Gene Expression Analysis Uncovers Possible Resistance Mechanisms to Bortezomib in Mantle Cell Lymphoma That Differ from Mechanisms Identified in Other Lymphoid Malignancies.

Bortezomib, a potent inhibitor of the 26S proteasome, has remarkable activity against some lymphoid malignancies, particularly multiple myeloma and mantle cell lymphoma (MCL). The observed 30–50% clinical response rate to bortezomib as a single-agent in relapsed MCL is believed to be mediated mainly by inhibition of the NFkB signaling pathway. Recently, Hsp27 has been shown to confer resistance to bortezomib in a lymphoid cell line. However, much less is known about the mechanisms underlying anti-tumor activity of or resistance to bortezomib in MCL. To address these questions we studied 10 MCL cell lines with t(11;14)(q13;q32) as in vitro models. IC50 values were measured by the MTT cytotoxicity assay for both bortezomib and the BAY 11-7082 compound, a specific inhibitor of the NFkB pathway. The cell lines showed different profiles of response to bortezomib and were grouped according to their IC50 values as sensitive (S) (Granta 519, Jeko-1, SP-49, UPN-1), intermediate (In) (HBL-2, JVM-2, Z-138) and resistant (R) (Mino, NCEB-1, SP-53). The mean IC50 for bortezomib in the R group was 3 times higher than the mean IC50 of the S group. The cell lines also showed very different profiles of response to the NFkB-specific inhibitor BAY 11-7082, but grouped differently, suggesting that other mechanisms, in addition to the NFkB pathway, participate in the anti-neoplastic effect of proteasome inhibition in MCL. Resistance to both drugs had no correlation with P-glycoprotein activity as measured by the rodhamine efflux assay. We performed gene expression profiling on Affymetrix U133A 2.0 arrays of all 10 cell lines. Using GeneSpring software (Agilent), we normalized expression to the mean of the S group and identified 79 transcripts that showed a dose response behavior; that is, genes whose mean expression was 1.5x higher in the In versus the S group and 1.5x higher in the R versus the In group. Conversely, 55 transcripts followed the opposite trend, being down-regulated at least 1.5x in both comparisons. This list contained genes important in apoptosis control and in transmembrane transport, but was most notable for an overexpression of stress response genes in the R group. Notably, expression of Hsp70 was almost 10-fold higher in the R versus S group. We therefore analyzed expression of the HSP70 as well as HSP27 and HSP90 proteins by Western blotting. Hsp70 protein was highly expressed in all MCL cell lines and there was no discernable correlation with bortezomib resistance for any of the HSP-proteins analyzed. Other resistance mechanisms appear more important in MCL and we are currently testing further candidate genes for their ability to confer resistance to bortezomib in MCL.