KMT2A-rearranged acute myeloid leukemias (AML) have an intermediate to adverse prognosis. It was shown in a mouse model that the oncogene Myb contributes to AML maintenance in the KMT2A-MLLT3/NRasG12D genetic background (Zuber et al., 2011). Interestingly, the Myb-regulated genetic network mostly overlaps with KMT2A-MLLT3 target genes, especially with leukemic stem cell-associated gene sets. However, canonical KMT2A target genes are not included. Hence, MYB seems not to act as a co-factor for KMT2A complexes. SKI is a nuclear co-regulator and an oncoprotein, which is overexpressed in AML and several solid tumors (Bonnon & Atanasoski, 2012). Overexpression (OE) of SKI is prognostic in AML and correlates with shorter overall survival. Our group showed that SKI OE in AML is partially regulated by MYB (Frech et al., 2018). As a MYB target gene, we wanted to know if SKI can mediate MYB oncogenic activity in KMT2A-MLLT3/NRasG12D-positive AML. We adopted the Zuber mouse model and performed an shRNA-mediated Ski knockdown (KD) in murine KMT2A-MLLT3/NRasG12D-positive AML cells (RN2). Mice were transplanted with RN2 cells carrying two different anti-Ski shRNAs or an anti-Renilla shRNA as control. The expansion of RN2 cells in mice was monitored by luciferase-based bioimaging. Upon disease onset, shRNA expression was induced by doxycycline treatment. The course of the disease was further monitored by bioimaging. Bone marrow cells were isolated and analyzed via flow cytometry. The expression of KMT2A target genes was analyzed by RT-qPCR in doxycycline-induced RN2 cells. Ski KD induced a decreased viability of the RN2 cells in vitro. Moreover, SKI KD led to the eradication of RN2 cells in the mice in vivo and was associated with longer overall survival (log-rank p<0.0005). In line with the SKI OE mouse model of Singbrant et al. (2014), Ski KD led vice versa in our model to a decrease of myeloid cells and an increase of B cells. In contrast to Myb KD in the RN2 cells, Ski KD led to a lower expression of canonical KMT2A target genes, implicating that Ski may be part of KMT2A complexes in AML. In summary, SKI seems to be crucial for KMT2A-MLLT3/NRasG12D-driven AML and may be a potential target for new therapeutic strategies.

Acute myeloid leukemia (AML) arises through clonal expansion of transformed myeloid progenitor cells. The SKI proto-oncogene is highly upregulated in different solid tumors and leukemic cells, but little is known about its transcriptional regulation during leukemogenesis. MYB is an important hematopoietic transcription factor involved in proliferation as well as differentiation and upregulated in most human acute leukemias. Here, we find that MYB protein binds within the regulatory region of the SKI gene in AML cells. Reporter gene assays using MYB binding sites present in the SKI gene locus show MYB-dependent transcriptional activation. SiRNA-mediated depletion of MYB in leukemic cell lines reveals that MYB is crucial for SKI gene expression. Consistently, we observed a positive correlation of MYB and SKI expression in leukemic cell lines and in samples of AML patients. Moreover, MYB and SKI both were downregulated by treatment with histone deacetylase inhibitors. Strikingly, differentiation of AML cells induced by depletion of MYB is attenuated by overexpression of SKI. Our findings identify SKI as a novel MYB target gene, relevant for the MYB-induced differentiation block in leukemic cells.

Introduction: Acute promyelocytic leukemia (APL) patients are successfully treated via differentiation therapy with all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). Attempts to apply ATRA-based differentiation therapy to non-APL acute myeloid leukemia (AML) patients have not been effective so far (Johnson & Redner, 2015). Furthermore, 10-30% of the APL patients suffer an early death (ED) within 30 days, due to hemorrhages, infections, differentiation syndrome and thrombosis. Different risk factors have been identified, but the underlying mechanisms and successful treatment of ED in APL patients still have not been established (Kwaan et al., 2014, Lehmann et al., 2017). Hence, it is highly important to identify novel risk factors to understand the mechanistic processes in APL ED patients.

