David Weng

Birinapant (TL32711), a Bivalent SMAC Mimetic, Targets TRAF2-Associated cIAPs, Abrogates TNF-Induced NF-κB Activation, and Is Active in Patient-Derived Xenograft Models

The acquisition of apoptosis resistance is a fundamental event in cancer development. Among the mechanisms used by cancer cells to evade apoptosis is the dysregulation of inhibitor of apoptosis (IAP) proteins. The activity of the IAPs is regulated by endogenous IAP antagonists such as SMAC (also termed DIABLO). Antagonism of IAP proteins by SMAC occurs via binding of the N-terminal tetrapeptide (AVPI) of SMAC to selected BIR domains of the IAPs. Small molecule compounds that mimic the AVPI motif of SMAC have been designed to overcome IAP-mediated apoptosis resistance of cancer cells. Here, we report the preclinical characterization of birinapant (TL32711), a bivalent SMAC-mimetic compound currently in clinical trials for the treatment of cancer. Birinapant bound to the BIR3 domains of cIAP1, cIAP2, XIAP, and the BIR domain of ML-IAP in vitro and induced the autoubiquitylation and proteasomal degradation of cIAP1 and cIAP2 in intact cells, which resulted in formation of a RIPK1:caspase-8 complex, caspase-8 activation, and induction of tumor cell death. Birinapant preferentially targeted the TRAF2-associated cIAP1 and cIAP2 with subsequent inhibition of TNF-induced NF-κB activation. The activity of a variety of chemotherapeutic cancer drugs was potentiated by birinapant both in a TNF-dependent or TNF-independent manner. Tumor growth in multiple primary patient–derived xenotransplant models was inhibited by birinapant at well-tolerated doses. These results support the therapeutic combination of birinapant with multiple chemotherapies, in particular, those therapies that can induce TNF secretion. Mol Cancer Ther; 13(4); 867–79. ©2014 AACR.

Abstract LB-406: Phase 1 study of the Smac mimetic TL32711 in adult subjects with advanced solid tumors and lymphoma to evaluate safety, pharmacokinetics, pharmacodynamics, and antitumor activity

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FLnnIntroduction : TL32711 is a small molecule Smac mimetic that potently and specifically antagonizes inhibitor of apoptosis proteins (IAPs), resulting in caspase-dependent apoptosis and inactivation of NF-kB signaling. In preclinical studies, single agent tumor regression was observed for multiple tumor types and potent anti-tumor activity was observed when TL32711 was combined with specific chemotherapies and death receptor ligands. This first-in-human study assesses the safety, pharmacokinetic (PK), and pharmacodynamic (PD) profile and anti-tumor activity of single agent TL32711.nnMethods : Using a standard 3+3 dose escalation design, subjects with refractory solid tumors and lymphoma received weekly intravenous TL32711 for 3 weeks on/ 1 week off, with disease assessment every 2 cycles. PK analysis was conducted using an intensive sampling approach. PD assays included measurement of cIAP1 and cIAP2 levels in lysates from serially collected PBMC and tumor tissue, serum levels of cleaved cytokeratin-18 (CK-18, requiring activated caspase-3) and activated caspase-3/7.nnResults : 27 patients were treated at doses 0.18–26 mg/m2 over 9 cohorts. TL32711 was well-tolerated, with no dose-limiting toxicities. Toxicities included reversible grade 2 lymphocytopenia and grade 1 rash at ≥11.5 mg/m2. TL32711 exhibited dose-proportional, uniform, non-accumulating PK, with a mean β-phase half-life of 35 hrs. Doses ≥2.88 mg/m2 achieved an AUC0-∞ exposure sufficient for single agent activity in preclinical tumor models. At doses ≥1.44 mg/m2, cIAP1 levels were suppressed >80% within 24 hrs and after 1 week >50% suppression was maintained. Based on PBMC drug levels, actual and predicted cIAP1 suppression was well-correlated. A dose-dependent increase in serum levels of cleaved CK-18 and activated caspase-3/7 was observed in patients treated at doses ≥2.88 mg/m2. At 11.5 mg/m2, > 90% cIAP1 suppression, increased activated caspase-8, and PARP cleavage in tumor lysates were observed in a tumor biopsy from a melanoma patient with progressive disease prior to TL32711, with stable disease after 2 cycles. One colon cancer subject with progressive disease after prior therapies, at 0.36 mg/m2 demonstrated tumor shrinkage in radiographic lesions, serum CEA decline, and elevated serum caspase-3/7. One colon cancer subject at 17.2 mg/m2 demonstrated serum CEA decline, elevated serum caspase-3/7, and a large photopenic lesion in a metastatic lesion within the first cycle.nnConclusions : TL32711 is well-tolerated, has dose-proportional PK, and demonstrates potent and sustained target inhibition and apoptotic pathway activation in tumor and surrogate tissues. Preliminary evidence of antitumor activity was observed in colon cancer and melanoma. Updated results of final dose escalation cohorts will be presented.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-406. doi:10.1158/1538-7445.AM2011-LB-406

