Molecular and cellular biology

Permanent URI for this collectionhttps://digital.lib.washington.edu/handle/1773/4943

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Nuclear speckle proteins modulate tau toxicity
(2026-04-20) LeBlanc, Katherine R.; Kraemer, Brian C
Alzheimer’s Disease (AD) and other tauopathies are neurodegenerative disorders with devastating consequences for cognition and memory. Pathogenic accumulation of tau can be modeled in Caenorhabditis elegans, which recapitulate human neurodegeneration including aging-dependent accumulation of phosphorylated tau, tau aggregation, neuronal dysfunction, and neuron degeneration. Using genetic tools to identify genes modulating tau pathology, we identified mutations in multiple nuclear speckle protein encoding genes that ameliorate tau-driven behavioral defects and prevent neurodegeneration. RNA sequencing reveals that these proteins, DIB-1, PRP-8, and ERH-1, have differential effects on gene expression, alternative splicing, and phosphorylated tau levels, highlighting how these proteins with similar roles may not share a single mechanism in alleviating tauopathy. Furthermore, levels of the human homologs of all three proteins are decreased in human Alzheimer’s disease samples, suggesting the translational relevance of this work. These findings point to nuclear proteins and mRNA dynamics as important drivers of tau toxicity and highlight the need to further investigate tau-mRNA interactions in neurodegenerative disease.
cJun Overexpression Sensitizes CAR-T Cells to PD-1 Axis Blockade by Preserving an Intratumoral PD-1⁺ Tcf1⁺ Stem-Like Reservoir
(2026-04-20) Snyder, Andrew; Srivastava, Shivani
T cells possess a unique capacity to recognize and kill cells expressing specific antigens,making them an attractive platform for cancer immunotherapy. Decades of iterative advances in immunology, synthetic biology, and genetic engineering have led to the development of adoptive T cell therapies, including chimeric antigen receptor (CAR) T cells, which have transformed how malignancies are treated. Despite the successes of CAR-T cells in hematological cancers, durable efficacy remains limited in solid tumors due to defects in CAR-T cell trafficking, exhaustion, and toxicities that constrain their therapeutic benefit. This thesis examines the immunological principles underlying T cell based therapies, the evolution of cancer immunotherapy, and the mechanisms that govern CAR-T cells response and failure in the solid tumor setting. Additionally, this thesis focuses on the role PD-1⁺ Tcf1⁺ stem-like T cells play in mediating responses to immune checkpoint blockade and examines whether CAR-T cells are capable of forming and maintaining this critical stem-like reservoir in the solid tumor microenvironment (TME). This work demonstrates that ROR1-targeting CAR-T cells rapidly downregulate Tcf1 in- vivo, undergo exhaustion, and fail to respond to PD-1 axis blockade. Overexpression of the AP- 1 transcription factor cJun enables the formation of an intratumoral PD-1⁺ Tcf1⁺ CAR-T cell reservoir, yet cJun overexpression alone is insufficient to overcome CAR-T cell exhaustion in the solid TME due to PD-1 induced post-transcriptional downregulation of cJun. PD-L1 blockade is able to restore cJun overexpression, promotes robust intratumoral CAR-T cell expansion culminating in dramatic tumor clearance. Collectively, these findings identify PD-1 as a negative regulator of cJun and demonstrate that, despite their MHC-independent design, CAR-T cells can be engineered to form intratumoral stem-like reservoirs that overcome resistance to checkpoint blockade. This work provides mechanistic insight into CAR-T cell failure in solid tumors and informs the rational design of next generation immunotherapies with improved durability and efficacy.
