Rewiring cancer drivers to activate apoptosis.

Genes that drive the proliferation, survival, invasion and metastasis of malignant cells have been identified for many human cancers1-4. Independent studies have identified cell death pathways that eliminate cells for the good of the organism5,6. The coexistence of cell death pathways with driver mutations suggests that the cancer driver could be rewired to activate cell death using chemical inducers of proximity (CIPs). Here we describe a new class of molecules called transcriptional/epigenetic CIPs (TCIPs) that recruit the endogenous cancer driver, or a downstream transcription factor, to the promoters of cell death genes, thereby activating their expression. We focused on diffuse large B cell lymphoma, in which the transcription factor B cell lymphoma 6 (BCL6) is deregulated7. BCL6 binds to the promoters of cell death genes and epigenetically suppresses their expression8. We produced TCIPs by covalently linking small molecules that bind BCL6 to those that bind to transcriptional activators that contribute to the oncogenic program, such as BRD4. The most potent molecule, TCIP1, increases binding of BRD4 by 50% over genomic BCL6-binding sites to produce transcriptional elongation at pro-apoptotic target genes within 15 min, while reducing binding of BRD4 over enhancers by only 10%, reflecting a gain-of-function mechanism. TCIP1 kills diffuse large B cell lymphoma cell lines, including chemotherapy-resistant, TP53-mutant lines, at EC50 of 1-10 nM in 72 h and exhibits cell-specific and tissue-specific effects, capturing the combinatorial specificity inherent to transcription. The TCIP concept also has therapeutic applications in regulating the expression of genes for regenerative medicine and developmental disorders.

Increased ACTL6A occupancy within mSWI/SNF chromatin remodelers drives human squamous cell carcinoma.

Mammalian SWI/SNF (BAF) chromatin remodelers play dosage-sensitive roles in many human malignancies and neurologic disorders. The gene encoding the BAF subunit actin-like 6a (ACTL6A) is amplified early in the development of many squamous cell carcinomas (SCCs), but its oncogenic role remains unclear. Here we demonstrate that ACTL6A overexpression leads to its stoichiometric assembly into BAF complexes and drives their interaction and engagement with specific regulatory regions in the genome. In normal epithelial cells, ACTL6A was substoichiometric to other BAF subunits. However, increased ACTL6A levels by ectopic expression or in SCC cells led to near saturation of ACTL6A within BAF complexes. Increased ACTL6A occupancy enhanced polycomb opposition genome-wide to activate SCC genes and facilitated the co-dependent loading of BAF and TEAD-YAP complexes on chromatin. Both mechanisms appeared to be critical and function as a molecular AND gate for SCC initiation and maintenance, thereby explaining the specificity of the role of ACTL6A amplification in SCCs.

NFIB is a governor of epithelial-melanocyte stem cell behaviour in a shared niche.

Adult stem cells reside in specialized niches where they receive environmental cues to maintain tissue homeostasis. In mammals, the stem cell niche within hair follicles is home to epithelial hair follicle stem cells and melanocyte stem cells, which sustain cyclical bouts of hair regeneration and pigmentation. To generate pigmented hairs, synchrony is achieved such that upon initiation of a new hair cycle, stem cells of each type activate lineage commitment. Dissecting the inter-stem-cell crosstalk governing this intricate coordination has been difficult, because mutations affecting one lineage often affect the other. Here we identify transcription factor NFIB as an unanticipated coordinator of stem cell behaviour. Hair follicle stem-cell-specific conditional targeting of Nfib in mice uncouples stem cell synchrony. Remarkably, this happens not by perturbing hair cycle and follicle architecture, but rather by promoting melanocyte stem cell proliferation and differentiation. The early production of melanin is restricted to melanocyte stem cells at the niche base. Melanocyte stem cells more distant from the dermal papilla are unscathed, thereby preventing hair greying typical of melanocyte stem cell differentiation mutants. Furthermore, we pinpoint KIT-ligand as a dermal papilla signal promoting melanocyte stem cell differentiation. Additionally, through chromatin-immunoprecipitation with high-throughput-sequencing and transcriptional profiling, we identify endothelin 2 (Edn2) as an NFIB target aberrantly activated in NFIB-deficient hair follicle stem cells. Ectopically induced Edn2 recapitulates NFIB-deficient phenotypes in wild-type mice. Conversely, endothelin receptor antagonists and/or KIT blocking antibodies prevent precocious melanocyte stem cell differentiation in the NFIB-deficient niche. Our findings reveal how melanocyte and hair follicle stem cell behaviours maintain reliance upon cooperative factors within the niche, and how this can be uncoupled in injury, stress and disease states.

Nfatc1 orchestrates aging in hair follicle stem cells.

