Conditionally Pathogenic Genetic Variants of a Hematopoietic Disease-Suppressing Enhancer
Authors: Soukup Alexandra , Johnson Kirby D. , Liu Peng , Matson Daniel R , Bresnick Emery H. 
Published: November 2021

Abstract
Human genetic variants are classified based on potential pathogenicity to guide clinical decisions. However, mechanistic uncertainties often preclude definitive categorization. Germline coding and enhancer variants within the hematopoietic regulator GATA2 create a bone marrow failure and leukemia predisposition (Soukup and Bresnick, 2020). The conserved murine enhancer (+9.5) promotes hematopoietic stem cell (HSC) genesis, and a single-nucleotide human variant in an Ets motif attenuates chemotherapy- and transplantation-induced hematopoietic regeneration in a cell-autonomous manner (Soukup et al., 2019). Since an epigenetically-silenced normal allele can exacerbate phenotypes of a pathogenic heterozygous variant, we engineered a bone marrow failure model harboring the Ets motif variant and a severe E-box:GATA enhancer mutation on the second allele (CH) that renders the enhancer chromatin inaccessible, to determine how perturbations of the hematopoietic system trigger pathogenesis.
Given the propensity of GATA2 deficiency syndrome patients to infection, we asked if CH mice could mount an appropriate response to the bacterial cell wall component LPS. In WT, LPS acts on HSCs to induce inflammatory signaling and proliferation (Caiado et al., 2021). Twenty-four hours after LPS (0.5 mg/kg) administration, CH LSK levels were 3.1-fold lower than WT (p = 0.009). Immunophenotypic LT-HSCs (LSKCD150 +CD34 -) were 1.9-fold lower in treated CH vs. WT mice (p = 0.019). LPS reduced all LSK subpopulations: CD34 -CD150 -, CD34 +CD150 -, and CD34 +CD150 + were 3.4-, 5.7-, and 5.3-fold lower, respectively (p = 0.007, 0.029, and 0.006). Thus, the CH mutation attenuated acute inflammation-induced HSPC expansion.
G-CSF is among the cytokines/chemokines induced by infection (Quinton et al., 2002). As G-CSF is used to mobilize bone marrow HSCs for collection prior to transplantation, we tested the following models: 1) +9.5 mutation abrogates HSPC expansion and/or mobilization in response to diverse stimuli; 2) +9.5 mutation permits expansion/mobilization, yet compromises function; 3) HSPC expansion, mobilization and function are +9.5 mutation-insensitive. After 8 doses of G-CSF, circulating neutrophil numbers increased 2.6-fold in WT (p = 0.003), while neutrophils did not increase in the CH mice. G-CSF treatment increased bone marrow HSCs 2.6-fold in WT mice, whereas the CH mutation abrogated this response (p &lt; 0.0001). As a metric of mobilization, splenic HSCs were quantified. After G-CSF treatment, WT splenic HSCs expanded 11-fold (p &lt; 0.0001), while CH HSCs were unchanged. To functionally analyze mobilized HSPCs, colony forming unit (CFU) assays were conducted with peripheral blood. CFU increased 11-fold in WT blood post-treatment (p &lt; 0.0001), and CH abrogated the increase.
To test whether G-CSF signaling is impacted by the CH mutation, we asked whether G-CSF treatment of bone marrow ex vivo induced STAT3 phosphorylation using a phospho-flow cytometry assay. While G-CSF induced pSTAT3 8.5-fold in WT Lin -Kit + cells (p &lt; 0.0001), the induction was attenuated to 4.9-fold in CH (p = 0.003). Signaling in Gr1 + granulocytes was indistinguishable between genotypes (p = 0.999). Thus, the CH mutation attenuated G-CSF-induced signaling and HSPC mobilization, abrogated HSPC expansion, and compromised HSPC function.
The Gata2 CH bone marrow failure model is characterized by relatively normal developmental hematopoiesis and adult steady-state hematopoiesis, yet the mice are vulnerable to stressors. As the CH mice were hypersensitive to myeloablation and acute inflammation and exhibited an attenuated response to a clinical HSC-mobilizing agent, GATA2-driven regenerative hematopoiesis mitigates damage resulting from chemotherapy and inflammation and promotes G-CSF-induced HSC mobilization. As the enhancer variant controlled HSC responsiveness to a therapeutically important HSC-mobilizing drug, these results link non-coding genome variation with mobilization efficacy. Based on the prominent GATA2 activities described herein, we are investigating how the full ensemble of macromolecular components constituting the GATA2 regulatory network generate a vital protective system conferring resilience to the hematopoietic system in the context of diverse stressors and whether genetic variants in genes encoding components of these pathways resemble or differ from those of GATA2 variants.

Disclosures
No relevant conflicts of interest to declare.