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H1299 cells stably co-transfected with KillerRed- and EGFP-expressing vectors (H1299-KillerRed-EGFP) were irradiated for 12 hours. Cells showed morphological changes following photobleaching and started to die, which could be visualized with GFP expression.

PDF file - 250K, The structures of OBP-301 and OBP-702

Supplementary Fig. S1: Schematic diagrams of the structures of replication-deficient and conditionally; Supplementary Fig. S2: The GFP fluorescence intensity of metastatic lymph nodes in HCT116-GFP-bearing mice after treatment with TelomKiller and laser irradiation. replicating adenovirus vectors; Supplementary Fig. S2: The GFP fluorescence intensity of metastatic lymph nodes in HCT116-GFP-bearing mice after treatment with TelomKiller and laser irradiation.

PDF file - 334K, The ID50 values of OBP-702 and Ad-p53 in SaOS-2 and MNNG/HOS cells

PDF file - 139K, Expression of endogenous p53 protein in SaOS-2 and MNNG/HOS cells

Tumor suppressor p53 is a multifunctional transcription factor that regulates diverse cell fates, including apoptosis and autophagy in tumor biology. p53 overexpression enhances the antitumor activity of oncolytic adenoviruses; however, the molecular mechanism of this occurrence remains unclear. We previously developed a tumor-specific replication-competent oncolytic adenovirus, OBP-301, that kills human osteosarcoma cells, but some human osteosarcoma cells were OBP-301–resistant. In this study, we investigated the antitumor activity of a p53-expressing oncolytic adenovirus, OBP-702, and the molecular mechanism of the p53-mediated cell death pathway in OBP-301–resistant human osteosarcoma cells. The cytopathic activity of OBP-702 was examined in OBP-301–sensitive (U2OS and HOS) and OBP-301–resistant (SaOS-2 and MNNG/HOS) human osteosarcoma cells. The molecular mechanism in the OBP-702–mediated induction of two cell death pathways, apoptosis and autophagy, was investigated in OBP-301–resistant osteosarcoma cells. The antitumor effect of OBP-702 was further assessed using an orthotopic OBP-301–resistant MNNG/HOS osteosarcoma xenograft tumor model. OBP-702 suppressed the viability of OBP-301–sensitive and -resistant osteosarcoma cells more efficiently than OBP-301 or a replication-deficient p53-expressing adenovirus (Ad-p53). OBP-702 induced more profound apoptosis and autophagy when compared with OBP-301 or Ad-p53. E1A-mediated miR-93/106b upregulation induced p21 suppression, leading to p53-mediated apoptosis and autophagy in OBP-702–infected cells. p53 overexpression enhanced adenovirus-mediated autophagy through activation of damage-regulated autophagy modulator (DRAM). Moreover, OBP-702 suppressed tumor growth in an orthotopic OBP-301–resistant MNNG/HOS xenograft tumor model. These results suggest that OBP-702–mediated p53 transactivation is a promising antitumor strategy to induce dual apoptotic and autophagic cell death pathways via regulation of miRNA and DRAM in human osteosarcoma cells. Mol Cancer Ther; 12(3); 314–25. ©2012 AACR.

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PDF file - 213K, Quantitative measurement of viral DNA replication in SaOS-2 cells infected with OBP-702 or OBP-301

PDF file - 852K, Immunohistochemistry of MNNG/HOS tumors

The epithelial-mesenchymal transition (EMT), a normal biological process by which epithelial cells acquire a mesenchymal phenotype, is associated with migration, metastasis, and chemoresistance in cancer cells, and with poor prognosis in patients with esophageal cancer. However, therapeutic strategies to inhibit EMT in tumor environments remain elusive. Here, we show the therapeutic potential of telomerase-specific replication- competent oncolytic adenovirus OBP-301 in human esophageal cancer TE4 and TE6 cells with an EMT phenotype. Transforming growth factor-β (TGF-β) administration induced the EMT phenotype with spindleshaped morphology, upregulation of mesenchymal markers and EMT transcription factors, migration, and chemoresistance in TE4 and TE6 cells. OBP-301 significantly inhibited the EMT phenotype via E1 accumulation. EMT cancer cells were susceptible to OBP-301 via massive autophagy induction. OBP-301 suppressed tumor growth and lymph node metastasis of TE4 cells co-inoculated with TGF-β-secreting fibroblasts. Our results suggest that OBP-301 inhibits the TGF-β-induced EMT phenotype in human esophageal cancer cells. OBP-301-mediated E1A overexpression is a promising antitumor strategy to inhibit EMT-mediated esophageal cancer progression.

