Search

Ki67 was evaluated by a board-certified pathologist on TMAs.

Purpose:The local immune infiltrate's influence on tumor progression may be closely linked to tumor-intrinsic factors. The study aimed to investigate whether integrating immunologic and tumor-intrinsic factors can identify patients from a low-risk cohort who may be candidates for radiotherapy (RT) de-escalation.Experimental Design:The SweBCG91RT trial included 1,178 patients with stage I to IIA breast cancer, randomized to breast-conserving surgery with or without adjuvant RT, and followed for a median of 15.2 years. We trained two models designed to capture immunologic activity and immunomodulatory tumor-intrinsic qualities, respectively. We then analyzed if combining these two variables could further stratify tumors, allowing for identifying a subgroup where RT de-escalation is feasible, despite clinical indicators of a high risk of ipsilateral breast tumor recurrence (IBTR).Results:The prognostic effect of the immunologic model could be predicted by the tumor-intrinsic model (Pinteraction = 0.01). By integrating measurements of the immunologic- and tumor-intrinsic models, patients who benefited from an active immune infiltrate could be identified. These patients benefited from standard RT (HR, 0.28; 95% CI, 0.09–0.85; P = 0.025) and had a 5.4% 10-year incidence of IBTR after irradiation despite high-risk genomic indicators and a low frequency of systemic therapy. In contrast, high-risk tumors without an immune infiltrate had a high 10-year incidence of IBTR despite RT treatment (19.5%; 95% CI, 12.2–30.3).Conclusions:Integrating tumor-intrinsic and immunologic factors may identify immunogenic tumors in early-stage breast cancer populations dominated by ER-positive tumors. Patients who benefit from an activated immune infiltrate may be candidates for RT de-escalation.

PURPOSE Both the performance characteristics of prostate-specific membrane antigen positron emission tomography and insurance approval improves with increasing prostate-specific antigen (PSA) level causing some physicians to delay post-radical prostatectomy salvage radiation therapy (sRT) after PSA failure. Yet, it is unknown for men with at most one high-risk factor (ie, pT3/4 or prostatectomy [p] Gleason score 8-10) whether a PSA level exists above which initiating sRT is associated with increased all-cause mortality (ACM)-risk and was investigated. METHODS Using a multinational database of 25,551 patients with pT2-4N0 or NXM0 prostate cancer, multivariable Cox regression analysis evaluated whether an association with a significant increase in ACM-risk existed when sRT was delivered above a prespecified PSA level beginning at 0.10 ng/mL and in 0.05 increments up to 0.50 ng/mL versus at or below that level. The model was adjusted for age at and year of radical prostatectomy, established prostate cancer prognostic factors, institution, and the time-dependent use of androgen deprivation therapy. RESULTS After a median follow-up of 6.00 years, patients who received sRT at a PSA level >0.25 ng/mL had a significantly higher ACM-risk (AHR, 1.49; 95% CI, 1.11 to 2.00; P = .008) compared with men who received sRT when the PSA was ≤0.25 mg/mL. This elevated ACM-risk remained significant for all PSA cutpoints up to 0.50 ng/mL but was not significant at PSA cutpoint values below 0.25 ng/mL. CONCLUSION Among patients with at most one high-risk factor, initiating sRT above a PSA level of 0.25 ng/mL was associated with increased ACM-risk.

Background Androgen deprivation therapy (ADT) with radiotherapy can benefit patients with localized prostate cancer. However, ADT can negatively impact quality of life and there remain no validated predictive models to guide its use. Methods Digital pathology image and clinical data from pre-treatment prostate tissue from 5,727 patients enrolled on five phase III randomized trials treated with radiotherapy +/- ADT were used to develop and validate an artificial intelligence (AI)-derived predictive model to assess ADT benefit with the primary endpoint of distant metastasis. After the model was locked, validation was performed on NRG/RTOG 9408 (n = 1,594) that randomized men to radiotherapy +/- 4 months of ADT. Fine-Gray regression and restricted mean survival times were used to assess the interaction between treatment and predictive model and within predictive model positive and negative subgroup treatment effects. Results In the NRG/RTOG 9408 validation cohort (14.9 years of median follow-up), ADT significantly improved time to distant metastasis (subdistribution hazard ratio [sHR] = 0.64, 95%CI [0.45–0.90], p = 0.01). The predictive model-treatment interaction was significant (p-interaction = 0.01). In predictive model positive patients (n = 543, 34%), ADT significantly reduced the risk of distant metastasis compared to radiotherapy alone (sHR = 0.34, 95%CI [0.19–0.63], p

