Fig 1: CDK4/6 knockdown results in enhanced MYC dependence CDK4/6 knockdown sensitizes HCT116 cells to MYC inhibition. Top, Western blotting analysis of MYC protein expression in CDK4/6‐kd and control cells after treatment with 50 μM 10058‐F4 or vehicle for 24 h. CDK, CDK4/6‐kd cells; Ctr, cells transfected with non‐targeting RNA duplexes. Bottom, CDK4/6‐kd or control cells were scored before and after treatment with 50 μM 10058‐F4 or vehicle for 24 h.Inhibition of MYC reverts key molecular and signaling responses to CDK4/6 inhibition. Left, incubation of HCT116 cells with increased doses of 10058‐F4 was paralleled by downregulation of MYC, GLS1, P‐mTOR, P‐S6K, and P‐Akt and upregulation of HIF‐1α. β‐actin signal was used as a Western blotting loading and transfer control. Right, dose‐dependent effects of 10058‐F4 on the proliferation of HCT116 cells treated with PD0332991 (2 μM). Statistically significant differences are indicated as P < 0.001 (***).MYC knockdown synergizes with CDK4/6 inhibition in its effects on HCT116 cell proliferation. Cells were counted 72 h and 96 h after siRNA transfection.Effect of MYC knockdown on the extracellular metabolic fluxes of CDK4/6‐kd (top) and PD0332991‐treated (bottom) cells. Glucose and glutamine consumption and lactate and glutamate production rates were obtained after 24 h of incubation with fresh media and normalized to cell number. Results are expressed as percentage to CDK4/6‐inhibited cells consumption and production rates.Effect of CDK4/6 and MYC combined knockdown on protein levels assessed by Western blotting. Cells were transfected with MYC siRNA 24 h before analysis.CDK4/6 knockdown sensitizes HCT116 cells to GLS1 inhibition. Top, Percentages of viable cells after incubation with BPTES (10 μM), BPTES (10 μM) + αKG (2 mM), under glutamine depletion or glutamine depletion + αKG (2 mM). Bottom, percentages of early apoptotic cells, assessed with the Annexin V‐PI assay.Data information: CDK4/6, CDK4/6‐kd cells; Control, cells transfected with non‐targeting RNA duplexes. Data are represented as mean ± SD (n = 3). Significance was determined by ANOVA and two‐tailed independent sample Student's t‐tests. Statistically significant differences between CDK4/6‐inhibited and control cells or MYC‐kd and control cells are indicated as P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***), while differences between treatment and the corresponding control are shown as P < 0.05 (#), P < 0.01 (##), and P < 0.001 (###) for CDK4/6‐kd and as P < 0.05 (¶), P < 0.01 (¶¶), and P < 0.001 (¶¶¶) for control cells. All experiments were performed 96 h after siRNA transfection or PD0332991 treatment. Source data are available online for this figure.
Fig 2: p38α interacts with and phosphorylates c-MYC. (a) In vitro binding assay between GST-p38α fusion protein and c-MYC recombinant protein. Bound proteins were analyzed by immunoblotting using anti-GST, anti-HIS, and anti-c-MYC antibodies. HIS-β-catenin was used as a positive control. (b) Co-immunoprecipitation of endogenous p38α and c-MYC in HT29 cells. (c) In vitro kinase assay showing c-MYC phosphorylation by p38α in the absence or presence of ralimetinib at the indicated concentrations. Statistical analysis was performed using Student’s t-test, * p < 0.05 vs. active p38α; ∆ p < 0.05 vs. active p38α + c-MYC. (d) In silico phosphorylation prediction meta-analysis. Four in silico prediction servers were used to identify MYC consensus phosphorylation sites: iPTMnet (https://research.bioinformatics.udel.edu/iptmnet; accessed on 14 April 2022), NETPHOS 3.1 (https://services.healthtech.dtu.dk/service.php?NetPhos-3.1; threshold ≥ 3, accessed on 14 April 2022), Phosphosite Plus (https://www.phosphosite.org/homeAction, only HTP data, accessed on 14 April 2022), and KynasePhos 2.0 (http://kinasephos2.mbc.nctu.edu.tw/, accessed on 13 December 2020). (e) MS/MS spectrum of the double-charged precursor ion of E54LLPTPPLSPSRRSGLCSPSYVAVTPFSLRG84. MB: MYC Boxed conserved domain. NLS: nuclear localization sequence. HLH: helix–loop–helix domain. LZ: leucine zip domain. The presented results are representative of at least three independent experiments. Detailed information about Western Blot can be found at supplementary materials.
