Mediator kinases CDK19 and CDK8, pleiotropic regulators of transcriptional reprogramming, are differentially regulated by androgen signaling, but both kinases are upregulated in castration-resistant prostate cancer (CRPC). Genetic or pharmacological inhibition of CDK8 and CDK19 reverses the castration-resistant phenotype and restores the sensitivity of CRPC xenografts to androgen deprivation in vivo. Prolonged CDK8/19 inhibitor treatment combined with castration not only suppressed the growth of CRPC xenografts but also induced tumor regression and cures. Transcriptomic analysis revealed that Mediator kinase inhibition amplified and modulated the effects of castration on gene expression, disrupting CRPC adaptation to androgen deprivation. Mediator kinase inactivation in tumor cells also affected stromal gene expression, indicating that Mediator kinase activity in CRPC molded the tumor microenvironment. The combination of castration and Mediator kinase inhibition downregulated the MYC pathway, and Mediator kinase inhibition suppressed a MYC-driven CRPC tumor model even without castration. CDK8/19 inhibitors showed efficacy in patient-derived xenograft models of CRPC, and a gene signature of Mediator kinase activity correlated with tumor progression and overall survival in clinical samples of metastatic CRPC. These results indicate that Mediator kinases mediated androgen-independent in vivo growth of CRPC, supporting the development of CDK8/19 inhibitors for the treatment of this presently incurable disease.
Jing Li, Thomas A. Hilimire, Yueying Liu, Lili Wang, Jiaxin Liang, Balazs Gyorffy, Vitali Sikirzhytski, Hao Ji, Li Zhang, Chen Cheng, Xiaokai Ding, Kendall R. Kerr, Charles E. Dowling, Alexander A. Chumanevich, Zachary T. Mack, Gary P. Schools, Chang-uk Lim, Leigh Ellis, Xiaolin Zi, Donald C. Porter, Eugenia V. Broude, Campbell McInnes, George Wilding, Michael B. Lilly, Igor B. Roninson, Mengqian Chen
Cancer-derived small extracellular vesicles (sEVs) are capable of modifying the tumor microenvironment and promoting tumor progression. Ovarian cancer (OvCa) is a lethal malignancy that preferentially spreads through the abdominal cavity. Thus, the secretion of such vesicles into the peritoneal fluid could be a determinant factor in the dissemination and behavior of this disease. We designed a prospective observational study to assess the impact of peritoneal fluid–derived sEVs (PFD-sEVs) in OvCa clinical outcome. For this purpose, 2 patient cohorts were enrolled: patients with OvCa who underwent a diagnostic or cytoreductive surgery and nononcological patients, who underwent abdominal surgery for benign gynecological conditions and acted as the control group. Systematic extraction of PFD-sEVs from surgical samples enabled us to observe significant quantitative and qualitative differences associated with cancer diagnosis, disease stage, and platinum chemosensitivity. Proteomic profiling of PFD-sEVs led to the identification of molecular pathways and proteins of interest and to the biological validation of S100A4 and STX5. In addition, unsupervised analysis of PFD-sEV proteomic profiles in high-grade serous ovarian carcinomas (HGSOCs) revealed 2 clusters with different outcomes in terms of overall survival. In conclusion, comprehensive characterization of PFD-sEV content provided a prognostic value with potential implications in HGSOC clinical management.
