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To dissect variant-function relationships in the KRAS oncoprotein, we performed deep mutational scanning (DMS) screens for both wild-type and KRAS G12D mutant alleles. We defined the spectrum of oncogenic potential for nearly all possible KRAS variants, identifying several novel transforming alleles and elucidating a model to describe the frequency of KRAS mutations in human cancer as a function of transforming potential, mutational probability, and tissue-specific mutational signatures. Biochemical and structural analyses of variants identified in a KRAS G12D second-site suppressor DMS screen revealed that attenuation of oncogenic KRAS can be mediated by protein instability and conformational rigidity, resulting in reduced binding affinity to effector proteins, such as RAF and PI3-kinases, or reduced SOS-mediated nucleotide exchange activity. These studies define the landscape of single amino acid alterations that modulate the function of KRAS, providing a resource for the clinical interpretation of KRAS variants and elucidating mechanisms of oncogenic KRAS inactivation for therapeutic exploitation.

Sequencing of human patient tumors has identified recurrent missense mutations in genes encoding core histones. We report that mutations that convert histone H3 amino acid 50 from a glutamate to a lysine (H3E50K) support an oncogenic phenotype in human cells. Expression of H3E50K is sufficient to transform human cells as evidenced by a dramatic increase in cell migration and invasion, and a statistically significant increase in proliferation and clonogenicity. H3E50K also increases the invasive phenotype in the context of co-occurring BRAF mutations, which are present in patient tumors characterized by H3E50K. H3E50 lies on the globular domain surface in a region that contacts H4 within the nucleosome. We find that H3E50K perturbs proximal H3 post-translational modifications globally and dysregulates gene expression, activating the epithelial to mesenchymal transition. Functional studies using S. cerevisiae reveal that, while yeast cells that express H3E50K as the sole copy of histone H3 show sensitivity to cellular stressors, including caffeine, H3E50K cells display some genetic interactions that are distinct from the characterized H3K36M oncohistone yeast model. Taken together, these data suggest that additional histone H3 mutations have the potential to be oncogenic drivers and function through distinct mechanisms that dysregulate gene expression.

Wilms tumor (WT) is the most common renal malignancy of childhood. Despite improvements in the overall survival, relapse occurs in ~15% of patients with favorable histology WT (FHWT). Half of these patients will succumb to their disease. Identifying novel targeted therapies remains challenging in part due to the lack of faithful preclinical in vitro models. Here we establish twelve patient-derived WT cell lines and demonstrate that these models faithfully recapitulate WT biology using genomic and transcriptomic techniques. We then perform loss-of-function screens to identify the nuclear export gene, XPO1, as a vulnerability. We find that the FDA approved XPO1 inhibitor, KPT-330, suppresses TRIP13 expression, which is required for survival. We further identify synergy between KPT-330 and doxorubicin, a chemotherapy used in high-risk FHWT. Taken together, we identify XPO1 inhibition with KPT-330 as a potential therapeutic option to treat FHWTs and in combination with doxorubicin, leads to durable remissions in vivo., (© 2024. The Author(s).)

The modern study of Wilms tumour was prompted nearly 50 years ago, when Alfred Knudson proposed the 'two-hit' model of tumour development. Since then, the efforts of researchers worldwide have substantially expanded our knowledge of Wilms tumour biology, including major advances in genetics - from cloning the first Wilms tumour gene to high-throughput studies that have revealed the genetic landscape of this tumour. These discoveries improve understanding of the embryonal origin of Wilms tumour, familial occurrences and associated syndromic conditions. Many efforts have been made to find and clinically apply prognostic biomarkers to Wilms tumour, for which outcomes are generally favourable, but treatment of some affected individuals remains challenging. Challenges are also posed by the intratumoural heterogeneity of biomarkers. Furthermore, preclinical models of Wilms tumour, from cell lines to organoid cultures, have evolved. Despite these many achievements, much still remains to be discovered: further molecular understanding of relapse in Wilms tumour and of the multiple origins of bilateral Wilms tumour are two examples of areas under active investigation. International collaboration, especially when large tumour series are required to obtain robust data, will help to answer some of the remaining unresolved questions., (© 2023. Springer Nature Limited.)

Developing synchronous bilateral Wilms tumor suggests an underlying (epi)genetic predisposition. Here, we evaluate this predisposition in 68 patients using whole exome or genome sequencing (n = 85 tumors from 61 patients with matched germline blood DNA), RNA-seq (n = 99 tumors), and DNA methylation analysis (n = 61 peripheral blood, n = 29 non-diseased kidney, n = 99 tumors). We determine the predominant events for bilateral Wilms tumor predisposition: 1)pre-zygotic germline genetic variants readily detectable in blood DNA [WT1 (14.8%), NYNRIN (6.6%), TRIM28 (5%), and BRCA-related genes (5%)] or 2)post-zygotic epigenetic hypermethylation at 11p15.5 H19/ICR1 that may require analysis of multiple tissue types for diagnosis. Of 99 total tumor specimens, 16 (16.1%) have 11p15.5 normal retention of imprinting, 25 (25.2%) have 11p15.5 copy neutral loss of heterozygosity, and 58 (58.6%) have 11p15.5 H19/ICR1 epigenetic hypermethylation (loss of imprinting). Here, we ascertain the epigenetic and genetic modes of bilateral Wilms tumor predisposition., (© 2023. The Author(s).)

Every year, approximately 600 infants, children, and adolescents are diagnosed with renal cancer in the United States. In addition to Wilms tumor (WT), which accounts for about 80% of all pediatric renal cancers, clear cell sarcoma of the kidney, renal cell carcinoma, malignant rhabdoid tumor, as well as more rare cancers (other sarcomas, rare carcinomas, lymphoma) and benign tumors can originate within the kidney. WT itself can be divided into favorable histology (FHWT), with a 5-year overall survival (OS) exceeding 90%, and anaplastic histology, with 4-year OS of 73.7%. Outcomes of the other pediatric renal cancers include clear cell sarcoma (5-year OS: 90%), malignant rhabdoid tumor (5-year OS: 10% for stages 3 and 4), and renal cell carcinoma (4-year OS: 84.8%). Recent clinical trials have identified novel biological prognostic markers for FHWT, and a series of Children's Oncology Group (COG) trials have demonstrated improving outcomes with therapy modification, and opportunities for further care refinement., (© 2023 Wiley Periodicals LLC.)

