Abstract
Recent reports suggest that BrCA1−/− and BrCA2−/− cells can be selectively targeted for cell death through abrogation of their PARP activity. It is postulated that as a result of PARP inhibition, accumulation of single strand DNA breaks (SSB) leads to the replication fork collapse and conversion of SSB to double strand DNA breaks (DSB). The inability of repair defective cells such as BrCA2−/− to repair the DSB would lead to cell death. Exploitation of DNA repair defects using PARP inhibitors (PI) thus represents a more specific and less toxic form of therapy for a number of haematological malignancies. Chromosomal instability (CI) syndromes that have inherent defects in double strand DNA repair also have a uniformly high incidence of transformation to acute leukaemia or lymphoma. In order to test the efficacy of PI therapy we analysed CI cell lines, myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML) cell lines and the potential for combination therapy with inhibitors of DNA methyltransferase (DNMTi) or histone deacetylase inhibitors (HDACi). We report that cells from CI syndromes; Blooms syndrome, Fanconi Anaemia (FancD2 and FancA), Ataxia telancgectasia and Nijmegen break syndrome display abnormal cell cycle profiles and excessive apoptosis in response to the PI’s PJ34 (3μM) and EB47 (45μM). In contrast, normal control cells displayed standard cell cycle profiles and no apoptosis in response to PI at equivalent concentrations. Clonogenic cytotoxicity assays showed that CI syndrome cells exhibit between 30–75% cell survival compared with 100% cell survival in control cells (p<0.05) in response to PI. The homologous recombination (HR) DNA repair component, rad51 forms foci in response to DNA damage. In HR compromised cells, rad51 foci fail to form. In response to PI, immunofluorescent studies show that CI syndrome cells demonstrate severely reduced rad51 foci formation (<5%) compared to control cells (15%). This confirms that PI targets the HR deficiencies in CI syndrome cells. Histone γH2AX, phosphorylated in response to DSB had greatly increased foci formation in CI syndrome cells compared to control cells as a result of unrepaired DNA damage (25.3 vs 9.3%)(p<0.05). CI syndromes have increased transformation potential to the MDS and AML. Addition of 3μM PJ34 to the myelomonocytoid leukaemic/myelodysplastic cell line, P39 exhibited significant apoptosis, with a cell survival fraction of 65% compared to 100% in control cells (p<0.01). Immunofluorescent studies revealed reduced rad51 foci formation (6.3 vs 15%) and increased γH2AX foci formation (17.6 vs 9.3%)(p<0.01). Strikingly, we were also able to reproduce similar PI responses in the Jurkat T-cell leukaemic cell line. We next explored the use of PI in combination with DNMTi or HDACi. Whilst 3μM PJ34 offered only additive effects on decitabine cytotoxicity, a sub-optimal concentration (1μM) of PJ34 behaved synergistically with HDACi potentiating the cytotoxic effect of 200nM MS275 by 55% compared to MS275 alone (p<0.05) in P39 cells. In conclusion, we have shown that in a panel of CI syndrome and leukaemic cells, PI demonstrates significant cytotoxic responses. We also show that PI acts synergistically in combination with HDACi. Parp inhibitors can potentially exploit DSB repair defects in leukaemic cells paving the way for a targeted therapy for MDS and leukaemia.
Wild-Type Tumor Repressor Protein 53 (TRP53) Promotes Ovarian Cancer Cell Survival