Miguel Quiralte, Arantzazu Barquín, Mónica Yagüe-Fernández, Paloma Navarro, Tatiana P. Grazioso, Elena Sevillano-Fernández, Juan F. Rodriguez-Moreno, Alejandra Balarezo-Saldivar, Héctor Peinado, Elena Izquierdo, Carlos Millán, Irene López-Carrasco, Mario Prieto, Rodrigo Madurga, Ismael Fernández-Miranda, Sergio Ruiz-Llorente, Jesús García-Donas
Gender-affirming hormone therapy (GAHT) is often prescribed to transgender (TG) adolescents to alleviate gender dysphoria, but the effect of GAHT on the growing skeleton is unclear. We found GAHT to improve trabecular bone structure via increased bone formation in young male mice and not to affect trabecular structure in female mice. GAHT modified gut microbiome composition in both male and female mice. However, fecal microbiota transfers (FMTs) revealed that GAHT-shaped gut microbiome was a communicable regulator of bone structure and turnover in male, but not in female mice. Mediation analysis identified 2 species of Bacteroides as significant contributors to the skeletal effects of GAHT in male mice, with Bacteroides supplementation phenocopying the effects of GAHT on bone. Bacteroides have the capacity to expand Treg populations in the gut. Accordingly, GAHT expanded intestinal Tregs and stimulated their migration to the bone marrow (BM) in male but not in female mice. Attesting to the functional relevance of Tregs, pharmacological blockade of Treg expansion prevented GAHT-induced bone anabolism. In summary, in male mice GAHT stimulated bone formation and improved trabecular structure by promoting Treg expansion via a microbiome-mediated effect, while in female mice, GAHT neither improved nor impaired trabecular structure.
Subhashis Pal, Xochitl Morgan, Hamid Y. Dar, Camilo Anthony Gacasan, Sanchiti Patil, Andreea Stoica, Yi-Juan Hu, M. Neale Weitzmann, Rheinallt M. Jones, Roberto Pacifici
Despite widespread utilization of immunotherapy, treating immune-cold tumors remains a challenge. Multiomic analyses and experimental validation identified the OTUD4/CD73 proteolytic axis as a promising target in treating immune-suppressive triple negative breast cancer (TNBC). Mechanistically, deubiquitylation of CD73 by OTUD4 counteracted its ubiquitylation by TRIM21, resulting in CD73 stabilization inhibiting tumor immune responses. We further demonstrated the importance of TGF-β signaling for orchestrating the OTUD4/CD73 proteolytic axis within tumor cells. Spatial transcriptomics profiling discovered spatially resolved features of interacting malignant and immune cells pertaining to expression levels of OTUD4 and CD73. In addition, ST80, a newly developed inhibitor, specifically disrupted proteolytic interaction between CD73 and OTUD4, leading to reinvigoration of cytotoxic CD8+ T cell activities. In preclinical models of TNBC, ST80 treatment sensitized refractory tumors to anti-PD-L1 therapy. Collectively, our findings uncover what we believe to be a novel strategy for targeting the immunosuppressive OTUD4/CD73 proteolytic axis in treating immune-suppressive breast cancers with the inhibitor ST80.
Yueming Zhu, Anupam Banerjee, Ping Xie, Andrey A. Ivanov, Amad Uddin, Qiao Jiao, Junlong Jack Chi, Lidan Zeng, Ji Young Lee, Yifan Xue, Xinghua Lu, Massimo Cristofanilli, William J. Gradishar, Curtis J. Henry, Theresa W. Gillespie, Manali Ajay Bhave, Kevin Kalinsky, Haian Fu, Ivet Bahar, Bin Zhang, Yong Wan
Development of effective strategies to manage the inevitable acquired resistance to osimertinib, a third-generation EGFR inhibitor for the treatment of EGFR-mutant (EGFRm) non–small cell lung cancer (NSCLC), is urgently needed. This study reports that DNA topoisomerase II (Topo II) inhibitors, doxorubicin and etoposide, synergistically decreased cell survival, with enhanced induction of DNA damage and apoptosis in osimertinib-resistant cells; suppressed the growth of osimertinib-resistant tumors; and delayed the emergence of osimertinib-acquired resistance. Mechanistically, osimertinib decreased Topo IIα levels in EGFRm NSCLC cells by facilitating FBXW7-mediated proteasomal degradation, resulting in induction of DNA damage; these effects were lost in osimertinib-resistant cell lines that possess elevated levels of Topo IIα. Increased Topo IIα levels were also detected in the majority of tissue samples from patients with NSCLC after relapse from EGFR tyrosine kinase inhibitor treatment. Enforced expression of an ectopic TOP2A gene in sensitive EGFRm NSCLC cells conferred resistance to osimertinib, whereas knockdown of TOP2A in osimertinib-resistant cell lines restored their susceptibility to osimertinib-induced DNA damage and apoptosis. Together, these results reveal an essential role of Topo IIα inhibition in mediating the therapeutic efficacy of osimertinib against EGFRm NSCLC, providing scientific rationale for targeting Topo II to manage acquired resistance to osimertinib.
