In this study, through searching in cancer-omics databases and immunohistochemistry validation with clinical samples, we showed that the expression of MYBL2, a key oncogenic transcriptional factor, was significantly upregulated correlatively with RRM2 in colorectal cancer (CRC). key oncogenic transcriptional factor, was significantly upregulated correlatively with RRM2 in colorectal 2C-I HCl cancer (CRC). Ectopic expression and knockdown experiments indicated that MYBL2 was essential for CRC cell proliferation, DNA synthesis, and cell cycle progression in an RRM2-dependent manner. Mechanistically, MYBL2 directly bound to the promoter of RRM2 gene and promoted its transcription during S-phase together with TAF15 and MuvB components. Notably, knockdown of MYBL2 sensitized CRC cells to treatment with MK-1775, a clinical trial drug for inhibition of WEE1, which is involved in a degradation pathway of RRM2. Finally, mouse xenograft experiments showed that the combined suppression of MYBL2 and WEE1 synergistically inhibited CRC growth with a low systemic toxicity in vivo. Therefore, we propose a new regulatory mechanism for RRM2 transcription for CRC proliferation, in which MYBL2 functions by constituting a dynamic S-phase transcription complex following the G1/early S-phase E2Fs complex. Doubly targeting the transcription and degradation machines of RRM2 could produce a synthetic inhibitory effect on RRM2 level with a novel potential for CRC treatment. . Mice were sacrificed on day 15, 24?h after the last dose. The tumors were harvested, weighted and photographed. The ALT and AST levels in the mouse sera were measured by Zhejiang Chinese Medical University Laboratory Animals Research Center. All animal procedures were approved by Laboratory Animals Welfare Ethics Review Committee of Zhejiang University (ZJU20170522). Statistical analysis All results were presented as the means??SD of three independent experiments. Students em t /em -tests and one-way ANOVA were used to analyze differences in expression among the groups. Pearsons 2 test was used to evaluate the correlations between the expression of RRM2 and TFs in CRC datasets. em P- /em values ?0.05 were considered significant, and F2RL2 statistical analyses were performed using GraphPad prims 7. Supplementary information Supplemental tables(22K, docx) Supplemental Figure Legends(16K, docx) Supplemental Figure 1(29M, tif) Supplemental Figure 2(26M, tif) Supplemental Figure 3(27M, tif) Supplemental Figure 4(26M, tif) Supplemental Figure 5(26M, tif) Acknowledgements We thank all patients involved in this study. Author contributions QL design and performance of experiments, data analysis and interpretation, and manuscript writing; LG and HQ, performance of experiments, data analysis and interpretation; XX and KX clinical sample collection, experiment performance, and data analysis; ML, RW, BH, LZ, LX, and JS molecular and cellular experiment performance; YD and 2C-I HCl CL bioinformatics analysis; JS 2C-I HCl design and manuscript writing. Funding This work was supported by National Natural Science Foundation of China (81972270, 81572384, 81372138, 81771518, and 81802351), National Science and Technology Major Project of China (2018ZX10302206-006-007), and National Key R&D 2C-I HCl Program of China (2016YFC1303401). Data availability The datasets analyzed during the current study are available in the Oncomine (https://www.oncomine.org/), The Cancer Genome Atlas (https://www.cancer.gov/) or Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) under the accession number “type”:”entrez-geo”,”attrs”:”text”:”GSE20916″,”term_id”:”20916″GSE20916, “type”:”entrez-geo”,”attrs”:”text”:”GSE8671″,”term_id”:”8671″GSE8671, and “type”:”entrez-geo”,”attrs”:”text”:”GSE35896″,”term_id”:”35896″GSE35896. Competing interests The authors declare no competing interests. Ethics statement All animal experiments were approved by Laboratory Animals Welfare Ethics Review Committee of Zhejiang University (ZJU20170522). Clinical samples from patients were obtained informed consent from patients and approved by the ethics committee of the Zhejiang University School of Medicine, China. Footnotes Edited by N Barlev Publishers note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. These authors contributed equally: Qian Liu, Lijuan Guo, Hongyan Qi. Contributor Information Xueping Xiang, Email: nc.ude.ujz@gnipeuxgnaix. Jimin Shao, Email: nc.ude.ujz@nimijoahs. Supplementary information The online version contains supplementary material available at 10.1038/s41419-021-03969-1..