Implications For Leukemogenesis Research Articles

Development and Clinical Applications of PI3K/AKT/mTOR Pathway Inhibitors as a Therapeutic Option for Leukemias.

Leukemias are hematological neoplasms characterized by dysregulations in several cellular signaling pathways, prominently including the PI3K/AKT/mTOR pathway. Since this pathway is associated with several important cellular mechanisms, such as proliferation, metabolism, survival, and cell death, its hyperactivation significantly contributes to the development of leukemias. In addition, it is a crucial prognostic factor, often correlated with therapeutic resistance. Changes in the PI3K/AKT/mTOR pathway are identified in more than 50% of cases of acute leukemia, especially in myeloid lineages. Furthermore, these changes are highly frequent in cases of chronic lymphocytic leukemia, especially those with a B cell phenotype, due to the correlation between the hyperactivation of B cell receptors and the abnormal activation of PI3Kδ. Thus, the search for new therapies that inhibit the activity of the PI3K/AKT/mTOR pathway has become the objective of several clinical studies that aim to replace conventional oncological treatments that have high rates of toxicities and low specificity with target-specific therapies offering improved patient quality of life. In this review we describe the PI3K/AKT/mTOR signal transduction pathway and its implications in leukemogenesis. Furthermore, we provide an overview of clinical trials that employed PI3K/AKT/mTOR inhibitors either as monotherapy or in combination with other cytotoxic agents for treating patients with various types of leukemias. The varying degrees of treatment efficacy are also reported.

Altered expression of imprinted genes in patients with cytogenetically normal‑acute myeloid leukemia: Implications for leukemogenesis and survival outcomes.

Genomic imprinting, an epigenetic mechanism that regulates gene expression from parental chromosomes, holds substantial relevance in multiple cancers, including hematopoietic malignancies. In the present study, the expression of a panel of 16 human imprinted genes in bone marrow samples from 64 patients newly diagnosed with cytogenetically normal-acute myeloid leukemia (CN-AML) were examined alongside peripheral blood samples from 85 healthy subjects. The validated findings of the present study revealed significant upregulation of seven genes [COPI coat complex subunit gamma 2 (COPG2), H19 imprinted maternally expressed transcript (H19), insulin like growth factor 2 (IGF2), PEG3 antisense RNA 1 (PEG3-AS1), DNA primase subunit 2 (PRIM2), solute carrier family 22 member 3 SLC22A3 and Zinc finger protein 215 (ZNF215)] in patients with CN-AML (P<0.001). Notably, the expression level of H19 exhibited an inverse association with the survival duration of the patients (P=0.018), establishing it as a predictive marker for two- and five-year survival in patients with CN-AML. Kaplan-Meier analysis demonstrated that patients with lower H19 expression had superior two- and five-year survival rates compared with those with higher H19 expression. The results of the present study highlighted the association between loss of imprinting and leukemogenesis in CN-AML, underscoring the significance of H19 imprinting loss as a prognostic indicator for unfavorable two- and five-year survival in CN-AML patients.

Rationale for Targeting Deregulated Metabolic Pathways as a Therapeutic Strategy in Acute Myeloid Leukemia.

Metabolic reprogramming is a common cancer cell phenotype as it sustains growth and proliferation. Targeting metabolic activities offers a wide range of therapeutic possibilities which are applicable to acute myeloid leukemia (AML). Indeed, in addition to the IDH1/2-mutated AML model which established the proof-of-concept for specifically targeting metabolic adaptations in AML, several recent reports have expanded the scope of such strategies in these diseases. This review highlights recent findings on metabolic deregulation in AML and summarizes their implications in leukemogenesis.

