Edwin J. Velazquez

Non-small-cell lung cancer cell lines A549 and NCI-H460 express hypoxanthine guanine phosphoribosyltransferase on the plasma membrane

In both males and females, lung cancer is one of the most lethal cancers worldwide and accounts for >30% of cancer-related deaths. Despite advances in biomarker analysis and tumor characterization, there remains a need to find suitable biomarker antigen targets for treatment in late-stage lung cancer. Previous research on the salvage pathway enzyme TK1 shows a unique relationship with cancer patients as serum levels are raised according to cancer grade. To expand this analysis, the other salvage pathway enzymes were evaluated for possible upregulation within lung cancer. Adenine phosphoribosyltransferase, deoxycytidine kinase, and hypoxanthine guanine phosphoribosyltransferase (HPRT) were assessed for their presentation on two non-small-cell lung cancer cell lines NCI-H460 and A549. In the present study, we show that deoxycytidine kinase and adenine phosphoribosyltransferase have no significant relationship with the membrane of NCI-H460 cells. However, we found significant localization of HPRT to the membrane of NCI-H460 and A549 cells. When treated with anti-HPRT antibodies, the average fluorescence of the cell population increased by 24.3% and 12.9% in NCI-H460 and A549 cells, respectively, in comparison with controls. To ensure that expression was not attributed to cytoplasmic HPRT, confocal microscopy was performed to visualize HPRT binding on the plasma membrane. After staining NCI-H460 cells treated with both fluorescent antibodies and a membrane-specific dye, we observed direct overlap between HPRT and the membrane of the cancer cells. Additionally, gold-conjugated antibodies were used to label and quantify the amount of HPRT on the cell surface using scanning electron microscopy and energy-dispersive analysis X-ray. Further confirming HPRT presence, the gold weight percentage of the sample increased significantly when NCI-H460 cells were exposed to HPRT antibody (P=0.012) in comparison with isotype controls. Our results show that HPRT is localized on the surface of these non-small-cell lung cancer cell lines.

Biomarker analysis and clinical relevance of TK1 on the cell membrane of Burkitt’s lymphoma and acute lymphoblastic leukemia

TK1 is an enzyme involved in DNA synthesis and repair. TK1 is usually found elevated in cancer patients’ serum, which makes it a useful tumor proliferation biomarker that strongly correlates with cancer stage, metastatic capabilities, and recurrence risk. In this study, we show that TK1 is upregulated and localizes on the plasma membrane of Burkitt’s lymphoma, acute promyelocytic leukemia, T cell leukemia, and acute lymphoblastic leukemia (ALL). Using flow cytometry, we confirmed that TK1 localizes on the surface of Raji, HL60, and Jurkat cell lines and on ALL clinical samples. Using fluorescent microscopy, we found a strong association of TK1 with the plasma membrane in Raji, HL60, and Jurkat cell lines. These findings were also confirmed by scanning electron microscopy. Our study also shows that this phenomenon does not occur on normal resting or proliferating lymphocytes. In addition, we show that membrane TK1 is found in all oligomeric forms ranging from monomer to tetramer and exhibits enzymatic activity. These findings suggest TK1 as a possible target for immunotherapy with the potential to be utilized in the treatment of hematological cancers.

Abstract 2149: Unusual expression of HPRT on the surface of the colorectal cancer cell lines HT29 and SW620

