PML-retinoic Acid Receptor Alpha Research Articles

Abstract C133: Aresenic Trioxide as targeted therapy for cancers harboring TRIM33-RET -fusion proteins

Abstract Chromosomal rearrangements are genomic events that can result in oncogenic gene fusions that are important drivers of tumerogenesis in multiple human cancers. Gene re-arrangements involving RET proto-oncogene were initially reported in papillary thyroid carcinoma. Recent genomic sequencing analysis have identified several rearrangements involving RET gene in other solid cancer types including lung cancers. The fusion protein resulting from the rearrangements usually contain the intact tyrosine kinase domain of RET fused to a partner protein which provides a constitutive dimerization domain. One such rearrangement involved Tripartite repeat motif containing protein 33 (TRIM33) where exons 1-14 of TRIM33 was fused with exon 12-19 of RET which was in frame. Rearrangements involving TRIM33 and RET have been reported in both thyroid cancer and lung cancer. Although these cancers respond to RET inhibitors, the responses are not durable, demonstrating the need for additional treatment approaches to these cancers. Arsenic, an ancient drug originally used in traditional Chinese medicine, shows remarkable anticancer activity in patients with acute promyelocytic leukemia (APL) and is now part of the standard therapy of this disease. Several studies have given new insight into the mechanism of action and specificity of AS2O3 in treatment of APL. AS2O3 promotes degradation of fusion protein, PML-RAR α, a fusion protein containing PML zinc finger protein and retinoic acid receptor alpha. The protein degradation is triggered by arsenic binding to cysteine residues in RBCC domain of PML and PML-RARα. Interestingly PML and TRIM33 both belong to same family of TRIM proteins and have highly conserved RBCC domains, suggesting that arsenic may also induced degradation of TRIM33-fusion proteins. To evaluate the effect of arsenic trioxide on TRIM33-RET fusion proteins, we first synthesized the in-frame fusion gene, and demonstrated that expression of TRIM33-RET fusion protein in primary cell lines leads to transformation. Cells transformed by TRIM33-RET fusion were highly sensitive to arsenic treatment, compared with similar cells expressing other gene fusions. Acute treatment with arsenic induced ubiquitiylation and degradation of TRIM33-RET fusion protein. The detailed molecular mechanism of arsenic-induced degradation of the TRIM33-RET fusion and possible role of such treatment in treating cancers with such rearrangements will be presented and discussed. These data suggest that arsenic trioxide, which is now used to target PML-RARa fusion protein in APML, may also be effective in treatment of solid-tumors driven by TRIM33-containing fusion proteins. Citation Format: Atul Kulkarni, Jeremy Tang, Kim Hirshfield, Lorna Rodriguez, Shridar Ganesan. Aresenic Trioxide as targeted therapy for cancers harboring TRIM33-RET -fusion proteins. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C133.

Targeting Epigenetic Misregulation in Synovial Sarcoma

Like many sarcomas, synovial sarcoma is driven by a characteristic oncogenic transcription factor fusion, SS18-SSX. In this issue of Cancer Cell, Su etal. elucidate the protein partners necessary for target gene misregulation and demonstrate a direct effect of histone deacetylase inhibitors on the SS18-SSX complex composition, expression misregulation, and apoptosis.

Rapid and reliable confirmation of acute promyelocytic leukemia by immunofluorescence staining with an antipromyelocytic leukemia antibody

The authors evaluated the utility of immunofluorescence staining with an antipromyelocytic leukemia (anti-PML) antibody for patients with a suspected diagnosis of new or relapsed acute promyelocytic leukemia (APL) and correlated the findings with the results of other established diagnostic modalities. Bone marrow (BM) and/or peripheral blood (PB) smears from 349 patients in whom the diagnosis of APL was considered were assessed with the anti-PML antibody using immunofluorescence. The study group included 199 patients with confirmed APL and 150 with other conditions. The results of conventional cytogenetics, reverse transcription polymerase chain reaction (RT-PCR), and fluorescence in situ hybridization (FISH) performed on these patients were correlated with the PML results. Among patients with confirmed APL, anti-PML antibody was positive in 182 of 184 BM and 32 of 33 PB smears. Conventional cytogenetics demonstrated t(15;17)(q22;q12) in 166 of 182 (91%) patients; 10 had a normal karyotype, 4 had insufficient mitoses to grow in culture, 1 was inconclusive, and 1 was 48, XX, +8, +8. Anti-PML staining was positive in 9 of 10 with a normal karyotype and in all 4 cases with insufficient mitoses. RT-PCR and FISH were positive for PML-retinoic acid receptor-alpha in 169 of 172 (98%) and 90 of 94 (96%) cases, respectively. Among the patients without APL, 148 of 150 (98.6%) were negative with anti-PML antibody. The sensitivity and specificity of the test were 98.9% and 98.7%, respectively. PML immunofluorescence staining is a rapid (<4 hours turnaround time) and reliable frontline diagnostic approach that can facilitate initiation of targeted therapy, particularly in clinical settings where cytogenetic and molecular testing are not readily available.

