Affimer-mediated locking of p21-activated kinase 5 in an intermediate activation state results in kinase inhibition.

Kinases are important therapeutic targets, and their inhibitors are classified according to their mechanism of action, which range from blocking ATP binding to covalent inhibition. Here, a mechanism of inhibition is highlighted by capturing p21-activated kinase 5 (PAK5) in an intermediate state of activation using an Affimer reagent that binds in the P+1 pocket. PAK5 was identified from a non-hypothesis-driven high-content imaging RNAi screen in urothelial cancer cells. Silencing of PAK5 resulted in reduced cell number, G1/S arrest, and enlargement of cells, suggesting it to be important in urothelial cancer cell line survival and proliferation. Affimer reagents were isolated to identify mechanisms of inhibition. The Affimer PAK5-Af17 recapitulated the phenotype seen with siRNA. Co-crystallization revealed that PAK5-Af17 bound in the P+1 pocket of PAK5, locking the kinase into a partial activation state. This mechanism of inhibition indicates that another class of kinase inhibitors is possible.

Development of resistance to FGFR inhibition in urothelial carcinoma via multiple pathways in vitro.

Alterations of fibroblast growth factor receptors (FGFRs) are common in bladder and other cancers and result in disrupted signalling via several pathways. Therapeutics that target FGFRs have now entered the clinic, but, in common with many cancer therapies, resistance develops in most cases. To model this, we derived resistant sublines of two FGFR-driven bladder cancer cell lines by long-term culture with the FGFR inhibitor PD173074 and explored mechanisms using expression profiling and whole-exome sequencing. We identified several resistance-associated molecular profiles. These included HRAS mutation in one case and reversible mechanisms resembling a drug-tolerant persister phenotype in others. Upregulated IGF1R expression in one resistant derivative was associated with sensitivity to linsitinib and a profile with upregulation of a YAP/TAZ signature to sensitivity to the YAP inhibitor CA3 in another. However, upregulation of other potential therapeutic targets was not indicative of sensitivity. Overall, the heterogeneity in resistance mechanisms and commonality of the persister state present a considerable challenge for personalised therapy. Nevertheless, the reversibility of resistance may indicate a benefit from treatment interruptions or retreatment following disease relapse in some patients. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

A transcriptional network of cell cycle dysregulation in noninvasive papillary urothelial carcinoma.

Human cancers display a restricted set of expression profiles, despite diverse mutational drivers. This has led to the hypothesis that select sets of transcription factors act on similar target genes as an integrated network, buffering a tumor's transcriptional state. Noninvasive papillary urothelial carcinoma (NIPUC) with higher cell cycle activity has higher risk of recurrence and progression. In this paper, we describe a transcriptional network of cell cycle dysregulation in NIPUC, which was delineated using the ARACNe algorithm applied to expression data from a new cohort (n = 81, RNA sequencing), and two previously published cohorts. The transcriptional network comprised 121 transcription factors, including the pluripotency factors SOX2 and SALL4, the sex hormone binding receptors ESR1 and PGR, and multiple homeobox factors. Of these 121 transcription factors, 65 and 56 were more active in tumors with greater and less cell cycle activity, respectively. When clustered by activity of these transcription factors, tumors divided into High Cell Cycle versus Low Cell Cycle groups. Tumors in the High Cell Cycle group demonstrated greater mutational burden and copy number instability. A putative mutational driver of cell cycle dysregulation, such as homozygous loss of CDKN2A, was found in only 50% of High Cell Cycle NIPUC, suggesting a prominent role of transcription factor activity in driving cell cycle dysregulation. Activity of the 121 transcription factors strongly associated with expression of EZH2 and other members of the PRC2 complex, suggesting regulation by this complex influences expression of the transcription factors in this network. Activity of transcription factors in this network also associated with signatures of pluripotency and epithelial-to-mesenchymal transition (EMT), suggesting they play a role in driving evolution to invasive carcinoma. Consistent with this, these transcription factors differed in activity between NIPUC and invasive urothelial carcinoma.

