PI3K/mTOR Dual Inhibitor VS-5584 Preferentially...

Tumor and Stem Cell Biology

PI3K/mTOR Dual Inhibitor VS-5584 PreferentiallyTargets Cancer Stem CellsVihren N. Kolev1, Quentin G.Wright1, Christian M.Vidal1, Jennifer E. Ring1, Irina M. Shapiro1,Jill Ricono2, David T.Weaver1, Mahesh V. Padval1, Jonathan A. Pachter1, and Qunli Xu1

Abstract

Cancer stem cells (CSC) have been implicated in diseaserecurrence, metastasis, and therapeutic resistance, but effectivetargeting strategies for these cells are still wanting. VS-5584 is apotent and selective dual inhibitor of mTORC1/2 and class IPI 3-kinases. Here, we report that VS-5584 is up to 30-fold morepotent in inhibiting the proliferation and survival of CSCcompared with non-CSC in solid tumor cell populations. VS-5584 preferentially diminished CSC levels in multiple mousexenograft models of human cancer, as evidenced by markedreduction of tumor-initiating capacity in limiting dilutionassays. Likewise, VS-5584 treatment ex vivo preferentiallyreduced CSC in surgically resected breast and ovarian patienttumors. In contrast, chemotherapeutics such as paclitaxel andcisplatin were less effective in targeting CSC than bulk tumorcells. Mechanistic investigations revealed that preferential tar-

geting of CSC required inhibition of multiple components ofthe PI3K–mTOR pathway: coordinate RNAi-mediated silencingof PI3Ka, PI3Kb, and mTOR phenocopied the effect of VS-5584, exhibiting the strongest preferential targeting of CSC,while silencing of individual PI3K isoforms or mTOR failed toreplicate the effect of VS-5584. Consistent with CSC ablation,VS-5584 delayed tumor regrowth following chemotherapy inxenograft models of small-cell lung cancer. Taken together, thepreferential targeting of CSC prompts a new paradigm forclinical testing of VS-5584: clinical trials designed with CSC-directed endpoints may facilitate demonstration of the thera-peutic benefit of VS-5584. We suggest that combining VS-5584with classic chemotherapy that debulks tumors may engender amore effective strategy to achieve durable remissions in patientswith cancer. Cancer Res; 75(2); 446–55. �2014 AACR.

IntroductionChemoresistance and cancer relapse represent significant chal-

lenges in cancer management, and are attributable in part to thepresence of subpopulations of cancer cells termed cancer stemcells (CSC) or tumor-initiating cells. CSC were first identified inacute myeloid leukemia (1, 2) and are operationally defined bytheir self-renewal and tumor initiation capacities. The presence ofCSC in solid tumors was first described in breast cancer (3) andhas since been identified in a wide variety of solid tumors, andimplicated in resistance to anticancer therapies, cancer recurrence,and metastasis. (4–7).

The phosphoinositide 3-kinase–mammalian target of rapamy-cin (PI3K–mTOR)pathway is one of themost frequently activatedsignaling pathways in cancer, playing a central role in tumori-genesis by regulating the proliferation, survival, differentiation,and migration of cancer cells as well as tumor angiogenesis (8).AKT andmTOR are the major effector kinases in the PI3K–mTORpathway. mTOR exists in two distinct protein complexes,

mTORC1 and mTORC2, each with different downstream sub-strates (9). The PI3K–mTOR pathway also plays a role in CSC(8, 10–14). Activation of the PI3K–mTORpathway in adult bloodcells through PTEN deletion led to the generation of leukemia-initiating cells (11). Similarly, activation of PI3K–mTOR signal-ing, achieved by knocking down PTEN, enriched breast CSC (13).

VS-5584 is a highly potent and selective dual PI3K–mTORinhibitor, exhibiting approximately equal low nanomolar poten-cy against mTOR kinase and all four class I isoforms of PI3K. VS-5584 was shown to target the mTORC1 and mTORC2 complexesas well as PI3K in cells, as evidenced by inhibition of phosphor-ylation of cellular targets of these kinases, including AKT (Ser473and Thr308) and ribosomal protein S6 (Ser240/244; ref. 15). VS-5584 shows no significant inhibitory activity against more than400 other protein and lipid kinases, including closely relatedlipid kinases such as DNA-PK and VSP34, underscoring the highselectivity of VS-5584 (15). Simultaneous inhibitionofmTORC1/2 and PI3K by VS-5584 is expected to more effectively shut downPI3K–mTOR signaling than isoform-selective PI3K or mTORinhibitors that block only a subset of these targets. This approachshould also overcome the feedback activation of PI3K signalingthat is thought to limit the effectiveness of rapamycin analogs(8, 9). It was reported recently that activation ofmTOR accountedfor the resistance of breast cancer to the PI3Ka-selective inhibitorBYL719 in the clinic, further arguing the importance of simulta-neous inhibition of mTOR and PI3K (16).