αE-Catenin as a Gatekeeper of β-Cell Identity and Growth
(2026-04-20) Andrade, Mark; Cirulli, Vincenzo
The pancreas is a vital organ that serves dual roles in digestion and metabolic regulation. Its exocrine compartment secretes digestive enzymes and bicarbonate into the duodenum, while its endocrine compartment - organized into the islets of Langerhans - produces hormones including insulin, glucagon, and somatostatin to regulate blood glucose and energy homeostasis. Both compartments arise during embryogenesis from a common pool of multipotent progenitor cells, whose fate decisions are orchestrated by key developmental signaling pathways, including Wnt and Sonic Hedgehog (SHH), that coordinate the sequential emergence of exocrine, ductal, and endocrine cell lineages including insulin-producing β-cells, glucagon-producing α-cells, and somatostatin-producing δ-cells. αE-catenin is a critical regulator of cell adhesion and intracellular signaling that has emerged as an important modulator of pancreatic cell fate. Prior work from our group demonstrated that loss of αE-catenin in early multipotent pancreatic progenitors aberrantly activates SHH signaling, resulting in a failure of progenitor cells to exit the progenitor state and commit to endocrine differentiation. Building on this, we investigated whether αE-catenin continues to play a regulatory role later in development, specifically within maturing β-cells at the stage when insulin expression is first initiated. We found that deleting αE-catenin specifically in developing β-cells leads to a striking expansion of β-cell mass driven by increased proliferation, as well as molecular evidence of endocrine lineage instability and enhanced cellular plasticity. These effects were also observed in human islets, underscoring the translational relevance of our findings. Collectively, this work positions αE-catenin as a key regulator of β-cell homeostasis and identity and suggests that modulating this pathway could offer new strategies for promoting islet regeneration and expansion in the treatment of diabetes.
Rehabilitating killers: leveraging immunomodulatory natural killer cell functions to orchestrate anti-tumor immunity
(2026-04-20) Avanessian, Shayan Ciamanto; Barry, Kevin C
Immunotherapies harness the immune system to kill cancer and have revolutionized cancer treatments. While durable immune responses against cancer rely on the adaptive arm of the immune system, it is the innate immune system that initiates and shapes its response. Seeking to better understand the immune features that support patient responses to immune checkpoint blockade (ICB) immunotherapies, previous work from our group identified a protective innate immune axis in the tumor microenvironment (TME), where natural killer (NK) cells support the maintenance of type one conventional dendritic cells (cDC1) through the production of the cDC1-formative growth factor Flt3L. The abundance of this NK cell–Flt3L–cDC1 axis correlates with increased overall survival and defines patient responsiveness to ICB therapy. These findings add to the growing body of literature emphasizing the important role NK cells play in orchestrating immune responses beyond cytotoxicity. Yet despite the clear clinical relevance, the mechanisms regulating NK cell production of Flt3L are currently unknown. To address this knowledge gap, my dissertation work investigates the molecular and cellular mechanisms that regulate Flt3L. Chapter 1 provides a detailed introduction into the role of the immune system in the cancer response, with particular attention given to NK cells and cDC1s. In Chapter 2, I describe a set of experiments that we performed to understand which signaling pathways in the tumor, and which subsets of NK cells, have a role in regulating Flt3L production and cDC1 abundance, and how this information can be used in the design of future immunotherapies. In Chapter 3, I present an investigation beyond the TME, where we explore a potential role for other cell populations in producing Flt3L, and supporting cDC1 abundance, in steady-state tissues. In Chapter 4, I discuss the findings of this dissertation work in the broader context of immunology and immunotherapies, and detail future work that will be crucial to more completely understand the mechanisms that support this protective immune axis.
Oncofetal Proteins Drive Aggressive Disease in Basal Pancreatic Cancer
(2026-04-20) Yamamoto, Naomi; Kugel, Sita
Pancreatic ductal adenocarcinoma (PDAC) is a deadly cancer arising from the exocrine pancreas. Unlike most other solid tumors, incidence and mortality from PDAC continues to rise in the United States. PDAC is typically diagnosed after it has metastasized to other organs, and the only treatment option for advanced disease is combination chemotherapy. Targeted small molecule inhibitors and immunotherapies have encountered rapid resistance and are not yet clinically approved. Identifying disease subtypes and targeting therapies to these subtypes remains of crucial importance to improve outcomes.We have found that the chromatin-associated protein high mobility group AT-hook 2 (HMGA2) is differentially regulated in the most lethal subtypes of PDAC. In addition to predicting worse overall survival, high levels of HMGA2 in primary tumors also correlate with shorter time to disease recurrence and chemoresistance. In preclinical studies, HMGA2 expression is sufficient to drive aggressive disease in human and murine models of PDAC and leads to increased translation. This phenotype can be specifically targeted with inhibitors of protein synthesis. Finally, HMGA2 levels lead to altered cytokine secretion from tumor cells, fundamentally reshaping the tumor microenvironment and potentially defining key targets to rewire the profound immunosuppression seen in these tumors. Together, these studies define the oncofetal protein HMGA2 as a driver of aggressive disease in PDAC and elucidate the intracellular and extracellular mechanisms that lead to this lethal phenotype. We propose that levels of HMGA2 can be used clinically to define subsets of PDAC patients who will respond to targeted and immune modulating therapies.
