Early T-cell Progenitors Research Articles

The Splenic Microenvironment Is a Novel Site of Relapse Following ABT-199 Treatment

T-cell acute lymphoblastic leukaemia (T-ALL) is an aggressive hematologic malignancy arising from the transformation of immune T-cell lymphocytes. Early T-cell progenitor (ETP-ALL) is a subgroup particularly associated with a poor prognosis and a high risk for relapse. While the leukaemia initially develops in the thymus it spreads in the blood to the bone marrow, lymph nodes and often the spleen. Interestingly, splenomegaly was previously associated with a poor prognosis in leukemic patients. Recently, it was shown that ETP-ALL is dependent on the expression of the anti-apoptotic protein BCL-2, and is sensitive to inhibition with ABT-199, a BCL-2 specific BH3 mimetic. However, one issue with targeted agents, like ABT-199, is the development of resistance to treatment. Our aim was to determine potential in vivosites of resistance/relapse following ABT-199 treatment using a xenograft model of ETP-ALL. We confirmed that the ETP-ALL LOUCY cell line is BCL-2 dependent and then labelled it with luciferase to enable visualisation of the leukaemia in vivo. Following establishment of the leukaemia in NOD/SCID gamma mice, as assessed by hCD45+, the mice were randomised to receive vehicle control or 50 mg/kg ABT-199 by oral gavage daily for two weeks. While the mice were initially sensitive to ABT-199, the leukaemia started to progress while on treatment. Interestingly, there appeared to be a selective redistribution of the leukaemia to the spleen following ABT-199 treatment. Indeed, LOUCY cells isolated from the spleen of the mice had a reduced BCL-2 dependence, as assessed by BH3 profiling. The reduced BCL-2 dependence correlated with reduced BCL-2 expression at both the mRNA and protein level. Next, we confirmed that human splenic fibroblasts (HSF) co-cultured with the LOUCY cell line in vitro also altered BCL-2 dependence and expression using BH3 profiling and Western blotting. To identify potential splenic cytokines involved in the regulation of BCL-2 protein expression in ETP-ALL we performed a screening cytokine array. Upon co-culture of the LOUCY cells with HSF there was an increased expression of IL-6, this was confirmed using ELISA. Using an IL-6 receptor antibody we confirmed that blocking IL-6 receptor reversed the change in BCL-2 dependence in the presence of the splenic microenvironment. Lastly, we confirmed in a T-ALL patient-derived xenograft, that is BCL-2 dependent, that the splenic microenvironment alters the mitochondrial apoptotic threshold. Currently, there are reports in the literature of ETP-ALL patients being treated with ABT-199. While there have been numerous studies lately describing cell autonomous events leading to ABT-199 resistance, our novel finding that the splenic microenvironment is a site of relapse is potentially of great clinical importance for BCL-2 dependent leukemia's. Disclosures Ni Chonghaile: AbbVie: Research Funding.

The Genetics and Mechanisms of T-Cell Acute Lymphoblastic Leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy derived from early T-cell progenitors. The recognition of clinical, genetic, transcriptional, and biological heterogeneity in this disease has already translated into new prognostic biomarkers, improved leukemia animal models, and emerging targeted therapies. This work reviews our current understanding of the molecular mechanisms of T-ALL.

Pediatric T-ALLs Developing into a Type 2 Relapse Originate from Cells That Carry the Potential of Variable Maturation into Subclones with Distinct Chromatin Landscapes

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy that is classified according to surface marker expression. In order to reveal the cells of origin in pediatric T-ALL and to understand mechanisms of relapse we used ATAC-Seq (Assay for Transposase-Accessible Chromatin Sequencing) to compare chromatin accessibility landscapes of healthy T-cell precursors to those of T-ALL cells obtained at initial diagnosis (INI) and relapse (REL).We have FACS sorted 7 differentiation stages of normal T-cell precursors contained in the thymus of infants undergoing cardiac surgery (DN2, DN3, ISP, DPCD3-, DPCD3+, CD4+ and CD8+) and subjected these to ATAC-Seq. Unsupervised learning by principal component analysis (PCA) clustered sorted populations according to the maturation stage, demonstrating that regulatory chromatin signatures of thymocytes are highly stage-specific and re-shaped during T-cell differentiation.We next compared normal T-cell precursors at different stages of maturation to pediatric T-ALLs and found fundamental differences with 30% of open chromatin regions to be more and 28% being less accessible in T-ALL (DESeq2, padj<.05), respectively. We next identified transcription factor (TF) binding sites in open chromatin regions of normal T-cell precursors and T-ALL cells by HOMER analysis. We found 76% of the accessible TF binding sites in double negative (DN2 and DN3) normal precursors to be also accessible in most (20/24) leukemias comparing to 17% of the sites accessible in the more mature (CD4+ and CD8+) stages. These data indicate that T-ALLs originate from cells with an epigenetic profile of early thymic progenitors.We then subjected the ATAC-seq data of all matched leukemia samples obtained at initial disease and at relapse to PCA. INI and REL samples derived from the same patient always clustered in close proximity and were separated according to the T-ALL driving fusion genes. A global analysis of differential accessibility revealed only 0.26% of ATAC-regions to be less- or more-accessible at relapse when compared to the matched initial samples (DESeq2, padj<.05). These data indicate chromatin accessibility to be largely determined by the cell of origin and to generally remain stable during progression from initial diagnosis to relapse. We then considered the 2 types of relapse separately, which we have previously characterized on the basis of subclonal mutation profiles (Kunz et al. Haematologica, 2015). These relapse types are defined by either clonal evolution of cells derived from the predominant clone at primary disease (type 1) or emergence and evolution of a minor initial clone showing a molecular profile that differs from the major initial clone (type 2). We found that the proportion of differentially accessible ATAC-regions between INI and REL is significantly higher in type 2 (1.3%) than in type 1 (0.006%) (DESeq2, padj<.05).Moreover, we trained the deconvolution algorithm CIBERSORT to recognize particular T-cell differentiation stages using ATAC-profiles of the 7 FACS-sorted healthy T-cell populations. We used regulatory chromatin landscape of non-sorted (total) thymus to assess the accuracy of deconvolution. Comparison of predicted fractions in total thymus to FACS measurements revealed highly accurate identification of the maturation stages (r2 = 0.95). CIBERSORT analysis confirmed that the profiles were largely preserved between INI and REL of each sample pair. Notably, however, while in T-ALLs that later developed into a type 1 relapse only one type of early T-cell progenitor dominated the deconvolution profile, T-ALLs that developed into a type 2 relapse showed heterogeneous profiles with contributions of progenitors at different maturation stages.In sum, these epigenomic analyses revealed that the chromatin landscape of normal T-cell precursors evolves in the course of thymic maturation and that the early maturation stages are the likely origin of T-ALL cells. Remarkably, pediatric T-ALLs that later develop a type 2 relapse consist of subclones with a variable profile of chromatin accessibility that define different stages of maturation. These data indicate that T-ALLs with the propensity to develop a type 2 relapse differ from type 1 in that they originate from early precursors that carry the potential of further development into different stages of maturation before the leukemia becomes apparent with a highly subclonal pattern. DisclosuresMuckenthaler:Novartis: Research Funding. Bourquin:Amgen: Other: Travel Support. Kulozik:bluebird bio: Consultancy, Honoraria.

