Acquired NF2 mutation confers resistance to TRK inhibition in an ex vivo LMNA::NTRK1-rearranged soft-tissue sarcoma cell model.

Genomic rearrangements of the neurotrophic receptor tyrosine kinase genes (NTRK1, NTRK2, and NTRK3) are the most common mechanism of oncogenic activation for this family of receptors, resulting in sustained cancer cell proliferation. Several targeted therapies have been approved for tumours harbouring NTRK fusions and a new generation of TRK inhibitors has already been developed due to acquired resistance. We established a patient-derived LMNA::NTRK1-rearranged soft-tissue sarcoma cell model ex vivo with an acquired resistance to targeted TRK inhibition. Molecular profiling of the resistant clones revealed an acquired NF2 loss of function mutation that was absent in the parental cell model. Parental cells showed continuous sensitivity to TRK-targeted treatment, whereas the resistant clones were insensitive. Furthermore, resistant clones showed upregulation of the MAPK and mTOR/AKT pathways in the gene expression based on RNA sequencing data and increased sensitivity to MEK and mTOR inhibitor therapy. Drug synergy was seen using trametinib and rapamycin in combination with entrectinib. Medium-throughput drug screening further identified small compounds as potential drug candidates to overcome resistance as monotherapy or in combination with entrectinib. In summary, we developed a comprehensive model of drug resistance in an LMNA::NTRK1-rearranged soft-tissue sarcoma and have broadened the understanding of acquired drug resistance to targeted TRK therapy. Furthermore, we identified drug combinations and small compounds to overcome acquired drug resistance and potentially guide patient care in a functional precision oncology setting. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Dietary methionine influences therapy in mouse cancer models and alters human metabolism.

Nutrition exerts considerable effects on health, and dietary interventions are commonly used to treat diseases of metabolic aetiology. Although cancer has a substantial metabolic component1, the principles that define whether nutrition may be used to influence outcomes of cancer are unclear2. Nevertheless, it is established that targeting metabolic pathways with pharmacological agents or radiation can sometimes lead to controlled therapeutic outcomes. By contrast, whether specific dietary interventions can influence the metabolic pathways that are targeted in standard cancer therapies is not known. Here we show that dietary restriction of the essential amino acid methionine-the reduction of which has anti-ageing and anti-obesogenic properties-influences cancer outcome, through controlled and reproducible changes to one-carbon metabolism. This pathway metabolizes methionine and is the target of a variety of cancer interventions that involve chemotherapy and radiation. Methionine restriction produced therapeutic responses in two patient-derived xenograft models of chemotherapy-resistant RAS-driven colorectal cancer, and in a mouse model of autochthonous soft-tissue sarcoma driven by a G12D mutation in KRAS and knockout of p53 (KrasG12D/+;Trp53-/-) that is resistant to radiation. Metabolomics revealed that the therapeutic mechanisms operate via tumour-cell-autonomous effects on flux through one-carbon metabolism that affects redox and nucleotide metabolism-and thus interact with the antimetabolite or radiation intervention. In a controlled and tolerated feeding study in humans, methionine restriction resulted in effects on systemic metabolism that were similar to those obtained in mice. These findings provide evidence that a targeted dietary manipulation can specifically affect tumour-cell metabolism to mediate broad aspects of cancer outcome.

HMGA1 regulates trabectedin sensitivity in advanced soft-tissue sarcoma (STS): A Spanish Group for Research on Sarcomas (GEIS) study.

