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Small Molecule Therapeutics

Single Agent and Synergistic Activity of the"First-in-Class" Dual PI3K/BRD4 Inhibitor SF1126with Sorafenib in Hepatocellular CarcinomaAlok R. Singh1, Shweta Joshi1, Adam M. Burgoyne2, Jason K. Sicklick3, Sadakatsu Ikeda2,Yuko Kono4, Joseph R. Garlich5, Guillermo A. Morales5, and Donald L. Durden1,5,6

Abstract

Deregulated PI3K/AKT/mTOR, Ras/Raf/MAPK, and c-Mycsignaling pathways are of prognostic significance in hepato-cellular carcinoma (HCC). Sorafenib, the only drug clinicallyapproved for patients with advanced HCC, blocks the Ras/Raf/MAPK pathway but it does not inhibit the PI3K/AKT/mTORpathway or c-Myc activation. Hence, there is an unmet med-ical need to identify potent PI3K/BRD4 inhibitors, which canbe used either alone or in combination with sorafenib to treatpatients with advanced HCC. Herein, we show that SF1126(pan PI3K/BRD4 inhibitor) as single agent or in combinationwith sorafenib inhibited proliferation, cell cycle, apoptosis,and multiple key enzymes in PI3K/AKT/mTOR and Ras/Raf/MAPK pathway in Hep3B, HepG2, SK-Hep1, and Huh7 HCCcell lines. We demonstrate that the active moiety of the

SF1126 prodrug LY294002 binds to and blocks BRD4 inter-action with the acetylated histone-H4 chromatin mark proteinand displaced BRD4 coactivator protein from the transcrip-tional start site of MYC in Huh7 and SK-Hep1 HCC cell lines.Moreover, SF1126 blocked expression levels of c-Myc in HCCcells. Treatment of SF1126 either alone or in combinationwith sorafenib showed significant antitumor activity in vivo.Our results establish that SF1126 is a dual PI3K/BRD4 inhib-itor. This agent has completed a phase I clinical trial inhumans with good safety profile. Our data support the poten-tial future consideration of a phase II clinical trial of SF1126, aclinically relevant dual "first-in-class" PI3K/BRD4 inhibitor inadvanced HCC, and a potential combination with sorafenib.Mol Cancer Ther; 15(11); 1–10. �2016 AACR.

IntroductionHuman hepatocellular carcinoma (HCC) is the most com-

mon primary malignancy of the liver, which represents the fifthmost common cancer and second leading cause of cancer-related mortality worldwide (1, 2). Prognosis of HCC is poor,and curative treatments (resection, liver transplantation, localablation) can only be applied to a limited number of patients asthe diagnosis is often made at an advanced stage of the disease(3). Sorafenib (Nexavar) is the first and only FDA-approveddrug that is clinically approved for patients with advanced HCC(4–6). Sorafenib is a tyrosine kinase inhibitor that is known to

potently inhibit VEGF receptor tyrosine kinases, viz. KDRand FLT4; serine/threonine kinases of RAF family, viz. c-Rafand B-Raf; and various growth factor receptors, specificallyplatelet-derived growth factor receptor (PDGFR; ref. 7). How-ever, the increased survival benefit related to sorafenib treat-ment is limited to about 3 months, emphasizing the need forthe development of new treatment strategies (8).

Recent publications indicate that HCC cell activation bydifferent factors is known to increase both Ras/Raf/MAPK andPI3K/AKT/mTOR signaling (9). Sorafenib, the only drug fortreatment of patients with advanced HCC inhibits Ras/Raf/MAPK pathway (8) but does not directly inhibit the PI3K/AKT/mTOR pathway, which also plays an important role inHCC proliferation. The PI3K pathway is known to be activatedin 30% to 50% of HCC cases (10). In HCC, somatic mutationof PIK3CA, enhancement of Akt and phosphorylated ribosomalprotein S6, and decrease of PTEN expression have beenreported (11–14). These observations suggest that combinedtargeting of the PI3K/AKT/mTOR and Ras/Raf/MAPK pathwaymight provide benefit in the treatment of HCC. In this regard,other laboratories have used PI3K/mTOR inhibitors PI-103 andPKI-587 in combination with sorafenib and found that thesedrugs can synergistically block Ras/Raf/MAPK and PI3K/mTORpathways (15, 16). However, these drugs are not slated to enterclinical trials for treatment of cancer patients (17–19), hencethere is an urgent need to find a PI3K drug that is in clinicaltrials and can be used to treat HCC patients.

SF1126, a pan-PI3K inhibitor, has shown antitumor and anti-angiogenic activity in a number of xenograft models (20–25).Furthermore, this drug has recently been shown to be safe (no

1Department of Pediatrics, Moores Cancer Center, University of Cali-fornia San Diego, La Jolla, California. 2Division of Hematology-Oncol-ogy,MooresCancerCenter,UniversityofCaliforniaSanDiego,LaJolla,California. 3Division of Surgical Oncology, Moores Cancer Center,University of California San Diego, La Jolla, California. 4Division ofHepatology, Department of Medicine, University of California SanDiego, La Jolla, California. 5SignalRx Pharmaceuticals, San Diego,California. 6Division of Pediatric Hematology-Oncology, UCSD RadyChildren's Hospital, University of California San Diego Health System,La Jolla, California.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

A.R. Singh, S. Joshi, and A.M. Burgoyne contributed equally to this article.

