Rational for a Combination of Bortezomib and Doxorubicin in the Treatment of Multiple Myeloma: A Pivotal Role for Mcl-1.

Bortezomib is a proteasome inhibitor for the treatment of relapsed/refractory multiple myeloma (MM). Mechanisms of resistance to Bortezomib are undefined. Myeloid cell leukemia-1 (Mcl-1) is an antiapoptotic protein, which protects tumor cells against spontaneous and chemotherapy-induced apoptosis. In MM, specific downregulation of Mcl-1 induces apoptosis. Here, we examined the role of Mcl-1 in Bortezomib- and doxorubicin-induced apoptosis. We demonstrate that Bortezomib, but not doxorubicin, triggers caspase-dependent generation of a 28 kDa Mcl-1-fragment, in several MM cell lines, including MM.1S cells. Conversely, transient transfection of MM.1S cells with a previously reported 28 kDa Mcl-1(128–350) fragment, but not with the Mcl-1(1–127) fragment, induces apoptosis. Therefore, both downregulation of full-length antiapoptotic Mcl-1, as well as Bortezomib-induced generation of Mcl-1(128–350) cleaved protein, contribute to MM cell apoptosis. To verify further these findings, we next compared effects triggered by Bortezomib, doxorubicin and melphalan in Mcl-1(wt/wt) and Mcl-1(Delta/null) murine embryonic fibroblasts (MEFs). Our results show that Bortezomib, but not doxorubicin or melphalan, triggers Mcl-1 cleavage in Mcl-1(wt/wt), but not Mcl-1(Delta/null) MEFs and induces sub-G(1) phase cells; caspase-3 and -9, and PARP cleavage as well as morphological signs of apoptosis. Interestingly, Bortezomib-induced generation of the Mcl-1(128–350) fragment was accompanied by the induction of the BH3-only protein NOXA and its increased binding to Mcl-1. Indeed, NOXA tightly binds Mcl-1 but not Bcl-2, Bcl-XL, or Bcl-w, thereby displacing Bak. In turn, free Bak induces apoptosis via permeabilization of organellar membranes and induction of caspase activation. Besides Mcl-1, Bcl-XL is also a guardian of Bak.Consistent with these data, our results show that Bortezomib induces apoptosis dependent on Mcl-1. In conclusion, our study demonstrates that lack of Mcl-1 and Mcl-1128–350 fragment confers resistance to Bortezomib and protects against Bortezomib-induced caspase-3 and PARP cleavage, highlighting the complexity of Mcl-1 post-translational regulation and its role in mitochondrial and caspase-3-mediated drug-induced apoptosis. Furthermore, our results identify a new mechanism of drug resistance, implicating a role for Mcl-1 not only as an antiapoptotic protein that opposes drug-induced apoptosis, but also as a proapoptotic cleaved protein enhancing mitochondrial/caspase activation and thereby leading to apoptosis. From a clinical point of view, our results suggest the potential utility of combining therapies that trigger Mcl-1-dependent and -independent pathways, that is Bortezomib and doxorubicin or Bortezomib with histone deacetylase inhibitors (Pei et al., 2004) and Bortezomib and doxorubicin with seliciclib, a small molecule cyclin-dependent kinase inhibitor (Raje et al., 2005). Recently, Orlowski et al. (2005) have combined these two drugs and demonstrated remarkably activity in patients with relapsed or refractory hematologic malignancies, supporting the potential utility of this therapeutic strategy.