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Supplementary tables 1-6 summarise the kinase panel screen data (1) mouse PK parameters (2) and further details of the anti-tumour activity (5) conferred by ASTX029, plus details of cell lines used in this study (3 and 4) and further details of the effects of ASTX029 and other MAPK inhibitors in models of MAPK resistance (6).

XIAP antagonism measurements in HEK293-XIAP-Caspase-9 xenografts

XIAP antagonism in A375 cells in the presence or absence of TNF-α

Preparation of 2-chloro-1-{6-[(4-fluorophenyl)methyl]-5-(hydroxymethyl)-3,3-dimethyl-1H,2H,3H-pyrrolo[3,2-b]pyridin-1-yl}ethan-1-one

Fold induction in secreted cytokine concentrations in PBMC supernatants after IAP antagonist treatment

Supplementary figures 1-7 show the effects of AST029 on ERK signalling in RAS-mutant cell lines (1) and tumour xenograft tissue (3), ASTX029 plasma PK linearity (2), ASTX029 activity in a cell line panel showing MAPK mutation status (4) the effects of ASTX029 on mouse body weight (5), PKPD effects of ASTX029 following b.i.d dosing to Colo205 tumour-bearing mice (6) and PD effects of ASTX029 following qd dosing to A375R tumour-bearing mice (7).

PDF file, 289KB, Western immunoblots and their densitometric analyses showing the pharmacodynamic response of GIST430 xenograft to 22 days of AT13387, imatinib or combination treatment.

PDF file, 56KB, Graphs showing the typical dose response of GIST882, GIST430 and GIST48 cell lines to AT13387 and imatinib.

Because of their roles in the evasion of apoptosis, inhibitor of apoptosis proteins (IAP) are considered attractive targets for anticancer therapy. Antagonists of these proteins have the potential to switch prosurvival signaling pathways in cancer cells toward cell death. Various SMAC-peptidomimetics with inherent cIAP selectivity have been tested clinically and demonstrated minimal single-agent efficacy. ASTX660 is a potent, non-peptidomimetic antagonist of cIAP1/2 and XIAP, discovered using fragment-based drug design. The antagonism of XIAP and cIAP1 by ASTX660 was demonstrated on purified proteins, cells, and in vivo in xenograft models. The compound binds to the isolated BIR3 domains of both XIAP and cIAP1 with nanomolar potencies. In cells and xenograft tissue, direct antagonism of XIAP was demonstrated by measuring its displacement from caspase-9 or SMAC. Compound-induced proteasomal degradation of cIAP1 and 2, resulting in downstream effects of NIK stabilization and activation of noncanonical NF-κB signaling, demonstrated cIAP1/2 antagonism. Treatment with ASTX660 led to TNFα-dependent induction of apoptosis in various cancer cell lines in vitro, whereas dosing in mice bearing breast and melanoma tumor xenografts inhibited tumor growth. ASTX660 is currently being tested in a phase I–II clinical trial (NCT02503423), and we propose that its antagonism of cIAP1/2 and XIAP may offer improved efficacy over first-generation antagonists that are more cIAP1/2 selective. Mol Cancer Ther; 17(7); 1381–91. ©2018 AACR.

Comparison of XIAP activity, cIAP1 activity and selectivity of clinical IAP antagonists

The majority of gastrointestinal stromal tumors (GIST) are characterized by activating mutations of KIT, an HSP90 client protein. Further secondary resistance mutations within KIT limit clinical responses to tyrosine kinase inhibitors, such as imatinib. The dependence of KIT and its mutated forms on HSP90 suggests that HSP90 inhibition might be a valuable treatment option for GIST, which would be equally effective on imatinib-sensitive and -resistant clones. We investigated the activity of AT13387, a potent HSP90 inhibitor currently being evaluated in clinical trials, in both in vitro and in vivo GIST models. AT13387 inhibited the proliferation of imatinib-sensitive (GIST882, GIST-T1) and -resistant (GIST430, GIST48) cell lines, including those resistant to the geldanamycin analogue HSP90 inhibitor, 17-AAG. Treatment with AT13387 resulted in depletion of HSP90 client proteins, KIT and AKT, along with their phospho-forms in imatinib-sensitive and -resistant cell lines, irrespective of KIT mutation. KIT signaling was ablated, whereas HSP70, a marker of HSP90 inhibition, was induced. In vivo, antitumor activity of AT13387 was showed in both the imatinib-sensitive, GIST-PSW, xenograft model and a newly characterized imatinib-resistant, GIST430, xenograft model. Induction of HSP70, depletion of phospho-KIT and inhibition of KIT signaling were seen in tumors from both models after treatment with AT13387. A combination of imatinib and AT13387 treatment in the imatinib-resistant GIST430 model significantly enhanced tumor growth inhibition over either of the monotherapies. Importantly, the combination of AT13387 and imatinib was well tolerated. These results suggest AT13387 is an excellent candidate for clinical testing in GIST in combination with imatinib. Mol Cancer Ther; 11(8); 1799–808. ©2012 AACR.

