BMP-ACVR1 Axis is Critical for Efficacy of PRC2 Inhibitors in B-Cell Lymphoma.

EZH2 is the catalytic subunit of the histone methyltransferase Polycomb Repressive Complex 2 (PRC2), and its somatic activating mutations drive lymphoma, particularly the germinal center B-cell type. Although PRC2 inhibitors, such as tazemetostat, have demonstrated anti-lymphoma activity in patients, the clinical efficacy is not limited to EZH2-mutant lymphoma. In this study, Activin A Receptor Type 1 (ACVR1), a type I Bone Morphogenetic Protein (BMP) receptor, is identified as critical for the anti-lymphoma efficacy of PRC2 inhibitors through a whole-genome CRISPR screen. BMP6, BMP7, and ACVR1 are repressed by PRC2-mediated H3K27me3, and PRC2 inhibition upregulates their expression and signaling in cell and patient-derived xenograft models. Through BMP-ACVR1 signaling, PRC2 inhibitors robustly induced cell cycle arrest and B cell lineage differentiation in vivo. Remarkably, blocking ACVR1 signaling using an inhibitor or genetic depletion significantly compromised the in vitro and in vivo efficacy of PRC2 inhibitors. Furthermore, high levels of BMP6 and BMP7, along with ACVR1, are associated with longer survival in lymphoma patients, underscoring the clinical relevance of this study. Altogether, BMP-ACVR1 exhibits anti-lymphoma function and represents a critical PRC2-repressed pathway contributing to the efficacy of PRC2 inhibitors.

Integrated multi-omics profiling to dissect the spatiotemporal evolution of metastatic hepatocellular carcinoma.

Comprehensive molecular analyses of metastatic hepatocellular carcinoma (HCC) are lacking. Here, we generate multi-omic profiling of 257 primary and 176 metastatic regions from 182 HCC patients. Primary tumors rich in hypoxia signatures facilitated polyclonal dissemination. Genomic divergence between primary and metastatic HCC is high, and early dissemination is prevalent. The remarkable neoantigen intratumor heterogeneity observed in metastases is associated with decreased T cell reactivity, resulting from disruptions to neoantigen presentation. We identify somatic copy number alterations as highly selected events driving metastasis. Subclones without Wnt mutations show a stronger selective advantage for metastasis than those with Wnt mutations and are characterized by a microenvironment rich in activated fibroblasts favoring a pro-metastatic phenotype. Finally, metastases without Wnt mutations exhibit higher enrichment of immunosuppressive B cells that mediate terminal exhaustion of CD8+ T cells via HLA-E:CD94-NKG2A checkpoint axis. Collectively, our results provide a multi-dimensional dissection of the complex evolutionary process of metastasis.

Phylogenomics Reveals High Levels of Incomplete Lineage Sorting at the Ancestral Nodes of the Macaque Radiation.

The genus Macaca includes 23 species assigned into 4 to 7 groups. It exhibits the largest geographic range and represents the most successful example of adaptive radiation of nonhuman primates. However, intrageneric phylogenetic relationships among species remain controversial and have not been resolved so far. In this study, we conducted a phylogenomic analysis on 16 newly generated and 8 published macaque genomes. We found strong evidence supporting the division of this genus into 7 species groups. Incomplete lineage sorting (ILS) was the primary factor contributing to the discordance observed among gene trees; however, we also found evidence of hybridization events, specifically between the ancestral arctoides/sinica and silenus/nigra lineages that resulted in the hybrid formation of the fascicularis/mulatta group. Combined with fossil data, our phylogenomic data were used to establish a scenario for macaque radiation. These findings provide insights into ILS and potential ancient introgression events that were involved in the radiation of macaques, which will lead to a better understanding of the rapid speciation occurring in nonhuman primates.

CoNet: Efficient Network Regression for Survival Analysis in Transcriptome-Wide Association Studies-With Applications to Studies of Breast Cancer.

