Abstract #5518: Bortezomib targets FANCD2 and enhances melphalan response in myeloma cells

We recently reported that Fanconi Anemia (FA)/BRCA DNA damage repair pathway genes are overexpressed and causative for resistance in multiple myeloma (MM) cell lines selected for resistance to melphalan (Chen et al., Blood 2005). Furthermore, others have found that proteasome function is required for FA/BRCA pathway activation (Jacquemont et al., Cancer Res 2007). In this study, we show that the enhanced sensitivity to melphalan seen when cells are first treated with bortezomib is linked to reduced expression and function of FA/BRCA pathway genes. We found that treatment of melphalan sensitive (8226/S and U266/S) and melphalan resistant (8226/LR5 and U266/LR6) MM cell lines with the proteasome inhibitor bortezomib causes a temporal decrease of FA/BRCA pathway gene expression, with maximal decreases seen 24 hours post-bortezomib treatment. Additionally, analysis of two drug resistant MM cell lines, the 8226/LR5 and U266/LR6 cell lines, revealed that FANCD2 gene and protein expression were decreased following treatment with low-dose (3nM) bortezomib, and remain attenuated with the combination of bortezomib plus melphalan. Bortezomib blocked melphalan-induced activation of FANCD2 in both melphalan resistant cell lines, as indicated by inhibition of monoubiquitination of FANCD2 protein. Using confocal microscopy, we found that melphalan-induced FANCD2 foci formation was also inhibited by bortezomib. Analysis of DNA damage and DNA damage repair, using the alkaline comet assay following treatment with low-dose bortezomib and melphalan, showed that bortezomib enhanced DNA damage following melphalan treatment at levels comparable to DNA damage seen when cells were depleted of FANCD2 using siRNA techniques. Collectively, these data show that bortezomib enhances melphalan response by reducing global FA/BRCA gene expression, blocking FANCD2 monoubiquitination and activation, and enhancing DNA damage, secondary to diminished DNA damage repair capacity mediated by the FA/BRCA pathway. Our findings provide evidence for targeting the FA/BRCA pathway to enhance chemotherapeutic response and reverse drug resistance in multiple myeloma patients. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 5518.