Leukemia Fusion Research Articles

Development Refractoriness of MLL-Rearranged Human B Cell Acute Leukemias to Reprogramming into Pluripotency

SummaryInduced pluripotent stem cells (iPSCs) are a powerful tool for disease modeling. They are routinely generated from healthy donors and patients from multiple cell types at different developmental stages. However, reprogramming leukemias is an extremely inefficient process. Few studies generated iPSCs from primary chronic myeloid leukemias, but iPSC generation from acute myeloid or lymphoid leukemias (ALL) has not been achieved. We attempted to generate iPSCs from different subtypes of B-ALL to address the developmental impact of leukemic fusion genes. OKSM(L)-expressing mono/polycistronic-, retroviral/lentiviral/episomal-, and Sendai virus vector-based reprogramming strategies failed to render iPSCs in vitro and in vivo. Addition of transcriptomic-epigenetic reprogramming “boosters” also failed to generate iPSCs from B cell blasts and B-ALL lines, and when iPSCs emerged they lacked leukemic fusion genes, demonstrating non-leukemic myeloid origin. Conversely, MLL-AF4-overexpressing hematopoietic stem cells/B progenitors were successfully reprogrammed, indicating that B cell origin and leukemic fusion gene were not reprogramming barriers. Global transcriptome/DNA methylome profiling suggested a developmental/differentiation refractoriness of MLL-rearranged B-ALL to reprogramming into pluripotency.

CATS (FAM64A) abnormal expression reduces clonogenicity of hematopoietic cells.

The CATS (FAM64A) protein interacts with CALM (PICALM) and the leukemic fusion protein CALM/AF10. CATS is highly expressed in leukemia, lymphoma and tumor cell lines and its protein levels strongly correlates with cellular proliferation in both malignant and normal cells. In order to obtain further insight into CATS function we performed an extensive analysis of CATS expression during differentiation of leukemia cell lines. While CATS expression decreased during erythroid, megakaryocytic and monocytic differentiation, a markedly increase was observed in the ATRA induced granulocytic differentiation. Lentivirus mediated silencing of CATS in U937 cell line resulted in somewhat reduced proliferation, altered cell cycle progression and lower migratory ability in vitro; however was not sufficient to inhibit tumor growth in xenotransplant model. Of note, CATS knockdown resulted in reduced clonogenicity of CATS-silenced cells and reduced expression of the self-renewal gene, GLI-1. Moreover, retroviral mediated overexpression of the murine Cats in primary bone marrow cells lead to decreased colony formation. Although our in vitro data suggests that CATS play a role in cellular processes important for tumorigenesis, such as cell cycle control and clonogenicity, these effects were not observed in vivo.

Discovery of Novel Inhibitors Targeting the Menin-Mixed Lineage Leukemia Interface Using Pharmacophore- and Docking-Based Virtual Screening.

Disrupting the interaction between mixed lineage leukemia (MLL) fusion protein and menin provides a therapeutic approach for MLL-mediated leukemia. Here, we aim to discover novel inhibitors targeting the menin-MLL interface with virtual screening. Both structure-based molecular docking and ligand-based pharmacophore models were established, and the models used for compound screening show a remarkable ability to retrieve known active ligands from decoy molecules. Verified by a fluorescence polarization assay, five hits with novel scaffolds were identified. Among them, DCZ_M123 exhibited potent inhibitory activity with an IC50 of 4.71 ± 0.12 μM and a KD of 14.70 ± 2.13 μM, and it can effectively inhibit the human MLL-rearranged leukemia cells MV4;11 and KOPN8 with GI50 values of 0.84 μM and 0.54 μM, respectively.

New insights into transcriptional and leukemogenic mechanisms of AML1-ETO and E2A fusion proteins.

