Targeting Phosphatidylinositol 3-Kinase Signaling Pathway For

Published OnlineFirst August 23, 2017; DOI: 10.1158/1535-7163.MCT-17-0326

Small Molecule Therapeutics Molecular Cancer Therapeutics Targeting Phosphatidylinositol 3-Kinase Signaling Pathway for Therapeutic Enhancement of Vascular-Targeted Photodynamic Therapy Daniel Kraus1, Pratheeba Palasuberniam1, and Bin Chen1,2

Abstract

Vascular-targeted photodynamic therapy (PDT) selectively the strongest synergism, followed in order by combinations disrupts vascular function by inducing oxidative damages to with pan-PI3K inhibitor BKM120 and p110a isoform-selective the vasculature, particularly endothelial cells. Although effec- inhibitor BYL719. Combination treatments of PDT and tive tumor eradication and excellent safety proﬁle are well BEZ235 exhibited a cooperative inhibition of antiapoptotic demonstrated in both preclinical and clinical studies, incom- Bcl-2 family protein Mcl-1 and induced more cell apoptosis plete vascular shutdown and angiogenesis are known to cause than each treatment alone. In addition to increasing treatment tumor recurrence after vascular-targeted PDT. We have explored lethality, BEZ235 combined with PDT effectively inhibited therapeutic enhancement of vascular-targeted PDT with PI3K PI3K pathway activation and consequent endothelial cell pro- signaling pathway inhibitors because the activation of PI3K liferation after PDT alone, leading to a sustained growth inhi- pathway was involved in promoting endothelial cell survival bition. In the PC-3 prostate tumor model, combination treat- and proliferation after PDT. Here, three clinically relevant ments improved treatment outcomes by turning a temporary small-molecule inhibitors (BYL719, BKM120, and BEZ235) of tumor regrowth delay induced by PDT alone to a more long- the PI3K pathway were evaluated in combination with verte- lasting treatment response. Our study strongly supports the porﬁn-PDT. Although all three inhibitors were able to syner- combination of vascular-targeted PDT and PI3K pathway inhi- gistically enhance PDT response in endothelial cells, PDT bitors, particularly mTOR inhibitors, for therapeutic enhance- combined with dual PI3K/mTOR inhibitor BEZ235 exhibited ment. Mol Cancer Ther; 1–10. 2017 AACR.

Introduction gland and readily accessible by laser fibers. Preclinical studies demonstrate that vascular-targeted PDT with photosensitizers Photodynamic therapy (PDT) combines a photosensitizer, such as verteporfin and Tookad effectively causes tumor destruc- laser light matching the absorption of photosensitizer, and oxy- tion by inducing endothelial cell injuries (5–7) and consequent gen to produce reactive oxygen species (ROS)-dependent thera- disruption of vascular function (8–10). Multicenter clinical trials peutic effects (1). Vascular-targeted PDT is a PDT regimen that have confirmed the clinical efficacy of vascular-targeted PDT and preferentially targets abnormal blood vessels through targeted its excellent safety profile (11). A recent phase III randomized delivery of photosensitizer molecules to the vasculature (2). As a clinical trial showed that, compared with active surveillance (the modality for disrupting existing blood vessels, vascular-targeted standard treatment for low-risk localized prostate cancers), vas- PDT has been clinically used for the treatment of diseases with cular-targeted PDT with photosensitizer padeliporfin (Tookad- abnormally activated vascularization including age-related mac- soluble, WST11) significantly prevented cancer progression and ular degeneration (AMD) and port-wine stain birthmarks (3, 4). It increased the number of cancer-free patients at 24 months after is also being actively explored for the treatment of various PDT (12). However, despite such promising outcomes, tumor types of cancers. recurrence was detected after vascular-targeted PDT with verte- Vascular-targeted PDT has been particularly investigated for porfin or Tookad in animal tumor models (10, 13). Close to 40% treating localized prostate cancer, a disease that is in a confined prostate cancer patients treated with padeliporfin-PDT had resid- ual tumors in the treated lobes (14), and about 30% patients still showed disease progression after PDT (12). Clearly, a therapeutic 1Department of Pharmaceutical Sciences, Philadelphia College of Pharmacy, enhancement strategy is needed for vascular-targeted PDT. University of the Sciences, Philadelphia, Pennsylvania. 2Department of Radiation We have shown that PDT response can be significantly Oncology, Perelman School of Medicine, University of Pennsylvania, Philadel- enhanced by PI3K pathway inhibitors (15, 16). As the most phia, Pennsylvania. frequently altered cell signaling pathway in human cancers, the Note: Supplementary data for this article are available at Molecular Cancer PI3K signaling pathway plays an important role in promoting Therapeutics Online (http://mct.aacrjournals.org/). tumor cell survival and progression (17). PI3K enzymes convert Corresponding Author: Bin Chen, University of the Sciences, 600 South 43rd phosphatidylinositol-4,5-biphosphate (PIP2) in the cell mem- Street, Philadelphia, PA 19104. Phone: 215-596-7481; Fax: 215-895-1161; E-mail: brane to phosphatidylinositol-3,4,5-triphosphate (PIP3), which [email protected] acts as a second message to activate downstream cell signaling doi: 10.1158/1535-7163.MCT-17-0326 molecules such as Akt (protein kinase B) and mTOR by inducing a 2017 American Association for Cancer Research. cascade of protein phosphorylation. Because abnormal activation

www.aacrjournals.org OF1

Kraus et al.

