Central to cellular proliferative, survival, and metabolic responses is the serine/threonine kinase mTOR, which is activated in many human cancers. mTOR is present in distinct complexes that are either modulated by AKT (mTORC1) or are upstream and regulatory of it (mTORC2). Governance of mTORC2 activity is poorly understood. Here, we report a transmembrane molecule in hematopoietic progenitor cells that physically interacts with and inhibits RICTOR, an essential component of mTORC2. Upstream of mTORC2 (UT2) negatively regulates mTORC2 enzymatic activity, reducing AKTS473, PKCα, and NDRG1 phosphorylation and increasing FOXO transcriptional activity in an mTORC2-dependent manner. Modulating UT2 levels altered animal survival in a T cell acute lymphoid leukemia (T-ALL) model that is known to be mTORC2 sensitive. These studies identify an inhibitory component upstream of mTORC2 in hematopoietic cells that can reduce mortality from NOTCH-induced T-ALL. A transmembrane inhibitor of mTORC2 may provide an attractive target to affect this critical cell regulatory pathway.
•UT2 is a transmembrane inhibitor of mTORC2 in hematopoietic progenitors•UT2 directly binds RICTOR, limiting the kinase activity of mTORC2 on pAKTS473•UT2 modulates the outcome of an mTORC2-dependent hematopoietic cancer
In this article, Scadden and colleagues show that an undefined transmembrane molecule upstream of mTORC2 (UT2) negatively regulates AKT signaling via modulation of mTORC2. Their work points to the value of models that can be used to examine niche contributions to oncogenesis and reveals a previously unrecognized transmembrane modulator of a critical pathway with therapeutic implications for cancers such as T-ALL.
Relapsed pediatric acute lymphoblastic leukemia (ALL) has high rates of treatment failure. Epigenetic regulators have been proposed as modulators of chemoresistance, here we sequence genes encoding epigenetic regulators in matched diagnosis-remission-relapse ALL samples. We find significant enrichment of mutations in epigenetic regulators at relapse with recurrent somatic mutations in SETD2, CREBBP, MSH6, KDM6A and MLL2, mutations in signaling factors are not enriched. Somatic alterations in SETD2, including frameshift and nonsense mutations, are present at 12% in a large de novo ALL patient cohort. We conclude that the enrichment of mutations in epigenetic regulators at relapse is consistent with a role in mediating therapy resistance.
We used an in vivo short hairpin RNA (shRNA) screening approach to identify genes that are essential for MLL-AF9 acute myeloid leukemia (AML). We found that Integrin Beta 3 (Itgb3) is essential for murine leukemia cells in vivo, and for human leukemia cells in xenotransplantation studies. In leukemia cells, Itgb3 knockdown impaired homing, downregulated LSC transcriptional programs, and induced differentiation via the intracellular kinase, Syk. In contrast, loss of Itgb3 in normal HSPCs did not affect engraftment, reconstitution, or differentiation. Finally, we confirmed that Itgb3 is dispensable for normal hematopoiesis and required for leukemogenesis using an Itgb3 knockout mouse model. Our results establish the significance of the Itgb3 signaling pathway as a potential therapeutic target in AML.
Mislocated enzymatic activity of DOT1L has been proposed as a driver of leukemogenesis in mixed lineage leukemia (MLL). The characterization of EPZ004777, a potent, selective inhibitor of DOT1L is reported. Treatment of MLL cells with the compound selectively inhibits H3K79 methylation and blocks expression of leukemogenic genes. Exposure of leukemic cells to EPZ004777 results in selective killing of those cells bearing the MLL gene translocation, with little effect on non-MLL-translocated cells. Finally, in vivo administration of EPZ004777 leads to extension of survival in a mouse MLL xenograft model. These results provide compelling support for DOT1L inhibition as a basis for targeted therapeutics against MLL.
