Elena A. Federzoni

A novel TNFR1-triggered apoptosis pathway mediated by class IA PI3Ks in neutrophils

The most common form of neutrophil death is apoptosis. In the present study, we report surprising differences in the molecular mechanisms used for caspase activation between FAS/CD95-stimulated and TNF receptor 1 (TNFR1)-stimulated neutrophils. Whereas FAS-induced apoptosis was followed by caspase-8 activation and required Bid to initiate the mitochondrial amplification loop, TNF-α-induced apoptosis involved class IA PI3Ks, which were activated by MAPK p38. TNF-α-induced PI3K activation resulted in the generation of reactive oxygen species, which activated caspase-3, a mechanism that did not operate in neutrophils without active NADPH oxidase. We conclude that in neutrophils, proapoptotic pathways after TNFR1 stimulation are initiated by p38 and PI3K, but not by caspase-8, a finding that should be considered in anti-inflammatory drug-development strategies.

MicroRNA-29b is involved in the Src-ID1 signaling pathway and is dysregulated in human lung adenocarcinoma

The c-Src kinase regulates cancer cell invasion through inhibitor of DNA binding/differentiation 1 (ID1). Src and ID1 are frequently overexpressed in human lung adenocarcinoma. The current study aimed at identifying microRNAs (miRNAs) involved in the Src-ID1 signaling in lung cancer. Incubation of lung cancer cells with the Src inhibitor saracatinib led to the upregulation of several miRNAs including miR-29b, which was the most highly upregulated miRNA with predicted binding to the ID1 3′-untranslated region (UTR). Luciferase reporter assays confirmed direct binding of miR-29b to the ID1 3′-UTR. Expression of miR-29b suppressed ID1 levels and significantly reduced migration and invasion. Expression of antisense-miR-29b (anti-miR-29b), on the other hand, enhanced ID1 mRNA and protein levels, and significantly increased lung cancer cell migration and invasion, a hallmark of the Src-ID1 pathway. The ectopic expression of ID1 in miR-29b-overexpressing cells was able to rescue the migratory potential of these cells. Both, anti-miR-29b and ID1 overexpression diminished the effects of the Src inhibitors saracatinib and dasatinib on migration and invasion. Saracatinib and dasatinib decreased c-Myc transcriptional repression on miR-29b and led to increased ID1 protein levels, whereas forced expression of c-Myc repressed miR-29b and induced ID1. In agreement, we showed direct recruitment of c-Myc to the miR-29b promoter. miR-29b was significantly downregulated in primary lung adenocarcinoma samples compared with matched alveolar lung tissue, and miR-29b expression was a significant prognostic factor for patient outcome. These results suggest that miR-29b is involved in the Src-ID1 signaling pathway, is dysregulated in lung adenocarcinoma and is a potential predictive marker for Src kinase inhibitors.

Inhibition of GSK3B Bypass Drug Resistance of p53-Null Colon Carcinomas by Enabling Necroptosis in Response to Chemotherapy

Purpose: Evasion from chemotherapy-induced apoptosis due to p53 loss strongly contributes to drug resistance. Identification of specific targets for the treatment of drug-resistant p53-null tumors would therefore increase the effectiveness of cancer therapy. Experimental Design: By using a kinase-directed short hairpin RNA library and HCT116p53KO drug-resistant colon carcinoma cells, glycogen synthase kinase 3 beta (GSK3B) was identified as a target whose silencing bypasses drug resistance due to loss of p53. p53-null colon cancer cell lines with different sets of mutations were used to validate the role of GSK3B in sustaining resistance and to characterize cell death mechanisms triggered by chemotherapy when GSK3B is silenced. In vivo xenograft studies were conducted to confirm resensitization of drug-resistant cells to chemotherapy upon GSK3 inhibition. Colon cancer samples from a cohort of 50 chemotherapy-treated stage II patients were analyzed for active GSK3B expression. Results: Downregulation of GSK3B in various drug-resistant p53-null colon cancer cell lines abolished cell viability and colony growth after drug addition without affecting cell proliferation or cell cycle in untreated cells. Cell death of 5-fluorouracil (5FU)–treated p53-null GSK3B-silenced colon carcinoma cells occurred via PARP1-dependent and AIF-mediated but RIP1-independent necroptosis. In vivo studies showed that drug-resistant xenograft tumor mass was significantly reduced only when 5FU was given after GSK3B inhibition. Tissue microarray analysis of colon carcinoma samples from 5FU-treated patients revealed that GSK3B is significantly more activated in drug-resistant versus responsive patients. Conclusions: Targeting GSK3B, in combination with chemotherapy, may represent a novel strategy for the treatment of chemotherapy-resistant tumors. Clin Cancer Res; 19(14); 3820–31. ©2013 AACR.

