Preclinical Research Articles

Abstract B026: Identification of small RNAs that function as multi-target therapeutics and as tools for novel target discovery in prostate cancer

Abstract Introduction: A major contributor to prostate cancer (PCa) mortality is the development of resistance to single agents targeting androgen receptor (AR) signaling. We have developed an unbiased shRNA-based negative selection screen to identify small RNAs (shRNAs and siRNAs) that inhibit PCa cell growth and survival (toxic sRNAs). Computational analyses of the most toxic sRNAs identified revealed that each can simultaneously target multiple known AR-coregulatory mRNAs. AR-coregulators modulate AR transcriptional response and are essential to AR signaling. We have proposed that the identified toxic sRNAs inhibit androgen signaling through seed-mediated targeting of androgen regulatory gene networks, resulting in inhibition of cell growth and viability. The goal of the current studies was to begin to analyze the broad therapeutic potential of these toxic sRNAs for PCa. We focused on analyzing i) their potential to thwart cell growth and/or viability when delivered to preclinical models of PCa, and ii) the targetome of the toxic sRNAs as a mean to uncover novel AR-coregulators that represent a targetable axis within androgen signaling. Methods: i) cationic lipid nanoparticles (LNPs), that efficiently deliver the toxic siRNAs to PCa cell lines, were used to deliver toxic siRNAs to organoids grown from PCa patient derived xenografts (PDX) models. 3D image analysis using the Ominer® software package was used to determine organoid count and size, the numbers of nuclei per organoid, and the fraction of dead cells to discriminate between cytotoxic and cytostatic effects. ii) to identify novel AR-coregulators, toxic siRNA direct targets were identified by sequencing of transcripts captured by biotinylated toxic siRNAs. Directly bound targets, were subjected to pathway enrichment and network analysis followed by identification of hub genes (Cytoscape). The effect of individually silencing potential AR-coregulators on the expression of androgen responsive genes was analyzed using Nanostring. Results and Conclusions: i) The effect of the toxic siRNAs on growth and viability of the PCa PDX-derived organoids will be presented and discussed as compelling preliminary data for therapeutic testing in more complex pre-clinical models (PCa PDXs). ii) Based on bioinformatic and downstream analysis of the direct targets of two toxic siRNAs, we selected 12 potentially novel AR- coregulators, with varied known functions including chromatin modifiers, transcriptional regulators, etc. Silencing of several of these potential AR-coregulators significantly affect expression of numerous AR responsive genes. Future experiments will focus on analyzing their interaction and functional association with the AR (i.e. effect of AR binding to chromatin). In conclusion, we have developed an assay that allowed the identification of toxic sRNAs for PCa cells and demonstrated their potential as therapeutics and as a tool for discovery of potential novel targets for PCa. Citation Format: Joshua M Corbin, Hamilton Young, Stefan Wilhelm, Adam S Asch, Chao Xu, Maria J Ruiz Echevarria. Identification of small RNAs that function as multi-target therapeutics and as tools for novel target discovery in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B026.

CD40 agonist on patient-derived xenograft mice for the treatment of B-cell acute lymphoblastic leukemia

Abstract Purpose: Cluster of differentiation 40 (CD40) is expressed on B-cell acute lymphoblastic leukemia (B-ALL) cases. However, the effect of CD40 activation on B-ALL cells has never been tested in vivo. Experimental Design: The aim of our preclinical study was to investigate the therapeutic potential of a CD40 agonist in the treatment of B-ALL using patient-derived xenograft (PDX) mouse models. Results: Intravenous administration of the CD40 agonist significantly impeded B-ALL cell proliferation and growth in vivo, accompanied by rapid activation of the extracellular signal-regulated kinase (ERK) pathway, leading to the induction of apoptosis and disruption of cell cycle progression. Co-treatment with a specific inhibitor of ERK further demonstrated that CD40 stimulation induced the pro-apoptosis of B-ALL cells in an ERK-dependent manner. Proteomic analysis revealed alterations in key signaling pathways associated with B-ALL expansion and maintenance. Moreover, the CD40 agonist markedly reduced the frequency of leukemia-initiating cells and leukemia development in PDX mice. Our study showed that the CD40 agonist can be associated with chemotherapeutic agents such as vincristine and dexamethasone, and this combination showed improved effectiveness. Additionally, the CD40 agonist was more effective on pre-B-ALL (EGIL B-III) that expressed CD40, than on common B-ALL (EGIL B-II) that lacked CD40 expression. Conclusion: These findings suggest that CD40 agonists are promising immunotherapeutic candidates for pediatric B-ALL, warranting further clinical investigations to improve patient outcomes in CD40-expressing B-ALL.

