Large tandem duplications in cancer result from transcription and DNA replication collisions.

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 in TP53, CDK12, and SPOP. Upon inactivating CDK12, cells display significantly more TRCs, R-loops, and large TDs. Inhibition of G2/M checkpoint proteins, such as WEE1, CHK1, and ATR, selectively inhibits the growth of cells deficient in CDK12. Our data suggest that large TDs in cancer form due to TRCs, and their presence can be used as a biomarker for prognosis and treatment.

Immunotherapeutic Targeting and PET Imaging of DLL3 in Small-Cell Neuroendocrine Prostate Cancer.

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.

Treatment of Prostate Cancer with CD46-targeted 225Ac Alpha Particle Radioimmunotherapy.

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.

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.

TROP2 Expression Across Molecular Subtypes of Urothelial Carcinoma and Enfortumab Vedotin-resistant Cells.

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 with >1400 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.

Heterogeneity in NECTIN4 Expression Across Molecular Subtypes of Urothelial Cancer Mediates Sensitivity to Enfortumab Vedotin.

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.

An integrated functional and clinical genomics approach reveals genes driving aggressive metastatic prostate cancer.

Genomic sequencing of thousands of tumors has revealed many genes associated with specific types of cancer. Similarly, large scale CRISPR functional genomics efforts have mapped genes required for cancer cell proliferation or survival in hundreds of cell lines. Despite this, for specific disease subtypes, such as metastatic prostate cancer, there are likely a number of undiscovered tumor specific driver genes that may represent potential drug targets. To identify such genetic dependencies, we performed genome-scale CRISPRi screens in metastatic prostate cancer models. We then created a pipeline in which we integrated pan-cancer functional genomics data with our metastatic prostate cancer functional and clinical genomics data to identify genes that can drive aggressive prostate cancer phenotypes. Our integrative analysis of these data reveals known prostate cancer specific driver genes, such as AR and HOXB13, as well as a number of top hits that are poorly characterized. In this study we highlight the strength of an integrated clinical and functional genomics pipeline and focus on two top hit genes, KIF4A and WDR62. We demonstrate that both KIF4A and WDR62 drive aggressive prostate cancer phenotypes in vitro and in vivo in multiple models, irrespective of AR-status, and are also associated with poor patient outcome.

Molecular Imaging of Prostate Cancer Targeting CD46 Using ImmunoPET.

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.