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The cell cycle is driven by cyclin-dependent kinases (Cdks). The decision whether the cell cycle proceeds is made during G1 phase, when Cdk4/6 functions. Cyclin-dependent kinase inhibitor 2 (Cdkn2) is a specific inhibitor of Cdk4/6, and their interaction depends on D84 in Cdkn2 and R24/31 in Cdk4/6. This knowledge is based mainly on studies in mammalian cells. Here, we comprehensively analyzed Cdk4/6 and Cdkn2 in invertebrates and found that Cdk4/6 was present in most of the investigated phyla, but the distribution of Cdkn2 was rather uneven among and within the phyla. The positive charge of R24/R31 in Cdk4/6 was conserved in all analyzed species in phyla with Cdkn2. The presence of Cdkn2 and the conservation of the positive charge were statistically correlated. We also found that Cdkn2 has been tightly linked to Fas associated factor 1 (Faf1) during evolution. We discuss potential interactions between Cdkn2 and Cdk4/6 in evolution and the possible cause of the strong conservation of the microsynteny., (© 2024 The Author(s). Genes to Cells published by Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Mammalian cells make the decision to divide at the G 1 -S transition in response to diverse signals impinging on the retinoblastoma protein Rb, a cell cycle inhibitor and tumor suppressor. Passage through the G 1 -S transition is initially driven by Rb inactivation via phosphorylation and by Rb's decreasing concentration in G 1 . While many studies have identified the mechanisms of Rb phosphorylation, the mechanism underlying Rb's decreasing concentration in G 1 was unknown. Here, we found that Rb's concentration decrease in G 1 requires the E3 ubiquitin ligase UBR5. UBR5 knockout cells have increased Rb concentration in early G 1 , exhibited a lower G 1 -S transition rate, and are more sensitive to inhibition of cyclin-dependent kinase 4/6 (Cdk4/6). This last observation suggests that UBR5 inhibition can strengthen the efficacy of Cdk4/6 inhibitor-based cancer therapies.

PROteolysis TArgeting Chimeras (PROTACs) have been considered the next blockbuster therapies. However, due to their inherent limitations, the efficacy of PROTACs is frequently impaired by limited tissue penetration and particularly insufficient cellular internalization into their action sites. Herein, based on the ultra-pH-sensitive and enzyme-sensitive nanotechnology, a type of polymer PROTAC conjugated and pH/cathepsin B sequential responsive nanoparticles (PSRNs) are deliberately designed, following the construction of the PROTAC for Cyclin-dependent kinase 4 and 6 (CDK4/6). Colorectal cancer (CRC) which hardly responds to many treatments even immune checkpoint blockades was selected as the tumor model in this study. As a result, PSRNs were found to maintain nanostructure (40 nm) in circulation and efficiently accumulated in tumors via enhanced permeation and retention effect. Then, they were dissociated into unimers (<10 nm) in response to an acidic tumor microenvironment, facilitating tumor penetration and cellular internalization. Eventually, the CDK4/6 degrading PROTACs were released intracellularly following the cleavage of cathepsin B. Importantly, PSRNs led to the enhanced degradation of target protein in vitro and in vivo. The degradation of CDK4/6 also augmented the efficacy of immune checkpoint blockades, through the upregulation of programmed cell death-ligand 1 (PD-L1) expression in cancer cells and the suppression of regulatory T cells cell proliferation in tumor microenvironment. By combination with α-PD-1, an enhanced anti-tumor outcome is well achieved in CT26 tumor model. Overall, our study verifies the significance of precise intracellular delivery of PROTACs and introduces a promising therapeutic strategy for the targeted combination treatment of CRC., (© 2024. The Author(s).)

Background: Copy number alterations (CNA) are acquired during the evolution of cancers from their early stage to metastatic stage. This study aims at analysing the clinical value of the identified metastasis-associated CNAs both in metastatic breast cancers (mBCs) and early breast cancers (eBCs)., Methods: Single-nucleotide polymorphism (SNP)-array was performed on 926 biopsies from mBC patients, enrolled in SAFIR02-BREAST prospective trial. CNA profiles of eBCs from The Cancer Genome Atlas Breast Invasive Carcinoma (n = 770), Molecular Taxonomy of Breast Cancer International Consortium (n = 1620) and PACS04 trial (n = 243) cohorts were used as references for comparing mBCs and eBCs CNA profiles. Overall survival was the considered survival endpoint., Results: Among the twenty-one genes frequently altered in ER + /HER2- mBCs: focal amplification of TERT was associated with poor outcome in the ER + /HER2- mBC population. Among the ER + /HER2- mBCs patients for whom CDK4/6 inhibitors information before biopsies collection was available: we identified seven genes on post-treatment biopsies, including the cyclin-dependent kinase 4 (CDK4), which was amplified in 9.8% of the ER + /HER2- mBCs pretreated population, as compared to 1.5% in the ER + /HER2- mBCs unpretreated population (P = 2.82E-04) as well as the 3 eBC populations. CDK4 amplification was associated with poor outcome in the ER + /HER2- eBCs., Conclusions: This study provides insights into the biology of mBCs and identifies clinically useful genomic features for future improvement of breast cancer patient management., (© 2024. The Author(s).)

Small cell lung cancer (SCLC) is characterized by rapid development of chemoresistance and poor outcomes. Cyclin-dependent kinase 4/6 inhibitors (CDK4/6is) are widely used in breast cancer and other cancer types. However, the molecular mechanisms of CDK4/6 in SCLC chemoresistance remain poorly understood. Here, Rb1 flox/flox , Trp53 flox/flox , Pten flox/flox (RTP) and Rb1 flox/flox , Trp53 flox/flox , Myc LSL/LSL (RPM) spontaneous SCLC mouse models, SCLC cell line-derived xenograft (CDX) models, and SCLC patient-derived xenograft (PDX) models are established to reveal the potential effects of CDK4/6is on SCLC chemoresistance. In this study, it is found that CDK4/6is palbociclib (PD) or ribociclib (LEE) combined with chemotherapeutic drugs significantly inhibit SCLC tumor growth. Mechanistically, CDK4/6is do not function through the classic Retionblastoma1 (RB) dependent axis in SCLC. CDK4/6is induce impair autophagy through the AMBRA1-lysosome signaling pathway. The upregulated AMBRA1 protein expression leads to CDK6 degradation via autophagy,  and the following TFEB and TFE3 nuclear translocation inhibition leading to the lysosome-related genes levels downregulation. Moreover, it is found that the expression of CDK6 is higher in SCLC tumors than in normal tissue and it is associated with the survival and prognosis of SCLC patients. Finally, these findings demonstrate that combining CDK4/6is with chemotherapy treatment may serve as a potential therapeutic option for SCLC patients., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Purpose: Elacestrant significantly prolonged progression-free survival (PFS) with manageable safety versus standard-of-care (SOC) endocrine therapy (ET) in patients with estrogen receptor-positive (ER+), HER2- metastatic breast cancer and tumors harboring estrogen receptor 1 (ESR1) mutation following ET plus a cyclin-dependent kinase 4/6 inhibitor (ET+CDK4/6i). In patients with ESR1-mutated tumors, we evaluated the efficacy and safety of elacestrant versus SOC based on prior ET+CDK4/6i duration and in clinical subgroups with prior ET+CDK4/6i ≥12 months., Patients and Methods: EMERALD, an open-label phase III trial, randomly assigned patients with ER+, HER2- metastatic breast cancer who had received 1-2 prior lines of ET, mandatory CDK4/6i, and ≤1 chemotherapy to elacestrant (345 mg daily) or SOC (aromatase inhibitor or fulvestrant). PFS was assessed across subgroups in post hoc exploratory analyses without adjustment for multiple testing., Results: In patients with ESR1-mutated tumors and prior ET+CDK4/6i ≥12 months, the median PFS for elacestrant versus SOC was 8.6 versus 1.9 months (HR, 0.41; 95% confidence interval, 0.26-0.63). In this population, the median PFS (in months) for elacestrant versus SOC was 9.1 versus 1.9 (bone metastases), 7.3 versus 1.9 (liver and/or lung metastases), 9.0 versus 1.9 (<3 metastatic sites), 10.8 versus 1.8 (≥3 metastatic sites), 5.5 versus 1.9 (PIK3 catalytic subunit α mutation), 8.6 versus 1.9 (tumor protein p53 gene mutation), 9.0 versus 1.9 (HER2-low), 9.0 versus 1.9 (ESR1D538G-mutated tumors), and 9.0 versus 1.9 (ESR1Y537S/N-mutated tumors). Subgroup safety was consistent with the overall population., Conclusions: The duration of prior ET+CDK4/6i ≥12 months in metastatic breast cancer was associated with a clinically meaningful improvement in PFS for elacestrant compared with SOC and was consistent across all subgroups evaluated in patients with ER+, HER2-, ESR1-mutated tumors., (©2024 The Authors; Published by the American Association for Cancer Research.)

