Karyotype Acute Myeloid Leukemia Research Articles

Mir-10a Regulates Autophagy and Cell Cycle in Normal Karyotype Acute Myeloid Leukaemia

Aim: Normal karyotype (NK) Acute Myeloid Leukemia (AML) accounts for approximately half of all AML cases. Almost two-thirds of these carry the Nucleophosmin 1(NPM1) gene mutation (NPM1c+). MicroRNAs (miRNA/miRs), short RNA molecules that act as post-transcriptional regulators of gene expression, have multiple roles in regulating key gene signalling networks in cancer, including in NK-AML. miR-10a is significantly overexpressed in NK-AML compared to normal bone marrow, particularly in those carrying the NPM1c+ mutation in which it is expressed at levels ~20-fold higher than in NPM1 wildtype (NPM1wt) NK-AML. We have previously shown that repression of miR-10a in the NPM1c+ AML cell line OCI-AML3 leads to inhibited proliferation and initiation of apoptosis. Here we aimed to further delineate the role of miR-10a in NK-AML.Results: miR-10a was shown to directly bind to and downregulate key members of the p53-mediated tumour suppressor gene network, including the CDKN1A (p21) inhibitor Transcription Factor AP2-gamma (TFAP2C) and Retinoblastoma (RB1) regulator Rb1-Inducible Coiled-Coil 1 (RB1C11). Inhibition of miR-10a resulted in significant repression of the cell cycle inhibitor p21, upregulation of RB1CC1, decrease in the expression of autophagy marker LC3-II, and the induction of apoptosis in serum-deprived cells. The individual modulation of these miR-10a target genes via siRNA (CDKN1A) and overexpression (RB1CC1) had similar effects, triggering apoptosis and also sensitizing OCI-AML3 cells to chemotherapy agents cytarabine and daunorubicin in transfected cells.Due to miR-10a’s potential role in regulating the central p53-p21-Rb1 tumour suppressor signalling axis in AML, miR-10a levels were quantified by QPCR in primary NK-AML patient samples and correlated to clinical outcome. Analysis of 61 patients from the German SAL AML2003 randomized clinical trial who received post-induction chemotherapy + allogeneic bone marrow transplantation (BMT) in a risk-adapted manner demonstrated that high miR-10a expression (> median expression) was associated with significantly poorer relapse-free survival (hazard ratio (HR) = 5.6, p < 0.001) compared those patient samples with low miR-10a expression. This is consistent with these patients being more resistant to post-induction chemotherapy and/or upfront BMT, and suggests that these patients may benefit from alternate therapy.To investigate the mechanism of miR-10a dysregulation in NPM1c+ AML cells, we assayed methylation of the miR-10a promoter in NPM1c+ (OCI-AML3) versus NPM1wt(HL60, MV4) cell lines. Consistent with the observed upregulation of miR-10a in NPM1c+ AML cells, hypomethylation of the miR-10a promoter was observed in OCI-AML3 cells (<5% CpG methylation) compared to the HL60 and MV4 (60% and 92% CpG methylation, respectively; p < 0.01), which was mirrored in NPM1c+ and NPM1WT primary patient samples. Exposure of the NPM1WT AML cells to hypomethylating agents decitabine and azacitidine resulted in reduced DNA methylation of the miR-10a promoter and a corresponding 20-100-fold upregulation of miR-10a (p < 0.001 for both lines). There was no significant association between miR-10a promoter hypomethylation with mutations of epigenetic modifiers such as DNA Methytransferanse 3A (DNMT3A; which are highly enriched in NPM1c+ AML) in primary patient specimens.Conclusion: Overexpression of the pro-survival miRNA miR-10a in NPM1c+ AML is epigenetically regulated, and results in modulation of the p53-p21-Rb1 tumour suppressor signalling axis in NK-AML. This may have clinical significance and identifies miR-10a as a potential novel biomarker and drug target for AML. DisclosuresNo relevant conflicts of interest to declare.

Azacitidine (AZA) Prolongs Overall Survival in Older Patients with Acute Myeloid Leukemia (AML) with Poor Prognostic Karyotypes Compared with Conventional Care Regimens (CCR)

Azacitidine (AZA) Prolongs Overall Survival in Older Patients with Acute Myeloid Leukemia (AML) with Poor Prognostic Karyotypes Compared with Conventional Care Regimens (CCR)

Diagnostic and Prognostic Implications of Prohibitin Overexpression in Normal Karyotype Acute Myeloid Leukemia

