Proteome turnover, the process by which proteins are continuously synthesized and degraded, is a crucial biological process for gene expression regulation, cell state maintenance, cellular homeostasis, and response to stimuli. This dissertation outlines novel methods to quantify proteome turnover in both mouse-derived organoid disease models and in vivo using stable isotope labeling combined with mass spectrometry-based methods. In Chapter 1, we review recent LC-MS/MS techniques for measuring proteome turnover, highlighting their applications and limitations. Chapter 2 focuses on a systematic analysis of proteome turnover in an organoid model of pancreatic ductal adenocarcinoma (PDA) using dynamic Stable Isotope Labeling of Organoids (dSILO). This study reveals faster proteome turnover in metastatic organoids compared to primary tumors and identifies several differentially regulated protein complexes in metastatic tumors, particularly from the mitochondrial respiratory chain. In Chapter 3, we present the exploration of various methods for quantifying in vivo proteome turnover in mice. We employed an isotopic pulse-labeling strategy, dynamic Stable Isotopic Labeling of Mammals (dSILAM), then compared four combinations of mass spectrometry-based data acquisition and half-life modeling methods. We uncovered moderate differences in coverage, reproducibility, and half-life estimations between datasets, although further optimization is required for robust conclusions. Chapter 4 discusses potential limitations and future directions for all of the work described herein. Overall, our findings contribute to the optimization of proteomic workflows for studying protein turnover, which can be applied to enhance our understanding of cellular physiology and the molecular mechanisms underlying disease.

Altered RAS signaling pathways drive uncontrolled cell proliferation, migration, and survival, ultimately leading to tumor development and progression Effective inhibition of RAS signaling holds the potential to benefit roughly a quarter of human cancer patients whose tumors are driven by RAS mutations. In pancreatic ductal adenocarcinoma (PDAC), the third leading cause of cancer related mortality in the US, over 90% of patient cases are driven by activating mutations in KRAS. Here we assessed two divergent approaches to target aberrant RAS signaling for PDAC treatment.In Part I we investigated the therapeutic potential of the RAS(ON) multi-selective inhibitor RMC-7977, a highly potent compound blocking the active GTP-bound forms of KRAS, HRAS, and NRAS, showing affinity for both mutant and wild type (WT) variants. We evaluated RMC-7977 in a comprehensive range of preclinical PDAC models and, following direct RAS inhibition, we observed broad and pronounced anti-tumor activity, at exposures that were well-tolerated in vivo. Pharmacological analyses revealed divergent responses to RMC-7977 in tumor versus normal tissues. Treated tumors exhibited waves of apoptosis along with sustained proliferative arrest whereas normal tissues underwent only transient decreases in proliferation, with no evidence of apoptosis. In the autochthonous KPC model, RMC-7977 treatment resulted in a profound extension of survival followed by on-treatment relapse. Analysis of relapsed tumors identified Myc copy number gain as a prevalent candidate resistance mechanism, which could be overcome by combinatorial TEAD inhibition in vitro. Together, these data established a strong preclinical rationale for the use of broad-spectrum RAS-GTP inhibition in the setting of PDAC and identified a promising candidate combination therapeutic regimen to overcome monotherapy resistance. In Part II, we investigated the therapeutic potential of combined MEK and autophagy pathways inhibition in preclinical models of PDAC. Consistent with previously published work, MEK inhibition increased the dependency of PDAC cells on autophagy, and the combination of Trametinib (a MEK inhibitor) and Hydroxychloroquine (an autophagy inhibitor) demonstrated synergistic anti-proliferative effects in murine PDAC cell lines. We added to this body of work by evaluating the combination in the clinically predictive genetically engineered mouse models (GEMMs). Trametinib/Hydroxychloroquine treatment led to significant tumor regressions, MAPK signaling inhibition, and reduced cell proliferation in the KPC mice, as well as improved survival in the KPF/FC model. Single-cell RNA sequencing analysis of treated KPC tumors revealed both treatment sensitive and resistant malignant cell populations, with the sensitive cells showing upregulated MAPK and autophagy pathway activities. Analysis of fibroblast population additionally revealed that inflammatory CAFs (iCAFs) are diminished in response to treatment, likely due to disrupted paracrine signaling mediated by IL1A secretion from malignant cells. Together, these data provided a preclinical rationale for the use of the Trametinib and Hydroxychloroquine combination in PDAC treatment. However, recent updates from ongoing clinical trials have reported limited clinical activity of this combination. The datasets generated here may help identify key discrepancies between preclinical models and clinical samples that contribute to the lack of translation, and guide the development of improved therapeutic combinations.

