FOXP3 Can Modulate TAL1 Transcriptional Activity Through Interaction with LMO2

SHORT COMMUNICATION FOXP3 can modulate TAL1 transcriptional activity through interaction with LMO2

V Fleskens1, M Mokry2, AM van der Leun1, S Huppelschoten1, CEGM Pals1, J Peeters1, S Coenen1, BA Cardoso3, JT Barata3, J van Loosdregt1,2 and PJ Coffer1,2

T-cell acute lymphoblastic leukemia (T-ALL) frequently involves aberrant expression of TAL1 (T-cell acute lymphocytic leukemia 1) and LMO2, oncogenic members of the TAL1 transcriptional complex. Transcriptional activity of the TAL1-complex is thought to have a pivotal role in the transformation of thymocytes and is associated with a differentiation block and self-renewal. The transcription factor Forkhead Box P3 (FOXP3) was recently described to be expressed in a variety of malignancies including T-ALL. Here we show that increased FOXP3 levels negatively correlate with expression of genes regulated by the oncogenic TAL1-complex in human T-ALL patient samples as well as a T-ALL cell line ectopically expressing FOXP3. In these cells, FOXP3 expression results in altered regulation of cell cycle progression and reduced cell viability. Finally, we demonstrate that FOXP3 binds LMO2 in vitro, resulting in decreased interaction between LMO2 and TAL1, providing a molecular mechanism for FOXP3-mediated transcriptional modulation in T-ALL. Collectively, our ﬁndings provide initial evidence for a novel role of FOXP3 as a tumor suppressor in T-ALL through modulation of TAL1 transcriptional activity.

Oncogene (2016) 35, 4141–4148; doi:10.1038/onc.2015.481; published online 21 December 2015

INTRODUCTION differential expression of TAL1 target genes involved in T-ALL 12–15 T-cell acute lymphoblastic leukemia (T-ALL) is a malignancy of development. immature cells of the T-cell lineage arrested during different The transcription factor Forkhead Box P3 (FOXP3) was initially stages of intrathymic development, with deregulated control described as the key protein required for the differentiation of cellular processes including cell cycle, proliferation and and functional maintenance of regulatory T-cells (Tregs), differentiation.1 This transformation of thymocytes is a multistep which have a critical role in the establishment of immune homeostasis.16,17 Although high FOXP3 levels are mainly detected process generally caused by genetic alterations including 18 chromosomal rearrangements, commonly resulting in aberrant in Tregs, expression is not restricted to these cells. In addition 2 FOXP3 is induced in non-regulatory T-cells upon T-cell receptor- expression of key regulatory transcription factors. One of the 19,20 most prevalent oncogenic transcription factors in T-ALL is the mediated activation, it was recently described to be expressed in immature T-cells as early as the pre-double-positive stage21 and basic helix-loop-helix transcription factor TAL1 (T-cell acute it is expressed in non-hematopoietic cells of various origins lymphocytic leukemia 1). In ~ 60% of T-ALL cases, aberrant including breast, lung and prostate tissue.22 Furthermore, FOXP3 expression and activity of TAL1 results in an arrest at the double- – was recently demonstrated to be differentially expressed in positive stage of thymocyte development.2 5 TAL1-mediated various malignancies including breast, prostate and gastric cancer, regulation of gene transcription occurs through formation where it is associated with tumor progression and prognosis.23–28 of a transcription complex minimally consisting of TAL1, basic Depending on cellular context of the malignancy, FOXP3 can be helix-loop-helix E-proteins and non-DNA-binding LIM-domain only 29 6,7 either downregulated or aberrantly expressed, raising questions (LMO) proteins. LMO proteins function as bridging molecules, about the role FOXP3 has in tumorigenesis. of which the importance is illustrated in transgenic mice Given the crucial role for FOXP3 in T lymphocytes, it is expressing Tal1 and Lmo2 in developing thymocytes. Although not surprising that FOXP3 expression has been detected in in Tal1 and Lmo2 single transgenic mice T-ALL was induced with malignancies with a T-lymphocytic origin.23,30 However, a 8,9 long latency, oncogenic potential was enhanced in double potential role for FOXP3 in regulation of T-cell leukemogenesis, transgenic mice, in which disease onset was accelerated and and the molecular mechanisms by which it could impinge on 8 aggressive T-ALL developed with high prevalence. Furthermore, transformation, are currently poorly understood. Here we aberrant activation of LMO2 is frequently detected in T-ALL, where provide evidence that FOXP3 can inhibit TAL1-complex function it is associated with thymocyte self-renewal.10 During healthy through formation of a competitive interaction with LMO2 and thymocyte development, both TAL1 and LMO2 expressions modulate cellular processes including cell cycle progression and are repressed;11 however, deregulated expression results in apoptosis, suggesting a putative role for FOXP3 as a tumor formation of an oncogenic TAL1-complex responsible for suppressor in T-ALL.