Here, we report that in addition to exhibiting potent anticanceractivity in a broad set of tumor models, VS-5584 preferentiallytargets CSC as demonstrated using a panel of orthogonal CSCassays, including the limiting dilution tumor-initiating assay, a

1Verastem Inc., Needham, Massachusetts. 2Molecular Response, SanDiego, California.

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

Corresponding Authors: Jonathan A. Pachter, Verastem Inc., 117 Kendrick Street,Needham, MA 02494. Phone: 781-292-4215; E-mail: [email protected]; andQunli Xu, Verastem Inc., E-mail: [email protected].

doi: 10.1158/0008-5472.CAN-14-1223

�2014 American Association for Cancer Research.

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gold standard for CSC. Consistent with the observation that VS-5584 preferentially abolishes CSC, VS-5584 delayed tumorregrowth in small-cell lung xenograft models after cessation oftreatment with cisplatin. These results provide compelling ratio-nale for the clinical development of VS-5584 as a CSC targetingagent.

Materials and MethodsCell lines, primary human tumor tissues, compounds, andreagents

All cancer cell lines used were obtained from the ATCCwith theexception of SUM159, which was acquired from Asterand. Celllines were authenticated by STR (short tandem repeat) analysis ateither ATCC or IDEXX Radil. V12H-Ras–transformed humanmammary epithelial cells (HMLER) were obtained from theBroad Institute and maintained as described previously (17).Primary human breast and ovarian tumor specimens wereobtained from Tissue Solutions Ltd. after patients' consent andinstitutional IRB approval. VS-5584 was synthesized by S�Bio(15). Other compounds were purchased from Selleckchem.Reverse-Phase Protein Microarray (RPMA) was conducted atTheranostics Health Inc.

Aldefluor assaysAn imaging-based Aldefluor assay was conducted using the

Aldefluor Assay Kit (STEMCELL Technologies) with the follow-ing modifications: cells were plated in collagen-treated plates.Following compound treatment, Aldefluor reagents supplemen-ted with 10 ng/mL Hoechst 33342 dye were added. Plates wereincubated at 37�C for 20 minutes. After washing, Aldefluorþ

and total cells (in buffer containing Hoechst 33342) werequantified using Celigo (Nexcelom Bioscience). ALDH inhibitordiethylamino benzaldehyde (DEAB)–treated cells were usedas a negative control. FACS-based Aldefluor assay was con-ducted following the manufacturer's instructions (STEMCELLTechnologies).

Side Population assaysHoechst 33342 exclusion (Side Population) assay was carried

out as previously described (18).

HMLER assayHMLER cells were treated with compounds for 4 days. Follow-

ing a 4-day compound wash-off and cell recovery period, cellswere stained with PE-labeled anti-CD24, APC-labeled anti-CD44antibody (BD Biosciences), and 7-AADþ live cells were subject toFACS analysis. APC-labeled IgG 2bK and PE-labeled IgG2aK (BDBiosciences) were used as isotype controls for CD44 and CD24,respectively.

Tumorsphere assayTo determine tumorsphere forming efficiency, cells from tissue

culture or dissociated tumorswere plated in tumorsphere formingmedium as previously described (17). Spheres were enumeratedusing Celigo.

Apoptosis assays and siRNA transfectionStandard methods were used for Annexin V and caspase-3/7

assays and siRNA transfection with details in SupplementaryMaterials and Methods.

In vivo tumor xenograft studies, cell dissociation, and limitingdilution assay

For the SCLC PDX study, 105 cells of p2 SCLC PDXact tumor(Molecular Response) admixedwithCultrex ECM(Trevigen)weresubcutaneously injected into NOD-SCID mice (Harlan). Whentumors reached an average size of 200 mm3, mice were random-ized with 5 mice per group and treated with vehicle control,cisplatin (i.p. once a week for 2 weeks), or cisplatin followed byVS-5584 (orally, three times weekly for 4 weeks). Details of othertumor xenograft studies are in Supplementary Materials andMethods. To dissociate single cells, tumor tissue was minced intosmaller pieces and incubated with Liberase (Roche Applied Sci-ence) or dispase for 1 hour at 37�C under agitation. Mouse cellswere removed from xenograft human tumors using mouse anti-bodies and magnetic beads.

For the limiting dilution assay, cells dissociated from xenografttumors were admixed with Matrigel and injected into the mam-mary fat pad (for MDA-MB-231 and MCF-7 breast tumors) orsubcutaneously (NCI-H841 tumors) of immunodeficient SHrNmice (Harlan). Tumor-initiating frequency (TIF) was calculatedusing the ELDA Software (19).

