Development and Pilot Screen of Novel High Content Assay for Down Regulators of Expression of Heterogenous Nuclear Ribonuclear Protein H2.

BACKGROUND/AIMS: Despite recent advances in melanoma drug discovery, the average overall survival of patients with late-stage metastatic melanoma is approximately 3 years, suggesting a need for new approaches and melanoma therapeutic targets. Previously we identified heterogeneous nuclear ribonucleoprotein H2 as a potential target of anti-melanoma compound 2155-14 (Palrasu et al., Cell Physiol Biochem 2019;53:656-686). In the present study, we endeavored to develop an assay to enable a high throughput screening campaign to identify drug-like molecules acting via down regulation of heterogeneous nuclear ribonucleoprotein H2 that can be used for melanoma therapy and research. METHODS: We established a cell-based platform using metastatic melanoma cell line WM266-4 expressing hnRNPH2 conjugated with green fluorescent protein to enable assay development and screening. High Content Screening assay was developed and validated in 384 well plate format, followed by miniaturization to 1,536 well plate format. RESULTS: All plate-based QC parameters were acceptable: %CV = 6.7±0.3, S/B = 21±2.1, Z' = 0.75±0.04. Pilot screen of FDA-approved drug library (n=1,400 compounds) demonstrated hit rate of 0.5%. Two compounds demonstrated pharmacological response and were authenticated by western blot analysis. CONCLUSION: We developed a highly robust HTS-amenable high content screening assay capable of monitoring down regulation of hnRNPH2. This assay is thus capable of identifying authentic down regulators of hnRNPH1 and 2 in a large compound collection and, therefore, is amenable to a large-scale screening effort.

A multiplex preclinical model for adenoid cystic carcinoma of the salivary gland identifies regorafenib as a potential therapeutic drug.

Adenoid cystic carcinomas (ACC) are rare salivary gland cancers with a high incidence of metastases. In order to study this tumor type, a reliable model system exhibiting the molecular features of this tumor is critical, but none exists, thereby inhibiting in-vitro studies and the analysis of metastatic behavior. To address this deficiency, we have coupled an efficient method to establish tumor cell cultures, conditional reprogramming (CR), with a rapid, reproducible and robust in-vivo zebrafish model. We have established cell cultures from two individual ACC PDX tumors that maintain the characteristic MYB translocation. Additional mutations found in one ACC culture also seen in the PDX tumor. Finally, the CR/zebrafish model mirrors the PDX mouse model and identifies regorafenib as a potential therapeutic drug to treat this cancer type that mimic the drug sensitivity profile in PDX model, further confirming the unique advantages of multiplex system.

Conditional reprogramming and long-term expansion of normal and tumor cells from human biospecimens.

Historically, it has been difficult to propagate cells in vitro that are derived directly from human tumors or healthy tissue. However, in vitro preclinical models are essential tools for both the study of basic cancer biology and the promotion of translational research, including drug discovery and drug target identification. This protocol describes conditional reprogramming (CR), which involves coculture of irradiated mouse fibroblast feeder cells with normal and tumor human epithelial cells in the presence of a Rho kinase inhibitor (Y-27632). CR cells can be used for various applications, including regenerative medicine, drug sensitivity testing, gene expression profiling and xenograft studies. The method requires a pathologist to differentiate healthy tissue from tumor tissue, and basic tissue culture skills. The protocol can be used with cells derived from both fresh and cryopreserved tissue samples. As approximately 1 million cells can be generated in 7 d, the technique is directly applicable to diagnostic and predictive medicine. Moreover, the epithelial cells can be propagated indefinitely in vitro, yet retain the capacity to become fully differentiated when placed into conditions that mimic their natural environment.