ABCB1 , also known as MDR1 , is a gene that encodes P-glycoprotein (P-gp), a membrane-associated ATP-dependent transporter. P-gp is widely expressed in many healthy tissues-in the gastrointestinal tract, liver, kidney, and at the blood-brain barrier. P-gp works to pump xenobiotics such as toxins and drugs out of cells. P-gp is also commonly upregulated across multiple cancer types such as ovarian, breast, and lung. Overexpression of ABCB1 has been linked to the development of chemotherapy resistance across these cancers. In vitro work across a wide range of drug-sensitive and -resistant cancer cell lines has shown that upon treatment with chemotherapeutic agents such as doxorubicin, cisplatin, and paclitaxel, ABCB1 is upregulated. This upregulation is caused in part by a variety of genetic and epigenetic mechanisms. This includes single-nucleotide variants that lead to enhanced P-gp ATPase activity without increasing ABCB1 RNA and protein levels. In this review, we summarize current knowledge of genetic and epigenetic mechanisms leading to ABCB1 upregulation and P-gp-enhanced ATPase activity in the setting of chemotherapy resistance across a variety of cancers.

This review highlights the role of several immunomodulating elements contributing to the tumor microenvironment of various pediatric renal tumors including Wilms tumor. The roles of innate and adaptive immune cells in renal tumors are summarized as well as immunomodulatory cytokines and other proteins. The expression and the predictive role of checkpoint modulators like PD-L1 and immunomodulating proteins like glypican-3, B7-H3, COX-2 are highlighted with a translational view toward potential therapeutic innovations. We further discuss the current state of preclinical models in advancing this field of study. Finally, examples of clinical trials of immunomodulating strategies such as monoclonal antibodies and chimeric antigen receptor T (CAR-T) cells for relapsed/refractory/progressive pediatric renal tumors are described., (© 2022 The Authors. Pediatric Blood & Cancer published by Wiley Periodicals LLC.)

This study comprehensively evaluated the landscape of genetic and epigenetic events that predispose to synchronous bilateral Wilms tumor (BWT). We performed whole exome or whole genome sequencing, total-strand RNA-seq, and DNA methylation analysis using germline and/or tumor samples from 68 patients with BWT from St. Jude Children's Research Hospital and the Children's Oncology Group. We found that 25/61 (41%) of patients evaluated harbored pathogenic or likely pathogenic germline variants, with WT1 (14.8%), NYNRIN (6.6%), TRIM28 (5%) and the BRCA-related genes (5%) BRCA1, BRCA2, and PALB2 being most common. Germline WT1 variants were strongly associated with somatic paternal uniparental disomy encompassing the 11p15.5 and 11p13/ WT1 loci and subsequent acquired pathogenic CTNNB1 variants. Somatic coding variants or genome-wide copy number alterations were almost never shared between paired synchronous BWT, suggesting that the acquisition of independent somatic variants leads to tumor formation in the context of germline or early embryonic, post-zygotic initiating events. In contrast, 11p15.5 status (loss of heterozygosity, loss or retention of imprinting) was shared among paired synchronous BWT in all but one case. The predominant molecular events for BWT predisposition include pathogenic germline variants or post-zygotic epigenetic hypermethylation at the 11p15.5 H19/ICR1 locus (loss of imprinting). This study demonstrates that post-zygotic somatic mosaicism for 11p15.5 hypermethylation/loss of imprinting is the single most common initiating molecular event predisposing to BWT. Evidence of somatic mosaicism for 11p15.5 loss of imprinting was detected in leukocytes of a cohort of BWT patients and long-term survivors, but not in unilateral Wilms tumor patients and long-term survivors or controls, further supporting the hypothesis that post-zygotic 11p15.5 alterations occurred in the mesoderm of patients who go on to develop BWT. Due to the preponderance of BWT patients with demonstrable germline or early embryonic tumor predisposition, BWT exhibits a unique biology when compared to unilateral Wilms tumor and therefore warrants continued refinement of its own treatment-relevant biomarkers which in turn may inform directed treatment strategies in the future., Competing Interests: Conflict of Interest Statement: The authors have no conflicts of interest to declare.

Patients with DNA double-strand breakage repair disorders are at increased risk of malignancy which is often difficult to treat given underlying sensitivity to chemotherapy and radiotherapy, lending an important role to hematopoietic stem cell transplantation. The choice of conditioning regimen used must balance reducing risk of rejection with minimizing excessive toxicity from myeloablative chemotherapy or ionizing radiation. We describe successful engraftment following a nonmyeloablative hematopoietic stem cell transplantation in a patient with Ligase IV syndrome and numerous pretransplant complications including malignancy, cardiac failure, and secondary hemophagocytic lymphohistiocytosis. Congruent with prior reports, a reduced intensity regimen appears efficacious in Ligase IV syndrome patients., Competing Interests: The authors declare no conflict of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

The high frequency of aberrant PI3K pathway activation in hormone receptor-positive (HR + ) breast cancer has led to the development, clinical testing, and approval of the p110α-selective PI3K inhibitor alpelisib. The limited clinical efficacy of alpelisib and other PI3K inhibitors is partially attributed to the functional antagonism between PI3K and estrogen receptor (ER) signaling, which is mitigated via combined PI3K inhibition and endocrine therapy. We and others have previously demonstrated chromatin-associated mechanisms by which PI3K supports cancer development and antagonizes ER signaling through the modulation of the H3K4 methylation axis, inhibition of KDM5A promoter H3K4 demethylation and KMT2D/MLL4-directed enhancer H3K4 methylation. Here we show that inhibition of the H3K4 histone methyltransferase MLL1 in combination with PI3K inhibition impairs HR + breast cancer clonogenicity and cell proliferation. While combined PI3K/MLL1 inhibition reduces PI3K/AKT signaling and H3K4 methylation, MLL1 inhibition increases PI3K/AKT signaling through the dysregulation of gene expression associated with AKT activation. These data reveal a feedback loop between MLL1 and AKT whereby MLL1 inhibition reactivates AKT. We show that combined PI3K and MLL1 inhibition synergizes to cause cell death in in vitro and in vivo models of HR + breast cancer, which is enhanced by the additional genetic ablation of the H3K4 methyltransferase and AKT target KMT2D/MLL4. Together, our data provide evidence of a feedback mechanism connecting histone methylation with AKT and may support the preclinical development and testing of pan-MLL inhibitors., Significance: Here the authors leverage PI3K/AKT-driven chromatin modification to identify histone methyltransferases as a therapeutic target. Dual PI3K and MLL inhibition synergize to reduce clonogenicity and cell proliferation, and promote in vivo tumor regression. These findings suggest patients with PIK3CA-mutant, HR + breast cancer may derive clinical benefit from combined PI3K/MLL inhibition., Competing Interests: No disclosures were reported., (© 2022 The Authors; Published by the American Association for Cancer Research.)

