Molecular medicine and mechanisms of disease

Permanent URI for this collectionhttps://digital.lib.washington.edu/handle/1773/49182

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Characterizing the role of tumor-specific B cells in Merkel cell carcinoma disease control
(2026-04-13) Rodriguez Chevez, Haroldo Jose; Nghiem, Paul; Taylor, Justin J
Merkel cell carcinoma (MCC) is a rare neuroendocrine skin cancer with high recurrence and mortality rates. MCC is primarily driven by truncation and clonal integration of the Merkel cell polyomavirus (MCPyV) DNA into the host cell’s chromosomes. Viral integration leads to constitutive expression of the immunogenic T-antigen (T-Ag) oncoproteins, small and truncated large T-Ag, which promote MCPyV-driven MCC (VP-MCC). A second, less common form of non-viral MCC (VN-MCC) arises from accumulation of UV-mediated DNA mutations that affect tumor-suppressor genes. Independently of its origin, MCC is highly immunogenic and often recognized by T & B lymphocytes. Tumor-infiltrating and circulating cancer-specific T cells in MCC patients have been shown to be key promoters of tumor control. In contrast, the role B cells may play in anti-MCC tumor immunity remains unknown.In Chapter 1, we 1) describe MCC biology and therapy resistance, the most pressing issue in the field; 2) highlight the opportunity that MCC presents to investigate total and cancer-specific B cell responses across patients—the latter being extremely difficult to assess in most solid tumors; and 3) address how B cells may be harnessed to develop novel therapies aimed at improving MCC treatments for patients with refractory disease. In Chapter 2, we characterized total and cancer-specific B cell responses in 47 blood samples and 19 unmatched tumors from VP-MCC patients. MCC patient blood data revealed circulating B cell phenotypes that correlate with MCC progression only in female patients, and independently of specificity for MCC viral oncoproteins. In contrast, data from B cells in tumors revealed a strong association between high frequencies of viral oncoprotein-specific antibody-secreting cells and long-term MCC control. These MCC-specific antibody-secreting cells are primarily derived from germinal center B cells, whose detection in tumors also associated with improved disease control. In line with these findings, we identified higher frequencies of follicular helper CD4+ T cells in VP-MCC tumors from patients with better MCC outcomes. Finally, we demonstrated in vitro that B cells engineered to be specific for viral oncoproteins increase the sensitivity of oncoprotein-specific CD4+ T cells by over 50-fold. Together, these results suggest that synergy of viral oncoprotein-specific B and CD4+ T cell responses may promote MCC anti-tumor immunity. Given the association between MCC-specific antibody-secreting B cells and patient outcomes, Chapter 3 explores the T-antigen epitopes recognized by oncoprotein-specific antibodies in VP-MCC patient blood. Mapping of these antibodies revealed an association between preferential binding (immunodominance) against conformational epitopes on the “commonT” domain shared by all T-Ag isoforms and better MCC control. Importantly, this observation was lost when antibody binding against T-Ag linear epitopes was assessed. In line with these results, we detail a patient with progressive VP-MCC and no oncoprotein-specific serum antibody immunodominance, whose tumor was marked by a large expansion of antibody-secreting cells against unique domains of the large T-Ag and no detectable B cells against commonT. These results suggest that B cell binding to specific oncoprotein epitopes impact their ability to promote anti-tumor immunity. The importance of the adaptive immune response against viral oncoproteins in MCC led us to explore in Chapter 4 whether the length of truncated large T-Ag in VP-MCC tumors associates with patient outcomes. The truncation site of large T-Ag is clonal within a given patient’s tumor and results in an oncoprotein length between 228 to 787 amino acids. We found that most patients present with tumors in which truncated large T-Ag is under 350 amino acids. Importantly, analysis of T-antigen DNA sequences from 40 MCC patients with associated clinical data revealed that patients with longer large T-Ag (above 350 amino acids) were significantly less likely to recur and survived longer. Together, these data suggest that increased large T-Ag length may promote MCC immunogenicity as evidenced by lower frequency at presentation and improved disease control after diagnosis. Chapter 5 describes a patient with a lymph node invaded by VP-MCC and Chronic Lymphocytic Leukemia (CLL), a B cell malignancy. Interestingly, this patient has remained MCC- and CLL-free for over 10 years following tumor surgical excision and local radiation. Single-cell RNA-sequencing revealed that malignant B cells from CLL may enhance MCC tumor growth via cytokine-enhanced cell proliferation. This observation highlights a potential mechanism by which aberrant B cells may promote MCC tumorigenesis and progression. Finally, Chapters 6 and 7 describe additional immune mechanisms of MCC control and propose mechanistic studies to probe the role of B cells in MCC anti-tumor immunity. In Chapter 6, we show that certain subsets of myeloid cells in MCC tumors associate with progressive disease. Chapter 7 speculates how the newly developed “SLAP” MCC mouse can be used to test MCC-specific B cell responses in tumor control. Specifically, we propose using classic immunological approaches such as genetic knockout, depletion of immune cell types, and adoptive transfer of engineered B cells specific for the T-antigens to address the role of B cells in MCC. Collectively, our work provides the first in-depth analysis of cancer-specific B cell responses in MCC. By integrating clinical outcomes, immunophenotyping, antibody specificity, and viral oncoprotein structure, these studies reveal that the quality and epitope specificity of B cell responses may have a key role in controlling anti-tumor immunity. We conclude by summarizing how our findings lay the groundwork for mechanistic studies in MCC mouse models that may form the basis for future therapeutic strategies leveraging B cells against solid cancers.
