Survival In Preclinical Models Research Articles

EXTH-80. A NOVEL BISAMINOQUINOLINE DERIVATIVE NANOPARTICLE (BAQ13-NP) FOR THE TREATMENT OF GLIOBLASTOMA

Abstract Aminoquinolines, such as hydroxycholoquine, are known to inhibit autophagy and slow cancer cell growth, improving survival in preclinical models of glioblastoma. Unfortunately, they failed to demonstrate benefit in clinical trials due to a lack of sufficient potency and BBB penetration. We have developed a novel bisaminoquinoline derivative packaged in a nanoparticle (BAQ13-NP) with improved potency and intratumoral accumulation after systemic administration, confirmed by distribution and toxicity studies in animals. In this study, we evaluated the impact of BAQ13-NP on tumor cell autophagy and survival using patient-derived glioma cell lines and murine models. GBM patient-derived cell lines (n=4) and murine glioma cells (GL261, CT-2A) were treated with BAQ13-NP (1-4 µM) in culture, demonstrating growth restriction to less than 50% of control at 5-7 days (p<0.01) by MTT assay. Cells treated with BAQ13-NP also demonstrated reduced autophagy with significant increases in accumulation of LC3B and p62 (p<0.01) and reduced IL-6 dependent STAT3 phosphorylation in tumor cells, T cells, and myeloid cells (p<0.01). In addition, treatment of tumor cells with BAQ13-NP reduced IL-6 production over 50% (p<0.01). Mice with orthotopic GL261 or CT2A tumors were treated with BAQ13-NP by tail vein injection (25 mg/kg q2 days) starting 7 days post implantation. Significantly increased survival was observed in BAQ13-NP treated GL261 (HR 0.75, p=0.030) and CT2A (HR 0.78, p=0.033) bearing mice compared to vehicle control treated animals. Reduced tumor size at specific intervals was also observed with BAQ13-NP treatment, corelating with improved survival. BAQ13-NP can inhibit autophagy and slow glioma tumor growth, improving survival in preclinical models. Given the excellent distribution and low toxicity of this novel agent when delivered intravenously, we believe it has great potential to improve outcomes for GBM patients. Our drug has recently received IND approval and will soon be tested in a phase I clinical trial.

PRMT5 maintains tumor stem cells to promote pediatric high-grade glioma tumorigenesis.

Pediatric high-grade gliomas (PHGG) are aggressive, undifferentiated CNS tumors with poor outcomes, for which no standard-of-care drug therapy currently exists. Through a knockdown screen for epigenetic regulators, we identified PRMT5 as essential for PHGG cell growth. We hypothesized that, similar to its effect in normal cells, PRMT5 promotes self-renewal of stem-like PHGG tumor initiating cells (TICs) essential for tumor growth. We conducted in vitro analyses, including limiting dilution studies of self-renewal, to determine the phenotypic effects of PRMT5 KD. We performed ChIP-Seq to identify PRMT5-mediated epigenetic changes and performed gene set enrichment analysis to identify pathways that PRMT5 regulates. Using an orthotopic xenograft model of PHGG, we tracked survival and histological characteristics resulting from PRMT5 KD or administration of a PRMT5 inhibitor ± radiation therapy (RT). In vitro, PRMT5 KD slowed cell cycle progression, tumor growth and self-renewal, and altered chromatin occupancy at genes associated with differentiation, tumor formation and growth. In vivo, PRMT5 KD increased survival and reduced tumor aggressiveness; however, pharmacological inhibition of PRMT5 with or without RT did not improve survival. PRMT5 KD epigenetically reduced TIC self-renewal, leading to increased survival in preclinical models. Pharmacological inhibition of PRMT5 enzymatic activity may have failed in vivo due to insufficient reduction of PRMT5 activity by chemical inhibition, or this failure may suggest that non-enzymatic activities of PRMT5 are more relevant. Implications: PRMT5 maintains and promotes the growth of stemlike cells that initiate and drive tumorigenesis in pediatric high grade glioma.

Current Scenario and Future Perspectives of Porcine Corneal Xenotransplantation.

Corneal diseases represent a significant cause of blindness worldwide, with corneal transplantation being an effective treatment to prevent vision loss. Despite substantial advances in transplantation techniques, the demand for donor corneas exceeds the available supply, particularly in developing countries. Cornea xenotransplantation has emerged as a promising strategy to address the worldwide scarcity, notably using porcine corneas. In addition to the inherent immune privilege of the cornea, the low cost of porcine breeding and the anatomical and physiological similarities between humans and pigs have made porcine corneas a viable alternative. Nonetheless, ethical concerns, specifically the risk of xenozoonotic transmission and the necessity for stringent biosafety measures, remain significant obstacles. Moreover, the success of xenotransplantation is compromised by innate and adaptive immune responses, which requires meticulous consideration and further studies. Despite these challenges, recent breakthroughs have further contributed to reducing immunogenicity while preserving the corneal architecture. Advances in genetic engineering, such as the use of CRISPR-Cas9 to eliminate critical porcine antigens, have shown promise for mitigating immune reactions. Additionally, new immunosuppressive protocols, such as have techniques like decellularization and the use of porcine-derived acellular matrices, have greatly increased graft survival in preclinical models. Future research must focus on refining immunomodulatory strategies and improving graft preparation techniques to ensure the long-term survival and safety of porcine corneal xenotransplantation in clinical trials in humans.

