DN3 Stage Research Articles

Ezh2 Loss Promotes the Transformation of Early T Cell Precursor Leukemia Via Suppressing Critical Genes for T-Cell Differentiation

Early T cell precursor (ETP) acute lymphoblastic leukemia (ALL) has been identified as a new pathologic entity with poor outcome in patients with T-ALL. In contrast to cortical T-ALL, ETP-ALL has been characterized by the activating mutations in genes regulating cytokine signaling and the inactivating mutations in the polycomb repressor complex 2 (PRC2) component genes including EZH2. EZH2 catalyzes trimethylation of histone H3 at lysine 27 (H3K27me3) that mediates silencing of critical genes for cell proliferation and differentiation. Given that loss-of-function mutations in EZH2 are often found in ETP-ALL, EZH2 may play a tumor suppressor role in the pathogenesis of ETP-ALL. To determine how EZH2 dysfunction promotes the development of ETP-ALL in vivo, we generated a novel mouse model of ETP-ALL by utilizing Ezh2 and p53 conditional knockout mice. We harvested bone marrow cells from Cre-ERT (WT), Cre-ERT;Ezh2flox/flox (Ezh2 KO), Cre-ERT;Trp53flox/flox (p53 KO) and Cre-ERT;Trp53flox/flox;Ezh2flox/flox (DKO) mice, and transplanted them into lethally-irradiated Ly5.1+ recipient mice. We deleted p53 and Ezh2 via administration of tamoxifen 4 weeks post-transplantation. We observed that p53 KO mice died dominantly due to CD3+ CD8+ cortical T-ALL (median survival; 137 days), as previously reported. While 2 out of 8 DKO mice developed CD8+ cortical T-ALL, we found that 6 out of 8 DKO mice developed CD4- CD8- (DN) T-ALL with a longer latency (median survival; 189 days). At the time of sacrifice, DKO mice showed significantly enlarged thymus due to the expansion of c-Kit+ CD44+ CD25- DN1 and c-Kit+ CD44+ CD25+ DN2 cells, which were markedly compromised in differentiation into DN3 and further down to CD4+ CD8+ (DP) cells. Histological analysis demonstrated the expansion of cytoplasmic CD3+ (cyCD3) tumor cells in the thymus of DKO mice with DN T-ALL. Thus, DKO mice faithfully recapitulated the phenotypic features of human ETP-ALL, indicating that the loss of Ezh2 is prerequisite to the development of c-Kit+ CD44+ CD25+ cyCD3+ DN ALL in mice lacking p53.To understand the underlying molecular mechanism in the pathogenesis of Ezh2 loss-induced ETP-ALL, we performed gene expression analysis in DN1, DN2 and DN3 cells isolated from WT and DKO mice at pre-disease and ETP-ALL stages. Since NOTCH1 activating mutations are not frequently found in patients with ETP-ALL, we observed no significant difference in activation of Notch1-target genes between WT and DKO ETP-ALL cells. In contrast, Ras-target genes were significantly activated in DKO ETP-ALL DN2 cells relative to WT DN2 cells. Thus, our ETP-ALL mouse model shows active Ras signaling but lacks Notch1 activation, consistent with the molecular features of human ETP-ALL. Furthermore, we found that the genes critical for T-cell commitment, including Tcf7 or Bcl11b, were transcriptionally repressed in DKO ETP-ALL DN2 cells compared to WT DN2 cells, while the signature genes of both HSCs and myeloid cells were retained in DKO ETP-ALL DN2 cells. Taken together, Ezh2 and p53 deletions cooperate to activate the function of HSCs and impede the transcriptional program of T-cell differentiation at the DN2 stage with sustaining myeloid potential. To determine how H3K27me3 modification contributed to induce ETP-ALL in the absence of Ezh2, we performed H3K27me3-chromatin immunoprecipitation (ChIP) sequencing in WT and DKO ETP-ALL DN1/2 cells. We found that H3K27me3 marks were lost or kept at low levels at the promoter regions of T-cell differentiation regulators in ETP-ALL cells. Thus, we sought to determine whether altered DNA hypermethylation contributed to silencing the expression of T-cell differentiation regulators. The transduction of either Tcf7 or Bcl11b expression alone was not sufficient to induce differentiation of DKO DN1/2 cells in vitro, however, we found that treatment of decitabine, a demethylating agent, clearly induced the differentiation of DKO DN1/2 cells beyond the DN3 stage in vitro, implying that Ezh2 loss and p53 loss cooperatively induced aberrant DNA hypermethylation, thereby impeding the differentiation of DN1/2 cells. In conclusion, we demonstrated that combined deletion of Ezh2 and p53 altered the epigenetic regulation to an extent not seen in either deletion alone, and induced highly penetrant ETP-ALL characterized by the molecular profile similar to that in patients with ETP-ALL harboring mutations in the PRC2 components. DisclosuresNo relevant conflicts of interest to declare.

