Silencing Immunoglobulin Gene Enhancers as a Potential Treatment Strategy for Multiple Myeloma

Background: Primary translocations involving the immunoglobulin heavy chain (IgH) locus on chromosome 14q32 occur in 60–75% of myeloma cases. This results in oncogenes from translocated chromosomes being overexpressed by linkage to the strong IgH enhancers. Specifically inhibiting the activity of a single translocated oncogene product (e.g. FGFR3 in t(4;14)+ myeloma) has been shown to decrease proliferation and increase apoptosis only in myeloma cells harboring the relevant translocation. Several recurrent translocations have been identified in myeloma, and in many cases the partner loci are non-recurrent or unidentified. No single translocation partner has been found in more than 15–20% of myelomas, and more than one overexpressed gene on the partner locus can be pathogenic (e.g. FGFR3 and MMSET). This limits the widespread applicability and potential effectiveness of therapies that directly target only one IgH-translocated oncogene. We hypothesize that silencing the IgH enhancers is a potential treatment strategy for all IgH translocation-positive myelomas, regardless of the oncogenes involved in the translocation. We further hypothesize that by specifically targeting the B/plasma cell specific transcription factors Oct-2 and Bob-1 that partially drive the IgH enhancer, a therapy could be designed with minimal toxicity to normal tissues apart from B-lineage cells.

Methods: Expression of Oct-2 and Bob-1 genes and proteins were assayed with qRT-PCR and Western blot in 6 myeloma cell lines harbouring different (or no) IgH translocations, in AutoMACS-purified CD138+ and CD138− bone marrow cells from 36 patients, and in bone marrow core biopsies from 30 patients with immunohistochemistry. Using RNA interference (RNAi), we inhibited the expression of Bob-1 in KMS-11, a myeloma cell line that overexpresses fibroblast growth factor receptor 3 (FGFR3) due to a t(4;14) translocation. The effects of Bob-1 RNAi on FGFR3 expression, cell viability and induction of apoptosis were assayed by MTS and Annexin V/propidium iodide flow cytometry assays respectively.

Results: Oct-2 and Bob-1 are ubiquitously expressed in myeloma cell lines and patient plasma cells, irrespective of translocation status. Both mRNA and protein expression are much greater in CD138+ myeloma bone marrow cells than in CD138− cells, and vary among patients. We will present updated results on a larger patient cohort at the meeting, along with clinical correlations including the prognostic significance of Oct-2 and Bob-1 expression levels.

In keeping with decreased activity of the IgH enhancer, a decrease in the level of FGFR3 protein was observed following Bob-1 RNAi knockdown in KMS-11. Furthermore, Bob-1 knockdown led to a reduction in cell viability, and an increase in apoptosis.

Discussion: Overall, these results demonstrate the potential of Bob-1 and possibly Oct-2 as therapeutic targets for the treatment of translocation-positive myeloma, due to both their contribution to overexpression of translocated oncogenes, as well as their selective and high expression in the plasma cell population. Clinically applicable approaches to Bob-1 or Oct-2 inhibition are under consideration in our laboratory.