Nav1.7 as a chondrocyte regulator and therapeutic target for osteoarthritis.

Osteoarthritis (OA) is the most common joint disease. Currently there are no effective methods that simultaneously prevent joint degeneration and reduce pain1. Although limited evidence suggests the existence of voltage-gated sodium channels (VGSCs) in chondrocytes2, their expression and function in chondrocytes and in OA remain essentially unknown. Here we identify Nav1.7 as an OA-associated VGSC and demonstrate that human OA chondrocytes express functional Nav1.7 channels, with a density of 0.1 to 0.15 channels per µm2 and 350 to 525 channels per cell. Serial genetic ablation of Nav1.7 in multiple mouse models demonstrates that Nav1.7 expressed in dorsal root ganglia neurons is involved in pain, whereas Nav1.7 in chondrocytes regulates OA progression. Pharmacological blockade of Nav1.7 with selective or clinically used pan-Nav channel blockers significantly ameliorates the progression of structural joint damage, and reduces OA pain behaviour. Mechanistically, Nav1.7 blockers regulate intracellular Ca2+ signalling and the chondrocyte secretome, which in turn affects chondrocyte biology and OA progression. Identification of Nav1.7 as a novel chondrocyte-expressed, OA-associated channel uncovers a dual target for the development of disease-modifying and non-opioid pain relief treatment for OA.

Genomic heterogeneity as a barrier to precision oncology in urothelial cancer.

Precision oncology relies on the accurate molecular characterization of individual patients with cancer at the time of treatment initiation. However, tumor molecular profiles are not static, and cancers continually evolve because of ongoing mutagenesis and clonal selection. Here, we performed genomic analyses of primary tumors, metastases, and plasma collected from individual patients to define the concordance of actionable genomic alterations and to identify drivers of metastatic disease progression. We observed a high degree of discordance of actionable genomic alterations, with 23% discordant between primary and metastatic disease sites. Among chromatin-modifying genes, ARID1A mutations, when discordant, were exclusive to the metastatic tumor samples. Our findings indicate that the high degree of lesion-to-lesion genomic heterogeneity may be a barrier to precision oncology approaches for bladder cancer and that circulating tumor DNA profiling may be preferred to tumor sequencing for a subset of patients.

FOXA1 repression drives lineage plasticity and immune heterogeneity in bladder cancers with squamous differentiation.

Cancers arising from the bladder urothelium often exhibit lineage plasticity with regions of urothelial carcinoma adjacent to or admixed with regions of divergent histomorphology, most commonly squamous differentiation. To define the biologic basis for and clinical significance of this morphologic heterogeneity, here we perform integrated genomic analyses of mixed histology bladder cancers with separable regions of urothelial and squamous differentiation. We find that squamous differentiation is a marker of intratumoral genomic and immunologic heterogeneity in patients with bladder cancer and a biomarker of intrinsic immunotherapy resistance. Phylogenetic analysis confirms that in all cases the urothelial and squamous regions are derived from a common shared precursor. Despite the presence of marked genomic heterogeneity between co-existent urothelial and squamous differentiated regions, no recurrent genomic alteration exclusive to the urothelial or squamous morphologies is identified. Rather, lineage plasticity in bladder cancers with squamous differentiation is associated with loss of expression of FOXA1, GATA3, and PPARG, transcription factors critical for maintenance of urothelial cell identity. Of clinical significance, lineage plasticity and PD-L1 expression is coordinately dysregulated via FOXA1, with patients exhibiting morphologic heterogeneity pre-treatment significantly less likely to respond to immune checkpoint inhibitors.

The immune subtypes and landscape of sarcomas.

