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Index of Supplementary Data

Supplementary Table 3: Transcriptomic analyses of the human patient samples A. TCGA Interactome B. SU2C Interactome C. Protein activity TCGA D. Protein Activity SU2C

Prioritizing treatments for individual patients with cancer remains challenging, and performing coclinical studies using patient-derived models in real time is often unfeasible. To circumvent these challenges, we introduce OncoLoop, a precision medicine framework that predicts drug sensitivity in human tumors and their preexisting high-fidelity (cognate) model(s) by leveraging drug perturbation profiles. As a proof of concept, we applied OncoLoop to prostate cancer using genetically engineered mouse models (GEMM) that recapitulate a broad spectrum of disease states, including castration-resistant, metastatic, and neuroendocrine prostate cancer. Interrogation of human prostate cancer cohorts by Master Regulator (MR) conservation analysis revealed that most patients with advanced prostate cancer were represented by at least one cognate GEMM-derived tumor (GEMM-DT). Drugs predicted to invert MR activity in patients and their cognate GEMM-DTs were successfully validated in allograft, syngeneic, and patient-derived xenograft (PDX) models of tumors and metastasis. Furthermore, OncoLoop-predicted drugs enhanced the efficacy of clinically relevant drugs, namely, the PD-1 inhibitor nivolumab and the AR inhibitor enzalutamide.Significance:OncoLoop is a transcriptomic-based experimental and computational framework that can support rapid-turnaround coclinical studies to identify and validate drugs for individual patients, which can then be readily adapted to clinical practice. This framework should be applicable in many cancer contexts for which appropriate models and drug perturbation data are available.This article is highlighted in the In This Issue feature, p. 247

Supplementary Table 4: OncoMatch A. OncoMatch TCGA B. OncoMatch SU2C

Detailed Materials and Methods

Supplementary Table 6. OncoLoop summary table

Supplementary Table 2: Transcriptomic analyses of the GEMMs A. GEMMs Interactome B. Protein activity C. Cluster analyses D. Pathway analyses

Figure S1: Genomic alterations in prostate cancer represented in the GEMMs (related to Fig. 2). Figure S2: Additional phenotypic analyses of the GEMMs (related to Fig. 2). Figure S3: Phenotypic analysis of allograft and organoid models (related to Fig. 2). Figure S4: Additional transcriptomic analyses of the GEMMs (related to Fig. 3). Figure S5: Analyses of AR activity in GEMMS (related to Fig. 3). Figure S6: Regulatory sub-networks of the GEMM clusters (related to Fig. 3). Figure S7. MR-match of prostate cancer cells lines to human PCa (related to Figs. 5). Figure S8. Drug perturbation protein activity profiles from DU145 cells (related to Figs. 5, 6, 7). Figure S9. LNCaP Pharmacotyping to patients and GEMMs (related to Figs. 5). Figure S10: Additional validation of drug candidates (related to Figs 6, 7). Figure S11: Additional validation of drug candidates (related to Figs 6).

Supplementary Table 7. Transcriptomic analyses of the PDX models A. PDX interactome B. OncoTreat for drugs to PDX using DU145 cell perturbation data

Supplementary Table 5. Drug perturbation and OncoTreat analysis A. Summary of drugs and concentrations used B. Drug perturbation data for DU145 C. OncoTreat for drugs to SU2C patients using DU145 D. OncoTreat for drugs to GEMMs using DU145

Supplementary Table 1: Phenotypic analysis of the GEMMs

Video of mouse procedures for post-surgical steps

Video of mouse surgical procedures for transurethral delivery of Adenovirus to bladder

Video of mouse procedures for intravesical delivery of 4-hydroxy tamoxifen into bladder

