Browsing by Subject "Pharmacology"

Osteoporosis and low bone density affect an estimated 54 million adults of 50 years and over in the United States, resulting in $19 billion in costs for osteoporosis-related bone breaks. Current treatments include the use of antiresorptive and anabolic drugs to decrease the rate of bone resorption and increase the rate of bone formation, respectively. However, these current treatments are unable to completely normalize skeletal integrity. As bone diseases become increasingly prevalent, there is an urgent need to identify novel therapies to improve quality of life and reduce economic burden on the healthcare system.

To identify novel therapeutic targets, we must first begin to understand the cellular complexity of the bone marrow niche and how cellular function is regulated within the bone tissue. Bone-resident cells, such as skeletal progenitors and their descendants, are critically influenced by extrinsic signals derived from the local microenvironment. Previous studies have identified hypoxia as a key microenvironment factor in bone. Thus, the ability to target the hypoxic bone marrow niche presents an attractive and untapped potential for regenerative medicine.

Much of the work investigating the role of hypoxia and HIF signaling have focused on mature osteoblast and chondrocyte populations. In contrast, studies investigating the contribution of HIF signaling on skeletal progenitors and marrow adipocyte populations are scarce. In this dissertation, I investigate the role of hypoxia and HIF signaling in skeletal lineage cells, chiefly skeletal progenitor cells and marrow adipogenic lineage cells. Using cellular, genetic, and pharmacological-based approaches, I characterize the roles of HIF-1α and HIF-2α in both homeostatic and pathological contexts in the aforementioned cell populations.

First, I propose an optimized cell-based system to investigate the function of skeletal progenitors in vitro. Here, I highlight the limitations of current in vitro isolation techniques and introduce a relatively simple method of bone marrow stromal cell purification using hypoxia. Using this system, I assess how skeletal progenitors respond to hypoxic cues and interrogate skeletal progenitor cell differentiation and functional responses in my subsequent research. Next, using genetic and pharmacological approaches, I investigate the role of HIF-2α in bone formation following radiation-injury where I identify HIF-2α as a negative regulator of bone recovery. Additionally, with the assistance of my collaborators, I develop and characterize a bone-targeting nanocarrier to ameliorate radiation-induced bone loss. Lastly, I detail early work I conducted to investigate the role of HIF signaling in marrow adipogenic lineage cells. Here, I establish and characterize animal models to determine how hypoxia and HIF signaling influences adipogenic lineage commitment and expansion in an early and mature marrow adipogenic population.

In summary, this dissertation aims to expand our limited understanding on how the hypoxic bone microenvironment and HIF signaling regulate skeletal lineage cells in vivo, with a special focus on skeletal progenitor and marrow adipogenic populations. In terms of boarder impacts, understanding the signaling networks that regulate bone homeostasis and recovery processes will not only expand our basic understanding of the molecular mechanisms underlying skeletal development, but also provide novel insights for developing therapies to treat bone loss.

Temporal lobe epilepsy is a common and devastating disorder that features recurrent seizures and is often associated with pathologic anxiety and hippocampal sclerosis. An episode of prolonged seizures (status epilepticus) is thought to promote development of human temporal lobe epilepsy years later. A chemical-genetic approach established proof of concept that transiently inhibiting the receptor tyrosine kinase, TrkB, following status epilepticus prevented epilepsy, anxiety-like behavior and hippocampal damage in a mouse model, providing rationale for developing a therapeutic targeting TrkB signaling. To circumvent the undesirable consequence that global inhibition of TrkB exacerbates neuronal degeneration following status epilepticus, we sought to identify both the TrkB-activated signaling pathway mediating these pathologies and a compound that uncouples TrkB from the responsible signaling effector. To accomplish these goals, we used genetically modified mice and a model of seizures and epilepsy induced by a chemoconvulsant. Genetic inhibition of TrkB-mediated phospholipase C gamma 1 (PLC gamma 1) signaling suppressed seizures induced by a chemoconvulsant, leading to design of a peptide (pY816) that inhibited the interaction of TrkB with PLC gamma 1. We demonstrate that pY816 selectively inhibits TrkB-mediated activation of PLC gamma 1 both in vitro and in vivo. Treatment with pY816 prior to administration of a chemoconvulsant suppressed seizures in a dose- and time-dependent manner. Treatment with pY816 initiated after chemoconvulsant-evoked status epilepticus and continued for just three days suppressed seizure-induction of epilepsy, anxiety-like behavior and hippocampal damage assessed months later. This study elucidates the signaling pathway by which TrkB activation produces diverse neuronal activity-driven pathologies and demonstrates therapeutic benefits of an inhibitor of this pathway in an animal model in vivo. A strategy of uncoupling a receptor tyrosine kinase from a signaling effector may prove useful in diverse diseases in which excessive receptor tyrosine kinase signaling contributes.

Inositol phosphates (IPs) are versatile metabolites that play important roles in multiple cellular processes. They have been considered signaling messengers that relay extracellular signals via a wave of their production and allosteric regulation of downstream targets. In addition to this classical role, recent studies have revealed that certain IPs can also function as protein structural cofactors. However, except for the two plant hormone receptors TIR1 and COI1, these IP binding proteins have neither sequences nor functions in common. Therefore, to test whether other cellular proteins are also subjected to this type of regulation and whether an IP binding motif exists, more proteins that bind IPs in a similar manner need to be identified. Via a proteome-wide biochemical screen, two yeast proteins were found to contain IP₆ as an integral component. One is the N-terminal acetyltransferase A complex (NatA), and the other one is Tif31 (or Clu1). IP₆ binding was also observed in NatC, another N-terminal acetyltransferase. The bioinformatics analysis and mutagenesis study showed that tandem tetratricopeptide repeats (TPRs), the only common structural element of NatA and Tif31, were responsible for coordinating IP₆. This mechanism of IP_{6 binding is conserved in the fly homologs of these proteins.}

