Blast crisis of chronic myeloid leukemia is associated with poor survival and the accumulation of genomic lesions. Using whole-exome and/or RNA sequencing of patients at chronic phase (CP, n = 49), myeloid blast crisis (MBC, n = 19), and lymphoid blast crisis (LBC, n = 20), we found 25 focal gene deletions and 14 fusions in 24 patients in BC. Deletions predominated in LBC (83% of structural variants). Transcriptional analysis identified the upregulation of genes involved in V(D)J recombination, including RAG1/2 and DNTT in LBC. RAG recombination is a reported mediator of IKZF1 deletion. We investigated the extent of RAG-mediated genomic lesions in BC. Molecular hallmarks of RAG activity; DNTT-mediated nucleotide insertions and a RAG-binding motif at structural variants were exclusively found in patients with high RAG expression. Structural variants in 65% of patients in LBC displayed these hallmarks compared with only 5% in MBC. RAG-mediated events included focal deletion and novel fusion of genes associated with hematologic cancer: IKZF1, RUNX1, CDKN2A/B, and RB1. Importantly, 8/8 patients with elevated DNTT at CP diagnosis progressed to LBC by 12 months, potentially enabling early prediction of LBC. This work confirms the central mutagenic role of RAG in LBC and describes potential clinical utility in CML management.
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Hehlmann R. How I treat CML blast crisis. Blood. 2012;120:737–47.
Hochhaus A, Larson RA, Guilhot F, Radich JP, Branford S, Hughes TP, et al. Long-term outcomes of imatinib treatment for chronic myeloid leukemia. N Engl J Med. 2017;376:917–27.
Branford S, Wang P, Yeung DT, Thomson D, Purins A, Wadham C, et al. Integrative genomic analysis reveals cancer-associated mutations at diagnosis of CML in patients with high-risk disease. Blood. 2018;132:948–61.
Chen Z, Cortes JE, Jorgensen JL, Wang W, Yin CC, You MJ, et al. Differential impact of additional chromosomal abnormalities in myeloid vs lymphoid blast phase of chronic myelogenous leukemia in the era of tyrosine kinase inhibitor therapy. Leukemia. 2016;30:1606–9.
Funck T, Barnkob MB, Holm N, Ohm-Laursen L, Mehlum CS, Moller S, et al. Nucleotide composition of human ig nontemplated regions depends on trimming of the flanking gene segments, and terminal deoxynucleotidyl transferase favors adding cytosine, not guanosine, in most VDJ rearrangements. J Immunol. 2018;201:1765–74.
Di Noia JM, Neuberger MS. Molecular mechanisms of antibody somatic hypermutation. Annu Rev Biochem. 2007;76:1–22.
Roth DB. V(D)J recombination: mechanism, errors, and fidelity. Microbiol Spectr. 2014;2:MDNA3-0041-2014. https://doi.org/10.1128/microbiolspec.MDNA3-0041-2014.
Zhang M, Swanson PC. V(D)J recombinase binding and cleavage of cryptic recombination signal sequences identified from lymphoid malignancies. J Biol Chem. 2008;283:6717–27.
Teng G, Maman Y, Resch W, Kim M, Yamane A, Qian J, et al. RAG represents a widespread threat to the lymphocyte genome. Cell. 2015;162:751–65.
Bassing CH, Swat W, Alt FW. The mechanism and regulation of chromosomal V(D)J recombination. Cell 2002;109:S45–55.
Bolland DJ, Koohy H, Wood AL, Matheson LS, Krueger F, Stubbington MJ, et al. Two mutually exclusive local chromatin states drive efficient V(D)J recombination. Cell Rep. 2016;15:2475–87.
Ji Z, Sheng Y, Miao J, Li X, Zhao H, Wang J, et al. The histone methyltransferase Setd2 is indispensable for V(D)J recombination. Nat Commun. 2019;10:3353.