Abstract A25: Phase 1 PK/PD analysis of the Smac-mimetic TL32711 demonstrates potent and sustained cIAP1 suppression in patient PBMCs and tumor biopsies.

The Smac mimetic TL32711 differentially targets members of the inhibitor of apoptosis proteins (cIAP1, cIAP2 and XIAP) involved in the blockade of apoptosis. TNFα-activated pro-survival signaling pathway is maintained by the cIAPs. TL32711 causes rapid autoubiquitinylation and proteosomal degradation of the cIAP1 converting TNFα signaling from pro-survival to pro-apoptotic and potently inhibiting TNFα driven canonical NF-kB signaling. TL32711 also relieves inhibition of caspase-3 and -7 by XIAP to further potentiate apoptosis. The pharmacokinetics (PK) and pharmacodynamics (PD) of TL32711 have been studied in human tumor xenografts, patient plasma/PBMCs and Phase 1 tumor biopsy samples. In mice bearing the MDA-MB-231 xenograft, TL32711 is rapidly and extensively taken up into the tumor (tumor/plasma AUC ratio >22) and is eliminated slowly with a half-life of 96 hrs (20 hrs in plasma). A PK/PD link model was used to characterize the relationship between TL32711 tumor concentrations and cIAP1 suppression. cIAP1 suppression was dose and time dependent with cIAP1 levels reduced to 70% inhibition maintained 7–14 days post treatment following a single IV bolus dose (5 mg/kg). TL32711 had a potent effect on tumor cIAP1 levels (EC50 24 ng/g) and caused significant tumor growth inhibition and regressions at doses ≥2.5 mg/kg q3D. Efficacy has also been evaluated in primary human melanoma tumors, recently derived from patients and transplanted into nude mice. Significant tumor growth inhibition was observed in 5/6 primary melanoma tumor xenografts with mean Day 7 tumor concentrations of 187, 579 and 2658 ng/g at 15, 30 and 60 mg/kg respectively. TL32711 PK/PD (drug concentration analysis and cIAP1 degradation in PBMCs and tumor biopsies) has also been investigated in patients as part of the single agent Phase I study. Following weekly, 30 min IV infusions TL32711 plasma PK was dose proportional and non-accumulating (0.18 to 47 mg/m2). Plasma PK was tri-exponential with a long terminal t1/2 (73–79 hrs). The target AUC in plasma for therapeutic activity (71 h.ng/mL) based on the MDA-MB-231 model was achieved in patients at dose >2.88 mg/m2 (Mean AUC 86 h.ng/mL). This exposure was associated with marked uptake and retention in PBMCs (t1/2 = 29–35hrs) and resulted in prolonged cIAP1 suppression over 7 days. A dose related increase in PBMC PARP cleavage and plasma caspase-3 activity was also observed indicative of apoptosis pathway activation. TL32711 PK/PD was also assessed in tumor biopsy samples from patients 4 hrs to 6 days post treatment (11.5 to 17.2 mg/m2). TL32711 is extensively taken up into the tumor with levels >350 ng/g on day 6, significantly in excess of the EC50 for cIAP1 inhibition. Estimated tumor exposure at 35 to 47 mg/m2 was also in excess of the measured drug levels observed at 15 to 30 mg/kg in the primary human tumor xenograft models in mice. Together these PK/PD data show that TL32711 results in potent and sustained cIAP1 suppression over 7 days at tolerable dose levels with evidence of apoptosis pathway activation and promising early signs of efficacy in patients with solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A25.

