The Oncolytic Adenovirus XVir-N-31 Joins Forces with CDK4/6 Inhibition Augmenting Innate and Adaptive Antitumor Immunity in Ewing Sarcoma.

PURPOSE: Ewing sarcoma (EwS) is a highly malignant pediatric tumor characterized by a non-T-cell-inflamed immune-evasive phenotype. When relapsed or metastasized, survival is poor, emphasizing the need for novel treatment strategies. Here, we analyze the novel combination approach using the YB-1-driven oncolytic adenovirus XVir-N-31 and CDK4/6 inhibition to augment EwS immunogenicity. EXPERIMENTAL DESIGN: In vitro, viral toxicity, replication, and immunogenicity were studied in several EwS cell lines. In vivo tumor xenograft models with transient humanization were applied to evaluate tumor control, viral replication, immunogenicity, and dynamics of innate as well as human T cells after treatment with XVir-N-31 combined with CDK4/6 inhibition. Furthermore, immunologic features of dendritic cell maturation and T-cell-stimulating capacities were assessed. RESULTS: The combination approach significantly increased viral replication and oncolysis in vitro, induced HLA-I upregulation, and IFNγ-induced protein 10 expression and enhanced maturation of monocytic dendritic cells with superior capacities to stimulate tumor antigen-specific T cells. These findings were confirmed in vivo showing tumor infiltration by (i) monocytes with antigen-presenting capacities and M1 macrophage marker genes, (ii) TReg suppression in spite of adenovirus infection, (iii) superior engraftment, and (iv) tumor infiltration by human T cells. Consequently, survival was improved over controls with signs of an abscopal effect after combination treatment. CONCLUSIONS: The joint forces of the YB-1-driven oncolytic adenovirus XVir-N-31 and CDK4/6 inhibition induce therapeutically relevant local and systemic antitumor effects. Innate as well as adaptive immunity against EwS is boosted in this preclinical setting, pointing toward high therapeutic potential in the clinic.

No Improvement of Survival for Alveolar Rhabdomyosarcoma Patients After HLA-Matched Versus -Mismatched Allogeneic Hematopoietic Stem Cell Transplantation Compared to Standard-of-Care Therapy.

Background: Patients with stage IV alveolar rhabdomyosarcoma (RMA) have a 5-year-survival rate not exceeding 30%. Here, we assess the role of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for these patients in comparison to standard-of-care regimens. We also compare the use of HLA-mismatched vs. HLA-matched grafts after reduced vs. myeloablative conditioning regimens, respectively. Patients and Methods: In this retrospective analysis, we compare event-free survival (EFS), overall survival (OS), and toxicity of HLA-mismatched vs. -matched transplanted patients in uni- and multivariate analyses (total: n = 50, HLA-matched: n = 15, HLA-mismatched: n = 35). Here, the factors age at diagnosis, age at allo-HSCT, sex, Oberlin score, disease status at allo-HSCT, and HLA graft type are assessed. For 29 primarily transplanted patients, three matched non-transplanted patients per one transplanted patient were identified from the CWS registry. Outcomes were respectively compared for OS and EFS. Matching criteria included sex, age at diagnosis, favorable/unfavorable primary tumor site, and metastatic sites. Results: Median EFS and OS did not differ significantly between HLA-mismatched and -matched patients. In the mismatched group, incidence of acute GvHD was 0.87 (grade III-IV: 0.14) vs. 0.80 in HLA-matched patients (grade III-IV: 0.20). Transplant-related mortality (TRM) of all patients was 0.20 and did not differ significantly between HLA-mismatched and -matched groups. A proportion of 0.58 relapsed or progressed and died of disease (HLA-mismatched: 0.66, HLA-matched: 0.53) whereas 0.18 were alive in complete remission (CR) at data collection. Multivariate and competing risk analyses confirmed CR and very good partial response (VGPR) status prior to allo-HSCT as the only decisive predictor for OS (p < 0.001). Matched-pair survival analyses of primarily transplanted patients vs. matched non-transplanted patients also identified disease status prior to allo-HSCT (CR, VGPR) as the only significant predictor for EFS. Here, OS was not affected, however. Conclusion: In this retrospective analysis, only a subgroup of patients with good response at allo-HSCT survived. There was no survival benefit of allo-transplanted patients compared to matched controls, suggesting the absence of a clinically relevant graft-versus-RMA effect in the current setting. The results of this analysis do not support further implementation of allo-HSCT in RMA stage IV patients.

Tumor Targeting with Bacterial Shiga Toxin B Subunit in Genetic Porcine Models for Colorectal Cancer and Osteosarcoma.

The B subunit of bacterial Shiga toxin (STxB) is nontoxic and has low immunogenicity. Its receptor, the glycosphingolipid Gb3/CD77, is overexpressed on the cell surface of human colorectal cancer. We tested whether genetic porcine models, closely resembling human anatomy and pathophysiology, can be used to exploit the tumor-targeting potential of STxB. In accordance with findings on human colorectal cancer, the pig model APC1311 bound STxB in colorectal tumors, but not in normal colon or jejunum, except for putative enteroendocrine cells. In primary tumor cells from endoscopic biopsies, STxB was rapidly taken up along the retrograde intracellular route to the Golgi, whereas normal colon organoids did not bind or internalize STxB. Next, we tested a porcine model (TP53LSL-R167H) for osteosarcoma, a tumor entity with a dismal prognosis and insufficient treatment options, hitherto not known to express Gb3. Pig osteosarcoma strongly bound StxB and expressed the Gb3 synthase 1,4-galactosyltransferase (A4GALT). Primary osteosarcoma cells, but not normal osteoblasts, rapidly internalized fluorescently labeled STxB along the retrograde route to the Golgi. Importantly, six of eight human osteosarcoma cell lines expressed A4GALT mRNA and showed prominent intracellular uptake of STxB. The physiologic role of A4GALT was tested by CRISPR/Cas9 mutagenesis in porcine LLC-PK1 kidney epithelial cells and RNAi in MG-63 human osteosarcoma cells. A4GALT deficiency or knockdown abolished STxB uptake and led to significantly reduced cell migration and proliferation, hinting toward a putative tumor-promoting role of Gb3. Thus, pig models are suitable tools for STxB-based tumor targeting and may allow "reverse-translational" predictions on human tumor biology.