Corresponding Author: Donald L. Durden, Moores Cancer Center, University ofCalifornia San Diego, 3855 Health Sciences Drive, La Jolla, CA 92093. Phone:858-534-3355; Fax: 858-822-0022; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-15-0976

�2016 American Association for Cancer Research.

MolecularCancerTherapeutics

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Published OnlineFirst August 5, 2016; DOI: 10.1158/1535-7163.MCT-15-0976

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dose-limiting toxicity or hepatotoxicity) and have considerableefficacy in B-cell malignancies and a variety of solid tumors in aphase I clinical trial (26). SF1126 is an RGDS-conjugatedLY294002 prodrug, which is designed to exhibit increased solu-bility and bind to specific integrins within the tumor compart-ment, resulting in enhanced delivery of the active compound tothe tumor vasculature and tumor (27). A recent study has dem-onstrated that LY294002, the active moiety of SF1126, has beencocrystallized in the active site of BRD4 and inhibits BET bromo-domain binding to acetylated lysine-binding sites within chro-matin (28). The bromodomain and extraterminal domain(BET) proteins recently emerged as important therapeutictargets in several types of hematopoietic cancers (29–32).Bromodomains are protein motifs that primarily bind toacetylated lysine residues, including those on histone tails(33). The BET family proteins (BRD2, BRD3, BRD4, BRDT)contain two amino-terminal bromodomains and have recent-ly been recognized in the literature as a therapeutic strategy totarget c-Myc (32). c-Myc transcription factor is frequentlyupregulated in a variety of human cancers (34), includingliver cancer and targeting c-Myc is considered as a novelapproach to treat HCC (35). Recent studies have shown thatBRD4 is overexpressed in HCC tissues and it promotes tumorgrowth and epithelial mesenchymal transition in HCC (36,37). Recent studies have shown that JQ1 reduced HCC cellgrowth by suppressing BRD4 using MYC-dependent or -inde-pendent pathway (37, 38). Focusing on the novel dual inhib-itory activity of SF1126 toward PI3K and BRD4, the aim ofthis study was to evaluate the antiproliferative effect ofSF1126 and sorafenib as single agents and in combinationevaluate their effects on the PI3K/AKT/mTOR, Ras/Raf/MAPK,and Myc signaling pathways in preparation for a phase II trialin HCC.

Materials and MethodsTissue culture, cell lines, and reagents

The human HCC cell lines, Hep3B, HepG2, SK-Hep1 wereobtained from ATCC. The Huh7 cell line was obtained fromJapanese Collection of Research Bio resources. All cell lineswere authenticated by short tandem repeat DNA profiling at therespective cell banks and were maintained as recommended bythe manufacturers. These cells were cultured in DMEM (Invi-trogen) supplemented with 10% FBS, 2 mmol/L glutamine and1% penicillin–streptomycin at 37�C in a 5% CO2 atmosphere.SF1126 was provided by SignalRx Pharmaceuticals. JQ1 was agift from James Bradner (Dana-Farber Cancer Institute, Boston,MA). Antibodies specific for AKT, pAKT, ERK, pERK, pP70S6K,P70S6K, and tri-methyl histone H3 (Lys4) were obtained fromCell Signaling Technology. Normal rabbit IgG, protein A/Gagarose beads, and c-Myc antibodies were from Santa CruzBiotechnology, and anti-BRD4 antibody was obtained fromBethyl Laboratories.

Cell viability and synergy analysisHep3B, HepG2, SK-Hep1, and Huh7 cells (4 � 104 cells/

well) were plated in 96-well plates in DMEM containing 10%FBS and 2 mmol/L L-glutamine. Cells were incubated overnightand treated with DMSO (0.1% final) or different concentrationsof SF1126 or sorafenib alone and in combination (1:1) ofsorafenib þ SF1126 for 48 hours. AlamarBlue (Roche) was

added and plates were incubated at 37�C in 5% CO2 for 6hours. Fluorescence signals were read as emission at 590 nmafter excitation at 560 nm. IC50 values were calculated byplotting fluorescence intensity versus drug concentration. Syn-ergy studies were performed on these cell lines using Chou–Talalay method reported earlier (39) as described in Supple-mentary Methods.

Cell cycle and apoptosis studiesFor apoptosis studies, cells were treated with inhibitor for 24

hours, followed by caspase-3 activity assay using kit (Roche) orstaining with Annexin V FITC antibody and propidium iodide(PI) according to the manufacturer's instructions (BD Pharmin-gen) as reported earlier (25). For cell-cycle analysis, DNAcontent was analyzed with FACSCalibur flow cytometer (BDBiosciences).

Western blot analysisFor all Western blots, 2 � 106 cells were plated in 10-cm

tissue culture dishes, were allowed to adhere for 36 hours, andwere treated with different concentrations of SF1126 (1, 5, 10,20 mmol/L), sorafenib (1, 10 mmol/L) either alone or incombination (0.5 mmol/L each or 0.25 mmol/L each) for 30minutes. Whole cell lysates were prepared using RIPA buffercontaining protease inhibitor cocktail (Roche Molecular Bio-chemicals). Clarified lysates were resolved in 10% SDS-PAGE,transferred to PVDF membrane, and probed for differentantibodies.

