Cedric Tremblay

Modeling T-cell acute lymphoblastic leukemia induced by the SCL and LMO1 oncogenes

Deciphering molecular events required for full transformation of normal cells into cancer cells remains a challenge. In T-cell acute lymphoblastic leukemia (T-ALL), the genes encoding the TAL1/SCL and LMO1/2 transcription factors are recurring targets of chromosomal translocations, whereas NOTCH1 is activated in >50% of samples. Here we show that the SCL and LMO1 oncogenes collaborate to expand primitive thymocyte progenitors and inhibit later stages of differentiation. Together with pre-T-cell antigen receptor (pre-TCR) signaling, these oncogenes provide a favorable context for the acquisition of activating Notch1 mutations and the emergence of self-renewing leukemia-initiating cells in T-ALL. All tumor cells harness identical and specific Notch1 mutations and Tcrbeta clonal signature, indicative of clonal dominance and concurring with the observation that Notch1 gain of function confers a selective advantage to SCL-LMO1 transgenic thymocytes. Accordingly, a hyperactive Notch1 allele accelerates leukemia onset induced by SCL-LMO1 and bypasses the requirement for pre-TCR signaling. Finally, the time to leukemia induced by the three transgenes corresponds to the time required for clonal expansion from a single leukemic stem cell, suggesting that SCL, LMO1, and Notch1 gain of function, together with an active pre-TCR, might represent the minimum set of complementing events for the transformation of susceptible thymocytes.

HES1 is a novel interactor of the Fanconi anemia core complex

Fanconi anemia (FA) proteins are thought to play a role in chromosome stability and repair of DNA cross-links; however, these functions may not fully explain the developmental abnormalities and bone marrow failure that are characteristic of FA individuals. Here we associate the FA proteins with the Notch1 developmental pathway through a direct protein-protein interaction between the FA core complex and the hairy enhancer of split 1 (HES1). HES1 interaction with FA core complex members is dependent on a functional FA pathway. Cells depleted of HES1 exhibit an FA-like phenotype that includes cellular hypersensitivity to mitomycin C (MMC) and lack of FANCD2 monoubiquitination and foci formation. HES1 is also required for proper nuclear localization or stability of some members of the core complex. Our results suggest that HES1 is a novel interacting protein of the FA core complex.

ZEB2 drives immature T-cell lymphoblastic leukaemia development via enhanced tumour-initiating potential and IL-7 receptor signalling

Early T-cell precursor leukaemia (ETP-ALL) is a high-risk subtype of human leukaemia that is poorly understood at the molecular level. Here we report translocations targeting the zinc finger E-box-binding transcription factor ZEB2 as a recurrent genetic lesion in immature/ETP-ALL. Using a conditional gain-of-function mouse model, we demonstrate that sustained Zeb2 expression initiates T-cell leukaemia. Moreover, Zeb2-driven mouse leukaemia exhibit some features of the human immature/ETP-ALL gene expression signature, as well as an enhanced leukaemia-initiation potential and activated Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signalling through transcriptional activation of IL7R. This study reveals ZEB2 as an oncogene in the biology of immature/ETP-ALL and paves the way towards pre-clinical studies of novel compounds for the treatment of this aggressive subtype of human T-ALL using our Zeb2-driven mouse model.

SCL, LMO1 and Notch1 Reprogram Thymocytes into Self- Renewing Cells

The molecular determinants that render specific populations of normal cells susceptible to oncogenic reprogramming into self-renewing cancer stem cells are poorly understood. Here, we exploit T-cell acute lymphoblastic leukemia (T-ALL) as a model to define the critical initiating events in this disease. First, thymocytes that are reprogrammed by the SCL and LMO1 oncogenic transcription factors into self-renewing pre-leukemic stem cells (pre-LSCs) remain non-malignant, as evidenced by their capacities to generate functional T cells. Second, we provide strong genetic evidence that SCL directly interacts with LMO1 to activate the transcription of a self-renewal program coordinated by LYL1. Moreover, LYL1 can substitute for SCL to reprogram thymocytes in concert with LMO1. In contrast, inhibition of E2A was not sufficient to substitute for SCL, indicating that thymocyte reprogramming requires transcription activation by SCL-LMO1. Third, only a specific subset of normal thymic cells, known as DN3 thymocytes, is susceptible to reprogramming. This is because physiological NOTCH1 signals are highest in DN3 cells compared to other thymocyte subsets. Consistent with this, overexpression of a ligand-independent hyperactive NOTCH1 allele in all immature thymocytes is sufficient to sensitize them to SCL-LMO1, thereby increasing the pool of self-renewing cells. Surprisingly, hyperactive NOTCH1 cannot reprogram thymocytes on its own, despite the fact that NOTCH1 is activated by gain of function mutations in more than 55% of T-ALL cases. Rather, elevating NOTCH1 triggers a parallel pathway involving Hes1 and Myc that dramatically enhances the activity of SCL-LMO1 We conclude that the acquisition of self-renewal and the genesis of pre-LSCs from thymocytes with a finite lifespan represent a critical first event in T-ALL. Finally, LYL1 and LMO1 or LMO2 are co-expressed in most human T-ALL samples, except the cortical T subtype. We therefore anticipate that the self-renewal network described here may be relevant to a majority of human T-ALL.

