Browsing by Author "Coarfa, Cristian"
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Item Beyond Autoantibodies: Biologic Roles of Human Autoreactive B Cells in Rheumatoid Arthritis Revealed by RNA?Sequencing(Arthritis and Rheumatism, 2017-06) Mahendra, Ankit; Yang, Xingyu; Abnouf, Shaza; Adolacion, Jay R.T.; Park, Daechan; Soomro, Sanam; Roszik, Jason; Coarfa, Cristian; Romain, Gabrielle; Wanzeck, Keith; Bridges, Louis Jr. S.; Aggarwal, Amita; Qiu, Peng; Agarwal, Sandeep K.; Mohan, Chandra; Varadarajan, NavinObjective:To obtain the comprehensive transcriptome profile of human citrulline?specific B cells from patients with rheumatoid arthritis (RA). Methods:Citrulline? and hemagglutinin?specific B cells were sorted by flow cytometry using peptide–streptavidin conjugates from the peripheral blood of RA patients and healthy individuals. The transcriptome profile of the sorted cells was obtained by RNA?sequencing, and expression of key protein molecules was evaluated by aptamer?based SOMAscan assay and flow cytometry. The ability of these proteins to effect differentiation of osteoclasts and proliferation and migration of synoviocytes was examined by in vitro functional assays. Results:Citrulline?specific B cells, in comparison to citrulline?negative B cells, from patients with RA differentially expressed the interleukin?15 receptor ? (IL?15R?) gene as well as genes related to protein citrullination and cyclic AMP signaling. In analyses of an independent cohort of cyclic citrullinated peptide–seropositive RA patients, the expression of IL?15R? protein was enriched in citrulline?specific B cells from the patients’ peripheral blood, and surprisingly, all B cells from RA patients were capable of producing the epidermal growth factor ligand amphiregulin (AREG). Production of AREG directly led to increased migration and proliferation of fibroblast?like synoviocytes, and, in combination with anti–citrullinated protein antibodies, led to the increased differentiation of osteoclasts. Conclusion:To the best of our knowledge, this is the first study to document the whole transcriptome profile of autoreactive B cells in any autoimmune disease. These data identify several genes and pathways that may be targeted by repurposing several US Food and Drug Administration–approved drugs, and could serve as the foundation for the comparative assessment of B cell profiles in other autoimmune diseases.Item CAMKK2 Promotes Prostate Cancer Progression through Independent Intrinsic and Extrinsic Roles(2021-05) Pulliam, Thomas Loyd; Zhang, Xiaoliu Shaun; Frigo, Daniel E.; Umetani, Michihisa; Chung, Sang-Hyuk; Coarfa, CristianCalcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) is emerging as a promising therapeutic target due to its roles in driving several pathological conditions including metabolic disorders, osteoporosis, Alzheimer’s disease and multiple cancer types. Recently, it has been discovered that CAMKK2 also regulates the immune response to infection and promotes an immunosuppressive tumor microenvironment to extrinsically aide certain cancers. CAMKK2 is a calcium/calmodulin-activated master kinase with a restricted expression profile, but when expressed, it can potentially drive pathological conditions. This dissertation will cover CAMKK2’s intrinsic function in prostate cancer and an emerging mechanism of resistance that could guide the development of future rationale treatment combinations. Prostate cancers overcome CAMKK2 knockout (KO) or knockdown (KD) by increasing angiogenesis to sustain tumor growth in vivo. We discovered this was due to a change in expression of two angiogenesis regulatory genes, thrombospondin-1 and angiopoietin-2, both previously reported as mediators of a pro-angiogenic “switch” necessary to promote cancer progression. We leveraged a well-known clinical inhibitor of angiogenesis, bevacizumab, with CAMKK2 KO in preclinical animal models of castration-resistant prostate cancer (CRPC) to profoundly reduce tumor growth and increase overall survival. This dissertation will also cover CAMKK2’s extrinsic role in aiding prostate cancer progression via immune suppression and a possible new role via macrophage-mediated EMT. Systemic Camkk2 KO in host mice slowed tumor growth macrophage-mediated EMT. Systemic Camkk2 KO in host mice slowed tumor growth in two separate syngeneic prostate cancer models. Decreased activated T-cells and greater infiltration