The role of TET proteins in hematopoietic malignancy


Recent studies have revealed a new layer of epigenetic regulation in stem cells and differentiated cell types whereby methyl groups at the 5-position of cytosine bases (5mC) can be modified by a novel family of α-keto-glutarate (α-KG)-dependent enzymes: Ten-Eleven-Translocation (TET) proteins, TET1, 2 and 3. All three TET proteins catalyze the conversion of 5mC to 5-hydroxymethylcytosine (5hmC), a DNA modification that is shown to be enriched at enhancers, promoters and gene bodies of actively expressed genes. The presence of 5hmC has been proposed by several studies to contribute to both passive and active DNA demethylation in the mammalian genome. Decreased expression of TET proteins and loss of 5hmC is a hallmark of many cancers, suggesting a critical role for the maintenance of this epigenetic modification in normal cellular function. The TET gene family was first identified because of the involvement of TET1 as a fusion partner of MLL in acute myeloid leukemia (AML). Studies in large cohorts of myeloid dysplastic syndrome (MDS) and AML patients revealed that somatic mutations in TET2 are found in 10 – 50% of cases. Our lab and other groups have generated knockout mouse models of Tet2 to confirm that loss of function promotes aberrant stem cell self-renewal and hematopoietic malignancy. In addition, we have found that Tet1 deletion in mice promotes lymphomagenesis. These studies highlight the non-redundant role of TET proteins to act as tumor suppressors of hematopoietic malignancy. Our current goal is to determine how modulation of the levels of 5mC and 5hmC predispose hematopoietic stem and progenitor cell to transformation. We are currently modeling the role of TET proteins in B, T and myeloid malignancy using conditional deletion and transgenic RNAi mouse models for inducible and reversible loss-of-function studies. Our goal is to identify novel and potential therapeutic targets in stem, progenitor and mature cells of TET proteins that regulate hematopoietic transformation and maintenance of aberrant DNA methylation.

Role of Microenvironmental factors in T-ALL progression

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive adult and pediatric blood cancer. A quarter of childhood and half of adult T-ALL patients relapse within 5 years of treatment and, despite advances in chemotherapy protocols, receive a dismal prognosis. Recent attempts to introduce targeted therapies were limited by both toxicity and resistance, further highlighting the urgent need for novel innovative therapies. While much is known about cell-intrinsic factors promoting leukemia, little is understood about the role of the microenvironment in leukemia progression. Several lines of evidence support our hypothesis that leukemia-initiating cells (LIC) require a specialized microenvironment to survive, and that disrupting this niche may be a promising therapeutic strategy. Our recent work identified CXCL12-producing vascular endothelial cells as the first component of a T-ALL niche in the bone marrow. We demonstrated that in murine and xenograft models targeting CXCL12/CXCR4 signaling after disease onset dramatically reduced T-ALL burden, suggesting a potential new therapeutic paradigm for treatment of this aggressive cancer.

LncRNA mapping in leukemia

Acute myeloid and lymphoid leukemias are aggressive diseases, and treatment outcomes for patients remain dismal. Long non-coding RNAs (lncRNAs) have emerged as important regulators of cellular processes in normal and disease states, and we recently demonstrated that lncRNA LUNAR1 is an important regulator of downstream targets of oncogenic NOTCH1 signaling in human T-cell acute lymphoblastic leukemia (T-ALL) (Trimarchi et al Cell 2014). LncRNA drivers of acute myeloid leukemia (AML) remain largely unknown. Consequently we aim to use an integrative approach to identify functional lncRNAs that contribute to T-ALL and AML pathogenesis. We assembled a T-ALL and AML transcriptome based on whole transcriptome sequencing of human leukemia cell lines, normal hematopoietic cells, and a large number of patient samples. Thousands of novel lncRNAs were annotated based on sequence length >200 bp, the presence of active histone marks (H3K4me3 and H3K27ac), and low protein coding potential. Differential expression analysis revealed many lncRNAs that are overexpressed in leukemic cells compared to normal hematopoietic cells, including LUNAR1. We are also developing methods to identify and characterize protein complexes associated with lncRNAs implicated in leukemias. These findings will help us elucidate novel molecular mechanisms that drive leukemias and identify new targets for therapeutic intervention.

