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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.

Sacituzumab govitecan (SG) is an antibody-drug conjugate (ADC) targeting TROP2, which has recently been approved for treatment-refractory metastatic urothelial cancer (UC). However, the variability of TROP2 expression across different bladder cancer (BC) subtypes, as well as after enfortumab vedotin (EV) exposure, remains unknown. Using gene expression data from four clinical cohorts with1400 patient samples of muscle-invasive BC and a BC tissue microarray, we found that TROP2 mRNA and protein are highly expressed across basal, luminal, and stroma-rich subtypes, but depleted in the neuroendocrine subtype. In addition, TROP2 mRNA levels are correlated with NECTIN4 mRNA but are more highly expressed than NECTIN4 mRNA in patient cohorts and BC cell lines. Moreover, CRISPR/Cas9-mediated knockdown of TROP2 demonstrates that its expression is one factor governing SG sensitivity. After prolonged EV exposure, cells can downregulate NECTIN4, leading to EV resistance, but retain TROP2 expression and remain sensitive to SG, suggesting nonoverlapping resistance mechanisms to these ADCs. While our findings warrant further validation, they have significant implications for biomarker development, patient selection, and treatment sequencing in the clinic as well as clinical trial design and stratification for metastatic BC patients. PATIENT SUMMARY: In this report, we investigated the expression levels of the drug target TROP2 across different molecular subtypes of bladder cancer in multiple patient cohorts and cell lines. We found high levels of TROP2 in most subtypes except in the neuroendocrine subtype. Overall, TROP2 gene expression is higher than NECTIN4 gene expression, and cells resistant to enfortumab vedotin (EV), a NECTIN4-targeting antibody-drug conjugate, remain sensitive to sacituzumab govitecan (SG). Our findings suggest that SG may be effective across most bladder cancer subtypes, including the bladder cancers previously treated with EV.

Effective treatments for de novo and treatment-emergent small-cell/neuroendocrine (t-SCNC) prostate cancer represent an unmet need for this disease. Using metastatic biopsies from patients with advanced cancer, we demonstrate that delta-like ligand 3 (DLL3) is expressed in de novo and t-SCNC and is associated with reduced survival. We develop a PET agent, [89Zr]-DFO-DLL3-scFv, that detects DLL3 levels in mouse SCNC models. In multiple patient-derived xenograft models, AMG 757 (tarlatamab), a half-life–extended bispecific T-cell engager (BiTE) immunotherapy that redirects CD3-positive T cells to kill DLL3-expressing cells, exhibited potent and durable antitumor activity. Late relapsing tumors after AMG 757 treatment exhibited lower DLL3 levels, suggesting antigen loss as a resistance mechanism, particularly in tumors with heterogeneous DLL3 expression. These findings have been translated into an ongoing clinical trial of AMG 757 in de novo and t-SCNC, with a confirmed objective partial response in a patient with histologically confirmed SCNC. Overall, these results identify DLL3 as a therapeutic target in SCNC and demonstrate that DLL3-targeted BiTE immunotherapy has significant antitumor activity in this aggressive prostate cancer subtype. Significance: The preclinical and clinical evaluation of DLL3-directed immunotherapy, AMG 757, and development of a PET radiotracer for noninvasive DLL3 detection demonstrate the potential of targeting DLL3 in SCNC prostate cancer.

Despite the abundance of somatic structural variations (SVs) in cancer, the underlying molecular mechanisms of their formation remain unclear. Here, we use 6,193 whole-genome sequenced tumors to study the contributions of transcription and DNA replication collisions to genome instability. After deconvoluting robust SV signatures in three independent pan-cancer cohorts, we detect transcription-dependent replicated-strand bias, the expected footprint of transcription-replication collision (TRC), in large tandem duplications (TDs). Large TDs are abundant in female-enriched, upper gastrointestinal tract and prostate cancers. They are associated with poor patient survival and mutations inTP53,CDK12, andSPOP. Upon inactivating or suppressingCDK12, cells display significantly more TRCs and R-loops. Inhibition of WEE1, a cell cycle regulator that promotes DNA repair, selectively inhibits the growth of cells with loss of CDK12. Our data suggest that large TDs in cancer form due to TRC, and their presence can be used as a biomarker for prognosis and treatment.

Figure S11: Antitumor activity of [225Ac]DOTA-YS5 in subcutaneous DU145 tumors in nude mice. Tumor volume, survival analysis, and weight measurements after the injections of [225Ac]DOTA-YS5 in DU145 xenografts (n=6).