Abstract PR004: Second generation RNA Polymerase I inhibitor PMR-116 targets ribsomal RNA synthesis to potently treat a broad spectrum of malignancies

Abstract Purpose and rational for the study: The purpose of the study was to develop novel 2nd generation ribosomal RNA synthesis inhibitors for cancer treatment. Ribosome biogenesis (RiBi) is essential for cell growth and proliferation. Transcription of ribosomal RNA genes by Pol I is a critical step in RiBi and is tightly regulated by tumor suppressors and oncoproteins. Indeed, the ability of the potent oncoprotein, MYC, to drive RiBi is necessary for its oncogenic activity (Bywater et al., Cancer Cell, 2011). These data suggested that targeting RiBi, might be a promising strategy to treat malignancies, especially those driven by MYC. To test this, we developed the 1st small molecule inhibitor of Pol I transcription, CX-5461 (Drgyin et al., Cancer Research 2011). While CX-5461 has shown promising activity in pre-clinical studies (Bywater et al., Cancer Cell, 2011; Rebello et al., Clin Cancer Res., 2016; Hein et al., Blood, 2017) and in early Phase trials (Khot et al., Cancer Discovery, 2019; Hilton et al., Nat Commun. 2022) its mechanism of action remains controversial with evidence of off-target activity inducing DNA damage through inhibition of TOPO2a (Cameron et al., Biomedicines, 2024; Koh et al., Nature Genetics, 2024). Thus, to determine if selective inhibition of Pol I transcription in the absence of DNA damage can be used to therapeutical treat cancer in vivo, we developed a series of more selective 2nd generation Pol I inhibitors with our lead compound being PMR-116. Methods and summary of data: We demonstrate that PMR-116 is selective for inhibition of Pol I over Poll II transcription and functions by stalling the Pol I complex at the rDNA promoter. PMR-116, exhibits improved drug-like properties and pharmacokinetics compared to 1st generation CX-5461. As a single agent PMR-116 efficiently treats a range of pre-clinical models of hematologic and solid tumors including lymphoma, AML, hepatocellular carcinoma and prostate cancer. MYC expression positively correlates with sensitivity to PMR-116 in human cancer cell lines and high MYC tumors in vivo are exceptionally sensitive to PMR-116. In contrast to CX-5461, the therapeutic response to PMR-116 occurs in the absence of DNA damage suggesting that DNA damage is not an obligatory response to Pol I transcription inhibition but rather an off-target effect of the 1st generation drug. Small molecule drug combination screens in AML cells demonstrate that PMR-116 works synergistically to reduce cell viability with a range of drugs including carfilzomib and quisinostat used for treatment of hematologic malignancies. Conclusions: Our data establishes inhibition of RNA polymerase I as a bone fide therapeutic target for cancer therapy and demonstrated that PMR-116 is a promising new broad- spectrum anti-cancer drug particularly for those tumors driven by MYC, which are highly refractory to standard therapies. Based on these data we have launched a Phase I clinical trial of PMR-116 in patients with advanced solid tumors (CTRN12620001146987). Citation Format: Rita Ferreira, Katherine M. Hannan, Konstantin Panov, Thejanni Udumanne, Amee J George, Zaka Yuen, Perlita Poh, Eric Kusnadi, Alisee Huglo, Mitchell Lawrence, Mustapha Haddach, Denis Drygin, Luc Furic, Ross D Hannan, Nadine Hein. Second generation RNA Polymerase I inhibitor PMR-116 targets ribsomal RNA synthesis to potently treat a broad spectrum of malignancies [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr PR004.

Abstract I008: Antisense Oligonucleotides as Therapeutics for Difficult-to-Drug Targets in Oncology

Abstract Many potential oncology targets are difficult to modulate by conventional classes of agents. Interfering with these targets at the level of RNA is a novel approach for difficult-to target proteins, as well as for non-coding RNAs, emerging targets that are part of the “dark matter’ of the genome. Antisense oligonucleotide (ASO) therapeutics are short sequences of 12- 30 DNA/RNA residues, designed to bind the cognate transcribed RNA. The ASO is chemically modified to increase stability, improve binding affinity, optimize pharmacokinetic properties and reduce immunostimulatory potential. The hybridization of ASO to cognate RNA results in cleavage of bound RNA by RNAse H1 and subsequent degradation. Several ASOs are marketed for treatment of rare diseases, but to date none have been approved for oncology indications. The long-noncoding RNA (lncRNA) MALAT1 is an example of an oncology target uniquely suited for an ASO therapeutic. This lncRNA is not translated and resides mostly in the nucleus, limiting access for most therapeutic approaches. MALAT1 is highly upregulated in various cancers, appears to play a role in epithelial to mesenchymal transition, and is associated with disease progression in patients. Inhibition of MALAT1 in preclinical breast cancer models, either genetic or pharmacologic using a murine ASO, leads to changes in tumor architecture and reduction in metastasis. FLM-7523 is a 16-mer ASO designed to bind to human MALAT1. It is a generation 2.5 design based on the constrained ethyl (cEt) modification to the 3 residues on the 3’ and 5’ ends of the ASO, and administered in a saline solution without any delivery vehicle. Preclinical studies have shown that FLM-7523 effectively inhibits MALAT1 expression in in vitro and in vivo models, and GLP toxicology studies have been completed to support a First-in-Human clinical trial. The signal transduction molecule STAT3 is another oncology target of interest that has, to date, been difficult to modulate. Danvatirsen is a 16-mer generation 2.5 ASO targeting human STAT3 mRNA and is currently in clinical trials. Preclinical in vivo studies in murine tumor models have demonstrated a reduction in STAT3 mRNA and protein following treatment with a STAT3 targeting ASO, along with tumor growth inhibition. Combination treatments with immune checkpoint inhibitors, including anti-PD-1 and anti-PD-L1, demonstrate improved tumor growth inhibition relative to monotherapy. In clinical trials, tumor uptake of danvatirsen is observed in post-treatment biopsies, with corresponding reduction in STAT3 protein. Danvatirsen is generally well tolerated with the most common adverse events being increased liver function tests and decreased myeloid hematologic parameters, both of which are easily monitored, manageable, and reversible. Clinical responses have been observed with the most robust being in combination with a PD-L1 inhibitor in recurrent/metastatic head and neck squamous cell carcinoma. In conclusion, ASOs are novel therapies that can inhibit oncology targets not tractable by traditional approaches. Citation Format: Andrew Denker. Antisense Oligonucleotides as Therapeutics for Difficult-to-Drug Targets in Oncology [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr I008.

