Chlorambucil Cytotoxicity in Malignant B Lymphocytes Is Synergistically Increased by 2-(Morpholin-4-yl)-benzo[h]chomen-4-one (NU7026)-Mediated Inhibition of DNA Double-Strand Break Repair via Inhibition of DNA-Dependent Protein Kinase

Abstract

Chlorambucil (CLB) treatment is used in chronic lymphocytic leukemia (CLL) but resistance to CLB develops in association with accelerated repair of CLB-induced DNA damage. Phosphorylated histone H2AX (␥H2AX) is located at DNA doublestrand break (DSB) sites; furthermore, it recruits and retains damage-responsive proteins. This damage can be repaired by nonhomologous DNA end-joining (NHEJ) and/or homologous recombinational repair (HR) pathways. A key component of NHEJ is the DNA-dependent protein kinase (DNA-PK) complex. Increased DNA-PK activity is associated with resistance to CLB in CLL. We used the specific DNA-PK inhibitor 2-(morpholin-4-yl)-benzo[h]chomen-4-one (NU7026) to sensitize CLL cells to chlorambucil. Our results indicate that in a CLL cell line (I83) and in primary CLL-lymphocytes, chlorambucil plus NU7026 has synergistic cytotoxic activity at nontoxic doses of NU7026. CLB treatment results in G 2 /M phase arrest, and NU7026 increases this CLB-induced G 2 /M arrest. Moreover, a kinetic time course demonstrates that CLB-induced DNA-PK activity was inhibited by NU7026, providing direct evidence of the ability of NU7026 to inhibit DNA-PK function. DSBs, visualized as ␥H2AX, were enhanced 24 to 48 h after CLB and further increased by CLB plus NU7026, suggesting that the synergy of the combination is mediated by NU7026 inhibition of DNA-PK with subsequent inhibition of DSB repair.

Effects of dna-Dependent Protein Kinase Inhibition by NU7026 on dna Repair and Cell Survival in Irradiated Gastric Cancer Cell Line N87

“…The resulting G2/M accumulation observed in the combination of radiation and NU7026 is congruent with previous studies combining NU7026 with chlorambucil or etoposide 12,18,19 . The contribution of apoptosis to cytotoxicity in solid tumours is usually small, unlike that in hematogenous tumour models.…”

“…The specificity of this compound was confirmed when no effect on cell survival and dna dsb repair was observed in NU7026-treated dnapk-deficient cell lines 11 . In vitro testing of NU7026 in combination with chlorambucil demonstrated dna-pk inhibitor-mediated sensitization of primary chronic lymphocytic leukemia cells 12 . We hypothesized that the use of dna-pk inhibitor in combination with ionizing radiation in gastric cancer cells might increase dna dsb, which might in turn increase cell killing by increased radiosensitivity.…”

“…Inhibition of dna-pk has previously been shown to sensitize chronic lymphocytic leukemia cells to cytotoxic agents 12 . The combination of specific NU7026 dna-pk inhibition and ionizing radiation has been demonstrated to increase dna dsb and cell kill in human glioma, multiple myeloma, and ovarian cancer cell lines [15][16][17] .…”

“…The current hypothesis proposes that dna-dsb repair is mediated by dna-pk via the nehj mechanism and occurs predominantly in the G2/M phase of the cell cycle 12 . The resulting G2/M accumulation observed in the combination of radiation and NU7026 is congruent with previous studies combining NU7026 with chlorambucil or etoposide 12,18,19 .…”

DNA repair of myeloma plasma cells correlates with clinical outcome: the effect of the nonhomologous end-joining inhibitor SCR7

“…Three repair inhibitors were used: two of them modify NHEJ (NU7026 and SCR7) [39][40][41] and IR-1 disrupts HR (supplemental Materials and methods). 42 To investigate the effect of the combination treatment of melphalan with an inhibitor, we first selected the optimal concentrations of the inhibitors that, when administered alone, showed minimal decrease in cell viability.…”