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Telomere biology disorders (TBDs) present heterogeneously, ranging from infantile bone marrow failure associated with very short telomeres to adult-onset interstitial lung disease (ILD) with normal telomere length. Yield of genetic testing and phenotypic spectra for TBDs caused by the expanding list of telomere genes in adults remain understudied. Thus, we screened adults aged ≥18 years with a personal and/or family history clustering hematologic disorders and/or ILD enrolled on The University of Chicago Inherited Hematologic Disorders Registry for causative variants in 13 TBD genes. Sixteen (10%) of 153 probands carried causative variants distributed among TERT (n = 6), TERC (n = 4), PARN (n = 5), or RTEL1 (n = 1), of which 19% were copy number variants. The highest yield (9 of 22 [41%]) was in families with mixed hematologic and ILD presentations, suggesting that ILD in hematology populations and hematologic abnormalities in ILD populations warrant TBD genetic testing. Four (3%) of 117 familial hematologic disorder families without ILD carried TBD variants, making TBD second to only DDX41 in frequency for genetic diagnoses in this population. Phenotypes of 17 carriers with heterozygous PARN variants included 4 (24%) with hematologic abnormalities, 67% with lymphocyte telomere lengths measured by flow cytometry and fluorescence in situ hybridization at or above the 10th percentile, and a high penetrance for ILD. Alternative etiologies for cytopenias and/or ILD such as autoimmune features were noted in multiple TBD families, emphasizing the need to maintain clinical suspicion for a TBD despite the presence of alternative explanations.

Clinical utility of gene panel-based testing for hereditary myelodysplastic syndrome/acute leukemia predisposition syndromes

Acute myeloid leukemia (AML) is a common and fatal form of hematopoietic malignancy. Overexpression and/or mutations of FLT3 have been shown to occur in the majority of cases of AML. Our analysis of a large-scale AML patient cohort (N = 562) indicates that FLT3 is particularly highly expressed in some subtypes of AML, such as AML with t(11q23)/MLL-rearrangements or FLT3-ITD. Such AML subtypes are known to be associated with unfavorable prognosis. To treat FLT3-overexpressing AML, we developed a novel targeted nanoparticle system: FLT3 ligand (FLT3L)-conjugated G7 poly(amidoamine) (PAMAM) nanosized dendriplex encapsulating miR-150, a pivotal tumor suppressor and negative regulator of FLT3. We show that the FLT3L-guided miR-150 nanoparticles selectively and efficiently target FLT3-overexpressing AML cells and significantly inhibit viability/growth and promote apoptosis of the AML cells. Our proof-of-concept animal model studies demonstrate that the FLT3L-guided miR-150 nanoparticles tend to concentrate in bone marrow, and significantly inhibit progression of FLT3-overexpressing AML in vivo, while exhibiting no obvious side effects on normal hematopoiesis. Collectively, we have developed a novel targeted therapeutic strategy, using FLT3L-guided miR-150–based nanoparticles, to treat FLT3-overexpressing AML with high efficacy and minimal side effects. Cancer Res; 76(15); 4470–80. ©2016 AACR.

It is generally assumed that gain- and loss-of-function manipulations of a functionally important gene should lead to the opposite phenotypes. We show in this study that both overexpression and knockout of microRNA (miR)-126 surprisingly result in enhanced leukemogenesis in cooperation with the t(8;21) fusion genes AML1-ETO/RUNX1-RUNX1T1 and AML1-ETO9a (a potent oncogenic isoform of AML1-ETO). In accordance with our observation that increased expression of miR-126 is associated with unfavorable survival in patients with t(8;21) acute myeloid leukemia (AML), we show that miR-126 overexpression exhibits a stronger effect on long-term survival and progression of AML1-ETO9a-mediated leukemia stem cells/leukemia initiating cells (LSCs/LICs) in mice than does miR-126 knockout. Furthermore, miR-126 knockout substantially enhances responsiveness of leukemia cells to standard chemotherapy. Mechanistically, miR-126 overexpression activates genes that are highly expressed in LSCs/LICs and/or primitive hematopoietic stem/progenitor cells, likely through targeting ERRFI1 and SPRED1, whereas miR-126 knockout activates genes that are highly expressed in committed, more differentiated hematopoietic progenitor cells, presumably through inducing FZD7 expression. Our data demonstrate that miR-126 plays a critical but 2-faceted role in leukemia and thereby uncover a new layer of miRNA regulation in cancer. Moreover, because miR-126 depletion can sensitize AML cells to standard chemotherapy, our data also suggest that miR-126 represents a promising therapeutic target.

TNF signaling inactivation sensitizes AML cells to NF-kB inhibition but protects healthy hematopoietic stem progenitor cells from this treatment., Leukemic stem cells (LSCs) isolated from acute myeloid leukemia (AML) patients are more sensitive to nuclear factor κB (NF-κB) inhibition-induced cell death when compared with hematopoietic stem and progenitor cells (HSPCs) in in vitro culture. However, inadequate anti-leukemic activity of NF-κB inhibition in vivo suggests the presence of additional survival/proliferative signals that can compensate for NF-κB inhibition. AML subtypes M3, M4, and M5 cells produce endogenous tumor necrosis factor α (TNF). Although stimulating HSPC with TNF promotes necroptosis and apoptosis, similar treatment with AML cells (leukemic cells, LCs) results in an increase in survival and proliferation. We determined that TNF stimulation drives the JNK–AP1 pathway in a manner parallel to NF-κB, leading to the up-regulation of anti-apoptotic genes in LC. We found that we can significantly sensitize LC to NF-κB inhibitor treatment by blocking the TNF–JNK–AP1 signaling pathway. Our data suggest that co-inhibition of both TNF–JNK–AP1 and NF-κB signals may provide a more comprehensive treatment paradigm for AML patients with TNF-expressing LC.

TET1 was first identified as a fusion partner of the histone H3 Lys4 (H3K4) methyltransferase MLL (mixed-lineage leukemia) in acute myeloid leukemia (AML), and then was discovered as the founding member of the Ten-Eleven-Translocation (TET) family of DNA hydroxylases which are capable of converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Our group has previously demonstrated that TET1 plays an oncogenic role in MLL-rearranged AML (Huang H, et al. PNAS 2013; 110(29):11994-9) and also other TET1-overexpressing AMLs (e.g., t(8;21) AML and AMLs carrying FLT3-ITD and/or NPM1 mutations) (Jiang X, et al. Nature Communications. 2017; 8(1):2099). The expression of the TET1 protein and the global level of its enzymatic product, 5hmC, are significantly up-regulated in MLL-rearranged leukemia, whereas the opposite has been reported in other cancers where TET1 functions as a tumor suppressor. Therefore, a comprehensive identification of all critical targets of TET1 in AML is important for us to better understand the role and underlying molecular mechanism of TET1 in AML. To this end, we cultured murine inducible MLL-ENL cells and performed stable isotope labeling by amino acids in cell culture (SILAC)-based proteomic profiling in parallel with RNA-seq to systematically explore the functional targets of TET1 in a genome-wide and unbiased way. Gene ontology (GO) analysis of target genes indicated enrichment in genes associated with DNA replication (FDR Chromatin immunoprecipitation (ChIP) assays demonstrated that TET1 binds directly to the CpG islands in the promoters of the MCM genes, suggesting that the regulation of these genes by TET1 may occur at the transcriptional level. However, 5hmC sequencing revealed low abundance of 5hmC around these genomic regions, and more importantly, the abundance was not influenced by TET1 knockdown. In addition, catalytically inactive TET1 showed similar effects to wild-type TET1 on promoting cell cycle progression and DNA replication, suggesting that TET1 regulates MCM genes and cell cycle in a manner independent of its catalytic activity. Interestingly, we found that KAT8, an acetyltransferase that specifically catalyzes histone H4 lysine 16 acetylation (H4K16Ac) and was reported to bind to TET1, plays a role in the regulation of TET1 on transcription of MCM genes. Direct binding of KAT8 with TET1 was confirmed in AML cells. Depletion of TET1 reduced H4K16Ac abundance in the MCM promoters where TET1 bind, suggesting that the binding of KAT8 to such genomic regions is owing to its recruitment by TET1. Similar to TET1 knockdown, KAT8 knockdown also decreased MCM genes expression at both RNA and protein levels and resulted in defects in cell cycle progression and DNA replication. Based on the above data, we speculated that combined inhibition of TET1 and KAT8 could exert potent inhibitory effect on DNA replication in TET1-overexpressing AML cells. Indeed, Tet1 knockout sensitized MLL-AF9-transformed mouse bone marrow (BM) progenitor cells to MG149, a KAT8 inhibitor. In addition, TET1 inhibitor (U514321) synergized with MG149 in inhibiting the viability and growth of human AML cells. Furthermore, co-treatment of MG149 and U514321 exhibited synergistic effect on inhibiting colony-forming ability of MLL-AF9-transformed mouse BM progenitors. Collectively, our findings reveal a catalytic-independent novel function of TET1 on regulating DNA replication in AML cells through cooperating with KAT8 and highlight the therapeutic implication of targeting both TET1 and KAT8 in treating TET1-overexpressing AMLs. Disclosures Chen: Genovel Biotech Corp: Other: scientific founder and Chairman.

