Leighton Daigh

All Publications

• Uncoupling of mTORC1 from E2F activity maintains DNA damage and senescence. Nature communications Daigh, L. H., Saha, D., Rosenthal, D. L., Ferrick, K. R., Meyer, T. 2024; 15 (1): 9181

  Abstract

  DNA damage is a primary trigger for cellular senescence, which in turn causes organismal aging and is a promising target of anti-aging therapies. Most DNA damage occurs when DNA is fragile during DNA replication in S phase, but senescent cells maintain DNA damage long-after DNA replication has stopped. How senescent cells induce DNA damage and why senescent cells fail to repair damaged DNA remain open questions. Here, we combine reversible expression of the senescence-inducing CDK4/6 inhibitory protein p16INK4 (p16) with live single-cell analysis and show that sustained mTORC1 signaling triggers senescence in non-proliferating cells by increasing transcriptional DNA damage and inflammation signaling that persists after p16 is degraded. Strikingly, we show that activation of E2F transcriptional program, which is regulated by CDK4/6 activity and promotes expression of DNA repair proteins, repairs transcriptionally damaged DNA without requiring DNA replication. Together, our study suggests that senescence can be maintained by ongoing mTORC1-induced transcriptional DNA damage that cannot be sufficiently repaired without induction of protective E2F target genes.

  View details for DOI 10.1038/s41467-024-52820-6

  View details for PubMedID 39448567

  View details for PubMedCentralID 5796526

• Clinical CDK4/6 inhibitors induce selective and immediate dissociation of p21 from cyclin D-CDK4 to inhibit CDK2. Nature communications Pack, L. R., Daigh, L. H., Chung, M., Meyer, T. 2021; 12 (1): 3356

  Abstract

  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.

  View details for DOI 10.1038/s41467-021-23612-z

  View details for PubMedID 34099663

• Putting the brakes on the cell cycle: mechanisms of cellular growth arrest. Current opinion in cell biology Pack, L. R., Daigh, L. H., Meyer, T. n. 2019; 60: 106–13

  Abstract

  Precise regulation of cellular proliferation is critical to tissue homeostasis and development, but misregulation leads to diseases of excess proliferation or cell loss. To achieve precise control, cells utilize distinct mechanisms of growth arrest such as quiescence and senescence. The decision to enter these growth-arrested states or proliferate is mediated by the core cell-cycle machinery that responds to diverse external and internal signals. Recent advances have revealed the molecular underpinnings of these cell-cycle decisions, highlighting the unique nature of cell-cycle entry from quiescence, identifying endogenous DNA damage as a quiescence-inducing signal, and establishing how persistent arrest is achieved in senescence.

  View details for DOI 10.1016/j.ceb.2019.05.005

  View details for PubMedID 31252282

• Stochastic Endogenous Replication Stress Causes ATR-Triggered Fluctuations in CDK2 Activity that Dynamically Adjust Global DNA Synthesis Rates. Cell systems Daigh, L. H., Liu, C., Chung, M., Cimprich, K. A., Meyer, T. 2018

  Abstract

  Faithful DNA replication is challenged by stalling of replication forks during Sphase. Replication stress is further increased in cancer cells or in response to genotoxic insults. Using live single-cell image analysis, we found that CDK2 activity fluctuates throughout an unperturbed Sphase. We show that CDK2 fluctuations result from transient ATR signals triggered by stochastic replication stress events. In turn, fluctuating endogenous CDK2 activity causes corresponding decreases and increases in DNA synthesis rates, linking changes in stochastic replication stress to fluctuating global DNA replication rates throughout Sphase. Moreover, cells that re-enter the cell cycle after mitogen stimulation have increased CDK2 fluctuations and prolonged Sphase resulting from increased replication stress-induced CDK2 suppression. Thus, our study reveals a dynamic control principle for DNA replication whereby CDK2 activity is suppressed and fluctuates throughout Sphase to continually adjust global DNA synthesis rates in response to recurring stochastic replication stress events.

  View details for PubMedID 29909278

• Inadvertent omission of a specimen integrity comment- an overlooked post-analytical error. Clinical chemistry and laboratory medicine Bowen, R. A., Daigh, L. H. 2024

  View details for DOI 10.1515/cclm-2023-1445

  View details for PubMedID 38205628

• Pediatric acquired factor VIII deficiency presenting as hemarthrosis. Pediatric blood & cancer Daigh, L. H., Chien, M. C., Lo, C. Y. 1800: e29530

  View details for DOI 10.1002/pbc.29530

  View details for PubMedID 34913591

• Stress-mediated exit to quiescence restricted by increasing persistence in CDK4/6 activation. eLife Yang, H. W., Cappell, S. D., Jaimovich, A., Liu, C., Chung, M., Daigh, L. H., Pack, L. R., Fan, Y., Regot, S., Covert, M., Meyer, T. 2020; 9

  Abstract

  Mammalian cells typically start the cell-cycle entry program by activating cyclin-dependent protein kinase 4/6 (CDK4/6). CDK4/6 activity is clinically relevant as mutations, deletions, and amplifications that increase CDK4/6 activity contribute to the progression of many cancers. However, when CDK4/6 is activated relative to CDK2 remained incompletely understood. Here we developed a reporter system to simultaneously monitor CDK4/6 and CDK2 activities in single cells and found that CDK4/6 activity increases rapidly before CDK2 activity gradually increases, and that CDK4/6 activity can be active after mitosis or inactive for variable time periods. Markedly, stress signals in G1 can rapidly inactivate CDK4/6 to return cells to quiescence but with reduced probability as cells approach S phase. Together, our study reveals a regulation of G1 length by temporary inactivation of CDK4/6 activity after mitosis, and a progressively increasing persistence in CDK4/6 activity that restricts cells from returning to quiescence as cells approach S phase.