Molecular modeling of SF1126/LY294002 in BRD4 BD1 siteand BRD4 binding assays

The crystallographic atomic coordinates of BRD4-BD1 cocrys-tallized with JQ1 (PDB code 3MXF) were obtained from theProtein Data Bank (40). To model the binding of LY294002 andJQ1 at the key acetyl-lysine recognition pocket, the PDB file wasimported into LeadIT (BioSolveIT GmbH), all water moleculeswere kept, residues around JQ1within a grid of 7 Å3 were selectedand used for in silico docking calculations. The 3D structures ofLY294002 and JQ1 (all hydrogens included) were docked usingLeadIT's standard parameters. Compounds LY294002 and JQ1were tested for BRD4-1 and BRD4-2 activity by using Histone H4peptide (1-21) K5/8/12/16Ac-Biotin as a ligand in alpha screenbinding assay. The test was performed in collaboration withReaction Biology.

RNA extraction and reverse transcription PCRTotal RNA was extracted using the Qiagen RNAeasy Kit

(Qiagen) and reverse transcribed using iscript cDNA Syn-thesis Kit (Bio-Rad). Amplification of cDNA was performedwith 1� SYBR Green supermix (Bio-Rad) on an CFX96 Realtime system (Bio-Rad). cDNAs were amplified using specificc-Myc primers and primer sequences will be available uponrequest.

Chromatin immunoprecipitation studiesHuh7 and SK-Hep1 cells were treated with SF1126 (10 mmol/L),

JQ1 (1 mmol/L), or vehicle control for 24 hours. Cells werethen harvested for chromatin immunoprecipitation (ChIP) assayas previously reported (32) and described in SupplementaryMethods. ChIP and input DNA were analyzed by real-time PCR

Singh et al.

Mol Cancer Ther; 15(11) November 2016 Molecular Cancer TherapeuticsOF2




analysis as described before (24) using previous publishedprimers against the MYC transcriptional start site (41) and anegative region upstream of MYC (32). Fold enrichment overcontrol antibody and over the negative region upstream of MYCwas determined from duplicate PCR reactions according to

the formula 2ðCtMYC�Ct control antibodyÞ or 2ð

Ct MYC� Ct negative regionÞ,

respectively.

Animal studiesAll procedures involving animals were approved by the

University of California San Diego Animal Care Committee.Eight million SK-Hep1 cells or 10 million Huh-7 cells in 100-mLPBS were injected subcutaneously into the right flank of NSGmouse. Tumor dimensions were recorded regularly using Ver-nier calipers. Tumor volume was measured using the followingformula: volume ¼ 0.5 � length � (width)2. For SF1126 andsorafenib combination studies, treatment was initiated whentumors reached approximately 50 mm3. After 15 days ofimplantation of SK-Hep1 and 25 days of Huh-7 tumor implan-tation, mice were divided into four groups (n ¼ 7–8 mice pergroup). Mice in group 1 were treated with acidified water(vehicle control), Group 2 treated with SF1126 (50 mg/kg/day), Group 3 treated with sorafenib (25 mg/kg), and Group 4,

SF1126 þ sorafenib (50 and 25 mg/kg, respectively). Mice ineach group were treated for 6 days a week till the termination ofthe experiment.

Statistical analysisThe Student t test was used to evaluate differences observed

between experimental groups and to compare tumor volumedifferences between SF1126 treated, combination treatment ofSF1126 and sorafenib, and vehicle-treated controls.

ResultsSF1126 and sorafenib as single agents or in combinationinhibited HCC proliferation in a dose-dependent fashion

To test the effect of SF1126 on HCC cell proliferation, wechose four different established HCC cell lines, Hep3B,HepG2, SK-Hep1, and Huh7. SF1126 as a single agent inhib-ited proliferation of all the cell lines tested (Fig. 1). The IC50 ofSF1126 for Hep3B, HepG2, SK-Hep1, and Huh7 cells wasfound to be 5.05, 6.89, 3.14, and 2.14 mmol/L, respectively(Fig. 1A–D). Importantly, these IC50 concentrations are wellwithin the pharmacokinetic levels of SF1126 achieved in thehuman phase I trial (26). Next, we evaluated the potency of thecombination of SF1126 and sorafenib in HCC proliferation.

Figure 1.

SF1126 and sorafenib as single agents or in combination inhibited HCC proliferation. A–D, 4 � 104 cells (Hep3B, HepG2, SK-Hep1, and Huh7) were grown in96-well plates overnight, then treated with SF1126 or sorafenib (0.0978–100 mmol/L) alone and in combination of SF1126þ sorafenib (1:1) for 48 hours followed byaddition of Alamar Blue as described in Materials and Methods.

Synergistic Activity of SF1126 and Sorafenib in HCC

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SF1126 and sorafenib inhibit cell-cycle progression by inducing apoptosis. Hep3B, HepG2, SK-Hep1, and Huh7 cells were treated with SF1126 or sorafenib (1, 5,and 10 mmol/L concentrations) and in combination of SF1126 þ sorafenib (1:1) at 0.5 and 2.5 mmol/L concentrations and were incubated for 24 hours.Cell-cycle distribution was determined by DNA staining with PI and flow cytometry. A–D, left shows the fraction of cells in each phase (M, S, and G1 phase) ofthe cell cycle in Hep 3B, Hep G2, SK-Hep1, and Huh7, respectively. Right, the fraction of cells in G1 phase in all the cell lines. Data are shown as mean � SEM.� , P < 0.05; �� , P < 0.01; and �� , P < 0.001 vs. vehicle, t test.

