Inertial effect of cell state velocity on the quiescence-proliferation fate decision.

Energy landscapes can provide intuitive depictions of population heterogeneity and dynamics. However, it is unclear whether individual cell behavior, hypothesized to be determined by initial position and noise, is faithfully recapitulated. Using the p21-/Cdk2-dependent quiescence-proliferation decision in breast cancer dormancy as a testbed, we examined single-cell dynamics on the landscape when perturbed by hypoxia, a dormancy-inducing stress. Combining trajectory-based energy landscape generation with single-cell time-lapse microscopy, we found that a combination of initial position and velocity on a p21/Cdk2 landscape, but not position alone, was required to explain the observed cell fate heterogeneity under hypoxia. This is likely due to additional cell state information such as epigenetic features and/or other species encoded in velocity but missing in instantaneous position determined by p21 and Cdk2 levels alone. Here, velocity dependence manifested as inertia: cells with higher cell cycle velocities prior to hypoxia continued progressing along the cell cycle under hypoxia, resisting the change in landscape towards cell cycle exit. Such inertial effects may markedly influence cell fate trajectories in tumors and other dynamically changing microenvironments where cell state transitions are governed by coordination across several biochemical species.

Treatment of Sulfur Mustard Corneal Injury by Augmenting the DNA Damage Response (DDR): A Novel Approach.

Sulfur mustard (SM) is a highly reactive organic chemical has been used as a chemical warfare agent and terrorist threat since World War I. The cornea is highly sensitive to SM toxicity and exposure to low vapor doses can cause incapacitating acute injuries. Exposure to higher doses can elicit persistent secondary keratopathies that cause reduced quality of life and impaired or lost vision. Despite a century of research, there are no specific treatments for acute or persistent ocular SM injuries. SM cytotoxicity emerges, in part, through DNA alkylation and double-strand breaks (DSBs). Because DSBs can naturally be repaired by DNA damage response pathways with low efficiency, we hypothesized that enhancing the homologous recombination pathway could pose a novel approach to mitigate SM injury. Here, we demonstrate that a dilithium salt of adenosine diphosphoribose (INV-102) increases protein levels of p53 and Sirtuin 6, upregulates transcription of BRCA1/2, enhances γH2AX focus formation, and promotes assembly of repair complexes at DSBs. Based on in vitro evidence showing INV-102 enhancement of DNA damage response through both p53-dependent and p53-independent pathways, we next tested INV-102 in a rabbit preclinical model of corneal injury. In vivo studies demonstrate a marked reduction in the incidence and severity of secondary keratopathies in INV-102-treated eyes compared with vehicle-treated eyes when treatment was started 24 hours after SM vapor exposure. These results suggest DNA repair mechanisms are a viable therapeutic target for SM injury and suggest topical treatment with INV-102 is a promising approach for SM as well as other conditions associated with DSBs. SIGNIFICANCE STATEMENT: Sulfur mustard gas corneal injury currently has no therapeutic treatment. This study aims to show the therapeutic potential of activating the body's natural DNA damage response to activate tissue repair.

YAP localization mediates mechanical adaptation of human cancer cells during extravasation in vivo.

Biophysical profiling of primary tumors has revealed that individual tumor cells fall along a highly heterogeneous continuum of mechanical phenotypes. One idea is that a subset of tumor cells is "softer" to facilitate detachment and escape from the primary site, a step required to initiate metastasis. However, it has also been postulated that cells must be able to deform and generate sufficient force to exit into distant sites. Here, we aimed to dissect the mechanical changes that occur during extravasation and organ colonization. Using multiplexed methods of intravital microscopy and optical tweezer based active microrheology, we obtained longitudinal images and mechanical profiles of cells during organ colonization in vivo. We determined that cells were softer, more liquid like upon exit of the vasculature but stiffened and became more solid like once in the new organ microenvironment. We also determined that a YAP mediated mechanogenotype influenced the global dissemination in our in vivo and in vitro models and that reducing mechanical heterogeneity could reduce extravasation. Moreover, our high throughput analysis of mechanical phenotypes of patient samples revealed that this mechanics was in part regulated by the external hydrodynamic forces that the cancer cells experienced within capillary mimetics. Our findings indicate that disseminated cancer cells can keep mutating with a continuum landscape of mechano-phenotypes, governed by the YAP-mediated mechanosensing of hydrodynamic flow.

