Quantitative Proteomics of the Cancer Cell Line Encyclopedia.

Proteins are essential agents of biological processes. To date, large-scale profiling of cell line collections including the Cancer Cell Line Encyclopedia (CCLE) has focused primarily on genetic information whereas deep interrogation of the proteome has remained out of reach. Here, we expand the CCLE through quantitative profiling of thousands of proteins by mass spectrometry across 375 cell lines from diverse lineages to reveal information undiscovered by DNA and RNA methods. We observe unexpected correlations within and between pathways that are largely absent from RNA. An analysis of microsatellite instable (MSI) cell lines reveals the dysregulation of specific protein complexes associated with surveillance of mutation and translation. These and other protein complexes were associated with sensitivity to knockdown of several different genes. These data in conjunction with the wider CCLE are a broad resource to explore cellular behavior and facilitate cancer research.

Cancer-Germline Antigen Expression Discriminates Clinical Outcome to CTLA-4 Blockade.

CTLA-4 immune checkpoint blockade is clinically effective in a subset of patients with metastatic melanoma. We identify a subcluster of MAGE-A cancer-germline antigens, located within a narrow 75 kb region of chromosome Xq28, that predicts resistance uniquely to blockade of CTLA-4, but not PD-1. We validate this gene expression signature in an independent anti-CTLA-4-treated cohort and show its specificity to the CTLA-4 pathway with two independent anti-PD-1-treated cohorts. Autophagy, a process critical for optimal anti-cancer immunity, has previously been shown to be suppressed by the MAGE-TRIM28 ubiquitin ligase in vitro. We now show that the expression of the key autophagosome component LC3B and other activators of autophagy are negatively associated with MAGE-A protein levels in human melanomas, including samples from patients with resistance to CTLA-4 blockade. Our findings implicate autophagy suppression in resistance to CTLA-4 blockade in melanoma, suggesting exploitation of autophagy induction for potential therapeutic synergy with CTLA-4 inhibitors.

A Cancer Cell Program Promotes T Cell Exclusion and Resistance to Checkpoint Blockade.

Immune checkpoint inhibitors (ICIs) produce durable responses in some melanoma patients, but many patients derive no clinical benefit, and the molecular underpinnings of such resistance remain elusive. Here, we leveraged single-cell RNA sequencing (scRNA-seq) from 33 melanoma tumors and computational analyses to interrogate malignant cell states that promote immune evasion. We identified a resistance program expressed by malignant cells that is associated with T cell exclusion and immune evasion. The program is expressed prior to immunotherapy, characterizes cold niches in situ, and predicts clinical responses to anti-PD-1 therapy in an independent cohort of 112 melanoma patients. CDK4/6-inhibition represses this program in individual malignant cells, induces senescence, and reduces melanoma tumor outgrowth in mouse models in vivo when given in combination with immunotherapy. Our study provides a high-resolution landscape of ICI-resistant cell states, identifies clinically predictive signatures, and suggests new therapeutic strategies to overcome immunotherapy resistance.

Defining a Cancer Dependency Map.

Most human epithelial tumors harbor numerous alterations, making it difficult to predict which genes are required for tumor survival. To systematically identify cancer dependencies, we analyzed 501 genome-scale loss-of-function screens performed in diverse human cancer cell lines. We developed DEMETER, an analytical framework that segregates on- from off-target effects of RNAi. 769 genes were differentially required in subsets of these cell lines at a threshold of six SDs from the mean. We found predictive models for 426 dependencies (55%) by nonlinear regression modeling considering 66,646 molecular features. Many dependencies fall into a limited number of classes, and unexpectedly, in 82% of models, the top biomarkers were expression based. We demonstrated the basis behind one such predictive model linking hypermethylation of the UBB ubiquitin gene to a dependency on UBC. Together, these observations provide a foundation for a cancer dependency map that facilitates the prioritization of therapeutic targets.

High-performance 4-nm-resolution X-ray tomography using burst ptychography.