Cellular and Molecular Dissection of the CD8+ T-Cell Response to Kaposi Sarcoma: Implications for Immunotherapeutic Strategies
(2026-02-05) Tiamiyu, Iyabode Limot; Warren, Edus H
Kaposi sarcoma-associated herpesvirus (KSHV) is one of two known tumorigenic human herpesviruses and the etiologic agent of Kaposi sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD), malignancies that predominantly arise in the context of T-cell deficiency or dysfunction. There are no approved vaccines, antiviral drugs, or immunotherapies that can prevent or eliminate KSHV infection. The impact of this therapeutic gap is most pronounced in sub-Saharan Africa (SSA), where the burden of KS morbidity and mortality is highest, KSHV is endemic, and HIV-1, the greatest risk factor for KS, is highly prevalent. All other human herpesviruses elicit potent and durable antiviral T-cell responses to control infection. However, studies of circulating T-cell responses to KSHV in the blood suggest that this response is heterogeneous, infrequent, and low-intensity, with the identity of their peptide-MHC targets poorly defined. Given the consistent expression of KSHV in KS tumors, we hypothesized that KSHV-specific T cells would be frequently recruited to KS tumors. We therefore analyzed the TCR repertoire of tumor biopsies from 144 Ugandan adults with KS, 106 of whom were people living with HIV (PLWH). Clusters of T cells with predicted shared specificity for uncharacterized antigens comprised approximately 25% of the T-cell repertoire in the KS tumor microenvironment, representing 4,283 unique αβ TCRs potentially recognizing KSHV- or HIV-encoded peptide antigens. From 25 reconstructed TCRs, we identified two ORF6-specific, HLA-B*45:01-restricted TCRs, one HLA-B*57:03-restricted TCR with specificity for ORF59, and one HLA-A*66:01-restricted TCR recognizing ORF57. The ORF6- and ORF59-specific TCRs were detected in 27-29 and 30 tumors from 14 HLA-B*45:01+ and 10 HLA-B*57:03+ individuals, respectively. Therefore, these TCRs provide the first evidence of a shared, i.e., “public,” T-cell response to KSHV. Primary CD8+ T cells engineered with KSHV-specific TCRs from KS tumor-infiltrating lymphocytes displayed high-avidity, MHC-restricted recognition of two distinct cell types infected with KSHV, B cells (PEL) and endothelial cells (iTIME.219). In parallel, we characterized three novel HIV-specific TCRs within KS tumors from PLWH, two targeting Nef71-79 and one recognizing Vpr34-42, and confirmed the presence of a previously described public HIV-specific TCR recognizing Pol982-990. HIV-specific T cells remained detectable in KS tumors from individuals who had been on antiretroviral therapy for up to five months. Together, these findings demonstrate that KS tumors recruit polyclonal, high-avidity CD8+ T cells recognizing multiple lytic KSHV and in PLWH, HIV antigens. The identification of CD8+ T cells capable of killing diverse KSHV-infected cell types will provide the foundation for rational vaccine design and TCR-based immunotherapeutic strategies to prevent or treat KS.
CRISPR Screening in Primary Human B Cells to Improve B Cell Therapies
(2026-02-05) Ito, Cade Ellis; James, Richard G.
Pooled CRISPR screening in primary human B cells has been limited by inefficient lentiviraltransduction and high vector copy numbers that compromise single-cell resolution genetic perturbation. This study established a robust CRISPR screening platform by integrating CD20- targeted Nipah pseudotyped lentiviral vectors for single-copy sgRNA delivery with recombinant Cas9 protein electroporation, achieving efficient transduction while maintaining single vector copy number per cell. Essential gene screens validated the platform's technical feasibility, and application to an epigenetic regulator screen revealed that the RARA-NCOR2-TBL1XR1 repressor complex negatively regulates plasma cell differentiation, with disruption of individual components promoting expression of plasma cell surface markers CD38 and CD138. Furthermore, CRISPR screening for natural killer (NK) cell mediated killing revealed HLA-E as an important factor in preventing B cell lysis. This versatile CRISPR screening platform enables systematic investigation of genetic regulators controlling B cell differentiation, survival, and NK tolerance, with applications spanning fundamental immunology and translational cell therapy development.
Characterizing the role of tumor-specific B cells in Merkel cell carcinoma disease control
(2026-02-05) Rodriguez Chevez, Haroldo Jose; Nghiem, Paul; Taylor, Justin J
Merkel cell carcinoma (MCC) is a rare neuroendocrine skin cancer with high recurrence and mortality rates. MCC is primarily driven by truncation and clonal integration of the Merkel cell polyomavirus (MCPyV) DNA into the host cell’s chromosomes. Viral integration leads to constitutive expression of the immunogenic T-antigen (T-Ag) oncoproteins, small and truncated large T-Ag, which promote MCPyV-driven MCC (VP-MCC). A second, less common form of non-viral MCC (VN-MCC) arises from accumulation of UV-mediated DNA mutations that affect tumor-suppressor genes. Independently of its origin, MCC is highly immunogenic and often recognized by T & B lymphocytes. Tumor-infiltrating and circulating cancer-specific T cells in MCC patients have been shown to be key promoters of tumor control. In contrast, the role B cells may play in anti-MCC tumor immunity remains unknown.In Chapter 1, we 1) describe MCC biology and therapy resistance, the most pressing issue in the field; 2) highlight the opportunity that MCC presents to investigate total and cancer-specific B cell responses across patients—the latter being extremely difficult to assess in most solid tumors; and 3) address how B cells may be harnessed to develop novel therapies aimed at improving MCC treatments for patients with refractory disease. In Chapter 2, we characterized total and cancer-specific B cell responses in 47 blood samples and 19 unmatched tumors from VP-MCC patients. MCC patient blood data revealed circulating B cell phenotypes that correlate with MCC progression only in female patients, and independently of specificity for MCC viral oncoproteins. In contrast, data from B cells in tumors revealed a strong association between high frequencies of viral oncoprotein-specific antibody-secreting cells and long-term MCC control. These MCC-specific antibody-secreting cells are primarily derived from germinal center B cells, whose detection in tumors also associated with improved disease control. In line with these findings, we identified higher frequencies of follicular helper CD4+ T cells in VP-MCC tumors from patients with better MCC outcomes. Finally, we demonstrated in vitro that B cells engineered to be specific for viral oncoproteins increase the sensitivity of oncoprotein-specific CD4+ T cells by over 50-fold. Together, these results suggest that synergy of viral oncoprotein-specific B and CD4+ T cell responses may promote MCC anti-tumor immunity. Given the association between MCC-specific antibody-secreting B cells and patient outcomes, Chapter 3 explores the T-antigen epitopes recognized by oncoprotein-specific antibodies in VP-MCC patient blood. Mapping of these antibodies revealed an association between preferential binding (immunodominance) against conformational epitopes on the “commonT” domain shared by all T-Ag isoforms and better MCC control. Importantly, this observation was lost when antibody binding against T-Ag linear epitopes was assessed. In line with these results, we detail a patient with progressive VP-MCC and no oncoprotein-specific serum antibody immunodominance, whose tumor was marked by a large expansion of antibody-secreting cells against unique domains of the large T-Ag and no detectable B cells against commonT. These results suggest that B cell binding to specific oncoprotein epitopes impact their ability to promote anti-tumor immunity. The importance of the adaptive immune response against viral oncoproteins in MCC led us to explore in Chapter 4 whether the length of truncated large T-Ag in VP-MCC tumors associates with patient outcomes. The truncation site of large T-Ag is clonal within a given patient’s tumor and results in an oncoprotein length between 228 to 787 amino acids. We found that most patients present with tumors in which