Tumor Suppressor RASSF2 Is Downregulated By The RUNX1-ETO Fusion Protein In t(8;21)+ Acute Myeloid Leukemia

Abstract Introduction The t(8;21) chromosomal translocation is one of the most common chromosomal translocations associated with acute myeloid leukemia (AML), present in greater than 10% of de novo AML cases. Most of these t(8;21) AML cases are classified as FAB subtype M2. This translocation results in the formation of a stable fusion protein made up of portions of the RUNX1 (aka AML1) and ETO (aka MTG8 and RUNX1T1) proteins. RUNX1 is a transcription factor that is essential for regulating the differentiation of hematopoietic cells, and the fusion protein retains its DNA-binding domain. Additionally, ETO contains four Nervy homology (NH) domains which facilitate a number of protein-protein interactions, notably with the NCOR2/SMRT co-repressor complex. The identification of individual genes or biological pathways which are specifically disrupted in the presence of RUNX1-ETO will provide further molecular insight into the pathogenesis of t(8;21)+ AML and lead to the possibility for improved treatment for these patients. Methods/Results We analyzed publicly available gene expression microarray datasets (Oncomine, TCGA) to search for genes whose expression was significantly altered in the blood of t(8;21)+ AML patients as compared to non-t(8;21) FAB subtype M2 AML and to CD34+ cells in healthy controls. One such gene that was consistently significantly downregulated in t(8;21)+ patients was Ras-association domain family member 2 (RASSF2). RASSF2 is a putative tumor suppressor that is capable of mediating apoptosis (in a Ras dependent manner) through its interactions with the MST1/2 kinases and the cancer-specific apoptotic protein Par-4. RASSF2 has previously been shown to be frequently downregulated via hypermethylation in a wide variety of solid tumors, however little is known about its function in leukemia. Here we demonstrate that RASSF2 is a potentially interesting target for downregulation by the RUNX1-ETO fusion protein. Gene expression analysis by RT-qPCR in leukemia cell lines confirmed that RASSF2 is significantly downregulated in both Kasumi-1 and SKNO t(8;21)+ cell lines as compared to a similar non-t(8;21) HL-60 line. We found that exogenous expression of AML1-ETO in HL-60 leukemia cells induces a rapid downregulation of RASSF2, further supporting that it is a target of this leukemogenic fusion protein. Over-expression of RASSF2 in leukemia cells significantly inhibits their proliferative capability, indicating an important biological effect of RASSF2 in blood cells. Finally, over-expression of RASSF2 significantly inhibits the long-term self-renewal capability of RUNX1-ETO expressing hematopoietic cells as measured by their serial replating ability in a colony formation assay. Discussion Based on the analysis of patient data and our own experiments it appears that RASSF2 is a direct target for downregulation by the AML1-ETO fusion protein. Due to its potential involvement as a mediator of apoptosis in important oncogenic signaling pathways RASSF2 is a strong candidate for further investigation in the context of t(8;21)+ AML pathogenesis. In particular, it will be interesting to continue to investigate the relationship between RASSF2 and apoptotic protein Par-4, as several lines of evidence suggest Par-4 to be therapeutically relevant due to its ability to selectively induce apoptosis in cancer cells. Disclosures: No relevant conflicts of interest to declare.

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Identification of Sensitizing Targets to the Hypomethylating Agent 5-Azacytidine in Acute Myeloid Leukemia Cells Using RNAi-Based Functional Genomics Screening

Blood ◽

10.1182/blood.v112.11.2665.2665 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2665-2665

Author(s):

Raoul Tibes ◽

Ashish Choudhary ◽

Amanda Henrichs ◽

Sadia Guled ◽

Irma Monzon ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Growth Inhibition ◽

