Expression Of Thymidine Kinase Research Articles

Serum bta-miRNA-375 as a potential biomarker for the early diagnosis of enzootic bovine leukosis.

To identify a biomarker for the early diagnosis of enzootic bovine leukosis (EBL) caused by bovine leukemia virus (BLV), we investigated the expression of a microRNA, bta-miR-375, in cattle serum. Using quantitative reverse-transcriptase PCR analysis, we measured bta-miR-375 levels in 27 samples from cattle with EBL (EBL cattle), 45 samples from animals infected with BLV but showing no clinical signs (NS cattle), and 30 samples from cattle uninfected with BLV (BLV negative cattle). In this study, we also compared the kinetics of bta-miR-375 with those of the conventional biomarkers of proviral load (PVL), lactate dehydrogenase (LDH), and thymidine kinase (TK) from the no-clinical-sign phase until EBL onset in three BLV-infected Japanese black (JB) cattle. Bta-miR-375 expression was higher in NS cattle than in BLV negative cattle (P < 0.05) and greater in EBL cattle than in BLV negative and NS cattle (P < 0.0001 for both comparisons). Receiver operating characteristic curves demonstrated that bta-miR-375 levels distinguished EBL cattle from NS cattle with high sensitivity and specificity. In NS cattle, bta-miR-375 expression was increased as early as at 2 months before EBL onset-earlier than the expression of PVL, TK, or LDH isoenzymes 2 and 3. These results suggest that serum miR-375 is a promising biomarker for the early diagnosis of EBL.

Abstract PO1-15-06: Serum thymidine kinase activity as a “real-time” clinical biomarker of tumor response to CDK4/6 inhibition in hormone receptor positive (HR+), HER2 negative (HER2-) metastatic breast cancer

Abstract Background: CDK4/6 inhibitors (CDK4/6i) plus endocrine therapy (ET) are standard first-line (1L) treatment for hormone receptor positive (HR+), HER2-negative (HER2-) metastatic breast cancer (MBC). Despite general improvements in long-term outcome, not all patients benefit equally from these drugs. The average duration of 1L CDK4/6i therapy is ~2 years. However, 10-15% of patients will progress within 6 months of therapy initiation, while others will derive durable clinical benefit of years. Differences in treatment efficacy cannot be easily explained by clinical-pathological features. No predictive biomarkers are currently available to determine tumor sensitivity to CDK4/6i. There is an unmet need for a complementary diagnostic to identify patients that will or will not respond to these treatments in order to better inform therapy management. Thymidine kinase (TK) is an E2F-regulated enzyme that plays a key role in DNA replication during cell division. The expression and activity of TK is strongly linked to the cell cycle. TK is first expressed in late G1, peaks during S-phase, and then is degraded during mitosis. Because of the restricted expression of TK (only during S phase of the cell cycle) combined with the mechanism of action of CDK4/6i, the measurement of TK activity (TKa) may serve as an ideal pharmacodynamic biomarker to assess a tumor’s response to CDK4/6i-based therapy. DiviTum® TKa is an FDA cleared assay that can quantitate TKa from human serum (sTKa) and is intended to be used as a disease progression monitoring tool for patients with HR+, HER2- MBC. TK IMPACT is a prospective, single arm trial designed to assess the impact of incorporation of DiviTum® TKa on the physician’s decision regarding subsequent timing of routine disease monitoring modalities in patients with advanced HR+, HER2- MBC receiving 1L ET plus CDK 4/6i (NCT04968964). As a part of this ongoing study, DiviTum® TKa values are serially assessed relative to imaging findings and other circulating tumor biomarkers, such as CA15-3 and ctDNA. The primary endpoint is any physician-reported intended change in imaging testing interval post TKa by study cohort, within the first 48-week period of study participation, assessments for which are ongoing. Here we report real-world data and examples of the clinical utility of sTKa as a companion diagnostic from patients treated at Washington University Siteman Cancer Center, St. Louis, MO. Methods: Patients with HR+, HER2- MBC receiving 1L ET + any FDA-approved CDK 4/6i (palbociclib, ribociclib, or abemaciclib) had blood serum samples collected at baseline, weeks 2, 4, 6, 8, every 4 weeks up to week 24, and then every 12 weeks thereafter until disease progression. Samples were analyzed for TKa levels in real-time using the DiviTum® TKa assay. Optional repeat TKa within 2-4 wks (+/- 3 days) is permitted in case of rising TKa. Other standard monitoring assessments were carried out according to institutional guidelines and/or treating clinician discretion. Results: Pre-treatment and early on-therapy TKa levels predicted response to CDK4/6i therapy more accurately than a patient’s clinical characteristics. Rising TKa levels also identified patients with clinically occult disease progression ahead of imaging, and in some cases where radiographic interpretation and serum tumor markers such as CA15-3 were ambiguous. Measurement of TKa levels following CDK4/6i dose reduction was able to confirm continued optimal therapy response at the lower dose. A rise in TKa levels aligned with the re-emergence of ESR1 mutation as identified by ctDNA in one patient and may herald molecular progression. Conclusion: sTKa was able to predict tumor sensitivity and resistance (both intrinsic and acquired) to CDK4/6i + ET therapy in patients with HR+, HER2- MBC. We believe these findings help support the role of sTKa as a non-invasive pharmacodynamic biomarker that can assess in real-time a tumor’s response to CDK4/6i based therapy. Citation Format: Nusayba Bagegni, Amy Williams, Isabella Grigsby, Mattias Bergqvist, Katherine Clifton, Cynthia Ma. Serum thymidine kinase activity as a “real-time” clinical biomarker of tumor response to CDK4/6 inhibition in hormone receptor positive (HR+), HER2 negative (HER2-) metastatic breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-15-06.

