The deregulation of the cell cycle is important in the leukemic transformation and in the continued discovery of more efficient targeted therapies. After elucidation of the role of the RNA editor ADAR1 in the malignant reprogramming of Chronic Myeloid Leukemia (CML) progenitors to leukemia stem cells (LSCs) during the chronic phase to blast crisis stages of the disease, current research seeks to uncover the mechanism of action of ADAR1 in this process. To study the potential role ADAR1 plays in the changes to cell cycle during this disease progression, a novel Fucci (fluorescent ubiquitination-based cell cycle indicator) system was introduced into the CML cell line K562 through stable transduction of both Fucci vectors. This allows for the visualization of the cell cycle via FACS (fluorescence activated cell sorting), and fluorescent live imaging. This system was validated for use in therapeutic CML research through preliminary evaluation of ADAR1 activity in specific cell cycle stages as well as the effects of a potentially novel CML therapy, 8-azaadenosine, on the cell cycle. Completion of this work shows successful creation of a novel generation of Fucci cell line; which will aid in the clarification of the changes which occur in the cell cycle during CML disease progression, as well as the effects of CML targeted therapies on cell cycle regulation though quantitative and qualitative analysis.

Head and neck squamous cell carcinoma (HNSCC) is pernicious disease with majority of cases presenting as oral squamous cell carcinoma (OSCC). OSCC is characterized by tumor heterogeneity, locoregional metastases and resistance to existing treatments. OSCC five-year overall survival rates are currently ~ 50 % with few therapeutic options available. Cancers are typically associated with dysregulated signaling pathways that normally have vital roles in embryonic development and hemostasis. We have focused on two homeostatic pathways, protein N-glycosylation and the canonical Wnt/β-catenin signaling, which have been shown to converge on each other’s activities. These pathways, when left unchecked, drive early pathogenesis and/or metastasis of a range of cancers, including OSCC. Aberrant activation of the DPAGT1 gene, encoding a key regulatory enzyme that functions at the first committed step in the protein N-glycosylation pathway, has been shown to be associated with OSCC. In OSCC, high DPAGT1 expression drives hyper-glycosylation of E-cadherin and loss of intercellular adhesion. Here, we provide evidence that ectopic expression of DPAGT1 in indolent OSCC CAL27 cells induced epithelial-to-mesenchymal transition (EMT) by upregulation of a key transcription factor, ZEB1, and mesenchymal protein, vimentin, coincident with increased cell migration. In contrast, partial knockdown of DPAGT1 in metastatic OSCC HSC-3 cells reduced expression of the EMT markers, diminished cell migration and enhanced intercellular adhesion. Further, inhibition of the DPAGT1 enzyme, GPT, with tunicamycin interfered with orthotopic tongue tumor growth and metastasis in nude mice coincident with diminished expression of vimentin- positive cells invading the tongue stroma. One mechanism responsible for increased DPAGT1 expression in OSCC is transcriptional activation by β-catenin. We now show that blocking transcriptional activity of β-catenin by interfering with its interaction with the cAMP-responsive element binding (CREB)-binding protein (CBP) using a small molecule inhibitor, ICG-001, inhibited cell proliferation and mesenchymal cell phenotypes in cellular models. In addition, ICG-001 abrogated tumor growth and metastases in zebrafish and murine models. Microarray analyses of ICG-001 gene expression signature revealed inhibition of genes involved in cell proliferation, survival, stemness, as well as N-glycosylation, but upregulation of genes functioning in cell adhesion and cell polarity. Importantly, the ICG-001 inhibition-associated transcriptional signature tracked with advanced tumor grade and poor survival in human patients. Our studies provide the first evidence that aberrant activation of DPAGT1 and β-catenin signaling promotes aggressive traits of OSCC cells and suggest that targeting the β-catenin/CBP interaction in the nucleus may provide an effective novel strategy for OSCC therapy.

