Epigenetically Enhanced MED12L in ETO2-GLIS2 Positive Pediatric Acute Megakaryoblastic Leukemia Is Associated with Resistance to the CDK8 Inhibitors

Abstract Introduction. ETO2-GLIS2 (aka CBFA2T3-GLIS2) is the most common (30%) alteration in pediatric de novo acute megakaryoblastic leukemia (AMKL). These patients have poor response to induction therapy, a high incidence of relapse (~90%), and dismal 5-year survival rates (<20%). Previous studies suggest that ETO2-GLIS2 induces leukemia through abnormal enhancer formation as a single oncogenic "hit". Because ETO2-GLIS2 expression induces formation of leukemia-specific neo-superenhancer (SE) elements, we hypothesize that mediator (MED) proteins are involved in linking neo-SE elements to distal gene expression, and thus could be a therapeutic target. Here, we analyzed expression of MED-family genes in ETO2-GLIS2 positive AMKL patients in combination with enhancer-histone marks, and evaluated impact of MED-kinase inhibition with selective CDK8 inhibitors (CDKi) against cell viability. Methods. To address our hypothesis, we analyzed published RNA sequencing dataset (Smith et al. 2020) for human MED-genes expression from a pan-pediatric AML cohort (N=1476). The cohort consisted of subgroups expressing fusions of ETO2-GLIS2 (N=40), CBFB-MYH11 (N=174), DEK-NUP214 (N=49), KMT2A-ELL (N=50), KMT2A-MLLT10 (N=86), KMT2A-MLLT3 (N=114), KMT2A-MLLT4 (N=49), NUP98-NSD1 (N=107), RUNX1-RUNX1T1 (N=210) and no detectable fusions (N=526), compared to normal bone marrow (NBM) samples (N=71). Enrichment of histone marks overlapping MED-genes was analyzed from published chromatin immunoprecipitation (ChIP) sequencing in ETO2-GLIS2 positive M-07e AMKL line (Thirant et al. 2017). We tested efficacy of CDK8 inhibition with BI-1347 and CCT251545 against M-07e cells to determine their activity in the context of marked MED12L overexpression. Results. We examined expression of 29 MED-genes comprising the 4 major (head, middle, tail, and kinase) MED-modules. MED gene expression was variable across AML subtypes and NBM. However, MED genes were more commonly over-expressed in the ETO2-GLIS2 group, in particular MED 17, MED1, MED10, MED27, and MED12L (paralog of MED12) were upregulated in the subgroup. Most notably, we noted exceptional upregulation of MED12L (FC 4.9, log2), compared to NBM (Figure 1). Because MED12/12L plays an intrinsic biological role in establishing oncogenic enhancer-expression loops in hematopoietic stem or leukemic cells, we investigated the overlap of enhancer bound histones such as H3K27ac and H3K4me1 to MED12L in M-07e cells, compared to umbilical cord blood-derived normal megakaryoblasts (MK) (S004BT; Blueprint epigenome database). We found enrichment of H3K4me1 at MED12L transcription start site (TSS) and upstream promoter both in MK and M-07e cells. In addition, we observed a large region of H3K27ac enrichment spanning 89 Kb (16 kb upstream and 73 kb downstream) across MED12L TSS in M-07e cells, suggesting neo-enhancer activity at this locus. Considering the dependency of MED12 on CDK8 for MED-kinase activities (Klatt et al. 2020), we treated M-07e cells with CDK8i(s), to test our hypothesis if perturbation of epigenetically enhanced MED12L expression can impact leukemic growth. However, we observed a poor correlation between M-07e cell viability and IC 50 of BI-1347 (IC 50: 0.87 µM, R 2: 0.36) or CCT251545 (IC 50: 0.4 µM, R 2: 0.48). In contrast, ETO2-GLIS2 negative MV4-11 AML cells were susceptible to both BI-1347 (IC 50: 0.44 µM, R 2: 0.88) and CCT251545 (IC 50: 0.12 µM, R 2: 0.87). Given the inefficacy of CDK8i against M-07e, and cooperativity of bromodomain extra-terminal (BET)-BRD4 and MED12/12L in forming enhancer complexes, we tested possible inhibitory impact of BRD4 inhibitor JQ1 on MED12L expression and leukemic growth in target cells. We observed effective reduction in M-07e cell viability (IC 50:0.3 µM, R 2: 0.93) with concomitant reduction not only in BRD4 protein-expression, but diminished MED12L protein expression at IC 50 and higher doses of JQ1. Conclusion. Our findings revealed that MED12L is highly overexpressed and overlapped with strong neo-enhancer chromatin-marks in ETO2-GLIS2 positive cells, while maintaining resistance to CDK8i. Future studies will contribute to deeper insights into the preferential recruitment and role of MED12L in ETO2-GLIS2 bound enhancers and potential mechanisms of resistance to CDK8 inhibition in the disease. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Pediatric Acute Megakaryoblastic Leukemia without Down Syndrome: A Retrospective Study by the International Berlin-Frankfurt-Munster Study Group (I-BFMSG)

Blood ◽

10.1182/blood.v124.21.3670.3670 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3670-3670

Author(s):

Hiroto Inaba ◽

Yinmei Zhou ◽

Oussama Abla ◽

Souichi Adachi ◽

Anne Auvrignon ◽

...

