The context-dependent epigenetic and organogenesis programs determine 3D vs. 2D cellular fitness of MYC-driven cancer.

3D cellular-specific epigenetic and transcriptomic reprogramming is critical to organogenesis and tumorigenesis. Here we dissect the distinct cell fitness in 2D (normoxia vs. chronic hypoxia) vs 3D (normoxia) culture conditions. We identify over 600 shared essential genes and additional context-specific fitness genes and pathways. Knockout of the VHL-HIF1 pathway results in incompatible fitness defects under normoxia vs. 1% oxygen or 3D culture conditions. Moreover, deletion of each of the mitochondrial respiratory electron transport chain complex has distinct fitness outcomes. Notably, multicellular organogenesis signaling pathways including TGFß-SMAD specifically constrict the uncontrolled cell proliferation in 3D while inactivation of epigenetic modifiers (Bcor, Kmt2d, Mettl3 and Mettl14) has opposite outcomes in 2D vs. 3D. We further identify a 3D-dependent synthetic lethality with partial loss of Prmt5 due to a reduction of Mtap expression resulting from 3D-specific epigenetic reprogramming. Our study highlights unique epigenetic, metabolic and organogenesis signaling dependencies under different cellular settings.

SAMP: Identifying Antimicrobial Peptides by an Ensemble Learning Model Based on Proportionalized Split Amino Acid Composition.

It is projected that 10 million deaths could be attributed to drug-resistant bacteria infections in 2050. To address this concern, identifying new-generation antibiotics is an effective way. Antimicrobial peptides (AMPs), a class of innate immune effectors, have received significant attention for their capacity to eliminate drug-resistant pathogens, including viruses, bacteria, and fungi. Recent years have witnessed widespread applications of computational methods especially machine learning (ML) and deep learning (DL) for discovering AMPs. However, existing methods only use features including compositional, physiochemical, and structural properties of peptides, which cannot fully capture sequence information from AMPs. Here, we present SAMP, an ensemble random projection (RP) based computational model that leverages a new type of features called Proportionalized Split Amino Acid Composition (PSAAC) in addition to conventional sequence-based features for AMP prediction. With this new feature set, SAMP captures the residue patterns like sorting signals at around both the N-terminus and the C-terminus, while also retaining the sequence order information from the middle peptide fragments. Benchmarking tests on different balanced and imbalanced datasets demonstrate that SAMP consistently outperforms existing state-of-the-art methods, such as iAMPpred and AMPScanner V2, in terms of accuracy, MCC, G-measure and F1-score. In addition, by leveraging an ensemble RP architecture, SAMP is scalable to processing large-scale AMP identification with further performance improvement, compared to those models without RP. To facilitate the use of SAMP, we have developed a Python package freely available at https://github.com/wan-mlab/SAMP.

A Review for Artificial Intelligence Based Protein Subcellular Localization.

Proteins need to be located in appropriate spatiotemporal contexts to carry out their diverse biological functions. Mislocalized proteins may lead to a broad range of diseases, such as cancer and Alzheimer's disease. Knowing where a target protein resides within a cell will give insights into tailored drug design for a disease. As the gold validation standard, the conventional wet lab uses fluorescent microscopy imaging, immunoelectron microscopy, and fluorescent biomarker tags for protein subcellular location identification. However, the booming era of proteomics and high-throughput sequencing generates tons of newly discovered proteins, making protein subcellular localization by wet-lab experiments a mission impossible. To tackle this concern, in the past decades, artificial intelligence (AI) and machine learning (ML), especially deep learning methods, have made significant progress in this research area. In this article, we review the latest advances in AI-based method development in three typical types of approaches, including sequence-based, knowledge-based, and image-based methods. We also elaborately discuss existing challenges and future directions in AI-based method development in this research field.

Procyanidin alleviates ferroptosis and inflammation of LPS-induced RAW264.7 cell via the Nrf2/HO-1 pathway.

Inflammation is a common occurrence in many medical conditions and is a natural defense mechanism of the human body. Ferroptosis, an iron-dependent form of cell death related to lipid peroxide build-up, has been found to be involved in inflammation. The anti-inflammatory effects of procyanidin, however, are not yet fully understood. Through network pharmacology and bioinformatics analysis, it was suggested that procyanidin could modulate ferroptosis and cause anti-inflammatory effects on RAW264.7 cells. This was further evidenced through molecular docking, molecular dynamics, and in vitro experiments. The results indicated that procyanidin could diminish inflammation in LPS-induced RAW264.7 cells by regulating ferroptosis via the Nrf2/HO-1/Keap-1 pathway. In conclusion, procyanidin supplementation might be an effective way to reduce inflammation by decreasing the release of inflammatory cytokines and suppressing ferroptosis.

