Multi-omics analyses of Mixed-lineage Acute Leukemia (MLL) focusing on the importance of glycosylation-a pilot study

Mixed-lineage Acute Leukemia (MLL) is one of the most high-risk forms of pediatric cancer. Although the long-term survival rates of pediatric acute lymphoblastic leukemia have increased over the past 40 years, the current chemotherapeutic treatment schemes often fail in the treatment of MLL. The biological mechanisms resulting in drug resistance, however, are still not fully understood and there is an urgent need to identify novel diagnostic and therapeutical targets. We have established the first integrated multi-omics investigation of primary patient MLL samples and control precursor B bonemarrow cells from healthy donors, mapping their proteome, transcriptome and glycome. 4–6 million cells from 3 normal bone marrow (BM) and 2 MLL samples were used for analysis on our multi-omics platform. Porous-Graphitised Carbon (PGC) nanoLC-ESIMS/MS was used for Glycomics analyses after the enzymatic and chemical release of N- and O-glycans, respectively. The proteome was explored using RP-LC-ESI-MS/MS analyses, performed after offline high-pH fractionation, in addition to RNA-seq analyses. Overall, 4225 proteins were identified across the patient MLL and control BM cells, of which 216 were overexpressed in MLL (p 2). Preliminary analyses of the RNA-seq data matched well with the proteomics findings, revealing significant alterations in expression of glycoprotein signalling receptors and extracellular matrix proteins as well as various transcription factors. High-pH fractionation also allowed us to identify numerous important glycosyltransferases and expression changes thereof on protein and RNA– seq level. N- and O-glycomics revealed overall lower levels of α2–6 sialylated N- and O-glycans in MLL cells (correlating with ST6Gal1 transcript and ST6Gal1 protein levels) as well as an increase in Core 2 type O-glycans (correlating with GCNT1 transcript level). Patient MLL cells exhibit distinct N- and O-glycan fingerprints, along with specific alterations of receptor tyrosine kinase signalling networks. In addition to wellknown MLL glycoprotein markers, our integrated multiomics workflow identified a number of diagnostic/therapeutic protein candidates that have not previously been described.