Keyword(s):

Down Syndrome ◽

Retrospective Study ◽

Large Scale ◽

Conflicts Of Interest ◽

Central Nervous System Involvement ◽

Acute Megakaryoblastic Leukemia ◽

Hematopoietic Stem ◽

Megakaryoblastic Leukemia ◽

Group I ◽

Significant Difference

Abstract Acute megakaryoblastic leukemia (AMKL) comprises up to 10% of childhood acute myeloid leukemia (AML) cases. However, no large-scale studies have comprehensively evaluated the clinical characteristics and outcomes of patients with AMKL. We performed a large-scale international retrospective study of pediatric patients (diagnosed at age ≤18 years) with de novoAMKL without Down syndrome treated from 1989 to 2009. The study included 490 patients with AMKL, which comprises 7.9% of the pediatric AML patients treated by 19 members of the I-BFMSG. At diagnosis, the median age of patients was 1.5 years (range, 0.0–16.5 years), median white blood cell count was 12.0×109/L (range, 0.6–188.0×109/L), 23 (4.7%) patients showed central nervous system involvement, and both sexes were equally represented. Complete remission (CR) was achieved in 417 (85.1%) patients, and 5-year event-free (EFS) and overall survival (OS) rates were 43.7%±2.7% and 49.0%±2.7%, respectively. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) was performed in 206 (42.0%) patients. The 5-year disease-free survival and OS rates for patients who received allo-HSCT in first CR (n=112, 56.3%±5.2%, and 57.7%±5.2%, respectively) and for those who did not receive transplantation in first CR (n=298, 50.2%±3.6% and 55.2%±3.5%) were not significantly different (P=0.12 and P=0.57). Complete cytogenetic data were available for 372 (75.9%) patients: diploid (n=49, 13.2%), hypodiploid (n=18, 4.8%), pseudodiploid (n=119, 32.0%), 47–50 chromosomes (n=142, 38.2%), and >50 chromosomes (n=44, 11.8%). Chromosome gain occurred in 195 (52.4%) patients: +21 was the most common (n=106, 28.5%), followed by +19 (n=93, 25.0%), and +8 (n=77, 20.7%). Losses occurred in 65 cases (17.5%), in the decreasing order of –7 (n=13, 3.5%), –9 (n=9, 2.4%), –13, and –15 (n=7 each, 1.9%). Structural chromosomal aberrations were observed in 278 (74.7%) patients, most commonly t(1;22) (n=51, 13.7%) and 11q23 rearrangements (n=38, 10.2%) with t(9;11) in 21 patients. Other abnormalities included 7p (n=43, 11.6%) and 13q (n=31, 8.3%; 16 with deletions) breakpoints. Patients with t(1;22) were significantly younger at diagnosis (P<0.001; median 0.6 years, range 0.0–6.3 years) and more often female (P=0.05; 64.7%) while those with –7 were significantly older (P=0.006; median 4.2 years, range 0.4–15.0 years) than those with other cytogenetic subgroups. Multiple regression analysis for EFS and OS with clinical and cytogenetic features showed that treatment period (1989–1999 vs. 2000–2009), normal cytogenetics, –7, t(9;11), 13q-, and –15 were associated with significantly worse outcomes than those for other subgroups, whereas abnormalities in 7p were associated with better outcomes (Table). Patients with –13 and 9p abnormalities other than t(9;11) had a poorer EFS and OS, respectively. Patients with +21 or t(1;22) did not show a significant difference in survival rates. This international study on the largest cohort of AMKL patients analyzed to date shows heterogeneity in cytogenetic findings and identifies some subgroups with a particularly dismal outcome. The advent of newer methods to evaluate genetic lesions can help identify therapeutic targets for improving the outcome in this subgroup of patients. Disclosures No relevant conflicts of interest to declare.

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Receptor-driven invasion profiles in diffuse intrinsic pontine glioma (DIPG)

Neuro-Oncology Advances ◽

10.1093/noajnl/vdab039 ◽

2021 ◽

Author(s):

Anju Karki ◽

Noah E Berlow ◽

Jin-Ah Kim ◽

Esther Hulleman ◽

Qianqian Liu ◽

...

Keyword(s):

