The effects of immortalization on the N-glycome and proteome of CDK4-transformed lung cancer cells.

Biological experiments are often conducted in vitro using immortalized cells due to their accessibility and ease of propagation compared to primary cells and live animals. However, immortalized cells may present different proteomic and glycoproteomic characteristics from the primary cell source due to the introduction of genes that enhance proliferation (e.g. CDK4) or enable telomere lengthening. To demonstrate the changes in phenotype upon CDK4-transformation, we performed LC-MS/MS glycomic and proteomic characterizations of a human lung cancer primary cell line (DTW75) and a CDK4-transformed cell line (GL01) derived from DTW75. We observed that the primary and CDK4-transformed cells expressed significantly different levels of sialylated, fucosylated, and sialofucosylated N-glycans. Specifically, the primary cells expressed higher levels of hybrid- and complex-type sialylated N-glycans, while CDK4-transformed cells expressed higher levels of complex-type fucosylated and sialofucosylated N-glycans. Further, we compared the proteomic differences between the cell lines and found that CDK4-transformed cells expressed higher levels of RNA-binding and adhesion proteins. Further, we observed that the CDK4-transformed cells changed N-glycosylation after 31 days in cell culture, with a decrease in high-mannose and increase in fucosylated, sialylated, and sialofucosylated N-glycans. Identifying these changes between primary and CDK4-transformed cells will provide useful insight when adapting cell lines that more closely resemble in vivo physiological conditions.

Combined SET7/9 and CDK4 inhibition act synergistically against osteosarcoma.

Osteosarcoma is the most common malignant bone tumor. It has a poor prognosis because of a lack of therapeutic targets and strategies. The SET domain-containing lysine-specific methyltransferase, SET7/9, has various functions in different cancer types in tissue-type and signaling context-dependent manners. The role of SET7/9 in osteosarcoma cells is currently controversial and its potential as a therapeutic candidate in osteosarcoma is unknown. In the present study, SET7/9 inhibition or ablation suppressed osteosarcoma cell proliferation by causing G1 arrest. Mechanistically, SET7/9 inhibition disrupted the interaction between cyclin-dependent kinase 4 (CDK4) and cyclin D1, which affected CDK4-cyclin D1 complex function, leading to decreased phosphorylation of retinoblastoma protein. CDK4 was overexpressed in osteosarcoma tissues and was closely related to a poor prognosis in patients with osteosarcoma. We therefore hypothesized that SET7/9 inhibition might increase the sensitivity of osteosarcoma cells to CDK4 inhibitors, potentially decreasing the risk of adverse effects of CDK4 inhibitors. The combination of SET7/9 and CDK4 inhibition enabled dose reductions of both inhibitors and had a synergistic effect against osteosarcoma growth in vivo. Collectively, these findings indicate that SET7/9 plays an oncogenic role in osteosarcoma by regulating CDK4-cyclin D1 complex interaction and function. The combination of SET7/9 and CDK4 inhibition may thus provide a novel effective therapeutic strategy for osteosarcoma with no significant toxicity.

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.

Exemestane plus everolimus and palbociclib in metastatic breast cancer: clinical response and genomic/transcriptomic determinants of resistance in a phase I/II trial.

The landscape of cyclin-dependent kinase 4/6 inhibitor (CDK4/6i) resistance is still being elucidated and the optimal subsequent therapy to overcome resistance remains uncertain. Here we present the final results of a phase Ib/IIa, open-label trial (NCT02871791) of exemestane plus everolimus and palbociclib for CDK4/6i-resistant metastatic breast cancer. The primary objective of phase Ib was to evaluate safety and tolerability and determine the maximum tolerated dose/recommended phase II dose (100 mg palbociclib, 5 mg everolimus, 25 mg exemestane). The primary objective of phase IIa was to determine the clinical benefit rate (18.8%, n = 6/32), which did not meet the predefined endpoint (65%). Secondary objectives included pharmacokinetic profiling (phase Ib), objective response rate, disease control rate, duration of response, and progression free survival (phase IIa), and correlative multi-omics analysis to investigate biomarkers of resistance to CDK4/6i. All participants were female. Multi-omics data from the phase IIa patients (n = 24 tumor/17 blood biopsy exomes; n = 27 tumor transcriptomes) showed potential mechanisms of resistance (convergent evolution of HER2 activation, BRAFV600E), identified joint genomic/transcriptomic resistance features (ESR1 mutations, high estrogen receptor pathway activity, and a Luminal A/B subtype; ERBB2/BRAF mutations, high RTK/MAPK pathway activity, and a HER2-E subtype), and provided hypothesis-generating results suggesting that mTOR pathway activation correlates with response to the trial's therapy. Our results illustrate how genome and transcriptome sequencing may help better identify patients likely to respond to CDK4/6i therapies.

