Dickkopf-1 Can Lead to Immune Evasion in Metastatic Castration-Resistant Prostate Cancer

Abstract

Purpose: Metastatic castration-resistant prostate cancer (mCRPC) with low androgen receptor (AR) and without neuroendocrine signaling, termed double-negative prostate cancer (DNPC), is increasingly prevalent in patients treated with AR signaling inhibitors and is in need of new biomarkers and therapeutic targets.

Methods: Candidate genes enriched in DNPC were determined using differential gene expression analysis of discovery and validation cohorts of mCRPC biopsies. Laboratory studies were carried out in human mCRPC organoid cultures, prostate cancer (PCa) cell lines, and mouse xenograft models. Epigenetic studies were carried out in a rapid autopsy cohort.

Results: Dickkopf-1 (DKK1) expression is increased in DNPC relative to prostate-specific antigen (PSA)-expressing mCRPC in the Stand Up to Cancer/Prostate Cancer Foundation discovery cohort (11.2 v 0.28 reads per kilobase per million mapped reads; q < 0.05; n = 117) and in the University of Washington/Fred Hutchinson Cancer Research Center cohort (9.2 v 0.99 fragments per kilobase of transcript per million mapped reads; P < .0001). DKK1 expression can be regulated by activated Wnt signaling in vitro and correlates with activating canonical Wnt signaling mutations and low PSA mRNA in mCRPC biopsies (P < .05). DKK1 hypomethylation was associated with increased DKK1 mRNA expression (Pearson r = -0.66; P < .0001) in a rapid autopsy cohort (n = 7). DKK1-high mCRPC biopsies are infiltrated with significantly higher numbers of quiescent natural killer (NK) cells (P < .005) and lower numbers of activated NK cells (P < .0005). Growth inhibition of the human PCa model PC3 by the anti-DKK1 monoclonal antibody DKN-01 depends on the presence of NK cells in a severe combined immunodeficient xenograft mouse model.

Conclusion: These results support DKK1 as a contributor to the immunosuppressive tumor microenvironment of DNPC. These data have provided the rationale for a clinical trial targeting DKK1 in mCRPC (ClinicalTrials.gov identifier: NCT03837353).