Cell Lines ◽

Myeloid Leukemia ◽

Statistical Significance ◽

Molecular Targets ◽

Leukemia Cells ◽

Hypomethylating Agents ◽

Acute Myeloid Leukemia Cells ◽

Acute Myeloid

Abstract • Hypomethylating agents like 5-Azacytidine (5Aza) have become an effective therapy for myelodysplastic syndromes (MDS) and show promise in acute myeloid leukemia (AML). In AML, complimentary mechanisms including epigenetic silencing of growth controlling genes, i.e. tumor suppressors, and activation of kinases contribute to malignant transformation. In order to enhance the therapeutic potential of epigenetic therapies, we developed a high-throughput RNA interference (HT-RNAi) platform for large-scale transient gene silencing in acute myeloid leukemia cells. This assay allows for the first time to individually silence hundreds or thousands of genes in combination with 5Aza to identify molecular targets whose inhibition enhances the anti-leukemic effect of hypomethylating agents. As part of assay development for HT-RNAi, ten AML cell lines were used to determine the median inhibitory concentration (IC50) of 5Aza for each AML cell lines. Furthermore, the ten cell lines were tested with a panel of cationic lipid transfection reagents at varying weight to volume (wt:vol) ratios to determine the optimal siRNA transfection conditions. Results from these studies identified two AML cell lines TF1 and ML4, which were advanced into kinome-epigenetic RNAi screens. Using a lipid-based method, cells were reverse transfected for 48hrs with 2 different siRNA sequences per gene targeting a total of 572 kinases. After 48hrs, 5Aza at the calculated IC25 was added for an additional 72 hrs and cell proliferation was measured using a luminescence-based assay. Data was background corrected and analyzed using the B-score method to report the strength and statistical significance of growth inhibition compared to controls. A B-score of <−2 indicates statistical significance with p<0.05 (>95% confidence); a B-score <−1.5 provides >87% confidence and was used as lowest cutoff given that screens are focused and contain validated siRNA to kinases. Analysis of two independent RNAi kinome screens, one in TF1 and the other in ML4, in combination with 5Aza, identified six and eleven kinases respectively whose silencing by two different siRNA sequences (2× coverage) potentiated the effects of 5Aza at B-score <−1.5. In ML4 cells 2 kinases were highly significant with a B-score for both siRNA <−2. Six kinases were common targets in both cell lines with significant growth inhibition at a B-score for both siRNA of at least <−1.5 making these kinases potential important modifiers of response to 5Aza. In summary, initial kinome RNAi screens in myeloid cells identified specific kinases as potential sensitizing targets to hypomethylating agents. Moreover, functional genomic RNAi screens provide a fast and attractive approach to identify molecular targets in AML for the rational development of combination therapies with hypomethylating agents as well as other drug classes.

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

Blood ◽

10.1182/blood.v114.22.1275.1275 ◽

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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Hypomethylation of Tumor Suppressor Genes in Acute Myeloid Leukemia: Characteristic of Cell Lines with MLL Abnormalities?.

Blood ◽

10.1182/blood.v114.22.365.365 ◽

2009 ◽

Vol 114 (22) ◽

pp. 365-365

Author(s):

Hilmar Quentmeier ◽

Sonja Röhrs ◽

Wilhelm G Dirks ◽

Claus Meyer ◽

Rolf Marschalek ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Tumor Suppressor ◽

