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The in-frame internal tandem duplication (ITD) of the FLT3 gene is an important negative prognostic factor in acute myeloid leukemia (AML). FLT3-ITD is constitutive active and partially retained in the endoplasmic reticulum (ER). Recent reports show that 3′UTRs function as scaffolds that can regulate the localization of plasma membrane proteins by recruiting the HuR-interacting protein SET to the site of translation. Therefore, we hypothesized that SET could mediate the FLT3 membrane location and that the FLT3-ITD mutation could somehow disrupt the model, impairing its membrane translocation. Immunofluorescence and immunoprecipitation assays demonstrated that SET and FLT3 co-localize and interact in FLT3-WT cells but hardly in FLT3-ITD. SET/FLT3 interaction occurs before FLT3 glycosylation. Furthermore, RNA immunoprecipitation in FLT3-WT cells confirmed that this interaction occurs through the binding of HuR to the 3′UTR of FLT3. HuR inhibition and SET nuclear retention reduced FLT3 in the membrane of FLT3-WT cells, indicating that both proteins are involved in FLT3 membrane trafficking. Interestingly, the FLT3 inhibitor midostaurin increases FLT3 in the membrane and SET/FLT3 binding. Therefore, our results show that SET is involved in the transport of FLT3-WT to the membrane; however, SET barely binds FLT3 in FLT3-ITD cells, contributing to its retention in the ER.

Purpose of Study: Diffuse Midline Gliomas (DMG) are aggressive pediatric brain tumors that arise in the brainstem of children, with a peak of incidence between 5-10 years old. The median survival of DMG patients is only 9 months, being the leading cause of pediatric death caused by a brain tumor. On this project we set to characterize the efficacy of Delta-24-RGDOX, an oncolytic adenovirus based on Delta-24-RGD platform, which has demonstrated safety and a therapeutic benefit in different pediatric tumors, armed with the ligand of OX40. The binding of OX40 to OX40L leads to the co-stimulation of CD4 and CD8 cells, generating effector and memory T cells. Therefore, the aim of this project is to improve the antitumor effect of the virus, providing a greater co-stimulation in the tumor. Experimental Procedures: Murine and human DMG cell lines were used. Viral protein expression was measured by western blot, viral replication was analyzed using a method based on hexon detection and the oncolytic effect by MTS assay. OX40L expression was measured by flow cytometry and qPCR. For in vivo experiments, cells were injected in the pons of mice using a screw-guided system. The adenovirus was administered once into the tumor using the same procedure. Tumor immune populations were analyzed by flow cytometry. Results: We first confirmed the oncolytic effect of Delta-24-RGDOX in DMG murine and human cell lines in vitro. The virus was able to infect the cells, produce viral proteins, and to cause cell death in a dose-dependent manner. In addition, we observed effective viral replication in human cell lines but not in mouse cells, as previously described. We then measured OX40L expression in infected cells both at protein and mRNA level, observing that at 50 PFU/cell almost 100% of cells expressed the ligand on their membrane. More importantly, the OX40L was functional as it activated CD8 lymphocytes in vitro. Once we confirmed that the viral administration in vivo was safe, we assess the efficacy of the virus in murine DMG models. We observed a significant survival benefit in mice bearing NP53 tumor treated with Delta-24-RGDOX, which lead to 30% of long-term survivors (P=0.003, median OS PBS 25.5 days vs 35.5 days for treated mice). More importantly, we administered the virus in an already stablished tumor model using XFM cells and also obtained a significant improve of survival (P=0.018, median OS PBS 9 days vs 12.5 treated mice). Finally, we analyzed the immune mechanisms underlying the survival benefit, both in the tumor and in spleen. We observed a significant increase of activated immune cells in the tumor microenvironment 7 days after the viral administration. In addition, splenocytes from virus-treated mice were also significantly more activated. Conclusions: These data show that Delta-24-RGDOX adenovirus expresses a functional OX40L that can modulate the immune response, leading to a significantly improved survival outcome in DMG models. Citation Format: Virginia Laspidea, Sara Labiano, Sumit Gupta, Hong Jiang, Iker Ausejo-Mauleon, Daniel de la Nava, Marc García-Moure, Javier Marco-Sanz, Reyes Hernández-Osuna, Oren J Becher, Juan Fueyo, Ana Patiño-García, Candelaria Gomez-Manzano, Marta M Alonso. Delta-24-RGDOX oncolytic adenovirus improves the survival by modulating the immune system in DMG models [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 2001.