Flow cytometry-assisted quantification of cell cycle arrest in cancer cells treated with CDK4/6 inhibitors.

Cyclin-dependent kinase 4 (CDK4) and CDK6 inhibitors (i.e., palbociclib, abemaciclib, and ribociclib) are well known for their capacity to mediate cytostatic effects by promoting cell cycle arrest in the G1 phase, thus inhibiting cancer cell proliferation. Cytostatic effects induced by CDK4/6 inhibitors can be transient or lead to a permanent state of cell cycle arrest, commonly defined as cellular senescence. Induction of senescence is often associated to metabolic modifications and to the acquisition of a senescence-associated secretory phenotype (SASP) by cancer cells, which in turn can promote or limit antitumor immunity (and thus the efficacy of CDK4/6 inhibitors) depending on SASP components. Thus, although accumulating evidence suggests that anti-cancer effects of CDK4/6 inhibitors also depend on the promotion of antitumor immune responses, assessing cell cycle arrest and progression in cells treated with palbociclib remains a key approach for investigating the efficacy of CDK4/6 inhibitors. Here, we describe a method to assess cell cycle distribution simultaneously with active DNA replication by flow cytometry in cultured hormone receptor-positive breast cancer MCF7 cells.

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.

Effect of CDK4/6 Inhibitors on Tumor Immune Microenvironment.

Cancer is an abnormal proliferation of cells that is stimulated by cyclin-dependent kinases (CDKs) and defective cell cycle regulation. The essential agent that drive the cell cycle, CDK4/6, would be activated by proliferative signals. Activated CDK4/6 results in the phosphorylation of the neuroblastoma protein (RB) and the release of the transcription factor E2F, which promotes the cell cycle progression. CDK4/6 inhibitor (CDK4/6i) has been currently a research focus, which inhibits the CDK4/6-RB-E2F axis, thereby reducing the cell cycle transition from G1 to S phase and mediating the cell cycle arrest. This action helps achieve an anti-tumor effect. Recent research has demonstrated that CDK4/6i, in addition to contributing to cell cycle arrest, is also essential for the interaction between the tumor cells and the host immune system, i.e., activating the immune system, strengthening the tumor antigen presentation, and reducing the number of regulatory T cells (Treg). Additionally, CDK4/6i would elevate the level of PD-L1, an immunosuppressive factor, in tumor cells, and CDK4/6i in combination with anti-PD-L1 therapy would more effectively reduce the tumor growth. Our results showed that CDK4/6i caused autophagy and senescence in tumor cells. Herein, the impact of CDK4/6i on the immune microenvironment of malignant tumors was mainly focused, as well as their interaction with immune checkpoint inhibitors in affecting anti-tumor immunity.

Discovery of (4-Pyrazolyl)-2-aminopyrimidines as Potent and Selective Inhibitors of Cyclin-Dependent Kinase 2.

CDK2 is a critical regulator of the cell cycle. For a variety of human cancers, the dysregulation of CDK2/cyclin E1 can lead to tumor growth and proliferation. Historically, early efforts to develop CDK2 inhibitors with clinical applications proved unsuccessful due to challenges in achieving selectivity over off-target CDK isoforms with associated toxicity. In this report, we describe the discovery of (4-pyrazolyl)-2-aminopyrimidines as a potent class of CDK2 inhibitors that display selectivity over CDKs 1, 4, 6, 7, and 9. SAR studies led to the identification of compound 17, a kinase selective and highly potent CDK2 inhibitor (IC50 = 0.29 nM). The evaluation of 17 in CCNE1-amplified mouse models shows the pharmacodynamic inhibition of CDK2, measured by reduced Rb phosphorylation, and antitumor activity.

CDK4/6 inhibitors in lung cancer: current practice and future directions.