BACKGROUND: Considering the molecular heterogeneity of sarcomas and their immunologically quiet character, immunotherapy (e.g., immune checkpoint inhibitors) plays a viable role in only a subset of these tumors. This study aimed to determine the immune subtypes (IMSs) of sarcomas for selecting suitable patients from an extremely heterogeneous population. RESULTS: By performing consensus clustering analysis of the gene expression profiles of 538 patients with sarcomas in online databases, we stratified sarcomas into three IMSs characterized by different immune cell features, tumor mutational burdens (TMBs), gene mutations, and clinical outcomes. IMS1 showed an immune "hot" and immunosuppressive phenotype, the highest frequencies of CSMD3 mutation but the lowest frequencies of HMCN1 and LAMA2 mutations; these patients had the worst progression-free survival (PFS). IMS2 was defined by a high TMB and more gene mutations, but had the lowest frequency of MND1 mutations. IMS3 displayed the highest MDN1 expression level and an immune "cold" phenotype, these patients had the worst PFS. Each subtype was associated with different expression levels of immunogenic cell death modulators and immune checkpoints. Moreover, we applied graph learning-based dimensionality reduction to the immune landscape and identified significant intra-cluster heterogeneity within each IMS. Finally, we developed and validated an immune gene signature with good prognostic performance. CONCLUSIONS: Our results provide a conceptual framework for understanding the immunological heterogeneity of sarcomas. The identification of immune-related subtypes may facilitate optimal selection of sarcoma patients who will respond to appropriate therapeutic strategies.

Convergence of oncogenic cooperation at single-cell and single-gene levels drives leukemic transformation.

Cancers develop from the accumulation of somatic mutations, yet it remains unclear how oncogenic lesions cooperate to drive cancer progression. Using a mouse model harboring NRasG12D and EZH2 mutations that recapitulates leukemic progression, we employ single-cell transcriptomic profiling to map cellular composition and gene expression alterations in healthy or diseased bone marrows during leukemogenesis. At cellular level, NRasG12D induces myeloid lineage-biased differentiation and EZH2-deficiency impairs myeloid cell maturation, whereas they cooperate to promote myeloid neoplasms with dysregulated transcriptional programs. At gene level, NRasG12D and EZH2-deficiency independently and synergistically deregulate gene expression. We integrate results from histopathology, leukemia repopulation, and leukemia-initiating cell assays to validate transcriptome-based cellular profiles. We use this resource to relate developmental hierarchies to leukemia phenotypes, evaluate oncogenic cooperation at single-cell and single-gene levels, and identify GEM as a regulator of leukemia-initiating cells. Our studies establish an integrative approach to deconvolute cancer evolution at single-cell resolution in vivo.

14-3-3 epsilon is an intracellular component of TNFR2 receptor complex and its activation protects against osteoarthritis.

OBJECTIVES: Osteoarthritis (OA) is the most common joint disease; however, the indeterminate nature of mechanisms by which OA develops has restrained advancement of therapeutic targets. TNF signalling has been implicated in the pathogenesis of OA. TNFR1 primarily mediates inflammation, whereas emerging evidences demonstrate that TNFR2 plays an anti-inflammatory and protective role in several diseases and conditions. This study aims to decipher TNFR2 signalling in chondrocytes and OA. METHODS: Biochemical copurification and proteomics screen were performed to isolate the intracellular cofactors of TNFR2 complex. Bulk and single cell RNA-seq were employed to determine 14-3-3 epsilon (14-3-3ε) expression in human normal and OA cartilage. Transcription factor activity screen was used to isolate the transcription factors downstream of TNFR2/14-3-3ε. Various cell-based assays and genetically modified mice with naturally occurring and surgically induced OA were performed to examine the importance of this pathway in chondrocytes and OA. RESULTS: Signalling molecule 14-3-3ε was identified as an intracellular component of TNFR2 complexes in chondrocytes in response to progranulin (PGRN), a growth factor known to protect against OA primarily through activating TNFR2. 14-3-3ε was downregulated in OA and its deficiency deteriorated OA. 14-3-3ε was required for PGRN regulation of chondrocyte metabolism. In addition, both global and chondrocyte-specific deletion of 14-3-3ε largely abolished PGRN's therapeutic effects against OA. Furthermore, PGRN/TNFR2/14-3-3ε signalled through activating extracellular signal-regulated kinase (ERK)-dependent Elk-1 while suppressing nuclear factor kappa B (NF-κB) in chondrocytes. CONCLUSIONS: This study identifies 14-3-3ε as an inducible component of TNFR2 receptor complex in response to PGRN in chondrocytes and presents a previously unrecognised TNFR2 pathway in the pathogenesis of OA.

