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Drug resistance in cancer is often linked to changes in tumor cell state or lineage, but the molecular mechanisms driving this plasticity remain unclear. Using murine organoid and genetically engineered mouse models, we investigated the causes of lineage plasticity in prostate cancer and its relationship to antiandrogen resistance. We found that plasticity initiates in an epithelial population defined by mixed luminal-basal phenotype and that it depends on increased Janus kinase (JAK) and fibroblast growth factor receptor (FGFR) activity. Organoid cultures from patients with castration-resistant disease harboring mixed-lineage cells reproduce the dependency observed in mice by up-regulating luminal gene expression upon JAK and FGFR inhibitor treatment. Single-cell analysis confirms the presence of mixed-lineage cells with increased JAK/STAT (signal transducer and activator of transcription) and FGFR signaling in a subset of patients with metastatic disease, with implications for stratifying patients for clinical trials.

To gain insight into how ERG translocations cause prostate cancer, we performed single cell transcriptional profiling of an autochthonous mouse model at an early stage of disease initiation. Despite broad expression of ERG in prostate epithelial cells, we observed enhanced proliferation primarily in an intermediate subpopulation with a basal identity based on transcriptomics but with luminal morphology and cytokeratin expression. Through a series of lineage tracing and primary prostate epithelial transplantation experiments, we find that tumor initiating activity resides in a subpopulation of basal cells that also express luminal genes such asTmprss2andNkx3.1, but not in the larger population of classical luminal cells. Upon ERG activation, these basal-luminal hybrid cells expand into a highly proliferative population of intermediate basal-luminal identity then transition to fully differentiated luminal cells that reflect the lineage of ERG-positive cancers. These findings help resolve a preexisting debate about the cell of origin of ERG-driven prostate cancer and narrow the focus for future mechanistic studies to a basal-luminal subpopulation of tumor initiating cells.

Supplemental Methods. Supplemental Table 1: ââ,¬â€¹Genomic Data Characterization by Center. Supplemental Table 2: ââ,¬â€¹Gene Panels Submitted by Each Center. Figure S1: Number of putative germline SNPs per sample, before and after uniform germline filtering. Figure S2ââ,¬â€¹. Distribution of total somatic mutation burden per sample stratified by sequencing panel. Figure S3: ââ,¬â€¹Log-scale comparison of mutation frequencies at hotspot sites between GENIE (data aggregated from all sequencing panels) and cancerhotspots.org (CHS) using a binomial test. Figure S4:ââ,¬â€¹ Comparison of mutation frequencies at hotspot sites in each GENIE sequencing panel with cancerhotspots.org (CHS) using a binomial test.

Table S4

Supplementary Figures and Legends

Supplementary Figure S1 - PDF file 1194K, Gamma-H2AX foci and Comet assay normalized of LNCaP plus or minus androgen normalized to time 0

Supplementary Table S2 - PDF file 250K, Defining AR-regulated DNA repair genes in LNCaP

To study genetic factors influencing the progression and therapeutic responses of advanced prostate cancer, we developed a fast and flexible system that introduces genetic alterations relevant to human disease directly into the prostate glands of mice using tissue electroporation. These electroporation-based genetically engineered mouse models (EPO-GEMM) recapitulate features of traditional germline models and, by modeling genetic factors linked to late-stage human disease, can produce tumors that are metastatic and castration-resistant. A subset of tumors with Trp53 alterations acquired spontaneous WNT pathway alterations, which are also associated with metastatic prostate cancer in humans. Using the EPO-GEMM approach and an orthogonal organoid-based model, we show that WNT pathway activation drives metastatic disease that is sensitive to pharmacologic WNT pathway inhibition. Thus, by leveraging EPO-GEMMs, we reveal a functional role for WNT signaling in driving prostate cancer metastasis and validate the WNT pathway as therapeutic target in metastatic prostate cancer.Significance:Our understanding of the factors driving metastatic prostate cancer is limited by the paucity of models of late-stage disease. Here, we develop EPO-GEMMs of prostate cancer and use them to identify and validate the WNT pathway as an actionable driver of aggressive metastatic disease.This article is highlighted in the In This Issue feature, p. 890

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AACR GENIE Data Guide

Supplementary Tables and Figures

mp3 file (37.5 MB). "Personalized Medicine: Late-breaking Science from AACR Journals," Saturday, March 31, 2012. Hosted by Lewis C. Cantley, Co-Editor-in-Chief of Cancer Discovery, this press conference held during the AACR Annual Meeting covers the following topics:Lack of Oxygen Influenced Tumor Behavior and Patient Outcome in Intermediate-risk Prostate CancerNovel Technology Allows for Noninvasive Imaging of Prostate Cancer (Ulmert et al., April issue of Cancer Discovery) EGFR Mutation Unique to Glioblastoma May Explain Lack of Response to Traditional EGFR Inhibitors