Hu J, Zhang Y, Zhao L, Frock RL, Du Z, Meyers RM, et al. Chromosomal loop domains direct the recombination of antigen receptor genes. Cell. 2015;163:947–59.
Heinaniemi M, Vuorenmaa T, Teppo S, Kaikkonen MU, Bouvy-Liivrand M, Mehtonen J, et al. Transcription-coupled genetic instability marks acute lymphoblastic leukemia structural variation hotspots. eLife. 2016;5:e13087. https://doi.org/10.7554/eLife.13087.
Shimazaki N, Tsai AG, Lieber MR. H3K4me3 stimulates the V(D)J RAG complex for both nicking and hairpinning in trans in addition to tethering in cis: implications for translocations. Mol cell. 2009;34:535–44.
Kirkham CM, Scott JNF, Wang X, Smith AL, Kupinski AP, Ford AM, et al. Cut-and-run: a distinct mechanism by which V(D)J recombination causes genome instability. Mol Cell. 2019;74:584–97. e9.
Kuo TC, Schlissel MS. Mechanisms controlling expression of the RAG locus during lymphocyte development. Curr Opin Immunol. 2009;21:173–8.
Corcoran AE. Immunoglobulin locus silencing and allelic exclusion. Semin Immunol. 2005;17:141–54.
Fisher MR, Rivera-Reyes A, Bloch NB, Schatz DG, Bassing CH. Immature lymphocytes inhibit Rag1 and Rag2 transcription and V(D)J recombination in response to DNA double-strand breaks. J Immunol. 2017;198:2943–56.
Mullighan CG, Miller CB, Radtke I, Phillips LA, Dalton J, Ma J, et al. BCR-ABL1 lymphoblastic leukaemia is characterized by the deletion of Ikaros. Nature. 2008;453:110–4.
Iacobucci I, Storlazzi CT, Cilloni D, Lonetti A, Ottaviani E, Soverini S, et al. Identification and molecular characterization of recurrent genomic deletions on 7p12 in the IKZF1 gene in a large cohort of BCR-ABL1-positive acute lymphoblastic leukemia patients: on behalf of Gruppo Italiano Malattie Ematologiche dell’Adulto Acute Leukemia Working Party (GIMEMA AL WP). Blood. 2009;114:2159–67.
Raschke S, Balz V, Efferth T, Schulz WA, Florl AR. Homozygous deletions of CDKN2A caused by alternative mechanisms in various human cancer cell lines. Genes Chromosomes Cancer. 2005;42:58–67.
Marculescu R, Le T, Bocskor S, Mitterbauer G, Chott A, Mannhalter C, et al. Alternative end-joining in follicular lymphomas’ t(14;18) translocation. Leukemia. 2002;16:120–6.
Heyer EE, Deveson IW, Wooi D, Selinger CI, Lyons RJ, Hayes VM, et al. Diagnosis of fusion genes using targeted RNA sequencing. Nat Commun. 2019;10:1388.
Roberts KG, Li Y, Payne-Turner D, Harvey RC, Yang YL, Pei D, et al. Targetable kinase-activating lesions in Ph-like acute lymphoblastic leukemia. N Engl J Med. 2014;371:1005–15.
Curry JD, Schulz D, Guidos CJ, Danska JS, Nutter L, Nussenzweig A, et al. Chromosomal reinsertion of broken RSS ends during T cell development. J Exp Med. 2007;204:2293–303.
Antoszewska-Smith J, Pawlowska E, Blasiak J. Reactive oxygen species in BCR-ABL1-expressing cells—relevance to chronic myeloid leukemia. Acta Biochim Polonica. 2017;64:1–10.
Koptyra M, Falinski R, Nowicki MO, Stoklosa T, Majsterek I, Nieborowska-Skorska M, et al. BCR/ABL kinase induces self-mutagenesis via reactive oxygen species to encode imatinib resistance. Blood. 2006;108:319–27.
