Corepressor ETO2 Research Articles

Embryonic erythropoiesis and hemoglobin switching require transcriptional repressor ETO2 to modulate chromatin organization.

The underlying mechanism of transcriptional co-repressor ETO2 during early erythropoiesis and hemoglobin switching is unclear. We find that absence of ETO2 in mice interferes with down-regulation of PU.1 and GATA2 in the fetal liver, impeding a key step required for commitment to erythroid maturation. In human β-globin transgenic Eto2 null mice and in human CD34+ erythroid progenitor cells with reduced ETO2, loss of ETO2 results in ineffective silencing of embryonic/fetal globin gene expression, impeding hemoglobin switching during erythroid differentiation. ETO2 occupancy genome-wide occurs virtually exclusively at LDB1-complex binding sites in enhancers and ETO2 loss leads to increased enhancer activity and expression of target genes. ETO2 recruits the NuRD nucleosome remodeling and deacetylation complex to regulate histone acetylation and nucleosome occupancy in the β-globin locus control region and γ-globin gene. Loss of ETO2 elevates LDB1, MED1 and Pol II in the locus and facilitates fetal γ-globin/LCR looping and γ-globin transcription. Absence of the ETO2 hydrophobic heptad repeat region impairs ETO2-NuRD interaction and function in antagonizing γ-globin/LCR looping. Our results reveal a pivotal role for ETO2 in erythropoiesis and globin gene switching through its repressive role in the LDB1 complex, affecting the transcription factor and epigenetic environment and ultimately restructuring chromatin organization.

SCL/TAL1 cooperates with Polycomb RYBP-PRC1 to suppress alternative lineages in blood-fated cells

During development, it is unclear if lineage-fated cells derive from multilineage-primed progenitors and whether active mechanisms operate to restrict cell fate. Here we investigate how mesoderm specifies into blood-fated cells. We document temporally restricted co-expression of blood (Scl/Tal1), cardiac (Mesp1) and paraxial (Tbx6) lineage-affiliated transcription factors in single cells, at the onset of blood specification, supporting the existence of common progenitors. At the same time-restricted stage, absence of SCL results in expansion of cardiac/paraxial cell populations and increased cardiac/paraxial gene expression, suggesting active suppression of alternative fates. Indeed, SCL normally activates expression of co-repressor ETO2 and Polycomb-PRC1 subunits (RYBP, PCGF5) and maintains levels of Polycomb-associated histone marks (H2AK119ub/H3K27me3). Genome-wide analyses reveal ETO2 and RYBP co-occupy most SCL target genes, including cardiac/paraxial loci. Reduction of Eto2 or Rybp expression mimics Scl-null cardiac phenotype. Therefore, SCL-mediated transcriptional repression prevents mis-specification of blood-fated cells, establishing active repression as central to fate determination processes.

3-Deazaneplanocin A (DZNep), an Inhibitor of S-Adenosylmethionine-dependent Methyltransferase, Promotes Erythroid Differentiation

EZH2, a core component of polycomb repressive complex 2 (PRC2), plays a role in transcriptional repression through histone H3 Lys-27 trimethylation and is involved in various biological processes, including hematopoiesis. It is well known that 3-deazaneplanocin A (DZNep), an inhibitor of S-adenosylmethionine-dependent methyltransferase that targets the degradation of EZH2, preferentially induces apoptosis in various hematological malignancies, suggesting that EZH2 may be a new target for epigenetic treatment. Because PRC2 participates in epigenetic silencing of a subset of GATA-1 target genes during erythroid differentiation, inhibition of EZH2 may influence erythropoiesis. To explore this possibility, we evaluated the impact of DZNep on erythropoiesis. DZNep treatment significantly induced erythroid differentiation of K562 cells, as assessed by benzidine staining and quantitative RT-PCR analysis for representative erythroid-related genes, including globins. When we evaluated the effects of DZNep in human primary erythroblasts derived from cord blood CD34-positive cells, the treatment significantly induced erythroid-related genes, as observed in K562 cells, suggesting that DZNep induces erythroid differentiation. Unexpectedly, siRNA-mediated EZH2 knockdown had no significant effect on the expression of erythroid-related genes. Transcriptional profiling of DZNep-treated K562 cells revealed marked up-regulation of SLC4A1 and EPB42, previously reported as representative targets of the transcriptional corepressor ETO2. In addition, DZNep treatment reduced the protein level of ETO2. These data suggest that erythroid differentiation by DZNep may not be directly related to EZH2 inhibition but may be partly associated with reduced protein level of hematopoietic corepressor ETO2. These data provide a better understanding of the mechanism of action of DZNep, which may be exploited for therapeutic applications for hematological diseases, including anemia.