ResultsPreferential targeting of CSC by VS-5584 in vitro

In a previous screening of more than 300,000 compoundsusing an HMLE CSC assay (17), a number of PI3K–mTORpathway inhibitors were found to preferentially target CSC. Thishistoric observation prompted our initial interest in exploring thePI3K–mTOR pathway to target CSC.We therefore determined theeffect of VS-5584, a highly potent and selective dual inhibitor ofPI3K–mTOR(15), onCSC inmultiple orthogonal CSCassays thathave been validated by limiting dilution implantation in mice.

CSC have high aldehyde dehydrogenase (ALDH) activity,which can be measured by the Aldefluor enzymatic assay (20).Aldefluorþ cells from several breast cancer cell lines have beenshown to display CSC characteristics (21). We also show thatAldefluorþ SUM159 triple-negative breast cancer cells displayenhanced self-renewal and tumor-initiating capability relative toAldefluor� cells in immunodeficient mice (Supplementary Fig.S1). To quantify CSC that harbor high ALDH activity, we devel-oped an imaging-based Aldefluor assay. As shown in Fig. 1A,although VS-5584 dose-dependently reduced both Aldefluorþ

andAldefluor� cell populations, it was farmore potent against theAldefluorþ CSC within the SUM159 cell line. A similar preferen-tial effect of VS-5584 on Aldefluorþ CSC was observed using theestrogen receptor–positive (ERþ) MCF7 and carcinosarcomaHs578T cell lines (Fig. 1A), indicating that VS-5584 exerts anapproximately 10- to 30-fold more potent inhibitory effect onCSC than bulk tumor cells across these cell lines. In contrast,exposure to the cytotoxic agents paclitaxel or cisplatin increasedthe percentage of Aldefluorþ cells (Fig. 1B), consistent with CSCbeing resistant to conventional chemotherapy. Importantly, VS-5584 attenuated paclitaxel-induced enrichment of Aldefluorþ

cells (Fig. 1C).A tumorsphere assay can be used tomeasure the self-renewal of

CSC (22). SUM159 and MCF-7 tumorspheres were treated withVS-5584, paclitaxel, or combination of both agents, and the effectof these treatments on CSC was determined in a secondarytumorsphere assay. Tumorsphere forming efficiency wasdecreased by VS-5584 but increased by paclitaxel. Strikingly,

VS-5584 Preferentially Targets Cancer Stem Cells

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Figure 1.VS-5584 preferentially targets CSC in vitro. A, VS-5584 markedly reduced the viability of Aldefluorþ cells but had weaker effect against Aldefluor� cells.SUM159, MCF7, and Hs578T breast cancer cells were treated with VS-5584 for 2 days and an imaging-based Aldefluor assay was carried out. B, paclitaxeland cisplatin enriched for Aldefluorþ CSC. SUM159 cells were treated with paclitaxel or cisplatin for 2 days followed by an Aldefluor assay. C, VS-5584attenuated paclitaxel-induced Aldefluorþ cells. (Continued on the following page.)

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combination of VS-5584 and paclitaxel almost completely abol-ished tumorsphere formation, indicating a marked reduction ofCSC (Fig. 1D).

Enhanced drug efflux is another attribute of CSC. Thus CSC,when assayed by their ability to exclude the Hoechst 33342 dye,are found to reside in a side population (SP) with greater dyeexclusion capacity (18). As with the Aldefluor assay, we firstvalidated that SP of SUM159 cells indeed exhibited elevatedtumor-initiating capability relative to non-SP (SupplementaryFig. S2). To determine the effect of VS-5584 on SP CSC, SUM159

cells were cultured under hypoxic conditions (1% O2) to enrichfor CSC (23). VS-5584 markedly reduced the proportion of SPcellswith less than1%SP remaining following treatmentwith 100nmol/L VS-5584 and no detectable SP CSC following 1 mmol/LVS-5584 treatment. Similarly, VS-5584 also reduced the propor-tion of SP cells in MCF-7 cells (Fig. 1E).

In the HMLER cell line model, the CD44hi/CD24lo subpopu-lation has been demonstrated to possess characteristics of CSC(17). A distinct CD44hi/CD24lo population was most evident inthe HMLER cells that survived paclitaxel treatment, and this

Figure 2.VS-5584 induced apoptosis preferentially in CSC. A, SUM159 cells were treated with VS-5584 or vehicle for 24 hours, costained with Annexin V and Aldefluorreagents. B–D, SUM159 cells were first FACS-sorted into Aldefluorþ and Aldefluor� cells (B) or SP and non-SP cells (C and D) and treated with VS-5584 or paclitaxelfor 24 hours. Apoptosis induction was analyzed using either an Annexin V (C) or caspase-3/7 (B and D) assay.