Supplementary Data: Methods and tables.

Purpose:Radiopharmaceutical therapy is changing the standard of care in prostate cancer and other malignancies. We previously reported high CD46 expression in prostate cancer and developed an antibody–drug conjugate and immunoPET agent based on the YS5 antibody, which targets a tumor-selective CD46 epitope. Here, we present the preparation, preclinical efficacy, and toxicity evaluation of [225Ac]DOTA-YS5, a radioimmunotherapy agent based on the YS5 antibody.Experimental Design:[225Ac]DOTA-YS5 was developed, and its therapeutic efficiency was tested on cell-derived (22Rv1, DU145), and patient-derived (LTL-545, LTL484) prostate cancer xenograft models. Biodistribution studies were carried out on 22Rv1 tumor xenograft models to confirm the targeting efficacy. Toxicity analysis of the [225Ac]DOTA-YS5 was carried out on nu/nu mice to study short-term (acute) and long-term (chronic) toxicity.Results:Biodistribution study shows that [225Ac]DOTA-YS5 agent delivers high levels of radiation to the tumor tissue (11.64% ± 1.37%ID/g, 28.58% ± 10.88%ID/g, 29.35% ± 7.76%ID/g, and 31.78% ± 5.89%ID/g at 24, 96, 168, and 408 hours, respectively), compared with the healthy organs. [225Ac]DOTA-YS5 suppressed tumor size and prolonged survival in cell line–derived and patient-derived xenograft models. Toxicity analysis revealed that the 0.5 μCi activity levels showed toxicity to the kidneys, likely due to redistribution of daughter isotope 213Bi.Conclusions:[225Ac]DOTA-YS5 suppressed the growth of cell-derived and patient-derived xenografts, including prostate-specific membrane antigen–positive and prostate-specific membrane antigen–deficient models. Overall, this preclinical study confirms that [225Ac]DOTA-YS5 is a highly effective treatment and suggests feasibility for clinical translation of CD46-targeted radioligand therapy in prostate cancer.

PDF file - 37K, Drug list identified in silico. List of drugs identified in silico to inhibit breast cancer cell lines that overexpress ZNF217. Correlation of ZNF217 expression in the cell line panel with the drug panel of ~50,000 drugs generated by the NCI Developmental Therapeutics Program (dtp.nci.nih.gov) identified several drugs that selectively inhibited growth of cells, assessed by GI50, expressing high levels of ZNF217 with a low drug concentration

PDF file - 29K, Overlapping adult stem cell signature genes. List of overlapping genes between our dataset (this study) and our adult stem cell signature, which is downregulated in many epithelial cancers relative to normal tissue. Primary MECs overexpressing Znf217 significantly repressed genes of the adult stem cell signature (P=1.89x10-10)

PDF file - 12MB, Movie of SCp2 cells infected with vector (left movie) or Znf217 (right movie) following a scratch with a pipette tip. This movie ran for 20.25 hours

PDF file - 2.4MB

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.

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.

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.

PDF file - 233K, This file contains supplemental materials and methods, including cell lines, antibodies and staining procedures used in this study

PDF file - 50K, Human microarray summary. Summary of correlations between ZNF217 and selected genes from ChIP-chip and gene expression microarray datasets. ZNF217 expression levels correlated with expression of K19 and K8/18, genes that have ZNF217 enriched at their promoters, in human breast cancer cell lines, tumors, and after knockdown in MCF7 cells

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

PDF file - 46K, Gene ontology for microarray gene sets. List of gene ontology (GO) terms from the microarray gene expression datasets of primary MECs � Znf217 using DAVID software

PDF file - 117K, Microarray target genes from MCF7 siRNA. Target genes identified by microarray expression analysis from MCF7 cells � Znf217 siRNA

CDCP1 mRNA is overexpressed in basal/squamous subtypes of bladder cancer, related to Figure 1A-D. Pairwise comparisons of CDCP1 expression between bladder cancer consensus subtypes using the Wilcoxon rank-sum test across four different datasets are shown.

