Chicago, Sept 9, 2017

Society of Hematopathology Workshop Molecular Genetics of Hematopoietic Neoplasms

Kojo S. J. Elenitoba-Johnson MDPeter Nowell MD Professor

Director, Division of Precision and Computational DiagnosticsDepartment of Pathology and Laboratory Medicine

University of Pennsylvania

Novel Insights into the Molecular Pathogenesis of T-cell Lymphomas

Disclosure

GENOMENON: Co-Founder

OutlineBackground

• WHO classification of mature T cell lymphoma

Recent updates in genetics of T cell lymphoma

• PTCL/AITL• CTCL/SS• ALCL

• Pathobiologic and therapeutic implications

• Conclusions

OutlineBackground

• WHO classification of mature T cell lymphoma

Recent updates in genetics of T cell lymphoma

• PTCL/AITL• CTCL/SS• ALCL

• Pathobiologic and therapeutic implications

• Conclusions

2016 World Health Organization

Mature T-cell neoplasms

Cutaneous

Primary cutaneous CD8+ aggressive epidermotropic

cytotoxic TCL*

Primary cutaneous CD4+ small/medium T cell

lymphoproliferative disorder*

Primary cutaneous γδ TCL

Primary cutaneous ALCL

Primary cutaneous CD30+ T-cell Disorders

Sézary syndrome

Mycosis fungoides (MF)

ExtranodalNK/TCL nasal type

Enteropathy-associated TCL

Hepatosplenic TCL

Subcutaneous Panniculitis-Like TCL

Systemic EBV+ T-cell lymphoproliferative

disorder of childhood

Hydroa vacciniforme-like

NodalPeripheral TCL-NOS

Anaplastic large Cell lymphoma (ALK +)

Anaplastic Large Cell lymphoma (ALK -)

AngioimmunoblasticTCL

LeukemicAdult T-cell

leukemia/lymphoma

T-cell prolymphocyticleukemia

T-cell large granular lymphocytic leukemia

*Provisional entity.

Aggressive NK-cell leukemia

Chronic lymphoproliferativedisorder of NK cells*

Monomorphic epitheliotropic intestinal T-

cell lymphoma*

Primary cutaneous acralCD8+ TCL*

Follicular T cell lymphoma*

Nodal peripheral T cell lymphoma with TFH

phenotype*

Breast implant-associated ALCL*

Relative frequencies of mature T-cell lymphomas

International T-Cell Lymphoma Project

1. Vose JM et al. J Clin Oncol. 2008;26(25):4124-4130.

Subclassification of PTCL and Survival

International T-Cell Lymphoma Project. J Clin Oncol. 2008;26(25):4124-4130.

Molecular diagnostic signatures of TCL subgroups

8

Iqbal J et al., Blood Rev 2016

Diagnostic and biologic gray zones in PTCL

Markers to consider:

• Morphologic, immunophenotypic and genetic overlap

PTCL-NOS

AITL

ALCL, ALK-negative

Herrera AF et al. Cancer. 2014;120(13):1993-1999.

Herrera AF et al. Cancer. 2014;120(13):1993-1999.

OutlineBackground

• WHO classification of mature T cell lymphoma

Recent updates in genetics of T cell lymphoma

• PTCL/AITL• CTCL/SS• ALCL

• Pathobiologic and therapeutic implications

• Conclusions

Peripheral T-cell Lymphoma/Angioimmunoblastic T-cell Lymphoma

Mutational landscape of PTCL and AITL

12

Cell-type specific mutations in AITL

13 Nguyen and Chiba, Blood Cancer J 2017

Angioimmunoblastic T-cell Lymphoma

Cortes JR and Palomero T. Current Opinions in Hematology 2016

Summary

16

Mutations in RHOA 70% of AITL; 20%PTCL

Loss of function of TET2 and DNMT3A frequent

IDH2 R172H is highly enriched in AITL and associates with TET2mutations

Mutations targeting the TCR signaling pathway (CD28, Fyn) contribute to PTCL/AITL pathogenesis

Current model for pathogenesis of AITL suggests initial TET2/DNMT3A mutation followed by RHOA or IDH mutation with lineage commitment to TFH cells