NatA is one of the enzymes that acetylate the α-amino groups at protein N-termini. This widespread protein modification affects a wide range of cellular processes. IP₆ was shown to be essential for yeast NatA in vitro thermostability and for some but not all functions of the protein in cells grown under temperature stress. Other multiple phosphate-containing molecules including IP₅ species and the bacterial alarmone ppGpp were found to bind NatA and partially compensate for the lack of IP₆. IP₆ also binds the human NatA homolog. This binding is crucial for hNatA complex formation, in vitro and in vivo activities, and ability to rescue NatA-deficient phenotypes when it is expressed in yeast. Therefore, IP₆ acts as a molecular glue that brings hNatA (and hNatE) subunits together. The other protein found in our screen, Tif31, is important for normal mitochondrial morphology and distribution. In cells that cannot produce IP₄, IP₅ and IP₆, Tif31 levels were significantly decreased and these cells exhibited severe mitochondrial aggregation. Tif31 mutants that cannot bind IP₆ showed a reduction in cellular levels, a shift to high molecular weight complexes or aggregates, and inability to rescue tif31δ mitochondrial phenotype. This study established the vital role of IP₆ and IP₅ in maintaining Tif31 stability and Tif31-mediated regulation of mitochondrial distribution.

Collectively, this dissertation discovered two proteins that use IP₆ as a structural cofactor. For the first time, a conserved IP₆ binding motif has been shown to be present in certain TPR-containing proteins. Via tight binding to these proteins, IP₆ stabilizes their structures or subunit interaction. This research provides mechanistic evidence for the interplay between IP biology and N-terminal acetylation as well as between IP biology and mitochondrial morphology.

Normal development and homeostasis require dynamic and tight regulation of epithelial architecture. Abnormal epithelial physiology is associated with various pathological conditions including cancers, and may be induced by changes in epithelial polarity, morphology and/or movement. Among the signaling pathways modulating epithelial physiology are those downstream of integrins and receptor tyrosine kinases (RTKs). Although roles of multiple integrins and RTKs in epithelium homeostasis have been established, the identity of signals regulating the functions of these surface receptors and the pathways connecting them to the regulation of epithelial architecture remain largely unknown. In this dissertation, I have identified the Abl family of non-receptor tyrosine kinases (Abl and Arg) as regulators of beta1 integrin and Met receptor tyrosine kinase signaling.

Abl family kinases are hyper-activated in multiple solid tumors and implicated in epithelial polarity regulation. Dysfunction of beta1 integrin is also associated with carcinoma development. To study the role of the Abl family member Arg in epithelial cell polarity, I have taken advantage of a three-dimensional (3D) cell culture system, where Madin Darby canine kidney type II (MDCKII) cells grown in collagen gels develop into polarized cyst structures. I have found that expression of active Arg kinase results in the formation of cysts with inverted apical polarity and that active Arg modulates epithelial polarity by regulating beta1 integrin and small GTPases pathways. In addition, I have shown that Arg regulates the Rap1-beta1 integrin pathway independently of the Rac1 pathway which promotes basal laminin assembly. I have also found that Abl family kinases function downstream of Met and that Abl kinase hyperactivity correlates with Met activation in a mouse mammary tumor model. Abl kinases are activated by HGF which is the ligand for Met, and active Abl kinases are recruited to the Met receptor and promote its tyrosine phosphorylation. Using fluorescence resonance energy transfer (FRET), I have found that Abl kinases regulate RhoA GTPase activity which contributes to actomyosin contractility induced by Met receptor activation. Further, Abl kinases positively regulate Met-dependent migration and invasion induced by HGF in several breast cancer cell lines.

In conclusion, I have identified novel functions of the Abl kinases in epithelial architecture regulation: modulation of epithelial polarity by targeting beta1 integrin function and promotion of Met signaling required for migration and invasion. This has important implications as it suggests potential roles for Abl kinases in carcinoma initiation mediated by beta1 integrin dysfunction, and development of Abl kinases inhibitors for treatment of cancers driven by hyper-activation of HGF-Met signaling.

Conventional therapy for malignant glioma (MG) fails to specifically target tumor cells. In contrast, immunotherapy offers an exquisitely precise approach, and substantial evidence indicates that if appropriately redirected, T cells can eradicate large, well-established tumors. Even the latest generation of redirected T cell therapies are limited, however, in that they require a centralized manufacturing infrastructure with heavily trained laboratory personnel to genetically modify each patient’s own T cells, use viral transduction which poses uncertain risks, are limited to the initial subset of T cells manipulated and infused, and still face uncertainty as to the optimal T cell phenotype to infuse. This dissertation reports the rational development and clinical translation of a fully-human, bispecific antibody (hEGFRvIII-CD3 bi-scFv) that overcomes these limitations through a recombinant antibody approach that effectively redirects any human T cell to lyse MG cells expressing a tumor-specific mutation of the epidermal growth factor receptor (EGFRvIII).

Chapters one, two and three provide an overview of T cell based immunotherapy of cancer and advances in antibody engineering. Also included is a discussion of the current standard-of-care therapy for MG, other immunotherapeutic approaches for MG, and relevant targets and their therapeutic potential for the treatment of MG.

Chapter four details the rational development of a fully-human, anti-human bispecific antibody, hEGFRvIII-CD3 bi-scFv, for immunotherapy of MG. By generating a panel of fully human bispecific single chain variable fragments (bi-scFvs) and testing their specificity through successive stages of screening and refinement, a highly-expressed and easily purified construct with high-affinity to both CD3 and EGFRvIII target antigens was obtained (hEGFRvIII-CD3 bi-scFv). In vitro, hEGFRvIII-CD3 bi-scFv re-directed naïve human T cells to upregulate cell surface activation markers, secrete pro-inflammatory cytokines, and proliferate in response to antigen-bearing targets. Each of these anti-tumor effects were robust and occurred exclusively in the presence of target antigen, illustrating the specificity of the approach. Using MG cell lines expressing EGFRvIII and patient derived MG with endogenous drivers and levels of EGFRvIII expression, bispecific antibody induced specific lysis was assessed. In each case, hEGFRvIII-CD3 bi-scFv was both potent and antigen-specific, mediating significant target-specific lysis at exceedingly low antibody concentrations. Tumor growth and survival was assessed in xenogenic subcutaneous and orthotopic models of human MG, respectively. In both these models, well-engrafted, patient-derived MG was effectively treated. Intravenous administration of hEGFRvIII-CD3 bi-scFv resulted in significant regression of tumor burden in the subcutaneous models and significantly extended survival in the orthotopic models.

