Authors: Katherine A Freitas, Julia A Belk, Elena Sotillo...

T cells are the major arm of the immune system responsible for controlling and regressing cancers. To identify genes limiting T cell function, we conducted genome-wide CRISPR knockout screens in human chimeric antigen receptor (CAR) T cells. Top hits were and , components of the Mediator kinase module. Targeted deletion enhanced antitumor activity and sustained the effector phenotype in CAR- and T cell receptor-engineered T cells, and inhibition of CDK8/19 kinase activity increased expansion of nonengineered T cells. -deficient T cells manifested increased core Meditator chromatin occupancy at transcriptionally active enhancers-most notably for STAT and AP-1 transcription factors-and increased expression and interleukin-2 sensitivity. These results implicate Mediator in T cell effector programming and identify the kinase module as a target for enhancing potency of antitumor T cell responses.

Topics: Humans; Cyclin-Dependent Kinase 8; Cyclin-Dependent Kinases; Mediator Complex; T-Lymphocytes; Transcription Factors; Receptors, Chimeric Antigen; Genome-Wide Association Study; Cyclin C; Genetic Testing; Immunotherapy, Adoptive; Neoplasms

PubMed: 36356142
DOI: 10.1126/science.abn5647

Authors: Kai-Ti Chang, Jan Jezek, Alicia N Campbell...

syndrome is a haploinsufficiency developmental disorder characterized by intellectual disability, heart malformation, and hypotonia. MED13L controls transcription by tethering the cyclin C-Cdk8 kinase module (CKM) to the Mediator complex. In addition, cyclin C has CKM-independent roles in the cytoplasm directing stress-induced mitochondrial fragmentation and regulated cell death. Unstressed patient fibroblasts exhibited aberrant cytoplasmic cyclin C localization, mitochondrial fragmentation, and a 6-fold reduction in respiration. In addition, the fibroblasts exhibited reduced mtDNA copy number, reduction in mitochondrial membrane integrity, and hypersensitivity to oxidative stress. Finally, transcriptional analysis of mutant fibroblasts revealed reduced mRNA levels for several genes necessary for normal mitochondrial function. Pharmacological or genetic approaches preventing cyclin C-mitochondrial localization corrected the fragmented mitochondrial phenotype and partially restored organelle function. In conclusion, this study found that mitochondrial dysfunction is an underlying defect in cells harboring the allele and identified cyclin C mis-localization as the likely cause. These results provide a new avenue for understanding this disorder.

PubMed: 35198885
DOI: 10.1016/j.isci.2022.103823

Review

Authors: Jan Ježek, Daniel G J Smethurst, David C Stieg...

The class I cyclin family is a well-studied group of structurally conserved proteins that interact with their associated cyclin-dependent kinases (Cdks) to regulate different stages of cell cycle progression depending on their oscillating expression levels. However, the role of class II cyclins, which primarily act as transcription factors and whose expression remains constant throughout the cell cycle, is less well understood. As a classic example of a transcriptional cyclin, cyclin C forms a regulatory sub-complex with its partner kinase Cdk8 and two accessory subunits Med12 and Med13 called the Cdk8-dependent kinase module (CKM). The CKM reversibly associates with the multi-subunit transcriptional coactivator complex, the Mediator, to modulate RNA polymerase II-dependent transcription. Apart from its transcriptional regulatory function, recent research has revealed a novel signaling role for cyclin C at the mitochondria. Upon oxidative stress, cyclin C leaves the nucleus and directly activates the guanosine 5'-triphosphatase (GTPase) Drp1, or Dnm1 in yeast, to induce mitochondrial fragmentation. Importantly, cyclin C-induced mitochondrial fission was found to increase sensitivity of both mammalian and yeast cells to apoptosis. Here, we review and discuss the biology of cyclin C, focusing mainly on its transcriptional and non-transcriptional roles in tumor promotion or suppression.

PubMed: 30621145
DOI: 10.3390/biology8010003

Authors: Kun Wang, Ruilan Yan, Katrina F Cooper...

Mitochondria are dynamic organelles that undergo constant fission and fusion cycles. In response to cellular damage, this balance is shifted dramatically toward fission. Cyclin C-Cdk8 kinase regulates transcription of diverse gene sets. Using knockout mouse embryonic fibroblasts (MEFs), we demonstrate that cyclin C directs the extensive mitochondrial scission induced by the anticancer drug cisplatin or oxidative stress. This activity is independent of transcriptional regulation, as Cdk8 is not required for this activity. Furthermore, adding purified cyclin C to unstressed permeabilized MEF cultures induced complete mitochondrial fragmentation that was dependent on the fission factors Drp1 and Mff. To regulate fission, a portion of cyclin C translocates from the nucleus to the cytoplasm, where it associates with Drp1 and is required for its enhanced mitochondrial activity in oxidatively stressed cells. In addition, although HeLa cells regulate cyclin C in a manner similar to MEF cells, U2OS osteosarcoma cultures display constitutively cytoplasmic cyclin C and semifragmented mitochondria. Finally, cyclin C, but not Cdk8, is required for loss of mitochondrial outer membrane permeability and apoptosis in cells treated with cisplatin. In conclusion, this study suggests that cyclin C connects stress-induced mitochondrial hyperfission and programmed cell death in mammalian cells.