Multiplex Methods for Profiling and Programming Protein Degradation
(2026-02-05) Suiter, Chase Cameron; Shendure, Jay
This thesis focuses on the development of multiplex experimental methods for profiling and programming intracellular protein degradation. In addition, it explores the synergistic application of deep-learning models for understanding and engineering protein function at scale.Chapter 1 provides an overview of the mechanisms governing proteome homeostasis and outlines the technological gap that has historically limited our ability to study these systems. I review how the convergence of multiplex cellular assays and deep learning models offers a new paradigm for dissecting endogenous regulatory networks and engineering novel protein functions. Chapter 2 presents COMET (COmbinatorial Mapping of E3 Targets), a pooled assay for mapping E3 ubiquitin ligases to their substrates. By coupling combinatorial libraries of dual-fluorescent reporters with E3-targeting CRISPR guides, we enabled the many-by-many measurement of E3-dependent changes in protein abundance within a single experiment. I apply COMET to identify substrates of SCF complex E3 ubiquitin ligases as well as map the E3s mediating degradation of short-lived transcription factors, revealing that proteolytic regulation is often characterized by complex, many-to-many connectivity rather than simple one-to-one relationships. Finally, I demonstrate the use of deep-learning-based structural prediction models for the in silico validation of COMET-nominated E3–substrate pairs, pointing toward a future where computational nomination guides experimental validation. Chapter 3 shifts from mapping endogenous degradation to programming it. Here, we demonstrate a multiplex framework for the discovery of functional de novo designed “proximity handles.” We designed a library of binders targeting various effector proteins and characterized them using LABEL-seq, a multiplex RNA-barcoded protein abundance assay. This approach identified hundreds of designs capable of mediating the stabilization or degradation of a target protein. We further validated a subset of designs in orthogonal assays, demonstrated handle-mediated degradation of the endogenous oncoprotein MCL1, and applied these handles to remodel mitochondrial organization. This study establishes a generalizable pipeline linking computational protein design to high-throughput cell-based readouts. To conclude, Chapter 4 discusses future directions at the interface of multiplex technology and deep learning, specifically focusing on the potential for closed-loop design-build-test-learn cycles and the expansion of these methods to enzyme engineering.
Epistasis and pleiotropy in viral protein evolution
(2026-02-05) Yu, Timothy; Bloom, Jesse D
Viruses evolve under Darwinian selection, and forecasting their evolution requires understanding which mutations confer fitness advantages. Over the past decade, advances in high-throughput experiments have made it possible to measure the phenotypic effects of all mutations to key viral proteins. Yet we continue to fall short when predicting which viral mutations will rise in frequency. The main problem is that the effect of a mutation is not fixed—it depends on the genetic background in which it occurs and on an immune context that is often unknown and dynamic. A mutation that is deleterious in one background may become tolerated in another. A mutation that benefits one phenotype but harms another can create conflicts that constrain selection. These phenomena, known as epistasis and pleiotropy, complicate efforts to make accurate viral forecasts. In chapter 2, we examine how viral mutations combine to escape antibodies in human sera. We introduce a simple biophysical model that explains how the effect of a mutation on antibody escape depends on epistatic interactions with other mutations. We then show how these mutation effects can be inferred directly from deep mutational scanning datasets. In chapter 3, we investigate how pleiotropy constrains viral protein evolution. We use deep mutational scanning to measure the effects of mutations to human influenza virus hemagglutinin on three phenotypes: cell entry, acid stability, and serum antibody neutralization. By quantifying mutation effects across multiple phenotypes, we identify viral mutations that are beneficial in one context but deleterious in another, revealing evolutionary trade-offs. Finally, in chapter 4, we compare the effects of viral mutations to H3, H5, and H7 influenza virus hemagglutinin to explore how mutation effects differ across three sequentially divergent but structurally conserved proteins.
