TAL1 Expression Research Articles

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

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

Suppression of super-enhancer-driven TAL1 expression by KLF4 in T-cell acute lymphoblastic leukemia.

TAL1 is one of the most frequently dysregulated genes in T-ALL and is overexpressed in about 50% of T-ALL cases. One of the molecular mechanisms of TAL1 overexpression is abnormal mutations in the upstream region of the TAL1 promoter that introduce binding motifs for the MYB transcription factor. MYB binding at this location creates a 5' TAL1 super-enhancer (SE), which leads to aberrant expression of TAL1 and is associated with unfavorable clinical outcomes. Although targeting TAL1 is considered to be an attractive therapeutic strategy for patients with T-ALL, direct inhibition of transcription factors is challenging. Here, we show that KLF4, a known tumor suppressor in leukemic cells, suppresses SE-driven TAL1 expression in T-ALL cells. Mechanistically, KLF4 downregulates MYB expression by directly binding to its promoter and inhibits the formation of 5' TAL1 SE. In addition, we found that APTO-253, a small molecule inducer of KLF4, exerts an anti-leukemic effect by targeting SE-driven TAL1 expression in T-ALL cells. Taken together, our results suggest that the induction of KLF4 is a promising strategy to control TAL1 expression and could be a novel treatment for T-ALL patients with a poor prognosis.

GATA2-TAL1 Axis Is Essential for Human NK Cell Development and Function

Introduction: Natural killer (NK) cells, as the major subset of innate lymphocytes, produce proinflammatory cytokines and mediate anti-tumor cytotoxicity (1-3). Since NK cells are not limited by clonotypic receptors, they can be utilized in cell therapies against a broad spectrum of malignancies. Irrespective of the clinical potentials, the transcriptional regulation of the development and functions of human NK cells is far from fully understood. A dysregulated NK cell development is a hallmark of GATA2 haploinsufficiency (4)(eg. GATA2 T354M mutation). The specific loss of the CD56 bright NK cell population is a significant striking feature in GATA2-deficient patients (4), with or without reduced total CD56 dim NK cells (4, 12, 13). Consequently, the effector functions of these NK cells are significantly reduced, including inflammatory cytokine production and anti-tumor cytotoxicity (4, 12). However, a mechanistic understanding of GATA2 functions is lacking. In humans, GATA2 forms a core heptad complex with six other transcription factors (TF), including TAL1, regulating more than 1000 target genes. However, the unique functions of the GATA2-TAL1 axis in immature and mature NK cell has not been established. Based on these, we hypothesize that GATA2-dependent and independent functions of TAL1 play an essential role in the development and functions of human NK cells. Results: To explore the mechanism of co-transcriptional regulation of GATA2 and TAL1, we transduced NK-92 cells with lentiviral vectors containing GATA2 WT or GATA2 T354M plasmid and quantified TAL1 mRNA levels by qPCR and protein levels by WB. TAL1 is upregulated in GATA2 T354M-expressing NK-92 cells both at mRNA ( Fig. 1A) and protein levels ( Fig. 1B). TAL1 is a TF activating GTPase of immunity-associated protein (GIMAP) family super-enhancer regions in HSC and T cells (11). As TAL1 target genes, we further quantify GIMAP mRNA levels by qPCR. The result indicates that genes from the GIMAP family were notably upregulated in GATA2 T354M-expressing NK-92 cells ( Fig. 1C). Knocking down TAL1 results in the significant downregulation of TAL1 and GIMAPs in GATA2 T354M-expressingcells ( Fig. 1D). To further validate the GATA2-TAL1 axis, sorted NK cells from PBMCs of three GATA2 T354M patients and five age-matched healthy controls (HC) were analyzed with single-cell RNA-seq. We identified the genes from GIMAP family were significantly upregulated in all three GATA2 T354M patients. The representative data from one patient is shown in Fig. 1E. To explore how GATA2 and TAL1 mediate transcriptional regulation, we performed GATA2 CUT&Tag experiments with human NK cells, in which TAL1 motif was predominately enriched in 63.2% of GATA2 targets among NK cells through HOMER motif enrichment analyses ( Fig. 1F). On the other hand, the TAL1 ChIP-seq data from the K562 cells (download from ENCFF101DBG) reveals that the GATA2 motif is enriched in approximately 44% of TAL1 targets ( Fig. 1G). Moreover, both GATA2 and TAL1 expression in NK cells are modified by H3K27Me3 instead of H3K4Me3, suggesting a repressive methylenation signature in their expression ( Fig. 1H). Conclusion: Based on this study, we uncover a novel role for the GATA2-TAL1 axis in human NK cells. we define the transcriptional regulation role of GATA2 and TAL1 complex and provide direct evidence for the mechanism of NK cell deficiency among GATA2 T354M patients. Importantly, through our current study, we will determine the unique role of TAL1 in NK development and function and its transcriptional network.

