LLC: un approccio di tipo funzionale

attraverso l’inibizione di BTK

Antonella Zucchetto, PhDCRO National Cancer Institute Aviano, Italy

Chronic lymphocytic leukemia

➢A common malignancy of CD5+ B cells that is characterized by an accumulation of small, mature-appearing lymphocytes in the blood, bone marrow and lymphoid tissues

➢ CLL cells are highly dependent on their microenvironment and B cell receptor signalling for survival and growth.

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Niiro H., Nat Rev Immunol. 2002

B cell receptor (BCR) signalling

•B cell receptor (BCR) signalling is indispensablefor normal B celldevelopment and adaptiveimmunity

•Mechanism of activation of BCR signalling include continuous BCR stimulation by microbial antigens and/or autoantigens and by ligand-independent tonic BCR signalling

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BTK

PI3K

antigen

BCR signaling controls integrin activation

Inside-out integrin activation

•Normal B cell differentiation is controlled by antigen-driven BCR stimulation by the follicular dendritic cells (FDC)-presented antigen, generating signals that result in inside-out integrin activation and enhanced adhesion on VCAM-1

•B cells with a competent BCR are retained in GC microenvironment and are protected from apoptosis

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➢ heterodimeric integrin formed by non-covalentassociation of α₄ (CD49d; 155kDa) and β₁ (CD29; 150kDa) subunits

➢ It functions as a matrix and cell receptor throughinteractions with fibronectin and VCAM-1

➢Key role in CLL microenvironmental interactions

α₄(CD49d)

β₁(CD29)

The VLA-4 integrin (CD49d/CD29)

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30%cutoff

CD49d-

CD49d+

➢ Expressed by ~40% CLL cases (30% cut-off)

➢Expression associated with bad prognosisin the context of chemoimmunotherapy

T. Robak and P. Smolewski, Blood 2015

BCR inhibitors

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BTK inhibitor ibrutinib

covalent/irreversible binding

BTK inhibition

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Ibrutinib in CLL: first reports

Ibrutinib was superior (longer PFS and OS) to ofatumumab (RESONATE clinical trial, NCT1578707 and to chlorambucil (RESONATE-2 Clinical Trial, NCT01722487) in

previously treated (RESONATE) and untreated (RESONATE-2) CLL patients

Burger JA et al, NEJM 2015

Byrd JC et al, NEJM 2014

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Ibrutinib in CLL and TP53 disruption

Stilgenbauer S et al, Blood 2014

TP53 mutations predict poor response tochemo-immunotherapy in CLL

ibrutinib induces durable responses in CLL with del17p or TP53 aberrations

RESONATE clinical trial, NCT1578707

CLL8 clinical trial, NCT00281918

Brown JR et al. Leukemia 2018

Phase-2 clinical trial, NCT01500733Farooqui MZH et al. The Lancet Oncology 2015

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Guidelines for TP53 analysis in clinical practice

When What

iwCLL Before treatment 17p deletion

ERIC Before treatment TP53 mutation

BCSH Before treatment 17p deletion and TP53 mutation

NCCN Before treatment 17p deletion and TP53 mutation

ESMO Before treatment 17p deletion and TP53 mutation

Hallek et al. Blood. 2008Oscier et al. Br J Haematol. 2013Pospisilova et al. Leukemia. 2012Zelenetz et al J Natl Cancer Inst 2015Eichhorts et al, Ann Oncol 2015

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Ibrutinib in CLL: first reports

Byrd JC et al. N Eng J Med 2013

Ibrutinib causes redistribution of malignant B cells from tissue sites into the peripheral blood and concomitant rapid shrinkage of affected tissue sites

Herman S et al. Leukemia 2014

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What about VLA-4 inside-out activation?

Ibrutinib and CLL cell adhesion

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• LDV small molecule to mimic the bindingof natural ligand(curve from 0,1 nM to 10 µM)

• Conformationally sensitive mAb(HUTS-21 mAb→mapped to the hybrid domain of β1)

Flow-cytometry based methods (Chigaev et al, 2009):

Chigaev and Sklar, 2012. Front Immunology

The different VLA-4 conformations can be quantified

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CD49d and BCR-induced inside-out activation

Receptor Occupancy = 1/(1 + 10(log EC

50– log[LDV]))

RO=1.0 (100%)RO=0 (0%)

No Receptor Occupancy

100% Receptor Occupancy

Chigaev et al. J. Biol. Chem. 2009

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CD49d and BCR-induced inside-out activation

HUTS-21 MFI at the increasing

LDV concentrations

CROAVIANOTissino E. et al., J Exp Med 2018

in-vitro in-vivo

CLL cell adhesion

BCR-induced VLA-4 inside-out activation and ibrutinib

▪ Italian cohort (22 CD49d+, 15 CD49d- cases)

▪ NIH cohort (21 CD49d+, 13 CD49d- cases)

▪ Mayo Clinic cohort (12 CD49d+, 18 CD49d- cases)

Impact of CD49d expression on:

• Redistribution lymphocytosis

• Lymph node masses (SPD)

• Progression-Free Survival (PFS)

101 cases(55 CD49d+, 46 CD49d-)

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BCR-induced VLA-4 inside-out activation: clinical implications

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BCR-induced VLA-4 inside-out activation: clinical implications

Tissino E. et al., J Exp Med 2018

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BCR-induced VLA-4 inside-out activation: clinical implications

CD49d+ CLL display reducedibrutinib-induced LN shrinkage(12 months)

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BCR-induced VLA-4 inside-out activation: clinical implications

Tissino E. et al., J Exp Med 2018

CD49d expression is correlated with shorter PFS

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BCR-induced VLA-4 inside-out activation: clinical implications

