MICU1 loss-of-function variants in human patients are associated with proximal muscle weakness and myopathy. Mitochondrial Ca2+ levels are basally elevated when MICU1, the gatekeeper of the mitochondrial Ca2+ uniporter, is absent. The importance of regulating mitochondrial Ca2+ in skeletal muscle has generally been studied in mature muscle fibers. How satellite cells are impacted by mitochondrial Ca2+ dysregulation is poorly understood. We investigated Micu1 deletion specifically in Pax7+ satellite cells to address this gap in knowledge. Colony-forming activity in vitro was unaffected in Micu1-deficient satellite cells, but colony sizes were smaller. Although satellite cell homeostasis was not significantly affected 1 month following Micu1 deletion, the regenerative response post-injury was significantly impaired. Satellite cell self-renewal from Micu1-deficient donor cells in transplant recipients was also heavily compromised. Our data suggest that properly gating mitochondrial Ca2+ import via the uniporter is integral to satellite cell activation from quiescence in response to muscle injury.

Oscillatory p53 expression occurs in individual cells responding to DNA breaks. While the majority of cells exhibit the same qualitative response, quantitative features of the oscillations (e.g., amplitude or period) can be highly variable between cells, generating heterogeneity in downstream cell fate responses. Since heterogeneity can be detrimental to therapies based on DNA damage, methods to induce synchronization of p53 oscillations across cells in a population have the potential to generate more predictable responses to DNA-damaging treatments. Using mathematical modeling and time-lapse microscopy, we demonstrated that p53 oscillations can be synchronized through the phenomenon of phase resetting. Surprisingly, p53 oscillations were synchronized over a wider range of damage-induction frequencies than predicted computationally. Recapitulating the range of synchronizing frequencies required, non-intuitively, a less robust oscillator. We showed that p53 phase resetting altered the expression of downstream targets responsible for cell fate depending on target mRNA stability. This study demonstrates that p53 oscillations can be phase reset and highlights the potential of driving p53 dynamics to reduce cellular variability and synchronize cell fate responses to DNA damage.

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.

Hyperproliferation driven by the protooncogene MYC may lead to tumor suppressor p53 activating DNA damage that has been presumed to derive from hypertranscription and over-replication. Here, we report that excessive MYC-topoisome (MYC/topoisomerase 1/topoisomerase 2) activity acutely damages DNA-activating pATM and p53. In turn, MYC is shut off and degraded, releasing TOP1 and TOP2A from MYC topoisomes in vitro and in vivo. To manage the topological and torsional stress generated at its target genes, p53 assembles a separate topoisome. Because topoisomerase activity is intrinsically DNA damaging, p53 topoisomes provoke an initial burst of DNA damage. Because p53, unlike MYC, upregulates the DNA-damage response (DDR) and activates tyrosyl-DNA-phosphodiesterase (TDP) 1 and TDP2, it suppresses further topoisome-mediated damage. The physical coupling and activation of TOP1 and TOP2 by p53 creates a tool that supports p53-target expression while braking MYC-driven proliferation in mammalian cells.

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.

primer sequences for qPCR of NF-kB regulated genes, and for chromatin immunoprecipitation

Ovarian cancer cell line characteristics are listed with respect to source, tissue site, p53 mutation status, and histology at diagnosis.

RELA Chromatin immunoprecipitation, PCR

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.

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.

Immunofluorescence image acquisition and analysis procedures

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.

The size of the satellite cell pool is reduced in estradiol (E 2 )-deficient female mice and humans. Here, we use a combination of in vivo and in vitro approaches to identify mechanisms, whereby E 2 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 G 0 /G 1 to S and G 2 /M phases, compared with cells from ovary-intact mice, associated with changes in two key satellite cell cycle regulators, ccna2 and p16 INK4a . Further, freshly isolated satellite cells treated with E 2 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 E 2 treatment compared with vehicle-treated cells. Together, these results identify a novel mechanism for preservation of the satellite cell pool by E 2 via promotion of satellite cell cycling.

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.

