Supplemental Table S1: Inhibition of HDAC class I and class II family by CUDC-101 (IC50 in nm)

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1 Supplemental Table S1: Inhibition of HDAC class I and class II family by CUDC-101 (IC50 in nm) Class I Class II HDAC1 HDAC2 HDAC3 HDAC8 HDAC4 HDAC5 HDAC6 HDAC7 HDAC9 HDAC For HDAC specificity assay, purified GST- (human HDAC1, 4, 5, 6, 7 and10) or His- (human HDAC 2, 3 and 9) tagged E. coli. expressed enzymes were used. For HDAC8, SIRT1, SIRT2 and SIRT3, full length purified E. coli. expressed enzymes were used. Substrate for HDAC8 is diacetylated peptide (Arg-His-Lys(Ac)- Lys(Ac)), which based on amino acid residues of p53 protein. For other HDAC enzymes, a single acetylated peptide (Arg-His-Lys- Lys(Ac)) was used.

2 Supplemental Figure S1: Inhibition of EGFR-T790M by CUDC-101 EGFR kinase activity was measured using the HTScan EGF receptor kinase assay kit (Cell Signaling Technology). Briefly, EGFR T790M mutant fusion protein was incubated with synthetic biotinylated peptide substrate and different concentrations of CUDC-101 and erlotinib, in the presence of 400uM ATP. Phosphorylated substrate was captured in strapavidin coated 96-well plates. Levels of phosphorylation were monitored using antiphospho-tyrosin and europium labeled secondary antibodies (DELFIA, Perkin Elmer) at a concentration of 1:1000 dilution. Enhancement solution was added at the end of the assay and enzyme activity measured in a Wallac Victor II 1420 microplate reader at 615nM.

3 Supplemental Figure S2: CUDC-101 increases acetylation of histones in cultured cancer cells A. H292 cells were treated with various concentrations of CUDC-101, vorinostat or DMSO as indicated for 24 hrs. Cells were fixed in 4% paraformaldehyde before immunocytochemistry analysis was performed. A Li-Cor Odyssey machine was used for detection of staining results. Results indicate that CUDC-101 increases the level of acetylated histone3 protein. B. MDA-MB-468 cells were treated with various concentrations of CUDC-101, vorinostat as indicated for 24 hrs. Cells were fixed in 4% paraformaldehyde before immunocytochemistry analysis was performed. A Li-Cor Odyssey machine was used for detection and quantification of staining results. Quantification of the results reveals the relative intensities of acetylated histone-4 and total tubulin protein increase in a dose-dependent-manner with the treatment of CUDC-101.

4 Supplemental Figure S3: CUDC-101 increases acetylation of p53 in cultured cancer cells A. H292 cells were treated with 1 μm of CUDC-101, vorinostat or DMSO (control) as indicated for 24 hrs before immunocytochemistry analyses were performed. Results indicate that CUDC-101 increases the level of acetylated p53 protein. B. H292, HCT-116 and SkBr-3 cells were treated with 1 μm of CUDC-101, vorinostat or DMSO as indicated for 24 hrs. Immunocytochemistry analysis was performed and a Li-Cor Odyssey machine was used for detection and quantification of staining results. Bar graph shows the relative intensities of acetylated p53 and total p53 protein levels.

5 Supplemental Figure S4: CUDC-101 inhibits EGFR auto-phosphorylation in cultured cancer cells A431 cell line Control CUDC-101 p-egfr EGFR EGF A431 cells were cultured and starved for 8 hours before treatment with DMSO (control) or 1 μm of CUDC-101 as indicated for 1 hour. EGF was added to culture medium for 5 minutes to stimulate the phosphorylation of EGFR as indicated (+). Results show that CUDC-101 inhibits EGFR phosphorylation.