SummaryTargeted therapy for T-cell lymphoma

Cortes JR and Palomero T. Current Opinions in Hematology 2016

Cutaneous T-cell LymphomaMycosis Fungoides/Sezary Syndrome

• Malignancy of post-thymic T-cells characterized by triad of erythroderma, lymphadenopathy, pruritus

• Complex karyotypes without recurrent structural variants

19

SÉZARY SYNDROME

Whole Genome Sequencing Confirms Complexity of Sézary Syndrome Genomes

20Kiel M, et al. Nat Commun 2015

Exome Sequencing Identifies Regions of Recurrent Aneuploidy in 74 Sézary Patients• 1p36.11• 3p21.31• 9p21.3• 10p11.22• 10q23.31• 10q21.2• 13q14.2• 17p• 19p

TP53

CDKN2A21

RB1

PTEN

Loss of Function of ARID1A in Sézary Genomes (25/62; 40%)

22

ARID1a inactivation detected by immunocytochemistry

Loss of Function of TET1/2 in Sézary Genomes (38/62; 61%)

24

Epigenetic Modifiers are Targeted by Aneuploidy and Mutation in Sézary Genomes

25

Choi J et al Nat Genet. 2015 Sep;47(9):1011-9Wang L et al Nat Genet. 2015 Dec;47(12):1426-34da Silva Almeida AC et al Nat Genet. 2015 Dec;47(12):1465-70Ungewickell A et al Nat Genet. 2015 Sep;47(9):1056-60.

DNMT EZH2BMI1PRC

HDAC

KDM6B

TRX ARID, SMARMLL, SETD

CDKN2A/BTET1/2

H3K27Me2/3

H3K4Me2/3

Xp16

p19

MLL

CCNDCDK4CCNDCDK6

p53 p21

E2F

RB1

X

Cell cycle progression

Cell cycle arrest

STAT3

εεδ γ

CD4

TCR/CD3

PLCγ1LA

T

MYC

ARID1A

ZAP70

IP3 DAG

[CA++]

Calcineurin

NFAT

RAS

MAPK

c-FOSAP-1

c-JUN

PKCθ CARMA1MALT1

BCL10

IKKNFκBNFκB

STAT5

ZEB1

~15%

~40%

~40%

~25%

~40%

~12%

~47%

~50%

NCOR~50%

~92%

~45%

~67%

~11%

~9%

IL-2

JAK1/3

RII

TGFβ

RI

Smad 2

P

P

Smad 3

P

PP

PDCD1~36%

~25%

~26%

RB1MDM2

TRAF3

DDDD

TNF

TRAF2

kinase

TRADD TRADD

DD

RIP

A20

R2R2

R2~18%

~25%

FAS-L

Apoptosis

FAS

ATM

Genotoxic stress

~30%

P

P

P

TAB2

TAK1

TAB1

P

P

P

CD80/86

PP

Lck

* CTLA4-CD28

GRB2 Y

ITK

Loss of Function

Gain of Function

Growth ArrestCell Proliferation

PI3

K

PIP2 PIP3

PTEN

AKT

~36%

STAT3P

STAT5P

IL2R

• Genomic complexity

• Somatic mutations in chromatin remodelers and epigenetic modifiers (ARID1A, TET2, DNMT3A)

• Mutations in oncogenic drivers coexist in neoplastic cells (JAK1/3, STAT3/5)

• Mutations in tumor suppressors (p53, p16, PTEN)

28

Summary

Anaplastic Large Cell Lymphoma

Rearrangements in ALK- ALCL

73 ALK- ALCL• 22/73 (30%) DUSP22 translocated• 6/73 (8%) TP63 translocated• Mutually exclusive• 45 were ALK/DUSP22/TP63 triple negative

Parillia Castellar ER et al Blood 2014

Primary cutaneous CD30-positive T-cell

lymphoproliferative disorders

● 2nd most common group of cutaneous T-cell

lymphomas (CTCL): 30% of cases

● 2 different types:

CD30H&E► Primary cutaneous anaplastic large

cell lymphoma (c-ALCL)

► Lymphomatoid papulosis (LyP)