AKT inhibitors have promising activity in AKT1E17K-mutant estrogen receptor (ER)–positive metastatic breast cancer, but the natural history of this rare genomic subtype remains unknown. Utilizing AACR Project GENIE, an international clinicogenomic data-sharing consortium, we conducted a comparative analysis of clinical outcomes of patients with matched AKT1E17K-mutant (n = 153) and AKT1–wild-type (n = 302) metastatic breast cancer. AKT1-mutant cases had similar adjusted overall survival (OS) compared with AKT1–wild-type controls (median OS, 24.1 vs. 29.9, respectively; P = 0.98). AKT1-mutant cases enjoyed longer durations on mTOR inhibitor therapy, an observation previously unrecognized in pivotal clinical trials due to the rarity of this alteration. Other baseline clinicopathologic features, as well as durations on other classes of therapy, were broadly similar. In summary, we demonstrate the feasibility of using a novel and publicly accessible clincogenomic registry to define outcomes in a rare genomically defined cancer subtype, an approach with broad applicability to precision oncology.Significance:We delineate the natural history of a rare genomically distinct cancer, AKT1E17K-mutant ER-positive breast cancer, using a publicly accessible registry of real-world patient data, thereby illustrating the potential to inform drug registration through synthetic control data.See related commentary by Castellanos and Baxi, p. 490.

The supplementary figures provide additional data demonstrating that ERG represses RTK-PI3K signaling through direct transcriptional repression of IRS2 across multiple model systems. Additional data is provided showing the biologic role of ERG and PI3K signaling in promoting cell proliferation and migration.

Purpose: Advanced non–small-cell lung cancer (NSCLC) patients harboring epidermal growth factor receptor (EGFR) mutations (deletion in exon 19 or L858R) show an impressive progression-free survival of 14 months when treated with erlotinib. However, the presence of EGFR mutations can only imperfectly predict outcome. We hypothesized that progression-free survival could be influenced both by the pretreatment EGFR T790M mutation and by components of DNA repair pathways.Experimental Design: We assessed the T790M mutation in pretreatment diagnostic specimens from 129 erlotinib-treated advanced NSCLC patients with EGFR mutations. The expression of eight genes and two proteins involved in DNA repair and four receptor tyrosine kinases was also examined.Results: The EGFR T790M mutation was observed in 45 of 129 patients (35%). Progression-free survival was 12 months in patients with and 18 months in patients without the T790M mutation (P = 0.05). Progression-free survival was 27 months in patients with low BRCA1 mRNA levels, 18 months in those with intermediate levels, and 10 months in those with high levels (P = 0.02). In the multivariate analysis, the presence of the T790M mutation (HR, 4.35; P = 0.001), intermediate BRCA1 levels (HR, 8.19; P < 0.0001), and high BRCA1 levels (HR, 8.46; P < 0.0001) emerged as markers of shorter progression-free survival.Conclusions: Low BRCA1 levels neutralized the negative effect of the T790M mutation and were associated with longer progression-free survival to erlotinib. We advocate baseline assessment of the T790M mutation and BRCA1 expression to predict outcome and provide alternative individualized treatment to patients based on T790M mutations and BRCA1 expression. Clin Cancer Res; 17(5); 1–9. ©2011 AACR.

Supplementary Methods Additional methodology of experimental methods.

Supplementary Figure 2. A) In vitro comparison of LNCaP and LNCaP-AR and DNA damage post-radiotherapy. B) In vivo correlation of baseline AR-output using bioluminescence to tumor progression.

Genomic rearrangements leading to the aberrant expression of ERG are the most common early events in prostate cancer and are significantly enriched for the concomitant loss of PTEN. Genetically engineered mouse models reveal that ERG overexpression alone is not sufficient to induce tumorigenesis, but combined loss of PTEN results in an aggressive invasive phenotype. Here, we show that oncogenic ERG repressed PI3K signaling through direct transcriptional suppression of IRS2, leading to reduced RTK levels and activity. In accordance with this finding, ERG-positive human prostate cancers had a repressed AKT gene signature and transcriptional downregulation of IRS2. Although overexpression of IRS2 activated PI3K signaling, promoting cell migration in a PI3K-dependent manner, this did not fully recapitulate the phenotype seen with loss of PTEN as PI3K signaling is not as robust as observed in the setting of loss of PTEN. Importantly, deletions of the PTEN locus, which promotes active PI3K signaling, were among the most significant copy-number alterations that co-occurred with ERG genomic rearrangements. This work provides insight on how initiating oncogenic events may directly influence the selection of secondary concomitant alterations to promote oncogenic signaling during tumor evolution.Significance:This work provides insight on how initiating oncogenic events may directly influence the selection of secondary concomitant alterations to promote tumorigenesis.