Abstract Background: Prohibitin (PHB) is a highly conserved protein, widely expressed in multiple cellular compartments such as mitochondria and nucleus. Recent studies have demonstrated that PHB played an important role in the regulation of mitochondrial function, cell proliferation and development. Our proteome analysis has revealed PHB was highly expressed in primary AML cells compared with normal cells. This finding prompted us to investigate whether PHB could serve as a potential diagnostic, prognostic, and therapeutic target in AML. Patients and Methods: PHB2 protein expression was assessed in 133 newly diagnosed normal karyotype AML (NK-AML) patients by using immunohistochemical staining (IHCS) on paraffin-embedded bone marrow sections. The degree of PHB2 protein expression was scored as 0-8 as a sum of a diffuseness score (0-5) and an intensity score (0-3). Overall survival (OS) and event-free survival (EFS) was analyzed according to PHB2 expression score. Also, we developed quantitative reverse transcriptase PCR (qRT-PCR) method to measure mRNA expression of PHB1 and PHB2 genes for the optimal algorithms for the discrimination of AML cells from the normal blood or hematopoietic cells. Because accumulation of beta-catenin in the nucleus has been identified in leukemic cells, we investigated the effect of beta-catenin accumulation on PHB expression. We monitored the expression levels of PHB with lithium chloride treatment, which blocks GSK-3beta activity and ultimately affects beta-catenin accumulation. For the development of anti-leukemic drug candidates targeting PHB in AML cells, we synthesized two potent chemical substances that can alkylate PHB: cyclohexylphenyl-chloroethyl urea (CCEU) and iodophenyl-chloroethyl urea (ICEU). We evaluated their therapeutic effects in the primary AML and AML cell lines. Results: In the PHB2 IHCS, using the cutoff as positivity score 4.0, PHB2 positive patients showed inferior OS (23.7% vs 50.0%; P = 0.026) and high hazard ratio (1.808; P = 0.028) compared to PHB2 negative patients (figure 1). Using the same cutoff, PHB2 positive patients showed inferior EFS (20.7% vs 47.1%; P= 0.024) and high hazard ratio (1.795; P = 0.026) (figure 2). In the qRT-PCR assay of PHB1 and PHB2, quantitative expression (ddCt) of PHB1 gene was higher in the control group: 0.31-28.32 (4.40+/-5.80; Mean+/-SD) versus 0.22-6.52 (1.83+/-1.56) in patient group. Meanwhile, PHB2 gene expression was higher in the NK-AML patient group: 0.15-3.49 (0.80+/-0.47) versus 0.00-0.46 (0.16+/-0.13) in control group. Using linear discrimination analysis, we derived a linear equation for discrimination between normal and AML cells. Estimating the prevalence of AML as 57.2/1,000,000, the sensitivity of newly developed algorithm was 93.6%, specificity was 76.9%, negative predictive value was 99.9%, and positive predictive value was 2.28% (figure 3). PHB protein expression was increased by inhibition of the MAPK pathway and decreased by activation of EGF signal. We identified the TCF-4/LEF-1 binding motif, CATCTG, in the promoter region of PHB by site-directed mutagenesis and ChIP assay. This beta-catenin-mediated activation of PHB expression was independent of c-MYC activation, a product of Wnt signaling. These data indicated that PHB was a direct target of beta-catenin and the increased level of PHB in leukemic cells can be regulated by Wnt signaling. Using CCEU and ICEU, time and dose dependent manner of proliferation suppression was observed in primary AML and AML cell lines. Notable morphological transformation of AML cells was observed when treated with 10-100 umol of CCEU and ICEU for 24 hours. The half maximal inhibitory concentration (IC50) was 25 umol for most AML cells. Conclusions: Overexpression of PHB2 was associated with poor prognosis in NK-AML. Newly developed discrimination algorithm using qRT-PCR showed the possibility as a rapid diagnostic tool for leukemic cell identification. Our functional study suggested that the elevated level of PHB in leukemic cells was the result of Wnt signals which were up-regulated in AML cells. Finally, this study developed novel alkylating chemotherapeutics targeting PHB for selective killing of leukemic cells. PHB2 expression status can provide clinically valuable information for the diagnosis, prediction of the prognosis, detection of minimal residual disease, and therapeutic targeting of NK-AML. Figure 1. Figure 1. Figure 2. Figure 2. Figure 3. Figure 3. Disclosures No relevant conflicts of interest to declare.

Whole Exome Sequencing Reveals Acquisition of Mutations Leading to the Onset of Donor Cell Leukemia after Hematopoietic Transplantation. a Model of Leukemogenesis

IntroductionDonor cell leukemia (DCL) is a rare complication of allogeneic stem cell transplantation (allo-SCT). The leukemic transformation of otherwise healthy donor stem cells provides a useful in vivo model to study the mechanisms involved in leukemogenesis.The objective of the present study is to study the dynamics of emergence of mutations that precede the development of DCL.Material and methodsWe report the case of a female patient diagnosed of acute lymphoblastic leukemia with t(1;19), who developed normal karyotype acute myeloid leukemia (AML) of donor origin 16 months after unrelated cord blood transplantation (UCBT).Whole exome sequencing(SureSelect-XT Human exon capture 50Mb Agilent Technologies) was performed by next generation sequencing (Hiseq 2000 Illumina) on bone marrow samples post allo-SCT, obtained in the days +98, +189, +350, +486 (DCL diagnosis) and + 569 (DCL relapse) as well as on the UCB unit used in SCT.The exome of bone marrow samples post allo-SCT were aligned to the human reference genome (NCBI build 37/hg19), non-synonymous variants in the coding regions of genes related to leukemia were selected and matched to the UCB exome to identify the acquired variants. Variants meeting such criteria were evaluated with three softwares (SIFT, Polyphen and Mutation Taster) to predict their functional effects. The UCB exome was aligned to the human reference genome (NCBI build 37/hg19).ResultsIn silico variants analysis revealed progressive emergence of multiple mutations related to the development of leukemia in bone marrow samples post allo-SCT (Figure 1). Interestingly three of the mutated genes (PTPN11, NRAS, MAP2K1) are part of the same cellular pathway (RAS-MAPK).In addition, mutations in leukemic subclones that disappear after chemotherapy were indentified, as well as the acquisition of new mutations in resistant subclones (Figure 2). Finally a mutation in SH2B3 gene, a gene that encodes for a regulatory protein of the JAK-STAT pathway, which links strongly to JAK2 inhibiting the activation of the pathway, was detected in the CBU.ConclusionsThe present study reveals the acquisition of additional somatic mutations in donor cells during leukemogenesis, including mutations not previously described in AML initiation (POU2F2, CA9, POU4F1, FMN2, TLR9, ELF5).Although the cause of DCL onset seems to be multifactorial, the infusion of a CBU with pre-leukemic potential due to mutation in SH2B3 in a context of residual toxicity in recipient as a result of pre-transplant chemotherapy, a post-transplant environment characterized by a decreased immune surveillance may well have played role in the case here reported.The study of a greater number of DCL cases by next generation sequencing could help to understand the process of leukemogenesis. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Feasibility of BAALC gene expression for detection of minimal residual disease and risk stratification in normal karyotype acute myeloid leukaemia.

High BAALC gene expression has been associated with poor prognosis in cytogenetically normal acute myeloid leukaemia (CN-AML) and has been suggested as a suitable marker for assessing minimal residual disease (MRD). The purpose of this study was to substantiate these findings by the analysis of a large data set of 632 diagnostic and follow-up samples in 142 intensively treated CN-AML patients. Paired diagnostic/relapse samples of 35 patients revealed stable high BAALC expression in 89%, irrespective of a high proportion of clonal evolution found in 49% of these cases. High BAALC expression, both directly after induction chemotherapy and within 3-6months after induction chemotherapy, correlated significantly with shorter event-free survival and overall survival. Moreover, 8 of 10 patients displaying high BAALC expression levels after completion of induction therapy as well as 5 of 5 patients exhibiting high BAALC expression levels within 3-6months after induction chemotherapy experienced relapse with a median of 197 and 101days, respectively, from sampling to relapse. Thus, BAALC expression-based MRD detection during therapy may be considered a strategy to identify patients at high risk of relapse.

