Targeting BRK-Positive Breast Cancers with Small …cancerres.aacrjournals.org/content/canres/early/2016/12/16/0008... · HER2 inhibitor or ER blockade to block breast cancer cell

Therapeutics, Targets, and Chemical Biology

Targeting BRK-Positive Breast Cancers withSmall-Molecule Kinase InhibitorsJie Jiang1, Fu Gui1, Zhixiang He1, Li Li1, Yunzhan Li1, Shunying Li2, Xinrui Wu1, Zhou Deng1,Xihuan Sun1, Xiaoxing Huang1,Wei Huang1, Shang Han1, Ting Zhang1, Zheng Wang1,Bo Jiao3, Siyang Song1, Hongrui Wang1, Lanfen Chen1, Dawang Zhou1, Qiang Liu2,Ruibao Ren3,4, Jianming Zhang5, and Xianming Deng1

Abstract

Approximately 80% of breast cancers overexpress the kinasebreast tumor kinase (BRK)/protein tyrosine kinase 6, which hasvarious oncogenic roles in breast cancer cell proliferation, surviv-al, and migration. However, BRK inhibitors have yet to beexplored as possible therapeutic tools. In this study, we used aparallel compound-centric approach to discover a new class ofpharmaceutical agents, exemplified by XMU-MP-2, as potent andselective BRK inhibitors. XMU-MP-2 exhibited target-specificinhibition of BRK kinase activity and disrupted signaling path-

ways mediated by this activity, thereby reducing proliferation inBRK-positive breast cancer cells. In mouse xenograft models,XMU-MP-2 repressed the growth of tumors driven by oncogenicBRK, including BRK-transformed Ba/F3 cells and BRK-positivebreast cancer cells. Notably, XMU-MP-2 cooperated strongly withHER2 inhibitor or ER blockade to block breast cancer cell prolif-eration in vitro and in vivo. Overall, our findings offer a preclinicalproof of concept for therapeutic targeting of the BRK kinase inbreast cancer. Cancer Res; 77(1); 1–12. �2016 AACR.

IntroductionProtein kinase inhibitors have been one of the major targeted

cancer therapeutics since the discovery of "Magic bullet" imatinibfor chronic myeloid leukemia (1). Albeit success of kinase inhi-bitorswidely used for variety of cancers, there aremany functionalimportant kinases left untouched because of either the uncertain-ty of pharmacologic relevance of those kinases as therapeutictargets in cancer, or lack of kinase-specific inhibitors. Of particularinterest is breast tumor kinase (BRK). There is ample evidence tosuggest the oncogenic role of BRK in variety cancers, yet none ofBRK kinase–directed approach exists in clinic (2, 3). We took

comprehensive translational approaches to address this challengespecifically targeting BRK in breast cancer.

BRK, also known as protein tyrosine kinase 6 (PTK6), is anonreceptor protein kinase originally cloned from metastaticbreast cancer (4, 5). Because of lacking canonical myristoyla-tion/palmitoylation or nuclear localization signals (6), BRK canbe localized to the nucleus, cytoplasm, and membrane. Theflexible intracellular localization extends binding partners andphosphorylation substrates of BRK. A broad range of substratesfor BRK make it involved in many signaling pathways that areessential for cancer cell proliferation, survival, and migration(3). A number of ligands, especially growth factors such as EGF(7) and insulin-like growth factors (IGF; ref. 8) that playimportant roles in tumor progression, lead to BRK activation,thereby phosphorylates a series of substrates such as STAT3 (9),STAT5a/b (10), and Akt (11), and enhances MAPK signaling(12). A vast amount of evidence has pointed to the potentialoncogenic function of BRK, such as the discrepant expressionlevels of BRK in cancer versus normal conditions. Although upto 80% breast cancer are found with the high-level expression(5-fold or more) of BRK, it is known to be low or undetectablein normal mammary tissue and benign lesions (13–16). How-ever, the questions remain whether the oncogenesis of BRK isdriven by its kinase enzymatic activity, or attributed to itsintracellular localization as an adapter by synergetic partner-ships with other tumorigenic factors.

Directly targeting kinase activity by using small molecules orknockdown the expressionof BRKwere reportedby several groups(2, 17–21); however, the question whether the small-moleculeBRK inhibitor canbe effectively used inbreast cancerwith elevatedBRK expression has not been thoroughly addressed both in celland in animal models. In addition, BRK expression is indepen-dent of ER/PR/HER2 expression, which may suggest a potentialtherapeutic opportunity whether the inhibition of BRK

1State Key Laboratory of Cellular Stress Biology, Innovation Center for CellSignaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian,China. 2Breast Tumor Center, Sun Yat-sen Memorial Hospital, Sun Yat-senUniversity, Guangzhou, China. 3State Key Laboratory for Medical Genomics,Shanghai Institute of Hematology, Collaborative Innovation Center of Hema-tology, Collaborative Innovation Center of System Biology, Ruijin HospitalAffiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China.4Department of Biology, Brandeis University, Waltham, Massachusetts. 5Cuta-neous Biology Research Center, Massachusetts General Hospital, Harvard Med-ical School, Boston, Massachusetts.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/)

J. Jiang, F. Gui, and Z. He contributed equally to this article.

Corresponding Authors: Xianming Deng, Xiamen University, South Xiang'anRoad, Xiang'an District, Xiamen 361102, China. Phone: 8659-2218-4180; Fax:8659-2218-1722; E-mail: [email protected]; Jianming Zhang, MassachusettsGeneral Hospital, Harvard Medical School, Boston, MA 02129. E-mail:[email protected]; and Ruibao Ren, Brandeis University, Waltham,MA 02453. E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-16-1038

�2016 American Association for Cancer Research.

