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MAY 2012 CANCER DISCOVERY | 425

A Small-Molecule Inhibitor of PI3K-p110 in Cancer research brief

Q1functional characterization of an isoform-selective inhibitor of Pi3K-p110b as a Potential anticancer agent

Jing Ni1,2, Qingsong Liu1,2, Shaozhen Xie1,2, Coby Carlson4, Thanh Von1,2, Kurt Vogel4, Steve Riddle4, Cyril Benes3, Michael Eck1,2, Thomas Roberts2, Nathanael Gray1,2, and Jean Zhao1,2

IntroductIonThe class Ia phosphatidylinositol-3-kinase (PI3K) path-

way is arguably the most important signaling pathway in cells because of its roles in the control of cell growth, sur-vival, and death (1, 2). The PI3K pathway is activated at the cell membrane by an important lipid signaling mol-ecule known as phosphatidylinositol 3,4,5-trisphosphate (PIP3). Under normal conditions, the level of PIP3 is tightly regulated by the activities of 2 enzymes, PI3K (a lipid ki-nase) and PTEN (a lipid phosphatase), which act as “on/off” switches in opposition to each other. In response to the extracellular signals mediated by receptor tyrosine kinases (RTK), G protein–coupled receptors (GPCR), or GTPases, class Ia PI3Ks are recruited to the cell membrane

AbstrAct

Authors’ Affiliations: 1Department of Cancer Biology, Dana-Farber Cancer Institute, 2Department of Biological Chemistry and Molecular Pharm-acology, Harvard Medical School, 3Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, Massachusetts; and 4Life Technologies Corporation, Madison, Wisconsin

J. Ni and Q. Liu contributed equally to this article.

Note: Supplementary data for this article are available at Cancer Discovery Online (http://www.cancerdiscovery.aacrjournals.org).

Corresponding Authors: Jean Zhao, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215. Phone: 617-632-2932; Fax: 617-632-3709; E-mail: [email protected]; or Nathanael Gray, Harvard Medical School, 250 Longwood Avenue, Boston, MA 02115. Phone: 617-582-8590; Fax: 617-582-8615; E-mail: [email protected]

doi: 10.1158/2159-8290.CD-12-0003

©2012 American Association for Cancer Research.

Genetic approaches have shown that the p110 isoform of class Ia phosphati-dylinositol-3-kinase (PI3K) is essential for the growth of PTEN-null tumors.

Thus, it is desirable to develop p110-specific inhibitors for cancer therapy. Using a panel of PI3K isoform-specific cellular assays, we screened a collection of compounds possessing activities against kinases in the PI3K superfamily and identified a potent and selective p110 inhibitor: KIN-193. We show that KIN-193 is efficacious specifically in blocking AKT signaling and tumor growth that are dependent on p110 activation or PTEN loss. Broad profiling across a panel of 422 human tumor cell lines shows that the PTEN mutation status of cancer cells strongly correlates with their response to KIN-193. Together, our data provide the first pharmacologic evidence that PTEN-deficient tumors are dependent on p110 in animals and suggest that KIN-193 can be pursued as a drug to treat tumors that are dependent on p110 while sparing other PI3K isoforms.

siGNificaNce: We report the first functional characterization of a p110-selective inhibitor, KIN-193, that is efficacious as an antitumor agent in mice. We show that this class of inhibitor holds great promise as a pharmacologic agent that could be used to address the potential therapeutic benefit of treating p110-dependent PTEN-deficient human tumors. Cancer Discov; 2(5); 425–33. ©2012 AACR.

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426 | CANCER DISCOVERY MAY 2012 www.aacrjournals.org

Ni et al.research brief Ni et al.research brief

and subsequently phosphorylate phosphatidylinositol-4, 5-bisphosphate (PIP2) to produce PIP3. This in turn activates the Ser/Thr kinase AKT and other downstream effectors to regulate multiple cellular functions, including prolifera-tion, survival, and migration. Class Ia PI3Ks are heterodi-meric lipid kinases consisting of a p110 catalytic subunit (p110, p110, or p110) and a p85 regulatory subunit. Although the expression of p110 is largely restricted to the immune system, p110 and p110 are commonly expressed in all tissues. The tumor suppressor PTEN catalyzes the dephosphorylation of PIP3 back to PIP2 and thereby antago-nizing PI3K activity.