Advances in science, medicine and engineering rely on breakthroughs in imaging, particularly for obtaining multiscale, three-dimensional information from functional systems such as integrated circuits or mammalian brains. Achieving this goal often requires combining electron- and photon-based approaches. Whereas electron microscopy provides nanometre resolution through serial, destructive imaging of surface layers1, ptychographic X-ray computed tomography2 offers non-destructive imaging and has recently achieved resolutions down to seven nanometres for a small volume3. Here we implement burst ptychography, which overcomes experimental instabilities and enables much higher performance, with 4-nanometre resolution at a 170-times faster acquisition rate, namely, 14,000 resolution elements per second. Another key innovation is tomographic back-propagation reconstruction4, allowing us to image samples up to ten times larger than the conventional depth of field. By combining the two innovations, we successfully imaged a state-of-the-art (seven-nanometre node) commercial integrated circuit, featuring nanostructures made of low- and high-density materials such as silicon and metals, which offer good radiation stability and contrast at the selected X-ray wavelength. These capabilities enabled a detailed study of the chip's design and manufacturing, down to the level of individual transistors. We anticipate that the combination of nanometre resolution and higher X-ray flux at next-generation X-ray sources will have a revolutionary impact in fields ranging from electronics to electrochemistry and neuroscience.

Lessons from the cancer genome.

Systematic studies of the cancer genome have exploded in recent years. These studies have revealed scores of new cancer genes, including many in processes not previously known to be causal targets in cancer. The genes affect cell signaling, chromatin, and epigenomic regulation; RNA splicing; protein homeostasis; metabolism; and lineage maturation. Still, cancer genomics is in its infancy. Much work remains to complete the mutational catalog in primary tumors and across the natural history of cancer, to connect recurrent genomic alterations to altered pathways and acquired cellular vulnerabilities, and to use this information to guide the development and application of therapies.

An interactive resource to identify cancer genetic and lineage dependencies targeted by small molecules.

The high rate of clinical response to protein-kinase-targeting drugs matched to cancer patients with specific genomic alterations has prompted efforts to use cancer cell line (CCL) profiling to identify additional biomarkers of small-molecule sensitivities. We have quantitatively measured the sensitivity of 242 genomically characterized CCLs to an Informer Set of 354 small molecules that target many nodes in cell circuitry, uncovering protein dependencies that: (1) associate with specific cancer-genomic alterations and (2) can be targeted by small molecules. We have created the Cancer Therapeutics Response Portal (http://www.broadinstitute.org/ctrp) to enable users to correlate genetic features to sensitivity in individual lineages and control for confounding factors of CCL profiling. We report a candidate dependency, associating activating mutations in the oncogene ß-catenin with sensitivity to the Bcl-2 family antagonist, navitoclax. The resource can be used to develop novel therapeutic hypotheses and to accelerate discovery of drugs matched to patients by their cancer genotype and lineage.

Punctuated evolution of prostate cancer genomes.

The analysis of exonic DNA from prostate cancers has identified recurrently mutated genes, but the spectrum of genome-wide alterations has not been profiled extensively in this disease. We sequenced the genomes of 57 prostate tumors and matched normal tissues to characterize somatic alterations and to study how they accumulate during oncogenesis and progression. By modeling the genesis of genomic rearrangements, we identified abundant DNA translocations and deletions that arise in a highly interdependent manner. This phenomenon, which we term "chromoplexy," frequently accounts for the dysregulation of prostate cancer genes and appears to disrupt multiple cancer genes coordinately. Our modeling suggests that chromoplexy may induce considerable genomic derangement over relatively few events in prostate cancer and other neoplasms, supporting a model of punctuated cancer evolution. By characterizing the clonal hierarchy of genomic lesions in prostate tumors, we charted a path of oncogenic events along which chromoplexy may drive prostate carcinogenesis.

Mapping the hallmarks of lung adenocarcinoma with massively parallel sequencing.

Lung adenocarcinoma, the most common subtype of non-small cell lung cancer, is responsible for more than 500,000 deaths per year worldwide. Here, we report exome and genome sequences of 183 lung adenocarcinoma tumor/normal DNA pairs. These analyses revealed a mean exonic somatic mutation rate of 12.0 events/megabase and identified the majority of genes previously reported as significantly mutated in lung adenocarcinoma. In addition, we identified statistically recurrent somatic mutations in the splicing factor gene U2AF1 and truncating mutations affecting RBM10 and ARID1A. Analysis of nucleotide context-specific mutation signatures grouped the sample set into distinct clusters that correlated with smoking history and alterations of reported lung adenocarcinoma genes. Whole-genome sequence analysis revealed frequent structural rearrangements, including in-frame exonic alterations within EGFR and SIK2 kinases. The candidate genes identified in this study are attractive targets for biological characterization and therapeutic targeting of lung adenocarcinoma.

