Relevant Mouse Model Research Articles

Clinically relevant mouse models of Charcot-Marie-Tooth type 2S.

Charcot-Marie-Tooth disease is an inherited peripheral neuropathy that is clinically and genetically heterogenous. Mutations in IGHMBP2, a ubiquitously expressed DNA/RNA helicase, have been shown to cause the infantile motor neuron disease spinal muscular atrophy with respiratory distress type 1 (SMARD1), and, more recently, juvenile-onset Charcot-Marie-Tooth disease type 2S (CMT2S). Using CRISPR-cas9 mutagenesis, we developed the first mouse models of CMT2S [p.Glu365del (E365del) and p.Tyr918Cys (Y918C)]. E365del is the first CMT2S mouse model to be discovered and Y918C is the first human CMT2S allele knock-in model. Phenotypic characterization of the homozygous models found progressive peripheral motor and sensory axonal degeneration. Neuromuscular and locomotor assays indicate that both E365del and Y918C mice have motor deficits, while neurobehavioral characterization of sensory function found that E365del mutants have mechanical allodynia. Analysis of femoral motor and sensory nerves identified axonal degeneration, which does not impact nerve conduction velocities in E365del mice, but it does so in the Y918C model. Based on these results, the E365del mutant mouse, and the human allele knock-in, Y918C, represent mouse models with the hallmark phenotypes of CMT2S, which will be critical for understanding the pathogenic mechanisms of IGHMBP2. These mice will complement existing Ighmbp2 alleles modeling SMARD1 to help understand the complex phenotypic and genotypic heterogeneity that is observed in patients with IGHMBP2 variants.

Bioprocessing of MIR17HG Results in Long and Short Noncoding RNAs with Targetable Tumor-Promoting Activity in Multiple Myeloma

Our recent studies demonstrated that multiple myeloma (MM) cells can become significantly addicted to and in turn therapeutically susceptible to the modulation of oncogenic noncoding RNAs (ncRNAs). Through large-scale CRISPR interference (CRISPRi) and CRISPR-Cas13d viability screens, we identified MIR17HG as a leading cell growth-dependency in 6 MM cell lines. MIR17HG is mainly known for producing the miRNA cluster miR-17-92, which includes six mature oncogenic miRNAs (-17, -18a, -19a, -20a, -19b, -92a). However, we observed that depleting MIR17HG pre-RNA with sgRNAs or antisense oligonucleotides (ASO) took >3 days to downregulate the miR-17-92 members, yet appreciable transcriptional changes were immediately produced followed by cell cycle arrest in G0-G1 in 10 MM cell lines and CD138+ cells from 13 MM patients. Moreover, antagonism of miR-17-92 using anti-miRs did not phenocopy the anti-MM activity of targeting the MIR17HG pre-RNA, and this activity was only partially rescued by ectopic expression of miR-17-92 mimics. Therefore, we reasoned that alternative transcripts generated at this locus could provide cell growth dependency. We analyzed our RNA-seq dataset of CD138+ cells from >300 MM patients and 70 MM cell lines for these transcripts and observed reads from regions of MIR17HG that are not included in miR-17-92. Using qRT-PCR and single molecule RNA FISH, we identified the expression of a nuclear-enriched, ~5k-nt long, polyA(+) lncRNA. This lncRNA, named lnc-17-92 was quickly downregulated within a few hours of exposure to ASOs targeting the MIR17HG pre-RNA. Using DROSHA-knockout MM cells, in which MIR17HG produces lnc-17-92 but not miR-17-92, we found that targeting the MIR17HG pre-RNA effectively antagonized MM cell growth. Using easy-to-manipulate cellular models (e.g., HCT-116), we observed stronger rescue by ectopic expression of lnc-17-92, as compared to miR-17-92, after targeting MIR17HG pre-RNA. Using RNA-protein pull down (RPPD) and RNA yeast-3-hybrid (Y3H) assays, we found that the transcription factor c-MYC and the epigenetic modulator WDR82 were binding partners for lnc-17-92. In fact, we found that lnc-17-92 mediated their protein-protein interaction by providing the chromatin scaffold for the assembly of a MYC-WDR82 complex at the promoter region of ACC1. This gene encodes the limiting enzyme for de novo lipogenesis, a metabolic pathway that we show is controlled by lnc-17-92 to promote MM cell growth. Using three different cellular models (P493-6, HMECMYC, and U226MYC), we found that targeting MIR17HG pre-RNA preferentially killed MYC+ cells. This could be explained both by lnc-17-92 acting as a chromatin scaffold for MYC and also the known role of miR-17-92 in fine-tuning the downstream transcriptional program of MYC. However, we also observed that lnc-17-92 protected MYC from proteasomal degradation and found that the miR-17-92 member miR-92a negatively regulated MYC protein expression via an, as yet, unknown mechanism. The concerted action of lnc-17-92 and miR-17-92 on MYC protein stability is under investigation and updates will also be presented. Finally, we explored MIR17HG as a therapeutic target, which includes targeting both its lncRNA and miRNAs. To develop clinically applicable inhibitors, we screened >80 fully phosphorothioated (PS), 2'-O-methoxyethyl (2'-MOE)-modified, lipid-conjugated ASOs that could either trigger RNase H-mediated degradation of MIR17HG pre-RNA (gapmeRs) or exert function via an RNase H-independent mechanism (blockmeRs). We identified an 18-mer tocopherol (T)-conjugated gapmeR G2-15b-T ("G") and an 18-mer tocopherol (T)-conjugated steric blocker SB9-19-T ("B"). We found they had potent anti-tumor effects both in vitro and in vivo in three pre-clinical animal models, including a clinically relevant PDX-NSG mouse model. Tumor growth inhibition ranged from 100% (regression) to 50% in these models. Inhibitors did not cause overt toxicity in the mice, as shown by blood cell count, clinical, and body weight analysis. These inhibitors are currently being developed for translation to clinical trials. Altogether, our studies characterize a novel oncogenic mechanism for MIR17HG and provide clinically applicable inhibitors.

