Urine-derived bladder cancer organoids (urinoids) as a tool for cancer longitudinal response monitoring and therapy adaptation.

BACKGROUND: Bladder cancer is one of the most common cancer types worldwide. Generally, research relies on invasive sampling strategies. METHODS: Here, we generate bladder cancer organoids directly from urine (urinoids). In this project, we establish 12 urinoid lines from 22 patients with non-muscle and muscle-invasive bladder tumours, with an efficiency of 55%. RESULTS: The histopathological features of the urinoids accurately resemble those of the original bladder tumours. Genetically, there is a high concordance of single nucleotide polymorphisms (92.56%) and insertions & deletions (91.54%) between urinoids and original tumours from patient 4. Furthermore, these urinoids show sensitivity to bladder cancer drugs, similar to their tissue-derived organoid counterparts. Genetic analysis of longitudinally generated tumoroids and urinoids from one patient receiving systemic immunotherapy, identify alterations that may guide the choice for second-line therapy. Successful treatment adaptation was subsequently demonstrated in the urinoid setting. CONCLUSION: Therefore, urinoids can advance precision medicine in bladder cancer as a non-invasive platform for tumour pathogenesis, longitudinal drug-response monitoring, and therapy adaptation.

Early invasion of the bladder wall by solitary bacteria protects UPEC from antibiotics and neutrophil swarms in an organoid model.

Recurrence of uropathogenic Escherichia coli (UPEC) infections has been attributed to reactivation of quiescent intracellular reservoirs (QIRs) in deep layers of the bladder wall. QIRs are thought to arise late during infection following dispersal of bacteria from intracellular bacterial communities (IBCs) in superficial umbrella cells. Here, we track the formation of QIR-like bacteria in a bladder organoid model that recapitulates the stratified uroepithelium within a volume suitable for high-resolution live-cell imaging. Bacteria injected into the organoid lumen enter umbrella-like cells and proliferate to form IBC-like bodies. In parallel, single bacteria penetrate deeper layers of the organoid wall, where they localize within or between uroepithelial cells. These "solitary" bacteria evade killing by antibiotics and neutrophils and are morphologically distinct from bacteria in IBCs. We conclude that bacteria with QIR-like properties may arise at early stages of infection, independent of IBC formation and rupture.

Patient-derived organoids as a predictive biomarker for treatment response in cancer patients.

Effective predictive biomarkers are needed to enable personalized medicine and increase treatment efficacy and survival for cancer patients, thereby reducing toxic side effects and treatment costs. Patient-derived organoids (PDOs) enable individualized tumour response testing. Since 2018, 17 publications have examined PDOs as a potential predictive biomarker in the treatment of cancer patients. We review and provide a pooled analysis of the results regarding the use of PDOs in individualized tumour response testing, focusing on evidence for analytical validity, clinical validity and clinical utility. We identify future perspectives to accelerate the implementation of PDOs as a predictive biomarker in the treatment of cancer patients.

Targeting G542X CFTR nonsense alleles with ELX-02 restores CFTR function in human-derived intestinal organoids.

BACKGROUND: Promoting full-length protein production is a requisite step to address some of the remaining unmet medical need for those with Cystic Fibrosis (CF) nonsense alleles. ELX-02 promotes read-through of mRNA transcripts bearing nonsense mutations, including the most common CF nonsense allele G542X, in several different preclinical models including human bronchial epithelial cells. Here we evaluate ELX-02 mediated read-through using the CFTR-dependent Forskolin-induced swelling (FIS) assay across a selection of G542X genotype patient derived organoids (PDOs). METHODS: CFTR functional restoration was evaluated in ELX-02 treated G542X homozygous and heterozygous PDOs in the CFTR-dependent FIS assay. CFTR mRNA abundance and integrity were evaluated by qPCR and Nanostring analysis while PDO protein was detected by capillary based size-exclusion chromatography. RESULTS: PDOs homozygous for G542X or heterozygous with a second minimally functional allele had significantly increased CFTR activity with ELX-02 in a dose-dependent fashion across a variety of forskolin induction concentrations. The functional increases are similar to those obtained with tezacaftor/ivacaftor in F508del homozygous PDOs. Increased CFTR C- and B-band protein was observed in accordance with increased function. In addition, ELX-02 treatment of a G542X/G542X PDO results in a 5-fold increase in CFTR mRNA compared with vehicle treated, resulting in normalization of CFTR mRNA as measured via Nanostring. CONCLUSIONS: These data with ELX-02 in PDOs are consistent with previous G542X model evaluations. These results also support the on-going clinical evaluation of ELX-02 as a read-through agent for CF caused by the G542X allele.