Aberrant activation of the class Ia PI3K signaling pathway is a common event in many types of cancer. Frequently observed mechanisms of PI3K pathway hyperactivation include gain-of-function mutations in p110, loss-of- function mutations or deletions in PTEN, and activation of RTKs (2). No activating mutations have been found in p110 so far with the exception of gene amplification in breast and ovarian cancers (3, 4). Interestingly, however, we have re-cently found that genetic ablation of p110, but not p110, is sufficient to inhibit tumor formation driven by Pten loss in the anterior prostate in a mouse prostate tumor model (5). Other recent studies have shown that certain PTEN-deficient human cancer cell lines are sensitive to inactivation of p110 rather than p110 (6, 7).

To investigate whether the dependence on p110 can be recapitulated with pharmacologic inhibitors of p110 kinase activity, several groups have been developing p110-specific inhibitors. However, only a few selective p110 inhibitors have been reported. Perhaps the best described p110-specific in-hibitor to date is TGX-221, which has been used in defining p110 as an important new target for antithrombotic agents (8), but none of these compounds has been reported for tumor studies in vivo. We sought to identify alternative compounds that are potent and selective p110 inhibitors with proper-ties suitable for use in tumor studies in vivo. Here we show that KIN-193 is a potent and selective p110 inhibitor when evaluated in a battery of biochemical and cellular assays. In addition, we show that this compound can inhibit the growth of tumors driven by p110 or PTEN loss in vivo. Together, our study has identified and characterized KIN-193 as a potential antitumor agent that can be used to treat tumors that are dependent on p110 while sparing other PI3K isoforms.

resultsTo screen for new selective PI3K inhibitors, we generated

a set of isogenic human mammary epithelial cell (HMEC) lines that stably express myristoylated (Myr)-tagged PI3K class Ia p110 isoforms (p110, p110, or p110), respec-tively, designated as HMEC-CA-p110, HMEC-CA-p110, and HMEC-CA-p110. In these cell lines, endogenous PI3K signaling is inactive under serum-free condition, whereas the ectopically expressed Myr-p110 isoforms are membrane- targeted and constitutively active as a result of N-terminal myristoylation (9, 10), thus driving the phosphorylation of AKT, a downstream target of PI3K (Supplementary Fig. S1, first lane vs. second lane). Notably, activation of p110 can also be achieved by N-terminal addition (11). We validated

the specificity of this system by monitoring the ability of well-characterized p110 isoform-specific inhibitors, for example, PIK-75 for p110 (12), TGX-221 for p110 (8), IC87114 for p110 (13), and a pan-inhibitor GDC-0941 (14), to inhibit phosphorylation of AKT at both Thr308 and Ser473 in a dose-dependent manner (Supplementary Fig. S1).

The high degree of sequence similarity among p110 catalytic isoforms of PI3K makes it extremely challenging to develop isoform-specific PI3K inhibitors de novo (15); we therefore assembled a collection of 19 compounds possessing activity against PI3Ks (Supplementary Table 1) for our study. To facilitate systematic analyses of these compounds, we used the BacMam gene delivery technology to express GFP-AKT in these isogenic HMEC cells, which enables a time-resolved fluorescence resonance energy transfer (TR-FRET)–based assay termed LanthaScreen (16). The phosphorylation status of AKT at both Thr308 and Ser473 was measured by the binding of terbium-labeled phospho-specific antibodies that undergo FRET with the green fluorescent protein–la-beled AKT (Supplementary Fig. S2A). The most promis-ing candidate to emerge from this profiling was KIN-193 (Fig. 1A and Supplementary Fig. S2B), a compound recently described as a p110-selective inhibitor (17). Interestingly, KIN-193 is a close structural analog of TGX-221, a p110 isoform-specific inhibitor that has been used in defining p110 as an important new target for antithrombotic agent (8) (Fig. 1B). KIN-193 has comparable selectivity and potency against p110 compared with TGX-221 as measured by AKT phosphorylation in HMECs through Western blot analysis (Supplementary Fig. S3).