Chronic Obstructive Pulmonary Disease (COPD) is a progressive respiratory disease characterized by airway inflammation and abnormal alveolar enlargement. It is the third leading cause of death around the world. Although extensive research efforts have been made, there is still no curable treatment available for lung tissue damage in patients with COPD. Therefore, it is of great significance to elucidate the mechanisms of tissue damage and repair in COPD. As the first barrier against environmental insults and pathogens, pulmonary epithelial cells play an essential role in regulating injury response and repair. However, how pulmonary epithelial cells contribute to irreversible alveolar destruction in COPD is not well understood. In this study, we elucidated the mechanisms of epithelial cell damage in both cigarette smoke-induced COPD and alpha1 antitrypsin deficiency (AATD)-associated genetic COPD. To investigate alveolar epithelial cell damage and repair in cigarette smoke-induced emphysema, a lineage tracing model was utilized to fluorescently label and chase alveolar type II (AT2) epithelial cells, the adult progenitor cells in the alveolar epithelium. An assessment of cigarette smoke-induced changes in cellular composition and regenerative capacity of the alveolar epithelial cells was performed. Cigarette smoke was found to impede the AT2-directed alveolar epithelial regeneration and repair process, and this impaired progenitor cell function was not restored after smoke cessation. Moreover, comparison analysis between stains that are sensitive and resistant to smoke-induced damage revealed that deficiency in lipid metabolism may contribute to the dysregulation of alveolar epithelial repair by AT2 cells. Restoring alveolar progenitor functions through lipid metabolism may serve as a novel therapeutic for alveolar destruction in smoke-induced COPD. To explore the mechanism of epithelial damage in AATD-associated genetic COPD, we utilized a PiZ (p.Glu342Lys) transgenic mouse model expressing human ZAAT protein. Morphometric analysis of PiZ lungs suggests that the accumulation of ZAAT polymers in the lung directly leads to the spontaneous development of emphysema. To investigate epithelial damage induced by zAAT accumulation, we isolated the epithelial cell population from the lung of PiZ mice. We identified epithelial-specific expression of cleaved caspase 3, indicating a direct cytotoxic effect of ZAAT in impairing epithelial function and inducing epithelial cell death. Future therapeutics could directly target the cytotoxicity of pulmonary epithelial cells in AATD to reduce lung tissue damage. Overall, our findings suggest that pulmonary epithelial damage plays an essential role in the pathogenesis of lung tissue damage in COPD. Future epithelial cell-based therapies may contribute to pulmonary re-epithelialization and tissue repair in both cigarette smoke-induced and AATD-associated COPD.

by Chan Tak Yee Bonita. / "July 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 121-137). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Indiana University-Purdue University Indianapolis (IUPUI) / Skeletal manifestations including short stature, osteoporosis, kyphoscoliosis, and tibial dysplasia cumulatively affect approximately 70% of patients with neurofibromatosis type 1 (NF1). Tibial pseudarthrosis, the chronic non-union of a spontaneous fracture, is a debilitating skeletal malady affecting young children with NF1. These non-healing fractures respond poorly to treatment and often require amputation of the affected limb due to limited understanding of the causative mechanisms. To better understand the cellular and molecular pathogenesis of these osseous defects, we have established a new mouse model which recapitulates a spectrum of skeletal pathologies frequently observed in patients with NF1. Nf1flox/-;Col2.3Cre mice, harboring Nf1 nullizygous osteoblasts on a Nf1+/- background, exhibit multiple osseous defects which are closely reminiscent of those found in NF1 patients, including runting (short stature), bone mass deficits, spinal deformities, and tibial fracture non-union. Through adoptive bone marrow transfer studies, we have demonstrated that the Nf1 haploinsufficient hematopoietic system pivotally mediates the pathogenesis of bone loss and fracture non-union in Nf1flox/-;Col2.3Cre mice. By genetic ablation of a single Nf1 allele in early myeloid development, under the control of LysMCre, we have further delineated that Nf1 haploinsufficient myeloid progenitors and osteoclasts are the culprit lineages mediating accelerated bone loss. Interestingly, conditional Nf1 haploinsufficiency in mature osteoclasts, induced by CtskCre, was insufficient to trigger enhanced lytic activity. These data provide direct genetic evidence for Nf1’s temporal significance as a gatekeeper of the osteoclast progenitor pool in primitive myelopoiesis. On the molecular level, we found that transforming growth factor-beta1 (TGF-β1), a primary mediator in the spatiotemporal coupling of bone remodeling, is pathologically overexpressed by five- to six- fold in both NF1 patients and in mice. Nf1 deficient osteoblasts, the principal source of TGF-β1 in the bone matrix, overexpress TGF-β1 in a gene dosage dependent fashion. Moreover, p21Ras dependent hyperactivation of the Smad pathway accentuates responses to pathological TGF-β1 signals in Nf1 deficient bone cells. As a proof of concept, we demonstrate that pharmacologic TβRI kinase inhibition can rescue bone mass defects and prevent tibial fracture non-union in Nf1flox/-;Col2.3Cre mice, suggesting that targeting TGF-β1 signaling in myeloid lineages may provide therapeutic benefit for treating NF1 skeletal defects.