Elucidation of the Role of LMO2 (LIM-only protein 2) in Erythroid Cells

AbstractAbstract 3443 Background:Developmental control mechanisms often utilize multimeric complexes containing transcription factors, coregulators, and additional non-DNA binding components. It is challenging to ascertain how such components contribute to complex function at endogenous loci. LMO2 (LIM-only protein 2) is a non-DNA binding transcriptional coregulator, and is an important regulator of hematopoietic stem cell development and erythropoiesis, as mice lacking this gene show defects in blood formation as well as fetal erythropoiesis (Warren et al. Cell. 1994). In the context of erythropoiesis, LMO2 has been demonstrated to be a part of multimetric complex, including master regulators of hematopoiesis (GATA-1 and SCL/TAL1), chromatin looping factor LDB1 and hematopoietic corepressor ETO2 (referred as GATA-SCL/TAL1 complex). As LMO2 controls hematopoiesis, its dysregulation is leukemogenic, and its influence on GATA factor function is still not evident, we investigated here the transcriptional regulatory mechanism via LMO2 in erythroid cells. Methods:For LMO2 knockdown, anti-LMO2 siRNA (Thermo Scientific Dharmacon) and pGIPZ lentiviral shRNAmir system (Open Biosystems) were used. Western blotting and Quantitative ChIP analysis were performed using antibodies for GATA-1, LMO2 (abcam), GATA-2, TAL1 and LDB1 (Santa Cruz). To obtain human primary erythroblasts, CD34-positive cells isolated from cord blood were induced in liquid suspension culture. For transcription profiling, human whole expression array was used (Agilent), and the data was analyzed with GeneSpring GX software. To induce erythroid differentiation of K562 cells, hemin was treated at a concentration of 30 uM for 24h. Results:siRNA-mediated LMO2 knockdown in hemin-treated K562 cells results in significantly decreased ratio of benzidine-staining positive cells, suggesting that LMO2 has an important role in the erythroid differentiation of K562 cells. Next, we conducted microarray analysis to characterize LMO2 target gene ensemble in K562 cells. In contrast to the predominantly repressive role of LMO2 in murine G1E-ER-GATA-1 cells (Fujiwara et al. PNAS. 2010), the analyses (n = 2) demonstrated that 177 and 78 genes were upregulated and downregulated (>1.5-fold), respectively, in the LMO2-knockdowned K562 cells. Downregulated gene ensemble contained prototypical erythroid genes such as HBB and SLC4A1 (encodes erythrocyte membrane protein band 3). To test what percentages of LMO2-regulated genes could be direct target genes of GATA-1 in K562 cells, we merged the microarray results with ChIP-seq profile (n= 5,749, Fujiwara et al. Mol Cell. 2009), and demonstrated that 26.4% and 23.1% of upregulated and downregulated genes, respectively, contained significant GATA-1 peaks in their loci. Furthermore, whereas LMO2 knockdown in K562 cells did not affect the expression of GATA-1, GATA-2 and SCL/TAL1 based on quantitative RT-PCR as well as Western blotting, the knockdown resulted in the significantly decreased chromatin occupancy of GATA-1, GATA-2, SCL/TAL1 and LDB1 at beta-globin locus control region and SLC4A1 locus.We subsequently analyzed the consequences of LMO2 knockdown in primary erythroblasts. Endogeneous LMO2 expression was upregulated along with the differentiation of cord blood cell-derived primary erythroblasts. shRNA-mediated knockdown of LMO2 in primary erythroblasts resulted in significant downregulation of HBB, HBA and SLC4A1. Conclusion:Our results suggest that LMO2 contributes to the expression of GATA-1 target genes in a context-dependent manner, through modulating the assembly of the components of GATA-SCL/TAL1 complex at endogeneous loci. Disclosures:No relevant conflicts of interest to declare.

