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

Abstract

Tumor microenvironment (TME) is commonly implicated in regulating the growth of tumors, but whether it can directly alter the genetics of tumors is not known. IntroductionMultiple myeloma (MM) is characterized by the growth of malignant plasma cells in the BM. Genomic instability and chromosome translocations are common and early hallmarks of this tumor. 1 In particular, aneuploidy as well as chromosome translocations that involve the immunoglobulin heavy chain (IgH) locus are commonly observed in MM as well as its precursor monoclonal gammopathy of undetermined significance. However, the mechanisms underlying genomic instability in MM or monoclonal gammopathy of undetermined significance are poorly understood. One candidate is activation-induced cytidine deaminase (AID), which is essential for somatic hypermutation and class switch recombination. 2,3 Mistargeting of AID has been implicated in oncogenic mutations and chromosome translocations in both lymphoid and nonlymphoid tumors. [3][4][5] However, prior studies have failed to detect AID in MM cell lines, implying that other pathways may be involved in mediating genomic instability in this tumor. 6 Several studies have emphasized the importance of tumor microenvironment (TME) in the biology of human MM. 7 Although the role for TME in regulating tumor growth is well established, whether and how it might directly alter the genetics of tumors are not known. Several studies have shown that tumor lesions in MM are highly infiltrated by dendritic cells (DCs). [8][9][10] In prior studies we had observed that the interaction between DCs and MM cells led to aberrant reexpression of BCL6 in MM cells, a gene typically silenced during normal plasma cell differentiation. 11 Therefore, we tested whether this interaction could also induce the expression of AID in tumor cells. Methods Tumor cell lines and patient samplesMM cell lines were purchased from ATCC (U266), or kindly provided by Dr Joshua Epstein (Little Rock, AR; OPM2, ARK, and CAG cells). Other tumor cells used were MCF-7 cells (breast cancer; gift from Dr Lyndsay Harris, Yale Cancer Center). Cells were cultured in RPMI 1640 with 10%-15% FBS. BM and peripheral blood specimens were obtained from patients with myeloma after informed consent was obtained in accordance with the Declaration of Helsinki under a protocol approved by the institutional review board of Yale University. Peripheral blood buffy coats from healthy donors were purchased from the New York Blood Center. Isolation of mononuclear cells, DCs, primary tumor cells, and DC subsetsPeripheral blood or BM mononuclear cells (MNCs) were isolated by density gradient centrifugation (Ficoll-Paque plus; Amersham Biosciences). DCs were generated from purified blood monocytes as described earlier. 11,12 In brief, monocytes isolated with CD14 microbeads (Miltenyi Biotec) were cultured in the presence of GM-CSF (20 ng/mL; Genzyne) and IL-4 (20 ng/mL; R&D Systems). DCs were typically used on day 5 or 6 of culture. For some experiments, DCs were matured by the use of...

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