Supplementary Table S1 Demographics of the training cohortsSupplementary Table S2 Immunscore modelSupplementary Table S3 Proliferative Index ModelSupplementary Table S4 Immunescore:Proliferative Index

Protocol for original SweBCG91RT trial

Large-scale sequencing efforts of thousands of tumor samples have been undertaken to understand the mutational landscape of the coding genome. However, the vast majority of germline and somatic variants occur within non-coding portions of the genome. These genomic regions do not directly encode for specific proteins, but can play key roles in cancer progression, for example by driving aberrant gene expression control. Here, we designed computational models to identify recurrently mutated non-coding regulatory regions that drive tumor progression. Application of this approach to whole-genome sequencing (WGS) data from a large cohort of metastatic castration-resistant prostate cancer (mCRPC) revealed a large set of recurrently mutated regions. We used (i)in silicaprioritization of functional non-coding mutations, (ii) massively parallel reporter assays, and (iii)in vivaCRISPR-interference (CRISPRi) screens in xenografted mice to systematically identify and validate driver regulatory regions that drive mCRPC. We discovered that one of these enhancer regions, GH22I030351, acts on a bidirectional promoter to simultaneously modulate expression of U2-associated splicing factor SF3A1 and chromosomal protein CCDC157. We found that both SF3A1 and CCDC157 are promoters of tumor growth in xenograft models of prostate cancer. We also nominated a number of transcription factors, including SOX6, to be responsible for higher expression of SF3A1 and CCDC157. Taken together, we have described and validated an integrative computational and experimental framework that enables systematic identification of non-coding regulatory regions that drive human cancers.Statement of SignificanceWe developed an integrated computational and experimental platform to identify and characterize non-coding driver regulatory regions in metastatic prostate cancer patient data. One found enhancer region, GH22I030351, was shown to act on a bidirectional promoter that simultaneously regulates previously uncharacterized genes SF3A1 and CCDC157 in a tumor-promoting manner.

Supplementary Figures

Metastases from primary prostate cancers to rare locations, such as the brain, are becoming more common due to longer life expectancy resulting from improved treatments. Epigenetic dysregulation is a feature of primary prostate cancer, and distinct DNA methylation profiles have been shown to be associated with the mutually exclusive SPOP-mutant or TMPRSS2-ERG fusion genetic backgrounds. Using a cohort of prostate cancer brain metastases (PCBM) from 42 patients, with matched primary tumors for 17 patients, we carried out a DNA methylation analysis to examine the epigenetic distinction between primary prostate cancer and PCBM, the association between epigenetic alterations and mutational background, and particular epigenetic alterations that may be associated with PCBM. Multiregion sampling of PCBM revealed epigenetic stability within metastases. Aberrant methylation in PCBM was associated with mutational background and PRC2 complex activity, an effect that is particularly pronounced in SPOP-mutant PCBM. While PCBM displayed a CpG island hypermethylator phenotype, hypomethylation at the promoters of genes involved in neuroactive ligand–receptor interaction and cell adhesion molecules such as GABRB3, CLDN8, and CLDN4 was also observed, suggesting that cells from primary tumors may require specific reprogramming to form brain metastasis. This study revealed the DNA methylation landscapes of PCBM and the potential mechanisms and effects of PCBM-associated aberrant DNA methylation.Significance:DNA methylation analysis reveals the molecular characteristics of PCBM and may serve as a starting point for efforts to identify and target susceptibilities of these rare metastases.

Metastases from primary prostate cancers to rare locations, such as the brain, are becoming more common due to longer life expectancy resulting from improved treatments. Epigenetic dysregulation is a feature of primary prostate cancer, and distinct DNA methylation profiles have been shown to be associated with the mutually exclusive SPOP-mutant or TMPRSS2-ERG fusion genetic backgrounds. Using a cohort of prostate cancer brain metastases (PCBM) from 42 patients, with matched primary tumors for 17 patients, we carried out a DNA methylation analysis to examine the epigenetic distinction between primary prostate cancer and PCBM, the association between epigenetic alterations and mutational background, and particular epigenetic alterations that may be associated with PCBM. Multiregion sampling of PCBM revealed epigenetic stability within metastases. Aberrant methylation in PCBM was associated with mutational background and PRC2 complex activity, an effect that is particularly pronounced in SPOP-mutant PCBM. While PCBM displayed a CpG island hypermethylator phenotype, hypomethylation at the promoters of genes involved in neuroactive ligand–receptor interaction and cell adhesion molecules such as GABRB3, CLDN8, and CLDN4 was also observed, suggesting that cells from primary tumors may require specific reprogramming to form brain metastasis. This study revealed the DNA methylation landscapes of PCBM and the potential mechanisms and effects of PCBM-associated aberrant DNA methylation. Significance: DNA methylation analysis reveals the molecular characteristics of PCBM and may serve as a starting point for efforts to identify and target susceptibilities of these rare metastases.