Gene Expression ◽

Growth Factor ◽

Protein Expression ◽

Cell Viability ◽

Rna Sequencing ◽

Cell Invasion ◽

Growth Factor Receptor ◽

Diffuse Intrinsic Pontine Glioma ◽

Sequencing Data ◽

Pontine Glioma

Abstract Background Diffuse intrinsic pontine glioma (DIPG) is a devastating pediatric cancer with unmet clinical need. DIPG is invasive in nature, where tumor cells interweave into the fiber nerve tracts of the pons making the tumor unresectable. Accordingly, novel approaches in combating the disease is of utmost importance and receptor-driven cell invasion in the context of DIPG is under-researched area. Here we investigated the impact on cell invasion mediated by PLEXINB1, PLEXINB2, platelet growth factor receptor (PDGFR)α, PDGFRβ, epithelial growth factor receptor (EGFR), activin receptor 1 (ACVR1), chemokine receptor 4 (CXCR4) and NOTCH1. Methods We used previously published RNA-sequencing data to measure gene expression of selected receptors in DIPG tumor tissue versus matched normal tissue controls (n=18). We assessed protein expression of the corresponding genes using DIPG cell culture models. Then, we performed cell viability and cell invasion assays of DIPG cells stimulated with chemoattractants/ligands. Results RNA-sequencing data showed increased gene expression of receptor genes such as PLEXINB2, PDGFRα, EGFR, ACVR1, CXCR4 and NOTCH1 in DIPG tumors compared to the control tissues. Representative DIPG cell lines demonstrated correspondingly increased protein expression levels of these genes. Cell viability assays showed minimal effects of growth factors/chemokines on tumor cell growth in most instances. Recombinant SEMA4C, SEM4D, PDGF-AA, PDGF-BB, ACVA, CXCL12 and DLL4 ligand stimulation altered invasion in DIPG cells. Conclusions We show that no single growth factor-ligand pair universally induces DIPG cell invasion. However, our results reveal a potential to create a composite of cytokines or anti-cytokines to modulate DIPG cell invasion.

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Epigenetic Control of C/EBPa by Noncoding RNAs.

Blood ◽

10.1182/blood.v114.22.3644.3644 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3644-3644

Author(s):

Annalisa Di Ruscio ◽

Alexander K Ebralidze ◽

Francesco D'Alò ◽

Maria Teresa Voso ◽

Giuseppe Leone ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Messenger Rna ◽

Noncoding Rna ◽

Gene Locus ◽

Conflicts Of Interest ◽

Lentiviral Vector ◽

Rt Pcr ◽

Hematopoietic Stem ◽

Factor C

Abstract Abstract 3644 Poster Board III-580 Little is currently known about the role of noncoding RNA transcripts (ncRNA) in gene regulation; although most, and perhaps all, gene loci express such transcripts. Our previous results with the PU.1 gene locus showed a shared transcription factor complex and chromatin configuration requirements for biogenesis of both messenger and ncRNAs. These ncRNAs were localized within the nuclear and cytoplasmic compartments. Disrupting ncRNAs in the cytoplasmic cellular fraction results in increased PU.1 mRNA and protein. Recently, we have focused on the C/EBPa gene locus and observed extensive noncoding transcription. The transcription factor C/EBPa plays a pivotal role in hematopoietic stem cell (HSC) commitment and differentiation. Expression of the C/EBPa gene is tightly regulated during normal hematopoietic development, and dysregulation of C/EBPa expression can lead to lung cancer and leukemia. However, little is known about how the C/EBPa gene is regulated in vivo. In this study, we characterize ncRNAs derived from the C/EBPa locus and demonstrate their functional role in regulation of C/EBPa gene expression. First, northern blot analysis and RT PCR determined a predominantly nuclear localization of the C/EBPa ncRNAs. Second, strand-specific quantitative RT PCR demonstrated a concordant expression of coding and noncoding C/EBPa transcripts. Next, we investigated the results of ablation of ncRNAs using a lentiviral vector containing ncRNA-targeting shRNAs on the expression of the C/EBPa gene. We have observed that reduced levels of ncRNAs leads to a significant downregulation of the expression of coding messenger RNA. These data strongly suggest that C/EBPa ncRNAs play an important role in maintaining optimal expression of the C/EBPa gene at different stages of hematopoiesis and makes targeting noncoding transcripts a novel and attractive tool in correcting aberrant gene expression levels. Disclosures: No relevant conflicts of interest to declare.

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Epigenetic Regulation of Lipid Signalling Pathways In Low-Risk MDS Patients During Azacitidine Treatment

Blood ◽

10.1182/blood.v116.21.233.233 ◽

2010 ◽

Vol 116 (21) ◽

pp. 233-233

Author(s):

Matilde Y Follo ◽

Sara Mongiorgi ◽

Cristina Clissa ◽

Carla Filì ◽

Chiara Colombi ◽

...

Keyword(s):