Role of transcriptional corepressor ETO2 in erythroid cells

Transcriptional corepressor ETO2 is a component of a protein complex containing master regulators of hematopoiesis, including GATA-1, SCL/TAL1, LMO2, and LDB1. To elucidate the role of ETO2 during erythroid differentiation, including the effects of ETO2 on GATA-1 targets, we performed gene expression profiling using K562 cells overexpressed with ETO2. The analysis demonstrated that 667 and 598 genes were upregulated and downregulated (more than twofold), respectively, in ETO2-overexpressing cells. ETO2-repressed genes included those encoding prototypical erythroid proteins. To test what percentages of ETO2-repressed genes could be direct target genes of GATA-1 in K562 cells, we merged the microarray results with ChIP-seq profile (n=5,749), demonstrating that 23.1% of ETO2-repressed genes contained significant GATA-1 in their loci. However, there was no significant enrichment of PU.1 target genes among ETO2-repressed genes. Gene ontology analysis among ETO2-repressed genes revealed significant enrichment of genes related to "oxygen transporter," corresponding to globin genes. Quantitative chromatin immunoprecipitation and ETO2 knockdown analyses confirmed that ETO2 directly regulates globin genes in K562 cells. Next, we evaluated the role of ETO2 in human primary erythroblasts, derived from cord blood CD34-positive cells. In an exvivo model of erythroid differentiation from CD34-positive cells, ETO2 protein level peaked at day 2-4 and almost diminished at the later stage of differentiation. Furthermore, short hairpin RNA-mediated knockdown and retroviral vector-mediated overexpression of ETO2 in primary erythroblasts suggested that ETO2 significantly represses HBB, HBA, and ALAS2 expression. In summary, ETO2 regulates GATA-1 target genes critical for erythroid differentiation, and the decrease of ETO2 levels during erythroid differentiation would contribute to the activation of these targets.

Distinct Ldb1/NLI complexes orchestrate γ-globin repression and reactivation through ETO2 in human adult erythroid cells

AbstractThe Ldb1/GATA-1/TAL1/LMO2 complex mediates long-range interaction between the β-globin locus control region (LCR) and gene in adult mouse erythroid cells, but whether this complex mediates chromatin interactions at other developmental stages or in human cells is unknown. We investigated NLI (Ldb1 homolog) complex occupancy and chromatin conformation of the β-globin locus in human erythroid cells. In addition to the LCR, we found robust NLI complex occupancy at a site downstream of the Aγ-globin gene within sequences of BGL3, an intergenic RNA transcript. In cells primarily transcribing β-globin, BGL3 is not transcribed and BGL3 sequences are occupied by NLI core complex members, together with corepressor ETO2 and by γ-globin repressor BCL11A. The LCR and β-globin gene establish proximity in these cells. In contrast, when γ-globin transcription is reactivated in these cells, ETO2 participation in the NLI complex at BGL3 is diminished, as is BCL11A occupancy, and both BGL3 and γ-globin are transcribed. In these cells, proximity between the BGL3/γ-globin region and the LCR is established. We conclude that alternative NLI complexes mediate γ-globin transcription or silencing through long-range LCR interactions involving an intergenic site of noncoding RNA transcription and that ETO2 is critical to this process.