Increased expression levels of miR-181 family members have been shown to be associated with favorable outcome in patients with cytogenetically normal acute myeloid leukemia. Here we show that increased expression of miR-181a and miR-181b is also significantly (P < .05; Cox regression) associated with favorable overall survival in cytogenetically abnormal AML (CA-AML) patients. We further show that up-regulation of a gene signature composed of 4 potential miR-181 targets (including HOXA7, HOXA9, HOXA11, and PBX3), associated with down-regulation of miR-181 family members, is an independent predictor of adverse overall survival on multivariable testing in analysis of 183 CA-AML patients. The independent prognostic impact of this 4-homeobox-gene signature was confirmed in a validation set of 271 CA-AML patients. Furthermore, our in vitro and in vivo studies indicated that ectopic expression of miR-181b significantly promoted apoptosis and inhibited viability/proliferation of leukemic cells and delayed leukemogenesis; such effects could be reversed by forced expression of PBX3. Thus, the up-regulation of the 4 homeobox genes resulting from the down-regulation of miR-181 family members probably contribute to the poor prognosis of patients with nonfavorable CA-AML. Restoring expression of miR-181b and/or targeting the HOXA/PBX3 pathways may provide new strategies to improve survival substantially.

MLLT11, an MLL fusion partner, is a poor prognostic biomarker for paediatric acute myeloid leukaemia (AML), adult normal cytogenetics AML, and adult myelodysplastic syndrome. MLLT11 is highly regulated during haematopoietic progenitor differentiation and development but its regulatory mechanisms have not been defined. In this study, we demonstrate by transfection experiments that MIR29B directly regulates MLLT11 expression in vitro. MIR29B expression level was also inversely related to MLLT11 expression in a cohort of 56 AML patients (P < 0·05). AML patients with low MIR29B/elevated MLLT11 expression had poor overall survival (P = 0·038). Therefore, MIR29B may be a potential prognostic biomarker for AML patients.

Clinical Genome Resource (ClinGen) is an NIH/NHGRI-funded effort dedicated to building an authoritative central resource that defines the clinical relevance of genes and variants for use in precision medicine and research. ClinGen has developed both gene and variant expert panels to adapt the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines for consistent and accurate variant classification of specific genes and diseases. Here, we describe a new effort initiated in 2018 and supported by the American Society of Hematology (ASH) in collaboration with ClinGen to develop expert panels. This effort was motivated by the increasing use of genomics in clinical hematology and the lack of resources containing expert interpretation of germline variation. This panel, named the ClinGen Myeloid Malignancy Variant Curation Expert Panel is focused on the curation and annotation of variants in genes associated with familial/inherited risk for myeloid malignancies. Our team consists of expert clinicians, clinical laboratory diagnosticians, and researchers interested in developing and implementing standardized protocols for sequence variant specific annotations of genes in inherited myeloid malignancies. The optimization of the ACMG/AMP guidelines encompasses disease-/gene-informed specifications or strength adjustments of existing rules, including defining gene-specific population frequency cutoffs, and specifying recommendations for the use of computational/predictive data, as supported by published functional and clinical data in addition to guidance on ACMG/AMP variant interpretation provided by the ClinGen effort. Our initial focus has been to organize sub-groups of teams to develop approaches for evaluating ACMG/AMP codes to interpret germline variants of the RUNX1 gene. Once the curation of RUNX1 variants is underway, we will extend our focus to include CEBPA, DDX41, ETV6, and GATA2. These efforts will be bolstered by encouraging submission of existing variant interpretations to ClinVar or other public variant databases by the Hematology community. In summary, the ClinGen Myeloid Malignancy Variant Curation Expert Panel aims to develop recommendations to optimize ACMG/AMP criteria for standardization of variant interpretation in myeloid leukemia genes and make expert-reviewed and interpreted variants available to the hematology community through ClinVar and the ClinGen website (www.clinicalgenome.org) to support patient care and research. Disclosures DiNardo: Karyopharm: Honoraria; Agios: Consultancy; Medimmune: Honoraria; Celgene: Honoraria; Bayer: Honoraria; Abbvie: Honoraria. Nichols:Incyte: Research Funding; Alpine Immune Sciences: Research Funding. Plon:Baylor Genetics: Membership on an entity's Board of Directors or advisory committees.

Gastric cancer causes nearly one million deaths worldwide per year. Although Helicobacter pylori infection is the main risk factor, in about 80% or more of gastric cancers, the molecular pathway underlying H. pylori infection leading to the development of gastric cancers remains unclear. Recently accumulating evidence suggests that microRNAs (miRNAs) may regulate diverse biological processes and may be important in tumorigenesis. miR-21 has been frequently observed to be aberrantly overexpressed in various tumors. Using TaqMan quantitative real-time PCR, we confirmed that miR-21 was significantly overexpressed in human gastric cancer tissues and cell lines. Remarkably, miR-21 was also significantly overexpressed in H. pylori-infected gastric mucosa, implying that overexpression of miR-21 in gastric cancer may be due in part to H. pylori infection. More importantly, we showed that forced expression of miR-21 significantly enhanced cell proliferation and invasion in AGS cells, a human gastric cancer cell line, whereas knockdown of miR-21 by inhibitor caused a significant reduction in cell proliferation and a significant increase in apoptosis. Furthermore, we demonstrated that knockdown of miR-21 significantly decreased cell invasion and migration of AGS cells. Finally, we showed that RECK, a known tumor suppressor in gastric cancer, is a bona fide target of miR-21. Taken together, miR-21 may be important in the initiation and progression of gastric cancers as an oncomiR, likely through regulating RECK. Our findings suggest a potential regulatory pathway in which H. pylori infection upregulates expression of miR-21, which in turn downregulates RECK, and then leads to the development of gastric cancer.

DNA cytosine methylation is one of the best-characterized epigenetic modifications that play important roles in diverse cellular and pathological processes. The mechanism underlying the dynamic regulation of the level and distribution of 5-methylcytosine (5mC) as well as the biological consequence of DNA methylation deregulation have been interesting research topics in recent years. TET1, first identified as a fusion partner of the histone H3 Lys4 (H3K4) methyltransferase MLL (mixed-lineage leukemia) in acute myeloid leukemia (AML), is the founding member of the Ten-Eleven-Translocation (TET) family of DNA hydroxylases which are capable of converting 5mC to 5hmC (5-hydroxymethylcytosine) and lead to gene activation. Our group has previously demonstrated that TET1 plays an oncogenic role in MLL-rearranged leukemia (Huang H, et al. PNAS 2013; 110(29):11994-9). The expression of the TET1 protein and the global level of its enzymatic product, 5hmC, are significantly up-regulated in MLL-rearranged leukemia, whereas the opposite has been reported in other cancers where TET1 functions as a tumor suppressor. Therefore, a global understanding of the targets of TET1 in MLL-rearranged leukemia would greatly help to understand the role of TET1 in this specific type of AML. To this end, we performed proteomics study in parallel with RNA-seq to systematically explore the functional targets of TET1 in a genome-wide and unbiased way. Stable isotope labeling by amino acids in cell culture (SILAC)-based proteomic profiling showed that when Tet1 was knocked down in MLL-ENL-estrogen receptor inducible (ERtm) mouse myeloid leukemia cells, a total of 123 proteins were down-regulated whereas 191 were up-regulated with a fold-change cutoff of 1.2 (Fig. 1A and B), representing positively and negatively regulated targets of TET1, respectively. Most of the proteins with altered expression upon Tet1 knock-down showed a corresponding change at the mRNA level as reflected by the RNA-seq data. Interestingly, gene ontology (GO) analysis indicated enrichment on genes associated with DNA replication and cell cycle progression. Among these genes, the minichromosome maintenance complex genes, including MCM2, MCM3, MCM4, MCM5, MCM6, and MCM7, showed significant downregulation when Tet1 expression was depleted. We further conducted chromatin immunoprecipitation (ChIP) assays and demonstrated that TET1 binds directly to the CpG islands in the promoters of these MCM genes, suggesting that the regulation of the MCM genes by TET1 may occur at the transcriptional level. The six main minichromosome maintenance proteins (MCM2-7) are recruited to DNA replication origins in early G1 phase of the cell cycle and constitute the core of the replicative DNA helicase. We showed that not only the total levels of the MCM2-7 proteins, but also their binding to chromatin (Fig. 1C), were decreased by shRNAs against TET1 in human leukemia cell lines. Examination on cell cycle distribution revealed a significant decrease in the S phase population upon TET1 knockdown (Fig. 1D), which could be phenocopied by silencing of individual MCM genes. Consistently, incorporation of 5-ethynyl-2'-deoxyuridine (EdU) into newly synthesized DNA in the S phase can be inhibited by TET1 shRNAs (Fig. 1E), indicating the inhibition on DNA replication by TET1 silencing. Furthermore, DNA combing assays suggest that TET1 knockdown inhibits new origin firing (Fig. 1F) but does not influence replication fork speed. Collectively, our findings reveal a novel role of TET1 on regulating DNA replication in MLL-rearranged leukemia through targeting of MCM genes and highlight the therapeutic implication of targeting the TET1/MCM signaling. Figure 1 Role of TET1 in regulate DNA replication by controlling expression of MCM genes Figure 1. Role of TET1 in regulate DNA replication by controlling expression of MCM genes Disclosures No relevant conflicts of interest to declare.

