Easyab Unravels Oncokinase Driven Regulation of Splicing Factors in AML
Authors: Jayavelu Ashok Kumar , Kuppusamy Maithreyan , Mokada Gopal Lavanya , Frenz Joris , Mann Matthias , Jung Noelle , Kulozik Andreas 
Published: December 2023

Acute myeloid leukemia (AML) is the most commonly diagnosed leukemia in adults. Gene mutations involved in various cellular functions are known to cause AML. A large proportion of AML is driven by aberrant activation of kinases and signal transducers such as FLT3, cKIT, PTPN11, JAK2, KRAS, NRAS, and to a lesser extent, JAK1, JAK3, and CSF3R. Treatment for AML has remained the same in the past five decades. Recent developments led to the availability of targeted therapies and enabled the practice of additional drugs like tyrosine kinase inhibitors. However, drug resistance and tolerance mechanism remain a challenge to finding a cure for the disease. Though we can identify and target kinase mutations individually, we have yet to learn how these mutations, like FLT3-ITD, change the cell's intrinsic signalling environment and cooperate with other oncogenes. Understanding these signalling behaviors is crucial for effective treatment outcomes and avoiding the development of drug resistance and tolerance mechanisms.

Protein post-translational modifications (PTM) such as phosphorylation, ubiquitination, methylation, control the activity, fate, and spatial location of the proteins, thereby controlling processes such as signal transduction. Mass spectrometry-based proteomics offers unbiased profiling of PTMs. However, comprehensive analyses of PTMs require conventional antibody-based enrichment approaches and large starting materials, laborious, low throughput, cost prohibitive, and time-consuming, thus limiting the application to cell line-based studies. It also limits application in exploring unbiased-global cell signaling in patient samples. To overcome these limitations, we developed a loss-less sample preparation method named ‘EasyAb’. We show that our short, high throughput and low-cost approach enables capturing PTM peptides from very small inputs.

As proof of principle, we enriched tyrosine-phosphorylated peptides from Molm13 and MV4-11 cell lines expressing FLT3-ITD kinase. Enrichment resulted in the ultra-deep identification of 4,416 tyrosine phosphosites, double the number of sites found in the conventional protocols, and took only one day of processing. About 40 % of the tyrosine phosphorylated proteins enriched are membrane proteins, including seven tyrosine phosphorylation sites on FLT3 kinase, of which FLT3 pY401 is a novel site discovered.

To test the applicability of the EasyAb protocol in patient samples, we performed phosphotyrosine profiling of AML samples expressing FLT3 WT (n=9) and FLT3-ITD (n=5) mutation. EasyAb enrichment for phosphotyrosine peptides resulted in identifying 6,339 phosphotyrosine sites, of which 2300 sites were quantified. Comparing FLT3WT and ITD samples revealed more than 500 phosphosites significantly regulated. The significantly changing sites include several known signalling molecules such as STAT5, SYK, BTK, GAB2, GRB2, and PTPN11. The data also revealed differential tyrosine phosphorylation of proteins involved in various cellular functions such as autophagy, apoptosis, cell cycle, DNA damage repair proteins, phosphatases, and kinases.

Interestingly, we also uncovered a class of proteins belonging to mRNA processing and splicing pathways distinctly tyrosine phosphorylated in FLT3-ITD samples. It is known that splicing factor mutations drive leukemia. Conversely, little is known about the regulatory mechanism of splicing factors by singling in cancer. To further investigate FLT3-ITD signalling regulation of splicing factors, we treated Molm13 and MV4-11 cells with Quizartinib and profiled tyrosine phosphorylation. Inhibition of FLT3-ITD lead to downregulation in tyrosine phosphorylation of several splicing factors. We have previously reported that splicing factor phosphorylation by JAK2 kinase plays a crucial role in Jak inhibitor persistence phenotype. Importantly, FLT3-ITD-mediated tyrosine phosphorylation-based regulation of splicing factors is unknown, and this process could be crucial for FLT3-ITD-mediated tumorigenesis. The functional significance of this novel regulatory mechanism is being studied. In conclusion, we have developed an effective tool for studying post-translational modifications that require antibody-based enrichment (tyrosine phosphorylation, ubiquitination, methylation, acetylation, and sumoylation) relatively quickly in primary patient samples.