SMARCB1 is a critical component of the BAF complex that is responsible for global chromatin remodeling. Loss of SMARCB1 has been implicated in the initiation of cancers such as malignant rhabdoid tumor (MRT), atypical teratoid rhabdoid tumor (ATRT), and, more recently, renal medullary carcinoma (RMC). These SMARCB1-deficient tumors have remarkably stable genomes, offering unique insights into the epigenetic mechanisms in cancer biology. Given the lack of druggable targets and the high mortality associated with SMARCB1-deficient tumors, a significant research effort has been directed toward understanding the mechanisms of tumor transformation and proliferation. Accumulating evidence suggests that tumorigenicity arises from aberrant enhancer and promoter regulation followed by dysfunctional transcriptional control. In this review, we outline key mechanisms by which loss of SMARCB1 may lead to tumor formation and cover how these mechanisms have been used for the design of targeted therapy.

Renal medullary carcinoma (RMC) is a rare renal malignancy that has been associated with sickle hemoglobinopathies. RMC is aggressive, difficult to treat, and occurs primarily in adolescents and young adults of African ancestry. This cancer is driven by the loss of SMARCB1, a tumor suppressor seen in a number of primarily rare childhood cancers (e.g., rhabdoid tumor of the kidney and atypical teratoid rhabdoid tumor). Treatment options remain limited due in part to the limited knowledge of RMC biology. However, significant advances have been made in unraveling the biology of RMC, from genomics to therapeutic targets, over the past 5 years. In this review, we will present these advances and discuss what new questions exist in the field.

Background: Adverse drug events (ADEs) are unintended side effects of drugs that cause substantial clinical and economic burdens globally. Not all ADEs are discovered during clinical trials; therefore, postmarketing surveillance, called pharmacovigilance, is routinely conducted to find unknown ADEs. A wealth of information, which facilitates ADE discovery, lies in the growing body of biomedical literature. Knowledge graphs (KGs) encode information from the literature, where the vertices and the edges represent clinical concepts and their relations, respectively. The scale and unstructured form of the literature necessitates the use of natural language processing (NLP) to automatically create such KGs. Previous studies have demonstrated the utility of such literature-derived KGs in ADE prediction. Through unsupervised learning of the representations (features) of clinical concepts from the KG, which are used in machine learning models, state-of-the-art results for ADE prediction were obtained on benchmark data sets., Objective: Due to the use of NLP to infer literature-derived KGs, there is noise in the form of false positive (erroneous) and false negative (absent) nodes and edges. Previous representation learning methods do not account for such inaccuracies in the graph. NLP algorithms can quantify the confidence in their inference of extracted concepts and relations from the literature. Our hypothesis, which motivates this work, is that by using such confidence scores during representation learning, the learned embeddings would yield better features for ADE prediction models., Methods: We developed methods to use these confidence scores on two well-known representation learning methods-DeepWalk and Translating Embeddings for Modeling Multi-relational Data (TransE)-to develop their weighted versions: Weighted DeepWalk and Weighted TransE. These methods were used to learn representations from a large literature-derived KG, the Semantic MEDLINE Database, which contains more than 93 million clinical relations. They were compared with Embedding of Semantic Predications, which, to our knowledge, is the best reported representation learning method using the Semantic MEDLINE Database with state-of-the-art results for ADE prediction. Representations learned from different methods were used (separately) as features of drugs and diseases to build classification models for ADE prediction using benchmark data sets. The methods were compared rigorously over multiple cross-validation settings., Results: The weighted versions we designed were able to learn representations that yielded more accurate predictive models than the corresponding unweighted versions of both DeepWalk and TransE, as well as Embedding of Semantic Predications, in our experiments. There were performance improvements of up to 5.75% in the F 1 -score and 8.4% in the area under the receiver operating characteristic curve value, thus advancing the state of the art in ADE prediction from literature-derived KGs., Conclusions: Our classification models can be used to aid pharmacovigilance teams in detecting potentially new ADEs. Our experiments demonstrate the importance of modeling inaccuracies in the inferred KGs for representation learning., (©Soham Dasgupta, Aishwarya Jayagopal, Abel Lim Jun Hong, Ragunathan Mariappan, Vaibhav Rajan. Originally published in JMIR Medical Informatics (https://medinform.jmir.org), 25.10.2021.)

Stroke patients have reduced aerobic capacity. Therefore, intensive structured exercise programs are needed. We report the case of a patient with stroke and cardiac disease who underwent early inpatient cardiac rehabilitation (CR). A 38-year-old male patient with atrial fibrillation, heart failure, and cerebral infarction underwent a symptom-limited exercise tolerance test (ETT) without any problems on day 45 after admission. He completed a 2-week inpatient program and an 8-week home-based CR program. Follow-up ETT showed increased exercise capacity. The present case might be the first to report a safely performed CR program in a patient with stroke and cardiac comorbidity in Korea. Systematic guidance is needed for post-stroke patients to receive safe and effective CR for the secondary prevention of stroke and cardiovascular risk.

Metastasis is a complex and poorly understood process. In pancreatic cancer, loss of the transforming growth factor (TGF)-β/BMP effector SMAD4 is correlated with changes in altered histopathological transitions, metastatic disease, and poor prognosis. In this study, we use isogenic cancer cell lines to identify SMAD4 regulated genes that contribute to the development of metastatic colonization. We perform an in vivo screen identifying FOSL1 as both a SMAD4 target and sufficient to drive colonization to the lung. The targeting of these genes early in treatment may provide a therapeutic benefit., Competing Interests: Declaration of interests W.C.H. is a consultant for Thermo Fisher Scientific, Solasta Ventures, MPM Capital, KSQ Therapeutics, iTeos, Tyra Biosciences, iTeos, Frontier Medicines, Jubliant Therapeutics, RAPPTA Therapeutics, and Paraxel. A.J.A. has consulted for Oncorus, Inc., Arrakis Therapeutics, and Merck & Co., Inc., and has research funding from Mirati Therapeutics and Deerfield, Inc. that is unrelated to this project. A.O.G. is a share and option holder of 10X Genomics. The remaining authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Renal medullary carcinomas (RMCs) are rare kidney cancers that occur in adolescents and young adults of African ancestry. Although RMC is associated with the sickle cell trait and somatic loss of the tumor suppressor, SMARCB1, the ancestral origins of RMC remain unknown. Further, characterization of structural variants (SVs) involving SMARCB1 in RMC remains limited., Methods: We used linked-read genome sequencing to reconstruct germline and somatic haplotypes in 15 unrelated patients with RMC registered on the Children's Oncology Group (COG) AREN03B2 study between 2006 and 2017 or from our prior study. We performed fine-mapping of the HBB locus and assessed the germline for cancer predisposition genes. Subsequently, we assessed the tumor samples for mutations outside of SMARCB1 and integrated RNA sequencing to interrogate the structural variants at the SMARCB1 locus., Results: We find that the haplotype of the sickle cell mutation in patients with RMC originated from three geographical regions in Africa. In addition, fine-mapping of the HBB locus identified the sickle cell mutation as the sole candidate variant. We further identify that the SMARCB1 structural variants are characterized by blunt or 1-bp homology events., Conclusions: Our findings suggest that RMC does not arise from a single founder population and that the HbS allele is a strong candidate germline allele which confers risk for RMC. Furthermore, we find that the SVs that disrupt SMARCB1 function are likely repaired by non-homologous end-joining. These findings highlight how haplotype-based analyses using linked-read genome sequencing can be applied to identify potential risk variants in small and rare disease cohorts and provide nucleotide resolution to structural variants., (© 2021. The Author(s).)