IncuCyte images from neutralization of TNF-α activity with anti-TNF-α experiment in MDA-MB-231 cells

Additional data from ASTX669 in vivo xenograft studies

Additional information to provide an insight into the full kinase selectivity profile of AT7519 and full details of its pharmakokinetic parameters

Cyclin-dependent kinases (CDK), and their regulatory cyclin partners, play a central role in eukaryotic cell growth, division, and death. This key role in cell cycle progression, as well as their deregulation in several human cancers, makes them attractive therapeutic targets in oncology. A series of CDK inhibitors was developed using Astex's fragment-based medicinal chemistry approach, linked to high-throughput X-ray crystallography. A compound from this series, designated AT7519, is currently in early-phase clinical development. We describe here the biological characterization of AT7519, a potent inhibitor of several CDK family members. AT7519 showed potent antiproliferative activity (40-940 nmol/L) in a panel of human tumor cell lines, and the mechanism of action was shown here to be consistent with the inhibition of CDK1 and CDK2 in solid tumor cell lines. AT7519 caused cell cycle arrest followed by apoptosis in human tumor cells and inhibited tumor growth in human tumor xenograft models. Tumor regression was observed following twice daily dosing of AT7519 in the HCT116 and HT29 colon cancer xenograft models. We show that these biological effects are linked to inhibition of CDKs in vivo and that AT7519 induces tumor cell apoptosis in these xenograft models. AT7519 has an attractive biological profile for development as a clinical candidate, and the tolerability and efficacy in animal models compare favorably with other CDK inhibitors in clinical development. Studies described here formed the biological rationale for investigating the potential therapeutic benefit of AT7519 in cancer patients. [Mol Cancer Ther 2009;8(2):324–32]

Supplementary Data from Biological characterization of AT7519, a small-molecule inhibitor of cyclin-dependent kinases, in human tumor cell lines

The MAPK signaling pathway is commonly upregulated in human cancers. As the primary downstream effector of the MAPK pathway, ERK is an attractive therapeutic target for the treatment of MAPK-activated cancers and for overcoming resistance to upstream inhibition. ASTX029 is a highly potent and selective dual-mechanism ERK inhibitor, discovered using fragment-based drug design. Because of its distinctive ERK-binding mode, ASTX029 inhibits both ERK catalytic activity and the phosphorylation of ERK itself by MEK, despite not directly inhibiting MEK activity. This dual mechanism was demonstrated in cell-free systems, as well as cell lines and xenograft tumor tissue, where the phosphorylation of both ERK and its substrate, ribosomal S6 kinase (RSK), were modulated on treatment with ASTX029. Markers of sensitivity were highlighted in a large cell panel, where ASTX029 preferentially inhibited the proliferation of MAPK-activated cell lines, including those with BRAF or RAS mutations. In vivo, significant antitumor activity was observed in MAPK-activated tumor xenograft models following oral treatment. ASTX029 also demonstrated activity in both in vitro and in vivo models of acquired resistance to MAPK pathway inhibitors. Overall, these findings highlight the therapeutic potential of a dual-mechanism ERK inhibitor such as ASTX029 for the treatment of MAPK-activated cancers, including those which have acquired resistance to inhibitors of upstream components of the MAPK pathway. ASTX029 is currently being evaluated in a first in human phase I–II clinical trial in patients with advanced solid tumors (NCT03520075).

