The Prostate 75:242^254 (2015)

Synergistic Co-Targeting of Prostate-Specif|c Membrane Antigen and Androgen Receptor in Prostate Cancer Jose D. Murga, Sameer M. Moorji, Amy Q. Han, Wells W. Magargal, Vincent A. DiPippo,* and William C. Olson Progenics Pharmaceuticals, Inc.,Tarrytown, NewYork

BACKGROUND. Antibody–drug conjugates (ADCs) are an emerging class of cancer therapies that have demonstrated favorable activity both as single agents and as components of combination regimens. Phase 2 testing of an ADC targeting prostate-specific membrane antigen (PSMA) in advanced prostate cancer has shown antitumor activity. The present study examined PSMA ADC used in combination with potent antiandrogens (enzalutamide and abiraterone) and other compounds. METHODS. Antiproliferative activity and expression of PSMA, prostate-specific antigen and androgen receptor were evaluated in the prostate cancer cell lines LNCaP and C4-2. Cells were tested for susceptibility to antiandrogens or other inhibitors, used alone and in combination with PSMA ADC. Potential drug synergy or antagonism was evaluated using the Bliss independence method. RESULTS. Enzalutamide and abiraterone demonstrated robust, statistically significant synergy when combined with PSMA ADC. Largely additive activity was observed between the antiandrogens and the individual components of the ADC (free drug and unmodified antibody). Rapamycin also synergized with PSMA ADC in certain settings. Synergy was linked in part to upregulation of PSMA expression. In androgen-dependent LNCaP cells, enzalutamide and abiraterone each inhibited proliferation, upregulated PSMA expression, and synergized with PSMA ADC. In androgen-independent C4-2 cells, enzalutamide and abiraterone showed no measurable antiproliferative activity on their own but increased PSMA expression and synergized with PSMA ADC nonetheless. PSMA expression increased progressively over 3 weeks with enzalutamide and returned to baseline levels 1 week after enzalutamide removal. CONCLUSIONS. The findings support exploration of clinical treatment regimens that combine potent antiandrogens and PSMA-targeted therapies for prostate cancer. Prostate 75: 242–254, 2015. # 2014 Wiley Periodicals, Inc. KEY WORDS:

PSMA ADC; enzalutamide; androgen receptor; rapamycin; prostate

INTRODUCTION Metastatic castration-resistant prostate cancer (CRPC) remains a deadly and incurable disease despite a growing number of treatment options. Life-extending therapies for metastatic CRPC currently include potent antiandrogens, immunotherapy, taxanebased chemotherapy, and a bone-seeking a-emitting radionuclide [1]. The approved products plus promising investigational agents have created opportunities for designing rational drug combinations as potential means to further improve treatment outcomes. ß 2014 Wiley Periodicals, Inc.

Conflicts of interest: All authors are current or former employees of Progenics Pharmaceuticals, Inc. and own or have owned stock in the company. The present address of Sameer M. Moorji is Promega Corporation, USA The present address of Amy Q. Han and William C. Olson are Regeneron Pharmaceuticals, Inc, Tarrytown, NY, USA 

Correspondence to: Vincent A. DiPippo, Progenics Pharmaceuticals, Inc., 777 Old Saw Mill River Road, Tarrytown, NY 10591. E-mail: [email protected] Received 4 July 2014; Accepted 27 August 2014 DOI 10.1002/pros.22910 Published online 18 October 2014 in Wiley Online Library (wileyonlinelibrary.com).

