Pb1790: looking ‘beneath’ the surface: proteomics and glyciproteomics reveal novel targets for car-t cell therapies in acute myeloid leukemia (aml)

Topic: 3. Acute myeloid leukemia - Biology & Translational Research Background: Acute myeloid leukemia (AML) is a particularly aggressive age-related malignancy characterized by abnormal hematopoietic differentiation and aberrant epigenetic driver mutations. Patients treated with the standard of care experience low response rates, high levels of relapse and an overall poor prognosis due to resistance. Chimeric antigen receptor (CAR) T cells directed towards cell surface proteins are being explored in AML but have shown limited success due to the expression of targeted proteins in healthy hematopoietic cells. To expose novel proteins in AML, a deeper annotation of the cell surface proteome is warranted. However, methods to enrich the cell surface proteome have not yet been translated to patient AML samples. Aims: Here, we looked deeper into the total proteome and further developed a method for the isolation of the cell surface proteome of AML patient samples using glycoproteomics. The human glycoproteome is known to have immunomodulating roles in cancer and therefore presents a promising group of proteins for therapeutic targeting. The upregulation of identified cell surface proteins in AML patients compared to healthy donors has been verified and we aim to generate target-specific antibody fragments (scFvs) for development of CAR-T cells. Methods: First, we performed LC/LC-MS mass spectrometry on the total proteome of 10 AML patients and 4 cell lines. Thereafter, LC/LC-MS mass spectrometry with TMT labelling was performed on isolated glycoproteomes from 4 cell lines and 7 AML patients. The expression of identified proteins was confirmed by mapping to the total proteome of AML patients and healthy donors (HD_CD34+ and HD_Lin-, Kramer et al. 2022) as well as the total proteome of AML patient leukemic stem cells (LSCs) and non-LSCs (Raffel et al. 2016). Results: We quantified a total of > 8,000 proteins from the total AML patient proteome including common lineage-restricted antigens (CD38, CD33, CD123, CLEC12A, CD180 and MRC1) and leukemia-associated antigens (CD96, CD44, CD70 and WT1). Using glycoproteomics, we successfully quantified up to 860 glycoproteins in adherent and suspension cell lines using only 10×106cells which is 5-fold lower than current methods. By implementing this method for AML patient samples, we reveal further novel targets (undisclosed) from the AML glycoproteome. Summary/Conclusion: We have performed a detailed characterization of the total AML patient proteome and developed a method for the enrichment of cell surface proteins which has not yet been done in AML patients. We identified known cell surface proteins upregulated in AML and LSCs compared to healthy donors and discovered novel undisclosed targets in AML from the patient glycoproteome. Using our methods, we have the potential to further expand the classes of therapeutic targets in AML beyond classical membrane proteins. Keywords: Acute myeloid leukemia, Targeted therapy, CAR-T, Proteomics