Timely and spatially controlled endosomal trafficking and signaling is important for cell proliferation, directed cell migration, and cell invasion, which are frequently misregulated in cancer cells. Cdc42-interacting protein-4 (CIP4) family adaptors promote endocytosis by inducing membrane invaginations via their Fer/CIP4 Homology-Bin/Amphyphysin/Rvs (F-BAR) domains, coupled with activation of the actin assembly machinery to promote vesicle fusion or motility. My thesis focuses on defining the roles of CIP4, and a related protein, Transducer of Cdc42-mediated actin assembly-1 (Toca-1), in regulating Epidermal Growth Factor Receptor (EGFR) endocytosis, EGFR trafficking, cancer cell motility, and invasion. In Chapter 2, I show that CIP4 and Toca-1 localize to early endosomes and promote EGFR trafficking from early endosomes to lysosomes for degradation, thus limiting extracellular signal-regulated kinase signaling from early endosomes and proliferation of A431 carcinoma cells. In Chapter 3, I provide novel evidence that depletion of Toca-1 results in defects in actin-based lamellipodial protrusions that are required for cell motility. The cause of these defects may relate to altered recruitment of the Abelson-interactor-1 and its effector Wiskott-Aldrich syndrome protein family verprolin-homologous protein to the lamellipodia in A431 cells depleted of Toca-1. Results in Chapter 4 identify CIP4 as a negative regulator of breast cancer invasiveness downstream of Src protein-tyrosine kinase. Src is a potent inducer of extracellular matrix (ECM)-degrading structures called invadopodia that function in tissue invasion by cancer cells. I found that CIP4 is a Src substrate that localizes to Src-induced invadopodia in MDA-MB-231 breast cancer cells. Interestingly, depletion of CIP4 results in enhanced ECM degradation, invadopodia formation, and invasiveness compared to control cells. Thus, CIP4 and Toca-1 are multifaceted regulators of EGFR downregulation, EGF-induced cell motility, and Src-induced cell invasion. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2010-08-25 11:44:46.934

<p>  </p> <p>Non-small cell lung cancer (NSCLC), the most common type of lung cancer, is essentially the leading cause of cancer related deaths in United States. Only 24% of NSCLC patients survive 5-years post diagnosis, largely attributed to the lack of efficient treatment strategies at the metastatic stage. Thus, understanding the biological mechanisms that promote NSCLC metastasis is critical for the development of effective cancer-specific therapeutic agents. The development of cancer metastasis is greatly driven and influenced by intercellular communication. Key mediators of cell-to-cell communication are extracellular vesicles (EVs). For the past years, the study of EVs released by tumor cells have gained attention, given their impact in modulating the tumor immunity, supporting tumorigenesis, and contributing to the development of metastasis. However, the mechanisms though which tumor EVs contribute to tumor development are still understudied. In this study, we isolated and characterized small EVs, also referred as exosomes, from NSCLC cell lines (H358, Calu6, H460, SKMES-1) and investigated their release, uptake, and impact in non-tumorigenic lung epithelial cells recipient cells (BEAS-2B and HBEC). Our results demonstrated that EVs from NSCLC can induce migration and invasion of non-tumorigenic epithelial cells, and impair epithelial barrier permeability, suggesting their role in supporting tumorigenesis and metastasis. Furthermore, we assessed the immunomodulatory effects of NSCLC EVs on anti-tumor immune cells, particularly T cells. Our findings revealed a suppressive effect of EVs derived from mutant KRASG12C NSCLC (H358) on T-cell proliferation, suggesting their contribution to immune evasion mechanisms in mutant KRAS tumors. To dissect the underlying mechanisms, we employed a dual approach utilizing genetic manipulation (shRNA knockdown) and a small molecule inhibitor (ARS-1620) targeting the oncogenic KRASG12C. Our data demonstrated that targeting KRASG12C impaired the EV-driven cancer phenotypes, highlighting the pivotal role of KRAS oncogenic signaling in tumorigenesis and immune suppression mediated by EVs. Overall, our study sheds light on the crucial involvement of tumor derived EVs in NSCLC progression, both in terms of promoting cellular migration and invasion, as well as dampening anti-tumor immune responses. By elucidating the mechanisms underlying EV-driven tumorigenesis and highlighting the therapeutic potential of targeting KRAS signaling, our findings pave the way for the development of novel and effective therapeutic agents for NSCLC.</p>