HMGA1 is a structural epigenetic chromatin factor that has been associated with tumor progression and drug resistance. Here, we reported the prognostic/predictive value of HMGA1 for trabectedin in advanced soft-tissue sarcoma (STS) and the effect of inhibiting HMGA1 or the mTOR downstream pathway in trabectedin activity. The prognostic/predictive value of HMGA1 expression was assessed in a cohort of 301 STS patients at mRNA (n = 133) and protein level (n = 272), by HTG EdgeSeq transcriptomics and immunohistochemistry, respectively. The effect of HMGA1 silencing on trabectedin activity and gene expression profiling was measured in leiomyosarcoma cells. The effect of combining mTOR inhibitors with trabectedin was assessed on cell viability in vitro studies, whereas in vivo studies tested the activity of this combination. HMGA1 mRNA and protein expression were significantly associated with worse progression-free survival of trabectedin and worse overall survival in STS. HMGA1 silencing sensitized leiomyosarcoma cells for trabectedin treatment, reducing the spheroid area and increasing cell death. The downregulation of HGMA1 significantly decreased the enrichment of some specific gene sets, including the PI3K/AKT/mTOR pathway. The inhibition of mTOR, sensitized leiomyosarcoma cultures for trabectedin treatment, increasing cell death. In in vivo studies, the combination of rapamycin with trabectedin downregulated HMGA1 expression and stabilized tumor growth of 3-methylcholantrene-induced sarcoma-like models. HMGA1 is an adverse prognostic factor for trabectedin treatment in advanced STS. HMGA1 silencing increases trabectedin efficacy, in part by modulating the mTOR signaling pathway. Trabectedin plus mTOR inhibitors are active in preclinical models of sarcoma, downregulating HMGA1 expression levels and stabilizing tumor growth.

FUS Contributes to Nerve Injury-Induced Nociceptive Hypersensitivity by Activating NF-κB Pathway in Primary Sensory Neurons.

Dysregulation of pain-associated genes in the dorsal root ganglion (DRG) is considered to be a molecular basis of neuropathic pain genesis. Fused in sarcoma (FUS), a DNA/RNA-binding protein, is a critical regulator of gene expression. However, whether it contributes to neuropathic pain is unknown. This study showed that peripheral nerve injury caused by the fourth lumbar (L4) spinal nerve ligation (SNL) or chronic constriction injury (CCI) of the sciatic nerve produced a marked increase in the expression of FUS protein in injured DRG neurons. Blocking this increase through microinjection of the adeno-associated virus (AAV) 5-expressing Fus shRNA into the ipsilateral L4 DRG mitigated the SNL-induced nociceptive hypersensitivities in both male and female mice. This microinjection also alleviated the SNL-induced increases in the levels of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) and glial fibrillary acidic protein (GFAP) in the ipsilateral L4 dorsal horn. Furthermore, mimicking this increase through microinjection of AAV5 expressing full-length Fus mRNA into unilateral L3/4 DRGs produced the elevations in the levels of p-ERK1/2 and GFAP in the dorsal horn, enhanced responses to mechanical, heat and cold stimuli, and induced the spontaneous pain on the ipsilateral side of both male and female mice in the absence of SNL. Mechanistically, the increased FUS activated the NF-κB signaling pathway by promoting the translocation of p65 into the nucleus and phosphorylation of p65 in the nucleus from injured DRG neurons. Our results indicate that DRG FUS contributes to neuropathic pain likely through the activation of NF-κB in primary sensory neurons.SIGNIFICANCE STATEMENT In the present study, we reported that fused in sarcoma (FUS), a DNA/RNA-binding protein, is upregulated in injured dorsal root ganglion (DRG) following peripheral nerve injury. This upregulation is responsible for nerve injury-induced translocation of p65 into the nucleus and phosphorylation of p65 in the nucleus from injured DRG neurons. Because blocking this upregulation alleviates nerve injury-induced nociceptive hypersensitivity, DRG FUS participates in neuropathic pain likely through the activation of NF-κB in primary sensory neurons. FUS may be a potential target for neuropathic pain management.

Differential metabolic secretion between muscular dystrophy mouse-derived spindle cell sarcomas and rhabdomyosarcomas drives tumor type development.

