2023 – INTRA- AND EXTRACELLULAR PROTEOME DYNAMICS DURING NORMAL AND MALIGNANT HEMATOPOIETIC STEM CELL EXPANSION

Delineating cell-intrinsic and -extrinsic drivers of hematopoietic stem cell (HSC) self-renewal is critical to improve efforts in ex vivo HSC expansion and to better understand leukemia cell biology. To characterize molecular changes at the proteome level, we applied miniaturized, multiplexed sample preparation protocols in combination with mass spectrometry (MS)-based quantitative proteomics to compare normal and Tet2-deficient HSCs, and data-independent acquisition (DIA)-MS to characterize the extracellular environment of HSCs in vivo and during ex vivo expansion. We show that both the cellular and secreted proteome accurately stratify HSCs based on functional potency and mutational status and identify novel molecular components not captured in transcriptomic analyses. On the pre-leukemia side, we reveal that Tet2-deficient HSCs have altered expression of extracellular matrix (ECM) proteins and that interaction with these proteins in artificial niches affects cellular function. Extracellular proteomic analysis further reveals that Tet2-deficient cells create a microenvironment is pro-inflammatory and pro-thrombotic even in young, asymptomatic animals. In HSC expansion assays, proteomics identifies the requirement for intact DNA repair pathways, specifically mismatch repair proteins, as key components of HSC clones capable of extensive self-renewal compared to unsuccessful expansion cultures. Analysis of the secretome of unsuccessful cultures further identifies mast cell proteins as predictive of failure to expand engraftable HSCs. Collectively these data highlight novel proteins to which transcriptomic studies are blind, and open new opportunities for HSC expansion and preleukemic biology, paving the way for future ex vivo and in vivo modulation of HSC function via manipulation of the cells and their extracellular environment.