The Lund Molecular Taxonomy Applied to Non-Muscle-Invasive Urothelial Carcinoma.

The precise classification of tumors into relevant molecular subtypes will facilitate both future research and optimal treatment. Here, the Lund Taxonomy system for molecular classification of urothelial carcinoma was applied to two large and independent cohorts of non-muscle-invasive tumors. Of 752 tumors classified, close to 100% were of the luminal subtypes, 95% urothelial-like (Uro; UroA, UroB, or UroC) and 5% genomically unstable. The obtained subtype structure organized the tumors into groups with specific and coherent gene mutation, genomic, and clinical profiles. The intrasubtype variability in the largest group of tumors, UroA, was caused by infiltration and proliferation, not considered as cancer cell type-defining properties. Within the UroA subtype, a HOXB/late cell-cycle gene expression polarity was found, strongly associated with FGFR3, STAG2, and TP53 mutations, as well as with chromosome 9 losses. Kaplan-Meier analyses identified the genomically unstable subtype as a progression high-risk group, also valid in the subgroup of T1 tumors. Almost all progression events occurred within 12 months in this subtype. Also, a general progression gene signature was derived that identifies high- and low-risk tumors. All findings were demonstrated in two independent cohorts. The Lund Taxonomy system is applicable to both non-muscle- and muscle-invasive tumors and may be a useful biological framework for translational studies.

Molecular profile of pure squamous cell carcinoma of the bladder identifies major roles for OSMR and YAP signalling.

Pure squamous cell carcinoma (SCC) is the most common pure variant form of bladder cancer, found in 2-5% of cases. It often presents late and is unresponsive to cisplatin-based chemotherapy. The molecular features of these tumours have not been elucidated in detail. We carried out whole-exome sequencing (WES), copy number, and transcriptome analysis of bladder SCC. Muscle-invasive bladder cancer (MIBC) samples with no evidence of squamous differentiation (non-SD) were used for comparison. To assess commonality of features with urothelial carcinoma with SD, we examined data from SD samples in The Cancer Genome Atlas (TCGA) study of MIBC. TP53 was the most commonly mutated gene in SCC (64%) followed by FAT1 (45%). Copy number analysis revealed complex changes in SCC, many differing from those in samples with SD. Gain of 5p and 7p was the most common feature, and focal regions on 5p included OSMR and RICTOR. In addition to 9p deletions, we found some samples with focal gain of 9p24 containing CD274 (PD-L1). Loss of 4q35 containing FAT1 was found in many samples such that all but one sample analysed by WES had FAT1 mutation or deletion. Expression features included upregulation of oncostatin M receptor (OSMR), metalloproteinases, metallothioneins, keratinisation genes, extracellular matrix components, inflammatory response genes, stem cell markers, and immune response modulators. Exploration of differentially expressed transcription factors identified BNC1 and TFAP2A, a gene repressed by PPARG, as the most upregulated factors. Known urothelial differentiation factors were downregulated along with 72 Kruppel-associated (KRAB) domain-containing zinc finger family protein (KZFP) genes. Novel therapies are urgently needed for these tumours. In addition to upregulated expression of EGFR, which has been suggested as a therapeutic target in basal/squamous bladder cancer, we identified expression signatures that indicate upregulated OSMR and YAP/TAZ signalling. Preclinical evaluation of the effects of inhibition of these pathways alone or in combination is merited.

Mutational landscape of non-muscle-invasive bladder cancer.

