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).

This article is featured in Highlights of This Issue, p. 1755

ERK1/2 are ubiquitously expressed protein serine/threonine kinases comprising a key component of the MAPK signaling pathway (1, 2). Their catalytic activity is regulated by the phosphorylation of two key residues in the activation loop (T185 and Y187 in ERK2) by the kinases MEK1/2 (3). This phosphorylation activates ERK and drives its translocation into the nucleus. ERK phosphorylates multiple cytoplasmic and nuclear substrates such as ribosomal S6 kinase (RSK) and c-Fos, which promote cell proliferation, survival, and differentiation (1, 4).

ERK activity is commonly upregulated in cancer, as a result of activating mutations within upstream components of the MAPK pathway, such as RAS and RAF (5–7). Although MAPK pathway inhibition has been clinically validated by the approval of BRAF and MEK inhibitors in the mutant BRAF indications, melanoma, non–small cell lung cancer (NSCLC), and thyroid cancer (8–10), limited activity has been observed in other mutant BRAF patients such as colorectal cancer or those with mutant RAS (11, 12). Moreover, the duration of response is often short-lived due to the rapid emergence of acquired resistance (13, 14). The recent early clinical activity of KRAS G12C inhibitors provides further therapeutic validation of the MAPK pathway, but these too may be limited by frequent relapse after response (15). A common feature of resistance is the reactivation of ERK and MAPK signaling. Because ERK is the primary downstream effector of the MAPK pathway, ERK inhibitors may be less susceptible to oncogenic bypass than other MAPK pathway inhibitors (16–18). Therefore, inhibition of ERK has the potential not only to overcome resistance, but also to improve efficacy, broadening the application of MAPK pathway inhibition to further indications both as single agent or in combination (19, 20).

Preclinical activity has been demonstrated with a number of ERK inhibitors and some clinical responses have been reported for ulixertinib (BVD-523), GDC-0994, and MK-8353 (21–27). These ERK inhibitors fall into two distinct classes. The first inhibit the kinase activity of ERK alone and have been termed “catalytic ERK inhibitors” (21, 22). In addition to inhibiting the kinase activity of ERK, the second class or “dual-mechanism ERK inhibitors” also prevent the phosphorylation of ERK itself (23, 28, 29). These dual-mechanism inhibitors alter the conformation of the glycine-rich loop of ERK, resulting in decreased phosphorylation of the activation loop and stabilization of ERK in its catalytically inactive form (26, 30). It has been suggested that dual-mechanism ERK inhibitors could have improved efficacy due to a more complete suppression of MAPK signaling and the prevention of ERK's nuclear accumulation, leading to increased inhibition of ERK-dependent gene expression.

We have previously described the discovery of lead compounds with a dual-mechanism profile, using fragment-based screening and subsequent structure-based drug design (30). Here we describe the discovery and characterization of the optimized clinical candidate from this series, ASTX029, which is currently being evaluated in a first-in-human (FIH) phase I–II clinical trial in patients with advanced solid tumors (ClinicalTrials.gov Identifier: NCT03520075).

Materials

ASTX029 (2R)-2-(6-{5-chloro-2-[(oxan-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide) was synthesized at Astex Pharmaceuticals. A solution of (R)-2-(6-(5-chloro-2-((oxan-4-yl)amino)pyrimidin-4-yl)-1-oxoisoindolin-2-yl)propanoic acid (ref. 30; 70 mg, 0.168 mmol), (S)-2-amino-2-(3-fluoro-5-methoxyphenyl)ethanol, HCl (41 mg, 0.185 mmol) and triethylamine (0.094 mL, 0.672 mmol) in DMF (1 mL) was treated with TBTU (65 mg, 0.202 mmol) and stirred at room temperature overnight. The mixture was diluted with ethyl acetate (20 mL), washed successively with 1 mol/L KHSO4 (10 mL), NaHCO3 (10 mL), brine (2 × 10 mL), and water (4 × 10 mL), dried (MgSO4) and evaporated. The residue was purified by chromatography (SiO2, 12 g column, 0%–5% EtOOH in EtOAc) to give a glass, which was triturated with ether (2 mL) to give a solid. The solid was collected by filtration, washed with ether (2 × 1 mL) and dried under vacuum at 50°C overnight to give the title compound (64.3 mg, 64.3%) as a cream solid. 1H NMR (400 MHz, DMSO-d6) δ 8.54 (d, J = 8.1 Hz, 1H), 8.44 (s, 1H), 8.04 (d, J = 1.6 Hz, 1H), 7.97 (dd, J = 7.9, 1.7 Hz, 1H), 7.74 (d, J = 7.9 Hz, 1H), 7.59 (d, J = 5.6 Hz, 1H), 6.76–6.66 (m, 3H), 4.99 (q, J = 7.2 Hz, 1H), 4.90 (t, J = 5.5 Hz, 1H), 4.81 (q, J = 6.6 Hz, 1H), 4.75 (d, J = 18.1 Hz, 1H), 4.61 (d, J = 18.1 Hz, 1H), 3.99–3.88 (m, 1H), 3.86 (dt, J = 11.3, 3.4 Hz, 2H), 3.76 (s, 3H), 3.61–3.49 (m, 2H), 3.44–3.33 (m, 2H), 1.91–1.78 (m, 2H), 1.60–1.47 (m, 2H), 1.45 (d, J = 7.2 Hz, 3H).

Vemurafenib, trametinib, and selumetinib were purchased from LC laboratories. All other reagents were purchased from Sigma unless otherwise stated.

Protein expression, purification, and crystallography

Full-length human ERK2 was expressed in Escherichia coli, purified and crystallized as described previously (30). X-ray diffraction data were collected using in-house X-ray sources.

