Supplementary Material. Targeted disruption of Myc-Max oncoprotein complex by a small molecule

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1 Supplementary Material Targeted disruption of Myc-Max oncoprotein complex by a small molecule Author: Seung H. Choi, Madhupriya Mahankali, Sang Jun Lee, Mitch Hull, H. Michael Petrassi, Arnab K. Chatterjee, Peter G. Schultz, Katherine A. Jones and Weijun Shen Inventory of Supplementary Material Supplementary figures and legends - Figure S1 page 2 - Figure S2 page 3 - Figure S3 page 4 - Figure S4 page 4 - Figure S5 page 5 - Figure S6 page 5 - Figure S7 page 6 Supplementary Experimental Procedures page 7 References page 11 1

2 Supplementary figures and legends Figure S1. High-throughput screening of Myc-Max PCA A Positive control F4 inhibits Myc-Max PCA activity. B Flow chart for hits identification and confirmation. C sajm589 is shown in a representative dot plot of a 50,000 small molecule library screening. 2

3 Figure S2. Dose response effect of sajm589 on cellular proliferation A Dose dependent inhibition of P493-6 cellular proliferation by sajm589. 1X10 5 of P493-6 cells were treated with indicated concentrations of sajm589 and numbers of live cells were counted each day and normalized to day 0 (100%). Tetracycline (0.1 µm) treatment is shown as a positive control. B P493-6 cellular proliferation in the presence with tetracyclin (Tet-On). C HepG2 cellular proliferation in the presence of sajm589. D, E, F Inhibition of cellular proliferation by sajm589 in time course in Ramos, HL-60 and KG1a cells respectively. Similar to A, 1X10 5 cells were initially plated and treated with different concentrations of sajm589. Numbers of live cells were counted each day and normalized to day 0 (100%). 3

4 Figure S3. Medicinal chemistry optimization of sajm589 A Structures of sajm589 analogs are shown. B The IC 50 values in biochemical PCA assay and P493-6 cell based assay for sajm589 analogs. P493-6 cells were treated with compounds for 48 h after which cell viability was measured. Cell viability was normalized with DMSO treatment. Figure S4. Effect of sajm589 in PCA of human Jun:Fos, Nrf2:Maf heterodimer, and yeast Gcn4 homodimer PCA was measured from HEK293 cells after transfection with each pair of PCA constructs. PCA was normalized with DMSO treatment. 4

5 Figure S5. sajm589 has no effect in MYC mrna expression level. MYC mrna levels were measured by qrt-pcr analysis in of P493-6 and Raji cells shown in A and B respectively, upon treatment with different concentrations of sajm589for 24 h with DMSO as vehicle control. Tetracycline (697 genes; -1 > Log 2 ) sajm589 (565 genes; -1 > Log 2 ) Figure S6. A high correlation in transcriptional profiles between sajm589 and tetracycline treatments in P493-6 cells. Total-RNAs from P493-6 cells with the presence of either sajm589 (20 µm, 24 hr) or tetracycline (0.1 µm, 24 hr) were subjected to quantitative NGS analysis with threshold set (-1 > Log 2 ). Numbers of down-regulated genes by treatments and its correlation displays in diagrams. 5

6 A Embedment 1 st 2 ed 3 rd 4 th Colony formation Days B sajm589 (µm) Figure S7. sajm589 inhibits anchorage independent growth of cancer cells. 2 x 10 3 of Burkitt lymphoma, Raji cells were embedded in agarose-matrix media. sajm589 was applied as shown in the experimental scheme in (A). Colony formation at 3 weeks was shown in (B) and quantified as the bar graph (Figure 4A). 6

