Childhood cancer mutagenesis caused by transposase-derived PGBD5.

Genomic rearrangements are a hallmark of most childhood tumors, including medulloblastoma, one of the most common brain tumors in children, but their causes remain largely unknown. Here, we show that PiggyBac transposable element derived 5 (Pgbd5) promotes tumor development in multiple developmentally accurate mouse models of Sonic Hedgehog (SHH) medulloblastoma. Most Pgbd5-deficient mice do not develop tumors, while maintaining normal cerebellar development. Ectopic activation of SHH signaling is sufficient to enforce cerebellar granule cell progenitor-like cell states, which exhibit Pgbd5-dependent expression of distinct DNA repair and neurodevelopmental factors. Mouse medulloblastomas expressing Pgbd5 have increased numbers of somatic structural DNA rearrangements, some of which carry PGBD5-specific sequences at their breakpoints. Similar sequence breakpoints recurrently affect somatic DNA rearrangements of known tumor suppressors and oncogenes in medulloblastomas in 329 children. This identifies PGBD5 as a medulloblastoma mutator and provides a genetic mechanism for the generation of oncogenic DNA rearrangements in childhood cancer.

p66ShcA promotes malignant breast cancer phenotypes by alleviating energetic and oxidative stress.

Significant efforts have focused on identifying targetable genetic drivers that support the growth of solid tumors and/or increase metastatic ability. During tumor development and progression to metastatic disease, physiological and pharmacological selective pressures influence parallel adaptive strategies within cancer cell sub-populations. Such adaptations allow cancer cells to withstand these stressful microenvironments. This Darwinian model of stress adaptation often prevents durable clinical responses and influences the emergence of aggressive cancers with increased metastatic fitness. However, the mechanisms contributing to such adaptive stress responses are poorly understood. We now demonstrate that the p66ShcA redox protein, itself a ROS inducer, is essential for survival in response to physiological stressors, including anchorage independence and nutrient deprivation, in the context of poor outcome breast cancers. Mechanistically, we show that p66ShcA promotes both glucose and glutamine metabolic reprogramming in breast cancer cells, to increase their capacity to engage catabolic metabolism and support glutathione synthesis. In doing so, chronic p66ShcA exposure contributes to adaptive stress responses, providing breast cancer cells with sufficient ATP and redox balance needed to withstand such transient stressed states. Our studies demonstrate that p66ShcA functionally contributes to the maintenance of aggressive phenotypes and the emergence of metastatic disease by forcing breast tumors to adapt to chronic and moderately elevated levels of oxidative stress.

The Deubiquitylase Otub1 Regulates the Chemotactic Response of Splenic B Cells by Modulating the Stability of the γ-Subunit Gng2.

The ubiquitin proteasome system performs the covalent attachment of lysine 48-linked polyubiquitin chains to substrate proteins, thereby targeting them for degradation, while deubiquitylating enzymes (DUBs) reverse this process. This posttranslational modification regulates key features both of innate and adaptative immunity, including antigen presentation, protein homeostasis and signal transduction. Here we show that loss of one of the most highly expressed DUBs, Otub1, results in changes in murine splenic B cell subsets, leading to a significant increase in marginal zone and transitional B cells and a concomitant decrease in follicular B cells. We demonstrate that Otub1 interacts with the γ-subunit of the heterotrimeric G protein, Gng2, and modulates its ubiquitylation status, thereby controlling Gng2 stability. Proximal mapping of Gng2 revealed an enrichment in partners associated with chemokine signaling, actin cytoskeleton and cell migration. In line with these findings, we show that Otub1-deficient B cells exhibit greater Ca2+ mobilization, F-actin polymerization and chemotactic responsiveness to Cxcl12, Cxcl13 and S1P in vitro, which manifests in vivo as altered localization of B cells within the spleen. Together, our data establishes Otub1 as a novel regulator of G-protein coupled receptor signaling in B cells, regulating their differentiation and positioning in the spleen.

Migration speed of captured breast cancer subpopulations correlates with metastatic fitness.