Somatic Mutations Reveal Lineage Relationships and Age-Related Mutagenesis in Human Hematopoiesis

SummaryMutation accumulation during life can contribute to hematopoietic dysfunction; however, the underlying dynamics are unknown. Somatic mutations in blood progenitors can provide insight into the rate and processes underlying this accumulation, as well as the developmental lineage tree and stem cell division numbers. Here, we catalog mutations in the genomes of human-bone-marrow-derived and umbilical-cord-blood-derived hematopoietic stem and progenitor cells (HSPCs). We find that mutations accumulate gradually during life with approximately 14 base substitutions per year. The majority of mutations were acquired after birth and could be explained by the constant activity of various endogenous mutagenic processes, which also explains the mutation load in acute myeloid leukemia (AML). Using these mutations, we construct a developmental lineage tree of human hematopoiesis, revealing a polyclonal architecture and providing evidence that developmental clones exhibit multipotency. Our approach highlights features of human native hematopoiesis and its implications for leukemogenesis.

30 Whole-genome sequencing of normal stem cells provides novel insights into human native hematopoiesis and leukaemia aetiology

IntroductionMature blood and immune cells are produced by the process of hematopoiesis, which is orchestrated by self-renewing hematopoietic stem cells (HSCs) and multipotent progenitor cells (MPPs) in the bone marrow. As people age, clonal expansion of mutated stem cells within the blood more commonly occur, which is associated with increased risk of developing haematological malignancies. However, the processes underlying mutation accumulation in normal stem cells and the clonal composition of the blood tissue within the human bone marrow compartment remain unknown.Material and methodsWe determined genome-wide mutation patterns in HSCs and downstream MPPs by sequencing clonally expanded primary cells derived from bone marrow of multiple human donors of different ages.Results and discussionsWe found that base substitutions accumulate gradually during life at a rate of approximately 16 novel mutations per year, while insertions and deletions occur more sporadically and at low numbers. The number and types of mutations were similar between HSCs and MPPs, suggesting that differentiation and quiescence status does not affect somatic mutation load. The majority of the base substitutions in blood progenitors could be attributed to a novel mutational signature, which also explains the majority of base substitutions in acute myeloid leukaemia (AML). Additionally, using base substitutions, we reconstructed the developmental hematopoietic phylogenetic tree and demonstrate that branching takes place early during development.ConclusionOur data demonstrate that developmental clones have differential contribution to the HSC and MPP compartments, and while these sub-clones exhibit multipotency, branches contributing predominantly to HSCs have an enriched presence in the megakaryocyte lineage. Together, our approach highlights novel features of human hematopoiesis and its implications for leukemogenesis.

Role of Granulocyte-Macrophage Colony-Stimulating Factor in Acute Myeloid Leukemia/Myelodysplastic Syndromes.

PurposeGranulocyte-macrophage colony-stimulating factor (GM-CSF) cytokine stimulates growth, differentiation, and function of myeloid progenitors. We aimed to study the role of GM-CSF gene expression, its protein, and antibodies in patients with acute myeloid leukemia/myelodysplastic syndromes (AML/MDS) and their correlation to disease behavior and treatment outcome. The study included 50 Egyptian patients with AML/MDS in addition to 20 healthy volunteers as control subjects.Patients and MethodsAssessment of GM-CSF gene expression was performed by quantitative real-time polymerase chain reaction. GM-CSF proteins and antibodies were assessed by enzyme-linked immunosorbent assay.ResultsThere was significant decrease in GM-CSF gene expression (P = .008), increase in serum level of GM-CSF protein (P = .0001), and increase in anti–GM-CSF antibodies (P = .001) in patients with AML/MDS compared with healthy control subjects. In addition, there was a significant negative correlation between serum levels of GM-CSF protein and initial peripheral blood blasts, percentage as well as response to therapy.ConclusionAny alteration in GM-CSF gene expression could have implications in leukemogenesis. In addition, GM-CSF protein serum levels could be used to predict outcome of therapy. GM-CSF antibodies may also play a role in the pathogenesis of AML/MDS. The use of these GM-CSF parameters for disease monitoring and as markers of disease activity needs further research.

