AbstractBackgroundAcute Myeloid Leukemia Research Articles

Next-generation sequencing-based genetic landscape and its clinical implications for Chinese acute myeloid leukemia patients

BackgroundAcute myeloid leukemia (AML) is a clinically and biologically heterogeneous disease. The survival of older patients is generally poor. In the current study, we sought to investigate the differences in molecular gene mutations between younger and older AML patients, and to identify those newly diagnosed AML patients who are more likely to respond to standard cytarabine and daunorubicin induction chemotherapy.MethodsWe retrospectively evaluated 179 patients who were newly diagnosed with non-M3 AML. A next-generation sequencing assay covering 34 genes was used to investigate recurrently mutated genes. The mutational status of fusion genes was determined by real time PCR.ResultsThe median age at diagnosis was 53 years (range 18–88 years). Sixty-eight patients were 60 years or older with a median age of 67 years (range 60–88 years). Eighteen patients (10.1%) carried t(8;21)(q22;q22.1) or RUNX1–RUNX1T1 gene fusion, and there was a significantly higher incidence in younger patients (p = 0.019). At least one non-synonymous gene mutation was detected in 159 patients (88.8%). The median number of gene mutations was two (range 0–6). The mean number of molecular gene mutations at diagnosis was higher in older patients than younger patients (2.5 vs 1.83, p = 0.003). Older patients had significantly higher incidences of ASXL1 (22.1% vs 13.5%, p = 0.025) and TP53 mutations (13.2% vs 3.6%, p = 0.034). In total, 78 patients received DA60 (daunorubicin 60 mg/m2 per day on days 1–3 and cytarabine 100 mg/m2 twice per day on days 1–7) as the induction therapy, and information was available on their response to induction treatment. Patients with RUNX1–RUNX1T1 gene fusion were significantly more likely to achieve complete remission (CR) after DA60 induction therapy (p = 0.026), as were patients without the ASXL1 mutation (p = 0.007).ConclusionOlder AML patients had a lower incidence of favorable cytogenetics and higher frequencies and burdens of molecular mutations that are associated with poor prognosis compared to younger patients. Patients with RUNX1–RUNX1T1 gene fusion or without the ASXL1 gene mutation had a better chance of achieving CR when treated with cytarabine and daunorubicin induction chemotherapy.

Circular RNA regulatory network reveals cell\u2013cell crosstalk in acute myeloid leukemia extramedullary infiltration

BackgroundAcute myeloid leukemia can develop as myoblasts infiltrate into organs and tissues anywhere other than the bone marrow, which called extramedullary infiltration (EMI), indicating a poor prognosis. Circular RNAs (circRNAs) are a novel class of non-coding RNAs that feature covalently closed continuous loops, suggesting their potential as micro RNA (miRNA) “sponges” that can participate in biological processes and pathogenesis. However, investigations on circRNAs in EMI were conducted rarely. In this study, the overall alterations of circRNAs and their regulatory network between EMI and non-EMI AML were delineated.MethodsCircRNA and whole genome microarrays derived from EMI and non-EMI AML bone marrow mononuclear cells were carried out. Functional analysis was performed via Gene Ontology and KEGG test methods. The speculated functional roles of circRNAs were based on mRNAs and predicted miRNAs that played intermediate roles. Integrated bioinformatic analysis was conducted to further characterize the circRNA/miRNA/mRNA regulatory network and identify the functions of distinct circRNAs. The Cancer Genome Atlas (TCGA) data were acquired to evaluate the poor prognosis of distinct target genes of circRNAs. Reverse transcription-quantitative polymerase chain reaction was conducted to identify the expression of has_circRNA_0004520. Connectivity map (CMap) analysis was further performed to predict potential therapeutic agents for EMI.Results253 circRNAs and 663 genes were upregulated and 259 circRNAs and 838 genes were downregulated in EMI compared to non-EMI AML samples. GO pathways were enriched in progress including cell adhesion (GO:0030155; GO:0007155), migration (GO:0016477; GO:0030334), signal transduction (GO:0009966; GO:0007165) and cell–cell communication. Overlapping circRNAs envolved in pathways related to regulate cell–cell crosstalk, 17 circRNAs were chosen based on their putative roles. 7 target genes of 17 circRNAs (LRRK1, PLXNB2, OLFML2A, LYPD5, APOL3, ZNF511, and ASB2) indicated a poor prognosis, while overexpression of PAPLN and NRXN3 indicated a better one based on data from TCGA. LY-294002, trichostatin A and SB-202190 were identified as therapeutic candidates for EMI by the CMap analysis.ConclusionTaken together, this study reveals the overall alterations of circRNA and mRNA involved in EMI and suggests potential circRNAs may act as biomarkers and targets for early diagnosis and treatment of EMI.