PURPOSE: Social determinants of health create barriers for pelvic cancer patients, and including qualitative measures in clinical research is imperative to assess all variables that contribute to rates of pelvic cancer. The aim of this study was to determine the barriers that pelvic cancer patients face in accessing healthcare, and to identify gaps in the literature that may direct future research. We also aim to advise healthcare professionals and clinical researchers on the importance of health disparities and inclusive patient education. METHODS: We used a grounded-theory thematic meta-analysis approach in our research to identify health disparities among pelvic cancer patients. We generated primary codes from all aspects of socioeconomic status, which served as PubMed boolean search terms to gather all literature pertaining to factors that impact healthcare access amongst pelvic cancer patients. We developed secondary codes as themes from the initial results for further focus on clinical experiences. We developed tertiary codes identifying intersections between clinical care themes specific to transgender healthcare access. RESULTS: Our total search for health disparities in pelvic cancer resulted in 348 papers which included cancer (41), physician education (35), race and ethnicity (29), surgical procedures (26), socioeconomic status (23), LGBTQIA+ (22), sexually transmitted diseases (20), diagnostic screenings (15), female reproductive health (15), sex and gender (15), transgender and gender non-binary health (11), imaging (10), robotics (9), ectomies (9), radiotherapies (9), sexual health and sexual education (8), patient education (7), physician surgical training (6), mental health (6), language (6), contraceptives (5), smoking (5), quality of life (3), adolescents (3), medical therapies (2), pelvic inflammatory disease (2), histology (2), hormones (2), pain management (1), and patient navigation (1). In decreasing order of literature papers found, this shows the paucity of research in topics about social determinants of health and post-clinical appointments. Most importantly, we found that lack of physician education on these topics, many cases of socio-cultural bias, and an absence of patient navigation that plays a role in the delay or absence of healthcare access for pelvic cancer patients. CONCLUSIONS: Our work emphasizes the impact healthcare providers and policies have for healthcare access and delivery of care. Our future research will find ways to address the health disparities that pelvic cancer patients face, develop effective strategies in diagnosing and treating pelvic cancer, and inform healthcare professionals and medical curricula on the importance of social determinants of health to create more inclusive patient-centered care. / 2024-01-30T00:00:00Z

Bispecific antibodies have recently taken the immune-oncology field by storm through its multi-targeting capabilities and unique mechanisms of action. In the past few decades, this therapeutic format has seen an exponential rise, and with every year, new variations of the basic bispecific concept emerge to precipitate new effects. These effects include cell-to-cell binding, enhanced agonism or antagonism, and target internalization. One of the most promising applications for bispecific antibodies is as a bispecific T-cell engager, which can induce inflammatory responses in previously immune-excluded tumors. This paper aims to provide a high-level overview of the functionality, formats, and clinical results of bispecific antibodies and place them in the context of current treatments for cancer.

OBJECTIVES: The objectives of the PREFERENCE Study are to characterize preferences of MGUS and SMM patients for early intervention according to attributes of the intervention, and to identify patient characteristics that predict intervention preference patterns. METHODS: We developed a survey instrument that contained a conjoint survey, a brief demographic survey, and validated survey instruments to assess numeracy, tolerance for uncertainty, and perceived risk of cancer. The survey was distributed to 301 patients with MGUS or SMM. We analyzed the conjoint data using conditional logistic regression models to investigate the attributes that drive patient decisions about early intervention. We next used latent profile analysis to investigate whether there are latent classes of patients defined by their choice preferences. We then estimated posterior probabilities to evaluate which patient characteristics predict class membership. RESULTS: We found that for every 10% increase in the ability of a treatment to prevent myeloma, there was an 86% increase in selecting the treatment strategy, and for every $100 increase in monthly cost, there was a 11% decrease in selecting the strategy. High personal inconvenience led to a 38% decrease in selecting a treatment strategy, and a high risk of side effects led to a 91% decrease in selecting a strategy. Latent profile analysis revealed three classes of patients. A class of patients who were willing to accept less efficacious therapies with a higher monthly cost to prioritize avoiding side effects, a class of patients who were willing to accept less efficacious therapies and some risk of side effects to keep monthly cost low, and a class of patients who were willing to accept some risk of side effects and a higher monthly cost to prioritize efficacious therapy. The posterior probabilities did not show a significant difference in class membership based on age, and participants with higher education and higher income were less likely to fall into the latent class prioritizing low cost. Results also showed that choosing efficacious therapy is not perfectly coupled with risk of multiple myeloma. CONCLUSION: These results emphasize that the “one-size-fits-all” approach in treatment of MM precursor conditions will not meet the needs and preferences of MGUS and SMM patients. In broad populations there are diverse preferences leading to a need for comprehensive intervention portfolios.