Micro-RNA Profiling of Exosomes from Marrow-Derived Mesenchymal Stromal Cells in Patients with Acute Myeloid Leukemia: Implications in Leukemogenesis

Gene regulatory networks in AML may be influenced by microRNAs (miRs) contained in exosomes derived from bone marrow mesenchymal stromal cells (MSCs). We sequenced miRs from exosomes isolated from marrow-derived MSCs from patients with AML (n = 3) and from healthy controls (n = 3; not age-matched). Known targets of mIRs that were significantly different in AML-derived MSC exosomes compared to controls were identified. Of the five candidate miRs identified by differential packaging in exosomes, only miR-26a-5p and miR-101-3p were significantly increased in AML-derived samples while miR-23b-5p, miR-339-3p and miR-425-5p were significantly decreased. Validation of the predicted change in gene expression of the potential targets was investigated by interrogating gene expression levels from public datasets of marrow-derived CD34-selected cells from patients with AML (n = 69) and healthy donors (n = 40). Two molecules with decreased gene expression in AML (EZH2 and GSK3β) were predicted by the miR profiling and have been previously implicated in AML while three molecules were increased in AML-derived cells and have not been previously associated with leukemogenesis (KRBA2, RRBP1 and HIST2H 2BE). In summary, profiling miRs in exosomes from AML-derived MSCs allowed us to identify candidate miRs with potential relevance in AML that could yield new insights regarding leukemogenesis or new treatment strategies.

Microenvironmental regulation of hematopoietic stem cells and its implications in leukemogenesis.

Hematopoietic stem cells (HSCs) are a population of cells in the bone marrow which can self-renew, differentiate into late lineage progenitors, or remain quiescent. HSCs exist alongside several cell types in the bone marrow microenvironment that comprise the stem cell niche. These cells regulate HSC function and can contribute to leukemogenesis. In this review we will discuss recent advances in this field. In the vascular niche, arteriolar and sinusoidal zones appear to play distinct roles in HSC function. Endothelial cells modulate HSC function via Notch and other signaling pathways. In the endosteal niche multiple cell types regulate HSCs. Osteoblasts promote HSC quiescence via secreted factors and possibly physical interactions, whereas adipocytes may oppose HSC quiescence. The balance of these opposing factors depends on metabolic cues. Feedback from HSC-derived cells, including macrophages and megakaryocytes also appears to regulate HSC quiescence. Dysfunction of the bone marrow microenvironment, including mesenchymal stem cell-derived stromal cells and the sympathetic nervous system can induce or alter the progression of hematologic malignancies. Many cell types in the bone marrow microenvironment affect HSC function and contribute to malignancy. Further understanding how HSCs are regulated by the microenvironment has clinical implications for stem cell transplantation and other therapies for hematologic malignancies.

Calcineurin and GSK-3 inhibition sensitizes T-cell acute lymphoblastic leukemia cells to apoptosis through X-linked inhibitor of apoptosis protein degradation.

The calcineurin (Cn)-nuclear factor of activated T cells signaling pathway is critically involved in many aspects of normal T-cell physiology; however, its direct implication in leukemogenesis is still ill-defined. Glycogen synthase kinase-3β (GSK-3β) has recently been reported to interact with Cn in neuronal cells and is implicated in MLL leukemia. Our biochemical studies clearly demonstrated that Cn was able to interact with GSK-3β in T-cell acute lymphoblastic leukemia (T-ALL) cells, and that this interaction was direct, leading to an increased catalytic activity of GSK-3β, possibly through autophosphorylation of Y216. Sensitivity to GSK-3 inhibitor treatment correlated with altered GSK-3β phosphorylation and was more prominent in T-ALL with Pre/Pro immunophenotype. In addition, dual Cn and GSK-3 inhibitor treatment in T-ALL cells promoted sensitization to apoptosis through proteasomal degradation of X-linked inhibitor of apoptosis protein (XIAP). Consistently, resistance to drug treatments in primary samples was strongly associated with higher XIAP protein levels. Finally, we showed that dual Cn and GSK-3 inhibitor treatment in vitro and in vivo is effective against available models of T-ALL, indicating an insofar untapped therapeutic opportunity.