Pharmacological activation of wild-type p53 in the therapy of leukemia.

The tumor suppressor p53 is inactivated by mutations in the majority of human solid tumors. Conversely, p53 mutations are rare in leukemias and are only observed in a small fraction of the patient population, predominately in patients with complex karyotype acute myeloid leukemia or hypodiploid acute lymphoblastic leukemia. However, the loss of p53 function in leukemic cells is often caused by abnormalities in p53-regulatory proteins, including overexpression of MDM2/MDMX, deletion of CDKN2A/ARF, and alterations in ATM. For example, MDM2 inhibits p53-mediated transcription, promotes its nuclear export, and induces proteasome-dependent degradation. The MDM2 homolog MDMX is another direct regulator of p53 that inhibits p53-mediated transcription. Several small-molecule inhibitors and stapled peptides targeting MDM2 and MDMX have been developed and have recently entered clinical trials. The clinical trial results of the first clinically used MDM2 inhibitor, RG7112, illustrated promising p53 activation and apoptosis induction in leukemia cells as proof of concept. Side effects of RG7112 were most prominent in suppression of thrombopoiesis and gastrointestinal symptoms in leukemia patients. Predictive biomarkers for response to MDM2 inhibitors have been proposed, but they require further validation both invitro and invivo so that the accumulated knowledge concerning pathological p53 dysregulation in leukemia and novel molecular-targeted strategies to overcome this dysregulation can be translated safely and efficiently into novel clinical therapeutics.

680 A common acute myeloid leukemia-associated DNMT3A mutation, when expressed ubiquitously in mice, causes reversible alopecia

In a study of normal karyotype acute myeloid leukemia (AML), our group found recurrent somatic mutations in the de novo DNA methyltransferase 3A (DNMT3A) gene in over 30% of normal karyotype AMLs. The most common change, a canonical DNMT3A R882H missense mutation, leads to the production of a hypomorphic protein that also dominantly represses the methyltransferase activity of wild type DNMT3A protein by inhibiting the ability of the wild type protein to form active homotetramers. To further study the biology of DNMT3A R882H, we generated mice bearing a doxycycline-inducible human DNMT3A R882H cDNA transgene that is expressed ubiquitously. Surprisingly, mice with the inducible DNMT3A R882H transgene, when treated with doxycycline, exhibit a high penetrance (21/36 treated mice), non-scarring alopecia. This alopecia was completely reversed within three weeks of the cessation of doxycycline. Histologic examination of the skin of mice overexpressing DNMT3A R882H revealed acanthosis of the epidermis, follicular plugging, and a mild dermal inflammatory infiltrate. Bone marrow transplantation from donor Dnmt3a-null mice or DNMT3A R882H transgenic mice does not cause alopecia in recipients, suggesting that hair loss is either due to a cell autonomous mechanism in the hair follicle, or from a local niche effect, rather than an abnormal inflammatory response in these mice. Accordingly, the expression of endogenous Dnmt3a in C57Bl/6 mice is ubiquitous in the hair follicles and interfollicular epidermis of newborn mice, but is restricted in adult mice, suggesting that it plays a role in the normal development of the hair follicle. Additional experiments to further define the roles of Dnmt3a and the highly related Dnmt3b in follicular development are currently underway.

Kinome Profiling Identifies Phospholipase C Gamma 1 (PLCγ1) As a Potential Target for t(8;21) AML

The t(8;21) (q22;q22) chromosomal translocation is one of the most frequent genetic alterations in acute myeloid leukemia (AML). Still about 30% of patients with t(8;21) AML struggle with relapse, despite intensive chemotherapy. We and others have shown that kinase activity profiling is able to identify new potential druggable targets. The aim of this study was to perform kinome profiling on 29 AML patients (cytogenetically normal, CN-AML, n=17 and t(8;21), n=12) and 4 normal bone marrow (NBM) samples to identify new potential druggable targets relevant to t(8;21) AML. We defined 130 peptides differentially phosphorylated between CN-AML, t(8;21) and NBM (p<0.05). With this technique, phospholipase C gamma 1 (PLCγ1_Y783) was identified as one of the highest phosphorylated peptide in t(8;21) AML. The knowledge about the role of PLCγ1 in leukemia progression is unknown. Therefore, we selected PLCγ1 to study its functional role in t(8;21) AML cell line (Kasumi-1) and also to test its efficacy as a potential target and provide proof of principle for this approach. PLCγ1 peptide phosphorylation was significantly higher in all t(8;21) AML samples when compared to NBM or to CN-AML (p<0.001 and p<0.05 respectively). mRNA expression of PLCγ1 in a publicly available paediatric AML database (http://r2.amc.nl) also showed significantly higher expression in t(8;21) AML compared to other AML karyotypes (p<0.001). PLCγ1 silencing with the specific short hairpin RNAs (two constructs, shRNAs PLCγ1-A and shRNAs PLCγ1-B), showed 50% and 80% decrease in PLCγ1 mRNA level compared with the control (scramble shRNA). These results were confirmed by PLCγ1 protein level analysis by western blotting. The shRNA-mediated silencing of PLCγ1 leads to suppression of the Kasumi-1 cell proliferation and viability at day 8 after transduction (p<0.05). The percentage of apoptosis in PLCγ1 supressing kasumi-1 cells at day 4 was two-fold higher than in scramble (p<0.01). To understand mechanistic insights of inhibited cell proliferation of PLCγ1 knock down cells, human phosphokinase microarray was performed and higher phosphorylation of Chk2_T68 and AMPK-α1_T183 were found in these cells. Higher Chk2 phosphorylation may responsible for cell cycle arrest and induced cell death of PLCγ1 supressing kasumi-1 cells. Similarly, higher phosphorylation of AMPK-α1 may inhibited cell proliferation of PLCγ1 knockdown cells. In conclusion kinome profiling is an elegant approach to identify interesting potential druggable targets. The results from proof of principle experiments strongly support the notion that suppression of PLCγ1 inhibited cell growth and induced apoptosis in Kasumi-1 cells. DisclosuresNo relevant conflicts of interest to declare.