Abstract Background: Acute myeloid leukemia (AML) accounts for 80% of acute leukemias and arises through clonal expansion and arrest of differentiation of hematopoietic progenitor cells in the bone marrow. Depending on the AML subtype and age of the patient, AML patients have a 5-year survival rate of about 25%. AML is a genetically heterogenous disease, where chromosomal aberrations, point mutations in critical oncogenes as well as aberrant expression of key regulatory factors of hematopoiesis collude during transformation. c-MYB is an important hematopoietic transcription factor involved in proliferation and differentiation of progenitor cells of the myeloid and lymphoid lineages. It was first described as a viral oncogene of avian leukemia viruses and is upregulated or mutated in many leukemic subtypes as well as solid tumors. c-MYB interacts with other transcription factors or co-factors, which are essential for its transcriptional activity. In this regard, c-MYB transactivation ability is inhibited by a histone deacetylases recruiting corepressor complex containing SKI, TIF1BETA, NCOR and mSIN3A. c-SKI is a proto-oncogene and an inhibitor of TGFβ signalling. However, it acts not only as a transcriptional co-repressor but also as a transcriptional co-activator. Like c-MYB, c-SKI is upregulated in different solid tumors and leukemias. Though SKI activity is well described, transcriptional regulation of the SKI gene itself still remains unknown. Here, we deliver insight into the transcriptional regulation of the human SKI gene via the transcription factor c-MYB. Methods: In silico analyses were performed to identify potential MYB binding sites in the SKI regulatory region. Chromatin immunoprecipitation (ChIP) assays were used to validate the interaction between MYB and the potential SKI regulatory regions. Reporter gene assays were engaged to analyze MYB regulatory potential regarding SKI expression. RNAi experiments were performed to further examine transcriptional regulation of SKI by MYB. Since MYB is known to be downregulated by the histone deacetylase inhibitor (HDACi) valproic acid (VPA), MYB and SKI protein levels were analyzed in AML cell lines treated with VPA via Western Blot. Correlated protein levels of MYB and SKI were examined in the myeloid leukemia cell lines HL60, U937, THP1, NB4 and K562 as well as in primary cells of AML patients (n=27). Correlation of MYB and SKI transcript levels were performed in three different data sets of primary AML samples. The first cohort (n=7) was analyzed via RT-qPCR. Cohort 2 consists of cDNA microarray data of AML patients (n=17). Furthermore, principal component analysis (PCA) of the gene expression profile of MYB and SKI of AML patients (n=542) were performed with the leukemia gene atlas (LGA). Results: In silico analyses revealed four putative MYB binding sites (MBS1-4) in the SKI regulatory region. Direct binding of MYB to the regulatory sites MBS2-4 of the SKI gene could be confirmed via ChIP experiments. Dual luciferase reporter gene assays comprising c-MYB binding sites present in the SKI gene locus further show c-MYB-dependent transcriptional activation of the reporter. RNAi experiments depleting c-MYB in leukemic cell lines resulted in the decrease of SKI protein levels and thereby reveal that c-MYB is essential for the induction of SKI gene expression. Accordingly, treatment of the AML cell lines with the HDACi VPA led to a decrease of MYB and consequently SKI protein levels. Consistently, we observed a positive correlation of MYB and SKI protein expression in leukemic cell lines and in samples of AML patients. Moreover, a highly positive correlation of MYB and SKI transcript levels could be observed in three different cohorts of AML patients, further confirming regulation of SKI expression by the transcription factor MYB. Conclusion: Our findings provide new insights in the transcriptional regulation of the proto-oncogene c-SKI by the oncogenic transcription factor c-MYB during leukemogenesis. c-MYB and c-SKI expression and functions are highly positively correlated in human AML suggesting that c-SKI is a mediator of c-MYB oncogenic potential. So far, various therapeutic approaches targeting MYB failed to be transferred to patients. In this regard, c-MYB and c-SKI represent promising marker and target proteins for novel HDACi-based therapeutic approaches in AML. Disclosures No relevant conflicts of interest to declare.

