The RNA-binding protein LRPPRC promotes resistance to CDK4/6 inhibition in lung cancer

Abstract

Kinase inhibitors against Cyclin Dependent Kinase 4 and 6 (CDK4/6i) are promising cancer therapeutic drugs. However, their effects are limited by primary or acquired resistance in virtually all tumor types. Here, we demonstrate that Leucine Rich Pentatricopeptide Repeat Containing (LRPPRC) controls CDK4/6i response in lung cancer by forming a feedback loop with CDK6. LRPPRC binds to CDK6-mRNA, increasing the stability and expression of CDK6. CDK6 and its downstream E2F Transcription Factor 1 (E2F1), bind to the LRPPRC promoter and elevate LRPPRC transcription. The activation of the LRPPRC-CDK6 loop facilitates cell cycle G1/S transition, oxidative phosphorylation, and cancer stem cell generation. Gossypol acetate (GAA), a gynecological medicine that has been repurposed as a degrader of LRPPRC, enhances the CDK4/6i sensitivity in vitro and in vivo. Our study reveals a mechanism responsible for CDK4/6i resistance and provides an enlightening approach to investigating the combinations of CDK4/6 and LRPPRC inhibitors in cancer therapy.

Fig. 1. GAA decreased CDK6 expression and increased CDK4/6i sensitivity in vitro.

a Schematic diagram of the screening process to search for chemical compounds which inhibited the expression of CDK6 protein and increased the efficacy of CDK4/6i. Eight compounds were screened out from 850 natural product compounds, with a coefficient of drug interaction score below 0.8 when combined with CDK4/6i ribociclib. Then, two of the eight compounds could reduce CDK6 expression in immunoblotting by about 50%. b Representative colony formation images of LUAD cell lines treated with indicated compounds. Normalized crystal violet staining intensity (mean; n = 3 biological replicates) was displayed in the lower right corner. c Dose-response curves of A549 and PC9 cells to CDK4/6i with or without GAA treatment (mean ± SEM.; n = 4 biological replicates). d Immunoblotting of indicated proteins in LUAD cells. LUAD cells were pretreated with ribociclib (Ribo), doxorubicin (DOX), and gossypol acetate (GAA) at different concentrations for 48 hours. e Schematic of generating LUAD cell lines with acquired resistance against CDK4/6i through continuous cell culture and stepwise dose escalation of ribociclib from 500 nM to 20 μM. Cell lines are listed below. Cells were first co-cultured with 500 nM ribociclib for one week, then co-cultured with 1 μM ribociclib for four weeks, 5 μM, 10 μM, 15 μM, and 20 μM ribociclib for six weeks sequentially and respectively. f Dose-response curves of parent cells (Parent) and acquired resistance cells (Re) to ribociclib (mean ± SEM, n = 4 biological replicates). g Dose-response curves of acquired resistance cells to ribociclib with or without GAA treatment (mean ± SEM, n = 3 biological replicates in H1299 cell lines and n = 4 biological replicates in H460 cell lines). Images b and d are representative results of n = 3 independent experiments with similar tendency. Source data are provided as a Source Data file.

Fig. 2. GAA regulated CDK6 expression and CDK4/6i sensitivity via LRPPRC.

a Fluorescence titration analysis of the binding between LRPPRC protein and GAA, where F_o is the fluorescence intensity of protein, F is the fluorescence intensity of protein after the addition of ligand. b, c Cellular thermal shift assay analysis of LRPPRC in A549 cells treated with DMSO or 40 μM GAA. d Immunoblotting of LRPPRC protein in A549 cells treated with ribociclib and GAA. e Immunoblotting of LRPPRC protein in A549 cells before and after LRPPRC knockout. f, g Representative images and quantified cell colonies of A549 cells (before and after LRPPRC knockout) treated with different concentrations of ribociclib (mean ± SEM.; n = 3 biological replicates). h Cell proliferation curves of A549 (before and after LRPPRC knockout) in the presence of ribociclib (Ribo, 5 μM; mean ± SD.; n = 3 biological replicates). i, j Tumor proliferation curves of A549 cells (before and after LRPPRC knockout) treated with ribociclib (mean ± SEM.; n = 5 mice in each group). The tumor formation rate in different groups was calculated. k IHC staining of LRPPRC in breast cancer patients responding or not responding to palbociclib treatment. l PFS analysis of breast patients who received palbociclib based on LRPPRC expression (n = 15 in LRPPRC- low arm, n = 13 in LRPPRC-high arm). PFS of patients who received first-line palbociclib treatment was analyzed separately (n = 6 in LRPPRC-low arm, n = 7 in LRPPRC- high arm). m Cell morphology imaging of A549 cells (before and after LRPPRC knockout) treated with DMSO or 5 μM GAA for 48 hours. n Dose-response curves of A549 cells to ribociclib before and after LRPPRC knockout (mean ± SEM.; n = 3 biological replicates). Immunoblotting results in images b and d were representative results of n = 2 and 3 independent experiments, respectively, with similar tendency. IHC images in k and cell morphology images in m were representative results of n = 2 independent experiments with similar tendency. Statistical significance in figure j was determined by Chi-square analysis. Statistical significance in figure l was determined by Kaplan-Meier analysis. Source data are provided as a Source Data file.