Novel Chemical Series of Anti-ST2 Small Molecules Suppress Allogeneic T Cells Proliferation While Increasing Treg Cells and Improve Gvhd and Survival In Vivo

Elevation of soluble Stimulation-2 (sST2) is considered a prognostic biomarker in several disease progression, such as cardiac allo-rejection, graft-versus-host disease (GVHD), and is associated with the response to tissue damage that secrets Interleukin-33 (IL-33), an alarmin. sST2 is an alternatively spliced isoform of the Interleukin 1 Receptor-Like 1 (IL1RL1) gene that also encodes the major membrane-bound ST2 (ST2) isoform expressed on tolerogenic immune cells including Foxp3+ regulatory T cells (Tregs), type 2 innate lymphoid cells (ILC2), type 2 helper T cells (Th2), monocytes, M2 macrophages, mast cells, eosinophils and neutrophils. IL-33 is the only known ligand that binds to both sST2 and ST2. Binding of IL-33 to ST2 activates intracellular signaling in ST2 expressing cells whereas sST2 functions as a decoy receptor to sequester the secreted free IL-33. After allogeneic hematopoietic cell transplantation (HCT), elevated sST2 levels are associated with patients with GVHD resistant to steroid therapy and with subsequent non-relapse related death. We hypothesized that therapeutic intervention to release IL-33 from sST2 may modulate the T cells responses for treating GVHD. As proof-of-concept, we have reported studies using either ST2 antibodies (Zhang et. al., Sci. Trans. Med. 2015) or small-molecule ST2 inhibitors (iST2-1 in Ramadan et. al., JCI Insight, 2018) in the murine GVHD models. Herein, we report a novel series of small-molecule ST2 inhibitors from our follow-up development of iST2-1. We show a representative compound, XY-52, effectively reduced sST2 levels, alleviated GVHD and improved survival in a murine GVHD model. Based on our previously discovered iST2-1, we recently developed 1-(furan-2ylmethyl)pyrrolidine-based ST2 Inhibitors and constructed the structure-activity relationship (SAR) of compounds against ST2/IL-33 binding (Yuan et. al., Bioorg. Med. Chem., 2022). That study led to identify 14e (Fig. 1A) that has an IC50 value of 7.77 mM against ST2/IL33 binding determined by the AlphaLISA assay. Despite the improvement of the IC50 value as compared to iST2-1, 14e has deficiency in solubility and metabolic stability (including a similar in vivo half-life as iST2-1) that required further optimization. To improve 14e, we first focused on replacing the furan group (C-ring) of 14e with the goal of eliminating the potential metabolic issue of the furan group. We found replacement of furan by either a pyrrole (ST2-J-100) or an alkylated pyrrole group (ST2-J-106, 107) retained the activity of the inhibitors. We then investigated the replacement of the chemical groups on A and B rings of 14e aiming to improve the solubility of the inhibitors while maintaining their activities. These modifications led to identify a new series of ST2 inhibitors represented by XY-52. In the mixed lymphocyte reaction (MLR) assay, we found XY-52 and analogs (XY-125, XY-146, XY-148, XY-150) inhibited CD4+, CD8+ T cell proliferation while increased Tregs (Fig. 1B). XY-52 has improved in vitro (t1/2= >60m in mouse, 41m in human liver microsome) and in vivo metabolic stability. In vivo PK study showed that XY-52 has higher tissue distribution including spleen, lung, skin, small intestine and liver than iST2-1 or Ruxolitinib (Fig. 1C). XY-52 was then selected to compare with 3c (previously reported) in a clinically relevant minor-mismatched GVHD mouse model (C57BL/6→C3H.SW). Using a 21-day prophylactic regimen of 40mg/kg/day (BID), we found that 3c reduced GVHD score (P=0.02) and extended survival (HR 3.90, P=0.02) in mice. Mice receiving XY-52 treatment had a more profound reduction in GVHD score (P<0.0001) and improvement in survival (HR, 31.22, P<0.0001) (Fig. 1D). Both 3c and XY-52 yielded lower plasma concentrations of sST2 and IFNg compared with the DMSO control (Fig. 1E) at Day 7 and 21 post-HCT. Ex vivo analysis of T cells in the gastrointestinal tract (GI) at day 21 post-HCT showed that XY-52 was more effective than 3c at reducing CD4+ IFNg+, CD8+ IFNγ+, CD4+ IL-17A+ T cells and increasing CD4+ Foxp3+ Tregs frequencies. In summary, XY-52 represents a promising ST2 inhibitor that reduces the plasma sST2 levels, CD4+, CD8+ effector T cells and increases CD4+ Foxp3+ Tregs frequencies in in vitro and in vivo GVHD models. Further improvement in the potency and the PK profile of XY-52 will allow us to develop effective ST2 inhibitors for use as a prophylactic or therapeutic regimen for GVHD therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract IA005: Generating T cell responses against pancreatic cancer

