B-Cell Progenitors Are Reduced in Hspa9 haploinsufficient Mice,

Abstract 3829

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis and an increased risk of acute myeloid leukemia (AML) development. In addition to erythroid and myeloid differentiation defects in MDS, a reduction in bone marrow B-cell progenitors exists in MDS patients. The genetic events leading to the development of MDS, reduction in B-cell progenitors, and the subsequent transformation to AML are poorly understood.

Heterozygous, interstitial deletions or loss of the long arm of chromosome 5 (5q) are among the most commonly acquired genetic abnormalities found in MDS, occurring in approximately 35% of patients. Within the commonly deleted regions of chromosome 5 are several genes implicated in MDS/AML development including RPS14, EGR1, CTNNA1, and HSPA9. HSPA9 mRNA expression levels have been shown to be 50% reduced in patients with deletions of the common region on chromosome 5, consistent with a haploinsufficient level of expression. shRNA knockdown of Hspa9 in a murine bone marrow transplant model resulted in a significant reduction in mature B-cells in the bone marrow, spleen, and peripheral blood of recipient mice, suggesting Hspa9 haploinsufficiency may contribute to B-cell alterations observed in MDS patients with chromosome 5 deletions.

In order to further test whether HSPA9 haploinsufficiency alters hematopoiesis in vivo, we created an Hspa9 heterozygous knockout mouse (Hspa9^+/−). Hspa9^+/− mice were born at normal Mendelian frequencies (N>100) and expressed 50% less protein in their spleen and bone marrow compared to wildtype littermates by Western blot. Breeding heterozygous mice yielded 24 Hspa9^+/+, 38 Hspa9^+/−, and 0 Hspa9^−/− mice, suggesting that homozygous knockout mice are embryonic lethal. Basal hematopoiesis in the bone marrow, spleen, and peripheral blood of Hspa9^+/− mice is normal at 2 (N=3–10 mice/genotype) and 6 (N=5 mice/genotype) months of age. Complete blood counts, flow cytometric analysis of lineage markers (B220, CD3e, Gr1, CD115, CD71, and Ter119), and hematopoietic organ cellularity do not differ between Hspa9^+/− and Hspa9^+/+ littermate controls. However, beginning at 2 months of age, Hspa9^+/− mice have a significant reduction in the number of pre B-cell progenitors compared to Hspa9^+/+ littermate control mice using methylcellulose cultures (14 vs 48 colonies/100,000 bone marrow cells plated, respectively, N=10 mice/genotype, p<0.0001). In addition, at 9 months of age, there is a significant reduction in splenic T-cells (CD3e+), but not B-cells, in Hspa9^+/− mice compared to Hspa9^+/+ mice measured by flow cytometry (35.3% vs 41.8% of cells, N=5–6 mice/genotype, p=0.03), further indicating a possible alteration in lymphocyte development or maturation in Hspa9^+/− mice. Ongoing, noncompetitive bone marrow transplants of Hspa9^+/− donor cells into Hspa9^+/+ recipient mice will define whether the reduction of B-cell progenitors is a hematopoietic cell intrinsic phenotype and address whether there is homeostatic expansion of mature B-cells in Hspa9^+/− mice.

In addition to the reduction in B-cell progenitors in MDS, recent gene expression profiling results indicate that del(5q) is specifically associated with a reduced expression of B-cell signaling genes in CD34+ MDS progenitor cells. Two of the most highly down-regulated gene sets in del(5q) MDS CD34+ cells compared to normal CD34+ cells included B-cell signaling genes (Leukemia. 2010 Apr;24(4):756–64.). Using independent Affymetrix U133plus2 gene expression profiling data from our institution, we confirm that the most down-regulated genes in CD34+ progenitor cells from del(5q) or non-del(5q) MDS patients compared to control CD34+ cells are also B-cell development genes using Gene Set Enrichment Analysis (GSEA)(p<0.001).

Collectively, the data suggest that loss of HSPA9 may contribute to the reduction of B-cell progenitors observed in patients with del(5q) associated MDS. Follow-up analysis of basal and stress hematopoiesis in aged Hspa9^+/− mice are ongoing and will be presented.