SMARCB1-mediated SWI/SNF complex function is essential for enhancer regulation.

SMARCB1 (also known as SNF5, INI1, and BAF47), a core subunit of the SWI/SNF (BAF) chromatin-remodeling complex, is inactivated in nearly all pediatric rhabdoid tumors. These aggressive cancers are among the most genomically stable, suggesting an epigenetic mechanism by which SMARCB1 loss drives transformation. Here we show that, despite having indistinguishable mutational landscapes, human rhabdoid tumors exhibit distinct enhancer H3K27ac signatures, which identify remnants of differentiation programs. We show that SMARCB1 is required for the integrity of SWI/SNF complexes and that its loss alters enhancer targeting-markedly impairing SWI/SNF binding to typical enhancers, particularly those required for differentiation, while maintaining SWI/SNF binding at super-enhancers. We show that these retained super-enhancers are essential for rhabdoid tumor survival, including some that are shared by all subtypes, such as SPRY1, and other lineage-specific super-enhancers, such as SOX2 in brain-derived rhabdoid tumors. Taken together, our findings identify a new chromatin-based epigenetic mechanism underlying the tumor-suppressive activity of SMARCB1.

Functionally distinct patterns of nucleosome remodeling at enhancers in glucocorticoid-treated acute lymphoblastic leukemia.

BACKGROUND: Precise nucleosome positioning is an increasingly recognized feature of promoters and enhancers, reflecting complex contributions of DNA sequence, nucleosome positioning, histone modification and transcription factor binding to enhancer activity and regulation of gene expression. Changes in nucleosome position and occupancy, histone variants and modifications, and chromatin remodeling are also critical elements of dynamic transcriptional regulation, but poorly understood at enhancers. We investigated glucocorticoid receptor-associated (GR) nucleosome dynamics at enhancers in acute lymphoblastic leukemia. RESULTS: For the first time, we demonstrate functionally distinct modes of nucleosome remodeling upon chromatin binding by GR, which we term central, non-central, phased, and minimal. Central and non-central remodeling reflect nucleosome eviction by GR and cofactors, respectively. Phased remodeling involves nucleosome repositioning and is associated with rapidly activated enhancers and induction of gene expression. Minimal remodeling sites initially have low levels of enhancer-associated histone modification, but the majority of these regions gain H3K4me2 or H3K27Ac to become de novo enhancers. Minimal remodeling regions are associated with gene ontologies specific to decreased B cell number and mTOR inhibition and may make unique contributions to glucocorticoid-induced leukemia cell death. CONCLUSIONS: Our findings form a novel framework for understanding the dynamic interplay between transcription factor binding, nucleosome remodeling, enhancer function, and gene expression in the leukemia response to glucocorticoids.

SWI/SNF-mutant cancers depend on catalytic and non-catalytic activity of EZH2.

Human cancer genome sequencing has recently revealed that genes that encode subunits of SWI/SNF chromatin remodeling complexes are frequently mutated across a wide variety of cancers, and several subunits of the complex have been shown to have bona fide tumor suppressor activity. However, whether mutations in SWI/SNF subunits result in shared dependencies is unknown. Here we show that EZH2, a catalytic subunit of the polycomb repressive complex 2 (PRC2), is essential in all tested cancer cell lines and xenografts harboring mutations of the SWI/SNF subunits ARID1A, PBRM1, and SMARCA4, which are several of the most frequently mutated SWI/SNF subunits in human cancer, but that co-occurrence of a Ras pathway mutation is correlated with abrogation of this dependence. Notably, we demonstrate that SWI/SNF-mutant cancer cells are primarily dependent on a non-catalytic role of EZH2 in the stabilization of the PRC2 complex, and that they are only partially dependent on EZH2 histone methyltransferase activity. These results not only reveal a shared dependency of cancers with genetic alterations in SWI/SNF subunits, but also suggest that EZH2 enzymatic inhibitors now in clinical development may not fully suppress the oncogenic activity of EZH2.

No improvement noted in overall or cause-specific survival for men presenting with metastatic prostate cancer over a 20-year period.

