Control over network chemistry and connectivity of hydrogels is critical for the generation of tunable material properties, including material degradation for applications such as tissue scaffolding and drug delivery. Here, the degradation of hydrogels employing different hydrolytically cleavable groups including benzamide and syringic acid-derived carbamates, kojic acid-derived carbonates, and kojic acid-derived esters under physiological conditions was studied. Tunability of the hydrogel network degradation was demonstrated by varying the hydrolytically degradable moiety, macromer functionality, and copolymerization with hydrolytically stable macromers. These hydrolytically labile macromers were introduced and cross-linked intracellularly to induce transient cellular quiescence in MCF10A cells, resulting in a highly tunable degradation mechanism that is shown to be capable of inducing reversible biostasis of cells with 60% of cells treated with the carbonate macromer returning to their proliferative state and rebounding in translational activity after 72 h, while the biological activity of the carbamate macromer-treated cells remained suppressed.

Over 50% of melanomas are driven by BRAFV600 mutations. Clinically, targeted inhibitors of mutant BRAF and MEK are the primary treatment for more than 50% of melanoma patients that are refractory to standard-of-care immunotherapy. While targeted therapies are initially clinically effective, a subset of residual cancer cells persist and inevitably drive tumor recurrence and drug resistance. While some cells persist due to pre-existing mechanisms (e.g. genetic mutations), others can non-genetically adapt to tolerate drug by rewiring their internal signaling cascades. Importantly, a key feature of these non-genetically adapted persister cells is that their drug-tolerant phenotype is acquired in response to drug pressure, making them extremely challenging to identify and characterize. Using cutting-edge long-term time-lapse microscopy and an automated single-cell tracking pipeline, we studied the behaviors of persister cells in drug-treated BRAFV600E melanoma populations. Notably, we found significant heterogeneity within persister populations. While all persister cells initially enter a state of drug-induced quiescence, a subset of these cells—termed “escapees”—can escape drug action and resume proliferation. By computationally reconstructing single-cell lineages from long-term time-lapse imaging experiments, we monitored key signaling pathways before, during, and after escape from drug. Using this approach, we identified a significant increase in mTorC1 signaling activity in persisters that would go on to escape drug. Additionally, mTorC1 signaling caused increases in protein translation and cell growth rates during the quiescent periods of these cells. The abundance of Cyclin D1, a key driver of proliferation, is extremely sensitive to cellular translation rates due to its short half-life. In order to determine if the observed increase in mTorC1-mediated growth and translation is driving the upregulation of Cyclin D1 levels past a threshold necessary for escape, we used CRISPR to tag Cyclin D1 at the endogenous locus. Importantly, we found that escapees significantly upregulate Cyclin D1 levels in the hours prior to their escape, compared to non-escapees. These findings have important clinical implications because they suggest that Cyclin D1 protein levels are a key bottleneck for cell-cycle re-entry under MAPK inhibition. Additionally, these findings implicate Cyclin D1 as one of the first markers that can distinguish future escapees from non-escapees in the quiescence period before these distinct cell-cycle fates emerge. Varuna Nangia, Humza Ashraf, Nasreen Marikar, Victor Passanisi, Christopher Ryland Ill, Timothy Hoffman, Sabrina Leigh Spencer. Non-genetic drug adaptation to MAPK pathway inhibitors in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6418.

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.

CDK2 is a core cell-cycle kinase that phosphorylates many substrates to drive progression through the cell cycle. CDK2 is hyperactivated in multiple cancers and is therefore an attractive therapeutic target. Here, we use several CDK2 inhibitors in clinical development to interrogate CDK2 substrate phosphorylation, cell-cycle progression, and drug adaptation in preclinical models. Whereas CDK1 is known to compensate for loss of CDK2 in Cdk2-/- mice, this is not true of acute inhibition of CDK2. Upon CDK2 inhibition, cells exhibit a rapid loss of substrate phosphorylation that rebounds within several hours. CDK4/6 activity backstops inhibition of CDK2 and sustains the proliferative program by maintaining Rb1 hyperphosphorylation, active E2F transcription, and cyclin A2 expression, enabling re-activation of CDK2 in the presence of drug. Our results augment our understanding of CDK plasticity and indicate that co-inhibition of CDK2 and CDK4/6 may be required to suppress adaptation to CDK2 inhibitors currently under clinical assessment.

