Journal: iScience

Article Title: CETSA-MS unveils novel targets engaged by rigosertib to promote anti-tumor activity and inflammatory responses

doi: 10.1016/j.isci.2025.112748

Figure Lengend Snippet: Rigosertib does not inhibit the interaction between active RAS with C-RAF RBD in NanoBiT assays and affects the RAS/RAF/MEK signaling pathway in HeLa cells (A) A NanoBiT cellular assay to monitor the interaction between C-RAF RBD and active RAS. BHK-21 cells transfected with SmBiT-C-RAF RBD and the indicated LgBiT-RAS G12V construct. After 48 h, cells were treated for 8 h with DMSO, rocaglamide (Roc, 100 nM), rigosertib (20 μM) or ON02180.Na (20 μM). Shown are relative luminescence unit (RLU) values relative to DMSO control and normalized to positive control from 3 independent experiments. Data are represented as mean ± SEM. ns: not significant p > 0.05, (∗) p ≤ 0.05, (∗∗) p ≤ 0.01, (∗∗∗) p ≤ 0.001, (∗∗∗∗) p ≤ 0.0001. (B) KRAS G12C binding to C-RAF RBD in a NanoBiT assay in BHK-21 cells treated 2, 4, or 8 h with increasing concentration of rigosertib. RAS G12C-binding compound ARS-1620 served as a positive control. Shown are mean RLU values for duplicates relative to DMSO control. Data are represented as a median. (C) A cell-free NanoBiT assay in lysates of HEK-293T transfected with SmBiT-C-RAF RBD and LgBiT-KRAS G12C. Cell lysates were incubated for 4 h with increasing concentrations of ARS-1620, rigosertib or ON02180.Na. Shown are RLU values relative to DMSO control from one representative experiment out of two. Data are represented as a mean with SD. ns: not significant p > 0.05, (∗) p ≤ 0.05, (∗∗) p ≤ 0.01, (∗∗∗) p ≤ 0.001, (∗∗∗∗) p ≤ 0.0001. (D) HeLa cells serum-starved and treated with rigosertib (RGS, 2 μM) for 2, 4, 8, or 24 h then stimulated with EGF at 100 ng/mL for 5 min. Active RAS pull-down and western blot analysis of pathway activation in total cell lysates. Shown is the result of one representative experiment. (E) Quantifications of (D) for EGF-stimulated cells relative to DMSO from 4 independent experiments. Two-way ANOVA, Tukey’s multiple comparisons test was used to determine significant differences. Data are represented as a mean with SEM; ns: not significant p > 0.05, (∗) p ≤ 0.05, (∗∗) p ≤ 0.01, (∗∗∗) p ≤ 0.001, (∗∗∗∗) p ≤ 0.0001.

Article Snippet: We first tested the hypothesis that rigosertib acts as a RAS mimetic by evaluating its direct binding of clinical-grade rigosertib obtained from Onconova Therapeutics, Inc. (currently under the name of Traws Pharma, Inc.) to the RBD of C-RAF (C-RAF RBD) using a thermal shift assay.

Techniques: Transfection, Construct, Control, Positive Control, Binding Assay, Concentration Assay, Incubation, Western Blot, Activation Assay

Journal: iScience

Article Title: CETSA-MS unveils novel targets engaged by rigosertib to promote anti-tumor activity and inflammatory responses

doi: 10.1016/j.isci.2025.112748

Figure Lengend Snippet: CETSA MS profiling of HL60, H358, and K562 cells (A) Schematic representation of the experimental workflow of the CETSA MS assay. (B and D) x axis represents effect size (amplitude, log2 fold-change), and y axis effect significance (−log10(pvalue)). Significance for the concentration (20 μM) response fit is based on Benjamini-Hochberg corrected p values from moderated t test. Only proteins with more than two tryptic peptides are shown. (C and E) Compound-induced protein thermal stability changes after 15 min incubation of lysed HL60, H358, and K562 cells with either 2 or 20 μM of rigosertib.