SynergyTargets: PSMA and Androgen Receptor CRPC often remains dependent on androgen receptor (AR) signaling despite castrate serum levels of testosterone. This dependence renders CRPC vulnerable to highly potent means of androgen suppression [2,3]. Enzalutamide competitively blocks binding of androgen to AR and inhibits AR’s translocation to the nucleus and subsequent interactions with DNA. Abiraterone is an inhibitor of cytochrome P450 17A1, which is critical for the synthesis of androgen in the testes, adrenals, and prostate tumors. In vitro studies have reported that abiraterone also antagonizes AR as a secondary mechanism of action [3–5]. In addition, deletions of phosphatase and tensin homolog (PTEN) and other mutations in the phosphoinositide 3-kinase (PI3K) pathway are common in CRPC. Laboratory studies have identified compensatory regulation of the AR and PI3K pathways, whereby inhibition of one pathway leads to cross-activation of the other [6,7]. AR signaling exerts pleiotropic effects on gene expression in prostate cancer. Androgen has opposing effects on the expression of prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA). PSA and PSMA are amongst the best-characterized secreted and cell-surface markers of prostate cancer, respectively. Androgen suppression decreases PSA and increases PSMA expression in vitro and in vivo [8–11]. PSMA is a type 2 integral membrane glycoprotein with carboxypeptidase activity and nearly universal expression in primary and metastatic prostate cancer [12,13]. Expression is highest in aggressive forms of disease and is prognostic for progression [14,15]. Interest in targeting PSMA is heightened by its abundant luminal expression on the neovasculature of several nonprostatic carcinomas but not on normal blood vessels [16,17]. Antibody–drug conjugates (ADCs) utilize the targeting power of monoclonal antibodies (mAbs) to selectively deliver chemotherapeutic drugs to cells that bear the cognate antigen. The approach is designed to maximize drug exposure at the site of disease while limiting systemic toxicity. Clinical application of this approach has accelerated in recent years, propelled by important technological advances and important clinical successes in both solid and liquid tumors [18,19]. Because of their targeted nature, ADCs can exhibit tolerability profiles that facilitate testing in combination with other therapies. PSMA ADC consists of a fully human IgG1 antiPSMA mAb that is conjugated to monomethylauristatin E (MMAE) through a valine-citrulline (vc) linker [20,21]. The dipeptide linker is designed to be stable in blood but efficiently cleaved within the lysosomes of tumor cells following ADC internalization. Cleavage of the traceless linker releases MMAE, which potently disrupts microtubule function. PSMA ADC contains on average four molecules of MMAE per mAb. The same drug–linker

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and drug-to-antibody ratio are used in the CD30-directed ADC brentuximab vedotin (ADCETRIS1, Seattle Genetics) [18]. PSMA ADC has completed accrual to a Phase 2 trial (clinicaltrials.gov identifier NCT01695044). The present study examined combinations of PSMA ADC with antiandrogens and other agents. Treatment of prostate cancer cells with enzalutamide or abiraterone significantly increased PSMA expression. The antiandrogens demonstrated striking synergy with PSMA ADC and largely additive activity with either of its components (i.e., unmodified PSMA mAb or free MMAE). In androgen-dependent cells, antiproliferative effects, upregulation of PSMA and synergy were observed at similar, clinically relevant concentrations of antiandrogens. In androgen-independent cells, the antiandrogens exhibited minimal antiproliferative activity but upregulated PSMA and potently synergized with PSMA ADC nonetheless. Amongst the PI3K pathway inhibitors, PSMA ADC showed the highest synergy with the mTOR inhibitor rapamycin. These studies provide a biological basis for potentially incorporating PSMA-targeted therapies into novel multidrug treatment regimens.

MATERIALS AND METHODS Materials LNCaP cells were obtained from American Type Culture Collection (ATCC). C4-2 is an androgenindependent subclone of LNCaP [22]. LNCaP and C4-2 are characterized by a T877A mutation in AR and loss of PTEN expression [23,24]. The cell lines allow investigations of androgen dependence in an isogenic background. Both cell lines were passaged in RPMI1640 (Mediatech, Inc.) supplemented with 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 100 mM nonessential amino acids, 1% penicillin/streptomycin and 10% fetal bovine serum (FBS, Life Technologies). Cells were drawn from a common bank and then used within 10 passages. PC-3 and 22Rv1 cells were obtained from ATCC and passaged as recommended by ATCC prior to use in limited testing. PSMA ADC was prepared as described [20]; enzalutamide and abiraterone (parent drug) were obtained from MedKoo Biosciences. Prednisone and prednisolone were purchased from Sigma; rapamycin was from EMD Millipore. GDC0941, MK-2206, and SB743921 were from Selleck Chemicals. Anti-AR (sc7305, 441) and anti-PSA (sc-7638, C-19) antibodies were obtained from Santa Cruz Biotechnology. Two murine anti-PSMA mAbs were used: MAB544 (Maine Biotechnology) for Westerns and 3.9 (Progenics) for flow cytometry. Anti-actin antibody (MAB1501) was from Millipore. Isotype-specific secondary IRDye antibodies were from LI-COR Biosciences. The Prostate

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Murga et al. Cell Viability Assay