truncated large T-Ag is under 350 amino acids. Importantly, analysis of T-antigen DNA sequences from 40 MCC patients with associated clinical data revealed that patients with longer large T-Ag (above 350 amino acids) were significantly less likely to recur and survived longer. Together, these data suggest that increased large T-Ag length may promote MCC immunogenicity as evidenced by lower frequency at presentation and improved disease control after diagnosis. Chapter 5 describes a patient with a lymph node invaded by VP-MCC and Chronic Lymphocytic Leukemia (CLL), a B cell malignancy. Interestingly, this patient has remained MCC- and CLL-free for over 10 years following tumor surgical excision and local radiation. Single-cell RNA-sequencing revealed that malignant B cells from CLL may enhance MCC tumor growth via cytokine-enhanced cell proliferation. This observation highlights a potential mechanism by which aberrant B cells may promote MCC tumorigenesis and progression. Finally, Chapters 6 and 7 describe additional immune mechanisms of MCC control and propose mechanistic studies to probe the role of B cells in MCC anti-tumor immunity. In Chapter 6, we show that certain subsets of myeloid cells in MCC tumors associate with progressive disease. Chapter 7 speculates how the newly developed “SLAP” MCC mouse can be used to test MCC-specific B cell responses in tumor control. Specifically, we propose using classic immunological approaches such as genetic knockout, depletion of immune cell types, and adoptive transfer of engineered B cells specific for the T-antigens to address the role of B cells in MCC. Collectively, our work provides the first in-depth analysis of cancer-specific B cell responses in MCC. By integrating clinical outcomes, immunophenotyping, antibody specificity, and viral oncoprotein structure, these studies reveal that the quality and epitope specificity of B cell responses may have a key role in controlling anti-tumor immunity. We conclude by summarizing how our findings lay the groundwork for mechanistic studies in MCC mouse models that may form the basis for future therapeutic strategies leveraging B cells against solid cancers.
Development of an RSV vaccine for infants derived from anti-idiotypic antibodies
(2025-10-02) Scharffenberger, Samuel; McGuire, Andrew
Respiratory syncytial virus (RSV) is a common pathogen that causes lower respiratory tract infections leading to significant morbidity and mortality at the extremes of age. Although vaccines have recently been approved for use in adults over 60, a vaccine that can prevent infection in the infant population is lacking. Current strategies to protect infants from RSV rely on passive immunity, either by the transfer of neutralizing monoclonal antibodies (mAbs) or maternal vaccination and transplacental antibody transfer. A vaccine that can rapidly elicit protective neutralizing antibodies and establish immunological memory in the infant population would fulfill a critical unmet need. A subset of antibodies that arise from the chromosomally encoded VH3-21/VL1-40 gene pairing are unique in that they are structurally pre-configured to bind to and neutralize RSV without the need to undergo affinity maturation, making them ideal candidates to be elicited through vaccination. Here, I describe a vaccine immunogen derived from anti-idiotypic monoclonal antibodies (ai-mAbs) that can recognize RSV-neutralizing VH3-21/VL1-40 B cell receptors from diverse pools of PBMCs. Additionally, I describe the characterization of a novel knock-in mouse model that expresses a bona fide germline VH3-21/VL1-40 RSV-neutralizing B cell receptor, ADI-19425, and demonstrate the utility of this model to evaluate the immunogenicity of the ai-mAb in pre-clinical vaccine/challenge studies. I found that immunization with the ai-mAb vaccine elicited robust on-target B cell activation and a durable neutralizing serum response that functioned to reduce RSV lung titers upon viral challenge. Collectively, this data illustrates the efficacy of the ai-mAb as a novel infant RSV vaccine candidate and supports its further evaluation in preclinical studies.
Editing Hematopoietic Stem Cells for HIV Treatment
(2025-08-01) Castelli, Jack Michele Pietro; Adair, Jennifer E
Advances in genome editing technologies have opened the door to one-time treatments for previously incurable, chronic diseases. This has accelerated interest in delivering gene therapies and other biologic drugs with durable therapeutic effects. Hematopoietic stem and progenitor cells (HSPC) are a promising target for gene therapy as they provide a steady supply of cells throughout the body over long periods of time. In this dissertation, I explore the use of CRISPR/Cas systems in HSPC to achieve site-specific gene knock-in and to produce antibodies against HIV in vivo. The editing approach described here has been optimized to target the endogenous locus for antibody expression in primates and does not rely on viral vectors. Following transplantation in an animal model, edited HSPC are capable of self-renewal and development into mature hematopoietic cell types. Anti-HIV antibodies are detected in these animals, demonstrating productive knock-in of our gene encoding DNA template. These findings are a proof-of-concept for non-viral HSPC gene editing as a platform for durable biologics production in the treatment of chronic diseases.
Single Cell Resolution of Chronic Graft-Versus-Host Disease
(2024-10-16) Boiko, Julie Renee; Hill, Geoffrey
Chronic graft-versus-host disease (cGVHD) remains a frequent and morbid outcome of allogeneic hematopoietic cell transplantation (HCT). Simply put, in cGVHD, the donor-derived immune system attacks healthy recipient tissue. Preceding tissue damage mediated by chemoradiotherapy and alloreactive T cells compromise central and peripheral tolerance mechanisms, leading to aberrant donor T cell and germinal center B cell differentiation, culminating in pathogenic macrophage infiltration and differentiation in target tissue, with ensuant fibrosis. This process results in a heterogeneous clinical syndrome frequently requiring prolonged immunosuppressive therapy.The first portion of this dissertation addresses the clinical problem that immune pathway-targeted cGVHD agents have sustained efficacy in only a minority of patients. This suggests that different patients’ cGVHD may be dominated by different immune pathways, and it would thus be beneficial to specifically discern druggable, dysregulated immunity. We used single cell RNA sequencing-based approaches in our informative preclinical cGVHD models to “reverse engineer” temporal IL-17 and CSF-1 signatures in mouse blood that could be used to interrogate patients. We defined distinct, non-intuitive IL-17 and CSF-1 signatures in mouse blood monocytes that could be identified in monocytes from approximately 50% of patients at the time of cGVHD diagnosis. These signatures can now be evaluated in clinical studies to help delineate potential responder and non-responders to therapeutics targeting these respective cGVHD pathways. The second portion considers the relationships of hematopoietic and non-hematopoietic cells in mediating cGVHD skin pathology. In lichenoid cGVHD patient skin biopsies, we conducted high-resolution spatial transcriptomics to dissect the geographical dimensionality of immune networks. We demonstrated varying degrees of immune cell infiltration characterized by T cells and previously unappreciated CSF-1R+ mononuclear cell subsets. Proximity analyses demonstrated adjacent monocyte-derived CSF1RintLYZhi macrophages and CD8+ T cells in juxtaposition to the keratinocyte layer. Keratinocytes in highly immune cell-infiltrated cGVHD skin showed fundamental profile changes associated with antigen presentation and immune cell recruitment. Furthermore, hematopoietic and nonhematopoietic cell gene expression and proximity analyses suggested high levels of epithelial-mesenchymal transition within keratinocytes and macrophage-mesenchymal transition within skin fibroblasts. These high resolution data highlight CSF-1R+ macrophage subsets as the early sine qua non lesion in human cutaneous cGVHD, with profound and previously unappreciated downstream effects on local keratinocyte and fibroblast differentiation that represent novel therapeutic targets.