PML has a predictive role in tumor cell permissiveness to interferon-sensitive oncolytic viruses

The oncotropic phenotypes of several viruses correlate with tumor-associated deficiencies within interferon (IFN) signaling pathways. This observation formed the conceptual basis for developing oncolytic viruses deleted for viral proteins that inhibit the host IFN-dependent antiviral response, such as herpes simplex virus type-1 infected cell protein-0 (ICP0) and vesicular stomatitis virus matrix protein. Many viruses have evolved means to disrupt promyelocytic leukemia protein (PML) nuclear bodies. For example, ICP0 promotes PML degradation to inhibit the antiviral activities of this IFN-stimulated gene. As PML is downregulated in a variety of tumors, we hypothesized ICP0-null herpes simplex type-1 viruses are selectively oncolytic in tumors with impaired PML expression. We illustrate that ICP0-null herpes simplex type-1 viruses target tumor cells that either possess impaired PML signaling or cannot upregulate PML because of impaired IFN responsiveness. Disruption of PML signaling through overexpression of the dominant-negative protein PML-retinoic acid receptor alpha in PML-positive cells renders them sensitive to oncolysis by ICP0-null herpes simplex virus type-1 and vesicular stomatitis virus M protein mutant viruses, whereas PML overexpression reverses this phenomenon. Together, these data illustrate that PML mediates an antiviral mechanism that predicts the tropism of IFN-sensitive oncolytic viruses. To our knowledge, these viruses are the first examples of anti-cancer therapeutics capable of targeting deficiencies in PML expression.

Cytoplasmic Function of Mutant Promyelocytic Leukemia (PML) and PML-Retinoic Acid Receptor-α

The promyelocytic leukemia (PML) tumor suppressor of acute promyelocytic leukemia (APL) regulates major apoptotic and growth-suppressive pathways. In APL, PML is involved in a chromosomal translocation generating the PML-retinoic acid receptor-alpha (RARalpha) fusion protein. Two missense mutations in the remaining PML alleles have been identified, which give rise to a truncated cytoplasmic PML protein (Mut PML). APL patients carrying these mutations display resistance to retinoic acid (RA) and very poor prognosis. Here we show that Mut PML associates with the cytoplasmic regions we refer to as PML-cytoplasmic bodies (PML-CBs). Mut PML interacts with PML-RARalpha in PML-CB and potentiates PML-RARalpha-mediated inhibition of RA-dependent transcription. Remarkably, Mut PML stabilizes PML-RARalpha and inhibits differentiation induced by pharmacological doses of RA. A mutant form of PML-RARalpha that accumulates in the cytoplasm inhibits RA-dependent transcription and differentiation, thus suggesting that cytoplasmic localization of PML-RARalpha may contribute to transformation. Finally, we show that the bcr3 PML-RARalpha form is predominantly cytoplasmic and accumulates in PML-CBs. Taken together, these findings reveal novel insights into the molecular mechanisms contributing to APL.

Cooperation of Cytokine Signaling with Chimeric Transcription Factors in Leukemogenesis: PML-Retinoic Acid Receptor Alpha Blocks Growth Factor-Mediated Differentiation

Cooperation of Cytokine Signaling with Chimeric Transcription Factors in Leukemogenesis: PML-Retinoic Acid Receptor Alpha Blocks Growth Factor-Mediated Differentiation

Acute promyelocytic leukemia: recent advances in therapy and molecular basis of response to arsenic therapies