Lung cancer is the leading cause of cancer-related deaths worldwide, and ∼85% of lung cancers are classified as nonsmall cell lung cancer (NSCLC). These malignancies can proliferate indefinitely, in part due to dysregulation of the cell cycle and the resulting abnormal cell growth. The specific activation of cyclin-dependent kinases 4 and 6 (CDK4/6) is closely linked to tumour proliferation. Approximately 80% of human tumours exhibit abnormalities in the cyclin D-CDK4/6-INK4-RB pathway. Specifically, CDK4/6 inhibitors either as monotherapy or combination therapy have been investigated in pre-clinical and clinical studies for the treatment of NSCLC, and promising results have been achieved. This review article focuses on research regarding the use of CDK4/6 inhibitors in NSCLC, including the characteristics and mechanisms of action of approved drugs and progress of pre-clinical and clinical research.

Cell cycle plasticity underlies fractional resistance to palbociclib in ER+/HER2- breast tumor cells.

The CDK4/6 inhibitor palbociclib blocks cell cycle progression in Estrogen receptor-positive, human epidermal growth factor 2 receptor-negative (ER+/HER2-) breast tumor cells. Despite the drug's success in improving patient outcomes, a small percentage of tumor cells continues to divide in the presence of palbociclib-a phenomenon we refer to as fractional resistance. It is critical to understand the cellular mechanisms underlying fractional resistance because the precise percentage of resistant cells in patient tissue is a strong predictor of clinical outcomes. Here, we hypothesize that fractional resistance arises from cell-to-cell differences in core cell cycle regulators that allow a subset of cells to escape CDK4/6 inhibitor therapy. We used multiplex, single-cell imaging to identify fractionally resistant cells in both cultured and primary breast tumor samples resected from patients. Resistant cells showed premature accumulation of multiple G1 regulators including E2F1, retinoblastoma protein, and CDK2, as well as enhanced sensitivity to pharmacological inhibition of CDK2 activity. Using trajectory inference approaches, we show how plasticity among cell cycle regulators gives rise to alternate cell cycle "paths" that allow individual tumor cells to escape palbociclib treatment. Understanding drivers of cell cycle plasticity, and how to eliminate resistant cell cycle paths, could lead to improved cancer therapies targeting fractionally resistant cells to improve patient outcomes.

Curcumin Enhances the Anti-Cancer Efficacy of CDK4/6 Inhibitors in Prostate Cancer.

OBJECTIVE: This study aimed to investigate the potential of combining cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors with curcumin (Cur), a natural compound known for its anti-aging properties, to enhance the anti-cancer efficacy in prostate cancer (PCa). METHODS: The cell viability was determined by cell counting kit-8 assay, colony forming assay and cell invasion. The cell cycle and mRNA levels of p16 (cyclin dependent kinase inhibitor 2A, CDKN2A), p21 (cyclin dependent kinase inhibitor 1A, CDKN1A) and Rb (RB transcriptional corepressor) were detected by flow cytometry and quantitative real-time polymerase chain reaction, respectively. SA-ß-gal staining and interleukin 6 (IL6) mRNA levels were used to evaluate cell aging. Western blot was used to detect mechanistic targets of rapamycin (mTOR) and signal transducer and activator of transcription 3 (STAT3) pathways. Moreover, Sphere formation assay and mRNA levels of aldehyde dehydrogenase (ALDH) 1A1, CD44 and Nanog were used to determine cell stemness. RESULTS: The combination of LY2835219 (LY, CDK4/6 inhibitor) and Cur exhibited a synergistic inhibitory effect on PCa cell proliferation (p < 0.01) and invasion (p < 0.01) and Rb gene expression (p < 0.05), as well as a synergistic promotive effect on p61 expression (p < 0.01), p21 expression (p < 0.01) and cell cycle G1 arrest in PCa cells (p < 0.05) compared with LY or Cur alone. LY and LY + Cur increased the SA-ß-gal-stained cells (p < 0.01). mTOR (p < 0.01) and STAT3 pathway (p < 0.01) were decreased by LY + Cur (p < 0.01). Furthermore, LY + Cur conditioned medium (CM) inhibited cell stemness by decreasing cell spheres (p < 0.05), ALDH1A1 (p < 0.01), CD44 (p < 0.01) and Nanog (p < 0.01) compared with LY CM. CONCLUSIONS: The findings of this study suggested that the combination of CDK4/6 inhibitor and curcumin may have clinical implications for the treatment of PCa.

