A Multiomic Single-Cell Atlas of Human Myelopoiesis Reveals Cellular and Molecular Drivers of Immunomodulatory Drug-Induced Neutropenia

Neutrophils are crucial innate immune cells which protect the host by killing infectious pathogens. Single cell RNA-sequencing analyses have previously described transcriptional heterogeneity of neutrophils and precursors, however the genome regulatory events underlying the transcriptional changes a...

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Published in:Blood 2023-11, Vol.142 (Supplement 1), p.935-935
Main Authors: Simoglou Karali, Christina, Riva, Simone, Clark, Sally-Ann, Wen, Sean, Sousos, Nikolaos, Louka, Eleni, Gür, E. Ravza, Hayder, Nawshad, Pierceall, William E., Thakurta, Anjan, Gandhi, Anita K., Hagner, Patrick R, Hughes, Jim, Mead, Adam J
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Language:English
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Summary:Neutrophils are crucial innate immune cells which protect the host by killing infectious pathogens. Single cell RNA-sequencing analyses have previously described transcriptional heterogeneity of neutrophils and precursors, however the genome regulatory events underlying the transcriptional changes are less well characterised. Understanding this is important as neutropenia can be caused by dysregulation of the genome regulatory events that orchestrate neutrophil maturation. A good example is the use of immunomodulatory drugs (IMiDs, lenalidomide [LEN] & pomalidomide [POM]) for the treatment of multiple myeloma (MM) which are now standard-of-care, but their use is frequently complicated by neutropenia because of neutrophil maturation impairment. This is due to Cereblon (CRBN)-driven substrate ubiquitination and Ikaros degradation, causing PU.1 downregulation. However, restoration of Ikaros levels cannot fully alleviate the IMiD-induced myeloid differentiation block suggesting other mechanisms might also contribute. Our aim was to build a multiomic atlas of human myelopoiesis and use this to interrogate the molecular drivers of IMiD-induced neutropenia. We developed an ex vivo myeloid cell differentiation assay using human mobilized peripheral blood CD34 + cells from healthy donors and optimised methods for single cell multiomic (combined RNA-seq & ATAC-seq) analysis. This platform was validated orthogonally (immunophenotype, morphology & functional) and employed to construct a multi-omic single-cell landscape of normal human neutrophil differentiation and it's perturbation by IMiDs. Single cell transcriptomes from >110,000 cells from untreated and IMiD-treated samples were analyzed, capturing the full spectrum of human myelopoiesis spanning from early myeloid progenitors through to neutrophil populations (band, segmented, mature) (Fig.1A-left). Ex vivo differentiated neutrophils retained transcriptional signatures reflective of effector functions (granule biogenesis, chemotactic activity, respiratory burst) and mapped to previously described human neutrophil populations in vivo. Ex vivo generated neutrophils showed expected functional properties of phagocytosis and neutrophil extracellular trap formation upon stimulation. Exposure to IMiDs caused a maturation arrest with a 40-50% (p
ISSN:0006-4971
1528-0020
DOI:10.1182/blood-2023-182041