1Department of Cell Biology, Centre of Molecular Medicine, University Medical Centre Utrecht, Utrecht, The Netherlands; 2Division of Pediatrics, Wilhelmina Children's Hospital, Utrecht, The Netherlands and 3Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Lisbon, Portugal. Correspondence: Professor PJ Coffer, Department of Cell Biology, Centre of Molecular Medicine, University Medical Centre Utrecht, Heidelberglaan 100, Utrecht 3584 CX, The Netherlands. E-mail: [email protected] Received 26 March 2015; revised 19 September 2015; accepted 6 November 2015; published online 21 December 2015 FOXP3 can modulate TAL1 activity in T-ALL V Fleskens et al 4142 RESULTS AND DISCUSSION FOXP3 expression negatively associates with TAL1 target gene FOXP3 expression in T-ALL expression in T-ALL To evaluate FOXP3 expression levels in T-ALL, publicly available Since disturbed transcriptional regulation in T-ALL commonly fi involves activation of the TAL1-complex, generally induced by microarray-pro ling data sets from 174 bone marrow samples of 2 newly diagnosed T-ALL patients (GSE13204)31 were analyzed. aberrant expression of TAL1, its binding partner LMO2, or both, To sufficiently discriminate between samples with low or high we investigated the role of FOXP3 in regulation of the TAL1- FOXP3 expression, samples in the lower FOXP3-expressing decile complex. Genes described to be regulated by TAL1 in T-ALL include ALDH12 (RALDH1), MYB, TOX, KIT and TRAF3, which are all were compared with those in the highest decile, each group reduced in FOXP3high samples (Figure 2a).12,13,34 Moreover, genes containing 17 samples, referred to as FOXP3low and FOXP3high, previously demonstrated to be repressed by TAL1; CD4, the pre-T- respectively (Figures 1a and b). To address whether FOXP3 may be cell receptor α-chain (pTα) PTCRA, CDKN2A and PTPRU,11,12,35,36 transcriptionally functional in T-ALL blasts, mRNA expression levels were increased in FOXP3high samples, further supporting an of Treg phenotypic markers and direct FOXP3 target genes IL-2 inhibitory role for FOXP3 in the regulation of the TAL1-complex. α 32 receptor -chain (CD25), CTLA4 and GITR were evaluated. All A list of previously described TAL1 target genes, which are three Treg cell markers were significantly increased in FOXP3high differentially expressed in FOXP3high versus FOXP3low samples is versus FOXP3low samples (Figure 1c). Correspondingly, PDE3B, depicted in Figure 2c. mRNA expression of TAL1, E2A and LMO2 PTPN22 and SKP2, genes previously described to be directly remained unchanged (Figure 2b), demonstrating that differential inhibited by FOXP3,25,33 were repressed in FOXP3high samples expression of TAL1 target genes could occur independently of (Figure 1d). Together, these findings suggest that FOXP3 can be changes in expression of TAL1-complex members. Using an expressed and functional in T-ALL. independent cohort of T-ALL expression profiles (GSE10609) we

Figure 1. FOXP3 and FOXP3 target gene expression in T-cell lymphatic leukemia. Publicly available microarray-proﬁling data sets of 174 newly diagnosed T-ALL patients included in the Microarray Innovations in Leukemia study (GSE13204)31 were obtained from the open access NIH Gene-expression Omnibus database. CEL ﬁles were processed by robust multi-array average method53 using the rma function from Bioconductor Affy library using standard settings. (a) Relative FOXP3 expression (probe 221334_at) of 174 T-ALL samples were ordered based on relative FOXP3 mRNA expression. The lowest and highest decile were used for further analysis, referred to FOXP3 low and FOXP3 high, respectively. Data are represented as median-centered Log2 values. (b–d) Relative mRNA expression in FOXP3 low versus FOXP3 high samples of; (b) FOXP3; (c)Treg cell phenotypic markers directly targeted by FOXP3; CD25, CTLA-4 and GITR; and (d) direct target genes repressed by FOXP3; PDE3B, PTPN22 and SKP2. Data represent individual values and median ± interquartile range. *Po0.05, **Po0.01 and ***Po0.001 (unpaired two-tailed Student's t-test).

