genetic causes of myeloproliferative...
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www.cemm.at
Genetic causes of myeloproliferative diseases:Stratification of patients andnew therapeutic targets
Robert KralovicsCeMM andDepartment of Laboratory Medicine,Medical University of Vienna
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대한혈액학회 Korean Society of Hematology
COI disclosureName of author : Robert Kralovics
I currently have, or I have had in the past two years, an affiliation or financial interest with business corporation(s):
(1) Consulting fees, patent royalties, licensing fees : Yes, AOP Orphan Pharmaceuticals, Pharma Essentia, Qiagen
(2) Research fundings: No
(3) Others No
Use the following slide to disclose any conflicts of interest
Form B: with conflict of interest to declare.
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Myeloproliferative Neoplasms
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MPN driver mutations active JAK/STAT signaling
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MPN: All Roads Lead to JAK-STAT
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The influence of mutations on PMF survival
• Significantly better survival of CALR mutation positive PMF (P<0.001)
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Genetic changes in MPN
Acute leukemia
Myelodysplastic
Normal
Myeloproliferative
phenotype
diagnosisof MPN
Germline predispositions
JAK2-GGCCTERTRBBP6LNKATG2B GSKIPdel-IL1RAP
Clonaldrivers
del20qdel13qTET2DNMT3A
MPNdriver mutations
JAK2MPLCALR
Progression associatedmutations
TP53 loss, MDM4 amplificationPRC2 complex loss of functionASXL1 lossCUX1 deletionsRUNX1 loss of functionSF3B1, Splicing factor mutations
CHIPphase
timetherapy
MPNphase
MDS/AMLphase
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Models of leukemic transformation in MPN
JAK2-V617F
RUNX1
JAK2-V617Fpositive
AML
FLT3/NPM1/NRAS
JAK2-V617Fnegative
AMLde novo like
TP53
JAK2-V617F
Mono-clonal Bi-clonal
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Exome sequencing in post-MPN AML
Elisa Rumi, Mario Cazzola Tiina Berg, Klaudia Bagienski
T cells as the control tissue
Chronic phase sample
Leukemic sample
Exomesequencing
Accelerated phase sample
14 x
Chronic phase MPN
Accelerated phase MPN
Secondary AML
Mutations causing MPN
Mutations causing disease progression
Mutations causing leukemic transformation
RNA-seq for fusions detection
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Clonal reconstruction in post-MPN AML
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Mutation landscape in post-MPN AML
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Mutation profiling in MPN• (Paired) whole exome sequencing (expensive, discovery base)
• Targeted re-sequencing:
• Skoda panel (104 genes)
• Illumina TruSight Myeloid Panel (55 genes)
• Welcome Trust panel (69 gene panel)
• RNA-seq (universal diagnostic platform?)
• Gene fusions, mutations, splicing abnormalities
• Loss of bi-allelic expression (X-based clonality, LOH)
• Differential expression (signatures of MPN subtypes & mutations, cellular composition of whole blood)
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Variant calling: TruSight and RNA-seq
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J Grinfeld et al. N Engl J Med 2018;379:1416-1430.
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Pontus Lundberg et al. Blood 2014;123:2220-2228
Clinical significance of mutations in MPN
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Clinical significance of mutations in MPN
Lundberg et al., Blood, 2014
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Clinical significance of mutations in MPN
Lundberg et al., Blood, 2014
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Genetic changes in MPN
Acute leukemia
Myelodysplastic
Normal
Myeloproliferative
phenotype
diagnosisof MPN
Germline predispositions
JAK2-GGCCTERTRBBP6LNKATG2B GSKIPdel-IL1RAP
Clonaldrivers
del20qdel13qTET2DNMT3A
MPNdriver mutations
JAK2MPLCALR
Progression associatedmutations
TP53 loss, MDM4 amplificationPRC2 complex loss of functionASXL1 lossCUX1 deletionsRUNX1 loss of functionSF3B1, Splicing factor mutations
CHIPphase
timetherapy
MPNphase
MDS/AMLphase
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”Deep” molecular response in MPN
patient
Follow-up time
(weeks)Sequencing
depth
Follow-up time
(weeks)%V617F(NGS)
03001 0 22756 0 42.5%03001 131 12135 131 * 4.9%03001 217 17641 217 1.7%
05010 0 13009 0 44.1%05010 68 13113 68 * 2.1%05010 171 15785 171 0.8%
time
mutational burden
JAK2-V617F
MPN (liquid tumor)
CurativeTherapy
CompleteMolecularResponse
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Genetic changes in MPN
Acute leukemia
Myelodysplastic
Normal
Myeloproliferative
phenotype
diagnosisof MPN
Germline predispositions
JAK2-GGCCTERTRBBP6LNKATG2B GSKIPdel-IL1RAP
Clonaldrivers
del20qdel13qTET2DNMT3A
MPNdriver mutations
JAK2MPLCALR
Progression associatedmutations
TP53 loss, MDM4 amplificationPRC2 complex loss of functionASXL1 lossCUX1 deletionsRUNX1 loss of functionSF3B1, Splicing factor mutations
CHIPphase
timetherapy
MPNphase
MDS/AMLphase
CURRATIVE THERAPY
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Mutant CALR as a target for therapy
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MPN-specific antigens: mutCALR
• mutCALR (35% PMF, 25% ET patients)• trafficked to the cell surface, secreted to serum• immunogenic in mice, rabbits, humans (?)