MiR-425 expression profiling in acute myeloid leukemia might guide the treatment choice between allogeneic transplantation and chemotherapy

BackgroundAcute myeloid leukemia (AML) is a highly heterogeneous disease. MicroRNAs function as important biomarkers in the clinical prognosis of AML.MethodsThis study identified miR-425 as a prognostic factor in AML by screening the TCGA dataset. A total of 162 patients with AML were enrolled for the study and divided into chemotherapy and allogeneic hematopoietic stem cell transplantation (allo-HSCT) groups.ResultsIn the chemotherapy group, patients with high miR-425 expression had significantly longer overall survival (OS) and event-free survival (EFS) compared with patients with low miR-425 expression. In multivariate analyses, high miR-425 expression remained independently predictive of a better OS (HR = 0.502, P = 0.005) and EFS (HR = 0.432, P = 0.001) compared with patients with low miR-425 expression. Then, all patients were divided into two groups based on the median expression levels of miR-425. Notably, the patients undergoing allo-HSCT had significantly better OS (HR = 0.302, P < 0.0001) and EFS (HR = 0.379, P < 0.0001) compared with patients treated with chemotherapy in the low-miR-425-expression group. Mechanistically, high miR-425 expression levels were associated with a profile significantly involved in regulating cellular metabolism. Among these genes, MAP3K5, SMAD2, and SMAD5 were predicted targets of miR-425.ConclusionsThe expression of miR-425 may be useful in identifying patients in need of strategies to select the optimal therapy between chemotherapy and allo-HSCT treatment regimens. Patients with low miR-425 expression may consider early allo-HSCT.

A novel miR-375-HOXB3-CDCA3/DNMT3B regulatory circuitry contributes to leukemogenesis in acute myeloid leukemia

BackgroundAcute myeloid leukemia (AML) is a heterogeneous group of hematopoietic malignancies due to sophisticated genetic mutations and epigenetic dysregulation. MicroRNAs (miRNAs), a class of small non-coding RNAs, are important regulators of gene expression in all biological processes, including leukemogenesis. Recently, miR-375 has been reported to be a suppressive miRNA in multiple types of cancers, but its underlying anti-leukemia activity in AML is largely unknown.MethodsQuantitative reverse transcriptase PCR (qRT-PCR) was used to measure the expression of miR-375 and HOXB3 in leukemic cells and normal controls. Targets of miR-375 were confirmed by western blot and luciferase assay. Phenotypic effects of miR-375 overexpression and HOXB3 knockdown were assessed using viability (trypan blue exclusion assay), colony formation/replating, as well as tumor xenograft assays in vivo.ResultsThe expression of miR-375 was substantially decreased in leukemic cell lines and primary AML blasts compared with normal controls, because DNA hypermethylation of precursor-miR-375 (pre-miR-375) promoter was discovered in leukemic cells but not in normal controls. Lower expression of miR-375 predicted poor outcome in AML patients. Furthermore, forced expression of miR-375 not only decreased proliferation and colony formation in leukemic cells but also reduced xenograft tumor size and prolonged the survival time in a leukemia xenograft mouse model. Mechanistically, overexpression of miR-375 reduced HOXB3 expression and repressed the activity of a luciferase reporter through binding 3′-untranslated regions (3’-UTR) of HOXB3 mRNA. Overexpression of HOXB3 partially blocked miR-375-induced arrest of proliferation and reduction of colony number, suggesting that HOXB3 plays an important role in miR-375-induced anti-leukemia activity. Knockdown of HOXB3 by short hairpin RNAs reduced the expression of cell division cycle associated 3 (CDCA3), which decreased cell proliferation. Furthermore, HOXB3 induced DNA methyltransferase 3B (DNMT3B) expression to bind in the pre-miR-375 promoter and enhanced DNA hypermethylation of pre-miR-375, leading to the lower expression of miR-375.ConclusionsCollectively, we have identified a miR-375-HOXB3-CDCA3/DNMT3B regulatory circuitry which contributes to leukemogenesis and suggests a therapeutic strategy of restoring miR-375 expression in AML.

INPP4B promotes cell survival via SGK3 activation in NPM1-mutated leukemia

BackgroundAcute myeloid leukemia (AML) with mutated nucleophosmin (NPM1) has been recognized as a distinct leukemia entity in the 2016 World Health Organization (WHO) classification. The genetic events underlying oncogenesis in NPM1-mutated AML that is characterized by a normal karyotype remain unclear. Inositol polyphosphate 4-phosphatase type II (INPP4B), a new factor in the phosphoinositide-3 kinase (PI3K) pathway-associated cancers, has been recently found a clinically relevant role in AML. However, little is known about the specific mechanistic function of INPP4B in NPM1-mutated AML.MethodsThe INPP4B expression levels in NPM1-mutated AML primary blasts and AML OCI-AML3 cell lines were determined by qRT-PCR and western blotting. The effect of INPP4B knockdown on OCI-AML3 leukemia cell proliferation was evaluated, using the Cell Counting Kit-8 and colony formation assay. After INPP4B overexpression or knockdown, the activation of serum and glucocorticoid-regulated kinase 3 (SGK3) and AKT was assessed. The effects of PI3K signaling pathway inhibitors on the levels of p-SGK3 in OCI-AML3 cells were tested. The mass of PI (3,4) P2 and PI (3) P was analyzed by ELISA upon INPP4B overexpression. Knockdown of SGK3 by RNA interference and a rescue assay were performed to confirm the critical role of SGK3 in INPP4B-mediated cell survival. In addition, the molecular mechanism underlying INPP4B expression in NPM1-mutated leukemia cells was explored. Finally, Kaplan–Meier survival analysis was conducted on the NPM1-mutated AML cohort stratified into quartiles for INPP4B expression in The Cancer Genome Atlas (TCGA) dataset.ResultsHigh expression of INPP4B was observed in NPM1-mutated AML. Knockdown of INPP4B repressed cell proliferation in OCI-AML3 cells, whereas recovered INPP4B rescued this inhibitory effect in vitro. Mechanically, INPP4B enhanced phosphorylated SGK3 (p-SGK3) status, but did not affect AKT activation. SGK3 was required for INPP4B-induced cell proliferation in OCI-AML3 cells. High levels of INPP4B were at least partially caused by the NPM1 mutant via ERK/Ets-1 signaling. Finally, high expression of INPP4B showed a trend towards lower overall survival and event-free survival in NPM1-mutated AML patients.ConclusionsOur results indicate that INPP4B promotes leukemia cell survival via SGK3 activation, and INPP4B might be a potential target in the treatment of NPM1-mutated AML.