Pulsed stimuli entrain p53 to synchronize single cells and modulate cell-fate determination.

Entrainment to an external stimulus enables a synchronized oscillatory response across a population of cells, increasing coherent responses by reducing cell-to-cell heterogeneity. It is unclear whether the property of entrainability extends to systems where responses are intrinsic to the individual cell, rather than dependent on coherence across a population of cells. Using a combination of mathematical modeling, time-lapse fluorescence microscopy, and single-cell tracking, we demonstrated that p53 oscillations triggered by DNA double-strand breaks (DSBs) can be entrained with a periodic damage stimulus, despite such synchrony not known to function in effective DNA damage responses. Surprisingly, p53 oscillations were experimentally entrained over a wider range of DSB frequencies than predicted by an established computational model for the system. We determined that recapitulating the increased range of entrainment frequencies required, non-intuitively, a less robust oscillator and wider steady-state valley on the energy landscape. Further, we show that p53 entrainment can lead to altered expression dynamics of downstream targets responsible for cell fate in a manner dependent on target mRNA stability. Overall, this study demonstrates that entrainment can occur in a biological oscillator despite the apparent lack of an evolutionary advantage conferred through synchronized responses and highlights the potential of externally entraining p53 dynamics to reduce cellular variability and synchronize cell-fate responses for therapeutic outcomes.

Inertial effect of cell state velocity on the quiescence-proliferation fate decision in breast cancer.

Energy landscapes can provide intuitive depictions of population heterogeneity and dynamics. However, it is unclear whether individual cell behavior, hypothesized to be determined by initial position and noise, is faithfully recapitulated. Using the p21-/Cdk2-dependent quiescence-proliferation decision in breast cancer dormancy as a testbed, we examined single-cell dynamics on the landscape when perturbed by hypoxia, a dormancy-inducing stress. Combining trajectory-based energy landscape generation with single-cell time-lapse microscopy, we found that initial position on a p21/Cdk2 landscape did not fully explain the observed cell-fate heterogeneity under hypoxia. Instead, cells with higher cell state velocities prior to hypoxia, influenced by epigenetic parameters, tended to remain proliferative under hypoxia. Thus, the fate decision on this landscape is significantly influenced by "inertia", a velocity-dependent ability to resist directional changes despite reshaping of the underlying landscape, superseding positional effects. Such inertial effects may markedly influence cell-fate trajectories in tumors and other dynamically changing microenvironments.

Coordination of MAPK and p53 dynamics in the cellular responses to DNA damage and oxidative stress.

In response to different cellular stresses, the transcription factor p53 undergoes different dynamics. p53 dynamics, in turn, control cell fate. However, distinct stresses can generate the same p53 dynamics but different cell fate outcomes, suggesting integration of dynamic information from other pathways is important for cell fate regulation. To determine how MAPK activities affect p53-mediated responses to DNA breaks and oxidative stress, we simultaneously tracked p53 and either ERK, JNK, or p38 activities in single cells. While p53 dynamics were comparable between the stresses, cell fate outcomes were distinct. Combining MAPK dynamics with p53 dynamics was important for distinguishing between the stresses and for generating temporal ordering of cell fate pathways. Furthermore, cross-talk between MAPKs and p53 controlled the balance between proliferation and cell death. These findings provide insight into how cells integrate signaling pathways with distinct temporal patterns of activity to encode stress specificity and drive different cell fate decisions.

