Adult Acute Myeloid Leukemia Samples Research Articles

Inhibition of NRF2 enhances the acute myeloid leukemia cell death induced by venetoclax via the ferroptosis pathway

Venetoclax, an inhibitor that selectively targets B cell lymphoma-2 (BCL-2) that has been approved for treating adult acute myeloid leukemia (AML) in combination with hypomethylating agents. However, its short duration of response and emergence of resistance are significant issues. In this study, we found that the sensitivity of AML cells to venetoclax was considerably enhanced by ML385, an inhibitor of the ferroptosis factor nuclear transcription factor erythroid 2-related factor 2 (NRF2). Using AML samples, we verified that NRF2 and its target gene ferritin heavy chain 1 (FTH1) were highly expressed in patients with AML and correlated with poor prognosis. Downregulation of NRF2 could inhibit FTH1 expression and significantly enhance the venetoclax-induced labile iron pool and lipid peroxidation. By contrast, NRF2 overexpression or administration of the reactive oxygen species inhibitor N-acetylcysteine and vitamin E could effectively suppress the anti-AML effects of ML385+venetoclax. Furthermore, the ferroptosis inducer erastin increased the anti-AML effects of venetoclax. Our study demonstrated that NRF2 inhibition could enhance the AML cell death induced by venetoclax via the ferroptosis pathway. Thus, the combination of ML385 with venetoclax may offer a favorable strategy for AML treatment.

RNAi-Mediated Screen of Primary AML Cells Nominates MDM4 as a Therapeutic Target in NK-AML with DNMT3A Mutations.

DNA-methyltransferase 3A (DNMT3A) mutations belong to the most frequent genetic aberrations found in adult acute myeloid leukemia (AML). Recent evidence suggests that these mutations arise early in leukemogenesis, marking leukemic progenitors and stem cells, and persist through consolidation chemotherapy, providing a pool for AML relapse. Currently, there are no therapeutic approaches directed specifically against this cell population. To unravel therapeutically actionable targets in mutant DNMT3A-driven AML cells, we have performed a focused RNAi screen in a panel of 30 primary AML samples, all carrying a DNMT3A R882 mutation. As one of the strongest hits, we identified MDM4 as a gene essential for proliferation of primary DNMT3AWT/R882X AML cells. We analyzed a publicly available RNA-Seq dataset of primary normal karyotype (NK) AML samples and found a trend towards MDM4 transcript overexpression particularly in DNMT3A-mutant samples. Moreover, we found that the MDM2/4 inhibitor ALRN-6924 impairs growth of DNMT3AWT/R882X primary cells in vitro by inducing cell cycle arrest through upregulation of p53 target genes. Our results suggest that MDM4 inhibition is a potential target in NK-AML patients bearing DNMT3A R882X mutations.

HMGA2 Gene Expression Profile Improves Risk Stratification in Acute Myeloid Leukemia

Abstract Introduction: In adult acute myeloid leukemia (AML), clinical outcome is mainly predicted by age, cytogenetics and specific gene mutations (Papaemmanuil et al ., N. Engl. J. Med., 2016; Dohner et al., Blood, 2017). The 2017 European LeukemiaNet (ELN) expert panel for AML diagnosis recommends screening for mutations in NPM1, CEBPA, RUNX1, FLT3, TP53, and ASXL1 genes inaddition to chromosomal anomalies (Dohner et al., Blood, 2017). The genetic and cytogenetic risk stratification guides AML therapy, but it remains insufficient to select the optimal therapy especially in the intermediate genetic risk category. Several gene expression signatures have been proposed to further refine AML risk stratification (Li et al ., J. Clin. Oncol., 2013; Ng et al ., Nature, 2016). However, they have not been widely adopted because of technical challenges in implementing large gene signatures in clinical settings. Accurate biomarkers are still needed to improve prognostic assessment in AML. Methods: We analyzed RNA sequencing data of 430 clinically annotated AML samples and identified HMGA2 asa new candidate prognostic marker. A RT-qPCR test was developed and validated in order to facilitate implementation in clinical laboratories. The association of HMGA2 expression with complete remission (CR), overall survival (OS), relapse-free survival (RFS) and cumulative incidence of relapse (CIR), was studied in a training cohort of 358 diagnostic AML samples from intensively treated patients. Multivariable models were adjusted for age, white blood cell counts, cytogenetic risk, NPM1 and FLT3 -ITD mutations and, except for CR prediction, hematopoietic stem cell transplantation as a time-dependent variable. The prognostic value of the HMGA2 test was validated in an independent cohort of 260 diagnostic AML samples from patients enrolled in the UK National Cancer Research Institute (NCRI) AML17 trial. Results: In the training cohort, HMGA2 was highly expressed in 22.3% of AML, mostly in patients with intermediate or adverse cytogenetics. A high expression level of HMGA2 (H+) was associated with lower CR frequency (58.8% vs 83.4%, p Next, we investigated whether the HMGA2 test could improve prognostic assessment in AML patients classified according to the 2017 ELN genetic risk stratification (Dohner et al., Blood, 2017). We observed that a positive HMGA2 test identified 45 of 87 (51.7%) ELN adverse risk patients who were unresponsive to current treatments. Importantly, the HMGA2 test was also positive in 43.5% of intermediate risk patients who were negative for NPM1, FLT3 -ITD and biallelic CEBPA mutations, and in 66.7% of patients with TP53, RUNX1 or ASXL1 mutations. This finding is clinically relevant, especially if screening for TP53, RUNX1 or ASXL1 mutations is not available. Consistent with results in the training cohort, H+ was a good predictor of poor survival and a higher CIR in the validation cohort (Fig. C-D). Multivariable analyses also confirmed that H+ was strongly and independently associated with a higher risk of primary refractory disease (CR: aOR = 3.98 [1.36-11.65], p= 0.01), lower OS (aHR = 2.03 [1.36-3.03], p Conclusion: High HMGA2 expression adds significant independent prognostic value to known clinical and genetic prognostic factors in AML, and is predictive of poor clinical outcomes with standard AML therapies. The HMGA2 test can complement the current AML tests to improve treatment orientation and be integrated in ongoing and future prospective clinical trials studying innovative therapies to increase survival of HMGA2 positive AML patients. Download : Download high-res image (149KB) Download : Download full-size image Figure 1 . Disclosures Wei: Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding. Fleming: Abbvie: Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Astellas: Membership on an entity's Board of Directors or advisory committees; BMS: Speakers Bureau; MSD: Speakers Bureau. Schwarer: BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Specialised Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Sauvageau: ExCellThera: Other: Chief Executive Officer and Chief Scientific Officer.

