Combined Analysis of Valproic Acid Induced MicroRNA and Gene Expression Changes in Acute Myeloid Leukemia.

Abstract Inhibitors of histone deacetylases (HDACs) like valproic acid (VPA) display activity in murine leukemia models and induce apoptosis and myeloid differentiation of acute myeloid leukemia (AML) blasts. While recently several studies examined the underlying VPA-mediated mechanisms, until today not many genes have been identified whose expression is altered by VPA treatment. Recently, microRNAs (miRs), a novel abundant class of negative gene regulators, have been shown to control a wide range of biological functions such as proliferation, differentiation and apoptosis by either translational repression or by mRNA cleavage or miR-mediated decay of the respective target mRNA. Furthermore, deregulated miR expression has been associated with various human cancers including leukemia. This led us to investigate whether VPA treatment of AML cells affects miR-expression which in turn might influence the level of miR target genes involved in VPA effects. First, we identified an in vitro miR VPA-response signature by profiling miR expression in 4 different myeloid leukemia cell lines following 48 hours of VPA treatment (Ambion microarray platform comprising 281 human miRs). In parallel, we profiled gene expression by using both cDNA microarrays and Affymetrix U133Aplus2.0 GeneChips. 13 miRs were found to be differentially expressed, 10 miRs were down-regulated and 3 miRs were up-regulated by VPA. Gene expression profiling revealed several hundred differentially regulated genes containing some known VPA influenced targets like e.g. cyclin-dependent kinase inhibitor CDKN1A coding for p21. To correlate miR and gene expression, we next searched for an enrichment of putative miR target genes of the VPA-regulated miRs in the VPA-induced gene expression pattern. Interestingly, there were several candidates for which miR expression in response to VPA inversely correlated with gene expression of the respective targets. These included genes involved in DNA damage checkpoint like e.g. CHEK1 which was found to be down-regulated in response to VPA and which is a predicted target of miR-15a and miR-16, both found to be up-regulated by VPA treatment. In addition, potential miR-targets included genes known to be regulated by HDAC inhibitors in cancer cells like e.g. the homeobox gene HOXA1 found to be up-regulated in response to VPA and being a putative target of miR-99a, found to be down-regulated by VPA. Our study is the first to show that VPA treatment significantly affects expression levels of several miRs in myeloid cell lines, and based on the correlation of VPA-induced miR and gene expression patterns we could identify putative miR-targets that included genes with tumorigenic relevance. While it remains to be determined whether VPA-induced miR-mediated mRNA cleavage or decay of the respective target mRNAs is involved in leukemogenesis, our data nevertheless provide new insights into VPA-induced mechanisms of myeloid differentiation and into deregulated miR expression in leukemia.

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Activation of C/EBPa Myeloid-Specific Transcription Factor By NAMPT/SIRT1-Triggered Deacetylation

Blood ◽

10.1182/blood.v126.23.2199.2199 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2199-2199

Author(s):

Bardia Samareh ◽

Masoud Nasri ◽

Inna Zimmer ◽

Olga Klimenkova ◽

Leonie Keller ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Transcription Factor ◽