Cell culture and reagents

The human cell lines A375 (RRID: CVCL_0132), A375-NRASQ61K (RRID:CVCL_JF22), WM-266-4 (RRID: CVCL_2765), NCI-H2122 (RRID: CVCL_1531), Calu-6 (RRID: CVCL_0236), NCI-H1355 (RRID: CVCL_1464), SW900 (RRID: CVCL_1731), PL-45 (RRID: CVCL_3567), HPAFII (RRID: CVCL_0313), and SKMel2 (RRID: CVCL_0069) were purchased from the ATCC, HCC-44 (RRID: CVCL_2060) and Capan-1 (RRID: CVCL_0237) cell lines from DSMZ (depositor A Gazdar and J Fogh, respectively). All other cell lines were purchased from the European Collection of Cell Cultures (ECACC). Cell panel cell lines were purchased by ChemPartner from ATCC, ECACC, DSMZ, JCRB, Riken, or CLS. All cell lines were authenticated by short tandem repeat PCR by the provider, certified Mycoplasma free on distribution and cultured for no longer than 6 months. Cells were cultured according to supplier recommendations and cell culture reagents purchased from Thermo Fisher Scientific. Patient-derived xenograft (PDX)-derived cell lines, MEXF535, MEXF622, MEXF1341, MEXF1539, MEXF1792, and MEXF2090 were established at Charles River. A375R were cultured as described previously in 2 μmol/L vemurafenib (31). A375-RS cells were generated by treatment of A375 cells for 24 hours with 0.05 mg/mL N-Nitroso-N-ethylurea (Sigma) followed by continuous culture in 0.5 μmol/L selumetinib for 2 weeks and clonal selection. Clonal A375-RS cells were maintained in 0.5 μmol/L selumetinib.

Kinase inhibition and binding

Inhibition of ERK2 kinase activity was determined as described previously (30). IC50 curves were generated using GraphPad Prism (RRID:SCR_002798) with the four-parameter logistic curve fit. Inhibitory activity against a panel of kinases and binding to MEK1 were determined using the SelectScreen kinase profiling service (Thermo Fisher Scientific).

Measurement of MEK-dependent phosphorylation of ERK

MEK1 phosphorylation of ERK2 on residues Thr202 and Tyr204 was measured in a time-resolved fluorescence energy transfer (TR-FRET) assay using a pERK assay kit (Cisbio). Following preincubation with compounds and ATP for 1 hour, unphosphorylated ERK2 (50 nmol/L) (Biaffin GmbH & Co KG) was incubated with MEK1 (2 nmol/L; Biaffin GmbH & Co KG) and ATP (21 μmol/L) in 50 mmol/L HEPES pH7.0, 10 mmol/L MgCl2, 1 mmol/L EGTA, 0.01% [volume for volume (v/v)] Brij-35, 2.5% (v/v) DMSO for 25 minutes. Reactions were stopped by the addition of Lysis Buffer (Cisbio) and incubated at 42°C for 2 hours. Two phospho-ERK1/2 (Thr202/Tyr204) antibodies, labelled with Eu3+-cryptate donor or d2 acceptor (Cisbio) were added. After overnight incubation, TR-FRET ratio was generated by measuring fluorescence at 665 nm (A counts) and 620 nm (B counts) upon excitation at 337 nm using a PHERastar plate reader (BMG Labtech).

Protein analysis

For Western blotting, equivalent amounts of cell or tumor lysates were resolved by SDS-PAGE and immunoblotted, as described previously (30) with antibodies specific for phospho-RSK, phospho-ERK1/2, ERK1/2, phospho-MEK1/2, MEK1/2, phospho-CRAF, Bim, cleaved caspase-3, cleaved PARP (Cell Signaling Technology), RSK (R&D Systems), β-actin, and α-tubulin (Abcam; Supplementary Materials and Methods). Phosphorylated RSK (pRSK) and ERK (pERK) levels were quantified by Mesoscale discovery assay (MSD) and ELISA, respectively (30).

Cell viability assays

Cell viability was measured in-house using Alamar Blue (Thermo Fisher Scientific; ref. 30). The cell line panel screen was performed at ChemPartner using the CellTiter-Glo luminescent Cell Viability assay (Promega) and viability of the PDX-derived NRAS-mutant melanoma cell lines was measured at Charles River using the CellTiter-Blue Cell Viability Assay (Promega). Cell viability was determined following 96 hours compound treatment to ensure that all cell lines (regardless of proliferation rate) had progressed through sufficient cell doublings to accurately assess proliferation effects.

Cell-cycle analysis

Cell-cycle analysis was performed at ChemPartner using propidium iodide (PI) staining. Cells were fixed in 70% ethanol, washed and incubated with 100 μg/mL RNase A (Sigma) in PBS and stained with 50 μg/mL PI (Life Technologies) before data were collected using a CytoFLEX (Beckman Coulter). For sub-G1 percentage determination, samples were treated as above and data collected in-house using a FACSMelody (Becton Dickinson). Data were analyzed using FlowJo V10 (FlowJo LLC, RRID:SCR_008520) with the cell-cycle plug-in.

Statistical analysis of the cell panel screen

Statistical analysis of the cell panel screen was conducted in R version 3.5.0 (32) and figures produced using the ggplot2 package version 3.1.0 (ref. 33; RRID:SCR_014601). Activity area, calculated as the area over the dose–response curve (34), was used to enable inclusion of insensitive cell lines (for which IC50 values could not be determined). ANOVA was used to determine significant associations between activity area and genomic features. P values were adjusted using the Benjamini and Hochberg multiple testing correction method and Cohen's d calculated to determine the effect size using the lsr package version 0.5 (35). The genomic features considered were cancer functional events (CFE; ref. 36) with the feature set consisting of disease type, as recorded by Cell Model Passports version 2019-06-21 (37), and pancancer CFEs occurring in three or more cell lines. CFEs with an identical pattern of occurrence were merged, and informative CpG islands were excluded to reduce the number of tests, resulting in a final set of 619 features across 437 cell lines. MAPK oncogenes are as defined by The Cancer Genome Atlas (37). Breast subtype classification of cell lines was as described previously (38).