PCAT18, as a novel differentially regulated long noncoding RNA in adult acute myeloid leukemia patients revealed by next-generation sequencing.

Long noncoding RNAs (lncRNAs) play significant roles in the carcinogenesis and progression of human tumors. The aim of this work was to evaluate the expression level of lncRNAs in adult acute myeloid leukemia (AML) patients and to explore the function of candidate lncRNA PCAT18 in clinical diagnosis and prognosis of AML. Noncoding RNA screening was performed by second-generation sequencing on adult newly diagnosed AML patients and healthy controls. GO and KEGG pathway analysis were performed for functional analysis of differentially regulated lncRNAs. The expression of candidate lncRNA PCAT18 was detected by real-time PCR. In K-562 and THP-1 cells, proliferation, and cell cycle analysis were performed using CCK-8 assay and Cell Cycle Assay Kit. The lncRNA expression profile of AML patients and healthy controls showed that six upregulated (>5-fold) and eight downregulated (<0.2-fold) lncRNAs. The candidate lncRNA PCAT18 showed higher expression in AML patients with NPM1 mutation and favorable-risk. RT-PCR revealed a significant increase of PCAT18 expression comparing to control cells. PCAT18 overexpression obviously promoted cell proliferation and PCAT18 knockdown decreased cell proliferation. The fraction of the S phases was increased in the PCAT18 overexpression group and decreased in the PCAT18 knockdown group. The candidate lncRNA PCAT18 in our study was firstly found upregulated in AML samples, that its overexpression promoted cell proliferation and G1/S transition. Further study of PCAT18 and its target mRNAs are needed to confirm the mechanism of PCAT18 in AML. PCAT18 may act as diagnostic and prognosis biomarker for AML.

ER, SOCS1, p15, E-cadherin and RARB are more Likely to be Members of the Methylator Phenotype of Adult and Childhood AML and Their Methylation is Primarly Regulated by an Overexpression of DNMT 3A

<strong>Objectives</strong>: The hypermethylator phenotype is used as a term for both acute myeloid leukemia (AML) and other tumors. We analyzed the methylation statuses of ten genes (DAP-kinase, SOCS1, ER, p15, Ecadherin, RARβ, p16, GSTP1, HIC1 and p73) and correlated them with the expression of DNA methyltransferases (DNMTs). <strong>Material and Method</strong>: We analyzed the methylation profiles using methylation specific PCR and COBRA and the expression of DNMTs (DNMT 1, 3A and 3B) by quantitative RT-PCR in 25 pediatric and 25 adult AML samples. <strong>Results</strong>: The ER, SOCS1, p15, E cadherin, and RARB genes methylated together significantly in the same patients and members of the methylator phenotype. <strong>Conclusion</strong>: Our study demonstrated that the gene DNMT3A was dramatically upregulated and significantly correlated with the methylator phenotype.

Limited Utility of Fluorescence In Situ Hybridization for Recurrent Abnormalities in Acute Myeloid Leukemia at Diagnosis and Follow-up

Acute myeloid leukemia (AML) is classified in part by recurrent cytogenetic abnormalities, often detected by both fluorescent in situ hybridization (FISH) and karyotype. The goal of this study was to assess the utility of FISH and karyotyping at diagnosis and follow-up. Adult AML samples at diagnosis or follow-up with karyotype and FISH were identified. Concordance was determined, and clinical characteristics and outcomes for discordant results were evaluated. Karyotype and FISH results were concordant in 193 (95.0%) of 203 diagnostic samples. In 10 cases, FISH detected an abnormality, but karyotype was normal. Of these, one had a FISH result with clinical significance. In follow-up cases, 17 (8.1%) of 211 showed FISH-positive discordant results; most were consistent with low-level residual disease. Clinically significant discordance between karyotype and AML FISH is uncommon. Consequently, FISH testing can safely be omitted from most of these samples. Focused FISH testing is more useful at follow-up, for minimal residual disease detection.