Structural changes in the nociceptive system induced by long-term conventional spinal cord stimulation in experimental painful diabetic polyneuropathy

BackgroundClinical studies suggest that long-term conventional spinal cord stimulation (LT-SCS) for painful diabetic peripheral neuropathy (PDPN) is initially effective but may decline in efficacy over time. Preclinical studies indicate that LT-SCS alleviates mechanical hypersensitivity and enhances hind paw blood flow in PDPN rats, suggesting nociceptive system plasticity. This study hypothesized that LT-SCS induces peripheral hind paw small-fiber sprouting and reduces central protein expression of glial and P2X4 brain-derived neurotrophic factor (BDNF) pathway markers.MethodsDiabetes was induced via Streptozotocin injection in 32 rats, with 16 developing PDPN and receiving a quadrupolar lead implant. LT-SCS was applied for 4 weeks, 12 hours per day. Pain behavior was assessed using the Von Frey test for mechanical hypersensitivity and the mechanical conflict avoidance system for motivational aspects of pain. Fiber sprouting was assessed via immunohistochemical analysis of nerve fibers in the hind paw skin. Protein expression in the spinal cord was assessed using western blotting.ResultsLT-SCS increased the baseline threshold of mechanical hypersensitivity in PDPN animals, consistent with previous findings, but showed no effects on motivational aspects of pain. Hind paw tissue analysis revealed significantly increased intraepidermal nerve fiber density of PGP9.5 fibers in LT-SCS animals compared with Sham-SCS animals. Protein analysis showed significantly decreased pro-BDNF expression in LT-SCS animals compared with Sham-SCS animals.ConclusionLT-SCS induces structural changes in both peripheral and central components of the nociceptive system in PDPN animals. These changes may contribute to observed behavioral modifications, elucidating mechanisms underlying LT-SCS efficacy in PDPN management.

Pathophysiology and preclinical relevance of experimental graft-versus-host disease in humanized mice

AbstractGraft-versus-host disease (GVHD) is a life-threatening complication of allogeneic hematopoietic cell transplantations (allo-HCT) used for the treatment of hematological malignancies and other blood-related disorders. Until recently, the discovery of actionable molecular targets to treat GVHD and their preclinical testing was almost exclusively based on modeling allo-HCT in mice by transplanting bone marrow and splenocytes from donor mice into MHC-mismatched recipient animals. However, due to fundamental differences between human and mouse immunology, the translation of these molecular targets into the clinic can be limited. Therefore, humanized mouse models of GVHD were developed to circumvent this limitation. In these models, following the transplantation of human peripheral blood mononuclear cells (PBMCs) into immunodeficient mice, T cells recognize and attack mouse organs, inducing GVHD. Thereby, humanized mice provide a platform for the evaluation of the effects of candidate therapies on GVHD mediated by human immune cells in vivo. Understanding the pathophysiology of this xenogeneic GVHD is therefore crucial for the design and interpretation of experiments performed with this model. In this article, we comprehensively review the cellular and molecular mechanisms governing GVHD in the most commonly used model of xenogeneic GVHD: PBMC-engrafted NOD/LtSz-PrkdcscidIL2rγtm1Wjl (NSG) mice. By re-analyzing public sequencing data, we also show that the clonal expansion and the transcriptional program of T cells in humanized mice closely reflect those in humans. Finally, we highlight the strengths and limitations of this model, as well as arguments in favor of its biological relevance for studying T-cell reactions against healthy tissues or cancer cells.

Abstract B008: Exploring the Dual Roles of RNAs as Oncogenic Drivers and Therapeutic Targets in Cancer: A Cross-Sectional Study