Spinal cord regeneration progresses via developmental and non-developmental mechanisms in X. tropicalis
(2026-02-05) Angell Swearer, Avery; Wills, Andrea
Human spinal cord regeneration is hampered by the inability of resident neural stem cells to regenerate developmentally derived neuron diversity and organization after injury. Unlike humans, the Xenopus tropicalis spinal cord is capable of functional regeneration after amputation, although the regenerative extent and mechanisms driving neural diversity and patterning remain unknown. During my dissertation work, I investigated if the X. tropicalis spinal cord regenerates cell diversity and patterning via the same developmental mechanisms with which it was originally made. First, I showed that the spinal cord dorsal/ventral axis is re-established by Shh signaling after injury in a similar manner to embryogenesis (Angell Swearer, Perkowski, et al. 2025). Next, I found that spinal cord regeneration deploys cell-type-specific developmental and non-developmental strategies to restore neuron diversity, in a divergence from the traditional regenerative paradigm (Angell Swearer et al. 2025, preprint). Overall, these results indicate that successful regeneration recruits a dynamic combination of developmental and non-developmental signals to complete functional healing.
Methods to direct and enrich hematopoietic stem cell engraftment post-transplantation
(2026-02-05) Petty, Nicholas E; Kiem, Hans-Peter
Hematopoietic stem cell transplantation (HSCT) was originally developed as a consequence of undesired toxicity against healthy hematopoietic stem cells (HSCs) while treating malignancies. However, since its inception 70 years ago, HSCT has become a well utilized and diverse tool for the treatment of not just malignant disorders, but monogenic diseases such as hemoglobinopathies and lysosomal storage diseases. The work presented herein aims to explore the engraftment and modification of HSCs through diverse compartments, from the hematopoietic system to the central nervous system. These studies include the modification of the CD33 and CD90 proteins on HSCs paired with their targeting by chimeric antigen receptor (CAR) T-cells in nonhuman primate (NHP) and murine models, as well as more focused studied investigating the engraftment and characterization of microglia-like cells (MLCs) in the NHP brain after HSCT. Finally, I present a snapshot of future directions for works investigating methods to enrich and functionalize MLC engraftment for future therapeutic development.
EBF1 Regulates Sensory Development in the Mammalian Cochlea
(2026-02-05) Powers, Kathryn Gorsuch; Bermingham-McDonogh, Olivia
Hair cell damage is a leading cause of sensorineural hearing loss. Unlike non-mammalian vertebrates, which regenerate hair cells throughout their lives, mammals lose the ability to spontaneously generate cochlear hair cells within the first postnatal week. Understanding the developmental programs that drive sensory development is essential for the design of effective regenerative therapeutics to fight hearing loss. However, large gaps remain in our understanding of the signals that direct cochlear development. Recent work in the Bermingham-McDonogh lab identified enrichment of EBF (Early B cell Factor) consensus DNA-binding motifs in the open chromatin of prosensory cells collected from embryonic mouse cochleae. Although Ebf genes are often expressed in overlapping patterns, this appears to not be the case for the cochlea. Single cell RNA-seq analysis revealed that Ebf1 shows strong expression in the developing cochlear epithelium, whereas Ebf2-4 show little to no expression. To determine how loss of EBF1 affects cochlear development, I generated conditional knockout mouse models. The work presented in this dissertation characterizes the role of EBF1 in cochlear development. First, Ebf1 conditional knockout mouse phenotypes were examined, with particular attention to how Ebf1 excision affects cochlear patterning, prosensory proliferation, sensory differentiation and maturation, and hearing. Second, a multiomic approach that combines single nucleus RNA-seq and single nucleus ATAC-seq into a single workflow was used to identify EBF1’s roles as a transcriptional activator and repressor within a regulatory network. This work provides valuable insight into how EBF1 regulates cell fate and proliferation to restrict sensory development in the mammalian cochlea, and in turn, offers valuable insight into a novel target for future regenerative strategies.