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combining with relevant target therapeutics could address thechallenge of the treatment for aggressive ER/PR/HER2 negativebreast cancers or overcome the drug resistance against ER/PR/HER2-targeted therapy.

In this study, we address those fundamental questions intargeting BRK-positive breast cancer by developing XMU-MP-2,a small-molecule inhibitor of BRK that selectively inhibits thekinase activity of BRK and its downstream signaling, results insignificant antiproliferation potency on oncogenic BRK trans-formed Ba/F3 cells and BRK overexpressing breast cancer cells.Remarkably, pharmacologic inhibition of BRK with XMU-MP-2exhibits substantial in vivo efficacy in both BRK transformed Ba/F3cell and BRK overexpressing breast cancer mouse xenograft mod-els.More interestingly, XMU-MP-2 exhibits strong synergismwiththe combination of HER2 inhibitor or ER modulator againstbreast cancer cell proliferation. Our findings suggest that phar-macologicmanipulationof BRK signalingmight be anew targetedintervention for breast cancer.

Materials and MethodsComputational analysis of TCGA and Curtis RNA-Seq data

A The Cancer Genome Atlas (TCGA; ref. 22) dataset corre-sponding to 414 breast cancer patients and a Curtis dataset (23)corresponding to 1,852 breast cancer patients were obtained fromthe Oncomine database (https://www.oncomine.org/). Patientswere clustered into two groups according to BRK mRNA expres-sion levels by using X-tile plot (24). Kaplan–Meier curves wereplotted using SPSS and the log-rank test and P values werecalculated to determine significance.

Cell cultureThe 293T, BT-474, BT-20, MCF7, T-47D, and MDA-MB-468

cell lines were obtained from the ATCC and authenticated byshort tandem repeat testing. All the cell lines were maintainedin appropriate medium as the manufacturer suggested. Thestable transformed Ba/F3 cells were cultured in RPMI1640without IL3. The tamoxifen-resistant MCF7-TR cell line wasincubated in estrogen-free 1640 with 10% estrogen-free FBSand containing 3 mmol/L 4-hydroxytamoxifen (Sigma-Aldrich)to maintain its resistance status. All the cell lines were tested formycoplasma contamination and were found to be negative.

Plasmid constructs, virus production, and infectionpBABE-Tel-BRK, pBABE-Tel-BRK(T264M), and pBABE-Tel-BRK

(Y447F) were constructed as described in the SupplementaryMethods. Oncogenic kinase transformed Ba/F3 cell lines wereestablished as previously described (25).

Molecular dockingThe molecular docking procedure was referred to the protocol

within LeDock (Lephar Research). The protein–ligand complexcrystal structure of imatinib bound to SRC-ABL tyrosine kinaseancestor (PDB code: 4CSV; ref. 26) was chosen as the template,which shared the highest sequence similarity (56.61%) with BRK(residues 191–445, UniProtKB, Q13882). The homology modelwas generated using SWISS-MODEL (27). Water molecules weredeleted and binding site was defined as a cuboid. The initial three-dimensional conformation of compounds was optimized in theAvogadro 1.1.1 (28) using UFF force field. Compounds weredocked in the defined binding site.

In vivo tumor modelsFemale nu/nu BALB/c mice at the age of 4 to 6 weeks were

obtained from Experimental Animal Centre of XiamenUniversityand acclimated for 1 week in the pathogen-free enclosure beforestarting the studies. Mice were maintained in 12-hour light/12-hour dark cycles with free access to food andwater. All procedureswere performed in compliance with the guidelines from theInstitutional Animal Care and Use Committee at ExperimentalAnimal Centre in Xiamen University (acceptance no. XMU-LAC20120030) and Harvard Medical School. For detailed pro-cedure, please see Supplementary Methods.

Drug combination studiesBT-474 cellswere seeded at the density of 5,000 cells/well in 96-

well plates and the single or combining agents then were addedsimultaneously at fixed ratios. Data were analyzed by CalcuSynsoftware (Biosoft), using the Chou–Talalay method (29).

Statistical analysisAll data were expressed as mean � SEM. The differences

between two groups were compared by using the two-tailedunpaired Student t-test or by the one-way or two-way ANOVAwith GraphPad Prism 5.0 (GraphPad). Statistical signi-ficant levels were �, P < 0.05; ��, P < 0.01, and ��, P < 0.001.

