Tankyrase Inhibition Blocks Wnt/B-Catenin Pathway And

Published OnlineFirst July 29, 2015; DOI: 10.1158/1078-0432.CCR-14-3081

Cancer Therapy: Preclinical Clinical Cancer Research Tankyrase Inhibition Blocks Wnt/b-Catenin Pathway and Reverts Resistance to PI3K and AKT Inhibitors in the Treatment of Colorectal Cancer Oriol Arques 1, Irene Chicote1, Isabel Puig1, Stephan P. Tenbaum1, Guillem Argiles 2,3, Rodrigo Dienstmann2,3,4, Natalia Fernandez 2,3, Ginevra Caratu5, Judit Matito5, Daniel Silberschmidt5, Jordi Rodon2,6, Stefania Landolﬁ7, Aleix Prat8, Eloy Espín9, Ramon Charco10, Paolo Nuciforo11, Ana Vivancos5, Wenlin Shao12, Josep Tabernero2,3, and Hector G. Palmer1

Abstract

Purpose: Oncogenic mutations in the KRAS/PI3K/AKT path- Results: Combination with NVP-TNKS656 promoted apopto- way are one of the most frequent alterations in cancer. Although sis in PI3K or AKT inhibitor-resistant cells with high nuclear PI3K or AKT inhibitors show promising results in clinical trials, b-catenin content. High FOXO3A activity conferred sensitivity to drug resistance frequently emerges. We previously revealed NVP-TNKS656 treatment. Thirteen of 40 patients presented high Wnt/b-catenin signaling hyperactivation as responsible for such nuclear b-catenin content and progressed earlier upon PI3K/AKT/ resistance in colorectal cancer. Here we investigate Wnt-mediated mTOR inhibition. Nuclear b-catenin levels predicted drug resistance in patients treated with PI3K or AKT inhibitors in response, whereas clinicopathologic traits, gene expression pro- clinical trials and evaluate the efﬁcacy of a new Wnt/tankyrase ﬁles, or frequent mutations (KRAS, TP53,orPIK3CA) did not. inhibitor, NVP-TNKS656, to overcome such resistance. Conclusions: High nuclear b-catenin content independently Experimental Design: Colorectal cancer patient-derived predicts resistance to PI3K and AKT inhibitors. Combined treat- sphere cultures and mouse tumor xenografts were treated with ment with a Wnt/tankyrase inhibitor reduces nuclear b-catenin, NVP-TNKS656, in combination with PI3K or AKT inhibitors.We reverts such resistance, and represses tumor growth. FOXO3A analyzed progression-free survival of patients treated with differ- content and activity predicts response to Wnt/b-catenin inhibi- ent PI3K/AKT/mTOR inhibitors in correlation with Wnt/b-cate- tion and together with b-catenin may be predictive biomarkers of nin pathway activation, oncogenic mutations, clinicopathologi- drug response providing a rationale to stratify colorectal cancer cal traits, and gene expression patterns in 40 colorectal cancer patients to be treated with PI3K/AKT/mTOR and Wnt/b-catenin baseline tumors. inhibitors. Clin Cancer Res; 22(3); 644–56. 2015 AACR.

Introduction 1Stem Cells and Cancer Laboratory, Vall d'Hebron Institute of Oncol- ogy, Barcelona, Spain. 2Medical Oncology Department, Vall d'Hebron Colorectal cancer is a leading cause of death worldwide (1), 3 University Hospital, Barcelona, Spain. Gastrointestinal and Endocrine mostly because conventional treatments or new target-directed Tumors Group, Vall d'Hebron Institute of Oncology, Barcelona, Spain. 4Sage Bionetworks, Fred Hutchinson Cancer Research Centre, Seattle, drugs are ineffective in patients presenting late-stage metastatic Washington. 5Cancer Genomics Group, Vall d'Hebron Institute of disease (2). It is therefore crucial to unmask the molecular 6 Oncology, Barcelona, Spain. Early Clinical Drug Development Group, mechanisms responsible for such resistance and to provide new Vall d'Hebron Institute of Oncology, Barcelona, Spain. 7Department of Pathology,Vall d'Hebron University Hospital, Universitat Autonoma de predictive biomarkers of drug response that could improve the Barcelona, Barcelona, Spain. 8Translational Genomics Group, Vall selection of patients sensitive to treatment. d'Hebron Institute of Oncology, Barcelona, Spain. 9General Surgery Activating mutations in genes encoding constituents of the Service,Vall d'Hebron University Hospital, Barcelona, Spain. 10Depart- ment of HBP Surgery and Transplantation, Vall d'Hebron University KRAS/PI3K/AKT signaling pathway can be considered one of the Hospital, Universitat Autonoma de Barcelona, Barcelona, Spain. most frequent cancer-causing genetic alterations in solid tumors, 11Molecular Oncology Group, Vall d'Hebron Institute of Oncology, including colorectal cancer (3). Thus, a new generation of drugs 12 Barcelona, Spain. Novartis Institutes for Biomedical Research, Inc., targeting PI3K or AKT activity is being tested in numerous clinical Cambridge, Massachusetts. trials with promising results in some tumor types. Unfortunately, Note: Supplementary data for this article are available at Clinical Cancer colorectal cancer patients show an enhanced resistance to these Research Online (http://clincancerres.aacrjournals.org/). drugs (4–8). Corresponding Author: Hector García Palmer, Vall d'Hebron Institute of Oncol- Active AKT phosphorylates FOXO proteins promoting their ogy, Passeig Vall d'Hebron 119-129, Barcelona 08035, Spain. Phone: 349-3489- sequestration in the cytoplasm and blocking their capacity to 4167; Fax: 349-3274-6708; E-mail: [email protected] induce the expression of target genes coding for proteins involved doi: 10.1158/1078-0432.CCR-14-3081 in cell-cycle arrest and apoptosis (9). Therefore, the efﬁcacy of 2015 American Association for Cancer Research. PI3K and AKT inhibitors can be mediated in part through nuclear

