14-methyl-20-oxa-5-7-14-26-tetraazatetracyclo(19.3.1.1(2-6).1(8-12))heptacosa-1(25)-2(26)-3-5-8(27)-9-11-16-21-23-decaene and Multiple-Myeloma

Carfilzomib (Kyprolis®), a second generation proteasome inhibitor, is FDA approved for single-agent use among relapsed/refractory multiple myeloma (MM). To enhance the therapeutic efficacy of carfilzomib, we sought to combine carfilzomib with other novel agents. TG02, a multi-kinase inhibitor, targets JAK2 and CDK9. The rationale for co-treatment with carfilzomib and TG02 is that both independently target Mcl-1 and most myeloma cells are dependent on this anti-apoptotic protein for survival. We observed at least additive effects using the combination treatment in MM cell lines and patient samples. To determine how the bone marrow environment affects the efficacy of the combination we conducted co-culture experiments with Hs-5 stromal cells. We also examined the mechanism of increased apoptosis by determining the affect on expression of the Bcl-2 family of proteins. We found that carfilzomib increases NOXA mRNA expression, as expected, and TG02 treatment caused a decrease in Mcl-1 protein but not mRNA levels. Consistent with this possibility, we find silencing CDK9 does not change carfilzomib sensitivity in the same manner as addition of TG02. Since changes in Mcl-1 protein occur in the presence of a proteasome inhibitor we hypothesize that regulation of Mcl-1 translation is the most likely mechanism. Taken together our data suggest that dual inhibition of Mcl-1 via decreased expression and the induction of its antagonist NOXA by the combination of carfilzomib and TG02 is active in myeloma and warrants further testing preclinically and in clinical trials. Moreover, regulation of Mcl-1 by TG02 is more complex than initially appreciated.

Topics: Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Heterocyclic Compounds, 4 or More Rings; Humans; Multiple Myeloma; Myeloid Cell Leukemia Sequence 1 Protein; Oligopeptides

To analyze the antimyeloma potential of TG02, an ERK5/CDK inhibitory drug.. Utilizing different multiple myeloma cell lines we determined the effect of TG02 over viability by MTT assays. The apoptotic effect over multiple myeloma patient samples was studied ex vivo by cytometry. The mechanism of action of TG02 was analyzed in the cell line MM1S, studying its effect on the cell cycle, the induction of apoptosis, and the loss of mitochondrial membrane potential by cytometry and Western blot. Two models of multiple myeloma xenograft were utilized to study the in vivo action of TG02.. TG02 potently inhibited proliferation and survival of multiple myeloma cell lines, even under protective bone marrow niche conditions, and selectively induced apoptosis of primary patient-derived malignant plasma cells. TG02 displayed significant single-agent activity in two multiple myeloma xenograft models, and enhanced the in vivo activity of bortezomib and lenalidomide. Signaling analyses revealed that the drug simultaneously blocked the activity of CDKs 1, 2, and 9 as well as the MAP kinase ERK5 in MM1S cells, leading to cell-cycle arrest and rapid commitment to apoptosis. TG02 induced robust activation of both the intrinsic and extrinsic pathways of apoptosis, and depletion of XIAP and the key multiple myeloma survival protein Mcl-1.. TG02 is a promising new antimyeloma agent that is currently in phase I clinical trials in leukemia and multiple myeloma patients.

Topics: Animals; Blotting, Western; Boronic Acids; Bortezomib; CDC2 Protein Kinase; Cell Cycle; Cell Line, Tumor; Cell Survival; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 9; Cyclin-Dependent Kinase-Activating Kinase; Cyclin-Dependent Kinases; Dose-Response Relationship, Drug; Drug Synergism; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Heterocyclic Compounds, 4 or More Rings; Humans; Lenalidomide; Mice, SCID; Mitogen-Activated Protein Kinase 7; Multiple Myeloma; Protein Kinase Inhibitors; Pyrazines; Reverse Transcriptase Polymerase Chain Reaction; Thalidomide; Xenograft Model Antitumor Assays