Comprehensive EBV-specific T cell profiling and multi-omics analysis of nasopharyngeal carcinoma CD8 T cells reveals novel cancer-associated phenotypes
(2024-09-09) Kumar, Nandita; Newell, Evan W
Nasopharyngeal carcinoma (NPC) is a high mortality, Epstein-Barr virus (EBV)-driven cancer with limited treatment options. Although EBV is highly antigenic and detectable in nearly all NPC tumor cells, it remains unclear why NPC tumors evade immune targeting despite high levels of immune infiltration. Here, we investigate the phenotypes of peripheral EBV-specific T cells with the purpose of identifying clinically relevant, cancer-associated EBV-specific T cell phenotypes. We developed a 34-marker mass cytometry and multiplexed MHC-I tetramer panel to detect and phenotype CD8 T cells of up to 55 antigen-specificities, including EBV and other known viral and non-viral epitopes. We used this approach to profile peripheral blood mononuclear cells (PBMCs) from treatment-naÃ¯ve NPC patients (n=51). Compared to other antigen-specific T cells detected in NPC patients and EBV-specific T cells in healthy individuals, EBV-specific T cells in NPC patients expressed high levels of activation markers (CD38, HLA-DR), co-inhibitory markers (CD39, TIGIT, PD-1), and migratory markers (ITB7, CD103). Using unsupervised clustering, we found that the frequency of EBV-specific T cells in a phenotypic cluster characterized by high CD103, CD39, CD71, HLA-DR, and CD38 expression positively correlated with plasma EBV-DNA titers and gross tumor volume. Remarkably, we observed that regardless of antigen specificity, NPC patients had a higher frequency of total CD8 T cells with this activated/exhausted cluster phenotype, and CD8 frequencies of this cluster were also positively correlated with plasma EBV-DNA titers stage (TNM), and EBV early antigen antibody titers. To investigate the gene expression profiles and TCR clonal diversity of this cancer-associated phenotype, we performed single-cell multi-omics analysis on peripheral CD8 T cells from NPC patients (n=17). We identified that cell subsets within this phenotype had distinct gene expression patterns indicative of potential associations with the tumor and we also assessed the clonality of these subpopulations. Finally, we assess connections between the NPC tumor microenvironment using multiplexed immunohistochemistry (mIHC) and bulk tumor gene expression and TCR sequencing. We found that peripheral activated/exhausted T cell phenotypes are correlated with mIHC tumor PD-1 scores, neutrophil levels identified using gene expression data, and have high TCR clonal sharing with NPC tumors. Overall, this research identified unique cancer-associated peripheral EBV-specific T cell phenotypes in the context of nasopharyngeal carcinoma that are positively correlated with advanced disease. Frequencies of peripheral T cells with this phenotype could also be useful as a blood-based biomarker for NPC.
Circulating disease-specific CD8 T cells in Merkel cell carcinoma and COVID-19: Prognostic significance and improved approaches for their detection
(2024-09-09) Jani, Saumya; Nghiem, Paul
Immune checkpoint inhibition (ICI) that targets the programmed death-1 (PD-1) pathway has made great strides in improving cancer patient outcomes and is the most successful immunotherapy to date, but only a subset of treated patients benefits. Predicting which patients are likely to respond to ICI and understanding the underlying mechanisms are of critical importance to prioritize patients for alternate or synergistic therapies and to avoid toxicities in patients who would not benefit. Because PD-1 pathway blockade targets T cells, it is feasible that patient-specific T cell characteristics could serve as predictive biomarkers of response. However, few studies can directly assess the numerically lower, but more functionally relevant population of cancer-specific T cells. This is because most cancers arise due to unique patient-specific mutations, which makes it extremely difficult to identify infrequent cancer-specific T cells. However, 80% of Merkel cell carcinoma (MCC) cases are virally-driven, persistently express conserved Merkel cell polyomavirus (MCPyV) oncoproteins, and have extraordinarily few amino acid-coding tumor mutations. This allows us to have a common set of target antigens that can be used to study MCPyV-specific T cells and gain insights that can be extended to other cancers. In chapter 1, we review limitations encountered in studying tumor-specific CD8 T cells, propose virus-driven MCC as a model tumor, summarize current knowledge of mechanisms of response and resistance to ICI. In chapter 2, we find that baseline abundance of MCPyV-specific CD8 T cells in blood can predict ICI response in two different clinical trials. However, this does not seem to be the case for intratumoral tumor-specific CD8 T cells. This difference may be attributable to our observation that tumor-specific CD8 T cells in the blood are less dysfunctional than their intratumoral counterparts. In chapter 3, we confirm our finding that baseline frequency of MCPyV-specific CD8 T cells associates with response to ICI in an independent patient cohort. And we additionally find that the frequency of CD39+CLA+ CD8 T cells can predict response to ICI. However, current methods to quantify cancer-specific T cells require freshly acquired (non-archival) tissue, labile expensive custom reagents (peptide-HLA tetramers), and significant technical expertise, and are therefore not well suited for routine clinical care. Thus, in chapters 4 and 5, we explore methods to identify cancer-specific CD8 T cells more easily. In chapter 4, we characterize MCPyV-specific CD8 T cells in blood and identify a set of 98 genes that are enriched in these cells compared to other T cells. We find that this gene set can accurately identify tumor-specific CD8 T cells in both MCC and various mutationally-driven cancers. In chapter 5, we explore the feasibility of generating a library of T cell receptors (TCR) specific to MCPyV oncoproteins and using this library in combination with existing TCR similarity algorithms to identify cancer-specific CD8 T cells. Given that significantly more TCR sequences were publicly available from patients with COVID infection than MCC, we tested the ability of TCR similarity algorithms to identify SARS-CoV-2-specific CD8 T cells in COVID vaccine recipients in chapters 6 and 7. In chapter 6, we compared the phenotype and repertoire of SARS-CoV-2 specific CD8 T cells from the breastmilk and blood of lactating individuals who received a spike-specific mRNA vaccination post-delivery. We used a TCR similarity algorithm, tcrdist3, to identify novel potential SARS-CoV-2-specific CD8 T cells. In chapter 7, we used tcrdist3 to track the frequency of SARS-CoV-2-specific CD8 T cells in longitudinal blood samples from patients with a history of COVID infection, who subsequently received SARS-CoV-2 vaccines. We observed large expansions in response to vaccination and identified novel, functionally-proven SARS-CoV-2-specific CD8 T cells. Being able to accurately track and identify novel COVID-specific CD8 T cells with TCR similarity metrics in these studies increases our confidence in using similar methods for identifying cancer-specific CD8 T cells and justify efforts to create libraries of TCRs that target common oncogenic viruses and mutations. Finally, in chapter 8, we investigate a case of secondary resistance to ICI, despite an abundance of tumor-specific CD8 T cells. We demonstrate that this patient’s tumor lacked HLA-I expression, preventing anti-tumor CD8 T cells from recognizing the tumor cells. Treatment with an intralesional stimulator of interferon genes (STING) agonist partially restored HLA-I expression via induction of an inflammatory tumor microenvironment, allowing to patient to experience an abscopal, partial response. The studies detailed in chapters 2, 3, and 8 have made key advances in identifying mechanisms of response and resistance to PD-(L)1 pathway blockade in Merkel cell carcinoma. Additionally, the studies detailed in chapters 4, 5, 6, and 7 have made important advances in translating the identified mechanisms to more common, mutationally-driven cancers via gene expression profiling and bulk T cell receptor sequencing. We believe that use of these methods will translate to improving outcomes for all patients with cancer.