Chapter five discusses challenges associated with intratumoral heterogeneity and details two mechanisms by which bispecific antibodies like hEGFRvIII-CD3 bi-scFv can induce epitope spreading, or an immunological response against tumor antigens other than those initially targeted. These mechanisms include: 1) re-activation of pre-existing T cell clones that have specificity for the tumor but fail to mount an immune response prior to bispecific antibody induced stimulation and 2) tumor cell death that results in release of tumor antigens and subsequent antigen uptake, processing and presentation by antigen presenting cells (APCs) leading to a secondary immune response. The chapter concludes with a discussion of a novel class of recombinant antibody molecules developed as part of this dissertation work, Bispecific Activators of Myeloid Cells (BAMs), that function to enhance phagocytosis and antigen presentation. BAM molecules may be useful in conjunction with other immunotherapeutic modalities to induce epitope spreading and combat intratumoral heterogeneity.

Chapter six describes research examining hEGFRvIII-CD3 bi-scFv in a unique human CD3 transgenic murine model. These studies have furthered the rationale for continued clinical translation of hEGFRvIII-CD3 bi-scFv as a safe and effective therapy for MG and have led to the discovery of a novel mechanism of drug delivery to brain tumors. The transgenic murine model was advantageous given that the CD3 binding portion of the fully-human bispecific antibody binds only to human CD3. Accordingly, the model provides a platform where the same molecule to be advanced to human studies can be tested pre-clinically in a pharmacologically responsive, fully-immunocompetent, syngeneic, murine glioma model. In vitro, hEGFRvIII-CD3 bi-scFv induced potent human CD3 transgenic T cell activation, pro-inflammatory cytokine secretion and proliferation exclusively in the presence of the highly-invasive and aggressive murine glioma, CT-2A, bearing EGFRvIII antigen (CT-2A-EGFRvIII). hEGFRvIII-CD3 bi-scFv mediated significant lysis of CT-2A-EGFRvIII at exceedingly low antibody concentrations. In vivo, hEGFRvIII-CD3 bi-scFv significantly reduced tumor growth in human CD3 transgenic mice with well-established, subcutaneous tumors and extended survival of human CD3 transgenic mice with well-established, orthotopic, MG. In the orthotopic setting, adoptive transfer of pre-activated human CD3 transgenic T cells significantly increased efficacy compared to human CD3 transgenic mice treated with hEGFRvIII-CD3 bi-scFv alone.

This led to the hypothesis that activated T cells, known to cross the blood-brain barrier (BBB) to perform routine immunosurveillance of the central nervous system (CNS), may bind to hEGFRvIII-CD3 bi-scFv intravascularly, via its CD3 receptor, and carry or “hitchhike” the large CD3 binding macromolecule to tumors located behind the BBB. Indeed, studies have revealed that adoptive transfer of activated T cells significantly increases the biodistribution of intravenously administered hEGFRvIII-CD3 bi-scFv to orthotopic glioma. Furthermore, blocking T cell extravasation, using natalizumab, for example, a drug used clinically to prevent the migration of T cells to the CNS in patients with multiple sclerosis, completely abrogates the increase in efficacy observed with the adoptive transfer of activated T cells. This newly uncovered hitchhiking mechanism of drug delivery to the CNS provides an important tool to enhance the immunotherapy of brain tumors and has potentially far-reaching consequences for the treatment of other CNS disorders, such as Alzheimer’s or Parkinson’s disease, where issues regarding drug delivery to the CNS are relevant. To begin to study this mechanism of drug delivery in disorders where the blood-brain barrier is intact, we have developed a novel transgenic murine model that expresses EGFRvIII at very low levels within neurons in the brain and have demonstrated that intravenously administered EGFRvIII-targeted recombinant antibody can accumulate in the CNS parenchyma, even in the presence of an intact BBB.

On the basis of these results, a series of clinical research development activities were conducted that have led to the initiation of a clinical study to test the hitchhiking mechanism of drug delivery in patients and ultimately to translate hEGFRvIII-CD3 bi-scFv therapy as a safe and effective treatment for patients with MG. These activities have resulted in a foundation in pre-clinical toxicology, clinical grade biologic manufacturing, clinical protocol development, and regulatory processes necessary to safely translate hEGFRvIII-CD3 bi-scFv therapy to the clinic.

This has involved conducing an extended single-dose toxicity study of hEGFRvIII-CD3 bi-scFv in animals to support studies in humans, the results of which are detailed in chapter seven. To assess for toxicity, human CD3 transgenic mice were administered hEGFRvIII-CD3 bi-scFv or vehicle as a control. Animals were observed for 14 days post-dosing with an interim necropsy on day two. Endpoints evaluated included clinical sings, body weights, feed consumption, clinical chemistries, hematology, urinalysis, and histopathology. There were no clinical observations, evidence of experimental autoimmune encephalomyelitis (EAE), or change in body weight or feed consumption noted during the study that would be associated with toxicity. Furthermore, no statistical difference was observed between drug- and control-receiving cohorts in hematological parameters or urinalysis and no pathological findings related to EGFRvIII-CD3 bi-scFv administration were observed. Statistical differences were observed between drug-treated and control-treated cohorts for some of the clinical chemistries assessed, such as hematocrit, calcium and phosphorus among the female, 14-day analysis cohorts.