Abstract The ultimate promise of tumor immunotherapy is dependent on the generation of an effective immune response against cancer, including CD4 and CD8 T cells. However, driving T cell responses in tumors such as pancreatic ductal adenocarcinoma (PDAC) requires overcoming significant hurdles in the tumor microenvironment (TME). Local and systemic suppression of dendritic cell function depresses endogenous T cell immunity against PDAC, which is further exacerbated by tumor cell intrinsic recruitment of suppressive myeloid cells and macrophages to the tumor site. To bypass these barriers, we’ve utilized agonistic anti-CD40 antibody, which mimics the endogenous CD40 ligand, to license and mature dendritic cells and re-educate tumor-associated myeloid cells towards a Th1/M1 phenotype in the PDAC TME. Using the KrasLSL-G12D/+,Trp53LSL-R172H/+,Pdx1-Cre (KPC) genetically engineered mouse model of pancreatic cancer, we show that combining standard-of-care chemotherapy with CD40 agonism promotes clonal T cell activation and expansion locally in the tumor site, resulting in regressions and cures of established tumors. This CD40-induced T cell response was further enhanced by the addition of dual immune checkpoint blockade (anti-PD-1 and anti-CTLA-4), but was independent of classical innate immune sensors such as Type I interferon receptor (IFNAR) and toll-like receptors (TLRs) previously thought to be critical for the generation of T cell immunity against tumors. These studies formed the preclinical rationale for trials investigating the use of agonistic CD40 therapy for patients with resectable or metastatic PDAC, and provided insight for immunological correlates of response in the clinical setting. More recently, we have reported on the negative impact of chemotherapy on the developing anti-tumor T cell response, emphasizing the need for additional investigations into the combination and sequencing of treatment modalities for enhanced outcomes in patients with PDAC. These studies reveal the potency of using an immunologically relevant mouse model to interrogate immune interventions in PDAC for patients with the greatest unmet clinical need. Citation Format: Katelyn T. Byrne. Generating T cell responses against pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr IA005.

Twist1 as a target for prevention of cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) represents an important clinical problem requiring novel approaches for both prevention and treatment. The transcription factor, Twist-related protein 1 (Twist1), has been identified as having a key mechanistic role in the development and progression of cSCC. Studies in relevant mouse models of cSCC have shown that Twist1 regulates epithelial-mesenchymal transition (EMT) and stemness driving progression and metastasis of cSCC. In addition, further research has shown that Twist1 regulates the balance between keratinocyte proliferation and differentiation and therefore impacts earlier stages of cSCC development. Through use of keratinocyte specific Twist1 knockout models, a role for this gene in keratinocyte stem cell homeostasis has been revealed. As a transcription factor, Twist1 regulates a large number of genes both in a positive, as well as a negative manner across several interdependent pathways. Studies in keratinocyte specific knockout models have shown that Twist1 upregulates the expression of genes involved in proliferation, stemness, and EMT while downregulating the expression of genes associated with differentiation. Furthermore, a number of compounds, including naturally occurring compounds, have been identified that target Twist1 and can block its effects in cancer cells and in keratinocytes in vivo. Collectively, the current understanding of Twist1 function in cSCC development and progression suggests that it represents a potential target for prevention and treatment of cSCC.

Reversing lysosome-ribosome circuit dysregulation mitigates C9FTD/ALS neurodegeneration and behaviors.

G4C2 repeat expansion in C9orf72 causes the most common familial frontotemporal dementia and amyotrophic lateral sclerosis (C9FTD/ALS). The pathogenesis includes haploinsufficiency of C9orf72, which forms a protein complex with Smcr8, as well as G4C2 repeat-induced gain of function including toxic dipeptide repeats (DPRs). The key in vivo disease-driving mechanisms and how loss- and gain-of-function interplay remain poorly understood. Here, we identified dysregulation of a lysosome-ribosome biogenesis circuit as an early and key disease mechanism using a physiologically relevant mouse model with combined loss- and gain-of-function across the aging process. C9orf72 deficiency exacerbates FTD/ALS-like pathologies and behaviors in C9ORF72 bacterial artificial chromosome (C9-BAC) mice with G4C2 repeats under endogenous regulatory elements from patients. Single nucleus RNA sequencing (snRNA-seq) and bulk RNA-seq revealed that C9orf72 depletion disrupts lysosomes in neurons and leads to transcriptional dysregulation of ribosomal protein genes, which are likely due to the proteotoxic stress response and resemble ribosomopathy defects. Importantly, ectopic expression of C9orf72 or its partner Smcr8 in C9FTD/ALS mutant mice promotes lysosomal functions and restores ribosome biogenesis gene transcription, resulting in the mitigation of DPR accumulation, neurodegeneration as well as FTD/ALS-like motor and cognitive behaviors. Therefore, we conclude that loss- and gain-of-function crosstalk in C9FTD/ALS converges on neuronal dysregulation of a lysosome-ribosome biogenesis circuit leading to proteotoxicity, neurodegeneration and behavioral defects.

Cardiac myosin-specific autoimmune Tcells contribute to immune-checkpoint-inhibitor-associated myocarditis.