Exciting therapeutic targets are emerging from CRISPR-based screens of high mutational-burden adult cancers. A key question, however, is whether functional genomic approaches will yield new targets in pediatric cancers, known for remarkably few mutations, which often encode proteins considered challenging drug targets. To address this, we created a first-generation pediatric cancer dependency map representing 13 pediatric solid and brain tumor types. Eighty-two pediatric cancer cell lines were subjected to genome-scale CRISPR-Cas9 loss-of-function screening to identify genes required for cell survival. In contrast to the finding that pediatric cancers harbor fewer somatic mutations, we found a similar complexity of genetic dependencies in pediatric cancer cell lines compared to that in adult models. Findings from the pediatric cancer dependency map provide preclinical support for ongoing precision medicine clinical trials. The vulnerabilities observed in pediatric cancers were often distinct from those in adult cancer, indicating that repurposing adult oncology drugs will be insufficient to address childhood cancers.

Purpose of Review: Pediatric renal tumors account for 7% of new cancer diagnoses in children. Here, we will review results from recently completed clinical trials informing the current standard of care and discuss targeted and immune therapies being explored for the treatment of high risk or relapsed/refractory pediatric renal malignancies., Recent Findings: Cooperative group trials have continued to make improvements in the care of children with pediatric tumors. In particular, trials that standardize treatment of rare cancers (e.g., bilateral Wilms tumor) have improved outcomes significantly. We have seen improvements in event free and overall survival in recently completed clinical trials for many pediatric renal tumors. Still, there are subsets of rarer cancers where outcomes remain poor and new therapeutic strategies are needed. Future trials aim to balance treatment toxicity with treatment efficacy for those with excellent outcomes while identifying novel therapeutics for those with poor outcomes.

Few therapies target the loss of tumor suppressor genes in cancer. We examine CRISPR-SpCas9 and RNA-interference loss-of-function screens to identify new therapeutic targets associated with genomic loss of tumor suppressor genes. The endosomal sorting complexes required for transport (ESCRT) ATPases VPS4A and VPS4B score as strong synthetic lethal dependencies. VPS4A is essential in cancers harboring loss of VPS4B adjacent to SMAD4 on chromosome 18q and VPS4B is required in tumors with co-deletion of VPS4A and CDH1 (E-cadherin) on chromosome 16q. We demonstrate that more than 30% of cancers selectively require VPS4A or VPS4B. VPS4A suppression in VPS4B-deficient cells selectively leads to ESCRT-III filament accumulation, cytokinesis defects, nuclear deformation, G2/M arrest, apoptosis, and potent tumor regression. CRISPR-SpCas9 screening and integrative genomic analysis reveal other ESCRT members, regulators of abscission, and interferon signaling as modifiers of VPS4A dependency. We describe a compendium of synthetic lethal vulnerabilities and nominate VPS4A and VPS4B as high-priority therapeutic targets for cancers with 18q or 16q loss., Competing Interests: Declaration of Interests F.V. and B.R.P. have received research support for this work from Novo Ventures. A.J.A. has consulted for Oncorus, Inc., Arrakis Therapeutics, and Merck & Co., Inc. and has research funding from Mirati Therapeutics and Deerfield, Inc. that is unrelated to this work. B.M.W. consults for BioLineRx, Celgene, G1 Therapeutics, and GRAIL and receives unrelated funding from Celgene and Eli Lilly. A.T. consults for Tango Therapeutics. W.C.H. consults for ThermoFisher, Solvasta Ventures, MPM Capital, KSQ Therapeutics, iTeos, Tyra Biosciences, Frontier Medicine, and Paraxel. K.S. has previously consulted for Novartis and Rigel Pharmaceuticals and receives funding through the Novartis/Dana-Farber Cancer Institute Drug Discovery Program unrelated to this project. T.R.G. is a consultant to GlaxoSmithKline, a founder of Sherlock Biosciences, and was formerly a consultant and equity holder in Foundation Medicine, acquired by Roche. T.R.G. receives research funding unrelated to this project from Bayer Healthcare. J.E.N., B.R.P., F.V., and A.J.A. are inventors on pending patent applications related to this work that are owned by the Broad Institute of MIT and Harvard and the Dana-Farber Cancer Institute., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Purpose: Rhabdoid tumors are devastating pediatric cancers in need of improved therapies. We sought to identify small molecules that exhibit in vitro and in vivo efficacy against preclinical models of rhabdoid tumor., Experimental Design: We screened eight rhabdoid tumor cell lines with 481 small molecules and compared their sensitivity with that of 879 other cancer cell lines. Genome-scale CRISPR-Cas9 inactivation screens in rhabdoid tumors were analyzed to confirm target vulnerabilities. Gene expression and CRISPR-Cas9 data were queried across cell lines and primary rhabdoid tumors to discover biomarkers of small-molecule sensitivity. Molecular correlates were validated by manipulating gene expression. Subcutaneous rhabdoid tumor xenografts were treated with the most effective drug to confirm in vitro results., Results: Small-molecule screening identified the protein-translation inhibitor homoharringtonine (HHT), an FDA-approved treatment for chronic myelogenous leukemia (CML), as the sole drug to which all rhabdoid tumor cell lines were selectively sensitive. Validation studies confirmed the sensitivity of rhabdoid tumor to HHT was comparable with that of CML cell lines. Low expression of the antiapoptotic gene BCL2L1 , which encodes Bcl-XL, was the strongest predictor of HHT sensitivity, and HHT treatment consistently depleted Mcl-1, the synthetic-lethal antiapoptotic partner of Bcl-XL. Rhabdoid tumor cell lines and primary-tumor samples expressed low BCL2L1 , and overexpression of BCL2L1 induced resistance to HHT in rhabdoid tumor cells. Furthermore, HHT treatment inhibited rhabdoid tumor cell line and patient-derived xenograft growth in vivo ., Conclusions: Rhabdoid tumor cell lines and xenografts are highly sensitive to HHT, at least partially due to their low expression of BCL2L1 . HHT may have therapeutic potential against rhabdoid tumors., (©2020 American Association for Cancer Research.)