Cells were plated at a density of 1  103 cells in 25 ml in 384-well white-colored microplates (Perkin Elmer) and cultured overnight. The next day, cells were treated with one or two drugs in a total volume of 50 ml. Cultures were incubated at 37°C for 7 days. Viability was assessed on days 0 and 7 using CellTiter-Glo (Promega) and an Analyst GT luminescence reader at 560 nm emission (Molecular Devices). The percent inhibition of proliferation was calculated as (Vu  Vt)/ (Vu  V0)  100, where Vu, Vt and V0 represent the viability values for untreated cells on day 7, treated cells on day 7, and cells prior to treatment on day 0, respectively. A value of 100% reflects complete inhibition of proliferation (Vt ¼ V0). Values of >100% reflect cytotoxic compounds or combinations, where Vt < V0. Flow Cytometry For short-term treatments (7 days), cells were plated on 24-well plates (BD Falcon) overnight, at a density of 200,000 cells per 500 ml. The next day, wells were exchanged with 500 ml of fresh media that included inhibitor, and incubated at 37°C until the indicated time point. For the long-term treatments (>7 days), cells were plated on T-25 flasks (BD Falcon) overnight, at a density of 500,000 cells per flask in 5 ml of media. Five milliliters of fresh media with or without treatment was exchanged the next day. On weekly splits thereafter, cells were detached with cell dissociation solution (Sigma), washed with fresh media, counted using Vi-CELL XR (Beckman Coulter), and split into two T-25 flasks for continued culture with or without inhibitor. Prior to analysis by flow cytometry, cells were detached, counted and suspended in PBS (PBS(), without Ca/Mg, Life Technologies) containing 0.3% bovine serum albumin (BSA, Sigma) and 0.1% sodium azide (VWR). Cells (100,000 in 100 ml) were placed in round-bottom 96-well plates (BD Falcon) and incubated for 30 min at room temperature with 1 mg/ml of anti-PSMA mAb 3.9 conjugated to phycoerythrin (PE). Cells then were washed twice with 200 ml PBS/BSA/azide buffer and read using a FACSCalibur instrument (BD Biosciences). A mouse IgG2b-PE conjugate of irrelevant specificity (Abcam) was included as an isotype control.

nology) and then centrifuged at 14,000g for 10 min at 4°C. The supernatant was quantitated for protein concentration using a BCA kit (Pierce/Thermo), resolved under reducing conditions using NuPAGE Novex 4–12% Bis-Tris gels (Life Technologies), and transferred to nitrocellulose using the iBlot 7-Minute Blotting System (Life Technologies). Membranes were blocked using Odyssey Blocking Reagent (LI-COR Biosciences), incubated with primary and secondary antibodies, and visualized using an Odyssey infrared imager (LI-COR Biosciences). Synergy Calculations and Statistical Methods Drug combinations were tested in 3–7 independent repeat cell viability assays unless otherwise indicated. Inhibition data were fit to a four-parameter logistic equation using GraphPad Prism. Potential nonadditive effects were evaluated by the Bliss independence method [25–27]. Briefly, the predicted Bliss value for the combination (Fc) was calculated as Fc ¼ Fa þ Fb  (Fa  Fb), where Fa and Fb represent the observed fractional growth inhibitions caused by compounds A and B used alone. Fc represents the Bliss value that would be expected if the compounds exhibited additive activity. Bliss differences were calculated by subtracting the predicted value (Fc) from the experimentally observed inhibition for each point in a matrix of drug–drug combinations. Synergy, additivity, and antagonism are associated with Bliss differences that are greater than zero, indistinguishable from zero, or less than zero, respectively. A matrix of Bliss differences was calculated in this way individually for each assay replicate. Mean Bliss differences and associated standard deviations were calculated from the replicate values and assessed for statistical significance from the null value of zero using two-sided t-tests with a significance level of 0.05. Statistical evaluations were not performed in cases where the fractional growth inhibition exceeded unity, since such values fall outside the Bliss framework. Bliss differences were reported as percentages, and values of less than 5% were considered to be of limited biological relevance and were excluded from synergy considerations. The pattern or clustering of positive Bliss differences within the matrix (e.g., Fig. 1) provides a secondary, qualitative measure of the scope and robustness of synergistic findings.