Abstract 5235: Identification of microRNA-3662 as a novel tumor suppressor miR

Abstract Background: Despite all progress, the prognosis of many acute myeloid leukemia (AML) patients remains poor. Identification of candidate genes for targeted treatment strategies may be beneficial for patient survival. Using online prediction programs, we identified miR-3662 as a novel tumor suppressor candidate in AML. Its predicted targets include numerous genes involved in hematopoietic differentiation and leukemogenesis, such as the oncogene BAALC. So far, miR-3662 has not been implicated in cancer biology. Aims: First, we strived to elucidate the role of miR-3662 in hematopoietic differentiation and leukemogenesis. Second, we aimed to experimentally validate BAALC as a miR-3662 target to explore the downstream biology of the tumor suppressor candidate. Methods &amp; Results: As tumor suppressor genes are often downregulated in cancer, we determined the expression of miR-3662 in peripheral blood blasts from cytogenetically normal (CN-) AML patients (n=10) and peripheral blood samples from non-cancer controls (n=20). Indeed, the non-cancer controls had an average 97-fold (P≤.001) higher miR-3662 expression compared to CN-AML patients, suggesting a downregulation of miR-3662 in leukemic cells. Next, we assessed the effects of forced miR-3662 expression and knock-down of miR-3662 on the growth and colony formation of hematopoietic progenitor cells (CD34+) from three healthy donors. While miR-3662 increased cell growth and colony formation, antagomiR-3662 reduced both parameters (miR-3662 vs. scramble: P≤.001; antagomiR-3662 vs. scramble: P≤.001). In contrast, colony forming assays with leukemia cell lines (OCI-AML3, KG1a) after stable infection with miR-3662 versus scramble reduced the cell growth and colony formation (miR-3662 vs. scramble: both P≤.001). To assess the effect of miR-3662 on leukemia cell survival, we performed caspase-3/7 assays with the leukemic blasts of five CN-AML patients infected with miR-3662 or scramble. miR-3662 increased caspase-3/7 activity (miR-3662 vs. scramble, P=.004), indicating miR-3662-mediated increased apoptosis. Thus, miR-3662 has tumor suppressive potential. Next we tested the effects of miR-3662 on the expression of its predicted target gene BAALC by overexpressing miR-3662 in two AML cell lines (KG1a, MV4-11) and blasts of CN-AML patients (n=10). miR-3662 reduced BAALC expression in both AML cell lines (KG1a: 70% reduction, MV4-11: 75% reduction, both P≤.001) and in the CN-AML patients (average reduction: 38.6%, P=.007). Finally, we performed luciferase assays with a BAALC 3′-UTR construct. Co-transfection with miR-3662 led to a 38% reduction of luciferase activity (miR-3662 vs. scramble, P≤.001), indicating that miR-3662 directly targets BAALC. Conclusion: miR-3662 is a novel tumor suppressor miR that has potential implications for leukemogenesis, as it increased colony formation and differentiation in CD34+ cells and decreased growth of leukemic cells. Citation Format: Sophia Maharry, Sujay Mehta, Sandya Liyanarachchi, Guido Marcucci, Clara D. Bloomfield, Albert de la Chapelle, Ann-Kathrin Eisfeld. Identification of microRNA-3662 as a novel tumor suppressor miR. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5235. doi:10.1158/1538-7445.AM2014-5235