MLL-rearranged leukemias exemplify malignancies with perturbations of the epigenetic landscape. Specific chromatin modifications that aid in the perpetuation of MLL-fusion gene driven oncogenic programs are being defined, presenting novel avenues for therapeutic intervention. Proof-of-concept studies have recently been reported, using small-molecule inhibitors targeting the histone methyltransferase DOT1L or the acetyl-histone binding protein BRD4 showing potent activity against MLL-rearranged leukemias in pre-clinical models. It is apparent that intensive efforts will be made toward the further development of small-molecule inhibitors targeting these, and other chromatin-associated protein targets. These studies may lead to the advent of a new generation of much-needed therapeutic modalities in leukemia and other cancers.
chromatin modifications; leukemia; epigenetics; targeted therapy; MLL
Leukemias and other cancers have been proposed to contain a subpopulation of cells that display characteristics of stem cells, and which maintain tumor growth. That most anti-cancer therapy is directed against the bulk of the tumor, and possibly spares the cancer stem cells, may lie at the heart of treatment failures with conventional modalities. Leukemia stem cells are fairly well described for acute myeloid leukemia (AML), but their existence and relevance for acute lymphoblastic leukemia (ALL) is less clear. Several reports describe subpopulations with primitive phenotypes in clinical ALL samples. However, it has also been suggested that the majority of leukemic subfractions can propagate leukemia in the appropriate experimental setting, and that their hierarchical organization is less strict than in AML. In addition, it is uncertain whether cancer stem cells arise from malignant transformation of a tissue-specific stem cell, or from committed progenitors or differentiated cells that re-acquire a stem cell-like program. In common childhood ALL, current evidence points towards the cell of origin being a committed lymphoid progenitor. In this review, we highlight recent findings relating to the question of leukemia stem cells in ALL.
The t(10;11)(p12;q23) translocation and the t(10;11)(p12;q14) translocation, which encode the MLL-AF10 and CALM-AF10 fusion oncoproteins respectively, are two recurrent chromosomal rearrangements observed in patients with acute myeloid leukemia and acute lymphoblastic leukemia. Here we demonstrate that MLL-AF10 and CALM-AF10 mediated transformation is dependent on the H3K79 methyltransferase Dot1l using genetic and pharmacological approaches in mouse models. Targeted disruption of Dot1l using a conditional knockout mouse model abolished in vitro transformation of murine bone marrow cells and in vivo initiation and maintenance of MLL-AF10 or CALM-AF10 leukemia.. Treatment of MLL-AF10 and CALM-AF10 transformed cells with EPZ004777, a specific small-molecule inhibitor of Dot1l, suppressed expression of leukemogenic genes such as Hoxa cluster genes and Meis1, and selectively suppressed proliferation of MLL-AF10 and CALM-AF10 transformed cells. Pretreatment with EPZ004777 profoundly decreased the in vivo spleen-colony forming ability of MLL-AF10 or CALM-AF10 transformed bone marrow cells. These results show that patients with leukemias bearing chromosomal translocations that involve the AF10 gene may benefit from small molecule therapeutics that inhibit H3K79 methylation.
MLL-AF10; CALM-AF10; MLL fusions; leukemia; Dot1l; EPZ004777
AKT activation is associated with many malignancies, where AKT acts, in part, by inhibiting FOXO tumor suppressors. We show a converse role for AKT/FOXOs in acute myeloid leukemia (AML). Rather than decreased FOXO activity, we observed that FOXOs are active in ∼40% of AML patient samples regardless of genetic subtype. We also observe this activity in human MLL-AF9 leukemia allele-induced AML in mice, where either activation of Akt or compound deletion of FoxO1/3/4 reduced leukemic cell growth, with the latter markedly diminishing leukemia-initiating cell (LIC) function in vivo and improving animal survival. FOXO inhibition resulted in myeloid maturation and subsequent AML cell death. FOXO activation inversely correlated with JNK/c-JUN signaling, and leukemic cells resistant to FOXO inhibition responded to JNK inhibition. These data reveal a molecular role for AKT/FOXO and JNK/c-JUN in maintaining a differentiation blockade that can be targeted to inhibit leukemias with a range of genetic lesions.
