Reduced HSPA9B Expression, a 5q31.2 Candidate Gene, in Primary Human CD34+ Cells Recapitulates Features of Ineffective Hematopoiesis Observed in MDS.

Abstract Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic disorders characterized by ineffective hematopoiesis. Deletions spanning chromosome 5q31.2 are among the most common karyotypic abnormalities in MDS, and evidence suggests that del(5q) maybe an early genetic event. We hypothesize that haploinsufficiency of one or more of the 28 candidate genes in the 5q31.2 commonly deleted segment (CDS) are important for MDS pathogenesis. Many genes in the CDS, including HSPA9B, have dose reduced mRNA levels in CD34+ cells from MDS patients with del(5q), and zebrafish carrying a heterozygous mutation in the orthologue of the human HSPA9B gene display increased apoptosis in blood cells. Therefore, we asked whether reduced HSPA9B expression in primary human hematopoietic cells can recapitulate key features of ineffective hematopoiesis (i.e. abnormal proliferation, apoptosis, and differentiation). To address this question, we purified human CD34+ hematopoietic progenitors from cord blood samples (>90% purity) and infected them with a shRNA expressing lentivirus that also carries the puromycin resistance gene. Cell culture densities were normalized after 4 days in selection medium, and 3 days later apoptosis (AnnexinV/7-AAD), cell cycle status (BrdU/7-AAD), and erythroid or myeloid differentiation was measured using flow cytometry. We performed 4–5 independent experiments using 5 individual shRNAs targeting HSPA9B (30% to >90% knockdown) and 2 control shRNAs. Cells transduced with control shRNAs expand 18.2 fold from days 4–7 in RBC unilineage differentiation culture medium (25ng/ml SCF, 10ng/ml IL-6, 10ng/ml IL-3 and 0.5U/ml Epo), vs. only a 0.8–5.2 fold expansion when HSPA9B is knocked down (N=5 for each shRNA, p≤0.007). The reduced cell numbers observed in HSPA9B knockdown cultures is associated with an increase in apoptosis and a decrease in the number of cells entering S-phase compared to control shRNA expressing cells. 8.2% of cells in control cultures were AnnexinV+/7AAD+ vs. 20–62% of cells in HSPA9B knockdown cultures (N=5 for each shRNA, p≤0.0004). Following 1 hour of BrdU exposure, 60% of cells in control cultures were in S-phase vs. only 22–48% of HSPA9B knockdown cultures (N=5 for each shRNA, p≤0.008). In addition, after 7 days in RBC unilineage differentiation culture medium, 58% of control shRNA expressing cells were CD71+/Gycophorin A+ vs. 7.1–32% of cells in the HSPA9B knockdown cultures (N=4 for each shRNA, p≤0.004). The decreased number of GPA+ cells in HSPA9B knockdown cultures was concomitant with retention of CD34+ expression on cells. Similar results were observed using myeloid unilineage culture conditions (10ng/ml SCF, 100ng/ml G-CSF, 20% FCS), where the number of cells expressing CD15+ was reduced from 47% in control cultures to 28–37% in HSPA9B knockdown cultures. A delay in erythroid and myeloid differentiation was confirmed by cell morphology. Lentiviral knockdown of the murine orthologue of HSPA9B (Hspa9a) in 2 independent transduction/transplantation experiments resulted in a loss of transduced cells over 2 months (2 shRNAs each) compared to a control shRNA, implicating a cell intrinsic defect when Hspa9a levels are reduced in vivo. Collectively, these results implicate that reduced HSPA9B expression in human CD34+ progenitor cells results in abnormal proliferation, increased apoptosis, and altered differentiation, key features of ineffective hematopoiesis, in a dose dependent manner.