Tumor immunological phenotype signature-based high-throughput screening for the discovery of combination immunotherapy compounds

Immune checkpoint blockade (ICB) therapy intends to only benefit a fraction of cancer patients, and combination immunotherapy with a compound is a promising treatment to overcome this limitation. Here, a tumor immunological phenotype (TIP) gene signature and high throughput sequencing-based high throughput screening (HTS2) were combined to identify combination immunotherapy compounds. We firstly defined a TIP gene signature, which expression pattern distinguishes “cold” tumors from “hot” tumors, and predicts ICB response in cancer patients. Then, after screening thousands of compounds, we identified that aurora kinase inhibitors, including ENMD-2076 and TAK-901, could reprogram the expression pattern of TIP genes from “cold” tumor to “hot” tumor in triple negative breast cancer (TNBC) cells. The treatment of aurora kinase inhibitors on TNBC cells dramatically up-regulates expression of Th1 type chemokine genes CXCL10 and CXCL11, which promotes effective T cells infiltrating into tumor microenvironment and significantly improves anti-PD-1 efficacy in inhibiting the tumor growth of TNBC in preclinical models. Mechanistically, these aurora kinase inhibitors are mainly through inhibiting AURKA-STAT3 signaling pathway to stimulate the expression of CXCL10 and CXCL11. Our study established a high throughput strategy to discover candidate compounds for combination immunotherapy, and suggested the therapeutic potential of combining aurora kinase inhibitors with checkpoint blockade immunotherapy for the treatment of TNBC. 2×10e5 MDA-MB-231 cells were plated in a 6-well plate for 24 h and then treated with ENMD-2076 at 2.5 or 5 µmol/L (or TAK901 at 1 or 2 µmol/L) for 24 h. 1×10e5 4T1 cells were plated in a 6-well plate for 24 h and then treated with ENMD-2076 at 1.25 or 2.5 µmol/L (or TAK901 at 1 or 2 µmol/L) for 24 h. Cells were harvested, and RNA was isolated using TRIzol (Invitrogen). The libraries were constructed by using the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB) and sequenced on HiSeq 2500 sequencing system (Illumina).