Because of their roles in the evasion of apoptosis, inhibitor of apoptosis proteins (IAP) are considered attractive targets for anticancer therapy. Antagonists of these proteins have the potential to switch prosurvival signaling pathways in cancer cells toward cell death. Various SMAC-peptidomimetics with inherent cIAP selectivity have been tested clinically and demonstrated minimal single-agent efficacy. ASTX660 is a potent, non-peptidomimetic antagonist of cIAP1/2 and XIAP, discovered using fragment-based drug design. The antagonism of XIAP and cIAP1 by ASTX660 was demonstrated on purified proteins, cells, and in vivo in xenograft models. The compound binds to the isolated BIR3 domains of both XIAP and cIAP1 with nanomolar potencies. In cells and xenograft tissue, direct antagonism of XIAP was demonstrated by measuring its displacement from caspase-9 or SMAC. Compound-induced proteasomal degradation of cIAP1 and 2, resulting in downstream effects of NIK stabilization and activation of noncanonical NF-κB signaling, demonstrated cIAP1/2 antagonism. Treatment with ASTX660 led to TNFα-dependent induction of apoptosis in various cancer cell lines in vitro, whereas dosing in mice bearing breast and melanoma tumor xenografts inhibited tumor growth. ASTX660 is currently being tested in a phase I–II clinical trial (NCT02503423), and we propose that its antagonism of cIAP1/2 and XIAP may offer improved efficacy over first-generation antagonists that are more cIAP1/2 selective. Mol Cancer Ther; 17(7); 1381–91. ©2018 AACR.

Whilst mitogen-activated protein kinase (MAPK) pathway inhibitors are approved therapies in mutant-BRAF driven cancers and have demonstrated a high response rate in the clinic, the duration of response is often short-lived. Approaches targeting the immune system have elicited durable responses and led to the approval of checkpoint inhibitors such as the anti-PD1 therapy, pembrolizumab, in indications where MAPK pathway activation is often observed, such as melanoma. Activation of the MAPK pathway has been associated with an immunosuppressive tumor microenvironment (TME). Further, preclinical studies have demonstrated that in addition to inhibiting MAPK activity in tumor cells, MAPK pathway inhibitors such as the BRAFV600E inhibitor dabrafenib or MEK inhibitor trametinib have promoted a more proinflammatory TME leading to upregulated antigen presentation on tumor cells, increased CD8+ T cell infiltration and tumor cell killing. Similar preclinical results were recently reported for a KRASG12C inhibitor, AMG 510, where treatment of in vivo models led to an increase in tumor infiltrating lymphocytes, macrophages, dendritic cells and an increase in active immune gene signatures leading to tumor immunity. We have recently described the discovery of ASTX029, which is currently undergoing clinical development in advanced solid tumors (NCT03520075). ASTX029 is a dual-mechanism ERK1/2 (ERK) inhibitor, inhibiting both the catalytic activity and phosphorylation of ERK, and shows potent inhibition of MAPK-activated tumor growth in preclinical models. Previous studies have demonstrated a difference in regulation of genes involved in response to type I interferon following treatment with dual-mechanism compared to catalytic ERK inhibitors. We investigated whether treatment with ASTX029 modulates antigen presentation and the TME in MAPK-activated tumors. Following treatment of cell lines in vitro, we observed ASTX029-dependent changes consistent with increased antigen presentation, including an increase in cell surface expression of MHC class I and the increase in gene expression of tumor-specific antigens gp100 and MART-1 following treatment of melanoma cells. We have previously demonstrated that ASTX029 has good bioavailability following oral dosing in mice. To further investigate the effect of ASTX029 on the TME, syngeneic tumors grown in immunocompetent mice dosed with ASTX029 were characterized in terms of immune cell composition of the TME and gene expression. Our results were consistent with increased immune activation, including increased interferon signaling and a change in the immune cell composition of the TME. These data demonstrate that treatment with ASTX029 leads to modulation of the TME and we hypothesize that to optimize therapeutic activity, ASTX029 could be partnered either with an immunomodulatory or tumor-directed agent. Citation Format: Chris Hindley, Andrea Biondo, Kim-Hien Dao, Matthew Davis, Lynsey Fazal, Roberta Ferraldeschi, Keisha Hearn, Vanessa Martins, Harpreet Saini, Martin Sims, Nicola Wallis, Nicola Wilsher, Harold Keer, John Lyons, Joanne Munck. Immune modulation by the dual-mechanism ERK inhibitor, ASTX029, in MAPK-activated tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1661.

