Biology subjects

Bradley, R. K.

Publications and source records attributed to Bradley, R. K..

12 recordsLinked to original sources

bioRxiv2026

A U1-U3 snRNA-snoRNA interaction couples SF3B1 mutation to chromatin-state rewiring and genome instability

Mutations in spliceosome factors such as SF3B1 are recurrent across human diseases, including myelodysplastic syndromes and leukemia1-4, yet splicing defects alone do not fully explain the widespread chromatin alterations and genome instability in mutant cells5. Here, by comprehensively mapping snRNA-directed RNA-RNA interactions, we identify two previously unrecognized interaction motifs in U1 snRNA beyond canonical 5' splice-site pairing6,7. These motifs enable U1 RNA to i) bind intronic and other chromatin-associated RNA (caRNA) regions outside of splice sites, and ii) base pair specifically with snoRNA. We uncover a U1-U3 snRNA-snoRNA interaction that recruits the H3K36 methyltransferase SETD2 to caRNA, promoting gene-body H3K36me3 and antagonizing H3K27me3 to modulate chromatin accessibility. The snRNA-snoRNA interface is essential for this previously unrecognized layer of chromatin and transcriptional regulation mediated through SETD2. SF3B1 mutation enhances U1-U3 binding and increases the association of the U1-U3 complex with caRNA, driving chromatin-accessibility remolding, R-loop formation, DNA damage, and copy-number abnormalities that promote tumorigenesis. A U1-specific 2'-O-methoxyethyl antisense oligonucleotide that selectively blocks U1-U3 pairing suppresses these genomic abnormalities, reduces leukemic infiltration, and prolongs survival in xenograft and patient-derived models, establishing pathological snRNA-snoRNA rewiring as a critical driver of SF3B1-mutant leukemogenesis.

cell biology↗

bioRxiv2025

Generative genomics accurately predicts future experimental results

Realizing AIs promise to accelerate biomedical research requires AI models that are both accurate and sufficiently flexible to capture the diversity of real-life experiments. Here, we describe a generative genomics framework for AI-based experimental prediction that mirrors the process of designing and conducting an experiment in the lab or clinic. We created GEM-1 (Generate Expression Model-1), an AI system that effectively models the enormous range of bulk and single-cell gene expression experiments performed by scientists and benchmarked its performance across multiple biological axes. GEM-1s prediction of future gene expression experiments-RNA-seq data deposited in public archives after our training data cutoff-yielded accuracy comparable to the best-possible performance estimated by comparing the results of matched lab experiments. Overall, our approach illustrates the transformative potential of generative genomics for applications ranging from predicting cellular perturbations in vitro to de novo generation of data from large clinical cohorts.

genomics↗

bioRxiv2025

Chorionic Gonadotropin Beta 7 is a marker of immune evasion in cancer

Human chorionic gonadotropin beta (beta-hCG) is an oncofetal antigen expressed by trophoblast cells of the placenta, with minimal expression in adult somatic tissues. Numerous studies have demonstrated that beta-hCG-encoding genes are expressed in various cancers, but expression of these genes (CGB3, CGB5, CGB7, and CGB8) across diverse cancers has not been systematically evaluated. Here, we report that CGB genes are more widely expressed across diverse cancer types than previously appreciated and that secreted beta-hCG is readily detected. In particular, CGB genes are expressed in the majority of urothelial bladder cancers, where CGB7 is most frequently expressed and significantly associated with an immunosuppressed tumor microenvironment, including decreased CD8+ T cell infiltration. Multiple CGB genes are associated with failure to respond to immune checkpoint inhibitor (ICI) therapy, and CGB7 is particularly strongly predictive of poor prognosis. Overall, our findings indicate that beta-hCG is a clinically accessible, predictive biomarker of immunotherapeutic response.

cancer biology↗

bioRxiv2023

DUX4 is a common driver of immune evasion and immunotherapy failure in metastatic cancers