Purpose: Development of a computational biomarker to predict, prior to treatment, the response to CDK4/6 inhibition (CDK4/6i) in combination with endocrine therapy in patients with breast cancer., Experimental Design: A mechanistic mathematical model that accounts for protein signaling and drug mechanisms of action was developed and trained on extensive, publicly available data from breast cancer cell lines. The model was built to provide a patient-specific response score based on the expression of six genes (CCND1, CCNE1, ESR1, RB1, MYC, and CDKN1A). The model was validated in five independent cohorts of 148 patients in total with early-stage or advanced breast cancer treated with endocrine therapy and CDK4/6i. Response was measured either by evaluating Ki67 levels and PAM50 risk of relapse (ROR) after neoadjuvant treatment or by evaluating progression-free survival (PFS)., Results: The model showed significant association with patient's outcomes in all five cohorts. The model predicted high Ki67 [area under the curve; AUC (95% confidence interval, CI) of 0.80 (0.64-0.92), 0.81 (0.60-1.00) and 0.80 (0.65-0.93)] and high PAM50 ROR [AUC of 0.78 (0.64-0.89)]. This observation was not obtained in patients treated with chemotherapy. In the other cohorts, patient stratification based on the model prediction was significantly associated with PFS [hazard ratio (HR) = 2.92 (95% CI, 1.08-7.86), P = 0.034 and HR = 2.16 (1.02 4.55), P = 0.043]., Conclusions: A mathematical modeling approach accurately predicts patient outcome following CDK4/6i plus endocrine therapy that marks a step toward more personalized treatments in patients with Luminal B breast cancer., (©2024 American Association for Cancer Research.)

Purpose: The National Cancer Institute-Children's Oncology Group Pediatric Molecular Analysis for Therapy Choice trial assigned patients age 1-21 years with relapsed or refractory solid tumors, lymphomas, and histiocytic disorders to phase II treatment arms of molecularly targeted therapies on the basis of genetic alterations detected in their tumor. Patients with tumors that harbored prespecified genomic alterations in the cyclinD-CDK4/6-INK4a-Rb pathway with intact Rb expression were assigned and treated with the cdk4/6 inhibitor palbociclib., Methods: Patients received palbociclib orally once daily for 21 days of 28-day cycles until disease progression, intolerable toxicity, or up to 2 years. The primary end point was objective response rate; secondary end points included safety/tolerability and progression-free survival., Results: Twenty-three patients (median age, 15 years; range, 8-21) were enrolled; 20 received protocol therapy and were evaluable for toxicity and response. Of the evaluable patients, the most common diagnoses were osteosarcoma (n = 9) and rhabdomyosarcoma (n = 6). A single actionable gene amplification was found in 19 tumors ( CDK4 , n = 11, CDK6 , n = 2, CCND3 , n = 6), with one tumor harboring two amplifications ( CDK4 and CCND2 ). Hematologic toxicities were the most common treatment-related events. No objective responses were seen. Two patients with tumors harboring CDK4 amplifications (neuroblastoma and sarcoma) had best response of stable disease for six and three cycles. Six-month progression was 10% (95% CI, 1.7 to 27.2)., Conclusion: The CDK4/6 inhibitor palbociclib at 75 mg/m 2 orally daily was tolerable in this heavily pretreated cohort. No objective responses were observed in this histology-agnostic biomarker-selected population with treatment-refractory solid tumors, demonstrating that pathway alteration alone is insufficient in pediatric cancers to generate a response to palbociclib monotherapy.

Combined CDK4/6 inhibitor (CDK4/6i) and endocrine therapy significantly improves outcome for patients with estrogen receptor-positive (ER+) metastatic breast cancer, but drug resistance and thus disease progression inevitably occur. Herein, we aimed to identify genomic alterations associated with combined CDK4/6i and endocrine therapy resistance, and follow the levels of specific mutations in longitudinal circulating tumor DNA (ctDNA) for early detection of progression. From a cohort of 86 patients with ER+ metastatic breast cancer we performed whole exome sequencing or targeted sequencing of paired tumor (N = 8) or blood samples (N = 5) obtained before initiation of combined CDK4/6i and endocrine therapy and at disease progression. Mutations in oncogenic genes at progression were rare, while amplifications of growth-regulating genes were more frequent. The most frequently acquired alterations observed were PIK3CA and TP53 mutations and PDK1 amplification. Longitudinal ctDNA dynamics of mutant PIK3CA or private mutations revealed increased mutation levels at progression in 8 of 10 patients (80%). Impressively, rising levels of PIK3CA-mutated ctDNA were detected 4-17 months before imaging. Our data add to the growing evidence supporting longitudinal ctDNA analysis for real-time monitoring of CDK4/6i response and early detection of progression in advanced breast cancer. Further, our analysis suggests that amplification of growth-related genes may contribute to combined CDK4/6i and endocrine therapy resistance., (© 2024 The Author(s). International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Breast cancer (BC) is marked by significant genetic, morphological and clinical heterogeneity. To capture this heterogeneity and unravel the molecular mechanisms driving tumor progression and drug resistance, we established a comprehensive patient-derived xenograft (PDX) biobank, focusing particularly on luminal (estrogen receptor, ER+) and young premenopausal patients, for whom PDX models are currently scarce. Across all BC subtypes, our efforts resulted in an overall success rate of 17% (26 established PDX lines out of 151 total attempts), specifically 15% in luminal, 12% in human epidermal growth factor receptor 2 positive (HER2+) and 35% in triple negative BC. These PDX mirrored morphologic and genetic features of BC from which they originated, serving as a reliable tool to investigate drug resistance and test therapeutic strategies. We focused on understanding resistance to CDK4/6 inhibitors (CDK4/6i), which are crucial in the treatment of patients with advanced luminal BC. Treating a sensitive luminal BC PDX with the CDK4/6i palbociclib revealed that, despite initial tumor shrinkage, some tumors might eventually regrow under drug treatment. RNA sequencing, followed by gene set enrichment analyses, unveiled that these PDXs have become refractory to CDK4/6i, both at biological and molecular levels, displaying significant enrichment in proliferation pathways, such as MTORC1, E2F and MYC. Using organoids derived from these PDX (PDxO), we observed that acquisition of CDK4/6i resistance conferred cross-resistance to endocrine therapy and that targeting MTORC1 was a successful strategy to overcome CDK4/6i resistance. Considered together, these results indicate that our PDX models may serve as robust tools to elucidate the molecular basis of BC disease progression and, by providing the possibility to simultaneously test different therapies on the same tumor, to surmount treatment resistance. While this approach is of course not feasible in the clinic, its exploitation in PDX may expedite the identification and development of more successful therapies for patients with advanced luminal BC. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland., (© 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.)

