Purine analogs are efficient chemotherapeutic drugs used in the treatment of chronic lymphocytic leukemia, the most common adult leukemia in Western countries. However, some patients do not respond to them or relapse after treatment. It is therefore essential to identify the causes of this chemoresistance and to find novel therapeutic strategies to circumvent them.
Purine analogs, like cladribine and fludarabine, are chemotherapeutic drugs chemically similar to natural constituents of DNA, our hereditary material. Their use in the treatment of chronic lymphocytic leukemia was a major therapeutic advance. But how these compounds, which are initially inactive, can they eradicate leukemic cells?
A prerequisite to the therapeutic action of purine analogs is their conversion inside the leukemic cell into an active form (Fig. 1). Three successive reactions are required for this activation, the first one playing a key role in the whole process. Once activated, purine analogs can be integrated in DNA instead of natural precursors. Nevertheless, the presence of fraudulent constituents in DNA will hinder DNA building, which will generate DNA damage. This DNA damage triggers a cell response leading to cell death by a process called apoptosis (a kind of cell suicide). Activation of purine analogs occurs preferentially inside lymphoid cells, which explains their use in the treatment of leukemia.
Mechanism of action of purine analogs and possible causes of resistance to these drugs. Purine analogs are drugs used in chemotherapy for leukemia. To exert their therapeutic action, they have to be converted inside the leukemic cell into an active compound by three successive reactions. The activated purine analog generates then DNA damage, which leads to cell death by a kind of cell suicide, called apoptosis. Several mechanisms can cause resistance to these drugs: (1) low entry of the drug into the cell, (2) low intracellular activation, (3) repair of DNA damage, or (4) defects in the apoptotic pathway.
Yet, despite their success, clinical use of purine analogs is limited by primary or acquired resistance. Several mechanisms can cause this resistance, called chemoresistance (Fig. 1): low activation of purine analogs, weak DNA damage (due for instance to increased damage repair), or defects in the apoptotic pathway.
The ultimate objective of our research is to find novel strategies to increase the clinical efficacy of purine analogs. Therefore, we focus on the mechanisms by which these chemotherapeutic drugs provoke eradication of leukemic cells. Indeed, it is essential to unravel the successive steps that lead from the activation of the purine analog to the induction of the cell death by apoptosis, in order to define which steps might be defective in chemoresistant patients and possibly to correct it.
In last years, we have particularly investigated the activation step of purine analogs, which is needed for their therapeutic action. Like any reaction inside the cell, this activation step involves the action of enzymes. Among them, deoxycytidine kinase, the enzyme that controls the first of the three reactions required for activation of purine analogs, plays a key role in their clinical efficacy. Recently, we identified a mechanism that modifies deoxycytidine kinase characteristics and allows increasing its activity, which might enhance the conversion of inactive purine analogs into their active form. Presently, we are examining which precise molecular event triggers the activation of deoxycytidine kinase. In addition, we are looking for molecules that could be combined to purine analogs in order to strengthen their therapeutic action.
Nucleoside analogs are chemotherapeutic agents widely used in the treatment of cancer or viral infections. Some of them are purine analogs, like the 2’-deoxyadenosine analogs fludarabine and cladribine (Fig. 1), while others are pyrimidine analogs. Fludarabine and cladribine are therapeutically active in chronic lymphoid malignancies, and especially in chronic lymphocytic leukaemia (CLL), the most common adult leukemia in Western countries. Despite remarkable efficacy, a sizeable proportion of patients with CLL either does not respond to cladribine or fludarabine, or relapses after treatment within a few years. Research to unravel the mechanisms leading to resistance to purine analogs and to find novel therapeutic strategies to counteract them is thus a major priority.