MiR-24 Promotes the Survival of Hematopoietic Cells

The microRNA, miR-24, inhibits B cell development and promotes myeloid development of hematopoietic progenitors. Differential regulation of cell survival in myeloid and lymphoid cells by miR-24 may explain how miR-24′s affects hematopoietic progenitors. MiR-24 is reported to regulate apoptosis, either positively or negatively depending on cell context. However, no role for miR-24 in regulating cell death has been previously described in blood cells. To examine miR-24′s effect on survival, we expressed miR-24 via retrovirus in hematopoietic cells and induced cell death with cytokine or serum withdrawal. We observed that miR-24 enhanced survival of myeloid and B cell lines as well as primary hematopoietic cells. Additionally, antagonizing miR-24 with shRNA in hematopoietic cells made them more sensitive to apoptotic stimuli, suggesting miR-24 functions normally to promote blood cell survival. Since we did not observe preferential protection of myeloid over B cells, miR-24′s pro-survival effect does not explain its promotion of myelopoiesis. Moreover, expression of pro-survival protein, Bcl-xL, did not mimic miR-24′s impact on cellular differentiation, further supporting this conclusion. Our results indicate that miR-24 is a critical regulator of hematopoietic cell survival. This observation has implications for leukemogenesis. Several miRNAs that regulate apoptosis have been shown to function as either tumor suppressors or oncogenes during leukemogenesis. MiR-24 is expressed highly in primary acute myelogenous leukemia, suggesting that its pro-survival activity could contribute to the transformation of hematopoietic cells.

Epigenomic analysis detects widespread gene-body DNA hypomethylation in chronic lymphocytic leukemia

We have extensively characterized the DNA methylomes of 139 patients with chronic lymphocytic leukemia (CLL) with mutated or unmutated IGHV and of several mature B-cell subpopulations through the use of whole-genome bisulfite sequencing and high-density microarrays. The two molecular subtypes of CLL have differing DNA methylomes that seem to represent epigenetic imprints from distinct normal B-cell subpopulations. DNA hypomethylation in the gene body, targeting mostly enhancer sites, was the most frequent difference between naive and memory B cells and between the two molecular subtypes of CLL and normal B cells. Although DNA methylation and gene expression were poorly correlated, we identified gene-body CpG dinucleotides whose methylation was positively or negatively associated with expression. We have also recognized a DNA methylation signature that distinguishes new clinico-biological subtypes of CLL. We propose an epigenomic scenario in which differential methylation in the gene body may have functional and clinical implications in leukemogenesis.

Aberrant induction of LMO2 by the E2A-HLF chimeric transcription factor and its implication in leukemogenesis of B-precursor ALL with t(17;19)

AbstractLMO2, a critical transcription regulator of hematopoiesis, is involved in human T-cell leukemia. The binding site of proline and acidic amino acid–rich protein (PAR) transcription factors in the promoter of the LMO2 gene plays a central role in hematopoietic-specific expression. E2A-HLF fusion derived from t(17;19) in B-precursor acute lymphoblastic leukemia (ALL) has the transactivation domain of E2A and the basic region/leucine zipper domain of HLF, which is a PAR transcription factor, raising the possibility that E2A-HLF aberrantly induces LMO2 expression. We here demonstrate that cell lines and a primary sample of t(17;19)-ALL expressed LMO2 at significantly higher levels than other B-precursor ALLs did. Transfection of E2A-HLF into a non-t(17;19) B-precursor ALL cell line induced LMO2 gene expression that was dependent on the DNA-binding and transactivation activities of E2A-HLF. The PAR site in the LMO2 gene promoter was critical for E2A-HLF-induced LMO2 expression. Gene silencing of LMO2 in a t(17;19)-ALL cell line by short hairpin RNA induced apoptotic cell death. These observations indicated that E2A-HLF promotes cell survival of t(17;19)-ALL cells by aberrantly up-regulating LMO2 expression. LMO2 could be a target for a new therapeutic modality for extremely chemo-resistant t(17;19)-ALL.

Integrative Analysis of NPM1 Mutation-Associated MicroRNA and Gene Expression Signatures Identifies Potential Leukemia-Relevant Genes in Acute Myeloid Leukemia.

