Mutant p53 reactivation restricts the protumorigenic consequences of wild type p53 loss of heterozygosity in Li-Fraumeni syndrome patient-derived fibroblasts.

The p53 tumor suppressor, encoded by the TP53 gene, serves as a major barrier against malignant transformation. Patients with Li-Fraumeni syndrome (LFS) inherit a mutated TP53 allele from one parent and a wild-type TP53 allele from the other. Subsequently, the wild-type allele is lost and only the mutant TP53 allele remains. This process, which is termed loss of heterozygosity (LOH), results in only mutant p53 protein expression. We used primary dermal fibroblasts from LFS patients carrying the hotspot p53 gain-of-function pathogenic variant, R248Q to study the LOH process and characterize alterations in various pathways before and after LOH. We previously described the derivation of mutant p53 reactivating peptides, designated pCAPs (p53 Conformation Activating Peptides). In this study, we tested the effect of lead peptide pCAP-250 on LOH and on its associated cellular changes. We report that treatment of LFS fibroblasts with pCAP-250 prevents the accumulation of mutant p53 protein, inhibits LOH, and alleviates its cellular consequences. Furthermore, prolonged treatment with pCAP-250 significantly reduces DNA damage and restores long-term genomic stability. pCAPs may thus be contemplated as a potential preventive treatment to prevent or delay early onset cancer in carriers of mutant p53.

Exposure to temporal variability promotes subsequent adaptation to new temporal regularities.

Noise is intuitively thought to interfere with perceptual learning; However, human and machine learning studies suggest that, in certain contexts, variability may reduce overfitting and improve generalizability. Whereas previous studies have examined the effects of variability in learned stimuli or tasks, it is hitherto unknown what are the effects of variability in the temporal environment. Here, we examined this question in two groups of adult participants (N = 40) presented with visual targets at either random or fixed temporal routines and then tested on the same type of targets at a new nearly-fixed temporal routine. Findings reveal that participants of the random group performed better and adapted quicker following a change in the timing routine, relative to participants of the fixed group. Corroborated with eye-tracking and computational modeling, these findings suggest that prior exposure to temporal variability promotes the formation of new temporal expectations and enhances generalizability in a dynamic environment. We conclude that noise plays an important role in promoting perceptual learning in the temporal domain: rather than interfering with the formation of temporal expectations, noise enhances them. This counterintuitive effect is hypothesized to be achieved through eliminating overfitting and promoting generalizability.

Sequential effect and temporal orienting in prestimulus oculomotor inhibition.

When faced with unfamiliar circumstances, we often turn to our past experiences with similar situations to shape our expectations. This results in the well-established sequential effect, in which previous trials influence the expectations of the current trial. Studies have revealed that, in addition to the classical behavioral metrics, the inhibition of eye movement could be used as a biomarker to study temporal expectations. This prestimulus oculomotor inhibition is found a few hundred milliseconds prior to predictable events, with a stronger inhibition for predictable than unpredictable events. The phenomenon has been found to occur in various temporal structures, such as rhythms, cue-association, and conditional probability, yet it is still unknown whether it reflects local sequential information of the previous trial. To explore this, we examined the relationship between the sequential effect and the prestimulus oculomotor inhibition. Our results (N = 40) revealed that inhibition was weaker when the previous trial was longer than the current trial, in line with findings of behavioral metrics. These findings indicate that the prestimulus oculomotor inhibition covaries with expectation based on local sequential information, demonstrating the tight connection between this phenomenon and expectation and providing a novel measurement for studying sequential effects in temporal expectation.

Contraction bias in temporal estimation.

When asked to compare the perceptual features of two serially presented objects, participants are often biased to over- or under-estimate the difference in magnitude between the stimuli. Overestimation occurs consistently when a) the two stimuli are relatively small in magnitude and the first stimulus is larger in magnitude than the second; or b) the two stimuli are relatively large in magnitude and the first stimulus is smaller in magnitude than the second; underestimation consistently occurs in the complementary cases. This systematic perceptual bias, known as the contraction bias, was demonstrated for a multitude of perceptual features and in various modalities. Here, we tested whether estimation of time-duration is affected by the contraction bias. In each trial of three experiments (n = 20 each), participants compared the duration of two visually presented stimuli. Findings revealed over- and under-estimation effects as predicted by the contraction bias. Here, we discuss this asymmetry and describe how these findings can be explained via a Bayesian inference framework.

Pseudo-mutant P53 is a unique phenotype of DNMT3A-mutated pre-leukemia.