Methylation of H3K79 is associated with chromatin at expressed genes, though it is unclear if this histone modification is required for transcription of all genes. Recent studies suggest that Wnt-responsive genes depend particularly on H3K79 methylation, which is catalyzed by the methyltransferase DOT1L. Human leukemias carrying MLL gene rearrangements show DOT1L-mediated H3K79 methylation and aberrant expression of leukemogenic genes. DOT1L inhibitors reverse these effects, but their clinical use is potentially limited by toxicity in Wnt-dependent tissues such as intestinal epithelium. Genome-wide positioning of the H3K79me2 mark in Lgr5+ mouse intestinal stem cells and mature intestinal villus epithelium correlated with expression levels of all transcripts and not with Wnt-responsive genes per se. Selective Dot1l disruption in Lgr5+ stem cells or in whole intestinal epithelium eliminated H3K79me2 from the respective compartments, allowing genetic evaluation of DOT1L requirements. The absence of methylated H3K79 did not impair health, intestinal homeostasis, or expression of Wnt target genes in crypt epithelium for up to 4 months, despite increased crypt cell apoptosis. Global transcript profiles in Dot1l-null cells were barely altered. Thus, H3K79 methylation is not essential for transcription of Wnt-responsive or other intestinal genes, and intestinal toxicity is not imperative when DOT1L is rendered inactive in vivo.
The mammalian target of rapamycin (mTOR)-pathway serves as a key sensor of cellular-energetic state, and functions to maintain tissue homeostasis. Hyperactivation of the mTOR pathway impairs hematopoietic stem cell (HSC) function and is associated with leukemogenesis. However, the roles of the unique mTOR complexes (mTORCs) in hematopoeisis and leukemogenesis have not been adequately elucidated. We deleted the mTORC1 component, Raptor (regulatory-associated protein of mTOR), in mouse HSC and its loss causes a non-lethal phenotype characterized by pancytopenia, splenomegaly, and the accumulation of monocytoid cells. Furthermore, Raptor is required for HSC regeneration, and plays largely non-redundant roles with Rictor (rapamycin-insensitive companion of mTOR), in these processes. Ablation of Raptor also significantly extends survival of mice in models of leukemogenesis evoked by Pten deficiency. These data delineate critical roles for mTORC1 in hematopoietic function and leukemogenesis, and inform clinical strategies based on chronic mTORC1 inhibition.
Notch behaves as a tumor suppressor in AML, and Notch activation induces cell cycle arrest, differentiation, and apoptosis of AML-initiating cells.
Notch signaling pathway activation is known to contribute to the pathogenesis of a spectrum of human malignancies, including T cell leukemia. However, recent studies have implicated the Notch pathway as a tumor suppressor in myeloproliferative neoplasms and several solid tumors. Here we report a novel tumor suppressor role for Notch signaling in acute myeloid leukemia (AML) and demonstrate that Notch pathway activation could represent a therapeutic strategy in this disease. We show that Notch signaling is silenced in human AML samples, as well as in AML-initiating cells in an animal model of the disease. In vivo activation of Notch signaling using genetic Notch gain of function models or in vitro using synthetic Notch ligand induces rapid cell cycle arrest, differentiation, and apoptosis of AML-initiating cells. Moreover, we demonstrate that Notch inactivation cooperates in vivo with loss of the myeloid tumor suppressor Tet2 to induce AML-like disease. These data demonstrate a novel tumor suppressor role for Notch signaling in AML and elucidate the potential therapeutic use of Notch receptor agonists in the treatment of this devastating leukemia.
New strategies to combat complex human disease require systems approaches to biology that integrate experiments from cell lines, primary tissues and model organisms. We have developed Pathprint, a functional approach that compares gene expression profiles in a set of pathways, networks and transcriptionally regulated targets. It can be applied universally to gene expression profiles across species. Integration of large-scale profiling methods and curation of the public repository overcomes platform, species and batch effects to yield a standard measure of functional distance between experiments. We show that pathprints combine mouse and human blood developmental lineage, and can be used to identify new prognostic indicators in acute myeloid leukemia. The code and resources are available at http://compbio.sph.harvard.edu/hidelab/pathprint
Hematopoietic malignant relapse still remains the major cause of death following allogeneic hematopoietic stem cell transplantation (HSCT). Although there has been a large focus on the immunologic mechanisms responsible for the graft-versus-tumor (GVT) effect or lack thereof, there has been little attention paid to investigating the biologic basis of hematologic malignant disease relapse following allogeneic HSCT. There are a large number of factors that are responsible for the biologic resistance of hematopoietic tumors following allogeneic HSCT. We have focused on 5 major areas including clonal evolution of cancer drug resistance, cancer radiation resistance, genomic basis of leukemia resistance, cancer epigenetics, and resistant leukemia stem cells. We recommend increased funding to pursue 3 broad areas that will significantly enhance our understanding of the biologic basis of malignant relapse after allogeneic HSCT, including: (1) genomic and epigenetic alterations, (2) cancer stem cell biology, and (3) clonal cancer drug and radiation resistance.
