Inhibition of GPX4 enhances CDK4/6 inhibitor activity in breast cancer.
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion Lumos
ORGANISM(S): Homo Sapiens (human)
SUBMITTER: 
Graeme Benstead-Hume
LAB HEAD: Jyoti Choudhary
PROVIDER: PXD043133 | Pride | 2023-06-20
REPOSITORIES: Pride
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Project description:Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors are approved for breast cancer treatment and show activity against other malignancies, including KRAS-mutant non-small cell lung cancer (NSCLC). However, the clinical efficacy of CDK4/6 inhibitors is limited due to frequent drug resistance and their largely cytostatic effects. Through a genome-wide cDNA screen, we identified that bromodomain-containing protein 4 (BRD4) overexpression conferred resistance to the CDK4/6 inhibitor palbociclib in KRAS-mutant NSCLC cells. Inhibition of BRD4, either by RNA interference or small-molecule inhibitors, synergized with palbociclib to induce senescence in NSCLC cells and tumors, and the combination prolonged survival in a KRAS-mutant NSCLC mouse model. Mechanistically, BRD4-inhibition enhanced cell cycle arrest and reactive oxygen species (ROS) accumulation, both of which are necessary for senescence induction; this in turn elevated GPX4, a peroxidase that suppresses ROS-triggered ferroptosis. Consequently, GPX4 inhibitor treatment selectively induced ferroptotic cell death in the senescent cancer cells, resulting in tumor regression. Co-targeting CDK4/6 and BRD4 also promoted senescence and ferroptosis vulnerability in pancreatic and breast cancer cells. Together, these findings reveal therapeutic vulnerabilities and effective combinations to enhance the clinical utility of CDK4/6 inhibitors.
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Project description:RNA-seq was performed to compare the transcriptional programmes of A375 cells treated with Palbociclib or GSK3326595 compared to untreated cells Cyclin dependent kinase 4/6 (CDK4/6) inhibitors are an established treatment in estrogen receptor positive breast cancer and are currently in clinical development in melanoma; a tumour that exhibits high rates of CDK4 activation. We analyzed melanoma cells with acquired resistance to the CDK4/6 inhibitor palbociclib, and demonstrated that palbociclib-mediated inhibition of PRMT5 is essential for sensitivity to CDK4/6 inhibitors. Mechanistically, by inhibiting PRMT5 activity, palbociclib alters MDM4 pre-mRNA splicing leading to decreased MDM4 protein expression and subsequent p53 activation. In turn, p53 increases p21 leading to inhibition of CDK2, the main kinase substituting for CDK4/6 and a key driver of resistance to palbociclib. Loss of the ability of palbociclib to regulate the PRMT5-MDM4 axis leads to resistance. Importantly, combining palbociclib with the PRMT5 inhibitor GSK3326595 enhances the efficacy of palbociclib in treatment naïve and resistant models and also delays the emergence of resistance. Our studies have uncovered a novel mechanism of action of CDK4/6 inhibitors in regulating the MDM4 oncogene and the tumor suppressor, p53. Furthermore, we have established that palbociclib inhibition of the PRMT5-MDM4 axis is essential for robust melanoma cell sensitivity and provide pre-clinical evidence that co-inhibition of CDK4/6 and PRMT5 is an effective and well tolerated therapeutic strategy. Overall our data provides a strong rationale for further investigation of novel combinations of CDK4/6 and PRMT5 inhibitors in not only melanoma but other tumour types including breast, pancreatic and esophageal carcinoma.
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Project description:RNA-seq was performed to compare the transcriptional programmes of palbociclib-resistant A375 and CHL1 cells compared to their parental counterparts Cyclin dependent kinase 4/6 (CDK4/6) inhibitors are an established treatment in estrogen receptor positive breast cancer and are currently in clinical development in melanoma; a tumour that exhibits high rates of CDK4 activation. We analyzed melanoma cells with acquired resistance to the CDK4/6 inhibitor palbociclib, and demonstrated that palbociclib-mediated inhibition of PRMT5 is essential for sensitivity to CDK4/6 inhibitors. Mechanistically, by inhibiting PRMT5 activity, palbociclib alters MDM4 pre-mRNA splicing leading to decreased MDM4 protein expression and subsequent p53 activation. In turn, p53 increases p21 leading to inhibition of CDK2, the main kinase substituting for CDK4/6 and a key driver of resistance to palbociclib. Loss of the ability of palbociclib to regulate the PRMT5-MDM4 axis leads to resistance. Importantly, combining palbociclib with the PRMT5 inhibitor GSK3326595 enhances the efficacy of palbociclib in treatment naïve and resistant models and also delays the emergence of resistance. Our studies have uncovered a novel mechanism of action of CDK4/6 inhibitors in regulating the MDM4 oncogene and the tumor suppressor, p53. Furthermore, we have established that palbociclib inhibition of the PRMT5-MDM4 axis is essential for robust melanoma cell sensitivity and provide pre-clinical evidence that co-inhibition of CDK4/6 and PRMT5 is an effective and well tolerated therapeutic strategy. Overall our data provides a strong rationale for further investigation of novel combinations of CDK4/6 and PRMT5 inhibitors in not only melanoma but other tumour types including breast, pancreatic and esophageal carcinoma.
