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XPR1 is the only annotated phosphate exporter protein in humans. Recent studies provide mechanistic clues to its cellular function; three posit non-export mechanisms to regulate phosphate homeostasis, while six present high-resolution cryo-EM data supporting a bona fide phosphate channel mechanism controlled by intracellular phosphate levels.

The use of a high-affinity VHL ligand allows the development of chimeric molecules that promote the association of ERRα or RIPK2 with the VHL E3 ubiquitin ligase complex, resulting in protein degradation. The current predominant therapeutic paradigm is based on maximizing drug-receptor occupancy to achieve clinical benefit. This strategy, however, generally requires excessive drug concentrations to ensure sufficient occupancy, often leading to adverse side effects. Here, we describe major improvements to the proteolysis targeting chimeras (PROTACs) method, a chemical knockdown strategy in which a heterobifunctional molecule recruits a specific protein target to an E3 ubiquitin ligase, resulting in the target's ubiquitination and degradation. These compounds behave catalytically in their ability to induce the ubiquitination of super-stoichiometric quantities of proteins, providing efficacy that is not limited by equilibrium occupancy. We present two PROTACs that are capable of specifically reducing protein levels by >90% at nanomolar concentrations. In addition, mouse studies indicate that they provide broad tissue distribution and knockdown of the targeted protein in tumor xenografts. Together, these data demonstrate a protein knockdown system combining many of the favorable properties of small-molecule agents with the potent protein knockdown of RNAi and CRISPR.

Conditional degron tags (CDTs) are a powerful tool for target validation that combines the kinetics and reversible action of pharmacological agents with the generalizability of genetic manipulation. However, successful design of a CDT fusion protein often requires a prolonged, ad hoc cycle of construct design, failure, and re-design. To address this limitation, we report here a system to rapidly compare the activity of five unique CDTs: AID/AID2, IKZF3d, dTAG, HaloTag, and SMASh. We demonstrate the utility of this system against 16 unique protein targets. We find that expression and degradation are highly dependent on the specific CDT, the construct design, and the target. None of the CDTs leads to efficient expression and/or degradation across all targets; however, our systematic approach enables the identification of at least one optimal CDT fusion for each target. To enable the adoption of CDT strategies more broadly, we have made these reagents, and a detailed protocol, available as a community resource. Conditional Degron Tags are a valuable tool to validate and study novel therapeutic targets. Here, the authors compared 5 orthogonal tags across 16 unique proteins and provide a panel of vectors for users to systematically screen the tags with their own protein of interest.

The objective of this work was to find a rapid, high-yield process to obtain an aqueous stable colloid suspension of cellulose nanocrystals/whiskers. Large quantities are required since these whiskers are designed to be extruded into polymers in the production of nano-biocomposites. Microcrystalline cellulose (MCC), derived from Norway spruce (Picea abies), was used as the starting material. The processing parameters have been optimized by using response surface methodology. The factors that varied during the process were the concentration of MCC and sulfuric acid, the hydrolysis time and temperature, and the ultrasonic treatment time. Responses measured were the median size of the cellulose particles/whiskers and yield. The surface charge as calculated from conductometric titration, microscopic examinations (optical and transmission electron microscopy), and observation of birefringence were also investigated in order to determine the outcome (efficiency) of the process. With a sulfuric acid concentration of 63.5% (w/w), it was possible to obtain cellulose nanocrystals/whiskers with a length between 200 and 400 nm and a width less than 10 nm in approximately 2 h with a yield of 30% (of initial weight).

XPR1 is the only annotated phosphate exporter protein in humans. Recent studies provide mechanistic clues to its cellular function; three posit non-export mechanisms to regulate phosphate homeostasis, while six present high-resolution cryo-EM data supporting a bona fide phosphate channel mechanism controlled by intracellular phosphate levels.

