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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).)

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).)

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.

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).)

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.)

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.)

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.)

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.)

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.

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.)

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.