Aryl hydrocarbon receptor nuclear translocator-like (ARNTL/BMAL1) is associated with bevacizumab resistance in colorectal cancer via regulation of vascular endothelial growth factor A

Abstract Background The identification of new biomarkers and the development of novel, targetable contexts of vulnerability are of urgent clinical need in drug-resistant metastatic colorectal cancer (mCRC). Aryl-Hydrocarbon-Receptor-Nuclear-Translocator-Like (ARNTL/BMAL1) is a circadian clock-regulated transcription factor promoting expression of genes involved in angiogenesis and tumour progression. We hypothesised that BMAL1 increases expression of the vascular endothelial growth factor A VEGFA gene and, thereby, confers resistance to anti-angiogenic therapy with bevacizumab (Beva), a clinically used antibody for neutralization of VEGFA. Methods PCR and immunohistochemistry were employed to assess BMAL1 expression in mice (C57BL/6 J Apcmin/ + ; BALB/c nu/nu xenografts) and CRC patients under combination chemotherapy with Beva. BMAL1 single nucleotide gene polymorphisms (SNPs) were analysed by DNA-microarray in clinical samples. BMAL1 functions were studied in human CRC cell lines using colorimetric growth, DNA-binding and reporter assays. Findings In murine CRCs, high BMAL1 expression correlated with poor preclinical response to Beva treatment. In CRC patients' tumours ( n  = 74), high BMAL1 expression was associated with clinical non-response to combination chemotherapy with Beva (* p  = .0061) and reduced progression-free survival (PFS) [* p  = .0223, Hazard Ratio (HR) = 1.69]. BMAL1 SNPs also correlated with shorter PFS ( rs7396943, rs7938307, rs2279287 ) and overall survival (OS) [ rs11022780, * p  = .014, HR = 1.61]. Mechanistically, Nuclear-Receptor-Subfamily-1-Group-D-Member-1 (NR1D1/REVERBA) bound a − 672 bp Retinoic-Acid-Receptor-Related-Orphan-Receptor-Alpha-responsive-element (RORE) adjacent to a BMAL1 DNA-binding motif ( E -box) in the VEGFA gene promoter, resulting in increased VEGFA synthesis and proliferation of human CRC cell lines. Interpretation BMAL1 was associated with Beva resistance in CRC. Inhibition of REVERBA-BMAL1 signalling may prevent resistance to anti-angiogenic therapy. Fund This work was in part supported by the European Commission Seventh Framework Programme (Contract No. 278981 [ANGIOPREDICT]).