Introduction: Patients with breast cancer, especially triple-negative breast cancer, have a poor prognosis. There is still no effective treatment for this disease. Due to resistance to traditional treatments such as chemotherapy and radiation therapy, there is a need to discover novel treatment strategies to treat this disease. Ribociclib is a selective CDK4/6 inhibitor. Approximately 20% of patients with HR+ breast cancer developed primary resistance to CDK4/6 inhibitors, and more than 30% experienced secondary resistance. Since most patients experience resistance during CDK4/6 inhibitor treatment, managing this disease is becoming more challenging. Many malignant tumors abnormally express microRNA (miR)-141, which participates in several cellular processes, including drug resistance, proliferation, epithelial-mesenchymal transition, migration, and invasion., Materials and Methods: In the present study, we cultured MDA-MB-231 and MCF-7 cells in DMEM-F12 medium. By performing MTT assay we determined the cytotoxic effects of ribociclib on breast cancer cells, as well as determining the IC50 of it. Then, we treated the cells with ribociclib at two time points: 24 h and 72 h. After that, RNA was isolated and reverse transcribed to cDNA. Finally, we performed qRT‒PCR to evaluate how ribociclib affects the expression level of desired genes., Results and Conclusion: We found that ribociclib can inhibit cell growth in a dose- and time-dependent manner. We examined the mRNA expression of 4 genes. After ribociclib treatment, the mRNA expression of CDK6 and MYH10 decreased (p < 0.01, p < 0.05). The mRNA expression of CDON increased (p<0.05), but no significant changes were observed in ZEB1 mRNA expression. Furthermore, the qRT‒PCR results for miR-141 showed that the expression of miR-141 increased (p<0.01) after 72 h of treatment with ribociclib., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Baghermanesh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Regulated cell cycle progression ensures homeostasis and prevents cancer. In proliferating cells, premature S phase entry is avoided by the E3 ubiquitin ligase anaphasepromoting complex/cyclosome (APC/C), although the APC/C substrates whose degradation restrains G1-S progression are not fully known. The APC/C is also active in arrested cells that exited the cell cycle, but it is not clear whether APC/C maintains all types of arrest. Here, by expressing the APC/C inhibitor, EMI1, we show that APC/C activity is essential to prevent S phase entry in cells arrested by pharmacological cyclin-dependent kinases 4 and 6 (CDK4/6) inhibition (Palbociclib). Thus, active protein degradation is required for arrest alongside repressed cell cycle gene expression. The mechanism of rapid and robust arrest bypass from inhibiting APC/C involves CDKs acting in an atypical order to inactivate retinoblastoma-mediated E2F repression. Inactivating APC/C first causes mitotic cyclin B accumulation which then promotes cyclin A expression. We propose that cyclin A is the key substrate for maintaining arrest because APC/C-resistant cyclin A, but not cyclin B, is sufficient to induce S phase entry. Cells bypassing arrest from CDK4/6 inhibition initiate DNA replication with severely reduced origin licensing. The simultaneous accumulation of S phase licensing inhibitors, such as cyclin A and geminin, with G1 licensing activators disrupts the normal order of G1-S progression. As a result, DNA synthesis and cell proliferation are profoundly impaired. Our findings predict that cancers with elevated EMI1 expression will tend to escape CDK4/6 inhibition into a premature, underlicensed S phase and suffer enhanced genome instability., Competing Interests: Competing interests statement:The authors declare no competing interest.

Purpose: Treatment paradigms for isocitrate dehydrogenase (IDH)-mutant gliomas are rapidly evolving. Although typically indolent and responsive to initial treatment, these tumors invariably recur at a higher grade and require salvage treatment. Homozygous deletion of the tumor suppressor gene CDKN2A/B frequently emerges at recurrence in these tumors, driving poor patient outcomes. We investigated the effect of CDK-Rb pathway blockade on IDH-mutant glioma growth in vitro and in vivo using CDK4/6 inhibitors (CDKi)., Experimental Design: Cell viability, proliferation assays, and flow cytometry were used to examine the pharmacologic effect of two distinct CDKi, palbociclib and abemaciclib, in multiple patient-derived IDH-mutant glioma lines. Isogenic models were used to directly investigate the influence of CDKN2A/B status on CDKi sensitivity. Orthotopic xenograft tumor models were used to examine the efficacy and tolerability of CDKi in vivo., Results: CDKi treatment leads to decreased cell viability and proliferative capacity in patient-derived IDH-mutant glioma lines, coupled with enrichment of cells in the G1 phase. CDKN2A inactivation sensitizes IDH-mutant glioma to CDKi in both endogenous and isogenic models with engineered CDKN2A deletion. CDK4/6 inhibitor administration improves survival in orthotopically implanted IDH-mutant glioma models., Conclusions: IDH-mutant gliomas with deletion of CDKN2A/B are sensitized to CDK4/6 inhibitors. These results support the investigation of the use of these agents in a clinical setting., (©2024 American Association for Cancer Research.)

Background: The 5-year survival rate of oesophageal squamous cell carcinoma (ESCC) is approximately 20%. The prognosis and drug response exhibit substantial heterogeneity in ESCC, impeding progress in survival outcomes. Our goal is to identify a signature for tumour subtype classification, enabling precise clinical treatments., Methods: Utilising pre-treatment multi-omics data from an ESCC dataset (n = 310), an enhancer methylation-eRNA-target gene regulation network was constructed and validated by in vitro experiments. Four machine learning methods collectively identified core target genes, establishing an Enhancer Demethylation-Regulated Gene Score (EDRGS) model for classification. The molecular function of EDRGS subtyping was explored in scRNA-seq (n = 60) and bulk-seq (n = 310), and the EDRGS's potential to predict treatment response was assessed in datasets of various cancer types., Findings: EDRGS stratified ESCCs into EDRGS-high/low subtypes, with EDRGS-high signifying a less favourable prognosis in ESCC and nine additional cancer types. EDRGS-high exhibited an immune-hot but immune-suppressive phenotype with elevated immune checkpoint expression, increased T cell infiltration, and IFNγ signalling in ESCC, suggesting a better response to immunotherapy. Notably, EDRGS outperformed PD-L1 in predicting anti-PD-1/L1 therapy effectiveness in ESCC (n = 42), kidney renal clear cell carcinoma (KIRC, n = 181), and bladder urothelial carcinoma (BLCA, n = 348) cohorts. EDRGS-low showed a cell cycle-activated phenotype with higher CDK4 and/or CDK6 expression, demonstrating a superior response to the CDK4/6 inhibitor palbociclib, validated in ESCC (n = 26), melanoma (n = 18), prostate cancer (n = 15) cells, and PDX models derived from patients with pancreatic cancer (n = 30)., Interpretation: Identification of EDRGS subtypes enlightens ESCC categorisation, offering clinical insights for patient management in immunotherapy (anti-PD-1/L1) and CDK4/6 inhibitor therapy across cancer types., Funding: This study was supported by funding from the National Key R&D Program of China (2021YFC2501000, 2020YFA0803300), the National Natural Science Foundation of China (82030089, 82188102), the CAMS Innovation Fund for Medical Sciences (2021-I2M-1-018, 2022-I2M-2-001, 2021-I2M-1-067), the Fundamental Research Funds for the Central Universities (3332021091)., Competing Interests: Declaration of interests The authors have declared that no conflict of interest exists., (Copyright © 2024. Published by Elsevier B.V.)

The comprehensive genomic analysis of the head and neck squamous cell carcinoma (HNSCC) oncogenome revealed the frequent loss of p16INK4A (CDKN2A) and amplification of cyclin D1 genes in most human papillomavirus-negative HNSCC lesions. However, cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors have shown modest effects in the clinic. The aberrant activation of the PI3K/mTOR pathway is highly prevalent in HNSCC, and recent clinical trials have shown promising clinical efficacy of mTOR inhibitors (mTORi) in the neoadjuvant and adjuvant settings but not in patients with advanced HNSCC. By implementing a kinome-wide CRISPR/Cas9 screen, we identified cell-cycle inhibition as a synthetic lethal target for mTORis. A combination of mTORi and palbociclib, a CDK4/6-specific inhibitor, showed strong synergism in HNSCC-derived cells in vitro and in vivo. Remarkably, we found that an adaptive increase in cyclin E1 (CCNE1) expression upon palbociclib treatment underlies the rapid acquired resistance to this CDK4/6 inhibitor. Mechanistically, mTORi inhibits the formation of eIF4G-CCNE1 mRNA complexes, with the consequent reduction in mRNA translation and CCNE1 protein expression. Our findings suggest that mTORi reverts the adaptive resistance to palbociclib. This provides a multimodal therapeutic option for HNSCC by cotargeting mTOR and CDK4/6, which in turn may halt the emergence of palbociclib resistance., Significance: A kinome-wide CRISPR/Cas9 screen identified cell-cycle inhibition as a synthetic lethal target of mTORis. A combination of mTORi and palbociclib, a CDK4/6-specific inhibitor, showed strong synergistic effects in HNSCC. Mechanistically, mTORis inhibited palbociclib-induced increase in CCNE1., (©2024 The Authors; Published by the American Association for Cancer Research.)