Relapse; Allogeneic stem cell transplant; Biology; Resistance; Cancer stem cells
Inactivation of Llgl1 enhances HSC self-renewal and fitness and is associated with unfavorable outcome in human AML.
A unique characteristic of hematopoietic stem cells (HSCs) is the ability to self-renew. Several genes and signaling pathways control the fine balance between self-renewal and differentiation in HSCs and potentially also in leukemia stem cells. Recently, studies have shed light on developmental molecules and evolutionarily conserved signals as regulators of stem cells in hematopoiesis and leukemia. In this study, we provide evidence that the cell fate determinant Llgl1 (lethal giant larvae homolog 1) plays an important role in regulation of HSCs. Loss of Llgl1 leads to an increase in HSC numbers that show increased repopulation capacity and competitive advantage after transplantation. This advantage increases upon serial transplantation or when stress is applied to HSCs. Llgl1−/− HSCs show increased cycling but neither exhaust nor induce leukemia in recipient mice. Llgl1 inactivation is associated with transcriptional repression of transcription factors such as KLF4 (Krüppel-like factor 4) and EGR1 (early-growth-response 1) that are known inhibitors of HSC self-renewal. Decreased Llgl1 expression in human acute myeloid leukemia (AML) cells is associated with inferior patient survival. Thus, inactivation of Llgl1 enhances HSC self-renewal and fitness and is associated with unfavorable outcome in human AML.
A key characteristic of hematopoietic stem cells (HSCs) is the ability to self-renew. Genetic deletion of β-catenin during fetal HSC development leads to impairment of self-renewal while β-catenin is dispensable in fully developed adult HSCs. Whether β-catenin is required for maintenance of fully developed CML leukemia stem cells (LSCs) is unknown. Here, we use a conditional mouse model to show that deletion of β-catenin after CML initiation does not lead to a significant increase in survival. However, deletion of β-catenin synergizes with imatinib (IM) to delay disease recurrence after imatinib discontinuation and to abrogate CML stem cells. These effects can be mimicked by pharmacologic inhibition of β-catenin via modulation of prostaglandin signaling. Treatment with the cyclooxygenase inhibitor indomethacin reduces β-catenin levels and leads to a reduction in LSCs. In conclusion, inhibiting β-catenin by genetic inactivation or pharmacologic modulation is an effective combination therapy with imatinib and targets CML stem cells.
Generation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming involves global epigenetic remodeling1. While several proteins are known to regulate chromatin marks associated with the distinct epigenetic states of cells before and after reprogramming2,3, the role of specific chromatin modifying enzymes in reprogramming remains to be determined. To address how chromatin-modifying proteins influence reprogramming, we used shRNAs to target genes in DNA and histone methylation pathways, and have identified positive and negative modulators of iPSC generation. While inhibition of the core components of the polycomb repressive complex 1 and 2, including the histone 3 lysine 27 methyltransferase Ezh2, reduced reprogramming efficiency, suppression of SUV39H1, YY1, and Dot1L enhanced reprogramming. Specifically, inhibition of the H3K79 histone methyltransferase Dot1L by shRNA or a small molecule accelerated reprogramming, significantly increased the yield of iPSC colonies, and substituted for Klf4 and c-Myc. Inhibition of Dot1L early in the reprogramming process is associated with a marked increase in two alternative factors, Nanog and Lin28, which play essential functional roles in the enhancement of reprogramming. Genome-wide analysis of H3K79me2 distribution revealed that fibroblast-specific genes associated with the epithelial to mesenchymal transition lose H3K79me2 in the initial phases of reprogramming. Dot1L inhibition facilitates the loss of this mark from genes that are fated to be repressed in the pluripotent state. These findings implicate specific chromatin-modifying enzymes as barriers to or facilitators of reprogramming, and demonstrate how modulation of chromatin-modifying enzymes can be exploited to more efficiently generate iPSCs with fewer exogenous transcription factors.
