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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.

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

Inactivation of PU.1 results in a myeloid differentiation block and contributes to the pathogenesis of acute myeloid leukemia (AML). A long list of PU.1 transcriptional targets with a direct function in myeloid development exits and clearly supports its role in cellular hematopoietic differentiation processes. Furthermore, we and others showed that PU.1 positively affects cell survival, for example by directly activating the anti-apoptotic genes BCL2A1 and BCL-XL. Recently, we demonstrated that the novel PU.1 target Hexokinase 3 supports acute promyelocytic leukemia (APL) cell survival. Surprisingly, our current data indicate that knocking down PU.1 in NB4 APL significantly increased resistance in apoptosis responses to anthracyclins. These findings were confirmed in MOLM-3 AML cells. Both cell lines showed an induction of the anti-apoptotic gene CFLAR (c-FLIP). c-FLIP blocks the activation of procaspase-8 at the death-inducing signalling complex (DISC) and is involved in AML chemotherapy resistance. Using PU.1 and c-FLIP double knockdown APL cells, we could significantly restore the sensitivity to anthracyclin treatment. This indicates that the PU.1 knockdown-dependent increase of c-FLIP is responsible for the resistance towards anthracyclin treatment. Moreover, we identified the pro-apoptotic kinase DAPK2 as novel transcriptional target of PU.1 in APL cells. Interestingly, we found that inhibiting DAPK2 in APL cells significantly attenuater arsenic trioxide (ATO)-induced cell death. Together, we link PU.1 to apoptosis and resistance against antracyclins and ATO via regulating c-FLIP and DAPK2. Our data indicate that low PU.1 level found in AML patients not only contribute to a block in differentiation but also increase resistance to cytotoxic therapies in AML. Citation Format: Mario P. Tschan, Aladin Haimovici, Daniel Brigger, Anna M. Schläfli, Deborah Shan, Martin F. Fey. Low PU.1 expression not only attenuates neutrophil differentiation of AML cells but also increases resistance to cytotoxic therapies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2348. doi:10.1158/1538-7445.AM2014-2348

1376 The myeloid key transcription factor CEBPA (CCAAT-enhancer binding protein alpha) is blocked by the mRNA binding protein calreticulin (CRT) thereby affecting myeloid differentiation. We previously reported that the unfolded protein response (UPR) is activated in 24.5% of AML patients, involving the up-regulation of endoplasmatic reticulum (ER) stressors, such as CRT. In addition, anthracycline treatment was shown to trigger the UPR and to specifically induce CRT cell membrane surface expression in some solid tumor types. This ecto-CRT is specifically targeting dendritic cells and initiates immunogenic cell death of cancer cells by acting as an “eat-me” signal. However, UPR induced translocation of CRT in leukemogenesis has hardly been investigated so far. This study aimed at analyzing anthracycline induced CRT exposure and extracellular release of CRT in leukemic cells as well as at elucidating the candidate pathways involved such as the UPR transducers PERK, ATF6 and IRE1. In leukemic cell lines, we observed that anthracycline treatment was inducing CRT expression on the cell membrane surface within 2 hours. This effect was caused by intracellular translocation, but not de novo synthesis. Exposure of CRT was decreased by Brefeldin A, which blocks the vesicle transport from the ER to the Golgi apparatus within 2 hours of treatment, suggesting that exocytosis is critical for CRT delocalization. By specific inhibition of the PERK pathway of the UPR using Salubrinal, we observed reduced CRT expression on the cell membrane. In contrast, blocking the ATF6 or the IRE1 pathway did not affect CRT exposure. Mean CRT levels on the cell membrane of blasts from 53 AML patients at diagnosis were significantly (21-fold; p<0.0005) increased compared to CRT levels of myeloid cells from healthy volunteers (n=75). Using an ELISA assay investigating serum from 104 AML patients at diagnosis and 86 healthy volunteers, we found that extracellular CRT was significantly (2.2-fold; p<0.0001) increased in AML patients compared to healthy controls. We observed a more favorable outcome in AML patients with increased CRT expression on the cell membrane, as well as in patients with higher levels of extracellular CRT in the serum. Finally, we identified a strong correlation between CRT serum levels and CRT expression on the cell surface of leukemic cells. Ongoing studies aim at elucidating the mechanisms of CRT secretion from the cell membrane to the serum. In conclusion, CRT translocation to the cell membrane and increasing levels of secreted CRT in the serum represent early events of anthracycline treatment in leukemic cells, and both are associated with a more favorable outcome in AML patients. Disclosures: No relevant conflicts of interest to declare.