Resistance to available hormone therapies in prostate cancer has been associated with alternative splicing of androgen receptor (AR) and specifically, the expression of truncated and constitutively active AR variant 7 (AR-V7). The transcriptional activity of steroid receptors, including AR, is dependent on interactions with the HSP90 chaperone machinery, but it is unclear whether HSP90 modulates the activity or expression of AR variants. Here, we investigated the effects of HSP90 inhibition on AR-V7 in prostate cancer cell lines endogenously expressing this variant. We demonstrate that AR-V7 and full-length AR (AR-FL) were depleted upon inhibition of HSP90. However, the mechanisms underlying AR-V7 depletion differed from those for AR-FL. Whereas HSP90 inhibition destabilized AR-FL and induced its proteasomal degradation, AR-V7 protein exhibited higher stability than AR-FL and did not require HSP90 chaperone activity. Instead, HSP90 inhibition resulted in the reduction of AR-V7 mRNA levels but did not affect total AR transcript levels, indicating that HSP90 inhibition disrupted AR-V7 splicing. Bioinformatic analyses of transcriptome-wide RNA sequencing data confirmed that the second-generation HSP90 inhibitor onalespib altered the splicing of at least 557 genes in prostate cancer cells, including AR. These findings indicate that the effects of HSP90 inhibition on mRNA splicing may prove beneficial in prostate cancers expressing AR-V7, supporting further clinical investigation of HSP90 inhibitors in malignancies no longer responsive to androgen deprivation. Cancer Res; 76(9); 2731–42. ©2016 AACR.

The MAPK pathway is frequently dysregulated in cancer, resulting in constitutive phosphorylation and activation of its downstream signaling node ERK1/2 (ERK). The direct targeting of activated ERK is being explored in several phase I clinical trials. A good understanding of the relationship between inhibition of pathway activity and inhibition of tumor growth allows dose scheduling in the clinic to maximise response to an inhibitor. We have recently described the development of a novel, potent and selective small molecule inhibitor of both ERK activity and phosphorylation using fragment-based drug discovery. Using this inhibitor as a tool compound, we explored the relationship between duration of ERK inhibition and inhibition of tumor growth. We examined the in vivo activity of our ERK inhibitor in a Colo205 xenograft model. Inhibition of the ERK pathway was monitored by measuring phosphorylation of a direct substrate of ERK, RSK90 (pRSK), and phosphorylated ERK itself (pERK) to determine pharmacodynamic profiles. We observed that pathway inhibition was consistent with the pharmacokinetic profile of the ERK inhibitor and that the duration of pathway inhibition was primarily determined by dosing frequency rather than the total dose of ERK inhibitor. High, single doses (50 or 40 mg/kg) of ERK inhibitor produce strong maximal pathway inhibition at 2 h post dose (pRSK levels 8% of control levels for a single dose of 40 mg/kg) but pRSK levels have returned to levels close to control values by 12 h post dose. In contrast, 3x equally spaced doses of 20 mg/kg have a lower maximal pathway inhibition at 2 h post dose (pRSK levels 29% of control) but extend pathway inhibition beyond 12 h (pRSK levels 21% of control at 14 h). Despite these two different pharmacodynamic profiles, we observed comparable tumor growth inhibition following daily dosing either at 1 × 50 mg/kg (maximal tumor growth inhibition of 21%) or 3 × 20 mg/kg (maximal tumor growth inhibition of 15%). These data suggest that a short duration of ERK inhibition rather than constant suppression can be sufficient to provide anti-tumor activity. This may allow flexibility in clinical dosing schedules to improve tolerability without a loss in efficacy. Citation Format: Christopher Hindley, Luke Bevan, Hannah Braithwaite, Aurélie Courtin, Lynsey Fazal, Roberta Ferraldeschi, Vanessa Martins, Sandra Muench, Nicola Wallis, Nicola Wilsher, John Lyons, Joanne Munck. Different pharmacodynamic profiles of ERK1/2 inhibition can elicit comparable anti-tumor activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3030.

