www.cemm.at Genetic causes of myeloproliferative diseases: Stratification of patients and new therapeutic targets Robert Kralovics CeMM and Department of Laboratory Medicine, Medical University of Vienna
www.cemm.at
Genetic causes of myeloproliferative diseases:Stratification of patients andnew therapeutic targets
Robert KralovicsCeMM andDepartment of Laboratory Medicine,Medical University of Vienna
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.
Myeloproliferative Neoplasms
MPN driver mutations active JAK/STAT signaling
MPN: All Roads Lead to JAK-STAT
The influence of mutations on PMF survival
• Significantly better survival of CALR mutation positive PMF (P<0.001)
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
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
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
Clonal reconstruction in post-MPN AML
Mutation landscape in post-MPN AML
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)
Variant calling: TruSight and RNA-seq
J Grinfeld et al. N Engl J Med 2018;379:1416-1430.
Pontus Lundberg et al. Blood 2014;123:2220-2228
Clinical significance of mutations in MPN
Clinical significance of mutations in MPN
Lundberg et al., Blood, 2014
Clinical significance of mutations in MPN
Lundberg et al., Blood, 2014
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
”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
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
Mutant CALR as a target for therapy
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
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
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
Frequently mutated genes in spliceosome
SF3B1 mutations cause splicing defects: neoantigens?
Neo-antigensfrom splicing defects
SF3B1-K700E
in frame
out of frame
RNA
Protein
Analysis of splicing junctions
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
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
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