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Abstract
p53 gene mutations are rarely detected at diagnosis in common haematological cancers such as multiple myeloma (MM), acute myeloid leukaemia (AML), chronic lymphocytic leukaemia (CLL) and Hodgkin's disease (HD), although their prevalence may increase with progression to more aggressive or advanced stages. Therapeutic induction of p53 might therefore be particularly suitable for the treatment of haematological malignancies. Some of the anti-tumour activity of current chemotherapeutics has been derived from activation of p53. However, until recently it was unknown whether p53 signalling is functional in certain haematological cancers including MM and if p53 activity is sufficient to trigger an apoptotic response. With the recent discovery of nutlins, which represent the first highly selective small molecule inhibitors of the p53–MDM2 interaction, pharmacological tools are now available to induce p53 irrespective of upstream signalling defects, and to functionally analyse the downstream p53 pathway in primary leukaemia and lymphoma cells. Combination therapy is emerging as a key factor, and development of non-genotoxic combinations seems very promising for tackling the problems of toxicity and resistance. This review will highlight recent findings in the research into molecules capable of modulating p53 protein activities and mechanisms that activate the p53 pathway, restoring response to therapy in haematological malignancies.
- Hematological malignancies
- p53
- MDM2
- nutlin
- apoptosis
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Introduction
The p53 tumour suppressor protein is a multi-functional transcription factor that regulates cellular processes affecting proliferation, DNA repair, cell cycle checkpoints and apoptosis.1–4 p53 is the most frequently altered gene in human cancer, being mutated in approximately 50% of all human solid tumours.5 In contrast to most solid tumours, the p53 gene is mutated in approximately 10–15% of both myeloid and lymphoid leukaemias at diagnosis6 and is found mainly in aggressive non-Hodgkin's lymphoma,7–12 progressive B-cell chronic lymphocytic leukaemia (B-CLL),13–19 and chronic prolymphocytic leukaemia (PLL).20 By molecular cytogenetic approaches, we and others found p53 mutations/deletions in about 10% of medullary myeloma cases,21–25 but higher incidences for patients with plasma cell leukaemia,26 or central nervous system myeloma.27 A strong correlation was also found between hemizygous p53 deletion and p53 protein expression in our study.28
The main function of p53 is to coordinate a highly conserved intracellular pathway, known as the p53 pathway, in response to different kinds of cellular stresses.29 30 In response to stress, the abundance of p53 increases, allowing it to orchestrate a multipronged response that involves both transcription-dependent and transcription-independent mechanisms.31 In the nucleus (transcription-dependent pathway), p53 governs apoptosis and/or cell cycle arrest by transactivating genes that code for pro-apoptotic members of the Bcl-2 protein family such as Bcl-2 associated X protein (Bax) and p53 up-regulated modulator of apoptosis (Puma) and/or for negative regulators of cell cycle progression such as p21.3 In the cytoplasm (transcription-independent pathway), p53 interacts with the mitochondria, favouring mitochondrial outer membrane permeabilisation (MOMP) which may lead to apoptosis.
In resting cells, p53 regulates its own expression by inducing the synthesis of the inhibitor murine double minute 2 (MDM2), which binds p53 and blocks DNA transactivation. MDM2 also has E3 ligase activity that promotes the ubiquitination and degradation of p53 through the 26S proteasome.32 Recognition of DNA damage leads to the stabilisation of p53 through post-translational modifications such as phosphorylation, acetylation and sumoylation, which interferes with the p53-MDM2 interaction.33 Studies have shown that p53 activating compounds that prevent disruption of the p53 pathway by blocking the p53-MDM2 interaction are efficacious in preclinical models.34
In cancer cells that retain wild type p53, disruption of the p53 pathway is a critical step in cancer progression. Since approximately half of all tumours retain wild type p53, it has been proposed that molecules which are able to activate the p53 response in tumour cells may be of therapeutic benefit. However, it has been shown that tumours often gain resistance via p53 mutations when treated with these compounds.35 Thus, therapeutic strategies should be aimed at targeting both wild type p53 and mutant p53.
