Therapeutic targeting of YOD1 disrupts the PAX-FOXO1-N-Myc feedback loop in rhabdomyosarcoma

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Abstract

Fusion-positive rhabdomyosarcoma (FP-RMS), driven by PAX-FOXO1, represents the subtype of RMS with the poorest prognosis. However, the oncogenic mechanisms and therapeutic strategies of PAX-FOXO1 remain incompletely understood. Here, we discovered that N-Myc, in addition to being a classic downstream target of PAX-FOXO1, can also activate its expression and form a transcriptional complex with PAX-FOXO1, thereby markedly amplifying oncogenic signaling. The reciprocal transcriptional activation of PAX3-FOXO1 and N-Myc is critical for FP-RMS malignancy. We further identified YOD1 as a deubiquitinating enzyme (DUB) that stabilizes both PAX-FOXO1 and N-Myc. Knocking down YOD1 or inhibiting it by G5 could suppress FP-RMS growth both in vitro and in vivo, through promoting the degradation of both PAX-FOXO1 and N-Myc. Collectively, our results identify that YOD1 promotes RMS progression by regulating the PAX3-FOXO1-N-Myc positive feedback loop, and highlight YOD1 inhibition as a promising therapeutic strategy that concurrently reduces the levels of both oncogenic proteins.

Authors

Wenwen Ying, Jiayi Yu, Xiaomin Wang, Jiayi Liu, Boyu Deng, Xuejing Shao, Jinhu Wang, Ting Tao, Ji Cao, Qiaojun He, Bo Yang, Yifan Chen, Meidan Ying

Differential effects of HDAC8 targeting on Foxp3⁺ T-regulatory cells and effector T-cells promote anti-tumor immunity

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Abstract

HDAC8, an evolutionarily distinct, X-linked, zinc-dependent class I histone/protein deacetylase, is implicated in developmental disorders, parasitic infections, myopathy, and cancers. Our study demonstrates the important role of HDAC8 in immune cells by conditional targeting of HDAC8 in murine T cells and application of selective HDAC8 inhibitors. Using flow cytometry, RNA-seq and ChIP-seq analyses, we demonstrate that knocking down or inhibiting HDAC8 impaired murine Treg suppressive function in vitro and in vivo, but promoted conventional host T cell responses, thereby limiting syngeneic tumor growth. Mechanistically, HDAC8 knockout downregulated Foxp3 expression, enhanced H3K27 acetylation levels and promoted IL-2, IL-6, Fas, and FasL expression in both Treg and conventional T-effector cells. Thus, our combined genetic and pharmacologic studies establish the central importance of HDAC8 in T cell responses and suggest that selective HDAC8 inhibitors represent a potential therapeutic approach in immuno-oncology.

Authors

Fanhua Kong, Yan Xiong, Liqing Wang, Rongxiang Han, Hossein Fazelinia, Jennifer Roof, Lynn A. Spruce, Aaron B. Beeler, Wayne W. Hancock

First-in-child phase I trial of p-STAT3 inhibitor WP1066 in pediatric brain tumor patients

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Abstract

BACKGROUND. WP1066 is an orally bioavailable, small molecule inhibitor of activated p-STAT3 that has demonstrated preclinical efficacy in pediatric brain tumor models. METHODS. In a first-in-child, single-center, single-arm 3+3 design Phase I clinical trial, ten patients were treated with WP1066 twice daily, Monday-Wednesday-Friday, for 14 days of each 28-day cycle to determine the maximum tolerated dose (MTD)/maximum feasible dose (MFD) of WP1066. Compassionate use treatment with WP1066 in three pediatric patients with H3.3 G34R/V-mutant high-grade glioma (HGG) is also described. RESULTS. There was no significant toxicity and the MFD was determined to be 8 mg/kg. Treatment-related adverse events were Grade 1-2 (diarrhea and nausea most common); there were no dose-limiting toxicities. Median progression-free and overall survival were 1.8 months and 4.9 months, respectively. One partial response was observed in a patient with pontine glioma. Among the H3.3 G34R/V-mutant HGG patients not on study, WP1066 was administered after upfront radiation to one patient for 17 months. At all dose levels tested, WP1066 suppressed p-STAT3 expression by peripheral blood mononuclear cells (PBMCs). Single cell RNA-seq analysis of PBMCs demonstrated increased CD4+ and CD8+ T cells, pro-inflammatory TNFA signaling, differentiation activity in myeloid cells, and downregulation of Tregs after WP1066 treatment, consistent with systemically inhibited STAT3 activity. CONCLUSIONS. WP1066 is safe, has minimal toxicity, and induces anti-tumor immune responses in pediatric brain tumor patients. Phase II investigation of WP1066 at the MFD in this patient population is warranted. TRIAL REGISTRATION. ClinicalTrials.gov NCT04334863. FUNDING. CURE Childhood Cancer (TJM) and Peach Bowl, Inc. (TJM)