Animal studies

The care and treatment of animals were in accordance with the United Kingdom Coordinating Committee for Cancer Research guidelines and the United Kingdom Animals (Scientific Procedures) Act 1986 (39, 40). Study protocols were approved by the University of Cambridge Animal Welfare and Ethical Review Board unless otherwise stated.

Studies were performed using male nude (BALB/cOlaHsd-Foxn1nu) or CB17 SCID (CB17/Icr-Prkdcscid/IcrIcoCrl) mice purchased from Envigo or Charles River. Colo205, A375, and A375R xenografts were prepared as described previously (30, 31). Calu-6, HCC44, HCT116, and MA-MEL-28 xenografts were established by subcutaneously injecting 5 × 106 cells in 100 μL of serum-free medium with or without Matrigel. The subcutaneous MEXF2090 PDX model was established in NMRI nude mice at Charles River in accordance with the German Animal Welfare Act (Tierschutzgesetz). Following excision from patients, tumor pieces were subcutaneously implanted into immunodeficient mice and serially passaged until stable growth established. Tumor fragments were removed from donor mice, placed in PBS containing 10% penicillin/streptomycin and implanted subcutaneously into the flank of recipient animals. Resistance of the A375R model was maintained by twice daily, oral (po) treatment with 50 mg/kg vemurafenib [suspended in 5% (v/v) DMSO, 0.95% (w/v) methylcellulose and administered at 10 mL/kg] until randomization. Tumor xenografts were measured using digital calipers and volumes calculated by applying the formula for ellipsoid. ASTX029 was formulated in 20% (v/v) PEG200 and 0.5% (w/v) methylcellulose and administered po at dose volume of 10 mL/kg. Blood samples were collected and plasma prepared by centrifugation as described previously (30).

For tumor growth inhibition studies, tumor-bearing animals were randomized into groups of 7 to 8 with an average tumor volume of 100–250 mm3 and treatment started on day 1. Control animals received vehicle. Statistical significance of difference between treated and control was analyzed using one-way ANOVA and two-way ANOVA with Dunnett multiple comparisons test (GraphPad Prism).

For pharmacodynamic analysis, tumors were excised and snap-frozen in liquid nitrogen.

Bioanalytical and pharmacokinetic analysis

ASTX029 was extracted from samples as described previously (30). All samples were centrifuged at 4,700 rpm at 4°C for 20 minutes. Supernatant was analysed by reverse-phase LC/MS-MS in positive ionisation mode.

ASTX029 is a potent and selective ERK inhibitor, with a dual mechanism, which prevents the phosphorylation of ERK by MEK

Previously, fragment screening and subsequent structure-based drug design led to the discovery of the ERK inhibitor, compound 27 (30). This lead compound was characterized as a highly potent and selective inhibitor of ERK, which also modulated ERK phosphorylation. The compound achieved good exposure in mice, which correlated with good antitumor activity in Colo205 (BRAFV600E-mutant colorectal cancer) xenograft studies (30). However, metabolic studies indicated oxidative metabolism of compound 27 mediated by CYP3A4. Optimization and a careful consideration of physicochemical properties, such as solubility, resulted in the discovery of ASTX029 (Fig. 1A), which is currently undergoing clinical development. The detailed optimization pathway that led to the identification of ASTX029 will be the subject of a medicinal chemistry publication.

Figure 1.

ASTX029 is a novel ERK1/2 inhibitor that inhibits ERK catalytic activity and the phosphorylation of ERK itself. Chemical structure of ASTX029 (A). Kinome selectivity profile of ASTX029 (B). X-ray crystal structure of ASTX029 bound to human ERK2 (PDB Code: 7AUV; C). Levels of ERK2 phosphorylation by MEK1 (measured by TR-FRET) when incubated with ERK inhibitors, ASTX029 and GDC-0994 and MEK inhibitor, trametinib (red squares). Blue bars represent extent of compound binding to MEK1 (D). Each data point is the mean of replicates (n ≥ 2) ± SD.

Figure 1.

ASTX029 is a novel ERK1/2 inhibitor that inhibits ERK catalytic activity and the phosphorylation of ERK itself. Chemical structure of ASTX029 (A). Kinome selectivity profile of ASTX029 (B). X-ray crystal structure of ASTX029 bound to human ERK2 (PDB Code: 7AUV; C). Levels of ERK2 phosphorylation by MEK1 (measured by TR-FRET) when incubated with ERK inhibitors, ASTX029 and GDC-0994 and MEK inhibitor, trametinib (red squares). Blue bars represent extent of compound binding to MEK1 (D). Each data point is the mean of replicates (n ≥ 2) ± SD.

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ASTX029 potently inhibited ERK2 with an IC50 value of 2.7 nmol/L and ERK1 by 52% at 3 nmol/L. The inhibitory activity of ASTX029 against a broad panel of kinases was determined and only 5 of a further 460 kinases (MAP2K6, MAP2K6S207E/T211E, PRKCN, PRKD1 and MAPK15) were inhibited with IC50 values <100 nmol/L (Fig. 1B; Supplementary Table S1).

Protein X-ray crystallography analysis revealed that ASTX029 has the same distinctive binding mode as described previously for advanced lead compound 27 (30). ASTX029 binds to the active site of ERK2, which is located between the N- and C-terminal domains of the kinase (Fig. 1C, PDB Code: 7AUV). The amino pyrimidine moiety of ASTX029 forms hydrogen bonds with Met108, a key residue in the so-called “hinge” region, which is also where the adenine of ATP binds to ERK2. ASTX029 adopts an extended conformation in the ATP-binding cleft and stabilizes an unusual, Tyr36 “in” conformation of the kinase P-loop via an extensive network of hydrogen bonds and nonpolar interactions, thus enabling ASTX029 to access a secondary pocket. X-ray crystallography studies by ourselves and others suggest that binding within this secondary pocket, enables ERK inhibitors to prevent the phosphorylation of ERK itself (30, 41). This was confirmed in an in vitro assay where ASTX029 was incubated with ERK2 in the presence of MEK1 and the phosphorylation of ERK2 quantified. The phosphorylation of ERK by MEK was reduced to 12% of control levels upon treatment with 100 nmol/L ASTX029, despite no direct binding of compound to MEK itself being observed (Fig. 1D). We confirmed that GDC-0994, an ERK inhibitor that binds within the ERK active site, but does not extend into the secondary pocket (21, 28), did not robustly prevent the phosphorylation of ERK by MEK. As expected, the MEK inhibitor, trametinib, bound directly to MEK while inhibiting the phosphorylation of ERK. Together these data suggest that the ASTX029-binding mode enables it to modulate the phosphorylation of ERK by MEK, potentially through an allosteric mechanism.

