Antimyeloma Action of the Multikinase Inhibitor TG02

Abstract 4062

Multiple myeloma (MM) is the second most common hematologic malignancy and remains incurable, despite recent advances in its treatment. Therefore, studies to develop new therapies are still necessary. MM is characterized by genetic instability with numerous chromosomal abnormalities and deregulation of several signalling pathways. Cell cycle control is also dysfunctional in this pathology, and altered functioning of cyclins has been indicated as a universal alteration in MM. Cyclins exert their action by controlling the activity of the cyclin-dependent kinases (CDKs). Therefore, CDKs represent promising novel therapeutic targets for MM. Several other kinases have important functions in MM, one of which - BMK or ERK5 - plays a key role in the proliferation and survival of the myelomatous clone. In this study we describe the preclinical activity of a novel multi-kinase inhibitor, TG02, in MM. This compound combines cell cycle regulatory and transcriptional CDK inhibition with activity against other oncogenic kinases, such as ERK5, JAK2, TYK2, FLT3 and TYRO3. Functional potency was assessed using MTT-based proliferation assays in a panel of MM cell lines. Maximal cytotoxicity was observed in the MM1S cell line with an IC₅₀ value after 24 hours of 64 nM. There were cell lines with a more resistant phenotype, like OPM2, with an IC₅₀ value after 24 hours of 4.7 μ M (although this was reduced to low nM by prolonged exposure). TG02 also inhibited the protective effect of the bone marrow microenvironment as it was able to induce growth inhibition of MM cells grown in direct coculture with bone marrow stromal cells (BMSC). To delineate the underlying mechanism of cytotoxicity induced by this compound, we performed cell cycle and apoptosis assays. There was a slight accumulation of cells in the G2-M phase of the cell cycle but the more marked effect was an increase in Annexin V-staining, indicative of apoptosis, which was detected as early as six hours after treatment of MM1S cells with 100 nM TG02. This process was associated with depletion of the anti-apoptotic proteins Mcl-1 and Bcl-XL, and cleavage of caspases-8,-9,-3 and -7 and PARP. Treatment with TG02 also induced a fall in mitochondrial membrane potential and the release of cytochrome c and AIF into the cytosol. We have also analyzed some substrates of the different kinases that TG02 targets, such as Rb (phosphorylated at Ser-807 by CDK2) and RNA pol II (phosphorylated by CDK9). TG02 dephosphorylated these two proteins in MM1S cells. TG02 was tested for in vivo anti-tumor activity using the human OPM2 MM xenograft model. In this model we observed that TG02 was able to decrease tumor volumes, and synergized with bortezomib. In conclusion, these results show a potent anti-myeloma activity of the multi-kinase inhibitor TG02, and supports additional preclinical studies together with its clinical development for the treatment of MM.