BACKGROUND: Prostate cancer mortality in the United States has declined by nearly 40% over the last 25 years. However, to the authors' knowledge, the contribution of prostate-specific antigen (PSA) screening for the early detection of prostate cancer remains unclear and controversial. In the current study, the authors attempted to determine whether improvements in survival over time among patients with metastatic prostate cancer have contributed to the decline in mortality. METHODS: Men aged ≥ 45 years who presented with de novo metastatic prostate cancer from 1988 to 2009 were identified within the California Cancer Registry. Overall survival and disease-specific survival were estimated using the Kaplan-Meier method. A multivariate analysis with Cox proportional hazards modeling was performed to adjust for different distributions of variables between groups. RESULTS: A total of 19,336 men presented with de novo metastatic prostate cancer during the study period. On multivariate analysis, overall survival was found to be better for men diagnosed from 1988 through 1992 and 1993 through 1998 than for men diagnosed in the most recent era (hazards ratio, 0.78; 95% confidence interval, 0.72-0.85 [P < .001] and HR, 0.79; 95% confidence interval, 0.74-0.86 [P < .001]). There was no improvement in disease-specific survival observed when comparing the most contemporary men (those diagnosed between 2004 and 2009) with those diagnosed between 1988 and 1997. CONCLUSIONS: In this analysis of men presenting with de novo metastatic prostate cancer, no consistent improvement in overall or disease-specific survival could be demonstrated over time. These data suggest that improvements in survival for patients with advanced disease have not contributed substantially to the observed drop in prostate cancer mortality over the PSA era and that stage migration secondary to PSA screening plays a more prominent role.

Urolithiasis risk factors in the bariatric population undergoing gastric bypass surgery.

BACKGROUND: Previous studies have suggested an increased risk of forming symptomatic urolithiasis after Roux-en-Y gastric bypass (RYGB) attributed to the development of hyperoxaluria. The objective of our investigation was to evaluate changes in the urine milieu after RYGB that might explain the increased risk of urolithiasis. METHODS: Patients underwent serum and urine chemistry tests 1 week before and 6 months after RYGB at a university hospital. The postoperative urolithiasis risk factors were compared with the preoperative values. Statistical analysis was performed using paired t tests. Significant changes were identified as P ≤ .05. RESULTS: A total of 38 patients (7 men and 31 women) submitted samples both before and after RYGB. The mean patient weight had decreased from 131 kg to 92 kg. The mean serum creatinine decreased from .83 to .72 mg/dL (P = .0004). Urinary changes known to increase the risk of urolithiasis include a decrease in volume (2-1.5 L/d, P = .03), an increase in calcium (139-182 mg/d, P = .04), and an increase in oxalate (38-48 mg/d, P < .001). The urinary supersaturation indexes for calcium oxalate (4.9-10.5, P < .001) increased. CONCLUSION: Our results confirm that patients undergoing RYGB develop changes in the urinary milieu predisposing them to forming urinary stones. Urolithiasis risk is multifactorial and is related to more than just hyperoxaluria. A patient's long-term risk of developing stones and the effect on renal function is unknown. Preoperative counseling of patients regarding their risk of forming stones and dietary counseling to minimize their risk of developing stones postoperatively is warranted.

ARID1A mutations in cancer: another epigenetic tumor suppressor?

UNLABELLED: Although disordered chromatin organization has long been recognized as a feature of cancer, the molecular underpinnings of chromatin structure, epigenetic regulation, and their relationships to transcription are only beginning to be understood. Cancer genome sequencing studies have revealed a novel theme: frequent mutation of epigenetic regulators. Among these, the ARID1A/BAF250A subunit of the SWI/SNF (BRG1-associated factors) chromatin remodeling complex has emerged as recurrently mutated in a broad array of tumor types. We review the genomic and functional data supporting classification of ARID1A as a tumor suppressor. SIGNIFICANCE: Mutations in chromatin remodeling complex genes are increasingly recognized in many cancer types. However, the mechanisms by which chromatin remodeling complexes contribute to gene expression and the cancer phenotype are poorly understood. Understanding how mutation of chromatin remodelers facilitates transformation may offer the potential for development and implementation of novel therapies for cancer.

Palliative care in urology.