  View details for DOI 10.7554/eLife.44571

  View details for PubMedID 32255427

• Altered G1 signaling order and commitment point in cells proliferating without CDK4/6 activity. Nature communications Liu, C., Konagaya, Y., Chung, M., Daigh, L. H., Fan, Y., Yang, H. W., Terai, K., Matsuda, M., Meyer, T. 2020; 11 (1): 5305

  Abstract

  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/CCDH1 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/CCDH1 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/CCDH1 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.

  View details for DOI 10.1038/s41467-020-18966-9

  View details for PubMedID 33082317

• Hepatic compartmentalization of exhausted and regulatory cells in HIV/HCV-coinfected patients JOURNAL OF VIRAL HEPATITIS Barrett, L., Trehanpati, N., Poonia, S., Daigh, L., Sarin, S., Masur, H., Kottilil, S. 2015; 22 (3): 281-288

  Abstract

  Accelerated intrahepatic hepatitis C virus (HCV) pathogenesis is likely the result of dysregulation within both the innate and adaptive immune compartments, but the exact contribution of peripheral blood and liver lymphocyte subsets remains unclear. Prolonged activation and expansion of immunoregulatory cells have been thought to play a role. We determined immune cell subset frequency in contemporaneous liver and peripheral blood samples from chronic HCV-infected and HIV/HCV-coinfected individuals. Peripheral blood mononuclear cells (PBMC) and biopsy-derived liver-infiltrating lymphocytes from 26 HIV/HCV-coinfected, 10 chronic HCV-infected and 10 HIV-infected individuals were assessed for various subsets of T and B lymphocytes, dendritic cell, natural killer (NK) cell and NK T-cell frequency by flow cytometry. CD8(+) T cells expressing the exhaustion marker PD-1 were increased in HCV-infected individuals compared with uninfected individuals (P = 0.02), and HIV coinfection enhanced this effect (P = 0.005). In the liver, regulatory CD4(+) CD25(+) Foxp3(+) T cells, as well as CD4(+) CD25(+) PD1(+) T cells, were more frequent in HIV/HCV-coinfected than in HCV-monoinfected samples (P < 0.001). HCV was associated with increased regulatory T cells, PD-1(+) T cells and decreased memory B cells, regardless of HIV infection (P ≤ 0.005 for all). Low CD8(+) expression was observed only in PD-1(+) CD8(+) T cells from HCV-infected individuals and healthy controls (P = 0.002) and was associated with enhanced expansion of exhausted CD8(+) T cells when exposed in vitro to PHA or CMV peptides. In conclusion, in HIV/HCV coinfection, ongoing HCV replication is associated with increased regulatory and exhausted T cells in the periphery and liver that may impact control of HCV. Simultaneous characterization of liver and peripheral blood highlights the disproportionate intrahepatic compartmentalization of immunoregulatory T cells, which may contribute to establishment of chronicity and hepatic fibrogenesis in HIV coinfection.

  View details for DOI 10.1111/jvh.12291

  View details for Web of Science ID 000350546300008

  View details for PubMedID 25174689

• Arginine-Rich Motifs Are Not Required for Hepatitis Delta Virus RNA Binding Activity of the Hepatitis Delta Antigen JOURNAL OF VIROLOGY Daigh, L. H., Griffin, B. L., Soroush, A., Mamedov, M. R., Casey, J. L. 2013; 87 (15): 8665-8674

  Abstract

  Hepatitis delta virus (HDV) replication and packaging require interactions between the unbranched rodlike structure of HDV RNA and hepatitis delta antigen (HDAg), a basic, disordered, oligomeric protein. The tendency of the protein to bind nonspecifically to nucleic acids has impeded analysis of HDV RNA protein complexes and conclusive determination of the regions of HDAg involved in RNA binding. The most widely cited model suggests that RNA binding involves two proposed arginine-rich motifs (ARMs I and II) in the middle of HDAg. However, other studies have questioned the roles of the ARMs. Here, binding activity was analyzed in vitro using HDAg-160, a C-terminal truncation that binds with high affinity and specificity to HDV RNA segments in vitro. Mutation of the core arginines of ARM I or ARM II in HDAg-160 did not diminish binding to HDV unbranched rodlike RNA. These same mutations did not abolish the ability of full-length HDAg to inhibit HDV RNA editing in cells, an activity that involves RNA binding. Moreover, only the N-terminal region of the protein, which does not contain the ARMs, was cross-linked to a bound HDV RNA segment in vitro. These results indicate that the amino-terminal region of HDAg is in close contact with the RNA and that the proposed ARMs are not required for binding HDV RNA. Binding was not reduced by mutation of additional clusters of basic amino acids. This result is consistent with an RNA-protein complex that is formed via numerous contacts between the RNA and each HDAg monomer.

  View details for DOI 10.1128/JVI.00929-13

  View details for Web of Science ID 000321590200035

  View details for PubMedID 23740973

  View details for PubMedCentralID PMC3719807