PO-072 ω3-Polyunsaturated fatty acids inhibit cell growth and invasion of human hypopharyngeal carcinoma cells

IntroductionT-cell acute lymphoblastic leukaemia (T-ALL) is an aggressive hematologic malignancy resulting from the transformation of T-cell progenitors. Early T-cell progenitor ALL (ETP-ALL) subgroup accounts for 5% to 10% of T-ALL cases and associated with a poor prognosis and a very high risk for relapse. T-ALL leukemias develop a dependence on anti-apoptotic BCL-2 family proteins that is linked to the maturation stage of the blasts. Typical T-ALL is mostly dependent on BCL-XL, whereas ETP-ALL is more dependent on BCL-2. In recent years, there has been the development of a series of specific BH3 mimetics (ABT-199, ABT-263, WEHI 539) that can inhibit the function of anti-apoptotic BCL-2 proteins and induce apoptosis. T-ALL patients have blasts disseminated in the blood, bone marrow and spleen. Each niche may provide distinct extrinsic signals that could offer a survival advantage to leukemic cells.Material and methodsWe aim to assess in both ETP-ALL (Loucy cell line) and typical T-ALL (CEM-CCRF cell line) if distinct microenvironments affect the BCL-2 family proteins expression and the sensitivity to BH3 mimetics. To study the anti-apoptotic BCL-2 dependencies we will use the cutting-edge technology mitochondrial BH3 profiling. In brief, BH3 profiling is a functional assay that measures the response of mitochondria to exposure of known concentrations of synthetic BH3 peptides by measuring loss of mitochondrial membrane potential or cytochrome c release.Results and discussionsWe found that ETP-ALL cell line was BCL-2 dependent and sensitive to ABT-199 (BCL-2 specific BH3 mimetic). While the Typical T-ALL cell line was BCL-XL dependent and sensitive to both ABT-263 (BCL-2,BCL-XL and BCL-w) and WEHI 539 (BCL-XL) BH3 mimetics. Interestingly, when the ETP–ALL cell line Loucy was co-cultured with the human splenic fibroblast (HSF) cell line we found a reduction in BCL-2 dependency, as assessed by BH3 profiling, and a reduced sensitivity to ABT-199 (BCL-2 specific BH3 mimetic). Remarkably, we found the inverse in the typical T-ALL cell line co-cultured with the HSF. We found an increase in BCL-2 dependence and an increase in sensitivity to ABT-199.ConclusionCurrently, we are trying to identify the signalling pathways which are activated to cause this switch in BCL-2 family dependence in the different subtypes of T-ALL. In addition, we are assessing the BCL-2 family dependence of a xenograft cell line isolated from the blood and spleen of animals to determine if this switiching of BCL-2 dependence occurs in vivo.

PO-068 Determining how distinct microenvironments alter BCL-2 family dependence in T-cell acute lymphoblastic leukaemia

IntroductionT-cell acute lymphoblastic leukaemia (T-ALL) is an aggressive hematologic malignancy resulting from the transformation of T-cell progenitors. Early T-cell progenitor ALL (ETP-ALL) subgroup accounts for 5% to 10% of T-ALL cases and associated with a poor prognosis and a very high risk for relapse. T-ALL leukemias develop a dependence on anti-apoptotic BCL-2 family proteins that is linked to the maturation stage of the blasts. Typical T-ALL is mostly dependent on BCL-XL, whereas ETP-ALL is more dependent on BCL-2. In recent years, there has been the development of a series of specific BH3 mimetics (ABT-199, ABT-263, WEHI 539) that can inhibit the function of anti-apoptotic BCL-2 proteins and induce apoptosis. T-ALL patients have blasts disseminated in the blood, bone marrow and spleen. Each niche may provide distinct extrinsic signals that could offer a survival advantage to leukemic cells.Material and methodsWe aim to assess in both ETP-ALL (Loucy cell line) and typical T-ALL (CEM-CCRF cell line) if distinct microenvironments affect the BCL-2 family proteins expression and the sensitivity to BH3 mimetics. To study the anti-apoptotic BCL-2 dependencies we will use the cutting-edge technology mitochondrial BH3 profiling. In brief, BH3 profiling is a functional assay that measures the response of mitochondria to exposure of known concentrations of synthetic BH3 peptides by measuring loss of mitochondrial membrane potential or cytochrome c release.Results and discussionsWe found that ETP-ALL cell line was BCL-2 dependent and sensitive to ABT-199 (BCL-2 specific BH3 mimetic). While the Typical T-ALL cell line was BCL-XL dependent and sensitive to both ABT-263 (BCL-2,BCL-XL and BCL-w) and WEHI 539 (BCL-XL) BH3 mimetics. Interestingly, when the ETP–ALL cell line Loucy was co-cultured with the human splenic fibroblast (HSF) cell line we found a reduction in BCL-2 dependency, as assessed by BH3 profiling, and a reduced sensitivity to ABT-199 (BCL-2 specific BH3 mimetic). Remarkably, we found the inverse in the typical T-ALL cell line co-cultured with the HSF. We found an increase in BCL-2 dependence and an increase in sensitivity to ABT-199.ConclusionCurrently, we are trying to identify the signalling pathways which are activated to cause this switch in BCL-2 family dependence in the different subtypes of T-ALL. In addition, we are assessing the BCL-2 family dependence of a xenograft cell line isolated from the blood and spleen of animals to determine if this switiching of BCL-2 dependence occurs in vivo.