A landscape of driver mutations in melanoma.

Despite recent insights into melanoma genetics, systematic surveys for driver mutations are challenged by an abundance of passenger mutations caused by carcinogenic UV light exposure. We developed a permutation-based framework to address this challenge, employing mutation data from intronic sequences to control for passenger mutational load on a per gene basis. Analysis of large-scale melanoma exome data by this approach discovered six novel melanoma genes (PPP6C, RAC1, SNX31, TACC1, STK19, and ARID2), three of which-RAC1, PPP6C, and STK19-harbored recurrent and potentially targetable mutations. Integration with chromosomal copy number data contextualized the landscape of driver mutations, providing oncogenic insights in BRAF- and NRAS-driven melanoma as well as those without known NRAS/BRAF mutations. The landscape also clarified a mutational basis for RB and p53 pathway deregulation in this malignancy. Finally, the spectrum of driver mutations provided unequivocal genomic evidence for a direct mutagenic role of UV light in melanoma pathogenesis.

Binding of TMPRSS2-ERG to BAF Chromatin Remodeling Complexes Mediates Prostate Oncogenesis.

Chromosomal rearrangements resulting in the fusion of TMPRSS2, an androgen-regulated gene, and the ETS family transcription factor ERG occur in over half of prostate cancers. However, the mechanism by which ERG promotes oncogenic gene expression and proliferation remains incompletely understood. Here, we identify a binding interaction between ERG and the mammalian SWI/SNF (BAF) ATP-dependent chromatin remodeling complex, which is conserved among other oncogenic ETS factors, including ETV1, ETV4, and ETV5. We find that ERG drives genome-wide retargeting of BAF complexes in a manner dependent on binding of ERG to the ETS DNA motif. Moreover, ERG requires intact BAF complexes for chromatin occupancy and BAF complex ATPase activity for target gene regulation. In a prostate organoid model, BAF complexes are required for ERG-mediated basal-to-luminal transition, a hallmark of ERG activity in prostate cancer. These observations suggest a fundamental interdependence between ETS transcription factors and BAF chromatin remodeling complexes in cancer.

An integrated approach to uncover drivers of cancer.

Systematic characterization of cancer genomes has revealed a staggering number of diverse aberrations that differ among individuals, such that the functional importance and physiological impact of most tumor genetic alterations remain poorly defined. We developed a computational framework that integrates chromosomal copy number and gene expression data for detecting aberrations that promote cancer progression. We demonstrate the utility of this framework using a melanoma data set. Our analysis correctly identified known drivers of melanoma and predicted multiple tumor dependencies. Two dependencies, TBC1D16 and RAB27A, confirmed empirically, suggest that abnormal regulation of protein trafficking contributes to proliferation in melanoma. Together, these results demonstrate the ability of integrative Bayesian approaches to identify candidate drivers with biological, and possibly therapeutic, importance in cancer.

Easy Access to Functionalized Indolines and Tetrahydroquinolines via a Photochemical Cascade Cyclization Reaction.

Herein, the development of a light-mediated synthesis of functionalized indolines and tetrahydroquinolines is reported. These structural motifs are considered as highly valuable targets, attributed to their widespread occurrence in pharmaceuticals and natural products. The gold-mediated approach offers a direct route to functionalized indolines in yields of up to 81% under mild photochemical conditions. Thereby, easily accessible Boc-protected N-aryl-allylamine and homoallylamine derivatives were reacted with sp3-hybridized haloalkanes in an intermolecular cascade cyclization reaction. A broad scope of substrates, including a variety of different substituents on the aromatic backbone as well as various haloalkanes, could be utilized. Indoline derivatives, which are functionalized in position 2, are also accessible by applying ortho-allylic anilines. Moreover, the synthetic appeal was demonstrated for a total synthesis of the anti-inflammatory agent AN669 in three reaction steps in an overall yield of 64%.