Supplemental Tables 1-7

Association between NECTIN4 mRNA expression and clinical characteristics in the Seiler cohort for (A) sex (p

Purpose:Carcinomas originate from epithelial tissues, which have apical (luminal) and basal orientations. The degree of luminal versus basal differentiation in cancer has been shown to be biologically important in some carcinomas and impacts treatment response.Experimental Design:Although prior studies have focused on individual cancer types, we used a modified clinical-grade classifier (PAM50) to subtype 8,764 tumors across 22 different carcinomas into luminal A, luminal B, and basal-like tumors.Results:We found that all epithelial tumors demonstrated similar gene expression–based luminal/basal subtypes. As expected, basal-like tumors were associated with increased expression of the basal markers KRT5/6 and KRT14, and luminal-like tumors were associated with increased expression of the luminal markers KRT20. Luminal A tumors consistently had improved outcomes compared with basal across many tumor types, with luminal B tumors falling between the two. Basal tumors had the highest rates of TP53 and RB1 mutations and copy number loss. Luminal breast, cervical, ovarian, and endometrial tumors had increased ESR1 expression, and luminal prostate, breast, cervical, and bladder tumors had increased androgen receptor (AR) expression. Furthermore, luminal B tumors had the highest rates of AR and ESR1 mutations and had increased sensitivity in vitro to bicalutamide and tamoxifen. Luminal B tumors were more sensitive to gemcitabine, and basal tumors were more sensitive to docetaxel.Conclusions:This first pan-carcinoma luminal/basal subtyping across epithelial tumors reveals global similarities across carcinomas in the transcriptome, genome, clinical outcomes, and drug sensitivity, emphasizing the biological and translational importance of these luminal versus basal subtypes.

Supplemental

Purpose:Autoantibody responses in cancer are of great interest, as they may be concordant with T-cell responses to cancer antigens or predictive of response to cancer immunotherapies. Thus, we sought to characterize the antibody landscape of metastatic castration-resistant prostate cancer (mCRPC).Experimental Design:Serum antibody epitope repertoire analysis (SERA) was performed on patient serum to identify tumor-specific neoepitopes. Somatic mutation–specific neoepitopes were investigated by associating serum epitope enrichment scores with whole-genome sequencing results from paired solid tumor metastasis biopsies and germline blood samples. A protein-based immunome-wide association study (PIWAS) was performed to identify significantly enriched epitopes, and candidate serum antibodies enriched in select patients were validated by ELISA profiling. A distinct cohort of patients with melanoma was evaluated to validate the top cancer-specific epitopes.Results:SERA was performed on 1,229 serum samples obtained from 72 men with mCRPC and 1,157 healthy control patients. Twenty-nine of 6,636 somatic mutations (0.44%) were associated with an antibody response specific to the mutated peptide. PIWAS analyses identified motifs in 11 proteins, including NY-ESO-1 and HERVK-113, as immunogenic in mCRPC, and ELISA confirmed serum antibody enrichment in candidate patients. Confirmatory PIWAS, Identifying Motifs Using Next-generation sequencing Experiments (IMUNE), and ELISA analyses performed on serum samples from 106 patients with melanoma similarly revealed enriched cancer-specific antibody responses to NY-ESO-1.Conclusions:We present the first large-scale profiling of autoantibodies in advanced prostate cancer, utilizing a new antibody profiling approach to reveal novel cancer-specific antigens and epitopes. Our study recovers antigens of known importance and identifies novel tumor-specific epitopes of translational interest.

Supplementary Data from Resistance to ATR Inhibitors Is Mediated by Loss of the Nonsense-Mediated Decay Factor UPF2

Supplementary Table from CUB Domain-Containing Protein 1 (CDCP1) Is a Target for Radioligand Therapy in Castration-Resistant Prostate Cancer, including PSMA Null Disease

Supplementary Figures S1-S9

Supplementary Figures 1-8 with legends

Effective treatments for de novo and treatment-emergent small-cell/neuroendocrine (t-SCNC) prostate cancer represent an unmet need for this disease. Using metastatic biopsies from patients with advanced cancer, we demonstrate that delta-like ligand 3 (DLL3) is expressed in de novo and t-SCNC and is associated with reduced survival. We develop a PET agent, [89Zr]-DFO-DLL3-scFv, that detects DLL3 levels in mouse SCNC models. In multiple patient-derived xenograft models, AMG 757 (tarlatamab), a half-life–extended bispecific T-cell engager (BiTE) immunotherapy that redirects CD3-positive T cells to kill DLL3-expressing cells, exhibited potent and durable antitumor activity. Late relapsing tumors after AMG 757 treatment exhibited lower DLL3 levels, suggesting antigen loss as a resistance mechanism, particularly in tumors with heterogeneous DLL3 expression. These findings have been translated into an ongoing clinical trial of AMG 757 in de novo and t-SCNC, with a confirmed objective partial response in a patient with histologically confirmed SCNC. Overall, these results identify DLL3 as a therapeutic target in SCNC and demonstrate that DLL3-targeted BiTE immunotherapy has significant antitumor activity in this aggressive prostate cancer subtype.Significance:The preclinical and clinical evaluation of DLL3-directed immunotherapy, AMG 757, and development of a PET radiotracer for noninvasive DLL3 detection demonstrate the potential of targeting DLL3 in SCNC prostate cancer.