The dystrophin gene (Dmd) is recognized for its significance in Duchenne muscular dystrophy (DMD), a lethal and progressive skeletal muscle disease. Some patients with DMD and model mice with muscular dystrophy (mdx) spontaneously develop various types of tumors, among which rhabdomyosarcoma (RMS) is the most prominent. By contrast, spindle cell sarcoma (SCS) has rarely been reported in patients or mdx mice. In this study, we aimed to use metabolomics to better understand the rarity of SCS development in mdx mice. Gas chromatography-mass spectrometry was used to compare the metabolic profiles of spontaneously developed SCS and RMS tumors from mdx mice, and metabolite supplementation assays and silencing experiments were used to assess the effects of metabolic differences in SCS tumor-derived cells. The levels of 75 metabolites exhibited differences between RMS and SCS, 25 of which were significantly altered. Further characterization revealed downregulation of nonessential amino acids, including alanine, in SCS tumors. Alanine supplementation enhanced the growth, epithelial mesenchymal transition, and invasion of SCS cells. Reduction of intracellular alanine via knockdown of the alanine transporter Slc1a5 reduced the growth of SCS cells. Lower metabolite secretion and reduced proliferation of SCS tumors may explain the lower detection rate of SCS in mdx mice. Targeting of alanine depletion pathways may have potential as a novel treatment strategy.NEW & NOTEWORTHY To the best of our knowledge, SCS has rarely been identified in patients with DMD or mdx mice. We observed that RMS and SCS tumors that spontaneously developed from mdx mice with the same Dmd genetic background exhibited differences in metabolic secretion. We proposed that, in addition to dystrophin deficiency, the levels of secreted metabolites may play a role in the determination of tumor-type development in a Dmd-deficient background.

Expression of Forkhead Box M1 and Anticancer Effects of FOXM1 Inhibition in Epithelioid Sarcoma.

Epithelioid sarcoma (ES) is a rare aggressive sarcoma that, unlike most soft-tissue sarcomas, shows a tendency toward local recurrence and lymph node metastasis. Novel antitumor agents are needed for ES patients. Forkhead box transcription factor 1 (FOXM1) is a member of the Forkhead transcription factor family and is associated with multiple oncogenic functions; FOXM1 is known to be overexpressed and correlated with pathogenesis in various malignancies. In this study, we immunohistochemically analyzed FOXM1 expression levels and their clinical, clinicopathologic, and prognostic significance in 38 ES specimens. In addition, to investigate potential correlations between FOXM1 downregulation and oncologic characteristics, we treated ES cell lines with thiostrepton, a naturally occurring antibiotic that inhibits both small interfering RNA (siRNA) and FOXM1. In the analyses using ES samples, all 38 specimens were diagnosed as positive for FOXM1 by immunohistochemistry. We separated specimens into high (n = 19) and low (n = 19) FOXM1-protein expression groups by staining index score, and into large (n = 12), small (n = 25), and unknown (n = 1) tumor-size groups using a cutoff of 5 cm maximum diameter. Although there were significantly more samples with high FOXM1 expression in the large tumor group (P = .013), there were no significant differences with respect to age (P = 1.00), sex (P = .51), primary site of origin (P = .74), histologic subtypes (P = 1.00), depth (P = .74), or survival rate (P = .288) between the high and low FOXM1-protein expression groups. In the in vitro experiments using ES cell lines, FOXM1 siRNA and thiostrepton successfully downregulated FOXM1 mRNA and protein expression. Furthermore, downregulation of FOXM1 inhibited cell proliferation, drug resistance against chemotherapeutic agents, migration, and invasion and caused cell cycle arrest in the ES cell lines. Finally, cDNA microarray analysis data showed that FOXM1 regulated cIAP2, which is one of the apoptosis inhibitors activated by the TNFα-mediated NF-κB pathway. In conclusion, the FOXM1 gene may be a promising therapeutic target for ES.

The molecular conversations of sarcomas: exosomal non-coding RNAs in tumor's biology and their translational prospects.