Disease-stabilizing treatment based on all-trans retinoic acid and valproic acid in acute myeloid leukemia \u2013 identification of responders by gene expression profiling of pretreatment leukemic cells

BackgroundAcute myeloid leukemia (AML) is an aggressive malignancy only cured by intensive therapy. However, many elderly and unfit patients cannot receive such treatment due to an unacceptable risk of treatment-related morbidity and mortality. Disease-stabilizing therapy is then the only possible strategy, one alternative being treatment based on all-trans retinoic acid (ATRA) combined with the histone deacetylase inhibitor valproic acid and possibly low-toxicity conventional chemotherapy.MethodsPrimary AML cells were derived from 43 patients included in two clinical studies of treatment based on ATRA, valproic acid and theophyllamine; low toxicity chemotherapy (low-dose cytarabine, hydroxyurea, 6-mercaptopurin) was also allowed. Pretreatment leukemic cells were analyzed by mutation profiling of 54 genes frequently mutated in myeloid malignancies and by global gene expression profiling before and during in vivo treatment.ResultsPatients were classified as responders and non-responders to the treatment, however response to treatment showed no significant associations with karyotype or mutational profiles. Significance analysis of microarray (SAM) showed that responders and non-responders significantly differed with regard to the expression of 179 different genes. The differentially expressed genes encoding proteins with a known function were further classified based on the PANTHER (protein annotation through evolutionary relationship) classification system. The identified genes encoded proteins that are involved in several important biological functions, but a main subset of the genes were important for transcriptional regulation. These pretherapy differences in gene expression were largely maintained during treatment. Our analyses of primary AML cells during in vivo treatment suggest that ATRA modulates HOX activity (i.e. decreased expression of HOXA3, HOXA4 and HOXA5 and their regulator PBX3), but altered function of DNA methyl transferase 3A (DNMT3A) and G-protein coupled receptor signaling may also contribute to the effect of the overall treatment.ConclusionsResponders and non-responders to AML stabilizing treatment based on ATRA and valproic acid differ in the pretreatment transcriptional regulation of their leukemic cells, and these differences may be important for the clinical effect of this treatment.Trial registrationsClinicalTrials.gov no. NCT00175812; EudraCT no. 2004–001663-22, registered September 9, 2005 and ClinicalTrials.gov no. NCT00995332; EudraCT no. 2007–2007–001995-36, registered October 14, 2009.

Acute myeloid leukemia and pregnancy: clinical experience from a single center and a review of the literature

BackgroundAcute myeloid leukemia (AML) accounts for more than two thirds of leukemia during pregnancy and has an incidence of 1 in 75,000 to 100,000. Its clinical management remains a challenging therapeutic task both for patient and medical team, given to the therapy-attributable risks for mother and fetus and the connected counseling regarding pregnancy continuation.MethodsWe provided a review of updated literature and a comprehensive description of five maternal/fetal outcomes of AML cases diagnosed concomitantly to pregnancy and treated at our Institution from 2006 to 2012.ResultsMedian age at AML diagnosis was 32 years (31–39). One diagnosis was performed in first trimester and the patient asked for therapeutic abortion before starting chemotherapy. Three cases were diagnosed in second/third trimester; in one case leukemia was diagnosed concomitantly with intrauterine fetal death, while the remaining two patients continued pregnancy and delivered a healthy baby by cesarean section. In only one of these two cases chemotherapy was performed during pregnancy (at 24 + 5 weeks) and consisted of a combination of daunorubicine and cytarabine. Therapy was well tolerated and daily fetus monitoring was performed. After completion of 30 weeks of gestation a cesarean section was carried out; the newborn had an Apgar score of 5/1'-7/5'-9/10', oxygen therapy was temporarily given and peripheral counts displayed transient mild leukopenia. One patient had diagnosis of myelodysplastic syndrome rapidly progressed to AML after delivery. Four out of the 5 described women are currently alive and disease-free. Three children were born and long-term follow-up has shown normal growth and development.ConclusionsThe treatment of AML occurring during pregnancy is challenging and therapeutic decisions should be taken individually for each patient. Consideration must be given both to the immediate health of mother and fetus and to long-term infant health. Our series confirmed the literature data: fetal toxicity of cytostatic therapy clusters during the first trimester; while chemotherapy can be administered safely during second/third trimester and combination of daunorubicin and cytarabine is recommended for induction.