Nearly 15% of acute myeloid leukemia (AML) cases are caused by aberrant expression of AML1-ETO, a fusion protein generated by the t(8;21) chromosomal translocation. Since its discovery, AML1-ETO has served as a prototype to understand how leukemia fusion proteins deregulate transcription to promote leukemogenesis. Another leukemia fusion protein, E2A-Pbx1, generated by the t(1;19) translocation, is involved in acute lymphoblastic leukemias (ALLs). While AML1-ETO and E2A-Pbx1 are structurally unrelated fusion proteins, we have recently shown that a common axis, the ETO/E-protein interaction, is involved in the regulation of both fusion proteins, underscoring the importance of studying protein-protein interactions in elucidating the mechanisms of leukemia fusion proteins. In this review, we aim to summarize these new developments while also providing a historic overview of the related early studies. A total of 218 publications were reviewed in this article, a majority of which were published after 2004.We also downloaded 3D structures of AML1-ETO domains from Protein Data Bank and provided a systematic summary of their structures. By reviewing the literature, we summarized early and recent findings on AML1-ETO, including its protein-protein interactions, transcriptional and leukemogenic mechanisms, as well as the recently reported involvement of ETO family corepressors in regulating the function of E2A-Pbx1. While the recent development in genomic and structural studies has clearly demonstrated that the fusion proteins function by directly regulating transcription, a further understanding of the underlying mechanisms, including crosstalk with other transcription factors and cofactors, and the protein-protein interactions in the context of native proteins, may be necessary for the development of highly targeted drugs for leukemia therapy.

S6K1 regulates hematopoietic stem cell self-renewal and leukemia maintenance

Hyperactivation of the mTOR pathway impairs hematopoietic stem cell (HSC) functions and promotes leukemogenesis. mTORC1 and mTORC2 differentially control normal and leukemic stem cell functions. mTORC1 regulates p70 ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding (eIF4E-binding) protein 1 (4E-BP1), and mTORC2 modulates AKT activation. Given the extensive crosstalk that occurs between mTORC1 and mTORC2 signaling pathways, we assessed the role of the mTORC1 substrate S6K1 in the regulation of both normal HSC functions and in leukemogenesis driven by the mixed lineage leukemia (MLL) fusion oncogene MLL-AF9. We demonstrated that S6K1 deficiency impairs self-renewal of murine HSCs by reducing p21 expression. Loss of S6K1 also improved survival in mice transplanted with MLL-AF9-positive leukemic stem cells by modulating AKT and 4E-BP1 phosphorylation. Taken together, these results suggest that S6K1 acts through multiple targets of the mTOR pathway to promote self-renewal and leukemia progression. Given the recent interest in S6K1 as a potential therapeutic target in cancer, our results further support targeting this molecule as a potential strategy for treatment of myeloid malignancies.

Deciphering “B-others”: Novel fusion genes driving B-cell acute lymphoblastic leukemia

Deciphering “B-others”: Novel fusion genes driving B-cell acute lymphoblastic leukemia

Correction: RUNX1B Expression Is Highly Heterogeneous and Distinguishes Megakaryocytic and Erythroid Lineage Fate in Adult Mouse Hematopoiesis

[This corrects the article DOI: 10.1371/journal.pgen.1005814.].

Context matters in MLL-AF9–driven leukemias

In this issue of Blood , Man et al demonstrate that the transforming activity of the leukemic fusion oncogene MLL-AF9 is regulated by inhibitor of DNA binding 1 (Id1), a dominant-negative regulator of E protein transcription factors. 1

T(8;21) Acute Myeloid Leukaemia Requires Active CCND-CDK4/6

Introduction: The leukaemic fusion oncoprotein RUNX1/ETO, which results from the t(8;21) translocation, establishes a novel transcriptional programme maintaining leukaemia.

The AS-RBM15 lncRNA enhances RBM15 protein translation during megakaryocyte differentiation.

Antisense RNAs regulate the transcription and translation of the corresponding sense genes. Here, we report that an antisense RNA, AS-RBM15, is transcribed in the opposite direction within exon 1 of RBM15 RBM15 is a regulator of megakaryocyte (MK) differentiation and is also involved in a chromosome translocation t(1;22) in acute megakaryocytic leukemia. MK terminal differentiation is enhanced by up-regulation of AS-RBM15 expression and attenuated by AS-RBM15 knockdown. At the molecular level, AS-RBM15 enhances RBM15 protein translation in a CAP-dependent manner. The region of the antisense AS-RBM15 RNA, which overlaps with the 5'UTR of RBM15, is sufficient for the up-regulation of RBM15 protein translation. In addition, we find that transcription of both RBM15 and AS-RBM15 is activated by the transcription factor RUNX1 and repressed by RUNX1-ETO, a leukemic fusion protein. Therefore, AS-RBM15 is a regulator of megakaryocyte differentiation and may play a regulatory role in leukemogenesis.