Immune checkpoint inhibitors (ICIs) are an effective therapy for various cancers; however, they can induce immune-related adverse events (irAEs) as a side effect. Myocarditis is an uncommon, but fatal, irAE caused after ICI treatments. Currently, the mechanism of ICI-associated myocarditis is unclear. Here, we show the development of myocarditis in A/J mice induced by anti-PD-1 monoclonal antibody (mAb) administration alone without tumor cell inoculation, immunization, or viral infection. Mice with myocarditis have increased cardiac infiltration, elevated cardiac troponin levels, and arrhythmia. Anti-PD-1 mAb treatment also causes irAEs in other organs. Autoimmune Tcells recognizing cardiac myosin are activated and increased in mice with myocarditis. Notably, cardiac myosin-specific Tcells are present in naive mice, showing a phenotype of antigen-experienced Tcells. Collectively, we establish a clinically relevant mouse model for ICI-associated myocarditis and find a contribution of cardiac myosin-specific Tcells to ICI-associated myocarditis development and pathogenesis.

Alterations of brain microstructures in a mouse model of prenatal opioid exposure detected by diffusion MRI

Growing opioid use among pregnant women is fueling a crisis of infants born with prenatal opioid exposure. A large body of research has been devoted to studying the management of opioid withdrawal during the neonatal period in these infants, but less substantive work has explored the long-term impact of prenatal opioid exposure on neurodevelopment. Using a translationally relevant mouse model of prenatal methadone exposure (PME), the aim of the study is to investigate the cerebral microstructural differences between the mice with PME and prenatal saline exposure (PSE). The brains of eight-week-old male offspring with either PME (n = 15) or PSE (n = 15) were imaged using high resolution in-vivo diffusion magnetic resonance imaging on a 9.4 Tesla small animal scanner. Brain microstructure was characterized using diffusion tensor imaging (DTI) and Bingham neurite orientation dispersion and density imaging (Bingham-NODDI). Voxel-based analysis (VBA) was performed using the calculated microstructural parametric maps. The VBA showed significant (p < 0.05) bilateral alterations in fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (AD), radial diffusivity (RD), orientation dispersion index (ODI) and dispersion anisotropy index (DAI) across several cortical and subcortical regions, compared to PSE. Particularly, in PME offspring, FA, MD and AD were significantly higher in the hippocampus, dorsal amygdala, thalamus, septal nuclei, dorsal striatum and nucleus accumbens. These DTI-based results suggest widespread bilateral microstructural alterations across cortical and subcortical regions in PME offspring. Consistent with the observations in DTI, Bingham-NODDI derived ODI exhibited significant reduction in PME offspring within the hippocampus, dorsal striatum and cortex. NODDI-based results further suggest reduction in dendritic arborization in PME offspring across multiple cortical and subcortical regions. To our best knowledge, this is the first study of prenatal opioid exposure to examine microstructural organization in vivo. Our findings demonstrate perturbed microstructural complexity in cortical and subcortical regions persisting into early adulthood which could interfere with critical neurodevelopmental processes in individuals with prenatal opioid exposure.

TARGETING TUMOR NECROSIS FACTOR (TNF) IN ATRIAL STRETCH-DEPENDENT ADVERSE ATRIAL REMODELING AND VALVULAR ATRIAL FIBRILLATION IN A MOUSE MODEL OF AORTIC REGURGITATION

TARGETING TUMOR NECROSIS FACTOR (TNF) IN ATRIAL STRETCH-DEPENDENT ADVERSE ATRIAL REMODELING AND VALVULAR ATRIAL FIBRILLATION IN A MOUSE MODEL OF AORTIC REGURGITATION

A MIR17HG-derived long noncoding RNA provides an essential chromatin scaffold for protein interaction and myeloma growth

AbstractLong noncoding RNAs (lncRNAs) can drive tumorigenesis and are susceptible to therapeutic intervention. Here, we used a large-scale CRISPR interference viability screen to interrogate cell-growth dependency to lncRNA genes in multiple myeloma (MM) and identified a prominent role for the miR-17-92 cluster host gene (MIR17HG). We show that an MIR17HG-derived lncRNA, named lnc-17-92, is the main mediator of cell-growth dependency acting in a microRNA- and DROSHA-independent manner. Lnc-17-92 provides a chromatin scaffold for the functional interaction between c-MYC and WDR82, thus promoting the expression of ACACA, which encodes the rate-limiting enzyme of de novo lipogenesis acetyl-coA carboxylase 1. Targeting MIR17HG pre-RNA with clinically applicable antisense molecules disrupts the transcriptional and functional activities of lnc-17-92, causing potent antitumor effects both in vitro and in vivo in 3 preclinical animal models, including a clinically relevant patient-derived xenograft NSG mouse model. This study establishes a novel oncogenic function of MIR17HG and provides potent inhibitors for translation to clinical trials.

Combination of Circulating Cell-Free DNA and Positron Emission Tomography to Distinguish Non-Small Cell Lung Cancer from Tuberculosis.