of the PI3K pathway is often involved in tumorigenesis and DMSO) was added into cell culture media at 1 hour before PDT. progression, this pathway has become a well-explored cancer The final concentration of DMSO was less than 0.1%. therapeutic target (17). A variety of selective and potent inhibitors targeting 4 different isoforms of class I PI3Ks (p110a, b, g, d) and Cell viability assay downstream signaling components have been developed and are Cell viability at different time points after treatment was showing promising results in clinical trials. examined by Alamar blue (resazurin from Sigma) assay where The interaction between PDT and these newly developed PI3K resazurin dye is metabolically converted to fluorescent resorufin pathway inhibitors is not well studied. Although beneficial effects by live cells. SVEC cells were implanted in multi-well cell culture were reported in previous studies combining PDT with early PI3K plates, allowed to attach for overnight, and treated with PI3K inhibitors such as wortmannin and LY294002 (18–20), these inhibitors alone, PDT alone, and combination treatments. At first-generation PI3K inhibitors are now considered of little clin- various time points after treatments, cells were incubated with ical relevance due to the lack of selectivity for PI3K enzymes (21). Alamar blue (180 mg/mL) for 4 hours and fluorescence intensity With the availability of clinically relevant inhibitors targeting was detected by a BioTek Synergy microplate reader with excita- different signaling molecules in the PI3K pathway, it is important tion at 540 Æ 20 nm and emission at 620 Æ 20 nm. Cell viability to evaluate their interactions with PDT in order to identify optimal was calculated by normalizing the fluorescence intensity of treated combination treatments. cells to that of control cells. Each experiment was repeated at least In this study, we assessed combination treatments of vertepor- three times. fin-PDT and three types of PI3K pathway inhibitors. These inhibitors, which have all progressed to clinical trials, include p110a Verteporfin uptake isoform-selective inhibitor BYL719 (22), pan-PI3K inhibitor Effects of PI3K inhibitors on the intracellular uptake of verte- BKM120 that inhibits all four isoforms of class I PI3Ks (23), and porfin were determined by flow cytometry. SVEC cells were dual PI3K/mTOR inhibitor BEZ235 that inhibits all four isoforms incubated with 400 ng/mL verteporfin for 15 minutes. Cells were of class I PI3Ks and mTOR kinases (24). Although all three trypsinized, rinsed with PBS, and resuspended in PBS for the inhibitors were able to synergistically enhance PDT response, analysis of verteporfin fluorescence with a FACSCalibur flow PDT combined with BEZ235 showed the strongest synergism. cytometer (BD Biosciences) in the FL3 channel (488 nm excita- PDT combined with BEZ235 increased endothelial cell apoptosis tion, 650 nm long pass emission). The fluorescence intensity of and caused sustained inhibition of cell proliferation, leading to a 20,000 cells in each sample was recorded. To determine the effects greater and more long-lasting therapeutic response than each of PI3K pathway inhibitors on verteporfin uptake, inhibitors were individual treatment both in vitro and in vivo. Our results dem- added at 45 minutes prior to adding verteporfin and verteporfin onstrate the importance of using clinically relevant PI3K pathway fluorescence was measured 15 minutes later. Experiments were therapeutics, particularly the mTOR inhibitors, for improving repeated three times. vascular-targeted PDT. Annexin V staining for apoptosis Materials and Methods Apoptotic cells were stained by Alexa Fluor 488-labeled fi fl Reagents Annexin V (Life Technologies) and quanti ed with ow cytome- Verteporfin (benzoporphyrin derivation in a lipid formula- try. SVEC cells were seeded in 60-mm cell culture dishes and fl tion) was kindly provided by QLT Inc. A stock PBS solution of allowed to grow to reach about 70% con uency. Cells were verteporfin was reconstituted according to the manufacturer's treated with PI3K inhibitors alone, PDT alone, and combination instruction and stored at 4C in the dark until use. BEZ235 was treatments. At various times after treatment, treated and untreated purchased from LC Laboratories, and BKM120 and BYL719 were control cells were trypsinized and resuspended in Annexin V kindly provided by Novartis. BEZ235, BKM120 and BYL719 were binding buffer (10 mmol/L HEPES, 140 mmol/L NaCl, Â 6 dissolved in DMSO, sterilized through filtration using 0.22-mm 2.5 mmol/L CaCl2, pH 7.4) at a concentration of 1 10 cells pore size filters, and stored in a À80C freezer. per mL. Floating cells collected from cell culture media were also included for the Annexin V staining. According to manufacturer's Cell culture instruction, 100-mL cell suspension was incubated with 3 mLof SV40 immortalized mouse endothelial cells (SVEC) and PC-3 Alexa Fluor 488-labeled Annexin V for 15 minutes at room human prostate tumor cells were obtained from ATCC in October temperature with constant rotation for apoptotic cell staining. 2010, expanded and frozen in the liquid nitrogen. Cells were not Cell fluorescence was measured with a FACSCalibur flow tested for mycoplasma. Cell culture was maintained in the cytometer (BD Biosciences) in the FL1 channel (488 nm excita- RPMI1640 (with glutamine) medium supplemented with 9% tion, 530 Æ 15 nm emission). For each sample, 20,000 cells FBS (Hyclone) and 100 U/mL penicillin/streptomycin (Media- were measured and the percentage of apoptotic cells (Annexin fi tech) at 37 C in a 5% CO2 incubator. Cells less than 20 passages V-positive) was quanti ed. Experiments were repeated three were used for experiments. times.

PDT treatment Western blot analysis Cells were incubated with verteporfin for 15 minutes and then Cells at 70%–80% confluence were treated and lysed on ice exposed to 5 mW/cm2 irradiance of 690-nm light for 100 seconds with the NP40 lysis buffer supplemented with protease and with verteporfin in cell culture media. Light intensity was mea- phosphatase inhibitors at various time points after treatment, sured by an optical power meter (Thorlabs Inc.). Light treatment as described previously (15). Cell lysates were separated by was performed using a diode laser system (High Power Devices SDS-PAGE and electrophoretically transferred to PVDF mem- Inc.). For combination treatments, PI3K inhibitors (dissolved in branes (Millipore). Blots were incubated with a primary

