Novel mutation in the PML/RARalpha chimeric gene exhibits dramatically decreased ligand-binding activity and confers acquired resistance to retinoic acid in acute promyelocytic leukemia.

OBJECTIVE: All-trans retinoic acid (RA) resistance in acute promyelocytic leukemia (APL) has been a serious clinical problem in differentiation-inducing therapy. However, the mechanisms underlying acquired RA resistance in APL patients are not well understood. MATERIALS AND METHODS: We recently established a spontaneous RA-resistant APL cell line (UF-1) from a patient and used this cell line as an excellent in vitro model for RA-resistant clinical situations. We investigated the structural and functional abnormalities of chimeric PML/RARalpha gene in UF-1 cells and preserved materials from the original patient. RESULTS: A novel point mutation was detected in the ligand-binding (E) domain of the RARalpha portion of the PML/RARalpha gene in UF-1 cells. This mutation resulted in amino acid substitution of Arg611 (CGG) for Trp611 (TGG) in the short-form PML/RARalpha protein, which corresponded to Arg276 in wild-type RARalpha. Importantly, the same mutation was also detected in the preserved materials from the original patient. COS-1 cells were transiently transfected with cDNA encoding wild-type and mutant PML/RARalpha constructed by site-directed mutagenesis and performed RA-binding assay. Interestingly, RA-binding activity was dramatically decreased in the mutant PML/RARalpha compared with that of the wild-type chimeric protein, suggesting that this single amino acid substitution is critical for RA binding. CONCLUSIONS: These results strongly suggest that a novel point mutation in the ligand-binding domain of the RARalpha portion (Arg611) of the chimeric PML/RARalpha gene decreased sensitivity to all-trans RA. We conclude that acquisition of the PML/RARalpha mutation is one possible mechanism for development of RA resistance in patients with APL in vivo.     

MEK inhibition induces caspases activation, differentiation blockade and PML/RARalpha degradation in acute promyelocytic leukaemia

The hallmark of acute promyelocytic leukaemia (APL) is the reciprocal translocation t(15;17), which leads to the expression of the promyelocytic leukaemia/retinoic acid receptor α (PML/RARα) fusion protein and a cell differentiation blockade at the promyelocytic stage. PML/RARα is directly targeted by all- trans -retinoic acid (ATRA), which degrades the oncoprotein and induces complete remission of malignancies. The aberrant function of PML/RARα, together with the constitutive activation of the mitogen-activated protein/extracellular signal-regulated kinase (MEK/ERK) signalling pathway, regulates the ability of haematopoietic cells to proliferate, differentiate, and escape from apoptotic episodes. The role of the MEK/ERK pathway in PML/RARα expression, differentiation, proliferation and apoptosis in APL cells was analysed using specific MEK inhibitors. The blockade of MEK/ERK pathway resulted in caspase-dependent degradation of PML/RARα, and attenuation of the cell differentiation induction. To our knowledge, this is the first report to show that PML/RARα was suppressed by MEK/ERK inhibition, through a mechanism dependent on caspase activation. ATRA co-operated with MEK inhibitor to increase degradation of PML/RARα and exhibited a convergence point in caspase activation with MEK inhibitors. Taken together, our data suggest a new role of MEK/ERK pathway in the pathogenesis of APL, thus supporting the use of MEK/ERK inhibitors as an efficient therapeutic strategy for this haematological malignancy.     

Combination of retinoic acid and tumor necrosis factor overcomes the maturation block in a variety of retinoic acid-resistant acute promyelocytic leukemia cells

Retinoic acid (RA) overcomes the maturation block in t(15:17) acute promyelocytic leukemia (APL), leading to granulocytic differentiation. Patients receiving RA alone invariably develop RA resistance. RA-resistant cells can serve as useful models for the development of treatments for both APL and other leukemias. Previously, we showed that RA and tumor necrosis factor (TNF) promote monocytic differentiation of the APL cell line NB4 and U937 monoblastic cells. Here, we report that combining TNF with RA leads to maturation of several RA-resistant APL cells along a monocytic pathway, whereas UF-1, a patient-derived RA-resistant cell line, showed characteristics of granulocytic differentiation. We found distinct differences in gene regulation between UF-1 cells and cells showing monocytic differentiation. Although IRF-7 was up-regulated by TNF and RA in all cells tested, expression of c-jun and PU.1 correlated with monocytic differentiation. Furthermore, synergistic induction of PU.1 DNA binding and macrophage colony-stimulating factor receptor (m-CSF-1R) mRNA was observed only in cells differentiating into monocytes. Using neutralizing antibodies against m-CSF-1R or its ligand, we found that inhibiting this pathway strongly reduced CD14 expression in response to RA and TNF, suggesting that this pathway is essential for their synergy in RA-resistant leukemia cells.     

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.     

Prognostic value of FLT3 mutations in patients with acute promyelocytic leukemia treated with all-trans retinoic acid and anthracycline monochemotherapy

Background

Fms-like tyrosine kinase-3 (FLT3) gene mutations are frequent in acute promyelocytic leukemia but their prognostic value is not well established.