Metabolic Profiling of Adult Acute Myeloid Leukemia (AML)

The role of metabolic dysfunction in the initiation and progression of AML is still to be determined. Next-generation sequencing studies in AML have identified somatic mutations in mitochondrially encoded subunits of the mitochondrial respiratory chain (MRC; N Engl J Med, 2013; 368:2059). Recurrent somatic mutations in nuclear MRC genes are yet to be reported, but recurrent founding mutations occur in nuclear genes encoding the TCA cycle enzymes Isocitrate Dehydrogenase 1 and 2 (IDH1, IDH2). IDH1 and IDH2 mutant AML samples display sensitivity to BCL-2 inhibition (Nat Med, 2015; 21:178), and reduced proliferative capacity has been observed for IDH1 mutant samples following glutaminase inhibition (Exp Hematol, 2014; 42:247). In glioma, IDH2 mutant tumors show enrichment of oxidative phosphorylation (OXPHOS) related gene sets (J Exp Clin Cancer Res, 2016;35:86). Additionally, germline succinate dehydrogenase (MRC Complex-II) mutations have been associated with hereditary paragangliomas (Science, 2000; 287:848). We therefore investigated the nature and frequency of rare germline and somatic mutations affecting nuclear MRC genes in an adult AML cohort, and profiled the metabolic phenotypes of primary BM AML samples.Whole-exome sequencing was performed on 145 diagnosis (Dx) adult BM AML samples. Variants were filtered against dbSNP137, 1000 genome and NHLBI-ESP with a focus on rare germline/somatic variants with a minor allele frequency < 0.005. A total of 62 variants affecting the 39 nuclear MRC Complex-I (CI) genes were found in 52 samples. Across the MRC (85 nuclear genes), we identified 140 variants in 95 samples. Confirmation of the germline/somatic status of identified variants is ongoing, with 20 / 21 variants tested being germline in origin. Case-control burden analysis (Am J Hum Genet, 2013; 92:841) was performed to compare variants in the AML cohort with those in an ethnically matched healthy control cohort (n = 329; PLoS Genet, 2011; 7:e1001372). This showed enrichment in the AML cases for variants across all 5 MRC complexes (p = 0.04; Burden analysis, Bonferroni adjusted), and for variants affecting the β-complex of CI (14 genes; p = 0.01; Burden analysis, Bonferroni adjusted). We further investigated the association of CI variants with genes commonly mutated in AML. This revealed a significant under-representation of IDH1 mutations in AML with CI variants (p = 0.036; Fisher’s exact; Figure 1).Additionally, for a panel of AML samples (n = 117), we used the BioMark (Fluidigm) qRT-PCR platform to profile the expression of all nuclear CI genes and a panel of AML oncogenes and tumor suppressors. This revealed a significant negative correlation (p < 0.05; Pearson’s correlation, Bonferroni adjusted) between the expression of all nuclear CI genes and that of IDH2, which is independent of underlying mutation or genetic alterations, and which is not observed for IDH1 expression. Finally, Seahorse metabolic profiling showed that IDH1 mutant samples were found to be utilizing their glycolytic pathway at maximal capacity with no glycolytic reserve while simultaneouslydisplaying significantly reduced glycolytic rates (p< 0.05; T-test, Bonferroni adjusted) when compared to IDH2 mutant AML samples, AML samples lacking recurrent AML mutations (WT-AML), and normal bone marrow (NBM) controls. Both IDH1 and IDH2 mutant samples showed significantly deficient OXPHOS reserve capacity when compared to NBM and WT-AML samples (p < 0.05; T-test, Bonferroni adjusted). Ongoing investigations will determine the metabolic phenotype of CI mutant samples.In summary, we observed significant enrichment of rare variants affecting the MRC and CI genes in an adult AML cohort compared to healthy controls, highlighting a potential role for altered cellular energetics in AML pre-disposition. The significant under-representation of somatic IDH1 mutations in AML with rare germline/somatic CI mutations raises the possibility that these CI variants induce functional consequences that mimic those associated with somatic IDH1 mutation. The distinct glycolytic profiles of IDH1 and IDH2 mutant samples, and the selective negative correlation of CI gene expression with IDH2 mRNA expression, suggests that distinct metabolic phenotypes maybe associated with perturbations to IDH1 and IDH2. Ongoing investigations will compare the metabolic phenotype of samples with CI variants to that of IDH1 and IDH2 mutant samples. [Display omitted] DisclosuresGill:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Molecular Profiling of High-Risk Pediatric Acute Myeloid Leukemia