Acute Leukemia ◽

Cell Lines ◽

Fusion Proteins ◽

Myeloid Leukemia ◽

Target Genes ◽

Leukemia Cell ◽

Hoxa Cluster ◽

Acute Myeloid

Abstract Abstract 365 Background: Translocations of the Mixed Lineage Leukemia (MLL) gene occur in a subset (5%) of acute myeloid leukemia (AML) and in mixed phenotype acute leukemia in infancy, a disease with extremely poor prognosis. Animal model systems show that MLL gain of function mutations may contribute to leukemogenesis. Wild-type MLL carries histone methyltransferase activity and affects specific target genes, such us HOXA cluster genes. While the more than three dozen MLL fusion proteins known today exert different specific functions, they finally induce transcription of individual target genes. Consequently, acute lymphoblastic leukemias (ALL) with MLL mutations (MLLmu) exhibit typical gene expression profiles including high-level expression of HOXA cluster genes. Aim of this study was to find a correlation between the MLL mutational status and tumor suppressor gene methylation/expression in acute leukemia cell lines. Results: Using MS-MLPA (methylation-specific multiplex ligation-dependent probe amplification assay), methylation of 24 different TSG was analyzed in 28 MLLmu and MLLwt acute leukemia cell lines. 1.8/24 TSG were methylated in MLLmu AML cells, 6.2/24 TSG were methylated in MLLwt AML cells. Hypomethylation and expression of the tumor suppressor genes (TSG) BEX2, IGSF4 and TIMP3 turned out to be characteristic of MLLmu acute myeloid leukemia (AML) cell lines. MLL wild-type (MLLwt) AML cell lines displayed hypermethylated TSG promoters resulting in transcriptional silencing. Demethylating agents and inhibitors of histone deacetylases restored expression of BEX2, IGSF4 and TIMP3 confirming epigenetic silencing of these genes in MLLwt cells. The positive correlation between MLL translocation, TSG hypomethylation and expression suggested that MLL fusion proteins were responsible for dysregulation of TSG expression in MLLmu cells. This concept was supported by our observation that Bex2 mRNA levels in MLL-ENL transgenic mouse cell lines required expression of the MLL fusion gene. Conclusion: These results suggest that the conspicuous expression of the TSG BEX2, IGSF4 and TIMP3 in MLLmu AML cell lines is the consequence of altered epigenetic properties of MLL fusion proteins. Disclosures: No relevant conflicts of interest to declare.

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Activities and Mechanism Based Combinations Of Omacetaxine In Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v122.21.1288.1288 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1288-1288

Author(s):

Rong Chen ◽

Bonnie Leung ◽

Yuling Chen ◽

William Plunkett

Keyword(s):

Acute Myeloid Leukemia ◽

Protein Synthesis ◽

Cell Lines ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Lymphocytic Leukemia ◽