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also identified FOXO3A as a determinant of response to Translational Relevance Wnt/b-catenin inhibitors and FOXO3A/b-catenin target genes To date, PI3K and AKT inhibitors are showing limited as better pharmacodynamic markers than the canonical TCF/ clinical benefit mostly due to unknown resistance mechan- b-catenin targets. isms and the lack of predictive biomarkers of drug re- Our data indicate that nuclear FOXO3A and b-catenin content sponse. We demonstrate that Wnt inhibitors can overcome and activity could be valuable predictive biomarkers of drug b-catenin–induced resistance to PI3K and AKT inhibitors in response and we propose an experimental-based rationale to colorectal cancer tumors. We also provide the experimental better guide the molecular selection of colorectal cancer patients evidence for a rational stratification of patients to be treated entering new clinical trials with PI3K/AKT and/or Wnt/b-catenin with PI3K/AKT and/or Wnt/b-catenin pathway inhibitors pathway inhibitors. using b-catenin and FOXO3A as predictive biomarkers of drug response. Such refined molecular selection of patients Materials and Methods could represent a significant improvement in response to Patients in clinical trials treatment and an important step forward in advancing Patients were enrolled in clinical trials with PI3K/AKT/mTOR colorectal cancer therapy. inhibitors carried out in the Vall d'Hebron University Hospi- tal (Barcelona, Spain; Clinical trial identifiers: 14-MC-JWAA, NCT01115751; B2151001, NCT00940498; CBEZ235A2101, NCT00620594; BKM1202101, NCT01723800; CBYL7192101, NCT01219699; INK1117-001, NCT01449370; PAM4743g, relocalization of FOXO proteins and consequent induction of NCT01090960; XL765-00, NCT00485719). Tumor response was apoptosis. We previously described that Wnt/b-catenin oncogenic assessed according to RECIST 1.0 or 1.1 (22, 23). We analyzed signaling confers resistance to FOXO3A-dependent apoptosis formalin-fixed paraffin-embedded (FFPE) tumor samples from promoted by PI3K or AKT-inhibitory drugs (10). Such resistance colorectal cancer patients at baseline before entering clinical trials was driven by nuclear b-catenin that impaired the capacity of with PI3K/AKT/mTOR inhibitors. FOXO3A to execute its apoptotic program. Thus, we hypothesized that reducing nuclear b-catenin content by Wnt inhibitors would overcome the resistance to PI3K or AKT inhibitors and combined Patient-derived cells treatments could be beneficial for treating colorectal cancer Written informed consent was signed by all patients. The patients. project was approved by the Research Ethics Committee of the Abnormal activation of the Wnt/b-catenin pathway by muta- Vall d'Hebron University Hospital (Barcelona, Spain; Approval tions in APC, CTNNB1/b-catenin, or AXIN2 is responsible for the ID: PR(IR)79/2009). Patient-derived cells were obtained as pre- initiation and progression of almost all colorectal cancers (11). viously described (24). Cells were injected subcutaneously in These mutations reduce the capacity of the Wnt pathway destruc- NOD-SCID mice or were seeded as sphere cultures. tion complex, formed by APC, AXIN, and GSK3b, to commit b-catenin to degradation. As a result, b-catenin accumulates in the Animals, xenotransplantation, and treatments nucleus, binds the TCF/LEF transcription factors, and induces the Experiments were conducted following the European Union's expression of Wnt target genes that play key roles in tumor animal care directive (2010/63/EU) and were approved by the progression (12). We and others have previously shown that Ethical Committee of Animal Experimentation of Vall d'Hebron binding of b-catenin to different transcription factors enhances Institute of Research (ID: 40/08 CEEA and 47/08/10 CEEA). the expression of alternative sets of target genes (13). FOXO3A is Xenografts were obtained as described in ref. (24). API2 one of these transcription factors, for which b-catenin acts as a (1 mg/kg in PBS-2% DMSO; Tocris Bioscience) was adminis- transcriptional coactivator (14). tered by intraperitoneal injection three times per week, NVP- As inappropriate activation of the Wnt/b-catenin pathway TNKS656 (100 mg/kg) was injected subcutaneously twice daily. was first linked to colon cancer three decades ago, there has been intense interest in developing effective inhibitors (15, 16). Gene expression It has been described that tankyrases promote AXIN1/2 parsy- Gene expression of 292 selected genes was profiled in base- lation and degradation through the proteasome (17). Recently, line tumors of patients treated with PI3K/ATK/mTOR inhibitors a new family of tankyrase inhibitors was shown to stabilize using the nCounter platform from Nanostring Technologies. AXIN1/2, enhancing the activity of the destruction complex and Differentially expressed genes were identified in tumors pre- reducing free b-catenin. These inhibitors are showing promis- senting high or low nuclear b-catenin content using Partek ing preclinical results as Wnt/b-catenin inhibitory drugs for the Genomics Suite Software. Lists were cut-off at fold change of treatment of Wnt-addicted tumors (18–20). 1.2 and a P value < 0.075 (two-tailed one-way ANOVA test). For Here, we present evidence that high nuclear b-catenin con- microarray analyses, we used a genome wide Human Gene tent is associated to resistance to PI3K and AKT inhibitors in the 1.0 ST Array (Affimetrix). Data were acquired using the Affime- context of clinical trials, whereas frequent mutations or clini- trix GeneChip/GeneTitan platforms. Genes were considered copathologic traits implicated in colorectal cancer progression differentially expressed in NVP-TNKS656 versus vehicle-treated do not. We demonstrate that combining these drugs with tumors at 1.5 fold change and P < 0.05 using a two-tailed one- NVP-TNKS656, a new therapeutic small-molecule inhibitor of way ANOVA test. Microarray data are deposited at ArrayExpress the Wnt/tankyrase pathway that reduces nuclear b-catenin (21), database (E-MTAB-2446). To perform qRT-PCR, RNA from overcomes such resistance and represses tumor growth in endpoint tumor xenografts was used to synthesize cDNA using colorectal cancer patient–derived xenograft (PDX) models. We Superscript-III reverse transcriptase with oligo-dT and random