Uncovering the Mechanisms that Control Unattached Kinetochore Clustering
(2026-02-05) Mallett, Darren R; Biggins, Sue
The equal partitioning of the genome during cell division is essential for the survival of an organism. Errors in this process are hallmarks of many human cancers. Central to ensuring faithful chromosome segregation are kinetochores, megadalton protein complexes that form on duplicated sister chromatids during cell division. Kinetochores bridge the connection between chromosomes and the microtubules of the mitotic spindle which generate the forces necessary to pull chromosomes to opposite sides of the cell. A challenge in this process is that sister chromatids must attach to microtubules from opposite spindle poles, a process called biorientation. Kinases play an essential role in this process. Mps1 is a kinase that halts the cell cycle until all kinetochores are bioriented through signaling the spindle assembly checkpoint (SAC). Mps1 phosphorylates Spc105 at its MELT motifs which recruits the Bub3:Bub1 and Mad1:Mad2 complexes to kinetochores to catalyze the formation of a cell cycle inhibitor. Kinetochores signaling the SAC also recruit Stu1 and Slk19, two spindle proteins involved in microtubule stability and crosslinking. Studies suggest Stu1 and Slk19 are involved in promoting the capture of kinetochores to microtubules, but how they are recruited and regulated remains unknown. Here, I investigated the role of Stu1 and Slk19 at unattached kinetochores and mechanisms that promote their recruitment. I found that Stu1 and Slk19 act to cluster unattached kinetochores and this depends on Mps1 activity. Unexpectedly, I found that Stu1 contains conserved MELT motifs like Spc105 that are phosphorylated by Mps1 to promote Slk19 binding. Preventing phosphorylation of the MELTs prevents Stu1 and Slk19 localization to kinetochores and disrupts kinetochore clustering. My work underscores the importance of Mps1 as the master regulator of unattached kinetochores. In addition to controlling kinetochore clustering and the SAC, phosphorylation plays important roles in kinetochore assembly and error correction, of which only a handful have been studied in detail. To further explore the importance of phosphorylation in kinetochore regulation, I have compiled a list of > 500 phosphorylation events that my colleagues and I have detected in vivo from the purification of kinetochore and kinetochore adjacent complexes by mass spectrometry. Using computational methods, I predicted the kinases that are likely responsible for each phosphorylation event. Together, these findings will further accelerate the discoveries of other key phosphorylation events at the kinetochore.
CD90 as a Target for In Vivo Gene and Cell Therapies
(2025-10-02) Thomas, Justin; Kiem, Hans-Peter
Hematopoiesis occurs through a hierarchical process of differentiation beginning with multipotent self-renewing hematopoietic stem cells (HSCs) that persist throughout the human lifespan and ending with transient terminally differentiated unipotent cells. This progression allows for a malleable hematopoietic system capable of responding to pathological and physical assaults to the body. The hierarchical differentiation of the hematopoietic system can be exploited in the clinic for benign and malignant hematology. A patients' pathological hematopoietic system can be cured by replacing their HSCs with new HSCs from a healthy donor (i.e. allogeneic transplantation). Furthermore, the advent of gene modification therapies allows for pathological mutations in a patient's HSCs to be treated by gene therapy and re-transfused into patients (i.e autologous transplantation). The best examples of this therapeutic platform being the treatment of hemoglobinopathies. The reliance of current ex vivo HSC gene therapy approaches on prolonged in vitro culture time, toxic myeloablative conditioning regimens to remove unmodified HSCs, and highly specialized infrastructure severely impact the accessibility of these therapie. Therefore, novel HSC-specific gene therapy platforms with simplified manufacturing protocols for in vivo applications are needed. Previous work refined the target for HSC gene therapy and identified a phenotypically defined subset of cells (CD34+CD90+) that is exclusively responsible for rapid recovery onset, robust long-term multilineage engraftment, and entire reconstitution of the bone marrow (BM) stem cell compartment. My long-term goal is to target this refined HSC subset ex vivo and in vivo for gene and cellular therapies. In this dissertation I develop CD90-recognizing chimeric molecules successfully engineered them onto viral vectors. First proof-of-concept studies in vitro showed that CD90-targeted lentiviral vectors (CD90-LVs) delivered their cargo, with high specificity, into CD90+ cell lines and primary human CD34+CD90+ HSCs. Encouraged by these results, we applied and evaluated the safety of CD90-targeted viral vectors for in vivo applications to transduce and edit human HSCs. Sufficient conditioning with low-dose chemotherapeutics before transplantation of gene-edited HSC is crucial for efficient ex vivo HSC gene therapy. This pretreatment, while genotoxic, enables the engraftment of gene-therapy cell products at therapeutic chimeric thresholds. In vivo gene therapy does not require pre-conditioning. Therefore, targeted, nongenotoxic selection platforms post-vector delivery can be leveraged to increase gene marked/corrected chimerism. Targeting the CD34+CD90+ HSC pool for these enrichment strategies would significantly improve gene modified chimerism. I hypothesized that using CD90-targeted gene and cellular therapies in vivo will not only efficiently deliver gene therapeutics to human HSCs with high target specificity, but gene-marked/edited HSCs can subsequently repopulate the hematopoietic hierarchy after anti-CD90-targeted selection (i.e., chimeric antigen receptor (CAR) T cells). In summary, CD90-LVs can be easily implemented into currently existing ex vivo HSC gene therapy approaches to replace the utilization of cytotoxic chemical transduction enhancers and increase the efficiency of HSC delivery. Furthermore, the ability to target LVs to HSCs in vivo will significantly enhance the accessibility of HSC gene therapy in areas with limited biomedical infrastructure. Additionally, protecting HSCs from CD90-targeted immunotherapies and discovering CD90-dependent signaling pathways will minimize off-target toxicities of potential therapies and open a new category of anti-tumor targets in the clinic. This strategy could also enrich gene-edited HSCs to treat sickle cell disease (SCD).
