Aberrant signaling from mutations at K171 in the kinase domain of IKKΒ is known to occur in multiple myeloma, spleen marginal zone lymphoma, and mantle cell lymphoma. Previous work demonstrated that constitutive kinase activation stimulates Signal Transducer and Activator of Transcription 3 (STAT3). Using mass spectrometry, we identified K147 as a site for K63-linked ubiquitination that only occurs in mutant IKKB K171E. The crosstalk between this IKKβ mutant and the Janus Kinases (JAKs) pathway was further investigated by using a IL-6-responsive cell line, which showed that IKKβ 171E mutants stimulates the release of IL-6 into conditioned media. This suggests that it triggers an autocrine loop in which IL-6 is secreted and binds to its own IL-6 receptor-gp130 complex, resulting in JAK activation. Lastly, proteins associated with K63-only-ubiquitinated IKKβ K171E were examined using mass spectrometry, and using proteomic analysis, activation of proteins and pathways involved in proliferative responses were found. Knowing that cancers harboring the K171E mutation on IKKβ can also activate STAT3 along with p44/42 MAPK (Erk1/2), the possibility of using MAPK and JAK inhibitors, or specific ubiquitination inhibitors as treatments should be investigated.

Inhibitor of kappaB kinase beta (IKKbeta) is the master regulatory kinase that modulates canonical nuclear factor kappaB (NFkappaB) inflammatory activation. Under inflammatory conditions, IKKbeta is phosphorylated at Ser177/Ser181 sites in the kinase domain, which activates the kinase function of the protein. IKKbeta, in turn, phosphorylates its downstream substrate IkappaBalpha, which is subsequently degraded. This event releases NFkappaB, which is normally sequestered in the cytoplasm, to translocate into the nucleus to activate transcription of inflammation-related genes.

In the first chapter of this dissertation, mutations of Lys171 located in the kinase domain of IKKbeta are shown to lead to increase the phosphorylation of Ser177/Ser181 sites and result in the upregulation of the kinase activity of IKKbeta. These mutations had been initially identified in tumor samples of Multiple Myeloma, Spleen Marginal Zone Lymphoma and Mantle Cell Lymphoma. Via site-directed mutagenesis, we constructed the K171E mutation in IKKbeta. This study further demonstrated that IKKbeta is conjugated to Lys63-linked ubiquitin chains, in which the activating K171E mutation upregulated this modification at Lys147. The main discovery of this project is that the K171E mutation in IKKbeta induces the activation of STAT3 in the absence of exogenous Interleukin-6 in HEK293T cells. Via LC-MS/MS, we identified various lysine ubiquitination sites in full length activated IKKbeta, in which some of these negatively regulate the phosphorylation of Ser177/Ser181. Ubiquitination of Lys147 in activated IKKbeta, located in proximity to the activation loop in the kinase domain, is critical to induce STAT3 activation.

The second chapter of this dissertation demonstrates that IKKbeta K171E relies on JAK, gp130, and TAK1 to signal to STAT3. In addition, we utilized a method termed Bio-ID proximity dependent biotinylation to identify proteins interacting with activated IKKBeta, in which UBC13 and LRPPRC were identified as interacting partners. Via siRNA, we showed that UBC13 and LRPPRC possibly negatively regulated STAT3 activation induced by IKKbeta K171E. Interestingly, IKKbeta K171E depends upon UBE2V2 cofactor of UBC13, previously demonstrated to be involved in modulation of DNA damage repair activation, to activate STAT3. Lastly, the oncogenic potential of K171E mutation is demonstrated by the transformation of murine myeloid 32D cells into Interleukin-3-independent phenotype. In conjunction, these results illustrate a novel mechanism by which the loss of Lys171 in IKKbeta induces the activation of STAT3 in the absence of cytokines and growth factors.