The transcription factor p53 regulates the cellular response to numerous stresses. A major activating stress for p53 is DNA damage, which can arise from several sources including chemotherapeutic drugs, gamma irradiation, and UV light. Once activated, it generates several downstream effects, including DNA repair, cell cycle arrest, and apoptosis. With the development of single-cell, time-lapse fluorescence microscopy technique, recent studies have shown that the dynamics of the tumor suppressor p53 encode information about stresses to which a cell responds and are decoded differentially to regulate appropriate responses, including cell cycle progression. Although it is known that distinct DNA damage repair pathways are activated when cells are damaged in different cell cycle phases, the dependence of p53 dynamics on cell cycle phase and how this relationship shapes p53-mediated cell fate decisions are poorly understood. In this study, we used long-term time-lapse fluorescence microscopy to track p53 dynamics as a function of cell cycle phase in individual cells in response to single-strand DNA damage induced by UV radiation. We found that p53 dynamics depend on both UV dose and cell cycle phase. We identify key mediators shaping the differential response, as well as downstream effects mediated by p53 dynamics. This study may help enhance cancer therapies based on the timing of administration of cell-cycle modulators and DNA damaging treatments.

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract We previously used a pulse-based in vitro assay to unveil targetable signalling pathways associated with innate cisplatin resistance in lung adenocarcinoma (Hastings et al., 2020). Here, we advanced this model system and identified a non-genetic mechanism of resistance that drives recovery and regrowth in a subset of cells. Using RNAseq and a suite of biosensors to track single-cell fates both in vitro and in vivo, we identified that early S phase cells have a greater ability to maintain proliferative capacity, which correlated with reduced DNA damage over multiple generations. In contrast, cells in G1, late S or those treated with PARP/RAD51 inhibitors, maintained higher levels of DNA damage and underwent prolonged S/G2 phase arrest and senescence. Combined with our previous work, these data indicate that there is a non-genetic mechanism of resistance in human lung adenocarcinoma that is dependent on the cell cycle stage at the time of cisplatin exposure. Introduction Lung adenocarcinoma (LUAD) is the most common form of lung cancer and the leading cause of cancer-related death in Australia. Less than 15% of patients have a targetable driver mutation and therefore cannot benefit from targeted therapy (Herbst et al., 2018). Consequently, the overwhelming majority of LUAD patients receive platinum-based chemotherapy as standard of care. The anti-tumour abilities of platinum compounds were first identified over 50 years ago with the discovery of cisplatin (Kelland, 2007). Since then, cisplatin and its derivatives have become one of the most successful groups of chemotherapeutics ever developed. Platinum therapy is essentially curative in testicular cancer, with survival rates > 90%, and is also a frontline treatment for small-cell lung cancer, ovarian, head and neck, bladder, and cervical cancers (Gonzalez-Rajal et al., 2020; Kelland, 2007). Unfortunately, response rates to platinum in LUAD are below 30%, due primarily to innate resistance (Herbst et al., 2018). Nearly 150 different mechanisms of platinum resistance have been identified to date (Stewart, 2007). The vast majority of these mechanisms have been derived from preclinical models that utilise continuous, high-dose-exposure models, well above what is physiologically achievable in patients. Unsurprisingly, the majority of these models have failed to translate into improved clinical outcomes. To overcome this, we recently demonstrated that analysis of an in vitro assay that accurately models the in vivo drug exposure kinetics for cisplatin could provide therapeutically relevant insights into the signalling dynamics associated with innate resistance (Hastings et al., 2020). Cisplatin is given to patients as a single bolus dose, reaching a peak plasma concentration of ~14 µM (5 µg/ml), which is then rapidly cleared by the kidneys within 2–4 hr (Andersson et al., 1996; Urien and Lokiec, 2004). We therefore mimicked this in vitro by pulsing cells for 2 hr with the maximum plasma concentration (Hastings et al., 2020). Once inside cells, platinum compounds can bind to DNA, RNA, and proteins (Gonzalez-Rajal et