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Flt3-ITD mutations in a mouse model

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LETTERTOTHEEDITORFlt3-ITDmutationsinamousemodelofradiation-induced

acutemyeloidleukaemia

Leukemiaadvanceonlinepublication,6January2012;doi:10.1038/leu.2011.377Acutemyeloidleukaemia(AML)isoneofthemostcommonmalignanciesseentooccurinhumanpopulationsexposedtoionisingradiation.1Mousemodelshavebeenwidelyusedforquantitativeandmechanisticstudiesofradiationleukaemogen-esis;further,thereisasimilarityinthehistopathologicalchangesfoundinhumanAMLandmouseAML.Themajorityofmouseradiation-inducedAMLs(rAMLs)carrydeletionsofchromosome2,andtheSfpi1/PU.1haematopoietictranscriptionfactorislocatedwithinthecommonlydeletedchromosome2region.2InmostrAMLsandrAMLcelllines(70%),Sfpi1/PU.1suffershemizygouslossaccompaniedbypointmutationsintheregionencodingtheETSDNA-bindingdomain,leadingtobasesubstitutionsattheR235residueoftheprotein.2-4Sfpi1/PU.1mutationshavebeenidentifiedinhumanAML,butthesearerare;incontrast,themostcommonmutationsinhumanAMLinvolvetheFlt3receptortyrosinekinase.Mostofthesemutationsareinternaltandemduplication(Flt3-ITD)of3--400bp5thatresultinligand-dependentdimerisationandreceptorphosphorylation.ThepresenceofaFlt3-ITDappearstoconferamoreseverephenotypeandapoorprognosisforAMLsufferers.Themajorityoftheremainingnon-ITDmutationsinFlt3occurinthesecondtyrosinekinasedomain,beingmostlypointmutationswithincodon835orthedeletionof836.6Inthisstudy,forthefirsttime,weidentifyFlt3mutationsinamurinemodelofrAML,providingadirectmechanisticlinkbetweenthemouseandhumanAML.Inall,30mouserAMLcaseswerescreenedforFlt3-ITDusingPCR,20onanF1CBA/HxC57BL/Liabackgroundand10onaninbredCBA/Hbackground.AllbutnineoftheCBA/HandrAMLshavebeendescribedpreviously,2,4thenewAMLsamplesweregeneratedfrommiceirradiatedwith3GyX-raysatMRCHarwell,Oxfordshire,UK.AnimalswerebredandmaintainedinaccordancewiththeUKAnimals(ScientificProcedures)Act1986.ForeachAML,DNAwasextractedfromleukaemicspleentissueusingaMagNApurecompactinstrument(RocheDiagnosticsGmbH,Mannheim,Germany).Flt3-ITDmutationsweredetectedusingPCRwithprimersdesignedtoamplifytheregioncoveringexons14and15,syntenictotheregioncontainingITDinhumanFLT3(F:50-GCAATTTAGGTACGAGAGTCAGC-30,R:50-CTTTTAGCATCTTCACCGCCACC-30;Sigma-Aldrich,Poole,UK).ThepresenceofFlt3-ITDwasindicatedbyanincreaseinampliconsizeandthenconfirmedbysequencing.Figure1aprovidesarepresentativeexampleof

PCR

Flt3-ITD:del2:nt 703:_333bpgagtacttctacgttgacttcaggaactatgaatatgaccttaagtgggagttcccgagagagaacttagagttt.....gccacggcctatggcattagtaaaacgggagtctcaattcaggtggcggtgaagatgctaaaagaEYFYVDFRDYEYDLKWEFPRENLEFGKVLGSGAFGVMNATAYGISKYGVSIQVAVKMLKAMLB, nt 2022acttctacgttgacttcagggactatg

acttctacgttgacttcaggaactatgaatatgaccAMLC, nt 2031

gtgaagatgctaaaagaaAML A, nt 2267AML B aa.598DFYVDFKDY

HFYVDFRDYEYAML C aa.601DVKMLKEAML Aaa.642nt 1990nt 2065

nt 2283nt 2220

aa.588AMLFAMLEAMLDAMLBAMLAAMLCBrainC57CBASample:

--CCCCCCCTT------+

+

-----+++

CBA

CBA

CBA

Figure1.(a)RepresentativeFlt3-ITDPCRanalysisofapanelofmurinerAMLsrunona2%agarosegel.Thenormalampliconsizeis333bp.PresenceofFlt3-ITDisindicatedbyanadditionallargerbandonthegel.Thegelisloadedasindicatedabovetheimage.‘CBA’and‘C57’refertonormalspleenDNAfromCBA/HandC57BL/Lia,respectively;‘Brain’referstobraintissuefromtheanimalinwhichAMLAdevelopedandrepresentsanormaltissuecontrol;andAMLsA--FrefertoindependentAMLsamples.ThegeneticalterationsidentifiedintheAMLsaresummarisedbeloweachsample(Flt3-ITD:þ,presenceandÀ,absence;del2:þ,presenceandÀ,absence;nucleotide703baseatnucleotide703ofSfpi1/PU.1.CiswildtypeandTismutant(theTalleleleadingtoarginine235beingconvertedtocysteineinSfpi1/PU.1protein).(b)CBAFlt3Exon14and15sequenceillustratingthethreeinsertionsitesandsequenceofFlt3ITDinAMLsA--C.ITDlengthsforeachare18,27and36bp,respectively.(c)PredictedFlt3protein

sequence

ofAMLsA--Cshowinginsertedaminoacids.Leukemia(2012)1-2&2012MacmillanPublishersLimitedAllrightsreserved0887-6924/12www.nature.com/leuanalysisofevents(Flt3-ITD)inmouserAML.Flt3-ITDs,ranginginsizefromapproximately10to30bp,wereidentifiedinthreecases.Ofthese,twoAMLswereheterozygousforthemutationandonehomozygous.DNAsequencingofPCRproductsconfirmedthepresenceofITDsinthesethreeAMLs:A,BandCof18,27and36bpinlength,respectively,atindependentinsertionsites(Figure1b).TheseITDswerepredictedbyBLASTtoleadto6--12novelaminoacidsbeinginsertedinframe(Figure1c).ThemajorityofrAMLsinthisstudy(23/30,77%)carrychromosome2deletionsasidentifiedbyloss-of-heterozygosityorinsituhybridisationmethods.2,7Ofthese,most(19/23,82%)carrySfpi1/PU.1mutationsaffectingR235(Table1).However,allFlt3-ITDoccurredinrAMLsinwhichnoSfpi1/PU.1involvementwasidentified,eitheraschromosome2deletionsorasSfpi1/PU.1exon5mutations.OutofsevenrAMLswithoutSfpi1/PU.1alterations,threehavepresentedwithFlt3-ITDs(B43%).TocheckforthepresenceofsmallFlt3-ITDmutationsinthoseAMLsthatwereassessedasnegativefortheITDmutationsusingPCRandgel-basedassays,Flt3exon14/15PCRproductsfromasampleof11AMLs(9fromCBA/Hanimalsand2fromCBAxC57BL/6Liaanimals;8withchromosome2deletionsand3withoutchromo-some2deletions)weresequenced.Noneofthesewasfoundtohaveanyexon14/15Flt3DNAsequencealterations.Therefore,noITDswerefoundinanyoftheserAMLswithchromosome2deletions(withorwithoutSfpi1/PU.1exon5mutations).StatisticalanalysisusingFisher’sexacttesttocompareFlt3-ITDandchromosome2deletionstatusindicatedthattheabsenceofAMLswithbothFlt3-ITDandchromosome2deletionswasstatisticallysignificant(P¼0.0086).ThesefindingssuggestthattheinvolvementofFlt3-ITDandSfpi1/PU.1ismutuallyexclusiveinthismodel.NophenotypicdifferencesbetweenAMLswiththeinvolvementofFlt3-ITDandSfpi1/PU.1havebeenobserved.ToruleoutthepossibilityofpointmutationswithinthesecondtyrosinekinasedomainofFlt3,wescreenedsamplesthatshowednoSfpi1/PU.1involvementusingPCRwithprimerscoveringexon20syntenictothemutatedregioninhumanAML(F:50-AGAAGAGGCTGGCAGAAGAA-30,R:50-CCGTAGGACCAGACGTCACT-30).Fragmentsweresequencedalongwithcorrespondingmatchedbrains(negativecontrols).NocasesofpointmutationswereidentifiedinanyoftherAMLs,indicatingthatFlt3-ITDistheonlyFlt3mutationpresentintheserAMLs.ThisstudyhasthereforeidentifiedFlt3-ITDmutationsinapproximately10%ofmouserAMLsforthefirsttime,butonlyincaseswithoutSfpi1/PU.1involvement.TheFlt3-ITDsareinthesizerangeidentifiedinhumans6andalthoughrareinoursamples,showgreaterparallelstohumanAML.Thethreeinsertionsarepredictedtoleadto6--12novelaminoacidsbeinginsertedinframeinFlt3withtwooftheseinsertionsinthetyrosine-richjuxtamembranedomain;theseITDsarethereforesimilartothoseseeninhumanAMLcases.8ThefindingsalsosuggestthatradiationcancauseFlt3-ITDmutationsdirectly.TherearethereforetwoidentifiedpathwaysleadingtorAMLinthemouse,themostcommoninvolvingloss/mutationofSfpi1/PU.1andtheotherinvolvingmutationofFlt3.FutureworkwillhelpestablishthemechanisticrelationshipbetweenthemouserAMLsandthehumandisease.TheFlt3-ITD-carryingAMLs,althoughrare,maybeofvalueinthepreclinicalevaluationofFLT3inhibitorsastherapeuticagentsformyeloidleukaemia.