T-cell egress from the thymus: Should I stay or should I go?

T‐cells bearing the αβTCR play a vital role in defending the host against foreign pathogens and malignant transformation of self. Importantly, T‐cells are required to remain tolerant to the host's own cells and tissues in order to prevent self‐reactive responses that can lead to autoimmune disease. T‐cells achieve the capacity for self/nonself discrimination by undergoing a highly selective and rigorous developmental program during their maturation in the thymus. This organ is unique in its ability to support a program of T‐cell development that ensures the establishment of a functionally diverse αβTCR repertoire within the peripheral T‐cell pool. The thymus achieves this by virtue of specialized stromal microenvironments that contain heterogeneous cell types, whose organization and function underpins their ability to educate, support, and screen different thymocyte subsets through various stages of development. These stages range from the entry of early T‐cell progenitors into the thymus, through to the positive and negative selection of the αβTCR repertoire. The importance of the thymus medulla as a site for T‐cell tolerance and the exit of newly generated T‐cells into the periphery is well established. In this review, we summarize current knowledge on the developmental pathways that take place during αβT‐cell development in the thymus. In addition, we focus on the mechanisms that regulate thymic egress and contribute to the seeding of peripheral tissues with newly selected self‐tolerant αβT‐cells.

Lack of Ikaros cripples expression of Foxo1 and its targets in naive T cells.

Ikaros is a transcription factor that regulates lymphocyte development from the level of the haematopoietic stem cell. Lack of Ikaros reduces the ability of progenitor cells to commit to the T-cell lineage, resulting in reduced numbers of early thymic T-cell progenitors and mature T cells. Mature CD4 T cells that lack Ikaros have defects in proliferation, T helper cell differentiation, cytokine expression and the ability to become anergic. A role for Ikaros in the naive T cell has not yet been identified. The receptors interleukin-7 receptor α (IL-7Rα) and l-selectin are important for ensuring survival and proper homing of naive T cells, respectively. Here we show that lack of Ikaros leads to reduced expression of these receptors in naive T cells, which impacts their ability to home and survive in response to IL-7. We define the mechanism underlying this phenotype as a requirement for Ikaros in maintenance of expression of Foxo1, a transcriptional regulator that is required for their expression. We also demonstrate that CD4 T cells lacking Ikaros are significantly crippled in their ability to become induced regulatory T cells, a phenotype also linked to reduced Foxo1 expression. Finally, we show that restoring Ikaros function to Ikaros-deficient CD4 T cells increases levels of Foxo1 message. Together, these studies define, for the first time, a role for Ikaros in naive T cells and establish it as the first transcriptional regulator required for maintaining levels of Foxo1 gene expression in these cells.

The E-Id Protein Axis Specifies Adaptive Lymphoid Cell Identity and Suppresses Thymic Innate Lymphoid Cell Development

Innate and adaptive lymphoid development is orchestrated by the activities of E proteins and their antagonist Id proteins, but how these factors regulate early Tcell progenitor (ETP) and innate lymphoid cell (ILC) development remains unclear. Using multiple genetic strategies, we demonstrated that E proteins E2A and HEB acted in synergy in the thymus to establish Tcell identity and to suppress the aberrant development of ILCs, including ILC2s and lymphoid-tissue-inducer-like cells. E2A and HEB orchestrated Tcell fate and suppressed the ILC transcription signature by activating the expression of genes associated with Notch receptors, Tcell receptor (TCR) assembly, and TCR-mediated signaling. E2A and HEB acted in ETPs to establish and maintain a T-cell-lineage-specific enhancer repertoire, including regulatory elements associated with the Notch1, Rag1, and Rag2 loci. On the basis of these and previous observations, we propose that the E-Id protein axis specifies innate and adaptive lymphoid cell fate.

Activation of the LMO2 Oncogene in T-ALL through a Somatically Acquired Neomorphic Promoter