Supplementary Figure Legends 1-4 from Preclinical Evaluation of Differentially Targeting Dual Virotherapy for Human Solid Cancer

Figure S5 shows heat maps from brain immune cells.

Supplementary Fig. S3 from Preclinical evaluation of synergistic effect of telomerase-specific oncolytic virotherapy and gemcitabine for human lung cancer

Table S1 shows KINOMEscan profiling of WP1066.

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Supplementary Table from HER3 Augmentation via Blockade of EGFR/AKT Signaling Enhances Anticancer Activity of HER3-Targeting Patritumab Deruxtecan in EGFR-Mutated Non–Small Cell Lung Cancer

Supplementary Figure from HER3 Augmentation via Blockade of EGFR/AKT Signaling Enhances Anticancer Activity of HER3-Targeting Patritumab Deruxtecan in EGFR-Mutated Non–Small Cell Lung Cancer

Supplementary Movie S1- MOV file 6383K, Time-lapse imaging of FUCCI-expressing CD133+ cells treated with OBP-301 or control on monolayer culture. Images were acquired every 20 min. The playback speed is 10, 800 x real time. Total imaging time = 48 hours. The cells in G0/ G1, S, or G2/M phases appear red, yellow, or green, respectively

Growth inhibition activity in 8 cell lines with different HER3 expression levels

Supplementary Figure legends

Supplementary Figure 3 from Preclinical Evaluation of Differentially Targeting Dual Virotherapy for Human Solid Cancer

Supplementary Figure 4 from Preclinical Evaluation of Differentially Targeting Dual Virotherapy for Human Solid Cancer

Cell surface expression of HER3 and HER2 on human cancer cell lines

Purpose:HER3 is a compelling target for cancer treatment; however, no HER3-targeted therapy is currently clinically available. Here, we produced U3-1402, an anti-HER3 antibody–drug conjugate with a topoisomerase I inhibitor exatecan derivative (DXd), and systematically investigated its targeted drug delivery potential and antitumor activity in preclinical models.Experimental Design:In vitro pharmacologic activities and the mechanisms of action of U3-1402 were assessed in several human cancer cell lines. Antitumor activity of U3-1402 was evaluated in xenograft mouse models, including patient-derived xenograft (PDX) models. Safety assessments were also conducted in rats and monkeys.Results:U3-1402 showed HER3-specific binding followed by highly efficient cancer cell internalization. Subsequently, U3-1402 was translocated to the lysosome and released its payload DXd. While U3-1402 was able to inhibit HER3-activated signaling similar to its naked antibody patritumab, the cytotoxic activity of U3-1402 in HER3-expressing cells was predominantly mediated by released DXd through DNA damage and apoptosis induction. In xenograft mouse models, U3-1402 exhibited dose-dependent and HER3-dependent antitumor activity. Furthermore, U3-1402 exerted potent antitumor activity against PDX tumors with HER3 expression. Acceptable toxicity was noted in both rats and monkeys.Conclusions:U3-1402 demonstrated promising antitumor activity against HER3-expressing tumors with tolerable safety profiles. The activity of U3-1402 was driven by HER3-mediated payload delivery via high internalization into tumor cells.

Summary of repeated dose toxicity studies in rats and monkeys

PDF file 1712K, Supplementary Methods Fig. S1. OBP-301 efficiently kills CD133+ cells from MKN7 cells resistant to conventional therapies. Fig. S2. CD133+ CS-like cells can produce CD133+ CS-like cells and CD133- non-CS-like cells. Fig. S3. CD133+ MKN7 cells produce larger colonies than CD133- MKN7 cells in vitro. Fig. S4. CD133+ cells have higher tumorigenicity in nude mice than CD133− cells. Fig. S5. Flow cytometry analysis shows that OBP-301 induces a significant increase in the percentage of both CD133+ CS-like and CD133- non-CS-like cells in S phase. Fig. S6. Expression of CD133 mRNA is associated with the population of CD133+ cells. Fig. S7. OBP-301 reduces the expression of CD133 mRNA. Fig. S8. CD133+ cells show higher expressions of p53, p21 and p27 proteins compared with CD133− cells. Fig. S9. Adenoviral E1A alters cell-cycle related proteins. Fig. S10. Experimental setup in vivo. Fig. S11. Experimental setup for combination therapy of OBP-301 plus cisplatin or paclitaxel in vitro. Fig. S12. The combination therapy of OBP-301 and chemotherapy is more effective than OBP-301 monotherapy on monolayer culture. Fig. S13. OBP-301 reduces the potentiality of stemness in CD133 positive cells. Fig. S14. The combination therapy of OBP-301 and chemotherapy is more effective against larger tumors. Fig. S15. A scheme for oncolytic adenovirus killing quiescent human cancer cells