Detailed procedures to accompany the videos

To study the progression of bladder cancer from non–muscle-invasive to muscle-invasive disease, we have developed a novel toolkit that uses complementary approaches to achieve gene recombination in specific cell populations in the bladder urothelium in vivo, thereby allowing us to generate a new series of genetically engineered mouse models (GEMM) of bladder cancer. One method is based on the delivery of adenoviruses that express Cre recombinase in selected cell types in the urothelium, and a second uses transgenic drivers in which activation of inducible Cre alleles can be limited to the bladder urothelium by intravesicular delivery of tamoxifen. Using both approaches, targeted deletion of the Pten and p53 tumor suppressor genes specifically in basal urothelial cells gave rise to muscle-invasive bladder tumors. Furthermore, preinvasive lesions arising in basal cells displayed upregulation of molecular pathways related to bladder tumorigenesis, including proinflammatory pathways. Cross-species analyses comparing a mouse gene signature of early bladder cancer with a human signature of bladder cancer progression identified a conserved 28-gene signature of early bladder cancer that is associated with poor prognosis for human bladder cancer and that outperforms comparable gene signatures. These findings demonstrate the relevance of these GEMMs for studying the biology of human bladder cancer and introduce a prognostic gene signature that may help to stratify patients at risk for progression to potentially lethal muscle-invasive disease.Significance:Analyses of bladder cancer progression in a new series of genetically engineered mouse models has identified a gene signature of poor prognosis in human bladder cancer.

Video of mouse surgical procedures for delivery of Adenovirus into the bladder lumen.

This file contains an index of all supplementary materials, and the supplementary figures, legends, and tables.

Purpose: Previously, FTY720 was found to possess potent anticancer effects on various types of cancer. In the present study, we aimed to first verify the role of Runx2 in prostate cancer progression and metastasis, and, subsequently, assessed if FTY720 could modulate Runx2 expression, thus interfering downstream events regulated by this protein.Experimental Design: First, the association between Runx2 and prostate cancer progression was assessed using localized prostate cancer specimens and mechanistic investigation of Runx2-induced cancer aggressiveness was then carried out. Subsequently, the effect of FTY720 on Runx2 expression and transcriptional activity was investigated using PC-3 cells, which highly expressed Runx2 protein. Last, the involvement of Runx2 in FTY720-induced anticancer effects was evaluated by modulating Runx2 expression in various prostate cancer cell lines.Results: Runx2 nuclear expression was found to be up-regulated in prostate cancer and its expression could be used as a predictor of metastasis in prostate cancer. Further mechanistic studies indicated that Runx2 accelerated prostate cancer aggressiveness through promotion of cadherin switching, invasion toward collagen I, and Akt activation. Subsequently, we found that FTY720 treatment down-regulated Runx2 expression and its transcriptional activity, as well as inhibited its regulated downstream events. More importantly, silencing Runx2 in PC-3 enhanced FTY720-induced anticancer effects as well as cell viability inhibition, whereas overexpressing Runx2 in 22Rv1 that expressed very low endogenous Runx2 protein conferred resistance in the same events.Conclusion: This study provided a novel mechanism for the anticancer effect of FTY720 on advanced prostate cancer, thus highlighting the therapeutic potential of this drug in treating this disease.

Supplementary Data from Suppression of Androgen-Independent Prostate Cancer Cell Aggressiveness by FTY720: Validating Runx2 as a Potential Antimetastatic Drug Screening Platform

Supplementary of differential gene expression and pathway analyses

Supplementary Figure S1 from Up-Regulation of TWIST in Prostate Cancer and Its Implication as a Therapeutic Target

