Poster Presentation 25th Annual Lorne Proteomics Symposium 2020

Unravelling the biological mechanisms of Acute Lymphoblastic Leukemia (ALL) drug-resistance with integrated multi-glycoconjugate analysis (#135)

Tiago Oliveira 1 , Andreia Filipa Ferreira de Almeida 1 , Kathirvel Alagesan 1 , Arun Everest-Dass 1 , Francis Jacob 2 , Maria Kavallaris 3 , Nicolle Packer 1 4 5 , Nora Heisterkamp 6 , Mark von Itzstein 1 , Daniel Kolarich 1 5
  1. Institute for Glycomics - Griffith University, Southport, QLD, Australia
  2. Glyco-Oncology, Ovarian Cancer Research, Department of Biomedicine, University Hospital Basel and University of Basel, Basel, Switzerland
  3. Children's Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia
  4. Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia
  5. ARC Centre for Nanoscale BioPhotonics, Macquarie University and Griffith University, North Ryde and Gold Coast, NSW and QLD, Australia
  6. Department of Systems Biology, Beckman Research Institute City of Hope, Monrovia, CA, USA

Acute Lymphoblastic Leukemias (ALL) are the most common type of children cancer diagnosed worldwide. Although ALL’s long-term survival rates have increased over the past 40 years, approximately 15% of infant and more than 50% of adult patients eventually relapse after a short remission period1. The biological mechanisms resulting in ALL's drug resistance, however, are still not fully understood. Changes in glycosylation have previously been linked to ALL drug resistance2, and the pharmacological inhibition of glycan‑binding proteins such as Galectin-1 sensitized ALL cells to the action of chemotherapeutical drugs3. In summary, glycosylation appears to play a relevant role in ALL pathogenesis and drug resistance.

In our quest to capture a comprehensive picture of the cell surface glycosylation, we devised a strategy to determine the glycolipidome, the N- and O-glycome as well as the proteome and glycoproteome, ideally from a single batch of starting material. After triphasic extraction using a Methanol:Chloroform:Water mixture, the glycan component of glycolipids and glycoproteins are enzymatically and chemically released from the respective fractions and analysed using porous graphitized carbon (PGC) nanoLC‑Electrospray tandem mass spectrometry (ESI‑MS/MS). The glycoproteome is determined after tryptic digestion and Hydrophilic Interaction Chromatography (HILIC) fractionation on microcrystalline cellulose prior analysis by reverse phased nanoLC‑ESI-MS/MS on an Orbitrap Fusion.

The current workflow was used to analyse CCRF-CEM T-ALL cell lines with different levels of 13-desoxyepothilone B (dEpoB) resistance, as well as primary ALL patient samples. Preliminary analysis allowed the identification of more 130 different proteins exclusively present on the dEpoB resistant CCRF-CEM cell lines, a tenth of which are glycoproteins. Moreover, 300 different proteins were present in ALL patient samples but not present in any of the normal bone marrow (BM) control cells. Also, ALL patients exhibited a distinct N- and O-glycan fingerprint when comparing to normal BM samples. Ultimately, the aim is to provide novel opportunities to tackle therapy‑resistance in ALL by mining unique molecular markers and identify drug resistance-associated pathways.

  1. Terwilliger, T. & Abdul-Hay, M. Acute lymphoblastic leukemia: a comprehensive review and 2017 update. Blood Cancer J 7, e577 (2017).
  2. Nakano, M., Saldanha, R., Gobel, A., Kavallaris, M. & Packer, N. H. Identification of Glycan Structure Alterations on Cell Membrane Proteins in Desoxyepothilone B Resistant Leukemia Cells. Molecular & Cellular Proteomics 10 (2011).
  3. Paz, H. et al. Treatment of B-cell precursor acute lymphoblastic leukemia with the Galectin-1 inhibitor PTX008. J Exp Clin Cancer Res 37, 67 (2018).