Oncology
Abstract
N6-Methyladenosine (m6A) is the most abundant posttranscriptional modification, and its contribution to cancer evolution has recently been appreciated. Renal cancer is the most common adult genitourinary cancer, approximately 85% of which is accounted for by the clear cell renal cell carcinoma (ccRCC) subtype characterized by VHL loss. However, it is unclear whether VHL loss in ccRCC affects m6A patterns. In this study, we demonstrate that VHL binds and promotes METTL3/METTL14 complex formation while VHL depletion suppresses m6A modification, which is distinctive from its canonical E3 ligase role. m6A RNA immunoprecipitation sequencing (RIP-Seq) coupled with RNA-Seq allows us to identify a selection of genes whose expression may be regulated by VHL-m6A signaling. Specifically, PIK3R3 is identified to be a critical gene whose mRNA stability is regulated by VHL in a m6A-dependent but HIF-independent manner. Functionally, PIK3R3 depletion promotes renal cancer cell growth and orthotopic tumor growth while its overexpression leads to decreased tumorigenesis. Mechanistically, the VHL-m6A–regulated PIK3R3 suppresses tumor growth by restraining PI3K/AKT activity. Taken together, we propose a mechanism by which VHL regulates m6A through modulation of METTL3/METTL14 complex formation, thereby promoting PIK3R3 mRNA stability and protein levels that are critical for regulating ccRCC tumorigenesis.
Authors
Cheng Zhang, Miaomiao Yu, Austin J. Hepperla, Zhao Zhang, Rishi Raj, Hua Zhong, Jin Zhou, Lianxin Hu, Jun Fang, Hongyi Liu, Qian Liang, Liwei Jia, Chengheng Liao, Sichuan Xi, Jeremy M. Simon, Kexin Xu, Zhijie Liu, Yunsun Nam, Payal Kapur, Qing Zhang
Abstract
Clear cell renal cell carcinoma (ccRCC) is characterized by dysregulated hypoxia signaling and a tumor microenvironment (TME) highly enriched in myeloid and lymphoid cells. Loss of the von Hippel Lindau (VHL) gene is a critical early event in ccRCC pathogenesis and promotes stabilization of HIF. Whether VHL loss in cancer cells affects immune cells in the TME remains unclear. Using Vhl WT and Vhl-KO in vivo murine kidney cancer Renca models, we found that Vhl-KO tumors were more infiltrated by immune cells. Tumor-associated macrophages (TAMs) from Vhl-deficient tumors demonstrated enhanced in vivo glucose consumption, phagocytosis, and inflammatory transcriptional signatures, whereas lymphocytes from Vhl-KO tumors showed reduced activation and a lower response to anti–programmed cell death 1 (anti–PD-1) therapy in vivo. The chemokine CX3CL1 was highly expressed in human ccRCC tumors and was associated with Vhl deficiency. Deletion of Cx3cl1 in cancer cells decreased myeloid cell infiltration associated with Vhl loss to provide a mechanism by which Vhl loss may have contributed to the altered immune landscape. Here, we identify cancer cell–specific genetic features that drove environmental reprogramming and shaped the tumor immune landscape, with therapeutic implications for the treatment of ccRCC.
Authors
Melissa M. Wolf, Matthew Z. Madden, Emily N. Arner, Jackie E. Bader, Xiang Ye, Logan Vlach, Megan L. Tigue, Madelyn D. Landis, Patrick B. Jonker, Zaid Hatem, KayLee K. Steiner, Dakim K. Gaines, Bradley I. Reinfeld, Emma S. Hathaway, Fuxue Xin, M. Noor Tantawy, Scott M. Haake, Eric Jonasch, Alexander Muir, Vivian L. Weiss, Kathryn E. Beckermann, W. Kimryn Rathmell, Jeffrey C. Rathmell
Abstract
Cancer-derived small extracellular vesicles (sEVs) are capable of modifying 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, two patient cohorts were enrolled, including OvCa cases who underwent a diagnostic or cytoreductive surgery, and non-oncological patients as controls, who underwent abdominal surgery for benign gynecological conditions. PFD-sEVs systematic extraction 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-sEVs proteomic profiles in high-grade serous ovarian carcinomas (HGSOC) revealed two clusters with different outcomes in terms of overall survival. In conclusion, comprehensive characterization of the PFD-sEVs content provided a prognostic value with potential implications in HGSOC clinical management.