Cell Lines ◽

Myeloid Leukemia ◽

Target Genes ◽

Leukemia Cell ◽

Myeloid Differentiation ◽

Granulocytic Differentiation ◽

Acute Myeloid ◽

Myeloid Leukemia Cell

Abstract Previously, we described new mechanism of G-CSF-triggered granulocytic differentiation of hematopoietic stem cells (HSCs) via activation of the enzyme Nicotinamide Phosphorybosyltransferase (NAMPT) leading to NAD+ production and activation of NAD+ -dependent protein deacetylase sirtuin 1 (SIRT1). We found, that upon stimulation of HSCs with NAMPT, SIRT1 bound to the key myeloid transcription factor C/EBPα followed by transcriptional induction of C/EBPα target genes G-CSFR and G-CSF and granulocytic differentiation. In the present work we investigated the mechanism of NAMPT/SIRT1-triggered deacetylation of C/EBPα. We found that C/EBPα is acetylated at the position Lys 161, which is evolutionarily conserved. Lys 161 is localized in the transactivation element III (TE-III) of the transactivation domain (TAD) of C/EBPα protein, which is responsible for recruitment of SWI/SNF and CDK2/CDK4. Western blot and DUOLINK analysis using rabbit polyclonal antibody specifically recognizing acetyl-Lys 161 of C/EBPα revealed predominantly nuclear localization of acetylated C/EBPα protein in acute myeloid leukemia cell lines NB4 and HL60 as well as in primary HSCs. Induction of myeloid differentiation of HSCs by treatment with G-CSF as well as ATRA-induced differentiation of NB4 cells resulted in the deacetylation of C/EBPα. NAMPT inhibition in NB4 and HL60 cell lines using specific inhibitor FK866 led to the dramatically elevated levels of acetylated C/EBPα and reduced amounts of total C/EBPα protein, which was in line with diminished mRNA expression of C/EBPα target genes (G-CSF, G-CSFR and ELANE). Interestingly, treatment of acute myeloid leukemia cell line HL60 with NAMPT or transduction of HL-60 cells with NAMPT-expressing lentiviral construct induced myeloid differentiation of these cells even without addition of ATRA. This was in line with time- and dose-dependent increase of total C/EBPα protein levels upon NAMPT treatment. Therefore, NAMPT overcomes transcriptional repression of C/EBPα in HL-60 cells by activation of positive CEBPA autoregulation. Taken together, we described a new mechanism of regulation of C/EBPα activities in hematopoiesis and leukemogenesis by its post-translational modification via NAMPT/SIRT1-triggered de-/acetylation. Disclosures No relevant conflicts of interest to declare.

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Identification of Distinct inv(16) Subclasses in Adult Acute Myeloid Leukemia Based on Gene Expression Profiling.

Blood ◽

10.1182/blood.v104.11.2037.2037 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2037-2037

Author(s):

Lars Bullinger ◽

Claudia Scholl ◽

Eric Bair ◽

Konstanze Dohner ◽

Stefan Frohling ◽

...

Keyword(s):

Gene Expression ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Target Genes ◽

Expression Patterns ◽

Rank Test ◽

Survival Times ◽

Strong Trend ◽

Candidate Target ◽

Acute Myeloid

Abstract Recurrent cytogenetic aberrations have been shown to constitute markers of diagnostic and prognostic value in acute myeloid leukemia (AML). However, even within the well-defined cytogenetic AML subgroup with an inv(16) we see substantial biological and clinical heterogeneity which is not fully reflected by the current classification system. To better characterize this cytogenetic group on the molecular level we profiled gene expression in a series of adult AML patients (n=26) with inv(16) using 42k cDNA microarrays. By unsupervised hierarchical clustering we observed that samples with inv(16) separated primarily into two different subgroups. These showed no significant differences regarding known risk factors like age, WBC, LDH, etc. However, these newly defined inv(16)-subgroups were characterized by distinct clinical behavior. There was a strong trend towards unfavorable outcome with shorter overall survival times in one group (P=0.09, log rank test). Since the primary translocation/inversion events themselves are not sufficient for leukemogenesis, distinct patterns of gene expression found within each of these cytogenetic groups may suggest alternative cooperating mutations and deregulated pathways leading to transformation. Therefore, we performed a supervised analysis to determine the characteristic gene expression patterns underlying the cluster-defined subgroups. This Significance Analysis of Microarrays (SAM) method identified 260 genes significantly differentially expressed between the two newly defined inv(16)-subgroups (false discovery rate = 0.002). High expression levels of JUN, JUNB, JUND, FOS and FOSB characterized the first inv(16) subgroup (having less favorable prognosis). FOS gene family members can dimerize with proteins of the JUN family, forming the transcription factor complex AP-1 which has been implicated in the regulation of cell proliferation, differentiation, and transformation. Among the second subgroup, the proto-oncogene ETS1,displayed elevated expression, possibly resulting from aberrant MEK/ERK pathway activation as these cases also showed an over-expression of MAP3K1 and MAP3K2. In conclusion, both supervised and unsupervised methods provide numerous insights into the pathogenesis of AML with inv(16), identifying clinically significant patterns of gene expression, as well as candidate target genes involved in leukemogenesis.