Colorectal cancer is one of the most common cancers in the world, affecting nearly 1.2 million people in the United States alone. The aim of this study is to investigate the salvage pathway enzyme HPRT as a possible biomarker in two colorectal cancer cell lines: HT29 and SW620. HPRT is a transferase in the purine salvage pathway that functions primarily by catalyzing the conversion of hypoxanthine to inosine monophosphate and guanine to guanosine monophosphate. Because of its role in proliferation and cell cycle regulation, we hypothesized an increase in HPRT expression within cancer cells, which could potentially lead to presentation on the surface of the cell. HPRT surface localization was assessed utilizing confocal microscopy, flow cytometry, and scanning electron microscopy. These techniques allowed us to visualize HPRT on the plasma membrane and quantify expression. There was statistically significant expression of HPRT on the surface of both HT29 and SW620 cells with a 28% and 58% fluorescent shift in the population, respectively. Confocal microscopy images revealed overlap between cells stained with a membrane dye and anti-HPRT FITC antibody, which indicates a direct relationship between HPRT and the plasma membrane of SW620 cells. To visualize the location of HPRT on the plasma membrane, anti-HPRT antibodies were labelled with gold and protein presence was quantified using an electron microscope, which measured the gold elemental weight percentage of each sample. When exposed to anti-HPRT antibody, the gold weight percentage of the samples significantly increased with 12.4% gold in SW620 cells and 11.2% gold in HT29 cells, indicating significant HPRT surface presence. The observed localization of HPRT to the plasma membrane was variable between the two different cancer cell lines. SW620 cells, which are faster growing and more aggressive, had significantly higher HPRT presentation that HT29 cells. These data collectively suggest that HPRT may be a possible biomarker target for the identification and possible treatment of colorectal cancer cells. Citation Format: Evita G. Weagel, Michelle H. Townsend, Michael D. Anderson, Edwin J. Velazquez, K Scott Weber, Richard A. Robison, Kim L. O9Neill. Unusual expression of HPRT on the surface of the colorectal cancer cell lines HT29 and SW620 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2149. doi:10.1158/1538-7445.AM2017-2149

Membrane expression of thymidine kinase 1 and potential clinical relevance in lung, breast, and colorectal malignancies

BackgroundLung, breast, and colorectal malignancies are the leading cause of cancer-related deaths in the world causing over 2.8 million cancer-related deaths yearly. Despite efforts to improve prevention methods, early detection, and treatments, survival rates for advanced stage lung, breast, and colon cancer remain low, indicating a critical need to identify cancer-specific biomarkers for early detection and treatment. Thymidine kinase 1 (TK1) is a nucleotide salvage pathway enzyme involved in cellular proliferation and considered an important tumor proliferation biomarker in the serum. In this study, we further characterized TK1’s potential as a tumor biomarker and immunotherapeutic target and clinical relevance.MethodsWe assessed TK1 surface localization by flow cytometry and confocal microscopy in lung (NCI-H460, A549), breast (MDA-MB-231, MCF7), and colorectal (HT-29, SW620) cancer cell lines. We also isolated cell surface proteins from HT-29 cells and performed a western blot confirming the presence of TK1 on cell membrane protein fractions. To evaluate TK1’s clinical relevance, we compared TK1 expression levels in normal and malignant tissue through flow cytometry and immunohistochemistry. We also analyzed RNA-Seq data from The Cancer Genome Atlas (TCGA) to assess differential expression of the TK1 gene in lung, breast, and colorectal cancer patients.ResultsWe found significant expression of TK1 on the surface of NCI-H460, A549, MDA-MB-231, MCF7, and HT-29 cell lines and a strong association between TK1’s localization with the membrane through confocal microscopy and Western blot. We found negligible TK1 surface expression in normal healthy tissue and significantly higher TK1 expression in malignant tissues. Patient data from TCGA revealed that the TK1 gene expression is upregulated in cancer patients compared to normal healthy patients.ConclusionsOur results show that TK1 localizes on the surface of lung, breast, and colorectal cell lines and is upregulated in malignant tissues and patients compared to healthy tissues and patients. We conclude that TK1 is a potential clinical biomarker for the treatment of lung, breast, and colorectal cancer.

Abstract 1949: Salvage pathway enzyme HPRT as a molecular marker for Burkitt’s Lymphoma

The aim of this study is to investigate the potential of Hypoxanthine phosphoribosyltransferase (HPRT) as a surface biomarker and target for future immunotherapies in Burkitt’s B-cell Lymphoma. B-cell malignancies are the most common type of childhood cancer. Development of immunotherapies could improve current treatment. HPRT is a cytosolic transferase involved in nucleotide production via the purine salvage pathway. Altered expression of TK1, a salvage pathway enzyme, is an indicator of prognosis and diagnosis in multiple cancer types due to active proliferation of cells and the resulting elevated nucleotide demand. It has previously been reported that some salvage pathway enzymes are found on the surface of Burkitt’s Lymphoma cells. HPRT presence on the surface of this B-cell lymphoma subtype could provide a target for adoptive cell transfer and other immunotherapies. The potential surface presentation of HPRT was assessed using flow cytometry, scanning electron microscopy (SEM), and cytoplasmic staining on both healthy lymphocytes and Burkitt’s Lymphoma cells (Raji). Flow cytometry experiments with HPRT antibodies and fluorescent-labeled secondary antibodies show that Raji cells exhibit an 81.4% (p-value .0001) positive fluorescence shift when compared to IgG controls (1.5%). Healthy lymphocytes had a fluorescence shift of 2.38% (p-value .9787). The presence of HPRT on the surface of both Raji cells and healthy lymphocytes was further confirmed using gold-labeled antibodies. Utilizing a scanning electron microscope, the presence of the protein on the surface was evaluated and quantified via increases in gold weight percentage of the sample. When treated with antibodies against HPRT, there was a significant increase in gold binding along with an increase in gold weight percentage. These results suggest a direct relationship between HPRT and the surface of Burkitt’s lymphoma cells, indicating HPRT as a potential target for future immunotherapeutic treatment in Burkitt’s B-cell lymphoma pediatric patients. Citation Format: Michelle H. Townsend, John Ellis Lattin, Michael D. Anderson, Abigail Felsted, Edwin Velazquez, Evita Weagel, Richard Robison, Kim L. O9Neill. Salvage pathway enzyme HPRT as a molecular marker for Burkitt’s Lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1949. doi:10.1158/1538-7445.AM2017-1949

