Jon Lewis

Abstract 2944: AT-IAP, a dual cIAP1 and XIAP antagonist with oral antitumor activity in melanoma models.

Melanoma is a highly aggressive malignancy with an exceptional ability to develop resistance and no curative therapy is available for patients with metastatic disease. Inhibitor of apoptosis proteins (IAP) play a key role in preventing cell death by apoptosis. In normal cell, IAPs are highly regulated by endogenous antagonists (e.g. SMAC) but in melanoma cell lines and in patient samples expression levels of IAPs are generally high and depleting IAPs by siRNA tended to reduce cell viability, with XIAP reduction being the most efficient [1]. Small molecule IAP antagonists have the ability to switch IAP-controlled pro-survival pathways towards apoptosis and cell death. Recent evidence suggests that a true dual antagonist of both cIAP1 and XIAP will promote an effective apoptotic response through generation of death-inducing ripoptosome complexes, with resultant caspase activation [2, 3]. We have used our fragment-based drug discovery technology PyramidTM to derive a non-peptidomimetic IAP antagonist, AT-IAP, which does not have an alanine warhead and has nanomolar cellular potency for both XIAP and cIAP1. Initial pharmacokinetic and pharmacodynamic modeling of AT-IAP in mice bearing the MDA-MB-231 cell line indicated that daily oral dosing of AT-IAP at 30 mg/kg ensures high concentrations of compound in tumor and plasma over a 24 h period with resultant inhibition of both XIAP and cIAP1 and induction of apoptosis markers (cleaved PARP and cleaved caspase-3). In this paper, we describe the characterization of AT-IAP in melanoma models. An in vitro cell line proliferation screen demonstrated that 36% of melanoma cell lines exhibited enhanced sensitivity to AT-IAP, which was improved on addition of exogenous 1 ng/ml TNF-α (92% of cell lines were sensitive to AT-IAP + TNF-α). Sensitivity of melanoma cells to AT-IAP has also been confirmed in a panel of 20 primary melanoma tumors in colony formation assays set up in the presence and absence of added TNF-α. Finally, a set of biomarkers has been identified and used to predict single agent activity of AT-IAP in a range of melanoma cell line and patient derived xenograft models. [1] Engesaeter et al., Cancer Biology & Therapy, 2011, 12 (1), 47 [2] Ndubaku et al., ACS Chem Biol., 2009, 4 (7), 557 [3] Meier, P., Nat Rev. Cancer, 2010, 10 (8), 561 Citation Format: Gianni Chessari, Ahn Maria, Ildiko Buck, Elisabetta Chiarparin, Joe Coyle, James Day, Martyn Frederickson, Charlotte Griffiths-Jones, Keisha Hearn, Steven Howard, Tom Heightman, Petra Hillmann, Aman Iqbal, Christopher N. Johnson, Jon Lewis, Vanessa Martins, Joanne Munck, Mike Reader, Lee Page, Anna Hopkins, Alessia Millemaggi, Caroline Richardson, Gordon Saxty, Tomoko Smyth, Emiliano Tamanini, Neil Thompson, George Ward, Glyn Williams, Pamela Williams, Nicola Wilsher, Alison Woolford. AT-IAP, a dual cIAP1 and XIAP antagonist with oral antitumor activity in melanoma models. [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 2944. doi:10.1158/1538-7445.AM2013-2944

Abstract 1287: The dual IAP antagonist, ASTX660, increases the anti-tumor activity of paclitaxel in preclinical models of triple-negative breast cancer in vivo