Despite advances in precision medicine, the clinical prospects for patients with ovarian and uterine cancers have not substantially improved. Here, we analyzed genome-scale CRISPR-Cas9 loss-of-function screens across 851 human cancer cell lines and found that frequent overexpression of SLC34A2-encoding a phosphate importer-is correlated with sensitivity to loss of the phosphate exporter XPR1, both in vitro and in vivo. In patient-derived tumor samples, we observed frequent PAX8-dependent overexpression of SLC34A2, XPR1 copy number amplifications and XPR1 messenger RNA overexpression. Mechanistically, in SLC34A2-high cancer cell lines, genetic or pharmacologic inhibition of XPR1-dependent phosphate efflux leads to the toxic accumulation of intracellular phosphate. Finally, we show that XPR1 requires the novel partner protein KIDINS220 for proper cellular localization and activity, and that disruption of this protein complex results in acidic \"vacuolar\" structures preceding cell death. These data point to the XPR1-KIDINS220 complex and phosphate dysregulation as a therapeutic vulnerability in ovarian cancer.

Modern biomedical research has unveiled many of the complicated processes that underlie life at the most basic of level of cells. This enterprise has shown reversible protein phosphorylation, mediated by kinases, is an integral process whose mis-regulation causes many diseases, including cancer. Current therapeutic strategies targeting kinases have been focused on inhibiting enzymatic activity, and this has led to many approved therapies that extend the life and livelihood of many people. This thesis explores the limitations of these strategies and a novel chemical biology tool to overcome them. In the first chapter, a brief history of kinases highlights how modern thinking focuses on kinase activity but ignores additional functions of protein kinases. The onco-kinase BCR/Abl is one such example: non-kinase roles of this protein are implicated in maintaining the disease and preventing cure even when the kinase activity is efficiently inhibited. A second example is the pseudokinase ROR2 and pseudokinases in general. These proteins share common structural features of kinases yet are enzymatically inactive and participate in important signaling programs within the cell. These two examples illustrate how inhibition is a limited paradigm of drug discovery. Chapter two exemplifies recent advances in a strategy to overcome the limitations of inhibition. This strategy is called proteolysis targeting chimera, or PROTAC, and is based on heterobifunctional small molecules which bind to the protein target and recruit it to E3 ubiquitin ligases. The target protein is then ubiquitinated and degraded. While previous iterations of PROTACs have been rather unimpressive, this chapter highlights the degradation of a protein kinase as well as a nuclear hormone receptor. These PROTAC molecules are unprecedented in their potency, selectivity, and drug-likeness. The chapter concludes by discussing the many recent examples of PROTACs and their application in research and, soon, therapeutic interventions. Chapter three then asks a basic and important question about PROTAC design. In designing potent degraders, minor structural changes in the molecule can lead to drastic effects on protein degradation. This chapter explores that phenomena, first by using a model system in which PROTAC geometry is finely tuned for degradation. It is shown that the discriminating factor between poor and potent PROTACs is the ability to form a stable ternary complex between the target, the PROTAC, and the E3 ligase. The best PROTACs induce protein:protein interactions between the target and E3 ligase, stabilizing the complex and leading to more potent degradation. In the second part of chapter three, PROTACs are explored which bind to many different kinase targets, but only degrade a subset of possible targets. Again, the discriminating factor between degraded and non-degraded proteins appear to be protein:protein interactions unique to the degraded proteins. This chapter offers biophysical explanations for commonly observed phenomena, and aids in developing design principles for PROTAC molecules. Having shown PROTAC molecules to be a strategy for potent protein degradation in chapter two and enhancing the understanding of that platform in chapter three, chapter four returns to the two examples listed above. First, potent degradation of BCR/Abl is achieved through a PROTAC designed to target the allosteric site. Next, these compounds are used in initial assays to explore functions of BCR/Abl that are affected by either inhibition or degradation of the protein. Finally, initial studies in patient-derived stem cells are presented. While the viability of these cells is reduced by PROTACs, more nuanced work must be done to highlight differences between degradation and inhibition of BCR/Abl. Second, initial efforts are made to develop ligands for the pseudokinase ROR2. While these compounds may not have activity on their own, they could be converted into PROTAC molecules which would deactivate all functions of ROR2. A thermal shift assay is used to identify potential ligands of ROR2 which bind with modest affinity. Future work will explore these compounds as well as developing high-throughput screens for pseudokinases in general. While previous iterations were limited in potency, this study demonstrates that PROTAC molecules can be versatile chemical tools. While outside the scope of this thesis, PROTACs also show promise as therapeutic interventions. By degrading the entire protein rather than just inhibiting one functionality, PROTACs may expand what is currently considered druggable. Many literature examples point to this possibility. With the first PROTAC molecules soon to enter clinical trials, this study highlights the reasons for the considerable excitement surrounding this technology.