Hair production is fueled by stem cells (SCs), which transition between cyclical bouts of rest and activity. Here, we explore why hair growth wanes with age. We show that aged hair follicle SCs (HFSCs) in mice exhibit enhanced resting and abbreviated growth phases and are delayed in response to tissue-regenerating cues. Aged HFSCs are poor at initiating proliferation and show diminished self-renewing capacity upon extensive use. Only modestly restored by parabiosis, these features are rooted in elevated cell-intrinsic sensitivity and local elevation in bone morphogenic protein (BMP) signaling. Transcriptional profiling presents differences consistent with defects in aged HFSC activation. Notably, BMP-/calcium-regulated, nuclear factor of activated T-cell c1 (NFATc1) in HFSCs becomes recalcitrant to its normal down-regulating cues, and NFATc1 ChIP-sequencing analyses reveal a marked enrichment of NFATc1 target genes within the age-related signature. Moreover, aged HFSCs display more youthful levels of hair regeneration when BMP and/or NFATc1 are inhibited. These results provide unique insights into how skin SCs age.

Absolute interdialytic weight gain is more important than percent weight gain for intradialytic hypotension in heavy patients.

AIM: Few published reports have mentioned the difference between absolute interdialytic weight gain (IDWG) and IDWG/DW (IDWG%), and subsequent effects on daily dialysis. The aim of present study was to evaluate the difference between absolute IDWG and IDWG% in new haemodialysis patients. METHOD: We retrospectively reviewed the records of 255 patients who recently received conventional haemodialysis for at least 1 year at the same centre from 1997 to 2008. The first 4 weeks after starting haemodialysis was defined as the pre-study period. Data were collected for 5-56 weeks. RESULTS: IDWG% value remained relatively constant in the first year of haemodialysis despite most patients having certain residual renal function. For haemodialysis outcomes, both absolute IDWG and IDWG% were significantly correlated with intradialytic hypotension (IDH) in men and heavy women. After dividing patients into four strata, which according to the gender and the median dry weight, stepwise multivariate linear regression analysis showed that absolute IDWG, rather than IDWG%, was an independent risk factor for IDH in heavy men (Beta = 0.585, P < 0.001) and heavy women (Beta= 0.458, P < 0.001). CONCLUSIONS: Absolute IDWG, rather than IDWG%, is an independent risk factor for IDH in heavy haemodialysis patients. Therefore, higher absolute IDWG needs to be strictly controlled despite the corresponding IDWG% possibly being relatively small in heavy haemodialysis patients.

Interdialytic weight gain does not influence the nutrition of new hemodialysis patients.

OBJECTIVE: The aim of the present study was to assess the relationship between interdialytic weight gain (IDWG) and nutrition markers in hemodialysis (HD) patients, by means of repeated measures analysis. METHODS: The records of 255 patients, who had recently received conventional HD for a minimum of 1 year, were retrospectively reviewed. Nutrition markers, including serum albumin, serum phosphate, blood urea nitrogen, and creatinine, were recorded at monthly intervals and subjected to repeated measures analysis. RESULTS: Patients with higher IDWG/dry weight (IDWG%) (>5%) had significantly lower body mass index throughout the study. Repeated measures analysis of variance indicated no significant difference in these nutrition markers for patients with different IDWG%. At the end of the study, neither IDWG nor IDWG% were found to be associated with albumin or phosphate, on linear regression analysis. CONCLUSIONS: There was no evidence of better nutrition in new HD patients with higher IDWG%. Although increased intake is promoted as critical for improving nutritional status in HD patients, it may be inappropriate to focus solely on the benefits of higher IDWG%, which can also lead to the development of hypertension, left ventricular hypertrophy, and intradialytic hypotension.

A CRISPR/Cas9-Engineered ARID1A-Deficient Human Gastric Cancer Organoid Model Reveals Essential and Nonessential Modes of Oncogenic Transformation.

