Publications

CDK2 activity crosstalk on the ERK kinase translocation reporter can be resolved computationally (Cell Systems 2025)

James Shirley — Wed, 15 Jan 2025 21:00:35 +0000

CDK2 activity crosstalk on the ERK kinase translocation reporter can be resolved computationally (Cell Systems 2025) James Shirley Wed, 01/15/2025 - 14:00 Publications Tim Hoffman , Chengzhe Tian , Varuna Nangia , Chen Yang , Sergi Regot , Luca Gerosa , Sabrina Leigh Spencer

The mitogen-activated protein kinase (MAPK) pathway integrates growth factor signaling through extracellular signal-regulated kinase (ERK) to control cell proliferation. To study ERK dynamics, many researchers use an ERK activity kinase translocation reporter (KTR). Our study reveals that this ERK KTR also partially senses cyclin-dependent kinase 2 (CDK2) activity, making it appear as if ERK activity rises as cells progress through the cell cycle. Through single-cell time-lapse imaging, we identified a residual ERK KTR signal that was eliminated by selective CDK2 inhibitors, indicating crosstalk from CDK2 onto the ERK KTR. By contrast, EKAREN5, a FRET-based ERK sensor, showed no CDK2 crosstalk. A related p38 KTR is also partly affected by CDK2 activity. To address this, we developed linear and non-linear computational correction methods that subtract CDK2 signal from the ERK and p38 KTRs. These findings will allow for more accurate quantification of MAPK activities, especially for studies of actively cycling cells.

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INX-315, a Selective CDK2 Inhibitor, Induces Cell Cycle Arrest and Senescence in Solid Tumors (Cancer Discovery 2024)

Anonymous — Fri, 01 Mar 2024 18:29:24 +0000

INX-315, a Selective CDK2 Inhibitor, Induces Cell Cycle Arrest and Senescence in Solid Tumors (Cancer Discovery 2024) Anonymous (not verified) Fri, 03/01/2024 - 11:29 Publications Lotte Watts and Sabrina Leigh Spencer

Cyclin-dependent kinase 2 (CDK2) is thought to play an important role in driving proliferation of certain cancers, including those harboring CCNE1 amplification and breast cancers that have acquired resistance to CDK4/6 inhibitors (CDK4/6i). The precise impact of pharmacologic inhibition of CDK2 is not known due to the lack of selective CDK2 inhibitors. Here we describe INX-315, a novel and potent CDK2 inhibitor with high selectivity over other CDK family members. Using cell-based assays, patient-derived xenografts (PDX), and transgenic mouse models, we show that INX-315 (i) promotes retinoblastoma protein hypophosphorylation and therapy-induced senescence (TIS) in CCNE1-amplified tumors, leading to durable control of tumor growth; (ii) overcomes breast cancer resistance to CDK4/6i, restoring cell cycle control while reinstating the chromatin architecture of CDK4/6i-induced TIS; and (iii) delays the onset of CDK4/6i resistance in breast cancer by driving deeper suppression of E2F targets. Our results support the clinical development of selective CDK2 inhibitors.

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Cells use multiple mechanisms for cell-cycle arrest upon withdrawal of individual amino acids (Cell Reports 2023)

Anonymous — Tue, 26 Dec 2023 07:00:00 +0000

Cells use multiple mechanisms for cell-cycle arrest upon withdrawal of individual amino acids (Cell Reports 2023) Anonymous (not verified) Tue, 12/26/2023 - 00:00 Publications Yao Rong , Alicia M. Darnell , Kiera M. Sapp , Matthew G. Vander Heiden , Sabrina Leigh Spencer

Amino acids are required for cell growth and proliferation, but it remains unclear when and how amino acid availability impinges on the proliferation-quiescence decision. Here, we used time-lapse microscopy and single-cell tracking of cyclin-dependent kinase 2 (CDK2) activity to assess the response of individual cells to withdrawal of single amino acids and found strikingly different cell-cycle effects depending on the amino acid. For example, upon leucine withdrawal, MCF10A cells complete two cell cycles and then enter a CDK2-low quiescence, whereas lysine withdrawal causes immediate cell-cycle stalling. Methionine withdrawal triggers a restriction point phenotype similar to serum starvation or Mek inhibition: upon methionine withdrawal, cells complete their current cell cycle and enter a CDK2-low quiescence after mitosis. Modulation of restriction point regulators p21/p27 or cyclin D1 enables short-term rescue of proliferation under methionine and leucine withdrawal, and to a lesser extent lysine withdrawal, revealing a checkpoint connecting nutrient signaling to cell-cycle entry.

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Multiple cancers escape from multiple MAPK pathway inhibitors and use DNA replication stress signaling to tolerate aberrant cell cycles (Science Signaling 2023)

Anonymous — Tue, 01 Aug 2023 17:21:58 +0000

Multiple cancers escape from multiple MAPK pathway inhibitors and use DNA replication stress signaling to tolerate aberrant cell cycles (Science Signaling 2023) Anonymous (not verified) Tue, 08/01/2023 - 11:21 Publications Tim Hoffman , Varuna Nangia , Riley Ill , Victor Passanisi , Claire Armstrong , Chen Yang , Sabrina Leigh Spencer

Many cancers harbor pro-proliferative mutations of the mitogen-activated protein kinase (MAPK) pathway. In BRAF-driven melanoma cells treated with BRAF inhibitors, subpopulations of cells escape drug-induced quiescence through a nongenetic manner of adaptation and resume slow proliferation. Here, we found that this phenomenon is common to many cancer types driven by EGFR, KRAS, or BRAF mutations in response to multiple, clinically approved MAPK pathway inhibitors. In 2D cultures and 3D spheroid models of various cancer cell lines, a subset of cells escaped drug-induced quiescence within 4 days to resume proliferation. These “escapee” cells exhibited DNA replication deficits, accumulated DNA lesions, and mounted a stress response that depended on the ataxia telangiectasia and RAD3-related (ATR) kinase. We further identified that components of the Fanconi anemia (FA) DNA repair pathway are recruited to sites of mitotic DNA synthesis (MiDAS) in escapee cells, enabling successful completion of cell division. Analysis of patient tumor samples and clinical data correlated disease progression with an increase in DNA replication stress response factors. Our findings suggest that many MAPK pathway–mutant cancers rapidly escape drug action and that suppressing early stress tolerance pathways may achieve more durable clinical responses to MAPK pathway inhibitors.