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坦度螺酮化学结构式

坦度螺酮化学结构式

坦度螺酮化学结构式

引言

坦度螺酮（Tandutinib）是一种具有潜在抗肿瘤活性的分子化合物。它属于一类被称为酪氨酸激酶抑制剂的药物，通过抑制酪氨酸激酶的活性来干扰细胞信号传导途径，从而抑制肿瘤细胞的生长和扩散。本文将详细介绍坦度螺酮的化学结构式、药理作用机制以及相关的研究进展。

化学结构式

坦度螺酮的化学结构式如下所示：

坦度螺酮的化学名为N-(5-((5-氟-2-氧代苯基)氨基)-1,3,4-噻二唑-2-基)苯胺。它的化学式为C18H13FN4OS，相对分子量为358.39。从结构式可以看出，坦度螺酮由苯胺基团和噻二唑环组成，并且在苯胺基团上有一个氟和一个氧代苯基取代基。

药理作用机制

坦度螺酮作为酪氨酸激酶抑制剂，主要通过抑制细胞内的酪氨酸激酶活性来发挥抗肿瘤作用。酪氨酸激酶是一类激酶酶家族，它在细胞信号传导途径中起着重要的调节作用。酪氨酸激酶的异常活化与多种肿瘤的发生和发展密切相关。

坦度螺酮通过与酪氨酸激酶结合，竞争性地阻断其活性位点，从而抑制其催化活性。这种抑制作用可以阻断细胞内信号传导途径中酪氨酸激酶的参与，进而干扰肿瘤细胞的生长和扩散。此外，坦度螺酮还可能通过调节细胞周期、诱导细胞凋亡、抑制血管生成等多种机制发挥抗肿瘤作用。

研究进展

近年来，对坦度螺酮的研究已经取得了一些进展。以下是一些相关研究的概述：

1. 抗肿瘤活性研究：多个研究表明，坦度螺酮具有抗肿瘤活性，对多种肿瘤细胞系具有抑制作用。例如，在乳腺癌、肺癌和胰腺癌等肿瘤模型中，坦度螺酮能够抑制肿瘤细胞的增殖和迁移能力。

2. 耐药性研究：一些研究表明，坦度螺酮可能对某些耐药性肿瘤具有反转作用。例如，在结直肠癌细胞中，坦度螺酮可以增强顺铂等化疗药物的敏感性。

3. 临床试验：目前，坦度螺酮已经进入了临床试验阶段。一些早期的临床试验结果显示，坦度螺酮具有一定的抗肿瘤活性，并且耐受性良好。然而，仍需要更多的临床试验来评估其疗效和安全性。 结论

PML-RARa融合基因BCR3亚型急性早幼粒细胞白血病的临床研究

PML-RARa融合基因BCR3亚型急性早幼粒细胞白血病的临床研究

PML-RARa融合基因BCR3亚型急性早幼粒细胞白血病的临床研究

王欣;刘林;陈建斌;王建渝;肖青

【摘 要】Objective To assess the efficacy of combination therapy with all-trans-retinoic acid (ATRA ) and arsenic trioxide (ATO) in treating patients

with acute promyelocytic leukemia (APL) .Methods A retrospective study

was conducted to evaluate the efficacy of combining ATO with ATRA

based induction therapy ,followed by 3 courses of consolidation

chemotherapy and 2-year sequential ATO/ATRA maintenance therapy in

newly diagnosed APL ,and the efficacy between high risk group and

low/inter-mediate risk group ,also between different PML-RARa isoform

sub-group were compared .Results In high risk group and low/in-termediate risk group ,the complete remission(CR)rates were 70 .0% and

