Extracellular DEK Treatment Mimics Hypoxic Blockade of Extra Physiologic Oxygen Stress in Human and Mouse Hematopoietic Cells
Authors: Ropa James , Cooper Scott , Beasley Lindsay , Markovitz David M , Broxmeyer Hal E. , Capitano Maegan L. 
Published: November 2021

Abstract
Hematopoietic stem cell (HSC) and progenitor cell (HPC) self-renewal, proliferation, survival, and function are regulated by extracellular signals from cytokines and chemokines. DEK, a nuclear regulator of chromatin availability, was recently shown to also function as an extracellular protein that regulates HSC and HPC by inducing signaling through the CXC chemokine receptor 2 (CXCR2), enhancing pools of long-term stem cells while decreasing pools of functional HPC [Capitano et al., JCI, 2019, 129(6):2555-70]. Due to differing effects of extracellular (ec)DEK treatment on HSC growth and function compared to the effects on HPC, we hypothesized that DEK differentially regulates HSC compared to HPC. To explore this, we pulse-treated purified human cord blood (CB) CD34+ cells for 18 hours with recombinant human ecDEK protein. We then isolated by flow cytometry stringently immunophenotypically-defined human HSC, multipotent progenitors (MPP), common myeloid progenitors (CMP), and granulocyte-macrophage progenitors (GMP) and performed mRNA-sequencing to profile ecDEK-dependent transcriptomes of these separate populations of HSC/HPC. Interestingly, there are widely different ranges of effects on the transcriptomes of the different isolated HSC/HPC populations. CMP had the most differentially expressed (DE) genes induced by DEK treatment, followed by HSC and MPP, and GMP had relatively few changes, suggesting DEK treatment may primarily affect HSC and earlier HPC. HSC exhibited the strongest magnitude of changes in DE genes. Fast gene set enrichment analysis (FGSEA) revealed that GMP showed upregulation of gene programs associated with leukocyte migration and downregulation of gene programs regulated by MYC, CMP showed upregulation of gene programs associated with myeloid cell development and downregulation of lysine methyltransferase activity, and MPP and HSC both upregulated cytokine signaling responses in response to DEK treatment, though HSC also exhibited a downregulation of genes associated with cell cycle. Despite the identified unique changes upon DEK treatment in these different HSC/HPC populations, we were most interested to discover that all examined cell types demonstrated upregulation of genes associated with hypoxic responses in cells, and a concordant downregulation of genes that are frequently downregulated by hypoxic exposure following DEK treatment. This suggested to us that DEK may stimulate overlapping pathways related to hypoxia responses and/or response to exposure to extra physiologic oxygen. In fact, the effects on HSC and HPC induced by ecDEK is similar with previous work demonstrating that isolating HSC/HPC at 3% O 2 compared to ambient air conditions (~21% O 2) leads to a preserved pool of functional HSC but a lower number of functional HPC, due to a phenomenon termed extracellular physiologic shock/stress (EPHOSS) [Mantel et al., Cell, 2015, 161(7):1553-65]. To explore whether DEK regulates similar stress response pathways that are associated with EPHOSS, we assessed the effects of ecDEK treatment on HSC/HPC isolated at 3% O 2. Mice were treated with DEK in vivo, then bone marrow cells were isolated at 3% O 2 in a hypoxia chamber. Cells were kept at 3% O 2 or split to ambient air to allow equilibration to a higher oxygen tension and then assessed for HSC and functional HPC numbers. In contrast to untreated HSC/HPC isolated at 3% O 2, ecDEK treated cells isolated at 3% O 2 did not exhibit further increases in LT-HSC numbers or decreases in pools of functional HPC over ecDEK treatment alone identified by colony assays. Isolation at 3% O 2 followed by in vitro treatment with DEK showed similar results. This suggests ecDEK may neutralize the effects of EPHOSS by acting on similar gene programs that are induced by ambient air exposure. We also demonstrate that ecDEK prevents the ex vivo apoptosis of CB HSC/HPC populations, again suggesting that ecDEK is preventing the cellular stress associated with ambient air exposure and ex vivo growth, resulting in a preservation of HSC. Taken together, these data show that ecDEK acts as an extracellular signaling protein that prevents cellular stress by activating gene programs that overlap with hypoxia associated gene programs. This has important implications for the role of ecDEK in normal and diseased hematopoiesis, as well as clinical implications wherein ecDEK may be used to mimic the HSC preserving effects of isolation at 3% O 2.

Disclosures
Markovitz: University of Michigan: Patents & Royalties: H84T BanLec and of the H84T-driven CAR construct.