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.

In 2005, a single recurrent gain-of-function mutation in the JAK2 tyrosine kinase (JAK2V617F) was discovered in the majority of patients with the blood disease myeloproliferative neoplasms (MPNs), with this discovery allowing precise diagnosis and catalysing the development of several therapeutic JAK2 inhibitors. In 2013, somatic CALR mutations were identified in most JAK2-unmutated patients. To identify kinases important for survival of CALR-mutant cells, I developed a novel method (KISMET) that utilizes the full range of kinase selectivity data available from each inhibitor and thus takes advantage of off- target noise that limits conventional small-interfering RNA or inhibitor screens. KISMET successfully identified known essential kinases in haematopoietic and non- haematopoietic cell lines and identified the mitogen activated protein kinase (MAPK) pathway as required for growth of the CALR-mutated MARIMO cells. Expression of mutant CALR in murine or human haematopoietic cell lines was accompanied by myeloproliferative leukemia protein (MPL)-dependent activation of MAPK signaling, and MPN patients with CALR mutations showed increased MAPK activity in CD34 cells, platelets and megakaryocytes. Although CALR mutations resulted in protein instability and proteosomal degradation, mutant CALR was able to enhance megakaryopoiesis and pro-platelet production from human CD34+ progenitors. These data link aberrant MAPK activation to the MPN phenotype and identify it as a potential therapeutic target in CALR-mutant positive MPNs. In collaboration with the group of David Ron we found that mutant CALR accumulates in the nucleus (compared to wild type CALR that is mainly located in the cytoplasm). This was a highly unexpected and controversial finding and the work on this topic is still ongoing. Deregulation of the JAK/STAT signaling pathway is found in cancer with the transcription factor STAT5AB controlling leukemic cell survival and disease progression. As mutations in STAT5B, but not STAT5A have been frequently described in hematopoietic tumors, I used BCR/ABL as model systems to investigate the contribution of STAT5A or STAT5B for leukemogenesis. The aim was to identify essential pathways driven by either STAT5A or STAT5B and thereby identifying new therapeutic approaches for chronic myeloid leukemia (CML). RNA-seq profiling revealed a marked enhancement of IFN-alpha and IFN-gamma signatures in Stat5b-/- cells. Inhibition of interferon responses rescued BCR/ABL+ colony formation of Stat5b-/- cells.