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hexamer primers (Life Technologies). A 7900HT qPCR System and/or NVP-BKM120 (2.4 mmol/L, Selleck Chemicals) for was used with Power SYBR-green (Applied Biosystems) and another 48 hours prior apoptosis analysis. Proportions of specific primer pairs. Relative gene expression was determined apoptotic cells were determined using the Annexin V-eGFP by the comparative Ct method (25) and significance was (BioVision) kit. Dead cells were detected as DAPI negative determined using an unpaired t test with Welch correction. (1 mg/mL, Roche). Cells were analyzed by flow cytometry using See details in Supplementary Materials and Methods. a Navios Flow Cytometer (Beckman Coulter). To measure apoptosis by immunofluorescence in sphere cul- Genotyping tures, cells suspended in culture media were mixed 1:1 with fi Tumor samples from colorectal cancer patients in clinical trials Matrigel (BD Biosciences), xed for 1 hour in 4% PFA, permea- bilized with PBS/1% Triton X-100 at room temperature for with PI3K/AKT/mTOR inhibitors were genotyped by sequencing the amplified product of a multiplexed PCR reaction (Amplicon 3 hours, and blocked overnight at 4 C in PBS/1% Triton sequencing) as described in Supplementary Materials and Meth- X-100/3% BSA. Samples were incubated for 24 hours with pri- ods. Frequent mutations in 57 oncogenes and tumor suppressor mary antibodies (Supplementary Table S3). Secondary anti- m genes were interrogated (Supplementary Table S1). Microsatellite bodies and Hoechst 33342 (5 g/mL) were incubated overnight instability was analyzed using the MSI-Analysis System (Pro- at room temperature. mega). Purified patient-derived cells were genotyped straight after surgical resection of patients' tumors by Sequenom or Haloplex Cell culture platforms. Genotyping by Sequenom (CLIA panel) was per- Cell lines were cultured under standard conditions. DLD1F formed as previously described (10). Haloplex Target Enrichment cells are DLD1 derivates expressing pcDNA-FOXO3A(3A)ER System (Agilent Technologies) was used to capture the complete (30). HT29F cells express pLHCX-HA-FOXO3A(3A):ER. All coding regions of 388 oncogenes and tumor suppressor genes parental cell lines were originally obtained from ATCC. Cell (Supplementary Table S2). lines were authenticated by short-tandem repeat analysis by Three PDX models were genotyped by Exome sequencing. the cell bank. Patients provided written informed consent for somatic and germline DNA analysis. Mutations were called with VarScan2 Western blot analysis software, either using the mpileup2snp or somatic commands, The detailed protocol for protein extraction is described in depending on the availability of normal tissue (26). Nontumoral Supplementary Materials and Methods. Western blot analysis was tissue was not available for PDX-P2, thus, common SNPs were performed as described in ref. (10) using specific antibodies filtered according to the 1000 genome catalogue (27). SIFT and (Supplementary Table S3). Polyphen-2 helped predicting functionality of the identified mutations. Complete Exome sequencing data from PDX-P2, TCF/LEF1 reporter assays P5, and P30 are available at the SRA database at NCBI (BioProject DLD1 cell line was stably transfected with a vector (7TGP, ID: PRJNA242531). obtained at Addgene) expressing eGFP controlled by a pro- moter containing seven TCF/LEF transcription factor–binding Immunohistochemistry and immunofluorescence sites (7xTOP; ref. 31). Cells were treated with NVP-TNKS656 Samples from paraffin-embedded tissues were stained as 100 nmol/L (Novartis) for 7 days and eGFP accumulation was described in ref. 24 using the following antibodies (Supple- measured by flowcytometryusingaNaviosFlowCytometer mentary Table S3). Nuclei were stained with Hoechst 33342 (Beckman Coulter). (5 mg/mL; Sigma-Aldrich). Pictures of the immunofluorescent þ signal were captured using a NIKON C2 confocal microscope Statistical analysis and analyzed with MBF ImageJ software using criteria previ- We analyzed progression-free survival (PFS) of patients by ously described (28, 29). the Kaplan–Meier method and compared the curves using a b -Catenin immunohistochemistry was done using the Dako log-rank (Mantel–Cox) test. We used Pearson correlation test to Autostainer Plus Staining System. For visualization, EnVision comparetimeonpreviouslineoftreatmentversustimeon FLEX detection system (DAKO) was used. Sections were counter- treatment with PI3K/AKT/mTOR inhibitors of patients ana- stained with hematoxylin, dehydrated, cleared, and mounted lyzed for nuclear b-catenin content and to correlate apoptosis b for examination. -Catenin staining was evaluated by a patho- versus nuclear b-catenin or FOXO3A content in sphere cell logist as described in Supplementary Materials and Methods. cultures treated with NVP-TNKS656, API2, and NVP-BKM120. DLD1F and HT29F cells were seeded on glass coverslips Pearson correlation test was also used to compare nuclear and treated for 6 hours with 4-hydroxytamoxifen 100 nmol/L FOXO3A content in patients and corresponding PDX tumors, fi (4-OHT, Sigma-Aldrich). Cells were xed in 4% para-formalde- or in primary tumors versus liver metastases or to compare with fl hyde (PFA) and immuno uorescent staining was performed as SLC2A3 mRNA levels. described previously (10). Differences in apoptosis of treated sphere cell cultures; levels of AXIN1, b-catenin, phosphoS6, Ki67, and cleaved caspase-3 Apoptosis assays expression in tumor xenografts; mRNA expression of NVP- Patient-derived cells were seeded in suspension as sphere TNKS656 target genes by qRT-PCR; and apoptosis of DLD1F or cultures on low attachment multiwell dishes, whereas cell lines HT29F cell lines, were analyzed by an unpaired t test comparing were seeded in adherent multiwell cell culture dishes. Cells were the means of two groups of values. Fisher exact test served to pretreated with NVP-TNKS656 (100 nmol/L, Novartis) or DMSO analyze the differences in response among patients regarding for 48 hours and then with API2 (20 mmol/L, Tocris Bioscience) nuclear b-catenin content, mutations affecting PIK3CA, KRAS,

