Magali Humbert

Deregulated expression of Kruppel-like factors in acute myeloid leukemia.

The known participation of Kruppel-like transcription factors (KLF) in cellular differentiation prompted us to investigate their expression in acute myeloid leukemia (AML) blast cells that are typically blocked in their differentiation. We determined the expression patterns of KLFs with a putative role in myeloid differentiation in a large cohort of primary AML patient samples, CD34+ progenitor cells and granulocytes from healthy donors. We found that KLF2, KLF3, KLF5 and KLF6 are significantly lower expressed in AML blast and CD34+ progenitor cells as compared to normal granulocytes. Moreover, we found markedly increased KLF levels in acute promyelocytic leukemia patients who received oral ATRA. Accordingly, we observed a strong induction of KLF5/6 upon ATRA-treatment in NB4 and HT93 APL but not in ATRA-resistant NB4-R cells. Lastly, knocking down KLF5 or KLF6 in NB4 cells significantly attenuated neutrophil differentiation. In conclusion, we found a significant repression of KLF transcription factors in primary AML samples as compared to mature neutrophils and further show that KLF5 and KLF6 are functionally involved in neutrophil differentiation of APL cells.

Protective autophagy is involved in resistance towards MET inhibitors in human gastric adenocarcinoma cells.

MET, also known as hepatocyte growth factor receptor (HGFR), is a receptor tyrosine kinase with an important role, both in normal cellular function as well as in oncogenesis. In many cancer types, abnormal activation of MET is related to poor prognosis and various strategies to inhibit its function, including small molecule inhibitors, are currently in preclinical and clinical evaluation. Autophagy, a self-digesting recycling mechanism with cytoprotective functions, is induced by cellular stress. This process is also induced upon cytotoxic drug treatment of cancer cells and partially allows these cells to escape cell death. Thus, since autophagy protects different tumor cells from chemotherapy-induced cell death, current clinical trials aim at combining autophagy inhibitors with different cancer treatments. We found that in a gastric adenocarcinoma cell line GTL-16, where MET activity is deregulated due to receptor overexpression, two different MET inhibitors PHA665752 and EMD1214063 lead to cell death paralleled by the induction of autophagy. A combined treatment of MET inhibitors together with the autophagy inhibitor 3-MA or genetically impairing autophagy by knocking down the key autophagy gene ATG7 further decreased cell viability of gastric cancer cells. In general, we observed the induction of cytoprotective autophagy in MET expressing cells upon MET inhibition and a combination of MET and autophagy inhibition resulted in significantly decreased cell viability in gastric cancer 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.

Inhibition of damage-regulated autophagy modulator-1 (DRAM-1) impairs neutrophil differentiation of NB4 APL cells

The damage-regulator autophagy modulator 1 (DRAM-1) is a lysosomal protein that positively regulates autophagy in a p53-dependent manner. We aimed at analyzing the role of DRAM-1 in granulocytic differentiation of APL cells. We observed a significant increase of DRAM-1 expression during all-trans retinoic acid (ATRA)-induced neutrophil differentiation of NB4 APL cells but not in ATRA-resistant NB4-R2 cells. Next, knocking down DRAM-1 in NB4 APL cells was sufficient to impair neutrophil differentiation. Given that DRAM-1 is a transcriptional target of p53, we tested if DRAM-1 is regulated by the p53 relative p73. Indeed, inhibiting p73 prevented neutrophil differentiation and DRAM-1 induction of NB4 cells. In conclusion, we show for the first time that p73-regulated DRAM-1 is functionally involved in neutrophil differentiation of APL cells.

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.

The actin-binding protein CORO1A is a novel PU.1 (SPI1)- and CEBPA-regulated gene with significantly lower expression in APL and CEBPA-mutated AML patients

