Downregulation Of Deoxycytidine Kinase In Cytarabine-Resistant Mantle Cell Lymphoma Cells Confers Cross-Resistance To Nucleoside Analogs Fludarabine, Gemcitabine and Cladribine, But Not To Other Classes Of Anti-Lymphoma Agents

Mantle cell lymphoma (MCL) is an aggressive type of B-cell non-Hodgkin lymphoma associated with poor prognosis. In recent years several studies brought evidence that implementation of high-dose cytarabine (ara-C) into induction therapy, e.g. by sequential chemotherapy by CHOP and DHAP regimens, induced higher response rate and prolonged progression-free survival compared to R-CHOP-only. Based on these results, implementation of ara-C into induction therapy became standard of care for all newly diagnosed younger MCL patients. Despite considerable improvement, however, many MCL patients relapse even after ara-C-based first-line regimen. There is no second-line standard-of-care for relapsed/refractory MCL. Currently available treatment approaches include fludarabine, gemcitabine, cisplatin, temsirolimus, bortezomib, bendamustine and many investigational agents, e.g. ibrutinib.

By long-term co-culture of 5 cytarabine-sensitive MCL cell lines (Jeko-1, Mino, Rec-1, Hbl-2 and Granta-519, designated as CTRL cell lines) with increasing doses of ara-C (up to 50uM) we derived 5 respective ara-C-resistant (R) MCL subclones. Gene expression analyses of R subclones compared to CTRL cell lines by IlluminaHumanRef-12 BeadChips revealed that the only deregulated (namely downregulated) gene accross all 5 datasets was deoxycytidine-kinase (DCK). Marked downregulation of DCK was confirmed by western blotting. In vitro proliferation assay by WST-8 revealed cross-resistance of R subclones to all tested nucleoside analogs (namely gemcitabine, fludarabine and cladribine; 20-1000x compared to sensitivity of respective CTRL cell lines). Importantly, in vitro sensitivity of R subclones to the other tested anti-tumor agents (i.e. other than antinucleotides) including bortezomib, bendamustine, temsirolimus, cisplatin, etoposide, doxorubicin, ibrutinib, ABT-737, olaparib, roscovitine, homoharringtonine, vorinostat and TRAIL was not significantly changed compared to CTRL cell lines. Similarly, R subclones retained in vitro sensitivity to anti-CD20 monoclonal antibodies rituximab and ofatumumab as determined by standard ⁵¹Cr release assays. Experimental therapy of Jeko-1 and Mino-xenografted mice (immunodeficient NSG mice, each cohort comprising 8 animals) with single-agent fludarabine, gemcitabine, cisplatin, temsirolimus, bendamustine and rituximab confirmed the anticipated loss of anti-tumor activity (as measured by overall survival of experimental animals) of the nucleoside analogs in mice transplanted with R subclones compared to mice transplanted with CTRL cell lines. Anti-tumor activity of cisplatin, temsirolimus, bendamustine and rituximab remained comparable between experimental therapy of R subclone and CTRL cell line xenografts.

In conclusion our data show that acquired resistance of MCL cells to ara-C is associated with marked downregulation of mRNA and protein expression of DCK, enzyme of the nucleotide salvage pathway responsible for phosphorylation (=activation) of most nucleoside analogs used in anti-cancer therapy. Indeed, all R subclones (compared to CTRL cell lines) were cross-resistant to fludarabine, gemcitabine and cladribine, but remained sensitive to other classes of anti-lymphoma agents, including genotoxic drugs and targeted agents. Despite the fact that these preclinical data need definite confirmation on primary patient samples, the results do suggest that nucleoside analogs should not be used for the therapy of MCL patients, who relaps after ara-C-based first-line regimen.

Financial Support: IGA-MZ: NT13201-4/2012, GA-UK 446211, UNCE 204021, PRVOUK-27/LF1/1, PRVOUK P24/LF1/3, SVV-2013-266509