Unpacking the Phosphoproteome: towards clinical application of phosphoproteomics in acute myeloid leukemia

Due to the importance of RTKs in AML leukemogenesis and the current challenges in improving AML patient survival with KI treatment, phosphoproteomics may provide an additional layer of information of functional relevance to bridge the gap between a patient’s mutational status and their variable response to KI treatment. Therefore, the aim of this thesis is to explore the potential of pY phosphoproteomics in guiding AML patient selection for treatment with KIs. Chapter 2 focusses on the evaluation of the Integrated Inferred Kinase Activity Analysis, INKA. The utility of INKA is shown in multiple settings relevant for cancer research; (i) cancer cell lines with known oncogenes, (ii) cell lines in a differential setting (wild-type versus mutant, +/- drug), (iii) pre- and on-treatment tumor needle biopsies, (iv) cancer cell panel with available drug sensitivity data, and (v) patient-derived tumor xenografts with INKA-guided drug selection and testing. To expand on previous pY phosphoproteomics experiments, chapter 3 investigates tyrosine kinase signaling in 16 AML cell lines, using INKA in combination with functional testing, to evaluate the potential for individualized identification of signaling pathways and active driver kinases in the context of AML. Validation using KIs is further correlated with target kinase INKA activation metrics. In chapter 4 we explore the impact of delay of mononuclear cell isolation on the phosphorylation profiles of four clinical AML samples. Here we provide data for a range of time points and evaluate their effect on the stability of the phosphoproteome. Based on the results we formulate a recommendation as to the time frame within which samples should be processed. In chapter 5 we explore the AML phosphoproteome in 35 clinical samples, including both FLT3-ITD mutant- and FLT3WT samples in relation to their in-vitro response to two FLT3 inhibitors, midostaurin and gilteritinib. Data is provided on differential phosphorylation between FLT3-WT and FLT3-ITD samples and drug responders vs. non-responders. The prime candidate for drug resistance in these samples is tested as a combination therapy. The general discussion places our findings in the light of current literature and gives a view on future perspectives and next steps towards application of phosphoproteomics into clinical practice.