Leukemia Cells ◽

Bh3 Mimetics ◽

Abl Kinase ◽

Acute Myeloid

Abstract Omacetaxine, an inhibitor of translation, was recently granted accelerated approval for the treatment of chronic myeloid leukemia (CML). Omacetaxine blocks translation elongation by competing with the incoming aminoacyl-tRNAs for binding to the A-site cleft in the peptidyl-transferase center. Our previous studies showed that by transiently inhibiting translation, omacetaxine reduced the expression of the key, short-lived oncoproteins Bcr/Abl and Mcl-1, leading to cell death in the CML cells. This action sensitized the cells to the Abl kinase inhibitor and killed the CML cells synergistically. Further, as omacetaxine acts in a different mechanism than the Abl kinase inhibitors, it overcame resistance to TKI that was associated with kinase domain mutations. These studies paved the foundation for the clinical development of omacetaxine in CML. We also demonstrated that omacetaxine was active in chronic lymphocytic leukemia by translational inhibition of Mcl-1 expression. In contrast to normal tissues, the fact that the leukemia cells are critically dependent on the oncogene activity for survival provided a biologic context for a positive therapeutic index. As the biological features of acute myeloid leukemia (AML) rely largely on the overexpressed oncoproteins or constitutively activated kinases, we hypothesized that omacetaxine would have therapeutic benefit in AML either alone or in mechanism based combinations. To test this hypothesis, first, we compared omacetaxine to AC220, a potent FLT3 inhibitor, in AML cell lines OCI-AML3 and MV4-11. OCI-AML3 cells harbor the signature mutation of NPM1, whereas MV4-11 is a widely used model for the internal tandem duplications of FLT3 (FLT3-ITD), a common FLT3 mutation that constitutively activates the receptor tyrosine kinase. AC220 was selectively toxic to the MV4-11 cells, but had no effect on the viability of OCI-AML3. This is consistent with the biological context of MV4-11, but not OCI-AML3, that is addicted to the sustained activity of FLT3 for survival. In contrast, omacetaxine induced apoptosis in both cell lines with IC50s less than 100 nM. Protein synthesis was inhibited in both lines, measured by the incorporation of tritiated leucine. Apoptosis was induced rapidly within 24 h by omacetaxine, whereas AC220 required 72 h to kill the leukemia cells. These results indicated a common dependence on the continued protein synthesis in the AML lines, suggesting a potentially broad application of omacetaxine in AML patients with diverse genetic backgrounds. Over-expression of the anti-apoptotic protein Mcl-1 is associated with AML disease maintenance and resistant to therapy. Both Mcl-1 and FLT3 turn-over rapidly and are vulnerable targets of transient translation inhibition. Immunoblots showed that omacetaxine reduced the levels of both FLT3 and Mcl-1 in the MV4-11 cells. This activity augmented the effect of AC220 on FLT3 kinase, and induced synergistic apoptosis. Same synergistic combination was observed with omacetaxine and sunitinib, an inhibitor of FLT3, KIT and PDGF-R. Dose reduction index derived from these analyses showed that omacetaxine greatly potentiated the activity of both AC220 and sunitinib, resulting in profound apoptosis. Both Bcl-2 and Mcl-1 are pro-survival proteins that regulate apoptosis by interacting with the BH3 motifs of their pro-apoptotic partners. BH3 mimetics, such as ABT-199, bind with high affinity to Bcl-2 and block this interaction, but not to Mcl-1. Resistance to BH3 mimetics in AML cells is associated with upregulation of Mcl-1. Since ABT-199 inhibits Bcl-2 but spares Mcl-1, and omacetaxine reduces Mcl-1 without affecting Bcl-2 expression, we hypothesized that their combination would target the two parallel arms of apoptosis control and kill the AML cells synergistically. Indeed, omacetaxine reduced Mcl-1 in the OCI-AML3 cells, leading to loss of mitochondrial membrane potential and apoptosis. ABT-199 blocked Bcl-2 function and also induced the intrinsic pathway of apoptosis. Their combination induced greater mitochondrial damage and apoptosis than either drug alone. The median effect analysis showed that they potentiate each other and exhibited strong synergy. Taken together, these results demonstrated that omacetaxine is active in AML cells alone and in mechanism based combinations. These actions provide rationale that warrants investigation in the clinic. Disclosures: No relevant conflicts of interest to declare.

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Inhibition Of LSD1 As a Therapeutic Strategy For The Treatment Of Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v122.21.3964.3964 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3964-3964 ◽

Cited By ~ 14

Author(s):