Distribution of colorectal cancer cases according to SMAD4 expression status and immune response to the tumor in validation cohort

Supplementary Figure legends for Supplementary Figure 1 and 2

Kaplan-Meier graphs in the validation cohort with number of subjects at risk showing colorectal cancer-specific survival according to SMAD4 status.

Supplementary Material: Case Studies of Recent Therapeutic Breakthroughs PDF file 98K, This describes four recent breakthroughs (3 in oncology and 1 in cystic fibrosis)

Clinical trials have established the benefit of androgen deprivation therapy (ADT) combined with radiotherapy in prostate cancer. ADT sensitizes prostate cancer to radiotherapy-induced death at least in part through inhibition of DNA repair machinery, but for unknown reasons, adjuvant ADT provides further survival benefits. Here, we show that androgen receptor (AR) expression and activity are durably upregulated following radiotherapy in multiple human prostate cancer models in vitro and in vivo. Moreover, the degree of AR upregulation correlates with survival in vitro and time to tumor progression in animal models. We also provide evidence of AR pathway upregulation, measured by a rise in serum levels of AR-regulated hK2 protein, in nearly 20% of patients after radiotherapy. Furthermore, these men were three-fold more likely to experience subsequent biochemical failure. Collectively, these data demonstrate that radiotherapy can upregulate AR signaling after therapy to an extent that negatively affects disease progression and/or survival. Cancer Res; 75(22); 4688–96. ©2015 AACR.

Kaplan-Meier graphs with number of subjects at risk comparing recurrence-free survival (RFS) by (a) TIL status, or (b) PLA status, with arrows indicating patients whose tumors were also SMAD4 loss.

Supplementary Methods, Results and Figure Legends from Antibody-Based Profiling of the Phosphoinositide 3-Kinase Pathway in Clinical Prostate Cancer

Clinical, pathological, and molecular characteristics of colorectal cancer cases according to SMAD4 expression status in validation cohort

Purpose: As kinase inhibitors transition from the laboratory to patients, it is imperative to develop biomarkers that can be used in the clinic. The primary objectives are to identify patients most likely to benefit from molecularly targeted therapies and to document modulation of the drug target. Constitutive activation of the phosphoinositide 3-kinase (PI3K) pathway and its downstream effectors, as a result of PTEN loss or by other mechanisms, occurs in a high proportion of prostate cancers, making it an ideal template for the design of clinical trials involving PI3K pathway inhibitors. Prostate cancers also present unique organ-specific challenges, in that tumors are heterogeneous and diagnostic tissue is extremely limited.Experimental Design: Working within these limitations, we have developed a set of immunohistochemical assays that define activation of the PI3K pathway in clinical samples.Results and Conclusions: Using both univariate and multivariate analyses, we show that loss of PTEN is highly correlated with the activation of AKT, and this, in turn, is associated with the phosphorylation of S6, one of its main effectors. These three antibodies are potentially able to define a molecular signature of PTEN loss and/or AKT pathway activation in prostate cancer.

Ordinal logistic regression analyses to assess the association of SMAD4 expression status with lymphocytic reaction patterns in validation cohort