Ncrna-a3 Epigenetically Regulates TAL1 and Its Target Genes to Drive Erythroid Differentiation

Erythropoiesis is the development of enucleated red blood cells from multipotent hematopoietic stem cells. This complex process is tightly regulated by the dynamic changes in occupancy of tissue specific transcription factors (TFs) that activate and repress genes appropriately. TAL1, a critical regulator is expressed throughout the development of mature erythroblasts from multi-progenitors. Loss of TAL1 leads to defective erythropoiesis. As opposed to TAL1's role in erythropoiesis, itis an extraneous factor in developing lymphoid cells and aberrant expression of TAL1 in T-cells is associated with T-ALL. The differential regulation during normal and malignant hematopoiesis remains largely unclear. We found that a distil RNA - ncRNA-a3, is transcribed from erythroid specific enhancer, 50kbp downstream of the TAL1 TSS. We hypothesized that ncRNA-a3 plays an important role in regulating TAL1 and its target gene transcription in an erythroid specific manner. To test this hypothesis, we examine the regulatory potential of ncRNA-a3 towards the activation of TAL1 expression in the context of hematopoiesis and leukemogenesis. We used RNA interference to deplete ncRNA-a3 expression across several hematopoietic and leukemic cells. The decrease in ncRNA-a3 resulted in significant reduction of TAL1 transcript and protein levels in hematopoietic cells while TAL1 transcripts showed little or no reduction in leukemic cells. Subsequently, transcriptomic profile changes in erythroleukemic K562 cells showed downregulation of erythroid specific targets of TAL1. To ascertain the role of ncRNA-a3 in erythropoiesis, we used siRNA against ncRNA-a3 in primary CD34+ cells and subjected cells to differentiation. Quantification of early and late erythroblasts by colony forming assay and flow cytometry indicated impaired erythroid differentiation and maturation in orthochromatic and polychromatic stages. Further, RNA-seq analysis of ncRNA-a3 knockdown cells revealed defects in globin gene function, heme biosynthesis and nucleosome condensation compared to control CD34+ cells during time course EPO induced erythroid differentiation. Mechanistically, we observed ncRNA-a3s occupancy at TAL1 regulatory regions from ChIRP-qPCR across TAL1 locus. NG Capture-C data depicting changes in interaction frequencies indicated that ncRNA-a3 maintains canonical interactions at the TAL1 locus. Knockdown of ncRNA-a3 negatively impacted the chromatin accessibility in TAL1 and TAL1 target loci. Perturbation of chromatin landscape affected TAL1 and TAL1 binding partner - P300 and BRG1 occupancy at TAL1 target sites. To distinguish reduced TAL1 binding as a consequence of lower levels of TAL1, from RNA dependent binding of TAL1, we examined TAL1, P300 and BRG1 chromatin occupancy through Cut&Run in conjunction with RNase treatment. Effective depletion of global RNAs resulted in reduced enrichment of TAL1, P300 and BRG1 bound fragments indicating possible RNA dependent binding of TAL1 and its cofactors. TAL1 coimmunoprecipitation using RNase treatment (1) and ncRNA-a3 KD (2), demonstrated that TAL1s interaction with P300 and BRG1 is RNA dependent, but ncRNA-a3 independent. RNA-IP results further confirmed that ncRNA-a3 is associated with TAL1 through its interacting epigenetic cofactors P300 and BRG1. Together, our data indicate that although RNA dependent binding of TAL1 and its epigenetic cofactors is cell type or target locus specific, ncRNA-a3 modulates the occupancy of TAL1, P300 and BRG1 at the TAL1 locus during erythroid specific TAL1 transcription.

Determining mechanisms of T cell leukemogenesis using E2A −/−mice

Abstract T cell acute lymphoblastic leukemia (T-ALL) is a hematological malignancy caused by the transformation of T-lymphocytes during their development. 60% of pediatric T-ALLs have ectopic expression of Tal1, a gene that promotes leukemogenesis in part by altering the function of E2A. Consistent with this, mice with germline deletion of E2A develop T-ALL that mirrors human Tal1 +T-ALL. In this study, we used mice that delete E2A at different stages in T cell development to investigate the mechanisms causing T-ALL. Mice carrying a VavCre transgene and floxed alleles of E2A (VcKO) delete E2A early in T cell development, have reduced numbers of thymocytes prior to transformation, and develop T-ALL with an average latency of 18 weeks. Mice with an LckCre transgene (LcKO) delete E2A later than VcKO mice, have replete thymi, and rarely develop T-ALL. From this, we hypothesized that 1) E2A regulates a gene program in early T cell development that is antagonistic to transformation, or 2) Having a replete, competitive thymus suppresses transformation. Pre-leukemic VcKO thymocytes showed significantly more differentially expressed genes than LcKO thymocytes, including Notch1 and Myc, and are enriched for cancer associated pathways. To determine if the lack of competition in the VcKO thymus contributes to leukemogenesis, we reconstituted VcKO thymi by transplanting wild type bone marrow into sublethally irradiated VcKO mice. BM transplant significantly increased the number and frequency of DP thymocytes and increased leukemia latency. Together, these data suggest that intrinsic loss of E2A promotes leukemogenesis by changing the gene program of early developing thymocytes and by creating a non-competitive environment that supports transformation.

Hemato-vascular specification requires arnt1 and arnt2 genes in zebrafish embryos.