Western Blotting Cells (300,000) were incubated overnight in 6-well plates (BD Falcon) in 4 ml of 10% FBS RPMI1640 media prior to addition of inhibitors. Plates were incubated at 37°C for an additional 7 days, washed once with 5 ml of cold PBS() and placed on ice. Cells were lysed with 130 ml of RIPA Lysis Buffer (Santa Cruz BiotechThe Prostate

RESULTS PSMA ADC Synergized With Antiandrogens and Rapamycin Inhibitors were tested for antiproliferative activity individually and in combination across a range of

SynergyTargets: PSMA and Androgen Receptor

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Fig. 1. Combinations of PSMA ADCwith enzalutamide, abiraterone, or rapamycin.Percentinhibitionvalues and Bliss differences are shown for LNCaP cells (A) and C4-2 cells (B).Each data pointrepresents themean of three to seven independent assays.Bliss differences are depicted via heat maps of values that are positive (green), negative (red), or near zero (yellow). Bliss parameters that are significantly different from zero (P < 0.05) are highlighted via bold text and red borders, and the corresponding percent inhibition values are highlighted with shading and red text.NA ¼ not applicable. The Prostate

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concentrations in a matrix fashion. Inhibition data were compared with Bliss predictions, and the differences between the experimental and predicted results were calculated for each point in the matrix. Positive differences indicate supra-additive or synergistic effects. Negative differences indicate antagonism. Results were assessed for statistical significance as described above. Mean inhibition data and Bliss differences are shown in Figure 1 for PSMA ADC combined with enzalutamide, abiraterone, or rapamycin. Heat maps illustrate Bliss differences that are positive (green), negative (red), or near zero (yellow). Boxes are used to indicate statistically significant Bliss differences and the corresponding percent inhibitions. PSMA ADC exhibited similar single-agent activity against LNCaP and C4-2 cells, with IC50 values of approximately 200 pM in each case. These results are in line with prior reports [20,21]. Enzalutamide, abiraterone, and rapamycin each inhibited proliferation of LNCaP cells but had minimal antiproliferative effects on C4-2 cells when used as single agents (Fig. 1). Despite a lack of direct antiproliferative activity in C4-2 cells, enzalutamide, abiraterone, and rapamycin all significantly enhanced the activity of PSMA ADC. Bliss differences ranged to nearly 40% and were statistically significant across a range of inhibitor concentrations and levels of inhibition (Fig. 1B). No significant antagonism was observed for these combinations under any condition. Statistically significant synergy also was observed for ADC/enzalutamide and ADC/abiraterone combinations in LNCaP cells (Fig. 1A); however, the breadth and magnitude were more limited as compared to C4-2 cells. Similarly, Bliss differences were generally positive across the matrix of concentrations for the ADC/rapamycin combination in LNCaP cells; however, no value reached statistical significance. As an initial step towards dissecting mechanisms of synergy, the antiandrogens, and rapamycin were combined with free MMAE and with unmodified PSMA mAb. Free MMAE showed subnanomolar potency (IC50  0.5 nM) against both LNCaP and C4-2 cells, as expected [21]. When paired with enzalutamide or abiraterone, free MMAE exhibited additive activity in LNCaP cells and additive to weakly synergistic activity on C4-2 (Supplementary Fig. S1). Similar results were obtained when docetaxel, another microtubule inhibitor, was combined with enzalutamide. There were only sporadic instances of statistically significant synergy for the docetaxel/enzalutamide combination (Supplementary Fig. S1). The rapamycin/MMAE combination exhibited additive effects in LNCaP and moderate synergy in C4-2 cells (Supplementary Fig. S2). Thus, rapamycin synergized less potently with MMAE as compared The Prostate

with PSMA ADC on both cell lines. However, the rapamycin/MMAE combination appeared to be as or more potently synergistic than the enzalutamide/ MMAE or abiraterone/MMAE combination in C4-2 cells. Weak to moderate synergy was observed between rapamycin and enzalutamide in both cell lines. In C4-2 cells, noninhibitory concentrations of enzalutamide enhanced the weak antiproliferative activity of rapamycin (Supplementary Fig. S2). Unmodified PSMA mAb was inactive both alone and in combination with enzalutamide, abiraterone or rapamycin (Supplementary Fig. S3). Due to the very limited activity of the individual agents or combinations, assays were performed only twice in some cases. Thus, the robust synergies observed upon combining PSMA ADC with antiandrogens are specific to the conjugate and are not reproduced with either of its individual components. Combinations of PSMA ADC and antiandrogens were also tested on 22Rv1 cells. Compared to C4-2 and LNCaP cells, 22Rv1 have lower levels of PSMA expression and reduced susceptibility to PSMA ADC [21]. In these cells, the synergy results were mixed, with PSMA ADC exhibiting synergy when combined with abiraterone but not with enzalutamide (Supplementary Fig. S4). PSMA-negative cells were not included in the present combinational study. However, PSMA ADC is taken up nonspecifically by PSMA-negative cells via pinocytosis or other processes and then releases free MMAE, resulting in cell-cycle arrest and cell death [20,21]. The process is inefficient in PSMA-negative cells (typically requiring 1,000-fold higher concentrations of ADC); however, the mechanism of cell killing is indistinguishable between PSMA-positive and -negative cells. For this reason, the studies with free MMAE in LNCaP and C4-2 (Supplementary Fig. S1) provide models for the effects of PSMA ADC on PSMA-negative cells, without the complication of varying the cellular background. Collectively, the studies with free MMAE and with 22Rv1 cells strengthen the view that the strong synergies between PSMA ADC and antiandrogens are linked to PSMA expression. As a control, a mock combination was prepared and assessed for synergy by adding PSMA ADC to the assay plate via two separate additions. As expected, the mock combination did not show statistically significant synergy or antagonism in either cell line (data not shown). Antiandrogens Reversibly Increased PSMA Expression in aTime- and Dose-Dependent Manner Additional studies examined treatment-induced changes in PSMA expression as a potential correlate of