To produce hEGFRvIII-CD3 bi-scFv and autologous activated T cells to be administered to patients for clinical study, chemistry, manufacturing and control protocols for the production of clinical grade hEGFRvIII-CD3 bi-scFv and autologous activated T cells were developed and implemented. The data presented in chapter eight describe optimized manufacturing processes and rationale for the selection and implementation of in-process and release analytical methods. This work includes the generation of a stable Chinese hamster ovary (CHO) cell line that expresses high levels of hEGFRvIII-CD3 bi-scFv, the generation and certification of a current Good Manufacturing Practice (cGMP) master cell bank (MCB), optimization and scale up of upstream and downstream manufacturing procedures, and development of standard operating procedures (SOPs) for the manufacture and assessment of clinical grade hEGFRvIII-CD3 bi-scFv and autologous activated T cells. Together, these have allowed for the production of clinical grade antibody and autologous patient derived cells within Duke University Medical Center. The production of recombinant antibodies for use in the clinic is a complex endeavor often performed in industry with teams of highly skilled scientists who test and optimize manufacturing protocols using a large, well-established manufacturing infrastructure. The successful production of clinical grade recombinant antibody at an academic center, therefore, represents a significant achievement and would likely be of interest to other academic-based researchers and clinicians embarking on similar clinical endeavors.

Chapter nine describes a clinical protocol for a phase 0 study of hEGFRvIII-CD3 bi-scFv in patients with recurrent EGFRvIII-positive glioblastoma (GBM). The protocol details intravenous administration of single doses of radiolabeled hEGFRvIII-CD3 bi-scFv with and without pre-administration of radiolabeled autologous activated T cells in a given patient. This will allow for imaging studies that will reveal the pharmacokinetics of the recombinant antibody both with and without adoptive transfer of autologous activated T cells. Endpoints include an assessment of the: intracerebral tumor localization of 124iodine (I)-labeled hEGFRvIII-CD3 bi-scFv with and without prior administration of 111indium (In)-labeled autologous T cells; percentage of patients with unacceptable toxicity; percentage of patients alive or alive without disease progression six months after study drug infusion; median progression-free survival; 111-In-autologous T cell intracerebral tumor localization; and percentage of patients who are EGFRvIII-positive at recurrence.

Chapter 10 concludes with a discussion of ongoing and anticipated future pre-clinical and clinical research. Together, these data presented in this dissertation have been submitted to the US Food and Drug Administration (FDA) in support of an Investigational New Drug (IND) application permit for clinical studies of hEGFRvIII-CD3 bi-scFv at Duke University Medical Center. This clinical study of the hitchhiking mechanism of drug delivery and the pharmacokinetics of hEGFRvIII-CD3 bi-scFv may have far reaching implications for disorders of the CNS where drug access past the BBB is relevant and will advance our understanding of hEGFRvIII-CD3 bi-scFv therapy in patients, guiding future clinical study of the molecule as a safe and effective form of immunotherapy for patients with EGFRvIII-positive GBM and other cancers.

Cardiovascular disease and cancer are the two leading causes of death in the U.S. Open heart surgery involving cardiopulmonary bypass (CPB) is performed in over a million patients worldwide in order to treat patients with severe ischemic cardiovascular disease, among other cardiac pathologies. Due to fulminant activation of the hemostatic system, CPB surgery requires administration of highly potent anticoagulation to prevent thrombosis during the procedure, followed by rapid neutralization to minimize the risk of post-operative bleeding. Since the 1950s, unfractionated heparin (UFH) has remained the standard anticoagulant for CPB surgery because of both its potency and reversibility with protamine. Unfortunately, UFH has several limitations that contribute to patient morbidity associated with CPB, creating an unmet clinical need for anticoagulant alternatives to UFH for CPB that can overcome its drawbacks while still maintaining robust potency and antidote-control. A similar clinical need exists for improved therapies that combat metastatic disease, which is ultimately responsible for the vast majority of cancer deaths, a fact that is particularly true for highly aggressive solid tumors such as pancreatic cancer.

Nucleic acid aptamers are an emerging class of therapeutics that are especially attractive as anticoagulants because their activity can be readily reversed by administration of either sequence specific antidotes comprised of complementary oligonucleotides or by “universal” antidotes that include certain cationic nucleic acid binding polymers (NABPs). Our lab has generated several antidote-controllable RNA aptamers that specifically inhibit individual coagulation factors, which have shown promising efficacy as anti-thrombotic agents in pre-clinical and clinical studies for indications other than CPB. Nevertheless, none of these aptamers when used alone can sufficiently match the anticoagulant intensity of UFH needed for CPB. 11F7t is one such aptamer which binds to exosites on both Factor (F)X and FXa and inhibits several procoagulant steps important for clot formation, but does not occlude the protease’s active site. As such, 11F7t’s mechanism of action is distinct from small molecule catalytic site inhibitors of FXa which are used clinically for oral thromboprophylaxis but are potent enough to facilitate anticoagulation during invasion procedures like CPB. In the first part of this work, we demonstrate that 11F7t and catalytic site inhibitors of FXa reciprocally and potently enhance anticoagulation in purified reaction mixtures and in plasma. Furthermore, such combinations prevent clot formation as effectively as UFH in human blood circulated within an extracorporeal oxygenator circuit that mimics CPB, while limiting thrombin generation and immunogenic platelet activation, which contribute to complications associated with UFH-facilitated CPB. Finally, we show that addition of GD-FXaS195A, a Gla-domainless FXa variant with an alanine substitution at the catalytic serine, can promptly neutralize the anticoagulant effects of both FXa inhibitors. GD-FXaS195A closely mimics a therapeutic (Andexanet Alfa) currently in late-stage clinical trials as a universal antidote specific for FXa inhibitors. Thus, our findings suggest promising avenues for developing improved alternatives to UFH for potent, antidote-controllable CPB anticoagulation.