Supplementary materials and methods for xenograft studies and IHC

Purpose:Patients with central nervous system (CNS) tumors are typically treated with radiotherapy, but this is not curative and results in the upregulation of phosphorylated STAT3 (p-STAT3), which drives invasion, angiogenesis, and immune suppression. Therefore, we investigated the combined effect of an inhibitor of STAT3 and whole-brain radiotherapy (WBRT) in a murine model of glioma.Experimental Design:C57BL/6 mice underwent intracerebral implantation of GL261 glioma cells, WBRT, and treatment with WP1066, a blood–brain barrier–penetrant inhibitor of the STAT3 pathway, or the two in combination. The role of the immune system was evaluated using tumor rechallenge strategies, immune-incompetent backgrounds, immunofluorescence, immune phenotyping of tumor-infiltrating immune cells (via flow cytometry), and NanoString gene expression analysis of 770 immune-related genes from immune cells, including those directly isolated from the tumor microenvironment.Results:The combination of WP1066 and WBRT resulted in long-term survivors and enhanced median survival time relative to monotherapy in the GL261 glioma model (combination vs. control P < 0.0001). Immunologic memory appeared to be induced, because mice were protected during subsequent tumor rechallenge. The therapeutic effect of the combination was completely lost in immune-incompetent animals. NanoString analysis and immunofluorescence revealed immunologic reprograming in the CNS tumor microenvironment specifically affecting dendritic cell antigen presentation and T-cell effector functions.Conclusions:This study indicates that the combination of STAT3 inhibition and WBRT enhances the therapeutic effect against gliomas in the CNS by inducing dendritic cell and T-cell interactions in the CNS tumor.

A phase I dose-escalation study of telomerase-specific oncolytic adenovirus, OBP-301 (Telomelysin), is now under way in the United States to assess feasibility and to characterize its pharmacokinetics in patients with advanced solid tumors. The present preclinical study investigates whether OBP-301 and a chemotherapeutic agent that is commonly used for lung cancer treatment, gemcitabine, are able to enhance antitumor effects in vitro and in vivo. The antitumor effects of OBP-301 infection and gemcitabine were evaluated by 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt assay. In vivo antitumor effects of intratumoral injection of OBP-301 in combination with systemic administration of gemcitabine were assessed on nu/nu mice s.c. xenografted with human lung tumors. OBP-301 infection combined with gemcitabine resulted in very potent synergistic cytotoxicity in human lung cancer cells. The three human lung cancer cell lines treated with OBP-301 for 24 hours tended to accumulate in S phase compared with controls. The proportion of cells in S phase increased from 43.85% to 56.41% in H460 cells, from 46.72% to 67.09% in H322 cells, and from 38.22% to 57.67% in H358 cells. Intratumoral injection of OBP-301 combined with systemic administration of gemcitabine showed therapeutic synergism in human lung tumor xenografts. Our data suggest that the combination of OBP-301 and gemcitabine enhances the antitumor effects against human lung cancer. We also found that the synergistic mechanism may be due to OBP-301–mediated cell cycle accumulation in S phase. These results have important implications for the treatment of human lung cancer.[Mol Cancer Ther 2009;8(4):980–7]

Supplementary Figure 1 from Preclinical Evaluation of Differentially Targeting Dual Virotherapy for Human Solid Cancer