PU.1 is linking the glycolytic enzyme HK3 in neutrophil differentiation and survival of APL cells

The transcription factor PU.1 is a master regulator of myeloid differentiation and function. On the other hand, only scarce information is available on PU.1-regulated genes involved in cell survival. We now identified the glycolytic enzyme hexokinase 3 (HK3), a gene with cytoprotective functions, as transcriptional target of PU.1. Interestingly, HK3 expression is highly associated with the myeloid lineage and was significantly decreased in acute myeloid leukemia patients compared with normal granulocytes. Moreover, HK3 expression was significantly lower in acute promyelocytic leukemia (APL) compared with non-APL patient samples. In line with the observations in primary APL patient samples, we observed significantly higher HK3 expression during neutrophil differentiation of APL cell lines. Moreover, knocking down PU.1 impaired HK3 induction during neutrophil differentiation. In vivo binding of PU.1 and PML-RARA to the HK3 promoter was found, and PML-RARA attenuated PU.1 activation of the HK3 promoter. Next, inhibiting HK3 in APL cell lines resulted in significantly reduced neutrophil differentiation and viability compared with control cells. Our findings strongly suggest that HK3 is: (1) directly activated by PU.1, (2) repressed by PML-RARA, and (3) functionally involved in neutrophil differentiation and cell viability of APL cells.

CEBPA-dependent HK3 and KLF5 expression in primary AML and during AML differentiation

The basic leucine zipper transcription factor CCAAT/enhancer binding protein alpha (CEBPA) codes for a critical regulator during neutrophil differentiation. Aberrant expression or function of this protein contributes to the development of acute myeloid leukemia (AML). In this study, we identified two novel unrelated CEBPA target genes, the glycolytic enzyme hexokinase 3 (HK3) and the krüppel-like factor 5 (KLF5) transcription factor, by comparing gene profiles in two cohorts of CEBPA wild-type and mutant AML patients. In addition, we found CEBPA-dependent activation of HK3 and KLF5 transcription during all-trans retinoic acid (ATRA) mediated neutrophil differentiation of acute promyelocytic leukemia (APL) cells. Moreover, we observed direct regulation of HK3 by CEBPA, whereas our data suggest an indirect regulation of KLF5 by this transcription factor. Altogether, our data provide an explanation for low HK3 and KLF5 expression in particular AML subtype and establish these genes as novel CEBPA targets during neutrophil differentiation.

Transcriptional regulation of MIR29B by PU.1 (SPI1) and MYC during neutrophil differentiation of acute promyelocytic leukaemia cells