A Role for ETO-2 In Megakaryocytic Inhibition by Cyclic AMP Agonists

AbstractAbstract 289The regulation of megakaryopoiesis by cyclic AMP (cAMP) signaling forms the basis for treatment approaches to thrombocytosis in patients with myeloproliferative neoplasms. Anagrelide, the longstanding treatment of choice, is an inhibitor of the cAMP catabolizing enzyme phosphodiesterase III, and acts to lower platelet counts by inhibiting early stages of megakaryocytic differentiation. Essential for programming megakaryocytic differentiation is the transcriptionally activating pentameric complex consisting of GATA-1, SCL/TAL-1, an E-protein (either E2A or the similar HEB), and the bridging factors Lmo2 and Ldb1. This complex becomes repressive upon the recruitment of the co-repressor ETO-2 (also called MTG16 and CBFA2T3) by either SCL or E-protein components. ETO-2 has also been described as an A-kinase floating protein (AKAP). AKAPs bind the regulatory subunits of Protein kinase A (PKA) and tether the kinase within a specific compartment of the cell, ensuring proximity to appropriate substrates following kinase activation. The current study addresses the hypothesis that elevations in cAMP, such as those induced by anagrelide, inhibit megakaryocytic differentiation through the activation of a novel PKA signaling pathway involving ETO-2 and other components of the pentameric complex. Using peripheral blood mobilized human CD34+ progenitors cultured under conditions promoting megakaryopoiesis, we have confirmed that 500 nM anagrelide or 10 μ M forskolin, a direct adenylyl cyclase agonist, suppress upregulation of CD41 by two to three-fold as well as polyploidization, as determined by FACS on day 6 cultures. Pre-treatment of cultures with 10 μ M H89, a specific inhibitor of PKA, prevented the inhibitory effect of forskolin on megakaryopoiesis, as reflected by CD41 upregulation at day 6. Analysis of the kinetics of PKA activation, by immunoblotting for phospho-CREB and for phospho-PKA substrates (Cell Signaling), surprisingly showed only transient PKA induction by forskolin, with signals returning to baseline level after 4 hours. Additionally, washout experiments in which cells were treated with forskolin for only the first 4 hours of megakaryocytic culture showed severely blunted CD41 upregulation on day 6. These findings argue that elevations in cAMP inhibit megakaryocytic differentiation via activation of PKA and that a brief window of PKA activation during early megakaryopoiesis suffices for a stable blockade of differentiation. To determine a potential role for ETO-2, expression levels were analyzed at various times during megakaryocytic culture. In contrast to the rapid downregulation described in fetal murine megakaryocyte culture (Hamlett I. et al. Blood 112:2738-2749, 2008), human CD34+ progenitors showed no downregulation of ETO-2 mRNA or protein during megakaryocytic differentiation, nor were levels significantly affected by forskolin treatment. To study the functional role of ETO-2, we utilized shRNA knockdown in CD34+ hematopoietic progenitor cells undergoing megakaryocytic culture. In contrast to cells transduced with control vector, cells expressing shRNA targeting ETO-2 showed resistance to the inhibitory effects of elevated cAMP (n = 3). In examining potential targets within the pentameric complex, we found that elevations in cAMP reproducibly lowered the protein and mRNA levels of E2A by 50% while not affecting mRNA levels of another E protein, HEB. Altogether, these data suggest that cAMP mediates inhibition of megakaryopoiesis via activation of PKA, which utilizes ETO-2, possibly as an AKAP, to modulate the composition of an essential activating complex by downregulating E2A. Interestingly, E2A+/− mice are impaired in their ability to generate megakaryocytic progenitors from bipotent megakaryocyte-erythroid precursors (Semerad CL et al. PNAS 106:1930-1935, 2009). This novel signaling pathway contributes to an understanding of the mechanism of action of anagrelide and also further elucidates the basic regulatory circuitry governing determination of the megakaryocytic lineage. Disclosures:No relevant conflicts of interest to declare.

Discovering Hematopoietic Mechanisms through Genome-wide Analysis of GATA Factor Chromatin Occupancy

GATA factors interact with simple DNA motifs (WGATAR) to regulate critical processes, including hematopoiesis, but very few WGATAR motifs are occupied in genomes. Given the rudimentary knowledge of mechanisms underlying this restriction and how GATA factors establish genetic networks, we used ChIP-seq to define GATA-1 and GATA-2 occupancy genome-wide in erythroid cells. Coupled with genetic complementation analysis and transcriptional profiling, these studies revealed a rich collection of targets containing a characteristic binding motif of greater complexity than WGATAR. GATA factors occupied loci encoding multiple components of the Scl/TAL1 complex, a master regulator of hematopoiesis and leukemogenic target. Mechanistic analyses provided evidence for crossregulatory and autoregulatory interactions among components of this complex, including GATA-2 induction of the hematopoietic corepressor ETO-2 and an ETO-2-negative autoregulatory loop. These results establish fundamental principles underlying GATA factor mechanisms in chromatin and illustrate a complex network of considerable importance for the control of hematopoiesis.