ResultsBRK exhibits prognostic significance in breast cancer

We firstly examined the correlation between the expressionlevel of BRK and the clinical prognoses of breast cancer patients.Adopting the best cut-off percentile of BRK mRNA expressionlevel calculated by the X-tile plot, the TCGA dataset, corre-sponding to 414 breast cancer patients, was clustered into thehigh-BRK expression group (10.14%) and the low-BRK expres-sion group (89.86%; Fig. 1A). Kaplan–Meier analysis of theTCGA dataset revealed a significant negative correlationbetween patient overall survival and BRK mRNA expressionlevels, highlighting the prognostic significance of BRK (Fig. 1B).A Curtis BRK expression dataset (23), which represents 1,852breast cancer patients, was used to further validate the potentialprognostic significance of the BRK expression profile in breastcancer patients. When the best cut-off percentile of BRK mRNAexpression level divides Curtis dataset into two groups (high/low: 11.34%/88.66%), the consistent result confirmed ourprevious observation: the higher the BRK mRNA expressionlevels, the lower the patient survival rate (Fig. 1C and D). Themodest negative correlation between BRK overexpression andcancer progress, complementing earlier studies (3, 15), hasagain urged us to realize the potential oncogenic role of BRKplays in breast cancer and prompted us to take the challenge ofseeking a small-molecule inhibitor of BRK for combating BRK-positive breast cancer.

XMU-MP-2 identification and optimizationIn an effort to identify small molecules that can selectively

inhibit BRK kinase activity, we applied a novel, parallel, com-pound-centric strategy (30). We screened an in-house compoundlibrary designed to target the ATP-binding site against a panel of28 diverse tyrosine kinases using a cell-based differential cyto-toxicity assay (Fig. 2A; ref. 31). In contrast to the traditional, linearprocess of inhibitor discovery, this high-throughput kinase

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inhibitor profiling method enables a parallel approach by inter-rogating compounds against a panel of kinases in a single screen.On the basis of the identified lead scaffold with the core structureof 1,3-disubstituent-1,2,3,4-tetrahydropyrimido -[4,5-d]-pyrimi-dine, XMU-MP-2 was developed with iterative rounds of medic-inal chemistry optimization (Fig. 2B). XMU-MP-2 blocked pro-liferation and survival of BRK-transformed Ba/F3 cells with theIC50 of 29.7 nmol/L, while exhibiting a sufficient differentialcytotoxicity window against the wild-type Ba/F3 cells with theIC50 of 4,802nmol/L (Fig. 2C). The enzymatic kinase assay furtherconfirmed the binding of XMU-MP-2 to BRK with the IC50 of 5.4nmol/L by using the HotSpot technology (Fig. 2D; ref. 32).Moreover, XMU-MP-2 exhibited the best activity among 28 tyro-sine kinases profiled (Supplementary Table S1), suggesting XMU-MP-2 is a potent and selective inhibitor of BRK.

We next investigated the ability of XMU-MP-2 to inhibit BRKactivation and downstream signaling in BRK-transformed Ba/F3cells. Compound 4f (Supplementary Fig. S1), a derivative of 4-anilino-pyrido[2,3-b]indoles, was used as the control, which wasreported with the IC50 of 3.2 nmol/L of inhibiting BRK in abiochemical assay (18). In BRK-transformed Ba/F3 cells, XMU-MP-2 inhibited the autophosphorylation of BRK at the site ofY342 in a dose-dependent manner and the maximum inhibitionwas observed starting at the concentration of 500 nmol/L (Fig.2E). Furthermore, phosphorylation levels of STAT3Y705 andSTAT5Y694, which were reported to be the downstream of BRK(9, 10), were also abolished with increasing concentration ofXMU-MP-2. BRK was reported to induce prolonged activation of

the MAPK pathway, and promoted cell proliferation in breastcancer (12). The Inhibition of ERK1/2 phosphorylation byXMU-MP-2 was observed but less potent than that of STAT3/STAT5. Comparing with 4f, XMU-MP-2 showed an improvedinhibitory effect against BRK activation at the concentrationaround 1 mmol/L. To further elucidate the biological effects ofBRK inhibition brought by XMU-MP-2 on the growth andsurvival of oncogenic BRK-transformed Ba/F3 cells, we per-formed the cell apoptosis analysis. XMU-MP-2 induced signif-icant cell apoptosis, as assessed by the apoptotic marker cas-pase-3 and PARP activity (Fig. 2F), and Annexin-V-FLUOS/PIstaining (Fig. 2G and Supplementary Fig. S2A). As expected,XMU-MP-2 did not affect the survival of the parental Ba/F3 cells(Supplementary Fig. S2B). Together, these results establish thatXMU-MP-2 is a potent and selective inhibitor to suppress BRKkinase activity and its downstream signaling.

XMU-MP-2 induces differential apoptosis in mutantBRK–transformed Ba/F3 cells

To evaluate selectivity of XMU-MP-2, we sought to use achemical-genetic approach by engineering an "inhibitor-resis-tant" mutant of BRK. One potential drug-resistant mutation isthe so-called kinase "gatekeeper" mutation (33–35), as it is wellknown that the size of the gatekeeper residue defines the acces-sibility of the ATP-binding pocket in protein kinase. In BRK(T264M)-Ba/F3 cells, the introduction of the gatekeepermutationT264M in BRK leads to an enhanced kinase activity indicated byincreased phosphorylation of BRKY342 and activities of the

Figure 1.

