BEX1, a Marker for Acute Myeloid Leukemia Cell Lines with MLL Rearrangements.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3013-3013
Author(s):  
Hilmar Quentmeier ◽  
Wilhelm G. Dirks ◽  
Robert Geffers ◽  
Cord C. Uphoff ◽  
Hans G. Drexler
Keyword(s):  
Gene Expression ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Hox Genes ◽  
Expression Profiles ◽  
Pcr Analysis ◽  
Wild Type ◽  
Acute Leukemias ◽  
Acute Myeloid

Abstract Patients with acute leukemias carrying MLL rearrangements have a poor prognosis. The tumor cells show characteristic gene expression profiles with increased levels of selected HOX genes (1). We have shown that acute lymphoblastic leukemia (ALL) cell lines with and without MLL rearrangements could likewise be recognized by analysis of HOX gene expression (2). In contrast, MLL wild-type (MLLwt) and mutant (MLLmu) acute myeloid leukemia (AML) cell lines could not be distinguished by analysis of HOX genes, because even wild-type cell lines had a high expression background (2). It was our aim to find out whether MLLwt and MLLmu AML cell lines could be discriminated on the basis of gene expression - other than HOX genes. We performed gene expression analysis with pooled RNAs of MLLmu (n=8) and MLLwt (n=8) cell lines applying high density oligonucleotide Genechips from Affymetrix (HG-U133A). Defensin alpha4 (83x), defensin beta1 (32x), cathepsinG (9x) and FLT3 (7x) genes were overexpressed in MLLmu cell lines, stabilin1 (82x) and galectin10 (55x) in MLLwt cell lines. PCR analysis with individual (non-pooled) cDNAs of the 16 cell lines showed that none of the above genes was exclusively expressed by MLLmu or MLLwt cells. Thus, pooling RNAs has a major disadvantage: many PCRs have to be performed to establish faithful expression profiles for individual samples. BEX1 finally proved to be a gene that was exclusively overexpressed in one group of cell lines. It had been an interesting candidate already after oligonucleotide chip analysis, being both overexpressed (18x) in MLLmu cell lines, and - in contrast to the genes listed above - not a marker of myeloid differentiation. BEX1 is reportedly expressed in brain, testis and ovary, but not in peripheral blood leukocytes, lymph node and bone marrow (3). By RT-PCR analysis we showed that 7/8 MLLmu and 0/8 MLLwt cell lines expressed BEX1. Screening a panel of 54 hematopoetic cell lines gave the same result: BEX1 expression was restricted to MLLmu AML cell lines: 8/11 (73%) MLLmu AML cell lines expressed BEX1, but 43 other hematopoetic cell lines (including Hodgkin′s disease, anaplastic large cell lymphoma, ALL and MLLwt AML cell lines) tested negative. BEX1 expression may depend on the type of MLL rearrangement or the histological background of the cells, as MLLmu ALL cell lines (0/5) also tested negative. It has been shown in the mouse system, that BEX family members may be involved in cell signalling processes. Thus it will be interesting to elucidate whether BEX1 also participates in proliferative/antiapoptotic signalling processes in MLLmu AML cell lines.

FLT3 ligand causes autocrine signaling in acute myeloid leukemia cells

Blood ◽  
2004 ◽  
Vol 103 (1) ◽  
pp. 267-274 ◽  
Author(s):  
Rui Zheng ◽  
Mark Levis ◽  
Obdulio Piloto ◽  
Patrick Brown ◽  
Brenda R. Baldwin ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Conditioned Media ◽  
Autocrine Signaling ◽  
Wild Type ◽  
Acute Leukemias ◽  
Type Receptor ◽  
Acute Myeloid

Abstract The FLT3 receptor tyrosine kinase is highly expressed in most acute leukemias and frequently mutated in acute myeloid leukemia (AML). The mutated form of the receptor is constitutively activated and known to play an important role in AML, but the activation state of the overexpressed wild-type (wt) receptor is, at present, unknown. In this study, we examined the activation state of the wild-type receptor in AML. We found that the wild-type receptor was constitutively phosphorylated/activated in 8 of 12 primary AML samples and 4 of 13 leukemia cell lines. To explain why wtFLT3 is often activated, we investigated the expression of its ligand, FL, by these same cells. Coexpression of FL with FLT3 was a universal finding in both primary AML samples and leukemic-derived cell lines. To further prove that autocrine signaling was accounting for the activation, we showed that conditioned media but not fresh media was able to activate FLT3. In addition, an antibody that blocks binding of ligand to the receptor blocks FLT3 activation. Finally, depletion of FL from conditioned media is able to block the activation of FLT3. Taken together, these findings represent strong evidence that wtFLT3 is often constitutively activated in AML and thus, like its mutated form, might contribute to the altered signaling that characterizes leukemogenesis.