New role for the (pro)renin receptor in T-cell development

AbstractThe (pro)renin receptor (PRR) was originally thought to be important for regulating blood pressure via the renin-angiotensin system. However, it is now emerging that PRR has instead a generic role in cellular development. Here, we have specifically deleted PRR from T cells. T-cell–specific PRR-knockout mice had a significant decrease in thymic cellularity, corresponding with a 100-fold decrease in the number of CD4+ and CD8+ thymocytes, and a large increase in double-negative (DN) precursors. Gene expression analysis on sorted DN3 thymocytes indicated that PRR-deficient thymocytes have perturbations in key cellular pathways essential at the DN3 stage, including transcription and translation. Further characterization of DN T-cell progenitors leads us to propose that PRR deletion affects thymocyte survival and development at multiple stages; from DN3 through to DN4, double-positive, and single-positive CD4 and CD8. Our study thus identifies a new role for PRR in T-cell development.

Disregulated expression of the transcription factor ThPOK during T-cell development leads to high incidence of T-cell lymphomas

The transcription factor T-helper-inducing POZ/Krueppel-like factor (ThPOK, encoded by the Zbtb7b gene) plays widespread and critical roles in T-cell development, particularly as the master regulator of CD4 commitment. Here we show that mice expressing a constitutive T-cell-specific ThPOK transgene (ThPOK(const) mice) develop thymic lymphomas. These tumors resemble human T-cell acute lymphoblastic leukemia (T-ALL), in that they predominantly exhibit activating Notch1 mutations. Lymphomagenesis is prevented if thymocyte development is arrested at the DN3 stage by recombination-activating gene (RAG) deficiency, but restored by introduction of a T-cell receptor (TCR) transgene or by a single injection of anti-αβTCR antibody into ThPOK(const) RAG-deficient mice, which promotes development to the CD4(+)8(+) (DP) stage. Hence, TCR signals and/or traversal of the DN (double negative) > DP (double positive) checkpoint are required for ThPOK-mediated lymphomagenesis. These results demonstrate a novel link between ThPOK, TCR signaling, and lymphomagenesis. Finally, we present evidence that ectopic ThPOK expression gives rise to a preleukemic and self-perpetuating DN4 lymphoma precursor population. Our results collectively define a novel role for ThPOK as an oncogene and precisely map the stage in thymopoiesis susceptible to ThPOK-dependent tumor initiation.

291. A Novel Safety Assay for Retroviral Vectors That Reproduces Lmo2 Proto-Oncogene Insertional Activation Events

Gene therapies for X-linked severe combined immunodeficiency (SCID-X1) and Wiskott-Aldrich syndrome (WAS) with gammaretroviral vectors have caused a number of cases of acute lymphoid leukemias due to insertional activation of oncogenes, mostly LMO2. Transduction of murine bone marrow hematopoietic cells and subsequent culture for myeloid immortalization or transplant into recipient mice detected the oncogenic activity of the spleen focus-forming virus (SFFV) vector, which was used in the WAS trial, but failed to detect the oncogenic activity of the SCID-X1 gene therapy vector MFG-gc. The lack of the most relevant Lmo2 insertion by gammaretroviral vectors in these assays demands more relevant and sensitive vector safety assays. It has been shown that murine early thymic progenitor cells (ETP), when cultured on OP9-DL1 stroma cells, progress through the DN1, DN2, DN3, DN4, DP and SP differentiation stages, and that overexpression of LMO2 in ETP cells caused a differentiation block at the DN2 stage (CD4-CD8-CD25+CD44+). We tested whether transduction of ETP cells with gammaretroviral or lentiviral vectors could reproduce Lmo2 integrations and induce DN2 differentiation block. We tested three different gamma-retroviral vectors MSCV-GFP, SFFV-GFP and MFG-gc and two lentiviral vectors Cl20-SFFV-mCherry and Cl20i4r-SFFV-mCherry. The two self-inactivating lentiviral vectors contain a single internal SFFV long terminal repeat but differ in that one contains the cHS400 chromatin insulators. We transduced ETP cells with these vectors and cultured them on OP9-DL1 cells for up to 40 days. The range of vector copy number was 1.07-10.78 for the MSCV-GFP vector, 0.24-1.22 for the SFFV-GFP vector, 0.59-7.98 for the MFG-gc vector, 3.97-13.04 for the Cl20-SFFV-mCherry vector and 4.57-18.21 for the Cl20i4r-SFFV-mCherry vector at days 10-11. Between days 20-35, a distinct DN2-blocked cell subpopulation was clearly detected by flow cytometry in all the 20 gamma-retroviral groups and also in 3/9 lentiviral groups. None of the 6 mock groups had any evidence of DN2 blockade. Insertion site mapping of sorted DN2 cells showed that in the 10 MSCV-GFP groups, 3 had Lmo2 insertions, 3 had Mef2c insertions, and 3 had both Lmo2 and Mef2c insertions; in the 6 SFFV-GFP groups, 3 had Lmo2 insertions; in the 4 MFG-gc groups, 3 had Lmo2 insertions; both Cl20-SFFV-mCherry groups had Mef2c insertions and the one Cl20i4r-SFFV-mCherry group had Lmo2 insertion. These insertions occurred either in the introns or within +/- 50kb window of the gene. Transplant of the DN2-blocked cells from selected MSCV-GFP groups into recipient mice led to acute lymphoid leukemias. These results show that the transduction of ETP cells and the resultant DN2 blockade represent a more relevant and sensitive assay for vector safety assessment. Our result also suggests that an ETP-like subpopulation may be present in the bone marrow of SCID-X1 and WAS patients and explain the enhanced propensity of these disorders to oncogenic transformations.

