CLL Stromal cell protect Conventional therapies Versteckspiel im Wald by Friedrich Eduard Meyerheim ( ) 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: From: PE Patten et al. Blood. 2008;111: Messmer BT, et al. J Clin Invest. 115(3): , 03/2005

Microenvironment in CLL:  -SMA and CD14/68 + cells From: J Rual et al. Clinical Cancer Research 12, , 2006 Bhattacharya and Mertens et al., Leukemia (2011) 25, 722–726 CLL#1 CLL#2 CLL#3 normal tonsil

CLL in vitro model: BMSC co-cultures Standardized CLL-stroma co- culture conditions for drug testing Ideal for testing drug combinations

From: Sivina et al., Leukemia 26: , 08/2012

IN VITRO MODEL : Nurselike cells CXCR4 † CXCL12 CXCL13 CXCR5 ¶ CD31, plexin-B1 CD38, CD100 ‡ BAFF, APRIL BAFF-R, BCMA, TACI* * Nishio M et al. Blood 106: , 2005 ¶ Burkle A et al. Blood 110: , 2007 † Burger JA et al. Blood 96, , 2000 ‡ Deaglio S et al. Blood 105(8): , 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 Jan 13;117(2): 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 Oct 15;114(16):

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 Asp 171 in TM-IV and Asp 262 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= mg/kg n= mg/kg n= mg/kg n= mg/kg n=4Overalln=21 No. of Evaluable Patients Evaluable patients with, n (%): Complete Response Partial Response (PR) Stable Disease (SD) Progressive Disease Relapsed Disease Treatment Failure 0 2 (67) 0 1 (33) (100) (100) (57) 2 (29) 1 (14) (67) (33) 0 8 (44) 2 (11) 7 (39) 0 1 (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 Jun 15;87(12):

Targeting of BCR signaling in CLL BCR-associated kinases are targets of new drugs in preclinical and clinical development BCR-associated kinases are targets of new drugs in preclinical and clinical development SYK (Spleen tyrosine kinase) inhibitors: fostamatinib (R788) SYK (Spleen tyrosine kinase) inhibitors: fostamatinib (R788) BTK (Bruton’s tyrosine kinase) inhibitors: ibrutinib (formerly: PCI-32765) BTK (Bruton’s tyrosine kinase) inhibitors: ibrutinib (formerly: PCI-32765) PI3 kinases: Isoform- Selective Inhibitor of PI 3- Kinases, formerly: 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 Mean % Change from Baseline Cycle ALC SPD 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 chemotaxis CXCL12CXCL12CXCL13CXCL13 CtrlCtrl PCI (10 nM) PCI (100 nM) PCI (1000 nM) (% of input) Migrated cells (% of input) means of 6 patients ± SEM, *p≤0.05 compared to Medium MediumMedium AMD3100AMD3100 * * + chemokine MediumMedium CtrlCtrl * * PCI (10 nM) PCI (100 nM) PCI (1000 nM) S. Ponader et al., Blood 119: , 2012

GS-1101 (CAL-101) inhibits CLL cell chemotaxis towards CXCL12 and CXCL13 Hoellenriegel J et al.; Blood 118(13): , 09/2011

GS-1101 (CAL-101) antagonizes CLL cell migration beneath marrow stroma cells (pseudoemperipolesis) Hoellenriegel J et al.; Blood 118(13): , 09/2011

PCI-32765: effects on adhesion and migration PCI-32765: effects on adhesion and migration From: Rooij et al., Blood 119: , 2012 VCAM-1 adhesion assay Chemotaxis assay

TCL1 MOUSE MODEL OF CLL EFFECTS OF BTK INHIBITOR IBRUTINIB Vehicle controlIbrutinib 25mg/kg/day Ctrl (n=4) 2.5 (n=3) 25 (n=4) 25 (n=4) Outliers Body weight (g) Ibrutinib (mg/kg/day) P=0.356 P= P= mg/kg/d25mg/kg/day Control + Ibrutinib Spleen weight (g) P=0.64 P=0.05 P=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: , 2012

BCR-RELATED BIOMARKER: CCL3, CCL4 (MIP-1α,β) BCR-RELATED BIOMARKER: CCL3, CCL4 (MIP-1α,β) CCL3 CCL4 pg/mL time (days) pre-treatment S. Ponader et al., Blood 119: , 2012 pre-treatment Ibrutinib trial GS-1101 trial Hoellenriegel J et al.; Blood 118(13): , 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 Oct 15;114(16):

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