Design and Synthesis of New bis-oxindole and Spiro(triazole-oxindole) as CDK4 Inhibitors with Potent Anti-breast Cancer Activity.

BACKGROUND: Since CDKs have been demonstrated to be overexpressed in a wide spectrum of human malignancies, their inhibition has been cited as an effective technique for anticancer drug development. METHODS: In this context, new bis-oxindole/spiro-triazole-oxindole anti-breast cancer drugs with potential CDK4 inhibitory effects were produced in this work. The novel series of bis-oxindole/spirotriazole- oxindole were synthesized from the reaction of bis-oxindole with the aniline derivatives then followed by 1,3-dipolar cycloaddition of hydrazonoyl chloride. RESULTS: The structure of these bis-oxindole/spiro-triazole-oxindole series was proven based on their spectral analyses. Most bis-oxindole and bis-spiro-triazole-oxindole compounds effectively inhibited the growth of MCF-7 (IC50 = 2.81-17.61 µM) and MDA-MB-231 (IC50 = 3.23-7.98 µM) breast cancer cell lines with low inhibitory activity against normal WI-38 cells. While the reference doxorubicin showed IC50 values of 7.43 µM against MCF-7 and 5.71 µM against the MDA-MB-231 cell line. Additionally, compounds 3b, 3c, 6b, and 6d revealed significant anti-CDK4 activity (IC50 = 0.157- 0.618 µM) compared to palbociclib (IC50 = 0.071 µM). Subsequent mechanistic investigations demonstrated that 3c was able to trigger tumor cell death through the induction of apoptosis. Moreover, it stimulated cancer cell cycle arrest in the G1 phase. Furthermore, western blotting disclosed that the 3c-induced cell cycle arrest may be mediated through p21 upregulation. CONCLUSION: According to all of the findings, bis-oxindole 3c shows promise as a cancer treatment targeting CDK4.

To Quiesce or Senesce, That Is the Question for Dedifferentiated Liposarcoma.

Dedifferentiated liposarcoma (DDLPS) has an appealing therapeutic target due to its CDK4 amplification on chromosome 12q. The understanding of geroconversion from quiescent cells to senescent cells defines a patient's response to CDK4 inhibitors. This new observation will inform not only the ongoing phase III clinical trial of abemaciclib, but all future clinical trials in DDLPS. See related article by Gleason et al., p. 703.

CDK4/6 Inhibition With Lerociclib is a Potential Therapeutic Strategy for the Treatment of Pediatric Sarcomas.

BACKGROUND: Sarcomas are a heterogenous collection of bone and soft tissue tumors. The heterogeneity of these tumors makes it difficult to standardize treatment. CDK 4/6 inhibitors are a family of targeted agents which limit cell cycle progression and have been shown to be upregulated in sarcomas. In the current preclinical study, we evaluated the effects of lerociclib, a CDK4/6 inhibitor, on pediatric sarcomas in vitro and in 3D bioprinted tumors. METHODS: The effects of lerociclib on viability, proliferation, cell cycle, motility, and stemness were assessed in established sarcoma cell lines, U-2 OS and MG-63, as well as sarcoma patient-derived xenografts (PDXs). 3D printed biotumors of each of the U-2 OS, MG-63, and COA79 cells were utilized to study the effects of lerociclib on tumor growth ex vivo. RESULTS: CDK 4/6, as well as the intermediaries retinoblastoma protein (Rb) and phosphorylated Rb were identified as targets in the four sarcoma cell lines. Lerociclib treatment induced cell cycle arrest, decreased proliferation, motility, and stemness of sarcoma cells. Treatment with lerociclib decreased sarcoma cell viability in both traditional 2D culture as well as 3D bioprinted microtumors. CONCLUSIONS: Inhibition of CDK 4/6 activity with lerociclib was efficacious in traditional 2D sarcoma cell culture as well as in 3D bioprints. Lerociclib holds promise and warrants further investigation as a novel therapeutic strategy for management of these heterogenous groups of tumors.

CDK4/6 Inhibition Sensitizes Intracranial Tumors to PD-1 Blockade in Preclinical Models of Brain Metastasis.