Exosomes mediate cell-to-cell crosstalk involving a variety of biomolecules through an intricate signaling network. In recent years, the pivotal role of exosomes and their non-coding RNAs cargo in the development and progression of several cancer types clearly emerged. In particular, tumor bulk and its microenvironment co-evolve through cellular communications where these nanosized extracellular vesicles are among the most relevant actors. Knowledge about the cellular, and molecular mechanisms involved in these communications will pave the way for novel exosome-based delivery of therapeutic RNAs as well as innovative prognostic/diagnostic tools. Despite the valuable therapeutic potential and clinical relevance of exosomes, their role on sarcoma has been vaguely reported because the rarity and high heterogeneity of this type of cancer. Here, we dissected the scientific literature to unravel the multifaceted role of exosomal non-coding RNAs as mediator of cell-to-cell communications in the sarcoma subtypes.

Cell-cycle phase progression analysis identifies three unique phenotypes in soft tissue sarcoma.

PURPOSE: Loddo et al. (Br J Cancer 100:959-70, 2009) established the prognostic significance of cell cycle markers and "Cell-Cycle Phenotypes" in breast carcinoma. This study aims to 1) identify prognostic cell-cycle markers in sarcoma, and 2) assess the prognostic potential of specific cell-cycle phenotypes in sarcoma. METHODS: Tissue samples from 128 soft tissue sarcomas were stained for four cell cycle-specific markers: Mcm2, Geminin, Plk1, and H3S10ph. Only primary soft tissue tumors (liposarcoma, leiomyosarcoma, synovial sarcoma, and undifferentiated pleomorphic sarcoma) were included in the analysis. Any tumor coming from a recurrent or metastatic lesion were excluded from the analysis. Three cell-cycle phenotypes (I, II, III) were derived from marker expression patterns. Prognostic significance was evaluated in a subset of primary soft tissue sarcomas using Cox regression for survival analysis. RESULTS: Compared to phenotype I, the phenotype III tumors had a decreased 5-year overall survival (HR 6.81 [2.36-19.61]; p = < 0.001), 5-year disease-free survival (HR 1.07 (1.02-1.18); p = 0.004), and 5-year metastasis-free survival (HR 4.34 [1.58-11.93]; p = 0.004). High expression of Plk1 was associated with decreased 5-year overall survival (HR: 4.04 CI [1.21-6.67; p = 0.02) and 5-year metastasis-free survival (HR: 2.91 CI [1.15-7.37]; p = 0.03). Geminin was also found to have a decreased 5-year overall survival (HR:2.84 CI [1.21-6.67]; p = 0.02). No statistical difference in prognostication were noted between phenotypes and the AJCC system. CONCLUSIONS: We identified three unique sarcoma cell cycle phenotypes that have prognostic significance. This performs similarly to the AJCC staging system.

Targeting the EphA2 pathway: could it be the way for bone sarcomas?

Bone sarcomas are malignant tumors of mesenchymal origin. Complete surgical resection is the cornerstone of multidisciplinary treatment. However, advanced, unresectable forms remain incurable. A crucial step towards addressing this challenge involves comprehending the molecular mechanisms underpinning tumor progression and metastasis, laying the groundwork for innovative precision medicine-based interventions. We previously showed that tyrosine kinase receptor Ephrin Type-A Receptor 2 (EphA2) is overexpressed in bone sarcomas. EphA2 is a key oncofetal protein implicated in metastasis, self-renewal, and chemoresistance. Molecular, genetic, biochemical, and pharmacological approaches have been developed to target EphA2 and its signaling pathway aiming to interfere with its tumor-promoting effects or as a carrier for drug delivery. This review synthesizes the main functions of EphA2 and their relevance in bone sarcomas, providing strategies devised to leverage this receptor for diagnostic and therapeutic purposes, with a focus on its applicability in the three most common bone sarcoma histotypes: osteosarcoma, chondrosarcoma, and Ewing sarcoma.

Subclassification of epithelioid sarcoma with potential therapeutic impact.