7 Supplemental Experimental Procedures Cell lines and culture HEK293 and HCT116 cells were cultured in DMEM, supplemented with 2 mm L-Glutamine and 10% Fetal Bovine Serum in 5% CO 2 at 37 C. P493-6 cells were grown in RPMI 1640 with 10% tetracycline-approved FBS. Ramos and KG1a cells were grown in RPMI1640 with 10 % FBS and HL-60 cells were grown in Iscove s Modified Dulbecco s Medium with 10% FBS. All the cell lines were maintained in 5% CO 2 at 37 C. PCA construction General PCA principle and methods were explained previously 1,2. In detail, Myc-Max PCA cell line was established in HEK293 cell. B-HLH-LZ domain from Myc ( a.a) was fused to the C-terminus of split Gaussia luciferase (GLuc2) to generate FLAG-Myc:GLuc2 fusion protein under CMV-driven mammalian expression vector with IRES-Neo (FLAG-Myc:GLuc2-IRES-Neo) for stable protein expression and selection. Full-length Max (1-151 a.a) including B-HLHZ-LZ domain was fused to the N-terminus of split Gaussia luciferase (GLuc1) to generate HA-Max:GLuc1 fusion protein in CMV-driven mammalian expression vector with IRES-Puro (FLAG-Myc:GLuc2- IRES-Puro) for stable protein expression and selection. For Max-Max homodimer interaction, fulllength Max was fused to GLuc2 to generate Max:GLuc1 fusion protein in pcdna3 (pcdna3- Max:GLuc1). These constructs were co-transfected in 293 cells and double selected with G418 (100 ug/ml)/puromycin (1 ug/ml) in DMEM media containing 10% FBS for two weeks and maintained in the selection media. High throughput screening by PCA assay PCA assay was optimized with reported Myc-Max inhibitor F4, and miniaturized to well format for high throughput screening with a Z factor > 0.75 and good assay window (~ 8-7

8 fold). Cell lysate from HEK293 Myc/Max-GLuc reporter cell line was dispensed (3 µl/well) in a white solid bottom 1536-well plate where 20 nl of compounds (20 µm final concentration) or DMSO control were pre-spotted. The plates were incubated at 37 o C for 3 h followed by addition of 3 µl of renilla Glo substrate, incubated for 30 min at room temperature and the luciferase activity was recorded in Viewlux. Compounds that showed > 30% inhibition were identified as hits and confirmed in triplicates, followed by 8-point dose response confirmation. Cell viability assays P493-6, Ramos and HL60 cells were seeded at a density of 3000 cells/well in a 384 well plate and KG1a cells were seeded at 5000 cells/well. Cells were treated with compounds of interest in 12-point dose response and incubated for 2 days at 37 C in 5% CO 2 and 95% relative humidity. Cell viability was measured by luminescence upon addition of 10 µl of cell titer Glo reagent. cdna synthesis and quantitative Real-time PCR Total RNA was extracted using TRIzol reagent (Life Technologies, Carlsbad, CA) and treated with DNase (Life Technologies, Carlsbad, CA). Reverse transcription (Qiagen, Hilden, Germany) was performed with 1µg of total RNA. Quantitative real-time PCR was utilized to evaluate expression levels from the cdna, using absolute SYBR Green real-time PCR master mix (Thermo Fisher Scientific, Waltham, MA) and a real-time PCR thermocycler (Eppendorf, Hamburg, Germany). Expression level of each gene was measured and normalized with 18S ribosomal RNA or GAPDH expression. Immunoprecipitation Immunoprecipitation assay was described previously 2,3. In detail, PCA cells cultured in a 100 mm dishes were lysed in immunoprecipitation (IP) buffer (50 mm HEPES-NaOH, ph 7.9, 300 mm 8

9 NaCl, 1% NP-40, 10 mm MgCl 2 and 15% glycerol) with protease inhibitors. Protein extracts were clarified by centrifugation (14,000 x g for 15 min at 4 C) and incubated with anti-ha affinity gel (Sigma, St. Louis, MO) in a ratio of 20 µl slurry (50% beads in slurry) to 1 ml of total lysate for 4 h on a rotisserie at 4 C. The beads were washed four times at 4 C in IP-wash buffer containing 25 mm HEPES-NaOH ph 7.9, 300 mm NaCl and 0.2% NP-40 and 50 µl of 1x SDS sample buffer was added to the slurry and heated to 95 o C to elute bound proteins. For endogenous proteins, Max monoclonal antibody (Santa Cruz, Dallas, TX) was mixed with 1 ml of clarified lysate (5 mg/ml of total protein) and incubated overnight on a rotisserie at 4 C. Immune complex was captured by addition of 50 µl of protein A/G agarose (Santa Cruz, Dallas, TX) with additional 2 h incubation. Washing and elution were performed as described above. Electrophoretic mobility shift assay (EMSA) Gel swift assay was performed using LightShift Chemiluminescent EMSA Kit (Thermo Fisher) according to the manufacturer s instruction. The monitoring probe has DNA sequence containing a E-box (5 -biotin-gctcagggaccacgtggtcggggatc-3 ). Biolayer interferometry assay (BLI) for small molecule binding Binding assays for small molecule were conducted using an Octet Red instrument (ForteBio, Menlo Park, CA, USA) in a solid black 96-well micro-plate at 30 C. Biotin tagged Myc peptide ( ) was immobilized on a streptavidin tip by incubating the sensor in 200 µl of peptide solution (1 µg/ml) for five minutes. Kinetics assay consisted of the following steps; equilibration in buffer (PBS % Tween 20; 1% BSA; 0.05% azide) for three to four minutes to establish a baseline signal, association with different concentrations of sajm589 for five minutes, followed by dissociation in the same buffer as the baseline. Data analysis was done using ForteBio Data Analysis 7.0 (ForteBio, Menlo Park, CA, USA). 9

