Impaired NHEJ function in multiple myeloma

Abstract

Multiple Myeloma (MM) is characterized by multiple chromosomal aberrations. To assess the contribution of DNA repair to this phenotype, ionizing radiation was used to induce DNA double strand breaks in three MM cell lines. Clonogenic survival assays showed U266 (SF4= 15.3+6.4%) and RPMI 8226 (SF4=12.6.0+1.7%) were radiation sensitive while OPM2 was resistant (SF4=78.9 +4.1%). Addition of the DNA-PK inhibitor NU7026 showed the expected suppression in radiation survival in OPM2 but increased survival in both radiation sensitive cell lines. To examine NHEJ repair in these lines, the ability of protein extracts to support in vitro DNA repair was measured. Among the three MM cell lines analyzed, RPMI 8226 demonstrated impaired blunt ended DNA ligation using a ligation mediated PCR technique. In a bacterial based functional assay to rejoin a DNA break within the β-galactosidase gene, RPMI 8226 demonstrated a four fold reduction in rejoining fidelity compared to U266, with OPM2 showing an intermediate capacity. Ionizing radiation induced a robust γ-H2AX response in OPM2 but only a modest increase in each radiation sensitive cell line perhaps related to the high level of γ-H2AX in freshly plated cells. Examination of γ-H2AX foci in RPMI 8226 cells confirmed data from Western blots where a significant number of foci were present in freshly plated untreated cells which diminished over 24 h of culture. Based on the clonogenic survival and functional repair assays, all three cell lines exhibited corrupt NHEJ repair. We conclude that suppression of aberrant NHEJ function using the DNA-PK inhibitor NU7026 may facilitate access of DNA ends to an intact homologous recombination repair pathway, paradoxically increasing survival after irradiation. These data provide insight into the deregulation of DNA repair at the site of DNA breaks in MM that may underpin the characteristic genomic instability of this disease.

DNA repair pathways in human multiple myeloma

“…[90][91][92] Using an in vitro DNA repair assay, NHEJ activity was found to be corrupted in one (RPMI8226) and functional in two (U266, OPM2) MM cell lines. 93 A link between NHEJ defect and radiation sensitivity was unclear, since both RPMI8266 and U266 cells were radiationsensitive, and addition of a DNA-PKcs inhibitor was toxic in untreated cells but remained innocuous to irradiated cells. On the contrary, OPM2 cells were radiation-resistant, and the DNAPKcs inhibitor abrogated radiation resistance while exhibiting no toxicity to untreated OPM2 cells.…”

“…On the contrary, OPM2 cells were radiation-resistant, and the DNAPKcs inhibitor abrogated radiation resistance while exhibiting no toxicity to untreated OPM2 cells. 93 Presumably, the inactivation of a specific DNA repair pathway may be compensated by the activity of alternative pathways, as discussed above. A recent report shows an increased affinity of Ku80 for DNA ends, and constitutive activation of NHEJ in MM cell lines compared with healthy peripheral blood mononuclear cells.…”

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

“…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] . To our knowledge, no studies have so far correlated dna-pk inhibition with dna dsbs, apoptosis, and ultimately, cell survival in any human gastric cancer cell line.…”

Dendritic cell–mediated activation-induced cytidine deaminase (AID)–dependent induction of genomic instability in human myeloma

“…16 Briefly, slides were washed again in PBS-1% BSA and then incubated for 1 hour at 37°C with anti-phospho-H2AX Ser 139 Ab (1:500; Millipore) in PBS/1%BSA/0.5% Tween-20. Slides were washed thoroughly and then incubated with a Alexa Fluor 488 goat anti-mouse IgG 1 secondary Ab (Invitrogen) at 1:500 dilutions for 30 minutes at 37°C in PBS/1%BSA/0.5% Tween-20.…”