Cancer immune evasion contributes to checkpoint immunotherapy failure in many patients with metastatic cancers. The embryonic transcription factor DUX4 was recently characterized as a suppressor of interferon-{gamma} signaling and antigen presentation that is aberrantly expressed in a small subset of primary tumors. Here, we report that DUX4 expression is a common feature of metastatic tumors, with [~]10-50% of advanced bladder, breast, kidney, prostate, and skin cancers expressing DUX4. DUX4 expression is significantly associated with immune cell exclusion and decreased objective response to PD-L1 blockade in a large cohort of urothelial carcinoma patients. DUX4 expression is a significant predictor of survival even after accounting for tumor mutational burden and other molecular and clinical features in this cohort, with DUX4 expression associated with a median reduction in survival of over one year. Our data motivate future attempts to develop DUX4 as a biomarker and therapeutic target for checkpoint immunotherapy resistance.

cancer biology↗

bioRxiv2022

Recursive splicing discovery using lariats in total RNA sequencing

Recursive splicing is a noncanonical splicing mechanism in which an intron is removed in segments via multiple splicing reactions. Relatively few recursive splice sites have been identified with high confidence in human introns, and more comprehensive analyses are needed to better characterize where recursive splicing happens and whether or not it has a regulatory function. In this study, we use an unbiased approach utilizing intron lariats to search for recursive splice sites in constitutive introns and alternative exons in the human transcriptome. We find evidence for recursive splicing in a broader range of intron sizes than previously reported and detail a new location for recursive splicing at the distal ends of cassette exons. Additionally, we identify evidence for the conservation of these recursive splice sites among higher vertebrates and the use of these sites to influence alternative exon exclusion. Together, our data demonstrate the prevalence of recursive splicing and its potential influence on gene expression through alternatively spliced isoforms.

genomics↗

bioRxiv2022

Activation of targetable inflammatory immune signaling is seen in Myelodysplastic Syndromes with SF3B1 mutations

BackgroundMutations in the SF3B1 splicing factor are commonly seen in Myelodysplastic syndromes (MDS) and Acute Myeloid Leukemia (AML), yet the specific oncogenic pathways activated by missplicing have not been fully elucidated. Inflammatory immune pathways have been shown to play roles in pathogenesis of MDS, though the exact mechanisms of their activation in splicing mutant cases are not well understood. MethodsRNA-seq data from SF3B1 mutant samples was analyzed and functional roles of IRAK4 isoforms were determined. Efficacy of IRAK4 inhibition was evaluated in pre-clinical models of MDS/AML ResultsRNA-seq splicing analysis of innate immune mediators in SF3B1 mutant MDS samples revealed retention of full-length exon 6 of interleukin-1 receptor-associated kinase 4 (IRAK4), a critical downstream mediator that links the Myddosome to inflammatory NF-kB activation. Exon 6 retention leads to a longer isoform, encoding a protein (IRAK4-Long) that contains the entire death domain and kinase domain, leading to maximal activation of NF-kB. Cells with wild-type SF3B1 contain smaller IRAK4 isoforms that are targeted for proteosomal degradation. Expression of IRAK4-Long in SF3B1 mutant cells induces TRAF6 activation leading to K63-linked ubiquitination of CDK2, associated with a block in hematopoietic differentiation. Inhibition of IRAK4 with CA-4948, leads to reduction in NF-kB activation, inflammatory cytokine production, enhanced myeloid differentiation in vitro and reduced leukemic growth in xenograft models. ConclusionsSF3B1 mutation leads to expression of a therapeutically targetable, longer, oncogenic IRAK4 isoform in AML/MDS models.

cancer biology↗

bioRxiv2021

Nonsense-mediated mRNA decay utilizes complementary mechanisms to suppress mRNA and protein accumulation

Nonsense-mediated mRNA decay (NMD) is an essential, highly conserved quality control pathway that detects and degrades mRNAs containing premature termination codons (PTCs). Although the essentiality of NMD is frequently ascribed to its prevention of truncated protein accumulation, the extent to which NMD actually suppresses proteins encoded by NMD-sensitive transcripts is less well-understood than NMD-mediated suppression of mRNA. Here, we describe a reporter system that permits accurate quantification of both mRNA and protein levels via stable integration of paired reporters encoding NMD-sensitive and NMD-insensitive transcripts into the AAVS1 safe harbor loci in human cells. We use this system to demonstrate that NMD suppresses proteins encoded by NMD-sensitive transcripts by up to [~]8-fold more than the mRNA itself. Our data indicate that NMD limits the accumulation of proteins encoded by NMD substrates by mechanisms beyond mRNA degradation, such that even when NMD-sensitive mRNAs escape destruction, their encoded proteins are still effectively suppressed.