al., 2020); however, the binding to DNA, which forms platinum-DNA adducts, is thought to be the primary mechanism for their tumour-specific killing. Intra-strand DNA-platinum adducts are repaired by base excision and nucleotide excision repair during G1 (Slyskova et al., 2018). Inter-strand crosslinks (ICLs) are removed largely by the Fanconi anaemia (FA) pathway (Smogorzewska, 2019), which generates single- and double-strand breaks that are resolved by either the high-fidelity homologous recombination (HR) pathway during S phase (Karanam et al., 2012) or by the error-prone non-homologous end joining (NHEJ) pathway during G1 and G2 phase (Enoiu et al., 2012; Slyskova et al., 2018). Consequently, targeting DNA repair pathways has become a major focus for enhancing platinum chemotherapies. For example, cells with defective HR repair have been shown to be highly sensitive to combination therapy with cisplatin and PARP inhibitors in a number of cancer types, including ovarian and breast (Tutt et al., 2018). However, correlation between cisplatin sensitivity and impaired DNA repair has often failed to translate clinically in LUAD (Mamdani and Jalal, 2016). In contrast, we have recently identified TGF-β (Marini et al., 2018) and P70S6K (Hastings et al., 2020) as key mediators of innate platinum resistance in LUAD. We now build upon these previous results and identify in this research advance that a sub-population of cells are capable of continued proliferation despite exposure to pulsed cisplatin. Using a combination of cell cycle, DNA damage, and replication biosensors together with real-time single-cell fate tracking, we identified that these proliferative cells were enriched in late G1/early S phase at the time of cisplatin exposure and were able to sufficiently repair their DNA over multiple generations and rounds of replication. These results increase our understanding of the complexities underlying non-genetic resistance and recovery mechanisms in LUAD, while also highlighting mechanistic issues with a number of current clinical trials focused on combination therapy with cisplatin. Results Cells remain equally sensitive upon re-exposure to pulsed cisplatin In our previous work (Hastings et al., 2020), we identified several targetable signalling pathways that were associated with resistance to cisplatin in LUAD cells. In this work, our goal was to analyse the innate mechanisms that enable cell survival after the initial exposure to cisplatin. To assess this, we analysed how cells respond to a subsequent dose of cisplatin, following recovery from an initial exposure event. To do this, we pulsed cells with cisplatin (5 µg/ml) and followed their response by time-lapse imaging. Cells were allowed to recover for 21–42 days (depending on their base rate of proliferation), before being challenged again with cisplatin, which equates to the approximate time patients normally receive a second dose in the clinic (Figure 1A). We utilised three LUAD cell lines: A549 (wild-type p53), NCI-H1573 (p53R248L mutant), and NCI-H1299 (p53 null), which were all engineered to stably express histone H2B fused to mCherry, allowing real-time quantitation of cell number and nuclear size. The initial pulse of cisplatin blocked the proliferation of A549 and NCI-H1573 cells, and significantly reduced NCI-H1299 cell numbers over a 3-day period. This was mirrored in colony formation assays, with both A549 and NCI-H1573 showing strong suppression of colony outgrowth, while p53-null H1299 cells were impacted to a lesser degree (Figure 1—figure supplement 1A). Interestingly, there was a less noticeable effect on cell confluence (Figure 1B). Subsequent visual and quantitative analysis of cells by immunofluorescence revealed a corresponding two- to sixfold increase in total cell and nuclear area across all three cell lines (Figure 1C, D), accounting for the reduced impact on confluence. Over the following 7–21 days of recovery (42 days for NCI-H1573 due to slower rate of proliferation), cells eventually recovered to their pre-pulse size and normal cell cycle distribution (Figure 1C, D, Figure 1—figure supplement 1B). In all three cell lines, subsequent cisplatin pulse of recovered cultures resulted in a near identical response to the initial pulse, both in terms of inhibition of cell number, reduced confluence, and increased cell size (Figure 1B–D). Based on these results, and our previous observation that all cells contained significantly increased levels of cisplatin-DNA adducts (Hastings et al., 2020), we concluded that cells surviving the first exposure remained equally sensitive to cisplatin