HIV evolution during ART failures revealed by using long-read sequencing and bioinformatics tools
(2024-02-12) WANG, SHIYI; Torbett, Bruce E
HIV resistance often leads to antiretroviral therapy (ART) failures, involving two crucial mutation categories: drug-resistance mutations (DRMs) and compensatory mutations. DRMs reside in HIV enzyme active sites (protease, reverse transcriptase, and integrase), hindering drug binding, while compensatory mutations restore enzyme stability and function, compensating for DRMs. With the increase of drug potency, more compensatory mutations are involved in compensating for one DRM, forming complex mutational patterns. However, the interplay between DRMs and compensatory mutations remains elusive. In this thesis work, I combined a long-read sequencing approach and bioinformatic tools to unveil the complex mutational patterns driving HIV resistance development. Long-read sequencing yielded 4.5kb gag-pol sequences from individual HIV genomes within clinical serum samples, preserving co-varying mutations critical for pattern identification. Mutational patterns were inferred based on pairwise correlations detected in the sequencing data and quantified using a custom bioinformatic tool. I utilized Hamming-distance-based phylogenetic analysis (HDBPA) and paired post-ART HIVs with their pre-ART most recent common ancestors (MRCAs) based on sequence similarity. In this way, I divided mutations in mutational patterns into different categories (mutations inherited from pre-ART MRCA, and mutations acquired during ART) and revealed the order of mutation development. I demonstrated the utility of this approach by studying the HIV evolution in two PWHs facing ART failures. The findings revealed different mutational patterns selected and enriched during ART and inferred evolutionary pathways taken by HIVs during resistance development. Alongside substitution mutation involved in HIV evolution, I participated in a collaborative study, aiming to measure linkage disequilibrium between recombination events and SNVs. The findings revealed novel correlations between p6Gag insertions and Gag cleavage site mutations in drug-resistant HIV genomes. Taken together, my work deciphered mutational patterns and recombination events driving HIV evolution during ART using long-read sequencing and custom bioinformatics tools. The findings of this study indicated interactions both within HIV proteins and among proteins, which could guide anti-viral drug design. The methods introduced could be used for identifying complex mutational patterns required for resistance development and revealing the order of mutation development in HIV as well as other fast-evolving viruses and bacteria.
Immunomodulation of Influenza A Virus Infection by Fibroblast-Derived Versican
(2023-09-27) Brune, Jourdan E.; Frevert, Charles W
Viral and bacterial lung infections place a significant burden on public health. Versican, an extracellular matrix (ECM) chondroitin sulfate proteoglycan, is a critical coordinator of the innate immune response. Versican’s potential as an immunomodulatory molecule makes it a promising therapeutic target for controlling the host's immune response to lung infection. However, versican’s contribution to lung inflammation, injury, and immune cell activity during influenza A virus (IAV) infection represents a critical knowledge gap. In the following work, we characterized the spatiotemporal accumulation of versican in the lungs of wild-type (C57BL/6J, WT) mice in response to IAV and found that versican accumulation correlates with mouse acute lung injury scores and pulmonary inflammatory cell infiltration. We found that versican expression is increased in both mononuclear phagocytic cells and stromal cells in the lungs in response to IAV in WT mice and discovered that the expression of versican in stromal cells is partially mediated by type I interferon receptor signaling. These data from WT mice raised intriguing questions about the role of versican in the early host response to IAV. To rigorously investigate the potential of versican to modulate lung inflammation, we developed a complete protocol combining spectral flow cytometry with in vivo compartmental analysis, for the precise localization of multiple immune cell populations participating in the pulmonary host response to IAV. The lungs have high autofluorescence (AF) compared to other tissues, which complicates the analysis of pulmonary leukocytes by flow cytometry. In the following work, we described changes in the AF characteristics of control and IAV-infected lungs during the transition from innate to adaptive immunity. We meticulously assembled a broad panel of 20 antibody-fluorophore conjugates compatible with these AF characteristics and spectral flow cytometry. Additionally, our studies validated that incorporating multiple heterogeneous AF signatures from the lungs improves the resolution and identification of fluorescence signals, particularly when alveolar macrophages are a component of the immune response. The methodology presented includes a robust gating strategy for the identification of B cells, T cells (cytotoxic T, T helper, and T cells), NK cells, macrophages (alveolar, recruited and monocyte-macrophages), monocytes (Ly6Clo and Ly6Chi), dendritic cells (CD103+ and CD11b+), neutrophils, and eosinophils in combination with dual in vivo CD45 labeling to facilitate identification of these immune cell populations in four pulmonary compartments. Finally, our studies implemented informed gating and dimensionality reduction algorithms to visualize the recruitment and migration of leukocytes from the vasculature, across the lung interstitium, and into the alveolar airways at 9 days post-infection with IAV. Our robust spectral flow cytometry panel and in vivo intravascular CD45 labeling technique were applied to studies investigating the role of versican in lung inflammation, injury, and immune cell activity during IAV infection. To address our central hypothesis that fibroblast-derived versican is pro-inflammatory and enhances the innate immune response to IAV infection, we generated a tamoxifen-inducible mouse strain that is deficient in fibroblast-derived versican (B6. Col1a2-CreERT+/-/Vcantm1.1Cwf, Col1a2/Vcan-/-). We reported that fibroblast-derived versican plays a critical role in neutrophil, monocyte, mono-macrophage, dendritic cell, and eosinophil migration into the lungs and airways early in IAV infection. Intriguingly, fibroblast-derived versican deficiency had the most substantial impact on neutrophil emigration into the lungs. We found that Col1a2/Vcan-/- mouse lung fibroblasts (mLFs) have reduced cell-associated hyaluronan (HA) and that neutrophils were less adhesive to the versican/HA ECM of Col1a2/Vcan-/- mLFs compared to WT controls. These findings suggest that fibroblast-derived versican and associated HA are necessary for the adhesion of neutrophils to the lung fibroblasts as they transit into the lung interstitium and airways from the pulmonary vasculature. Our findings demonstrate that fibroblast-derived versican is a key integrator of the early host immune response to IAV.
