A Small Molecule RAS-Mimetic Disrupts RAS Association with Effector Proteins to Block Signaling

Abstract

Oncogenic activation of RAS genes via point mutations occurs in 20%-30% of human cancers. The development of effective RAS inhibitors has been challenging, necessitating new approaches to inhibit this oncogenic protein. Functional studies have shown that the switch region of RAS interacts with a large number of effector proteins containing a common RAS-binding domain (RBD). Because RBD-mediated interactions are essential for RAS signaling, blocking RBD association with small molecules constitutes an attractive therapeutic approach. Here, we present evidence that rigosertib, a styryl-benzyl sulfone, acts as a RAS-mimetic and interacts with the RBDs of RAF kinases, resulting in their inability to bind to RAS, disruption of RAF activation, and inhibition of the RAS-RAF-MEK pathway. We also find that ribosertib binds to the RBDs of Ral-GDS and PI3Ks. These results suggest that targeting of RBDs across multiple signaling pathways by rigosertib may represent an effective strategy for inactivation of RAS signaling.

Keywords: MAPK; PI3K; RAF; RAS; RAS-binding domain; rigosertib.

Figure 1. Rigosertib Binds to the RBDs of c-RAF and B-RAF

(A and B) Recombinant GST-RBD (AA 1–149) of c-RAF (A) or kinase domain (KD) of c-RAF (AA 306–648) (B) were subjected to DSF in the presence of DMSO (control), rigosertib (RGS), or PLX4032 (PLX). GST-RBD and KD reactions were performed in triplicate and duplicate, respectively. (C) Binding of RGS to GST-RBD. GST-RBD was mixed with total cell lysates and incubated with RGS-biotin conjugate and streptavidin-agarose beads. The precipitates were subjected to western blot analysis using GST-specific antibodies. (D) GST-KD was mixed with total cell lysates and incubated with RGS-biotin conjugate and streptavidin-agarose beads. The precipitates were processed as described for GST-RBD. (E and F) Microscale thermophoretic analysis of RGS interaction with c-RAF and B-RAF RBDs. N-terminally labeled recombinant GST-tagged B-RAF and c-RAF RBD proteins were incubated with increasing concentrations of RGS and the binding reactions subjected to MST. RGS binds to the B-RAF and c-RAF RBDs with K_d values of 0.71 nM and 0.18 nM, respectively. See also Figures S1, S4, and S5.

Figure 2. NMR Analysis of the B-RAF RBD-Rigosertib Interaction: Chemical Shift Perturbation Studies and NMR Structure

(A) Expanded view of the ¹H-¹⁵N HSQC spectra of the titration of RGS with ¹⁵N-labeled B-RAF RBD. Overlaid ¹H-¹⁵N HSQC spectra of ¹⁵N-labeled B-RAF RBD with increasing concentrations of RGS: 0 (black), 0.25 (red), 0.5 (green), 1 (blue), 2 (magenta) mM. (B) Surface representation of the B-RAF RBD (apo structure) showing the residues affected by RGS. Residues that show significant NMR chemical shifts (>3σ from mean) are labeled and shown in cyan. (C and F) Superimposition of ten lowest energy complex structures of the B-RAF RBD (153–228) with RGS for complex I (blue/magenta) (C) and complex II (green/ red) (F). (D and G) Ribbon plot showing the lowest energy structure of the B-RAF RBD with RGS in complex I (D) and complex II (G). Residues interacting with RGS are denoted in stick and gray color. Dotted line denotes hydrogen bonding. (E and H) Superimposition of the lowest energy complex structures of B-RAF RBD:RGS with c-RAF RBD:Ras X-ray co-crystal structure (PBD: 4G0N). ComplexI (blue) with RGS (magenta) and c-RAF RBD:Ras complex (cyan) is shown in (E). Complex II (green) with RGS (red) and c-RAF RBD:Ras complex (cyan) is shown in (H). Backbone RMSD between B-RAF RBD in complex I and complex II with c-RAF RBD in the crystal structure (PDB: 4G0N) is 1.15 Å. See also Figure S2 and Table S1.

Figure 3. Rigosertib Interacts with the β1 Strand, β2 Strand, and α1 Helix of the B-RAF RBD, Interferes with the Binding of Active RAS with the RAF-RBD, and Inhibits RAS-Mediated Activation of RAF Kinase Activity

