Téma: Drug resistance and pharmacology
Číslo abstraktu: P971
Mutations in the TP53 gene are usually associated with very poor prognosis in hematological malignancies, including strong resistance to therapy. The concept of synthetic lethality based on inhibition of Chk1 kinase represents a promising approach for elimination of highly aggressive cancer cells. After this inhibition, the blockade of all three major cell cycle checkpoints is presumed, ie, the G1/S (owing to TP53 mutation), and S together with G2/M (both through the Chk1 inhibition). This should result in uncontrolled cell proliferation followed by mitotic catastrophe and cell death. DNA-PK is a kinase responsible for DNA damage signaling within the nonhomologous end-joining DNA repair. It has been reported that especially high risk cancer cells harboring TP53 mutations can be protected from chemotherapy through enhanced activation of this pathway. The sensitization to chemotherapeutic drugs using DNA-PK inhibition has been reported in CLL.
To analyze potential sensitization of B-cell leukemia and lymphoma cells with TP53 mutations to nucleoside analog fludarabine using: (i) Chk1 inhibition in growing cell lines, (ii) Chk1 inhibition in non-dividing CLL cells, (iii) DNA-PK inhibition in growing cell lines, (iiii) DNA-PK inhibition in non-dividing CLL cells.
Following cultures were used: 15 B-cell leukemia or B-cell lymphoma permanent cell lines with 10 of them having TP53 mutation and 24 primary CLL cultures with 12 of them having TP53 mutation (PBMNC containing >85 % of CLL lymphocytes). The inhibitors were following: SCH900776 (racemic isoform, 200 nmol) for Chk1 and NU7026 (10 µmol) for DNA-PK. After 2h pre-incubation with inhibitors, fludarabine was administered at four different concentrations (assessed individually for cell lines - see Results, and 25; 6.25; 1.6 and 0.4 µg/ml for CLL cultures). Cell viability was measured by the metabolic WST-1 assay (Roche). The sensitization effect was evaluated if appearent in at least two bordering concentrations using two-way analysis of variance (ANOVA).
The detailed TP53 status (deletion and/or mutation presence) was assessed for all used cell lines and primary cultures by I-FISH and by the yeast functional analysis with subsequent sequencing. Concentrations of fludarabine ranged from 20 to 0.125 µg/ml in the tested cell lines, with those having TP53 defects being obviously more resistant. The Chk1 inhibitor on its own exhibited none or only negligible effect on cell viability (≥ 90 % in comparison with untreated control). The significant sensitization effect (p<0.01) was observed in 5/10 (50 %) mut-TP53 cell lines and 1 out of 5 (20%) wt-TP53 cell lines. By contrast, no sensitization effect was observed in tested primary CLL cultures. The DNA-PK inhibitor on its own reduced a viability in most of the cultures (average 73 %). The sensitization effect (p<0.01) was apparent in 2/10 (20 %) mut-TP53 cell lines and none out of 5 wt-TP53 cell lines. Similarly to cell lines, also some primary CLL cultures responded to DNA-PK sensitization: positive effect was observed in 4/12 (33 %) TP53-defected samples and none out of 12 wt cultures. Using western blot for phosphorylated histone H2AX (γH2AX), we verified that both Chk1 and DNA-PK sensitization was mediated through enhanced accumulation of DNA damage (dsDNA breaks).
Summary / Conclusion:
Our work shows that both Chk1 kinase inhibition and DNA-PK inhibition may represent feasible approach how to eliminate some aggressive leukemia and lymphoma cells. Some other cultures remain, however, inert to the sensitization. Concerning primary non-dividing CLL cells, we confirm that the DNA-PK approach might be promising. Our preliminary data suggests that Chk-1 inhibition should be tested in CLL cells after these are stimulated to proliferation. The work was supported by the project FNUSA-ICRC (CZ.1.05/1.1.00/02.0123), MUNI/A/0784/2012, and NT13519-3.
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Keywords: Chronic lymphocytic leukemia, Lymphoma, p53, Resistance
Datum přednesení příspěvku: 15. 6. 2013