The PML/RAR alpha oncoprotein is a direct molecular target of retinoic acid in acute promyelocytic leukemia cells

Acute promyelocytic leukemia (APL) is characterized by the translocation, t(15;17) and the expression of a PML/RAR alpha fusion protein that is diagnostic of the disease. There is evidence that PML/RAR alpha protein acts as a dominant negative inhibitor of normal retinoid receptor function and myeloid differentiation. We now show that the PML/RAR alpha fusion product is directly downregulated in response to retinoic acid (tRA) treatment in the human APL cell line, NB4. tRA treatment induces loss of PML/RAR alpha at the protein level but not at the level of mRNA, as determined by Northern blots, by Western blots, and by ligand binding assays and in binding to RA-responsive DNA elements. We present evidence that this regulation is posttranslational. This evidence suggests that tRA induces synthesis of a protein that selectively degrades PML/RAR alpha. We further show that this loss of PML/ RAR-alpha is not limited to the unique APL cell line. NB4, because PML/RAR alpha protein is selectively downregulated by tRA when expressed in the transfected myeloid cell line U937. The loss of PML/RAR alpha may be directly linked to tRA-induced differentiation, because in a retinoid-resistant subclone of NB4, tRA does not decrease PML/RAR alpha protein expression. In NB4 cells, the specific downregulation of the fusion protein decreases the ratio of PML/RAR alpha to wild-type RAR alpha. Because the ratio of expression of PML/RAR alpha to wild-type RAR alpha and PML may be important in maintaining the dominant negative block of myelocytic differentiation, these data suggest a molecular mechanism for restoration by tRA normal myeloid differentiation in APL cells.     

Prolonged molecular remission after PML/RAR alpha-positive autologous peripheral blood stem cell transplantation in acute promyelocytic leukemia: is relevant pretransplant minimal residual disease in the graft?

The contribution of residual malignant cells contaminating the autologous graft with the occurrence of post-transplant relapse in acute myeloid leukemia (AML) is still unclear. The presence of a specific molecular marker (the PML/RAR alpha rearrangement) in acute promyelocytic leukemia (APL) offers the opportunity to investigate better the pathogenesis of disease recurrence after transplant. We report an APL patient who received high-dose chemotherapy and peripheral blood stem cell (PBSC) autograft in second hematologic remission. Two leukaphereses that tested PML/RAR alpha positive by RT-PCR were obtained during the post-reinduction hematopoietic recovery, while the patient also tested PCR positive in the BM, and was reinfused after myeloablative chemotherapy (BUCY4), when the patient had spontaneously converted to PCR negative in the marrow. At present, he remains in continuous molecular and hematologic remission 22 months after PBSC transplantation. This is the second report of an APL patient who was transplanted in molecular remission with a PML/RAR alpha-positive PBSC autograft. As in the previous report, the prolonged clinical and molecular remission experienced post-transplant suggests that autologous PBSC infusion is still worthy of consideration for patients with APL in spite of the detection of PML/RAR alpha-positive cells in the PBSC collections. Possible underlying mechanisms and the potential role of molecular monitoring of the graft, as well as the host, before and after transplant, in patients with APL undergoing autologous HSCT are also discussed.     

A PMLRARalpha transgene initiates murine acute promyelocytic leukemia

The malignant cells of acute promyelocytic leukemia (APL) contain a reciprocal chromosomal translocation that fuses the promyelocytic leukemia gene (PML) with the retinoic acid receptor alpha gene (RAR alpha). To test the hypothesis that the chimera PMLRAR alpha plays a role in leukemogenesis, we expressed a PMLRAR alpha cDNA in myeloid cells of transgenic mice. PMLRAR alpha transgenic mice exhibited impaired neutrophil maturation early in life, which progressed at a low frequency over the course of several months to overt APL. Both the preleukemic state and the leukemia could be transplanted to nontransgenic mice, and the transplanted preleukemia could progress to APL. The APL recapitulated features of the human disease, including a response to retinoic acid. Retinoic acid caused the leukemic cells to differentiate in vitro and in vivo, eliciting remissions of both the preleukemic state and APL in mice. Our results demonstrate that PMLRAR alpha impairs neutrophil differentiation and initiates the development of APL. The transgenic mice described here provide an apparently accurate model for human APL that includes clear evidence of tumor progression. The model should be useful for exploring the molecular pathogenesis of APL and the mechanisms of the therapeutic response to retinoic acid, as well as for preclinical studies of therapeutic regimens.     