RAS mutations are frequently found among acute myeloid leukemia patients (AML), generating a constitutively active signaling protein changing cellular proliferation, differentiation and apoptosis. We have previously shown that treatment of AML patients with high-dose cytarabine is preferentially beneficial for those harboring oncogenic RAS. On the basis of a murine AML cell culture model, we ascribed this effect to a RAS-driven, p53-dependent induction of differentiation. Hence, in this study we sought to confirm the correlation between RAS status and differentiation of primary blasts obtained from AML patients. The gene expression signature of AML blasts with oncogenic NRAS indeed corresponded to a more mature profile compared to blasts with wildtype RAS, as demonstrated by gene set enrichment analysis (GSEA) and real-time PCR analysis of myeloid ecotropic viral integration site 1 homolog (MEIS1) in a unique cohort of AML patients. In addition, in vitro cell culture experiments with established cell lines and a second set of primary AML cells showed that oncogenic NRAS mutations predisposed cells to cytarabine (AraC) driven differentiation. Taken together, our findings show that AML with inv(16) and NRAS mutation have a differentiation gene signature, supporting the notion that NRAS mutation may predispose leukemic cells to AraC induced differentiation. We therefore suggest that promotion of differentiation pathways by specific genetic alterations could explain the superior treatment outcome after therapy in some AML patient subgroups. Whether a differentiation gene expression status may generally predict for a superior treatment outcome in AML needs to be addressed in future studies.

Abstract Abstract 2410 Acute myeloid leukemia (AML) with deletion of chromosome 7 (−7) or 7q (del7q) has a poor prognosis. Using gene expression analysis, we previously identified the nuclear oncogene Ski as being up-regulated in AML, especially in AML with −7/del7q. We demonstrated that the transcriptional corepressor Ski acts as an inhibitor of vitamin A induced myeloid differentiation through interaction with N-CoR recruiting histone deacetylases (HDAC) (Ritter et al., Leukemia 2006). HDAC inhibitors such as valproic acid (VPA) promote histone acetylation, induce apoptosis and cell growth arrest in tumor cells (e.g. Kraemer et al., Trends Endocrinol Metab 2001). As Ski interacts with HDACs the aim of our investigation was to test the effect of HDAC inhibitors to cellular differentiation, apoptosis and Ski expression in primary AML cells. Treatment of the AML cell line HL60 expressing Ski with the HDAC inhibitor VPA enhances expression of the myeloid differentiation markers CD11b and CD11c as well as apoptosis. To address whether this effect is also observed in primary AML cells, we isolated mononuclear cells from blood or bone marrow of 12 AML patients (first diagnosis or relapse) and treated these cells with the HDAC inhibitor VPA. After harvesting, Ski protein expression was determined by Western blot. Flow cytometry was used to analyse expression of the differentiation markers CD11b and CD11c and apoptosis after propidium iodide staining. Of six AMLs with Ski protein expression, four responded either with differentiation or apoptosis, whereas none of six primary AMLs without Ski expression showed an effect to VPA compared to untreated control cells. To test whether other HDAC inhibitors would also reveal this effect we treated primary AML cells with further HDAC inhibitors (TSA, SAHA, LBH589) and confirmed our observation that HDAC inhibitors renders AML cells expressing Ski sensitive to differentiation. In parallel, we observed that VPA down regulates Ski in AML cells as well as in melanoma cell lines with high Ski protein levels. Our goal was to elucidate the molecular background of Ski reduction by VPA. Treatment of melanoma cells expressing Ski with VPA and/or the proteasomal inhibitor MG132 revealed that decrease of Ski depends on proteasomal degradation. The ring finger protein Arkadia (RNF111) is an E3-ligase of Ski (Nagano et al., J Biol Chem 2007) and we tested whether Arkadia is involved in Ski reduction after VPA addition. First we demonstrated that Arkadia expression is inversely associated with Ski expression in several AML cell lines. Furthermore the expression of Arkadia is induced by VPA on protein and RNA level while Ski protein is down regulated in melanoma cells. We also found that knockdown of Arkadia using RNAi impairs reduction of Ski by VPA in melanoma cells. Taken together our data suggest that high Ski expression in AML cells could be a molecular marker for VPA therapy. Thereby, VPA reduces the expression of the oncogene Ski as VPA induces expression of the E3-ligase Arkadia which abolish Ski by proteasomal degradation. Disclosures: No relevant conflicts of interest to declare.