The objective of this work was to find a rapid, high-yield process to obtain an aqueous stable colloid suspension of cellulose nanocrystals/whiskers. Large quantities are required since these whiskers are designed to be extruded into polymers in the production of nano-biocomposites. Microcrystalline cellulose (MCC), derived from Norway spruce (Picea abies), was used as the starting material. The processing parameters have been optimized by using response surface methodology. The factors that varied during the process were the concentration of MCC and sulfuric acid, the hydrolysis time and temperature, and the ultrasonic treatment time. Responses measured were the median size of the cellulose particles/whiskers and yield. The surface charge as calculated from conductometric titration, microscopic examinations (optical and transmission electron microscopy), and observation of birefringence were also investigated in order to determine the outcome (efficiency) of the process. With a sulfuric acid concentration of 63.5% (w/w), it was possible to obtain cellulose nanocrystals/whiskers with a length between 200 and 400 nm and a width less than 10 nm in approximately 2 h with a yield of 30% (of initial weight).

The DNA-incorporating nucleoside analogs azacytidine (AZA) and decitabine (DEC) have clinical efficacy in blood cancers, yet the precise mechanism by which these agents kill cancer cells has remained unresolved - specifically, whether their anti-tumor activity arises from conventional DNA damage or DNA hypomethylation via DNA methyltransferase 1 (DNMT1) inhibition. This incomplete mechanistic understanding has limited their broader therapeutic application, particularly in solid tumors, where early clinical trials showed limited efficacy. Here, through the assessment of drug sensitivity in over 600 human cancer models and comparison to a non-DNA-damaging DNMT1 inhibitor (GSK-3685032), we establish DNA hypomethylation, rather than DNA damage, as the primary killing mechanism of AZA and DEC across diverse cancer types. In further support of an epigenetic killing mechanism, CRISPR drug modifier screens identified a core set of chromatin regulators, most notably the histone deubiquitinase USP48, as AZA and DEC protective factors. We show that USP48 is recruited to newly hypomethylated CpG islands and deubiquitinates non-canonical histones, establishing USP48 as a key molecular link between the two components of epigenetic gene regulation: DNA methylation and chromatin modification. Furthermore, loss of , which occurs naturally through biallelic deletions in human cancers, sensitized both hematologic and solid tumors to DNMT1 inhibition and . Our findings elucidate the epigenetic mechanism of action of AZA and DEC and identify a homeostatic link between DNA methylation and chromatin state, revealing new therapeutic opportunities for DNMT1 inhibitors in solid tumors.

Fusions between protein-coding genes are common oncogenic drivers across cancers, typically pairing a proto-oncogene with partner that does not independently drive cancer. In all therapeutically actionable fusions, the proto-oncogene is the drug target, the contributions to oncogenicity of the fusion partner have largely been ignored. We studied the role of BRAF fusion partners and found that they are necessary for transformation. In the setting of KIAA1549::BRAF, the most common fusion protein across brain tumors, we found that KIAA1549 is necessary for the oncogenicity of KIAA1549::BRAF and engenders a striking and specific dependency on the protein O-mannosyltransferase complex (POMT1/2). Specifically, we show that genetic silencing or pharmacologic inhibition of the protein O-mannosyltransferase complex (POMT1/2) reverses fusion-induced transformation, thereby representing a novel and MAPK independent therapeutic target. Furthermore, POMT1/2 is required to glycosylate and enable maturation of the K::B fusion protein. These findings represent a proof-of-concept for targeting the partners in oncogenic fusions as a potential cancer therapeutic strategy.