Spectroscopic, electrochemical, and kinetic trends in Fe(III)-thiolate disproportionation near physiologic pH.

In addition to its primary oxygen-atom-transfer function, cysteamine dioxygenase (ADO) exhibits a relatively understudied anaerobic disproportionation reaction (ADO-Fe(III)-SR → ADO-Fe(II) + ½ RSSR) with its native substrates. Inspired by ADO disproportionation reactivity, we employ [Fe(tacn)Cl3] (tacn = 1,4,7-triazacyclononane) as a precursor for generating Fe(III)-thiolate model complexes in buffered aqueous media. A series of Fe(III)-thiolate model complexes are generated in situ using aqueous [Fe(tacn)Cl3] and thiol-containing ligands cysteamine, penicillamine, mercaptopropionate, cysteine, cysteine methyl ester, N-acetylcysteine, and N-acetylcysteine methyl ester. We observe trends in UV-Vis and electron paramagnetic resonance (EPR) spectra, disproportionation rate constants, and cathodic peak potentials as a function of thiol ligand. These trends will be useful in rationalizing substrate-dependent Fe(III)-thiolate disproportionation reactions in metalloenzymes.

Dementia Screening in Rural Kenya: The Prevalence and Impact of Screening Positive for Dementia.

INTRODUCTION: In Kenya, there is a lack of data on the number of people with dementia. In this article, we aim to estimate the number of community-dwelling older adults (aged 60 years and above) that are potentially living with dementia in rural Kenya. METHODS: Recruitment of older adults occurred through adopting a convenience approach based on the catchment areas served by ten trained community health workers. Screening was conducted using the Brief Community Screening Instrument for Dementia (CSI-D), in which prevalence ratios were reported. Regression analyses were run to understand the association between screening outcome and wellbeing, social isolation, and employment status (adjusted for age, sex, literacy, geography, and social status). RESULTS: Of the 3,546 older adults who were screened for dementia, 652 screened positive (PR = 0.18, 95% CIs: 0.17-0.20). Back estimating screen positives based on established sensitivity and specificity of the tool against a gold standard (clinical diagnosis), yielded a prevalence of 9.4% (0.09, 95% CIs: 0.08-0.11). Screening positive for dementia was associated with poorer quality of life (B = -0.17, p < 0.001) and loneliness (B = 0.28, p < 0.001). CONCLUSION: There are potentially 258,000 older adults living with dementia in Kenya, who likely have poorer outcomes. We need to encourage a timely diagnosis and develop better ways to support people living with dementia in Kenya and other resource-limited settings.

Bone Endosteal Mimics Regulates Breast Cancer Development and Phenotype.

Bone is a frequent site for metastatic development in various cancer types, including breast cancer, with a grim prognosis due to the distinct bone environment. Despite considerable advances, our understanding of the underlying processes leading to bone metastasis progression remains elusive. Here, we applied a bioactive three-dimensional (3D) model capable of mimicking the endosteal bone microenvironment. MDA-MB-231 and MCF7 breast cancer cells were cultured on the scaffolds, and their behaviors and the effects of the biomaterial on the cells were examined over time. We demonstrated that close interactions between the cells and the biomaterial affect their proliferation rates and the expression of c-Myc, cyclin D, and KI67, leading to cell cycle arrest. Moreover, invasion assays revealed increased invasiveness within this microenvironment. Our findings suggest a dual role for endosteal mimicking signals, influencing cell fate and potentially acting as a double-edged sword, shuttling between cell cycle arrest and more active, aggressive states.

Motivators for family carers of persons with dementia in Kenya.