scholarly journals Transcriptomic Profiles of MV4-11 and Kasumi 1 Acute Myeloid Leukemia Cell Lines Modulated by Epigenetic Modifiers Trichostatin A and 5-Azacytidine

2020 ◽  
Author(s):  
Mat Jusoh Siti Asmaa ◽  
Hamid Ali Al-Jamal ◽  
Abdul Rahim Hussein ◽  
Badrul Hisham Yahaya ◽  
Azlan Husin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Trichostatin A ◽  
Leukemia Cell ◽  
Leukemic Cell ◽  
Enrichment Score ◽  
Acute Leukemias ◽  
Acute Myeloid

Background: Acute myeloid leukemia (AML) is the most common form of acute leukemias in adults which is clinically and molecularly heterogeneous. Several risk and genetic factors have been widely investigated to characterize AML. However, the concomitant epigenetic factors in controlling the gene expression lead to AML transformation was not fully understood. This study was aimed to identify epigenetically regulated genes in AML cell lines induced by epigenetic modulating agents, Trichostatin A (TSA) and 5-Azacytidine (5-Aza). Materials and Methods: MV4-11 and Kasumi 1 were treated with TSA and/or 5-Aza at IC50 concentration. Gene expression profiling by microarray was utilized using SurePrint G3 Human Gene Expression v3. Gene ontology and KEGG pathway annotations were analyzed by DAVID bioinformatics software using EASE enrichment score. mRNA expression of the differentially expressed genes were verified by quantitative real time PCR. Results: Gene expression analysis revealed a significant changes in the expression of 24,822, 15,720, 15,654 genes in MV4-11 and 12,598, 8828, 18,026 genes in Kasumi 1, in response to TSA, 5-Aza and combination treatments, respectively, compared to non-treated (p<0.05). 7 genes (SOCS3, TUBA1C, CCNA1, MAP3K6, PTPRC, STAT6 and RUNX1) and 4 genes (ANGPTL4, TUBB2A, ADAM12 and PTPN6) shown to be predominantly expressed in MV4-11 and Kasumi 1, respectively (EASE<0.1). The analysis also revealed phagosome pathway commonly activated in both cell lines. Conclusion: Our data showed a distinct optimal biological characteristic and pathway in different types of leukemic cell lines. These finding may help in the identification of cell-specific epigenetic biomarker in the pathogenesis of AML.  

scholarly journals DOT1L As a Therapeutic Target for the Treatment of DNMT3A-Mutant Acute Myeloid Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 614-614
Author(s):  
Rachel E. Rau ◽  
Min Luo ◽  
Benjamin Rodriguez ◽  
Mira Jeong ◽  
Allison Rosen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Myeloid Leukemia ◽  
Hox Genes ◽  
Time Dependent ◽  
Mutant Cells ◽  
Acute Myeloid

