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Resistance to the BCR-ABL tyrosine kinase inhibitor imatinib poses a pressing challenge in treating chronic myeloid leukemia (CML). This resistance is often caused by point mutations in the ABL kinase domain or by overexpression of LYN. The second-generation BCR-ABL inhibitor INNO-406 is known to inhibit most BCR-ABL mutants and LYN efficiently. Knowledge of its full target spectrum would provide the molecular basis for potential side effects or suggest novel therapeutic applications and possible combination therapies. We have performed an unbiased chemical proteomics native target profile of INNO-406 in CML cells combined with functional assays using 272 recombinant kinases thereby identifying several new INNO-406 targets. These include the kinases ZAK, DDR1/2 and various ephrin receptors. The oxidoreductase NQO2, inhibited by both imatinib and nilotinib, is not a relevant target of INNO-406. Overall, INNO-406 has an improved activity over imatinib but a slightly broader target profile than both imatinib and nilotinib. In contrast to dasatinib and bosutinib, INNO-406 does not inhibit all SRC kinases and most TEC family kinases and is therefore expected to elicit fewer side effects. Altogether, these properties may make INNO-406 a valuable component in the drug arsenal against CML. [ABSTRACT FROM AUTHOR]

Introduction: The first line of defense against invading pathogens is the innate immune system. Recognition of an intruder results in transcriptional up-regulation of several genes, including cytokines. The production of these potent immune mediators needs to be tightly monitored as aberrant cytokine expression results in tissue damage or development of autoimmune diseases. It becomes increasingly evident that post-transcriptional regulation of gene expression also strongly impacts on cytokine production, especially during the resolution phase of the immune response. One means to post-transcriptionally control cytokine expression is by modulating the stability of their mRNA via adenosine/uridine (AU)-rich elements (ARE), which are found within the 3’untranslated region (UTR) of several cytokine mRNAs. Although several ARE-interacting proteins (ARE-BPs) have been described, it is not clear how they regulate individual AREs of distinct cytokine mRNAs during the immune response. Methods: Here we propose to use affinity purification coupled to mass spectrometry to analyze the dynamic interactions of selected cytokine ARE elements with ARE-BPs during an immune response. We chose to study the human interferon-beta (IFN-b) mRNA, which contains two instability determining sequences, an ARE in the 3’UTR and a coding region instability element (CIRD) within the open reading frame. We used 3’biotinylated IFN-b ARE RNA oligomers as affinity reagent to isolate ARE-BPs from total cell extracts of mock-treated or polyinosinic:polycytidylic acid (p(I:C)) stimulated cells. Isolated interacting proteins were analyzed via 1D shot gun mass spectrometry analysis. Results: We could identify the human antigen R (HuR) as the most prominent IFN-b ARE binding protein. Previous studies have pointed to a role for HuR mRNA binding in antagonizing the rapid decay of some ARE-containing mRNAs, which correlates with reports of IFN-b message stabilization during the immune response. Conclusion: The stability of cytokine mRNAs is highly regulated, as deregulation of cytokine production can have detrimental effects. Our work will shed new light on the dynamic interactions of cytokine ARE elements and their ARE-BPs during an immune response. [Copyright &y& Elsevier]