Enhanced tumor control and survival in preclinical models with adoptive cell therapy preceded by low-dose radiotherapy.

Effective infiltration of chimeric antigen receptor T (CAR-T) cells into solid tumors is critical for achieving a robust antitumor response and improving therapeutic outcomes. While CAR-T cell therapies have succeeded in hematologic malignancies, their efficacy in solid tumors remains limited due to poor tumor penetration and an immunosuppressive tumor microenvironment. This study aimed to evaluate the potential of low-dose radiotherapy (LDRT) administered before T-cell therapy to enhance the antitumor effect by promoting CAR-T cell infiltration. We hypothesized that combining LDRT with T-cell therapy would improve tumor control and survival compared to either treatment alone. We investigated this hypothesis using two NSG mouse models bearing GSU or CAPAN-2 solid tumors. The mice were treated with engineered CAR-T cells targeting guanyl cyclase-C (GCC) or mesothelin as monotherapy or in combination with LDRT. Additionally, we extended this approach to a C57BL/6 mouse model implanted with MC38-gp100+ cells, followed by adoptive transfer of pmel+ T cells before and after LDRT. Tumor growth and survival outcomes were monitored in all models. Furthermore, we employed atomic force microscopy (AFM) in a small cohort to assess the effects of radiotherapy on tumor stiffness and plasticity, exploring the role of tumor nanomechanics as a potential biomarker for treatment efficacy. Our results demonstrated enhanced tumor control and prolonged survival in mice treated with LDRT followed by T-cell therapy across all models. The combination of LDRT with CAR-T or pmel+ T-cell therapy led to superior tumor suppression and survival compared to monotherapy, highlighting the synergistic impact of the combined approach. Additionally, AFM analysis revealed significant changes in tumor stiffness and plasticity in response to LDRT, suggesting that the nanomechanical properties of the tumor may be predictive of therapeutic response. The findings of this study highlight the transformative potential of incorporating LDRT as a precursor to adoptive T-cell therapy in solid tumors. By promoting CAR-T and pmel+ T-cell infiltration into the tumor microenvironment, LDRT enhanced tumor control and improved survival outcomes, offering a promising strategy to overcome the challenges associated with CAR-T therapy in solid tumors. Additionally, the changes in tumor nanomechanics observed through AFM suggest that tumor stiffness and plasticity could be biomarkers for predicting treatment outcomes. These results support further investigation into the clinical application of this combined approach to improve the efficacy of cell-based therapies in patients with solid tumors.

Integrin α6-containing extracellular vesicles promote lymphatic remodelling for pre-metastatic niche formation in lymph nodes via interplay with CD151.

Heterogeneous extracellular vesicles (EVs) from various types of tumours are acknowledged for inducing the formation of pre-metastatic "niches" in draining lymph nodes (LNs) to promote lymphatic metastasis. In order to identify the specific subpopulations of EVs involved, we performed high-resolution proteomic analysis combined with nanoflow cytometry of bladder cancer (BCa) tissue-derived EVs to identify a novel subset of tumour-derived EVs that contain integrin α6 (ITGA6+EVs) and revealed the positive correlation of ITGA6+EVs with the formation of pre-metastatic niche in draining LNs and lymphatic metastasis in multicentre clinical analysis of 820-case BCa patients. BCa-derived ITGA6+EVs induced E-selectin (SELE)-marked lymphatic remodelling pre-metastatic niche and promoted metastasis in draining LNs through delivering cargo circRNA-LIPAR to lymphatic endothelial cells in vivo and in vitro. Mechanistically, LIPAR linked ITGA6 to the switch II domain of RAB5A and sustained RAB5A GTP-bound activated state, thus maintaining the production of ITGA6+EVs loaded with LIPAR through endosomal trafficking. ITGA6+EVs targeted lymphatic vessels through ITGA6-CD151 interplay and released LIPAR to induce SELE overexpression-marked lymphatic remodelling pre-metastatic niche. Importantly, we constructed engineered-ITGA6 EVs to inhibit lymphatic pre-metastatic niche, which suppressed lymphatic metastasis and prolonged survival in preclinical models. Collectively, our study uncovers the mechanism of BCa-derived ITGA6+EVs mediating pre-metastatic niche and provides an engineered-EV-based strategy against BCa lymphatic metastasis.

Peritoneal Infusion of Oxygen Microbubbles Alters the Metabolomic Profile of the Lung and Spleen in Acute Hypoxic Exposure.