Koptyra M, Cramer K, Slupianek A, Richardson C, Skorski T. BCR/ABL promotes accumulation of chromosomal aberrations induced by oxidative and genotoxic stress. Leukemia. 2008;22:1969–72.
Tsai AG, Lu H, Raghavan SC, Muschen M, Hsieh CL, Lieber MR. Human chromosomal translocations at CpG sites and a theoretical basis for their lineage and stage specificity. Cell. 2008;135:1130–42.
Score J, Calasanz MJ, Ottman O, Pane F, Yeh RF, Sobrinho-Simoes MA, et al. Analysis of genomic breakpoints in p190 and p210 BCR-ABL indicate distinct mechanisms of formation. Leukemia. 2010;24:1742–50.
Dong Y, Liu F, Wu C, Li S, Zhao X, Zhang P, et al. Illegitimate RAG-mediated recombination events are involved in IKZF1 Delta3-6 deletion in BCR-ABL1 lymphoblastic leukaemia. Clin Exp Immunol. 2016;185:320–31.
Nacheva EP, Brazma D, Virgili A, Howard-Reeves J, Chanalaris A, Gancheva K, et al. Deletions of immunoglobulin heavy chain and T cell receptor gene regions are uniquely associated with lymphoid blast transformation of chronic myeloid leukemia. BMC Genom. 2010;11:41.
Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29:15–21.
Chen X, Schulz-Trieglaff O, Shaw R, Barnes B, Schlesinger F, Kallberg M, et al. Manta: rapid detection of structural variants and indels for germline and cancer sequencing applications. Bioinformatics. 2016;32:1220–2.
Bailey TL, Boden M, Buske FA, Frith M, Grant CE, Clementi L, et al. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res. 2009;37:W202–8.
Alexandrov LB, Nik-Zainal S, Wedge DC, Aparicio SA, Behjati S, Biankin AV, et al. Signatures of mutational processes in human cancer. Nature. 2013;500:415–21.
Diaz-Gay M, Vila-Casadesus M, Franch-Exposito S, Hernandez-Illan E, Lozano JJ, Castellvi-Bel S. Mutational Signatures in Cancer (MuSiCa): a web application to implement mutational signatures analysis in cancer samples. BMC Bioinform. 2018;19:224.
Law CW, Alhamdoosh M, Su S, Dong X, Tian L, Smyth GK, et al. RNA-seq analysis is easy as 1-2-3 with limma, Glimma and edgeR. F1000 Res. 2016;5:1408–34.
Robinson JT, Thorvaldsdottir H, Winckler W, Guttman M, Lander ES, Getz G, et al. Integrative genomics viewer. Nat Biotechnol. 2011;29:24–6.
Radich JP, Dai H, Mao M, Oehler V, Schelter J, Druker B, et al. Gene expression changes associated with progression and response in chronic myeloid leukemia. Proc Natl Acad Sci USA. 2006;103:2794–9.
Motea EA, Berdis AJ. Terminal deoxynucleotidyl transferase: the story of a misguided DNA polymerase. Biochim Biophys Acta 2010;1804:1151–66.
Yu W, Nagaoka H, Misulovin Z, Meffre E, Suh H, Jankovic M, et al. RAG expression in B cells in secondary lymphoid tissues. Cold Spring Harb symposia Quant Biol. 1999;64:207–10.
Klemm L, Duy C, Iacobucci I, Kuchen S, von Levetzow G, Feldhahn N, et al. The B cell mutator AID promotes B lymphoid blast crisis and drug resistance in chronic myeloid leukemia. Cancer Cell. 2009;16:232–45.
Swaminathan S, Klemm L, Park E, Papaemmanuil E, Ford A, Kweon SM, et al. Mechanisms of clonal evolution in childhood acute lymphoblastic leukemia. Nat Immunol. 2015;16:766–74.
Casellas R, Basu U, Yewdell WT, Chaudhuri J, Robbiani DF, Di Noia JM. Mutations, kataegis and translocations in B cells: understanding AID promiscuous activity. Nat Rev Immunol. 2016;16:164–76.