Urological malignancies, especially prostate cancer, are relatively common, but patients may live many years before eventually dying of the disease. Caring for these patients is an important role for urologists, although medical training often does not adequately prepare urologists for the palliative care of patients with advanced malignancies. Palliative care is no longer equated with end-of-life care, but rather integrated throughout illness, even when cure is impossible. This article focuses on the various palliative treatments available for the 3 most common urological malignancies: prostate, bladder, and renal cancers.

Active surveillance for localized prostate cancer--current practices and recommendations.

Prostate cancer is now the most commonly diagnosed solid tumor in American men, due in part to widespread screening and aggressive diagnostic practices. Prostate cancer autopsy studies show the uniquely high prevalence rates of small, indolent tumors in men dying of other causes. These findings have led to increased concern for the over detection and overtreatment of prostate cancer. Active surveillance for prostate cancer allows one to limit prostate cancer treatment with concomitant risks of treatment-related morbidity to the men who will benefit the most from aggressive therapies. Several tools have been developed in treated and surveyed men to assist physicians in selecting men with potentially indolent tumors amenable to active surveillance. Recent published results describe institutional experiences with active surveillance and delayed selective therapy for men with low-grade, early prostate cancer. Although median follow-up from these studies is relatively short, the outcomes appear favorable. Data from these reports provide information for selecting men for this approach, as well as for following them over time and determining triggers for further intervention. Ongoing clinical trials with watchful waiting and active surveillance for prostate cancer will ultimately provide improved evidence for managing early, localized disease.

Adhesion- and degranulation-promoting adapter protein is required for efficient thymocyte development and selection.

Adhesion- and degranulation-promoting adapter protein (ADAP) is required in TCR-induced activation and proliferation of peripheral T cells. Loss of ADAP also impairs TCR-initiated inside-out activation of the integrin LFA-1 (CD11a/CD18, alphaLbeta2). In this study, we demonstrate that ADAP-deficient CD4/CD8 double-positive (DP) cells have a diminished ability to proliferate, and that these DP thymocytes up-regulate CD69 poorly in vivo. Moreover, in both MHC class I- and class II-restricted TCR transgenic models, loss of ADAP interferes with both positive and negative selection. ADAP deficiency also impairs the ability of transgene-bearing DP thymocytes to form conjugates with Ag-loaded presenting cells. These findings suggest that ADAP is critical for thymocyte development and selection.

Impaired degranulation but enhanced cytokine production after Fc epsilonRI stimulation of diacylglycerol kinase zeta-deficient mast cells.

Calcium and diacylglycerol are critical second messengers that together effect mast cell degranulation after allergen cross-linking of immunoglobulin (Ig)E-bound FcepsilonRI. Diacylglycerol kinase (DGK)zeta is a negative regulator of diacylglycerol-dependent signaling that acts by converting diacylglycerol to phosphatidic acid. We reported previously that DGKzeta-/- mice have enhanced in vivo T cell function. Here, we demonstrate that these mice have diminished in vivo mast cell function, as revealed by impaired local anaphylactic responses. Concordantly, DGKzeta-/- bone marrow-derived mast cells (BMMCs) demonstrate impaired degranulation after Fc epsilonRI cross-linking, associated with diminished phospholipase Cgamma activity, calcium flux, and protein kinase C-betaII membrane recruitment. In contrast, Ras-Erk signals and interleukin-6 production are enhanced, both during IgE sensitization and after antigen cross-linking of Fc epsilonRI. Our data demonstrate dissociation between cytokine production and degranulation in mast cells and reveal the importance of DGK activity during IgE sensitization for proper attenuation of Fc epsilonRI signals.

The SLP-76 family of adapter proteins.

Adapter molecules are multidomain proteins lacking intrinsic catalytic activity, functioning instead by nucleating molecular complexes during signal transduction. The SLP-76 family of adapters includes SH2 domain-containing leukocyte phosphoprotein of 76kDa (SLP-76), B cell linker protein (BLNK), and cytokine-dependent hematopoietic cell linker (Clnk). These proteins are critical for integration of numerous signaling cascades downstream of immunotyrosine-based activation motif (ITAM)-bearing receptors and integrins in diverse hematopoietic cell types. Mutations in genes encoding SLP-76 family adapters result in severe phenotypes, underscoring the critical role these proteins play in cellular development and function by directing formation of signaling complexes in a temporally- and spatially-specific manner.