In patients with ER + metastatic breast cancer (mBC), the first-line treatment involves the combination of endocrine therapy (ET) and CDK4/6 inhibitors (CDK4/6i). However, a significant group of patients experiences disease progression, emphasizing the urgent clinical need to identify novel anti-tumor therapies. We previously generated breast cancer cells resistant to the combination of fulvestrant (ER downregulator) and abemaciclib (CDK4/6 inhibitor) from MCF7 and T47D (MCF7-FAR and T47D-FAR). RNA-seq-based Gene Set Enrichment Analysis (GSEA) revealed hyper-activation of EGFR, HER2, and AKT signaling in both MCF7-FAR and T47D-FAR. Modulating EGFR or ERBB2 expression through loss- and gain-of-function experiments altered tumor sensitivity to fulvestrant and abemaciclib in parental and FAR spheroids, affecting ERK and AKT/S6 pathways. Cetuximab treatment overcame tumor resistance to fulvestrant and abemaciclib in FAR and EGFR-overexpressing breast cancer spheroids and xenografts. Likewise, patient-derived organoids (PDOs) from individuals with ER + mBC, progressing on palbociclib, exhibited up-regulation of EGFR and HER2 pathways. In conclusion, our findings suggest that inhibiting EGFR and HER2 pathways might overcome resistance to ET + CDK4/6i in selected patients with ER + mBC., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Servetto A. reports honoraria from Eli Lilly, MSD, and Janssen and travel support from Bristol-Myers Squibb and AstraZeneca. Formisano L. declares the following competing interests: consultant and advisory board for Seagen, Amgen, BMS, MSD, Jansen, and Pierre Fabre Pharma. Bianco R. declares the following competing interests: consultant and advisory board for BMS, MSD, Pfizer, AstraZeneca, Lilly, and Novartis. The remaining authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Abemaciclib (ABM), a cyclin-dependent kinase 4/6 inhibitor, shows pharmacological effects in cell cycle arrest. Epithelial-mesenchymal transition is an important cellular event associated with pathophysiological states such as organ fibrosis and cancer progression. In the present study, we evaluated the contribution of factors associated with cell cycle arrest to ABM-induced epithelial-mesenchymal transition. Treatment with 0.6 µM ABM induced both cell cycle arrest and epithelial-mesenchymal transition-related phenotypic changes. Interestingly, the knockdown of cyclin-dependent kinase 4/6, pharmacological targets of ABM or cyclin D1, which forms complexes with cyclin-dependent kinase 4/6, resulted in cell cycle arrest at the G1-phase and induction of epithelial-mesenchymal transition, indicating that downregulation of cyclin-dependent kinase 4/6-cyclin D1 complexes would mimic ABM. In contrast, knockdown of the Rb protein, which is phosphorylated by cyclin-dependent kinase 4/6, had no effect on the expression level of α-smooth muscle actin, an epithelial-mesenchymal transition marker. Furthermore, ABM-induced epithelial-mesenchymal transition was not affected by Rb knockdown, suggesting that Rb is not involved in the transition process. Our study is the first to suggest that cyclin-dependent kinase 4/6-cyclin D1 complexes, as pharmacological targets of ABM, may contribute to ABM-induced epithelial-mesenchymal transition, followed by clinical disorders such as organ fibrosis and cancer progression. This study suggests that blocking epithelial-mesenchymal transition might be a promising way to prevent negative side effects caused by a medication (ABM) without affecting its ability to treat the disease., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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., (©2024 American Association for Cancer Research.)

Cell cycle events are coordinated by cyclin-dependent kinases (CDKs) to ensure robust cell division. CDK4/6 and CDK2 regulate the growth 1 (G 1 ) 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 G 2 -arrested cells to prevent cell cycle exit after the S phase. This mechanism revealed a role for CDK4/6 in maintaining the G 2 state, challenging the notion that the cell cycle is irreversible and that cells do not require mitogens after passing the restriction point. Exit from G 2 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.

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., (©2024 The Authors; Published by the American Association for Cancer Research.)

Background: In estrogen receptor-positive metastatic breast cancer, ESR1 mutations (ESR1 m ) are a common mechanism of acquired resistance to aromatase inhibitors (ArIh). However, the impact ESR1 alterations have on CDK4/6 inhibitor (CDK4/6i) sensitivity has not been established. Analyses of CDK4/6i trials suggest that the endocrine therapy partner and specific ESR1 allele may affect susceptibility. We analyzed a real-world data set to investigate CDK4/6i efficacy in ESR1 m metastatic breast cancer and associated clinical factors., Methods: ESR1 m were identified by analysis of circulating-tumor deoxyribonucleic acid. The GuardantINFORM database contains genomic information from tumors linked with claims data. Patients who started a CDK4/6i within 30 days of sequencing were categorized as having ESR1 m or non- ESR1 -mutant (non-ESR1 m ) breast cancer. Data were analyzed to determine the real-world time-to-next-treatment, defined as the start of a breast cancer treatment to initiation of the subsequent treatment., Results: One hundred forty-five patients with ESR1 m and 612 with non-ESR1 m metastatic breast cancer were analyzed. ESR1 m and non-ESR1 m tumors had similar real-world time-to-next-treatment on CDK4/6i regimens (hazard ratio, 1.02; 95% confidence interval, 0.82 to 1.23). Duration on therapy in the first-line and second-line plus treatment settings were comparable regardless of ESR1 status. We stratified treatment duration by concurrent endocrine therapy, and patients with ESR1 m had worse outcomes on ArIh but comparable real-world time-to-next-treatment on fulvestrant., Conclusions: These data suggest ESR1 variants are not associated with pan-CDK4/6i resistance and are consistent with the hypothesis that CDK4/6 blockade combined with a selective estrogen receptor degrader is potentially an effective option for ESR1 m metastatic breast cancer.

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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., (© 2023. Springer Nature Limited.)

Despite the development of predictive biomarkers to shape treatment paradigms and outcomes, de novo EGFR TKI resistance advanced non-small cell lung cancer (NSCLC) remains an issue of concern. We explored clinical factors in 332 advanced NSCLC who received EGFR TKI and molecular characteristics through 65 whole exome sequencing of various EGFR TKI responses including; de novo (progression within 3 months), intermediate response (IRs) and long-term response (LTRs) (durability > 2 years). Uncommon EGFR mutation subtypes were significantly variable enriched in de novo resistance. The remaining sensitizing EGFR mutation subtypes (exon 19 del and L858R) accounted for 75% of de novo resistance. Genomic landscape analysis was conducted, focusing in 10 frequent oncogenic signaling pathways with functional contributions; cell cycle, Hippo, Myc, Notch, Nrf2, PI-3-Kinase/Akt, RTK-RAS, TGF-β, p53 and β-catenin/Wnt signaling. Cell cycle pathway was the only significant alteration pathway among groups with the FDR p-value of 6 × 10 -4 . We found only significant q-values of < 0.05 in 7 gene alterations; CDK6, CCNE1, CDK4, CCND3, MET, FGFR4 and HRAS which enrich in de novo resistance [range 36-73%] compared to IRs/LTRs [range 4-22%]. Amplification of CDK4/6 was significant in de novo resistance, contrary to IRs and LTRs (91%, 27.9% and 0%, respectively). The presence of co-occurrence CDK4/6 amplification correlated with poor disease outcome with HR of progression-free survival of 3.63 [95% CI 1.80-7.31, p-value < 0.001]. The presence of CDK4/6 amplification in pretreatment specimen serves as a predictive biomarker for de novo resistance in sensitizing EGFR mutation., (© 2022. The Author(s).)

Standard radiation therapy (RT) does not reliably provide locoregional control for women with multinode-positive breast cancer and triple-negative breast cancer (TNBC). We hypothesized that CDK4/6 inhibition (CDK4/6i) would increase the radiosensitivity not only of estrogen receptor-positive (ER+) cells, but also of TNBC that expresses retinoblastoma (RB) protein. We found that CDK4/6i radiosensitized RB WT TNBC (n = 4, radiation enhancement ratio [rER]: 1.49-2.22) but failed to radiosensitize RB-null TNBC (n = 3, rER: 0.84-1.00). RB expression predicted response to CDK4/6i + RT (R2 = 0.84), and radiosensitization was lost in ER+/TNBC cells (rER: 0.88-1.13) after RB1 knockdown in isogenic and nonisogenic models. CDK4/6i suppressed homologous recombination (HR) in RB WT cells but not in RB-null cells or isogenic models of RB1 loss; HR competency was rescued with RB reexpression. Radiosensitization was independent of nonhomologous end joining and the known effects of CDK4/6i on cell cycle arrest. Mechanistically, RB and RAD51 interact in vitro to promote HR repair. CDK4/6i produced RB-dependent radiosensitization in TNBC xenografts but not in isogenic RB1-null xenografts. Our data provide the preclinical rationale for a clinical trial expanding the use of CDK4/6i + RT to difficult-to-control RB-intact breast cancers (including TNBC) and nominate RB status as a predictive biomarker of therapeutic efficacy.

