Targeting B cell receptor signaling in cancer: preclinical ......Targeting B cell receptor signaling in cancer: preclinical and clinical advances. Jan Burger Innovations in Hematology

Targeting B cell receptor signaling in cancer: preclinical and clinical advances

Jan Burger

Innovations in Hematology

Dan Tel Aviv Hotel, Tel Aviv, Israel

May 29, 2018

Targets in the BCR signaling pathway

Burger & O’Brien Nat Rev Clin Onc 2018

VH D JH C

NN

Somatic mutations

1/51 1/27 1/6

B-Cell Diversity: IGHV

Rearrangement and Mutation

VH in chronic lymphocytic leukemia (CLL)

– U-CLL, with 98% or more sequence homology with the corresponding consensus germline sequence

– M-CLL, with less than 98% sequence homology

Su

rviv

ing

(%

)

Survival of CLL Patients With Mutated vsUnmutated IGHV

All patients (N=84) Stage-A CLL patients (n=62)

Su

rviv

ing

(%

)

0 50 100 150 200 250 3000

20

40

60

80

100

Months

P=0.0008

Months

P=0.001

0 50 100 150 200 250 3000

20

40

60

80

100

Mutated

UnmutatedUnmutated

Mutated

Hamblin TJ et al. Blood. 1999;94:1848-1854.

In CLL:

Sites of proliferation =

sites of BCR activation

CLL lymph nodes

From: Soma LA et al,

Human Pathology. 2006;37:152-159

Proliferation

centers

Burger & O’Brien Nat Rev Clin Onc 2018

Fro

m: Y

He

rish

an

ue

t a

l., B

loo

d. 2

01

1

• Pro-B cells to pre-B cells : generation of μ heavy chain by rearranging VDJ segments

• The pre-B cell receptor (pre-BCR) μ heavy chain paired with the surrogate light chain (λ5 and VpreB)

• Mature B cells express IgM and IgD

• Subsequent B cell development occurs in the secondary lymphoid organs (SLOs)

• In the germinal centre (GC), the enzyme activation- induced cytidine deaminase (AID) introduces mutations in IGHV and IGLV to diversify the Ig repertoire (somatic hypermutation)

• AID also mediates class switch recombinationto generate IgA, IgG or IgE

• Selected GC B cells give rise to memory B cells and long-lived plasma cells

• Plasma cells home to the bone marrow or reside in SLOs, where they secrete antibodies

Burger & Wiestner, NRC 2018

• Centroblasts: somatic hypermutation in the dark zone

• Centrocytes in the light zone: antigen selection and affinity maturation

• Differentiation into memory B cells or plasma cells

• CLL cells proliferate in proliferation centers, in close contact with T cells and nurselike cells

• Antigen triggers BCR signaling in CLL cells in SLO

Antigen-dependent and oncogenic BCR signaling in B cell malignancies

a) Chronic infections can lead to clonal B cell expansion. Autoantigen can stimulate polyreactive BCRs. Autonomous BCR signalling,

b) Antigen-dependent chronic active BCR signalling is increased by oncogenic mechanisms. Mutations in CD79A and CD79B contribute to the activation of BCR signalling in ABC-DLBCL.

c) Oncogenic tonic BCR signaling. In Burkitt lymphoma (BL), expression of MYC and transcription factor 3 (TCF3) cooperate to promote tonic BCR signalling; MYC upregulates the microRNAs miR-19a and miR-19b, which in turn decrease expression of the PI3K negative regulator PTEN.

Burger & Wiestner, NRC 2018

The B cell receptor signaling pathway

Burger & Wiestner, NRC 2018

IgM and IgD receptors are highly expressed in CLL

subsets

M-C

LL

U-C

LL

CD38

neg

CD38

pos

ZAP70

neg

ZAP70

pos

del13

q,tri1

2,neg

del11

q,del

17p

0

100

200

300

MF

IR

**** ns ** *

IgMIgM IgD

M-C

LL

U-C

LL

CD38

neg

CD38

pos

ZAP70

neg

ZAP70

pos

del13

q,tri1

2,neg

del11

q,del

17p

0

50

100

150

200

MF

IR

** ns *** ns

IgD

Ten Hacken E, J Immunol. 2016

IgD stimulation induces strong HS1 protein activation

anti-IgM

(minutes)