Topics: Animals; Apoptosis; Cyclin C; Dynamins; HeLa Cells; Humans; Mice; Mitochondria; Mitochondrial Dynamics; Protein Transport; Signal Transduction; Stress, Physiological

PubMed: 25609094
DOI: 10.1091/mbc.E14-08-1315

Review

Authors: Wu Xu, Jun-Yuan Ji

Appropriately controlled gene expression is fundamental for normal growth and survival of all living organisms. In eukaryotes, the transcription of protein-coding mRNAs is dependent on RNA polymerase II (Pol II). The multi-subunit transcription cofactor Mediator complex is proposed to regulate most, if not all, of the Pol II-dependent transcription. Here we focus our discussion on two subunits of the Mediator complex, cyclin-dependent kinase 8 (CDK8) and its regulatory partner Cyclin C (CycC), because they are either mutated or amplified in a variety of human cancers. CDK8 functions as an oncoprotein in melanoma and colorectal cancers, thus there are considerable interests in developing drugs specifically targeting the CDK8 kinase activity. However, to evaluate the feasibility of targeting CDK8 for cancer therapy and to understand how their dysregulation contributes to tumorigenesis, it is essential to elucidate the in vivo function and regulation of CDK8-CycC, which are still poorly understood in multi-cellular organisms. We summarize the evidence linking their dysregulation to various cancers and present our bioinformatics and computational analyses on the structure and evolution of CDK8. We also discuss the implications of these observations in tumorigenesis. Because most of the Mediator subunits, including CDK8 and CycC, are highly conserved during eukaryotic evolution, we expect that investigations using model organisms such as Drosophila will provide important insights into the function and regulation of CDK8 and CycC in different cellular and developmental contexts.

Topics: Amino Acid Sequence; Animals; Cell Transformation, Neoplastic; Cyclin C; Cyclin-Dependent Kinase 8; Drosophila melanogaster; Gene Expression Regulation, Neoplastic; Humans; Mediator Complex; Molecular Sequence Data; Neoplasms; Phylogeny; Protein Conformation; RNA Polymerase II; Transcription, Genetic

PubMed: 22035865
DOI: 10.1016/j.jgg.2011.09.002

Authors: Margarita Jiménez-Palomares, José Francisco López-Acosta, Pablo Villa-Pérez...

Activation of pancreatic β-cell proliferation has been proposed as an approach to replace reduced functional β-cell mass in diabetes. Quiescent fibroblasts exit from G0 (quiescence) to G1 through pRb phosphorylation mediated by cyclin C/cdk3 complexes. Overexpression of cyclin D1, D2, D3, or cyclin E induces pancreatic β-cell proliferation. We hypothesized that cyclin C overexpression would induce β-cell proliferation through G0 exit, thus being a potential therapeutic target to recover functional β-cell mass. We used isolated rat and human islets transduced with adenovirus expressing cyclin C. We measured multiple markers of proliferation: [(3)H]thymidine incorporation, BrdU incorporation and staining, and Ki67 staining. Furthermore, we detected β-cell death by TUNEL, β-cell differentiation by RT-PCR, and β-cell function by glucose-stimulated insulin secretion. Interestingly, we have found that cyclin C increases rat and human β-cell proliferation. This augmented proliferation did not induce β-cell death, dedifferentiation, or dysfunction in rat or human islets. Our results indicate that cyclin C is a potential target for inducing β-cell regeneration.

Topics: Animals; Cell Differentiation; Cell Proliferation; Cell Survival; Cells, Cultured; Cyclin C; Humans; Insulin-Secreting Cells; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Rats; Rats, Wistar

PubMed: 25564474
DOI: 10.1152/ajpendo.00260.2014

Authors: Jan Jezek, Kun Wang, Ruilan Yan...

The cyclin C-Cdk8 kinase has been identified as both a tumor suppressor and an oncogene depending on the cell type. The genomic locus encoding cyclin C () is often deleted in aggressive anaplastic thyroid tumors. To test for a potential tumor suppressor role for cyclin C, alone, or in combination with a previously described thyroid tumor suppressor , was deleted late in thyroid development. Although mice harboring individual or deletions exhibited modest thyroid hyperplasia, the double mutant demonstrated dramatic thyroid expansion resulting in animal death by 22 weeks. Further analysis revealed that tissues exhibited a reduction in signal transducer and activator of transcription 3 (Stat3) phosphorylation at Ser727. Further analysis uncovered a post-transcriptional requirement of both Pten and cyclin C in maintaining the levels of the p21 and p53 tumor suppressors (also known as CDKN1A and TP53, respectively) in thyroid tissue. In conclusion, these data reveal the first tumor suppressor role for cyclin C in a solid tumor model. In addition, this study uncovers new synergistic activities of Pten and cyclin C to promote quiescence through maintenance of p21 and p53.

