MDM2 Antagonist Nutlin-3 Enhances Bortezomib-Mediated Mitochondrial Apoptosis In TP53-Mutated Mantle Cell Lymphoma

Abstract 3920

Mantle cell lymphoma (MCL) is a subtype of B-cell lymphoma and frequently resistant to standard chemotherapeutic agents, and the aggressive blastoid MCL cases often possess mutant (mt-) TP53. Although a proteasome inhibitor bortezomib demonstrates single agent efficacy in MCL, more than 50% of the relapsed or refractory MCL are not enough sensitive to bortezomib. Therefore, development of rationally designed combinations of bortezomib and other antineoplastic agents is needed. We previously reported a dose- and time- dependent inhibition of cellular proliferation in wt-TP53 MCL cells by an MDM2 inhibitor nutlin-3, and its synergistic effects when combined with bortezomib in both mt-TP53- and wt-TP53- bearing MCL cells (Tabe et al., Clin Cancer Res. 2009).

In this study, we investigated the molecular mechanism of the synergistical cytotoxic effects of nutlin-3/bortezomib in the mt-TP53 MCL cells which are intrinsically resistant to bortezomib. The MCL cell lines with wild-type and mutant TP53 (wt-TP53: Z-138, Granta-519, mt-TP53: MINO) have been utilized. First, we established the IC₅₀ of nutlin-3 and bortezomib (nutlin-3: 19.1 μM for MINO, 8.2 μM for Granta 519, 1.0 μM for Z-138, bortezomib: 21.8 nM for MINO, 3.7 nM for Granta, 5.0 nM for Z138, MTS assay at 48 hours). The combination of nutlin-3 and bortezomib synergistically induced cytotoxicity more prominently in the bortezomib resistant and mt-TP53 bearing MINO cells (combination index: 0.05 for MINO, 0.71 for Granta 519, 0.12 for Z-138, Calcusyn software). The combination of nutlin-3/bortezomib caused G₀/G₁ cell cycle arrest in the bortezomib sensitive wt-TP53 Granta 519 and Z138, but not in MINO. In contrast, nutlin-3/bortezomib induced marked increase in the sub-G₁ fraction in MINO, but only minimal or no further increase in Granta 519 and Z138, which was confirmed by annexin V/PI staining. BH3-only protein NOXA expression was increased by bortezomib in all tested cells, and the nutlin-3/bortezomib enhanced NOXA accumulation in MINO but not in Granta 519 and Z138 (western blot). On the other hand, anti-apoptotic Mcl-1 has been upregulated by bortezomib in Granta 519 and Z138. In MINO cells, Mcl-1 upregulation was induced only by nutlin-3/bortezomib, which was coimmunoprecipitated with NOXA along with the induction of caspase-3 and -9 cleavage products. We then, assess the subsequent conformational change of proapoptotic BAX/BAK by the nutlin-3/bortezmib combination in MINO (flow cytometric analysis). Taken together, our findings indicate that mitochondrial apoptotic pathway may be an important mechanism contributing to synergistic apoptosis induction by the nutlin-3/bortezomib combination in MCL cells expressing mt-TP53 and intrinsically resistant to bortezomib. The nutlin-3/bortezomib combination may be a potential efficient therapeutic strategy for the chemorefractory MCL.