Mutations in ARID1A rank among the most common molecular aberrations in human cancer. However, oncogenic consequences of ARID1A mutation in human cells remain poorly defined due to lack of forward genetic models. Here, CRISPR/Cas9-mediated ARID1A knockout (KO) in primary TP53-/- human gastric organoids induced morphologic dysplasia, tumorigenicity, and mucinous differentiation. Genetic WNT/ß-catenin activation rescued mucinous differentiation, but not hyperproliferation, suggesting alternative pathways of ARID1A KO-mediated transformation. ARID1A mutation induced transcriptional regulatory modules characteristic of microsatellite instability and Epstein-Barr virus-associated subtype human gastric cancer, including FOXM1-associated mitotic genes and BIRC5/survivin. Convergently, high-throughput compound screening indicated selective vulnerability of ARID1A-deficient organoids to inhibition of BIRC5/survivin, functionally implicating this pathway as an essential mediator of ARID1A KO-dependent early-stage gastric tumorigenesis. Overall, we define distinct pathways downstream of oncogenic ARID1A mutation, with nonessential WNT-inhibited mucinous differentiation in parallel with essential transcriptional FOXM1/BIRC5-stimulated proliferation, illustrating the general utility of organoid-based forward genetic cancer analysis in human cells. SIGNIFICANCE: We establish the first human forward genetic modeling of a commonly mutated tumor suppressor gene, ARID1A. Our study integrates diverse modalities including CRISPR/Cas9 genome editing, organoid culture, systems biology, and small-molecule screening to derive novel insights into early transformation mechanisms of ARID1A-deficient gastric cancers.See related commentary by Zafra and Dow, p. 1327.This article is highlighted in the In This Issue feature, p. 1307.

Atg19 mediates a dual interaction cargo sorting mechanism in selective autophagy.

Autophagy is a catabolic membrane-trafficking mechanism conserved in all eukaryotic cells. In addition to the nonselective transport of bulk cytosol, autophagy is responsible for efficient delivery of the vacuolar enzyme Ape1 precursor (prApe1) in the budding yeast Saccharomyces cerevisiae, suggesting the presence of a prApe1 sorting machinery. Sequential interactions between Atg19-Atg11 and Atg19-Atg8 pairs are thought responsible for targeting prApe1 to the vesicle formation site, the preautophagosomal structure (PAS), and loading it into transport vesicles, respectively. However, the different patterns of prApe1 transport defect seen in the atg11Delta and atg19Delta strains seem to be incompatible with this model. Here we report that prApe1 could not be targeted to the PAS and failed to be delivered into the vacuole in atg8Delta atg11Delta double knockout cells regardless of the nutrient conditions. We postulate that Atg19 mediates a dual interaction prApe1-sorting mechanism through independent, instead of sequential, interactions with Atg11 and Atg8. In addition, to efficiently deliver prApe1 to the vacuole, a proper interaction between Atg11 and Atg9 is indispensable. We speculate that Atg11 may elicit a cargo-loading signal and induce Atg9 shuttling to a specific PAS site, where Atg9 relays the signal and recruits other Atg proteins to induce vesicle formation.

Chemical Inhibitors of a Selective SWI/SNF Function Synergize with ATR Inhibition in Cancer Cell Killing.

SWI/SNF (BAF) complexes are a diverse family of ATP-dependent chromatin remodelers produced by combinatorial assembly that are mutated in and thought to contribute to 20% of human cancers and a large number of neurologic diseases. The gene-activating functions of BAF complexes are essential for viability of many cell types, limiting the development of small molecule inhibitors. To circumvent the potential toxicity of SWI/SNF inhibition, we identified small molecules that inhibit the specific repressive function of these complexes but are relatively nontoxic and importantly synergize with ATR inhibitors in killing cancer cells. Our studies suggest an avenue for therapeutic enhancement of ATR/ATM inhibition and provide evidence for chemical synthetic lethality of BAF complexes as a therapeutic strategy in cancer.

Dominant-negative SMARCA4 mutants alter the accessibility landscape of tissue-unrestricted enhancers.

Mutation of SMARCA4 (BRG1), the ATPase of BAF (mSWI/SNF) and PBAF complexes, contributes to a range of malignancies and neurologic disorders. Unfortunately, the effects of SMARCA4 missense mutations have remained uncertain. Here we show that SMARCA4 cancer missense mutations target conserved ATPase surfaces and disrupt the mechanochemical cycle of remodeling. We find that heterozygous expression of mutants alters the open chromatin landscape at thousands of sites across the genome. Loss of DNA accessibility does not directly overlap with Polycomb accumulation, but is enriched in 'A compartments' at active enhancers, which lose H3K27ac but not H3K4me1. Affected positions include hundreds of sites identified as superenhancers in many tissues. Dominant-negative mutation induces pro-oncogenic expression changes, including increased expression of Myc and its target genes. Together, our data suggest that disruption of enhancer accessibility represents a key source of altered function in disorders with SMARCA4 mutations in a wide variety of tissues.

TOP2 synergizes with BAF chromatin remodeling for both resolution and formation of facultative heterochromatin.