<p>Triple Negative Breast Cancer (TNBC) is the most aggressive type of breast cancer. TNBC patients are resistant to virtually all target therapies and suffer a higher post-chemotherapy relapse with a worse overall survival compared with other types of breast cancers. Therefore, the development of an effective therapy is urgently needed. PKM2 plays a prominent role in mediating<b> </b>tumor glycolysis and PKM2 is often overexpressed in human cancers. However, whether PKM2 mediated glycolysis is necessary for cancer cell growth is questionable. Here, I have found that inhibition of PKM2 does not affect TNBC cell growth due to a metabolic switch from glycolysis to fatty acid oxidation (FAO). We show that PKM2 directly interacts with EZH2 to coordinately mediate epigenetic silencing of SLC16A9, transporter of a key player in FAO, Carnitine. Inhibition of either PKM2 or EZH2 increases levels of SLC16A9 and intracellular Carnitine to promote FAO and thereby sustains cancer cell growth. Direct inhibition of EZH2 using a clinically tested EZH2 inhibitor, GSK126, is able to elicit a previously unidentified vulnerability to a clinically tested FAO inhibitor, Etomoxir. As a result, combined GSK126-Etomoxir treatment synergistically abolishes TNBC xenograft tumor growth in vivo. Together, this study uncovers PKM2-EZH2 mediated metabolic reprogramming that leads to a new drug combination therapy by dual targeting of EZH2 and FAO for effective treatment of TNBC.<b> </b></p> <p> </p> <p>Furthermore, Dendritic Cell (DC) vaccination has shown promise in treating cancer patients. However, the <i>in vitro</i> generation of a fully functional DC remains a big challenge in this field. EZH2 inhibition has shown to be able to create an immunologically ‘hot’ tumors. Nonetheless, the role of EZH2 in regulation of DC function is still unclear. I found that the expression levels of EZH2 and its functional maker, H3K27Me3, are enhanced following maturation from immature DC (iDC) into two functional DCs, α-type 1-polarized-DC (αDC) and gold standard DC (sDC). Moreover, inhibition of EZH2 by GSK126 treatment elicits a dependency of sDC on FAO. These results suggest that EZH2 plays a role in maturation of DC through metabolic reprogramming, which may also provide new DC based immunotherapy of TNBC. </p>

<p>Epigenetic mechanisms, including DNA methylation, play an important role in regulation of stem cell fate and tumorigenesis. The Ten-Eleven-Translocation 2 (TET2) is a core enzyme for DNA demethylation by catalyzing the conversion of 5-methylcytosine (5mC) to 5-hydromethylcytosine (5hmC). It has been shown that TET2 is the main regulator of hematopoietic stem cell homeostasis and loss of TET2 is highly associated with hematopoietic malignancies. Our previous work has also shown that loss of TET2 expression is linked to promotion of an epithelial-mesenchymal-transition phenotype and expansion of a breast cancer stem cell-like population with skewed asymmetric cell division in vitro; however, the in vivo role that TET2 plays in regulation of mammary stem cell (MaSC) fate and development of mammary pathology has yet to be determined. Here, using our newly established mammary-specific Tet2-knockout mouse model, the data reveals for the first time that TET2 plays a pivotal role in mammary gland development via directing MaSC to luminal lineage commitment in vivo. Furthermore, we find that TET2 coordinates with FOXP1 to target and demethylate FOXA1, GATA3, and ESR1, key transcription factors that orchestrate mammary luminal lineage specification and endocrine response and are often silenced by DNA methylation in aggressive human breast cancers. Finally, loss of TET2 expression leads to promotion of mammary tumor development with defective luminal cell differentiation and tamoxifen resistance in a PyMT;Tet2 deletion breast cancer mouse model. As a result, this study provides a previously unidentified role for TET2 in governing luminal lineage specification and endocrine response that underlies resistance to anti-estrogen treatments.</p>