Abstract Mutations of the DNA methyltransferase, DNMT3A, occur in approximately 20% of adult patients with acute myeloid leukemia (AML), and portend a poor prognosis. The most common of these mutations results in a dominant negative loss of function. Our lab observed that upon conditional inactivation of Dnmt3a in the murine hematopoietic system, Dnmt3a–/– hematopoietic stem cells (HSCs) expanded dramatically while their differentiation was inhibited, consistent with a pre-leukemic state. The likely mechanism by which Dnmt3a loss contributes to leukemogenesis is altered DNA methylation and the attendant gene expression changes, however our current understanding is incomplete. In analyses of gene expression data, we observed that murine Dnmt3a–/– HSCs markedly overexpress the histone 3, lysine 79 (H3K79) methyltransferase, Dot1l. This is of interest given the known functional interplay between DNA methylation and histone modifications. Additionally, DOT1L plays a critical role in leukemia with MLL-rearrangements, lesions that essentially never occur concomitantly with DNMT3A mutations in AML. The mutual exclusion of these lesions combined with the observed overexpression of Dot1l in our murine model, led us to postulate that MLL-rearrangements and DNMT3A mutations are distinct epigenetic aberrations that converge on a common mechanism resulting in dysregulated gene expression, specifically mediated by H3K79 methylation (H3K79me). Therefore, in the pathogenesis of DNMT3A-mutant AML, like in MLL-rearranged leukemia, DOT1L-induced H3K79me may play a central role, and may represent a viable therapeutic target. Throughout the genome of normal HSCs, expansive regions with low DNA methylation (canyons) exist. These canyons span conserved domains frequently containing transcription factors. In our Dnmt3a-/- model, canyon borders, particularly flanking genes frequently dysregulated in human leukemia such as HOX genes, are highly prone to DNA methylation loss when Dnmt3a is deleted, resulting in canyon expansion. However, not all canyons expand with Dnmt3a loss. We found a close association between canyon behavior and the associated histone marks, with expanding canyons characterized by a lack of the repressive histone mark, H3K27me. To determine if in Dnmt3a-mediated malignant hematopoiesis, H3K79me also correlates with altered DNA methylation, we performed chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) for H3K79 di-methylation (H3K79me2) and aligned these data with whole genome DNA methylation data. This revealed that H3K79me2 specifically coats canyons that lose methylation with Dnmt3a loss, including the HoxA and HoxB clusters, but is not present at canyons without methylation loss. This strong correlation between H3K79me and DNA hypomethylation with Dnmt3a loss suggests a functional interaction. To examine whether this also occurred in human samples with DNMT3A mutations, we analyzed TCGA data, which confirmed many canyon borders as regions with marked DNA methylation loss. Further, many canyon-associated genes, including HOX genes are significantly changed in human DNMT3A-mutant AML. To explore the role of H3K79me, and specifically of DOT1L in human DNMT3A-mutant AML, we utilized the DNMT3A-mutant human AML cell lines OCIAML2 and OCIAML3. These cell lines were found to have increased total H3K79me compared to DNMT3A-wild type controls, consistent with the increased Dot1l expression in Dnmt3a–/– HSCs. We then tested the in vitro efficacy of two selective DOT1L inhibitors, SYC-522 (Anglin. J Med Chem. 2011) and the Epizyme compound, EPZ004777 (Daigle. Cancer Cell. 2011), against DNMT3A-mutant cells. Both compounds led to a dose- and time-dependent inhibition of proliferation and induction of apoptosis in the DNMT3A-mutant cell lines at concentrations comparable to those used for MLL-rearranged cell lines. With treatment, DNMT3A-mutant cells also had evidence of induction of differentiation with increased expression of the mature monocyte marker, CD14. Importantly, oncogenic HOX genes overexpressed in DNMT3A-mutant AML were repressed in a time-dependent fashion with DOT1L inhibitor treatment. In conclusion, our data suggest that DOT1L may be a novel, immediately actionable therapeutic target for the treatment of DNMT3A-mutant AML. Disclosures Rau: Epizyme: Honoraria.

Acute Myeloid Leukemia With Biallelic CEBPA Gene Mutations and Normal Karyotype Represents a Distinct Genetic Entity Associated With a Favorable Clinical Outcome

2010 ◽  
Vol 28 (4) ◽  
pp. 570-577 ◽  
Author(s):  
Annika Dufour ◽  
Friederike Schneider ◽  
Klaus H. Metzeler ◽  
Eva Hoster ◽  
Stephanie Schneider ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Myeloid Leukemia ◽  
Clinical Outcome ◽  
Myeloid Leukemia ◽  
Expression Profiles ◽  
Gene Mutations ◽  
Gene Expression Profiles ◽  
Similar Frequency ◽  
Favorable Clinical Outcome ◽  
Acute Myeloid