MicroRNAs (miRNAs) are small, noncoding RNA molecules that regulate growth and differentiation. miRNAs are frequently located at cancer-specific fragile sites in the human genome, such as chromosome 7q. The nuclear oncogene SKI is up-regulated in acute myeloid leukemia (AML) with -7/del7q. Here we asked whether loss of miRNAs on chromosome 7q may explain this up-regulation. miR-29a expression was found to be down-regulated in AML with -7/del7q. Forced expression of miR-29a down-regulated Ski and its target gene, Nr-CAM, whereas miR-29a inhibition induced Ski expression. Luciferase assays validated a functional binding site for miR-29a in the 3' untranslated region of SKI. Finally, in samples of AML patients, we observed an inverse correlation of Ski and miR-29a expression, respectively. In conclusion, up-regulation of Ski in AML with -7/del7q is caused by loss of miR-29a. miR-29a may therefore function as an important tumor suppressor in AML by restraining expression of the SKI oncogene.

Acute myeloid leukemia (AML) patients with internal tandem duplication (ITD) mutations in the Fms-like tyrosine-3 (FLT3) gene have a dismal prognosis. Here we report compassionate-use results with the multikinase and FLT3-ITD inhibitor sorafenib for the treatment of relapsed or refractory FLT3-ITD-positive AML. Sorafenib induced clinically meaningful and very rapid responses in all 6 patients treated either before (n = 2), after (n = 3), or both before and after (n = 1) allogeneic stem cell transplantation (allo-SCT). Sorafenib-induced remissions facilitated allo-SCT in 2 of the 3 refractory patients. Two of the 4 patients who were treated after allo-SCT survived 216 and 221 days, respectively, whereas the other 2 remain in ongoing complete molecular remission. Sorafenib response was associated with an inhibition of the antiapoptotic FLT3-ITD target Stat-5 in vivo. Together, sorafenib monotherapy before or after allo-SCT has remarkable clinical activity in poor risk FLT3-ITD-positive AML and deserves further evaluation in prospective clinical trials. (Blood. 2009; 113: 6567-6571)

Abstract Abstract 1968 Poster Board I-991 Introduction: AML patients with deletion of chromosome 7 (−7) or deletion of 7q (−7q) have a poor prognosis. We have found that the nuclear oncogene SKI is overexpressed in AML, especially in AML with −7/−7q. SKI acts in AML as a repressor of retinoic acid induced myeloid differentiation (Ritter et al., (2006) Leukemia). As we found SKI up regulated in AML, we asked how SKI expression may be regulated. The aim of our study was to find a molecular background for increased SKI level. On chromosome 7 is a cluster of micro-RNAs (miRNAs) localized particularly around the fragile site 7q32 (Calin et al., (2003) PNAS). Therefore we investigated whether there exists a link between expression of miRNAs localized on chromosome 7 and up regulation of SKI expression in AML. Methods: We used micro RNA profiling analysis, FACS, Western blot, RQ-PCR and luciferase assays to determine the role of miRNA29a in regulating SKI expression. Results: We found that the expression of miRNA25, miRNA29a, miRNA183 and miRNA335 was downregulated in AML patients with -7/-7q. Transfection studies with these four miRNAs in HL60 cells revealed in FACS that miRNA29a inhibits SKI expression (60,4%) compared to nonsense control (100%) and other miRNAs (miRNA25: 91%, miRNA183: 101%, miRNA335: 93%). Western blot experiments confirmed that miRNA29a reduces SKI level in HL60 cells. In keeping, miRNA29a also represses expression of the SKI target gene Nr-CaM in IFB melanoma cells. Knock down of miRNA29a using miRNA29a inhibitor molecules induces SKI expression in the high miRNA29a and low SKI expressing cell line NW1539. Luciferase assays in NW1539 and HeLa transfected with 3′UTR-constructs and HeLa cells cotransfected with miRNA29a demonstrated that miRNA29a binds to 3′UTR of SKI in vitro. Furthermore, comparison of SKI and miRNA29a expression of AML patient samples indicates that miRNA29a expression is associated with low SKI level in vivo. Conclusion: Our data show that miRNA29a which is located on 7q32 regulates expression of the oncogene SKI in vitro and in vivo. We suggest the deletion of miRNA29a as mechanism for up regulation of SKI in AML with -7/-7q and thus propose that in AML, this effect may contribute to the tumor suppressive function of miRNA29a. Disclosures: No relevant conflicts of interest to declare.