Requirement for Lyl1 in a model of Lmo2-driven early T-cell precursor ALL.

Lmo2 is an oncogenic transcription factor that is frequently overexpressed in T-cell acute lymphoblastic leukemia (T-ALL), including early T-cell precursor ALL (ETP-ALL) cases with poor prognosis. Lmo2 must be recruited to DNA by binding to the hematopoietic basic helix-loop-helix factors Scl/Tal1 or Lyl1. However, it is unknown which of these factors can mediate the leukemic activity of Lmo2. To address this, we have generated Lmo2-transgenic mice lacking either Scl or Lyl1 in the thymus. We show that although Scl is dispensable for Lmo2-driven leukemia, Lyl1 is critical for all oncogenic functions of Lmo2, including upregulation of a stem cell-like gene signature, aberrant self-renewal of thymocytes, and subsequent generation of T-cell leukemia. Lyl1 expression is restricted to preleukemic and leukemic stem cell populations in this model, providing a molecular explanation for the stage-specific expression of the Lmo2-induced gene expression program. Moreover, LMO2 and LYL1 are coexpressed in ETP-ALL patient samples, and LYL1 is required for growth of ETP-ALL cell lines. Thus, the LMO2-LYL1 interaction is a promising therapeutic target for inhibiting self-renewing cancer stem cells in T-ALL, including poor-prognosis ETP-ALL cases.

The Fanconi Anemia Core Complex Acts as a Transcriptional Co-regulator in Hairy Enhancer of Split 1 Signaling

Mutations in one of the 13 Fanconi anemia (FA) genes cause a progressive bone marrow failure disorder associated with developmental abnormalities and a predisposition to cancer. Although FA has been defined as a DNA repair disease based on the hypersensitivity of patient cells to DNA cross-linking agents, FA patients develop various developmental defects such as skeletal abnormalities, microphthalmia, and endocrine abnormalities that may be linked to transcriptional defects. Recently, we reported that the FA core complex interacts with the transcriptional repressor Hairy Enhancer of Split 1 (HES1) suggesting that the core complex plays a role in transcription. Here we show that the FA core complex contributes to transcriptional regulation of HES1-responsive genes, including HES1 and the cyclin-dependent kinase inhibitor p21cip1/waf1. Chromatin immunoprecipitation studies show that the FA core complex interacts with the HES1 promoter but not the p21cip1/waf1 promoter. Furthermore, we show that the FA core complex interferes with HES1 binding to the co-repressor transducin-like-Enhancer of Split, suggesting that the core complex affects transcription both directly and indirectly. Taken together these data suggest a novel function of the FA core complex in transcriptional regulation.

Loss-of-function mutations of Dynamin 2 promote T-ALL by enhancing IL-7 signalling.

Mutations in the DYNAMIN2 (DNM2) gene are frequently detected in human acute T-cell lymphoblastic leukemia (T-ALL), although the mechanisms linking these mutations to disease pathogenesis remain unknown. Using an ENU-based forward genetic screen for mice with erythroid phenotypes, we identified a heterozygous mouse line carrying a mutation in the GTPase domain of Dnm2 (Dnm2V265G) that induced a microcytic anemia. In vitro assays using the V265G mutant demonstrated loss of GTPase activity and impaired endocytosis that was comparable to other DNM2 mutants identified in human T-ALL. To determine the effects of DNM2 mutations in T-ALL, we bred the Dnm2V265G mice with the Lmo2 transgenic mouse model of T-ALL. Heterozygous Dnm2 mutants lacking the Lmo2 transgene displayed normal T-cell development, and did not develop T-ALL. In contrast, compound heterozygotes displayed an accelerated onset of T-ALL compared with mice carrying the Lmo2 oncogene alone. The leukemias from these mice exhibited a more immature immunophenotype and an expansion in leukemic stem cell numbers. Mechanistically, the Dnm2 mutation impaired clathrin-mediated endocytosis of the interleukin (IL)-7 receptor resulting in increased receptor density on the surface of leukemic stem cells. These findings suggest that DNM2 mutations cooperate with T-cell oncogenes by enhancing IL-7 signalling.

