclear protein, PML.
Retinoic Acid (RA) treatment induces disease remission of Acute Promyelocytic Leukaemia (APL) patients by triggering terminal differentiation of neoplastic cells. RA-sensitivity in APL is mediated by ...its oncogenic protein, which results from the recombination of the PML and the RA receptor alpha (RAR alpha) genes (PML/RAR alpha fusion protein). Ectopic expression of PML/RAR alpha into haemopoietic cell lines results in increased response to RA-induced differentiation. By structure-function analysis of PML/RAR alpha-mediated RA-differentiation, we demonstrated that fusion of PML and RAR alpha sequences and integrity of the PML dimerization domain and of the RAR alpha DNA binding region are required for the effect of PML/RAR alpha on RA-differentiation. Indeed, direct fusion of the PML dimerization domain to the N- or C-terminal extremities of RAR alpha retained full biological activity. All the biologically active PML/RAR alpha mutants formed high molecular weight complexes in vivo. Functional analysis of mutations within the PML dimerization domain revealed that the capacity to form PML/RAR alpha homodimers, but not PML/RAR alpha-PML heterodimers, correlated with the RA-response. These results suggest that targeting of RAR alpha sequences by the PML dimerization domain and formation of nuclear PML/RAR alpha homodimeric complexes are crucial for the ability of PML/RAR alpha to mediate RA-response.
The anti-epileptic drug valproic acid (VPA) acts as an inhibitor of histone deacetylases. In combination with retinoic acid (RA), VPA triggers myeloid differentiation of primary acute myeloid ...leukemia (AML) blasts in vitro. In vivo, VPA posses an antineoplastic activity as indicated by pre-clinical studies in murine models of leukemia, renal and lung metastatic tumors. Therefore, we have designed a phase II clinical study in which VPA was combined with RA (VPA-RA) in the AML treatment. Eigth chemotherapy-resistant or high risk AML patients not eligible for additional intensive therapy (median age 61.5 yrs), were treated at the Hematology Units of the Universities “La Sapienza” and “Tor Vergata” Rome-Italy. VPA (DepakinSanofi-Wintrop) was administrated from day 1 to day 28, at the initial dosage of 10 mg/kg/die p.o. with dose escalation until optimal VPA plasma levels (80–110ug/ml). RA (Vesanoid Roche) at the dosage of 45 mg/m2 p.o./d, divided in two administrations, was added once the optimal VPA plasma levels were reached or at day 14 and continued until day 28. Four patients had a history of MDS, three patients had a FAB M0, M1 and M2 de novo AMLs, while the remaining case was a myeloid blast crisis (FAB M0) of a Ph+ve CML. Cytogenetic characterization in the other patients revealed normal karyotype in one case, a pseudodiploid der(12) in one, hyperdiploid (+8) in one, complex K with a 7q- alteration in one, while in the three remaining cases the karyotype was not evaluable. Pre-treatment leukemic infiltration ranged from 22% to 95%. VPA plasma level >60mg/ml was reached between 8 to 28 days (median 14.5 days). In three patients, VPA-RA treatment induced hyperleukocytosis (>50x 109/l) at day 16, 21 and 24, respectively, that was treated with chemotherapy (HU in two cases and low dose Ara-C in 1 case). Hematological improvement (≥50% decrease in packed red blood cell or platelet transfusion requirement) was observed in one case, a stable disease in five cases and disease progression in two cases. Peripheral blood and/or bone marrow samples were collected at day 0,3,7,14,21,28 for morphologic, immunophenotypic, cytogenetic and molecular studies. All patients showed features of myeloid-monocytic and/or erythroid differentiation of the leukemic clone, as revealed by morphologic, cytochemical, immunophenotypic analyses and by Q-RT-PCR of myeloid gene expression (GATA 1, MPO, CSF2Rb, etc.). Of note that high degree of myeloid differentiation correlated with early achievement of therapeutic VPA plasma levels and histone hyper-acetylation, as measured by immunocytochemistry and immunoblotting using antiacetylated histone H3 and H4 antibodies. Finally, differentiation of the leukemic clone was proven by FISH analysis showing the presence of the +8 and 7q- in maturing elements in patients whose leukemia blasts carried these cytogenetic lesions. The VPA-RA combination is a well tolerated treatment that induces phenotypic changes of the leukemic clone through chromatine remodelling. Further studies are needed to optimise this regimen with the aim of improving clinical response in leukemia patients.
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