Abstract 1939: TL32711, a novel Smac mimetic, exerts significant antitumor efficacy in primary pancreatic adenocarcinoma model

Introduction. Pancreatic cancer is highly resistant to treatment. Inhibitors of apoptosis (IAPs) were overexpressed in pancreatic cancer cells and IAPs downregulation were shown to induce sensitivity to death receptor signaling, cytotoxic agents and radiation. TL32711, a novel Smac mimetic, is a potent IAP antagonist that induces caspase-dependent apoptosis and NFkB canonical pathway inactivation. Here, we investigated the efficacy of TL32711 using a patient-derived primary pancreatic cancer explant model that mirrors the disease9s biological heterogeneity. Methods. Effect of TL32711 alone and with TRAIL was evaluated in Panc1 by immunoblotting and Trypan blue staining. Dose escalation studies were performed in 2 primary pancreatic tumors at i.p. 30 mg/kg, 45 mg/kg and 60 mg/kg twice weekly and tumor volume were measured for 28 days. No significant toxicity was observed in tumor-bearing mice at all dose levels. An additional 6 primary pancreatic tumors were evaluated at 60 mg/kg. HE 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1939. doi:1538-7445.AM2012-1939

Loss of expression of the uvomorulin gene in compaction-defective embryonal carcinoma cells

The lack of uvomorulin protein in the 6B(NG)C25 subline of H6 embryonal carcinoma cells is due to loss of expression of the uvomorulin gene, without evidence of gross alteration in the gene itself. This suggests that the dominant mutation in 6B(NG)C25 may involve atrans-acting factor involved in the regulation of uvomorulin gene expression.