Transcriptome-wide association studies (TWASs) aim to detect associations between genetically predicted gene expression and complex diseases or traits through integrating genome-wide association studies (GWASs) and expression quantitative trait loci (eQTL) mapping studies. Most current TWAS methods analyze one gene at a time, ignoring the correlations between multiple genes. Few of the existing TWAS methods focus on survival outcomes. Here, we propose a novel method, namely a COx proportional hazards model for NEtwork regression in TWAS (CoNet), that is applicable for identifying the association between one given network and the survival time. CoNet considers the general relationship among the predicted gene expression as edges of the network and quantifies it through pointwise mutual information (PMI), which is under a two-stage TWAS. Extensive simulation studies illustrate that CoNet can not only achieve type I error calibration control in testing both the node effect and edge effect, but it can also gain more power compared with currently available methods. In addition, it demonstrates superior performance in real data application, namely utilizing the breast cancer survival data of UK Biobank. CoNet effectively accounts for network structure and can simultaneously identify the potential effecting nodes and edges that are related to survival outcomes in TWAS.

The roles of ETS transcription factors in liver fibrosis.

E26 transformation specific or E twenty-six (ETS) protein family consists of 28 transcription factors, five of which, named ETS1/2, PU.1, ERG and EHF, are known to involve in the development of liver fibrosis, and are expected to become diagnostic markers or therapeutic targets of liver fibrosis. In recent years, some small molecule inhibitors of ETS protein family have been discovered, which might open up a new path for the liver fibrosis therapy targeting ETS. This article reviews the research progress of ETS family members in the development liver fibrosis as well as their prospect of clinical application.

Characterization of the evolution trajectory and immune profiling of new histologic patterns in lung adenocarcinoma.

In lung adenocarcinoma (LUAD), the appearance of morphologically diverse tumor regions, termed histological patterns, is closely associated with disease progression and lymph node metastasis. However, the molecular characteristics of the histological patterns in LUAD and the underlying molecular evolutionary mechanisms between the histological patterns in primary tumors and lymph node metastases are poorly understood. Here, we re-analyzed the large TCGA-LUAD dataset and depicted a comprehensive profiling of the genome and transcriptome across the histological patterns in LUAD. Tumor phylogenetic trajectory analysis suggested that the complex glands is more apt to metastasize to the lymph node. Further deconvolution of the tumor microenvironment demonstrated that the complex glands had a higher infiltration of cancer-associated fibroblasts (CAFs). Single-cell transcriptome profiling of complex glands pattern identified a novel CAF subtype co-expressing fibroblast activation protein-alpha (FAP) and stimulator of interferon genes (STING). Moreover, our data demonstrated that FAP is an important downstream effector of STING in CAFs. In summary, our results provide the basis for the development of innovative therapeutic guidelines and intervention strategies for LUAD patients.

Joint analysis of scATAC-seq datasets using epiConv.

BACKGROUND: Technical improvement in ATAC-seq makes it possible for high throughput profiling the chromatin states of single cells. However, data from multiple sources frequently show strong technical variations, which is referred to as batch effects. In order to perform joint analysis across multiple datasets, specialized method is required to remove technical variations between datasets while keep biological information. RESULTS: Here we present an algorithm named epiConv to perform joint analyses on scATAC-seq datasets. We first show that epiConv better corrects batch effects and is less prone to over-fitting problem than existing methods on a collection of PBMC datasets. In a collection of mouse brain data, we show that epiConv is capable of aligning low-depth scATAC-Seq from co-assay data (simultaneous profiling of transcriptome and chromatin) onto high-quality ATAC-seq reference and increasing the resolution of chromatin profiles of co-assay data. Finally, we show that epiConv can be used to integrate cells from different biological conditions (T cells in normal vs. germ-free mouse; normal vs. malignant hematopoiesis), which reveals hidden cell populations that would otherwise be undetectable. CONCLUSIONS: In this study, we introduce epiConv to integrate multiple scATAC-seq datasets and perform joint analysis on them. Through several case studies, we show that epiConv removes the batch effects and retains the biological signal. Moreover, joint analysis across multiple datasets improves the performance of clustering and differentially accessible peak calling, especially when the biological signal is weak in single dataset.

The effectiveness of portable ultrasound-guided resuscitative endovascular balloon occlusion of the aorta for stopping iliac artery hemorrhage during first aid pre-hospital: a randomized control animal trial.