Abstract 363▪▪This icon denotes an abstract that is clinically relevant.MicroRNAs (miRs) have been shown to control a wide range of biological functions such as differentiation, proliferation and apoptosis, either by translational repression, mRNA cleavage or miR mediated decay of the respective target mRNA. Deregulated miR expression has been associated with various human cancers, including acute myeloid leukemia (AML), a disease characterized by the accumulation of acquired genetic alterations in hematopoietic progenitor cells that lead to altered self-renewal, proliferation and differentiation. Mutations of the nucleophosmin (NPM1) gene could be identified as the most common genetic alteration in AML, mainly occurring in cytogenetically normal karyotype (CN-AML) cases. Furthermore, while NPM1 mutated cases show a favorable prognosis (in the absence of FLT3-ITD) and have been shown to possess a distinct gene expression profile (GEP), so far the biology underlying this aberration has still not been fully understood.In previous work, we profiled the miR expression in a cohort of 91 AML cases comprising all major cytogenetic and molecular genetic subgroups. Significance Analysis of Microarrays (SAM) revealed a distinct miR-signature associated with NPM1 mutation (NPM1mut) in CN-AML as also shown by other groups: 66 miRs were differentially expressed in NPM1mut compared to NPM1 wild-type (NPM1wt) cases. The vast majority of these miRs was strongly upregulated in NPM1mut CN-AML, whereas only few miRs were downregulated compared to NPM1wt cases. Therefore, overexpression of a distinct set of miRs seems to be an important characteristic of NPM1mut CN-AML, and the resulting deregulated expression of target genes of these NPM1mut signature miRs might contribute to leukemogenesis. To identify putative target genes of NPM1mut-associated miRs, we performed an integrative analysis of miR-expression and NPM1mut-related gene expression data in our cohort. First, we generated target gene lists for the core 33 overexpressed miRs of the NPM1mut signature by using the miRGator database. This resulted in a theoretical NPM1mut associated GEP. Then, a comparison of the theoretical with the measured NPM1mut GEP was performed in order to find putative targets whose mRNA levels are directly affected by the respective miRs. This approach revealed several promising candidate genes with known implication in tumorigenesis and/or leukemogenesis like APP, CCND1, IRF2, BCL2L1, MLL and KIT. Interestingly, these genes are putative targets of not only one, but several miRs (4 to 15) of the NPM1mut signature, thereby pointing towards a synergistic effect of these miRs. Validation of individual miR-target gene relations was carried out by qRT-PCR in cell lines transfected with the respective miR mimics, supplemented by Western Blot and 3'UTR-luciferase-reporter assays. This validation was successful, not only for already known miR-target gene connections, but also for novel candidates including e.g. CCND1, a cell cycle regulator, and interferon regulatory factor-2 (IRF2). IRF2 is known to show dysregulated expression in the majority of AML cases and has recently been described to be essential for preserving the self-renewal and multilineage differentiation capacity of hematopoietic stem cells (Sato et al., Nat Med 2009).Thus, our approach of combining miR expression information and GEP in NPM1mut CN-AML led to the identification of promising target genes with potential implication in leukemogenesis. Additional functional analyses of relevant miRs and target genes are currently in progress to further illuminate the mechanism of NPM1mut AML pathogenesis. Disclosures:No relevant conflicts of interest to declare.

The aldo-keto reductase AKR1C3 contributes to 7,12-dimethylbenz(a)anthracene-3,4-dihydrodiol mediated oxidative DNA damage in myeloid cells: Implications for leukemogenesis

