ASN007 is a selective ERK1/2 inhibitor with preferential activity against RAS-and RAF-mutant tumors

Abstract

Inhibition of the extracellular signal-regulated kinases ERK1 and ERK2 (ERK1/2) offers a promising therapeutic strategy in cancers harboring activated RAS/RAF/MEK/ERK signaling pathways. Here, we describe an orally bioavailable and selective ERK1/2 inhibitor, ASN007, currently in clinical development for the treatment of cancer. In preclinical studies, ASN007 shows strong antiproliferative activity in tumors harboring mutations in BRAF and RAS (KRAS, NRAS, and HRAS). ASN007 demonstrates activity in a BRAF^V600E mutant melanoma tumor model that is resistant to BRAF and MEK inhibitors. The PI3K inhibitor copanlisib enhances the antiproliferative activity of ASN007 both in vitro and in vivo due to dual inhibition of RAS/MAPK and PI3K survival pathways. Our data provide a rationale for evaluating ASN007 in RAS/RAF-driven tumors as well as a mechanistic basis for combining ASN007 with PI3K inhibitors.

Keywords: ASN007; ERK; KRAS; PI3K; RAF/RAS-driven cancers; biomarker; combinational therapy; kinase inhibitor; lymphoma; solid tumors.

Graphical abstract

Figure 1

Structural and biochemical characterization of ASN007 (A) Molecular structure of ASN007, MW = 473. (B) Dose-response curves of ERK1/2 inhibitors (ASN007 or SCH772894) in a homogeneous time-resolved fluorescence (HTRF)-based ERK1/2 enzymatic activity assay. (C) Kinome tree dendrogram showing the biochemical kinase profiling assay (KinaseProfiler, Eurofiler), which measures the kinase inhibitory activity of ASN007 in a radiometric enzymatic assay in a panel of 335 purified kinases. Hit kinases reported are marked with a red circle and the size of the circle indicates the relative potency of ASN007 for each kinase. ERK1/2 kinases are illustrated by a blue circle. Green dots indicate kinases against which ASN007 does not have significant activity at 1 μM. The dendrogram was generated using the web tool TREESpot, provided by DiscoverX. (D) IC₅₀ values (nM) for ASN007 against the top 22 kinases, which showed >75% inhibition at 1 μμM, identified from the primary kinome screening. (E) Dose-dependent effect of ASN007 treatment on phosphorylation of ERK1/2 targets. Representative western blot analysis shows inhibition of RSK1 (Ser380) phosphorylation after 4h of ASN007 treatment in a colorectal adenocarcinoma cell line, HT-29. (F) Dose-response curve of ASN007 inhibition of RSK phosphorylation in the human colon cancer HT-29 BRAF^V600E mutant cell line, measured by an ELISA test.

Figure 2

Antiproliferative efficacy of ASN007 in solid tumor and lymphoma cell lines (A) The IC₅₀ values of ASN007 were compared with two other ERK1/2 inhibitors (ulixertinib/BVD-523 and ravoxertinib/GDC-0994) in a panel of solid tumor cell lines with or without mutations in the RAS/RAF/MEK/ERK pathway. Cells were treated with increasing concentrations of the specified ERK1/2 inhibitor for 72 h before cell viability assessment. (B) ASN007 was more effective against lymphoma cell lines harboring mutations in the RAS/RAF pathway. Each bar represents the mean ± SD of three independent experiments. (C) Dot plot comparing IC₅₀ values of ASN007 in cell lines with or without mutations in the RAS/RAF/MAPK pathway. Black dots illustrate IC₅₀ values for lymphoma cell lines and red dots illustrate solid tumor cell lines. Differences between groups were calculated with Student’s t test. ∗∗∗∗p < 0.0001.

Figure 3

In vivo activity of ASN007 in mouse xenografts and PDX models (A) MIA PaCa-2, PANC-1, SK-N-AS, and HCT116 tumor cells were implanted, and when tumors reached ~100 mm³ in size, mice were treated with either vehicle or ASN007 at the indicated doses and schedules. BID, twice per day; PO, per os (oral) administration; QD, once per day. Error bars represent means ± SEMs (n > 8 per treatment group). (B) Activity of two different doses of ASN007 in a mantle cell lymphoma xenograft model using the MINO cell line. Error bars represent means ± SEMs (n >8 per treatment group). (C) Representative western blot analysis of total and phosphorylated ERK1/2 and RSK in tumor extracts from the MINO xenograft at the end of treatment (75 mg/kg, QD, or 40 mg/kg, BID). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. (D) PDX model derived from a melanoma patient before (ST052B) and after relapsing on vemurafenib treatment (ST052C). In the ST052C model, treatment with ASN007 (25 mg/kg or 50 mg/kg PO, BID) remained effective while therapy with the BRAF inhibitor dabrafenib showed no efficacy. Error bars represent means ± SEMs (n > 5 per treatment group). (E) Tumor growth inhibition by ASN007 (40 mg/kg PO, BID) in a panel of colorectal cancer PDX models treated for 22 to 35 days, depending on the model. Each bar represents 1 model. The color of the bar denotes the mutation status of BRAF and KRAS genes; magenta = BRAF mutant, green = KRAS mutant, and white = wild type for both BRAF and KRAS. In all in vivo efficacy studies except for the HCT116 model, the besylate (salt correction factor 1.33) or mandelate salt form (salt correction factor 1.32) of ASN007 was used. However, the freebase form of ASN007 was used for the study involving the HCT116 model.

Figure 4

The combination of ASN007 and the PI3K inhibitor copanlisib results in enhanced antiproliferative activity in vitro and enhanced tumor growth inhibition in vivo (A) Drug matrix heatmap of the enhanced antiproliferative effect when ASN007 is combined with the PI3K inhibitor copanlisib in an MCL cell line, MINO. Heatmap grids correspond to the mean of three independent experiments measured by the MTS assay. (B) Western blot analysis of total and phosphorylated levels of rS6 and MSK after 72 h of treatment with ASN007 (0.3 μM), copanlisib (0.05 μM), or the combination of both compounds in MINO cells. Blots are representative of three independent experiments. (C) Combination of ASN007 and copanlisib results in enhanced efficacy in MINO and two lung carcinoma (A549, NCI-H1975) xenograft tumor models. Error bars represent means ± SEMs (n > 8 per treatment group). (D) Combination of ASN007 and copanlisib results in enhanced efficacy in an MCL PDX mouse model (PDX44685). Doses administered: ASN007 (40 mg/kg PO, BID in all four models); copanlisib (14 mg/kg for MINO, NCI-H1975, and PDX 44685, and 10 mg/kg for A549, 2 days on followed by 5 days off, intraperitoneally [i.p.]). Error bars represent means ± SEMs (n > 8 per treatment group).