Many cancers harbor pro-proliferative mutations of the mitogen-activated protein kinase (MAPK) pathway and many targeted inhibitors now exist for clinical use, but drug resistance remains a major issue. We recently showed that BRAF-driven melanoma cells treated with BRAF inhibitors can non-genetically adapt to drug within 3-4 days to escape quiescence and resume slow proliferation. Here we show that this phenomenon is not unique to melanomas treated with BRAF inhibitors but rather is widespread across many clinical MAPK inhibitors and cancer types driven by EGFR, KRAS, and BRAF mutations. In all treatment contexts examined, a subset of cells can escape drug-induced quiescence within four days to resume proliferation. These escapee cells broadly experience aberrant DNA replication, accumulate DNA lesions, spend longer in G2-M cell cycle phases, and mount an ATR-dependent stress response. We further identify the Fanconi anemia (FA) DNA repair pathway as critical for successful mitotic completion in escapees. Long-term cultures, patient samples, and clinical data demonstrate a broad dependency on ATR- and FA-mediated stress tolerance. Together, these results highlight the pervasiveness with which MAPK-mutant cancers are able to rapidly escape drug and the importance of suppressing early stress tolerance pathways to potentially achieve more durable clinical responses to targeted MAPK pathway inhibitors.

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.

CDK2 is a core cell-cycle enzyme that phosphorylates a variety of substrates to drive progression through the cell cycle. CDK2 is hyperactivated in multiple cancers and is therefore an attractive therapeutic target. Here, application of a novel small molecule CDK2/4/6 inhibitor, PF-06873600, enabled in-depth interrogation of CDK2 substrate phosphorylation, cell-cycle progression, and drug adaptation in preclinical models. CDK2-specific inhibition leads to rapid loss of substrate phosphorylation, but unexpectedly, CDK2 substrate phosphorylation rebounds within several hours. Whereas CDK1 is known to compensate for loss of CDK2 in Cdk2-/- mice, here we demonstrate an early dependence on CDK4/6 activity. CDK2 inhibition reveals latent CDK4/6 activity throughout S phase that backstops the proliferative program by maintaining Rb1 hyperphosphorylation, active E2F transcription, and Cyclin A2 expression, enabling re-activation of CDK2. Our results augment our understanding of CDK plasticity and indicate that co-inhibition of CDK2 and CDK4/6 is required to suppress adaptation to CDK2 inhibitors. Citation Format: Mansi Arora, Justin Moser, Timothy E. Hoffman, Lotte P. Watts, Mingwei Min, Monica Musteanu, Yao Rong, Jordan Schneider, C Ryland Ill, Varuna Nangia, Manuel Sanclemente, John Lapek, Lisa Nguyen, Sherry Niessen, Stephen Dann, Todd van Arsdale, Mariano Barbacid, Nichol Miller, Sabrina L. Spencer. Cells rapidly adapt to CDK2 inhibitors via plasticity of the CDK2/4/6-Rb-E2F axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5992.

In nature, some organisms survive extreme environments by inducing a biostatic state wherein cellular contents are effectively vitrified. Recently, a synthetic biostatic state in mammalian cells is achieved via intracellular network formation using bio-orthogonal strain-promoted azide-alkyne cycloaddition (SPAAC) reactions between functionalized poly(ethylene glycol) (PEG) macromers. In this work, the effects of intracellular network formation on a 3D epithelial MCF10A spheroid model are explored. Macromer-transfected cells are encapsulated in Matrigel, and spheroid area is reduced by ≈50% compared to controls. The intracellular hydrogel network increases the quiescent cell population, as indicated by increased p21 expression. Additionally, bioenergetics (ATP/ADP ratio) and functional metabolic rates are reduced. To enable reversibility of the biostasis effect, a photosensitive nitrobenzyl-containing macromer is incorporated into the PEG network, allowing for light-induced degradation. Following light exposure, cell state, and proliferation return to control levels, while SPAAC-treated spheroids without light exposure (i.e., containing intact intracellular networks) remain smaller and less proliferative through this same period. These results demonstrate that photodegradable intracellular hydrogels can induce a reversible slow-growing state in 3D spheroid culture.

