Journal: Redox Biology

Article Title: Targeting ALDH1A1 to enhance the efficacy of KRAS-targeted therapy through ferroptosis

doi: 10.1016/j.redox.2024.103361

Figure Lengend Snippet: ALDH1A1 promotes resistance to KRAS G12C inhibitors relying on enzyme activity. ( A) Venn plot displayed 36 candidates from the intersection of differentially expressed genes detected by RNA sequencing between the DMSO and the KRAS G12C inhibitors (sotorasib or adagraisb) treatment in MIAPACA2 and H2122 cells. The cells were treated for 72 h with IC50 (sotorasib: MIAPACA2, IC50 = 40 nM; H2122, IC50 = 100 nM; adagrasib: MIAPACA2, IC50 = 60 nM; H2122, IC50 = 180 nM). (B) The volcano plots showed that ALDH1A1 was upregulated in both cells treated with KRAS G12C inhibitors (|log2FC| > 0.4 and p < 0.05). (C) qRT-PCR and WB verified the ALDH1A1 upregulation in twelve KRAS G12C mutant cell lines treated with sotorasib or adagraisb for 72 h (pancreatic adenocarcinoma cell line MIAPACA2; bladder urothelial carcinoma cell line UMUC3; esophageal squamous cell carcinoma cell line KYSE410; lung adenocarcinoma cell lines H2122, LU65, LU99, SW1573, and H1792; colorectal adenocarcinoma cell lines JVE-015, TT1TKB, SW837, and SW1463) (n = 3). (D-E) Relative ALDH1A1 activity enhanced in MIAPACA2 and H2122 cells in response to sotorasib or adagraisb (sotorasib: MIAPACA2, IC50 = 40 nM, IC90 = 1000 nM, Peak serum concentration = 10000 nM; H2122, IC50 = 100 nM, IC90 = 5000 nM, Peak serum concentration = 10000 nM; adagrasib: MIAPACA2, IC50 = 60 nM, IC90 = 1500 nM, Peak serum concentration = 6000 nM; H2122, IC50 = 180 nM, IC90 = 5000 nM, Peak serum concentration = 6000 nM, 72 h), determined via ALDH Activity Assay Kit (D) and ALDEFLUOR assay (E) (representative flow cytometry dot plots are shown) (n = 3). (F) Crucial active site 193K for ALDH1A1 was mutated from K to Q/R, blocking the ALDH1A1 activity but not the protein expression. (G-H) MIAPACA2 and H2122 stable cell lines were established with the ALDH1A1 knockout (KO) and overexpression (OE) with or without mutants (K193Q/R). The ALDH1A1 enzyme activity was decreased extremely in the ALDH1A1 -KO, K193Q/R group and increased in ALDH1A1 -OE (representative flow cytometry dot plots are shown) (n = 3). (I) Relative viability of MIAPACA2 and H2122 cells showed that ALDH1A1 KO and loss of enzyme function (K193Q/R) increased the effect of gradient concentrations of KRAS G12C inhibitors for 72 h, while ALDH1A1 OE did the opposite (n = 3). (J) Relative viability of MIAPACA2 and H2122 cells showed that ALDH1A1 inhibitor CM10 (500 nM) increased the effect of gradient concentrations of KRAS G12C inhibitors for 72 h (n = 3). Data were analyzed by two-way ANOVA or Student’s t-test and were presented by mean ± SD.

Article Snippet: Various doses of KRAS-targeted inhibitors, RSL3, IKE, fer-1, Z -VAD, DFO, necro, CM10, ATRA, AGN193109, were used to treat the cells for 72 h. The Cell Counting Kit-8 (TargetMol) was used to gauge the activity of cellular dehydrogenases as an indicator of cell viability.

Techniques: Activity Assay, RNA Sequencing Assay, Quantitative RT-PCR, Mutagenesis, Concentration Assay, Flow Cytometry, Blocking Assay, Expressing, Stable Transfection, Knock-Out, Over Expression

Journal: Redox Biology

Article Title: Targeting ALDH1A1 to enhance the efficacy of KRAS-targeted therapy through ferroptosis