96 .9% (P=0 .04) ,respectively ;the 3 years overall sur-vival rates(OS) and

DNMT3A R882突变在成人急性髓系白血病中的研究进展

DNMT3A R882突变在成人急性髓系白血病中的研究进展

DNMT3A R882突变在成人急性髓系白血病中的研究进展

王蕾;杨艳丽

【摘 要】DNA methyltransferase 3A (DNMT3A), one of important DNA

methyltransferases, is closely related to the occurrence and development

of hematologic malignancies. In recent years, studies have founded that

there are more frequently DNMT3A R882 mutations in adult acute myeloid

leukemia (AML), which becomes a hot topic. DN-MT3A R882 mutation

decreases the DNMT3A enzyme activity, makes genome-wide showing

DNA hypomethylation and promotes the occurrence and development of

adult AML by altering the structure of DNMT3A. The long-term pres-ence

of DNMT3A R882 mutations in complete remission (CR) of adult AML is a

biological marker of poor prognosis, and it also indicates that routine

monitoring of minimal residual disease (MRD) can not be used as a

molecular marker of R882 mutation. Now this paper reviews the advances

儿童核心结合因子相关性急性髓系白血病中C-KIT基因突变分析

儿童核心结合因子相关性急性髓系白血病中C-KIT基因突变分析

儿童核心结合因子相关性急性髓系白血病中C-KIT基因突变分析

吴珺;于水;陆爱东;左英熹;贾月萍;张乐萍

【摘 要】目的 分析C-KIT基因突变与儿童核心结合因子相关性急性髓系白血病(CBF-AML)临床特点及预后的关系.方法 回顾分析CBF-AML患儿中C-KIT基因突变情况,分析伴随C-KIT基因突变患儿的临床和实验室特点及预后.结果 86例CBF-AML患儿中,35例(40.7％)C-KIT基因突变阳性.inv(16)/CBFβ-MYH11患儿中C-KIT基因8号外显子突变检出率明显高于t(8;21)/RUNX1-RUNX1T1患儿,差异有统计学意义(P<0.01).与阴性组相比,C-KIT阳性组患儿在性别、年龄、初诊白细胞计数、初诊骨髓幼稚细胞比例、伴随其他基因、髓外白血病、第一疗程完全缓解率等方面的差异均无统计学意义(P>0.05).C-KIT基因突变阳性及阴性组患儿5年无事件生存率分别为82.8％ 和76.4％,总生存率分别为89.3％和85.9％,组间差异均无统计学意义(P>0.05).结论 C-KIT基因突变在儿童CBF-AML中发生率高,对CBF-AML患儿的预后无明显影响.

【期刊名称】《临床儿科杂志》

【年(卷),期】2019(037)003

【总页数】5页(P177-181)

【关键词】核因子;急性髓系白血病;C-KIT基因;预后;儿童

【作 者】吴珺;于水;陆爱东;左英熹;贾月萍;张乐萍 【作者单位】北京大学人民医院儿科 北京 100044;北京大学人民医院儿科 北京

100044;北京大学人民医院儿科 北京 100044;北京大学人民医院儿科 北京

100044;北京大学人民医院儿科 北京 100044;北京大学人民医院儿科 北京

100044

【正文语种】中 文

核心结合因子相关性急性髓系白血病（core binding factor-acute myeloid

leukemia, CBF-AML）是指一组以染色体[t(8;21)(q22;q22)]或[inv(16)(p13q22)/t(16;16)(p13;q22)]异常为特征的白血病亚型，其相应形成的融合基因为RUNX1-RUNX1T1（既往命名为AML1-ETO）和CBFβ-MYH11。CBF-AML是儿童AML中最常见的一种亚型，发生率约20%～22%[1]。CBF-AML患儿对化疗敏感性好，完全缓解（complete remission，CR）率及总生存（overall survival，OS）率高，总体预后相对较好[2]。