Fig. 3. LRPPRC directly modulated CDK6 expression and controlled cell cycle G1/S transition.

a Immunoblotting of indicated proteins in different A549 cell colonies. b Heatmap of relative abundance of indicated transcripts detected by hybridization chip in A549 cells before and after LRPPRC knockdown. c Meta-gene analysis showed the distribution of LRPPRC-binding sites along with a normalized transcript. d The RIP/Input signal mRNAs determined by two RIP-sequencing biological replicates. e Mapping of RIP-seq reads back to the genomic locus of indicated transcripts. f RIP/Input signals of indicated mRNAs quantified by PCR (mean ± SEM, n = 3 biological replicates). g Relative enrichment of CDK6 mRNA by LRPPRC antibody in the presence of ssDNA R14 or RTT (mean ± SEM, n = 3 biological replicates). h Quantification of CDK6 mRNA in in different A549 cell colonies (mean ± SEM, n = 4 biological replicates). i Representative quantified CDK6 mRNA degradation rate in different A549 cells colonies (mean ± SEM, n = 4 technical replicates). j Normalized G0 and G1 cell percentage in different A549 cell colonies after nocodozol was added (mean ± SEM.; n = 3 biological replicates). The Flow cytometry gating strategy was shown in supplemental Figure S10a. k, l Representative EDU staining images of different A549 cell colonies. The percentage of EDU-positive cells was quantified by Flow cytometry (mean ± SEM.; n = 3 biological replicates). The Flow cytometry gating strategy was shown in supplemental Figure S10c. m Representative quantified CDK6 mRNA degradation rate in A549 cells before and after GAA (5 μM) treatment (mean ± SEM, n = 4 technical replicates). n Quantification of the CDK6 mRNA in cells treated with GAA for 48 hours (mean ± SEM, n = 4 biological replicates). o, p Flow cytometry analysis of PI or EDU staining of LUAD cells treated with GAA, ribociclib (Ribo), or combination (Combi) (mean ± SEM.; n = 3 biological replicates). Immunoblotting results in a, EDU staining images in k, and the PCR results in images i and m were representative results of n = 3 independent experiments with similar tendency. Statistical significance in figures f, g, h, l, n, o, and p was determined by a one-way ANOVA analysis. Source data are provided as a Source Data file.

Fig. 4. CDK6 and E2F1 promoted LRPPRC expression by enhancing transcription.

a Volcano plot showed the quantitative dynamics of proteins in H1299 and H1299-Re quantified by non-labeled MS (n = 3 biological replicates). b Immunoblotting of indicated protein in parental cells (Par) and acquired-resistant cells (Res). c Quantification of protein and mRNA level of CDK6 and LRPPRC in parental and acquired-resistant cells (mean ± SEM, n = 3 biological replicates). d KEGG Pathway enrichment (performed using KOBAS software) of differentially expressed proteins in H1299 and H1299-Re. e Mapping of CDK6, E2F1, or H3K27AC Chip-seq reads back to the genomic locus of LRPPRC. The figure was obtained from Cistrome database. f Quantification of LRPPRC promoter in Chip products using CDK6 antibody, E2F1 antibody, histone H3 antibody (Anti-H3, positive control), and non-immune antibody (Anti-NC, negative control) in A549 cells (mean ± SEM, n = 4 biological replicates). g Diagram of the binding sites in LRPPRC promoter interacted with E2F1 protein. The conserved E2F1 binding sequences were also shown. h EMSA experiment testing the interaction between E2F1 and LRPPRC promoter. i Quantification of LRPPRC mRNA in A549 cell before and after knockdown of either CDK6 or E2F1 (mean ± SEM, n = 4 biological replicates). j, k Immunoblotting of LRPPRC in A549 cells transfected with indicated siRNAs or released from nocodazole treatment at different time points. l, m Correlation analysis of indicated mRNAs in different cancer types. The correlation score, sample number, and P value were obtained from the online analysis website TIMER (https://cistrome.shinyapps.io/timer/). n, o Correlation analysis of indicated mRNAs in LUAD samples provided by the GEO dataset (GSE101929). The Immunoblotting results in figure b, j, and k were representative results of n = 3 independent experiments with the same tendency. The statistical significance of results in figures c, f, and i was determined by a one-way ANOVA analysis. The statistical significance of the correlation analysis in figure l-o was determined by a two-tailed nonparametric Spearman correlation analysis. Source data are provided as a Source Data file.