Administration of oxygen microbubbles (OMBs) has been shown to increase oxygen and decrease carbon dioxide in systemic circulation, as well as reduce lung inflammation and promote survival in preclinical models of hypoxia caused by lung injury. However, their impact on microenvironmental oxygenation remains unexplored. Herein, we investigated the effects of intraperitoneal administration of OMBs in anesthetized rats exposed to hypoxic ventilation (FiO2 = 0.14). Blood oxygenation and hemodynamics were evaluated over a 2 h time frame, and then organ and tissue samples were collected for hypoxic and metabolic analyses. Data showed that OMBs improved blood SaO2 (~14%) and alleviated tissue hypoxia within the microenvironment of the kidney and intestine at 2 h of hypoxia. Metabolomic analysis revealed OMBs induced metabolic differences in the cecum, liver, kidney, heart, red blood cells and plasma. Within the spleen and lung, principal component analysis showed a metabolic phenotype more comparable to the normoxic group than the hypoxic group. In the spleen, this shift was characterized by reduced levels of fatty acids and 2-hydroxygluterate, alongside increased expression of antioxidant enzymes such as glutathione and hypoxanthine. Interestingly, there was also a shuttle effect within the metabolism of the spleen from the tricarboxylic acid cycle to the glycolysis and pentose phosphate pathways. In the lung, metabolomic analysis revealed upregulation of phosphatidylethanolamine and phosphatidylcholine synthesis, indicating a potential indirect mechanism through which OMB administration may improve lung surfactant secretion and prevent alveolar collapse. In addition, cell-protective purine salvage was increased within the lung. In summary, oxygenation with intraperitoneal OMBs improves systemic blood and local tissue oxygenation, thereby shifting metabolomic profiles of the lung and spleen toward a healthier normoxic state.

STING agonist 8803 reprograms the immune microenvironment and increases survival in preclinical models of glioblastoma.

STING agonists can reprogram the tumor microenvironment to induce immunological clearance within the central nervous system. Using multiplexed sequential immunofluorescence (SeqIF) and the Ivy Glioblastoma Atlas, STING expression was found in myeloid populations and in the perivascular space. The STING agonist 8803 increased median survival in multiple preclinical models of glioblastoma, including QPP8, an immune checkpoint blockade-resistant model, where 100% of mice were cured. Ex vivo flow cytometry profiling during the therapeutic window demonstrated increases in myeloid tumor trafficking and activation, alongside enhancement of CD8+ T cell and NK effector responses. Treatment with 8803 reprogrammed microglia to express costimulatory CD80/CD86 and iNOS, while decreasing immunosuppressive CD206 and arginase. In humanized mice, where tumor cell STING is epigenetically silenced, 8803 therapeutic activity was maintained, further attesting to myeloid dependency and reprogramming. Although the combination with a STAT3 inhibitor did not further enhance STING agonist activity, the addition of anti-PD-1 antibodies to 8803 treatment enhanced survival in an immune checkpoint blockade-responsive glioma model. In summary, 8803 as a monotherapy demonstrates marked in vivo therapeutic activity, meriting consideration for clinical translation.

Restoring Mitochondrial Function and Muscle Satellite Cell Signaling: Remedies against Age-Related Sarcopenia.

Sarcopenia has a complex pathophysiology that encompasses metabolic dysregulation and muscle ultrastructural changes. Among the drivers of intracellular and ultrastructural changes of muscle fibers in sarcopenia, mitochondria and their quality control pathways play relevant roles. Mononucleated muscle stem cells/satellite cells (MSCs) have been attributed a critical role in muscle repair after an injury. The involvement of mitochondria in supporting MSC-directed muscle repair is unclear. There is evidence that a reduction in mitochondrial biogenesis blunts muscle repair, thus indicating that the delivery of functional mitochondria to injured muscles can be harnessed to limit muscle fibrosis and enhance restoration of muscle function. Injection of autologous respiration-competent mitochondria from uninjured sites to damaged tissue has been shown to reduce infarct size and enhance cell survival in preclinical models of ischemia-reperfusion. Furthermore, the incorporation of donor mitochondria into MSCs enhances lung and cardiac tissue repair. This strategy has also been tested for regeneration purposes in traumatic muscle injuries. Indeed, the systemic delivery of mitochondria promotes muscle regeneration and restores muscle mass and function while reducing fibrosis during recovery after an injury. In this review, we discuss the contribution of altered MSC function to sarcopenia and illustrate the prospect of harnessing mitochondrial delivery and restoration of MSCs as a therapeutic strategy against age-related sarcopenia.

