by ecancer reporter Clare Sansom

The kinase inhibitor imatinib (Glivec™) has proved to be a landmark in the development of personalised medicine for cancer.

This drug is directed against the oncogene BCR-ABL, which is a constitutively active tyrosine kinase that is produced by the chromosome fusion characteristic of that disease known as the “Philadelphia chromosome”.

Prospects for patients with CBL have greatly improved since this drug was introduced into the clinic in 2001. However, the efficacy of the drug is very often compromised by the development of mutations in its target protein, particularly the so-called “gatekeeper mutation”, T315I.

One way of overcoming drug resistance that is very often effective is the simultaneous prescription of combinations of drugs.

The rationale for this approach has recently become more evident with studies of drug effects on the transcriptome and proteome that have shown that even apparently simple drugs affect proteins other than their intended targets. This implies that drugs with an appropriate range of “off-target” interactions might have a synergistic effect when prescribed together.

Although no currently marketed kinase inhibitor is effective against imatinib-resistant CML when used alone, it has been thought that efficacy might be improved by combining several inhibitors. Giulio Superti-Furga from the Center for Molecular Medicine, Vienna, Austria and his co-workers have now tested this hypothesis and discovered a strong synergy between the kinase inhibitors danusertib and bosutinib that is directed against CML cells bearing the “gatekeeper mutation” in BCR-ABL.

Superti-Furga and his colleagues first selected eight kinase inhibitors that are in clinical use for cancer, and tested each pair of drugs together against a BCR-ABL^T315I cell line.

They used the Bliss additivity model to evaluate whether each combination of drugs was more potent than expected. This screen highlighted a significant degree of synergy between danusertib and bosutinib, with the drug combination showing up to 40% more potentcy than was predicted by that model. Furthermore, synergy was also observed in other cells bearing this mutation, including some taken from the peripheral blood of a patient with advanced CML.

There was no synergy when the drugs were tested against cells bearing wild type BCR-ABL.

The researchers then used a chemical proteomics technique to determine the “off-target” effects of these two kinases that might lead to the observed synergy. They modified each of the eight kinases in the study to form an analogue that could be immobilised on sepharose beads without compromising its activity, and tested these against a panel of 68 human protein kinases. Danusertib was found to bind to 37 of the kinases, and bosutinib to forty.

The screen picked up a number of previously known interactions, such as the propensity of both bosutinib and dasatinib to bind to the Src and Csk kinases. More interestingly, a number of kinases implicated in the Mapk signalling pathway, including Mapk1 (Erk2), Map2k1 (Mek1), Map2k2 (Mek2), Map3k3 and Pyk2 (Ptk2b), were found to bind to either bosutinib or dasatinib but not to the other kinases.

Superti-Furga and his colleagues then analysed changes in global gene expression in BCR-ABL^T315I cells caused by administration of either bosutinib, dasatinib or the combination of both drugs. The effect of both drugs together on the transcriptome was much stronger than that of each drug alone; 645 genes were significantly upregulated and 584 significantly downregulated by danusertib and bosutinib together.

The set of genes downregulated by this drug combination contained many that are targets of the transcription factor c-Myc.

Further analysis suggested that the inhibition of kinases in the Mapk pathway causes a decrease in the activity of c-Myc. This result was validated when the cells were treated with small molecule inhibitors of Mapk kinases and with JQ1, a recently developed compound that downregulates c-Myc through inhibition of the protein BRD4, and a similar pattern of up- and downregulated genes was observed.

Taken together, these results suggest that the kinase inhibitors danusertib and bosutinib might prove a successful treatment for refractory, imatinib-resistant CML if prescribed together. More generally, this study proves that drug combinations may be effective through synergy between non-obvious “off target” interactions, and that the mechanism of action of such drug combinations can be elucidated through transcriptomics and proteomics. 

Reference

Winter, G.E., Rix, U., Carlson, S.M. and 11 others (2012). Systems-pharmacology dissection of a drug synergy in imatinib-resistant CML. Nature Chemical Biology, published online ahead of print 30 September 2012. doi: 10.1038/NChemBio.1085