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  • P-III-0948

Intra- and extracellular proteome dynamics during normal and malignant hematopoietic stem cell expansion

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Cell Biology Insights

Poster

Intra- and extracellular proteome dynamics during normal and malignant hematopoietic stem cell expansion

Thema

  • Cell Biology Insights

Mitwirkende

Maria Jassinskaja (York / GB), Helena Kooi (York / GB; Amsterdam / NL), Daniel Bode (York / GB), Sudip Ghosh (Lund / SE), Monika Gonka (York / GB), Juan Antonio Rubio Lara (York / GB), Alyssa Cull (York / GB), Lilia Cabrera Cosme (York / GB), Rachel Popplewell (York / GB), Alexander J. Hogg (York / GB), Elise Bennett (York / GB), Joanna Milek (York / GB), Fiona Bain (York / GB), Sophie Davies (York / GB), Samuel Elberfeld (York / GB), Adam Wilkinson (Oxford / GB), Satoshi Yamazaki (Tokyo / JP), Jenny Hansson (Lund / SE), David G. Kent (York / GB)

Abstract

Delineating 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. Loss-of-function mutations in the TET2 gene are frequently observed in myeloproliferative neoplasms (MPNs) and confer HSCs with increased self-renewal potential leading to clonal outgrowth of mutant cells. Relatively few genes mediating MPN initiation and progression have been identified to date and transcriptomic approaches have had limited success in identifying novel therapeutic targets. The low number of HSCs within mouse and human tissues has historically precluded global characterization of the cellular and extracellular proteome of HSCs; however, parallel advances in ex vivo HSC expansion and low-input proteomics puts us on the cusp of breaking through this decades-old barrier. Here, our aim was to perform proteomic profiling of freshly isolated or ex vivo-expanded normal and Tet2-deficient HSCs, and to characterize soluble factors in the cells" microenvironment in vivo and in culture to generate a comprehensive molecular map of intra- and extracellular self-renewal drivers in normal and pre-malignant HSCs. 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 normal and pre-leukemic HSCs in vivo and during ex vivo expansion. We show that 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 that is pro-inflammatory and pro-thrombotic even in young, asymptomatic animals. In HSC expansion assays, proteomics identifies the requirement for intact DNA repair pathways as key components of HSC clones capable of extensive self-renewal compared to unsuccessful expansion cultures. In line with this, functional assays demonstrate that DNA repair enzyme Parp1 is a key player in ex vivo HSC expansion. Analysis of the secretome of unsuccessful cultures further identifies mast cell proteins as predictive of failure to expand engraftable HSCs. Collectively, our study highlights novel proteins to which transcriptomic studies are blind, and open new opportunities for HSC expansion and pre-leukemic biology, paving the way for future ex vivo and in vivo modulation of HSC function via manipulation of the cells and their extracellular environment.

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