Singh et al.





All four cell lines were treated with different concentrations ofSF1126 and sorafenib. The combination of SF1126 and sor-afenib resulted in an increased inhibition of HCC proliferation(Fig. 1). As expected, differences were noted in the sensitivityfor each cell line for each single agent and combinationtreatment. For Hep3B cells, IC50 for sorafenib and combina-tion of SF1126 and sorafenib was found to be 6.52 and 2.74mmol/L, respectively (Fig. 1A). For HepG2 cells, IC50 forsorafenib and combination of drugs was found to be 8.6 and1.79 mmol/L, respectively (Fig. 1B). For SK-Hep1 cells, IC50 forsorafenib and combination of SF1126 and sorafenib wasfound to be 7.6 and 0.52 mmol/L, respectively (Fig. 1C). ForHuh 7 cells, IC50s are 8.08 and 0.65 mmol/L for sorafenib aloneor in combination with SF1126, respectively (Fig. 1D). Anal-ysis of the enhanced cytotoxic effect in combination versusSF1126 or sorafenib alone was performed using the Chou–Talalay method for calculating the combination index (CI),with values <1 suggesting synergism. Combination of SF1126and sorafenib in Hep G2, SK-Hep1, and Huh7 cells resulted inCI values of 0.6, 0.7, and 0.5, which is consistent with syn-ergistic inhibition of proliferation. For Hep 3B, the CI value is1.049 suggests an additive effect of the drug combination(Supplementary Table S1).

SF1126 and sorafenib suppresses proliferation of HCC cells byinducing cell-cycle arrest and induced apoptosis

To study the growth-inhibitory mechanisms of SF1126 andsorafenib onHCC cells, we evaluated the effect of these inhibitorson cell-cycle progression and apoptosis. Cell-cycle analysisrevealed that SF1126 or sorafenib as single-agent treatmentresulted in cell-cycle arrest with a proportional increase inG0–G1 and a decrease in the number of cells in the S-phase inHep 3B, Hep G2, SK-Hep1, and Huh7 cells (Fig. 2A–D). Thecombined targeting of SF1126 and sorafenib on SK-Hep1 cellsresulted in substantial increase in the number of cells in G0–G1

phase with very few cells in S-phase (Fig. 2A–D).To determine whether the cell-cycle arrest phenotype was

associated with the induction of apoptosis, Annexin-V FITCstaining was performed. Hep3B, HepG2, SK-Hep1, and Huh7cells were incubated with various concentrations of SF1126and sorafenib for 48 hours and stained with Annexin V andPI. Table 1 and Supplementary Fig. S1A show that SF1126 andsorafenib at 1 and 10 mmol/L concentrations, respectively, dosedependently increase apoptotic cells in Sk-Hep1, Huh-7, and

Hep3B cell lines. Interestingly, HepG2 cells are not sensitive tothe apoptosis-inducing effects of SF1126 or sorafenib eitheralone or in combination (Supplementary Fig. S1A; Table 1).Supplementary Fig. S1B shows the Annexin-V FITC analysis ofSF1126 and sorafenib-treated SK-Hep1 cells.

To further validate these results, cell death assay and caspase-3assays were performed on SK-Hep1 and Huh7 cells. Supplemen-tary Fig. S2A and S2B revealed that Huh 7 and SK-Hep1 cellstreated with SF1126 either alone or in combination with sorafe-nib had increased cell death. The results shown in SupplementaryFig. S2C and S2D illustrate caspase-3 activity assay performed onHuh7 and SK-Hep1 cells suggesting that our combined treatmentof SF1126 and sorafenib induce apoptosis in both cells. Takentogether, these results suggest that SF1126and sorafenib inhibitedcell proliferation through the induction ofG1 arrest and apoptosisin a dose-dependent manner in all the cell lines tested whiledifferences in sensitivity exist.

Combination of SF1126 and sorafenib inhibits multiple keyenzymes in PI3K/AKT/mTOR and Ras/Raf/MAPK signalingpathways

SF1126 as a single agent is known to inhibit the PI3K/AKT/mTOR pathway in various cell lines (23, 27, 42). SF1126 at 1and 10 mmol/L concentrations significantly suppressed phos-phorylation of AKT, p70S6K, and 4EBP1 in Hep3B, HepG2, SK-Hep1, and Huh7 cells in a dose-dependent manner as deter-mined by Western blot analysis (Fig. 3). SF1126 supressedphosphorylation of ERK in Hep3B and Huh 7 cells, whereasthere was no effect or mild effect of this drug on pERK levelsin SK-Hep1 and HepG2 cells, respectively (Fig. 3). As expected,sorafenib showed no effect on phosphorylation of AKT,p70S6K, and 4EBP1 in HepG2, SK-Hep1, and Huh7 cells(Fig. 3B–D). Interestingly, sorafenib increased PI3K signalingin Hep3B cells (Fig. 3A). Similar to this result, a previous reporthas demonstrated that blockage/inhibition of only one of themain pathways PI3K/AKT/mTOR or Ras/Raf/MAPK, separately,can result in activation of the other pathway (16). We nexttested whether sorafenib alone or in combination with SF1126can block the key enzymes of PI3K/AKT/mTOR and Ras/Raf/MAPK pathway. As shown in Fig. 3, treatment of cells withcombination of SF1126 and sorafenib at 0.5 and 2.5 mmol/Lsuppress phosphorylation of AKT, p70S6K, 4EBP1, and ERK inall the cell lines. These results suggest that combined treatmentof SF1126 and sorafenib blocked multiple key enzymes in