(Continued.) SUM159 cells were treated with 100 nmol/L VS-5584 and 10 nmol/L paclitaxel alone or in combination and analyzed as in B. D, VS-5584 alone or incombination with paclitaxel decreased tumorsphere forming efficiency. SUM159 and MCF-7 tumorspheres were treated with 100 nmol/L VS-5584, 10 nmol/Lpaclitaxel alone or in combination. Results of secondary tumorsphere assay are shown. E, preferential targeting of SP CSC by VS-5584. SUM159 and MCF-7cells were cultured under hypoxia (1% O2) and treated with VS-5584 for 2 days. Cells were incubated with Hoechst 33342 dye and SP was determined byFACS. F, VS-5584 preferentially targeted CD44hi/CD24lo cells. HMLER cells were treated with VS-5584 or paclitaxel for 4 days. Following a 4-day recovery period,cells were costained with CD44 and CD24 antibodies and subjected to FACS analysis.






helped to set the gate for FACS analysis across all samples.Whereas paclitaxel treatment significantly increased the percent-age of CD44hi/CD24lo cells compared to the control sample, VS-5584abolished theCD44hi/CD24lo cellswith greater than10-foldreduction (Fig. 1F). Taken together, these data demonstrate thatVS-5584has a strongpreferential inhibitory effect onCSC indirectcontrast with the cytotoxic agents paclitaxel and cisplatin, suggest-ing that CSC have an increased dependency on PI3K–mTORsignaling compared with non-CSC.

Preferential induction of apoptosis by VS-5584 in CSCBecause VS-5584 dramatically reduced the proportion of CSC-

containing population, we surmised that VS-5584 might inducestronger apoptosis in CSC. To test this hypothesis, SUM159 cellswere treated with VS-5584 or DMSO control for 24 hours and thepercentage of Annexin V–positive apoptotic cells was determinedfor both the Aldefluorþ and Aldefluor� subpopulations. VS-5584(500 nmol/L) induced apoptosis in 8% of Aldefluorþ cells com-

pared with 2% of Aldefluor� cells (Fig. 2A), indicating that VS-5584 selectively induced apoptosis in CSC. In addition, SUM159cells were sorted for Aldefluorþ and Aldefluor� cells and eachsubpopulation was treated with VS-5584 for 24 hours. Apoptosiswas assessed by a caspase-3/7 assay. VS-5584 induced 2-foldgreater caspase activation in Aldefluorþ cells compared withAldefluor� cells (Fig. 2B). Similarly, VS-5584 caused more pro-nounced apoptosis induction in SP cells as comparedwithnon-SPcells (Fig. 2C and D). In contrast, paclitaxel induced slightly moreapoptosis in non-CSC than CSC (Fig. 2B–D). Altogether, theseresults indicate that VS-5584 preferentially induces apoptosis inCSC relative to non-CSC.

Preferential targeting of CSC by VS-5584 in the MDA-MB-231triple-negative breast cancer model in vivo

We subsequently determined whether similar preferential tar-geting of CSC occurs in vivo. We first used MDA-MB-231 triple-negative human breast cancer cells implanted orthotopically in

Figure 3.VS-5584 preferentially abolishes CSC in two human breast tumor xenograft models in vivo. A, experimental design. Rx, denotes treatment with either compoundor vehicle. B–D, mice bearing MDA-MB-231 tumors were treated with either vehicle or 25 mg/kg VS-5584 once a day for 7 days. Cells were dissociated fromtumors and subject to CSCassays shown inA. B, Aldefluor assaydata showing that VS-5584preferentially reducedAldefluorþCSC inMDA-MB-231 tumor xenografts.Shown is a scatter plot of percent Aldefluorþ cells. � , P ¼ 0.015, unpaired t test. C, limiting dilution assay showing that VS-5584 reduced tumor-initiatingcapability of CSC in vivo. Cells dissociated from MDA-MB-231 tumors were re-implanted in limiting dilutions into the mammary fat pad of SHrN immunodeficientmice. The number of mice that grew tumors at week 8 out of total of 4 mice per group (in parenthesis) is tabulated. TIF was calculated using the ELDA software.D–G, mice bearing MCF7 tumors were dosed orally with vehicle (control), 20 mg/kg VS-5584, or 5 mg/kg everolimus daily for 10 days. Cells were dissociatedfrom tumors and subject to a panel of CSC assays as shown in A. D, Aldefluor assay results showing preferential reduction of CSC by VS-5584 (�� , P ¼ 0.007)but not everolimus. E, secondary tumor sphere assay showing that VS-5584 but not everolimus significantly suppressed self-renewing capability of CSC(�� , P ¼ 0.001). F, in vivo limiting dilution assay showing that VS-5584 reduced, whereas everolimus increased TIF. G, results of RPMA showing that both VS-5584and everolimus reduced pS6 (Ser240/244) levels in MCF-7 tumors.