Epithelioid sarcoma is a rare and aggressive mesenchymal tumour, the genetic hallmark of which is the loss of expression of SMARCB1, a key member of the SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodelling complex. Hampered by its rarity, epithelioid sarcoma has received little research attention and therapeutic options for this disease remain limited. SMARCB1-deficient tumours also include malignant rhabdoid tumour, atypical teratoid and rhabdoid tumour, epithelioid malignant peripheral nerve sheath tumour, and poorly differentiated chordoma. Histologically, it can be challenging to distinguish epithelioid sarcoma from malignant rhabdoid tumour and other SMARCB1-deficient tumours, whereas methylation profiling shows that they represent distinct entities and facilitates their classification. Methylation studies on SMARCB1-deficient tumours, although not including epithelioid sarcomas, reported methylation subgroups which resulted in new clinical stratification and therapeutic approaches. In addition, emerging evidence indicates that immunotherapy, including immune checkpoint inhibitors, represents a promising therapeutic strategy for SMARCB1-deficient tumours. Here, we show that some epithelioid sarcomas share methylation patterns of malignant rhabdoid tumours indicating that this could help to distinguish these entities and guide treatment. Using gene expression data, we also showed that the immune environment of epithelioid sarcoma is characterised by a predominance of CD8+ lymphocytes and M2 macrophages. These findings have potential implications for the management of patients with epithelioid sarcoma. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Brain region-specific synaptic function of FUS underlies the FTLD-linked behavioural disinhibition.

Synaptic dysfunction is one of the earliest pathological processes that contribute to the development of many neurological disorders, including Alzheimer's disease and frontotemporal lobar degeneration. However, the synaptic function of many disease-causative genes and their contribution to the pathogenesis of the related diseases remain unclear. In this study, we investigated the synaptic role of fused in sarcoma, an RNA-binding protein linked to frontotemporal lobar degeneration and amyotrophic lateral sclerosis, and its potential pathological role in frontotemporal lobar degeneration using pyramidal neuron-specific conditional knockout mice (FuscKO). We found that FUS regulates the expression of many genes associated with synaptic function in a hippocampal subregion-specific manner, concomitant with the frontotemporal lobar degeneration-linked behavioural disinhibition. Electrophysiological study and molecular pathway analyses further reveal that fused in sarcoma differentially regulates synaptic and neuronal properties in the ventral hippocampus and medial prefrontal cortex, respectively. Moreover, fused in sarcoma selectively modulates the ventral hippocampus-prefrontal cortex projection, which is known to mediate the anxiety-like behaviour. Our findings unveil the brain region- and synapse-specific role of fused in sarcoma, whose impairment might lead to the emotional symptoms associated with frontotemporal lobar degeneration.

Case of a CIC::DUX4 fusion gene in a vascular neoplasm extends the spectrum of CIC-rearranged sarcomas.

CIC-rearranged sarcomas comprise a group of exceptionally aggressive round-cell sarcomas. These tumors most commonly demonstrate CIC::DUX4 fusion and show similar histopathology to Ewing sarcomas, though lesions mimicking vascular neoplasms have recently been described. Here, we describe a case of a patient with CIC::DUX4 fusion sarcoma identified using RNA-based molecular testing who was initially diagnosed with an endothelial neoplasm. The tumor showed extensive vasoformative growth, complete WT1 negativity, and global positive staining for ERG, CD31, and DUX4 by immunohistochemistry. Methylation testing of the tumor clustered more closely with angiosarcomas than with CIC-rearranged sarcomas. Our findings suggest that CIC::DUX4 fused neoplasms may demonstrate a more diverse phenotypic range than previously appreciated and offer evidence that both molecular and immunohistochemical studies are needed for accurate diagnosis.

Prognostic evaluation of the Ki-67 labeling system in histological grading of non-small round cell sarcoma: a supplementary analysis of a randomized controlled trial, JCOG1306.