Introduction N 6-methyladenosine (m6A) is the most abundant internal modification in messenger RNA (mRNA) mainly occurring at consensus motif of G[G>A]m6AC[U>A>C]. Despite the functional importance of m6A modification in various fundamental bioprocesses, the studies of m6A modification in cancer, especially in leukemia have largely been limited.Fat mass and obesity-associated protein (FTO), the first RNA demethylase,was known to be robustly associated with increased body mass and obesity in humans. However, the impact of FTO, especially as a RNA demethylase, in cancer development and progression has yet to be investigated. Acute myeloid leukemia (AML) is one of the most common and fatal forms of hematopoietic malignancies with distinct geneticabnormalities and variable response to treatment.Here, we aim to definethe roleof FTO as an m6A demethylase in AML. Methods To access the potential effect of FTO, we analyzed its expression in AML patients with distinct genetic mutations. To determine the influence of FTO on transformation capacity/ cell viability and leukemogenesis, colony-forming/replating assay (CFA), MTT assays, cell apoptosis and bone marrow transplantation (BMT) were carried out. To identify potential targets of FTO, transcriptome-wide m6A-seq and RNA-seq were performed. To evaluate the function of FTO on m6A modification and mRNA metabolism,m6A dot blot, gene-specific m6A qPCR assays and RNA stability assays were conducted. To elucidate whether FTO-mediated regulation of its targets depends on its demethylase activity, gene-specific m6A qPCR assays and luciferase reporter and mutagenesis assays were carried out. To investigate the potential roles of FTO and its targets in hematopoiesis, ATRA-induced APL cell differentiation was used. Results In analysis of AML datasets, we found FTO is highly expressed in AMLs with t(11q23)/MLL-rearrangements, t(15;17)/PML-RARA, FLT3-ITD and/or NPM1 mutations. Lentivirus-induced expression of wild-type FTO, but not mutant FTO (carrying two point mutations, H231A and D233A , which disrupt its enzymatic activity), significantly enhanced colony forming activities, promoted cell proliferation/transformation, restricted cell apoptosis and decreased global mRNA m6A levelin vitro. Forced expression of Fto significantly (p Via transcriptome-wide m6A-sequencingand RNA-sequencing (RNA-Seq) assays in MONOMAC-6 AML cells with or without overexpression or knockdown of FTO, we identified two functionally critical targets of FTO, ASB2 and RARA.Forced expression of wild-type FTO, but not mutant FTO, reduced expression of RARA and ASB2. Forced expression of either ASB2 or RARA largely recapitulated the phenotypes caused by FTO knockdown. Moreover, the effects of overexpression or knockdownof FTO can be largely rescued by that of RARA or ASB2, indicating that they are functional important targets of FTO. Forced expression and knockdown of FTO reduced and increased, respectively, the m6A levels on ASB2 and RARA mRNA transcripts,and shortened and prolonged, respectively, the half-life of ASB2 and RARAmRNA transcripts in AML cells.Importantly, FTO reduced luciferase activity ofASB23'UTR, RARA3'UTR or RARA5'UTR constructs with intact m6A sites, while mutations in the m6A sites abrogated the inhibition, demonstrating that FTO-mediated gene regulation relies on its demethylase activity. Upon ATRA treatment, FTO was significantly down-regulated, while RARA and ASB2were up-regulated in NB4 APL cells. Forced expression of FTO noticeably suppressed, while depletion of FTO enhanced, ATRA-induced cell differentiation.Forced expression of either RARA or ASB2 could also substantially enhance NB4 cell differentiation. Conclusions In summary, we provide compelling in vitro and in vivo evidence demonstrating that FTO, an m6A demethylase, plays a critical oncogenic role in cell transformation and leukemogenesis as well as in ATRA-mediated differentiation of leukemic cells, through reducing m6A levels in mRNA transcripts of its critical target genes such as ASB2 and RARA and thereby triggering corresponding signaling cascades. Our study highlights the functional importance of the m6A modification machinery in leukemia. Disclosures No relevant conflicts of interest to declare.

Although PBX proteins are known to increase DNA-binding/transcriptional activity of HOX proteins through their direct binding, the functional importance of their interaction in leukemogenesis is unclear. We recently reported that overexpression of a 4-homeobox-gene signature (ie, PBX3/HOXA7/HOXA9/HOXA11) is an independent predictor of poor survival in patients with cytogenetically abnormal acute myeloid leukemia (CA-AML). Here we show that it is PBX3, but not PBX1 or PBX2, that is consistently coexpressed with HOXA9 in various subtypes of CA-AML, particularly MLL-rearranged AML, and thus appears as a potential pathologic cofactor of HOXA9 in CA-AML. We then show that depletion of endogenous Pbx3 expression by shRNA significantly inhibits MLL-fusion–mediated cell transformation, and coexpressed PBX3 exhibits a significantly synergistic effect with HOXA9 in promoting cell transformation in vitro and leukemogenesis in vivo. Furthermore, as a proof of concept, we show that a small peptide, namely HXR9, which was developed to specifically disrupt the interactions between HOX and PBX proteins, can selectively kill leukemic cells with overexpression of HOXA/PBX3 genes. Collectively, our data suggest that PBX3 is a critical cofactor of HOXA9 in leukemogenesis, and targeting their interaction is a feasible strategy to treat presently therapy resistant CA-AML (eg, MLL-rearranged leukemia) in which HOXA/PBX3 genes are overexpressed.

Compound K (20-O-beta-D-glucopyranosyl-20(S)-protopanaxadiol, CK), an intestinal bacterial metabolite of ginseng protopanaxadiol saponins, has been shown to inhibit cell growth in a variety of cancers. However, the mechanisms are not completely understood, especially in colorectal cancer (CRC). A xenograft tumor model was used first to examine the anti-CRC effect of CK in vivo. Then, multiple in vitro assays were applied to investigate the anticancer effects of CK including antiproliferation, apoptosis and cell cycle distribution. In addition, a qPCR array and western blot analysis were executed to screen and validate the molecules and pathways involved. We observed that CK significantly inhibited the growth of HCT-116 tumors in an athymic nude mouse xenograft model. CK significantly inhibited the proliferation of human CRC cell lines HCT-116, SW-480, and HT-29 in a dose- and time-dependent manner. We also observed that CK induced cell apoptosis and arrested the cell cycle in the G1 phase in HCT-116 cells. The processes were related to the upregulation of p53/p21, FoxO3a-p27/p15 and Smad3, and downregulation of cdc25A, CDK4/6 and cyclin D1/3. The major regulated targets of CK were cyclin dependent inhibitors, including p21, p27, and p15. These results indicate that CK inhibits transcriptional activation of multiple tumor-promoting pathways in CRC, suggesting that CK could be an active compound in the prevention or treatment of CRC.

5-hydroxymethylcytosine (5hmC), also called the "sixth DNA base", is involved in the DNA demethylation process which generally leads to gene activation. Formation of 5hmC is catalyzed by the Ten-Eleven-Translocation (TET) family proteins, with TET1 being the founding member. The expression of TET1 protein and the global level of its enzymatic product, 5hmC, is markedly reduced in a wide range of solid tumors, including melanoma, prostate, breast, lung, and liver cancer, suggesting that TET1 functions as a tumor suppressor in these types of cancers. However, a recent study from our group demonstrated that TET1 expression and the associated 5hmC levels are significantly up-regulated in MLL -rearranged leukemia, revealing the oncogenic role of TET1 in this type of acute myeloid leukemia (AML) (Huang H, et al. PNAS 2013; 110(29):11994-9). In support of this, another study from a different group showed that high 5hmC level is an independent predictor of poor overall survival in patients with AML (Kroeze LI, et al. Blood 2014; 124(7):1110-8). However, how TET1, as a critical methylcytosine dioxygenase, plays its oncogenic role in AML, especially in MLL -rearranged leukemia, is still unclear. To address this issue, we performed stable isotope labeling by amino acids in cell culture (SILAC)-based proteomic profiling to systematically explore the functional targets of TET1 in a genome-wide and unbiased way. When TET1 was knocked down in MLL-ENL-estrogen receptor inducible (ERtm) mouse myeloid leukemia cells, 123 proteins were found downregulated whereas 191 were upregulated with a fold-change cutoff of 1.2. The expression changes of a set of these genes were confirmed by quantitative PCR in MLL-ENL-ERtm cells and mice samples with TET1 knock-down or depletion. After taking into account the correlation of TET1 and its candidate targets in several sets of AML patient samples, we focused on IDH1 and PSIP1, which represent the negatively- and positively-regulated targets by TET1, respectively. IDH1 encodes an isocitrate dehydrogenase whose mutations are frequently found in AML, whereas the PSIP1 protein is shown to be required for both MLL-dependent transcription and leukemic transformation. Chromatin immunoprecipitation (CHIP) assays suggest that TET1 directly binds to the CpG islands in the promoters of these two genes. Forced expression of Idh1 in leukemic bone marrow cells collected from mice developed MLL-AF9-driven AML significantly inhibited the colony-forming capacity of these cells, which mimics the effect of TET1 knock-out. We are now further investigating the functions and underlying molecular mechanisms of IDH1 and PSIP1 in AML using both in vitro and in vivo models. Considering the important roles of IDH1 and PSIP1 in AML, our findings will provide new insight into the mechanisms underlying the oncogenic role of TET1 in MLL -rearranged leukemia and may ultimately lead to the development of targeted therapy of AML. Disclosures No relevant conflicts of interest to declare.

