Multiple myeloma (MM), the second most common hematopoietic malignancy, is an incurable plasma cell neoplasm representing approximately 2% of all cancer deaths. The proteasome inhibitor, bortezomib (Bz), has been widely used to treat MM, and emerging next-generation proteasome inhibitors are being evaluated. Despite advances in therapies, patients treated with Bz eventually relapse due to the development of drug resistance. Thus, identifying signatures that distinguish Bz sensitive from resistant MM cells will more accurately predict Bz sensitivity and will aid in the development of novel approaches to reverse resistance.
MM populations are characterized by phenotypic heterogeneity suggesting the possibility that certain MM subpopulations are associated with Bz resistance. In order to characterize these populations in greater detail, we have utilized isogenic pairs of Bz sensitive and resistant MM lines derived from the genetically engineered iMycCa/Bcl-xl mouse model of plasma cell malignancy. The iMycCa/Bcl-xl mouse is a highly robust model that very closely resembles human MM based on gene expression profiling (GEP), chromosomal abnormalities and progression of disease and response to therapy (Boylan, et al. Cancer Res (2007) 67: 4069). We have employed this mouse model because of its striking similarity to human MM, and MM cells isolated from these mice can be readily infected by lentivirus as well as adoptively transferred into syngeneic, immunocompetent recipients establishing a powerful system to directly test biologically relevant hypotheses relating to Bz resistance.
Isogenic pairs of Bz sensitive and resistant lines were extensively characterized using GEP, quantitative PCR and flow cytometry. We observed increased expression of germinal B cell markers Pax5, AID and Bcl-6 and reduced expression of the plasma cell marker CD93 in Bz resistant lines compared to Bz sensitive pairs suggesting that Bz resistance is associated with germinal center-like B cell characteristics. Remarkably, we detect within the same cell co-expression of both CD138/Pax5 and CD138/AID demonstrating the tremendous plasticity of these cells and their intermediate B cell differentiation phenotype. Recently, both AID and Bcl-6 have been identified as crucial mediators of resistance to tyrosine kinase inhibitors in leukemia cells (Duy, et al. Nature (2011) 473: 384; Klemm, et al. Cancer Cell (2009) 16: 232) raising an intriguing possibility that these proteins thought to be restricted to germinal center B cells could play a role in Bz resistance in MM. These observations have led us to more closely characterize the apparent shift in developmental stage resulting from Bz-driven expression changes or selection. Time course analysis of gene and protein expression following low-dose Bz treatment in the absence of cell death indicates that Bz increases the expression and promotes the stabilization of Pax5, AID and Bcl-6. This demonstrates a drug-induced change in markers associated with B cell differentiation relating to the ultimate emergence of the resistance phenotype. We are now determining whether similar B cell differentiation phenotypes emerge in a recently published human drug trial where GEP was completed prior to and 48 hours after Bz treatment in patients (Shaughnessy, et al. Blood (2011), ahead of print). Thus, we will present phenotypic signatures correlating to Bz resistance in cell lines derived from our mouse model of MM and suggest that targeting B cell differentiation may represent a rational therapeutic approach to overcoming Bz resistance.
Stessman:Millennium: The Takeda Oncology Company: Research Funding. Mansoor:Millennium: The Takeda Oncology Company: Research Funding. Van Ness:Millennium: The Takeda Oncology Company: Research Funding.
Asterisk with author names denotes non-ASH members.