Klotho (KL), an anti-aging protein, attracted interest in the aging field because of the dramatic phenotype of KL deficient mice and its connection to signaling pathways implicated in aging. The KLVS variant consists of the F352V (KLV) and C370S (KLS) substitutions. It was detected in genome wide association studies (GWAS) that linked it to alterations in longevity and disease risk. The molecular mechanism(s) underlying these associations are unknown. To understand how KL increases the risk of age-related diseases, the studies in this dissertation investigated whether expression of the KLVS variant, when compared to wildtype (KLWT), displays differences in processing, protein-protein interactions and enzymatic activity. Differences in processing were evaluated by studying changes in shedding, half-life and plasma membrane localization of KL variants. The decrease in KLV shedding, as measured by the intracellular: extracellular ratio, were explained by a decreased half-life. This decreased half-life is potentially due to decreased KLV plasma membrane localization, which is attenuated by co-expression of dominant negative dynamin, suggesting a role of endocytosis in these differences. To assess whether there are changes in KLVS protein-protein interactions, differences in dimerization were measured by Blue Native gel electrophoresis and cross-linking. KLV dimerization was increased while KLS and KLVS variants decreased dimerization. Co-immunoprecipitation of tagged KL assessed whether these changes were due to alterations in homodimerization. The presence of KLVS in dimers decreased the levels of immunoprecipitated KL suggesting KLVS decreases homodimerization. Changes in heterodimerization of KLVS with fibroblast growth factor receptor (FGFR) 1c were also investigated through co-immunoprecipitation. KLVS increased heterodimerization with FGFR1c. Addition of FGF23, for which KL is a co-receptor, showed that KLVS increases FGF signaling downstream of FGFR1c. To determine differences in enzymatic activity of KLVS, 4-metylumbelliferyl-beta-D-glucuronide was used to measure alterations in glucuronidase activity. Results showed that KLVS had decreased enzymatic activity compared to KLWT. These findings are the first to show that KLVS leads to differences in function as demonstrated by decreased homodimerization and enzymatic activity and increased heterodimerization with FGFR1c. Given the association of KLVS with disease and longevity, these results suggest that these functions are integral in KL's anti-aging role in humans.

Breast cancer (BC) is one of the deadliest forms of cancers with high incidence and mortality rates, especially in women. Encouragingly, targeted therapies have improved the overall<br>survival and quality of life in patients with various subtypes of BC. Unfortunately, these first-line therapies often fail due to inherent as well as acquired resistance of cancer cells. Treatment evading cancer cells can exhibit systemic dormancy in patients over a long period of time without manifesting any symptoms. In a suitable environment, these undetected disseminated tumor cells can relapse in the form of metastasis. Therefore, it is essential to understand the mechanisms of<br><div>BC recurrence and to develop durable therapeutic interventions to improve patient’s survival. In this dissertation work, we studied fibroblast growth factor receptors (FGFR), as therapeutic targets to treat the recurrence of drug-resistant and immune-dormant BC metastasis. <br></div><div><br></div><div>The HER2 subtype of BC is characterized by the overexpression of human epidermal growth factor receptor 2 (HER2), which drives elevated downstream signaling promoting tumorigenesis. Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate in which an anti-HER2 antibody targets HER2 overexpressing tumor cells and delivers a highly potent microtubule inhibitor. Using novel models of minimal residual disease (MRD) following T-DM1 treatments, we found that epithelial to mesenchymal transition is a critical process for cells to persist the TDM1 treatments. The upregulation of FGFR1 may facilitate insensitivity to T-DM1. Our data also showed that FGFR1 overexpression in HER2+ tumors leads to a higher incidence of recurrence, and these recurrent tumors show sensitivity towards covalent inhibition of FGFR. <br></div><div><br></div><div>In addition to drug-induced MRD in the primary tumor sites, disseminated tumor cells (DTCs) can