Design and Methods

We evaluated FLT3-internal tandem duplication and FLT3-D835 mutations in patients treated with all-trans retinoic acid and anthracycline-based chemotherapy enrolled in two subsequent trials of the Programa de Estudio y Tratamiento de las Hemopatías Malignas (PETHEMA) and Hemato-Oncologie voor Volwassenen Nederland (HOVON) groups between 1996 and 2005.

Results

FLT3-internal tandem duplication and FLT3-D835 mutation status was available for 306 (41%) and 213 (29%) patients, respectively. Sixty-eight (22%) and 20 (9%) patients had internal tandem duplication and D835 mutations, respectively. Internal tandem duplication was correlated with higher white blood cell and blast counts, lactate dehydrogenase, relapse-risk score, fever, hemorrhage, coagulopathy, BCR3 isoform, M3 variant subtype, and expression of CD2, CD34, human leukocyte antigen-DR, and CD11b surface antigens. The FLT3-D835 mutation was not significantly associated with any clinical or biological characteristic. Univariate analysis showed higher relapse and lower survival rates in patients with a FLT3-internal tandem duplication, while no impact was observed in relation to FLT3-D835. The prognostic value of the FLT3-internal tandem duplication was not retained in the multivariate analysis.

Conclusions

FLT3-internal tandem duplication mutations are associated with several hematologic features in acute promyelocytic leukemia, in particular with high white blood cell counts, but we were unable to demonstrate an independent prognostic value in patients with acute promyelocytic leukemia treated with all-trans retinoic acid and anthracycline-based regimens.     

PML protein expression in hematopoietic and acute promyelocytic leukemia cells

Acute promyelocytic leukemia (APL) is thought to be caused by the t(15,17) translocation that fuses the PML gene to that of the retinoic acid receptor alpha (RAR alpha) and generates a PML/RAR alpha fusion protein. Yet, paradoxically, APL cells are exquisitely sensitive to retinoic acid (RA), as they terminally differentiate upon RA exposure. In this report, we have examined the expression of PML and PML/RAR alpha in normal and APL cells. By immunofluorescence or immunocytochemistry, we show that PML has a speckled nuclear pattern of expression that contrasts with that of PML/RAR alpha (mostly a micropunctuated nuclear pattern or a cytoplasmic localization). The APL- derived cell line NB4 that expresses both the PML and PML/RAR alpha genes also shows the fine micropunctuated nuclear pattern, suggesting a dominant effect of the fusion protein over the localization of wild- type PML. RA treatment of NB4 cells or clones expressing PML/RAR alpha gradually leads to a PML pattern before apparent morphologic maturation. In 14 untreated APL patients, the PML-reactive proteins were cytoplasmic (by immunocytochemistry) or both cytoplasmic and nuclear with a micropunctuated pattern (by immunofluorescence). Strikingly, in 4 patients, after 1 to 2 weeks of RA therapy, the speckled nuclear PML pattern reappeared concomitant with the onset of differentiation. These results establish that fusion of PML to RAR alpha results in an altered localization of PML that is reverted upon RA treatment. This observation, which highlights the importance of PML, is likely to be a key to unravelling the molecular mechanism of both leukemogenesis and RA-induced differentiation of APL.     

Mutations of the PML tumor suppressor gene in acute promyelocytic leukemia

The promyelocytic leukemia (PML) tumor suppressor of acute promyelocytic leukemia (APL) is essential for a number of proapoptotic and growth-suppressive pathways as well as for the activity of differentiating agents such as retinoic acid (RA). In human APL, the dose of PML is reduced to heterozygosity given that one allele is involved in the chromosomal translocation while the status of the remaining PML allele is unknown. We have therefore used single-strand conformational polymorphism (SSCP) and sequencing analysis to screen DNA from APL patients for mutations at the PML locus. We identified DNA sequence variations resulting in a truncated PML protein in APL cases that displayed RA resistance and a very poor prognosis. Mutation analysis also led to the identification of aberrant PML sequence variations in other hematopoietic malignancies. Complete functional loss of PML is therefore selected by the APL phenotype and associates with poor prognosis and RA unresponsiveness.     

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.     

Hemophagocytic syndrome associated with retinoic acid syndrome in acute promyelocytic leukemia

A 56-year-old woman with an acute promyelocytic leukemia (APL) developed a severe all-trans-retinoic (ATRA) syndrome on day 17 of treatment. Shortly after, she presented a picture of pancytopenia, hepatosplenomegaly, increased triglycerides, ferritin, and liver enzymes. A bone marrow biopsy showed abundant macrophages and no evidence of leukemia. Tests for secondary hemophagocytic syndrome (HPS) were negative. A diagnosis of HPS was made. Treatment with dexamethasone and high-dose immunoglobulins was unsuccessful. Consolidation chemotherapy with idarubicin and ATRA rapidly reversed the HPS. The HPS in this patient could be related to the release of macrophage-stimulating cytokines by APL cells during ATRA syndrome.