We next determined the target spectrum of KIN-193 against the PI3K superfamily as well as the kinome. An in vitro kinase assay showed that KIN-193 is highly potent in the inhibition of p110’s kinase activity (IC50 of 0.69 nmol/L)and has 200-, 20-, and 70-fold selectivity over p110, p110, and p110γ isoforms, respectively (Fig. 1C). KIN-193 also exhibited selectivity of approximately 80-fold over PI3K-C2 and DNA-PK and more than 1,000-fold over other PI3K-related kinases (Fig. 1C). An inhibitor-kinase interaction profiling of KIN-193 against a panel of 433 kinases using the KinomeScan approach (DiscoverX) showed that KIN-193 is highly selec-tive in its interaction with PI3Ks (Fig. 1D and Supplementary Table 2). Together, these data suggest that KIN-193 is a selective kinase inhibitor that targets the p110 isoform of PI3K.

Recent studies have shown that certain PTEN-deficient tumors are critically dependent on p110’s activity (5–7). To determine whether KIN-193 selectively targets PTEN-deficient tumors, we tested the effect of KIN-193 on cell proliferation on a large panel of 422 cancer cell lines using high-through-put tumor cell line profiling (18). As shown in Figure 2A, 35% of cell lines with PTEN mutations (20 of 57) and 16% of cell lines with wild-type PTEN (58 of 365) were sensitive to KIN-193 with a threshold of EC50 less than 5 µmol/L. The statisti-cal analysis suggested that cell lines harboring mutations in PTEN exhibited significantly higher sensitivity to KIN-193 (P = 0.0014, 2-tailed Fisher exact test; Fig. 2A and Supplementary Table 3). We further evaluated the effect of KIN-193 along with other pan- or isoform-selective PI3K inhibitors on PI3K signaling on a number of PTEN-null cancer cell lines,





A Small-Molecule Inhibitor of PI3K-p110 in Cancer research briefA Small-Molecule Inhibitor of PI3K-p110 in Cancer research brief

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Figure 1. Identification of KIN-193 as a p110-specific inhibitor. a, distribution of IC50 values for selected small-molecule inhibitors against various PI3K p110 isoforms in isogenic HMEC lines expressing CA-p110, CA-p110, or CA-p110 using BacMam-enabled LanthaScreen assays. IC50 values were measured by inhibition of AKT phosphorylation. b, the chemical structure of KIN-193. c, in vitro kinase assay measurements of IC50 values for KIN-193 against recombinant PI3Ks and phosphatidylinositol-3 kinase-related kinases (PIKK) using SelectScreen (Invitrogen). D, KinomeScan profile of KIN-193 against 433 kinases. The dendrograms were generated with DiscoverX TREEspot Version 4 software. Targets retaining less than 5% residual activity (red circles) or more than 35% (green circles) (compared with DMSO controls) after KIN-193 (10 µmol/L) treatment. Circle size is proportional to binding affinity.

including HCC70, MDA-MB-468, BT549, and PC3 cell lines (Supplementary Fig. S4). Our results show that both KIN-193 and GDC-0941 significantly inhibited AKT phosphorylation, whereas PIK-75 and IC87114 had much less effect (Fig. 2B). Taken together, these data suggest that KIN-193 strongly im-pairs PI3K signaling in PTEN-deficient cancer cells.

To facilitate in vivo efficacy studies of KIN-193, we performed pharmacokinetic analyses of KIN-193 and found that intraperitoneal (i.p.) delivery to be the optimal route to achieve robust in vivo exposure (Supplementary Fig. S5A). To determine the pharmacodynamics of KIN-193 in tumors in vivo, we engineered rat fibroblast (Rat1) cells to express both p53DD, a dominant negative mutant of

p53 (10), and a constitutively activated myr-p110 (Rat1-CA-p110) to enable these cells to form xenograft tumors in mice. For comparison, we also generated an isogenic Rat1 cell line expressing p53DD and myr-p110 (Rat1-CA-p110), which is also tumorigenic in vivo. We introduced Rat1-CA-p110 and Rat1-CA-p110 cells subcutaneously into the contralateral flanks of athymic mice such that tu-mors driven by activated p110 or p110 would be exposed to identical conditions and that concern about animal-to-animal variability could be eliminated. When tumors reached a volume of approximately 500 mm3, the tumor-bearing mice received a single i.p. injection of KIN-193 (10 mg/kg). The plasma concentration of KIN-193 was highest






