Combination Bortezomib (PS341, Velcade) and Rituximab Treatment Affects Multiple Survival and Death Pathways To Promote Apoptosis in Mantle Cell Lymphoma.

Abstract

Mantle cell lymphoma (MCL) is a distinct histologic subtype of B cell non-Hodgkin’s lymphoma that is associated with an aggressive clinical course and a particularly poor prognosis. The mechanisms that contribute to resistance of MCL to chemotherapy are not clear, however, recent work examining the consequences of ubiquitin-proteasome pathway inhibition on cell cycle (p21, p27) and key survival/death networks (NFkB, p53, Bcl2) has provided rationale for exploring combination regimens that include tumor-specific reagents (rituximab) and the 26S proteasome inhibitor bortezomib. In this study, we examined the effects of combination treatment with bortezomib and rituximab on MCL patient samples and three patient-derived cell lines (Jeko, Mino, SP53). Cells treated with bortezomib (10 – 100nM) for 4 hours demonstrated proteasome inhibition that persisted for 24 hours but returned to baseline activity at 48 hours after treatment. Despite transient proteasome inhibition, combination therapy with bortezomib (10–100nM for 4hrs) and rituximab (1 mg/ml immobilized with 20 mg/ml goat anti-human IgG) resulted in synergistic induction of apoptosis that persisted for as long as 72 hours after treatment. While bortezomib (100 nM) induced apoptosis in 18.3 ± 6.5% and rituximab induced apoptosis in 24.5 ± 4.5% of MCL cells, combination treatment resulted in 57.4 ± 5.1% apoptosis at 48 hours (p ≤ 0.02). Pretreatment of MCL cells with the broad spectrum caspase inhibitor zVAD-FMK (10 mM) showed that bortezomib-induced cell death occurred by caspase-dependent mechanisms, however, when immobilized rituximab was added, cell death occurred via caspase dependent and independent pathways. Single agent bortezomib (10 nM) or rituximab treatment of Mino and Jeko lines resulted in decreased levels of nuclear NFkB complex(s) capable of binding p65 consensus oligonucleotides (28% and 21% reduction, respectively), while combination treatment resulted in enhanced reduction of detectable nuclear NFkB (36% reduction, p ≤ 0.0007). Similar trends were observed with primary MCL cells. Experiments with an IKK inhibitor (PS1145, Millenium Pharmaceuticals) resulted in nuclear NFkB reduction without equivalent induction of apoptosis which led us to hypothesize that other pro-death pathways might be operable with combination treatment. Western blot analysis of BCL2-family members revealed that combination treatment of MCL lines resulted in near complete elimination of Bcl-xL protein while Bcl-2 protein levels remained unchanged. The pro-death gene product Bax was induced in a synergistic fashion with combined bortezomib and rituximab treatment. Finally, we have developed a reliable preclinical animal model utilizing the severe combined immune deficient (SCID) mouse engrafted with three patient-derived MCL cell lines. Each cell line results in a characteristic pattern of tumor burden and highly reproducible time to develop advanced disease. We are currently evaluating combination therapy with bortezomib and rituximab in this preclinical animal model. Our preclinical evaluation provides clear rationale for pursuing combination strategies that inhibit the proteasome in combination with tumor-specific immunotherapy in patients with MCL.