Constitutive degradation of PML/RARalpha through the proteasome pathway mediates retinoic acid resistance

PML/RARalpha is the leukemogenetic protein of acute promyelocytic leukemia (APL). Treatment with retinoic acid (RA) induces degradation of PML/RARalpha, differentiation of leukaemic blasts, and disease remission. However, RA resistance arises during RA treatment of APL patients. To investigate the phenomenon of RA resistance in APL, we generated RA-resistant sublines from APL-derived NB4 cells. The NB4.007/6 RA-resistant subline does not express the PML/RARalpha protein, although its mRNA is detectable at levels comparable to those of the parental cell line. In vitro degradation assays showed that the half-life of PML/RARalpha is less than 30 minutes in NB4.007/6 and longer than 3 hours in NB4. Treatment of NB4.007/6 cells with the proteasome inhibitors LLnL and lactacystin partially restored PML/RARalpha protein expression and resulted in a partial release of the RA-resistant phenotype. Similarly, forced expression of PML/RARalpha, but not RARalpha, into the NB4/007.6 cells restored sensitivity to RA treatment to levels comparable to those of the NB4 cells. These results indicate that constitutive degradation of PML/RARalpha protein may lead to RA resistance and that PML/RARalpha expression is crucial to convey RA sensitivity to APL cells.     

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.     

G-CSF signaling can differentiate promyelocytes expressing a defective retinoic acid receptor: evidence for divergent pathways regulating neutrophil differentiation

Several lines of investigation suggest that granulocyte colony-stimulating factor (G-CSF) augments all-trans retinoic acid (ATRA)-induced neutrophil differentiation in acute promyelocytic leukemia (APL). We sought to characterize the relationship between G-CSF- and ATRA-mediated neutrophil differentiation. We established a G-CSF receptor-transduced promyelocytic cell line, EPRO-Gr, derived from the granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent EPRO cell line harboring a dominant-negative retinoic acid receptor alpha (RARalpha). In EPRO-Gr, neutrophil differentiation occurs either in GM-CSF upon addition of ATRA or upon induction with G-CSF alone. Transient transfection of EPRO-Gr cells with a RARE-containing reporter plasmid demonstrates increased activity in the presence of ATRA, but not G-CSF, while STAT3 phosphorylation occurs only in response to G-CSF. This suggests that ATRA-mediated differentiation of EPRO-Gr cells occurs via a RARE-dependent, STAT3-independent pathway, while G-CSF-mediated differentiation occurs via a RARE-independent, STAT3-dependent pathway. ATRA and G-CSF thus regulate differentiation by divergent pathways. We characterized these pathways in the APL cell line, NB4. ATRA induction of NB4 cells resulted in morphologic differentiation and up-regulation of C/EBPepsilon and G-CSFR, but not in STAT3 phosphorylation. The addition of G-CSF with ATRA during NB4 induction resulted in STAT3 phosphorylation but did not enhance differentiation. These results may elucidate how G-CSF and ATRA affect the differentiation of primary and ATRA-resistant APL cells.     

Histone deacetylase inhibitor but not arsenic trioxide differentiates acute promyelocytic leukaemia cells with t(11;17) in combination with all-trans retinoic acid

Acute promyelocytic leukaemia (APL) with t(11;17)/PLZF-RARalpha responds poorly to all-trans retinoic acid (ATRA) and arsenic trioxide (As2O3), in contrast to APL with t(15;17)/PML-RARalpha. Molecular studies have shown that histone deacetylase (HDAC) recruited by PLZF-RARalpha is associated with the ATRA resistance. Here, we analysed in vitro the differentiation of APL cells with t(11;17) using ATRA, As203, granulocyte colony-stimulating factor (G-CSF), HDAC inhibitor trichostatin A (TSA), or combinations of these. Although 1 microM ATRA, which stimulated the differentiation of APL cells with t(15;17), was insufficient to induce differentiation, 3 microM ATRA induced terminal differentiation into granulocytes. As203 alone or in combination with ATRA induced neither differentiation nor apoptosis. However, the combination of TSA and 1 microM ATRA had a potent differentiating effect, although TSA alone had little effect. The combination of 1 microM ATRA and G-CSF did not induce differentiation. These results indicate that APL cells with t(11;17) need a higher concentration of ATRA than those with t(15;17) to differentiate and suggest that HDAC inhibitor is a promising differentiation enhancer in APL with t(11;17).     