Background: Ovarian cancer (OC) is one of the most lethal gynecologic cancers. Growing evidence has proven that CDK4/6 plays a key role in tumor immunity and the prognosis of many cancers. However, the expression and function of CDK4/6 in OC remain unclear. Therefore, we aimed to explore the influence of CDK4/6 in OC, especially on immunity., Methods: We analyzed CDK4/6 expression and prognosis using The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO) and Genotype Tissue Expression (GTEx) data. Subsequently, we used the cytoHubba plug-in of Cytoscape software and starBase to identify the noncoding RNAs (ncRNAs) regulating CDK4/6. Finally, we verified the effect of CDK4/6 on immunity in OC cell lines and animal models., Results: CDK4/6 expression was higher in OC tissues than in normal ovarian tissues, and the high expression levels of CDK4/6 contributed to the immunosuppressive state of OC and were thus related to the poor prognosis of OC patients. This was also in general agreement with the results of OC cell line and animal experiments. Mechanistically, the CDK4/6 inhibitor palbociclib increased the secretion of interferon (IFN)-γ and the interferon-stimulated gene (ISG) response, thereby upregulating the expression of antigen-presenting molecules; this effect was partly dependent on the STING pathway and thus activated immunity in OC. Additionally, according to public data, the LRRC75A-AS1-hsa-miR-330-5p axis could inhibit the immune response of OC patients by upregulating CDK4/6, leading to a poor prognosis., Conclusion: CDK4/6 affects the immune microenvironment of OC and correlates with the prognosis of OC patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Liu, Huang, Cui, Zhou, Qin, Zhang, Li, Li, Guo, Yang, Li, Fan, Li, Fu, Liu, Hu, Xiao, Wang, Dou, Wang, Li, Yang, Liu, Peng, Qin, You, Lu and Sun.)

Background: There is no effective therapy for patients with malignant pleural mesothelioma (MPM) who progressed to platinum-based chemotherapy and immunotherapy., Methods: We aimed to investigate the antitumor activity of CDK4/6 inhibitors using in vitro and in vivo preclinical models of MPM., Results: Based on publicly available transcriptomic data of MPM, patients with CDK4 or CDK6 overexpression had shorter overall survival. Treatment with abemaciclib or palbociclib at 100 nM significantly decreased cell proliferation in all cell models evaluated. Both CDK4/6 inhibitors significantly induced G1 cell cycle arrest, thereby increasing cell senescence and increased the expression of interferon signalling pathway and tumour antigen presentation process in culture models of MPM. In vivo preclinical studies showed that palbociclib significantly reduced tumour growth and prolonged overall survival using distinct xenograft models of MPM implanted in athymic mice., Conclusions: Treatment of MPM with CDK4/6 inhibitors decreased cell proliferation, mainly by promoting cell cycle arrest at G1 and by induction of cell senescence. Our preclinical studies provide evidence for evaluating CDK4/6 inhibitors in the clinic for the treatment of MPM., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitors are considered a breakthrough in cancer therapy. Currently approved for breast cancer treatment, CDK4/6 inhibitors are extensively tested in other cancer subtypes. Frequently observed side effects include hematological abnormalities such as reduced numbers of neutrophils, erythroid cells and platelets that are associated with anemia, bleeding and a higher risk of infections. In order to understand whether the adverse effects within the hematopoietic system are related to CDK4 or CDK6 we generated transgenic mice that lack either CDK4 or CDK6 in adult hematopoiesis. Anemia and perturbed erythroid differentiation are associated with the absence of CDK6 but did not manifest in CDK4- deficient mice. Total CDK6 knockout mice accumulate the most dormant fraction of hematopoietic stem cells due to an impaired exit of the quiescent state. We recapitulated this finding by deleting CDK6 in adult hematopoiesis. In addition, unlike total CDK6 knockout, all stem cell fractions were affected and increased in numbers. The deletion of CDK6 was also accompanied by neutropenia which is frequently seen in patients receiving CDK4/6 inhibitors. This was not the case in the absence of CDK4; CDK4 deficiency resulted in elevated numbers of myeloid progenitors without translating into numeric changes of differentiated myeloid cells. By using Cdk4fl/fl and Cdk6fl/fl mice we assign side effects of CDK4/6 inhibitors predominantly to the absence of CDK6. These mice represent a novel and powerful tool that will enable to study the distinct functions of CDK4 and CDK6 in a tissue-dependent manner.

CDK4/6 inhibitors (CDK4/6i) combined with endocrine therapy have shown impressive efficacy in estrogen receptor-positive advanced breast cancer. However, most patients will eventually experience disease progression on this combination, underscoring the need for effective subsequent treatments or better initial therapies. Here, we show that triple inhibition with fulvestrant, CDK4/6i and AKT inhibitor (AKTi) durably impairs growth of breast cancer cells, prevents progression and reduces metastasis of tumor xenografts resistant to CDK4/6i-fulvestrant combination or fulvestrant alone. Importantly, switching from combined fulvestrant and CDK4/6i upon resistance to dual combination with AKTi and fulvestrant does not prevent tumor progression. Furthermore, triple combination with AKTi significantly inhibits growth of patient-derived xenografts resistant to combined CDK4/6i and fulvestrant. Finally, high phospho-AKT levels in metastasis of breast cancer patients treated with a combination of CDK4/6i and endocrine therapy correlates with shorter progression-free survival. Our findings support the clinical development of ER, CDK4/6 and AKT co-targeting strategies following progression on CDK4/6i and endocrine therapy combination, and in tumors exhibiting high phospho-AKT levels, which are associated with worse clinical outcome., (© 2021. The Author(s).)

Intrinsic resistance to CDK4/6 inhibitors hinders their clinical utility in cancer treatment. Furthermore, the predictive markers of CDK4/6 inhibitors in gastric cancer (GC) remain incompletely described. Here, we found that PAX6 expression was negatively correlated with the response to palbociclib in vitro and in vivo in GC. We observed that the PAX6 expression level was negatively correlated with the overall survival of GC patients and further showed that PAX6 can promote GC cell proliferation and the cell cycle. The cell cycle is regulated by the interaction of cyclins with their partner serine/threonine cyclin-dependent kinases (CDKs), and the G1/S-phase transition is the main target of CDK4/6 inhibitors. Therefore, we tested whether PAX6 expression was correlated with the GC response to palbociclib. We found that PAX6 hypermethylates the promoter of LATS2 and inactivates the Hippo pathway, which upregulates cyclin D1 (CCND1) expression. This results in a suppressed response to palbociclib in GC. Furthermore, we found that the induction of the Hippo signaling pathway or treatment with a DNA methylation inhibitor could overcome PAX6-induced palbociclib resistance in GC. These findings uncover a tumor promoter function of PAX6 in GC and establish overexpressed PAX6 as a mechanism of resistance to palbociclib., (© 2021 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Since their discovery as drivers of proliferation, cyclin-dependent kinases (CDKs) have been considered therapeutic targets. Small molecule inhibitors of CDK4/6 are used and tested in clinical trials to treat multiple cancer types. Despite their clinical importance, little is known about how CDK4/6 inhibitors affect the stability of CDK4/6 complexes, which bind cyclins and inhibitory proteins such as p21. We develop an assay to monitor CDK complex stability inside the nucleus. Unexpectedly, treatment with CDK4/6 inhibitors-palbociclib, ribociclib, or abemaciclib-immediately dissociates p21 selectively from CDK4 but not CDK6 complexes. This effect mediates indirect inhibition of CDK2 activity by p21 but not p27 redistribution. Our work shows that CDK4/6 inhibitors have two roles: non-catalytic inhibition of CDK2 via p21 displacement from CDK4 complexes, and catalytic inhibition of CDK4/6 independent of p21. By broadening the non-catalytic displacement to p27 and CDK6 containing complexes, next-generation CDK4/6 inhibitors may have improved efficacy and overcome resistance mechanisms.

