HSPA9 Gene Regulates Erythroid Maturation in Human Hematopoietic Progenitor cells

Abstract

Background and HypothesisMyelodysplastic syndromes (MDS) is the most common form of adult myeloid blood cancers within the United States. A high fraction of patients with MDS can further progress to acute myeloid leukemia (AML) without intervention. MDS is a heterogeneous group of blood cancers characterized by abnormal hematopoietic cell maturation, increased apoptosis of bone marrow cells, and peripheral blood cytopenias. Low blood cell counts in MDS patients result in morbidity and mortality due to dysregulation of hemostasis and infection. Up to 25% of MDS patients acquire an interstitial deletion on chromosome 5q, also known as Del(5q). The HSPA9 gene is located in the commonly deleted region and is associated with patients that progress from MDS to AML.We hypothesize that that inhibition of the Del(5q) candidate HSPA9 gene can disrupt the erythroid maturation, contributing to the peripheral blood cytopenia observed in MDS patients.Results Knockdown of HSPA9 inhibited cell growth, increased apoptosis, inhibited erythroid maturation, and increased TP53 levels in human CD34+ cells. In order to test the effects of HSPA9 knockdown in human CD34+, we first utilized the siRNA that specifically targets the HSPA9 gene. HSPA9 specific siRNA reduced the HSPA9 protein expression level to approximately 50% compared to control siRNA. Knockdown of HSPA9 by siRNA inhibited cell growth, increased apoptosis, inhibited erythroid maturation, and increased TP53 levels in human CD34+ cells. Pharmacologic inhibition of HSPA9 induced apoptosis, decreased erythroid maturation and increased TP53 levels in human CD34+ cells. MKT‐077 is an HSPA9 inhibitor and it reduced HSPA9 expression, inhibited cell growth, increased apoptosis, inhibited erythroid maturation and induced TP53 expression in a dose‐dependent manner in human CD34+ cells. Reduction of TP53 reversed the inhibition of erythroid maturation caused by HSPA9 knockdown. We constructed two TP53 shRNAs which were able to decrease TP53 levels by about 80% and 50%, respectively, compared to control shRNA. We co‐transduced lentiviruses containing shRNA targeting HSPA9 and TP53 into human CD34+ cells using double antibiotic selection. The erythroid inhibition following HSPA9 knockdown was significantly decreased and reversed following simultaneous knockdown of TP53 with shTP53. ConclusionPrevious studies showed that Del(5q) MDS bone marrow samples display an induction of TP53 in erythroid cells and upregulation of TP53 target gene expression. Our study focuses on the effects of HSPA9 on erythroid maturation, as well as cell growth and apoptosis in human CD34+ hematopoietic progenitor cells. We observed that inhibition of HSPA9 (by siRNA, short hairpin RNA and MKT‐077), a gene on the proximal CDR of chromosome 5, caused cell growth inhibition, apoptosis induction and erythroid maturation inhibition in human CD34+ cells. These phenotypes were associated with increased TP53 levels. We also found that inhibition of TP53 in human CD34+ cells could reverse erythroid inhibition caused by HSPA9 inhibition. In conclusion, our study indicates that inhibition of HSPA9 gene can inhibit erythroid maturation, which may contribute to the peripheral blood cytopenia seen in Del(5q) MDS patients. Our data also suggests the inhibition of erythroid maturation via HSPA9 inhibition may be regulated through a TP53‐dependent mechanism.Support or Funding InformationThis work is supported by West Virginia School of Osteopathic Medicine Faculty Startup Funding (T. Liu).