Disruption of the mTOR-eIF4F Axis by Selectively Targeting PI3K d and - PowerPoint PPT Presentation

Disruption of the mTOR-eIF4F Axis by Selectively Targeting PI3K d and Proteasome Potently Inhibits Cap Dependent Translation of c-Myc in Aggressive Lymphomas Changchun Deng, M.D., Ph.D. Mark Lipstein, B.S. Luigi Scotto, Ph.D. Michael Mangone, Ph.D. Owen A. O’Connor , M.D., Ph.D. Columbia University Medical Center Department of Medicine Center for Lymphoid Malignancies

Therapeutic Strategy Targeting c-Myc in Cancer Is Urgently Needed • C-MYC rearrangement is a risk factor for poor survival in diffuse large B cell lymphoma (DLBCL) Savage et al. Blood 2009; Barrans et al. JCO 2010; Copie-Bergman et al. Blood 2015 • C-MYC expression is a risk factor for poor survival in DLBCL Green TM et al., J Clin Oncol. 2012; Johnson NA, et al., J Clin Oncol. 2012; Hu S et al., Blood. 2013 • However, no drugs specifically targeting the activity of c-Myc have been approved for any cancer. • C-Myc is a master transcription factor, and lacks enzymatic activity • Structurally, c-Myc lacks globular functional domains for small molecule targeting • The extended interaction between the c-Myc and Max offers no apparent site for positioning a small-molecule inhibitor. • Targeting the BET bromodomains is a promsing strategy for c-Myc driven cancer McKeown and Bradner, CSH Perspective 2014

Potential Strategies to Silence the Translation of c-Myc through Targeting the mTOR-eIF4F Axis Carfilzomib TGR-1202 (Cfz) (TG) PI3K d Bortezomib Idelalisib (Bz) (Cal) eIF4E 4EBP1 AKT Proteasome Amino acids mTOR ? ? P P 4EBP1 eIF4E (eIF4F) eIF4G c-Myc Translation eIF4A Suraweera, A., et al., Mol Cell, 2012 Quy, P.N., et al., J Biol Chem, 2013 Hutter, G., et al., Leukemia, 2012 Dibble CC and Cantley LC. Zhang, Y., et al., Nature, 2014 Trends Cell Biol, 2015

PI3K d Inhibitors and Proteasome Inhibitors Synergistically Inhibit DLBCL Observed Inhibition in the DLBCL cell line LY10 Carfilzomib (nM) Bortfezomib (nM) TGR-1202 ( m M) Cal-101 ( m M)

Dual Inhibition of PI3K d and Proteasome Is Most Synergistic with TG&Cfz Followed by Cal&Cfz > TG&Bz > Cal&Bz Excess Over BLISS (EOB) in the DLBCL cell line LY10 Carfilzomib (nM) Bortfezomib(nM) TGR-1202 ( m M) Cal-101 ( m M)

Dual Inhibition of PI3K d and Proteasome Is Most Synergistic with TG&Cfz Followed by Cal&Cfz > TG&Bz > Cal&Bz L y 1 0 1 0 0 O b s e rv e d % In h ib itio n 7 5 5 0 2 5 0 0 2 5 5 0 7 5 1 0 0 E x p e c te d % In h ib itio n

TGR-1202&Carfilzomib Is the Most Synergistic Combination in DLBCL, Mantle Cell Lymphoma, and Multiple Myeloma L y 1 S U D H L -2 S U D H L -4 1 0 0 1 0 0 1 0 0 7 5 7 5 7 5 Observed Inhibition (%) 5 0 5 0 5 0 2 5 2 5 2 5 0 0 0 0 2 5 5 0 7 5 1 0 0 0 2 5 5 0 7 5 1 0 0 0 2 5 5 0 7 5 1 0 0 J e k o -1 Z 1 3 8 M M .1 s 1 0 0 1 0 0 1 0 0 7 5 7 5 7 5 5 0 5 0 5 0 2 5 2 5 2 5 0 0 0 0 2 5 5 0 7 5 1 0 0 0 2 5 5 0 7 5 1 0 0 0 2 5 5 0 7 5 1 0 0 Expected Inhibition (%)

TGR-1202&Carfilzomib Is the Most Synergistic Combination in T Cell Lymphoma H 9 HH 1 0 0 1 0 0 7 5 7 5 Observed Inhibition (%) 5 0 5 0 2 5 2 5 0 0 0 2 5 5 0 7 5 1 0 0 0 2 5 5 0 7 5 1 0 0 P F 3 8 2 P 1 2 1 0 0 1 0 0 7 5 7 5 5 0 5 0 2 5 2 5 0 0 0 2 5 5 0 7 5 1 0 0 0 2 5 5 0 7 5 1 0 0 Expected Inhibition (%)