6 Supplemental Table S2: CUDC-101 displays weak activities on other kinases Kinase % Inhibition of Control Kinase % Inhibition of Control Kinase % Inhibition of Control Abl 57 FGFR4 6 p38γ kinase 10 Akt1-2 FLT-3 85 PAK1 2 AMPKα 32 Fyn 1 PAK2 5 AurA 21 IGF1R 3 PDGFRβ 39 Etk -3 IKKα -3 PDK1 2 Brk 40 IRAK4 9 Pim1 32 CaMK2α 7 IRK -5 Pim2 2 CaMK4-5 JNK1 19 PKA 3 CDK1-5 JNK2 30 PKCα 0 CDK2-14 JNK3 12 PKCβ1-1 CDK5-4 KDR 64 PKCβ2-3 CHK1-1 Lck 56 PKCγ 0 CHK2 11 Lyn 95 PKG1β 7 CK2 17 MAPKAPK2 1 PKG2 11 c-met 24 MARK1 8 PYK2 8 CSK 15 MARK2 0 RAF-1 17 DAPK1 8 MARK4 8 RET 93 DAPK2 13 MEK1-3 ROCK2-3 EphB4 20 MKK6 8 RSK2 10 ERK1 1 NEK2-2 SGK1 6 ERK2 16 NEK4 15 Src 12 FGFR2 73 p38α kinase 9 Syk 1 FGFR3 15 p38δ kinase -4 TrkA 11 Kinase KDR Lyn Lck Abl-1 FGFR2 FLT3 RET IC50 (nm) In order to assess the selectivity of the EGFR and HER2 kinase activities, CUDC-101 was evaluated against a panel of 69 kinases. Standard kinase activity assays with appropriate reference compounds were used for each of the evaluated kinases. An initial assessment for inhibitory activity was made using single dose (5uM) of CUDC-101. An inhibition of greater than 50% of reference compound activity was considered to be a potentially significant effect and for those targets IC50s were measured. For the 69 enzyme kinases tested, 5 um of CUDC -101 inhibited Abl, FGFR2, Flt-3, KDR (VEGFR2), Lyn, Lck and Ret at a level of higher than 50% of reference compounds. Subsequently, the inhibition of KDR, Lyn, Lck, Abl-1, FGFR2, Flt-3 and Ret by CUDC-101 was determined with IC50 equal to 0.86uM, 0.84uM, 15.8uM, 2.89uM, 3.43uM, 1.5uM and 3.2uM, respectively. Additionally the IC50 of CUDC-101 against Src was determined to be 11 um. Overall, the results show that CUDC-101 potently inhibits EGFR and HER2 kinases (IC50s of 4 and 20 nm, respectively) at concentrations that are at least 40 fold lower than the other kinases evaluated in this

7 panel.

8 Supplemental Figure S5: CUDC-101 rapidly and durably inhibits Akt signaling BT-474 cells were cultured and treated with DMSO or 1 μm of CUDC-101 or vorinostat as indicated. Cell extracts were prepared after 0, 3, 8, 24 or 30 hours of treatment as indicated and Western analyses were performed. This time course studies were designed to monitor molecular events before massive protein degradation occurred due to apoptosis. CUDC- 101 showed a rapid and prolonged inhibition of Akt signaling in cultured cancer cells, with no subsequent reactivation during 30 hours of continuous exposure. Vorinostat also showed similar late-stage inhibition of Akt signaling, but the latter effect was observed only after 8 hours of treatment.

9 Supplemental Figure S6: CUDC-101 reduces estrogen receptor alpha protein levels BT-474 cells were treated with varying concentrations of CUDC-101, vorinostat, lapatinib or erlotinib as indicated for 0, 2, 7 or 24 hours before Western analyses were performed. Results show a significant reduction in estrogen receptor-α levels in CUDC-101 treated cells. Weaker effects are seen in vorinostat treated cells.

10 Supplemental Figure S7: CUDC-101 induces no significant change in body weight in treated animals A C % Body Weight Change E Body Weight Change (%) Vehicle CUDC-101 Vorinostat Erlotinib 25 mg/kg Days same experiment as Fig. 4A, HepG2 Cell Line Vehicle CUDC-101 Lapatinib Days same experiment as Fig. 4B, upper right, MDA-MB-468 cell line Body Weight Change (%) Vehicle CUDC-101 Paclitaxel C + P Days Same experiment as Fig. 4B, lower right panel, MDA-MB-468 cell line B Body Weight Change (%) Vehicle CUDC Days same experiment as Fig. 4B, upper left panel, A549 cell line D Body Weight Change (%) Vehicle CDUC Days Same experiment as Fig. 4B, lower left panel, Cal-27 cell line F

11 A-E. These results depict body weight results for the same experiment shown in Fig. 4. No significant weight loss was observed in tumor explanted animals treated with CUDC-101. Body weights were determined twice weekly for the duration of the study. F. Body weight results of the rat toxicity study. This was a 2-cycle study, with each cycle lasting 7 days and separated by a nondosing period of 7 days. CUDC-101 formulations were administered on study days 1 to 7 and 15 to 21 by intravenous infusion over a 30-minute period with different dose levels as indicated. Male and female animals were indicated as solid and dash lines respectively.