445. A Delectable Lentiviral Vector System for Testing Oncogene Addiction in Hematological Malignancies

Mouse models of hematological malignancies can be established by overexpressing oncogenes in hematopoietic cells followed by transplantation. It is often unclear whether the oncogene is only necessary for initiating the malignancies or if it is also required for maintenance, known as oncogene addiction. An addicted oncogene could then serve as target for therapy. We have developed a dual lentiviral vector system to distinguish these possibilities. We chose the oncogene LMO2 as our model because it is involved in a number of leukemias caused by gamma-retroviral vector insertion in gene therapy of SCID-X1 and Wiskott-Aldrich syndrome. Our previous studies showed that overexpression of LMO2 using a gamma-retroviral vector in early thymic progenitors (ETP) caused a differentiation block at the DN2 stage of development and subsequent transplant of these cells gave rise to leukemia with 100% penetrance in mice. We inserted the LoxP sequence into the 3’ U3 region of a self-inactivating CL20 lentiviral backbone and subcloned a MSCV-LMO2-i-GFP expression cassette into the vector (Fig 1A) to make a CL20-tLMO2-i-GFP vector. To deliver Cre recombinase in an inducible fashion, we constructed a second lentiviral vector in which a MSCV-CreERT2-i-mCherry expression cassette is inserted. Vectors were made by transiently transfecting 293T cells and the unconcentrated titer of these vectors were 2E6 and 3E6 TU/ml, respectively. We first tested these vectors in NIH3T3 cells. NIH3T3 cells transduced with these two vectors were subsequently sorted for GFP+mCherry+ double positive cells. We then treated the cells with various concentrations of 4-hydroxytamoxifen (4-OHT) to induce nuclear entry of Cre recombinase in order to delete the MSCV-LMO2-i-GFP expression cassette. Seven days later, 79.3% of the cells lost GFP expression in the group that was treated with 100nM 4-OHT (Fig 1B), indicating efficient cassette deletion. The loss of LMO2 protein is also confirmed by Western blot (Fig 1C). We then transduced ETP cells with the CL20-tLMO2-i-GFP vector and cultured the cells on an OP9-DL1 stroma expressing the Delta-like ligand DL1 to induce differentiation. At day 14, 90.5% of the transduced cells were blocked at the DN2 stage, demonstrating that this deletable lentiviral vector was able to express sufficient levels of LMO2 to induce the DN2 block (Fig 1D). These cells have been transplanted into sublethally irradiated Rag2-/-IL2rg-/- mice to establish the leukemia. One leukemia has been established with a GFP+ tumor, enlarged spleen of 0.534g and an erythroid phenotype as analyzed via flow cytometry. Tumor cells have been transduced with CreERT2 vector for secondary transplant and treatment with tamoxifen to establish whether LMO2 is required for tumor maintainence. In summary, we have established a dual lentiviral vector system that can be used conveniently to study oncogene addiction in hematological malignancies. View Large Image | Download PowerPoint Slide

T cell development is regulated by the coordinated function of two Lck promoters (IRM7P.701)

Abstract Lck is a T cell lineage-specific tyrosine kinase that is critical for T cell development and activation. Expression of an identical Lck protein can be mediated by two promoters, the proximal and distal lck promoters, but the function of these promoters during T cell development has not been directly assessed. We generated mutant mice by inactivating either one of the two lck promoters. Lck-PROX mice, in which only the proximal promoter is functional, had normal levels of Lck protein and thymic development through the CD4-CD8- double negative (DN) stage of T cell development, but reduced levels of Lck later in development and a dramatic reduction of mature single positive thymic T cells. In contrast, Lck-DIST mice, in which only the distal promoter was functional, were deficient in Lck and severely defective in early T cell development, with a block at the DN3 stage equivalent to the defect seen in complete Lck knockouts. Interestingly, the small number of peripheral T cells that developed in Lck-DIST mice expressed Lck protein and proliferated normally in response to TCR/CD3 cross-linking, while those of Lck-PROX mice were deficient in Lck and defective in response to TCR stimulation. These results define the coordinated requirements for proximal and distal lck promoters during T cell development and represent a unique direct demonstration of the requirement for alternative promoters in allowing developmental stage-specific regulation of a single protein product.

Activation of the Hypoxia-Inducible Factor Pathway Expands Early Thymic Progenitors