Multimodal approaches combining drugs that differentially function is the most popular regimen for treating human cancer. Understanding the molecular mechanisms underlying the synergistic, potentiative, and antagonistic effects of drug combinations could facilitate the discovery of novel efficacious combinations. We previously showed that telomerase-specific replication-competent adenovirus (Telomelysin, OBP-301), in which the human telomerase reverse transcriptase promoter controls the adenoviral E1 gene expression, induces a selective antitumor effect in human cancer cells. Here, using E1-deleted replication-deficient adenovirus expressing the p53 tumor suppressor gene (Advexin, Ad-p53) and OBP-301, we investigate how these adenoviruses that kill tumor cells with different mechanisms could work in combination on human cancer. We found that E1-deficient Ad-p53 could kill cancer cells more efficiently in the presence of OBP-301 than Ad-p53 alone or OBP-301 alone, because Ad-p53 could become replication-competent by being supplied adenoviral E1 from coinfected OBP-301 in trans. Ad-p53 plus OBP-301 induced high levels of p53 protein expression without p21 induction, resulting in apoptotic cell death documented by active caspase-3 expression with a cytometric bead array and an increased subdiploid apoptotic fraction of the cell cycle. For in vivo evaluation, nude mice xenografted with human lung tumors received intratumoral injection of OBP-301 and/or Ad-p53. Analysis of the growth of implanted tumors showed an enhanced antitumor effect in combination therapy. Our data show that Ad-p53 in combination with OBP-301 induces not only oncolytic but also apoptotic cancer cell death and enhances antitumor activity in vitro and in vivo, providing potential merits as a multimodal treatment for human cancer. Mol Cancer Ther; 9(6); 1884–93. ©2010 AACR.

Purpose: Because chemoradiotherapy selectively targets proliferating cancer cells, quiescent cancer stem–like cells are resistant. Mobilization of the cell cycle in quiescent leukemia stem cells sensitizes them to cell death signals. However, it is unclear that mobilization of the cell cycle can eliminate quiescent cancer stem–like cells in solid cancers. Thus, we explored the use of a genetically-engineered telomerase-specific oncolytic adenovirus, OBP-301, to mobilize the cell cycle and kill quiescent cancer stem–like cells.Experimental Design: We established CD133+ cancer stem–like cells from human gastric cancer MKN45 and MKN7 cells. We investigated the efficacy of OBP-301 against quiescent cancer stem–like cells. We visualized the treatment dynamics of OBP-301 killing of quiescent cancer stem–like cells in dormant tumor spheres and xenografts using a fluorescent ubiquitination cell-cycle indicator (FUCCI).Results: CD133+ gastric cancer cells had stemness properties. OBP-301 efficiently killed CD133+ cancer stem–like cells resistant to chemoradiotherapy. OBP-301 induced cell-cycle mobilization from G0–G1 to S/G2/M phases and subsequent cell death in quiescent CD133+ cancer stem–like cells by mobilizing cell-cycle–related proteins. FUCCI enabled visualization of quiescent CD133+ cancer stem–like cells and proliferating CD133− non–cancer stem–like cells. Three-dimensional visualization of the cell-cycle behavior in tumor spheres showed that CD133+ cancer stem–like cells maintained stemness by remaining in G0–G1 phase. We showed that OBP-301 mobilized quiescent cancer stem–like cells in tumor spheres and xenografts into S/G2/M phases where they lost viability and cancer stem–like cell properties and became chemosensitive.Conclusion: Oncolytic adenoviral infection is an effective mechanism of cancer cell killing in solid cancer and can be a new therapeutic paradigm to eliminate quiescent cancer stem–like cells. Clin Cancer Res; 19(23); 6495–505. ©2013 AACR.

Supplementary Figures S1-S5; Supplementary Table S1.

Purpose:EGFR-tyrosine kinase inhibitor (TKI) is a standard first-line therapy for activated EGFR-mutated non–small cell lung cancer (NSCLC). Treatment options for patients with acquired EGFR-TKI resistance are limited. HER3 mediates EGFR-TKI resistance. Clinical trials of the HER3-targeting antibody–drug conjugate patritumab deruxtecan (HER3-DXd) demonstrated its anticancer activity in EGFR-mutated NSCLC; however, the mechanisms that regulate HER3 expression are unknown. This study was conducted with the aim to clarify the mechanisms underlying HER3 regulation in EGFR-mutated NSCLC tumors and explored the strategy for enhancing the anticancer activity of HER3-DXd in EGFR-mutated NSCLC.Experimental Design:Paired tumor samples were obtained from 48 patients with EGFR-mutated NSCLC treated with EGFR-TKI(s). HER3 expression was immunohistochemically quantified with H-score, and genomic alteration and transcriptomic signature were tested in tumors from pretreatment to post-EGFR-TKI resistance acquisition. The anticancer efficacy of HER3-DXd and osimertinib was evaluated in EGFR-mutated NSCLC cells.Results:We showed augmented HER3 expression in EGFR-mutated tumors with acquired EGFR-TKI resistance compared with paired pretreatment samples. RNA sequencing revealed that repressed PI3K/AKT/mTOR signaling was associated with HER3 augmentation, especially in tumors from patients who received continuous EGFR-TKI therapy. An in vitro study also showed that EGFR-TKI increased HER3 expression, repressed AKT phosphorylation in multiple EGFR-mutated cancers, and enhanced the anticancer activity of HER3-DXd.Conclusions:Our findings help clarify the mechanisms of HER3 regulation in EGFR-mutated NSCLC tumors and highlight a rationale for combination therapy with HER3-DXd and EGFR-TKI in EGFR-mutated NSCLC.