Differential requirement for adapter proteins Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa and adhesion- and degranulation-promoting adapter protein in FcepsilonRI signaling and mast cell function.

The adapter molecule Src homology 2 (SH2) domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) is essential for FcepsilonRI-mediated signaling, degranulation and IL-6 production in mast cells. To test the structural requirements of SLP-76 in mast cell signaling and function, we have studied the functional responses of murine bone marrow-derived mast cells (BMMCs) expressing mutant forms of SLP-76. We found that the N-terminal tyrosines as well as the central proline-rich region of SLP-76 are required for participation of SLP-76 in FcepsilonRI-mediated signaling and function. The C-terminal SH2 domain of SLP-76 also contributes to optimal function of SLP-76 in mast cells. Another adapter molecule, adhesion- and degranulation-promoting adapter protein (ADAP), is known to bind the SH2 domain of SLP-76, and cell line studies have implicated ADAP in mast cell adhesion and FcepsilonRI-induced degranulation. Surprisingly, we found that mast cells lacking ADAP expression demonstrate no defects in FcepsilonRI-induced adhesion, granule release, or IL-6 production, and that ADAP-deficient mice produce a normal passive systemic anaphylactic response. Thus, failure to bind ADAP does not underlie the functional defects exhibited by SLP-76 SH2 domain mutant-expressing mast cells.

Roles of the proline-rich domain in SLP-76 subcellular localization and T cell function.

The adaptor protein Src homology (SH)2 domain-containing and leukocyte-specific phosphoprotein of 76 kDa (SLP-76) is critical for signal transduction in multiple hematopoietic lineages. It links proximal and distal T cell receptor signaling events through its function as a molecular scaffold in the assembly of multimolecular signaling complexes. Here we studied the functional roles of sub-domains within the SLP-76 proline-rich region, specifically the Gads binding domain and the recently defined P1 domain. To gain a further understanding of the functions mediated by this region, we used three complementary approaches as follows: reconstitution of SLP-76-deficient cells with functional domain deletion mutants, blocking molecular associations through the expression of a dominant negative protein fragment, and directed localization of SLP-76 to assess the role of the domains in SLP-76 recruitment. We find the Gads binding domain and the P1 domain are both necessary for optimal SLP-76 function, and in the absence of these two regions, SLP-76 is functionally inert. Furthermore, we provide direct evidence that SLP-76 localization and, in turn, function are dependent upon association with Gads.

SLP-76 regulates Fcgamma receptor and integrin signaling in neutrophils.

While the contribution of intracellular adaptor proteins to lymphocyte activation has been well studied, the function of these molecules in innate immune effector cells such as neutrophils has not been extensively addressed. Here we demonstrate a critical role for the adaptor molecule SH2 domain-containing leukocyte-specific phosphoprotein of 76 kDa (SLP-76) in FcgammaR and integrin signaling. Stimulation of these receptors induces tyrosine phosphorylation and cytoplasmic relocalization of SLP-76 in freshly isolated murine neutrophils. Neutrophils lacking SLP-76 demonstrate decreased FcgammaR-induced calcium flux and reactive oxygen intermediate (ROI) production in response to immune complex stimulation. More dramatically, SLP-76-/- neutrophils fail to produce ROI, spread, or activate critical downstream regulators in response to integrin ligation. These results provide genetic evidence for a critical role of SLP-76 in the regulation of neutrophil function.

Regulation of hematopoietic cell development and activation by adapter proteins.

Adapter proteins, molecules with modular domains that mediate intermolecular interactions, play critical roles in the regulation of signaling events in all cell types. A major focus of our laboratory has been to examine the role of adapter molecules in hematopoietic cell development and activation. This review will describe the approaches we are taking to identify such proteins and to determine the mechanisms by which they exert their functions. This work represents the enormous efforts of the students and postdocs who have committed themselves to these projects, as well as the important collaborations we have developed with other investigators at the University of Pennsylvania and elsewhere.