Lineage commitment of haematopoietic cells is tightly regulated through an intricate network of molecular pathways and cascades of genes including microRNAs (miRNAs). Moreover, recent studies show that abnormal expression of miRNAs directly contributes to haematological malignancies, such as acute myeloid leukaemia (AML), chronic myeloid leukaemia (CML) and myelodysplastic syndromes (MDS) (Fabbri et al, 2008; Starczynowski et al, 2011). miRNAs are 19–25 nucleotide long, non-protein coding RNAs that regulate gene expression by binding to partially complementary sequences in the 3¢-untranslated regions (3¢-UTR) of their target mRNAs. Acute promyelocytic leukaemia (APL) is characterized by the t(15;17) translocation resulting in the oncogenic PML-RARA fusion gene. Pharmacologial doses of all-trans retinoic acid (ATRA) are used to directly target PML-RARA for degradation and, in combination with chemotherapy, have been successfully used in the treatment of APL patients (de The & Chen, 2010). A discrete number of miRNAs modulated upon ATRA treatment of APL cells in vitro have been published (Garzon et al, 2007; Saumet et al, 2009). Among them MIR29B, a key miRNA induced during neutrophil differentiation of APL cells. The tumour suppressor MIR29B plays a role in targeting leukaemic oncogenes such as DNA methyltransferase 3A/B (DNMT3A/DNMT3B) in AML cells (Garzon et al, 2009). In addition, high MIR29B levels were associated with a response to the demethylating agent decitabine in AML (Blum et al, 2010). The present study aimed to confirm a role for MIR29B in the neutrophil differentiation of APL cells and to analyse the transcriptional regulation of MIR29B during this process. We first measured MIR29B expression during ATRAinduced neutrophil differentiation of NB4 and HT93 APL cell lines. MIR29B was clearly induced in both APL cell lines (Fig 1A). Furthermore, MIR29B expression was markedly induced in an APL patient undergoing ATRA therapy (Fig 1B). We then measured MIR29B in blast cells of primary AML patients of different French-American-British (FAB) subtypes (M0-M4) as well as in mature neutrophils from healthy donors. We observed significantly lower MIR29B expression levels in AML patient samples as compared to normal granulocytes from healthy individuals (Fig 1C). Lastly, to further define the role of MIR29B in neutrophil differentiation, we inhibited its expression by antisense MIR29B (antiMIR29B) in HT93 APL cells. Neutrophil differentiation in anti-MIR29B expressing HT93 cells was significantly reduced compared to control cells as evidenced by lower CD11b levels (Fig 1D,E). As a control for the functionality of our antisense MIR29B, we analysed the protein expression of the reported MIR29B target gene DNMT3B. We found markedly elevated DNMT3B protein levels in anti-MIR29B expressing AML cells indicating successful inhibition of MIR29B (Fig 1F). In general, our results indicated that MIR29B expression was not only suppressed in APL but generally in AML patients, indicating that the low expression is associated with an immature myeloid phenotype. In summary, these data suggest that MIR29B is associated with granulocytic differentiation and further support a tumour suppressor function of MIR29B in AML. To assess transcriptonal regulation of MIR29B, we analysed its promoter region for myeloid transcription factor binding sites. We identified two putative responsive elements of the myeloid master regulator PU.1 (SPI1) upstream of the MIR29B2/MIR29C (MIR29B2/C) gene (Fig 2A). First, to test if PU.1 activates MIR29B2/C transcription, we cloned a 1Æ4-kb putative MIR29B2/C promoter carrying the two PU.1 response elements and a shorter 0Æ7-kb construct encompassing only one PU.1 binding element into luciferase reporter vectors. PU.1 specifically increased promoter activity of both constructs to the same levels in a dose-dependent manner (Fig 2B). Our experiments indicate that the PU.1 binding site between the MIR29B2 and MIR29C coding regions is the major PU.1 responsive element. Next, we showed direct binding of PU.1 to the MIR29B2/C promoter in vivo using chromatin immunoprecipitation (ChIP) assays (Fig 2C). To analyse PU.1-dependent MIR29B induction during neutrophil differentiation, we assessed its regulation in HT93 PU.1 knockdown cells upon ATRA treatment. Indeed, inhibiting PU.1 in HT93 cells resulted in 50% reduction of ATRAinduced MIR29B expression (Fig 2D, left panel). Successful PU.1 knockdown was confirmed by real-time quantitative polymerase chain reaction and Western blotting in HT93 shPU.1_1 and _2 knockdown cells (Figs 2D, right panel and 2E). PU.1 induced MIR29B by directly interacting with its promoter and we confirmed an essential role for PU.1 in MIR29B2/C regulation upon myeloid differentiation by ChIP, luciferase reporter and PU.1 knockdown experiments. Chang et al (2008) placed the transcriptional start site of MIR29B2/C within a conserved regulatory region located 20 kb upstream of the MIR29B2/C cluster on chromosome 1. However, we identified a functional PU.1 binding site in the proximal MIR29B2/C promoter, indicating additional regulatory eleCorrespondence

The RNA binding proteins RBM38 and DND1 are repressed in AML and have a novel function in APL differentiation

The RNA binding proteins RBM binding motif protein 38 (RBM38) and DEAD END 1 (DND1) selectively stabilize mRNAs by attenuating RNAse activity or protecting them from micro(mi)RNA-mediated cleavage. Furthermore, both proteins can efficiently stabilize the mRNA of the cell cycle inhibitor p21(CIP1). Since acute myeloid leukemia (AML) differentiation requires cell cycle arrest and RBM38 as well as DND1 have antiproliferative functions, we hypothesized that decreased RBM38 and DND1 expression may contribute to the differentiation block seen in this disease. We first quantified RBM38 and DND1 mRNA expression in clinical AML patient samples and CD34(+) progenitor cells and mature granulocytes from healthy donors. We found significantly lower RBM38 and DND1 mRNA levels in AML blasts and CD34(+) progenitor cells as compared to mature neutrophils from healthy donors. Furthermore, the lowest expression of both RBM38 and DND1 mRNA correlated with t(8;21). In addition, neutrophil differentiation of CD34(+) cells in vitro with G-CSF (granulocyte colony stimulating factor) resulted in a significant increase of RBM38 and DND1 mRNA levels. Similarly, neutrophil differentiation of NB4 acute promyelocytic leukemia (APL) cells was associated with a significant induction of RBM38 and DND1 expression. To address the function of RBM38 and DND1 in neutrophil differentiation, we generated two independent NB4RBM38 as well as DND1 knockdown cell lines. Inhibition of both RBM38 and DND1 mRNA significantly attenuated NB4 differentiation and resulted in decreased p21(CIP1) mRNA expression. Our results clearly indicate that expression of the RNA binding proteins RBM38 and DND1 is repressed in primary AML patients, that neutrophil differentiation is dependent on increased expression of both proteins, and that these proteins have a critical role in regulating p21(CIP1) expression during APL differentiation.