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the mouse mammary fat pad. After tumors reached a volume ofapproximately 200 mm3, VS-5584 was administered orally at 25mg/kg once daily for 9 days. Tumors were harvested and disso-ciated into single cells and subjected toCSCassayswithout furthercompound treatment (Fig. 3A). Results of the Aldefluor assayshowed that the percentage of Aldefluorþ cells was relatively lowwith an average of 0.7% in control tumors, VS-5584 treatmentcaused significant reduction of the proportion of Aldefluorþ CSCto 0.2% (P ¼ 0.015, Fig. 3B, Supplementary Fig. S5). A morerigorous and functional test for CSC is the limiting dilution assay.Accordingly, cells dissociated from either VS-5584- or vehicle-treated tumors were injected into immunodeficient mice in lim-iting dilutions. Cells from VS-5584–treated tumors displayed a 7-fold reduction of TIF, confirming reduction of CSC in tumors byVS-5584 treatment (Fig. 3C).

Differential effects of VS-5584 and everolimus on CSC in theERþ MCF7 breast cancer model in vivo

Similar to our observations using the MDA-MB-231 model,VS-5584 treatment also reduced the proportion of CSC in theMCF7 ERþ breast tumor xenograft model, as evidenced by asignificant decrease in the percentage of Aldefluorþ cells,tumorsphere forming efficiency, and TIF in a limiting dilutionassay (Fig. 3D–F).

Because VS-5584 inhibits multiple nodes of the PI3K–mTORsignaling pathway, we reasoned that inhibition of CSC by VS-5584 might involve effects on more than one of these pathwaycomponents. To test this hypothesis, we investigated effects of thepotent mTORC1 inhibitor everolimus on CSC in the same MCF7xenograft model. In contrast with VS-5584, oral daily dosing ofeverolimus at 5 mg/kg, shown to inhibit mTORC1 activity intumors as evidenced by reduction of S6 phosphorylation (Fig.3G), did not reduce the proportion of CSC, as measured bymultiple assays (Fig. 3D–F). Collectively, these data suggest thatinhibition of components of the PI3K–mTORpathway other than

mTORC1 might be important for the preferential reduction ofCSC observed with VS-5584.

The role of PI3K isoforms and mTOR in the preferentialtargeting of CSC by VS-5584

To further assess the contribution of individual PI3K isoformsandmTOR inmediating the CSC effect of VS-5584, SUM159 cellswere transfected with siRNA specific for PI3Ka, PI3Kb, or mTORindividually or in combination. Two different siRNA sequenceswere used per gene. An Aldefluor assay was carried out 3 days aftersiRNA transfection. PI3Kd and PI3Kg were not examined becausethey aremostly expressed in leukocytes and their role in epithelialcells is not established (24). Single isoform knockdown of PI3Ka,PI3Kb or mTOR resulted in a moderate increase in the percentageof Aldefluorþ cells compared with scrambled control siRNA.Importantly, double knockdown of PI3Ka and PI3Kb, PI3Ka andmTOR, or PI3Kb and mTOR decreased the percentage ofAldefluorþ CSC to some extent, whereas triple knockdown ofPI3Ka, PI3Kb, and mTOR, which mirrors the kinase inhibitionprofile of VS-5584, showed themost striking 70% reduction of theproportion of Aldefluorþ cells (Fig. 4A). Effective target knock-down by all siRNAs was confirmed by Western blot analysis (Fig.4B). These data further support the notion that inhibition ofmultiple components of the PI3K–mTOR pathway by VS-5584 iscritical for the observed preferential targeting of CSC.

