Evidence for a role of RUNX1 as recombinase cofactor for TCRβ rearrangements and pathological deletions in ETV6-RUNX1 ALL.

Publisher: Nature Publishing Group Country of Publication: England NLM ID: 101563288 Publication Model: Electronic Cited Medium: Internet ISSN: 2045-2322 (Electronic) Linking ISSN: 20452322 NLM ISO Abbreviation: Sci Rep Subsets: MEDLINE

Original Publication: London : Nature Publishing Group, copyright 2011-

Gene Deletion*; Core Binding Factor Alpha 2 Subunit/*physiology ; Gene Rearrangement, beta-Chain T-Cell Antigen Receptor/*genetics ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/*genetics ; Proto-Oncogene Proteins c-ets/*genetics ; Receptors, Antigen, T-Cell, alpha-beta/*genetics ; Repressor Proteins/*genetics; Animals ; B-Lymphocytes ; Core Binding Factor Alpha 2 Subunit/genetics ; Lymphocyte Count ; Mice, Knockout ; T-Lymphocytes ; Thymus Gland/pathology ; ETS Translocation Variant 6 Protein

T-cell receptor gene beta (TCRβ) gene rearrangement represents a complex, tightly regulated molecular mechanism involving excision, deletion and recombination of DNA during T-cell development. RUNX1, a well-known transcription factor for T-cell differentiation, has recently been described to act in addition as a recombinase cofactor for TCRδ gene rearrangements. In this work we employed a RUNX1 knock-out mouse model and demonstrate by deep TCRβ sequencing, immunostaining and chromatin immunoprecipitation that RUNX1 binds to the initiation site of TCRβ rearrangement and its homozygous inactivation induces severe structural changes of the rearranged TCRβ gene, whereas heterozygous inactivation has almost no impact. To compare the mouse model results to the situation in Acute Lymphoblastic Leukemia (ALL) we analyzed TCRβ gene rearrangements in T-ALL samples harboring heterozygous Runx1 mutations. Comparable to the Runx1 ^+/- mouse model, heterozygous Runx1 mutations in T-ALL patients displayed no detectable impact on TCRβ rearrangements. Furthermore, we reanalyzed published sequence data from recurrent deletion borders of ALL patients carrying an ETV6-RUNX1 translocation. RUNX1 motifs were significantly overrepresented at the deletion ends arguing for a role of RUNX1 in the deletion mechanism. Collectively, our data imply a role of RUNX1 as recombinase cofactor for both physiological and aberrant deletions.