and were therefore unlikely to have acquired resistance or arisen from an intrinsically resistant sub-clonal population within each cell line. Figure 1 with 1 supplement see all Download asset Open asset Rechallenging lung adenocarcinoma (LUAD) cells with cisplatin results in similar response profiles. (A) Schematic of rechallenging experiments. Briefly, cells stably expressing H2B-mCherry were pulsed with 5 µg/ml cisplatin for 2 hr. Cell proliferation (nuclear number and cell size) was then tracked for up to 4 days. Cells were then allowed to recover for 21 (A549 and NCI-H1299) or 42 days (NCI-H1573), re-culturing once confluent, before being re-pulsed with cisplatin. (B) Cell confluence and cell number were tracked for up to 4 days using IncuCyte based time-lapse imaging. Shown are the mean ± SD of n = 3 biological repeats. (C) Immunofluorescence of cells at 72 hr post-cisplatin treatment. Nuclei: cyan; Phalloidin-Alexa 647: red, scale bar = 10 µm. (D) Quantification of cell size and nuclear size from (A), with a minimum of n = 200 cells analysed per condition. Shown are the mean ± SD. Statistical significance was determined by one-way ANOVA (****p<0.0001, n.s = not significant). To assess this, we analysed the variability of cell and nuclear size after the initial pulse of cisplatin at 3–7 days post exposure. Visual analysis identified several colonies of cells whose size was similar to that of untreated control cells (Figure 2A, B). We hypothesised that these cells were able to proliferate and outgrow the non-proliferative (arrested) cells over the 21-to-42-day period. In support, a significant increase in senescence associated beta-galactosidase (β-gal) staining was observed both visually and by flow cytometry (C12FDG) in A549 and to a lesser extent H1573 and H1299 cell lines (Figure 2—figure supplement 1A, B). Increased levels of the cyclin-dependent kinase inhibitor p16, which is commonly linked with senescence, was associated with larger cells in NCI-H1573 and H1299 cells, while in A549 cells, which are null for p16 (Kawabe et al., 2000), p21 was similarly increased (Figure 2—figure supplement 1C). These data suggest that in each cell line the larger cells have a lower proliferative capacity and are likely senescent. Figure 2 with 1 supplement see all Download asset Open asset Variable cell size and clonal outgrowth in post-cisplatin-treated cells. (A) Representative images from cells treated as per Figure 1A showing control (Cont.), proliferative (Prolif.), and arrested (Arrest) cells. Scale bars = 100 µm. (B) Cell and nuclear size were calculated on sub-populations of cells that appeared to form clonal outgrowths. Quantification of cell size and nuclear size from (A), with a minimum of n = 100 cells analysed per condition. Shown are the mean ± SD. Statistical significance was determined by one-way ANOVA (****p<0.0001, n.s = not significant). (C) LeGO RGB colour-guided clonal cell tracking system was used to track clonal dynamics after cisplatin pulse treatment. Cells were treated as per Figure 1A, with clonal identification and quantification (D), measured at 3 and 21 days post-cisplatin exposure using Image J/Fiji (42 days post-cisplatin exposure for NCI-H1573). To test whether the surviving population arose from a subset of proliferating cells, we utilised the LeGO RGB colour-guided clonal cell tracking system (Weber et al., 2011). Briefly, each cell line was co-transfected with three different lentiviral vectors containing either a red, green, or blue fluorescent protein. Each cell randomly received a variable amount of each plasmid resulting in a unique colour code for each cell. Quantitative colour analysis of untreated control cells revealed that up to 64 unique colours could be detected in A549 and NCI-H1299 cells and up to 46 colours in NCI-H1573 cells (Figure 2—figure supplement 1D). After pulsed exposure to cisplatin, single-colour colony outgrowths were clearly visible in all three cell lines at 3–7 days post exposure, which was maintained at 21 for A549 and NCI-H1299 or 42 days for NCI-H1573 cells (Figure 2C, D). Colour diversity in cisplatin-recovered cells correlated with the colony formation assay data (Figure 1—figure supplement 1A), with NCI-H1299 (p53 null) cells displaying a greater variability in recovered clonal colours compared to A549 (p53 wt) and NCI-H1573 (p53 mutant) cells (Figure 2C, Figure 2—figure supplement 1D). Taken together, these results indicate that repopulation of the culture after the initial pulse exposure is primarily driven by a small fraction of cells, with the total number of clones impacted by p53 