ASTX029 potently inhibits ERK catalytic activity and modulates the phosphorylation of ERK in MAPK-activated cancer cells

The dual mechanism of ASTX029 was investigated further in cell lines with activating mutations in the MAPK pathway. Effects on the catalytic activity of ERK were determined by measuring the phosphorylation levels of the ERK substrate, RSK. Following a 2-hour treatment, ASTX029 inhibited the phosphorylation of RSK in a dose-dependent manner in both A375 (BRAFV600E-mutant melanoma) and HCT116 (KRASG13D-mutant colorectal) cells with IC50 values of 3.3 and 4 nmol/L, respectively (Fig. 2A).

Figure 2.

ASTX029 inhibits ERK catalytic activity and the phosphorylation of ERK in cells. Levels of pRSK (A) and pERK (B; measured by MSD assay and ELISA, respectively) in A375 and HCT116 cells after 2 hours treatment with the indicated concentrations of ASTX029. Each data point represents mean ± SEM; n = 3. Levels of MAPK pathway proteins (C) and markers of apoptosis (D) in A375 and HCT116 cells over 72 hours after treatment with 20 and 100 nmol/L ASTX029, respectively. ASTX029 concentrations used are approximately equivalent to 5× the proliferation IC50 values determined for each cell line. Cell-cycle profiles following treatment of A375 and HCT116 cells with the indicated concentrations of ASTX029 for 24 hours (E). Mean ± SD is shown; n = 2. Proportion of cells in the sub-G1 phase of A375 and HCT116 cells treated for 48 hours with the indicated concentrations with ASTX029, approximately equivalent to 5× and 100× the proliferation IC50 values determined for each cell line (F). Mean ± SD is shown; n = 3; **, P < 0.01; ****, P < 0.0001.

Figure 2.

ASTX029 inhibits ERK catalytic activity and the phosphorylation of ERK in cells. Levels of pRSK (A) and pERK (B; measured by MSD assay and ELISA, respectively) in A375 and HCT116 cells after 2 hours treatment with the indicated concentrations of ASTX029. Each data point represents mean ± SEM; n = 3. Levels of MAPK pathway proteins (C) and markers of apoptosis (D) in A375 and HCT116 cells over 72 hours after treatment with 20 and 100 nmol/L ASTX029, respectively. ASTX029 concentrations used are approximately equivalent to 5× the proliferation IC50 values determined for each cell line. Cell-cycle profiles following treatment of A375 and HCT116 cells with the indicated concentrations of ASTX029 for 24 hours (E). Mean ± SD is shown; n = 2. Proportion of cells in the sub-G1 phase of A375 and HCT116 cells treated for 48 hours with the indicated concentrations with ASTX029, approximately equivalent to 5× and 100× the proliferation IC50 values determined for each cell line (F). Mean ± SD is shown; n = 3; **, P < 0.01; ****, P < 0.0001.

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In agreement with the observations in cell-free assays, 2-hour treatment of A375 and HCT116 cells with ASTX029 resulted in a decrease in pERK levels with a maximum inhibition of 93% and 94% relative to control levels, respectively (Fig. 2B).

The time-dependent effects of ASTX029 treatment on ERK catalytic activity and pERK levels were investigated further (Fig. 2C). A375 and HCT116 cells were treated with 20 and 100 nmol/L, respectively, which were approximately equivalent to 5× the proliferation IC50 values (to ensure near complete inhibition of ERK signaling. The maximal decrease in pRSK levels was observed 2 hours after treatment. Levels gradually increased thereafter but remained below those observed in untreated cells up to 72 hours. A similar maximal decrease in pERK levels was observed at 2 hours in both cell lines, but this was more sustained in A375 cells, while levels returned in HCT116 cells by 72 hours. ASTX029 treatment also inhibited phosphorylation of the ERK substrate, CRAF. The ERK-dependent phosphorylation of CRAF at S289/296/301 is known to inhibit CRAF activity and therefore acts as a negative feedback mechanism within the MAPK pathway (42). ASTX029 treatment led to an increase in the phosphorylation of MEK in HCT116 cells over time. This is likely to be a direct effect of alleviating the inhibition of CRAF and thereby inducing feedback activation. No modulation of phosphorylated MEK (pMEK) levels was observed in A375 cells (Fig. 2C). Similar effects on ERK signaling were observed in KRAS-mutant NSCLC (HCC-44 and Calu-6), KRAS-mutant pancreatic (Capan-1), and NRAS-mutant melanoma (MA-MEL-28) cells (Supplementary Fig. S1).

ASTX029 treatment resulted in a dose-dependent cell-cycle arrest in the G1-phase (Fig. 2E). An increase in the fraction of cells in the sub-G1-phase (Fig. 2F) along with an increase in apoptotic markers such as cleaved PARP and Bim (Fig. 2D) suggested compound treatment induced apoptosis.

ASTX029 inhibits ERK catalytic activity and modulates the phosphorylation of ERK in vivo

The pharmacokinetic parameters of ASTX029 were determined following intravenous (0.5 mg/kg) and oral (5 mg/kg) administration to mice. ASTX029 showed good oral bioavailability (42%) and a moderate clearance of 22 mL/minute/kg (Supplementary Table S2).