BACKGROUND: Soft tissue sarcoma (STS) has various histological types and is rare, making it difficult to evaluate the malignancy of each histological type. Thus, comprehensive histological grading is most important in the pathological examination of STS. The Fédération Nationale des Centres de Lutte Contre le Cancer (FNCLCC) grading system is most commonly used in daily pathological analysis of STS. Among the FNCLCC grading system parameters, mitotic count is a key morphological parameter reflecting the proliferative activity of tumor cells, although its reproducibility may be lacking. Here, we compared the prognostic utility of the conventional and modified FNCLCC grading systems in JCOG1306. METHODS: We analyzed 140 patients with non-small round cell sarcoma. We performed Ki-67 immunostaining using open biopsy specimens before preoperative chemotherapy in all patients. We assessed histological grade in individual cases by conventional FNCLCC grading (tumor differentiation, mitotic count, and necrosis) and modified FNCLCC grading using the Ki-67 labeling index instead of mitotic count. We conducted univariable and multivariable Cox regression analyses to investigate the influence of grade on overall survival. RESULTS: In univariable analysis, prognosis was worse for patients with conventional FNCLCC Grade 3 tumors compared with Grade 1 or 2 tumors (hazard ratio [HR] 4.21, 95% confidence interval [CI] 1.47-12.05, P = 0.008). Moreover, prognosis was worse in patients with modified FNCLCC Grade 3 tumors compared with Grade 1 or 2 tumors (HR 4.90, 95% CI 1.64-14.65, P = 0.004). In multivariable analysis including both conventional and modified FNCLCC grading, the modified grading more strongly affected overall survival (HR 6.70, 95% CI 1.58-28.40, P = 0.010). CONCLUSIONS: The modified FNCLCC grading system was superior to the conventional system in predicting the prognosis of patients with non-small round cell sarcoma according to this supplementary analysis of data from the randomized controlled trial JCOG1306.

Mitochondrial apoptotic priming is a key determinant of cell fate upon p53 restoration.

Reactivation of p53 in established tumors typically results in one of two cell fates, cell cycle arrest or apoptosis, but it remains unclear how this cell fate is determined. We hypothesized that high mitochondrial priming prior to p53 reactivation would lead to apoptosis, while low priming would lead to survival and cell cycle arrest. Using a panel of Kras-driven, p53 restorable cell lines derived from genetically engineered mouse models of lung adenocarcinoma and sarcoma (both of which undergo cell cycle arrest upon p53 restoration), as well as lymphoma (which instead undergo apoptosis), we show that the level of mitochondrial apoptotic priming is a critical determinant of p53 reactivation outcome. Cells with high initial priming (e.g., lymphomas) lacked sufficient reserve antiapoptotic capacity and underwent apoptosis after p53 restoration. Forced BCL-2 or BCL-XL expression reduced priming and resulted in survival and cell cycle arrest. Cells with low initial priming (e.g., lung adenocarcinoma and sarcoma) survived and proceeded to arrest in the cell cycle. When primed by inhibition of their antiapoptotic proteins using genetic (BCL-2 or BCL-XL deletion or BAD overexpression) or pharmacologic (navitoclax) means, apoptosis resulted upon p53 restoration in vitro and in vivo. These data demonstrate that mitochondrial apoptotic priming is a key determining factor of cell fate upon p53 activation. Moreover, it is possible to enforce apoptotic cell fate following p53 activation in less primed cells using p53-independent drugs that increase apoptotic priming, including BH3 mimetic drugs.

P16 and HPV status in head and neck sarcomas and sarcomatoid carcinomas.