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Figure 2. Effects of KIN-193 on PTEN-deficient cancer cells. a, effects of KIN-193 on the proliferation of a panel of 422 cancer cell lines. IC50 values of KIN-193 are ordered from low to high. Pink bars indicate cell lines known to be mutant for PTEN. The subset of cell lines with IC50 values <5 µmol/L were defined as KIN-193–sensitive cell lines. Thirty-five percent of cell lines with PTEN mutations (20 of 57) versus 16% of cell lines with wild-type PTEN (58 of 365) were sensitive to KIN-193 (P = 0.0014, Fisher exact test). b, effects of KIN-193, GDC-0941, PIK-75, and IC87114 on AKT phosphorylation in PTEN-deficient cell lines as indicated. Representative Western blots are shown. Bar graphs represent mean SD of Western blot quantitations of AKTT308 (n = 3). *P , 0.05, **P , 0.01 (one-way analysis of variance followed by Dunnett’s multiple comparison test). DMSO, dimethyl sulfoxide.






at 1 hour postinjection and declined to undetectable lev-els by 4 hours (Supplementary Fig. S5B). Concentrations of KIN-193 in both the CA-p110- and CA-p110-driven tumors paralleled the plasma concentrations (Fig. 3A). Analyses of tumor lysates harvested at various time points after KIN-193 injection revealed that the phosphorylation of AKT was significantly reduced at 1 hour after KIN-193 injection in Rat1-CA-p110 tumors but remained unchanged in Rat1-CA-p110 tumors (Fig. 3A). These in vivo pharmacokinetic and pharmacodynamic properties suggest that KIN-193 holds promise as an effective in vivo p110-specific inhibitor.

To evaluate the efficacy of KIN-193 in blocking tumor growth in vivo, we generated additional cohorts of mice bear-ing tumors driven by Rat1 cell expressing CA-p110 or CA-p110. When tumors size reached approximately 500 mm3, these mice were grouped and administered with vehicle con-trol or KIN-193 by i.p. injection (20 mg/kg) once or twice daily. Although administration of KIN-193 significantly impaired Rat1-CA-p110-driven tumor growth in a dose-dependent manner, KIN-193 had little effect on the growth of Rat1-CA-p110-derived xenograft tumors (Fig. 3B), demonstrating the

specific antitumor effect of KIN-193 on p110-driven tumors in vivo. Remarkably, all mice receiving KIN-193, either 1 or 2 doses daily, maintained their body weight throughout the en-tire treatment course of 13 days (Fig. 3C), indicating that KIN-193 is well tolerated in mice.

We next assessed the antitumor activity of KIN-193 on the growth of PTEN-deficient tumors in vivo using co-horts of mice bearing PTEN-deficient tumor xenografts (HCC70 and PC3) and PTEN wild-type tumor xenografts (HCC1954). KIN-193 significantly inhibited tumor growth of both HCC70 and PC3 tumors but failed to block the growth of HCC1954 tumors (Fig. 4A–C). However, HCC1954, a wild-type PTEN cancer cell line harboring an activated mutant p110, responded to treatment with the pan-PI3K inhibitor, GDC-0941 (Fig. 4C). Concurrently, immunohistochemistry analyses of tumor specimens iso-lated from tumor-bearing mice at 4 days after treatment revealed that KIN-193 dramatically reduced levels of both AKT phosphorylation and Ki67 signal in xenograft tumors of both PTEN-deficient cancer cell lines, HCC70 and PC3 (Fig. 4D, 4E). In contrast, a pan-PI3K inhibitor, GDC-0941, but not KIN-193, blocked AKT phosphorylation and cell

Ni et al Figure. 3

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cFigure 3. In vivo characterization of the anticancer potential of KIN-193. a, pharmacokinetics and pharmacodynamics of KIN-193 in mice. Concentrations of KIN-193 in tumors (red lines) and relative pAKTT308 phosphorylation values (black columns) in Rat1-CA-p110 or Rat1-CA-p110 tumors in tumor-bearing mice at 0, 1, 4, 8, and 24 hours after KIN-193 i.p. injections. The data are presented as mean 6 SEM (n = 6). **P , 0.01 (one-way analysis of variance followed by Dunnett’s multiple comparison test). b, in vivo effect of KIN-193 on CA-p110- or CA-p110-driven tumors. Tumor growth curves of Rat1-CA-p110 and Rat1–CA-p110 tumors treated with vehicle control or KIN-193 (20 mg/kg, per day or twice a day). The data are presented as mean 6 SEM (n = 8). *P , 0.05, **P , 0.01 (Student t test). c, body weight measurements of mice in control or KIN-193-treated groups throughout the treatment course.