Abstract 2957: Senescence of stromal, endothelial, and myeloid cells in the tumor microenvironment has distinct effects on cancer progression

Abstract Accumulation of senescent cells, undermining the function of vital organs, is a culprit of aging. The effects of senescence in different types of cells on tumor growth and metastasis remain incompletely characterized. Cell senescence has been shown to result from inactivation of Telomerase (TERT), an enzyme that maintains telomere length and has obscure roles in regulating gene expression and supporting mitochondrial function. As we reported previously, knockout (KO) of Tert gene in mesenchymal stromal cells (MSC) of Pdgfra+ or Pdgfrb+ lineages leads to premature replicative stromal cell senescence in adipose tissue. To assess the effect of senescence in endothelial and myeloid cells, we also created mice with Tert KO driven by the Tie2e promoter or the LysM promoter, respectively. Here, to investigate the effect of these three KOs on cancer, we used syngeneic graft models of breast, prostate, and pancreatic carcinoma. Overall, Tert KO in cells of Pdgfra+, Pdgfrb+, Tie2e+, or LysM+ lineage had a suppressive effect on tumor growth. The strongest tumor suppression effect of Tert KO was observed in female mice orthotopically grafted with E0771 breast cancer cells. Intermediate suppressive effects of Tert KO were observed in mice orthotopically grafted with KPC pancreatic ductal adenocarcinoma cells, whereas the effect on RM1 prostate cancer cells grafts in male mice was the least significant. MSC Tert KO and myeloid Tert KO also suppressed the growth of breast and prostate cells grafts. Importantly, spontaneous metastases to the liver were induced by Tie2e-Tert KO in all mice with pancreatic KPC grafts. Analysis of adipose tissue in this endothelial senescence model revealed vasculature abnormality, leakiness, and tissue hypoxia, which provides a mechanistic explanation for increased tumor dissemination. In contrast, LysM-Tert KO did not promote KPC metastases, consistent with the lack of contribution of myeloid cells to the vasculature. Pdgfrb-Tert KO in the KPC model resulted in liver metastases observed for some but not all mice, consistent with the role of MSC in supporting vascular integrity. To test an independent model of replicative MSC senescence, we exhausted their pool by several cycles of ganciclovir treatment in mice expressing thymidine kinase under the control of Pdgfrb promoter. Upon KPC grafting in this model, reduced primary pancreatic tumor growth was also observed and liver metastases were also induced. We conclude that senescence of distinct cells in the tumor microenvironment affects early and late stages of cancer progression differently. Specifically, our data indicate that vascular cell senescence suppresses tumor growth but promotes metastases. These findings are important to consider as new approaches designed to mitigate cell senescence are investigated in the context of cancer. Citation Format: Joseph Rupert, Zhanguo Gao, Yongmei Yu, Mikhail Kolonin. Senescence of stromal, endothelial, and myeloid cells in the tumor microenvironment has distinct effects on cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2957.