In musculoskeletal oncology, limb salvage surgery is now the standard of care for most patients with bone or soft tissue tumors of the extremities. There has been an increased interest in quantifying functional outcomes following limb salvage with both physician reported and patient reported outcome tools utilized. However, due to the rarity of the disease and lack of prospective data, there remains significant gaps in the literature surrounding functional outcomes in this population. This thesis proposes minimal important difference values for commonly used functional outcome measures and created a predictive model for postoperative function for musculoskeletal oncology patients following lower extremity limb salvage and endoprosthetic reconstruction. The data for this thesis was retrieved from the Prophylactic Antibiotic Regimens in Tumor Surgery (PARITY) trial database and included patients undergoing lower extremity surgical resection and endoprosthetic reconstruction for bone or soft tissue tumors. Utilizing this data, we provided answers to two important clinical questions 1) establishing minimal important difference (MID) values for commonly utilized functional outcome scoring systems and 2) determine patient and tumor factors predictive of postoperative function. We developed both anchor-based and distribution-based MID values for both the Musculoskeletal Tumor Society Score and the Toronto Extremity Salvage Score, the two most utilized functional outcome tools in the field. Secondly, we characterized the longitudinal changes in function following endoprosthetic reconstruction and identified patient and tumor predictors of postoperative function. On average, patient function improved significantly from their preoperative baseline to 1-year follow-up, exceeding the predefined MID values. Older age, poor preoperative function, and endoprosthetic reconstruction for soft tissue sarcomas were associated with worse outcomes; reconstruction for giant cell tumors were associated with better post-operative function. Overall, these two studies aim to provide a deeper and more meaningful understanding of functional status in musculoskeletal oncology patients. / Thesis / Master of Science (MSc)

<p>The incidence of urinary bladder carcinoma is increasing and many patients die every year of this disease despite assumed radical therapy. Thus, there is a need for improved methods of diagnosis and therapy. Radionuclide targeting is based on achieving specific delivery of radioactive nuclides to tumour cells with minimal damage to surrounding normal tissues. Two possible target structures are the epidermal growth factor (EGF) receptor and the related receptor HER-2.</p><p>Cellular binding and retention of ¹²⁵I-EGF-dextran conjugates was investigated in two bladder carcinoma cell lines. The conjugate bound specifically to the EGF receptor with delayed maximum binding, limited intracellular degradation and prolonged cellular retention compared to ¹²⁵I-EGF.</p><p>EGF was labelled using different radionuclides and methods. All the labelled variants bound specifically to the tumour cells although the cellular binding patterns and retention varied considerably. ¹¹¹In-DTPA-EGF had highest cellular retention and in decreasing order ²¹¹At-benzoyl-EGF and ¹²⁵I-labelled EGF.</p><p>Bladder cancer spheroids bound both ¹²⁵I-EGF-dextran as well as ¹²⁵I-EGF. Conjugate binding increased during a 48 h incubation period and was most prominent in the outer cell layers. The length of the dextran chain appeared not to alter the binding pattern.</p><p>The expression of EGF receptors and HER-2 in metastases and primary bladder carcinoma tumours was investigated. Both receptors were expressed in the majority of metastases and primary tumours.</p><p>Targeting the EGF receptor and/or HER-2 in urinary bladder carcinoma is an exciting new concept.</p>

<p>Tyrosine phosphorylation is a central mechanism in cellular signalling leading to proliferation, migration or differentiation. Protein tyrosine phosphorylation is regulated by the coordinated actions of protein tyrosine kinases and protein tyrosine phosphatases. This thesis investigates the involvement of tyrosine phosphatases in contact-induced growth inhibition of cells. Furthermore, it describes the structure and function of the extracellular domain of the receptor-like tyrosine phosphatase DEP- 1. </p><p>Tyrosine phosphatases, negatively regulating tyrosine kinases, have been suggested being involved in contact-induced growth inhibition of cells. Both endogenous EGF-receptors and transfected PDGF receptors showed a decreased ligand-induced tyrosine phosphorylation in cells of dense cultures. This difference was found to be due to increased receptor-directed tyrosine phosphatase activity in dense cultures. </p><p>Density enhanced tyrosine phosphatase-1 (DEP-1) is a receptor-like tyrosine phosphatase. It was found that DEP-1 contains chondroitin sulfate chains, thus identifying DEP-1 as a proteoglycan. Furthermore, DEP-1 was found to interact with a heparan sulfate proteoglycan. </p><p>No ligands have been identified for DEP-1. We have established a biacore-based assay for the identification of molecules interacting with the extracellular domain of DEP-1. A library of cell conditioned media was screened with the biacore assay. One of the samples was found to contain DEP-1 interacting molecules. Purification of the ligand has been initiated. </p><p>In an attempt to identify modulators of DEP-1 activity, Matrigell™ , a preparation of extracellular matrix was investigated. Stimulation with Matrigel™ was found to increase the specific activity of DEP-1 through interactions between the extracellular domain of DEP-1 and Matrigel™ component(s). </p>