Abstract RNAs have emerged as pivotal molecules in the complex landscape of cancer biology, acting as both drivers of oncogenesis and therapeutic targets. This cross-sectional experimental design research explores the dual roles of RNAs, particularly focusing on the interplay between non-coding RNAs (ncRNAs), messenger RNAs (mRNAs), and their regulatory networks in various cancer types. The study systematically analyzes RNA expression profiles across a broad spectrum of cancer samples, employing high-throughput sequencing technologies combined with bioinformatics tools to identify key RNA species that contribute to tumorigenesis. The experimental design includes the collection of tumor and adjacent normal tissues from patients diagnosed with breast, lung, and colorectal cancers. RNA extraction and sequencing are performed to generate comprehensive expression profiles. Differential expression analysis is conducted to pinpoint RNAs that are significantly dysregulated in cancerous tissues compared to their normal counterparts. Subsequently, functional assays, including RNA interference (RNAi) and CRISPR/Cas9-mediated gene editing, are utilized to validate the oncogenic or tumor-suppressive roles of these RNAs in cancer cell lines. To further elucidate the mechanistic pathways, the study integrates data from RNA-binding protein immunoprecipitation (RIP) and crosslinking immunoprecipitation (CLIP) assays, providing insights into the RNA-protein interactions that influence cancer progression. Additionally, the research evaluates the therapeutic potential of targeting these RNAs, using small interfering RNAs (siRNAs) and antisense oligonucleotides (ASOs) in preclinical cancer models. Preliminary findings highlight the significance of long non-coding RNAs (lncRNAs) and micro RNAs (miRNAs) in modulating key signaling pathways involved in cell proliferation, apoptosis, and metastasis. This study underscores the importance of RNA-based diagnostics and therapeutics, paving the way for innovative treatment strategies in cancer management. Citation Format: Fathelrahman M Gameel. Exploring the Dual Roles of RNAs as Oncogenic Drivers and Therapeutic Targets in Cancer: A Cross-Sectional Study [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B008.

Abstract B013: Investigation of a novel lncRNA, TROLL-9, in breast cancer formation and progression

Abstract Breast cancer (BC) is the most diagnosed cancer and the second leading cancer-related cause of mortality among women in the US. Despite advances in treatment, BC remains a challenge due to its metastatic potential. The most aggressive BC subtype, triple negative (TN), often harbors mutations in the tumor suppressor gene, p53. One of the effects of mutant p53 involves the inhibition of the tumor and metastatic suppressive functions of TAp63, a p53 family member. Loss of TAp63 leads to the formation of metastatic mammary adenocarcinomas in mice, in part through the regulation of a group of nine long non-coding RNAs (lncRNAs), that we identified and named TAp63 regulated oncogenic lncRNAs (TROLLs). We previously characterized two TROLLs, TROLL-2 and TROLL-3, as markers of tumor progression in multiple cancer types via the activation of the AKT pathway. Currently we are investigating the role of another member, TROLL-9. To investigate the biological role of TROLL-9, we performed in vivo experiments using preclinical mouse models. Specifically, MCF-DCIS, MCF-CA1D, and MDA-MB-231 triple negative breast cancer (TNBC) cells constitutively expressing TROLL-9 were generated and utilized for two mouse models: orthotopic xenografts for primary tumor growth and spontaneous metastasis. The former was designed to identify the potential role of TROLL-9 in tumor formation, whereas the latter involved the resection of a primary tumor and monitoring for metastases via bioluminescence imaging. Notably, TROLL-9 overexpression led to increased formation of lung and livers metastases in all these models, suggesting a role for TROLL-9 in the metastatic cascade. In order to elucidate the mechanism of function of TROLL-9, two complementary approaches were used. Firstly, pulldown using MS2-TRAP followed by mass spectrometry (MS) was performed and led to the identification of putative protein interactors. In parallel, Laser Capture Microscopy (LCM) of the lung metastases followed by RNA-sequencing was used to assess for any gene expression changes due to TROLL-9 overexpression. Integration of the two datasets is currently being performed to unveil the network of genes and proteins perturbed by TROLL-9. Furthermore, to understand the relevance in BC patients, TROLL-9 expression levels were analyzed in the TCGA BC dataset. Interestingly, differences in TROLL-9 levels across BC subtypes were observed, thus supporting its putative role in TNBC. Overall, our data suggest a role for TROLL-9 in BC progression. Our findings provide evidence for further characterizing its mechanism of function as a potential therapeutic target for the treatment of invasive BC, particularly TNBC harboring TP53 mutations. Citation Format: Avani A Deshpande, Marco Napoli, John H Lockhart, Christina L Carr, Jaden R Baldwin, Elsa R Flores. Investigation of a novel lncRNA, TROLL-9, in breast cancer formation and progression [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B013.