proliferation in HCC1954 tumor xenografts (Fig. 4F). We conclude that KIN-193, a p110-selective inhibitor, can specifically suppress both the PI3K pathway activation and oncogenic transformation induced by PTEN deficiency.

dIscussIonAccumulating evidence has suggested that distinct PI3K

isoforms are specifically involved in a variety of different dis-ease conditions, including cancer, metabolic disorders, im-munity, and cardiovascular dysfunction (18–21). Previous reports have shown that p110 is important in thrombo-sis and that a selective p110 small molecular inhibitor, TGX-221, prevents platelet aggregation in an extracorporeal circulation model (8). Recently our group and others have provided compelling evidence that p110 is involved in PTEN loss–induced tumorigenesis (5–7). Additional aspects of p110 isoform dependency of PTEN-deficient cancer cell lines were presented at the 2012 Cold Spring Harbor conference on PTEN Pathways and Targets (22). However, no p110-specific inhibitors have been described in tumor studies in vivo. Here we show for the first time that a p110-selective inhibitor, KIN-193, can block both the signaling and

tumor growth driven by PTEN loss, providing the first phar-macologic evidence for tumor dependence on p110 kinase activity and suggesting that PTEN-null tumors would be an appropriate genetic background to deploy these inhibitors. Notably, IC50 values for KIN-193 differ with the system of study, for example, it is approximately 1 nmol/L in vitro and 100 to 500 nmol/L in cell culture (Figs. 1A and 1C). It can reach as high as 1 μmol/L in vivo. Although enzymatic assays are useful, they are poor predictors of whether bona fide cel-lular selectivity will be achieved. We show that in cell culture, we can achieve selectivity of p110 over p110 and p110 (Fig. 1A). In mice we have only shown that KIN-193 inhibits the PI3K signaling and tumor growth driven by activated p110 but not p110. However, because the concentrations in vivo are in the range that other PI3K family members (e.g., p110 and DNA-PK) may be inhibited, we cannot exclude that they contribute to the antiproliferative effects.

The waterfall profiling of cancer cell lines for sensitivity to KIN-193 is particularly interesting for two notions. First, although there is a significant correlation between PTEN mutation and sensitivity to KIN-193, not all PTEN-null cell lines are impacted by treatment with KIN-193. This is per-haps not surprising. Our prior finding of the importance of

Figure 4. In vivo effect of KIN-193 on PTEN-deficient tumors. a–c, xenograft tumor growth curves of HCC70, PC3, and HCC1954 treated with vehicle or KIN-193 (i.p., 20 mg/kg, twice a day) or GDC-0941 [orally, 150 mg/kg, per day (HCC1954 tumors)] as indicated. The data are represented as mean 6 SEM (n = 10). *P , 0.05, **P , 0.01 (Student t test). D–f, immunohistochemistry analyses of pAKTS473 and Ki67 on xenograft tumor samples of HCC70, PC3, and HCC1954 treated with vehicle, KIN-193 (i.p., 20 mg/kg, twice a day), or GDC-0941 (orally, 150 mg/kg, per day) for 4 days. Tumors were collected 1 hour after the last dosing, fixed, and subjected to immunohistochemistry analysis. Scale bar, 10 µm.

Ki67pAkt S473

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Compounds and ReagentsPIK-75 and TGX-221 were from Chemdea. IC87114 was from

Selleck Chemicals. GDC-0941 and KIN compounds were pur-chased from MedChemexpress. Anti-PTEN, anti-p110, anti-p110, anti-phospho-AKT (Thr308), anti-phospho-AKT (Ser473), and anti-AKT were all from Cell Signaling Technology. Anti-p85 antibody was from Upstate Biotechnology. Anti-vinculin and anti– -tubulin antibodies were from Sigma. Anti-Ki67 antibody was from Vector Labs.