High expression levels of BRK areassociated with poor survival rates inbreast cancer patients. A and B, 414breast cancer patients, from the TCGAdataset, were clustered into twogroups according to the expressionlevels of BRK (low BRK expressiongroup: 89.86%; high BRK expressiongroup: 10.14%). Kaplan–Meier analysisrevealed a significant reversecorrelation between patient overallsurvival rates and BRK mRNAexpression levels. C and D, Curtisdataset (N ¼ 1,852) was used as anindependent validation dataset (lowBRK expression group: 88.65%; highBRK expression group: 11.35%).

Pharmacologic Inhibition of BRK Combats Breast Cancer

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Figure 2.

The lead scaffold identification and optimization of XMU-MP-2 as a potent BRK inhibitor. A, Heatmap representation of the compound-centric high-throughputscreening on a panel of 28 tyrosine kinase–transformed Ba/F3 cells. Each column represents one chemical entity and each row represents one specifickinase-transformed Ba/F3 cell. Green, inhibitory activity. The spot of the lead scaffold is highlighted. B, The chemical structure of XMU-MP-2. C, Dose–responsecurves of BRK-transformed and parental Ba/F3 cells from 48-hour antiproliferative assays. Results are shown as mean � SEM (n ¼ 3). D, XMU-MP-2 inhibitsBRK enzymatic activity with the presence of 10 mmol/L ATP in vitro. Each concentration point was performed in duplicate. E, XMU-MP-2 inhibited BRK-mediatedsignaling pathways in BRK-transformed Ba/F3 cell line. Cells were treated with either DMSO or XMU-MP-2 for 4 hours. F and G, BRK Ba/F3 cells weretreated with indicated dose of XMU-MP-2 for 24 hours. Induction of cell apoptosis was assessed by immunoblotting caspase-3 and PARP cleavage (F) orAnnexin-V-FLUOS/PI double staining (G). 4f was used as the control. NS, not significant. �� , P < 0.001.

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downstream effectors (Fig. 3A). As predicted, XMU-MP-2 wasmuch less potent with the IC50 of 340.4 nmol/L against theproliferation of BRK(T264M)-transformed Ba/F3 cells to com-pare with the wild-type BRK–transformed Ba/F3 cells (Fig. 3B).The downstream signaling of BRK also showed robust resistanceto XMU-MP-2 inhibition (Fig. 3C).

To further understand the inhibition mechanism of XMU-MP-2 on BRK, we generated an additional mutant transformed BRK(Y447F)-Ba/F3 cell line, accordingly. Elevating phosphorylationlevel of Y342 was observed (Fig. 3A), which is consistent with thereport that mutation of Y447F exhibited an approximately 2.5-fold increase in kinase activity as compared with the wild-typeBRK (36). As the mutation of Y447F is outside the ATP-bindingpocket of BRK, the similar inhibitory potency of XMU-MP-2 isexpected. Indeed, XMU-MP-2 maintained the same range ofinhibitory potency with the IC50 of 91.0 nmol/L against BRK(Y447F)-Ba/F3 cells (Fig. 3B), inhibited the phosphorylation ofBRK in a dose-dependent manner, as well as the phosphoryla-tion of downstream effectors, STAT3Y705, STAT5Y694, and ERK1/2T202/Y204 (Fig. 3C), and induced significant apoptosis (Supple-mentary Fig. S3). Together, these data suggest that XMU-MP-2 is"on-target" to BRK, inhibiting the growth and survival of onco-genic BRK-transformed Ba/F3 cells through suppressing theBRK activation and downstream signaling.

XMU-MP-2 analogue structure–activity relationsWe further elucidated the binding mode of XMU-MP-2 to BRK

by using the homology modeling approach. The SRC-ABL tyro-sine kinase ancestor (PDB code: 4CSV) with the highest sequenceidentity of 56.61%was used as the template (26), because no BRKcrystal structure is available. Themolecular docking suggested thatXMU-MP-2 is in a type II binding mode (37), forming twohydrogen bonds in the hinge binding region with Met267, andtwohydrogenbonds through its amidemoietywithGlu235 in thec-Helix and Asp330 in DFG motif (Fig. 4A). In addition, thehydroxyl group of XMU-MP-2 is potentially able to interact withArg195 in the b-sheet via a hydrogen bond. Certainly, the gate-keeper residue M264 in the engineered mutant form of BRK(T264M) is predicted to clash with XMU-MP-2, because theincreased residue size of Met267 caused steric hindrance. Thisobservation provides a mechanistic basis for understanding thesignificant potency drop of XMU-MP-2 against BRK(T264M)-Ba/F3 cells in our previous experiments.

Next, the structure–activity relationship (SAR) study revealedthat theN-substituent (R2) varied frommethyl-(1a) to ethyl-(1b),isopropyl-(1c), and cyclopentyl-(1d) groups resulted in thereduced antiproliferative activities against both BRK-Ba/F3 celland BRK-positive breast cancer cell BT-474 (Fig. 4C), exhibitingthe positive correlation between BRK inhibitory effect and

Figure 3.