Klein IA, Resch W, Jankovic M, Oliveira T, Yamane A, Nakahashi H, et al. Translocation-capture sequencing reveals the extent and nature of chromosomal rearrangements in B lymphocytes. Cell. 2011;147:95–106.
Papaemmanuil E, Rapado I, Li Y, Potter NE, Wedge DC, Tubio J, et al. RAG-mediated recombination is the predominant driver of oncogenic rearrangement in ETV6-RUNX1 acute lymphoblastic leukemia. Nat Genet. 2014;46:116–25.
Slaper-Cortenbach IC, Admiraal LG, Kerr JM, van Leeuwen EF, von dem Borne AE, Tetteroo PA. Flow-cytometric detection of terminal deoxynucleotidyl transferase and other intracellular antigens in combination with membrane antigens in acute lymphatic leukemias. Blood. 1988;72:1639–44.
Costi R, Crucitti GC, Pescatori L, Messore A, Scipione L, Tortorella S, et al. New nucleotide-competitive non-nucleoside inhibitors of terminal deoxynucleotidyl transferase: discovery, characterization, and crystal structure in complex with the target. J Med Chem. 2013;56:7431–41.
Hamilton E, Infante JR. Targeting CDK4/6 in patients with cancer. Cancer Treat Rev. 2016;45:129–38.
Churchman ML, Low J, Qu C, Paietta EM, Kasper LH, Chang Y, et al. Efficacy of retinoids in IKZF1-mutated BCR-ABL1 acute lymphoblastic leukemia. Cancer Cell. 2015;28:343–56.
Gong X, Du J, Parsons SH, Merzoug FF, Webster Y, Iversen PW, et al. Aurora A kinase inhibition is synthetic lethal with loss of the RB1 tumor suppressor gene. Cancer Discov. 2019;9:248–63.
Branford S, Kim DDH, Apperley JF, Eide CA, Mustjoki S, Ong ST, et al. Laying the foundation for genomically-based risk assessment in chronic myeloid leukemia. Leukemia. 2019;33:1835–50.
Mill CP, Fiskus W, DiNardo CD, Qian Y, Raina K, Rajapakshe K, et al. RUNX1-targeted therapy for AML expressing somatic or germline mutation in RUNX1. Blood. 2019;134:59–73.
Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015;43:e47.
This work was funded by the National Health and Medical Research Council of Australia APP1027531 and APP1117718 (S.B.), APP1135949 (T.P.H.) and APP1023059 (H.S.S.), the Ray and Shirl Norman Cancer Research Trust and the Royal Adelaide Hospital Research Foundation. T.P.H. and H.S.S have the financial support of Cancer Council SA’s Beat Cancer Project on behalf of its donors and the State Government of South Australia through the Department of Health. ACRF Cancer Genomics Facility was established with funding from Therapeutic Innovation Australia and the Australian Cancer Research Foundation (ACRF). We would like to thank Verity Saunders for preparation of some patient samples and David Yeung and Deborah White for critically reading the manuscript.
Conflict of interest
SB: Member of the advisory board of Qiagen, Novartis and Bristol-Myers Squibb, and consultant for Cepheid and received honoraria from Qiagen, Novartis, Bristol-Myers Squibb, and Cepheid. Research support from Novartis. TPH: Holds a consultancy role and has received research funding and honoraria from Novartis, Bristol-Myers Squibb, and Ariad. HSS received honoraria from Celgene. NS received travel support from Novartis, Bristol-Myers Squibb, Amgen, and Janssen. Other authors declare no conflicts of interest.
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Thomson, D.W., Shahrin, N.H., Wang, P.P.S. et al. Aberrant RAG-mediated recombination contributes to multiple structural rearrangements in lymphoid blast crisis of chronic myeloid leukemia. Leukemia (2020). https://doi.org/10.1038/s41375-020-0751-y