Bone marrow microenvironment–derived signals induce Mcl-1 dependence in multiple myeloma

Abstract

Multiple myeloma is highly dependent on the bone marrow microenvironment until progressing to very advanced extramedullary stages of the disease such as plasma cell leukemia. Stromal cells in the bone marrow secrete a variety of cytokines that promote plasma cell survival by regulating antiapoptotic members of the Bcl-2 family including Mcl-1, Bcl-x, and Bcl-2. Although the antiapoptotic protein on which a cell depends is typically consistent among normal cells of a particular phenotype, Bcl-2 family dependence is highly heterogeneous in multiple myeloma. Although normal plasma cells and most multiple myeloma cells require Mcl-1 for survival, a subset of myeloma is codependent on Bcl-2 and/or Bcl-x We investigated the role of the bone marrow microenvironment in determining Bcl-2 family dependence in multiple myeloma. We used the Bcl-2/Bcl-x inhibitor ABT-737 to study the factors regulating whether myeloma is Mcl-1 dependent, and thus resistant to ABT-737-induced apoptosis, or Bcl-2/Bcl-x codependent, and thus sensitive to ABT-737. We demonstrate that bone marrow stroma is capable of inducing Mcl-1 dependence through the production of the plasma cell survival cytokine interleukin-6 (IL-6). IL-6 upregulates Mcl-1 transcription in a STAT3-dependent manner, although this occurred in a minority of the cells tested. In all cells, IL-6 treatment results in posttranslational modification of the proapoptotic protein Bim. Phosphorylation of Bim shifts its binding from Bcl-2 and Bcl-x to Mcl-1, an effect reversed by MEK inhibition. Blocking IL-6 or downstream signaling restored Bcl-2/Bcl-x dependence and may therefore represent a clinically useful strategy to enhance the activity of Bcl-2 inhibitors.

Phosphorylation alters Bim‐mediated Mcl‐1 stabilization and priming

“…Our model was further validated by our studies of the S69E mutation. We previously reported that phosphorylation of Bim at S69 in multiple myeloma cells was associated with increased Bim binding to Mcl‐1 . When we applied our densitometry‐based analysis to RPCI‐WM1 cells overexpressing the S69E form of Bim, we observed a strong stabilization of Mcl‐1 despite relatively low Bim overexpression (Fig.…”

“…We've shown that Bim plays a role in stabilizing Mcl‐1 in myeloma cells, even in cells that were strongly dependent on Bcl‐2/x L for survival (Fig. C) . Mcl‐1 is overexpressed in many cancers including myeloma, frequently due to chromosome 1q amplification .…”

“…30 μg of whole‐cell lysate was used as input along with the entirety of the eluted bound fraction for SDS/PAGE and immunoblotting. Bim binding patterns in the immunoprecipitation experiments were quantified as previously described .…”

“…Based on previous literature and kinase prediction software, there are several putative Bim phosphorylation sites ( Fig. 2B) [31,37,[40][41][42][43][44]. To determine which sites are phosphorylated and the functional consequences of Bim phosphorylation in RPCI-WM1, we utilized site-directed mutagenesis to create phospho-mimetic and unphosphorylateable mutants of several likely candidates.…”

“…Additionally, the interplay between Bim‐mediated stabilization of Mcl‐1 and Bcl‐2 protein dependence remains unexplored. Given our previous observation that phosphorylation of Bim is associated with increased binding to Mcl‐1 , we hypothesized that Bim phosphorylation can result in increased or decreased stabilization of Mcl‐1. Here we show that Bim stabilizes Mcl‐1 in several cell lines, and that the ability of Bim to stabilize Mcl‐1 is altered by introduction of various phosphorylation site mutations.…”