Thalidomide promotes degradation of SALL4, a transcription factor implicated in Duane Radial Ray Syndrome, part 1
Frequently used to treat morning sickness, the drug thalidomide led to the birth of thousands of children with severe birth defects. Despite their teratogenicity, thalidomide and related IMiD drugs are now a mainstay of cancer treatment, however, the molecular basis underlying the pleiotropic biology and characteristic birth defects remains unknown. Here we show that IMiDs disrupt a broad transcriptional network through induced degradation of several C2H2 zinc finger transcription factors, including SALL4, a member of the Spalt-like family of developmental transcription factors. Strikingly, heterozygous loss of function mutations in SALL4 result in a human developmental condition that phenocopies thalidomide induced birth defects such as absence of thumbs, phocomelia, defects in ear and eye development, and congenital heart disease. We find that thalidomide induces degradation of SALL4 exclusively in humans, primates and rabbits, but not in rodents or fish, providing a mechanistic link for the species-specific pathogenesis of thalidomide syndrome.
Sample Processing Protocol
H9 hESC, Kelly, SK-N-DZ and MM1s cells were treated with DMSO, 1 µM pomalidomide, 5 µM lenalidomide or 10 µM thalidomide in biological triplicates (DMSO) or biological duplicates (pomalidomide, lenalidomide, thalidomide) for 5 hours and cells harvested by centrifugation. Lysis buffer (8 M Urea, 50 mM NaCl, 50 mM 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (EPPS) pH 8.5, 1x Roche protease inhibitor and 1x Roche PhosphoStop was added to the cell pellets and cells were homogenized by 20 passes through a 21 gauge (1.25 in. long) needle to achieve a cell lysate with a protein concentration between 0.5 – 4 mg mL-1. The homogenized sample was clarified by centrifugation at 20,000 x g for 10 minutes at 4 °C. A micro-BCA assay (Pierce) was used to determine the final protein concentration in the cell lysate. 200 µg protein for each sample were reduced and alkylated as previously described(An et al., 2017). Proteins were precipitated using methanol/chloroform. In brief, four volumes of methanol were added to the cell lysate, followed by one volume of chloroform, and finally three volumes of water. The mixture was vortexed and centrifuged at 14,000 x g for 5 minutes to separate the chloroform phase from the aqueous phase. The precipitated protein was washed with three volumes of methanol, centrifuged at 14,000 x g for 5 min, and the resulting washed precipitated protein was allowed to air dry. Precipitated protein was resuspended in 4 M Urea, 50 mM HEPES pH 7.4, followed by dilution to 1 M urea with the addition of 200 mM EPPS pH 8 for digestion with LysC (1:50; enzyme:protein) for 12 hours at RT. The LysC digestion was diluted to 0.5 M Urea, 200 mM EPPS pH 8 and then digested with trypsin (1:50; enzyme:protein) for 6 hours at 37°C. Tandem mass tag (TMT) reagents (Thermo Fisher Scientific) were dissolved in anhydrous acetonitrile (ACN) according to manufacturer’s instructions. Anhydrous ACN was added to each peptide sample to a final concentration of 30% v/v, and labeling was induced with the addition of TMT reagent to each sample at a ratio of 1:4 peptide:TMT label. The 10-plex labeling reactions were performed for 1.5 hours at RT and the reaction quenched by the addition of 0.3% hydroxylamine for 15 minutes at RT. The sample channels were combined at a 1:1:1:1:1:1:1:1:1:1 ratio, desalted using C18 solid phase extraction cartridges (Waters) and analyzed by LC-MS for channel ratio comparison. Samples were then combined using the adjusted volumes determined in the channel ratio analysis and dried down in a speed vacuum. The combined sample was then resuspended in 1% formic acid, and acidified (pH 2−3) before being subjected to desalting with C18 SPE (Sep-Pak, Waters). Samples were then offline fractionated into 96 fractions by high pH reverse-phase HPLC (Agilent LC1260) through an aeris peptide xb-c18 column (phenomenex) with mobile phase A containing 5% acetonitrile and 10 mM NH4HCO3 in LC-MS grade H2O, and mobile phase B containing 90% acetonitrile and 10 mM NH4HCO3 in LC-MS grade H2O (both pH 8.0). The 96 resulting fractions were then pooled in a non-continuous manner into 24 fractions or 48 fractions and every fraction was used for subsequent mass spectrometry analysis. Data were collected using an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) coupled with a Proxeon EASY-nLC 1200 LC pump (Thermo Fisher Scientific). Peptides were separated on a 50 cm and 75 μm inner diameter Easyspray column (ES803, Thermo Fisher Scientific). Peptides were separated using a 3 hour gradient of 6 – 27% acetonitrile in 1.0% formic acid with a flow rate of 300 nL/min. Each analysis used an MS3-based TMT method as described previously(McAlister et al., 2014). The data were acquired using a mass range of m/z 350 – 1350, resolution 120,000, AGC target 1 x 106, maximum injection time 100 ms, dynamic exclusion of 90 seconds for the peptide measurements in the Orbitrap. Data dependent MS2 spectra were acquired in the ion trap with a normalized collision energy (NCE) set at 35%, AGC target set to 1.8 x 104 and a maximum injection time of 120 ms. MS3 scans were acquired in the Orbitrap with a HCD collision energy set to 55%, AGC target set to 1.5 x 105, maximum injection time of 150 ms, resolution at 50,000 and with a maximum synchronous precursor selection (SPS) precursors set to 10.
Data Processing Protocol
Proteome Discoverer 2.2 (Thermo Fisher) was used for .RAW file processing and controlling peptide and protein level false discovery rates, assembling proteins from peptides, and protein quantification from peptides. MS/MS spectra were searched against a Uniprot human database (September 2016) with both the forward and reverse sequences. Database search criteria are as follows: tryptic with two missed cleavages, a precursor mass tolerance of 20 ppm, fragment ion mass tolerance of 0.6 Da, static alkylation of cysteine (57.02146 Da), static TMT labeling of lysine residues and N-termini of peptides (229.16293 Da), and variable oxidation of methionine (15.99491 Da). TMT reporter ion intensities were measured using a 0.003 Da window around the theoretical m/z for each reporter ion in the MS3 scan. Peptide spectral matches with poor quality MS3 spectra were excluded from quantitation (summed signal-to-noise across 10 channels > 200 and precursor isolation specificity < 0.5). Reporter ion intensities were normalized and scaled using in house scripts and the R framework(Team, 2013). Statistical analysis was carried out using the limma package within the R framework(Ritchie et al., 2015).
Eric Fischer, Dana-Farber Cancer Institute
Eric S. Fischer, Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA., Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA ( lab head )
Donovan KA, An J, Nowak RP, Yuan JC, Fink EC, Berry BC, Ebert BL, Fischer ES. Thalidomide promotes degradation of SALL4, a transcription factor implicated in Duane Radial Ray Syndrome. Elife. 2018 7 PubMed: 30067223