TNFR2/14-3-3ε signaling complex instructs macrophage plasticity in inflammation and autoimmunity.

TNFR1 and TNFR2 have received prominent attention because of their dominance in the pathogenesis of inflammation and autoimmunity. TNFR1 has been extensively studied and primarily mediates inflammation. TNFR2 remains far less studied, although emerging evidence demonstrates that TNFR2 plays an antiinflammatory and immunoregulatory role in various conditions and diseases. Herein, we report that TNFR2 regulates macrophage polarization, a highly dynamic process controlled by largely unidentified intracellular regulators. Using biochemical copurification and mass spectrometry approaches, we isolated the signaling molecule 14-3-3ε as a component of TNFR2 complexes in response to progranulin stimulation in macrophages. In addition, 14-3-3ε was essential for TNFR2 signaling-mediated regulation of macrophage polarization and switch. Both global and myeloid-specific deletion of 14-3-3ε resulted in exacerbated inflammatory arthritis and counteracted the protective effects of progranulin-mediated TNFR2 activation against inflammation and autoimmunity. TNFR2/14-3-3ε signaled through PI3K/Akt/mTOR to restrict NF-κB activation while simultaneously stimulating C/EBPß activation, thereby instructing macrophage plasticity. Collectively, this study identifies 14-3-3ε as a previously unrecognized vital component of the TNFR2 receptor complex and provides new insights into the TNFR2 signaling, particularly its role in macrophage polarization with therapeutic implications for various inflammatory and autoimmune diseases with activation of the TNFR2/14-3-3ε antiinflammatory pathway.

Bach2 Deficiency Promotes Intestinal Epithelial Regeneration by Accelerating DNA Repair in Intestinal Stem Cells.

Epithelial regeneration is critical for barrier maintenance and organ function after intestinal injury, although the repair mechanisms are unclear. Here, we found that Bach2 deficiency promotes intestinal epithelial cell proliferation during homeostasis. Moreover, genetic inactivation of Bach2 in mouse intestinal epithelium facilitated crypt regeneration after irradiation, resulting in a reduction in mortality. RNA-sequencing analysis of isolated crypts revealed that Bach2 deficiency altered the expression of numerous genes, including those regulating double-strand break repair. Mechanistic characterizations indicated that Bach2 deletion facilitated DNA repair in intestinal crypt cells, as evidenced by faster resolution of γ-H2AX and 53BP1 foci in Bach2-/- crypt cells, compared with Bach2+/+ control. Together, our studies highlight that Bach2 deficiency promotes intestinal regeneration by accelerating DNA repair in intestinal stem cells after radiation damage.

AKT1 E17K Inhibits Cancer Cell Migration by Abrogating ß-Catenin Signaling.

Mutational activation of the PI3K/AKT pathway is among the most common pro-oncogenic events in human cancers. The clinical utility of PI3K and AKT inhibitors has, however, been modest to date. Here, we used CRISPR-mediated gene editing to study the biological consequences of AKT1 E17K mutation by developing an AKT1 E17K-mutant isogenic system in a TP53-null background. AKT1 E17K expression under the control of its endogenous promoter enhanced cell growth and colony formation, but had a paradoxical inhibitory effect on cell migration and invasion. The mechanistic basis by which activated AKT1 inhibited cell migration and invasion was increased E-cadherin expression mediated by suppression of ZEB1 transcription via altered ß-catenin subcellular localization. This phenotypic effect was AKT1-specific, as AKT2 activation had the opposite effect, a reduction in E-cadherin expression. Consistent with the opposing effects of AKT1 and AKT2 activation on E-cadherin expression, a pro-migratory effect of AKT1 activation was not observed in breast cancer cells with PTEN loss or expression of an activating PIK3CA mutation, alterations which induce the activation of both AKT isoforms. The results suggest that the use of AKT inhibitors in patients with breast cancer could paradoxically accelerate metastatic progression in some genetic contexts and may explain the frequent coselection for CDH1 mutations in AKT1-mutated breast tumors. IMPLICATIONS: AKT1 E17K mutation in breast cancer impairs migration/invasiveness via sequestration of ß-catenin to the cell membrane leading to decreased ZEB1 transcription, resulting in increased E-cadherin expression and a reversal of epithelial-mesenchymal transition.