Abstract 3233: EP102: Pharmacological Inhibition of METTL3 causes tumor growth inhibition and prolongs survival in preclinical models of NSCLC, ovarian and squamous cell carcinoma

Abstract METTL3 is the RNA methyltransferase predominantly responsible for the addition of N-6-methyladenosine (m6A), the most abundant epigenetic modification to mRNA. The prevalence of m6A and the activity and expression of METTL3 have been linked to the appearance and progression of numerous hematological and solid tumor cancers. EPICS has discovered and optimized small molecule inhibitors of METTL3 (“M3i”). The aim of the current study is to evaluate the efficacy of M3i in various CDX mouse tumor models. M3i compound potency was evaluated in vitro by measuring enzymatic inhibition of METTL3/14 by scintillation proximity assay (IC50 = 2 nM). The anti-proliferative effects of M3i were measured in cell viability assays following a 72h treatment of A549 (NSCLC, IC50 = 190 nM), SKOV3 (ovarian, IC50 = 9 nM) and FaDu (hypopharyngeal squamous cell carcinoma, IC50 = 24 nM) cells. Complementary data in all three cell lines demonstrates a clear concentration-response for a target engagement biomarker (m6A; IC50 = 2-8 nM, across cell lines) as well as M3i-induced changes in biomarkers of oncogenic drivers. M3i (30 mg/kg, IP, QD) was tested in vivo in mouse models using FaDu (subcutaneous implant) in addition to A549 and SKOV3 (both orthotopic implants); all animal models were performed by LabCorp (MI, USA). Daily M3i treatment significantly prolonged survival in all three disease models consistent with efficacy on tumor growth inhibition as measured by imaging (A549, SKOV3) or direct measurement of tumor volume in the case of subcutaneous implant (FaDu). M3i has previously been shown to be effective in mouse CDX and PDX models of AML. The current results extend the application of METTL3 inhibitors to include the treatment of various types of solid tumors. The efficacy of M3i as a single-agent on tumor growth inhibition and survival advances the validation of METTL3 as a cancer target and provides additional evidence for the applicability of mRNA epigenetic mechanisms to the treatment of solid tumors. Citation Format: Graeme Fraser, Catherine Sorlet, Nicolas Parmentier, Elodie Brunel, Christian Coremans, Sarah Rorive, Anne-France Hartiel, Killian Oukoloff, Guillaume Dutheuil. EP102: Pharmacological Inhibition of METTL3 causes tumor growth inhibition and prolongs survival in preclinical models of NSCLC, ovarian and squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3233.

Abstract 4127: Combined inhibition of RAF, MEK, and FAK attenuates melanoma brain metastases and prolongs survival in preclinical models

Abstract Despite promising results from recent FDA-approved therapies, many advanced melanoma patients develop resistance to both immunotherapy and targeted therapy. A common resistance mechanism to targeted therapy is upregulation of the PI3K/AKT signaling pathway, which has also been shown to promote the development of melanoma brain metastases. Historically, AKT inhibitors have failed in the clinic due to their limited efficacy or intolerable toxicity. Proteomic analysis comparing non-metastatic vs brain metastatic primary tumors in mice revealed focal adhesion kinase (FAK) as an AKT1 specific effector and a potential alternative therapeutic target. FAK is a non-receptor tyrosine kinase that localizes primarily to focal adhesions to regulate cell migration. To determine whether targeting FAK alone or in combination with the RAF/MEK inhibitor avutometinib reduces brain metastases and prolongs survival, we utilized both autochthonous and syngeneic melanoma mouse models. Mice with either subcutaneous tumors or established brain metastases were treated with FAK inhibitor, RAF/MEK inhibitor, or the combination of FAK and RAF/MEK inhibitors. Each cohort was assessed for tumor onset, growth, metastasis, and survival. Our results show that combined RAF/MEK/FAK inhibition significantly delays tumor onset, causes regression of established tumors, prevents the development of brain metastases, promotes the regression of established brain metastases, and prolongs survival. In addition, patient-derived BRAF V600E melanoma xenograft mouse models resistant to the BRAF inhibitor dabrafenib and the MEK inhibitor trametinib, were sensitive to combined RAF/MEK/FAK blockade. The addition of the BRAF inhibitor encorafenib to these models further enhanced the effect on tumor growth. These results support the initiation of a clinical trial evaluating the efficacy of the RAF/MEK inhibitor avutometinib in combination with the FAK inhibitor defactinib in patients with brain metastases from cutaneous melanoma. Additionally, we are assessing non-canonical roles of FAK in modulating the tumor microenvironment to determine whether avutometinib and defactinib also enhance the efficacy of immune checkpoint inhibition in this disease. Citation Format: Karly A. Stanley, Jared D. Almazan, Tursun Turapov, David A. Kircher, Gennie L. Parkman, MiKaela N. Field, Katie M. Culver, Christopher M. Stehn, Silvia Coma, Jonathan A. Pachter, Howard Colman, Sheri L. Holmen. Combined inhibition of RAF, MEK, and FAK attenuates melanoma brain metastases and prolongs survival in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4127.