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The intensities of canonical senescence biomarkers integrate the duration of cell-cycle withdrawal (Nature Communications 2023)

Anonymous — Thu, 27 Jul 2023 06:00:00 +0000

The intensities of canonical senescence biomarkers integrate the duration of cell-cycle withdrawal (Nature Communications 2023) Anonymous (not verified) Thu, 07/27/2023 - 00:00 Publications Humza Ashraf , Brianna Fernandez , Sabrina Leigh Spencer

Senescence, a state of permanent cell-cycle withdrawal, is difficult to distinguish from quiescence, a transient state of cell-cycle withdrawal. This difficulty arises because quiescent and senescent cells are defined by overlapping biomarkers, raising the question of whether quiescence and senescence are truly distinct states. To address this, we used single-cell time-lapse imaging to distinguish slow-cycling quiescent cells from bona fide senescent cells after chemotherapy treatment, followed immediately by staining for various senescence biomarkers. We found that the staining intensity of multiple senescence biomarkers is graded rather than binary and primarily reflects the duration of cell-cycle withdrawal, rather than senescence per se. Together, our data suggest that quiescence and senescence are not distinct cellular states but rather fall on a continuum of cell-cycle withdrawal, where the intensities of canonical senescence biomarkers reflect the likelihood of cell-cycle re-entry.

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RNA binding by the glucocorticoid receptor attenuates dexamethasone-induced gene activation (Scientific Reports 2023)

Anonymous — Fri, 09 Jun 2023 18:53:09 +0000

RNA binding by the glucocorticoid receptor attenuates dexamethasone-induced gene activation (Scientific Reports 2023) Anonymous (not verified) Fri, 06/09/2023 - 12:53 Publications Nickolaus C Lammer , Humza Ashraf , Daniella A Ugay , Sabrina Leigh Spencer , Mary A Allen , Robert T Batey , Deborah S Wuttke

The glucocorticoid receptor (GR) is a ligand-activated transcription factor that regulates a suite of genes through direct binding of GR to specific DNA promoter elements. GR also interacts with RNA, but the function of this RNA-binding activity remains elusive. Current models speculate that RNA could repress the transcriptional activity of GR. To investigate the function of the GR-RNA interaction on GR's transcriptional activity, we generated cells that stably express a mutant of GR with reduced RNA binding affinity and treated the cells with the GR agonist dexamethasone. Changes in the dexamethasone-driven transcriptome were quantified using 4-thiouridine labeling of RNAs followed by high-throughput sequencing. We find that while many genes are unaffected, GR-RNA binding is repressive for specific subsets of genes in both dexamethasone-dependent and independent contexts. Genes that are dexamethasone-dependent are activated directly by chromatin-bound GR, suggesting a competition-based repression mechanism in which increasing local concentrations of RNA may compete with DNA for binding to GR at sites of transcription. Unexpectedly, genes that are dexamethasone-independent instead display a localization to specific chromosomal regions, which points to changes in chromatin accessibility or architecture. These results show that RNA binding plays a fundamental role in regulating GR function and highlights potential functions for transcription factor-RNA interactions.

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Intracellular Crowding by Bio-Orthogonal Hydrogel Formation Induces Reversible Molecular Stasis (Advanced Materials 2022)

Anonymous — Tue, 07 Jun 2022 06:00:00 +0000

Intracellular Crowding by Bio-Orthogonal Hydrogel Formation Induces Reversible Molecular Stasis (Advanced Materials 2022) Anonymous (not verified) Tue, 06/07/2022 - 00:00 Publications Laura J. Macdougall , Timothy E. Hoffman , Bruce E. Kirkpatrick , Benjamin D. Fairbanks , Christopher N. Bowman , Sabrina L. Spencer , and Kristi S. Anseth

To survive extreme conditions, certain animals enter a reversible protective stasis through vitrification of the cytosol by polymeric molecules such as proteins and polysaccharides. In this work, synthetic gelation of the cytosol in living cells is used to induce reversible molecular stasis. Through the sequential lipofectamine-mediated transfection of complementary poly(ethylene glycol) macromers into mammalian cells, intracellular crosslinking occurs through bio-orthogonal strain-promoted azide–alkyne cycloaddition click reactions. This achieves efficient polymer uptake with minimal cell death (99% viable). Intracellular crosslinking decreases DNA replication and protein synthesis, and increases the quiescent population by 2.5-fold. Real-time tracking of single cells containing intracellular crosslinked polymers identifies increases in intermitotic time (15 h vs 19 h) and decreases in motility (30 µm h⁻¹ vs 15 µm h⁻¹). The cytosol viscosity increases threefold after intracellular crosslinking and results in disordered cytoskeletal structure in addition to the disruption of cellular coordination in a scratch assay. By incorporating photodegradable nitrobenzyl moieties into the polymer backbone, the effects of intracellular crosslinking are reversed upon exposure to light, thereby restoring proliferation (80% phospho-Rb+ cells), protein translation, and migration. Reversible intracellular crosslinking provides a novel method for dynamic manipulation of intracellular mechanics, altering essential processes that determine cellular function.

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