Systems Biology for Drug Target Discovery in Acute Myeloid Leukemia.

Combining new therapeutics with all-trans-retinoic acid (ATRA) could improve the efficiency of acute myeloid leukemia (AML) treatment. Modeling the process of ATRA-induced differentiation based on the transcriptomic profile of leukemic cells resulted in the identification of key targets that can be used to increase the therapeutic effect of ATRA. The genome-scale transcriptome analysis revealed the early molecular response to the ATRA treatment of HL-60 cells. In this study, we performed the transcriptomic profiling of HL-60, NB4, and K562 cells exposed to ATRA for 3-72 h. After treatment with ATRA for 3, 12, 24, and 72 h, we found 222, 391, 359, and 1032 differentially expressed genes (DEGs) in HL-60 cells, as well as 641, 1037, 1011, and 1499 DEGs in NB4 cells. We also found 538 and 119 DEGs in K562 cells treated with ATRA for 24 h and 72 h, respectively. Based on experimental transcriptomic data, we performed hierarchical modeling and determined cyclin-dependent kinase 6 (CDK6), tumor necrosis factor alpha (TNF-alpha), and transcriptional repressor CUX1 as the key regulators of the molecular response to the ATRA treatment in HL-60, NB4, and K562 cell lines, respectively. Mapping the data of TMT-based mass-spectrometric profiling on the modeling schemes, we determined CDK6 expression at the proteome level and its down-regulation at the transcriptome and proteome levels in cells treated with ATRA for 72 h. The combination of therapy with a CDK6 inhibitor (palbociclib) and ATRA (tretinoin) could be an alternative approach for the treatment of acute myeloid leukemia (AML).

CDK4/6 activity is required during G2 arrest to prevent stress-induced endoreplication.

Cell cycle events are coordinated by cyclin-dependent kinases (CDKs) to ensure robust cell division. CDK4/6 and CDK2 regulate the growth 1 (G1) to synthesis (S) phase transition of the cell cycle by responding to mitogen signaling, promoting E2F transcription and inhibition of the anaphase-promoting complex. We found that this mechanism was still required in G2-arrested cells to prevent cell cycle exit after the S phase. This mechanism revealed a role for CDK4/6 in maintaining the G2 state, challenging the notion that the cell cycle is irreversible and that cells do not require mitogens after passing the restriction point. Exit from G2 occurred during ribotoxic stress and was actively mediated by stress-activated protein kinases. Upon relief of stress, a significant fraction of cells underwent a second round of DNA replication that led to whole-genome doubling.

LncRNA BlncAD1 Modulates Bovine Adipogenesis by Binding to MYH10, PI3K/Akt Signaling Pathway, and miR-27a-5p/CDK6 Axis.

Research on adipogenesis will help to improve the meat quality of livestock. Long noncoding RNAs (lncRNAs) are involved in mammalian adipogenesis as epigenetic modulators. In this study, we analyzed lncRNA expression during bovine adipogenesis and detected 195 differentially expressed lncRNAs, including lncRNA BlncAD1, which was significantly upregulated in mature bovine adipocytes. Gain- and loss-of-function experiments confirmed that BlncAD1 promoted the proliferation, apoptosis, and differentiation of bovine preadipocytes. RNA pull-down revealed that the nonmuscle myosin 10 (MYH10) is a potential binding protein of BlncAD1. Then, we elucidated that loss of BlncAD1 caused increased ubiquitination of MYH10, which confirmed that BlncAD1 regulates adipogenesis by enhancing the stability of the MYH10 protein. Western blotting was used to demonstrate that BlncAD1 activated the PI3K/Akt signaling pathway. Bioinformatic analysis and dual-luciferase reporter assays indicated that BlncAD1 competitively absorbed miR-27a-5p. The overexpression and interference of miR-27a-5p in bovine preadipocytes displayed that miR-27a-5p inhibited proliferation, apoptosis, and differentiation. Further results suggested that miR-27a-5p targeted the CDK6 gene and that BlncAD1 controlled the proliferation of bovine preadipocytes by modulating the miR-27a-5p/CDK6 axis. This study revealed the complex mechanisms of BlncAD1 underlying bovine adipogenesis for the first time, which would provide useful information for genetics and breeding improvement of Chinese beef cattle.