SynergyTargets: PSMA and Androgen Receptor synergy. Flow cytometry and Western blotting were used to assess cell-surface and total PSMA, respectively. Enzalutamide and abiraterone increased cell-surface levels of PSMA in a dose-dependent manner in both cell lines (Fig. 2A–D). PSMA expression was approximately doubled at antiandrogens concentrations above 1 mM at 7 days post-treatment. In contrast, rapamycin increased PSMA expression in C4-2 cells only (Fig. 2E and F). Low nanomolar concentrations of rapamycin were sufficient to induce a greater than twofold increase in PSMA expression in C4-2 cells. To probe the dynamics of expression, C4-2 cells were cultured in enzalutamide (1 mM) for 3 weeks prior to continued culture in the absence of drug for an additional 8 days. Cell-surface PSMA was measured by flow cytometry. PSMA expression increased steadily over time in the presence of enzalutamide. After 21 days, PSMA expression on treated cells was nearly fourfold greater than expression on untreated cells passaged in parallel. PSMA expression returned to baseline levels within 7 days of culture in the absence of enzalutamide (Fig. 2G). Separate treatments of enzalutamide on PSMA-negative (PC-3) or low expressing PSMA (22Rv1) cells showed no increase or minimal increases of PSMA, respectively (data not shown). Western blotting was used as an independent test of PSMA expression. Doublet PSMA bands were observed in both LNCaP and C4-2 cells. The two bands may represent known splice variants or different glycoforms of PSMA (reviewed in [12,13]). Treatmentinduced increases in PSMA were also apparent by Western blotting (Fig. 3). The magnitude of increase was approximately fivefold for enzalutamide-treated LNCaP cells as determined by serial dilution of lysates and semi-quantitative analysis of the Western blots (data not shown). Enzalutamide (10 mM), abiraterone (10 mM) and rapamycin (100 nM) upregulated PSMA to similar extents in C4-2 cells. The magnitude of PSMA expression in the presence of the antiandrogens approached but seldom exceeded the expression seen in cells cultured in charcoal-stripped serum (data not shown). The flow cytometry and Western blot analyses were each repeated in two independent assays that yielded similar results. Expression analyses conducted following treatment with PSMA ADC were uninformative, perhaps due to the potent cytotoxicity and steep dose–response curve of the compound (data not shown). Treatment-Induced Changes in AR and PSA Levels of AR and PSA were also evaluated pre- and post-treatment by Western blotting. Actin was probed as a measure of total cell loading. As expected, antiandrogen-induced upregulation of PSMA was accompa-