PURPOSE: This study aimed at to comparing the effectiveness of portable ultrasound guided REBOA vs. traditional manual extracorporeal compression in stopping iliac artery hemostasis. METHODS: Twelve swine were included in this study (treatment group vs. control group, 6:6). A biopsy device was used to create an iliac artery rupture and hemorrhage in each swine. After 30 s of bleeding, the treatment group received REBOA under the guidance of ultrasound, whereas the control group received traditional manual extracorporeal compression. General physiological conditions were recorded at 0 s (baseline, T1), 30 s (initiation of therapies to stop bleeding, T2), 10 min (T3) and 30 min (T4) after bleeding. Intraperitoneal and retroperitoneal hemorrhage and specimens of iliac artery were collected after all swine were euthanized. RESULTS: One swine was excluded because of accidental death not related to the experiment; thus, 11 swine were analyzed in this study. The general physiological characteristics of the two groups showed no difference at T1. Hemorrhagic shock occurred in both groups. After the hemostatic procedure was performed, systolic pressure, diastolic pressure and heart rate first increased significantly between T2 and T3, and then became stable between T3 and T4; these indicators in the control group deteriorated over time. The total blood loss in the treatment group (1245.23 ± 190.07 g) was much significantly less than that in the control group (2605.63 ± 291.67 g) with p < 0.001. CONCLUSIONS: Performing REBOA under the guidance of portable ultrasound is an effective way to stop bleeding. It suggests a potential alternative method for iliac artery hemostasis in the pre-hospital setting.

Induction of senescence-associated secretory phenotype underlies the therapeutic efficacy of PRC2 inhibition in cancer.

The methyltransferase Polycomb Repressive Complex 2 (PRC2), composed of EZH2, SUZ12, and EED subunits, is associated with transcriptional repression via tri-methylation of histone H3 on lysine 27 residue (H3K27me3). PRC2 is a valid drug target, as the EZH2 gain-of-function mutations identified in patient samples drive tumorigenesis. PRC2 inhibitors have been discovered and demonstrated anti-cancer efficacy in clinic. However, their pharmacological mechanisms are poorly understood. MAK683 is a potent EED inhibitor in clinical development. Focusing on MAK683-sensitive tumors with SMARCB1 or ARID1A loss, we identified a group of PRC2 target genes with high H3K27me3 signal through epigenomic and transcriptomic analysis. Multiple senescence-associated secretory phenotype (SASP) genes, such as GATA4, MMP2/10, ITGA2 and GBP1, are in this group besides previously identified CDKN2A/p16. Upon PRC2 inhibition, the de-repression of SASP genes is detected in multiple sensitive models and contributes to decreased Ki67+, extracellular matrix (ECM) reorganization, senescence associated inflammation and tumor regression even in CDKN2A/p16 knockout tumor. And the combination of PRC2 inhibitor and CDK4/6 inhibitor leads to better effect. The genes potential regulated by PRC2 in neuroblastoma samples exhibited significant enrichment of ECM and senescence associated inflammation, supporting the clinical relevance of our results. Altogether, our results unravel the pharmacological mechanism of PRC2 inhibitors and propose a combination strategy for MAK683 and other PRC2 drugs.

Amelioration of cognitive impairment using epigallocatechin-3-gallate in ovariectomized mice fed a high-fat diet involves remodeling with Prevotella and Bifidobacteriales.

