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As tumor development relies on a coordination of angiogenesis and tumor growth, an efficient antitumor strategy should target both the tumor and its associated vessels. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a tumor-selective manner. Additionally, thrombospondin-1, a naturally occurring inhibitor of angiogenesis, and a recombinant protein containing functional domains of thrombospondin-1, 3TSR, have been shown to be necessary and sufficient to inhibit tumor angiogenesis. Here, we show that a combination of a TRAIL receptor 2 agonist antibody, Lexatumumab, and 3TSR results in a significantly enhanced and durable tumor inhibition. We further observed that 3TSR induces apoptosis in primary endothelial cells by up-regulating the expression of TRAIL receptors 1 and 2 in a CD36 and Jun NH2-terminal kinase-dependent manner leading to the activation of both intrinsic and extrinsic apoptotic machineries. The modulation of these pathways is critical for 3TSR-induced apoptosis as disrupting either via specific inhibitors reduced apoptosis. Moreover, 3TSR attenuates the Akt survival pathway. These studies indicate that 3TSR plays a critical role in regulating the proapoptotic signaling pathways that control growth and death in endothelial cells and that a combination of TRAIL and 3TSR acts as a double hit against tumor and tumor-associated vessels. [Cancer Res 2009;69(9):3856–65]

Supplementary Figure 1 from A Double Hit to Kill Tumor and Endothelial Cells by TRAIL and Antiangiogenic 3TSR

Supplementary Figure Legends 1-3 from A Double Hit to Kill Tumor and Endothelial Cells by TRAIL and Antiangiogenic 3TSR

Immune checkpoint blockers (ICBs) have failed in all Phase III glioblastoma trials. Here, we found that ICBs induce cerebral edema in some patients and mice with glioblastoma. Through single-cell RNA sequencing, intravital imaging, and T cell blocking studies in mice, we demonstrated that this edema results from an inflammatory response following anti-PD1 antibody treatment that disrupts the blood-tumor-barrier. Used in lieu of immunosuppressive corticosteroids, the angiotensin receptor blocker losartan prevented this ICB-induced edema and reprogrammed the tumor microenvironment, curing 20% of mice which increased to 40% in combination with standard of care treatment. Using a bihemispheric tumor model, we identified a “hot” tumor immune signature prior to losartan+anti-PD1 therapy that predicted long-term survival. Our findings provide the rationale and associated biomarkers to test losartan with ICBs in glioblastoma patients.One-Sentence SummaryLosartan prevents immunotherapy-associated edema and enhances the outcome of immunotherapy in glioblastoma.

Brain metastases are refractory to therapies that control systemic disease in patients with human epidermal growth factor receptor 2 (HER2+) breast cancer, and the brain microenvironment contributes to this therapy resistance. Nutrient availability can vary across tissues, therefore metabolic adaptations required for brain metastatic breast cancer growth may introduce liabilities that can be exploited for therapy. Here, we assessed how metabolism differs between breast tumors in brain versus extracranial sites and found that fatty acid synthesis is elevated in breast tumors growing in brain. We determine that this phenotype is an adaptation to decreased lipid availability in brain relative to other tissues, resulting in a site-specific dependency on fatty acid synthesis for breast tumors growing at this site. Genetic or pharmacological inhibition of fatty acid synthase (FASN) reduces HER2+ breast tumor growth in the brain, demonstrating that differences in nutrient availability across metastatic sites can result in targetable metabolic dependencies.

Chimeric antigen receptor (CAR)-T cells have revolutionized the treatment of multiple types of hematological malignancies, but have shown limited efficacy in patients with glioblastoma (GBM) or other solid tumors. This may be largely due to the immunosuppressive tumor microenvironment (TME) that compromises CAR-T cells’ delivery and antitumor activity. We previously showed that blocking vascular endothelial growth factor (VEGF) signaling can normalize tumor vessels in murine and human tumors, including GBM, breast, liver, and rectal carcinomas. Moreover, we demonstrated that vascular normalization can improve the delivery of CD8+ T cells and the efficacy of immunotherapy in breast cancer models in mice. In fact, the US FDA (Food and drug administration) has approved seven different combinations of anti-VEGF drugs and immune checkpoint blockers for liver, kidney, lung and endometrial cancers in the past 3 years. Here, we tested the hypothesis that anti-VEGF therapy can improve the delivery and efficacy of CAR-T cells in immunocompetent mice bearing orthotopic GBM tumors. We engineered two syngeneic mouse GBM cell lines (CT2A and GSC005) to express EGFRvIII—one of the most common neoantigens in human GBM—and CAR T cells to recognize EGFRvIII. We found that treatment with the anti-mouse VEGF antibody (B20) improved CAR-T cell infiltration and distribution throughout the GBM TME, delayed tumor growth, and prolonged survival of GBM-bearing mice compared with EGFRvIII-CAR-T cell therapy alone. Our findings provide compelling data and a rationale for clinical evaluation of anti-VEGF agents with CAR T cells for GBM patients.