Protocol for Application, Standardization and Validation of the Forskolin-Induced Swelling Assay in Cystic Fibrosis Human Colon Organoids.

This protocol describes the isolation, handling, culture of, and experiments with human colon stem cell organoids in the context of cystic fibrosis (CF). In human colon organoids, the function of cystic fibrosis transmembrane conductance regulator (CFTR) protein and its rescue by CFTR modulators can be quantified using the forskolin-induced swelling assay. Implementation procedures and validation experiments are described for six CF human colon organoid lines, and representative CFTR genotypes are tested for basal CFTR function and response to CFTR-modulating drugs. For complete details on the use and execution of this protocol, please refer to Dekkers et al (2016) and Berkers and van Mourik (2019).

CRISPR-Based Adenine Editors Correct Nonsense Mutations in a Cystic Fibrosis Organoid Biobank.

Adenine base editing (ABE) enables enzymatic conversion from A-T into G-C base pairs. ABE holds promise for clinical application, as it does not depend on the introduction of double-strand breaks, contrary to conventional CRISPR/Cas9-mediated genome engineering. Here, we describe a cystic fibrosis (CF) intestinal organoid biobank, representing 664 patients, of which ~20% can theoretically be repaired by ABE. We apply SpCas9-ABE (PAM recognition sequence: NGG) and xCas9-ABE (PAM recognition sequence: NGN) on four selected CF organoid samples. Genetic and functional repair was obtained in all four cases, while whole-genome sequencing (WGS) of corrected lines of two patients did not detect off-target mutations. These observations exemplify the value of large, patient-derived organoid biobanks representing hereditary disease and indicate that ABE may be safely applied in human cells.

Mouse and human urothelial cancer organoids: A tool for bladder cancer research.

Bladder cancer is a common malignancy that has a relatively poor outcome. Lack of culture models for the bladder epithelium (urothelium) hampers the development of new therapeutics. Here we present a long-term culture system of the normal mouse urothelium and an efficient culture system of human bladder cancer cells. These so-called bladder (cancer) organoids consist of 3D structures of epithelial cells that recapitulate many aspects of the urothelium. Mouse bladder organoids can be cultured efficiently and genetically manipulated with ease, which was exemplified by creating genetic knockouts in the tumor suppressors Trp53 and Stag2. Human bladder cancer organoids can be derived efficiently from both resected tumors and biopsies and cultured and passaged for prolonged periods. We used this feature of human bladder organoids to create a living biobank consisting of bladder cancer organoids derived from 53 patients. Resulting organoids were characterized histologically and functionally. Organoid lines contained both basal and luminal bladder cancer subtypes based on immunohistochemistry and gene expression analysis. Common bladder cancer mutations like TP53 and FGFR3 were found in organoids in the biobank. Finally, we performed limited drug testing on organoids in the bladder cancer biobank.

Oncogenic hijacking of the stress response machinery in T cell acute lymphoblastic leukemia.

Cellular transformation is accompanied by extensive rewiring of many biological processes leading to augmented levels of distinct types of cellular stress, including proteotoxic stress. Cancer cells critically depend on stress-relief pathways for their survival. However, the mechanisms underlying the transcriptional initiation and maintenance of the oncogenic stress response remain elusive. Here, we show that the expression of heat shock transcription factor 1 (HSF1) and the downstream mediators of the heat shock response is transcriptionally upregulated in T cell acute lymphoblastic leukemia (T-ALL). Hsf1 ablation suppresses the growth of human T-ALL and eradicates leukemia in mouse models of T-ALL, while sparing normal hematopoiesis. HSF1 drives a compact transcriptional program and among the direct HSF1 targets, specific chaperones and co-chaperones mediate its critical role in T-ALL. Notably, we demonstrate that the central T-ALL oncogene NOTCH1 hijacks the cellular stress response machinery by inducing the expression of HSF1 and its downstream effectors. The NOTCH1 signaling status controls the levels of chaperone/co-chaperone complexes and predicts the response of T-ALL patient samples to HSP90 inhibition. Our data demonstrate an integral crosstalk between mediators of oncogene and non-oncogene addiction and reveal critical nodes of the heat shock response pathway that can be targeted therapeutically.