Okuda, T. et al. Expression of a knocked-in AML1-ETO leukemia gene inhibits the establishment of normal definitive hematopoiesis and directly generates dysplastic hematopoietic progenitors. Blood 91, 3134–3143 (1998). (PMID: 10.1182/blood.V91.9.3134)
Hecht, J. et al. Evolution of a core gene network for skeletogenesis in chordates. PLoS Genet 4, e1000025, https://doi.org/10.1371/journal.pgen.1000025 (2008). (PMID: 10.1371/journal.pgen.1000025183694442265531)
de Bruijn, M. & Dzierzak, E. Runx transcription factors in the development and function of the definitive hematopoietic system. Blood 129, 2061–2069, https://doi.org/10.1182/blood-2016-12-689109 (2017). (PMID: 10.1182/blood-2016-12-68910928179276)
Sood, R., Kamikubo, Y. & Liu, P. Role of RUNX1 in hematological malignancies. Blood 129, 2070–2082, https://doi.org/10.1182/blood-2016-10-687830 (2017). (PMID: 10.1182/blood-2016-10-687830281792795391618)
Harada, Y. & Harada, H. Molecular pathways mediating MDS/AML with focus on AML1/RUNX1 point mutations. J Cell Physiol 220, 16–20, https://doi.org/10.1002/jcp.21769 (2009). (PMID: 10.1002/jcp.2176919334039)
Grossmann, V. et al. The molecular profile of adult T-cell acute lymphoblastic leukemia: mutations in RUNX1 and DNMT3A are associated with poor prognosis in T-ALL. Genes Chromosomes Cancer 52, 410–422, https://doi.org/10.1002/gcc.22039 (2013). (PMID: 10.1002/gcc.2203923341344)
Mok, M. M. et al. RUNX1 point mutations potentially identify a subset of early immature T-cell acute lymphoblastic leukaemia that may originate from differentiated T-cells. Gene 545, 111–116, https://doi.org/10.1016/j.gene.2014.04.074 (2014). (PMID: 10.1016/j.gene.2014.04.07424792891)
Grossmann, V. et al. Prognostic relevance of RUNX1 mutations in T-cell acute lymphoblastic leukemia. Haematologica 96, 1874–1877, https://doi.org/10.3324/haematol.2011.043919 (2011). (PMID: 10.3324/haematol.2011.043919218281183232273)
Roumier, C. et al. M0 AML, clinical and biologic features of the disease, including AML1 gene mutations: a report of 59 cases by the Groupe Francais d’Hematologie Cellulaire (GFHC) and the Groupe Francais de Cytogenetique Hematologique (GFCH). Blood 101, 1277–1283, https://doi.org/10.1182/blood-2002-05-1474 (2003). (PMID: 10.1182/blood-2002-05-147412393381)
Tsai, S. C. et al. Biological Activities of RUNX1 mutants predict secondary Acute Leukemia Transformation from Chronic Myelomonocytic Leukemia and Myelodysplastic Syndromes. Clin Cancer Res 21, 3541–3551, https://doi.org/10.1158/1078-0432.CCR-14-2203 (2015). (PMID: 10.1158/1078-0432.CCR-14-220325840971)
Jamil, A., Theil, K. S., Kahwash, S., Ruymann, F. B. & Klopfenstein, K. J. TEL/AML-1 fusion gene. its frequency and prognostic significance in childhood acute lymphoblastic leukemia. Cancer Genet Cytogenet 122, 73–78 (2000). (PMID: 10.1016/S0165-4608(00)00272-7)
Inaba, H., Greaves, M. & Mullighan, C. G. Acute lymphoblastic leukaemia. Lancet 381, 1943–1955, https://doi.org/10.1016/S0140-6736(12)62187-4 (2013). (PMID: 10.1016/S0140-6736(12)62187-423523389)
Alpar, D. et al. Clonal origins of ETV6-RUNX1(+) acute lymphoblastic leukemia: studies in monozygotic twins. Leukemia 29, 839–846, https://doi.org/10.1038/leu.2014.322 (2015). (PMID: 10.1038/leu.2014.32225388957)
Papaemmanuil, E. et al. RAG-mediated recombination is the predominant driver of oncogenic rearrangement in ETV6-RUNX1 acute lymphoblastic leukemia. Nat Genet 46, 116–125, https://doi.org/10.1038/ng.2874 (2014). (PMID: 10.1038/ng.2874244137353960636)
Eastman, Q. M., Leu, T. M. & Schatz, D. G. Initiation of V(D)J recombination in vitro obeying the 12/23 rule. Nature 380, 85–88, https://doi.org/10.1038/380085a0 (1996). (PMID: 10.1038/380085a08598914)
Bassing, C. H., Swat, W. & Alt, F. W. The mechanism and regulation of chromosomal V(D)J recombination. Cell 109(Suppl), S45–55 (2002). (PMID: 10.1016/S0092-8674(02)00675-X)
Dik, W. A. et al. New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling. The Journal of experimental medicine 201, 1715–1723, https://doi.org/10.1084/jem.20042524 (2005). (PMID: 10.1084/jem.20042524159281992213269)
Cieslak, A. et al. RUNX1-dependent RAG1 deposition instigates human TCR-delta locus rearrangement. The. Journal of experimental medicine 211, 1821–1832, https://doi.org/10.1084/jem.20132585 (2014). (PMID: 10.1084/jem.2013258525135298)
Turka, L. A. et al. Thymocyte expression of RAG-1 and RAG-2: termination by T cell receptor cross-linking. Science 253, 778–781 (1991). (PMID: 10.1126/science.1831564)