Introduction: Myelodysplastic syndrome (MDS) and acute leukemia (AL) are a clinically diverse and genetically heterogeneous group of hematologic malignancies. Familial forms of MDS/AL have been increasingly recognized in recent years, and can occur as a primary event or secondary to genetic syndromes, such as inherited bone marrow failure syndromes (IBMFS). It is critical to confirm a genetic diagnosis in patients with hereditary predisposition to hematologic malignancies in order to provide prognostic information and cancer risk assessment, and to aid in identification of at-risk or affected family members. In addition, a molecular diagnosis can help tailor medical management including informing the selection of family members for allogeneic stem cell transplantation donors. Until recently, clinical testing options for this diverse group of hematologic malignancy predisposition genes were limited to the evaluation of single genes by Sanger sequencing, which is a time consuming and expensive process. To improve the diagnosis of hereditary predisposition to hematologic malignancies, our CLIA-licensed laboratory has recently developed Next-Generation Sequencing (NGS) panel-based testing for these genes. Methods: Thirty six patients with personal and/or family history of aplastic anemia, MDS or AL were referred for clinical diagnostic testing. DNA from the referred patients was obtained from cultured skin fibroblasts or peripheral blood and was utilized for preparing libraries with the SureSelectXT Enrichment System. Libraries were sequenced on an Illumina MiSeq instrument and the NGS data was analyzed with a custom bioinformatic pipeline, targeting a panel of 76 genes associated with IBMFS and/or familial MDS/AL. Results: Pathogenic and highly likely pathogenic variants were identified in 7 out of 36 patients analyzed, providing a positive molecular diagnostic rate of 20%. Overall, 6 out of the 7 pathogenic changes identified were novel. In 2 unrelated patients with MDS, heterozygous pathogenic sequence changes were identified in the GATA2 gene. Heterozygous pathogenic changes in the following autosomal dominant genes were each identified in a single patient: RPS26 (Diamond-Blackfan anemia 10), RUNX1 (familial platelet disorder with propensity to myeloid malignancy), TERT (dyskeratosis congenita 4) and TINF2 (dyskeratosis congenita 3). In addition, one novel heterozygous sequence change (c.826+5_826+9del, p.?) in the Fanconi anemia associated gene FANCA was identified. . The RNA analysis demonstrated this variant causes skipping of exon 9 and results in a premature stop codon in exon 10. Further review of the NGS data provided evidence of an additional large heterozygous multi-exon deletion in FANCA in the same patient. This large deletion was confirmed using array-CGH (comparative genomic hybridization). Conclusions: This study demonstrates the effectiveness of using NGS technology to identify patients with a hereditary predisposition to hematologic malignancies. As many of the genes associated with hereditary predisposition to hematologic malignancies have similar or overlapping clinical presentations, analysis of a diverse panel of genes is an efficient and cost-effective approach to molecular diagnostics for these disorders. Unlike Sanger sequencing, NGS technology also has the potential to identify large exonic deletions and duplications. In addition, RNA splicing assay has proven to be helpful in clarifying the pathogenicity of variants suspected to affect splicing. This approach will also allow for identification of a molecular defect in patients who may have atypical presentation of disease. Disclosures No relevant conflicts of interest to declare.

Acute myeloid leukemia (AML) is one of the most common and fatal forms of hematopoietic malignancies. With standard chemotherapies, only 30-50% of younger (aged MicroRNAs (miRNA) are a class of small, non-coding RNAs that play important roles in post-transcriptional gene regulation. We recently reported that miR-150 functions as a pivotal tumor-suppressor gatekeeper in MLL-rearranged and other subtypes of AML, through targeting FLT3 and MYB directly, and the MYC/LIN28/HOXA9/MEIS1 pathway indirectly. Our data showed that MLL-fusion proteins up-regulate FLT3 level through inhibiting the maturation of miR-150. Therefore, our findings strongly suggest a significant clinical potential of restoration of miR-150 expression/function in treating FLT3 -overexpressing AML. In the present study, we first analyzed FLT3 expression patterns and prognostic impact in a large cohort of AML patients (n=562). We found that FLT3 is aberrantly highly expressed in FAB M1/M2/M5 AML or AML with t(11q23)/MLL -rearrangements, FLT3 -ITD or NPM1 mutations, and that increased expression of FLT3 is an independent predictor of poor prognosis in patients with FLT3 -overexpressing AML. To treat FLT3 -overexpressing AML, we developed a novel targeted nanoparticle system consisting of FLT3 ligand (FLT3L)-conjugated G7 poly(amidoamine) (PAMAM) dendriplexes encapsulating miR-150 oligos (see Figure 1A). In FLT3 -overexpressing cell lines, the uptake ratios of the G7-FLT3L dendrimers were much higher (50.3~97.1%) than the G7-histone 2B (H2B) control nanoparticles (4.3~33.2%). And the uptake only took minutes. By integrating the miR-150 oligo with G7-FLT3L dendrimers, we constructed the G7-FLT3L-miR-150 dendriplexes, which significantly reduced the viability and increased the apoptosis of MONOMAC-6 cells carrying t(9;11) in a dose-dependent manner. To increase the stability of miR-150 oligos, we incorporated a 2'-o -methyl (2'-O Me) modification into the miRNA oligos. Indeed, the G7-FLT3L nanoparticles carrying 2'-O Me modified miR-150 exhibited a more sustained inhibition on cell growth. In order to further investigate the in vivo therapeutic effects of the miR-150 nanoparticles, we used a MLL -rearranged leukemia model. We transplanted wild-type recipient mice with primary mouse leukemic cells bearing the MLL-AF9 fusion. After the onset of leukemia, the mice were treated with G7-Flt3L or G7-NH2 control nanoparticles complexed with 2'-O Me-modified miR-150 oligos. In these treated animals, G7-Flt3L-miR-150 nanoparticles tended to be enriched in the bone marrow. The G7-Flt3L-miR-150 nanoparticles showed the best therapeutic effect (with median survival of 86 days), as compared with the control group (Ctrl; PBS treated; with median survival of 54 days) or the G7-NH2-miR-150 treated group (with median survival of 63 days). Nanoparticles carrying miR-150 mutant oligos showed no anti-leukemia effect at all. Notably, the G7-Flt3L-miR-150 treatment almost completely blocked MLL-AF9 -induced leukemia in 20% of the mice (Fig. 1B). Furthermore, the G7-Flt3L-miR-150 nanoparticles showed a synergistic effect with JQ1, a small-molecule inhibitor of the MYC pathway, in treating AML in vivo (Fig. 1C). Collectively, we have developed a novel targeted therapeutic strategy to treat FLT3-overexpressing AML, such as MLL-rearranged leukemias, which are resistant to currently available therapies, with both high specificity and efficacy. Disclosures No relevant conflicts of interest to declare.

MicroRNAs (miRNAs) have been implicated in the pathogenesis of various types of cancers. We reported previously that miR-126 likely functioned as an oncogene in acute myeloid leukemia (AML) (Li Z, et al. PNAS. 2008 Oct 7; 105:15535-40), which was confirmed by others (de Leeuw DC, et al. Cancer Research. 2014 Apr 1,74:2094-105; Dorrance AM, et al. Leukemia. 2015). However, it was also reported that miR-126 knockdown in normal hematopoietic stem/progenitor cells (HSPCs) resulted in expansion of long-term HSCs (Lechman ER, et al. Cell Stem Cell. 2012 Dec 7,11:799-811). Thus, the role of miR-126 in normal and malignant hematopoiesis warrants further investigation. Here we show that miR-126 is particularly overexpressed in t(8;21) AML and increased expression of miR-126 is associated with poor prognosis in patients with t(8;21) AML. To determine the function of miR-126 in leukemogenesis, we conducted both gain- and loss-of-function in vivo studies of miR-126 in t(8;21) AML models via mouse bone marrow transplantation (BMT). Surprisingly, we found that both overexpression and knockout of miR-126 accelerated leukemogenesis by the t(8;21) fusion genes, AML1-ETO (AE) or AML1-ETO9a (AE9a; a potent oncogenic isoform of AE). For example, forced expression of miR-126 (by retroviral transduction?) significantly accelerated AE9a-mediated leukemogenesis (median survival of AE9a+miR-126 vs. AE9a: 234 days vs. 317 days, p To assess the impact of miR-126 overexpression and depletion on long-term self-renewal of t(8;21) leukemia stem/initiating cells (LSCs/LICs), we performed serial mouse BMT assays with leukemic BM cells collected from the prior generation of BMT recipients as donor cells. In the secondary mouse BMT assay, we found that the AE9a+miR-126 group showed similar leukemia development to the miR-126KO+AE9a group (median survival: 79 days vs. 81 days, p =0.22), and both groups had significantly shorter survival (p 10 fold), and both were significantly higher (p Our mechanistic studies indicate that miR-126 overexpression activates genes that are highly expressed in LSCs/LICs and/or primitive HSPCs through directly targeting ERRFI1 and SPRED 1, which in turn activate the MAPK signaling pathway. On the other hand, miR-126 knockout activates genes that are highly expressed in committed, more differentiated hematopoietic cells and triggers the WNT/β-catenin signaling pathway by inducing FZD7 expression. We show that ERRFI1, SPRED 1 and FZD7 are bona fide targets of miR-126, and their increased expression is associated favorable survival of t(8;21) AML patients. Overall, our data show that both gain- and loss-of-function of a single miRNA (e.g., miR-126) can promote tumorigenesis and enhance long-term self-renewal/progression of LSCs/LICs, through targeting distinct gene signaling and thus being associated with different biological consequences. As miR-126 depletion can sensitize AML cells to standard chemotherapy, our data also suggest that miR-126 represents a promising therapeutic target. Disclosures No relevant conflicts of interest to declare.