OF2 Mol Cancer Ther; 2017 Molecular Cancer Therapeutics

Targeting PI3K Pathway for Enhancing Vascular-Targeted PDT

antibody followed by incubation with horseradish peroxidase- Results conjugated secondary antibody (Cell Signaling Technology). Effects of PI3K inhibitors on endothelial cell viability and PI3K Primary antibodies include phospho-Akt (p-Akt), Akt, Ser473 pathway signaling phospho-ribosome protein S6 (p-S6), S6, PARP, Ser235/236 Effects of BYL719 (a p110a isoform–selective inhibitor), caspase-3 (casp-3), caspase-9 (casp-9), B-cell lymphoma 2 (Bcl-2), BKM120 (a pan-PI3K inhibitor), and BEZ235 (a dual pan- B-cell lymphoma-extra large (Bcl-X ), myeloid cell leukemia-1 L PI3K/mTOR inhibitor) on cell viability and PI3K pathway sig- (Mcl-1), and b-actin antibodies (from Cell Signaling Technology). naling were evaluated in SVEC endothelial cells. Figure 1 shows Blots were treated with Super Signal West Dura extended duration that different types of PI3K inhibitors had significantly different substrate (Thermo Scientific) and immunoreactive bands were effects on the cell viability (Fig. 1A) and PI3K signaling (Fig. 1B). captured with a GE Amersham Imager 600 (GE Healthcare Bio- BEZ235 potently reduced SVEC cell viability at all concentrations Sciences). The density of immunoreactive bands was quantified examined (P < 0.001). Its inhibitory effect on cell viability was with the NIH ImageJ software. much stronger than BKM120, which was slightly effective only at Tumor model and treatments higher concentrations (250 and 500 nmol/L). BYL719 showed no fi Subcutaneous PC-3 human prostate tumors in male athymic signi cant inhibition on cell viability at all doses examined. The nude mice (NCR, nu/nu, 6–8 weeks old) from Charles River reduction in cell viability induced by different types of PI3K Laboratories were used. Tumor implantation and PDT treatment inhibitors appeared to correlate with the inhibition on PI3K were described previously (25). Tumors at a size of about 5 mm in pathway activity shown by Western blot analysis. Treatment of diameter were used for experiments and randomly distributed SVEC cells with BEZ235 (500 nmol/L) led to almost complete to different groups. BEZ235 was orally administered at a dose of dephosphorylation of two PI3K downstream signaling molecules 40 mg/kg body weight once a day for 24 days. For vascular- Akt and S6 for up to 24 hours, indicating a potent and sustained targeted PDT, PC-3 tumors were externally illuminated with an inhibition of PI3K pathway signaling. Compared with BEZ235, irradiance of 50 mW/cm2 light at 690 nm for 600 seconds, less inhibition of PI3K signaling was induced by BKM120 and resulting in a total light dose of 30 J/cm2, at 15 minutes after pathway activity started to recover at 24 hours after treatment. intravenous injection of 0.5 mg/kg dose of verteporfin. Animals BYL719 induced the least inhibitory effect on PI3K pathway were anesthetized by intraperitoneal injection of a mixture of signaling, as substantial p-Akt and p-S6 signal remained detect- ketamine (120 mg/kg) and xylazine (12 mg/kg) during PDT able after treatment. treatment. BEZ235 was given at 1 hour before PDT for the fi combination therapy. Tumor size was measured every 4 days after Effects of PI3K pathway inhibitors on vertepor n-PDT and fi treatment and tumor volume was calculated using the formula vertepor n uptake of 0.52 Â length Â width Â height. Animal body weight was also Effects of three types of PI3K inhibitors on cell viability reduc- fi monitored to indicate treatment-associated toxicity. All animal tion induced by vertepor n PDT were examined in SVEC procedures were performed according to a protocol approved by cells. Figure 2A and B shows the cell viability after PDT (200 or fi the Institutional Animal Care and Use Committee (IACUC). 400 ng/mL vertepor n) alone and PDT in combination with PI3K inhibitors. BEZ235 combined with either 200 or 400 ng/mL Statistical and combination index analysis verteporfin PDT caused significantly more reduction in cell via- Two-way ANOVA test followed by multiple comparisons was bility than PDT alone, indicating therapeutic enhancement. used to determine statistical difference. Statistical significance was BKM120 was also able to significantly increase the effectiveness accepted at P < 0.05. The mode of interaction (antagonistic of 400 ng/mL verteporfin PDT, and 200 ng/mL verteporfin PDT at additive, synergistic) between PDT and PI3K inhibitors was deter- 250 and 500 nmol/L concentrations. BYL719 only increased the mined using CompuSyn software based on the Chou–Talalay response of PDT with 400 ng/mL verteporfin, but not PDT with method for drug combination (26). 200 ng/mL verteporfin.

Figure 1. Effects of PI3K inhibitors on endothelial cell viability and PI3K pathway signaling. A, SVEC cells were treated with different PI3K inhibitors for 48 hours, and cell viability was determined by Alamar blue assay. Data are presented as mean Æ SD from at least three independent experiments. ÃÃ, P < 0.01; ÃÃÃ, P < 0.001, compared with untreated control. ÃÃÃ shown on the right of dose–response curve indicates P < 0.001 at all concentrations examined. B, SVEC cell lysates were collected at various time points after treatments with PI3K inhibitors and probed for PI3K pathway signaling molecules by Western blot analysis.

www.aacrjournals.org Mol Cancer Ther; 2017 OF3

Kraus et al.

Figure 2. Verteporfin-PDT in combination with PI3K inhibitors synergistically reduced endothelial cell viability. Effects of PDT with 200 (A) or 400 (B) ng/mL verteporfin alone or PDT in combination with PI3K inhibitors on SVEC cell viability determined by Alamar blue assay at 48 hours after treatment. Data are presented as mean Æ SD from at least three independent experiments. ÃÃ, P < 0.01; ÃÃÃ, P < 0.001, compared with PDT alone, which is the data point at 0 concentration. ÃÃÃ shown on the right of dose–response curve indicates P < 0.001 at all concentration examined. Combination index analysis showing the mode of interaction between each PI3K inhibitor and PDT with 200 (C) or 400 (D) ng/mL verteporfin. Combination index of 1 indicates additive interaction and combination indexes of less than 1 and more than 1 are synergistic and antagonistic, respectively. ÃÃ, P < 0.01; ÃÃÃ, P < 0.001, compared with the index of 1.