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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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Identifying Small Molecules That Overcome HoxA9-Mediated Differentiation Arrest in Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v120.21.3513.3513 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3513-3513

Author(s):

David B. Sykes ◽

Mark K Haynes ◽

Nicola Tolliday ◽

Anna Waller ◽

Julien M Cobert ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Acute Myeloid Leukemia ◽

Small Molecules ◽

Cell Line ◽

Cell Lines ◽

Myeloid Leukemia ◽

Myeloid Differentiation ◽

Prostacyclin Receptor ◽

Acute Myeloid

Abstract Abstract 3513 AML in adults is a devastating disease with a 5-year survival rate of 25%. We lack new treatments for AML, and the chemotherapy standard of care remains unchanged in thirty years. One success story in the treatment of AML has been the discovery of drugs that trigger the differentiation of leukemic blasts in the small subset of patients with acute promyelocytic leukemia. However, differentiation therapy is unfortunately not available for the remaining 90% of non-APL acute myeloid leukemia patients. Understanding and targeting the mechanism of differentiation arrest in AML has been under investigation for more than four decades. There is growing evidence to support the role of the homeobox transcription factors in normal hematopoietic differentiation as well as malignant hematopoiesis. The persistent, and inappropriate, expression of the homeobox gene HoxA9 has been described in the majority of acute myeloid leukemias. This implicates HoxA9 dysregulation as a common pathway of differentiation arrest in myeloid leukemias and suggests that by understanding and targeting this pathway, one might be able to overcome differentiation arrest. In cultures of primary murine bone marrow, constitutive expression of HoxA9 blocks myeloid differentiation and results in the outgrowth of immature myeloid cell lines. The mechanism by which HoxA9 causes differentiation arrest is not known and no compounds exist that inhibit HoxA9. We developed a murine cell line model in which the cells were blocked in differentiation by a conditional version of HoxA9. In this system, an estrogen-dependent ER-HoxA9 protein was generated by fusion with the estrogen receptor hormone-binding domain. When expressed in cultures of primary murine bone marrow, immortalized myeloblast cell lines can grow indefinitely in the presence of stem cell factor and beta-estradiol. Upon removal of beta-estradiol, and inactivation of HoxA9, these cell lines undergo synchronous and terminal myeloid differentiation. We took advantage of an available transgenic mouse model in which GFP was expressed downstream of the lysozyme promoter, a promoter expressed only in mature neutrophils and macrophages. Cell lines derived from the bone marrow of this lysozyme-GFP mouse were GFP-negative at baseline and brightly GFP-positive upon differentiation. In this manner, we generated a cell line with a built-in reporter of differentiation. These cells formed the basis of a high-throughput screen in which cells were incubated with small molecules for a period of four days in 384-well plate format. The cells were assayed by multi-parameter flow cytometry to assess for toxicity and differentiation. Compounds that triggered green fluorescence were scored as “HITS” and their pro-differentiation effects confirmed by analysis of morphology and cell surface markers. Given the availability of cells and the simple and reliable assay, we performed both a pilot screen of small molecules at The Broad Institute as well as an extensive screen of the NIH Molecular Libraries Small Molecule Repository. The screen of more than 350,000 small molecules was carried out in collaboration with the University of New Mexico Center for Molecular Discovery. We have identified one lead class of compounds - prostacyclin agonists – capable of promoting myeloid differentiation in this cell line model of AML. Using a parallel cell line derived from a prostacyclin receptor knock-out mouse, we confirmed that activity was due to signaling through the prostacyclin receptor. The role of prostacyclin signaling in myeloid differentiation has not been previously described. Analysis of gene expression demonstrated that the expression of the prostacyclin receptor is seen in ∼60% of in primary human AML samples. This is a potentially exciting finding as prostacyclin agonists (e.g. treprostinil) are clinically relevant as well as FDA-approved. Their potential role in the treatment of acute myeloid leukemia is unknown. Here we present the details of our high-throughput flow cytometry system and preliminary identification of pro-differentiation agents in AML. If successful, we anticipate that one of these small molecules may offer insight into a mechanism for overcoming differentiation arrest, and may also translate into a novel, clinically relevant treatment for acute myeloid leukemia. Disclosures: Sklar: IntelliCyt: Founder of IntelliCyt, the company that sells the HyperCyt high-throughput flow cytometry system. Other. Zon:Fate Therapeutics: Founder Other.

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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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Downregulation of c-Myc is critical for valproic acid-induced growth arrest and myeloid differentiation of acute myeloid leukemia

Leukemia Research ◽

10.1016/j.leukres.2007.03.012 ◽

2007 ◽

Vol 31 (10) ◽

pp. 1403-1411 ◽

Cited By ~ 38

Author(s):

Yun-Chih Cheng ◽

Hsiupen Lin ◽

Ming-Jer Huang ◽

Jyh-Ming Chow ◽

Shufan Lin ◽

...