Zinc Finger Nuclease (ZFN) Targeted Homologous Recombination Assay Detects Elevated Inter Homologous Recombination Activity and Copy Neutral Loss of Heterozygosity (CN-LOH) in Internal Tandem Duplication Mutated FLT3 (FLT3-ITD) Acute Myeloid Leukaemia (AML)

Mutations in the FMS-like tyrosine kinase 3 (FLT3) receptor whether it is internal tandem duplication (ITD) of its juxtamembrane domain or point mutations in its kinase domain are one of the most common mutations in normal karyotype acute myeloid leukaemia (NK-AML). The presence of FLT3-ITD mutation in NK-AML results in a more aggressive disease, resistance to therapy and poor survival. Acquired copy neutral loss of heterozygosity (CN-LOH) also referred to as uniparental disomy (UPD) is a common phenomenon of myeloid malignancies where an oncogenic allele is duplicated on the other chromosome. The use of single nucleotide phenotype analysis (SNP-A) karyotyping detects CN-LOH at the FLT3 locus, 13q associated with a FLT3-ITD mutation in NK-AML that results in an even more aggressive disease compared to NK-AML + FLT3-ITD without CN-LOH. It has been proposed that CN-LOH is the result of a homologous recombination (HR) DNA repair event. However, the underlying mechanisms that confer CN-LOH have yet to be determined.To elucidate the mechanisms that produce CN-LOH in NK-AML we developed a Zinc Finger Nuclease (ZFN) inter homologous recombination (iHR) targeting assay. The assay, based on the DR-GFP reporter developed by Pierce et al (1999), (Genes Dev. 13(20):2633-8) relies on the sequential targeting of two overlapping, but disrupted GFP repeat sequences to the adeno associated virus integration site 1 (AAVS1) on separate 19q alleles. Productive recombination between heterologous chromosomes in the region of 19q would result in restoration of GFP expression. The AML cell lines NB4 and THP-1 were transfected with a ZFN specific for AAVS1 integration (CompZr, Sigma) and the PZDonor AAVS1 puromycin derived construct DR1-Puro (Figure 1A,C) that possesses a 5' GFP sequence interrupted by an 18bp recognition sequence for the rare restriction enzyme, I-SCE I. Puromycin selected cells were then transfected with pZDonor derived DR2-Blast (Figure 1B,D) possessing the 3' GFP sequence and a blastocidin resistance gene. Puromycin and blastocidin selected cells were verified for correct integration of the plasmids to separate AAVS1 loci using location specific PCR and FISH, Figure 1F-H. Clones with correct integration were then expanded and transfected with FLT3-ITD, WT FLT3 expressing or empty vectors. Transfected cells were treated with an I-SCE I expression viral supernatant to induce a double strand break in DR1-Puro. Cells were cultured for a further 5 days before FACS analysis. FLT3-ITD transfected cells demonstrated GFP positive cells (inter HR events, 0.15% compared to WT-FLT3 and empty vector transfected cells (0.15% vs 0%, p<0.05, n=3) (Figure 1I). Furthermore, we were able to abolish inter HR events with co-culture with the FLT3 inhibitor, ACC220. To verify that GFP positive cells in this assay were the result of HR and not false positives, hot start PCR using primers designed at the DR1 locus was carried out on FACS sorted GFP positive cells. PCR products from GFP positive cells could only be digested by Bcg 1 and not I-SCE I in primary AML confirming the presence of iHR dependent GFP positive cells.Evaluation of HR events in primary AML was also investigated using ZFN targeted integration. Using the PZDonor AAVS1 puromycin derived construct HR substrate, PZD-DR1/DR2-Puro (Figure 1C), primary FLT3-ITD demonstrated significantly increased single locus targeted HR activity compared to primary WT FLT3, (0.62 vs 0.17%, p<0.01, n=22). Spontaneous HR activity (without I-SCE I cleavage) was observed in more than half the primary FLT3-ITD and was not seen in primary WT FLT3. Moreover, HR activity was significantly inhibited in primary AML with ACC220 (0.65% vs 0.1%, p<0.01, n=3) correlating with the reduction in RAD51 expression.The findings that FLT3-ITD mutation directly confers iHR activity provide a strong proof of principle that constitutive FLT3-ITD kinase activity augments HR activity, shifting the cellular milieu in favour of illegitimate recombination events such as CN-LOH that are selected for leukaemic progression. As both JAK2 and p53 mutations demonstrate enhanced HR repair and acquire CN-LOH at 9p and 17p respectively, we have identified a common mechanism of mutagenesis that furthers significantly our understanding of leukaemic progression and relapse in NK-AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Helpful Tool or Oversimplification? Concept of the Monosomal Karyotype from the Clinical and Cytogenetic Point of View.

Helpful Tool or Oversimplification? Concept of the Monosomal Karyotype from the Clinical and Cytogenetic Point of View.

Normal karyotype acute myeloid leukemia patients with CEBPA double mutation have a favorable prognosis but no survival benefit from allogeneic stem cell transplant.

Normal karyotype acute myeloid leukemia (NK-AML) with CCAAT/enhancer binding protein α (CEBPA) mutations is known to have a more favorable prognosis. However, direct comparison of the clinical significance according to consolidation therapy has not been widely performed in patients with NK-AML. A total of 404 patients with NK-AML who received intensive induction chemotherapy were included in the present study. Diagnostic samples from the patients were evaluated for CEBPA mutations by direct sequencing. CEBPA single (sm) or double mutation (dm) was observed in 27 (6.7%) and 51 (12.6%) patients, respectively. CEBPA (dm) was associated with GATA2 (mut), and it was less frequently associated with FLT3-ITD(pos), NPM1 (mut), and DNMT3A (mut) in comparison with CEBPA (wild) or CEBPA (sm) (all p values <0.05). On multivariate analysis, CEBPA (dm) (p = 0.007, OR 39.593) was an independent risk factor for achievement of complete remission (CR). With a median follow-up of 40.1months, CEBPA (dm) showed a favorable overall survival (OS), event-free survival (EFS), and lower relapse incidence (RI) in comparison with CEBPA (wild) (all p values <0.005). Comparison of clinical outcome analyses (consolidation chemotherapy vs. allogeneic hematopoietic cell transplantation (HCT)) demonstrated the role of consolidation treatment in patients with CEBPA (dm). Allogeneic HCT was associated with lower EFS and RI and a trend of higher non-relapse mortality. However, there was no statistically significant difference in OS. In conclusion, CEBPA (dm) was associated with other molecular mutations. Consolidation chemotherapy alone may overcome higher relapse rates by reducing the treatment mortality and increasing survival after relapse events in patients with CEBPA (dm) in NK-AML.