Purpose CEBPA mutations are found as either biallelic (biCEBPA) or monoallelic (moCEBPA). We set out to explore whether the kind of CEBPA mutation is of prognostic relevance in cytogenetically normal (CN) acute myeloid leukemia (AML). Patients and Methods Four hundred sixty-seven homogeneously treated patients with CN-AML were subdivided into moCEBPA, biCEBPA, and wild-type (wt) CEBPA patients. The subgroups were analyzed for clinical parameters and for additional mutations in the NPM1, FLT3, and MLL genes. Furthermore, we obtained gene expression profiles using oligonucleotide microarrays. Results Only patients with biCEBPA had an improved median overall survival when compared with patients with wtCEBPA (not reached v 20.4 months, respectively; P = .018), whereas patients with moCEBPA (20.9 months) and wtCEBPA had a similar outcome (P = .506). Multivariable analysis confirmed biCEBPA, but not moCEBPA, mutations as an independent favorable prognostic factor. Interestingly, biCEBPA mutations, compared with wtCEBPA, were never associated with mutated NPM1 (0% v 43%, respectively; P < .001) and rarely associated with FLT3 internal tandem duplication (ITD; 5% v 23%, respectively; P = .059), whereas patients with moCEBPA had a similar frequency of mutated NPM1 and a significantly higher association with FLT3-ITD compared with patients with wtCEBPA (44% v 23%, respectively; P = .037). Furthermore, patients with biCEBPA showed a homogeneous gene expression profile that was characterized by downregulation of HOX genes, whereas patients with moCEBPA showed greater heterogeneity in their gene expression profiles. Conclusion Biallelic disruption of the N and C terminus of CEBPA is required for the favorable clinical outcome of CEBPA-mutated patients and represents a distinct molecular subtype of CN-AML with a different frequency of associated gene mutations. These findings are of great significance for risk-adapted therapeutic strategies in AML.

scholarly journals Prognostic Importance ofMN1Transcript Levels, and Biologic Insights FromMN1-Associated Gene and MicroRNA Expression Signatures in Cytogenetically Normal Acute Myeloid Leukemia: A Cancer and Leukemia Group B Study

2009 ◽  
Vol 27 (19) ◽  
pp. 3198-3204 ◽  
Author(s):  
Christian Langer ◽  
Guido Marcucci ◽  
Kelsi B. Holland ◽  
Michael D. Radmacher ◽  
Kati Maharry ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Expression Profiles ◽  
Microrna Expression ◽  
Wild Type ◽  
Prognostic Importance ◽  
Group B ◽  
Leukemia Group ◽  
Tandem Duplications ◽  
Acute Myeloid

PurposeTo determine the prognostic importance of the meningioma 1 (MN1) gene expression levels in the context of other predictive molecular markers, and to derive MN1 associated gene– and microRNA–expression profiles in cytogenetically normal acute myeloid leukemia (CN-AML).Patients and MethodsMN1 expression was measured in 119 untreated primary CN-AML adults younger than 60 years by real-time reverse-transcriptase polymerase chain reaction. Patients were also tested for FLT3, NPM1, CEBPA, and WT1 mutations, MLL partial tandem duplications, and BAALC and ERG expression. Gene- and microRNA-expression profiles were attained by performing genome-wide microarray assays. Patients were intensively treated on two first-line Cancer and Leukemia Group B clinical trials.ResultsHigher MN1 expression associated with NPM1 wild-type (P < .001), increased BAALC expression (P = .004), and less extramedullary involvement (P = .01). In multivariable analyses, higher MN1 expression associated with a lower complete remission rate (P = .005) after adjustment for WBC; shorter disease-free survival (P = .01) after adjustment for WT1 mutations, FLT3 internal tandem duplications (FLT3-ITD), and high ERG expression; and shorter survival (P = .04) after adjustment for WT1 and NPM1 mutations, FLT3-ITD, and WBC. Gene- and microRNA-expression profiles suggested that high MN1 expressers share features with high BAALC expressers and patients with wild-type NPM1. Higher MN1 expression also appears to be associated with genes and microRNAs that are active in aberrant macrophage/monocytoid function and differentiation.ConclusionMN1 expression independently predicts outcome in CN-AML patients. The MN1 gene- and microRNA-expression signatures suggest biologic features that could be exploited as therapeutic targets.

The Critical Role of Peptidyl-Prolyl cis/trans Isomerase, Pin1 in Acute Myeloid Leukemia with C/EBPalpha Mutations.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2213-2213
Author(s):  
J. Pulikkan ◽  
A. Peer Zada ◽  
M. Geletu ◽  
V. Dengler ◽  
Daniel G. Tenen ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Dominant Negative ◽  
Pcr Analysis ◽  
Wild Type ◽  
Rt Pcr ◽  
Promoter Assay ◽  
Type C ◽  
Acute Myeloid

