Oncolytic effect of SARS-CoV-2 in a patient with mycosis fungoides (MF): a case report

The most common variant of cutaneous T-cell lymphomas (CTCL) is mycosis fungoides (MF).The spontaneous regression (SR) of MF is rare. Here, we are reporting an interesting case of refractory MF after COVID-19. The SARS-CoV-2 could be an essential component in the improvement of clinical features related to MF.

Strategies to package recombinant Adeno-Associated Virus expressing the N-terminal gasdermin domain for tumor treatment

Pyroptosis induced by the N-terminal gasdermin domain (GSDMNT) holds great potential for anti-tumor therapy. However, due to the extreme cytoxicity of GSDMNT, it is challenging to efficiently produce and deliver GSDMNT into tumor cells. Here, we report the development of two strategies to package recombinant adeno-associated virus (rAAV) expressing GSDMNT: 1) drive the expression of GSDMNT by a mammal specific promoter and package the virus in Sf9 insect cells to avoid its expression; 2) co-infect rAAV-Cre to revert and express the double-floxed inverted GSDMNT. We demonstrate that these rAAVs can induce pyroptosis and prolong survival in preclinical cancer models. The oncolytic-viruses induce pyroptosis and evoke a robust immune-response. In a glioblastoma model, rAAVs temporarily open the blood-brain barrier and recruit tumor infiltrating lymphocytes into the brain. The oncolytic effect is further improved in combination with anti-PD-L1. Together, our strategies efficiently produce and deliver GSDMNT into tumor cells and successfully induce pyroptosis, which can be exploited for anti-tumor therapy.

745 Oncolytic adenovirus expressing 4-1BBL demonstrates a significant increase of survival in Diffuse Midline Glioma models

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. Neuro Oncol 2017;19(9):1279–1280.Grasso CS, Tang Y, Truffaux N, Berlow NE, Liu L, Debily MA, Quist MJ, Davis LE, Huang EC, Woo PJ, Ponnuswami A, Chen S, Johung TB, Sun W, Kogiso M, Du Y, Qi L, Huang Y, Hütt-Cabezas M, Warren KE, Le Dret L, Meltzer PS, Mao H, Quezado M, van Vuurden DG, Abraham J, Fouladi M, Svalina MN, Wang N, Hawkins C, Nazarian J, Alonso MM, Raabe EH, Hulleman E, Spellman PT, Li XN, Keller C, Pal R, Grill J, Monje M. Functionally defined therapeutic targets in diffuse intrinsic pontine glioma. Nat Med 2015;21(6):555–9.Martínez-Vélez N, Garcia-Moure M, Marigil M, González-Huarriz M, Puigdelloses M, Gallego Pérez-Larraya J, Zalacaín M, Marrodán L, Varela-Guruceaga M, Laspidea V, Aristu JJ, Ramos LI, Tejada-Solís S, Díez-Valle R, Jones C, Mackay A, Martínez-Climent JA, García-Barchino MJ, Raabe E, Monje M, Becher OJ, Junier MP, El-Habr EA, Chneiweiss H, Aldave G, Jiang H, Fueyo J, Patiño-García A, Gomez-Manzano C, Alonso MM. The oncolytic virus Delta-24-RGD elicits an antitumor effect in pediatric glioma and DIPG mouse models. Nat Commun 2019;10(1):2235.Garcia-Moure M, Gonzalez-Huarriz M, Labiano S, Guruceaga E, Bandres E, Zalacain M, Marrodan L, de Andrea C, Villalba M, Martinez-Velez N, Laspidea V, Puigdelloses M, Gallego Perez-Larraya J, Iñigo-Marco I, Stripecke R, Chan JA, Raabe EH, Kool M, Gomez-Manzano C, Fueyo J, Patiño-García A, Alonso MM. Delta-24-RGD, an oncolytic adenovirus, increases survival and promotes proinflamatory immune landscape remodeling in models of AT/RT and CNS-PNET. Clin Cancer Res 2021;27(6):1807–1820.Chester C, Sanmamed MF, Wang J, Melero I. Immunotherapy targeting 4-1BB: mechanistic rationale, clinical results, and future strategies. Blood 2018;131(1):49–57.Yonezawa A, Dutt S, Chester C, Kim J, Kohrt HE. Boosting cancer immunotherapy with anti-CD137 antibody therapy. Clin Cancer Res 2015;21(14):3113–20.Marigil M, Martinez-Velez N, Domínguez PD, Idoate MA, Xipell E, Patiño-García A, Gonzalez-Huarriz M, García-Moure M, Junier MP, Chneiweiss H, El-Habr E, Diez-Valle R, Tejada-Solís S, Alonso MM. Development of a DIPG orthotopic model in mice using an implantable guide-screw system. PLoS One 2017;12(1):e0170501.

