Therapeutic Targeting of MLL Degradation Pathways in MLL-Rearranged Leukemia.

Chromosomal translocations of the mixed-lineage leukemia (MLL) gene with various partner genes result in aggressive leukemia with dismal outcomes. Despite similar expression at the mRNA level from the wild-type and chimeric MLL alleles, the chimeric protein is more stable. We report that UBE2O functions in regulating the stability of wild-type MLL in response to interleukin-1 signaling. Targeting wild-type MLL degradation impedes MLL leukemia cell proliferation, and it downregulates a specific group of target genes of the MLL chimeras and their oncogenic cofactor, the super elongation complex. Pharmacologically inhibiting this pathway substantially delays progression, and it improves survival of murine leukemia through stabilizing wild-type MLL protein, which displaces the MLL chimera from some of its target genes and, therefore, relieves the cellular oncogenic addiction to MLL chimeras. Stabilization of MLL provides us with a paradigm in the development of therapies for aggressive MLL leukemia and perhaps for other cancers caused by translocations.

Regulation of MLL/COMPASS stability through its proteolytic cleavage by taspase1 as a possible approach for clinical therapy of leukemia.

Chromosomal translocations of the Mixed-lineage leukemia 1 (MLL1) gene generate MLL chimeras that drive the pathogenesis of acute myeloid and lymphoid leukemia. The untranslocated MLL1 is a substrate for proteolytic cleavage by the endopeptidase threonine aspartase 1 (taspase1); however, the biological significance of MLL1 cleavage by this endopeptidase remains unclear. Here, we demonstrate that taspase1-dependent cleavage of MLL1 results in the destabilization of MLL. Upon loss of taspase1, MLL1 association with chromatin is markedly increased due to the stabilization of its unprocessed version, and this stabilization of the uncleaved MLL1 can result in the displacement of MLL chimeras from chromatin in leukemic cells. Casein kinase II (CKII) phosphorylates MLL1 proximal to the taspase1 cleavage site, facilitating its cleavage, and pharmacological inhibition of CKII blocks taspase1-dependent MLL1 processing, increases MLL1 stability, and results in the displacement of the MLL chimeras from chromatin. Accordingly, inhibition of CKII in a MLL-AF9 mouse model of leukemia delayed leukemic progression in vivo. This study provides insights into the direct regulation of the stability of MLL1 through its cleavage by taspase1, which can be harnessed for targeted therapeutic approaches for the treatment of aggressive leukemia as the result of MLL translocations.

TGF-ß Promotes the Postselection Thymic Development and Peripheral Function of IFN-γ-Producing Invariant NKT cells.

IFN-γ-producing invariant NKT (iNKT)1 cells are lipid-reactive innate-like lymphocytes that are resident in the thymus and peripheral tissues where they protect against pathogenic infection. The thymic functions of iNKT1 cells are not fully elucidated, but subsets of thymic iNKT cells modulate CD8 T cell, dendritic cell, B cell, and thymic epithelial cell numbers or function. In this study, we show that a subset of murine thymic iNKT1 cells required TGF-ß-induced signals for their postselection development, to maintain hallmark TGF-ß-induced genes, and for expression of the adhesion receptors CD49a and CD103. However, the residency-associated receptor CD69 was not TGF-ß signaling-dependent. Recently described CD244+ c2 thymic iNKT1 cells, which produce IFN-γ without exogenous stimulation and have NK-like characteristics, reside in this TGF-ß-responsive population. Liver and spleen iNKT1 cells do not share this TGF-ß gene signature, but nonetheless TGF-ß impacts liver iNKT1 cell phenotype and function. Our findings provide insight into the heterogeneity of mechanisms guiding iNKT1 cell development in different tissues and suggest a close association between a subset of iNKT1 cells and TGF-ß-producing cells in the thymus that support their development.

Not All H3K4 Methylations Are Created Equal: Mll2/COMPASS Dependency in Primordial Germ Cell Specification.

The spatiotemporal regulation of gene expression is central for cell-lineage specification during embryonic development and is achieved through the combinatorial action of transcription factors/co-factors and epigenetic states at cis-regulatory elements. Here, we show that in addition to implementing H3K4me3 at promoters of bivalent genes, Mll2 (KMT2B)/COMPASS can also implement H3K4me3 at a subset of non-TSS regulatory elements, a subset of which shares epigenetic signatures of active enhancers. Our mechanistic studies reveal that association of Mll2's CXXC domain with CpG-rich regions plays an instrumental role for chromatin targeting and subsequent implementation of H3K4me3. Although Mll2/COMPASS is required for H3K4me3 implementation on thousands of loci, generation of catalytically mutant MLL2/COMPASS demonstrated that H3K4me3 implemented by this enzyme was essential for expression of a subset of genes, including those functioning in the control of transcriptional programs during embryonic development. Our findings suggest that not all H3K4 trimethylations implemented by MLL2/COMPASS are functionally equivalent.