Over one million cases of gastric cancer are diagnosed each year globally, and the metastatic disease continues to have a poor prognosis. A significant proportion of gastric tumors have defects in the DNA damage response pathway, creating therapeutic opportunities through synthetic lethal approaches. Several small-molecule inhibitors of ATR, a key regulator of the DNA damage response, are now in clinical development as targeted agents for gastric cancer. Here, we performed a large-scale CRISPR interference screen to discover genetic determinants of response and resistance to ATR inhibitors (ATRi) in gastric cancer cells. Among the top hits identified as mediators of ATRi response were UPF2 and other components of the nonsense-mediated decay (NMD) pathway. Loss of UPF2 caused ATRi resistance across multiple gastric cancer cell lines. Global proteomic, phosphoproteomic, and transcriptional profiling experiments revealed that cell-cycle progression and DNA damage responses were altered in UPF2-mutant cells. Further studies demonstrated that UPF2-depleted cells failed to accumulate in G1 following treatment with ATRi. UPF2 loss also reduced transcription–replication collisions, which has previously been associated with ATRi response, thereby suggesting a possible mechanism of resistance. Our results uncover a novel role for NMD factors in modulating response to ATRi in gastric cancer, highlighting a previously unknown mechanism of resistance that may inform the clinical use of these drugs.Significance:Loss of NMD proteins promotes resistance to ATR inhibitors in gastric cancer cells, which may provide a combination of therapeutic targets and biomarkers to improve the clinical utility of these drugs.

FigureS3

Purpose:We recently identified CD46 as a novel therapeutic target in prostate cancer. In this study, we developed a CD46-targeted PET radiopharmaceutical, [89Zr]DFO-YS5, and evaluated its performance for immunoPET imaging in murine prostate cancer models.Experimental Design:[89Zr]DFO-YS5 was prepared and its in vitro binding affinity for CD46 was measured. ImmunoPET imaging was conducted in male athymic nu/nu mice bearing DU145 [AR−, CD46+, prostate-specific membrane antigen–negative (PSMA−)] or 22Rv1 (AR+, CD46+, PSMA+) tumors, and in NOD/SCID gamma mice bearing patient-derived adenocarcinoma xenograft, LTL-331, and neuroendocrine prostate cancers, LTL-331R and LTL-545.Results:[89Zr]DFO-YS5 binds specifically to the CD46-positive human prostate cancer DU145 and 22Rv1 xenografts. In biodistribution studies, the tumor uptake of [89Zr]DFO-YS5 was 13.3 ± 3.9 and 11.2 ± 2.5 %ID/g, respectively, in DU145 and 22Rv1 xenografts, 4 days postinjection. Notably, [89Zr]DFO-YS5 demonstrated specific uptake in the PSMA- and AR-negative DU145 model. [89Zr]DFO-YS5 also showed uptake in the patient-derived LTL-331 and -331R models, with particularly high uptake in the LTL-545 neuroendocrine prostate cancer tumors (18.8 ± 5.3, 12.5 ± 1.8, and 32 ± 5.3 %ID/g in LTL-331, LTL-331R, and LTL-545, respectively, at 4 days postinjection).Conclusions:[89Zr]DFO-YS5 is an excellent PET imaging agent across a panel of prostate cancer models, including in both adenocarcinoma and neuroendocrine prostate cancer, both cell line- and patient-derived xenografts, and both PSMA-positive and -negative tumors. It demonstrates potential for clinical translation as an imaging agent, theranostic platform, and companion biomarker in prostate cancer.

The p values from pairwise comparisons amongst the molecular subtypes for each cohort using the Wilcoxon rank-sum test are shown.

