Alcalay, M

Bernard, L

Bodini, M

Cignetti, A

Dellino, Gi

Iacobucci, I

Lavorgna, S

Lo Coco, F

Luzi, L

Martinelli, G

Melloni, Gem

Ottone, T

Pelicci, Pg

Riva, L

Ronchini, C

Russo, A

Tarella, C

Volorio, S

Blood Cancer Journal

PubMed

LO COCO, FRANCESCO

LAVORGNA, SERENA

OTTONE, TIZIANA

Acute promyelocytic leukemias share cooperative mutations with other myeloid-leukemia subgroups

C. Ronchini

M. Bodini

F. Lo Coco

S. Lavorgna

T. Ottone

G. Martinelli

I. Iacobucci

C. Tarella

A. Cignetti

S. Volorio

L. Bernard

A. Russo

G.E.M. Melloni

L. Luzi

M. Alcalay

G.I. Dellino

P.G. Pelicci

AIR Universita degli studi di Milano

OPENLETTER TO THE EDITORAcute promyelocytic leukemias share cooperative mutations withother myeloid-leukemia subgroupsBlood Cancer Journal (2013) 3, e147; doi:10.1038/bcj.2013.46;published online 13 September 2013Acute promyelocytic leukemias (APLs) are characterized by theexpression of the PML-RARA oncogene, which is a product of the15;17 chromosomal translocation. Two lines of evidence suggestthat this genetic alteration is involved in the initiation ofpromyelocytic leukemogenesis in vivo: i) the t(15;17) oftenrepresents the only cytogenetic abnormality in an otherwise-normal karyotype; ii) PML-RARA is able to initiate leukemogenesisin mouse models on expression in hematopoietic stem cells(HSCs) or progenitors, giving rise to a disease that recapitulatesclinical and morphological features of human APLs.1 APL onset inmice, however, occurs after a long latency and with a lowpenetrance, suggesting that, like other cancers, APL is a multistepdisease, whereby PML-RARA cooperates with secondary mutationsfor the full development of the leukemia phenotype. Notably, inthe pre-leukemic phase of the disease, PML-RARA expressioninduces DNA damage in HSCs/progenitors and activates DNArepair, thus imparting a mutator phenotype that might contributeto disease progression.2,3Next-generation sequencing represents a powerful tool for thediscovery of genetic alterations at high resolution. To identifygene mutations that might cooperate with PML-RARA in theleukemogenic process, we performed whole-exome sequencingof 5 leukemias that developed in PML-RARA transgenic mice(mouse APLs; mAPLs) and 11 patients’ leukemias expressingPML-RARA (human APLs; hAPLs) (Supplementary Table 1). For thescope of this investigation, we only considered non-synonymoussingle-nucleotide variants (SNVs) and small insertions/deletions(indels) occurring in the tumor DNA with an allelic frequencyX25% (corresponding to a 450% frequency of cells carrying themutation in the tumor sample, assuming the majority ofmutations to be heterozygous). These experimental conditionsallow identiﬁcation of SNVs with a validation rate of 100%, asshown by an independent sequencing approach (Sanger sequen-cing; unpublished). We identiﬁed a total of 18 mutations in the5 mAPLs (16 SNVs and 2 indels) and 73 mutations in the 11 hAPLs(59 SNVs and 14 indels) (Table 1 and Supplementary Table 2). Wevalidated all the 16 mAPL SNVs by Sanger sequencing (the 2 indelswere not ampliﬁable for technical reasons), 18/18 randomlyselected human SNVs and 1/1 indel (Supplementary Table 2).We next combined the results of our sequencing analyses withthose previously published for APLs (1 mAPL and 24 hAPLs),4–7obtaining a data set of 41 APL samples (6 mAPLs and 35 hAPLs).Analysis of this data set showed a total of 270 mutations affecting248 human genes (231 SNVs, 34 indels