Following fluorescence activated cell sorting FACS

Following fluorescence activated cell sorting (FACS) for dTomato-reporter expression, HSPCs were maintained for ten days in liquid culture using StemSpan medium (StemCell Technologies, Grenoble, France) supplemented with 1% penicillin/streptomycin, 1% l-glutamine (Biochrom AG, Berlin, Germany) and the following recombinant human cytokines: 100ng/mlM-CSF, 50ng/ml FLT3-L, 20ng/ml SCF, and 20ng/ml IL-6 (all PeproTech, Hamburg, Germany). For flow cytometry, the following SAR 405 were used: anti-human CD11b APC (ICRF44) (eBioscience, Frankfurt am Main, Germany); anti-human CD14 APC (555399) (BD Pharmingen, Heidelberg, Germany). Colony formation assays were performed two days after transduction with 5000 FACS-purified reporter-positive cells per 35mm2 culture dish using MethoCult™ H4230 methylcellulose (StemCell Technologies) supplemented with 1% penicillin/streptomycin (Biochrom AG), as well as a recombinant human cytokine cocktail containing 100ng/ml SCF, 20ng/mlG-CSF, 20ng/ml GM-CSF, 20ng/ml IL-3, and 20ng/ml IL-6 (PeproTech). Granulocyte/macrophage colony-forming units (CFU-GM) were determined after 14days of cultivation under standard conditions.
Results & discussion To study the impact of GABP during myeloid differentiation and self-renewal of primary human hematopoietic stem/progenitor cells (HSPCs), GABP function was impaired by ectopic overexpression of a GABPβ1 subunit lacking the TAD (GABPB1.ΔTAD, shown schematically in Fig. 1A) (Ripperger et al., 2015). GABPB1.ΔTAD ectopic overexpression was performed since knockdown efficiencies of shRNAs targeting GABP subunits were not sufficient in HSPCs (data not shown). Initially, effects of GABPB1.ΔTAD ectopic overexpression were studied in the BCR-ABL1+ K-562 cell line in combination with imatinib treatment to confirm the dominant-negative action of the mutant protein (Fig. 1B). A similar proliferation behavior in terms of elevated sensitivity to imatinib upon GABPB1.ΔTAD ectopic overexpression was observed as compared to our previous investigations applying shRNA-mediated knockdown of GABPA in K-562 cells (Manukjan et al., 2015). Hence, the approach is feasible to be used in primary HSPCs particularly as overexpression efficiency could be proven by qPCR (Fig. 1C). Subsequently, liquid culture assays on mobilized peripheral blood (mPB) CD34+ cells were performed after transduction with the empty vector control or GABPB1.ΔTAD. CD34+ cells were achieved by leukapheresis of AML patients at remission. Upon transduction with GABPB1.ΔTAD, CD11b and CD14 expressions were significantly decreased in mPB CD34+ HSPCs after ten days in liquid culture containing a myeloid cytokine cocktail (Fig. 2A). CD11b and CD14 are defined cell-surface markers of the mature myelo-monocytic compartment. The significant reduction of CD11b+ and CD14+ cells indicates that functional GABP is necessary to perform proper myelopoiesis in human CD34+ HSPCs. In this context, GABP was recently shown to directly regulate expression of ITGAM (integrin alpha M), which codes for CD11b (Ripperger et al., 2015). Remarkably, after 14days in liquid culture containing a myeloid cytokine cocktail, fewer total cell numbers were detected in the GABPB1.ΔTAD group (Fig. 2B). This confirms the lowered myelopoietic potential of mPB CD34+ HSPCs with functionally impaired GABP. In addition, we performed colony formation assays focusing on granulocyte/macrophage (GM) colonies. Upon ectopic overexpression of GABPB1.ΔTAD, colony forming capacity of CD34+ HSPCs derived from mPB and healthy bone marrow (BM) was significantly decreased (Fig. 2C). Apart from GABP\'s effect on myeloid differentiation, these results indicate its crucial role for human hematopoietic self-renewal potential. To address the question whether GABP also contributes to the propagation of human leukemic stem/progenitor cells, we tested CD34+ cells obtained from five CML patients at diagnosis. Cytogenetic and molecular genetic analyses showed the presence of BCR-ABL1 fusion and indicated high tumor cell burden. Colony formation assays were performed with CD34+ cells from each individual and showed remarkably decreased colony forming capacity (>80%) upon overexpression of GABPB1.ΔTAD (Fig. 3). Noteworthy, colony forming capacities were heterogeneous among individual patient samples. This observation did not correlate with the BCR-ABL1/ABL1 ratio, cytogenetic aberrations, sex, age at diagnosis or any other known traits. It remains speculative whether this is due to pre-programmed cell-extrinsic effects or if undetected genomic alterations may be responsible. Moreover, by applying qPCR on colony forming cells maintained in methylcellulose for 14days, initially present overexpression of GABPB1.ΔTAD was lacking (data not shown). In line with this, expression of the dTomato reporter was decreased in GABPB1.ΔTAD overexpressing cells to nearly undetectable levels indicating transgene silencing in those cells (Zhang et al., 2007). This hints towards a negative selective pressure on cells overexpressing GABPB1.ΔTAD confirming the necessity of functional GABP during leukemic stem cell maintenance.