USP1 Expression Driven by EWS::FLI1 Transcription Factor Stabilizes Survivin and Mitigates Replication Stress in Ewing Sarcoma.

In this study, we identify USP1 as a transcriptional target of EWS::FLI1 and demonstrate the requisite function of USP1 in Ewing sarcoma (EWS) cell survival in response to endogenous replication stress. EWS::FLI1 oncogenic transcription factor drives most EWS, a pediatric bone cancer. EWS cells display elevated levels of R-loops and replication stress. The mechanism by which EWS cells override activation of apoptosis or cellular senescence in response to increased replication stress is not known. We show that USP1 is overexpressed in EWS and EWS::FLI1 regulates USP1 transcript levels. USP1 knockdown or inhibition arrests EWS cell growth and induces cell death by apoptosis. Mechanistically, USP1 regulates Survivin (BIRC5/API4) protein stability and the activation of caspase-9 and caspase-3/7 in response to endogenous replication stress. Notably, USP1 inhibition sensitizes cells to doxorubicin and etoposide treatment. Together, our study demonstrates that USP1 is regulated by EWS::FLI1, the USP1-Survivin axis promotes EWS cell survival, and USP1 inhibition sensitizes cells to standard of care chemotherapy. IMPLICATIONS: High USP1 and replication stress levels driven by EWS::FLI1 transcription factor in EWS are vulnerabilities that can be exploited to improve existing treatment avenues and overcome drug resistance.

Embedded Bioprinting of Breast Tumor Cells and Organoids Using Low-Concentration Collagen-Based Bioinks.

Bioinks for 3D bioprinting of tumor models should not only meet printability requirements but also accurately maintain and support phenotypes of tumor surrounding cells to recapitulate key tumor hallmarks. Collagen is a major extracellular matrix protein for solid tumors, but low viscosity of collagen solution has made 3D bioprinted cancer models challenging. This work produces embedded, bioprinted breast cancer cells and tumor organoid models using low-concentration collagen I based bioinks. The biocompatible and physically crosslinked silk fibroin hydrogel is used to generate the support bath for the embedded 3D printing. The composition of the collagen I based bioink is optimized with a thermoresponsive hyaluronic acid-based polymer to maintain the phenotypes of both the noninvasive epithelial and invasive breast cancer cells, as well as cancer-associated fibroblasts. Mouse breast tumor organoids are bioprinted using optimized collagen bioink to mimic in vivo tumor morphology. A vascularized tumor model is also created using a similar strategy, with significantly enhanced vasculature formation under hypoxia. This study shows the great potential of embedded bioprinted breast tumor models utilizing a low-concentration collagen-based bioink for advancing the understanding of tumor cell biology and facilitating drug discovery research.

Etiology of oncogenic fusions in 5,190 childhood cancers and its clinical and therapeutic implication.

Oncogenic fusions formed through chromosomal rearrangements are hallmarks of childhood cancer that define cancer subtype, predict outcome, persist through treatment, and can be ideal therapeutic targets. However, mechanistic understanding of the etiology of oncogenic fusions remains elusive. Here we report a comprehensive detection of 272 oncogenic fusion gene pairs by using tumor transcriptome sequencing data from 5190 childhood cancer patients. We identify diverse factors, including translation frame, protein domain, splicing, and gene length, that shape the formation of oncogenic fusions. Our mathematical modeling reveals a strong link between differential selection pressure and clinical outcome in CBFB-MYH11. We discover 4 oncogenic fusions, including RUNX1-RUNX1T1, TCF3-PBX1, CBFA2T3-GLIS2, and KMT2A-AFDN, with promoter-hijacking-like features that may offer alternative strategies for therapeutic targeting. We uncover extensive alternative splicing in oncogenic fusions including KMT2A-MLLT3, KMT2A-MLLT10, C11orf95-RELA, NUP98-NSD1, KMT2A-AFDN and ETV6-RUNX1. We discover neo splice sites in 18 oncogenic fusion gene pairs and demonstrate that such splice sites confer therapeutic vulnerability for etiology-based genome editing. Our study reveals general principles on the etiology of oncogenic fusions in childhood cancer and suggests profound clinical implications including etiology-based risk stratification and genome-editing-based therapeutics.

A Sequence Obfuscation Method for Protecting Personal Genomic Privacy.