In the second part of this work, we identify a novel application of NABPs for therapeutic inhibition of pancreatic cancer metastasis. Beyond their utility as universal antidotes for aptamers, our lab previously discovered that a subset of NABPs can also serve as anti-inflammatory agents by capturing extracellular nucleic acids and associated protein complexes that promote pathological activation of toll-like receptors (TLRs) in diseases such as systemic lupus erythematosus, sepsis, and influenza infection. Nucleic acid-mediated TLR signaling also facilitates tumor progression and metastasis in several cancers, including pancreatic cancer (PC). In addition, extracellular DNA and RNA circulate on or within lipid microvesicles, such as microparticles or exosomes, which also promote metastasis by inducing pro-tumorigenic signaling in cancer cells and pre-conditioning secondary sites for metastatic establishment. Here we explore the use of an NABP, the 3rd generation polyamidoamine dendrimer (PAMAM-G3), as an anti-metastatic agent. We show that PAMAM-G3 not only inhibits nucleic acid-mediated activation of TLRs and invasion of PC tumor cells in vitro, but also can directly bind extracellular microvesicles to neutralize their pro-invasive effects as well. Moreover, we demonstrate that PAMAM-G3 dramatically reduces liver metastases in a syngeneic murine model of PC. Our findings identify a promising therapeutic application of NABPs for combating metastatic disease in PC and potentially other malignancies.

β-Adrenergic receptors (βAR) are one of the key modulators of cardio-pulmonary functions and belong to a large family of membrane proteins, termed as G-protein coupled receptors (GPCRs). β-blockers (βAR antagonists) and βAR agonists are the mainstay treatments for heart failure and asthma respectively, which reflects the significance of βARs as therapeutic targets. The binding of catecholamines (e.g. adrenaline) to βARs activates intracellular transducer proteins such as hetero trimeric GTP binding proteins (G-proteins) or β-arrestins (βarr), which results in the regulation of cardiac output and bronchodilation.

The bifurcated signaling pathways initiated by G-protein and β-arrestin downstream of βAR, as well as other members in the GPCR family can be selectively activated, a phenomenon termed as ‘biased agonism’. Biased ligands, which can pharmacologically separate these pathways, are of major therapeutic interest due to their potential for improving the specificity of drug actions. For βAR, biased agonism towards β-arrestin is expected to render cardo-protective benefits, while selective activation of G proteins is hypothesized to subdue major side effects from current asthma therapy. Therefore, elucidation of how βARs can preferentially interact with their transducers is at the core of developing the next generation therapeutics, beyond conventional β-blockers and agonists.

Thus far, the exact mechanism behind GPCR biased agonism remains obscure. The leading hypothesis in the field is that GPCRs adopt distinct conformations that preferentially couple to G proteins or β-arrestins. In order to test this hypothesis, we developed and established a G protein biased mutant β2AR (Chapter 2), since efficacious biased ligands for this receptor are yet to be found. Subsequent assessment of GPCR kinase (GRK)-mediated phosphorylation states of this mutant receptor and phosphorylation rescue experiments revealed unexpected findings that contradict the initial hypothesis (Chapter 3). Next, we initiated a biophysical characterization of this mutant β2AR (Chapter 4) to comprehend the conformational and structural basis for its apparent biased phenotype. The cumulative insight gained from experiments described in chapters 2-4 highlight the underappreciated role of GRKs in determining GPCR biased agonism – the mutant β2AR is biased towards G protein due to conformational selection against GRKs, rather than β-arrestins. Furthermore, to obtain a comprehensive understanding of biased agonism, we devised a strategy to map the interface between β2AR-β-arrestin, which can also be used to form stable complexes for further biophysical characterizations (Chapter 5). In summary, this dissertation improves the current understanding of the molecular mechanism behind biased agonism at the prototypical GPCR, β2AR.

The steroid hormone progesterone has complex physiologic effects. In typical development and function, cells respond to progesterone in a dose- and tissue-specific manner. Despite the wide range of physiologic concentrations, canonical effects of progesterone have been characterized in the context of a high physiologic dose (10nM+), relevant during uterine cycling. This narrow focus has produced a gap in knowledge, particularly as it relates to the effects of post-menopausal low concentration progestins (0.1-0.3nM). Given that healthy tissues possess regulatory mechanisms to sense and respond to progesterone in a non-linear dose-specific manner, we hypothesized that breast malignancies would also display discontinuous dose-specific dynamic responses. Our results show that treatment with low dose progestins (0.1-0.3nM) drives proliferation in T47D human breast cancer cells, while high dose progestins (10nM+) inhibit proliferation. Using both unbiased and targeted approaches, we found that low dose progestins facilitate cell cycle entry by enhanced expression of CCND1 (cyclin D1) and SGK1 (serum and glucocorticoid related kinase 1), which are required for initiation of the downstream molecular cascade including phosphorylation of retinoblastoma protein (Rb) and expression E2F1. Expression of CCND1 and SGK1 mRNA are proximal responses to low dose progestin treatment, but transcriptional activation is not mediated by canonical progesterone receptor (PR) activity. Future work is needed to identify previously unexplored mechanisms of PR action in the context of low dose progestin treatments. In summary, these results challenge the assumption of dose response linearity to progestins and show unique functional and molecular effects of low dose progestin treatment. Of potential concern, our findings suggest that certain breast cancers, especially those expressing high levels of PR, may be accelerated by normal post-menopausal circulating concentrations of progestins (0.1-0.3nM). However, these findings also offer a sound rationale for the clinical therapeutic use of high dose progestins for patients with PR+ breast cancer.

Metabolism is known to be driven by intrinsic genetic programs as well as contextual factors within the environment. Individual genetic and environmental determinants of metabolic state have been extensively characterized, both within normal physiological processes as well as in the context of disease states such as cancer. However, it is becoming increasingly appreciated that the inevitable interaction between these differential sources of metabolic regulation can dramatically influence cellular phenotypes, a phenomenon commonly referred to as gene-environment interaction. These interactions can create substantial heterogeneity between individuals, particularly in the context of tumor metabolism which can ultimately impede the development and efficacy of many clinical therapies. Characterization of these relationships can therefore improve the predictive applicability of targeted therapeutic approaches, as well as contribute to the identification of novel treatment strategies that can circumvent the biological limitations imposed by gene-environment interactions. Using metabolomic, genetic, and pharmacological approaches, in this dissertation I examine the metabolic consequences of environmental alterations in defined genetic settings. I provide in-depth characterization of the relative predictability of cellular responsiveness to nutrient availability in the context of genetic deletion of the metabolic enzyme MTAP, results of which demonstrate potential implications in previously-identified metabolic vulnerabilities in MTAP-deleted cancers. I additionally examine how perturbation of energetic demand, via either pharmacological inhibition of the Na+/K+ ATPase or with the physiological stimulus of exercise, impacts metabolic processes in diverse biological contexts. This work collectively illustrates the exceptional heterogeneity in metabolic adaptation to environmental alterations, and provides support for the future development of lifestyle modifications and repurposing of common pharmacological agents as therapeutic modalities in cancer treatment.