Background: Several landmark genomic profiling studies have dramatically advanced our understanding of the origin, progression and clonal evolution of adult acute myeloid leukemia (AML) and directly impacted clinical care. However, very little is known about the mutational landscape of pediatric AML, a distinct entity that shares few genetic and clinical characteristics with adult AML. To investigate potential drivers of high-risk pediatric AML, comprehensive genomic profiling was performed on high-risk AML samples as part of a prospective clinical next-generation sequencing program.Methods:Samples obtained from patients with known high-risk features at diagnosis or with refractory or relapsed AML were selected for molecular profiling. Comprehensive testing included whole-exome sequencing (WES) of matched tumor (bone marrow or chloroma tissue) and normal tissue (peripheral blood or buccal swab) samples and transcriptome analysis (RNAseq). Targeted sequencing of 467 cancer-associated genes was used when tumor tissue was limited. Sequencing was performed on Illumina's HiSeq 2500 with 150X and 500X average coverage for WES and targeted sequencing, respectively. Variants were filtered to select alterations in cancer-related genes or genes relevant for patient care.Results:Fifteen patients with AML (mean age 7.7 yrs; range 0.75-19 yrs) met high-risk criteria (high-risk features at diagnosis = 4, relapsed disease = 8, refractory disease = 3) and were selected for profiling. WES and RNAseq were performed on 11 samples, WES only on 3 samples and targeted DNA sequencing on 1 sample. The median number of variants was 60 (range 14- 5950) per case. After filtering, 54 mutations were identified in 35 genes with a mean of 3.6 mutated genes per patient sample (range 0-14); two samples only carried a fusion gene with no other genetic alterations. At least one driver genetic alteration was detected in each patient sample. Thirteen samples carried mutations in at least one gene known to be altered in AML (e.g. IDH1, WT1, TP53, NRAS) (mean, 2; range, 1-6) and 5 samples carried novel mutations in 15 genes not previously implicated in AML (e.g. CARD9, CHD9, Axin1). Mutations in 11 AML related genes were detected in more than one sample including NRAS in 4, TP53 in 3 and KRAS, PTPN11 PHF6, JAK3 in 2 samples each; genes not previously implicated in AML were only mutated in single patients. Of note, mutations in genes encoding members of the RAS pathway occurred in 60% of cases (9/15 samples). RNAseq identified gene fusions in 7/11 samples (63%). Four fusions involving KMT2A and core binding factor genes were also detected by FISH while three fusions were detected by RNAseq only: NUP98-NSD1 in two patients and CBFA2T3-GLIS2 in one patient. Samples carrying driver gene fusions had the lowest number of mutated genes (0-1) compared to samples lacking a gene fusion (1-5 mutated genes), with one exception of a patient with history of infant ALL who later developed KMT2A-AFF1AML with the highest number of mutated genes (n=14). There was no correlation between the number of mutated genes and age, clinical characteristics, initial risk classification at diagnosis or intensity of therapy prior to sequencing.Conclusion:Our study provides an initial overview of the genetic alterations that characterize high-risk, chemo-resistant pediatric AML. Analysis of the data highlights the overall low genetic complexity of high-risk AML despite the aggressive clinical behavior and exposure to intense chemotherapy, including stem cell transplant. Of interest, similar to adult AML, we found that mutations leading to aberrant activation of the RAS pathway were also very frequent in our cohort of pediatric high-risk AML, while genes typically mutated early in the process of leukemogenesis in adult AML, such as NPM1, DNMT3A, FLT3, IDH1, IDH2 were not affected. Such findings suggest that distinct, age-specific mechanisms of leukemogenesis might exist. Furthermore, our data also highlights the important role of RNA sequencing in complementing current standard diagnostic tools, allowing the identification of driver fusion genes in samples for which no other driver event is detected. Larger studies, preferably including diagnostic samples and utilizing broader approaches, are needed to better understand the mechanisms responsible for the initiation and progression of childhood AML. DisclosuresNo relevant conflicts of interest to declare.

UNC2025, a Small Molecule MerTK and Flt3 Tyrosine Kinase Inhibitor, Decreases Disease Burden, Prolongs Survival, and Promotes Sensitivity to Chemotherapy in Xenograft Models of Acute Leukemia