Zhen Chen, Karin A. Vallega, Dongsheng Wang, Zihan Quan, Songqing Fan, Qiming Wang, Ticiana Leal, Suresh S. Ramalingam, Shi-Yong Sun
Fibrosis following tissue injury is distinguished from normal repair by the accumulation of pathogenic and apoptosis-resistant myofibroblasts (MFs), which arise primarily by differentiation from resident fibroblasts. Endogenous molecular brakes that promote MF dedifferentiation and clearance during spontaneous resolution of experimental lung fibrosis may provide insights that could inform and improve the treatment of progressive pulmonary fibrosis in patients. MAPK phosphatase 1 (MKP1) influences the cellular phenotype and fate through precise and timely regulation of MAPK activity within various cell types and tissues, yet its role in lung fibroblasts and pulmonary fibrosis has not been explored. Using gain- and loss-of-function studies, we found that MKP1 promoted lung MF dedifferentiation and restored the sensitivity of these cells to apoptosis — effects determined to be mainly dependent on MKP1’s dephosphorylation of p38α MAPK (p38α). Fibroblast-specific deletion of MKP1 following peak bleomycin-induced lung fibrosis largely abrogated its subsequent spontaneous resolution. Such resolution was restored by treating these transgenic mice with the p38α inhibitor VX-702. We conclude that MKP1 is a critical antifibrotic brake whose inhibition of pathogenic p38α in lung fibroblasts is necessary for fibrosis resolution following lung injury.
Sean M. Fortier, Natalie M. Walker, Loka R. Penke, Jared D. Baas, Qinxue Shen, Jennifer M. Speth, Steven K. Huang, Rachel L. Zemans, Anton M. Bennett, Marc Peters-Golden
The mammalian SUMO-targeted E3 ubiquitin ligase Rnf4 has been reported to act as a regulator of DNA repair, but the importance of RNF4 as a tumor suppressor has not been tested. Using a conditional-knockout mouse model, we deleted Rnf4 in the B cell lineage to test the importance of RNF4 for growth of somatic cells. Although Rnf4–conditional-knockout B cells exhibited substantial genomic instability, Rnf4 deletion caused no increase in tumor susceptibility. In contrast, Rnf4 deletion extended the healthy lifespan of mice expressing an oncogenic c-myc transgene. Rnf4 activity is essential for normal DNA replication, and in its absence, there was a failure in ATR-CHK1 signaling of replication stress. Factors that normally mediate replication fork stability, including members of the Fanconi anemia gene family and the helicases PIF1 and RECQL5, showed reduced accumulation at replication forks in the absence of RNF4. RNF4 deficiency also resulted in an accumulation of hyper-SUMOylated proteins in chromatin, including members of the SMC5/6 complex, which contributes to replication failure by a mechanism dependent on RAD51. These findings indicate that RNF4, which shows increased expression in multiple human tumor types, is a potential target for anticancer therapy, especially in tumors expressing c-myc.
Joonyoung Her, Haiyan Zheng, Samuel F. Bunting
Clarkson disease, or monoclonal gammopathy–associated idiopathic systemic capillary leak syndrome (ISCLS), is a rare, relapsing-remitting disorder featuring the abrupt extravasation of fluids and proteins into peripheral tissues, which in turn leads to hypotensive shock, severe hemoconcentration, and hypoalbuminemia. The specific leakage factor(s) and pathways in ISCLS are unknown, and there is no effective treatment for acute flares. Here, we characterize an autonomous vascular endothelial defect in ISCLS that was recapitulated in patient-derived endothelial cells (ECs) in culture and in a mouse model of disease. ISCLS-derived ECs were functionally hyperresponsive to permeability-inducing factors like VEGF and histamine, in part due to increased endothelial nitric oxide synthase (eNOS) activity. eNOS blockade by administration of N(γ)-nitro-l-arginine methyl ester (l-NAME) ameliorated vascular leakage in an SJL/J mouse model of ISCLS induced by histamine or VEGF challenge. eNOS mislocalization and decreased protein phosphatase 2A (PP2A) expression may contribute to eNOS hyperactivation in ISCLS-derived ECs. Our findings provide mechanistic insights into microvascular barrier dysfunction in ISCLS and highlight a potential therapeutic approach.