Introduction: Tissue from pediatric solid tumors is in high demand for use in high-impact research studies, making the allocation of tissue from an anatomic pathology laboratory challenging. We designed, implemented, and assessed an interdepartmental process to optimize tissue allocation of pediatric solid tumors for both clinical care and research., Methods: Oncologists, pathologists, surgeons, interventional radiologists, pathology technical staff, and clinical research coordinators participated in the workflow design. Procedures were created to address patient identification and consent, prioritization of protocols, electronic communication of requests, tissue preparation, and distribution. Pathologists were surveyed about the value of the new workflow., Results: Over a 5-year period, 644 pediatric solid tumor patients consented to one or more studies requesting archival or fresh tissue. Patients had a variety of tumor types, with many rare and singular diagnoses. Sixty-seven percent of 1768 research requests were fulfilled. Requests for archival tissue were fulfilled at a significantly higher rate than those for fresh tissue (P > .001), and requests from resection specimens were fulfilled at a significantly higher rate than those from biopsies (P > .0001). In an anonymous survey, seven of seven pathologists reported that the process had improved since the introduction of the electronic communication model., Conclusions: A collaborative and informed model for tissue allocation is successful in distributing archival and fresh tissue for clinical research studies. Our workflows and policies have gained pathologists' approval and streamlined our processes. As clinical and research programs evolve, a thoughtful tissue allocation process will facilitate ongoing research., (© 2020 Wiley Periodicals, Inc.)

Androgen-receptor (AR) inhibitors, including enzalutamide, are used for treatment of all metastatic castration-resistant prostate cancers (mCRPCs). However, some patients develop resistance or never respond. We find that the transcription factor CREB5 confers enzalutamide resistance in an open reading frame (ORF) expression screen and in tumor xenografts. CREB5 overexpression is essential for an enzalutamide-resistant patient-derived organoid. In AR-expressing prostate cancer cells, CREB5 interactions enhance AR activity at a subset of promoters and enhancers upon enzalutamide treatment, including MYC and genes involved in the cell cycle. In mCRPC, we found recurrent amplification and overexpression of CREB5. Our observations identify CREB5 as one mechanism that drives resistance to AR antagonists in prostate cancers., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

The MRI-derived porosity index (PI) is a non-invasively obtained biomarker based on an ultrashort echo time sequence that images both bound and pore water protons in bone, corresponding to water bound to organic collagenous matrix and freely moving water, respectively. This measure is known to strongly correlate with the actual volumetric cortical bone porosity. However, it is unknown whether PI may also be able to directly quantify bone organic composition and/or mechanical properties. We investigated this in human cadaveric tibiae by comparing PI values to near infrared spectral imaging (NIRSI) compositional data and mechanical compression data. Data were obtained from a cohort of eighteen tibiae from male and female donors with a mean ± SD age of 70 ± 21 years. Biomechanical stiffness in compression and NIRSI-derived collagen and bound water content all had significant inverse correlations with PI ( r  = -0.79, -0.73, and -0.95 and p  = 0.002, 0.007, and <0.001, respectively). The MRI-derived bone PI alone was a moderate predictor of bone stiffness ( R 2  = 0.63, p  = 0.002), and multivariate analyses showed that neither cortical bone cross-sectional area nor NIRSI values improved bone stiffness prediction compared to PI alone. However, NIRSI-obtained collagen and water data together were a moderate predictor of bone stiffness (R 2  = 0.52, p  = 0.04). Our data validates the MRI-derived porosity index as a strong predictor of organic composition of bone and a moderate predictor of bone stiffness, and also provides preliminary evidence that NIRSI measures may be useful in future pre-clinical studies on bone pathology.

BET-bromodomain inhibition (BETi) has shown pre-clinical promise for MYC-amplified medulloblastoma. However, the mechanisms for its action, and ultimately for resistance, have not been fully defined. Here, using a combination of expression profiling, genome-scale CRISPR/Cas9-mediated loss of function and ORF/cDNA driven rescue screens, and cell-based models of spontaneous resistance, we identify bHLH/homeobox transcription factors and cell-cycle regulators as key genes mediating BETi's response and resistance. Cells that acquire drug tolerance exhibit a more neuronally differentiated cell-state and expression of lineage-specific bHLH/homeobox transcription factors. However, they do not terminally differentiate, maintain expression of CCND2, and continue to cycle through S-phase. Moreover, CDK4/CDK6 inhibition delays acquisition of resistance. Therefore, our data provide insights about the mechanisms underlying BETi effects and the appearance of resistance and support the therapeutic use of combined cell-cycle inhibitors with BETi in MYC-amplified medulloblastoma.

Focal amplification of chromosome 1q23.3 in patients with advanced primary or relapsed urothelial carcinomas is associated with poor survival. We interrogated chromosome 1q23.3 and the nearby focal amplicon 1q21.3, as both are associated with increased lymph node disease in patients with urothelial carcinoma. Specifically, we assessed whether the oncogene MCL1 that resides in 1q21.3 and the genes that reside in the 1q23.3 amplicon were required for the proliferation or survival of urothelial carcinoma. We observed that suppressing MCL1 or the death effector domain-containing protein (DEDD) in the cells that harbor amplifications of 1q21.3 or 1q23.3, respectively, inhibited cell proliferation. We also found that overexpression of MCL1 or DEDD increased anchorage independence growth in vitro and increased experimental metastasis in vivo in the nonamplified urothelial carcinoma cell line, RT112. The expression of MCL1 confers resistance to a range of apoptosis inducers, while the expression of DEDD led to resistance to TNFα-induced apoptosis. These observations identify MCL1 and DEDD as genes that contribute to aggressive urothelial carcinoma. IMPLICATIONS: These studies identify MCL1 and DEDD as genes that contribute to aggressive urothelial carcinomas., (©2019 American Association for Cancer Research.)