Following a single oral dose of 75 mg/kg ASTX029 to mice bearing Colo205 (BRAFV600E-mutant colorectal) tumor xenografts, maximal plasma, and tumor exposure was reached within 0.5 hours and was detectable up to 24 hours (Fig. 3A). This correlated with a dramatic decrease in pRSK within tumors which was maximal 2 hours after dosing, with levels reduced to 3% of control (Fig. 3B). Levels gradually increased thereafter, being restored to basal levels by 24 hours. This modulation was dose dependent over the range 25–150 mg/kg (Fig. 3C). Plasma pharmacokinetics were linear within this dose range (Supplementary Fig. S2). In addition to inhibiting ERK catalytic activity, ASTX029 treatment also resulted in a decrease in tumor pERK levels. This was maximal at 1-hour post-dose, with pERK levels reduced to 35% of control. Levels then increased with time and were fully restored to untreated levels by 24 hours (Fig. 3B). Following ASTX029 treatment an increase in Bim, cleaved caspase 3 and cleaved PARP was observed, suggesting induction of apoptosis in the tumors (Fig. 3D). Similar modulation of ERK signaling was also observed in xenograft tumors with KRAS (Calu-6 and HCC44, NSCLC; HCT116, colorectal cancer) or NRAS (MA-MEL-28, melanoma) mutations (Supplementary Fig. S3).

Figure 3.

Orally administered ASTX029 modulates pharmacodynamic markers of MAPK signaling in a Colo205 subcutaneous xenograft model. BALB/c nude mice bearing Colo205 subcutaneous xenografts were treated with a single oral dose of ASTX029. Mean plasma and tumor pharmacokinetic profile (A) and tumor levels of pRSK and pERK (measured by MSD assay and ELISA, respectively; B) after dosing with 75 mg/kg ASTX029. Tumor levels of pRSK after single doses of 25 to 150 mg/kg of ASTX029 (C). Each data point represents mean ± SD; n = 3. Tumor levels of apoptosis proteins Bim, cleaved caspase 3 and cleaved PARP after single 75 mg/kg oral dose of ASXT029 (D). Each sample represents tumors taken from individual mice.

Figure 3.

Orally administered ASTX029 modulates pharmacodynamic markers of MAPK signaling in a Colo205 subcutaneous xenograft model. BALB/c nude mice bearing Colo205 subcutaneous xenografts were treated with a single oral dose of ASTX029. Mean plasma and tumor pharmacokinetic profile (A) and tumor levels of pRSK and pERK (measured by MSD assay and ELISA, respectively; B) after dosing with 75 mg/kg ASTX029. Tumor levels of pRSK after single doses of 25 to 150 mg/kg of ASTX029 (C). Each data point represents mean ± SD; n = 3. Tumor levels of apoptosis proteins Bim, cleaved caspase 3 and cleaved PARP after single 75 mg/kg oral dose of ASXT029 (D). Each sample represents tumors taken from individual mice.

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ASTX029 inhibits the proliferation of human cancer cell lines harboring MAPK-activating mutations

To investigate the therapeutic potential of ASTX029 further, its activity was initially tested in a small panel of human cancer cell lines harboring activating mutations within BRAF, KRAS, or NRAS. These included BRAFV600-mutant colorectal and melanoma cell lines, KRAS-mutant NSCLC and pancreatic cell lines and NRAS-mutant melanoma cell lines (Supplementary Table S3). Proliferation of these cell lines was inhibited with IC50 values ranging from 1.8 to 380 nmol/L (A375: 3.4 nmol/L, HCT116: 28 nmol/L; Fig. 4A). A more in-depth analysis of features of sensitivity was carried out by testing the effect of ASTX029 in a wider panel of 437 human cancer cell lines with varied genetic backgrounds (Supplementary Table S4). ASTX029 potently inhibited the proliferation of a substantial proportion of the cell lines, with 109 of 437 having an IC50 value less than 150 nmol/L. We demonstrated a significant difference in ASTX029 sensitivity (measured as activity area), between cell lines with and without CFE oncogene mutations in the MAPK pathway (Wilcoxon rank-sum test P < 0.001; Fig. 4B; Supplementary Fig. S4A), with those that are wild type for genes in the MAPK pathway, exhibiting significantly less sensitivity to ASTX029. ANOVA confirmed that the presence of MAPK pathway oncogene mutations significantly associated with sensitivity to ASTX029 (BRAF and KRASPadj < 0.001, FLT3Padj = 0.05, and NRASPadj = 0.06; Fig. 4C). The cancer types with the greatest sensitivity were melanoma, acute myeloid leukemia (AML) and colorectal cancer (Fig. 4C–E); an unsurprising observation, given the prevalence of functional MAPK mutations in these indications. Ten of the 16 profiled AML cell lines had functional mutations in MAPK oncogenes and were highly sensitive to ASTX029 (Fig. 4E and F). This stratification was also observed in all “other” indications (i.e., the remaining cell lines screened after excluding melanoma, AML and colorectal cancer; Fig. 4E). For example, although basal-like breast cancers were largely resistant to ASTX029, 4 of the 17 cell lines harbored functional mutations in MAPK pathway oncogenes and were significantly more sensitive to ASTX029 than those without such a mutation (Supplementary Fig. S4B). Together these data highlight the therapeutic potential of ASTX029 in MAPK-activated hematologic malignancies and solid tumors.

Figure 4.

ASTX029 inhibits the proliferation of human cancer cell lines harboring MAPK-activating mutations. Effects on proliferation of ASTX029 treatment on cells harboring BRAF, KRAS, or NRAS mutations (A). Data points represent mean ± SD. Analysis of ASTX029 437 cell panel screen data showing a significant difference in activity area when cell lines grouped by the presence of CFE mutations in MAPK oncogenes (B) and significant association by ANOVA of 619 features across cell lines (FDR<10%, signed Cohen's d > 0) between sensitivity to ASTX029 and MAPK oncogene CFE mutations (BRAF, KRAS, NRAS, FLT3; C). Mean activity area of cell lines by cancer type (D). Number of cell lines indicated for each cancer type. Cancer types significantly associated (ANOVA FDR<10%) with ASTX029 sensitivity when grouped by MAPK oncogene mutation status (E). Activity areas of acute myeloid leukemia cell lines in panel annotated with MAPK oncogene CFE mutation status (F).