Table 1. Annexin-V FITC analysis of SF1126 and sorafenib-treated HCC cells

Apoptosis Cells Control SF 1 mmol/L SF 10 mmol/L Sora 1 mmol/L Sora 10 mmol/L SF þ Sora 0.5 mmol/L SF þ Sora 2.5 mmol/L

Early stage Hep3B 0.2 � 0.05 11.3 � 1.3 43.9 � 3.7 10.4 � 1.6 18.5 � 2.5 45 � 4.3 59.6 � 2.9HepG2 0.2 � 0.01 3.7 � 0.5 4.4 � 0.7 0.6 � 0.2 0.5 � 0.07 4.5 � 1.1 4.8 � 0.6Sk-Hep1 0.2 � 0.03 6.8 � 0.8 16.8 � 1.8 1.6 � 0.1 11.6 � 2.1 40.1 � 2.0 39.8 � 1.7Huh 7 0.2 � 0.1 11.8 � 2.1 15.9 � 2.3 4.7 � 0.3 16.4 � 2.4 19.5 � 1.8 28.6 � 1.5

Late stage Hep3B 0.0 29.1 � 3.6 31.6 � 3.3 41.2 � 2.7 34.1 � 3.1 29 � 2.5 26 � 1.1HepG2 0.0 15.1 � 2.1 9.8 � 1.1 7.0 � 0.6 8.9 � 0.8 10.9 � 1.6 10.4 � 0.9Sk-Hep1 0.0 8.6 � 0.4 11.7 � 0.9 2.0 � 0.03 15.1 � 1.7 8.69 � 0.3 44.3 � 3.2Huh 7 0.0 31.7 � 3.3 35.1 � 2.4 20.9 � 1.6 17.3 � 1.9 33.9 � 2.8 24.6 � 1.7

Total apoptotic cells Hep3B 0.2 � 0.05 40.4 � 4.2 75.5 � 5.6 51.6 � 3.5 52.6 � 4.8 74 � 5.3 85.6 � 3.7HepG2 0.2 � 0.01 18.8 � 2.4 14.2 � 1.7 7.6 � 0.9 9.4 � 1.1 15.4 � 2.2 15.2 � 1.3Sk-Hep1 0.2 � 0.03 15.4 � 1.3 28.5 � 2.0 3.6 � 0.2 26.7 � 2.4 48.79 � 2.8 84.1 � 2.6Huh 7 0.2 � 0.1 43.5 � 3.8 51.0 � 3.9 25.6 � 1.1 33.7 � 2.9 53.4 � 3.0 53.2 � 2.2

NOTE: Induction of apoptosis in Hep3B, HepG2, SK-Hep1, andHuh7 cells treatedwith different concentrations of SF1126 and sorafenib, either alone or in combination,was determined by flow cytometric analysis using Annexin V/PI staining. Table shows the percentage of cells in early and late stage of apoptosis. Data are presentedas mean � SEM of three independent experiments.Abbreviations: SF, SF1126; Sora, sorafenib.






PI3K/AKT/mTOR and Ras/Raf/MAPK pathway (Fig. 3). This iscorrelated with greater antitumor activity in vivo.

Theactivemoiety of SF1126, LY294002binds to theactive site ofin BRD4 (BD1), inhibits BRD4 binding to acetyl lysine of H4chromatin mark protein, and displaces BRD4 from the MYCtranscriptional start site in HCC

Herein, we investigated the molecular interaction betweenLY294002, the active moiety of SF1126, and the BRD4 bromo-domain binding domain 1 (BD1; ref. 28). We utilized in silicomodelingof BRD4-BD1with LY294002 comparedwith JQ1 (PDBcode: 3MXF) tomap the comparative binding orientation and freeenergy of binding (DG�, kcal/mol) of these two smallmolecules inthe BRD4-BD1 active site (Fig. 4A, left). We discovered thatdocking LY294002 (BRD4-BD1 IC50 ¼ 5.3 mmol/L) and JQ1(BRD4-BD1 IC50 ¼ 33 nmol/L) into the binding sites of 3MXFpredicted the expected binding conformations of these two com-pounds as found in their corresponding BRD4-BD1 crystal struc-tures. The predicted binding affinity trend for LY294002 and JQ1was in accordance when docked in 3MXF (binding scores ¼�14.808 and �24.956 kcal/mol, respectively). Alpha screenbinding assay performed in collaboration with Reaction Biologyshowed BRD4 activity of LY294002 and JQ1using K5/8/12/16Ac-Biotin as a ligand (Fig. 4A, bottom) of 5 and 33 nmol/L, respec-tively, for BD1. Because the literature supports c-MYC as a clin-ically relevant target for the treatment of HCC (35), we firstdetermined whether SF1126 can block c-Myc expression in HCCcells. Fig. 4B–E showed that SF1126 significantly decreased c-Mycexpression in Hep3B, HepG2, SK-Hep1, and Huh7 cell lines. Inconsistence with these results, SF1126 blocked c-MYC protein