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.

Neurocognitive consequences of blast-induced traumatic brain injury (bTBI) pose significant concerns for military service members and veterans with the majority of "invisible injury." However, the underlying mechanism of such mild bTBI by low-intensity blast (LIB) exposure for long-term cognitive and mental deficits remains elusive. Our previous studies have shown that mice exposed to LIB result in nanoscale ultrastructural abnormalities in the absence of gross or apparent cellular damage in the brain. Here we tested the hypothesis that glutamatergic hyperexcitability may contribute to long-term learning deficits. Using brain slice electrophysiological recordings, we found an increase in averaged frequencies with a burst pattern of miniature excitatory postsynaptic currents (mEPSCs) in hippocampal CA3 neurons in LIB-exposed mice at 1-and 7-days post injury, which was blocked by a specific NMDA receptor antagonist AP5. In addition, cognitive function assessed at 3 months post LIB exposure by automated home-cage monitoring showed deficits in dynamic patterns of discrimination learning and cognitive flexibility in LIB-exposed mice. Collected hippocampal tissue was further processed for quantitative global-proteomic analysis. Advanced data-independent acquisition for quantitative tandem mass spectrometry analysis identified altered expression of proteins involved in synaptic plasticity and serine protease inhibitors in LIB-exposed mice. Some were correlated with the ability of discrimination learning and cognitive flexibility. These findings show that acute glutamatergic hyperexcitability in the hippocampus induced by LIB may contribute to long-term cognitive dysfunction and protein alterations. Studies using this military-relevant mouse model of mild bTBI provide valuable insights into developing a potential therapeutic strategy to ameliorate hyperexcitability-modulated LIB injuries.

Despite the increasing number of effective anti-cancer therapies, successful treatment is limited by the development of drug resistance. While the contribution of genetic factors to drug resistance is undeniable, little is known about how drug-sensitive cells first evade drug action to proliferate in drug. Here we track the responses of thousands of single melanoma cells to BRAF inhibitors and show that a subset of cells escapes drug via non-genetic mechanisms within the first three days of treatment. Cells that escape drug rely on ATF4 stress signalling to cycle periodically in drug, experience DNA replication defects leading to DNA damage, and yet out-proliferate other cells over extended treatment. Together, our work reveals just how rapidly melanoma cells can adapt to drug treatment, generating a mutagenesis-prone subpopulation that expands over time.

Despite increasing numbers of effective anti-cancer therapies, successful treatment is limited by the development of drug resistance. While the contribution of genetic factors to drug resistance is undeniable, little is known about how drug-sensitive cells first evade drug action to proliferate in drug. Here we track the response of thousands of single melanoma cells to BRAF inhibitors and show that a subset escapes drug within the first 3 days of treatment. Cell-cycle re-entry occurs via a non-genetic mechanism involving activation of mTORC1 and ATF4, validated in cultures of patient biopsies. These escapees cycle periodically in drug, incur significant DNA damage, and out-proliferate non-escapees over extended treatment. Our work reveals a mutagenesis-prone, expanding subpopulation of early drug escapees that may seed development of permanent drug resistance.

Alzheimer’s disease (AD) is the leading cause of dementia and one of the most common causes of death worldwide. As an age-dependent multifactorial disease, the causative triggers of AD are rooted in spontaneous declines in cellular function and metabolic capacity with increases in protein stressors such as the tau protein. This multitude of age-related processes that cause neurons to change from healthy states to ones vulnerable to the damage seen in AD are difficult to simultaneously investigate and even more difficult to quantify. Here we aimed to diminish these gaps in our understanding of neuronal vulnerability in AD development by using simulation methods to theoretically quantify an array of cellular stress responses and signaling molecules. This temporally-descriptive molecular signature was produced using a novel multimethod simulation approach pioneered by our laboratory for biological research; this methodology combines hierarchical agent-based processes and continuous equation-based modeling in the same interface, all while maintaining intrinsic distributions that emulate natural biological stochasticity. The molecular signature was validated for a normal organismal aging trajectory using experimental longitudinal data from Caenorhabditis elegans and rodent studies. In addition, we have further predicted this aging molecular signature for cells impacted by the pathogenic tau protein, giving rise to distinct stress response conditions needed for cytoprotective aging. Interestingly, our simulation experiments showed that oxidative stress signaling (via daf-16 and skn-1 activities) does not substantially protect cells from all the early stressors of aging, but that it is essential in preventing a late-life degenerative cellular phenotype. Together, our simulation experiments aid in elucidating neurodegenerative triggers in the onset of AD for different genetic conditions. The long-term goal of this work is to provide more detailed diagnostic and prognostic tools for AD development and progression, and to provide more comprehensive preventative measures for this disease.

