Deregulation of DNA Double-Strand Break Repair in Multiple Myeloma: Implications for Genome Stability

Ana B. Herrero,Jesús San Miguel,Norma C. Gutierrez,Miria Ricchetti

doi:10.1371/journal.pone.0121581

Abstract

Multiple myeloma (MM) is a hematological malignancy characterized by frequent chromosome abnormalities. However, the molecular basis for this genome instability remains unknown. Since both impaired and hyperactive double strand break (DSB) repair pathways can result in DNA rearrangements, we investigated the functionality of DSB repair in MM cells. Repair kinetics of ionizing-radiation (IR)-induced DSBs was similar in MM and normal control lymphoblastoid cell lines, as revealed by the comet assay. However, four out of seven MM cell lines analyzed exhibited a subset of persistent DSBs, marked by γ-H2AX and Rad51 foci that elicited a prolonged G2/M DNA damage checkpoint activation and hypersensitivity to IR, especially in the presence of checkpoint inhibitors. An analysis of the proteins involved in DSB repair in MM cells revealed upregulation of DNA-PKcs, Artemis and XRCC4, that participate in non-homologous end joining (NHEJ), and Rad51, involved in homologous recombination (HR). Accordingly, activity of both NHEJ and HR were elevated in MM cells compared to controls, as determined by in vivo functional assays. Interestingly, levels of proteins involved in a highly mutagenic, translocation-promoting, alternative NHEJ subpathway (Alt-NHEJ) were also increased in all MM cell lines, with the Alt-NHEJ protein DNA ligase IIIα, also overexpressed in several plasma cell samples isolated from MM patients. Overactivation of the Alt-NHEJ pathway was revealed in MM cells by larger deletions and higher sequence microhomology at repair junctions, which were reduced by chemical inhibition of the pathway. Taken together, our results uncover a deregulated DSB repair in MM that might underlie the characteristic genome instability of the disease, and could be therapeutically exploited.

Highlights

Multiple myeloma (MM) is a clonal disorder of B-cells at the last stage of differentiation
To analyze double strand break (DSB) formation and repair we first monitored the phosphorylation of H2AX, a sensitive marker of DSBs [23], after treatment with 2 Gy of ionizing radiation (IR). γH2AX signal was quantified by flow cytometry in 7 MM cells lines and compared to 5 cell lines (3 lymphoblastoid cell lines obtained from normal lymphocytes, HeLa and HCT116), that were used as repair-proficient controls (Fig. 1A)
Whereas γH2AX signal decreased with a fast kinetics in controls and U266 cells, and with an intermediate kinetics in IM9 and H929 cells, the reduction of γH2AX was slower in OPM2, JJN3, MM1S and especially in RPMI-8226, which suggests a defect in DSB repair at least in these 4 MM cell lines

Summary

Introduction

Multiple myeloma (MM) is a clonal disorder of B-cells at the last stage of differentiation. Genome instability is a prominent feature of MM cells, and includes ploidy changes, deletions, amplifications and chromosomal translocations mainly involving the IGH locus on PLOS ONE | DOI:10.1371/journal.pone.0121581. Aberrant DSB Repair in Multiple Myeloma chromosome 14q32 [1]. The underlying molecular mechanisms for the generation of this instability are unclear. Numerical chromosome abnormalities may be generated by centrosome amplification or alterations in the spindle assembly checkpoint (SAC) [2,3]. Structural abnormalities, such as chromosomal deletions or translocations, might arise from alterations in the repairing of DNA double strand breaks (DSBs)

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Results

Conclusion