Approximately 10% of human acute leukemias are involved in chromosomal translocations between the mixed lineage leukemia (MLL) gene and over 50 partner genes. MLL-rearranged leukemias occur preferentially in infant and young children and are often associated with poor outcome. MicroRNAs (miRNAs) are an abundant class of small noncoding RNAs which repress gene expression and mRNA stability by base pairing with target mRNAs usually at the 3’-untranslated regions (UTRs). The ten-eleven translocation 1 (TET1), the founding member of the TET family of enzymes (TET1/2/3) that convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), was first identified in MLL-rearranged leukemia. But its definitive role in leukemia was unclear until our recent report published in PNAS (Huang H. et al. 2013). In contrast to the frequent repression and tumor-suppressor roles of the three TET genes observed in various cancers, we showed that TET1 is a direct target of MLL-fusion proteins and significantly up-regulated in MLL-rearranged leukemia, leading to a global increase of 5hmC level. Furthermore, Tet1 plays an indispensable oncogenic role in MLL-rearranged leukemia, through coordination with MLL-fusion proteins in regulating their critical co-targets including Hoxa/Meis1/Pbx3 genes. However, whether TET1 is also post-transcriptionally regulated by miRNAs in hematopoietic cells remains unknown. In the present report, through genome-wide miRNA expression profiling assays, we found that miR-26a and miR-29a were expressed at a significantly lower level in MLL-rearranged AML than in normal controls. The down-regulation of miR-26a and miR-29a is, at least in part, attributed to the transcriptional repression mediated by MLL-fusion proteins and MYC. Interestingly, both miR-26a and miR-29a target TET1 directly at the post-transcriptional level. More importantly, we showed that miR-26a or miR-29a significantly inhibited MLL-fusion-mediated cell transformation in vitro and leukemogenesis in vivo down regulating expression of Tet1 and its downstream target genes. Thus, our data suggest that the transcriptional repression of miR-26a and miR-29a is required for the aberrant overexpression and potent oncogenic role of TET1 in MLL-rearranged leukemia, and that miR-26a and miR-29a play important tumor-suppressor role in leukemogenesis. Taken together, our data reveals a previously unappreciated signaling pathway involving the MLL-fusion/Myc⊣miR-26a/miR-29a⊣Tet1 circuit in MLL-rearranged leukemia. Our data not only provides novel insight into our understanding of the complex molecular mechanisms underlying the pathogenesis of MLL-rearranged leukemia, but also may lead to the development of novel, more effective therapeutic strategies to treat this type of dismal disease. Disclosures No relevant conflicts of interest to declare.

Acute myeloid leukemia (AML) is one of the most common and fatal forms of hematopoietic malignancies with diverse chromosomal and molecular abnormalities. The majority of AML patients do not survive more than 5 years. Advanced genomic studies reveal that both genetic and epigenetic abnormalities frequently occur in de novo AML. However, it remains a challenge to understand the complicated genetic/epigenetic regulatory networks and identify the functionally important nodes in these networks. There is an urgent need to develop effective therapeutic strategies based on these new insights. The ten-eleven translocation (Tet) proteins are important epigenetic regulators, which can convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and lead to DNA demethylation. Among the three TET family members (TET1/2/3), TET2 was identified as a tumor suppressor in myeloid malignancies. Our lab recently reported that TET1 is highly expressed in MLL/KMT2A (Mixed Lineage Leukemia)-rearranged AML, a subtype of AML with poor prognosis. It is a direct target activated by MLL-fusions, and functions as an essential oncogene (Huang et al., PNAS, 2013). However, the function and regulatory pathway(s) of TET1 in AML remain poorly understood. MicroRNAs (miRNAs) are a class of small, non-coding RNAs that play important roles in posttranscriptional gene regulation. Dysregulation of miRNAs is frequently observed in AML. Results of our profiling assays show that miR-22 is widely down-regulated in all major subtypes of de novo AML (Jiang et al., Cancer Cell, 2012), implying a tumor suppressor function. However, an oncogenic role for miR-22 was recently reported in myelodysplastic syndromes (MDS) and breast cancer, in which TET2 was repressed by miR-22 as its direct target gene. Here we show that, amongst a group of miRNAs (e.g. miR-495 and miR-150, etc.) whose expression levels are repressed in AML, miR-22 exhibits the most potent and consistent inhibition on MLL-AF9-induced transformation of mouse bone marrow (BM) progenitor cells. Moreover, forced expression of miR-22 dramatically inhibits cell transformation and leukemogenesis induced by multiple fusion genes, such as MLL-fusions and RUNX1/AML1-ETO9a. Furthermore, the maintenance of various subtypes of AML (e.g., those induced by MLL-fusion, AML1-ETO9a or FLT3-ITD/NPM1c+) is also dependent on the repression of miR-22. Thus, our data demonstrate a potent tumor-suppressor role of miR-22 in AML. Surprisingly, our analysis of three (in-house and outside) large-scale AML datasets revealed that TET2 (and likely also TET3) expression levels exhibited a significant positive correlation, whereas only TET1 exhibited a significant negative correlation (r Further, through a series of data analyses followed by experimental validations and functional studies, we show that a set of critical oncogenes, including CRTC1, FLT3 and MYCBP, are functionally important direct target genes of miR-22 in AML and thus, miR-22 negatively regulates the CREB and MYC signaling pathways. Our proof-of-concept study shows that miR-22 RNA oligos formulated with dendritic nanoparticles significantly inhibit leukemia progression and extend the overall median survival of MLL-AF9-induced leukemic mice from 29 days to 54 days (n=10 per group, p Taken together, our results demonstrate a potent tumor-suppressor role of miR-22 in AML, and suggest the potential clinical application of miR-22-nanoparticles in treating AML. We also identified a TET1⊣miR-22⊣CREB/MYC regulatory pathway, which is critical in AML pathogenesis (see Fig. 1). Our findings also highlight potential distinct genetic/epigenetic mechanisms underlying de novo AML and MDS. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

A critical area of cancer biology is the study of the deregulation of noncoding RNAs called microRNAs (miRNAs). Acute leukemia represents one of the most deadly cancers in the United States. One subset of leukemia with a poor to intermediate clinical outcome are chromosomal translocations involving Mixed Lineage Leukemia (MLL). As MLL-translocations are sufficient to drive leukemogenesis, and few additional mutations are observed in patients, it is imperative to understand the biology driving leukemogenesis. Previously, we and others have shown that several miRNAs are deregulated in MLL-rearranged Acute Myeloid Leukemia (AML). To identify miRNAs that are driving leukemogenesis we performed messenger RNA and miRNA expression profiling on primary patient samples and identified microRNA-9 (miR-9) as specifically overexpressed in MLL-rearranged AML. We further confirmed this observation using publically available microRNA sequencing data from the Cancer Genome Atlas (TCGA) and several AML cell lines. After showing that MLL directly binds and regulates miR-9 we show that depletion of MLL-fusion expression leads to the loss of miR-9 expression. Using publically available Illumina 450K methylation data from TCGA, we show that there is no significant difference in modified cytosine between miR-9 high and miR-9-low patients, suggesting that expression of miR-9 is likely not be regulated by DNA methylation machinery in AML patients. We show that miR-9 in the presence of MLL-AF9 (a common MLL-fusion) promotes colony growth over multiple passages while blocking miR-9 using a miR-9 sponge remarkably inhibits MLL-fusion-mediated cell transformation. Furthermore, we show that miR-9 increases proliferation and reduces apoptosis of human MLL-rearranged leukemic cells in vitro using MTT and Caspase 3/7 assays. We then show that co-transfection of miR-9 with MLL-AF9 in a bone marrow transplantation assay results in a higher leukemia burden in vivo compared to MLL-AF9 alone and promotes an immature cellular phenotype. Using microarray data we found several putative miR-9 targets by identifying genes that had an inverse correlation to miR-9. Next, we verified several genes were being inhibited by miR-9 such as Ras homology gene family member H (RHOH) and Ring1- and YY1-binding protein (RYBP). To understand the role of miR-9 in context with other miRNAs we did an association analysis of the top 300 differentially expressed miRNAs in the TCGA dataset. We found interestingly, that two of the miR-9 genes, miR-9-1 and miR-9-2, are highly correlated with each other across all the patients although they are located on distinct chromosomes. We also found that several other miRs were either negatively (e.g., miR-130a and miR-221) or positively (e.g., miR-191 and miR-642) associated with miR-9 expression, suggesting that these miRs might be operating either cooperatively or antagonistically in a complex circuitry. To support this hypothesis we found in univariate analysis that miR-9 itself was not a good predictor of patient survival but was a better predictor when combined with other miRs including the miR-181 family. Together this suggests that miR-9 is an important and critical regulator of MLL-rearranged AML and is a very good candidate for potential therapeutic targeting. Disclosures: No relevant conflicts of interest to declare.