TGR-1202 and Carfilzomib Are Synergistic in Primary CLL and MCL Cells but Not Toxic to Healthy Lymphocytes T G 7 .5 m M + C fz C a l 7 .5 m M + B z C fz B z T G 2 .5 m M + C fz T G 5 m M + C fz C a l 5 m M + B z C a l 2 .5 m M + B z a . b . C L L # 1 C L L # 1 1 0 0 1 0 0 C e ll V ia b ility (% ) 7 5 7 5 5 0 5 0 2 5 2 5 0 0 Healthy lymphocytes 2 .5 5 .0 7 .5 2 .5 5 .0 7 .5 c . d . C L L # 2 C L L # 2 PBMC - 24HR PBMC - 48HR 1 0 0 1 0 0 C e ll V ia b ility (% ) PBMC - 72HR 7 5 7 5 120% 5 0 5 0 100% 2 5 2 5 80% 0 0 Viability 2 .5 5 .0 7 .5 2 .5 5 .0 7 .5 60% e . f. M C L M C L 40% 1 0 0 1 0 0 C e ll V ia b ility (% ) 20% 7 5 7 5 5 0 5 0 0% 2 5 2 5 0 0 2 .5 5 .0 7 .5 2 .5 5 .0 7 .5 [C fz ] n M [B z ] n M

TGR-1202 and Carfilzomib Synergistically Induce Apoptosis in Lymphoma Cell Lines and Primary Lymphoma Cells PARP Cleavage (A:LY7) (C: PF382) PARP PARP b-actin b-Actin (D: CLL-1) PARP (B: LY10) b-actin PARP (E: CLL-2) b-Actin PARP b-actin Caspase 3/7 Activity (F: LY10) Cal-Bort - Cal+Bortb TG-Cfz - TG+Cfz 10 Fold Change / Control 8 6 4 2 0

TGR-1202 and Carfilzomib Synergistically Inhibit Phosphorylation of 4EBP1 and Expression of c-Myc (A: DLBCL) (B: T-ALL) P-Akt P-4EBP1 4EBP1 c-Myc HIF1a Actin GAPDH (C:CLL-1) (D: CLL-2) P-4EBP1 4EBP1 c-Myc B-Actin (E: MM) P-4EBP1 4EBP1 C-Myc B-Actin

TGR-1202 and Carfilzomib in Combination Inhibit Cap Dependent Translation of c-Myc in DLBCL 150% (B) (A) MYC % control mRNA level of 100% c-Myc 50% Actin 0% (C) (D) MYC % control 150% mRNA level of 100% C-Myc 50% B-Actin 0% (F) R/F Luc (%control) (E) Cap dependent translation of Myc 100% p=0.0013 LucR LucF 50% PolioV IRES MYC UTR 0%

TGR-1202 and Carfilzomib in Combination Inhibit the c-Myc Transcription Program in DLBCL Gene Set 52 : SCHUHMACHER Gene Set 70 : DANG RES Running Enrichment Score (RES) Gene Set 29 : SCHLOSSER Gene Set 32 : KIM RES Gene List Index Gene List Index

Overexpression of eIF4E Suppresses the Synergistic Cytotoxicity of TGR-1202 and Carfilzomib and Increases the Protein Level of c-Myc 100% No Transduction GFP-EV 80% GFP-eIF4E Viability 60% 40% 20% 0% TG5 m M & TG5 m M & TG5 m M & Cfz5nM Cfz6nM Cfz7nM TG5 m M & TG5 m M & TG5 m M & Control Cfz7nM Cfz5nM Cfz6nM c-Myc eIF4E B-Actin

Silence c-Myc through Simultaneous Targeting of the PI3K d and Proteasome Pathways TGR-1202 PI3K d Translation mTOR pp-4EBP1 eIF4F c-Myc Proteasome Carfilzomib • Optimize c-Myc-silencing therapy by targeting phosphorylation of 4EBP1 • Phase I/II clinical trial of TGR-1202 and carfilzomib in relapsed and refractory lymphoma

Thank you! Owen A. O’Connor, M.D., Ph.D. Mark Lipstein, B.S. Luigi Scotto, Ph.D. Michael Mangone, Ph.D. Xavier O. Jirau Serrano, B.S. Center for Lymphoid Malignancies Columbia University Medical Center Shirong Li, Ph.D. & Suzanne Lentzsch, M.D., Ph.D. Hematology & Oncology, CUMC Nicholas Tatonetti, Ph.D. Biomedical informatics, CUMC Charles Karan, Ph.D. Sulzberger Columbia Genome Center, CUMC Funding from: Lymphoma Research Fund Irving Institute (CUMC) Pilot Award TG Therapeutics Amgen/Onyx