Article Snippet: We first tested the hypothesis that rigosertib acts as a RAS mimetic by evaluating its direct binding of clinical-grade rigosertib obtained from Onconova Therapeutics, Inc. (currently under the name of Traws Pharma, Inc.) to the RBD of C-RAF (C-RAF RBD) using a thermal shift assay.

Techniques: Concentration Assay, Incubation

Journal: iScience

Article Title: CETSA-MS unveils novel targets engaged by rigosertib to promote anti-tumor activity and inflammatory responses

doi: 10.1016/j.isci.2025.112748

Figure Lengend Snippet: Rigosertib induces ROS production and activates the JNK signaling by targeting the ERO1A and NQO2 (A) Human recombinant NQO2 protein was incubated with DMSO, rigosertib (2, 10, 20, or 100 μM) and imatinib (1 or 100 μM) for 10 min. The enzymatic reactions were initiated by adding 300 μM menadione as a substrate along with 500 μM NMEH as a cosubstrate. The enzymatic activity of NQO2 was determined by measuring the absorbance at 595 nm of the reduced MTT. Data are represented as a mean with SD. (B) Quantification of the NQO2 enzymatic activity immediately after incubation with rigosertib or imatinib. Shown is the result of 3 experiments and ordinary one-way ANOVA, Dunnett’s multiple comparisons test was used to determine significant differences. Data are represented as a median; ns: not significant p > 0.05, (∗) p ≤ 0.05, (∗∗) p ≤ 0.01, (∗∗∗) p ≤ 0.001, (∗∗∗∗) p ≤ 0.0001. (C) Quantification from 3 independent experiments of the live cell imaging of the effect of single ERO1A or NQO2 knock-down on ROS generation in H358 cells treated with rigosertib 2 μM. Significance was determined using two-way ANOVA test. Data are represented as a mean with SD; ns: not significant p > 0.05, (∗) p ≤ 0.05, (∗∗) p ≤ 0.01, (∗∗∗) p ≤ 0.001, (∗∗∗∗) p ≤ 0.0001. (D) Western blot of the lysates that were used for (C) to confirm the ERO1A and NQO2 knockdown. (E) Western blot analysis of the effect of rigosertib on the phosphorylated JNK levels in H358-shERO1A or -shNQO2 knockdown cells from 2 to 24 h. (F) Western blot analysis of the effect of ERO1A and NQO2 single or double knockdown in H358 cells treated with rigosertib 2 μM. (G) Comparison of the levels of phosphorylated Thr183/Tyr185 residues of SAPK/JNK between double and single knockdown of ERO1A and NQO2 after 24 h rigosertib treatment from 3 independent experiments. Two-way ANOVA, Tukey’s multiple comparisons test was used to determine significant differences. Data are represented as a median; ns: not significant p > 0.05, (∗) p ≤ 0.05, (∗∗) p ≤ 0.01, (∗∗∗) p ≤ 0.001, (∗∗∗∗) p ≤ 0.0001.

Article Snippet: We first tested the hypothesis that rigosertib acts as a RAS mimetic by evaluating its direct binding of clinical-grade rigosertib obtained from Onconova Therapeutics, Inc. (currently under the name of Traws Pharma, Inc.) to the RBD of C-RAF (C-RAF RBD) using a thermal shift assay.

Techniques: Recombinant, Incubation, Activity Assay, Live Cell Imaging, Knockdown, Western Blot, Comparison