The third chapter of this dissertation entails a review article describing that the misregulated expression of members of the ubiquitination cascade contributes to cancer progression and metastasis. Ubiquitination largely serves as a degradation mechanism of proteins. Ubiquitin signaling is further involved in additional cellular processes such as the activation of NFκB inflammatory response and DNA damage repair. In this review, we highlight the E2 ubiquitin conjugating enzymes, E3 ubiquitin ligases and Deubiquitinases that support the proliferation, migration, stemness and metastasis of a plethora of cancers, including their contribution to poor clinical prognosis and modulation of pluripotent cancer stem cells attributed to metastasis. We further describe mutations in E3 ubiquitin ligases that support the proliferation and adaptation to hypoxia of some cancers. Thus, this review illustrates how tumors exploit the members of the vast ubiquitin signaling pathways to support oncogenic signaling for the purposes of survival and metastasis.

The fourth chapter of this dissertation entails a review article related to mutations in Fibroblast Growth Factor Receptors (FGFRs) in cancer and developmental disorders. This comprehensive review describes approximately 200 different mutations in FGFRs in diverse cancers, in which some of these mutations have been found to contribute to developmental syndromes. In addition, this review shows the occurrence of chromosomal translocations that result in fusion proteins involving FGFR’s in cancers, and it illustrates the aberrant signaling mechanisms activated by these oncogenes.

Fibroblast Growth Factor Receptors (FGFRs) are critical for cell proliferation and differentiation. Mutation and/or translocation of FGFRs lead to aberrant signaling that often results in developmental syndromes or cancer growth. As sequencing of human tumors becomes more frequent, so does the emergence of FGFR translocations and fusion proteins. The research conducted in this work will focus on a frequently identified fusion protein between FGFR3 and transforming acidic coiled-coil containing protein 3 (TACC3). As detailed in this dissertation, it is apparent that the fused coiled-coil TACC3 domain results in constitutive phosphorylation of key activating FGFR3 tyrosine residues. The presence of the TACC coiled-coil domain leads to increased and altered levels of FGFR3 activation, fusion protein phosphorylation, MAPK pathway activation, nuclear localization, cellular transformation, and IL3-independent proliferation. Introduction of K508R FGFR3 kinase dead mutation abrogates these effects, except for nuclear localization which is due solely to the TACC3 domain. We further demonstrate that the oncogenic effects initiated by FGFR3-TACC3 are dependent on the overactivation of the MAPK pathway and localization of FGFR3-TACC3 to the secretory pathway or the plasma membrane. The activation of the MAPK pathway is essential for cell transformation but involvement in the cell cycle via the canonical TACC3 pathways is not. Additionally, we have shown that kinase inhibitors for MEK (Trametinib) and FGFR (BGJ398) are effective in blocking cell transformation and MAPK pathway upregulation. The need for precision medicine is evidenced by the different effects these inhibitors have against various FGFR3-TACC3 breakpoints. The existence of FGFR3-TACC3 fusions in human cancers creates additional challenges and opportunities for identifying effective treatment strategies. The development of such personalized medicines will be essential in treating patients who harbor oncogenic drivers such as FGFR3-TACC3.

Prostate cancer is the second most common cause of cancer death for U.S. men. While androgen deprivation therapy is effective for treating many patients, some patients’ prostate cancer nevertheless progress into a lethal form which does not depend on androgen. The mechanism behind androgen-independent prostate cancer remains unknown; however, previous data suggests cancer stem cells are a potential cause. To confirm the presence of cancer stem cells in prostate cancer and to explore the timing of prostate cancer stem cell detection, patient needle biopsy samples confirmed as containing prostate cancer were stained with six stem cell markers. Stem cell markers CD133, LGR5, ALDH7A1, CD44, Nanog and Oct4 were found to react with the exact structures used by pathologists to diagnose prostate cancer in H&E staining procedures. The six markers are positively expressed in all available patient needle biopsy samples with the exception of CD44. CD44 is not expressed in one of the samples. The expression of six stem cell markers confirms that the adenocarcinoma structures in prostate cancer are composed of stem cell-like cells. The stem cell nature of cancer cells in early prostate cancer provides insight to a new treatment, differentiation therapy, to target the disease.