Abstract CCAAT enhancer binding protein alpha (C/EBPα) is a myeloid specific transcription factor that coordinates cellular differentiation and cell cycle arrest. Loss of C/EBPα expression or function in leukemic blasts contributes to a block in myeloid cell differentiation. C/EBPα is mutated in around 9% of acute myeloid leukemia (AML). The mutations reported in C/EBPα are frame shift mutations and point mutations at basic region Leucine zipper. The mutant form of C/EBPα ie C/EBPα-p30 exhibits dominant negative function over the wild type protein. The role of peptidyl-prolyl cis/trans isomerase, Pin1 in tumorogenesis and its overexpression in many cancers led us to investigate its role in acute myeloid leukemia with C/EBPα mutation. Here we show that Pin1 is upregulated in patients with acute myeloid leukemia by affymetrix analysis. By quantitative Real-Time RT-PCR analysis, we show C/EBPα-p30 could induce Pin1 transcription, while the wild type C/EBPα downregulates Pin1 expression. Luciferase promoter assay for the Pin1 promoter shows that wild type C/EBPα is able to block Pin1 promoter activity. Mean while, C/EBPα-p30 couldn’t block Pin1 promotor activity. By silencing Pin1 by RNA Interference as well as with inhibitor against Pin1 (PiB) we could show myeloid differentiation in human CD34+ cord blood cells as well as in Kasumi-6 cells as assessed by FACS analysis with granulocytic markers. We investigated the mechanism underlying the dominant negative action of C/EBPα-p30 over the wild type protein. We report that Pin1 increases the transcriptional activity of the oncogene c-jun. We also show that c-jun blocks the DNA binding and transactivation of C/EBPα protein as assessed by gel shift assay and promoter assay respectively. We have previously shown that c-jun expression is high in AML patients with C/EBPα mutation and c-jun could block C/EBPα function by protein-protein interaction. Quantitative Real-Time RT-PCR analysis shows that inhibition of Pin1 by the inhibitor PiB downregulates c-jun mRNA expression. In conclusion, inhibition of Pin1 leads to granulocytic differentiation. Our results show Pin1 as a novel target in treating AML patients with C/EBPα mutation.

Long-Term Expansion and Self Renewal Is Contained in the CD34+, but Not the CD34- Subfraction of Nucleophosmin Mutated Acute Myeloid Leukemia Cells.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1349-1349
Author(s):  
Carolien Woolthuis ◽  
Lina Han ◽  
Djoke van Gosliga ◽  
Philip Kluin ◽  
Edo Vellenga ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Hox Genes ◽  
Mutant Protein ◽  
Self Renewal ◽  
Cd34 Cells ◽  
Stromal Layer ◽  
Acute Myeloid

Abstract Mutations in the nucleophosmin (NPM) gene are found in about 30% of cases of acute myeloid leukemia (AML) and lead to a dislocation of the nucleophosmin protein from the nucleus to the cytoplasm (NPMc+ AML). NPMc+ AML shows distinctive biological and clinical features, including a unique gene expression profile, a distinct microRNA signature, low percentage of CD34+ cells, increased incidence of Flt3-ITD (about 40% of cases), good response to induction chemotherapy and (in the absence of Flt3-ITD) a favourable prognosis. Despite significant progress in the characterization of the NPMc+ AML subgroup, questions remain about the leukemia-initiating cell. Distinct features of NPMc+ AML, including multilineage involvement and overexpression of HOX-genes, may point to an early progenitor as the leukemia-initiating cell, but the characteristic low percentage of CD34+ cells may point to a more differentiated leukemic stem cell in NPMc+ AML. To gain more insight in the leukemia-initiating cell in AML with mutated NPM, NPMc+ AML cells were sorted based on the expression of CD34 (n=8, the percentage of CD34+ in the total AML fraction varied between 0.06 and 37%). Western blotting, using an antibody that specifically recognizes the nucleophosmin mutant protein revealed that the NPM mutant protein is expressed in both CD34+ and CD34− cells, proving that the CD34+ NPMc+ AML cells belong to the leukemic clone. This was verified by sequencing the NPM gene in CD34+ and CD34− AML cells. Importantly, culture of sorted CD34+ and CD34− NPMc+ AML cells on a stromal layer revealed that the CD34+ but not the CD34− cells of NPMc+ AML were capable of expanding and initiating long-term growth. In the first 5 weeks of culture an at least 16 fold (range 16–208) expansion of CD34+ AML cells was seen in 5 out of 6 NPMc+ AML cases. This expansion was associated with the formation of cobblestone areas (CAs) under the stromal layer within 3 weeks after plating. The NPMc+ AML cells which expanded in culture were able to expand further after replating in 4 out of 5 investigated cases (fold expansion range 1.6–2.5), indicative of the self renewal capacity of these CD34+ NPMc+ AML cells. Gene expression analysis of CD34+ and CD34− NPMc+ AML cells of 4 cases analyzed thus far revealed the presence of the characteristic HOX-overexpression profile in both CD34+ and CD34− NPMc+ AML cells. In summary, this study shows that the NPM mutation is not only present in CD34−, but also in CD34+ cells of NPMc+ AML and that the properties of long-term expansion and self renewal belong exclusively to the CD34+ subfraction of NPMc+ AML.

