Despite aggressive, multimodal therapies, the prognosis of patients with refractory or recurrent rhabdomyosarcoma (RMS) has not improved in four decades. Because RMS resembles skeletal muscle precursor cells, differentiation-inducing therapy has been proposed for patients with advanced disease. In RAS-mutant PAX fusion-negative RMS (FN-RMS) preclinical models, MEK1/2 inhibition (MEKi) induces differentiation, slows tumor growth, and extends survival. However, the response is short-lived. A better understanding of the molecular mechanisms regulating FN-RMS differentiation could improve differentiation therapy. In this study, we identified a role in FN-RMS differentiation for ASAP1, an ADP ribosylation factor (ARF) GTPase-activating protein (GAP) with both proinvasive and tumor-suppressor functions. We found that ASAP1 knockdown inhibited differentiation in FN-RMS cells. Interestingly, knockdown of the GTPases ARF1 or ARF5, targets of ASAP1 GAP activity, also blocked differentiation of FN-RMS. We discovered that loss of ARF pathway components blocked myogenic transcription factor expression. Therefore, we examined the effects on transcriptional regulators. MEKi led to the phosphorylation and inactivation of WW domain-containing transcriptional regulator 1 (WWTR1; TAZ), a homolog of the pro-proliferative transcriptional co-activator YAP1, regulated by the Hippo pathway. However, loss of ASAP1 or ARF1 blocked this inactivation, which inhibits MEKi-induced differentiation. Finally, MEKi-induced differentiation was rescued by dual knockdown of ASAP1 and WWTR1. This study shows that ASAP1 and ARF1 are necessary for myogenic differentiation, providing a deeper understanding of differentiation in FN-RMS and illuminating an opportunity to advance differentiation therapy. Implications: ASAP1 and ARF1 regulate MEKi-induced differentiation of FN-RMS cells by modulating WWTR1 (TAZ) activity, supporting YAP1/TAZ inhibition as a FN-RMS differentiation therapy strategy.

PURPOSE:Corneal cross-linking (CXL) is the standard of care in patients with keratoconus but presents unique challenges in children and developmentally delayed patients. We present our clinical decision-making algorithm, CXL surgical technique, and outcomes in these groups. METHODS:A retrospective chart review was undertaken at a tertiary referral center of all patients who underwent CXL for keratoconus at University of Pittsburgh Medical Center (UPMC) Children's Hospital of Pittsburgh between October 1, 2017, and April 1, 2021. Demographic information along with preoperative, intraoperative, and postoperative ophthalmic examination findings were collected. The main outcome measures were indications of CXL, postoperative complications, and visual acuity (VA). RESULTS:Forty-eight eyes of 34 patients [21 patients (30 eyes) with developmental delay (DD) and 13 patients (18 eyes) with no DD (NDD)] underwent epithelium-off, standard CXL. General anesthesia was used for CXL in all patients except for 3 with NDD. A temporary central tarsorrhaphy was performed in all patients with DD and 7 patients with NDD. The remaining got a bandage contact lens. There were no immediate postoperative complications. A trend toward improvement in VA was noted postoperatively. The mean logMAR VA (with habitual correction) was 0.67 preoperatively and 0.57 postoperatively ( P = 0.3) in DD and 0.52 and 0.36, respectively ( P = 0.13), in NDD. CONCLUSIONS:This retrospective review presents a technique for assessment and treatment of keratoconus in children and those with DD. Our technique ensures timely diagnosis and provides a safe method for CXL in these groups. Temporary central tarsorrhaphy is a well-tolerated option to reduce postoperative pain.