Dissecting the epigenetic landscape in Leukemia

data_miniT cell Acute Lymphoblastic Leukemia (T-ALL) and Myeloid Leukemia both constitute aggressive diseases, with T-ALL specifically presenting increased incidence in adolescents and young adults. Despite recent progress on initial disease remission, up to 25% of patients experience disease recurrence. While Notch1 activating mutations are found in more than 50% of patients with T-ALL, its interaction partners and the epigenetic changes in humans leading to target gene activation during oncogenesis remain largely unknown. Current Notch-related investigational drugs include use of antibodies against members of the Notch1 pathway or . Secretase Inhibitors (.SI) exhibiting gastroinstestinal toxicity, and can lead to goblet cell metaplasia requiring glucocorticoid treatment. We believe that development of therapeutic agents targeting tumor-specific epigenetic changes may represent an attractive strategy for leukemia. In order to fully map the molecular interactions, we take into consideration the epigenetic modifications as well as the genetic status of the disease with an emphasis on mutations of epigenetic modulators. Using high throughput screen techniques, such ChIP- and RNA-seq, exome sequencing, and RNAi-based functional genetic screens, we identify important epigenetic signatures and their associated expression changes in mouse models of leukemia and primary samples. We make use of leukemic cells with specific characteristic and mutations in order to dissect the molecular mechanism of action of specific chromatin enzymes. Moreover, we generate proof-of-principle knock-in and knock-out mouse models as well as xenograft human:mouse models to in-vivo examine our findings. Our studies specifically in myeloid leukemia and the discovery and characterization of the role of ten-eleven translocation 2 (TET2) in myeloid homeostasis and transformation, constitute a highlight of this effort. Moreover we dissected genetically and in the molecular level the tumor suppressor role of EZH2 in T-ALL. Current studies in the lab focus on the role of Myc in leukemia compared to normal hematopoietic development.
The main focus of the lab includes characterization of the epigenetic landscape in leukemia, studying the combinatorial action and mutational status of epigenetic enzymes and the functions of the non-coding part of the genome in disease and normal development.

The role of the fbw7 ubiquitin ligase in stem cell function and leukemia

linseyWe would like to understand how the E3 Ligase, Fbw7, as a regulator of post-transcriptional regulation of protein stability, can regulate hematopoietic stem cell fate decisions. Fbw7 is a potent tumor suppressor as it regulates several proto-oncogenes such as c-Myc, Notch and CyclinE. Work from our lab has concluded that Fbw7 indeed has a physiological role in hematopoiesis. We have confirmed the tumor suppressor role of Fbw7 as conditional ablation of Fbw7 in the hematopoietic compartment leads to T-cell acute lymphoblastic leukemia. However, a significant percentage of mice succumb to anemia before tumor development occurs suggesting an alternative function for Fbw7 in normal hematopoiesis. Indeed, we observed a severe loss of hematopoietic stem cells upon deletion of Fbw7 in hematopoietic compartment. Our lab has addressed the contribution of two well characterized Fbw7 substrates, c-Myc and Notch, to the Fbw7-/- HSC defect. We found that relative c-Myc protein stability (and not Notch) was essential for regulating HSC self-renewal and differentiation. Interestingly, Fbw7 was dispensible for self-renewing embryonic stem cells. This work demonstrated, for the first time, that the ubiquitin proteasome system is a novel regulator of HSC function. Furthermore, it suggested that the Fbw7:substrate interaction can be interpreted differently in stem cells from different ages. At least two interesting questions have emerged from these studies; how do Fbw7:substrate interactions impact stem cell fate decisions over time (i.e during aging)? Do Fbw7:substrate interactions contribute to stem cell malignancies (i.e Chronic Myelogenous Leukemia)?

