Abstract 2808: Mechanism of action of the multikinase inhibitor TG02 in chronic lymphocytic leukemia
Authors: Chen Rong , Tsai Jennifer , Chen Yuling , Burrows Francis , Wierda William , Plunkett William 
Published: August 2012

Abstract
TG02 is small molecular multikinase inhibitor with a unique inhibition spectrum of therapeutic targets. It potently inhibits Cdk9 and Cdk7, the Cdks involved in transcription regulation, with IC50 values of 3 nM and 37 nM, respectively. Cdk1 and 2 are inhibited at IC50s of 9 and 5 nM. Kinases in other signaling pathways are also targets of TG02, such as JAK2 and FLT3 (IC50 19 nM for both). It is currently in Phase I clinical trials in hematological malignancies based on its unique target profile and the encouraging preclinical results. In this study, we investigated its activity in an indolent tumor type: chronic lymphocytic leukemia (CLL). CLL is characterized by the dependence on the continuous expression of anti-apoptotic proteins for survival. Our previous studies showed that inhibiting Cdk9 and Cdk7 blocked the activity of RNA polymerase II (RNA Pol II), which resulted in the depletion of the short-lived anti-apoptotic protein Mcl-1 and induction of apoptosis in primary CLL cells in vitro. This action was confirmed during clinical trials of Cdk9 inhibitors. In this study, we first confirmed the activity of TG02 as a Cdk7/9 inhibitor. Our results showed that the phosphorylation of Ser2 sites (Cdk9 substrate) of RNA Pol II C-terminal domain repeat was significantly reduced at 4 hr by 0.5 μM TG02. Greater concentrations were required to inhibit the phosphorylation at Ser5 sites (Cdk7 substrate), consistent with a higher IC50 for Cdk7. These actions led to a decrease in RNA Pol II mediated transcription, measured by tritiated uridine incorporation. Mcl-1 expression was reduced rapidly at both the mRNA and protein levels. This was associated with loss of mitochondrial membrane potential and induction of apoptosis. To differentiate the targets that contribute to the induction of apoptosis, toxicity of TG02 was compared to SNS-032 (Cdk9 inhibitor), Sunitinib (FLT3 inhibitor) and TG-101348 (JAK2 inhibitor). The pattern of TG-02 induced cell death was similar to that of SNS-032, whereas sunitinib induced rapid necrosis, and TG-101348 was much less potent. Thus, this data indicated that inhibition of Cdk9 played a major role for the toxicity of TG02 in CLL. While in these experiments CLL cells were cultured in RPMI media supplemented with 10% autologous plasma to mimic the in vivo conditions, our experiments showed that human plasma proteins impact on the potency of TG02. Compared to 10% FBS, the IC50 for cell death at 24 h was about 4-fold and 18-fold greater in 10% and 50% human plasma, respectively, indicating a substantial plasma protein binding for TG02. Finally, BAX/BAK double knockout MEF cells were resistant to TG02, suggesting that TG02 requires BAX/BAK to induce apoptosis, consistent with a mitochondrial pathway of cell death. Taken together, these studies suggested that among the multiple targets of TG02, inhibition of Cdk7/9 and transcriptional down-regulation of Mcl-1 was the major cytotoxic pathway activated by TG02 in CLL.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2808. doi:1538-7445.AM2012-2808