Journal: iScience

Article Title: CETSA-MS unveils novel targets engaged by rigosertib to promote anti-tumor activity and inflammatory responses

doi: 10.1016/j.isci.2025.112748

Figure Lengend Snippet: Rigosertib promotes cell death induction and caspase-1 activity to trigger IL-18 secretion in patient-derived lung cancer organoids (A) Fluorescent staining of the expression of the luminal epithelial marker cytokeratin 7 (CK7), the lung ciliated cell marker acetylated alpha-tubulin (Ac-Tub), and nuclei (DAPI) of organoids derived from human lung cancer organoids (LPTO54, LPTO85-KRAS G12D, and PDXO344-KRAS G12C) and normal (P2N and P4N). Organoids were treated with DMSO or RGS 2 μM for 72 h. (B) Cell viability of organoids derived from human lung cancer organoids (LPTO54, LPTO85, and PDXO344) and normal (P2N and P4N) after 72 h rigosertib treatment. Ordinary one-way ANOVA, Tukey’s multiple comparisons test was used to determine significant differences. Data are represented as a mean with SD; ns: not significant p > 0.05, (∗) p ≤ 0.05, (∗∗) p ≤ 0.01, (∗∗∗) p ≤ 0.001, (∗∗∗∗) p ≤ 0.0001. (C) Analysis of NLRP3 inflammasome activation marker caspase-1 after 48 h rigosertib treatment of the organoids. Two-way ANOVA, Šídák’s multiple comparisons test was used to determine significant differences. Data are represented as a mean with SD; ns: not significant p > 0.05, (∗) p ≤ 0.05, (∗∗) p ≤ 0.01, (∗∗∗) p ≤ 0.001, (∗∗∗∗) p ≤ 0.0001. (D and E) Western blot analysis of cell death activation markers after 72 h rigosertib treatment of organoids. Vinculin levels are detected as a loading control for each blot and presented below. Activation of pyroptotic death was measured by immunoblotting of activated GSDMD (p20). Activation of apoptosis was measured by immunoblotting of active caspase-3 (p17). (F) Quantification of (D and E) from 3 independent experiments. Two-way ANOVA, Tukey’s multiple comparisons test was used to determine significant differences. Data are represented as a median; ns: not significant p > 0.05, (∗) p ≤ 0.05, (∗∗) p ≤ 0.01, (∗∗∗) p ≤ 0.001, (∗∗∗∗) p ≤ 0.0001.

Article Snippet: We first tested the hypothesis that rigosertib acts as a RAS mimetic by evaluating its direct binding of clinical-grade rigosertib obtained from Onconova Therapeutics, Inc. (currently under the name of Traws Pharma, Inc.) to the RBD of C-RAF (C-RAF RBD) using a thermal shift assay.

Techniques: Activity Assay, Derivative Assay, Staining, Expressing, Marker, Activation Assay, Western Blot, Control

Journal: iScience

Article Title: CETSA-MS unveils novel targets engaged by rigosertib to promote anti-tumor activity and inflammatory responses

doi: 10.1016/j.isci.2025.112748

Figure Lengend Snippet: Rigosertib does not inhibit the interaction between active RAS with C-RAF RBD in NanoBiT assays and affects the RAS/RAF/MEK signaling pathway in HeLa cells (A) A NanoBiT cellular assay to monitor the interaction between C-RAF RBD and active RAS. BHK-21 cells transfected with SmBiT-C-RAF RBD and the indicated LgBiT-RAS G12V construct. After 48 h, cells were treated for 8 h with DMSO, rocaglamide (Roc, 100 nM), rigosertib (20 μM) or ON02180.Na (20 μM). Shown are relative luminescence unit (RLU) values relative to DMSO control and normalized to positive control from 3 independent experiments. Data are represented as mean ± SEM. ns: not significant p > 0.05, (∗) p ≤ 0.05, (∗∗) p ≤ 0.01, (∗∗∗) p ≤ 0.001, (∗∗∗∗) p ≤ 0.0001. (B) KRAS G12C binding to C-RAF RBD in a NanoBiT assay in BHK-21 cells treated 2, 4, or 8 h with increasing concentration of rigosertib. RAS G12C-binding compound ARS-1620 served as a positive control. Shown are mean RLU values for duplicates relative to DMSO control. Data are represented as a median. (C) A cell-free NanoBiT assay in lysates of HEK-293T transfected with SmBiT-C-RAF RBD and LgBiT-KRAS G12C. Cell lysates were incubated for 4 h with increasing concentrations of ARS-1620, rigosertib or ON02180.Na. Shown are RLU values relative to DMSO control from one representative experiment out of two. Data are represented as a mean with SD. ns: not significant p > 0.05, (∗) p ≤ 0.05, (∗∗) p ≤ 0.01, (∗∗∗) p ≤ 0.001, (∗∗∗∗) p ≤ 0.0001. (D) HeLa cells serum-starved and treated with rigosertib (RGS, 2 μM) for 2, 4, 8, or 24 h then stimulated with EGF at 100 ng/mL for 5 min. Active RAS pull-down and western blot analysis of pathway activation in total cell lysates. Shown is the result of one representative experiment. (E) Quantifications of (D) for EGF-stimulated cells relative to DMSO from 4 independent experiments. Two-way ANOVA, Tukey’s multiple comparisons test was used to determine significant differences. Data are represented as a mean with SEM; ns: not significant p > 0.05, (∗) p ≤ 0.05, (∗∗) p ≤ 0.01, (∗∗∗) p ≤ 0.001, (∗∗∗∗) p ≤ 0.0001.