0 2 5 0 2 5

2 5 2 5100

150

200

250

300

350

HS

1 p

ho

sp

ho

ryla

tio

n

(% u

ntr

eate

d c

on

tro

l)

Minutes of anti-IgM

Minutes of anti-IgD

**

anti-IgM

(minutes)

anti-IgD

(minutes)

anti-IgD

(minutes)

P-HS1

(Y397)

HS1

IgM and IgD: upstream signaling responses

Ten Hacken E, J Immunol. 2016

IgM induces prolonged pathway activation

P-ERK

ERK

0 2 5 15 30 0 2 5 15 30

anti-IgM

(minutes)

anti-IgD

(minutes)

5 15 302 60

100

200

300

400

BCR stimulation (minutes)

ER

K p

ho

sp

ho

ryla

tio

n

(%u

ntr

ea

ted

co

ntr

ol)

* *

*

IgM

IgD

IgM and IgD: downstream signaling responses

Ten Hacken E, J Immunol. 2016

IgM signaling exclusively induces CCL3 and CCL4 secretion

an

ti-I

gM

an

ti-I

gD

0 1000 2000 3000 4000

Untreated

0.5 µg/mL

1 µg/mL

5 µg/mL

10 µg/mL

0.5 µg/mL

1 µg/mL

5 µg/mL

10 µg/mL

CCL3 (pg/mL)

0 2000 4000 6000 8000 10000

Untreated

0.5 µg/mL

1 µg/mL

5 µg/mL

10 µg/mL

0.5 µg/mL

1 µg/mL

5 µg/mL

10 µg/mL

CCL4 (pg/mL)an

ti-I

gM

an

ti-I

gD

IgM and IgD: CCL3 and CCL4 chemokine secretion

Ten Hacken E, J Immunol. 2016

IgM induces CCL3 secretion through BCL6 down-regulation

3 6 9 12 240

500

1000

1500

BCR stimulation (hours)

CC

L3

(pg

/mL

)

anti-IgM

anti-IgD

BCL6

GAPDH

anti-IgM stimulation

(hours)

0 3 6 9 12 24

Untreated

(hours)

3 6 9 12 24

3 6 9 12 24

anti-IgD stimulation

(hours)

0 3 6 9 12 24

Untreated

(hours)

BCL6

GAPDH

BCL

6

IgM and IgD: mechanism of CCL3 production

Ten Hacken E et al., J Immunol. 2016 Sep 15;197(6):2522-31

Ten Hacken E, J Immunol. 2016

Variable CCL3 protein expression in CLL lymph nodes

• In 42 cases: 24 CCL3 positive (56%) and 19 CCL3 negative (44%) cases. • Membranous staining, impression of secretion of CCL3 into the

environment• Prolymphocytes and paraimmunoblasts particularly in the proliferation

centers were positive

Hartmann EM,… & Rosenwald A. Leukemia& Lymphoma 12:1-9, 10/2015

• CCL3 positive cases (B) shows more prominent T cell infiltrates (CD3, CD4 and CD8) • CCL3 positive cases (B) have higher number of CD57 positive cells and a higher

proliferation fraction (Ki-67-pos.)

Hartmann EM,… & Rosenwald A. Leukemia& Lymphoma 12:1-9, 10/2015

Association between CCL3 and T cell densityCCL3-neg. CCL3-pos. CCL3-neg. CCL3-pos.

BCR-RELATED BIOMARKERS:CCL3, CCL4 (MIP-1α,β)

CCL3

CCL4

pg

/mL

time (days)

pre-treatment

S. Ponader et al., Blood 119: 1182-9, 2012

pre-treatment

Ibrutinib trial

Idelalisib trial

Hoellenriegel J et al.; Blood 118(13):3603-12, 09/2011

Heavy water labeling of CLL cells prior to ibrutinib therapy

MDACC

KineMed, Inc.