This review will highlight the activation of wild type p53 and reactivation of mutant p53 by the small molecule inhibitors of the p53-MDM2 interaction and the molecules which bind to the core domain of p53 to stabilise p53. Effective combination of these small molecule inhibitors that show a synergistic response in inducing apoptosis will also be reviewed. In addition, we will discuss the molecular mechanisms associated with p53-mediated apoptosis in haematological malignancies.
Activation of the p53 pathway in cells harbouring wild type p53
The p53–MDM2 feedback loop is a key signalling pathway of p53,36 and therefore the most intensely studied route to activate p53 in tumour cells has been in the development of agents that block the p53–MDM2 interaction (figure 1).37 38 The activity of MDM2 inhibitors depends on p53 activation in cells expressing wild type p53 and therefore haematological malignancies, such as acute myeloid leukaemia (AML), B-CLL and multiple myeloma (MM), that express wild type p53 are potentially attractive tumour types. Small molecule inhibitors which have been reported to induce apoptosis in different haematological malignancies are discussed below.
Schematic representation of strategies to activate the p53 signalling pathway through p53-transcription-dependent (1) and transcription-independent (2) pathways in haematological malignancies. p53 functions in the nucleus to regulate pro-apoptotic and anti-apoptotic targets, whereas cytoplasmic p53 directly activates Bcl2 family proteins to permeabilise mitochondria and initiate apoptosis. (1) p53-transcription-dependent pathway: p53 is normally bound by MDM2 and targeted for degradation. Pharmacological dissociation of this interaction leads to activation of p53, resulting in its up-regulation and trans-activation of pro-apoptotic p53 target genes. This dissociation can occur through phosphorylation of p53 by genotoxic agents such as doxorubicin, etoposide and melphalan (A) or through direct dissociation of the p53–MDM2 interaction with small molecule inhibitors such as nutlin, MI-63 or MI-219 which bind MDM2 (B) or RITA which binds p53 (C). Small molecules such as CP31398, PRIMA-1 and MIRA-1 can induce mutant p53 to adopt a wild type conformation leading to its activation (D). Nutlin and PRIMA-1 can also activate p53 in a p53-independent manner by inhibiting the p73-MDM2 interaction, although the detailed mechanism of this pathway is unclear (E). Activated p53 induces up-regulation of p53 target genes, including MDM2 and p21 which are associated with negative feedback inhibition of p53 and cell cycle arrest, respectively, as well as pro-apoptotic targets including Puma, Bax and Bak. Activated p53 also leads to a down-regulation of anti-apoptotic proteins, Bcl2 and survivin. Pifithrin-α (PFT-α) inhibits transcription of p53-transcriptional targets in p53-mediated apoptosis (F). (2) p53-transcription-independent pathway: Activated p53 may result in mitochondrial translocation from the nucleus which may be inhibited by PFT-μ (G). p53 can directly interact with Bcl-XL, leading to the release of Bax from Bcl-XL sequestration (H). This event triggers Bax oligomerisation, resulting in MOMP and release of cytochrome C, which in turn activates caspase-3 through activation of capsase-9 and finally induces apoptosis (I).