status. To confirm these results in vivo, we injected A549, NCI-H1573, or NCI-H1299 cells subcutaneously into the flanks of nude mice and allowed establishment of tumours (150 mm3) before administering a single treatment of carboplatin (60 mg/kg). Mice harvested at 3 days post treatment were analysed by imunohistochemistry (IHC) for cell size and proliferating cell nuclear antigen (PCNA)-positive staining, with all cell lines showing a significant increase in cell size (Figure 3A, B). Similar to the in vitro results, active proliferation (PCNA positivity) in A549 cells was strongly suppressed after carboplatin exposure, indicating that the majority of cells were not proliferating. In p53 mutant NCI-1573, there was no significant reduction in PCNA in vivo despite significant reduction in cell numbers in vitro cells. In contrast, NCI-H1299 cells, which lack p53, did not show any significant decrease in PCNA staining in vivo (Figure 3A, B). BrdU pulse labelling of in vitro-treated cells showed similar results, with strong suppression of active BrdU incorporation in A549 cells, with partial and no significant inhibition seen in NCI-H1573 and NCI-H1299 cells, respectively (Figure 3—figure supplement 1). In summary, these in vitro and in vivo data suggest that in LUAD cells exposure to pulsed cisplatin results in a significant proportion of cells increasing in size, with presence of functional p53 correlating with increased senescence and reduced active replication in enlarged cells. Interestingly, in all three lines, a sub-population of cells remain at a normal (stable) cell size, maintained their proliferative capacity, and drove repopulation of the cell culture. Despite this, these proliferative cells remained equally sensitive to subsequent cisplatin treatment, indicating a non-genetic mechanism of resistance. Figure 3 with 1 supplement see all Download asset Open asset In vivo validation of cisplatin effects on cell size and proliferation. (A) A549 and NCI-H1299 cells were injected subcutaneously with 2 × 106 cells into the flanks of nude mice (n = 5). Carboplatin (60 mg/kg) was delivered by a single tail-vein injection, and tumours were harvested at 3 days post treatment and analysed by IHC for cell size and PCNA-positive staining. Scale bar = 50 µm. (B) Quantification of IHC images from (A) (control n = 300, carbo n = 400). Shown are the mean ± SD. Statistical significance was determined by unpaired two-tailed Students t-test (****p<0.0001, n.s = not significant). Differential RNAseq analysis of cisplatin-treated sub-populations To better understand the potential mechanism driving the difference between cells that become enlarged and senescent compared to those that maintain stable size and proliferative capacity, we performed RNAseq analysis on each unique population. Briefly, A549 cells were pulsed with cisplatin, harvested at 72 hr, and sorted into stable or enlarged cells based on size. This was determined by forward scatter (FSC) and side scatter (SSC) parameters, with gates established based on the size of untreated control cells (Figure 4—figure supplement 1A). Pre- and post-sorted cells were then processed for RNAseq analysis (Figure 4A, B). Two-way hierarchical clustering indicated that there were clear differences in gene expression between cisplatin-treated cells that maintained a stable size compared to untreated control and enlarged cisplatin-treated cells (Figure 4C, Supplementary files 1–4). To better understand these effects, we undertook a more detailed bioinformatic analysis using Ingenuity Pathway Analysis (IPA). Strong upregulation of the CDK inhibitor p21 was present in both pre-sorted cisplatin-treated and post-sorted enlarged cells, matching the early flow data (Figure 2—figure supplement 1C). This corresponded with upregulation of p53, CHK, and G2/M cell cycle checkpoint signalling, and a corresponding reduction in DNA replication and increase in senescence pathways (Figure 4D, E), correlating with the increased β-gal and reduced proliferation observed above (Figure 1B, Figure 2—figure supplement 1B). Importantly, stable (size) cisplatin-treated cells were significantly different from untreated controls, indicating that these cells were impacted by cisplatin exposure, similar to our previous reports where all cells contained detectible cisplatin-DNA adducts post-pulsed exposure (Hastings et al., 2020). Notably, these stable cells displayed a decrease in EIF2, mTOR, and p70S6K signalling, with a corresponding increase in TREM1, GP6, and IL-17F signalling pathways (Figure 4F, Supplementary files 3 and 4). Four-way comparative analysis