Abstract 194: Overcoming adaptive resistance to anti-VEGF therapy by targeting CD5L
Authors: LaFargue Christopher J. , Amero Paola , Noh Kyunghee , Mangala Selanare , Lu Chunhua , Cho Minsoon , Pradeep Sunila , Wan Yihong , Hu Wei , Rupaimoole Rajesha , Coleman Robert L. , Zhang Ningyan , An Zhiqiang , Lopez-Bernstein Gabriel , Franciscis Vittorio de , Sood Anil K. 
Published: August 2019

Abstract
Purpose: To identify mechanisms responsible for the development of adaptive resistance to anti-vascular endothelial growth factor (VEGF) therapy, and to develop novel agents to reverse its occurrence.
Methods: Gene expression profiling was performed on endothelial cells isolated from tumors sensitive or resistant to anti-VEGF therapy in a syngeneic ovarian cancer mouse model. A series of in vitro (lentiviral transfection, tube formation, siRNA knockdown, luciferase promoter assays) and in vivo (including Tie2; PPARγ KO mice) experiments were then used to elucidate the upstream and downstream effectors of CD5L, as well as the efficacy of CD5L inhibition in pre-clinical orthotopic mouse models.
Results: We identified substantially increased CD5L expression in endothelial cells isolated from anti-VEGF therapy resistant vs. sensitive tumors. Ectopic expression of CD5L in RF24 endothelial cells resulted in increased tube formation, cell migration, and cell proliferation, whereas siCD5L treatment resulted in an overall decrease. Observing that a PPARγ binding site was present in the CD5L promoter, we found that ectopic expression of PPARγ into RF24 cells results in increased CD5L expression, whereas siPPARγ treatment decreased it. Ectopic PPARγ expression increased luciferase activity but decreased activity with siPPARγ treatment when using a reporter assay for CD5L promoter. Conversely, mutation of the PPARγ binding site in the promoter construct lacked any increase in activity after ectopic PPARγ expression. IPA analysis identified HIF1α as playing an integral role in the PPARγ /CD5L activation pathway. Indeed, hypoxic conditions resulted in an increase of both PPARγ and CD5L in RF24 cells. HIF1α stabilizers (cobalt chloride) and inhibitors (YC-1 and topotecan) also showed an increase and decrease in PPARγ and CD5L expression, respectively. RPPA analysis of anti-VEGF resistant tumor endothelial cells implicated an upregulation of the PI3K pathway. While exogenous CD5L treatment led to increased p-AKT, the addition of the PI3K inhibitor LY294002 negated this effect and also decreased tube formation and cell migration. Next, we injected both WT B6 and Tie2:PPARγ KO mice with the ID8 mouse ovarian cancer cell line and treated with B20 until moribund. Mice harboring the PPARγ KO had significantly increased survival as well as decreased tumor weight, number of nodules, Ki67 expression, and blood vessel density. Our non-syngeneic model was then treated with B20 and either CD5L RNA aptamer or vehicle. The aptamer group treated with CD5L aptamer displayed significantly decreased tumor weight, number of nodules, Ki67 expression, and blood vessel density.
Conclusions: This study identifies a pathway centered on CD5L as an important driver of adaptive resistance to anti-angiogenic therapy.
Citation Format: Christopher J. LaFargue, Paola Amero, Kyunghee Noh, Selanare Mangala, Chunhua Lu, Minsoon Cho, Sunila Pradeep, Yihong Wan, Wei Hu, Rajesha Rupaimoole, Robert L. Coleman, Ningyan Zhang, Zhiqiang An, Gabriel Lopez-Bernstein, Vittorio de Franciscis, Anil K. Sood. Overcoming adaptive resistance to anti-VEGF therapy by targeting CD5L [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 194.