Supplementary Figure from CUB Domain-Containing Protein 1 (CDCP1) Is a Target for Radioligand Therapy in Castration-Resistant Prostate Cancer, including PSMA Null Disease

Purpose:With the improvement in overall survival with 177Lu-PSMA 617, radioligand therapy (RLT) is now a viable option for patients with metastatic castration-resistant prostate cancer (mCRPC). However, responses are variable, in part due to low PSMA expression in 30% of patients. Herein, we evaluated whether the cell surface protein CUB domain-containing protein 1 (CDCP1) can be exploited to treat mCRPC with RLT, including in PSMA-low subsets.Experimental Design:CDCP1 levels were evaluated using RNA sequencing from 119 mCRPC biopsies. CDCP1 levels were assessed in 17 post–enzalutamide- or abiraterone-treated mCRPC biopsies, 12 patient-derived xenografts (PDX), and prostate cancer cell lines. 4A06, a recombinant human antibody that targets the CDCP1 ectodomain, was labeled with Zr-89 or Lu-177 and tested in tumor-bearing mice.Results:CDCP1 expression was observed in 90% of mCRPC biopsies, including small-cell neuroendocrine (SCNC) and adenocarcinomas with low FOLH1 (PSMA) levels. Fifteen of 17 evaluable mCRPC biopsies (85%) demonstrated membranous CDCP1 expression, and 4 of 17 (23%) had higher CDCP1 H-scores compared with PSMA. CDCP1 was expressed in 10 of 12 PDX samples. Bmax values of approximately 22,000, 6,200, and 2,800 fmol/mg were calculated for PC3, DU145, and C4–2B human prostate cancer cells, respectively. 89Zr-4A06 PET detected six human prostate cancer xenografts, including PSMA-low tumors. 177Lu-4A06 significantly suppressed growth of DU145 and C4–2B xenografts.Conclusions:The data provide the first evidence supporting CDCP1-directed RLT to treat mCRPC. Expanded studies are warranted to determine whether CDCP1 is a viable drug target for patients with mCPRC.

Purpose:Enfortumab vedotin (EV) is an antibody–drug conjugate (ADC) targeting NECTIN4 (encoded by the PVRL4/NECTIN4 gene) approved for treatment-refractory metastatic urothelial cancer. Factors that mediate sensitivity or resistance to EV are unknown. In this study, we sought to (i) examine heterogeneity of NECTIN4 gene expression across molecular subtypes of bladder cancer and (ii) determine whether NECTIN4 expression mediates EV sensitivity or resistance.Experimental Design:Molecular subtyping and NECTIN4 expression data from seven muscle-invasive bladder cancer clinical cohorts (n = 1,915 total specimens) were used to assess NECTIN4 expression across molecular subtypes. The outcome of the transcriptomic analysis was relative NECTIN4 expression in the consensus molecular subtypes of bladder cancer. Expression of NECTIN4 was validated in bladder cancer cell lines. NECTIN4 was stably overexpressed or knocked down in basal and luminal bladder cancer cell lines and EV drug sensitivity assays were performed, as measured by cell proliferation and clonogenic assays.Results:NECTIN4 expression is heterogenous across molecular subtypes of bladder cancer and significantly enriched in luminal subtypes. NECTIN4 expression is positively correlated with luminal markers GATA3, FOXA1, and PPARG across all cohorts. NECTIN4 expression is both necessary and sufficient for EV sensitivity in luminal and basal subtypes of urothelial bladder cancer cells. Downregulation of NECTIN4 leads to EV resistance.Conclusions:Sensitivity to EV is mediated by expression of NECTIN4, which is enriched in luminal subtypes of bladder cancer. These findings may have implications for biomarker development, patient selection, and the inclusion of molecular subtyping in ongoing and future EV clinical trials.See related commentary by Teo and Rosenberg, p. 4950

Supplemental Movies 1a-1d: 22Rv1-LacZ and 22Rv1-DLL3 cells co-cultures. Time-lapse movies of 22Rv1-LacZ and 22Rv1-DLL3 cells (labeled with NuclightRed) co-cultured with T cells and either NT control BiTE® or AMG 757. T cells were plated at an E:T ratio of 5:1. Images were captured every 6 hours at 10x magnification. a. 22Rv1-LacZ cells and T cells treated with NT control BiTE®. b. 22Rv1-DLL3 cells and T cells treated with NT control BiTE®. c. 22Rv1-LacZ cells and T cells treated with AMG 757. d. 22Rv1-DLL3 cells and T cells treated with AMG 757.

Supplementary Figure from Resistance to ATR Inhibitors Is Mediated by Loss of the Nonsense-Mediated Decay Factor UPF2