of tumor supporting M2 macrophages in Camkk2 wild-type (WT) mice suggests CAMKK2 plays a functional role in regulating the immune system to support prostate cancer. Finally, this dissertation will cover problems plaguing the currently accepted inhibitor of CAMKK2, STO-609, and efforts to design a novel selective and potent inhibitor of CAMKK2 to take its place. With our collaborators, we have used the approach of hinge-binder scaffold modification to discover a new CAMKK2 inhibitor, YL-36, with a 10 fold increase in potency over STO-609 as well as vastly improved selectivity. Together, we solidify CAMKK2’s role as an oncogenic driver of prostate cancer intrinsically, provide insights into CAMKK2’s emerging tumor extrinsic roles in driving cancer, and finally describe a combinatorial approach of treatment with angiogenesis and CAMKK2 inhibitors simultaneously to increase the efficacy of CAMKK2 inhibitors once they reach the clinic.Item Developing microRNA-509-3p as an inhibitor of metastatic progression and cisplatin sensitizer for osteosarcoma(2017-08-03) Patil, Sagar Laxman 1986-; Gunaratne, Preethi H.; Briggs, James M.; Coarfa, Cristian; Bark, Steven J.Osteosarcoma (OS) is the most common primary pediatric malignancy of the bone. Systemic chemotherapy, which benefits patients with localized OS to increase survival to 70%, has had minimal effects on metastatic OS with patient survival rates of < 30%. MicroRNAs (miRNAs) are non-coding RNAs that can target and silence hundreds of genes regulating the majority of biological processes. Here, we report, microRNA-509-3p as a strong tumor suppressor of OS, which inhibited migration of primary OS cell lines, HOS (p53mut/-) and SaOS2 (p53-/-); their metastatic derivatives 143B (p53mut/-) and LM7 (p53-/-), respectively; and the primary cell line U2OS (p53wt/wt). Overexpression of miR-509-3p inhibited proliferation and invasion of HOS and 143B and sensitized cells to cisplatin. miR-509-3p failed to sensitize U2OS (p53wt/wt) to cisplatin treatment. miR-509-3p inhibited migration by directly targeting ARHGAP1 only in HOS cell line while in 143B cell line knockdown of ARHGAP1 alone by siRNA had no effect on migration inhibition. These results are consistent with published findings that the action of microRNAs and their downstream targets are highly cell-type specific. Reverse Phase Protein Array (RPPA) analysis uncovered AXL, as significantly down-regulated upon miR-509-3p overexpression in HOS and 143B cells. Previous studies have shown that knockdown of AXL in 143B cells inhibits migration and invasion. RPPA data also showed significant down-regulation of AXL and ATM, both established to play a role in acquired cisplatin resistance, in miR-509-3p+cisplatin treated cells. We propose that AXL is a critical downstream effector of miR-509-3p-mediated inhibition of migration in 143B and cisplatin sensitization in 143B and HOS. In addition, to develop biocompatible microRNA delivery system, we optimized cysteamine-functionalized gold nanoparticle (cAuNP) based microRNA delivery system earlier developed in our lab. Here we, established optimal cAuNP:miR-509-3p ratio and incubation time for optimal delivery into LM7 OS cells. Lastly, as an out of box approach to treat OS we explored possibility of converting OS to fat by modulating peroxisome proliferator-activated receptor gamma (PPAR), a master regulator of adipogenesis through a combination of miR-130b inhibitor, diabetes drug, and unsaturated fatty acids. We, showed that linoleic acid can induce lipid droplet formation in HOS cells.Item Discovery of Immunogenic Neopeptides from Chimeric RNAs to Develop Peptide and mRNA Vaccines for Lung Cancer Treatment(2023-12) Castillo, Micah Becal; Gunaratne, Preethi H.; Briggs, James M.; Antunes, Dinler A.; Coarfa, Cristian; Abbas, Hussein A.Non–small cell lung cancer (NSCLC) accounts for nearly 80% of lung cancer cases and exhibits a median survival of less than one year following diagnosis. A relatively unexplored avenue of immunotherapy for specific cancers may lie in chimeric RNAs that separate cancerous cells from their non-cancerous neighbors. RNA from fusion genes resulting from deletions, duplications, inversions, translocations, or aberrant splicing events, can be used as vaccine targets due to the neoantigenic peptide junctions formed in the translated proteins. RNA sequencing data from Lung Adenocarcinoma and Squamous Cell