IKKβ, which is the main protein kinase in the canonical NF-κB signaling, regulates cytokine production, inflammation, cell proliferation, survival and the responses to cellular stress. In the previous research by Gallo LH. et al. [1] showed that IKKβ K171E mutant undergoes the K-63 linked ubiquitination at K147 site as well as it activates STAT3 signaling pathway. We aimed to investigate the mechanism why which the mutant IKKβ is mediating the signaling crosstalk, by investigating interacting proteins, specifically an E3 ubiquitin ligase that catalyzing the K-63linked ubiquitination of the mutant IKKβ. We utilized a new technique called BioID that enables the selective capture of proximal interacting proteins by the mutant biotin ligase fused to a protein of interest, the IKKβ K171E 4KR. Here, the results of the identified interacting proteins including E3 ubiquitin ligases which may be involved in the signaling crosstalk are shown. This project has potential to characterize the mechanism by identifying the E3 ligase, which is a promising cancer therapeutics for patients expressing activated mutants of IKKβ which were originally identified in the hematopoietic cancers patients.

Chromosomal translocations have been a focus in human cancer research. Since the exponential advancement in sequencing technology, a number of actionable fusion proteins have emerged as oncogenic drivers, serving as potential targets for personalized treatments. As the second most common form of liver cancer, Intrahepatic Cholangiocarcinoma (ICC) is as a universally fatal disease with limited treatment options. Recent studies have revealed that translocation of Fibroblast Growth Factor Receptor 2 (FGFR2) to Periphilin 1 (PPHLN1) occurs in 16% ICC cases and FGFR2-PPHLN1 alone is sufficient in driving carcinogenesis. The clinically discovered FGFR2-PPHLN1 preserves the N-terminal structure of FGFR2 containing the extracellular domain, transmembrane domain, as well as an intact kinase domain; joined at the C-terminus of FGFR2 is the PPHLN1 moiety that contains a coiled-coil domain. In this study, we demonstrate that functions of FGFR2-PPHLN1 are achieved through constitutive receptor phosphorylation, thereby activating the canonical MAPK/ERK, JAK/STAT3 and PI3K/AKT pathways. Furthermore, we show that the introduction of an activating mutation in the kinase domain N549/550K on the fusion showed an even higher activation of these pathways, while the kinase dead K517/518R mutation completely abolished the signaling activities. These findings collectively reinforce the essential function of the kinase activity of the FGFR2 moiety. We also show that the oncogenicity of FGFR2-PPHLN1 is dependent upon the coiled-coil PPHLN1 domain. Finally, we determine the localization of this fusion protein and its entrance into the secretory pathway on its transforming ability by engineering a myristylation (Myr) signal, introducing a Myr G2A mutation, as well as deletion of the delta signal.

Fibroblast Growth Factor Receptors (FGFRs) are critical for cell proliferation and differentiation. Mutation and/or translocation of FGFRs lead to aberrant signaling that often results in developmental syndromes or cancer growth. As sequencing of human tumors becomes more frequent, so does the emergence of FGFR translocations and fusion proteins. The research conducted in this work will focus on a frequently identified fusion protein between FGFR3 and transforming acidic coiled-coil containing protein 3 (TACC3). Through titanium dioxide-based phosphopeptide enrichment (TiO2)-liquid chromatography (LC)-high mass accuracy tandem mass spectrometry (MS/MS), it is apparent that the fused coiled-coil TACC3 domain results in constitutive phosphorylation of key activating FGFR3 tyrosine residues. Fusion of FGFR3 and TACC3 also results in MAPK pathway activation, nuclear localization, and cell transformation. Introduction of K508R FGFR3 kinase dead mutation abrogates these effects, except for nuclear localization which is due solely to the TACC3 domain. FGFR3-TACC3 also produces IL-3 independent growth and cell proliferation. Taken together, these results suggest that FGFR3 kinase activity is essential for the oncogenic effects of the FGFR3-TACC3 fusion protein and could serve as a therapeutic target.

