HB9 Represses Hematopoietic Stem Cell Proliferation and Induces Senescence
Authors: Ingenhag Deborah , Auer Franziska , Borkhardt Arndt , Hauer Julia 
Published: October 2019

Abstract
Introduction: HB9 is a transcription factor encoded by homeobox gene B9 (HLXB9). It is physiologically expressed during early embryonic development as well as in pancreatic beta- and motor neuronal cell development. Ectopic HB9 expression is found in infant acute myeloid leukemia with translocation t(7;12), accounting for up to one third of infant AML cases with a poor 3-year EFS of 0% irrespective of the treatment approach. We previously showed that HB9 regulates cell-cell interaction/adhesion (Wildenhain et al. Leukemia, 2010) in hematopoietic cells and influences the prostaglandin signalling pathway (Wildenhain & Ingenhag et al. JBC, 2012). In this study we focussed on the oncogenic potential of HB9 in hematopoiesis.
Methods: To investigate the oncogenic influence of HB9 expression on hematopoiesis, we developed an in vivo murine transplantation model. HB9-transduced lineage negative (Lin-) murine HSCs were transplanted into lethally irradiated wild-type mice and we monitored hematopoietic reconstitution and leukemia emergence by serial retroorbital bleedings for up to one year. Final analysis included comprehensive flow cytometric analysis of all hematopoietic compartments, with respect to dissemination of blast cells and cellular distribution. In vitro studies included proliferation as well as cell cycle analysis. Senescent phenotype was characterized by senescence-associated beta-galactosidase staining and cellular morphology. Knockdown of p53 was obtained via transfection of siRNA.
Results: Transplantation of HB9- or mock-transduced murine Lin- cells into lethally irradiated wild-type recipient mice (n=10) showed >80% donor chimerism and HB9-transduced Lin- cells gave rise to all hematopoietic lineages (B-lineage: CD19+, T-lineage: CD3+, NK-lineage: Nk1.1+, granulocytic lineage: Gr-1+, Monocytic lineage: CD11b+) in the peripheral blood, indicating no lineage-related preference of HB9-expressing HSCs. Reconstitution of peripheral blood cell compartments in HB9 transplanted mice, however, was significantly decreased in all three lineages (CD3+: 9.5-fold, CD19+: 34.7-fold , Gr+: 1.8-fold) compared to the control group with respect to copy number, mRNA and protein expression. We did not observe an accumulation of hematopoietic stem (LT-HSC, ST-HSC, MPP) and precursor cells subsets (CLP, MEP, CMP, GMP) in the bone marrow of mice transplanted with HB9-positive Lin- cells. Finally, mice transplanted with HB9-transduced Lin- cells did not develop leukemia after 12 months follow-up. The decreased reconstitution capacity of HB9 expressing HSCs led us to the assumption that HB9 represses cellular proliferation in vivo.
Thus we performed proliferation studies in vitro. Ectopic expression of HB9 in the murine NIH3T3 cell line revealed a complete inhibition of cell proliferation compared to mock control (n=3). The same effect was observed in human HT1080 cell line. Cell cycle analysis revealed a significant decrease of the S-phase (2-fold, p<0.05), stalling the cells in G1 and G2 phase of the cell cycle. In both cell line models HB9-transduced cells developed a senescent phenotype being multinuclear, flattened and enlarged. Staining for senescence-associated β-galactosidase activity was positive in HB9-transduced cells in contrast to complete absence in mock-transduced cells. Immunoblot analysis revealed that the HB9 dependent cell cycle arrest was mediated via p53-induced upregulation of p21. Knockdown experiments using p53-targeting siRNAs confirmed that the p53-signalling is responsible for the growth arrest because p53-knockdown was able to reverse the effect.
Conclusion:In our study HB9 represses hematopoietic stem cell proliferation in vivo and induces a senescent phenotype in vitro. Senescence is an evasion mechanism in response to aberrant oncogene expression and induction of senescence is the first evidence for an oncogenic potential of HB9. Future studies elucidating the signal pattern of HB9-induced senescence will shed new light on the pathomechanism and potential therapeutic targets in the treatment of translocation t(7;12) positive AML.

Disclosures
No relevant conflicts of interest to declare.