Early thymic progenitors enter the thymus and are exposed to regional hypoxia while they develop in a step-wise manner to mature functional T-cells. Therefore, hypoxia might represent an important component of the highly specialized thymic microenvironment. On the molecular level the hypoxia-inducible factor pathway controls the cellular response to hypoxia. In this pathway, the von-Hippel-Lindau protein (pVHL) continuously mediates the destruction of the transcription factor hypoxia-inducible factor-1α (HIF-1α) under normoxic conditions. Under hypoxia HIF-1α degradation is inhibited leading to the activation of HIF-1α target genes.Others used lck-Cre transgene-mediated conditional in vivo deletion of the Vhl gene to study the role of the oxygen-sensing pathway in developing thymocytes and found normal numbers of early double-negative (DN; CD4-CD8-) thymocytes (Biju et al., Mol Cell Biol, 2004). However, lck-Cre deletion initiates at the DN3 (CD25+CD44-) stage leaving the Vhl locus of very early DN1 (CD25-CD44+), DN2 (CD25+CD44+) and DN3 thymocytes unaltered. Therefore, we here used the ubiquitous hematopoietic deleter strain vav-Cre to investigate the role of pVHL in very early thymocytes (vav-Cre;VhlloxP;loxP mice). Using a PCR-based strategy we confirmed complete deletion of the Vhl gene in this model. We observed unaltered DN1 and DN2 progenitor numbers, however in contrast to the published lck-cre-mediated system we consistently observed an up to twofold expansion of the DN3 cellular compartment. As the hypoxia-inducible factor pathway was shown to modulate NOTCH1 signaling we studied Notch1 expression on Vhl-deficient thymocytes. Strikingly, Notch1 expression was significantly increased on expanded Vhl null DN3 thymocytes. At the DN3 developmental stage selection of cells with an accurately re-arranged T-cell receptor β-locus occurs. Thus, we analyzed pre- and post-β-selection DN3 cells by CD28 staining. Interestingly, we found both pre- and post-β-selection DN3 subpopulations expanded. In order to investigate whether the progenitor expansion is mediated by the lack of HIF-1α inhibition in the Vhl-deficient context we studied DN3 thymocytes in a conditional hematopoietic HIF-1α gain-of-function model (vav-Cre;HIF1dPA). Overexpression of HIF-1α, which is insensitive to pVHL-mediated degradation in vav-Cre;HIF1dPAmice, also resulted in expanded DN3 thymocytes.In summary, we describe novel conditional models to genetically alter the hypoxia-inducible factor pathway within very early thymic progenitors. Genetic Vhl loss led to an expansion of DN3 thymocytes. This DN3 expansion is most likely due to the absence of HIF-1α-inhibition, because HIF-1α overexpression phenocopied the Vhl-deficient DN3 thymocyte expansion. DisclosuresDührsen:Celgene: Honoraria, Research Funding.

GATA-3 dose-dependent checkpoints in early T cell commitment.

GATA-3 expression is crucial for T cell development and peaks during commitment to the T cell lineage, midway through the CD4(-)CD8(-) (double-negative [DN]) stages 1-3. We used RNA interference and conditional deletion to reduce GATA-3 protein acutely at specific points during T cell differentiation in vitro. Even moderate GATA-3 reduction killed DN1 cells, delayed progression to the DN2 stage, skewed DN2 gene regulation, and blocked appearance of the DN3 phenotype. Although a Bcl-2 transgene rescued DN1 survival and improved DN2 cell generation, it did not restore DN3 differentiation. Gene expression analyses (quantitative PCR, RNA sequencing) showed that GATA-3-deficient DN2 cells quickly upregulated genes, including Spi1 (PU.1) and Bcl11a, and downregulated genes, including Cpa3, Ets1, Zfpm1, Bcl11b, Il9r, and Il17rb with gene-specific kinetics and dose dependencies. These targets could mediate two distinct roles played by GATA-3 in lineage commitment, as revealed by removing wild-type or GATA-3-deficient early T lineage cells from environmental Notch signals. GATA-3 worked as a potent repressor of B cell potential even at low expression levels, so that only full deletion of GATA-3 enabled pro-T cells to reveal B cell potential. The ability of GATA-3 to block B cell development did not require T lineage commitment factor Bcl11b. In prethymic multipotent precursors, however, titration of GATA-3 activity using tamoxifen-inducible GATA-3 showed that GATA-3 inhibits B and myeloid developmental alternatives at different threshold doses. Furthermore, differential impacts of a GATA-3 obligate repressor construct imply that B and myeloid development are inhibited through distinct transcriptional mechanisms. Thus, the pattern of GATA-3 expression sequentially produces B lineage exclusion, T lineage progression, and myeloid-lineage exclusion for commitment.

The multifaceted roles of Bcl11b in thymic and peripheral T cells: impact on immune diseases.

The transcription factor Bcl11b is expressed in all T cell subsets and progenitors, starting from the DN2 stage of T cell development, and it regulates critical processes implicated in the development, function, and survival of many of these cells. Among the common roles of Bcl11b in T cell progenitors and mature T cell subsets are the repression of the innate genetic program and, to some extent, expression maintenance of TCR-signaling components. However, Bcl11b also has unique roles in specific T cell populations, suggesting that its functions depend on cell type and activation state of the cell. In this article, we provide a comprehensive review of the roles of Bcl11b in progenitors, effector T cells, regulatory T cells, and invariant NKT cells, as well as its impact on immune diseases. While emphasizing common themes, including some that might be extended to skin and neurons, we also describe the control of specific functions in different T cell subsets.

Clinical Spectrum of SCID: The Key is in the Thymus?

Clinical Spectrum of SCID: The Key is in the Thymus?

