Mechanisms of action and resistance to all-trans retinoic acid (ATRA) and arsenic trioxide (As2O3) in acute promyelocytic leukemia
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Since the introduction of all-trans retinoic acid (ATRA) and arsenic trioxide (As2O3) for the treatment of acute promyelocytic leukemia (APL), the overall survival rate has improved dramatically. However, relapse/refractory patients showing resistance to ATRA and/or As2O3 are recognized as a clinically significant problem. Genetic mutations resulting in amino acid substitution in the retinoic acid receptor alpha (RARα) ligand binding domain (LBD) and the PML-B2 domain of PML-RARα, respectively, have been reported as molecular mechanisms underlying resistance to ATRA and As2O3. In the LBD mutation, ATRA binding with LBD is generally impaired, and ligand-dependent co-repressor dissociation and degradation of PML-RARα by the proteasome pathway, leading to cell differentiation, are inhibited. The PML-B2 mutation interferes with the direct binding of As2O3 with PML-B2, and PML-RARα SUMOylation with As2O3 followed by multimerization and degradation is impaired. To overcome ATRA resistance, utilization of As2O3 provides a preferable outcome, and recently, a synthetic retinoid Am80, which has a higher binding affinity with PML-RARα than ATRA, has been tested in the clinical setting. However, no strategy attempted to date has been successful in overcoming As2O3 resistance. Detailed genomic analyses using patient samples harvested repeatedly may help in predicting the prognosis, selecting the effective targeting drugs, and designing new sophisticated strategies for the treatment of APL.
KeywordsAPL PML-RARα ATRA Arsenic trioxide (As2O3) Drug resistance
Almost two decades ago, the prognosis of acute promyelocytic leukemia (APL) was critically poor due to fatal coagulation disorders at diagnosis [1, 2]. Even with conventional chemotherapy using anthracyclines, more than 70 % of APL patients showed poor prognosis [3, 4]. After introduction of all-trans retinoic acid (ATRA) in the clinical setting in combination with conventional chemotherapy, the prognosis of APL has improved dramatically, with the result that more than 85 % of patients now achieve complete remission (CR) and nearly 70 % of patients can be cured [5, 6, 7, 8]. Since 1994, the marked effectiveness of As2O3 in APL patients, even in relapsed patients after combination therapy with ATRA, has been confirmed [9, 10, 11, 12]. When As2O3 is utilized as a single agent, ~70 % of patients can be cured, whereas nearly 90 % of patients can be cured if As2O3 is utilized in combination with ATRA [13, 14]. Although outcomes of APL treatment with ATRA and/or As2O3 in combination with conventional chemo-drugs have improved, relapsed/refractory patients are still observed in the clinical setting and drug resistance to ATRA and As2O3 has been recognized as a critical problem.
Mechanisms of action of molecular targeting drugs to APL cells
Wild-type RARα is a nuclear hormone receptor that binds to consensus sequence DR5 (five bases spaced between two AGGTCA motifs) in target gene promoters, normally as heterodimer with retinoid X receptor (RXR) [31, 32, 33]. Without ligands, ATRA and 9-cis retinoic acid, RAR-RXR heterodimer induces transcription repression throughout chromatin remodeling by recruiting transcription co-repressors, such as N-CoR/SMRT large protein complexes, that contain histone deacetylases (HDACs) [27, 34, 35, 36, 37] and histone methyltransferases [38, 39, 40]. In the presence of ligand (~10−7 M), the co-repressor complexes dissociate from RAR-RXR, and transcriptional de-repression and activation are induced [34, 35, 36, 37, 41]. PML-RARα binds to DR5 of target gene promoters primarily as a homodimer, but also as a heterodimer with RXR [42, 43], and induces transcription repression by recruiting N-CoR/SMRT complexes and polycomb group repressive complex 1 and 2 (PRC1/2) [39, 40], which contain histone methyl transferases, in the absence of ligands  (Fig. 1). PML-RARα can be SUMOylated at K160 of the PML protein to recruit death domain-associated protein (DAXX), resulting in the transcriptional repression of target genes . Even in the presence of physiological concentration of ligand (10−7 M), the co-repressor complex still binds with PML-RARα and the transcriptional repression cannot be dissolved. In the presence of pharmacological concentration of ATRA (10−6 M), transcription activation can be induced by dissociation of co-repressor complexes from PML-RARα and proteasome-dependent PML-RARα degradation [45, 46, 47].
Molecular mechanisms of drug resistance in APL cells
From the molecular mechanisms of ATRA and As2O3 effectiveness as indicated above, several mechanisms of drug resistance have been speculated . In this section, we outline the molecular mechanisms of resistance that are thought to be significant from the clinical perspective.