Immune checkpoint blockers (ICBs) have revolutionized the treatment of some solid cancers but have failed to benefit the majority of glioblastoma (GBM) patients. Two reasons underlying limited ICB benefit are: 1) immune-related adverse events, and 2) resistance conferred by the tumor microenvironment. Here, we show that ICBs induce cerebral edema in patients and GBM mouse models. This edema results from an inflammatory response to ICB treatment that disrupts the blood-tumor-barrier, as confirmed by intravital imaging, mechanistic blocking studies, and single-cell RNA sequencing. Losartan – a commonly prescribed antihypertensive agent – controls ICB-induced edema, reprograms the immunosuppressive tumor microenvironment, and improves survival under ICB therapy. In combination with a standard of care regimen in mice mimicking clinical treatment (surgical resection, chemoradiation), losartan increases the percent of long-term surviving (cured) mice under ICB therapy from 16% to 43%. Finally, a bihemispheric “resect-and-response” model to establish predictive biomarkers from the tumor microenvironment reveals that cured mice have an immunostimulatory (“hot”) immune tumor compartment prior to therapy. These results provide the basis for clinical testing of adding to losartan to ICB treatment for GBM patients.

Brain metastases are refractory to therapies that otherwise control systemic disease in patients with human epidermal growth factor receptor 2 (HER2+) breast cancer, and the unique brain microenvironment contributes to this therapy resistance. Nutrient availability can vary across tissues, therefore metabolic adaptations required for breast cancer growth in the brain microenvironment may also introduce liabilities that can be exploited for therapy. Here, we assessed how metabolism differs between breast tumors growing in the brain versus extracranial sites and found that fatty acid synthesis is elevated in breast tumors growing in the brain. We determine that this phenotype is an adaptation to decreased lipid availability in the brain relative to other tissues, which results in a site-specific dependency on fatty acid synthesis for breast tumors growing at this site. Genetic or pharmacological inhibition of fatty acid synthase (FASN) reduces HER2+ breast tumor growth in the brain, demonstrating that differences in nutrient availability across metastatic sites can result in targetable metabolic dependencies.

Brain metastases are refractory to therapies that otherwise control systemic disease in patients with human epidermal growth factor receptor 2 (HER2+) breast cancer, and the unique brain microenvironment contributes to this therapy resistance. Nutrient availability can vary across tissues, therefore metabolic adaptations required for breast cancer growth in the brain microenvironment may also introduce liabilities that can be exploited for therapy. Here, we assessed how metabolism differs between breast tumors growing in the brain versus extracranial sites and found that fatty acid synthesis is elevated in breast tumors growing in the brain. We determine that this phenotype is an adaptation to decreased lipid availability in the brain relative to other tissues, which results in a site-specific dependency on fatty acid synthesis for breast tumors growing at this site. Genetic or pharmacological inhibition of fatty acid synthase (FASN) reduces HER2+ breast tumor growth in the brain, demonstrating that differences in nutrient availability across metastatic sites can result in targetable metabolic dependencies. Citation Format: Gino B. Ferraro, Ahmed Ali, Alba Luengo, David P. Kodack, Amy Deik, Keene L. Abbott, Divya Bezwada, Landry Blanc, Brendan Prideaux, Xin Jin, Jessica M. Possada, Jiang Chen, Christopher R. Chin, Zohreh Amoozgar, Raphael Ferreira, Ivy Chen, Kamila Naxerova, Christopher Ng, Anna M. Westermark, Mark Duquette, Sylvie Roberge, Costas A. Lyssiotis, Dan G. Duda, Todd R. Golub, Shawn M. Davidson, Dai Fukumura, Véronique A. Dartois, Clary B. Clish, Matthew G. Vander Heiden, Rakesh K. Jain. Fatty acid synthesis is required for breast cancer brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 90.