A genome-scale gain-of-function screening platform nominates genetic modulators for CAR T cell therapy enhancement
(2023-08-14) Curtis, Benjamin C; Jensen, Michael C; Oda, Shannon K
Chimeric antigen receptor (CAR) T cell therapy has revolutionized cancer care through genetic reprogramming, and next-generation cell engineering aims to encode increasingly complex genetic programs to further modulate T cell behavior, and ultimately potentiate anti-tumor activity. Effective selection of coding sequences for subsequent clinical development requires one or multiple prioritization regimens, but scalable gene nomination platforms for T cell therapy are currently lacking. In this work, I describe the creation of a novel genome-scale, gain-of-function screening platform, powered by CRISPR activation (CRISPRa) technology, and designed for pooled screening in primary human CAR T cells. To achieve this, established CRISPRa designs were first optimized for fidelity, potency, and durability in T cell lines. Second, restrictive primary T cell requirements were overcome through cell manufacturing featuring transposon-based gene delivery coupled to CAR-restricted expansion (EP-TICLE). To overcome abbreviated CRISPRa activity, CRISPRa expression was reinforced through construct optimization that included dual divergent hybrid promoters (EF1α-HTLV)2 and intronization of the dCas9 coding cassette. Finally, to prevent library distortion, gRNAs were delivered in trans via lentivirus immediately prior to landscape initiation. This manufacturing schema was trialed at genome-scale and nominated modulators of CAR T cell homeostatic persistence (MYC, STAT5A, STAT5B) and cytokine-free survival (CSF2RB, TNFRSF1A, TNFRSF1B). Follow-up studies suggested that this platform would tolerate a druggable-ON switch (ERT2) to regulate landscape initiation. In sum, this work demonstrates the utility of a novel cell manufacturing and gain-of-function screening system for next-generation therapy development. Insights into achieving sustained dCas9 expression may also offer a blueprint for orthogonal CRISPR-based screening systems or for in vivo CRISPR applications.
Developing Targeting Techniques for the Advancement of In Vivo Gene Therapies
(2023-08-14) Berckmueller, Kurt; Kiem, Hans-Peter
Hematopoietic Stem Cell (HSC) gene therapy is a promising route to curing patients with a variety of hematologic diseases and disorders. HSC gene therapy is currently performed ex vivo and requires rare cleanroom infrastructure, which is a significant obstacle to patients in resource impoverished areas. Application of gene therapy agents directly in the patient (in vivo) would overcome this bottleneck. We have previously identified the CD34+CD90+ subset to be exclusively responsible for short- and long-term engraftment. However, purification and enrichment of this subset is laborious and expensive. HSC-specific delivery agents for the direct modification of rare HSCs are currently lacking. Here, we developed novel targeted viral vectors to specifically transduce CD90-expressing HSCs. Anti-CD90 single chain variable fragments (scFvs) were engineered onto measles- and VSVG-pseudotyped lentiviral vectors that were knocked out for native targeting. We further developed a custom hydrodynamic titration methodology to assess the loading of surface-engineered capsids, measure antigen recognition of the scFv, and predict the performance on cells. Engineered vectors formed with minimal impairment in the functional titer maintained their ability to fuse with the target cells and showed highly specific recognition of CD90 on cells ex vivo. Most importantly, targeted vectors selectively transduced human HSCs with secondary colony-forming potential. However, agents recognizing only a single marker on target cells are often insufficient due to epitope sharing in between on- and off-target tissues. The Baker Lab previously designed a system of protein switches known as Co-LOCKR, which enable the specific recognition of two antigens simultaneously. Co-LOCKR targeted CAR T cells highly specifically killed tumor cells in vitro with virtually no off-target effects. Therefore, we further designed and evaluated Co-LOCKR-targeted viral vectors for ex vivo as well as in vivo applicability and comprehensively evaluated the on-target specificity in a murine tumor model. Together this work lays a foundation and provides a robust toolset for the exploration of in vivo gene therapies.
Mitochondrial Redox Adaptations Enable Alternative Aspartate Synthesis in SDH-Deficient Cells
(2023-08-14) Hart, Madeleine Louisa; Sullivan, Lucas B
The oxidative tricarboxylic acid (TCA) cycle is a central mitochondrial pathway integrating catabolic conversions of NAD+ to NADH and anabolic production of aspartate, a key amino acid for cell proliferation. Several TCA cycle components are implicated in tumorigenesis, including loss of function mutations in subunits of succinate dehydrogenase (SDH), also known as complex II of the electron transport chain (ETC). Mechanistic understanding of how proliferating cells tolerate the metabolic defects of SDH loss is still lacking. Here, we identify that SDH supports cell proliferation through aspartate synthesis but, unlike other ETC impairments, SDH inhibition is not ameliorated by electron acceptor supplementation. Interestingly, we find aspartate production and cell proliferation are restored to SDH-impaired cells by concomitant inhibition of ETC complex I. We determine that the benefits of complex I inhibition in this context depend on decreasing mitochondrial NAD+/NADH, which drives SDH-independent aspartate production. We also find that genetic loss or restoration of SDH selects for cells with concordant complex I activity, establishing distinct modalities of mitochondrial metabolism for maintaining aspartate synthesis. Collectively, these data identify a metabolically beneficial mechanism for complex I loss in proliferating cells and reveal that compartmentalized redox changes can impact cellular fitness.