A synthetic lethality screen reveals ING5 as a genetic dependency of catalytically dead Set1A/COMPASS in mouse embryonic stem cells.

Embryonic stem cells (ESCs) are defined by their ability to self-renew and the potential to differentiate into all tissues of the developing organism. We previously demonstrated that deleting the catalytic SET domain of the Set1A/complex of proteins associated with SET1 histone methyltransferase (Set1A/COMPASS) in mouse ESCs does not impair their viability or ability to self-renew; however, it leads to defects in differentiation. The precise mechanisms by which Set1A executes these functions remain to be elucidated. In this study, we demonstrate that mice lacking the SET domain of Set1A are embryonic lethal at a stage that is unique from null alleles. To gain insight into Set1A function in regulating pluripotency, we conducted a CRISPR/Cas9-mediated dropout screen and identified the MOZ/MORF (monocytic leukaemia zinc finger protein/monocytic leukaemia zinc finger protein-related factor) and HBO1 (HAT bound to ORC1) acetyltransferase complex member ING5 as a synthetic perturbation to Set1A. The loss of Ing5 in Set1AΔSET mouse ESCs decreases the fitness of these cells, and the simultaneous loss of ING5 and in Set1AΔSET leads to up-regulation of differentiation-associated genes. Taken together, our results point toward Set1A/COMPASS and ING5 as potential coregulators of the self-renewal and differentiation status of ESCs.

SET1A/COMPASS and shadow enhancers in the regulation of homeotic gene expression.

The homeotic (Hox) genes are highly conserved in metazoans, where they are required for various processes in development, and misregulation of their expression is associated with human cancer. In the developing embryo, Hox genes are activated sequentially in time and space according to their genomic position within Hox gene clusters. Accumulating evidence implicates both enhancer elements and noncoding RNAs in controlling this spatiotemporal expression of Hox genes, but disentangling their relative contributions is challenging. Here, we identify two cis-regulatory elements (E1 and E2) functioning as shadow enhancers to regulate the early expression of the HoxA genes. Simultaneous deletion of these shadow enhancers in embryonic stem cells leads to impaired activation of HoxA genes upon differentiation, while knockdown of a long noncoding RNA overlapping E1 has no detectable effect on their expression. Although MLL/COMPASS (complex of proteins associated with Set1) family of histone methyltransferases is known to activate transcription of Hox genes in other contexts, we found that individual inactivation of the MLL1-4/COMPASS family members has little effect on early Hox gene activation. Instead, we demonstrate that SET1A/COMPASS is required for full transcriptional activation of multiple Hox genes but functions independently of the E1 and E2 cis-regulatory elements. Our results reveal multiple regulatory layers for Hox genes to fine-tune transcriptional programs essential for development.

Nore1 inhibits age-associated myeloid lineage skewing and clonal hematopoiesis but facilitates termination of emergency (stress) granulopoiesis.

Age-associated bone marrow changes include myeloid skewing and mutations that lead to clonal hematopoiesis. Molecular mechanisms for these events are ill defined, but decreased expression of Irf8/Icsbp (interferon regulatory factor 8/interferon consensus sequence binding protein) in aging hematopoietic stem cells may contribute. Irf8 functions as a leukemia suppressor for chronic myeloid leukemia, and young Irf8-/- mice have neutrophilia with progression to acute myeloid leukemia (AML) with aging. Irf8 is also required to terminate emergency granulopoiesis during the innate immune response, suggesting this may be the physiologic counterpart to leukemia suppression by this transcription factor. Identifying Irf8 effectors may define mediators of both events and thus contributors to age-related bone marrow disorders. In this study, we identified RASSF5 (encoding Nore1) as an Irf8 target gene and investigated the role of Nore1 in hematopoiesis. We found Irf8 activates RASSF5 transcription and increases Nore1a expression during emergency granulopoiesis. Similar to Irf8-/- mice, we found that young Rassf5-/- mice had increased neutrophils and progressed to AML with aging. We identified enhanced DNA damage, excess clonal hematopoiesis, and a distinct mutation profile in hematopoietic stem cells from aging Rassf5-/- mice compared with wildtype. We found sustained emergency granulopoiesis in Rassf5-/- mice, with repeated episodes accelerating AML, also similar to Irf8-/- mice. Identifying Nore1a downstream from Irf8 defines a pathway involved in leukemia suppression and the innate immune response and suggests a novel molecular mechanism contributing to age-related clonal myeloid disorders.

