Identification of a Selective Small-Molecule Inhibitor of Breast Cancer Stem Cells—Probe 2

Excerpt

Cancer stem cells (CSCs), which drive tumor growth, are known to be resistant to standard chemotherapy and radiation treatment. This raises a significant unmet need to find therapies that can target CSCs within tumors because these cells are responsible for recurrence, the primary cause of patient mortality. However, one of the technical challenges of working with CSCs is that they are not stable outside the tumor environment and are not easy to grow in culture media. Hence, stable sibling cell lines that were induced into epithelial-to-mesenchymal transdifferentiation (EMT) to stably propagate CSC-enriched populations were used to screen a library of 300,718 compounds from the Molecular Libraries Small Molecule Repository (MLSMR). Several classes of selective inhibitors of CSCs were identified. The use of isogenic control cell lines for the secondary validation assays minimized the probability of false hits advancing along the critical path to probe development. Of these, 19 compounds were chosen based on their selectivity, potency, and chemical tractability and were retested in the primary screen and secondary assays. Three scaffolds were prioritized for structure-activity relationship studies. In this report, we describe the development of the chemical probe ML245 that displayed greater than 14-fold selective inhibition of the breast CSC-like cell line (HMLE_sh_ECad, IC₅₀=0.536 μM) over the isogenic control cell line (HMLE_sh_GFP). Furthermore, the probe (ML245) was screened against a panel of 68 biological targets that are commonly used in drug discovery for lead profiling and eight were identified as binding targets in a primary biochemical assay at a single dose of 10 μM, Adenosine (A₁, A_2A, A₃), Opiate κ, and Serotonin 5-HT_2B G-protein coupled receptors, norepinephrine and dopamine transporters, and GABA_A receptor. To further characterize the targets of ML245, gene expression studies were conducted to identify potential target pathways and targets mediating the response. Gene expression profiling revealed that ML245 regulated the expression of several pro-apoptotic/mitochondrial maintenance factors (such as caspase recruitment domain family member 10, activating transcription factor 4, mitochondrial ribosomal protein MRPL12), and DNA-modifying enzymes (such as Early Growth Response 1, zinc finger-containing proteins ZNF295 and CGRRF1, and TBP-associated factor 1D), in the CSC-like cell line and not the isogenic control cell line. Although the link to regulation of cancer is unknown, upregulation of the gene CGRRF1 by the probe (ML245) and other CSC-selective inhibitors is exciting due to its function of determining cell-cycle arrest and may have a role in inhibiting the progression of Burkitt’s lymphoma. More studies are needed to understand the direct target of the probe (ML245) and its method of action for inhibiting CSC-like cells.