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TP53, APC, or tumor histologic TNM status. P values lower than was reduced upon combination with NVP-TNKS656 (Fig. 2A 0.05 were considered significant in all tests. and B). Tankyrase inhibition significantly reduced nuclear b-catenin, API2 decreased phosphor-S6 content, and both diminished proliferation (Fig. 2C, Supplementary Fig. S5). Results PDX-P30, derived from a liver metastasis, presented high Effective pharmacologic inhibition of Wnt/b-catenin and amounts of both nuclear b-catenin and FOXO3A (Fig. 2A). PI3K/AKT pathways reduces tumor growth It was also resistant to API2, but NVP-TNKS656 treatment We hypothesized that reducing nuclear b-catenin content alone reduced tumor growth rate equally to the drug combi- could be sufficient to sensitize colorectal cancer tumors to the nation (Fig. 2B). Tankyrase but not AKT inhibition promoted treatment with PI3K or AKT inhibitors. Consequently, we apoptosis, whereas the number of proliferative cells was not blocked the Wnt/b-catenin signaling using the tankyrase inhib- affected (Supplementary Fig. S5). This was the only model itor NVP-TNKS656 (21) in colorectal cancer PDX models. We where NVP-TNKS656 alone showed an effect on tumor selected five PDX models with high (P2, P7, P19, P22, and P30) growth, probably due to high endogenous amounts of and five with low nuclear b-catenin content (P5, P6, P31, P33, FOXO3A that might have induced apoptosis when nuclear and P34; Fig. 1A and B). Sphere cell cultures derived from b-catenin was reduced by NVP-TNKS656. NVP-TNKS656 xenograft tumors of each model were treated with API2 or NVP- increased AXIN1 protein levels in all subcutaneous tumors BKM120, inhibiting AKT or PI3K activity, respectively, alone or confirming its activity as a Wnt/tankyrase pathway inhibitor in combination with NVP-TNKS656 (Fig. 1C; Supplementary (ref. 21; Fig. 2D). Table S4). API2 or NVP-BKM120 induced significantly less apoptosis in cells with high rather than low nuclear b-catenin FOXO3A/b-catenin target genes are pharmacodynamic content by measuring the proportion of Annexin V-positive markers of response to Wnt/tankyrase-inhibitory drugs cells. Combination with NVP-TNKS656 significantly increases Although NVP-TNKS656 reduced the high nuclear b-catenin apoptosis in cells with high as opposed to low nuclear b-cate- content observed in both PDX-P2 and PDX-P30, treatment nin content. Similar results were observed by using NVP- only reduced tumor growth rate in the latter model (Fig. 2 and XAV939, another inhibitor of tankyrase activity (Supplemen- Supplementary Fig. S5B). Contrarily, NVP-TNKS656 did not tary Fig. S1). Alternative measurement of apoptosis by cleaved affect tumor growth in PDX-P5 model. We studied whether caspase-3 showed equivalent results (Supplementary Fig. S2). the high FOXO3A content observed in the metastatic PDX-P30 Furthermore, apoptosis induced by API2 or NVP-BKM120 model (Fig. 2A) could determine the repression by NVP- treatment showed a significant inverse correlation with nuclear TNKS656ofadistinctsetofWnt/b-catenin target genes and b-catenin content (Fig. 1D). Such correlation was lost when its enhanced sensitivity to treatment. RNA from tumors of API2 or NVP-BKM120 was combined with NVP-TNKS656. PDX-P2, PDX-P30, and PDX-P5 models was analyzed at the Mutations in PIK3CA, KRAS,orTP53 genes or tumor site did endpoint of the in vivo experiments (Fig. 2B). The three models not condition a differential response of sphere cell cultures to showed a distinct gene expression pattern that was modified PIK3 or AKT inhibition (Supplementary Fig. S3). Only one out by NVP-TNKS656 treatment (Fig. 3A; Supplementary Tables of the 10 PDX models presented microsatellite instability S7–S9). The prometastatic S100A4 gene was repressed in (MSI), preventing the possibility to evaluate its impact on drug both PDX-P2 and PDX-P30 models (Fig. 3B). Four NVP- response (Supplementary Table S5). TNKS656–repressed genes in PDX-P2, two in PDX-P5, and yet Our data indicate that nuclear b-catenin content conditions none in PDX-P30 were direct TCF/b-catenin targets (Supple- drug response in patient-derived sphere cell cultures, whereas mentary Table S10). Instead, two genes in PDX-P2, one in frequent mutations in colorectal cancer do not (Fig. 1C and D and PDX-P5, and 9 in PDX-P30 were FOXO3A/b-catenin targets Supplementary Fig. S3; Supplementary Table S5). (Supplementary Table S11), many of them formally associated We further investigated these results in vivo. Cells from three with metastasis (10, 32–34). The regulation of some of these PDX models with known nuclear b-catenin and FOXO3A status NVP-TNKS656–repressed genes was further confirmed by qRT- and with limited response to AKT or PI3K inhibition in vitro PCR in the same tumor xenograft samples (Fig. 3C). Any gene (P2, P5, and P30), were injected subcutaneously into NOD-SCID evaluated in PDX-P5 model was significantly regulated by mice (Figs. 1B and D and 2A). Treatment with NVP-TNKS656 NVP-TNKS656, a result in line with its low nuclear b-catenin caused a systemic reduction of nuclear b-catenin content and content and its lack of tumor growth response (Fig. 2B). Our function in skin and intestine, tissues where the Wnt pathway data suggest that FOXO3A/b-catenin targets could be better tightly controls homeostasis, but showed no major negative side pharmacodynamic markers than TCF/b-catenin target genes effects (Supplementary Fig. S4). for evaluating therapeutic Wnt/tankyrase pathway inhibition. API2 alone or in combination with NVP-TNKS656 did not repress tumor growth in PDX-P5, with low basal nuclear b-catenin and FOXO3A content, probably representing a case of a colorectal FOXO3A determines the response to Wnt/b-catenin pathway cancer tumor resistant to AKT inhibition due to mechanisms inhibitors independent of nuclear b-catenin accumulation (Fig. 2A and NVP-TNKS656 treatment was particularly effective in a B). Exome sequencing revealed a mutation in the AKT2 gene that tumor with high endogenous nuclear b-catenin and FOXO3A could explain the lack of reduction of phosphor-S6 or tumor content, promoting apoptosis, reducing tumor growth rate growth upon API2 treatment in this model (Supplementary Fig. (Fig. 2) and preferentially repressing the expression of FOXO3A S5; Supplementary Table S6). instead of TCF target genes (Fig. 3). The repression of the PDX-P2 presented high nuclear b-catenin and low FOXO3A Wnt/b-catenin pathway by NVP-TNKS656 alone did not pro- amounts, was resistant to API2 alone, and yet tumor growth rate mote apoptosis in DLD1 or HT29 colon cancer cells but

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Figure 1. Colorectal cancer patient–derived cells with high amounts of nuclear b-catenin present high sensitivity to API2 or NVP-BKM120 in combination with NVP-TNKS656. A, representative pictures of immunofluorescence and confocal microscopy of histologic sections of the indicated PDX models with high (left) or low (right) nuclear b-catenin content. Inserts show magnification to better visualize b-catenin sub-cellular localization. Dashed lines delineate nuclei. Nuclei were stained with Hoechst 33342 (blue). Scale bars, 100 mm; magnifications 50 mm. B, column scatter plot showing the amount of nuclear b-catenin measured by immunofluorescence and confocal microscopy in 10 primary tumors and liver metastases from which sphere cell cultures were derived and used to test drug response. Horizontal lines indicate arithmetic mean values, and error bars show SD. C, column scatter plot showing the apoptosis induced in sphere cell cultures of these 10 patient-derived models treated as indicated. Data, fold change of apoptotic cells induced by the treatment compared with cells treated with vehicle. Horizontal lines indicate arithmetic mean values, and error bars show SEM. P values correspond to unpaired t tests. The original percentage of Annexin V–positive cells is shown in Supplementary Table S4. D, scatter plots representing the apoptosis induced by API2 (top left) and NVP-BKM120 (top right) or API2 þ NVP-TNKS656 (bottom left) and NVP-BKM120 þ NVP-TNKS656 (bottom left) in sphere cell cultures of these 10 patient-derived models versus the histologic amount of nuclear b-catenin in the original patient's tumors. Data, fold change of apoptotic cells induced by the treatment compared to cells treated with vehicle. P values correspond to Pearson correlation test. B and D, b-catenin relative units (r.u.) were calculated as described in Materials and Methods.

