CLL

Stromal cell

protect

Conventional therapies

Ve

rste

cksp

iel i

m W

ald

b

y F

ried

rich

Ed

ua

rd M

eye

rhe

im (

18

08

-18

79

)

The “outside” (microenvironment) of the CLL cell: new insights into disease

biology and new therapeutic targets

Jan Burger, MD PhD

Department of Leukemia

MD Anderson Cancer Center

Microenvironment in CLL Larger proliferating CLL

cells form proliferation centers (pseudofollicles), a hallmark finding in CLL

Within pseudofollicles, CLL cells are in close contact with accessory cell (stomal cells, T cells)

From: Soma LA et al, Human Pathology. 2006;37:152-159

From: PE Patten et al. Blood. 2008;111:5173-81

Messmer BT, et al.

J Clin Invest. 115(3): 755-764, 03/2005

Microenvironment in CLL: a-SMA and CD14/68+ cells

From: J Rual et al. Clinical Cancer Research 12, 5622-31, 2006

Bh

att

ach

ary

a a

nd

Me

rte

ns

et

al.,

Le

uke

mia

(2

011

) 2

5,

72

2–

72

6

CLL#1

CLL#2

CLL#3

normaltonsil

CLL in vitro model: BMSC co-cultures

• Standardized CLL-stroma co-culture conditions for drug testing

• Ideal for testing drug combinations

Fro

m:

Siv

ina

et a

l., L

euke

mia

26:

1812

-20,

08/

2012

IN VITRO MODEL : Nurselike cells

CXCR4†

CXCL12CXCL13CXCR5¶

CD31, plexin-B1

CD38, CD100‡

BAFF, APRIL

BAFF-R, BCMA, TACI*

* Nishio M et al. Blood 106:1012-20, 2005¶ Burkle A et al. Blood 110:3316-25, 2007

† Burger JA et al. Blood 96, 2655-63, 2000‡ Deaglio S et al. Blood 105(8):3042-50, 2005

# Burger JA et al. Blood. 113:3050-8, 2009

?antigen

BCR#

The lymph node microenvironment promotes BCR signaling

CLL cells isolated from lymph nodes showed upregulation of CCL3 and CCL4

LN signature GEPs have a remarkable similarity to GEP of CLL cells after co-cultured with nurselike cells, including upregulation of CCL3, CCL4, EGR2, EGR3, and MYC

From: Y Herishanu et al., Blood. 2011 Jan 13;117(2):563-74

CCL3

CCL4

Summary: molecular interactions in the CLL microenvironment

The moleculoar interactions between CLL cells and their microenvironment are complex

Soluble factors, BCR signaling and cell-cell interactions are important

Chemokine receptors and BCR-associated kinases are current drug targets

From: Burger JA et al., Blood. 2009 Oct 15;114(16):3367-75

Targeting the microenvironment in CLL: Plerixafor (AMD3100)

Plerixafor is a CXCR4 antagonist and blocks HIV-1 entry into T cells

Plerixafor is a bicyclam Plerixafor binds to Asp171

in TM-IV and Asp262 in TM-VI of CXCR4

From: Gerlach LE et al., J. Biol. Chem. 276:14153, 2001

Results: Best Confirmed Response*

0.08 mg/kg

n=3

0.16 mg/kg

n=4

0.24 mg/kg

n=3

0.32 mg/kg

n=7

0.42 mg/kg

n=4

Overalln=21

No. of Evaluable Patients 3 3 2 7 3 18

Evaluable patients with, n (%):

Complete ResponsePartial Response (PR)Stable Disease (SD)Progressive DiseaseRelapsed DiseaseTreatment Failure

02 (67)

01 (33)

00

000

3 (100)00

000

2 (100)00

04 (57)2 (29)1 (14)

00

02 (67)

000

1 (33)

0 8 (44)2 (11)7 (39)

01 (6)

* Responses were assessed based on definitions provided by the National Cancer Institute-sponsored Working Group guidelines for chronic lymphocytic leukemia. Cheson BD, et al; Blood. 1996 Jun 15;87(12):4990-7.

Targeting of BCR signaling in CLL

• BCR-associated kinases are targets of new drugs in preclinical and clinical development

• SYK (Spleen tyrosine kinase) inhibitors: fostamatinib (R788)

• BTK (Bruton’s tyrosine kinase) inhibitors: ibrutinib (formerly: PCI-32765)

• PI3 kinases: Isoform-Selective Inhibitor of PI 3-Kinases, GS-110 (formerly: CAL-101)

From: Nat Rev Immunol 2:945

Pattern of Response: Blood Lymphocytes vs Lymph Nodes

12

Mea

n %

Cha

nge

from

Bas

elin

e

Cycle

ALCSPD

SCR(61) 1(60) 2(57) 3(52) 4(50) 5(51) 6(47) 7(46) 8(48) 9(43) 10(38) 11(31)

SPD- sum of products of lymph node dimension

Inhibition of chemotaxisCXCL12 CXCL13

Ctrl

PCI-327

65 (1

0 nM

)

PCI-327

65 (1

00 n

M)

PCI-327

65 (1

000

nM)

Mig

rate

d c

ells

(%

of i

np

ut)

means of 6 patients ± SEM, *p≤0.05 compared to Medium

Med

ium

AMD31

00

**

+ chemokine

Med

iumCtrl

+ chemokine

**

PCI-327

65 (1

0 nM

)

PCI-327

65 (1

00 n

M)

PCI-327

65 (1

000

nM)

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

GS-1101 (CAL-101) inhibits CLL cell chemotaxis towards CXCL12 and CXCL13

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

GS-1101 (CAL-101) antagonizes CLL cell migration beneath marrow stroma cells (pseudoemperipolesis)

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

PCI-32765: effects on adhesion and migration

From: Rooij et al., Blood 119: 2590-2594, 2012

VCAM-1adhesion assay

Chemotaxisassay

TCL1 MOUSE MODEL OF CLLEFFECTS OF BTK INHIBITOR IBRUTINIB

Vehicle control Ibrutinib 25mg/kg/day

0

10

20

Ctrl(n=4) 2.5

(n=3)25

(n=4)25

(n=4)Outliers

Bo

dy

we

igh

t (g

)

Ibrutinib (mg/kg/day)

P=0.356

P=0.13730 P=0.028

2.5 mg/kg/d 25mg/kg/day

Control + Ibrutinib

0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6S

ple

en

we

igh

t (g

)P=0.64

P=0.05P=0.01

Ctrl(n=4) 2.5

(n=3)25

(n=4)25

(n=4)Outliers

Ibrutinib (mg/kg/day)

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

BCR-RELATED BIOMARKER: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

GS-1101 trial

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

Key effects of inhibitors of BCR-associated kinases Btk, Syk, PI3Kδ

Inhibitors of BCR signaling and block survival and proliferation,

but also homing and tissue retention of CLL cells

Effects on either pathway differ between patients

Adapted after: Burger JA et al., Blood. 2009 Oct 15;114(16):3367-75

Summary and outlook

Chemokine receptors and adhesion molecules are essential for tissue homing and migration of CLL cells

CXCR4 and BCR-associated kinases (SYK, BTK, PI3Kδ) are targeted in clinical trials in CLL

These therapies “mobilize” CLL cells from the tissues into the blood

There is promising clinical activity of these new therapeutic approaches