Identification of the resistance mechanism to CDK4/6 inhibition and novel overcoming combination therapy with RNR inhibition for chemo-resistant bladder cancer

The identification of 42 down-regulated protein candidates in treated T24 cells and 46 in RT112 cells compared to the PBS control with 18 overlapping proteins (Supplementary Table S1); At the different time points, 2596, 3674 and 1994 genes were identified as differentially expressed genes (DEGs) (Supplementary Table S2, 3, 4); Identified activation of enormous genes that may confer acquired resistance to CDK4/6 inhibition after 7 days of CDK4/6 inhibition by a CRISPR-dCas9 activation functional genomics analysis, to further narrow down the functional candidates, we applied LFC＞1 and FDR＜0.001 and identified 365 potential candidates in this study (Supplementary Table S5); To further investigate the potential biological interaction network of RRM2 in clinical bladder cancer specimen, we inquired RRM2 in the TCGA cohorts for mRNA co-expression.

Through this analysis, we identified the top 10 correlated genes: KIF18B, SKA3, FANCA, MYBL2, CENPA, FAM72B, IQGAP3, E2F1, FAM72D and CDT1 ranked by Spearman's Correlation (Supplementary Table S6); The informaton of prospectively collected 20 MIBC bladder cancer specimens from patients who received radical cystectomy in our hospital. Patients were divided into 3 subgroups according to therapy naïve, chemotherapy responsive and chemotherapy resistance (Supplementary Table S7)