RARα fusion proteins in APL
In very limited cases with APL phenotype, RARα translocations with X-genes other than PML (PLZF , NuMA , NPM , STAT5b [21, 53], FIP1L1 , PRKAR1A [23, 24], and BCOR ) resulting in the production of X-RARα fusion protein have been reported (Fig. 1). PML-RARα forms mainly homodimers, and it has been reported that homodimerization of PML-RARα is critical for the pathogenesis of APL [42, 43]. Sternsdorf et al.  indicated that forced homodimerization of RARα induces ALP-like leukemia in a mouse model, indicating that the dimerization domain of the fusion protein may be critical to the induction of leukemogenesis by X-RARα. In fact, homodimerization through specific domains (coiled-coil; PML-, NPM-, and STAT5b-, POZ/BTB; PLZF-, RIIA; PRKAR1A-, and so on) has been confirmed in all X-RARα proteins. Interestingly, in PML-, PRKAR1A- , and BCOR-RARα , heterodimerization with RXR is also important for transformation and/or RARE binding.
Since those chimeric proteins all hold RARα DNA binding domain (DBD) and ligand binding domain (LBD), ATRA responsiveness is speculated in all cases. However, ATRA resistance has been confirmed clinically in cases showing PLZF-RARα [18, 34, 41] and STAT5b-RARα [21, 53, 55] fusions. One explanation for ATRA resistance is that the N-CoR/SMRT-corepressor complex interacts with PLZF, even in the presence of pharmacological concentration of ATRA, such that transcriptional de-repression cannot occur at RARα target gene promoters [34, 41]. The molecular mechanisms of ATRA resistance in STAT5b-RARα-expressing cells has not been fully explicated. Wild-type Stat5b is localized in cytoplasm, but STAT5b-RARα aberrantly localizes in nucleus . STAT5b is a component of the janus kinase (JAK)-STAT signaling pathway, and phosphorylation of STAT5b by JAK causes homodimerization and translocation into the nucleus, where it acts as a transcription factor . Aberrant transcription regulation of STAT5b target genes in addition to RARα target genes by STAT5b-RARα may be related to ATRA resistance.
On the other hand, As2O3 resistance in clinical setting was observed in patients expressing PLZF- [57, 58], STAT5b- , and BCoR-RARα . The As2O3-binding C–C motif is confirmed in PML-B2 domain, and As2O3 binding is critical for the multimerization followed by PML-RARα degradation [29, 30, 42]. Lack of As2O3 binding sites in X-RARα protein may be one explanation of loss of As2O3 responsiveness. However, no direct effect of As2O3 on RARα has been reported.
Mechanisms of resistance to ATRA
To overcome ATRA resistance, a number of therapeutics has been tested in vitro and in vivo. Several clinical reports indicated that As2O3 rescue most of relapsed/refractory patients treated with ATRA/chemotherapy [9, 10, 11, 12, 68]. Am80, a synthetic retinoid that shows higher binding affinity with PML-RARα than ATRA, is utilized in the clinical setting [69, 70, 71]. Am80 is approximately 10 times more potent than ATRA as an in vitro inducer of differentiation in NB-4 and HL60 cells, and is chemically more stable than ATRA [72, 73]. Histone deacetylase (HDAC) inhibitors , such as sodium butyrate (NaF), valproic acid (VPA), and trichostatin A (TSA), have been utilized with ATRA and are expected to transcriptionally activate PML-RARα target genes to inhibit co-repressors complexes that contain HDACs [75, 76, 77]. Another approach to overcoming the resistance uses other molecular targeting therapeutics, such as gemtuzumab ozogamicin (GO), an anti-CD33 monoclonal antibody linked with calicheamicins [78, 79].
Molecular mechanisms of resistance to As2O3
Even for relapsed/refractory patients following treatment with ATRA/chemotherapy, As2O3 therapy is highly effective, with a complete remission rate of more than 80 % [80, 81, 82]. Although the CR rate is high even in relapsed patients, resistance to As2O3 treatment has been recognized as a clinically critical problem. Information on As2O3 resistance remains limited compared with that on ATRA resistance.
Although the overall survival of APL has been significantly prolonged since the introduction of ATRA and As2O3, relapse/refractory disease due to ATRA and/or As2O3 resistance remains a serious clinical problem. Additional genetic mutations in PML-RARα and another gene, such as FLT3-ITD or TP53 [66, 85], may contribute to disease progression and drug resistance in APL. Detailed genomic analyses using clinical samples harvested repeatedly from patients may help for predicting prognosis, selecting effective targeting drugs, understanding molecular backgrounds, and designing sophisticated new therapeutic strategies.
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