A novel pan-PI3K inhibitor KTC1101 synergizes with anti-PD-1 therapy by targeting tumor suppression and immune activation

BackgroundPhosphoinositide 3-kinases (PI3Ks) are critical regulators of diverse cellular functions and have emerged as promising targets in cancer therapy. Despite significant progress, existing PI3K inhibitors encounter various challenges such as suboptimal bioavailability, potential off-target effects, restricted therapeutic indices, and cancer-acquired resistance. Hence, novel inhibitors that overcome some of these challenges are needed. Here, we describe the characterization of KTC1101, a novel pan-PI3K inhibitor that simultaneously targets tumor cell proliferation and the tumor microenvironment. Our studies demonstrate that KTC1101 significantly increases the anti-PD-1 efficacy in multiple pre-clinical mouse models.MethodsKTC1101 was synthesized and characterized employing chemical synthesis, molecular modeling, Nuclear Magnetic Resonance (NMR), and mass spectrometry. Its target specificity was confirmed through the kinase assay, JFCR39 COMPARE analysis, and RNA-Seq analysis. Metabolic stability was verified via liver microsome and plasma assays, pharmacokinetics determined by LC–MS/MS, and safety profile established through acute toxicity assays to determine the LD50. The antiproliferative effects of KTC1101 were evaluated in a panel of cancer cell lines and further validated in diverse BALB/c nude mouse xenograft, NSG mouse xenograft and syngeneic mouse models. The KTC1101 treatment effect on the immune response was assessed through comprehensive RNA-Seq, flow cytometry, and immunohistochemistry, with molecular pathways investigated via Western blot, ELISA, and qRT-PCR.ResultsKTC1101 demonstrated strong inhibition of cancer cell growth in vitro and significantly impeded tumor progression in vivo. It effectively modulated the Tumor Microenvironment (TME), characterized by increased infiltration of CD8+ T cells and innate immune cells. An intermittent dosing regimen of KTC1101 enhanced these effects. Notably, KTC1101 synergized with anti-PD-1 therapy, significantly boosting antitumor immunity and extending survival in preclinical models.ConclusionKTC1101's dual mechanism of action—directly inhibiting tumor cell growth and dynamically enhancing the immune response— represents a significant advancement in cancer treatment strategies. These findings support incorporating KTC1101 into future oncologic regimens to improve the efficacy of immunotherapy combinations.

A humanized IL-2 mutein expands Tregs and prolongs transplant survival in preclinical models.

Long-term organ transplant survival remains suboptimal, and life-long immunosuppression predisposes transplant recipients to an increased risk of infection, malignancy, and kidney toxicity. Promoting the regulatory arm of the immune system by expanding Tregs may allow immunosuppression minimization and improve long-term graft outcomes. While low-dose IL-2 treatment can expand Tregs, it has a short half-life and off-target expansion of NK and effector T cells, limiting its clinical applicability. Here, we designed a humanized mutein IL-2 with high Treg selectivity and a prolonged half-life due to the fusion of an Fc domain, which we termed mIL-2. We showed selective and sustainable Treg expansion by mIL-2 in 2 murine models of skin transplantation. This expansion led to donor-specific tolerance through robust increases in polyclonal and antigen-specific Tregs, along with enhanced Treg-suppressive function. We also showed that Treg expansion by mIL-2 could overcome the failure of calcineurin inhibitors or costimulation blockade to prolong the survival of major-mismatched skin grafts. Validating its translational potential, mIL-2 induced a selective and sustainable in vivo Treg expansion in cynomolgus monkeys and showed selectivity for human Tregs in vitro and in a humanized mouse model. This work demonstrated that mIL-2 can enhance immune regulation and promote long-term allograft survival, potentially minimizing immunosuppression.

VEGF and Other Gene Therapies Improve Flap Survival-A Systematic Review and Meta-Analysis of Preclinical Studies.

Surgical flaps are basic tools in reconstructive surgery. Their use may be limited by ischemia and necrosis. Few therapies address or prevent them. Genetic therapy could improve flap outcomes, but primary studies in this field present conflicting results. This systematic review and meta-analysis aimed to appraise the efficacy of external gene delivery to the flap for its survival in preclinical models. This review was registered with PROSPERO (CRD42022359982). PubMed, Embase, Web of Science, and Scopus were searched to identify studies using animal models reporting flap survival outcomes following any genetic modifications. Random-effects meta-analysis was used to calculate mean differences in flap survival with accompanying 95% CI. The risk of bias was assessed using the SYRCLE tool. Subgroup and sensitivity analyses were performed to ascertain the robustness of primary analyses, and the evidence was assessed using the GRADE approach. The initial search yielded 690 articles; 51 were eventually included, 36 of which with 1576 rats were meta-analyzed. VEGF gene delivery to different flap types significantly improved flap survival area by 15.66% (95% CI 11.80-19.52). Other interventions had smaller or less precise effects: PDGF-13.44% (95% CI 3.53-23.35); VEGF + FGF-8.64% (95% CI 6.94-10.34); HGF-5.61% (95% CI 0.43-10.78); FGF 3.84% (95% CI 1.13-6.55). Despite considerable heterogeneity, moderate risk of bias, and low quality of evidence, the efficacy of VEGF gene therapy remained significant in all sensitivity analyses. Preclinical data indicate that gene therapy is effective for increasing flap survival, but further animal studies are required for successful clinical translation.