Ryan G. Kruger ◽

Helai Mohammad ◽

Kimberly Smitheman ◽

Monica Cusan ◽

Yan Liu ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Cell Surface ◽

Growth Inhibition ◽

Cell Lines ◽

Myeloid Leukemia ◽

Stem Cell Differentiation ◽

Ethical Review Process ◽

Acute Myeloid

Abstract Lysine specific demethylase 1 (LSD1) is a histone H3K4me1/2 demethylase found in various transcriptional co-repressor complexes. These complexes include Histone Deacetylases (HDAC1/2) and Co-Repressor for Element-1-Silencing Transcription factor (CoREST). LSD1 mediated H3K4 demethylation can result in a repressive chromatin environment that silences gene expression. LSD1 has been shown to play a role in development in various contexts. LSD1 can interact with pluripotency factors in human embryonic stem cells and is important for decommissioning enhancers in stem cell differentiation. Beyond embryonic settings, LSD1 is also critical for hematopoietic differentiation. LSD1 is overexpressed in multiple cancer types and recent studies suggest inhibition of LSD1 reactivates the all-trans retinoic acid receptor pathway in acute myeloid leukemia (AML). These studies implicate LSD1 as a key regulator of the epigenome that modulates gene expression through post-translational modification of histones and through its presence in transcriptional complexes. The current study describes the anti-tumor effects of a novel LSD1 inhibitor (GSK2879552) in AML. GSK2879552 is a potent, selective, mechanism-based, irreversible inhibitor of LSD1. Screening of over 150 cancer cell lines revealed that AML cells have a unique requirement for LSD1. While LSD1 inhibition did not affect the global levels of H3K4me1 or H3K4me2, local changes in these histone marks were observed near transcriptional start sites of putative LSD1 target genes. This increase in the transcriptionally activating histone modification correlated with a dose dependent increase in gene expression. Treatment with GSK2879552 promoted the expression of cell surface markers, including CD11b and CD86, associated with a differentiated immunophenotype in 12 of 13 AML cell lines. For example, in SKM-1 cells, increases in cell surface expression of CD86 and CD11b occurred after as early as one day of treatment with EC50 values of 13 and 7 nM respectively. In a separate study using an MV-4-11 engraftment model, increases in CD86 and CD11b were observed as early as 8 hours post dosing. GSK2879552 treatment resulted in a potent anti-proliferative growth effect in 19 of 25 AML cell lines (average EC50 = 38 nM), representing a range of AML subtypes. Potent growth inhibition was also observed on AML blast colony forming ability in 4 out of 5 bone marrow samples derived from primary AML patient samples (average EC50 = 205 nM). The effects of LSD1 inhibition were further characterized in an in vivo mouse model of AML induced by transduction of mouse hematopoietic progenitor cells with a retrovirus encoding MLL-AF9 and GFP. Primary AML cells were transplanted into a cohort of secondary recipient mice and upon engraftment, the mice were treated for 17 days. After 17 days of treatment, control treated mice had 80% GFP+ cells in the bone marrow whereas treated mice possessed 2.8% GFP positive cells (p<0.012). The percentage of GFP+ cells continued to decrease to 1.8% by 1-week post therapy. Remarkably, in a preliminary assessment for survival, control-treated mice succumbed to AML by 28 days post transplant, while treated mice showed prolonged survival. Together, these data demonstrate that pharmacological inhibition of LSD1 may provide a promising treatment for AML by promoting differentiation and subsequent growth inhibition of AML blasts. GSK2879552 is currently in late preclinical development and clinical trials are anticipated to start in 2014. All studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed the Institutional Animal Care and Use Committee either at GSK or by the ethical review process at the institution where the work was performed. Disclosures: Kruger: GlaxoSmithKline Pharmaceuticals: Employment. Mohammad:GlaxoSmithKline Pharmaceuticals: Employment. Smitheman:GlaxoSmithKline Pharmaceuticals: Employment. Liu:GlaxoSmithKline Pharmaceuticals: Employment. Pappalardi:GlaxoSmithKline Pharmaceuticals: Employment. Federowicz:GlaxoSmithKline Pharmaceuticals: Employment. Van Aller:GlaxoSmithKline Pharmaceuticals: Employment. Kasparec:GlaxoSmithKline Pharmaceuticals: Employment. Tian:GlaxoSmithKline Pharmaceuticals: Employment. Suarez:GlaxoSmithKline Pharmaceuticals: Employment. Rouse:GlaxoSmithKline Pharmaceuticals: Employment. Schneck:GlaxoSmithKline Pharmaceuticals: Employment. Carson:GlaxoSmithKline Pharmaceuticals: Employment. McDevitt:GlaxoSmithKline Pharmaceuticals: Employment. Ho:GlaxoSmithKline Pharmaceuticals: Employment. McHugh:GlaxoSmithKline Pharmaceuticals: Employment. Miller:GlaxoSmithKline Pharmaceuticals: Employment. Johnson:GlaxoSmithKline Pharmaceuticals: Employment. Armstrong:Epizyme Inc.: Has consulted for Epizyme Inc. Other. Tummino:GlaxoSmithKline Pharmaceuticals: Employment.