(A) Alignment of the three RAF RBDs showing the residues in contact with RGS that are highlighted in cyan. (B) Effect of RBD mutations on c-RAF-RBD:RAS interaction. HeLa cells (WT) or HeLa cells expressing a mutant form of RAS (G12D) were serum starved for 18 hr and stimulated with EGF for 5 min. The level of GTP-bound RAS in the cell lysates was tested for its ability to bind to WT c-RAF RBD and the indicated mutants. The same mutants were examined for their ability to bind to RGS using MST. K_d values for the mutants are provided below the blot. (C) Four representative titration curves are shown. (D) A431 cells were serum starved for 18 hr and treated with EGF and cell lysates analyzed for the level of RAS-GTP binding to the c-RAF RBD that was pre-incubated with DMSO or increasing concentrations of RGS for overnight. (E) Exponentially growing HeLa cells were treated overnight with DMSO, RGS, or PLX4032 (PLX) and the cell lysates incubated for 2–4 hr at 4° C with agarose beads linked to GST-Ras^G12D. After extensive washing, the complexes were examined by immunoblot analysis using B-RAF or c-RAF antibodies. The levels of RAF proteins in the whole cell extracts are also shown. (F and G) HeLa (F) or A431 (G) cells were serum starved for 18 hr in the presence of DMSO, RGS, or PLX, treated with EGF and the levels of RAS-GTP in cell lysates were determined using GST-RAF1-RBD and glutathione beads in pull-down assays. (H) HeLa cells expressing N-RAS (G12D) were treated as in (F). The level of binding of G12D-N-RAS-GTP with the c-RAF RBD is shown. (I and J) HeLa (I) and A431(J) cell lysates from (F) and (G) were subjected to immunoprecipitation with anti-c-RAF antibodies and the immunoprecipitates analyzed for RAF kinase activity using kinase-inactive recombinant MEK protein as a substrate. See also Figure S2 and Table S1.

Figure 4. Rigosertib Interferes with Growth Factor-Induced RAF Dimerization and MEK/ERK Activation

(A–F) HeLa (A) or A431 (B) cells were serum starved for 18 hr in the presence of DMSO, RGS, or PLX, treated with EGF and cell lysates subjected to immunoprecipitation with anti-c-RAF antibodies and the level of associated B-RAF determined by western blot analysis. The level of ERK, phospho-ERK, MEK, phospho-MEK, total c-RAF and B-RAF, and GAPDH (loading control) were also determined by western blot analysis. The effect of DMSO, RGS, or PLX on EGF-induced RAF dimerization and/or activation of MEK and ERK in HeLa (C) cells transfected with a vector control or HA-tagged, N-RAS (G12D) expression vector HCT-116 (D) and A549 (E) and WM1617 (F) cells were determined as described above. See also Figure S3.

Figure 5. Treatment of Human Tumor Cells with Rigosertib Inhibits c-RAF^Ser338 Phosphorylation and Inhibits PLK-1:RAF Interaction

(A) HCT116 cells were serum-starved in the presence of DMSO, RGS, or PLX, stimulated with EGF and the lysates subjected to immunoprecipitation using a c-RAF-specific antibody. The immunoprecipitates were subjected to western blot analysis using c-RAF and c-RAF^Ser338 antisera. (B and C) HCT 116 cells were blocked at the G₁/S boundary using a double thymidine block, released into growth medium containing RGS or nocadazole (NOCDZ), and the levels of c-RAF^Ser338, PLK1, and histone H3^Ser10 were determined by western blot analysis. Note the accumulation of c-RAF^Ser338 in cells treated with nocadazole as the cells progress toward mitosis, which is drastically reduced in RGS-treated cells. (D) Cell lysates isolated from RGS- or nocodazole-treated cells were subjected to immunoprecipitation using a PLK1-specific antibody and the level of associated c-RAF^Ser338 determined by western blot analysis.

Figure 6. Rigosertib Inhibits RAS-Mediated Transformation and Tumor Growth

(A) Rigosertib inhibits transformation of chicken fibroblasts (CEFs) by mutant RAS, p110α-E545K, p110α-H1047R, PI3Kβ, PI3Kγ, and PI3Kδ. CEFs were transfected with RCAS virus expressing the indicated oncogene in the presence of increasing concentrations of RGS. The plates were overlaid with nutrient agar every other day for 2–3 weeks post-infection. Transformation values are represented in graphical form. (B) The absolute level of colony formation by each of these constructs in the absence or presence of RGS. (C) Growth of HCT116 xenograft tumors in vehicle or RGS-treated ncr/ncr mice as measured using Transferrin-Vivo 750 imaging agent. Fluorescence units are shown. (D) Florescence images of the mice on day 16. (E) Tumor extracts derived from placebo and RGS-treated mice were subjected to western blot analysis using the indicated antibodies. (F) Average values of each phospho protein are shown in graphical form. See also Figure S6.

Figure 7. Treatment with Rigosertib Suppresses the Growth of RAS-Driven Pancreatic Intraepithelial Neoplasia

(A and B) Average number of PanINs per section (A) and total number of PanINs by grade (B) in vehicle- and RGS-treated animals. (C) Representative hematoxylin & eosin (H&E) and immunohistochemical staining of representative pancreatic sections harvested from three vehicle- and RGS-treated mice. Scale bar, 100 mm. Average levels of ERK and AKT^Ser473 phosphorylation and caspase-3 cleavage in PanINs are also represented graphically. All values represent mean ± SD; n ≥ 3 for each treatment group. *p ≤ 0.05; **p ≤ 0.005. See also Figure S5.