Analysis of the Contribution of 6-mer Seed Toxicity to HIV-1-Induced Cytopathicity.

HIV-1 (HIV) infects CD4+ T cells, the gradual depletion of which can lead to AIDS in the absence of antiretroviral therapy (ART). Some cells, however, survive HIV infection and persist as part of the latently infected reservoir that causes recurrent viremia after ART cessation. Improved understanding of the mechanisms of HIV-mediated cell death could lead to a way to clear the latent reservoir. Death induced by survival gene elimination (DISE), an RNA interference (RNAi)-based mechanism, kills cells through short RNAs (sRNAs) with toxic 6-mer seeds (positions 2 to 7 of sRNA). These toxic seeds target the 3' untranslated region (UTR) of mRNAs, decreasing the expression of hundreds of genes critical for cell survival. In most cells under normal conditions, highly expressed cell-encoded nontoxic microRNAs (miRNAs) block access of toxic sRNAs to the RNA-induced silencing complex (RISC) that mediates RNAi, promoting cell survival. HIV has been shown to inhibit the biogenesis of host miRNAs in multiple ways. We now report that HIV infection of cells deficient in miRNA expression or function results in enhanced RISC loading of an HIV-encoded miRNA HIV-miR-TAR-3p, which can kill cells by DISE through a noncanonical (positions 3 to 8) 6-mer seed. In addition, cellular RISC-bound sRNAs shift to lower seed viability. This also occurs after latent HIV provirus reactivation in J-Lat cells, suggesting independence of permissiveness of cells to viral infection. More precise targeting of the balance between protective and cytotoxic sRNAs could provide new avenues to explore novel cell death mechanisms that could be used to kill latent HIV. IMPORTANCE Several mechanisms by which initial HIV infection is cytotoxic to infected cells have been reported and involve various forms of cell death. Characterizing the mechanisms underlying the long-term survival of certain T cells that become persistent provirus reservoirs is critical to developing a cure. We recently discovered death induced by survival gene elimination (DISE), an RNAi-based mechanism of cell death whereby toxic short RNAs (sRNAs) containing 6-mer seed sequences (exerting 6-mer seed toxicity) targeting essential survival genes are loaded into RNA-induced silencing complex (RISC) complexes, resulting in inescapable cell death. We now report that HIV infection in cells with low miRNA expression causes a shift of mostly cellular RISC-bound sRNAs to more toxic seeds. This could prime cells to DISE and is further enhanced by the viral microRNA (miRNA) HIV-miR-TAR-3p, which carries a toxic noncanonical 6-mer seed. Our data provide multiple new avenues to explore novel cell death mechanisms that could be used to kill latent HIV.

Regulation of the imprinted Dlk1-Dio3 locus by allele-specific enhancer activity.

Genomic imprinting is a critical developmental process characteristic of parent of origin-specific gene expression. It is well accepted that differentially DNA-methylated regions (DMRs) and enhancers are two major classes of cis-elements determining parent of origin-specific gene expression, with each recruiting different sets of transcription factors. Previously, we identified the AF4/FMR2 (AFF) family protein AFF3 within the transcription elongation complex SEC-L3. Here, we report that AFF3 can specifically bind both gametic DMRs (gDMRs) and enhancers within imprinted loci in an allele-specific manner. We identify the molecular regulators involved in the recruitment of AFF3 to gDMRs and provide mechanistic insight into the requirement of AFF3 at an enhancer for the expression of an ∼200-kb polycistronic transcript within the imprinted Dlk1-Dio3 locus. Our data suggest that the heterochromatic environment at the gDMR reinforces silencing of its related enhancer by controlling the binding and activity of AFF3 in an allele-specific manner. In summary, this study provides molecular details about the regulation of dosage-critical imprinted gene expression through the regulated binding of the transcription elongation factor AFF3 between a DMR and an enhancer.

Epigenetic repression of the Igk locus by STAT5-mediated recruitment of the histone methyltransferase Ezh2.