With the technological advances in recent decades, determining whole genome sequencing of a person has become feasible and affordable. As a result, large-scale individual genomic sequences are produced and collected for genetic medical diagnoses and cancer drug discovery, which, however, simultaneously poses serious challenges to the protection of personal genomic privacy. It is highly urgent to develop methods which make the personal genomic data both utilizable and confidential. Existing genomic privacy-protection methods are either time-consuming for encryption or with low accuracy of data recovery. To tackle these problems, this paper proposes a sequence similarity-based obfuscation method, namely IterMegaBLAST, for fast and reliable protection of personal genomic privacy. Specifically, given a randomly selected sequence from a dataset of genomic sequences, we first use MegaBLAST to find its most similar sequence from the dataset. These two aligned sequences form a cluster, for which an obfuscated sequence was generated via a DNA generalization lattice scheme. These procedures are iteratively performed until all of the sequences in the dataset are clustered and their obfuscated sequences are generated. Experimental results on benchmark datasets demonstrate that under the same degree of anonymity, IterMegaBLAST significantly outperforms existing state-of-the-art approaches in terms of both utility accuracy and time complexity.

The nuclear receptor ERR cooperates with the cardiogenic factor GATA4 to orchestrate cardiomyocyte maturation.

Estrogen-related receptors (ERR) α and γ were shown recently to serve as regulators of cardiac maturation, yet the underlying mechanisms have not been delineated. Herein, we find that ERR signaling is necessary for induction of genes involved in mitochondrial and cardiac-specific contractile processes during human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) differentiation. Genomic interrogation studies demonstrate that ERRγ occupies many cardiomyocyte enhancers/super-enhancers, often co-localizing with the cardiogenic factor GATA4. ERRγ interacts with GATA4 to cooperatively activate transcription of targets involved in cardiomyocyte-specific processes such as contractile function, whereas ERRγ-mediated control of metabolic genes occurs independent of GATA4. Both mechanisms require the transcriptional coregulator PGC-1α. A disease-causing GATA4 mutation is shown to diminish PGC-1α/ERR/GATA4 cooperativity and expression of ERR target genes are downregulated in human heart failure samples suggesting that dysregulation of this circuitry may contribute to congenital and acquired forms of heart failure.

Special Issue on Bioinformatics and Machine Learning for Cancer Biology.

Cancer is a leading cause of death worldwide, claiming millions of lives each year [...].

Genomic profiling identifies genes and pathways dysregulated by HEY1-NCOA2 fusion and shines a light on mesenchymal chondrosarcoma tumorigenesis.

Mesenchymal chondrosarcoma is a rare, high-grade, primitive mesenchymal tumor. It accounts for around 2-10% of all chondrosarcomas and mainly affects adolescents and young adults. We previously described the HEY1-NCOA2 as a recurrent gene fusion in mesenchymal chondrosarcoma, an important breakthrough for characterizing this disease; however, little study had been done to characterize the fusion protein functionally, in large part due to a lack of suitable models for evaluating the impact of HEY1-NCOA2 expression in the appropriate cellular context. We used iPSC-derived mesenchymal stem cells (iPSC-MSCs), which can differentiate into chondrocytes, and generated stable transduced iPSC-MSCs with inducible expression of HEY1-NCOA2 fusion protein, wildtype HEY1 or wildtype NCOA2. We next comprehensively analyzed both the DNA binding properties and transcriptional impact of HEY1-NCOA2 expression by integrating genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) and expression profiling (RNA-seq). We demonstrated that HEY1-NCOA2 fusion protein preferentially binds to promoter regions of canonical HEY1 targets, resulting in transactivation of HEY1 targets, and significantly enhances cell proliferation. Intriguingly, we identified that both PDGFB and PDGFRA were directly targeted and upregulated by HEY1-NCOA2; and the fusion protein, but not wildtype HEY1 or NCOA2, dramatically increased the level of phospho-AKT (Ser473). Our findings provide a rationale for exploring PDGF/PI3K/AKT inhibition in treating mesenchymal chondrosarcoma. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Improving bulk RNA-seq classification by transferring gene signature from single cells in acute myeloid leukemia.

The advances in single-cell RNA sequencing (scRNA-seq) technologies enable the characterization of transcriptomic profiles at the cellular level and demonstrate great promise in bulk sample analysis thereby offering opportunities to transfer gene signature from scRNA-seq to bulk data. However, the gene expression signatures identified from single cells are typically inapplicable to bulk RNA-seq data due to the profiling differences of distinct sequencing technologies. Here, we propose single-cell pair-wise gene expression (scPAGE), a novel method to develop single-cell gene pair signatures (scGPSs) that were beneficial to bulk RNA-seq classification to transfer knowledge across platforms. PAGE was adopted to tackle the challenge of profiling differences. We applied the method to acute myeloid leukemia (AML) and identified the scGPS from mouse scRNA-seq that allowed discriminating between AML and control cells. The scGPS was validated in bulk RNA-seq datasets and demonstrated better performance (average area under the curve [AUC] = 0.96) than the conventional gene expression strategies (average AUC$\le$ 0.88) suggesting its potential in disclosing the molecular mechanism of AML. The scGPS also outperformed its bulk counterpart, which highlighted the benefit of gene signature transfer. Furthermore, we confirmed the utility of scPAGE in sepsis as an example of other disease scenarios. scPAGE leveraged the advantages of single-cell profiles to enhance the analysis of bulk samples revealing great potential of transferring knowledge from single-cell to bulk transcriptome studies.