During embryonic development, a subset of cells in the mesoderm germ layer are specified as hemato-vascular progenitor cells, which then differentiate into endothelial cells and hematopoietic stem and progenitor cells. In zebrafish, the transcription factor npas4l (cloche) is required for the specification of hemato-vascular progenitor cells. However, it is unclear whether npas4l is the sole factor at the top of the hemato-vascular specification cascade. Here, we show that arnt1 and arnt2 genes are required for hemato-vascular specification. We found that arnt1;arnt2 double mutant zebrafish embryos, but not arnt1 or arnt2 single mutants, lack blood cells and most endothelial cells. arnt1/2 mutants have reduced or absent expression of etsrp and tal1, the earliest known endothelial and hematopoietic transcription factor genes. We found that Npas4l binds both Arnt1 and Arnt2 proteins in vitro, consistent with the idea that PAS domain-containing bHLH transcription factors act in a multimeric complex to regulate gene expression. Our results demonstrate that npas4l, arnt1 and arnt2 act together to regulate endothelial and hematopoietic cell fate, where each gene is necessary, but not sufficient, to drive hemato-vascular specification.

Abstract 3155: Gene expression profiles reveal distinct regulatory activities of transcription factors GATA1 and TAL1 upon AML relapse

Abstract The purpose of this study is to identify key regulatory pathways that potentially drive abnormal gene expression program in relapsed Acute Myeloid Leukemia (AML) patients, by integrative computational analyses on multi-omics molecular profiles. Relapsed AML remains a clinical challenge. Epigenetic heterogeneity may contribute to transcriptional dysregulation and disease progression in AML. However, what specific transcriptional programs and potential regulatory mechanisms contribute to disease relapse are not yet well understood. To characterize the global transcriptional landscapes in relapsed AML, we integrated genomics data from two cohorts of matched diagnosis and relapse patient specimens. We identified 5,416 differentially expressed genes (DEGs) between diagnosis and relapse in Cohort I. Unsupervised clustering yielded three distinct DEG groups: group A, B and C genes that were predominantly (88%) down-regulated, divergently regulated, or predominantly (65%) up-regulated, respectively, upon relapse. The expression pattern of all DEGs separated the patients into two clusters, most robustly by Group B genes. Interestingly, the majority of DEGs did not associate with changes in gene promoter methylation. Similar patterns were observed in Cohort II. We used Binding Analysis for Regulation of Transcription (BART) to identify transcriptional regulators (TRs) that potentially regulated the DEGs not associated with DNA methylation changes, and assessed the differential expression of identified TRs during disease progression. PU.1 was identified as a potential TR for Group A genes and was down-regulated upon relapse. GATA1 and TAL1 were identified as regulating Group B genes and were up-regulated in patient cluster1 and down-regulated in cluster2, consistent with the expression pattern of Group B genes. RBBP5 was a top predicted TR for Group C genes and was up-regulated upon relapse. We next validated the potential functionality of those predicted factors. In NSG mice transplanted with a human AML specimen, TAL1 and GATA1 were downregulated in AML cells collected four weeks after chemotherapy treatment, and were inferred as TRs for the down-regulated genes, similar to the patient data. PU.1 was inferred as regulating the up-regulated genes. Furthermore, we found that the level of differential expression of TAL1, GATA1, and PU.1 in each patient specimen associated with the correlation of DEG profiles between the patient specimen and TR perturbation in human-derived hematopoietic cell lines. Our results support the possibility that in some AML patients, TRs with roles in hematopoiesis and leukemia might contribute to disease relapse. Further mechanistic studies deciphering the molecular and phenotypic events facilitated by these TRs will yield significant insight into disease biology and possible therapeutic targeting approaches in relapsed AML. Citation Format: Zhenjia Wang, Yaseswini Neelamraju, Cem Meydan, Nicholas Dunham, Jorge Gandara, Tak Lee, Subhash Prajapati, Franck Rapaport, Caroline Sheridan, Paul Zumbo, Michael Becker, Lars Bullinger, Martin Carroll, Richard D’Andrea, Richard Dillon, Ross Levine, Christopher E. Mason, Ari Melnick, Donna Neuberg, Stefan Bekiranov, Chongzhi Zang, Francine E. Garrett-Bakelman. Gene expression profiles reveal distinct regulatory activities of transcription factors GATA1 and TAL1 upon AML relapse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3155.

TSPAN32 suppresses chronic myeloid leukemia pathogenesis and progression by stabilizing PTEN

We report herein that TSPAN32 is a key node factor for Philadelphia (Ph+) leukemia pathogenesis. We found that TSPAN32 expression was repressed by BCR-ABL and ectopic TSPAN32 expression upon Imatinib treatment inhibited the proliferation of Ph+ cell lines. Tspan32 overexpression significantly prevented BCR-ABL induced leukemia progression in a murine model and impaired leukemia stem cell (LSC) proliferation. LSCs represent an obstacle for chronic myeloid leukemia (CML) elimination, which continually replenish leukemia cells and are associated with disease relapse. Therefore, the identification of essential targets that contribute to the survival and self-renewal of LSCs is important for novel curative CML. Mechanistically, TSPAN32 was shown to interact with PTEN, increased its protein level and caused a reduction in PI3K-AKT signaling activity. We also found that TSPAN32 was repressed by BCR-ABL via the suppression of an important transcription factor, TAL1. Ectopic expression of TAL1 significantly increased TSPAN32 mRNA and protein level, which indicated that BCR-ABL repressed TSPAN32 transcription by decreasing TAL1 expression. Overall, we identified a new signaling axis composed of “BCR-ABL-TAL1-TSPAN32-PTEN-PI3K-AKT”. Our findings further complement the known mechanisms underlying the transformation potential of BCR-ABL in CML pathogenesis. This new signaling axis also provides a potential means to target PI3K-AKT for CML treatment.

