1. CLL Stromal cell protect Conventional therapies Versteckspiel im Wald by Friedrich Eduard Meyerheim (1808-1879) 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

2. 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

3. Microenvironment in CLL:  -SMA and CD14/68 + cells From: J Rual et al. Clinical Cancer Research 12, 5622-31, 2006 Bhattacharya and Mertens et al., Leukemia (2011) 25, 722–726 CLL#1 CLL#2 CLL#3 normal tonsil

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

5. From: Sivina et al., Leukemia 26:1812-20, 08/2012

6. 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 106:1012-20, 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 #

7. 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

8. 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

9. 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

10. 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=4Overalln=21 No. of Evaluable Patients 3327318 Evaluable patients with, n (%): Complete Response Partial Response (PR) Stable Disease (SD) Progressive Disease Relapsed Disease Treatment Failure 0 2 (67) 0 1 (33) 00000 3 (100) 00000 2 (100) 000 4 (57) 2 (29) 1 (14) 000 2 (67) 000 1 (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. 1996 Jun 15;87(12):4990-7.

11. 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

12. 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

13. Inhibition of chemotaxis CXCL12CXCL12CXCL13CXCL13 CtrlCtrl PCI-32765 (10 nM) PCI-32765 (100 nM) PCI-32765 (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-32765 (10 nM) PCI-32765 (100 nM) PCI-32765 (1000 nM) S. Ponader et al., Blood 119: 1182-9, 2012

14. GS-1101 (CAL-101) inhibits CLL cell chemotaxis towards CXCL12 and CXCL13 Hoellenriegel J et al.; Blood 118(13):3603-12, 09/2011

15. GS-1101 (CAL-101) antagonizes CLL cell migration beneath marrow stroma cells (pseudoemperipolesis) Hoellenriegel J et al.; Blood 118(13):3603-12, 09/2011

16. PCI-32765: effects on adhesion and migration PCI-32765: effects on adhesion and migration From: Rooij et al., Blood 119: 2590-2594, 2012 VCAM-1 adhesion assay Chemotaxis assay

17. TCL1 MOUSE MODEL OF CLL EFFECTS OF BTK INHIBITOR IBRUTINIB Vehicle controlIbrutinib 25mg/kg/day 0 10 20 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=0.137 30 P=0.028 2.5 mg/kg/d25mg/kg/day Control + Ibrutinib 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 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: 1182-9, 2012

18. 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: 1182-9, 2012 pre-treatment Ibrutinib trial GS-1101 trial Hoellenriegel J et al.; Blood 118(13):3603-12, 09/2011

19. 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

20. 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