Identification of a modular super-enhancer in murine retinal development.

Super-enhancers are expansive regions of genomic DNA comprised of multiple putative enhancers that contribute to the dynamic gene expression patterns during development. This is particularly important in neurogenesis because many essential transcription factors have complex developmental stage- and cell-type specific expression patterns across the central nervous system. In the developing retina, Vsx2 is expressed in retinal progenitor cells and is maintained in differentiated bipolar neurons and Müller glia. A single super-enhancer controls this complex and dynamic pattern of expression. Here we show that deletion of one region disrupts retinal progenitor cell proliferation but does not affect cell fate specification. The deletion of another region has no effect on retinal progenitor cell proliferation but instead leads to a complete loss of bipolar neurons. This prototypical super-enhancer may serve as a model for dissecting the complex gene expression patterns for neurogenic transcription factors during development. Moreover, it provides a unique opportunity to alter expression of individual transcription factors in particular cell types at specific stages of development. This provides a deeper understanding of function that cannot be achieved with traditional knockout mouse approaches.

Alzheimer's disease-associated U1 snRNP splicing dysfunction causes neuronal hyperexcitability and cognitive impairment.

Recent proteome and transcriptome profiling of Alzheimer's disease (AD) brains reveals RNA splicing dysfunction and U1 small nuclear ribonucleoprotein (snRNP) pathology containing U1-70K and its N-terminal 40-KDa fragment (N40K). Here we present a causative role of U1 snRNP dysfunction to neurodegeneration in primary neurons and transgenic mice (N40K-Tg), in which N40K expression exerts a dominant-negative effect to downregulate full-length U1-70K. N40K-Tg recapitulates N40K insolubility, erroneous splicing events, neuronal degeneration and cognitive impairment. Specifically, N40K-Tg shows the reduction of GABAergic synapse components (e.g., the GABA receptor subunit of GABRA2), and concomitant postsynaptic hyperexcitability that is rescued by a GABA receptor agonist. Crossing of N40K-Tg and the 5xFAD amyloidosis model indicates that the RNA splicing defect synergizes with the amyloid cascade to remodel the brain transcriptome and proteome, deregulate synaptic proteins, and accelerate cognitive decline. Thus, our results support the contribution of U1 snRNP-mediated splicing dysfunction to AD pathogenesis.

Targeting the spliceosome through RBM39 degradation results in exceptional responses in high-risk neuroblastoma models.

Aberrant alternative pre-mRNA splicing plays a critical role in MYC-driven cancers and therefore may represent a therapeutic vulnerability. Here, we show that neuroblastoma, a MYC-driven cancer characterized by splicing dysregulation and spliceosomal dependency, requires the splicing factor RBM39 for survival. Indisulam, a "molecular glue" that selectively recruits RBM39 to the CRL4-DCAF15 E3 ubiquitin ligase for proteasomal degradation, is highly efficacious against neuroblastoma, leading to significant responses in multiple high-risk disease models, without overt toxicity. Genetic depletion or indisulam-mediated degradation of RBM39 induces significant genome-wide splicing anomalies and cell death. Mechanistically, the dependency on RBM39 and high-level expression of DCAF15 determine the exquisite sensitivity of neuroblastoma to indisulam. Our data indicate that targeting the dysregulated spliceosome by precisely inhibiting RBM39, a vulnerability in neuroblastoma, is a valid therapeutic strategy.

YAP/TAZ maintain the proliferative capacity and structural organization of radial glial cells during brain development.

The Hippo pathway regulates the development and homeostasis of many tissues and in many species. It controls the activity of two paralogous transcriptional coactivators, YAP and TAZ (YAP/TAZ). Although previous studies have established that aberrant YAP/TAZ activation is detrimental to mammalian brain development, whether and how endogenous levels of YAP/TAZ activity regulate brain development remain unclear. Here, we show that during mammalian cortical development, YAP/TAZ are specifically expressed in apical neural progenitor cells known as radial glial cells (RGCs). The subcellular localization of YAP/TAZ undergoes dynamic changes as corticogenesis proceeds. YAP/TAZ are required for maintaining the proliferative potential and structural organization of RGCs, and their ablation during cortical development reduces the numbers of cortical projection neurons and causes the loss of ependymal cells, resulting in hydrocephaly. Transcriptomic analysis using sorted RGCs reveals gene expression changes in YAP/TAZ-depleted cells that correlate with mutant phenotypes. Thus, our study has uncovered essential functions of YAP/TAZ during mammalian brain development and revealed the transcriptional mechanism of their action.