The aryl hydrocarbon receptor (AhR) is a nuclear receptor that facilitates a wide transcriptional response and causes a variety of adaptive and maladaptive physiological functions. Such functions are entirely dependent on the type of ligand activating it, and therefore, the nuances in the activation of this receptor at the single-cell level have become a research interest for different pharmacological and toxicological applications. Here, we investigate the activation of the AhR by diverse classes of compounds in a Hepa1c1c7-based murine hepatoma cell line. The exogenous compounds analyzed produced different levels of ultrasensitivity in AhR activation as measured by XRE-coupled EGFP production and analyzed by both flow cytometric and computational simulation techniques. Interestingly, simulation experiments reported herein were able to reproduce and quantitate the natural single-cell stochasticity inherent to mammalian cell lines as well as the ligand-specific differences in ultrasensitivity. Classical AhR modulators 2,3,7,8-tetrachlorodibenzodioxin (10 − 1 –10 5 pM), PCB-126 (10 − 1 –10 7 pM), and benzo[a]pyrene (10 − 1 –10 7 pM) produced the greatest levels of single-cell ultrasensitivity and most maximal responses, while consumption-based ligands indole-3-carbinol (10 3 –10 9 pM), 3,3′-diindolylmethane (10 3 –10 8 pM), and cannabidiol (10 3 –10 8 pM) caused low-level AhR activation in more purely graded single-cell fashions. All compounds were tested and analyzed over a 24 h period for consistency. The comparative quantitative results for each compound are presented within. This study aids in defining the disparity between different types of AhR modulators that produce distinctly different physiological outcomes. In addition, the simulation tool developed for this study can be used in future studies to predict the quantitative effects of diverse types of AhR ligands in the context of pharmacological therapies or toxicological concerns.

•Multi-species PBPK models have been developed and validated by experimental data.•Repeated, dietarily-relevant oral doses were assessed to predict internal exposures.•Humans see less benzoic acid compared to rats due to faster metabolism/excretion.•These comparisons present very little pharmacokinetic interspecies variation.

Research in biogerontology has largely focused on the complex relationship between mitochondrial dysfunction and biological aging. In particular, the mitochondrial free radical theory of aging (MFRTA) has been well accepted. However, this theory has been challenged by recent studies showing minimal increases in reactive oxygen species (ROS) as not entirely deleterious in nature, and even beneficial under the appropriate cellular circumstances. To assess these significant and nonintuitive observations in the context of a functional system, we have taken an in silico approach to expand the focus of the MFRTA by including other key mitochondrial stress response pathways, as they have been observed in the nematode Caenorhabditis elegans. These include the mitochondrial unfolded protein response (UPRmt), mitochondrial biogenesis and autophagy dynamics, the relevant DAF-16 and SKN-1 axes, and NAD(+)-dependent deacetylase activities. To integrate these pathways, we have developed a multilevel hybrid-modeling paradigm, containing agent-based elements among stochastic system-dynamics environments of logically derived ordinary differential equations, to simulate aging mitochondrial phenotypes within a population of energetically demanding cells. The simulation experiments resulted in accurate predictions of physiological parameters over time that accompany normal aging, such as the declines in both NAD(+) and ATP and an increase in ROS. Additionally, the in silico system was virtually perturbed using a variety of pharmacological (e.g., rapamycin, pterostilbene, paraquat) and genetic (e.g., skn-1, daf-16, sod-2) schemes to quantitate the temporal alterations of specific mechanistic targets, supporting insights into molecular determinants of aging as well as cytoprotective agents that may improve neurological or muscular healthspan.