LMO2is a crucial regulator of normal hematopoiesis but is progressively silenced from the early T-cell progenitor stage of thymic development. Aberrant LMO2 expression leads to a block in differentiation, increased self-renewal and aggressive T-cell acute lymphoblastic leukemia (T-ALL). Although ≈50% of T-ALL patients overexpress LMO2, this can be attributed to a cytogenetic lesion in only ≈10% of patients, leaving a significant portion mechanistically unaccounted for. We recently discovered somatic mutations that create an oncogenic super-enhancer driving TAL1 expression (Mansour et al., 2014, Science). Therefore, we investigated whether comparable mutations may be causing dysregulated LMO2 expression.Aberrant H3K27ac marks indicative of active chromatin were identified prior to and encompassing the non-coding exon 2 of the LMO2 gene by ChIP-seq in DU.528 and PF-382 T-ALL cell lines, both of which exhibit upregulated LMO2 expression but lack chromosomal lesions at this locus. Sequencing across this peak revealed a heterozygous 20bp duplication in PF-382 cells and a heterozygous 1bp deletion in DU.528 cells, located close to a region recently described as an intermediate promoter. Both indels generated a de novo MYB consensus motif. MYB ChIP-seq showed that ≥96% of reads aligned to the mutant rather than the wild-type allele, suggesting that MYB was preferentially recruited to the mutant allele. Heterozygous mutations were also detected in diagnostic samples from 3% (5/159) of pediatric and 6% (10/164) of adult T-ALL patients. Absence of the mutations in 7 available patient-matched remission samples confirmed that they were somatic. The mutations were densely distributed around highly conserved native ETS1, MYB and GATA motifs; 5 patients had an additional MYB site, 3 both a MYB and an ETS1 site, 1 an ETS1 site alone, and 4 had new RUNX1 binding sites. This suggests that this region may have a regulatory role that is exploited by the mutations to constitutively activate LMO2.All 6 mutant-positive patients with available RNA showed LMO2 overexpression as determined by qRT-PCR, consistent with the hypothesis that these mutations activate gene expression. Furthermore, monoallelic LMO2 expression could be demonstrated in DU.528 cells and 3 of 4 informative T-ALL samples using a heterozygous germline SNP. Expression of the mutations in luciferase reporter assays also indicated that they all markedly activated luciferase activity to between 2x and 57x compared to the wild-type sequence.To assess causality between the mutations and LMO2 dysregulation, we used CRISPR/Cas9 genome-editing with a guide RNA designed to target the mutant MYB site in PF-382 cells. Reduction of LMO2 expression to ≤10% of PF-382 mutant activity was observed in a clone where the mutant allele had been reverted to wild-type and in another clone with a single T>C substitution disrupting the mutant MYB binding site. Interestingly, 2 clones that increased the distance between the native and the mutant MYB sites also resulted in a reduction in LMO2 expression to 19% and 25% of PF-382 activity, suggesting that there are functionally limiting spatial constraints on the mutant MYB site in relation to other neighboring transcription factor binding sites. This was further validated by the lack of reduction in LMO2 expression in a clone where the sequence between the two MYB sites was altered but the spacing distance was unchanged.In conclusion, we have identified and functionally validated a novel recurrent mutation hotspot occurring in a non-coding site whereby introduction of additional binding sites for a number of different transcription factors drives monoallelic LMO2 overexpression from a neomorphic promoter in a substantial proportion of both adult and pediatric T-ALL patients. This mechanism of oncogene activation may be relevant to a wide variety of human cancers. DisclosuresFielding:Baxalta: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees.

Constitutive Expression of MEF2C, LYL1, or LMO2 in CD34+ HSCs Blocks the in Vitro Differentiation before the T-Cell Commitment Point and Could be Oncogenic in Early T-Cell Progenitor ALL

Background:T-cell development in the thymus is a complex process that depends on sequential transcriptional and epigenetic events that induce T-cell lineage commitment and simultaneously suppress alternative cell fates. In T-cell acute lymphoblastic leukemia (T-ALL), aberrantly expressed oncogenes result in the arrest of developing thymocytes, which can lead to the acquisition of secondary mutations, uncontrolled proliferation and disease progression. MEF2C is often expressed as a result of chromosomal rearrangements in immature, early T-cell progenitor ALL (ETP-ALL), but is also expressed in normal thymocyte progenitors before T-cell commitment (in the ETP stage). As the only hematopoietic lineage, thymocytes that have passed the T-cell commitment checkpoint (as well as mature T-cells) do no longer express MEF2C.Aims:We aimed to investigate the effect of constitutive MEF2C expression on early T-cell development. OP9-DL1 co-cultures have been most useful for mimicking in vitro T-cell development starting with hematopoietic stem cells (HSCs) derived from human cord blood or bone marrow. We also aimed to investigate the impact of MEF2C in comparison to LYL1 and LMO2; two T-ALL oncogenes also highly expressed at the ETP stage.Methods:We have utilized the OP9-DL1 in vitro co-culture system to gradually differentiate CD34+ HSCs from umbilical cord blood into the T-cell lineage. HSCs in this co-culture will recapitulate in vivo T-cell development as measured by incremental acquisition of surface markers CD7, CD5, CD1a, and reach the CD4, CD8 double-positive (DP) stage. We generated gene expression profiles of 11 subsequent in vitro stages of differentiation to help us match them to in vivo development stages. We investigated in vitro T-cell differentiation of HSCs after lentiviral transduction with MEF2C or control vectors, as well as with other transcriptional regulators LYL1 and LMO2 that are expressed at the ETP stage.Results:The major change in gene expression of subsequent early T-cell differentiation stages defines two distinct T-cell differentiation clusters that correlate with in vivo pre- and post-T-cell commitment profiles. We found that T-cell commitment occurs in CD7+ CD5+ cells before the acquisition of CD1a surface expression. Expression of control vectors in HSCs does not affect the in vitro T-cell differentiation, but MEF2C expression blocks differentiation into the direction of T-cells as measured by the failure of most cells to acquire CD7 as the first marker. Instead, with increased passage number cells gradually lose CD34 expression and eventually disappear from the co-culture. Similar effects were observed for the expression of LYL1 and LMO2; LYL1 expression arrests the cells at the most immature CD7+ ETP stage and prevents the transition towards CD7+ CD5+ cells, whereas LMO2 expressing cells reach the CD7+ CD5+ stage but fail to acquire CD1a as a marker of T-cell commitment.Summary/Conclusion:The gene expression profiles of 11 human in vitro T-cell differentiation subsets has enabled us to pinpoint T-cell commitment to a stage in which cells have acquired CD7 and CD5, just prior to the acquisition of CD1a. MEF2C, LYL1, and LMO2, expressed in ETP-ALL as well as in normal thymocyte progenitors, do not allow the transition to T-cell commitment when constitutively expressed. These proteins each result in the arrest of in vitro differentiating T-cells at different ETP stages, all before the T-cell commitment as marked by CD1a expression. Constitutive expression of MEF2C, LYL1, or LMO2 in very early thymocyte progenitors is incompatible with development into and beyond the T-cell commitment checkpoint and these proteins could therefore play important roles in the pathogenesis of ETP-ALL. DisclosuresNo relevant conflicts of interest to declare.