Background: Paclitaxel-mediated secretion of inflammatory mediators, including TNFα, potentially creates paracrine and autocrine signaling loops that can reduce paclitaxel-induced apoptosis with resultant cancer cell survival (paclitaxel resistance). One mechanism of cancer cell survival is the expression of inhibitor of apoptosis proteins (IAPs): Cellular IAP (cIAP) is involved in inflammatory pro-survival NF-κB activation, blocking the activation of effector caspases 3 and 7, while X-linked IAP (XIAP) directly binds the effector caspases 3, 7 and 9, inhibiting the full activation of the apoptotic pathway. Antagonism of IAPs in the setting of paclitaxel treatment may enhance the cancer cell death by switching the chemotherapy-induced inflammatory TNFα signaling from pro-survival to apoptosis. ASTX660 is an orally bioavailable dual antagonist of cIAP and XIAP, currently being investigated in a single-agent Phase 1/2 clinical trial in patients with advanced solid tumors and lymphomas (NCT02503423). Here, we characterize the activity of ASTX660 in triple-negative breast cancer (TNBC) preclinical models as a single agent and in combination with paclitaxel. Results: We first investigated the viability of 21 TNBC cell lines treated with ASTX660 in vitro and found that 43% were sensitive either to ASTX660 alone (MDA-MB-231 and HCC38) or in the presence of exogenous TNFα (HCC1806, Hs578T, BT549, HCC1395, DU4475, MDA-MB-453 and mouse EMT6). In HCC1806 xenografts in mice, both ASTX660 (daily oral treatment) and paclitaxel (weekly intravenous treatment) as single agents caused moderate tumor growth inhibition but not regression. ASTX660 and paclitaxel combination treatment, however, caused regression and all tumors achieved a partial response by Day 19 of treatment. An increase in circulating acute-phase-proteins in animals was also observed on paclitaxel treatment. Conclusions: Our study suggests that paclitaxel treatment causes an inflammatory response which could qualitatively change the tumor environment. Inhibition of IAPs in such inflammatory environment induced by paclitaxel may lead to rapid apoptotic cell death. Thus, addition of IAP antagonist may be a promising approach to enhance the TNBC response to paclitaxel therapy. Citation Format: Tomoko Smyth, Yoko Nakatsuru, Ryoto Fujita, George Ward, Luke Bevan, Jon Lewis, Nicola Wallis, John Lyons. The dual IAP antagonist, ASTX660, increases the anti-tumor activity of paclitaxel in preclinical models of triple-negative breast cancer in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1287.

Abstract 1794: The HSP90 inhibitor, AT13387, combined with erlotinib improves response in EGFR-driven xenograft models of NSCLC

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Although Epidermal Growth Factor Receptor (EGFR)-driven non-small cell lung cancer (NSCLC) can be successfully treated with EGFR inhibitors such as erlotinib, relapse is common due to the development of resistance. Methods to improve response and delay resistance are therefore of value. Inhibition of the chaperone, HSP90, leads to the depletion of many client proteins, including EGFR, and has the capacity to simultaneously affect many signalling pathways, offering an alternative strategy for targeting EGFR-driven disease. AT13387 is a potent, second generation HSP90 inhibitor currently being tested in Phase II clinical trials. Here we investigated the effects of combining AT13387 and erlotinib in models of EGFR-driven NSCLC. Results: AT13387 single-agent treatment was effective in EGFR-driven NSCLC xenograft models, significantly inhibiting tumor growth of both HCC827 and NCI-H1650 xenograft tumors when dosed once weekly (see table). As expected, treatment with erlotinib caused significant tumor regression in all HCC827 tumors, but was less effective in the NCI-H1650 model; its PTEN status conferring a reduction in sensitivity. However, combination of AT13387 with erlotinib led to a further enhancement of tumor growth inhibition over either of the monotherapies in both of these models. All HCC827 tumors treated with the combination regressed further (by at least 80%), and regression was also now observed in all NCI-H1650 tumors. These data demonstrate that combination with AT13387 has the potential to increase response, despite the differing initial sensitivities to erlotinib of these models. The combination was well tolerated. View this table: Conclusions: AT13387 was shown to improve response when combined with erlotinib in EGFR-driven xenograft models. These data suggest that there is therapeutic potential in an upfront combination of an HSP90 inhibitor, such as AT13387, with erlotinib and support clinical investigation of such a combination. Citation Format: Tomoko Smyth, Jon Lewis, Keisha Hearn, Neil Thompson, John Lyons, Nicola G. Wallis. The HSP90 inhibitor, AT13387, combined with erlotinib improves response in EGFR-driven xenograft models of NSCLC. [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 1794. doi:10.1158/1538-7445.AM2014-1794

Abstract A55: Potent, dual cIAP1/XIAP antagonists induce apoptosis in a melanoma stem cell population.