The Wilms Tumor-1 (WT1) rs2234593 variant is a prognostic factor in normal karyotype acute myeloid leukemia

The single-nucleotide polymorphism (SNP) within Wilms tumor-1 (WT1) exon 7, rs16754, has been arguably reported to be implicated in acute myeloid leukemia (AML) prognosis. We assessed the potential association of selected WT1 SNPs as well as WT1 mutations in normal karyotype (NK)-AML and evaluated the prognostic value of these normal gene variants. Diagnostic samples from a series of 474 young adult NK-AML patients were used to genotype five WT1 SNPs using TaqMan assays and to directly sequence WT1 exons 7 and 9. Analysis of five WT1 gene variants showed an association of rs2234593 allele C with WT1 Ex7 mutation. Prognostic study of the same variants identified rs2234593 significantly associated with relapse and overall survival (OS). Patients with rs2234593AA/AC showed significantly higher 10-year OS (50 vs 36 %, hazard ratio (HR) = 0.69 (0.52–0.90), p = 0.006) and lower cumulative incidence of relapse (CIR) (36 vs 51 %, HR = 0.62 (0.45–0.86), p = 0.004) compared to those with rs2234593CC. The effect of AA genotype on CIR remained significant after adjustment for basic covariates including FLT3 internal-tandem duplication (FLT3-ITD) and nucleophosmin 1 (NPM1) mutations (HR = 0.60 (0.41–0.89), p = 0.009), with some evidence of improved survival (HR = 0.75 (0.55–1.03), p = 0.07). A multivariate analysis showed WT1 Ex7-mutant as the major relapse predictor, with a tendency for rs2234593-A effect after allowing for Ex7 mutation (p = 0.07). No adjusted risk benefit was found for previously reported rs16754-G. In conclusion, WT1 normal gene variant rs2234593 is associated with mutational status of WT1 Ex7 and is a further prognostic marker independent from FLT3-ITD and NPM1 mutations in NK-AML.

T-LAK cell-originated protein kinase presents a novel therapeutic target in FLT3-ITD mutated acute myeloid leukemia.

Gain-of-function mutations of FLT3 (FLT3-ITD), comprises up to 30% of normal karyotype acute myeloid leukemia (AML) and is associated with an adverse prognosis. Current FLT3 kinase inhibitors have been tested extensively, but have not yet resulted in a survival benefit and novel therapies are awaited. Here we show that T-LAK cell-originated protein kinase (TOPK), a mitotic kinase highly expressed in and correlated with more aggressive phenotype in several types of cancer, is expressed in AML but not in normal CD34+ cells and that TOPK knockdown decreased cell viability and induced apoptosis. Treatment of AML cells with TOPK inhibitor (OTS514) resulted in a dose-dependent decrease in cell viability with lower IC50 in FLT3-mutated cells, including blasts obtained from patients relapsed after FLT3-inhibitor treatment. Using a MV4-11-engrafted mouse model, we found that mice treated with 7.5 mg/kg IV daily for 3 weeks survived significantly longer than vehicle treated mice (median survival 46 vs 29 days, P < 0.001). Importantly, we identified TOPK as a FLT3-ITD and CEBPA regulated kinase, and that modulating TOPK expression or activity resulted in significant decrease of FLT3 expression and CEBPA phosphorylation. Thus, targeting TOPK in FLT3-ITD AML represents a novel therapeutic approach for this adverse risk subset of AML.

Favorable outcome of patients with normal karyotype acute myeloid leukemia harboring FLT3-ITD and treated with cladribine added to daunorubicin and cytarabine induction

Favorable outcome of patients with normal karyotype acute myeloid leukemia harboring FLT3-ITD and treated with cladribine added to daunorubicin and cytarabine induction

CEBPA copy number variations in normal karyotype acute myeloid leukemia: Possible role of breakpoint-associated microhomology and chromatin status in CEBPA mutagenesis

Copy number variations (CNV) in CEBPA locus represent heterogeneous group of mutations accompanying acute myeloid leukemia (AML). The aim of this study was to characterize different CEBPA mutation categories in regard to biological data like age, cytology, CD7, and molecular markers, and identify possible factors affecting their etiology. We report here the incidence of 12.6% of CEBPA mutants in the population of 262 normal karyotype AML (NK-AML) patients. We confirmed that double mutant AMLs presented uniform biological features when compared to single CEBPA mutations and accompanied mostly younger patients. We hypothesized that pathogenesis of distinct CEBPA mutation categories might be influenced by different factors. The detailed sequence analysis revealed frequent breakpoint-associated microhomologies of 2 to 12bp. The analysis of distribution of microhomology motifs along CEBPA gene showed that longer stretches of microhomology at the mutational junctions were relatively rare by chance which suggests their functional role in the CEBPA mutagenesis. Additionally, accurate quantification of CEBPA transcript levels showed that double CEBPA mutations correlated with high-level CEBPA expression, whereas single N-terminal CEBPA mutations were associated with low-level CEBPA expression. This might suggest that high-level CEBPA expression and/or accessibility of CEBPA locus contribute to B-ZIP in-frame duplications.

Cytoplasmic nucleophosmin has elevated T199 phosphorylation upon which G2/M phase progression is dependent.