Conflict of interest statement

The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated unless otherwise noted. Relationships are self-held unless noted. I = Immediate Family Member, Inst = My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO's conflict of interest policy, please refer to www.asco.org/rwc or ascopubs.org/po/author-center. Open Payments is a public database containing information reported by companies about payments made to US-licensed physicians (Open Payments). David R. WiseHonoraria: OncLive Consulting or Advisory Role: Best Doctors, Leap Therapeutics, GLG, Guidepoint Global, Pfizer, Foundation Medicine, Silverlight, Alphasights Travel, Accommodations, Expenses: PfizerJeffrey A. SchneiderStock and Other Ownership Interests: Abbott Laboratories, AstraZeneca, AbbVie, CVS Health, MerckBrigit McLaughlinEmployment: Bristol Myers Squibb Stock and Other Ownership Interests: Bristol Myers SquibbRyan BrennanEmployment: Flatiron Health Stock and Other Ownership Interests: RocheKatie L. ThorenResearch Funding: Sebia (Inst), The Binding Site (Inst)Wassim AbidaHonoraria: CARET Consulting or Advisory Role: Clovis Oncology, Janssen Pharmaceuticals, MORE Health, ORIC Pharmaceuticals, Daiichi Sankyo Research Funding: AstraZeneca (Inst), Zenith Epigenetics (Inst), Clovis Oncology (Inst), GlaxoSmithKline (Inst) Travel, Accommodations, Expenses: GlaxoSmithKline, Clovis Oncology, ORIC PharmaceuticalsAngelo M. De MarzoConsulting or Advisory Role: Cepheid Research Funding: Janssen Research & DevelopmentSrinivasan YegnasubramanianStock and Other Ownership Interests: Brahm Astra Therapeutics, Digital Harmonic Consulting or Advisory Role: Cepheid Research Funding: Cepheid (Inst), Celgene (Inst), Bristol Myers Squibb (Inst), Janssen Pharmaceuticals (Inst) Patents, Royalties, Other Intellectual Property: Patent: “COMPARE-MS: A novel technique for rapid, sensitive, and accurate detection of DNA methylation”; Patent: “Agents for reversing epigenetic silencing of genes”; Patent: “DNA methylation markers for metastatic prostate cancer”; Patent pending: “Induction of synthetic lethality with epigenetic therapy”; Patent: “Bisulfite-converted duplexes for the strand-specific detection and quantification of rare mutations”Michael HaasEmployment: Leap Therapeutics Stock and Other Ownership Interests: Leap Therapeutics Research Funding: Leap TherapeuticsMichael H. KageyEmployment: Leap Therapeutics, Biogen (I) Stock and Other Ownership Interests: Syros Pharmaceuticals Consulting or Advisory Role: Medical Research Commercialisation Fund Patents, Royalties, Other Intellectual Property: Patents pending and granted for discovery-based work at Leap Therapeutics (current employer), Tensha Therapeutics (acquired by Roche), and the Whitehead Institute (licensed to Syros Pharmaceuticals)Walter NewmanEmployment: Leap Therapeutics Leadership: Leap Therapeutics Stock and Other Ownership Interests: Leap Therapeutics Patents, Royalties, Other Intellectual Property: May be a co-patentee on Leap Therapeutics intellectual propertyCynthia A. SirardEmployment: Leap Therapeutics Leadership: Leap Therapeutics Stock and Other Ownership Interests: Leap Therapeutics Patents, Royalties, Other Intellectual Property: Pending patent application No. 62/260,959 Travel, Accommodations, Expenses: Leap TherapeuticsMichael J. MorrisConsulting or Advisory Role: Astellas Pharma, Bayer AG, Endocyte, Advanced Accelerator Applications, Blue Earth Diagnostics, Tokai Pharmaceuticals, Tolmar Pharmaceuticals, ORIC Pharmaceuticals Research Funding: Bayer AG (Inst), Sanofi (Inst), Endocyte (Inst), Progenics (Inst), Corcept Therapeutics (Inst), Roche (Inst), Genentech (Inst) Travel, Accommodations, Expenses: Bayer AG, EndocyteYu ChenStock and Other Ownership Interests: ORIC Pharmaceuticals Honoraria: Merck Patents, Royalties, Other Intellectual Property: Invention related to glucocorticoid inhibitors for treatment of prostate cancer (SK2013-045)Steven M. LarsonStock and Other Ownership Interests: Ymabs Therapeutics, Elucida Consulting or Advisory Role: Prescient Therapeutics Research Funding: Wilex, Regeneron Pharmaceuticals, Ymabs Therapeutics Patents, Royalties, Other Intellectual Property: Synthesis and utilization of 17-methyl and 17-cyclo-propylmethyl-3, 14-di-hydroxy-4, 5-epoxy 6 b-fluoromorphines (foxy and cyclofoxy) as (18F)-labeled opiate ligands for positron emission transaxial tomography (Rice KC, Pert CB, Burke TR Jr, Larson SM, Eckelman WC, Channing MA; October 4, 1988; US Patent No. 4,775,759); Antigen-specific composition and in vivo methods for detecting and localizing an antigenic site and for radiotherapy (Larson SM, Finn R, Carrasquillo JA, Reynolds JC, Neumann RD, Graham MC, Pentlow KS; February 9, 1993; US Patent No. 5,185,142); Non-invasive imaging and quantification of specific antigen and uses thereof (Smith-Jones P, Larson SM; January 6, 2003; Provisional Patent Application Registration No. 28,325); Single chain Fv polynucleotide or peptide construct of anti-ganglioside GD2 antibodies, cells expressing same and related methods (Cheung NK, Larson SM, Guo HF, Rivlin K, Sadelain M; US Patent No. 6,451,995); Small-molecule HSP90 inhibitors, chiosis, gabriela (Huazhong H, Llauger-Bufi L, Joungnam K, Larson S, Smith-Jones P; November 16, 2010, US Patent No. 7,834,181); Multi-specific antibodies with affinity for human A33 antigen and DOTA metal complex and uses thereof (Cheal S, Hong X, Larson SM, Cheung NK; February 9, 2015; US Patent No. 62,113,988); Systems and methods for determining optimum patient-specific antibody dose for tumor targeting (Zanzonico P, Cheal SM, Larson SM, Osborne J, Fung Edward K; May 22, 2015, US Patent No. 62,165699)Peter S. NelsonConsulting or Advisory Role: Janssen Oncology, Astellas Pharma, Roche, Genentech, Bristol Myers Squibb Research Funding: Genomic Health (Inst), Expert Testimony: Veneble-Fitzpatrick Law Firm Travel, Accommodations, Expenses: Janssen OncologyHoward I. ScherLeadership: Asterias Biotherapeutics Stock and Other Ownership Interests: Asterias Biotherapeutics Honoraria: Research to Practice Consulting or Advisory Role: Janssen Biotech, Amgen, Janssen Research & Development, Menarini Silicon Biosystems, WIRB-Copernicus Group, ESSA, Sanofi, Ambry Genetics, Konica Minolta, Pfizer, Bayer AG Research Funding: Janssen Pharmaceuticals (Inst), Illumina (Inst), Epic Sciences (Inst), Menarini Silicon Biosystems (Inst), Thermo Fisher Scientific (Inst) Travel, Accommodations, Expenses: Asterias Biotherapeutics, Menarini Silicon Biosystems, Amgen, WIRB-Copernicus Group, Konica Minolta, ESSA, Prostate Cancer Foundation, Sanofi, Bayer AG, Phosplatin TherapeuticsCharles L. SawyersLeadership: Novartis Stock and Other Ownership Interests: Novartis, Agios, Blueprint Medicines, BeiGene, ORIC Pharmaceuticals, Foghorn, PMV Pharma, KSQ Therapeutics, Petra Pharma Consulting or Advisory Role: Novartis, Blueprint Medicines, Agios, BeiGene, ORIC Pharmaceuticals, Foghorn, PMV Pharma, KSQ Therapeutics, Petra Pharma, Arsenal, Housey Pharmaceuticals Patents, Royalties, Other Intellectual Property: Xtandi, Apalutamide No other potential conflicts of interest were reported.