ATH-1105, a small-molecule positive modulator of the neurotrophic HGF system, is neuroprotective, preserves neuromotor function, and extends survival in preclinical models of ALS.

Amyotrophic lateral sclerosis (ALS), a progressive and fatal neurodegenerative disorder, primarily affects the motor neurons of the brain and spinal cord. Like other neurodegenerative conditions, ongoing pathological processes such as increased inflammation, excitotoxicity, and protein accumulation contribute to neuronal death. Hepatocyte growth factor (HGF) signaling through the MET receptor promotes pro-survival, anti-apoptotic, and anti-inflammatory effects in multiple cell types, including the neurons and support cells of the nervous system. This pleiotropic system is therefore a potential therapeutic target for treatment of neurodegenerative disorders such as ALS. Here, we test the effects of ATH-1105, a small-molecule positive modulator of the HGF signaling system, in preclinical models of ALS. In vitro, the impact of ATH-1105 on HGF-mediated signaling was assessed via phosphorylation assays for MET, extracellular signal-regulated kinase (ERK), and protein kinase B (AKT). Neuroprotective effects of ATH-1105 were evaluated in rat primary neuron models including spinal motor neurons, motor neuron-astrocyte cocultures, and motor neuron-human muscle cocultures. The anti-inflammatory effects of ATH-1105 were evaluated in microglia- and macrophage-like cell systems exposed to lipopolysaccharide (LPS). In vivo, the impact of daily oral treatment with ATH-1105 was evaluated in Prp-TDP43A315T hemizygous transgenic ALS mice. In vitro, ATH-1105 augmented phosphorylation of MET, ERK, and AKT. ATH-1105 attenuated glutamate-mediated excitotoxicity in primary motor neurons and motor neuron- astrocyte cocultures, and had protective effects on motor neurons and neuromuscular junctions in motor neuron-muscle cocultures. ATH-1105 mitigated LPS-induced inflammation in microglia- and macrophage-like cell systems. In vivo, ATH-1105 treatment resulted in improved motor and nerve function, sciatic nerve axon and myelin integrity, and survival in ALS mice. Treatment with ATH-1105 also led to reductions in levels of plasma biomarkers of inflammation and neurodegeneration, along with decreased pathological protein accumulation (phospho-TDP-43) in the sciatic nerve. Additionally, both early intervention (treatment initiation at 1 month of age) and delayed intervention (treatment initiation at 2 months of age) with ATH-1105 produced benefits in this preclinical model of ALS. The consistent neuroprotective and anti-inflammatory effects demonstrated by ATH-1105 preclinically provide a compelling rationale for therapeutic interventions that leverage the positive modulation of the HGF pathway as a treatment for ALS.

Iscalimab Combined With Transient Tesidolumab Prolongs Survival in Pig-to-Rhesus Monkey Renal Xenografts.

To evaluate the clinically relevant anti-CD40 antibody iscalimab for baseline immunosuppression in a preclinical pig-to-rhesus renal xenograft model. CD40/CD40L co-stimulation blockade-based immunosuppression has been more successful than calcineurin-based protocols in prolonging xenograft survival in preclinical models. GGTA1 knockout/CD55 transgenic pig kidneys were transplanted into rhesus monkeys (n=6) receiving an iscalimab-based immunosuppressive regimen. Two grafts were lost early (22 and 26 days) because of ectatic donor ureters with otherwise normal histology. The other recipients survived 171, 315, 422, and 439 days with good renal function throughout the posttransplant course. None of the recipients experienced serious infectious morbidity. It may be reasonable to evaluate an iscalimab-based immunosuppressive regimen in clinical renal xenotransplantation.

PI3K inhibition circumvents resistance to SHP2 blockade in metastatic triple-negative breast cancer

The protein tyrosine phosphatase SHP2 activates oncogenic pathways downstream of most receptor tyrosine kinases (RTK) and has been implicated in various cancer types, including the highly aggressive subtype of triple-negative breast cancer (TNBC). Although allosteric inhibitors of SHP2 have been developed and are currently being evaluated in clinical trials, neither the mechanisms of the resistance to these agents, nor the means to circumvent such resistance have been clearly defined. The PI3K signaling pathway is also hyperactivated in breast cancer and contributes to resistance to anticancer therapies. When PI3K is inhibited, resistance also develops for example via activation of RTKs. We therefore assessed the effect of targeting PI3K and SHP2 alone or in combination in preclinical models of metastatic TNBC. In addition to the beneficial inhibitory effects of SHP2 alone, dual PI3K/SHP2 treatment decreased primary tumor growth synergistically, blocked the formation of lung metastases, and increased survival in preclinical models. Mechanistically, transcriptome and phospho-proteome analyses revealed that resistance to SHP2 inhibition is mediated by PDGFRβ-evoked activation of PI3K signaling. Altogether, our data provide a rationale for co-targeting of SHP2 and PI3K in metastatic TNBC.