Human papillomavirus (HPV)-positive oropharyngeal carcinoma is a distinct type of head and neck carcinoma with improved prognosis. p16 immunostaining is often used as a surrogate marker for HPV infection in this particular setting. The aim of this study is to estimate the prevalence of p16 staining and HPV infection in head and neck sarcomatoid carcinomas as well as head and neck sarcomas. 21 sarcomatoid carcinomas and 28 head and neck sarcomas were tested for p16 positivity using immunohistochemical staining, and for high-risk HPV infection using In situ hybridization (ISH). 24 % of sarcomatoid carcinomas and 21 % of sarcomas were positive for p16 staining. All 49 cases were negative for HPV ISH. The results confirm that p16 staining is not specific and may not be associated with HPV infection in non-oropharyngeal head and neck sites. They also indicate that non-oropharyngeal head and neck sarcomatoid carcinomas are not likely to be HPV related.

Differential cyclin-E1 expression in CIC-rearranged sarcoma.

CIC-rearranged sarcoma (CRS) is a group of high-grade undifferentiated small round cell sarcomas examined as a separate entity in the current WHO classification; since it shows more aggressive clinical behavior and distinct morphological and molecular features compared to Ewing sarcoma (ES). As CCNE1 expression is associated with tumor growth in CIC::DUX4 sarcomas, we aimed to demonstrate the value of cyclin E1 expression in CRS. Cyclin E1 immunohistochemistry and break-apart FISH for EWSR1 and CIC gene rearrangements were performed on 3-mm tissue microarrays composed of 40 small round cell tumors. Five cases were classified as CRS, whereas 22 were ES and 13 were unclassified (EWSR1-/CIC-). Among all three diagnostic groups, we found cyclin E1 expression level to be higher in CRS (80 %) and unclassified groups (61.5 %) compared to ES (4.5 %, p < 0.001). In addition, high cyclin E1 expression levels were associated with higher mean age at diagnosis, presence of atypical histology and myxoid stroma, low CD99 expression, and presence of metastasis at diagnosis. The sensitivity and specificity of high cyclin E1 expression in detecting non-ES cases were 95.5 % and 66.7 %, respectively. However, the correlation between cyclin E1 expression level and survival was not statistically significant. This is the first study that shows cyclin E1 immunohistochemical expression in EWSR1-negative undifferentiated small cell sarcomas, particularly CRS.

Insertion of the CXXC domain of KMT2A into YAP1: An unusual mechanism behind the formation of a chimeric oncogenic protein.

Most neoplasia-associated gene fusions are formed through the fusion of the 5'-part of one gene with the 3'-part of another. We here describe a unique mechanism, by which a part of the KMT2A gene through an insertion replaces part of the YAP1 gene. The resulting YAP1::KMT2A::YAP1 (YKY) fusion was verified by RT-PCR in three cases of sarcoma morphologically resembling sclerosing epithelioid fibrosarcoma (SEF-like sarcoma). In all cases, a portion (exons 4/5-6) encoding the CXXC domain of KMT2A was inserted between exon 4/5 and exon 8/9 of YAP1. The inserted sequence from KMT2A thus replaced exons 5/6-8 of YAP1, which encode an important regulatory sequence of YAP1. To evaluate the cellular impact of the YKY fusion, global gene expression profiles from fresh frozen and formalin-fixed YKY-expressing sarcomas were compared with control tumors. The effects of the YKY fusion, as well as YAP1::KMT2A and KMT2A::YAP1 fusion constructs, were further studied in immortalized fibroblasts. Analysis of differentially upregulated genes revealed significant overlap between tumors and cell lines expressing YKY, as well as with previously reported YAP1 fusions. Pathway analysis of upregulated genes in cells and tumors expressing YKY revealed an enrichment of genes included in key oncogenic signaling pathways, such as Wnt and Hedgehog. As these pathways are known to interact with YAP1, it seems likely that the pathogenesis of sarcomas with the YKY fusion is linked to distorted YAP1 signaling.

Disaggregation-Activated pan-COX Imaging Agents for Human Soft tissue Sarcoma.

