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