Purpose of the work: Pediatric High Grade Gliomas (pHGGs), including Diffuse Midline Gliomas (DMGs), are very aggressive solid tumors developed during childhood with a poor overall survival underscoring the need for effective treatments. The adenovirus Delta-24RGD and the imipridone ONC201 have demonstrated safety and effectiveness in preclinical models and clinical trials. Thus, we evaluated the therapeutic efficacy of the Delta-24-RGD/ONC201 combination and analyzed possible changes in the tumor microenvironment in preclinical models of pHGGs and DMGs. Experimental procedures: A battery of pHGGs (SF188 and CHLA-03-AA), human DMG (SU-DIPG IV), and murine DMG cell lines (XFM and NP-53) were used. We assessed the viral replication by hexon titration, the protein expression amount (E1A, fiber, and mTORC1 key proteins)by immunoblotting, and oxygen consumption by Seahorse Analyses. Viability was measured by MTS. CHLA-03-AA and XFM were engrafted orthotopically in athymic nude mice and Balb/c mice, respectively. To assess in vivo efficacy, the animals were treated with Delta-24-RGD (107 PFU/animal) or PBS, followed by ONC201administration (125 mg/kg/twice/weekly). The tumor immune microenvironment was evaluated by flow cytometry. Results: First, the potential adverse interactions between both agents were checked in vitro, showing that ONC201 cotreatment did not interfere with the Delta-24-RGD replication capacity. As expected, ONC201 treatment led to a reduction in oxygen consumption, which was also observed in the combination treatment. Evaluation of cytotoxicity in a panel of cell lines showed that the combination was significantly better than either agent alone. In vivo, CHLA-03-AA bearing mice treated with the combination showed a significant increase in the median overall survival (PBS: 48 days; ONC201: 54.5 days; Delta-24-RGD: 62 days; Delta-24-RGD/ONC201: 95 days (P=0.0008)), leading to 20%long-term survivors, free of disease. Evaluation of the combination in an immunocompetent orthotopic DMG model also revealed the superior efficacy of the Delta-24-RGD/ONC201 (PBS: 11 days; ONC201: 12 days; Delta-24-RGD: 12 days; ONC201+Delta-24-RGD: 15 days (P=0.02)). Of importance, the combination treatment resulted in the reshaping of the tumor microenvironment towards a proinflammatory phenotype (T cells, NK cells, macrophages, granulocytes, monocytes, dendritic cells, and microglia). Conclusions: Our data indicate that the combination Delta-24-RGD/ONC201 generates a pro-inflammatory microenvironment that ultimately leads to a superior therapeutic effect in preclinical pHGG and DMGs models. Citation Format: Daniel de la Nava, Javier Marco-Sanz, Virginia Laspidea, Sara Labiano, Iker Ausejo-Mauleon, Reyes Hernandez-Osuna, Sabine Mueller, Javad Nazarian, Joshua E. Allen, Oren Becher, Juan Fueyo, Candelaria Gomez-Manzano, Ana Patiño-Garcia, Marta M. Alonso. The adenovirus Delta-24-RGD in combination with ONC201 provides a therapeutic benefit and a proinflammatory recruitment in preclinical models of pHGGs and DMGs [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 2000.

BackgroundDiffuse Midline Gliomas (DMG) are aggressive pediatric brain tumors that arise in the brainstem of children between 5–10 years old. DMGs are the leading cause of pediatric death caused by a brain tumor, with a median survival of only 9 months.1 2 We have previously shown that the administration of the oncolytic adenovirus Delta-24-RGD is safe and lead to an increase in long-term survivors in murine models.3 4 In order to further increase the antitumor effect of Delta-24-RGD by boosting the immune response, we have constructed a new adenovirus, Delta-24-ACT, which incorporates the 4-1BBL (CD137L) into its backbone. 4-1BB is a costimulatory receptor that promotes the survival and expansion of activated T cells and NK cells and the generation and maintenance of memory CD8+ T cells, among other functions.5 6MethodsMurine and human DMG cell lines were used. 4-1BBL expression was assessed in infected cells by flow cytometry and immunofluorescence. Viral protein expression was measured by western blot, viral replication was analyzed using a method based on hexon detection and the oncolytic effect by MTS assay. For in vivo experiments, cells were injected in the pons of mice using a screw-guided system.7 A single administration of the adenovirus was injected intratumorally using the same procedure. The tumor immune populations were analyzed by flow cytometry.ResultsWe first confirmed by flow cytometry that DMG cells infected with Delta-24-ACT expressed 4-1BBL in their membrane in a dose-dependent manner. Afterwards, we analyzed the oncolytic effect of Delta-24-ACT in vitro. Delta-24-ACT was able to express viral early and late proteins in murine and human DMG cell lines and to replicate efficiently in human cells. In addition, the virus caused cell death in a dose-dependent manner. In vivo, Delta-24-ACT administration demonstrated to be safe and to produce a significant survival benefit in murine DMG models, obtaining 30–50% of long-term survivors depending on the model. More importantly, Delta-24-ACT generated immune memory, as long-term survivors were disease-free after cell rechallenge. On the other hand, we analyzed immune infiltration 7 or 10 days after the viral administration into the tumor and observed a significant increase of tumor infiltration in treated mice, which showed an activated state.ConclusionsDelta-24-ACT administration into DMG murine tumor models significantly increases the recruitment and activation of immune cells, which leads to long term survivors and immunological memory.ReferencesCooney T, Lane A, Bartels U, Bouffet E, Goldman S, Leary S, Foreman NK, Packer RJ, Broniscer A, Minturn JE, Shih C, Chintagumpala M, Hassall T, Gottardo NG, Dholaria H, Hoffman L, Chaney B, Baugh J, Doughman R, Leach JL, Jones BV, Fouladi M, Warren KE, Monje M. Contemporary survival endpoints: an International diffuse Intrinsic pontine glioma registry study. 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