Background: Estrogen deficiency and a high-fat diet (HFD) are both risk factors for Alzheimer's disease (AD). HFD can accelerate cognitive impairment in estrogen-deficient patients, but there is currently no effective treatment. Epigallocatechin-3-galate (EGCG) is widely studied for its anti-inflammatory, anti-cancer, and anti-neurodegeneration effects. Nevertheless, whether EGCG can ameliorate cognitive impairment in HFD-fed estrogen-deficient mice has not been studied. Methods and Results: Ovariectomized (OVX) mice fed an HFD (HFOVX) for 8 weeks experienced impaired object recognition and spatial memory, but this damage was significantly attenuated by the administration of EGCG at a dose of 45 mg/kg. Through 16S rRNA gene sequencing, we found that HFOVX changed the diversity and structure of the gut microbiota in mice, which could be restored with EGCG. Further analysis showed that HFOVX exposure not only resulted in a decrease of Alloprevotella in Bacteroidetes, Lactobacillaceae in Firmicutes, and Prevotella in Bacteroidetes but also in an increase of Bifidobacteriales in Actinobacteria. EGCG effectively reversed the decrease of Prevotella and inhibited the increase of Bifidobacteriales but had no effect on the decrease of Alloprevotella or Lactobacillaceae or on the increase of Enterorhabdus in HFOVX mice. Additionally, using Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, we found that EGCG significantly reversed the five functional gut microbiota genes elevated by HFOVX, including iron complex transport system substrate-binding protein, iron complex transport system permease protein, 3-oxoacyl- [acyl-carrier protein] reductase, transketolase, and 8-oxo-dGTP diphosphatase. Conclusions: We concluded that EGCG improved cognitive impairment in mice with estrogen deficiency exacerbated by an HFD involved a rebuilding of the disrupted gut microbiota composition.

Cancer-associated dynamics and potential regulators of intronic polyadenylation revealed by IPAFinder using standard RNA-seq data.

Intronic polyadenylation (IpA) usually leads to changes in the coding region of an mRNA, and its implication in diseases has been recognized, although at its very beginning status. Conveniently and accurately identifying IpA is of great importance for further evaluating its biological significance. Here, we developed IPAFinder, a bioinformatic method for the de novo identification of intronic poly(A) sites and their dynamic changes from standard RNA-seq data. Applying IPAFinder to 256 pan-cancer tumor/normal pairs across six tumor types, we discovered 490 recurrent dynamically changed IpA events, some of which are novel and derived from cancer-associated genes such as TSC1, SPERD2, and CCND2 Furthermore, IPAFinder revealed that IpA could be regulated by factors related to splicing and m6A modification. In summary, IPAFinder enables the global discovery and characterization of biologically regulated IpA with standard RNA-seq data and should reveal the biological significance of IpA in various processes.

NAD+ supplement potentiates tumor-killing function by rescuing defective TUB-mediated NAMPT transcription in tumor-infiltrated T cells.

Although tumor-infiltrating lymphocytes (TILs) maintain their ability to proliferate, persist, and eradicate tumors, they are frequently dysfunctional in situ. By performing both whole-genome CRISPR and metabolic inhibitor screens, we identify that nicotinamide phosphoribosyltransferase (NAMPT) is required for T cell activation. NAMPT is low in TILs, and its expression is controlled by the transcriptional factor Tubby (TUB), whose activity depends on the T cell receptor-phospholipase C gamma (TCR-PLCγ) signaling axis. The intracellular level of NAD+, whose synthesis is dependent on the NAMPT-mediated salvage pathway, is also decreased in TILs. Liquid chromatography-mass spectrometry (LC-MS) and isotopic labeling studies confirm that NAD+ depletion led to suppressed glycolysis, disrupted mitochondrial function, and dampened ATP synthesis. Excitingly, both adoptive CAR-T and anti-PD1 immune checkpoint blockade mouse models demonstrate that NAD+ supplementation enhanced the tumor-killing efficacy of T cells. Collectively, this study reveals that an impaired TCR-TUB-NAMPT-NAD+ axis leads to T cell dysfunction in the tumor microenvironment, and an over-the-counter nutrient supplement of NAD+ could boost T-cell-based immunotherapy.

Inhibition of the FACT Complex Targets Aberrant Hedgehog Signaling and Overcomes Resistance to Smoothened Antagonists.