Figure 4.

ASTX029 inhibits the proliferation of human cancer cell lines harboring MAPK-activating mutations. Effects on proliferation of ASTX029 treatment on cells harboring BRAF, KRAS, or NRAS mutations (A). Data points represent mean ± SD. Analysis of ASTX029 437 cell panel screen data showing a significant difference in activity area when cell lines grouped by the presence of CFE mutations in MAPK oncogenes (B) and significant association by ANOVA of 619 features across cell lines (FDR<10%, signed Cohen's d > 0) between sensitivity to ASTX029 and MAPK oncogene CFE mutations (BRAF, KRAS, NRAS, FLT3; C). Mean activity area of cell lines by cancer type (D). Number of cell lines indicated for each cancer type. Cancer types significantly associated (ANOVA FDR<10%) with ASTX029 sensitivity when grouped by MAPK oncogene mutation status (E). Activity areas of acute myeloid leukemia cell lines in panel annotated with MAPK oncogene CFE mutation status (F).

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ASTX029 demonstrates in vivo antitumor activity in a range of MAPK-activated xenograft models

Antitumor effects of ASTX029 were further investigated in a range of MAPK-activated tumor xenograft models (Fig. 5). Daily administration of ASTX029 was tested at doses up to 75 mg/kg or every other day at 150 mg/kg. Median bodyweight loss of up to 7% was observed at high doses but the mice showed no other notable signs of adverse effects (Supplementary Fig. S5).

Figure 5.

ASTX029 confers antitumor activity in subcutaneous tumor xenografts with MAPK pathway activating mutations. Each data point represents mean ± SEM from 8 animals unless otherwise stated. Dashed lines represent the mean volume of all tumors on day 1 and 50% mean volume on day 1. ASTX029 was orally administered to mice bearing Colo205 (A), A375 (B), Calu-6 (n = 7 mice; C), HCC44 (D), HCT116 (E) or MA-MEL-28 (F) xenografts or MEXF2090 PDXs (G). Vemurafenib was orally administered at 50 mg/kg twice daily. Vehicle was administered orally every day to control animals. *, P < 0.05; ***, P < 0.001. Animals were dosed for the duration of the experiments.

Figure 5.

ASTX029 confers antitumor activity in subcutaneous tumor xenografts with MAPK pathway activating mutations. Each data point represents mean ± SEM from 8 animals unless otherwise stated. Dashed lines represent the mean volume of all tumors on day 1 and 50% mean volume on day 1. ASTX029 was orally administered to mice bearing Colo205 (A), A375 (B), Calu-6 (n = 7 mice; C), HCC44 (D), HCT116 (E) or MA-MEL-28 (F) xenografts or MEXF2090 PDXs (G). Vemurafenib was orally administered at 50 mg/kg twice daily. Vehicle was administered orally every day to control animals. *, P < 0.05; ***, P < 0.001. Animals were dosed for the duration of the experiments.

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Once daily oral administration of ASTX029 to Colo205 (BRAFV600E-mutant colorectal cancer) tumor-bearing mice significantly inhibited tumor growth in a dose-dependent manner over the range of 25 to 75 mg/kg, with tumor regressions observed at 75 mg/kg (P < 0.0001 vs. control; Fig. 5A; Supplementary Table S5). Administering ASTX029 at 150 mg/kg every other day also resulted in tumor regression (P < 0.0001). The antitumor activity of 150 mg/kg every other day ASTX029 was equivalent to that of 75 mg/kg ASTX029 every day (Fig. 5A), despite the different pharmacodynamic profiles over a 48-hour period (Supplementary Fig. S6), indicating that, as observed previously, continuous target inhibition is not required for antitumor activity (27). Significant antitumor activity of ASTX029 was also observed in Calu-6 and HCC44 KRAS-mutant lung, HCT116 KRAS-mutant colorectal cancer, A375 BRAFV600E-mutant melanoma, and MA-MEL-28 NRAS-mutant melanoma xenografts models and an MEXF2090 NRAS-mutant melanoma PDX model on once daily dosing of 75 mg/kg ASTX029 (Fig. 5B–G; Supplementary Table S5). These data demonstrate the broad efficacy of ASTX029 in models from various indications and with different mechanisms of MAPK pathway activation.

Models of acquired BRAF inhibitor and MEK inhibitor resistance are highly sensitive to ASTX029

The clinical efficacy of therapeutic agents targeting the MAPK pathway, such as RAF and MEK inhibitors is limited by the development of acquired resistance, often through the reactivation of ERK signaling (14). We investigated the potential of ASTX029 to overcome such resistance in two cell lines where resistance had been generated by continuous culture in the presence of vemurafenib [A375R, (31)] or selumetinib (A375-RS) and one where a clinically relevant mutation had been introduced (A375 NRASQ61K; refs. 16, 43). The A375 NRASQ61K cell line was less sensitive to vemurafenib (30-fold) and selumetinib (23-fold) than the parental A375 cell line but had similar sensitivity to trametinib (Supplementary Table S6). The A375R (31) and A375-RS cell lines were less sensitive to all three MAPK pathway inhibitors. In contrast, all cell lines remained highly sensitive to ASTX029. Proliferation of A375 NRASQ61K was inhibited with an IC50 of 12 nmol/L (a 3.5-fold increase in the IC50 value relative to the parental cell line), when treated with ASTX029, while proliferation of A375R and A375RS were inhibited with IC50 values of 7.2 and 8.1 nmol/L, respectively (no significant difference to the IC50 values of the parental cell line; Fig. 6A; Supplementary Table S6).

Figure 6.