Non-muscle-invasive bladder cancer (NMIBC) includes stage Ta and stage T1 tumors and carcinoma in situ (CIS). Grading of Ta tumors subdivides these lesions into papillary urothelial neoplasms of low malignant potential and low- and high-grade noninvasive papillary urothelial carcinoma. CIS is by definition high-grade and the majority of stage T1 tumors are of high-grade. This pathologic heterogeneity is associated with divergent clinical outcome, with significantly worse prognosis for patients with T1 tumors or CIS. A wealth of molecular information has accumulated on NMIBC including mutational data that ranges from the whole chromosome level to next generation sequence data at nucleotide level. This has not only identified key genes that are mutated in NMIBC, but also provides insight into the processes that shape their mutational landscape. Although molecular analyses cannot yet provide definitive personal prognostic information, many differences between these entities promise improved disease management in the future. Most information is available for Ta and T1 samples and this is the focus of this review.

The Warburg effect as a therapeutic target for bladder cancers and intratumoral heterogeneity in associated molecular targets.

Bladder cancer is the 10th most common cancer worldwide. For muscle-invasive bladder cancer (MIBC), treatment includes radical cystectomy, radiotherapy, and chemotherapy; however, the outcome is generally poor. For non-muscle-invasive bladder cancer (NMIBC), tumor recurrence is common. There is an urgent need for more effective and less harmful therapeutic approaches. Here, bladder cancer cell metabolic reprogramming to rely on aerobic glycolysis (the Warburg effect) and expression of associated molecular therapeutic targets by bladder cancer cells of different stages and grades, and in freshly resected clinical tissue, is investigated. Importantly, analyses indicate that the Warburg effect is a feature of both NMIBCs and MIBCs. In two in vitro inducible epithelial-mesenchymal transition (EMT) bladder cancer models, EMT stimulation correlated with increased lactate production, the end product of aerobic glycolysis. Protein levels of lactate dehydrogenase A (LDH-A), which promotes pyruvate enzymatic reduction to lactate, were higher in most bladder cancer cell lines (compared with LDH-B, which catalyzes the reverse reaction), but the levels did not closely correlate with aerobic glycolysis rates. Although LDH-A is expressed in normal urothelial cells, LDH-A knockdown by RNAi selectively induced urothelial cancer cell apoptotic death, whereas normal cells were unaffected-identifying LDH-A as a cancer-selective therapeutic target for bladder cancers. LDH-A and other potential therapeutic targets (MCT4 and GLUT1) were expressed in patient clinical specimens; however, positive staining varied in different areas of sections and with distance from a blood vessel. This intratumoral heterogeneity has important therapeutic implications and indicates the possibility of tumor cell metabolic coupling.

An integrated multi-omics analysis identifies prognostic molecular subtypes of non-muscle-invasive bladder cancer.

The molecular landscape in non-muscle-invasive bladder cancer (NMIBC) is characterized by large biological heterogeneity with variable clinical outcomes. Here, we perform an integrative multi-omics analysis of patients diagnosed with NMIBC (n = 834). Transcriptomic analysis identifies four classes (1, 2a, 2b and 3) reflecting tumor biology and disease aggressiveness. Both transcriptome-based subtyping and the level of chromosomal instability provide independent prognostic value beyond established prognostic clinicopathological parameters. High chromosomal instability, p53-pathway disruption and APOBEC-related mutations are significantly associated with transcriptomic class 2a and poor outcome. RNA-derived immune cell infiltration is associated with chromosomally unstable tumors and enriched in class 2b. Spatial proteomics analysis confirms the higher infiltration of class 2b tumors and demonstrates an association between higher immune cell infiltration and lower recurrence rates. Finally, the independent prognostic value of the transcriptomic classes is documented in 1228 validation samples using a single sample classification tool. The classifier provides a framework for biomarker discovery and for optimizing treatment and surveillance in next-generation clinical trials.

Stage-stratified molecular profiling of non-muscle-invasive bladder cancer enhances biological, clinical, and therapeutic insight.