During B lymphopoiesis, recombination of the locus encoding the immunoglobulin κ-chain complex (Igk) requires expression of the precursor to the B cell antigen receptor (pre-BCR) and escape from signaling via the interleukin 7 receptor (IL-7R). By activating the transcription factor STAT5, IL-7R signaling maintains proliferation and represses Igk germline transcription by unknown mechanisms. We demonstrate that a STAT5 tetramer bound the Igk intronic enhancer (E(κi)), which led to recruitment of the histone methyltransferase Ezh2. Ezh2 marked trimethylation of histone H3 at Lys27 (H3K27me3) throughout the κ-chain joining region (J(κ)) to the κ-chain constant region (C(κ)). In the absence of Ezh2, IL-7 failed to repress Igk germline transcription. H3K27me3 modifications were lost after termination of IL-7R-STAT5 signaling, and the transcription factor E2A bound E(κi), which resulted in acquisition of H3K4me1 and acetylated histone H4 (H4Ac). Genome-wide analyses showed a STAT5 tetrameric binding motif associated with transcriptional repression. Our data demonstrate how IL-7R signaling represses Igk germline transcription and provide a general model for STAT5-mediated epigenetic transcriptional repression.

SPOROS: A pipeline to analyze DISE/6mer seed toxicity.

microRNAs (miRNAs) are (18-22nt long) noncoding short (s)RNAs that suppress gene expression by targeting the 3' untranslated region of target mRNAs. This occurs through the seed sequence located in position 2-7/8 of the miRNA guide strand, once it is loaded into the RNA induced silencing complex (RISC). G-rich 6mer seed sequences can kill cells by targeting C-rich 6mer seed matches located in genes that are critical for cell survival. This results in induction of Death Induced by Survival gene Elimination (DISE), through a mechanism we have called 6mer seed toxicity. miRNAs are often quantified in cells by aligning the reads from small (sm)RNA sequencing to the genome. However, the analysis of any smRNA Seq data set for predicted 6mer seed toxicity requires an alternative workflow, solely based on the exact position 2-7 of any short (s)RNA that can enter the RISC. Therefore, we developed SPOROS, a semi-automated pipeline that produces multiple useful outputs to predict and compare 6mer seed toxicity of cellular sRNAs, regardless of their nature, between different samples. We provide two examples to illustrate the capabilities of SPOROS: Example one involves the analysis of RISC-bound sRNAs in a cancer cell line (either wild-type or two mutant lines unable to produce most miRNAs). Example two is based on a publicly available smRNA Seq data set from postmortem brains (either from normal or Alzheimer's patients). Our methods (found at https://github.com/ebartom/SPOROS and at Code Ocean: https://doi.org/10.24433/CO.1732496.v1) are designed to be used to analyze a variety of smRNA Seq data in various normal and disease settings.

Ezh2 Represses Transcription of Innate Lymphoid Genes in B Lymphocyte Progenitors and Maintains the B-2 Cell Fate.

Lymphocyte lineage specification and commitment requires the activation of lineage-specific genes and repression of alternative lineage genes, respectively. The mechanisms governing alternative lineage gene repression and commitment in lymphocytes are largely unknown. In this study, we demonstrate that Ezh2, which represses gene expression through methylation of histone 3 lysine 27, was essential for repression of numerous genes, including genes encoding innate lymphocyte transcription factors, specifically in murine B lymphocyte progenitors, but these cells maintained their B lymphocyte identity. However, adult Ezh2-deficient B lymphocytes expressed Lin28b, which encodes an RNA-binding protein associated with fetal hematopoietic gene expression programs, and these cells acquired a fetal B-1 lymphocyte phenotype in vitro and in vivo. Therefore, Ezh2 coordinates the repression of multiple gene programs in B lymphocytes and maintains the adult B-2 cell fate.

RACK1 evolved species-specific multifunctionality in translational control through sequence plasticity within a loop domain.

Receptor of activated protein C kinase 1 (RACK1) is a highly conserved eukaryotic protein that regulates several aspects of mRNA translation; yet, how it does so, remains poorly understood. Here we show that, although RACK1 consists largely of conserved ß-propeller domains that mediate binding to several other proteins, a short interconnecting loop between two of these blades varies across species to control distinct RACK1 functions during translation. Mutants and chimeras revealed that the amino acid composition of the loop is optimized to regulate interactions with eIF6, a eukaryotic initiation factor that controls 60S biogenesis and 80S ribosome assembly. Separately, phylogenetics revealed that, despite broad sequence divergence of the loop, there is striking conservation of negatively charged residues amongst protists and dicot plants, which is reintroduced to mammalian RACK1 by poxviruses through phosphorylation. Although both charged and uncharged loop mutants affect eIF6 interactions, only a negatively charged plant - but not uncharged yeast or human loop - enhances translation of mRNAs with adenosine-rich 5' untranslated regions (UTRs). Our findings reveal how sequence plasticity within the RACK1 loop confers multifunctionality in translational control across species.