Coagulation and inflammation are intimately linked processes that protect the body from a wide range of insults. However, dysregulation of this system contributes to morbidity and mortality in infection, autoimmune disease, and countless other disease processes. To successfully intervene on this axis, we need to understand the molecular drivers of thromboinflammation and design biocompatible pharmaceuticals to combat them. Towards that effort, this dissertation first explores the clinical landscape of COVID-19, with specific focus on characterization of the molecular drivers of COVID-19-associated coagulopathy. Through analysis of coagulative profiles and clinical data, fibrinolytic suppression and endothelial injury are identified as primary drivers of thrombosis and respiratory distress in COVID-19. Next, this dissertation describes efforts to reduce the toxicity of the anti-inflammatory scavenging polymer polyamidoamine (PAMAM). We show that the density of cationic surface charges underpins the direct cellular and systemic toxicity of these polymers and present novel PAMAM variants with a mix of cationic and neutral surface groups that resolve the toxicity of the original cationic polymers while retaining their scavenging properties. Together, these data highlight the importance of thromboinflammation in human disease and advance the translational potential of a new class of anti-inflammatory agents.

Cells thrive on sustaining order and balance to maintain proper homeostatic functions. However, the primary machinery involved in protein quality control including chaperones, ubiquitin proteasome system, and autophagy all decline in function and expression with age. Failures in protein quality control lead to enhanced protein misfolding and aggregation. Efficient elimination of misfolded proteins by the proteasome system is critical for cellular proteostasis. However, inadequate proteasome capacity can lead to aberrant aggregation of misfolded proteins and inclusion body formation, which is a hallmark of numerous neurodegenerative diseases. Due to the post-mitotic nature of neurons, they are more susceptible to the collapse in proteostasis correlated with age.

Here, we propose a cell based model of aggresome clearance using a reversible proteasome inhibitor, MG132, to identify the precise molecular machinery involved in proper processing of inclusions. It is known that once misfolded proteins are aggregated, the proteasome system can no longer degrade them. Furthermore, the continuous accumulation of aggregates often leads to aggresome formation, which results in amalgamated inclusion bodies that are simply too large for autophagosomes to engulf and degrade. Although, studies have shown that aggresomes can eventually be cleared by autophagy, the molecular mechanisms underlying this process remain unclear.

Our research reveals that regardless of impaired proteolysis, proteasomes can still stimulate autophagy-dependent aggresome clearance by producing unanchored lysine (K)63-linked ubiquitin chains via the deubiquitinating enzyme Poh1. Unanchored ubiquitin chains activate ubiquitin-binding histone deacetylase 6, which mediates actin-dependent disassembly of aggresomes. This crucial de-aggregation of aggresomes allows autophagosomes to efficiently engulf and eliminate the protein aggregates. Interestingly, the canonical function of Poh1 involves the cleavage of ubiquitin chains en bloc from proteasomal substrates prior to their degradation by the 20S core, which requires intact 26S proteasomes. In contrast, here we present evidence that during aggresome clearance, 20S proteasomes dissociate from protein aggregates, while Poh1 and selective subunits of 19S proteasomes are retained as an efficient K63 deubiquitinating enzyme complex. The dissociation of 20S proteasome components requires the molecular chaperone Hsp90. Hsp90 inhibition suppresses 26S proteasome remodeling, unanchored ubiquitin chain production, and aggresome clearance. Ultimately, we hope to apply these molecular markers of inclusion body processing to identify the underlying lesion in aggregate prone neurodegenerative disease.

The intestine is a multifunctional organ that must perform dichotomous roles in order to maintain health. While it is the primary site of absorption of dietary nutrients, it must also serve as a barrier to both the multitude of microorganisms which reside in the intestinal lumen (the microbiota) and foreign compounds (xenobiotics) which can be toxic to the host. Moreover, the microbiota are required for normal physiology, regulating immunological development, metabolism and behavior. Understanding how the intestine maintains homeostasis and responds to insult in the face of a chemically and microbially complex and dynamic environment is not only a fundamental question of biology, but has important implications for human health. We used zebrafish in order to better understand how the intestine responds to xenobiotics (Chapter 2) and transduces signals from the microbiota to the immune system (Chapter 3).

In Chapter 1, I introduce the complex and reciprocal interactions between xenobiotics, the microbiota, and the host. I highlight examples whereby the microbiota modulates the activity and toxicity of pharmaceuticals, with relevance to diseases of different organ systems. I also describe mechanisms by which the intestine responds to xenobiotic toxicity, and finally advocate for the use of novel model organisms to improve our understanding of these complex interactions.

In Chapter 2, I present our work using the NSAID Glafenine to explore how the intestine responds to xenobiotic challenge. Using transgenic zebrafish and high resolution in vivo imaging, we demonstrate loss epithelial cells in a live animal following xenobiotic challenge. Moreover, Glafenine causes intestinal inflammation, which is potentiated by microbial dysbiosis. We also show that Glafenine can directly alter microbiota composition. Glafenine treatment resulted in activation of the unfolded protein response (UPR), and while pharmacological inhibition of the UPR sensor Ire1a suppressed Glafenine-induced IEC loss, this was associated with increased inflammation and mortality. Ultimately, we demonstrate that Glafenine-induced intestinal toxicity is likely due to off-target inhibition of multidrug resistance (MDR) efflux pumps, as other MDR inhibitors were able to elicit similar phenotypes. Collectively, our findings revealed that (i) MDRs serve an evolutionarily conserved role in maintenance of intestinal homeostasis and (ii) IEC delamination is a protective mechanism which serves to limit inflammation and promote animal survival.

