Distinct leukemia phenotypes in transgenic mice and different corepressor interactions generated by promyelocytic leukemia variant fusion genes PLZF–RARα and NPM–RARα

Acute promyelocytic leukemia (APL) is characterized by a specific chromosome translocation involving RARalpha and one of four fusion partners: PML, PLZF, NPM, and NuMA genes. To study the leukemogenic potential of the fusion genes in vivo, we generated transgenic mice with PLZF-RARalpha and NPM-RARalpha. PLZF-RARalpha transgenic animals developed chronic myeloid leukemia-like phenotypes at an early stage of life (within 3 months in five of six mice), whereas three NPM-RARalpha transgenic mice showed a spectrum of phenotypes from typical APL to chronic myeloid leukemia relatively late in life (from 12 to 15 months). In contrast to bone marrow cells from PLZF-RARalpha transgenic mice, those from NPM-RARalpha transgenic mice could be induced to differentiate by all-trans-retinoic acid (ATRA). We also studied RARE binding properties and interactions between nuclear corepressor SMRT and various fusion proteins in response to ATRA. Dissociation of SMRT from different receptors was observed at ATRA concentrations of 0.01 microM, 0.1 microM, and 1.0 microM for RARalpha-RXRalpha, NPM-RARalpha, and PML-RARalpha, respectively, but not observed for PLZF-RARalpha even in the presence of 10 microM ATRA. We also determined the expression of the tissue factor gene in transgenic mice, which was detected only in bone marrow cells of mice expressing the fusion genes. These data clearly establish the leukemogenic role of PLZF-RARalpha and NPM-RARalpha and the importance of fusion receptor/corepressor interactions in the pathogenesis as well as in determining different clinical phenotypes of APL.     

Overexpression of wild-type retinoic acid receptor alpha (RARalpha) recapitulates retinoic acid-sensitive transformation of primary myeloid progenitors by acute promyelocytic leukemia RARalpha-fusion genes.

Retinoic acid receptor alpha (RARalpha) is the target of several chromosomal translocations associated with acute promyelocytic leukemias (APLs). These rearrangements fuse RARalpha to different partner genes creating the chimeric proteins: PML-RARalpha, PLZF-RARalpha, and NPM-RARalpha. Although the vast majority of APLs respond to retinoic acid therapy, those associated with PLZF-RARalpha are resistant. We have used retroviruses to express PML-RARalpha, PLZF-RARalpha, NPM-RARalpha, RARalpha403 (a dominant negative mutant of RARalpha), and wild-type RARalpha in murine bone marrow progenitors and found that all of these constructs blocked differentiation and led to the immortalization of myeloid progenitors. This cellular transformation is specific to an alteration of the RARalpha pathway because overexpression of RARbeta, RARgamma, or RXRalpha did not result in similar growth perturbations. Pharmacological doses of RA induced differentiation and inhibited proliferation of cells transformed with either of the APL fusion genes, including PLZF-RARalpha, whereas physiological retinoic acid concentrations were sufficient to reverse the phenotype of cells transformed with wild-type RARalpha. The cellular responses to retinoic acid were accompanied by a sharp decrease in the amount of the RARalpha-fusion proteins expressed in the cells. Our findings suggest that the oncogenicity of RARalpha-fusion proteins results from their nature to behave as unliganded RARalpha in the presence of physiological concentrations of retinoic acid.     

Retinoic acid (RA) and As2O3 treatment in transgenic models of acute promyelocytic leukemia (APL) unravel the distinct nature of the leukemogenic process induced by the PML-RARalpha and PLZF-RARalpha oncoproteins

Acute promyelocytic leukemia (APL) is associated with chromosomal translocations always involving the RARalpha gene, which variably fuses to one of several distinct loci, including PML or PLZF (X genes) in t(15;17) or t(11;17), respectively. APL in patients harboring t(15;17) responds well to retinoic acid (RA) treatment and chemotherapy, whereas t(11;17) APL responds poorly to both treatments, thus defining a distinct syndrome. Here, we show that RA, As(2)O(3), and RA + As(2)O(3) prolonged survival in either leukemic PML-RARalpha transgenic mice or nude mice transplanted with PML-RARalpha leukemic cells. RA + As(2)O(3) prolonged survival compared with treatment with either drug alone. In contrast, neither in PLZF-RARalpha transgenic mice nor in nude mice transplanted with PLZF-RARalpha cells did any of the three regimens induce complete disease remission. Unexpectedly, therapeutic doses of RA and RA + As(2)O(3) can induce, both in vivo and in vitro, the degradation of either PML-RARalpha or PLZF-RARalpha proteins, suggesting that the maintenance of the leukemic phenotype depends on the continuous presence of the former, but not the latter. Our findings lead to three major conclusions with relevant therapeutic implications: (i) the X-RARalpha oncoprotein directly determines response to treatment and plays a distinct role in the maintenance of the malignant phenotype; (ii) As(2)O(3) and/or As(2)O(3) + RA combination may be beneficial for the treatment of t(15;17) APL but not for t(11;17) APL; and (iii) therapeutic strategies aimed solely at degrading the X-RARalpha oncoprotein may not be effective in t(11;17) APL.     