Models of acquired BRAF inhibitor and MEK inhibitor resistance are sensitive to ASTX029. Effects on proliferation of vemurafenib, selumetinib, trametinib and ASTX029 in A375, A375-NRASQ61K, A375R and A375-RS cell lines (A). Data points represent mean ± SD. Effects on MAPK signaling of a 2 hours treatment with 17 nmol/L ASTX029 (A), 470 nmol/L vemurafenib (V), 220 nmol/L selumetinib (S) or 3.6 nmol/L trametinib (T) in A375, A375-NRASQ61K, A375R and A375-RS cells (B). Concentrations of compounds used were equivalent to 5× the A375 cell proliferation IC50 value for each compound. Antitumor activity of ASTX029 in A375R xenografts (C). Each data point represents mean ± SEM from 8 (ASTX029) or 7 (vemurafenib and vehicle) animals. Animals were dosed for the duration of the experiment. Vehicle consisting of 5% DMSO/0.95% methylcellulose was administered orally every day to control animals. ***, P < 0.001; ns, not significant. Effects on A375 cell proliferation of ASTX029 and vemurafenib in combination (D). Data points represent mean ± SD; n = 3.

Figure 6.

Models of acquired BRAF inhibitor and MEK inhibitor resistance are sensitive to ASTX029. Effects on proliferation of vemurafenib, selumetinib, trametinib and ASTX029 in A375, A375-NRASQ61K, A375R and A375-RS cell lines (A). Data points represent mean ± SD. Effects on MAPK signaling of a 2 hours treatment with 17 nmol/L ASTX029 (A), 470 nmol/L vemurafenib (V), 220 nmol/L selumetinib (S) or 3.6 nmol/L trametinib (T) in A375, A375-NRASQ61K, A375R and A375-RS cells (B). Concentrations of compounds used were equivalent to 5× the A375 cell proliferation IC50 value for each compound. Antitumor activity of ASTX029 in A375R xenografts (C). Each data point represents mean ± SEM from 8 (ASTX029) or 7 (vemurafenib and vehicle) animals. Animals were dosed for the duration of the experiment. Vehicle consisting of 5% DMSO/0.95% methylcellulose was administered orally every day to control animals. ***, P < 0.001; ns, not significant. Effects on A375 cell proliferation of ASTX029 and vemurafenib in combination (D). Data points represent mean ± SD; n = 3.

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Consistent with this, while MAPK signaling in A375 NRASQ61K cells was unaffected by vemurafenib or selumetinib treatment, levels of pRSK and pERK decreased on treatment with ASTX029 (Fig. 6B). In A375R and A375RS cells, vemurafenib, selumetinib and trametinib all failed to inhibit MAPK signaling to the same extent as in the parental A375 cell line, whilst decreases in pRSK and pERK levels were observed on ASTX029 treatment.

Antitumor activity of ASTX029 was demonstrated in vivo in the A375R xenograft model (Fig. 6C). A375R tumors developed in mice while undergoing treatment with vemurafenib at 50 mg/kg twice daily, a level that causes significant tumor growth inhibition in the parental A375 tumors (Fig. 5B). Replacement of vemurafenib treatment with 75 mg/kg every day ASTX029 treatment, led to significant tumor regression. The inhibition of MAPK signaling in ASTX029-treated A375R tumors was also confirmed (Supplementary Fig. S7). Overall, these data demonstrate that ASTX029 can overcome acquired resistance to MAPK inhibitors targeting upstream components of the pathway.

Furthermore, we confirmed that in the sensitive A375 cell line, the addition of ASTX029 to vemurafenib enhanced antiproliferative effects (Fig. 6D).

As the downstream effector of the MAPK pathway, ERK is considered an attractive cancer target. Therapeutically, ERK inhibitors have the potential not only to address resistance to the upstream BRAF and MEK inhibitors, but also to improve clinical response in a wide range of cancers with MAPK pathway aberrations, either as single-agent or combination therapy. (44–46). Using fragment-based screening and subsequent structure-based drug design, we identified the novel dual-mechanism orally bioavailable ERK inhibitor, ASTX029, with potent activity in human tumor cells lines and in vivo xenograft models. In contrast to the majority of ERK inhibitors currently undergoing clinical development, ASTX029 inhibits the catalytic activity of ERK and also prevents the phosphorylation of ERK. This distinctive profile could provide further clinical options for MAPK pathway modulation.

We and others have suggested that the differing abilities of ERK inhibitors to prevent the phosphorylation of ERK is related to their binding modes (28, 30). The mechanism by which this distinctive ERK-binding mode leads to a decrease in the phosphorylation of ERK has not previously been defined. The excellent selectivity profile of ASTX029 confirmed that this was not the result of direct MEK inhibition. However, it remained unclear whether the decrease in pERK levels observed was due to modulation of the phosphorylation of ERK by MEK or the modulation of ERK-directed phosphatases known as DUSP proteins. A cell-free assay confirmed that ASTX029 prevented the phosphorylation of ERK by MEK, but did not directly inhibit MEK activity suggesting that the decrease in pERK levels observed on ASTX029 treatment is indeed driven by the induction of a conformational change within ERK that prevents its phosphorylation.

The dual mechanism was confirmed in cell lines and xenograft models, where reductions in phosphorylation of both RSK, an ERK substrate, and ERK itself were observed on treatment with ASTX029. This contrasts with “catalytic” ERK inhibitors, which inhibit the phosphorylation of ERK substrates, but do not inhibit ERK phosphorylation (28, 29). Indeed, on treatment of cells with “catalytic” ERK inhibitors, a significant increase in pERK levels is observed (22, 27), which are most profound in mutant-KRAS cell lines due to the loss of negative feedback within the MAPK pathway upon the alleviation of CRAF inhibition (47, 48). The inhibition of ERK phosphorylation conferred by ASTX029 significantly delayed this rebound in pERK levels. Consistent with previous observations that feedback activation is greater in KRAS-mutant than BRAF-mutant cell lines (47, 48), ASTX029 conferred an increase in pMEK levels in HCT116, but not A375 cells. The increased feedback activation in HCT116 cells rationalizes why modulation of pERK is less sustained in this cell line compared with A375. Despite the restoration of pERK to basal levels by 16 hours in HCT116 cells, ERK catalytic activity remained inhibited by ASTX029 even at 72 hours, as demonstrated by the reduced pRSK levels.

