Eugenie S. Kleinerman, MD

https://openworks.mdanderson.org/legendsandlegacieschapters/1009/thumbnail.jp

10: Eugenie S. Kleinerman, MD

https://openworks.mdanderson.org/legendsandlegaciesbook/1012/thumbnail.jp

Epigenetic Regulation of Apoptosis and Cell Cycle in Osteosarcoma

The role of genetic mutations in the development of osteosarcoma, such as alterations in p53 and Rb, is well understood. However, the significance of epigenetic mechanisms in the progression of osteosarcoma remains unclear and is increasingly being investigated. Recent evidence suggests that epigenetic alterations such as methylation and histone modifications of genes involved in cell cycle regulation and apoptosis may contribute to the pathogenesis of this tumor. Importantly, understanding the molecular mechanisms of regulation of these pathways may give insight into novel therapeutic strategies for patients with osteosarcoma. This paper serves to summarize the described epigenetic mechanisms in the tumorigenesis of osteosarcoma, specifically those pertaining to apoptosis and cell cycle regulation

Tumor Vessel Development and Expansion in Ewing's Sarcoma: A Review of the Vasculogenesis Process and Clinical Trials with Vascular-Targeting Agents

Ewing's sarcoma accounts for a disproportionately high portion of the overall pediatric mortality rate compared to its rare incidence in the pediatric population. Little progress has been made since the introduction of traditional chemotherapies, and understanding the biology of the tumor is critical for developing new therapies. Ewing's sarcomas rely on a functional vascular supply, which is formed by a combination of angiogenesis and vasculogenesis. Recent insights into the molecular regulation of bone marrow (BM) cell participation in vascular development have identified VEGF, SDF-1α, and DLL4 as critical players in the vasculogenesis process. Clinical trials using vascular targeting agents, specifically targeting VEGF or DLL4, are underway

Knockdown of autophagy-related protein 5, ATG5, decreases oxidative stress and has an opposing effect on camptothecin-induced cytotoxicity in osteosarcoma cells

BACKGROUND: Autophagy induction can increase or decrease anticancer drug efficacy. Anticancer drug-induced autophagy induction is poorly characterized in osteosarcoma (OS). In this study, we investigated the impact of autophagy inhibition on camptothecin (CPT)-induced cytotoxicity in OS. METHODS: Autophagy-inhibited DLM8 and K7M3 metastatic murine OS cell lines were generated by infection with lentiviral shRNA directed against the essential autophagy protein ATG5. Knockdown of ATG5 protein expression and inhibition of autophagy was confirmed by immunoblot of ATG5 and LC3II proteins, respectively. Metabolic activity was determined by MTT assay and cell viability was determined by trypan blue exclusion. Acridine orange staining and immunoblotting for LC3II protein expression were used to determine autophagy induction. Oxidative stress was assessed by staining cells with HE and DCFH-DA followed by flow cytometry analysis. Mitochondrial membrane potential was determined by staining cells with TMRE followed by flow cytometry analysis. Immunoblotting was used to detect caspase activation, Parp cleavage and p53 phosphorylation. RESULTS: Autophagy inhibition caused a greater deficit in metabolic activity and cell growth in K7M3 cells compared to DLM8 cells. K7M3 cells exhibited higher basal autophagy levels than DLM8 cells and non-transformed murine MCT3 osteoblasts. Autophagy inhibition did not affect CPT-induced DNA damage. Autophagy inhibition decreased CPT-induced cell death in DLM8 cells while increasing CPT-induced cell death in K7M3 cells. Autophagy inhibition reduced CPT-induced mitochondrial damage and CPT-induced caspase activation in DLM8 cells. Buthionine sulfoximine (BSO)-induced cell death was greater in autophagy-competent DLM8 cells and was reversed by antioxidant pretreatment. Camptothecin-induced and BSO-induced autophagy induction was also reversed by antioxidant pretreatment. Significantly, autophagy inhibition not only reduced CPT-induced oxidative stress but also reduced basal oxidative stress. CONCLUSIONS: The results of this study indicate that autophagy inhibition can have an opposing effect on CPT-induced cytotoxicity within OS. The cytoprotective mechanism of autophagy inhibition observed in DLM8 cells involves reduced CPT-induced oxidative stress and not reduced DNA damage. Our results also reveal the novel finding that knockdown of ATG5 protein reduces both basal oxidative stress and drug-induced oxidative stress

Production of VEGF165 by Ewing's sarcoma cells induces vasculogenesis and the incorporation of CD34+ stem cells into the expanding tumor vasculature

The Ewing's sarcoma cell line TC71 overexpresses vascular endothelial growth factor isoform 165 (VEGF165), a potent proangiogenic molecule that induces endothelial cell proliferation, migration, and chemotaxis. CD34+ bone marrow stem cells can differentiate into endothelial and hematopoietic cells. We used a transplant model to determine whether CD34 + cells migrate from the bone marrow to Ewing's sarcoma tumors and participate in the neovascularization process that supports tumor growth. We also examined the role of VEGF165 in CD34+ cell migration. Human umbilical cord CD34+ cells were transplanted into sublethally irradiated severe combined immunodeficient mice. Seven days later, the mice were injected subcutaneously with TC71 tumor cells. Tumors were excised 2 weeks later and analyzed by immunohistochemistry. The tumor sections expressed both human VE-cadherin and mouse CD31, indicating involvement of donor-derived human cells in the tumor vessels. To determine the role of VEGF165 in the chemoattraction of CD34+ cells, we generated two VEGF 165-deficient TC71 clones, a stable anti-sense VEGF165 cell line (Clone 17) and a VEGF165 siRNA-inhibited clone (TC/siVEGF7-1). The resulting VEGF165-deficient tumor cells had normal growth rates in vitro, but had delayed growth when implanted into mice. Immunohistochemical analysis revealed decreased infiltration of CD34+ cells into both VEGF165-deficient tumors. These data show that bone marrow stem cells contribute to the growing tumor vasculature in Ewing's sarcoma and that VEGF165 is critical for the migration of CD34+ cells from the bone marrow into the tumor. © 2006 Wiley-Liss, Inc.Fil: Lee, Tim H.. University of Texas; Estados UnidosFil: Bolontrade, Marcela Fabiana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. University of Texas; Estados UnidosFil: Worth, Laura L.. University of Texas; Estados UnidosFil: Guan, Hui. University of Texas; Estados UnidosFil: Ellis, Lee M.. University of Texas; Estados UnidosFil: Kleinerman, Eugenie S.. University of Texas; Estados Unido

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