Supplementary Tables 1-6, Figures 1-4 from Central Role of c-Myc during Malignant Conversion in Human Hepatocarcinogenesis

Supplementary Figures 1-3, Tables 1-3, Methods from Definition of Ubiquitination Modulator COP1 as a Novel Therapeutic Target in Human Hepatocellular Carcinoma

Supplementary Figure Legends 1-4 from Central Role of c-Myc during Malignant Conversion in Human Hepatocarcinogenesis

Upregulation of c-MYC in cancer stem cell enriched populations (S1); The doxycycline (Dox)-controlled mCherry marker expression in c-MYC inducible hepatoma cell lines (S2); The doxycycline (Dox) dose-dependent effects of c-MYC induction on CSC properties (S3); The doxycycline (Dox) dose-dependent effects of c-MYC induction on self-renewal potential (S4); c-MYC switch-on and switch-off effects on self-renewal of PLC cells (S5); Effect of p53 knockdown on self-renewal of hepatoma cell lines with doxycycline (Dox) regulated c-MYC expression (S6); p53 knockdown increases the growth rate of HepG2 cells with doxycycline (Dox) regulated c-MYC expression (S7).

Supplementary Table 1 from Loss of Hepatocyte Growth Factor/c-Met Signaling Pathway Accelerates Early Stages of <i>N</i>-nitrosodiethylamine–Induced Hepatocarcinogenesis

Supplementary Table S1. List of 88 genes commonly deregulated by siRNA-mediated knockdown of HDAC2 in Huh7 and HepG2 cells.

S1. Correlation of prognostic probability and risk scores for mouse HCCs. S2. Glycolytic gene expression signature in mouse liver. S3. Glycolytic expression signature in mouse HCC tumors from 9 models. S4. Correlation of hepatic stem cell probability with SOH probability. S5. Expression of hepatic stem cell markers in 9 mouse models. S6. Expression of Krt-7 in Sav1 KO liver issues. S7. Expression of downstream target genes in mouse tumors. S8. Expression of Pdcd1 and Cd274 and their correlation with IFNG6 score in mouse HCCs in nine models. S9. Regulation of Cd276 by Yap1.

Background The lung intratumor microbiome influences lung cancer tumorigenesis and treatment responses, but detailed data on the extent, location, and effects of microbes within lung tumors are missing, information needed for improved prognosis and treatment. Methods To address this gap, we developed a novel spatial meta-transcriptomic method simultaneously detecting the expression level of 1,811 host genes and 3 microbe targets (bacteria, fungi, and cytomegalovirus). After rigorous validation, we analyzed the spatial meta-transcriptomic profiles of tumor cells, T cells, macrophages, other immune cells, and stroma in surgically resected tumor samples from 12 patients with early-stage lung cancer. Results Bacterial burden was significantly higher in tumor cells compared with T cells, macrophages, other immune cells, and stroma. This burden increased from tumor-adjacent normal lung and tertiary lymphoid structures to tumor cells to the airways, suggesting that lung intratumor bacteria derive from the latter route of entry. Expression of oncogenic β-catenin was strongly correlated with bacterial burden, as were tumor histological subtypes and environmental factors. Conclusions Intratumor bacteria were enriched with tumor cells and associated with multiple oncogenic pathways, supporting a rationale for reducing the local intratumor microbiome in lung cancer for patient benefit. Trial registration number NCT00242723 , NCT02146170 .