The resolution and formation of facultative heterochromatin are essential for development, reprogramming, and oncogenesis. The mechanisms underlying these changes are poorly understood owing to the difficulty of studying heterochromatin dynamics and structure in vivo. We devised an in vivo approach to investigate these mechanisms and found that topoisomerase II (TOP2), but not TOP1, synergizes with BAF (mSWI/SNF) ATP-dependent chromatin remodeling complexes genome-wide to resolve facultative heterochromatin to accessible chromatin independent of transcription. This indicates that changes in DNA topology that take place through (de-)catenation rather than the release of torsional stress through swiveling are necessary for heterochromatin resolution. TOP2 and BAF cooperate to recruit pluripotency factors, which explains some of the instructive roles of BAF complexes. Unexpectedly, we found that TOP2 also plays a role in the re-formation of facultative heterochromatin; this finding suggests that facultative heterochromatin and accessible chromatin exist at different states of catenation or other topologies, which might be critical to their structures.

Factors affecting perceptions of family function in caregivers of children with attention deficit hyperactivity disorders.

BACKGROUND: Attention deficit and hyperactivity disorder (ADHD) is the most common neurobehavioral disorder of childhood. ADHD has been shown to persist into adulthood in 30%-70% of cases. The long-term and escalating nature of ADHD creates an increasing burden on families because of the influence of hyperactivity and impulsivity on academic achievement and social interaction. There is a lack of information on factors influencing function in the families of children with ADHD. PURPOSE: The purpose of this study was to test theoretically derived relationships among family demographic characteristics; family factors such as support, hardiness, and caregiver health; and family-functioning outcomes. METHODS: This study used a cross-sectional study and structural equation modeling approach. A self-report questionnaire collected information from 122 caregivers on demographics, income, employment, and marital status data as well as on personal health, family support, family hardiness, and family function statuses as determined, respectively, using the Duke Health Profile, Family APGAR score, Family Hardiness Index, and Family Assessment Device. RESULTS: Structural equation modeling provided a reasonable fit to the data using AMOS (χ = .249, df = 1, p = .613, minimum discrepancy C = .249), goodness-of-fit index (.999), adjusted goodness of fit index (.990), normed fit index (.999), comparative fit index (1.0), and root mean square error of approximation (.000). Results indicated a 55.6% probability of becoming the construct model, with family hardiness and family support directly affecting family function and caregiver health. Family support functioned as a mediator in the relationship between family hardiness and family function. CONCLUSIONS/IMPLICATIONS FOR PRACTICE: The findings of this study help nurses improve professional assessments and interventions for families of children with ADHD by highlighting the importance of increased family support, promoting family hardiness, and promoting caregivers' health to improved family function.

Health-related quality of life in patients undergoing cholecystectomy.

This large-scale prospective cohort study of a Taiwan population applied generalized estimating equations to evaluate predictors of health-related quality of life (HRQOL) after open cholecystectomy (OC) and laparoscopic cholecystectomy (LC) procedures performed between February 2007 and November 2008. The Gastrointestinal Quality of Life Index and Short Form-36 were used in a preoperative assessment and in 3(rd) month and 6(th) month postoperative assessments of 38 OC and 259 LC patients. The HRQOL of the cholecystectomy patients were significantly improved at 3 months and 6 months postsurgery (p<0.05). At 3 months postsurgery, HRQOL improvement was significantly larger in LC patients than in OC patients. Patient characteristics, clinical characteristics, and health care quality were also significantly related to HRQOL improvement (p<0.05). Additionally, after controlling for related variables, preoperative health status was significantly and positively associated with each subscale of the Gastrointestinal Quality of Life Index and Short Form-36 throughout the 6 months (p<0.05). Patients should be advised that their postoperative HRQOL may depend not only on their postoperative health care but also on their preoperative functional status.

Sarm1, a negative regulator of innate immunity, interacts with syndecan-2 and regulates neuronal morphology.

Dendritic arborization is a critical neuronal differentiation process. Here, we demonstrate that syndecan-2 (Sdc2), a synaptic heparan sulfate proteoglycan that triggers dendritic filopodia and spine formation, regulates dendritic arborization in cultured hippocampal neurons. This process is controlled by sterile α and TIR motif-containing 1 protein (Sarm1), a negative regulator of Toll-like receptor 3 (TLR3) in innate immunity signaling. We show that Sarm1 interacts with and receives signal from Sdc2 and controls dendritic arborization through the MKK4-JNK pathway. In Sarm1 knockdown mice, dendritic arbors of neurons were less complex than those of wild-type littermates. In addition to acting downstream of Sdc2, Sarm1 is expressed earlier than Sdc2, which suggests that it has multiple roles in neuronal morphogenesis. Specifically, it is required for proper initiation and elongation of dendrites, axonal outgrowth, and neuronal polarization. These functions likely involve Sarm1-mediated regulation of microtubule stability, as Sarm1 influenced tubulin acetylation. This study thus reveals the molecular mechanism underlying the action of Sarm1 in neuronal morphogenesis.