The histone 3 lysine 79 (H3K79) methyltransferase Dot1l has been implicated in the development of leukemias bearing translocations of the Mixed Lineage Leukemia (MLL) gene. We identified the MLL-fusion targets in an MLL-AF9 leukemia model, and conducted epigenetic profiling for H3K79me2, H3K4me3, H3K27me3 and H3K36me3 in hematopoietic progenitor and leukemia stem cells (LSC). We found abnormal profiles only for H3K79me2 on MLL-AF9 fusion target loci in LSC. Inactivation of Dot1l lead to down-regulation of direct MLL-AF9 targets and an MLL-translocation associated gene expression signature, while global gene expression remained largely unaffected. Suppression of MLL-translocation associated gene expression corresponded with dependence of MLL-AF9 leukemia on Dot1l in vivo. These data point to DOT1L as a potential therapeutic target in MLL-rearranged leukemia.
A key characteristic of hematopoietic stem cells (HSC) is the ability to self-renew. Several genes and signaling pathways control the fine balance between self-renewal and differentiation in HSC and potentially also in leukemic stem cells. Besides pathways such as Wnt signaling, Hedgehog signaling and Notch signaling, transcription factors (FoxOs) and cell fate determinants may also play a role in stem cells. While some of these pathways seem to be dispensable for maintenance of adult HSC, there may be a distinct requirement in leukemia stem cells for leukemic self-renewal. Here we will focus on self-renewal related signaling in myeloid leukemia stem cells and its therapeutic relevance.
Leukemia stem cell; Self-renewal; Hedgehog; Wnt; Notch; FoxO
Characterization of gene expression programs and pathways important for normal and cancer stem cells has become an active area of investigation. Microarray analysis of various cell populations provides an opportunity to assess genomewide expression programs to define cellular identity and to potentially identify pathways activated in various stem cells. Here we describe methods to isolate a leukemia stem cell population, amplify RNA, and perform microarray analyses.
Stem cells; Leukemia stem cells; Gene expression profiling; Microarray; MLL-AF9; Leukemia; Gene expression; RNA amplification
Increasing use of high throughput genomic scale assays requires effective visualization and analysis techniques to facilitate data interpretation. Moreover, existing tools often require programming skills, which discourages bench scientists from examining their own data. We have created iCanPlot, a compelling platform for visual data exploration based on the latest technologies. Using the recently adopted HTML5 Canvas element, we have developed a highly interactive tool to visualize tabular data and identify interesting patterns in an intuitive fashion without the need of any specialized computing skills. A module for geneset overlap analysis has been implemented on the Google App Engine platform: when the user selects a region of interest in the plot, the genes in the region are analyzed on the fly. The visualization and analysis are amalgamated for a seamless experience. Further, users can easily upload their data for analysis—which also makes it simple to share the analysis with collaborators. We illustrate the power of iCanPlot by showing an example of how it can be used to interpret histone modifications in the context of gene expression.
Fusion oncogenes in acute myeloid leukemia (AML) promote self-renewal from committed progenitors, thereby linking transformation and self-renewal pathways. Like most cancers, AML is a genetically and biologically heterogeneous disease, but it is unclear whether transformation results from common or overlapping genetic programs acting downstream of multiple mutations, or by the engagement of unique genetic programs acting cooperatively downstream of individual mutations. This distinction is important, because the involvement of common programs would imply the existence of common molecular targets to treat AML, no matter which fusion oncogenes are involved. Here we demonstrate that the ability to promote self-renewal is a generalized property of leukemia-associated oncogenes. Disparate oncogenes initiated overlapping transformation and self-renewal gene expression programs, the common elements of which were defined in established leukemia stem cells from an animal model as well as from a large cohort of patients with differing AML subtypes, where they strongly predicted pathobiological character. Notably, individual genes commonly activated in these programs could partially phenocopy the self-renewal function of leukemia-associated oncogenes in committed murine progenitors. Further, they could generate AML following expression in murine bone marrow. In summary, our findings reveal the operation of common programs of self-renewal and transformation downstream of leukemia-associated oncogenes, suggesting mechanistically common therapeutic approaches to AML are likely to be possible, regardless of the identity of the driver oncogene involved.