Analysis of lifespan extending compounds using growth kinetics and replicative lifespan in Saccharomyces cerevisiae
(2023-08-14) Kiflezghi, Michael G.; Kaeberlein, Matt
Advances in modern medicine have facilitated stunning rises in life expectancy over the last two centuries. This longer life, however, is concomitant with a gradual decline in health and the onset of multiple age-associated pathologies including heart disease, neurodegeneration and cancer. Risk of developing and dying from these age-associated diseases increases dramatically with age suggesting a common underlying cause. Targeting the aging process itself with clinical intervention may allow for amelioration of the onset and progression of multiple age-associated pathologies simultaneously. The mechanistic Target of Rapamycin (mTOR) signaling pathway has emerged as an important clinical target in this regard with its dysregulation being observed in multiple pathologies including many cancers, autoimmune disorders, heart failure, neurodegeneration and type 2 diabetes. Importantly, genetic or pharmacological inhibition of mTOR signaling results in increased life-span across evolutionary distant organisms suggesting a conserved role for this signaling pathway in longevity regulation. A novel yeast-based system to identify putative mTOR inhibitors is described. Utilizing differential growth kinetics of WT and mutant strains sensitized to mTOR perturbations, this system successfully discriminates between allosteric (i.e. rapamycin) and ATP competitive mTOR inhibitors. A number of nutraceutical compounds were screened and of these, caffeine was confirmed to be an mTOR inhibitor (Chapter two). Next a screen of natural products and natural product mixtures were screened for effects on growth rate, mTOR-mediated growth inhibition, and replicative lifespan (RLS). No mTOR inhibitors were identified but two treatments, berberine and green tea extract significantly reduced RLS. Pterocarpus marsupium extract (PME), a plant extract with a long history of use in Ayurvedic medicine, extended cellular lifespan (Chapter three). Constituents of this extract include several compounds with reported health span and life span extending properties: epicatechin, quercetin, berberine, and pterostilbene. Of these PME constituents, quercetin and epicatechin had no effect on cellular life span, while berberine decreased cellular life span. We describe the lifespan and outgrowth kinetics of pterostilbene-treated cells and report on dose-dependent effects on longevity, potent cytotoxicity, and a possible role for mitochondrial function in mediating these phenotypes (Chapter Four).
Designing a Next-generation Epstein-Barr Virus Vaccine
(2023-01-21) Malhi, Harman; Mcguire, Andrew T
Epstein-Barr virus (EBV) is a cancer-associated pathogen responsible for 165,000 deaths per year. EBV is also the etiological agent of infectious mononucleosis and is associated with multiple sclerosis and rheumatoid arthritis. Thus, an EBV vaccine could alleviate significant morbidity and mortality. EBV is orally transmitted and has tropism for both epithelial cells and B cells. Therefore, a vaccine would need to prevent infection of both cell types in the oral cavity. I describe how the passive transfer of AMMO1, a dual-tropic neutralizing monoclonal antibody targeting the viral gH/gL glycoprotein complex, prevents experimental EBV infection in humanized mice and rhesus macaques, suggesting that gH/gL is an attractive vaccine candidate. Spurred by these findings, we produced and evaluated the immunogenicity of several nanoparticle immunogens displaying gH/gL with distinct valencies and geometries. After one or two immunizations, all nanoparticles elicited superior binding and neutralizing titers relative to monomeric gH/gL. Antibodies elicited by a computationally designed self-assembling nanoparticle that displays 60 copies of the gH/gL protein conferred protection against a lethal dose of EBV in a humanized mouse challenge model, whereas antibodies elicited by monomeric gH/gL did not. Taken together, these data motivate further development of nanoparticle vaccine candidates for EBV which target the gH/gL glycoprotein.
The role of mTOR in mitochondrial disease
(2022-09-23) Bornstein, Rebecca Lois; Morgan, Philip G
Genetic mitochondrial diseases (GMD) impact over 1:4,000 live births and are a common underlying cause in both inherited metabolic disorders and inherited neurological diseases. No effective treatments exist for GMDs, and the mechanisms linking the primary genetic defects to pathology are unknown. Inhibition of the mechanistic target of rapamycin (mTOR), a master intracellular nutrient sensing-signaling kinase complex, with rapamycin significantly attenuates disease in multiple GMD models. In particular, rapamycin treatment attenuates neurodegeneration, metabolic dysfunction, and significantly extends survival of the Ndufs4(KO) mouse model of Leigh syndrome (LS), the most common pediatric presentation of GMD. Factors mediating the benefits of mTOR inhibition have previously been unknown. Studies targeting discrete pathways downstream of mTOR Ndufs4(KO) mouse have not recapitulated the effects of rapamycin treatment. The role of mTOR in metabolism is highly pleiotropic, and mTOR inhibition produces significant metabolic changes in the Ndufs4(KO) mouse that could mediate the benefits of rapamycin. Conversely, upstream signaling and cell-specific mTOR activity may define the role of mTOR in disease. In this work we reveal a novel model for the role of mTOR signaling in LS, representing a potential avenue for clinical therapy development, and explore the relationship between mTOR, metabolism, and mitochondrial disease. First, we determined that PI3Kγ-mTOR signaling participates in leukocyte proliferation, neuroinflammation, and disease in the Ndufs4(KO) mouse, and that pharmacological depletion of leukocytes robustly prevents disease in the Ndufs4(KO) model. Thus, we believe LS has an mTOR-associated immunologic origin, which is amenable to pharmacological targeting. Second, we show that these immunologic interventions rescue both neurodegeneration and major metabolic sequelae of disease, indicating that mTOR-mediated immune processes disrupt metabolism in the Ndufs4(KO) mouse. Finally, we characterize the metabolic effects of mTOR inhibition in control and Ndufs4(KO) animals, finding that mTOR inhibition decreases glycolytic flux and significantly alters systemic glucose regulation. Given the known role of glucose metabolism in leukocyte function, these changes are expected to have secondary effects on immune proliferation. Taken together, our findings support a mechanistic role for the immune system in driving disease pathogenesis in LS, but also reveal metabolic changes resulting from mTOR inhibition which could play a role in attenuating disease either through direct effects or by contributing impaired leukocyte proliferation. Future research will further probe the causal relationship between mTOR, glucose metabolism, and immune activity in GMDs.