The ubiquitously expressed protein tyrosine phosphatase SHP2 is required for signalling downstream of receptor tyrosine kinases (RTKs) and plays a role in regulating many cellular processes. Recent advances have shown that genetic knockdown and pharmacological inhibition of SHP2 suppresses RAS/MAPK signalling and inhibits proliferation of RTK-driven cancer cell lines. SHP2 is now understood to act upstream of RAS and plays a role in KRAS-driven cancers, an area of research which is rapidly growing. Considering that RTK deregulation often leads to a wide range of cancers and the newly appreciated role of SHP2 in KRAS-driven cancers, SHP2 inhibitors are therefore a promising therapeutic approach. SHP2 contains two N-terminal tandem SH2 domains (N-SH2, C-SH2), a catalytic phosphatase domain and a C-terminal tail. SHP2 switches between “open” active and “closed” inactive forms due to autoinhibitory interactions between the N-SH2 domain and the phosphatase domain. Historically, phosphatases were deemed undruggable as there had been no advancements with active site inhibitors. We hypothesised that fragment screening would be highly applicable and amenable to this target to enable alternative means of inhibition through identification of allosteric binding sites. Here we describe the first reported fragment screen against SHP2. Using our fragment-based PyramidTM approach, screening was carried out on two constructs of SHP2; a closed autoinhibited C-terminal truncated form (phosphatase and both SH2 domains), as well as the phosphatase-only domain. A combination of screening methods such as X-ray crystallography and NMR were employed to identify fragment hits at multiple sites on SHP2, including the tunnel-like allosteric site reported by Chen et al, 2016. Initial fragment hits had affinities for SHP2 in the range of 1mM as measured by ITC. Binding of these hits was improved using structure-guided design to generate compounds which inhibit SHP2 phosphatase activity and are promising starting points for further optimization. Citation Format: Philip J. Day, Valerio Berdini, Juan Castro, Gianni Chessari, Thomas G. Davies, James E. Day, Satoshi Fukaya, Chris Hamlett, Keisha Hearn, Steve Hiscock, Rhian Holvey, Satoru Ito, Yasuo Kodama, Kenichi Matsuo, Yoko Nakatsuru, Nick Palmer, Amanda Price, Tadashi Shimamura, Jeffrey D.St. Denis, Nicola G. Wallis, Glyn Williams, Christopher N. Johnson. Fragment-based drug discovery to identify small molecule allosteric inhibitors of SHP2 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1039.

Non-small cell lung cancer (NSCLC) molecular profiling is a key factor in treatment selection. Although, patients with NSCLC tumors harboring EGFR or ALK mutations can benefit from personalized therapies, there are currently no approved targeted therapies for KRAS mutant tumors which occur in 25% to 30% of patients with NSCLC. The constitutive activation of the MAPK pathway in these tumors provides a rationale for targeting effectors such as MEK1/2 (MEK) or ERK1/2 (ERK). Inhibitor of MEK kinase have been tested clinically in KRAS-mutant NSCLC but results have been disappointing, possibly because compensatory signaling such as the reactivation of ERK is triggered following the inhibition of MEK, leading to cancer cell survival. Therefore, targeting ERK directly represents an attractive therapeutic approach. As previously described, we have developed a novel, potent and selective ERK inhibitor identified by fragment-based drug discovery which has potent activity in vitro and in vivo. Here, we demonstrate the activity of this lead compound in KRAS-mutant NSCLC models. Our novel ERK inhibitor was tested in a panel of 440 human cancer cell lines of which the KRAS NSCLC population was identified as particularly sensitive. 62% of the KRAS-mutant NSCLC cell lines tested, exhibited antiproliferative IC50s ranging from 1 nM to 500 nM. This lead compound also inhibited ERK downstream signaling in KRAS NSCLC models both in vitro and in vivo. Indeed, the phosphorylation level of the ERK substrate, RSK, was strongly decreased in HCC-44 and Calu-6 xenograft tumors 2h after oral administration of the lead compound at 50 mg/kg. Levels of pRSK remained below those of untreated tumors for up to 16h in HCC-44 tumors and 24h in Calu-6 tumors. We also confirmed that, the ERK inhibitor conferred a decrease in phosphorylation of ERK itself in both models. The inhibition of ERK signaling corresponded to significant anti-tumor activity in these models with a daily oral administration of 50 mg/kg compound leading to significant tumor regression in subcutaneous models of HCC-44 (18.3% T/C) and Calu-6 (8.9% T/C) xenograft tumors. This work demonstrates the in vitro and in vivo activity of a novel, highly potent, selective ERK inhibitor in models of KRAS-mutant NSCLC. These data support the further optimisation of this series of compounds for clinical development. Citation Format: Aurelie Courtin, Luke Bevan, Tom Heightman, Birikiti Kidane, Justyna Kucia-Tran, John Lyons, Sandra Muench, Alpesh Shah, Lukas Stanczuk, Neil Thompson, Nicola Wallis, Nicola Wilsher, Joanne Munck. A novel ERK1/2 inhibitor has potent activity in KRAS-mutant non-small cell lung cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5781.