The genetic alterations contributing to migration proficiency, a phenotypic hallmark of metastatic cells required for colonizing distant organs, remain poorly defined. Here, we used single-cell magneto-optical capture (scMOCa) to isolate fast cells from heterogeneous human breast cancer cell populations, based on their migratory ability alone. We show that captured fast cell subpopulations retain higher migration speed and focal adhesion dynamics over many generations as a result of a motility-related transcriptomic profile. Upregulated genes in isolated fast cells encoded integrin subunits, proto-cadherins and numerous other genes associated with cell migration. Dysregulation of several of these genes correlates with poor survival outcomes in people with breast cancer, and primary tumors established from fast cells generated a higher number of circulating tumor cells and soft tissue metastases in pre-clinical mouse models. Subpopulations of cells selected for a highly migratory phenotype demonstrated an increased fitness for metastasis.

Protein arginine methyltransferase 1 regulates B cell fate after positive selection in the germinal center in mice.

Positively selected germinal center B cells (GCBC) can either resume proliferation and somatic hypermutation or differentiate. The mechanisms dictating these alternative cell fates are incompletely understood. We show that the protein arginine methyltransferase 1 (Prmt1) is upregulated in murine GCBC by Myc and mTORC-dependent signaling after positive selection. Deleting Prmt1 in activated B cells compromises antibody affinity maturation by hampering proliferation and GCBC light zone to dark zone cycling. Prmt1 deficiency also results in enhanced memory B cell generation and plasma cell differentiation, albeit the quality of these cells is compromised by the GCBC defects. We further demonstrate that Prmt1 intrinsically limits plasma cell differentiation, a function co-opted by B cell lymphoma (BCL) cells. Consistently, PRMT1 expression in BCL correlates with poor disease outcome, depends on MYC and mTORC1 activity, is required for cell proliferation, and prevents differentiation. Collectively, these data identify PRMT1 as a determinant of normal and cancerous mature B cell proliferation and differentiation balance.

A Compendium of Syngeneic, Transplantable Pediatric High-Grade Glioma Models Reveals Subtype-Specific Therapeutic Vulnerabilities.

Pediatric high-grade gliomas (pHGG) are lethal, incurable brain tumors frequently driven by clonal mutations in histone genes. They often harbor a range of additional genetic alterations that correlate with different ages, anatomic locations, and tumor subtypes. We developed models representing 16 pHGG subtypes driven by different combinations of alterations targeted to specific brain regions. Tumors developed with varying latencies and cell lines derived from these models engrafted in syngeneic, immunocompetent mice with high penetrance. Targeted drug screening revealed unexpected selective vulnerabilities-H3.3G34R/PDGFRAC235Y to FGFR inhibition, H3.3K27M/PDGFRAWT to PDGFRA inhibition, and H3.3K27M/PDGFRAWT and H3.3K27M/PPM1DΔC/PIK3CAE545K to combined inhibition of MEK and PIK3CA. Moreover, H3.3K27M tumors with PIK3CA, NF1, and FGFR1 mutations were more invasive and harbored distinct additional phenotypes, such as exophytic spread, cranial nerve invasion, and spinal dissemination. Collectively, these models reveal that different partner alterations produce distinct effects on pHGG cellular composition, latency, invasiveness, and treatment sensitivity. SIGNIFICANCE: Histone-mutant pediatric gliomas are a highly heterogeneous tumor entity. Different histone mutations correlate with different ages of onset, survival outcomes, brain regions, and partner alterations. We have developed models of histone-mutant gliomas that reflect this anatomic and genetic heterogeneity and provide evidence of subtype-specific biology and therapeutic targeting. See related commentary by Lubanszky and Hawkins, p. 1516. This article is highlighted in the In This Issue feature, p. 1501.

K27M in canonical and noncanonical H3 variants occurs in distinct oligodendroglial cell lineages in brain midline gliomas.

Canonical (H3.1/H3.2) and noncanonical (H3.3) histone 3 K27M-mutant gliomas have unique spatiotemporal distributions, partner alterations and molecular profiles. The contribution of the cell of origin to these differences has been challenging to uncouple from the oncogenic reprogramming induced by the mutation. Here, we perform an integrated analysis of 116 tumors, including single-cell transcriptome and chromatin accessibility, 3D chromatin architecture and epigenomic profiles, and show that K27M-mutant gliomas faithfully maintain chromatin configuration at developmental genes consistent with anatomically distinct oligodendrocyte precursor cells (OPCs). H3.3K27M thalamic gliomas map to prosomere 2-derived lineages. In turn, H3.1K27M ACVR1-mutant pontine gliomas uniformly mirror early ventral NKX6-1+/SHH-dependent brainstem OPCs, whereas H3.3K27M gliomas frequently resemble dorsal PAX3+/BMP-dependent progenitors. Our data suggest a context-specific vulnerability in H3.1K27M-mutant SHH-dependent ventral OPCs, which rely on acquisition of ACVR1 mutations to drive aberrant BMP signaling required for oncogenesis. The unifying action of K27M mutations is to restrict H3K27me3 at PRC2 landing sites, whereas other epigenetic changes are mainly contingent on the cell of origin chromatin state and cycling rate.