Androgen-independent metastatic prostate cancer is the main obstacle in the treatment of this cancer. Unlike a majority of solid cancers, prostate cancer usually shows poor response to chemotherapeutic drugs. In this study, we have shown a potential novel target, TWIST, a highly conserved bHLH transcription factor, in the treatment of prostate cancer. Using malignant and nonmalignant prostate tissues, we found that TWIST expression was highly expressed in the majority (90%) of prostate cancer tissues but only in a small percentage (6.7%) of benign prostate hyperplasia. In addition, the TWIST expression levels were positively correlated with Gleason grading and metastasis, indicating its role in the development and progression of prostate cancer. Furthermore, down-regulation of TWIST through small interfering RNA in androgen-independent prostate cancer cell lines, DU145 and PC3, resulted in increased sensitivity to the anticancer drug taxol-induced cell death which was associated with decreased Bcl/Bax ratio, leading to activation of the apoptosis pathway. More importantly, inactivation of TWIST suppressed migration and invasion abilities of androgen-independent prostate cancer cells, which was correlated with induction of E-cadherin expression as well as morphologic and molecular changes associated with mesenchymal to epithelial transition. These results were further confirmed on the androgen-dependent LNCaP cells ectopically expressing the TWIST protein. Our results have identified TWIST as a critical regulator of prostate cancer cell growth and suggest a potential therapeutic approach to inhibit the growth and metastasis of androgen-independent prostate cancer through inactivation of the TWIST gene.

To study the progression of bladder cancer from non–muscle-invasive to muscle-invasive disease, we have developed a novel toolkit that uses complementary approaches to achieve gene recombination in specific cell populations in the bladder urothelium in vivo, thereby allowing us to generate a new series of genetically engineered mouse models (GEMM) of bladder cancer. One method is based on the delivery of adenoviruses that express Cre recombinase in selected cell types in the urothelium, and a second uses transgenic drivers in which activation of inducible Cre alleles can be limited to the bladder urothelium by intravesicular delivery of tamoxifen. Using both approaches, targeted deletion of the Pten and p53 tumor suppressor genes specifically in basal urothelial cells gave rise to muscle-invasive bladder tumors. Furthermore, preinvasive lesions arising in basal cells displayed upregulation of molecular pathways related to bladder tumorigenesis, including proinflammatory pathways. Cross-species analyses comparing a mouse gene signature of early bladder cancer with a human signature of bladder cancer progression identified a conserved 28-gene signature of early bladder cancer that is associated with poor prognosis for human bladder cancer and that outperforms comparable gene signatures. These findings demonstrate the relevance of these GEMMs for studying the biology of human bladder cancer and introduce a prognostic gene signature that may help to stratify patients at risk for progression to potentially lethal muscle-invasive disease. Significance: Analyses of bladder cancer progression in a new series of genetically engineered mouse models has identified a gene signature of poor prognosis in human bladder cancer.

Purpose: There is epidemiologic evidence that high garlic consumption decreases the incidence of prostate cancer, and compounds isolated from garlic have been shown to have cancer-preventive and tumor-suppressive effects. Recent in vitro studies in our laboratory have shown that garlic-derived organosulfur compound S-allylmercaptocysteine suppresses invasion and cell motility of androgen-independent prostate cancer cells via the up-regulation of cell-adhesion molecule E-cadherin. S-allylmercaptocysteine is therefore a potential antimetastatic drug with broad clinical applications that we tested in vivo for the first time in this study.Experimental Design: We used a newly established fluorescent orthotopic androgen-independent prostate cancer mouse model to assess the ability of S-allylmercaptocysteine to inhibit tumor growth and dissemination.Results: We showed that oral S-allylmercaptocysteine not only inhibited the growth of primary tumors by up to 71% (P < 0.001) but also reduced the number of lung and adrenal metastases by as much as 85.5% (P = 0.001) without causing notable toxicity. This metastatic suppression was accompanied by a 91% reduction of viable circulating tumor cells (P = 0.041), suggesting that S-allylmercaptocysteine prevents dissemination by decreasing tumor cell intravasation.Conclusions: Our results provide in vivo evidence supporting the potential use of S-allylmercaptocysteine as an E-cadherin up-regulating antimetastatic agent for the treatment of androgen-independent prostate cancer. This is the first report of the in vivo antimetastatic properties of garlic, which may also apply to other cancer types.

