Oncogenic YAP sensitizes cells to CHK1 inhibition via CDK4/6 driven G1 acceleration
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PROVIDER: PRJNA1259509 | ENA |
REPOSITORIES: ENA
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Project description:Replication stress is a driver of genomic instability, contributing to carcinogenesis by causing DNA damage and mutations. While YAP, the downstream co-activator of the Hippo signaling pathway, plays a crucial role in regulating cell growth and differentiation, it is unclear whether it generates replication stress exploitable for therapy. Here, we report that oncogenic YAP shortens the G1 phase through increased CDK4/6 activity, leading to early S-phase entry. This causes origin under-licensing, an overall reduced rate of DNA replication, and, untypically, an accelerated speed of individual replication forks. CHK1 inhibition in cells expressing oncogenic YAP results in DNA damage during S-phase, which is not due to premature CDK1 activation or mitotic entry. Sensitivity to CHK1 inhibition depends on the YAP-TEAD interaction and involves a global increase in transcription and an increase in transcription-replication conflicts (TRCs). Replication stress from oncogenic YAP can be mitigated by restoring G1 length through partial CDK4/6 inhibition or by reducing YAP-induced hypertranscription. Our findings suggest a potential therapeutic strategy for targeting YAP-dependent cancers by exploiting their vulnerability to replication stress.
2025-07-01 | GSE296501 | GEO
Project description:The retinoblastoma tumor suppressor protein (Rb) regulates early G1 phase checkpoints, including the DNA damage response, as well as cell cycle exit and differentiation. The widely accepted model of G1 cell cycle progression proposes that cyclin D:Cdk4/6 partially inactivates the Rb tumor suppressor during early G1 phase by progressive multi-phosphorylation, termed hypo-phosphorylation, resulting in release of E2F transcription factors. However, this model remains largely unproven biochemically and the biologically active form(s) of Rb remains unknown. Here we find that Rb is un-phosphorylated in G0 cells and becomes exclusively mono-phosphorylated throughout all of early G1 phase by cyclin D:Cdk4/6. Early G1 phase mono-phosphorylated Rb is composed of 14 independent isoforms that are all targeted by the E1a oncoprotein, but each shows a preferential binding pattern to specific E2F1-4 transcription factors. At the late G1 Restriction Point, cyclin E:Cdk2 inactivates Rb by a quantum hyper-phosphorylation (>12 phosphates/Rb). Cells undergoing a DNA damage response activate cyclin D:Cdk4/6 to generate mono-phosphorylated Rb that regulates global transcription. In contrast, a non-phosphorylatable ?Cdk-Rb allele was non-functional for regulating a DNA damage response, but functional for driving cell cycle exit and differentiation during myogenesis. These observations fundamentally change our understanding of G1 cell cycle progression and show that there is no progressive multi-phosphorylation or hypo-phosphorylation inactivation of Rb during early G1 phase by cyclin D:Cdk4/6. Instead, cyclin D:Cdk4/6 generates functionally active, mono-phosphorylated Rb that is the only Rb isoform present in cells during early G1 phase. Global transcriptional analysis of murine embryonic fibroblasts (MEFs) with conditional deletion of the endogenous RB gene by treatment with cell permeable TAT-Cre. Comparison to unaltered MEFs and MEFs with physiological level of exogenous wildtype or phospho-mutant RB expressed at time of RB gene deletion.
2014-04-03 | E-GEOD-56453 | biostudies-arrayexpress
Project description:By selective and reversible inhibition of CDK4/CDK6, we have developed a strategy to both inhibit proliferation and enhance cytotoxic killing of cancer cells. Induction of prolonged early-G1 arrest (pG1) by CDK4/CDK6 inhibition halts gene expression in early-G1 and prevents expression of genes programmed for other cell cycle phases. S-phase synchronization upon removal of the early-G1 block (pG1-S) fails to completely restore scheduled gene expression. Consequently, coordinate loss of IRF4 and gain of Bim and Noxa expression sensitize myeloma tumor cells to bortezomib-induced apoptosis in pG1 and more profoundly in pG1-S in vitro. Induction of pG1 and pG1-S by CDK4/CDK6 inhibition augments tumor-specific bortezomib killing in myeloma xenografts. Inhibition of CDK4/CDK6 in combination therapy thus represents a novel mechanism-based cancer therapy. PD 0332991 (PD) is the only known specific and reversible CDK4/CDK6 inhibitor. Gene expression was measured in myeloma MM1.S cells treated with PD (0.25 uM) in triplicate for 12, 24 or 36 h, or in cells released from G1, induced by 24hPD, for 4 or 18 h.