Cells are subjected to oxidative stress during the initiation and progression of tumors, and this imposes selective pressure for cancer cells to adapt mechanisms to tolerate these conditions. Here, we examined the dependency of cancer cells on glutathione (GSH), the most abundant cellular antioxidant. While cancer cell lines displayed a broad range of sensitivities to inhibition of GSH synthesis, the majority were resistant to GSH depletion. To identify cellular pathways required for this resistance, we carried out genetic and pharmacologic screens. Both approaches revealed that inhibition of deubiquitinating enzymes (DUBs) sensitizes cancer cells to GSH depletion. Inhibition of GSH synthesis, in combination with DUB inhibition, led to an accumulation of polyubiquitinated proteins, induction of proteotoxic stress, and cell death. These results indicate that depletion of GSH renders cancer cells dependent on DUB activity to maintain protein homeostasis and cell viability and reveal a potentially exploitable vulnerability for cancer therapy., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Malignant rhabdoid tumors (MRT) are highly aggressive pediatric cancers that respond poorly to current therapies. In this study, we screened several MRT cell lines with large-scale RNAi, CRISPR-Cas9, and small-molecule libraries to identify potential drug targets specific for these cancers. We discovered MDM2 and MDM4 , the canonical negative regulators of p53, as significant vulnerabilities. Using two compounds currently in clinical development, idasanutlin (MDM2-specific) and ATSP-7041 (MDM2/4-dual), we show that MRT cells were more sensitive than other p53 wild-type cancer cell lines to inhibition of MDM2 alone as well as dual inhibition of MDM2/4. These compounds caused significant upregulation of the p53 pathway in MRT cells, and sensitivity was ablated by CRISPR-Cas9-mediated inactivation of TP53 . We show that loss of SMARCB1, a subunit of the SWI/SNF (BAF) complex mutated in nearly all MRTs, sensitized cells to MDM2 and MDM2/4 inhibition by enhancing p53-mediated apoptosis. Both MDM2 and MDM2/4 inhibition slowed MRT xenograft growth in vivo , with a 5-day idasanutlin pulse causing marked regression of all xenografts, including durable complete responses in 50% of mice. Together, these studies identify a genetic connection between mutations in the SWI/SNF chromatin-remodeling complex and the tumor suppressor gene TP53 and provide preclinical evidence to support the targeting of MDM2 and MDM4 in this often-fatal pediatric cancer. SIGNIFICANCE: This study identifies two targets, MDM2 and MDM4, as vulnerabilities in a deadly pediatric cancer and provides preclinical evidence that compounds inhibiting these proteins have therapeutic potential., (©2019 American Association for Cancer Research.)

Currently, clinical determination of pathologic fracture risk in the hip is conducted using measures of defect size and shape in the stance loading condition. However, these measures often do not consider how changing lesion locations or how various loading conditions impact bone strength. The goal of this study was to determine the impact of defect location on bone strength parameters in both the sideways fall and stance-loading conditions. We recruited 20 female subjects aged 48-77 years for this study and performed MRI of the proximal femur. Using these images, we simulated 10-mm pathologic defects in greater trochanter, superior, middle, and inferior femoral head, superior, middle, and inferior femoral neck, and lateral, middle, and medial proximal diaphysis to determine the effect of defect location on change in bone strength by performing finite element analysis. We compared the effect of each osteolytic lesion on bone stiffness, strength, resilience, and toughness. For the sideways fall loading, defects in the inferior femoral head (12.21%) and in the greater trochanter (6.43%) resulted in the greatest overall reduction in bone strength. For the stance loading, defects in the mid femoral head (-7.91%) and superior femoral head (-7.82%) resulted in the greatest overall reduction in bone strength. Changes in stiffness, yield force, ultimate force, resilience, and toughness were not found to be significantly correlated between the sideways fall and stance-loading for the majority of defect locations, suggesting that calculations based on the stance-loading condition are not predictive of the change in bone strength experienced in the sideways fall condition. While stiffness was significantly related to yield force (R 2  > 0.82), overall force (R 2  > 0.59), and resilience (R 2  > 0.55), in both, the stance-loading and sideways fall conditions for most defect locations, stiffness was not significantly related to toughness. Therefore, structure-dependent measure such as stiffness may not fully explain the post-yield measures, which depend on material failure properties. The data showed that MRI-based models have the sensitivity to determine the effect of pathologic lesions on bone strength., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Renal medullary carcinoma (RMC) is a rare and deadly kidney cancer in patients of African descent with sickle cell trait. We have developed faithful patient-derived RMC models and using whole-genome sequencing, we identified loss-of-function intronic fusion events in one SMARCB1 allele with concurrent loss of the other allele. Biochemical and functional characterization of these models revealed that RMC requires the loss of SMARCB1 for survival. Through integration of RNAi and CRISPR-Cas9 loss-of-function genetic screens and a small-molecule screen, we found that the ubiquitin-proteasome system (UPS) was essential in RMC. Inhibition of the UPS caused a G2/M arrest due to constitutive accumulation of cyclin B1. These observations extend across cancers that harbor SMARCB1 loss, which also require expression of the E2 ubiquitin-conjugating enzyme, UBE2C . Our studies identify a synthetic lethal relationship between SMARCB1 -deficient cancers and reliance on the UPS which provides the foundation for a mechanism-informed clinical trial with proteasome inhibitors., Competing Interests: AH, YT, JW, WK, BK, MD, GK, GS, TH, JL, XY, MT, MG, AS, RD, AW, BM, KL, PK, AT, CC, CC, AC, BC, KJ, BV, DS, OG, SS, DR, PG, SC, EM, CR, KL, JB No competing interests declared, PB, RB is a consultant for Novartis (Cambridge, MA), PC is an adviser for Pfizer, Inc (Groton, CT), CK is a Scientific Founder, member of the Board of Director, Scientific Advisory Board member, Shareholder, and Consultant for Foghorn Therapeutics, Inc (Cambridge, MA). Disclosure information for CK is also found at: http://www.kadochlab.org, WH is a consultant for Thermo Fisher, Aju IB, MPM Capital and Paraxel. W.C.H. is a founder and shareholder and serves on the scientific advisory board of KSQ Therapeutics, (© 2019, Hong et al.)

Renal medullary carcinoma (RMC) is one of the most aggressive renal cell carcinomas. It predominantly afflicts young adults and adolescents with sickle cell trait and other sickle hemoglobinopathies, and is refractory to targeted and antiangiogenic therapies used in patients with clear-cell renal cell carcinoma. Platinum-based cytotoxic chemotherapy is the mainstay for RMC treatment. On the basis of recent advances in the diagnosis, management, and clinical trial development for RMC, a panel of experts met in October 2017 and developed updated consensus recommendations to inform clinicians, researchers, and patients. Because RMC often aggressively recurs while patients are still recovering from nephrectomy, upfront chemotherapy should be considered for most patients, including those with localized disease. After safety and dosing information has been established in adults, phase II and III trials enrolling patients with RMC should allow patients aged 12 years and older to be accrued. Patients with the very rare unclassified renal cell carcinoma with medullary phenotype variant should be included in RMC trials. Medical providers should be aware that RMC can afflict subjects of all races, and not only those of African descent, and that the presence of sickle cell trait, or of other sickle hemoglobinopathies, can affect drug responses and toxicity., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The prevalence of metabolic bone diseases particularly osteoporosis and its precursor, osteopenia, continue to grow as serious global health issues today. On a worldwide perspective, 200million people suffer from osteoporosis and in 2005, over 2million fracture incidents were estimated due to osteoporosis in the United States. Currently, osteoporosis and other metabolic bone diseases are evaluated primarily through dual energy X-ray absorptiometry, and rarely by bone biopsy with tetracycline labeling or Technetium-99m ( 99m Tc) based bone scintigraphy. Deficiencies in these methods have prompted the use of more precise methods of assessment. This review highlights the use of 18 F-sodium fluoride (NaF) with PET (NaF-PET), NaF-PET/CT, or NaF-PET/MRI in the evaluation of osteoporosis and osteopenia in the lumbar spine and hip. This imaging modality provides a molecular perspective with respect to the underlying metabolic alterations that lead to osseous disorders by measuring bone turnover through standardized uptake values. Its sensitivity and ability to examine the entire skeletal system make it a more superior imaging modality compared to standard structural imaging techniques. Further research is needed to determine its accuracy in reflecting the efficacy of therapeutic interventions in metabolic bone diseases., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Alternative splicing of mRNA precursors represents a key gene expression regulatory step and permits the generation of distinct protein products with diverse functions. In a genome-scale expression screen for inducers of the epithelial-to-mesenchymal transition (EMT), we found a striking enrichment of RNA-binding proteins. We validated that QKI and RBFOX1 were necessary and sufficient to induce an intermediate mesenchymal cell state and increased tumorigenicity. Using RNA-seq and eCLIP analysis, we found that QKI and RBFOX1 coordinately regulated the splicing and function of the actin-binding protein FLNB, which plays a causal role in the regulation of EMT. Specifically, the skipping of FLNB exon 30 induced EMT by releasing the FOXC1 transcription factor. Moreover, skipping of FLNB exon 30 is strongly associated with EMT gene signatures in basal-like breast cancer patient samples. These observations identify a specific dysregulation of splicing, which regulates tumor cell plasticity and is frequently observed in human cancer., Competing Interests: JL, PC, CC, KL, JH, AG, JK, NI, JD, SL, CD, KH, AH, OG, SG, JG, DR, JZ, AB, RW, WH No competing interests declared, (© 2018, Li et al.)