PURPOSE: Brain metastases are associated with high morbidity and are often resistant to immune checkpoint inhibitors. We evaluated whether CDK4/6 inhibitor (CDKi) abemaciclib can sensitize intracranial tumors to programmed cell death protein 1 (PD-1) inhibition in mouse models of melanoma and breast cancer brain metastasis. EXPERIMENTAL DESIGN: Treatment response was evaluated in vivo using immunocompetent mouse models of brain metastasis bearing concurrent intracranial and extracranial tumors. Treatment effect on intracranial and extracranial tumor-immune microenvironments (TIME) was evaluated using immunofluorescence, multiplex immunoassays, high-parameter flow cytometry, and T-cell receptor profiling. Mice with humanized immune systems were evaluated using flow cytometry to study the effect of CDKi on human T-cell development. RESULTS: We found that combining abemaciclib with PD-1 inhibition reduced tumor burden and improved overall survival in mice. The TIME, which differed on the basis of anatomic location of tumors, was altered with CDKi and PD-1 inhibition in an organ-specific manner. Combination abemaciclib and anti-PD-1 treatment increased recruitment and expansion of CD8+ effector T-cell subsets, depleted CD4+ regulatory T (Treg) cells, and reduced levels of immunosuppressive cytokines in intracranial tumors. In immunodeficient mice engrafted with human immune systems, abemaciclib treatment supported development and maintenance of CD8+ T cells and depleted Treg cells. CONCLUSIONS: Our results highlight the distinct properties of intracranial and extracranial tumors and support clinical investigation of combination CDK4/6 and PD-1 inhibition in patients with brain metastases. See related commentary by Margolin, p. 257.

MDM2-p53 in liposarcoma: The need for targeted therapies with novel mechanisms of action.

Well-differentiated and dedifferentiated liposarcomas (WDLPS and DDLPS) are rare tumors that arise from lipocytes in soft tissue. There is a high unmet need in patients with these liposarcomas given poor outcomes, particularly for DDLPS. WDLPS and DDLPS share important genetic and histological characteristics - most notably, the amplification of the 2 genes MDM2 and CDK4. Both genes are considered oncogenes because of their ability to shut down tumor suppressor pathways. There are multiple therapeutic approaches that aim to target MDM2 and CDK4 activity for the purpose of restoring intrinsic tumor suppressor cellular response and terminating oncogenesis. However, current understanding of the molecular mechanisms involved in WDLPS and DDLPS pathology is limited. In recent years, significant efforts have been made to refine and implement targeted therapy for this patient population. The use of patient-derived cell and tumor xenograft models has been an important tool for recapitulating WDLPS and DDLPS biology. These models also offer valuable insights for drug development and drug combination studies. Here we offer a review of the current understanding of WDLPS and DDLPS biology and its therapeutic implications.

Beyond cell cycle regulation: The pleiotropic function of CDK4 in cancer.

CDK4, along with its regulatory subunit, cyclin D, drives the transition from G1 to S phase, during which DNA replication and metabolic activation occur. In this canonical pathway, CDK4 is essentially a transcriptional regulator that acts through phosphorylation of retinoblastoma protein (RB) and subsequent activation of the transcription factor E2F, ultimately triggering the expression of genes involved in DNA synthesis and cell cycle progression to S phase. In this review, we focus on the newly reported functions of CDK4, which go beyond direct regulation of the cell cycle. In particular, we describe the extranuclear roles of CDK4, including its roles in the regulation of metabolism, cell fate, cell dynamics and the tumor microenvironment. We describe direct phosphorylation targets of CDK4 and decipher how CDK4 influences these physiological processes in the context of cancer.

[Prognostic model for assessing the human glioma cell malignancy grade based on MDM2, MELK, SOX2, CDK4, DR5 and OCT4 gene expression]. / Prognosticheskaya model' dlya otsenki stepeni zlokachestvennosti kul'tury kletok gliomy cheloveka na osnovanii issledovaniya ekspressii paneli genov MDM2, MELK, SOX2, CDK4, DR5 i OCT4.