Whole Transcriptome Sequencing (RNAseq) As a Comprehensive, Cost-Efficient Diagnostic Tool for Acute Myeloid Leukemia

Abstract Background Acute myeloid leukemia (AML) is caused by cooperating oncogenic driver mutations that induce uncontrolled proliferation in combination with maturation arrest in myeloid precursor cells. The majority of oncogenic drivers are somatically acquired. Recurrent genetic lesions are used for molecular classification and prognosis of AML. In addition, expression levels of selected genes contribute to AML subclassification (CD34, KIT) and prognostication (e.g. EVI1). Actionable aberrations and pathways are molecular targets for personalized pharmacotherapy. Structures, incidences, and mutual associations of recurrent genetic aberrations have been elucidated by large whole genome/exome sequencing efforts. However, 10-20% of AML appear to be caused by hitherto unrecognized or individual driver lesions. Current AML diagnostics are poorly standardized and comprise a combination of morphology, flow cytometry, cytogenetics, targeted gene amplification with electrophoresis, sequence analysis, and qRT-PCR. We here test the hypothesis that whole transcriptome sequencing (RNAseq) without parallel germ-line sequencing could potentially be used as a single and cost-efficient platform for AML diagnosis and prognostication. Methods Poly(A)+ RNA was isolated from 100 cryopreserved AML samples with a blast count of 10-99% (median: 75%). These samples were obtained from 97 patients (2 diagnosis-relapse pairs, 1 de novo AML with subsequent tAML). RNA was sequenced at a depth of 59x106 paired-end reads per sample with median read length of 126 bp through a ISO17025-accredited Illumina HiSeq 2500 pipeline. After alignment against the GRCh38 reference genome, single nucleotide variants (SNV) and small indels were called by VarScan with a threshold of 20% aberrant reads. Variants were filtered for known polymorphisms occurring at &gt;5% in defined ethnic subpopulations of the 1000 Genomes and Genome of the Netherlands Projects. Internal tandem duplications (ITD) were identified by frequency and distribution of soft clipped reads. Detection of fusion genes was performed on raw reads using STAR-Fusion and FusionCatcher. Geneexpression levels were measured relative to HBMS as a housekeeping gene by counting the number of reads aligned to gene exons normalized to the sum of exon lengths. HAMLET was developed and implemented as an integrated RNAseq pipeline to measure relevant recurrent AML-associated aberrations, i.e. SNV and small indels in recurrently mutated genes, the FLT3-ITD, AML-associated gene fusions, and EVI1 overexpression. Results A total of 221 SNV/indels (15 homozygous) were detected at expected or higher frequencies in NPM1 (33%), FLT3 (ITD+TKD: 37%), DNMT3A (30%), TET2 (27%), IDH1 (14%), IDH2 (14%), RUNX1 (23%), CEBPA (4%), KIT (7%), WT1 (8%), ASXL1 (11%), and TP53 (3%). Sensitivities to detect SNV/small indels and FLT3-ITD were 96% and 94%, respectively, with no apparent relation to the blast count. Fusion genes relevant to the WHO AML classification were found with a sensitivity of 100% when compared to metaphase cytogenetics and FISH. Specifically, 10 cases of CBFB-MYH11, 3 RUNX1-RUNX1T1, 2 PML-RARA, 1 FUS-ERG, 1 KMT2A-MLLT3, and 1 DEK-NUP214 fusion events were correctly detected. In addition, 3 variant KMT2A translocations with MLLT1, MLLT4, and MLLT6 as fusion partners were correctly called. The specificity of HAMLET for recurrent SNV, indels, and fusion genes was 100%. Finally, RNAseq quantitatively detected variability in gene expression as exemplified for EVI1 overexpression and confirmed by qRT-PCR (r=0.85). Conclusions In conclusion, RNAseq analysis of AML samples without concomitant germ-line sequencing detects molecular information with relevance for classification, prognosis, and targeted therapy with 96% overall sensitivity and 100% specificity. HAMLET is currently optimized to further improve the sensitivity to detect SNV/small indel and ITD events. With a cost price of €980,- per case and full accreditation for diagnostic application of the RNAseq raw data, RNAseq facilitates comprehensive and cost-effective AML diagnostics in a single assay. Additional predicted benefits of this RNAseq approach to be explored include identification of non-recurrent individual drivers, neoantigens, and minor histocompatibility antigens, a potential to replace conventional HLA typing, and as a potential alternative for flow cytometry. Disclosures Janssen: GenomeScan BV: Employment.