Cyclin-dependent kinases (CDK), and their regulatory cyclin partners, play a central role in eukaryotic cell growth, division, and death. This key role in cell cycle progression, as well as their deregulation in several human cancers, makes them attractive therapeutic targets in oncology. A series of CDK inhibitors was developed using Astex's fragment-based medicinal chemistry approach, linked to high-throughput X-ray crystallography. A compound from this series, designated AT7519, is currently in early-phase clinical development. We describe here the biological characterization of AT7519, a potent inhibitor of several CDK family members. AT7519 showed potent antiproliferative activity (40-940 nmol/L) in a panel of human tumor cell lines, and the mechanism of action was shown here to be consistent with the inhibition of CDK1 and CDK2 in solid tumor cell lines. AT7519 caused cell cycle arrest followed by apoptosis in human tumor cells and inhibited tumor growth in human tumor xenograft models. Tumor regression was observed following twice daily dosing of AT7519 in the HCT116 and HT29 colon cancer xenograft models. We show that these biological effects are linked to inhibition of CDKs in vivo and that AT7519 induces tumor cell apoptosis in these xenograft models. AT7519 has an attractive biological profile for development as a clinical candidate, and the tolerability and efficacy in animal models compare favorably with other CDK inhibitors in clinical development. Studies described here formed the biological rationale for investigating the potential therapeutic benefit of AT7519 in cancer patients. [Mol Cancer Ther 2009;8(2):324–32]

The RAS-RAF-MEK-ERK signalling cascade is activated through mutations in RAS or RAF in over 30% of cancers. The successful development of inhibitors of BRAF and MEK kinases has led to effective treatment particularly of melanomas whose tumor growth is driven by activating mutations in BRAF such as V600E. Despite these successes, resistance emerges after several months, leading to increased signaling through ERK1/2. This has prompted the development of direct inhibitors of ERK1/2, several of which are in early clinical trials. The majority of clinical ERK1/2 inhibitors are ATP competitive, blocking ERK1/2 catalytic phosphorylation of downstream substrates such as RSK, but do not modulate phosphorylation of ERK1/2 by MEK. Crystal structural studies performed by us and others on the pERK1/2 modulating inhibitor SCH772984 suggested that it induces a conformational change in the glycine-rich loop of ERK2, which leads to Tyr36 becoming tucked under the loop and creating a new binding pocket. We hypothesized that this binding mode might underlie the ability of SCH772984 to block the phosphorylation of ERK1/2, and initiated a fragment-based approach to develop novel, orally bioavailable inhibitors that elicit a similar conformational change and also modulate the phosphorylation of ERK1/2. Using screening methods including high-throughput X-ray crystallography and biophysical assays, we identified fragments binding to both the hinge and the inducible pocket of ERK2. Progressive rounds of structure-guided fragment optimization and growing led to an understanding of inhibitor structure determinants required to induce the conformational change in ERK2. These efforts, together with iterative optimization in a screening cascade including measurement of pRSK and pERK levels and antiproliferative activity in RAS and BRAF mutant cells, led to the discovery of a novel series of pERK modulating ERK1/2 inhibitors. The lead compound shows low nanomolar potency in biochemical ERK1/2 assays and an excellent kinome selectivity profile. In BRAF and RAS mutant cell lines, the lead shows low nanomolar cell proliferation IC50 values, while sparing cell lines not driven by the MAPK pathway. The lead exhibits robust antitumor activity upon oral dosing in a range of subcutaneous xenograft models including the mutant BRAF colorectal line Colo205, providing a promising basis for further optimization towards clinical pERK1/2 modulating ERK1/2 inhibitors. Citation Format: Tom D. Heightman, Valerio Berdini, Hannah Braithwaite, Ildiko Buck, Megan Cassidy, Juan Castro, Aurélie Courtin, James Day, Charlotte East, Lynsey Fazal, Brent Graham, Charlotte Griffiths-Jones, John Lyons, Vanessa Martins, Sandra Muench, Joanne Munck, David Norton, Marc O'Reilly, Nick Palmer, Puja Pathuri, Mike Reader, David Rees, Sharna Rich, Caroline Richardson, Harpreet Saini, Neil Thompson, Nicola Wallis, Hugh Walton, Nicola Wilsher, Alison Woolford, Chris Murray. Fragment-based discovery of a highly potent, orally bioavailable ERK1/2 inhibitor that modulates the phosphorylation and catalytic activity of ERK1/2 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B161.