Complete hematologic response of early T-cell progenitor acute lymphoblastic leukemia to the γ-secretase inhibitor BMS-906024: genetic and epigenetic findings in an outlier case.

Notch pathway antagonists such as γ-secretase inhibitors (GSIs) are being tested in diverse cancers, but exceptional responses have yet to be reported. We describe the case of a patient with relapsed/refractory early T-cell progenitor acute lymphoblastic leukemia (ETP-ALL) who achieved a complete hematologic response following treatment with the GSI BMS-906024. Whole-exome sequencing of leukemic blasts revealed heterozygous gain-of-function driver mutations in NOTCH1, CSF3R, and PTPN11, and a homozygous/hemizygous loss-of-function mutation in DNMT3A. The three gain-of-function mutations were absent from remission marrow cells, but the DNMT3A mutation persisted in heterozygous form in remission marrow, consistent with an origin for the patient's ETP-ALL from clonal hematopoiesis. Ex vivo culture of ETP-ALL blasts confirmed high levels of activated NOTCH1 that were repressed by GSI treatment, and RNA-seq documented that GSIs downregulated multiple known Notch target genes. Surprisingly, one potential target gene that was unaffected by GSIs was MYC, a key Notch target in GSI-sensitive T-ALL of cortical T-cell type. H3K27ac super-enhancer landscapes near MYC showed a pattern previously reported in acute myeloid leukemia (AML) that is sensitive to BRD4 inhibitors, and in line with this ETP-ALL blasts downregulated MYC in response to the BRD4 inhibitor JQ1. To our knowledge, this is the first example of complete response of a Notch-mutated ETP-ALL to a Notch antagonist and is also the first description of chromatin landscapes associated with ETP-ALL. Our experience suggests that additional attempts to target Notch in Notch-mutated ETP-ALL are merited.

A monoallelic-to-biallelic T-cell transcriptional switch regulates GATA3 abundance.

Protein abundance must be precisely regulated throughout life, and nowhere is the stringency of this requirement more evident than during T-cell development: A twofold increase in the abundance of transcription factor GATA3 results in thymic lymphoma, while reduced GATA3 leads to diminished T-cell production. GATA3 haploinsufficiency also causes human HDR (hypoparathyroidism, deafness, and renal dysplasia) syndrome, often accompanied by immunodeficiency. Here we show that loss of one Gata3 allele leads to diminished expansion (and compromised development) of immature T cells as well as aberrant induction of myeloid transcription factor PU.1. This effect is at least in part mediated transcriptionally: We discovered that Gata3 is monoallelically expressed in a parent of origin-independent manner in hematopoietic stem cells and early T-cell progenitors. Curiously, half of the developing cells switch to biallelic Gata3 transcription abruptly at midthymopoiesis. We show that the monoallelic-to-biallelic transcriptional switch is stably maintained and therefore is not a stochastic phenomenon. This unique mechanism, if adopted by other regulatory genes, may provide new biological insights into the rather prevalent phenomenon of monoallelic expression of autosomal genes as well as into the variably penetrant pathophysiological spectrum of phenotypes observed in many human syndromes that are due to haploinsufficiency of the affected gene.

The expansion of thymopoiesis in neonatal mice is dependent on expression of high mobility group a 2 protein (Hmga2).

Cell number in the mouse thymus increases steadily during the first two weeks after birth. It then plateaus and begins to decline by seven weeks after birth. The factors governing these dramatic changes in cell production are not well understood. The data herein correlate levels of High mobility group A 2 protein (Hmga2) expression with these temporal changes in thymopoiesis. Hmga2 is expressed at high levels in murine fetal and neonatal early T cell progenitors (ETP), which are the most immature intrathymic precursors, and becomes almost undetectable in these progenitors after 5 weeks of age. Hmga2 expression is critical for the initial, exponential expansion of thymopoiesis, as Hmga2 deficient mice have a deficit of ETPs within days after birth, and total thymocyte number is repressed compared to wild type littermates. Finally, our data raise the possibility that similar events occur in humans, because Hmga2 expression is high in human fetal thymic progenitors and falls in these cells during early infancy.

Epigenetics in T-cell acute lymphoblastic leukemia.

Normal T-cell development is a strictly regulated process in which hematopoietic progenitor cells migrate from the bone marrow to the thymus and differentiate from early T-cell progenitors toward mature and functional T cells. During this maturation process, cooperation between a variety of oncogenes and tumor suppressors can drive immature thymocytes into uncontrolled clonal expansion and cause T-cell acute lymphoblastic leukemia (T-ALL). Despite improved insights in T-ALL disease biology and comprehensive characterization of its genetic landscape, clinical care remained largely similar over the past decades and still consists of high-dose multi-agent chemotherapy potentially followed by hematopoietic stem cell transplantation. Even with such aggressive treatment regimens, which are often associated with considerable side effects, clinical outcome is still extremely poor in a significant subset of T-ALL patients as a result of therapy resistance or hematological relapses. Recent genetic studies have identified recurrent somatic alterations in genes involved in DNA methylation and post-translational histone modifications in T-ALL, suggesting that epigenetic homeostasis is critically required in restraining tumor development in the T-cell lineage. In this review, we provide an overview of the epigenetic regulators that could be implicated in T-ALL disease biology and speculate how the epigenetic landscape of T-ALL could trigger the development of epigenetic-based therapies to further improve the treatment of human T-ALL.