doi: 10.1016/j.redox.2024.103361

Figure Lengend Snippet: Inhibition of ALDH1A1 confers sensitivity to KRAS G12C inhibitors in PDOs and in vivo. (A) Flow chart of KRAS G12C mutant PAAD and LUAD patient-derived organoids (PDOs) Establishment. (B) HE staining images of PAAD and LUAD patient tissues and PDOs (scale bars, 50 μm). (C) Top: WB showed the successful construction of ALDH1A1 KO and OE of PAAD and LUAD PDOs. Below: representative brightfield images of PAAD and LUAD PDOs (scale bars, 100 μm). (D) Representative brightfield images of PAAD PDOs with ALDH1A1 KO or OE, treated with KRAS G12C inhibitors for 72 h (scale bars, 100 μm). (E) Luminescence measurement showed the relative viability of PAAD with different concentrations of sotorasib and adagrasib for 72 h. ALDH1A1 KO caused more severe death in PDOs, which could be abolished by overexpression. (F – G) Representative brightfield images (F) and luminescence measurement (G) of PAAD PDOs incubated with or without CM10 (1000 nM), and treated with sotorasib (300 nM) or adagrasib (300 nM) for 72 h (scale bars, 100 μm). (H) Representative brightfield images of LUAD PDOs with ALDH1A1 KO or OE, treated with KRAS G12C inhibitors for 72 h (scale bars, 100 μm). (I) Luminescence measurement showed the relative viability of PAAD with different concentrations of sotorasib and adagrasib for 72 h. (J – K) Representative brightfield images (J) and luminescence measurement (K) of LUAD PDOs incubated with or without CM10 (1000 nM), and treated with sotorasib (300 nM) or adagrasib (300 nM) for 72 h (scale bars, 100 μm). (L) Schematic design of xenografted tumor assay. ( M – O) Tumor images (M), growth curves (N), and tumor weight at the end of the treatment (O) showed that ALDH1A1 OE rescued the ALDH1A1 KO-induced sensitivity to KRAS G12C inhibitors in vivo. (P) Top: Administration of sotorasib, adagrasib, and CM10 for nude mice. Below: Tumor images of each group, growth curves (Q), and Tumor weight (R) showed that CM10 combined with KRAS G12C inhibitors repressed the tumor growth derived from MIAPACA2 cells more than KRAS inhibitors treatment alone. Data were analyzed by two-way ANOVA or Student's t-test and were presented by mean ± SD.

Article Snippet: Various doses of KRAS-targeted inhibitors, RSL3, IKE, fer-1, Z -VAD, DFO, necro, CM10, ATRA, AGN193109, were used to treat the cells for 72 h. The Cell Counting Kit-8 (TargetMol) was used to gauge the activity of cellular dehydrogenases as an indicator of cell viability.

Techniques: Inhibition, In Vivo, Mutagenesis, Derivative Assay, Staining, Over Expression, Incubation

Journal: Redox Biology

Article Title: Targeting ALDH1A1 to enhance the efficacy of KRAS-targeted therapy through ferroptosis

doi: 10.1016/j.redox.2024.103361

Figure Lengend Snippet: ALDH1A1 confers resistance to KRAS-targeted inhibitors by inhibiting ferroptosis . (A) Cell viability of MIAPACA2 and H2122 cells ( ALDH1A1 -NC, KO) treated with sotorasib or adagrasib alone, or combined with fer-1 (500 nM) or DFO (5000 nM) or Z -VAD (500 nM) or necrosulfonamide (250 nM) for 72 h (n = 3). Only fer-1 and DFO reversed the KRAS inhibitors-induced cell death and showed no difference in ALDH1A1 KO groups. (B–C) Co-treatment with fer-1 (500 nM) or DFO (5000 nM) abolished the KRAS G12C inhibitors-enhanced lipid peroxidation (B) and MDA (C) levels of MIAPACA2 and H2122 cells (IC50, 72 h) (n = 3). (D – G) Lipid peroxidation (D) and MDA (E) levels in MIAPACA2 and H2122 cells increased in ALDH1A1 -KO, K193Q/R groups, and CM10-treated group (500 nM) (F–G), under the treatment of sotorasib or adagrasib (IC50, 72 h) . ALDH1A1 -OE reverses the increase. (n = 3) (H) CM10 and KRAS inhibitors combination increased the intracellular ferrous iron level. (n = 3) (I) DFO rescued mitochondrial shrinkage. CM10 exacerbated and mitochondrial shrinkage (MIAPACA2 cell) caused by sotorasib or adagrasib (IC50, 72 h), displayed by transmission electron microscopy images (scale bars, 10 μm). Data were analyzed by Student's t-test and were presented by mean ± SD.

Article Snippet: Various doses of KRAS-targeted inhibitors, RSL3, IKE, fer-1, Z -VAD, DFO, necro, CM10, ATRA, AGN193109, were used to treat the cells for 72 h. The Cell Counting Kit-8 (TargetMol) was used to gauge the activity of cellular dehydrogenases as an indicator of cell viability.

