Mechanisms of Ibrutinib Sensitivity and Resistance in CLL Y. Lynn Wang, MD, PhD, FCAP Dept. of Pathology University of Chicago October 24, 2014

Mechanisms of Ibrutinib Sensitivity and Resistance in CLL

Y. Lynn Wang, MD, PhD, FCAPDept. of Pathology

University of ChicagoOctober 24, 2014

Outline

• Ibrutinib targets in vivo CLL proliferation through inhibition of B-cell receptor signaling activity

• BTKC481S confers resistance to ibrutinib as a consequence of loss of irreversible drug binding

• PLCg2 mutations confers resistance to ibrutinib via re-activation of BCR-signaling

• Overcome ibrutinib resistance: Exploration of other kinase inhibitors

Outline





BTK is more active in treatment-naive CLL patients than in normal individuals

4

Normal donors

259

259

CLL patients

89 92 95 96

123

11

03127

2078208

45 64 67 74

82

76

11

03127

2078208

t-BTK

GAPDH

t-BTK

GAPDH

A

Controls

ly7+IgM

ly7+IgM

CLL patients Normal donors

ly745 64 67 74

76

4771

47782078

208259

ly789 92 95 96

123

4

7714778

2078208

259

82

p-BTK

GAPDH

p-BTK

GAPDH

C

B

CLL(N=11)

Normal(N=5)

p-B

TK

rel

ativ

e ex

pres

sion

p-B

TK

rel

ativ

e ex

pres

sion

CLL(N=1

Normal(N=5)

D

Cheng S…Wang YL. Leukemia, 2014, 28, 649-57

Study Design

• In vivo: Prospective collection of serial samples from patients undergoing clinical trial of ibrutinib

• Enrolled a total of 16 patients– 12

previously treated

– 4 treatment naïve

What is the effect of ibrutinib on apoptosis induction?

Does ibrutinib induce apoptosis?-An ex vivo study

“Apoptosis induced by PCI-32765 in primary CLL cells was significant compared with vehicle treatment alone at 10 uM PCI-32765”

Blood, 117, 6287, 2011

The Cmax values in humans range from 80-200 nM

Cytopenia was uncommon in patients treated at 420 mg/day. Grade 3&4 neutropenia was more common in the 840 mg group-ASCO 2011

In vivo apoptosis was largely absent in the peripheral blood of CLL patients treated with ibrutinib

8

BCL2

XIAP

MCL1

PARP

Cleaved Caspase-3

GAPDH

CLL 085

D28

CLL 195 CLL 043 CLL 011

pre 4hpre D28 pre D28 pre D28

Again, apoptosis induction may not be the predominant effect of ibrutinib in CLL patients

Cutoff 15%


What is the effect of ibrutinib on CLL cell proliferation?

In vivo CLL proliferation is targeted by BTK inhibition: Ki-67 was reduced over the treatment course

10Cheng S…Wang YL. Leukemia, 2014, 28, 649-57

11

CLL proliferation is targeted by BTK inhibition in an in vitro model of cell proliferation


12

CLL proliferation is targeted by BTK inhibition in an in vitro model of cell proliferation


What is the effect of ibrutinib on signal transduction?

B-cell Receptor Signaling

15

Activity of BTK (pY223) was inhibited in treated patients


BCR signaling in CLL

• CLL in vivo proliferation is mediated by the pathway LYN-SYK-BTK-PLC2 and downstream signaling through AKT and ERK.

• The data highlight the role of cell proliferation in CLL that has been thought mainly as a disease with apoptotic defects.

Outline





The Case • The patient is a 61 year old woman first diagnosed with

CLL in 2000 when she presented with lymphadenopathy. • In September of 2010, she was enrolled in the phase Ib

trial (NCT01105247) of ibrutinib. She achieved an excellent response, with normalization of her CBC, (ALC=3600), but some residual lymphadenopathy remained on CT scan.

• In March of 2012, 19 months after ibrutinib initiation, her ALC and lymphadenopathy rapidly increased.

• In May of 2012, the ibrutinib dose was escalated from 540 to 840 mg. Over the next four weeks, the patient continued to demonstrate disease progression.

