Biology subjects

Tan, D.

Publications and source records attributed to Tan, D..

34 records · Page 2Linked to original sources

bioRxiv2023

A transcriptome-wide meta-analysis reveals lack of cancer-cell intrinsic determinants of response to immune checkpoint blockade

Immune-checkpoint therapy (ICB) has conferred significant and durable clinical benefit to some cancer patients. However, most patients do not respond to ICB, and reliable biomarkers of ICB response are needed to improve patient stratification. Here, we performed a transcriptome-wide meta-analysis across 1,486 tumors from ICB-treated patients and tumors with expected ICB outcomes based on microsatellite status. Using a robust transcriptome deconvolution approach, we inferred cancer and stroma-specific gene expression differences and identified cell-type specific features of ICB response across cancer types. Consistent with current knowledge, stromal expression of CXCL9, CXCL13, and IFNG were the top determinants of favorable ICB response. In addition, we identified a group of potential immune-suppressive genes, including FCER1A, associated with poor response to ICB. Strikingly, PD-L1 expression in stromal cells, but not cancer cells, is correlated with ICB response across cancer types. Furthermore, the unbiased transcriptome-wide analysis failed to identify cancer-cell intrinsic features of ICB response conserved across tumor types. Overall, our results challenge the prevailing dogma that cancer cells present tissue-agnostic molecular markers that modulate immune activity and ICB treatment response. These results have implications for the development of improved ICB treatments and diagnostics.

cancer biology↗

bioRxiv2023

Quantification of circulating tumor DNA using deep learning

Quantification of circulating tumor DNA (ctDNA) levels in blood enables non-invasive surveillance of cancer progression. Fragle is an ultra-fast deep learning-based method for ctDNA quantification directly from cell-free DNA fragment length profiles. We developed Fragle using low-pass whole genome sequence (lpWGS) data from multiple cancer types and healthy control cohorts, demonstrating high accuracy, and improved lower limit of detection in independent cohorts as compared to existing tumor-naive methods. Uniquely, Fragle is also compatible with targeted sequencing data, exhibiting high accuracy across both research and commercial targeted gene panels. We used this method to study longitudinal plasma samples from colorectal cancer patients, identifying strong concordance of ctDNA dynamics and treatment response. Furthermore, prediction of minimal residual disease in resected lung cancer patients demonstrated significant risk stratification beyond a tumor-naive gene panel. Overall, Fragle is a versatile, fast, and accurate method for ctDNA quantification with potential for broad clinical utility.

bioinformatics↗

bioRxiv2023

The BTB-ZF gene Bm-mamo regulate pigmentation in caterpillars

The color pattern of insects is one of the most diverse adaptive evolutionary phenotypes. However, the molecular regulation of this color pattern is not fully understood. In this study, we found that the transcription factor Bm-mamo is responsible for black dilute (bd) allele mutations in the silkworm. Bm-mamo belongs to the BTB zinc finger family and is orthologous to mamo in Drosophila melanogaster. This gene has a conserved function in gamete production in Drosophila and silkworms and has evolved a pleiotropic function in the regulation of color patterns in caterpillars. Using RNAi and clustered regularly interspaced short palindromic repeats (CRISPR) technology, we showed that Bm-mamo is a repressor or has dark melanin patterns in the larval epidermis. Using in vitro binding assays and gene expression profiling in wild-type and mutant larvae, we also showed that Bm-mamo likely regulates the expression of related pigment synthesis and cuticular protein genes in a coordinated manner to mediate its role in color pattern formation. This mechanism is consistent with the dual role of this transcription factor in regulating both the structure and shape of the cuticle and the pigments that are embedded within it. This study provides new insight into the regulation of color patterns as well as into the construction of more complex epidermis features in some insects.