While arsenic has long been known as a poison and environmental carcinogen, its dramatic effect in the treatment of acute promyelocytic leukemia (APL) has made its mechanism of action a topic of intense interest. This paper reviews recent findings that reveal why a traditional poison has become a magical potion for a major type of APL, which is characterized by a balanced chromosomal translocation t(15;17). Daily IV infusion of arsenic trioxide (As2O3; ATO) for 30 to 40 days can lead to complete remission in about 85% of patients with newly diagnosed or relapsed APL. Oral preparations of ATO and tetra-arsenic tetra-sulfide (As4S4) seem to be as effective as parenteral ATO, with similar toxicity profiles. The combination of all-trans retinoic acid and ATO in patients with newly diagnosed APL has yielded more durable remission than monotherapy. The mechanism of arsenic cytotoxicity is thought to involve posttranslational modification followed by degradation of the PML-retinoic acid receptor-alpha (PML-RARalpha) fusion protein; targeting of PML to nuclear bodies with restoration of its physiologic functions; and production of reactive oxygen species (ROS) by NADPH oxidase in leukemic cells or collapse of the mitochondrial transmembrane potential. The understanding of arsenic cytotoxicity has stimulated modifications that promise to improve efficacy, such as interfering with ROS scavenging or boosting of ROS production to enhance the cytotoxicity, and adding cAMP or interferons to ATO regimens. Recent advances in the clinical use of arsenic, the mechanism of arsenic-mediated cytotoxicity, and modulations of ATO to increase its efficacy and expand its clinical spectrum are reviewed.

Translocation products in acute myeloid leukemia activate the Wnt signaling pathway in hematopoietic cells.

The acute myeloid leukemia (AML)-associated translocation products AML1-ETO, PML-retinoic acid receptor alpha (RARalpha), and PLZF-RARalpha encode aberrant transcription factors. Several lines of evidence suggest similar pathogenetic mechanisms for these fusion proteins. We used high-density oligonucleotide arrays to identify shared target genes in inducibly transfected U937 cells expressing AML1-ETO, PML-RARalpha, or PLZF-RARalpha. All three fusion proteins significantly repressed the expression of 38 genes and induced the expression of 14 genes. Several of the regulated genes were associated with Wnt signaling. One of these, plakoglobin (gamma-catenin), was induced on the mRNA and protein level by all three fusion proteins. In addition, primary AML blasts carrying one of the fusion proteins significantly overexpressed plakoglobin. The plakoglobin promoter was cloned and shown to be induced by AML1-ETO, with promoter activation depending on the corepressor and histone deacetylase binding domains. The induction of plakoglobin by AML fusion proteins led to downstream signaling and transactivation of TCF- and LEF-dependent promoters, including the c-myc promoter, which was found to be bound by plakoglobin in vivo after AML1-ETO expression. beta-Catenin protein levels and TCF and LEF target genes such as c-myc and cyclin D1 were found to be induced by the fusion proteins. On the functional level, a dominant negative TCF inhibited colony growth of AML1-ETO-positive Kasumi cells, whereas plakoglobin transfection into myeloid 32D cells enhanced proliferation and clonal growth. Injection of plakoglobin-expressing 32D cells into syngeneic mice accelerated the development of leukemia. Transduction of plakoglobin into primitive murine hematopoietic progenitor cells preserved the immature phenotype during colony growth, suggesting enhanced self-renewal. These data provide evidence that activation of Wnt signaling is a common feature of several balanced translocations in AML.

Cooperation of cytokine signaling with chimeric transcription factors in leukemogenesis: PML-retinoic acid receptor alpha blocks growth factor-mediated differentiation.

We utilized a mouse model of acute promyelocytic leukemia (APL) to investigate how aberrant activation of cytokine signaling pathways interacts with chimeric transcription factors to generate acute myeloid leukemia. Expression in mice of the APL-associated fusion, PML-RARA, initially has only modest effects on myelopoiesis. Whereas treatment of control animals with interleukin-3 (IL-3) resulted in expanded myelopoiesis without a block in differentiation, PML-RARA abrogated differentiation that normally characterizes the response to IL-3. Retroviral transduction of bone marrow with an IL-3-expressing retrovirus revealed that IL-3 and promyelocytic leukemia-retinoic acid receptor alpha (PML-RARalpha) combined to generate a lethal leukemia-like syndrome in <21 days. We also observed that a constitutively activated mutant IL-3 receptor, beta(c)V449E, cooperated with PML-RARalpha in leukemogenesis, whereas a different activated mutant, beta(c)I374N, did not. Analysis of additional mutations introduced into beta(c)V449E showed that, although tyrosine phosphorylation of beta(c) is necessary for cooperation, the Src homology 2 domain-containing transforming protein binding site is dispensable. Our results indicate that chimeric transcription factors can block the differentiative effects of growth factors. This combination can be potently leukemogenic, but the particular manner in which these types of mutations interact determines the ability of such combinations to generate acute myeloid leukemia.