Antitumor activity of U3-1402 in HER3-positive HCC1569 and MDA-MB-453 xenograft models

Supplementary Methods, Figures 1-10 from Telomerase-Dependent Oncolytic Adenovirus Sensitizes Human Cancer Cells to Ionizing Radiation via Inhibition of DNA Repair Machinery

Purpose: EGFR-tyrosine kinase inhibitor (TKI) is a standard first-line therapy for activated EGFR-mutated non–small cell lung cancer (NSCLC). Treatment options for patients with acquired EGFR-TKI resistance are limited. HER3 mediates EGFR-TKI resistance. Clinical trials of the HER3-targeting antibody–drug conjugate patritumab deruxtecan (HER3-DXd) demonstrated its anticancer activity in EGFR-mutated NSCLC; however, the mechanisms that regulate HER3 expression are unknown. This study was conducted with the aim to clarify the mechanisms underlying HER3 regulation in EGFR-mutated NSCLC tumors and explored the strategy for enhancing the anticancer activity of HER3-DXd in EGFR-mutated NSCLC. Experimental Design: Paired tumor samples were obtained from 48 patients with EGFR-mutated NSCLC treated with EGFR-TKI(s). HER3 expression was immunohistochemically quantified with H-score, and genomic alteration and transcriptomic signature were tested in tumors from pretreatment to post-EGFR-TKI resistance acquisition. The anticancer efficacy of HER3-DXd and osimertinib was evaluated in EGFR-mutated NSCLC cells. Results: We showed augmented HER3 expression in EGFR-mutated tumors with acquired EGFR-TKI resistance compared with paired pretreatment samples. RNA sequencing revealed that repressed PI3K/AKT/mTOR signaling was associated with HER3 augmentation, especially in tumors from patients who received continuous EGFR-TKI therapy. An in vitro study also showed that EGFR-TKI increased HER3 expression, repressed AKT phosphorylation in multiple EGFR-mutated cancers, and enhanced the anticancer activity of HER3-DXd. Conclusions: Our findings help clarify the mechanisms of HER3 regulation in EGFR-mutated NSCLC tumors and highlight a rationale for combination therapy with HER3-DXd and EGFR-TKI in EGFR-mutated NSCLC.

Purpose: HER3 is a compelling target for cancer treatment; however, no HER3-targeted therapy is currently clinically available. Here, we produced U3-1402, an anti-HER3 antibody–drug conjugate with a topoisomerase I inhibitor exatecan derivative (DXd), and systematically investigated its targeted drug delivery potential and antitumor activity in preclinical models. Experimental Design: In vitro pharmacologic activities and the mechanisms of action of U3-1402 were assessed in several human cancer cell lines. Antitumor activity of U3-1402 was evaluated in xenograft mouse models, including patient-derived xenograft (PDX) models. Safety assessments were also conducted in rats and monkeys. Results: U3-1402 showed HER3-specific binding followed by highly efficient cancer cell internalization. Subsequently, U3-1402 was translocated to the lysosome and released its payload DXd. While U3-1402 was able to inhibit HER3-activated signaling similar to its naked antibody patritumab, the cytotoxic activity of U3-1402 in HER3-expressing cells was predominantly mediated by released DXd through DNA damage and apoptosis induction. In xenograft mouse models, U3-1402 exhibited dose-dependent and HER3-dependent antitumor activity. Furthermore, U3-1402 exerted potent antitumor activity against PDX tumors with HER3 expression. Acceptable toxicity was noted in both rats and monkeys. Conclusions: U3-1402 demonstrated promising antitumor activity against HER3-expressing tumors with tolerable safety profiles. The activity of U3-1402 was driven by HER3-mediated payload delivery via high internalization into tumor cells.