Pre-leukemic clones carrying DNMT3A mutations have a selective advantage and an inherent chemoresistance, however the basis for this phenotype has not been fully elucidated. Mutations affecting the gene TP53 occur in pre-leukemic hematopoietic stem/progenitor cells (preL-HSPC) and lead to chemoresistance. Many of these mutations cause a conformational change and some of them were shown to enhance self-renewal capacity of preL-HSPC. Intriguingly, a misfolded P53 was described in AML blasts that do not harbor mutations in TP53, emphasizing the dynamic equilibrium between wild-type (WT) and "pseudo-mutant" conformations of P53. By combining single cell analyses and P53 conformation-specific monoclonal antibodies we studied preL-HSPC from primary human DNMT3A-mutated AML samples. We found that while leukemic blasts express mainly the WT conformation, in preL-HSPC the pseudo-mutant conformation is the dominant. HSPC from non-leukemic samples expressed both conformations to a similar extent. In a mouse model we found a small subset of HSPC with a dominant pseudo-mutant P53. This subpopulation was significantly larger among DNMT3AR882H-mutated HSPC, suggesting that while a pre-leukemic mutation can predispose for P53 misfolding, additional factors are involved as well. Treatment with a short peptide that can shift the dynamic equilibrium favoring the WT conformation of P53, specifically eliminated preL-HSPC that had dysfunctional canonical P53 pathway activity as reflected by single cell RNA sequencing. Our observations shed light upon a possible targetable P53 dysfunction in human preL-HSPC carrying DNMT3A mutations. This opens new avenues for leukemia prevention.

The Spatiotemporal Link of Temporal Expectations: Contextual Temporal Expectation Is Independent of Spatial Attention.

Temporal expectation is the ability to construct predictions regarding the timing of events, based on previously experienced temporal regularities of different types. For example, cue-based expectations are constructed when a cue validly indicates when a target is expected to occur. However, in the absence of such cues, expectations can be constructed based on contextual temporal information, including the onset distribution of the event and recent prior experiences, both providing implicit probabilistic information regarding the timing of the event. It was previously suggested that cue-based temporal expectation is exerted via synchronization of spatially specific neural activity at a predictable time of a target, within receptive fields corresponding to the expected location of the target. Here, we tested whether the same theoretical model holds for contextual temporal effects. Participants (n = 40, 25 females) performed a speeded spatial-cuing detection task with two-thirds valid spatial cues. The hazard-rate function of the target was modulated by varying the foreperiod-the interval between the spatial cue and the target-among trials and was manipulated between groups by changing the interval distribution. Reaction times were analyzed using both frequentist and Bayesian generalized linear mixed models, accounting for hazard and sequential effects. Results showed that the effects of contextual temporal structures on reaction times were independent of spatial attention. This suggests that the spatiotemporal mechanisms, thought to account for cue-based expectation, cannot explain other sources of temporal expectations. We conclude that expectations based on contextual structures have different characteristics than cue-based temporal expectation, suggesting reliance on distinct neural mechanisms.SIGNIFICANCE STATEMENT Temporal expectation is the ability to predict an event onset based on temporal regularities. A neurophysiological model suggested that temporal expectation relies on the synchronization of spatially specific neurons whose receptive fields represent the attended location. This model predicts that temporal expectation would be evident solely within the locus of spatial attention. Existing evidence supported this model for expectation based on associations between a temporal cue and a target, but here we show that it cannot account for temporal expectation that is based on contextual information, that is, the distribution of intervals and recent priors. These findings reveal the existence of different predictive mechanisms for cued and contextual temporal predictions, with the former depending on spatial attention and the latter nonspatially specific.

Prestimulus inhibition of eye movements reflects temporal expectation rather than time estimation.

Eye movements are inhibited prior to the occurrence of temporally predictable events. This 'oculomotor inhibition effect' has been demonstrated with various tasks and modalities. Specifically, it was shown that when intervals between cue and target are fixed, saccade rate prior to the target is lower than when they are varied. However, it is still an open question whether this effect is linked to temporal expectation to the predictable target, or to the duration estimation of the interval preceding it. Here, we examined this question in 20 participants while they performed an implicit temporal expectation and an explicit time estimation task. In each trial, following cue onset, two consecutive grating patches were presented, each preceded by an interval. Temporal expectation was manipulated by setting the first interval duration to be either fixed or varied within each block. Participants were requested to compare either the durations of the two intervals (time estimation), or the tilts of the two grating patches (temporal expectation). Saccade rate, measured prior to the first grating, was lower in the fixed relative to the varied condition of both tasks. This suggests that the inhibition effect is elicited by target predictability and indicates that it is linked to temporal expectation, rather than to time estimation processes. Additionally, this finding suggests that the oculomotor inhibition is independent of motor readiness, as it was elicited even when no response was required. We conclude that the prestimulus oculomotor inhibition effect can be used as a marker of temporal expectation, and discuss its potential underlying mechanisms.

Pre-target oculomotor inhibition reflects temporal orienting rather than certainty.

Recent studies suggested that eye movements are linked to temporal predictability. These studies manipulated predictability by setting the cue-target interval (foreperiod) to be fixed or random throughout the block. Findings showed that pre-target oculomotor behavior was reduced in the fixed relative to the random condition. This effect was interpreted as reflecting the formation of temporal expectation. However, it is unknown whether the effect is driven by target-specific temporal orienting, or rather a result of a more context-dependent state of certainty that participants may experience during blocks with a high predictability rate. In this study we dissociated certainty and orienting in a tilt-discrimination task. In each trial, a temporal cue (fixation color change) was followed by a tilted grating-patch. The foreperiod distribution was varied between blocks to be either fully fixed (same foreperiod in 100% of trials), mostly fixed (80% of trials with one foreperiod and 20% with another) or random (five foreperiods in equal probabilities). The two hypotheses led to different prediction models which were tested against the experimental data. Results were consistent with the orienting hypothesis and inconsistent with the certainty hypothesis, supporting the link between oculomotor inhibition and temporal orienting and its validity as a temporal expectations marker.