Weighted gene co-expression network analysis identifies key hub genes and pathways in acute myeloid leukemia.

Introduction: Acute myeloid leukemia (AML) is the most common type of leukemia in adults. However, there is a gap in understanding the molecular basis of the disease, partly because key genes associated with AML have not been extensively explored. In the current study, we aimed to identify genes that have strong association with AML based on a cross-species integrative approach. Methods: We used Weighted Gene Co-Expression Network Analysis (WGCNA) to identify co-expressed gene modules significantly correlated with human AML, and further selected the genes exhibiting a significant difference in expression between AML and healthy mouse. Protein-protein interactions, transcription factors, gene function, genetic regulation, and coding sequence variants were integrated to identify key hub genes in AML. Results: The cross-species approach identified a total of 412 genes associated with both human and mouse AML. Enrichment analysis confirmed an association of these genes with hematopoietic and immune-related functions, phenotypes, processes, and pathways. Further, the integrated analysis approach identified a set of important module genes including Nfe2, Trim27, Mef2c, Ets1, Tal1, Foxo1, and Gata1 in AML. Six of these genes (except ETS1) showed significant differential expression between human AML and healthy samples in an independent microarray dataset. All of these genes are known to be involved in immune/hematopoietic functions, and in transcriptional regulation. In addition, Nfe2, Trim27, Mef2c, and Ets1 harbor coding sequence variants, whereas Nfe2 and Trim27 are cis-regulated, making them attractive candidates for validation. Furthermore, subtype-specific analysis of the hub genes in human AML indicated high expression of NFE2 across all the subtypes (M0 through M7) and enriched expression of ETS1, LEF1, GATA1, and TAL1 in M6 and M7 subtypes. A significant correlation between methylation status and expression level was observed for most of these genes in AML patients. Conclusion: Findings from the current study highlight the importance of our cross-species approach in the identification of multiple key candidate genes in AML, which can be further studied to explore their detailed role in leukemia/AML.

Dormancy Is a Feature of Therapy Resistant Mouse and Human Leukemia-Initiating Cells in T-ALL

Addressing therapy failure and relapse is critical in improving outcomes for pediatric T-cell acute lymphoblastic leukemia (T-ALL) patients. Relapse is thought to represent a failure to effectively eliminate leukemia-initiating cells (L-ICs), which survive chemotherapy and self-renew, regenerating the full complement of leukemic cell populations. Results from mouse T-ALL models show that the double negative-3 (DN3) thymic progenitors are enriched for L-IC activity; however, the L-IC remains an undefined subset of this population. We used single cell RNA-sequencing (scRNA-seq) to profile leukemic development in vivo, with a focus on uncovering DN3 heterogeneity. Integrated UMAP clustering of scRNA-seq libraries from wild type (WT), Tal1/Lmo2 preleukemic (PL), and Tal1/Lmo2 T-ALL (Leuk) thymi revealed an expansion of DN3 progenitors in PL and Leuk samples with decreases in mature (single positive) thymocytes. Unlike WT thymocytes, where Notch1 target gene expression was restricted to DN3 cells, Notch1-Myc pathway activation was maintained throughout thymocyte development in Tal1/Lmo2 transgenic mice. Re-clustering of DN3 cells revealed the presence of dormant and proliferative DN3 cells in PL and Leuk thymus. Dormant DN3 signatures resembled quiescent, Notch1-active DN3a (pre-β-selection) thymocytes and proliferative DN3 cells were transcriptionally similar to DN3b thymocytes. Dormant mouse DN3 cells were enriched for signatures of quiescent, therapy resistant ALL patient cells and cells obtained from ALL patients at minimal residual disease (Ebinger et al., Cancer Cell 2016), suggesting that dormant leukemic DN3 cells resemble human L-ICs that mediate drug resistance and relapse. To test this, we employed in vivo nucleosome labeling to profile dormant DN3 cells throughout leukemia development and functionally evaluate their response to chemotherapy. To assess cell divisional history, WT, Tal1, Lmo2, or Tal1/Lmo2 mice harboring the tet-inducible H2B-GFP reporter allele were subjected to in vivo doxycycline pulse (labeling), followed by a two-week chase. Analysis of pulse-chased thymocytes revealed low frequencies of label retaining (GFPHI) DN3 cells in WT mice. However, label retention was significantly increased in Lmo2 DN3 thymocytes (2.9-fold), consistent with other Lmo2 models (Tremblay et al., Nat. Commun. 2018). Tal1 expression also promoted DN3 label retention (6.0-fold) and Tal1/Lmo2 co-expression resulted in further increases in DN3 GFPHI cells (25-fold), indicating that Tal1 and Lmo2 cooperate to promote DN3 dormancy. DN3 GFPHI cells were present at similar frequency in mice with overt T-ALL and in line with scRNA-seq data, GFPHI cells were observed nearly exclusively as a subset of the leukemic DN3a population. Treatment with Vincristine, DeXamethasone, and L-asparaginase (VXL) led to a further increase in GFPHI DN3 frequency in Lmo2 and Tal1 preleukemic mice (3.1- and 9.7-fold, respectively), suggesting that cell cycle restricted L-ICs may evade conventional chemotherapy and mediate relapse. We then tested whether dormancy is a feature of the L-IC in T-ALL patients. Analysis of relapsed pediatric T-ALL cells revealed that the CD7+CD1a- L-IC population (Chiu et al., Blood 2010) is cell cycle restricted compared to CD7+CD1a+ leukemic cells and that VXL treatment enriched for CD7+CD1a- cells in xenografted mice, suggesting that cell cycle restriction may be an important feature of both mouse and human T-ALL L-ICs. Analysis of transcriptomes to reveal mediators of dormancy that may be exploited to chemosensitize L-ICs will be discussed.

