The purpose of this study was to analyze the regulatory gene cancer network of the tumor biomarker Thymidine Kinase 1 (TK1) across all cancers and identify the genes with meaningful correlations that interact and drive tumorigenesis along with TK1. In addition, we sought to find a feasible mechanism for TK1 membrane expression in cancer cells. Using data from The Cancer Genome Atlas (TCGA), the pathway commons protein interaction database, the Biogrid database and the R graphite library, we analyzed the correlation of mRNA expression levels of all proteins that were involved in TK1 metabolic pathways or physically interacted with TK1. By searching the pathway commons protein interaction database and data drawn from the graphite library in R, we found that 1495 proteins were involved with TK1 metabolism. Using Dijkstra's algorithm we found that 294 proteins had 1-2 degrees of interaction with TK1. Analysis of the Biogrid database showed that 191 proteins physically interact with TK1. The genes with higher average Pearson's correlation values across all 25 cancers had the following functions: cell cycle regulation, DNA synthesis, replication and repair, transcription factors (E2F family), transport of mono and dicarboxylate for energy production pathways, transport of nucleosides, pyrimidine metabolism, DNA methylation of tumor suppressor genes, mRNA stabilization, aging, regulation of cell proliferation, control of cyclin kinases, apoptosis, and formation of channels on cell membranes. Among the genes that had significant correlation values with TK1 (P<0.001) were: RRM2(2), CDK1, CDK2, CDK4(0), CDC20(0), CDC25A, MKI67(0), MCM3, E2Fs, CD6, PXMP2, DTYMK(1), BRCA1, EZH2(0), NUDT1(3), SLC29A2(2), SLC25A10(2), SLC16A3(3), SLC25A19(3), PRIM1, CARHSP1(0), NKIRAS2(0), METTL23(0) CENPB(0), FAF1(0), COPS6 (0), TRIM28 (0), MSH2(0), AAMP (0). Further analysis revealed that 476 of the proteins from the common pathways and Biogrid had transmembrane (tm) domains. Although it had a moderate correlation value, SEZ6L protein, which is overexpressed on the cell membrane of lung, colon and gastric cancers was a physical interactor of TK1. Additional analysis showed that the nucleoside transporter SLC29A2, that transports both adenosine and thymidine, had a tm domain and 2 degrees of interaction with TK1. Like the purinosome, we hypothesize that TK1 in malignant cells may be complexing with other pyrimidine synthesis proteins. Moreover, the physical interaction with other proteins containing tm domains could provide a path for TK1 membrane expression. Overexpression, secretion and membrane association of TK1 may be an evolutionary advantage that supply enough enzymes to modify the nucleotide pool balance necessary for DNA replication in tumor proliferating cells. Future directions will focus on the TK1 protein-protein interactions found in this analysis. Citation Format: Edwin J. Velazquez, David M. Bellini, Rachel A. Skabelund, Tyler B. Humpherys, Jonathan R. Skidmore, Brett E. Pickett, Stephen R. Piccolo, Kim L. O'Neill. Bioinformatic analysis of the tumor biomarker thymidine kinase 1: Elucidating its cancer gene network and membrane expression across all cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2134.

Our research explores the potential of a panel of monoclonal antibodies targeting the tumor proliferation biomarker Thymidine Kinase 1 (TK1) for both clinical and therapeutic applications. Cancer biomarkers have become a critical component of precision medicine. TK1 is a well-known tumor proliferation biomarker that is released into the serum, is up-regulated in malignant tissues and can be found on the cell membrane of several cancer types. Notwithstanding the versatility of TK1 as a tumor biomarker, there are a limited number of available antibodies for the detection and quantification of TK1. Moreover, to the date TK1 antibody-based therapies are not being tested in the clinical setting. Thus, the generation of more sensitive TK1 antibodies could increase the availability, accuracy and options of current TK1-based diagnostics and antibody-based immuno-cell therapies. Six peptide sequences across the TK1 molecule were selected. The antibodies were generated using hybridoma technology. Seventeen clones were evaluated in ELISA with a calibration dose-response curve. The calibration curves showed R squares ranging from 0.9738-0.9980 with a 10-15 %CV. The limit of detection and quantification (LOQ, LOD) were obtained. The clones 3B2E11, 9C10, 8G2, 5F7G11, 3B4 and 3G7 showed the lowest values, being 3B2E11 the most sensitive with a LOD of 18.6 ng/ml and a LOQ of 64 ng/ml. Western blot data showed specific binding to recombinant TK1 and to multiple forms of TK1 in cell lysates, and serum samples for 14 of the 17 clones. Although, differences in the binding patterns were found in cell lysates. Flow cytometry was performed to analyze TK1 surface expression. Antibodies 8G2, 3B4 and 57G11 showed consistent binding across 4 cancer cell types in a similar percentage to the commercial TK1 antibody (Abcam91651) with a maximum percentage of binding in lung of (95.6%) followed by prostate (72.2%), colon (62.4%) and breast (49.1%). No significant binding for either the commercial or the custom TK1 antibodies was found on normal mono nuclear cells (MNC). The clones 8G2 and 3B4 were selected for testing their potential as therapeutic agents in antibody-dependent cell-mediated cytotoxicity (ADCC) experiments. Around 50% and 42% increased killing of A549 cells was observed with antibodies 8G2B and 3B4 respectively 48 hrs. after adding the antibodies when compared with isotype controls (p <0.05). Based on dose response curves 2.5 ug/ml for the clone 8G2B and 5-7 ug/ml for the clone 3B4 were the minimum concentrations required to show significant specific cell death in A549 cells. The antibodies developed have shown capacity for detection and quantification of TK1 in serum and on the membrane of cancer cells. Moreover, our in vitro ADCC experiments provide more evidence that membrane associated TK1 has potential as an immunotherapeutic target. Citation Format: Edwin J. Velazquez, Taylor D. Brindley, Gajendra Shrestha, Rachel A. Skabelund, Corbin M. Lee, Zachary D. Ewel, Eliza E. Bitter, Michelle H. Townsend, Kelsey B. Bennion, Kai Li Ong, Kiara V. Whitley, Richard A. Robison, Scott K. Weber, Kim L. O'Neill. Generation and characterization of a panel of monoclonal antibodies against the tumor biomarker Thymidine Kinase 1 for research, clinical and therapeutic applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2987.

No abstract