Background: Alpelisib, a PI3Kα-selective inhibitor and degrader, plus fulvestrant showed efficacy in hormone receptor-positive, HER2-negative, PIK3CA-mutated advanced breast cancer in SOLAR-1; limited data are available in the post-cyclin-dependent kinase 4/6 inhibitor setting. BYLieve aimed to assess alpelisib plus endocrine therapy in this setting in three cohorts defined by immediate previous treatment; here, we report results from cohort A., Methods: This ongoing, phase 2, multicentre, open-label, non-comparative study enrolled patients with hormone receptor-positive, HER2-negative, advanced breast cancer with tumour PIK3CA mutation, following progression on or after previous therapy, including CDK4/6 inhibitors, from 114 study locations (cancer centres, medical centres, university hospitals, and hospitals) in 18 countries worldwide. Participants aged 18 years or older with an Eastern Cooperative Oncology Group performance status of 2 or less, with no more than two previous anticancer treatments and no more than one previous chemotherapy regimen, were enrolled in three cohorts. In cohort A, patients must have had progression on or after a CDK4/6 inhibitor plus an aromatase inhibitor as the immediate previous treatment. Patients received oral alpelisib 300 mg/day (continuously) plus fulvestrant 500 mg intramuscularly on day 1 of each 28-day cycle and on day 15 of cycle 1. The primary endpoint was the proportion of patients alive without disease progression at 6 months per local assessment using Response Evaluation Criteria in Solid Tumors, version 1.1, in patients with a centrally confirmed PIK3CA mutation. This trial is registered with ClinicalTrials.gov, NCT03056755., Findings: Between Aug 14, 2017, and Dec 17, 2019 (data cutoff), 127 patients with at least 6 months' follow-up were enrolled into cohort A. 121 patients had a centrally confirmed PIK3CA mutation. At data cutoff, median follow-up was 11·7 months (IQR 8·5-15·9). 61 (50·4%; 95% CI 41·2-59·6) of 121 patients were alive without disease progression at 6 months. The most frequent grade 3 or worse adverse events were hyperglycaemia (36 [28%] of 127 patients), rash (12 [9%]), and rash maculopapular (12 [9%]). Serious adverse events occurred in 33 (26%) of 127 patients. No treatment-related deaths were reported., Interpretation: BYLieve showed activity of alpelisib plus fulvestrant with manageable toxicity in patients with PIK3CA-mutated, hormone receptor-positive, HER2-negative advanced breast cancer, after progression on a CDK4/6 inhibitor plus an aromatase inhibitor., Funding: Novartis Pharmaceuticals., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Although the cyclin-dependent kinases CDK4 and CDK6 play fundamental roles in cancer, the specific pathways and downstream targets by which they exert their tumorigenic effects remain elusive. In this study, we uncover distinct and novel functions for these kinases in regulating tumor formation and metastatic colonization in various solid tumors, including those of the breast, prostate, and pancreas. Combining in vivo CRISPR-based CDK4 and CDK6 gene editing with pharmacologic inhibition approaches in orthotopic transplantation and patient-derived xenograft preclinical models, we defined clear functions for CDK4 and CDK6 in facilitating tumor growth and progression in metastatic cancers. Transcriptomic profiling of CDK4/6 CRISPR knockouts in breast cancer revealed these two kinases to regulate cancer progression through distinct mechanisms. CDK4 regulated prometastatic inflammatory cytokine signaling, whereas CDK6 mainly controlled DNA replication and repair processes. Inhibition of CDK6 but not CDK4 resulted in defective DNA repair and increased DNA damage. Multiple CDK6 DNA replication/repair genes were not only associated with cancer subtype, grades, and poor clinical outcomes, but also facilitated primary tumor growth and metastasis in vivo . CRISPR-based genomic deletion of CDK6 efficiently blocked tumor formation and progression in preestablished cell- and patient-derived xenograft preclinical models of breast cancer, providing a potential novel targeted therapy for these deadly tumors. SIGNIFICANCE: In-depth transcriptomic analysis identifies cyclin-dependent kinases CDK4 and CDK6 as regulators of metastasis through distinct signaling pathways and reveals the DNA replication/repair pathway as central in promoting these effects., (©2020 American Association for Cancer Research.)

Intrinsic or acquired resistance to clinically approved CDK4/6 inhibitors has emerged as a major obstacle that hinders their utility beyond ER + breast cancer. In this study, CDK4/6-dependent and -resistant models were employed to identify functional determinants of response to pharmacologic CDK4/6 inhibitors. In all models tested, the activation of RB and inhibition of CDK2 activity emerged as determinants of sensitivity. While depleting CDK4 and 6 was sufficient to limit proliferation in specific resistance settings, RB loss rendered cells completely independent of these kinases. The main downstream target in this context was the activation status of CDK2, which was suppressed with CDK4/6 inhibition in an RB-dependent fashion. Protein levels of p27 were associated with plasticity/rigidity of the cell cycle and correlated with sensitivity to CDK4/6 inhibition. Exogenous overexpression and pharmacologic induction of p27 via inhibition of SKP2 and targeting the MEK/ERK pathway enhanced the cytostatic effect of CDK4/6 inhibitors. Mice bearing ER + xenografts displayed a durable antitumor response to palbociclib; however, over the course of treatment, few cells retained RB phosphorylation, which was associated with limited p27 protein levels as determined by multispectral imaging. Similarly, combination treatment of palbociclib with a MEK inhibitor in pancreatic cancer PDX models upregulated p27 and further enhanced the in vivo tumor response to palbociclib. Collectively, these results suggest that the cell cycle plasticity, which enables tumor models to evade palbociclib-mediated activation of RB, could be targeted using a clinically applicable CDK2 inhibitor. SIGNIFICANCE: This work provides a mechanistic insight toward understanding the functional roles of multiple cell cycle regulators that drive plasticity and sensitivity to CDK4/6 inhibition., (©2020 American Association for Cancer Research.)

Breast cancer (BC) is the most common cause of cancer-related death in women worldwide. Therapies targeting molecular pathways altered in BC had significantly enhanced treatment options for BC over the last decades, which ultimately improved the lives of millions of women worldwide. Among various molecular pathways accruing substantial interest for the development of targeted therapies are cyclin-dependent kinases (CDKs)-in particular, the two closely related members CDK4 and CDK6. CDK4/6 inhibitors indirectly trigger the dephosphorylation of retinoblastoma tumor suppressor protein by blocking CDK4/6, thereby blocking the cell cycle transition from the G1 to S phase. Although the CDK4/6 inhibitors abemaciclib, palbociclib, and ribociclib gained FDA approval for the treatment of hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative BC as they significantly improved progression-free survival (PFS) in randomized clinical trials, regrettably, some patients showed resistance to these therapies. Though multiple molecular pathways could be mechanistically responsible for CDK4/6 inhibitor therapy resistance, one of the most predominant ones seems to be the fibroblast growth factor receptor (FGFR) pathway. FGFRs are involved in many aspects of cancer formation, such as cell proliferation, differentiation, and growth. Importantly, FGFRs are frequently mutated in BC, and their overexpression and/or hyperactivation correlates with CDK4/6 inhibitor resistance and shortened PFS in BC. Intriguingly, the inhibition of aberrant FGFR activity is capable of reversing the resistance to CDK4/6 inhibitors. This review summarizes the molecular background of FGFR signaling and discusses the role of aberrant FGFR signaling during cancer development in general and during the development of CDK4/6 inhibitor resistance in BC in particular, together with other possible mechanisms for resistance to CDK4/6 inhibitors. Subsequently, future directions on novel therapeutic strategies targeting FGFR signaling to overcome such resistance during BC treatment will be further debated.

Objective: Kaempferol has been reported to play an anti-tumor role in various human cancers, while its role in gallbladder cancer (GBC) is unclear., Materials and Methods: We found that kaempferol significantly inhibited the growth, invasion and migration, meanwhile induced apoptosis through cells arrested at G0/G1 phase of GBC cell lines, including GBC-SD and SGC996 cells in vitro., Results: Kaempferol promoted the release of cytochrome C from the mitochondria to cytoplasm, the activation of c-caspase-3 and c-caspase-9 and increased the expression levels of pro-apoptotic factor Bax, meanwhile decreased the expression levels of anti-apoptotic factor Bcl-2. In addition, the expression levels of CDK4, CDK6 and cyclin D1, which are members of the CDK4/CDK6/cyclin D1 signaling pathway, were also decreased by kaempferol. Moreover, kaempferol could efficiently prevent tumor progression of GBC in the xenograft in vivo., Conclusions: Our results demonstrated that kaempferol suppressed GBC progression through activation of the CDK4/CDK6/cyclin D1 signaling pathway, suggesting that it might be a potential anti-tumor agent for clinical treatment of GBC.