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Figure 2. NVP-TNKS656 stabilizes AXIN1 and reduces both, nuclear b-catenin and tumor growth alone or in combination with the AKT inhibitor API2 in colorectal cancer PDX models. A, representative pictures of double immunofluorescent staining and confocal microscopy to detect b-catenin (red) and FOXO3A (green) in histologic sections of subcutaneous xenografted tumors from patients P2, P5, and P30. Right panels show magnifications to visualize the amounts of nuclear b-catenin and FOXO3A. Scale bar, 100 mm; magnifications, 20 mm. Nuclei were stained with Hoechst 33342 (blue). Arrowheads point to b-catenin localized exclusively in cell membranes in tumors from P5 model. B, tumor cells derived from the three indicated patients were injected subcutaneously in NOD-SCID mice and treated as indicated. A minimum of 5 mice with tumors in both flanks was treated in each group. The graphs represent the fold change calculated by comparing the tumor volume at each given time point to the volume at the first day of treatment. Error bars and SD are shown for tumor volume fold change of all tumors. Unpaired t tests were used to compare the area under the curve generated for each growing tumor along the experiment and grouped by treatment. Asterisks indicate significant differences (P < 0.05). C, representative pictures showing immunofluorescent staining of b-catenin in tumor xenografts from PDX-P2 model growing in mice treated with vehicle or NVP-TNKS656. Inserts show magnifications to better visualize b-catenin reduction from cytoplasm and nuclei upon tankyrase inhibition. Scale bars, 100 mm; inserts, 50 mm. D, three xenograft tumors per group of treated mice were processed for the analysis of AXIN1 by Western blot analysis (top). b-Tubulin was used as loading control. Blots were quantified using ImageJ software (bottom). Protein expression levels are represented as relative units (r.u.) versus vehicle treated samples. Bars indicate SD. P values correspond to unpaired t tests.

enhanced the apoptosis induced by exogenous nuclear their correspondent original patient samples (Supplementary Fig. FOXO3A-ER (Fig. 4). These data confirm the capacity of nuclear S6). We also observed that nuclear FOXO3A content positively b-catenin to confer resistance to FOXO3A-induced apoptosis correlated with the expression of SLC2A3 mRNA, a FOXO3A/ (10) and, therefore, the therapeutic value of reducing nuclear b-catenin target gene (10) that was repressed upon NVP-TNKS656 b-catenin by tankyrase inhibitors in FOXO3A-active cancer treatment in vivo (Fig. 3). cells. Interestingly, we observed that paired primary tumors and Furthermore, NVP-TNKS656 promoted apoptosis in patient- liver metastases accumulated similar nuclear FOXO3A derived sphere cell cultures proportionally to the amount of amounts, showing that its activation could be durable and nuclear FOXO3A detected in their correspondent patients' tumors occur prior progression to metastatic stages (Supplementary (Fig. 5A and B). Apoptosis was also proportional to FOXO3A Fig. S7). content when sphere cultures were treated with a different tan- Finally, by profiling 130 colorectal cancer cases, we identified a kyrase inhibitor, NVP-XAV939 (Supplementary Fig. S1). Similarly distinctive population of patients with tumors presenting high to b-catenin (10), PDX models and primary sphere cultures expression of FOXO3A/b-catenin target genes (Fig. 5C). The faithfully preserved the levels of nuclear FOXO3A detected in expression of TCF/b-catenin target genes also identified such a

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Figure 3. Reduction of nuclear b-catenin content by NVP-TNKS656 regulatesgeneexpressionincolorectalcancerPDXmodels.A,threetumorxenograftsfrom mice injected with PDX-P2, PDX-P30, or PDX-P5 treated with vehicle or NVP-TNKS656 were analyzed for gene expression at the end point of the experiments. Gene clustering diagrams shown were calculated using robust multiarray normalized expression values from genome-wide microarray analysis. Triplicates of each treatment are indicated as colored rectangles (R1, R2, and R3). Significance was calculated using two-tailed ANOVA with a significance cutoff of P 0.05. Gene expression scaling was indicated from low (blue) to high (red). B, Venn diagrams showing the number of genes downregulated by NVP-TNKS656 in all PDX models and genes that belong to the TCF/b-catenin (44) or FOXO3A/b-catenin (10) transcriptional programs. Common genes are indicated on the right and enumerated in brackets. Asterisks indicate genes involved in metastasis. C, relative mRNA expression levels of selected genes was confirmed by qRT-PCR in the same xenograft tumors from the three PDX models treated with vehicle (blue bars) or NVP-TNKS656 (red bars). Expression values are shown as normalized DCt.Errorbars SD of triplicate values obtained in three independent measurements are shown. P values correspond to unpaired t tests.

population but showed lower signal. Interestingly, FOXO3A/ aiming to homogenize the study cohort. There was no signif- b-catenin and TCF/b-catenin target genes clustered separately icant difference in PFS between patients treated with different among all cases profiled. Samples were also evaluated for their PI3K, AKT, or dual PI3K/mTOR drug subtypes (Fig. 6A, bot- MSI and mutational status of KRAS, BRAF, and PIK3CA, all tom). We performed a double-blinded evaluation of nuclear molecular features relevant for colorectal cancer tumors. Any b-catenin content by two independent pathologists using obvious correlation was observed between them and gene expres- immunohistochemistry and immunofluorescence on all base- sion signatures distinctive of active FOXO3A/b-catenin transcrip- line tumor samples (Fig. 6B and Supplementary Fig. S8). We tion (Fig. 5C; Supplementary Table S12). classified tumors into two histologic categories: high or low, These data suggest that patients with tumors that are active depending on the number of cells positive for nuclear b-catenin for FOXO3A/b-catenin transcription can be identified by accumulation. Out of 40 cases, 13 were high and 27 were low transcriptional profiling, whereby reduction of nuclear b-cate- in content. nin by Wnt inhibitors could promote FOXO3A-dependent After the first clinical diagnosis of disease progression by apoptosis. computerized axial tomography (CT) scan, all 13 patients presenting tumors with high nuclear b-catenin content had Comparative analysis of b-catenin as potential biomarker of progressed despite treatment with PI3K/AKT/mTOR pathway resistance to PI3K and AKT-inhibitory drugs inhibitors. Contrarily, 9 of 27 patients with tumors presenting We decided to compare the potency of nuclear b-catenin in low b-catenin continued treatment after CT scan evaluation, predicting resistance to PI3K or AKT inhibitors with the most showing some degree of stabilized disease (Fig. 6C, top). Such frequent mutations or histological traits observed in colorectal longer lasting response to PI3K/AKT/mTOR pathway inhibitors cancer tumors. Limited availability of samples from clinical did not correlate with better response to the previous line of trials only permitted the study of tumors at baseline from a treatment, ruling out the possibility that those 9 patients were cohort of 40 colorectal cancer patients treated with PI3K/AKT/ either more sensitive to antitumoral drugs in general or had mTOR pathway inhibitors in several phase I clinical trials slower tumor growth independently of treatment [Figs. 6C (Fig. 6A, top; Supplementary Table S13). We selected tumors (bottom) and D]. Contrary to nuclear b-catenin content, rele- from patients treated with half the maximum tolerated dose vant oncogenic mutations in PIK3CA, KRAS, APC,orTP53

