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 Tcrβ 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.

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.

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.

IntroductionFanconi anemia (FA) is a congenital form of aplastic anemia and is transmitted through an autosomal and X-linked recessive mode. FA is manifested by bone marrow failure, congenital abnormalities, and a predisposition to malignancy. 1 The primary clinical phenotype and major cause of death in patients is the progressive depletion of hematopoietic stem cells (HSCs) leading to bone marrow (BM) failure. This progressive loss of HSCs has been linked to a defect in HSC self-renewal and maintenance. 2,3 Currently, FA is defined by 13 complementation groups and cloned genes (A through N). 4 Eight FA proteins (FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL/ PHF9, and FANCM) bind together in a nuclear complex that is termed the FA core complex. This complex, through the E3 ubiquitin ligase activity of FANCL, mediates monoubiquitination of FANCD2 and FANCI in response to DNA cross-link damage and during normal S phase. [5][6][7][8] Mutations in any of the core complex components disrupt the structural integrity of the complex and prevent activation of the FA pathway as measured by FANCD2 monoubiquitination. Despite the identification of so many genes, a clear picture of how this pathway relates to the major phenotypic feature of FA such as progressive hematopoietic stem cell loss remains elusive.Several pathways involved in mesoderm patterning and formation such as Notch, were found to be involved in HSC self-renewal. [9][10][11] Notch1 through constitutive expression of its intracellular domain, NICD, as well as overexpression of its downstream effector hairy and enhancer of split homologue 1 (HES1) has been shown to increase HSC numbers in vivo, increase HSC self-renewal, reduce HSC cycling, and preserve the long-term reconstitution ability of primitive hematopoietic cells. 10,[12][13][14] In FA, HSCs were shown to have reduced selfrenewal and reconstitution abilities and increased cycling activity, 2,3,15 suggesting a link between Notch1/HES1 and FA pathways. In an attempt to elucidate the molecular basis of the HSC defect in FA, we found that HES1 directly interacts with several members of the FA core complex, notably FANCA, FANCF, FANCG, and FANCL.HES1 is a member of the highly conserved family of Hairyrelated basic helix-loop-helix (bHLH) proteins. There are 7 described members in the mammalian HES family. Among these, HES1 and HES5 are the only members known to be involved specifically in Notch1 signaling in neural cells and in bone marrow. 16,17 HES1 is a repressor-type bHLH that represses expression of its own gene 18,19 (autoregulatory mechanism) and antagonizes bHLH activators. 20 In this study, we provide evidence, using several approaches, that HES1 is a novel interactor of the FA core complex. We also show that HES1 is required for cellular resistance to mitomycin C (MMC), FANCD2 monoubiquitination, proper cellular localization of FANCA and FANCL, and protein stability of several FA core complex members. Methods Cell lines and culture conditions293T, HeLa, PD430 (FA-A), and FancC ϩ/ϩ and FancC Ϫ/...