demonstrate dormant phenotype via maintaining an equilibrium with immunemediated tumor clearance. Factors affecting such equilibrium may contribute to the recurrence of breast cancers metastasis. We show that such immune-mediated dormancy can be modeled with the 4T07 tumors. These tumors display immune-exclusion phenotypes in metastatic pulmonary organs. The inhibition of FGFR modulates the immune cell compositions of pulmonary organs favoring anti-tumor immunity. However, inhibition of FGFR may also affect T cell receptor downstream signaling, resulting in the inhibition of cytolytic T cell’s function. Finally, we report that combination therapy using the FGFR kinase inhibitor and an immune checkpoint blockade showed effective targeting of metastatic 4T07 tumors. <br></div><div><br></div><div>FGFR signaling as a therapeutic target in various tumors has been an active focus of cancer research. In this dissertation work, we have expanded our understanding of the role of FGFR in the recurrence of drug-resistant breast cancers as well as in the maintenance of an immune evasive microenvironment promoting pulmonary growth of tumors. Moreover, we presented evidence that it is possible to repurpose FGFR targeted therapy alone or in combination with checkpoint blockades to target recurrent metastatic BCs. In the future, our novel models of minimal residual diseases and systemic immune dormancy may act as valuable biological tools to expand our understanding of the minimal residual disease and dormant tumor cells.</div>

<p dir="ltr">Metastatic breast cancer (BC) remains a dauting therapeutic challenge due to the heterogeneity and cellular plasticity that exists. Because of these, BC resistance to targeted therapies and immune checkpoint blockade (ICB) present major challenges in the clinical setting. As a result, incomplete clearance of BC during a therapeutic regimen can lead to the persistence of minimal residual disease (MRD) which greatly contributes to tumor relapse. Here we develop a powerful in vivo model of lung metastasis in which we can achieve robust pulmonary tumor regression in response to the fibroblast growth factor receptor (FGFR) inhibitor, pemigatinib.</p><p dir="ltr">To enhance the efficacy of ICB, tumors must first be converted from an immune “cold” environment to an immune “hot” environment. Using our in vivo model of lung metastasis, we demonstrated that pemigatinib can significantly increase the presence of infiltrating T-cells into the lungs while suppressing the presence of MDSCs both locally in the lungs and systemically. Taken together, pemigatinib is an ideal candidate to prime these immune “cold” tumors for combination with ICB.</p><p dir="ltr">Upon establishment of MRD by pemigatinib in our in vivo model we observe upregulation of an alternate growth factor receptor, platelet-derived growth factor receptor (PDGFR). Functionally, upon FGFR inhibition, there is increased response to pulmonary fibroblast derived PDGF ligand, fueling survival of MRD. We demonstrated that knockdown of PDGFR significantly delayed tumor growth reinitiation in an in vitro 3D culture following pemigatinib as well as delayed tumor relapse in our pulmonary metastasis model.</p><p dir="ltr">To limit cellular plasticity and reduce survival of MRD, we propose a novel dual-targeted approach utilizing pemigatinib, in conjunction with inhibition of DNMT1 using the reversible inhibitor GSK3484862. We used our in vivo model of lung metastasis after treatment with pemigatinib as a model of cellular plasticity to targeted therapy. This combination therapy prevented growth factor plasticity and delayed tumor recurrence. Through prevention of PDGFR upregulation induced by pemigatinib.</p><p dir="ltr">In the present dissertation works, our study demonstrates pemigatinib’s robust ability to increase infiltrating T-cells in addition to its strong antitumor effects on pulmonary tumors. Despite the robust effects of pemigatinib, acquired mechanism of resistance through upregulation of PDGFR allows survival of MRD and are supported by PDGF secreting fibroblasts. Using an approach of limiting cellular plasticity through DNA methylation inhibition combined with pemigatinib, we achieved a more durable therapeutic response. Our findings underscore the significance of understanding adaptive responses to targeted therapies and provide a tangible therapeutic strategy to prolong treatment response in metastatic breast cancer.</p>