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Abstract In advanced hepatocellular carcinoma (HCC), RNA helicase DDX5 regulates the Wnt/β-catenin-ferroptosis axis, influencing the efficacy of the multi-tyrosine kinase inhibitor (mTKI) sorafenib. DDX5 inhibits Wnt/β-catenin signaling, preventing sorafenib-induced ferroptosis escape. Sorafenib/mTKIs reduce DDX5 expression, correlating with poor patient survival post-sorafenib treatment. Notably, DDX5-knockout in HCC cells activates Wnt/β-catenin signaling persistently. Herein, we investigate the mechanistic impact of Wnt/β-catenin activation resulting from DDX5 downregulation in the progression and treatment of HCC. RNAseq analyses identified shared genes repressed by DDX5 and upregulated by sorafenib, including Wnt signaling genes, NF-κB-inducing kinase (NIK) essential for non-canonical NF-κB (p52/RelB) activation, and cytoprotective transcription factor NRF2. We demonstrate, Wnt/β-catenin activation induced NIK transcription, leading to non-canonical NF-κB activation, which subsequently mediated NRF2 transcription. Additionally, DDX5 deficiency extended NRF2 protein half-life by inactivating KEAP1 through p62/SQSTM1 stabilization. In a preclinical HCC mouse model, NRF2 knockdown or DDX5 overexpression restricted tumor growth upon sorafenib treatment, via induction of ferroptosis. Importantly, DDX5-knockout HCC cells exhibited elevated expression of Wnt signaling genes, NIK, p52/RelB, and NRF2-regulated genes, regardless of sorafenib treatment. Transcriptomic analyses of HCCs from TCGA and the Stelic Animal Model (STAM) of non-alcoholic steatohepatitis revealed elevated expression of these interconnected pathways in the context of DDX5 downregulation. In conclusion, DDX5 deficiency triggers Wnt/β-catenin signaling, promoting p52/RelB and NRF2 activation, thereby enabling ferroptosis evasion upon sorafenib treatment. Similarly, independent of sorafenib, DDX5 deficiency in liver tumors enhances activation and gene expression of these interconnected pathways, underscoring the clinical relevance of DDX5 deficiency in HCC progression and therapeutic response.

The present study focused on comparing the gene expression profiles of different mouse models of prostate cancer, focusing on the TRAMP transgenic model and its derived cell lines and extending the comparisons to relevant genetically engineered mouse models and human prostate cancer datasets. Employing RNA sequencing, we examined different levels of prostate cancer aggressiveness from the original TRAMP cells to the TRAMP-C2 (TC2) derived cell line and extending to the aggressive TC2-Ras (TC2R) cells and tumors. TC2R acquire the ability to grow in bone tissue upon implantation, unlike the parental TC2 cells. Analysis identified upregulated genes in cell cycle regulation, immune response, and mitotic processes in TRAMP compared to wild-type tissues. TC2 cells exhibited unique gene profiles enriched in ECM organization and tissue development pathways, while TC2R cells showed increased cytokine signaling and motility genes, with decreased ECM and immune response pathways. In vivo TC2R models demonstrated enhanced ECM organization and receptor tyrosine kinase signaling in tumors, notably enriching immune processes and collagen degradation pathways in intratibial tumors. Comparative analysis among mouse and human datasets showed overlaps, particularly in pathways relating to mitotic cycle regulation, ECM organization, and immune interactions. A gene signature identified in TC2R tumors correlated with aggressive tumor behavior and poor survival in human datasets. Further immune cell landscape analysis of TC2R tumors revealed altered T cell subsets and macrophages, confirmed in single-cell RNA-seq from human samples. TC2R models thus hold significant promise in helping advance preclinical therapeutics, potentially contributing to improved prostate cancer patient outcomes.

Abstract Severe heterogeneity within glioblastoma has spurred the notion that disrupting the interplay between multiple elements on immunosuppression is at the core of meaningful anti-tumor responses. T cell immunoreceptor with Ig and ITIM domains (TIGIT) and its glioblastoma-associated antigen, CD155, form a highly immunosuppressive axis in glioblastoma and other solid tumors, yet targeting of TIGIT, a functionally heterogeneous receptor on tumor-infiltrating immune cells, has largely been ineffective as monotherapy, suggesting that disruption of its inhibitory network might be necessary for measurable responses. It is within this context that we show that the usurpation of the TIGIT − CD155 axis via engineered synNotch-mediated activation of induced pluripotent stem cell-derived natural killer (NK) cells promotes transcription factor-mediated activation of a downstream signaling cascade that results in the controlled, localized blockade of CD73 to disrupt purinergic activity otherwise resulting in the production and accumulation of immunosuppressive extracellular adenosine. Such “decoy” receptor engages CD155 binding to TIGIT, but tilts inhibitory TIGIT/CD155 interactions toward activation via downstream synNotch signaling. Usurping activities of TIGIT and CD73 promotes the function of adoptively transferred NK cells into intracranial patient-derived models of glioblastoma and enhances their natural cytolytic functions against this tumor to result in complete tumor eradication. In addition, targeting both receptors, in turn, reprograms the glioblastoma microenvironment via the recruitment of T cells and the downregulation of M2 macrophages. This study demonstrates that TIGIT/CD155 and CD73 are targetable receptor partners in glioblastoma. Our data show that synNotch-engineered pluripotent stem cell-derived NK cells are not only effective mediators of anti-glioblastoma responses within the setting of CD73 and TIGIT/CD155 co-targeting, but represent a powerful allogeneic treatment option for this tumor.