Coronins are highly conserved regulators of the actin cytoskeleton [reviewed in (Chan et al, 2011)]. In neutrophils, CORO1A is fundamental in cell adhesion/migration and is functional in activating the respiratory burst [reviewed in (Chan et al, 2011)]. CORO1A has been linked to inhibition of neutrophil apoptosis in the context of chronic inflammation (Moriceau et al, 2009). Currently, no direct transcriptional activators of CORO1A have been identified, but given the leucocyte-specific expression of CORO1A (Fig S1), we hypothesized that the myeloid master regulator PU.1 (SPI1) drives CORO1A expression. Firstly, we found that Coro1a is highly induced in Pu.1-restored 503 Pu.1 knockout myeloid cells (Table SI). We then investigated whether low PU.1 (SPI1) levels associate with low CORO1A expression. To this end, we quantified CORO1A message levels in acute myeloid leukaemia (AML), a disease where haematopoietic transcription factors, such as PU.1 and CEBPA, are commonly deregulated [reviewed in (Gombart et al, 2002; Rosenbauer et al, 2004)]. CORO1A and PU.1 mRNA levels were determined by quantitative real-time polymerase chain reaction (qPCR) in a total of 101 primary AML (French-American-British type M0-M4) patient samples and 25 granulocyte samples from healthy donors (Fig 1A). In general, CORO1A expression was significantly lower in AML patient samples as compared to granulocytes from healthy donors. A more detailed analysis revealed that CORO1A expression was particularly low in acute promyelocytic leukaemia (APL) t(15;17) as compared to non-APL patient samples (P < 0 0001). Significantly lower CORO1A mRNA expression was also found in CEBPA-mutated AML (Fig 1B). These data led to the hypothesis that CORO1A expression is regulated by PU.1 and CEBPA, while repressed by PML-RARA. Importantly, we identified five PU.1 and one CEBPA putative binding sites in the CORO1A promoter (Fig 1C). Chromatin immunoprecipitation (ChIP) revealed in vivo binding of PU.1 at 462, 846, 4708/ 4559 but not at 2574 and of CEBPA at 6700 (Figs 1 D,E). Wang et al (2010) recently showed that PU.1 binding motifs, including one in the CORO1A promoter, coexist with retinoic acid responsive elements resulting in PML-RARA mediated inhibition of PU.1-regulated promoters. Accordingly, we found in vivo binding of PML-RARA to three of the four regions of the CORO1A promoter bound by PU.1 (Fig 1D). To further show that PU.1 directly activates CORO1A transcription, we activated PU.1 in PU.1-oestrogen receptor (ER) expressing NB4 cells resulting in a significant CORO1A mRNA increase (Fig 1F). Induction of CSF3R, a direct PU.1-regulated gene (Smith et al, 1996), was measured as a positive control (Fig S2A). Similarly, a significant 3-fold transcriptional activation of CORO1A upon CEBPA activation was only seen in CEBPA wild-type (K562-CEBPA-p42-ER), but Correspondence

A specific expression profile of LC3B and p62 is associated with nonresponse to neoadjuvant chemotherapy in esophageal adenocarcinomas.

Paclitaxel is a powerful chemotherapeutic drug, used for the treatment of many cancer types, including esophageal adenocarcinomas (EAC). Autophagy is a lysosome-dependent degradation process maintaining cellular homeostasis. Defective autophagy has been implicated in cancer biology and therapy resistance. We aimed to assess the impact of autophagy on chemotherapy response in EAC, with a special focus on paclitaxel. Responsiveness of EAC cell lines, OE19, FLO-1, OE33 and SK-GT-4, to paclitaxel was assessed using Alamar Blue assays. Autophagic flux upon paclitaxel treatment in vitro was assessed by immunoblotting of LC3B-II and quantitative assessment of WIP1 mRNA. Immunohistochemistry for the autophagy markers LC3B and p62 was applied on tumor tissue from 149 EAC patients treated with neoadjuvant chemotherapy, including pre- and post-therapeutic samples (62 matched pairs). Tumor response was assessed by histology. For comparison, previously published data on 114 primary resected EAC cases were used. EAC cell lines displayed differing responsiveness to paclitaxel treatment; however this was not associated with differential autophagy regulation. High p62 cytoplasmic expression on its own (p ≤ 0.001), or in combination with low LC3B (p = 0.034), was associated with nonresponse to chemotherapy, regardless of whether or not the regiments contained paclitaxel, but there was no independent prognostic value of LC3B or p62 expression patterns for EAC after neoadjuvant treatment. p62 and related pathways, most likely other than autophagy, play a role in chemotherapeutic response in EAC in a clinical setting. Therefore p62 could be a novel therapeutic target to overcome chemoresistance in EAC.