Abstract 3522 MLL (mixed lineage leukemia) gene rearrangements account for about 10% of human acute leukemias, including ∼80% of infant acute leukemia. At present, the majority of patients die within two years of diagnosis, and a more effective therapeutic strategy is thus urgently needed. MLL fusion proteins induce aberrant expression of a group of homeobox gene super-family members, including HOXA and co-factors such as MEIS1 and PBX3. Overexpression of individual HOXA genes can induce myeloproliferation and block differentiation. Co-expression of Meis1 and Hoxa9 is sufficient to transform normal hematopoietic progenitor cells and to induce a rapidly fatal leukemia in transplanted mice, and their aberrant overexpression is required for the induction and maintenance of MLL-rearranged leukemia. Although Pbx proteins are known to interact with Hox proteins, thereby increasing the DNA-binding affinity of the latter and enhancing the transcription of downstream target genes, little is known about the interaction between Pbx proteins and Hox proteins (e.g., Hoxa9) in cell transformation and leukemogenesis. Recently, we showed that increased expression of a four-homeobox-gene signature (HOXA7, HOXA9, HOXA11 and PBX3) was an independent predictor of shorter overall survival in patients with cytogenetically abnormal acute myeloid leukemia (CA-AML) (Li Z., et al., Blood. 2012). Our analysis of the expression profiles of three independent large-scale patient sets showed that PBX3 was the only member of the PBX family that was consistently co-expressed with HOXA9 in various subtypes of CA-AML, particularly in MLL-rearranged AML; in contrast, both PBX1 and PBX2 tended to exhibit an inverse correlation of expression with HOXA9 in CA-AML. We then investigated the role of PBX3 in CA-AML, because its function in leukemia was unclear. We found a similar pattern of co-expression of Hoxa9 and Pbx3 in MLL fusion-mediated mouse leukemia models. We then showed that depletion of Pbx3 (but not Pbx1 and Pbx2) by Pbx3 shRNA dramatically inhibited MLL-AF9 induced transformation/immortalization of mouse normal bone marrow progenitor cells (about 50% reduction in colony numbers and about 70% reduction of number of cells in each colony). Furthermore, we demonstrated that forced expression of PBX3 exhibited a significantly synergistic effect with HOXA9 in promoting cell transformation/immortalization in vitro. In mouse bone marrow reconstitution/transplantation assay, the PBX3+HOXA9 mice developed leukemia significantly faster than HOXA9 alone with overall median survival of 71 days versus 140 days (P Disclosures: No relevant conflicts of interest to declare.

Abstract 3499 MicroRNAs (miRNAs), a class of small, non-coding RNAs, are important for posttranscriptional gene regulation in both health and disease. Expression of miRNAs is under stringent regulation at both transcriptional and post-transcriptional levels. Disturbance at either level could cause dysregulation of miRNAs. However, though altered expression of many miRNAs has been reported in various cancers, including acute myeloid leukemia (AML), their dysregulational mechanisms and pathologic functions remain less well understood. Here we report that mature miR-150 level is significantly downregulated in most AML samples, including those with rearrangements of the mixed lineage leukemia (MLL) gene. Strikingly, we found that despite the over 75% decrease of mature miR-150, its primary and precursor transcript abundance is increased to 2∼4 fold in human MLL-associated AML, relative to normal controls. Interestingly, we show that while MLL fusion proteins can bind to the promotor region of miR-150 and promote its primary transcription, they also negatively regulate the maturation process of miR-150 through the MYC/LIN28 functional axis. MiR-150 has been implicated as either an oncogene or a tumor suppressor in various types of solid tumors. However, its function in the pathogenesis of AML is unknown. Here we showed that ectopic expression of miR-150 dramatically inhibited cell growth and promoted apoptosis of human MLL-associated leukemic cells. Furthermore, using colony-forming/replating assays, we found that co-transduction of miR-150 and MLL-AF9 (a fusion gene resulting from t(9;11)) into mouse bone marrow (BM) progenitor cells, caused a significant reduction in colonies (down to 1∼10%; p Previous studies have shown that there is an autoregulatory feedback loop between FLT3/MYB and HOXA9/MEIS1, and the latter two are critical downstream targets of MLL fusion proteins. In addition, FLT3 has been identified as an upstream regulator of MYC, while MYC is also a downstream target of MLL fusion proteins and an upstream regulator of Lin28. These previous findings together with the data we reported above suggest that there is a critical MLL-fusion/MYC/LIN28-miR-150-FLT3/MYB/HOXA9/MEIS1 regulatory circuit in MLL-associated leukemia (see Fig. 1). In this circuit, MLL fusion proteins function as the driver, and their presence leads to the significant up-regulation of all six downstream genes, MYC, LIN28, FLT3, MYB, HOXA9, and MEIS1, as well as the primary transcription of miR-150. The up-regulation of MYC/LIN28 results in the blockade of the miR-150 maturation process. This in turn leads to the release of miR-150 inhibition on FLT3 and MYB expression, which would enhance the expression of HOXA9, MEIS1, MYC, and LIN28, and further enhance/maintain the blockade of miR-150 maturation. As a result, the cells reach and maintain high levels of MYC/LIN28/FLT3/MYB/HOXA9/MEIS1, and thereby transform the cells and lead to leukemogenesis. Our further systematic studies confirmed the existence/fidelity of this regulatory circuit in MLL-associated leukemia. Taken together, we revealed a previously unappreciated regulatory circuit. Our findings may advance our understanding of the complex molecular mechanisms underlying the development and maintenance of MLL-associated leukemia, and may also provide new strategies to treat MLL-associated leukemia, a disease that is presently treatment resistant, and likely also other subtypes of AML (as miR-150 is down-regulated in all subtypes of AML), or even other types of cancer that also utilize at least part of the signaling circuit we have described herein. Disclosures: No relevant conflicts of interest to declare.

Abstract 525 Acute leukemia represents one of the most deadly cancers in the United States. Clinical treatments in leukemia have progressed significantly through the use of therapies targeted specifically to chromosomal translocations. The success of these therapies has provided a model for future treatment in various cancers. However, there are various subtypes of leukemia where five-year survival and relapse rates have poor clinical outcome, indicating that new therapies are needed. A particular leukemia subtype, namely mixed lineage leukemia (MLL)-rearranged leukemia that is a result of chromosomal rearrangements leading to fusions between MLL and partner genes, is associated with a dismal outcome. Therapeutic targeting of MLL rearrangements has proven challenging as there have been dozens of described rearrangements. An emerging hallmark of cancer is the deregulation of non-coding RNAs called microRNAs (miRs). We hypothesized that MLL-associated leukemias have aberrant microRNA expression. We performed microRNA microarray analyses on leukemia patient samples and showed that microRNA-9 (miR-9) is highly upregulated in MLL-associated leukemias. We discovered that expression of miR-9 enhances cell transformation in vitro and tumorigenesis in vivo, and the opposite is true when miR-9 function is inhibited by anti-miR sponges. Interestingly, we observed that mice transplanted with both MLL-AF9 and miR-9 had a significantly higher amount of blast-like cells and immature cells in the bone marrow and fewer in the peripheral blood compared to mice transplanted with MLL-AF9 alone. Furthermore, inhibition of miR-9 function causes a significant defect in stem cell self-renewal and myeloid differentiation. Thus, this suggests that miR-9 has a critical role in stem cell potentiation and myeloid promotion in MLL-associated leukemias. To investigate target genes of miR-9, we correlated microarray expression of miR-9 and those of its putative target genes predicted with multiple prediction algorithms. We identified six potential target genes that exhibit a significantly inverse correlation of expression with miR-9 and are inhibited in MLL-associated leukemia. These identified genes are Transforming growth factor beta-induced protein (TGFBI), E-cadherin (CDH1), Nuclear factor-kappa-B p105 subunit (NFkB1), Ras homolog gene family member H (RHOH), Ring1 and YY1-binding protein (RYBP) and Foxhead Box O3 (FOXO3), We demonstrated that all these targets genes are inhibited upon expression of miR-9, in vitro and in vivo and blocking miR-9 function rescues their expression. As these target genes affect multiple cell processes such as proliferation, stem cell differentiation, chromatin remodeling, and cellular migration, it appears that miR-9 is a potential master regulator during MLL-associated tumorigenesis. Disclosures: No relevant conflicts of interest to declare.

