Acute promyelocytic leukaemia, all-trans-retinoic acid and arsenic.
Acute promyelocytic leukaemia (APL) was recognised as a distinct subtype of acute myeloid leukaemia (AML) in 1957 and affects 4-11% of children with AML. Its features include abnormal promyelocytes in bone marrow, coagulopathy (DIC) and fibrinolysis. In 1988 it was successfully treated with high doses of all-trans-retinoic acid (ATRA), which produces complete clinical remission in about 80% of patients . The treatment produces terminal differentiation of leukaemic blast cells. However, the effect is usually temporary and many patients relapse: treatment may then be continued with ATRA and a chemotherapeutic agent such as idarubicin, and this can produce over 90% remission  .
In APL about 90% of affected individuals have a reciprocal chromosome 15;17 translocation in which the t(15;17) breakpoint occurs near the retinoid receptor RAR [alpha] gene (Figure 1). This gene codes for one of the family of intracellular retinoid receptor proteins (RAR and RXR, which each have three subtypes, [alpha] , [beta] [gamma]) and which, on binding a retinoid, dimerise and bind to the nuclear DNA causing a change in gene expression. The chromosomal translocation results in a fusion between RAR [alpha] and another gene whose product is 'PML', generating a fusion product PML-RAR [alpha] , a chimaeric oncoprotein that has been extensively studied over the last 10 years. The fusion protein seems to block promyelocytic differentiation in a dominant-negative manner by interfering with either retinoic acid- or PML-mediated transcription pathways. However, myelocytic differentiation can take place in the presence of pharmacological rather than physiological levels of ATRA. It has also been reported that ATRA induces a specific degradation of PML-RAR [alpha] protein possibly through a proteasome pathway. This has led to the hypothesis that retinoids induce APL cells to mature by selectively targeting PML-RAR [alpha] .
In normal cells, PML is shown by histology to be localised within subnuclear structures called 'nuclear bodies' (or 'PML oncogenic domains'). A mammalian cell nucleus typically contains 10-30 PML nuclear bodies, but these are disrupted by the presence of RAR [alpha] -PML. Treatment with ATRA reconstitutes the normal PML nuclear body pattern in APL cells. The nuclear bodies contain, in addition to PML (in normal individuals), several important regulatory proteins including SUMO-1 (see next page), CREB-binding protein, retinoblastoma-susceptibility gene product (pRB), the death-domain-associated protein (Daxx), and p53. These proteins are involved with a number of cellular processes such as senescence, apoptosis and transcriptional regulation. Nuclear body components can contact chromatin by interacting with a heterochromatin protein, thereby providing a link between nuclear bodies and the chromatin. Inclusion of the PML-RAR [alpha] fusion protein disrupts the nuclear bodies .
Despite an excellent initial response to ATRA, APL cells develop resistance and relapse occurs in APL patients treated with ATRA alone. Originally, as mentioned above, traditional chemotherapy was then used, but it was shown that arsenic trioxide ([As.sub.2][O.sub.3]) is a very effective treatment for APL even in patients who had relapsed after ATRA treatment. Arsenic trioxide seems to induce apoptosis in APL cells .
[FIGURE 1 OMITTED]
Arsenic specifically induces the degradation of PML-RAR [alpha] in APL (as well as in its normal PML counterpart). It enhances the conjugation onto PML or PML-RAR [alpha] of the ubiquitin-like peptide SUMO ('small ubiquitin-like modifiers') - a process called 'SUMOylation'. It was shown that arsenic-induced PML SUMOylation triggers polyubiquitination and proteasome-dependent degradation .
A digression: proteasomes, the cellular dustbins
Cells do not store proteins or amino acids, and the majority of the proteins in a cell are turned over, although at different rates, and unwanted amino acids are degraded. In addition, it is necessary to eliminate potentially harmful abnormal proteins and get rid of superfluous regulatory proteins. There are various ways of doing this, including the lysosomes. However, a major way of targeting and removing proteins is the proteasome system which requires a small (76-amino acid residue) protein called ubiquitin (because of its ubiquitous presence in cells). Covalent binding of ubiquitin to a protein marks it out for degradation. The process of binding ubiquitin requires energy (ATP) and in fact up to 50 ubiquitin units may be added in tandem. It is not completely clear how proteins are identified for ubiquitination and destruction, but it seems that some have more 'unstable' amino-terminal ends than others and this may identify them to the ubiquitinating enzymes.