Authors

Robert C. Castellino, Hope L. Mumme, Andrea T. Franson, Bing Yu, M. Hope Robinson, Kavita Dhodapkar, Dolly Aguilera, Matthew J. Schniederjan, Rohali Keesari, Zhulin He, Manoj Bhasin, Waldemar Priebe, Amy B. Heimberger, Tobey J. MacDonald

An oral lichen planus-like mouse model driven by IFN-γ signaling and cytotoxic CD8⁺ T cells

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Abstract

Oral lichen planus (OLP) is a recalcitrant inflammatory disease with potential for malignant transformation, involving a cytotoxic CD8+ T cells-mediated basal keratinocyte apoptosis. However, it lacks an appropriate mouse model for study. Here we developed an OLP-like mouse model using topical oxazolone to induce a delayed-type hypersensitivity-mediated oral lichenoid reaction. Histological and ultrastructural analysis confirmed hallmark pathological features of OLP, including band-like CD8+ T cell infiltration and basal cell damage, and the presence of Civatte bodies. Comparative transcriptomic analysis revealed significant similarity between RNA-seq profiles of the mouse model and human OLP lesions, highlighting shared upregulated genes and enriched pathways, particularly those related to IFN-γ signaling and cytotoxic T cell activity. Functional studies demonstrated that the OLP phenotype depended on IFN-γ, with local priming by IFN-γ intensifying the disease through upregulation of major histocompatibility complex class I. Additionally, the absence of Langerhans cells exacerbated disease severity in vivo. Therapeutic evaluation showed that the JAK inhibitors baricitinib and ruxolitinib effectively reduced disease burden and provided mechanistic insights. In conclusion, this OLP-like mouse model recapitulates key immunopathological and transcriptomic features of human OLP, offering a robust platform for dissecting disease mechanisms and evaluating novel therapeutic strategies.

Authors

Zhenlai Zhu, Tinglan Yang, Peng Peng, Kang Li, Wen Qin, Chen Zhang, Shuyan Wang, Yuanyuan Wang, Minghui Wei, Erle Dang, Meng Fu, Hao Guo, Wen Yin, Shuai Shao, Qing Liu

Rare epigenetic alterations are conserved across hematopoietic differentiation stages after mycobacterial infection

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Abstract

Infection leads to durable cell-autonomous changes in hematopoietic stem and progenitor cells (HSPCs), resulting in production of innate immune cells with heightened immunity. The mechanisms underlying this phenomenon, termed central trained immunity, remain poorly understood. We hypothesized that infection induces histone modifications leading to changes in chromatin accessibility that are conserved during differentiation from HSPCs to myeloid progenitors and monocytes. We conducted genome-wide surveillance of histone marks H3K27ac and H3K4me3 and chromatin accessibility in hematopoietic stem cells, multipotent progenitor 3, granulocyte-monocyte progenitors, monocytes and macrophages of naïve and Mycobacterium avium infected mice. Interferon signaling pathways and related transcription factor binding motifs including IRFs, NF-κB, and CEBP showed increased activating histone marks and chromatin accessibility across cell types. However, histone marks and increased chromatin accessibility were conserved at only a few loci, notably Irf1 and Gbp6. Knock out of IRF1 disrupted enhanced mitochondrial respiration and bacterial killing in human monocyte cell lines, while GBP6 KO monocyte cell lines showed dysregulated mitochondrial respiration. In summary, this study identifies IRF1 and GBP6 as two key loci at which infection-induced systemic inflammation leads to epigenetic changes that are conserved from HSPCs to downstream monocytes, providing a mechanistic avenue for central trained immunity.