Supplementary Figure from Drug-Induced Epigenomic Plasticity Reprograms Circadian Rhythm Regulation to Drive Prostate Cancer toward Androgen Independence

In prostate cancer, androgen receptor (AR)–targeting agents are very effective in various disease stages. However, therapy resistance inevitably occurs, and little is known about how tumor cells adapt to bypass AR suppression. Here, we performed integrative multiomics analyses on tissues isolated before and after 3 months of AR-targeting enzalutamide monotherapy from patients with high-risk prostate cancer enrolled in a neoadjuvant clinical trial. Transcriptomic analyses demonstrated that AR inhibition drove tumors toward a neuroendocrine-like disease state. Additionally, epigenomic profiling revealed massive enzalutamide-induced reprogramming of pioneer factor FOXA1 from inactive chromatin sites toward active cis-regulatory elements that dictate prosurvival signals. Notably, treatment-induced FOXA1 sites were enriched for the circadian clock component ARNTL. Posttreatment ARNTL levels were associated with patients’ clinical outcomes, and ARNTL knockout strongly decreased prostate cancer cell growth. Our data highlight a remarkable cistromic plasticity of FOXA1 following AR-targeted therapy and revealed an acquired dependency on the circadian regulator ARNTL, a novel candidate therapeutic target.Significance:Understanding how prostate cancers adapt to AR-targeted interventions is critical for identifying novel drug targets to improve the clinical management of treatment-resistant disease. Our study revealed an enzalutamide-induced epigenomic plasticity toward prosurvival signaling and uncovered the circadian regulator ARNTL as an acquired vulnerability after AR inhibition, presenting a novel lead for therapeutic development.See related commentary by Zhang et al., p. 2017.This article is highlighted in the In This Issue feature, p. 2007

Supplementary Data from Drug-Induced Epigenomic Plasticity Reprograms Circadian Rhythm Regulation to Drive Prostate Cancer toward Androgen Independence

Supplemental Figure Legends

Supplementary Figure 8 - PDF file 140K, Supplemental data demonstrating that DNAPKcs is required for DNA repair in the presence of androgen

Supplementary Figure 5,6 - PDF file 143K, Supplemental data demonstrating that DNAPKcs knockdown is maintained in growth studies and ATM expression and activity is regulated by DNAPKcs knockdown and inhibition

Direct comparison of various BET inhibitors in prostate cancer cells.

Materials and Methods for Supplemental experiments

Supplementary Figure 1,2 - PDF file 56K, Supplemental data demonstrating that scheduling of IR and ADT treatment does not alter cell survival and DNA damage induces AR mediated gene transcription in a dose dependent fashion

Purpose:Despite recent approvals for checkpoint inhibitors and antibody–drug conjugates targeting NECTIN4 or TROP2, metastatic bladder cancer remains incurable and new treatment strategies are urgently needed. CUB domain–containing protein 1 (CDCP1) is a cell surface protein and promising drug target for many cancers. This study aimed to determine whether CDCP1 is expressed in bladder cancer and whether CDCP1 can be targeted for treatment with radiolabeled antibodies.Experimental Design:CDCP1 expression was evaluated in four bladder cancer datasets (n = 1,047 biopsies). A tissue microarray of primary bladder cancer biopsies was probed for CDCP1 by IHC. CDCP1 expression was evaluated in patient-derived xenografts and cell lysates by immunoblot, flow cytometry, and saturation binding assays. Tumor detection in mouse bladder cancer models was tested using 89Zr-labeled 4A06, a monoclonal antibody targeting the ectodomain of CDCP1. 177Lu-4A06 was applied to mice bearing UMUC3 or HT-1376 xenografts to evaluate antitumor effects (CDCP1 expression in UMUC3 is 10-fold higher than HT-1376).Results:CDCP1 was highest in the basal/squamous subtype, and CDCP1 was expressed in 53% of primary biopsies. CDCP1 was not correlated with pathologic or tumor stage, metastatic site, or NECTIN4 and TROP2 at the mRNA or protein level. CDCP1 ranged from 105 to 106 receptors per cell. Mechanism studies showed that RAS signaling induced CDCP1 expression. 89Zr-4A06 PET detected five human bladder cancer xenografts. 177Lu-4A06 inhibited the growth of UMUC3 and HT-1376 xenografts, models with high and moderate CDCP1 expression, respectively.Conclusions:These data establish that CDCP1 is expressed in bladder cancer, including TROP2 and NECTIN4-null disease, and suggest that bladder cancer can be treated with CDCP1-targeted radiotherapy.

