Abstract
Multiple myeloma (MM) is a malignant plasma cell tumor that arises secondarily from monoclonal gammopathy of uncertain significance (MGUS) due to accumulation of genetic abnormalities. Apolipoprotein B mRNA editing enzyme catalytic polypeptide-like (APOBEC) is a family of DNA cytosine deaminases that play critical roles in innate and acquired immunity. APOBEC proteins catalyze cytosine to uracil deamination and eventually induce C to T mutations in DNA. Recent genome-wide analysis revealed that APOBEC-induced signature DNA mutations accumulate during progression of MM and are associated with poor prognosis (Bolli et. al., Nat Commun. 2014). We previously found that one of the APOBEC3 proteins, APOBEC3B (A3B), induces genomic DNA mutations in a human cell culture model (Shinohara et. al., Sci Rep. 2012) and hypothesized that A3B might induce DNA mutations during onset and progression of MGUS and MM.
A deletion polymorphism that removes the entire A3B gene is reported to affect breast cancer prognosis, therefore we first investigated the frequency of A3B deletion allele in 88 Japanese MM/MGUS patients and in healthy controls. 41 patients (46.6%) showed wild type, 42 patients (47.7%) heterozygous, and 5 patients (5.7%) had homozygous deletion allele. We could not detect a significant difference between MM/MGUS patients and healthy controls, suggesting that A3B may not contribute to disease initiation. In contrast, real-time PCR analysis revealed a considerably high expression of A3B mRNA in 23 bone marrow specimens from MM/MGUS patients, except for two samples with homozygous deletion allele. We sorted myeloma cells using anti-CD138 beads and the enriched myeloma cell samples exhibited even higher A3B mRNA expression in 9 MM/MGUS patients (relative quantity to peripheral blood cells [PBMC]: range 1.06 to 55.14, median 7.67).
Next, we investigated the APOBEC3 family protein expression profile in six myeloma cell lines: U266, RPMI8226, SKMM1, AMO1, OPM2 and THK72. As expected, all of these cell lines demonstrated remarkably high levels of A3B expression by real-time PCR analysis (relative quantity to PBMC: range 1.22 to 489.4, median 62.47). Western blot analysis using an anti-A3B antibody that we newly generated also confirmed the high A3B protein expression in these myeloma cell lines. In addition, fluorescent immunostaining analysis confirmed high expression of A3B protein localized in the nucleus. Because previous studies showed that A3B expression is activated via the NF-kB signaling pathway, we investigated the regulation of A3B transcription in myeloma cells by p65 knockdown through shRNA lentivirus, resulting in suppression of A3B at protein level.
To investigate the mutagenic potential of aberrantly expressed A3B in myeloma cell lines, we evaluated somatic mutations in genomic DNA by differential DNA denaturation PCR (3D-PCR) using stable A3B knock-down cells by shRNA lentivirus simultaneously transduced with a fluorescent gene, mCherry. Interestingly, we detected that scramble shRNA-transduced cells lost fluorescence much faster than those transduced with shRNA against A3B. 3D-PCR amplified mCherry fragments more efficiently at a lower denaturation temperature, indicating that these fragments include more AT-rich sequences. We cloned these DNA fragments and sequenced about 4000 bp in total, and detected prominently more TC > TT mutations in the mCherry sequences from control shRNA-transduced cells (C to T mutation, 3.001%) than in those from A3B shRNA-tranduced cells (C to T mutation, 0.0024%). Additionally, the amplified mCherry sequences from scramble shRNA-transduced cells contained various deletions which may be repaired by microhomology-mediated end joining. Since DNA double strand breaks (DSB) are constitutively activated in myeloma cells, we also examined whether aberrantly expressed A3B can induce DSBs. A3B knockdown in RPMI8226 decreased gH2AX signals by fluorescent immunostaining and Western blot analysis, suggesting that A3B induces DSBs.
The present study clearly shows that myeloma cells express aberrantly high levels of A3B and suggests that A3B might play crucial roles in clonal evolution or genomic instability of MM/MGUS albeit A3B may not contribute to initiation of MGUS/MM. Whether A3B accelerates chemoresistance or disease progression remains to be elucidated, however, A3B might be a potential therapeutic target in MM/MGUS.
Disclosures
Iida: Janssen Pharmaceuticals: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Takaori-Kondo:Mochida Pharmaceutical: Research Funding; Takeda Pharmaceutical: Research Funding; Pfizer: Research Funding; Kyowa Kirin: Research Funding; Astellas Pharma: Research Funding; Eisai: Research Funding; Alexion Pharmaceuticals: Research Funding; Chugai Pharmaceutical: Research Funding; Janssen Pharmaceuticals: Speakers Bureau; Merck Sharp and Dohme: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Shionogi: Research Funding; Toyama Chemical: Research Funding; Cognano: Research Funding.
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