Preferential targeting of CSC by VS-5584 in primary humantumor tissue samples

Having demonstrated that VS-5584 preferentially reduced CSCin both cell line models and in vivo tumor xenografts, we deter-mined if ex vivo treatment with VS-5584 preferentially targets CSCin surgically removed primary human tumor specimens. Ex vivotreatment of breast tumor tissue fragments with VS-5584 for 5days reduced the proportion of both CD44hi/CD24lo (Fig. 5A)and Aldefluorþ (Fig. 5B) CSC. The modest effect on the tumor ofpatient 3 (Fig. 5A and B) may be explained by a sample qualityissue, as there were significantly fewer live cells available for FACSanalysis compared with tumors of patients 1 and 2 (Fig. 5A). Wereasoned that other carcinomas, such as ovarian cancer, mightdepend on similar CSC signaling pathways. Accordingly, ex vivotreatment with VS-5584 was undertaken using ovarian tumortissue, surgically removed from 2 patients who had received priortaxane- and platinum-based chemotherapy. Ex vivo treatmentwith 100 nmol/L VS-5584 for 5 days reduced the proportion ofCD44hi/CD117hi cells, previously validated as a CSC marker inovarian cancer (25), in both ovarian cancer patient tumor speci-mens (Fig. 5C). To further evaluate the effect of VS-5584 ontumor-initiating cells, 10,000 cells dissociated from the abovecontrol- or VS-5584–treated ovarian tumor specimens wereimplanted subcutaneously into immunodeficient mice. After17 weeks, mice injected with cells from control-treated ovariantumor specimens 1 and 2 developed tumors, whereas no tumorsformed in mice injected with cells from VS-5584–treated tumorspecimens (Fig. 5D). Thus, the results from ex vivo treatment ofprimary patient tumor specimens are in agreement with ourfindings with cell lines and xenograft tumors, and indicate thatVS-5584 preferentially reduces CSC populations.

Effects of VS-5584 on tumor regrowth after chemotherapyCSC have been postulated to account for tumor recurrence

after chemotherapy (4). We therefore hypothesized that, by

Figure 4.Inhibition of PI3Ka, PI3Kb, and mTOR is required for strong preferentialtargeting of CSC. A, SUM159 cells were transfected with siRNA against PI3Ka,PI3Kb, and mTOR individually or in combination. Aldefluorþ cells as apercentage of control, averaged from two independent experiments, isshown (Y axis). B, confirmation of siRNA knockdown of PI3K isoforms andmTOR. Lysates from cells transfected with various siRNA were subjected toWestern blot analysis. b-actin served as a loading control.






preferentially eliminating CSC, VS-5584 might delay tumorregrowth after initial treatment with chemotherapy. Small-celllung cancer (SCLC) represents a logical setting to test this hypoth-esis asmost patientswith SCLC initially respond to chemotherapybut subsequently experience tumor recurrence (26). The effect ofVS-5584on tumor-initiatingCSCof SCLCwas, therefore, assessedin the NCI-H841 SCLC xenograft model in vivo. VS-5584 treat-ment caused significant tumor growth inhibition (P¼ 0.003, Fig.6A) and reduced the proportion of CSC in NCI-H841 tumors asevidenced by a significant decrease in the percentage of SP cells (P¼ 0.006; Fig. 6B). Furthermore, cells dissociated from NCI-H841tumors of VS-5584–treatedmice showed a striking 67-fold reduc-tion in tumor-initiating frequency when injected in limitingdilutions into immunodeficient mice, indicating a marked deple-tion of CSC by VS-5584 in xenograft tumors (Fig. 6C).

Subsequently, we sought to determine if VS-5584 can delaytumor regrowth after initial debulking by cisplatin, a standard-of-care first-line chemotherapeutic agent, in human SCLC xenograftmodels. Before in vivo experiments, we confirmed that cisplatin oretoposide was not effective in depleting CSC of SCLC cell lines; infact, both agents enriched CSC, whereas VS-5584 depleted CSC invitro (Supplementary Fig. S3). In the NCI-H69 SCLC xenograftstudy, weekly i.p. dosing of 5mg/kg cisplatin for 2 weeks inducedinitial tumor regression, but these tumors regrew quickly aftercessation of cisplatin treatment. Single agent VS-5584 at 15mg/kgdosed orally once daily caused significant inhibition of NCI-H69 tumor growth (P < 0.05). In combination, VS-5584 substan-tially delayed the regrowth of NCI-H69 tumors following cessa-

tion of cisplatin treatment (Fig. 6D). Patient-derived–xenograft(PDX)models, which preserve the histology and heterogeneity ofpatient tumors, may be more relevant models for CSC research(27, 28). We, therefore, tested VS-5584 in a SCLC PDX modelestablished from a lymph nodemetastasis of a patient with SCLC.In keeping with our observations with the NCI-H69 model, VS-5584 administered following cessation of cisplatin dosingdelayed tumor regrowth (Fig. 6E). These results are consistentwith the suppression of cisplatin-resistant CSC by VS-5584,resulting in a longer delay of tumor regrowth after cessation ofcisplatin treatment.