The acute promyelocytic leukaemia specific PML and PLZF proteins localize to adjacent and functionally distinct nuclear bodies

Acute promyelocytic leukaemia is characterized by translocations that involve the retinoic acid receptor alpha (RAR alpha) locus on chromosome 17 and the PML locus on 15 or the PLZF locus on 11. The resulting abnormal translocation products encode for PML/RAR alpha or PLZF/RAR alpha fusion proteins. There is increasing experimental evidence that the APL-specific fusion proteins have similar biologic activities on differentiation and survival and that both components of the fusion proteins (PML or PLZF and RAR alpha) are indispensable for these biological activities. The physiologic function of PML or PLZF or whether PML and PLZF contribute common structural or functional features to the corresponding fusion proteins is not known. We report here immunofluorescence studies on the cellular localization of PLZF and PLZF/RAR alpha and compare it with the localization of PML and PML/RAR alpha. PLZF localizes to nuclear domains of 0.3-0.5 microns, approximately 14 per cell in the KG1 myeloid cell line. These PLZF-bodies are morphologically similar to the domains reported for PML (PML-NBs). There is tight spatial relationship between about 30% of PLZ-NBs and PML-NBs: they partially overlap. However, PML and PLZF do not form soluble complexes in vivo. PLZF- and PML-NBs are functionally distinct. Adenovirus E4-ORF3 protein expression alters the structure of the PML-NBs and interferon increases the number of PML-NBs and neither has any effect on PLZF NBs. The localization of PLZF/RAR alpha is different to that of PLZF and RAR alpha. The nuclear distribution pattern of PLZF/RAR alpha is one of hundreds of small dots (microspeckles) less than 0.1 micron. Expression of PLZF/RAR alpha did not provoke disruption of the PML-NBs. Co-expression of PML/RAR alpha and PLZF/RAR alpha in U937 cells revealed apparent colocalization. Overall the results suggest that the PML- and PLZF-NBs are distinct functional nuclear domains, but that they may share common regulatory pathways and/or targeting sequences, as revealed by the common localization of their corresponding fusion proteins.     

Treatment results of intermittent and cyclic regimen with ATRA and chemotherapy in childhood acute promyelocytic leukemia. Children's Cancer and Leukemia Study Group

An intermittent and cyclic regimen with All-Trans Retinoic Acid (ATRA) and intensive chemotherapy was conducted due to pharmacokinetic studies on ATRA for acute promyelocytic leukemia (APL) in children. We have treated 17 children with APL using ATRA for remission induction followed by an intermittent schedule of ATRA plus intensive chemotherapy (APL-ATRA protocol). There were 10 males and 7 females. The median age was 9.0 years old. The median baseline white blood cell count was 12.1 x 10(3)/microliter, hemoglobin 7.8 g/dl, platelet 4.5 x 10(4) microliters at diagnosis. Sixteen patients showed t(15; 17) translocation. RT-PCR analysis was available in 15 patients and showed PML/RAR alpha rearrangement in all patients. Overall, 13 or 17 newly diagnosed patients (88%) achieved complete remission and EFS was 67%. Compared to the control (same chemotherapy without ATRA regimen), remission induction and EFS were significantly increased. The toxicity of ATRA consisted of retinoic acid syndrome in 1 and pseudotumor cerebli in another. Other toxicities included headache, chelitis, gastrointestinal trouble and bone pain. These results suggest that intermittent and cyclic regimen with ATRA and intensive chemotherapy (APL-ATRA protocol) is highly effective for APL patients.     

Infrequent alterations of the RAR alpha gene in acute myelogenous leukemias, retinoic acid-resistant acute promyelocytic leukemias, myelodysplastic syndromes, and cell lines

Retinoids are important regulators of cell growth and differentiation in vitro and in vivo and they exert their biologic activities by binding to nuclear retinoic acid receptors (RARs; alpha, beta, and gamma) and retinoid X receptors (RXRs; alpha, beta, and gamma). All-trans retinoic acid (RA) induces complete remission in patients with acute promyelocytic leukemia (APL) presumably by binding directly to RAR alpha of APL cells. Leukemic blasts from APL patients initially responsive to RA can become resistant to the agent. HL-60 myeloblasts cultured with RA have developed mutations of the ligand-binding region of RAR alpha and have become resistant to RA. Furthermore, insertion of an RAR alpha with an alteration in the ligand-binding region into normal murine bone marrow cells can result in growth factor-dependent immortalization of the early hematopoietic cells. To determine if alterations of the ligand binding domain of RAR alpha might be involved in several malignant hematologic disorders, the mutational status of this region (exons 7, 8, and 9) was examined in 118 samples that included a variety of cell lines and fresh cells from patients with myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML), including 20 APL patients, 5 of whom were resistant to RA and 1 who was refractory to RA at diagnosis, using polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) analysis and DNA sequencing. In addition, 7 of the 20 APLs were studied for alterations of the other coding exons of the gene (exons 2 through 6). No mutations of RAR alpha were detected. Although the sensitivity of PCR-SSCP analysis is less than 100%, these findings suggest that alterations of RAR alpha gene are rare and therefore other mechanisms must be involved in the onset of resistance to retinoids and in the lack of differentiation in disorders of the myeloid lineage.     