Abstract 1239: The expression of salvage pathway enzymes in non-small cell lung cancer cells

The aim of this study is to evaluate the expression of the salvage pathway enzymes DCK, APRT, and HPRT in lung cancer cells to determine if they could serve as biomarkers for lung cancer diagnosis and potential treatment. In both men and women, lung cancer is the most lethal cancer in the world, and accounts for more than 30% of cancer-related deaths. We chose to evaluate the salvage pathway enzymes due to an established relationship between the serum biomarker Thymidine Kinase 1 (TK1) and lung cancer. Two non-small cell lung carcinoma cell lines were utilized for this analysis (NCI-H460 and A549) along with cancer tissue and healthy tissue from 27 lung squamous carcinoma patients. The surface localization of these enzymes was determined utilizing flow cytometry, confocal microscopy, and scanning electron microscopy, while upregulation within tissue was assessed using immunohistochemistry (IHC). Throughout our investigation, we found no significant expression of DCK or APRT on the surface of non-small cell lung cancer cells, but found a significant presence of HPRT on the plasma membrane of both NCI-H460 and A549 cells. The average population florescence of cells treated with HPRT antibodies increased by 24.3% and 12.9% in NCI-H460 and A549 cells, respectively, in comparison to controls. To ensure expression was not attributed to cytoplasmic HPRT, confocal microscopy was performed to visualize HPRT binding on the plasma membrane. After staining NCI-H460 cells treated with both fluorescent antibodies and a membrane-specific dye, we observed direct overlap between HPRT and the membrane of the cancer cells. Additionally, gold conjugated antibodies were used to label and quantify the amount of HPRT on the cell surface using scanning electron microscopy and EDAX. Further confirming HPRT presence, the gold weight percentage of the sample increased significantly when NCI-H460 cells were exposed to HPRT antibody (p-value 0.012) in comparison to isotype controls. Finally, the general upregulation of the protein was observed in patient tissue samples, with approximately 44% of lung cancer tissues showing significant HPRT upregulation when compared to healthy tissue samples. This differential upregulation shows an altered HPRT expression within some patients, which may help explain the presence of this presumed cytosolic enzyme on the surface of lung cancer cells. These results strongly indicate a unique relationship between cancer cells and HPRT and suggest HPRT as a possible biomarker for the detection and treatment of non-small cell lung cancers. Citation Format: Michelle H. Townsend, Evita G. Weagel, Michael D. Anderson, Edwin Velazquez, Richard A. Robison, Kim L. O9Neill. The expression of salvage pathway enzymes in non-small cell lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1239. doi:10.1158/1538-7445.AM2017-1239

Abstract 5619: Development of a TK1 specific chimeric antigen receptor T-cell for the treatment of non-small-cell lung cancer