Gene Expression ◽

Promoter Methylation ◽

Lower Risk ◽

Conflicts Of Interest ◽

Stem Cell Differentiation ◽

Epigenetic Therapy ◽

Signalling Pathways ◽

Molecular Profile ◽

Hematopoietic Stem ◽

Lipid Signalling

Abstract Abstract 233 Azacitidine, a DNA methyltransferase inhibitor currently used for the treatment of higher-risk myelodysplastic syndromes (MDS) patients, was shown to delay the evolution into acute myeloid leukemia (AML) and prolong overall survival (Fenaux P et al, Lancet Oncol 2009). In addition, azacitidine has recently been shown to potentially be a feasible and effective treatment even for patients with lower-risk MDS (Musto P et al, Cancer 2010). Lipid signalling pathways are involved in many important biological processes, such as cell growth, differentiation and apoptosis and play a role in the progression of MDS towards AML (Follo MY et al, J Cell Biochem 2010). Moreover, we recently demonstrated that phosphoinositide-phospholipase C beta1 (PI-PLCbeta1) promoter gene is hyper-methylated in higher-risk MDS and that azacitidine treatment can induce an increase in the level of PI-PLCbeta1 splicing variants as well as a down-regulation of activated Akt (Follo MY et al, Leukemia 2008; Follo MY et al, PNAS 2009). In fact, responding patients showed an increase in PI-PLCbeta1 expression in correlation with the therapeutic response, whereas their PI-PLCbeta1 promoter methylation was reduced. Furthermore, the decrease of promoter methylation anticipated the hematologic response, since the variations in PI-PLCbeta1 gene expression were observed prior to the clinical outcome. Stemming from these data, we further investigated the role of inositide signalling pathways during the epigenetic therapy, focusing on the effect of azacitidine on lipid signal transduction pathways in lower-risk MDS patients. The study included 25 patients (IPSS risk: low or intermediate-1) treated with azacitidine (75mg/m2 subcutaneous daily for 5 consecutive days every 28 days, for a total of 8 courses). For each patient we followed the effect of azacitidine in correlation to both PI-PLCbeta1 promoter methylation and gene expression, as well as the molecular profile of key molecules involved in the regulation of methylation processes, such as histone deacetylases (HDACs), methyl-CpG binding domain proteins (MBDs), and transcription factors correlated to hematopoietic stem cell differentiation and proliferation. Our results show that 8/25 (34%) of our lower-risk MDS patients, showing hematologic improvements after azacitidine therapy, had a significant increase in PI-PLCbeta1 expression, as compared with the amount of the pre-treatment period, thus confirming the involvement of this molecule in the response to demethylating agents. As for the remaining patients, mainly showing a stable disease, we observed slight increases or almost constant levels of PI-PLCbeta1 expression. Moreover, ongoing analyses are trying to disclose whether lower-risk MDS patients responding to azacitidine show a specific molecular epigenetic profile during the regulation of methylation processes. Taken together, our data suggest a correlation between azacitidine treatment and PI-PLCbeta1 signalling even in lower-risk MDS, thus hinting at a role for PI-PLCbeta1 in the evaluation of patients likely to respond to azacitidine and paving the way for the development of innovative therapeutic strategies in lower-risk MDS patients. Disclosures: No relevant conflicts of interest to declare.

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Sequential Acquisition of AML1-ETO and c-Kit Mutation Leads to Formation of Human t(8;21) Acute Myelogenous Leukemia Stem Cells,

Blood ◽

10.1182/blood.v118.21.3584.3584 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3584-3584

Author(s):

Takahiro Shima ◽

Yoshikane Kikushige ◽

Toshihiro Miyamoto ◽

Koichi Akashi

Keyword(s):

Stem Cells ◽

Acute Myelogenous Leukemia ◽

Conflicts Of Interest ◽

Critical Role ◽

Myelogenous Leukemia ◽

Target Cells ◽

Hematopoietic Stem ◽

Kit Mutation ◽

Acute Myelogenous

Abstract Abstract 3584 The 8;21 translocation, one of the most general chromosomal abnormalities in acute myelogenous leukemia (AML), encodes the AML1-ETO chimeric fusion gene. Because AML1-ETO can inhibit the CBF complex to transactivate myeloid-lineage genes in a dominant negative fashion, the high level expression of this gene plays a critical role in inhibiting differentiation of target cells, which leads to progression of AML. We, however, have reported that patients maintaining a long-term remission retain AML1-ETO expression at a very low level that can be detected by nested RT-PCR. The AML1-ETO transcripts in these patients were derived from a small fraction of t(8;21)+ hematopoietic stem cells (HSCs) capable of multilineage differentiation (PNAS 2000). In fact, previous data shown that AML1/ETO knock-in or AML1/ETO transgenic mice did not develop AML. These data suggest that acquisition of the AML1-ETO fusion is not sufficient to develop t(8;21) AML. Since t(8;21) AML cells frequently possess constitutive active mutation of c-Kit, we hypothesized that the c-Kit mutation may work as a second oncogenic hit in t(8;21)+ HSCs to transform into AML. To test the hypothesis, we extensively analyzed the existence of c-Kit mutation within AML1-ETO+ HSCs from patients maintaining remission for a long-term. CD34+CD38− HSCs were purified from the bone marrow of patients in long-term remission, and were cultured in vitro to form colonies. These HSC-derived colonies were picked up, and tested for the presence ofAML1-ETO and c-Kit mutation. Five t(8;21) AML patients with c-Kit mutation were enrolled in this study. All of 1020 blastic colonies at diagnosis were positive for both AML1-ETO and c-Kit mutation. In 7187 colonies formed in the culture of remission marrow, almost 1% (89 colonies) of these colonies expressed AML1-ETO. Surprisingly, none of these colonies possessed c-Kit mutation, indicating that AML1-ETO+ clones in remission are not identical to these in t(8;21) AML. Accordingly, it is highly likely that HSCs first acquire AML1-ETO, and a fraction of these cells additionally mutated c-Kit, resulting in transformation into AML stem cells. This is the first clear-cut evidence that human HSCs transform into AML via multi-step oncogenesis in vivo. Disclosures: No relevant conflicts of interest to declare.