LanthaScreen Cellular AssayExperiments were performed according to the manufacturer’s in-

structions (Invitrogen) and a previous report (16). Briefly, a 10% BacMam GFP-AKT virus (10% v/v) solution was incubated with HMECs overnight with 0.53 Enhancer Solution. The next day, transduced cells were harvested and seeded in a white 384-well plate (Corning #3570) at a density of 5,000 cells/well in 36 μL of Opti-MEM reduced serum medium. The next day, cells were treated with serial dilutions of com-pounds (4 µL of 103 solution in 1% dimethyl sulfoxide) for 1.5 hours. After compound treatment, the assay medium was removed and cells were lysed through the addition of LanthaScreen lysis buffer in 20 μL volume supplemented with protease and phosphatase inhibitor cock-tails and either Tb- anti-pAKTT308 or Tb-anti-pAKTS473 antibody (2 nmol/L final concentration) for 2 hours at room temperature. The TR-FRET signal (emission ratio 520 nm/495 nm) was read on an EnVision fluorescence plate reader from PerkinElmer. Compounds were tested in duplicate and the data presented are from at least two independent experiments. Curve fitting analysis and IC50 value determination were performed using GraphPad Prism 4 (GraphPad Software).

Ambit in Vitro KinomeScan Kinase Selectivity ProfileKIN-193 was profiled at a concentration of 10 µmol/L against a

diverse panel of 433 kinases by Ambit Biosciences. Scores for primary screen hits are reported as percent of the dimethyl sulfoxide control (% control). For kinases in which no score is shown, no measurable bind-ing was detected. The lower the score, the lower the Kd is likely to be such that scores of zero represent strong hits. Scores are related to the probability of a hit but are not strictly an affinity measurement. At a screening concentration of 10 µmol/L, a score of less than 10% implies that the false-positive probability is less than 20% and the Kd is most likely less than 1 µmol/L. A score between 1% and 10% implies that the false-positive probability is less than 10%, although it is difficult to as-sign a quantitative affinity from a single-point primary screen. A score of less than 1% implies that the false-positive probability is less than 5% and the Kd is most likely less than 1 µmol/L.

High-Throughput Cell Viability AssayCell viability was determined as previously described (18). Briefly,

cells were seeded in medium containing 5% FBS at a density en-suring cell growth throughout drug treatment (approximately 15% for most cell lines). Drug treatment was started 24 hours postseed-ing and continued for 72 hours. Cell were fixed and stained using Syto60 (Invitrogen), a red fluorescent DNA stain. The relative cell number was calculated by taking the ratio of the relative fluorescence intensity from drug-treated wells over untreated wells after back-ground subtraction (cell-free wells). Nine doses of KIN-193 were used in twofold dilution steps ranging from 5.12 µmol/L to 0.02 µmol/L. IC50, corresponding to 50% cell number compared with control (un-treated) wells, determined using a fixed top and bottom sigmoidal fitting algorithm implemented in PipelinePilot (Accelerys).

Western Blot AnalysisTumor samples or cells were lysed in ice-cold RIPA buffer (1%

NP40, 0.1% sodium dodecyl sulfate, 0.5% sodium deoxycholate

p110 in PTEN-loss driven tumorigenesis was based on a defined genetic mouse model, whereas human cancer lines are far more complex in their genetic makeups. Because loss of PTEN simply removes the “brakes” on the PI3K path-way, the dependence of PTEN-null tumors on p110 may be altered by coexisting mutations of the tumor. Thus, if PTEN-null tumor cells also harbor a p110 gain-of-function mutation or an upstream mutation that primarily drives p110 activation, then the tumor might depend on p110, not p110. It is also possible that the presence of other on-cogenic mutations downstream of PI3K (e.g., AKT) or in PI3K-independent pathways may render PTEN-null tumors less reliant on p110. Recent studies showed that p110 signals downstream of certain GPCRs or integrins (5, 23). It also has been proposed that p110 is responsible for the basal lipid kinase activity that can be enhanced in the ab-sence of PTEN to drive transformation (5, 12, 24). Therefore, only those PTEN-null tumors in which the PI3K pathway is activated by certain GPCRs or integrins that drive p110 activation or perhaps through the background PI3K activity contributed by p110 are expected to remain dependent on p110. The second feature of the profiling is perhaps more interesting. There are a number of the cell lines that respond to KIN-193 that are not PTEN-null by mutation. Although some of these lines may have lost PTEN expression by other means (e.g., epigenetic alterations), it is possible that there are PTEN-independent mechanisms that activate p110 in tumors.