Non-small cell lung cancer (NSCLC) holds high metabolic tumor burden and circulating cell-free DNA (cfDNA) levels, and the relationship between metabolic tumor burden and cfDNA in NSCLC and the underlying mechanism of their interaction therein remain poorly characterized. Our aim was to evaluate the clinical value of cfDNA and metabolic tumor burden by positron emission tomography-computed tomography (PET/CT) for NSCLC differential diagnosis from tuberculosis in patients with solitary pulmonary nodules. Metabolic tumor burden values in humans (subjects with NSCLC, subjects with tuberculosis, and healthy control subjects) and relevant mouse models were detected by preoperative 18F-fluorodeoxyglucose PET (18F-FDG PET/CT) and [3H]-2-deoxy-DG uptake, respectively. The cfDNA levels were detected by quantifying serum cfDNA fragments from the ALU (115 bp) gene using reverse transcription-polymerase chain reaction. RNA sequence was performed to determine the underlying target genes and knocked down or inhibited the target genes in vivo and in vitro to determine the mechanism therein. Metabolic tumor burden correlated with serum cfDNA levels in NSCLC subjects but not in tuberculosis subjects or healthy controls. Mouse models showed a similar phenomenon. In addition, the RNA sequence showed that glucose transporter 1 (GLU1), factor-related apoptosis ligand (FasL), caspase 8, and caspase 3 were significantly increased in NSCLC mouse tumors compared with those in tuberculosis mouse masses. Inhibiting the metabolic tumor burden by blocking or knocking down GLU1 markedly reduced the expression of FasL, the phosphorylation of caspase 8/caspase 3, and serum cfDNA levels/apoptosis percentage in vivo and in vitro. Furthermore, the use of a combination of cfDNA and metabolic tumor burden allowed better ability to distinguish NSCLC subjects from those with tuberculosis or healthy controls than either method used alone. Metabolic tumor burden promotes the formation of circulating cfDNA through GLU1-mediated apoptosis in NSCLC, and the combination of cfDNA and metabolic tumor burden could be valuable for distinguishing NSCLC from tuberculosis.

Effect of savirin in the prevention of biofilm-related Staphylococcus aureus prosthetic joint infection.

Background: Most of the arthroplasty surgery failure due to prosthetic joint infections (PJI) is caused by biofilm-associated Staphylococcus aureus. In a recent experimental study, savirin has been used to prevent and treat S. aureus skin infections in animal models. We explored the application of savirin in a PJI mouse model to determine its utility as an adjunct therapy to prevent PJI. Materials and methods: The in-vitro antibacterial and antibiofilm activity of savirin, with or without antibiotics (cefazolin, rifampicin, and vancomycin), against S. aureus were investigated using broth microdilution and crystal violet staining method, respectively. The effect of savirin treatment on the expression of the key biofilm-related genes (icaA, icaD, eno, fib, ebps, and agr) in S. aureus was studied using quantitative reverse transcriptase polymerase chain reaction (qRTPCR). The in-vivo efficacy of savirin alone and with cefazolin to prevent S. aureus PJI was determined using a clinically relevant PJI mouse model. Mice were randomized into five groups (n = 8/group): 1) infected K-wire savirin treated group, 2) infected K-wire cefazolin treated group, 3) infected K-wire savirin plus cefazolin treated group, 4) infected K-wire PBS treated group, 5) sterile K-wire group. Savirin was administered subcutaneously immediately post-surgery and intravenous cefazolin was given on day seven. Results: Savirin inhibited planktonic and biofilm in-vitro growth of S. aureus, showed enhanced inhibitory activity when combined with antibiotics, and down-regulated the expression of key S. aureus biofilm-related genes (icaA, icaD, eno, fib, ebps, and agr). Savirin significantly reduced bacterial counts on joint implants in comparison with the PBS treated control, while savirin plus cefazolin reduced bacterial counts on both implants and peri-prosthetic tissues. Conclusion: Savirin adjuvant therapy may prevent biofilm formation and S. aureus PJI. This study gives baseline data for using savirin for the prevention as well as treatment of S. aureus PJI in future animal studies.

The BET Protein Inhibitor JQ1 Decreases Hypoxia and Improves the Therapeutic Benefit of Anti-PD-1 in a High-Risk Neuroblastoma Mouse Model.

Anti-programmed death 1 (PD-1) is a revolutionary treatment for many cancers. The response to anti-PD-1 relies on several properties of tumor and immune cells, including the expression of PD-L1 and PD-1. Despite the impressive clinical benefit achieved with anti-PD-1 in several cancers in adults, the use of this therapy for high-risk neuroblastoma remains modest. Here, we evaluated the therapeutic benefit of anti-PD-1 in combination with JQ1 in a highly relevant TH-MYCN neuroblastoma transgenic mouse model. JQ1 is a small molecule inhibitor of the extra-terminal domain (BET) family of bromodomain proteins, competitively binding to bromodomains. Using several neuroblastoma cell lines in vitro, we showed that JQ1 inhibited hypoxia-dependent induction of HIF-1α and decreased the expression of the well-known HIF-1α downstream target gene CA9. Using MRI relaxometry performed on TH-MYCN tumor-bearing mice, we showed that JQ1 decreases R2* in tumors, a parameter associated with intra-tumor hypoxia in pre-clinical settings. Decreasing hypoxia by JQ1 was associated with improved blood vessel quality and integrity, as revealed by CD31 and αSMA staining on tumor sections. By analyzing the immune landscape of TH-MYCN tumors in mice, we found that JQ1 had no major impact on infiltrating immune cells into the tumor microenvironment but significantly increased the percentage of CD8+ PD-1+, conventional CD4+ PD-1+, and Treg PD-1+ cells. While anti-PD-1 monotherapy did not affect TH-MYCN tumor growth, we showed that combinatorial therapy associating JQ1 significantly decreased the tumor volume and improved the therapeutic benefit of anti-PD-1. This study provided the pre-clinical proof of concept needed to establish a new combination immunotherapy approach that may create tremendous enthusiasm for treating high-risk childhood neuroblastoma.