levels in all the cell lines as shown by Western blot analysis(Fig. 3). It was previously shown that BET bromodomain inhi-bitors displace BRD4 from theMYC transcriptional start site, sowenext investigated whether SF1126 is able to displace BRD4 fromthe MYC promoter. Using ChIP with a BRD4 antibody followedby quantitative real-time PCR with primers designed against theMYC transcriptional start site, we observed marked BRD4 local-ization to theMYC transcriptional start site (>10-fold enrichmentin comparison with a nonspecific locus upstream of MYC and>100-fold enrichment in comparison with a nonspecific controlantibody). This BRD4binding to theMYC transcriptional start sitewas significantly abrogated by treatment with SF1126 by 10-foldin comparison with a nonspecific locus upstream of MYC and by45-fold in comparison with a nonspecific control antibody (Fig. 4F and G). The BRD4 inhibitor JQ1 was used as a positive controlwith similar efficacy. These results demonstrate that SF1126 is adual PI3K/BRD4 inhibitor that targets multiple signaling nodesdysregulated in HCC.

Antitumor activity of SF1126 and sorafenib in humanxenografts

The antitumor activity of SF1126 in vivo was investigated in axenograft model using SK-Hep1 and Huh7 cells. Fig. 5A and Cillustrate the antitumor efficacy of SF1126 either alone or incombination with sorafenib in SK-Hep1 and Huh-7 cells, respec-tively. Mice bearing 50 mm3 tumors were treated subcutaneouslywith SF1126 at 50 mg/kg, six times a week for 3 weeks. Thecombined treatment of SF1126 (50 mg/kg, six times a week;administered subcutaneously) and sorafenib (administered intra-peritoneally; 25mg/kg, six times aweek) significantly blocked SK-

Figure 3.

Combination of SF1126 and sorafenib inhibits multiple key enzymes in PI3K/AKT/mTOR and Ras/Raf/MAPK signaling pathways. A–D, effect of SF1126 or sorafenibalone and in combination on Hep 3B, Hep G2, SK-Hep1, and Huh7 hepatocellular carcinoma cells. Cells were treated with SF1126 and sorafenib (1 and10 mmol/L concentrations) alone or in combination (0.5 and 2.5 mmol/L concentrations each) for 30 minutes followed by Western blot analysis. The membranewas probed with antibodies for pAKT(S473), AKT, pP70S6K(Thr 389), P70S6K, p4EBP1(Thr 37/46), 4EBP1, pERK(Thr 202/Tyr204), ERK, c-MYC, and b-actin.

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Hep1 and Huh 7 HCC tumor growth in vivo (Fig. 5A and C).SF1126 either alone or with sorafenib produced tumor growthinhibitionwithno evidenceof toxicity, asmeasuredbyweight lossrelative to control animals or drug related lethality (data notshown). Interestingly, the levels of c-MYC, pAKT, and pERK weredecreased in tumors excised from the SF1126 or sorafenib or drugcombination treatedmice at 1hour after drug administration (Fig.5B and D). These results suggest that combined SF1126 andsorafenib treatment suppresses HCC tumor growth through thedownregulation of c-MYC, PI3K/AKT/mTOR, and Ras/Raf/MAPKpathways in vivo.

DiscussionThis study was focused on the effect of SF1126 alone or in

combination with sorafenib in preclinical HCC models. We

evaluated HCC proliferation, cell cycle, apoptosis, phosphor-ylation of the key enzymes in the PI3K/mTOR/Ras/Raf path-ways, and antitumor activity in vivo. The conceptual frameworkfor the investigation SF1126 alone or in combination withsorafenib in HCC emerged from the literature in that: (i) PI3Kpathway is known to be activated in 30% to 50% of HCC cases(14); (ii) Sorafenib, the only drug FDA approved for use inadvanced HCC treatment, does not directly inhibit the PI3K/AKT/mTOR pathway; (iii) the MYC oncogene has been impli-cated in the pathogenesis and prognosis of HCC. This led to thehypothesis that a novel dual PI3K/BRD4 inhibitor, SF1126used alone or in combination with sorafenib would demon-strate potent antitumor activity in HCC. Importantly, SF1126 isan IND-enabled drug currently in clinical trials that has shownantitumor and antiangiogenic activity in a number of xenograftmodels and has recently showed considerable efficacy in B-cell

Figure 4.