Leveraging Systematic Functional Analysis to Benchmark an In Silico Framework Distinguishes Driver from Passenger MEK Mutants in Cancer.

Despite significant advances in cancer precision medicine, a significant hurdle to its broader adoption remains the multitude of variants of unknown significance identified by clinical tumor sequencing and the lack of biologically validated methods to distinguish between functional and benign variants. Here we used functional data on MAP2K1 and MAP2K2 mutations generated in real-time within a co-clinical trial framework to benchmark the predictive value of a three-part in silico methodology. Our computational approach to variant classification incorporated hotspot analysis, three-dimensional molecular dynamics simulation, and sequence paralogy. In silico prediction accurately distinguished functional from benign MAP2K1 and MAP2K2 mutants, yet drug sensitivity varied widely among activating mutant alleles. These results suggest that multifaceted in silico modeling can inform patient accrual to MEK/ERK inhibitor clinical trials, but computational methods need to be paired with laboratory- and clinic-based efforts designed to unravel variabilities in drug response. SIGNIFICANCE: Leveraging prospective functional characterization of MEK1/2 mutants, it was found that hotspot analysis, molecular dynamics simulation, and sequence paralogy are complementary tools that can robustly prioritize variants for biologic, therapeutic, and clinical validation.See related commentary by Whitehead and Sebolt-Leopold, p. 4042.

Modeling biological and genetic diversity in upper tract urothelial carcinoma with patient derived xenografts.

Treatment paradigms for patients with upper tract urothelial carcinoma (UTUC) are typically extrapolated from studies of bladder cancer despite their distinct clinical and molecular characteristics. The advancement of UTUC research is hampered by the lack of disease-specific models. Here, we report the establishment of patient derived xenograft (PDX) and cell line models that reflect the genomic and biological heterogeneity of the human disease. Models demonstrate high genomic concordance with the corresponding patient tumors, with invasive tumors more likely to successfully engraft. Treatment of PDX models with chemotherapy recapitulates responses observed in patients. Analysis of a HER2 S310F-mutant PDX suggests that an antibody drug conjugate targeting HER2 would have superior efficacy versus selective HER2 kinase inhibitors. In sum, the biological and phenotypic concordance between patient and PDXs suggest that these models could facilitate studies of intrinsic and acquired resistance and the development of personalized medicine strategies for UTUC patients.

Oncogenic ERG Represses PI3K Signaling through Downregulation of IRS2.

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.

CD97 is a critical regulator of acute myeloid leukemia stem cell function.

Despite significant efforts to improve therapies for acute myeloid leukemia (AML), clinical outcomes remain poor. Understanding the mechanisms that regulate the development and maintenance of leukemic stem cells (LSCs) is important to reveal new therapeutic opportunities. We have identified CD97, a member of the adhesion class of G protein-coupled receptors (GPCRs), as a frequently up-regulated antigen on AML blasts that is a critical regulator of blast function. High levels of CD97 correlate with poor prognosis, and silencing of CD97 reduces disease aggressiveness in vivo. These phenotypes are due to CD97's ability to promote proliferation, survival, and the maintenance of the undifferentiated state in leukemic blasts. Collectively, our data credential CD97 as a promising therapeutic target on LSCs in AML.

PYK2 Is Involved in Premalignant Acinar Cell Reprogramming and Pancreatic Ductal Adenocarcinoma Maintenance by Phosphorylating ß-CateninY654.

