Gene expression profiling identifies IL-13 receptor alpha 2 chain as a therapeutic target in prostate tumor cells overexpressing adrenomedullin

Human adrenomedullin (AM) is a 52 amino acid peptide, which shares homology with the calcitonin gene-related peptide. Overexpression of AM in the prostate carcinoma cell line PC-3 results in growth inhibition with a 20% (for human AM) and 35% (for rat AM) increase in doubling time compared to parental or mock-transfected cells. We demonstrate by gene expression profiling that AM overexpression results in the dysregulation of approximately 100 genes. Examples of such genes include many involved in the formation of the cytoskeleton, cell adhesion and the extracellular matrix, as well as regulators of the cell cycle and apoptosis, cytokines and transcription factors. Several genes related to cell growth arrest, such as GADD45, IGF-BP6 and RUNX-3, are upregulated by AM. Interestingly, interleukin-13 receptor alpha 2 (IL-13R alpha 2) transcripts were significantly increased in clones overexpressing AM, which was confirmed by semiquantitative RT-PCR analysis. In addition, PC-3 cells treated with AM showed an overexpression of IL-13R alpha 2, which was abolished when cells were preincubated with an anti-AM blocking antibody. When PC-3 cells overexpressing AM and the IL-13R alpha 2 were treated with the highly specific IL13-PE38 cytotoxin, which binds to this receptor, a concentration-dependent inhibition of protein synthesis was observed. The IC(50) (concentration of cytotoxin inhibiting protein synthesis by 50%) ranged from 1 to 4 ng/ml. This cytotoxicity was specific as it was neutralized by the excess of IL-13 and confirmed by clonogenic assays. This study describes a novel AM-induced mechanism of tumor sensitization through the upregulation of functional IL-13R alpha 2 chain, an ideal target for the highly specific recombinant chimeric cytotoxin IL13-PE38

Integration of Machine Learning and Mechanistic Models Accurately Predicts Variation in Cell Density of Glioblastoma Using Multiparametric MRI

Glioblastoma (GBM) is a heterogeneous and lethal brain cancer. These tumors are followed using magnetic resonance imaging (MRI), which is unable to precisely identify tumor cell invasion, impairing effective surgery and radiation planning. We present a novel hybrid model, based on multiparametric intensities, which combines machine learning (ML) with a mechanistic model of tumor growth to provide spatially resolved tumor cell density predictions. The ML component is an imaging data-driven graph-based semi-supervised learning model and we use the Proliferation-Invasion (PI) mechanistic tumor growth model. We thus refer to the hybrid model as the ML-PI model. The hybrid model was trained using 82 image-localized biopsies from 18 primary GBM patients with pre-operative MRI using a leave-one-patient-out cross validation framework. A Relief algorithm was developed to quantify relative contributions from the data sources. The ML-PI model statistically significantly outperformed (p \u3c 0.001) both individual models, ML and PI, achieving a mean absolute predicted error (MAPE) of 0.106 ± 0.125 versus 0.199 ± 0.186 (ML) and 0.227 ± 0.215 (PI), respectively. Associated Pearson correlation coefficients for ML-PI, ML, and PI were 0.838, 0.518, and 0.437, respectively. The Relief algorithm showed the PI model had the greatest contribution to the result, emphasizing the importance of the hybrid model in achieving the high accuracy

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Identification of a novel role of IL-13Rα2 in human Glioblastoma multiforme: interleukin-13 mediates signal transduction through AP-1 pathway

Abstract Background Previously, we have demonstrated that Interleukin 13 receptor alpha 2 (IL-13Rα2) is overexpressed in approximate 78% Glioblastoma multiforme (GBM) samples. We have also demonstrated that IL-13Rα2 can serve as a target for cancer immunotherapy in several pre-clinical and clinical studies. However, the significance of overexpression of IL-13Rα2 in GBM and astrocytoma and signaling through these receptors is not known. IL-13 can signal through IL-13R via JAK/STAT and AP-1 pathways in certain cell lines including some tumor cell lines. Herein, we have investigated a role of IL-13/IL-13Rα2 axis in signaling through AP-1 transcription factors in human glioma samples in situ. Methods We examined the activation of AP-1 family of transcription factors (c-Jun, Fra-1, Jun-D, c-Fos, and Jun-B) after treating U251, A172 (IL-13Rα2 +ve) and T98G (IL-13Rα2 −ve) glioma cell lines with IL-13 by RT-qPCR, and immunocytochemistry (ICC). We also performed colorimetric ELISA based assay to determine AP-1 transcription factor activation in glioma cell lines. Furthermore, we examined the expression of AP-1 transcription factors in situ in GBM and astrocytoma specimens by multiplex-immunohistochemistry (IHC). Student t test and ANOVA were used for statistical analysis of the results. Results We have demonstrated up-regulation of two AP-1 transcription factors (c-Jun and Fra-1) at mRNA and protein levels upon treatment with IL-13 in IL-13Rα2 positive but not in IL-13Rα2 negative glioma cell lines. Both transcription factors were also overexpressed in patient derived GBM specimens, however, in contrast to GBM cell lines, c-Fos is also overexpressed in patient derived specimens. Astrocytoma specimens showed lesser extent of immunostaining for IL-13Rα2 and three AP-1 factors compared to GBM specimens. By transcription factor activation assay, we demonstrated that AP-1 transcription factors (C-Jun and Fra-1) were activated upon treatment of IL-13Rα2 + GBM cell lines but not IL-13Rα2 − GBM cell line with IL-13. Our results demonstrate functional activity of AP-1 transcription factor in GBM cell lines in response to IL-13. Conclusions These results indicate that IL-13/IL-13Rα2 axis can mediate signal transduction in situ via AP-1 pathway in GBM and astrocytoma and may serve as a new target for GBM immunotherapy

Hodgkin lymphoma therapy with interleukin-4 receptor–directed cytotoxin in an infiltrating animal model

Hodgkin lymphoma represents unique clinicopathologic features because Hodgkin and Reed-Sternberg (H-RS) cells produce a variety of cytokines, express a variety of cytokine receptors, and are surrounded by numerous nonmalignant immunoreactive cells. We found that receptors for interleukin-4 (IL-4R) are highly expressed in H-RS cells. To target interleukin-4 receptor (IL-4R), we used a recombinant protein fusing circularly permuted human IL-4 and Pseudomonas exotoxin termed IL438-37-PE38KDEL, or IL-4 cytotoxin. The cytotoxic effect of IL-4 cytotoxin on H-RS cell lines was determined to be moderate to high in vitro. We developed an infiltrating model of Hodgkin disease (HD) by injecting an adherent population of HD-MyZ cells subcutaneously into the flanks of beige/nude/X-linked immunodeficient mice. The animal model exhibited spontaneous metastasis of H-RS cells to lymph nodes and dissemination to vital organs, including the lungs. Intraperitoneal or intratumoral treatment of these mice with IL-4 cytotoxin resulted in regression of the primary tumor mass and a decrease in the incidence of lymph node metastasis. Mice injected with HD-MyZ cells demonstrated 203% prolonged survival (mean survival, 63 days) compared with control (mean survival, 31 days) when they received systemic IL-4 cytotoxin treatment. Because numerous H-RS cell lines express receptors for IL-4, IL-4 cytotoxin may be a unique agent for the treatment of Hodgkin lymphoma