The inhibitor of apoptosis proteins (IAP) are key regulators of anti-apoptotic and pro-survival signaling pathways. Overexpression of IAPs occurs in various cancers and has been associated with tumor progression and resistance to treatment. IAP antagonists activate the E3 ligase function of cIAP1 and stimulate rapid autoubiquitylation and proteosomal degradation of both cIAP1 and cIAP2. Elimination of these proteins leads to a switch in TNFα signalling from being pro-survival to being pro-apoptotic. However, a strong pro-apoptotic effect from cIAP loss cannot be achieved without sustained antagonism of XIAP-mediated caspase inhibition. Therefore, a best in class profile for IAP antagonists requires potent dual antagonism of cIAP1 and XIAP. Astex has used fragment based-drug discovery to develop a second generation of IAP antagonists, which are non-peptidomimetic and do not contain an alanine as a warhead. This series has the ability not only to efficiently degrade cIAP1 but also to potently antagonize XIAP, delivering a dual cIAP1/XIAP inhibitory profile which is not apparent in the first generation of IAP antagonists based on an alanine warhead. Here, we report the structural understanding of the unique molecular profile of the series together with the enhanced activity of these compounds in melanoma cancer stem cells (CSC). CSC populations are more resistant to apoptosis than the bulk cell population and they have been associated with resistance to cancer therapy, relapse and cancer progressions. Blockade of the apoptotic pathway by up-regulation of anti-apoptotic factors has been implicated in conferring resistance in CSC fractions and increased XIAP expression has also been reported in these cells. We have analysed the CD133+ population of three melanoma cell lines (SK-MEL-2, SK-MEL-5 and SK-MEL-28) and measured activation of caspase-3 (NucView™ cell staining) after treatment with IAP antagonists in presence of TNFα. Our potent dual cIAP1/XIAP antagonists (XIAP EC50 35 nM in cells). The enhanced XIAP potency of our compounds is overriding the resistance in CSC subpopulations, highlighting the importance of dual antagonism in promoting efficient induction of apoptosis. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A55. Citation Format: Gianni Chessari, Maria Ahn, Keisha Hearn, Christopher N. Johnson, Jon Lewis, Neil Thompson, George Ward, Pamela Williams. Potent, dual cIAP1/XIAP antagonists induce apoptosis in a melanoma stem cell population. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A55.

Abstract B298: The controlled local expression of IL-12 as an immunotherapeutic treatment of glioma through the use of the Rheoswitch Therapeutic System® (RTS®) platform.

The challenges of developing immunotherapies to treat glioma include the immune- privileged status of the CNS and the physiological processes that contribute to the suppression of immune responses in the brain. The paucity of dendritic cells (DC) in the brain combined with lack of lymphatic drainage, the production of inflammatory mediators, and the physical blood-brain barrier provide several challenges in eliciting tumor specific responses. We have previously assessed the safety and biologic activity of two different RheoSwitch Therapeutic System® (RTS®) controlled IL-12 expression-based therapeutic candidates in Phase I clinical trials for the treatment of metastatic melanoma: AD-RTS-hIL-12 (AD) and DC-RTS-hIL-12 (DC), along with the activator ligand veledimex, also known as INXN 1001 (AL). Based upon preliminary favorable results observed for these two product candidates, we chose to study the activity of RTS-mIL-12 delivered intratumorally by either a dendritic cell or an adenoviral vector-based approach in an orthotopic murine glioma model. The ability and the extent of AL to cross the blood brain barrier (BBB) were assessed in both C57B6 mice and cynomolgus monkeys. The results in mice demonstrated that AL crosses the BBB (peak and trough level of 67-16 ng/mL in cerebral spinal fluid (CSF) over the dose range of 450-900 mg/m2/day) which exceeded tumor AL levels previously shown to induce the production of mIL-12 and resulted in a >50% tumor growth inhibition in the 4T1 mouse model. AL also crossed the BBB in a similar fashion in the cynomolgus monkey. The effects of DC and AD on survival were assessed in the mouse orthotopic GL261 glioma model where each animal received 1 x 105 GL261 glioma cells via intracranial injection (i.c.) on Day 0. In this study murine dendritic cells were transduced at increasing multiplicity of infection (MOI) of 100, 500, 1,000, 5,000, or 10,000 of AD vector particles (vp)/cell. The transduced DCs were intratumorally (i.t.) administered at 1 x 106 DC /animal, on Day 5. AD was administered i.t. at 1 x 108, 5 x 108, 1 x 109, or 5 x 109 vp/animal on Day 5. AL was administered ad libitum in the chow starting on Day 4 (∼675 mg/m2/day) and continued for the duration of the study. DC + AL or AD + AL both clearly demonstrated a dose-related increase in survival benefit without exhibiting an adverse safety profile. All animals treated with DC > 5000 MOI + AL or AD 5 x 109 vp + AL survived throughout the duration of the study (100% survival at 75 days) with no adverse clinical signs observed. In contrast, the mean survival in the control groups was 22 ± 3 days. Additional studies in this model are ongoing to determine the optimal dose and schedule. This novel regulated immunotherapeutic approach could potentially be translated into an effective clinical regimen for the treatment of glioma. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B298. Citation Format: John A. Barrett, Lei Sun, Charles Reed, Laurent Humeau, Francois Lebel, Beth A. Hollister, Kay R. Meshaw, Jon Lewis. The controlled local expression of IL-12 as an immunotherapeutic treatment of glioma through the use of the Rheoswitch Therapeutic System® (RTS®) platform. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B298.

