Three-dimensional CRISPR screening reveals epigenetic interaction with anti-angiogenic therapy

Michael Y He,Marc G Achen,Ruofei Liu,Zoe L Grant,Mark A Dawson,Yih-Chih Chan,Omer Gilan,Leigh Coultas,Michael M Halford,Niko Thio,Steven A Stacker,James P Roy

doi:10.1038/s42003-021-02397-3

Abstract

Angiogenesis underlies development, physiology and pathogenesis of cancer, eye and cardiovascular diseases. Inhibiting aberrant angiogenesis using anti-angiogenic therapy (AAT) has been successful in the clinical treatment of cancer and eye diseases. However, resistance to AAT inevitably occurs and its molecular basis remains poorly understood. Here, we uncover molecular modifiers of the blood endothelial cell (EC) response to a widely used AAT bevacizumab by performing a pooled genetic screen using three-dimensional microcarrier-based cell culture and CRISPR–Cas9. Functional inhibition of the epigenetic reader BET family of proteins BRD2/3/4 shows unexpected mitigating effects on EC survival and/or proliferation upon VEGFA blockade. Moreover, transcriptomic and pathway analyses reveal an interaction between epigenetic regulation and anti-angiogenesis, which may affect chromosomal structure and activity in ECs via the cell cycle regulator CDC25B phosphatase. Collectively, our findings provide insight into epigenetic regulation of the EC response to VEGFA blockade and may facilitate development of quality biomarkers and strategies for overcoming resistance to AAT.

Highlights

Angiogenesis underlies development, physiology and pathogenesis of cancer, eye and cardiovascular diseases
We demonstrated that endothelial cell (EC) proliferation and baseline gene expression in complete growth medium were comparable between 2D and 3D cultures (Fig. 1c; Supplementary Fig. 2b and Supplementary Data 1)
We examined siRNAmediated knockdown of all candidate genes plus BRD4 in the endothelial cell-multicolor competition assay (EC-MCA), which served as a complementary system to the CRISPR screen to assess the EC response to bevacizumab (Fig. 3b)

Summary

Spinner flask 1

Incubation Transduction Incubation Puromycin selection + serum reduction for three days. At three well-separated concentrations tested (1, 3, and 10 μM), the addition of NSC 663284 at a high concentration (10 μM) to cotreatment further reduced the number of viable cells (Fig. 7a), whereas NSC 663284 itself did not alter the EC response to bevacizumab when BETi was not present (Fig. 7b), suggesting that CDC25 activity, as well as CENP-A deposition, were involved in the altered EC response caused by co-treatment with bevacizumab plus BETi. As for Class II HDAC signaling, which was upregulated in palivizumab plus BETi, we assessed the effect of Class IIa HDAC inhibition on EC survival and/or proliferation with palivizumab plus JQ1. Concentrations of TMP195 (0.3, 1, and 3 μM) changed the number of viable cells compared with palivizumab plus JQ1 (Fig. 7a) These results suggest the involvement of chromosomal regulation via CDC25B in the altered EC response caused by co-treatment with BETi and bevacizumab

Discussion

Methods

Code availability