Fig. 5. LRPPRC-CDK6 inhibition suppressed OXPHOS and eliminated cancer stem cells.

a Heatmap of relative abundance of indicated mRNAs in A549 cells before and after LRPPRC knockout. b, c Representative images and quantification of colony formation of A549 cells treated with metformin, ribociclib (Ribo), or combination (Combi) (mean ± SEM.; n = 3 biological replicates). d Heatmap of relative abundance of indicated mRNAs in A549 cells treated with GAA. e OCR (mean ± SEM.; n = 5 biological replicates) and ECAR (mean ± SEM.; n = 4 biological replicates) analysis of A549 cells before and after GAA treatment. f Overview of changes in central carbon metabolism after GAA treatment. Metabolite changes were given as folds of control (mean ± SEM.; n = 3 biological replicates). g Representative images and number quantification of tumorspheres of A549 before and after LRPPRC knockout (mean ± SEM.; n = 3 biological replicates). h, i Quantification of CD133⁺, or CD44⁺CD133⁺ cells in cells with LRPPRC knockout or GAA treatment (mean ± SEM.; n= 3 biological replicates). j Representative flow cytometry images of parental cells (H1299) and acquired CDK4/6i-resistant cells (H1299-Re). The percentage of CD44⁺CD133⁺ cells was quantified (mean ± SEM.; n = 3 biological replicates). k Representative images and number quantification of tumorspheres of parental and acquired CDK4/6i-resistant cells (mean ± SEM.; n = 3 biological replicates). l, m Quantification of CSC mRNA markers in parental and acquired resistant cells (mean ± SEM, n = 4 biological replicates). n Representative images of tumorspheres generated from H1299-Re cells treated with DMSO or GAA. The number of tumorspheres per 1000 cells was quantified (mean ± SEM.; n = 3 biological replicates). o Quantification of CD44⁺CD133⁺ cells in H1299-Re cells treated with different concentrations of GAA (mean ± SEM.; n = 3 biological replicates). p, q Quantification of CSC mRNA markers in acquired resistant cells treated with different concentrations of GAA (mean ± SEM, n = 3 biological replicates). The Flow cytometry gating strategy in figure h, i, j, and o was shown in supplemental Figure S10d. The statistical significance of quantification results in figure c, i, o, p, and q was determined by a one-way ANOVA analysis. Statistical significance in figure h, j, l, m, and n was determined by a two-tailed unpaired Student’s t-test. Source data are provided as a Source Data file.

Fig. 6. GAA inhibited LRPPRC-CDK6 feedback loop and increased CDK4/6i sensitivity in vivo.

a Schematic diagram of GAA treatment in mice bearing A549 cells generated subcutaneous tumor. b Tumor volume of subcutaneous A549 cells treated with indicated drugs (mean ± SEM.; n = 1 experiment; n = 6 mice in GAA treatment group (GAA) and n = 7 mice in other groups). c Kaplan–Meier simulated survival analysis of mice inoculated with A549 cells and treated with indicated compounds. d, e IHC images and quantification of IHC intensity of indicated proteins in A549 cells xenografts treated with different drugs. f Schematic diagram of GAA treatment with mice model bearing subcutaneous tumor generated from clinical LUAD samples. g Tumor volume of subcutaneous LUAD tissues in the mice treated with indicated drugs (mean ± SEM.; n = 1 experiment; n = 7 mice in the control group (NC), n = 6 mice in GAA treatment group (GAA), n = 5 mice in ribociclib treatment group (Ribo), and n = 6 mice in the combination treatment group (Combi)). h Kaplan–Meier simulated survival analysis of mice inoculated with LUAD tissues and treated with indicated compounds. i IHC images and quantification of IHC intensity of indicated proteins in PDX xenografts treated with different drugs. The statistical significance of quantification results in figure b was determined by a two-way ANOVA analysis for comparison at the endpoint. IHC images d and i were representative results of n = 3 independent experiments. The statistical significance of quantification results in figure g was determined by a two-way ANOVA analysis for comparison at day 25. The statistical significance of quantification results in figure c and h was determined by Kaplan-Meier analysis. Statistical significance of quantification results in figure e and i was determined by a Chi-square test. Source data are provided as a Source Data file. j Schematic diagram of the principle of this work. LRPPRC binds to CDK6 mRNA and promotes CDK6 expression. CDK6 and its downstream E2F1 bind to the promoter of LRPPRC and promote its expression, forming a positive feedback loop. The combination of CDK4/6i and LRPPRC inhibitor GAA arrests cells in the G0/G1 phase and eliminates tumor stem cells.