and 5 splice junctionmutations—SJMs), with a low frequency of mutations per case(B7.1 in hAPLs and B3.5 in mAPLs). In three cases (two mAPLsand one hAPL), we found no SNVs or indels with a frequencyhigher than 25%. Surprisingly, two hAPL samples showed severalhundreds of low-frequency mutations, whose signiﬁcance remainsunclear (not shown).Of the 248 mutated genes, 9 were found in X2 patients(recurrent mutations), and 8 showed a signiﬁcantly highermutation rate (qo0.005; FLT3; WT1; KRAS; CALR; CSMD1; DDR2;REV3L and TCERG1L; Table 2 and Supplementary Table 3). FLT3 andKRAS have been already described as cooperators of PML-RARA inmouse models of APLs,8 whereas WT1 is infrequently mutatedin hAPLs.9 Of the remaining ﬁve, the DDR2 (discoidin domainreceptor 2) tyrosine kinase is mutated in a small subset ofsquamous cell lung cancer. Notably, DDR2 mutations are criticaloncogenic events for these tumors and confer high sensitivity tothe multi-targeted kinase inhibitor dasatinib.10 We found a total of25 mutations affecting these eight genes (APL driver mutations),with FLT3 and WT1 being the most frequently involved (Table 2).We next investigated the APL speciﬁcity of the identiﬁedmutations, as compared with other subgroups of acute myeloidleukemias (AMLs). First, we generated a data set of mutationsin all the available AML samples (n¼ 206; 196 previouslypublished samples7,11–14 and 10 new samples from this study(Supplementary Tables 1 and 2)) and divided the AML samples indifferent genetic/cytogenetic subgroups: i) samples with muta-tions of nucleophosmin (NPM1þ : 58 cases); ii) normal karyotypewithout NPM1 mutations (NK-NPM1 : 58 cases); iii) complexkaryotype (n¼ 22); iv) translocations or inversions affectingCBFB/MYH11 (n¼ 11); v) t(8;21) RUNX1/RUNX1T1 (n¼ 7); vi)trisomy 8 (n¼ 8); vii) MLL-X translocations (n¼ 10). Twenty-sevencases did not fall into any of these categories (‘Other’).As for PML-RARA, indirect evidence from mouse modelssuggests that mutated NPM115 and fusion proteins of CBFB,RUNX1 and MLL are initiating mutations for AMLs (reviewed inMcCormack et al.8).AML mutations were separately analyzed in each of theAML subgroups. We found a total of 1360 mutated genes(B9 mutations per case), of which 153 were recurrently mutated(X2 patients in the same subgroup) and 40 showed asigniﬁcantly higher mutation rate (qo0.005; Table 2 andSupplementary Table 3). Notably, included in the 40 geneswere 21 of the 22 signiﬁcantly mutated genes identiﬁed in arecent analysis of 200 AMLs (also part of our data set7), andadditional mutated genes critical for AMLs (BCOR, ASXL1,GATA2, SUZ12 and DDX41) or for selected epithelial cancers(CTCF, PLCE1 and CHD4). The most frequently mutated geneswere also signiﬁcantly associated with speciﬁc AML subgroups:FLT3, IDH1, DNMT3A and PTPN11 with NPM1þ AMLs; RUNX1,CEBPA and GATA2 with NK-NPM1 AMLs; TP53 with AMLs withcomplex karyotypes; KIT with CBFB/MYH11 AMLs; IDH1 withAMLs with trisomy 8 (Table 2). Moreover, mutations in SF3B1,PTPN11, DNAH9, are present in both human and mouseleukemias.We then analyzed the distribution of the signiﬁcantly mutatedAPL and AML genes (n¼ 44) across all samples (n¼ 239). Twenty-eight genes (B64%) were mutated in more than one cytogeneticsubgroup, covering 383 of the 416 mutations identiﬁed in allsamples (B92%). In the remaining 16 genes (B36%), mutationswere instead associated with a speciﬁc subgroup, correspondingto just 33 of the identiﬁed mutations (B8%) and suggesting