Introduction: The pathogenesis of thymic epithelial tumors remains largely unknown. We previously identified GTF2I L424H as the most frequently recurrent mutation in thymic epithelial tumors. Nevertheless, the precise role of this mutation in tumorigenesis of thymic epithelial cells is unclear.Methods: To investigate the role of GTF2I L424H mutation in thymic epithelial cells in vivo, we generated and charac-terized a mouse model in which the Gtf2i L424H mutation was conditionally knocked-in in the Foxn1 thorn thymic epithelial cells. Digital spatial profiling was performed on thymomas and normal thymic tissues with GeoMx-mouse whole transcriptome atlas. Immunohistochemistry staining was performed using both mouse tissues and human thymic epithelial tumors.Results: We observed that the Gtf2i mutation impairs development of the thymic medulla and maturation of medullary thymic epithelial cells in young mice and causes tumor formation in the thymus of aged mice. Cell cycle -related pathways, such as E2F targets and MYC targets, are enriched in the tumor epithelial cells. Results of gene set variation assay analysis revealed that gene signatures of cortical thymic epithelial cells and thymic epithelial pro-genitor cells are also enriched in the thymomas of the knock-in mice, which mirrors the human counterparts in The Cancer Genome Atlas database. Immunohistochemistry results revealed similar expression pattern of epithelial cell markers between mouse and human thymomas.Conclusions: We have developed and characterized a novel thymoma mouse model. This study improves knowledge of the molecular drivers in thymic epithelial cells and provides a tool for further study of the biology of thymic epithelial tumors and for development of novel therapies.(c) 2022 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Abstract Hepatocellular carcinoma (HCC) is a heterogeneous disease. Mouse models are commonly used as preclinical models to study hepatocarcinogenesis, but how well these models recapitulate molecular subtypes of human HCC is unclear. Here, integration of genomic signatures from molecularly and clinically defined human HCC (n = 11) and mouse models of HCC (n = 9) identified the mouse models that best resembled subtypes of human HCC and determined the clinical relevance of each model. Mst1/2 knockout (KO), Sav1 KO, and SV40 T antigen mouse models effectively recapitulated subtypes of human HCC with a poor prognosis, whereas the Myc transgenic model best resembled human HCCs with a more favorable prognosis. The Myc model was also associated with activation of β-catenin. E2f1, E2f1/Myc, E2f1/Tgfa, and diethylnitrosamine (DEN)-induced models were heterogeneous and were unequally split into poor and favorable prognoses. Mst1/2 KO and Sav1 KO models best resemble human HCC with hepatic stem cell characteristics. Applying a genomic predictor for immunotherapy, the six-gene IFNγ score, the Mst1/2 KO, Sav1 KO, SV40, and DEN models were predicted to be the least responsive to immunotherapy. Further analysis showed that elevated expression of immune-inhibitory genes (Cd276 and Nectin2/Pvrl2) in Mst1/2 KO, Sav1 KO, and SV40 models and decreased expression of immune stimulatory gene (Cd86) in the DEN model might be accountable for the lack of predictive response to immunotherapy. Implication: The current genomic approach identified the most relevant mouse models to human liver cancer and suggests immunotherapeutic potential for the treatment of specific subtypes. Mol Cancer Res; 16(11); 1713–23. ©2018 AACR.

The majority of hepatocellular carcinoma develops in the background of chronic liver inflammation caused by viral hepatitis and alcoholic or nonalcoholic steatohepatitis. However, the impact of different types of chronic inflammatory microenvironments on the phenotypes of tumors generated by distinct oncogenes is largely unresolved. To address this issue, we generated murine liver tumors by constitutively active AKT‐1 (AKT) and β‐catenin (CAT), followed by induction of chronic liver inflammation by 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC) and carbon tetrachloride. Also, the impact of DDC‐induced chronic liver inflammation was compared between two liver tumor models using a combination of AKT‐CAT or AKT‐NRASG12V. Treatment with DDC and carbon tetrachloride significantly facilitated the adenoma‐to‐carcinoma conversion and accelerated the growth of AKT‐CAT tumors. Furthermore, DDC treatment altered the morphology of AKT‐CAT tumors and caused loss of lipid droplets. Transcriptome analysis of AKT‐CAT tumors revealed that cellular growth and proliferation were mainly affected by chronic inflammation and caused up‐regulation of Cxcl16, Galectin‐3, and Nedd9, among others. Integration with transcriptome profiles from human hepatocellular carcinomas further demonstrated that AKT‐CAT tumors generated in the context of chronic liver inflammation showed enrichment of poor prognosis gene sets or decrease of good prognosis gene sets. In contrast, DDC had a more subtle effect on AKT‐NRASG12V tumors and primarily enhanced already existent tumor characteristics as supported by transcriptome analysis. However, it also reduced lipid droplets in AKT‐NRASG12V tumors. Conclusion: Our study suggests that liver tumor phenotype is defined by a combination of driving oncogenes but also the nature of chronic liver inflammation. (Hepatology 2016;63:1888‐1899)