3D chromatin remodeling potentiates transcriptional programs driving cell invasion.

The contribution of deregulated chromatin architecture, including topologically associated domains (TADs), to cancer progression remains ambiguous. CCCTC-binding factor (CTCF) is a central regulator of higher-order chromatin structure that undergoes copy number loss in over half of all breast cancers, but the impact of this defect on epigenetic programming and chromatin architecture remains unclear. We find that under physiological conditions, CTCF organizes subTADs to limit the expression of oncogenic pathways, including phosphatidylinositol 3-kinase (PI3K) and cell adhesion networks. Loss of a single CTCF allele potentiates cell invasion through compromised chromatin insulation and a reorganization of chromatin architecture and histone programming that facilitates de novo promoter-enhancer contacts. However, this change in the higher-order chromatin landscape leads to a vulnerability to inhibitors of mTOR. These data support a model whereby subTAD reorganization drives both modification of histones at de novo enhancer-promoter contacts and transcriptional up-regulation of oncogenic transcriptional networks.

Clinical Characteristics of the End-of-Life Phase in Children with Life-Limiting Diseases: Retrospective Study from a Single Center for Pediatric Palliative Care.

BACKGROUND: Data on the end-of-life phase of children receiving palliative care are limited. The purpose of this study is to investigate the spectrum of symptoms of terminally ill children, adolescents, and young adults, depending on their underlying disease. METHODS: Findings are based on a 4.5-year retrospective study of 89 children who received palliative care before they died, investigating the symptomatology of the last two weeks before death. RESULTS: In this study, the most common clinical symptomatology present in children undergoing end-of-life care includes pain, shortness of breath, anxiety, nausea, and constipation. Out of 89 patients included in this study, 47% suffered from an oncological disease. Oncological patients had a significantly higher symptom burden at the end of life (p < 0.05) compared to other groups, and the intensity of symptoms increased as the underlying disease progressed. The likelihood of experiencing pain and nausea/vomiting was also significantly higher in oncological patients (p = 0.016). CONCLUSIONS: We found that the underlying disease is associated with marked differences in the respective leading clinical symptom. Therefore, related to these differences, symptom management has to be adjusted according to the underlying disease, since the underlying disorder seems to exert an influence on the severity of symptoms and thereby on the modality and choice of treatment. This study is intended to aid underlying disease-specific symptom management at the end-of-life care for children, adolescents, and young adults, with a specific focus on end-of-life care in a home environment.

STAT1 potentiates oxidative stress revealing a targetable vulnerability that increases phenformin efficacy in breast cancer.

Bioenergetic perturbations driving neoplastic growth increase the production of reactive oxygen species (ROS), requiring a compensatory increase in ROS scavengers to limit oxidative stress. Intervention strategies that simultaneously induce energetic and oxidative stress therefore have therapeutic potential. Phenformin is a mitochondrial complex I inhibitor that induces bioenergetic stress. We now demonstrate that inflammatory mediators, including IFNγ and polyIC, potentiate the cytotoxicity of phenformin by inducing a parallel increase in oxidative stress through STAT1-dependent mechanisms. Indeed, STAT1 signaling downregulates NQO1, a key ROS scavenger, in many breast cancer models. Moreover, genetic ablation or pharmacological inhibition of NQO1 using ß-lapachone (an NQO1 bioactivatable drug) increases oxidative stress to selectively sensitize breast cancer models, including patient derived xenografts of HER2+ and triple negative disease, to the tumoricidal effects of phenformin. We provide evidence that therapies targeting ROS scavengers increase the anti-neoplastic efficacy of mitochondrial complex I inhibitors in breast cancer.

Copy number and transcriptome alterations associated with metastatic lesion response to treatment in colorectal cancer.