Abstract 5583: Integrative genomic analyses on pediatric acute myeloid leukemia

Abstract Background Acute myeloid leukemia (AML) is a complex and heterogeneous disease leading to a wide range of response variability to conventional therapy, excessive treatment related toxicity, and an overall poor outcome. The NCI supported Therapeutically Applicable Research to Generate Effective Treatments (TARGET) Initiative provided initial support for large-scale genomics to identify novel disease-associated genomic and epigenomic alterations that could be used to develop novel, targeted therapies for pediatric AML. Patients Diagnostic (Dx), remission (Rm), and relapse (Rel) samples were obtained from over 200 children with AML treated on the most recent Children's Oncology Group clinical trials. Data analyses were performed using Partek Genomics Suite 6.6 and Ingenuity Pathway Analysis. Results Methylation profiling (Illumina HumanMethylation27) identified 603 CpG sites significantly differentially methylated between Dx and Rm. In addition, 514 CpG sites were significantly differentially methylated between matched Rel and Rm (p&amp;lt;0.05 FDR, change in β≥0.25). Pathway analysis using genes associated with these CpG sites identified significant (p&amp;lt;0.01) over-representation of the WNT/β-catenin signaling pathway in Dx vs. Rm and in Rel vs. Rm. Gene expression profiling (Affymetrix Human Gene 1.1 ST) revealed 1,142 significantly differentially expressed between Dx and normal bone marrow obatined from the blood bank. Similarly, there were 1,242 significantly differentially expressed genes between Rel and normal bone marrow (p&amp;lt;0.05 FDR, fold-change≥2.00). Many expression differences were coordinated with methylation changes. Pathway analysis revealed 100 significantly (p&amp;lt;0.05) over-represented pathways in Dx vs. normal, Rel vs. normal, or both. Copy-number data analysis (Affymetrix Genome-Wide Human SNP 6.0) identified 180,379 significantly different regions in 254 Dx vs matched Rm. Similarly, 4,707 regions a significantly different in 49 Rel vs matched Rm (p&amp;lt;0.01). Many regions were distinct to either Dx or Rel groups. Whole-genome sequencing (Complete Genomics, Inc) was performed on 100 cases with matched Dx and Rm pairs and on 86 cases with matched Dx, Rel, and Rm samples. The frequencies of either somatic or germline variants were not significantly different when comparing Dx vs. Rel samples. Pathway analysis compared 1,528 mutated genes in the pediatric (TARGET) cohort with 1,963 mutated genes in adult de novo AML (TCGA cohort). Of the 85 and 80 significantly (p&amp;lt;0.05) over-represented canonical pathways, respectively, there are 44 pathways in common and many could be targeted by existing drugs. Conclusion A combination of genomic approaches have identified genes and pathways that appear deregulated in pediatric AML. Many changes are specific to Dx or Rel sample groups, or to pediatric vs. adult AML. Further study into these genes and pathways may reveal candidate targets for therapeutic intervention. Citation Format: Daniel H. Wai, Rhonda E. Ries, Michael Considine, David W. Lee, Sun-Mei Liu, Nicole M. O'Connor, Oliver Pepper, Ranjan Bista, Eiman Aleem, David O. Azorsa, Tanja M. Davidsen, Toni Farley, Jaime Guidry-Auvil, Jason E. Farrar, Malcolm A. Smith, Daniela S. Gerhard, MIchael F. Ochs, Spyro Mousses, Soheil Meshinchi, Robert J. Arceci. Integrative genomic analyses on pediatric acute myeloid leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5583. doi:10.1158/1538-7445.AM2014-5583

Metformin suppresses pediatric acute myeloid leukemia cell viability and clonogenicity

Background Acute myeloid leukemia (AML) accounts for 15-20% of childhood leukemias. Although remission is achieved following treatment with front-line chemotherapy, nearly half of the patients are faced with disease relapse associated with chemoresistance. Therefore, therapies that could sustain the remission phase in pediatric AML are urgently needed. Metformin is a widely used antidiabetic drug known to exhibit anti-cancer effect in several malignancies. Metformin is well-tolerated at very high doses, and its safety has also been demonstrated in clinical trials in diabetic children.

Significance of gain and loss of chromosomal abnormalities in AML: an Indian experience

Background Acute myeloid leukemia (AML) is a heterogeneous group of disorder. Recurrent translocations are generally recognized to be a major parameter for prognostication in AML. Recurrent chromosomal translocation t(15;17),t (8;21) and inv(16) have good prognosis, whereas, loss and gain of different chromosomes and/or chromosome segments play a vital role by different mechanism in leukemogenesis. Cytogenetics is one of the most powerful independent prognostic indicators in AML. It serves to identify biologically distinct subsets of disease and has been widely adopted to provide the framework for riskadapted treatment approaches. The aim of the present study was to appraise the clinical significance of numerical and structural chromosomal abnormalities in AML patients in terms of loss and gain of chromosomal material.

Critical Role Of Interleukin Receptor Signaling In Acute Myeloid Leukemia Identified Using An RNAi Functional Screen