RUNX1B Expression Is Highly Heterogeneous and Distinguishes Megakaryocytic and Erythroid Lineage Fate in Adult Mouse Hematopoiesis.

The Core Binding Factor (CBF) protein RUNX1 is a master regulator of definitive hematopoiesis, crucial for hematopoietic stem cell (HSC) emergence during ontogeny. RUNX1 also plays vital roles in adult mice, in regulating the correct specification of numerous blood lineages. Akin to the other mammalian Runx genes, Runx1 has two promoters P1 (distal) and P2 (proximal) which generate distinct protein isoforms. The activities and specific relevance of these two promoters in adult hematopoiesis remain to be fully elucidated. Utilizing a dual reporter mouse model we demonstrate that the distal P1 promoter is broadly active in adult hematopoietic stem and progenitor cell (HSPC) populations. By contrast the activity of the proximal P2 promoter is more restricted and its upregulation, in both the immature Lineage- Sca1high cKithigh (LSK) and bipotential Pre-Megakaryocytic/Erythroid Progenitor (PreMegE) populations, coincides with a loss of erythroid (Ery) specification. Accordingly the PreMegE population can be prospectively separated into “pro-erythroid” and “pro-megakaryocyte” populations based on Runx1 P2 activity. Comparative gene expression analyses between Runx1 P2+ and P2- populations indicated that levels of CD34 expression could substitute for P2 activity to distinguish these two cell populations in wild type (WT) bone marrow (BM). Prospective isolation of these two populations will enable the further investigation of molecular mechanisms involved in megakaryocytic/erythroid (Mk/Ery) cell fate decisions. Having characterized the extensive activity of P1, we utilized a P1-GFP homozygous mouse model to analyze the impact of the complete absence of Runx1 P1 expression in adult mice and observed strong defects in the T cell lineage. Finally, we investigated how the leukemic fusion protein AML1-ETO9a might influence Runx1 promoter usage. Short-term AML1-ETO9a induction in BM resulted in preferential P2 upregulation, suggesting its expression may be important to establish a pre-leukemic environment.

CD34-Negative is Highly Associated with T (15;17), T (V; 11q23) and the NPM1-Mutation Subtypes in 343 Newly Diagnosed Patients with Acute Myeloid Leukemia

Objective: Recent reports found that several cytogenetic or molecular subtypes of acute myeloid leukemia (AML) are associated with CD34-positive. However, the status of CD34-negative in AML needs to be explored. In this study, we aimed to explore the prevalence of the CD34-negative patients and its association with molecular genetics status in a large consecutive AML cohort. Methods: A group of 343 consecutive newly diagnosed AML patients was retrospectively analyzed in our center. CD34 expression was detected by flow cytometry and considered negative when it was expressed in less than 20% of the bone marrow blast cells. The karyotype was analyzed by the G-banding technique. Leukemic fusion genes and mutated genes were detected by PCR method. Results: CD34-negative was found in 143 (41.7%) of the 343 patients. According to FAB classification, the percentages of CD34-negative patients were higher in the M3 and M5 (100% and 70%, respectively) and lower in the M2 and M4 subtypes (30.3% and 21.2%, respectively). According to the WHO classification, the percentage of CD34-negative patients was higher in those with t(15; 17), t(v; 11q23), and the NPM1-mutation (100%, n=37; 100%, n=7; and 81.7%, n=71, respectively) and lower in those with t(8;21) and AML with MDS-related changes (8.6%, n=35 and 5.0%, n=20, respectively). The patients with t(15; 17), t(v; 11q23) and the NPM1-mutation consisted of 71.3% (102/143) of the CD34-negative population and 6.5% (13/200) of the CD34-positive population (p<0.0001). A CD34-negative phenotype was associated with risk subgroups according to cytogenetics alone and when combining cytogenetics and molecular analysis (p=0.025 and p<0.0001, respectively). The sensitivity, specificity, positivepredictive value and negative-predictive value of the CD34-negative to t (15;17), t (v; 11q23) and the NPM1-mutation were 88.7%, 82.0%, 71.3% and 93.5%, respectively. Conclusions: The prevalence of the CD34-negative patients is very common in newly diagnosed AML. CD34- negative is highly associated with t (15;17), t(v; 11q23) and the NPM1-mutation in AML patients, which provide the evidence about the association of immunophenotype and molecular genetics.