The aldo-keto reductase AKR1C3, has been shown to regulate myelopoiesis via its ability to metabolise prostaglandin D 2 (PGD 2). Other studies have demonstrated the oxidative activation of polycyclic aromatic hydrocarbon (PAH) procarcinogens by AKR1C3 in cell-free systems. This is the first study that addresses whether AKR1C3 mediates carcinogen activation within intact living cells following manipulation of AKR1C3 by molecular intervention. Quantitative RT-PCR identified AKR1C3 as the predominant AKR1C isoform expressed in acute myeloid leukemia (AML). Exposure of K562 and KG1a myeloid cell lines to the known AKR1C3 substrate 7,12-dimethylbenz(a)anthracene-3,4-dihydrodiol (7,12-DMBA-3,4-diol) resulted in both single strand DNA breaks and oxidative DNA damage as measured using conventional and FPG-modified comet assays respectively. PGD 2-keto reductase activity was shown to be correlated with relative AKR1C3 expression and together with quantitative real time PCR was used to validate the RNAi-knockdown of AKR1C3 in K562 cells. Knockdown of AKR1C3 did not alter single strand DNA breaks following 7,12-DMBA-3,4-diol exposure but significantly decreased oxidative DNA damage. A similar interrelationship between AKR1C3 activity and 7,12-DMBA-3,4-diol mediated oxidative DNA damage but not single strand breaks was observed in KG1a cells. Finally, AKR1C3 knockdown also resulted in spontaneous erythroid differentiation of K562 cells. Since K562 cells are a model of AML blast crisis of chronic myeloid leukemia (CML) the data presented here identify AKR1C3 as a novel mediator of carcinogen-induced initiation of leukemia, as a novel regulator of erythroid differentiation and paradoxically as a potential new target in the treatment of CML.

The Role of MLL in DNA Damage Checkpoint and Its Implication in Leukemogenesis

Cell cycle checkpoints are implemented to safeguard our genome and the deregulation of which results in human cancers. Hence, it is of great significance to discover and investigate novel key constituents of the mammalian DNA damage response network. Human chromosome band 11q23 translocation disrupting the MLL gene leads to poor prognostic leukemias. MLL is a transcription co-activator that maintains HOX gene expression. The importance of HOX gene deregulation in MLL leukemogenesis has been intensively investigated. However, physiological murine MLL leukemia knockin models have indicated that incurred HOX gene aberration alone is insufficient to initiate MLL leukemia. Thus, additional signaling pathway must be involved, which remains to be discovered. Our recent studies demonstrated an intimate relationship between MLL and the cell cycle(Takeda et al. 2006, Genes & Development , 20, 2397–2409; Liu et al. 2007, Genes & Development , 21, 2385–2398). More importantly, our studies uncovered a critical role of MLL in executing the S phase checkpoint. We showed:Over-expression of MLL induces an S phase block.MLL accumulates in the S phase upon DNA damage.MLL deficiency results in radioresistant DNA synthesis (RDS) and chromatid-type chromosomal abnormalities, two signature characteristics of S phase checkpoint defects.We further determined the underlying mechanisms concerning the DNA damage-induced MLL accumulation. Our data showed that MLL is phosphorylated after DNA damage, which in turn blocks its degradation by SCFSkp2 in the S phase and results in the ultimate accumulation. Our data revealed the link between MLL and the S phase checkpoint, which provides novel insights into the mammalian cell cycle checkpoint network and human leukemia pathogenesis. Future studies utilizing murine leukemia models will be performed to examine whether MLL translocation compromises the S phase checkpoint and if the resulted dysfunction contributes to MLL leukemogenesis.

PEBP2-β/CBF-β–dependent phosphorylation of RUNX1 and p300 by HIPK2: implications for leukemogenesis

The heterodimeric transcription factor RUNX1/PEBP2-beta (also known as AML1/CBF-beta) is essential for definitive hematopoiesis. Here, we show that interaction with PEBP2-beta leads to the phosphorylation of RUNX1, which in turn induces p300 phosphorylation. This is mediated by homeodomain interacting kinase 2 (HIPK2), targeting Ser(249), Ser(273), and Thr(276) in RUNX1, in a manner that is also dependent on the RUNX1 PY motif. Importantly, we observed the in vitro disruption of this phosphorylation cascade by multiple leukemogenic genetic defects targeting RUNX1/CBFB. In particular, the oncogenic protein PEBP2-beta-SMMHC prevents RUNX1/p300 phosphorylation by sequestering HIPK2 to mislocalized RUNX1/beta-SMMHC complexes. Therefore, phosphorylation of RUNX1 appears a critical step in its association with and phosphorylation of p300, and its disruption may be a common theme in RUNX1-associated leukemogenesis.