genetics↗

bioRxiv2023

Host-cell Interactions of Engineered T cell Micropharmacies

Genetically engineered, cytotoxic, adoptive T cells localize to antigen positive cancer cells inside patients, but tumor heterogeneity and multiple immune escape mechanisms have prevented the eradication of most solid tumor types. More effective, multifunctional engineered T cells are in development to overcome the barriers to the treatment of solid tumors, but the interactions of these highly modified cells with the host are poorly understood. We previously engineered prodrug-activating enzymatic functions into chimeric antigen receptor (CAR) T cells, endowing them with an orthogonal killing mechanism to conventional T-cell cytotoxicity. These drug-delivering cells, termed Synthetic Enzyme-Armed KillER (SEAKER) cells, demonstrated efficacy in mouse lymphoma xenograft models. However, the interactions of an immunocompromised xenograft with such complex engineered T cells are distinct from those in an immunocompetent host, precluding an understanding of how these physiologic processes may affect the therapy. Here, we also expand the repertoire of SEAKER cells to target solid-tumor melanomas in syngeneic mouse models using specific targeting with TCR-engineered T cells. We demonstrate that SEAKER cells localize specifically to tumors, and activate bioactive prodrugs, despite host immune responses. We additionally show that TCR-engineered SEAKER cells are efficacious in immunocompetent hosts, demonstrating that the SEAKER platform is applicable to many adoptive cell therapies.

immunology↗

bioRxiv2022

Design of a systemic small molecule clinical STING agonist using physics-based simulations and artificial intelligence

The protein STING (stimulator of interferon genes) is a central regulator of the innate immune system and plays an important role in antitumor immunity by inducing the production of cytokines such as type I interferon (IFN). Activation of STING stems from the selective recognition of endogenous cyclic dinucleotides (CDNs) by the large, polar, and flexible binding site, thus posing challenges to the design of small molecule agonists with drug-like physicochemical properties. In this work we present the design of SNX281, a small molecule STING agonist that functions through a unique self-dimerizing mechanism in the STING binding site, where the ligand dimer approximates the size and shape of a cyclic dinucleotide while maintaining drug-like small molecule properties. SNX281 exhibits systemic exposure, STING-mediated cytokine release, strong induction of type I IFN, potent in vivo antitumor activity, durable immune memory, and single-dose tumor elimination in mouse models via a Cmax-driven pharmacologic response. Bespoke computational methods - a combination of quantum mechanics, molecular dynamics, binding free energy simulations, and artificial intelligence - were developed during the course of the project to design SNX281 by explicitly accounting for the unique self-dimerization mechanism and the large-scale conformational change of the STING protein upon activation. Over the course of the project, we explored millions of virtual molecules while synthesizing and testing only 208 molecules in the lab. This work highlights the value of a multifaceted computationally-driven approach anchored by methods tailored to address target-specific problems encountered along the project progression from initial hit to the clinic.

cancer biology↗

bioRxiv2021

Reference genomes of 545 silkworms enable high-throughput exploring genotype-phenotype relationships

The silkworm Bombyx mori is a domestic insect for silk production and a lepidopteran model. The currently available genomes limit a full understanding of its genetic and phenotypic diversity. Here we assembled long-read genomes of 545 domestic and wild silkworms and constructed a high-resolution pan-genome dataset. We found that the silkworm population harbors extremely variable genomes containing 7,308 new gene families, 4,260 (22%) core gene families, and 3,432,266 non-redundant SVs. We deciphered a series of causal genes and variants associated with domestication, breeding, and ecological adaptation traits, and experimentally validated two of those genes using CRISPR-Cas9 or RNA interference. This unprecedented large-scale genomic resource allows for high-throughput screening of interesting traits for functional genomic research and breeding improvement of silkworms and may serve as a guideline for traits decoding in other species.

genomics↗

bioRxiv2021

In vitro Evaluation of Lytic Phages for Carbapenem-Resistant Klebsiella pneumoniae Isolates

Klebsiella pneumoniae is a dominant cause of community-acquired and nosocomial infections, specifically among immunocompromised individuals. The increasing occurrence of multidrug-resistant (MDR) isolates has significantly impacted the effectiveness of antimicrobial agents. As antibiotic resistance is becoming prevalent, the use of bacteriophages to treat pathogenic bacterial infections is becoming revitalized. Elucidating the details of phage-bacteria interactions will provide insights into phage biology and the better development of phage therapies. In this study, a total of 22 K. pneumoniae isolates were assessed for their genetic and phenotypic relatedness by multi-locus sequence typing (MLST), endonuclease S1 nuclease pulsed-field gel electrophoresis (S1-PFGE), and in vitro antibiotic susceptibility testing. In addition, the beta-lactamase gene (blaKPC) was characterized to determine the spread and outbreak of K. pneumoniae carbapenemase (KPC)-producing enterobacterial pathogens. Using these K. pneumoniae isolates, three phages were isolated and characterized to evaluate the application of lytic phages against these 22 ST11 K. pneumoniae isolates. In vitro inhibition assays with three phages and K. pneumoniae strain ZS15 demonstrated the strong lytic potential of the phages, however, followed by the rapid growth of phage-resistant mutants. Together, this data adds more comprehensive knowledge to known phage biology and further emphasizes their complexity and future challenges to overcome prior to using phages for controlling this important MDR bacterium.

