A novel kinome-screen method to identify drug targets in MPN

Kinases are the class of proteins most often associated with cancer. The elucidation of kinases contributing to disease maintenance promises the identification of potential drug targets. The activity of most kinase inhibitors correlates with biochemical parameters that can be measured in vitro. Knowledge of these parameters allows the analysis of off-target effects of kinase inhibitors on each of the 518 known kinases of the human kinome. To date, the off-targets (often referred to as noise) have been a huge disadvantage for every kinase-inhibitor based drug-target screen. I intend to develop a novel, cost- and time-saving method to screen for essential/addicted kinases in cancer cells by making use of the noise. I propose to use the known off-target effects of 202 kinase inhibitors on 300 kinases to develop an algorithm that will output a list of 300 kinases ranked according to their importance for the cells survival/proliferation. The reliability of the method will be tested on several human cancer cell lines with known kinase addiction as well as on Ba/F3 cells expressing and depending on distinct human kinases. When using this algorithm on a preliminary kinome screen of 19 human cell lines which have been reported to be addicted to a total of 7 distinct kinases (ABL1, JAK2, JAK3, FLT3, ALK, ErbB2, PDGFRa) the correct addicted kinase was ranked in average at position 2.3 out of 300 kinases. Since the presented method does not depend on specific inhibitors and can be further extended by including additional inhibitors with known off-target profiles, it is a promising alternative to existing screening methods. The myeloproliferative neoplasias (MPN) comprise several clonal hematologic diseases thought to arise from a transformation in a hematopoietic stem cell. The main clinical features of the diseases are the overproduction of mature, functional blood cells and a long clinical course. Although improving MPN-associated phenotypes and slightly prolonging life-span, clinical trials of JAK2 inhibitors have not met the high expectations. My screeinig-method will be applied to JAK2V617F+ MPN cell lines with the aim of identifying one or more kinases essential for the proliferation and/or survival of JAK2V617F+ cells thereby uncovering additional drug targets. Preliminary screening data on five distinct JAK2V617F dependent cell lines indicate that the p21-activated kinase 1 (PAK1) is an essential kinase in MPN. I will validate my top-ranked candidates by pharmacologic inhibition and shRNA mediated knock-down experiments, studies on primary MPN patient samples and MPN mouse models. Back in Vienna at the CeMM I intend to apply the acquired knowledge and newly established method to screen for potential kinase drug targets other than BCR-ABL1 in CML.

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