thatsubgroup speciﬁcity might be due to their low frequency. Indeed,we found a signiﬁcant correlation between frequency of muta-tions per gene and numbers of subgroups where it is mutated(Spearman’s coefﬁcient value of 0.93). As regards APLs, 5 of the 8Citation: Blood Cancer Journal (2013) 3, e147; doi:10.1038/bcj.2013.46& 2013 Macmillan Publishers Limited All rights reserved 2044-5385/13www.nature.com/bcjsigniﬁcantly mutated genes were also found in other AMLcytogenetic subgroups (FLT3; WT1; KRAS; CSMD1 and DDR2),covering a total of 100 mutations, while 3 (CALR; REV3L andTCERG1L) were only found in APLs, covering 6 mutations.Three genes that were found signiﬁcantly mutated in otherAML subgroups were also mutated in APLs (PHF6, FAM5C andPTPN11).Together, these results imply that different myeloid leukemias,including APLs, share the same subset of cooperating mutations,and are consistent with a scenario whereby speciﬁc initiatingmutations interact with a common pool of highly heterogeneous,yet phenotypically equivalent, cooperating mutations. Indirectevidence, however, suggests that the pool of cooperatingmutations in AMLs is not yet entirely deﬁned, and that sequencingof additional AMLs is needed. In fact, due to the limited size ofsome samples (for example, in selected AML subgroups such asthose with rearrangements of CBFB, RUNX1 or MLL), it is likely thatour statistical analyses do not allow the identiﬁcation of all thedriver mutations in AML. Among all the mutated genes (n¼ 1559),B3% (n¼ 45) were mutated at statistically signiﬁcant frequency(driver mutations). Among the others (passenger mutations),however, we identiﬁed mutations that have been causallyimplicated in the pathogenesis of AMLs (for example, ETV6,JAK2, NOTCH1, NUMA1, PRDM16, CBL, CBFB, CHIC2, ELF4, NSD1 andPDGFRB) or other cancers (for example, PTEN, MYC, ARID1A, SF3B1,EGFR, NF1, THRAP3, MED12, KDR, IKZF1, DAXX and SETD2).Table 1. Mutations identified by next-generation sequencing in APLsCases Mutations StudynsSNVs Indels SJMs TotalMouse APLsmAPL#Mi1 0 0 0 0 PresentmAPL#Mi2 1 0 0 1 PresentmAPL#Mi3 9 1 0 10 PresentmAPL#Mi4 6 1 0 7 PresentmAPL#Mi5 0 0 0 0 PresentmAPL 3 0 0 3 Wartman et al.4Total mutations (per Pt.) 19 (3.16) 2 (0.33) 0 21 (3.50) 6 cases totalHuman APLshAPL#Mi1 5 0 0 5 PresenthAPL#Mi2 13 3 0 16 PresenthAPL#Mi3 2 1 0 3 PresenthAPL#Mi4 3 3 0 6 PresenthAPL#Mi5 0 0 0 0 PresenthAPL#Mi6 5 1 0 6 PresenthAPL#Mi7 12 2 0 14 PresenthAPL#Mi8 1 1 0 2 PresenthAPL#Mi9 4 1 0 5 PresenthAPL#Mi10 7 1 0 8 PresenthAPL#Mi11 7 1 0 8 PresenthAPL#1 5 2 0 7 Greif et al.5hAPL#2 3 0 0 3 Greif et al.5hAPL#3 4 0 0 4 Greif et al.5hAPL 12 0 0 12 Welch et al.6TCGA-AB-2803 12 1 0 13 TCGA7TCGA-AB-2804 7 1 0 8 TCGA7TCGA-AB-2823 0 1 0 1 TCGA7TCGA-AB-2840 0 1 0 1 TCGA7TCGA-AB-2841 4 0 0 4 TCGA7TCGA-AB-2862 7 0 0 7 TCGA7TCGA-AB-2872 9 1 0 10 TCGA7TCGA-AB-2897 5 0 1 6 TCGA7TCGA-AB-2905 15 2 0 17 TCGA7TCGA-AB-2906 9 1 0 10 TCGA7TCGA-AB-2980 3 1 0 4 TCGA7TCGA-AB-2982 1 1 0 2 TCGA7TCGA-AB-2991 10 0 0 10 TCGA7TCGA-AB-2994 6 1 0 7 TCGA7TCGA-AB-2997 9 1 0 10 TCGA7TCGA-AB-2998 4 1 0 5 TCGA7TCGA-AB-2999 9 0 0 9 TCGA7TCGA-AB-3001 8 0 2 10 TCGA7TCGA-AB-3007 5 1 1 7 TCGA7TCGA-AB-3012 6 2 1 9 TCGA7Total in hAPLs (per Pt.) 212 (6.06) 32 (0.91) 5 (0.14) 249 (7.11) 35 casesTotal hþmAPLs (per Pt.) 231 (5.63) 34 (0.83) 5 (0.12) 270 (6.59) 41 casesAbbreviations: APL, acute promyelocytic leukemia; h, human; indels, small insertion/deletions; m, mouse; nsSNV, non-synonymous single nucleotide variant;per