OS08.8.A Integrin-specific CAR T cells for the treatment of glioblastoma

Abstract Background Current standard of care for glioblastoma patients has limited therapeutic efficacy and novel innovative treatment strategies are urgently needed. One such strategy is chimeric antigen receptor (CAR) T cell therapy that has shown great success in hematological malignancies. αv integrins are overexpressed in several neoplasms and have already been used as therapeutic targets for small molecule inhibitors and antibodies, which did not cause major toxicities. Herein, we propose αv integrins as an ideal target for CAR T cell therapy in glioblastoma. Material and Methods CAR T cells targeting specific heterodimers (αvβ3, αvβ5 or αvβ8) were generated by transducing primary human T cells from healthy donors with a lentiviral vector expressing a second-generation CAR. Activity and specificity of CAR T cells was determined by co-culture assays with different glioma cells. Efficacy of CAR T cells to control tumor growth in vivo was investigated in clinically relevant orthotopic xenograft glioma mouse models. Additionally, we generated CAR T cells from T cells from a glioblastoma patient and measured their activity against the patient’s autologous tumor cells. Results All newly generated integrin-targeting CAR T cells exerted strong anti-glioma activity in vitro. Long-term and repetitive killing assays as well as cytokine-release measurements demonstrated highest activity of αvβ5 and αvβ8 integrin-specific CAR T cells. Antigen specificity of these cells was confirmed, as glioma cells with a CRISPR/Cas9-mediated knockout of the target antigen were resistant to CAR T cell-mediated cytotoxicity. Intratumoral injection of αvβ5 or αvβ8 CAR T cells significantly prolonged the survival and cured a substantial fraction of glioma-bearing mice in two different xenograft models. When used in a patient-derived setting, matched CAR T cells exerted strong anti-glioma activity. Conclusion We show strong and integrin-specific anti-glioma activity of CAR T cells developed from healthy donor T cells and glioblastoma-patient-derived T cells in vitro. αvβ5- and αvβ8-specific CAR T cells exerted the best therapeutic activity in two different xenograft glioma models in vivo. These data support the evaluation of integrin-specific CAR T cells as a therapeutic strategy in clinical neuro-oncology.

9. Novel murine model of heterotopic ossification induced by spinal cord injury

<h3>BACKGROUND CONTEXT</h3> Heterotopic ossification (HO) is a painful and debilitating condition causing abnormal bone formation in the soft tissues reported in up to 50% of polytrauma patients with spinal cord injury (SCI) and limb damage. In the absence of knowledge regarding the pathogenesis of HO, prophylactic treatment with NSAIDs, bisphosphonates, and radiation therapy do little to relieve the chronic pain and joint contractures resulting from HO or prevent its recurrence after surgical intervention to remove ectopic bone. A lack of clinically relevant animal models has been a major obstacle to understanding the pathogenesis of SCI-induced HO. <h3>PURPOSE</h3> The goal of this research is to characterize a clinically relevant mouse model of SCI-induced HO based on the hypothesis that simultaneous SCI and musculotendinous injury (MTI) can cause HO. <h3>METHODS</h3> Four-week-old C57BL/6J wild type mice were subjected to spinal cord transection between T10 and T11 using a pair of spring scissors via posterior open incision. The quadriceps were then crushed using a surgical clamp and a No. 15 surgical blade used to induce a 2mm MTI at the quadriceps insertion into patella in the LEFT hindlimb. The RIGHT hindlimb remained intact as internal procedural control. During the postoperative course, urine was expelled from the paraplegic using twice daily bladder massage. Soft food and water were placed in petri dishes on the bottom of the cages. Buprenorphine SR at 1mg/kg was administered subcutaneously on Day 0 and Day 4 to provide postoperative analgesia. All mice were euthanized at postoperative Day 21 when both hindlegs were dis-articulated at hip and ankle and fixed in formaldehyde overnight. High resolution micro-CT scans of the specimens were captured at the spatial resolution of 8µm. The volume of ectopic bone around the knee joint was quantified in a 3D reconstructed model. Undecalcified samples were then dehydrated, infiltrated, and embedded in low temperature MMA, and sectioned at 5µm. Consecutive sections were assessed by Von Kossa and Toluidine Blue staining to show ossification of peri-articular soft tissues, alkaline phosphatase (ALP) staining for osteoblasts, and tartrate-resistant acid phosphatase (TRAP) staining for osteoclasts. Mann-Whitney U test was used for statistical analysis of ectopic bone volumes. <h3>RESULTS</h3> Of the seven mice euthanized at three weeks post SCI/MTI, two sustained patellar dislocation in the LEFT knee and were excluded from analyses. Quantitative microCT analysis of the remaining five revealed ectopic bone deposits in the peri-articular region of all LEFT knees but none in the RIGHT knees. The HO volume of the LEFT hindlimb was consistently higher than that of RIGHT hindlimb of each mouse (25th - 75th percentile 0.02 - 0.34 vs 0.00 - 0.00, P=0.09). Histological evaluation confirmed ectopic bone deposited in the peri-articular soft tissues of the LEFT knees while the musculotendinous tissue was intact on the RIGHT side. <h3>CONCLUSIONS</h3> This mouse model represents a novel, reproducible and clinically relevant preclinical model of SCI-induced HO that will facilitate investigation of pathogenic mechanisms and assessment of novel therapeutics for this condition. <h3>FDA DEVICE/DRUG STATUS</h3> This abstract does not discuss or include any applicable devices or drugs.