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Efficiency of Leukemic Stem Cell Separation From Patients with Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v118.21.4997.4997 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4997-4997

Author(s):

Lu Zhang ◽

Susanne Hofmann ◽

Lars Bullinger ◽

Marlies Goetz ◽

Markus Wiesneth ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Cell Sorting ◽

Peripheral Blood ◽

Recovery Rate ◽

Mononuclear Cells ◽

Separation Efficiency ◽

Conflicts Of Interest ◽

Cell Number ◽

Hematopoietic Stem

Abstract Abstract 4997 Leukemic stem cells (LSC) are the source for leukemic disease self-renewal and account for disease relapse after treatment. Therefore LSCs probably represent a critical target for therapeutic options. Xenograft models confirmed repeatedly that LSCs from AML patients reside mainly in CD34+CD38- compartment of leukemic blasts which makes the pure and efficient separation of this population mandatory to identify new therapeutic drugs to target LSC in different AML subtypes. We separated this subpopulation out of primary AML peripheral blood mononuclear cells (PBMC) samples with fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) and compared the efficiency of both methods. In order to profile gene expression of LSCs and hematopoietic stem cells (HSC) MicroArrays were performed using GeneChip Human Genome U133 Plus 2.0 from Affymetrix. The CD34+CD38- subpopulation was separated from PBMCs of 12 AML patients and 5 healthy volunteers using FACS. Concerning the 12 primary AML samples, the ratio of CD34+CD38- cells ranges between 0.79% and 86.2% using 1–5×107 PBMC for separation. After sorting, the purity of those AML samples increased to 88.4–98.4% while 2×104-3.6×106 cells were obtained. MACS was used to separate 2 representative samples, in which the CD34+CD38- subpopulation was rather small (sample1: 0.78%) or large (sample2: 86.1%). Those sorted subpopulations were compared to the samples sorted via FACS. In order to evaluate separation efficiency in a standardized manner, we defined the recovery rate: (CD34+CD38- cell number obtained /total CD34+CD38- cell number) × 100%. The total CD34+CD38- cell number was calculated through a pre-sorting FACS analysis. For sample 1, MACS resulted in a recovery rate of 4.2–6.4% with a purity of 86.6–90.3%, which is inferior to the recovery rate of 17% and the purity of 92.1% using FACS. For Sample 2, MACS resulted in a recovery rate of 0.4% with a purity of 98.8%, compared to the recovery rate of 11.6% with a purity of 98.1% by FACS. Comparing both methods it is obvious that the purity doesn't differ a lot, but the yield is much higher using FACS. This could represent a powerful tool, when managing rare samples. Finally, by comparing purity and yield, we showed that FACS is the adequate separation method. At the moment MicroArrays are being performed in order to investigate the gene expression profile for 12–15 AML patients and 5 HVs. Taken together, we showed a widely efficient method to routinely separate LSCs from patients with different subtypes of AML. Microarrays, that have been performed, represent a method that allows the comparison of the characteristics of LSCs in different AML subtypes and also of LSCs from bone-marrow with LSCs from peripheral blood and with HVs. These array data analyses are ongoing and will be presented. Disclosures: No relevant conflicts of interest to declare.

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Favorable Outcome of Children with Acute Megakaryoblastic Leukemia Treated with the AIEOP AML 2002/01 Protocol

Blood ◽

10.1182/blood.v120.21.3586.3586 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3586-3586

Author(s):

Franco Locatelli ◽

Maria Caterina Putti ◽

Andrea Pession ◽

Franca Fagioli ◽

Giuseppe Menna ◽

...

Keyword(s):