<p>The DNA <i>double-strand break</i> (DSB) is a critical lesion which, if not completely restored, can have serious biological consequences. The <i>relative biological effectiveness</i> (RBE) of many severe end-points are closely related to radiation quality, with increased effectiveness at elevated ionization density. Data presented provide information about the influence of radiation quality on the initial processes causing DNA damage, and the mechanisms leading to its restoration. Such information will increase the understanding of radiation action mechanisms in mammalian cells. </p><p>Human cells were irradiated with accelerated ions having <i>linear energy transfer</i> (LET) values in the range 40-225 keV/μm, and ⁶⁰Co-photons. Detailed analyses of the DNA fragment distributions were performed in the size-range 5 kilobasepairs to 6 megabasepairs by pulsed-field gel electrophoresis. </p><p>A non-random fragmentation of DNA was evident, with an elevated number of small and medium-sized fragments for ion irradiation, and the total number of breaks increased by 80-110% when these fragments were included in the analyses. The RBE for DSB induction was 1.2-1.5. A two-fold increase of the number of breaks induced per nitrogen ion passing the cell nuclues was found when LET was increased from 80 to 225 keV/μm, indicating a possible role of particle track structure in DSB induction. Furthermore, the ability to repair DNA was closely related to radiation quality, with an increased proportion of unrejoined breaks for densely ionizing radiation. Surprisingly, the majority of breaks were rapidly rejoined even following exposure to high-LET radiation. The proportion of breaks restored by the slow phase showed a five-fold increase for the highest LET tested, compared with photons. The results presented nominates the complexity of breaks as one determining factor for reduced reparability reported following high-LET exposure.</p>

<p>Stem Cell Factor (SCF) can function as a survival factor, a mitogen or a chemoattractant depending on cell type. Binding of SCF to c-Kit induces dimerization and subsequent autophosphorylation of the receptor. This thesis describes the intracellular signal transduction elicited by c-Kit. </p><p>In the search for signal transduction molecules binding to activated c-Kit, we identified the adaptor proteins Grb2 and Grb7 as interacting partners. Grb2 could associate to Tyr-703 in the kinase insert as well as to Tyr-936 in the C-terminal tail of c-Kit. However, Grb7 could only bind to Tyr-936. </p><p>Tyr-568 in c-Kit is essential for SCF induced association and activation of Src family kinases (SFK). A mutated receptor that could not activate SFK (Y568F or Y568/570F) showed reduced Shc phosphorylation, Ras GTP loading, Erk activation and induction of <i>c-fos</i>. However, activation of SFK is not essential for the mitogenic response as measured by DNA synthesis.</p><p>Tyr-900 in c-Kit was identified as a SFK dependent phosphorylation site. The adaptor protein Crk and the p85 subunit of PI3’-kinase could associate with phosphorylated Tyr-900. In addition, we could demonstrate a constitutive complex between Crk and p85, suggesting indirect binding of Crk to Tyr-900 via p85. Mutation of Tyr-900 (Y900F) led to a reduced phosphorylation of Crk-II, loss of the second wave of Erk phosphorylation and a reduced mitogenic response. In addition the mutated receptor showed an increased ligand-induced degradation as compared to the wild-type receptor.</p><p>There exist two splice forms of c-Kit that differ in the presence or absence of four amino acids in the extracellular juxtamembrane region. These splice forms bind SCF with similar affinity but display striking differences in signaling characteristics, <i>e.g.</i> in phosphorylation kinetics, ligand-induced c-Kit degradation and activation of Erks. However, other pathways are activated similarly by both splice forms, such as the Ser/Thr kinase Akt which lies downstream of PI3’-kinase. In this study we show that differential phosphorylation of the various tyrosine residues occurs. Interestingly, Tyr-568 is more efficiently phosphorylated in the shorter form leading to stronger binding of SFK, whereas the PI3’-kinase binding site showed a similar degree of phosphorylation consistent with the data on Akt activation.</p>