Early T cell differentiation lessons from T-cell acute lymphoblastic leukemia.

T cells develop from bone marrow-derived self-renewing hematopoietic stem cells (HSC). Upon entering the thymus, these cells undergo progressive commitment and differentiation driven by the thymic stroma and the pre-T cell receptor (pre-TCR). These processes are disrupted in T-cell acute lymphoblastic leukemia (T-ALL). More than 70% of recurring chromosomal rearrangements in T-ALL activate the expression of oncogenic transcription factors, belonging mostly to three families, basic helix-loop-helix (bHLH), homeobox (HOX), and c-MYB. This prevalence is indicative of their importance in the T lineage, and their dominant mechanisms of transformation. For example, bHLH oncoproteins inhibit E2A and HEB, revealing their tumor suppressor function in the thymus. The induction of T-ALL, nonetheless, requires collaboration with constitutive NOTCH1 signaling and the pre-TCR, as well as loss-of-function mutations for CDKN2A and PTEN. Significantly, NOTCH1, the pre-TCR pathway, and E2A/HEB proteins control critical checkpoints and branchpoints in early thymocyte development whereas several oncogenic transcription factors, HOXA9, c-MYB, SCL, and LYL-1 control HSC self-renewal. Together, these genetic lesions alter key regulatory processes in the cell, favoring self-renewal and subvert the normal control of thymocyte homeostasis.

The Fanconi anemia pathway has a dual function in Dickkopf-1 transcriptional repression

Significance Fanconi anemia (FA) is a devastating disease associated with a progressive bone marrow failure (BMF) and clonal proliferation of primitive hematopoietic cells that leads to leukemia. In an effort to understand the molecular basis of BMF and leukemogenesis in FA, we recently uncovered a unique function of proteins associated with FA in transcriptional regulation that translates into elevated levels of the signaling molecule Dickkopf-1 (DKK1). Overproduction of DKK1 has been shown to alter functions in hematopoiesis and to promote hematologic malignancies. Thus, our findings represent a crucial step in the development of strategies aimed at preventing BMF and/or clonal hematopoiesis in patients with FA. Fanconi anemia (FA) is an inherited bone marrow failure syndrome associated with a progressive decline in hematopoietic stem cells, developmental defects, and predisposition to cancer. These various phenotypic features imply a role of FA proteins in molecular events regulating cellular homeostasis. Interestingly, we previously found that the Fanconi C protein (FANCC) interacts with the C-terminal-binding protein-1 (CtBP1) involved in transcriptional regulation. Here we report that FANCC with CtBP1 forms a complex with β-catenin, and that β-catenin activation through glycogen synthase kinase 3β inhibition leads to FANCC nuclear accumulation and FA pathway activation, as measured by the Fanconi D2 protein (FANCD2) monoubiquitination. β-catenin and FANCC nuclear entry is defective in FA mutant cells and in cells depleted of the Fanconi A protein or FANCD2, suggesting that integrity of the FA pathway is required for FANCC nuclear activity. We also report that FANCC with CtBP1 acts as a negative regulator of Dickkopf-1 (DKK1) expression, and that a FA disease-causing mutation in FANCC abrogates this function. Our findings reveal that a defective FA pathway leads to up-regulation of DKK1, a molecule involved in hematopoietic malignancies.

The clonal evolution of leukemic stem cells in T-cell acute lymphoblastic leukemia.

Purpose of reviewRecent genome sequencing studies have identified a broad spectrum of gene mutations in T-cell acute lymphoblastic leukemia (T-ALL). The purpose of this review is to outline the latest advances in our understanding of how these mutations contribute to the formation of T-ALL. Recent findingsAberrant expression of transcription factors that control hematopoiesis can induce an aberrant stem cell-like program in T-cell progenitors, allowing the emergence of an ancestral or preleukemic stem cell (pre-LSC). In contrast, gain-of-function mutations of genes involved in signaling pathways regulating T-cell development, such as NOTCH1, interleukin-7, KIT and FLT3, are insufficient per se to initiate T-ALL but promote pre-LSC growth independent of the thymic niche. Loss-of-function mutations of epigenetic regulators, such as DNMT3A, have been identified in T-ALL, but their role in leukemogenesis remains to be defined. SummaryRelapse is associated with clonal evolution from a population of pre-LSCs that acquire the whole set of malignant mutations leading to a full-blown T-ALL. Understanding the genetic events that underpin the pre-LSC will be crucial for reducing the risk of relapse.