<p>Breast cancer is the most frequently diagnosed malignancy and the second most lethal cancer in women. Metastasis in breast cancer is invariably responsible for patient death and is comprised of many steps, of which proliferation in vital organs is responsible for morbidity and mortality due to vital organ failure. Patients with the metastatic disease are limited to chemotherapy, which non-specifically targets proliferating cells. Despite it being initially effective, chemotherapy is associated with high toxicity and many patients develop resistance. Thus, there is an urgent need to characterize the biology of metastatic breast cancer to develop targeted therapies for the late-stage disease.</p> <p>EGFR is a member of the ErbB family of receptor tyrosine kinases, which have particular relevance in breast tumorigenesis. Clinical studies show that high expression levels of EGFR in the primary mammary tumors correlate with poor prognosis and decreased survival of breast cancer patients due to metastasis. Patient data is supported by experimental and pre-clinical studies, which describe various signaling pathways that mediate the oncogenic effects of EGFR, such as the MAPK, STAT3, and PI3K pathways. Despite these well-documented roles of EGFR, clinical trials evaluating EGFR inhibitors (EGFRi) in metastatic breast cancer have been unanimously unsuccessful in improving patient prognosis, and the mechanisms that contribute to this intrinsic resistance are unknown.</p> <p>To characterize the signaling events that govern EGFR behavior in metastatic breast cancer resistant to EGFRi, we utilized multiple pre-clinical breast cancer progression series and patient-derived cells that display the intrinsic resistance phenomenon. In these models, EGFR functions as a pro-apoptotic molecule whose ligand-mediated activation results in growth inhibition and/or apoptosis of metastatic breast cancer cells. Here we show that in the later stages of metastasis, increased nuclear translocation of EGFR leads to increased physical access to STAT1 and STAT3 molecules residing in the nucleus. Indeed, an EGFR mutant that is defective in endocytosis is unable to elicit STAT1/3 phosphorylation. Additionally, specific inhibition of nuclear EGFR function using the EGFR kinase inhibitor gefitinib linked to a nuclear localization signal (NLS-gefitinib) prevents EGF-induced STAT1/3 phosphorylation. Altogether, these findings implicate nuclear localization of EGFR in downstream STAT1/3 signaling in metastatic breast cancer.</p> <p>Subsequently, we examined the involvement of nuclearly-activated STAT1/3 signaling in the apoptotic function of EGFR. NLS-gefitinib treatment or genetic/pharmacologic inhibition of STAT1/3 efficiently blocks EGF-induced apoptosis in metastatic breast cancer cells resistant to EGFRi. These findings were utilized therapeutically by activating EGFR with EGF treatment while simultaneously blocking the downstream proliferative MAPK:ERK1/2 pathway using the MEK1/2 inhibitor trametinib. EGF + trametinib combination preserved STAT1 signaling while effectively blocking the MAPK pathway, thus potentiating EGF-mediated apoptosis in metastatic breast cancer cells. Importantly, combined administration of trametinib and EGF resulted in STAT1-mediated apoptosis of primary mammary tumor cells, which respond to EGF in a proliferative fashion. These data provide a novel approach of targeting metastatic breast cancer by biasing EGFR signaling towards nuclear activation of STAT1/3 signaling resulting in apoptosis.</p> Our studies herein also examined the role of STAT3 in primary mammary tumor cells overexpressing EGFR. Depletion of STAT3 expression normalized the transformed phenotype of these cells <i>in vitro</i> and resulted in smaller mammary tumors <i>in vivo</i>. These results implicate STAT3 in EGFR-driven breast tumorigenesis localized to the mammary tissues. Further, systemic dissemination of breast cancer is associated with activation of the JAK1/2:STAT3 signaling axis. Despite the involvement of STAT3 in EGFR-mediated oncogenesis in the primary tumor setting, targeting JAK1/2:STAT signaling with the JAK1/2 inhibitor ruxolitinib proved ineffective in inhibiting the growth and invasion of metastatic cells derived from these primary tumors. These results are in agreement with the role of STAT1/3 in driving the pro-apoptotic function of EGFR in metastatic breast cancer cells. Altogether, these investigations provide a plausible explanation for the inability of JAK1/2 inhibitors to effectively target metastatic breast cancer in clinical and experimental investigations. Further, these findings indicate that the development of therapeutics or molecular tools that efficiently activate STAT1/3 signaling in metastatic breast cancer may represent an important concept for eradicating tumors resistant to targeted therapies.