Systematic Classification of Mixed-Lineage Leukemia Fusion Partners Predicts Additional Cancer Pathways.

Chromosomal translocations of the human mixed-lineage leukemia (MLL) gene have been analyzed for more than 20 yr at the molecular level. So far, we have collected about 80 direct MLL fusions (MLL-X alleles) and about 120 reciprocal MLL fusions (X-MLL alleles). The reason for the higher amount of reciprocal MLL fusions is that the excess is caused by 3-way translocations with known direct fusion partners. This review is aiming to propose a solution for an obvious problem, namely why so many and completely different MLL fusion alleles are always leading to the same leukemia phenotypes (ALL, AML, or MLL). This review is aiming to explain the molecular consequences of MLL translocations, and secondly, the contribution of the different fusion partners. A new hypothesis will be posed that can be used for future research, aiming to find new avenues for the treatment of this particular leukemia entity.

Leukaemia: Breaking tradition with PARP inhibition.

Espositoet al. report the mechanisms of PARP inhibitor (PARPi) sensitivity in cells expressing certain leukaemia fusion oncoproteins and highlight the use of PARPis as a promising novel approach for treating acute myeloid leukaemia.

The Molecular Mechanism of Transcriptional Activation By MLL-AEP Fusion Proteins

Chromosomal translocations generate a variety of mixed lineage leukemia (MLL) fusion genes, which cause aggressive leukemia. Although >70 different fusion partners have been identified, the majority of the cases are caused by the chimeric genes of MLL and a component of the AEP co-activator complex (hereafter referred to as AEP), which comprises of AF4 family proteins (e.g. AF4, AF5Q31), ENL family proteins (e.g. ENL, AF9), and the P-TEFb elongation factor. MLL-AEP fusion proteins constitutively activate their target genes by recruiting AEP components to their target chromatin, whereas wild-type MLL recruits AEP in a context-dependent manner. In the hematopoietic lineage, MLL fusion proteins aberrantly activate a subset of genes implicated in the hematopoietic stem cell (HSC) program, such as HOXA9 and MEIS1. Constitutive expression of these HSC program genes in hematopoietic progenitors has been shown to induce leukemia in a mouse model. It has been speculated that MLL-AEP activates transcription of those HSC program genes by aberrantly activating transcription elongation. However, it is largely unclear how AEP activates transcription.Using an extensive structure/function analysis, we revealed that a serine-rich domain of the AF4 family proteins, termed pSER, is an essential functional component of MLL-AEP-dependent gene activation and leukemic transformation. Through biochemical purification, we have identified Selectivity Factor 1 (SL1) as a novel factor associated with the pSER domain. SL1 comprises TBP and four TBP-associated factors (TAF1A, TAF1B, TAF1C, TAF1D), and is known as a core component of the pre-initiation complex (PIC) of RNA polymerase I (RNAP1). In the presence of UBF, SL1 forms a PIC on the promoters of ribosomal RNA genes, to drive RNAP1-dependent transcription. However, its role in RNAP2-dependent transcription was unknown. The initiation of RNAP2-dependent transcription in eukaryotes occurs through the loading of TBP to the promoter, via a direct association with the TATA element or through as-yet-unidentified mechanisms. Our results demonstrate that AEP facilitates the initiation of RNAP2-dependent transcription via the loading of TBP onto the TATA element, through SL1 activity. MLL-AEP fusion proteins utilize this TBP-loading function to activate transcription initiation in leukemic transformation. The wild-type AEP complex activates gene expression in the same manner in the physiological conditions. Taken together, our results unveil a novel role of SL1 as a TBP-loading factor in RNAP2-dependent gene activation, and a previously unknown transcription initiation mechanism involving AEP, which is more important than its transcription elongation activities for leukemic transformation. These findings greatly advance our understanding of the molecular mechanism of MLL fusion-dependent leukemic transformation, which was previously interpreted simply as mis-regulated transcription elongation. DisclosuresYokoyama:Dainipon Sumitomo Pharma Co., Ltd.: Research Funding.