To determine the mode of interaction between PDT and three BEZ235 combined with verteporfin-PDT enhanced types of PI3K inhibitors, combination index analysis was per- mitochondria-mediated apoptosis formed using ComboSyn software based on the Chou–Talalay Because BEZ235 combined with verteporfin-PDT exhibited the method for drug interaction assessment. According to this meth- strongest synergism, this combination was studied further to od, combination index of 1 indicates additive interaction, and determine therapeutic enhancement mechanisms. Annexin V indexes of less than 1 and more than 1 are synergistic and staining was performed to assess whether combination treatments antagonistic, respectively (26). Combination index analysis increased endothelial cell apoptosis. Figure 4A shows flow cyt- demonstrates a strong synergistic interaction between BEZ235 ometer histograms of Annexin V staining at 1, 4, and 24 hours and PDT with 200 ng/mL verteporfin (Fig. 2C). However, 200 after BEZ235 (12.5 nmol/L) alone, 400 ng/mL verteporfin PDT ng/mL verteporfin PDT combined with either BKM120 or BYL719 alone, and combination treatments. Quantification of apoptotic only showed additive effects. Synergism was detected when cells based on positive Annexin V staining is shown in Fig. 4B. 400 ng/mL verteporfin PDT was combined with any one of three BEZ235 alone did not induce apoptosis in SVEC cells, whereas PI3K inhibitors, except with BYL719 at 500 nmol/L concentration PDT alone caused a robust endothelial cell apoptosis. The com- (Fig. 2D). Notably, PDT combined with BEZ235 exhibited the bination of BEZ235 and PDT induced significantly more cell strongest synergism, followed in order by combinations with apoptosis than PDT alone at 4 and 24 hours after treatment. BKM120 and BYL719. To examine effects of treatments on PI3K signaling and apo- Effects of PI3K inhibitors on verteporfin intracellular uptake ptotic pathways, SVEC cells were lysed at 1–24 hours after treat- were determined by flow cytometry. Figure 3 shows that BEZ235 ment and probed for p-S6, caspase-9 (marker of intrinsic mito- caused a slight, but statistically significant, increase in the uptake chondrial apoptotic pathway), caspase-3 (a general executioner of verteporfin whereas BKM120 and BYL719 had no significant caspase), PARP (a substrate of activated executioner caspases), effect on verteporfin uptake. and Bcl-2 family proteins by Western blot analysis (Fig. 5A).

OF4 Mol Cancer Ther; 2017 Molecular Cancer Therapeutics

Targeting PI3K Pathway for Enhancing Vascular-Targeted PDT

proliferation versus p-S6 signaling showed that p-S6 signaling activation occurred prior to cell proliferation increase, suggesting the involvement of PI3K signaling activation in cell regrowth after PDT. PDT in combination with BEZ235 resulted in a complete inhibition of PDT-induced p-S6 elevation and continuous inhibition of SVEC cell proliferation.

BEZ235 combined with vascular-targeted PDT resulted in a more durable treatment response in prostate tumors Effects of vascular-targeted PDT alone, BEZ235 alone and combination treatments on tumor growth were examined in the PC-3 prostate tumor model (Fig. 6D). BEZ235 (40 mg/kg, daily, orally) alone significantly inhibited PC-3 tumor growth. Although PDT alone (30 J/cm2 light at 15 minutes after intravenous injection of 0.5 mg/kg verteporfin) was effective in inhibiting tumor growth, tumor regrowth occurred after an initial decrease in tumor volume. The combination of BEZ235 and PDT caused a significantly greater reduction in tumor volume and more sustained inhibition of tumor growth than each individual Figure 3. treatment. There was no significant difference in the body weight Effects of PI3K inhibitors on verteporfin intracellular uptake in SVEC cells. of animals in different treatment groups (Fig. 6E). Cells were incubated with 400 ng/mL verteporfin for 15 minutes, trypsinized and resuspended in PBS for the flow cytometry analysis of verteporfin fluorescence. PI3K inhibitors were added at 45 minutes before adding verteporfin. Data Discussion ÃÃÃ are presented as mean Æ SD from three independent experiments. , P < 0.001, Vascular-targeted PDT with photosensitizer verteporfin has compared with cells without PI3K inhibitor treatment, which was been approved for AMD and is being intensively investigated as normalized to 100%. a cancer therapy (27). We previously reported the intravascular localization of verteporfin and PDT-induced vascular effects Western blot band intensity was quantified and shown in Fig. 5B– including vascular permeability increase, thrombus formation, H. BEZ235 alone induced a potent and continuous inhibition of and vascular dysfunction, which lead to tumor destruction p-S6, whereas PDT alone caused a transient upregulation of p-S6 (8, 28). However, as we and others have shown, incomplete followed by a downregulation at 24 hours after treatment (Fig. occlusion of blood vessels after PDT as well as subsequent 5B). Combination treatments with BEZ235 completely inhibited angiogenesis-mediated neovascularization often result in vascular PDT-induced p-S6 upregulation. PDT alone activated the cleavage function resumption and disease recurrence (13, 29, 30). Because of caspase-9 (Fig. 5C), caspase-3 (Fig. 5D), and PARP (Fig. 5E), endothelial cell survival and proliferation are primarily involved indicating mitochondria-mediated apoptosis, whereas BEZ235 in these post-PDT events that lead to tumor recurrence, we attempt alone had no effect on the activation of apoptotic pathway. PDT in to target prosurvival signaling pathways in endothelial cells for combination with BEZ235 resulted in significantly more mito- therapeutic enhancement of vascular-targeted PDT. We have chondria-mediated apoptosis than PDT alone, as indicated by shown that verteporfin-PDT in combination with PI3K pathway greater caspase-9, caspase-3, and PARP cleavage after the combi- inhibitors enhanced endothelial cell death and increased the nation therapy. PDT alone increased the level of antiapoptotic inhibition of endothelial cell proliferation (15, 16). As more Bcl-2 family protein Mcl-1 (Fig. 5F), which was statistically PI3K pathway inhibitors targeting different signaling mole- significant by 4 hours after treatment. PDT-induced Mcl-1 eleva- cules/nodes are developed for clinical applications, we performed tion was completely suppressed by BEZ-235. Bcl-2 level did not this study to evaluate the effects of three types of PI3K pathway show significant change after either individual or combination inhibitors on verteporfin-PDT to identify the optimal combina- treatments (Fig. 5G). PDT alone induced Bcl-XL level reduction, tion treatments. although this was not statistically significant (Fig. 5H). There We found that inhibitors targeting different PI3K signaling was no significant difference in the Bcl-XL level between PDT alone molecules exhibited distinct effects on endothelial cell viability and combination treatments. and PDT response. Although p110a is considered the most important PI3K isoform in endothelial cells (31), p110a BEZ235 combined with verteporfin-PDT resulted in a sustained isoform-selective inhibitor BYL719 at concentrations used in this inhibition of PI3K pathway signaling and cell proliferation study was unable to inhibit SVEC cell survival and only partially Effects of PDT alone, BEZ235 alone and combination treat- inhibited PI3K signaling, suggesting that endothelial cells resort to ments on cell proliferation and PI3K signaling pathway marker p- other isoforms for downstream signaling when p110a is blocked. S6 were monitored daily for up to 5 days after treatment. PDT with We have shown that SVEC cells also express p110b isoform, albeit either 200 or 400 ng/mL verteporfin significantly inhibited endo- at a lower level than p110a (15). Inhibiting all isoforms of class I thelial cell proliferation (Fig. 6A and B). However, SVEC cells PI3Ks by a pan-inhibitor BKM120 did lead to more effective resumed proliferation shortly after PDT, particularly after the inhibition of SVEC cell viability and stronger inhibition of PI3K lower dose PDT. The level of p-S6 was increased after PDT with signaling. However, it is the dual PI3K/mTOR inhibitor BEZ235 either 200 or 400 ng/mL verteporfin, which occurred sooner after that caused the most effective inhibition of cell viability and the the lower dose PDT (Fig. 6C). Comparing the time course of cell strongest PI3K signaling suppression. The same trend was also

