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D. Ingenhag et al.
HLXB9, also known as MNX1 (motor neuron and pan- creas homeobox 1), belongs to the ANTP class of home- obox genes.5 It is located on chromosome 7q36, spanning 5.8 kb and comprising 3 exons. The corresponding 401 aa protein is named HB9; this is highly conserved and func- tions as a transcription factor.6 Physiologically, HB9 is expressed during embryogenesis and is essential for the formation of the dorsal pancreatic bud and B-cell matura- tion.7-9 In addition, HB9 plays an important role in neu- ronal development by promoting motor neuron differenti- ation.10,11 A deregulated HB9 expression has been found in several tumor types. In poorly differentiated hepatocellu- lar carcinomas, microarray analyses identified HB9 as the strongest differentially expressed gene compared to non- neoplastic hepatic controls.12 Also in transcriptome analy- sis of prostate cancer biopsies from African-Americans, HB9 was the most highly up-regulated protein coding gene compared to matched benign tissues.13
In hematopoietic neoplasias, HB9 is aberrantly highly expressed in translocation t(7;12) acute myeloid leukemia (AML), which accounts for up to 30% of infant AML.14,15 Translocation t(7;12) AML patients have a very dismal prognosis, with a 3-year event-free survival of 0%, regard- less of the treatment approach.15,16 Since its first descrip- tion in 2000, aberrant HB9 expression remains the only known molecular hallmark of translocation t(7;12) AML,17,18 but only poor functional data exist regarding its oncogenic properties and how, if at all, aberrant HB9 expression influences hematopoiesis, thereby contributing to leukemogenesis. Early expression studies reported HB9 expression in healthy CD34+ hematopoietic stem and pro- genitor cells (HSPCs),19 but could not be validated by stud- ies of our and other groups.15,20,21 Hence, a physiological function of HB9 in HSPCs remains a subject of debate. Morphologically, translocation t(7;12) AML blast cells are less differentiated (FAB subtype M0 or M2), accompanied by expression of stem cell markers like CD34 and CD117,15,22 indicating a very early differentiation block. Gene expression profiling of HB9+ blast cells revealed a modulation of cell-cell interaction and cell adhesion.22 In previous studies, we had used the AML cell line HL-60 for stable HB9 overexpression to identify potential HB9 target genes by combined ChIP-on-chip and expression analy- ses.21 As HL-60 cells represent an already transformed AML cell line model, harboring several genetic aberrations like loss of p53 and MYC replication,23 it is difficult to come to any conclusions about the oncogenic potential of HB9 and its influence on primary hematopoietic cells in vivo with respect to translocation t(7;12) leukemogenesis.
Thus, in our current study, we evaluated the oncogenic potential of HB9 by its effect on proliferation and cell cycle regulation. Furthermore, we performed for the first time in vivo hematopoietic reconstitution experiments to investigate the influence of HB9 expression on hematopoietic cell differentiation with regard to transloca- tion t(7;12) AML.
Methods
Cell cycle analysis
3x105 cells were washed twice with PBS and resuspended in hypotonic buffer solution, containing 0.1% Triton-X 100, 0.1% sodium-citrate and 50 μg/mL propidium iodide. After resuspen- sion, cells were incubated for 10 minutes in the dark at room tem-
perature and immediately analyzed by flow cytometry (FACSCalibur, BD Biosciences, Heidelberg, Germany).
β-galactosidase staining
Six days after transduction, cells were stained for β-galactosi-
dase activity using the Senescence Cells Histochemical Staining Kit (Sigma-Aldrich, Taufkirchen, Germany) according to the man- ufacturer’s instructions. A total of 300 cells were counted for each replicate and the frequency of positive cells was determined. Images were taken using an Axiovert 200 microscope (Zeiss, Jena, Germany).
Bone marrow transplantation
Bone marrow cells were harvested from femurs and tibiae of 8-10-week old male C57BL/6 mice and Lineage+ cells were deplet- ed using the Lineage Cell Depletion Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Lin– cells were cultured in Stemspan SFEM (Stemcell Technologies, Cologne, Germany), supplemented with 1% penicillin-streptomycin, 100 ng/mL SCF, 100 ng/mL FLT3L, 100 ng/mL TPO, 25 ng/mL IL6 and 25 ng/mL IL11 (PeproTech, Hamburg, Germany ). Twenty-four hours (h) after isolation, Lin– cells were lentivirally transduced (MOI 50), together with hexa- dimethrine bromide (5 mg/mL; Sigma-Aldrich) on RetroNectin (Takara, Frankfurt, Germany) coated plates, and spinoculated at 1000 g for 2 h at 32°C. One day after transduction, Lin– cells were transplanted via tail-vein injection into myeloablative-irradiated 8- 10-week old female B6.SJL-Ptprc Pepc/BoyJ mice.
Isolation of CD34+ cord blood cells
The use of primary human CD34+ cord blood cells for this study was approved by the ethics committee of the medical faculty of the Heinrich-Heine-University, Duesseldorf, and was carried out in accordance with the Declaration of Helsinki. Cord blood sam- ples were obtained from healthy donors after informed consent (José Carreras Stem Cell Bank, University Hospital Duesseldorf, Germany) and mononuclear cells were enriched by density gradi- ent centrifugation with Ficoll Paque Plus (GE Healthcare, Frankfurt, Germany). CD34+ cells were enriched using CD34 MicroBead Kit and MACS technology (Miltenyi Biotec) according to the manufacturer’s instructions and cultivated in X-Vivo 20 (Lonza, Cologne, Germany) supplemented with 100 ng/mL SCF, 100 ng/mL FLT3L, 100 ng/mL TPO and 20 ng/mL IL3 (Peprotech).
Statistical analysis
Statistical significance was determined from adequately pow- ered sample sizes of similar variation using two-tailed unpaired Student t-tests and was defined as P≤0.05 (*), P≤0.01 (**) and P≤0.001 (***). Sample sizes are given in the figure legends.
Other methods
The details of other methods, including virus production, immunofluorescence, siRNA transfection, cell preparation and flow cytometric analysis, as well as western blot, PCR, quantita- tive Reverse Transcriptase-PCR and RNA-Seq-analysis are given in the Online Supplementary Appendix.
The RNA-Seq data generated in this study can be found with accession number GSE117060 in the NCBI GEO database.
Results
Aberrant HB9 expression induces premature senescence
In order to investigate the influence of HB9 on cellular proliferation and cell cycle, human HT1080 and murine
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haematologica | 2019; 104(1)