Small molecule inhibition of lysine-specific demethylase 1 (LSD1) and histone deacetylase (HDAC) alone and in combination in Ewing sarcoma cell lines.

Ewing Sarcoma (ES) is characterized by recurrent translocations between EWSR1 and members of the ETS family of transcription factors. The transcriptional activity of the fusion oncoprotein is dependent on interaction with the nucleosome remodeling and deactylase (NuRD) co-repressor complex. While inhibitors of both histone deacetylase (HDAC) and lysine-specific demethylase-1 (LSD1) subunits of the NuRD complex demonstrate single agent activity in preclinical models, combination strategies have not been investigated. We selected 7 clinically utilized chemotherapy agents, or active metabolites thereof, for experimentation: doxorubicin, cyclophosphamide, vincristine, etoposide and irinotecan as well as the HDAC inhibitor romidepsin and the reversible LSD1 inhibitor SP2509. All agents were tested at clinically achievable concentrations in 4 ES cell lines. All possible 2 drug combinations were then tested for potential synergy. Order of addition of second-line conventional combination therapy agents was tested with the addition of SP2509. In two drug experiments, synergy was observed with several combinations, including when SP2509 was paired with topoisomerase inhibitors or romidepsin. Addition of SP2509 after treatment with second-line combination therapy agents enhanced treatment effect. Our findings suggest promising combination treatment strategies that utilize epigenetic agents in ES

Long-distance placement of substrate RNA by H/ACA proteins

The structural basis for accurate placement of substrate RNA by H/ACA proteins is studied using a nonintrusive fluorescence assay. A model substrate RNA containing 2-aminopurine immediately 3′ of the uridine targeted for modification produces distinct fluorescence signals that report the substrate's docking status within the enzyme active site. We combined substrate RNA with complete and subcomplexes of H/ACA ribonucleoprotein particles and monitored changes in the substrate conformation. Our results show that each of the three accessory proteins, as well as an active site residue, have distinct effects on substrate conformations, presumably as docking occurs. Interestingly, in some cases these effects are exerted far from the active site. Application of our data to an available structural model of the holoenzyme, enables the functional role of each accessory protein in substrate placement to come into view