www.aacrjournals.org Mol Cancer Ther; 2017 OF5

Kraus et al.

Figure 4. Combination treatments of BEZ235 (12.5 nmol/L) and PDT (400 ng/mL verteporﬁn) enhanced endothelial cell apoptosis. SVEC cells were treated as indicated and stained with Alexa Fluor 488-labeled Annexin V at 1, 4, and 24 hours after treatment. A, Annexin V staining histograms after different treatments. B, The percentage of apoptotic cells (Annexin V-positive) was quantiﬁed and shown. Data shown are mean Æ SD from three independent experiments. ^^, P < 0.01; ^^^, P < 0.001, compared with controls at corresponding time points. Ã, P < 0.05; ÃÃÃ, P < 0.001, compared with PDT alone.

found in the enhancement of PDT response by PI3K inhibitors. apoptosis, inhibition of PDT-induced Mcl-1 upregulation by BEZ235 showed the highest efficacy for enhancing PDT response BEZ235 likely led to increased mitochondrial apoptosis after the and BYL719 had the lowest effectiveness. These results indicate combination therapy. This proposed mechanism, yet to be cor- the redundancy of different PI3K isoforms in endothelial cell roborated, is supported by some published results. For instance, signaling and the necessity of inhibiting all isoforms for better cell inhibition of Mcl-1 by BEZ235 has been shown to sensitize tumor growth inhibition and PDT enhancement. More importantly, our cells to undergo apoptosis induced by chemotherapy (32). data suggest the importance of mTOR signaling in determining Increase of Mcl-1 level was also reported after PDT with photofrin endothelial cell survival and PDT response. We are currently in tumor cells, and inhibition of Mcl-1 elevation by celecoxib was determining how selective inhibition of mTOR only affects the shown to enhance apoptosis (35). Antiapoptotic protein Bcl-XL PDT response. was found reduced after verteporfin-PDT. Bcl-XL is known as a Strong synergism between BEZ235 and verteporfin-PDT in cell target of PDT with phthalocyanine Pc 4 for the initiation of viability reduction prompted us to determine whether combina- apoptosis (36). Bcl-XL level reduction after verteporfin-PDT sug- tion treatments enhanced cell death. BEZ235 alone is not effective gests its involvement in verteporfin-PDT-mediated apoptosis as for inducing apoptosis in tumor cells (32). As shown in this study, well. However, Bcl-XL is unlikely to play an important role in the it did not induce apoptosis in endothelial cells either. In fact, increased apoptosis after combination treatments because its level inability to induce cell death is a major problem limiting the was not modulated by BEZ235. clinical application of PI3K inhibitors as cancer monotherapy In addition to promoting cell death, our results demonstrate because tumor cells are able to survive the cytostatic effect and that BEZ235 combined with verteporfin-PDT resulted in more develop drug resistance eventually (33). In contrast, verteporfin- sustained inhibition of endothelial cell proliferation and durable PDT alone effectively induced endothelial cell apoptosis through treatment response, which provides another advantage to this mitochondria-mediated pathway, as shown in the current and combinatorial approach. Although verteporfin-PDT alone effec- previous studies (25, 34). Importantly, our data demonstrate that tively inhibited endothelial cell viability and tumor growth, its combining cytostatic BEZ235 with cytotoxic PDT significantly therapeutic effects were nevertheless transient. Thus, increasing enhanced therapeutic lethality by enhancing mitochondrial apo- treatment response durability is an important issue that needs ptosis pathway, which provides a strong rationale for the com- to be addressed for vascular-targeted PDT. Fine adjustments of bination therapy. PDT dosimetry parameters such as light and photosensitizer Probing for antiapoptotic Bcl-2 family proteins revealed an doses are known to increase PDT response and treatment dura- interesting mechanistic cooperation between PDT and BEZ235 in bility by optimizing photochemical reactions in the tumor micro- enhancing endothelial cell apoptosis. Mcl-1 level was significantly environment (37–39). Rational combinations of PDT and other upregulated after PDT, a possible consequence of increased pro- treatment modalities have been shown to enhance treatment tein translation due to PDT-induced p-S6 activation. By strongly outcomes (40). Our results here demonstrate that the activation suppressing p-S6 and consequent Mcl-1 protein translation, of PI3K pathway signaling after PDT represents a therapeutic BEZ235 effectively inhibited PDT-induced Mcl-1 upregulation. target for the enhancement of vascular-targeted PDT. We found Because increased Mcl-1 may protect cells from mitochondrial that the PI3K pathway signaling was activated after sublethal and

OF6 Mol Cancer Ther; 2017 Molecular Cancer Therapeutics

Targeting PI3K Pathway for Enhancing Vascular-Targeted PDT

Figure 5. Combination treatments of BEZ235 and verteporfin-PDT enhanced mitochondrial apoptosis in endothelial cells. A. Effects of BEZ235, PDT, and combination treatments on PI3K signaling and apoptotic pathway markers. SVEC cells were treated with BEZ235 (12.5 nmol/L) alone, PDT (400 ng/mL verteporfin) alone, and combination treatments. Cells were lysed at 1 to 24 hours after treatments and probed for various markers by Western blot analysis. Control cells received no treatment. The quantification of Western blot band intensity showing changes of p-S6/S6 (B), caspase-9 cleavage (C), caspase-3 cleavage