Acute promyelocytic leukemia with a PLZF-RARalpha fusion protein

In most cases of acute promyelocytic leukemia (APL), a fusion of the promyelocytic leukemia (PML) and the retinoic acid receptor-alpha (RARalpha) genes occurs, resulting in the expression of a PML-RARalpha chimeric protein. In approximately 1% of the cases of APL, variant chromosomal aberrations may be found fusing RARa with other genes. Four variant mutations have been described, and the t(11;17)(q21;q23) translocation generating a promyelocyte leukemia zinc finger (PLZF)-RARalpha fusion gene is the most common. PLZF-RARalpha-positive APL forms a clinically distinct group because unlike PML-RARalpha-positive leukemia, it does not respond to retinoic acid with terminal granulocytic differentiation of the cells, and remissions cannot be achieved with retinoids alone. At the molecular level, this has been explained by the retinoic acid-insensitive binding of corepressor proteins to the PLZF part of the fusion protein, leading to sustained repression of target genes that are important for cellular differentiation. Targeting of the PLZF-RARalpha-bound corepressor complexes using a combination of all-trans retinoic acid (ATRA) and deacetylase inhibitors has shown that the repression of target genes can be relieved, allowing differentiation of the cells. In addition, when a combination of retinoic acid and the hematopoietic growth factor granulocyte colony-stimulating factor (G-CSF) is applied, the cells may be forced to undergo terminal differentiation, both in vitro and in vivo. This suggests that signals from the activated G-CSF receptor may induce the release of corepressor proteins from PLZF. Together, these findings indicate that PLZF-RARalpha-positive leukemia is not completely resistant to differentiation induction if the proper costimuli are given.     

PLZF-RAR alpha fusion proteins generated from the variant t(11;17)(q23;q21) translocation in acute promyelocytic leukemia inhibit ligand-dependent transactivation of wild-type retinoic acid receptors.

Recently, we described a recurrent variant translocation, t(11;17)(q23;q21), in acute promyelocytic leukemia (APL) which juxtaposes PLZF, a gene encoding a zinc finger protein, to RARA, encoding retinoic acid receptor alpha (RAR alpha). We have now cloned cDNAs encoding PLZF-RAR alpha chimeric proteins and studied their transactivating activities. In transient-expression assays, both the PLZF(A)-RAR alpha and PLZF(B)-RAR alpha fusion proteins like the PML-RAR alpha protein resulting from the well-known t(15;17) translocation in APL, antagonized endogenous and transfected wild-type RAR alpha in the presence of retinoic acid. Cotransfection assays showed that a significant repression of RAR alpha transactivation activity was obtained even with a very low PLZF-RAR alpha-expressing plasmid concentration. A "dominant negative" effect was observed when PLZF-RAR alpha fusion proteins were cotransfected with vectors expressing RAR alpha and retinoid X receptor alpha (RXR alpha). These abnormal transactivation properties observed in retinoic acid-sensitive myeloid cells strongly implicate the PLZF-RAR alpha fusion proteins in the molecular pathogenesis of APL.     

Modeling acute promyelocytic leukemia in the mouse: new insights in the pathogenesis of human leukemias

Acute promyelocytic leukemia (APL) is characterized by the expansion of malignant myeloid cells blocked at the promyelocytic stage of differentiation and is associated with reciprocal chromosomal translocations always involving the retinoic acid receptor alpha (RARalpha) gene on chromosome 17. As a consequence of the translocation, RARalpha variably fuses to the PML, PLZF, NPM, NuMA, and Stat5b genes (X genes), respectively, leading to the generation of RARalpha-X and X-RARalpha fusion genes. The aberrant chimeric proteins encoded by these genes, as well as the inactivation of the X and RARalpha functions, may exert a crucial role in leukemogenesis. To define the molecular genetics of APL and the contribution of each molecular event in APL pathogenesis, we have generated transgenic mice harboring X-RARalpha and/or RARalpha-X genes as well as mice where the various X genes have been inactivated by homologous recombination. Here we show that while the X-RARalpha fusion gene is crucial for leukemogenesis, the presence of RARalpha-X and the inactivation of X function are critical in modulating the onset as well as the phenotype of the leukemia.     

