p53 is the major tumor suppressor and the most frequently inactivated gene in cancer. p53 could be disabled either by mutations or by upstream negative regulators, including, but not limited to MDM2 and MDMX. p53 activity is required for the prevention as well as for the eradication of cancers. Restoration of p53 activity in mouse models leads to the suppression of established tumors of different origin. These findings provide a strong support to the anti-cancer strategy aimed for p53 reactivation. In this review, we summarize recent progress in the development of small molecules, which restore the tumor suppressor function of wild-type p53 and discuss their clinical advance. We discuss different aspects of p53-mediated response, which contribute to suppression of tumors, including non-canonical p53 activities, such as regulation of immune response. While targeting p53 inhibitors is a very promising approach, there are certain limitations and concerns that the intensive research and clinical evaluation of compounds will hopefully help to overcome.

The p53-family member TAp73 is known to function as a tumor suppressor and regulates genomic integrity, cellular proliferation, and apoptosis; however, its role in tumor angiogenesis is poorly understood. Here we demonstrate that TAp73 regulates tumor angiogenesis through repression of proangiogenic and proinflammatory cytokines. Importantly, loss of TAp73 results in highly vascularized tumors, as well as an increase in vessel permeability resulting from disruption of vascular endothelial-cadherin junctions between endothelial cells. In contrast, loss of the oncogenic p73 isoform ΔNp73 leads to reduced blood vessel formation in tumors. Furthermore, we show that up-regulated ΔNp73 levels are associated with increased angiogenesis in human breast cancer and that inhibition of TAp73 results in an accumulation of HIF-1α and up-regulation of HIF-1α target genes. Taken together, our data demonstrate that loss of TAp73 or ΔNp73 up-regulation activates the angiogenic switch that stimulates tumor growth and progression.

The repression of repetitive elements is an important facet of p53's function as a guardian of the genome. Paradoxically, we found that p53 activated by MDM2 inhibitors induced the expression of endogenous retroviruses (ERVs) via increased occupancy on ERV promoters and inhibition of two major ERV repressors, histone demethylase LSD1 and DNA methyltransferase DNMT1. Double-stranded RNA stress caused by ERVs triggered type I/III interferons expression and antigen processing and presentation. Pharmacological activation of p53 in vivo unleashed the interferon program, promoted T cell infiltration and significantly enhanced the efficacy of checkpoint therapy in a xenograft tumor model. Furthermore, MDM2 inhibitor ALRN-6924 induced a viral mimicry pathway and tumor inflammation signature genes in melanoma patients.Our results identify ERV expression as the central mechanism whereby p53 induction overcomes tumor immune evasion and transforms tumor microenvironment to a favorable phenotype, providing a rationale for the synergy of MDM2 inhibitors and immunotherapy. Significance:We found that p53 activated by MDM2 inhibitors induced the expression of ERVs, in part due via epigenetic factors LSD1 and DNMT1. Induction of IFN response caused by ERV de-repression upon p53-targeting therapies provides a possibility to overcome resistance to immune checkpoint blockade and potentially transform 'cold' tumors into 'hot'.

Human skeletal muscle characteristics such as fiber type composition, fiber size and myonuclear content are widely studied in clinical and sports related contexts. Being aware of the methodological and biological variability of the characteristics is a critical aspect in study design and outcome interpretation, but comprehensive data on the variability of morphological features in human skeletal muscle is currently limited. Accordingly, in the present study, m. vastus lateralis biopsies (10 per subject) from young and healthy individuals, collected in a systematic manner, were analyzed for various characteristics using immunohistochemistry (n=7) and SDS-PAGE (n=25). None of the analyzed parameters; fiber type % (FT%), type I and II CSA (fCSA), percentage fiber type area (fCSA%), myosin heavy chain composition (MyHC%), type IIX content, myonuclear content or myonuclear domain varied in a systematic manner longitudinally along the muscle or between the two legs. The average within subject coefficient of variation for FT%, fCSA, fCSA%, and MyHC% ranged between 13-18%, but was only 5% for fiber specific myonuclear content, which reduced the variability for myonuclear domain size to 11-12%. Pure type IIX fibers and type IIX MyHC were randomly distributed and present in <24% of the analyzed samples, with the average content being 0.1 and 1.1%, respectively. In conclusion, leg or longitudinal orientation does not seem to be an important aspect to consider when investigating human vastus lateralis characteristics. However, single muscle biopsies should preferably not be used when studying fiber type and fiber size related aspects given the notable sample to sample variability.