XMU-MP-2 is "on-target" to BRK.A, Theinhibition of XUM-MP-2 on BRK andBRKmutants stably transformed Ba/F3cells. All transformed Ba/F3 cell lineswere IL3 independent. Both BRKmutations Y447F and T264Msignificantly boosted thephosphorylation of BRK and itsdownstream signaling pathways.B, Dose–response curves of theantiproliferation assay for stablytransformed Ba/F3 cells with the wild-type and mutant BRKs, T264M, andY447F. The gatekeeper mutant BRKT264M significantly elevated IC50 at thecellular proliferation level. C, Impact ofXMU-MP-2 on the BRK-mediatedsignaling pathways in Ba/F3 cellsexpressingBRK T264MandY447F after4-hour compound treatment. Clearly,the gatekeeper mutation T264Mshowed much robust resistance to theinhibition of XMU-MP-2.






antiproliferative potency against BRK-positive cancer cells. Inconjunction with molecular docking study, the gradual decreaseof antiproliferative activities of these analogues was due to thesteric hindrance between N-substituents and phenyl group ofF381 (Fig. 4B). A comparison of potency between compound1e and 1a demonstrated that the presence of the toluene methylgroup (R1) is an important structural element for achieving potentinhibition against BRK. This observation suggests that the methylgroup favors the twisted conformation required for high affinitybinding of XMU-MP-2 to BRK. The "flag-methyl effect" was alsoimplicated in the Abl-bound conformations of imatinib, niloti-nib, and AP24534 (38–40). Together, these results elucidate thekey structure features for achieving potent inhibition against BRK.

XMU-MP-2 inhibits BRK-positive breast cancer cellproliferation

To investigate whether XMU-MP-2 is able to inhibit breastcancer cell proliferation through blocking BRK activity, a panelof different breast cancer cell lines including BRK-positive cells, T-47D,MCF7, BT-474, and BT-20, and BRK-negative cell,MDA-MB-468were tested (Fig. 5A). As anticipated, XMU-MP-2 inhibited theproliferation of BRK-positive breast cancer cells with IC50 valuesranged from 200 to 500 nmol/L (Fig. 5B). In contrast, XMU-MP-2

lost its antiproliferative potency against the BRK-negative breastcancer cell MDA-MB-468 (with the IC50 of 4302 nmol/L; Fig. 5C).In addition, XMU-MP-2 demonstrated negligible cytotoxicityagainst nontumorigenic cell lines L-02, GES-1, and MCF 10A(Supplementary Table S2). XMU-MP-2 also significantly inhib-ited colony formation (Fig. 5D) and induced cell apoptosis (Fig.5E and F), whereas the control compound 4f did not showappreciable inhibition efficacy even at the concentration of 10mmol/L (Fig. 5E and F). These results established that XMU-MP-2selectively inhibits the proliferation of BRK-positive breast cancercells.

We further examined whether the antiproliferative potency ofXMU-MP-2 against BRK-positive cancer cells was accompanied byinhibition of cellular BRK tyrosine kinase activity. Consistent withthe results of the cell proliferation assay, the autophosphorylationlevels of BRKY342 decreased significantly at the concentration of500 nmol/L in BT-474 cells and 1,000 nmol/L in BT-20 cells,respectively (Fig. 5G). Similarly, the phosphorylation of theBRK downstream effectors STAT3Y705, STAT5Y694, AktS473, andERK1/2T202/Y204 were also dramatically abolished (Fig. 5G). Tofurther verify the on-target effect of XMU-MP-2, BRK stablyknockdown BT-474 cells were established (SupplementaryFig. S4A). Depletion of BRKby shRNA in BT-474 cells significantly

Figure 4.

Molecular docking and structure–activity relationship of XMU-MP-2 against BRK.A, The bindingmode of XMU-MP-2 to the homologymodel of BRK kinase domain byusing SRC-ABL tyrosine kinase ancestor (PDB code: 4CSV) as the template. BRK kinase is shown in cartoon representation. Compounds are labeled in colorby atoms. The hydrogen bonds are labeled as dashed lines. The key amino acid residues for the binding are labeled as carbon. The black arrow points to theDFG-motif. B, Cartoon view of the binding mode of compound 1d with BRK. The steric interaction between compound 1d and the residue Phe331 is pointedto by the red arrow. C, Structure–activity relationship for BRK. The orhto-methyl substitution (R1) of the 6-aryl ring and N-methyl substitution (R2) wereessential to achieve potent cellular inhibitory activity against BRK.

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Figure 5.