Acute myeloid leukemia (AML) and relapsed or refractory acute lymphoblastic leukemia (ALL) are extremely difficult to treat and new therapies are needed to improve survival and decrease toxicities associated with conventional therapy. The MerTK receptor tyrosine kinase is aberrantly expressed in 30% of pediatric B-ALL patient samples, 50% of pediatric T-ALL samples, and 80-90% of pediatric and adult AML samples and inhibition of MerTK using shRNA prolonged median survival time in ALL and AML xenograft models. These data validate MerTK as a potential therapeutic target in acute leukemias. In AML, internal tandem duplication (ITD) mutations of the FMS-like tyrosine kinase 3 (Flt3) receptor are observed in ~20-30% of adult and ~15% of pediatric AML patient samples and are associated with poor prognosis. Flt3 inhibitors have demonstrated therapeutic efficacy in clinical studies but resistance to current Flt3-targeted TKIs and unacceptable adverse side effects have been reported, indicating a need for new therapeutic agents directed against Flt3. Here we report a novel dual MerTK/Flt3-selective small molecule TKI as a potential therapy for acute leukemias with MerTK expression and/or activating Flt3 mutation. UNC2025 mediated potent inhibition of MerTK and Flt3 in enzymatic (Ki = 0.16 nM and 0.59 nM, respectively) and cell-based (IC50 = 2.7 nM and 14 nM, respectively) assays and had limited off-target activity against 300 other kinases. Treatment with 100-200 nM UNC2025 inhibited MerTK-dependent pro-survival signaling pathways, induced apoptosis in 40-90% of cells, and reduced colony-forming potential by 80-100% in leukemia cell line cultures. Similarly, a MerTK-expressing leukemic patient sample exhibited reduced MerTK-dependent pro-survival signaling, induction of apoptosis, and decreased colony formation in semi-solid medium in response to UNC2025. In contrast, a MerTK-negative leukemic patient sample did not respond to treatment, suggesting that UNC2025-mediated anti-leukemia effects are a consequence of MerTK inhibition. UNC2025 is orally bioavailable and inhibits MerTK phosphorylation/activation in leukemic blasts in the bone marrow. Treatment with UNC2025 resulted in a dose-dependent reduction in tumor burden and increased median survival from 27 days after inoculation with tumor cells to 70 days in an orthotopic B-ALL xenograft model of minimal residual disease (p < 0.0001) and from 27.5 to 45 days in a similar model of existent disease (p < 0.0001). In a MerTK-expressing patient-derived xenograft (PDX) model of AML, short-term treatment with UNC2025 resulted in a significant decrease in peripheral disease burden (9.3% blasts versus 35.6% for vehicle-treated animals, p = 0.0225) and both the fraction of splenic blasts (28.5% versus 75.7%, p = 0.0005) and spleen mass (170 mg versus 834 mg, p < 0.0001) were dramatically decreased. UNC2025 was even more effective in a FLT3-ITD mutant, MerTK-expressing AML PDX model. In this case, treatment with UNC2025 resulted in stable disease regression (0.24% peripheral blasts after 4 weeks of treatment versus 11.4% pre-treatment, p = 0.0094) and prolonged median survival from 16 days post-treatment to > 107 days (p = 0.0114). In addition, in the B-ALL xenograft model, treatment with UNC2025 and methotrexate, a chemotherapy currently in clinical use for treatment of pediatric ALL, resulted in reduced tumor burden and increased tumor-free survival relative to mice treated with either agent alone, suggesting that addition of UNC2025 to standard therapies may be a particularly effective treatment strategy and/or may allow for chemotherapy dose-reduction and decreased toxicity. The very high potency, relative selectivity, oral bioavailability, and target inhibition and therapeutic efficacy mediated by UNC2025 in murine models, both alone and in combination with chemotherapy, support continued development of a dual MerTK/Flt3 inhibitor for treatment of patients with acute leukemia. DisclosuresDeRyckere:Meryx, Inc.: Equity Ownership. Wang:University of North Carolina Chapel Hill: UNC2025 Patents & Royalties; Meryx, Inc.: Equity Ownership. Frye:Meryx, Inc.: Equity Ownership; University of North Carolina Chapel Hill: UNC2025 Patents & Royalties. Earp:Meryx, Inc.: Equity Ownership; University of North Carolina Chapel Hill: UNC2025 Patents & Royalties. Graham:Merys, Inc.: Equity Ownership.

Repetitive genomic elements and overall DNA methylation changes in acute myeloid and childhood B-cell lymphoblastic leukemia patients.

Aberrant epigenetic regulation is a hallmark of neoplastic cells. Increased DNA methylation of individual genes' promoter regions and decreases in overall DNA methylation level are both generally observed in cancer. In solid tumors, this global DNA hypomethylation is related to reduced methylation of repeated DNA elements (REs) and contributes to genome instability. The aim of the present study was to assess methylation level of LINE-1 and ALU REs and total 5-methylcytosine (5metC) content in adult acute myeloid leukemia (AML) (n = 58), childhood B-cell acute lymphoblastic leukemia (ALL) (n = 32), as the most frequent acute leukemias in two age categories and in normal adult bone marrow and children's blood samples. DNA pyrosequencing and ELISA assays were used, respectively. Global DNA hypomethylation was not observed in leukemia patients. Results revealed higher DNA methylation of LINE-1 in AML and ALL samples compared to corresponding normal controls. Elevated methylation of ALU and overall 5metC level were also observed in B-cell ALL patients. Differences of REs and global DNA methylation between AML cytogenetic-risk groups were observed, with the lowest methylation levels in intermediate-risk/cytogenetically normal patients. B-cell ALL is characterized by the highest DNA methylation level compared to AML and controls and overall DNA methylation is correlated with leukocyte count.