Understanding the molecular determinants that underpin the clinical heterogeneity of non-muscle-invasive bladder cancer (NMIBC) is essential for prognostication and therapy development. Stage T1 disease in particular presents a high risk of progression and requires improved understanding. We present a detailed multi-omics study containing gene expression, copy number, and mutational profiles that show relationships to immune infiltration, disease recurrence, and progression to muscle invasion. We compare expression and genomic subtypes derived from all NMIBCs with those derived from the individual disease stages Ta and T1. We show that sufficient molecular heterogeneity exists within the separate stages to allow subclassification and that this is more clinically meaningful for stage T1 disease than that derived from all NMIBCs. This provides improved biological understanding and identifies subtypes of T1 tumors that may benefit from chemo- or immunotherapy.

Monitoring of urothelial cancer disease status after treatment by digital droplet PCR liquid biopsy assays.

OBJECTIVES: Real-time monitoring of disease status would be beneficial for timely decision making in the treatment of urothelial cancer (UC), and may accelerate the evaluation of clinical trials. Use of cell free tumor DNA (cftDNA) as a biomarker in liquid biopsy is minimally invasive and its successful use has been reported in various cancer types, including UC. The objective of this study was to evaluate the use of digital droplet PCR (ddPCR)-based assays to monitor UC after treatment. METHOD AND MATERIALS: Blood, urine and matching formalin fixed, paraffin embedded diagnostic specimens were collected from 20 patients diagnosed with stage T1 (n = 2) and T2/T3 (n = 18) disease. SNaPshot assays, Sanger sequencing and whole exome sequencing were used to identify tumor-specific mutations, and somatic mutation status was confirmed using patient-matched DNAs extracted from buffy coats and peripheral blood mononucleocytes. The ddPCR assays of the tumor-specific mutations were used to detect the fractional abundance of cftDNA in plasma and urine. RESULTS: SNaPshot and Sanger sequencing identified point mutations in 70% of the patients that were assayable by ddPCR. Cases of remission and relapse monitored by assays for PIK3CA E542K and TP53 Y163C mutations in plasma and urine concurred with clinical observations up to 48 months from the start of chemotherapy. A new ddPCR assay for the telomerase reverse transcriptase (TERT) promoter (-124) mutation was developed. The TERT assay was able to detect mutations in cases below the limit of detection by SNaPshot. Whole exome sequencing identified a novel mutation, CNTNAP4 G727*. A ddPCR assay designed to detect this mutation was able to distinguish mutant from wild-type alleles. CONCLUSIONS: The study demonstrated that ddPCR assays could be used to detect cftDNA in liquid biopsy monitoring of the post-therapy disease status in patients with UC. Overall, 70% of the patients in our study harbored mutations that were assayable by ddPCR.

ETV5 links the FGFR3 and Hippo signalling pathways in bladder cancer.

Activating mutations of fibroblast growth factor receptor 3 (FGFR3) are common in urothelial carcinoma of the bladder (UC). Silencing or inhibition of mutant FGFR3 in bladder cancer cell lines is associated with decreased malignant potential, confirming its important driver role in UC. However, understanding of how FGFR3 activation drives urothelial malignant transformation remains limited. We have previously shown that mutant FGFR3 alters the cell-cell and cell-matrix adhesion properties of urothelial cells, resulting in loss of contact-inhibition of proliferation. In this study, we investigate a transcription factor of the ETS-family, ETV5, as a putative effector of FGFR3 signalling in bladder cancer. We show that FGFR3 signalling induces a MAPK/ERK-mediated increase in ETV5 levels, and that this results in increased level of TAZ, a co-transcriptional regulator downstream of the Hippo signalling pathway involved in cell-contact inhibition. We also demonstrate that ETV5 is a key downstream mediator of the oncogenic effects of mutant FGFR3, as its knockdown in FGFR3-mutant bladder cancer cell lines is associated with reduced proliferation and anchorage-independent growth. Overall this study advances our understanding of the molecular alterations occurring during urothelial malignant transformation and indicates TAZ as a possible therapeutic target in FGFR3-dependent bladder tumours.