The MAPK pathway is commonly hyper-activated in human cancers due to the occurrence of oncogenic mutations in RAF, RAS and the upregulation of RTKs. The therapeutic potential of MAPK pathway inhibition has been demonstrated by the clinical efficacy of RAF and MEK1/2 (MEK) inhibitors in the treatment of BRAF-mutant melanoma. However, response to such agents is short-lived due to the onset of resistance mechanisms, which in the majority of cases result in the reactivation of ERK1/2 (ERK) signalling. Therefore, the direct targeting of ERK is an attractive therapeutic approach to overcoming the limitations of RAF or MEK inhibitors. Here, we describe a novel, potent, and selective ERK inhibitor, which inhibits both ERK catalytic activity and also the phosphorylation of ERK by MEK. Using fragment-based drug discovery we have developed a selective ERK inhibitor, which inhibits in vitro ERK catalytic activity with a low nM IC50 value. This lead compound has strong antiproliferative effects in a wide range of MAPK-activated cell lines, including the BRAF-mutant cell lines A375 (melanoma) and Colo205 (colorectal), the KRAS-mutant cell lines HCT116 (colorectal), Calu6 (lung) and Panc05.04 (pancreatic), and the NRAS-mutant cell line Ma-mel-27 (melanoma). The lead compound potently inhibits ERK cell signalling. The potent (nM) inhibition of RSK phosphorylation (a direct ERK substrate) was confirmed in A375 (BRAF-mutant melanoma) cells, using MSD analysis. In addition to inhibiting downstream ERK signalling, we demonstrated by ELISA and Western blotting that the lead compound confers a decrease in phospho-ERK levels in both BRAF-mutant and KRAS-mutant cell lines. We investigated the biochemical mechanism of the modulation of ERK phosphorylation in vitro and demonstrated that the compound prevents the phosphorylation of ERK by MEK (at key ERK activation loop residues, T202/Y204), without directly inhibiting MEK activity. The compound was profiled in a range of subcutaneous xenograft models including A375 (BRAF-mutant melanoma) and Calu-6 (KRAS-mutant lung). Once-daily oral dosing of the lead compound conferred significant antitumor activity in a range of in vivo efficacy studies. The compound potently inhibited the phosphorylation of downstream ERK substrates (including RSK) in tumor xenograft tissue. There was a clear relationship between in vivo compound concentrations and the modulation of ERK substrate phosphorylation. Furthermore, as was demonstrated in vitro, we confirmed that in addition to inhibiting ERK catalytic activity the compound potently inhibited the phosphorylation of ERK itself, in both KRAS and BRAF-mutant tumor xenografts. Here, we characterize a novel, highly potent, selective ERK inhibitor, which inhibits both ERK catalytic activity and also the upstream phosphorylation of ERK by MEK. These data support the further optimization of this series of compounds for clinical development. Citation Format: Joanne M. Munck, Valerio Berdini, Luke D. Bevan, Hannah Braithwaite, Ildiko M. Buck, Megan Cassidy, Juan Castro, Aurelie Courtin, James E. Day, Charlotte East, Lynsey Fazal, Brent Graham, Charlotte M. Griffiths-Jones, Tom D. Heightman, Chris J. Hindley, Birikiti Kidane, Justyna Kucia-Tran, John F. Lyons, Vanessa Martins, Sandra Muench, Chris W. Murray, David Norton, Marc O'Reilly, Nick Palmer, Puja Pathuri, Mike Reader, David C. Rees, Sharna J. Rich, Caroline J. Richardson, Harpreet K. Saini, Alpesh Shah, Lukas Stanczuk, Neil T. Thompson, Hugh Walton, Nicola E. Wilsher, Alison J. Woolford, Nicola G. Wallis. Characterization of a novel ERK1/2 inhibitor, which modulates the phosphorylation and catalytic activity of ERK1/2 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B154.

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.