Cancer stem cells are pivotal players in tumors initiation, growth, and metastasis. While several markers have been identified, there remain challenges particularly in heterogeneous malignancies like adult soft tissue sarcomas, where conventional markers are inherently overexpressed. Here, we designed BODIPY scaffold fluorescence probes (BD-IMC-1, BD-IMC-2) that activate via disaggregation targeting for cyclooxygenase (COX), a potential marker for CSCs in sarcoma in clinical pathology. Based on their structures, BD-IMC-1 showcased higher susceptibility to disaggregation compared to BD-IMC-2, consistent with their selective interaction with COX. Notably, the BD-IMC-1 revealed positive cooperativity binding to COX-2 at sub-micromolar ranges. Both probes showed significant fluorescence turn-on upon LPS or PMA triggered COX-2 upregulation in live RAW264.7, HeLa, and human sarcoma cell line (Saos-LM2) up to 2-fold increase with negligible toxicity. More importantly, the BD-IMC-1 demonstrated their practical imaging for COX-2 positive cells in paraffin-fixed human sarcoma tissue. Considering the fixed tissues are most practiced pathological sample, our finding suggests a potential of disaggregation activated chemosensor for clinical applications.

Geometrical analysis identified morphological features of hydrogel-induced cancer stem cells in synovial sarcoma model cells.

Cancer stem cells (CSCs) has been a key target to cure cancer patients completely. Although many CSC markers have been identified, they are frequently cancer type-specific and those expressions are occasionally variable, which becomes an obstacle to elucidate the characteristics of the CSCs. Here we scrutinized the relationship between stemness elevation and geometrical features of single cells. The PAMPS hydrogel was utilized to create the CSCs from mouse myoblast C2C12 and its synovial sarcoma model cells. qRT-PCR analysis confirmed the significant increase in expression levels of Sox2, Nanog, and Oct3/4 on the PAMPS gel, which was higher in the synovial sarcoma model cells. Of note, the morphological heterogeneity was appeared on the PAMPS gel, mainly including flat spreading, elongated spindle, and small round cells, and the Sox2 expression was highest in the small round cells. To examine the role of morphological differences in the elevation of stemness, over 6,400 cells were segmented along with the Sox2 intensity, and 12 geometrical features were extracted at single cell level. A nonlinear mapping of the geometrical features by using uniform manifold approximation and projection (UMAP) clearly revealed the existence of relationship between morphological differences and the stemness elevation, especially for C2C12 and its synovial sarcoma model on the PAMPS gel in which the small round cells possess relatively high Sox2 expression on the PAMPS gel, which supports the strong relationship between morphological changes and the stemness elevation. Taken together, these geometrical features can be useful for morphological profiling of CSCs to classify and distinguish them for understanding of their role in disease progression and drug discovery.

Negative MAPK-ERK regulation sustains CIC-DUX4 oncoprotein expression in undifferentiated sarcoma.

Transcription factor fusions (TFFs) are present in ∼30% of soft-tissue sarcomas. TFFs are not readily "druggable" in a direct pharmacologic manner and thus have proven difficult to target in the clinic. A prime example is the CIC-DUX4 oncoprotein, which fuses Capicua (CIC) to the double homeobox 4 gene, DUX4. CIC-DUX4 sarcoma is a highly aggressive and lethal subtype of small round cell sarcoma found predominantly in adolescents and young adults. To identify new therapeutic targets in CIC-DUX4 sarcoma, we performed chromatin immunoprecipitation sequencing analysis using patient-derived CIC-DUX4 cells. We uncovered multiple CIC-DUX4 targets that negatively regulate MAPK-ERK signaling. Mechanistically, CIC-DUX4 transcriptionally up-regulates these negative regulators of MAPK to dampen ERK activity, leading to sustained CIC-DUX4 expression. Genetic and pharmacologic MAPK-ERK activation through DUSP6 inhibition leads to CIC-DUX4 degradation and apoptotic induction. Collectively, we reveal a mechanism-based approach to therapeutically degrade the CIC-DUX4 oncoprotein and provide a precision-based strategy to combat this lethal cancer.