Ararat J. Ablooglu, Wei-Sheng Chen, Zhihui Xie, Abhishek Desai, Subrata Paul, Justin B. Lack, Linda A. Scott, A. Robin Eisch, Arkadiusz Z. Dudek, Samir M. Parikh, Kirk M. Druey
Renal interstitial fibrosis is an important mechanism in the progression of chronic kidney disease (CKD) to end-stage kidney disease. However, we lack specific treatments to slow or halt renal fibrosis. Ribosome profiling identified upregulation of a secreted micropeptide, C4orf48 (Cf48), in mouse diabetic nephropathy. Cf48 RNA and protein levels were upregulated in tubular epithelial cells in human and experimental CKD. Serum Cf48 levels were increased in human CKD and correlated with loss of kidney function, increasing CKD stage, and the degree of active interstitial fibrosis. Cf48 overexpression in mice accelerated renal fibrosis, while Cf48 gene deletion or knockdown by antisense oligonucleotides significantly reduced renal fibrosis in CKD models. In vitro, recombinant Cf48 (rCf48) enhanced TGF-β1–induced fibrotic responses in renal fibroblasts and epithelial cells independently of Smad3 phosphorylation. Cellular uptake of Cf48 and its profibrotic response in fibroblasts operated via the transferrin receptor. RNA immunoprecipitation–sequencing identified Cf48 binding to mRNA of genes involved in the fibrotic response, including Serpine1, Acta2, Ccn2, and Col4a1. rCf48 binds to the 3′UTR of Serpine1 and increases mRNA half-life. We identify the secreted Cf48 micropeptide as a potential enhancer of renal fibrosis that operates as an RNA-binding peptide to promote the production of extracellular matrix.
Jiayi Yang, Hongjie Zhuang, Jinhua Li, Ana B. Nunez-Nescolarde, Ning Luo, Huiting Chen, Andy Li, Xinli Qu, Qing Wang, Jinjin Fan, Xiaoyan Bai, Zhiming Ye, Bing Gu, Yue Meng, Xingyuan Zhang, Di Wu, Youyang Sia, Xiaoyun Jiang, Wei Chen, Alexander N. Combes, David J. Nikolic-Paterson, Xueqing Yu
Neurofibromatosis type 1 (NF1) is caused by mutations in the NF1 gene that encodes neurofibromin, a RAS GTPase–activating protein. Inactivating NF1 mutations cause hyperactivation of RAS-mediated signaling, resulting in the development of multiple neoplasms, including malignant peripheral nerve sheath tumors (MPNSTs). MPNSTs are an aggressive tumor and the main cause of mortality in patients with NF1. MPNSTs are difficult to resect and refractory to chemo- and radiotherapy, and no molecular therapies currently exist. Immune checkpoint blockade (ICB) is an approach to treat inoperable, undruggable cancers like MPNST, but successful outcomes require an immune cell–rich tumor microenvironment. While MPNSTs are noninflamed “cold” tumors, here, we converted MPNSTs into T cell–inflamed “hot” tumors by activating stimulator of IFN genes (STING) signaling. Mouse genetic and human xenograft MPNST models treated with a STING agonist plus ICB exhibited growth delay via increased apoptotic cell death. This strategy offers a potential treatment regimen for MPNSTs.
Bandarigoda N. Somatilaka, Laasya Madana, Ali Sadek, Zhiguo Chen, Sanjay Chandrasekaran, Renee M. McKay, Lu Q. Le
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