The t(8;21) translocation is the most common cytogenetic abnormality in acute myeloid leukemia (AML). Although t(8;21) AML patients have a relatively favorable prognosis, relapse is a frequent occurrence, underscoring the need to develop novel therapeutic approaches. Here, we showed that t(8;21) AML is characterized by frequent mutation and overexpression of CCND2. Analysis of 19 AML cell lines showed that t(8;21) AML cells had lower IC50 values for the selective CDK4/6 inhibitors palbociclib and abemaciclib than non-t(8;21) AML cells. CDK4/6 inhibitors caused cell cycle arrest at G1 phase and impaired cell proliferation in t(8;21) AML cells. CDK4/6 inhibition decreased MAP-ERK and PI3K-AKT-mTOR signaling pathway activity, induced LC3B-I to LC3B-II conversion, and enhanced autophagosome formation, suggesting autophagy induction. Treatment of t(8;21) AML cells with the autophagy inhibitors chloroquine (CQ) or LY294002 in combination with the CDK4/6 inhibitor abemaciclib significantly increased the percentage of apoptotic (Annexin V positive) cells, whereas CQ or LY294002 single treatment had no significant effects. The effectiveness of co-inhibiting CDK4/6 and autophagy was confirmed in primary t(8;21) AML cells. The results suggest that the combination of CDK4/6 and autophagy inhibitors had a synergistic effect on inducing apoptosis, suggesting a novel therapeutic approach for the treatment of t(8;21) AML.

BACKGROUNDAlthough CDK4/6 inhibitors are an established treatment for hormone receptor-positive, HER2-negative metastatic breast cancers, their benefit in other malignancies remains limited.METHODSWe investigated factors associated with clinical outcomes from CDK4/6 inhibitor-based therapy among patients with G1/S phase cell-cycle alterations (CDK4/6 amplifications, CCND1/2/3 amplifications, or CDKN2A/B alterations).RESULTSOverall, 2457 patients with diverse solid tumors that underwent clinical-grade, next-generation sequencing (182-465 genes) and therapy outcome of (non-breast cancer) patients treated with matched CDK4/6 inhibitors were analyzed. G1/S phase cell-cycle alterations occurred in 20.6% (507 of 2457) of patients; 99% of those patients (n = 501) harbored ≥1 characterized co-alteration (median, 4; range, 0-24). In 40 patients with G1/S phase cell-cycle alterations given CDK4/6 inhibitors as part of their regimen, significantly longer median progression-free survival (PFS) was observed when CDK4/6 inhibitor-based therapies matched a larger proportion of tumor alterations, often because CDK4/6 inhibitors were administered together with other drugs that were matched to genomic co-alterations, hence achieving a high matching score (high vs. low [≥50% vs. <50%] matching score, PFS, 6.2 vs. 2.0 months, P < 0.001 [n = 40] [multivariate]) and higher rate of stable disease ≥6 months or an objective response (57% vs. 21%, P = 0.048).CONCLUSIONIn summary, in cell-cycle-altered cancers, matched CDK4/6 inhibitors, as part of an individualized regimen targeting a majority of genomic alterations, was independently associated with longer PFS.TRIAL REGISTRATIONClinicalTrials.gov NCT02478931.FUNDINGJoan and Irwin Jacobs Fund, National Cancer Institute (P30 CA023100, R01 CA226803), and the FDA (R01 FD006334).

Cyclin-dependent kinase (CDK) 4/6 inhibitors have emerged in the treatment of metastatic hormone receptor (HR)-positive and human epidermal growth factor receptor 2 (HER2)-negative breast cancer. However, most patients will eventually present disease progression, highlighting the inevitable resistance of cancer cells to CDK4/6 inhibition. Several studies have suggested that resistance mechanisms involve aberrations of the molecules that regulate the cell cycle, and the re-wiring of the cell to escape CDK4/6 dependence and turn to alternative pathways. Loss of retinoblastoma function, overexpression of CDK 6, upregulation of cyclin E, overexpression of CDK 7, and dysregulation of several signaling pathways, notably the PI3/AKT/mTOR pathway, have been implicated in the development of resistance to CDK4/6 inhibitors. Overlap with endocrine resistance mechanisms might be possible. Combinational therapeutic strategies should be explored in order to prevent resistance and optimize the management of patients after progression under CDK 4/6 inhibition.

Psoriasis is a frequent, inflammatory skin disease characterized by keratinocyte hyperproliferation and a disease-related infiltration of immune cells. Here, we identified a novel proinflammatory signaling pathway driven by cyclin-dependent kinase 4 (CDK4) and CDK6 and the methyltransferase EZH2 as a valid target for psoriasis therapy. Delineation of the pathway revealed that CDK4/6 phosphorylated EZH2 in keratinocytes, thereby triggering a methylation-induced activation of STAT3. Subsequently, active STAT3 resulted in the induction of IκBζ, which is a key proinflammatory transcription factor required for cytokine synthesis in psoriasis. Pharmacological or genetic inhibition of CDK4/6 or EZH2 abrogated psoriasis-related proinflammatory gene expression by suppressing IκBζ induction in keratinocytes. Importantly, topical application of CDK4/6 or EZH2 inhibitors on the skin was sufficient to fully prevent the development of psoriasis in various mouse models by suppressing STAT3-mediated IκBζ expression. Moreover, we found a hyperactivation of the CDK4/6-EZH2 pathway in human and mouse psoriatic skin lesions. Thus, this study not only identifies a novel psoriasis-relevant proinflammatory pathway, but also proposes the repurposing of CDK4/6 or EZH2 inhibitors as a new therapeutic option for patients with psoriasis.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly solid tumors in the world. Aerobic glycolysis is among the characteristic features of pancreatic cancer. However, the regulatory process of aerobic glycolysis in pancreatic cancer is too complicated, and the underlying mechanism remains unexplained. Reportedly, CDK4/6 inhibitors repress breast cancer cell proliferation by modulating glucose metabolism. Here, we reveal that the CDK4/6 inhibitor, PD0332991 stabilized FBP1 to hinder aerobic glycolysis in pancreatic cancer. We also show that the CDK4/6-E2 F1 signaling pathway mediated an increase in MAGED1 expression, promoting FBP1 degradation in pancreatic cancer. We, therefore, might have identified a novel mechanism by which the CDK4/6 inhibitor, PD0332991 blocks the Warburg effect of pancreatic cancer by stabilizing FBP1., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Cell-cycle entry relies on an orderly progression of signaling events. To start, cells first activate the kinase cyclin D-CDK4/6, which leads to eventual inactivation of the retinoblastoma protein Rb. Hours later, cells inactivate APC/C CDH1 and cross the final commitment point. However, many cells with genetically deleted cyclin Ds, which activate and confer specificity to CDK4/6, can compensate and proliferate. Despite its importance in cancer, how this entry mechanism operates remains poorly characterized, and whether cells use this path under normal conditions remains unknown. Here, using single-cell microscopy, we demonstrate that cells with acutely inhibited CDK4/6 enter the cell cycle with a slowed and fluctuating cyclin E-CDK2 activity increase. Surprisingly, with low CDK4/6 activity, the order of APC/C CDH1 and Rb inactivation is reversed in both cell lines and wild-type mice. Finally, we show that as a consequence of this signaling inversion, Rb inactivation replaces APC/C CDH1 inactivation as the point of no return. Together, we elucidate the molecular steps that enable cell-cycle entry without CDK4/6 activity. Our findings not only have implications in cancer resistance, but also reveal temporal plasticity underlying the G1 regulatory circuit.

