New High-Throughput Screening Identifies Compounds That Reduce Viability Specifically in Liver Cancer Cells That Express High Levels of SALL4 by Inhibiting Oxidative Phosphorylation

Abstract

Background & aims: Some oncogenes encode transcription factors, but few drugs have been successfully developed to block their activity specifically in cancer cells. The transcription factor SALL4 is aberrantly expressed in solid tumor and leukemia cells. We developed a screen to identify compounds that reduce the viability of liver cancer cells that express high levels of SALL4, and we investigated their mechanisms.

Methods: We developed a stringent high-throughput screening platform comprising unmodified SNU-387 and SNU-398 liver cancer cell lines and SNU-387 cell lines engineered to express low and high levels of SALL4. We screened 1597 pharmacologically active small molecules and 21,575 natural product extracts from plant, bacteria, and fungal sources for those that selectively reduce the viability of cells with high levels of SALL4 (SALL4^hi cells). We compared gene expression patterns of SALL4^hi cells vs SALL4-knockdown cells using RNA sequencing and real-time polymerase chain reaction analyses. Xenograft tumors were grown in NOD/SCID gamma mice from SALL4^hi SNU-398 or HCC26.1 cells or from SALL4^lo patient-derived xenograft (PDX) cells; mice were given injections of identified compounds or sorafenib, and the effects on tumor growth were measured.

Results: Our screening identified 1 small molecule (PI-103) and 4 natural compound analogues (oligomycin, efrapeptin, antimycin, and leucinostatin) that selectively reduced viability of SALL4^hi cells. We performed validation studies, and 4 of these compounds were found to inhibit oxidative phosphorylation. The adenosine triphosphate (ATP) synthase inhibitor oligomycin reduced the viability of SALL4^hi hepatocellular carcinoma and non-small-cell lung cancer cell lines with minimal effects on SALL4^lo cells. Oligomycin also reduced the growth of xenograft tumors grown from SALL4^hi SNU-398 or HCC26.1 cells to a greater extent than sorafenib, but oligomycin had little effect on tumors grown from SALL4^lo PDX cells. Oligomycin was not toxic to mice. Analyses of chromatin immunoprecipitation sequencing data showed that SALL4 binds approximately 50% of mitochondrial genes, including many oxidative phosphorylation genes, to activate their transcription. In comparing SALL4^hi and SALL4-knockdown cells, we found SALL4 to increase oxidative phosphorylation, oxygen consumption rate, mitochondrial membrane potential, and use of oxidative phosphorylation-related metabolites to generate ATP.

Conclusions: In a screening for compounds that reduce the viability of cells that express high levels of the transcription factor SALL4, we identified inhibitors of oxidative phosphorylation, which slowed the growth of xenograft tumors from SALL4^hi cells in mice. SALL4 activates the transcription of genes that regulate oxidative phosphorylation to increase oxygen consumption, mitochondrial membrane potential, and ATP generation in cancer cells. Inhibitors of oxidative phosphorylation might be used for the treatment of liver tumors with high levels of SALL4.

Keywords: Chemical-Genetic Screen; HCC; Metabolic Vulnerability; Metabolism.