MED12 Regulates HSC-Specific Enhancers Independently of Mediator Kinase Activity to Control Hematopoiesis.

Hematopoietic-specific transcription factors require coactivators to communicate with the general transcription machinery and establish transcriptional programs that maintain hematopoietic stem cell (HSC) self-renewal, promote differentiation, and prevent malignant transformation. Mediator is a large coactivator complex that bridges enhancer-localized transcription factors with promoters, but little is known about Mediator function in adult stem cell self-renewal and differentiation. We show that MED12, a member of the Mediator kinase module, is an essential regulator of HSC homeostasis, as in vivo deletion of Med12 causes rapid bone marrow aplasia leading to acute lethality. Deleting other members of the Mediator kinase module does not affect HSC function, suggesting kinase-independent roles of MED12. MED12 deletion destabilizes P300 binding at lineage-specific enhancers, resulting in H3K27Ac depletion, enhancer de-activation, and consequent loss of HSC stemness signatures. As MED12 mutations have been described recently in blood malignancies, alterations in MED12-dependent enhancer regulation may control both physiological and malignant hematopoiesis.

Cohesin loss alters adult hematopoietic stem cell homeostasis, leading to myeloproliferative neoplasms.

The cohesin complex (consisting of Rad21, Smc1a, Smc3, and Stag2 proteins) is critically important for proper sister chromatid separation during mitosis. Mutations in the cohesin complex were recently identified in a variety of human malignancies including acute myeloid leukemia (AML). To address the potential tumor-suppressive function of cohesin in vivo, we generated a series of shRNA mouse models in which endogenous cohesin can be silenced inducibly. Notably, silencing of cohesin complex members did not have a deleterious effect on cell viability. Furthermore, knockdown of cohesin led to gain of replating capacity of mouse hematopoietic progenitor cells. However, cohesin silencing in vivo rapidly altered stem cells homeostasis and myelopoiesis. Likewise, we found widespread changes in chromatin accessibility and expression of genes involved in myelomonocytic maturation and differentiation. Finally, aged cohesin knockdown mice developed a clinical picture closely resembling myeloproliferative disorders/neoplasms (MPNs), including varying degrees of extramedullary hematopoiesis (myeloid metaplasia) and splenomegaly. Our results represent the first successful demonstration of a tumor suppressor function for the cohesin complex, while also confirming that cohesin mutations occur as an early event in leukemogenesis, facilitating the potential development of a myeloid malignancy.

TET1 is a tumor suppressor of hematopoietic malignancy.

The methylcytosine dioxygenase TET1 ('ten-eleven translocation 1') is an important regulator of 5-hydroxymethylcytosine (5hmC) in embryonic stem cells. The diminished expression of TET proteins and loss of 5hmC in many tumors suggests a critical role for the maintenance of this epigenetic modification. Here we found that deletion of Tet1 promoted the development of B cell lymphoma in mice. TET1 was required for maintenance of the normal abundance and distribution of 5hmC, which prevented hypermethylation of DNA, and for regulation of the B cell lineage and of genes encoding molecules involved in chromosome maintenance and DNA repair. Whole-exome sequencing of TET1-deficient tumors revealed mutations frequently found in non-Hodgkin B cell lymphoma (B-NHL), in which TET1 was hypermethylated and transcriptionally silenced. Our findings provide in vivo evidence of a function for TET1 as a tumor suppressor of hematopoietic malignancy.

Contrasting roles of histone 3 lysine 27 demethylases in acute lymphoblastic leukaemia.