Abstract B011: RNA-based therapeutics to target the p53 pathway in cancer

Abstract Mutations in the TP53 gene and activation of the PI3K/AKT pathway are the two most frequent genetic alterations across multiple human tumors. We discovered a crucial mechanism of crosstalk between these two events consisting of two oncogenic lncRNAs, TROLL-2 and TROLL-3, whose expression is induced by mutant p53. We demonstrated that these lncRNAs drive tumor and metastasis formation in several orthotopic models, including breast cancer, lung adenocarcinoma, and melanoma. The investigation of these mouse models and a pan-cancer analysis of human cancers showed that these lncRNAs are markers of tumor progression and lead to the phosphorylation and subsequent activation of AKT by promoting the cytoplasmic localization of the scaffold protein WDR26. Notably, we have now proven that TROLL-2 and TROLL-3 are also involved in the response to cancer therapies. Indeed, the downregulation of these lncRNAs via siRNAs bypasses the AKT-driven resistance to chemotherapeutics (e.g. Adriamycin, docetaxel, and paclitaxel) in triple-negative breast cancer cells, to inhibitors of mutant KRAS (e.g. sotorasib) and EGFR (e.g. erlotinib) in lung adenocarcinoma cells, and to inhibitors of PARP (e.g. niraparib) in ovarian cancer cells. To target these lncRNAs in a clinically relevant manner, we designed LNA GapmeRs, also known as antisense oligonucleotides (ASOs), to downregulate these two lncRNAs directly. The efficacy of the ASOs was confirmed in ex vivo specimens of tumors and metastases of lung adenocarcinoma and ovarian cancer patients. These clinical specimens were also analyzed via single-cell RNA sequencing and phospho-proteomics to unveil the common vs. unique downstream effects of targeting each lncRNA. When administered together, the ASOs targeting TROLL-2 and TROLL-3 effectively decreased the levels of these lncRNAs, which in turn caused WDR26 sequestration in the nucleus, thus preventing the activation of the AKT pathway and the proliferation of these cancer specimens. Furthermore, we confirmed that the antitumor activities of these ASOs were also maintained in vivo, as proven by the decreased tumor proliferation observed in breast cancer xenograft models. Finally, to improve the delivery of these ASOs in vivo, the usage of non-viral systems, including lipid-based and metallic-based nanoparticles, is being tested in both ex vivo and in vivo preclinical models. Overall, our results unveiled TROLL-2 and TROLL-3 as suitable targets to counteract tumor growth and promote the efficacy of chemotherapeutics and targeted therapies in ex vivo and in vivo cancer models. Citation Format: Marco Napoli, Ashley K Lu, Avani A Deshpande, Jaden R Baldwin, Christina L Carr, Michael R Dunne, Omar K Farha, Michelle H Teplensky, Elsa R Flores. RNA-based therapeutics to target the p53 pathway in cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B011.

Abstract A025: RNA-Based Therapeutics to target the p53 pathway in high-grade serous ovarian cancer: TAp63-Regulated Oncogenic LncRNAs (TROLLs) as novel therapeutic targets in cancer

Abstract High-grade serous ovarian cancer (HGSOC) is the most common and malignant type of ovarian cancer with a high frequency of p53 mutation and hyperactivation of the AKT pathway. Even though roughly 85% of patients achieve remission after initial surgical debulking and chemotherapy, four out of five HGSOC patients will experience disease recurrence, resulting in a 5-year survival rate of 43%. We have identified and characterized two TAp63 Regulated Oncogenic Long non-coding RNAs (TROLL-2 and TROLL-3) whose transcription is induced by mutant p53 and high expression level correlate with a more aggressive ovarian cancer grade and phosphorylation of AKT. In-depth analysis demonstrated that TROLL-2 and TROLL-3 directly activate AKT in tumor cells, thus linking p53 alterations to AKT activity. The use of AKT inhibitors in the clinic is limited because of frequent adverse effects, likely due to the constitutive expression of AKT in all cells. TROLL-2 and TROLL-3 expression is limited to tumor cells and provides a more specific target for decreasing AKT activation, potentially minimizing off-target effects. We have optimized reduction of TROLL-2 and TROLL-3 expression using antisense oligonucleotides (ASOs) in an ex vivo perfusion platform for 3D culture of microtumors from primary patient derived tumors. Samples show maximum reduction of proliferation (EdU) and phosphorylation of AKT when TROLL-2 and TROLL-3 ASOs are used concurrently. We also performed phospho-proteomics and single cell RNAseq to compare individual ASO-mediated knockdown of each lncRNA to combined targeted ASO treatments. We have developed primary patient derived xenograft models using ovarian tumors orthotopically implanted into NSG mice generating a relevant preclinical model to determine the efficacy of ASO treatment. To reduce the rapid degradation and clearance of ASOs in circulation, ASOs were encapsulated into lipid nanoparticles and metal-organic frameworks (MOFs) and we are currently testing these in vivo. Since AKT activation is associated with increased staging and resistance to platinum and targeted therapies in ovarian cancer patients, our data provides preclinical rationale for the implementation of ASOs and the use of new delivery methods as a relevant translational therapy and novel method to exploit the specificity of TROLL-2 and TROLL-3 expression in HGSOC. Citation Format: Ashley Lui, Marco Napoli, Jaden R Baldwin, Christina L Carr, Angie Rivera, Duy Nguyen, W. Gregory Sawyer, Michael R Dunne, Erin George, Omar K Farha, Michelle Teplensky, Elsa R Flores. RNA-Based Therapeutics to target the p53 pathway in high-grade serous ovarian cancer: TAp63-Regulated Oncogenic LncRNAs (TROLLs) as novel therapeutic targets in cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A025.