The molecular pathogenesis of acute promyelocytic leukaemia: implications for the clinical management of the disease

Acute promyelocytic leukaemia (APL) is characterised by chromosomal rearrangements of 17q21, leading to fusion of the gene encoding retinoic acid receptor alpha (RARalpha) to a number of alternative partner genes (X), the most frequent of which are PML (>95%), PLZF (0.8%) and NPM (0.5%). Over the last few years, it has been established that the X-RARalpha fusion proteins play a key role in the pathogenesis of APL through recruitment of co-repressors and the histone deacetylase (HDAC)-complex to repress genes implicated in myeloid differentiation. Paradoxically, the X-RARalpha fusion protein has the potential to mediate myeloid differentiation at pharmacological doses of its ligand (all trans-retinoic acid (ATRA)), which is dependent on the dissociation of the HDAC/co-repressor complex. Arsenic compounds have also been shown to be promising therapeutic agents, leading to differentiation and apoptosis of APL blasts. It is now apparent that the nature of the RARalpha-fusion partner is a critical determinant of response to ATRA and arsenic, underlining the importance of cytogenetic and molecular characterisation of patients with suspected APL to determine the most appropriate treatment approach. Standard protocols involving ATRA combined with anthracycline-based chemotherapy, lead to cure of approximately 70% patients with PML-RARalpha-associated APL. Patients at high risk of relapse can be identified by minimal residual disease monitoring. The challenge for future studies is to improve complete remission rates through reduction of induction deaths, particularly due to haemorrhage, identification of patients at high risk of relapse who would benefit from additional therapy, and identification of a favourable-risk group, for which treatment intensity could be reduced, thereby reducing risks of treatment toxicity and development of secondary leukaemia/myelodysplasia. With the advent of ATRA and arsenic, APL has already provided the first example of successful molecularly targeted therapy; it is hoped that with further understanding of the pathogenesis of the disease, the next decade will yield further improvements in the outlook for these patients.     

Role of P-glycoprotein in all-trans retinoic acid (ATRA) resistance in acute promyelocytic leukaemia cells: analysis of intracellular concentration of ATRA

We analysed the relationship between all-trans retinoic acid (ATRA) resistance and P-glycoprotein (P-gp)-associated multidrug resistance (MDR) in acute promyelocytic leukaemia (APL). There was no difference in the intracellular ATRA accumulation between NB4 cells and an MDR1 cDNA-transduced NB4 subline and between ATRA-resistant NB4 cells (NB4/RA) and an MDR1 cDNA-transduced NB4/RA subline. PSC833, a MDR modifier, did not increase the intracellular accumulation of ATRA or affect the expression of CD11b, the nitroblue tetrazolium (NBT) reduction activity, the proportion of apoptotic cells or the morphology of these four ATRA-treated cell lines. Similar results were obtained in the analysis of APL cells from five patients relapsed after ATRA-induced complete remission.     

Histone deacetylase inhibitors induce caspase-dependent apoptosis and downregulation of daxx in acute promyelocytic leukaemia with t(15;17)

Histone deacetylase (HDAC) appears to play an important role in the pathogenesis of acute promyelocytic leukaemia (APL) as it is recruited by both PML-RARalpha and PLZF/RAR alpha in leukaemic cells with t(15;17) and t(11;17) respectively. Recent studies have demonstrated that HDAC inhibitors can be therapeutically used in various neoplastic disorders including APL. Cell differentiation was considered the major mechanism of the anti-leukaemic effects of HDAC inhibitors in APL. However, most of these studies either evaluated the effect of HDAC inhibitors in combination with all-trans retinoic acid (ATRA) or focused on the less common form of APL with t(11;17). To investigate the cellular effects of HDAC inhibitors, including sodium butyrate, trichostatin A, and suberoylanilide hydroxamic acid (SAHA), we used two APL cell lines, NB4 and the ATRA-resistant derivative NB4.306. Moreover, primary cells from five patients with cytogenetic evidence for t(15;17) were also studied. Our results demonstrated that HDAC inhibitors induce distinct caspase-dependent apoptosis in APL, which showed both concentration-and time-dependence. In addition, changes in the apoptosis-regulatory proteins, daxx, bcl-2 and bax were analysed. HDAC inhibitors induced downregulation of daxx, but no significant changes were detected in bcl-2 or bax. In conclusion, apoptosis induced by HDAC inhibitors in APL could provide an effective strategy for treatment of patients with t(15;17).     

Molecular pathogenesis of acute promyelocytic leukaemia and APL variants

It has been 12 years since the simultaneous discovery of the unique sensitivity of acute promyelocytic leukaemia (APL) to differentiation therapy with all-trans retinoic acid (ATRA) and the discovery that the retinoic acid receptor alpha (RARalpha) gene was rearranged in APL. Nearly 98% of cases of APL are associated with t(15;17) chromosomal translocation and fusion of the PML gene to that encoding RARalpha to yield an abnormal receptor with the capability of de-regulating gene expression in the haematopoietic cell, causing differentiation block and eventually the development of leukaemia. Since this original discovery, four other translocations were described in APL. In each of these the RARalpha gene is fused to different partner genes, all yielding aberrant nuclear receptors. These fusion proteins share in common the ability to repress rather than activate retinoic acid targets, one so strongly that the result is an ATRA-resistant form of the disease. In addition each of the partner proteins is important for normal cell growth and development. In this chapter we explore the biology of the RARalpha, the fusion proteins created in APL and the normal forms of the partner proteins. Through continued study of this disease it is hoped that novel treatments, potentially more applicable to other forms of leukaemia, may arise.     