2012-04-06 | E-GEOD-35728 | biostudies-arrayexpress
Project description:The retinoblastoma tumor suppressor protein (Rb) regulates early G1 phase checkpoints, including the DNA damage response, as well as cell cycle exit and differentiation. The widely accepted model of G1 cell cycle progression proposes that cyclin D:Cdk4/6 partially inactivates the Rb tumor suppressor during early G1 phase by progressive multi-phosphorylation, termed hypo-phosphorylation, resulting in release of E2F transcription factors. However, this model remains largely unproven biochemically and the biologically active form(s) of Rb remains unknown. Here we find that Rb is un-phosphorylated in G0 cells and becomes exclusively mono-phosphorylated throughout all of early G1 phase by cyclin D:Cdk4/6. Early G1 phase mono-phosphorylated Rb is composed of 14 independent isoforms that are all targeted by the E1a oncoprotein, but each shows a preferential binding pattern to specific E2F1-4 transcription factors. At the late G1 Restriction Point, cyclin E:Cdk2 inactivates Rb by a quantum hyper-phosphorylation (>12 phosphates/Rb). Cells undergoing a DNA damage response activate cyclin D:Cdk4/6 to generate mono-phosphorylated Rb that regulates global transcription. In contrast, a non-phosphorylatable ?Cdk-Rb allele was non-functional for regulating a DNA damage response, but functional for driving cell cycle exit and differentiation during myogenesis. These observations fundamentally change our understanding of G1 cell cycle progression and show that there is no progressive multi-phosphorylation or hypo-phosphorylation inactivation of Rb during early G1 phase by cyclin D:Cdk4/6. Instead, cyclin D:Cdk4/6 generates functionally active, mono-phosphorylated Rb that is the only Rb isoform present in cells during early G1 phase.
2014-04-03 | GSE56453 | GEO
Project description:By selective and reversible inhibition of CDK4/CDK6, we have developed a strategy to both inhibit proliferation and enhance cytotoxic killing of cancer cells. Induction of prolonged early-G1 arrest (pG1) by CDK4/CDK6 inhibition halts gene expression in early-G1 and prevents expression of genes programmed for other cell cycle phases. S-phase synchronization upon removal of the early-G1 block (pG1-S) fails to completely restore scheduled gene expression. Consequently, coordinate loss of IRF4 and gain of Bim and Noxa expression sensitize myeloma tumor cells to bortezomib-induced apoptosis in pG1 and more profoundly in pG1-S in vitro. Induction of pG1 and pG1-S by CDK4/CDK6 inhibition augments tumor-specific bortezomib killing in myeloma xenografts. Inhibition of CDK4/CDK6 in combination therapy thus represents a novel mechanism-based cancer therapy.
2012-04-07 | GSE35728 | GEO
Project description:MEK5/ERK5 inhibition sensitizes NRAS-mutant melanoma to MAPK-targeted therapy by preventing Cyclin D/CDK4-mediated G1/S progression
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Project description:Despite the advent of immune-oncological therapies, patients with advanced NRAS-mutant melanoma still have a significantly worse prognosis than their BRAF-mutant counterparts. This is mainly due to a high propensity for resistance to available therapies targeting the RAS/RAF/MEK/ERK mitogen-activated protein kinase (MAPK) pathway (MAPKi). Preclinical studies and mouse models have implicated the stress-activated MEK5/ERK5 MAPK cascade as a major resistance pathway activated by MAPKi-based targeted therapy in NRAS-mutant melanoma. Accordingly, MAPKi/ERK5i co-inhibition was capable of triggering a sustained cell cycle arrest in NRAS-mutant melanoma cells, but the key mediator(s) of its vigorous anti-proliferative effect remain elusive. Here, we further investigated the mechanism of MAPKi/ERK5i-induced cell cycle arrest in NRAS-mutant melanoma cells using both genetic methods and pharmacological inhibitors. Transcriptome analysis of human NRAS-mutant melanoma cells established that MAPKi/ERK5i-induced a near-complete shutdown of the mitotic machinery as consequence of a sustained G1 cell cycle arrest. This arrest was not only observed in diverse treatment-naïve melanoma cells but could also be induced in cells that already had developed resistance to therapeutic MEK inhibition (MEKi) and was accompanied by suppression of Cyclin D1 and E2F-mediated gene expression. Forced expression of Cyclin D1 and its effector kinase CDK4 restored cell cycle progression and mitotic gene expression in NRAS-mutant melanoma cells exposed to MEKi/ERK5i, implying Cyclin D/CDK4 activity as major target of combined MEKi/ERK5i. These findings suggest Cyclin D/CDK4 dependency as a major vulnerability of NRAS-mutant melanoma that could effectively be targeted by combined MAPKi/ERK5i.