Evaluation of a six-minute walk test in the DE50-MD canine model of Duchenne muscular dystrophy and its effect on blood-borne biomarkers

Background Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease caused by mutations in the dystrophin gene resulting in cycles of muscle degeneration, inflammation and regeneration. The 6-minute walk test (6MWT) is a key functional outcome measure for DMD patient clinical trials and has been adapted for use in animal models of the disease. The DE50-MD dog model of DMD closely reflects the DMD patient phenotype prior to loss of ambulation. For pre-clinical trials using this model, functional outcome measures must be established. Methods This longitudinal study compared distance walked in a 6MWT by DE50-MD and WT control dogs and assessed the utility of the 6MWT as a functional biomarker. Dogs underwent two 6MWTs conducted approximately 48-hours apart, at 3, 6, 9, 12, 15 and 18 months of age. In addition, we evaluated the stability of selected blood-borne biomarkers in 12-month old DE50-MD and WT dogs 0, 3, 6, 24 and 48 hours following a 6MWT. Results DE50-MD dogs exhibited significantly shorter 6-minute walk distance (6MWD) than WT dogs at all timepoints (P<0.05), with no difference in 6MWD between the first and second 6MWT. C-C motif chemokine ligand 2 (CCL2), myomesin-3 (MYOM3) and myostatin (MSTN) were biomarkers of the DE50-MD phenotype that remained unchanged in DE50-MD dogs following the 6MWT, while creatine kinase (CK) activity significantly increased 3-hours following the test in DE50-MD dogs but remained unchanged in WT dogs. Conclusions The 6MWT effectively discriminates DE50-MD from WT dogs aged 3-18 months and a single 6MWT is sufficient for future studies. Serum MYOM3, CCL2 and MSTN are good biomarkers of the DE50-MD phenotype that are unaffected by this relatively low level exertion.

CardiLect: A combined cross‐species lectin histochemistry protocol for the automated analysis of cardiac remodelling

AbstractBackgroundCardiac remodelling, a crucial aspect of heart failure, is commonly investigated in preclinical models by quantifying cardiomyocyte cross‐sectional area (CSA) and microvascular density (MVD) via histological methods, such as immunohistochemistry. To achieve this, optimized protocols are needed, and the species specificity is dependent on the antibody used. Lectin histochemistry offers several advantages compared to antibody‐based immunohistochemistry, including as cost‐effectiveness and cross‐species applicability. Direct comparisons between the two methods are lacking from the literature.Methods and resultsIn this study, we compared antibody‐ and lectin‐based methods for the histological assessment of cardiomyocyte CSA (with the use of anti‐laminin and wheat germ agglutinin [WGA]) and microvascular density (utilizing anti‐CD31 and isolectin B4 [ILB4]) using different embedding and antigen/carbohydrate retrieval techniques. Here, we describe a detailed, easy‐to‐use combined lectin histochemistry protocol (WGA and ILB4, ‘CardiLect’ protocol) for the histological assessment of cardiac remodelling. The lectin‐based approach has been evaluated on a cross‐species basis, and its efficacy has been demonstrated in zebrafish, rodents, large animals and human samples. We provide an ImageJ script (‘CardiLect Analyser’) for automated image analysis, validated in a preclinical heart failure model by correlating histological parameters with echocardiographic findings. CSA showed a significant positive correlation with left ventricular (LV) mass (P = 0.0098, rS = 0.7545) and significant negative correlation with markers of systolic function, such as ejection fraction (EF) (P = 0.0402, rS = −0.6364). Microvascular density showed significant negative correlation with LV mass (P = 0.0055, rS = −0.7622) and significant positive correlation with EF (P = 0.0106, rS = 0.7203).ConclusionsThe described combined lectin histochemistry protocol with the provided ImageJ script is an easy‐to‐use, cost‐effective, cross‐species approach for the histological assessment of cardiac remodelling.

Overcoming immunosuppression in cancer: how ketogenic diets boost immune checkpoint blockade

AbstractImmune checkpoint blockade (ICB) is now part of the standard of care in the treatment of many forms of cancer, yet it lacks efficacy in some patients, necessitating adjunct therapies to support the anti-tumor immune response. Ketogenic diets (KDs), i.e., high-fat low-carbohydrate diets, have been shown to have antiproliferative and immunomodulatory effects in various preclinical cancer studies. Here, we review current knowledge of the complex interplay of KDs and the anti-tumor immune response in the context of ICB therapy, to update our understanding of diet-induced immunometabolic reprogramming in cancer. Preclinical cancer studies have revealed increased activation of and infiltration by tumor-fighting immune cells, especially CD8+ T cells, but also M1 macrophages and natural killer cells, in response to a KD regimen. In contrast, immune-suppressive cells such as regulatory CD4+ T lymphocytes, M2 macrophages, and myeloid-derived suppressor cells were reported to be decreased or largely unaffected in tumors of KD-fed mice. KDs also showed synergism with ICB therapy in several preclinical tumor studies. The observed effects are ascribed to the ability of KDs to improve immune cell infiltration and induce downregulation of immune-inhibitory processes, thus creating a more immunogenic tumor microenvironment. The studies reviewed herein show that altering the metabolic composition of the tumor microenvironment by a KD can boost the anti-tumor immune response and diminish even immunotherapy-resistant as well as immunologically "cold" tumors. However, the exact underlying mechanisms remain to be elucidated, requiring further studies before KDs can be successfully implemented as an adjunct tumor therapy to improve survival rates for cancer patients.