While studies in gnotobiotic mice and zebrafish have demonstrated that the microbiota are required for normal development of the innate immune system, the underlying host and microbial signals which mediate these effects remain largely unknown. We had previously demonstrated that motility of gut commensal bacteria in zebrafish was important for successful colonization of some strains and stimulation of the normal host innate immune response to colonization. In Chapter 3, we describe how microbiota colonization is associated with changes in the PMN transcriptome in addition to promoting systemic abundance and distribution of myeloid cells. Intriguingly, the only pattern recognition receptors found to be differentially expressed in PMNs were the Flagellin receptors tlr5a and tlr5b. Colonization of zebrafish larvae with bacteria lacking Flagellin resulted in attenuated PMN transcriptional activation compared to larvae colonized with isogenic wild type (WT) bacteria. We subsequently demonstrated that direct exposure to purified Flagellin can potently induce transcriptional activation in zebrafish PMNs. These findings identify how the presence of the microbe associated molecular pattern (MAMP) Flagellin serves as a bacterial cue from the microbiota which promotes PMN activation. In Chapter 4, I offer perspectives as to how the Glafenine-zebrafish model system can be used to more deeply investigate host-microbiota-xenobiotic interactions, and genetic, biochemical and computational analyses can help delineate mechanisms by which MDR efflux pumps function in the maintenance of intestinal homeostasis. Moreover, I propose the use of bacterial screens as well as inflammatory and infectious challenge assays in order to better understand the functional outcomes of PMN transcriptional activation elicited by microbiota-derived signals such as Flagellin.

Projecting neurons of the central nervous system extend their axons through considerable distances from the cell soma. This extreme architecture requires local regulation driven by proteins whose functions are essential for axonal maintenance and survival. Pathways involved in axon survival represent an important question in neurodegenerative conditions like glaucoma. The loss of visual acuity in glaucoma is caused by the degeneration of retinal ganglion cells (RGCs), that transmit visual signals from the retina to the brain. The principal site of RGC axonal insult occurs where the axons exit the eye, weaving through the honeycomb structure of the glial lamina (GL), at the optic nerve head (ONH). While much is understood about the ONH, the specific molecular pathways involved in local regulation of this crucial area is not fully understood. Furthermore, it is not known how these pathways may be altered in glaucoma, which hinders potential therapeutic intervention. Here, we utilize wild-type and glaucomatous mice to decipher the specific proteomic signature of the GL. By Isolating healthy and glaucomatous GL and RL samples, we performed mass spectrometry proteomics analysis to find proteins specifically enriched in the murine GL. Results from wild-type mice showed enrichment of translation proteins in the GL. At the time this thesis is written, proteomics results on glaucomatous mice are not yet available.

Tumor recurrence following initial treatment is the leading cause of death among breast cancer patients. Epigenetic mechanisms are critical for regulation of gene expression and to facilitate appropriate responses to environmental cues. However, it is increasingly appreciated that epigenetic dysregulation directly promotes therapeutic resistance and tumor progression. While genetic alterations have been shown to promote tumor progression, the contribution of non-genetic drivers of recurrence remains unexplored. In the current work, we utilized genetically engineered mouse models of breast cancer recurrence to evaluate the contribution of epigenetic plasticity to tumor recurrence and chemoresistance. First, we found that recurrent tumors undergo dramatic epigenetic and transcriptional reprogramming, partially through acquisition of an epithelial-to-mesenchymal transition (EMT). EMT promoted epigenetic silencing of tumor suppressor Par-4 through a unique, bivalent histone configuration. This bivalent configuration conferred plasticity to Par-4, and Par-4 silencing was reversed with epigenetic inhibitors of EHZ2 and HDAC. Further, Par-4 re-expression sensitized recurrent tumors to commonly utilized microtubule-targeting chemotherapeutics through altered cytoskeletal regulation. Second, we found that recurrent tumor epigenetic and transcriptional rewiring conferred sensitivity to G9a inhibitors. G9a inhibition promoted recurrent tumor cell necroptosis through demethylation of genes involved in a pro-inflammatory cytokine program. Further, knockout of G9a protein delayed the time until mammary tumors recurred in vivo. Collectively, our studies demonstrate that epigenetic dysregulation is a key feature of breast cancer progression, and pharmacologic strategies designed to target epigenetic enzymes underlying these processes may be of clinical value in the treatment of recurrent breast cancer.

The dramatic expansion of genomic sequencing methodologies, applications and efforts has empowered our abilities to deepen the conceptual understanding of complex biological processes, including diseases like cancer. Through our accumulated understanding of cancer genomics, targeted therapies, which inhibit the specific driver oncogenes and pathophysiological processes that underlie cancer progression, have been developed. However, in modern precision oncology and therapeutics, cancer drug resistance, both primary and secondary, has greatly limited the potential of targeted therapies to improve patients’ lives. Here, we systematically define combination treatment strategies by using unbiased pharmacological and functional genetic screening approaches to overcome the persistent problem of primary drug resistance in two cancer contexts: (1) epidermal growth factor receptor (EGFR)-driven triple-negative breast cancer (TNBC) and (2) PIK3CA mutant gastric cancer. Particularly, in the first context, using a candidate drug screen, we discovered that inhibition of cyclin-dependent kinase (CDK) 12 dramatically sensitizes diverse models of TNBC to EGFR blockade. Instead of functioning through CDK12’s well-established transcriptional roles, this combination therapy drives cell death through the 4E-BP1-dependent suppression of the translation and consequent stability of driver oncoproteins, including MYC. Further, with mechanistic intent, using a genome-wide CRISPR/Cas9 screen, we identified the CCR4-NOT complex as a major determinant of sensitivity to the combination therapy whose loss renders 4E-BP1 unresponsive to drug-induced dephosphorylation, rescuing MYC translational suppression and stability. Thus, by revealing a long debated EGFR dependence in TNBC, we have identified a therapeutic approach that functions through the cooperative regulation of translation-coupled oncoprotein stability and holds promising translational potential for the treatment of this difficult-to-treat disease subtype. In the second context, despite extensive molecular characterization of gastric cancer, personalized treatment approaches to improve patient survival outcomes are still lacking. Motivated by this unmet need, we performed drug sensitizer screens with a PI3K-alpha isoform-specific inhibitor, BYL719, in multiple PIK3CA wild-type (WT) and mutant cell lines, including those derived from gastric cancers, head and neck squamous cell carcinomas (HNSCCs), and colorectal cancers using a miniaturized CRISPR/Cas9 library targeting key druggable nodes of cellular survival pathways. This work led to the promising findings that intrinsic resistance to PI3K-alpha inhibition specifically in gastric cancer may be mediated by BCL-xL and NEDD9. Sensitization to PI3K-alpha inhibition by BCL-xL specific inhibitor revealed a novel targeted approach for the treatment of EBV+ PIK3CA mutant gastric cancers, thereby overcoming a perplexing obstacle to the effective targeting of PI3K oncogenic dependency in this cancer subtype. Collectively, our work demonstrated the ability and applicability of screening approaches to define the determinants of primary drug resistance in precision cancer therapies across diverse cancer contexts.