further highlighted key differences between each sub-population, with stable cells all showing strong upregulation of BRCA1, ATM, and DNA replication signalling pathways, which were heavily suppressed in non-proliferative cells (Figure 4G). Similarly, Gene Set Enrichment Analysis (GSEA) identified strong enrichment for cell cycle, HR-directed repair, ATR, and the Fanconi pathway in stable compared to enlarged cells (Figure 4—figure supplement 1B, C). In summary, these data suggest that the cisplatin-treated stable cells are considerably different from enlarged and untreated control cells. Specifically, stable cells do not undergo significant p53/p21-dependent cell cycle checkpoint arrest but do show prominent upregulation of DNA repair pathways involving HR/BRCA1 and ATM/ATR. Figure 4 with 1 supplement see all Download asset Open asset Comparative RNAseq analysis of FACS-sorted cisplatin-treated cells. (A) Schematic (created with BioRender.com) describing treatment, sorting, and analysis pipeline. (B) Representative example of pre- and post-sorted control and cisplatin-treated cells. (C) Hierarchical clustering of three independent biological repeat experiments of all altered genes identified by RNAseq. (D–F) Volcano plots displaying significantly downregulated (blue) or upregulated (orange) genes and subsequent Ingenuity Pathway Analysis (IPA) canonical pathway analysis. Predictions of inhibition (blue) or activation (orange) or no change (white) states are based on the IngenuityKnowledge Base, which compares the expected change with experimental observation to all known upstream canonical regulatory pathways. Variable stringent p-value (>1.3) and z-score (>0.5) cut-offs were used to limit pathways to top 7–8 most significant hits. (G) Hierarchical clustering of IPA comparative canonical pathway analysis. Cell cycle and p53 status at the time of exposure correlates with cell fate outcomes The above RNAseq data indicated that there were strong cell cycle-dependent differences between proliferative and non-proliferative cisplatin-treated cells. To better understand these differences, we utilised the FUCCI biosensor system to enable real-time cell cycle status of individual cells, as published in the previous paper (Hastings et al., 2020). Briefly, asynchronous A549 cells stably expressing FUCCI were pulsed with or without cisplatin and then followed by time-lapse microscopy for 72 hr. Individual cells were manually tracked and scored for cell cycle status and cell fate as previously described (Caldon and Burgess, 2019; Hastings et al., 2020). The majority of control cells divided at least two times within the 72 hr time period (Figure 5A, Figure 5—video 1). In contrast and as demonstrated previously (Hastings et al., 2020), cisplatin-treated cells showed a range of cell cycle perturbations (Figure 5B, Figure 5—figure supplement 1A), including a significant and prolonged S/G2 phase arrest (Figure 5—figure supplement 1B), which correlated with a reduced number of total divisions (Figure 5—figure supplement 1C). Combining this with additional scoring of cisplatin-treated cells that underwent multiple (two or more) divisions within the 72 hr period revealed an enrichment for cells that were in late G1 and early S phase at the time of cisplatin exposure (Figure 5B). Unbiased analysis of an additional 400 cisplatin-treated A549 cells found a significant increase in the number of divisions arising from cells that were in G1/S or early S phase at the time of exposure compared to G1 phase cells (Figure 5C). Furthermore, the overwhelming majority of cells in late S or G2/M only completed one division during the 72 hr period. Taken together, these data suggest that there is a cell cycle-dependent mechanism driving the ability of A549 cells to continue to proliferate after cisplatin exposure. Figure 5 with 4 supplements see all Download asset Open asset Cell cycle and p53 status at the time of exposure correlates with cell fate outcomes. (A, B) The fate of asynchronously growing FUCCI expressing A549 individual control (n = 50) and cisplatin pulsed cells (n = 100) was tracked by time-lapse microscopy, with images taken every 30 min for 72 hr. Cisplatin-treated cell analysis was slightly biased for cells that underwent multiple divisions. (C) Violin plots of the number of divisions from cells in (A, B), based on the cell cycle phase at the time of cisplatin exposure. Statistical significance was determined by one-way ANOVA with Tukey’s correction for multiple comparisons (**p<0.01, ****p<0.0001, ns = not significant). (D) Schematic (created with BioRender.com) of optical