2019-08-23 | GSE133567 | GEO
Project description:CDK4/6 inhibitor, Palbociclib, has shown a high success in breast cancer subtypes however it is often accompanied by a dependency on mutation status of cell lines. Consequently, there has been speculation on the breast cancer subtypes which should be offered treatment with specific CDK4/6 inhibitors and the response that can be expected. A common observation has been the induction of a cell programme known as "senescence" whereby cells experience a complete halt in mitosis, however, remaining metabolically active. Therapy-induced senescence markers and its effect on the tumour microenvironment as well as the diversity of this cell state remain unappreciated. In the following proteomics, phosphoproteomics and secretome datasets we focus on MDA-MB-231 cells, a triple negative cell line, to better understand the senescence-like cell state which arises after treatment. Ultimately, the dataset encompasses the population that arises upon Palbociclib treatment and highlights potential signatures and biomarkers of the cell state which could provide information on the use of CDK4/6 inhibitors in triple negative breast cancer subtypes.
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Project description:Intrinsic or acquired resistance to clinically approved CDK4/6 inhibitors have emerged as a major obstacle that hinders their utility beyond ER+ breast cancer. Palbociclib-sensitive ER+ breast cancer models and pancreatic ductal adenocarcinoma (PDAC) models were employed to identify functional determinants of response. 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 setting, RB loss renders cells completely CDK4/6 independent. The main down-stream target in this context is the activation status of CDK2 which is suppressed with CDK4/6 inhibition in an RB-dependent fashion. The P27KIP1 protein levels are associated with plasticity/rigidity of the cell cycle and correlates with sensitivity to CDK4/6 inhibiion. Exogenous overexpression and pharmacological induction of P27KIP1 by targeting the MEK/ERK pathway enhances the cytostatic effect of palbociclib. Similar to cell-culture data, in vivo experiments revealed that combination treatment of palbociclib and trametinib in mice bearing PDAC PDXs elicited a robust anti-proliferative effect with a corresponding increase in p27 expression. Mice bearing MCF7 xenografts displayed a durable response in the presence of palbociclib; however, over the course of treatment few cells begin to evade the negative cell-cycle regulation. Based on multispectral staining, the MCF7 tumor cells that underwent RB inactivation in the presence of palbociclib harbored low p27 expression. Finally, we demonstrate that the cell-cycle plasticity that enables tumor models to evade the palbociclib mediated RB activation could be potently targeted using a clinically applicable CDK2 inhibitor.
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Project description:Ferroptosis is associated with lipid hydroperoxides generated by oxidation of polyunsaturated acyl chains. Lipid hydroperoxides are reduced by glutathione peroxidase 4 (GPX4) and GPX4 inhibitors induce ferroptosis. However, the therapeutic potential of triggering ferroptosis in cancer cells with polyunsaturated fatty acids is unknown. We identified conjugated linoleates including α-eleostearic acid (αESA) as novel ferroptosis inducers. αESA did not alter GPX4 activity but was incorporated into cellular lipids and promoted lipid peroxidation and cell death in diverse cancer cell types. αESA-triggered death was mediated by acyl-CoA synthetase long-chain isoform 1, which promoted αESA incorporation into neutral lipids including triacylglycerols. Interfering with triacylglycerol biosynthesis suppressed ferroptosis triggered by αESA but not by GPX4 inhibition. Orally administered tung oil, naturally rich in αESA, limited tumor growth and metastasis with transcriptional changes consistent with ferroptosis. Overall, these findings illuminate a novel approach to ferroptosis, complementary to GPX4 inhibition, with therapeutic potential.