● No consistent genetic abnormalities

31

NPM1 TYK2

• Nucleophosmin 1• Multiple functions particularly in

nucleolus associating with ribonucleolar proteins

• Non-receptor tyrosine protein kinase• First member of JAK family• Signal transduction by interferons

and interleukins

33

Translocations involving TYK2 were found

in clinical samples by FISH

34

17.2% of CD30+

cutaneous LPDs

0

5

10

15

20

25

Lymphomatoidpapulosis

Primarycutaneous

anaplastic largecell lymphoma

Other mature T-cell lymphoma

% o

ccur

renc

e

3/15

2/14

0/151

35

Tyk2 mediates multiple important inflammatory

cytokine responses

IL-12

p35subunit

p40subunit

IL-23

INFα/β

p40subunit

p19subunit

Tyk2

Jak2

Jak2Tyk2 Jak1

Tyk2

STAT 1-5

P

kinasepseudokinaseSH2FERMN- -C1 1187

pSTAT5

CD30+LPD NPM1-TYK2 -

CD30+LPD NPM1-TYK2 +

Expression of pSTAT5 in NPM1-TYK2+ lymphoma

Ectopic expression of NPM1-TYK2 protein in

HEK293FT cells activates STAT proteins

37

In vivo modeling of NPM1-TYK2 fusion

38

hNPM1-TYK2

Elenitoba-Johnson Lab Unpublished

39

Knockdown of TYK2 in MyLa cells decreases cell proliferation

Targeting NPM1-TYK2 fusion protein in MyLa

cells decreases proliferation

0 1 2 3 40

5 0

1 0 0

L o g 1 0 (J A K in h ib ito r I , n M )

Re

lati

ve

gro

wth

IC50 = 130 nM

40

Elenitoba-Johnson Lab,Unpublished

NDI-031301 inhibits downstream signaling

pathways of NPM1-TYK2 and PCM1-JAK2

NPM-TYK2

β-actin

p-STAT1 (Y701)

p-STAT4 (Y693)

STAT1

NID-031301 (3 µM) - - - -+ + + +

30 min 1 h 3 h 6 h

p-NPM-TYK2 (Y1054/1055)

STAT4

p-STAT3 (Y705)

p-STAT5 (Y694)

STAT3

STAT5

MyLa

PCM1-JAK2

- - - -+ + + +

30 min 1 h 3 h 6 h

p-PCM1-JAK2 (Y1007/1008)

β-actin

p-STAT3 (Y705)

p-STAT5 (Y694)

STAT3

STAT5

Mac-1

Elenitoba-Johnson Lab,Unpublished

Cell line IC50 (μM)

HH NR

MyLa (NPM1-TYK2) 3.407

Hut-78 (JAK1 and JAK3 mutation) 3.050

H9 (JAK1 and JAK3 mutation) 3.337

Mac1 (PCM1-JAK2) 3.516

MJ 2.8240

5 0

1 0 0

1 5 0

Re

lati

ve

gro

wth

(%

of

co

ntr

ol)

H H

M y L a

H u t-7 8

H 9

M a c -1

M J

NDI-031301 inhibits TYK2

● Proliferation assay (72h)

NR: not reached10 100 1,000 10,0000

NDI-031301 (nM)