XMU-MP-2 inhibits the growth of BRK-positive breast cancer cells. A, The expression profile of BRK in human breast cancer cell lines. B, Dose–responsecurves of BRK-positive breast cancer cell lines BT-474, BT-20, MCF7, and T-47D. C, The BRK-negative cell MDA-MB-468 in 48-hour triplicate cellproliferation assays. D, Anti-colony formation of XMU-MP-2 and 4f on BT-474. E, XMU-MP-2 induced cell apoptosis in BRK-positive breast cancer cells BT-474and BT-20. F, XMU-MP-2 induced caspase-3/7 cleavage and PARP activation in BRK-positive breast cancer cells BT-474 and BT-20. G, Effects of XMU-MP-2on BRK-mediated signaling pathways in BT-474 and BT-20 cells. H, BRK knockdown (1# and 4#) attenuated the potency of XMU-MP-2 against cell proliferationin BT-474 cells.






decreased colony formation (Supplementary Fig. S4B and S4C).Meanwhile the depletion of BRK by shRNA in BT-474 cells alsosufficiently attenuated the inhibitory activity of XMU-MP-2 tocompare with that of sh-Control treatment (Fig. 5H). Meanwhile,although the BT-474 cells are also HER2-overexpression andEGFR-positive and XMU-MP-2 shows quite potent inhibitoryeffects on TEL-EGFR- or TEL-HER2-transformed Ba/F3 cells,depletion of EGFR or HER2 by shRNA in BT-474 cells did notsufficiently attenuate the inhibitory activity of XMU-MP-2 (Sup-plementary Fig. S5). Taken together, these data show a positivecorrelation between the inhibition of BRK tyrosine kinase activityand XMU-MP-2 efficacy, thereby providing strong evidence tosupport the notion that directly targeting BRK by XMU-MP-2 isthe essential mechanism for inhibiting BRK-positive breast cancercell proliferation in vitro.

XMU-MP-2 suppresses tumor growth in vivoTo extend the potential preclinical application of BRK-specific

inhibitors, the pharmacokinetics of XMU-MP-2 was evaluated inBALB/c mice at the dose of 1 mg/kg via intravenous injection.XMU-MP-2 was found to have the half-life (T1/2) of 0.9 hour,clearance (Cl) of 39.1 mL/min/kg, and volume of distribution(Vd) of 1.7 L/kg, representing moderate pharmacokinetic prop-erties. The BRK-positive mouse xenograft models were used tofurther evaluate the in vivo antitumor activity of XMU-MP-2. Itshowed that XMU-MP-2 treatment led to substantial repression oftumor growth in a dose-dependent manner against both wild-type BRK- and BRK(Y447F)-driven tumors without significantbodyweight loss or gross signs of toxicity (Fig. 6A and B). The dataobtained from cellular apoptosis analysis by flow cytometry(Fig. 6C and Supplementary Fig. S6), immunohistochemicalstaining (Fig. 6D), and TUNEL staining (Supplementary Fig.S7) indicated that XMU-MP-2 dose-dependently induced cellapoptosis in vivo. Furthermore, the phosphorylation of BRKY342

was abrogated by XMU-MP-2 in both tumors (Fig. 6E). XMU-MP-2 showed negligible antitumor effect in the kinase gatekeepermutant xenograft model of BRK(T264M)-Ba/F3 as predicted(Supplementary Fig. S8).

The in vivo antitumor efficacy for XMU-MP-2 was furtherexamined in the xenograft mouse model of BRK-positive breastcancer cells BT-474. Administration of XMU-MP-2 for 14 daysresulted in cancer cell apoptosis (Fig. 6F) and significant tumorshrinkage (Fig. 6G). More importantly, we observed that a singledose of XMU-MP-2 (40 mg/kg) via intravenously injection hasbeen capable of inducing significant apoptosis as assessed byTUNEL staining (Fig. 6I). Together, these results clearly indicatethat BRK-specific kinase inhibitor XMU-MP-2 exhibits potenttherapeutic efficacy against BRK-positive tumors in vivo.

Synergistic effects of BRK inhibition on combating HER2-positive or ER-positive breast cancer

The synergism of BRK and HER2 was reported that the dualknockdown of BRK and HER2 resulted in a significant prolifer-ation reduction of breast cancer cells (41). Although XMU-MP-2showed quite potent inhibitory effects against TEL-EGFR- or TEL-HER2-transformed Ba/F3 cells, it was much less efficient inhibit-ing EGFR or HER overexpression breast cancer cell lines, such asMDA-MB-468 (with the IC50 of 4,302 nmol/L; Fig. 5C), andMCF-7 (with the IC50 of 543 nmol/L). On the basis of chemicalstructure and modeling data, we predicted that XMU-MP-2 is atype II kinase inhibitor. We intended to explore the combinatory

effect of XMU-MP-2 with a type I HER2 inhibitor, CP-724,714(42). In BT-474 cells, which showed overexpression of both BRKand HER2 (Fig. 7A and B), the combination index (CI) value ofXMU-MP-2 and CP-724,714 was 0.29 (Fig. 7A), revealing theexistence of strong synergy between XUM-MP-2 and CP-724,714.