IFN-γ–Producing and IL-17–Producing γδ T Cells Differentiate at Distinct Developmental Stages in Murine Fetal Thymus

γδ T cells develop at the double-negative (DN) 2 and DN3 stages and acquire functions to produce IL-17 and IFN-γ in fetal thymus. However, the relationship between differentiation stages and their functions was unclear. In this study, we found that, although IFN-γ-producing and IL-17-producing γδ T cells developed from DN2 cells, only IFN-γ-producing γδ T cells developed from DN3 cells, indicating the direct generation of IL-17-producing γδ T cells from the DN2 stage, not through the DN3 stage. Single-cell analysis revealed that DN2 cells contained heterogeneous γδ T cell precursors with or without an ability to develop IL-17 producers. Inactivation of B cell leukemia/lymphoma 11b, a zinc finger transcription factor responsible for transition from early to late stages of DN2 cells, completely abrogated the development of IL-17-producing γδ T cells, although a unique subset of IFN-γ-producing γδ T cells expressing a high level of promyelocytic leukemia zinc finger was able to develop. Thus, our results reveal that γδ T cells are functionally differentiated to IFN-γ and IL-17 producers at different developmental stages in fetal thymus.

Ectopic TLX1 Expression Accelerates Malignancies in Mice Deficient in DNA-PK

The noncluster homeobox gene HOX11/TLX1 (TLX1) is detected at the breakpoint of the t(10;14)(q24;q11) chromosome translocation in patients with T cell acute lymphoblastic leukemia (T-ALL). This translocation results in the inappropriate expression of TLX1 in T cells. The oncogenic potential of TLX1 was demonstrated in IgHμ-TLX1Tg mice which develop mature B cell lymphoma after a long latency period, suggesting the requirement of additional mutations to initiate malignancy. To determine whether dysregulation of genes involved in the DNA damage response contributed to tumor progression, we crossed IgHμ-TLX1Tg mice with mice deficient in the DNA repair enzyme DNA-PK (PrkdcScid/Scid mice). IgHµ-TLX1TgPrkdcScid/Scid mice developed T-ALL and acute myeloid leukemia (AML) with reduced latency relative to control PrkdcScid/Scid mice. Further analysis of thymi from premalignant mice revealed greater thymic cellularity concomitant with increased thymocyte proliferation and decreased apoptotic index. Moreover, premalignant and malignant thymocytes exhibited impaired spindle checkpoint function, in association with aneuploid karyotypes. Gene expression profiling of premalignant IgHµ-TLX1TgPrkdcScid/Scid thymocytes revealed dysregulated expression of cell cycle, apoptotic and mitotic spindle checkpoint genes in double negative 2 (DN2) and DN3 stage thymocytes. Collectively, these findings reveal a novel synergy between TLX1 and impaired DNA repair pathway in leukemogenesis.

Dietary Restriction and Fasting Arrest B and T Cell Development and Increase Mature B and T Cell Numbers in Bone Marrow

Dietary restriction (DR) delays ageing and extends life span. Both long- and short-term DR, as well as short-term fasting provide robust protection against many “neuronal and surgery related damaging phenomena” such as Parkinson’s disease and ischemia-reperfusion injury. The exact mechanism behind this phenomenon has not yet been elucidated. Its anti-inflammatory actions prompted us to thoroughly investigate the consequences of DR and fasting on B and T cell compartments in primary and secondary lymphoid organs of male C57Bl/6 mice. In BM we found that DR and fasting cause a decrease in the total B cell population and arrest early B cell development, while increasing the number of recirculating mature B cells. In the fasting group, a significant reduction in peripheral B cell counts was observed in both spleen and mesenteric lymph nodes (mLN). Thymopoiesis was arrested significantly at double negative DN2 stage due to fasting, whereas DR resulted in a partial arrest of thymocyte development at the DN4 stage. Mature CD3+ T cell populations were increased in BM and decreased in both spleen and mLN. Thus, DR arrests B cell development in the BM but increases the number of recirculating mature B cells. DR also arrests maturation of T cells in thymus, resulting in depletion of mature T cells from spleen and mLN while recruiting them to the BM. The functional relevance in relation to protection against organ damage needs to be determined.

N-Myc Is Overexpressed In Both Murine and Human Early T-Cell Precursor Leukemia and Is Sufficient To Initiate this Leukemia In Multipotent Primitive Arf-/- thymocytes