Dissecting the role of CDK8 mis-splicing in SF3B1-mutant MDS
(2025-10-02) Bonner, Elizabeth; Lee, Stanley C
Somatic SF3B1 mutations are believed to arise early in the pathogenesis of clonal myeloid disorders such as myelodysplastic syndromes (MDS). Studies show that SF3B1 mutations bias hematopoietic stem and progenitor cells (HSPCs) toward the myeloid and erythroid lineages and impair terminal erythroid maturation. However, a direct mechanistic link between mutant SF3B1 and cell fate choice in primitive HSPCs has yet to be established. Recent studies suggest that mutant SF3B1 alters transcription via impaired transcriptional elongation and lead to altered transcriptional cell states of primitive progenitors. Given the central role of transcriptional regulators in modulating lineage-specific gene expression programs and cell fate determination in HSPCs, we hypothesize that mutant SF3B1 promotes aberrant splicing of core transcriptional cofactors, which, in turn, disrupt gene regulatory networks and drive biased hematopoietic differentiation. To investigate this, we analyzed SF3B1-mutant MDS patient data and identified CDK8 as a candidate driver. Using primary CD34+ HSPCS and preclinical modeling our study identifies CDK8 as an important regulator of HSPC homeostasis and cell fate determination in SF3B1-mutant MDS. CDK8 depletion not only biases HSPCs towards myelomonocytic and erythroid lineages but also mirrors phenotypes observed in SF3B1-mutant MDS. This finding directly connects an SF3B1-mutant splicing event to skewed hematopoietic differentiation, shedding new light on MDS etiology.
Decoding the Innate Immune Response to Group B Streptococcus Infections at the Maternal-Fetal Interface
(2025-10-02) Manuel, Gygeria; Adams Waldorf, Kristina
Bacterial infections, including Group B Streptococcus (GBS) in pregnancy are a common cause of early preterm birth and severe neonatal morbidity and mortality. The virulence factors expressed by a particular GBS strain are one important factor in determining invasion; host defenses at the maternal-fetal interface are likely equally important in clearing GBS before invasion can occur. The central hypothesis of the dissertation is that differences in immune cell composition and recruitment, expression of immune cell checkpoints, and cell death profile govern GBS infection at the maternal-fetal interface and clinical outcomes. I found that an early GBS infection would induce the expression of immune checkpoint proteins in the placental chorioamniotic membranes and decidua to counteract the cytokine and chemokine response of immune infiltration. Moreover, bacterial invasion into the amniotic fluid was associated with IL-1 signaling and inflammatory host cell death (pyroptosis, ferroptosis) at the maternal-fetal interface. In contrast, a resolved GBS infection was linked to a mild-inflammatory response of cytotoxic granules, likely produced by uterine NK cells and/or CD8+ T cells. Surprisingly, there was a prominent inflammatory signature in the uterine muscle and preterm labor rate in the resolved infection group. Future studies should focus on understanding how preterm birth therapeutics might support the function of innate immune cells in the maternal decidua to combat pathogens inducing preterm birth.
