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Autophagy describes a process of membrane trafficking where specialized compartments (autophagosomes) engulf damaged or dispensable organelles and target them to lysosomic degradation. Thus, autophagy has cytoprotective functions e.g. during starvation, but autophagy can also lead to caspase-independent cell death (PCD type II). ATG5 is a central player in autophagy and inactivating ATG5 severely impairs normal development. In hematopoiesis, ATG5 is essential for maturation of B lymphocytes as well as for T cell survival and proliferation. In recent years, several differentiation and death-inducing agents were shown to activate autophagy in acute myeloid leukemia (AML) cell line models, but the mechanisms involved are still poorly defined. We therefore decided to investigate the role of ATG5 and autophagy in all-trans retinoic acid (ATRA)-induced neutrophil differentiation of AML cells. We found that ATG5 mRNA was upregulated 5.9-, 3.8- and 3.4-fold after 6 days of ATRA-treatment in HL60, NB4 and HT93 AML cells, respectively. In contrast, PMA-induced macrophage differentiation of HL60 and U937 cells only slightly induced ATG5 mRNA by 1.3- and 1.5-fold, respectively, indicating a specific role for ATG5 in granulocyte differentiation. In line with the above observation in leukemic cell lines, we found that ATG5 mRNA levels were increased in 5/5 APL patients upon ATRA therapy. In addition, ATG5-ATG12 conjugates, hallmarks of the autophagic process, were markedly activated in ATRA-treated NB4 and HL60 cells compared to control cells as measured by Western blotting. In agreement, we found increased autophagic activity during myeloid differentiation as evidenced by two additional autophagy markers, i.e. conversion of light chain 3 B (LC3B)-I into LC3B-II and degradation of sequestosome 1 (SQSTM1, p62) protein by Western blotting. Inhibition of autophagy during ATRA treatment of NB4 and HL60 cells with 3-Methyladenine or Bafilomycin A significantly impaired neutrophil differentiation by 80% as measured by CD11b surface expression. Similarly, lentivirus-driven short hairpin (sh)RNA-mediated silencing of ATG5 in NB4 cells resulted in an 85% reduction of ATRA-induced neutrophil differentiation as measured by CD11b surface expression and by quantitative RT-PCR of the myeloid differentiation markers GCSFR, C/EBPε and lactotransferrin. Interestingly, inhibition of autophagy increased overall cell death during the myeloid differentiation process rather than reducing it as measured by reduction of tetrazolium salt (XTT assay). Enhanced cell death and reduced myeloid differentiation upon blocking ATG5 would suggest that autophagy is needed for maintaining myeloid differentiation. Further support for our hypothesis that ATG5 is needed for neutrophil development stems from our survey of ATG5 mRNA expression in primary hematopoietic cells. We found significantly higher ATG5 mRNA levels in granulocytes and macrophages (n=7) as compared to CD34+ hematopoietic progenitor cells from healthy donors (n=4; p=0.0424) as well as compared to primary AML patient samples at diagnosis (n=76; p=0.0003). ATG5 mRNA expression in CD34+ and AML patients was not significantly different (p=0.6669). In summary, we show a correlation of high ATG5 expression with terminal myeloid differentiation and of low ATG5 expression with a myeloid leukemic phenotype. Using chemical inhibitors of autophagy as well as RNAi technology to knockdown ATG5, we further provide evidence that ATG5 and consequently autophagy are essential for neutrophil development.

PU.1 is a hematopoietic transcriptional regulator that is necessary for the development of both myeloid and B cells. To identify new PU.1 target genes in neutrophil development PU.1 was introduced into mouse 503 PU.1-null cells using lentiviral gene transfer and microarray analyses of two independent 503 PU.1-rescued and parental 503 cells were compared. The BCL2A1 gene was found to be more than 50-fold induced in 503 PU.1- restored as compared to the parental 503-null cells. BCL2A1 (also known as BFL-1/A1) is an anti-apoptotic member of the BCL2 family. BCL2A1 was initially identified as a tissue-specific BCL2-related factor that is induced by different reagents such as granulocyte macrophage colony-stimulating factor (GM-CSF) or all-trans retinoic acid (ATRA) during myeloid differentiation. Upregulation of BCL2A1 in granulocytes may promote a time-dependent survival. To follow up on our microarray findings we evaluated loss of PU.1 function in human NB4 acute promyelocytic leukemia (APL) cells using lentivector delivered, short hairpin (sh) RNAs targeting PU.1. Knockdown efficacy upon ATRA-treatment in the two shPU.1 expressing NB4 cell lines was 67 and 30%, respectively. Silencing of PU.1 markedly reduced BCL2A1 mRNA induction upon ATRA-treatment from 167-fold in control cells to 47- and 112-fold in the two PU.1 knockdown NB4 