Figure 2. FOXP3 levels negatively correlate with TAL1 target gene expression. (a–c) Relative mRNA expression in FOXP3 low versus FOXP3 high samples as described in Figure 1a. (a) Heat map of relative expression levels of TAL1 target genes, arranged according to induction or repression by TAL1. Samples are ordered based on FOXP3 levels. (b) Relative mRNA expression of TAL1-complex members TAL1, LMO2 and E2A. Individual values and median ± interquartile range are represented. (c) Differential expression levels of previously described TAL1 target genes, up- or downregulation in FOXP3 high versus FOXP3 low samples are indicated. (d) HEK293 cells were transfected using PEI (Polysciences, Eppelheim, Germany) with a P4.2-promoter luciferase reporter construct, TAL1, LMO2 and E47, with or without Flag-Foxp3. Luciferase activity was measured with a Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) on a Centro LB 960 Micorplate Luminometer (Berthold Technologies, Bad Wildbad, Germany). pRLTK Renilla and pMT2 Flag-Foxp3 were described previously.44 Foxp3 was cloned from MIGR1-Foxp3 (provided by Sakaguchi32 into pcDNA3.47 P4.2-promoter luciferase reporter and pcDNA3 E47 were provided by Brandt.37 LMO2 and TAL1 were amplified from cDNA from the TAIL7 cell line54 and subcloned into pcDNA3.1 and pMT2-Flag. Data represent mean ± s.e.m. values of five independent experiments. (e–f) Amphotrophic Phoenix cells were transfected with pMX control:IRES:GFP (Control) or pMX FOXP3:IRES:GFP (FOXP3) (provided by Mutis55) using PEI. Virus supernatants (48 and 72 h post transfection) were used for two rounds of transduction of CCRF-CEM cells in retronectin-coated plates (5 μg/ml). CEM-Control and CEM-FOXP3 cells were purified by FACS sorting of GFP+ cells using a FACSAria II cell sorter (BD Bioscience, San Jose, CA, USA). Cells were subjected to (e) western blot analysis, utilizing antibodies against FOXP3 (eBioscience, San Diego, CA, USA), TAL1 and LMO2 (Abcam, Cambridge, UK) and HSP90 (Professor I Braakman, UMC Utrecht, Utrecht, The Netherlands) or (f) quantitative PCR with reverse transcription analysis of TAL1 target gene expression. Quantitative PCR with reverse transcription data are normalized for β2M and the comparative Ct method was used to quantify obtained data. Data are representative of four independent experiments. Utilized primers are listed in Supplementary Table 1. Mean ± s.e.m. *Po0.05, **Po0.01; NS, not significant (unpaired two-tailed Student's t-test).

Figure 3. FOXP3 expression results in a growth disadvantage in T-ALL cells by modulation of cell cycle progression and apoptosis. (a) GO-Term analysis of genes with reduced expression (P ⩽ 0.001) in FOXP3 high versus FOXP3 low samples, using the Database for Annotation Visualization and Integrated Discovery.56,57 (b) GFP+ CEM-FOXP3 cells were analyzed for FOXP3 expression levels by flow cytometry at indicated time-points using a FACS CANTOII analyzer. (c) CEM-control and CEM-FOXP3 cells were ethanol-fixed and subjected to RNAse A treatment (40 μg/ml), followed by permeabilization with the Foxp3 staining kit (eBioscience). Next, cells were stained for FOXP3, DNA content was stained propidium iodide and cell cycle analysis of FOXP3+ cells was performed by flow cytometry. (d) Quantification of results depicted in c.(e) Flow cytometric analysis of apoptosis levels of GFP+ CEM-Control and CEM-FOXP3 cells using the Annexin V:PE apoptosis Detection kit (BD Bioscience), with a quantitative representation depicted in (f). Data are representative of four independent experiments, mean ± s.e.m. are depicted. *Po0.05, **Po0.01 (Mann–Whitney test).