Cell cycle dysregulation caused by aberrant cyclin D1 and CDK4 expression is a major determinant for proliferation of cancer cells in mantle cell lymphoma (MCL). Inhibition of CDK4/6 induces G1 arrest of MCL cells in patients, appearing to deepen and prolong the clinical response to partner agents. This article reviews aberrations of cell cycle genes in MCL cells and clinical trials of CDK4/6 inhibitors for MCL. Integrative longitudinal functional genomics is discussed as a strategy to discover genomic drivers for resistance in cancer cells and cancer-immune interactions that potentially contribute to the clinical response to palbociclib combination therapy in MCL., Competing Interests: Conflicts of interest The authors have no conflicts of interest to declare., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Palbociclib, ribociclib, and abemaciclib belong to the third generation of cyclin-dependent kinases inhibitors (CDKis), an established therapeutic class for advanced and metastatic breast cancer. Interindividual variability in the therapeutic response of CDKis has been reported and some individuals may experience increased and unexpected toxicity. This narrative review aims at identifying the factors potentially concurring at this variability for driving the most appropriate and tailored use of CDKis in the clinic. Specifically, concomitant medications, pharmacogenetic profile, and pathophysiological conditions could influence absorption, distribution, metabolism, and elimination pharmacokinetics. A personalized therapeutic approach taking into consideration all factors potentially contributing to an altered pharmacokinetic/pharmacodynamic profile could better drive safe and effective clinical use.

Laboratory research and pharmacoepidemiology provide support for metformin as a potential antitumor agent. However, the lack of a clear understanding of the indications of metformin limits its efficacy. Here, we performed a genome-wide CRISPR knockout negative screen to identify potential targets that might synergize with metformin. Next-generation sequencing of pooled genomic DNAs isolated from surviving cells after 18 days of metformin treatment (T18) compared to those of the untreated cells at day 0 (T0) yielded candidate genes. Knockdown of a group of cyclin-dependent kinases (CDKs), including CDK1, CDK4, and CDK6, confirmed the results of the screen. Combination treatment of the CDKs inhibitor abemaciclib with metformin profoundly inhibited tumor viability in vitro and in vivo. Although cell cycle parameters were not further altered under the combination treatment, investigation of the metabolome revealed significant changes in cell metabolism, especially with regard to fatty acid oxidation, the tricarboxylic acid cycle and aspartate metabolism. Such changes appeared to be mediated through inhibition of the mTOR pathway. Collectively, our study suggests that the combination of CDKs inhibitor with metformin could be recognized as a potential therapy in future clinical applications.

Lysosomes are degradation and signaling organelles that adapt their biogenesis to meet many different cellular demands; however, it is unknown how lysosomes change their numbers for cell division. Here, we report that the cyclin-dependent kinases CDK4/6 regulate lysosome biogenesis during the cell cycle. Chemical or genetic inactivation of CDK4/6 increases lysosomal numbers by activating the lysosome and autophagy transcription factors TFEB and TFE3. CDK4/6 interact with and phosphorylate TFEB/TFE3 in the nucleus, thereby inactivating them by promoting their shuttling to the cytoplasm. During the cell cycle, lysosome numbers increase in S and G2/M phases when cyclin D turnover diminishes CDK4/6 activity. These findings not only uncover the molecular events that direct the nuclear export of TFEB/TFE3, but also suggest a mechanism that controls lysosome biogenesis in the cell cycle. CDK4/6 inhibitors promote autophagy and lysosome-dependent degradation, which has important implications for the therapy of cancer and lysosome-related disorders., (© 2020 Yin et al.)

Background: Germ cell tumours (GCTs) are the most common solid malignancies in young men. Although high cure rates can be achieved, metastases, resistance to cisplatin-based therapy and late toxicities still represent a lethal threat, arguing for the need of new therapeutic options. In this study, we analysed the potential of cyclin-dependent kinase 4/6 (CDK4/6) inhibitors palbociclib and ribociclib (PaRi) as molecular drugs to treat cisplatin-resistant and -sensitive paediatric and adult GCTs., Methods: Ten GCT cell lines, including cisplatin-resistant subclones and non-malignant controls, were treated with PaRi and screened for changes in viability (triphenyl tetrazolium chloride (XTT) assay), apoptosis rates (flow cytometry, caspase assay), the cell cycle (flow cytometry), the transcriptome (RNA-sequencing, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and on protein level (western blot). Expression profiling was performed on paediatric and adult GCT tissues (expression microarrays, qRT-PCR, immunohistochemistry, 'The Cancer Genome Atlas' database)., Results: We demonstrate that adult GCTs highly express CDK4, while paediatric GCTs strongly express CDK6 instead. Thus, both GCT types are potentially treatable by PaRi. GCTs presented as highly sensitive towards PaRi, which caused a decrease in viability, cell cycle arrest and apoptosis. Although GCTs mainly arrested in the G1/G0 phase, some embryonal carcinoma cell lines were able to bypass the G1/S checkpoint and progressed to the G2/M phase. We found that upregulation of CDK3 and downregulation of many mitosis regulation factors, like the HAUS genes, might be responsible for bypassing the G1/S checkpoint and termination of mitosis, respectively. We postulate that GCT cells do not tolerate these alterations in the cell cycle and eventually induce apoptosis., Conclusion: Our study highlights PaRi as therapeutic options for cisplatin-resistant and -sensitive paediatric and adult GCTs.

Cyclin-dependent kinases 4 and 6 (CDK4/6) phosphorylate and inhibit retinoblastoma (RB) family proteins. Hyperphosphorylated RB releases E2F transcription factors, activating a transcriptional program that initiates S phase. Due to the critical role that this pathway has in regulating cell cycle progression, inhibiting CDK4/6 is an attractive therapeutic strategy. Indeed, CDK4/6 inhibitors in combination with antiestrogens produce a significant benefit in patients with ER + /HER2 - breast cancer. Clinical trials are currently investigating if the use of CDK4/6 inhibitors alone or in combination can be extended to other cancer types. Inhibition of CDK4/6 can result in different cell fates such as quiescence, senescence, or apoptosis. Senescence is a stress response that can be induced by stimuli that include oncogenic activation, chemotherapy, irradiation, and targeted therapies such as CDK4/6 inhibitors. Senescent cells undergo a stable cell cycle arrest and produce a bioactive secretome that remodels their microenvironment and engages the immune system. In this review, we analyze the therapeutic relevance of senescence induction by CDK4/6 inhibitors. We also discuss how different therapies, including checkpoint inhibitors and drugs targeting MEK or PI3K, can be used in combination with CDK4/6 inhibitors to reinforce or exploit senescence. Recently, a lot of effort has been put into identifying compounds that selectively kill senescent cells (termed senolytics). Thus, sequential treatment with senolytics might be an additional strategy to potentiate the antitumor effects of CDK4/6 inhibitors.

We describe a potential role for thymidine kinase-1, a general marker of cellular proliferation, to act as a prognostic biomarker in patients receiving CDK4/6 inhibitors for advanced hormone receptor-positive, HER2-negative breast cancer, with early data suggesting that it may also provide early indication of treatment response.

Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors, which block the transition from the G1 to S phase of the cell cycle by interfering with Rb phosphorylation and E2F release, have shown potent antitumor activity and manageable toxicity in HR+/HER2- breast cancer patients. Some clinical trials involving CDK4/6 inhibitors in other tumors have achieved preliminary impressive efficacy. Whether CDK4/6 inhibitors possess great potential as broad-spectrum antitumor drugs and how to maximize their clinical benefits remain uncertain. TCGA database analysis showed that CDK4/6 genes and related genes are widely expressed among various tumors, and high or moderate expression of CDK4/6 genes commonly indicates poor survival. CDK4/6 gene expression is significantly higher in COAD, ESCA, STAD, LIHC, and HNSC, suggesting that CDK4/6 inhibitors could be more efficacious in those tumors. Moreover, network analysis with the STRING database demonstrated that CDK4/6-related proteins were co-expressed or co-occurred with the classical tumor signaling pathways, such as the cell cycle pathway, RAS pathway, PI3K pathway, Myc pathway, and p53 pathway. The extensive antitumor effects of CDK4/6 inhibitors may be achieved by synergizing or antagonizing with other signaling molecule inhibitors, and combination therapy might be the most effective treatment strategy. This article analyzed the feasibility of expanding the application of CDK4/6 inhibitors at the genetic level and further summarized the associated clinical/preclinical studies to collect supportive evidence. This is the first study that presents a theoretical foundation for CDK4/6 inhibitor precision therapy via combined analysis of comprehensive gene information and clinical research results.