Resistance to Cytarabine in Mantle Cell Lymphoma Is Mediated By Down-Regulation of Deoxycytidine Kinase at the Protein Level

INTRODUCTION:

The addition of high-dose cytarabine to mantle cell lymphoma (MCL) treatment regimens has significantly prolonged survival of patient subgroups, but relapses are common and are usually associated with treatment resistance. High-dose cytarabine is effective due to the improved retention of ara-CTP by target cells, but likewise toxic, causing mainly hematological side effects. Thus, understanding the molecular mechanism(s) responsible for resistance and to identify predictive markers for resistance and/or sensitizing agents would be of great clinical value. In an attempt to elucidate those mechanisms and to create a tool for drug discovery investigations, we established a unique and molecularly reproducible cytarabine resistant model from the Z138 MCL cell line. Using molecular profiling, we confirm that down-regulation of the deoxycytidine kinase (dCK) protein is key to development of resistance. The MCL resistance model was carefully characterized by screening with annotated compound libraries focused on (i) chemotherapeutics to identify potential cross-resistance and/or sensitivity, and (ii) epigenetic pathways to investigate sensitivity, but also to select individual candidates for sensitization of cytarabine resistant cells. Furthermore, we investigated the hypothesis that the levels of dCK at diagnosis can be used to predict cytarabine resistance through measurement of event-free survival using the Nordic MCL 2/3 cohort, where patients are treated with a combinatorial protocol including high-dose cytarabine.

MATERIAL AND METHODS:

The first resistant sub-clone defined as Z138 Cytarabine Resistant (Z138-CytR) was established by continuous exposure of wild type Z138 Cytarabine Naïve Sensitive cells (Z138-CytNS) to increasing concentrations (0.005 - 0.3 µM) of cytarabine. Using this model, we could identify the approximate time to resistance development, and utilize this information for developing a novel highly reproducible time-controlled cytarabine resistant model. Molecular changes were investigated by protein and gene expression analyses. Utilizing drug libraries, the cell model was further used to identify substances with growth reducing effect on cytarabine resistant cells.

RESULTS AND CONCLUSION:

Gene expression profiling revealed that major transcriptional changes occur during the initial phase of adaptation to cellular growth in cytarabine containing media, and only few genes are deregulated upon development of resistance. Instead, resistance to cytarabine was shown to be mediated by down-regulation of the dCK protein, responsible for activation of nucleoside analogue prodrugs. Consequently, cytarabine resistant cells showed cross-resistance to other nucleoside analogues including gemcitabine, cladribine and fludarabine. Of major importance, using drug libraries, we identify substances with growth reducing effect on cytarabine resistant cells. Further investigations are needed to pinpoint compounds that can prevent the down-regulation, or possibly restore dCK protein levels. The possibility to predict cytarabine resistance in diagnostic samples was assessed, but analysis show that the majority of patients have moderate to high expression of dCK at diagnosis, corresponding well to the initial successful response to cytarabine-containing treatment protocols.