NADH Dehydrogenase Subunit 4 (ND4) Mutations in Pediatric Acute Myeloid Leukemia

AbstractAbstract 1380NADH dehydrogenase subunit 4 (ND4) is encoded by mitochondrial DNA and is an integral component of Complex I, one of the core enzymatic complexes critical for mitochondrial oxidative phosphorylation and regulation of the balance between NADH and NAD+. ND4 mutations have recently been described in adult acute myeloid leukemia (AML). In the current study, we investigated the frequency and prognostic impact of ND4 mutations in 289 pediatric leukemia patients (<= 18 years). Total cellular DNA was isolated from bone marrow or peripheral blood samples at diagnosis (n=289) and at complete remission (n=6) for children treated uniformly within multicenter treatment trials AML-Berlin-Frankfurt-Münster (BFM, n=180) and Dutch Childhood Oncology Group (DCOG, n=109).ND4 mutations were detected by direct sequencing in 13 of 289 (4.5 %) pediatric AML patients. Mutations occurred throughout the ND4 sequence, and included missense mutations (n=10), deletions (n=2) and a nonsense mutation. The most commonly detected mutations were S86N (n=2), delA 11,032–11,038 (n=2), and F50L (n=2). All other mutations were detected in single cases. Four (30.8 %) ND4 mutations were heteroplasmic (i.e. both wild-type and mutated ND4 were detected) and 9 (69.2 %) were homoplasmic (i.e. only mutated ND4 was detected), which is similar to the distribution we previously observed for adult AML patients (37.9% and 62.1%, respectively). Of the 4 heteroplasmic mutations detected in the pediatric AML cohort, 3 are predicted to result in a truncated ND4 protein. The remaining heteroplasmic mutation, which results in an L72P substitution, is predicted to be damaging (PolyPhen2 score = 0.999). Thus all 4 heteroplasmic mutations are expected to interfere with ND4 protein function. In contrast, 3 of the 9 (33.3 %) homoplasmic mutations are within transmembrane regions and only 1 (11.1 %) is predicted to be damaging (S459Y, PolyPhen2 score = 0.906). The 11 predicted transmembrane domains (TMD) of ND4 may be important for mitochondrial proton transport. However, like in adult AML, the presence of ND4 mutations affecting or not affecting a TMD had no impact on pediatric AML patient outcome.Non-tumoral DNA available through samples collected in routine follow-up examinations during complete remission allowed determination of mutation origin (e.g. somatic or germ-line) in 6 cases. Interestingly, the homoplasmic substitutions resulting in F50L, S86N and A131T were each defined to be germline mutations in both adult and pediatric AML samples. The heteroplasmic one base-pair deletion in a stretch of seven adenine residues (11,032–11,038) detected in two pediatric leukemia samples was determined to be somatic in the one case for whom a sample obtained during complete remission was available for analyses. Patient characteristics including age, FAB-subtype, WBC count, cytogenetic subgroup or presence of FLT3-ITD were similar regardless of ND4 mutation status.In accordance with our earlier observations in adult AML, comparison of ND4mutated with ND4wildtype patients demonstrated no significant difference on overall survival (OS, P=.67). In the adult study, a survival advantage was observed for patients with somatic heteroplasmic ND4 mutations. No survival advantage was observed for children with heteroplasmic ND4 mutations, possibly due to limited numbers of ND4mutated patients treated in the BFM and DCOG study groups.Gene expression profiles (GEP) for ND4mutated (n=11) and ND4wild-type (n=188) pediatric AML patients revealed no significant differences. However, 8 probe sets were found to be differentially regulated when GEP for heteroplasmic ND4mutated (n=4) and ND4wildtype (n=187) were compared. Two of these probe sets annotated the SETDB2 (CLLD8, KMT1F) gene, which encodes a histone H3 methyltransferase. Quantitative RT-PCR validated the lower SETDB2 expression as a characteristic of ND4mutated cases (P=.02). SETDB2 contributes to several important cellular functions, including heterochromatin formation, chromatin condensation and transcriptional repression.In summary, ND4 mutations were not predictive for outcome in pediatric AML, but were significantly associated with decreased SETDB2 expression, providing a link between mitochondrial gene mutation and epigenetic control of gene expression. Disclosures:No relevant conflicts of interest to declare.

POU4F1 is associated with t(8;21) acute myeloid leukemia and contributes directly to its unique transcriptional signature

The t(8;21)(q22;q22) translocation, present in ~5% of adult acute myeloid leukemia (AML) cases, produces the AML1/ETO fusion protein. Dysregulation of the POU domain-containing transcription factor POU4F1 is a recurring abnormality in t(8;21) AML. Here, we show that POU4F1 over-expression is highly correlated with, but not caused by AML1/ETO. AML1/ETO markedly increases the self-renewal capacity of myeloid progenitors from murine bone marrow or fetal liver and drives expansion of these cells in liquid culture. POU4F1 is neither necessary nor sufficient for these AML1/ETO-dependent properties, suggesting that it contributes to leukemia through novel mechanisms. To identify targets of POU4F1, we performed gene expression profiling in primary mouse cells with genetically defined levels of POU4F1 and identified 140 differentially expressed genes. This expression signature was significantly enriched in human t(8;21) AML samples and was sufficient to cluster t(8;21) AML samples in an unsupervised hierarchical analysis. Among the most highly differentially expressed genes, half are known AML1/ETO targets, implying that the unique transcriptional signature of t(8;21) AML is, in part, attributable to POU4F1 and not AML1/ETO itself. These genes provide novel candidates for understanding the biology and developing therapeutic approaches for t(8;21) AML.