Purpose:SMAD4 has shown promise in identifying patients with colorectal cancer at high risk of recurrence or death.Experimental Design: A discovery cohort and independent validation cohort were classified by SMAD4 status. SMAD4 status and immune infiltrate measurements were tested for association with recurrence-free survival (RFS). Patient-derived xenografts from SMAD4-deficient and SMAD4-retained tumors were used to examine chemoresistance.Results:The discovery cohort consisted of 364 patients with stage I–IV colorectal cancer. Median age at diagnosis was 53 years. The cohort consisted of 61% left-sided tumors and 62% stage II/III patients. Median follow-up was 5.4 years (interquartile range, 2.3–8.2). SMAD4 loss, noted in 13% of tumors, was associated with higher tumor and nodal stage, adjuvant therapy use, fewer tumor-infiltrating lymphocytes (TIL), and lower peritumoral lymphocyte aggregate (PLA) scores (all P < 0.04). SMAD4 loss was associated with worse RFS (P = 0.02). When stratified by SMAD4 and immune infiltrate status, patients with SMAD4 loss and low TIL or PLA had worse RFS (P = 0.002 and P = 0.006, respectively). Among patients receiving 5-fluorouracil (5-FU)-based systemic chemotherapy, those with SMAD4 loss had a median RFS of 3.8 years compared with 13 years for patients with SMAD4 retained. In xenografted mice, the SMAD4-lost tumors displayed resistance to 5-FU. An independent cohort replicated our findings, in particular, the association of SMAD4 loss with decreased immune infiltrate, as well as worse disease-specific survival.Conclusions:Our data show SMAD4 loss correlates with worse clinical outcome, resistance to chemotherapy, and decreased immune infiltrate, supporting its use as a prognostic marker in patients with colorectal cancer.

Supplementary Figures from Antibody-Based Profiling of the Phosphoinositide 3-Kinase Pathway in Clinical Prostate Cancer

Kaplan-Meier graph with number of subjects at risk comparing (a) SMAD4, (b) TIL, and (c) PLA status, separately, as well as (d) SMAD4 and TIL, and (e) SMAD4 and PLA, together, with recurrence-free survival (RFS) among only the MMR-proficient patients.

Supplementary Figures S1-S3; Supplementary Tables S1-S14.

Related Article from Developing Standards for Breakthrough Therapy Designation in Oncology

List of most significantly differentially expressed genes following ERG knock-out.

Supplementary Figures 1 - 3

Supplementary Figure 1. In vitro experiments demonstrating increase AR protein level post-radiotherapy; A) Nuclear AR staining, B) 18F-FDHT uptake. C) Isodose distribution of multibeam animal EBRT.

In July 2012, Congress passed the Food and Drug Administration Safety and Innovation Act (FDASIA). The Advancing Breakthrough Therapies for Patients Act was incorporated into a Title of FDASIA to expedite clinical development of new, potential “breakthrough” drugs or treatments that show dramatic responses in early-phase studies. Using this regulatory pathway, once a promising new drug candidate is designated as a “Breakthrough Therapy”, the U.S. Food and Drug Administration (FDA) and sponsor would collaborate to determine the best path forward to abbreviate the traditional three-phase approach to drug development. The breakthrough legislation requires that an FDA guidance be drafted that details specific requirements of the bill to aid FDA in implementing requirements of the Act. In this article, we have proposed criteria to define a product as a Breakthrough Therapy, and discussed critical components of the development process that would require flexibility in order to enable expedited development of a Breakthrough Therapy. Clin Cancer Res; 19(16); 4297–304. ©2013 AACR.

Transcriptional activity of the androgen receptor (AR) is crucial for growth and survival of prostate cancer even upon development of resistance to androgen ablation and antiandrogen therapies. Therefore, novel therapies that can suppress AR transcriptional activity when conventional hormone therapies fail are needed. Here, we show that histone deacetylase (HDAC) inhibitors, including SAHA (vorinostat) and LBH589, which are currently being tested in clinic, could be such a therapy. HDAC inhibitors block the AR-mediated transcriptional activation of many genes, including the TMPRSS2 gene involved in fusion with ETS family members in a majority of prostate cancers. Genetic knockdown of either HDAC1 or HDAC3 can also suppress expression of AR-regulated genes, recapitulating the effect of HDAC inhibitor treatment. Whereas HDAC inhibitor treatment can lower androgen receptor protein levels in prostate cancer cells, we show that independent of AR protein levels, HDAC inhibitors block AR activity through inhibiting the assembly of coactivator/RNA polymerase II complex after AR binds to the enhancers of target genes. Failed complex assembly is associated with a phase shift in the cyclical wave of AR recruitment that typically occurs in response to ligand treatment. HDAC inhibitors retain the ability to block AR activity in castration-resistant prostate cancer models and, therefore, merit clinical investigation in this setting. The HDAC-regulated AR target genes defined here can serve as biomarkers to ensure sufficient levels of HDAC inhibition. [Cancer Res 2009;69(3):958–66]

Supplementary Table 1, Figures 1-4 from Murine Cell Lines Derived from Pten Null Prostate Cancer Show the Critical Role of PTEN in Hormone Refractory Prostate Cancer Development

Supplementary Figure 3 from Histone Deacetylases Are Required for Androgen Receptor Function in Hormone-Sensitive and Castrate-Resistant Prostate Cancer

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