4'-Ethynyl-2'-Deoxycytidine (EdC) Preferentially Targets Lymphoma and Leukemia Subtypes by Inducing Replicative Stress.

Anticancer nucleosides are effective against solid tumors and hematological malignancies, but typically are prone to nucleoside metabolism resistance mechanisms. Using a nucleoside-specific multiplexed high-throughput screening approach, we discovered 4'-ethynyl-2'-deoxycytidine (EdC) as a third-generation anticancer nucleoside prodrug with preferential activity against diffuse large B-cell lymphoma (DLBCL) and acute lymphoblastic leukemia (ALL). EdC requires deoxycytidine kinase (DCK) phosphorylation for its activity and induced replication fork arrest and accumulation of cells in S-phase, indicating it acts as a chain terminator. A 2.1Å co-crystal structure of DCK bound to EdC and UDP reveals how the rigid 4'-alkyne of EdC fits within the active site of DCK. Remarkably, EdC was resistant to cytidine deamination and SAMHD1 metabolism mechanisms and exhibited higher potency against ALL compared to FDA approved nelarabine. Finally, EdC was highly effective against DLBCL tumors and B-ALL in vivo. These data characterize EdC as a pre-clinical nucleoside prodrug candidate for DLBCL and ALL.

MYC regulates fatty acid metabolism through a multigenic program in claudin-low triple negative breast cancer.

BACKGROUND: Recent studies have suggested that fatty acid oxidation (FAO) is a key metabolic pathway for the growth of triple negative breast cancers (TNBCs), particularly those that have high expression of MYC. However, the underlying mechanism by which MYC promotes FAO remains poorly understood. METHODS: We used a combination of metabolomics, transcriptomics, bioinformatics, and microscopy to elucidate a potential mechanism by which MYC regulates FAO in TNBC. RESULTS: We propose that MYC induces a multigenic program that involves changes in intracellular calcium signalling and fatty acid metabolism. We determined key roles for fatty acid transporters (CD36), lipases (LPL), and kinases (PDGFRB, CAMKK2, and AMPK) that each contribute to promoting FAO in human mammary epithelial cells that express oncogenic levels of MYC. Bioinformatic analysis further showed that this multigenic program is highly expressed and predicts poor survival in the claudin-low molecular subtype of TNBC, but not other subtypes of TNBCs, suggesting that efforts to target FAO in the clinic may best serve claudin-low TNBC patients. CONCLUSION: We identified critical pieces of the FAO machinery that have the potential to be targeted for improved treatment of patients with TNBC, especially the claudin-low molecular subtype.

N-terminal sequence analysis of proteins and peptides.

Automated N-terminal sequence analysis involves a series of chemical reactions that derivatize and remove one amino acid at a time from the N-terminus of purified peptides or intact proteins. At least several picomoles of a purified protein or 10 to 20 pmol of a purified peptide with an unmodified N-terminus is required to obtain useful sequence information. In recent years, the demand for N-terminal sequencing has decreased substantially as some applications for protein identification and characterization can now be more effectively performed using mass spectrometry. However, N-terminal sequencing remains the method of choice for verifying the N-terminal boundary of recombinant proteins, determining the N-terminus of protease-resistant domains, identifying proteins isolated from species where most of the genome has not yet been sequenced, and mapping modified or crosslinked sites in proteins that prove to be refractory to analysis by mass spectrometry.