BACKGROUND & AIMS: Identification and validation of new functionally relevant and pharmacologically actionable targets for pancreatic ductal adenocarcinoma (PDAC) remains a great challenge. Premalignant acinar cell reprogramming (acinar-to-ductal metaplasia [ADM]) is a precursor of pancreatic intraepithelial neoplasia (PanIN) lesions that can progress to PDAC. This study investigated the role of proline-rich tyrosine kinase 2 (PYK2) in mutant Kras-induced and pancreatitis-associated ADM and PanIN formation, as well as in PDAC maintenance. METHODS: Genetically engineered mouse models of mutant Kras (glycine 12 to aspartic acid) and Pyk2 deletion were used for investigating the role of PYK2 in PDAC genesis in mice. In vitro ADM assays were conducted using primary pancreatic acinar cells isolated from mice. Immunohistochemistry, immunofluorescence, and a series of biochemical experiments were used to investigate upstream regulators/downstream targets of PYK2 in pancreatic carcinogenesis. PDAC cell line xenograft experiments were performed to study the role of PYK2 and its downstream target in PDAC maintenance. RESULTS: PYK2 was increased substantially in ADM lesions induced by mutant Kras or inflammatory injury. Pyk2 deletion remarkably suppressed ADM and PanIN formation in a mutant Kras-driven and pancreatitis-associated PDAC model, whereas PYK2 knockdown substantially inhibited PDAC cell growth in vitro and in nude mice. This study uncovered a novel yes-associated protein 1/transcriptional co-activator with PDZ binding motif/signal transducer and activator of transcription 3/PYK2/ß-catenin regulation axis in PDAC. Our results suggest that PYK2 contributes to PDAC genesis and maintenance by activating the Wnt/ß-catenin pathway through directly phosphorylating ß-cateninY654. CONCLUSIONS: The current study uncovers PYK2 as a novel downstream effector of mutant KRAS signaling, a previously unrecognized mediator of pancreatitis-induced ADM and a novel intervention target for PDAC.

Aberrant Expression of ERG Promotes Resistance to Combined PI3K and AR Pathway Inhibition through Maintenance of AR Target Genes.

On the basis of our previous work defining the molecular rationale for combined targeting of the PI3K and AR pathways in PTEN loss prostate cancer, the first clinical trial was recently reported demonstrating a significant benefit for combination therapy in patients with metastatic prostate cancer. In this phase II trial, loss of PTEN was a biomarker predictive of response to combined AKT and AR inhibition. Given that PTEN loss prostate cancers are significantly enriched for ERG genomic rearrangements, we evaluated how the aberrant expression of ERG may impact response to PI3K/AR-targeted therapy. Here, we show that overexpression of ERG in the setting of Pten loss promotes resistance to combined PI3K and AR pathway inhibition with associated maintenance of AR target gene expression. Importantly, following AR knockout in the setting of ERG overexpression, there is maintenance of a subset of AR lineage-specific target genes, making AR dispensable in this context. This has important clinical implications as even in the setting of the androgen-regulated TMPRSS2:ERG genomic rearrangement, ERG expression is never abolished following AR inhibition and may allow for cell survival following AR (lineage)-targeted therapies.

Genomic landscape of inverted urothelial papilloma and urothelial papilloma of the bladder.

Inverted urothelial papilloma (IUP) and urothelial papilloma (UP) are rare urothelial neoplasms that typically follow a benign clinical course. Oncogenic mutations in FGFR3, HRAS, and the TERT promoter have been reported in these entities but no comprehensive molecular analysis has been performed. We sought to characterize the genomic landscape of IUP and UP using whole-exome and targeted next-generation sequencing. In IUP, 10 of 11 tumors harbored oncogenic hotspot mutations in HRAS and the remaining tumor had an oncogenic KRAS mutation. None of the IUP tumors harbored TERT promoter or FGFR3 mutations. In UP, 8 of 11 tumors had oncogenic KRAS mutations and two had oncogenic HRAS mutations. One UP tumor had oncogenic mutations in FGFR3, PIK3CA, and the TERT promoter, and arose in a patient with recurrent non-invasive papillary urothelial carcinomas. In contrast to urothelial carcinoma, the APOBEC mutational signature was not present in any IUP and UP tumors, and oncogenic alterations in chromatin remodeling genes were uncommon in both IUP and UP. The current study suggests that IUP and UP are driven primarily by RAS pathway activation and lack the more common genomic features of urothelial cancers. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Genomic Differences Between "Primary" and "Secondary" Muscle-invasive Bladder Cancer as a Basis for Disparate Outcomes to Cisplatin-based Neoadjuvant Chemotherapy.