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Project description:Ferroptosis is a regulated form of necrotic cell death caused by iron-dependent phospholipid peroxidation. It can be induced by inhibiting glutathione peroxidase 4 (GPX4), the key enzyme for efficiently reducing peroxides within phospholipid bilayers. Recent data suggest that cancer cells undergoing EMT (dedifferentiation) and those resistant to standard therapy expose a high vulnerability toward ferroptosis. Although recent studies have begun to identify and characterize the metabolic and genetic determinants underlying ferroptosis, many mechanisms that dictate ferroptosis sensitivity remain unknown. Here, we show that low cell density sensitizes primary mammary epithelial and breast cancer cells to ferroptosis induced by GPX4 inhibition, whereas high cell density confers resistance. These effects occur irrespective of oncogenic signaling, cellular phenotype and expression of the fatty acid ligase acyl-CoA synthetase long chain family member 4 (ACSL4). By contrast, we show that a massive accumulation of neutral triacylglycerides (TAG) enriched with polyunsaturated fatty acids (PUFA) is induced at low cell density. In addition, de novo lipogenesis and desaturation pathways were found to be reduced at low cell density, indicative of increased fatty acid uptake. Our study suggests that PUFA-mediated toxicity is limited by the enrichment in TAGs that in turn might pose a vulnerability towards ferroptosis. Conclusively, cell density regulates lipid metabolism of breast epithelial and cancer cells, which results in a ferroptosis-sensitive cell state with the potential to be exploited therapeutically during metastatic dissemination.
2020-11-13 | PXD016276 | Pride
Project description:One potential resistance mechanism to CDK4/6 inhibitors in breast cancer could be induction of cellular senescence in normal host tissues. The aim of our study was to identify CDK4/6 inhibition-induced changes to host tissues that impact metastasis. Using mouse models, we found that pre-treatment with palbociclib can increase metastatic seeding of mouse mammary cancer cells in the lungs and that this can be mitigated by eliminating senescent host cells. We describe palbociclib-induced gene expression changes in lungs that correlate with this effect and reveal altered intra-lung immune populations. Senescent endothelial cells are identifiable within lung metastases of mice pre-treated with palbociclib. Using in vitro models, we show that palbociclib treatment increases endothelial cell senescence and affects macrophage invasion and migration. These studies describe how CDK4/6 inhibition-induced cellular senescence in host tissues affects metastasis and disease progression in breast cancer, which remain key obstacles to achieving long-term survival.
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Project description:CDK4/6 inhibitors are highly effective against ER+/HER2- breast cancer (BC); however, intrinsic and acquired resistance is common. Elucidating the molecular features of sensitivity and resistance to CDK4/6 inhibitors may lead to the identification of predictive biomarkers and novel therapeutic targets, paving the way toward improving patient outcomes. Parental BC cells and their endocrine-resistant derivatives (EndoR) were used in this study. Derivatives with acquired resistance to palbociclib (PalboR) were generated from parental and estrogen deprivation-resistant MCF7 and T47D cells. Transcriptomic and proteomic analyses were performed in palbociclib-sensitive and PalboR lines. We found that parental and EndoR BC lines showed varying degrees of sensitivity to palbociclib. Transcriptomic analysis of these cell lines identified an association between high Interferon (IFN) signaling and reduced sensitivity to CDK4/6 inhibitors, thus an ‘IFN-Related Palbociclib-Resistance Signature’ (IRPS) was derived. In two neoadjuvant trials of a CDK4/6 inhibitor plus endocrine therapy, IRPS and other IFN-related signatures were highly enriched in patients with tumors exhibiting intrinsic resistance to CDK4/6 inhibition. PalboR derivatives displayed a dramatic activation of IFN/STAT1-signaling compared to their short-term treated or untreated counterparts. In primary ER+/HER2- tumors, the IRPS score was significantly higher in lumB than lumA subtype and correlated with increased gene expression of immune checkpoints, endocrine resistance, and poor prognosis. Our study demonstrates that aberrant IFN-signaling is associated with intrinsic resistance to CDK4/6 inhibitors. Experimentally, acquired resistance to palbociclib is associated with activation of the IFN-pathway, which warrants additional studies to clarify its involvement in resistance to CDK4/6 inhibitors.
2021-12-04 | GSE150997 | GEO
Project description:Malignant pleural mesothelioma (MPM) is an aggressive cancer with limited therapeutic options. In this study, we evaluated the impact of CDK4/6 inhibition by palbociclib in established MPM cell lines using a variety of approaches including RNA sequencing. Palbociclib used alone sufficed to strongly and durably inhibit the proliferation of 4 of 6 cell lines. In a parallel study, the efficacy of palbociclib was confirmed in a large panel of patient-derived cell lines indicating a unique sensitivity of MPM to CDK4/6 inhibition. Importantly, insensitivity to palbociclib was mostly explained by the lack of active T172-phosphorylated CDK4, which was associated with the high p16 levels that accompany RB1 defects or inactivation, and also (unexpectely) CCNE1 overexpression in the presence of WT RB1. Prolonged treatment with palbociclib irreversibly inhibited proliferation despite re-induction of cell cycle genes upon drug washout. A senescence-associated secretory phenotype (SASP) including various potentially immunogenic components was also irreversibly induced. Our study strongly supports the clinical evaluation of CDK4/6 inhibitory drugs for MPM treatment including in several combinatorial therapies.
2022-12-20 | GSE195568 | GEO