Impact of Protein Citrullination by Periodontal Pathobionts on Oral and Systemic Health: A Systematic Review of Preclinical and Clinical Studies

Background: This review synthesizes the role of Porphyromonas gingivalis (P. gingivalis) and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) in modulating immune responses through citrullination and assesses its impact on periodontitis and systemic conditions. Methods: A systematic review was conducted on preclinical and clinical studies focusing on P. gingivalis- and A. actinomycetemcomitans-induced citrullination and its effects on immune responses, particularly inflammatory pathways, and systemic diseases. The search included PubMed, Scopus, Google Scholar, Web of Science, and gray literature. Quality and risk of bias were assessed using OHAT Rob Toll and QUIN-Tool. The review is registered in PROSPERO (ID: CRD42024579352). Results: 18 articles published up to August 2024 were included. Findings show that P. gingivalis and A. actinomycetemcomitans citrullination modulates immune responses, leading to neutrophil dysfunction and chronic inflammation. Key mechanisms include citrullination of antimicrobial peptides, CXCL10, histone H3, α-enolase, and C5a, impairing neutrophil activation and promoting NET formation. Conclusions: This review suggests that P. gingivalis and A. actinomycetemcomitans citrullination modulates immune responses and may influence periodontitis and systemic conditions like RA. Beyond ACPA production, these pathogens affect key proteins such as H3, C5a, and CXCL10, as well as antimicrobial peptides, NET formation, and phagocytosis. These interactions lead to neutrophil dysfunction and potentially affect other cells, subsequently disrupting local and systemic inflammatory responses.

Non-invasive vagus nerve stimulation in anti-inflammatory therapy: mechanistic insights and future perspectives

Non-invasive vagus nerve stimulation (VNS) represents a transformative approach for managing a broad spectrum of inflammatory and autoimmune conditions, including rheumatoid arthritis and inflammatory bowel disease. This comprehensive review delineates the mechanisms underlying VNS, emphasizing the cholinergic anti-inflammatory pathway, and explores interactions within the neuro-immune and vagus-gut axes based on both clinical outcomes and pre-clinical models. Clinical applications have confirmed the efficacy of VNS in managing specific autoimmune diseases, such as rheumatoid arthritis, and chronic inflammatory conditions like inflammatory bowel disease, showcasing the variability in stimulation parameters and patient responses. Concurrently, pre-clinical studies have provided insights into the potential of VNS in modulating cardiovascular and broader inflammatory responses, paving the way for its translational application in clinical settings. Innovations in non-invasive VNS technology and precision neuromodulation are enhancing its therapeutic potential, making it a viable option for patients who are unresponsive to conventional treatments. Nonetheless, the widespread adoption of this promising therapy is impeded by regulatory challenges, patient compliance issues, and the need for extensive studies on long-term efficacy and safety. Future research directions will focus on refining VNS technology, optimizing treatment parameters, and exploring synergistic effects with other therapeutic modalities, which could revolutionize the management of chronic inflammatory and autoimmune disorders.

Production of Value-Added Arabinofuranosyl Nucleotide Analogues from Nucleoside by an In Vitro Enzymatic Synthetic Biosystem

Arabinofuranosyl nucleotide analogue (arabinoside) and the derived compounds, a family of nucleoside analogues, exhibit diverse, typically biological activities and are widely used as antibacterial, antiviral, anti-inflammatory, and antitumor drugs in both clinical and preclinical trials. Despite their long and rich history in medicinal chemistry, the biosynthesis of arabinoside has only been sporadically designed and studied and has remained a challenging task. In this study, an in vitro synthetic enzymatic biosystem was designed and constructed for the production of arabinoside from low-cost nucleoside, based on a phosphorolysis -isomerization-dephosphorylation enzymatic cascade conversion routes. The enzymatic system achieves the biosynthesis of arabinoside by isomerizing the ribose part of nucleoside to arabinose. The reaction conditions affecting the yield of arabinoside were investigated and optimized, including meticulous enzyme selection, key enzyme dosage, the concentration of orthophosphate, and reaction time. Under the optimized conditions, we achieved the production of 0.12 mM of arabinofuranosylguanine from 0.5 mM of guanosine, representing 24% of the theoretical yield. Furthermore, this biosystem also demonstrated the capability to produce other arabinosides, such as vidarabine, spongouridine, and hypoxanthine arabinofuranoside from corresponding nucleosides. Overall, our biosynthesis approach provides a pathway for the biosynthesis of arabinoside.

The abscopal effects of sonodynamic therapy in cancer

AbstractThe abscopal effect is a phenomenon wherein localised therapy on the primary tumour leads to regression of distal metastatic growths. Interestingly, various pre-clinical studies utilising sonodynamic therapy (SDT) have reported significant abscopal effects, however, the mechanism remains largely enigmatic. SDT is an emerging non-invasive cancer treatment that uses focussed ultrasound (FUS) and a sonosensitiser to induce tumour cell death. To expand our understanding of abscopal effects of SDT, we have summarised the preclinical studies that have found SDT-induced abscopal responses across various cancer models, using diverse combination strategies with nanomaterials, microbubbles, chemotherapy, and immune checkpoint inhibitors. Additionally, we shed light on the molecular and immunological mechanisms underpinning SDT-induced primary and metastatic tumour cell death, as well as the role and efficacy of different sonosensitisers. Notably, the observed abscopal effects underscore the need for continued investigation into the SDT-induced ‘vaccine-effect’ as a potential strategy for enhancing systemic anti-tumour immunity and combating metastatic disease. The results of the first SDT human clinical trials are much awaited and are hoped to enable the further evaluation of the safety and efficacy of SDT, paving the way for future studies specifically designed to explore the potential of translating SDT-induced abscopal effects into clinical reality.