Rare cancers pose unique challenges to research due to their low incidence. Barriers include a scarcity of tissue and experimental models to enable basic research and insufficient patient accrual for clinical studies. Consequently, an understanding of the genetic and cellular features of many rare cancer types and their associated vulnerabilities has been lacking. However, new opportunities are emerging to facilitate discovery of therapeutic targets in rare cancers. Online platforms are allowing patients with rare cancers to organize on an unprecedented scale, tumor genome sequencing is now routinely performed in research and clinical settings, and the efficiency of patient-derived model generation has improved. New CRISPR/Cas9 and small-molecule libraries permit cancer dependency discovery in a rapid and systematic fashion. In parallel, large-scale studies of common cancers now provide reference datasets to help interpret rare cancer profiling data. Together, these advances motivate consideration of new research frameworks to accelerate rare cancer target discovery., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Although renal medullary carcinoma (RMC) is a rare subtype of kidney cancer, it is particularly devastating in that it is nearly uniformly lethal. No established guidelines exist for the diagnosis and management of RMC. In April 2016, a panel of experts developed clinical guidelines on the basis of a literature review and consensus statements. The goal was to propose recommendations for standardized diagnostic and management approaches and to establish an international clinical registry and biorepository for RMC. Published data are limited to case reports and small retrospective reviews. The RMC Working Group prepared recommendations to inform providers and patients faced with a low level of medical evidence. The diagnosis of RMC should be considered in all patients younger than 50 years with poorly differentiated carcinoma that arises from the renal medulla. These patients should be tested for sickle cell hemoglobinopathies, and if positive, SMARCB1/INI1 loss should be confirmed by immunohistochemistry. The majority of patients with RMC are diagnosed with metastatic disease. Upfront radical nephrectomy should be considered in patients with good performance status and low metastatic burden or after response to systemic therapy. Currently, cytotoxic, platinum-based chemotherapy provides the best, albeit brief, palliative clinical benefit. Vascular endothelial growth factor-directed therapies and mammalian target of rapamycin inhibitors are ineffective in RMC as monotherapy. Therapeutic trials of novel agents are now available for RMC, and every effort should be made to enroll patients in clinical studies.

Identifying therapeutic targets in rare cancers remains challenging due to the paucity of established models to perform preclinical studies. As a proof-of-concept, we developed a patient-derived cancer cell line, CLF-PED-015-T, from a paediatric patient with a rare undifferentiated sarcoma. Here, we confirm that this cell line recapitulates the histology and harbours the majority of the somatic genetic alterations found in a metastatic lesion isolated at first relapse. We then perform pooled CRISPR-Cas9 and RNAi loss-of-function screens and a small-molecule screen focused on druggable cancer targets. Integrating these three complementary and orthogonal methods, we identify CDK4 and XPO1 as potential therapeutic targets in this cancer, which has no known alterations in these genes. These observations establish an approach that integrates new patient-derived models, functional genomics and chemical screens to facilitate the discovery of targets in rare cancers.

Importance: Pediatric cancers represent a unique case with respect to cancer genomics and precision medicine, as the mutation frequency is low, and targeted therapies are less available. Consequently, it is unknown whether clinical sequencing can be of benefit., Objective: To assess the feasibility of identifying actionable alterations and making individualized cancer therapy (iCat) recommendations in pediatric patients with extracranial solid tumors., Design, Setting, and Participants: Clinical sequencing study at 4 academic medical centers enrolling patients between September 5, 2012, and November 19, 2013, with 1 year of clinical follow-up. Participants were 30 years or younger with high-risk, recurrent, or refractory extracranial solid tumors. The data analysis was performed October 28, 2014., Interventions: Tumor profiling performed on archived clinically acquired specimens consisted of mutation detection by a Sequenom assay or targeted next-generation sequencing and copy number assessment by array comparative genomic hybridization. Results were reviewed by a multidisciplinary expert panel, and iCat recommendations were made if an actionable alteration was present, and an appropriate drug was available., Main Outcomes and Measures: Feasibility was assessed using a 2-stage design based on the proportion of patients with recommendations., Results: Of 100 participants (60 male; median [range] age, 13.4 [0.8-29.8] years), profiling was technically successful in 89 (89% [95% CI, 83%-95%]). Median (range) follow-up was 6.8 (2.0-23.6) months. Overall, 31 (31% [95% CI, 23%-41%]) patients received an iCat recommendation and 3 received matched therapy. The most common actionable alterations leading to an iCat recommendation were cancer-associated signaling pathway gene mutations (n = 10) and copy number alterations in MYC/MYCN (n = 6) and cell cycle genes (n = 11). Additional alterations with implications for clinical care but not resulting in iCat recommendations were identified, including mutations indicating the possible presence of a cancer predisposition syndrome and translocations suggesting a change in diagnosis. In total, 43 (43% [95% CI, 33%-53%]) participants had results with potential clinical significance., Conclusions and Relevance: A multi-institution clinical genomics study in pediatric oncology is feasible and a substantial proportion of relapsed or refractory pediatric solid tumors have actionable alterations., Trial Registration: clinicaltrials.gov Identifier: NCT01853345.