Estradiol deficiency reduces the satellite cell pool by impairing cell cycle progression.

The size of the satellite cell pool is reduced in estradiol (E2)-deficient female mice and humans. Here, we use a combination of in vivo and in vitro approaches to identify mechanisms, whereby E2 deficiency impairs satellite cell maintenance. By measuring satellite cell numbers in mice at several early time points postovariectomy (Ovx), we determine that satellite cell numbers decline by 33% between 10 and 14 days post-Ovx in tibialis anterior and gastrocnemius muscles. At 14 days post-Ovx, we demonstrate that satellite cells have a reduced propensity to transition from G0/G1 to S and G2/M phases, compared with cells from ovary-intact mice, associated with changes in two key satellite cell cycle regulators, ccna2 and p16INK4a. Further, freshly isolated satellite cells treated with E2 in vitro have 62% greater cell proliferation and require less time to complete the first division. Using clonal and differentiation assays, we measured 69% larger satellite cell colonies and enhanced satellite cell-derived myoblast differentiation with E2 treatment compared with vehicle-treated cells. Together, these results identify a novel mechanism for preservation of the satellite cell pool by E2 via promotion of satellite cell cycling.

Progress and challenges in understanding the regulation and function of p53 dynamics.

The dynamics of p53 expression provide a mechanism to increase differentiation between cellular stresses and specificity in appropriate responses. Here, we review recent advances in our understanding of the molecular mechanisms regulating p53 dynamics and the functions of the dynamics in the regulation of p53-dependent cell stress responses. We also compare dynamic encoding in the p53 system with that found in other important cell signaling systems, many of which can interact with the p53 network. Finally, we highlight some of the current challenges in understanding dynamic cell signaling within a larger cellular network context.

Rucaparib Treatment Alters p53 Oscillations in Single Cells to Enhance DNA-Double-Strand-Break-Induced Cell Cycle Arrest.

DNA double strand breaks induce oscillatory expression of the transcription factor p53 that is dependent on ataxia telangiectasia mutated (ATM) activity and the rate of double strand break resolution. Although p53 dynamics are known to play a role in the regulation of cell fate determination, the consequences of the variability in dynamics associated with differences in repair rates and utilized repair pathways are unknown. Using single-cell time-lapse microscopy, we found that disruption of specific repair pathways has distinct impacts on p53 dynamics. The small-molecule rucaparib, an inhibitor of the alternative end-joining-associated protein poly (ADP-ribose) polymerase (PARP), increased p53 pulse duration, altering the temporal expression of multiple p53 target genes. As a result, combination treatments of the radiomimetic drug neocarzinostatin with rucaparib drove prolonged growth arrest beyond that of DNA damage alone. This study highlights how pharmacological manipulation of DNA repair pathways may be used to alter p53 dynamics to enhance therapeutic regimens.

FUBP1 and FUBP2 enforce distinct epigenetic setpoints for MYC expression in primary single murine cells.

Physiologically, MYC levels must be precisely set to faithfully amplify the transcriptome, but in cancer MYC is quantitatively misregulated. Here, we study the variation of MYC amongst single primary cells (B-cells and murine embryonic fibroblasts, MEFs) for the repercussions of variable cellular MYC-levels and setpoints. Because FUBPs have been proposed to be molecular "cruise controls" that constrain MYC expression, their role in determining basal or activated MYC-levels was also examined. Growing cells remember low and high-MYC setpoints through multiple cell divisions and are limited by the same expression ceiling even after modest MYC-activation. High MYC MEFs are enriched for mRNAs regulating inflammation and immunity. After strong stimulation, many cells break through the ceiling and intensify MYC expression. Lacking FUBPs, unstimulated MEFs express levels otherwise attained only with stimulation and sponsor MYC chromatin changes, revealed by chromatin marks. Thus, the FUBPs enforce epigenetic setpoints that restrict MYC expression.