Early T-cell precursor–ALL (ETP-ALL) is a subtype of T-cell acute lymphoblastic leukemia that displays both myeloid and lymphoid phenotypic features and has a relatively poor prognosis. Our previous work has shown that activating mutations in the Il7 receptor (Il7r) and Lmo2 overexpression in CD4-CD8- thymocytes cause ETP-ALL in mice via DN2 thymocyte differentiation block. To further understand the molecular mechanisms responsible for initiation and maintenance of ETP-ALL, we compared expression profiles of seven murine ETP leukemias induced by Lmo2 and Il7r mutant vectors and four preleukemic blocked DN2 Arf-/- thymocyte populations to normal murine DN2 Arf-/- thymocytes. This analysis and qRT-PCR identified N-Myc as one of the top upregulated genes in either Lmo2 or Il7r mutant samples. Importantly in pediatric ETP-ALL N-Myc RNA is expressed at high levels in 11/13 human cases compared to 26/72 T-ALL cases corroborating our murine model with the human disease. To investigate if N-Myc itself was sufficient to induce ETP leukemia, immature murine Arf-/- thymocytes were transduced with a retroviral vector expressing the N-Myc cDNA and YFP. After 20 days in culture a block at the DN2 stage of thymocyte development was observed that recapitulated the Lmo2 and the Il7r receptor mutant phenotype. When sublethally irradiated Rag2-/-, γc-/- recipient mice were transplanted with N-Myc transduced Arf-/- thymocytes 5/5 of the primary recipients developed ETP leukemia originating from the thymocyte graft with a median latency of 60 days (Figure 1). These mice had elevated white blood cell counts between 17-85x103/µl and enlarged spleens ranging in weight from 0.42g-0.67g. Flow cytometry analysis showed that their spleen, thymus and peripheral blood contained greater than 70% YFP+ Gr1+ cells. These N-Myc induced leukemias expressed both T-cell (cytoplasmic CD3) and myeloid (Gr1 and Mac1) markers and displayed other features typical of ETP disease. Having established that N-Myc is sufficient to initiate ETP leukemia in the murine model, we next tested if N-Myc is critical in the maintenance of ETP leukemia induced by Il7r mutants. ETP leukemic cells arising from the Il7r 241-242TC mutant were transduced with a mir30 lentiviral vector that contains a short hairpin N-Myc inhibiting sequence and mCherry as a reporter gene (shN-Myc). This vector has been shown to reduce N-Myc mRNA levels by five fold in N-Myc overexpressing cells. The transduced cells were analyzed and sorted for leukemic+ (GFP) scrambled+ (mCherry) or leukemic+(GFP) shN-Myc+(mCherry) double positive cells. Analysis of the cells showed the mCherry mean fluorescence intensity (MFI) of the leukemic cells transduced with shN-Myc was approximately double that of the scrambled transduced cells, demonstrating at least equal transduction efficiency of the two vectors. Sorted double positive leukemic cells were transplanted into sublethally irradiated WT recipients. All animals succumbed to leukemia and were subsequently analyzed by flow cytometry for expression of scrambled and shN-Myc vector expression. Analysis of tumor cells showed a significant decrease in the mCherry expression of the cells containing the shN-Myc compared to the scrambled control indicating that sustained N-Myc expression is selected for during the maintenance of ETP leukemia and in the future a tetracycline control element will be incorporated into the shN-Myc to confirm this (Figure 2). Furthermore we plan to test therapeutics against N-Myc such as bromodomain and Aurora kinase A inhibitors in vitro and plan to expand this in vivo. This demonstrates the utility of this novel mouse model combined with gene expression profiling to elucidate the signaling networks in ETP leukemia to develop targeted therapy for this aggressive disease. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Abstract 3851: NUP98-PHF23 (NP23) binds H3K4me3 and causes a wide spectrum of leukemias, including AML, T-ALL, and pre-B1 ALL.

Abstract Nucleoporin 98 (NUP98) fusions have been associated with acute myeloid leukemia (AML), chronic myeloid leukemia, myelodysplastic syndrome, and T-cell acute lymphoblastic leukemia (T-ALL). An AML patient with a t(11;17)(p15;p13) chromosome translocation was found to have a novel gene fusion between NUP98 and plant homeodomain (PHD) finger 23 (PHF23). PHF23 has been reported to act as a reader of tri-methylated histone 3 lysine 4 (H3K4me3) marks, suggesting that PHF23 may function in chromatin regulation and that the NP23 fusion protein may play a role in aberrant chromatin modification. To determine the oncogenic potential of NP23, we generated mice that expressed the NP23 fusion in hematopoietic tissues. We characterized two founder lines (C10 and B10) that expressed the NP23 fusion. The C10 line principally developed AML that closely resembled the human disease, with increased blasts in the blood and bone marrow, widespread organ infiltration, and myeloid immunophenotype. Onset of disease was as early as 4.5 months, and 70 percent of the NP23 mice succumbed to leukemia by 12 months of age. The B10 line developed a wider spectrum of leukemias with similar age of onset and penetrance. The B10 mice predominantly developed T-ALL and AML. Interestingly, four B10 mice developed a previously unreported form of ALL that was of pre-B1 (B220−/CD19+/AA4.1+) origin, indicating that the NP23 protein is oncogenic in multiple hematopoietic lineages. Gene expression arrays and RQ-PCR revealed Hoxa5, a7, a9 and a10 to be overexpressed from 10-1000 fold in both AML and T-ALL, as well as in hematopoietic tissues from clinically healthy NP23 transgenic mice. Premalignant, clinically healthy mice showed aberrant hematopoietic stem and progenitor cell (HSPC) populations. Bone marrow lineage negative (Linneg) cells were decreased by half that of WT littermates, and LSK (Linneg/Sca1+/cKit+) cells were further reduced by 3-fold. NP23 thymi were dramatically smaller in size with an average 5-fold reduction in thymocytes, which was associated with a partial differentiation block of thymocytes at the DN2 stage. B2 pre-B-cells were significantly reduced, by 50%, compared to WT controls. B1-progenitor B-cells (Linneg/CD19+/AA4.1+) in the bone marrow were reduced by 30% in the bone marrow and mature B1 B-cells (B220−/CD19+/IgM+) of the peritoneal cavity were further decreased to half that of WT. We also performed genome wide NP23 and H3K4me3 ChIP-seq which identified a core set of H3K4me3 marked genes bound by the NP23 fusion protein that were overexpressed in both myeloid and T-cell lineages. The NP23 oncogene is capable of promoting cell lineage-independent leukemic transformation, and will be useful in identifying oncoproteins and transformation pathways that involve chromatin deregulation and HSPC gene signatures such as those of the HOXA cluster. Citation Format: Sheryl M. Gough, Fan Lee, Yang Jo Chung, Robert L. Walker, Fan Yang, Jack Zhu, Yi Ning, Paul S. Meltzer, Peter D. Aplan. NUP98-PHF23 (NP23) binds H3K4me3 and causes a wide spectrum of leukemias, including AML, T-ALL, and pre-B1 ALL. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3851. doi:10.1158/1538-7445.AM2013-3851