Abstract Reduced expression of the RNA helicase DDX5 associated with increased hepatocellular carcinoma (HCC) tumor grade and poor patient survival following treatment with sorafenib. While immunotherapy is the first-line treatment for HCC, sorafenib and other multi-tyrosine kinase inhibitors (mTKIs) are widely used when immunotherapy is contra-indicated or fails. Herein, we elucidate the role of DDX5 in sensitizing HCC to sorafenib, offering new therapeutic strategies. Treatment of various human HCC cell lines with sorafenib/mTKIs downregulated DDX5 in vitro and in preclinical HCC models. Conversely, DDX5 overexpression reduced the viability of sorafenib-treated cells via ferroptosis, suggesting a role for DDX5 in sorafenib sensitivity. RNAseq of wild-type vs. DDX5-knockdown cells treated with or without sorafenib identified a set of common genes repressed by DDX5 and upregulated by sorafenib. This set significantly overlaps with Wnt signaling genes, including Disheveled-1 (DVL1), an indispensable Wnt activator and prognostic indicator of poor survival for sorafenib-treated patients. DDX5-knockout (DDX5KO) HCC cells exhibited DVL1 induction, Wnt/β-catenin pathway activation, and ferroptosis upon inhibition of canonical Wnt signaling. Consistently, xenograft HCC tumors exhibited reduced growth by inhibition of Wnt/β-catenin signaling via induction of ferroptosis. Significantly, overexpression of DDX5 in HCC xenografts repressed DVL1 expression and increased ferroptosis, resulting in reduced tumor growth by sorafenib. We conclude that DDX5 downregulation by sorafenib mediates adaptive resistance by activating Wnt/β-catenin signaling, leading to ferroptosis escape. Conversely, overexpression of DDX5 in vivo enhances the anti-tumor efficacy of sorafenib by suppressing Wnt/β-catenin activation and induction of ferroptosis. Thus, DDX5 overexpression in combination with mTKIs is a promising therapeutic strategy for HCC.

Summary: TIGIT is a receptor on human natural killer (NK) cells. Here, we report that TIGIT does not spontaneously induce inhibition of NK cells in glioblastoma (GBM), but rather acts as a decoy-like receptor, by usurping binding partners and regulating expression of NK activating ligands and receptors. Our data show that in GBM patients, one of the underpinnings of unresponsiveness to TIGIT blockade is that by targeting TIGIT, NK cells do not lose an inhibitory signal, but gains the potential for new interactions with other, shared, TIGIT ligands. Therefore, TIGIT does not define NK cell dysfunction in GBM. Further, in GBM, TIGIT+ NK cells are hyperfunctional. In addition, we discovered that 4-1BB correlates with TIGIT expression, the agonism of which contributes to TIGIT immunotherapy. Overall, our data suggest that in GBM, TIGIT acts as a regulator of a complex network, and provide new clues about its use as an immunotherapeutic target.

Immunometabolic reprogramming due to adenosine produced by CD73 (encoded by the 5’-ectonucleotidase gene NT5E) is a recognized immunosuppressive mechanism contributing to immune evasion in solid tumors. Adenosine is not only known to contribute to tumor progression, but it has specific roles in driving dysfunction of immune cells, including natural killer (NK) cells. Here, we engineered human NK cells to directly target the CD73-adenosine axis by blocking the enzymatic activity of CD73. In doing so, the engineered NK cells not only impaired adenosinergic metabolism driven by the hypoxic uptake of ATP by cancer cells in a model of non-small-cell lung cancer, but also mediated killing of tumor cells due to the specific recognition of overexpressed CD73. This resulted in a ‘single agent’ immunotherapy that combines antibody specificity, blockade of purinergic signaling, and killing of targets mediated by NK cells. We also showed that CD73-targeted NK cells are potent in vivo and result in tumor arrest, while promoting NK cell infiltration into CD73+ tumors and enhancing intratumoral activation.

There is a great need to improve the outlook for people facing urinary bladder cancer, especially for patients with invasive urothelial carcinoma (InvUC) which is lethal in 50% of cases. Improved outcomes for patients with InvUC could come from advances on several fronts including emerging immunotherapies, targeted therapies, and new drug combinations; selection of patients most likely to respond to a given treatment based on molecular subtypes, immune signatures, and other characteristics; and prevention, early detection, and early intervention. Progress on all of these fronts will require clinically relevant animal models for translational research. The animal model(s) should possess key features that drive success or failure of cancer drugs in humans including tumor heterogeneity, genetic-epigenetic crosstalk, immune cell responsiveness, invasive and metastatic behavior, and molecular subtypes (e.g., luminal, basal). Experimental animal models, while essential in bladder cancer research, do not possess these collective features to accurately predict outcomes in humans. These key features, however, are present in naturally-occurring InvUC in pet dogs. Canine InvUC closely mimics muscle-invasive bladder cancer in humans in cellular and molecular features, molecular subtypes, immune response patterns, biological behavior (sites and frequency of metastasis), and response to therapy. Thus, dogs can offer a highly relevant animal model to complement other models in research for new therapies for bladder cancer. Clinical treatment trials in pet dogs with InvUC are considered a win-win-win scenario; the individual dog benefits from effective treatment, the results are expected to help other dogs, and the findings are expected to translate to better treatment outcomes in humans. In addition, the high breed-associated risk for InvUC in dogs (e.g., 20-fold increased risk in Scottish Terriers) offers an unparalleled opportunity to test new strategies in primary prevention, early detection, and early intervention. This review will provide an overview of canine InvUC, summarize the similarities (and differences) between canine and human InvUC, and provide evidence for the expanding value of this canine model in bladder cancer research.

Many microbial lineages have not yet been cultured, which hampers our understanding of their physiology. Here, Wurch et al. use single-cell genomics to infer cultivation conditions for the isolation of a tiny ectosymbiotic nanoarchaeon and its crenarchaeota host from a geothermal spring.