Structure of a natural purine (2’-deoxyadenosine) and of two synthetic purine analogs (cladribine and fludarabine) active in hematologic malignancies
Fludarabine and cladribine are prodrugs. To exert their antileukemic effects, these purine analogs, which mimic 2’-deoxyadenosine in term of uptake and metabolism, have to enter lymphocytes and undergo three successive phosphorylations that convert them into an active triphophate form. The latter inhibits various enzymes involved in DNA synthesis, including ribonucleotide reductase, and can be incorporated in replicating or repairing DNA, causing DNA breaks. All these effects result in DNA damage, up-regulation of the tumor suppressor p53 and apoptosis by mechanisms that are not yet entirely understood. To improve our understanding of the mechanisms by which fludarabine and cladribine induce apoptosis, we study their effects in CLL cell lines as well as in freshly isolated CLL lymphocytes. So, we recently analyzed the effects of cladribine and fludarabine on proteins involved in the regulation of cell cycle, and especially the p53-p21 axis, and we currently investigate their effects on various signaling pathways that could play a role in purine analog-induced cell death.
Resistance to purine analogs may arise from several defects: (1) inefficient cellular uptake, (2) low intracellular activation, (3) repair of induced DNA damage, and (4) defective induction of apoptosis. To overcome chemoresistance, combination therapy can be beneficial. Some years ago, we have shown that combination of cladribine with DNA damaging agents, such as cyclophosphamide derivatives, resulted in synergic cytotoxicity in CLL cells, due to inhibition of DNA repair. This in vitro study has provided the rationale for a clinical trial of this combination, which has given encouraging results. To identify new mechanisms that could be involved in the chemoresistance of CLL lymphocytes to purine analogs, we have more recently initiated microarray analyses aimed at comparing genes induced or repressed by cladribine or fludarabine in sensitive and refractory CLL patients. This led to the observation that the p53-dependent gene PLK2 (polo-like kinase 2) was the most highly activated at early time points in chemosensitive CLL samples, whereas PLK2 response was abolished in chemoresistant samples. Therefore, it was proposed that testing PLK2 activation after in vitro incubation of CLL cells with fludarabine or cladribine could be used to investigate the functional integrity of DNA-damage pathways in CLL cells and to predict clinical sensitivity to these drugs. Nevertheless, the precise role of the PLK2 protein in purine analog-induced apoptosis remains to unravel.
The first and limting step in the activation of fludarabine and cladribine as well as of several other nucleoside analogs used in anticancer and antiviral therapy is catalyzed by deoxycytidine kinase (dCK), which plays thus an essential role in their therapeutic efficacy (Fig. 2). dCK is preferentially expressed in lymphoid cells, which explains the clinical success of purine analogs against lymphoproliferative disorders, such as CLL. We are interested in this enzyme because diminished activity of dCK is a potential cause of resistance to nucleoside analogs.
Role of deoxycytidine kinase in the therapeutic action of nucleoside analogs. Deoxycytidine kinase (dCK) catalyzes the first ant rate-limiting step of the activation of several anticancer and antiviral nucleoside analogs (NA) into their active triphosphate form. In lymphoid cells, a high ratio dCK/5’-NT favors the net accumulation of phosphorylated derivatives and explains the success of NA against lymphoproliferative disorders. 5’-NT, 5’-nucleotidase; NMPK, nucleoside monophosphate kinase; NDPK, nucleoside diphosphate kinase.
Using HEK293T cells overexpressing dCK, we have been the first to demonstrate that dCK is a phosphoprotein and that dCK activity is strongly correlated to phosphorylation of Ser-74. Using an anti-phospho-Ser-74 antibody, we also demonstrated that endogenous dCK is phosphorylated on Ser-74 in CLL lymphocytes and that genotoxic agents increases Ser-74 phosphorylation, in close parallel with changes in dCK activity. This finding led to the hypothesis that increasing dCK via phosphorylation of Ser-74 might constitute a new therapeutic strategy to enhance activation and efficacy of nucleoside analogs. This hypothesis was investigated in cell lines and it was confirmed that increasing dCK activity via Ser-74 phosphorylation could enhance activation and cytotoxicity of pyrimidine, but not purine, analogs. We are now deciphering the signaling pathways involved in the regulation of Ser-74 phosphorylation and hence in the regulation of dCK activity.
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PURINE ANALOGS IN LEUKEMIA