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Supplementary Figure Legends from Aflibercept Exerts Antivascular Effects and Enhances Levels of Anthracycline Chemotherapy In vivo in Human Acute Myeloid Leukemia Models

Supplementary Figures 7-8 from Aflibercept Exerts Antivascular Effects and Enhances Levels of Anthracycline Chemotherapy In vivo in Human Acute Myeloid Leukemia Models

We examined whether potent vascular endothelial growth factor (VEGF) blockade mediated by aflibercept, a decoy VEGF receptor (VEGFR) 1/2 moiety with stronger affinity for VEGF than bevacizumab, resulted in antileukemia effects and enhanced the efficacy of systemic chemotherapy. The efficacy of aflibercept alone and in combination with doxorubicin was evaluated in human VEGF-expressing acute myeloid leukemia (AML) cell lines and primary cells xenotransplanted into immunodeficient mice. Aflibercept reduced primary VEGF/VEGFR-positive AML colony formation growth in vitro and inhibited AML xenograft growth up to 93% in association with antiangiogenic and antiproliferative effects, hypoxia, and VEGF sequestration in multiple models. High VEGF-A expression by AML cells promoted in vivo xenograft growth and aflibercept sensitivity. Aflibercept therapy slowed disease progression in two systemic human AML xenograft models and reduced peripheral leukemia disease in a primary relapsed AML model in NOD/SCID/IL2Rγnull mice. Combination aflibercept and doxorubicin enhanced antitumor effects in local xenograft models. Sequential aflibercept followed by doxorubicin resulted in progressive anthracycline accumulation in marrow and extramedullary AML sites and resulted in 2-fold higher drug levels 24 hours after administration. In contrast, tissues (tumor, plasma, marrow) treated with chemotherapy only showed progressive drug clearance over time. Combination aflibercept and doxorubicin also resulted in vascular narrowing, decreased vessel number, and perivascular apoptosis. These data suggest that inefficient drug delivery by leukemia-associated vasculature may mediate chemoresistance and support further clinical evaluation of combination aflibercept and anthracycline therapy in refractory/relapsed AML patients. Mol Cancer Ther; 9(10); 2737–51. ©2010 AACR.

Supplementary Tables 1-3 from Aflibercept Exerts Antivascular Effects and Enhances Levels of Anthracycline Chemotherapy In vivo in Human Acute Myeloid Leukemia Models

Supplementary Methods and References from Aflibercept Exerts Antivascular Effects and Enhances Levels of Anthracycline Chemotherapy In vivo in Human Acute Myeloid Leukemia Models

Supplementary Figures 1-6 from Aflibercept Exerts Antivascular Effects and Enhances Levels of Anthracycline Chemotherapy In vivo in Human Acute Myeloid Leukemia Models

Retinoic acid receptor-related orphan receptor γ (RORc, RORγ, or NR1F3) is the nuclear receptor master transcription factor that drives the function and development of IL-17-producing T helper cells (Th17), cytotoxic T cells (Tc17), and subsets of innate lymphoid cells. Activation of RORγ+ T cells in the tumor microenvironment is hypothesized to render immune infiltrates more effective at countering tumor growth. To test this hypothesis, a family of benzoxazines was optimized to provide LYC-55716 (37c), a potent, selective, and orally bioavailable small-molecule RORγ agonist. LYC-55716 decreases tumor growth and enhances survival in preclinical tumor models and was nominated as a clinical development candidate for evaluation in patients with solid tumors.

Integration of signaling and metabolic pathways enables and sustains lymphocyte function. Whereas metabolic changes occurring during T cell activation are well characterized, the metabolic demands of differentiated T lymphocytes are largely unexplored. In this study, we defined the bioenergetics of Th17 effector cells generated in vivo. These cells depend on oxidative phosphorylation (OXPHOS) for energy and cytokine production. Mechanistically, the essential role of OXPHOS in Th17 cells results from their limited capacity to increase glycolysis in response to metabolic stresses. This metabolic program is observed in mouse and human Th17 cells, including those isolated from Crohn disease patients, and it is linked to disease, as inhibiting OXPHOS reduces the severity of murine colitis and psoriasis. These studies highlight the importance of analyzing metabolism in effector lymphocytes within in vivo inflammatory contexts and suggest a therapeutic role for manipulating OXPHOS in Th17-driven diseases.

Adoptive cell transfer (ACT) therapy using tumor infiltrating lymphocytes (TILs) or T cells modified to express chimeric antigen receptors is a promising anti-cancer approach. Evidence suggests that Th17 or Tc17 cells display superior anti-tumor activity over unpolarized T cells commonly used in ACT clinical trials. As an approach to improve the potency and persistence of ACT, synthetic RORɣ agonists were designed to bolster the generation of type 17 cells. In vitro stimulation in the presence of RORɣ agonists modulates expression of costimulatory and coinhibitory molecules and increases IL-17A, IL-22 and GM-CSF production while maintaining IFNɣ production. Additional investigation revealed that treating tumor-specific Th0, Th17, Tc0 or Tc17 T cells with a RORɣt agonist in vitro significantly enhanced their ability to regress large and established EG.7 lymphoma and B16F10 melanoma tumors in vivo. Animals receiving RORɣ agonist-treated T cells had increased frequency of transferred cells, elevated IL-17A levels and decreased PD-1 expression in tumors and spleen compared to control animals. Previous studies have also shown that systemic delivery of an RORɣ agonist can also enhance anti-tumor immune response in syngeneic pre-clinical tumor models. By enhancing cytokine/chemokine production, promoting survival as well as decreasing PD-1 expression, RORɣ agonist molecules provide an effective means to enhance the potency of adoptively transferred T cell products.