Targeting Mcl-1 for Multiple Myeloma (MM) Therapy: Drug-Induced Generation of Mcl-1 Fragment Mcl-1^128–350 Triggers MM Cell Death Via c- Jun Upregulation

Abstract 3959

Myeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic protein of the Bcl-2 family located on chromosome 1q21. Gains of 1q21 are unfavorable genetic prognostic factors for Multiple Myeloma (MM) patients. Our present data confirm significant overexpression of Mcl-1 gene in tumor versus plasma cells derived from healthy donors. Moreover, a significant link between Mcl-1 overexpression and poor prognosis was found in a data set of 590 MM patients, supporting a key role for Mcl-1 in MM survival and drug resistance. Mcl-1 is therefore a potential therapeutic target in MM. Strategies to inhibit Mcl-1 activity in MM and other malignancies may not only include induction of proteasome-dependent Mcl-1 degradation and modulation of Mcl-1 interaction with BH3-only proteins, but also induction of proteasome- independent Mcl-1 degradation. Indeed, our own and other previous data demonstrate that in contrast to full length Mcl-1 a caspase- induced 28kD Mcl-1 fragment, Mcl-1D^128–350, inhibits MM cell proliferation and triggers apoptosis. We therefore performed a large-scale drug screen for agents which induce generation of Mcl-1D^128–350 in MM cells. Our results show that bortezomib and staurosporine (STS) but not melphalan, doxorubicin or thalidomide induce generation of Mcl-1D^128–350. We next sought to identify molecular sequelae downstream of Mcl-1D^128–350 which mediate its anti-proliferative and pro-apoptotic effect in MM cells. Surprisingly, we observed nuclear accumulation of drug- induced Mcl-1D^128–350, enhanced AP-1 reporter activity as well as elevated protein levels of c-Jun. Conversely, drug- induced c-Jun upregulation was blocked after introduction of point mutations (A127D, A157D) into the two highly conserved Mcl-1 caspase-cleavage sites Asp127 and Asp157. Gene profiles on MM cells transfected with either Mcl-1wt or Mcl-1D^128–350 identified a group of differentially expressed genes associated with MM cell proliferation, survival and drug resistance. Consistent with these data, treatment with bortezomib or STS triggered c-Jun upregulation followed by apoptosis in Mcl-1wt/wt but not Mcl-1d/null murine embryonic fibroblasts (MEFs). Importantly, STS-induced c-Jun upregulation and cell death was restored in Mcl-1d/null MEFs transfected with a plasmid carrying Mcl-1wt. In ongoing studies we are analyzing genome sequencing data in MM cells for the existence of point mutations in the Mcl-1 caspase cleavage sites. The presence of such mutations could represent a potential new mechanism of drug resistance. Taken together, our data add another facet to the complex cellular functions of Mcl-1 in MM and derive a promising therapeutic and potential prognostic role for Mcl-1D^128–350 in MM.