microbiology↗

bioRxiv2021

A robust field-based method to screen heat tolerance in wheat

Wheat is highly sensitive to elevated temperatures, particularly during pollen meiosis and early-to-mid grain filling. The impact of heat stress greatly depends on the plant developmental stage. Thus, germplasm ranking for heat tolerance in field trials may be confounded by variations in developmental phase between genotypes at the time of heat events. A photoperiod-extension method (PEM) was developed allowing screening of 35 diverse genotypes at matched developmental phase despite phenological variations. Paired trials were conducted to compare the new PEM against conventional field screening in plots. In the PEM, plants were sown in single rows or small plots. Artificial lighting was installed at one end of each row or plot to extend day length, inducing a gradient of flowering times with distance from the lights. Individual stems or plot quadrats of each genotype were tagged at flowering. Late-sown plants received more heat shocks during early to mid grain filling than earlier sowings, suffering reductions in both individual grain weight (IGW) and yield. IGW was reduced by 1.5 mg for each additional post-flowering day with temperature > 30{degrees}C. Significant genotypic differences in heat tolerance ranking were observed between PEM versus conventional plot screening. Strong correlations between trials experiencing similar degree of heat were found both for IGW and for total grain weight with the PEM either with individual-stem tagging (e.g. average r of 0.59 and 0.54, respectively for environments with moderate postflowering heat) or quadrat tagging (r of 0.53 and 0.47). However, correlations for IGW and yield in these environments were either poor or negative for conventional trials (e.g. average r of 0.11 and 0.12, respectively for environments with moderate postflowering heat). Accordingly, a PCA grouped genotypes consistently for heir performance across environments with similar heat stress in PEM trials but not in conventional trials. In this study, most consistent genotype ranking for heat tolerance was achieved with the PEM with tagging and harvesting individual spikes at matched developmental phase. The PEM with quadrat sampling provided slightly less consistent rankings but appears overall more suitable for high-throughput phenotyping. The method promises to improve the efficiency of heat tolerance field screening, particularly when comparing genotypes of different maturity types.

plant biology↗

bioRxiv2020

Quantitative Control of Noise in Mammalian Gene Expression by Dynamic Histone Regulations

Fluctuation ( noise) in gene expression is critical for mammalian cellular processes. Numerous mechanisms contribute to its origins, yet large noises induced by single transcriptional activator species remain to be experimentally understood. Here, we combined the dynamic regulation of transcriptional activator binding, histone regulator inhibitors, and single-cell quantification of chromatin accessibility, mRNA, and protein to probe putative mechanisms. Using a light-induced expression system, we show that the transcriptional activator forms a positive feedback loop with histone acetyltransferases CBP/p300. It generates epigenetic bistability in H3K27ac, which contributes to large noise. Disable of the positive feedback loop by CBP/p300 and HDAC4/5 inhibitors also reduces heterogeneity in endogenous genes, suggesting a universal mechanism. We showed that the noise was reduced by pulse-wide modulation of transcriptional activator binding due to alternating the system between high and low monostable states. Our findings could provide a mechanism-based approach to modulate noise in synthetic and endogenous gene expressions.

molecular biology↗

bioRxiv2020

A Fast Lysine Cross-linker DOPA Enables Mass Spectrometry Analyses of Protein Unfolding and Weak Protein-protein Interactions

Chemical cross-linking of proteins coupled with mass spectrometry analysis (CXMS) has become a widely used method for protein structure analysis. Central to this technology are chemical cross-linkers. The most popular cross-linkers are N-hydroxysuccinimide (NHS) esters, which react with protein amino groups relatively slowly over 10 minutes or more while in competition with the hydrolysis reaction of NHS esters. To improve the speed of cross-linking, we developed a new class of amine-selective and non-hydrolyzable di-ortho-phthalaldehyde (DOPA) cross-linkers. DOPA can cross-link proteins in 10 seconds under near physiological conditions, which is 60 times faster than the NHS ester cross-linker DSS. DOPA also works at low pH, low temperature, or in the presence of high concentrations of denaturants such as 8 M urea or 6 M guanidine hydrochloride. Further, DOPA-mediated pulse cross-linking captured the dynamic conformational changes associated with RNase A unfolding. Lastly, DOPA outperformed DSS at capturing weak but specific protein-protein interactions.