Ectopic retroviral expression of LMO2, but not IL2Rγ, blocks human T-cell development from CD34+ cells: implications for leukemogenesis in gene therapy

The occurrence of leukemia in a gene therapy trial for SCID-X1 has highlighted insertional mutagenesis as an adverse effect. Although retroviral integration near the T-cell acute lymphoblastic leukemia (T-ALL) oncogene LIM-only protein 2 (LMO2) appears to be a common event, it is unclear why LMO2 was preferentially targeted. We show that of classical T-ALL oncogenes, LMO2 is most highly transcribed in CD34+ progenitor cells. Upon stimulation with growth factors typically used in gene therapy protocols transcription of LMO2, LYL1, TAL1 and TAN1 is most prominent. Therefore, these oncogenes may be susceptible to viral integration. The interleukin-2 receptor gamma chain (IL2Rgamma), which is mutated in SCID-X1, has been proposed as a cooperating oncogene to LMO2. However, we found that overexpressing IL2Rgamma had no effect on T-cell development. In contrast, retroviral overexpression of LMO2 in CD34+ cells caused severe abnormalities in T-cell development, but B-cell and myeloid development remained unaffected. Our data help explain why LMO2 was preferentially targeted over many of the other known T-ALL oncogenes. Furthermore, during T-cell development retrovirus-mediated expression of IL2Rgamma may not be directly oncogenic. Instead, restoration of normal IL7-receptor signaling may allow progression of T-cell development to stages where ectopic LMO2 expression causes aberrant thymocyte growth.

Aberrant transcriptional regulation of the MLL fusion partner EEN by AML1-ETO and its implication in leukemogenesis

AbstractThe EEN (extra eleven nineteen) gene, located on chromosome 19p13, was cloned as a fusion with MLL from a patient with acute myeloid leukemia (AML) with translocation t(11;19)(q23;p13). In this study, we characterized the genomic structure of the EEN gene, including its 5′ regulatory region and transcription start site (TSS). We found that Sp1 could bind to the guanine-cytosine (GC)–stretch of the EEN promoter and was critical for the normal EEN expression, whereas the leukemia-associated fusion protein AML1-ETO could aberrantly transactivate the EEN gene through an AML1 binding site. Of note, overexpressed EEN showed oncogenic properties, such as transforming potential in NIH3T3 cells, stimulating cell proliferation, and increasing the activity of transcriptional factor AP-1. Retroviral transduction of EEN increased self-renewal and proliferation of murine hematopoietic progenitor cells. Moreover, Kasumi-1 and HL60-cell growth was inhibited with down-regulation of EEN by RNAi. These findings demonstrate that EEN might be a common target in 2 major types of AML associated with MLL or AML1 translocations, and overexpression of EEN may play an essential role in leukemogenesis.

Aberrant Transcriptional Regulation of the MLL Fusion Partner EEN Gene by AML1-ETO and Its Implication in Leukemogenesis.

EEN (extra eleven nineteen) gene located on chromosome 19p13 was cloned as a fusion with MLL from an acute myeloid leukemia (AML) patient with translocation t(11;19)(q23;p13). In this study, we characterized the genomic structure of EEN gene including its 5′ regulatory region and transcription start site (TSS). We found that Sp1 could bind to the GC-stretch of EEN promoter and was critical for the normal EEN expression, while the leukemia associated fusion protein AML1-ETO could aberrantly transactivate EEN gene through an AML1 binding site. Of note, overexpressed EEN showed oncogenic properties, such as transforming potential in NIH3T3 cells, stimulating cell proliferation and increasing the activity of transcriptional factor AP-1. Retroviral transduction of EEN increased self-renewal and proliferation of murine hematopoietic progenitor cells. Moreover, Kasumi-1 and HL60 cell growth was inhibited with down regulation of EEN by RNAi. These findings demonstrate that EEN might be a common target in two major types of AML associated with MLL or AML1 translocations, and over-expression of EEN may play an essential role in leukemogenesis.