Tissino E. et al., J Exp Med 2018

In the context of RR CLL cases

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BCR-induced VLA-4 inside-out activation: clinical implications

Tissino E. et al., J Exp Med 2018

Brown JR et al. Leukemia 2018

Ibrutinib in CLL: follow-ups

RESONATE clinical trial, NCT1578707

No lymphocytosis is associated with shorter PFS

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Ibrutinib in CLL: follow-ups

O’Brian S et al. Blood 2018

Ahn IE et al. Blood 2018

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Ibrutinib in CLL: follow-ups

•only a low percentage of patients achieve a very low burden of disease, and undetectable MRD is unlikely

•25-40% of patients will experience treatment discontinuation (mainly due to adverseevents or disease progression)

•CLL progression is associated with mutations in BTK (loss of ibrutinib covalent binding) or PLCG2 (activating mutations)

mutation at C481

no covalent binding

resistance to ibrutinib

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What happens to residual CLL cells during ibrutinib treatment?

Ibrutinib effects on CLL phenotype and signaling

IOSI-EMA-001 (TN or RR; TP53 mut and/or del)IOSI-EMA-003 (RR; TP53 wt)

Baseline Week 96Week 72Week 48Week 24Week 2 ProgressionEnd of treatment

Yearly

Davide Rossi, Oncology Institute of Southern Switzerland

CLLBCR

Signaling

B-cell/CLL markers

AdhesionHoming

Immunophenotype Phospho-proteins and NF-kB activation

▪Basal levels

▪After activation with BCR-dependentand BCR-independent stimuli

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-50

0

50

100

150

200

250

300

w0 w2 w24 w48 w72

CD49d

CD11b

CD184/CXCR4

CD29

ITGB7

CD49e

CD54

Adhesion/Homing

BCRSignaling

B-cell/CLL markers

Ibrutinib effects on CLL phenotype

-40

-20

0

20

40

60

80

100

wo w2 w24 w48 w72

CD79b

IgD

IgM

-100

-80

-60

-40

-20

0

20

wo w2 w24 w48 w72

CD305

CD307b

FCmR

-100

-80

-60

-40

-20

0

20

wo w2 w24 w48 w72CD38

CD20

CD43

CD5

CD23

med

ian

% M

FI c

han

gefr

om

bas

elin

em

edia

n%

MFI

ch

ange

fro

mb

asel

ine

med

ian

% M

FI c

han

gefr

om

bas

elin

e

med

ian

% M

FI c

han

gefr

om

bas

elin

e

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Ibrutinib effects on CLL signaling: BCR-dependent

w0 w2 w24 w48 w72IgM - + - + - + - + - +

w0 w2 w24 w48 w72IgM - + - + - + - + - +

p-BTK p-PLCɤ2

p-ERK p-AKT

w0 w2 w24 w48 w72IgM - + - + - + - + - +

w0 w2 w24 w48 w72IgM - + - + - + - + - + CRO

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Weeks on ibrutinib Weeks on ibrutinib

Ibrutinib effects on CLL signaling : BCR-independent

p-ERK p-AKT

w0 w2 w24 w48 w72CD40L - + - + - + - + - +

w0 w2 w24 w48 w72CD40L - + - + - + - + - + CRO

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p-BTK p-PLCɤ2

w0 w2 w24 w48 w72CD40L - + - + - + - + - +

w0 w2 w24 w48 w72CD40L - + - + - + - + - +

Weeks on ibrutinib Weeks on ibrutinib

Ibrutinib effects on CLL signaling

Canonical NF-kB pathway Non canonical NF-kB pathway

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Weeks on ibrutinib Weeks on ibrutinib

Progression

Summary: an hypothesis

Pre-treatment

• Retention to capability to respond to chemokines• Retention of the integrin activation• CLL cells competent for adhesion• CLL cells competent for BCR signaling• by-pass mechanisms that keep ongoing signaling

ibrutinibDiseaseburden

Residual disease(infrequent MRD negativity)

Clonal evolutionResistance mutations

Combinationtherapies

(Ib+-CD20Ib+ -BCL-2)

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Clinical and Experimental Onco-Hematology Unit CRO, Aviano

CROB, Rionero in VultureGiovanni D’Arena

Pietro BulianMassimo DeganFrancesca M. RossiMichele BertonAlessandra BraidaIlaria CattarossiHillary ChivilòPaola NanniEva Zaina

University of Tor Vergata, RomeGiovanni Del PoetaEnrico Santinelli

Division of Hematology, University of Eastern PiedmontGianluca Gaidano

Division of Hematology, University of CataniaFrancesco Di RaimondoAnnalisa Chiarenza

Maggiore General Hospital, University of TriesteGabriele Pozzato

Division of Hematology, University of UdineFrancesco Zaja

Valter Gattei

Dania BenedettiTamara BittoloRiccardo BombenTiziana D’AgaroFederico PozzoFabrizio SerraErika TissinoElena VendraminiFilippo Vit

MDACC, University of Texas, Houston, TXJan BurgerElisa ten Hacken

Hematology Brench, NIH, Bethesda, MDAdrian Wiestner Sarah Herman

Inhye E. Ahn

Mayo Clinic, Rochester, MNTait Shanafelt Kari Chaffee

Ayed Ayed

Paracelsus Medical University, Salzburg, AustriaTanja Hartmann Elisabeth Bayer

Andrea HärzschelEva Szenes

University of New Mexico, Albuquerque, NMAlexandre ChigaevLarry Sklar

Institute of Oncology of Southern SwitzerlandDavide Rossi Gabriela Forestieri

Catholic University of the Sacred Heart, RomeLuca Laurenti

Acknowledgments

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