Abstract 7221: A compound targeting the circadian clock protein CRY2 enhances therapeutic efficacy of bevacizumab in a colorectal cancer (CRC) xenograft model
Authors: Lo Jae Ho , Soni Shivani , Smbatyan Goar , Torres-Gonzalez Lesly , Mittal Pooja , Yang Yan , Battaglin Francesca , Chan Priscilla , Pan Yuanzhong , Algaze Sandra , Jayachandran Priya , Ashouri Karam , Wong Alexandra , Zhang Wu , Millstein Joshua , Singh Indrakant K. , Roussos Torres Evanthia T. , Mumenthaler Shannon M. , Kay Steve A. , Lenz Heinz-Josef 
Published: March 2024

Abstract
Anti VEGF A antibody Bevacizumab combined with standard chemotherapy has improved outcomes for CRC patients. However, a subset of CRC patients acquires resistance to anti-VEGF treatment and underlying mechanism is poorly understood. Our recent study reported that circadian clock transcription factor BMAL1 drives transcription of VEGFA conferring resistance to bevacizumab treatment. Our rational is to stabilize negative regulator of circadian clock CRY protein to inhibit BMAL1 activity in a negative feedback loop. In this study, we evaluated whether CRY stabilizers SHP656 and SHP1705 in combination with bevacizumab can synergistically enhance anti-tumor effect in CRC mouse models.
Methods: In vitro tube formation assay was performed with HUVECs cells to investigate the effect of CRY stabilizer on tube formation. For in vivo experiments, 106 HCT116 human CRC cells were injected subcutaneously into the right flank of 8-week-old nude mice. Mice (5mice/group) were randomized into: A. vehicle (2% carboxymethylcellulose PO 5 days/week; control IgG IP, 2 days/week); B. bevacizumab (5mg/kg IP, twice/week); C. 10mg/kg SHP656; D. 30mg/kg SHP656; E. 30mg/kg SHP1705; F. 10mg/kg SHP656 + bevacizumab; G. 30mg/kg SHP656 + bevacizumab; G. 30mg/kg SHP1705 + bevacizumab. SHP 656 & 1705 was given PO; 5days/week. Tumor volume was measured three times a week. The survival curve was plotted using Kaplan and Meier method and assessed for statistical significance with log-rank test.
Results: The in vitro tube formation assay showed significant inhibition of tube formation in HUVEC cells treated with SHP1705 plus bevacizumab compared to treated with bevacizumab alone. In in vivo study, the tumors treated with 30mg/kg SHP1705 + bevacizumab significantly suppressed tumor growth compared to bevacizumab monotherapy (one way-ANOVA, P &lt;0.05). 30mg/kg SHP1705 + bevacizumab significantly improved survival of the mice (Log-rank test, P &lt;0.05). No significant survival benefit was observed with bevacizumab alone. 3/5 mice treated with SHP1705 + bevacizumab showed decreased tumor volume and achieved complete regression for more than 4 months without additional treatment. In combination with bevacizumab, higher dose of SHP656 (30mg/kg) delayed tumor growth and extended survival compared with bevacizumab monotherapy (though did not reach statistical significance) whereas the lower dose of 10mg/kg did not show anti-tumor effect, demonstrating a dose dependent response.
Conclusion: Our study shows that CRY stabilizers in combination with bevacizumab significantly enhances anti-tumor efficacy in CRC mouse models. Although mechanism of action of SHP compounds on angiogenesis pathway still needs further investigation, these findings may support development of novel treatment strategies modulating circadian clock to overcome bevacizumab resistance in CRC patients.
Citation Format: Jae Ho Lo, Shivani Soni, Goar Smbatyan, Lesly Torres-Gonzalez, Pooja Mittal, Yan Yang, Francesca Battaglin, Priscilla Chan, Yuanzhong Pan, Sandra Algaze, Priya Jayachandran, Karam Ashouri, Alexandra Wong, Wu Zhang, Joshua Millstein, Indrakant K. Singh, Evanthia T. Roussos Torres, Shannon M. Mumenthaler, Steve A. Kay, Heinz-Josef Lenz. A compound targeting the circadian clock protein CRY2 enhances therapeutic efficacy of bevacizumab in a colorectal cancer (CRC) xenograft model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7221.