e21175 Background: Epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) is a standard first-line therapy for activated EGFR-mutated non-small-cell lung cancer (NSCLC). Treatment options for patients with acquired EGFR-TKI resistance are limited. HER3 mediates EGFR-TKI resistance and is hence a promising target for anticancer treatment. Patritumab deruxtecan (HER3-DXd) is an antibody drug conjugate comprised of a fully human anti-HER3 IgG1 monoclonal antibody, covalently linked to a topoisomerase 1 inhibitor payload, an exatecan derivative, via a tetrapeptide-based cleavable linker. Clinical trials of theHER3-DXd demonstrated its anticancer activity in EGFR-mutated NSCLC. However, the genomic background that regulates HER3 expression is unknown. This study was conducted with the aim to clarify the genomic background for HER3 regulation in EGFR-mutated NSCLC tumors and to explore the strategy for enhancing the anticancer activity of HER3-DXd. Methods: 48 paired samples were obtained before EGFR-TKI treatment and after the acquisition of EGFR-TKI resistance from patients with EGFR-mutated NSCLC. HER3 expression was immunohistochemically quantified with H-score, and genomic alteration and transcriptomic signature were tested in tumors from pre-treatment to post-EGFR-TKI resistance acquisition. For statistical tests, linear regression analysis was performed to investigate whether a factor, including EGFR-TKI administration, affected HER3 expression. Pearson correlation coefficients were calculated to explore the relationships between HER3 expression level and other genomic expression in the tumors. Results: We showed augmented HER3 expression in EGFR-mutated tumors with acquired EGFR-TKI resistance compared to paired pretreatment samples (mean H-score 100.8 vs. 155.9, paired t-test p = 0.0007). Although genomic alterations including EGFR secondary T790M mutation did not correlate with HER3 augmentation, RNA sequencing revealed that repressed PI3K/AKT/mTOR signaling was associated with HER3 augmentation (GSEA; normalized enrichment score -1.78, p = 0.004). Multivariable regression analysis of HER3 augmentation revealed that re-biopsy under continuing EGFR-TKI treatment independently effected HER3 augmentation. Indeed, an in-vitro study also showed that EGFR-TKI increased HER3 expression via repressed AKT phosphorylation in multiple EGFR-mutated cancers, and it finally enhanced the anticancer activity of HER3-DXd. Conclusions: Our findings highlight a rationale for combination therapy with HER3-DXd and EGFR-TKI in EGFR-mutated NSCLC that is currently being evaluated in a clinical trial for patients with EGFR-mutated NSCLC tumors, not only with regard to an acquired resistance to EGFR-TKI but also in EGFR-TKI-naïve tumors (NCT04676477).

Glioblastoma is highly enriched with macrophages, and osteopontin (OPN) expression levels correlate with glioma grade and the degree of macrophage infiltration; thus, we studied whether OPN plays a crucial role in immune modulation. Quantitative PCR, immunoblotting, and ELISA were used to determine OPN expression. Knockdown of OPN was achieved using complementary siRNA, shRNA, and CRISPR/Cas9 techniques, followed by a series of in vitro functional migration and immunological assays. OPN gene–deficient mice were used to examine the roles of non-tumor-derived OPN on survival of mice harboring intracranial gliomas. Patients with mesenchymal glioblastoma multiforme (GBM) show high OPN expression, a negative survival prognosticator. OPN is a potent chemokine for macrophages, and its blockade significantly impaired the ability of glioma cells to recruit macrophages. Integrin α(v)β(5) (ITGαvβ5) is highly expressed on glioblastoma-infiltrating macrophages and constitutes a major OPN receptor. OPN maintains the M2 macrophage gene signature and phenotype. Both tumor-derived and host-derived OPN were critical for glioma development. OPN deficiency in either innate immune or glioma cells resulted in a marked reduction in M2 macrophages and elevated T cell effector activity infiltrating the glioma. Furthermore, OPN deficiency in the glioma cells sensitized them to direct CD8(+) T cell cytotoxicity. Systemic administration in mice of 4-1BB–OPN bispecific aptamers was efficacious, increasing median survival time by 68% (P < 0.05). OPN is thus an important chemokine for recruiting macrophages to glioblastoma, mediates crosstalk between tumor cells and the innate immune system, and has the potential to be exploited as a therapeutic target.