CircPRMT5 promotes progression of osteosarcoma by recruiting CNBP to regulate the translation and stability of CDK6 mRNA.

Research has demonstrated that circular RNAs (circRNAs) exert critical functions in the occurrence and progression of numerous malignant tumors. CircPRMT5 was recently reported to be involved in the pathogenesis of cancers. However, the potential role of circPRMT5 in osteosarcoma needs further investigation. In present study, our results suggested that circPRMT5 was highly upregulated in osteosarcoma cells and mainly localizes in the cytoplasm. CircPRMT5 promoted the proliferation, migration and invasion capacities of osteosarcoma cells, and suppressed cell apoptosis. Knockdown of circPRMT5 exerted the opposite effects. Mechanically, circPRMT5 promoted the binding of CNBP to CDK6 mRNA, which enhanced the stability of CDK6 mRNA and facilitated its translation, thereby promoting the progression of osteosarcoma. Knockdown of CDK6 reversed the promoting effect of circPRMT5 on osteosarcoma cells. These findings suggest that circPRMT5 promotes osteosarcoma cell malignant activity by recruiting CNBP to regulate the translation and stability of CDK6 mRNA. Thus, circPRMT5 may represent a promising therapeutic target for osteosarcoma.

Comprehensive Transcriptomic Analysis of EWSR1::WT1 Targets Identifies CDK4/6 Inhibitors as an Effective Therapy for Desmoplastic Small Round Cell Tumors.

Desmoplastic small round cell tumors (DSRCT) are a type of aggressive, pediatric sarcoma characterized by the EWSR1::WT1 fusion oncogene. Targeted therapies for DSRCT have not been developed, and standard multimodal therapy is insufficient, leading to a 5-year survival rate of only 15% to 25%. Here, we depleted EWSR1::WT1 in DSRCT and established its essentiality in vivo. Transcriptomic analysis revealed that EWSR1::WT1 induces unique transcriptional alterations compared with WT1 and other fusion oncoproteins and that EWSR1::WT1 binding directly mediates gene upregulation. The E-KTS isoform of EWSR1::WT1 played a dominant role in transcription, and it bound to the CCND1 promoter and stimulated DSRCT growth through the cyclin D-CDK4/6-RB axis. Treatment with the CDK4/6 inhibitor palbociclib successfully reduced growth in two DSRCT xenograft models. As palbociclib has been approved by the FDA for the treatment of breast cancer, these findings demonstrate the sensitivity of DSRCT to palbociclib and support immediate clinical investigation of palbociclib for treating this aggressive pediatric cancer. SIGNIFICANCE: EWSR1::WT1 is essential for desmoplastic small round cell tumors and upregulates the cyclin D-CDK4/6-RB axis that can be targeted with palbociclib, providing a targeted therapeutic strategy for treating this deadly tumor type.

PRMT5 is an actionable therapeutic target in CDK4/6 inhibitor-resistant ER+/RB-deficient breast cancer.

CDK4/6 inhibitors (CDK4/6i) have improved survival of patients with estrogen receptor-positive (ER+) breast cancer. However, patients treated with CDK4/6i eventually develop drug resistance and progress. RB1 loss-of-function alterations confer resistance to CDK4/6i, but the optimal therapy for these patients is unclear. Through a genome-wide CRISPR screen, we identify protein arginine methyltransferase 5 (PRMT5) as a molecular vulnerability in ER+/RB1-knockout breast cancer cells. Inhibition of PRMT5 blocks the G1-to-S transition in the cell cycle independent of RB, leading to growth arrest in RB1-knockout cells. Proteomics analysis uncovers fused in sarcoma (FUS) as a downstream effector of PRMT5. Inhibition of PRMT5 results in dissociation of FUS from RNA polymerase II, leading to hyperphosphorylation of serine 2 in RNA polymerase II, intron retention, and subsequent downregulation of proteins involved in DNA synthesis. Furthermore, treatment with the PRMT5 inhibitor pemrametostat and a selective ER degrader fulvestrant synergistically inhibits growth of ER+/RB-deficient cell-derived and patient-derived xenografts. These findings highlight dual ER and PRMT5 blockade as a potential therapeutic strategy to overcome resistance to CDK4/6i in ER+/RB-deficient breast cancer.

DNA hypomethylation activates Cdk4/6 and Atr to induce DNA replication and cell cycle arrest to constrain liver outgrowth in zebrafish.