RasGRP1, but Not RasGRP3, Is Required for Efficient Thymic β-Selection and ERK Activation Downstream of CXCR4

T cell development is a highly dynamic process that is driven by interactions between developing thymocytes and the thymic microenvironment. Upon entering the thymus, the earliest thymic progenitors, called CD4−CD8− ‘double negative’ (DN) thymocytes, pass through a checkpoint termed “β-selection” before maturing into CD4+CD8+ ‘double positive’ (DP) thymocytes. β-selection is an important developmental checkpoint during thymopoiesis where developing DN thymocytes that successfully express the pre-T cell receptor (TCR) undergo extensive proliferation and differentiation towards the DP stage. Signals transduced through the pre-TCR, chemokine receptor CXCR4 and Notch are thought to drive β-selection. Additionally, it has long been known that ERK is activated during β-selection; however the pathways regulating ERK activation remain unknown. Here, we performed a detailed analysis of the β-selection events in mice lacking RasGRP1, RasGRP3 and RasGRP1 and 3. We report that RasGRP1 KO and RasGRP1/3 DKO deficient thymi show a partial developmental block at the early DN3 stage of development. Furthermore, DN3 thymocytes from RasGRP1 and RasGRP1/3 double knock-out thymi show significantly reduced proliferation, despite expression of the TCRβ chain. As a result of impaired β-selection, the pool of TCRβ+ DN4 is significantly diminished, resulting in inefficient DN to DP development. Also, we report that RasGRP1 is required for ERK activation downstream of CXCR4 signaling, which we hypothesize represents a potential mechanism of RasGRP1 regulation of β-selection. Our results demonstrate that RasGRP1 is an important regulator of proliferation and differentiation at the β-selection checkpoint and functions downstream of CXCR4 to activate the Ras/MAPK pathway.

Computational modelling of T-cell formation kinetics: output regulated by initial proliferation-linked deferral of developmental competence

Bone-marrow-derived progenitors must continually enter the thymus of an adult mouse to sustain T-cell homeostasis, yet only a few input cells per day are sufficient to support a yield of 5 × 10(7) immature T-cells per day and an eventual output of 1-2 × 10(6) mature cells per day. While substantial progress has been made to delineate the developmental pathway of T-cell lineage commitment, still little is known about the relationship between differentiation competence and the remarkable expansion of the earliest (DN1 stage) T-cell progenitors. To address this question, we developed computational models where the probability to progress to the next stage (DN2) is related to division number. To satisfy differentiation kinetics and overall cell yield data, our models require that adult DN1 cells divide multiple times before becoming competent to progress into DN2 stage. Our findings were subsequently tested by in vitro experiments, where putative early and later-stage DN1 progenitors from the thymus were purified and their progression into DN2 was measured. These experiments showed that the two DN1 sub-populations divided with similar rates, but progressed to the DN2 stage with different rates, thus providing experimental evidence that DN1 cells increase their commitment probability in a cell-intrinsic manner as they undergo cell division. Proliferation-linked shifts in eligibility of DN1 cells to undergo specification thus control kinetics of T-cell generation.

The TAL1 Complex Represses the FBXW7 Tumor Suppressor Through Mir-223 in Human T-Cell Acute Lymphoblastic Leukemia