Fig 3: High protein expression levels of p38α and c-MYC are potential predictive biomarkers for therapy efficacy in CRC patients. (a,b) Kaplan–Meier curve of progression-free survival (PFS) (a) and disease-free survival (DFS) (b) in CRC patients as a function of p38α (MAPK14) and c-MYC (MYC) protein levels based on clinical data of 581 CRC patients retrieved from TCGA PanCancer Atlas.
Fig 4: Effects of p38α/ERK crosstalk on c-MYC expression in CRC cells. (a) Left panel: Classification of the CRC cell lines used in this study by their microsatellite instability (MIN) and chromosome instability (CIN) phenotypes and the mutational status of critical cancer genes. Right panel: Immunoblotting showing c-MYC protein levels in HT29, HCT116, and Caco2 cells treated with SB202190 (10 μM) for up to 72 h. (b) Immunoblotting showing c-MYC protein levels in HT29 cells treated with PD98059 (20 μM) for up to 72 h. (c) Immunoblotting showing c-MYC protein levels in HT29 cells treated with SB202190 (10 μM) and/or PD98059 (20 μM) for 24 h. (d) Left panel: Densitometric analysis of c-MYC protein levels detected by immunoblotting in HCT116 cells transfected with p38α-specific and/or MEK-specific siRNAs. Right panel: Densitometric analysis of p38α and MEK protein levels detected by immunoblotting to show silencing efficiency in HCT116 cells transfected with p38α-specific or MEK-specific siRNAs, respectively. (a–d) β-actin was used for normalization; MEK1 activation (p-MEK1) and p38 activity (p-MK2) were analyzed to check treatment efficacy. (d) Statistical analysis was performed using Student’s t-test; * p < 0.05 vs. cells treated with control siRNAs, # p < 0.05: vs. single silenced cells. The presented results are representative of at least three independent experiments. Detailed information about Western Blot can be found at supplementary materials.
Fig 5: Pharmacological inhibition of p38α and MEK as a synthetic lethality approach. (a) Immunoblotting showing c-MYC protein levels in HT29 and HCT116 cells treated for 24 h with SB202190 (10 μM) or ralimetinib (10 μM). (b) Immunoblotting showing c-MYC protein levels in HT29 and HCT116 cells treated for up to 48 h with ralimetinib (10 μM) and/or PD98059 (20 μM). (c) Immunoblotting showing c-MYC protein levels in HT29 and HCT116 cells treated for up to 48 h with ralimetinib (10 μM) and/or trametinib (1 nM). (d) Quantification of cell death by trypan blue staining in HT29 and HCT116 cells treated with ralimetinib (10 μM) and/or trametinib (1 nM) for 48 h. (e) Proliferative index of HT29 and HCT116 cells treated with ralimetinib (10 μM) and/or trametinib (1 nM) for 48 h, as determined by WST-1 assay. (f) Cell viability assay on HT29 and HCT116 cells treated with ralimetinib (10 μM) and/or trametinib (1 nM) for 48 h. (g) Graphs summarizing the percentage of Ki67-positive cells, as determined by flow cytometry analysis, in HT29 and HCT116 cells treated with SB202190 (10 μM) and/or PD98059 (20 μM) or with ralimetinib (10 μM) and/or trametinib (1 nM) for 48 h. (h) Graphs summarizing the percentage of apoptotic cells (early + late), as determined by flow cytometry analysis of annexin V staining, in HT29 and HCT116 cells treated as in g. (i) RT-PCR analysis of the mRNA levels of the c-MYC target genes p21, Cyclin E, Cyclin A, and cdc25 in HT29 cells treated with ralimetinib (10 μM) and/or trametinib (1 nM) for 24 h. (j) Immunoblotting showing c-MYC and cleaved PARP protein levels in two lines of patient-derived CRC-SCs grown as tumorspheres (#9 and #40) treated with ralimetinib (10 μM) and/or trametinib (1 nM) for 48 h. (a–c,j) β-actin was used for normalization. (d,e,g–i) Statistical analysis was performed using Student’s t-test; * p < 0.05: vs. untreated cells, # p < 0.05: vs. corresponding single treatment; cl.PARP: cleaved PARP; p1: patient 1-derived CRC-SC; p2: patient 2-derived CRC-SC. The presented results are representative of at least three independent experiments. Detailed information about Western Blot can be found at supplementary materials.
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