Supplementary Table 2. Clinical Characteristics of the Evaluable Paired Biopsy Cohort

Drugs targeting the cell cycle–regulatory cyclin-dependent kinase (CDK) 4 and 6 have been approved for the treatment of hormone receptor–positive breast cancer, and inhibitors targeting other cell-cycle CDKs are currently in clinical trials. Another class of CDKs, the transcription-associated CDKs, including CDK7, CDK8, CDK9, CDK12 and CDK13, are critical regulators of gene expression. Recent evidence suggests several novel functions of these CDKs, including regulation of epigenetic modifications, intronic polyadenylation, DNA-damage responses, and genomic stability. Here, we summarize our current understanding of the transcriptional CDKs, their utility as biomarkers, and their potential as therapeutic targets.Significance:CDK inhibitors targeting CDK4 and CDK6 have been approved in hormone receptor–positive breast cancer, and inhibitors targeting other cell-cycle CDKs are currently in clinical trials. Several studies now point to potential therapeutic opportunities by inhibiting the transcription-associated CDKs as well as therapeutic vulnerabilities with PARP inhibitors and immunotherapy in tumors deficient in these CDKs.

Supplementary Methods - PDF file 79K, Additional materials and methods not described in main manuscript

PDF file - 410K, Dose responses, transcriptional, biological, biochemical, and in vivo supplemental data

CDCP1 mRNA is not correlated NECTIN4 or TROP2 mRNA, related to Figure 2C. Spearman correlation analyses were performed between CDCP1 and NECTIN4 or TROP2 for the Seiler, Sjodahl 2017, and Sjodahl 2012 datasets, with p values and correlation coefficients shown. Points are colored by consensus subtype as in Figure 1.

De-recruitment of BRD2/3/4 from the SRSF1 and U2AF1 promoter by JQ1.

Supplemental Materials and Methods

Antibodies and PCR Primers

Supplementary Figure S1: Evaluation of pre-amplication. Supplementary Figure S2: Algorithm for CTC positivity. Supplementary Figure S3: CTC isolation and gene analysis methodology. Supplementary Figure S4: Validation of gene expression. Supplementary Figure S5: CTC status is not predictive of overall survival. Supplementary Figure S6: miCTC score flowsheet. Supplementary Table 1: primer tables. Supplementary Table 2: Ct values.

Figure S1. Validation of Project Achilles data, evaluation of TPRKB expression profile across human tissues and ability to knock down TPRKB across cell lines. Figure S2. Cell lines with different types of TP53 alterations show dramatic reductions in cell proliferation upon TPRKB knockdown. Figure S3. Cell lines harboring mutant TP53 show reduced proliferation with TPRKB knockdown. Figure S4. Knockdown of EKC/KEOPS members PRPK, OSGEP and LAGE3 have variable effects across cell lines not related to TP53 status. Figure S5. PRPK specifically stabilizes TPRKB expression.

Supplementary Table 1. Clinical Characteristics of the Whole Genome Sequencing Cohort

Supplementary Figure 4. Overlap of positive and negative correlations.

Supplementary Tables - PDF file 753K, Supplementary Tables S1-S15 include transcriptome and exome sequencing results for the FGFR fusion positive index cases, and summaries of sequencing cohorts

AR- and ERG-positive PDX model displays sensitivity to JQ1.

Supplemental Table S1: CDK12 and CDK13 inhibitors and their properties, including in vitro potencies and structures.

Supplementary Figure Legends - PDF file 66K, Figure legends corresponding to supplemental figures

Enzalutamide resistant VCaP prostate cancer cells.