(D), PARP cleavage (E), Mcl-1/actin (F), Bcl-2/actin (G), and Bcl-XL/actin (H) after treatments. Band intensity ratios of p-S6/S6, cleaved/total (uncleaved plus cleaved), and each Bcl-2 family protein/actin were normalized to untreated control and shown. Data represent mean Æ SE from at least three independent experiments. ^, P < 0.05; ^^, P < 0.01; ^^^, P < 0.001, compared with untreated control, which was normalized to 100%. Ã, P < 0.05; ÃÃÃ, P < 0.001, compared with PDT alone. therapeutic doses of PDT with 200 and 400 ng/mL verteporfin, in the PI3K pathway including the loss of PTEN tumor suppressor respectively. The activation of PI3K signaling led to subsequent and activating mutations in PI3Ks often occur in human cancers endothelial cell proliferation, which occurred sooner after the particularly prostate and breast cancers (33), combining vascular- sublethal dose of PDT. Blocking the PI3K signaling activation with targeted PDT with a PI3K inhibitor ensure the maximum eradi- BEZ235 transformed a transient growth inhibition induced by cation of tumor cells by targeting both tumor vasculature and PDT alone to a sustained suppression of endothelial cell prolif- tumor cells. eration. The durable therapeutic response as a result of cooper- It is interesting to note that BEZ235 increased verteporfin ative inhibition of PI3K signaling provides another rationale for uptake in SVEC cells, which provides additional benefit for the the combination of PDT and PI3K pathway inhibitors. combination therapy. Although the exact mechanism remains to A pilot study in the PC-3 prostate tumor model confirmed that be determined, increased verteporfin uptake induced by BEZ235 PDT combined with BEZ235 led to a more durable tumor is likely due to the interaction with ATP-binding cassette subfam- response than individual treatment, demonstrating the effective- ily G2 (ABCG2) transporters. As a type of efflux pump involved in ness of this therapeutic enhancement approach in vivo. We pre- transporting a variety of exogenous substances and endogenous viously showed that endothelial cells were more sensitive to PDT metabolites out of cells, the ABCG2 transporter is known for in combination with BEZ235 than PC-3 prostate tumor cells pumping out verteporfin (41). PDT with verteporfin has been (15, 16). Thus, enhanced tumor response after the combination shown to cause ABCG2 transporter downregulation (42), further therapy is presumably attributed to the enhanced vascular suggesting the association between ABCG2 transporters and ver- response. In addition to targeting tumor vasculature, PDT com- teporfin. BEZ235 was recently identified as an ABCG2 substrate as bined with BEZ235 also enables a direct inhibition of PC-3 tumor well (43). Thus, competing for the same efflux transporter pos- cells. The PC-3 cells do not express PTEN, a negative regulator of sibly led to increased verteporfin uptake when it was combined the PI3K pathway that terminates the pathway signaling by with BEZ235. Furthermore, some small molecule kinase inhibi- converting PIP3 to PIP2 (15). Because of the loss of PTEN function, tors have been shown to inhibit ABCG2 activity by directly PC-3 cells exhibit activated PI3K signaling and are responsive to inhibiting transporter ATP-binding sites, thereby reducing the PI3K pathway inhibitors such as BEZ235 (16). Because mutations efflux of ABCG2 substrates (44). A kinase inhibitor erlotinib was

www.aacrjournals.org Mol Cancer Ther; 2017 OF7

Kraus et al.

Figure 6. Combination treatments of BEZ235 and verteporfin-PDT resulted in sustained inhibition of endothelial cell proliferation and tumor growth. A–C, Effects of verteporfin-PDT alone, BEZ235 alone, and combination treatments on endothelial cell proliferation and the PI3K signaling. SVEC cells were treated with 200- (A) or 400- (B) ng/mL verteporfin PDT alone, BEZ235 (12.5 nmol/L) alone, and combination treatments. Cell proliferation was determined by Alamar blue assay every day for 5 days after treatment. Data were normalized to untreated control on Day 1. Data are presented as mean Æ SD from at least three independent experiments. Cells lysates were prepared at corresponding time points after each individual and combination treatments and probed for p-S6/S6 by Western blot analysis (C). D and E, Effects of verteporfin-PDT alone, BEZ235 alone, and combination treatments on PC-3 tumor growth (D)and animal body weight (E). Mice bearing subcutaneous PC-3 tumors were treated as indicated and tumor volume was monitored every 4 days. Each group included six to eight animals.

recently shown to increase verteporfin intracellular uptake (45). It Disclosure of Potential Conflicts of Interest is not yet known whether BEZ235 directly inhibits ABCG2 activ- No potential conflicts of interest were disclosed. ity. Interestingly, BYL719 and BKM120 had no effect on the uptake of verteporfin. Because PDT in combination with BYL719 Authors' Contributions or BKM120 also showed synergistic effects, it suggests that Conception and design: D. Kraus, B. Chen increased verteporfin uptake is just an additional benefit, but not Development of methodology: D. Kraus, B. Chen indispensable, for the combination of verteporfin-PDT and PI3K Acquisition of data (provided animals, acquired and managed patients, pathway inhibitors. provided facilities, etc.): D. Kraus, P. Palasuberniam, B. Chen In summary, we have shown in this study synergistic Analysis and interpretation of data (e.g., statistical analysis, biostatistics, fi computational analysis): D. Kraus, B. Chen interactions between vertepor n-PDT and three newly devel- Writing, review, and/or revision of the manuscript: D. Kraus, P. Palasuberniam, oped PI3K therapeutics that are in clinical trials. Particularly, B. Chen PDT combined with a dual PI3K/mTOR inhibitor BEZ235 Study supervision: B. Chen exhibited the strongest synergism in reducing endothelial cell viability. The combination therapy overcomes the lim- Acknowledgments itation of being cytostatic for BEZ235 alone and having We would like to thank Novartis for providing PI3K pathway inhibitors, QLT short-lived effectiveness for PDT alone through a cooperative Inc. for providing verteporfin, and Dr. Babasola Fateye, Dr. Xue Yang, and inhibition of PI3K signaling and apoptotic pathways. As a Matthew Mansi for assistance and helpful discussions. result, PDT in combination with BEZ235 led to more lethal The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked and long-lasting therapeutic effects. These results demon- advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate strate the importance of PI3K signaling pathway in deter- this fact. mining PDT treatment outcomes, and support the use of clinically relevant PI3K pathway inhibitors for enhancing Received April 12, 2017; revised July 10, 2017; accepted August 7, 2017; vascular-targeted PDT. published OnlineFirst August 23, 2017.