Thrombotic events are known to be associated with various cancers and recent research has implicated parts of the coagulation systemin promoting cancer progression. In particular, thrombin has been studied for its mitogenic effects in 2D cultures as well as in cancer progression in vivo in animal models however, conflicting results exist. Studies of proliferation in response to thrombin stimulation, of pancreatic cancer cells or pancreatic cancer-associated fibroblasts (CAFs) in vitro, that utilize a3D culture platform are significantly limited. In this study, PDAC cancer cells and cancer-associated fibroblast (CAF) cells were exposed to thrombin using a microfluidic device that mimics in vivo conditions. The cells used herein were cultured in a microfluid device, suspended inside of a 3D collagen matrix, and exposed to daily stimulation of 1 U/mL of thrombin in serum-free media for one hour. The findings of this study are that there is no statistically significant effect, promotive or inhibitory, on the proliferation of the cells used in this study, these results were unexpected. At the end of this paper, a review of potential reasons as to why no significant effect was seen on the cells is presented.

<p>Breast cancer is the predominant cancer diagnosed among women, and the second most deadly cancer. The vast majority of cancer-related deaths is caused by the metastatic spread of cancer from the primary tumor to a distant site in the body. Therefore, new strategies which minimize breast cancer metastasis are imperative to improve patient survival. Cancer cells which acquire anchorage independence, or the ability to survive without extracellular matrix attachment, and metabolic flexibility have increased potential to metastasize. In the present studies, the ability to survive detachment and subsequent metabolic changes were determined in human Harvey-<i>ras</i> transformed MCF10A-<i>ras</i> breast cancer cells. Detachment resulted in reduced viability in a time-dependent manner with the lowest cell viability observed at forty hours. In addition, decreased cell viability was observed in both glutamine and glucose depleted detached conditions, suggesting a dependence on both nutrients for detached survival. Compared to attached cells, detached cells had reduced total pool sizes of pyruvate, lactate, α-ketoglutarate, fumarate, malate, alanine, serine, and glutamate, suggesting the metabolic stress which occurs under detached conditions. However, intracellular citrate and aspartate pools were unchanged, demonstrating a preference to maintain these pools in detached conditions. Compared to attached cells, detached cells had suppressed glutamine metabolism, as determined by decreased glutamine flux into the TCA cycle and reduced mRNA abundance of glutamine metabolizing enzymes. Further, detached glucose anaplerosis through pyruvate dehydrogenase activity was decreased, while pyruvate carboxylase (PC) expression and activity were increased. A switch in metabolism was observed away from glutamine anaplerosis to a preferential utilization of PC activity to replenish the TCA cycle, determined by reduced PC mRNA abundance in detached cells treated with a cell-permeable analog of α-ketoglutarate, the downstream metabolite of glutamine which enters the TCA cycle. These results suggest that detached cells elevate PC to increase flux of carbons into the TCA cycle when glutamine metabolism is reduced. </p> <p>Vitamin D is recognized for its role in preventing breast cancer progression, and recent studies suggest that regulation of energy metabolism may contribute to its anticancer effects. Vitamin D primarily acts on target tissue through its most active metabolite, 1α,25-dihydroxyvitamin D (1,25(OH)₂D). The present work investigated 1,25(OH)₂D’s effects on viability of detached cells through regulation of energy metabolism. Treatment of MCF10A-<i>ras</i> cells with 1,25(OH)₂D resulted in decreased viability of detached cells. While 1,25(OH)₂D treatment did not affect many of the glucose metabolism outcomes measured, including intracellular pyruvate and lactate pool sizes, glucose flux to pyruvate and lactate, and mRNA abundance of enzymes involved in glucose metabolism, 1,25(OH)₂D treatment reduced detached PC expression and glucose flux through PC. A reduction in glutamine metabolism was observed with 1,25(OH)₂D treatment, although no 1,25(OH)₂D target genes were identified. Further, PC depletion by shRNA decreased cell viability in detached conditions with no additional effect with 1,25(OH)₂D treatment. Moreover, PC overexpression resulted in increased detached cell viability and inhibited 1,25(OH)₂D’s negative effects on viability. These results suggest that 1,25(OH)₂D reduces detached cell viability through regulation of PC. Collectively this work identifies a key metabolic adaptation where detached cells increase PC expression and activity to compensate for reduced glutamine metabolism and that 1,25(OH)₂D may be utilized to reverse this effect and decrease detached cell viability. These results contribute to an increased understanding of metastatic processes and the regulation of these processes by vitamin D, which may be effective in preventing metastasis and improve breast cancer patient survival.</p>