An Optimised Live Attenuated Influenza Vaccine Ferret Efficacy Model Successfully Translates H1N1 Clinical Data

Between 2013 and 2016, the A/H1N1pdm09 component of the live attenuated influenza vaccine (LAIV) produced instances of lower-than-expected vaccine effectiveness. Standard pre-clinical ferret models, using a human-like vaccine dose and focusing on antigenic match to circulating wildtype (wt) strains, were unable to predict these fluctuations. By optimising the vaccine dose and utilising clinically relevant endpoints, we aimed to develop a ferret efficacy model able to reproduce clinical observations. Ferrets were intranasally vaccinated with 4 Log10 FFU/animal (1000-fold reduction compared to clinical dose) of seven historical LAIV formulations with known (19–90%) H1N1 vaccine efficacy or effectiveness (VE). Following homologous H1N1 wt virus challenge, protection was assessed based on primary endpoints of wt virus shedding in the upper respiratory tract and the development of fever. LAIV formulations with high (82–90%) H1N1 VE provided significant protection from wt challenge, while formulations with reduced (19–32%) VE tended not to provide significant protection. The strongest correlation observed was between reduction in wt shedding and VE (R2 = 0.75). Conversely, serum immunogenicity following vaccination was not a reliable indicator of protection (R2 = 0.37). This demonstrated that, by optimisation of the vaccine dose and the use of non-serological, clinically relevant protection endpoints, the ferret model could successfully translate clinical H1N1 LAIV VE data.

An antimicrobial blue light prototype device controls infected wounds in a preclinical porcine model

Abstract We developed a translational prototype antimicrobial blue light (ABL) device for treating skin wounds with ABL. Partial-thickness surgical wounds were created in live swine, an animal whose skin is considered the most like human skin, then heavily contaminated and left untreated for 24 hours with methicillin-resistant Staphylococcus aureus (MRSA). ABL treatment stabilized and reduced MRSA infection by greater than four orders of magnitude (>99.99%; p<0.0001) compared with untreated wounds in the same animal, after only two daily treatments. These data support further development of such devices for controlling infection in skin wounds. ABL, with or without concomitant administration of negative pressure, antimicrobials, or photosensitizers, could play an important role in modern wound care by reducing the amount, duration, and cost of antibiotics needed, helping reduce AMR. No such device for treating human cutaneous wounds currently exists. This deserves further development and study.

Minimally Invasive Transthoracic Intramyocardial Cellular Transplantation Under Echocardiographic Guidance for Myocardial Impairment

Abstract Objective: To explore the approach of minimally invasive transthoracic intramyocardial cellular transplantation under echocardiographic guidance to promote ischemic myocardial repair in a preclinical big-animal study. Methods: Female Guangxi Bama miniature pigs (weight: 25–30 kg) were randomly allocated into the sham group, untreated myocardial infarction (MI) group (MI group), the MI and surgical intramyocardial injection (SIM) group (MI-SIM group), and the MI and transthoracic echocardiography-guided percutaneous intramyocardial injection (TTEPIM) group (MI-TTEPIM group) (n = 4 each) using a lottery method. A swine MI model was established in the 3 groups excluding the sham group, and human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CM) labeled with the herpes simplex virus type-1 thymidine kinase reporter gene (hiPS-CMTK+) were transplanted by SIM in MI-SIM group and TTEPIM in MI-TTEPIM group. The operation time, postoperative recovery time of animals and volume of blood loss were collected for comparison between MI-SIM group and MI-TTEPIM group. 9-(4-[18F] fluoro-3-(hydroxymethyl) butyl) guanine positron emission tomography/computed tomography imaging was performed to track the hiPS-CMTK+ in vivo. Cardiac function and morphology were evaluated by echocardiography. Results: The operation time and postoperative recovery time of MI-TTEPIM group were significantly shorter than those of MI-SIM group ((28.3 ± 3.6) min vs. (97.0 ± 6.7) min, P < 0.001; (1.3 ± 0.3) d vs. (7.5 ± 0.9) d, P < 0.001). MI-TTEPIM also showed significantly lesser volume of blood loss during cell transplantation than MI-SIM group ((4.3 ± 0.8) mL vs. (47.0 ± 4.1) mL, P < 0.001). The transplanted cells could be traced more accurately in vivo in MI-TTEPIM than in MI-SIM. The circumferential strain of intervention region in the MI-TTEPIM group (–25.07% ± 0.27%) was significantly higher than that of the MI-SIM (–20.39% ± 0.67%) and MI groups (–19.68% ± 0.67%), respectively (P < 0.01). Conclusion: A minimally invasive TTEPIM protocol with stem cells for treating the ischemic myocardium was established in this study. Transplantation of hiPS-CMTK+ with this method could promote the recovery of the circumferential strain of the ischemic myocardium. The findings of this study lay a foundation for the clinical transformation of this auxiliary means of treatment in the future.