Article Snippet: Rigosertib (RGS) was provided by Onconova Therapeutics as dry powder and stored at −20°C.

Techniques: Transfection, Construct, Control, Positive Control, Binding Assay, Concentration Assay, Incubation, Western Blot, Activation Assay

Journal: iScience

Article Title: CETSA-MS unveils novel targets engaged by rigosertib to promote anti-tumor activity and inflammatory responses

doi: 10.1016/j.isci.2025.112748

Figure Lengend Snippet: CETSA MS profiling of HL60, H358, and K562 cells (A) Schematic representation of the experimental workflow of the CETSA MS assay. (B and D) x axis represents effect size (amplitude, log2 fold-change), and y axis effect significance (−log10(pvalue)). Significance for the concentration (20 μM) response fit is based on Benjamini-Hochberg corrected p values from moderated t test. Only proteins with more than two tryptic peptides are shown. (C and E) Compound-induced protein thermal stability changes after 15 min incubation of lysed HL60, H358, and K562 cells with either 2 or 20 μM of rigosertib.

Article Snippet: Rigosertib (RGS) was provided by Onconova Therapeutics as dry powder and stored at −20°C.

Techniques: Concentration Assay, Incubation

Journal: iScience

Article Title: CETSA-MS unveils novel targets engaged by rigosertib to promote anti-tumor activity and inflammatory responses

doi: 10.1016/j.isci.2025.112748

Figure Lengend Snippet: Rigosertib induces ROS production and activates the JNK signaling by targeting the ERO1A and NQO2 (A) Human recombinant NQO2 protein was incubated with DMSO, rigosertib (2, 10, 20, or 100 μM) and imatinib (1 or 100 μM) for 10 min. The enzymatic reactions were initiated by adding 300 μM menadione as a substrate along with 500 μM NMEH as a cosubstrate. The enzymatic activity of NQO2 was determined by measuring the absorbance at 595 nm of the reduced MTT. Data are represented as a mean with SD. (B) Quantification of the NQO2 enzymatic activity immediately after incubation with rigosertib or imatinib. Shown is the result of 3 experiments and ordinary one-way ANOVA, Dunnett’s multiple comparisons test was used to determine significant differences. Data are represented as a median; ns: not significant p > 0.05, (∗) p ≤ 0.05, (∗∗) p ≤ 0.01, (∗∗∗) p ≤ 0.001, (∗∗∗∗) p ≤ 0.0001. (C) Quantification from 3 independent experiments of the live cell imaging of the effect of single ERO1A or NQO2 knock-down on ROS generation in H358 cells treated with rigosertib 2 μM. Significance was determined using two-way ANOVA test. Data are represented as a mean with SD; ns: not significant p > 0.05, (∗) p ≤ 0.05, (∗∗) p ≤ 0.01, (∗∗∗) p ≤ 0.001, (∗∗∗∗) p ≤ 0.0001. (D) Western blot of the lysates that were used for (C) to confirm the ERO1A and NQO2 knockdown. (E) Western blot analysis of the effect of rigosertib on the phosphorylated JNK levels in H358-shERO1A or -shNQO2 knockdown cells from 2 to 24 h. (F) Western blot analysis of the effect of ERO1A and NQO2 single or double knockdown in H358 cells treated with rigosertib 2 μM. (G) Comparison of the levels of phosphorylated Thr183/Tyr185 residues of SAPK/JNK between double and single knockdown of ERO1A and NQO2 after 24 h rigosertib treatment from 3 independent experiments. Two-way ANOVA, Tukey’s multiple comparisons test was used to determine significant differences. Data are represented as a median; ns: not significant p > 0.05, (∗) p ≤ 0.05, (∗∗) p ≤ 0.01, (∗∗∗) p ≤ 0.001, (∗∗∗∗) p ≤ 0.0001.