Characterization of megakaryocyte GATA1-interacting proteins: the corepressor ETO2 and GATA1 interact to regulate terminal megakaryocyte maturation

AbstractThe transcription factor GATA1 coordinates timely activation and repression of megakaryocyte gene expression. Loss of GATA1 function results in excessive megakaryocyte proliferation and disordered terminal platelet maturation, leading to thrombocytopenia and leukemia in patients. The mechanisms by which GATA1 does this are unclear. We have used in vivo biotinylated GATA1 to isolate megakaryocyte GATA1-partner proteins. Here, several independent approaches show that GATA1 interacts with several proteins in the megakaryocyte cell line L8057 and in primary megakaryocytes. They include FOG1, the NURD complex, the pentameric complex containing SCL/TAL-1, the zinc-finger regulators GFI1B and ZFP143, and the corepressor ETO2. Knockdown of ETO2 expression promotes megakaryocyte differentiation and enhances expression of select genes expressed in terminal megakaryocyte maturation, eg, platelet factor 4 (Pf4). ETO2-dependent direct repression of the Pf4 proximal promoter is mediated by GATA-binding sites and an E-Box motif. Consistent with this, endogenous ETO2, GATA1, and the SCL pentameric complex all specifically bind the promoter in vivo. Finally, as ETO2 expression is restricted to immature megakaryocytes, these data suggest that ETO2 directly represses inappropriate early expression of a subset of terminally expressed megakaryocyte genes by binding to GATA1 and SCL.

Dynamic Change in the Stochiometry of ETO2 and SCL Governs the Transition to Terminal Differentiation in Erythroid Progenitors.

As highly proliferative erythroid progenitors commit to terminal differentiation, they also progressively undergo growth arrest. To determine the mechanisms underlying the appropriate timing of erythroid gene expression and those associated with growth cessation, we analyzed the dynamical composition of the multiprotein complex nucleated by the bHLH transcription factor SCL, a crucial regulator of erythropoiesis that absolutely requires interaction with other factors to activate transcription. In progenitor cells, the SCL complex marks a subset of erythroid specific genes (alpha-globin, P4.2, glycophorin A) that are transcribed later in differentiating cells, conducting cells toward terminal maturation. To unravel the regulation of transcription by SCL, we used tagging/proteomics approaches in two differentiation-inducible erythroid cell lines, coupled with binding assays to immobilized DNA templates and chromatin immunoprecipitation. Our analyses reveal that the core complex comprised of known proteins (SCL, GATA-1, LMO2, Ldb1 and E2A) and two additional E protein family members, HEB and E2-2, did not change with differentiation. Strikingly, this complex recruits HDAC1-2 in undifferentiated cells which were exchanged with TRRAP, a chromatin remodelling factor, upon differentiation, suggesting an epigenetic regulation of erythroid differentiation mediated by the core SCL complex. Finally, we identified the corepressor ETO2 targeted via this complex through direct interaction with E2A/HEB. In vivo, ETO2 represses the transcription of SCL target genes both in transient assays and in chromatin. During erythroid differentiation, ETO2 remains associated with the SCL complex bound to erythroid promoters. However, the stoichiometry of ETO2 and SCL/HEB changes as SCL and HEB levels increase with erythroid differentiation, both in nuclear extracts and on DNA. To determine the functional consequence of this imbalance in activator to co-repressor ratio, we delivered ETO2 siRNA in primary hematopoietic cells and found an accelerated onset of SCL target genes on induction of erythroid differentiation, and conversely, these genes were decreased following ectopic ETO2 expression. Strikingly, inhibition of ETO2 expression in erythroid progenitors arrests cell proliferation, indicating that ETO2 is required for their expansion. We therefore analyzed gene expression in purified erythroid progenitors and differentiating erythroid cells (E1-E5) and found an inverse correlation between the mRNA levels of ETO2 and cyclin-dependent kinase inhibitors. Moreover, ETO2 siRNA treatment of primary erythroid progenitors results in increased p21 CDKI and Gfi1b expression, as assessed by real-time PCR. Finally, we show by chromatin immunoprecipitation that Gfi-1b, p21 and p27, are direct targets of the SCL- ETO2 complex. We therefore conclude that ETO2 regulates the erythroid lineage fate by repressing SCL marked erythroid genes in undifferentiated cells, and by controlling the expansion of erythroid progenitors. Our study elucidates the dual function of ETO2 in the erythroid lineage and sheds light on epigenetic mechanisms coordinating red blood cell proliferation and differentiation.

ETO-2 associates with SCL in erythroid cells and megakaryocytes and provides repressor functions in erythropoiesis.