The vertebrate visual system involves a series of complex steps which cover a number ofdiverse anatomical structures. Light first enters the eye and is projected onto the retina, a thin layer of tissue that lines the posterior of the eye and is comprised of millions of specialized neurons. Rods and cones initiate the process light signaling upon absorption of photons, with stimulation of retinal ganglion cells (RGCs) representing the final step of intraretinal signaling. RGC somas localize to the innermost layers of the retina, while their axons converge at the optic nerve head and bundle together to form the optic nerve carrying visual information to the brain. The RGC structure is unique in that the soma and dendrites are compartmentalized inside of the eye whereas the RGC axons exist primarily outside of the eye as they project via the optic nerve to synaptic targets in the CNS. In the proximal region of the optic nerve is a short, unmyelinated region of axons running between an astrocytic meshwork termed the glial lamina (GL). Seminal studies have shown that mitochondria are particularly abundant in the GL compared to other compartments of RGCs, potentially suggesting a particularly high demand for ATP production in this region. Of relevance, the GL is the first region of axonal degeneration in glaucoma, suggesting an inherent susceptibility of this axonal compartment to cellular stress. The focus of this dissertation centers on elucidating the local landscape and regulation of the GL in healthy mice and disease models. Our earliest work is a comprehensive analysis of the enrichment of mitochondria in the GL in wild type mice, improving upon the low-resolution histological studies on which this concept was based. Using complementary immunofluorescence and electron microscopy techniques, we confirm that mitochondria are more abundant in the GL compared to the retrolaminar (RL) optic nerve, but show that the overall mitochondrial accumulation arises only because of differential mitochondrial abundance in the largest diameter RGC axons. We also show that the mitochondrial accumulation is established by postnatal day 6- v 9, preceding the onset of axonal myelination. Therefore, the enrichment of mitochondria in the GL is not a direct consequence of the unique absence of myelin in this region. The remainder of our work is an exploration of differences between the GL and RL in mitochondrial and axonal morphology as well as in proteomic signatures. In preliminary studies, we have observed distinct mitochondrial morphologies between the two compartments, with small differences in mitochondrial length and cristae structure. We also have found preliminary evidence of unprecedented inter-axonal fusion events between RGC axons of the GL, a phenomenon we term short axonal merging sites (SAMS). SAMS are characterized as two individual axons that run parallel and exhibit focal breakdown of their plasma membranes with apparent fusion between the two. While additional experiments are required to eliminate the possibility that SAMS are non-physiological artifacts of tissue fixation, should we confirm their presence it would open a new direction exploring the functional significance of the fusion events in light signaling to the central nervous system and in the propagation of pathology in optic nerve disorders. Finally, we describe our early efforts proving the feasibility of characterizing the compartment-specific proteomes of optic nerve tissue and of RGC mitochondria specifically. We also describe preliminary studies designed to compare the GL proteomes of DBA/2J mice with early glaucoma to control non-glaucomatous littermates. Completion of this analysis may identify key biological differences between the GL and RL and highlight cellular processes that may subject the GL to early axonal degeneration in optic neuropathies like glaucoma. It is our hope that this work will help to identify pathways that may be targeted pharmacologically to combat RGC neurodegeneration in glaucoma and other blinding diseases.

X chromosome inactivation (XCI) is a mammalian dosage compensation phenomenon by which expression of X-linked genes is equalized between females and males. Initiation of XCI depends on Xist long noncoding RNA (lncRNA), which triggers transcriptional silencing of one of the two X chromosomes in female cells, except for genes that escape XCI. The inactive state of the X chromosome is maintained throughout subsequent cell divisions with continuous Xist expression. Proper maintenance of XCI in somatic cells is essential for female health, and its disruption has been observed in many diseases. However, whether Xist is required for maintaining XCI and how XCI alterations lead to diseases still remain open questions. Emerging evidence has revealed that the hematopoietic system consists of cells with variable XCI patterns and is highly sensitive to Xist depletion. This makes the hematopoietic system a good model for studying the functional role of Xist during XCI maintenance. By deleting Xist in hematopoietic stem cells (HSCs) of mice, I showed that Xist loss leads to lineage-specific cell cycle and differentiation defects in hematopoietic progenitor cells of female mice. At the molecular level, I showed that Xist loss results in upregulated expression of several X-linked genes, large proportion of which are XCI escape genes. In addition, many transcriptionally upregulated X-linked genes have been implicated in cell cycle- and immune-related functions. Furthermore, transcriptional upregulation of X-linked genes in Xist-deficient cells is accompanied with a series of epigenetic changes on the inactive X chromosome (Xi) including reduced density of repressive histone marks, increased density of active histone modifications, and enhanced chromatin accessibility which accommodates increased binding of a transcription factor, Ying Yang 1 (YY1). Collectively, these findings suggest a critical role of Xist in regulating expression level of X-linked genes with essential hematopoietic functions during XCI maintenance.