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Figure 4. NVP-TNKS656 sensitizes colorectal cancer cell lines to the apoptosis induced by FOXO3A. A, immunofluorescence and confocal microscopy to detect the translocation of exogenous FOXO3A(A3):ER from cytoplasm to nucleus of DLD1F and HT29F colon cancer cells upon 4-OHT treatment. Scale bar, 10 mm. B, Western blots showing the amount of exogenous FOXO3A(A3):ER in the nucleus and stabilization of AXIN1 in the cytoplasm of DLD1F and HT29F cells upon indicated treatments. The amounts of b-tubulin and Lamin A/C were evaluated to confirm the purity of nuclear and cytoplasmic extracts and as loading control. C, Representative pictures of DLD1F cells expressing fluorescent green protein (eGFP) under the control of seven upstream TCF/b-catenin–binding sites (7xTOP-eGFP) upon 5 days of NVP-TNKS656 treatment (left). Scale bar, 200 mm. 7xTOP-eGFP activity after treating DLD1F cells with vehicle or NVP-TNKS656 was assessed by flow cytometry (right). Bars indicate SD of experimental triplicates. D, apoptosis was measured in DLD1F and HT29F cells upon indicated treatments. Data, fold change of apoptotic cells induced by the treatment compared with cells treated with vehicle. Bars show SD of five replicates in six independent experiments. P values correspond to unpaired t tests.

genes, the site of tumor samples (primary tumor or metastasis) (Supplementary Fig. S9G–S9I). However, results were not statis- or TNM stage (T3 or T4) at the time of diagnosis did not tically significant as expected from the small number of cases correlate with any significant difference in response to PI3K/ available for the analyses. AKT/mTOR pathway inhibitors (Fig. 6C and D and Supple- To confirm the higher risk of progression in the b-catenin mentary Fig. S9A–S9F; Supplementary Tables S13 and S14). high–group, we performed a Cox Proportional Hazards analysis Only 2 of 28 samples analyzed presented MSI, preventing the on a subset of our cohort (n ¼ 27) with complete annotation for possibility to evaluate its impact on drug response (Supple- variables potentially linked to outcome: age, gender, therapy mentary Table S13). (AKT, PI3Ka, pan-PI3K, PI3K-mTOR inhibitor), number of Equivalent analyses were performed separately in patients prior treatment lines, presence of liver metastasis, and molecular treated with PKI-587 (n ¼ 11), BEZ235 (n ¼ 9), or NVP-BKM120 profile (PIK3CA, KRAS, TP53, APC mutations, PTEN loss). Even (n ¼ 6). We observed that cases with high nuclear b-catenin after multivariate adjustment, patients whose tumors had high presented a shorter PFS upon PKI-587 or BEZ235 treatment nuclear b-catenin content still displayed a significantly worse

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Figure 5. High FOXO3A activity in colorectal cancer patient-derived cells determines the apoptosis induced by NVP-TNKS656. A, representative pictures of double immunofluorescence and confocal microscopy to detect b-catenin (red) and FOXO3A (green) in models PDX-P33 and PDX-P34. Arrowheads point to cancer cells in PDX-P33 presenting high nuclear FOXO3A and b-catenin accumulation. PDX-P33 presented high and PDX-P34 low nuclear FOXO3A and b-catenin content. Bottom panels show magnifications to better visualize b-catenin and FOXO3A subcellular localization. White dotted lines delineate nuclei stained with Hoechst 33342 (blue). Scale bars, 100 mm; magnifications, 50 mm. B, Scatter plot comparing apoptosis induced by NVP-TNKS656 in xenograft-derived sphere cell cultures versus the histological amount of nuclear FOXO3A in the corresponding PDX models. (Continued on the following page.)

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Therapeutic Wnt Pathway Inhibition in Colorectal Cancer

Figure 6. High nuclear b-catenin content is associated with resistance to PI3K/AKT/mTOR inhibitors in colorectal cancer patients. A, Table summarizing molecular targets, drugs, and number of patients (40 in total) in each clinical trial whose baseline tumors were profiled. Kaplan–Meier analysis representing PFS of patients separated by drug subtype administered: dual PI3K/mTOR, PI3K or AKT inhibitors. Number of patients is shown for each cohort (n). P value was calculated by log-rank (Mantel–Cox) test. B, representative pictures of double immunofluorescence staining and confocal microscopy to detect b-catenin (red) and a-catenin (green) in colorectal cancer tumor sections of patients treated with PI3K or AKT inhibitors. Tumors present low (top image) or high (bottom image) nuclear b-catenin content (Scale bar, 100 mm). Panels on the right show magnifications with fluorescent channels split to better visualize b-catenin subcellular localization (Scale bar, 20 mm). All sections were counterstained with Hoechst 33342 (blue) to detect nuclei. Dashed lines delineate nuclei. C, chart representing the time that each patient received a PI3K/mTOR, PI3K or AKT inhibitor and the time on the corresponding previous line of treatment (top). The 40 patients studied are split in those with tumors presenting high and those with low nuclear b-catenin accumulation. Pink vertical bar indicates the latest period of time when disease progression was evaluated by computerized axial tomography (CAT) scan. Green horizontal bars correspond to patients who had progressed to treatments with PI3K/mTOR, PI3K or AKT inhibitors at the time of first CAT scan after initiating the clinical trial. Red bars show the time on treatment for those patients who had not progressed to treatment at the time of first CAT scan. Bottom, scatter plot representing the time on treatment with PI3K/mTOR, PI3K, or AKT inhibitors versus the time on previous line of treatment for all 40 colorectal cancer patients. Their level of nuclear b-catenin is indicated. P value corresponds to Pearson correlation test. D, Kaplan–Meier curves showing progression-free survival (PFS) of patients presenting tumors with high or low nuclear b-catenin content and treated with PI3K/mTOR, PI3K or AKT inhibitors (left) or the correspondent previous line of therapy (right). Number of patients (n) is shown for each cohort. P value, HR and 95% confidence interval (CI) are shown and calculated by the log-rank (Mantel–Cox) test.

outcome (PFS HR 3.96, 95% confidence interval, 1.03–15.27; log- (Supplementary Fig. S10A). The oncogenic mutations detected rank P 0.0158; Supplementary Table S15). did not correlate with any of the gene expression clusters Concerning gene expression patterns, we could analyze 31 of observed. We finally observed that the expression profile of a the initial 40 baseline samples and observed that tumors respond- reduced set of genes could also help identifying tumors presenting ing to PI3K/AKT/mTOR pathway inhibitors clustered together high or low nuclear b-catenin content, cross-validating the initial