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nied by downregulation of PSA (Fig. 3). In contrast, rapamycin increased PSA expression. This result is consistent with prior observations for rapamycin or its analogs [28,29]. The co-upregulation of PSMA and PSA by rapamycin contrasts with the opposing effects on PSMA and PSA exerted by antiandrogens. AR protein levels were affected modestly by treatment, consistent with prior findings [30,31] (Fig. 3). Enzalutamide and other AR antagonists have been shown to impede translocation of AR to the nucleus, an essential step in its activation of gene transcription [30,31]. Additive to Weakly Synergistic Effects for Other Drug Combinations In exploratory studies, an additional group of compounds was screened for activity alone and in combination with PSMA ADC. These agents include the Akt inhibitor MK-2206 [32]; the PI3K inhibitor GDC-0941 [33]; prednisone; and SB-743921 [34], an inhibitor of the kinesin spindle protein (ksp) Eg5. MK2206 and GDC-0941 are selective inhibitors of upstream components of the PI3K/mTOR pathway. Interest in combining these agents with PSMA ADC is heightened by the report of additive or better activity between a PI3K inhibitor and a PSMA-targeted immunotoxin that acts via inhibiting protein synthesis [35]. Prednisone is a component of abiraterone- and docetaxel-based treatment regimens in prostate cancer. SB743931 was included in our study based on gene expression profiling within the cBio cancer genomics database [36], which revealed coordinated expression (P ¼ 0.000004) of the genes for PSMA and Eg5 within primary and metastatic prostate cancer tumors. Each of these compounds showed additive to weakly synergistic activity when combined with PSMA ADC (Fig. 4). That is, Bliss differences trended towards synergy for most conditions and reached statistical significance in a few isolated cases. No significant antagonism was observed for any of these combinations. Similar results were obtained when prednisone was replaced with its active metabolite, prednisolone (data not shown). Table I provides an overview of the synergies observed in this study. For each drug:drug combination, the breadth of synergy is represented as the percentage of evaluable conditions that resulted in statistically significant synergy across the matrix of drug concentrations. Combinations are sorted, highest to lowest, according to the breadth of synergy observed in LNCaP cells. The ADC/enzalutamide and ADC/abiraterone combinations showed the broadest synergy in LNCaP cells. Next were PSMA ADC combinations with prednisone or SB-743921. For the ADC/SB-743921 combination, the data should be The Prostate

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Fig. 2. Flow cytometry analysis of PSMA expression. PSMA expression was measured by flow cytometry on cells treated for 7 days with varying concentrations of enzalutamide, abiraterone, or rapamycin (A^F ) or treated with1 mM enzalutamide for varying times (G). In A^D, anti-PSMA stainingis shown as a function of antiandrogens concentrations as follows: green (0.125 mM), pink (0.5 mM), cyan (1 mM) and orange (5 mM). In E and F, the green, pink and cyan histograms represent rapamycin concentrations of 1, 10, and 100 nM, respectively. Filled purple histograms denote untreated cells, and the blue line depicts background staining with isotype-control antibody. (G) Time-course of PSMA expression in enzalutamide-treated C4-2 cells.Vertical bars depict mean fluorescence intensity (MFI) values on the left axis, and the red line represents the fold increase in PSMA expression in treated cells relative to untreated cells on the right axis. (PR ¼ days post-enzalutamide removal.)

The Prostate

SynergyTargets: PSMA and Androgen Receptor

Fig. 3. Western blot analysis. LNCaP (A) or C4-2 (B) cells were left untreated (Unt) or treated for 7 days with10 mM enzalutamide (Enza),10 mM abiraterone (Abi), or100 nM rapamycin (Rapa) prior to Western blot analysis of protein expression in whole-cell extracts.

interpreted with caution, since several drug–drug combinations resulted in >100% inhibition and thus were not evaluable for Bliss differences. Further testing of this combination over a more focused range of concentrations is warranted. Most combinations did not show significant synergy in LNCaP cells. Synergy was more widespread in C4-2 cells. The ADC/enzalutamide and ADC/abiraterone combinations exhibited a high frequency of synergistic conditions in C4-2 cells, as did rapamycin in combination with PSMA ADC or MMAE. Other combinations (e.g., PSMA ADC in combination with MK-2206 or GDC0941) also exhibited significant synergy in C4-2 but not LNCaP cells (Table I). Combinations that did not show significant synergy in either LNCaP or C4-2 cells were those that involved PSMA mAb and the PSMA ADC mock combination. DISCUSSION The principal finding of the present study is the robust synergy observed between agents that target PSMA and AR. Synergistic inhibition of tumor cell growth was associated with an upregulation of PSMA by antiandrogens, even in cells that were refractory to treatment with antiandrogens alone. The findings support combining a PSMA-targeted agent with potent antiandrogens for prostate cancer therapy. Both enzalutamide and abiraterone synergized with PSMA ADC over a range of concentrations. Synergy was observed on cells that were both responsive and unresponsive to treatment with antiandrogens alone. In androgen-dependent LNCaP cells, the pharmacologic effects of antiandrogens (antiproliferative effects, effects on gene expression, and synergy with PSMA ADC) were observed over a similar range of clinically relevant concentrations. In androgen-independent

