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