Loss of the USP22 deubiquitylase confers resistance to chemotherapy in small cell lung cancer
(2025-10-02) Best, Scott Ryan; MacPherson, David
Small cell lung cancer (SCLC) responds exceptionally well to cytotoxic chemotherapy. However, relapse with the emergence of chemoresistant disease is rapid and accompanied by poor treatment outcomes. To understand the genetic basis of chemoresistance in SCLC, we applied in vivo CRISPR deletion screening to patient-derived xenograft (PDX) models. Top screen hits included genes encoding components of the transcriptional co-activator SAGA (Spt-Ada-Gcn5 acetyltransferase) complex. We demonstrate that deletion of the SAGA deubiquitylase USP22 conferred cisplatin/etoposide resistance in two chemosensitive PDX models, and that restoring expression in a PDX model harboring homozygous truncating mutation of USP22 re-sensitized tumors to chemotherapy. USP22 loss increased gene body histone H2A-K119 monoubiquitylation in genes encoding key regulators of neuronal differentiation and suppressed neural and neuroendocrine gene expression including targets of ASCL1. Chemoresistance following USP22 loss reflected attenuated DNA damage-driven phosphorylation events and apoptosis, in conjunction with increased expression of glycolysis and hypoxia-related genes. Glycolysis program upregulation may reflect a targetable vulnerability, as inhibition of GLUT1 re-sensitized USP22-null tumors to chemotherapy.
Disentangling the relationship between pathology and cellular vulnerability in Alzheimer's disease
(2025-10-02) Rachleff, Victoria Mallett; Keene, C. Dirk; Lein, Ed
Alzheimer's Disease (AD) is the most common cause of dementia affecting approximately 57 million people worldwide. Currently, no effective preventions, treatments, or cures exist. This work ventures to study AD from a new perspective; approaching AD as a cellular disorder that preferentially affects unique subpopulations of cells over time as the disease progresses. The goal of this work is to identify cells vulnerable to dysregulation and cell death at the earliest stages of disease and investigate the role of pathogenic protein accumulation in these processes. The overarching hypothesis of this work was that selectively vulnerable populations of cells preferentially accumulate pathologic peptides and can be identified transcriptomically based on changes in relative abundance and cell state associated with disease progression. This dissertation leverages the massive data collection effort of the multi-center Seattle Alzheimer's Disease Brain Cell Atlas (SEA-AD) initiative, which includes single-nucleus RNA sequencing (snRNA-seq) and quantitative neuropathology data from a clinical cohort spanning a spectrum of AD pathology. Additionally, high resolution multiomic data, including spatial transcriptomics and pathologic peptide-based single cell RNA sequencing on a subset of SEA-AD donors enables direct assessment of the relationship between protein pathology and cellular vulnerability. Upon completion, the findings generated from this work will serve as a resource for mapping the cellular changes in AD and will identify opportunities for therapeutic intervention at the earliest stages of disease.
Thermosensory Neurons Mediate Tactile-Dependent Locomotion Modulation in C. elegans
(2025-10-02) Rosero, Manuel; Bai, Jihong
Behavioral plasticity is the ability of animals to modify their behavior, a capacity essential for survival in constantly changing environments. This flexibility arises from experience-dependent changes in neuronal activity that modulate the flow of information through neural circuits. These changes are supported by molecular and circuit mechanisms that promote behavioral adaptation by modulating a wide range of neuronal functions, including excitability, synaptic strength, and connectivity. Disruption of these mechanisms can contribute to a wide range of neurological and psychiatric disorders that severely impair behavior. Despite their importance, the fundamental cellular and molecular principles of neuronal function and behavioral plasticity remain poorly understood, partly because the complexity of mammalian systems makes it difficult to dissect these mechanisms at the cellular and molecular levels. To bypass these limitations, neuroscientists have turned to simpler vertebrate and invertebrate animal models to gain insight into the molecular and circuit mechanisms that enable behavioral plasticity. One such model is the nematode Caenorhabditis elegans, which offers powerful genetic tools, and a well-mapped nervous system ideally suited for dissecting these mechanisms. In this dissertation, I investigated the molecular and circuit mechanisms that enable context-dependent behavioral plasticity in the nematode C. elegans. Using behavioral assays and genetic tools, I uncovered a novel role for the thermosensory neuron AFD in modulating locomotion based on tactile experience across different contexts. Genes in a cGMP signaling pathway are required in AFD for tactile-dependent modulation. Although these genes have previously been implicated in AFD's role in thermosensation, our findings indicate that they function in tactile modulation independently of thermal signaling. This conclusion is supported by the observation that tactile-dependent modulation persists even in the absence of AFD's thermosensory apparatus, suggesting that AFD does not act as a sensory neuron in this context. Rather, its function appears to depend on electrical synapses with the AIB interneuron, indicating that AFD's connectivity is essential for its role in tactile-dependent behavior. Together, these findings reveal a previously unrecognized circuit logic in which AFD contributes to behavioral plasticity by integrating information from other sensory modalities. This work advances our understanding of how molecular signaling and circuit architecture shape behavior and highlights C. elegans as a powerful model for dissecting the molecular basis of neuronal plasticity.