Cumulative Incidence ◽

Conflicts Of Interest ◽

White Blood Cells ◽

Favorable Outcome ◽

Induction Treatment ◽

Published Data ◽

High Dose ◽

Acute Megakaryoblastic Leukemia ◽

Megakaryoblastic Leukemia ◽

Excellent Outcome

Abstract Abstract 3586 Introduction: Acute megakaryoblastic leukemia (AMKL) is a biologically heterogeneous variant of acute myeloid leukemia (AML), known as AML-M7 according to the FAB classification. The incidence of AML-M7 among pediatric AML in previously published studies ranged between 7 and 15% of the whole number. Differently from children with Down Syndrome (DS)-AMKL who have an excellent outcome with specifically designed, low-intensity chemotherapy, some studies have reported that the outcome of children with non-DS-AMKL is poor (Ribeiro et al, 1993; Athale et al, 2001). We analyzed the clinical/biological characteristics and outcome of children with non-DS-AMKL treated with the AIEOP AML 2002/01 Protocol. Patients and Methods: Between 12/2002 and 06/2011, 44 patients (pts) were included in the analysis. They were assigned to the high-risk (HR) group. Treatment was administered as follows: pts were given 2 courses of 7-day induction therapy (idarubicin, cytarabine and etoposide); if they obtained first complete remission (CR1), they proceeded to receive 2 consolidation courses based on high-dose cytarabine (HD-Ara-c), combined with either etoposide during the first course (AVE 3+4) or mitoxantrone during the second course (HAM 3+2). At the end of consolidation, pts with AMKL in CR1 were eligible to be treated with allogeneic (ALLO) HSCT from a HLA-identical relative, if available, or from alternative donors, namely unrelated donors or HLA-mismatched relatives. We analyzed CR, early death (ED) and induction failure (IF) rate of pts with AMKL; moreover, we calculated the cumulative incidence of relapse and the probability of both overall survival (OS) and event-free survival (EFS). Results: AMKL was diagnosed in 44 of 482 (9.1%) children and adolescents below 18 years of age without DS enrolled in the AIEOP AML 2002/01 protocol. Twenty-one pts were males and 23 females. Median age at diagnosis was 1.6 years (range 0.3–15), with 12/44 (27%) pts aged < 1 year. The median count of white blood cells (WBC) at diagnosis was 18,910×103/ml (range 2,490–59,000). Involvement of central nervous system (CNS) at diagnosis was documented in 5/44 (11%), whereas extramedullary disease in sites other than CNS was found in 3/44 pts (7%). Cytogenetic results were available in 34/44 (77%) pts; they showed t(1;22)(p13;q13) in 3/34 (9%) pts, either as sole abnormality or together with other numerical and structural changes and 11q23 rearrangements in 4/34 (12%). Complex karyotype was present in 5/34 (15%) pts. Three out of 44 pts (7%) showed FLT3-ITD mutation. The induction treatment outcome of pts with AMKL was as follows: the CR rate was 86%, while ED and IF rates were 5% and 9%, respectively. The cumulative incidence of leukemia recurrence was 27% [standard error (SE) 8.7]. Thirty out of 44 pts with AMKL (68%) received an ALLO HSCT in first CR. With a median follow up of 57 months (range 3–130), the 8-year OS and EFS of AMKL were 58.4% (SE 7.9) and 55.6% (SE 7.7), respectively, these values being similar to those of children with other variants of AML. Conclusions: Although AMKL is generally considered an AML variant characterized by a poor prognosis, our data indicate that an intensive treatment strategy including a wide use of HSCT in first CR results in a favorable outcome, more than 50% of pts being alive and disease-free. The main cause of treatment failure remains leukemia recurrence; once relapsed, children with AMKL have a dismal probability of being rescued by second-line treatment. Overall, our results compare favorably with previously published data on AMKL, including those recently reported by Rubnitz et al (Lancet Oncol 2010;11:543–52). Disclosures: No relevant conflicts of interest to declare.

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Disease-Relevant Expression Of TRAF6 In Mouse HSC Results In An MDS-Like Disease

Blood ◽

10.1182/blood.v122.21.101.101 ◽

2013 ◽

Vol 122 (21) ◽

pp. 101-101

Author(s):

Jing Fang ◽

Xiaona Liu ◽

Brenden Barker ◽

Lyndsey Bolanos ◽

Yue Wei ◽

...

Keyword(s):

Gene Expression ◽

Conflicts Of Interest ◽

Early Stage ◽

Bone Marrow Failure ◽

Gene Signature ◽

Sublethal Dose ◽

Hematopoietic Tissue ◽

Hematopoietic Stem ◽

Disease Evolution ◽

Cd34 Cells

Abstract Overexpression of immune-related genes is widely reported in Myelodysplastic Syndrome (MDS), and chronic immune stimulation increases the risk for developing MDS. We find that TNF receptor associated factor 6 (TRAF6), an innate immune protein, is overexpressed approximately 2-fold in CD34+ cells from 40% of MDS patients, and may explain immune pathway activation in the MDS-initiating hematopoietic stem/progenitor cell (HSPC). In support of these observations and our hypothesis that TRAF6 is important in the pathophysiology of MDS, a gene expression analysis revealed that TRAF6 controls an MDS gene signature in human cells. We, and others, have previously shown that retroviral overexpression of TRAF6 in mouse HSPC results in MDS and Acute Myeloid Leukemia (AML). However, interpretations of these findings are hampered by supra-physiological levels of TRAF6 (>10-fold overexpression) and the stress associated with HSPC transduction/transplantation. To investigate the consequences of TRAF6 overexpression to MDS, we generated a transgenic mouse model overexpressing TRAF6 from a hematopoietic-specific Vav promoter. Expression of TRAF6 in HSPC was approximately 2-fold higher as compared to endogenous TRAF6 and in line with MDS patient CD34+ cells. By 15 months of age, half of Vav-TRAF6 mice succumbed to a hematologic disease resembling MDS associated with bone marrow failure (BMF). In contrast to the retroviral overexpression approach, Vav-TRAF6 mice did not develop AML. Examination of sick mice revealed stage-specific disease evolution. Initially, all Vav-TRAF6 mice exhibit an inversion of myeloid/lymphoid proportions. For Vav-TRAF6 mice that develop a fatal disease, they present with a hypocellular marrow, dysplasic myeloid cells, and neutropenia. A subset of mice also display anemia with nucleated red blood cells, poikilocytosis, and extramedullular erythropoiesis. In support of a BMF phenotype, HSPC from Vav-TRAF6 mice form fewer colonies in methylcellulose. To investigate the consequences of an acute exposure to pathogen, early-stage Vav-TRAF6 mice were treated with a single sublethal dose of lipopolysaccharide (LPS). Unlike wild-type (WT) mice, Vav-TRAF6 mice developed a rapid and reversible anemia, suggesting environmental factors can influence the severity of the disease. To gain insight into the mechanism contributing to BMF, gene expression profiling was performed in WT and Vav-TRAF6 HSPC. One of the enriched pathways consisted of AKT activation and FOXO downregulation. Consistent with the microarray analysis, AKT is constitutively phosphorylated at Thr308 in hematopoietic tissue from Vav-TRAF6 mice. SOD2, a transcriptional target of FoxO3a that is suppressed by activated AKT, is decreased in Vav-TRAF6 HSPC. Given that AKT/FOXO regulate reactive oxygen species (ROS) in cells, we investigated ROS levels in HPSC from Vav-TRAF6 and WT mice. Intracellular ROS is significantly elevated in BM cells from Vav-TRAF6 mice, and restored to normal levels when AKT was inhibited. In conclusion, we propose the potential role of TRAF6 in the development of MDS-associated BMF, partly due to constitutive activation of AKT and subsequent ROS elevation in HSPC cells. Disclosures: No relevant conflicts of interest to declare.

