ABSTRACT
The anti-apoptotic protein Mcl-1 plays a major role in multiple myeloma (MM) cell survival as well as bortezomib- and microenvironmental forms of drug resistance in this disease. Consequently, there is a critical need for strategies capable of targeting Mcl-1-dependent drug resistance in MM. The present results indicate that a regimen combining Chk1 with MEK1/2 inhibitors effectively kills cells displaying multiple forms of drug resistance stemming from Mcl-1 up-regulation in association with direct transcriptional Mcl-1 down-regulation and indirect disabling of Mcl-1 anti-apoptotic function through Bim up-regulation and increased Bim/Mcl-1 binding. These actions release Bak from Mcl-1, accompanied by Bak/Bax activation. Analogous events were observed in both drug-naïve and acquired bortezomib-resistant MM cells displaying increased Mcl-1 but diminished Bim expression, or cells ectopically expressing Mcl-1. Moreover, concomitant Chk1 and MEK1/2 inhibition blocked Mcl-1 up-regulation induced by IL-6/IGF-1 or co-culture with stromal cells, effectively overcoming microenvironment-related drug resistance. Finally, this regimen down-regulated Mcl-1 and robustly killed primary CD138+ MM cells, but not normal hematopoietic cells. Together, these findings provide novel evidence that this targeted combination strategy could be effective in the setting of multiple forms of Mcl-1-related drug resistance in MM.
Subject(s)
Drug Resistance, Neoplasm/drug effects , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Multiple Myeloma/drug therapy , Multiple Myeloma/enzymology , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Protein Kinase Inhibitors/therapeutic use , Protein Kinases/metabolism , Apoptosis/drug effects , Apoptosis Regulatory Proteins/metabolism , Bcl-2-Like Protein 11 , Benzamides/pharmacology , Benzamides/therapeutic use , Boronic Acids/pharmacology , Bortezomib , Cell Line, Tumor , Checkpoint Kinase 1 , Cytoprotection/drug effects , Down-Regulation/drug effects , Humans , Intercellular Signaling Peptides and Proteins/pharmacology , Membrane Proteins/metabolism , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/pathology , Mitogen-Activated Protein Kinase Kinases/metabolism , Protein Binding/drug effects , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins/metabolism , Pyrazines/pharmacology , Syndecan-1/metabolism , Up-Regulation/drug effects , bcl-2 Homologous Antagonist-Killer Protein/metabolism , bcl-2-Associated X Protein/metabolismABSTRACT
PURPOSE: Preclinical studies in human melanoma cell lines and murine xenograft tumor models suggest that the proteasome inhibitor bortezomib enhances the activity of the cytotoxic agent dacarbazine. We performed a phase I trial of bortezomib and dacarbazine in melanoma, soft tissue sarcoma, and amine precursor uptake and decarboxylation tumors. The primary objective was to identify recommended phase II doses for the combination. EXPERIMENTAL DESIGN: Bortezomib and dacarbazine were both administered intravenously once weekly. All patients received prophylactic antiemetics. Dose escalation proceeded using a standard 3 + 3 design. Response was assessed according to NCI RECIST v1.0. RESULTS: Twenty eight patients were enrolled to six dose levels. Bortezomib 1.6 mg/m(2) and dacarbazine 580 mg/m(2) are the recommended phase II weekly doses. The combination was generally well tolerated. Among 15 patients with melanoma there was one durable complete response in a patient with an exon-11 cKIT mutation, and one partial response. Among 12 patients with soft tissue sarcoma there was one partial response. CONCLUSIONS: Bortezomib 1.6 mg/m(2) and dacarbazine 580 mg/m(2) administered intravenously once weekly is well tolerated and has at least minimal activity in melanoma and soft tissue sarcoma.
Subject(s)
Antineoplastic Agents/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Boronic Acids/therapeutic use , Dacarbazine/therapeutic use , Melanoma/drug therapy , Pyrazines/therapeutic use , Sarcoma/drug therapy , Adult , Aged , Antineoplastic Agents/adverse effects , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Boronic Acids/adverse effects , Bortezomib , Dacarbazine/adverse effects , Dose-Response Relationship, Drug , Female , Humans , Lung Neoplasms/diagnostic imaging , Lung Neoplasms/secondary , Male , Medication Adherence , Melanoma/diagnostic imaging , Middle Aged , Pyrazines/adverse effects , Radiography , Treatment Outcome , Young AdultABSTRACT
Effects of Chk1 and MEK1/2 inhibition were investigated in cytokinetically quiescent multiple myeloma (MM) and primary CD138(+) cells. Coexposure to the Chk1 and MEK1/2 inhibitors AZD7762 and selumetinib (AZD6244) robustly induced apoptosis in various MM cells and CD138(+) primary samples, but spared normal CD138(-) and CD34(+) cells. Furthermore, Chk1/MEK1/2 inhibitor treatment of asynchronized cells induced G(0)/G(1) arrest and increased apoptosis in all cell-cycle phases, including G(0)/G(1). To determine whether this regimen is active against quiescent G(0)/G(1) MM cells, cells were cultured in low-serum medium to enrich the G(0)/G(1) population. G(0)/G(1)-enriched cells exhibited diminished sensitivity to conventional agents (eg, Taxol and VP-16) but significantly increased susceptibility to Chk1 ± MEK1/2 inhibitors or Chk1 shRNA knock-down. These events were associated with increased γH2A.X expression/foci formation and Bim up-regulation, whereas Bim shRNA knock-down markedly attenuated lethality. Immunofluorescent analysis of G(0)/G(1)-enriched or primary MM cells demonstrated colocalization of activated caspase-3 and the quiescent (G(0)) marker statin, a nuclear envelope protein. Finally, Chk1/MEK1/2 inhibition increased cell death in the Hoechst-positive (Hst(+)), low pyronin Y (PY)-staining (2N Hst(+)/PY(-)) G(0) population and in sorted small side-population (SSP) MM cells. These findings provide evidence that cytokinetically quiescent MM cells are highly susceptible to simultaneous Chk1 and MEK1/2 inhibition.