Efficacy of fluorescence in situ hybridization for detecting PML/RARA gene fusion in treated and untreated acute promyelocytic leukemia

OBJECTIVE: To test the efficacy of fluorescence in situ hybridization (FISH) for detection of fusion of the promyelocytic leukemia (PML) and retinoic acid receptor alpha (RARA) genes in patients with treated or untreated acute promyelocytic leukemia (APL). DESIGN: We conducted a retrospective blind study on a series of stored bone marrow specimens from normal subjects and patients with APL. MATERIAL AND METHODS: Conventional cytogenetic and FISH analyses were done on interphase and metaphase cells in specimens from 31 normal subjects and 19 patients with untreated or treated APL. RESULTS: From 25 of the normal specimens, we calculated a normal cutoff of 10% for interphase cells and 0% for metaphase cells. With use of these criteria, the other six specimens from normal subjects showed normal findings, and each of the seven specimens from patients with untreated APL was abnormal by FISH analysis. The specimens from four patients in clinical relapse or with residual APL were abnormal. Of the eight specimens from patients in clinical remission, three were abnormal; two of these patients had a relapse within 8 months, and the other patient had received 1 month of chemotherapy and was entering remission. Of the other five patients in remission, four had normal FISH results and have now been in remission for 2.5 to 10 years. The other patient in remission with normal FISH results had a relapse within 6 months. PML/RARA fusion was detectable in three patients with hypogranular APL and in three with a cytogenetic variant of the t(15;17). CONCLUSION: The results of this study suggest that FISH with PML and RARA probes can be used to diagnose APL and may be useful for monitoring treated patients.     

A case of extrahepatic portal vein aneurysm with massive thrombosis: diagnosis with reconstruction images from helical CT scans

Acute promyelocytic leukemia (APL) originate from chromosomal translocations generating two types of fusion proteins both involving the retinoic acid receptor alpha (RAR alpha) and either the gene PML (t(15;17)) or PLZF (t(11;17)). Recent publications cast a new light on the detailed molecular mechanism underlying the oncogenic activity of these fusion proteins which block myeloid terminal differentiation by recruiting histone deacetylases to the promoters of target genes through co-repressor proteins. They also explain the different responses to treatment by all-trans retinoic acid (ATRA) of these two variants which are otherwise clinically indistinguishable.     

Recent advances in the treatment of acute leukemia

This review briefly summarizes literature considered noteworthy in the field of adult acute leukemia published during 1996. Does intensity remains a controversial issue in both acute myelogenous and lymphoblastic leukemia. The most convincing data showing efficacy of high dose fractionated chemotherapy was presented in patients with Burkitt's lymphoma/leukemia; the remainder of clinical studies failed to show a definitive advantage to high-dose therapy. Numerous studies addressed the role of the multidrug resistant phenotype and, at least in adult disease, demonstrated that the presence of this particular phenotype was a poor prognostic indicator. In the pediatric population, the significance of multidrug resistance expression appeared less clear. Discrepancies between protein expression and function were also evaluated in clinical samples and outcomes reported in large clinical series. Among the most interesting scientific investigations were those focused on the molecular mechanisms involved in the specific translocations t(15;17) and t(8;21) in acute myelogenous leukemia and t(12;21) in acute lymphoblastic leukemia. The genes PML and AML1, and ETO were examined in normal hematopoietic progenitors and their fusions proteins, PML/RAR alpha and AML1/ETO, measured in patients in clinical remission, and important data were presented concerning these proteins and measurement of minimal residual disease. Provocative data were also presented suggesting that retinoic acid may induce synthesis of a protein that selectively degrades PML/RAR alpha, and that interferons may regulate PML/RAR alpha expression.     

Trivalent antimonials induce degradation of the PML-RAR oncoprotein and reorganization of the promyelocytic leukemia nuclear bodies in acute promyelocytic leukemia NB4 cells

Acute promyelocytic leukemia (APL) is characterized by a specific t(15;17) chromosomal translocation that fuses the genes encoding the promyelocytic leukemia protein (PML) and the retinoic acid receptor (RAR). The resulting PML-RAR protein induces a block in the differentiation of the myeloid progenitor cells, which can be released by retinoic acid (RA) in vitro and in vivo. The RA-induced differentiation of APL blasts is paralleled by the degradation of the fusion protein and the relocation of wild-type PML from aberrant nuclear structures to its normal localization in nuclear bodies. Recently, arsenic trioxide (As2O3) treatment was proposed as an alternative therapy in APL, because it can induce complete remission in both RA-sensitive and -resistant APL patients. Intriguingly, As2O3 was also shown to induce degradation of the PML-RAR chimera and to reorganize PML nuclear bodies. Here we show that trivalent antimonials also have striking effects on RA-sensitive and RA-resistant APL cells. Treatment of the APL-derived NB4 cells and the RA-resistant subclone NB4R4 with antimony trioxide or potassium antimonyl tartrat triggers the degradation of the fusion protein and the concomitant reorganization of the PML nuclear bodies. In addition, as reported for As2O3, the antimonials provoke apoptosis of NB4 and NB4R4 cells. The mechanism of antimony action is likely to be similar to that of As2O3, notably both substances induce the attachment of the ubiquitin-like SUMO-1 molecule to the PML moiety of PML-RAR. From these data, we propose that, in analogy to As2O3, antimonials might have a beneficial therapeutic effect on APL patients, perhaps with less toxicity than arsenic.