To date, the array of PI3K inhibitors that are in preclinical and clinical development consists largely of pan inhibitors, and patients with PTEN-deficient tumors are potential candidates for such PI3K-targeted therapy. However, isoform-specific molecules are emerging in the clinic. The promising early clinical results of the p110-selective inhibitor CAL-101 in treating lymphoid malignancies sug-gest that isoform-selective inhibitors may have efficacy and safety advantages over pan-PI3K inhibitors (25). This study identifies KIN-193 as a selective and efficacious p110 inhibitor and shows its potent anticancer activity in PTEN-deficient tumor models, providing a starting point from which to develop orally bioavailable compounds that could ultimately be used to assess the potential therapeutic benefit of treating p110-dependent tumors.

MethodsCell Culture

Cancer cell lines (HCC1954, HCC70, BT549, BT474, and PC3) were obtained from the American Type Culture Collection. The MDA-MB-468 cell line was from MD Anderson Cancer Center. These cells were frozen after receiving and freshly thawed cells were used at early passage, and no authentication was done by the authors. These cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 100 μg/mL penicillin-streptomycin. HMEC derivative cell lines were cultured as previously described (10). Rat1 cells and its derived cell lines were maintained in DMEM supple-mented with 10% FBS and 100 μg/mL penicillin–streptomycin. Rat1 stable cell lines were generated by serial infection with retroviral su-pernatants carrying p53DD and Myr-HA-p110 or Myr-HA-p110 in the presence of 8 μg/mL polybrene. The cells were then selected with blasticidin 5 μg/mL and puromycin 2 μg/mL.






authors’ contributionsConception and design: J. Ni, Q. Liu, C. Benes, M. Eck, N. Gray, J. ZhaoDevelopment of methodology: J. Ni, Q. Liu, C. Carlson, S. Riddle, J. ZhaoAcquisition of data: J. Ni, Q. Liu, S. Xie, C. Carlson, C. Benes, N. GrayAnalysis and interpretation of data: J. Ni, Q. Liu, S. Xie, C. Carlson, C. Benes, N. Gray, J. ZhaoWriting, review, and/or revision of the manuscript: J. Ni, Q. Liu, S. Xie, C. Carlson, C. Benes, M. Eck, T. Roberts, N. Gray, J. ZhaoAdministrative, technical, or material support: J. Ni, K. Vogel, N. Gray, J. ZhaoStudy supervision: N. Gray, J. ZhaoTechnical assistance: T. Von

acknowledgmentsWe thank Dr. Roderick Bronson and the Dana-Farber/Harvard

Cancer Center Rodent Histopathology Core for histopathologic analyses.

Grant supportThis work was supported by NIH grants CA030002 and P01

CA089021 (T. Roberts); CA134502, P50 CA089393-08S1, and P01 CA142536 (J. Zhao); CA148164-01 (J. Zhao, N. Gray, M. Eck, and T. Roberts); and 1U54HG006097-01 (C. Benes). T. Roberts and J. Zhao are supported by a Stand Up To Cancer Dream Team Translational Research Grant, a Program of the Entertainment Industry Foundation (SU2C-AACR-DT0209). C. Benes received a research grant from AstraZeneca.

Received January 3, 2012; revised February 24, 2012; accepted February 24, 2012; published OnlineFirst April 12, 2012.

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3. Brugge J, Hung MC, Mills GB. A new mutational AKTivation in the PI3K pathway. Cancer Cell 2007;12:104–7.

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5. Jia S, Liu Z, Zhang S, Liu P, Zhang L, Lee SH, et al. Essential roles of PI(3)K-p110beta in cell growth, metabolism and tumorigenesis. Nature 2008;454:776–9.

6. Wee S, Wiederschain D, Maira SM, Loo A, Miller C, deBeaumont R, et al. PTEN-deficient cancers depend on PIK3CB. Proc Natl Acad Sci U S A 2008;105:13057–62.

7. Torbett NE, Luna-Moran A, Knight ZA, Houk A, Moasser M, Weiss W, et al. A chemical screen in diverse breast cancer cell lines reveals genetic enhancers and suppressors of sensitivity to PI3K isoform-selective inhibition. Biochem J 2008;415:97–110.

8. Jackson SP, Schoenwaelder SM, Goncalves I, Nesbitt WS, Yap CL, Wright CE, et al. PI 3-kinase p110beta: a new target for antithrom-botic therapy. Nat Med 2005;11:507–14.