Encouraged by this observation, we extended our drug com-bination search to ER- andBRK-positive breast cancer. As themostcommon subtype, about 75% of all breast cancers are ER-positive(3). Tamoxifen is widely used for the treatment of ER-positivebreast cancer in postmenopausal women (43).Wewere delightedto see that XMU-MP-2 also exhibited strong synergism (CI¼0.38)with 4-hydroxytamoxifen against ER-positive breast cancer cellsMCF7 under estrogen-free medium mimicking the postmeno-pausal condition (Fig. 7C). Moreover, the elevated expressionlevel of BRK was observed in our 4-hydroxytamoxifen-inducedMCF7-resistant cell line (MCF7-TR; Supplementary Fig. S9).XMU-MP-2 alone exhibited remarkable inhibition againstMCF7-TR (Fig. 7D). Taken together, the strong synergism of thecombination of BRK with other breast cancer relevant therapeu-tics, such as HER2 inhibitors or ER modulators may represent anew approach for targeting breast cancer in the future.

DiscussionAmong more than 500 human kinases, only a handful of

kinases are targeted by chemical and biological drugs currentlyavailable on the market (44). Nonreceptor tyrosine kinase BRK isone of a few kinases widely reported associated with varietytumors yet without direct-targeted therapeutics available. Thechallenges of targeting BRK lie in the following two aspects. First,despite intensively scientific efforts to understand the physiologicand pathologic functions of BRK in the past two decades, muchremains unknown about its role in the promotion of tumorigen-esis, especially the role of BRK enzymatic activity in cancer (3). Forinstance, in a RNA interference experience, specifically knock-down BRK in breast carcinoma cells results in a significantsuppression of cell proliferation. However, introduction of exog-enous kinase-inactive mutant BRK can promote human breastcancer cell T-47D proliferation (20). This observation cast someambiguity on targeting BRK kinase activity directly for cancertherapy. It could be argued that BRK functions as a kinaseindependent "adapter" in tumorigenesis (3). However, it couldalso be argued that the overexpression of exogenous BRK neu-tralizes cellular internal checkingmachinery, consequently ampli-fies endogenous BRK kinase activity or signaling pathways medi-ated byBRK. Second, because the highly conserved structure of theATP-binding pocket among human kinases, to develop a BRK-specific kinase inhibitor is the key challenge in medicinal chem-istry. Currently, most kinase selectivity profiling of small-mole-cule inhibitors was based on single kinase biochemical assays invitro (30).However, inadequate knowledge of inhibitor specificitycertainly limits our capability to explore the therapeutic potentialof targeted kinase. For example, dasatinib, a reported potent BRKinhibitor, also strongly inhibits SRMS (32, 45, 46).Unfortunately,SRMS is the key kinase to phosphorylate BRK at Y447, andnegatively regulates BRK kinase activity (47). It would not besurprised to see limited response when dasatinib was used to treatbreast cancer (48).

To bear those challenges inmind,wefirst seek the correlation ofthe expression levels of BRK and the clinical prognoses in twoindependent searches with total number of 2,266 breast cancer

Jiang et al.





Figure 6.

XMU-MP-2 substantially suppresses tumor growth in mouse xenograft models. A–E, The in vivo antitumor efficacy of XMU-MP-2 in BRK- and BRK(Y447F)-driven tumors. A, The tumor volume and body weight of BRK Ba/F3 and BRK Y447F Ba/F3 xenograft models. B, BRK- and BRK(Y447F)-driventumors were harvested on day 14 for the weight measurement. XMU-MP-2 significantly reduced the tumor size without affecting animal weights.Annexin-V-FLUOS/PI double-staining (C), immunohistochemical analysis and hematoxylin and eosin staining (D), immunoblot analysis of expressionand phosphorylation of BRK (E) indicated that the inhibition of BRK kinase activity by XMU-MP-2 inducing significant cell apoptosis in tumors.Scale bar, 50 mm. F–I, The in vivo antitumor efficacy of XMU-MP-2 in BT-474 xenograft model. TUNEL staining was assayed on BT-474 tumors frommice sacrificed at day 14th and XMU-MP-2 induces significant breast tumor apoptosis (F). The tumor volume (G) was significantly reduced by XMU-MP-2and the body weight (H) was not affected. I, A single dose of XMU-MP-2 induces significant apoptosis in the BT-474 breast tumor xenograft model shownby TUNEL staining. Tumor samples were obtained at 6 hours postadministration.






patients. The statistical analysis consistently suggests that thelower overall survival of breast cancer patients displaying thehigher BRK mRNA expression levels. These data lined with pre-liminary positive progress reported fromother groups (13, 14, 49)prompt us to develop small-molecule therapeutics targeting BRK.Meantime, we also intended to use those newly developed selec-tive BRK inhibitor as the chemical probe deciphering the signalingpathways mediated by BRK.

Our next effort was to build a cell-based high-throughputscreening platform to search valuable BRK-specific lead com-pounds. This compound screening platform consists of a panelof 28 individual oncogenic tyrosine kinases transformed Ba/F3cells. Such a cellular screen should provide anunbiasedmethod toidentify compounds that target any protein essential to oncogenic

kinase transformation, simply by the comparison of the differ-ential cytotoxicity of each compound to different oncogenickinase transformed and parental Ba/F3 cells. The problematiccompounds with selectivity and cellular permeability issues arealso partially eliminated in the initial developmental stage.