Coordinating epigenomic inheritance and cell cycle progression is essential for organogenesis. UHRF1 connects these functions during development by facilitating maintenance of DNA methylation and cell cycle progression. Here, we provide evidence resolving the paradoxical phenotype of uhrf1 mutant zebrafish embryos which have activation of pro-proliferative genes and increased number of hepatocytes in S-phase, but the liver fails to grow. We uncover decreased Cdkn2a/b and persistent Cdk4/6 activation as the mechanism driving uhrf1 mutant hepatocytes into S-phase. This induces replication stress, DNA damage and Atr activation. Palbociclib treatment of uhrf1 mutants prevented aberrant S-phase entry, reduced DNA damage, and rescued most cellular and developmental phenotypes, but it did not rescue DNA hypomethylation, transposon expression or the interferon response. Inhibiting Atr reduced DNA replication and increased liver size in uhrf1 mutants, suggesting that Atr activation leads to dormant origin firing and prevents hepatocyte proliferation. Cdkn2a/b was downregulated pro-proliferative genes were also induced in a Cdk4/6 dependent fashion in the liver of dnmt1 mutants, suggesting DNA hypomethylation as a mechanism of Cdk4/6 activation during development. This shows that the developmental defects caused by DNA hypomethylation are attributed to persistent Cdk4/6 activation, DNA replication stress, dormant origin firing and cell cycle inhibition.

MYC induces CDK4/6 inhibitors resistance by promoting pRB1 degradation.

CDK4/6 inhibitors (CDK4/6i) show anticancer activity in certain human malignancies, such as breast cancer. However, their application to other tumor types and intrinsic resistance mechanisms are still unclear. Here, we demonstrate that MYC amplification confers resistance to CDK4/6i in bladder, prostate and breast cancer cells. Mechanistically, MYC binds to the promoter of the E3 ubiquitin ligase KLHL42 and enhances its transcription, leading to RB1 deficiency by inducing both phosphorylated and total pRB1 ubiquitination and degradation. We identify a compound that degrades MYC, A80.2HCl, which induces MYC degradation at nanomolar concentrations, restores pRB1 protein levels and re-establish sensitivity of MYC high-expressing cancer cells to CDK4/6i. The combination of CDK4/6i and A80.2HCl result in marked regression in tumor growth in vivo. Altogether, these results reveal the molecular mechanisms underlying MYC-induced resistance to CDK4/6i and suggest the utilization of the MYC degrading molecule A80.2HCl to potentiate the therapeutic efficacy of CDK4/6i.

Long noncoding RNA LINC00675 drives malignancy in acute myeloid leukemia via the miR-6809 -CDK6 axis.

Hematological malignancies such as acute myeloid leukemia (AML) have a low cure rate and a high recurrence rate. Long noncoding RNAs (LNCs) are essential regulators of tumorigenesis and progression. The role of lncRNA LINC00675 in AML has rarely been reported. This study revealed elevated LINC00675 expression in AML that promotes proliferation and inhibits apoptosis. Mechanistically, LINC00675 combines with miR-6809 to promote the expression of CDK6 in vitro and in vivo. Immune-checkpoint genes were expressed more highly in LINC00675-high patients. A high level of LINC00675 expression may make patients more susceptible to palbociclib treatments. In conclusion, our study demonstrated that LINC00675 is an oncogenic lncRNA that enhances the malignancy of AML by upregulating CDK6 expression through miR-6809 sponging, providing a new perspective and feasible target for the diagnosis and treatment of AML.

Cracking the Genomic Code of CDK4/6 Inhibitor Resistance.

The therapeutic approach to metastatic hormone receptor-positive, human epidermal growth factor-2-negative metastatic breast cancer (HR+/HER2- MBC) has evolved rapidly over recent years. The cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) have become first-line targeted agents of choice, in combination with an antiestrogen. Simultaneously, the clinical landscape of therapeutic options has been rapidly shifting, with novel antiestrogens, signal transduction inhibitors, and next-generation CDK inhibitors in various stages of development. Given these dynamic changes, understanding the genomic and molecular landscape of resistance to currently available antiestrogen therapy and CDK4/6 inhibitors represents a major focus of translational breast cancer research globally. See related article by Goetz et al., p. 2233.