Authors

Brandon T. Tran, Pamela N. Luna, Ruoqiong Cao, Duy T. Le, Apoorva Thatavarty, Laure Maneix, Bailee N. Kain, Scott Koh, Andre Catic, Katherine Y. King

Thrombomodulin protects against acute vascular and multiorgan injury in Sickle Cell disease

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Abstract

Vaso-occlusive episodes (VOEs) in the setting of hyperhemolysis can rapidly evolve into multiorgan failure in sickle cell disease (SCD). Although the mechanisms for rapid progression to multiorgan failure are unclear, a systemic vasculopathy with thrombotic microangiopathy-type features has been described. Reduced thrombomodulin (TM) function is implicated in some thrombotic microangiopathy syndromes. We observed a greater decline in platelet count and hemoglobin concentration and increase in vascular injury biomarkers within 24-hours of admission for a VOE in 12 SCD patients with versus 12 without multiorgan failure. We observed decreased TM expression on the lung and kidney vasculature of three additional SCD patients with multiorgan failure and an autopsy performed compared to a non-SCD control. Transgenic SCD mice challenged with cell-free hemoglobin had reduced TM function, increased vascular injury biomarkers, and reduced renal cortical blood flow. Infusion of recombinant TM 2- or 24-hours after the challenge restored cortical blood flow, mitigated increases in vascular injury, complement activation, and tubular injury biomarkers, and protected against acute kidney and lung injury. We demonstrated that impaired TM function may be involved in the systemic vasculopathy of SCD-related multiorgan failure and infusion of recombinant TM may restore vascular function and protect against acute organ damage.

Authors

Guohui Ren, Dustin R. Fraidenburg, Suman Setty, Jiwang Chen, Janae Gonzales, Maria Armila Ruiz, Zalaya Ivy, Najmeh Eskandari, Richard D. Minshall, James P. Lash, Victor R. Gordeuk, Santosh L. Saraf

Nectin-4 reduces T cell effector function and is a therapeutic target in pancreatic cancer

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Abstract

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis and current therapies show limited efficacy. Ligands and receptors of the TIGIT axis were analyzed using multicolor flow cytometry of tumor and blood samples, immunohistochemistry from primary tumors, and single-cell RNA sequencing from primary tumors and liver metastasis from patients with various stages of PDAC. The effect of soluble and plate-bound Nectin-4 on T cell function was tested in vitro. Further, patient-derived PDAC organoids were treated with the standard of care therapies FOLFIRINOX, gemcitabine plus paclitaxel, or the antibody-drug conjugate enfortumab vedotin. TIGIT expression was increased on tumor-infiltrating conventional and regulatory T cells compared with T cells from matched blood. Nectin-4, but not CD155 expression was associated with poor outcome. Nectin-4 was exclusively expressed by tumor cells and correlated with low immune infiltration. Notably, Nectin-4 inhibited T cell effector cytokine production in vitro. Targeting Nectin-4 with the antibody-drug conjugate enfortumab vedotin inhibited tumor growth in multiple patient-derived PDAC organoids. Collectively, our data underscores Nectin-4 as a novel therapeutic target and provides the rationale to test this agent in PDAC patients.

Authors

Max Heiduk, Carolin Beer, Sarah Cronjaeger, Emily A. Kawaler, Ulrich Sommer, Franziska Baenke, David Digomann, Loreen Natusch Bufe, Charlotte Reiche, Jessica Glück, Franziska Hoffmann, Sungsik Kim, Daniel E. Stange, Diane M. Simeone, Jürgen Weitz, Lena Seifert, Adrian M. Seifert

CoREST complex inhibition alters RNA splicing to promote neoantigen expression and enhance tumor immunity

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Abstract

Epigenetic macromolecular enzyme complexes tightly regulate gene expression at the chromatin level and have recently been found to colocalize with RNA splicing machinery during active transcription; however, the precise functional consequences of these interactions are uncertain. Here, we identify unique interactions of the CoREST repressor complex (LSD1-HDAC1-CoREST) with components of the RNA splicing machinery and their functional consequences in tumorigenesis. Using mass spectrometry, in vivo binding assays, and cryo-EM we find that CoREST complex-splicing factor interactions are direct and perturbed by the CoREST complex selective inhibitor, corin, leading to extensive changes in RNA splicing in melanoma and other malignancies. Moreover, these corin-induced splicing changes are shown to promote global effects on oncogenic and survival-associated splice variants leading to a tumor-suppressive phenotype. Using machine learning models, MHC IP-MS, and ELISpot assays we identify thousands of neopeptides derived from unannotated splice sites which generate corin-induced splice-neoantigens that are demonstrated to be immunogenic in vitro. Corin is further shown to reactivate the response to immune checkpoint blockade, effectively sensitizing tumors to anti-PD1 immunotherapy. These data position CoREST complex inhibition as a unique therapeutic opportunity which perturbs oncogenic splicing programs while also creating tumor-associated neoantigens that enhance the immunogenicity of current therapeutics.

