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Contribution of Pol nu in CLL progression
marker determining CLL patients’ outcome and fludarabine chemoresistance. By stimulating in vitro proliferation of pri- mary CLL samples, we also revealed that POLN is the only up-regulated 3R gene among 82 genes analyzed, suggesting that higher expression of POLN could be important for CLL proliferation and progression, independently of external treatment. In an independent approach, using two cell lines established sequentially from the same CLL patient which reflected clinical progression of the disease, we found that the most striking difference between the two cell lines was, again, the relatively enhanced expression of POLN in the more aggressive CLL clone. Cell survival and DNA syn- thesis experiments with CLL cell lines and murine homozygous, heterozygous and knockout MEF for POLN demonstrated that relatively high expression of POLN con- ferred higher viability following nucleotide pool starvation by fludarabine, the backbone of therapeutic treatment in CLL. The link between fludarabine resistance and expres- sion of POLN was further confirmed in the high POLN- expressing CLL cells by showing that 30% POLN deple-
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tion by a small interfering RNA in these cells resulted in a mild but significant decrease in fludarabine sensitivity (data not shown). Collectively, these results suggest a possible role of POLN in the natural course of CLL and in cell survival upon external stress affecting the dNTP pool balance. This led us to reason that these two mechanisms might be mutually associated as endogenous replicative stress fre- quently results in dNTP pool starvation when replication origins are over-used. Upon nucleotide starvation by flu- darabine, a high POLN level allowed globally higher DNA replication efficiency and, conversely, POLN depletion led to increased perturbation of replication dynamics. Importantly, rescue experiments by supplementing POLN- depleted cells with deoxyribonucleosides in the presence of fludarabine showed a complete restoration of the rate of DNA synthesis.
POLN encodes a low fidelity A-family Pol ν, whose cel- lular role still remains elusive although some repair activity has been proposed.22,23 Taking into consideration the high sequence homology between Pol ν and the polymerase
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Figure 5. POLN and DNA synthesis in the presence of replicative stress due to a limited dNTP pool. (A) Flow cytometry cell cycle analysis of MEF POLN+/+ and MEF POLN+/- cell lines in the control condition or treated with fludarabine for 4 hours (h). Delta (D) is calculated as the difference of percent of cells present in the EdU+ gate by control condition (representative of 3 experiments). (B) Flow cytometry DNA synthesis analy- sis of MEF POLN+/+ and MEF POLN+/- cell lines, control and treated with hydroxyurea for 4 h. Delta (D) is cal- culated as difference of percent of cells present in the EdU+ gate by control condition, (representative of 3 experiments). (C) Flow cytometry DNA synthesis analy- sis of MEF POLN+/+ and MEF POLN+/- cell lines, control and treated with fludarabine for 8 h at 30 mM concen- tration and supplemented or not with deoxynucleo- sides (dNs). Delta (D) is calculated as a fold change of EdU incorporation by control condition (representative of 3 experiments).
haematologica | 2018; 103(6)
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