SF1126 displaces BRD4 from the MYC transcriptional start site in HCC. A, top, molecular modeling diagram depicting molecular interaction between LY294002, theactive moiety of SF1126, and the BRD4 bromodomain BD1. The figure shows LY294002 and JQ1 docked at the key acetyl-lysine recognition pocket of BRD4-BD1.LY294002 is displayed with brown carbons and JQ1 with magenta carbons. Figure shows the expected hydrogen bond interaction between the carbonyl ofLY294002 and N140. Water molecules are not displayed for clarity but they were included in the in silico docking experiments. Bottom, alpha screen binding assayshowing BRD4 activity of LY294002 and JQ1 using Histone H4 peptide (1–21) K5/8/12/16Ac-Biotin as a ligand. B–E, Hep3B, HepG2, SK-Hep1, and Huh7 cells weretreated with 10 and 20 mmol/L of SF1126 for 24 hours followed by RNA isolation and real time PCR analysis of c-Myc. c-Myc levels are normalized expressedrelative to Gapdh expression. Data are shown as mean � SEM. � , P < 0.05; �� , P < 0.01; and �� , P < 0.001 versus control, t test. F, BRD4 binding to the MYCtranscriptional start site is abrogated by SF1126. Huh-7 andSK-Hep1 cellswere treatedwith SF1126 (10mmol/L), JQ1 (1mmol/L), or vehicle control for 24 hours and thenproceeded for ChIP analysis as described in Materials and Methods. Purified immunoprecipitated DNA and input DNA was amplified by quantitative real-timePCR and analyzed for enriched binding of BRD4 to theMYC transcriptional start site in comparison with a nonspecific locus upstream ofMYC (F) and in comparisonwith nonspecific control antibody (G). Data were normalized and plotted as percentage of vehicle control binding. Asterisks denote P � 0.05 in comparisonwith vehicle control by unpaired Student t test.






malignancies in a phase I clinical trial (26). A recent report thatthe active moiety of SF1126, LY294002 can be cocrystalized inthe acetyl-lysine binding pocket of BRD4 prompted us toevaluate the effect SF1126 on the potential BRD4–MYC inter-action. We observe in Huh7 and SK-Hep1 HCC cells thatSF1126 displaced BRD4 coactivator protein from the transcrip-tional start site of MYC. Our molecular modeling and BRD4binding studies demonstrate LY294002 binding to the BRD4BD1 bromodomain. These findings suggest a novel and inter-esting function of this drug as it targets both PI3K and MYCtranscription in HCC via the inhibition of BRD4 interactionwith MYC enhancer–promoter elements.

Our cell viability assay experiments indicated that SF1126 orsorafenib, as single agents, inhibited HCC cell proliferation. Acombination of SF1126 and sorafenib resulted in synergisticinhibition of HCC cell proliferation in HepG2, SK-hep1, andHuh 7 cells. The synergistic inhibition by the drug combinationtreatment suggests that both the Ras/Raf/MAPK and PI3K/AKT/mTORpathways play an important role inHCC cell proliferation.In addition, the results of cell-cycle analysis and apoptosis assaysshowed that G1 arrest and apoptosis were induced by SF1126treatment alone and these effectswere augmented in combinationwith sorafenib. These results suggest that the antiproliferativeeffects of these agents are due to G1 arrest and apoptosis in theHCC cell lines and that the combination increased G1 arrest andapoptosis in vitro.

Western blot experiments demonstrated potent inhibition ofkey enzymes of both Ras/Raf/MAPK and PI3K/AKT/mTOR path-ways when cells were treated with SF1126 and sorafenib. SF1126monotherapy strongly inhibited phosphorylation of S6K, 4EBP1,and AKT (Ser473). Sorafenib, as single agent, inhibited ERKphosphorylation, but interestingly it stimulated AKT (Ser473)phosphorylation. These results confirm the previous findings thatinhibition of one of the canonical pathways may cause stimula-tion of the pathway in HCC cells (15, 16). This supports thehypothesis that monotherapy in HCC treatment may not beefficacious. Presumably, sorafenib may induce other signals, suchas HGF/HGFR, that further stimulates the PI3K/AKT/mTOR path-way. Indeed, sorafenib stimulated HGF secretion in HCC cellspromoted c-Met, S6K, and 4EBP1 phosphorylation (43).Increased HCC activation and mobility by HGF were reportedto be mediated through PI3K signaling (44).

Recent studies have found that the Ras/Raf/MAPK and PI3K/AKT/mTOR are the dominant signaling pathways activatedin HCC cells (45). Several attempts have been made to targetboth of these pathways as therapy. In one study, everolimus,known rapamycin analogue, and AEE788, EGF/VEGF inhibitor,blocked tumor growth in Huh7 HCC cells in combinationcompared with everolimus monotherapy (46). In anotherstudy, Newell and colleagues used sorafenib and rapamycinto target mTOR and Ras/Raf/MAPK signaling and decreasedproliferation and induced apoptosis in HCC cell lines. In a

Figure 5.

Antitumor effect of SF1126 and sorafenib in human xenografts. A and C, 8 � 106 SK-Hep1 cells (A) or 10 � 106 Huh7 (C) cells were injected subcutaneously in NSGmice. After 15 days of SK-Hep1 or 25 days of Huh 7 tumor implantation when tumor volume reached up to approximately 50 mm3 mice were divided in to fourgroups (n ¼ 7–8 mice per group). Mice in group 1, treated with acidified water (vehicle control); Group 2, treated with SF1126 (50 mg/kg/day), injectedsubcutaneously; Group 3, treated with sorafenib (25 mg/kg) injected intraperitoneally; and Group 4, SF1126 þ sorafenib (50 and 25 mg/kg, respectively), 6 days aweek for 3 weeks. Arrow indicates the treatment start date (P � 0.001). B and D, one hour after the last treatment, the SK-Hep1 (B) or Huh 7 (D) HCCxenografts were removed and the expression level of pAKT, pERK, and c-MYC were analyzed byWestern blot analysis. Data in A and C are shown as mean� SEM.� , P < 0.05; �� , P < 0.01; �� , P < 0.001 versus vehicle control, t test.