Identifying a novel role for the master regulator Tal1 in the Endothelial to Hematopoietic Transition

Progress in the generation of Hematopoietic Stem and Progenitor Cells (HSPCs) in vitro and ex vivo has been built on the knowledge of developmental hematopoiesis, underscoring the importance of understanding this process. HSPCs emerge within the embryonic vasculature through an Endothelial-to-Hematopoietic Transition (EHT). The transcriptional regulator Tal1 exerts essential functions in the earliest stages of blood development, but is considered dispensable for the EHT. Nevertheless, Tal1 is expressed with its binding partner Lmo2 and it homologous Lyl1 in endothelial and transitioning cells at the time of EHT. Here, we investigated the function of these genes using a mouse embryonic-stem cell (mESC)-based differentiation system to model hematopoietic development. We showed for the first time that the expression of TAL1 in endothelial cells is crucial to ensure the efficiency of the EHT process and a sustained hematopoietic output. Our findings uncover an important function of Tal1 during the EHT, thus filling the current gap in the knowledge of the role of this master gene throughout the whole process of hematopoietic development.

Single-cell transcriptome analysis of embryonic and adult endothelial cells allows to rank the hemogenic potential of post-natal endothelium

Hematopoietic stem cells (HSCs) are crucial for the continuous production of blood cells during life. The transplantation of these cells is one of the most common treatments to cure patient suffering of blood diseases. However, the lack of suitable donors is a major limitation. One option to get HSCs matching perfectly a patient is cellular reprogramming. HSCs emerge from endothelial cells in blood vessels during embryogenesis through the endothelial to hematopoietic transition. Here, we used single-cell transcriptomics analysis to compare embryonic and post-natal endothelial cells to investigate the potential of adult vasculature to be reprogrammed in hematopoietic stem cells. Although transcriptional similarities have been found between embryonic and adult endothelial cells, we found some key differences in term of transcription factors expression. There is a deficit of expression of Runx1, Tal1, Lyl1 and Cbfb in adult endothelial cells compared to their embryonic counterparts. Using a combination of gene expression profiling and gene regulatory network analysis, we found that endothelial cells from the pancreas, brain, kidney and liver appear to be the most suitable targets for cellular reprogramming into HSCs. Overall, our work provides an important resource for the rational design of a reprogramming strategy for the generation of HSCs.

Hirsutine, a novel megakaryopoiesis inducer, promotes thrombopoiesis via MEK/ERK/FOG1/TAL1 signaling

BackgroundThrombocytopenia (TP) remains a challenge in clinical hematology. TP may have serious consequences, such as recurrent skin and mucosal bleeding and increased risk of intracranial and internal organ hemorrhage. However, effective and safe therapeutic drugs for the long-term management of TP are still lacking. PurposeThis study aimed to identify more effective active compounds for TP therapy. MethodsLiquid chromatography-mass spectrometry-nuclear magnetic resonance analysis was used to confirm the medicinal species and chemical structure of Hirsutine (HS). The proliferation of HS was examined by Cell Counting Kit (CCK-8) assay on cells lines. The effect of HS on megakaryocyte differentiation was analyzed by evaluating the expression of CD41, CD42b, and DNA ploidy via flow cytometry (FCM). The morphology of megakaryocytes and intermediate cells was observed using an optical microscope. K562 cells were then stained with Giemsa and benzidine. qRT-PCR was used to examine the mRNA expression of GATA-1, GATA-2, FOG-1, TAL-1, RUNX-1, NF-E2, and KLF-1 in K562 cells. Protein levels of the transcription factors were analyzed by western blotting. An MEK inhibitor was used to verify the relationship between the MEK/ERK signaling pathway and CD41/CD42b (FCM), FOG-1, and TAL-1. The Kunming thrombocytopenia mouse model was established by X-ray irradiation (4 Gy) and used to test HS activity and related hematopoietic organ index in vivo. Finally, computer simulations of molecular docking were used to predict the binding energies between HS-MEK and HS-ERK. ResultsWe preliminarily identified HS by screening a plant-sourced compound library for natural compounds with megakaryocytic differentiation and maturation (MKD/MKM)-promoting activity. We found that HS not only enhanced MKD/MKM of K562 and Meg01 cells, but also suppressed the decline of peripheral platelet levels in X-ray-induced myelosuppressive mice. In addition, HS promoted MKD via activation of MEK-ERK-FOG1/TAL1 signaling, which may be the key molecular mechanism of HS action in TP treatment. Molecular docking simulations further verified that HS targets the signaling protein MEK with high-affinity. ConclusionIn this study, we report for the first time that hirsutine boosts MKD/MKM through the MEK/ERK/FOG1/TAL1 signaling pathway and thus represents a promising treatment option for TP.