Genomic Subtypes of Non-invasive Bladder Cancer with Distinct Metabolic Profile and Female Gender Bias in KDM6A Mutation Frequency.

Bladder cancer incurs a higher lifetime treatment cost than other cancers due to frequent recurrence of non-invasive disease. Improved prognostic biomarkers and localized therapy are needed for this large patient group. We defined two major genomic subtypes of primary stage Ta tumors. One of these was characterized by loss of 9q including TSC1, increased KI67 labeling index, upregulated glycolysis, DNA repair, mTORC1 signaling, features of the unfolded protein response, and altered cholesterol homeostasis. Comparison with muscle-invasive bladder cancer mutation profiles revealed lower overall mutation rates and more frequent mutations in RHOB and chromatin modifier genes. More mutations in the histone lysine demethylase KDM6A were present in non-invasive tumors from females than males.

Polycomb Repressor Complex 1 Member, BMI1 Contributes to Urothelial Tumorigenesis through p16-Independent Mechanisms.

Urothelial carcinoma (UC) causes significant morbidity and remains the most expensive cancer to treat because of the need for repeated resections and lifelong monitoring for patients with non-muscle-invasive bladder cancer (NMIBC). Novel therapeutics and stratification approaches are needed to improve the outlook for both NMIBC and muscle-invasive bladder cancer. We investigated the expression and effects of B Lymphoma Mo-MLV Insertion Region 1 (BMI1) in UC. BMI1 was found to be overexpressed in most UC cell lines and primary tumors by quantitative real-time polymerase chain reaction and immunohistochemistry. In contrast to some previous reports, no association with tumor stage or grade was observed in two independent tumor panels. Furthermore, upregulation of BMI1 was detected in premalignant bladder lesions, suggesting a role early in tumorigenesis. BMI1 is not located within a common region of genomic amplification in UC. The CDKN2A locus (which encodes the p16 tumor suppressor gene) is a transcriptional target of BMI1 in some cellular contexts. In UC cell lines and primary tissues, no correlation between BMI1 and p16 expression was observed. Retroviral-mediated overexpression of BMI1 immortalized normal human urothelial cells (NHUC) in vitro and was associated with induction of telomerase activity, bypass of senescence, and repression of differentiation. The effects of BMI1 on gene expression were identified by expression microarray analysis of NHUC-BMI1. Metacore analysis of the gene expression profile implicated downstream effects of BMI1 on α4/ß1 integrin-mediated adhesion, cytoskeleton remodeling, and CREB1-mediated transcription.

Towards a Next-Generation Sequencing Diagnostic Service for Tumour Genotyping: A Comparison of Panels and Platforms.

Detection of clinically actionable mutations in diagnostic tumour specimens aids in the selection of targeted therapeutics. With an ever increasing number of clinically significant mutations identified, tumour genetic diagnostics is moving from single to multigene analysis. As it is still not feasible for routine diagnostic laboratories to perform sequencing of the entire cancer genome, our approach was to undertake targeted mutation detection. To optimise our diagnostic workflow, we evaluated three target enrichment strategies using two next-generation sequencing (NGS) platforms (Illumina MiSeq and Ion PGM). The target enrichment strategies were Fluidigm Access Array custom amplicon panel including 13 genes (MiSeq sequencing), the Oxford Gene Technologies (OGT) SureSeq Solid Tumour hybridisation panel including 60 genes (MiSeq sequencing), and an Ion AmpliSeq Cancer Hotspot Panel including 50 genes (Ion PGM sequencing). DNA extracted from formalin-fixed paraffin-embedded (FFPE) blocks of eight previously characterised cancer cell lines was tested using the three panels. Matching genomic DNA from fresh cultures of these cell lines was also tested using the custom Fluidigm panel and the OGT SureSeq Solid Tumour panel. Each panel allowed mutation detection of core cancer genes including KRAS, BRAF, and EGFR. Our results indicate that the panels enable accurate variant detection despite sequencing from FFPE DNA.