BACKGROUND: Therapeutic resistance is the main cause of death in metastatic colorectal cancer. To investigate genomic plasticity, most specifically of metastatic lesions, associated with response to first-line systemic therapy, we collected longitudinal liver metastatic samples and characterized the copy number aberration (CNA) landscape and its effect on the transcriptome. METHODS: Liver metastatic biopsies were collected prior to treatment (pre, n = 97) and when clinical imaging demonstrated therapeutic resistance (post, n = 43). CNAs were inferred from whole exome sequencing and were correlated with both the status of the lesion and overall patient progression-free survival (PFS). We used RNA sequencing data from the same sample set to validate aberrations as well as independent datasets to prioritize candidate genes. RESULTS: We identified a significantly increased frequency gain of a unique CN, in liver metastatic lesions after first-line treatment, on chr18p11.32 harboring 10 genes, including TYMS, which has not been reported in primary tumors (GISTIC method and test of equal proportions, FDR-adjusted p = 0.0023). CNA lesion profiles exhibiting different treatment responses were compared and we detected focal genomic divergences in post-treatment resistant lesions but not in responder lesions (two-tailed Fisher's Exact test, unadjusted p ≤ 0.005). The importance of examining metastatic lesions is highlighted by the fact that 15 out of 18 independently validated CNA regions found to be associated with PFS in this study were only identified in the metastatic lesions and not in the primary tumors. CONCLUSION: This investigation of genomic-phenotype associations in a large colorectal cancer liver metastases cohort identified novel molecular features associated with treatment response, supporting the clinical importance of collecting metastatic samples in a defined clinical setting.

Dual targeting of polyamine synthesis and uptake in diffuse intrinsic pontine gliomas.

Diffuse intrinsic pontine glioma (DIPG) is an incurable malignant childhood brain tumor, with no active systemic therapies and a 5-year survival of less than 1%. Polyamines are small organic polycations that are essential for DNA replication, translation and cell proliferation. Ornithine decarboxylase 1 (ODC1), the rate-limiting enzyme in polyamine synthesis, is irreversibly inhibited by difluoromethylornithine (DFMO). Herein we show that polyamine synthesis is upregulated in DIPG, leading to sensitivity to DFMO. DIPG cells compensate for ODC1 inhibition by upregulation of the polyamine transporter SLC3A2. Treatment with the polyamine transporter inhibitor AMXT 1501 reduces uptake of polyamines in DIPG cells, and co-administration of AMXT 1501 and DFMO leads to potent in vitro activity, and significant extension of survival in three aggressive DIPG orthotopic animal models. Collectively, these results demonstrate the potential of dual targeting of polyamine synthesis and uptake as a therapeutic strategy for incurable DIPG.

Histone H3.3G34-Mutant Interneuron Progenitors Co-opt PDGFRA for Gliomagenesis.

Histone H3.3 glycine 34 to arginine/valine (G34R/V) mutations drive deadly gliomas and show exquisite regional and temporal specificity, suggesting a developmental context permissive to their effects. Here we show that 50% of G34R/V tumors (n = 95) bear activating PDGFRA mutations that display strong selection pressure at recurrence. Although considered gliomas, G34R/V tumors actually arise in GSX2/DLX-expressing interneuron progenitors, where G34R/V mutations impair neuronal differentiation. The lineage of origin may facilitate PDGFRA co-option through a chromatin loop connecting PDGFRA to GSX2 regulatory elements, promoting PDGFRA overexpression and mutation. At the single-cell level, G34R/V tumors harbor dual neuronal/astroglial identity and lack oligodendroglial programs, actively repressed by GSX2/DLX-mediated cell fate specification. G34R/V may become dispensable for tumor maintenance, whereas mutant-PDGFRA is potently oncogenic. Collectively, our results open novel research avenues in deadly tumors. G34R/V gliomas are neuronal malignancies where interneuron progenitors are stalled in differentiation by G34R/V mutations and malignant gliogenesis is promoted by co-option of a potentially targetable pathway, PDGFRA signaling.

Stalled developmental programs at the root of pediatric brain tumors.

Childhood brain tumors have suspected prenatal origins. To identify vulnerable developmental states, we generated a single-cell transcriptome atlas of >65,000 cells from embryonal pons and forebrain, two major tumor locations. We derived signatures for 191 distinct cell populations and defined the regional cellular diversity and differentiation dynamics. Projection of bulk tumor transcriptomes onto this dataset shows that WNT medulloblastomas match the rhombic lip-derived mossy fiber neuronal lineage and embryonal tumors with multilayered rosettes fully recapitulate a neuronal lineage, while group 2a/b atypical teratoid/rhabdoid tumors may originate outside the neuroectoderm. Importantly, single-cell tumor profiles reveal highly defined cell hierarchies that mirror transcriptional programs of the corresponding normal lineages. Our findings identify impaired differentiation of specific neural progenitors as a common mechanism underlying these pediatric cancers and provide a rational framework for future modeling and therapeutic interventions.