Abstract 3336: The Smac Mimetic Birinapant Synergistically Induces Apoptosis in Combination with Type I Interferons and GM-CSF.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnThe Inhibitor of Apoptosis (IAP) protein family is able to inhibit cell death and promote survival signaling triggered by a wide variety of stimuli. Two members of this family in particular, cIAP1 and cIAP2, play key roles in enabling tumor necrosis factor alpha (TNFα)-induced nuclear factor-kappa beta (NF-kB) activation and inflammatory responses. Birinapant (TL32711) is a bivalent Smac Mimetic that antagonizes multiple IAP family members resulting in the conversion of TNFα-induced survival signaling into an apoptotic response, an effect which is more prominent in tumor cells than their normal counterparts. Combining birinapant with agents capable of increasing the levels of TNFα in the tumor microenvironment represents a novel approach to cancer treatment. We have tested the ability of interferon-alpha/-beta (IFN-α, IFN-β) and granulocyte macrophage colony stimulating factor (GM-CSF), both known to be immunomodulatory agents, for their ability to stimulate TNFα production by peripheral blood mononuclear cells (PBMC). Treatment with each cytokine induced the production of TNFα by cultured PBMC as measured by ELISA. Supernatants of IFN-α/-β and GM-CSF-treated PBMC cultures were able to sensitize resistant tumor cells to Smac Mimetics in a TNFα-dependent manner. Furthermore, the combination of birinapant and either IFN-β or GM-CSF synergistically inhibited the development of RENCA tumors in a syngeneic mouse prevention model. Due to the highly aggressive nature of the syngeneic tumors, this model is performed by simultaneous implantation of tumor cells and initiation of dosing of test agents. Activity in this model is measured by prevention of tumor outgrowth. Birinapant in combination with IFN-β resulted in only 5 of 18 measurable tumors, versus 19 of 20 tumors in vehicle-treated mice, 16 of 20 tumors in IFN-β alone-treated mice and 13 of 20 tumors in birinapant alone-treated mice. In addition, when tumor growth occurred it was delayed in mice treated with the combination. Similarly, the birinapant and GM-CSF combination treatment resulted in 9 of 20 tumors, versus 20 of 20 tumors in the vehicle-treated mice, 20 of 20 tumors in the GM-CSF alone-treated mice, and 16 of 20 tumors in the birinapant alone-treated mice. In addition when tumors did develop in the combination-treated mice, their growth was substantially delayed: 4 of 10 mice treated with the combination of birinapant and GM-CSF were still on study on day 85 post initiation - approximately 40 days post dosing - with no measureable tumor. The combination of birinapant with immunomodulatory agents that induce TNF represents a potential novel therapeutic strategy.nnCitation Format: Christopher A. Benetatos, Jennifer M. Burns, Ernest C. Borden, Daniel Lindner, Yasuhiro Mitsuuchi, Mark A. Mckinlay, Gurpreet Singh Kapoor, Eric M. Neiman, Martin E. Seipel, Guangyao Yu, Martin Graham, David Weng, Stephen M. Condon, C. Glenn Begley, Srinivas Chunduru. The Smac Mimetic Birinapant Synergistically Induces Apoptosis in Combination with Type I Interferons and GM-CSF. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3336. doi:10.1158/1538-7445.AM2013-3336

Abstract 3333: Birinapant, a novel bivalent Smac mimetic drug, is superior to monovalent Smac mimetics in inhibition of NF-kB by targeting TRAF2-bound cIAP1 and cIAP2.

A variety of Smac-mimetic drugs have been developed to target the inhibitor of apoptosis (IAP) proteins including X chromosome-linked IAP (XIAP), cellular IAP proteins (cIAP1 and cIAP2) and ML-IAP whose gene mutations, amplifications and chromosomal translocations have been implicated in various malignancies. The IAPs also play an important role in multiple signaling pathways including the TNFα-mediated NF-kB and MAPK pathways (inflammatory responses) and pattern recognition receptor signaling pathways (innate immunity). Smac-mimetics have been designed to mimic the IAP-binding motif of the second mitochondria-derived activator of caspase (Smac). The IAP-binding motif consists of four amino acids (AVPI) that serve as an endogenous IAP antagonist. Structurally different Smac-mimetic compounds have been published and showed different binding affinity to IAP proteins. These differences in structure and binding have important consequences in terms of biological activity. Here, we present evidence that bivalent Smac-mimetics are superior to monovalent Smac-mimetics in their ability to inhibit NF-kB in cells stimulated with TNFα. Over 300 monovalent and 300 bivalent Smac-mimetics, including compounds whose structures have been published, were tested for ability to degrade cIAP1 and cIAP2, and inhibit NF-kB in cell-based assays. Inhibition of NF-kB by bivalent Smac-mimetics correlated (R²=0.78) with the degradation of cIAP1 over a range of four logs, while monovalent Smac-mimetics did not (R²=0.20). Furthermore, monovalent Smac-mimetics degraded TRAF2-bound cIAP1 and cIAP2 less effectively (1/10-1/100 fold) compared to bivalent Smac-mimetics. While bivalent Smac-mimetics effectively degraded cIAP1 and cIAP2, birinapant (TL32711), a bivalent Smac-mimetic currently in Phase 2 clinical trials, was unique in its ability to preferentially degrade TRAF2-bound cIAP1 and cIAP2. Inhibition of NF-kB by birinapant was further characterized by the ImageStream cytometry, which showed that the nuclear translocation of p65 in response to TNFα stimulation was blocked in both HeLa and HL-60 cells. These data demonstrate that bivalent Smac-mimetics are superior to monovalent Smac-mimetics in degrading TRAF2-bound cIAPs, in inhibition of NF-kB and efficiently mediating cell death. Furthermore, the unique profile of birinapant versus other bivalent Smac-mimetics may explain its preclinical and clinical safety profile. Citation Format: Yasuhiro Mitsuuchi, Stephen M. Condon, Eric M. Neiman, Christopher A. Benetatos, Martin E. Seipel, Gurpreet Singh Kapoor, Angeline C. Mufalli, Guangyao Yu, Orla Maguire, Hans Minderman, Mark A. Mckinlay, Martin Graham, David Weng, Srinivas Chunduru. Birinapant, a novel bivalent Smac mimetic drug, is superior to monovalent Smac mimetics in inhibition of NF-kB by targeting TRAF2-bound cIAP1 and cIAP2. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3333. doi:10.1158/1538-7445.AM2013-3333

