Wednesday, February 5, 2025
2/5/2025
PROJECT SUMMARY Transcription factors and cellular signaling mechanisms establish and maintain regulatory networks that allow the hematopoietic system to respond to infection, inflammation and other stresses. These networks mediate the expansion, mobilization, and differentiation of hematopoietic stem and progenitor cells (HSPCs). As exemplified by human germline variation in genes encoding HSPC-regulatory factors, such as GATA2 and RUNX1, genetic variation can disrupt HSPC activities, leading to recurring infection, cytopenia, and/or bone marrow failure. However, the mechanisms underlying these defective responses are incompletely understood. We have modeled human pathogenic variants causing GATA2 deficiency syndrome, where patients present with recurring infections and cytopenias with progression to MDS and AML. This model utilizes mice with a single-nucleotide variant from GATA2 deficiency patients, in conjunction with a severely impaired Gata2 +9.5 enhancer in the second allele (compound heterozygous; CH), mimicking epigenetic silencing and allele-specific expression correlating with disease presentation. We demonstrated that Gata2 variation alters steady-state HSPC levels, blocks HSPC expansion and differentiation in response to chemotherapy, attenuates long-term repopulating activity following bone marrow transplantation, leads to bone marrow failure following chronic inflammation by the viral mimetic polyI:C, alters HSPC response to the bacterial cell wall component LPS, and attenuates HSPC mobilization in response to pro-inflammatory cytokines, including G-CSF. We hypothesize that pathogenic clinical variants disrupt GATA2- and infection/inflammatory-instigated networks essential for HSPC expansion and differentiation, and we shall utilize global approaches to generate a rigorous foundation for elucidating how variants impact networks in the context of sterile- and infection-induced inflammation. Aim 1 will develop global insights into GATA2-dependent mechanisms that regulate HSPCs in response to bacterial infection and sterile inflammation. Barcoding cell surface markers coupled with single cell RNA-sequencing (CITE-seq), will be performed on HSPCs to determine pathways altered by the pathogen response, immunophenotypic populations affected, and how clinical variants disrupt the networks. The contribution of the niche to HSPC function will also be analyzed. Aim 2 will determine how fungal infection alters networks governing HSPC expansion, mobilization, and inflammation, and the requirement of GATA2 in these processes. We will generate libraries of differentially expressed transcripts in HSPC populations following infection with the pathogen Aspergillus fumigatus. Inflammatory cytokines will be quantified via multiplex cytokine analysis. We will test if networks responsible for HSPC response to fungal infection remain intact in GATA2 deficiency genetic models. These pilot studies will leverage existing models to generate systems and omic datasets to elucidate how germline genetic variation creates a predisposition to bone marrow failure.
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