HSPA9 is located on chromosome 5q31.2 in humans, a region that is commonly deleted in patients with myeloid malignancies [del(5q)], including myelodysplastic syndrome (MDS). HSPA9 expression is reduced by 50% in patients with del(5q)-associated MDS, consistent with haploinsufficient levels. Zebrafish mutants and knockdown studies in human and mouse cells have implicated a role for HSPA9 in hematopoiesis. To comprehensively evaluate the effects of Hspa9 haploinsufficiency on hematopoiesis, we generated an Hspa9 knockout mouse model. Although homozygous knockout of Hspa9 is embryonically lethal, mice with heterozygous deletion of Hspa9 (Hspa9+/-) are viable and have a 50% reduction in Hspa9 expression. Hspa9+/- mice have normal basal hematopoiesis and do not develop MDS. However, Hspa9+/-mice have a cell-intrinsic reduction in bone marrow colony-forming unit-PreB colony formation without alterations in the number of B-cell progenitors invivo, consistent with a functional defect in Hspa9+/- B-cell progenitors. We further reduced Hspa9 expression (<50%) using RNA interference and observed reduced B-cell progenitors invivo, indicating that appropriate levels (≥50%) of Hspa9 are required for normal B lymphopoiesis invivo. Knockdown of Hspa9 in an interleukin 7 (IL-7)-dependent mouse B-cell line reduced signal transducer and activator of transcription 5 (Stat5) phosphorylation following IL-7 receptor stimulation, supporting a role for Hspa9 in Stat5 signaling in B cells. Collectively, these data imply a role for Hspa9 in B lymphopoiesis and Stat5 activation downstream of IL-7 signaling.
Bibliographical noteFunding Information:
We thank Deepta Bhattacharya for kindly providing the IL-7Rα antibody, as well as helpful scientific discussions, and Charles Mullighan for kindly providing the B7 cells. This work was supported by the National Cancer Institute and the National Heart Lung Blood Institute of the National Institutes of Health under Award no. F31CA165702 (to K Krysiak) and Research Project Grant no. R01HL109336 (to MJ Walter), respectively, as well as by a Howard Hughes Medical Institute Physician-Scientist Early Career Award (to MJ Walter) and a Siteman Cancer Biology Pathway Fellowship (to K Krysiak). Technical assistance was provided by the Alvin J. Siteman Cancer Center Flow Cytometry Core, which provided cell sorting; Tissue Procurement Core, which provided RNA processing for microarray analysis; Biostatistics Core, which provided informatics support; and GTAC for microarray hybridization, and are supported by an NCI Cancer Center Support Grant (no. P30CA91842 ). Additional technical assistance was provided by the Hope Center Viral Vectors Core, which is supported by a Neuroscience Blueprint Interdisciplinary Center Core Award (no. P30NS057105).