From the analysis of purine analog activation to the development of new therapeutic strategies in chronic lymphocytic leukemia
(2016)
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- Chronic lymphocytic leukemia (CLL), the most common leukemia in Europe, is a disease of the elderly characterized by the progressive accumulation of B lymphocytes in bone marrow, lymphoid organs and peripheral blood. Despite recent major advances in its treatment, CLL is still considered as an incurable disease. Among the different therapeutic options for CLL treatment are the purine analogs fludarabine and cladribine. These chemotherapeutic agents are prodrugs, requiring intracellular conversion into their triphosphate form to be active. Triphosphate analogs exert cytotoxicity by inducing DNA damage, which leads to up-regulation of p53 and activation of apoptotic cell death. Despite remarkable efficacy of purine analogs, chemoresistance and toxicity limit their clinical use. Therefore, different strategies, including combination therapy, are explored aiming to increase their effectiveness. In this thesis, we are interested in the combination of fludarabine or cladribine with aphidicolin, a DNA polymerase inhibitor, which was found to increase the activity of deoxycytidine kinase (dCK), the rate-limiting enzyme of purine analog activation. Hypothesis was that aphidicolin could promote the activation of purine analogs and thereby their efficacy. However, aphidicolin fails to increase the conversion of fludarabine or cladribine into their active triphosphate form. Yet, preliminary experiments showed that aphidicolin was able to potentiate fludarabine and cladribine cytotoxicity in primary CLL cells, suggesting a still unidentified mechanism of sensitization to purine analogs. In our first study, we investigated the mechanism by which aphidicolin sensitizes CLL cells to purine analogs. We found that aphidicolin enhanced the DNA damage induced by purine analogs, as demonstrated by up-regulation of two DNA damage markers, p53 and γH2AX. On the other hand, we observed that γH2AX disappearance that occurs after removal of purine analogs was slowed down by aphidicolin, suggesting that aphidicolin inhibits the repair of purine analog-induced DNA damage. As aphidicolin also prevented DNA repair after UV irradiation, which is known to occur via NER (nucleotide excision repair), we examined whether NER could be activated by purine analogs. We observed that fludarabine induced nuclear import of XPA, an indispensable factor for NER. Moreover, XPA siRNA sensitized cell lines to undergo apoptosis in response to fludarabine, indicating that efficacy of fludarabine could be limited by NER and that inhibition of this pathway could enhance purine analog efficacy. Furthermore, as suggested by analysis of a large number of CLL samples, this strategy might be beneficial regardless of CLL prognostic markers. In our second study, we intended to identify the protein kinase responsible for dCK activation in response to aphidicolin and various other genotoxic stresses in lymphoblastoid cell lines. Prior to this work, it had been demonstrated that aphidicolin induces phosphorylation of dCK at Ser-74, which increases its activity, and that Ser-74 could be phosphorylated by the DNA damage-activated kinase ATM after ionizing radiation. By using ATM-deficient cell lines and various kinase inhibitors, we established that ATM was not involved in dCK activation by aphidicolin. In addition, we identified the kinase ATR, another DNA damage-activated kinase, as a key regulator of dCK activity in response to agents that cause replication stress such as aphidicolin, UV light and cladribine. We conclude that ATR could activate dCK in all the situations that generate DNA single-strand breaks, including stalled replication forks and double-strand break resection, while ATM activates dCK in response to DNA double strand breaks
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Starczewska, E. (2016). From the analysis of purine analog activation to the development of new therapeutic strategies in chronic lymphocytic leukemia. https://hdl.handle.net/2078.5/69241