PLAsurface stain MPL/CALR complexUT7-TPO CRISP/Cas9 mutCALR
anti-CALRanti-mutCALR
anti-MPLanti-mutCALR
wtmutCALR
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C-terminal domain of mutCALR is immunogenic
(RR)KMSPARPRTSCREACLQGWTEA (24/22 aa peptide)Recombinant del52 and ins5
Hybridoma fusion
Hybridoma mAb7 mAb clones
polyclonal Ab: SAT601Monoclonal 15 clones
Affinity purification (w/peptide)
mutant-C-termType 1 mutation
Type 2 mutation
52 bp deletion
5 bp insertionmutant-C-term
STOP
STOP
5bp
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Specificity of antibodies: FACS
Characterization of the rabbit/mouse IgG2a
CB16 showed the best binding profile in this readouts.
1°Ab: 0,125µg Ab / test
UT7/TPO cells
a-mouse /rabbit-PE
Purified Abs (IgG)
CALR
coun
ts
CALRmut/mut
CALRwt/wt
isotype mIgG2a
CB_1 CB_2 CB_3 CB_4 CB_5
CB_7 CB_10 CB_11 CB_15 CB_16
CB_17 CB_18 CB_20 CB_21 CB_22
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Frequently mutated genes in spliceosome
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SF3B1 mutations cause splicing defects: neoantigens?
Neo-antigensfrom splicing defects
SF3B1-K700E
in frame
out of frame
RNA
Protein
Analysis of splicing junctions
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Predicted peptide derived for splice-in eventsGene name Novel peptide (aa) Novel aa lengthUBL7 GPHQCPLVSSQGRPSPMISSAKPYSMPFRPLGSPAFRASGSPSCSSYVTWASRTMS* 56
CBY1 HLAHSWIDQPGRWSWAWNTDPRLSESTAESHLMEKELDELRISRKRK* 47TBRG1 DLRFSSEENMQEKENGGRCSQAGSAHCPGSLRTACVPHRTRGSNSI* 46FLT3LG HPLPCSLSPPAAAAELGTQWDPGLLLPTQPHLLRLRCQNP* 40TTI1 VVLFFQRNSQSLPKWMRMTPVQMWSHHCHCRSK* 33ACOT11 RLCLRFSRPKSTADLDTFKTPETLS* 25MAP3K7 VCAFLSQCCMVLNHCHIILLPTQ* 23RWDD4 FFISDGTRSITLYL* 14OXA1L FCIFQVQSLPAA* 12
PPM1M MVFTGSQSWPRC* 12
TMCC2 PLCPCRRPWSPA* 12ZFYVE27 VFLILQVHGTQ* 11C7orf43 LCLPPGRAV* 9EFEMP2 APLPWHRH* 9ZNF397* TFPFCFL 7PRPF38A* LTSLFY 6VWA7* VPFPNP 6SIRPD* VLPLSA 6
CC2D1A* PRPPQ 5
HINT2 AQFQ* 4CALR RRRRQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWTEA* 51JAK2-V617F HLVLNYGVCFCGDENILVQ 1MPL-W515K AVLGLLLLRKQFPAHYRRL 1MPL-W515L AVLGLLLLRLQFPAHYRRL 1
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New targets for therapy• Mutant CALR is a neoantigen
• Anti-mutant CALR antibodies: ADCC, ADC, CAR-T
• SF3B1 mutated MPN: splicing defects result in novel protein sequences
• MPN associated neoantigens: targeted vaccines
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CeMM RK labHarini NivarthiFiorella SchischlikRoland JägerElisabeth FuchsRouchen Jia
Acknowledgements
CeMMChristoph Bock University of Pavia
Mario CazzolaIlaria CasettiDaniela PietraElisa Rumi
Med.Univ. ViennaHeinz GisslingerBettina GisslingerMartin Schalling
Ludwig Cancer ResearchBrusselsStefan Constantinescu