Gastric cancer patients positive for peritoneal cytology are at increased risk of tumor recurrence, but although a certain proportion of cytology‐positive patients relapse rapidly with aggressive progression, others survive longer with conventional chemotherapies. This heterogeneity makes it difficult to stratify patients for more intensive therapy and poses a substantial challenge for the implementation of precision medicine. We developed a new approach to identify biologically malignant subpopulations in cytology‐positive gastric cancer patients, using a green fluorescent protein (GFP)‐expressing attenuated adenovirus in which the telomerase promoter regulates viral replication (TelomeScan, OBP‐401). The fluorescence emitted from TelomeScan‐positive cells was successfully quantified using a multi‐mode microplate reader. We then analyzed clinical peritoneal washes obtained from 68 gastric cancer patients and found that patients positive for TelomeScan had a significantly worse prognosis. In 21 cytology‐positive patients, the median survival time of those who were TelomeScan positive (235 days) was significantly shorter than that for those who were TelomeScan negative (671 days; P = 0.0062). This fluorescent virus‐guided cytology detects biologically malignant cancer cells from the peritoneal washes of gastric cancer patients and may thus be useful for both therapy stratification and precision medicine approaches based on genetic profiling of disseminated cells.

Surgery and radiation therapy are the standard treatment options for meningiomas, but these treatments are not always feasible. Expression profiling was performed to determine the presence of therapeutic actionable biomarkers for prioritization and selection of agents. Meningiomas (n = 115) were profiled using a variety of strategies including next-generation sequencing (592-gene panel: n = 14; 47-gene panel: n = 94), immunohistochemistry (n = 8–110), and fluorescent and chromogenic in situ hybridization (n = 5–70) to determine mutational and expression status. The median age of patients in the cohort was 60 years, with a range spanning 6–90 years; 52% were female. The most frequently expressed protein markers were EGFR (93%; n = 44), followed by PTEN (77%; n = 110), BCRP (75%; n = 8), MRP1 (65%, n = 23), PGP (62%; n = 84), and MGMT (55%; n = 97). The most frequent mutation among all meningioma grades occurred in the NF2 gene at 85% (11/13). Recurring mutations in SMO and AKT1 were also occasionally detected. PD-L1 was expressed in 25% of grade III cases (2/8) but not in grade I or II tumors. PD-1 + T cells were present in 46% (24/52) of meningiomas. TOP2A and thymidylate synthase expression increased with grade (I = 5%, II = 22%, III = 62% and I = 5%, II = 23%, III = 47%, respectively), whereas progesterone receptor expression decreased with grade (I = 79%, II = 41%, III = 29%). If predicated on tumor expression, our data suggest that therapeutics directed toward NF2 and TOP2A could be considered for most meningioma patients.

// Sherise D. Ferguson 1 , Shouhao Zhou 2 , Joanne Xiu 3 , Yuuri Hashimoto 1 , Nader Sanai 4 , Lyndon Kim 5 , Santosh Kesari 6 , John de Groot 7 , David Spetzler 2 and Amy B. Heimberger 1 1 Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 2 Department of Biostatistics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 3 Caris Life Sciences, Phoenix, AZ, USA 4 Division of Neurosurgical Oncology, Barrow Neurological Institute, Phoenix, AZ, USA 5 Department of Neurological Surgery and Medical Oncology, Thomas Jefferson University Hospital, Philadelphia, PA, USA 6 Department of Translational Neurosciences and Neurotherapeutics, Pacific Neuroscience Institute and John Wayne Cancer Institute at Providence Saint John’s Health Center, Santa Monica, CA, USA 7 Department of Neuro-Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Correspondence to: Sherise D. Ferguson, email: sdferguson@mdanderson.org Keywords: ependymoma, chemoresistance, molecular profiling, DNA repair Received: July 20, 2017 Accepted: October 05, 2017 Published: December 15, 2017 ABSTRACT Background: After surgery and radiation, treatment options for ependymoma are few making recurrence a challenging issue. Specifically, the efficacy of chemotherapy at recurrence is limited. We performed molecular profiling on a cohort of ependymoma cases in order to uncover therapeutic targets and to elucidate the molecular mechanisms contributing to treatment resistance. Results: This ependymoma cohort showed minimal alterations in gene amplifications and mutations but had high expression rates of DNA synthesis and repair enzymes such as RRM1 (47%), ERCC1 (48%), TOPO1 (62%) and class III β-tublin (TUBB3) (57%), which are also all associated with chemoresistance. This cohort also had high expression rates of transporter proteins that mediate multi-drug resistance including BCRP (71%) and MRP1 (43%). Subgroup analyses showed that cranial ependymomas expressed the DNA synthesis enzyme TS significantly more frequently than spinal lesions did (57% versus 15%; p = 0.0328) and that increased TS expression was correlated with increased tumor grade ( p = 0.0009). High-grade lesions were also significantly associated with elevated expression of TOP2A ( p = 0.0092) and TUBB3 ( p = 0.0157). Materials and Methods: We reviewed the characteristics of 41 ependymomas (21 cranial, 20 spinal; 8 grade I, 11 grade II, 22 grade III) that underwent multiplatform profiling with immunohistochemistry, next-generation sequencing, and in situ hybridization. Conclusions: Ependymomas are enriched with proteins involved in chemoresistance and in DNA synthesis and repair, which is consistent with the meager clinical effectiveness of conventional systemic therapy in ependymoma. Adjuvant therapies that combine conventional chemotherapy with the inhibition of chemoresistance-related proteins may represent a novel treatment paradigm for this difficult disease.