Hedgehog signaling is aberrantly activated in hematologic malignancies and solid tumors, and targeting it is a promising therapeutic strategy against these cancers. Resistance to clinically available hedgehog-targeted Smoothened inhibitor (SMOi) drugs has become a critical issue in hedgehog-driven cancer treatment. Our previous studies identified inhibition of BET and CDK7 as two epigenetic/transcriptional-targeted therapeutic strategies for overcoming SMOi resistance, providing a promising direction for anti-hedgehog drug development. To uncover additional strategies for inhibiting aberrant hedgehog activity, here we performed CRISPR-Cas9 screening with an single-guide RNA library targeting epigenetic and transcriptional modulators in hedgehog-driven medulloblastoma cells, combined with tumor dataset analyses. Structure specific recognition protein 1 (SSRP1), a subunit of facilitates chromatin transcription (FACT) complex, was identified as a hedgehog-induced essential oncogene and therapeutic target in hedgehog-driven cancer. The FACT inhibitor CBL0137, which has entered clinical trials for cancer, effectively suppressed in vitro and in vivo growth of multiple SMOi-responsive and SMOi-resistant hedgehog-driven cancer models. Mechanistically, CBL0137 exerted anti-hedgehog activity by targeting transcription of GLI1 and GLI2, which are core transcription factors of the hedgehog pathway. SSRP1 bound the promoter regions of GLI1 and GLI2, while CBL0137 treatment substantially disrupted these interactions. Moreover, CBL0137 synergized with BET or CDK7 inhibitors to antagonize aberrant hedgehog pathway and growth of hedgehog-driven cancer models. Taken together, these results identify FACT inhibition as a promising epigenetic/transcriptional-targeted therapeutic strategy for treating hedgehog-driven cancers and overcoming SMOi resistance. SIGNIFICANCE: This study identifies FACT inhibition as an anti-hedgehog therapeutic strategy for overcoming resistance to Smoothened inhibitors and provides preclinical support for initiating clinical trials of FACT-targeted drug CBL0137 against hedgehog-driven cancers.

HDL cholesterol: A potential mediator of the association between urinary cadmium concentration and cardiovascular disease risk.

Evidence from previous studies has shown that exposure to metals is associated with cardiovascular disease (CVD). However, the association between metal mixtures and CVD risk and the potential mechanisms in epidemiologic studies remain unclear. The data of 14,795 adults who participated in the U.S. National Health and Nutrition Examination Survey (NHANES) 1999-2016 were analyzed. Multivariate logistic regression was performed to investigate the associations between urinary metal levels and CVDs. Weighted quantile sum (WQS) regression was performed to examine the effects of mixed metals on CVDs. Multivariate linear regression and mediation analysis were conducted to explore the associations between metals and blood lipids. Urinary cadmium (Cd) was significantly associated with an increased total CVD risk and with individual CVD risk. The odds ratio (OR) for CVD in the highest quartile of the WQS index was 1.43 (95% confidence interval [CI]: 1.19, 1.71). One augmented urinary Cd concentration unit (Log10) was associated with a 0.93 mg/dL decrease in HDL cholesterol, a 1.34 mg/dL increase in LDL cholesterol and a 1.30 mg/dL increase in total cholesterol in the fully adjusted model. Mediation analysis showed that HDL cholesterol mediated 4.91% of the association between urinary Cd and the prevalence of CVD. Our findings suggest that urinary Cd and metal mixtures were significantly and positively associated with CVD. The downregulation of HDL cholesterol might play a significant role in mediating Cd exposure-associated CVD risk increases.

Systematically Characterizing A-to-I RNA Editing Neoantigens in Cancer.

A-to-I RNA editing can contribute to the transcriptomic and proteomic diversity of many diseases including cancer. It has been reported that peptides generated from RNA editing could be naturally presented by human leukocyte antigen (HLA) molecules and elicit CD8+ T cell activation. However, a systematical characterization of A-to-I RNA editing neoantigens in cancer is still lacking. Here, an integrated RNA-editing based neoantigen identification pipeline PREP (Prioritizing of RNA Editing-based Peptides) was presented. A comprehensive RNA editing neoantigen profile analysis on 12 cancer types from The Cancer Genome Atlas (TCGA) cohorts was performed. PREP was also applied to 14 ovarian tumor samples and two clinical melanoma cohorts treated with immunotherapy. We finally proposed an RNA editing neoantigen immunogenicity score scheme, i.e. REscore, which takes RNA editing level and infiltrating immune cell population into consideration. We reported variant peptide from protein IFI30 in breast cancer which was confirmed expressed and presented in two samples with mass spectrometry data support. We showed that RNA editing neoantigen could be identified from RNA-seq data and could be validated with mass spectrometry data in ovarian tumor samples. Furthermore, we characterized the RNA editing neoantigen profile of clinical melanoma cohorts treated with immunotherapy. Finally, REscore showed significant associations with improved overall survival in melanoma cohorts treated with immunotherapy. These findings provided novel insights of cancer biomarker and enhance our understanding of neoantigen derived from A-to-I RNA editing as well as more types of candidates for personalized cancer vaccines design in the context of cancer immunotherapy.