Recent studies confirmed a critical importance of c-Met signaling for liver regeneration by modulating redox balance. Here we used liver-specific conditional knockout mice (MetKO) and a nutritional model of hepatic steatosis to address the role of c-Met in cholesterol-mediated liver toxicity. Liver injury was assessed by histopathology and plasma enzymes levels. Global transcriptomic changes were examined by gene expression microarray, and key molecules involved in liver damage and lipid homeostasis were evaluated by Western blotting. Loss of c-Met signaling amplified the extent of liver injury in MetKO mice fed with high-cholesterol diet for 30days as evidenced by upregulation of liver enzymes and increased synthesis of total bile acids, aggravated inflammatory response and enhanced intrahepatic lipid deposition. Global transcriptomic changes confirmed the enrichment of networks involved in steatosis and cholestasis. In addition, signaling pathways related to glutathione and lipid metabolism, oxidative stress and mitochondria dysfunction were significantly affected by the loss of c-Met function. Mechanistically, exacerbation of oxidative stress in MetKO livers was corroborated by increased lipid and protein oxidation. Western blot analysis further revealed suppression of Erk, NF-kB and Nrf2 survival pathways and downstream target genes (e.g. cyclin D1, SOD1, gamma-GCS), as well as up-regulation of proapoptotic signaling (e.g. p53, caspase 3). Consistent with the observed steatotic and cholestatic phenotype, nuclear receptors RAR, RXR showed increased activation while expression levels of CAR, FXR and PPAR-alpha were decreased in MetKO. Collectively, our data provide evidence for the critical involvement of c-Met signaling in cholesterol and bile acids toxicity.

DNA methyltransferase 1 (DNMT1) is an essential regulator maintaining both epigenetic reprogramming during DNA replication and genome stability. We investigated the role of DNMT1 in the regulation of postnatal liver histogenesis under homeostasis and stress conditions. We generated Dnmt1 conditional knockout mice (Dnmt1Δalb) by crossing Dnmt1fl/fl with albumin‐cyclization recombination transgenic mice. Serum, liver tissues, and primary hepatocytes were collected from 1‐week‐old to 20‐week old mice. The Dnmt1Δalb phenotype was assessed by histology, confocal and electron microscopy, biochemistry, as well as transcriptome and methylation profiling. Regenerative growth was induced by partial hepatectomy and exposure to carbon tetrachloride. The impact of Dnmt1 knockdown was also analyzed in hepatic progenitor cell lines; proliferation, apoptosis, DNA damage, and sphere formation were assessed. Dnmt1 loss in postnatal hepatocytes caused global hypomethylation, enhanced DNA damage response, and initiated a senescence state causing a progressive inability to maintain tissue homeostasis and proliferate in response to injury. The liver regenerated through activation and repopulation from progenitors due to lineage‐dependent differences in albumin‐cyclization recombination expression, providing a basis for selection of less mature and therefore less damaged hepatic progenitor cell progeny. Consistently, efficient knockdown of Dnmt1 in cultured hepatic progenitor cells caused severe DNA damage, cell cycle arrest, senescence, and cell death. Mx1‐cyclization recombination‐driven deletion of Dnmt1 in adult quiescent hepatocytes did not affect liver homeostasis. Conclusion: These results establish the indispensable role of DNMT1‐mediated epigenetic regulation in postnatal liver growth and regeneration; Dnmt1Δalb mice provide a unique experimental model to study the role of senescence and the contribution of progenitor cells to physiological and regenerative liver growth. (Hepatology 2016;64:582‐598)