Oculomotor inhibition precedes temporally expected auditory targets.

Eye movements are inhibited prior to the onset of temporally-predictable visual targets. This oculomotor inhibition effect could be considered a marker for the formation of temporal expectations and the allocation of temporal attention in the visual domain. Here we show that eye movements are also inhibited before predictable auditory targets. In two experiments, we manipulate the period between a cue and an auditory target to be either predictable or unpredictable. The findings show that although there is no perceptual gain from avoiding gaze-shifts in this procedure, saccades and blinks are inhibited prior to predictable relative to unpredictable auditory targets. These findings show that oculomotor inhibition occurs prior to auditory targets. This link between auditory expectation and oculomotor behavior reveals a multimodal perception action coupling, which has a central role in temporal expectations.

Correction: Cancer therapeutic approach based on conformational stabilization of mutant p53 protein by small peptides.

[This corrects the article DOI: 10.18632/oncotarget.10516.].

Immune deficiency augments the prevalence of p53 loss of heterozygosity in spontaneous tumors but not bi-directional loss of heterozygosity in bone marrow progenitors.

p53 loss of heterozygosity (LOH) is a frequent event in tumors of somatic and Li-Fraumeni syndrome patients harboring p53 mutation. Here, we focused on resolving a possible crosstalk between the immune-system and p53 LOH. Previously, we reported that p53 heterozygous bone-marrow mesenchymal progenitor cells undergo p53 LOH in-vivo. Surprisingly, the loss of either the wild-type p53 allele or mutant p53 allele was detected with a three-to-one ratio in favor of losing the mutant allele. In this study, we examined whether the immune-system can affect the LOH directionality in bone marrow progenitors. We found that mesenchymal progenitor cells derived from immune-deficient mice exhibited the same preference of losing the mutant p53 allele as immune-competent matched cells, nevertheless, these animals showed a significantly shorter tumor-free survival, indicating the possible involvement of immune surveillance in this model. Surprisingly, spontaneous tumors of p53 heterozygous immune-deficient mice exhibited a significantly higher incidence of p53 LOH compared to that observed in tumors derived of p53 heterozygous immune-competent mice. These findings indicate that the immune-system may affect the p53 LOH prevalence in spontaneous tumors. Thus suggesting that the immune-system may recognize and clear cells that underwent p53 LOH, whereas in immune-compromised mice, those cells will form tumors with shorter latency. In individuals with a competent immune-system, p53 LOH independent pathways may induce malignant transformation which requires a longer tumor latency. Moreover, this data may imply that the current immunotherapy treatment aimed at abrogating the inhibition of cellular immune checkpoints may be beneficial for LFS patients.

Cancer therapeutic approach based on conformational stabilization of mutant p53 protein by small peptides.

The p53 tumor suppressor serves as a major barrier against malignant transformation. Over 50% of tumors inactivate p53 by point mutations in its DNA binding domain. Most mutations destabilize p53 protein folding, causing its partial denaturation at physiological temperature. Thus a high proportion of human tumors overexpress a potential potent tumor suppressor in a non-functional, misfolded form. The equilibrium between the properly folded and misfolded states of p53 may be affected by molecules that interact with p53, stabilizing its native folding and restoring wild type p53 activity to cancer cells. To select for mutant p53 (mutp53) reactivating peptides, we adopted the phage display technology, allowing interactions between mutp53 and random peptide libraries presented on phages and enriching for phage that favor the correctly folded p53 conformation. We obtained a large database of potential reactivating peptides. Lead peptides were synthesized and analyzed for their ability to restore proper p53 folding and activity. Remarkably, many enriched peptides corresponded to known p53-binding proteins, including RAD9. Importantly, lead peptides elicited dramatic regression of aggressive tumors in mouse xenograft models. Such peptides might serve as novel agents for human cancer therapy.

Mutant p53 attenuates the anti-tumorigenic activity of fibroblasts-secreted interferon beta.

Mutations in the p53 tumor suppressor protein are highly frequent in tumors and often endow cells with tumorigenic capacities. We sought to examine a possible role for mutant p53 in the cross-talk between cancer cells and their surrounding stroma, which is a crucial factor affecting tumor outcome. Here we present a novel model which enables individual monitoring of the response of cancer cells and stromal cells (fibroblasts) to co-culturing. We found that fibroblasts elicit the interferon beta (IFNß) pathway when in contact with cancer cells, thereby inhibiting their migration. Mutant p53 in the tumor was able to alleviate this response via SOCS1 mediated inhibition of STAT1 phosphorylation. IFNß on the other hand, reduced mutant p53 RNA levels by restricting its RNA stabilizer, WIG1. These data underscore mutant p53 oncogenic properties in the context of the tumor microenvironment and suggest that mutant p53 positive cancer patients might benefit from IFNß treatment.