Summary

● Recurrent TYK2 translocations in cutaneous T-cell lymphomas

● NPM1 as one of the fusion partners

● NPM1-TYK2 translocation results in constitutive activation of TYK2 kinase

● NPM1-TYK2 fusion protein: oncogenic effects through STATs activation

● TYK2 may be a novel therapeutic target in a subset of CD30+

lymphoproliferative disorders

43

Velusamy et al. BLOOD 2015

● Evidence for causation and sufficiency in vivo is pending

Deregulated JAK/STAT signaling in ALCL, ALK -ve

Crescenzo R, et al. Cancer Cell April 2015

Anaplastic large cell lymphoma

45Crescenzo R, et al. Cancer Cell April 2015

Subclassification of ALK- ALCL

ALK- ALCL

DUSP22Rearrangement

TYK2 RearrangementVAV

Rearrangement

TP63Rearrangement

Functional Proteogenomics of ALCL

• Glycosylation is a common posttranslational modification

• Glycoproteins are expressed oncell surface and secreted

• Most CD markers recognizeglycoproteins

Nature Medicine 2009, 15: 828

48

Glycoproteomics for identification of biomarkers and therapeutic targets

Glycoproteomic Profiling BySolid Phase Extraction of Glyoproteins

LC-MS/MS analysis

PNGase F

∆0.98406 amu

49

Peptide N Glycosidase F

Hydrogen atom = 1.00782580

T/NK-cell B-cell

NLPHL

tFL/GCB

MCL

PMBL

DLBCL

BL

CHL

MM

ALCL, ALK-

T-ALL

NK

ALCL, ALK+

MF

SS

HH

Hut

-78

DE

LS

UD

-HL-

1M

ac-1

Mac

-2A

Kar

pas

299

SR

-786

SU

P-M

2JU

RK

ATYT N

K-9

2N

K-9

2MI

U-2

66K

MS

-12

KM

S-1

1R

PM

I 822

6FL

-518

DE

VK

arpa

s11

06P

NC

EB

-1G

rant

a51

9R

ec-1

FL-2

18O

CI-L

y2S

UD

-HL-

4B

JA-B

Ram

osO

CI-L

y1R

aji

FL-1

8FL

-318

KM

H-2

Med

-B1

L123

6L4

28

ALCL NK MM MCL CHLBL/tFL

N-glycoproteomic profiles classify lymphoid neoplasia

● IL2Rα (CD25)

● IL31Rβ (Oncostatin M receptor)

T/NK-cell B-cell

DE

LS

UD

-HL-

1

HH

Hut

-78

Mac

-1M

ac-2

A

Kar

pas

299

SR

-786

SU

P-M

2JU

RK

ATYT N

K-9

2N

K-9

2MI

U-2

66K

MS

-12

KM

S-1

1R

PM

I 822

6FL

-518

DE

VK

arpa

s11

06P

NC

EB

-1G

rant

a51

9R

ec-1

FL-2

18O

CI-L

y2S

UD

-HL-

4B

JA-B

Ram

osO

CI-L

y1R

aji

FL-1

8FL

-318

KM

H-2

Med

-B1

L123

6L4

28

ALCL,ALK+

IL2RαIL31Rβ

Potential novel biomarkers

A distinct cytokine signature is characteristic of ALK+ ALCL

IL31Rβ

Cou

nt

DEL Karpas 299 SR786 SU-DHL-1

SupM2 MAC2A Jurkat

8.75 2.16 2.46 4.76

13.7 1.02 1.02

L428

KM

H2

RA

JI

Kar

pas

299

SU

-DH

L-1

HH

HU

T78

HT1

080

YT NK

-92M

I

BJA

B

DE

L

SR

786

MA

C2A

Jurk

at

Sup

M2

MA

C1

HT1

080

GAPDH

IL31Rb

Tonsil ALK-, IL31Rβ-

ALK-, IL31Rβ+

ALK+, IL31Rβ+

IL31Rβ + IL31Rβ -

ALK + 21 0

ALK - 14 21

Χ2 = 20.16p<0.001

ALCL, ALK+

● Cell lines ● 56 primary biopsies of ALCL

IL31Rβ is selectively expressed in ALK+ALCL

LentiviralVector

Packaged Pooled Lentiviral sgRNA

LibraryBarcoded

Pooled Plasmid sgRNA Library

TransducedTarget Cells

Barcode Representation

Freq

uenc

y80-90% of Sequences Within 1 Order of Magnitude

Quantitative Identification of

Enriched or Depleted sgRNA

Corresponding to Gene Targets

BarcodedsgRNA

Amplification

Designs: sgRNAsgRNA expression: U6, U6-Tet, H1 or H1-TetMarkers: GFP, RFP, PuroR, Promoters: UbiC, EF1a, CMV

PCR & Cloning

Target CellTransduction

CRISPR-Cas9 sgRNA genome-wide vulnerability

14, 250 sgRNAs

Cytokine receptor pathways are exquisite vulnerability targets in ALK+ALCL

Markov Chain Monte Carlo Simulation

IL6-STAT3 IL2-STAT5

0

2 0

4 0

6 0

8 0

Pe

rce

nta

ge

of

tum

or

gro

wth

ScrambleshRNA

IL31RβshRNA

p <0.01

Per

cent

age

of tu

mor

gro

wth

IL31Rβ knockdown abrogates tumor growth in ALK+ALCL, xenotransplants

ScrambleshRNA

IL31RβshRNA

IL31Rβ contributes to oncogenesis in ALK+ALCL

IL31Rβ

β-actin

WT

Scr

ambl

e sh

RN

A

IL31

Rβ

shR

NA

Rolland D et al., PNAS 2017

56

Cytokine/receptor – JAK- STAT signaling plays a prominent role in ALCL pathogenesis