Indiana University-Purdue University Indianapolis (IUPUI) / Signal Transducer and Activator of Transcription 3 (STAT3) is known to be related to bone metabolism. Mutation of STAT3 causes a rare disorder in which serum levels of IgE are elevated. This causes various skeletal problems similar to osteoporosis. To examine the effect of STAT3 in the osteoclast, we obtained two osteoclast specific STAT3 knockout mouse models: one using the CTSK promoter to drive Cre recombinase and another using a TRAP promoter. Examination of these mice at 8 weeks of age revealed a decreased trabecular bone volume in CTSK specific STAT3 knockout mice along with a slight decrease in osteoclast number in both CTSK and TRAP specific STAT3 knockout females. We also noticed changes in bone mineral density and bone mechanical strength in females. These data suggest that STAT3 plays a part in the function of the osteoclast.

Indiana University-Purdue University Indianapolis (IUPUI) / Autonomic innervation of the heart begins in utero and continues during the neonatal phase of life. A balance between the sympathetic and parasympathetic arms of the autonomic nervous system is required to regulate heart rate as well as the force of each contraction. Our lab studies the development of sympathetic innervation of the early postnatal heart in a conditional knockout (cKO) of Src homology protein tyrosine phosphatase 2 (Shp2). Shp2 is a ubiquitously expressed non-receptor phosphatase involved in a variety of cellular functions including survival, proliferation, and differentiation. We targeted Shp2 in post-migratory neural crest (NC) lineages using our novel Periostin-Cre. This resulted in a fully penetrant mouse model of diminished cardiac sympathetic innervation and concomitant bradycardia that progressively worsen. Shp2 is thought to mediate its basic cellular functions through a plethora of signaling cascades including extracellular signal-regulated kinases (ERK) 1 and 2. We hypothesize that abrogation of downstream ERK1/2 signaling in NC lineages is primarily responsible for the failed sympathetic innervation phenotype observed in our mouse model. Shp2 cKOs are indistinguishable from control littermates at birth and exhibit no gross structural cardiac anomalies; however, in vivo electrocardiogram (ECG) characterization revealed sinus bradycardia that develops as the Shp2 cKO ages. Significantly, 100% of Shp2 cKOs die within 3 weeks after birth. Characterization of the expression pattern of the sympathetic nerve marker tyrosine hydroxylase (TH) revealed a loss of functional sympathetic ganglionic neurons and reduction of cardiac sympathetic axon density in Shp2 cKOs. Shp2 cKOs exhibit lineage-specific suppression of activated pERK1/2 signaling, but not of other downstream targets of Shp2 such as pAKT (phosphorylated-Protein kinase B). Interestingly, restoration of pERK signaling via lineage-specific expression of constitutively active MEK1 (Mitogen-activated protein kinase kinase1) rescued TH-positive cardiac innervation as well as heart rate. These data suggest that the diminished sympathetic cardiac innervation and the resulting ECG abnormalities are a result of decreased pERK signaling in post-migratory NC lineages.

Indiana University-Purdue University Indianapolis (IUPUI) / Neurofibromatosis Type 1 (NF1) is a genetic disorder resulting from mutations in the NF1 tumor suppressor gene. Neurofibromin is the protein product of NF1 and functions as a negative regulator of Ras activity in both hematopoietic and vascular wall cells, which are critical for maintaining blood vessel homeostasis. NF1 patients are predisposed to chronic inflammation and premature cardiovascular disease, including development of large arterial aneurysms, which may result in sudden death secondary to their rupture. However, the molecular pathogenesis of NF1 aneurysm formation is completely unknown. Utilizing a novel model of Nf1 murine aneurysm formation, we demonstrate that heterozygous inactivation of Nf1 (Nf1+/-) results in enhanced aneurysm formation with myeloid cell infiltration and increased reactive oxygen species in the vessel wall. Using cell lineage-restricted transgenic mice, we show that loss of a single Nf1 allele in myeloid cells is sufficient to recapitulate the Nf1+/- aneurysm phenotype in vivo. Additionally, oral administration of simvastatin, a statin with antioxidant and anti-inflammatory effects, significantly reduced aneurysm formation in Nf1+/- mice. Finally, the antioxidant apocynin was administered orally and also resulted in a significant reduction of Nf1+/- aneurysms. These data provide genetic and pharmacologic evidence that neurofibromin-deficient myeloid cells are the central cellular triggers for aneurysm formation in a novel model of NF1 vascular disease, implicated oxidative stress as the key biochemical mechanisms of NF1 aneurysm formation and provide a potential therapeutic target for NF1 vasculopathy.