veriﬁed that FOXP3 expression can be detected in a proportion of modulate TAL1-complex activity, we co-transfected FOXP3. T-ALL samples (Supplementary Figures 1A–D), and that increased Expression of TAL1, E47 and LMO2 was sufﬁcient to induce strong FOXP3 expression can associate with reduced TAL1-complex P4.2-promoter activity, and TAL1-complex-mediated luciferase transcriptional output (Supplementary Figures 2A–D). activity was decreased upon co-expression of FOXP3 (Figure 2d). To further investigate the potential regulation of TAL1 transcriptional activity by FOXP3, we utilized a T-ALL cell line FOXP3 can inhibit TAL1 transcriptional activity (CCRF-CEM) endogenously expressing both TAL1 and LMO2 but During early hematopoietic development, the TAL1-complex not FOXP3 (Figure 2e). CCRF-CEM were transduced with either induces expression of the erythrocyte membrane protein band FOXP3:IRES:GFP or control virus, generating CEM-FOXP3 and CEM- 37 42, encoded by P4.2. To investigate TAL1-complex activity we Control cells, respectively, and sorted based on GFP expression. utilized the P4.2-promoter luciferase reporter. HEK293 cells were Importantly, TAL1 and LMO2 protein expression levels were co-transfected with the P4.2 reporter and plasmids encoding TAL1, unaffected by overexpression of FOXP3 (Figure 2e). To verify that E2A protein E47 and LMO2. To verify that FOXP3 could indeed FOXP3 can negatively modulate TAL1-complex activity, we

Figure 4. FOXP3 inhibits TAL1-complex formation through a competitive interaction with LMO2. (a) HEK293 cells were transfected with Flag- Foxp3 and HA-LMO2 as indicated and lysed with NP-40 lysis buffer (50 mM Tris-HCl pH 7.5, 0.5% Nonidet p40 (US Biologicals, Salem, MA, USA), 150 mM NaCl, 10 mM EDTA, supplemented with 1% HALT protease inhibitor cocktail (Thermo Scientific, Waltham, MA, USA). Lysates were incubated with anti-Flag-M2 affinity gel or anti-HA coupled beads (Sigma-Aldrich, Saint Louis, MI, USA) and immunoblots were analyzed with antibodies against Flag and HA (Sigma). (b) Representative confocal microscopy images of CEM-Control and CEM-FOXP3 cells, performed as described previously.47 Cells were probed with antibodies against FOXP3 (green) and endogenous TAL1 (yellow) or LMO2 (red). 4,6-diamidino- 2-phenylindole (DAPI) was used to visualize the nucleus (blue). (c, e) Protein–protein interactions in CEM-Control and CEM-FOXP3 cells were visualized with an in situ proximity ligation assay (PLA) (Sigma) as described previously.58 Punctate staining (red) indicates protein–protein interactions, green staining indicates FOXP3 and DAPI was used to visualize the nucleus (blue). (c) Cells were probed with mouse-anti-FOXP3 and rabbit-anti-LMO2 antibodies to visualize FOXP3:LMO2 interaction, anti-mouse IgG-AF488 was used to indicate FOXP3 expression. (d) Lysates from HEK293 cells transfected with Flag-TAL1, HA-LMO2 and a titration of Foxp3 were incubated with anti-HA-coupled beads. Immunoblots were probed with antibodies against Flag and HA. (e) Cells were probed with mouse-anti-TAL1 and rabbit-anti-LMO2 antibodies to visualize TAL1:LMO2 interaction, and co-stained with rat-anti-FOXP3 followed by anti-rat IgG-AF488 to indicate FOXP3 expression. Confocal samples were analyzed with a x63 objective on a Zeiss LSM 710 microscope (Carl Zeiss, Jena, Germany). Data represent at least three individual experiments. analyzed mRNA expression of various TAL1 target genes (Figure 1d) and CDKN2A (Figures 2a and f). Interestingly, in differentially expressed in FOXP3high T-ALL patient samples CEM-FOXP3 cells, a proportion of cells did not express FOXP3 and (Figure 2f). FOXP3 significantly reduced expression of TAL1 the population of FOXP3+ cells decreased over time (Figure 3b), target genes, including oncogenes MYB and KIT.Moreover, suggesting a growth disadvantage for FOXP3-expressing cells. TAL1-mediated repression of PTCRA, PTPRU and the cell cycle To verify that FOXP3 may function as a cell cycle regulator, cell regulator CDKN2A was reverted. Taken together, these data cycle profiling was performed on CEM-FOXP3 and control cells, further suggest that FOXP3 can repress TAL1-complex transcrip- using flow cytometry. Expression of FOXP3 resulted in modest tional activity. change in cell cycle distribution, with an apparent arrest in S-phase (Figures 3c and d). Although significant and reproducible, FOXP3 negatively modulates cell cycle progression and survival of these changes in cell cycle distribution are likely not exclusively T-ALL Cells responsible for the observed growth disadvantage of these cells. To investigate whether FOXP3 expressed in T-ALL could modulate FOXP3 was recently reported to not only exert a tumor cellular processes, we performed GO-term analysis evaluating suppressive function through inhibition of cell cycle progression genes differentially expressed in FOXP3high versus FOXP3low in breast cancer25,38 but also through modulation of cell survival, samples (Po0.0001). Although no enrichment was observed in by inducing apoptosis in gastric cancer.24 To address whether upregulated genes (data not shown), genes repressed in FOXP3 may affect cell survival, cells were stained with Annexin V FOXP3high samples displayed enrichment for cell cycle regulation and 7-AAD. An increase in apoptotic cells was observed upon (Figure 3a). This was supported by the observation that expression of FOXP3, with almost a doubling of cells in early FOXP3-induced expression of cell cycle regulator SKP2 apoptosis (Figures 3e and f). Inhibition of proliferation by FOXP3

