DNA polymerase ν gene expression influences fludarabine resistance in chronic lymphocytic leukemia independently of p53 status

Abstract

Alteration in the DNA replication, repair or recombination processes is a highly relevant mechanism of genomic instability. Despite genomic aberrations manifested in hematologic malignancies, such a defect as a source of biomarkers has been underexplored. Here, we investigated the prognostic value of expression of 82 genes involved in DNA replication-repair-recombination in a series of 99 patients with chronic lymphocytic leukemia without detectable 17p deletion or TP53 mutation. We found that expression of the POLN gene, encoding the specialized DNA polymerase ν (Pol ν) correlates with time to relapse after first-line therapy with fludarabine. Moreover, we found that POLN was the only gene up-regulated in primary patients' lymphocytes when exposed in vitro to proliferative and pro-survival stimuli. By using two cell lines that were sequentially established from the same patient during the course of the disease and Pol ν knockout mouse embryonic fibroblasts, we reveal that high relative POLN expression is important for DNA synthesis and cell survival upon fludarabine treatment. These findings suggest that Pol ν could influence therapeutic resistance in chronic lymphocytic leukemia. (Patients' samples were obtained from the CLL 2007 FMP clinical trial registered at: clinicaltrials.gov identifer: 00564512).

Trial registration: ClinicalTrials.gov NCT00564512.

Copyright © 2018 Ferrata Storti Foundation.

Figures

Figure 1.

POLN gene expression as an independent prognostic marker of time to progression in chronic lymphocytic leukemia (CLL). (A) Unsupervised clustering of CLL and healthy donor (HD) samples based on 3R gene expression data. (B) POLN gene expression in CLL primary samples in comparison to HD samples. (C) Kaplan-Meier graphical estimates of time to progression (TTP) according to POLN expression level. The cut-off between low and high expression is 11.9×10−2. The expression number ranges in under-expressed and over-expressed groups are 0.008–0.118 and 0.119–0.272, respectively.

Figure 2.

Proliferation and 3R gene expression profiles of primary sample from patients with chronic lymphocytic leukemia (CLL). Peripheral mononuclear cells from CLL patients were cultured in vitro in the absence or presence of proliferation stimuli (interleukin-2/DSP30) for six days. (A) CFSE-labeled unstimulated/non-proliferating and stimulated/proliferating primary CLL lymphocytes. (B) Unsupervised clustering according to 3R gene expression in non-proliferating and proliferating purified CLL cells. Out of 82 3R genes, only one gene was up-regulated upon proliferation (POLN), 10 genes were down-regulated and the other 71 genes remained stably expressed.

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.

Figure 4.

POLN and fludarabine chemoresistance in vitro. (A) MTS viability assay of MEC-1 and MEC-2 cell lines in the absence or presence of fludarabine for a treatment period of 24 hours (h). The percentage of viable cells is expressed as the mean ± Standard Deviation of three independent experiments. The fludarabine EC50 value for the MEC-1 cell line was 239 μM and that for the MEC-2 cell line was 433 μM. (B) Flow cytometry cell cycle analysis of MEC-1 and MEC-2 cell lines, control and treated with fludarabine for 2 h. Delta (Δ) is calculated as the difference of percent of cells present in the EdU+ gate by control condition (representative of three experiments). (C) DNA combing in nanochannels. The length of replicated domains is expressed in kilobase pairs (kbp). The number of tracks measured in all conditions was 60. The concentration of fludarabine used was 600 μM; the duration of treatment was 4 h. Medians in the dot plot are indicated by red lines.

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 (Δ) 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 analysis of MEF POLN+/+ and MEF POLN+/− cell lines, control and treated with hydroxyurea for 4 h. Delta (Δ) is calculated as difference of percent of cells present in the EdU+ gate by control condition, (representative of 3 experiments). (C) Flow cytometry DNA synthesis analysis of MEF POLN+/+ and MEF POLN+/− cell lines, control and treated with fludarabine for 8 h at 30 μM concentration and supplemented or not with deoxynucleosides (dNs). Delta (Δ) is calculated as a fold change of EdU incorporation by control condition (representative of 3 experiments).