Apoptosis is a highly conserved form of programmed cell death in multicellular organisms where a cell activates caspase proteases to trigger its own demise. It has critical functions under physiological conditions in removing superfluous or damaged cells from a healthy organism. However, dysregulated apoptosis plays an important role in diseases: improper or elevated apoptosis leads to diseases of cell loss, such as diabetes or neurodegeneration, while deficiencies in apoptosis can contribute to tumor cell growth and survival.

While the importance of apoptosis during late embryogenesis is well-established, its function during the earliest stages of development, namely the exit of embryonic stem cells (ESC) from the pluripotent state, is unclear. Here, I discovered that apoptosis plays a critical role in removing slow-differentiating murine ESC from the total cell population. This is initiated by p53-dependent upregulation of the Fas death receptor on straggling ESCs, which then triggers apoptosis specifically in these cells. An inability to initiate apoptosis, by transient knockdown or genetic deletion of components of this pathway, causes retention of slow-differentiating ESCs and a global delay in differentiation, both in vitro and in an in vivo teratoma model. Thus, apoptosis is crucial in promoting the efficient differentiation of ESCs.

While apoptosis is important for normal development, elevated levels due to cellular stress can cause inappropriate cell loss and degeneration, leading to disease. One such example is retinitis pigmentosa, an inherited disease where rod photoreceptors are progressively lost from the neural retina, eventually leading to blindness. There is increasing evidence that accumulation of unfolded proteins within the endoplasmic reticulum (ER) of rod photoreceptors causes ER stress and subsequent cell death. Here, I utilize a novel small molecule inhibitor to show that inhibition of IRE1α, an ER transmembrane kinase/endoribonuclease that can signal both adaptation and apoptosis in response to ER stress, preserves rod photoreceptor viability and function in two rodent models of ER stress-induced photoreceptor degeneration.

These findings reveal new insights into the role of apoptosis under both normal and stressed conditions, and may have implications for human diseases such as cancer and neurodegeneration.

A critical regulator of the unfolded protein response (UPR), the IRE1α kinase/endoribonuclease promotes adaptation or apoptosis depending on the level of upstream endoplasmic reticulum (ER) stress. The UPR is implicated in multiple cancer types, but whether IRE1α signaling is beneficial or detrimental to tumor growth remains controversial. We hypothesized that pancreatic neuroendocrine tumors (PanNETs), which are highly secretory neoplasms prone to protein-folding stress, would be one type of cancer that would be particularly sensitive to changes in UPR signaling. We used a series of genetic and pharmacologic approaches to modulate IRE1α in xenograft and spontaneous genetic (RIP-Tag2) mouse models of PanNET. We found that IRE1α signaling is carefully titrated in PanNETs in vivo such that hyperactivating or inhibiting its enzymatic activity cripples tumor growth and survival. Importantly, we found that a monoselective IRE1α kinase inhibitor dramatically decreased tumor burden and prolonged animal survival in two preclinical PanNET models by eliminating the adaptive signals that would otherwise counteract an ER stress-induced apoptotic cascade. Here, I also present preliminary data suggesting that PanNETs may be similarly sensitive to modulation of PERK, a second regulatory arm of the UPR, as demonstrated by decreased tumor burden in response to both genetic and pharmacological inhibition of PERK. Our results provide a strong rationale for therapeutically targeting IRE1α in PanNETs and other cancers that experience high levels of ER stress, as well as further investigations of the effects of targeting PERK.

The control of cell number depends on the balance between pro- and anti-death signals. The Drosophila PDGF/VEGF Receptor (PVR) provides an anti-apoptotic signal that is necessary for the survival of both embryonic hemocytes and the hemocyte-like Kc cell line, thus providing a model system for uncovering other mammalian PDGF/VEGF receptors regulated signaling pathways for hematopoietic cell survival. RNAi silencing of PVR in Kc cell leads to inactivation of both Ras/Erk and Akt/Tor, and reactivation of one of these pathways or inhibiting a pro-death regulator, leads to restoration of cell numbers. To identify other pathways, which involved in hemocyte survival, a genome-wide RNAi screen to identify suppressors and enhancers of the PVR RNAi-mediated cell number reduction had been conducted previously. The screen provided the basis for this thesis project, as we subsequently characterized the biological and molecular roles of genes identified in the screen. Among the suppressors identified are many known tumor suppressors and negative regulators of the Akt/Tor and Ras/Erk pathways, and as well as heterodimeric nuclear hormone receptor- ecdysone receptor (EcR) and ultraspiracle (usp). Loss of EcR or usp suppresses cell death in a PVR-silenced background, and expression of dominant negative EcR rescues hemocyte survival in PVR null mutant embryos. Ecdysone stimulation in Kc cells triggers cell death, possibly by the induction of reaper and E93. When trying to assess the relationship of EcR/usp or ecdysone signaling with the PVR signaling pathway, biochemistry data suggest that both function in a parallel fashion. Interestingly, PVR is required for ecdysone-induced upregulation of EcR, suggesting a regulatory balance between pro-survival PVR- and pro-death ecdysone signaling that stabilizes cell lifespan. These studies suggest a novel role of ecdysone signaling in Drosophila embryonic development and blood cell survival, and emphasize parallels with the homologous vertebrate rexinoid receptor (RXR) that acts in pro-death signaling and tumor suppression. In addition, novel PVR suppressors identified in the screen, and their mammalian counterparts, may play a role in cell number control and tumor suppression.

The endoplasmic reticulum (ER) is the main site in the cell for the folding and processing of secreted proteins. Various physiological and pathological processes can overwhelm the protein folding capacity of the ER. This condition, referred to as "ER stress, typically triggers the activation of the Unfolded Protein Response Pathway (UPR). The activation of this pathway results in expansion of the ER, an increase in chaperone proteins, inhibition of cap-dependent translation, and if homeostasis is not reestablished, apoptosis.

ER stress-induced apoptosis, secondary to a buildup of misfolded proteins, leads to cell loss in a number of human degenerative diseases including ALS, Parkinson, and type II diabetes. In contrast, cancer cells and viruses manipulate the cytoprotective aspects of the UPR, aberrantly promoting cell survival. Therefore, understanding how to manipulate the UPR's cytoprotective/cytotoxic threshold could lead to new treatment approaches for a wide variety of unrelated diseases.

Despite its important role in such a wide variety of diseases, the mechanisms underlying ER stress-induced apoptosis are poorly understood. Utilizing an unbiased biochemical system, I have identified the ER stress-induced pro-apoptotic effectors that signal Bax and Bak at the mitochondrion, BID and CRK, and the mechanisms behind their activation up irremediable ER stress.

In addition, the study of viral manipulation of host machinery has historically provided great insight into cellular biology. Viral infection is a biologically relevant system in which to study the cytoprotective/cytotoxic threshold of the UPR. A large lytic virus, such as Kaposi's Sarcoma Herpes Virus (KSHV or HHV-8), must be able to increase the folding capacity of the ER in order to accommodate the acute influx of viral protein during replication, while coordinately inhibiting the apoptotic consequences of intense ER stress. In this study, I have determined that KSHV inhibits UPR signaling through the IRE1 transmembrane kinase/endoribonuclease during lytic infection and is able to expand the ER through a novel function. These findings reveal new insights into the cytoprotective/cytotoxic regulation of the UPR and novel functions of lytic KSHV. These findings have implications for many diseases aside from viral infection, including type II diabetes, cancer, and neurodegeneration.

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