Evaluation of the In Vitro Activity of the Multikinase Inhibitor BAY 43-9006 (Sorafenib) in Primary Cells from Patients with Myelofibrosis with Myeloid Metaplasia (MMM).

Abstract

Background: Myelofibrosis with myeloid metaplasia (MMM) currently has limited therapeutic options. BAY 43-9006 (sorafenib; Bayer Pharmaceuticals) is a oral multikinase inhibitor with both inhibitory properties against the Raf/ MEK survival pathway and VEGF that are a potential therapeutic targets for the myeloproliferation and apoptotic resistance seen in this disorder. We evaluated the activity of this agent through inhibition of aberrant in vitro myeloid colony growth, as we have previously successfully utilized in this disorder (Leukemia2003;17(5):849). Methods: Peripheral blood mononuclear cells (PBMC) were isolated and plated on the Methocult™ (StemCell Technologies) colony assay media. Dilutions of BAY 43-9006 (0–10 μM) (Phase I trial sustained concentrations of 10 μM (JCO2005;23:965) was added for either a 24 hour incubation, or for 14 days. Myeloid colonies were then quantified according to standards established by the manufacturer. Subsequent assays were performed combining BAY 43-9006 with the pro-apoptotic agents arsenic trioxide (ATO) or adaphostin (ADA) (both at 1.25μM). Finally, immunoblotting was performed on protein extracted from treated primary MMM cells to quantify the downregulation of Mcl-1 (shown to be crucial for the pro-apoptotic effects of BAY 43-9006; Yu et. al. Oncogene 2005). Results: PBMC were isolated from 12 MMM patients and 10 controls (normal n=5, other myeloproliferative (MPD) n=5). Twenty-four hour exposure of cells to the agent was insufficient to suppress aberrant colony growth. Continuous exposure (for 14 days) of the agent led to a median inhibitory concentration-50% (IC50) of 12 μM for MMM (compared to 7.1μM for normals, and 5μM for MPD controls). Although no specificity could be deduced from these latter results, 4 MMM patients had an IC50 of <10μM (range 1.25 to 8.6μM). In contrast, the other 8 patients were quite resistant IC50 of > 10μM (higher than clinically achievable ranges). Subsequently we evaluated whether there might be benefit to use of BAY43-9006 in combination with a pro-apoptotic agent (i.e. ATO or ADA). No significant activity was seen combining ADA with BAY43-9006. When BAY43-9006 was combined with ATO, the median IC 50 for the BAY compound decreased to 2μM (range 1.25 to 16), however there was not a significant increase in colony inhibition over ATO alone. In order to evaluate whether the lack of activity of BAY43-9006 observed in colony formation assays was secondary to a lack of downregulation of the established target Mcl-1, we immunoblotted for Mcl-1 (after in vitro incubation) in a subset of MMM patients. Western blots demonstrated successful downregulation of Mcl-1 by Bay 43-9006 in MMM samples, even in samples with clear in vitro resistance to the drug (i.e. IC50 > 10 μM). Conclusions: The multi-kinase inhibitor BAY43-9006 appears to have minimal activity against primary cells isolated from MMM patients. Although, suppression of aberrant colony growth was noted in a subset of patients the sensitivities would not support specificity when compared to controls. Although BAY 43-9006 was observed to down regulate the anti-apoptotic target Mcl-1, in patient samples, no detectable inhibition of colony formation was observed in 8/12 MMM samples. These preliminary results do not appear to support a clinical trial of BAY 43-9006 in MMM, however combination of this multi-kinase inhibitor with other pro-apoptotic agents should be evaluated.