PDF file - 91K

Supplementary Figure 7 - PDF file 79K, Supplemental data further demonstrating that androgen deprivation sensitizes CRPC cells to IR

Supplementary Figures - PDF file 639K, Supplementary Figures S1-S9 show the phenotypic effects and signaling pathways of FGFR fusions in vitro and in vivo

Supplementary Data from PLX038: A Long-Acting Topoisomerase I Inhibitor With Robust Antitumor Activity in ATM-Deficient Tumors and Potent Synergy With PARP Inhibitors

Supplementary Table 1 - PDF file 282K, Supplemental table listing primer sequences utilized throughout study

Supplementary Figure 3,4 - PDF file 443K, Supplemental data demonstrating that exposure to IR promotes cell cycle arrest and AR activity regulates genes required for DNA damage repair

Therapeutic resistance in metastatic castration-resistant prostate cancer (mCRPC) can be accompanied by treatment-emergent small-cell neuroendocrine carcinoma (t-SCNC), a morphologically distinct subtype. We performed integrative whole-genome and -transcriptome analysis of mCRPC tumor biopsies including paired biopsies after progression, and multiple samples from the same individual. t-SCNC was significantly less likely to have amplification of AR or an intergenic AR-enhancer locus, and demonstrated lower expression of AR and its downstream transcriptional targets. Genomic and transcriptional hallmarks of t-SCNC included biallelic loss of RB1, elevated expression levels of CDKN2A and E2F1, and loss of expression of the AR and AR-responsive genes including TMPRSS2 and NKX3-1. We identified three tumors that converted from adenocarcinoma to t-SCNC and demonstrate spatial and temporal intrapatient heterogeneity of metastatic tumors harboring adenocarcinoma, t-SCNC, or mixed expression phenotypes, with implications for treatment strategies in which dual targeting of adenocarcinoma and t-SCNC phenotypes may be necessary.Implications:The t-SCNC phenotype is characterized by lack of AR enhancer gain and loss of RB1 function, and demonstrates both interindividual and intraindividual heterogeneity.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/17/6/1235/F1.large.jpg.

Tumor protein 53 (TP53; p53) is the most frequently altered gene in human cancer. Identification of vulnerabilities imposed by TP53 alterations may enable effective therapeutic approaches. Through analyzing short hairpin RNA (shRNA) screening data, we identified TP53RK-Binding Protein (TPRKB), a poorly characterized member of the tRNA-modifying EKC/KEOPS complex, as the most significant vulnerability in TP53-mutated cancer cell lines. In vitro and in vivo, across multiple benign-immortalized and cancer cell lines, we confirmed that TPRKB knockdown in TP53-deficient cells significantly inhibited proliferation, with minimal effect in TP53 wild-type cells. TP53 reintroduction into TP53-null cells resulted in loss of TPRKB sensitivity, confirming the importance of TP53 status in this context. In addition, cell lines with mutant TP53 or amplified MDM2 (E3-ubiquitin ligase for TP53) also showed high sensitivity to TPRKB knockdown, consistent with TPRKB dependence in a wide array of TP53-altered cancers. Depletion of other EKC/KEOPS complex members exhibited TP53-independent effects, supporting complex-independent functions of TPRKB. Finally, we found that TP53 indirectly mediates TPRKB degradation, which was rescued by coexpression of PRPK, an interacting member of the EKC/KEOPS complex, or proteasome inhibition. Together, these results identify a unique and specific requirement of TPRKB in a variety of TP53-deficient cancers.Implications:Cancer cells with genomic alterations in TP53 are dependent on TPRKB.

Supplementary Table S1: Patient 1 reversion alleles. Supplementary Table 2: DNA read identifiers supporting reversion alleles. Supplementary Table S3: Gene targets of the SeqCap library used for cfDNA sequencing. Supplementary Figure S1: Frequent loss of heterozygosity in Patient 1's tumor DNA. Supplementary Figure S2: A single copy of chromosome 13 at the BRCA2 locus is present in Patient 1's tumor DNA. Supplementary Figure S3: Mutation signature three is present in patient 1's solid tumor. Supplementary Figure S4: A single copy of chromosome 13 at the BRCA2 locus is present in Patient 2's tumor DNA. Supplementary Figure S5: Patient 2's DNA coverage depth at residue 259. Supplementary Figure S6: Nucleotide and inferred protein sequences for patient 2's reversion alleles. Supplementary Figure S7: Reversion indels were significantly longer than the background distribution of indels. Supplementary Methods.

Supplementary Table 1. Primers and Oligos Sequences Supplementary Table 2. shRNA and siRNA Sequences Supplementary Table 3. Antibody list