10 Ubiquitination assay Ubiquitination assay was described previously 2,3. Briefly, to assay ubiquitination of endogenous Myc protein, P493-6 cells (4 x 10 6 ) were treated with either sajm589 (20 µm) and/or MG-132 (5 µm) for 12 h. Cells were lysed with 1 ml of IP buffer and clarified by centrifugation (14,000 x g for 15 min at 4 C). 10% of clarified lysates were saved for input and the remaining lysates were incubated with 4 µg of anti-cmyc antibody (Santa Cruz, Dallas, TX) to immunoprecipitate Myc protein complex. After overnight incubation with rotation at 4 C, protein A/G was added to capture immune complex with additional incubation for 2 h at 4 C. Protein A/G was washed three times with IP-wash buffer and SDS sample buffer was added to elute the captured proteins. Isolated protein complex was resolved in SDS-PAGE and probed with indicated anti-sera. Determination of protein half-life To measure the half-life of endogenous cmyc protein, P493-6 cells (1 x 10 6 ) were treated with cycloheximide (CHX, 50 µg/ml final concentration) and incubated for indicated times. Cells were lysed in RIPA buffer (50 mm Tris-Cl, ph 7.7, 150 mm NaCl, 1 mm EDTA, 1% NP-40 (v/v), 0.1% SDS (w/v), 0.1% Sodium Deoxycholate (w/v)). After centrifugation (14,000 x g for 15 min at 4 C), soluble fractions were subjected to SDS-PAGE and immunoblot analysis, and quantified by ImageJ ( to calculate the half-life. Western blots Following antibodies were used for immunoblot assays. cmyc, Max and Ubiquitin (Santa Cruz, Dallas, TX), FLAG and HA (Sigma, St. Louis, MO), GAPDH (Cell Signaling, Danvers, MA). These antibodies were probed for overnight in TBS with 0.1% Tween 20 (v/v) and 5% skim milk (w/v). RNA Sequence analysis 10

11 High-throughput RNA sequencing was carried out at the Next Generation Sequencing Core, the Salk Institute using Illumina HiSeq 2500 device. Cell proliferation assays P493-6, Ramos and HL-60 cells at a density of 1 x 10 5 cells/well and KG1a cells at 2 x 10 5 cells/well were seeded in a 24-well plate and treated with 0, 10, 20 or 50 µm sajm589. For P493-6 cells, 0.1 µg/ml tetracycline was used as positive control. Following compound addition, the cell suspension was mixed well to ensure the homogenous distribution of the compound. Twenty microliter samples were taken at different time points (0, 24, 48, 72 and 96 h) of incubation, mixed with equal amount of trypan blue dye. Live cells were counted in Biorad automatic cell counter. Anchorage-independent assays The assay was described previously 1. Briefly, 2 x 10 3 of Burkitt lymphoma, Raji cells were embedded in agarose-matrix media (1 ml of RPMI 1680 containing 10% FBS and 0.4% agarose) on the top of the supporting media (2 ml of RPMI 1680 containing 10% FBS and 0.8% agarose). After solidification at RT for 30min, the plates were incubated in 5% CO 2 at 37 C. The compound was applied in 200 µl volume at indicated concentrations. Reference for Supplemental Experimental Procedures (1) Choi, S. H.; Estaras, C.; Moresco, J. J.; Yates, J. R., 3rd; Jones, K. A. Genes & development 2013, 27, (2) Michnick, S. W.; Ear, P. H.; Manderson, E. N.; Remy, I.; Stefan, E. Nat Rev Drug Discov 2007, 6, 569. (3) Choi, S. H.; Wright, J. B.; Gerber, S. A.; Cole, M. D. Genes & development 2010, 24,