window-based longitudinal in vivo imaging of FUCCI A549 cells. (E) Representative 3D projection images from mice imaged at days 1 and 7 with carboplatin (Carbo) or control (Saline). (F) Quantification of the proportion of red (G1), yellow (G1/S), and green (S/G2-M) cells found in tumours (n = 3) from day 0 to 7. Our above data indicated that loss of p53 increased the number of clones capable of regrowth after cisplatin exposure. To assess this in greater detail, we analysed asynchronous FUCCI expressing NCI-H1299 (p53 null) cells pulsed with or without (Control) cisplatin. Treatment with cisplatin significantly reduced the number of overall divisions in H1299 cells (Figure 5—figure supplement 2A–C); however, compared to A549 cells, this reduction was not as pronounced, in line with the cell proliferation data observed above (Figure 1B). Single-cell fate tracking of NCI-H1299 cells suggested a small bias for higher rates of division in cells that were in late G1 and early S phase at the time of treatment (Figure 5—figure supplement 2B, C); however, this trend was not significant (Figure 5—figure supplement 2D). We further assessed p53 loss by reanalysing our previously published data on siRNA knockdown of p53 in A549 cells (Hastings et al., 2020). Notably, depletion of p53 in A549 cells resulted in a corresponding increase in the number of divisions completed after pulsed exposure to cisplatin, supporting the NCI-H1299 data (Figure 5—figure supplement 2E). To validate these results in vivo, we implanted FUCCI expressing A549, NCI-H1573, or NCI-H1299 cells under optical windows in mice (Figure 5D). Tumours were allowed to establish before mice were given a single dose of carboplatin. Individual mice were then repeatedly imaged over 7 days post treatment. Notably, prior to cisplatin treatment, approximately 70–80% of cells from each line were in G1 phase (Figure 5E, F, Figure 5—figure supplement 3A, B). Similar to in vitro results, we observed an increase in proportion of S/G2 phase cells at day 1 in all cell lines, indicating that cells were arrested in S/G2. In A549 cells, the percentage of S/G2 cells reduced gradually from day 3 to 7, resulting in over 90% of A549 cells in a G1 like state (red) at 7 days post treatment (Figure 5E, F). In contrast, the percentage of S/G2 cells in both NCI-H1573 or NCI-H1299 increased until day 3, before returning to pre-treatment levels by day 7 (Figure 5—figure supplement 3A, B). This mirrored our above in vitro data, where the presence of wild-type p53 (in A549 cells) corresponded with a higher rate of G2-exit and senescence compared to p53 null and mutant cells (Hastings et al., 2020), suggesting that the results we observed in vitro are recapitulated in vivo. Cisplatin treatment during late G1 early S phase correlates with multiple divisions The above data indicated that in p53 wild-type cells those in late G1 and early S phase at the time of exposure had a greater capacity to undergo multiple division compared to cells G1 and S-G2/M phase. To assess this in greater detail, we synchronised and released FUCCI expressing A549 cells into either G1 or early S phase using either palbociclib or thymidine, respectively (Figure 6A, B, Figure 6—video 1, Figure 6—video 2). We combined these synchronisations with pulsed exposure to cisplatin at various points following release to target G1, early or late S phase populations, which were then monitored by time-lapse microscopy. To target G1, cells were pulsed with cisplatin upon release from palbociclib (Cis at G1). This resulted in the majority of cells undergoing a prolonged S/G2 phase and then exiting back into a G1-like state without undergoing mitosis (G2-exit; Figure 6C), a state we described previously (Hastings et al., 2020). Notably, only 7/50 cells completed a single division, and no cells underwent multiple (two or more) divisions during the 72 hr time period (Figure 6A–D; proliferative). In contrast, targeting cells in G1/S (Cis at G1/S) using either palbociclib or thymidine synchronisation with cisplatin resulted in significantly more cells (13/50 and 16/50, respectively) completing two or more divisions (Figure 6A–D; proliferative). Finally, nearly all cells targeted in late S phase (Cis at S) completed the first mitosis and then underwent prolonged S/G2 arrest and G2-exit, with only 4 out of 50 cells completing two divisions within the 72 hr period (Figure 6B–D). Taken together, these data indicate that cells in late G1 and early S at the time of cisplatin exposure have a greater capacity to continue proliferating. Figure 6 with 2 supplements see all Download asset Open