Unbiased, systematic surfaceome profiling of Acute Leukemia to identify novel immunotherapy targets.

UNLABELLED: Cell surface proteomics provides a direct topological assessment of the outer membrane of cells and enables the capture of low abundance proteins that may be missed by whole cell proteomics. Here we present an unbiased atlas of the whole cell and surface proteomes of 25 commonly used leukemic cell lines, encompassing both lymphoid and myeloid lineages, and a variety of driver mutations. Paired-wise analysis highlights recurrent surface proteins that are not detected by whole cell proteomics. Coupling this dataset to RNA-sequencing, we also discovered genes where protein and RNA abundances are discordant. In KMT2A -rearranged AML, CD70 expression was increased across cell lines and validated in primary patient samples, supporting CD70 as a candidate therapeutic target in this disease. Several proteins are enriched in the surface proteomes but lack surface annotation, adding to the growing list of potential non-canonical cell surface proteins. These findings reveal a substantial pool of proteins absent from conventional surface annotations, including RNA-binding proteins, an emerging class of candidate immunotherapeutic targets. KEY POINTS: Direct surface proteomics identifies leukemia cell-surface proteins not reliably predicted by transcriptomic or whole-proteome profilingSurface profiling reveals genotype-specific therapeutic targets, including CD70 in KMT2A-rearranged AML.