molecular biology↗

bioRxiv2020

Discovery of synthetic lethal and tumor suppressive paralog pairs in the human genome

CRISPR knockout screens have accelerated the discovery of important cancer genetic dependencies. However, traditional CRISPR-Cas9 screens are limited in their ability to assay the function of redundant or duplicated genes. Paralogs in multi-gene families constitute two-thirds of the protein-coding genome, so this blind spot is the rule, not the exception. To overcome the limitations of single gene CRISPR knockout screens, we developed paired guide RNAs for Paralog gENetic interaction mapping (pgPEN), a pooled CRISPR/Cas9 approach which targets over a thousand duplicated human paralogs in single knockout and double knockout configurations. We applied pgPEN to two cell lineages and discovered that over 10% of human paralogs exhibit synthetic lethality in at least one cellular context. We recovered known synthetic lethal paralogs such as MAP2K1/MAP2K2, important drug targets such as CDK4/CDK6, and numerous other synthetic lethal pairs such as CCNL1/CCNL2. In addition, we identified ten tumor suppressive paralog pairs whose compound loss promotes cell growth. These findings identify a large number of previously unidentified essential gene families and nominate new druggable targets for oncology drug discovery. HighlightsO_LIComprehensive genetic interaction mapping of 1,030 human duplicated paralogs using a dual targeting CRISPR/Cas9 approach C_LIO_LIDuplicated paralogs are highly enriched for genetic interactions C_LIO_LISynthetic lethal paralogs include CCNL1/CCNL2, CDK4/CDK6, and GSK3A/GSK3B C_LIO_LITumor suppressor paralog pairs include CDKN2A/CDKN2B and FBXO25/FBXO32 C_LI

genomics↗

bioRxiv2020

The origins and consequences of UPF1 variants in pancreatic adenosquamous carcinoma

Pancreatic adenosquamous carcinoma (PASC) is a rare and aggressive subtype of pancreatic cancer whose mutational origins are poorly understood. An early study reported somatic mutations in UPF1, which encodes a core component of the nonsense-mediated mRNA decay (NMD) pathway, as a common signature of PASC, but subsequent studies did not observe these lesions in other PASC cohorts. The corresponding controversy about whether UPF1 mutations are important contributors to PASC has been exacerbated by a paucity of functional studies of these lesions. Here, we systematically assessed the potential roles of UPF1 mutations in PASC. We modeled two reported UPF1 mutations to find no consistent effects on pancreatic cancer growth, acquisition of adenosquamous features, UPF1 splicing, UPF1 protein levels, or NMD efficiency. We subsequently discovered that ~40% of UPF1 mutations reportedly present in PASCs are identical to standing genetic variation in the human population, suggesting that they are likely non-pathogenic inherited variation rather than pathogenic mutations. Our data suggest that UPF1 is not a common functional driver of PASC and motivate further attempts to identify unique genetic features defining these malignancies.

cancer biology↗

bioRxiv2018

Transgenic mice expressing tunable levels of DUX4 develop characteristic facioscapulohumeral muscular dystrophy-like pathophysiology ranging in severity

