characterizing the sos1 transcript in b-all• jorge, r. et al. hsos1 contains a new amino-terminal...
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CharacterizingtheSos1transcriptinB-ALL
DipM.ShuklaMS2
FarrarLabT35NIHSummerImmunologyResearchProgram
WhatisB-cellAcuteLymphoblasticLeukemia(B-ALL)?
NormalBloodSmear B-ALLBloodSmear
ProjectOverviewSBxStat5b-ca
Leukemia
LeukemiaDevelopment:
SB-inducedSos1truncations:
~70days
ProjectOverviewPax5+/- xEbf1+/-
(PEOnly)SBxPax5+/- xEbf1+/-
(SBxPE)
LeukemiaLeukemia
SBxStat5b-ca
Leukemia
LeukemiaDevelopment:
SB-induced SpontaneousSpontaneousSos1truncations:
~70days ~300days~200days
Sos1HumanPathogenesis
• NoonanSyndrome– developmentaldisordercharacterizedbyshortstature,facialdysmorphia,congenitalheartdefects,andskeletalanomalies.• Cause:activatedRAS-MAPKsignaling
• ~20%ofpatientsinRobertsetal.2007study
• Hereditarygingivalfibromatosistype1–characterizedbybenigngingivaloutgrowth• Cause:cytosineinsertioninexon21->frameshiftandtruncation(lossofPRmotif=Grb2-bindingsites)
-RTK-CytokineReceptorsà Sos1à Ras activation-GPCR
Aims
• Aim1:Characterizethetruncated,Sos1 transcriptinotherleukemiamodels:• SBxPax5+/- xEBF1+/- (SleepingBeauty)• Pax5+/- xEBF1+/- (non-SleepingBeauty)
• Aim2:Investigatepost-transcriptionalregulationoftheN-terminallytruncated,3’overexpressedSos1 transcript.• Luciferaseassay
Aim1:5’Sos1RACE
5’RACEPrimer
750bp
Aim1:5’Sos1RNASequencingAnalysis
SCALE
39
33
24635
170ßPooledSample
Exons
RepresentativeIndividualMice
Normalexpression
Increasedexpression
Increasedexpression
Aim1:5’F420015M19RikRNASequencing
SCALE
10
14
112
21
65ßPooledSample
RepresentativeIndividualMice
Increasedexpression
Increasedexpression
Aim1:PCR-BasedValidationofRACEResults
PCRDesign
F420015M19RikFullLength
Sos1FullLength
F420015M19Rik– Sos1Fusion
Aim1:PCR-BasedValidationofRACEResults
Aim1:Sos1 LocusMap,Chr.17
~43Kb
~84Kb
F420015M19Rik
Aim1:3’Sos1SequencingAnalysis
SCALE
200
200
200
200
200ßPooledSample
RepresentativeIndividualMice
Aim1:3’Sos1RACE
3’RACEPrimer
~1500bp
~1083bpsof3’UTR
Aim2:Sos13’UTRCharacterization3’UTRLuciferaseAssay
(FireflyOnly)
Vectorsize: ~7.4Kb ~8.4Kb ~11.8Kb
3’UTRLuciferaseAssay(Normalized)
3’UTRLuciferaseAssay(RenillaOnly)
Conclusions:TheTruncationDefined
FutureDirections
• PursuesequencingoftheSos1 locusonChr.17.• Cloneandexpressthefulltruncatedtranscript• Functionalimpactof:RIKENandthe3’UTRtruncation
• InvestigatehumanB-ALLforthepresenceofatruncatedSos1transcript
Acknowledgements
FarrarLabMembers:• MichaelA.Farrar,PI• LynnM.Heltemes-Harris• GreyHubbard• CaseyKaterndahl• DavidOwen
Funding:• NIHT35SummerImmunologyProgram
References
• http://www.cancer.org/cancer/leukemiainchildren/detailedguide• http://omim.org/entry/182530• Heltemes-Harris,L.M.etal. SleepingBeautytransposonscreenidentifiessignalingmodulesthatcooperatewithSTAT5activationtoinduceB-cellacutelymphoblasticleukemia.Oncogene 35,3454–3464(2016).
• Heltemes-Harris,L.M.etal. Ebf1 orPax5 haploinsufficiency synergizeswithSTAT5activationtoinitiateacutelymphoblasticleukemia.TheJournalofExperimentalMedicine 208, 1135–1149(2011).
• Jorge,R.etal. hSos1ContainsaNewAmino-terminalRegulatoryMotifwithSpecificBindingAffinityforItsPleckstrin HomologyDomain.JournalofBiologicalChemistry 277, 44171–44179(2002).
• Rojas,J.M.,Oliva,J.L.&Santos,E.MammalianSonofSevenless GuanineNucleotideExchangeFactors:OldConceptsandNewPerspectives.Genes&Cancer 2, 298–305(2011).
• Tartaglia,M.etal. Gain-of-functionSOS1mutationscauseadistinctiveformofNoonansyndrome.NatureGenetics 39, 75–79(2007).
miRNATargetSitesin3’Sos1UTR
mmu-miR-3105-3pmmu-miR-6407mmu-miR-7242-3pmmu-miR-5623-5pmmu-miR-7013-3pmmu-miR-3084-5pmmu-miR-126b-5pmmu-miR-494-3pmmu-miR-666-3pmmu-miR-124-3p
miRNAsTargeting:WithinTruncationOnly
mmu-miR-880-5pmmu-miR-7061-5pmmu-miR-6981-3pmmu-miR-6945-5pmmu-miR-705mmu-miR-7008-5pmmu-miR-6993-5pmmu-miR-6935-5pmmu-miR-6922-5pmmu-miR-7674-5p
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