Acute leukemias, such as acute lymphoblastic leukemia (ALL), are aggressive cancers characterized by the rapid proliferation of malignant hematopoietic cells. Throughout the last 60 years, childhood ALL’s long-term survival rates increased from less than 10% to more than 90%. Despite these improvements, certain subtypes remain hard to manage (e.g. mixed lineage leukemia, MLL-r), and even new therapies frequently fail. Therefore, identifying leukemia-cell restricted antigens in these ALL subtypes remains crucial in the quest to develop novel diagnostic tools and specific treatments. Traditionally, ALL research has mostly been focusing on genomics and transcriptomics efforts, mainly due to the limited amounts of patient material, and technical difficulties throughout sample processing. The potential encompassed in the use of other -omics technologies has remained unexplored in the context of ALL. For the work presented in this thesis, I have focused on undertaking the first comprehensive characterisation of glycocalyx alterations that occur in ALL and particularly in MLL-r in primary, patient derived cancer cells. This work supports the concept that the glycocalyx of ALL and MLL-r cells undergoes dramatic alterations that clearly differentiate these cells from healthy precursor B- (pre-B) cells. These findings might open doors towards novel potential diagnostic and therapeutic targets. The studies encompassed in chapters III, IV and V were performed in collaboration with Prof. Eleonora Heisterkamp’s team at the Beckman Research Institute (City of Hope, CA, USA). I have performed the first multi-omics analyses of primary patient MLL-r cells by integrating data from the transcriptome, glycome, and proteome of these cells. These results revealed that MLL-r cells exhibit distinct glycosylation features that differentiate them from healthy pre-B cells, which I was able to correlate with alterations at the transcript level of relevant glycosyltransferases. In depth proteome analyses revealed an overall good correlation between proteomics and transcriptomics findings, but also uncovered numerous examples where significant changes were just found in one but not the other approach. Nevertheless, this integrated approach used for the systematic evaluation of MLL-r allowed to obtain significant data for putative novel diagnostic/therapeutic protein markers and revealed important features of the disease that remained elusive until now. I was also able to apply the developed integrated multi-omics workflow to investigate the protective role of the surrounding microenvironment and its impact in environmentmediated drug resistance (EMDR) of pre-B ALL cells, which remains a major obstacle for the efficacy of chemotherapeutics in patients. To date the relevance of glycoconjugates for the development of EMDR has been largely unexplored. I explored a long-term co-culture system using human pre-B ALL cells and mitotically inactivated supporting murine stromal cells (OP9 cells), where pre-B ALL cells were put under a selective pressure to survive in the presence of vincristine, a widely used chemotherapeutic drug. I have performed a multi-omics analyses to understand the effect vincristine-resistance has on the cells' glycocalyx. These results demonstrated both glycome-wide and glycoprotein site-specific alterations, which could potentially be employed to identify emerging drug-resistance at an earlier stage or possibly serve as treatment targets in pre-B ALL. Throughout these studies, I have observed a significant modulation of the sialylation profile on pre-B ALL cells in patients and during EMDR development. Unsurprisingly, changes in sialylation have frequently been linked with development and progression of many cancer types but remain largely unexplored in the context of pre-B ALL. I investigated the impact of the major sialyltransferase, ST6Gal1, on the glycome of pre-B ALL cells. ST6Gal1 is the transferase known to be largely responsible for attaching sialic acids in an a2-6 linkage onto N-glycans. Surprisingly, these results demonstrated that a ST6GAL1 knockout did not ablate the production of a2-6 sialylated N-glycans, unless these N-glycans carried a core fucose residue. Demonstrating for the first time how core-fucosylation regulates a2-6 sialylation also allowed me to unravel the existence of ST6Gal1 independent, alternative a2-6 sialylation pathways that are specific for non-fucosylated N-glycans. I demonstrated that ST6GalNAc3-6 are capable to produce a2-6 sialylated N-glycans in the absence of core-fucose and that ST6Gal1 is required to introduce a2-6 sialylation on corefucosylated N-glycans. These results challenge long standing dogmas in glycobiology while delivering a novel understanding of hitherto unknown mechanism that regulate protein glycosylation, which will have a significant impact on our understanding of glycosylation changes in health and disease.