EVI1 overexpression in distinct subtypes of pediatric acute myeloid leukemia

Overexpression of the ecotropic virus integration-1 (EVI1) gene (EVI1+), localized at chromosome 3q26, is associated with adverse outcome in adult acute myeloid leukemia (AML). In pediatric AML, 3q26 abnormalities are rare, and the role of EVI1 is unknown. We studied 228 pediatric AML samples for EVI1+ using gene expression profiling and RQ-PCR. EVI1+ was found in 20/213 (9%) of children with de novo AML, and in 4/8 with secondary AML. It was predominantly found in MLL-rearranged AML (13/47), monosomy 7 (2/3), or FAB M6/7 (6/10), and mutually exclusive with core-binding factor AML, t(15;17), and NPM1 mutations. Fluorescent in situ hybridization (FISH) was performed to detect cryptic 3q26 abnormalities. However, none of the EVI1+ patients harbored structural 3q26 alterations. Although significant differences in 4 years pEFS for EVI1+ and EVI1- pediatric AML were observed (28%+/-11 vs 44%+/-4, P=0.04), multivariate analysis did not identify EVI1+ as an independent prognostic factor. We conclude that EVI1+ can be found in approximately 10% of pediatric AML. Although EVI1+ was not an independent prognostic factor, it was predominantly found in subtypes of pediatric AML that are related with an intermediate to unfavorable prognosis. Further research should explain the role of EVI1+ in disease biology in these cases. Remarkably, no 3q26 abnormalities were identified in EVI1+ pediatric AML.

POU4F1 Is Associated with t(8;21) AML and Contributes Directly to Its Unique Transcriptional Signature.

Abstract 2623Poster Board II-599The t(8;21)(q22;q22) translocation, present in 10–15% of adult acute myeloid leukemia (AML) cases produces the AML1/ETO (AE) fusion protein. The molecular mechanism of AE action is not well understood. Using Affymetrix RNA expression array analysis on RNA isolated from whole bone marrow (BM) aspirates from patients with de novo AML (n=113), we identified POU4F1 as an expression outlier uniquely associated with t(8;21). All t(8;21) positive patients in this cohort (n=4) had POU4F1 expression levels exceeding 2 SD above the mean for this cohort. None of the t(8;21) negative AML samples or normal CD34+ BM samples (n=5) had POU4F1 levels above this threshold. POU4F1 is a POU domain containing transcription factor that has been studied most extensively in neurons of the caudal CNS and peripheral sensory nervous system. Pou4f1 null mice die peripartum from motor neuron failure. Given the strong correlation between POU4F1 expression and t(8;21), we hypothesized that POU4F1 is a direct transcriptional target of AE and a mediator of its activity. We found that transient or stable over-expression of AE in primary mouse BM or human cell lines did not increase the levels of POU4F1 when analyzed by qRT-PCR, implying that POU4F1 over-expression in t(8;21) AML is AE independent. AE is known to cause increased self renewal of hematopoietic progenitor cells in vitro. To determine whether Pou4f1 may contribute to this phenotype we retrovirally transduced murine BM cells with AE, Pou4f1, or empty vector. We then performed weekly clonal serial replating of myeloid progenitors in cytokine enriched methocellulose media. Progenitors transduced with AE had robust replating activity (15–20/24 CFUs replated for at least five generations in three independent experiments), whereas cells transduced with Pou4f1 did not (1-4/24 CFUs replated; P <0.05). We next used Pou4f1 null mice to determine whether Pou4f1 is required for AE induced progenitor self renewal. We repeated the serial replating assay using Pou4f1 +/+, +/−, or −/− E14.5-16.5 fetal liver cells (3 independent experiments, n=3 mice per genotype). Regardless of Pou4f1 genotype, cells transduced with AE retained replating capacity for five generations. Taken together these results indicate Pou4f1 is neither necessary nor sufficient for enhanced self renewal of hematopoietic progenitors in vitro. To further elucidate the role of POU4F1 in t(8;21) AML, we investigated what effect Pou4f1 had on the transcriptional profile of hematopoietic cells. Pou4f1 +/+ or −/− fetal liver cells were retrovirally transduced to produce cells with high (MSCV-IRES-Pou4f1-GFP in +/+), normal (MSCV-IRES-GFP in +/+), or no (MSCV-IRES-GFP in −/−) Pou4f1 expression. GFP positive cells were sorted and RNA was prepared. Affymetrix MOE430v2.0 GeneChip microarrays were run and comparisons were made between the gene expression profiles of the three experimental groups (each group n=3). As expected, since Pou4f1 is not normally expressed in hematopoietic cells, the gene expression profiles between Pou4f1 wildtype and null cells showed no significant differences. Comparisons of the cells expressing high levels of Pou4f1 to Pou4f1 wildtype or null, however, revealed 114 differentially expressed genes (FDR<0.25). Using Gene Set Enrichment Analysis on our AML data, we found that the human homologs of these 114 genes were significantly enriched in t(8;21) positive compared to t(8;21) negative patients (FDR q value = 0.018), implying that the Pou4f1 gene expression signature is conserved from mice to humans and is retained throughout leukemic transformation. Remarkably, unsupervised clustering performed on the expression profiles of the 114 Pou4f1 signature genes alone was sufficient to separately group the t(8;21) patients from all the other AML patients. Of these 114 genes, the expression of eight (PPAPDC1B, PELI2, FAM69B, ROBO1, SOX4, CAV1, H2AFV, PLXDC2) was significantly increased (P<0.01 by t-test) up to 33-fold (CAV1) in t(8;21) positive vs. negative patients. In this cross species analysis we identified a POU4F1 gene expression signature that distinguishes between t(8;21) positive and negative AML samples. These surprising results directly refute the assumption that the gene expression profile of t(8;21) AML is solely attributable to AE and suggest that POU4F1 is an important independent contributor to the pathophysiology of these leukemias. Disclosures:No relevant conflicts of interest to declare.