PRL2 Phosphatase Regulate Early Stage T-Cell Development through Fine-Tuning SCF/Kit Signaling

The phosphatase of regenerating liver (PRL) family of phosphatases, consisting of PRL1, PRL2, and PRL3, represents an intriguing group of proteins being validated as biomarkers and therapeutic targets in human cancer. We have been investigating the role of PRL2 in normal / malignant hematopoiesis and found that PRL2 is important for HSC self-renewal (Kobayashi et al., Stem Cells, 2014). The receptor tyrosine kinase KIT can balance quiescence for HSC maintenance and proliferation for progeny supply. The defects seen in the PRL2-deficient hematopoietic and testis cells recapitulate the phenotype of c-Kit mutant mice, suggesting that the SCF/KIT signaling may be impaired in the absence of PRL2 (Kobayashi et al., Stem Cells, 2014; Dong et al., JBC, 2013). Given that KIT also plays critical role in maintaining postnatal T-lymphopoiesis in thymus, we hypothesized that PRL2 is important for T cell development.Here we report that loss of PRL2 impairs T-lymphopoiesis both in vitro and in vivo. PRL2 deficiency resulted in marked reduction of splenocyte and thymocyte counts compared to wild type (WT) mice. While we observed modest increase in the frequency of early T cell progenitor (ETP), DN2, and DN3 cells in PRL2 deficient thymus, T-cell reconstitution was dramatically decreased after HSC transplantation. T-cell number in the peripheral blood (PB) of recipient mice repopulated with PRL2-null HSCs was 30 times less than that of the WT HSCs (WT: 2288.6±579.8/µl vs PRL2 null: 69.5±22.1/µl, p<0.00001). Although the frequency of donor-derived thymocytes in recipient thymus was 91±6.1% in WT, PRL2 null HSCs contributed only 7.1±4.9% (p<0.00001) in the recipient thymus. By detailed fractionation, surprisingly, chimerism in ETP was comparable between WT and PRL2 null cells (WT: 91.8±10.1% vs PRL2 null: 59.6±13.5%, p<0.01). Importantly, the chimerism of PRL2-null thymocytes fell down to 10% in gated DN2, whereas WT HSCs consistently contributed around 90%, suggesting that the DN1-to-DN2 transition requires PRL2. Next, we evaluated the in vitro T-cell generation by utilizing the Delta-Like1 (DLL1) expressing OP9 (DL-OP9) stromal cells. While wild type KSLs produced massive amount of T-cells (fold increase: 33,000±3371) 22 days following plating onto the DL-OP9, PRL2 null KSLs only generated limited amount of T-cells (fold increase: 1765±665, p<0.0001), demonstrating that PRL2 is important for T-cell proliferation. We also monitored the generation of ETPs from KSLs in DL-OP9 cultures and observed significant expansion of ETPs derived from WT KSLs compared to that of the PRL2 null KSLs (fold increase: 183.8±14.4 vs 12.5±4.3, p<0.001). However, when sorted DN3 cells from WT and PRL2 KO thymus were plated onto DL-OP9, we saw similar increase in cell expansion, suggesting PRL2 regulate early T-cell development.WhilePRL2 is a dual specificity protein phosphatase, its substrates are unknown. To identifyPRL2 substrates in hematopoietic cells, we performed a protein phosphatase substrate trap assay. We utilized a GST-tagged PRL2/CS-DA mutant, in which the catalyticsite cysteine was mutated to serine, so that PRL2 binds to its substrates better, but is unable todephosphorylate them. We found that the mutant PRL2/CS-DA showed enhanced association with KIT than WT PRL2 in Kasumi-1 cells, suggesting that KIT is a potential PRL2 substrate. The PRL2 and KIT interaction was further confirmed by the Immunoprecipitation (IP) assay in 293T cells expressing KIT. We also detected the association of PRL2 with SHP2, CBL and PLC-g in Kasumi-1 cells, which are important regulators of KIT activation and stability. Moreover, PRL2 KO hematopoietic progenitor cells show decreased KIT phosphorylation at tyrosine 703 following SCF stimulation, suggesting that PRL2 may modulate KIT activation in these cells. To evaluate the impact of SCF signal strength on T-cell proliferation, we cultured sorted lympho-primed multipotent progenitor cells (LMPPs) from WT and KO mice onto DLL-Fc coated plates with graded doses of SCF (0.2, 1, 5, 25 ng/ml). The total number of cells generated from SCF treated WT LMPPs was significantly higher than that of the KO LMPPs in a dosage dependent manner. KO exhibited 6 times less sensitive to SCF than WT, indicating that PRL2 fine-tunes SCF signal intensity in early T-cell.Taken together, we have identified a critical role for PRL2 in T-cell proliferation and maintenance through fine-tuning SCF/KIT signaling. DisclosuresNo relevant conflicts of interest to declare.