Background Acute myeloid leukemia (AML) is one of the most common hematologic malignancies affecting both children and adults. Targeted therapy represents a promising approach in AML with the potential for increased efficacy and reduced toxicity. However, the molecular abnormalities in AML are extraordinarily heterogeneous. Numerous genetic changes are reported which require careful functional analyses to distinguish “driver mutations” from “passengers.” Because cytokine/growth factor receptors contribute to cancer pathogenesis by regulating growth, differentiation, senescence and survival, we designed a functional screen focusing on these molecules. This RNAi-based screen was designed to reveal the functional and prognostic relevance of cytokine/growth factor receptors in leukemia. Using this approach, we identified several interleukin receptors that play critical roles in promoting leukemia cell growth. Importantly, we show the novel roles of two interleukin receptors, IL2Rγ and IL1R1, in promoting AML cell growth. Methods and Results We designed an siRNA-based functional screen targeting 188 growth factor receptors that were found highly expressed in mononuclear cells of 140 primary leukemia samples by gene expression microarray analysis. We tested 72 leukemia patient samples with AML, ALL and MPN and 10 cell lines for dependence on these receptors. Cells were electroporated with siRNAs and cell viability was quantified after 72 hrs. Novel candidate targets found in AML patient samples include IL17R, IL9R, IL4R IL1R1, IL2Rγ, IL2Rα and IL15Rα. We found a variety of genetic abnormalities in these target genes, including splice variation (IL2Rα) and intron retention (IL15Rα). We found that IL2Rγ is a potential target in a JAK3A572V mutation positive AML cell line (CMK). Validation experiments demonstrated that knockdown of IL2Rγ significantly reduces the viability of CMK cells (90% decrease) and abrogates phosphorylation of JAK3 and downstream signaling molecules, JAK1, STAT5, MAPK and pS6 ribosomal protein. Intriguingly, the absence of IL2Rγ in murine bone marrow completely abrogated the clonogenic potential of JAK3A572V as compared to IL2Rγ wild-type marrow. These effects were rescued by co-expressing IL2Rγ with JAK3A572V but not by co-expressing IL2Rγ with JAK3Y100C, an inactivating mutation. Additionally, we found that IL2Rγ contributes to constitutive JAK3 mutant signaling by increasing JAK3 protein levels and phosphorylation. Conversely, mutant but not wild type JAK3 increased the expression of IL2Rγ, indicating IL2Rγ contributes to constitutive JAK3 signaling through a feedback mechanism. Similarly, IL2Rγ receptor is critical for transforming potential of additional JAK3 activating mutations such as JAK3M511I and JAK3A573V. Overall these results demonstrate an oncogenic potentiating role of IL2Rγ. Additionally, we found that silencing IL1R1 reduces the viability of 25% of AML primary samples. Most IL1R1 knockdown-sensitive AML samples exhibit monocytic and myelomonocytic features; however, no correlation with particular somatic mutations has thus far been observed. Confirming the importance of IL1R1 using a genetic model, we found that the absence of IL1R1 in murine bone marrow leads to the significant ablation of clonogenic potential (80% reduction) induced with AML1-ETO9a, NRASG12D and MLL-ENL oncogenes as compared to wild-type marrow in a ligand-dependent manner. Similarly knocking down IL1R1 in CD34+ AML cells reduces cell growth and phosphorylation of p38. Conversely, the presence of exogenous IL1 promotes AML cell survival by increasing p38 phosphorylation, which is significantly inhibited with p38 inhibitors such as Doramapimod. These results support a novel role for IL1R1-mediated signaling in promoting AML cell growth and targeting of this pathway in AML. Conclusions RNAi-based functional screening for leukemia cell dependence on cytokine/growth factor receptors led to the identification of novel oncogenic pathophysiological mechanisms. Specifically, (A) IL2Rγ is essential for the growth of leukemia cells harboring activating JAK3 mutations and (B) IL1R1 is involved in regulating growth and survival of AML cells, particularly with monocytic differentiation. These findings underscore the importance of interleukin receptors in leukemia pathogenesis and suggest that targeting these pathways in AML will be beneficial. Disclosures: Tyner: Incyte Corporation: Research Funding. Bagby:NIH: Membership on an entity’s Board of Directors or advisory committees.

IGN523: A Therapeutic Anti-CD98 Antibody With Multiple Mechanisms Of Action Demonstrates Anti-Tumor Efficacy

Abstract Background Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults resulting in &gt;10,000 deaths per year in the US. Limited treatment options and poor clinical response to current standard of care (SOC) necessitate development of more efficacious therapeutics. CD98 is a heterodimeric cell surface protein that is implicated in the pathogenesis of many hematological and solid malignancies and its elevated expression is correlated with poor prognosis and patient outcome. Previous studies demonstrated that CD98 functions both in integrin signaling and amino acid transport processes, which support the proliferation, anchorage-independence, invasion, and metastasis of tumor cells. Here we demonstrate that IGN523, a humanized monoclonal antibody targeting CD98, possesses multiple mechanisms of action (MOAs) and elicits potent anti-tumor activity against a variety of leukemic xenograft models. Methods CD98 expression was assessed on CD34+/CD33- progenitors and more mature CD34+/CD33+ precursors from AML patient bone marrow samples by flow cytometry. Anti-tumor efficacy of IGN523 treatment was evaluated across 11 lymphoma or leukemic xenograft models, including 5 AML, established in NOD/SCID, CB17, or NSG mice. Animals were treated with either IGN523 alone or in combination with SOC anti-tumor agents and dose-responses were performed in selected models. IGN523’s ability to elicit NK-cell mediated antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent tumor cell lysis was determined. Additional in vitro MOA studies investigated IGN523’s effect upon lysosomal and mitochondrial membrane permeabilization, amino acid transport, tumor cell proliferation and apoptosis. Results Flow cytometry data demonstrate that CD98 is differentially expressed on CD34+/CD33- and CD34+/CD33+ AML bone marrow cells compared to healthy donors. In AML patients, CD98 expression was significantly increased on the CD34+/CD33+ cells versus the CD34+/CD33- population. Leukemic tumors were established in mice (100-200mm3 starting volumes) prior to weekly treatment with IGN523 dosed at 15 or 30 mg/kg. IGN523 elicited robust and significant tumor growth inhibition (TGI; p&lt;0.01) in the majority of models including Ramos (87% TGI), HL-60 (70% TGI), KG-1 (53% TGI) and OCI-AML-3 (74% TGI). A well-defined IGN523 dose response (0.1 – 30 mg/kg) was also observed. IGN523 was at least as efficacious as SOC anti-tumor agents (e.g. cytarabine). In vitro data established that IGN523 elicits strong ADCC activity, induces lysosomal membrane permeabilization, and inhibits essential amino acid transport function. Moreover, IGN523 impedes cell proliferation/survival signaling cascades through reduction of PRAS40 and p70s6K phosphorylation, ultimately resulting in caspase-mediated apoptosis. Conclusion The combination of increased CD98 expression on AML cells, pre-clinical efficacy of IGN523 in xenograft models, and identification of diverse MOAs provide strong scientific rationale for therapeutic use in patients. Clinical trials are therefore warranted to evaluate the efficacy and safety of IGN523 in AML. Disclosures: Hayes: Igenica, Inc.: Employment. Chinn:Igenica, Inc.: Employment. Cantor:Igenica, Inc.: Consultancy. Velilla:Igenica, Inc.: Employment. Ginsberg:Igenica, Inc.: Consultancy. van der Horst:Igenica, Inc.: Employment.