T-cell acute lymphoblastic leukaemia (T-ALL) is a haematological malignancy with a dismal overall prognosis, including a relapse rate of up to 25%, mainly because of the lack of non-cytotoxic targeted therapy options. Drugs that target the function of key epigenetic factors have been approved in the context of haematopoietic disorders, and mutations that affect chromatin modulators in a variety of leukaemias have recently been identified; however, 'epigenetic' drugs are not currently used for T-ALL treatment. Recently, we described that the polycomb repressive complex 2 (PRC2) has a tumour-suppressor role in T-ALL. Here we delineated the role of the histone 3 lysine 27 (H3K27) demethylases JMJD3 and UTX in T-ALL. We show that JMJD3 is essential for the initiation and maintenance of T-ALL, as it controls important oncogenic gene targets by modulating H3K27 methylation. By contrast, we found that UTX functions as a tumour suppressor and is frequently genetically inactivated in T-ALL. Moreover, we demonstrated that the small molecule inhibitor GSKJ4 (ref. 5) affects T-ALL growth, by targeting JMJD3 activity. These findings show that two proteins with a similar enzymatic function can have opposing roles in the context of the same disease, paving the way for treating haematopoietic malignancies with a new category of epigenetic inhibitors.

The ubiquitin ligase FBXW7 modulates leukemia-initiating cell activity by regulating MYC stability.

Sequencing efforts led to the identification of somatic mutations that could affect the self-renewal and differentiation of cancer-initiating cells. One such recurrent mutation targets the binding pocket of the ubiquitin ligase Fbxw7. Missense FBXW7 mutations are prevalent in various tumors, including T cell acute lymphoblastic leukemia (T-ALL). To study the effects of such lesions, we generated animals carrying regulatable Fbxw7 mutant alleles. Here, we show that these mutations specifically bolster cancer-initiating cell activity in collaboration with Notch1 oncogenes but spare normal hematopoietic stem cell function. We were also able to show that FBXW7 mutations specifically affect the ubiquitylation and half-life of c-Myc protein, a key T-ALL oncogene. Using animals carrying c-Myc fusion alleles, we connected Fbxw7 function to c-Myc abundance and correlated c-Myc expression to leukemia-initiating activity. Finally, we demonstrated that small-molecule-mediated suppression of MYC activity leads to T-ALL remission, suggesting an effective therapeutic strategy.

Mechanisms of epigenetic regulation of leukemia onset and progression.

Over the past decade, it has become clear that both genetics and epigenetics play pivotal roles in cancer onset and progression. The importance of epigenetic regulation in proper maintenance of cellular state is highlighted by the frequent mutation of chromatin modulating factors across cancer subtypes. Identification of these mutations has created an interest in designing drugs that target enzymes involved in DNA methylation and posttranslational modification of histones. In this review, we discuss recurrent genetic alterations to epigenetic modulators in both myeloid and lymphoid leukemias. Furthermore, we review how these perturbations contribute to leukemogenesis and impact disease outcome and treatment efficacy. Finally, we discuss how the recent advances in our understanding of chromatin biology may impact treatment of leukemia.

Interleukin-1R-associated kinase 2 is a novel modulator of the transforming growth factor beta signaling cascade.

The transforming growth factor beta (TGFbeta) pathway orchestrates an extensive transcriptional program that is important for many processes in the cell. For example, TGFbeta regulates cell cycle, migration, and epithelial-to-mesenchymal transition. The TGFbeta pathway has a dual role in cancer: it is involved in early-stage tumor suppression but also contributes to tumor progression by promoting invasion. To identify the novel genes involved in TGFbeta pathway signaling, we have performed a functional genetic loss-of-function screen. We screened a small interfering RNA library targeting 700 kinases and kinase-related genes in a TGFbeta-responsive reporter assay. Several genes were identified that upon knockdown could repress the reporter signal; among these are the two cellular receptors for TGFbeta. In addition to these two known components of the TGFbeta pathway, several genes were identified that were previously not linked to the TGFbeta signaling. Knockdown of one of these genes, the IRAK2 kinase, resulted not only in an impaired TGFbeta target gene response but also in a reduction of the nuclear accumulation and phosphorylation of SMAD2. In addition, suppression of interleukin-1R-associated kinase 2 expression led to a partial override of a TGFbeta-induced cell cycle arrest. Our data show that interleukin-1R-associated kinase 2 is a novel and critical component of TGFbeta signaling.

Candidate biomarkers of response to an experimental cancer drug identified through a large-scale RNA interference genetic screen.