12 Supplemental Table S3: In vitro studies of CUDC-101 and its interactions with key receptors, channels and enzymes Summary of binding assays Protein % Inhibition of Control Protein % Inhibition of Control Protein % Inhibition of Control A1 34 AMPA 17 P2Y 12 A2A 55 Kainate 8 Rolipram 30 A3 78 NMDA 37 5-HT -3 α1 11 H1-1 σ 12 α2 11 H2 28 Glucocorticoid -20 β1 0 H3 7 Estrogen 1 β2 15 I1 0 Progesterone 3 AT1-3 I2 26 Androgen 0 AT2 3 LTB4 7 TRH1 3 BZD (central) 19 LTD4 6 V1a 8 B1 1 MC4-3 V2 2 B2-1 M 28 Ca 2+ channel (L, DHP) 17 CB1-1 NK1 93 Ca 2+ channel (L, diltiazem) 0 CB2-5 NK2 76 Ca 2+ channel (L, verapamil) 6 CCK1-3 NK3 38 K + ATP channel -4 CCK2 7 Y -15 K + V channel -3 CRF1-9 N 2 SK + ca Channel -1 D1 8 Opioid 65 Na + channel 12 D2S 5 ORL1 7 Cl - channel 13 D3-3 PPARγ 5 NE transporter 52 D4.4 2 PCP 21 DA transporter 47 ETA 10 EP4-3 GABA transporter 6 ETB 11 IP -3 Choline transporter 60 GABA 1 P2X 13 5-HT transporter 9 Summary of enzyme activity assays Enzyme % Inhibition of Control Enzyme % Inhibition of Control Enzyme % Inhibition of Control PDE1 53 COMT 15 Tyrosine hydroxylase 21 PDE2 75 ATPase 1 Acetylcholinesterase -20 PDE3 21 MAO-A -5 GABA Transaminase 5 PDE5 96 MAO-B 5 PNMT 4 % Stimulation of % Stimulation of Enzyme Enzyme Control Control Adenylyl cyclase 2 Guanylyl cyclase 5

13 We evaluated the potential interaction of CUDC-101 with major proteins, enzymes and channels known to be clinically relevant. Using standard binding competition or enzyme activity assays, the first screen was performed with a high concentration and single dose (10uM) of CUDC-101 against a panel of 86 targets. The percent inhibition by CUDC-101 relative to the specific binding of reference ligands to each target protein was obtained. Among the 86 proteins, enzymes and channels we studied, 10uM CUDC -101 inhibited the binding of labeled ligands to adenosine receptor A2A, A3, NK1, NK2, opioid receptor, NE transporter and choline transporter at a level of more than 50% of the control. The inhibition of NK1 receptor binding by CUDC-101 was shown to be greater than 90%. Subsequently, the binding affinity of CUDC-101 to NK1 was determined to have a ki of 2.3uM. CUDC-101, at 10uM, also inhibits PDE1 and PDE5 at a level of more than 50% of that of reference compound. These weak activities of CUDC-101 against other targets are predicted to be of minor concern for its future clinical development.

14 Supplemental Table S4: CUDC-101 displays a favorable safety profile in in vivo toxicity studies A 2-cycle 28-day dose toxicity study of CUDC-101 in Sprague-Dawley rats and Beagle dogs Maximum Tolerance Dose (MTD) Rats 75 mg/kg/day or 450 mg/m2/day Dogs 40 mg/kg/day or 800 mg/m2/day Exposure, AUC (um X h) 47.7 to 91.9 (male) 33.5 to 38.7 (male) 73.9 to (female) 44.2 to 61.1 (female) Mortality No death occurred No death occurred Food consumption and body weight Normal Normal Clinical Observation Normal Mild and transient emesis and soft feces Hematology Normal Non-adversely and transiently higher total white blood cells and neutrophil counts Serum Chemistry Histopathology Electrocardiographic Assessment Mild and reversible lower cholesterol and serum potassium s Normal Not available Normal Mild and reversible changes: 1. Interstitial inflammation in lung 2. Atrophy of Peyer s pathes in lymphoid tissues (female) No drug related effects were noted Summary of toxicity study results of CUDC-101 in Sprague-Dawley rats and Beagle dogs. Animals were administered by intravenous infusion with indicated dose of CUDC-101 for two seven day cycles (study days 1 to 7 and 15 to 21), in which both cycles were followed by a 7 day recovery. Clinical examinations were performed daily. Individual body weights were recorded at least twice weekly. Food consumption was recorded daily and weekly in dogs and rats respectively. Clinical hematology and serum chemistry analysis were performed prior to the initiation of dose administration, on the day following the last infusion for each dosing cycle (study days 8 and 22) and prior to the scheduled primary necropsy for histopathological analysis (day 29).

15 Supplemental Figure S8: CUDC-101 inhibits HIF1-α induction Control CUDC-101 vorinostat erlotinib HIF1-α Tubulin HCT-116 cells were cultured and treated with 1μM of CUDC-101, vorinostat or erlotinib as indicated in the presence of 100mM of CoCl2 to induce the expression of HIF-1α. CUDC- 101 effectively represses HIF-1α induction.

16 Supplemental Figure S9: CUDC-101 down-regulates EGFR protein in H1975 NSCLC cells Control CUDC-101 erlotinib μm EGFR Tubulin H1975 NSCLC cells were cultured and treated with 0.3 or 1 μm of CUDC-101 or erlotinib as indicated for 31 hours. CUDC-101 effectively represses EGFR expression.