Carcinoma patients was analyzed for chimeric RNA content. The KIF5B [exon 1-15] -RET [exon 12-19] fusion was found and chosen as the primary candidate fusion for this study based on previous studies that established it to be an actionable cancer driver. The affinity of the neopeptide sequences formed by the translated junction of KIF5B-RET to Major Histocompatibility Complex (MHC) Class I molecules was assessed using the in-silico prediction pipelines MHCNuggets and MixMHCPred 2. HLA-C*07:02 was found to be the highest affinity binder to 9-mer peptides within the KIF5B-RET junction region. I then investigated binding affinity from a structural approach using HLA-Arena to dock peptides and calculate binding energies, revealing NNDVKEDPK as the strongest binder. Furthermore, peptides predicted as strong binders tended to have regions with positive electrostatic potentials oriented toward the binding pocket of HLA-C*07:02. The immunogenicity of these neoantigenic peptides to HLA-C*07:02 matched Peripheral Blood Mononuclear Cells (PBMCs) was assessed by the IFN-γ Enzyme Linked Immunosorbent Spot (ELISpot) assay. Effects across donors were identified, as PBMC-isolated CD8+ T cells from donor 1 and 2 showed the highest responses to KEDPKWEFP and a pool of all junction peptides, respectively. The Single-cell 5’ gene expression and T Cell Receptor (TCR) mapping assay then identified a subset of TCR sequences responsive to peptide stimulation, along with critical positions and glycine residues in the CDR3-α and CDR3-β chains that may be responsible for this heightened immune response. This study establishes a robust pipeline to discover chimeric RNAs from RNA-sequencing performed on tumor samples, extract and validate immunogenic neoantigens from fusion junctions, and design vaccines for the treatment of cancer.Item Regulation of Nutrient Uptake in Prostate Cancer(2017-05) White, Mark Anthony 1987-; Frigo, Daniel E.; Zhang, Xiaoliu Shaun; Lin, Chin-Yo; Gilbertson, Scott R.; Coarfa, CristianProstate cancer is a hormone-driven malignancy that relies on the function of the androgen receptor (AR). AR is a transcription factor that regulates the expression of many downstream targets, some of which can facilitate an important hallmark of cancer; metabolic reprogramming. Metabolic reprogramming allows the cancer to maintain an aberrant metabolism that supports uncontrolled cellular growth and survival. This reprogramming if often initiated by signaling pathways essential for growth and survival. There are therapies available that target AR signaling but they inevitably fail. Therefore, I sought to identify new potential targets that are downstream of AR and other oncogenic signals in prostate cancer and define the mechanism through which they are regulated. First, I investigated how two glutamine transporters, SLC1A4 and SLC1A5 (Solute Carrier Family 1A, members 4 and 5) were regulated in glutamine-addicted prostate cancer cells. I found that the transporters were hormone-responsive but not direct targets of AR. Downstream of AR they are regulated via mammalian target of rapamycin (mTOR) signaling and selectively regulated via MYC. Importantly I determined that SLC1A4 and SLC1A5 represented a central node of several oncogenic signaling pathways that controlled overall cell growth, making them promising targets for prostate cancer therapy. Next, I investigated the regulation of glucose uptake through SLC2A12 (GLUT12 (glucose transporter 12)). I found that SLC2A12 is a direct target of AR and is required for prostate cancer cell growth. GLUT12 is also regulated through calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2)-5’-AMP-activated protein kinase (AMPK) signaling. CaMKK2-AMPK activity promotes GLUT12 translocation to the plasma membrane via modulation of TBC1D4 (TBC1 Domain Family Member 4) and also regulation of TBC1D4 expression. Taken together my findings demonstrate that SLC1A4, SLC1A5, and SLC2A12 all have the potential to be prostate cancer therapeutic targets due to their modulation by major oncogenic signaling pathways and their functional role in cancer cell growth. Their essential role in cancer cell growth and easily accessible location on the cell surface suggest these proteins may be readily druggable. Thus, my findings highlight the utility of targeting pathogenic metabolism as a therapy and provide potential starting points for future translational research.