TAL1 cooperates with PI3K/AKT pathway activation in T-cell acute lymphoblastic leukemia.

TAL1 is ectopically expressed in about 30% of T-cell acute lymphoblastic leukemia (T-ALL) due to chromosomal rearrangements leading to the STIL-TAL1 fusion genes or due to non-coding mutations leading to a de novo enhancer driving TAL1 expression. Analysis of sequence data from T-ALL cases demonstrates a significant association between TAL1 expression and activating mutations of the PI3K-AKT pathway. We investigated the oncogenic function of TAL1 and the possible cooperation with PI3K-AKT pathway activation using isogenic pro-T-cell cultures ex vivo and in vivo leukemia models. We found that TAL1 on its own suppressed T-cell growth, in part by affecting apoptosis genes, while the combination with AKT pathway activation reduced apoptosis and was strongly driving cell proliferation ex vivo and leukemia development in vivo. As a consequence, we found that TAL1+AKTE17K transformed cells are more sensitive to PI3K-AKT pathway inhibition compared to AKTE17K transformed cells, related to the negative effect of TAL1 in the absence of activated PI3K-AKT signaling. We also found that both TAL1 and PI3K-AKT signaling increased the DNA-repair signature in T cells resulting in synergy between PARP and PI3K-AKT pathway inhibition. In conclusion, we have developed a novel mouse model for TAL1+AKTE17K driven T-ALL development and have identified a vulnerability of these leukemia cells to PI3K-AKT and PARP inhibitors.

Metabolic Changes in Venetoclax Resistance Are Determined By Differentiation State in T-Cell Acute Lymphoblastic Leukemia

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 chemoresistance and a high risk for relapse. 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 1,2. However, one issue with a targeted agent, such as ABT-199, is the development of acquired resistance. Interestingly, there have been numerous high impact papers connecting ABT-199 resistance to altered oxidative phosphorylation (OXPHOS) 3,4. While there are relatively few studies into T-ALL metabolism, there is evidence that aerobic glycolysis, the conversion of glucose to lactate, is greater in proliferating T-cells than in T-ALL and that NOTCH signalling can drive mitochondrial OXPHOS 5. A recent study showed that the transcription factor RUNX2 altered T-ALL metabolism, increasing both glycolysis and OXPHOS and enhancing leukemic cell migration 6. However, there has been relatively little research into the metabolic profile of T-ALL at distinct stages of differentiation. The aim of this study was to determine the role of ABT-199 resistance in altering metabolism and determine if that was due to the differentiation state of the T-ALL.ABT-199 R LOUCY cells were generated by chronic exposure to increasing concentrations of ABT-199 administered every two days. The EZH2 KO Jurkat cell lines were previously generated through CRISPR-Cas9 engineering 7. In order to assess the metabolic profile, cells were attached to a 96 well plate using CellTak and the extracellular acidification rate (ECAR) and oxidative phosphorylation (OXPHOS) was measured on a Seahorse Bioscience XF96 Extracellular Flux Analyzer. Anti-apoptotic dependence was measured using BH3 profiling and cell death by Annexin V/propidium iodide staining. The mitochondrial structure was visualized using transmission electron microscopy.Previously, we generated ABT-199 resistant ETP-ALL LOUCY cells (ABT-199 R) following continuous exposure to ABT-199 over a prolonged period of several months 8. The ABT-199 R cells showed dependence on BCL-XL for survival and sensitivity to the BCL-XL inhibitor WEHI 539. The ABT-199 R cells showed evidence of differentiation to a more mature T-cell. The ABT-199 R cells had increased surface CD3 (sCD3) expression and CD1A expression, along with increased expression of TAL1 and LMO2 genes compared to parental LOUCY cells. Interestingly, the ABT-199 R cells showed enhanced basal respiration, ATP production and max respiration compared to the parental cells. Indeed, analysis of the expression of OXPHOS complexes showed increased expression of complexes I-IV in the ABT-199 R cells, compared to the parental controls. Indeed, the parental LOUCY cells appeared to have reduced cristae number and length compared to the ABT-199R cells.Next, we assessed if inhibiting OXPHOS with a series of inhibitors (oligomycin, rotenone, antimycin) could sensitize the ABT-199 R LOUCY cells to ABT-199. However, we did not detect any changes to sensitivity of ABT-199. This led us to hypothesize that perhaps the changes in OXPHOS were due differentiation state of the LOUCY cells. We confirmed that more typical T-ALL cell lines (JURKAT and CEM-CCRF) had higher OXPHOS than the ETP-ALL cell line LOUCY and had higher expression of the OXPHOS complexes I-IV by Western blotting.To assess if de-differentiation of a more typical T-ALL cell line would cause a reduction in OXPHOS we turned to the EZH2 knockout (K/O) Jurkat cells 7. We found that EZH2 KO2 showed a reduction in the differentiation markers CD1A and CD3 on the cell surface and TAL1 gene expression, compared to WT control Jurkats. Next, we assessed the OXPHOS and found that the de-differentiated EZH2 cells had reduced OXPHOS compared to the parental controls, with altered mitochondrial structure. Suggesting, that de-differentiation of typical T-ALL cell line reduces OXPHOS.In this study we show that metabolic phenotype is linked to the maturation stage of T-ALL. We believe that the altered metabolism identified in ABT-199 resistance is linked to the selection of a more mature cell type. Highlighting, that altered metabolism may not be a driver of resistance to ABT-199 but a consequence of the maturation stage of the resistant cell. DisclosuresDi Grande: Novartis: Current Employment. Leon: BenevolentAI: Current Employment. Mansour: Astellas: Consultancy, Honoraria; Janssen: Consultancy. Bond: Haematology Association of Ireland Award funded by Novartis: Research Funding. Ni Chonghaile: AbbVie: Research Funding.