Clinical relevance of Wilms tumor 1 gene mutations in childhood acute myeloid leukemia

AbstractWilms tumor 1 (WT1) mutations have recently been identified in approximately 10% of adult acute myeloid leukemia (AML) with normal cytogenetics (CN-AML) and are associated with poor outcome. Using array-based comparative genome hybridization in pediatric CN-AML samples, we detected a WT1 deletion in one sample. The other WT1 allele was mutated. This prompted us to further investigate the role of WT1 aberrations in childhood AML. Mutations were found in 35 of 298 (12%) diagnostic pediatric AML samples. In 19 of 35 (54%) samples, more than one WT1 aberration was found: 15 samples had 2 different mutations, 2 had a homozygous mutation, and 2 had a mutation plus a WT1 deletion. WT1 mutations clustered significantly in the CN-AML subgroup (22%; P < .001) and were associated with FLT3/ITD (43 vs 17%; P < .001). WT1 mutations conferred an independent poor prognostic significance (WT1 mutated vs wild-type patients: 5-year probability of overall survival [pOS] 35% vs 66%, P = .002; probability of event-free survival 22% vs 46%, P < .001; and cumulative incidence of relapse or regression 70% vs 44%, P < .001). Patients with both a WT1 mutation and a FLT3/ITD had a dismal prognosis (5-year pOS 21%). WT1 mutations occur at a significant rate in childhood AML and are a novel independent poor prognostic marker.

Nucleophosmin Gene Mutations Identify a Favorable Risk Group in Childhood Acute Myeloid Leukemia with a Normal Karyotype.

Exon 12 gene mutations in nucleophosmin (NPM1) were recently discovered in approximately 30% of adult acute myeloid leukemia (AML) samples, and cluster in the normal karyotype subgroup (NK-AML). NPM1-mutated adult NK-AML has a favorable outcome (pOS in the 40-50% range), but in case a FLT3 internal tandem duplication (FLT3/ITD) is also present outcome is worse with 25–30% pOS. In pediatric AML, NPM1 mutations are less frequent (6–8%; Cazzaniga, Blood 2005 & Brown, Blood 2007). No studies have specifically addressed pediatric NK-AML, a subgroup lacking favorable prognostic cytogenetic aberrations and therefore mostly stratified in the intermediate risk arm of pediatric AML treatment protocols. We screened 292 newly diagnosed AML samples, and detected NPM1 mutations in 25 cases (8.6%). We also screened 46 initial diagnosis-relapse pairs, and no clonal instability was observed, which suggests that NPM1 mutations may be used for minimal residual disease detection. In contrast to adults, where type A mutations (TCTG-insertion) are most frequent (80%), in our cohort type B (CATG-insertion) mutations were found in 39% and type A in 23%. In the NK-AML cohort (n=98), 20% was NPM1-mutated, which was age dependent: <3 years, 0%; 3–10 years, 19%; >10 years, 29% (p=0.04). None of the 10 FAB M5 cases was NPM1 mutated (p=0.09). NPM1 mutations had an independent favorable prognostic impact on outcome in patients with NK-AML (5-year pEFS 77% vs. 41% for wild type patients; p=0.003), irrespective of FLT3 mutational status. In fact, NPM1-mutated patients with a FLT3/ITD did better than patients without an ITD, although this was not statistically significant (5-year pEFS 90% vs. 63%, respectively; p=0.48). In NK-AML without NPM1 mutations, patients with FLT3/ITD positive AML did significantly worse than wild type FLT3 AML patients (5-year pEFS 18% vs. 52%, p=0.002). The differential prognostic impact of FLT3/ITD between the NPM1-mutated vs. the wild type patients was not caused by differences in the FLT3/ITD allelic ratio or ITD length, nor was there a relationship with the type of NPM1 mutations. Multivariate analysis, including age, white blood cell count, NPM1 and FLT3 status and stem cell transplantation as time-dependent co-variable, showed that only NPM1 mutations had independent prognostic significance for pEFS (RR 0.34, p=0.02). We conclude that the incidence of NPM1 mutations increases with age, and that NPM1 mutations define a subgroup with favorable prognosis in pediatric NK-AML. Our data suggest that these molecular abnormalities allow stratification of children with NK-AML. However, different from adult NK-AML, we observed that all children with NPM1 mutations did well, irrespective of FLT3 status. Therefore, treatment in the ‘good risk' arm should be considered for children with NPM1-mutated NK-AML.