Arsenic trioxide is a potent inhibitor of the interaction of SMRT corepressor with Its transcription factor partners, including the PML-retinoic acid receptor alpha oncoprotein found in human acute promyelocytic leukemia.

The SMRT corepressor complex participates in transcriptional repression by a diverse array of vertebrate transcription factors. The ability to recruit SMRT appears to play a crucial role in leukemogenesis by the PML-retinoic acid receptor alpha (RARalpha) oncoprotein, an aberrant nuclear hormone receptor implicated in human acute promyelocytic leukemia (APL). Arsenite induces clinical remission of APL through a incompletely understood mechanism. We report here that arsenite is a potent inhibitor of the interaction of SMRT with its transcription factor partners, including PML-RARalpha. Arsenite operates, in part, through a mitogen-activated protein (MAP) kinase cascade culminating in phosphorylation of the SMRT protein, dissociation of SMRT from its nuclear receptor partners, and a relocalization of SMRT out of the nucleus into the cytoplasm of the cell. Conversely, inhibition of this MAP kinase cascade attenuates the effects of arsenite on APL cells. Our results implicate SMRT as an important biological target for the actions of arsenite in both normal and neoplastic cells.

The aberrant fusion proteins PML-RARα and PLZF-RARα contribute to the overexpression of cyclin A1 in acute promyelocytic leukemia

Cyclin A1 is a newly discovered cyclin that is overexpressed in certain myeloid leukemias. Previously, the authors found that the frequency of cyclin A1 overexpression is especially high in acute promyelocytic leukemia (APL). In this study, the authors investigated the mechanism of cyclin A1 overexpression in APL cells and showed that the APL-associated aberrant fusion proteins (PML–retinoic acid receptor alpha [PML-RARα] or PLZF-RARα) caused the increased levels of cyclin A1 in these cells. The ectopic expression of either PML-RARα or PLZF-RARα in U937 cells, a non-APL myeloid cell line, led to a dramatic increase of cyclin A1 messenger RNA and protein. This elevation of cyclin A1 was reversed by treatment with all-trans retinoic acid (ATRA) in cells expressing PML-RARα but not PLZF-RARα. ATRA also greatly reduced the high levels of cyclin A1 in the APL cell lines NB4 and UF-1. No effect of ATRA on cyclin A1 levels was found in the ATRA-resistant NB4-R2 cells. Further studies using ligands selective for various retinoic acid receptors suggested that cyclin A1 expression is negatively regulated by activated RARα. Reporter assays showed that PML-RARα led to activation of the cyclin A1 promoter. Addition of ATRA inhibited PML-RARα–induced cyclin A1 promoter activity. Taken together, our data suggest that PML-RARα and PLZF-RARα cause the high-level expression of cyclin A1 seen in acute promyelocytic leukemia.

The aberrant fusion proteins PML-RARα and PLZF-RARα contribute to the overexpression of cyclin A1 in acute promyelocytic leukemia

AbstractCyclin A1 is a newly discovered cyclin that is overexpressed in certain myeloid leukemias. Previously, the authors found that the frequency of cyclin A1 overexpression is especially high in acute promyelocytic leukemia (APL). In this study, the authors investigated the mechanism of cyclin A1 overexpression in APL cells and showed that the APL-associated aberrant fusion proteins (PML–retinoic acid receptor alpha [PML-RARα] or PLZF-RARα) caused the increased levels of cyclin A1 in these cells. The ectopic expression of either PML-RARα or PLZF-RARα in U937 cells, a non-APL myeloid cell line, led to a dramatic increase of cyclin A1 messenger RNA and protein. This elevation of cyclin A1 was reversed by treatment with all-trans retinoic acid (ATRA) in cells expressing PML-RARα but not PLZF-RARα. ATRA also greatly reduced the high levels of cyclin A1 in the APL cell lines NB4 and UF-1. No effect of ATRA on cyclin A1 levels was found in the ATRA-resistant NB4-R2 cells. Further studies using ligands selective for various retinoic acid receptors suggested that cyclin A1 expression is negatively regulated by activated RARα. Reporter assays showed that PML-RARα led to activation of the cyclin A1 promoter. Addition of ATRA inhibited PML-RARα–induced cyclin A1 promoter activity. Taken together, our data suggest that PML-RARα and PLZF-RARα cause the high-level expression of cyclin A1 seen in acute promyelocytic leukemia.