Alternative Super-Enhancer States Determine MYC Sensitivity to Notch and Brd4 Inhibitors in T Lymphoblastic Leukemia/Lymphoma

Gain-of-function NOTCH1 mutations are oncogenic drivers in a high fraction of T-cell lymphoblastic leukemia/lymphoma (T-LL). These mutations variously cause increased production or stabilization of the free intracellular domain of NOTCH1, which regulates gene expression by forming a transcription complex with the DNA-binding factor RBPJ and coactivators of the MAML family. Using expression profiling and ChIP-seq, we have shown that NOTCH1/RBPJ complexes activate most target genes by binding to super-enhancers, large regulatory elements that switch on transcription through long-range interactions with gene promoters. MYC is a critical target of Notch in normal and malignant pre-T cells, but how Notch regulates MYC is unknown. To understand which regulatory element(s) regulate MYC expression, we used chromatin conformation capture (3C) assays to test the interaction between putative enhancer(s) and the MYC promoter in T-LL cell lines, and reporter gene assays to confirm enhancer function of candidate sites. We identified a distal site located >1 Mb 3’ of human and murine MYC termed the Notch-dependent MYC enhancer (NDME) that binds Notch transcription complexes and physically interacts with the MYC proximal promoter. An ~1 kb DNA fragment containing this site activates a luciferase reporter gene in a Notch-dependent fashion in T-LL cells but not in heterologous cell types. The Notch binding site lies within a large enhancer region (>600 kb in breadth) containing multiple discrete H3K27ac peaks. Remarkably, acute changes in Notch activation produce rapid changes in H3K27 acetylation across the entire enhancer region and the MYC promoter that correlate with NOTCH1/RBPJ complex binding and MYC expression. T-LL cells selected for resistance to gamma-secretase inhibitors (GSIs) exhibit epigenetic silencing of the NDME and loss of NDME looping interactions with the MYC promoter, yet maintain MYC expression. 3C analysis of GSI resistant cells shows preferential interaction between the MYC promoter and a more 3’ enhancer element recently described as a BRD4-dependent regulator of MYC expression in acute myeloid leukemia cells. In line with this observation, BRD4 antagonists are potent inhibitors of MYC expression in GSI resistant T-LL cells but not GSI-sensitive cells. We also studied a case of Notch-mutated early T-cell progenitor acute lymphoblastic leukemia (ETP-ALL). ChIP-Seq analysis of the leukemic blasts revealed an “AML-like” MYC enhancer chromatin state, and as predicted from our analysis of cell lines, the blasts rapidly down-regulated MYC in response to BRD4 inhibitor but not in response to GSI. These findings suggest that specific MYC chromatin states predict responsiveness to Notch and BRD4 inhibitors, and provide a rationale for use of Notch and BRD4 inhibitor combinations in Notch-mutated leukemias. DisclosuresNo relevant conflicts of interest to declare.

Maturation stage of T-cell acute lymphoblastic leukemia determines BCL-2 versus BCL-XL dependence and sensitivity to ABT-199.

Acute lymphoblastic leukemia (ALL) is a hematopoietic malignancy derived from immature B-lymphoid and T-lymphoid cells (T-ALL). In T-ALL, there is an early T-cell progenitor (ETP) subgroup that has a very high risk for relapse. In this study, we used mitochondrial BH3 profiling to determine antiapoptotic protein dependencies in T-ALL. We found that T-ALL cell lines and primary patient samples are dependent upon BCL-XL, except when the cancer bears an ETP phenotype, in which case it is BCL-2 dependent. These distinctions directly relate to differential sensitivity to the BH3 mimetics ABT-263 and ABT-199, both in vitro and in vivo. We thus describe for the first time a change of antiapoptotic protein dependence that is related to the differentiation stage of the leukemic clone. Our findings demonstrate that BCL-2 is a clinically relevant target for therapeutic intervention with ABT-199 in ETP-ALL. ETP T-ALL is a treatment-resistant subtype of T-ALL for which novel targeted therapies are urgently needed. We have discovered, through BH3 profiling, that ETP-ALL is BCL-2 dependent and is very sensitive to in vitro and in vivo treatment with ABT-199, a drug well tolerated in clinical trials.

NUP98-PHF23 and NUP98-HOXD13 oncogenes confer aberrant self-renewal potential to thymocyte progenitors