An Early Thymic Precursor Phenotype Predicts Outcome Exclusively in HOXA-Overexpressing Adult T-ALL: A GRAALL Study

▪Introduction: Gene expression studies have consistently identified a HOXA positive (HOXAPos) subgroup of T-cell acute lymphoblastic leukemia (T-ALL) (Ferrando et al, Cancer Cell 2002, Soulier et al, Blood 2005, Homminga et al, Cancer Cell 2011). It is however unclear if HOXAPos T-ALL constitutes a distinct and homogeneous clinical entity, and the biological consequences of HOXA over-expression have not been systematically examined.Methods: We identified and characterized the biological characteristics and clinical outcome of 55 HOXAPos cases among a cohort of 209 adult T-ALL patients who were uniformly treated as part of the Group for Research on Adult Acute Lymphoblastic Leukemia (GRAALL)-2003 and -2005 studies.Results: HOXAPos patients had higher rates of an early thymic precursor (ETP)-like immunophenotype (38% v 13.9%, p = 0.0008), early bone marrow chemoresistance (59.3% v 40.8%, p = 0.026) and positive minimal residual disease (MRD, 51.5% v 23.5%, p = 0.01) than the HOXANeg group. These differences were due to a particularly high frequency of chemoresistant ETP-ALL among HOXAPos cases harboring leukemic fusion proteins that trans-activate the HOXA locus (e.g. PICALM-MLLT10, SET-NUP214).Strikingly, the presence of an ETP-like immunophenotype conferred marked differences in outcome within the HOXAPos group (5 year event-free survival (EFS) 25% for HOXAPos ETP v 52.2% for HOXAPos non-ETP, p = 0.02), which were mirrored by corresponding increases in cumulative incidence of relapse (CIR, 57.1% v 25%, p = 0.01, Figure 1). In contrast, these survival differences were not seen in the HOXANeg patients, where ETP and non-ETP cases had similar 5 year EFS (54.9% v 50%, p = 0.73) and CIR (34.5% v 41.2%, p = 0.57).Multivariate analysis revealed that early bone marrow chemosensitivity was the clinico-biological covariate that had the strongest prognostic interaction with HOXA status. HOXA positivity conferred significant decreases in both the EFS and CIR of chemoresistant patients (p = 0.053 and 0.039 respectively), that was independent of white blood cell count (WCC), stem cell transplant (SCT), ETP phenotype, EGIL classification, and our recently reported risk classifier that integrates the prognostic effects of mutations of NOTCH1, FBXW7, RAS and PTEN (Trinquand et al, J Clin Oncol 2013). There were corresponding marked survival differences within the HOXAPos cohort between chemoresistant and chemosensitive cases. These disparities were not seen in the HOXANeg group, indicating that the prognostic value of chemosensitivity in adult T-ALL is specific to HOXAPos patients.Discussion: Our data show that clinico-biological phenotype is intimately linked to the underlying mechanism of HOXA locus deregulation, and we identify HOXA overexpression as a novel prognostic variable in ETP-ALL. Multivariate analysis suggests that this poor outcome is strongly related to intrinsic treatment resistance, and that this effect is exclusive to the HOXAPos cohort. Patients in the GRAALL-2003 and -2005 studies received enhanced induction and/ or salvage therapy in the event of poor early treatment response. Our results suggest that pediatric regimen-based intensification provides significant survival benefits for HOXANeg chemoresistant cases. In contrast, these modifications are inadequate for therapeutic rescue of the majority of HOXAPos chemoresistant ETP-ALL. The dramatically inferior prognosis of this group mandates consideration for alternative treatments in future clinical trials. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Mircrorna-550 Functions As a Critical Tumor Suppressor in Acute Myeloid Leukemia