On the basis of the lead compound from a screen of in-housecompound library designed for novel ATP-site-directed kinaseinhibitors, a highly potent BRK kinase inhibitor XMU-MP-2 wasdeveloped with mild cytotoxicity. A remarkable characteristic ofXMU-MP-2 was its selectivity for BRK among various types ofkinases investigated, including SRC and FRK (SupplementaryTable S1). Kinase selectivity of an inhibitor was related to thenumber of hinge hydrogen bonds between the kinase and thekinase inhibitor and the space usage in the ATP-binding pocket

Figure 7.

Synergistic effects of BRK inhibition in drug combination with XMU-MP-2 for BRK-positive breast cancer cells. A and B, Antiproliferative effects of the combinationof XMU-MP-2 with CP-724,714 on BRKþ/HER2þ breast cancer cells BT-474. BT-474 cells were treated with indicated doses of XMU-MP-2, CP-724,714, or acombination for 48 hours. C and D, Antiproliferative effects of combining XMU-MP-2 with 4-hydroxytamoxifen (4-OH-Tam) on BRKþ/ERþ breast cancer cells MCF7(C) and MCF7-TR (D). After 3-day treatment with indicated doses of XMU-MP-2, 4-OH-Tam, or combination, cell viability was assayed by MTS.

Jiang et al.





(50). Several developed BRK inhibitors showed three hydrogenbonds with the hinge region of BRK in the predicted bindingmode (17, 18). On the basis of molecular docking studies, XMU-MP-2 is predicted to form five hydrogen bonds with the BRKkinase. Encouraged by this theoretical understanding of com-pound-protein binding mode, we used XMU-MP-2 as a toolcompound to explore the 1,3-disubstituent-1,2,3,4-tetrahydro-pyrimido [4,5-d]-pyrimidine scaffold against BRK. By thoroughlyexamining the inhibitory potency of XMU-MP-2 against twoimportant BRK mutations, that is, the gatekeeper mutation ofT264M and the autoinhibition mutation of Y447F, we confirmedthat XMU-MP-2 not only directly targeted on BRK, but alsodisplayed substantial efficacy against autoinhibition mutantY447F-driven Ba/F3 cells in vitro (Fig. 2B and C) and xenografttumors in vivo (Fig. 5A–E). XMU-MP-2provides a valuable startingpoint for further medicinal chemistry efforts aimed at developingBRK-targeted therapeutics.

Recently, several small-molecule inhibitors of BRK werereported with biochemical activities and limited cellular results(17–19). However, these compounds have not been used toinvestigate the impact of BRK inhibition on cancer cell signaling,nor in vivo potential therapeutic efficacy on BRK-positive cancer.With XMU-MP-2 as the chemical probe, we verified that STAT3and STAT5 are the substrates of BRK, and ERK1/2 signalingpathway is affected as well. XMU-MP-2 induced caspase-3/7cleavage and PARP activation and eventually led to cell death inBRK-positive breast cancer cells BT-474 and BT-20. More impor-tantly, XMU-MP-2 substantially suppressed the tumor growth inwild-type BRK and BRK(Y447F) Ba/F3 xenograft mouse modelsvia abrogating the BRK pathway, but not for drug-resistant BRK(T264M)model. These data clearly demonstrate that BRK can be adruggable target for cancer therapy.

The potential therapeutic significance of targeting BRK wasfurther demonstrated in our drug combination studies. The strongsynergistic effects of BRK inhibition combing with the HER2inhibitor or the ER modulator in relevant breast cancer cells

suggest the importance of breakdown of BRK kinase activity oncombating HER2-positive or ER-positive breast cancer. This mayrepresent a novel approach to improving therapeutic effects forBRK-positive breast cancer.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: J. Jiang, L. Li, D. Zhou, Q. Liu, R. Ren, J. Zhang, X. DengDevelopment of methodology: J. Jiang, F. Gui, L. Li, J. Zhang, X. DengAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. Jiang, F. Gui, Z. He, L. Li, Y. Li, S. Li, X. Wu,X. Sun, X. Huang, W. Huang, S. Han, T. Zhang, Z. Wang, L. Chen, R. RenAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J. Jiang, F.Gui, L. Li, S. Li, Z.Deng, X. Sun,H.Wang,Q.Liu, R. Ren, J. Zhang, X. DengWriting, review, and/or revision of the manuscript: J. Jiang, F. Gui, X. Sun,B. Jiao, S. Song, Q. Liu, R. Ren, J. Zhang, X. DengAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J. Jiang, L. Chen, J. Zhang, X. DengStudy supervision: D. Zhou, Q. Liu, J. Zhang, X. Deng

AcknowledgmentsWe thankDr. J. Han for providing the cDNAs of BRK and Tel, andChemAxon

for giving academic license to access free software products.

Grant SupportThis work was supported by grants from the National Natural Science

Foundation of China (nos. 81422045, U1405223, and 21272195 to X. Deng,81230055 to R. Ren, 21402165 to W. Huang, and 81603131 to L. Li), theChina's 1000 Young Talents Program to X. Deng, and the FundamentalResearch Funds for the Central Universities of China (nos. 2013121032 and20720160064 to X. Deng).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received April 21, 2016; revised September 8, 2016; accepted September 30,2016; published OnlineFirst October 10, 2016.

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