BRAF(V600E) mutation exerts an essential oncogenic function in many tumors, including papillary thyroid carcinoma (PTC). Although BRAF(V600E) inhibitors are available, lack of response has been frequently observed. To study the mechanism underlying intrinsic resistance to the mutant BRAF(V600E) selective inhibitor vemurafenib, we established short-term primary cell cultures of human metastatic/recurrent BRAF(V600E)-PTC, intrathyroidal BRAF(V600E)-PTC, and normal thyroid (NT). We also generated an early intervention model of human BRAF(V600E)-PTC orthotopic mouse. We find that metastatic BRAF(V600E)-PTC cells elicit paracrine-signaling which trigger migration of pericytes, blood endothelial cells and lymphatic endothelial cells as compared to BRAF(WT)-PTC cells, and show a higher rate of invasion. We further show that vemurafenib therapy significantly suppresses these aberrant functions in non-metastatic BRAF(V600E)-PTC cells but lesser in metastatic BRAF(V600E)-PTC cells as compared to vehicle treatment. These results concur with similar folds of down-regulation of tumor microenvironment-associated pro-metastatic molecules, with no effects in BRAF(WT)-PTC and NT cells. Our early intervention preclinical trial shows that vemurafenib delays tumor growth in the orthotopic BRAF(WT/V600E)-PTC mice. Importantly, we identify high copy number gain of MCL1 (chromosome 1q) and loss of CDKN2A (P16, chromosome 9p) in metastatic BRAF(V600E)-PTC cells which are associated with resistance to vemurafenib treatment. Critically, we demonstrate that combined vemurafenib therapy with BCL2/MCL1 inhibitor increases metastatic BRAF(V600E)-PTC cell death and ameliorates response to vemurafenib treatment as compared to single agent treatment. In conclusion, short-term PTC and NT cultures offer a predictive model for evaluating therapeutic response in patients with PTC. Our PTC pre-clinical model suggests that combined targeted therapy might be an important therapeutic strategy for metastatic and refractory BRAF(V600E)-positive PTC.

Pancreatic ductal adenocarcinoma (PDAC) is highly resistant to chemotherapy, partly due to the presence of a dense-fibrotic stroma and adaptive metabolism. Telmisartan is an angiotensin II type receptor 1 (AT1) antagonist with partial peroxisome proliferator-activated receptor gamma (PPARγ) agonistic activity used for treatment of hypertension. The aim of this study was to determine the effects of telmisartan on the viability of PDAC cells and tumor progression. In panels of 4 murine and 8 human PDAC cells, the telmisartan IC50 was lower in cells with a low steady-state expression of PPARγ and a mesenchymal cell morphology. In contrast, losartan—a selective AT1 inhibitor—did not affect the viability of PDAC cells. The siRNA knockdown of PPARγ enhanced the sensitivity of telmisartan and stimulated epithelial-mesenchymal transition, which was accompanied by an increase in Wnt signaling. PPARγ regulates glucose metabolism and autophagy. We thus assessed effects of telmisartan on bioenergetics and autophagy of PDAC cells. In PPARγ-knockdown and -scrambled cells telmisartan significantly reduced glucose uptake, without affecting ATP production, but increased respiratory capacity, which can maintain the production of ATP during hypoglycemia. Immunoblotting revealed that PPARγ knockdown compared to scramble cells had increased levels of phosphorylated-AMP-activated protein kinase (p-AMPK) and increased expression of LC3A/B—structural proteins of autophagosomal membranes—which implies higher levels of autophagy. We also compared effects of telmisartan treatment on LC3A/B expression to well-established autophagy modulators, chloroquine and verapamil. Under nutrient-rich conditions and as expected, chloroquine and verapamil treatment induced LC3A/B accumulation, consistent with active autophagic flux in these cells. Telmisartan treatment decreased the levels of LC3A/B in both scramble and PPARγ knockdown cells and decreased the formation of LC3A/B positive granules in other PDAC cell lines. Telmisartan can also induce the accumulation of the signal adaptor protein p62 (SQSTM1), even in the presence of verapamil, which is also consistent with autophagy inhibition. Telmisartan did not prevent the accumulation LC3A/B in the presence of chloroquine, implying that telmisartan acts after the autophagosome-lysosome fusion step. To assess the effects of telmisartan in vivo, we used an orthotopic PDAC model. Telmisartan monotherapy inhibited the growth of primary tumors, decreased the incidence of liver metastasis, and significantly improved the survival of mice. Hence, telmisartan can reduce autophagy and the viability of PDAC cells, and PDAC progression. Because telmisartan is an FDA-approved drug, our findings provide the scientific rationale for testing its efficacy in the prevention of PDAC progression. Citation Format: Jelena Grahovac, Shiwei Han, Hao Liu, Mark Duquette, Alba Luengo, Daniel Schanne, Andrew S. Liss, Matthew G. Vander Heiden, Rakesh K. Jain, Yves Boucher. The angiotensin receptor blocker and partial PPARγ agonist telmisartan inhibits the growth of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B06.