Nutlin
One of the most promising wild type p53 reactivating agents is nutlin (the active form of nutlin is nutlin-3A or nutlin-3), a highly specific, non-genotoxic MDM2 antagonist that functions as a competitive inhibitor of the p53-MDM2 interaction.34 Nutlin is a cis-imidazoline analogue that binds MDM2 in the p53 pocket, thus preventing p53 degradation. In preclinical studies, nutlin displayed an increased potential for the treatment of human cancers harbouring wild type p53.34 37–39
Treatment of MM cells with nutlin specifically increased the protein levels of p53 and of its transcriptional targets, MDM2 and p21 in the samples retaining wild type p53.40 Similar to this finding, we have also demonstrated nutlin-induced activation of the p53 pathway resulting in a p53-dependent apoptosis in MM cells lines and MM primary samples.41 Ex vivo experiments in AML,42–45 B-CLL,46–49 Hodgkin lymphomas,50 51 mantle cell lymphoma (MCL),52 53 Kaposi's sarcoma-associated herpesvirus lymphomas (KSHV),54 55 and acute lymphoblastic leukaemia (ALL)56 showed that inhibition of MDM2 by nutlin effectively triggers cell cycle arrest and/or apoptosis. The dependence of the activity of nutlin on p53 status was recently shown in a cohort of more than 100 B-CLL patients.49 Experiments using ALL cell lines and primary ALL samples have also shown that nutlin exhibits cytotoxic activity against wild type p53 ALL cells only. Interestingly, this study showed that nutlin-mediated apoptosis was significantly co-related with MDM2 over-expression, that is, nutlin potently killed wild type p53 ALL cells over-expressing MDM2.56
MI-63 and MI-219
MI-63 is another inhibitor of the p53-MDM2 interaction that binds MDM2 with high affinity leading to activation of the p53 pathway and selective inhibition of cell growth in cancer cell lines with wild type p53.57 58 MI-63 effectively induced apoptosis ex vivo in CLL patient samples with functional p53.58 However, it has a poor pharmacokinetic profile and is unsuitable for evaluation in vivo.
Shangary et al59 recently designed MI-219 as a potent, specific and orally available small molecule inhibitor of the p53-MDM2 interaction. Like nutlin, MI-219 blocks the p53-MDM2 interaction, activates the p53 pathway and selectively inhibits cell growth in cancer cell lines including haematological malignancies expressing wild type p53. Interestingly, MI-219 induced cell cycle arrest in both cancer and normal cells but apoptosis was only induced in cancer cells. MI-219 also activated p53, inhibited cell proliferation, and induced apoptosis in xenograft tumours.5
RITA (reactivation of p53 and induction of tumour cell apoptosis)
RITA, a furanic compound identified in a cell-based screen, binds with high affinity to the p53 NH2-terminal domain. This binding induces a conformational change in p53 that reduces the p53–MDM2 interaction and p53 ubiquitylation, leading to p53 accumulation, down-regulation of MDM2, and induction of the p53-dependent apoptotic pathway.60 By evaluating the effects of RITA on leukaemic cells isolated from AML and CLL patients Nahi et al61 described RITA induced apoptosis accompanied by induction of intracellular p53 in cells harbouring wild type p53.
Attempts to reactivate mutant p53
Although only a minority of haematological malignancies harbours mutant p53 at diagnosis, the percentage of p53 deletions and/or mutations tends to increase with time in patients treated with chemotherapy. Unlike wild type p53, mutant p53 is normally expressed at high levels in tumour cells, likely due to an inability to bind to such proteins as MDM2 and induce a p53 negative feedback loop. Studies have shown that conformational changes induced in mutant p53 are reversible and can be restored to a wild type p53 conformation. Thus, restoration of wild type p53 function from mutant p53 in human tumours led several groups to evaluate p53 reactivating small molecules.