Abstract 3857: Smac mimetic TL32711 and TRAIL synergistically induce apoptosis of germinal center B lymphoma cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, ILnnTL32711, a Smac mimetic in clinical testing, potently targets Inhibitor of Apoptosis Proteins (IAPs, including cIAPs and XIAP) to unblock intrinsic and extrinsic pathways, enabling caspase-dependent apoptosis via multiple signals including TRAIL (Tumor Necrosis Factor-Related Apoptosis Inducing Ligand). TL32711 also inactivates canonical and activates non-canonical NF-κB signaling through cIAPs. We investigated the pro-apoptotic effects of TL32711, alone and in combination with TRAIL to activate the extrinsic pathway, in a panel of B cell lymphoma cell lines representing germinal center/follicular (GC) vs activated B cell (ABC) histologies. We hypothesized that the efficacy of this potential combination therapeutic strategy might differ between GC and ABC lymphoma types, as ABC are reported to be NF-kB dependent. We used the following EBV negative cell lines: WSU- FSCCL t(14:18)+ follicular lymphoma (FL), FC-TxFL2 t(14:18)+ transformed FL and SU-DHL4 GC-type diffuse large B cell lymphoma (DLBCL) as examples of GC origin lymphomas. U2932 and TMD8 served as ABC-type DLBCL. Apoptosis was determined by annexin V staining after 48 hr incubation (TL32711 added 2-3 hr after TRAIL). The contribution of the different apoptotic pathways was determined by caspase-3 (common), -8 (extrinsic), and-9 (intrinsic) activity, assessed by flow cytometric methods and Western blot. TL32711 had little effect as a single agent on any of these B cell lymphoma cell lines at ≤ 100nM, though apoptosis was induced at 10-20 µM in GC types. TRAIL alone (10-25 ng/ml) induced apoptosis in the 3 GC lines, but had little effect on the 2 ABC lines. Combination of TL32711 and TRAIL at low concentrations showed marked apoptosis in GC lines, with minimal to no effect for each agent alone. In the combination-treated GC types, caspases -3, -8 and -9 were significantly induced, confirming activation of both pathways. We had previously shown in FSCCL and TxFL2 that caspase-8 induction also cleaved tBID, activating the intrinsic apoptotic pathway via caspase-9. GC subtypes, including FL, transformed FL and GC-DLBCL were sensitive to TRAIL and TL32711 at clinically achievable concentrations for TL32711, with supra-additive combination effects. ABC-type DLBCL were not sensitive to either agent alone or in combination. We are investigating whether ABC-DLBCL are truly dependent on canonical NF-κB activation for survival or have other mechanisms of resistance to TRAIL and IAP inhibition. These data suggest that clinical trials of TL32711 may focus on FL and GC lymphoma in combination with TRAIL or TRAIL-agonists.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3857. doi:1538-7445.AM2012-3857