Along with these increased inhibitory effects on MAPK signaling, dual-mechanism inhibitors also inhibit the nuclear translocation of ERK and enhance suppression of ERK-dependent gene expression (28). These effects resulted in increased potency in cells and suggest that dual-mechanism ERK inhibitors have potential for improved clinical efficacy.

Clinical responses have been observed for ERK inhibitors in BRAF-mutant tumors such as melanoma, NSCLC, and NRAS-mutant melanoma (23–25). Bioinformatic analysis of a cell line panel confirmed that cell lines with MAPK pathway–activating mutations were most sensitive to ASTX029 treatment. These included those which represent currently clinically underserved patient populations such as KRAS-mutant NSCLC and pancreatic cancer. Overall, these data demonstrate the tumor agnostic therapeutic potential of ASTX029 in MAPK-activated cancers and highlight further patient populations in solid and hematologic malignancies that may benefit from ERK inhibition.

Clinical activity of approved MAPK inhibitors is often limited by acquired resistance. We demonstrated that ASTX029 has activity in models of resistance to BRAF and MEK inhibitors. In cell proliferation assays, while the dose–response curves of vemurafenib, trametinib, and selumetinib shifted to the right in models of acquired resistance when compared with the parental cell line, no such shift was observed for ASTX029 (Fig. 6A). We therefore conclude that the mechanisms of acquired resistance to the RAF and MEK inhibitors, did not lead to resistance to ASTX029. This is consistent with previous observations that ERK inhibitors confer more robust inhibition of MAPK signaling, in the presence of upstream resistance mechanisms, than MEK inhibitors (19, 49) and highlights the therapeutic potential of dual-mechanism ERK inhibitors, such as ASTX029 in overcoming MAPK-inhibitor resistance. Although clinical resistance mechanisms are yet to be reported, preclinical data suggest that resistance to ERK inhibitors can occur via upstream activation of the MAPK pathway or the amplification or mutation of ERK itself (28, 50). As for other MAPK inhibitors, combination therapies, such as the BRAF inhibitor/ASTX029 combination demonstrated here, may ultimately prove to be essential in maximizing the clinical response to ERK inhibitors (50).

ASTX029 is currently being tested in a FIH phase I–II clinical trial in patients with advanced solid tumors (ClinicalTrials.gov Identifier: NCT03520075). As a dual-mechanism ERK inhibitor, its profile is distinct from many other ERK inhibitors investigated in the clinic. The differences in inhibition of MAPK signaling combined with the enhanced suppression of ERK-dependent gene expression observed preclinically may confer advantages over “catalytic” ERK inhibitors, but the impact of this different profile will only become apparent with further clinical investigation. Overall, these data highlight the therapeutic potential of ASTX029 for the treatment of patients with MAPK-driven tumors, including those with acquired resistance to BRAF and MEK inhibitors.

J.M. Munck reports personal fees from Astex Pharmaceuticals outside the submitted work. V. Berdini reports a patent for WO 2017/068412 pending and issued. C. East is employed by Astex Pharmaceuticals. R. Ferraldeschi reports personal fees from Astex Pharmaceuticals outside the submitted work; and R. Ferraldeschi is a former employee of Astex Pharmaceuticals. T.D. Heightman reports a patent for WO2017068412A1 pending; and T.D. Heightman is a former employee of Astex Pharmaceuticals which has a commercial interest in the discovery of ERK1/2 inhibitors. C.J. Hindley is a senior research associate at Astex Pharmaceuticals. C.W. Murray is an employee of Astex Pharmaceuticals. D. Norton reports a patent for WO 2017/068412 pending and issued. M. O'Reilly reports other support from Astex Pharmaceuticals during the conduct of the study; in addition, M. O'Reilly has a patent for WO 2017/068412 pending and issued. M. Reader reports a patent for WO 2017/068412 pending and issued. D.C. Rees is an employee of Astex. S.J. Rich reports is an employee of Astex. N.T. Thompson reports other support from Healx Ltd and personal fees from Astex Pharmaceuticals outside the submitted work; and is a member of the CanSAR scientific advisory board supported by Cancer Research UK and ICR. No financial compensation for this role. A.J.-A. Woolford reports a patent for WO 2017/068412 pending and issued. N.G. Wallis reports a patent for WO 2017/068412 pending and issued; and is an employee of Astex Pharmaceuticals. No disclosures were reported by the other authors.

J.M. Munck: Formal analysis, supervision, investigation, writing–original draft, project administration. V. Berdini: Formal analysis, visualization. L. Bevan: Formal analysis, investigation. J.L. Brothwood: Formal analysis, visualization. J. Castro: Formal analysis, investigation. A. Courtin: Formal analysis, investigation. C. East: Formal analysis, investigation. R. Ferraldeschi: Conceptualization. T.D. Heightman: Supervision, project administration. C.J. Hindley: Formal analysis, investigation, visualization, writing–review and editing. J. Kucia-Tran: Formal analysis, investigation, visualization, writing–review and editing. J.F. Lyons: Conceptualization, supervision, writing–review and editing. V. Martins: Formal analysis, investigation. S. Muench: Formal analysis, investigation. C.W. Murray: Conceptualization, supervision, project administration. D. Norton: Investigation. M. O'Reilly: Formal analysis, supervision, project administration. M. Reader: Investigation. D.C. Rees: Conceptualization, supervision. S.J. Rich: Formal analysis, investigation. C.J. Richardson: Formal analysis, visualization, project administration, writing–review and editing. A.D. Shah: Formal analysis, investigation. L. Stanczuk: Formal analysis, investigation. N.T. Thompson: Conceptualization, supervision. N.E. Wilsher: Formal analysis, project administration, writing–review and editing. A.J.-A. Woolford: Investigation. N.G. Wallis: Conceptualization, supervision, visualization, writing–original draft, project administration, writing–review and editing.

We are grateful to Keisha Hearn and Tomoko Smyth for useful discussion of the article and Anne Cleasby for PDB deposition.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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