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Supplementary Tables and Figures from Cabozantinib Exhibits Potent Antitumor Activity in Colorectal Cancer Patient-Derived Tumor Xenograft Models via Autophagy and Signaling Mechanisms

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posted on 2023-04-03, 15:00 authored by Aaron J. Scott, John J. Arcaroli, Stacey M. Bagby, Rachel Yahn, Kendra M. Huber, Natalie J. Serkova, Anna Nguyen, Jihye Kim, Andrew Thorburn, Jon Vogel, Kevin S. Quackenbush, Anna Capasso, Anna Schreiber, Patrick Blatchford, Peter J. Klauck, Todd M. Pitts, S. Gail Eckhardt, Wells A. Messersmith

Supplemental Table 1: Molecular and clinical characteristics of the 10 CRC explants used in the PDX model experiments. Seven explants have a KRAS mutation, 4 have a PIK3CA mutation, and none have a BRAF mutation. Over half of the explants came from patients who had previously received treatment for mCRC. Supplemental Table 2: Using DCE-MRI measurements, treatment with cabozantinib demonstrated significant decrease in vascularity on day 28 compared to baseline and day 7. Regorafenib treatment did not demonstrate a decrease in vascularity. Supplemental Table 3: [18F] FDG-PET imaging revealed significantly reduced FDG avidity on days 7 and days 28 of cabozantinib treatment compared to baseline, whereas FDG avidity did not diminish in the regorafenib treated explants. Supplemental Table 4: To better understand the potential metabolic effects of cabozantinib, we analysed signalling pathways involved in metabolism using the KEGG database. Receptor tyrosine kinase (RTK) assay revealed a decrease in many of the pathways responsible for cell metabolism after 3 days of cabozantinib treatment in three of the sensitive CRC explants. Supplemental Figure 1: Cabozantinib and regorafenib treatment in a Humanized HGF mouse model. Cabozantinib had greater antitumor effects when compared to regorafenib in the hHGF scid mice in CRC203 and CRC020. Similar results were observed when compared to athymic nude mice for CRC203 (REG TGII: 39.3, Cabo TGII: 14.93) and CRC020 (REG TGII: 17.5, Cabo TGII: -5.68). Supplemental Figure 2: Changes in TIE2 and VEGFR2 expression were variable and did not demonstrate a sustained decrease after treatment with regorafenib. Supplemental Figure 3: After 7 days of regorafenib treatment, there was no significant reduction in activation of p-AXL, AKT, and pS6 compared to control, and there was a variable trend in p-MET and p-RET levels as measured by RTK assay. Supplemental Figure 4: Regorafenib treatment led to decreased ATG3, LC3A/B, and beclin-1 in the CRC098 explant; however, levels of these enzymes increased in the CRC162 explant. Supplemental Figure 5: In vitro analysis of caspase 3/7 expression in cell lines HCT116 (a, c) and HT29 (b, d) after cabozantinib and crizotinib treatment showed significant increase in autophagy index relative to control*** and to regorafenib###. Caspase 3/7 expression was measured via fluorescence using the CYTO-ID® Autophagy Detection Kit. Crizotinib combined with SBI-020695, an anti-ULK-1 agent, did not result in increased caspase 3/7 expression, whereas caspase 3/7 expression significantly increased with cabozantinib plus SBI-020695.

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University of Colorado Cancer Center

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ARTICLE ABSTRACT

Antiangiogenic therapy used in treatment of metastatic colorectal cancer (mCRC) inevitably succumbs to treatment resistance. Upregulation of MET may play an essential role to acquired anti-VEGF resistance. We previously reported that cabozantinib (XL184), an inhibitor of receptor tyrosine kinases (RTK) including MET, AXL, and VEGFR2, had potent antitumor effects in mCRC patient-derived tumor explant models. In this study, we examined the mechanisms of cabozantinib sensitivity, using regorafenib as a control. The tumor growth inhibition index (TGII) was used to compare treatment effects of cabozantinib 30 mg/kg daily versus regorafenib 10 mg/kg daily for a maximum of 28 days in 10 PDX mouse models. In vivo angiogenesis and glucose uptake were assessed using dynamic contrast-enhanced (DCE)-MRI and [18F]-FDG-PET imaging, respectively. RNA-Seq, RTK assay, and immunoblotting analysis were used to evaluate gene pathway regulation in vivo and in vitro. Analysis of TGII demonstrated significant antitumor effects with cabozantinib compared with regorafenib (average TGII 3.202 vs. 48.48, respectively; P = 0.007). Cabozantinib significantly reduced vascularity and glucose uptake compared with baseline. Gene pathway analysis showed that cabozantinib significantly decreased protein activity involved in glycolysis and upregulated proteins involved in autophagy compared with control, whereas regorafenib did not. The combination of two separate antiautophagy agents, SBI-0206965 and chloroquine, plus cabozantinib increased apoptosis in vitro. Cabozantinib demonstrated significant antitumor activity, reduction in tumor vascularity, increased autophagy, and altered cell metabolism compared with regorafenib. Our findings support further evaluation of cabozantinib and combinational approaches targeting autophagy in colorectal cancer. Mol Cancer Ther; 17(10); 2112–22. ©2018 AACR.

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