PURPOSE: A major impediment in the optimal selection of cancer patients for the most effective therapy is the lack of suitable biomarkers that foretell the response of a patient to a given drug. In the present study, we have used large-scale RNA interference-based genetic screens to find candidate biomarkers of resistance to a new acyl sulfonamide derivative, R3200. This compound inhibits the proliferation of tumor cells in vitro and in vivo, but its mechanism of action is unknown. EXPERIMENTAL DESIGN: We used a large-scale RNA interference genetic screen to identify modulators of the efficacy of R3200. We searched for genes whose suppression in an in vitro cell system could cause resistance to the anticancer effects of R3200. RESULTS: We report here that knockdown of either RBX1 or DDB1 causes resistance to the anticancer effects of R3200, raising the possibility that these two genes may have utility as biomarkers of response to this drug in a clinical setting. Interestingly, both RBX1 and DDB1 are part of an E3 ubiquitin ligase complex. CONCLUSIONS: We propose that suppression of the activity of a RBX1 and DDB1-containing E3 ligase complex leads to the stabilization of certain proteins, the increased abundance of which is in turn responsible for resistance to R3200. Moreover, our data suggest that RBX1 and DDB1 could potentially be developed into biomarkers of resistance to acyl sulfonamide-based cancer drugs. This will require clinical validation in a series of patients treated with R3200.

A large scale shRNA barcode screen identifies the circadian clock component ARNTL as putative regulator of the p53 tumor suppressor pathway.

BACKGROUND: The p53 tumor suppressor gene is mutated in about half of human cancers, but the p53 pathway is thought to be functionally inactivated in the vast majority of cancer. Understanding how tumor cells can become insensitive to p53 activation is therefore of major importance. Using an RNAi-based genetic screen, we have identified three novel genes that regulate p53 function. RESULTS: We have screened the NKI shRNA library targeting 8,000 human genes to identify modulators of p53 function. Using the shRNA barcode technique we were able to quickly identify active shRNA vectors from a complex mixture. Validation of the screening results indicates that the shRNA barcode technique can reliable identify active shRNA vectors from a complex pool. Using this approach we have identified three genes, ARNTL, RBCK1 and TNIP1, previously unknown to regulate p53 function. Importantly, ARNTL (BMAL1) is an established component of the circadian regulatory network. The latter finding adds to recent observations that link circadian rhythm to the cell cycle and cancer. We show that cells having suppressed ARNTL are unable to arrest upon p53 activation associated with an inability to activate the p53 target gene p21(CIP1). CONCLUSIONS: We identified three new regulators of the p53 pathway through a functional genetic screen. The identification of the circadian core component ARNTL strengthens the link between circadian rhythm and cancer.

Miz1 and HectH9 regulate the stability of the checkpoint protein, TopBP1.

The Myc-associated zinc-finger protein, Miz1, activates transcription of the p21cip1 gene in response to UV irradiation. Miz1 associates with topoisomerase II binding protein1 (TopBP1), an essential activator of the Atr kinase. We show here that Miz1 is required for the recruitment of a fraction of TopBP1 to chromatin, for the protection of TopBP1 from proteasomal degradation and for Atr-dependent signal transduction. TopBP1 that is not bound to chromatin is degraded by the HectH9 (Mule, ARF-BP1 and HUWE1) ubiquitin ligase. Myc antagonizes the binding of TopBP1 to Miz1; as a result, expression of Myc leads to dissociation of TopBP1 from chromatin, reduces the amount of total TopBP1 and attenuates Atr-dependent signal transduction. Our data show that Miz1 and Myc affect the activity of the Atr checkpoint through their effect on TopBP1 chromatin association and stability.

An shRNA barcode screen provides insight into cancer cell vulnerability to MDM2 inhibitors.

The identification of the cellular targets of small molecules with anticancer activity is crucial to their further development as drug candidates. Here, we present the application of a large-scale RNA interference-based short hairpin RNA (shRNA) barcode screen to gain insight in the mechanism of action of nutlin-3 (1). Nutlin-3 is a small-molecule inhibitor of MDM2, which can activate the p53 pathway. Nutlin-3 shows strong antitumor effects in mice, with surprisingly few side effects on normal tissues. Aside from p53, we here identify 53BP1 as a critical mediator of nutlin-3-induced cytotoxicity. 53BP1 is part of a signaling network induced by DNA damage that is frequently activated in cancer but not in healthy tissues. Our results suggest that nutlin-3's tumor specificity may result from its ability to turn a cancer cell-specific property (activated DNA damage signaling) into a weakness that can be exploited therapeutically.