Identification of Three Major Sentrinization Sites in PML

Acute promyelocytic leukemia arises following a reciprocal chromosome translocation t(15;17), which generates PML-retinoic acid receptor alpha fusion proteins (PML-RARalpha). We have shown previously that wild type PML, but not PML-RARalpha, is covalently modified by the sentrin family of ubiquitin-like proteins (Kamitani, T., Nguyen, H. P., Kito, K., Fukuda-Kamitani, T., and Yeh, E. T. H. (1998) J. Biol. Chem. 273, 3117-3120). To understand the mechanisms underlying the differential sentrinization of PML versus PML-RARalpha, extensive mutational analysis was carried out to determine which Lys residues are sentrinized. We show that Lys65 in the RING finger domain, Lys160 in the B1 Box, and Lys490 in the nuclear localization signal contributes three major sentrinization sites. The PML mutant with Lys to Arg substitutions in all three sites is expressed normally, but cannot be sentrinized. Furthermore, the triple substitution mutant is localized predominantly to the nucleoplasm, in contrast to wild type PML, which is localized to the nuclear bodies. Thus, sentrinization of PML, in the context of the RING finger and the B1 box, regulates nuclear body formation. Furthermore, we showed that sentrinization of PML-RARalpha could be restored by overexpression of sentrin, but not by retinoic acid treatment. These studies provide novel insight into the pathobiochemistry of acute promyelocytic leukemia and the sentrinization pathway.

An immunofluorescent assay for acute promyelocytic leukemia cells.

Sequential treatment with all-trans retinoic acid followed by chemotherapy significantly improves the long-term survival of patients who have acute promyelocytic leukemia (APL). Consequently, a simple and accurate test is needed to establish the diagnosis of APL and to identify those patients having a relapse of the disease. We describe an accurate, 2-hour indirect immunofluorescent assay for identifying APL cells in bone marrow specimens. The assay uses the PML (PG-M3) murine monoclonal antibody that is directed against the amino-terminal portion of the PML gene product. We observed a distinctive, finely speckled pattern of fluorescence in the NB4 cell line (a positive control), as well as in 15 clinical specimens that were confirmed to have APL by cytogenetic, cytochemical, and immunophenotypic studies, including four cases of microgranular variant of APL. By contrast, a coarse globular pattern of fluorescence was observed in 53 other clinical specimens that did not contain APL. When we performed dilution studies using artificial mixtures of APL cells with normal bone marrow cells, we detected as few as 5% APL cells in the mixture. Finally, there was complete concordance between the immunofluorescent assay and a polymerase chain reaction-based assay for the PML-retinoic acid receptor alpha chimeric gene in 12 other clinical specimens. We conclude that the immunofluorescent assay for PML protein is a rapid, sensitive, and accurate method for determining the presence of APL cells in clinical specimens. This assay therefore should be considered as a cost-effective alternative to other diagnostic tests, such as karyotyping or polymerase chain reaction, for the diagnostic evaluation of APL.

SMRT corepressor interacts with PLZF and with the PML-retinoic acid receptor alpha (RARalpha) and PLZF-RARalpha oncoproteins associated with acute promyelocytic leukemia.

Retinoic acid receptors (RARs) are hormone-regulated transcription factors that control key aspects of normal differentiation. Aberrant RAR activity may be a causal factor in neoplasia. Human acute promyelocytic leukemia, for example, is tightly linked to chromosomal translocations that fuse novel amino acid sequences (denoted PML, PLZF, and NPM) to the DNA-binding and hormone-binding domains of RARalpha. The resulting chimeric receptors have unique transcriptional properties that may contribute to leukemogenesis. Normal RARs repress gene transcription by associating with ancillary factors denoted corepressors (also referred to as SMRT, N-CoR, TRAC, or RIP13). We report here that the PML-RARalpha and PLZF-RARalpha oncoproteins retain the ability of RARalpha to associate with corepressors, and that this corepressor association correlates with certain aspects of the leukemic phenotype. Unexpectedly, the PLZF moiety itself can interact with SMRT corepressor. This interaction with corepressor is mediated, in part, by a POZ motif within PLZF. Given the presence of POZ motifs in a number of known transcriptional repressors, similar interactions with SMRT may play a role in transcriptional silencing by a variety of both receptor and nonreceptor transcription factors.