biochemistry↗

bioRxiv2020

Deficiency of the splicing factor RBM10 limits EGFR inhibitor response in EGFR mutant lung cancer

Molecularly targeted cancer therapy has improved outcomes for cancer patients with targetable oncoproteins, such as mutant epidermal growth factor receptor (EGFR) in lung cancer. Yet, long-term patient survival remains limited because treatment responses are typically incomplete. One potential explanation for the lack of complete and durable responses is that oncogene-driven cancers with activating mutations in the EGFR often harbor additional co-occurring genetic alterations. This hypothesis remains untested for most genetic alterations that co-occur with mutant EGFR. Here, we report the functional impact of inactivating genetic alteration of the mRNA splicing factor RBM10 that co-occur with mutant EGFR. RBM10 deficiency decreased EGFR inhibitor efficacy in patient-derived EGFR mutant tumor models. RBM10 modulated mRNA alternative splicing of the mitochondrial apoptotic regulator Bcl-x to regulate tumor cell apoptosis during treatment. Genetic inactivation of RBM10 diminished EGFR inhibitor-mediated apoptosis by decreasing the ratio of Bcl-xS-(pro-apoptotic)-to-Bcl-xL(anti-apoptotic) Bcl-x isoforms. RBM10 deficiency was a biomarker of poor response to EGFR inhibitor treatment in clinical samples. Co-inhibition of Bcl-xL and mutant EGFR overcame resistance induced by RBM10 deficiency. This study sheds light on the role of co-occurring genetic alterations, and on the impact of splicing factor deficiency in the modulation of sensitivity to targeted kinase inhibitor cancer therapy.

cancer biology↗

bioRxiv2019

An Improved Human smORF Annnotation Workflow Combining De Novo Transcriptome Assembly and Ribo-Seq

Protein-coding small open reading frames (smORFs) are emerging as an important class of genes, however, the coding capacity of smORFs in the human genome is unclear. By integrating de novo transcriptome assembly and Ribo-Seq, we confidently annotate thousands of novel translated smORFs in three human cell lines. We find that smORF translation prediction is noisier than for annotated coding sequences, underscoring the importance of analyzing multiple experiments and footprinting conditions. These smORFs are located within non-coding and antisense transcripts, the UTRs of mRNAs, and unannotated transcripts. Analysis of RNA levels and translation efficiency during cellular stress identifies regulated smORFs, providing an approach to select smORFs for further investigation. Sequence conservation and signatures of positive selection indicate that encoded microproteins are likely functional. Additionally, proteomics data from enriched human leukocyte antigen complexes validates the translation of hundreds of smORFs and positions them as a source of novel antigens. Thus, smORFs represent a significant number of important, yet unexplored human genes.

genomics↗

bioRxiv2018

Proliferation Saturation Index in an adaptive Bayesian approach to predict patient-specific radiotherapy responses

PurposeRadiotherapy prescription dose and dose fractionation protocols vary little between individual patients having the same tumor grade and stage. To personalize radiotherapy a predictive model is needed to simulate radiation response. Previous modeling attempts with multiple variables and parameters have been shown to yield excellent data fits at the cost of nonidentifiability and clinically unrealistic results.\n\nMaterials and MethodsWe develop a mathematical model based on a proliferation saturation index (PSI) that is a measurement of pre-treatment tumor volume-to-carrying capacity ratio that modulates intrinsic tumor growth and radiation response rates. In an adaptive Bayesian approach, we utilize an increasing number of data points for individual patients for predicting response to subsequent radiation doses.\n\nResultsModel analysis shows that using PSI as the only patient-specific parameter, model simulations can fit longitudinal clinical data with high accuracy (R2=0.84). By analyzing tumor response to radiation using daily CT scans early in the treatment, response to the remaining treatment fractions can be predicted after two weeks with high accuracy (c-index=0.89).\n\nConclusionThe PSI model may be suited to forecast treatment response for individual patients and offer actionable decision points for mid-treatment protocol adaptation. The presented work provides an actionable image-derived biomarker prior to and during therapy to personalize and adapt radiotherapy.

