A Small Molecule that Binds and Inhibits the ETV1 Transcription Factor Oncoprotein

Members of the ETS transcription factor family have been implicated in several cancers, where they are often dysregulated by genomic derangement. ETS variant 1 (ETV1) is an ETS factor gene that undergoes chromosomal translocation in prostate cancers and Ewing\u27s sarcomas, amplification in melanomas, and lineage dysregulation in gastrointestinal stromal tumors. Pharmacologic perturbation of ETV1 would be appealing in these cancers; however, oncogenic transcription factors are often deemed “undruggable” by conventional methods. Here, we used small-molecule microarray (SMM) screens to identify and characterize drug-like compounds that modulate the biological function of ETV1. We identified the 1,3,5-triazine small molecule BRD32048 as a top candidate ETV1 perturbagen. BRD32048 binds ETV1 directly, modulating both ETV1-mediated transcriptional activity and invasion of ETV1-driven cancer cells. Moreover, BRD32048 inhibits p300-dependent acetylation of ETV1, thereby promoting its degradation. These results point to a new avenue for pharmacological ETV1 inhibition and may inform a general means to discover small molecule perturbagens of transcription factor oncoproteins

A Small Molecule That Binds and Inhibits the ETV1 Transcription Factor Oncoprotein

Members of the ETS transcription factor family have been implicated in several cancers, where they are often dysregulated by genomic derangement. ETS variant 1 (ETV1) is an ETS factor gene that undergoes chromosomal translocation in prostate cancers and Ewing sarcomas, amplification in melanomas, and lineage dysregulation in gastrointestinal stromal tumors. Pharmacologic perturbation of ETV1 would be appealing in these cancers; however, oncogenic transcription factors are often deemed “undruggable” by conventional methods. Here, we used small-molecule microarray screens to identify and characterize drug-like compounds that modulate the biologic function of ETV1. We identified the 1,3,5-triazine small molecule BRD32048 as a top candidate ETV1 perturbagen. BRD32048 binds ETV1 directly, modulating both ETV1-mediated transcriptional activity and invasion of ETV1-driven cancer cells. Moreover, BRD32048 inhibits p300-dependent acetylation of ETV1, thereby promoting its degradation. These results point to a new avenue for pharmacologic ETV1 inhibition and may inform a general means to discover small molecule perturbagens of transcription factor oncoproteins.National Cancer Institute (U.S.) (Initiative for Chemical Genetics Contract N01-CO-12400)National Cancer Institute (U.S.) (Cancer Target Discovery and Development Network RC2 CA148399

Requirements for selective recruitment of Ets proteins and activation of mb-1/Ig-α gene transcription by Pax-5 (BSAP)

Pax-5, a member of the paired domain family of transcription factors, is a key regulator of B lymphocyte-specific transcription and differentiation. A major target of Pax-5-mediated activation is the mb-1 gene, which encodes the essential transmembrane signaling protein Ig-α. Pax-5 recruits three members of the Ets family of transcription factors: Ets-1, Fli-1 and GABPα (with GABPβ1), to assemble ternary complexes on the mb-1 promoter in vitro. Using the Pax-5:Ets-1:DNA crystal structure as a guide, we defined amino acid requirements for transcriptional activation of endogenous mb-1 genes using a novel cell-based assay. Mutations in the β-hairpin/β-turn of the DNA-binding domain of Pax-5 demonstrated its importance for DNA sequence recognition and activation of mb-1 transcription. Mutations of amino acids contacting Ets-1 in the crystal structure reduced or blocked mb-1 promoter activation. One of these mutations, Q22A, resulted in greatly reduced mb-1 gene transcript levels, concurrent with the loss of its ability to recruit Fli-1 to bind the promoter in vitro. In contrast, the mutation had no effect on recruitment of the related Ets protein GABPα (with GABPβ1). These data further define requirements for Pax-5 function in vivo and reveal the complexity of interactions required for cooperative partnerships between transcription factors

The Autophagy-Related Beclin‑1 Protein Requires the Coiled-Coil and BARA Domains To Form a Homodimer with Submicromolar Affinity

Beclin-1 (BECN1) is an essential component of macroautophagy. This process is a highly conserved survival mechanism that recycles damaged cellular components or pathogens by encasing them in a bilayer vesicle that fuses with a lysosome to allow degradation of the vesicular contents. Mutations or altered expression profiles of BECN1 have been linked to various cancers and neurodegenerative diseases. Viruses, including HIV and herpes simplex virus 1 (HSV-1), are also known to specifically target BECN1 as a means of evading host defense mechanisms. Autophagy is regulated by the interaction between BECN1 and Bcl-2, a pro-survival protein in the apoptotic pathway that stabilizes the BECN1 homodimer. Disruption of the homodimer by phosphorylation or competitive binding promotes autophagy through an unknown mechanism. We report here the first recombinant synthesis (3–5 mg/L in an <i>Escherichia coli</i> culture) and characterization of full-length, human BECN1. Our analysis reveals that full-length BECN1 exists as a soluble homodimer (<i>K</i>_D ∼ 0.45 μM) that interacts with Bcl-2 (<i>K</i>_D = 4.3 ± 1.2 μM) and binds to lipid membranes. Dimerization is proposed to be mediated by a coiled-coil region of BECN1. A construct lacking the C-terminal BARA domain but including the coiled-coil region exhibits a homodimer <i>K</i>_D 3.5-fold weaker than that of full-length BECN1, indicating that both the BARA domain and the coiled-coil region of BECN1 contribute to dimer formation. Using site-directed mutagenesis, we show that residues at the C-terminus of the coiled-coil region previously shown to interact with the BARA domain play a key role in dimerization and mutations weaken the interface by ∼5-fold

The Creston, California, meteorite fall and the origin of L chondrites

It has been proposed that all L chondrites resulted from an ongoing collisional cascade of fragments that originated from the formation of the similar to 500 Ma old asteroid family Gefion, located near the 5:2 mean-motion resonance with Jupiter in the middle Main Belt. If so, L chondrite pre-atmospheric orbits should be distributed as expected for that source region. Here, we present contradictory results from the orbit and collisional history of the October 24, 2015, L6 ordinary chondrite fall at Creston, CA (here reclassified to L5/6). Creston's short 1.30 +/- 0.02 AU semimajor axis orbit would imply a long dynamical evolution if it originated from the middle Main Belt. Indeed, Creston has a high cosmic ray exposure age of 40-50 Ma. However, Creston's small meteoroid size and low 4.23 +/- 0.07 degrees inclination indicate a short dynamical lifetime against collisions. This suggests, instead, that Creston originated most likely in the inner asteroid belt and was delivered via the nu(6) resonance. The U-Pb systematics of Creston apatite reveals a Pb-Pb age of 4,497.1 +/- 3.7 Ma, and an upper intercept U-Pb age of 4,496.7 +/- 5.8 Ma (2 sigma), circa 70 Ma after formation of CAI, as found for other L chondrites. The K-Ar (age similar to 4.3 Ga) and U,Th-He (age similar to 1 Ga) chronometers were not reset at similar to 500 Ma, while the lower intercept U-Pb age is poorly defined as 770 +/- 320 Ma. So far, the three known L chondrites that impacted on orbits with semimajor axes a AU all have high (>3 Ga) K-Ar ages. This argues for a source of some of our L chondrites in the inner Main Belt. Not all L chondrites originate in a continuous population of Gefion family debris stretching across the 3:1 mean-motion resonance.Peer reviewe