Keyword(s):

Valproic Acid ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Growth Arrest ◽

Myeloid Differentiation ◽

Acute Myeloid

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Valproic Acid Plus Retinoic Acid Induce Myeloid Differentiation in Chemotherapy-Resistant Acute Myeloid Leukemia Patients.

Blood ◽

10.1182/blood.v104.11.1805.1805 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1805-1805 ◽

Cited By ~ 1

Author(s):

C. Nervi ◽

F. Lo Coco ◽

S. Minucci ◽

A. Careddu tech. ◽

R. Fiorini ◽

...

Keyword(s):

Valproic Acid ◽

Acute Myeloid Leukemia ◽

Retinoic Acid ◽

Plasma Levels ◽

Myeloid Leukemia ◽

Intensive Therapy ◽

Myeloid Differentiation ◽

Fish Analysis ◽

Leukemic Clone ◽

Acute Myeloid

Abstract The anti-epileptic drug valproic acid (VPA) acts as an inhibitor of histone deacetylases. In combination with retinoic acid (RA), VPA triggers myeloid differentiation of primary acute myeloid leukemia (AML) blasts in vitro. In vivo, VPA posses an antineoplastic activity as indicated by pre-clinical studies in murine models of leukemia, renal and lung metastatic tumors. Therefore, we have designed a phase II clinical study in which VPA was combined with RA (VPA-RA) in the AML treatment. Eigth chemotherapy-resistant or high risk AML patients not eligible for additional intensive therapy (median age 61.5 yrs), were treated at the Hematology Units of the Universities “La Sapienza” and “Tor Vergata” Rome-Italy. VPA (Depakin[Sanofi-Wintrop]) was administrated from day 1 to day 28, at the initial dosage of 10 mg/kg/die p.o. with dose escalation until optimal VPA plasma levels (80–110ug/ml). RA (Vesanoid [Roche]) at the dosage of 45 mg/m2 p.o./d, divided in two administrations, was added once the optimal VPA plasma levels were reached or at day 14 and continued until day 28. Four patients had a history of MDS, three patients had a FAB M0, M1 and M2 de novo AMLs, while the remaining case was a myeloid blast crisis (FAB M0) of a Ph+ve CML. Cytogenetic characterization in the other patients revealed normal karyotype in one case, a pseudodiploid [der(12)] in one, hyperdiploid (+8) in one, complex K with a 7q- alteration in one, while in the three remaining cases the karyotype was not evaluable. Pre-treatment leukemic infiltration ranged from 22% to 95%. VPA plasma level >60mg/ml was reached between 8 to 28 days (median 14.5 days). In three patients, VPA-RA treatment induced hyperleukocytosis (>50x 109/l) at day 16, 21 and 24, respectively, that was treated with chemotherapy (HU in two cases and low dose Ara-C in 1 case). Hematological improvement (≥50% decrease in packed red blood cell or platelet transfusion requirement) was observed in one case, a stable disease in five cases and disease progression in two cases. Peripheral blood and/or bone marrow samples were collected at day 0,3,7,14,21,28 for morphologic, immunophenotypic, cytogenetic and molecular studies. All patients showed features of myeloid-monocytic and/or erythroid differentiation of the leukemic clone, as revealed by morphologic, cytochemical, immunophenotypic analyses and by Q-RT-PCR of myeloid gene expression (GATA 1, MPO, CSF2Rb, etc.). Of note that high degree of myeloid differentiation correlated with early achievement of therapeutic VPA plasma levels and histone hyper-acetylation, as measured by immunocytochemistry and immunoblotting using antiacetylated histone H3 and H4 antibodies. Finally, differentiation of the leukemic clone was proven by FISH analysis showing the presence of the +8 and 7q- in maturing elements in patients whose leukemia blasts carried these cytogenetic lesions. The VPA-RA combination is a well tolerated treatment that induces phenotypic changes of the leukemic clone through chromatine remodelling. Further studies are needed to optimise this regimen with the aim of improving clinical response in leukemia patients.

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Synergistic Antileukemic Interactions Between Valproic Acid and Cytarabine: A Potential Means to Improve the Treatment of Pediatric Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v114.22.2084.2084 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2084-2084

Author(s):

Chengzhi Xie ◽

Holly Edwards ◽

Lisa Polin ◽

Hui Zhou ◽

Xuanlin Wang ◽

...