Identification of Novel Fusion Genes and Characterization of Transcriptome Features in T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is a clonal malignancy of immature T cells, accounting for 15% of pediatric ALL and 25% of adult ALL. Significant therapeutic progress has been made in pediatric T-ALL, while the outcome remains poor in adult T-ALL. Therefore, it is important to further characterize the molecular pathogenesis of T-ALL. Recently, the next-generation sequencing approach allowed systematic identification of molecular markers in pediatric T-ALL.Here, by performing RNA-sequencing and other genomic analysis, we investigated genomic landscape in 130 T-ALL cases. 36 distinct gene fusions were identified, including 18 novel ones. Four types of gene abnormalities were identified among novel fusions. Of note, newly discovered ZBTB16-ABL1 and TRA-SALL2 fusions, and the involvement of NKX2-1, were recurrent events. Functional studies revealed the recurrent ZBTB16-ABL1 fusion as a leukemogenic driver. Among 48 genes with mutation rates>3%, 6 were newly found in T-ALL. Moreover, aberrant mRNA isoform of SLC17A9 gene was found in majority of cases with high expression of TAL1 . Although most fusions were mutually exclusive, they co-existed with mutations. These genetic abnormalities were correlated to deregulated gene expression markers in three transcriptome-based subgroups. The G1 group mainly harbored TLX1 or TLX3 high expression, and over-expression of HOXA gene family. The transcriptome landscape of G2 group was closely associated with NUP family genes-containing fusions. The transcriptional factors important for early hematopoiesis were also found highly expressed in this group. Notably, 91.7% of our ETP cases were clustered in G2 group. With regard to G3, all patients with STIL-TAL1 and LMO1/2-TRA were located in this group, as cases with variant fusions involving TAL1 or STIL did. The genes with transcriptional aberration related to STIL-TAL1 were found over-expressed in most cases of G3 group. Finally, we compared genomic landscapes between adult and pediatric T-ALL patients. The types of fusions seemed more diverse in children than in adults, and the mutation rates in the genes of signaling pathways and epigenetic factors, both of which been frequently seen in ETP-ALL, were much higher in adults than in children. Our data suggest that the two T-ALL age groups might represent distinct disease entities, which could explain, at least in part, their distinct clinical outcomes. This study shed new lights on the leukemogenesis of T-ALL. DisclosuresNo relevant conflicts of interest to declare.

ARID5B Activates the TAL1-Induced Core Regulatory Circuit and the MYC Oncogene in T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is a malignant disorder characterized by the clonal proliferation of T-cell precursors. The most frequent molecular abnormality in T-ALL is the dysregulation of transcription factor genes, including the overexpression of TAL1/SCL . TAL1 induces a stem cell-like transcriptional regulatory program with its regulatory partners (RUNX1, GATA3 and MYB) in T-ALL cells. However, critical factors downstream of the TAL1 complex in T-ALL cells have not been fully characterized. Here, we performed RNA-seq analysis after genetic knockdown of TAL1 and each of its regulatory partners (E2A, HEB, LMO1, GATA3, RUNX1 and MYB) in a T-ALL cell line (Jurkat). We selected genes that were significantly downregulated after the knockdown of all these factors and were also directly bound by TAL1, HEB, GATA3, RUNX1 and MYB based on chromatin immunoprecipitation sequencing (ChIP-seq) analysis. We further filtered genes that were associated with “super-enhancers” based on the enrichment of histone H3 lysine 27 acetylation (H3K27ac). One of identified genes was ARID5B, which has not been previously implicated in T-ALL. ChIP-seq analysis showed DNA binding of the TAL1 transcriptional complex at a putative enhancer upstream of the ARID5B gene. A chromatin-chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) for the cohesin protein demonstrated a cis-regulatory interaction between the putative enhancer and the ARID5B transcriptional start site. The ARID5B enhancer locus is associated with a super-enhancer in multiple T-ALL samples but not in normal thymus, suggesting that this gene is abnormally activated in T-ALL. The shRNA knockdown experiment showed that ARID5B is required for the survival and growth of T-ALL cells in vitro. Using ChIP-seq and RNA-seq analysis for ARID5B, we discovered that ARID5B frequently co-occupies its target genes with TAL1 and co-ordinately controls their expression. Notably, ARID5B positively regulates expression of TAL1 and its regulatory partners (GATA3, RUNX1 and MYB). Additionally, we identified that ARID5B directly binds to the MYC enhancer, which has previously been reported to be activated by NOTCH1 in T-ALL cells, and activates the expression of this oncogene. Finally, transgenic study using zebrafish model indicated that forced expression of ARID5B in immature thymocytes results in thymus retention, radio-resistance and tumor formation in zebrafish. Taken together, our results indicate that ARID5B reinforces the oncogenic transcriptional program by upregulating the core regulatory circuit, the extended circuitry and the MYC oncogene in T-ALL, thereby contributing to T-cell leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