Pt., per patient; SJM, splice junction mutation; TCGA, The Cancer Genome Atlas.Letter to the Editor2Blood Cancer Journal & 2013 Macmillan Publishers LimitedCONFLICT OF INTERESTThe authors declare no conﬂict of interest.ACKNOWLEDGEMENTSLR was supported by a Reintegration AIRC/Marie Curie International Fellowship inCancer Research. This work was supported by grants from Fondazione Cariplo to LR;Associazione Italiana Ricerca sul Cancro (AIRC) and Italian Ministry of Health to PGP;European Commission (FP7) to GM. We thank Paola Dalton for critical review of themanuscript.L Riva1,9, C Ronchini1,9, M Bodini1, F Lo-Coco2,3, S Lavorgna2,3,T Ottone2,3, G Martinelli4, I Iacobucci4, C Tarella5, A Cignetti5,S Volorio6, L Bernard7, A Russo7, GEM Melloni1, L Luzi6,7,M Alcalay7,8, GI Dellino7,8 and PG Pelicci7,81Center for Genomic Science of IIT@SEMM, Istituto Italiano diTecnologia, at the IFOM-IEO Campus, Milan, Italy;2Department of Biomedicine and Prevention, University of ‘Rome TorVergata’, Rome, Italy;3Laboratorio di Neuro-Oncoematologia, Fondazione Santa Lucia,Rome, Italy;4Institute of Hematology ‘Sera`gnoli’, Department of Experimental,Diagnostic and Specialty Medicine, University of Bologna,Bologna, Italy;5University Division of Hematology and Cell Therapy, MaurizianoHospital and University of Torino, Torino, Italy;6IFOM, FIRC Institute of Molecular Oncology Foundation, Milan, Italy;7Department of Experimental Oncology, European Institute ofOncology, IEO, Milan, Italy and8Dipartimento di Scienze della salute, Universita` degli Studi diMilano, Milan, ItalyE-mail: chiara.ronchini@iit.it or piergiuseppe.pelicci@ieo.eu9These authors contributed equally to this work.REFERENCES1 Grisolano JL, Wesselschmidt RL, Pelicci PG, Ley TJ. Altered myeloid developmentand acute leukemia in transgenic mice expressing PML-RAR alpha under controlof cathepsin G regulatory sequences. Blood 1997; 89: 376–387.2 Viale A, De Franco F, Orleth A, Cambiaghi V, Giuliani V, Bossi D et al. Cell-cyclerestriction limits DNA damage and maintains self-renewal of leukaemia stem cells.Nature 2009; 457: 51–56.Table 2. Genes with a significantly higher mutation rate in APLs and AMLsMutatedGene PML-RARA NPM1+ NK-NPM1- Complexkaryotype CBFB/ MYH11 MLL-X Trisomy 8 RUNX1/ RUNX1T1 Other  Totala TCGAbFLT3 9 32 9 1 3 2 1 1 5 63 xCSMD1 3 1 0 1 0 0 0 0 1 6WT1 3 5 5 0 1 1 0 0 2 17 xDDR2 2 0 1 0 0 0 0 0 0 3KRAS 2 3 2 2 0 0 1 0 1 11 xCALR 2 0 0 0 0 0 0 0 0 2REV3L 2 0 0 0 0 0 0 0 0 2TCERG1L 2 0 0 0 0 0 0 0 0 2FAM5C 1 2 0 1 0 0 0 0 1 5 xPHF6 1 1 2 1 0 0 1 0 1 7 xPTPN11 1 7 0 2 1 0 0 0 0 11 xASXL1 0 0 3 0 0 0 2 0 1 6CEBPA 0 2 12 1 0 0 1 0 3 19 xDNMT3A 0 29 15 3 0 0 5 0 4 56 xEZH2 0 0 1 0 0 0 0 0 3 4 xGATA2 0 1 6 0 0 0 0 0 0 7GRIK2 0 2 0 0 0 1 0 0 0 3IDH1 0 12 2 1 0 0 5 0 3 23 xIDH2 0 5 8 1 0 0 3 0 4 21 xKIT 0 1 0 1 3 0 0 2 1 8 xNRAS 0 5 4 2 2 1 0 0 2 16 xPHACTR1 0 0 0 1 0 0 0 0 2 0PLCE1 0 0 1 0 0 0 0 1 2 4RAD21 0 3 1 0 0 0 0 1 0 5 xRUNX1 0 0 12 1 0 0 2 0 7 22 xSMC1A 0 4 0 0 0 1 1 1 0 7 xSMC3 0 3 2 1 0 0 1 1 0 8 xSTAG2 0 3 3 0 0 0 0 0 1 7 xSUZ12 0 0 1 2 0 0 0 0 0 3TET2 0 2 9 1 0 0 1 1 4 18 xTP53 0 0 1 12 0 0 0 0 2 15 xU2AF1 0 1 2 0 0 0 2 0 3 8 xBCOR 0 0 3 0 0 0 0 0 0 3CHD4 0 0 0 2 0 0 0 0 0 2CTCF 0 0 2 0 0 0 0 0 0 2DDX41 0 0 2 0 0 0 0 0 0 2found  in ≥2 AML subgroups  found in only one AML subgroup associated to a specific AML subgroup with q≥0.01 associated to a specific AML subgroup with q≥0.05  FAM57B 0 2 0 0 0 0 0 0 0 2GIGYF2 0 0 0 0 0 0 0 2 0 2GJB3 0 0 0 2 0 0 0 0 0 2KDM3B 0 0 0 2 0 