Jennifer M. Burns

Birinapant, a Smac-Mimetic with Improved Tolerability for the Treatment of Solid Tumors and Hematological Malignancies

Birinapant (1) is a second-generation bivalent antagonist of IAP proteins that is currently undergoing clinical development for the treatment of cancer. Using a range of assays that evaluated cIAP1 stability and oligomeric state, we demonstrated that 1 stabilized the cIAP1-BUCR (BIR3-UBA-CARD-RING) dimer and promoted autoubiquitylation of cIAP1 in vitro. Smac-mimetic 1-induced loss of cIAPs correlated with inhibition of TNF-mediated NF-κB activation, caspase activation, and tumor cell killing. Many first-generation Smac-mimetics such as compound A (2) were poorly tolerated. Notably, animals that lack functional cIAP1, cIAP2, and XIAP are not viable, and 2 mimicked features of triple IAP knockout cells in vitro. The improved tolerability of 1 was associated with (i) decreased potency against cIAP2 and affinity for XIAP BIR3 and (ii) decreased ability to inhibit XIAP-dependent signaling pathways. The P2 position of 1 was critical to this differential activity, and this improved tolerability has allowed 1 to proceed into clinical studies.

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.

Sensitization of human bladder tumor cells to TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis with a small molecule IAP antagonist

Urothelial carcinoma of the bladder accounts for approximately 5% of all cancer deaths in humans. The large majority of bladder tumors are non-muscle invasive at diagnosis, but even after local surgical therapy there is a high rate of local tumor recurrence and progression. Current treatments extend time to recurrence but do not significantly alter disease survival. The objective of the present study was to investigate the tumoricidal potential of combining the apoptosis-inducing protein TNF-related apoptosis-inducing ligand (TRAIL) with a small molecule inhibitor of apoptosis proteins (IAP) antagonist to interfere with intracellular regulators of apoptosis in human bladder tumor cells. Our results demonstrate that the IAP antagonist Compound A exhibits high binding affinity to the XIAP BIR3 domain. When Compound A was used at nontoxic concentrations in combination with TRAIL, there was a significant increase in the sensitivity of TRAIL-sensitive and TRAIL-resistant bladder tumor lines to TRAIL-mediated apoptosis. In addition, modulation of TRAIL sensitivity in the TRAIL-resistant bladder tumor cell line T24 with Compound A was reciprocated by XIAP small interfering RNA-mediated suppression of XIAP expression, suggesting the importance of XIAP-mediated resistance to TRAIL in these cells. These results suggest the potential of combining Compound A with TRAIL as an alternative therapy for bladder cancer.

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 B163: Pharmacokinetic analysis and preclinical evaluation of the SMAC mimetic TL32711 in an orthotopic human breast tumor xenograft model

Inhibitor of Apoptosis Proteins (IAPs) are cellular proteins that prevent cell death by apoptosis and are frequently overexpressed in cancer cells resulting in elevated levels leading to cancer survival and progression. Currently we are developing a class of compounds called SMAC mimetics that reverse the effects of IAPs including XIAP, cIAP‐1 and cIAP‐2 and ML‐IAP causing cancer cells to undergo apoptosis in a broad range of tumor types. TL32711 is a SMAC mimetic which has demonstrated potent and selective activity in several human tumor cell lines as well as in vitro inhibition of IAP target proteins. To determine translational effects in vivo , we evaluated PK/PD and single agent antitumor activity of TL32711 in an orthotopic human breast tumor xenograft model. Single agent toxicity as well as tumor growth inhibition, delay and regression were monitored in the study. The designated endpoint for the efficacy experiment was a mean control tumor volume of approximately 1 cm 3 . TL32711 administered by intravenous injection on a q3dx5 schedule was well tolerated up to 10 mg/kg alone, the highest dose tested. The minimal effective dose was determined to be 1.25 mg/kg. When tumors were allowed to grow to 260 mm 3 prior to initiation of treatment, the treatment effect was not diminished. PK/PD analysis of TL32711 found a longer half‐life of TL32711 in tumor compared to normal tissue and a time and dose dependent inhibition of the target protein cIAP‐1. The effects on the cIAP‐1 target were in good agreement with the tissues levels measured using a LC/MS method. TL32711 was not found to cross the blood brain barrier in this analysis. Results from these studies confirm translational effects of TL32711 in a preclinical model of human breast cancer. In addition, PK/PD analysis demonstrated dose and time dependent target inhibition in vivo and decreased half life in tumor tissue. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B163.

