ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets

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1 Supplementary Information ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets Andrew J Souers 1, Joel D Leverson 1, Erwin R Boghaert 1, Scott L Ackler 1, Nathaniel D Catron 1, Jun Chen 1, Brian D Dayton 1, Hong Ding 1, Sari H Enschede 1, Wayne J Fairbrother 2, David C S Huang 3,4, Sarah G Hymowitz 2, Sha Jin 1, Seong Lin Khaw 3,4, Peter J Kovar 1, Lloyd T Lam 1, Jackie Lee 2, Heather L Maecker 2, Kennan C Marsh 1, Kylie D Mason 3 5, Michael J Mitten 1, Paul M Nimmer 1, Anatol Oleksijew 1, Chang H Park 1, Cheol-Min Park 1,7, Darren C Phillips 1, Andrew W Roberts 3 5, Deepak Sampath 2, John F Seymour 4,6, Morey L Smith 1, Gerard M Sullivan 1, Stephen K Tahir 1, Chris Tse 1, Michael D Wendt 1, Yu Xiao 1, John C Xue 1, Haichao Zhang 1, Rod A Humerickhouse 1, Saul H Rosenberg 1 & Steven W Elmore 1 1 AbbVie Inc., 1 North Waukegan Road, North Chicago, Illinois, USA. 2 Genentech, Inc., South San Francisco, California, USA. 3 Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia. 4 Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Melbourne, Victoria, Australia. 5 Department of Clinical Hematology and Bone Marrow Transplantation, Royal Melbourne Hospital, Parkville, Victoria, Australia. 6 Department of Hematology, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia. 7 Present address: Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University, Singapore. Correspondence should be addressed to A.J.S. (andrew.souers@abbvie.com).

2 Supplementary Figures O O O S O NH O N +O H N O N N H N O N Cl Indole-tethered ligand BCL-2 FRET K i <.1 nm BCL-X L FRET K i > 66 nm Supplementary Figure 1. Chemical structure and selected TR-FRET binding K i values for indolecontaining analog of 2 used for X-ray structure analysis with BCL-2. Chemical structure is 4-[4-({4'- chloro-3-[2-(dimethylamino)ethoxy]biphenyl-2-yl}methyl)piperazin-1-yl]-2-(1h-indol-5-yloxy)-n-({3- nitro-4-[(tetrahydro-2h-pyran-4-ylmethyl)amino]phenyl}sulfonyl)benzamide. % Specific apoptosis FL5.12-BCL-2 FL5.12-BCL-X L Navitoclax ABT-199 Supplementary Figure 2. ABT-199 induces apoptosis in FL5.12-BCL-2 cells to a greater magnitude than FL5.12-BCL-X L cells. Specific apoptosis as assessed by quantifying the fraction of sub-g/g1 cells by FACS and normalizing to control-treated samples. FL5.12-BCL-2 and FL5.12-BCL-X L cells were incubated with 1 M ABT-199 or 1 M navitoclax for 24 h. All data represent the means of three separate experiments with error bars indicating the standard error of the mean.

3 MCL-1-NOXA complexes Percentage complex remaining Concentration ABT-199 (µm) Supplementary Figure 3. ABT-199 does not disrupt MCL-1:NOXA interactions in HeLa cells. Quantitative measure of relevant protein complexes as measured by Luciferase activity in presented. HeLa cells stably transfected with a GAL4-driven luciferase reporter and expressing GAL4DBD-MCL-1 and VP16AD-NOXA fusion proteins were incubated with increasing concentrations of ABT-199 for 24 h before assessing luciferase activity as a quantitative measure of the relevant protein complexes. All data represent the means of triplicate experiments with error bars denoting standard deviation. 12 Percent cell viability Concentration (log M) ABT-199 Navitoclax Staurosporine Supplementary Figure 4. ABT-199 does not kill Bak / Bax / mouse embryonic fibroblasts. Cell viability of Bak / Bax / mouse embryonic fibroblasts following incubation with increasing concentrations of ABT-199, navitoclax, or staurosporine for 48 h. All data represent the mean of three separate experiments with error bars indicating the standard error of the mean.