Enhancer Reprogramming within Pre-existing Topologically Associated Domains Promotes TGF-ß-Induced EMT and Cancer Metastasis.

Transcription growth factor ß (TGF-ß) signaling-triggered epithelial-to-mesenchymal transition (EMT) process is associated with tumor stemness, metastasis, and chemotherapy resistance. However, the epigenomic basis for TGF-ß-induced EMT remains largely unknown. Here we reveal that HDAC1-mediated global histone deacetylation and the gain of specific histone H3 lysine 27 acetylation (H3K27ac)-marked enhancers are essential for the TGF-ß-induced EMT process. Enhancers gained upon TGF-ß treatment are linked to gene activation of EMT markers and cancer metastasis. Notably, dynamic enhancer gain or loss mainly occurs within pre-existing topologically associated domains (TADs) in epithelial cells, with minimal three-dimensional (3D) genome architecture reorganization. Through motif enrichment analysis of enhancers that are lost or gained upon TGF-ß stimulation, we identify FOXA2 as a key factor to activate epithelial-specific enhancer activity, and we also find that TEAD4 forms a complex with SMAD2/3 to mediate TGF-ß signaling-triggered mesenchymal enhancer reprogramming. Together, our results implicate that key transcription-factor (TF)-mediated enhancer reprogramming modulates the developmental transition in TGF-ß signaling-associated cancer metastasis.

Protein tyrosine phosphatase receptor type R (PTPRR) antagonizes the Wnt signaling pathway in ovarian cancer by dephosphorylating and inactivating ß-catenin.

Despite a lack of mutations, accumulating evidence supports an important role for the Wnt/ß-catenin pathway in ovarian tumorigenesis. However, the molecular mechanism that contributes to the aberrant activation of the Wnt signaling cascade in ovarian cancer has not been fully elucidated. Here, we found that protein tyrosine phosphatase receptor type R (PTPRR) suppressed the activation of the Wnt/ß-catenin pathway in ovarian cancer. We performed an shRNA-based biochemical screen, which identified PTPRR as being responsible for tyrosine dephosphorylation of ß-catenin on Tyr-142, a key site controlling the transcriptional activity of ß-catenin. Of note, PTPRR was down-regulated in ovarian cancers, and ectopic PTPRR re-expression delayed ovarian cancer cell growth both in vitro and in vivo Using a proximity-based tagging system and RNA-Seq analysis, we identified a signaling nexus that includes PTPRR, α-catenin, ß-catenin, E-cadherin, and AT-rich interaction domain 3C (ARID3C) in ovarian cancer. Immunohistochemistry staining of human samples further suggested that PTPRR expression is inversely correlated with disease prognosis. Collectively, our findings indicate that PTPRR functions as a tumor suppressor in ovarian cancer by dephosphorylating and inactivating ß-catenin. These results suggest that PTPRR expression might have utility as a prognostic marker for predicting overall survival.

XAB2 depletion induces intron retention in POLR2A to impair global transcription and promote cellular senescence.