© 2016 Macmillan Publishers Limited, part of Springer Nature. Oncogene (2016) 4141 – 4148 FOXP3 can modulate TAL1 activity in T-ALL V Fleskens et al 4146 was recently shown in human conventional T-cells, where can repress transcriptional activity of the oncogenic TAL1- activation-induced FOXP3 acts as a negative regulator of complex, through a competitive interaction with LMO2, enabling proliferation.39 Furthermore, FOXP3 was reported to exert the FOXP3 to counteract TAL1-complex-mediated regulation of same inhibitory function in breast cancer cells, by inducing target genes. expression of SKP2.25 In gastric cancer, FOXP3 mediates an anti- It remains unclear why FOXP3 is differentially expressed in proliferative effect by promotion of apoptosis, both through T-ALL samples, and how this is regulated. Transcriptional induction of pro-apoptotic genes, and inhibition of anti-apoptotic regulation of FOXP3 expression involves multiple transcription genes.24 Combined, these findings indicate that FOXP3-mediated factors acting in response to a variety of extracellular signals transcriptional regulation may result in decreased cell survival in including T-cell receptor stimulation (reviewed in Josefowicz T-ALL, through inhibition of cell cycle progression and promotion et al.48). The finding that in conventional T-cells receptor activation of apoptosis. induces FOXP3 expression39 suggests that an analogous mechanism may be responsible for FOXP3 induction in T-ALL. In FOXP3 interacts with LMO2 to inhibit formation of the addition, it has been demonstrated that FOXP3 expression can be TAL1-complex positively modulated by NOTCH signaling,49 an oncogenic path- 2 FOXP3 transcriptional output is generally determined by the way frequently subjected to activating mutations in T-ALL. formation of macro-molecular complexes, and it has been Recently, it was suggested that in T-ALL FOXP3 expression indeed 50 described to interact with a variety of proteins to regulate gene involves NOTCH signaling. However, further investigation into transcription.40,41 Examples of such interactions include a the mechanisms regulating FOXP3 expression in T-ALL is required. cooperative association with nuclear factor of activated T-cells Notably, in adult T-cell leukemia/lymphoma, FOXP3 expression resulting in repression of nuclear factor of activated T-cells- is associated with poor prognosis and decreased patient AP1-mediated gene transcription,42 modulation of TH17 lineage survival.30 In contrast to the immature origin of T-ALL cells, adult specification through an inhibitory interaction with RORγt,43 as T-cell leukemia/lymphoma is a malignancy of post-thymic T-cells, well as association with TCF1 enabling Wnt signaling to suggested to be derived from Treg cells, indicated by the modulate Treg cell suppressive function.44 LMO proteins mainly phenotypical and functional resemblance with these cells.51 function as bridging molecules to enable complex formation Whereas in these cells FOXP3 is the key transcription factor between transcription factors,7 and have been demonstrated to orchestrating the transcriptional program resulting in a suppres- form interactions with members of the Forkhead Box family sive phenotype, in T-ALL aberrantly activated oncogenic transcrip- FOXK1 and FOXO1.45,46 In T-ALL, formation of protein–protein tion factors are the main driving force of gene expression.1,2 It is interactions is an important mechanism to regulate TAL1 thus likely that FOXP3 mediates transcriptional regulation through transcriptional activity, illustrated by the finding that TAL1 must different mechanisms in these malignancies, explaining how form multiprotein