BCL6 Dose Not Negatively Regulate DNA Damage Response Genes in Multiple Myeloma Cells

Background: A proto-oncogene BCL6 is a known transcriptional repressor and a master regulator of germinal center B cell program. It represses expression of DNA damage response genessuch as p53, ATR, CHEK1 and p21, which helps B cells to survive and expand during antigen receptor-diversification reactions. It also plays a pivotal role in the formation of germinal centers and lymphomagenesis. BCL6 is down-regulated in normal plasma cells while it is aberrantly expressed in bone marrow residing myeloma cells. Although the role of BCL6 in B cell lymphomas has been intensively studied, its role in plasma cell neoplasms remains to be elucidated. In this study we asked whether BCL6 plays a role in DNA damage response of myeloma cells.

Methods: Bone marrow samples obtained from 36 of primary plasma cell dyscrasia patients (5 cases of MGUS, 30 of multiple myeloma (MM) and 1 plasma cell leukemia (PCL)) were subjected to the study after informed consent. This study was approved by IRBs of Gunma University Hospital and Nishigunma National Hospital. CD138-positive bone marrow plasma cells were isolated as a purity of >95% using magnetic beads and RNAs were extracted. Expression levels of BCL6, p53, ATR, CHEK1 and p21 were quantified by real time PCR using Taqman-probes. For retroviral infection, BCL6 was cloned into pMY-IRES-GFP vector and transfected to PlatA cells using lipofection reagents. 48 hours later, supernatants were collected and centrifuged for 16 hours and the pellets were used for infection. GFP positive cells were collected and used for following experiments. For γH2AX foci formation analysis, the cells were given ionized irradiation at doses of 0, 3, 5 and 10Gy, and used after an hour incubation. Cells were also exposed to different concentration of etoposide (0, 1, 5, 10, 50, 100μM) for 30minute, then washed with fresh media and incubated for an hour. Cells were stained with a FITC-labeled anti-γH2AX antibody as reported (Muslimovic et al, Nat. Protoc. 2008). For cell cycle analysis by flow cytometry, EdU uptake and 7AAD DNA staining were performed according to a manufacture’s protocol (Life Technologies). Correlation of expression levels between each of genes was assessed using Spearman’s non-parametric correlation analysis.

Results: Median of BCL6 expression levels of MM and PCL cells (Qty median=1.47, range 0.09-17.2) was not significantly different from that of MGUS cells (Qty median=1.84, range 0.8-4.2, p=0.51). In marked contrast to germinal center B cells, expression levels of BCL6 and p53 were positively correlated in MM, PCL and MGUS (r=0.457, p=0.007). The correlation between expression of BCL6 and ATR did not reach statistical significance (r=0.323, p=0.062). ATR and p53 were also positively correlated (r=0.549, p=0.001). The expression level of p21, a well-known target of p53, was positively correlated to that of p53 (r=0.681, p=0.03), which supports our data qualification. We also examined mRNA expression levels of BCL6 in MM cell lines, RPMI8226, KMS11, KMS12PE, KMS12BM, KMS18, KMS26, ARH 77 and U266. U266, KMS12PE, KMS26 expressed little amount of BCL6 compared to the patient samples. The other five cell lines did not express BCL6. In order to study BCL6 functions in MM cells, we retrovirally expressed BCL6 in the KMS12BM cell line. The expression level of BCL6 was comparable to the patient samples after the retroviral expression (QTy 6.70). Since BCL6 is known to be a transcriptional repressor and supposed to directly repress p53, ATR, CHEK1 and p21 in B cells, we analyzed expression levels of these genes. Intriguingly, p53, ATR, CHEK1 and p21 are not repressed by overexpression of BCL6 in KMS12BM. These results suggest that alternative regulatory mechanisms of transcriptional regulation by BCL6 are present in MM cells. For further evaluation of the DNA damage response by BCL6 expression, we irradiated or treated these cells with etoposide and analyzed for γH2AX foci formation, a hallmark of DNA double strand break. No differences in the foci formation between mock-infected and BCL6-infected-KSM12BM were detected either with irradiation or etoposide exposure. We also studied cell cycle progressionin these cells. Flow cytometry analysis showed no significant difference between these cells.

Conclusion: Unlike B cells in germinal centers, BCL6 expression in myeloma cells did not repress DNA damage response gene expression.