Reduction of Hspa9 Expression, a Del(5q) Candidate Gene, in a Murine Bone Marrow Transplantation Model Recapitulates Key Feature of Ineffective Hematopoiesis Observed in MDS.

Abstract 946

Deletions spanning chromosome 5q31.2 are among the most common karyotypic abnormalities in MDS, and evidence suggests that haploinsufficiency of del(5q) genes maybe an early genetic event. To test whether haploinsufficiency of del(5q31.2) genes contribute to ineffective hematopoiesis observed in MDS (i.e. abnormal proliferation, apoptosis, and differentiation), we reduced the expression of HSPA9 (Hspa9 for the murine orhologue), one of the 28 genes, in primary human hematopoietic cells and a murine transplantation model using lentiviral mediated gene silencing.

We purified human CD34⁺ hematopoietic progenitors from cord blood samples (>90% purity) and infected them individually with 5 unique lentiviral shRNAs targeting HSPA9 (each achieving 30% to >90% knockdown) and 2 control shRNAs. HSPA9 knockdown cells expanded an average of 6 fold less than control cells from days 4-7 in erythroid unileage differentiation culture conditions (SCF, IL-6, IL-3, and EPO) (p≤0.007). The reduced cell numbers observed in HSPA9 knockdown cultures was associated with an increase in apoptosis and a decrease in the number of cells in S-phase compared to control shRNA expressing cells (p≤0.008) (increased membrane depolarization and active Caspase-3 expression were also observed with HSPA9 knockdown). In addition, after 7 days, there was a significant reduction in maturing erythroid precursors (p≤0.004), but not myeloid or megakaryocytic precursors, in HSPA9 knockdown cultures vs. control cultures. These results suggest that reduced expression of HSPA9 in primary hematopoietic cells recapitulates key features of ineffective erythropoiesis observed in MDS.

We extended these studies using a bone marrow lentiviral transduction/transplantation mouse model. Bone marrow progenitors were infected with a shRNA expressing lentivirus with yellow fluorescence protein (YFP) as a reporter. Loss of Hspa9 shRNA transduced peripheral blood cells occurred over 4-months, necessitating evaluation of mice from 3-8 weeks post-transplant. Two independent transplantation cohorts were performed and analyzed (control shRNA, n=8 mice; Hspa9 shRNA, n=9 mice). The percent of YFP donor leukocytes was significantly reduced in bone marrow (36.3% vs. 27.9%, p≤0.02), spleen (41.6% vs. 19.5%, p≤0.001), and peripheral blood (39.5% vs. 29.5%, p≤0.007) in mice receiving Hspa9 knockdown cells, consistent with in vitro results. The absolute number of YFP+, Ter119^High/CD71+ erythroid cells was reduced by 2.5 fold in the bone marrow (2.21×10⁶ vs. 8.97×10⁵, p≤0.001), 8.9 fold in the spleen (3.46×10⁶ vs. 3.87×10⁵, p≤0.002), and 4.4 fold in the peripheral blood (20.9×10³/μl vs. 4.80×10³/μl, p≤0.01), whereas the total number of myeloid cells (CD11b⁺/Gr-1⁻ monocytes and CD11b⁺/Gr-1⁺ neutrophils) in the three compartments was no different in mice receiving control vs. Hspa9 knockdown cells, respectively. Furthermore, the absolute number of reticulocytes, polychromatic normoblasts, and basophillic normoblasts (defined by flow cytometry) in Hspa9 knockdown recipients were significantly reduced in the bone marrow, spleen, and peripheral blood vs. control (p≤0.01 for all comparisons). The absolute number of B-lymphocytes (B220⁺), but not T-lymphocytes (CD3e⁺), were reduced by 2.4 fold in the bone marrow (1.16×10⁷ vs. 4.81×10⁶, p≤0.001), 3.4 fold in the spleen (4.26×10⁸ vs. 1.24×10⁸, p≤0.001), and 3.5 fold in the peripheral blood (1.06×10³/μl vs. 3.05×10²/μl p≤0.004). These results suggest that erythroid and B-cell progenitors are preferentially affected by Hspa9 knockdown, both findings that are also observed in patients with MDS. Following a 24-hour BrdU labeling in vivo, the percent of YFP+ cells in S-phase was significantly reduced in Hspa9 knockdown cells vs. control cells (21.3% vs. 27.2%, respectively, p≤0.02) in the bone marrow. Furthermore, the absolute number of YFP+ progenitor cells (lineage⁻/Sca-1⁺/C-kit⁺) in the bone marrow was reduced by 3.9 fold (2.59×10⁴ vs. 6.59×10³, p≤0.03), indicating that Hspa9 knockdown alters the early progenitor pool.

Collectively, these results suggest that haploinsufficiency of HSPA9 alone contributes to ineffective erythropoiesis, but that other cooperating genes are necessary for clonal dominance to occur in MDS. Because HSPA9 has been shown to physically bind and sequester p53 in the cytoplasm, we are testing whether the apoptosis and cell cycle abnormalities are p53 dependent using p53-/- donor murine bone marrow cells in a transduction/transplantation model.