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Large Conserved Domains Of Low DNA Methylation Maintained By 5-Hydroxymethycytosine and Dnmt3a

Blood ◽

10.1182/blood.v122.21.2406.2406 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2406-2406

Author(s):

Mira Jeong ◽

Deqiang Sun ◽

Min Luo ◽

Yun Huang ◽

Myunggon Ko ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Hox Genes ◽

Conflicts Of Interest ◽

P Value ◽

Genomic Feature ◽

Conserved Domains ◽

Hematopoietic Stem ◽

Genome Wide ◽

The Impact

Abstract Identification of recurrent leukemia-associated mutations in genes encoding regulators of DNA methylation such as DNMT3A and TET2 have underscored the critical importance of DNA methylation in maintenance of normal physiology. To gain insight into how DNA methylation exerts the central role, we sought to determine the genome-wide pattern of DNA methylation in the normal precursors of leukemia cells: the hematopoietic stem cell (HSC), and investigate the factors that affect alterations in DNA methylation and gene expression. We performed whole genome bisulfite sequencing (WGBS) on purified murine HSCs achieving a total of 1,121M reads, resulting in a combined average of 40X coverage. Using Hidden Markov Model we identified 32,325 under-methylated regions (UMRs) with average proportion of methylation ≤ 10% and by inspecting the UMR size distribution, we discovered exceptionally large “methylation Canyons” which span highly conserved domains frequently containing transcription factors and are quite distinct from CpG islands and shores. Methylation Canyons are a distinct genomic feature that is stable, albeit with subtle differences, across cell-types and species. Canyon-associated genes showed a striking pattern of enrichment for genes involved in transcriptional regulation (318 genes, P=6.2 x 10-123), as well as genes containing a homeobox domain (111 genes, P=3.9 x 10-85). We compared Canyons with TF binding sites as identified from more than 150 ChIP-seq data sets across a variety of blood lineages (>10)19 and found that TF binding peaks for 10 HSC pluripotency TFs are significantly enriched in entirety of Canyons compared with their surrounding regions. Low DNA methylation is usually associated with active gene expression. However, half of Canyon genes associated with H3K27me3 showed low or no expression regardless of their H3K4me3 association while H3K4me3-only Canyon genes were highly expressed. Because DNMT3A is mutated in a high frequency of human leukemias24, we examined the impact of loss of Dnmt3a on Canyon size. Upon knockout of Dnmt3a, the edges of the Canyons are hotspots of differential methylation while regions inside of Canyon are relatively resistant. The methylation loss in Dnmt3a KO HSCs led Canyon edge erosion, Canyon size expansion and addition of 861 new Canyons for a total of 1787 Canyons. Canyons marked with H3K4me3 only were most likely to expand after Dnmt3a KO and the canyons marked only with H3K27me3 or with both marks were more likely to contract. This suggests Dnmt3a specifically is acting to restrain Canyon size where active histone marks (and active transcription) are already present. WGBS cannot distinguish between 5mC and 5hmC, so we determined the genome-wide distribution of 5hmC in WT and Dnmt3a KO HSCs using the cytosine-5-methylenesulphonate (CMS)-Seq method in which sodium bisulfate treatment convert 5hmC to CMS; CMS-containing DNA fragments are then immunoprecipitated using a CMS specific antiserum. Strikingly, 5hmC peaks were enriched specifically at the borders of Canyons. In particular, expanding Canyons, typically associated with highest H3K4me3 marking, were highly enriched at the edges for the 5hmC signal suggesting a model in which Tet proteins and Dnmt3a act concomitantly on Canyon borders opposing each other in alternately effacing and restoring methylation at the edges, particularly at sites of active chromatin marks. Using Oncomine data, we tested whether Canyon-associated genes were likely to be associated with hematologic malignancy development and found Canyon genes were highly enriched in seven signatures of genes over-expressed in Leukemia patients compared to normal bone marrow; in contrast, four sets of control genes were not similarly enriched. Further using TCGA data, we found that expressed canyon genes are significantly enriched for differentially expressed genes between patients with and without DNMT3A mutation (p value<0.05) Overall, 76 expressed canyon genes, including multiple HOX genes, are significantly changed in patients with DNMT3A mutation (p=0.0031). Methylation Canyons, the novel epigenetic landscape we describe may provide a mechanism for the regulation of hematopoiesis and may contribute to leukemia development. Disclosures: No relevant conflicts of interest to declare.

