Hspa9 Haploinsufficiency Induces a Cell-Intrinsic Defect in Mouse B-Cell Progenitors
Authors: Krysiak Kilannin , Tibbitts Justin , Chen Tim H , Walter Matthew J. 
Published: October 2019

Abstract
Abstract 1714
Patients with myelodysplastic syndromes (MDS) have a clonal hematopoietic stem cell disorder that results in dysplastic hematopoietic cells in their bone marrow as well as peripheral blood cytopenias. In addition to the commonly described erythroid and myeloid differentiation defects associated with MDS, a reduction in bone marrow B-cell progenitors exists in patients. The genetic events contributing to the reduction in B-cell progenitors remain poorly understood.
The most common cytogentic abnormality identified in patients with MDS, occurring in approximately 35% of patients, is heterozygous interstitial deletion or loss of the long arm of chromosome 5 (5q). The interstitial deletions on chromosome 5 are single copy losses, and no biallelic disruptions of genes in deleted regions have been identified, implicating haploinsufficiency as the underlying genetic mechanism. We, and others, have shown that the levels of HSPA9 mRNA expression are reduced ∼50% in patients with del(5q) when compared to MDS patients without del(5q), consistent with a haploinsufficient phenotype. To model haploinsufficiency, we used shRNA to achieve ∼50% knockdown of Hspa9 in a murine bone marrow transplant model. This model showed a significant reduction in mature B-cells in the bone marrow, spleen, and peripheral blood of recipient mice, implicating HSPA9 haploinsufficiency may contribute to the B-cell alterations observed in MDS patients with del(5q).
To further evaluate HSPA9 haploinsufficiency in vivo, we created a mouse model with a heterozygous deletion of Hspa9 (Hspa9+/−) and confirmed a 50% reduction in Hspa9 protein levels in bone marrow and spleen of these mice by Western blot. Hspa9+/− mice are born at normal Mendelian frequencies (N>100), however, breeding heterozygous mice suggests Hspa9−/− mice are embryonic lethal (24 Hspa9+/+:38 Hspa9+/−:0 Hspa9−/−). No significant differences in mature lineage markers, complete blood counts, and hematopoietic organ cellularity, have been identified up to 12 months of age. However, as early as 2 months of age, the numbers of bone marrow CFU-preB colonies as assessed by methylcellulose assay, are significantly reduced in Hspa9+/− mice compared to Hspa9+/+ littermates (14 vs 48 colonies/100,000 bone marrow cells plated, respectively, N=10 mice/genotype, p<0.0001). We performed noncompetitive bone marrow transplants of Hspa9+/− or Hspa9+/+ donor cells into Hspa9+/+ recipient mice and confirmed that the reduction of B-cell progenitors is a hematopoietic cell intrinsic phenotype (N=7–9 mice/genotype, p=0.002). We also confirmed that the Hspa9+/− bone marrow microenvironment did not contribute to the phenotype as transplantation of Hspa9+/+ donor bone marrow cells into Hspa9+/− recipients did not alter the number of CFU-preB colonies (N=5).
Total frequencies of common lymphoid progenitors and B-cell precursors (Hardy fractions A, B/C, D, E and F) as assessed by flow cytometry are no different in Hspa9+/− and Hspa9+/+ mice. Therefore, we hypothesize that early Hspa9+/− B-cells may have an intrinsic signaling defect which can be compensated for in vivo. Early B-cell maturation is dependent on intracellular signaling mediated through cell surface receptors in response to environmental cytokines. Consistent with our hypothesis, we showed that Hspa9+/− CFU-preB in vitro colony formation is partially rescued by increasing concentrations of IL7 while Hspa9+/+ colony numbers remain unchanged (fold change in colony formation from 10ng/mL to 50ng/mL IL7 was 1.80 for Hspa9+/− vs. 0.80 for Hspa9+/+, p=0.03, N=6 mice/genotype). Supplementation of the media with another cytokine that contributes to early B-cell maturation, Flt3 ligand, does not alter Hspa9+/− or Hspa9+/+ CFU-preB colony formation, further implicating altered IL7 signaling. We are currently investigating the downstream responses to IL7 stimulation in B-cell progenitors from Hspa9+/− mice.
Collectively, these data implicate loss of HSPA9 as a contributing factor in the reduction of B-cell progenitors observed in patients with del(5q) associated MDS.

Disclosures:
No relevant conflicts of interest to declare.