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C4-2 cells, effects on gene expression and synergy were uncoupled from any appreciable antiproliferative activity of either enzalutamide or abiraterone. Thus, these antiandrogens remain pharmacologically active in C4-2; however, the cells have adopted compensatory survival mechanisms that allow them to proliferate in the presence of ongoing AR blockade. Little to no synergy was observed between the antiandrogens and the components of the ADC (i.e., free MMAE and unmodified PSMA mAb). Free MMAE showed additive to weakly synergistic effects when combined with the antiandrogens. The weak synergy may reflect the impairment of AR nuclear localization and signaling by microtubule inhibitors [37,38]. Consistent with this view, there was modest synergy between the antiandrogens and docetaxel. The unmodified mAb showed no antiproliferative activity either alone or in combination with antiandrogens. Therefore, the strong synergy observed with PSMA ADC appeared specific to the conjugate and is neither a pass-through effect of its components nor generalizable to microtubule inhibitors as a class. In terms of maximizing drug–drug synergies, the findings suggest that PSMA ADC delivers greater effects as compared with docetaxel when paired with enzalutamide or abiraterone. PSMA expression was doubled after 7 days’ treatment with enzalutamide, and fourfold higher after 21 days. The magnitude of PSMA upregulation by enzalutamide or abiraterone approached that induced by charcoal-stripped serum, which is depleted of a range of hormones, cytokines, and growth factors [39]. The findings suggest near-maximal androgen suppression in our in vitro system. The time course of expression was monitored in cells treated with enzalutamide. PSMA expression increased with continued treatment over three weeks and then rapidly returned to baseline upon removal of enzalutamide. The findings have implications for combining and sequencing potent antiandrogens and PSMA-targeted therapies in the clinic. In particular, sequential treatment may miss the benefits attainable with combination therapy, unless the antiandrogens have a markedly more persistent effect on PSMA expression in vivo. There are a limited number of prostate cancer cell lines that remain differentiated and express key markers of disease. For example, PC-3 and DU 145 are two widely used lines that fail to express AR, PSA or PSMA at the level of either RNA or protein. LNCaP and C4-2 retain expression of these markers and provide a system for evaluating the effects of varying androgen dependence in a common cellular background. These features made these cells attractive choices for our studies. In addition, LNCaP or ARtransfected LNCaP lines are commonly used in studies The Prostate

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Fig. 4. Combinations of PSMA ADCwith prednisone or inhibitors of Akt,PI3Kor kinesin spindle protein.Percentinhibitionvalues and Bliss differences are shown for LNCaP cells (A) and C4-2 cells (B).Each data point represents the mean of four or five independent assays.Bliss differences are depicted via heat maps of values that are positive (green), negative (red), or near zero (yellow). Bliss parameters that are significantly different from zero (P < 0.05) are highlighted via bold text and red borders, and the corresponding percent inhibition values are highlightedwith shading andred text.NA ¼ not applicable. The Prostate

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TABLE I. Summary of Synergies Observed for Different Drug Combinations LNCaP

Combination PSMA ADC þ enzalutamide PSMA ADC þ abiraterone PSMA ADC þ prednisone PSMA ADC þ SB 743921 Rapamycin þ enzalutamide Docetaxel þ enzalutamide PSMA ADC þ rapamycin MMAE þ rapamycin MMAE þ enzalutamide PSMA ADC þ MK-2206 PSMA ADC þ GDC0941 MMAE þ abiraterone PSMA mAb þ abiraterone PSMA mAb þ enzalutamide PSMA mAb þ rapamycin PSMA ADC þ PSMA ADC

Number of synergistic conditions in drug:drug matrix

Number of evaluable conditions in drug:drug matrix

6 5 2 1 2 1 0 0 0 0 0 0 0 0 0 0

24 24 24 12 35 26 24 19 24 25 25 23 36 36 26 24

of potent antiandrogens [30,31]. More recently, an F876L point mutation was shown to impart resistance to enzalutamide in laboratory studies and was detected in approximately 10% of subjects treated with ARN-509, a potent AR antagonist [40]. Finally, several PSMA-expressing lines are available as well-characterized patient-derived xenografts [41,42]. Such models are being utilized in ongoing studies designed to examine the applicability of our findings to in vivo settings. As an AR antagonist, enzalutamide is well suited for mechanistic studies in vitro. Its mechanism of action includes preventing AR from translocating into the nucleus [30,31]. In vitro studies such as those described here may be less suitable for studying abiraterone’s primary mechanism of action as an inhibitor of CYP17A1, which is not expressed at appreciable levels in LNCaP cells [43]. However, abiraterone may also act through a secondary mechanism of action involving downregulation of AR expression [4,5]. Thus, although consistent findings were observed for enzalutamide and abiraterone in the present study, the abiraterone results should be interpreted with caution. The PI3K/mTOR pathway is a central survival pathway that mediates resistance to AR inhibitors in certain settings [6]. PI3K pathway inhibitors have established preclinical activity in prostate cancer, and several agents are undergoing clinical investiga-