To date, colorectal cancer (CRC) remains one of the most prevalent malignancies in the United States. Health benefits derived from medicinal plants have been investigated for years. Ginseng has been shown to induce diverse pharmacological effects mediated predominantly by saponins, a type of ginsenoside contained in ginseng. Compound K (20-O-beta-D-glucopyranosyl-20(S)-protopanaxadiol, CK), an intestinal bacterial metabolite of ginseng protopanaxadiol saponins, has been shown to inhibit cell growth in a variety of tumors. However, the detailed mechanism, especially the effects of CK on cell cycle regulation in CRC cells, has not been examined. In addition, it is uncertain whether CK can inhibit colorectal tumor growth in vivo. In this study, we observed that CK significantly inhibited the growth of HCT-116 cells in athymic nude mouse xenograft models. Quantitative analysis revealed that CK significantly inhibited xenograft tumor growth from the 3rd week after CK administration (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1685. doi:1538-7445.AM2012-1685

Altered expression of microRNAs (miRNAs, a class of small regulatory RNAs) is associated with various types of cancers, including acute myeloid leukemia (AML). In our previous bead-based miRNA expression profiling assay, we observed that miR-181a, b, c, and d were all expressed at a higher level in the favorable prognosis subtypes of cytogenetically abnormal AML (CA-AML) carrying t(8;21), inv(16), or t(15;17) than in an intermediate- to poor-risk subtype of CA-AML harboring MLL rearrangements. Indeed, we found that increased expression of miR-181a or miR-181b was significantly (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2309. doi:1538-7445.AM2012-2309

Abstract 2453 Acute Leukemia represents one of the most deadly cancers in the United States. Clinical treatments in leukemia have progressed significantly through the use of therapies targeted specifically to chromosomal translocations. The success of these therapies has provided a model for future treatment in various cancers. However, there are various subtypes of leukemia where five-year survival and relapse rates have poor clinical outcome, indicating that new therapies are needed. A particular leukemia subtype, namely mixed lineage leukemia (MLL)-rearranged leukemia that is a result of chromosomal rearrangements leading to fusions between MLL and partner genes, is associated with a dismal outcome. Therapeutic targeting of MLL rearrangements has proven challenging as there have been dozens of described rearrangements. We have performed both messenger RNA (mRNA) and microRNA (a class of small non-coding RNA) microarray analyses on over 100 leukemia patient samples and have identified that microRNA-9 (miR-9) is highly upregulated in MLL-associated leukemias. Through correlating expression of miR-9 and those of its predicted target genes, we identified TGFbeta-induced protein (TGFBI) as a potential target of miR-9 that exhibited a significantly (P Disclosures: No relevant conflicts of interest to declare.

Abstract 3462 Acute myeloid leukemia (AML) bearing MLL (mixed lineage leukemia) translocations are associated with poor survival, and only fewer than 50% of the patients survive longer than 5 years. Thus, an improved strategy leading to a higher cure rate is urgently needed to treat MLL-associated AML. MicroRNAs (miRNAs), a class of small non-coding RNAs, have been postulated to be important gene expression regulators in all biology including human leukemia. Through large-scale, genome-wide miRNA expression profiling assays, we determined that miR-495 is significantly down-regulated in the majority of human AML samples, particularly, in those with MLL rearrangements. More interestingly, through correlating the expression signature of miR-495 with clinical outcome of AML patients, we revealed that a low expression level of miR-495 is a predictor of poor prognosis in most AML patients. Our further qPCR assays confirmed that the expression of miR-495 is even more significantly downregulated in MLL-rearranged AML primary patient samples and cell lines. Through in vitro colony-forming/replating assays and in vivo bone marrow transplantation studies, we found that forced expression of miR-495 significantly inhibits the capacity of the MLL-AF9 fusion gene to support colony formation in mouse bone marrow progenitor cells in vitro and to induce leukemia in vivo. In leukemia cell lines, overexpression of miR-495 greatly inhibits the viability of the cells, while increasing apoptosis. Furthermore, by using 3 algorithms for miR-495 3'UTR binding sites, we identified several well-known MLL leukemia-related genes, e. g. BMI1, MEF2C, BID and MEIS1, as potential targets of miR-495. Results of qPCR revealed that forced expression of miR-495 significantly inhibits the expression levels of these genes in leukemia cell lines, mouse bone marrow progenitor cells, as well as mouse peripheral blood cells with MLL fusion genes. Therefore we hypothesize that miR-495 may function as a tumor suppressor in AML with MLL rearrangements by targeting essential tumor-related genes. Further studies will focus on: 1) effects of miR-495 on the functions of target genes studied in vitro and in vivo; 2) the epigenetic mechanisms and the signaling pathways involved in regulating the expression level of miR-495 in human leukemia. Disclosures: No relevant conflicts of interest to declare.

Abstract 236 Altered expression of microRNAs (miRNAs, a class of small regulatory RNAs) is associated with various types of cancers, including acute myeloid leukemia (AML). We showed previously that increased expression of miR-181a with or without miR-181b was associated with a favorable prognosis for patients with cytogenetically normal AML (CN-AML). However, the prognostic value of miR-181 expression in cytogenetically abnormal AML (CA-AML) remains elusive, even though CA-AML represents the majority of human AML. To investigate the association of expression signatures of miR-181 family members and of their potential target genes with outcome in patients with primary CA-AML, we employed two independent sets of 86 CA-AML patients to investigate the association of expression signatures of miR-181 family members with outcome. We also used four independent sets of 454 CA-AML patients to identify and validate a prognostic signature of miR-181 targets. In addition, we investigated the biological functions of miR-181a/b and target(s) in leukemia cell lines and in a leukemia mouse model. As with CN-AML, we found that both miR-181a and miR-181b expression signatures are significantly (P Disclosures: No relevant conflicts of interest to declare.

Abstract 2603 Poster Board II-579 We have consistently shown that elevated expression of AF1q, an MLL fusion partner, is a poor prognostic biomarker for pediatric acute myeloid leukemia (AML), adult AML with normal cytogenetics (NC-AML), and adult myelodysplastic syndrome (MDS). However, the underlying mechanism of how AF1q is regulated in normal and abnormal hematopoiesis is still unclear. Our previous studies suggest that AF1q is highly regulated during hematopoietic cell differentiation and development and it is known that genes related to cell development and differentiation are likely to be regulated by various microRNAs. We used a variety of the web based programs to identify microRNA candidates that may potentially regulate AF1q based on the predicted targeting efficiency. We found the strongest predicted binder to the AF1q 3′untranslated region (3′UTR) was miR-29b, a member of the miR-29 family which has recently been characterized to regulate a member of the Bcl-2 family protein, Mcl-1 and other leukemia related oncogenes. We found that MiR-29b expression had a significantly inverse correlation (p Disclosures: No relevant conflicts of interest to declare.

Chromosome translocations are among the most common genetic abnormalities in human leukemia. The mixed lineage leukemia (MLL) gene was identified as a common target of chromosomal translocations associated with human acute leukemias; it is located on human chromosome 11 band q23 and on mouse chromosome 9. More than 50 different loci are rearranged in11q23 leukemias involving MLL, resulting in either acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL). In general, MLL rearrangements are associated with a poor prognosis. MLL-ELL and MLL-ENL resulting from t(11;19)(q23;p13.1) and t(11;19)(q23;p13.3) respectively are two common examples of these rearrangements. These two fusions are frequently involved in human AML, while MLL-ENL is also involved in human ALL. There is a common observation that important biological properties are often conserved across species. Cross-species sequence comparison has been widely used to infer gene function, but it is becoming apparent that sequence similarity is not always proportional to functional similarity. To determine the function of a gene precisely, therefore, we need to investigate not only its sequence characteristics but also its expression characteristics. Model organisms have contributed substantially to our understanding of the etiology of human disease and the development of new treatment methodologies. However, although genetically engineered mouse leukemia models have been established for many years, there are few systematic studies to identify and study the genes that exhibit similar abnormal expression patterns in both human leukemia and mouse leukemia model cells. To perform an interspecies gene expression comparative study in leukemia, we used the serial analysis of gene expression (SAGE) technique to examine gene expression profiles between MLL-ELL or MLL-ENL myeloid leukemia progenitor cells and normal myeloid progenitor cells in both humans and mice. We obtained 484,303 total SAGE tags for the nine samples and a total of 103,899 unique SAGE tags from five human and 60,993 from four mouse samples. We identified 88 genes that appeared to be significantly deregulated (32 up- and 56 down-regulated) in both human and murine MLL-ELL and/or MLL-ENL leukemia. Fifty-seven genes have not been reported previously. A large-scale quantitative real-time PCR (qPCR) assay was performed to validate the candidate genes, and 84% (36/43) of the tested SAGE candidate genes were confirmed. The most up-regulated genes include several HOX genes (e.g., HOX A5, HOXA9 and HOXA10) and a HOX cofactor MEIS1; their overexpression is a hallmark of MLL-rearrangement leukemia. The top down-regulated genes include LTF, LCN2, MMP9, S100A8, S100A9, PADI4, TGFBI and CYBB. Remarkably, up-regulated genes have a much higher percentage of enrichment in Gene Ontology (GO) terms related to gene expression and transcription, whereas down-regulated genes are more enriched in GO terms related to apoptosis, signal transduction and response. Thus, the up-regulation of genes responsible for gene expression and transcription but down-regulation of genes responsible for apoptosis, signal transduction and response, can promote cell proliferation and inhibit apoptosis, and thereby contribute to the development of leukemia. We showed that the CpG islands of several significantly down-regulated genes including LIF, TGFBI and G0S2 are hypermethylated. We also examined the expression of microRNAs from the mir-17–92 cluster, which are overexpressed in human MLL-rearrangement leukemias, and showed that seven individual microRNAs (i.e., miR-17-5p, miR-17-3p, miR-18a, miR-19a, miR-20a, miR-19b and miR-92) within this cluster are also overexpressed in mouse MLL-rearrangement leukemia cells. Nineteen putative targets (i.e., APP, RASSF2, SH3BP5, DBN1, ELK3, FLT1, GNAI1, HIF1A, ITGA6, MN1, POU4F1, RB1, RGL1, RNF167, SASH1, SLC24A3, TNFRSF21, WWP1 and YES1) of these microRNAs were reported and/or confirmed by our qPCR to be down-regulated in MLL-rearrangement leukemias. We further confirmed both APP and RASSF2 as direct targets of miR-17 through luciferase reporter and mutagenesis assay. The identification and validation of gene expression changes in MLL-rearrangement human and murine leukemia provides important insights into the genetic pathways that are important for MLL fusion-induced leukemogenesis.

Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults. It is estimated that 13,410 cases will be diagnosed and 8,990 will die of AML in the United States in 2007 (http://seer.cancer.gov). AML is a genetically diverse hematopoietic malignancy with variable response to treatment. Expression profiling of protein-coding genes using DNA microarray in AML has resulted in inconsistent data from different laboratories. Therefore, further validation of these observations in large cohorts and in independent studies is definitely required before clinical application becomes feasible. Recently, Golub and colleagues described a new, bead-based flow cytometric microRNA (miRNAs, miRs) expression profiling method that could successfully classify tumors. MiRNAs are endogenous ∼22 nucleotide non-coding RNAs, which can function as oncogenes and tumor suppressors. To provide new insights into the complex genetic alterations in leukemogenesis and to identify novel markers for diagnosis and treatment of AML, we performed a genome-wide analysis of miRNA expression profiles using the bead-based method on 54 AML samples with common translocations including t(15;17), t(8;21), inv(16), and 11q23 rearrangement, along with normal controls. In both unsupervised and supervised hierarchical cluster analyses, we observed that t(15;17) samples grouped together as one cluster, as do the 11q23 rearrangement samples. Interestingly, t(8;21) and inv(16), both CBF (core-binding factor) AMLs, grouped together as a unique cluster. Forty-one miRNAs exhibited significantly differential expression between different subtypes of AMLs, and/or between AMLs and normal controls. Notably, expression signature of a minimal number of two, three, and seven miRNAs could be used for class prediction of CBF, t(15;17), and 11q23 rearrangement AMLs, respectively, with an overall diagnostic accuracy of 94–96%. We further showed that overexpression of the two discriminatory miRNAs in CBF AML is associated with epigenetic regulation, rather than DNA copy number amplification. Moreover, several important target genes of these discriminatory miRNAs have also been validated. We are currently exploring the role of these discriminatory miRNAs and their critical target genes in the development of AML using in vitro and in vivo models. This work will enhance our understanding of the biological role of these miRNAs and their targets in leukemogenesis.

Human acute leukemias include acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). It is estimated that 5,200 and 13,410 cases will be diagnosed with and 1,420 and 8,990 will die of ALL and AML, respectively, in the United States in 2007. Although remarkable progress has been made in the past decades in the treatment and in the understanding of the biology of acute leukemias, the 5-year overall survival rate of patients with AML is only approximately 22%, which is much lower than that of ALL patients (65%; http://seer.cancer.gov). One of the most exciting recent findings is the discovery of an abundant class of small (∼22 nt), non-(protein-)coding RNAs, called microRNAs (miRNAs, miRs), which can function as oncogenes and tumor suppressors, whose deregulation is clearly associated with the development of cancer. To understand the distinct mechanisms in leukemogenesis between ALL and AML and to identify novel markers for diagnosis and treatment of acute leukemia, we have performed a large-scale miRNA expression profiling assay with a bead-based flow cytometric method and identified 27 differentially expressed miRNAs. Among them, miR-128a and b are significantly overexpressed while let-7b and miR-223 are significantly down-regulated in ALL compared to AML. They are the most discriminatory miRNAs between ALL and AML. Using the expression signatures of any two of the four most significantly discriminatory miRNAs in diagnosis of ALL and AML resulted in an accuracy rate of 97–100%. The differential expression patterns of these four miRNAs were validated further through quantitative real-time PCR on 98 acute leukemia samples covering most of the common cytogenetic subtypes of AML and B- and T-cell ALL, along with 10 normal controls. Furthermore, we found that overexpression of miR-128a and b in ALL was at least partly associated with hypomethylation, rather than amplification of DNA locus copy. Moreover, several important target genes of these four miRNAs have also been validated. We are currently exploring the role of these four miRNAs and their critical target genes in leukemogenesis and in the determination of lineage fate during leukemia development using in vitro and in vivo models. This work will enhance our understanding of the biological role of these miRNAs and their targets in leukemogenesis, and in determining the lineage fate of acute leukemia.

Chromosome translocations are among the most common genetic abnormalities in human leukemia. Their abnormally expressed genes identify specific markers for their clinical diagnosis. Important biological properties are often conserved across species. However, although genetically engineered mouse leukemia models are well-established, few systematic studies have validated the genes that exhibit similar abnormal expression patterns in both human and mouse leukemia models. MLL-ELL and MLL-ENL fusion genes resulting from t(11;19)(q23;p13.1) and t(11;19)(q23;p13.3), respectively, are frequently involved in human acute leukemia, and in retrovirus-mediated mouse leukemia models. We used the SAGE technique to compare gene expression profiles between MLL-ELL or MLL-ENL myeloid leukemia progenitor cells and normal myeloid progenitor cells in both human and mouse. We analyzed four patient samples (two with each fusion) and two retrovirally-induced mouse leukemias containing either MLL-ELL or MLL-ENL fusions, and a leukemia cell line with an MLL-ELL fusion. 484,303 SAGE tags were identified from the nine samples, yielding 103,899 unique tags in human and 60,993 in mouse samples. We identified 40 candidate genes that appear to be abnormally expressed in both human and murine MLL-ELL leukemias (2 up- and 38 down-regulated), and 72 in both human and murine MLL-ENL leukemias (23 up and 49 down). 25 candidate genes are down-regulated in both types of leukemias, and many of them can bind with and/or regulate other candidate genes in the candidate list. For example, LCN2 can bind directly with and positively regulate MMP9; MMP9 and TMSB4X may positively regulate FOS; FOS and JUNB can bind directly and positively regulate each other. JUNB may inhibit proliferation and promote apoptosis, and it was reported that inactivation of JunB in LT-HSC leads to MPD while its inactivation in committed myeloid progenitors also predisposes to leukemia evolution. LCN2 may also positively regulate apoptosis. Meanwhile, some important candidate genes are observed only in one type of leukemia. For example, both PXN and ARHGEF1 are down-regulated only in MLL-ELL leukemias. PXN can bind directly with ARHGEF1, and the latter may inhibit proliferation. Similarly, MYB is significantly upregulated only in MLL-ENL leukemias, which was reported to play a role in MLL-ENL-mediated transformation. Taken together, some common pathways may exist in the development of both types of leukemias, whereas each may also have their own pathway. The deregulation of the important candidate genes may contribute to leukemogenesis through inhibiting apoptosis while promoting proliferation of hematopoietic cells. We have validated the expression patterns of the candidate genes, and are studying the functions and pathways of the validated candidate genes. Our studies will provide important insights into the complex functional pathways related to MLL rearrangements in the development of acute myeloid leukemia, which may lead to more effective therapy for these leukemias.

MicroRNAs (miRNAs), are small non-coding RNA molecules known to be important regulators of cancer biology. Notably, we and others have shown that miRNAs play important roles in Acute Myeloid Leukemia (AML), a heterogeneous malignancies with multiple chromosomal and molecular abnormalities. Patients with chromosomal rearrangements involving mixed lineage leukemia (MLL), the mammalian homology of trithorax gene, are associated with poor survival. Previously, we have found that MLL-rearranged AML drives aberrant expression of several miRNAs, most notably microRNA-9 (miR-9). Expression of miR-9 with MLL-AF9, a common MLL-translocation, was sufficient to promote transformation normal hematopoietic progenitor cells in vitro and leukemogenesis in vivo. We previously found that miR-9 reduces expression of several genes but we did not know which genes were critical tumor suppressors. We found that the polycomb group member RING1- and YY1-Bindin Protein (RYBP) was consistently inhibited upon miR-9 expression. To assess the regulation of RYBP we used publically available data from the Cancer Genome Atlas (TCGA) and looked at genome-wide Illumina 450K methylation data. We did not find a strong correlation with methylation and RYBP expression, suggesting that expression of RYBP is likely not regulated by the DNA methylation machinery in patients. Upon looking at copy number alterations we found that a small population of AML patients contained either homozygous or heterozygous loss of RYBP, suggesting a potential role of RYBP in leukemia pathogenesis. To assess the role of RYBP we did a series of in vitro experiments. We found that expression of RYBP was sufficient to attenuate colony-forming growth driven by MLL- AF9. Furthermore, RYBP expression was able to reduce proliferation, increase apoptosis, and significantly reduce immature cell population. To determine the role of RYBP expression in vivo, we transplanted lethally irradiated mice with progenitors retrovirally transduced with MLL-AF9 compared to MLL-AF9 and RYBP. We found that expression of RYBP was sufficient to reduce leukemia burden in vivo as well as induce differentiation as shown by flow cytometry and histological analysis. Thus, this demonstrates that RYBP is a functional tumor suppressor in MLL-rearranged AML. In conclusion, we have demonstrated that chromosomal rearrangements involving MLL, the mammalian homology of trithorax, downregulates a member of the polycomb complex through upregulation of miR-9. Disclosures No relevant conflicts of interest to declare.