Certain genetic events accompany transformation of MM to more aggressive disease. For example, there is a predominance of new and promiscuous chromosomal translocations into the switch region of the immunoglobulin heavy chain (IgH) gene on chromosome 14 (i.e. at 14q32). Development of chromosomal translocations involves the process of DNA double strand break repair (DSBR) by non-homologous end joining (NHEJ). DNA protein kinase (DNA-PK) is the principal DNA repair enzyme mediating DNA DSBR. It is made up of a catalytic subunit (DNA-PKcs) and a regulatory subunit (the Ku70/Ku86 heterodimer). Interestingly, the majority (86% to 100%) of freshly isolated patient MM cells express a variant form of Ku86 protein (Ku86v), which has been associated with abnormalities in DNA repair. Since, the combined effects of CD40 plus interleukin-4 (IL-4) are required for normal IgH isotype class switch recombination (CSR), and this process involves DNA DSBR, NHEJ and DNA-PK; we hypothesized that CD40 and/or IL-4 activation of MM cells could induce abnormalities in DNA DSBR, which could lead eventually to genomic instability and clonal evolution. In this study, we first showed that RPMI 8226 and SGH-MM5 MM cell lines (but not the CESS Epstein-Barr virus (EBV) immortalized normal B cell line) that are optimally triggered via CD40 and/or IL-4 demonstrate abnormal decoupling of IL-4 signal transduction from CD40. Specifically, CD40 alone was sufficient to trigger growth of tumor cell lines, suggesting that biological sequelae mediated by CD40 could be dysregulated in MM cells. Whether this process involves Ku86v is presently being investigated. We further demonstrate that CD40 triggering induced both DNA DSBs as well as new (acquired) karyotypic abnormalities in the SGH-MM5 MM cell line. These complex karyotypic changes included at least 5 new and clonal chromosomal translocations and deletions. Since, normal IgH isotype CSR is accompanied by induction of activation induced cytidine deaminase (AID) expression, we next demonstrated that CD40 triggering of MM cell lines (without IL-4) was sufficient to upregulate AID expression. These data suggest that DNA DSBs induced by CD40 were part of IgH isotype CSR rather than CD40 induced apoptosis of tumor cells. This is an important distinction to make because CD40 has been demonstrated to induce apoptosis by both p53 -dependent and -independent pathways. In order to confirm that CD40-triggered MM cells did not undergo apoptosis, we performed annexin V/propidium iodide (PI) staining on CD40-triggered MM cells. We showed that MM cell lines not only remained viable after CD40 triggering, but also demonstrated G1 cell cycle exit. In conclusion, our present study shows that CD40 alone could act as an inducer of genomic instability in MM cell lines, and lead to clonal evolution. Since CD40 ligand (CD40L) is naturally expressed during inflammation and T cell activation, it is tempting to speculate that the normal inflammatory process could potentially participate in clonal evolution and even myelomagenesis in vivo.

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