asset Cell cycle-dependent regulation of cisplatin response. (A, B) Schematic of palbociclib (Palbo) and thymidine (Thy) protocols used to synchronise FUCCI expressing A549 cells in G1, G1/S, and S phase prior to 2 hr cisplatin pulse treatment. Specifically, G1 cells were released from palbociclib and pulsed immediately with cisplatin (Palbo + Cis at G1). G1/S cells were pulsed with cisplatin at 6 hr post release from palbociclib (Palbo + Cis at G1/S), or alternatively G1/S phase cells targeted by treatment with cisplatin 2 hr prior to release from thymidine (Thy + Cis at G1/S). Finally, S phase cells targeted by pulsing with cisplatin at 4 hr post-thymidine release (Thy + Cis at S). The fate of individual cells (n = 50) was tracked by time-lapse microscopy, with images taken every 30 min for 72 hr. (C) Quantification of cell fate outcomes from (A), including G1 arrest before mitosis (G1 ABM), G1 arrest after mitosis (G1 AAM), death before mitosis (DBM), and death after mitosis (DAM) and proliferative (Prolif.). (D) Quantification of the total number of cell divisions observed in each condition (n = 50). Mean is shown, statistical significance was determined by one-way ANOVA with Tukey’s correction for multiple comparisons (****p<0.0001, *p<0.05). Disruption of DNA repair reduces ability of early S phase cells to proliferate A major target of cisplatin is DNA, with intra-strand crosslinks and ICLs disrupting replication and repair, leading to stalled replication forks and the formation of double-strand breaks (Gonzalez-Rajal et al., 2020). We therefore hypothesised that cells in late G1/early S phase were able to repair cisplatin-induced DNA damage during the first cell cycle more efficiently than cells in early G1 or late S phase, thereby allowing them to continue proliferation. To test this, we engineered A549 cells to stably co-express a truncated form of 53BP1 fused to mApple (Apple-53BP1trunc), which has previously been shown to bind double-strand break sites co-marked with γH2A.X but lacks any of the functional domains of 53BP1 (Yang et al., 2015). We combined this with a PCNA chromobody, where we replaced GFP with mNeonGreen, to mark sites of active DNA replication (Figure 7A, inset). Cells were synchronised with thymidine and pulsed with cisplatin 2 hr prior to release to enrich for early S phase-targeted cells (as per Figure 6B), and then tracked by 4D live*cell imaging. Individual cells were divided into either those in G1, early S, or mid/late S based on the pattern of PCNA foci (cyan), and then tracked through time (Burgess et al., 2012; Charrasse et al., 2017). In control cells, a small number (<20) of 53BP1-positive foci (red hot) were observed as cells underwent the first round of replication (Figure 7A, B, Figure 7—video 1). Daughter and grand-daughter cells then displayed several (<5) large foci during G1 (up to 5 µm2), which were resolved as cells entered S phase and began replicating (Figure 7C, white arrow). Cells that were in G1 at the time of cisplatin exposure entered S phase and rapidly accumulated a large number (~100) of 53BP1-positive foci; these slowly reduced over the remainder of the time lapse (Figure 7A–C), which corresponded with an increase in the average size of the foci (~1 µm2). In c

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.

Supraphysiological MYC levels are oncogenic. Originally considered a typical transcription factor recruited to E-boxes (CACGTG), another theory posits MYC a global amplifier increasing output at all active promoters. Both models rest on large-scale genome-wide ”-omics’. Because the assumptions, statistical parameter and model choice dictates the ‘-omic’ results, whether MYC is a general or specific transcription factor remains controversial. Therefore, an orthogonal series of experiments interrogated MYC’s effect on the expression of synthetic reporters. Dose-dependently, MYC increased output at minimal promoters with or without an E-box. Driving minimal promoters with exogenous (glucocorticoid receptor) or synthetic transcription factors made expression more MYC-responsive, effectively increasing MYC-amplifier gain. Mutations of conserved MYC-Box regions I and II impaired amplification, whereas MYC-box III mutations delivered higher reporter output indicating that MBIII limits over-amplification. Kinetic theory and experiments indicate that MYC activates at least two steps in the transcription-cycle to explain the non-linear amplification of transcription that is essential for global, supraphysiological transcription in cancer.

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.