Four specimens collected

19

-100 0 100 200 300 400 500 600 7000

50

100

150

200

250

300

350

400

450

Day

ALC

Re

lap

sed

S

2

Pre

Re

spo

nd

ing

Re

lap

sed

S

1

Ibru

tinib

initi

atio

n

Furman RR…Wang YL, NEJM, 2014, 370, 2352-4

A novel BTK mutation is identified by RNA-Seq which is present only in relapse samples

20

RelapsedBefore relapse

Pre-Rx

Responding

Relapse 1

Relapse 2

G C T G C C T C G C A G C C T C

Responding

Pre-Rx Relapse s1

Relapse s2

Nucleotide composition at position 1634 of the BTK gene

CLL Numbers of the reads %A

Specimens A C G T

Pre-Rx 0 0 0 84 0Responding 0 0 0 54 0Relapse S1 102 8 0 6 88Relapse S2 67 5 0 1 92


What is the impact of the mutation on ibrutinib binding?

The mutation disrupts the covalent binding of ibrutinib to BTK

22

Probe-labeled BTK

Ibrutinib (nM) - 1 10 100 - 1 10 100 - 1 10 100

No DNA

Total BTK

-actin

WT C481S C481A

WT C481S

Covalent bond


WT BTK(IC50 =2.2nM)

BTK C481S (IC50 =1006nM)

Inhi

bitio

n of

BT

K p

hosp

hory

latio

n (%

)

10 10 100 1000

Ibrutinib (nM)

What is functional consequence of BTKC481S at the molecular

level?

The mutation restores the BCR signaling pathway

24

p-BTK(Y223)

p-ERK1/2(T202/Y204)

Before relapse RelapsedPre-RxResponding 1 2

p-AKT(S473)

GAPDH

3210-1-2-3

Pre-Rx Responding 1 2

RelapsedBefore relapse

EGR1

PAICSDUSP2

KLF10

CD83

CASP3

NFKBIEDDX21

GFI1

IRF4

CTLA4

GNPDA1

Ly9

RGS10

CCL4LPLPDGFAEGR3SLAMF7NAB2LILRA4NR4A3

DDIT3

OAS3TXNRD1

BCR signature

BC

R S

ign

atu

re S

core

***

***

*

* ***

Pre-Rx Responding Relapse1 Relapse2

• Pre-Rx• Responding• Relapse 1• Relapse 2

Cheng S…Wang YL, Leukemia, 2014, epub

What is the functional consequence of BTK C481S at the

cellular level?

The mutation leads to increased cellular proliferation in vivo

26

CD19

Ki6

7

Pre-Rx Responding

Before Relapse

1

Relapsed

2


The mutation leads to increased cellular proliferation in vitro that did not respond well to ibrutinib

27

Iso 0 nM 250nM 500nM

7-AAD

Brd

U

+CLL+Stroma + ibrutinib

Befo

re Relap

seR

elapsed

1R

espo

nd

ing

2P

re-Rx


BTK Mutation Is Recurrent

Woyach JA…Byrd JC. NEJM, 2014, 370, 2286-94

Outline





Reactivation of BCR signaling in patient with PLCg2R665W mutation


Reactivation of BCR signaling in patient with PLCg2R665W mutation


Outline





Ibrutinib-resistance CLL cells remain sensitive to other BCR inhibitors

33Cheng S…Wang YL, Leukemia, 2014, epub

34

Ibrutinib-resistance CLL cells remain sensitive to idelalicib


Acknowledgement

• Wang Lab at Cornell

Shuhua Cheng, PhD

Chunyan Yang, MDPin Lu, MD, PhD Ailin Guo, MD, PhDAnnie Ma, PhDZibo Song, PhDJoelle Racchumi

Acknowledgement

• Cornell CLL

Research CenterDr. Morton Coleman

Dr. Richard Furman

• MSKCCDr. Christina Leslie

Menu Setty

• PharmacyclicsDr. Betty Chang

• Harvard UniversityDr. Hao Wu

Thank you!

Documents

Mechanisms of Ibrutinib Sensitivity and Resistance in CLL Y. Lynn Wang, MD, PhD, FCAP Dept. of Pathology University of Chicago October 24, 2014