Integrative score based on CDK6, PD-L1 and TMB predicts response to platinum-based chemotherapy and PD-1/PD-L1 blockade in muscle-invasive bladder cancer.

BACKGROUND: Cyclin-dependent kinase 6 (CDK6) was proved to be an important regulator in the progression of cell cycle and has been a promising therapeutic target in cancer treatment. However, the clinical significance of CDK6 in muscle-invasive bladder cancer (MIBC) remains obscure. Herein, we attempt to explore the clinical relevance of CDK6 and assess the feasibility of the integrative model to predict immune checkpoint blockade (ICB) response. METHODS: This study enrolled 933 patients with muscle-invasive bladder cancer (MIBC) from Zhongshan Hospital (ZSHS), The Cancer Genome Atlas (TCGA), Chemo, IMvigor210 and UC-GENOME cohorts. Kaplan-Meier survival and Cox regression analyses were performed to assess clinical outcomes based on CDK6 expression. RESULTS: High CDK6 expression conferred poor prognosis and superior response to platinum-based chemotherapy but inferior response to ICB in MIBC. Furthermore, the integrative model named response score based on CDK6, PD-L1 and TMB could better predict the response to ICB and chemotherapy. Patients with higher response scores were characterised by inflamed immune microenvironment and genomic instability. CONCLUSIONS: CDK6 expression was correlated with prognosis and therapy response in MIBC. Integration of CDK6, PD-L1 and TMB could better identify patients who were most likely to benefit from ICB and chemotherapy.

IGF2BP2 Drives Cell Cycle Progression in Triple-Negative Breast Cancer by Recruiting EIF4A1 to Promote the m6A-Modified CDK6 Translation Initiation Process.

IGF2BP2 is a new identified N6-methyladenosine (m6A) reader and associated with poor prognosis in many tumors. However, its role and related mechanism in breast cancer, especially in triple-negative breast cancer (TNBC), remains unclear. In this study, it is found that IGF2BP2 is highly expressed in TNBC due to the lower methylation level in its promoter region. Functional and mechanical studies displayed that IGF2BP2 could promote TNBC proliferation and the G1/S phase transition of the cell cycle via directly regulating CDK6 in an m6A-dependent manner. Surprising, the regulation of protein levels of CDK6 by IGF2BP2 is related to the changes in translation rate during translation initiation, rather than mRNA stability. Moreover, EIF4A1 is found to be recruited by IGF2BP2 to promote the translation output of CDK6, providing new evidence for a regulatory role of IGF2BP2 between m6A methylation and translation. Downregulation of IGF2BP2 in TNBC cell could enhance the sensitivity to abemaciclib, a CDK4/6 inhibitor. To sum up, our study revealed IGF2BP2 could facilitate the translation output of CDK6 via recruiting EIF4A1 and also provided a potential therapeutic target for the diagnosis and treatment of TNBC, as well as a new strategy for broadening the drug indications for CDK4/6 inhibitors.

Targeting Transcriptional Regulation with a CDK9 Inhibitor Suppresses Growth of Endocrine- and Palbociclib-Resistant ER+ Breast Cancers.

The combination of endocrine therapy and CDK4/6 inhibitors such as palbociclib is an effective and well-tolerated treatment for estrogen receptor-positive (ER+) breast cancer, yet many patients relapse with therapy-resistant disease. Determining the mechanisms underlying endocrine therapy resistance is limited by the lack of ability to fully recapitulate inter- and intratumor heterogeneity in vitro and of availability of tumor samples from women with disease progression or relapse. In this study, multiple cell line models of resistant disease were used for both two-dimensional (2D)- and three-dimensional (3D)-based inhibitor screening. The screens confirmed the previously reported role of pro-proliferative pathways, such as PI3K-AKT-mTOR, in endocrine therapy resistance and additionally identified the transcription-associated cyclin-dependent kinase CDK9 as a common hit in ER+ cell lines and patient-derived organoids modeling endocrine therapy-resistant disease in both the palbociclib-sensitive and palbociclib-resistant settings. The CDK9 inhibitor, AZD4573, currently in clinical trials for hematologic malignancies, acted synergistically with palbociclib in these ER+in vitro 2D and 3D models. In addition, in two independent endocrine- and palbociclib-resistance patient-derived xenografts, treatment with AZD4573 in combination with palbociclib and fulvestrant resulted in tumor regression. Tumor transcriptional profiling identified a set of transcriptional and cell-cycle regulators differentially downregulated only in combination-treated tumors. Together, these findings identify a clinically tractable combination strategy for overcoming resistance to endocrine therapy and CDK4/6 inhibitors in breast cancer and provide insight into the potential mechanism of drug efficacy in targeting treatment-resistant disease. SIGNIFICANCE: Targeting transcription-associated CDK9 synergizes with CDK4/6 inhibitor to drive tumor regression in multiple models of endocrine- and palbociclib-resistant ER+ breast cancer, which could address the challenge of overcoming resistance in patients.

