Keisha Hearn

Inhibition of HSP90 by AT13387 Delays the Emergence of Resistance to BRAF Inhibitors and Overcomes Resistance to dual BRAF and MEK Inhibition in Melanoma Models

Emergence of clinical resistance to BRAF inhibitors, alone or in combination with MEK inhibitors, limits clinical responses in melanoma. Inhibiting HSP90 offers an approach to simultaneously interfere with multiple resistance mechanisms. Using the HSP90 inhibitor AT13387, which is currently in clinical trials, we investigated the potential of HSP90 inhibition to overcome or delay the emergence of resistance to these kinase inhibitors in melanoma models. In vitro, treating vemurafenib-sensitive cells (A375 or SK-MEL-28) with a combination of AT13387 and vemurafenib prevented colony growth under conditions in which vemurafenib treatment alone generated resistant colonies. In vivo, when AT13387 was combined with vemurafenib in a SK-MEL-28, vemurafenib-sensitive model, no regrowth of tumors was observed over 5 months, although 2 of 7 tumors in the vemurafenib monotherapy group relapsed in this time. Together, these data suggest that the combination of these agents can delay the emergence of resistance. Cell lines with acquired vemurafenib resistance, derived from these models (A375R and SK-MEL-28R) were also sensitive to HSP90 inhibitor treatment; key clients were depleted, apoptosis was induced, and growth in 3D culture was inhibited. Similar effects were observed in cell lines with acquired resistance to both BRAF and MEK inhibitors (SK-MEL-28RR, WM164RR, and 1205LuRR). These data suggest that treatment with an HSP90 inhibitor, such as AT13387, is a potential approach for combating resistance to BRAF and MEK inhibition in melanoma. Moreover, frontline combination of these agents with an HSP90 inhibitor could delay the emergence of resistance, providing a strong rationale for clinical investigation of such combinations in BRAF-mutated melanoma. Mol Cancer Ther; 13(12); 2793–804. ©2014 AACR.

Emergence of resistance to tyrosine kinase inhibitors in non-small-cell lung cancer can be delayed by an upfront combination with the HSP90 inhibitor onalespib

Background:Tyrosine kinase inhibitors, such as crizotinib and erlotinib, are widely used to treat non-small-cell lung cancer, but after initial response, relapse is common because of the emergence of resistance through multiple mechanisms. Here, we investigated whether a frontline combination with an HSP90 inhibitor could delay the emergence of resistance to these inhibitors in preclinical lung cancer models.Methods:The HSP90 inhibitor, onalespib, was combined with either crizotinib or erlotinib in ALK- or EGFR-activated xenograft models respectively (H2228, HCC827).Results:In both models, after initial response to the monotherapy kinase inhibitors, tumour relapse was observed. In contrast, tumour growth remained inhibited when treated with an onalespib/kinase inhibitor combination. Analysis of H2228 tumours, which had relapsed on crizotinib monotherapy, identified a number of clinically relevant crizotinib resistance mechanisms, suggesting that HSP90 inhibitor treatment was capable of suppressing multiple mechanisms of resistance. Resistant cell lines, derived from these tumours, retained sensitivity to onalespib (proliferation and signalling pathways were inhibited), indicating that, despite their resistance to crizotinib, they were still sensitive to HSP90 inhibition.Conclusions:Together, these preclinical data suggest that frontline combination with an HSP90 inhibitor may be a method for delaying the emergence of resistance to targeted therapies.

Discovery of a Potent Nonpeptidomimetic, Small-Molecule Antagonist of Cellular Inhibitor of Apoptosis Protein 1 (cIAP1) and X-Linked Inhibitor of Apoptosis Protein (XIAP).

XIAP and cIAP1 are members of the inhibitor of apoptosis protein (IAP) family and 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. Structure-based drug design (SBDD) guided by structural information from X-ray crystallography, computational studies, and NMR solution conformational analysis was successfully applied to a fragment-derived lead resulting in AT-IAP, a potent, orally bioavailable, dual antagonist of XIAP and cIAP1 and a structurally novel chemical probe for IAP biology.

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 2688: The HSP90 inhibitor, onalespib (AT13387), delays the emergence of resistance to erlotinib in an EGFR-driven xenograft model

Background: Kinase inhibitors have been used successfully in the clinic, but relapse is common due to the emergence of resistance. Inhibition of HSP90 leads to the depletion of oncogenic ‘client’ proteins and the simultaneous inhibition of many signalling pathways. As such, the use of HSP90 inhibitors to overcome resistance has been widely investigated. Onalespib (AT13387) is a potent, second generation HSP90 inhibitor, which has been studied in many preclinical models of kinase inhibitor resistance. Recent findings indicate that an upfront combination of onalespib and either vemurafenib or crizotinib, in models of mutant BRAF melanoma or ALK-translocated non-small cell lung cancer (NSCLC), can delay the emergence of resistance to these therapies. Here we have extended this work to a combination of onalespib and erlotinib in an EGFR-driven NSCLC model. Methods: Mice bearing HCC827 tumor xenografts were dosed with either 12.5 mg/kg erlotinib po qd, or a combination of 12.5 mg/kg erlotinib and 55 mg/kg onalespib ip qw for 52 weeks. Results: Initially both the erlotinib single-agent and onalespib/erlotinib combination treatments significantly inhibited tumor growth of the EGFR-driven NSCLC HCC827 xenograft model; all tumors in the monotherapy and combination groups regressed rapidly and achieved complete response (CR) (median times to CR were 79 and 56 days for monotherapy and combination treatments respectively). However, after 20 weeks of continuous treatment, tumor relapse was observed in one of the xenografts dosed with erlotinib only and this was followed by regrowth of a further 2 tumors in this group over the 52-week study period, with these tumors reaching 50% of the original tumor volumes by 26 and 46 weeks. Of the remaining tumors, 5 out of 6 were detectable at the end of the study. In comparison, tumor growth remained inhibited in the group treated with the erlotinib/onalespib combination; no tumors were observed to regrow over the course of the study and none (of 9) were palpable when treatments were terminated. This suggests that while treatment with monotherapy erlotinib in this model leads to relapse, as seen in the clinic, combination treatment with onalespib can delay the emergence of resistance. Conclusions: These data demonstrate that an upfront combination of onalespib and erlotinib can delay the emergence of resistance in an EGFR-driven xenograft model, suggesting there is therapeutic potential for an upfront erlotinib/onalespib combination in the clinic. Onalespib is currently being tested in a Phase 2 clinical trial in combination with crizotinib in ALK-positive NSCLC. The preclinical data presented here, alongside our previous data on combinations of onalespib with vemurafenib or crizotinib, suggest that the concept of using an HSP90 inhibitor combination upfront in the clinic to delay resistance could be extended to further targeted therapies. Citation Format: Aurelie Courtin, Tomoko Smyth, Keisha Hearn, John Lyons, Neil Thompson, Nicola G. Wallis. The HSP90 inhibitor, onalespib (AT13387), delays the emergence of resistance to erlotinib in an EGFR-driven xenograft model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2688. doi:10.1158/1538-7445.AM2015-2688

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 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