The cytoplasmic mutant of nucleophosmin (NPMc) is found approximately in one-third of acute myeloid leukemia (AML) cases and is highly associated with normal karyotype. Whereas previous studies have focused on wtNPM in centrosome duplication, we further elucidate the role of NPM in the cell cycle by utilizing the increased cytoplasmic load of NPMc. Overexpression of NPMc causes increased phosphorylation of NPM on T199 and, to a lesser degree, S4. T199 phosphorylation is dependent on cdk2 but activators of cdk2 were not elevated. Upon inhibition of cdk2, NPMc-overexpressing cells demonstrate a greater G2/M phase arrest than wtNPM or GFP counterparts. However, the number of cells with 2 centrosomes did not increase concordantly. This suggests that the arrest was caused by a delay in centrosome duplication, most likely due to the inhibition of centrosome duplication caused by unphosphorylated NPMc. Overall, these results suggest that the phosphorylation of T199 is important in the mitotic progression of NPMc-expressing cells. This further supports the hypothesis that NPMc is associated with normal karyotypes in AML because the higher cytoplasmic load of NPM can better suppress centrosome overduplication which would otherwise result in unequal segregation of chromosomes during mitosis, leading to aneuploidy and other genomic instabilities.

Azacitidine in Older Patients with Acute Myeloid Leukemia (AML) and Adverse Karyotype. Subanalisis from the Alma Study

BackgroundAdverse karyotype and age are associated with poor prognosis in older AML patients (pts). Although complete remission (CR) can be achieved with intensive chemotherapy (IC) in pts with adverse cytogenetics, overall survival (OS) and leukemia free-survival (LFS) are poor (Knipp S, et al. Cancer 2007). In addition, low doses of ARA-C (LDAC) did not offer survival benefit in older pts with poor cytogenetics as compared to hydroxyurea and best supportive care (BSC) (Burnett A, et al. Cancer 2007). Azacitidine has shown a survival advantage in older pts with newly diagnosed AML in a recent phase 3 trial, as compared to conventional care regimens (BSC/LDAC/IC)(Dombret H, et al. EHA 2014).AimTo evaluate the role of azacitidine and factors associated with OS in pts with newly diagnosed AML and adverse karyotype from the ALMA study (Ramos F, et al. Blood 2012;120:abstract 3593).MethodsRetrospective, multicenter study, of pts with newly diagnosed AML and adverse karyotype who received front-line azacitidine in Spain. Response to azacitidine was evaluated by ELN-2010 criteria. Overall survival was evaluated by Kaplan-Meier method and log-rank test.ResultsThirty-nine pts were identified. Median age: 71y (52-83). Baseline characteristics are shown in table 1. All pts received azacitidine (75 mg/m2 sc x7 days) as first line treatment. Median time from AML diagnosis to therapy was 16 days (1-88). 56% of pts had bone marrow (BM) blasts >30% at diagnosis (median: 32%; range: 20-90). Five out of 39 (13%) pts had leucocyte count (WBC) >15x109/L and 20/39 (51%) had ECOG performance status >1. Complex karyotype (CK) and monosomal karyotype (MK) was present in 18/39 pts each (46%). Within the subset showing CK, 14/18 (78%) had MK. Complete remission (CR) and CRi was 23.1% and 5.1% respectively (ORR=28.2%). Median courses of therapy to best response: 3 (1-11). After 17m median follow-up, median OS (95% CI) was 7m (3.1-8.8). Estimated 1-year survival was 29.8%. There were no differences in median OS in pts with MK vs. rest of pts with CK (median OS 9m [95% CI: 5.3-12.6] vs. 6.5m [3.1-8.8], respectively; p=0.854). Monosomal karyotype, CK with >5 abnormalities, BM blasts >30% and ECOG status >1 did not achieve statistically significance in multivariate analysis. Only WBC at baseline (>10x109/L or >15x109/L; threshold not relevant) had an impact on OS in both univariate (3m [95% CI: 4.9-13] vs. 9m [4.9-13] if WBC <10x10e /L; p=0.014) and multivariate analysis (HR=3.3, [95% CI 1.1-9.5], p=0.025; Table 2). Most frequent causes of death were disease progression and infection (82%).Table 2Multivariate analysis for survivalParameterHR95% CIp-valueWBC >10x109/L3.341.17-9.580.025BM blast >30%1.080.46-2.540.855MK0.950.33-2.740.919CK1.610.58-4.510.361ECOG >11.840.81-4.170.144ConclusionAlbeit retrospective and non-comparative, azacitidine seems to improve median and 1y survival as compared to historic AML data in pts with adverse cytogenetics. An elevated WBC >10x109/L at diagnosis was the only parameter associated to adverse outcome in this set of pts.Table 1Baseline characteristicsAge, median (range)71 (52-83)ECOG performance status 0-1 2-319 (48.7%) 20 (51.3%)WBC (x109/L), median (range) WBC >10x109/L, N (%)2.2 (0.9-89.2) 5 (12.8%)BM blasts, median; (range) BM blasts >30%, N (%)32 (20-90) 22(56.4%)Complex karyotype (CK), N (%)18 (46.2)Monosomal karyotype (MK), N (%)18 (46.2%)Both CK and MK, N (%)14/18 (77.8%)Prior MDS, N (%)11 (28.2%)Karyotype, N (%) Complex >3 Complex >5 Chrom 7 abn Chrom 3 abn -7 only Chrom 5 and 7 abn Chrom 5 abn Other6 (15.4%) 11 (28.2%) 4 (10.3%) 4 (10.3%) 8 (20.5%) 2 (5.1%) 2 (5.1%) 2 (5.1%)*Excludes pts with chromosome 5 and 7 abnormality DisclosuresFalantes:Celgene: Consultancy. Off Label Use: Use of azacitidine in AML with blast count >30% This paper deals with both on-label and off-label use of azacitidine in AML. Xicoy:Celgene: Honoraria. Ramos:Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees.

