Evasive Resistance To Vegf Blockade Mediated By Autocrine Il-6/Stat3 Signaling In Xenograft Models Of Human Cancer

Blockade of the VEGF pathway and subsequent inhibition of tumor angiogenesis has been shown to prolong the survival of patients with certain types of solid tumors. However, the extent and duration of the survival benefit is often limited by emergence of resistance. To date, mechanisms of resistance and relevant biomarkers have not been clearly defined. To study mechanisms of anti-VEGF resistance, we used the VEGF inhibitor ziv-aflibercept (also known as VEGF Trap) and derived a resistant tumor line from sensitive A431 human squamous cell carcinomas. A431 tumor cells were implanted subcutaneously into the flank of CB17 SCID mice. When tumors reached approximately 100 -150 mm3, animals were continuously (up to 7 weeks) treated with ziv-aflibercept. Initially, A431 tumors were very responsive to ziv-aflibercept, but resistant outgrowth was observed at 6-7 weeks post treatment start. Outgrowing tumors were harvested, in vivo passaged in the presence of ziv-aflibercept and cell lines were isolated from these resistant tumor variants (A431-V) as well as from in vivo passaged control treated tumors (A431-P). Inherent resistance of A431-V tumors to ziv-aflibercept was confirmed. Using several screening approaches, such as PathScan RTK Signaling Antibody Array, isotope-labeled mass spectrometry and ELISA based assays, the IL-6/STAT3 pathway was identified as one of the most up-regulated signaling pathways in the resistant A431-V model compared to sensitive A431-P cells. IL-6-dependent STAT3 activation was confirmed using the IL-6 receptor blocking antibody sarilumab, which dramatically reduced the constitutive phosphorylation of STAT3 in A431-V cells and tumors. To determine if IL-6 plays a role in anti-VEGF resistance, we assessed the effects of ziv-aflibercept alone or in combination with sarilumab in established subcutaneous A431-V tumors. A431-V tumor resistance to VEGF blockade was abrogated by inhibition of IL-6 signaling; compared to control-treated tumors, combination treatment resulted in 91% tumor growth inhibition 14 days post treatment start, while single agent ziv-aflibercept treatment resulted in 57% tumor growth inhibition. These findings were corroborated in Du145 human prostate tumors with high levels of endogenous IL-6/STAT3 signaling. Combined VEGF and IL-6 blockade showed enhanced anti-tumor activity in DU145 tumor bearing mice compared to single agents. Thus, blockade of the IL-6/STAT3 pathway warrants further investigation in overcoming resistance to anti-VEGF therapy. These data underscore that advanced knowledge of predictive biomarkers as well as resistance mechanisms is crucial for successful patient and treatment selection. Citation Format: Alexander P. Adler, Alexandra Eichten, Li Zhang, Jia Su, Ella Ioffe, George D. Yancopoulus, Douglas MacDonald, Christopher Daly, Xunbao Duan, Gavin Thurston. Evasive resistance to VEGF blockade mediated by autocrine IL-6/STAT3 signaling in xenograft models of human cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1378. doi:10.1158/1538-7445.AM2015-1378