Opportunities for targeted vulnerabilities

57

Summary

Anaplastic large cell lymphoma

Outline• Background

– WHO classification of mature T cell lymphoma

• Recent updates in genetics of T cell lymphoma

– PTCL/AITL– CTCL/SS– ALCL

• Pathobiologic and therapeutic implications

• Conclusions

Recurrent large structural alterations in T-cell neoplasms

59

Disease entity Structural Abnormality Frequency

ALK+ ALCL NPM1-ALK

Various-ALK

84%

16%

T-PLL TRA-TCL1A

TRA-MTCP1

70-80%

10%

HSTL i(7q)(q10) >80%

EATL 9q gains

16q12.1 loss

~70%

~30%

ALK- ALCL, c-ALCL IRF4/DUSP22 translocations

TYK2/ NPM1-TYK2

VAV rearrangements

PCM-JAK2

25%

17%/4%

4%

rare

PTCL P53-related genes 6%

F-PTCL ITK-SYK 18-38%

AITL ITK SYK rare

Recurrent somatic gene mutations in T-cell neoplasms

60

LeukemiasALK-ALCL

Lymphomas

Gene Disease entity FrequencyJAK1/JAK3 T-PLL

NKTCL/EATL40%20-35%

STAT3 T-LGLLGD HSTCL/EATLCLPD-NK

27-40%9%30%

STAT5B T-PLLGD HSTCL/EATLT-LGLL

36%36%rare

CD28 AITL, MF/SS 11%, rare

FYN AITL; PTCL, NOS 3% ,rare

PKCG1 AITL, MF/SS, PTCL, NOS

12%,20%,15%

PRKCB NKTCL 33%

CARD11 MF/SS, NKTCL 15%,24%

TNFRSF1B MF/SS, NKTCL 6%, rare

TET2 AITL, F-PTCL 33-47%, 58%

IDH2 AITL ~25%

DNMT3A AITL; PTCL, NOS 11% overall

SETD2 EATL I/II 32%

RHOA AITL; PTCL, NOS 67%; 18%

CCR4 MF/SS 7%

JAK/STAT

Co-stimulatoryTCR signaling

Epigenetic

NF-κB

Other

Impact on therapeutic decisions

Lymphoma Genetic features Therapeutic relevance

ALCL ALK ALK inhibitorcALCL TYK2 TYK2 inhibitorAITL ITK/SYK SYK inhibitorT-PLL/ENKTCL JAK3 JAK3 inhibitorT-PLL STAT5B STAT5 inhibitorT-LGLL, NK-LPD STAT3 STAT3 inhibitor

AITL TET2, IDH2, DNMT3A Epigenetic modulatorsPTCL, NOS DNMT3A Epigenetic modulatorsF-PTCL TET2 Epigenetic modulators

61

Conclusions

• Genetic basis of T-cell lymphomas reveals complexity within each entity

• Profound implications for taxonomy

• Biologic subcategories and prognosis

• Targeted therapeutics

Acknowledgements

Department of Pathology,

University of Pennsylvania

Megan S. Lim

Delphine Rolland

Ozlem Onder

Thirunavukkarasu Velusamy

Mark J. Kiel

Anagh A. Sahasrabuddhe

Catherine A. Dixon

Nathanael G. Bailey (Univ of Pittsburgh)

Bryan L. Betz

Noah A. Brown

Alexandra C. Hristov

Department of Internal Medicine,

University of Michigan

Ryan A. Wilcox

Department of Hematopathology,

MD Anderson Cancer CenterRoberto N. Miranda

L. Jeffrey Medeiros

Department of Pathology,

Seoul National University Hospital

Yoon K. Jeon

Department of Pathology,

Henry Ford Health SystemKedar V. Inamdar 63

Department of Pathology,

University of Michigan

QUESTIONS?

64