▪Background:Acute Myeloid leukemia (AML) is one of the most common and fatal form of hematologic malignancies. Recurring chromosomal aberrations and gene mutations have been shown to contribute to AML pathogenesis and clinical outcomes. However, no effective therapy is available to selectively target the cytogenetic and molecular abnormalities, except for PML-RARA in acute promyelocytic leukemia (APL) which can be targeted by all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). As a result, the majority of AML patients are suffering from unsatisfied treatment of standard chemotherapy, associated with high rate of relapse and inferior survival. Thus, better understanding of the molecular mechanisms underlying the pathogenesis and drug resistance of AML, and more effective treatments based on such understanding, are urgently needed.MiRNAs are a class of non-coding RNAs which post-transcriptionally regulate targeted gene expression. They usually consist of 20~24 nucleotides. MiRNAs are closely involved in almost all physiological and pathological processes. The widespread dysregulation of miRNA expressions have been shown to be correlated with various types of malignancies, including AML.In our recent publication, we show through Exiqon miRNA microarray analysis that several miRNAs are significantly down-regulated in most subtypes of de novo AML; miR-550 is one of them (Jiang et al., Cancer Cell, 2012). However, its role and regulatory mechanism in AML have been poorly elucidated. The present study is to investigate the biological functions and molecular mechanisms of miR-550 in AML.Methods:The expression levels of miR-550 were analyzed in multiple AML cell lines, AML patients' bone marrow (BM) mononuclear cells (MNC) and normal MNC control samples by using Taqman miRNA assay qPCR kit. Cell viability and proliferation assays, i.e., MTT assays, were performed in human AML cell lines with stable ectopic expression of miR-550 or control plasmids induced by retrovirus. Cell apoptosis and cell cycle were assessed via flow cytometry analysis. To determine the influence of miR-550 on the transformation capacity of mouse BM progenitor cells transduced with leukemic fusion genes, e.g. MLL-AF9 and AML-ETO9a (AE9a), colony-forming/replating assay (CFA) was carried out. To evaluate the effect of restoration of miR-550 expression/function in AML progression in vivo, we retrovirally infected leukemic blast cells carrying MLL-AF9 with miR-550 or empty vector, and performed secondary BM transplantation by i.v. injecting recipient mice with these donor cells. To identify potential target genes of miR-550, two independent AML patient datasets were analyzed and the correlation patterns between miR-550 and the candidate targets were shown.Results:Consistent with the results of our previous miRNA array, the expression level of miR-550 was significantly down-regulated in most AML patient samples and AML cell lines as compared with normal controls. In AML cell lines, retrovirus induced enforced expression of miR-550 resulted in G1-phase arrest, increased apoptosis, and inhibited cell growth and viability. In mouse BM progenitor cells, forced expression of miR-550 dramatically attenuated colony-forming capacity driven by MLL-AF9 or AE9a. Overexpression of miR-550 significantly inhibited progression of AML induced by MLL-AF9 (MLL-AF9+miR-550, with medium survival of 33 days; MLL-AF9, with medium survival of 27 days; P =0.01) in vivo.We further analyzed in two independent AML patient datasets the expressional correlation between miR-550 and its potential gene targets predicted by miRanda, miRWalk, PITA and Targetscan, etc. 77 candidate target genes inversely correlated with miR-550 in expression. Amongst these genes, FOXE1, IGFBP5 and KSR2 etc. have been shown to be oncogenes and are closely related with AML pathology. Therefore, these genes are candidate oncogenic targets that we will focus on in the future.Conclusions:The above results suggest that miR-550 is an important tumor suppressor in AML. Through targeting a series of oncogenes, miR-550 represses the viability and proliferation of leukemic cells, promotes apoptosis and differentiation, and inhibits cell transformation. Our study indicates that down-regulation of miR-550 likely plays a critical role in AML pathogenesis and restoration of miR-550 might hold great potential in treating AML in the future. DisclosuresNo relevant conflicts of interest to declare.