Authors
Miguel Quiralte, Arantzazu Barquín, Mónica Yagüe Fernández, Paloma Navarro, Tatiana P. Grazioso, Elena Sevillano, Juan F. Rodriguez Moreno, Alejandra Balarezo-Saldivar, Héctor Peinado, Elena Izquierdo, Carlos Millán, Irene López Carrasco, Mario Prieto, Rodrigo Madurga de Lacalle, Ismael Fernández-Miranda, Sergio Ruiz-Llorente, Jesús García-Donas
Abstract
BACKGROUND Precise stratification of patients with non–small cell lung cancer (NSCLC) is needed for appropriate application of PD-1/PD-L1 blockade therapy.METHODS We measured soluble forms of the immune-checkpoint molecules PD-L1, PD-1, and CTLA-4 in plasma of patients with advanced NSCLC before PD-1/PD-L1 blockade. A prospective biomarker-finding trial (cohort A) included 50 previously treated patients who received nivolumab. A retrospective observational study was performed for patients treated with any PD-1/PD-L1 blockade therapy (cohorts B and C), cytotoxic chemotherapy (cohort D), or targeted therapy (cohort E). Plasma samples from all patients were assayed for soluble immune-checkpoint molecules with a highly sensitive chemiluminescence-based assay.RESULTS Nonresponsiveness to PD-1/PD-L1 blockade therapy was associated with higher concentrations of these soluble immune factors among patients with immune-reactive (hot) tumors. Such an association was not apparent for patients treated with cytotoxic chemotherapy or targeted therapy. Integrative analysis of tumor size, PD-L1 expression in tumor tissue (tPD-L1), and gene expression in tumor tissue and peripheral CD8+ T cells revealed that high concentrations of the 3 soluble immune factors were associated with hyper or terminal exhaustion of antitumor immunity. The combination of soluble PD-L1 (sPD-L1) and sCTLA-4 efficiently discriminated responsiveness to PD-1/PD-L1 blockade among patients with immune-reactive tumors.CONCLUSION Combinations of soluble immune factors might be able to identify patients unlikely to respond to PD-1/PD-L1 blockade as a result of terminal exhaustion of antitumor immunity. Our data suggest that such a combination better predicts, along with tPD-L1, for the response of patients with NSCLC.TRIAL REGISTRATION UMIN000019674.FUNDING This study was funded by Ono Pharmaceutical Co. Ltd. and Sysmex Corporation.
Authors
Hidetoshi Hayashi, Kenji Chamoto, Ryusuke Hatae, Takashi Kurosaki, Yosuke Togashi, Kazuya Fukuoka, Megumi Goto, Yasutaka Chiba, Shuta Tomida, Takayo Ota, Koji Haratani, Takayuki Takahama, Junko Tanizaki, Takeshi Yoshida, Tsutomu Iwasa, Kaoru Tanaka, Masayuki Takeda, Tomoko Hirano, Hironori Yoshida, Hiroaki Ozasa, Yuichi Sakamori, Kazuko Sakai, Keiko Higuchi, Hitoshi Uga, Chihiro Suminaka, Toyohiro Hirai, Kazuto Nishio, Kazuhiko Nakagawa, Tasuku Honjo
Abstract
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 suppresses the growth of CRPC xenografts but also induces tumor regression and cures. Transcriptomic analysis revealed that Mediator kinase inhibition amplifies and modulates the effects of castration on gene expression, disrupting CRPC adaptation to androgen deprivation. Mediator kinase inactivation in tumor cells also affects stromal gene expression, indicating that Mediator kinase activity in CRPC molds the tumor microenvironment. The combination of castration and Mediator kinase inhibition downregulates the MYC pathway, and Mediator kinase inhibition suppresses a MYC-driven CRPC tumor model even without castration. CDK8/19 inhibitors show efficacy in patient-derived xenograft models of CRPC, and a gene signature of Mediator kinase activity correlates with tumor progression and overall survival in clinical samples of metastatic CRPC. These results indicate that Mediator kinases mediate androgen-independent in vivo growth of CRPC, supporting the development of CDK8/19 inhibitors for the treatment of this presently incurable disease.