IMMU-06. DELTA-24-RGD EXPRESSING POSITIVE IMMUNE MODULATORS SHOW ANTI-DIPG EFFECT AND INCREASE TUMOR IMMUNE INFILTRATION

Diffuse Intrinsic Pontine Gliomas (DIPG) are aggressive pediatric brain tumors that arise in the pons of children, being the leading cause of pediatric death caused by cancer. We have previously demonstrated that Delta-24-RGD administration is safe and efficacious in DIPG preclinical models, indicating that it could be a good candidate as therapeutic approach for DIPG. However, our data underscore that there is still room to improve the anti-DIPG effect obtained with Delta-24-RGD. For that purpose, we have constructed three new virus by engineering Delta-24-RGD with different T cell activators: 4-1BBL (Delta-24-ACT), OX40-L (Delta-24-RGDOX) and GITRL (Delta-24-GREAT), to further increase the immune response generated by the viral effect. In vitro, the three virus were able to infect murine and human DIPG cell lines, produce oncolytic effect in a dose-dependent manner and express the corresponding functional ligand (4-1BBL, OX40L or GITRL) in the membrane of infected cells (almost 100% of cells expressing them at 10 MOIs). As expected, viral replication was optimal in human cell lines but semipermissive in murine cells. In vivo, the intratumoral administration of armed oncolytic viruses was safe and significantly increased survival of mice bearing orthotopic DIPG murine tumors, leading to long-term survivors. In addition, we analyzed the effect of the virus in the tumor microenvironment by flow cytometry and immunohistochemistry, which indicated that there was a significant increase of immune infiltration in brains of treated mice. Moreover, the immune infiltrated showed a functional active phenotype. Although deeper characterization is needed, these data show that the incorporation of a positive immune modulator into Delta-24-RGD could improve the oncolytic effect of the virus by boosting the immune response, while maintaining a safe profile in immunocompetent models offering a feasible option treatment for DIPG.

Lonidamine potentiates the oncolytic efficiency of M1 virus independent of hexokinase 2 but via inhibition of antiviral immunity

Background Viruses are obligate parasites that depend on host cells to provide the energy and molecular precursors necessary for successful infection. The main component of virus-induced metabolic reprogramming is the activation of glycolysis, which provides biomolecular resources for viral replication. However, little is known about the crosstalk between oncolytic viruses and host glycolytic processes. Methods A MTT assay was used to detect M1 virus-induced cell killing. Flow cytometry was used to monitor infection of M1 virus expressing the GFP reporter gene. qPCR and western blotting were used to detect gene expression. RNA sequencing was performed to evaluate gene expression under different drug treatments. Scanning electron microscopy was performed to visualize the endoplasmic reticulum (ER). Caspase activity was detected. Last, a mouse xenograft model was established to evaluate the antitumor effect in vivo. Most data were analyzed with a two-tailed Student’s t test or one-way ANOVA with Dunnett’s test for pairwise comparisons. Tumor volumes were analyzed by repeated measures of ANOVA. The Wilcoxon signed-rank test was used to compare nonnormally distributed data. Results Here, we showed that the glucose analog 2-deoxy-d-glucose (2-DG) inhibited infection by M1 virus, which we identified as a novel type of oncolytic virus, and decreased its oncolytic effect, indicating the dependence of M1 replication on glycolysis. In contrast, lonidamine, a reported hexokinase 2 (HK2) inhibitor, enhanced the infection and oncolytic effect of M1 virus independent of HK2. Further transcriptomic analysis revealed that downregulation of the antiviral immune response contributes to the lonidamine-mediated potentiation of the infection and oncolytic effect of M1 virus, and that MYC is the key factor in the pool of antiviral immune response factors inhibited by lonidamine. Moreover, lonidamine potentiated the irreversible ER stress-mediated apoptosis induced by M1 virus. Enhancement of M1′s oncolytic effect by lonidamine was also identified in vivo. Conclusions This research demonstrated the dependence of M1 virus on glycolysis and identified a candidate synergist for M1 virotherapy.