Abstract 2278: The SMAC-mimetic birinapant regulates autocrine TNF production by caspase-8:RIPK1 complex via p38MAPK pathway

Birinapant (TL32711), a SMAC-mimetic currently in clinical trials, antagonizes multiple members of the inhibitor of apoptosis (IAP) protein family to promote apoptosis in cancer cells through the formation of a caspase-8:RIPK1 complex and induction of autocrine tumor necrosis factor (TNF). Evidently, birinapant-resistant cells do not produce TNF upon drug treatment. However, the mechanism by which birinapant-sensitive cells produce TNF is not clear. Owing to the fact that mitogen-activated protein kinases (MAPKs) regulate a variety of cellular processes, we explored the role of MAPKs in birinapant-induced TNF production. Western blot analyses on a panel of birinapant-sensitive (SK-OV-3, EVSA-T, TOV-21G: all with an IC50 10μM) cell lines showed that birinapant induced rapid and robust phosphorylation of p38MAPK in sensitive cells, which corresponded to increased TNF production as assessed by ELISA. However, resistant cells were not affected at multiple time points. The p38MAPK inhibitor, LY2228820, reduced the levels of birinapant-induced TNF (2-fold with 5nM birinapant after 24h) in birinapant-sensitive cancer cells (SK-OV-3), whereas inhibitors of Erk1/2 (PD98059) and JNK (SP600125) had no effect on TNF production. Interestingly, RNAi-mediated inhibition of p38α (MAPK14) isoform significantly inhibited birinapant-induced TNF production (∼3-fold with 5nM birinapant after 24h) in SK-OV-3 cells while inhibition of p38β (MAPK11) isoform upregulated TNF production (∼3-fold), suggesting that there may be a balance between p38MAPK isoforms to regulate TNF production. Pharmacological and RNAi-mediated inhibition of RIPK1 and caspase-8 activities in SK-OV-3 cells resulted in reduced birinapant-induced p38MAPK phosphorylation and TNF production. Furthermore, commercially available pooled siRNAs targeting different isoforms of cFLIP, a catalytically-inactive homologue of caspase-8, upregulated p38MAPK phosphorylation (∼4-fold) and TNF production (∼8 to 26-fold) in birinapant-resistant cancer cell lines (T24, HT1376 & IGROV-1), and conferred differential sensitivity to the drug treatment (T24-IC50: 7nM; HT1376-IC50: >10nM; IGROV-1-IC50: >1μM). Experiments involving cFLIP long (cFLIP-L) and short (cFLIP-S) isoform-specific siRNAs showed that inhibition of cFLIP-L, but not cFLIP-S, in T24 & HT1376 cells induced p38MAPK phosphorylation and TNF production, and conferred sensitivity to birinapant. However, the combined inhibition of cFLIP-L and cFLIP-S in IGROV-1 cells was required to induce p38MAPK phosphorylation, TNF production and apoptosis. Collectively, these observations indicate that birinapant-induced autocrine TNF production is triggered by the activation of the caspase-8/RIPK1/p38MAPK axis, and that cFLIP isoforms may confer a context-dependent negative regulatory effect. Citation Format: Gurpreet Singh Kapoor, Christopher A. Benetatos, Yasuhiro Mitsuuchi, Eric M. Neiman, Guangyao Yu, Mark A. Mckinlay, Jennifer Burns, John Silke, Stephen M. Condon, Srinivas K. Chunduru. The SMAC-mimetic birinapant regulates autocrine TNF production by caspase-8:RIPK1 complex via p38MAPK pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2278. doi:10.1158/1538-7445.AM2014-2278

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

Abstract A225: Dose scheduling and efficacy analysis of the SMAC mimetic TL32711 in primary melanoma tumor xenotransplant models.