PRMT5 is essential for B cell development and germinal center dynamics.

Mechanisms regulating B cell development, activation, education in the germinal center (GC) and differentiation, underpin the humoral immune response. Protein arginine methyltransferase 5 (Prmt5), which catalyzes most symmetric dimethyl arginine protein modifications, is overexpressed in B cell lymphomas but its function in normal B cells is poorly defined. Here we show that Prmt5 is necessary for antibody responses and has essential but distinct functions in all proliferative B cell stages in mice. Prmt5 is necessary for B cell development by preventing p53-dependent and p53-independent blocks in Pro-B and Pre-B cells, respectively. By contrast, Prmt5 protects, via p53-independent pathways, mature B cells from apoptosis during activation, promotes GC expansion, and counters plasma cell differentiation. Phenotypic and RNA-seq data indicate that Prmt5 regulates GC light zone B cell fate by regulating transcriptional programs, achieved in part by ensuring RNA splicing fidelity. Our results establish Prmt5 as an essential regulator of B cell biology.

Integration of Distinct ShcA Signaling Complexes Promotes Breast Tumor Growth and Tyrosine Kinase Inhibitor Resistance.

The commonality between most phospho-tyrosine signaling networks is their shared use of adaptor proteins to transduce mitogenic signals. ShcA (SHC1) is one such adaptor protein that employs two phospho-tyrosine binding domains (PTB and SH2) and key phospho-tyrosine residues to promote mammary tumorigenesis. Receptor tyrosine kinases (RTK), such as ErbB2, bind the ShcA PTB domain to promote breast tumorigenesis by engaging Grb2 downstream of the ShcA tyrosine phosphorylation sites to activate AKT/mTOR signaling. However, breast tumors also rely on the ShcA PTB domain to bind numerous negative regulators that limit activation of secondary mitogenic signaling networks. This study examines the role of PTB-independent ShcA pools in controlling breast tumor growth and resistance to tyrosine kinase inhibitors. We demonstrate that PTB-independent ShcA complexes predominately rely on the ShcA SH2 domain to activate multiple Src family kinases (SFK), including Src and Fyn, in ErbB2-positive breast cancers. Using genetic and pharmacologic approaches, we show that PTB-independent ShcA complexes augment mammary tumorigenesis by increasing the activity of the Src and Fyn tyrosine kinases in an SH2-dependent manner. This bifurcation of signaling complexes from distinct ShcA pools transduces non-redundant signals that integrate the AKT/mTOR and SFK pathways to cooperatively increase breast tumor growth and resistance to tyrosine kinase inhibitors, including lapatinib and PP2. This study mechanistically dissects how the interplay between diverse intracellular ShcA complexes impacts the tyrosine kinome to affect breast tumorigenesis.Implications: The ShcA adaptor, within distinct signaling complexes, impacts tyrosine kinase signaling, breast tumor growth, and resistance to tyrosine kinase inhibitors. Mol Cancer Res; 16(5); 894-908. ©2018 AACR.

H3.3K27M Cooperates with Trp53 Loss and PDGFRA Gain in Mouse Embryonic Neural Progenitor Cells to Induce Invasive High-Grade Gliomas.

Gain-of-function mutations in histone 3 (H3) variants are found in a substantial proportion of pediatric high-grade gliomas (pHGG), often in association with TP53 loss and platelet-derived growth factor receptor alpha (PDGFRA) amplification. Here, we describe a somatic mouse model wherein H3.3K27M and Trp53 loss alone are sufficient for neoplastic transformation if introduced in utero. H3.3K27M-driven lesions are clonal, H3K27me3 depleted, Olig2 positive, highly proliferative, and diffusely spreading, thus recapitulating hallmark molecular and histopathological features of pHGG. Addition of wild-type PDGFRA decreases latency and increases tumor invasion, while ATRX knockdown is associated with more circumscribed tumors. H3.3K27M-tumor cells serially engraft in recipient mice, and preliminary drug screening reveals mutation-specific vulnerabilities. Overall, we provide a faithful H3.3K27M-pHGG model which enables insights into oncohistone pathogenesis and investigation of future therapies.