9. Zhao JJ, Gjoerup OV, Subramanian RR, Cheng Y, Chen W, Roberts TM, et al. Human mammary epithelial cell transformation through the activation of phosphatidylinositol 3-kinase. Cancer Cell 2003;3:483–95.

10. Zhao JJ, Liu Z, Wang L, Shin E, Loda MF, Roberts TM. The oncogenic properties of mutant p110alpha and p110beta phosphatidylinositol

in 13 phosphate-buffered saline) containing protease inhibitor cocktail (Roche) and phosphatase inhibitor (Thermo) and lysates were clarified by centrifugation. For experiments with compound treatments, the 80% confluent cells in 6-well plates were treated with the indicated compound for 1 hour, then the cells were lysed in SDS loading/lysis buffer (4% sodium dodecyl sulfate, 20% glycerol, 120 mmol/L Tris pH 6.8, and pyronin Y). Equal amounts of lysates were resolved on sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels and transferred to nitrocellulose membranes. After incubation with blocking buffer (50% LICOR blocking buffer in phosphate-buffered saline) for 1 hour, the membranes were incubated with primary antibody overnight at 4°C and then incu-bated with fluorescently labeled secondary antibodies (Rockland Immunochemicals) for 1 hour at room temperature. The protein signal was detected using an Odyssey scanner (LI-COR).

in Vivo Pharmacokinetics and Pharmacodynamic AssaysApproximately 6- to 8-week-old female nude mice (Taconic)

were injected subcutaneously with Rat1-Myr-HA-p110 (Rat1-CAp110) cells (1 3 106 cells in 40% matrigel) (BD Biosciences) in one flank (Site 1) and Rat1-Myr-HA-p110 (Rat1-CAp110) cells (0.5 3 106 cells in 10% matrigel) in the contralateral flank (Site 2). When tumors grew to approximately 500 mm3, mice were dosed once by i.p. injection with KIN-193 formulated in 7.5% NMP (Sigma), 40% PEG400 (Sigma), 52.5% dH20 at 0.1 mL/10 g body weight, and 10 mg/kg. Tumors were collected at 0, 1, 4, 8, and 24 hours after compound administration and blood samples were ob-tained by direct heart puncture. Serum was separated and stored at –80°C. The drug concentrations in serum and tumor samples were assessed by liquid chromatography–tandem mass spectroscopy analysis by the DMPK group. The phosphorylation status of AKT in tumor samples was assessed by Western blot analysis and quanti-fied using ImageJ software with statistical calculations performed using Prism software.

Xenograft Tumor GrowthEach flank of athymic nude mice was injected subcutaneously

with 1 3 106 cells resuspended in 30% matrigel. KIN-193 was given by i.p. injection either once or twice a day at 20 mg/kg. GDC-0941 (0.1% methylose, 0.1% Tween 80) was given by gavage at 150 mg/kg once per day. Rat1-CA-p110, Rat1-CA-p110, HCC70, and HCC1954 xenograft tumor growth was assessed in female nude mice. PC3 xeno-graft tumor growth was assessed in male nude mice. Tumor-bearing animals were treated with KIN-193, GDC-0941, or vehicle control as described previously. Tumor volumes were calculated using the formula (length × width2)/2. All the animal experiments were done in accordance with National Institutes of Health animal use guidelines and protocols approved by the Dana-Farber Cancer Institute Animal Care and Use Committee.

Immunohistochemical StainingDeparaffinized tissue sections were heated in 10 mmol sodium

citrate buffer for 20 minutes using a microwave oven. Primary anti-bodies were incubated on slides overnight at 4°C. Sections were then incubated with biotinylated secondary antibody (Vector Labs) and ABC solution (Vector Labs). Tissues were stained by diaminobenzi-dine (Vector Labs) followed by Meyer’s hematoxylin counterstaining (BioGenex Laboratories).

Disclosure of Potential conflicts of interestM. Eck, T. Roberts, and J. Zhao are consultants for Novartis

Pharmaceuticals, Inc. M. Eck is a consultant/advisory board mem-ber for Novartis Institutes for Biomedical Research. No potential conflicts of interest were disclosed by the other authors.






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as a Potential Anticancer Agentβof PI3K-p110Functional Characterization of an Isoform-Selective Inhibitor

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