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Lgr4-Mediated Potentiation Of Wnt/β-Catenin Signaling Promotes MLL Leukemogenesis Via An Rspo3/Wnt3a-Gnaq Pathway In Leukemic Stem Cells

Blood ◽

10.1182/blood.v122.21.887.887 ◽

2013 ◽

Vol 122 (21) ◽

pp. 887-887 ◽

Cited By ~ 3

Author(s):

Hangyu Yi ◽

Jianlong Wang ◽

Maria Kavallaris ◽

Jenny Yingzi Wang

Keyword(s):

Gene Expression ◽

Stem Cells ◽

G Protein ◽

Colony Formation ◽

Conflicts Of Interest ◽

Active Form ◽

Leukemic Stem Cells ◽

Western Blots ◽

Hematopoietic Stem

Abstract Although the clinical importance of aberrant Wnt/β-catenin signaling has been recognized in various cancers, including MLL-rearranged acute myeloid leukemia (MLL AML), its key tractable pathway components have not yet been discovered in leukemic stem cells (LSC). Our studies have identified an Rspo3/Wnt3a-Lgr4-Gnaq pathway, which significantly potentiates β-catenin signaling in MLL LSC. Genetic and pharmacological targeting of this pathway impairs LSC self-renewal and survival, inhibiting MLL-AF9-induced leukemia progression in vivo. Gene expression analysis of AML patient samples (Nucleic Acids Res, 41:D1034-9, 2013) revealed an approximately 3-fold increase (p=0.00002) in expression of leucine-rich repeat-containing G protein-coupled receptor 4 (Lgr4) in leukemic cells from patients with MLL AML compared to normal human hematopoietic stem cells (HSC). As recent studies have highlighted a critical link between R-spondin (Rspo)/Lgr4 and Wnt/β-catenin signaling pathways, we hypothesized that up-regulation of Lgr4 is associated with aberrant activation of β-catenin signaling in MLL LSC. We have previously demonstrated that β-catenin is highly expressed in HSC transformed by MLL-AF9 and is lower in HSC transduced with leukemic oncogenes such as Hoxa9/Meis1, while increased β-catenin expression is correlated with a poor survival rate in mice. In this study, western blots confirmed high levels of Lgr4 expression in HSC expressing MLL-AF9 compared to Hoxa9/Meis1. ShRNA-mediated stable knockdown of Lgr4 markedly reduced colony formation of HSC expressing MLL-AF9 by 55-65% (p=0.0001) and significantly prolonged mouse survival (p=0.0019) through its inhibition of endogenous β-catenin expression. This deficient phenotype could be rescued by expression of a constitutively active form of β-catenin. Furthermore, ectopic expression of Lgr4 alone was not sufficient for triggering the leukemic transformation of HSC but conferred a growth advantage in vivo to HSC expressing Hoxa9/Meis1 and significantly accelerated the onset of Hoxa9/Meis1-induced AML in mice (p=0.0011). These data support an oncogenic role of Lgr4 in promoting tumor formation through activation of β-catenin signaling. As Lgr4 has recently been identified as a receptor for the Rspo family of secreted proteins (Rspo1–Rspo4), we sought to determine if Rspo is a positive regulator of β-catenin signaling in MLL AML. We found that only the combination of Rspo3 and Wnt3a potently enhanced β-catenin signaling in HSC expressing MLL-AF9 whereas Rspo and Wnt3a alone or the combination of Wnt3a with other Rspo had no effects on β-catenin activity. Depletion of Lgr4 completely abolished Rspo3/Wnt3a-induced β-catenin signaling, suggesting Rspo3/Wnt3a potentiating β-catenin signaling through Lgr4. Next, we assessed if Lgr4 signals through G protein pathways. By testing G protein alpha inhibitors in MLL LSC, we demonstrated that G protein alpha-q (Gnaq) was required for maintenance of stem cell properties by chemical suppression of the Gnaq-activated β-catenin pathway with a Gnaq selective inhibitor, which exhibited a 3-fold decrease in colony formation (p=0.0001) and a 4-fold reduction in cell number (p=0.0009), and was sufficient to induce substantial cell differentiation and apoptosis. Treatment with Gnaq inhibitor abolished the effect of Lgr4 on β-catenin transactivation, implicating an Lgr4-Gnaq-β-catenin signaling pathway in MLL LSC. Microarray analysis of gene expression confirmed enrichment of genes related to cancer cell proliferation, migration and growth, as well as enrichment of Wnt target genes in LSC expressing Lgr4. Taken together, we report here an Rspo3/Wnt3a-Lgr4-Gnaq-β-catenin signaling circuit in MLL leukemogenesis. Interference with components of the circuit can block β-catenin signaling and perturb leukemia development. Thus, our findings provide potential therapeutic targets in treating LSC-based hematological malignancy driven by Wnt/β-catenin signaling. Disclosures: No relevant conflicts of interest to declare.

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