Overexpression of primary microRNA 221/222 in acute myeloid leukemia

BackgroundAcute myeloid leukemia (AML) is a hematopoietic malignancy with a dismal outcome in the majority of cases. A detailed understanding of the genetic alterations and gene expression changes that contribute to its pathogenesis is important to improve prognostication, disease monitoring, and therapy. In this context, leukemia-associated misexpression of microRNAs (miRNAs) has been studied, but no coherent picture has emerged yet, thus warranting further investigations.MethodsThe expression of 636 human miRNAs was compared between samples from 52 patients with AML and 13 healthy individuals by highly specific locked nucleic acid (LNA) based microarray technology. The levels of individual mature miRNAs and of primary miRNAs (pri-miRs) were determined by quantitative reverse transcriptase (qRT) PCR. Transfections and infections of human cell lines were performed using standard procedures.Results64 miRNAs were significantly differentially expressed between AML and controls. Further studies on the clustered miRNAs 221 and 222, already known to act as oncogenes in other tumor types, revealed a deficiency of human myeloid cell lines to process vector derived precursor transcripts. Moreover, endogenous pri-miR-221/222 was overexpressed to a substantially higher extent than its mature products in most primary AML samples, indicating that its transcription was enhanced, but processing was rate limiting, in these cells. Comparison of samples from the times of diagnosis, remission, and relapse of AML demonstrated that pri-miR-221/222 levels faithfully reflected the stage of disease.ConclusionsExpression of some miRNAs is strongly regulated at the posttranscriptional level in AML. Pri-miR-221/222 represents a novel molecular marker and putative oncogene in this disease.

SNS-032 inhibits mTORC1/mTORC2 activity in acute myeloid leukemia cells and has synergistic activity with perifosine against Akt

BackgroundAcute myeloid leukemia (AML) is a heterogeneous disorder with aberrant regulation of a variety of signal pathways. Therefore, simultaneous targeting of two or even more deregulated signal transduction pathways is needed to overcome drug resistance. Previously, it was reported that SNS-032, a selective cyclin-dependent kinase inhibitor, is an effective agent for treatment of AML; however, the molecular mechanisms of SNS-032-induced cell death of AML cells are not yet fully understood. The aim of the study was to characterize the effects in vitro of SNS-032, used alone and in combination with an Akt inhibitor perifosine, against AML cells and to identify the mechanism involved.ResultsSNS-032 significantly induced cytotoxicity in human AML cell lines and blasts from patients with newly diagnosed or relapsed AML. However, Kasumi-1 cells and some of leukemic samples (14.9%) from AML patients were resistant to SNS-032-mediated cell death. Western blot analysis showed that SNS-032 strongly inhibited the phosphorylation of mammalian target of rapamycin (mTOR) on Ser 2448 and Ser2481, and that removal of SNS-032 resulted in partial recovery of cell death and reactivation of phosphorylation of mTOR. Moreover, exogenous insulin-like growth factor-1 (IGF-1) did not reverse SNS-032-induced cell growth inhibition and downregualtion of phosphor-mTOR at Ser2448 and Ser2481 although slight suppression of IGF-1R expression was triggered by the agent. Furthermore, SNS-032 at a lower concentration (60–80 nM) enhanced AML cell cytotoxicity induced by perifosine, an Akt inhibitor. Importantly, SNS-032 treatment reduced colony formation ability of AML cells, which was significantly increased when two agents were combined. This combination therapy led to almost complete inhibition of Akt activity.ConclusionWe conclude that SNS-032 might directly target mammalian target of rapamycin complex 1 (mTORC1)/mTORC2. Our results further provide a rationale for combining SNS-032 with perifosine for the treatment of AML.