Keyword(s):

Valproic Acid ◽

Clinical Trials ◽

Acute Myeloid Leukemia ◽

Cell Lines ◽

Myeloid Leukemia ◽

Caspase 3 ◽

Active Form ◽

Acute Leukemias ◽

Induction Of Apoptosis ◽

Acute Myeloid

Abstract Abstract 2084 Poster Board II-61 Acute myeloid leukemia (AML) accounts for one fourth of acute leukemias in children, but it is responsible for more than half of the leukemia deaths in this patient population. In contrast to the tremendous success in the treatment of acute lymphoblastic leukemia over the last three decades, resulting in a >80% cure rate, improvements in AML therapy have been limited. Resistance to cytarabine (ara-C), the most active drug in the treatment of AML, is a major cause of treatment failure in this disease. Therefore, new therapies for children with AML need to be developed to overcome drug resistance, decrease relapse rate, and reduce short- and long-term adverse effects of treatment. Histone deacetylase inhibitors (HDACIs) possess antitumor activity and are currently being tested in clinical trials for the treatment of a variety of different cancers. Valproic acid (VPA), an FDA-licensed drug for treating both children and adults with epilepsy, also acts as an HDACI and can induce apoptosis in leukemic cells but not normal cells. In this study, we hypothesized that VPA synergizes with ara-C in antileukemic activity by inducing apoptosis in AML cells. To model this concept and to provide the basis for future clinical studies, we examined the effects of VPA on sensitivities to ara-C in 8 AML cell lines derived from patients (4 were children) with different subtypes of AML and in AML blasts collected at the time of diagnosis from 10 children with de novo AML treated at Children's Hospital of Michigan. We demonstrated synergistic antileukemic interactions between ara-C and VPA in all of the AML cell lines and additive to synergistic antileukemic interactions between the two drugs in the patient samples by standard isobolograms and calculation of combination indexes. It is interesting to note that MV4-11 [which harbors t(4;11)] and Kasumi-1 [which harbors t(8;21)] cells were substantially more sensitive to VPA than the other AML cell lines. Analogous to the Kasumi-1 cells, diagnostic blasts from t(8;21) AML cases (n=4) were significantly more sensitive to VPA than blasts from non-t(8;21) AML cases (n=6) (mean VPA IC50 0.51 mM vs 1.95 mM, p=0.0095) and showed median 53.9-fold increased ara-C sensitivities when combined with VPA at concentrations of 0.5 mM or lower. By contrast, non-t(8;21) AML blasts only showed median 2.1-fold increased ara-C sensitivities when combined with 0.5 mM VPA (p=0.048). In a pilot experiment, treatment of SCID mice with K562 xenograft tumors with combined Palmo-ara-C and VPA resulted in a 31% T/C and a 0.8 gross log cell kill compared to treatments with Palmo-ara-C (67% T/C) or VPA alone (100% T/C), establishing unambiguous in vivo synergy. Real-time RT-PCR analyses revealed changes in transcript levels for hENT1 and cytidine deaminase in Kasumi-1 cells post VPA and ara-C treatment alone or in combination. However, these changes would antagonize ara-C sensitivity in Kasumi-1 cells, suggesting that the effects of VPA or ara-C alone or in combination on expression of genes related to ara-C transport and metabolism do not contribute to the observed synergistic effects in AML cells. Interestingly, ara-C and VPA co-treatment resulted in synergistic induction of apoptosis and S-phase arrest in Kasumi-1 cells determined by flow cytometry analysis with annexin V and PI staining. The synergy between ara-C and VPA in induction of apoptosis in Kasumi-1 cells was accompanied by synergistic activation of caspase-3, induction of both total and acetylated p53 (ac-p53), and release of the active form of Bax determined by caspase-3 assays, co-immunoprecipitation, and Western blotting. Collectively, these results suggest that VPA enhances ara-C sensitivity in Kasumi-1 cells most likely by modulating levels of total and ac-p53 proteins and then release of the active form of Bax to trigger apoptosis. Based on our laboratory results, VPA has been incorporated into a treatment arm for high risk AML patients enrolled in the St. Jude Children's Research Hospital (SJCRH) clinical trial AML08: “A Randomized Trial of Clofarabine Plus Cytarabine Versus Conventional Induction Therapy and of Natural Killer Cell Transplantation Versus Conventional Consolidation Therapy in Patients with Newly Diagnosed Acute Myeloid Leukemia”. The results of our study provide compelling evidence to support the use of VPA in combination with ara-C in clinical trials for treating different risk groups of pediatric AML. Disclosures: No relevant conflicts of interest to declare.

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