cancer biology↗

bioRxiv2018

Whole-Genome Genomics Correlates of Response To Anti-PD1 Therapy in Relapsed/Refractory Natural Killer/T Cell Lymphoma

AbstractThis study aims to identify recurrent genetic alterations in relapsed or refractory (RR) natural-killer/T-cell lymphoma (NKTL) patients who have achieved complete response (CR) with programmed cell death 1 (PD-1) blockade therapy. Seven of the eleven patients treated with pembrolizumab achieved CR while the remaining four had progressive disease (PD). Using whole genome sequencing (WGS), we found recurrent clonal structural rearrangements (SR) of the PD-L1 gene in four of the seven (57%) CR patients pretreated tumors. These PD-L1 SRs consist of inter-chromosomal translocations, tandem duplication and micro-inversion that disrupted the suppressive function of PD-L1 3UTR. Interestingly, recurrent JAK3-activating (p.A573V) mutations were also validated in two CR patients tumors that did not harbor the PD-L1 SR. Importantly, these mutations were absent in the four PD cases. With immunohistochemistry (IHC), PD-L1 positivity could not discriminate patients who archived CR (range: 6%-100%) from patients who had PD (range: 35%-90%). PD-1 blockade with pembrolizumab is a potent strategy for RR NKTL patients and genomic screening could potentially accompany PD-L1 IHC positivity to better select patients for anti-PD-1 therapy.

genomics↗

bioRxiv2018

A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9-DNMT3A constructs

AbstractDNA methylation is a covalent modification of the genome that plays important roles in genome regulation and vertebrate development. Although detection of this modification in the genome has been possible for several decades, the ability to deliberately and specifically manipulate local DNA methylation states in the genome has been extremely limited. Consequently, this has impeded the direct determination of the consequence of DNA methylation on transcriptional regulation and transcription factor binding in the native chromatin context. Thus, highly specific targeted epigenome editing tools are needed to address this outstanding question. Recent adaptations of genome editing technologies, such as the fusion of the DNMT3A methyltransferase catalytic domain to catalytically inactive Cas9 (dC9-D3A), have aimed to provide new tools for altering DNA methylation at desired loci. Here, we performed a deeper analysis of the performance of these tools, revealing consistent off-target binding events and DNA methylation deposition in the genome, limiting the capacity of these tools to unambiguously assess the functional consequences of DNA methylation. To address this, we developed a modular dCas9-SunTag (dC9Sun-D3A) system that can recruit multiple DNMT3A catalytic domains to a target site for editing DNA-methylation. dC9Sun-D3A is tunable, specific and exhibits much higher induction of DNA methylation at target sites than the dC9-D3A direct fusion protein. Importantly, genome-wide characterization of dC9Sun-D3A binding sites and DNA methylation revealed minimal off-target protein binding and induction of DNA methylation with dC9Sun-D3A, compared to pervasive off-target binding and methylation by the dC9-D3A direct fusion construct. Furthermore, we used dC9Sun-D3A to test the impact of DNA methylation upon the DNA binding of CTCF and NRF1 upon targeted methylation of their core binding sites, demonstrating the binding sensitivity of these proteins to DNA methylation in situ. Overall, this modular dC9Sun-D3A system enables precise DNA methylation deposition with the lowest amount of off-target DNA methylation reported to date, allowing accurate functional determination of the role of DNA methylation at single loci.

molecular biology↗

bioRxiv2018

Cryo-EM structure of an early precursor of large ribosomal subunit reveals a half assembled intermediate

Assembly of eukaryotic ribosome is a complicated and dynamic process that involves a series of intermediates. How the highly intertwined structure of 60S large ribosomal subunits is established is unknown. Here, we report the structure of an early nucleolar pre-60S ribosome determined by cryo-electron microscopy at 3.7 [A] resolution, revealing a half assembled subunit. Domains I, II and VI of 25S/5.8S rRNA tightly pack into a native-like substructure, but domains III, IV and V are not assembled. The structure contains 12 assembly factors and 19 ribosomal proteins, many of which are required for early processing of large subunit rRNA. The Brx1-Ebp2 complex would interfere with the assembly of domains IV and V. Rpf1, Mak16, Nsa1 and Rrp1 form a cluster that consolidates the joining of domains I and II. Our structure reveals a key intermediate on the path to the establishment of the global architecture of 60S subunits.

biophysics↗