Androgen-independent metastatic prostate cancer is the main obstacle in the treatment of this cancer. Unlike a majority of solid cancers, prostate cancer usually shows poor response to chemotherapeutic drugs. In this study, we have shown a potential novel target, TWIST, a highly conserved bHLH transcription factor, in the treatment of prostate cancer. Using malignant and nonmalignant prostate tissues, we found that TWIST expression was highly expressed in the majority (90%) of prostate cancer tissues but only in a small percentage (6.7%) of benign prostate hyperplasia. In addition, the TWIST expression levels were positively correlated with Gleason grading and metastasis, indicating its role in the development and progression of prostate cancer. Furthermore, down-regulation of TWIST through small interfering RNA in androgen-independent prostate cancer cell lines, DU145 and PC3, resulted in increased sensitivity to the anticancer drug taxol-induced cell death which was associated with decreased Bcl/Bax ratio, leading to activation of the apoptosis pathway. More importantly, inactivation of TWIST suppressed migration and invasion abilities of androgen-independent prostate cancer cells, which was correlated with induction of E-cadherin expression as well as morphologic and molecular changes associated with mesenchymal to epithelial transition. These results were further confirmed on the androgen-dependent LNCaP cells ectopically expressing the TWIST protein. Our results have identified TWIST as a critical regulator of prostate cancer cell growth and suggest a potential therapeutic approach to inhibit the growth and metastasis of androgen-independent prostate cancer through inactivation of the TWIST gene.

Introduction: Chemotherapy (both intravesical and systemic) can reduce the risk of recurrence and progression in various stages of bladder cancer. However, recurrence after treatment failure is associated with an increased risk of progression. There are currently no established methods for predicting patient-specific responses to treatment prior to drug selection. In our studies, we have developed a novel protocol for efficient establishment of cell lines from primary human bladder tumors, which enables in vitro drug sensitivity assays using chemotherapeutic agents. Methods: Using an Institutional Review Board-approved tissue acquisition protocol, informed consent was obtained prior to specimen acquisition for all samples. Specimens were obtained during standard transurethral resection of papillary bladder tumors. Following generation of a single-cell suspension, epithelial cells were isolated using immunomagnetic cell separation and used for establishment of adherent cell cultures using a novel protocol. We performed immunohistochemistry on parental tissue as well as cultured cells to confirm that the urothelial cancer phenotype was maintained during serial passaging. For sensitivity assays, cultured cells were passaged and treated with chemotherapeutic agents, followed by assessment of cell viability using MTT assays. Results: To date, seven specimens from patients with papillary urothelial carcinoma have been obtained, resulting in the establishment of six independent adherent cell lines. All established lines have been serially passaged (as high as P10) without significant decline in growth rate, and maintained expression of CK7, uroplakin III, p53, and Ki67 in patterns similar to parental tissue. Cells from line #7 were treated with mitomycin C, docetaxel, gemcitabine, and rapamycin at three different equivalent concentrations, resulting in a unique sensitivity profile that was reproduced in a replicate experiment performed at a subsequent passage. Conclusions: We have established a novel protocol for culture and rapid expansion of primary cells from human bladder tumors for assays of drug response. Ultimately, we envision that this approach will provide a basis for the design of patient-specific therapeutic regimens for bladder cancer. Citation Format: LaMont Barlow, Chee Wai Chua, Ming Lei, G. Joel DeCastro, Ketan Badani, Mitchell Benson, James McKiernan, Michael Shen. An individualized approach to bladder cancer treatment using patient-derived cell lines to predict response to chemotherapeutic agents. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3729. doi:10.1158/1538-7445.AM2014-3729