Inhibitor of Apoptosis Proteins (IAPs) are cellular proteins that prevent cell death by apoptosis and are frequently overexpressed in cancer cells resulting in elevated levels leading to tumor survival and progression. SMAC mimetics are a new class of apoptosis-inducing agents that relieve the effects of IAPs including XIAP, cIAP1 and cIAP2 resulting in apoptosis of sensitive tumor cells from a broad range of tumor types. TL32711 is a potent SMAC mimetic currently in Phase 1 clinical trials. To date, a weekly IV infusion schedule has proved to be well tolerated in patients, resulting in sustained cIAP1 suppression throughout the dosing interval. Initial pharmacokinetics modeling of TL32711 in mice bearing the MDA-MB-231 tumor indicated a potential efficacy benefit may be possible with a biweekly dosing schedule. The objectives of the current study were to 1) evaluate the efficacy of TL32711 as a single agent in primary human melanoma tumor xenograft models, 2) assess the efficacy and tolerability of TL32711 in combination with carboplatin and paclitaxel and 3) determine if a biweekly dosing schedule is more effective than weekly administration. Significant tumor growth inhibition was observed in 5 of 6 of the primary melanoma tumor xenografts evaluated following treatment with single agent TL32711 (30 mg/kg, IP). Combining TL32711 with carboplatin and paclitaxel resulted in a further enhancement in anti-tumor efficacy with tumor regressions noted in 4 of the 6 models without any marked changes in tolerability ( 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 A225.

Abstract 1806: Birinapant, a bivalent SMAC-mimetic, promotes efficient cellular IAP E3 ligase activity and formation of a pro-apoptotic RIPK1:caspase-8 complex while monovalent IAP inhibitors are less efficient - implications for therapeutic utility

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnThe second mitochondria-derived activator of caspases (SMAC) is thought to exert its pro-apoptotic activity as a homodimeric protein. Both monovalent and bivalent peptidomimetics of the SMAC tetrapeptide are being developed for cancer therapy. Birinapant/TL32711 is a bivalent SMAC-mimetic that targets the inhibitor of apoptosis (IAP) proteins whose gene abnormalities have been implicated in various cancers. Owing to structural differences between bivalent SMAC-mimetics and monovalent IAP-inhibitors, we sought to compare and contrast the biochemical activity of birinapant with several monovalent IAP-inhibitors including a monovalent- version of birinapant/TL32711, MV711. Previous studies have shown that both bivalent and monovalent agents promote auto-ubiquitylation and subsequent degradation of cIAP1 and cIAP2, which triggers tumor necrosis factor receptor (TNFR)-mediated cell death in certain tumor cell lines. However, birinapant showed substantial differences from IAP-inhibitors in degrading TRAF2-associated cIAP1 and cIAP2. Here we show that MV711 was less efficient at degrading cIAP1 by a factor of 7-fold (16 vs. 118 nM, birinapant vs. MV711, respectively) and inhibiting TNF-mediated NF-κB activation by 220-fold (9 vs. 1985 nM, respectively). In addition, a linker-lengthened variant of birinapant was less able to inhibit NF-κB activation by 71-fold (9 vs. 642 nM, respectively). We also studied the effect of birinapant or IAP-inhibitor treatment on SKOV-3, MDA-MB-231 and EVSA-T cancer cell lines in vitro. Comparable cIAP1 BIR3 domain binding constants and IC50 values for the degradation of cIAP1 and cIAP2 (ΔcIAP1/2) were observed for these two classes of agents, and both birinapant and IAP-inhibitors showed dose-dependent induction of cell death. However, despite such comparable potencies, the IAP-inhibitors did not completely kill SKOV-3 or MDA-MB-231 cells even with concentrations >100-times their ΔcIAP1/2 IC50 values. Birinapant revealed the highest suppression of cancer cell growth in the cell lines tested, even after the agent was removed, whereas the cell lines treated with the IAP-inhibitors showed rapid restoration of cell proliferation within 24 h following removal of the agents. These results suggested that monovalent IAP-inhibitors require maintenance of high steady state levels of drug to effectively suppress tumor growth in vivo. In agreement with their inability to induce cell death, IAP-inhibitors were less efficient in stimulating the formation of a RIPK1:caspase-8 complex when compared to birinapant in EVSA-T or SKOV-3 cells. These observations may be partly attributed to the reduced ability of IAP-inhibitors to degrade TRAF2-associated cIAP1 which serves a central role in the activation of NF-κB via TNFR.nnCitation Format: Yasuhiro Mitsuuchi, Christopher A. Benetatos, Thomas Haimowitz, Yijun Deng, Angeline C. Mufalli, Martin E. Seipel, Jennifer M. Burns, Gurpreet S. Kapoor, C. Glenn Begley, Stephen M. Condon. Birinapant, a bivalent SMAC-mimetic, promotes efficient cellular IAP E3 ligase activity and formation of a pro-apoptotic RIPK1:caspase-8 complex while monovalent IAP inhibitors are less efficient - implications for therapeutic utility. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1806. doi:10.1158/1538-7445.AM2014-1806