Abstract 2018: Discovery of potent dual inhibitors of both XIAP and cIAP1 using fragment based drug discovery

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL XIAP and cIAP1 are members of the inhibitor of apoptosis (IAP) protein family. Both proteins have the ability to attenuate apoptosis induced through intrinsic and extrinsic stimuli via inhibition of caspase-3, -7, -8 and -9. The defining feature of both XIAP and cIAP1 is the presence in their protein sequence of 3 Baculoviral IAP Repeat (BIR) domains, which are necessary for their antiapoptotic activity. The mitochondrial protein SMAC uses its N-terminal region (AVPI) to interact with BIR domains and deactivate the antiapoptotic function of IAPs. Several companies and academic groups have active programs developing SMAC peptidomimetic compounds based on the AVPI motif. In general, those compounds have the tendency to be cIAP1 selective like their tetrapeptide progenitor (AVPI IC50 values for XIAP-BIR3 and cIAP1-BIR3 are 0.3 uM and 0.016 uM respectively). Using our fragment-based screening approach, PyramidTM, we identified a non-peptidomimetic chemotype which binds with similar potency to the BIR3 domain of both XIAP and cIAP1. Hit optimisation using a structure based approach led to the discovery of potent true dual XIAP and cIAP1 antagonists with good in vivo physico-chemical profile and no P450 or hERG liabilities. Dual XIAP/cIAP1 inhibitors have potential for more effective apoptosis and less toxicity associated with cytokine production. Compounds were initially characterised in fluorescence polarisation binding assays using XIAP-BIR3 or cIAP1-BIR3 domains. Robust induction of apoptosis was observed in two sensitive breast cancer cell lines (EC50s well below 0.1 uM in EVSA-T and MDA-MB-231); whilst HCT116 cells (colon cancer) were insensitive (unless exogenous TNF-α was added). This in vitro cell line killing was demonstrated to correlate closely with cIAP1 antagonism and hence a parallel cell assay was established to measure XIAP antagonism. An engineered HEK293 cell line was stably co-transfected with full length FLAG-tagged human XIAP cDNA and full length (untagged) human caspase-9 cDNA. Inhibition of caspase-9 binding to XIAP was measured in immunoprecipitation assays. This gave us a sensitive read-out for XIAP antagonism in cells which could be plotted against the most sensitive cell killing read-out (from the EVSA-T cell line) to establish relative XIAP vs cIAP1 selectivities and to select dual antagonists of both IAPs. Potent compounds (HEK293-EC50 <0.01 uM and EVSA-T-EC50 <0.01 uM) were further characterised in PKPD studies in mice bearing MDA-MB-231 xenografts. Compounds with good oral exposure achieved high concentration in tumor over 24h periods which ensured excellent inhibition of both XIAP and cIAP1 with consequent reduction of cIAP1 levels and induction of apoptosis markers (PARP, Caspase-3). Finally, dual XIAP/cIAP1 inhibitors have been investigated in xenograft models (melanoma, breast and colorectal cancer) and have achieved significant efficacy at tolerated doses. Citation 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 2018. doi:1538-7445.AM2012-2018