Complete remission through blast cell differentiation in PLZF/RARalpha-positive acute promyelocytic leukemia: in vitro and in vivo studies

Acute leukemia with the t(11;17) expressing the PLZF-RARalpha gene fusion is a rare variant of acute promyelocytic leukemia (APL) that has been associated with poor clinical response to all-trans retinoic acid (ATRA) treatment. However, some recent reports have put into question the absolute refractoriness of this leukemia to ATRA. We describe here a patient with PLZF/RARalpha APL who was treated at relapse with ATRA and low-dose hydroxyurea. Complete hematologic remission was obtained through differentiation of leukemic blasts, as proven by morphologic, immunophenophenotypic, and genetic studies carried out in sequential bone marrow samples. Moreover, in vitro studies indicated that blast differentiation was potentiated by the addition of the histone deacetylase inhibitor tricostatin A, but not of hydroxyurea, to ATRA. Our findings indicate that the maturation block may be overcome and terminal differentiation obtained in this leukemia subset and support the view that sensitivity/refractoriness of this form to ATRA should be revisited.     

The acute promyelocytic leukemia-associated protein, promyelocytic leukemia zinc finger, regulates 1,25-dihydroxyvitamin D(3)-induced monocytic differentiation of U937 cells through a physical interaction with vitamin D(3) receptor.

Monocyte differentiation induced by 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) is interrupted during the course of acute promyelocytic leukemia (APL). One form of APL is associated with the translocation t(11;17), which joins the promyelocytic leukemia zinc finger (PLZF) and retinoic acid receptor alpha (RARalpha) genes. Because PLZF is coexpressed in the myeloid lineage with the vitamin D(3) receptor (VDR), the interplay between PLZF and VDR was examined. It was found that PLZF interacts directly with VDR. This occurred at least partly through contacts in the DNA-binding domain of VDR and the broad complex, tram-trak, bric-a-brac/pox virus zinc finger (BTB/POZ) domain of PLZF. Moreover, PLZF altered the mobility of VDR derived from nuclear extracts when bound to its cognate binding site, forming a slowly migrating DNA-protein complex. Overexpression of PLZF in a monocytic cell line abrogated 1,25(OH)(2)D(3) activation from both a minimal VDR responsive reporter and the promoter of p21(WAF1/CIP1), a target gene of VDR. Deletion of the BTB/POZ domain significantly relieved PLZF-mediated repression of 1,25(OH)(2)D(3)-dependent activation. In addition, stable, inducible expression of PLZF in U937 cells inhibited the ability of 1,25(OH)(2)D(3) to induce surface expression of the monocytic marker CD14 and morphologic changes associated with differentiation. These results suggest that PLZF may play an important role in regulating the process by which 1,25(OH)(2)D(3) induces monocytic differentiation in hematopoietic cells.     

The role of promyelocytic leukemia zinc finger and promyelocytic leukemia in leukemogenesis and development

Acute promyelocytic leukemia (APL) was originally distinguished by an extremely poor clinical outcome. In the past few years, however, important progress has been made in defining the molecular basis of APL pathogenesis and in optimizing its treatment to an extent that this leukemia is now considered curable. Two features are unique to this leukemia: its remission after retinoic acid (RA) treatment through induction of blast differentiation, and the presence in the leukemic blast of fusion proteins in which the retinoic acid receptor alpha (RARalpha) fuses to distinct partners. Here we review how a detailed analysis of the functions of two of these RARalpha partners, the promyelocytic leukemia (PML) and promyelocytic leukemia zinc finger (PLZF) proteins, has allowed a greater understanding of the molecular mechanisms implicated in APL pathogenesis.     

PML/RARalpha and FLT3-ITD induce an APL-like disease in a mouse model

Acute promyelocytic leukemia (APL) cells invariably express aberrant fusion proteins involving the retinoic acid receptor alpha (RARalpha). The most common fusion partner is promyelocytic leukemia protein (PML), which is fused to RARalpha in the balanced reciprocal chromosomal translocation, t(15;17)(q22:q11). Expression of PML/RARalpha from the cathepsin G promoter in transgenic mice causes a nonfatal myeloproliferative syndrome in all mice; about 15% go on to develop APL after a long latent period, suggesting that additional mutations are required for the development of APL. A candidate target gene for a second mutation is FLT3, because it is mutated in approximately 40% of human APL cases. Activating mutations in FLT3, including internal tandem duplication (ITD) in the juxtamembrane domain, transform hematopoietic cell lines to factor independent growth. FLT3-ITDs also induce a myeloproliferative disease in a murine bone marrow transplant model, but are not sufficient to cause AML. Here, we test the hypothesis that PML/RARalpha can cooperate with FLT3-ITD to induce an APL-like disease in the mouse. Retroviral transduction of FLT3-ITD into bone marrow cells obtained from PML/RARalpha transgenic mice results in a short latency APL-like disease with complete penetrance. This disease resembles the APL-like disease that occurs with long latency in the PML/RARalpha transgenics, suggesting that activating mutations in FLT3 can functionally substitute for the additional mutations that occur during mouse APL progression. The leukemia is transplantable to secondary recipients and is ATRA responsive. These observations document cooperation between PML/RARalpha and FLT3-ITD in development of the murine APL phenotype.     