OF8 Mol Cancer Ther; 2017 Molecular Cancer Therapeutics

Targeting PI3K Pathway for Enhancing Vascular-Targeted PDT

References 1. Agostinis P, Berg K, Cengel KA, Foster TH, Girotti AW, Gollnick SO, et al. 21. Rodon J, Dienstmann R, Serra V, Tabernero J. Development of PI3K Photodynamic therapy of cancer: an update. CA Cancer J Clin 2011;61: inhibitors: lessons learned from early clinical trials. Nat Rev Clin Oncol 250–81. 2013;10:143–53. 2. Chen B, Pogue BW, Hoopes PJ, Hasan T. Vascular and cellular targeting for 22. Furet P, Guagnano V, Fairhurst RA, Imbach-Weese P, Bruce I, Knapp M, photodynamic therapy. Crit Rev Eukaryot Gene Expr 2006;16:279–305. et al. Discovery of NVP-BYL719 a potent and selective phosphatidylino- 3. Brown SB, Mellish KJ. Verteporfin: a milestone in opthalmology and sitol-3 kinase alpha inhibitor selected for clinical evaluation. Bioorg Med photodynamic therapy. Expert Opin Pharmacother 2001;2:351–61. Chem Lett 2013;23:3741–8. 4. Qiu H, Zhou Y, Gu Y, Ang Q, Zhao S, Wang Y, et al. Monitoring micro- 23. Maira SM, Pecchi S, Huang A, Burger M, Knapp M, Sterker D, et al. circulation changes in port wine stains during vascular targeted photody- Identification and characterization of NVP-BKM120, an orally available namic therapy by laser speckle imaging. Photochem Photobiol 2012;88: pan-class I PI3-kinase inhibitor. Mol Cancer Ther 2012;11:317–28. 978–84. 24. Maira SM, Stauffer F, Brueggen J, Furet P, Schnell C, Fritsch C, et al. 5. Khurana M, Moriyama EH, Mariampillai A, Wilson BC. Intravital high- Identification and characterization of NVP-BEZ235, a new orally available resolution optical imaging of individual vessel response to photodynamic dual phosphatidylinositol 3-kinase/mammalian target of rapamycin treatment. J Biomed Opt 2008;13:040502. inhibitor with potent in vivo antitumor activity. Mol Cancer Ther 2008;7: 6. Madar-Balakirski N, Tempel-Brami C, Kalchenko V, Brenner O, Varon D, 1851–63. Scherz A, et al. Permanent occlusion of feeding arteries and draining veins 25. Li Z, Agharkar P, Chen B. Therapeutic enhancement of vascular-targeted in solid mouse tumors by vascular targeted photodynamic therapy (VTP) photodynamic therapy by inhibiting proteasomal function. Cancer Lett with Tookad. PLoS One 2010;5:e10282. 2013;339:128–34. 7. Gross S, Gilead A, Scherz A, Neeman M, Salomon Y. Monitoring photo- 26. Chou TC. Drug combination studies and their synergy quantification dynamic therapy of solid tumors online by BOLD-contrast MRI. Nat Med using the Chou-Talalay method. Cancer Res 2010;70:440–6. 2003;9:1327–31. 27. Huggett MT, Jermyn M, Gillams A, Illing R, Mosse S, Novelli M, et al. Phase 8. He C, Agharkar P, Chen B. Intravital microscopic analysis of vascular I/II study of verteporfin photodynamic therapy in locally advanced pan- perfusion and macromolecule extravasation after photodynamic vascular creatic cancer. Br J Cancer 2014;110:1698–704. targeting therapy. Pharm Res 2008;25:1873–80. 28. Chen B, Pogue BW, Goodwin IA, O'Hara JA, Wilmot CM, Hutchins JE, et al. 9. Huang Z, Haider MA, Kraft S, Chen Q, Blanc D, Wilson BC, et al. Magnetic Blood flow dynamics after photodynamic therapy with verteporfininthe resonance imaging correlated with the histopathological effect of Pd- RIF-1 tumor. Radiat Res 2003;160:452–9. bacteriopheophorbide (Tookad) photodynamic therapy on the normal 29. Moshfeghi DM, Kaiser PK, Grossniklaus HE, Sternberg P Jr, Sears JE, canine prostate gland. Lasers Surg Med 2006;38:672–81. Johnson MW, et al. Clinicopathologic study after submacular removal of 10. Koudinova NV, Pinthus JH, Brandis A, Brenner O, Bendel P, Ramon J, et al. choroidal neovascular membranes treated with verteporfin ocular photo- Photodynamic therapy with Pd-Bacteriopheophorbide (TOOKAD): suc- dynamic therapy. Am J Ophthalmol 2003;135:343–50. cessful in vivo treatment of human prostatic small cell carcinoma xeno- 30. Nowak-Sliwinska P, van Beijnum JR, van Berkel M, van den Bergh H, grafts. Int J Cancer 2003;104:782–9. Griffioen AW. Vascular regrowth following photodynamic therapy in the 11. Kawczyk-Krupka A, Wawrzyniec K, Musiol SK, Potempa M, Bugaj AM, chicken embryo chorioallantoic membrane. Angiogenesis 2010;13: Sieron A. Treatment of localized prostate cancer using WST-09 and WST-11 281–92. mediated vascular targeted photodynamic therapy-A review. Photodiag- 31. Graupera M, Guillermet-Guibert J, Foukas LC, Phng LK, Cain RJ, Salpekar nosis Photodyn Ther 2015;12:567–74. A, et al. Angiogenesis selectively requires the p110alpha isoform of PI3K to 12. Azzouzi AR, Vincendeau S, Barret E, Cicco A, Kleinclauss F, van der Poel control endothelial cell migration. Nature 2008;453:662–6. HG, et al. Padeliporfin vascular-targeted photodynamic therapy versus 32. Jebahi A, Villedieu M, Petigny-Lechartier C, Brotin E, Louis MH, Abeilard E, active surveillance in men with low-risk prostate cancer (CLIN1001 et al. PI3K/mTOR dual inhibitor NVP-BEZ235 decreases Mcl-1 expression PCM301): an open-label, phase 3, randomised controlled trial. Lancet and sensitizes ovarian carcinoma cells to Bcl-xL-targeting strategies, Oncol 2017;18:181–91. provided that Bim expression is induced. Cancer Lett 2014;348: 13. Chen B, Crane C, He C, Gondek D, Agharkar P, Savellano MD, et al. 38–49. Disparity between prostate tumor interior versus peripheral vasculature in 33. Fruman DA, Rommel C. PI3K and cancer: lessons, challenges and oppor- response to verteporfin-mediated vascular-targeting therapy. Int J Cancer tunities. Nat Rev Drug Discov 2014;13:140–56. 2008;123:695–701. 34. Granville DJ, Shaw JR, Leong S, Carthy CM, Margaron P, Hunt DW, et al. 14. Taneja SS, Bennett J, Coleman J, Grubb R, Andriole G, Reiter RE, et al. Final Release of cytochrome c, Bax migration, Bid cleavage, and activation of results of a phase I/II multicenter trial of WST11 vascular targeted photo- caspases 2, 3, 6, 7, 8, and 9 during endothelial cell apoptosis. Am J Pathol dynamic therapy for hemi-ablation of the prostate in men with unilateral 1999;155:1021–5. low risk prostate cancer performed in the United States. J Urol 2016;196: 35. Song J, Chen Q, Xing D. Enhanced apoptotic effects by downregulating 1096–104. Mcl-1: evidence for the improvement of photodynamic therapy with 15. Fateye B, Wan A, Yang X, Myers K, Chen B. Comparison between endo- Celecoxib. Exp Cell Res 2013;319:1491–504. thelial and tumor cells in the response to verteporfin-photodynamic 36. Xue LY, Chiu SM, Fiebig A, Andrews DW, Oleinick NL. Photodamage to therapy and a PI3K pathway inhibitor. Photodiagnosis Photodyn Ther multiple Bcl-xL isoforms by photodynamic therapy with the phthalocya- 2015;12:19–26. nine photosensitizer Pc 4. Oncogene 2003;22:9197–204. 16. Fateye B, Li W, Wang C, Chen B. Combination of phosphatidylinositol 3- 37. Rizvi I, Anbil S, Alagic N, Celli J, Zheng LZ, Palanisami A, et al. PDT dose kinases pathway inhibitor and photodynamic therapy in endothelial and parameters impact tumoricidal durability and cell death pathways in a 3D tumor cells. Photochem Photobiol 2012;88:1265–72. ovarian cancer model. Photochem Photobiol 2013;89:942–52. 17. Thorpe LM, Yuzugullu H, Zhao JJ. PI3K in cancer: divergent roles of 38. Zhou X, Pogue BW, Chen B, Demidenko E, Joshi R, Hoopes J, et al. isoforms, modes of activation and therapeutic targeting. Nat Rev Cancer Pretreatment photosensitizer dosimetry reduces variation in tumor 2015;15:7–24. response. Int J Radiat Oncol Biol Phys 2006;64:1211–20. 18. Bozkulak O, Wong S, Luna M, Ferrario A, Rucker N, Gulsoy M, et al. 39. Pogue BW, Elliott JT, Kanick SC, Davis SC, Samkoe KS, Maytin EV, et al. Multiple components of photodynamic therapy can phosphorylate akt. Revisiting photodynamic therapy dosimetry: reductionist & surrogate Photochem Photobiol 2007;83:1029–33. approaches to facilitate clinical success. Phys Med Biol 2016;61: 19. Volanti C, Hendrickx N, Van Lint J, Matroule JY, Agostinis P, Piette J. R57–89. Distinct transduction mechanisms of cyclooxygenase 2 gene activation in 40. Spring BQ, Rizvi I, Xu N, Hasan T. The role of photodynamic therapy in tumour cells after photodynamic therapy. Oncogene 2005;24:2981–91. overcoming cancer drug resistance. Photochem Photobiol Sci 20. Koon HK, Chan PS, Wong RN, Wu ZG, Lung ML, Chang CK, et al. Targeted 2015;14:1476–91. inhibition of the EGFR pathways enhances Zn-BC-AM PDT-induced apo- 41. Robey RW, Steadman K, Polgar O, Bates SE. ABCG2-mediated transport of ptosis in well-differentiated nasopharyngeal carcinoma cells. J Cell Bio- photosensitizers: potential impact on photodynamic therapy. Cancer Biol chem 2009;108:1356–63. Ther 2005;4:187–94.

