Knockdown of HSPA9 Induces Apoptosis and Increases TP53 Levels in Human CD34+ Hematopoietic Progenitor Cells
Authors: Liu Tuoen , Krysiak Kilannin , Shirai Cara Lunn , Ndonwi Matthew , Walter Matthew J. 
Published: October 2019

Abstract
HSPA9, a gene located on chromosome 5q31.2, is commonly deleted in patients with myelodysplastic syndromes (MDS). MDS patients with a deletion of the long arm of chromosome 5 [del(5q)] typically present with cytopenias, including anemia, and have increased levels of apoptosis in their bone marrow contributing to ineffective hematopoiesis. Recent evidence suggests that upregulation of TP53 in MDS bone marrow cells may contribute to the cytopenias and accererated apoptosis observed in patients. While the mechanisms of TP53 activation in MDS are likely to be multifactorial, gene haploinsufficiency has been shown to contribute. Previous reports have shown that knockdown of RPS14, a chromosome 5q33.1 gene, in human CD34+ cells (or heterozygous knockout in mouse bone marrow cells) results in upregulation of TP53 and an increase in apoptosis. It is not known whether additional del(5q) candidate genes contribute to TP53 activation in del(5q)-associated MDS. In order to determine whether HSPA9 gene deletion also results in TP53 activation, we used lentiviral shRNA vectors to knockdown the expression of HSPA9 in primary human CD34+ hematopoietic progenitor cells. The HSPA9 protein level was reduced to ~20% (sh960) and ~50% (sh433) compared to the control lentiviral shRNA (shGFP). Knockdown of HSPA9 significantly inhibited the growth (fold change sh960 compared to shGFP = 0.16, p<0.01; sh433 compared to shGFP = 0.39, p=0.06, N=3) and erythroid differentiation (CD71+ expression fold change sh960 compared to shGFP =0.26, p<0.001; sh433 compared to shGFP = 0.52, p<0.01, N=3) of human CD34+ cells grown in media promoting erythroid differentiation after 7 days in culture. In addition, knockdown of HSPA9 by sh960 significantly increased apoptosis (AnnexinV+ cells) in CD34+ cells compared to shGFP (fold change = 2, p<0.01, N=3). The increased apoptosis observed following HSPA9 knockdown was associated with increased TP53 expression (fold change sh960 compared to shGFP = 2 by intracellular flow cytometry, p<0.05, N=3), TP53 activity (sh960 compared to shGFP, p<0.05; sh433 compared to shGFP, p=0.06 by TP53 luciferase reporter assay), and increased mRNA expression of the pro-apoptotic TP53 target gene BAX (fold change sh960 compared to shGFP = 1.8 by Q-RT-PCR, p<0.05). Gene expression profiling of CD34+ cells transduced with sh433 vs. control knockdown revealed that 26 well-annotated TP53-induced genes are also expressed higher in HSPA9 knockdown cells compared to controls by gene set enrichment (FWER p=0.01), further implicating TP53 activation in HSPA9 knockdown cells. We show that treatment of primary human CD34+ cells with MKT-077, a rhodacyanine dye with inhibitory effects on HSPA9 protein by binding to its nucleotide binding domain, also results in dose-dependent growth inhibition, enhanced apoptosis, and reduced erythroid differentiation, similar to that observed following HSPA9 knockdown. HSPA9 has been shown to physically interact with TP53 in the cytoplasm, and knockdown of HSPA9 changes localization of TP53 from the cytoplasm to the nucleus in non-hematopoietic cells. The loss of cytoplasmic sequestration of TP53 by HSPA9 following HSPA9 knockdown is thought to contribute to many of the cellular phenotypes induced by HSPA9 knockdown. We show by immunoprecipitation that HSPA9 also interacts with TP53 in primary human CD34+ cells, providing a possible mechanism for regulation of TP53 by HSPA9 in hematopoietic cells. These findings indicate that HSPA9 knockdown may contribute to TP53 activation and increased apoptosis observed in del(5q)-associated MDS. The data also suggest that genetic (i.e., mutation or deletion) or functional inactivation of TP53 may be necessary for disease progression to occur in MDS patients with del(5q).

Disclosures
No relevant conflicts of interest to declare.