BACKGROUND: Cisplatin-based neoadjuvant chemotherapy (NAC) followed by radical cystectomy (RC) is the standard of care for patients with muscle-invasive bladder cancer (MIBC). It is unknown whether this treatment strategy is appropriate for patients who progress to MIBC after treatment for prior noninvasive disease (secondary MIBC). OBJECTIVE: To determine whether clinical and genomic differences exist between primary and secondary MIBC treated with NAC and RC. DESIGN, SETTING, AND PARTICIPANTS: Clinicopathologic outcomes were compared between 245 patients with clinical T2-4aN0M0-stage primary MIBC and 43 with secondary MIBC treated with NAC and RC at Memorial Sloan Kettering Cancer Center (MSKCC) from 2001 to 2015. Genomic differences were assessed in a retrospective cohort of 385 prechemotherapy specimens sequenced by whole-exome or targeted exon capture by the Cancer Genome Atlas or at MSKCC. Findings were confirmed in an independent validation cohort of 94 MIBC patients undergoing prospective targeted exon sequencing at MSKCC. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Pathologic response rates, recurrence-free survival (RFS), bladder cancer-specific survival (CSS), and overall survival (OS) were measured. Differences in somatic genomic alteration rates were compared using Fisher's exact test and the Benjamini-Hochberg false discovery rate method. RESULTS AND LIMITATIONS: Patients with secondary MIBC had lower pathologic response rates following NAC than those with primary MIBC (univariable: 26% vs 45%, multivariable: odds ratio=0.4 [95% confidence interval=0.18-0.84] p=0.02) and significantly worse RFS, CSS, and OS. Patients with secondary MIBC treated with NAC had worse CSS compared with cystectomy alone (p=0.002). In a separate genomic analysis, we detected significantly more likely deleterious somatic ERCC2 missense mutations in primary MIBC tumors in both the discovery (10.9% [36/330] vs 1.8% [1/55], p=0.04) and the validation (15.7% [12/70] vs 0% [0/24], p=0.03) cohort. CONCLUSIONS: Patients with secondary MIBC treated with NAC had worse clinical outcomes than similarly treated patients with primary MIBC. ERCC2 mutations predicted to result in increased cisplatin sensitivity were enriched in primary versus secondary MIBC. Prospective validation is still needed, but given the lack of clinical benefit with cisplatin-based NAC in patients with secondary MIBC, upfront RC or enrollment in clinical trials should be considered. PATIENT SUMMARY: A retrospective cohort study of patients with "primary" and "secondary" muscle-invasive bladder cancer (MIBC) treated with chemotherapy before surgical removal of the bladder identified lower response rates and shorter survival in patients with secondary MIBC. Tumor genetic sequencing of separate discovery and validation cohorts revealed that chemotherapy-sensitizing DNA damage repair gene mutations occur predominantly in primary MIBC tumors and may underlie the greater sensitivity of primary MIBC to chemotherapy. Prospective validation is still needed, but patients with secondary MIBC may derive greater benefit from upfront surgery or enrollment in clinical trials rather than from standard chemotherapy.

Tumor Evolution and Drug Response in Patient-Derived Organoid Models of Bladder Cancer.

Bladder cancer is the fifth most prevalent cancer in the U.S., yet is understudied, and few laboratory models exist that reflect the biology of the human disease. Here, we describe a biobank of patient-derived organoid lines that recapitulates the histopathological and molecular diversity of human bladder cancer. Organoid lines can be established efficiently from patient biopsies acquired before and after disease recurrence and are interconvertible with orthotopic xenografts. Notably, organoid lines often retain parental tumor heterogeneity and exhibit a spectrum of genomic changes that are consistent with tumor evolution in culture. Analyses of drug response using bladder tumor organoids show partial correlations with mutational profiles, as well as changes associated with treatment resistance, and specific responses can be validated using xenografts in vivo. Our studies indicate that patient-derived bladder tumor organoids represent a faithful model system for studying tumor evolution and treatment response in the context of precision cancer medicine.