Leukemia-associated fusion genes; activation of self-renewal; transcriptional dysregulation; common pathways; therapeutic targets
Mounting evidence suggests that malignant tumors are initiated and maintained by a subpopulation of cancerous cells with biological properties similar to those of normal stem cells. However, descriptions of stem-like gene and pathway signatures in cancers are inconsistent across experimental systems. Driven by a need to improve our understanding of molecular processes that are common and unique across cancer stem cells (CSCs), we have developed the Stem Cell Discovery Engine (SCDE)—an online database of curated CSC experiments coupled to the Galaxy analytical framework. The SCDE allows users to consistently describe, share and compare CSC data at the gene and pathway level. Our initial focus has been on carefully curating tissue and cancer stem cell-related experiments from blood, intestine and brain to create a high quality resource containing 53 public studies and 1098 assays. The experimental information is captured and stored in the multi-omics Investigation/Study/Assay (ISA-Tab) format and can be queried in the data repository. A linked Galaxy framework provides a comprehensive, flexible environment populated with novel tools for gene list comparisons against molecular signatures in GeneSigDB and MSigDB, curated experiments in the SCDE and pathways in WikiPathways. The SCDE is available at http://discovery.hsci.harvard.edu.
Genome-wide experiments are routinely conducted to measure gene expression, DNA-protein interactions and epigenetic status. Structured metadata for these experiments is imperative for a complete understanding of experimental conditions, to enable consistent data processing and to allow retrieval, comparison, and integration of experimental results. Even though several repositories have been developed for genomics data, only a few provide annotation of samples and assays using controlled vocabularies. Moreover, many of them are tailored for a single type of technology or measurement and do not support the integration of multiple data types.
We have developed eXframe - a reusable web-based framework for genomics experiments that provides 1) the ability to publish structured data compliant with accepted standards 2) support for multiple data types including microarrays and next generation sequencing 3) query, analysis and visualization integration tools (enabled by consistent processing of the raw data and annotation of samples) and is available as open-source software. We present two case studies where this software is currently being used to build repositories of genomics experiments - one contains data from hematopoietic stem cells and another from Parkinson's disease patients.
The web-based framework eXframe offers structured annotation of experiments as well as uniform processing and storage of molecular data from microarray and next generation sequencing platforms. The framework allows users to query and integrate information across species, technologies, measurement types and experimental conditions. Our framework is reusable and freely modifiable - other groups or institutions can deploy their own custom web-based repositories based on this software. It is interoperable with the most important data formats in this domain. We hope that other groups will not only use eXframe, but also contribute their own useful modifications.
This study evaluated the in vitro and in vivo pharmacological properties of TD-8954, a potent and selective 5-HT4 receptor agonist. TD-8954 had high affinity (pKi = 9.4) for human recombinant 5-HT4(c) (h5-HT4(c)) receptors, and selectivity (>2,000-fold) over all other 5-hydroxytryptamine (5-HT) receptors and non-5-HT receptors, ion channels, enzymes and transporters tested (n = 78). TD-8954 produced an elevation of cAMP in HEK-293 cells expressing the h5-HT4(c) receptor (pEC50 = 9.3), and contracted the guinea pig colonic longitudinal muscle/myenteric plexus preparation (pEC50 = 8.6). TD-8954 had moderate intrinsic activity in the in vitro assays. In conscious guinea pigs, subcutaneous administration of TD-8954 (0.03–3 mg/kg) increased the colonic transit of carmine red dye, reducing the time taken for its excretion. Following intraduodenal dosing to anesthetized rats, TD-8954 (0.03–10 mg/kg) evoked a dose-dependent relaxation of the esophagus. Following oral administration to conscious dogs, TD-8954 (10 and 30 μg/kg) produced an increase in contractility of the antrum, duodenum, and jejunum. In a single ascending oral dose study in healthy human subjects, TD-8954 (0.1–20 mg) increased bowel movement frequency and reduced the time to first stool. It is concluded that TD-8954 is a potent and selective 5-HT4 receptor agonist in vitro, with robust in vivo stimulatory activity in the gastrointestinal (GI) tract of guinea pigs, rats, dogs, and humans. TD-8954 may have clinical utility in patients with disorders of reduced GI motility.
constipation; serotonin; 5-HT4; prokinetic; TD-8954