0 0 0 0 2KRT13 0 2 0 0 0 0 0 0 0 2PCDHA13 0 0 2 0 0 0 0 0 0 2SCN1A 0 0 0 2 0 0 0 0 0 2TBX15 0 2 0 0 0 0 0 0 0 2TCEB3B 0 0 2 0 0 0 0 0 0 2aTotal number of mutations identified for each mutated gene. bSignificantly mutated genes identified by The Cancer Genome Atlas Research network (TCGA).Letter to the Editor3& 2013 Macmillan Publishers Limited Blood Cancer Journal3 Insinga A, Cicalese A, Faretta M, Gallo B, Albano L, Ronzoni S et al. DNA damage instem cells activates p21, inhibits p53, and induces symmetric self-renewingdivisions. Proc Natl Acad Sci USA 2013; 110: 3931–3936.4 Wartman LD, Larson DE, Xiang Z, Ding L, Chen K, Lin L et al. Sequencing a mouseacute promyelocytic leukemia genome reveals genetic events relevant for diseaseprogression. J Clin Invest 2011; 121: 1445–1455.5 Greif PA, Yaghmaie M, Konstandin NP, Ksienzyk B, Alimoghaddam K,Ghavamzadeh A et al. Somatic mutations in acute promyelocytic leukemia (APL)identiﬁed by exome sequencing. Leukemia 2011; 25: 1519–1522.6 Welch JS, Westervelt P, Ding L, Larson DE, Klco JM, Kulkarni S et al. Use ofwhole-genome sequencing to diagnose a cryptic fusion oncogene. JAMA 2011;305: 1577–1584.7 Cancer Genome Atlas Research Network. Genomic and epigenomic landscapesof adult de novo acute myeloid leukemia. N Engl J Med 2013; 368: 2059–2074.8 McCormack E, Bruserud O, Gjertsen BT. Review: genetic models of acute myeloidleukaemia. Oncogene 2008; 27: 3765–3779.9 Gaur GC, Ramadan SM, Cicconi L, Noguera NI, Luna I, Such E et al.Analysis of mutational status, SNP rs16754, and expression levels of Wilms tumor1 (WT1) gene in acute promyelocytic leukemia. Ann Hematol 2012; 91: 1855–1860.10 Hammerman PS, Sos ML, Ramos AH, Xu C, Dutt A, Zhou W et al. Mutations in theDDR2 kinase gene identify a novel therapeutic target in squamous cell lungcancer. Cancer Discov 2011; 1: 78–89.11 Greif PA, Dufour A, Konstandin NP, Ksienzyk B, Zellmeier E, Tizazu B et al.GATA2 zinc ﬁnger 1 mutations associated with biallelic CEBPA mutationsdeﬁne a unique genetic entity of acute myeloid leukemia. Blood 2012; 120:395–403.12 Greif PA, Eck SH, Konstandin NP, Benet-Pages A, Ksienzyk B, Dufour A et al.Identiﬁcation of recurring tumor-speciﬁc somatic mutations in acutemyeloid leukemia by transcriptome sequencing. Leukemia 2011; 25:821–827.13 Grossmann V, Tiacci E, Holmes AB, Kohlmann A, Martelli MP, Kern W et al.Whole-exome sequencing identiﬁes mutations of BCOR in acute myeloidleukemia with normal karyotype. Blood 2011; 118: 6153–6163.14 Yan XJ, Xu J, Gu ZH, Pan CM, Lu G, Shen Y et al. Exome sequencing identiﬁessomatic mutations of DNA methyltransferase gene DNMT3A in acute monocyticleukemia. Nat Genet 2011; 43: 309–315.15 Vassiliou GS, Cooper JL, Rad R, Li J, Rice S, Uren A et al. Mutant nucleophosminand cooperating pathways drive leukemia initiation and progression in mice. NatGenet 2011; 43: 470–475.This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy ofthis license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/Supplementary Information accompanies this paper on Blood Cancer Journal website (http://www.nature.com/bcj)Letter to the Editor4Blood Cancer Journal & 2013 Macmillan Publishers Limited

https://air.unimi.it/retrieve/handle/2434/229107/298692/357.%20Acute%20promyelocytic%20%20BLLOD%20CANCER%202013.pdf

Acute promyelocytic leukemias share cooperative mutations with other myeloid-leukemia subgroups

Abstract

Similar works

Full text

Available Versions

ART

AIR Universita degli studi di Milano