Small molecules such as PRIMA-1,62 CP3139863 and MIRA-1,64 that can activate mutant p53 in cell-based assays have already been identified (figure 1). Small peptide activators of mutant p53 have also been shown to be effective in animal models,65 however, the mechanism by which these compounds restore wild type conformation is still unclear. Studies using macromolecular nuclear magnetic resonance, however, have shown how a small peptide capable of binding the DNA-binding domain of mutant p53 proteins can help fold the protein into an active form.66
PRIMA-1 (p53 reactivation and induction of massive apoptosis)
A class of compounds able to restore the tumour suppressor function of mutant p53 is PRIMA-1.62 PRIMA-1 is a low molecular weight compound that can restore the wild type conformation from mutant p53 through specific DNA binding. Reactivation of mutant p53 by PRIMA-1 resulted in the induction of apoptosis and has shown anti-tumour activity in vivo.67 The sensitivity to PRIMA-1 was related to the levels of expression of mutant p53.67
The molecular mechanism by which PRIMA-1 induces mutant p53 conformational change and apoptosis has not been fully elucidated. PRIMA-1 induced cell death in CLL cells in vitro but no cytotoxic effects were observed in normal lymphocytes.68 PRIMA-1 showed cytotoxic effects on B-CLL cells from samples with and without hemizygous p53 deletion, however, the effects were much more pronounced in leukaemic cells with hemizygous p53 deletion. In addition, induction of apoptosis by PRIMA-1 was also shown in AML cells.69
CP-31398 and MIRA-1
Although its efficacy in primary leukaemic cells remains to be established, similar to nutlin, CP-31398 induced both cell cycle arrest and apoptosis, blocking the ubiquitination and degradation of p53.70 In contrast to nutlin, CP-31398 is not able to block the physical association between p53 and MDM2 in intact cells. Instead, it has been reported to stabilise the DNA binding core domain of p53. Thus, CP-31398 functions as a potential anti-cancer drug by rescuing the DNA binding activity, and consequently transcriptional activation of mutant p53.70
CP-31398 was found to stabilise exogenous p53 in p53 wild type (lymphoblastoid cell lines and solid tumours), mutant and p53 null human solid tumour cell lines, as well as in MDM2-null p53−/− mouse embryonic fibroblast.70 It has been suggested that CP-31398-mediated stabilisation of p53 may result from reduced ubiquitination, leading to high levels of transcriptionally active p53.
MIRA-1, a maleimide analogue, induces apoptosis in mutant p53 cells via restoration of p53-dependent transcriptional transactivation.64 Using sarcoma and lung carcinoma cell lines, Bykov et al64 described that MIRA-1 acted by shifting the equilibrium between the native and unfolded conformation of p53 towards the native conformation, leading to restoration of p53-mediated transactivation of target genes and induction of apoptosis in a mutant p53-dependent manner. The structural analogue, MIRA-3, showed anti-tumour activity in vivo against human mutant p53-expressing tumour xenografts in SCID mice.64
Nutlin
Recently, Drakos et al71 reported that nutlin activated the p53 pathway leading to cell cycle arrest and apoptosis in cultured anaplastic lymphoma kinase (ALK) positive anaplastic large cell lymphoma (ALK+ALCL) cells harbouring either a wild type or partially functional but mutated p53. Although cells harbouring mutant p53 are usually considered non-functional, expression of mutant p53 (codon 215 of exon-6, AGT [Ser]→TGT[Cys]) in these cells rendered p53 partially functional as it retained its ability to transactivate the target proapoptotic genes Bax and Puma.72 73
Synergistic effects of p53 activating drugs in inducing apoptosis in haematological malignancies
Drug synergism occurs when drugs interact in ways that enhance or magnify one or more effects, or side effects, of those drugs. Thus, combination of the non-genotoxic drug, nutlin, with other conventional chemotherapeutic drugs may offer a suitable and reliable therapy for patients with haematological malignancies. Since nutlin does not bind to p53 or induce post-translational modifications, nutlin can act in concert with conventional chemotherapeutics to activate p53, with the potential of improving efficacy or lowering the genotoxic burden by enabling dose reduction. Importantly, both the single agent and the combination effect of nutlin are selective for cancer versus normal cells, as shown by the lack of toxicity to peripheral blood mononuclear cells or bone marrow-derived haematopoietic progenitors and bone marrow stromal epithelium cells.40 Table 1 summarises the small molecules inducing apoptosis either individually or in combination discussed here.