Exploring the role of scaffold proteins in controlling helical cell shape in Helicobacter pylori
(2022-07-14) Sichel, Sophie Rae; Salama, Nina R
Helicobacter pylori infects the stomachs of 50% of the world’s population and can cause gastric cancer and ulcers. There are many factors that contribute to H. pylori’s ability to colonize the stomach including the helical shape of H. pylori cells. The helical shape of H. pylori cells is required for robust infection of the stomach and promotes pathology, however, how the helical shape of H. pylori cells is patterned and maintained is unresolved. Additionally, how the helical cell shape of H. pylori enhances colonization and increases disease severity is not completely understood. Previous work identified helical- cell-shape-promoting protein complexes containing the peptidoglycan-hydrolase (Csd1), a peptidoglycan precursor synthesis enzyme (MurF), a non-enzymatic homologue of Csd1 (Csd2), non-enzymatic transmembrane proteins (Csd5 and Csd7), and a cytoplasmic bactofilin (CcmA). Bactofilins are highly conserved, spontaneously polymerizing cytoskeletal proteins in bacteria. During my investigations, I sought to understand specifically how two of the scaffold proteins, CcmA and Csd5, were involved in generating the helical shape of H. pylori cells.Using a combination of CcmA deletion analysis, in vitro polymerization, and in vivo co- immunoprecipitation experiments we identified that the bactofilin domain and N-terminal region of CcmA are required for helical cell shape and the bactofilin domain of CcmA is sufficient for polymerization and interactions with transmembrane proteins Csd5 and Csd7. We also found that CcmA’s N-terminal region inhibits interaction with Csd7. Deletion of the N-terminal region of CcmA increases CcmA-Csd7 interactions and destabilizes the peptidoglycan-hydrolase Csd1. Using super-resolution microscopy, we found that Csd5 recruits CcmA to the cell envelope and promotes CcmA enrichment at the major helical axis. Additionally, we identified that in WT H. pylori and surprisingly, in ∆csd5 cells, tetrapeptides in the cell wall are organized in a helical band that wraps around the cell. The SH3 domain of Csd5 binds to tetrapeptides to direct Csd5 and CcmA to the region of the cell wall with increased levels of tetrapeptides. We hypothesize that together Csd5 and CcmA organize several other cell-shape-determining proteins and peptidoglycan synthesis machinery to coordinate increased levels of cell wall modification and synthesis where tetrapeptides are enriched to promote the curvature required to build a helical cell.
Probing the Role of SORL1 and Endolysosomal Network Dysfunction in Alzheimer’s Disease
(2022-01-26) Knupp, Allison; Young, Jessica E
Alzheimer’s disease (AD) is a progressive neurodegenerative disorder, and the most common cause of dementia among adults. There is currently no treatment that halts disease progression. There is ample pathological and biological evidence for endolysosomal network (ELN) dysfunction in AD, and emerging genetic studies repeatedly implicate ELN genes such as SORL1 as associated with increased AD risk. The SORL1 gene encodes the protein SORLA, a sorting receptor involved in retromer-related endosomal traffic. Many SORL1 genetic variants increase AD risk, and rare loss-of-function truncation mutations have been found to be causal of AD. To model the causal loss-of-function mutations, we used CRISPR/Cas9 technology to deplete SORL1 in human induced pluripotent stem cells (hiPSCs) to test the hypothesis that loss of SORL1 (SORL1 KO) contributes to AD pathogenesis by leading to dysfunction in ELN trafficking. We additionally used CRISPR/Cas9 to insert AD-risk variants in the VPS10 domain of SORL1 (SORL1Var) in hiPSCs to test the hypothesis that these VPS10 variants result in loss of SORLA function and lead to ELN dysfunction. We report that loss of SORL1 as well as SORL1 VPS10 variants in hiPSC-derived neurons leads to early endosome enlargement, a cellular phenotype that is indicative of ‘traffic jams’ and is now considered a hallmark cytopathology AD. We further report trafficking defects in the recycling and degradative pathways of the ELN in SORL1 KO neurons. Finally, we determine that retromer stabilizing small molecules reduce early endosome enlargement in both SORL1 KO and SORL1Var neurons. Collectively, and together with other recent observations, these findings suggest that SORL1 is a key and broad regulator of ELN trafficking in neurons, a conclusion that has both pathogenic and therapeutic implications. Moreover, demonstrating a partial rescue of cellular phenotypes in SORL1 deficient neurons will contribute to the development of new and precision treatments for AD.
Characterizing the WRN DNA Helicase in Prostate Cancer and Implications for Microsatellite Unstable Metastatic Prostate Cancers
(2021-10-29) Kohlbrenner, Emily; Nelson, Peter S
Prostate cancer is the most common non-skin malignancy in men worldwide and the second most common cause of cancer mortality in men. Metastatic prostate cancer (mPC) is highly heterogenous and enriched for aberrations in genes involved in DNA repair, the loss of which generates further genetic alterations and genomic instability that ultimately promotes tumorigenesis. The DNA Helicase-Exonuclease RECQL2 protein, commonly referred to as WRN, plays an integral role in DNA repair by regulating the dynamics of the replication fork. WRN is lost along the 8p chromosomal arm in 10% of prostate cancers; however, the role of WRN in mPC remains unclear. WRN has also been established as a promising target for synthetic lethality in mismatch repair deficient (MMRd) cancer cells with microsatellite instability (MSI), an aggressive subtype of metastatic disease that promotes oncogenesis via genome hypermutability. We aimed to identify the prognostic value of WRN-specific copy loss in mPC patient tumors as well as investigate the sensitivity of MSI-mPC cell models to engineered WRN knockdown. We first showed that adverse outcomes are associated with WRN copy number status in mPC, and connect mutual exclusivity between loss of WRN and mismatch repair deficient tumors using large scale clinical datasets. Then, we tested the sensitivity to WRN inhibitor NSC 19630 in the LUCaP PDX xenograft lines, and found marked sensitivity in tumor lines with DNA Repair Deficiency (DRD). Further, we demonstrated that MSI prostate cancer cells are indeed sensitive to WRN loss over time. Finally, using quantified confocal imaging, we showed that tertiary DNA secondary structures at GC rich regions, known as G-Quadruplexes, are associated with replicative stress in MMRd-MSI cells and are themselves a promising target for chemotherapeutics. Together, this work expands the knowledge of DRD heterogeneity in mPC and provides novel insight into the molecular mechanisms of WRN sensitivity in MSI cells.

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