2025-09-01 | GSE300191 | GEO
Project description:Splicing factor SF3B1 mutations are frequent somatic lesions in myeloid neoplasms that transform hematopoietic stem cells (HSCs) by inducing mis-splicing of target genes. However, the molecular and functional consequences of SF3B1 mutations in human HSCs remain unclear. Here, we identify the mis-splicing program in human HSCs as a targetable vulnerability by precise gene editing of SF3B1 K700E mutations in primary CD34+ cells. Mutant SF3B1 induced pervasive mis-splicing and reduced expression of genes regulating mitosis in single cell transcriptomics, critically BUBR1 and CDC27, leading to altered differentiation and delayed G2/M progression. Mutant SF3B1 mis-splicing or reduced expression of BUBR1 and CDC27 was sufficient to delay G2/M transit, leading to activation of CHK1, sensitizing cells to CHK1 inhibition. Clinical CHK1 inhibitor prexasertib selectively targeted SF3B1-mutant HSCs and abrogated engraftment in vivo. These findings identify mis-splicing of mitotic regulators in SF3B1-mutant HSCs as a targetable vulnerability engaged by pharmacological CHK1 inhibition.
2024-05-29 | GSE263300 | GEO
Project description:Splicing factor SF3B1 mutations are frequent somatic lesions in myeloid neoplasms that transform hematopoietic stem cells (HSCs) by inducing mis-splicing of target genes. However, the molecular and functional consequences of SF3B1 mutations in human HSCs remain unclear. Here, we identify the mis-splicing program in human HSCs as a targetable vulnerability by precise gene editing of SF3B1 K700E mutations in primary CD34+ cells. Mutant SF3B1 induced pervasive mis-splicing and reduced expression of genes regulating mitosis in single cell transcriptomics, critically BUBR1 and CDC27, leading to altered differentiation and delayed G2/M progression. Mutant SF3B1 mis-splicing or reduced expression of BUBR1 and CDC27 was sufficient to delay G2/M transit, leading to activation of CHK1, sensitizing cells to CHK1 inhibition. Clinical CHK1 inhibitor prexasertib selectively targeted SF3B1-mutant HSCs and abrogated engraftment in vivo. These findings identify mis-splicing of mitotic regulators in SF3B1-mutant HSCs as a targetable vulnerability engaged by pharmacological CHK1 inhibition.
2024-05-29 | GSE263299 | GEO
Project description:Genomic aberrations of Cyclin D1 (CCND1) and CDK4 in neuroblastoma indicate that dysregulation of the G1 entry checkpoint is an important cell cycle aberration in this pediatric tumor. Here we report that analysis of Affymetrix expression data of primary neuroblastic tumors shows an extensive over-expression of Cyclin D1 and CDK4 which correlates with histological subgroups and prognosis respectively. Immunohistochemical analysis demonstrated an over-expression of Cyclin D1 in neuroblasts and a low Cyclin D1 expression in all cell types in ganglioneuroma. This suggests an involvement of G1 regulating genes in neuronal differentiation processes which we further evaluated using RNA interference against Cyclin D1 and its kinase partner CDK4 in several neuroblastoma cell lines. This resulted in pRb pathway inhibition as shown by an almost complete disappearance of CDK4 specific pRb phosphorylation; reduction of E2F transcriptional activity and a decrease of Cyclin A protein levels. The Cyclin D1 and CDK4 knock-down resulted in a significant reduction in cell proliferation, a G1 specific cell cycle arrest and moreover an extensive neuronal differentiation. Affymetrix microarray profiling of siRNA treated cells revealed a shift in expression profile towards a neuronal phenotype. Several new potential downstream players are identified. We conclude that neuroblastoma functionally depend on over-expression of G1 regulating genes to maintain their undifferentiated phenotype. Experiment Overall Design: The Cell line IMR-32 at time point 0 and transiently transfected with siRNA against GFP, Cyclin D1 and CDK4 at time point 48 hours. All experiments are biological triplicates.
2008-10-25 | E-GEOD-8866 | biostudies-arrayexpress