Chromosomal translocations of neurotrophic receptor tyrosine kinases (NTRKs) lead to various pediatric cancers. While tyrosine kinase inhibitors such as Larotrectinib and Entrectinib remain a major course of treatment, relapse of tumor still occur, suggesting the need for new therapeutic targets. This work focuses on a novel translocation identified in cases of Infantile Fibrosarcoma, which contains the coiled-coil multimerization domain of Echinoderm Microtubule-like protein 4 (EML4) and the tyrosine kinase domain of Neurotrophic receptor tyrosine kinase 3 (NTRK3). Activation of EML4-NTRK3 relies on both the tyrosine kinase activity of NTRK3 and the salt bridge stabilization in the coiled-coil domain of EML4. As shown in focus formation assays, the tyrosine kinase activity of NTRK3 is essential for the biological activation of EML4-NTRK3. Furthermore, EML4-NTRK3 activates downstream signaling pathways MAPK/ERK, JAK/STAT3 and PKC/PLC. The importance of the salt bridge interactions within EML4-NTRK3 was shown as the disruption of all three salt bridge interactions blocks downstream activation, biological activity and the ability to heteromultimerize with EML4. This work also demonstrates that EML4-NTRK3 is localized in the cytoplasm and fails to associate with microtubules. Taken together, these data suggest several therapeutic strategies for Infantile Fibrosarcoma cases bearing EML4-NTRK3 fusion: inhibition of salt bridge interactions within EML4 and design of mimetic peptides of EML4 coiled coil.

Neurotrophic receptor tyrosine kinases (NTRKs) belong to the receptor tyrosine kinase (RTK) family. NTRKs are responsible for the activation of multiple downstream signaling pathways that regulate cell growth, proliferation, differentiation, and apoptosis. NTRK-associated mutations lead to aberrant activation of these downstream pathways and often result in oncogenesis. This study characterizes the NACC2-NTRK2 oncogenic fusion protein that leads to pediatric astrocytoma and glioblastoma. This fusion joins the broad-complex, tramtrack, and bric-a-brac (BTB) domain of Nucleus Accumbens-associated protein 2 (NACC2) with the transmembrane helix and tyrosine kinase domain of NTRK2. This work focuses on identifying domains critical to the fusion protein activity and possible methods to deactivate the fusion. NACC2-NTRK2 is able to transform NIH3T3 cells. Such activity depends on the NTRK2 kinase domain phosphorylation that activates signaling pathways including MAPK, JAK/STAT, and PLCγ. The activation of the NTRK2 kinase domain relies on the multimerization of the NACC2 BTB domain. A BTB domain charged pocket mutation and a monomer core mutation result in deactivation of the kinase domain and abrogates the ability to transform NIH3T3 cells, suggesting that BTB domain inhibition could be a potential treatment for the NACC2-NTRK2 induced cancer. The undefined region of NACC2 at residues 120-418 is responsible for forming stronger multimers. Once removed, the NACC2-NTRK2 multimer is vulnerable to SDS denaturation and leads to reduced activity of the fusion. Lastly, the removal of the transmembrane helix leads to higher activation of the NACC2-NTRK2 fusion. A protein stability assay indicates that transmembrane deletion prevents NACC2-NTRK2 degradation.

Single molecule Förster resonance energy transfer (smFRET) is a powerful biophysical structural method for investigating the structural dynamics and interactions of biomolecules at the single-molecule level. By utilizing fluorophores strategically placed on proteins, smFRET enables the measurement of distance changes between specific sites, providing insights into conformational changes, binding events, and molecular interactions. In the Komives lab, smFRET has been utilized to prove the dynamics of Nuclear Factor-kappa B (NFκB), a pivotal transcription factor involved in immune response and inflammation. Specifically, studies have focused on the NFκB p50/RelA heterodimer, revealing valuable insights into its conformational changes and DNA binding dynamics. The Komives lab was able to purify fulllength NFκB, including the intrinsically disordered transcription activation domain (TAD), and found that the TAD enhances the binding affinity of NFκB to DNA, with effect being more pronounced in binding to nonconsensus DNA sequences. However, a significant knowledge gap remains regarding the binding and dissociation mechanism of NFκB during DNA binding in the presence of multiple nearby binding sites. For my thesis I propose addressing this gap by combining smFRET techniques with optical tweezers using the Lumicks C-Trap system to visualize both the DNA substrate (lambda DNA) and the NFκB. This integrative approach holds promise for advancing our understanding of NFκB mediated transcriptional regulation and may offer new avenues for therapeutic intervention in diseases associated with dysregulated NFκB signaling pathways. I propose to do this by first establishing a working prism-based smFRET TIRF microscope, which hadn’t been working for two years prior to my work with it. Then using the established set up to investigate interdomain motions of fulllength NFκB, and combining this technique with Lumicks® C-Trap system to investigate the binding mechanism of NFκB.