DiscussionIntensive efforts have been devoted to targeting the PI3K–

mTOR pathway due to its critical importance in the proliferationand survival of cancer cells. Here, we report that, in addition toexhibiting broad and robust antitumor activity in animal models(15), VS-5584, a potent dual PI3K–mTOR kinase inhibitor, alsoexerts a preferential effect onCSCpresent in cell culture, in humantumor xenografts, and in surgically excised human tumor speci-mens. In stark contrast, the cytotoxic chemotherapeutic agentpaclitaxel, cisplatin, and etoposide increased the proportion ofCSC. Our observation that VS-5584 inhibited tumor regrowthafter effective treatment with cisplatin in SCLCmodels, includinga patient-derived primary tumor model, is consistent with thenotion that VS-5584 may delay tumor recurrence at least in partthrough the suppression of CSC. To our knowledge, this is the first

Figure 5.VS-5584 reduces the proportionof CSC in ex vivo human tumorspecimens. A–C, breast tumor tissuefrom three patients (A and B) orovarian tumor tissue from twopatients (C) was treated ex vivo with100 nmol/L VS-5584 for 5 days.Dissociated cells were subjected toCD44 andCD24FACS analysis (A) andAldefluor assay (B) or CD44 and CD117FACS analysis (C). Aldefluor assaywasnot performed on patient 2 tumor (B)due to insufficient number of cellsobtained after tumor dissociation. D,10,000 dissociated cells from ex vivotreated ovarian tumor tissue asdescribed in C were implantedsubcutaneously into immunodeficientmice. The number of mice that grewtumors atweek 17 out of total of 3miceper group (in parentheses) istabulated.

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comprehensive evaluation of the effect of a highly selective PI3K–mTOR inhibitor on CSC.

The PI3K–mTOR pathway has been shown to be important forthe proliferation of CSC in both solid tumors and leukemias (10–14). In prostate cancer, a CD133þ/CD44þ subpopulation withprogenitor/stem cell characteristics was shown to exhibit higherPI3K–mTOR pathway activity, and treatment with the PI3K–mTOR inhibitor BEZ-235 suppressed the proliferation of suchprostate CSC (10). PF-04691502, another dual PI3K–mTORinhibitor, was recently shown to inhibit the proliferation of CSCin vitro and to inhibit tumor growth in a colon CSC-derivedxenograft model (29). However, in these various studies, a criticalunanswered question remained whether PI3K–mTOR inhibitorsexert a general antiproliferative effect against both CSC and non-CSC, or whether the CSC aremore dependent on the PI3K–mTORpathway than are non-CSC. Interestingly, Korkaya and colleagues(13) reported that the AKT inhibitor perifosine reduced the

proportion of ALDHþ CSC as well as tumor-initiating cells inbreast tumor models, suggesting that perifosine has preferentialeffects on breast CSC. In light of the fact that perifosine wassubsequently reported to also inhibit EGFR and c-Met phosphor-ylation (30), the CSC effect of perifosine could be due to inhi-bition of these other molecular targets in addition to AKT.

VS-5584 is highly selective for class I PI3K isoforms andmTORkinase with no significant activity against more than 400 otherprotein and lipid kinases profiled (15).We provide clear evidencethat inhibition of mTORC1, mTORC2, and PI3K isoforms by VS-5584 confers a strong preferential inhibitory effect on CSC acrossdifferent carcinoma types, including breast, ovarian, and SCLC.Whereas VS-5584 preferentially abolished CSC in the MCF7breast tumor model, an mTORC1-selective inhibitor everolimusdid not reduce the proportion of CSC. Rapamycin, a closelyrelated mTORC1 inhibitor, also did not affect the self-renewalof mammary stem cells (13). These data, therefore, suggest that

Figure 6.VS-5584 targets SCLC CSC and delaystumor regrowth after chemotherapy inSCLC models. A–C, mice bearing NCI-H841 SCLC tumors were treated witheither vehicle or 20 mg/kg VS-5584three times weekly for 3 weeks. Cellswere dissociated from tumors andsubject to CSC assays. A, tumorvolume plot showing that VS-5584caused significant tumor growthinhibition (P ¼ 0.003). B, SP analysisshowing that VS-5584 significantlyreduced the proportion of SP cells intumors (P ¼ 0.006). C, in vivo limitingdilution assay showing that VS-5584caused a 67-fold reduction in TIF (P ¼5� 10�6). D and E, tumor weight plotsshowing that VS-5584 delayed tumorregrowth following weekly dosing of 5mg/kg cisplatin for 2 weeks. VS-5584was dosed orally at either 15 mg/kg ona once a day for 5 days weeklyschedule for 8 weeks starting onday 1 as a single-agent group (red) andin combination with cisplatin (green;D) or at 25 mg/kg on a three timesweekly schedule for 4 weeks startingon day 9 (E).






inhibiting PI3K isoforms,mTORC1, andmTORC2 simultaneous-ly may be important to exert a strong preferential effect on CSC.This hypothesis was further substantiated by our result that tripleknockdown of PI3Ka, PI3Kb, and mTOR by siRNA showed thestrongest preferential reduction of CSC, whereas knockdown ofPI3Ka, PI3Kb, or mTOR individually did not exert a preferentialeffect on CSC (Fig. 4).