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Repositioning of Quinacrine for Treatment of Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v124.21.3609.3609 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3609-3609

Author(s):

Anna Eriksson ◽

Albin Osterros ◽

Sadia Hassan ◽

Joachim Gullbo ◽

Linda Rickardson ◽

...

Keyword(s):

Gene Expression ◽

Acute Myeloid Leukemia ◽

Cytotoxic Activity ◽

Tumor Cells ◽

Cell Lines ◽

Myeloid Leukemia ◽

Lymphocytic Leukemia ◽

Ribosomal Biogenesis ◽

Rheumatic Drug ◽

Acute Myeloid

Abstract Background: A promising strategy for new drug discovery is ‘repositioning’, in which a new indication for an existing drug is identified. Using this approach, known on-patent, off-patent, discontinued and withdrawn drugs with unrecognized cancer activity, can be rapidly advanced into clinical trials for the new indication. We here report findings from a library screen of pharmacologically active and mechanistically annotated compounds in leukemia cells from patients aiming at the identification of repositioning candidates. Methods and results: The LOPAC®, 1280substance library (Sigma-Aldrich), with 1266 mechanistically annotated compounds, were investigated for cytotoxic activity by the fluorometric microculture cytotoxicity assay (FMCA) on tumor cells from 12 patients with leukemia (4 acute lymphocytic leukemia, 4 acute myeloid leukemia [AML], 4 chronic lymphocytic leukemia), as well as on peripheral blood mononuclear cells (PBMC) from 4 healthy donors. Sixty-eight compounds were identified as hits, defined as having a cytotoxic activity (less than 50% cell survival compared with controls) in all leukemia subgroups at the 10µM drug concentration used for screening. Only one of the hit compounds, quinacrine, showed higher activity in the leukemic cells than in normal PBMCs and was therefore selected for further preclinical evaluation focusing on AML. The aminoacridine quinacrine has a wide range of biological and therapeutical applications, and has been used for decades outside hemato-oncology, notably as an anti-protozoal and anti-rheumatic drug. Its side effects and toxicity are well characterized. Quinacrine showed significant cytotoxic activity in all four AML cell lines tested (HL-60, Kasumi-1, KG1a and MV4-11). In tumor cells from another 9 patients with AML, the cytotoxic effect (IC50 median 1.8, range 0.8-4 µM) was significantly superior to that in normal lymphocytes and clearly dose-dependent. Analysis of quinacrine data from the National Cancer Institute growth inhibitory screen in 60 cell lines (NCI 60 GI 50 data) was performed with the help of the NCI Cellminer database (http://discover.nci.nih.gov/cellminer/), and indicated leukemia sensitivity. To examine the ability of quinacrine to reverse diagnosis-specific gene expression, we utilized the Nextbio bioinformatics software, with its gene expression signatures of drug exposed myeloid leukemia cell cultures (HL60). These queries showed that myeloid leukemias had high reversibility scores. Moreover, gene enrichment and drug correlation data revealed a strong association to ribosomal biogenesis nucleoli. Translation initiation was observed including a high drug-drug correlation with ellipticine, a known inhibitor of RNA polymerase I (Pol-I). To validate the latter results, gene expression analysis of HL-60 cells exposed to quinacrine were obtained using the protocol described by Lamb et al (Science, 2006, 313, 1929), showing down regulation of Pol-1 associated RNA. Supporting these findings, quinacrine induced early inhibition of protein synthesis. Conclusions: The anti-protozoal and anti-rheumatic drug quinacrine has significant in vitro activity in AML. The anti-leukemic effect may be mediated by targeting ribosomal biogenesis. Considering its favorable and well-known safety profile, clinical studies of quinacrine in AML should be considered. Disclosures No relevant conflicts of interest to declare.