[FIGURE 2 OMITTED]
Ubiquitinated proteins are proteolytically degraded by a large multiprotein complex called the 26S proteasome. This is a large hollow cylinder formed from 28 protein subunits in stacked rings with 'caps' at each end (Figure 2). The cylinder has proteolytic activity in its core that indiscriminately hydrolyses unfolded proteins fed into the cylinder by the caps, which identify proteins to be degraded by their ubiquitin signals.
That was a small digression to explain ubiquitin and its action. SUMO is another small, rather similar protein in cells that can have a somewhat similar action to ubiquitin, but which also appears to have a role in protein localisation within the cell and perhaps other functions too. There are three versions of SUMO - 1, 2, and 3. The SUMO modification pathway resembles that of ubiquitin, but the enzymes involved in the two processes are different. The recognition sequence in target proteins for SUMOylation is usually but not always [PSI]-Lys-X-Glu/Asp, where [PSI] is a large hydrophobic residue and X represents any amino acid. SUMO-1 is linked to the target protein as a monomer (SUMO-2 and -3 are capable of forming polymers). Also present are SUMO proteases that can reverse the SUMOylation and so the process is dynamic. Nevertheless, SUMOylation (at least with SUMO-1) does not lead to degradation of the target protein: instead it appears to regulate certain protein:protein interactions, as well as subcellular or subnuclear localisation as mentioned above.
How [As.sub.2][O.sub.3] works
Arsenic trioxide is a component of antileukaemic drugs used in Chinese traditional medicine, and in 1998 it had been shown that it induced the degradation of the PML-RAR [alpha] fusion protein and the reorganisation of the nuclear bodies. In fact, arsenic trioxide specifically induces the degradation of the PML-RAR [alpha] fusion protein as well as normal PML. It enhances the conjugation onto PML or PML-RAR [alpha] of SUMO. PML contains three SUMOylation sites, each of them in protein domains that appear to be important for the formation of nuclear bodies, although only a single lysine residue (Lys 160) is sensitive to arsenic exposure. Other SUMOylated proteins (e.g. Daxx) accumulate on the nuclear bodies and this nuclear trafficking is regulated by the SUMOylation status of PML. Recent in vitro work  indicates that arsenic triggers SUMO-dependent polyubiquitination of PML and that this eventually leads to the degradation of PML-RAR [alpha] and thus differentiation of APL cells. It has also been shown that antimony compounds (e.g. the trioxide, or potassium antimony tartarate) can trigger the degradation of the fusion protein . Such compounds might be less toxic than arsenic ones.
There is another less frequently encountered form of APL in which the chromosome transition is t(11;17) rather than t(15;17) producing a different fusion protein, 'PLZF-RAR [alpha] ', and the prediction is that this form will not be susceptible to arsenic treatment. It was shown in vitro that cells containing PLZF-RAR [alpha] are not modified by SUMOylation and do not undergo apoptosis .
Thus the dramatic finding that ATRA therapy was successful in APL was the first example of differentiation therapy in the treatment of advanced cancer. However, a significant proportion of patients relapses (20-30%) after the initial remission and subsequently become resistant to ATRA treatment. The discovery that [As.sub.2][O.sub.3] successfully treats APL patients, both those who have become resistant to ATRA as well as those previously untreated with ATRA, is good news. It is satisfying that we can now see the molecular interactions in the cell, complicated though they are, that explain what is happening.
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Correspondence to: Professor Ed Wood, Centre for Bioscience, The Higher Education Academy, University of Leeds, Leeds LS2 9JT. (email: email@example.com)
E J WOOD Centre for Bioscience, The Higher Education Academy, University of Leeds, Leeds, UK
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|Title Annotation:||Leading Article|
|Date:||Jun 1, 2008|
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