BackgroundIn T-cell acute lymphoblastic leukemia (T-ALL), the homeobox (HOX) A cluster is activated directly by chromosomal translocations or indirectly by two fusion genes acting as upstream regulators, specifically NUP98-plant homeodomain finger 23 (PHF23) and NUP98-HOXD13. Interestingly, HOXA genes control the self-renewal activity of normal hematopoietic stem cells, suggesting a similar function in cancer stem cells (CSCs). This hypothesis is reinforced by the recent observations that two other oncogenic transcription factors in T-ALL, SCL and LMO1/2, confers self-renewal potential to non-self-renewing thymocyte progenitors.Methods and ResultsHere we used two transgenic mouse models ectopically expressing NUP98-PHF23 and NUP98-HOXD13 in hematopoietic stem/progenitor cells. Our results indicate that pre-leukemic thymocytes exhibit aberrant self-renewal potential as assessed by their engraftment in the thymus after intravenous transplantation. Furthermore, this activity is sustained over serial transplantations with 100-fold expansion of the most primitive thymocyte populations, i.e. DN1 and DN2. At the molecular level, engrafted thymocytes display a non-rearranged bTCR. Finally, purified early T-cell progenitors (ETP) and DN2 are the only cell types that can reconstitute the thymus of transplanted mice, i.e. in cells in which bTcr genes are in a germ line configuration.Conclusion and RelevanceWe therefore conclude that NUP98-PHF23 and NUP98-HOXD13 oncogenes reprogram ETP and DN2 thymocytes into self-renewing progenitors. Our results open possibilities of targeted therapy towards CSCs. BackgroundIn T-cell acute lymphoblastic leukemia (T-ALL), the homeobox (HOX) A cluster is activated directly by chromosomal translocations or indirectly by two fusion genes acting as upstream regulators, specifically NUP98-plant homeodomain finger 23 (PHF23) and NUP98-HOXD13. Interestingly, HOXA genes control the self-renewal activity of normal hematopoietic stem cells, suggesting a similar function in cancer stem cells (CSCs). This hypothesis is reinforced by the recent observations that two other oncogenic transcription factors in T-ALL, SCL and LMO1/2, confers self-renewal potential to non-self-renewing thymocyte progenitors. In T-cell acute lymphoblastic leukemia (T-ALL), the homeobox (HOX) A cluster is activated directly by chromosomal translocations or indirectly by two fusion genes acting as upstream regulators, specifically NUP98-plant homeodomain finger 23 (PHF23) and NUP98-HOXD13. Interestingly, HOXA genes control the self-renewal activity of normal hematopoietic stem cells, suggesting a similar function in cancer stem cells (CSCs). This hypothesis is reinforced by the recent observations that two other oncogenic transcription factors in T-ALL, SCL and LMO1/2, confers self-renewal potential to non-self-renewing thymocyte progenitors. Methods and ResultsHere we used two transgenic mouse models ectopically expressing NUP98-PHF23 and NUP98-HOXD13 in hematopoietic stem/progenitor cells. Our results indicate that pre-leukemic thymocytes exhibit aberrant self-renewal potential as assessed by their engraftment in the thymus after intravenous transplantation. Furthermore, this activity is sustained over serial transplantations with 100-fold expansion of the most primitive thymocyte populations, i.e. DN1 and DN2. At the molecular level, engrafted thymocytes display a non-rearranged bTCR. Finally, purified early T-cell progenitors (ETP) and DN2 are the only cell types that can reconstitute the thymus of transplanted mice, i.e. in cells in which bTcr genes are in a germ line configuration. Here we used two transgenic mouse models ectopically expressing NUP98-PHF23 and NUP98-HOXD13 in hematopoietic stem/progenitor cells. Our results indicate that pre-leukemic thymocytes exhibit aberrant self-renewal potential as assessed by their engraftment in the thymus after intravenous transplantation. Furthermore, this activity is sustained over serial transplantations with 100-fold expansion of the most primitive thymocyte populations, i.e. DN1 and DN2. At the molecular level, engrafted thymocytes display a non-rearranged bTCR. Finally, purified early T-cell progenitors (ETP) and DN2 are the only cell types that can reconstitute the thymus of transplanted mice, i.e. in cells in which bTcr genes are in a germ line configuration. Conclusion and RelevanceWe therefore conclude that NUP98-PHF23 and NUP98-HOXD13 oncogenes reprogram ETP and DN2 thymocytes into self-renewing progenitors. Our results open possibilities of targeted therapy towards CSCs. We therefore conclude that NUP98-PHF23 and NUP98-HOXD13 oncogenes reprogram ETP and DN2 thymocytes into self-renewing progenitors. Our results open possibilities of targeted therapy towards CSCs.

Loss of mTOR complex 1 induces developmental blockage in early T-lymphopoiesis and eradicates T-cell acute lymphoblastic leukemia cells

mTOR is an evolutionarily conserved kinase that plays a critical role in sensing and responding to environmental determinants. Recent studies have shown that fine-tuning of the activity of mTOR complexes contributes to organogenesis and tumorigenesis. Although rapamycin, an allosteric mTOR inhibitor, is an effective immunosuppressant, the precise roles of mTOR complexes in early T-cell development remain unclear. Here we show that mTORC1 plays a critical role in the development of both early T-cell progenitors and leukemia. Deletion of Raptor, an essential component of mTORC1, produced defects in the earliest development of T-cell progenitors in vivo and in vitro. Deficiency of Raptor resulted in cell cycle abnormalities in early T-cell progenitors that were associated with instability of the Cyclin D2/D3-CDK6 complexes; deficiency of Rictor, an mTORC2 component, did not have the same effect, indicating that mTORC1 and -2 control T-cell development in different ways. In a model of myeloproliferative neoplasm and T-cell acute lymphoblastic leukemia (T-ALL) evoked by Kras activation, Raptor deficiency dramatically inhibited the cell cycle in oncogenic Kras-expressing T-cell progenitors, but not myeloid progenitors, and specifically prevented the development of T-ALL. Although rapamycin treatment significantly prolonged the survival of recipient mice bearing T-ALL cells, rapamycin-insensitive leukemia cells continued to propagate in vivo. In contrast, Raptor deficiency in the T-ALL model resulted in cell cycle arrest and efficient eradication of leukemia. Thus, understanding the cell-context-dependent role of mTORC1 illustrates the potential importance of mTOR signals as therapeutic targets.

Enhancing T Lineage Production in Aged Mice: A Novel Function of Foxn1 in the Bone Marrow Niche

Foxn1 is essential for thymic organogenesis and T lymphopoiesis. Whereas reduced Foxn1 expression results in a decline in T lymphopoiesis, overexpression of Foxn1 in the thymus of a transgenic mouse model (Foxn1Tg) attenuates the age-associated decline in T lymphopoiesis. T lymphopoiesis begins with early T cell progenitors (ETP), derived from multipotent progenitors (MPP) in the bone marrow (BM). A decline in MPP and ETP numbers with age is thought to contribute to reduced T lymphopoiesis. Previously, we showed that reduced ETP number with age is attenuated in Foxn1 transgenic (Tg); whether the effect is initiated in the BM with MPP is not known. In this study, we report that Foxn1 is expressed in wild-type BM and overexpressed in Foxn1Tg. With age, the number of MPP in Foxn1Tg was not reduced, and Foxn1Tg also have a larger pool of hematopoietic stem cells. Furthermore, the Foxn1Tg BM is more efficient in generating MPP. In contrast to MPP, common lymphoid progenitors and B lineage cell numbers were significantly lower in both young and aged Foxn1Tg compared with wild type. We identified a novel population of lineage(neg/low), CD45(pos) EpCAM(pos), SCA1(pos), CD117(neg), CD138(neg), MHCII(neg) cells as Foxn1-expressing BM cells that also express Delta-like 4. Thus, Foxn1 affects both T lymphopoiesis and hematopoiesis, and the Foxn1 BM niche may function in skewing MPP development toward T lineage progenitors.