<p>Breast cancer remains a major cause of cancer-related deaths among women despite several advances in the field due to metastasis with a 5-year survival rate of less than 30% for metastatic breast cancer. Dissemination of tumor cells to metastatic sites begins as early as the primary tumor is diagnosed at just 4mm in size. These cells remain dormant for extended periods of time evading immune surveillance and later turn into therapy resistant metastases resulting in the poor prognosis in breast cancer patients. Hence, there is a <b>critical need </b>to improve our understanding of the metastatic programs in breast cancer and its contributors to develop better therapy options.</p><p>One such contributor is alcohol which is listed as a carcinogen by the National Toxicology Program. Alcohol consumption is a risk factor for several cancers and increases the risk of breast cancer incidence in a dose dependent manner. We have observed in preliminary studies, that alcohol consumption causes increased neutrophil extracellular trap (NET) formation in the lungs and outgrowth of previously dormant cancer cells in mice. Further, NETs increase cancer cell seeding and play a role in metastasis. Hence, we hypothesized that alcohol consumption breaks cancer cell dormancy by activating neutrophils.</p><p>In this study, we have broken cancer cell dormancy and generated a novel cell line, Alcohol-D2.OR, by inducing outgrowth of the dormant D2.OR cells in mice through alcohol consumption. Reinjection of the Alcohol-D2.OR cells, into alcohol-naïve mice results in aggressive outgrowth of the cells suggesting these cells are modified on a genetic level. Indeed, RNA sequencing analysis of the gene expression in the cells showed that these cells have significantly modified gene expression as well as modified morphology and surface protein expression than the parental D2.OR cells. Importantly, from our analysis we have identified a tumor suppressor, SPINK5 which was significantly downregulated in the alcohol line. Further, SPINK5 expression in cancer cells suppressed neutrophil activity in-vitro. Knockdown of SPINK5 in the parental D2.OR line resulted in outgrowth of the cells in-vivo with increased lung NETs highlighting the importance of this gene for maintenance of dormancy by suppression of neutrophil activity.</p><p>Hence, we have successfully identified a gene responsible for dormancy maintenance, SPINK5 which will aid in not only therapeutic intervention but also in identification of breast cancer patients likely to progress to metastasis. Further, the newly established Alcohol-D2.OR cells provide a novel tool to study other initiators of metastasis in breast cancer.</p><p>A common side-effect of most chemotherapeutic treatments is neutropenia, reduced neutrophils in circulation increasing susceptibility to infections. Hence, GM-CSF is often administered to patients to mobilize bone marrow neutrophils. However, neutrophils have been increasingly shown to promote distant metastases. Circulating disseminated cancer cells (DCCs), which are present as early as primary diagnosis, have been shown to activate neutrophils resulting in the release of neutrophil extracellular traps (NETs). These NETs alter the lung architecture providing a suitable environment for the seeding and growth of DCCs promoting lung metastases. One key player in neutrophil activation is spleen tyrosine kinase (SYK), an intracellular non-receptor kinase which is activated by the engagement of b-integrin on the neutrophil surface.</p><p>Using a chemical genetics approach we are able to specifically inhibit SYK in the murine host. Using our transgenic model of specific SYK inhibition as well as the FDA approved SYK inhibitor, fostamatinib, we see similar results of decreased lung metastases compared to controls. We also observed decreased neutrophil viability in-vitro in the presence of fibronectin, an effect that was not seen on plastic highlighting the importance of integrin mediated activity of SYK. We also observe decreased neutrophil and macrophage infiltration into the lungs upon host-specific SYK inhibition. Overall, these findings suggest a paracrine effect of SYK in stromal cells that promotes favorable tumor microenvironment (TME) and its inhibition may be a useful therapeutic option to combat DCCs from forming metastases.</p><p>Hence, through this work we address two mechanisms of neutrophil-mediated breast cancer metastasis and that therapeutic intervention by rescuing SPINK5 expression in cancer cells or inhibition of SYK in the tumor microenvironment can suppress pulmonary metastasis in breast cancer.</p>