A preclinical study to evaluate the combination of azeliragon, an inhibitor of the receptor for advanced glycation endproducts (RAGE), with radiation therapy as a potential treatment of glioblastoma.

e14056 Background: Systemic lymphopenia, which is independently associated with poor overall survival, is observed in approximately 30-50% of patients with glioblastoma (GBM) after chemoradiotherapy. We recently found that cranial irradiation of GBM tumors increases circulating myeloid-derived suppressor cells (MDSC) contributing to systemic lymphopenia. Additionally, we found that cranial radiation increases circulating levels of the RAGE ligands S100A8/9 and HMGB1, which have been reported to increase MDSC and their immunosuppressive effects through binding RAGE. We, therefore, hypothesized that targeting RAGE receptors on MDSC could prevent immunosuppression and improve survival in preclinical GBM models. Methods: We orthotopically implanted GBM cells (GL261 and CT2A) into immunocompetent C57BL6 mice. The GBM tumor-bearing mice were treated with five doses of fractionated radiation therapy (RT, 2 Gy/day for 5 consecutive days) to the whole head in combination with RAGE inhibitor azeliragon (0.1mg/mice/day i.p starting one day before RT for up to 10 days). We evaluated the immune cells in the tumor microenvironment (TME) and spleen at 14 days post-tumor implantation. We also evaluated the circulating immune cells and RAGE ligands (HMGB1 and S100A8/A9) in the plasma at 7, 14, and 21 days post-tumor implantation and monitored the mice for survival up to 60 days. Results: GBM tumor-bearing mice (GL261 & CT2A) treated with RT plus azeliragon had significantly improved survival when compared to RT alone or azeliragon alone. RT plus azeliragon significantly reduced the accumulation of granulocytic-MDSC (G-MDSC), monocytic-MDSC (M-MDSC), tumor-associated macrophages (TAM), and regulatory T cells (Treg), while increasing infiltrating CD8 T cells in the TME as compared to RT alone. RT alone significantly increased the accumulation of G-MDSC and M-MDSC in the spleen, which was significantly reduced with concurrent azeliragon. Concurrent administration of azeliragon with RT did not significantly affect the radiation-induced increase of circulating M-MDSC as well as plasma HMGB1 and S100A8/A9 concentration, though there was a modest reduction of circulating G-MDSC. Conclusions: Our study indicates that azeliragon, by blocking RAGE interaction with its ligands, helps to overcome the MDSC-mediated immunosuppression that occurs after RT. The combination of azeliragon with RT enhances survival in preclinical models. Clinical trials evaluating the safety and efficacy of azeliragon in combination with RT for newly diagnosed GBM are currently under development.

Abstract GS5-07: Estradiol represses anti-tumoral immune response to promote progression of triple-negative breast cancer brain metastases

