Mis-splicing Drives Loss of Function of p53E224D Point Mutation.

Background: Tp53 is the most commonly mutated gene in cancer. Canonical Tp53 DNA damage response pathways are well characterized and classically thought to underlie the tumor suppressive effect of Tp53. Challenging this dogma, mouse models have revealed that p53 driven apoptosis and cell cycle arrest are dispensable for tumor suppression. Here, we investigated the inverse context of a p53 mutation predicted to drive expression of canonical targets, but is detected in human cancer. Methods: We established a novel mouse model with a single base pair mutation (GAG>GAC, p53E221D) in the DNA-Binding domain that has wild-type function in screening assays, but is paradoxically found in human cancer in Li-Fraumeni syndrome. Using mouse p53E221D and the analogous human p53E224D mutant, we evaluated expression, transcriptional activation, and tumor suppression in vitro and in vivo. Results: Expression of human p53E224D from cDNA translated to a fully functional p53 protein. However, p53E221D/E221D RNA transcribed from the endogenous locus is mis-spliced resulting in nonsense mediated decay. Moreover, fibroblasts derived from p53E221D/E221D mice do not express a detectable protein product. Mice homozygous for p53E221D exhibited increased tumor penetrance and decreased life expectancy compared to p53 WT animals. Conclusions: Mouse p53E221D and human p53E224D mutations lead to splice variation and a biologically relevant p53 loss of function in vitro and in vivo.

Radiation-Induced Phosphorylation of a Prion-Like Domain Regulates Transformation by FUS-CHOP.

Chromosomal translocations generate oncogenic fusion proteins in approximately one-third of sarcomas, but how these proteins promote tumorigenesis is not well understood. Interestingly, some translocation-driven cancers exhibit dramatic clinical responses to therapy, such as radiotherapy, although the precise mechanism has not been elucidated. Here we reveal a molecular mechanism by which the fusion oncoprotein FUS-CHOP promotes tumor maintenance that also explains the remarkable sensitivity of myxoid liposarcomas to radiation therapy. FUS-CHOP interacted with chromatin remodeling complexes to regulate sarcoma cell proliferation. One of these chromatin remodelers, SNF2H, colocalized with FUS-CHOP genome-wide at active enhancers. Following ionizing radiation, DNA damage response kinases phosphorylated the prion-like domain of FUS-CHOP to impede these protein-protein interactions, which are required for transformation. Therefore, the DNA damage response after irradiation disrupted oncogenic targeting of chromatin remodelers required for FUS-CHOP-driven sarcomagenesis. This mechanism of disruption links phosphorylation of the prion-like domain of an oncogenic fusion protein to DNA damage after ionizing radiation and reveals that a dependence on oncogenic chromatin remodeling underlies sensitivity to radiation therapy in myxoid liposarcoma. SIGNIFICANCE: Prion-like domains, which are frequently translocated in cancers as oncogenic fusion proteins that drive global epigenetic changes, confer sensitivity to radiation via disruption of oncogenic interactions.

A Role for SMARCB1 in Synovial Sarcomagenesis Reveals That SS18-SSX Induces Canonical BAF Destruction.

Reduced protein levels of SMARCB1 (also known as BAF47, INI1, SNF5) have long been observed in synovial sarcoma. Here, we show that combined Smarcb1 genetic loss with SS18-SSX expression in mice synergized to produce aggressive tumors with histomorphology, transcriptomes, and genome-wide BAF-family complex distributions distinct from SS18-SSX alone, indicating a defining role for SMARCB1 in synovial sarcoma. Smarcb1 silencing alone in mesenchyme modeled epithelioid sarcomagenesis. In mouse and human synovial sarcoma cells, SMARCB1 was identified within PBAF and canonical BAF (CBAF) complexes, coincorporated with SS18-SSX in the latter. Recombinant expression of CBAF components in human cells reconstituted CBAF subcomplexes that contained equal levels of SMARCB1 regardless of SS18 or SS18-SSX inclusion. In vivo, SS18-SSX expression led to whole-complex CBAF degradation, rendering increases in the relative prevalence of other BAF-family subtypes, PBAF and GBAF complexes, over time. Thus, SS18-SSX alters BAF subtypes levels/balance and genome distribution, driving synovial sarcomagenesis. SIGNIFICANCE: The protein level of BAF component SMARCB1 is reduced in synovial sarcoma but plays a defining role, incorporating into PBAF and SS18-SSX-containing canonical BAF complexes. Reduced levels of SMARCB1 derive from whole-complex degradation of canonical BAF driven by SS18-SSX, with relative increases in the abundance of other BAF-family subtypes.See related commentary by Maxwell and Hargreaves, p. 2375.This article is highlighted in the In This Issue feature, p. 2355.

Genetic drivers and cells of origin in sarcomagenesis.

Sarcoma comprises a group of malignancies that includes over 100 individual disease entities. Type-specific genetic events initiate each tumor, occurring within a specific cellular context or circumstance. All sarcomas share a relationship with mesenchymal tissues of origin. Conceptual models for each specific route towards sarcomagenesis have developed over the years as clinical, cellular, and increasingly molecular observations have advanced hypotheses to be tested in the forward or reverse direction in experimental systems, often genetically engineered model organisms. This review considers the history of these discoveries in the context of technologies available at the time each was made and provides a comprehensive summary of the current knowledge of sarcoma genetics, including characteristic translocations, oncogene activation and loss of tumor suppressor gene events, and their putative cells of origin. Also considered are the interrelatedness of molecular clinical observations and genetic experiments in model systems to move this field of knowledge forward, as well as their implications for diagnostic and therapeutic paradigms for sarcoma. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Gene Expression in Torn Rotator Cuff Tendons Determined by RNA Sequencing.

BACKGROUND: Although the cause of rotator cuff tearing is likely multifactorial and a genetic predisposition has been proposed, the biochemical basis remains unknown. PURPOSE: To determine gene expression profiles in torn rotator cuff tendon tissue through use of RNA sequencing. STUDY DESIGN: Controlled laboratory study. METHODS: The supraspinatus tendon edge was biopsied in 24 patients undergoing arthroscopic rotator cuff repair for full-thickness supraspinatus rotator cuff tears. The supraspinatus tendon was also biopsied in 9 patients undergoing open reduction and internal fixation for a proximal humeral fracture (controls). Total RNA was extracted and sequenced. Differential gene expression was analyzed between the tear and control groups, and a secondary analysis was conducted between groups defined by an unbiased clustering. RESULTS: Tear and control transcriptomes demonstrated significant differential expression in more than 3000 genes. The identified differential genes were highlighted in pathways involved in inflammation in control patients and extracellular matrix generation in patients with tears. Secondary analysis using unsupervised and thus unbiased hierarchical clustering revealed 2 clusters (c2 and c3). Cluster c3 contained smaller (P < .001) and less retracted (P = .018) tears (ie, tears earlier in the progression of rotator cuff disease) with increased expression of hypoxia target genes. Cluster c2 contained larger, more retracted tears (ie, tears further in the progression of rotator cuff disease) with increased expression of endothelial cell markers and chronic inflammation target genes. Tears in c2 had significantly worse healing rates compared with tears in c3 (0% vs 89%; P = .007). CONCLUSION: Smaller, less retracted tears had increased expression of hypoxia target genes and improved healing, whereas larger, more retracted tears were associated with endothelial cell markers and worse healing. Thus, hypoxia may be the inciting event for tear development, whereas with tear enlargement, a chronic, inflammatory, angiogenic process may predominate. CLINICAL RELEVANCE: Identification of differential gene expression in rotator cuff tears may be a reliable tool to predict repair healing in the future.