4 ABT-199 EC (µm) Spearman r =.1223 NS, n = BCL-X L protein expression (ng per 3 µg total protein) Supplementary Figure 5. BCL-X L levels do not predict sensitivity to ABT-199 in Non-Hodgkin s Lymphoma cell lines. BCL-X L protein levels were assessed by quantitative immunoblotting and plotted against the corresponding ABT-199 EC 5 for each cell line. No significant correlation was observed between BCL-X L protein levels and ABT-199 EC 5 s.

5 a 3 b Weight (g) Vehicle ABT-199 (1) ABT-199 (5) ABT-199 (25) ABT-199 (12.5) Vehicle ABT-199 (1) ABT-199 (5) Time after first treatment (d) c 3 d Vehicle Vehicle ABT-199 (1) 15 ABT-199 (33) 1 R (1) B (12.5) + R (1) 1 ABT-199 (1) + R (1) ABT-199 (33) + B (12.5) + R (1) 5 B (25) + R (1) 5 ABT-199 (1) + B (25) + R (1) Supplementary Figure 6. Average body weight of xenografted mice does not vary following treatment with ABT-199 as single agent or in combination with chemotherapy. Mice were weighed either in groups (S6a, c, d) or as individuals (S6b) following the various treatments stated in the figure legend. The data were derived from the experiments illustrated in Fig. 4 in the manuscript. Supplementary panels S6a (RS4;11, ALL), S6b (Toledo, NHL), and S6d (Granta-519, MCL) correspond to Fig. 4a, 4b and 4e, respectively. Supplementary panel S6c (DoHH2, NHL) matches Fig. 4c and 4d. Each point represents either the average weight of an entire treatment group (S6a, S6c and S6d) or the mean weight of individual mice (S6b). The error bars in panel S6b reflect the standard error of the mean. The ordinate is the weight in g and the abscissa indicates the period following treatment in d. The dose is indicated in parentheses in the legends. ABT-199 was dosed orally, bendamustine (B) and rituximab (R) were dosed intravenously.

6 Platelet count ( 1 9 L -1 ) Dose (mg) Supplementary Figure 7. Prolonged dosing of ABT-199 does not cause thrombocytopenia. Platelet counts of first three patients (11 blue, 13 red, 14 black) during initial 12 weeks of continuing therapy with ABT-199 are presented. The graph shows the platelet counts for each patient from initial single dosing (week ; pale blue arrow, with dose for each subject listed by color) and through planned continuous dosing weeks The bars below the graph indicate the daily dose of ABT-199 for each patient during this time. For each, dosing continued beyond the week 12 assessment.

7 a b Tumor volume (mm 3 ) 2,5 2, 1,5 1, vehicle ABT-199 (2) ABT-199 (1) ABT-199 (5) ABT-199 (25) ABT-199 (12.5) ABT-199 (6.25) ABT-199 (3.125) ABT-199 (1.56) Tumor volume (mm 3 ) 3, 2, 1, vehicles ABT-199 (5) BR B R + ABT-199 (5) B R + ABT-199 (25) B R + ABT-199 (12.5) Time after first treatment (d) Time after first treatment (d) Supplementary Figure 8. ABT-199 inhibits xenograft growth as a single agent or in combination with rituximab and chemotherapy. The growth inhibition of Toledo (NHL, panel a) and Granta-519 (MCL, panel b) is presented. Each graph describes the change of tumor volume (mm 3, ordinate) in function of the time after initiation of treatment (days, abscissa). Each point on the curves represents the mean of 1 tumors. Error bars depict the standard error of the mean. ABT-199 was administered orally once a day for 21 d. The dose of ABT-199 in mg per kg body weight (mg kg -1 ) is indicated in the graph legend as the number between parentheses. The combination of bendamustine and rituximab (BR) used in experiment S7b consisted of bendamustine at 25 mg kg -1 and rituximab at 1 mg kg -1. Both agents were administered as a single intravenous dose on day.