Authors

Robert J. Fisher, Kihyun Park, Kwangwoon Lee, Katarina Pinjusic, Allison Vanasse, Christina S. Ennis, Parisa Farokh, Scott B. Ficarro, Jarrod A. Marto, Hanjie Jiang, Eunju Nam, Stephanie Stransky, Joseph Duke-Cohan, Melis A. Akinci, Anupa Geethadevi, Eric Raabe, Ana Fiszbein, Shadmehr Demehri, Simone Sidoli, Chad W. Hicks, Derin B. Keskin, Catherine J. Wu, Philip A. Cole, Rhoda M. Alani

Enhanced lipid metabolism serves as a metabolic vulnerability to polyunsaturated fatty acids in glioblastoma

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Abstract

Enhanced lipid metabolism, which involves the active import, storage, and utilization of fatty acids from the tumor microenvironment, plays a contributory role in malignant glioma transformation; thereby, serving as an important gain of function. In this work, through studies initially designed to understand and reconcile possible mechanisms underlying the anti-tumor activity of a high-fat ketogenic diet, we discovered that this phenotype of enhanced lipid metabolism observed in glioblastoma may also serve as a metabolic vulnerability to diet modification. Specifically, exogenous polyunsaturated fatty acids (PUFA) demonstrate the unique ability of short-circuiting lipid homeostasis in glioblastoma cells. This leads to lipolysis-mediated lipid droplet breakdown, an accumulation of intracellular free fatty acids, and lipid peroxidation-mediated cytotoxicity, which was potentiated when combined with radiation therapy. Leveraging this data, we formulated a PUFA-rich modified diet that does not require carbohydrate restriction, which would likely improve long-term adherence when compared to a ketogenic diet. The modified PUFA-rich diet demonstrated both anti-tumor activity and potent synergy when combined with radiation therapy in mouse glioblastoma models. Collectively, this work offers both a mechanistic understanding and a potentially translatable approach of targeting this metabolic phenotype in glioblastoma through diet modification and/or nutritional supplementation that may be readily integrated into clinical practice.

Authors

Shiva Kant, Yi Zhao, Pravin Kesarwani, Kumari Alka, Jacob F. Oyeniyi, Ghulam Mohammad, Nadia Ashrafi, Stewart F. Graham, C. Ryan Miller, Prakash Chinnaiyan

Urobiota analysis and genome-wide association study in pediatric recurrent urinary tract infections and vesicoureteral reflux

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Abstract

Urinary tract infections (UTIs) are the most common severe bacterial infections in young children, often associated with vesicoureteral reflux (VUR). To explore host genetic-microbiota interactions and their clinical implications, we analyzed the urinary microbiota (urobiota) and conducted genome-wide association studies (GWAS) for bacterial abundance traits in pediatric UTI and VUR patients from the RIVUR and CUTIE cohorts. We identified four urobiota community types based on relative abundance, characterized by the genera Enterococcus, Prevotella, Pseudomonas, and Escherichia/Shigella, and their associations with VUR, age, and toilet training. Children with VUR exhibited decreased microbial diversity and increased abundance of genera that included opportunistic pathogens, suggesting a disrupted urobiota. We detected genome-wide significant genetic associations with urinary bacterial relative abundances, in or near candidate genes including CXCL12, ABCC1, and ROBO1, which are implicated in urinary tract development and response to infection. We showed that Cxcl12 is induced 12 hours after uropathogenic bacterial infection in mouse bladder. The association with CXCL12 suggests a genetic link between UTI, VUR and cardiovascular phenotypes later in life. These findings provide the first characterization of host genetic influences on the pediatric urobiota in UTI and VUR, offering insights into the interplay between disease, host genetics and the urobiota composition.

Authors

Miguel Verbitsky, Pavan Khosla, Daniel Bivona, Atlas Khan, Yask Gupta, Heekuk Park, Tian H. Shen, Aryan Ghotra, Katherine Xu, Iman A. Ghavami, Priya Krithivasan, Jeremiah Martino, Tanya Sezin, Tze Y. Lim, Victoria Kolupaeva, Nita A. Limdi, Yuan Luo, Hakon Hakonarson, Simone Sanna-Cherchi, Krzysztof Kiryluk, Cathy L. Mendelsohn, Anne-Catrin Uhlemann, Jonathan Barasch, Ali G. Gharavi