CDK6 is a novel predictive and prognosis biomarker correlated with immune infiltrates in multiple human neoplasms, including small cell lung carcinoma.

The roles of cyclin-dependent kinase 6 (CDK6) in various cancers, including small cell lung carcinoma (SCLC), remain unclear. Here, 111,54 multi-center samples were investigated to determine the expression, clinical significance, and underlying mechanisms of CDK6 in 34 cancers. The area under the curve (AUC), Cox regression analysis, and the Kaplan-Meier curves were used to explore the clinical value of CDK6 in cancers. Gene set enrichment analysis and correlation analysis were performed to detect potential CDK6 mechanisms. CDK6 expression was essential in 24 cancer cell types. Abnormal CDK6 expression was observed in 14 cancer types (e.g., downregulated in breast invasive carcinoma; p < 0.05). CDK6 allowed six cancers to be distinguished from their controls (AUC > 0.750). CDK6 expression was a prognosis marker for 13 cancers (e.g., adrenocortical carcinoma; p < 0.05). CDK6 was correlated with several immune-related signaling pathways and the infiltration levels of certain immune cells (e.g., CD8+ T cells; p < 0.05). Downregulated CDK6 mRNA and protein levels were observed in SCLC (p < 0.05, SMD = - 0.90). CDK6 allowed the identification of SCLC status (AUC = 0.91) and predicted a favorable prognosis for SCLC patients (p < 0.05). CDK6 may be a novel biomarker for the prediction and prognosis of several cancers, including SCLC.

DNA damage induced by CDK4 and CDK6 blockade triggers anti-tumor immune responses through cGAS-STING pathway.

CDK4/6 are important regulators of cell cycle and their inhibitors have been approved as anti-cancer drugs. Here, we report a STING-dependent anti-tumor immune mechanism responsible for tumor suppression by CDK4/6 blockade. Clinical datasets show that in human tissues, CDK4 and CDK6 are over-expressed and their expressions are negatively correlated with patients' overall survival and T cell infiltration. Deletion of Cdk4 or Cdk6 in tumor cells significantly reduce tumor growth. Mechanistically, we find that Cdk4 or Cdk6 deficiency contributes to an increased level of endogenous DNA damage, which triggers the cGAS-STING signaling pathway to activate type I interferon response. Knockout of Sting is sufficient to reverse and partially reverse the anti-tumor effect of Cdk4 and Cdk6 deficiency respectively. Therefore, our findings suggest that CDK4/6 inhibitors may enhance anti-tumor immunity through the STING-dependent type I interferon response.

Long non-coding RNA H19 promotes proliferation in hepatocellular carcinoma cells via H19/miR-107/CDK6 axis.

Hepatocellular carcinoma (HCC) is the leading cause of cancer death worldwide; nevertheless, current therapeutic options are limited or ineffective for many patients. Therefore, elucidation of molecular mechanisms in HCC biology could yield important insights for the intervention of novel therapies. Recently, various studies have reported dysregulation of long non-coding RNAs (lncRNAs) in the initiation and progression of HCC, including H19; however, the biological function of H19 in HCC remains unclear. Here, we show that knockdown of H19 disrupted HCC cell growth, impaired the G1-to-S phase transition, and promoted apoptosis, while overexpression of H19 yielded the opposite results. Screening for expression of cell cycle-related genes revealed a significant downregulation of CDK6 at both RNA and protein levels upon H19 suppression. Bioinformatic analysis of the H19 sequence and the 3' untranslated region (3' UTR) of CDK6 transcripts showed several binding sites for microRNA-107 (miR-107), and the dual luciferase reporter assay confirmed their direct interaction with miR-107. Consistently, blockage of miR-107 activity alleviated the growth suppression phenotypes induced by H19 downregulation, suggesting that H19 serves as a molecular sponge for miR-107 to promote CDK6 expression and cell cycle progression. Together, this study demonstrates a mechanistic function of H19 in driving the proliferation of HCC cells and suggests H19 suppression as a novel approach for HCC treatment.