Small molecules targeting p53–MDM2 interaction for the activation of the p53 pathway in haematological malignancies
In vitro studies using human cell lines and primary samples harbouring wild type or mutant p53 showed p53-dependent synergistic or additive effects of nutlin with doxorubicin and cytosine arabinoside in killing myeloblasts in AML42; with doxorubicin, chlorambucil and fludarabine in B-CLL46–48; with doxorubicin in MCL53; and with melphalan and etoposide in MM.40 Treatment of Hodgkin/Reed–Sternberg (HRS) cells with nutlin and low concentrations of three conventional cytotoxic drugs, doxorubicin, etoposide or vincristine, significantly enhanced apoptosis in cells harbouring wild type p53, suggesting a synergistic response of nutlin with these drugs.50 However, in ALK+ALCL, nutlin induced synergistic responses with doxorubicin in cells harbouring either wild type or mutated but partially functional p53.71
A synergistic response of nutlin with recombinant TRAIL has been shown in p53 wild type AML cell lines and primary M4-type and M5-type AML, but not in p53-mutated AML.74 A synergistic cytotoxic response has also been shown by combining nutlin with TRAIL in wild type p53 expressing ALK+ALCL cells71 and AML cells,75 suggesting that the combined treatment of nutlin and TRAIL might offer a novel therapeutic strategy for cells harbouring wild type p53. Moreover, the same study in ALK+ALCL cells reported enhanced cytotoxic activity of nutlin when combined with doxorubicin in cells harbouring either potentially functional (wild type) or non-functional (mutant) p53.71
The molecular basis for these combined chemotherapeutic activities might be due to the fact that chemotherapeutic drugs are able to induce cell damage through p53-dependent and p53-independent mechanisms. For example, bortezomib, a proteasome inhibitor, showed a remarkable overall response rate of 35% in MM patients. However, treatment was associated with toxicity and development of drug resistance.76–78 Combination of bortezomib with nutlin might in principle elicit a higher response than each agonist used alone, combining the p53-dependent and p53-independent mechanisms. In this regard, Tabe et al53 described synergistic cytotoxic activity of nutlin with bortezomib in both wild type-p53 and mutant-p53 MCL cells. Thus, synergistic effects of bortezomib with nutlin may allow more flexibility in dosing patients on clinical trials.
In addition to nutlin, the small molecule activator of p53, RITA, has also been shown to act synergistically with fludarabine in CLL cells and with another p53-targeting drug, PRIMA-1 in AML cells.62 Co-incubation of PRIMA-1 with fludarabine resulted in a significant increase of cytotoxicity in the B-CLL samples with hemizygous p53 deletion. In addition, 100% of the hemizygous p53 deleted samples showed synergistic or additive responses, which were most pronounced at the highest dose level of PRIMA-1.64 The enhanced effect of combining PRIMA-1 with fludarabine in CLL cells can be explained by the fact that PRIMA-1 has a p53-independent cytotoxic effect and that fludarabine causes cytotoxicity by both p53 dependent and independent mechanisms.79
Molecular mechanisms associated with p53-mediated apoptosis
Activation of the p53 pathway can force the affected cell to cease proliferation or to enter an apoptotic route of self destruction which is triggered through p53-mediated transcriptional activation of pro-apoptotic target genes (transcription-dependent pathway) or via direct effects of p53 on the mitochondrial death pathway (transcription-independent pathway).42 43 47 80 81 Figure 1 illustrates the molecular mechanisms leading to cell survival (cell cycle arrest) or cell death (apoptosis) mediated by p53-activating small molecule inhibitors.
The conventional view of p53-mediated apoptosis has emphasised its role as a transcription factor which is achieved by inducing the expression of genes whose products then execute a function. Numerous target genes of p53 have been identified that may be involved in p53-dependent apoptosis.4 Prominent examples of p53-target genes are p21 for cell cycle arrest and Puma, Bax, phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1, also known as Noxa), BH3 interacting domain death agonist (BID), as well as death receptor 5 (DR5) for apoptosis.82 Central to p53-transcription-dependent apoptosis is the induction of genes encoding BH3-only members (Noxa and Puma) of the Bcl2 family, which indirectly promote Bax/Bak activation by inhibiting the function of Bcl2 or Bcl-XL. The proapoptotic multi-domain Bcl2 family member, Bax, which regulates the release of apoptogenic factors from the mitochondria, is also transcriptionally activated by p53. Several studies in haematological malignancies showed that nutlin-induced apoptosis was mediated by transcription-dependent pathways accompanied by the up-regulation of pro-apoptotic genes, Bax and Puma and the down-regulation of anti-apoptotic genes, Bcl-2 and survivin.41 43 45 47 50 51 Figure 1 illustrates strategies to activate p53-transcription-dependent pathways, as discussed above.