www.aacrjournals.org Mol Cancer Ther; 2017 OF9

Kraus et al.

42. Huang HC, Mallidi S, Liu J, Chiang CT, Mai Z, Goldschmidt R, et al. 44. Anreddy N, Gupta P, Kathawala RJ, Patel A, Wurpel JN, Chen ZS. Tyrosine Photodynamic therapy synergizes with irinotecan to overcome compen- kinase inhibitors as reversal agents for ABC transporter mediated drug satory mechanisms and improve treatment outcomes in pancreatic cancer. resistance. Molecules 2014;19:13848–77. Cancer Res 2016;76:1066–77. 45. Gallagher-Colombo SM, Miller J, Cengel KA, Putt ME, Vinogradov SA, 43. Lin F, de Gooijer MC, Hanekamp D, Chandrasekaran G, Buil LC, Thota N, et al. Busch TM. Erlotinib pretreatment improves photodynamic therapy of non- PI3K-mTOR pathway inhibition exhibits efﬁcacy against high-grade glioma small cell lung carcinoma xenografts via multiple mechanisms. Cancer Res in clinically relevant mouse models. Clin Cancer Res 2017;23:1286–98. 2015;75:3118–26.

OF10 Mol Cancer Ther; 2017 Molecular Cancer Therapeutics

Targeting Phosphatidylinositol 3-Kinase Signaling Pathway for Therapeutic Enhancement of Vascular-Targeted Photodynamic Therapy

Daniel Kraus, Pratheeba Palasuberniam and Bin Chen

Mol Cancer Ther Published OnlineFirst August 23, 2017.

Updated version Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-17-0326

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://mct.aacrjournals.org/content/early/2017/10/11/1535-7163.MCT-17-0326. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.