BackgroundAll types of facioscapulohumeral muscular dystrophy (FSHD) are caused by the aberrant myogenic activation of the somatically silent DUX4 gene, which initiates a cascade of cellular events ultimately leading to FSHD pathophysiology. Therefore, FSHD is a dominant gain-of-function disease that is amenable to modeling by DUX4 overexpression. However, there is large variability in the patient population. Typically, progressive skeletal muscle weakness becomes noticeable in the second or third decade of life, yet there are many genetically FSHD individuals who develop symptoms much later in life or remain relatively asymptomatic throughout their lives. Conversely, in rare cases, FSHD may present clinically prior to 5-10 yrs of age, ultimately manifesting as a very severe early onset form of the disease. Thus, there is a need to control the timing and severity of pathology in FSHD-like models.\n\nMethodsWe have recently described a line of conditional DUX4 transgenic mice, FLExDUX4, that develop a myopathy upon induction of human DUX4-fl expression in skeletal muscle. Here, we use the FLExDUX4 mouse crossed with the skeletal muscle-specific and tamoxifen inducible line ACTAl-MerCreMer to generate a highly versatile bi-transgenic mouse model with chronic, low-level DUX4-fl expression and mild pathology, that can be induced to develop more severe FSHD-like pathology in a dose-dependent response to tamoxifen. We identified conditions to reproducibly generate models exhibiting mild, moderate, or severe DUX4-dependent pathophysiology, and characterized their progression.\n\nResultsWe assayed DUX4-fl mRNA and protein levels, fitness, strength, global gene expression, histopathology, and immune response, all of which are consistent with an FSHD-like myopathic phenotype. Importantly, we identified sex-specific and muscle-specific differences that should be considered when using these models for preclinical studies.\n\nConclusionsThe ACTA1-MCM;FLExDUX4 bi-transgenic mouse model expresses a chronic low level of DUX4-fl and has mild pathology and detectable muscle weakness. The onset and progression of moderate to severe pathology can be controlled via tamoxifen injection to provide consistent and readily screenable phenotypes for assessing therapies targeting DUX4-fl mRNA and protein. Thus, these FSHD-like mouse models can be used to study a range of DUX4-fl expression and pathology dependent upon investigator need, through controlled mosaic expression of DUX4.

cell biology↗

bioRxiv2018

Quantitative proteomics reveals key roles for post-transcriptional gene regulation in the molecular pathology of FSHD

DUX4 is a transcription factor whose misexpression in skeletal muscle causes facioscapulohumeral muscular dystrophy (FSHD). While DUX4s transcriptional activity has been extensively characterized, the DUX4-induced proteome remains undescribed. Here, we report concurrent measurement of RNA and protein levels in DUX4-expressing cells via RNA-seq and quantitative mass spectrometry. DUX4 transcriptional targets were robustly translated, confirming the likely clinical relevance of proposed FSHD biomarkers. However, a multitude of mRNAs and proteins exhibited discordant expression changes upon DUX4 expression. Our dataset revealed unexpected proteomic, but not transcriptomic, dysregulation of diverse molecular pathways, including Golgi apparatus fragmentation, as well as extensive post-transcriptional buffering of stress response genes. Key components of RNA degradation machineries, including UPF1, UPF3B, and XRN1, exhibited suppressed protein, but not mRNA, levels, explaining the build-up of aberrant RNAs that characterizes DUX4-expressing cells. Our results provide a resource for the FSHD community and illustrate the importance of post-transcriptional processes to DUX4-induced pathology.

genomics↗

bioRxiv2018

RNA components of the spliceosome regulate tissue- and cancer-specific alternative splicing

Alternative splicing of pre-mRNAs plays a pivotal role during the establishment and maintenance of human cell types. Characterizing the trans-acting regulatory proteins that control alternative splicing in both healthy and malignant cells has therefore been the focus of much research. Recent work has established that even core protein components of the spliceosome, which are required for splicing to proceed, can nonetheless contribute to splicing regulation by modulating splice site choice. We here demonstrate that the RNA components of the spliceosome likewise influence alternative splicing decisions and contribute to the establishment of global splicing programs. Although these small nuclear RNAs (snRNAs), termed U1, U2, U4, U5, and U6 snRNA, are present in equal stoichiometry within the spliceosome, we found that their relative levels vary by an order of magnitude during development, across tissues, and between normal and malignant cells. Physiologically relevant perturbation of individual snRNAs drove widespread gene-specific differences in alternative splicing, but not transcriptome-wide splicing failure. Genes that were particularly sensitive to variations in snRNA abundance in a breast cancer cell line model were likewise preferentially mis-spliced within a clinically diverse cohort of invasive breast ductal carcinomas. As aberrant mRNA splicing is prevalent in many solid and liquid tumors, we propose that a full understanding of dysregulated pre-mRNA processing in cancers requires study of the RNA as well as protein components of the splicing machinery.

genomics↗