Multiple, distinct subpopulations of leukemic stem cells havepreviously been identified (4). In solid tumors, CSC have beenidentifiedusing a variety ofmarkers and functional attributes evenwithin the same type of cancer, e.g., breast or ovarian cancer.While these markers may identify overlapping states of CSC, amore plausible explanation is that CSC are also heterogeneous insolid tumors. An ideal CSC-targeting agent should optimallyreduce all CSC pools. The PI3K–mTOR dual inhibitor VS-5584appears to be such an agent, as we have demonstrated that VS-5584 reduced CSC based on multiple independent measure-ments, including Aldefluor, Hoechst-dye exclusion, and CSCsurface markers (CD44hi/CD24lo for breast cancer and CD44hi

/CD117hi for ovarian cancer). Furthermore, VS-5584 also prefer-entially reduced theproportionof cellswith self-renewal potentialas measured by tumorsphere assays and by tumor-initiatingcapability following limiting dilution re-implantation of cellsinto secondary immunodeficient mice.

Our findings have implications for the clinical development ofVS-5584. The observation that CSC exhibited 10- to 30- foldgreater sensitivity to VS-5584 than non-CSC (Fig. 1) suggests thatclinical trials designedwith CSC-directed endpointsmay facilitatedemonstration of efficacy at sub-MTD doses. One such clinicaltrial concept is to test VS-5584 in amaintenance setting followingcessation of front-line chemotherapy with survival endpoints,rather than conventional tumor shrinkage endpoints, whichprimarily assess targeting of the bulk tumor burden. Currentfirst-line chemotherapy generally consists of cytotoxic agents,such as taxanes (e.g., paclitaxel) and platinum agents (e.g., cis-platin). While these agents may effectively debulk tumors andcontrol disease initially, tumors invariably recur due to ineffectivecontrol of CSC. A CSC-targeting agent is expected to block CSC-mediated tumor recurrence. Our results generated using twoSCLC xenograft models (Fig. 6) provide proof-of-conceptthat inhibition of PI3K and mTOR kinase activities by VS-5584after debulking tumors with a cytotoxic agent may substant-

ially extend antitumor response and delay tumor regrowth.Our findings thus provide strong rationale for the clinical devel-opment of VS-5584, currently in a phase 1 clinical trial(NCT01991938), for the treatment of cancer with the goal ofachievingmore durable responses through the preferential target-ing of CSC.

Disclosure of Potential Conflicts of InterestC.M.Vidal, J.E. Ring, I.M. Shapiro,D.T.Weaver,M.V. Padval, J.A. Pachter, and

Q. Xu have ownership interest (including patents) in Verastem, Inc. Nopotential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: V.N. Kolev, J.E. Ring, D.T. Weaver, M.V. Padval,J.A. Pachter, Q. XuDevelopment ofmethodology: V.N. Kolev, Q.G.Wright, J.E. Ring, D.T.Weaver,M.V. PadvalAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): V.N. Kolev, Q.G. Wright, C.M. Vidal, J.E. Ring,J. RiconoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): V.N. Kolev, Q.G. Wright, C.M. Vidal, J.E. Ring,I.M. Shapiro, J.A. Pachter, Q. XuWriting, review, and/or revision of the manuscript: V.N. Kolev, Q.G. Wright,C.M. Vidal, I.M. Shapiro, D.T. Weaver, M.V. Padval, J.A. Pachter, Q. XuAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Q.G. WrightStudy supervision: J.A. Pachter, Q. Xu

AcknowledgmentsThe authors thank Dr. Robert Weinberg for critical reading of the article and

Verastem team for valuable advice throughout this work. The authors alsothank David Dombkowski at MGH for assistance with FACS analysis of SP andCD44hi/CD24lo cells.

The NCI-H69 xenograft study was conducted by TGen (Scottsdale, AZ).Animal husbandry and in vivo procedures for Figs 3, 5D, 6, and SupplementaryFigs. S1 and S2 were conducted with outstanding support of Sara Little atViviSource Laboratories (Waltham, MA).

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 April 23, 2014; revised October 23, 2014; accepted November 10,2014; published OnlineFirst November 28, 2014.

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2015;75:446-455. Published OnlineFirst November 28, 2014.Cancer Res Vihren N. Kolev, Quentin G. Wright, Christian M. Vidal, et al. Stem CellsPI3K/mTOR Dual Inhibitor VS-5584 Preferentially Targets Cancer

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