Glioma cell cultures are used in basic researches of tumor processes, personalized medicine for selecting treatment regimens depending on individual characteristics of patients and pharmacology for assessing the effectiveness of chemotherapy. Suppression of glioma culture growth without reduction of malignancy grade is common. Drug cancellation may be followed by substitution of precursor cells by more malignant clones. Therefore, analysis of culture cell malignancy grade is important. In the future, intraoperative analysis of glioma cell malignancy grade can be used to select individual therapy. OBJECTIVE: We analyzed the relationship between expression of marker genes TUBB3, CD133, CDK4, CDK6, CIRBP, DR4, DR5, EGFR, FGFR, FSHR, GDNF, GFAP, L1CAM, LEF1, MAP2, MDM2, MELK, NANOG, NOTCH2, OCT4, OLIG2, PDGFRA, PDGFA, PDGFB and SOX2 and glioma cell malignancy grade, as well as created appropriate prognostic model. MATERIAL AND METHODS: We analyzed expression of 25 marker genes in 22 samples of human glioma cultures using quantitative real-time PCR. Statistical analysis was performed using the IBM SPSS Statistics 26.0 software. We used the Kolmogorov-Smirnov and Shapiro-Wilk tests to assess distribution normality. Nonparametric Jonckheere-Terpstra and Spearman tests were applied. RESULTS: We obtained a prognostic model for assessing the grade III and IV glioma cell malignancy based on expression of marker genes MDM2, MELK, SOX2, CDK4, DR5 and OCT4. Predictive accuracy was 83% (Akaike information criterion -55.125).

Cyclin D1 extensively reprograms metabolism to support biosynthetic pathways in hepatocytes.

Cell proliferation requires metabolic reprogramming to accommodate biosynthesis of new cell components, and similar alterations occur in cancer cells. However, the mechanisms linking the cell cycle machinery to metabolism are not well defined. Cyclin D1, along with its main partner cyclin-dependent kinase 4 (Cdk4), is a pivotal cell cycle regulator and driver oncogene that is overexpressed in many cancers. Here, we examine hepatocyte proliferation to define novel effects of cyclin D1 on biosynthetic metabolism. Metabolomic studies reveal that cyclin D1 broadly promotes biosynthetic pathways including glycolysis, the pentose phosphate pathway, and the purine and pyrimidine nucleotide synthesis in hepatocytes. Proteomic analyses demonstrate that overexpressed cyclin D1 binds to numerous metabolic enzymes including those involved in glycolysis and pyrimidine synthesis. In the glycolysis pathway, cyclin D1 activates aldolase and GAPDH, and these proteins are phosphorylated by cyclin D1/Cdk4 in vitro. De novo pyrimidine synthesis is particularly dependent on cyclin D1. Cyclin D1/Cdk4 phosphorylates the initial enzyme of this pathway, carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), and metabolomic analysis indicates that cyclin D1 depletion markedly reduces the activity of this enzyme. Pharmacologic inhibition of Cdk4 along with the downstream pyrimidine synthesis enzyme dihydroorotate dehydrogenase synergistically inhibits proliferation and survival of hepatocellular carcinoma cells. These studies demonstrate that cyclin D1 promotes a broad network of biosynthetic pathways in hepatocytes, and this model may provide insights into potential metabolic vulnerabilities in cancer cells.

[Consensus on the clinical use of CDK4/6 inhibitors for the treatment of hormone receptor-positive human epidermal growth factor receptor 2-negative breast cancer (2023 edition)].

Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors have led transformative breakthrough of clinical therapy for hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER-2)-negative breast cancer patients. CDK4/6 inhibitors that have been marketed in China include Ribociclib, Palbociclib, Abemaciclib and Dalpiciclib. For HR-positive HER-2-negative locally advanced and metastatic breast cancer, CDK4/6 inhibitors combined with endocrine therapy have become standard regimen, which can prolong the survival of patients. In the adjuvant treatment stage of early breast cancer, CDK4/6 inhibitors have also achieved positive results and been approved for indications. At present, CDK4/6 inhibitors have been widely used in clinical practice in China. In order to further improve the standardized application of CDK4/6 inhibitors in China, the Breast Cancer Expert Committee of the National Center for Cancer Quality Control and the Professional Committee of Clinical Research of Cancer Drugs of the Chinese Anti-Cancer Association organized the related expert to update the consensus based on the "CDK4/6 inhibitor consensus on clinical application of in the treatment of hormone receptor positive human epidermal growth factor receptor 2 negative advanced breast cancer (2021 edition)" . The updated consensus systematically introduces the pharmacological characteristics, drug monitoring and adverse event management, etc., of CDK4/6 inhibitors to promote the accuracy of clinical decision-making with the ultimate goal to prolong the overall survival of patients and improve the quality of life.