PU.1 supports TRAIL-induced cell death by inhibiting NF-κB-mediated cell survival and inducing DR5 expression

The hematopoietic Ets-domain transcription factor PU.1/SPI1 orchestrates myeloid, B- and T-cell development, and also supports hematopoietic stem cell maintenance. Although PU.1 is a renowned tumor suppressor in acute myeloid leukemia (AML), a disease characterized by an accumulation of immature blast cells, comprehensive studies analyzing the role of PU.1 during cell death responses in AML treatment are missing. Modulating PU.1 expression in AML cells, we found that PU.1 supports tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis via two mechanisms: (a) by repressing NF-κB activity via a novel direct PU.1-RelA/p65 protein–protein interaction, and (b) by directly inducing TRAIL receptor DR5 expression. Thus, expression of NF-κB-regulated antiapoptotic genes was sustained in PU.1-depleted AML cells upon TRAIL treatment and DR5 levels were decreased. Last, PU.1 deficiency significantly increased AML cell resistance to anthracycline treatment. Altogether, these results reveal a new facet of PU.1s tumor suppressor function during antileukemic therapies.

Distinct TP73–DAPK2–ATG5 pathway involvement in ATO-mediated cell death versus ATRA-mediated autophagy responses in APL

We have previously demonstrated that the death‐associated protein kinase 2 (DAPK2) expression is significantly reduced in acute myeloid leukemia (AML), particularly in acute promyelocytic leukemia (APL) blast cells. In this study, we aimed at further understanding DAPK2 function and regulation during arsenic trioxide (ATO) cytotoxic or all‐trans retinoic acid (ATRA) differentiation therapy in APL cells. We found that the p53 family member transactivation domain‐p73 isoform (TAp73) binds to and activates the DAPK2 promoter, whereas the dominant‐negative ΔNp73 isoform inhibits DAPK2 transcription. Furthermore, the knocking down of tumor protein p73 (TP73) in NB4 cells resulted in reduced DAPK2 expression associated with decreased cell death and autophagy upon ATO and ATRA treatment, respectively. Moreover, the silencing of DAPK2 revealed that DAPK2 is an important downstream effector of p73 in ATO‐induced apoptosis but not autophagy responses of APL cells. In contrast, the p73–DAPK2 pathway is essential for ATRA‐induced autophagy that is mediated by an interaction of DAPK2 with the key autophagy‐related protein (ATG)5. Lastly, we show that DAPK2 binds and stabilizes the p73 protein; thus, we propose a novel mechanism by which ATO‐ or ATRA‐induced therapy responses initiate a positive p73–DAPK2 feedback loop.

Abstract 1909: Cytoprotective autophagy is involved in resistance towards MET inhibitors in human gastric adenocarcinoma cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, ILnnMET, also known as hepatocyte growth factor receptor, is a receptor tyrosine kinase (RTK) that plays an important role both in normal cellular function as well as in oncogenesis. In many different cancer types, abnormal activation of MET is related to poor prognosis and various strategies to inhibit its function, including small molecule inhibitors, are currently in preclinical and clinical evaluation. Although promising results have been obtained with a variety of different MET RTK inhibitors, different resistance mechanisms were found. We hypothesized that autophagy, a self-digesting recycling mechanism for cellular organelles and macromolecules with cytoprotective functions, might be involved in resistance towards MET inhibitors. Autophagy induced upon MET inhibition might support cancer cell survival. We therefore aimed at investigating the involvement of autophagy upon MET inhibition in GTL-16 gastric adenocarcinoma cells. First, we treated GTL-16 cells with the MET inhibitor PHA665752. We observed a marked induction of autophagy in these cells upon MET inhibition as measured by LC3I/LC3II conversion and GFP-LC3 dot formation. Similar results were seen in another gastric adenocarcinoma cell line, namely MKN-45. Next, to investigate if MET inhibition-mediated induction of autophagy is a protective mechanism, we treated GTL-16 cells with PHA665752 in combination with compounds that influence autophagic activity. Interestingly, a combination treatment of MET and autophagy inhibitors (3-Methyladenine, 3-MA) significantly (MWU, p<0.001) decreased cell viability as determined by an MTT assay. In contrast, using the autophagy activator lithium chloride in combination with MET inhibitors, cell viability was significantly increased (p<0.01). All data were confirmed using a second MET-inhibitor. To exclude that 3-MA autophagy independent functions account for the increased cell death during treatment with PHA665752, we inhibited autophagy by knocking down the key autophagy gene ATG7 in GTL-16 cells using lentiviral vectors expression small hairpin (sh)RNA targeting ATG7. These “autophagy knockdown” GTL-16 cells displayed a significant increase in cell death upon MET inhibition as compared to GTL-16 control cells expressing a scrambled control shRNA. In conclusion, our results point to a cytoprotective function of autophagy in response to MET inhibition. The use of MET in combination with autophagy inhibitors may represent a novel therapeutic option for the treatment of gastric carcinoma with aberrant MET activity.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1909. doi:1538-7445.AM2012-1909