Forskolin enhances the antitumor effect of oncolytic measles virus by promoting Rab27a dependent vesicular transport system

Background Measles vaccine strain viruses (MV-Edm) are an ideal platform for developing safe and effective oncolytic vectors. However, despite the promising pre-clinical data, understanding of determinants of efficacy and, thus, the interplay of the oncolytic virus with particular agents remains limited. Methods We investigated the potency of forskolin enhancing the antitumor effect of oncolytic measles virus by promoting Rab27a dependent vesicular transport system. Cells were infected with MV-Edm and the vesicles were observed by TEM. The oncolytic effects of MV-Edm/Forskolin were investigated in vitro. Results Here we demonstrate that the MV-Edm infection and spread in tumor, which are indispensable processes for the viral oncolysis, depend on the vesicular transport system of tumor cells. On the contrary, the tumor cells display a responsive mechanism to restrain the MV-Edm spread by down-regulating the expression of Rab27a, which is a key member of the vesicle transport system. Over-expression of Rab27a promotes the oncolytic efficacy of MV-Edm towards A549 tumor cells. Finally, we find a Rab27a agonist Forskolin, is capable of promoting the oncolytic effect of MV-Edm in vitro. Conclusions Our study reveals the important role of vesicle transporter Rab27a in the whole program of MV-Edm mediated oncolysis. We also provide a combined strategy of Forskolin and MV-Edm, which may exert a synergistic anti-tumor effect, for clinical treatment for patients with tumor.

Cytotoxic effect of unclassified group K rotaviruses on T98G and U87MG glioblastoma cells in vitro.

e15597 Background: Oncolytic virotherapy is developing intensively in modern oncology. Viruses demonstrate the ability to the direct oncolysis and to the stimulation of antitumor immune activity; this experiment was aimed at solving the question of the prevalence of one of them. Glial tumors are the most common brain tumors; oncolytic viruses show certain prospects in their treatment due to the ability to penetrate the blood-brain barrier. The aim of the study was to determine the possible oncolytic effect of new unclassified group K rotaviruses (RVK) on T98G and U87MG glioblastoma cells in vitro. Methods: T98G and U87MG cell cultures were received from Russian banks of cell lines of human and animal tissues. Standard culturing was performed with attenuated apatogenic RVK strains No. 100 and No. 228 at a concentration of 108, 107, 106 and 105 particles/mL. The cytotoxic effect was determined with MTT and Annexin V assays, cell morphology was evaluated by the light-optical method. Results: Both RVK strains demonstrated a dose-dependent cytotoxic activity; the maximal effect was observed in strain No.100 at a dose of 108 particles/mL on U87MG cells (predominantly apoptosis). Studies of cell morphology showed a pronounced effect of RVK on the cell culture: significant degenerative changes in cells, a tendency to a decrease in cluster size, a change in their shape and granularity. Cluster formation in culturing in the serum-free medium is considered in the literature as a property of cancer stem cells responsible in vivo for tumor recurrence and its chemo- and radio-resistance. T98G cells demonstrated morphological changes: nuclear segmentation, diffused cytoplasm, indistinct cell borders with signs of syncytium formation. Conclusions: The established oncolytic effect of RVK strain No. 100 in vitro on glioma cells, presumably with tumor stem cells, indicates a significant potential for the use of these rotaviruses in treatment of glial tumors.