Notch signaling in hematopoeisis

philmoHematopoiesis is a complex process that requires coordination between self-renewal, and differentiation of stem and progenitor cells to generate mature cells in the blood. Notch signaling has been implicated in the regulation of these diverse functions in the hematopoietic system and other tissues. Whereas the importance of Notch1 in lymphocyte development and oncogenic transformation has been well characterized, the relevance of Notch in the specification of other hematopoietic lineages and hematopoietic stem cell (HSC) function remains unclear.
We performed lineage tracing experiments in early hematopoietic progenitors to determine the fate of Notch receptor expressing cells within the hematopoietic system using transgenic mice with tamoxifen inducible CreER knocked into the locus of each Notch receptor. Crossing these animals to the ROSA26-tdRFP reporter permits the irreversible labeling of hematopoietic cells expressing a given Notch receptor and their progeny. To address whether these receptors were being activated, we analyzed Hes1-eGFP knock-in animals (Hes1GFP/+). Hes1, a bHLH repressor, is a well-characterized transcriptional target of intracellular Notch. We found that GFP expression in these mice faithfully recapitulates Notch signaling and that there are distinct subsets of hematopoietic progenitors that have activation of the Notch pathway. In addition, are using these and other novel genetic tools to further characterize Notch signaling within bone marrow niches using in vivo imaging.
Hes1 is also of special interest because it could be a key mediator of cell fates through its influence on transcription factor networks. For example, the promoters of the myeloid transcription factors PU.1 and CEBPa genes both contain Hes1 binding sites known as the .N-box.. Interestingly, their expression is increased upon Notch loss-of-function and decreased with Notch gain-of-function. Consequently, we have begun to identify the lineage-specific, direct targets of Hes1 during hematopoiesis using ChIP. By combining lineage tracing, Notch reporters, imaging, and deep sequencing we will complete the fragmented picture of Notch receptor expression and signaling during hematopoiesis. This work will provide insight into the normal developmental role of Notch in hematopoietic cells and highlight how Notch-Hes1 may act during oncogenic transformation.

Proteomic landscapes in cancer

labsitpicture_sCurrently, we are developing proteomic approaches to delineate the role of the ubiquitin proteasome system (UPS) in hematopoiesis, cancer and stem cell function. For that purpose, we mainly focus on the E3 ligase Fbw7, which regulates the degradation of several important oncogenes with central roles in cell division, growth and differentiation. We have recently shown that Fbw7 acts as a tumor suppressor of T-cell acute lymphoblastic leukemia (T-ALL) by targeting Notch1 (Thompson et al, J Exp Med. 2007). Furthermore, we have shown that, by targeting c-myc, Fbw7 regulates the quiescence and self-renewal capacity of hematopoietic stem cells (HSCs); however, it is dispensable for the pluripotency abilities of embryonic stem cells (ESC), but appears to acquire a role along with cell differentiation (Reavie et al, Nat Immunol 2010).
We are now working on the identification of novel Fbw7 substrates by in-vitro tandem purifications combined with mass spectrometry. We then want to understand the role that the newly identified Fbw7:substrate pairs have in the different considered systems. In addition, we are developing strategies to perform global mass spectrometry analysis of various types of cells in which Fbw7 has been deleted by genetic engineering in order to identify additional substrates by comparing them to their controls. For these series of experiments, we include in vivo and in vitro models aiming to define the tissue and function specificity of the landscape of Fbw7 substrates. After these experiments we should be able to answer question such as: .which are the Fbw7 susbtrates involved in stem cell differentiation? or which Fbw7 substrates are upregulated after Fbw7 malfunctioning in different types of cancer?. Altogether, the ultimate goal of our research is to identify pathways that are new potential therapeutic targets in leukemia and, possibly, in other types of cancer.