Before and after DUX4: deconstructing its web of silencers and defining its long-term impact on cellular processes
(2025-08-01) Paatela, Ellen; Tapscott, Stephen J
Facioscapulohumeral dystrophy (FSHD) is driven by a loss of epigenetic repression at the D4Z4 repeat region, leading to aberrant expression of early embryonic transcription factor Double Homeobox 4 (DUX4) and an embryonic transcriptional program in skeletal muscle. Mechanisms that initiate and maintain epigenetic repression at the D4Z4 are complex and incompletely understood. Here, I designed a functional silencing reporter system to identify specific D4Z4 sequences that drive silencing activity and discovered that one discrete sequence, termed D4Z4-S5, was sufficient for sequence-dependent silencer recruitment. This sequence drives silencing activity through both de novo DNA methylation and repressive histone modifications, driven specifically by several previously identified D4Z4 silencers: SETDB1, ATF7IP, SIN3A/3B, and LRIF1. The reporter system also showed promise as a robust platform for further discovery of FSHD therapeutics and disease modifiers, with candidate therapeutic p38 pathway inhibitors showing increased repression in D4Z4-S5. Our findings identify a key D4Z4 regulatory sequence that drives epigenetic repression and proposes a novel system for further screening of FSHD disease modifiers and therapeutics. The second focus of this work is based on the long-term effects of DUX4 in cancer. Expression of DUX4 has been identified in a wide variety of solid tissue cancers. DUX4 was recently found to be a driver of immune evasion and is associated with poor prognosis in response to immune-mediated cancer therapies due to a loss of antigen presentation machinery. Though the transcriptional activity of DUX4 has been well characterized, recent studies have implicated DUX4 in post-transcriptional modulation of cellular processes. Here, I describe the mechanism of DUX4-mediated translational suppression, leading to translational reprogramming. I also describe optimization attempts to develop a DUX4-lineage tracing system for further study of long-term effects of DUX4 activity in cancer and FSHD.
The Establishment and Maintenance of Centrosome Asymmetry in Neural Stem Cells
(2025-08-01) Segura, Roberto Carlos; Cabernard, Clemens
Asymmetric cell division (ACD) is used by stem cells to create diverse cell types while self-renewing the stem cell population. Nested within ACD is the biased segregation of organelles, which carries functional consequences on the functionality of sister cells from ACD. Centrosomes, which are the microtubule organizing centers of cells, are comprised of a pair of centrioles that differ by age and molecular composition. Biased segregation of molecularly distinct centrosomes could provide a mechanism to maintain stem cell fate, induce cell differentiation or both. However, the molecular mechanisms generating molecular and functional asymmetric centrosomes remain incompletely understood. The neural stem cell lineage in the developing Drosophila larval brain (neuroblasts) provide a robust model to address our mechanistic understanding of centrosome asymmetry within the context of ACD. Neuroblasts divide asymmetrically to form one neuroblast, which retains its stemness, and one ganglion mother cell (GMC), which will differentiate and divide once more to form neuronal and glial cells. Here, we show how to utilize Drosophila neuroblasts to study basic cell biology and ACD, and using Drosophila neuroblasts, we show that protein phosphatase 4 (Pp4) is functionally required for centrosome asymmetry.
Development of vaccines against MERS coronavirus and Merbecovirus prototype pathogens
(2025-08-01) Chao, Cara Wen-Yi; King, Neil P
Middle East Respiratory Syndrome is a severe respiratory infection caused by the MERS coronavirus (MERS-CoV), a member of the Merbecovirus subgenera in the Betacoronavirus genus. There are no licensed vaccines against MERS-CoV, furthermore our understanding of the members within the Merbecovirus subgenera is still within early stages. As global surveillance efforts continue to unveil new members within this subgenera, analysis of receptor utilization and viral tropism reveal the broad use of ACE2 and DPP4 receptor orthologues, some of which are capable of utilizing human receptors for cell entry. This underscores the need for not only continued viral surveillance to monitor for future outbreak potentials but also the development of vaccine therapeutics against MERS-CoV, which could help guide further advances to address broadly protective Merbecovirus vaccines.

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