Successful All-<Emphasis Type="Italic">trans</Emphasis> Retinoic Acid Treatment of Acute Promyelocytic Leukemia in a Patient with <Emphasis Type="Italic">NPM/RAR</Emphasis> Fusion

Acute promyelocytic leukemia (APL) is characterized by a reciprocal chromosomal translocation involving the gene for retinoic acid receptor alpha(RAR). Most APL patients have a t(15;17) translocation that generates the PML-RAR fusion gene, and such patients respond well to treatment with all-trans retinoic acid (ATRA). Some APL cases also involve rearrangements that fuse RAR to partner genes other than PML, including nucleophosmin (NPM), promyelocytic leukemia zinc finger (PLZF), nuclear mitotic apparatus (NUMA), and Stat5b, but the clinical characteristics of APL without PML-RAR have not been fully clarified. We describe a 64-year-old man with NPM-RAR-positive APL who was receiving hemodialysis therapy for chronic uremia. Complete remission was achieved with ATRA monotherapy and was maintained for 18 months with consolidation chemotherapy. These findings suggest that ATRA can be used to treat APL patients with NPM/RAR as well as APL with PML/RAR.     

Additive effects of PI3-kinase and MAPK activities on NB4 cell granulocyte differentiation: potential role of phosphatidylinositol 3-kinase γ

PURPOSE: In acute promyelocytic leukemia (APL) the chromosome translocation t(15;17) resulting in the PML-RARalpha fusion protein is responsible for a blockage of myeloid differentiation. In this study we investigated the expression of different Phosphatidylinositol 3-kinase (PI3K) isoforms during granulocyte differentiation of NB4 cells induced by all-trans-retinoic acid (ATRA), 9-cis-retinoic acid (9cisRA) or retinoic acid receptor (RAR) agonists. METHODS: NB4 cells were analysed for their ability to differentiate into granulocytic lineage by the use of ATRA, 9cisRA or RAR agonists. Expression of signalling proteins was investigated by western blot and real-time PCR. PI3K activity was determined by in vitro kinase assays. RESULTS: Co-treatment of NB4 cells with either LY294002 to inhibit PI3Ks or PD98059 in order to suppress MEK activity led to significant reduction of CD11b surface expression during ATRA, 9cisRA or the RARalpha agonist Ro40-6055 dependent NB4 cells granulocyte differentiation. We also show that only the G-protein coupled receptor activated PI3Kgamma isoform demonstrates up-regulated protein and mRNA expression during myeloid differentiation of NB4 cells via RARalpha and RARbeta-dependent mechanism. Furthermore, activation of MAPK cascade including phosphorylation of MEK increases during retinoid induced differentiation of NB4 cells. Interestingly, protein kinase assays of immunoprecipitated PI3Kgamma revealed a protein of about 50 kDa that is phosphorylated when NB4 cells were treated with the RARalpha agonist Ro40-6055. CONCLUSION: Collectively, our data suggest additive effects of PI3K and MAPK activity on ATRA-dependent NB4 cells granulocyte differentiation.     

Analysis of the molecular genetics of acute promyelocytic leukemia in mouse models

Acute promyelocytic leukemia (APL) is characterized by reciprocal chromosomal translocations that always Involve the retinoic acid receptor-alpha (RARalpha) gene on chromosome 17. RARalpha variably fuses to the PML, PLZF, NPM, NuMA, and STAT 5b genes (X genes), leading to the generation of X-RARalpha and RARalpha-X fusion genes. The aberrant X-RARalpha proteins retain the dimerization domains of their parental proteins and therefore can act as dominant negative oncogenic products on both RARalpha/RXR and X pathways. Studies in transgenic mice harboring X-RARalpha and RARalpha-X fusion genes and In mice lacking X genes have helped unravel the molecular mechanisms underlying APL leukemogenesis, which lead to the development of novel therapeutic strategies. Moreover, transgenic mouse models of APL were useful to test in vivo the efficacy of these novel therapeutic approaches as well as of drug combinations such as retinoic acid and As2O3 that were previously known to be effective as single agents in human APL.