complexes to enable target gene regulation.7 FOXP3 can play opposing roles in leukemias of different origin. Thus far, the majority of proteins identified to associate with the Although in recent years treatment outcome of patients with TAL-1 complex enhances transcriptional activity, but there is little T-ALL has improved, current treatment regimens of intensive knowledge concerning negative modulators of TAL1-mediated chemotherapy come along with severe side effects, and relapse regulation. Since LMO2 is necessary for TAL1 activity, we occurs in a significant number of patients. The desire for more hypothesized that the observed transcriptional inhibition of specific, and less toxic, treatment requires improved under- TAL1 by FOXP3 could be the result of competition for LMO2 standing of the molecular mechanisms underlying disease onset binding. and progression, to identify novel molecular targets to improve To investigate the association between FOXP3 and LMO2, T-ALL treatment and diagnostics. Our current study provides co-immunoprecipitation was performed on lysates from HEK293 evidence for a role for FOXP3, a key protein involved in T-cell cells ectopically expressing HA-LMO2 or Flag-Foxp3, showing a differentiation, as a novel molecular determinant of TAL1-complex clear interaction between both proteins (Figure 4a). To validate transcriptional outcome to intrinsically modulate the viability and this association can also occur in T-ALL cells, we utilized CEM- proliferative capacity of T-ALL cells. This study expands the FOXP3 or control cells to visualize the localization of FOXP3 growing understanding of T-ALL biology,47,52 and provides novel as well as endogenous LMO2 and TAL1, showing co-localization insight in the role of FOXP3 in T-ALL. of all three proteins in the nucleus (Figure 4b). To confirm the interaction of LMO2 with FOXP3 we performed an in situ Proximity Ligation Assay (PLA). For this, CEM-FOXP3 or control cells were CONFLICT OF INTEREST labeled with antibodies against FOXP3, LMO2 and TAL1 as The authors declare no conflict of interest. indicated followed by labeling with secondary PLA probes. A PLA signal can only be obtained when proteins of interest are in extremely close proximity (o40 nm), enabling the ACKNOWLEDGEMENTS detection of localized protein–protein interaction in cells We thank S Sakaguchi and T Mutis for providing us with plasmids and T Radstake for 47 using confocal microscopy. PLA verified the interaction between providing us with cells. VF and JvL were supported by a grant from the Dutch LMO2 and FOXP3, appearing as localized dots in the nucleus Arthritis Foundation (Rheumafonds), and BAC was supported by a fellowship from (Figure 4c). Fundação para a Ciência e a Tecnologia. This work was supported by grant from the To assess whether the FOXP3:LMO2 association can indeed Dutch Arthritis Foundation (Rheumafonds), and a fellowship from Fundação para a negatively affect formation of the TAL1-complex, we studied TAL1: Ciência e a Tecnologia. LMO2 association in presence or absence of FOXP3. Co- immunoprecipitation was performed on HEK293 lysates expres- REFERENCES sing Flag-TAL1, HA-LMO2 and increasing levels of FOXP3. A clear 1 Aifantis I, Raetz E, Buonamici S. Molecular pathogenesis of T-cell leukaemia and TAL1:LMO2 interaction was observed, which was inhibited by co- 8 – expression of FOXP3 in a dose-dependent manner (Figure 4d). lymphoma. Nat Rev Immunol 2008; : 380 390. 2 Van Vlierberghe P, Ferrando A. The molecular basis of T cell acute lymphoblastic Utilizing PLA to study the interaction between endogenous TAL1 leukemia. 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