cell lines, respectively (Figure A). Co-transfection of PU.1 with a human BCL2A1 promoter reporter resulted in a 7-fold activation, suggesting PU.1 can directly regulate BCL2A1. Co-transfection with NF-kappaB, used as positive control, induced the BCL2A1 promoter 14.5-fold. Moreover, in vivo binding of the transcription factor PU.1 to 2/8 putative PU.1 binding sites in the BCL2A1 promoter was shown by chromatin immunoprecipitation in HL60 promyelocytic cells further supporting a role for PU.1 regulation of BCL2A1. Evaluation of BCL2A1 and PU.1 mRNA expression in CD34+ hematopoietic progenitors, granulocytes, and primary acute myeloid leukemia (AML) cells was assessed using real-time quantitative RT-PCR. BCL2A1 and PU.1 mRNA levels were significantly lower in primary AML patient samples (n=80; p<0.0001) and in CD34+ progenitor cells (n=4; p=0.0095) than in granulocytes (n=6; Figure B). In line with this observation, we found that upon ATRA therapy BCL2A1 levels were increased in 5/5 APL patients and PU.1 mRNA levels in 4/5 APL cases, respectively. Altogether, these results clearly indicate that PU.1 and BCL2A1 are co-regulated during granulocyte differentiation. Lastly, we confirmed earlier data showing that ATRA-pretreatment of NB4 cells and thus induction of PU.1 and BCL2A1, rendered these cells less sensitive to arsenic trioxide (As2O3)- induced cell death. Conversely, NB4 PU.1 knockdown cells were markedly more sensitive to As2O3 -induced cell death upon ATRA-pretreatment than the parental NB4 control cells. The increase in sensitivity to As2O3 correlated with the lower BCL2A1 levels found in the PU.1 knockdown cells. In summary, we identified the anti-apoptotic BCL2A1 gene as direct, transcriptional target of PU.1 in myeloid leukemic cells. We hypothesize that PU.1-dependent induction of BCL2A1 is necessary for the survival of normal, terminally differentiated myeloid cells. Furthermore, aberrant expression of PU.1 in erythroleukemia may result in elevated BCL2A1 levels that support increased survival of erythroblasts in this particular type of leukemia. Figure Figure

The N-myc down-regulated gene 1 (NDRG1) is a stressed induced protein whose expression is associated with growth arrest and differentiation of tumor cells. Although the exact function of NDRG1 protein remains unknown, various studies support its role as a suppressor of tumor metastasis. In prostate, colon and breast cancer its expression is associated with a better disease prognosis and patient survival. In hematopoietic cells, NDRG1 was identified in a differential display screen for differentiation-related genes in human myelomonocytic U937 cells. In the present study, we sought to investigate the role of NDRG1 in myeloid differentiation. To this end we first evaluated NDRG1 mRNA expression in acute myeloid leukemia (AML; n=82) patient samples as well as in CD34+ progenitor cells (n=5) and neutrophils (n=6) of healthy donors using quantitative real-time RT-PCR. We found significantly higher NDRG1 mRNA levels in granulocytes as compared to CD34+ (p=0.0043) or AML blast cells (p<0.0001), whereas no significant difference between CD34+ progenitor and AML blast cells was seen (Figure A). Moreover, we found that NDRG1 mRNA levels were increased in 4/5 APL patients upon ATRA therapy. In contrast, the closest relative of NDRG1, NDRG2, did not show significantly different expression in these primary cells, thus indicating a unique role for NDRG1 in granulocyte differentiation. Next we examined NDRG1 expression using quantitative RT-PCR and Western blotting in two different cell line models for ATRA-induced neutrophil differentiation. ATRA treatment of NB4 and HT93 acute promyelocytic leukemia (APL) cells induced NDRG1 mRNA 2.3- and 14.3- fold, respectively. Increased NDRG1 mRNA expression was paralleled by an increase of NDRG1 protein as well as a decrease in c-myc protein. Earlier reports described that NDRG1 is also suppressed by c-myc suggesting that down-regulation of c-myc in our cell line models allowed for an increase of NDRG1. In line with these observations, lentivirus-driven short hairpin (sh)RNA-mediated silencing of NDRG1 diminished ATRA-induced neutrophil differentiation of NB4 and U937 cells as measured by CD11b, CD11c and CD18 surface expression. In NB4 NDRG1 knockdown versus non-targeting shRNA expressing cells mean fluorescent intensities (MFI) for CD11b, CD11c and CD18 upon six days of ATRA-treatment were 99±17 vs 146±7, 20±2 vs 32±10 and 19±2 vs 45±6, respectively (Figure B). Similarly, U937 NDRG1 knockdown versus control cells displayed the following MFIs for CD11b and CD18 upon neutrophil differentiation: 61±1 vs 102±2 and 11±4 vs 33±13, respectively. In conclusion, we report here for the first time an association of low NDRG1 levels with an immature hematopoietic cell phenotype. Using RNAi technology we further provide evidence that NDRG1 is functionally involved in neutrophil maturation. Figure Figure