Abstract 716: Depletion of stromal cells remodels tumor microenvironment and potentiates immune checkpoint blockade in pancreatic cancer

Abstract During carcinoma progression, mesenchymal stromal cells (MSCs) become recruited to tumors and contribute to the pool of cancer-associated fibroblasts (CAFs). Sub-populations of CAFs, have diverse and incompletely understood effects on disease progression and resistance to therapy. Adipose stromal cells (ASCs), the MSCs from fat tissue increasingly recruited by carcinomas in the context of obesity, have been shown to support cancer progression. Here, the roles of perivascular CAFs expressing platelet-derived growth factor receptor beta (Pdgfrb) and of CAFs expressing non-glycanated decorin (ngDCN), a marker of ASCs, were analyzed. We used mice orthotopically grafted with pancreatic ductal adenocarcinoma (KPC) cells. Ablation of cells expressing thymidine kinase under the control of Pdgfrb promoter with ganciclovir resulted in suppression of primary pancreatic tumor growth. However, ablation Pdgfrb+ cells induced spontaneous metastases to the liver. For depletion of ngDCN+ cells, we used a hunter-killer peptide D-CAN, which has been previously reported to induce apoptosis of ASCs and CAFs in mouse models. Here, D-CAN also had a negative effect on pancreatic tumor growth, however, there was no significant effect on metastatic dissemination. Single cell RNA sequencing of tumors demonstrated that targeting of Pdgfrb+ and ngDCN+ stromal cells had distinct effects on sub-populations of CAFs. Endothelial cell and cancer cell survival was decreased by depletion of either Pdgfrb+ or ngDCN+ stroma. However, pathway analysis revealed that depletion of Pdgfrb+ and ngDCN+ stromal cells has different effects on the markers of cancer cell aggressiveness. D-CAN treatment, and to a less extent Pdgfrb+ cell ablation, suppressed macrophage M2 polarization and increased infiltration of cytotoxic T-lymphocytes. This observation, confirmed in the MyCap graft model of prostate cancer, suggested that ablation of ngDCN+ stroma should synergize with immune checkpoint inhibitors. We tested the effect of D-CAN on the efficacy of anti-PDL1 antibody in the orthotopic KPC model. Compared to D-CAN and anti-PDL1 antibody alone, combination treatment had a synergistic effect on tumor growth. Importantly, liver metastases were suppressed by the D-CAN/anti-PDL1 antibody combination, but not by individual agents. We conclude that improved approaches to target mesenchymal stroma in tumors may be effective in combination with immunotherapy. Citation Format: Joseph Rupert, Alexes Daquinag, Lingyi Cai, Yulin Dai, Zhongming Zhao, Dimitris Anastassiou, Mikhail Kolonin. Depletion of stromal cells remodels tumor microenvironment and potentiates immune checkpoint blockade in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 716.

Engineering of Rhesus Monkey Pluripotent Stem Cells for Noninvasive Survey and Remote Control after Brain Allotransplantation

Advanced technologies of targeted differentiation and genetic engineering in pluripotent stem cells (PSCs) offer production of purposefully designed cells for transplantation. Here, we engineer rhesus monkey PSCs (rhPSCs) to produce safe and functional neural progenitors and neurons, which could be noninvasively surveyed and controlled after brain transplantation. RhPSCs expressing hM3Dq-mCherry exhibited normal karyotype and had the ability to equally self-renew and differentiate into functional neurons as that of the mCherry expressing control. After allotransplantation into monkey prefrontal cortex, the hM3Dq-mCherry expressing rhPSC-derived cortical progenitors survived and matured gradually, which could be long-term surveyed by [18F]-Fluorodeoxyglucose positron emission tomography ([18F]FDG-PET) and electroencephalogram (EEG) following clozapine-N-oxide (CNO) administration. Remote activation of transplanted neurons caused increased [18F]-Fluorodeoxyglucose uptake started at 3 months and reached a plateau 9-12 months post-transplantation. EEG analysis revealed a decrease in the high delta spectrum power while an increase in the beta power after CNO delivery. Notably, no seizure-like spikes were observed even after repeated CNO exposure. Magnetic resonance (MR) imaging found no overgrowth in all allografts. Expression of herpes simplex virus thymidine kinase (HSVtk), a Ganciclovir (GCV)-induced suicide gene in rhPSCs further assured the safety without sacrificing the functional outcome of brain grafts. Our study offers a feasible strategy for long-term noninvasive survey and remote control of brain-grafted neurons.

