The multi‐kinase inhibitor TG02 overcomes signalling activation by survival factors to deplete MCL1 and XIAP and induce cell death in primary acute myeloid leukaemia cells

Abstract

SummmaryThe novel multi-kinase inhibitor TG02 has selectivity against cell cycle and transcriptional cyclin dependent kinases (CDKs) as well as fms-like tyrosine kinase receptor-3 (FLT3). Inhibition of transcriptional CDKs preferentially depletes short-lived proteins such as MCL1. We evaluated the in vitro toxicity of TG02 to primary acute myeloid leukaemia (AML) cells in the presence of survival signalling pathway activation by cytokines and fibronectin. One hundred nanomolar TG02 induced a median decrease of 40% in bulk cell survival and 43% in the CD34 + CD38 À CD123 + subset. A 90% inhibitory concentration of 500 nmol/l indicated that TG02 toxicity is not halted by protective cell cycle arrest. Samples with FLT3 internal tandem duplication were not preferentially targeted. By flow cytometry, TG02 treatment caused loss of RNA Polymerase II serine 2 phosphorylation in patient samples, which correlated strongly with BAX activation (R 2 =0Á89), suggesting these as potential biomarkers for clinical studies. MCL1 and XIAP expression also decreased. Repeated brief exposure to TG02 in MOLM-13 cells did not result in compensatory up-regulation of survival protein expression. In conclusion, TG02 is potently cytotoxic towards CD34 + CD38À CD123 + and bulk AML cells, despite protective signalling pathway activation. This antitumour activity is most likely mediated by dephosphorylation of RNA Polymerase II leading to depletion of survival molecules such as MCL1 and XIAP, with subsequent BAX activation and apoptosis.

Complementary dynamic BH3 profiles predict co-operativity between the multi-kinase inhibitor TG02 and the BH3 mimetic ABT-199 in acute myeloid leukaemia cells

“…We showed that using either agent at 100 nM was overall less effective than the combination of 50 nM of each drug (Figure 5C). Another point of note is that whereas the toxicity of ABT-199 reaches a plateau at around 25 nM, the toxicity of TG02 continues to increase, and the majority of cells are eradicated at 100 nM, as previously reported [17]. …”

“…In this simplified scenario, TG02 as well as endogenous NOXA suppress the pro-survival function of MCL-1 and maybe of additional labile BCL-2 family pro-survival proteins (LBPP) [17, 20, 40]. ABT-199 and endogenous BAD suppress the pro-survival function of BCL-2 [14, 20, 40].…”

“…TG02 is a novel multi-kinase inhibitor which exerts greatest activity against the cyclin dependent kinase CDK9 (IC 50 3nM) [16]. CDK9 is permissive for transcription by phosphorylation of RNA polymerase II on serine 2 (RPIIS2), and in AML cells TG02 treatment causes rapid RPIIS2 dephosphorylation, such that RNA synthesis is strongly inhibited [16–18] and proteins with a short half-life, such as MCL-1, are rapidly downregulated [16, 17]. Our findings indicate the ability of TG02 to co-operate with BAD, the ability of ABT-199 to co-operate with NOXA and the ability of the two chemotherapeutic agents to co-operate with each other to induce cytochrome C release and apoptosis in AML cells.…”

Dual inhibition of Mcl-1 by the combination of carfilzomib and TG02 in multiple myeloma

“…TG02 is a pyrimidine-based multi-kinase inhibitor that has been shown to target CDKs, JAK2 and FLT3. [31][32][33] More interestingly, TG02 has been shown to increase Bax activation and decrease expression levels of anti-apoptotic protein Mcl-1. 33 Therefore, the combination of carfilzomib and TG02 would allow us to target Mcl-1 via 2 different mechanisms.…”

Mass Cytometry: A Highly Multiplexed Single-Cell Technology for Advancing Drug Development

“…Cell Signaling State Characterization. Antigen-or cytokineinduced phospho-signaling pathways (Hale and Nolan, 2006;Perez and Nolan, 2006) have been used as sensitive and early markers of cell activation in clinical studies to characterize cancers (Lim et al, 2012), measure intended targets (Strumberg et al, 2005;Crump et al, 2010;Brandwein et al, 2011;Pallis et al, 2012), and assess the off-target effects of drugs (Powers et al, 2011). However, it is increasingly recognized that a full appreciation of the nature and extent of cellular activation can be best assessed by simultaneously mapping several intersecting signaling pathways in single cells (Bonilla et al, 2008).…”