Techniques: Transmission Assay, Electron Microscopy

Journal: Redox Biology

Article Title: Targeting ALDH1A1 to enhance the efficacy of KRAS-targeted therapy through ferroptosis

doi: 10.1016/j.redox.2024.103361

Figure Lengend Snippet: ALDH1A1 inhibits ferroptosis through detoxifying aldehydes, promoting production of NADH and improving RARA function . (A – D) Measurement of retinal (A), 4-HNE (B), retinoic acid (C), and NADH (D) production in MIAPACA3 and H2122 cells with ALDH1A1 NC, inhibitor (CM10:500 nM), KO and OE under sotorasib or adagrasib treatment (IC50,IC90, Peak serum concentration, 72 h) (n = 3). (E) Relative viability showed retinoic acid receptor α (RARA) resisted KRAS G12C mutant cells to ferroptosis caused by RSL3, IKE, sotorasib and adagtasib (IC50), demonstrated by medications with RARA inhibitors AGN193109 (1000 nM) or activator ATRA (1000 nM) for 72 h. (n = 3) (F) Lipid peroxidation of KRAS G12C mutant cells increased when added AGN193109 (1000 nM) to sotorasib or adagrasib (IC50) for 72 h. (n = 3) (G) Dual-luciferase assays reflected significant RARA activity depression in ALDH1A1 -KO and CM10 (500 nM) treated groups (MIAPACA2 and H2122 cells) under KRAS G12C inhibitors medications (IC50). (n = 3) Data were analyzed by Student's t-test and were presented by mean ± SD.

Article Snippet: Various doses of KRAS-targeted inhibitors, RSL3, IKE, fer-1, Z -VAD, DFO, necro, CM10, ATRA, AGN193109, were used to treat the cells for 72 h. The Cell Counting Kit-8 (TargetMol) was used to gauge the activity of cellular dehydrogenases as an indicator of cell viability.

Techniques: Concentration Assay, Mutagenesis, Medications, Luciferase, Activity Assay

Journal: Redox Biology

Article Title: Targeting ALDH1A1 to enhance the efficacy of KRAS-targeted therapy through ferroptosis

doi: 10.1016/j.redox.2024.103361

Figure Lengend Snippet: ALDH1A1 inhibits ferroptosis through activation of the CREB1/GPX4 axis and promotion of lipid droplet formation depending on lower pH. (A) ALDH1A1 -KO or pharmacological inhibition (CM10:500 nM) increased intracellular pH (pHi) detected by BCECF-AM probe in MIAPACA2 and H2122 cells treated with KRAS G12C inhibitors, while ALDH1A1 OE did the opposite (n = 3). (B–C) Relative viability revealed the lower pHe rendered KRAS G12C mutant cells resistant to RSL3, IKE (B), sotorasib and adagrasib (C) (pHe = 7.5, 7.0, 6.5, 72 h) (n = 3). (D – E) Lipid peroxidation (D) and MDA (E) levels in MIAPACA2 and H2122 cells at indicating pH (pHe = 7.5, 7.0, 6.5) verified lower pHe inhibits ferroptosis (n = 3). (F) qRT-PCR and WB screened out that GPX4 upregulated by acidic conditions, not FSP1 and SLC7A11 (n = 3). (G) MIAPACA2 and H2122 cells were maintained at indicating pH (pHe = 7.5, 7.0, 6.5) for 72 h, and the WB results showed that lower pHe upregulated GPX4 expression. (H) ALDH1A1 KO reduced CREB1 phosphorylation and GPX4 expression, and ALDH1A1 OE activated CREB1/GPX4 axis, while HCL supplement abolished the reduction. (I – J) Representative pictures (MIAPACA2 and H2122 cells) (I) and quantification (J) of Lipi-Green-stained lipid droplet showed lower pHe promoted LD formation upon maintained at indicating pH for 24 h (pHe = 7.5, 7.0, 6.5) (scale bars, 10 μm) (n = 3). (K – L) ALDH1A1 KO and inhibition (CM10:500 nM, 72 h) repressed LD formation, and ALDH1A1 OE promoted it, while HCL supplement reversed the suppression. Representative pictures (MIAPACA2 and H2122 cells) (K) and quantification (L) of Lipi-Green-stained lipid droplet were shown (scale bars, 10 μm) (n = 3) . Data were analyzed by Student's t-test and were presented by mean ± SD. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Various doses of KRAS-targeted inhibitors, RSL3, IKE, fer-1, Z -VAD, DFO, necro, CM10, ATRA, AGN193109, were used to treat the cells for 72 h. The Cell Counting Kit-8 (TargetMol) was used to gauge the activity of cellular dehydrogenases as an indicator of cell viability.

Techniques: Activation Assay, Inhibition, Mutagenesis, Quantitative RT-PCR, Expressing, Staining