C4-2

% Synergistic conditions

Number of synergistic conditions in drug:drug matrix

Number of evaluable conditions in drug:drug matrix

% Synergistic conditions

25.0 20.8 8.3 8.3 5.7 3.8 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

10 9 6 2 5 1 11 6 3 2 2 1 0 0 0 0

24 24 24 8 36 24 24 18 18 23 24 18 36 36 36 15

41.7 37.5 25.0 25.0 13.9 4.2 45.8 33.3 16.7 8.7 8.3 5.6 0.0 0.0 0.0 0.0

tion [44–46]. PTEN loss is a common mechanism for PI3K activation in prostate cancer. Both LNCaP and C4-2 are PTEN deficient; however, there were important differences in pathway regulation between the two cells. LNCaP cells showed high sensitivity to rapamycin, and there was little advantage to combining rapamycin and PSMA ADC. In C4-2 cells, rapamycin alone had minimal effects on proliferation but markedly increased the expression of PSMA and potentiated the activity of PSMA ADC. Rapamycin’s primary mechanism of action is to decrease the global rate of mTOR-mediated protein synthesis in mammalian cells. The increase in PSMA protein level thus contrasts with this overall repression of translation. Rapamycin also synergized with free MMAE in C4-2 cells, but to a lesser extent than with PSMA ADC. Synergy between rapamycin analogs and a CD30targeted ADC also have been reported [47]. In this case, similar levels of synergy were noted for the antiCD30 ADC and free MMAE, with no reported effects on modulation of antigen expression by the rapalogs. Synergy was attributed to a decrease in the translation of antiapoptic factors as a result of mTOR inhibition. Compared with rapamycin, GDC0941 and MK-2206 synergized less strongly with PSMA ADC, highlighting the value in this context of blocking the pathway at downstream nodes. Additional studies will be needed to determine the effects of PI3K and Akt The Prostate

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inhibitors on PSMA expression, and cross-activation of other signaling pathways in these cells. Cross-talk between the AR and PI3K pathways has been well documented in prostate cancer [6,7]. Cotreatment with antiandrogens and rapamycin resulted in largely additive antiproliferative effects. AR blockade activates the PI3K pathway by relieving feedback inhibition of Akt. Similarly, PI3K pathway inhibition enhances AR signaling. One possible explanation for the additive rather than synergistic effects seen in our study is the blunting of rapamycin’s effects via activation of a feedback loop mediated by insulin receptor substrate 1 [48–51]. Evaluation of antiandrogens with other PI3K pathway inhibitors is warranted but was outside the scope of the present study. In our study, LNCaP cells responded to antiandrogen treatment via activation of the PI3K pathway. Several methods are available for assessing potential nonadditive effects of multidrug combinations. The relative merits of the different approaches are discussed elsewhere [52]. Combination Index and Loewe additivity methods compare the concentrations of drugs that are needed to produce a given effect when used alone or in combination. These other methods are predicated on the use of drugs that are active as single agents. Loewe additivity also is based on the assumption that the two drugs act competitively, and is poorly suited for combinations of drugs with differing mechanisms. However, the Bliss model is intended for drugs with independent mechanisms of action, as was typically the case here. The Bliss model also is suitable for cases where one or more drugs exhibit minimal activity alone, as was often the case in our study. CONCLUSIONS Collectively, our findings provide incentive for cotargeting PSMA and AR for potential prostate cancer therapy. PSMA expression and the activity of a PSMAtargeted agent were significantly increased in the presence of enzalutamide or abiraterone, even in settings where the antiandrogens showed minimal antiproliferative activity as single agents. Combination therapy thus appears to provide a potential means to maximize the therapeutic utility of potent antiandrogens and extend their duration of clinical benefit. In addition, PSMA ADC synergized with a PI3K/mTOR pathway inhibitor via a multimodal mechanism involving increased PSMA expression and disruption of microtubule function. Results presented here involving these combinations suggest the exploration of novel treatment strategies for advanced prostate cancer through further studies.

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SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s website.

Synergistic co-targeting of prostate-specific membrane antigen and androgen receptor in prostate cancer.

Antibody-drug conjugates (ADCs) are an emerging class of cancer therapies that have demonstrated favorable activity both as single agents and as compo...
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