Inhibition of IL-17A Protects against Thyroid Immune-Related Adverse Events while Preserving Checkpoint Inhibitor Antitumor Efficacy.

Immune checkpoint inhibitor (ICI) immunotherapy leverages the body's own immune system to attack cancer cells but leads to unwanted autoimmune side effects in up to 60% of patients. Such immune-related adverse events (IrAEs) may lead to treatment interruption, permanent organ dysfunction, hospitalization, and premature death. Thyroiditis is one of the most common IrAEs, but the cause of thyroid IrAEs remains unknown. In this study, we use a new, physiologically relevant mouse model of ICI-associated autoimmunity to identify a key role for type 3 immune cells in the development of thyroid IrAEs. Multiple lineages of IL-17A-producing T cells expand in thyroid tissue with ICI treatment. Intrathyroidal IL-17A-producing innate-like γδT17 cells were increased in tumor-free mice, whereas adaptive Th17 cells were also prominent in tumor-bearing mice, following ICI treatment. Furthermore, Ab-based inhibition of IL-17A, a clinically available therapy, significantly reduced thyroid IrAE development in ICI-treated mice with and without tumor challenge. Finally, combination of IL-17A neutralization with ICI treatment in multiple tumor models did not reduce ICI antitumor efficacy. These studies suggest that targeting Th17 and γδT17 cell function via the IL-17A axis may reduce IrAEs without impairing ICI antitumor efficacy and may be a generalizable strategy to address type 3 immune-mediated IrAEs.

Paternal alcohol exposures program intergenerational hormetic effects on offspring fetoplacental growth

Hormesis refers to graded adaptive responses to harmful environmental stimuli where low-level toxicant exposures stimulate tissue growth and responsiveness while, in contrast, higher-level exposures induce toxicity. Although the intergenerational inheritance of programmed hormetic growth responses is described in plants and insects, researchers have yet to observe this phenomenon in mammals. Using a physiologically relevant mouse model, we demonstrate that chronic preconception paternal alcohol exposures program nonlinear, dose-dependent changes in offspring fetoplacental growth. Our studies identify an inverse j-shaped curve with a threshold of 2.4 g/Kg per day; below this threshold, paternal ethanol exposures induce programmed increases in placental growth, while doses exceeding this point yield comparative decreases in placental growth. In male offspring, higher paternal exposures induce dose-dependent increases in the placental labyrinth layer but do not impact fetal growth. In contrast, the placental hypertrophy induced by low-level paternal ethanol exposures associate with increased offspring crown-rump length, particularly in male offspring. Finally, alterations in placental physiology correlate with disruptions in both mitochondrial-encoded and imprinted gene expression. Understanding the influence of ethanol on the paternally-inherited epigenetic program and downstream hormetic responses in offspring growth may help explain the enormous variation observed in fetal alcohol spectrum disorder (FASD) phenotypes and incidence.

Age-Dependent Behavioral and Metabolic Assessment of App NL-G-F/NL-G-F Knock-in (KI) Mice.

Mitochondria play a crucial role in Alzheimer's disease (AD) onset and progression. Traditional transgenic AD mouse models which were widely used in the past decades share a common limitation: The overexpression of APP and overproduction of amyloid-beta (Aβ) are accompanied by other APP peptide fragments, which could introduce artificial and non-clinically relevant phenotypes. Here, we performed an in-depth and time-resolved behavioral and metabolic characterization of a clinically relevant AD mouse model engineered to express normal physiological levels of APP harboring humanized Swedish (K670N/M671L), Beyreuther/Iberian (I716F), and Arctic (E693G) mutations (AppNL−G−F/NL−G−F), termed APP knock-in (APPKI) mice. Our result showed that APPKI mice exhibited fear learning deficits at 6-m age and contextual memory deficit at 12-m age. Histopathological analysis revealed mild amyloidosis (6E10) accompanied by microgliosis (Iba1) as early as 3 months, which progressed significantly together with significant astrocytosis at 6 and 12 m. We further analyzed hippocampal mitochondrial dysfunction by multiple assays, while 3-m APPKI mice brain mitochondrial function remains a similar level as WT mice. Significant mitochondrial dysfunction characterized by decreased ATP production and higher membrane potential with subsequent overproduction of reactive oxygen species (ROS) was observed in mitochondria isolated from 7-m APPKI mice hippocampal tissue. Morphologically, these mitochondria were larger in volume with a decreased level of mitochondrial fusion protein mitofusin-2 (MFN2). At 12 months, APPKI mice exhibit a significantly decreased total mitochondrial oxygen consumption rate (OCR) in isolated hippocampal mitochondria detected by high-resolution respirometry. These data indicate early mitochondrial dysfunction in the brain at pre-symptomatic age in the AppNL−G−F/NL−G−mice, which may play a key role in the progression of the disease. Moreover, the identified behavioral and bioenergetic alterations in this clinically relevant AD mouse model provide a valuable tool to optimize the temporal component for therapeutic interventions to treat AD.