OBJECTIVES: The number of people with dementia is on the rise in Kenya and across Africa. Although family carers act as the main providers of dementia care in Kenya, there is still a significant knowledge gap regarding why family members care for someone with dementia. This study explores perceived drivers of care for people with dementia in a rural Kenyan context. METHODS: Participants were recruited in Makueni County, Kenya. Primary data were derived from a focus group discussion (FGD) and five individual interviews with family carers of people with dementia. To complement interpretation, triangulation occurred through using data from FGDs with healthcare workers and members of the general public. All audio recordings were transcribed verbatim and inductive thematic analysis performed using NVIVO 12. RESULTS: Using the Positioning Theory, we sought to generate information pertaining to motivation for becoming a family carer. Five themes emerged from the analysis and included: (i) self-fulfillment, (ii) familial obligation, (iii) cultural and religious beliefs, (iv) reciprocity, and (v) societal pressures. These themes described the nature of care given to people with dementia, based on what the participants perceived as compelling and/or motivating factors. CONCLUSIONS: Our findings describe the unique motivators of family carers for people with dementia in Kenya. The ability to find meaning in the caregiving experience could contribute to development of effective support systems, interventions and policies for dementia carers with the aim of improving the overall quality of dementia care in Kenya.

Drawing improves memory in patients with hippocampal damage.

The hippocampus plays a critical role in the formation of declarative memories, and hippocampal damage leads to significant impairments in new memory formation. Drawing can serve as a form of multi-modal encoding that improves declarative memory performance relative to other multimodal encoding strategies such as writing. We examined whether, and to what extent, patients with hippocampal damage could benefit from the mnemonic strategy of drawing. Three patients with focal hippocampal damage, and one patient with both hippocampal and cortical lesions, in addition to 22 age-, sex-, and education-matched controls, were shown a list of words one at a time during encoding and instructed to either draw a picture or repeatedly write each word for 40 s. Following a brief filled delay, free recall and recognition memory for words from both encoding trial types were assessed. Controls showed enhanced recall and recognition memory for words drawn versus those that were written, an effect that was even more pronounced in patients with focal hippocampal damage. By contrast, the patient with both hippocampal and cortical lesions showed no drawing-mediated boost in either recall or recognition memory. These findings demonstrate that drawing is an effective encoding strategy, likely accruing from the engagement of extra-hippocampal processes including the integration of cortical-based motor, visual, and semantic processing, enabling more elaborative encoding.

IDLaS-NL - A platform for running customized studies on individual differences in Dutch language skills via the Internet.

We introduce the Individual Differences in Language Skills (IDLaS-NL) web platform, which enables users to run studies on individual differences in Dutch language skills via the Internet. IDLaS-NL consists of 35 behavioral tests, previously validated in participants aged between 18 and 30 years. The platform provides an intuitive graphical interface for users to select the tests they wish to include in their research, to divide these tests into different sessions and to determine their order. Moreover, for standardized administration the platform provides an application (an emulated browser) wherein the tests are run. Results can be retrieved by mouse click in the graphical interface and are provided as CSV file output via e-mail. Similarly, the graphical interface enables researchers to modify and delete their study configurations. IDLaS-NL is intended for researchers, clinicians, educators and in general anyone conducting fundamental research into language and general cognitive skills; it is not intended for diagnostic purposes. All platform services are free of charge. Here, we provide a description of its workings as well as instructions for using the platform. The IDLaS-NL platform can be accessed at www.mpi.nl/idlas-nl .

Intramedullary disseminated sporotrichosis in an immunocompetent patient: case report and review of the literature.

BACKGROUND: Disseminated sporotrichosis is a severe opportunistic infection that often affects immunocompromised patients after a cutaneous inoculation. Here we present a rare case of disseminated sporotrichosis discovered as a solitary intramedullary thoracic spinal cord lesion in an immunocompetent patient. CASE DESCRIPTION: A 37-year-old man presented with progressive lower limb weakness and sensory changes over 1 week. A spinal magnetic resonance imaging (MRI) revealed a contrast-enhancing intramedullary lesion centered at T10. The patient was afebrile and reported no history of trauma or cutaneous lesions. The lesion was unresponsive to a trial of corticosteroids. A thoracic laminectomy was performed and a biopsy obtained. A cutaneous lesion on the arm was concurrently discovered, which was also biopsied. Both the skin and spinal cord biopsies showed Sporothrix schenckii by macroscopic and microscopic morphology which were later confirmed by MALDI-TOF mass spectrometry. CONCLUSION: This is a rare case of intramedullary disseminated sporotrichosis affecting the central nervous system of an immunocompetent patient. This unusual presentation should be taken into consideration when such intramedullary lesions are encountered.