Role of Hypoxia-Inducible Factors in Regulating Right Ventricular Function and Remodeling during Chronic Hypoxia-induced Pulmonary Hypertension.

Pulmonary hypertension (PH) and right ventricular (RV) hypertrophy frequently develop in patients with hypoxic lung disease. Chronic alveolar hypoxia (CH) promotes sustained pulmonary vasoconstriction and pulmonary artery (PA) remodeling by acting on lung cells, resulting in the development of PH. RV hypertrophy develops in response to PH, but coronary arterial hypoxemia in CH may influence that response by activating HIF-1α (hypoxia-inducible factor 1α) and/or HIF-2α in cardiomyocytes. Indeed, other studies show that the attenuation of PH in CH fails to prevent RV remodeling, suggesting that PH-independent factors regulate RV hypertrophy. Therefore, we examined the role of HIFs in RV remodeling in CH-induced PH. We deleted HIF-1α and/or HIF-2α in hearts of adult mice that were then housed under normoxia or CH (10% O2) for 4 weeks. RNA-sequencing analysis of the RV revealed that HIF-1α and HIF-2α regulate the transcription of largely distinct gene sets during CH. RV systolic pressure increased, and RV hypertrophy developed in CH. The deletion of HIF-1α in smooth muscle attenuated the CH-induced increases in RV systolic pressure but did not decrease hypertrophy. The deletion of HIF-1α in cardiomyocytes amplified RV remodeling; this was abrogated by the simultaneous loss of HIF-2α. CH decreased stroke volume and cardiac output in wild-type but not in HIF-1α-deficient hearts, suggesting that CH may cause cardiac dysfunction via HIF-dependent signaling. Collectively, these data reveal that HIF-1 and HIF-2 act together in RV cardiomyocytes to orchestrate RV remodeling in CH, with HIF-1 playing a protective role rather than driving hypertrophy.

Small interfering RNAs based on huntingtin trinucleotide repeats are highly toxic to cancer cells.

Trinucleotide repeat (TNR) expansions in the genome cause a number of degenerative diseases. A prominent TNR expansion involves the triplet CAG in the huntingtin (HTT) gene responsible for Huntington's disease (HD). Pathology is caused by protein and RNA generated from the TNR regions including small siRNA-sized repeat fragments. An inverse correlation between the length of the repeats in HTT and cancer incidence has been reported for HD patients. We now show that siRNAs based on the CAG TNR are toxic to cancer cells by targeting genes that contain long reverse complementary TNRs in their open reading frames. Of the 60 siRNAs based on the different TNRs, the six members in the CAG/CUG family of related TNRs are the most toxic to both human and mouse cancer cells. siCAG/CUG TNR-based siRNAs induce cell death in vitro in all tested cancer cell lines and slow down tumor growth in a preclinical mouse model of ovarian cancer with no signs of toxicity to the mice. We propose to explore TNR-based siRNAs as a novel form of anticancer reagents.

EZH2 Regulates the Developmental Timing of Effectors of the Pre-Antigen Receptor Checkpoints.

The histone methyltransferase EZH2 is required for B and T cell development; however, the molecular mechanisms underlying this requirement remain elusive. In a murine model of lymphoid-specific EZH2 deficiency we found that EZH2 was required for proper development of adaptive, but not innate, lymphoid cells. In adaptive lymphoid cells EZH2 prevented the premature expression of Cdkn2a and the consequent stabilization of p53, an effector of the pre-Ag receptor checkpoints. Deletion of Cdkn2a in EZH2-deficient lymphocytes prevented p53 stabilization, extended lymphocyte survival, and restored differentiation resulting in the generation of mature B and T lymphocytes. Our results uncover a crucial role for EZH2 in adaptive lymphocytes to control the developmental timing of effectors of the pre-Ag receptor checkpoints.

A Beginner's Guide to Analysis of RNA Sequencing Data.

