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Purpose: Proteasome inhibition disrupts protein homeostasis and induces apoptosis. Up to 50% of patients with relapsed mantle cell lymphoma (MCL) respond to bortezomib. We used gene expression profiling to investigate the connection between proteasome inhibition, cellular response, and clinical efficacy., Experimental Design: We assessed transcriptional changes in primary tumor cells from five patients during treatment with bortezomib in vivo, and in 10 MCL cell lines exposed to bortezomib in vitro, on Affymetrix microarrays. Key findings were confirmed by western blotting., Results: MCL cell lines exposed to bortezomib in vitro showed upregulation of endoplasmic reticulum and oxidative stress response pathways. Gene expression changes were strongest in bortezomib-sensitive cells and these cells were also more sensitive to oxidative stress induced by H2O2. Purified tumor cells obtained at several timepoints during bortezomib treatment in 5 previously untreated patients with leukemic MCL showed strong activation of the antioxidant response controlled by NRF2. Unexpectedly, activation of this homeostatic program was significantly stronger in tumors with the best clinical response. Consistent with its proapoptotic function, we found upregulation of NOXA in circulating tumor cells of responding patients. In resistant cells, gene expression changes in response to bortezomib were limited and upregulation of NOXA was absent. Interestingly, at baseline, bortezomib-resistant cells displayed a relatively higher expression of the NRF2 gene-expression signature than sensitive cells (P < 0.001)., Conclusion: Bortezomib triggers an oxidative stress response in vitro and in vivo. High cellular antioxidant capacity contributes to bortezomib resistance., (©2011 AACR.)

Bortezomib induces remissions in 30%-50% of patients with relapsed mantle cell lymphoma (MCL). Conversely, more than half of patients' tumors are intrinsically resistant to bortezomib. The molecular mechanism of resistance has not been defined. We generated a model of bortezomib-adapted subclones of the MCL cell lines JEKO and HBL2 that were 40- to 80-fold less sensitive to bortezomib than the parental cells. Acquisition of bortezomib resistance was gradual and reversible. Bortezomib-adapted subclones showed increased proteasome activity and tolerated lower proteasome capacity than the parental lines. Using gene expression profiling, we discovered that bortezomib resistance was associated with plasmacytic differentiation, including up-regulation of IRF4 and CD38 and expression of CD138. In contrast to plasma cells, plasmacytic MCL cells did not increase immunoglobulin secretion. Intrinsically bortezomib-resistant MCL cell lines and primary tumor cells from MCL patients with inferior clinical response to bortezomib also expressed plasmacytic features. Knockdown of IRF4 was toxic for the subset of MCL cells with plasmacytic differentiation, but only slightly sensitized cells to bortezomib. We conclude that plasmacytic differentiation in the absence of an increased secretory load can enable cells to withstand the stress of proteasome inhibition. Expression of CD38 and IRF4 could serve as markers of bortezomib resistance in MCL. This study has been registered at http://clinicaltrials.gov as NCT00131976.

Bortezomib is more active against mantle cell lymphoma (MCL) than against most other lymphoma subtypes. Nevertheless, up to half of patients with MCL have bortezomib resistant disease. Factors contributing to intrinsic resistance to bortezomib have not been determined. Here we used a panel of eight bortezomib sensitive (median IC(50) 5.9 nM) and three relatively bortezomib resistant cell lines (median IC(50) 12.9 nM) to investigate differences in tumor biology that could determine sensitivity to bortezomib. Bortezomib effectively inhibited high baseline proteasome activity and induced a comparable degree of proteasome inhibition in both sensitive and resistant cells. At 10 nM, bortezomib induced the proapoptotic BH3-only protein Noxa in sensitive but not resistant cells. At higher concentrations of bortezomib, however, Noxa was also upregulated in resistant cells and this effect was sufficient to induce apoptosis. Silencing of Noxa with siRNA rescued these cells from apoptosis, arguing against a defect in Noxa regulation or function as the basis of bortezomib resistance. Bortezomib was equally effective against cells with high and low constitutive NF-kappaB signaling. Also, sensitive and resistant MCL cell lines showed comparable activation of the AKT pathway. We conclude that bortezomib can overcome classic mechanisms of resistance to apoptosis and that determinants of bortezomib sensitivity in MCL are due to differences in signaling or stress pathways upstream of Noxa.