Exposure to hypoxia causes stress erythropoiesis and downregulates immune response genes in spleen of mice

BackgroundThe spleen is the largest secondary lymphoid organ and the main site where stress erythropoiesis occurs. It is known that hypoxia triggers the expansion of erythroid progenitors; however, its effects on splenic gene expression are still unclear. Here, we examined splenic global gene expression patterns by time-series RNA-seq after exposing mice to hypoxia for 0, 1, 3, 5, 7 and 13 days.ResultsMorphological analysis showed that on the 3rd day there was a significant increase in the spleen index and in the proliferation of erythroid progenitors. RNA-sequencing analysis revealed that the overall expression of genes decreased with increased hypoxic exposure. Compared with the control group, 1380, 3430, 4396, 3026, and 1636 genes were differentially expressed on days 1, 3, 5, 7 and 13, respectively. Clustering analysis of the intersection of differentially expressed genes pointed to 739 genes, 628 of which were upregulated, and GO analysis revealed a significant enrichment for cell proliferation. Enriched GO terms of downregulated genes were associated with immune cell activation. Expression of Gata1, Tal1 and Klf1 was significantly altered during stress erythropoiesis. Furthermore, expression of genes involved in the immune response was inhibited, and NK cells decreased.ConclusionsThe spleen of mice conquer hypoxia exposure in two ways. Stress erythropoiesis regulated by three transcription factors and genes in immune response were downregulated. These findings expand our knowledge of splenic transcriptional changes during hypoxia.

The migratory pathways of the cells that form the endocardium, dorsal aortae, and head vasculature in the mouse embryo

BackgroundVasculogenesis in amniotes is often viewed as two spatially and temporally distinct processes, occurring in the yolk sac and in the embryo. However, the spatial origins of the cells that form the primary intra-embryonic vasculature remain uncertain. In particular, do they obtain their haemato-endothelial cell fate in situ, or do they migrate from elsewhere? Recently developed imaging techniques, together with new Tal1 and existing Flk1 reporter mouse lines, have allowed us to investigate this question directly, by visualising cell trajectories live and in three dimensions.ResultsWe describe the pathways that cells follow to form the primary embryonic circulatory system in the mouse embryo. In particular, we show that Tal1-positive cells migrate from within the yolk sac, at its distal border, to contribute to the endocardium, dorsal aortae and head vasculature. Other Tal1 positive cells, similarly activated within the yolk sac, contribute to the yolk sac vasculature. Using single-cell transcriptomics and our imaging, we identify VEGF and Apela as potential chemo-attractants that may regulate the migration into the embryo. The dorsal aortae and head vasculature are known sites of secondary haematopoiesis; given the common origins that we observe, we investigate whether this is also the case for the endocardium. We discover cells budding from the wall of the endocardium with high Tal1 expression and diminished Flk1 expression, indicative of an endothelial to haematopoietic transition.ConclusionsIn contrast to the view that the yolk sac and embryonic circulatory systems form by two separate processes, our results indicate that Tal1-positive cells from the yolk sac contribute to both vascular systems. It may be that initial Tal1 activation in these cells is through a common mechanism.

The transcription factor TAL1 and miR-17-92 create a regulatory loop in hematopoiesis

A network of gene regulatory factors such as transcription factors and microRNAs establish and maintain gene expression patterns during hematopoiesis. In this network, transcription factors regulate each other and are involved in regulatory loops with microRNAs. The microRNA cluster miR-17-92 is located within the MIR17HG gene and encodes six mature microRNAs. It is important for hematopoietic differentiation and plays a central role in malignant disease. However, the transcription factors downstream of miR-17-92 are largely elusive and the transcriptional regulation of miR-17-92 is not fully understood. Here we show that miR-17-92 forms a regulatory loop with the transcription factor TAL1. The miR-17-92 cluster inhibits expression of TAL1 and indirectly leads to decreased stability of the TAL1 transcriptional complex. We found that TAL1 and its heterodimerization partner E47 regulate miR-17-92 transcriptionally. Furthermore, miR-17-92 negatively influences erythroid differentiation, a process that depends on gene activation by the TAL1 complex. Our data give example of how transcription factor activity is fine-tuned during normal hematopoiesis. We postulate that disturbance of the regulatory loop between TAL1 and the miR-17-92 cluster could be an important step in cancer development and progression.