PRMT1-Mediated Methylation of DUSP4 Determines Megakaryocyte-Erythroid Lineage Choice By Regulating p38 Signaling

Protein arginine methyltransferase 1 (PRMT1) expression level is correlated with short survival rate of acute myeloid leukemia patients. PRMT1 contributes to leukemogenesis via direct interaction with leukemic fusion proteins such as AML1-ETO and MLL-EEN. Our previous studies have shown that PRMT1 blocks megakaryocyte (MK) terminal differentiation. Inhibition of PRMT1 enzymatic activity promotes apoptosis and differentiation of acute megakaryocytic leukemia (AMKL) cells. To understand the molecular mechanisms of PRMT1-mediated leukemogenesis, we apply bioorthognal profiling of protein methylation technology to identify PRMT1 substrates. A dual specificity protein phosphatase, DUSP4 was found a PRMT1 substrate. In literature, high expression of DUPS4 is linked to idiopathic CD4 lymphopenia and silencing of DUSP4 expression causes chemo-resistance in solid tumors and abnormal innate immune response to parasitic infections. To understand the role of DUSP4 in megakaryocytic differentiation, we ectopically expressed DUSP4 and found that it enhances megakaryocyte development. In liquid culture system, DUSP4 knockdown in human CD34+ cells crippled MK differentiation induced by TPO. In TPO+EPO culture system, DUSP4 knockdown in human CD34+ cells gave rise to more erythroid cells (CD71+) at the expense of MK cells (CD41+). Therefore, DUSP4 is essential for MK differentiation. To understand how PRMT1-mediated methylation of DUSP4 blocks MK differentiation, we mapped the DUSP4 methylation site. We found that the DUSP4 methylation mutant has a longer half-life compared to DUSP4 wild type. Further studies confirmed that DUSP4 protein stability is controlled by methylation-induced ubiquitylation.Although DUSP4 has been shown to dephosphorylate ERK, JNK and p38 kinases in overexpression systems, we found that during megakaryocytic differentiation DUSP4 specifically targets p38. These results are consistent with the findings in literature that p38 is required for erythroid differentiation, while ERK is required for megakaryocytic differentiation. Therefore, DUSP4 is responsible for turning off p38 signaling pathway, which pushes cells to erythroid lineage. Furthermore, we found that DUSP4 is associated with chromatin and dephosphorylates histone H3S10; while the methylation mutant DUSP4 locates in cytoplasm, therefore can still dephosphorylate p38 but not H3S10. Given that H3S10 phosphorylation may play a critical role in endomitosis, we also studied whether overexpression of DUSP4 antagonize aurora kinase activity to promote megakaryocytic differentiation in AMKL cell lines, containing OTT-Mal translocation or trisomy 21/down syndrome. We have tested an array of PRMT1 inhibitors to boost DUSP4 activity for potential curative therapy using AMKL leukemia mouse models. Our data support the notion that targeting PRMT1-DUSP4 pathway is a valid therapeutic approach for AMKL. DisclosuresNo relevant conflicts of interest to declare.

Impact of Cytogenetic and Molecular Markers on Disease-Free Survival of Allogeneic Hematopoietic Stem Cell Transplantation for Acute Myeloid Leukemia

