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Contribution of Pol nu in CLL progression
Many patients diagnosed with CLL survive for several years without needing any treatment while others devel- op an aggressive form of the disease and require immedi- ate therapeutic intervention. Apart from Binet and Rai staging, clinicians take into consideration several other biological characteristics, such as mutational status of the immunoglobulin heavy chain variable gene segment (IGHV) and genomic aberrations. Approximately 4 out of 5 CLL patients harbor at least one chromosomal aberra- tion in their leukemic clone and these, likewise, have a clear clinical impact on patients’ survival.6 Recent studies have also revealed other genomic defects in CLL, includ- ing mutations in genes involved in RNA processing, such as SF3B17 and, in general, in B-cell development,8 and mutations in non-coding regions that potentially affect the clinico-biological course of CLL.9 Nevertheless, few of these markers are used in routine clinical practice and even fewer of them have been able to demonstrate any predic- tive value in terms of response to chemotherapy, such as 17p-deletion6 and mutational status of the TP53 gene.10
Genomic instability is a prominent source of genetic diversity within tumors, generating a different cell popu- lation that can be subject to selection in a given microen- vironmental or therapeutic context. The past decade has seen a dramatic advance in our understanding of how genomic integrity is related to cancer and has revealed that replication stress in the S phase of the cell cycle is an important feature,11,12 placing studies on replication stress at the forefront of cancer research.13 The genomic instabil- ity in CLL may indicate the presence of underlying repli- cation stress. In addition, the backbone of the standard, first-line treatment for CLL patients is fludarabine,14 which leads to excessive replication stress by inhibiting ribonu- cleotide reductase, an enzyme involved in the production of precursors essential for DNA synthesis. In contrast to solid cancer, DNA replication defects as a source of mark- ers in hematologic malignancies have been poorly
Table 1. Clinical characteristics of the chronic lymphocytic leukemia patients.
explored. It was only recently that we revealed the impor- tance of the DNA replication checkpoint CHEK1 as a pre- dictor of survival in acute myeloid leukemia and resistance to therapy with cytarabine.15
Here, we hypothesized that mis-expression of DNA replication-repair-recombination (3R) genes could occur during CLL leukemogenesis and contribute to the evolu- tion of the pathology. We reasoned that a modified DNA replication program might, likewise, have an impact on the response to fludarabine which targets several steps in chromosome duplication. We also speculated that specific 3R gene expression signatures would be relevant in the context of relapse, representing potential predictors of the patients’ outcome. Our data show that, among more than 80 3R genes analyzed in a series of primary samples from 99 patients with CLL prior to treatment, only expression of the POLN gene encoding the specialized A-family DNA polymerase nu (Pol ν), with yet unknown function, can forecast the time to relapse after first-line therapy. We also demonstrate that relatively high POLN expression pro- tects CLL cells against replicative stress caused by fludara- bine by allowing efficient DNA replication in the presence of an unbalanced nucleotide pool.
Methods
Patients with chronic lymphocytic leukemia
RNA from patients’ samples were obtained from the CLL 2007 FMP clinical trial (clinicaltrials.gov identifier: 00564512).16 In accor- dance with the Declaration of Helsinki, informed consent to par- ticipation in the trial was obtained from each patient. Patients recruited for the trial were medically fit, previously untreated, diagnosed with Binet stage B or C CLL, under 65 years of age and without detected 17p deletion or TP53 mutation. Patients were randomized into two treatment arms: fludarabine-cyclophos- phamide-campath (FCCam) and fludarabine-cyclophosphamide- rituximab (FCR). The primary outcome, namely, progression-free survival at 36 months, was the same for both treatment arms (P=0.21). Relevant data regarding the cohort of patients are sum- marized in Table 1.
Cell lines and culture
MEF cells and culture
Primary mouse embryonic fibroblasts (MEF) were derived from e13.5 embryos with genotypes Poln+/+, Poln+/- and Poln-/- (C57BL/6J strain). Exon 2 containing the initiation codon was deleted in the knockout allele. Primary MEF were cultured in medium containing a high concentration of glucose, glutamax-Dulbecco modified Eagle medium (Invitrogen), 15% Hyclone fetal bovine serum (Thermoscientific), non-essential amino acids, sodium pyruvate, modified Eagle medium vitamin solution, and penicillin/strepto- mycin (Invitrogen) and was maintained in air-tight containers filled with a gaseous mixture of 93% N2, 5% CO2 and 2% O2 (Praxair) at 37°C. Immortalized MEF were cultured in medium containing a high concentration of glucose, glutamax-DMEM (Invitrogen), 10% fetal bovine serum (Atlanta Biologics) and peni- cillin/streptomycin, and maintained in a humidified incubator in 5% CO2 at 37°C.
Cell culture
MEC-1 and MEC-2 cell lines (DSMZ, Braunschweig, Germany) were cultured in Iscove modified Dulbecco medium with gluta- max supplemented with 10% fetal bovine serum and 1% peni- cillin/streptomycin at a density of 5x105 and 3x105 cells/mL for
Characteristic
Sex Female Male
Age
≤ or 57 years old > 57 years
Binet stage B
C
IGHV
Mutated
Unmutated
Cytogenetics Deletion 13q
Deletion 11q Trisomy 12
Category
N. (frequency, %)
30/99 (30) 69/99 (70)
51/99 (52)
48/99 (48)
78/99 (79) 21/99 (21)
43/99 (43)
56/99 (57)
52/99 (53) 42/99 (42) 5/99 (5) 21/99 (21) 78/99 (79) - 13/99 (14) 80/99 (80) 6/99 (6)
Detected Not present Unknown Detected Not present Unknown Detected Not present Unknown
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