Leveraging Automation toward Development of a High-Throughput Gene Expression Profiling Platform.

We previously developed a panel of one-step real-time quantitative reverse transcription PCR (one-step qRT-PCR; hereafter referred to as qRT-PCR) assays to assess compound efficacy. However, these high-cost, conventional qRT-PCR manual assays are not amenable to high-throughput screen (HTS) analysis in a time-sensitive and complex drug discovery process. Here, we report the establishment of an automated gene expression platform using in-house lysis conditions that allows the study of various cell lines, including primary T cells. This process innovation provides the opportunity to perform genotypic profiling in both immunology and oncology therapeutic areas with quantitative studies as part of routine drug discovery program support. This newly instituted platform also enables a panel screening strategy to efficiently connect HTS, lead identification, and lead optimization in parallel.

A novel SMAC mimetic APG-1387 exhibits dual antitumor effect on HBV-positive hepatocellular carcinoma with high expression of cIAP2 by inducing apoptosis and enhancing innate anti-tumor immunity.

Check point inhibitor anti-PD1 antibody produced some efficacy in Hepatocellular Carcinoma (HCC) patients previously treated with sorafenib. Unfortunately, HCC patients with hepatitis B virus (HBV) infection did not respond as well as uninfected patients. Previously, Second mitochondria-derived activator of caspases (SMAC) mimetics-the antagonist for inhibitor of apoptosis proteins (IAPs) can rapidly reduce serum hepatitis B virus DNA in animal model. APG-1387 is a novel SMAC-mimetic, small molecule inhibitor targeting inhibitor of apoptosis proteins (IAPs). In our study, firstly, we found that HCC patients with copy number alteration of cIAP1, cIAP2, and XIAP had a dismal prognosis. Then, we discovered that APG-1387 alone could induce apoptosis of PLC/PRF/5 which was HBV positive both in-vitro and in-vivo. Furthermore, we found that APG-1387 significantly up-regulated the expression of calreticulin and HLA-DR in PLC/PRF/5 via activating non-classic NF-κB pathway. Also, compared to vehicle group, APG-1387 increased NK cell counts by 5 folds in PLC/PRF/5 xenograft model. In-vitro, APG-1387 positively regulated T cells by reducing Treg differentiation and down-regulating PD1 expression in CD4 T cell. Moreover, APG-1387 had no impact on memory T cells. Consequently, our results suggest that APG1387 could be a good candidate to combine with anti-PD1 antibody treatment to overcome low responds of check point inhibitors in HBV positive HCC.

cIAP1/2-TRAF2-SHP-1-Src-MyD88 Complex Regulates Lipopolysaccharide-Induced IL-27 Production through NF-κB Activation in Human Macrophages.

The inhibitors of apoptosis (IAP) proteins, initially described in the context of apoptosis regulation as promoting cell survival, have recently emerged as key regulators of innate immune signaling. As a result, downregulation of IAP via Smac mimetics (SMM) has both survival and immunoregulatory effects. IAPs modulate cytokine production in murine models either as a single agent or in response to LPS. However, the role of SMM and the involvement of IAPs in primary human cells and in particular macrophages with respect to cytokine production and innate immune responses remain largely unknown. IL-27, a member of the IL-12 cytokine family produced by APCs such as macrophages, has broad immunoregulatory properties in both innate and adaptive immune responses. Herein, we show that cellular IAPs (cIAPs) positively regulate LPS-induced IL-27 production in both primary human monocytes and macrophages. Investigations for the signaling mechanism of cIAPs involvement in IL-27 production in human macrophages revealed that LPS-induced IL-27 production is regulated by a novel signaling complex comprising cIAP1/2, TNFR-associated factor 2 (TRAF2), SHP-1, Src, and MyD88 leading to p38, c-Jun N-terminal kinases (JNK) and Akt activation and NF-κB signaling. In cancer cells, SMM induce the production of cytokines by activating the noncanonical alternate NF-κB pathway. However, in human macrophages, SMM do not induce the production of TNF-α and other cytokines while inhibiting LPS-induced IL-27 production by inhibiting the classical NF-κB pathway. These signaling pathways may constitute novel therapeutic avenues for immune modulation of IL-27 and provide insight into the modulatory immune effects of SMM.