© 2020 by American Society of Clinical Oncology.

Figures

FIG 1.

Elevated Dickkopf-1 (DKK1) expression in lethal non-neuroendocrine low–androgen receptor (ARlow) and low–prostate-specific antigen (PSAlow) metastatic castration-resistant prostate cancer (mCRPC). (A) AR and PSA mRNA levels in 117 mCRPC biopsies. (B) Kaplan-Meier survival analysis of patients with ARlowPSAlow biopsies (n = 5) compared with the remainder of the cohort (n = 60). (C) Gene set enrichment analysis of differentially expressed ARlowPSAlow and ARhighPSAhigh using references gene sets. (D) DKK1 mRNA levels in ARlowPSAlow (n = 10) and AR PSAhigh (n = 10). (E) DKK1 mRNA levels in mCRPC biopsies. (F) DKK1 mRNA levels derived from publicly available RNA sequencing data from a Weill-Cornell Medical College mCRPC cohort. Adeno, adenocarcinoma; Adeno w/ NE diff, adenocarcinoma with neuroendocrine differentiation; EMT, epithelial-mesenchymal transition; FDR, false discovery rate; FPKM, fragments per kilobase of transcript per million mapped reads; NE, neuroendocrine; NES, normalized enrichment score; NS, not significant; RPKM, reads per kilobase per million mapped reads.

FIG 2.

Circulating Dickkopf-1 (DKK1) levels are elevated in patients with metastatic castration-resistant prostate cancer (mCRPC) and inversely correlate with tumoral [18F]dihydrotestosterone (FDHT) uptake. (A) Serum prostate-specific antigen (PSA) and (B) serum DKK1 quantitated in patients with mCRPC who underwent FDHT positron emission tomography (PET) imaging. Patients are grouped on the basis of total FDHT uptake of all metastatic lesions above (high) and below (low) the median for the cohort. (C) Plasma DKK1 protein quantitated in healthy control (HC) men and patients with mCRPC. (D) Plasma DKK1 protein quantitated in treatment-naive patients with metastatic castrate-sensitive prostate cancer and matched samples from onset of mCRPC.