The tumor suppressor gene DAPK2 is induced by the myeloid transcription factors PU.1 and C/EBPα during granulocytic differentiation but repressed by PML-RARα in APL

DAPK2 is a proapoptotic protein that is mostly expressed in the hematopoietic tissue. A detailed DAPK2 expression analysis in two large AML patient cohorts revealed particularly low DAPK2 mRNA levels in APL. DAPK2 levels were restored in APL patients undergoing ATRA therapy. PML‐RARA is the predominant lesion in APL causing transcriptional repression of genes important for neutrophil differentiation. We found binding of PML‐RARA and PU.1, a myeloid master regulator, to RARA and PU.1 binding sites in the DAPK2 promoter. Ectopic expression of PML‐RARA in non‐APL, as well as knocking down PU.1 in APL cells, resulted in a significant reduction of DAPK2 expression. Restoring DAPK2 expression in PU.1 knockdown APL cells partially rescued neutrophil differentiation, thereby identifying DAPK2 as a relevant PU.1 downstream effector. Moreover, low DAPK2 expression is also associated with C/EBPα‐mutated AML patients, and we found C/EBPα‐dependent regulation of DAPK2 during APL differentiation. In conclusion, we identified first inhibitory mechanisms responsible for the low DAPK2 expression in particular AML subtypes, and the regulation of DAPK2 by two myeloid transcription factors underlines its importance in neutrophil development.

Human DMTF1β antagonizes DMTF1α regulation of the p14(ARF) tumor suppressor and promotes cellular proliferation.

The human DMTF1 (DMP1) transcription factor, a DNA binding protein that interacts with cyclin D, is a positive regulator of the p14ARF (ARF) tumor suppressor. Our earlier studies have shown that three differentially spliced human DMP1 mRNAs, α, β and γ, arise from the human gene. We now show that DMP1α, β and γ isoforms differentially regulate ARF expression and promote distinct cellular functions. In contrast to DMP1α, DMP1β and γ did not activate the ARF promoter, whereas only β resulted in a dose-dependent inhibition of DMP1α-induced transactivation of the ARF promoter. Ectopic expression of DMP1β reduced endogenous ARF mRNA levels in human fibroblasts. The DMP1β- and γ-isoforms share domains necessary for the inhibitory function of the β-isoform. That DMP1β may interact with DMP1α to antagonize its function was shown in DNA binding assays and in cells by the close proximity of DMP1α/β in the nucleus. Cells stably expressing DMP1β, as well as shRNA targeting all DMP1 isoforms, disrupted cellular growth arrest induced by serum deprivation or in PMA-derived macrophages in the presence or absence of cellular p53. DMP1 mRNA levels in acute myeloid leukemia samples, as compared to granulocytes, were reduced. Treatment of acute promyelocytic leukemia patient samples with all-trans retinoic acid promoted differentiation to granulocytes and restored DMP1 transcripts to normal granulocyte levels. Our findings imply that DMP1α- and β-ratios are tightly regulated in hematopoietic cells and DMP1β antagonizes DMP1α transcriptional regulation of ARF resulting in the alteration of cellular control with a gain in proliferation.

Low DICER1 expression is associated with attenuated neutrophil differentiation and autophagy of NB4 APL cells

Successful myeloid differentiation depends on the expression of a series of miRNAs. Thus, it is hardly surprising that miRNAs are globally repressed in AML, a disease mainly characterized by a block in cellular myeloid differentiation. Studies investigating the mechanisms for low miRNA expression in AML has mostly focused on altered transcriptional regulation or deletions, whereas defective miRNA processing has received less attention. In this study, we report that the expression of the key miRNA processing enzyme DICER1 is down‐regulated in primary AML patient samples and healthy CD34+ progenitor cells as compared with granulocytes. In line with these findings, Dicer1 expression was induced significantly in AML cell lines upon neutrophil differentiation. The knocking down of DICER1 in AML cells significantly attenuated neutrophil differentiation, which was paralleled by decreased expression of miRNAs involved in this process. Moreover, we found that inhibiting DICER1 attenuated the activation of autophagy, a cellular recycling process that is needed for proper neutrophil differentiation of AML cells. Our results clearly indicate that DICER1 plays a novel role in neutrophil differentiation as well as in myeloid autophagy of AML cells.