Authors
Jing Li, Thomas A. Hilimire, Liu Yueying, Lili Wang, Jiaxin Liang, Balázs Győrffy, 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
Abstract
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 anti-cancer therapy, especially in tumors expressing c-myc.
Authors
Joonyoung Her, Haiyan Zheng, Samuel F. Bunting
Abstract
Despite widespread utilization of immunotherapy, challenge to treat immune-cold tumors needs to be resolved. 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 that inhibits 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 a novel strategy for targeting immunosuppressive OTUD4-CD73 proteolytic axis in treating immune-suppressive breast cancers with the inhibitor ST80.
Authors
Yueming Zhu, Anupam Banerjee, Ping Xie, Andrey A. Ivanov, Amad Uddin, Qiao Jiao, Junlong J. 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
Abstract
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 development of multiple neoplasms, including Malignant Peripheral Nerve Sheath Tumors (MPNSTs). MPNSTs are an aggressive tumor and the main cause of mortality in NF1 patients. 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 (TME). While MPNSTs are non-inflamed “cold” tumors, here, we turned MPNSTs into T cell-inflamed “hot” tumors by activating “stimulator of interferon genes” (STING) signaling. Mouse genetic and human xenograft MPNST models treated with STING agonist plus ICB exhibited growth delay via increased apoptotic cell death. This strategy offers a potential treatment regimen for MPNST.
Authors
Bandarigoda N. Somatilaka, Laasya Madana, Ali Sadek, Zhiguo Chen, Sanjay Chandrasekaran, Renee M. McKay, Lu Q. Le
Abstract
Macrophage immune checkpoint inhibitors, such as anti-CD47 antibodies, show promise in clinical trials for solid and hematologic malignancies. However, the best strategies to use these therapies remain unknown, and ongoing studies suggest they may be most effective when used in combination with other anticancer agents. Here, we developed a novel screening platform to identify drugs that render lung cancer cells more vulnerable to macrophage attack, and we identified therapeutic synergy exists between genotype-directed therapies and anti-CD47 antibodies. In validation studies, we found the combination of genotype-directed therapies and CD47 blockade elicited robust phagocytosis and eliminated persister cells in vitro and maximized anti-tumor responses in vivo. Importantly, these findings broadly applied to lung cancers with various RTK/MAPK pathway alterations—including EGFR mutations, ALK fusions, or KRASG12C mutations. We observed downregulation of β2-microglobulin and CD73 as molecular mechanisms contributing to enhanced sensitivity to macrophage attack. Our findings demonstrate that dual inhibition of the RTK/MAPK pathway and the CD47/SIRPa axis is a promising immunotherapeutic strategy. Our study provides strong rationale for testing this therapeutic combination in patients with lung cancers bearing driver mutations.
Authors
Kyle Vaccaro, Juliet Allen, Troy W. Whitfield, Asaf Maoz, Sarah Reeves, José Velarde, Dian Yang, Anna Meglan, Juliano Ribeiro, Jasmine Blandin, Nicole Phan, George W. Bell, Aaron Hata, Kipp Weiskopf
Abstract
Development of effective strategies to manage the inevitable acquired resistance to osimertinib, an approved 3rd generation EGFR inhibitor for the treatment of EGFR mutant (EGFRm) non-small cell lung cancer (NSCLC), is urgently needed. This study reported that the DNA topoisomerase II (Topo II) inhibitors, doxorubicin and etoposide (VP-16) 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 possessing elevated levels of Topo IIα. Topo IIα elevation was also detected in the majority of EGFRm NSCLC tissues relapsed from EGFR-TKI 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 response to undergo 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.
Authors
Zhen Chen, Karin A. Vallega, Dongsheng Wang, Zihan Quan, Songqing Fan, Qiming Wang, Ticiana Leal, Suresh S. Ramalingam, Shi-Yong Sun
Copyright © 2026 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)