tern of fluorescence histogram with or without CSFs.Nevertheless, the histogram uniformly had a single peak or two narrowly split peaks.Accordingly, there exists a continuous, but not discrete, gradient of hTERT promoter activity among individual cells.To monitor the changes in hTERT promoter activity during short-term culture, hTERT-Venus-transduced AML cells were maintained with CSFs (Figure 2B).In UPN5, the viable cell fraction, determined using a FSC vs. SSC dot plot, decreased progressively, whereas the Venus expression in this fraction increased transiently at day 6 and declined at day 10.In UPN15, both the viable cell fraction and its Venus expression increased at day 6.The viable cell fraction decreased thereafter, but the fluorescence intensity of this fraction still increased at day 10.This might be due to the gradual disappearance of the cell fraction with lower hTERT promoter activity.An interesting issue is whether leukemia cells showing high hTERT promoter activity are included in the leukemia stem cell (LSC) compartment.If most LSCs exit from the cell cycle as do hematopoietic stem cells (HSC), hTERT promoter activity is likely to be repressed in LSCs, and would not be a related parameter.In conclusion, we present a promising new method to monitor the hTERT promoter activity on a single living cell level.This method will help examine a relationship between hTERT promoter activity and replicative potential among primary leukemia cells.

Background: We evaluated, if preoperative, single-dose steroid application reduces nausea, pain and voice disturbances after thyroid surgery.Patients and methods: Sixty patients (m=16, w=44) with median age 55 years (range 18-80) entered a two-arm, randomized, doubleblinded, controlled study receiving either dexamethasone 8mg (D) (n=30) or NaCl 0.9% (C) (n=30) i/v 30 min before anaesthesia.At 6 timepoints (4-48 h) after the operation nausea (verbal response scale (0-3), use of antiemetic medication), pain (visual analogue scale (VAS), analgesic use) and vocal function was registered.Results: Groups did not differ in demographics.Nausea and antiemetic use was significantly less in group D compared to C.Over 48 h the difference in nausea and vomiting was significant (p<0.001;ANOVA repeated measures).There was a significant reduction in antiemetic use, too (D=16.1%/C=28.1%)(p<0.009).Mean scores for pain during the first 48 h after operation were 17.5/25.1 in group D and C, respectively (p<0.001) with a reduction in total opioid requirement (p<0.001).Changes in voice mean frequency over a 48 h period after surgery was significantly less in the dexamethasone group than in the placebo group (p< 0.02).No potential steroid related complications occurred.Conclusion: Preoperative single-dose steroid not only reduces postoperative nausea/vomiting and pain within 48 h after thyroid resection but decreases voice dysfunction, too.

Acute myeloid leukemia (AML) is a heterogeneous disease with multiple different cytogenetic and molecular aberrations contributing to leukemic transformation. We compared gene expression profiles of 4608 genes using cDNA-arrays from 20 AML patients (nine with −7/del7q and 11 with normal karyotype) with 23 CD34+ preparations from healthy bone marrow donors. SKI , a nuclear oncogene, was highly up regulated. In a second set of 183 AML patients analyzed with real-time PCR, the highest expression level of SKI in AML with −7/del7q could be confirmed. As previously described, Ski associates with the retinoic acid receptor (RAR) complex and can repress transcription. We wanted to investigate the interference of Ski with RAR α signaling in AML. Ski was co-immunoprecipitated and colocalized with RAR α . We also found that overexpression of wild-type Ski inhibited the prodifferentiating effects of retinoic acid in U937 leukemia cells. Mutant Ski, lacking the N-CoR binding, was no more capable of repressing RAR α signaling. The inhibition by wild-type Ski could partially be reverted by the histone deacetylase blocking agent valproic acid. In conclusion, Ski seems to be involved in the blocking of differentiation in AML via inhibition of RAR α signaling.