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The Role of TWIST1 Gene Expression and Hypoxic Microenvironment in Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v126.23.3839.3839 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3839-3839

Author(s):

Emilia Carolina Malafaia ◽

A. Mario Marcondes ◽

Ekapun Karoopongse ◽

Daniele Serehi ◽

Maria de Lourdes L. F. Chauffaille ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Leukemia Cells ◽

Twist1 Expression ◽

Disease Biology ◽

Acute Myeloid

Abstract TWIST1, a basic helix-loop-helix (bHLH) transcription factor, plays a critical role in mesodermal development and organogenesis. Overexpressed TWIST1 has been thoroughly related to epithelial-mesenchymal transition (EMT) in solid tumors (QIN Q et al., 2012) and has been described as an emerging risk factor in hematological neoplasms (MERINDOL et al., 2014). . Many questions remain to be addressed concerning to the role of TWIST1 in acute myeloid leukemia (AML). The understanding of TWIST1 in leukemia cells and its interaction with microenvironment can offer new insights in regards to disease biology and therapeutic targets for patients with AML. Objectives: 1) to evaluate the role of stroma contact and hypoxia in TWIST1 expression in myeloid cell lines. 2) To evaluate the functional impact of overexpressing TWIST1 on KG1a and PL21 cells. 3) To evaluate TWIST1 expression in primary cells of AML patients. Methods: In order to mimic bone marrow microenvironment, myeloid cells were co-cultured with mesenchymal HS5 cell line and PO2 1% was established with Smart -Trak¨ 2 (Sierra Instruments, Inc.) equipment. Quantitative mRNA was determined using TaqMan¨ Universal Master Mix (Applied Biosystems, Foster City, CA) and 3-step standard cycling conditions with sequence-specific primer TWIST1 normalized to the expression of β-actin. KG1a and PL21 cells were transduced with lentivirus vector carrying e-GFP ("enhanced green fluorescence protein") for stable expression of TWIST1. Transduced cells were sorted by FITC fluorochrome and then verified through western blot analysis with TWIST1 antibody. For quantification of apoptosis, cells were labeled with PE-conjugated antibody using annexin V-phycoerythrin and propidium iodide (BD Biosciences, USA). DAPI (4',6- diamidino-2-phenylindole dihydrochloride) was used to stain DNA and determine cell cycle information . Apoptosis and cell cycle were analyzed by FACS -Becton Dickinson Canto II (BD Biosciences). Statistical analysis was assessed with unpaired t test. Results: Hypoxia induced TWIST1 mRNA expression in OCIAML3, PL21, KG1a and ML1 cell lines (fold-increased 46.3, 29.8, 12.9 and 2.3 respectively). Cells expressing endogenous TWIST1 protein (OCIAML3 and ML1) showed resistance to apoptosis in a hypoxic microenvironment (normoxia versus hypoxia: OCI/AML3, 22.6 % vs 11.7% and ML1, 29.8% vs. 7.5%) in contrast, cells not expressing endogenous TWIST1 protein (KG1a and PL21) went to apoptosis in the same conditions. Thus, overexpressing TWIST1 in KG1a and PL21 induced apoptosis protection in hypoxia (KG1a unmodified vs. modified: 17.6 ± 6.3 vs. 2.8 ± 6.3, p=0.04; PL21 unmodified vs. modified: 26.9 ± 10.9 vs. 3.2 ± 0.6, p=0.04) (fig 1). We found increased TWIST1 mRNA levels in bone marrow samples of 23 AML patients (3.88 ± 1.59) compared with 5 healthy controls (0.54 ±0.25) (p= 0.02) (fig 2). Patients in the highest tertile of TWIST1 expression did not show differences in percentage of blasts in bone marrow and complete remission after treatment compared with patients in low and middle tertile. Conclusion: Our data suggest TWIST1 gene expression protects acute myeloid leukemia cells from apoptosis in a hypoxic microenvironment. Moreover, our results showed increased expression of TWIST1 in AML patients. Thus, TWIST1 is a potential gene involved in leukemogenesis and should be further explored to understand disease biology and potential therapeutic targets. Disclosures No relevant conflicts of interest to declare.

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