Deregulated Apoptotic Pathways Point to Effectiveness of IAP Inhibitor Therapy in Acute Myeloid Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1275-1275
Author(s):  
Sonja C Lück ◽  
Annika C Russ ◽  
Konstanze Döhner ◽  
Ursula Botzenhardt ◽  
Domagoj Vucic ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Myeloid Leukemia ◽  
Cell Line ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Gene Set Enrichment Analysis ◽  
Core Binding Factor ◽  
Binding Factor ◽  
Acute Myeloid

Abstract Abstract 1275 Poster Board I-297 Core binding factor (CBF) leukemias, characterized by translocations t(8;21) or inv(16)/t(16;16) targeting the core binding factor, constitute acute myeloid leukemia (AML) subgroups with favorable prognosis. However, 40-50% of patients relapse, and the current classification system does not fully reflect the heterogeneity existing within the cytogenetic subgroups. Therefore, illuminating the biological mechanisms underlying these differences is important for an optimization of therapy. Previously, gene expression profiling (GEP) revealed two distinct CBF leukemia subgroups displaying significant outcome differences (Bullinger et al., Blood 2007). In order to further characterize these GEP defined CBF subgroups, we again used gene expression profiles to identify cell line models similar to the respective CBF cohorts. Treatment of these cell lines with cytarabine (araC) revealed a differential response to the drug as expected based on the expression patterns reflecting the CBF subgroups. In accordance, the cell lines resembling the inferior outcome CBF cohort (ME-1, MONO-MAC-1, OCI-AML2) were less sensitive to araC than those modeling the good prognostic subgroup (Kasumi-1, HEL, MV4-11). A previous gene set enrichment analysis had identified the pathways Caspase cascade in apoptosis and Role of mitochondria in apoptotic signaling among the most significant differentially regulated BioCarta pathways distinguishing the two CBF leukemia subgroups. Thus, we concluded that those pathways might be interesting targets for specific intervention, as deregulated apoptosis underlying the distinct subgroups should also result in a subgroup specific sensitivity to apoptotic stimuli. Therefore, we treated our model cell lines with the Smac mimetic BV6, which antagonizes inhibitor of apoptosis (IAP) proteins that are differentially expressed among our CBF cohorts. In general, sensitivity to BV6 treatment was higher in the cell lines corresponding to the subgroup with good outcome. Time-course experiments with the CBF leukemia cell line Kasumi-1 suggested a role for caspases in this response. Interestingly, combination treatment of araC and BV6 in Kasumi-1 showed a synergistic effect of these drugs, with the underlying mechanisms being currently further investigated. Based on the promising sensitivity to BV6 treatment in some cell lines, we next treated mononuclear cells (mostly leukemic blasts) derived from newly diagnosed AML patients with BV6 in vitro to evaluate BV6 potency in primary leukemia samples. Interestingly, in vitro BV6 treatment also discriminated AML cases into two distinct populations. Most patient samples were sensitive to BV6 monotherapy, but about one-third of cases were resistant even at higher BV6 dosage. GEP of BV6 sensitive patients (at 24h following either BV6 or DMSO treatment) provided insights into BV6-induced pathway alterations in the primary AML patient samples, which included apoptosis-related pathways. In contrast to the BV6 sensitive patients, GEP analyses of BV6 resistant cases revealed no differential regulation of apoptosis-related pathways in this cohort. These results provide evidence that targeting deregulated apoptosis pathways by Smac mimetics might represent a promising new therapeutic approach in AML and that GEP might be used to predict response to therapy, thereby enabling novel individual risk-adapted therapeutic approaches. Disclosures Vucic: Genentech, Inc.: Employment. Deshayes:Genentech, Inc.: Employment.

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