Article Snippet: Rigosertib (RGS) was provided by Onconova Therapeutics as dry powder and stored at −20°C.

Techniques: Recombinant, Incubation, Activity Assay, Live Cell Imaging, Knockdown, Western Blot, Comparison

Journal: iScience

Article Title: CETSA-MS unveils novel targets engaged by rigosertib to promote anti-tumor activity and inflammatory responses

doi: 10.1016/j.isci.2025.112748

Figure Lengend Snippet: Rigosertib promotes cell death induction and caspase-1 activity to trigger IL-18 secretion in patient-derived lung cancer organoids (A) Fluorescent staining of the expression of the luminal epithelial marker cytokeratin 7 (CK7), the lung ciliated cell marker acetylated alpha-tubulin (Ac-Tub), and nuclei (DAPI) of organoids derived from human lung cancer organoids (LPTO54, LPTO85-KRAS G12D, and PDXO344-KRAS G12C) and normal (P2N and P4N). Organoids were treated with DMSO or RGS 2 μM for 72 h. (B) Cell viability of organoids derived from human lung cancer organoids (LPTO54, LPTO85, and PDXO344) and normal (P2N and P4N) after 72 h rigosertib treatment. Ordinary one-way ANOVA, Tukey’s multiple comparisons test was used to determine significant differences. Data are represented as a mean with SD; ns: not significant p > 0.05, (∗) p ≤ 0.05, (∗∗) p ≤ 0.01, (∗∗∗) p ≤ 0.001, (∗∗∗∗) p ≤ 0.0001. (C) Analysis of NLRP3 inflammasome activation marker caspase-1 after 48 h rigosertib treatment of the organoids. Two-way ANOVA, Šídák’s multiple comparisons test was used to determine significant differences. Data are represented as a mean with SD; ns: not significant p > 0.05, (∗) p ≤ 0.05, (∗∗) p ≤ 0.01, (∗∗∗) p ≤ 0.001, (∗∗∗∗) p ≤ 0.0001. (D and E) Western blot analysis of cell death activation markers after 72 h rigosertib treatment of organoids. Vinculin levels are detected as a loading control for each blot and presented below. Activation of pyroptotic death was measured by immunoblotting of activated GSDMD (p20). Activation of apoptosis was measured by immunoblotting of active caspase-3 (p17). (F) Quantification of (D and E) from 3 independent experiments. Two-way ANOVA, Tukey’s multiple comparisons test was used to determine significant differences. Data are represented as a median; ns: not significant p > 0.05, (∗) p ≤ 0.05, (∗∗) p ≤ 0.01, (∗∗∗) p ≤ 0.001, (∗∗∗∗) p ≤ 0.0001.

Article Snippet: Rigosertib (RGS) was provided by Onconova Therapeutics as dry powder and stored at −20°C.

Techniques: Activity Assay, Derivative Assay, Staining, Expressing, Marker, Activation Assay, Western Blot, Control