8 Supplementary Tables Supplementary Table 1. Binding affinity of navitoclax and compound 2 against BCL-2 and BCL-X L by fluorescence polarization assay. The lower limit of detection in the assay is 1 nm. Fluorescence polarization assay K i (nm) Compound BCL-2 BCL-X L Navitoclax < 1 < Supplementary Table 2. Binding affinity of navitoclax and ABT-199 for BCL-2 protein family members by TR-FRET. TR-FRET K i (nm) Compound BCL-2 BCL-X L MCL-1 BCL-W Navitoclax > ABT-199 <.1 48 >

9 Supplementary Table 3. Cell killing activity of navitoclax or ABT-199 against cell lines derived from NHL subtypes and acute myeloid and lymphoid leukemias. For DLBCL and follicular lymphoma (FL) lines, BCL2 copy number (CN), the presence or absence of t(14;18) translocation, and expression of BCL2, BCL-X L and MCL-1 proteins were determined by standard methods. Abbreviations: ABC Activated B cell subtype; GCB Germinal Center B cell subtype; PMBL Primary mediastinal B cell lymphoma. Cell TiterGlo cell viability assay BCL2 expression ng per 3 g protein BCL2L1 expression ng per 3 g protein MCL1 expression ng per 3 g protein navitoclax EC 5 ( M) ABT-199 EC 5 ( M) Tumor Type Sub-type Cell Line Mean s.e.m n Mean s.e.m n BCL2 CN t(14;18) status BCL2 status ABC SUDHL negative Low ABC U negative High ABC HBL negative High ABC OCI-Ly negative High GCB SUDHL positive High GCB OCI-Ly negative Low GCB OCI-Ly positive High GCB SUDHL negative Low GCB WSU-NHL positive High DLBCL GCB OCI-Ly negative Low GCB OCI-Ly positive High GCB HT negative Low GCB OCI-Ly positive High GCB OCI-Ly positive High GCB OCI-Ly positive High GCB SUDHL positive High nd Ri negative High nd NUDHL negative Low PMBL K negative Low PMBL U negative Low Karpas positive High WSU-DLCL positive High FL DoHH positive High SC positive High RL positive High ALL AML MCL Cell TiterGlo cell viability assay navitoclax EC 5 ( M) ABT-199 EC 5 ( M) SEM T-cell Molt RS4; KG-1a Pre-B-cell MV HBL Granta Rec Mino Z UPN JVM SP JVM Jeko

10 Supplementary Table 4. Data collection and refinement statistics (molecular replacement) for BCL- 2:navitoclax X-ray structure. BCL-2:navitoclax Data collection Space group P2(1) Cell dimensions a, b, c (Å) 34.77, 68.8, ( ) 9., 95.96, 9. Resolution (Å) R sym or R merge.6 (.2) I / I 12.8 (4.1) Completeness (%) 99.5 (96.7) Redundancy 4.2 (3.9) Refinement Resolution (Å) No. reflections R work / R free 16.9/22.2 No. atoms Protein 2324 Ligand/ion 35 Water 175 B-factors Protein Ligand/ion Water R.m.s. deviations Bond lengths (Å).1 Bond angles ( ).99 * Data from one crystal were used for molecular replacement *Values in parentheses are for highestresolution shell.

11 Supplementary Table 5. Data collection and refinement statistics (molecular replacement) for BCL- 2:2 X-ray structure. BCL-2:2 Data collection Space group P6(1) Cell dimensions a, b, c (Å) , , ( ) 9., 9., 12. Resolution (Å) R sym or R merge.8 (.38) I / I 1.3 (3.3) Completeness (%) 99.7 (99.2) Redundancy 7.1 (3.8) Refinement Resolution (Å) No. reflections 2997 R work / R free 18./21.2 No. atoms Protein 1191 Ligand/ion 279 Water 231 B-factors Protein Ligand/ion Water R.m.s. deviations Bond lengths (Å).1 Bond angles ( ).93 * Data from one crystal were used for molecular replacement *Values in parentheses are for highestresolution shell.

12 Supplementary Table 6. Data collection and refinement statistics (molecular replacement) for BCL- 2:indole analog of 2 X-ray structure. BCL-2:indole analog of 2 Data collection Space group P4(3) Cell dimensions a, b, c (Å) , , ( ) 9., 9., 9. Resolution (Å) R sym or R merge.7 (.187) I / I 14.7 (1.) Completeness (%) 99.9 (99.5) Redundancy 7.5 (6.2) Refinement Resolution (Å) No. reflections 3912 R work / R free 21.6/23.8 No. atoms Protein 2266 Ligand/ion 426 Water 255 B-factors Protein Ligand/ion Water R.m.s. deviations Bond lengths (Å).1 Bond angles ( ) 1.6 * Data from one crystal were used for molecular replacement *Values in parentheses are for highestresolution shell.