Purpose: Patients with central nervous system (CNS) tumors are typically treated with radiotherapy, but this is not curative and results in the upregulation of phosphorylated STAT3 (p-STAT3), which drives invasion, angiogenesis, and immune suppression. Therefore, we investigated the combined effect of an inhibitor of STAT3 and whole-brain radiotherapy (WBRT) in a murine model of glioma. Experimental Design: C57BL/6 mice underwent intracerebral implantation of GL261 glioma cells, WBRT, and treatment with WP1066, a blood–brain barrier–penetrant inhibitor of the STAT3 pathway, or the two in combination. The role of the immune system was evaluated using tumor rechallenge strategies, immune-incompetent backgrounds, immunofluorescence, immune phenotyping of tumor-infiltrating immune cells (via flow cytometry), and NanoString gene expression analysis of 770 immune-related genes from immune cells, including those directly isolated from the tumor microenvironment. Results: The combination of WP1066 and WBRT resulted in long-term survivors and enhanced median survival time relative to monotherapy in the GL261 glioma model (combination vs. control P < 0.0001). Immunologic memory appeared to be induced, because mice were protected during subsequent tumor rechallenge. The therapeutic effect of the combination was completely lost in immune-incompetent animals. NanoString analysis and immunofluorescence revealed immunologic reprograming in the CNS tumor microenvironment specifically affecting dendritic cell antigen presentation and T-cell effector functions. Conclusions: This study indicates that the combination of STAT3 inhibition and WBRT enhances the therapeutic effect against gliomas in the CNS by inducing dendritic cell and T-cell interactions in the CNS tumor.

Cancer-associated fibroblasts (CAFs) are strongly implicated in tumor progression, including in the processes of tumorigenesis, invasion, and metastasis. The targeting of CAFs using various therapeutic approaches is a novel treatment strategy; however, the efficacy of such therapies remains limited. Recently, near-infrared photoimmunotherapy (NIR-PIT), which is a novel targeted therapy employing a cell-specific mAb conjugated to a photosensitizer, has been introduced as a new type of phototherapy. In this study, we have developed a novel NIR-PIT technique to target CAFs, by focusing on fibroblast activation protein (FAP), and we evaluate the treatment efficacy in vitro and in vivo. Esophageal carcinoma cells exhibited enhanced activation of fibroblasts, with FAP over-expressed in the cytoplasm and on the cell surface. FAP-IR700-mediated PIT showed induced rapid cell death specifically for those cells in vitro and in vivo, without adverse effects. This novel therapy for CAFs, designed as local control phototherapy, was safe and showed a promising inhibitory effect on FAP(+) CAFs. PIT targeting CAFs via the specific marker FAP may be a therapeutic option for CAFs in the tumor microenvironment in the future.

Melanoma is molecularly and structurally heterogeneous, with some tumor cells existing under hypoxic conditions. Our cell growth assays showed that under controlled hypoxic conditions, BRAF(V600E) melanoma cells rapidly became resistant to vemurafenib. By employing both a three-dimensional (3D) spheroid model and a two-dimensional (2D) hypoxic culture system to model hypoxia in vivo, we identified upregulation of HGF/MET signaling as a major mechanism associated with vemurafenib resistance as compared with 2D standard tissue culture in ambient air. We further confirmed that the upregulation of HGF/MET signaling was evident in drug-resistant melanoma patient tissues and mouse xenografts. Pharmacologic inhibition of the c-Met/Akt pathway restored the sensitivity of melanoma spheroids or 2D hypoxic cultures to vemurafenib. Mol Cancer Ther; 15(10); 2442–54. ©2016 AACR.