Acute myeloid leukaemia (AML) remains the most prevalent myeloid malignancy with persistently poor outcomes despite recent therapeutic advances. Treatment failures and disease relapse highlights the urgent need for novel targeted therapies capable of inducing durable remissions and eliminating therapy-resistant leukaemia stem cells (LSCs). The Wnt/β-catenin signalling pathway, frequently dysregulated in AML, potentially represents such a target. However, targeting downstream pathway components poses significant toxicity risks, making cell surface-directed approaches associated with Wnt signalling more attractive for developing novel biomarkers and immunotherapeutic strategies. Wnt signalling target genes are already known to include cell surface antigens, but have not been explored in haematopoietic context. To identify signalling responsive surface proteins as potential druggable surface targets, we performed the first comprehensive characterisation of the Wnt-responsive surfaceome in myeloid cells using a Cell Surface Capture (CSC) enrichment strategy in combination with Wnt signalling modulation. Following confirmation of successful CSC glycoprotein enrichment in K562 cells to 20% of detected proteome by tandem mass spectrometry (MS/MS) experiments, we next investigated Wnt-responsive surface proteins in K562 cells following lentiviral mediated β-catenin knockdown (shRNA) and knockout (CRISPR/Cas9). Integrated CSC-LC/MS analysis coupled with differential expression profiling identified 37 significantly upregulated, and 74 signalling downregulated plasma membrane proteins in response to b-catenin ablation. Orthogonal western blot validation confirmed a significant downregulation of Differentiation-dependent protein A4 or proteolipid protein 2 (PLP2) following β-catenin depletion which appeared mainly post-transcriptional since mRNA levels remained unchanged with β-catenin knockdown. In addition to plasma membrane localisation, immunofluorescence studies also showed PLP2 to have cytosolic localisation, in keeping with previous reports as an endoplasmic reticulum localised protein. PLP2 knockdown in myeloid cell lines (K562 and NOMO1) significantly impaired proliferation and sustained the short-term self-renewal capacity and suppressed monocytic in primary cord blood derived CD34+ haematopoietic stem/progenitor cells. Taken together, these data propose PLP2 as a post-transcriptionally regulated surface antigen by Wnt/β-catenin signalling, and a novel regulator of haematopoetic cell growth and differentiation.

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2025
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