Antigen presentation plays a major role in tumor cell recognition and targeting by immune cells, and is critical to the success of many cancer immunotherapies. How the abnormal tumor microenvironment affects tumor cell antigen presentation is unclear. Hypoxia is a prevalent feature of the tumor microenvironment. Here, we showed that the expression of major histocompatibility complex class I (MHCI) is associated with regions of hypoxia in human breast tumors. The association between hypoxia and MHCI is independent of the breast tumor hormone receptor and HER2 expression status. In vitro studies revealed that hypoxia directly regulates the expression levels of MHCI along with other components of the antigen presentation machinery. Multiple kinase regulators of MHCI expression are responsive to hypoxia. These results suggest that hypoxia effects on cancer cell antigen presentation may be a potential mechanism of tumor immune evasion and treatment resistance. Citation Format: Mei Rosa Ng, Francesco Sabbatino, Mark Duquette, Kamila Naxerova, Mark Badeaux, Gino B. Ferraro, Shan M. Chin, Divya Bezwada, Elena F. Brachtel, Soldano Ferrone, Rakesh K. Jain. Hypoxia regulation of antigen presentation machinery expression in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-057.

Most women are diagnosed with epithelial ovarian cancer (EOC) at advanced stage, where therapies have limited effectiveness and the long-term survival rate is low. We evaluated the effects of combined antiangiogenic and chemotherapy treatments on advanced stage EOC. Treatment of EOC cells with a recombinant version of the thrombospondin-1 type I repeats (3TSR) induced more apoptotic cell death (36.5 ± 9.6%) in vitro compared to untreated controls (4.1 ± 1.4). In vivo, tumors were induced in an orthotopic, syngeneic mouse model of advanced stage EOC. Mice were treated with 3TSR (4 mg/kg per day) alone or in combination with chemotherapy drugs delivered with maximum tolerated dose or metronomic scheduling. Pretreatment with 3TSR induced tumor regression, normalized tumor vasculature, and improved uptake of chemotherapy drugs. Combination 3TSR and metronomic chemotherapy induced the greatest tumor regression (6.2-fold reduction in size compared to PBS-treated controls) and highest survival when treatment was initiated at advanced stage. 3TSR binding to its receptor, CD36 (cluster of differentiation 36), increased binding of CD36 and SHP-1, which significantly inhibited phosphorylation of the VEGF receptor. In this study, we describe a novel treatment approach and mechanism of action with 3TSR and chemotherapy that induces regression of advanced stage EOC and significantly improves survival.-Russell, S., Duquette, M., Liu, J., Drapkin, R., Lawler, J., Petrik, J. Combined therapy with thrombospondin-1 type I repeats (3TSR) and chemotherapy induces regression and significantly improves survival in a preclinical model of advanced stage epithelial ovarian cancer.

Aberrant activation of Janus kinase (JAK) / signal transducer and activator of transcription (STAT) 3 pathway has been implicated in tumor initiation, progression and metastasis which make it a potential therapeutic target in human malignancies. In vitro STAT3 inhibition exhibited anti-tumor effect in medulloblastoma (MB) cell lines; however, little has been reported about the mechanisms of STAT3 activation in medulloblastoma (MB). We found that STAT3 was constitutively activated in both medulloblastoma patient samples and 3 mice models that were clinically classified as SHH subgroup, Group 3 and Group 4 in pediatric MB patients. By using PCR arrays we investigated the early expression profile of local cytokines in host cerebellum during MB pathogenesis and found increased IL17B, IL6 and TNFα in the microenvironment of MB mice models. In vitro cytokine stimulation assay indicated that IL6 was the major activator of STAT3 in D283 MB cells. Confocal immunofluorescence staining of different cell components in the cerebellum in D283 xenografts showed that the endothelial cells and Purkinje cells were the two major sources of IL6 secretion. Our data provides molecular and cellular clue for the activation of STAT3 signaling during MB tumorigenesis and might help to develop more effective therapeutic targets for STAT3 blockade in MB. Citation Format: Shuang Yan, Sampurna Chatterjee, Trupti Vardam, Shuji Kitahara, Tai Hato, Sylvie Roberge, Vasileios Askoxylakis, Mark Duquette, Dai Fukumura, Lei Xu, Rakesh Jain. Microenvironmental Interleukin-6 induced activation of STAT3 signaling in medulloblastoma: Implications for molecular pathology and therapy. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A12.