Feinstein Institute

Burger J JCI Insight 2017; 2(2):e89904

Heavy water labeling of CLL cells prior to ibrutinib therapy:Effects of ibrutinib on birth and death rates

• CLL cell proliferation (“birth”) rates: before ibrutinib therapy 0.39% down to 0.05% on ibrutinib

• Death rates increased from 0.18% to 1.5%

• First direct in vivo measurements of ibrutinib’s anti-leukemia activity

• Profound inhibition of CLL cell proliferation

• Promotion of high rates of CLL cell death

Burger J JCI Insight 2017; 2(2):e89904

Role of tumour-infiltrating B cells in pancreatic ductal adenocarcinoma

Burger & Wiestner, NRC 2018

Clonal evolution on ibrutinib

Ahn, Underbayev, et al Blood 2017

Mutations in BTK & PLCG2 confer BTKi resistance

From Wiestner, Haematologica 2015

Chang, ASCO 2013; Woyach, NEJM 2014; Furman, NEJM 2014

Early clonal shifts during treatment with ibrutinib

Landau DA Nature Comm 2017

Ibrutinib-resistant patients

MDACC cohortPt # Age

(yrs)/

Gender

/

Rai

stage

Prior therapy Pre-ibrutinib

FISH cytogenetics

IGHV

(M, U)

Treatment Best

response

to

ibrutinib

Time to

PD on

ibrutinib

1 59/M

Rai III

FCR del (17p),

del (13q)

ND Ibrutinib PR 983

2 36/F

Rai IV

FCR,

R+HDMP

del (11q)* U Ibrutinib +

rituximab

PR 176

3 85/F

Rai IV

R, BR, CLB,

R+HDMP

del (17p),

del (13q), trisomy

12*

U Ibrutinib PR 554

4 58/M

Rai IV

FCR, FR,

CHOP, allo-

Tx, BR,

revlimid,

ofatumuma

b

del (17p),

del (11q),

del (13q)*

U Ibrutinib PR 669

5 58/M

Rai II

FCR, F, R, B del (11q),

del (13q)

U Ibrutinib PR 392

*Complex cytogenetics pre-ibrutinib:Pt 2: 46,XX,del(2q37),del(6q21q23), add(7q36),del(11q21q25)Pt 3: 45,X,add(X)(q24), add(6p23), add(12p13), del(17p11.2), -21Pt 4: 45,XY,del(6q13q23), add(8p21), del(11q21q24), -17,-18,+mar[5]

Clonal evolution in CLL patients during

ibrutinib therapy: Patient #1

Burger, Landau …. & Wu Nature Comm 2017

Clonal evolution during ibrutinib therapy:

transdifferentiation into histiocytic sarcoma

Burger, Landau …. & Wu Nature Comm 2017

Droplet-based detection of resistance subclonesat the time of ibrutinib treatment initiation

Conclusion: our analyses support the presence of resistant sub-clones at treatment initiation, in line with theoretical predictions

Burger JA Nature Comm 2017

SUMMARY• Ibrutinib and other BCR signaling inhibitors including

idelalisib results in major clinical benefit for patients with CLL

• However, this therapy also exerts strong selective pressure, which can promote the outgrowth of resistant subclones

• Long-term toxicity and resistance development are problems with long-term use of ibrutinib (plus: high costs)

• Intermittant therapy could help addressing these issues

Thank-

you!Collaborators:• Würzburg University: A Rosenwald, E

Hartmann• CLLGRF: F Caligaris-Cappio, N

Chiorazzi, Z Estrov, N Kay• MDACC: M Keating, W Wierda, S

O’Brien, H Kantarjian, V Gandhi, A Ferrajoli, K Balakrishnan

• UCSD: T Kipps, L Rassenti• UC Irvine: D Wodarz, N Komarova• DFCI, Broad I: C Wu, D Landau

My laboratory: Mariela Sivina, Julia Hoellenriegel, Stefan Koehrer, Ekaterina Kim, Elisa ten Hacken, ShubhchintanRandhawaFunding: CPRIT, MD Anderson Moonshot,Leukemia & Lymphoma Society

Dept. of Leukemia, MDACC