Lineage specification and cellular maturation require coordinated regulation of gene expression programs. In large part, this is dependent on the activator and repressor functions of protein complexes associated with tissue-specific transcriptional regulators. In this study, we have used a proteomic approach to characterize multiprotein complexes containing the key hematopoietic regulator SCL in erythroid and megakaryocytic cell lines. One of the novel SCL-interacting proteins identified in both cell types is the transcriptional corepressor ETO-2. Interaction between endogenous proteins was confirmed in primary cells. We then showed that SCL complexes are shared but also significantly differ in the two cell types. Importantly, SCL/ETO-2 interacts with another corepressor, Gfi-1b, in red cells but not megakaryocytes. The SCL/ETO-2/Gfi-1b association is lost during erythroid differentiation of primary fetal liver cells. Genetic studies of erythroid cells show that ETO-2 exerts a repressor effect on SCL target genes. We suggest that, through its association with SCL, ETO-2 represses gene expression in the early stages of erythroid differentiation and that alleviation/modulation of the repressive state is then required for expression of genes necessary for terminal erythroid maturation to proceed.

The Oncoprotein SCL/Tal-1 Associates with the Co-Repressor ETO-2 in Multiprotein Complexes in Erythroid Cells and Megakaryocytes.

The bHLH transcription factor SCL/Tal-1 is required for specification of hematopoietic stem cells and maturation of definitive erythroid cells and megakaryocytes. Additionally, ectopic SCL expression is the most common pathogenetic event in T-cell acute lymphoblastic leukaemia (T-ALL) (up to 60%). However, the mechanism of SCL action is not understood. Therefore, to begin to address this, we have isolated SCL-containing protein complexes in definitive erythroid cells and megakaryocytes. We have used a single step protein purification strategy where SCL is tagged to a sequence that can be biotinylated in vivo (biotag-SCL), and SCL-containing complexes purified by streptavidin beads and identified by mass spectrometry. First, to ensure that biotinylated SCL was functional, we were able to fully rescue both primitive and definitive haematopoietic development from SCL−/− ES cells in vitro with biotag SCL. Then, we expressed biotag-SCL in mouse erythroleukemic (MEL) and megakaryoblastic (L8057) cell lines and pulled-down SCL-containing protein complexes. Western Blot analysis of precipated proteins showed that known partners of SCL (including the transcription factors E2A, GATA1, LMO2 and Ldb1) were present in the pulled-down fractions, thereby validating the technique. Mass spectrometry analysis then led to the identification of several new candidate partners in MEL and L8057 cells. Two such proteins include ETO-2 (human homologue: MTG16), a member of the ETO family of co-repressor proteins involved in the pathogenesis of acute myeloid leukaemia, and SSDP-2, previously identified as a partner of Lbd1. Several approaches were then used to characterise the SCL/ETO-2 interaction. First, the interaction between the endogenous proteins was validated by co-immunoprecipitation and reverse co-immunoprecipitation in both MEL and L8057 nuclear extracts. Second, we showed that endogenous ETO-2 and SCL co-purify in the same high molecular weight fractions in gel-filtration profiles. Then, immunodepletion experiments demonstrated that the composition of the SCL/ETO-2 complexes is different in MEL and L8057 cells. In MEL cells, ETO-2 is part of at least two different complexes comprising other critical haematopoietic regulators including the oncoprotein Gfi-1b, whereas in L8057 cells these interactions are not detected. These results were then confirmed in primary erythroid cells and megakaryocytes. Finally, consistent with the repressor role of ETO-2, transactivation experiments in heterologous cells revealed that ETO-2 expression represses the activator function of a pentameric complex consisting of GATA1/SCL/E2A/LMO2/Ldb-1 on GATA-1 regulatory elements. Taken together, these data establish that SCL interacts with ETO-2 in erythroid cells and megakaryocytes, that the composition of SCL/ETO-2-containing complexes differ in these two lineages and that the SCL/ETO-2 complex may have a repressor activity on genes critical for haematopoietic differentiation. In this context, we also detect SCL binding to ETO-2 in an SCL-expressing T-ALL cell line. Though the exact role of the SCL-ETO-2 complex remains to be elucidated, it is plausible that ectopic T-cell SCL contributes to leukaemia by repressing genes indispensable for normal T cell differentiation. If confirmed, this would shed new light on the mechanism and possible therapeutics for SCL expressing T-ALL.