Abstract
Abstract 2421
Members of the nuclear factor-kB (NF-κB) family of transcription factors play important roles in cell-signaling involved in host-defense, immune responses, inflammation, and cancer. Multiple stimuli operating through different receptors are capable of activating NF-κB, including the Tumor Necrosis Factor (TNF) family cytokines, Toll-like receptors (TLRs), Nucleotide-binding Leucine Rich Repeat Proteins (NLRs), DNA-damaging agents, and others. Drugs that selectively block activation of NF-κB through one or more of these receptors could have activity against various types of cancer in which activation of NF-κB plays a role. We used a chemical biology strategy to identify novel chemical inhibitors of cell pathways responsible for NF-κB activation. Screening of a large chemical library (>330K compounds from NIH) was conducted using human 697 pre-B-cell leukemia cells with a stably integrated NF-κB-luciferase reporter gene and stimulating the Carma/Bcl-10/MALT pathway using protein kinase C (PKC) activators (PMA/Ionomycin). Confirmed hits were counter-screened against HEK293T-NF-κB-luciferase cells treated with TNF-a, with the goal to eliminate non-pathway specific inhibitors of NF-κB. Among the hit compounds, we identified oxadiazole- and oxazole-based chemical probes as potential cell lineage- and cell differentiation-specific inhibitors of NF-κB (“NLDSi” = NF-κB lineage, differentiation-specific inhibitors). Using a panel of cell lines with stably integrated NF-κB-driven luciferase reporter genes, NLDSi compounds suppressed PMA/Ionomycin-induced NF-κB activity in a concentration-dependent manner (IC50=0.1–1.0 μM) in the pre-B-cell leukemia cell-line 697, but not in the T-cell leukemia line JURKAT, the myeloma cell-line RPMI8226, or HEK293 epithelial cells. These compounds also suppressed NF-κB activity (measured by reporter gene) in 697 cells stimulated with BAFF or treated with doxorubicin. Examining NF-κB target gene expression by q-RT-PCR (mRNA level) and immunoblotting (protein level) showed that NLDSi compounds reduced PMA/Ionomycin-induced TRAF1 and A20 expression in 697 cells and in the pre-B-cell leukemia cell line REH, but not in several mature B-cell lines, including BJAB, Daudi, OCILy3, and OCILy10. NLDSi compounds also inhibited NF-κB target gene expression in pre-B-cell lines stimulated with BAFF or treated with lipopolysaccharide (LPS) or doxorubicin (Dox). Nevertheless we found these compounds could suppress of PMA/Ionomycin-induced expression of TRAF1 and A20 gene in the primary leukemia cells of patients with chronic lymphocytic leukemia (CLL). Taken together, the compounds described here appear to selectively suppress NF-κB activation in the B-cell lineage at specific stages of differentiation. As such, these small molecules could serve as chemical probes for uncovering novel targets that regulate NF-κB activity and potentially lead to development of therapies that selectively block certain types of NF-κB activation in pre-B cell ALL, CLL, or other hematologic malignancies. (Supported by NIH grants X01 MH077633-1, U54–005033, and P01-CA-81534).
Disclosures:
No relevant conflicts of interest to declare.
The transcriptional cofactor TRIM33 prevents apoptosis in B lymphoblastic leukemia by deactivating a single enhancer