Overexpression of BP1, a homeobox gene,is associated with resistance to all-trans retinoic acid in acute promyelocytic leukemia cells

BP1, a homeobox gene, is overexpressed in the bone marrow of 63% of acute myeloid leukemia patients. In this study, we compared the growth-inhibitory and cyto-differentiating activities of all-trans retinoic acid (ATRA) in NB4 (ATRA-responsive) and R4 (ATRA-resistant) acute promyelocytic leukemia (APL) cells relative to BP1 levels. Expression of two oncogenes, bcl-2 and c-myc, was also assessed. NB4 and R4 cells express BP1, bcl-2, and c-myc; the expression of all three genes was repressed after ATRA treatment of NB4 cells but not R4 cells. To determine whether BP1 overexpression affects sensitivity to ATRA, NB4 cells were transfected with a BP1-expressing plasmid and treated with ATRA. In cells overexpressing BP1: (1) proliferation was no longer inhibited; (2) differentiation was reduced two- to threefold; (3) c-myc was no longer repressed. These and other data suggest that BP1 may regulate bcl-2 and c-myc expression. Clinically, BP1 levels were elevated in all pretreatment APL patients tested, while BP1 expression was decreased in 91% of patients after combined ATRA and chemotherapy treatment. Two patients underwent disease relapse during follow-up; one patient exhibited a 42-fold increase in BP1 expression, while the other showed no change. This suggests that BP1 may be part of a pathway involved in resistance to therapy. Taken together, our data suggest that BP1 is a potential therapeutic target in APL. Rania T. Awwad and Khanh Do contributed equally to the present study.     

In vitro studies on cellular and molecular mechanisms of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia: As2O3 induces NB4 cell apoptosis with downregulation of Bcl-2 expression and modulation of PML-RAR alpha/PML proteins

It has been shown recently in China that arsenic trioxide (As2O3) is a very effective treatment for acute promyelocytic leukemia (APL). APL patients resistant to all-trans retinoic acid (ATRA) and conventional chemotherapy can still respond to AS2O3. In this study, we addressed the possible cellular and molecular mechanisms of this treatment by using NB4 cells as a model. The results show that: (1) As2O3 triggers relatively specific NB4 cell apoptosis at micromolar concentration, as proved by morphology, histogramic related nuclear DNA contents, and DNA gel eletrophoresis. (2) As2O3 does not influence bax, bcl-x, c-myc, and p53 gene expression, but downregulates bcl-2 gene expression at both mRNA and protein levels. (3) As2O3 induces a significant modulation of the PML staining pattern in NB4 cells and HL-60 cells. The micropunctates characteristic of PML-RAR alpha in NB4 cells dissappear after treatment with As2O3, whereas a diffuse PML staining occurs in the perinuclear cytoplasmic region. In addition, a low percentage of untreated NB4 cells exhibits an accumulation of PML positive particles in a compartment of cytoplasm. The percentage of these cells can be significantly increased after As2O3 treatment. A similar PML staining pattern is observed in apoptotic cells. (4) ATRA pretreatment does not influence As2O3-induced apoptosis. These results suggest that induction of cell apoptosis can be one of the mechanisms of the therapeutic effect of As2O3. Moreover, this apoptosis induction occurs independently of the retinoid pathway and may be mediated, at least partly, through the modulation of bcl-2, as well as PML-RAR alpha and/ or PML proteins.     

all-trans-Retinoic acid-induced expression and regulation of retinoic acid 4-hydroxylase (CYP26) in human promyelocytic leukemia

all-trans-Retinoic acid (ATRA) induces complete remission in majority of patients with acute promyelocytic leukemia (APL). However, accelerated metabolism of ATRA that is induced by chronic daily administration of oral ATRA has been implicated as one of the mechanisms leading to a reduced sensitivity or resistance to ATRA therapy. We investigated the expression and regulation of CYP26, a novel p450 enzyme, which is highly specific for ATRA, in promyelocytic leukemia cells (NB4 and HL-60). We found that treatment of NB4 cells with a pharmacological concentration of ATRA (1 microM) induced rapid and dose-dependent expression of CYP26 mRNA. The CYP26 expression returned to pretreatment levels in both cells after ATRA was removed from the media. Retinoic acid receptor-alpha (RARalpha) specific antagonist (CD2503) totally abolished the ATRA-induced expression of CYP26 mRNA in HL-60 and NB4 cells. Furthermore, HL-60R, a HL-60 subclone expressing nonfunctional RAR because of a point mutation in the ligand-binding domain of RARalpha, failed to show CYP26 mRNA expression in response to ATRA. ATRA-induced expression of CYP26 was restored in HL-60R cells retrovirally transduced with RARalpha, but not in those cells transduced with the other retinoid receptors. In conclusion, ATRA induces expression of CYP26 in myeloid and promyelocytic leukemia cells and this expression is modulated by RARalpha. The induction of CYP26 expression by ATRA treatment might be related to a substrate-driven feedback mechanism to regulate intracellular concentrations of ATRA and its over expression in some clones may be partly responsible for reduced sensitivity or resistance to ATRA therapy.     

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.