Cladribine Added to Daunorubicin and Cytarabine Induction Regimen Prolongs Survival of Patients with Complex but Not Monosomal Karyotype Acute Myeloid Leukemia – Retrospective Analysis of Polish Adult Leukemia Group (PALG)

Objectives:Cytogenetics is one of the most important prognostic factors in acute myeloid leukemia (AML). Two major, partially overlapping cytogenetic subsets of AML associated with an adverse prognosis, are AML with complex karyotype (CK) and AML with monosomal karyotype (MK). MK has been shown to constitute a cytogenetic feature that identifies AML patients (pts) with very poor outcomes when treated with currently available therapy. CK-AML pts are very heterogeneous cytogenetically, and treatment outcomes of these subsets are also influenced by TP53 alterations, complexity of the karyotype or presence of residual normal metaphases. However, impact of all these variables on outcome of CK-AML patients was so far analyzed separately.The aim of our study was to assess treatment outcome of CK-AML patients with or without MK regarding some cytogenetic and clinical features.Materials and methods: One hundred twenty five newly diagnosed AML patients with CK treated between January 2007 and January 2013 with PALG protocols (Holowiecki et al., JCO 2012) were included into the study. Cytogenetic analysis was performed on metaphases from bone marrow aspirates taken at diagnosis using standard banding techniques. Karyotypes were reported in accordance with the ISCN 2009. CK was defined as a presence of ≥ 3 unrelated abnormalities. MK was defined by the presence of an autosomal monosomy in conjunction with at least one other autosomal monosomy or structural abnormality in the absence of t(8;21), inv(16), t(15;17) (Breems et al., JCO 2008). FISH method was used to verify MK in all cases with monosomy as well as to assess 17p13 (TP53) deletion.Results:Clinical characteristics. The median age of all pts was 60 years (range 19-85 years). FISH verification proved the MK karyotype (MK+) in 75 out of 125 (60%) pts with CK-AML. Remaining pts had non-monosomal CK (MK-). Sixty six (55%) pts received intensive induction chemotherapy (IC) according to PALG protocols and 59 pts received non-intensive treatment with low-dose cytarabine (LDAC) or best supportive care (BSC). Baseline characteristics were generally balanced between MK+ and MK- groups in terms of age, WBC and PLT counts, hemoglobin level, percentage of bone marrow blasts at diagnosis and presence of residual normal metaphases in the karyotype. Higher proportions of MK+ pts had deletion of 17p13 (TP53) (47,9% vs. 20,4% of MK-; p=0.0017) as well as ≥5 cytogenetic aberrations (93,3% vs. 74%; p=0.004). Furthermore, there were no differences in the proportion of IC, LDAC and BSC strategies between both groups.Treatment Response and Outcome. With the median time of follow-up 7,7 months (mos), median OS in MK- pts was significantly longer compared to MK+ group (4,5 mos vs. 2,0 mos; p=0.0075). 66/125 CK-AML pts received standard IC according to PALG AML protocols (DA-36 pts; DAC-27 pts and DAF-3 pts). Only 7 pts underwent allogeneic stem cell transplantation (allo-SCT). In intensively treated pts the overall CR rate in MK- was about twice as high as in MK+ group (62,1% vs 32,4%; p=0.013). The median OS in MK- pts was longer compared to MK+ group (6,8 mos vs. 2,7 mos; p=0.016). Probability of 1-year OS in MK+ vs. MK- group was 14% vs. 30%. In multivariate analysis we have found that WBC >20 G/l, MK and ≥5 chromosomal abnormalities were independent prognostic factors associated with significantly shorter OS (Table 1). In contrast, allo-SCT was associated with survival benefit. Furthermore, we analyzed the impact of purine analogue containing induction regimens on treatment outcome. We found that addition of cladribine to the standard DA regimen improves OS in MK- but not in MK+ group (Figure 1).Conclusions: Monosomal karyotype, high WBC count and high complexity of the karyotype (≥5 aberrations) are associated with more aggressive clinical course and short survival in high-risk CK-AML. Cladribine added to standard DA induction regimen prolongs survival of pts with complex but not monosomal karyotype.*AW and EW equally contributed to the study. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Mutation in Wilms’ Tumor 1 Induces DNA Hypermethylation of PRC2 Targets, Blocks Myelomonocytic Differentiation, and Defines a Novel Subtype of AML Responsive to EZH2 Inhibition

Wilms’ Tumor 1 is a transcription factor found to be recurrently mutated (WT1mut) in 10% of normal karyotype acute myeloid leukemia (NK-AML), predominantly in young adults with intermediate-poor prognosis, often in association with FLT3-ITD and high white cell count. Mutations are usually heterozygous and consist of small insertions or deletions clustered around exons 7 and 9, which encode zinc-finger DNA binding domains. In general, mutations disrupt these DNA binding domains leading to a truncated protein that may act in a dominant negative fashion. Currently, the mechanism by which WT1mut contributes to leukemogenesis is unknown, and no lead drug targets linked to this mutation have been identified to date. Using a novel computational method based on Boolean implications that link the presence of a somatic mutation to CpG methylation on a site-by-site basis, we found that mutation in WT1 is strongly linked to DNA hypermethylation in AML patient samples. In order to validate these findings, we expressed mutant WT1 protein prematurely truncated at exon 7 in THP-1 AML cells (confirmed to be wildtype for WT1 at both alleles), and after 10 passages we measured DNA methylation by 450K bead-chip arrays. We found consistent upregulation of DNA methylation in mutant but not wildtype WT1-expressing cells when compared to parental THP-1 cells, validating WT1 mutation as an active driver of DNA hypermethylation. Additional methylome analysis of human hematopoietic stem and progenitor compartments (HSPC) including (HSC, MPP, L-MPP, CMP, and GMP) indicated that WT1mut induces predominantly de novo DNA methylation, as virtually all CpG sites induced by the mutant protein are unmethylated in normal HSPC. Strikingly, the pattern of methylation in both WT1mut patient samples and WT1mut-THP-1 cells was enriched for polycomb repressor complex 2 (PRC2) target genes (p<1.6E-87), implicating a role for this repressive chromatin-remodelling complex in WT1mut leukemogenesis. In keeping with this, gene expression analysis of WT1mut AMLs (but not other normal karyotype AMLs) showed marked repression of known hematopoietic PRC2 target genes (as defined by Chip-Seq), suggesting WT1mut may induce a differentiation block through deregulation and hypermethylation of PRC2 targets.To explore this possibility, we expressed WT1mut in purified normal cord blood CD34+ HSPC using lentiviral transduction and performed in vitro liquid culture differentiation assays in IL-3, SCF, FLT3L, and GM-CSF. We found that WT1mut (but not wildtype or empty vector) induced a myelomonocytic differentiation block with fewer cells expressing CD11b, CD11c, and CD14. Separately, we also showed that WT1mut induced a differentiation block in a TF-1 cell model of erythroid differentiation. These findings suggest a role for WT1mut in perturbing myeloid differentiation in early HSPC.To test the therapeutic implications of our findings, we asked whether inhibition of the major enzymatic histone trimethylase component of PRC2, EZH2, could reverse the differentiation block caused by WT1mut. Significantly, we found that treatment of primary WT1mut AML blasts with the selective EZH2 inhibitor GSK-126 induced upregulation of the mature myeloid markers CD11b, CD33, and CD14. In contrast, NK-AML without WT1mut or acute promyleocytic leukemia cells did not show a significant differentiation response.Our results indicate that mutation in WT1 defines a novel subgroup of DNA hypermethyated AML with de novo hypermethylation of PRC2 target genes that may clinically respond to selective EZH2 inhibitors through differentiation. Importantly, our methods show that genome-wide analysis of mutation-specific DNA methylation patterns may have a future role in determining epigenetic therapies for personalized medicine. DisclosuresNo relevant conflicts of interest to declare.