METTL9 derived circular RNA circ-METTL9 sponges miR-551b-5p to accelerate colorectal cancer progression by upregulating CDK6.

Circular RNAs (circRNAs) have been accepted to play key roles in the development and progression of mutiple cancers including colorectal cancer (CRC). Here, we identified circ-METTL9, derived from 2 to 4 exons of METTL9 gene, may promote CRC progression by accelerating cell cycle progression. However, the role and mechanism of circ-METTL9 in CRC remains unclear. Based on our data, the expression of circ-METTL9 was significantly upregulated in CRC tissues and markedly increased in advanced tumors in CRC patients. Functional experiments demonstrated that circ-METTL9 overexpression promoted CRC cells proliferation and migration in vitro, and simultaneously enhanced CRC tumor growth and metastasis in vivo. Mechanistically, RNA immunoprecipitation (RIP) assays proved that circ-METTL9 might be a miRNA sponge, and RNA pulldown assays showed the interaction between circ-METTL9 and miR-551b-5p. Notably, cyclin-dependent kinase 6 (CDK6), a key regulator in cell cycle, is a conserved downstream target of miR-551b-5p. Taken together, our findings highlight a novel oncogenic function of circ-METTL9 in CRC progression via circ-METTL9/miR-551b-5p/CDK6 axis, which may serve as a prognostic biomarker and therapeutic target for CRC patients.

[Biological, preclinical and clinical aspects of the association between radiation therapy and CDK4/6 inhibitors]. / Aspects biologiques, précliniques et cliniques de l'association de radiothérapie et d'inhibiteur de CDK 4/6.

Several clinical studies have shown that CDK4/6 inhibitors (CDK4/6i) improve survival in patients with metastatic or locally advanced HR-positive, HER-2-negative breast cancer (BC). The aim of this review was to synthesize the biological, preclinical and clinical aspects of the treatment of BC with CDK4/6i, with a focus on the combination of CDK4/6i and radiotherapy. The DNA damage induced after exposure of cells to ionizing radiation activates control pathways that inhibit cell progression in the G1 and G2 phases and induce a transient delay in progression in the S phase. These checkpoints are in particular mediated by cyclin-dependent kinases (CDK) 4/6 activated by cyclin D1. Several preclinical studies have shown that CDK4/6i could be used as radiosensitizers in non-small cell lung cancer, medulloblastoma, brainstem glioma and breast cancer. CDK4/6 inhibition also protected against radiation-induced intestinal toxicities by inducing redistribution of quiescent intestinal progenitor cells, making them less radiosensitive. Clinical data on the combination of CDK inhibitors and radiotherapy for both locoregional and metastatic irradiation are based on retrospective data. Nevertheless, the most optimal therapeutic sequence would be radiotherapy followed by palbociclib. Pending prospective clinical trials, the concomitant combination of the two treatments should be done under close supervision.

CDK6 is activated by the atypical cyclin I to promote E2F-mediated gene expression and cancer cell proliferation.

Cyclin-dependent kinases (CDKs), together with their cyclin partners, are the master cell cycle regulators. Remarkably, the cyclin family was extended to include atypical cyclins, characterized by distinctive structural features, but their partner CDKs remain elusive. Here, we conducted a yeast two-hybrid screen to identify new atypical cyclin-CDK complexes. We identified 10 new complexes, including a complex between CDK6 and cyclin I (CCNI), which was found to be active against retinoblastoma protein. CCNI upregulation increased the proliferation of breast cancer cells in vitro and in vivo, with a magnitude similar to that seen upon cyclin D upregulation, an effect that was abrogated by CDK6 silencing or palbociclib treatment. In line with these findings, CCNI downregulation led to a decrease in cell number and a reduction in the percentage of cells reaching S phase. Finally, CCNI upregulation correlated with the high expression of E2F target genes in large panels of cancer cell lines and tissue samples from breast cancer patients. In conclusion, we unveil CCNI as a new player in the pathways that activate CDK6, enriching the wiring of cell cycle control.