Indiana University-Purdue University Indianapolis (IUPUI) / The transcriptional repressor BCL6 has been shown to be essential for the differentiation of germinal center (GC) B cells and follicular T helper (TFH) cells. The interaction of TFH and GC B cells is necessary for the development of high affinity antibodies specific for an invading pathogen. Germline BCL6-deficient mouse models limit our ability to study BCL6 function in T cells due to the strong inflammatory responses seen in these mice. To overcome this, our lab has developed a new BCL6 conditional knockout (cKO) mouse using the cre/lox system, wherein the zinc finger region of the BCL6 gene is flanked by loxP sites. Mating to a CD4-Cre mouse allowed us to study the effects of BCL6 loss specifically in T cells, without the confounding effects seen in germline knockout models. Using this cKO model, we have reaffirmed the necessity of BCL6 for TFH differentiation, including its role in sustained CXCR5 surface expression, a signature marker for TFH cells. This model also allowed us to recognize the role of BCL6 in promoting the expression of PD-1, another key surface marker for TFH cells. Without BCL6, CD4+ T cells cannot express PD-1 at the high levels seen on TFH cells. Our discovery of DNMT3b as a target for BCL6 suggests BCL6-deficient T cells have increased DNA methyltransferase activity at the PD-1 promoter. This data establishes a novel pathway for explaining how BCL6, a transcriptional repressor, can activate genes. Experiments with the BCL6 cKO model have also established a role for BCL6 in naïve CD4+ T cell activation. Furthermore, we did not observe increased differentiation of other helper T cell subsets, in contrast to what has been reported elsewhere with germline BCL6-deficient models. Unexpectedly, we found decreased T helper type 2 (Th2) cells, whereas mouse models with a germline mutation of BCL6 have increased Th2 cells. These results indicate that BCL6 activity in non-T cells is critical for controlling T cell differentiation. Finally, using an HIV-1 gp120 immunization model, we have, for the first time, shown BCL6-dependent GCs to be limiting for antibody development and affinity maturation in a prime-boost vaccine scheme.

Indiana University-Purdue University Indianapolis (IUPUI) / Regulatory T (Treg) cells represent an important layer of immune-regulation indispensible for curtailing exuberant inflammatory responses and maintaining self-tolerance. Treg cells have translational potential for autoimmunity, inflammation, transplantation and cancer. Therefore, delineating the molecular underpinnings underlying the development, suppressor function and stability of Tregs is particularly warranted. The transcriptional repressor Bcl6 is a critical arbiter of helper T cell fate, promoting the follicular helper (Tfh) lineage while repressing Th1, Th2 and Th17 differentiation. Bcl6-deficient mice develop a spontaneous and severe Th2-type inflammatory disease including myocarditis and pulmonary vasculitis, suggesting a potential role for Bcl6 in Treg cell function. Bcl6-deficient Treg cells are competent in controlling Th1 responses, but fail to control Th2 inflammation in an airway allergen model. Importantly, mice with Bcl6 deleted specifically in the Treg lineage develop severe myocarditis, thus highlighting a critical role for Bcl6 in Treg-mediated control of Th2 inflammation. Bcl6-deficient Tregs display an intrinsic increase in Th2 genes and microRNA-21 (miR-21) expression. MiR-21 is a novel Bcl6 gene target in T cells and ectopic expression of miR-21 directs Th2 differentiation in non-polarized T cells. MiR-21 is up-regulated in mouse models of airway inflammation and also in human patients with eosinophilic esophagitis and asthma. Thus, miR-21 is a clinically relevant biomarker for Th2-type pathologies. Our results define a key function for Bcl6 in repressing Gata3 function and miR-21 expression in Tregs, and provide greater understanding of the control of Th2 inflammatory responses by Treg cells.