FIG 3.

Dickkopf-1 (DKK1) is modulated by canonical Wnt signaling and hypomethylation in advanced prostate cancer. (A) Quantitative real-time polymerase chain reaction (RT-qPCR) of mRNA[AE1] from LNCaP-abl cells with either induced stabilized (St.; hot spot mutant [mut]) b-catenin or APC knockdown. (B) RT-qPCR from LNCaP-abl and PC3 cells treated with Wnt inhibitor cyclic 13 for 24 hours at 10 mM. (C) mRNA levels of DKK1 in the Stand Up to Cancer/Prostate Cancer Foundation cohort of tumor samples. Wntmut tumors defined as pathogenic mutations in either APC, CTNNB1, RNF43, ZNRF3, or RSPO2. (D) Integrated DNA methylation and mRNA expression analyses of 25 anatomically distinct metastatic deposits from seven patients with metastatic castration-resistant prostate cancer (mCRPC) procured by rapid autopsy revealed hypomethylation of the DKK1 locus with associated high expression of DKK1 mRNA in cases with low androgen receptor (ARlow) and low prostate-specific antigen (PSAlow) expression. Heat map codes: methylation: blue, dense methylation; white, no methylation; mRNA expression: red, high expression; white, low expression. Statistical differences for RT-qPCR were calculated by unpaired t tests. (*)P < .05, (**)P < .01, (***)P < .001, where each point represents an individual biologic replicate. LN, lymph node; wt, wild type. [AE1]In Figure 3 legend, please confirm that the abbreviation RT-qPCR is expanded correctly.

FIG 4.

Dickkopf-1 (DKK1) can contribute to immune evasion through modulation of natural killer (NK) cell activity. (A) CIBERSORT2 immune cell composition analysis of the Stand Up to Cancer/Prostate Cancer Foundation metastatic castration-resistant prostate cancer cohort (n = 212) was carried out. DKK1 low and high groupings represent the bottom and top quartiles of DKK1 mRNA expression. (B) and (C) Mean tumor volumes and SEM are plotted. P values from two-way repeated-measures analysis of variance with Sidak’s multiple comparisons. Representative data from one of two independent experiments are shown. SCID, severe combined immunodeficient.

FIG A1.

(A) Comparison of Dickkopf-1 (DKK1) mRNA levels by treatment status the Stand Up to Cancer/Prostate Cancer Foundation (SU2C/PCF) metastatic castration-resistant prostate cancer (mCRPC) cohort. (B) Table of available preclinical models for the study of DKK1 and androgen receptor (AR)–negative prostate cancer. (C) DKK1 protein levels in DU145 cell culture medium analyzed with enzyme-linked immunosorbent assay in cells expressing control (sgNT) and DKK1-targeted (sgDKK1) CRISPR guides. (D) and (E) The correlation of [18F]dihydrotestosterone (FDHT) positron emission tomography (PET) uptake serum prostate-specific antigen (PSA) and serum DKK1. (F) Serum levels of DKK1 quantitated in healthy control men and patients with mCRPC. (G) mRNA levels of AXIN2 in the SU2C/PCF cohort of tumor samples. Wnt mutant tumors defined as pathogenic mutations in either APC, CTNNB1, RNF43, ZNRF3, or RSPO2. (H) Comparison of DKK1 mRNA levels by DKK1 amplification status in the SU2C/PCF mCRPC cohort. (I) Comparison of AXIN2 mRNA levels by PSA level and Wnt mutation status in the SU2C/PCF mCRPC cohort. Abi, abiraterone; Enz, enzalutamide; FHCRC, Fred Hutchinson Cancer Research Center; mut, mutated; NS, not significant; RPKM, reads per kilobase per million mapped reads; wt, wild type.