Abstract Background: Younger women with breast cancer have increased risk of development of brain metastases irrespective of the tumor subtype. We have shown that pre-menopausal levels of 17-β-Estradiol (E2) contributes to the promotion of brain metastases by influencing the tumor microenvironment. E2 promotes brain metastasis (BM) of estrogen receptor negative (ER¯) BC cells by inducing neuroinflammatory ER+ astrocytes in the brain niche to secrete pro-metastatic factors critical for early brain colonization. Ovarieoctomy (OVX) in combination with the aromatase inhibitor (Letrozole) prevented brain colonization of triple negative (TNBC) (ER-PR-HER2-) human xenografts (MDA231BR/NSG) and murine models (E0711/C57Bl6, 4T1/BALBc) through paracrine activation of EGFR and TRKB, pathways involved in increased invasion and early tumor initiation. Yet, the extent to which E2-depletion therapies can decrease progression of established BM in combination with current standard of care for brain metastasis remains unknown. Goal: Current standard of care (SOC) for patients with TNBC brain metastasis includes irradiation (SRS, whole brain) and immunotherapy (PD-1/PDL-1 inhibitors). The goal of this study was to assess how E2-depletion therapies affects brain immune function in the context of SOC for brain metastatic progression of TNBC. Results: To assess whether E2-depletion could decrease BM progression in a model that mimics standard of care for BM, TNBC E0771-GFP-luc cells were injected intracardially in syngeneic ovariectomized (OVX)-female C57Bl6 mice supplemented with pre-menopausal levels of E2. Seven days after injection (when cancer cells have colonized), mice received a single 15Gy dose brain irradiation and were randomized to continue receiving E2, E2 withdrawal (E2WD) or E2WD plus the aromatase-inhibitor letrozole (E2WD+LET). Brain metastatic burden significantly decreased in E2WD and E2WD+Letrozole treated mice as compared to E2-treated mice. Injection of E0711 cells in immunocompromised NSG mice or in the absence of brain irradiation abolished this effect, suggesting that E2-depletion therapies decrease BM progression through boosting radiation-induced anti-tumor immunity. Accordingly, there were no differences in BM progression in E2, E2WD or E2WD+let treated mice in a xenograft model (F2-7 TNBC cells) in NSG mice, even in the presence of brain irradiation. Immune-profiling of brains from OVX+E2, OVX and OVX+Let C57BL6 mice carrying BMs showed dynamic changes in immune populations at early and late stages of brain metastatic colonization. At early stages post brain colonization (3 days post ic injection) E2-treated mice showed a decreased fraction of CD11b+CD45Int CD206+ microglia/CNS macrophages as compared to OVX+LET-treated mice, without significant changes in the fraction of infiltrated lymphocytes, suggesting E2 represses early immunosurveillance through repression of microglia/CNS macrophage activation. At later stages of brain colonization (7 days post ic injection), E2-treated mice showed an increased fraction of proinflammatory microglia and decreased fraction of T and B cells as compared to OVX or OVX+let treated mice. While E2-depletion increased the recruitment of T cells to the brain niche, the fraction of CD279 (PD1+) brain T cells was similar among groups. Ongoing studies assess the efficacy of E2-depletion therapies in combination with brain radiation and PD-1 inhibitors to decrease metastatic burden and improve survival in preclinical models. Conclusion: Our results support the hypothesis that estradiol promotes brain metastatic progression by stimulating an immunosuppressive brain microenvironment. As such, FDA-approved E2-depletion therapies (aromatase inhibitors and selective-estrogen modulators) could be used in combination with brain irradiation and PD-1 inhibitors to promote a more effective anti-tumoral immune response. Citation Format: Diana Cittelly, Maria J. Contreras-Zarate, Karen ALvarez-Eraso, Vesna Tesic, Nicole Tsuji, Leanna Chafee, Sana Karam, D. Ryan Ormond, Peter Kabos. Estradiol represses anti-tumoral immune response to promote progression of triple-negative breast cancer brain metastases [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr GS5-07.

Regulation of bone homeostasis by MERTK and TYRO3

The fine equilibrium of bone homeostasis is maintained by bone-forming osteoblasts and bone-resorbing osteoclasts. Here, we show that TAM receptors MERTK and TYRO3 exert reciprocal effects in osteoblast biology: Osteoblast-targeted deletion of MERTK promotes increased bone mass in healthy mice and mice with cancer-induced bone loss, whereas knockout of TYRO3 in osteoblasts shows the opposite phenotype. Functionally, the interaction of MERTK with its ligand PROS1 negatively regulates osteoblast differentiation via inducing the VAV2-RHOA-ROCK axis leading to increased cell contractility and motility while TYRO3 antagonizes this effect. Consequently, pharmacologic MERTK blockade by the small molecule inhibitor R992 increases osteoblast numbers and bone formation in mice. Furthermore, R992 counteracts cancer-induced bone loss, reduces bone metastasis and prolongs survival in preclinical models of multiple myeloma, breast- and lung cancer. In summary, MERTK and TYRO3 represent potent regulators of bone homeostasis with cell-type specific functions and MERTK blockade represents an osteoanabolic therapy with implications in cancer and beyond.

Gene Therapy: Will the Promise of Optimizing Lung Allografts Become Reality?

Lung transplantation is the definitive therapy for patients living with end-stage lung disease. Despite significant progress made in the field, graft survival remains the lowest of all solid organ transplants. Additionally, the lung has among the lowest of organ utilization rates—among eligible donors, only 22% of lungs from multi-organ donors were transplanted in 2019. Novel strategies are needed to rehabilitate marginal organs and improve graft survival. Gene therapy is one promising strategy in optimizing donor allografts. Over-expression or inhibition of specific genes can be achieved to target various pathways of graft injury, including ischemic-reperfusion injuries, humoral or cellular rejection, and chronic lung allograft dysfunction. Experiments in animal models have historically utilized adenovirus-based vectors and the majority of literature in lung transplantation has focused on overexpression of IL-10. Although several strategies were shown to prevent rejection and prolong graft survival in preclinical models, none have led to clinical translation. The past decade has seen a renaissance in the field of gene therapy and two AAV-based in vivo gene therapies are now FDA-approved for clinical use. Concurrently, normothermic ex vivo machine perfusion technology has emerged as an alternative to traditional static cold storage. This preservation method keeps organs physiologically active during storage and thus potentially offers a platform for gene therapy. This review will explore the advantages and disadvantages of various gene therapy modalities, review various candidate genes implicated in various stages of allograft injury and summarize the recent efforts in optimizing donor lungs using gene therapy.