Introduction: Cytogenetics is an independent prognostic factor in acute myeloid leukemia (AML). Molecular genetics including leukemia fusion gene, gene mutation and gene over expression are recognized to have significant impact on survival in patients with AML as well. In present study, the impact of cytogenetic and molecular markers on disease-free survival (DFS) of allogeneic hematopoietic stem cell transplantation (HSCT) for AML was investigated. Methods: Between April 2012 and December 2014, consecutive 345 patients with AML who underwent allogeneic HSCT in our center were analyzed retrospectively. All patients were either in poor-risk or in good-risk/intermediate-risk but with persistent minimal residual disease. The median age was 19 (1.8 to 64) years old. Children (≤14 years) were 96 (27.8%) cases and adults (>14 years) were 249 (72.2%) cases. Male to female was 200:145. The median disease course was 6 (1-64) months. Leukocyte count at diagnosis was < 30 x 109/L in 230 (66.7%) patients (low leukocyte) and ≥30 x 109/L in 115 (33.3%) cases (high leukocyte). Transplants at CR1, ≥CR2, and advanced disease were 168 (48.7%), 53 (15.4%) and 124 (35.9%), respectively. Donor sources were identical sibling (IS) in 45 (13.0%) cases, unrelated (UR) in 71 (20.6%) cases and haploidentical (HI) in 229 (66.4%) cases. Myeloablative conditioning regimens were administered with either Busulfan (Bu) plus Cyclophosphamide (Cy)/Fludarabine (Flu)-based in 285 (82.6%) patients or total body irradiation (TBI) plus Cy/Flu-based in 60 (17.4%) patients. Antithymocyte globulin was used in unrelated and haploidentical HSCT. Unmanipulated bone marrow and peripheral blood stem cells (PBSC) for IS and HI HSCT and PBSC for UR transplant were applied as the grafts. Cyclosporine, short-term Methotrexate, and Mycophenolate mofetil were employed for GVHD prophylaxis. Results: Univariate analysis showed that DFS after allogeneic HSCT in AML was not associated with patient age (children vs. adults, 70.3% vs. 69.4%, p=0.6), leukocyte count at diagnosis (low leukocyte vs. high leukocyte, 68.8% vs. 71.3%, p=0.8), donor source (IS vs. UD vs. HI, 77.3% vs. 76.8% vs. 65.8%, p=0.21), and conditioning regimen (Bu-based vs. TBI-based, 70.1% vs. 67.3%, p=0.45). Multivariate analysis indicated that disease status before HSCT was the only impact factor on DFS (CR1 vs. ≥CR2 vs. advanced disease, 81.6% vs. 70.0% vs. 53.1%, p<0.0001). Therefore, total 221 of 345 patients with AML in complete remission pre-conditioning were analyzed for impact of cytogenetic and molecular markers on survival after HSCT. DFS rates were 79.1%, 80.4%, 74.1% in good-risk, intermediate-risk, poor-risk cytogenetics groups (p=0.81), respectively. According to gene mutations, the DFS rates were 100% in CEBPA+, 91.6% in IDH1+/NPM1+, 85.7% in Flt3-ITD+, 81.5% in c-KIT+, 75.0% in no mutation, 70.2% in MLL-PTD+/ASXL1+/TET2+, 54.3% in Flt3-ITD+ with other mutations (p=0.42). According to gene expression, the DFS rates were 100% in DEK-CAN+, 100% in HOX11+/EVI1+, 84.8% in no abnormal gene expression, 83.3% in CBFb-MYH11+, 78.5% in WT1+, 76.5% in MLL+, 74.9% in AML1-ETO+, 0% in TLS-ERG+ (p=0.004). Conclusions: Under our HSCT protocol, disease status before transplant for the patients with AML has significant impact on DFS but not patient age, leukocyte count at diagnosis, donor source and conditioning regimen. Allogeneic HSCT has attenuated the influence of cytogenetics on DFS in patients with AML. Our preliminary data have shown that patients with CEBPA+, IDH1+/NPM1+, DEK-CAN+, HOX11+/NPM1+ have favorable survival, but patients with both Flt3-ITD+ and other gene mutations or with TLS-ERG+ have poor survival after allogeneic HSCT in AML. DisclosuresNo relevant conflicts of interest to declare.

Clinical significance of monitoring E2A- PBX1 fusion gene expression in patients with allogeneic hematopoietic stem cell transplantation

目的观察伴有E2A-PBX1融合基因的急性淋巴细胞白血病（ALL）患者造血干细胞移植后E2A-PBX1融合基因表达水平的变化特点，初步探讨其临床意义。方法回顾性分析2010年12月至2015年1月于北京大学血液病研究所行异基因造血干细胞移植的10例E2A-PBX1融合基因阳性ALL患者临床资料，采用荧光实时定量PCR方法监测E2A-PBX1融合基因水平，分析其水平变化与疾病复发和治疗的相关性。结果10例E2A-PBX1融合基因阳性患者（其中4例为移植前阳性），移植后6例E2A-PBX1基因转阳，中位转阳时间90（75~180）d，第1次转阳基因中位水平为25.200%（0.022%~353.600%），从E2A-PBX1基因转阳至血液学复发间隔中位时间30（0~74）d，4例最终血液学复发，中位复发时间为164（75~240）d。6例E2A-PBX1融合基因转阳患者4例死亡，3例死于复发，1例死于弥漫性肺泡大出血。结论伴有E2A-PBX1融合基因ALL患者移植后一旦E2A-PBX1基因转阳，其水平迅速上升，病情进展迅速，预后差。