Since the first publications coining the term RNA-seq (RNA sequencing) appeared in 2008, the number of publications containing RNA-seq data has grown exponentially, hitting an all-time high of 2,808 publications in 2016 (PubMed). With this wealth of RNA-seq data being generated, it is a challenge to extract maximal meaning from these datasets, and without the appropriate skills and background, there is risk of misinterpretation of these data. However, a general understanding of the principles underlying each step of RNA-seq data analysis allows investigators without a background in programming and bioinformatics to critically analyze their own datasets as well as published data. Our goals in the present review are to break down the steps of a typical RNA-seq analysis and to highlight the pitfalls and checkpoints along the way that are vital for bench scientists and biomedical researchers performing experiments that use RNA-seq.

Translatome analyses capture of opposing tissue-specific brassinosteroid signals orchestrating root meristem differentiation.

The mechanisms ensuring balanced growth remain a critical question in developmental biology. In plants, this balance relies on spatiotemporal integration of hormonal signaling pathways, but the understanding of the precise contribution of each hormone is just beginning to take form. Brassinosteroid (BR) hormone is shown here to have opposing effects on root meristem size, depending on its site of action. BR is demonstrated to both delay and promote onset of stem cell daughter differentiation, when acting in the outer tissue of the root meristem, the epidermis, and the innermost tissue, the stele, respectively. To understand the molecular basis of this phenomenon, a comprehensive spatiotemporal translatome mapping of Arabidopsis roots was performed. Analyses of wild type and mutants featuring different distributions of BR revealed autonomous, tissue-specific gene responses to BR, implying its contrasting tissue-dependent impact on growth. BR-induced genes were primarily detected in epidermal cells of the basal meristem zone and were enriched by auxin-related genes. In contrast, repressed BR genes prevailed in the stele of the apical meristem zone. Furthermore, auxin was found to mediate the growth-promoting impact of BR signaling originating in the epidermis, whereas BR signaling in the stele buffered this effect. We propose that context-specific BR activity and responses are oppositely interpreted at the organ level, ensuring coherent growth.

CUX1 is a haploinsufficient tumor suppressor gene on chromosome 7 frequently inactivated in acute myeloid leukemia.

Loss of chromosome 7 and del(7q) [-7/del(7q)] are recurring cytogenetic abnormalities in hematologic malignancies, including acute myeloid leukemia and therapy-related myeloid neoplasms, and associated with an adverse prognosis. Despite intensive effort by many laboratories, the putative myeloid tumor suppressor(s) on chromosome 7 has not yet been identified.We performed transcriptome sequencing and SNP array analysis on de novo and therapy-related myeloid neoplasms, half with -7/del(7q). We identified a 2.17-Mb commonly deleted segment on chromosome band 7q22.1 containing CUX1, a gene encoding a homeodomain-containing transcription factor. In 1 case, CUX1 was disrupted by a translocation, resulting in a loss-of-function RNA fusion transcript. CUX1 was the most significantly differentially expressed gene within the commonly deleted segment and was expressed at haploinsufficient levels in -7/del(7q) leukemias. Haploinsufficiency of the highly conserved ortholog, cut, led to hemocyte overgrowth and tumor formation in Drosophila melanogaster. Similarly, haploinsufficiency of CUX1 gave human hematopoietic cells a significant engraftment advantage on transplantation into immunodeficient mice. Within the RNA-sequencing data, we identified a CUX1-associated cell cycle transcriptional gene signature, suggesting that CUX1 exerts tumor suppressor activity by regulating proliferative genes. These data identify CUX1 as a conserved, haploinsufficient tumor suppressor frequently deleted in myeloid neoplasms.

Loss of thymocyte competition underlies the tumor suppressive functions of the E2a transcription factor in T-ALL.

T lymphocyte acute lymphoblastic leukemia (T-ALL) is frequently associated with increased expression of the E protein transcription factor inhibitors TAL1 and LYL1. In mouse models, ectopic expression of TAL1 or LYL1 in T cell progenitors, or inactivation of E2A, is sufficient to predispose mice to develop T-ALL. How E2A suppresses thymocyte transformation is currently unknown. Here, we show that early deletion of E2a, prior to the DN3 stage, was required for robust leukemogenesis and was associated with alterations in thymus cellularity, T cell differentiation, and gene expression in immature CD4+CD8+ thymocytes. Introduction of wild-type thymocytes into mice with early deletion of E2a prevented leukemogenesis, or delayed disease onset, and impacted the expression of multiple genes associated with transformation and genome instability. Our data indicate that E2A suppresses leukemogenesis by promoting T cell development and enforcing inter-thymocyte competition, a mechanism that is emerging as a safeguard against thymocyte transformation. These studies have implications for understanding how multiple essential regulators of T cell development suppress T-ALL and support the hypothesis that thymocyte competition suppresses leukemogenesis.