Lineage-Specific Distribution of CALR EXON 9, JAK2V617F, MPLW515L/K, NPM1 and FLT3 Mutations in Myeloid Disorders

Introduction:Myeloid malignant disorders are clonal diseases arising in hematopoietic stem or progenitor cells. Several somatic mutations involved in these diseases are currently known and routine molecular testing involves screening genes of therapeutic and prognostic significance. Mutational analysis of FLT3 in combination with NPM1 can be used to predict outcome and direct therapy in normal karyotype acute myeloid leukemia (AML). JAK2, MPL and CALR mutation detection complements the molecular diagnostic testing menu for myeloproliferative neoplasm’s (MPN): polycytemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). In addition, these molecular markers are utilized for minimal residual disease (MRD) detection, e.g. following stem cell transplants. Little is currently known about the lineage-specific distribution of some of these markers. In this study we aimed to assess the distribution of common genetic mutations in multiple lineages (lymphoid, myeloid, monocyte, multipotent progenitors, myeloblast and erythroid) of MPN and AML utilizing fluorescent activated cell sorting (FACS).Method:Different cell lineage fractions (lymphoid (CD3+), mature (CD16+) and immature (CD16dim) granulocytes, monocyte (CD14+), erythroid(CD36+), multipotent progenitors (34+) and/ or myeloblasts (CD117+) of unseparated bone marrow and peripheral blood specimens of myeloid disorders were sorted on a BD Aria 2. The patient specimens selected were positive for either JAK2V617F, MPLW515L or CALR Exon9 insertion/ deletion (MPN’s) or for NPM1 and/or FLT3 mutations (AML; diagnostic, relapse and minimal residual disease (MRD)). Fractions were subsequently analyzed for the presence of the respective mutation by PCR and/ or bi- directional sequencing.Results:All FACS purified CD34+ progenitors, myeloid and erythroid cell fractions of MPL W515L (3) or CALR exon9 (12) positive MPN specimens demonstrated the presence of mutations, respectively. Interestingly, JAK2V617F was present in the sorted erythroid cell fraction in 5/6 MPN cases tested. However, the granulocyte cell and blast cell fraction of one polycythemia vera specimen tested negative for the presence of Jak2V617F. All lymphoid CD3+ T-cell fractions were negative.The NPM1 exon 12 mutation was uniformly detected in progenitors and all myeloid cell fractions of 3/3 diagnostic, 2/2 relapse and 1/8 MRD AML specimens. For 5/8 MRD cases all lineages tested negative. Surprisingly, for two MRD cases the mutation was observed only in the unseparated and myeloid lineages but not in CD34+ blast fraction. Similar to the above findings, FLT3 mutations were detected in multipotent progenitors, and/ or myeloblasts collections of 4/4 diagnostic specimens. However, the mutation was absent in the granulocyte and monocyte fraction of one case. No detectable signals were observed in the cell fractions of 5 MRD specimens and in the CD3+ lymphoid cell fractions of all AML cases.Conclusion:We conclude that CALR and MPL mutations are uniformly detectable in the unseparated bone marrow specimens of MPN’s as well as separated progenitor, erythroid, granulocyte and monocyte fractions. Interestingly, JAK2 mutations can be exclusively found in the erythroid lineage in PV, whereas it can be absent in the granulocyte and blast compartment. This finding may have implications on specimen processing to ensure that erythroids are retained for clinical Jak2 testing. In addition, our results support the hypothesis that CALR Exon9 mutations are early event driver mutations in comparison to JAK2V167F.Both NPM1 and FLT3 mutations, in AML, were detected in unseparated specimens as well as in the multipotent progenitors or myeloblasts at diagnosis and relapse. However, the NPM1 mutation was observed in unseparated specimens and granulocyte cell fractions of 2 residual disease cases, whereas it was surprisingly absent in the CD34+ cell lineage fractions. Conversely, FLT3-ITD was exclusively found in the progenitor cells and absent in the granulocyte lineage of one case at diagnosis.Our findings reported here may be able to assist assay development efforts for diagnostic and residual disease mutation detection in myeloid disorders. In addition, flow cytometric assessment of monitoring specimens prior to molecular analysis may be beneficial to decide if cell enrichment steps can give additional evidence for the presence of residual disease. DisclosuresBurnworth:HematoLogics Inc.: Employment. Bennington:HematoLogics Inc.: Employment. Fritschle:HematoLogics Inc.: Employment. Nguyen:HematoLogics Inc.: Employment. Verkamp:HematoLogics Inc.: Employment. Angela:Hematologics: Employment. Wentzel:HematoLogics Inc.: Employment. Broderson:HematoLogics Inc.: Employment. Loken:Hematologics: Employment, Equity Ownership. Wells:HematoLogics Inc.: Employment, Equity Ownership. Zehentner:HematoLogics Inc.: Employment, Equity Ownership.