(Continued.) FOXO3A relative units (r.u.) were calculated as described in methods. Apoptosis is represented as fold change of apoptotic cells induced by NVP-TNKS656 compared with the cells treated with vehicle. P values correspond to Pearson correlation test. Nuclear b-catenin content for each model is also indicated. C, Nanostring platform was used to analyze the expression of 31 FOXO3A/b-catenin and 21 TCF/b-catenin target genes in FFPE tumor samples from 130 colorectal cancer patients. Samples and genes were ordered by hierarchical clustering using uncentered Pearson correlation distance and complete linkage. MSI and mutational status of KRAS, PIK3CA and BRAF were evaluated as indicated in methods. Patients showing highest expression of FOXO3A/b-catenin and TCF/b-catenin target genes are highlighted with a yellow box. Gene expression scaling is shown from low (blue) to high (red).

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cut-off selected to catalog tumors histologically (Fig. 6B and treating patients with tankyrase inhibitors. Indeed, we identify Supplementary Figs. S8 and S10B). by gene expression profiling a population of patients presenting tumors with high FOXO3A/b-catenin activity who could potentially respond best to the treatment with these Discussion Wnt/b-catenin pathway inhibitors. The efficacy of several PI3K and AKT inhibitors is being tested However, very little is known about the determinants of in multiple clinical trials worldwide. Although initial results nuclear FOXO3A activation in colorectal cancer. It is well were promising in some tumor types, clinical responses are null described that oxidative stress promotes FOXO3A transloca- or limited in most colorectal cancer patients (4–8). A reason- tion from the cytoplasm to the nucleus and the consequent able hypothesis for such limited responses is the absence of a induction of genes involved in cell-cycle arrest, survival, apo- universal biomarker to select drug-sensitive patients or discard ptosis, and metastasis (9). Future investigations should reveal resistant cases. As activating mutations in PIK3CA gene confer the precise contribution of oxidative stress and other stimuli sensitivity to PI3K/AKT pathway inhibitors in preclinical assays, originated in the surrounding tumor stroma on determining they have been commonly used as inclusion criteria in clinical the final levels of activated FOXO3A. Our results suggest that trials. However, PIK3CA mutations showed conflicting results describing FOXO3A-activating factors would be of particular in predicting response to single-agent PI3K or AKT inhibitors in interest to understand the response to Wnt/b-catenin inhibi- early-phase clinical trials (7, 35–37). Similarly, we show here tors. The use of PDX models could be pivotal in these inves- that mutations in genes frequently altered in colorectal cancer, tigations since they preserve equivalent levels of FOXO3A including KRAS, TP53,orPIK3CA, do not predict response to than the original patients' tumors. In fact, the efficacy of PI3K/AKT pathway inhibitors. Interestingly, multivariate anal- Wnt/tankyrase inhibitors observed in cancer cell lines or genetic ysis indicates that APC mutations are a risk factor for patients mouse models has been modest (18–20), as they may not treated with these inhibitors (Supplementary Table S15). This recapitulate tumoral FOXO3A activity as consistently as PDX. would suggest that oncogenic activation of the Wnt/b-catenin Indeed, PDX are generally considered the best preclinical mod- pathway could be a mechanism of resistance to PI3K and AKT els to evaluate drug response as they faithfully recapitulate inhibitors. patient's disease preserving their molecular alterations and Indeed, we previously described that nuclear accumulation of histopathologic traits (24, 38, 39). b-catenin conferred resistance to PI3K and AKT inhibitors in colon Other alterations frequent in colorectal cancer could also be cancer cells (10). In the current study, we observed that colorectal relevant determinants of response to Wnt/b-catenin inhibitors. cancer patients with high nuclear b-catenin present a shorter PFS For instance, truncating mutations in APC gene are present in when treated with PI3K/AKT/mTOR pathway inhibitory drugs in more than 80% of colorectal cancer patients and constitutively the context of clinical trials. Such differential response contrasts activate the oncogenic Wnt/b-catenin pathway (3). Here we with the fact that all colorectal cancer patients in our cohort show that NVP-TNKS656 can reduce nuclear b-catenin content progressed to previous lines of treatment irrespective of their and repress tumor growth even in APC-mutant PDX models. nuclear b-catenin content. These results on clinical samples indi- These data suggest that the therapeutic potential of tankyrase cate that nuclear b-catenin accumulation could be an indepen- inhibitorscouldbeextendedtothemajorityofcolorectal dent predictive biomarker of resistance to PI3K and AKT inhibi- cancer tumors. This wide spectrum would contrast with the tors beyond other molecular alterations frequent in colorectal lack of activity in APC-mutated tumors expected from the cancer. treatment with NVP-LGK974. This is the first Wnt/b-catenin In accordance, we explored here the therapeutic potential of pathway inhibitor tested in clinical trials (NCT01351103), overcoming such resistance by reducing nuclear b-catenin content which blocks porcupine activity and the maturation of Wnt with a new Wnt/tankyrase inhibitor, NVP-TNKS656. We show ligands upstream in the oncogenic signaling. As tankyrases that it sensitizes patient-derived cells to PI3K or AKT inhibitors in parsylate and commit other proteins to degradation in addition vitro and in vivo, especially those with high accumulation of to AXIN1 and 2 (40, 41), it could be of interest to evaluate to nuclear b-catenin and, thus, high oncogenic Wnt/b-catenin path- what extent the antitumoral capacity of tankyrase inhibitors way activity. rely on affecting any other cell processes beyond Wnt/b-catenin Our results on Wnt/tankyrase inhibitors reveal a new ther- signaling. apeutic opportunity for the treatment of advanced colorectal Aiming to define clinically useful biomarkers to predict the cancer patients beyond their combination with PI3K or AKT response to PI3K/AKT and Wnt/b-catenin pathway inhibitors, inhibitors. Indeed, we previously showed that cancer cells we investigated the potential of gene expression signatures. Our with high proapoptotic FOXO3A transcriptional activity initial results suggest that specific gene expression profiles require high levels of nuclear b-catenin to preserve a viable could identify colorectal cancer tumors with high FOXO3A balance between survival and apoptosis (10). Here, we ob- and b-catenin content and transcriptional activity. In particular, served that NVP-TNKS656 treatment was particularly effective we observed that a particular set of genes was overexpressed in in cells and tumors with high endogenous nuclear b-catenin colorectal cancer tumors with high nuclear b-catenin content. and FOXO3A content, where the pharmacologic reduction of Our drug treatment experiments indicate that such Wnt/b-cate- nuclear b-catenin promoted FOXO3A-dependent apoptosis. nin activated tumors could be resistant to PI3K and AKT Therefore, it is expected that cancer cell survival in such endog- inhibitors. Furthermore, we observed a significant correlation enous FOXO3A-active scenario, more frequent in advanced between nuclear FOXO3A accumulation and mRNA expression metastatic colorectal cancer tumors (10), would rely on of one of its target genes, SLC2A3 (10). These results suggest Wnt/b-catenin pathway activity. This could represent an excep- that nuclear accumulation of FOXO3A and b-catenin observed tional opportunity to compromise cancer cell viability by by histology reflects their activation as transcription factors