Frequency of TERT Promoter Mutations in Prostate Cancer.

OBJECTIVE: Recently, recurrent mutations within the core promoter of the human telomerase reverse transcriptase (TERT) gene generating consensus binding sites for ETS transcription factor family members were described in melanomas and other malignancies (e.g. bladder cancer, hepatocellular carcinoma). These mutations were discussed as early drivers for malignant transformation. In prostate cancer (PrCa) TERT expression has been associated with a poor prognosis and higher risk for disease recurrence. The underlying mechanisms for high TERT expression in PrCa have still not been clarified. To date, data on TERT promoter mutation analysis in PrCa are sparse. Therefore, we performed sequence analysis of the core promoter region of the TERT gene in an unselected cohort of prostate tumors. METHODS: Sections from 167 formalin-fixed, paraffin-embedded and cryopreserved prostate tumors were microdissected and used for DNA isolation. The mutation hotspot region within the TERT core promoter (-260 to +60) was analyzed by direct Sanger sequencing or SNaPshot analysis. RESULTS: All cases were analyzed successfully. Mutations within the core promoter of the TERT gene were not detected in any of the cases with all tumors exhibiting a wild-type sequence. CONCLUSION: TERT core promoter mutations reported from several other malignancies were not detected in our unselected cohort of PrCa. These data indicate that alterations within the core promoter of the TERT gene do not play an important role in prostate carcinogenesis.

Molecular biology of bladder cancer: new insights into pathogenesis and clinical diversity.

Urothelial carcinoma of the bladder comprises two long-recognized disease entities with distinct molecular features and clinical outcome. Low-grade non-muscle-invasive tumours recur frequently but rarely progress to muscle invasion, whereas muscle-invasive tumours are usually diagnosed de novo and frequently metastasize. Recent genome-wide expression and sequencing studies identify genes and pathways that are key drivers of urothelial cancer and reveal a more complex picture with multiple molecular subclasses that traverse conventional grade and stage groupings. This improved understanding of molecular features, disease pathogenesis and heterogeneity provides new opportunities for prognostic application, disease monitoring and personalized therapy.

Molecular subtyping of invasive bladder cancer: time to divide and rule?

Therapeutic decisions for muscle-invasive bladder cancer (MIBC) are largely based on histopathologic characteristics. In this issue of Cancer Cell, Choi and colleagues report three molecular subtypes of MIBC with the potential to guide prognosis, patient stratification, and treatment.

Frequent inactivating mutations of STAG2 in bladder cancer are associated with low tumour grade and stage and inversely related to chromosomal copy number changes.

Inactivating mutations of STAG2 have been reported at low frequency in several cancers. In glioblastoma, the function of STAG2 has been related to maintenance of euploidy via its role in the cohesin complex. In a screen of a large series of bladder tumours and cell lines, we found inactivating mutations (nonsense, frameshift and splicing) in 67 of 307 tumours (21.8%) and 6 of 47 cell lines. Thirteen missense mutations of unknown significance were also identified. Inactivating mutation was associated with low tumour stage (P = 0.001) and low grade (P = 0.0002). There was also a relationship with female patient gender (P = 0.042). Examination of copy number profiles revealed an inverse relationship of mutation with both fraction of genome altered and whole chromosome copy number changes. Immunohistochemistry showed that in the majority of cases with inactivating mutations, STAG2 protein expression was absent. Strikingly, we identified a relatively large subset of tumours (12%) with areas of both positive and negative immunoreactivity, in only four of which a potentially function-altering mutation was detected. Regions of differential expression were contiguous and showed similar morphological phenotype in all cases. Microdissected positive and negative areas from one tumour showed an inactivating mutation to be present only in the negative area, suggesting intra-tumoral sub-clonal genomic evolution. Our findings indicate that loss of STAG2 function plays a more important role in non-invasive than that in muscle-invasive bladder cancer and suggest that cohesin complex-independent functions are likely to be important in these cases.