Macrophages Promote DNA Repair of Double Strand Break in Multiple Myeloma Cells By Non-Homologous End Joining(NHEJ), Nevertheless Decrease Its Accuracy

Multiple myeloma (MM) is a hematological malignancy of B cells, characterized by clonal proliferation of malignant plasma cells. DNA damage and genomic instability play an important role in the pathogenesis of MM. Based on the characteristics of high heterogeneity and genomic instability of MM, and the protective effect of MΦs on MM cells (MMCs), our study intended to further clarify whether MΦs affect MMCs DNA damage response (DDR) and DNA repair, and the relationship between MΦs and genomic instability of MMCs.

We found that the content of MΦs in bone marrow biopsy of MM patients was related to the results of cytogenetics (tested by FISH). The higher the content of MΦs, the more complicated the cytogenetic abnormalities of patients, especially in the IgH translocation, D13S319 locus deletion and RB1 deletion. In our study, MΦs were harvested from peripheral blood monocytes (PBMCs) , which were incubated for 7 days with M-CSF. Flow cytometry was used to detect M-CSF induced macrophages (MΦs) in vitro, and CD163 and CD206 were highly expressed in MΦs which implied that MΦs tended to be M2 type. The incubated MΦs were used in the following experiments. Our study showed that MΦs reduced the baseline γH2AX of MMCs, and contributed to MMCs surviving in the case of genomic instability detected by Western blot and immunofluorescence. We also confirmed that MΦs contribute to repairing the DNA damage in myeloma cells with the methods of comet assay. In the case of severe injury of MMCs' DNA, MΦs promoted the DDR and DNA damage repair.

We examined the effects of macrophages on HR and NHEJ using U2OS cells. HR repair was measured in U2OS-HR cells loaded with SCR reporter (HR reporter) while NHEJ repair was measured in U2OS-NHEJ cells loaded with vGEJ reporter (NHEJ Reporter). We found that macrophages increased NHEJ but had no sense on total HR. In order to detect NHEJ level in endogenous genes, we adopted paired gRNA-CRISPR/Cas9 system. AAVS1 and HBB were used as detection genes, and the sequence of about 250 bp near the NHEJ interface was sequenced by NGS. The results proclaimed that the MΦs co-culture group significantly increased the efficiency of NHEJ, and decreased proportion of accurate NHEJ repair. In addition, analysis of the length of base sequence loss showed that the probability of base loss >3 bp in the MΦs co-culture group was higher than that MMCs in the group cultured alone. In the HBB site, MΦs also prolonged the average length of base loss in NHEJ. Furthermore, we used gRNA-CRISPR/Cas9 technology to cause fixed-point cleavage in AAVS1 and HBB respectively, and detected translocation by PCR and NGS with comparing the translocation reads between different groups. The results showed that MΦs promoted the probability of chromosomal translocation, which was of great importance in MM's occurrence and progression.