XAB2 is a multi-functional protein participating processes including transcription, splicing, DNA repair and mRNA export. Here, we report POLR2A, the largest catalytic subunit of RNA polymerase II, as a major target gene down-regulated after XAB2 depletion. XAB2 depletion led to severe splicing defects of POLR2A with significant intron retention. Such defects resulted in substantial loss of POLR2A at RNA and protein levels, which further impaired global transcription. Treatment of splicing inhibitor madrasin induced similar reduction of POLR2A. Screen using TMT-based quantitative proteomics identified several proteins involved in mRNA surveillance including Dom34 with elevated expression. Inhibition of translation or depletion of Dom34 rescued the expression of POLR2A by stabilizing its mRNA. Immuno-precipitation further confirmed that XAB2 associated with spliceosome components important to POLR2A expression. Domain mapping revealed that TPR motifs 2-4 and 11 of XAB2 were critical for POLR2A expression by interacting with SNW1. Finally, we showed POLR2A mediated cell senescence caused by XAB2 deficiency. Depletion of XAB2 or POLR2A induced cell senescence by up-regulation of p53 and p21, re-expression of POLR2A after XAB2 depletion alleviated cellular senescence. These data together support that XAB2 serves as a guardian of POLR2A expression to ensure global gene expression and antagonize cell senescence.

CDK7 inhibition suppresses aberrant hedgehog pathway and overcomes resistance to smoothened antagonists.

The aberrant hedgehog (Hh) pathway plays important roles in multiple cancer types, therefore serving as a promising drug target. Current clinically available hedgehog-targeted drugs act mostly by antagonizing the upstream component smoothened; however, both primary and acquired resistance to FDA-approved smoothened inhibitor (SMOi) drugs have been described. We have recently demonstrated that the BET inhibitor effectively suppresses SMOi-resistant Hh-driven cancers through antagonizing transcription of GLI1 and GLI2, the core transcriptional factors of Hh pathway, suggesting epigenetic or transcriptional targeted therapy represents an anti-Hh therapeutic strategy that can overcome SMOi resistance. Here we performed an unbiased screening of epigenetic or transcriptional targeted small molecules to test their inhibitory effects on GLI1 and GLI2 transcription or cell viability of Hh-driven tumor lines. THZ1, a covalent inhibitor of cyclin-dependent kinase 7 (CDK7), is identified as the top hit in our screening. We then confirmed that antagonizing CDK7 by either small-molecule inhibitors or the CRISPR-Cas9 approach causes substantial suppression of GLI1 and GLI2 transcription, resulting in effective inhibition of Hh-driven cancers in vitro and in vivo. More importantly, antagonizing CDK7 retains inhibitory activity against Hh-driven cancers with almost all so-far described primary or acquired SMOi resistance. Furthermore, we reveal a synergy between CDK7 inhibition and BET inhibition on antagonizing aberrant Hh pathway and Hh-driven cancers that are either responsive or resistant to SMOi. Our results illustrate transcriptional inhibition through targeting CDK7 as a promising therapeutic strategy for treating Hh-driven cancers, especially those with primary or acquired resistance to SMOi drugs.

CaDrA: A Computational Framework for Performing Candidate Driver Analyses Using Genomic Features.

The identification of genetic alteration combinations as drivers of a given phenotypic outcome, such as drug sensitivity, gene or protein expression, and pathway activity, is a challenging task that is essential to gaining new biological insights and to discovering therapeutic targets. Existing methods designed to predict complementary drivers of such outcomes lack analytical flexibility, including the support for joint analyses of multiple genomic alteration types, such as somatic mutations and copy number alterations, multiple scoring functions, and rigorous significance and reproducibility testing procedures. To address these limitations, we developed Candidate Driver Analysis or CaDrA, an integrative framework that implements a step-wise heuristic search approach to identify functionally relevant subsets of genomic features that, together, are maximally associated with a specific outcome of interest. We show CaDrA's overall high sensitivity and specificity for typically sized multi-omic datasets using simulated data, and demonstrate CaDrA's ability to identify known mutations linked with sensitivity of cancer cells to drug treatment using data from the Cancer Cell Line Encyclopedia (CCLE). We further apply CaDrA to identify novel regulators of oncogenic activity mediated by Hippo signaling pathway effectors YAP and TAZ in primary breast cancer tumors using data from The Cancer Genome Atlas (TCGA), which we functionally validate in vitro. Finally, we use pan-cancer TCGA protein expression data to show the high reproducibility of CaDrA's search procedure. Collectively, this work demonstrates the utility of our framework for supporting the fast querying of large, publicly available multi-omics datasets, including but not limited to TCGA and CCLE, for potential drivers of a given target profile of interest.