Our current research explores the development and tumoricidal activity of chimeric antigen receptor T-cells targeting a new immunotherapeutic target, thymidine kinase 1 (TK1), against non-small lung cancer (NSCLC), both in vitro and in vivo. There has been recent success utilizing CAR T-cell therapy in clinical trials, but it has been mainly focused on the treatment of haematological malignancies targeting CD19. Lung cancer is the most common cause of cancer mortality globally and is responsible for more than one million of deaths every year. Moreover, NSCLC comprises approximately 85 % of all lung cancers. Clinical trials against NSCLC using engineered T-cells targeting NY-ESO-1, VEGFR2, MAGE-A3, and mesothelin are currently ongoing. In spite of efforts to find new molecular targets, CAR T-cell therapy still faces several challenges in the treatment of solid malignancies due to the lack of specific molecular targets. We have previously reported the up-regulation of TK1 in multiple malignant tissues including lung cancer tissues and the presence of TK1 on the cell surface of different NSCLC cell lines, such as H460 and A549. Flow cytometry, scanning electron microscopy and confocal microscopy showed evidence of TK1 on the surface of these cancer cells lines. We have built third generation TK1-CARs with lentiviral and retroviral vectors. The constructions include a single chain variable fragment for TK1, a CD28 and 4-1BB moieties connected with a CD3ζ signaling domain. Confirmation of the CAR expression on transduced human T cells was performed by flow cytometry and confocal microscopy, and approximately 50 % of the transduced T-cells expressed TK1-CARs. Upon co-culturing TK1-CAR T-cells there was a significant increase in T-cell activity and cancer cell lysis elevated as high as 48% in comparison to negative controls. Cytokine profiles revealed a significant increase of the levels of IL-2 and IFN-γ after 24 hrs of co-culturing, indicating T-cell activation. TK1-CAR T-cells, untransduced T-cells, and transduced T-cells with empty vectors were co-cultured with H460 cells and time-lapse videos were recorded, every 5 minutes between 12 and 24 hrs post transduction. Clustering of TK1-CAR T-cells around lung cancer cells and induction of cell death after T-cell synapsis with target cells was observed. Preliminary in vitro data has shown that TK1-CAR T-cells induce specific cell lysis in NSCLC cells. In vivo experiments using xenografts models in SNG mice will be performed. Statistical differences between survival curves of mice treated with TK1 CAR T-cells, untransduced T-cells and transduced T-cells with empty vectors are expected. Note: This abstract was not presented at the meeting. Citation Format: Edwin J. Velazquez, Kiara Vaden, Michelle H. Townsend, Evita G. Weagel, Scott Weber, Richard A. Robison, Kim L. O9Neill. Development of a TK1 specific chimeric antigen receptor T-cell for the treatment of non-small-cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5619. doi:10.1158/1538-7445.AM2017-5619

Abstract 1273: A novel molecular target for lung cancer

The purpose of this study is to provide further evidence that Thymidine Kinase I (TK1) is selectively expressed on the surface of lung cancer cells, and therefore could be utilized as a potential molecular target. TK1 is an enzyme in the pyrimidine salvage pathway whose expression is closely correlated with cell proliferation and cell turnover. It has previously been shown that upregulation of TK1 is an early event in the development of most cancers. Additionally TK1 serum levels in cancer patients correlate with tumor progression and cancer aggressiveness. Moreover TK1 levels in the original tumors have also been shown to correlate directly with tumor recurrence thus making TK1 a useful prognostic marker in clinical oncology. Recent studies in our lab have provided evidence that TK1 is localized on the plasma membrane in lung cancer cells. Using scanning electron microscopy (SEM), flow cytometry, confocal microscopy and tissue staining, we confirmed the presence of TK1 on the cell surface of H460 cells. Using normal lymphocytes as a comparative control we found TK1 expression on lung cancer cells to be significantly increased compared to control cells. For this investigation, we used three custom designed monoclonal antibodies against human TK1 conjugated with FITC, namely CB1, A72 and A74. Using flow cytometry we confirmed the presence of TK1 on the cell surface, our data shows that H460 cells stained positive for TK1 (12% for A72, 19% for A74, and 29% for CB1). Furthermore, confocal microscopy indicated a positive fluorescent signal for A72, A74 and CB1, suggesting the presence of TK1 on the plasma membrane. SEM images of lung cancer cells and normal lymphocytes stained with TK1 antibodies and gold labeling reported a positive gold signal on H460 cells with relatively no signal from human lymphocytes. Immunohistochemistry staining of carcinoma tissue array panels compared with normal tissue array panels indicated a statistical significant difference of expression levels of TK1 between normal lung tissue and lung carcinoma (P value Citation Format: Edwin J. Velazquez, Evita G. Weagel, Wei Meng, Michelle H. Townsend, Alex Cummonck, Craig Chandler, Michael R. Downey, Richard Robison, Kim L. O’Neill. A novel molecular target for lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1273.