Singh et al.





xenograft mouse model, the combination of rapamycin andsorafenib enhanced tumor necrosis and ulceration comparedwith sorafenib alone (47). Taken together, published reportsand unfavorable response of AZD6624, a novel MEK inhibitorin clinical settings (10), further provide evidence to our hypoth-esis that inhibiting only Ras/Raf/MAPK signaling pathway isnot efficacious in HCC treatment.

In this study, we found that SF1126 blocks both the Ras/Raf/MAPK and PI3K/AKT/mTOR pathways in the HCC cell lines.Moreover, SF1126 blocks c-Myc expression and displaces BRD4from the MYC transcriptional start site in HCC cell lines. More-over, the active moiety of SF1126, LY294002, blocks BRD4interaction with acetylated lysine residues Histone H4 peptide(1-21) K5/8/12/16Ac-Biotin, it has been cocrystallized in theactive site of BRD4 and we have executed extensive molecularmodeling of this chemotype and compared itwith JQ1 todevelopadditional more potent dual inhibitors of PI3K and BRD4 (48).Our data show that SF1126, either alone or in combination withsorafenib, significantly suppressed tumor growth in vivo in SK-Hep1 cells and Huh 7 cells. SF1126 exhibits potent dual inhibi-tion of PI3K and mTOR (mTORC1 and mTORC2). It has highbioavailability and biosafety. SF1126 has been used in preclinicalstudies to inhibit 50 human cancer types. Finally, several com-ponents are in place to rapidly translate our findings into a phaseII trial in HCC: (i) SF1126 has completed a phase I clinical trial inhumans with good safety profile where no MTD was reached andno hepatotoxicity observed (26); (ii) a well-established GMPmanufacturing and formulation plan has been established; (iii),a GMP drug supply and an active IND are in place to rapidlyadvance SF1126 into a phase II trial in advanced HCC; and (iv)based on the data generated in this report, 3 months ago wesubmitted a phase II clinical trial protocol to IND#744451 at theFDA to begin a phase II clinical trial of SF1126 in advanced HCC.In conclusion, we demonstrate that SF1126 has potent anti-HCCactivity in vitro and in vivo. Moreover, the application of SF1126 incombination with the FDA approved agent Sorafenib showssynergistic antitumor activity in vitro and in vivo, a result thatcorrelates with the inhibition of both canonical Ras/Raf/MAPKand PI3K/AKT/mTOR pathways in vivo. The results suggest theconsideration of a phase II clinical trial of SF1126 in advancedHCC and a potential combination phase I trial with sorafenib.

Disclosure of Potential Conflict of InterestJ.K. Sicklick reports receiving a commercial research grant from Novartis

Pharmaceuticals, Inc., Foundation Medicine, Inc., and Blueprint Medicines;is a consultant/advisory board member for Sirtex Medical Inc. (SIR-Spheresmicrospheres Advisory Board). Y. Kono has received speakers bureauhonoraria from Bayer and Wako and is a consultant/advisory board mem-ber for Bayer. J.R. Garlich has ownership interest (including patents) inSignalRx Pharmaceuticals. G.A. Morales is CEO at Innoventyx, LLC. D.L.Durden has ownership interest (including patents) in and is a consultant/advisory board member for SignalRx Pharmaceuticals. No potential con-flicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: A.R. Singh, S. Joshi, A.M. Burgoyne, J.K. Sicklick,Y. Kono, D.L. DurdenDevelopment of methodology: A.R. Singh, S. Joshi, A.M. Burgoyne,J.K. Sicklick, D.L. DurdenAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J.R. Garlich, D.L. DurdenAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A.R. Singh, S. Joshi, A.M. Burgoyne, J.K. Sicklick,D.L. DurdenWriting, review, and/or revision of the manuscript: A.R. Singh, S. Joshi, A.M.Burgoyne, J.K. Sicklick, S. Ikeda, Y. Kono, J.R.Garlich, G.A.Morales,D.L.DurdenAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J.R. Garlich, G.A. Morales, D.L. DurdenStudy supervision: D.L. Durden

AcknowledgmentsThe authors thank Muamera Zulcic for ordering and maintaining the mice

needed for the experiment.

Grant SupportThis work was supported by NIH grant RO1 CA94233-09, FDA RO1 grant

FD-004385, R41 CA192656 (to D.L. Durden), and grants from ALSF (Spring-board Grant) and Hyundai Hope on Wheels Foundation, Hope Grant and theOlivia Hudson Foundation NIH K08 CA168999 (to J. K. Sicklick) andP30CA023100 (to S. Lippmann).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received December 11, 2015; revised June 29, 2016; accepted July 8, 2016;published OnlineFirst August 15, 2016.

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Published OnlineFirst August 5, 2016.Mol Cancer Ther Alok R. Singh, Shweta Joshi, Adam M. Burgoyne, et al. Hepatocellular CarcinomaDual PI3K/BRD4 Inhibitor SF1126 with Sorafenib in Single Agent and Synergistic Activity of the ''First-in-Class''

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