Putz, G., Rosner, A., Nuesslein, I., Schmitz, N. & Buchholz, F. AML1 deletion in adult mice causes splenomegaly and lymphomas. Oncogene 25, 929–939, https://doi.org/10.1038/sj.onc.1209136 (2006). (PMID: 10.1038/sj.onc.120913616247465)
Niebuhr, B. et al. Runx1 is essential at two stages of early murine B-cell development. Blood 122, 413–423, https://doi.org/10.1182/blood-2013-01-480244 (2013). (PMID: 10.1182/blood-2013-01-48024423704093)
Kieback, E. et al. Thymus-Derived Regulatory T Cells Are Positively Selected on Natural Self-Antigen through Cognate Interactions of High Functional Avidity. Immunity 44, 1114–1126, https://doi.org/10.1016/j.immuni.2016.04.018 (2016). (PMID: 10.1016/j.immuni.2016.04.01827192577)
Taniuchi, I. et al. Differential requirements for Runx proteins in CD4 repression and epigenetic silencing during T lymphocyte development. Cell 111, 621–633 (2002). (PMID: 10.1016/S0092-8674(02)01111-X)
Ono, M. et al. Foxp3 controls regulatory T-cell function by interacting with AML1/Runx1. Nature 446, 685–689, https://doi.org/10.1038/nature05673 (2007). (PMID: 10.1038/nature0567317377532)
Yassai, M. et al. A molecular marker for thymocyte-positive selection: selection of CD4 single-positive thymocytes with shorter TCRB CDR3 during T cell development. J Immunol 168, 3801–3807 (2002). (PMID: 10.4049/jimmunol.168.8.3801)
Yassai, M. & Gorski, J. Thymocyte maturation: selection for in-frame TCR alpha-chain rearrangement is followed by selection for shorter TCR beta-chain complementarity-determining region 3. J Immunol 165, 3706–3712 (2000). (PMID: 10.4049/jimmunol.165.7.3706)
Lind, E. F., Prockop, S. E., Porritt, H. E. & Petrie, H. T. Mapping precursor movement through the postnatal thymus reveals specific microenvironments supporting defined stages of early lymphoid development. The Journal of experimental medicine 194, 127–134 (2001). (PMID: 10.1084/jem.194.2.127)
Wang, X. et al. Regulation of Tcrb recombination ordering by c-Fos-dependent RAG deposition. Nature immunology 9, 794–801, https://doi.org/10.1038/ni.1614 (2008). (PMID: 10.1038/ni.161418500346)
Thomson, D. W. et al. Aberrant RAG-mediated recombination contributes to multiple structural rearrangements in lymphoid blast crisis of chronic myeloid leukemia. Leukemia, https://doi.org/10.1038/s41375-020-0751-y (2020).
Neveu, B., Caron, M., Lagace, K., Richer, C. & Sinnett, D. Genome wide mapping of ETV6 binding sites in pre-B leukemic cells. Sci Rep 8, 15526, https://doi.org/10.1038/s41598-018-33947-1 (2018). (PMID: 10.1038/s41598-018-33947-1303413736195514)
Asnafi, V. et al. Analysis of TCR, pT alpha, and RAG-1 in T-acute lymphoblastic leukemias improves understanding of early human T-lymphoid lineage commitment. Blood 101, 2693–2703, https://doi.org/10.1182/blood-2002-08-2438 (2003). (PMID: 10.1182/blood-2002-08-243812446444)
Yannoutsos, N. et al. A cis element in the recombination activating gene locus regulates gene expression by counteracting a distant silencer. Nature immunology 5, 443–450, https://doi.org/10.1038/ni1053 (2004). (PMID: 10.1038/ni105315021880)
Naik, A. K., Byrd, A. T., Lucander, A. C. K. & Krangel, M. S. Hierarchical assembly and disassembly of a transcriptionally active RAG locus in CD4(+)CD8(+) thymocytes. The Journal of experimental medicine, https://doi.org/10.1084/jem.20181402 (2018).
Egawa, T., Tillman, R. E., Naoe, Y., Taniuchi, I. & Littman, D. R. The role of the Runx transcription factors in thymocyte differentiation and in homeostasis of naive T cells. The. Journal of experimental medicine 204, 1945–1957, https://doi.org/10.1084/jem.20070133 (2007). (PMID: 10.1084/jem.2007013317646406)
Chi, Y. et al. Loss of runx1 function results in B cell immunodeficiency but not T cell in adult zebrafish. Open Biol 8, https://doi.org/10.1098/rsob.180043 (2018).
Romana, S. P. et al. High frequency of t(12;21) in childhood B-lineage acute lymphoblastic leukemia. Blood 86, 4263–4269 (1995). (PMID: 10.1182/blood.V86.11.4263.bloodjournal86114263)
Hiebert, S. W. et al. The t(12;21) translocation converts AML-1B from an activator to a repressor of transcription. Mol Cell Biol 16, 1349–1355, https://doi.org/10.1128/mcb.16.4.1349 (1996). (PMID: 10.1128/mcb.16.4.13498657108231119)
Schatz, D. G. & Ji, Y. Recombination centres and the orchestration of V(D)J recombination. Nat Rev Immunol 11, 251–263, https://doi.org/10.1038/nri2941 (2011). (PMID: 10.1038/nri294121394103)