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Therapeutic Wnt Pathway Inhibition in Colorectal Cancer

inducing the expression of their corresponding target genes. activity who may benefit from combined treatments. Such Finally, we could identify, using Nanostring nCounter plat- molecular stratification of patients could represent a signifi- form, a population of colorectal cancer patients with tumors cant improvement in response to therapy and an important presenting high expression of FOXO3A/b-catenin and TCF/ step forward towards reverting the long-stalled scenario of b-catenin target genes. Our preclinical data indicate that these colorectal cancer therapy. patients could benefit from the treatment with Wnt/tankyrase inhibitors. Disclosure of Potential Conflicts of Interest fi Together, these results suggest that gene expression pro ling J. Tabernero is a consultant/advisory board member for Novartis. No could help to build complex predictive biomarkers of response potential conflicts of interest were disclosed by the other authors. to PI3K/AKT and Wnt/b-catenin pathway inhibitors. However, selecting a precise set of genes to build robust signatures would Disclaimer require evaluating whole gene expression patterns by micro- The funders had no role in study design, data collection and analysis, arrays or RNAseq in prospective studies with fresh-frozen decision to publish, or preparation of the manuscript. tumor samples. These expression profiles should be cross- compared with histologic evaluation of nuclear FOXO3A and Authors' Contributions b-catenin content and the response to treatment, to finally fi Conception and design: O. Arques, W. Shao, J. Tabernero, H.G. Palmer de ne those gene sets associated to such histologic and clinical Development of methodology: O. Arques, I. Chicote, I. Puig, R. Dienstmann, traits. These biomarker exploratory studies should ideally focus G. Caratu, J. Tabernero, H.G. Palmer on analyzing tumor biopsies taken from progressive lesions at Acquisition of data (provided animals, acquired and managed patients, the time of inclusion in clinical trials and not from archival provided facilities, etc.): O. Arques, I. Chicote, I. Puig, S.P. Tenbaum, G. Argiles, samples. In the particular case of nuclear FOXO3A, we have R. Dienstmann, N. Fernandez, G. Caratu, J. Matito, J. Rodon, S. Landolfi, A. Prat, observed that advanced metastatic tumors present the highest E. Espin, R. Charco, P. Nuciforo, A. Vivancos, J. Tabernero Analysis and interpretation of data (e.g., statistical analysis, biostatistics, proportion of cases activated for this transcription factor (10). computational analysis): O. Arques, S.P. Tenbaum, R. Dienstmann, Therefore, validating the use of nuclear FOXO3A accumulation D. Silberschmidt, J. Rodon, A. Prat, P. Nuciforo, A. Vivancos, J. Tabernero, and its associated gene expression signatures as potential bio- H.G. Palmer marker of sensitivity to Wnt/b-catenin pathway inhibitors Writing, review, and/or revision of the manuscript: O. Arques, G. Argiles, would require taking biopsies preferentially from metastatic R. Dienstmann, J. Rodon, E. Espin, P. Nuciforo, W. Shao, H.G. Palmer lesions that will actually be progressing at the time of patients' Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): O. Arques inclusion in clinical trials. Study supervision: O. Arques, H.G. Palmer Hence, we suggest combining histologic evaluation of FOXO3A and b-catenin with functional gene expression sig- Acknowledgments natures to build complex predictive biomarkers of response b The authors thank Dr. Alan Huang (Novartis Institutes for BioMedical to PI3K/AKT and Wnt/ -catenin pathway inhibitors in colo- Research, Inc) for providing the PI3K inhibitor NVP-BKM120 and Dr. Paul J. rectal cancer, which may help the design of future clinical Coffer (Utrecht, Netherlands) for kindly providing the pcDNA3-FOXO3A(A3): trials with this family of drugs. Similarly, current studies are ER expression plasmid. The authors also thank Javier Hernandez Losa for analyzing the expression of 50 selected genes by Nanostring evaluation of MSI status and Amanda Wren for her valuable assistance in nCounter platform to facilitate a more precise definition of the preparation of the English manuscript. breast cancer subtypes (42, 43), whose differential response to treatment is currently under validation in several clinical trials. Grant Support In the same line as our proposal, such gene expression sig- This work was supported by Instituto Carlos III - Fondo de Investiga- natures are being combined with clinical evaluation of hor- ciones Sanitarias - ISCIII (FIS-PI081356, FIS-PI11/00917, FIS-PI14/00103 mone receptors and HER2 protein levels by histopathologic and FIS-PI14/01239). O. Arques was supported by Agencia de Gestio d'Ajuts Universitaris i de Recerca - AGAUR; S.P. Tenbaum was supported techniques. by FERO Fellowship and Red Tematica de Investigacion Cooperativa del In summary, we propose combining gene expression pro- Cancer -RTICC (RD12/0036/0012), I. Puig was funded by the Fundacion filing and histology to define nuclear b-catenin and FOXO3A Científica de la Asociacion Espanola~ Contra el Cancer (AECC), and H.G. content and activity as predictive biomarkers of drug re- Palmer was supported by the Miguel Servet Program, Instituto de Salud sponse (Supplementary Fig. S11). We hypothesize that this Carlos III-ISCIII. molecular prescreening could establish three groups of The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked patients presenting tumors with: (i) low nuclear b-catenin advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate and FOXO3A activity, more suitable for the treatment with this fact. PI3K or AKT inhibitors alone, (ii) high b-catenin and FOXO3A activity who could benefitfromWnt/b-catenin inhibitors Received December 5, 2014; revised May 18, 2015; accepted July 2, 2015; alone and (iii) high nuclear b-catenin but low FOXO3A published OnlineFirst July 29, 2015.

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656 Clin Cancer Res; 22(3) February 1, 2016 Clinical Cancer Research

Tankyrase Inhibition Blocks Wnt/β-Catenin Pathway and Reverts Resistance to PI3K and AKT Inhibitors in the Treatment of Colorectal Cancer

Oriol Arqués, Irene Chicote, Isabel Puig, et al.

Clin Cancer Res 2016;22:644-656. Published OnlineFirst July 29, 2015.

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