[Establishment of Specialized Pediatric Palliative Home Care in the Medical Valley of the European Metropolitan Area Nuremberg]. / Praktische Umsetzung einer spezialisierten ambulanten pädiatrischen Palliativversorgung im Medical Valley der Europäischen Metropolregion Nürnberg.

Background As part of the 2007 health reform in Germany the structure of outpatient palliative care for children and adolescents was adopted for the first time and then implemented in Erlangen-Nuremberg in 2009. Methods The introduction of Pediatric Palliative Home Care (PPHC) at the Hospital for Children and Adolescents at the University of Erlangen-Nuremberg was retrospectively analyzed between the years 2009 to 2014. Referring medical records (paper-based and electronic) were evaluated systematically. Results Considering 69 patients within this study, 44 (63.8%) died during the investigated period and 61% of these Patients deceased at home. 60 patients (87%) had a written emergency plan, which was jointly developed with patients and particularly their parents and relatives in cooperation with the PPHC team. Over the years and with increasing experience, the number and duration of emergency hospitalization decreased. Even complex therapies, such as patient-controlled analgesia with PCA pump could be implemented on an outpatient basis. Conclusion The descriptive cohort study demonstrates that palliative care for children, despite the medical and structural complexity is possible in an ambulatory setting. It allows a similar, if not better care, compared to inpatient palliative care for children and adolescents, not only for the affected patients, but also for their families.

The Shc1 adaptor simultaneously balances Stat1 and Stat3 activity to promote breast cancer immune suppression.

Tyrosine kinase signalling within cancer cells is central to the establishment of an immunosuppressive microenvironment. Although tyrosine kinase inhibitors act, in part, to augment adaptive immunity, the increased heterogeneity and functional redundancy of the tyrosine kinome is a hurdle to achieving durable responses to immunotherapies. We previously identified the Shc1 (ShcA) scaffold, a central regulator of tyrosine kinase signalling, as essential for promoting breast cancer immune suppression. Herein we show that the ShcA pathway simultaneously activates STAT3 immunosuppressive signals and impairs STAT1-driven immune surveillance in breast cancer cells. Impaired Y239/Y240-ShcA phosphorylation selectively reduces STAT3 activation in breast tumours, profoundly sensitizing them to immune checkpoint inhibitors and tumour vaccines. Finally, the ability of diminished tyrosine kinase signalling to initiate STAT1-driven immune surveillance can be overcome by compensatory STAT3 hyperactivation in breast tumours. Our data indicate that inhibition of pY239/240-ShcA-dependent STAT3 signalling may represent an attractive therapeutic strategy to sensitize breast tumours to multiple immunotherapies.

Mouse cystic fibrosis transmembrane conductance regulator forms cAMP-PKA-regulated apical chloride channels in cortical collecting duct.

The cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in many segments of the mammalian nephron, where it may interact with and modulate the activity of a variety of apical membrane proteins, including the renal outer medullary potassium (ROMK) K(+) channel. However, the expression of CFTR in apical cell membranes or its function as a Cl(-) channel in native renal epithelia has not been demonstrated. Here, we establish that CFTR forms protein kinase A (PKA)-activated Cl(-) channels in the apical membrane of principal cells from the cortical collecting duct obtained from mice. These Cl(-) channels were observed in cell-attached apical patches of principal cells after stimulation by forskolin/3-isobutyl-1-methylxanthine. Quiescent Cl(-) channels were present in patches excised from untreated tubules because they could be activated after exposure to Mg-ATP and the catalytic subunit of PKA. The single-channel conductance, kinetics, and anion selectivity of these Cl(-) channels were the same as those of recombinant mouse CFTR channels expressed in Xenopus laevis oocytes. The CFTR-specific closed-channel blocker CFTR(inh)-172 abolished apical Cl(-) channel activity in excised patches. Moreover, apical Cl(-) channel activity was completely absent in principal cells from transgenic mice expressing the DeltaF508 CFTR mutation but was present and unaltered in ROMK-null mice. We discuss the physiologic implications of open CFTR Cl(-) channels on salt handling by the collecting duct and on the functional CFTR-ROMK interactions in modulating the metabolic ATP-sensing of ROMK.