The current study explored whether the marked hypertrophic response noted with a short-term unilateral concurrent exercise paradigm was associated with more prominent changes in myonuclei accretion, ribosome biogenesis, and capillarization compared with resistance exercise alone (RE). Ten men (age 25 ± 4 yr) performed aerobic and resistance exercise (AE + RE) for one leg while the other leg did RE. Muscle biopsies were obtained before and after 5 wk of training and subjected to fiber-type specific immunohistochemical analysis, and quantification of total RNA content and mRNA/rRNA transcript abundance. Type II fiber cross-sectional area (CSA) increased with both AE + RE (22%) and RE (16%), while type I fiber CSA increased mainly with AE + RE (16%). The change score tended to differ between legs for type I CSA ( P = 0.099), and the increase in smallest fiber diameter was greater in AE + RE than RE ( P = 0.029). The number of nuclei per fiber increased after AE + RE in both fiber types, and this increase was greater ( P = 0.027) than after RE. A strong correlation was observed between changes in number of nuclei per fiber and fiber CSA in both fiber types, for both AE + RE and RE ( r > 0.8, P < 0.004). RNA content increased after AE + RE (24%, P = 0.019), but the change-scores did not differ across legs. The capillary variables generally increased in both fiber types, with no difference across legs. In conclusion, the accentuated hypertrophic response to AE + RE was associated with more pronounced myonuclear accretion, which was strongly correlated with the degree of fiber hypertrophy. This suggests that myonuclear accretion could play a role in facilitating muscle hypertrophy also during very short training periods.

Because manual immunohistochemical analysis of features such as skeletal muscle fiber typing, capillaries, myonuclei, and fiber size-related parameters is time consuming and prone to user subjectivity, automatic computational methods could allow for faster and more objective evaluation. Here, we developed Muscle2View, a free CellProfiler-based pipeline that integrates all key fiber-morphological variables, including the novel quantification of the capillary-to-fiber interface, in one single tool. Provided that the images are of sufficient quality and the settings are configured for the specific study, the pipeline allows for automatic and unsupervised analysis of fiber borders, myonuclei, capillaries, and morphometric parameters in a fiber type-specific manner from large batches of images in <10 min/tissue sample. The novel identification of the capillary-to-fiber interface allowed for the calculation of microvascular factors such as capillary contacts (CC), individual capillary-to-fiber ratio (C/Fi), and capillary-to-fiber perimeter exchange (CFPE) index. When comparing the Muscle2View pipeline to manual or semiautomatic analysis, overall the results revealed strong correlations. For several variables, however, there were differences (5–15%) between values computed by manual counting and Muscle2View, suggesting that the methods should not necessarily be used interchangeably. Collectively, we demonstrate that the Muscle2View pipeline can provide unbiased and high-content analysis of muscle cross-sectional immunohistochemistry images. In addition to the classical morphological measurements, the Muscle2View can identify the complex capillary-to-fiber network and myonuclear density in a fiber type-specific manner. This robust analysis is done in one single run within a user-friendly and flexible environment based on the free and widely used image software CellProfiler. NEW & NOTEWORTHY Here, we developed a freely available CellProfiler-based pipeline termed Muscle2View, which provides unbiased, high-content analysis of muscle cross-sectional immunohistochemistry images. In addition to fiber typing, myonuclei counting, and the quantification of fiber type-specific morphological measurements, the Muscle2View pipeline can identify the complex capillary-to-fiber network from a batch of images within minutes. Thus, the Muscle2View is a viable tool for researchers aiming to quantify immunohistochemical variables from skeletal muscle biopsies.

Identification of the molecular mechanism of action (MoA) of bioactive compounds is a crucial step for drug development but remains a challenging task despite recent advances in technology. In this study, we applied multidimensional proteomics, sensitivity correlation analysis and transcriptomics to identify a common mechanism of action for the anticancer compounds RITA, aminoflavone (AF) and oncrasin-1 (Onc-1). Global thermal proteome profiling (TPP) revealed that the three compounds target mRNA processing and transcription, thereby attacking a cancer vulnerability -transcriptional addiction. This led to the preferential loss of expression of oncogenes involved in PDGF-, EGFR-, VEGF-, Insulin/IGF/MAPKK-, FGF-, Hedgehog-, TGF-beta-and PI3K-signaling pathways. Increased reactive oxygen species (ROS) level in cancer cells was a prerequisite for targeting the mRNA transcription machinery, thus conferring cancer-selectivity to these compounds. Furthermore, DNA repair factors involved in homologous recombination were among the most prominently repressed proteins. In cancer patient samples, RITA, AF and Onc-1 sensitized to poly(ADP-ribose) polymerase inhibitors both in vitro and ex vivo. These findings might pave a way for new synthetic lethal combination therapies. SignificanceFindings highlight agents that target transcriptional addiction in cancer cells and suggest combination treatments that target RNA processing and DNA repair pathways simultaneously as effective cancer therapies.

We describe a patient who presented with excessive daytime sleepiness (EDS) and was eventually diagnosed with anti-Ma2 encephalitis. Neurological examination disclosed somnolence, left palpebral ptosis, and vertical gaze paresis. A brain MRI showed high signal intensity in the hypothalamus and each hippocampus. Ma2 antibodies were found in the patient's serum, and fiberbronchoscopy disclosed a lung carcinoma. After three months of steroid treatment, the results of the patient's neurological exam became normal. We conclude that anti-Ma2 encephalitis may present with mostly isolated EDS and that it may respond to steroids despite old age and the presence of an untreated lung cancer.