Abstract 1296The oncogenic transcription factor TAL1/SCL is aberrantly expressed in over 40% of cases of human T-cell acute lymphoblastic leukemia (T-ALL) and causes T-ALL in murine transgenic models, emphasizing its importance in the molecular pathogenesis of this disease. However, the mechanism by which TAL1 leads to transformation of thymocytes is unclear. Dysregulation of miRNAs play an important role in tumorigenesis in diverse cancer types. A recent study identified miR-223 as the most abundant miRNA in T-ALL patient samples and was oncogenic by virtue of its ability to accelerate Notch-induced T-ALL in a murine model (Mavrakis et al. Nature Genetics 2011). However, the underlying mechanisms leading to dysregulated miRNA expression in T-ALL remain poorly understood.In order to explore the hypothesis that aberrant expression of miRNAs is mediated by the TAL1 oncogene in T-ALL, we generated high-resolution maps of the genome-wide occupancy of the TAL1 complex, including E2A, HEB, GATA3, LMO2 and RUNX1 by chromatin immunoprecipitation coupled to massively parallel DNA sequencing (ChIP-seq). Analysis of binding sites in two TAL1-positive T-ALL cell lines (Jurkat and CCRF-CEM cells) and two primary T-ALL samples identified 54 miRNAs where binding of the TAL1 complex was within 10 kb of either the transcriptional start sites or the start sites of genes that contain miRNAs in their intronic regions. To determine which of these miRNAs were not only directly bound, but also regulated by the TAL1 complex, we analyzed global changes in miRNAs after knockdown of TAL1 in Jurkat cells using two independent shRNAs. By miRNA expression profiling, we identified significant changes in expression of 25 miRNAs, of which nine were down-regulated on TAL1 knockdown (and thus positively regulated by TAL1) and 16 were up-regulated on TAL1 knockdown (and thus negatively regulated by TAL1). Of these 25 miRNAs, four (miR-223, miR181a*, miR-26a and miR-29c) were shown to be direct targets of the TAL1 complex based on our ChIP-seq data.We chose to focus on miR-223 because it exhibited the most dynamic down-regulation after TAL1 knockdown. ChIP-qPCR validated binding of the TAL1 complex to a region within 4 kb of the miR-223 transcriptional start site. Analysis of RNA polymerase II and CBP binding showed significant enrichment, and high levels of H3K4M3 and H3K79M2 modification were detected indicative of transcriptional initiation and elongation of this locus. Furthermore, expression of miR-223 was significantly higher in the TAL1-positive cell lines (n=13) as compared to the TAL1-low cells (n=10) (P<0.0001). miR-223 levels also closely mirrored TAL1 levels in murine thymic subsets, with marked down-regulation after the DN2 stage, suggesting miR-223 is a physiological target of TAL1 during normal thymic development, and that its overexpression in TAL1-positive T-ALL cells, arrested at the double-positive (DP) stage, is aberrant compared to their normal DP counterpart.To test the hypothesis that the growth inhibition observed after TAL1 knockdown is mediated by decreases in miR-223 expression, we retrovirally infected Jurkat and RPMI-8402 T-ALL cell lines with a miR-223 construct, such that miR-223 expression was no longer under the control of TAL1 in these cells. Forced expression of miR-223 partially rescued the growth inhibitory effects induced by TAL1 knockdown, in both a lentiviral and doxycycline-inducible shRNA system. Additionally, inhibition of mature miR-223 by lentiviral infection of a miR-223 shRNA construct led to significant growth inhibition of TAL1-positive cell lines through the induction of apoptosis. Thus, maintenance of miR-223 expression is required for optimal growth of TAL1-positive T-ALL cells.The highest ranked predicted target of miR-223 by Targetscan is the FBXW7 tumor suppressor, a ubiquitin ligase that is mutated in a significant proportion of T-ALL patients and targets oncogenes such as c-MYC, NOTCH and mTOR for degradation. Accordingly, overexpression of miR-223 in TAL1-low miR-223-low T-ALL cells markedly down-regulated FBXW7 protein expression. Furthermore, the up-regulation of FBXW7 protein expression observed on knockdown of TAL1 in TAL1-positive cell lines could be prevented by retroviral miR-223 expression. Thus, miR-223 is an important target of TAL1 and links the TAL1 oncogene to repression of the FBXW7 tumor suppressor. Disclosures:No relevant conflicts of interest to declare.

The protooncogene Vav1 regulates murine leukemia virus-induced T-cell leukemogenesis

Vav1 is expressed exclusively in hematopoietic cells and is required for T cell development and activation. Vav1-deficient mice show thymic hypocellularity due to a partial block during thymocyte development at the DN3 stage and between the double positive (DP) and single positive (SP) transition. Vav1 has been shown to play a significant role in several non-hematopoietic tumors but its role in leukemogenesis is unknown. To address this question, we investigated the role of Vav1 in retrovirus-induced T cell leukemogenesis. Infection of Vav1-deficient mice with the Moloney strain of murine leukemia virus (M-MuLV) significantly affected tumor phenotype without modulating tumor incidence or latency. M-MuLV-infected Vav1-deficient mice showed reduced splenomegaly, higher hematocrit levels and hypertrophic thymi. Notably, Vav1-deficient mice with M-MuLV leukemias presented with markedly lower TCRβ/CD3 levels, indicating that transformation occurred at an earlier stage of T cell development than in WT mice. Thus, impaired T cell development modulates the outcome of retrovirus-induced T cell leukemias, demonstrating a link between T cell development and T cell leukemogenesis.

Self-renewal of thymocytes in the absence of competitive precursor replenishment

Soon after transplantation of wild-type thymi into immunodeficient mice lacking functional T cell receptors, productive T cell development in the donor thymus ceases. This observation underlies one of the central dogmas of T cell biology: because thymocytes are seemingly short-lived, intrathymic T cell development depends on continuous import of lymphoid progenitors from the bone marrow. New work reinterprets the outcome of this classical experiment as being the result of competition for intrathymic niches specifically supporting the DN3 stage of early T cell development. Surprisingly, when this niche space is uncontested by immigrating host progenitors, development of T cells in the thymus grafts continues. These new findings suggest that early thymocytes do indeed have substantial self-renewing potential.