<p>  </p> <p>Breast cancer is a significant public health concern being the second leading cause of cancer-related death in women, with a projected 43,250 deaths in the US in 2022. However, cancer progression to metastatic sites is the primary cause of death in breast cancer patients. A hallmark of cancer is the dysregulation of cellular metabolism. Cancer cells have the ability to hijack their metabolism and drive cellular processes supporting cancer progression. As cancer cells continue through the metastatic cascade, they are challenged with various bioenergetic processes that can be supported by the influx of glucose. Thus, altering glycogen accumulation, where glucose is stored in cells, may be beneficial in supporting cancer progression. In this study, we aim to determine what drives glycogen accumulation in metastatic cells and if it is utilized to support cancer progression. We employed the non-metastatic MCF10A-<em>ras</em> and the metastatic MCF10CA1a cells for these studies. Our results demonstrate that metastatic MCF10CA1a have 20-fold accumulation of glycogen compared to the MCF10A-<em>ras</em> cells. Utilizing 13C6-glucose flux analysis, surprisingly, most of the glucose incorporated into glycogen of the MCF10CA1a cells was in the M+5 glucose labeling pattern instead of the expected M+6 pattern which occurs when glucose is directly converted to glycogen. We showed that glycogen was accumulated due to increased gluconeogenesis through cataplerosis (PEPCK) utilizing inhibitors of the enzyme. Additionally, in a pulse-chase experiment using 13C6-glucose flux analysis, there was an approximate 50% reduction in labeled glucose in glycogen, 3 hours after removing the label, suggesting that the MCF10CA1a cells also have a rapid turnover of glycogen. Glucose can be released through two mechanisms, glycogenolysis or glycophagy. Utilizing siRNAs to a rate limiting steps in each pathway, results suggest both glycogenolysis (PYGL) and glycophagy (GAA) are necessary to support cell migration, a critical step in metastasis of the MCF10CA1a cells. Thus, glycogen metabolism is dysregulated in the MCF10CA1a breast cancer cells such that they have increased glycogen accumulation and that glycogen is required to support cell migration. Further understanding the mechanism by which glucose is accumulated and released in a specific cancer and in specific steps or stressors in cancer progression may contribute to potential therapeutic targets to help mitigate metastasis, and potentially breast cancer mortality.</p>

<p>Prostate cancer is the most frequently diagnosed cancer and the second leading causes of cancer-related death in men in the United States. Despite high overall incidence, the disease is relatively well controlled due to slow progression and early detection. However, surgical resection and external beam radiation therapy, first lines of defense, are the only potentially curative options in the clinic. Radiation resistant and metastatic prostate cancer are treated with androgen signaling inhibition (ASI) therapy to target the major growth/proliferation signaling axis that drives prostate cancer cells, but resistance invariably develops. Further, as ASI therapeutic compounds become more potent and are approved for use as neoadjuvant therapeutic options, up to 25% of prostate cancer patients on ASI therapy develop neuroendocrine prostate cancer (NEPC) that is actually induced by the ASI therapy itself. NEPC is resistant to taxane and platinum-based therapies, has no curative or specific targeting options clinically, results in mean overall survival under 9 months in some patient cohorts, and represents a significant unmet clinical need.</p> <p>Protein arginine methyltransferase 5 (PRMT5) is a methyltransferase with histone (epigenetic) and non-histone (non-epigenetic) substrates. PRMT5 is a critical mediator of stemness-associated genes as well as epigenetic regulation of cell fate determination. Further, PRMT5 is a validated therapeutic target in multiple hematological and solid tumor malignancies with multiple clinical trials ongoing. The Hu lab has recently demonstrated that 1) PRMT5 drives androgen receptor (AR) expression in hormone naïve prostate cancer (HNPC) cells, 2) PRMT5 positively regulates DNA damage response gene expression to confer radiation resistance in prostate cancer cells, 3) PRMT5 cooperates with cofactor pICln to drive AR expression in  castration resistant prostate cancer (CRPC) cells, and 4) targeting PRMT5 inhibits development of radiation-induced NEPC development, and that PRMT5 is a valid therapeutic target for prevention of radiation-induced neuroendocrine differentiation (NED). </p> <p>The research presented in this thesis demonstrates that PRMT5 and MEP50 are required for ASI-induced NED in prostate cancer cells, that the PRMT5:MEP50 protein:protein interaction can be pharmacologically targeted, and that ASI-induced NED occurs in an AR-dependent manner. Further, this work contributes a novel class of PRMT5:MEP50 PPI inhibitors in addition to a single-cell, time-resolved model system for interrogating pharmacological targeting of ASI-induced NED <em>in vitro</em>.</p>