Abstract 6188: Targeted inhibition of the E3 ligase SCFSkp2/Cks1 has survival benefit and antitumor activity in osteosarcoma

Abstract Background: Osteosarcoma (OS) is a highly aggressive bone malignancy for which treatment has remained unchanged for years. Our previous studies indicate that the F-box protein SKP2 is overexpressed in OS and acts as a proto-oncogene. We recently reported that abolishing the interaction between SKP2 and its downstream substrate p27 using a Thr187Ala knock-in mutation leads to impaired tumor growth in an RB1/TP53 deficient transgenic OS mouse model. These data suggest that inhibiting the function of SKP2 may yield therapeutic benefits in OS. However, the precise mechanisms by which SKP2 promotes tumorigenesis in OS are not fully understood. Purposes: To determine the role of SCFSkp2/Cks1 in OS tumorigenesis and as a potential therapeutic target. Methods: We generated a genetically engineered mouse model of OS with a double knockout of Rb1 and p53 only within cells of the osteoblastic lineage (Osx1-Cre;Rb1lox/lox;Trp53lox/lox, DKO). The SKP2-knockout OS mouse model was further developed in the DKO background (Osx1-Cre;Rb1lox/lox;Trp53lox/lox; Skp2-/-, TKO). Mice of both genotypes were monitored for tumor growth, overall survival, and in vivo drug response. Early passaged OS cells and OS organoid culture was derived from mice tumors and used for in vitro analysis. Western Blot and IHC were used for protein level determination. RT-qPCR and RNA-seq were used for transcriptional level determination. Two small-molecule SCFSkp2/Cks1 inhibitors (C1 and Pevonedistat) were used for in vivo and in vitro testing. Results: All genotypes were born at the expected ratios and developmentally normal. DKO animals developed OS tumors with nearly full penetrance. SKP2 deletion significantly prolongs the overall survival (p&amp;lt;0.0001) and reduced the average tumor occurrence in OS mice (p&amp;lt;0.001). The in vivo tumor proliferation was also significantly delayed in the SKP2 deletion OS tumorigenesis (p=0.007). A histological analysis comparing tumors from two groups showed a decrease in proliferation (PCNA) and an increase in pro-apoptotic markers (TUNEL,etc) expression in SKP2-deleted OS. RT-qPCR and RNA-seq revealed upregulation of E2F1 downstream apoptotic-related target genes in Skp2-/- OS tumors. The transcriptional level analysis also revealed inhibition of cancer stemness markers and pathways in SKP2 deleted tumors and further proved by functional analyses. Two SCFSkp2/Cks1 inhibitors not only showed anti-tumoral activities both in vivo (p&amp;lt;0.05) and in organoid culture, but also synergistic with chemotherapy agents (ZIP synergy Score:9.34±1.24). Conclusions: Using genetic and pharmacologic approaches, we show that inhibiting SKP2 leads to significant antitumor activities in a clinically relevant OS mouse model. The strong survival benefit highlights SKP2 as a downstream actionable target in TP53/RB1-deficient OS, for which the targeted therapies have been disappointing to date. Citation Format: Jichuan Wang, Alexander Ferrena, Ranxin Zhang, Swapnil Singh, Hongling Zhao, Deyou Zheng, Hasibagan Borjihan, Waleed Al-Harden, Janet Tingling, Rui Yang, David S. Geller, Bang H. Hoang. Targeted inhibition of the E3 ligase SCFSkp2/Cks1 has survival benefit and antitumor activity in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6188.

Abstract 1495: Bioengineered RNAi molecules for cancer research and experimental therapy

Abstract RNAs have emerged as a novel group of medications, with distinct chemistry and pharmacology from conventional small molecules and proteins or antibodies. Indeed, some RNAi products (e.g., siRNAs) have been approved for clinical practice by the United State Food and Drug Administration while many others, including genome-derived miRNAs, are under active investigations. However, current research and development relies predominantly on the use of chemo-engineered RNA mimics made in vitro that are different from natural RNA molecules produced and folded in vivo. This is also in sharp contrast to protein research and therapy that primarily uses bioengineered or recombinant proteins produced and folded in vivo, instead of synthetic polypeptides or proteins made in vitro. Therefore, we have devoted large efforts to producing true biologic RNA molecules to better recapitulate the properties of natural RNA molecules for research and drug development. Herein I will present our one-of-a-kind RNA bioengineering technology that achieves high-yield, large-scale and cost-effective, in vivo fermentation production of biologic RNAs (BioRNAs) consisting of target RNAi molecules (e.g., miRNAs or siRNAs). Examples will be presented to demonstrate the precise release of payload RNAi molecules from BioRNAs in human cells and subsequently, modulate target gene expression and various cancer cellular processes. In addition, biologic RNAi prodrugs are effective to control tumor growth and metastasis in clinically relevant xenograft mouse models. Our results demonstrate that bioengineered RNA molecules are a novel class of agents for cancer research and drug development. Acknowledgements: This study was supported by grants R01CA225958, R01CA253230 and R35GM140835 from NIH. Citation Format: Ai-Ming Yu. Bioengineered RNAi molecules for cancer research and experimental therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1495.