International efforts to sequence cancer genomes now provide an overview of the major genetic alterations that occur in most human cancers. These studies have identified many highly recurrent alterations in specific cancer subtypes but have also identified mutations that occur at lower frequency and unstudied variants of known cancer-associated genes. To elucidate the function of such cancer alleles, we have developed several approaches to systematically interrogate genomic changes found in human tumors. In general, we have taken two complementary approaches. In the first approach, we focus on perturbing genes identified as mutated, amplified, or deleted by cancer genome annotation efforts, whereas in the second, we have taken an unbiased approach to identify genes that are essential for cancer cell proliferation or survival in cell lines that are extensively annotated to identify context-specific essential genes. These studies begin to allow us to define a cancer dependencies map., (© 2016 Howard et al.; Published by Cold Spring Harbor Laboratory Press.)

Central-line-associated bloodstream infections (CLABSIs) are a significant cause of preventable harm. A collaborative project involving a multifaceted intervention was used in the Michigan Keystone Project and associated with significant reductions in these infections. This intervention included the Comprehensive Unit-based Safety Program, a multifaceted approach to CLABSI prevention, and the monitoring and reporting of infections. The purpose of this study was to determine whether the multifaceted intervention from the Michigan Keystone program could be implemented in Connecticut and to evaluate the impact on CLABSI rates in intensive care units (ICUs). The primary outcome was the NHSN-defined rate of CLABSI. Seventeen ICUs, representing 14 hospitals and 104,695 catheter days were analyzed. The study period included up to four quarters (12 months) of baseline data and seven quarters (21 months) of postintervention data. The overall mean (median) CLABSI rate decreased from 1.8 (1.8) infections per 1,000 catheter days at baseline to 1.1 (0) at seven quarters postimplementation of the intervention. This study demonstrated that the multifaceted intervention used in the Keystone program could be successfully implemented in another state and was associated with a reduction in CLABSI rates in Connecticut. Moreover, even though the statewide baseline CLABSI rate in Connecticut was low, rates were reduced even further and well below national benchmarks., (© 2013 National Association for Healthcare Quality.)

The objective of this study was to investigate variations in UGT1A1 polymorphisms and haplotypes among African-American and Caucasian women and to assess whether variants other than UGT1A1*28 are associated with total serum bilirubin levels. The (TA)(n) repeats and 14 single nucleotide polymorphisms (SNPs) in the UGT1A1 gene were genotyped in 335 African Americans and 181 Caucasians. Total serum bilirubin levels were available in a subset of 125 women. Allele frequencies of all SNPs and (TA)(n) repeats were significantly different between African Americans and Caucasians. In Caucasians, three common haplotypes accounted for 71.8% of chromosomes, whereas five common haplotypes accounted for only 46.6% of chromosomes in African Americans. Mean total serum bilirubin levels were significantly lower (p = 0.005) in African Americans (0.36 mg/dl) than in Caucasians (0.44 mg/dl). The (TA)(n) repeats explained a significant amount of variation in total bilirubin levels (R(2) = 0.27, p < 0.0001), whereas other SNPs were less correlative. Thus, significant variations in UGT1A1 haplotype structure exist between African Americans and Caucasians in this relatively large cohort of women. The correlation of UGT1A1 with total bilirubin levels was mainly due to (TA)(n) repeats in Caucasians but a clear correlation was not observed in African Americans because of the high diversity of haplotypes and the small sample size. These data have implications for the design of epidemiologic studies of cancer susceptibility and pharmacogenetic studies for adverse drug reactions in populations of African ancestry.

The Sir2 histone deacetylase functions as a chromatin silencer to regulate recombination, genomic stability, and aging in budding yeast. Seven mammalian Sir2 homologs have been identified (SIRT1-SIRT7), and it has been speculated that some may have similar functions to Sir2. Here, we demonstrate that SIRT6 is a nuclear, chromatin-associated protein that promotes resistance to DNA damage and suppresses genomic instability in mouse cells, in association with a role in base excision repair (BER). SIRT6-deficient mice are small and at 2-3 weeks of age develop abnormalities that include profound lymphopenia, loss of subcutaneous fat, lordokyphosis, and severe metabolic defects, eventually dying at about 4 weeks. We conclude that one function of SIRT6 is to promote normal DNA repair, and that SIRT6 loss leads to abnormalities in mice that overlap with aging-associated degenerative processes.

The evolutionarily conserved spindle checkpoint is a key mechanism ensuring high-fidelity chromosome transmission. The checkpoint monitors attachment between kinetochores and mitotic spindles and the tension between sister kinetochores. In the absence of proper attachment or tension, the spindle checkpoint mediates cell cycle arrest prior to anaphase. Saccharomyces cerevisiae Mad1p is required for the spindle checkpoint and for chromosome transmission fidelity. Moreover, Mad1p associates with the nuclear pore complex (NPC) and is enriched at kinetochores upon checkpoint activation. Using partial mad1 deletion alleles we determined that the C-terminal half of Mad1p is necessary and sufficient for checkpoint activation in response to microtubule depolymerizing agents, high-fidelity transmission of a reporter chromosome fragment, and in vivo association with centromeres, but not for robust NPC association. Thus, spindle checkpoint activation and chromosome transmission fidelity correlate and these Mad1p functions likely involve kinetochore association but not robust NPC association. These studies are the basis for elucidating the role of protein complexes containing Mad1p in the spindle checkpoint pathway and in maintaining genome stability in S. cerevisiae and other systems.

Eight proteins, HRPs 1, 2, 3, 4, 5 and 6, lysozyme and histatin 6, are the major cationic components of the parotid salivas of normal healthy individuals. Histatins 2 and 4 appear to be further degradation products of the HRPs. Capillary electrophoresis separates all of these eight components, thus allowing future studies to correlate protein concentration with antimicrobial activity in health and disease.

Synthetic homologous peptides of L-histidine, ranging in length from 3 to 64 amino-acid residues, suppressed blastospore viability. Killing activity was dependent upon the peptide molecular size and concentration, and the time of cell exposure to the agent, but was independent of cell concentration in the range 10(5)-10(7) colony-forming units (c.f.u.) per ml. A 25 amino-acid residue polypeptide, similar to the human parotid salivary histidine-rich peptide (HRP-5), also affected yeast viability. Its killing effect was dependent upon the number of c.f.u. in the assay, as well as contact time with the blastospores and the final peptide concentration. HRP-5 inhibition increased with rising pH in the range 5-7.4, in contrast to poly-L-histidine and ketoconazole, which had optimal candidacidal activity at about pH 6. Poly-L-histidine, HRP-5, and ketoconazole each prevented conversion of blastospores to germ tubes, but their rank order of effectiveness varied with the assay selected. In N-acetylglucosamine-supplemented fetal calf serum, poly-L-histidine and HRP-5 were more effective inhibitors than ketoconazole, but the reverse was true in amino-acid-supplemented glucose beef-extract medium. Reduction of both germ-tube numbers and germ-tube size by HRP-5 was concentration-dependent.