Topics: Animals; Cell Line, Tumor; Cyclin C; Cyclin-Dependent Kinase Inhibitor p21; Mice; Mice, Knockout; PTEN Phosphohydrolase; STAT3 Transcription Factor; Thyroid Neoplasms

PubMed: 31331961
DOI: 10.1242/jcs.230029

Authors: Jana Trifinopoulos, Julia List, Thorsten Klampfl...

Despite major therapeutic advances in the treatment of acute lymphoblastic leukemia (ALL), resistances and long-term toxicities still pose significant challenges. Cyclins and their associated cyclin-dependent kinases are one focus of cancer research when looking for targeted therapies. We discovered cyclin C to be a key factor for B-cell ALL (B-ALL) development and maintenance. While cyclin C is not essential for normal hematopoiesis, CcncΔ/Δ BCR::ABL1+ B-ALL cells fail to elicit leukemia in mice. RNA sequencing experiments revealed a p53 pathway deregulation in CcncΔ/Δ BCR::ABL1+ cells resulting in the inability of the leukemic cells to adequately respond to stress. A genome-wide CRISPR/Cas9 loss-of-function screen supplemented with additional knock-outs unveiled a dependency of human B-lymphoid cell lines on CCNC. High cyclin C levels in B-cell precursor (BCP) ALL patients were associated with poor event-free survival and increased risk of early disease recurrence after remission. Our findings highlight cyclin C as a potential therapeutic target for B-ALL, particularly to enhance cancer cell sensitivity to stress and chemotherapy.

Topics: Humans; Tumor Suppressor Protein p53; Animals; Mice; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma; Cyclin C; Disease Progression; Gene Expression Regulation, Leukemic; Stress, Physiological

PubMed: 39385738
DOI: 10.3324/haematol.2024.285701

Authors: Haripriya Vittal Rao, Lakshmi Thirumangalakudi, Paula Grammas...

The extent to which neurons proceed into the cell cycle and the mechanisms whereby cell cycle re-entry leads to apoptosis vary in response to agonists. We previously showed upregulation of early G1 regulators in thrombin-treated neurons yet neurons did not proceed to S phase but to apoptosis. The objective of this study is to explore mechanisms which might prevent S phase entry and promote apoptosis in thrombin-treated neurons. Cultured rat brain neurons are exposed to thrombin (200nM) for 30min to 4.5h and the expression of cyclin C, cyclin dependent kinases (cdk1, cdk2, cdk3, cdk8) and the cell cycle inhibitor p27 assessed. Our data show a simultaneous decrease of both cyclin C and cdk3 proteins soon after thrombin treatment. The decrease in cyclin C also correlates with decreases in cdk1 and cdk2, at both mRNA and protein levels. There is no change in expression of cdk8 or the cell cycle inhibitor p27 in response to thrombin treatment. These results suggest that decreases in G1-S regulators cyclin C and cdks 3, cdk2 and cdk1 in response to thrombin could make conditions unfavorable for S phase entry and favor neuronal apoptosis.

Topics: Animals; Apoptosis; CDC2 Protein Kinase; Cell Cycle; Cell Cycle Proteins; Cells, Cultured; Cyclin C; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 3; Cyclin-Dependent Kinase 8; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cyclins; G1 Phase; Neurons; Rats; S Phase; Thrombin

PubMed: 19103257
DOI: 10.1016/j.neulet.2008.12.018

Review

Authors: Brittany Friedson, Katrina F Cooper

The Cdk8 kinase module (CKM) of the multi-subunit mediator complex plays an essential role in cell fate decisions in response to different environmental cues. In the budding yeast , the CKM consists of four conserved subunits (cyclin C and its cognate cyclin-dependent kinase Cdk8, Med13, and Med12) and predominantly negatively regulates a subset of stress responsive genes (SRG's). Derepression of these SRG's is accomplished by disassociating the CKM from the mediator, thus allowing RNA polymerase II-directed transcription. In response to cell death stimuli, cyclin C translocates to the mitochondria where it induces mitochondrial hyper-fission and promotes regulated cell death (RCD). The nuclear release of cyclin C requires Med13 destruction by the ubiquitin-proteasome system (UPS). In contrast, to protect the cell from RCD following SRG induction induced by nutrient deprivation, cyclin C is rapidly destroyed by the UPS before it reaches the cytoplasm. This enables a survival response by two mechanisms: increased ATP production by retaining reticular mitochondrial morphology and relieving CKM-mediated repression on autophagy genes. Intriguingly, nitrogen starvation also stimulates Med13 destruction but through a different mechanism. Rather than destruction via the UPS, Med13 proteolysis occurs in the vacuole (yeast lysosome) via a newly identified Snx4-assisted autophagy pathway. Taken together, these findings reveal that the CKM regulates cell fate decisions by both transcriptional and non-transcriptional mechanisms, placing it at a convergence point between cell death and cell survival pathways.

PubMed: 34683473
DOI: 10.3390/microorganisms9102152