Development of treatment and clinical results in childhood acute myeloid leukemia in the Slovak Republic

Background Acute myeloid leukemia (AML) in children is rare, and it is generally considered to be more resistant to chemotherapy than acute lymphoblastic leukemia. However, because of the gradual intensification of chemotherapy and better supportive care, outcomes have improved considerably over the past 20 years. The management of children with AML in the Slovak Republic was unified and centralized during the second half of the 1990s with the introduction of Berlin-Frankfurt-Munster (BFM) protocols. In 2000, the AML-BFM-1998 protocol was introduced, and data collection became centralized.

Curcumin reduces expression of Bcl-2, leading to apoptosis in daunorubicin-insensitive CD34+ acute myeloid leukemia cell lines and primary sorted CD34+ acute myeloid leukemia cells

BackgroundAcute myeloid leukemia (AML) is an immunophenotypically heterogenous malignant disease, in which CD34 positivity is associated with poor prognosis. CD34+ AML cells are 10-15-fold more resistant to daunorubicin (DNR) than CD34- AML cells. Curcumin is a major component of turmeric that has shown cytotoxic activity in multiple cancers; however, its anti-cancer activity has not been well studied in DNR-insensitive CD34+ AML cells. The aim of this study was to therefore to explore curcumin-induced cytotoxicity in DNR-insensitive CD34+ AML cell lines (KG1a, Kasumi-1), DNR-sensitive U937 AML cells, and primary CD34+ AML bone-marrow-derived cells.MethodsPrimary human CD34+ cells were isolated from peripheral blood mononuclear cells or bone marrow mononuclear cells using a CD34 MicroBead kit. The growth inhibitory effects of curcumin were evaluated by MTT and colony-formation assays. Cell cycle distribution was examined by propidium iodide (PI) assay. Apoptosis was analyzed by Wright-Giemsa, Hoechst 33342 and Annexin-V/PI staining assays. The change in mitochondrial membrane potential (MMP) was examined by JC-1 staining and flow cytometry. Expression of apoptosis-related proteins was determined by reverse transcription-polymerase chain reaction and Western blotting. Short interfering RNA (siRNA) against Bcl-2 was used in CD34+ KG1a and Kasumi-1 cells incubated with/without DNR.ResultsCurcumin inhibited proliferation and induced apoptosis and G1/S arrest in both DNR-insensitive KG1a, Kasumi-1 and DNR-sensitive U937 cells. Curcumin-induced apoptosis was associated with reduced expression of both Bcl-2 mRNA and protein, subsequent loss of MMP, and activation of caspase-3 followed by PARP degradation. Curcumin synergistically enhanced the cytotoxic effect of DNR in DNR-insensitive KG1a and Kasumi-1 cells, consistent with decreased Bcl-2 expression. Accordingly, siRNA against Bcl-2 increased the susceptibility of KG1a and Kasumi-1 cells to DNR-induced apoptosis. More importantly, curcumin suppressed Bcl-2 expression, selectively inhibited proliferation and synergistically enhanced the cytotoxicity of DNR in primary CD34+ AML cells, while showing limited lethality in normal CD34+ hematopoietic progenitors.ConclusionCurcumin down-regulates Bcl-2 and induces apoptosis in DNR-insensitive CD34+ AML cell lines and primary CD34+ AML cells.

Pre-apoptotic response to therapeutic DNA damage involves protein modulation of Mcl-1, Hdm2 and Flt3 in acute myeloid leukemia cells

BackgroundAcute myeloid leukemia (AML) cells are characterized by non-mutated TP53, high levels of Hdm2, and frequent mutation of the Flt3 receptor tyrosine kinase. The juxtamembrane mutation of FLT3 is the strongest independent marker for disease relapse and is associated with elevated Bcl-2 protein and p53 hyper-phosphorylation in AML. DNA damage forms the basic mechanism of cancer cell eradication in current therapy of AML.Hdm2 and pro-apoptotic Bcl-2 members are among the most intensely induced genes immediately after chemotherapy and Hdm2 is proposed a role in receptor tyrosine kinase regulation. Thus we examined the DNA damage related modulation of these proteins in relation to FLT3 mutational status and induction of apoptosis.ResultsWithin one hour after exposure to ionizing radiation (IR), the AML cells (NB4, MV4-11, HL-60, primary AML cells) showed an increase in Flt3 protein independent of mRNA levels, while the Hdm2 protein decreased. The FLT3 mutant MV4-11 cells were resistant to IR accompanied by presence of both Mcl-1 and Hdm2 protein three hours after IR. In contrast, the FLT3 wild type NB4 cells responded to IR with apoptosis and pre-apoptotic Mcl-1 down regulation. Daunorubicin (DNR) induced continuing down regulation of Hdm2 and Mcl-1 in both cell lines followed by apoptosis.ConclusionBoth IR and DNR treatment resulted in concerted protein modulations of Mcl-1, Hdm2 and Flt3. Cell death induction was associated with persistent attenuation of Mcl-1 and Hdm2. These observations suggest that defining the pathway(s) modulating Flt3, Hdm2 and Mcl-1 may propose new strategies to optimize therapy for the relapse prone FLT3 mutated AML patients.