Unbiased in-depth surfaceome profiling of Acute Myeloid Leukemia reveals novel immunotherapy targets beyond canonical membrane proteins Free

Background Despite recent therapeutic advances, 5-year survival for AML in adults remains ~35% (SEER estimate). Immunotherapies have shown limited efficacy in AML largely due to the lack of surface antigens that clearly distinguish malignant cells from normal hematopoietic counterparts. Conventional transcriptomics and proteomics have only found targets lacking clear distinction between AML, myeloid cells, and hematopoietic stem and progenitor cells (HSPCs). Here, we employed high-resolution surface proteomics of primary leukemia bone marrow aspirates to identify both tumor-enriched canonical surface proteins as well as a new class of “non-canonical” surface protein targets, i.e. typically intracellular proteins aberrantly presented on the AML cell surface.

Methods We used periodate-based cell surface-capture (CSC) and lysine-targeting (sulfo-NHS) labeling strategies, analyzed with both DDA and DIA proteomic methods, to profile surface proteins in newly diagnosed (n=35), relapsed (n=25), and remission (n=16) primary AML samples. An independent cohort (n=38) was used for target validation, with assessment in peripheral blood mononuclear cells and HSPCs for off-tumor expression. Plasma membrane localization of non-canonical targets was validated using flow cytometry and Western blotting. Interactors of canonical and non-canonical membrane proteins were evaluated through a recently-described multiscale photocatalytic proximity labeling strategy (Lin et al., Science (2024)). For promising AML-specific target DC-SIGN/CD209 we synthesized an MMAE-based antibody-drug conjugate (ADC).

Results Canonical cell surfaceome analysis revealed a distinctive expression of potential targets including CD209, CD300LB, LILRA1 amongst >150 significantly upregulated surface proteins in AML as compared to remission samples. Single-cell RNA-seq analysis of publicly available datasets supported AML specificity of these targets. Flow cytometry validated expression of CD209 across 5 cell line models and primary AML (n=15), with protein expression independent of genomic alterations. In non-canonical surfaceome analysis, we identified a preponderance of RNA binding proteins, consistent with our recent findings in other cell lines (Perr et al, Cell (2025)). However, we also identified many Golgi-, ER- and Mitochondrial-annotated proteins as possible AML-specific cell surface antigens given absence in remission sample surface proteomic data. Nine of these non-canonical surface antigens were confirmed to be surface-localized on AML cells by flow cytometry (n=13). Flow cytometry confirmed particularly strong expression of GORASP2 on the AML cell surface, whereas HSPC flow cytometry showed no expression of either CD209 or GORASP2. Further in silico analysis, Western blotting, and super-resolution imaging confirmed localized clusters of GORASP2, a canonical Golgi body protein, on the AML plasma membrane. Multiscale photocatalytic-based proximity labeling proteomics validated that GORASP2 interacted with a similar set of canonical membrane proteins as known surface antigens ITGB2 and CD209. An antibody-drug conjugate developed against CD209 demonstrated strong preclinical efficacy both in vitro and in vivo versus AML cell line models. The in vitro ADCcytotoxic assay targeting CD209 showed prominent efficacy with IC₅₀ ranging 1.5nM to 3nM in various AML cell lines. Ex vivo primary AML cytotoxicity assays showed killing in an antigen dependent manner. Treatment with CD209 ADC in Nomo-1 AML disseminated murine model suppressed tumor growth and improved overall survival compared to controls.

Conclusion Our study provides a comprehensive surfaceome landscape of AML, identifying both canonical and non-canonical membrane proteins as promising immunotherapy targets. The successful validation of these targets across multiple AML lines and primary samples, with absent expression on healthy cells or HSPCs, validates their potential as AML-specific immunotherapy targets. Preclinical efficacy of a CD209-targeting ADC underscores the ability to develop novel AML-selective therapies based on this data. This work will also drive fundamental investigation into new areas of AML tumor biology and surface protein trafficking.