p53 also has a direct apoptogenic role at the mitochondria83–85 where it can trigger MOMP and apoptosis in the absence of transcription (figure 1).84 85 This can occur through direct activation of Bax or Bak or through binding of p53 to Bcl2 family proteins, which blocks their activity. Kojima et al43 48 described mitochondrial-mediated transcription-independent pathways of apoptosis in AML42 and CLL47 by demonstrating the mitochondrial translocation of p53 induced by nutlin. The role of p53-mediated transcription-independent pathway of apoptosis was further emphasised by the use of a p53-transcriptional inhibitor, pifithrin-α (PFT-α). Studies showed that PFT-α inhibited nutlin-mediated transactivation of pro-apoptotic genes; however, surprisingly, PFT-α augmented apoptosis induced by nutlin in CLL80 and AML81 cells, suggesting that a non-transcriptional mechanism involving direct binding of p53 to mitochondrial anti-apoptotic proteins was the major route to apoptosis induction in these cells. This notion was further supported by studies in AML which showed that blocking the mitochondrial translocation of p53 by pifithrin-μ, an inhibitor of p53 protein translocation to mitochondria, resulted in inhibition of cytochrome C release, thus inhibiting nutlin-induced apoptosis.81 Taken together, studies in MM, CLL and AML have shown that p53-mediated apoptosis can be regulated by both p53-transcription-dependent41 80 81 and -independent pathways or mainly by the transcription-independent pathway.41 42 47
Conclusion and future perspectives
Activation of the p53 pathway in human tumours by non-genotoxic target-specific drugs is a promising strategy to improve cancer therapy. In this regard, stabilisation of p53 by MDM2 antagonists has clearly shown anti-tumour activities in some haematological malignancies. Substances including CP31398, PRIMA-1 and MIRA-1, that modify the conformation of mutant p53 to restore wild type p53 conformation, have been developed and shown to induce apoptosis in certain haematological cancers. Although several in vivo and in vitro studies have shown that nutlin is potentially effective as a single agent in cancer therapy, nutlin displays additive or synergistic effects when used in combination with conventional chemotherapeutic drugs. The effective combination of the conventional or novel chemotherapeutic drugs with non-genotoxic p53-activating agents such as nutlin should be further tested in haematological malignancies, especially those resistant to chemotherapies. Understanding the molecular mechanism associated with the pharmacological activation of the p53 pathway mediated by the recently developed small molecules offers new therapeutic avenues for the treatment of haematological malignancies.
Take-home messages
Small molecule inhibitors of the p53–MDM2 interaction such as nutlin and RITA can activate wild type p53 and induce apoptosis in different types of haematological malignancies.
Small molecules such as CP-31398, PRIMA-1 and MIRA-1 can bind to the core domain of p53 and help restore its wild type conformation, resulting in stabilisation of p53 and induction of apoptosis.
Although molecules such as nutlin and RITA can induce apoptosis alone, effective combination of these molecules with conventional chemotherapeutic drugs can exert synergistic cytotoxic responses in haematological cancers.
Understanding the molecular mechanisms mediating the anti-cancer activity of these small molecules which restore response to therapy will benefit therapeutic strategies for haematological cancers.
References
Footnotes
Funding This study was funded in part by Canadian Institute of Health Research (CIHR) and Leukemia & Lymphoma Society of Canada (LLSC).
Competing interests None.
Provenance and peer review Commissioned; not externally peer reviewed.