Cieslak, A., Payet-Bornet, D. & Asnafi, V. RUNX1 as a recombinase cofactor. Oncotarget 6, 21793–21794, https://doi.org/10.18632/oncotarget.5488 (2015). (PMID: 10.18632/oncotarget.5488263933994673123)
Chen, M. J., Yokomizo, T., Zeigler, B. M., Dzierzak, E. & Speck, N. A. Runx1 is required for the endothelial to haematopoietic cell transition but not thereafter. Nature 457, 887–891, https://doi.org/10.1038/nature07619 (2009). (PMID: 10.1038/nature07619191297622744041)
Seitz, V. et al. A new method to prevent carry-over contaminations in two-step PCR NGS library preparations. Nucleic Acids Res 43, e135, https://doi.org/10.1093/nar/gkv694 (2015). (PMID: 10.1093/nar/gkv694261523044787772)
Lee, T. I., Johnstone, S. E. & Young, R. A. Chromatin immunoprecipitation and microarray-based analysis of protein location. Nat Protoc 1, 729–748, https://doi.org/10.1038/nprot.2006.98 (2006). (PMID: 10.1038/nprot.2006.98174063033004291)
Dimitrova, L. et al. PAX5 overexpression is not enough to reestablish the mature B-cell phenotype in classical Hodgkin lymphoma. Leukemia 28, 213–216, https://doi.org/10.1038/leu.2013.211 (2014). (PMID: 10.1038/leu.2013.21123842424)
Bookout, A. L. & Mangelsdorf, D. J. Quantitative real-time PCR protocol for analysis of nuclear receptor signaling pathways. Nucl Recept Signal 1, e012, https://doi.org/10.1621/nrs.01012 (2003). (PMID: 10.1621/nrs.010121660418416604184)
Cartharius, K. et al. MatInspector and beyond: promoter analysis based on transcription factor binding sites. Bioinformatics 21, 2933–2942, https://doi.org/10.1093/bioinformatics/bti473 (2005). (PMID: 10.1093/bioinformatics/bti47315860560)
Klingenhoff, A., Frech, K., Quandt, K. & Werner, T. Functional promoter modules can be detected by formal models independent of overall nucleotide sequence similarity. Bioinformatics 15, 180–186 (1999). (PMID: 10.1093/bioinformatics/15.3.180)
Ho Sui, S. J. et al. oPOSSUM: identification of over-represented transcription factor binding sites in co-expressed genes. Nucleic Acids Res 33, 3154–3164, https://doi.org/10.1093/nar/gki624 (2005). (PMID: 10.1093/nar/gki624159332091142402)
Petit, A. et al. Oncogenetic mutations combined with MRD improve outcome prediction in pediatric T-cell acute lymphoblastic leukemia. Blood 131, 289–300, https://doi.org/10.1182/blood-2017-04-778829 (2018). (PMID: 10.1182/blood-2017-04-77882929051182)
Trinquand, A. et al. Toward a NOTCH1/FBXW7/RAS/PTEN-based oncogenetic risk classification of adult T-cell acute lymphoblastic leukemia: a Group for Research in Adult Acute Lymphoblastic Leukemia study. Journal of clinical oncology: official journal of the American Society of Clinical Oncology 31, 4333–4342, https://doi.org/10.1200/JCO.2012.48.5292 (2013). (PMID: 10.1200/JCO.2012.48.5292)
Bond, J. et al. Early Response-Based Therapy Stratification Improves Survival in Adult Early Thymic Precursor Acute Lymphoblastic Leukemia: A Group for Research on Adult Acute Lymphoblastic Leukemia Study. Journal of clinical oncology: official journal of the American Society of Clinical Oncology 35, 2683–2691, https://doi.org/10.1200/JCO.2016.71.8585 (2017). (PMID: 10.1200/JCO.2016.71.8585)
Bruggemann, M. et al. Standardized next-generation sequencing of immunoglobulin and T-cell receptor gene recombinations for MRD marker identification in acute lymphoblastic leukaemia; a EuroClonality-NGS validation study. Leukemia, https://doi.org/10.1038/s41375-019-0496-7 (2019).
Duez, M. et al. Vidjil: A Web Platform for Analysis of High-Throughput Repertoire Sequencing. PloS one 11, e0166126, https://doi.org/10.1371/journal.pone.0166126 (2016). (PMID: 10.1371/journal.pone.0166126278356905106020)
van Dongen, J. J. et al. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia 17, 2257–2317, https://doi.org/10.1038/sj.leu.2403202 (2003). (PMID: 10.1038/sj.leu.240320214671650)
Giudicelli, V., Chaume, D. & Lefranc, M. P. IMGT/GENE-DB: a comprehensive database for human and mouse immunoglobulin and T cell receptor genes. Nucleic Acids Res 33, D256–261, https://doi.org/10.1093/nar/gki010 (2005). (PMID: 10.1093/nar/gki01015608191)

0 (Core Binding Factor Alpha 2 Subunit)
0 (Proto-Oncogene Proteins c-ets)
0 (Receptors, Antigen, T-Cell, alpha-beta)
0 (Repressor Proteins)
0 (Runx1 protein, mouse)

Date Created: 20200624 Date Completed: 20201210 Latest Revision: 20231213

20231215

PMC7308335

10.1038/s41598-020-65744-0

32572036