Fig. 4: KRAS inhibitors enhance drug resistance by NLRX1-dependent mitophagy.
From: Cytosolic acetyl-coenzyme A is a signalling metabolite to control mitophagy
a–d, KRASi decreases Acly mRNA (a) and cytosolic AcCoA levels (b) and induces mitophagy (c,d). KPC cells were treated for 24 h and analysed using quantitative PCR with reverse transcription (RT–qPCR) (a); liquid chromatography coupled with MS (LC–MS) (b), normalized to cell numbers; immunoblotting (c); or flow cytometry for cells expressing mt-Keima reporter (d). n = 3 biological replicates. Data are mean ± s.e.m. Statistical analysis was performed using ordinary one-way ANOVA with Dunnett’s multiple-comparison test (a and d) and unpaired two-tailed Student’s t-tests (b). e–h, Nlrx1 knockout abolishes the KRASi-induced mitophagic response (e,f), and elevates ROS levels (g) and NADP+/NADPH ratio (h). KPC cells were used for immunoblot analyses (e), and cells expressing mt-Keima reporter were used for flow cytometry (f), CM-H2DCFDA staining (g) and the NADP+/NADPH colorimetric assay (h). n = 3 biological replicates. Data are mean ± s.e.m. Statistical analysis was performed using two-way ANOVA with Bonferroni’s multiple-comparison test. i, Nlrx1 depletion decreases KPC cell viability after MRTX1133 treatment. Dose–response curves for MRTX1133 treatment, based on 5-day CellTiter-Glo assays. Half-maximal inhibitory concentration (IC50) values are displayed at the top. n = 6 biological replicates. j, Nlrx1 knockout exacerbates the suppressive effect of MRTX1133 on tumour cell growth in vivo. NSG mice given subcutaneous injection of KPC cells were treated with vehicle or MRTX1133 (30 mg per kg, twice a day) when the tumour volume reached around 300 mm3. n = 5 mice per group. Data are mean ± s.e.m. Statistical analysis was performed using two-way ANOVA with Bonferroni’s multiple-comparison test. k,l, Impaired mitophagy and increased ROS production in Nlrx1-deficient tumours with MRTX1133 treatment. The mice were treated as in Fig. 4j, and after 6 d of treatment, tumours were collected for immunoblotting (k; n = 3 mice per group) or CM-H2DCFDA staining with counting by Image J (l; n = 30 images from 5 mice per group). Data are mean ± s.e.m. Statistical analysis was performed using two-way ANOVA with Bonferroni’s multiple-comparison test.