In vivo and in vitro characterization of the B1 and B2 zinc-binding domains from the acute promyelocytic leukemia protooncoprotein PML.

Acute promyelocytic leukemia (APL) has been ascribed to a chromosomal translocation event which results in a fusion protein comprising the PML protein and retinoic acid receptor alpha. PML is normally a component of a nuclear multiprotein complex which is disrupted in the APL disease state. Here, two newly defined cysteine/histidine-rich protein motifs called the B-box (B1 and B2) from PML have been characterized in terms of their effect on PML nuclear body formation, their dimerization, and their biophysical properties. We have shown that both peptides bind Zn2+, which induces changes in the peptides' structures. We demonstrate that mutants in both B1 and B2 do not form PML nuclear bodies in vivo and have a phenotype that is different from that observed in the APL disease state. Interestingly, these mutations do not affect the ability of wild-type PML to dimerize with mutant proteins in vitro, suggesting that the B1 and B2 domains are involved in an additional interaction central to PML nuclear body formation. This report in conjunction with our previous work demonstrates that the PML RING-Bl/B2 motif plays a fundamental role in formation of a large multiprotein complex, a function that may be common to those unrelated proteins which contain the motif.

Multimeric complexes of the PML-retinoic acid receptor alpha fusion protein in acute promyelocytic leukemia cells and interference with retinoid and peroxisome-proliferator signaling pathways.

The t(15;17) chromosomal translocation, specific for acute promyelocytic leukemia (APL), fuses the PML gene to the retinoic acid receptor alpha (RAR alpha) gene, resulting in expression of a PML-RAR alpha hybrid protein. In this report, we analyzed the nature of PML-RAR alpha-containing complexes in nuclear protein extracts of t(15;17)-positive cells. We show that endogenous PML-RAR alpha can bind to DNA as a homodimer, in contrast to RAR alpha that requires the retinoid X receptor (RXR) dimerization partner. In addition, these cells contain oligomeric complexes of PML-RAR alpha and endogenous RXR. Treatment with retinoic acid results in a decrease of PML-RAR alpha protein levels and, as a consequence, of DNA binding by the different complexes. Using responsive elements from various hormone signaling pathways, we show that PML-RAR alpha homodimers have altered DNA-binding characteristics when compared to RAR alpha-RXR alpha heterodimers. In transfected Drosophila SL-3 cells that are devoid of endogenous retinoid receptors PML-RAR alpha inhibits transactivation by RAR alpha-RXR alpha heterodimers in a dominant fashion. In addition, we show that both normal retinoid receptors and the PML-RAR alpha hybrid bind and activate the peroxisome proliferator-activated receptor responsive element from the Acyl-CoA oxidase gene, indicating that retinoids and peroxisome proliferator receptors may share common target genes. These properties of PML-RAR alpha may contribute to the transformed phenotype of APL cells.

The PML-retinoic acid receptor alpha translocation converts the receptor from an inhibitor to a retinoic acid-dependent activator of transcription factor AP-1.

We report here that the fusion of PML, a nuclear protein defined by the t(15;17) chromosomal translocation in acute promyelocytic leukemia, with retinoic acid receptor alpha (RAR alpha) changes the RAR alpha from a retinoic acid (RA)-dependent inhibitor to a RA-dependent activator of AP-1 transcriptional activity. The PML-RAR alpha chimera cooperates with c-Jun and, strikingly, with c-Fos to stimulate the transcription of both synthetic and natural reporter genes containing an AP-1 site. Stimulation is dependent on the concentration of RA and its dose-response curve is comparable to that for activation by RAR alpha of transcription on RA-responsive genes. Further, in the absence of RA, a circumstance in which RAR alpha has no effect on AP-1 activity, PML-RAR alpha is an inhibitor. Deletion of the dimerization, transactivation, or DNA-binding domains of c-Jun and removal of the PML dimerization domain in the PML-RAR alpha hybrid abrogates their transcriptional cooperatively. In view of the association between AP-1 activity and hemopoietic differentiation, we suggest that these properties of PML-RAR alpha could contribute to the leukemic phenotype and its response to RA.