Poly ADP Ribose Polymerase (PARP) Inhibitors Induce Apoptosis Alone or Synergistically with Histone Deacetylase Inhibitors in Primary Acute Myeloid Leukemic Patient Cells

Abstract

It has been previously reported that cells defective for double strand break (dsb) DNA repair can be selectively targeted for apoptosis through abrogation of their PARP activity. It has also been reported that leukemic cells have inherent defects in dsb DNA repair. Exploitation of DNA repair defects using PARP inhibitors (PI) thus represents a specific and less toxic form of therapy for a number of hematological malignancies. In order to test the efficacy of PI therapy we analysed primary cells from acute myeloid leukemia (AML) patients and the potential for combination therapy with inhibitors of DNA methyltransferase (DNMTi) or histone deacetylase inhibitors (HDACi). We report that from a panel of 12 AML patients, 2 AML patient cells demonstrated abnormal cell cycle profiles and apoptosis in response to the PI, KU-0058948 (1μM). In contrast, normal control cells displayed standard cell cycle profiles and no apoptosis in response to PI. Clonogenic cytotoxicity assays also showed that these PI sensitive AML patient cells exhibited between 45–55% 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 the 2 PI sensitive AML patients cells demonstrated severely reduced rad51 foci formation (<1%) compared to PI insensitive and normal control cells (15%). This confirmed that PI targets the HR deficiencies in PI sensitive cells. Histone H2AX, phosphorylated in response to DSB had greatly increased foci formation in PI sensitive cells compared to PI insensitive control cells as a result of unrepaired DNA damage (32.3 vs 19.3%)(p<0.05). We next explored the use of PI in combination with DNMTi or HDACi. Whilst KU-0058948 offered only additive effects on DNMTi cytotoxicity, a non-cytotoxic concentration of KU-0058948 (10nM) behaved synergistically with HDACi potentiating the cytotoxic effect of MS275 by 45% compared to MS275 alone (p<0.05). Furthermore, non-cytotoxic doses of MS275 (50nM) potentiated the cytotoxic effects of KU-0058948 in PI sensitive cells (25–30%) compared to KU-0058948 alone (P<0.05). In conclusion, we have shown that primary AML cells are sensitive to the cytotoxic actions of PI. We have also showed that PI acts synergistically in combination with HDACi. PARP inhibitors can potentially exploit dsb repair defects in leukemic cells paving the way for a targeted therapy for leukemia.