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Contribution of Pol nu in CLL progression
MEC-2 cells were able to sustain DNA synthesis more effi- ciently than the chemosensitive MEC-1 cells in the pres- ence of the drug.
To test this hypothesis in more detail, we evaluated DNA replication parameters in both cell lines upon fludarabine treatment by using an innovative genome-wide DNA combing nanofluidics-technology developed recently in our laboratory.21 By applying differential pressure between the entry and exit microchannels, individual DNA molecules are directed into and stretched on their way through the nanochannels. The strategy allows us to monitor the length of the in cellulo-replicated domains at the level of individual replicating DNA molecules. The results of this analysis showed that treating chemosensitive MEC-1 cells with flu- darabine reduced the length of the replicated domains. In contrast, the size of the replicated domains in MEC-2 cells was not decreased upon fludarabine treatment in compari- son to the untreated condition. Track length data (scatter and bar plots), representative photographs of tracks and their quantified histograms are presented in Figure 4C.
As we found that POLN expression is greater in the MEC-2 cell line than in the MEC-1 cell line, and since we observed relatively higher POLN expression to be a marker of negative patients’ outcome after fludarabine-based first- line therapy in our CLL cohort, we hypothesized that rela- tively higher POLN expression might contribute to enhanced DNA replication ability and cell survival upon flu- darabine treatment. To test this, we used independent MEF expressing normal (MEF+/+) and relatively low (MEF+/-) levels of POLN, as well as knockout POLN MEF (MEF-/-) (Online Supplementary Figure S5A). These cell lines were treated with fludarabine for 4 h, then exposed to EdU for 30 min and stained with propidium iodide. DNA synthesis effi- ciency was then assessed by flow cytometry. Analysis of cell cycle and quantification of the proportion of EdU-posi- tive cells confirmed that POLN-proficient cells (MEF+/+) could indeed sustain DNA synthesis more efficiently than MEF+/- or MEF-/- cells in the presence of fludarabine (Figure 5A and Online Supplementary Figures S4 and S5B). We did not observe any significant differences in fludarabine-medi- ated DNA replication inhibition between the MEF-/- and MEF+/- cell lines (Online Supplementary Figure S5B). This might be due to the poor expression of POLN in mouse fibroblasts, and consequently a non-significant differential expression between heterozygous and knock-out cells (Online Supplementary Figure S5A). Collectively, these results suggest that relatively high expression of POLN could con- tribute to higher replication efficiency and sustained cell survival upon fludarabine treatment.
POLN-mediated resistance to fludarabine is related to dNTP pool starvation
In order to investigate whether POLN could play a role in
limiting replication stress specifically upon decrease of
dNTP, we used the ribonucleotide-reductase inhibitor
hydroxyurea, which mimics the effect of fludarabine on the
dNTP pool. MEF+/+ and MEF+/- cells were treated with differ-
ent concentrations of hydroxyurea for 4 h and pulsed with
EdU for 30 min at the end of the treatment. DNA synthesis
profiles were analyzed by flow cytometry, schematically
could limit replication stress caused by fludarabine in the condition of dNTP starvation.
In order to confirm that fludarabine chemoresistance driven by POLN was specifically related to the condition of a reduced dNTP pool, we performed a rescue experiment by supplementing MEF cells with deoxynucleosides in the presence of fludarabine. MEF+/+ and MEF+/- cells were treated with a lower concentration of fludarabine (30 mM) for a longer period (8 h) in order to allow rescue of DNA synthe- sis with deoxynucleosides, which were added during the last 4 h of treatment. Finally, cells were pulsed with EdU for the last 30 min of the incubation period. Flow cytometry analysis showed no significant impact on the DNA synthe- sis rate in either cell line when the deoxynucleosides were added in the absence of fludarabine. As expected, in the absence of supplemental deoxynucleosides, inhibition of DNA synthesis by fludarabine was more pronounced in the MEF+/- cell line than in the MEF+/+ cell line (Figure 5C and Online Supplementary Figures S10 and S11). Importantly, we found that addition of deoxynucleosides could rescue DNA synthesis in the POLN+/- cell line abolishing the difference between two MEF cell lines (Figure 5C and Online Supplementary Figures S10 and S11). This result confirms that POLN reduces the impact of dNTP starvation on DNA synthesis. Collectively, these data suggest that a relatively high level of POLN might contribute to cell survival and provide advantages, not only upon endogenous replicative stress related to a pathology-associated dNTP pool imbal- ance, but also when CLL cells are treated with therapeutic agents that further affect dNTP metabolism, such as flu- darabine.
Discussion
In our study, we report that peripheral blood leukemic lymphocytes from CLL patients express POLN at a relative- ly high level and that the relative expression of POLN is a
Figure 3. 3R gene expression profile change during clinical progression of chronic lymphocytic leukemia (CLL) based on a CLL cellular model. MEC-1 and MEC-2 cell lines were established sequentially from leukemic lymphocytes obtained from the same CLL patient. MEC-1 was isolated at the beginning of the disease while MEC-2 was isolated at the onset of active disease and represents a more aggressive CLL subclone. Expression fold change for each 3R gene was calculated using the comparative Ct method.
represented in Online Supplementary Figure S7. We observed +/+
that the POLN-expressing MEF cells sustained DNA syn- thesis upon hydroxyurea treatment more efficiently than their POLN-deficient MEF+/- counterparts (Figure 5B and Online Supplementary Figures S8 and S9), implying that Pol ν
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