Approximately 50% of prostate tumors harbor the TMPRSS2-ERG translocation, but precisely how this mutation contributes to prostate cancer initiation and progression is unclear. In an effort to identify other causative mutations involved in prostate cancer, we previously performed an integrated genomic analysis of < 200 primary and advanced human prostate cancer samples and cell lines, including assessment of genomic copy-number alterations, mRNA expression, and focused exon sequencing. We identified a recurrent genomic loss, a focal region of chromosome 3p14.1-p13, which was significantly associated with TMPRSS2-ERG translocation. Comparison of copy-number and mRNA expression data implicated at least three genes in this region (FOXP1, RYBP, and SHQ1) as potential cooperative tumor suppressors, which may function in concert with TMPRSS2-ERG translocation. In addition to the genomic loss of SHQ1, we identified point mutations in both SHQ1 and its interacting partner DKC1/dyskerin in primary prostate tumors, leading us to focus on this SHQ1-dyskerin pathway as a potential tumor-suppressive mechanism. SHQ1 is a critical assembly factor for H/ACA-class snoRNA-containing snoRNPs (small nucleolar ribonucleoproteins), of which the core component is the RNA-modifying enzyme DKC1/dyskerin. Downstream targets of dyskerin-containing snoRNPs include the ribosome, splicesome, and telomerase RNPs. DKC1/dyskerin is mutated in the human syndrome dyskeratosis congenita (DC), a disease also caused by mutations in the telomerase complex, which results in bone marrow failure and increased incidence of various neoplasias. We found that, in both human prostate cancer cell lines and mouse fibroblasts in vitro, knockdown of SHQ1 led to increased growth and partial transformation, as evidenced by loss of anchorage-dependence. Additionally, loss of either SHQ1 or dyskerin in vitro led to a global impairment of snoRNA levels, confirming that snoRNA maturation is a major downstream target of the SHQ1-dyskerin pathway in prostate cancer cells. Strikingly, in a mouse model of prostate regeneration by sub-renal capsule implantation, SHQ1-loss in conjunction with ERG expression, but not SHQ1-loss alone, led to development of prostate intraepithelial neoplasia and a low incidence of invasive cancer. Finally, in an in vitro interaction assay, prostate cancer-derived mutations in either SHQ1 or dyskerin impaired their association, to a degree similar to that seen with mutations in dyskerin found in DC. These data, along with the identification of point mutations in both SHQ1 and DKC1/dyskerin in other human cancers, strongly implicate SHQ1 as a novel prostate cancer tumor suppressor gene, potentially acting via disruption of snoRNA maturation. This abstract is also presented as Poster C60. Citation Format: Phillip J. Iaquinta, Chee Wai Chua, Rosario Machado-Pinilla, Haley Hieronymus, John Wongvipat, U. Thomas Meier, Michael Shen, Charles L. Sawyers. The snoRNP assembly factor SHQ1 is a novel prostate cancer tumor suppressor gene [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr PR3.

Our laboratories have been investigating novel mechanisms of cancer progression, as well as new therapeutic approaches for early intervention and treatment of advanced disease by integrating analyses of human clinical data with molecular and functional studies of genetically engineered mutant (GEM) mice. In our analyses of prostate cancer, we have generated GEM mice that recapitulate the entire spectrum of disease progression from preinvasive lesions, termed prostate intraepithelial neoplasia (PIN), to castration-resistant and metastatic disease, which are the lethal forms of the disease. These GEM models are based on perturbation of the Akt/mTOR and MAP kinase signaling pathways, which are often co-activated in human prostate cancer and which function synergistically to promote castration-resistant metastatic prostate cancer. We have found that combinatorial inhibition of these signaling pathways in GEM mice that display castration-resistant metastatic prostate cancer results in abrogation of primary tumors, improved survival and reduced metastasis. Using computational approaches for comparative analyses of large-scale gene expression profiling data for mouse and human prostate cancer, we have been assembling molecular networks, called interactomes, to elucidate conserved cancer pathways. We have been investigating the consequences of drugs perturbations on the transcriptional network with the ultimate goal of identifying new druggable targets. In summary, our comparative investigations of prostate cancer in humans and GEM mice have revealed promising new molecular targets and new therapuetic approaches for the treatment of human cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr SY22-01. doi:10.1158/1538-7445.AM2011-SY22-01