TK1 expression influences pathogenicity by cell cycle progression, cellular migration, and cellular survival in HCC 1806 breast cancer cells.

Breast cancer is the most common cancer diagnosis worldwide accounting for 1 out of every 8 cancer diagnoses. The elevated expression of Thymidine Kinase 1 (TK1) is associated with more aggressive tumor grades, including breast cancer. Recent studies indicate that TK1 may be involved in cancer pathogenesis; however, its direct involvement in breast cancer has not been identified. Here, we evaluate potential pathogenic effects of elevated TK1 expression by comparing HCC 1806 to HCC 1806 TK1-knockdown cancer cells (L133). Transcriptomic profiles of HCC 1806 and L133 cells showed cell cycle progression, apoptosis, and invasion as potential pathogenic pathways affected by TK1 expression. Subsequent in-vitro studies confirmed differences between HCC 1806 and L133 cells in cell cycle phase progression, cell survival, and cell migration. Expression comparison of several factors involved in these pathogenic pathways between HCC 1806 and L133 cells identified p21 and AKT3 transcripts were significantly affected by TK1 expression. Creation of a protein-protein interaction map of TK1 and the pathogenic factors we evaluated predict that the majority of factors evaluated either directly or indirectly interact with TK1. Our findings argue that TK1 elevation directly increases HCC 1806 cell pathogenicity and is likely occurring by p21- and AKT3-mediated mechanisms to promote cell cycle arrest, cellular migration, and cellular survival.

IRF9 inhibits CyHV-3 replication by regulating the PI3K-AKT signalling pathway in common carp (Cyprinus carpio) epithelial cells

Interferon regulatory factor 9 (IRF9) is an important transcriptional regulator involved in innate and adaptive immunity. Cyprinid herpesvirus-3 (CyHV-3) is a virus causing widespread death and great economic loss in farmed common carp (Cyprinus carpio). However, the effect of IRF9 on CyHV-3 infection in common carp has not been reported. In this study, during CyHV-3 infection, IRF9 overexpression in common carp fin epithelial (CCF) cells significantly reduced the expression of viral factor thymidine kinase (TK) and open reading frame 72 (ORF72), and knockdown of IRF9 produced the opposite results (p < 0.05). In CCF cells. The IRF9 protein was expression in the nucleus and was rapidly induced in CCF cells by CyHV-3 infection. In addition, several genes associated with virus infection, including type I interferon (IFNI), IFN-stimulated gene 15 (ISG15), myxovirus resistance 1 (Mx1) and Viperin were induced in CCF cells overexpressing IRF9 upon CyHV-3 infection. IRF9 overexpression induced by CyHV-3 infection significantly increased the gene expression of Mx1 and phosphoinositide 3-kinase (PI3K) and the protein expression of protein kinase B (AKT) (p < 0.01). Interestingly, IRF9 did not significantly affect Mx1 gene expression when AKT protein levels remained unchanged during CyHV-3 infection of CCF cells. Furthermore, a significant resistance-related locus was found in the IRF9 sequence in “Longke-11” mirror carp (M11) and Yellow River carp (p < 0.05). These results indicated that IRF9 inhibited viral replication by upregulating the expression of Mx1 via the PI3K-AKT signalling pathway during CyHV-3 infection in CCF cells and provide some basis for the study of the antiviral molecular mechanisms of common carp.

Inhibition of human oral squamous cell carcinoma proliferation and migration by prodrug-activating suicide gene therapies.

Head and neck squamous cell carcinoma (HNSCC), which originates from mucosal epithelium in the oral cavity, pharynx and larynx, is the sixth most common malignancy in the world. The prognosis of HNSCC is not satisfactory due to metastasis, resulting in 5-year survival rates ranging from 65.9 to 67.2%. Previously, we developed a method to evaluate the effect prodrug-activating suicide gene (PA-SG) therapy on the proliferation of HNSCC. The present study investigated PA-SG therapy on metastatic HNSCC by wound-healing assay and our previously established method. HSC-3 cells with stable expression of suicide genes thymidine kinase (TK) or cytosine deaminase (CD) were treated with prodrugs ganciclovir (GCV) or 5-fluorocytosine (5-FC), respectively. Both GCV and 5-FC inhibited HSC-3 proliferation while the bystander effect of CD/5-FC was greater compared with that of TK/GCV. GCV showed a greater anti-migration effect compared with that of 5-FC. To the best of our knowledge, the present study is the first to evaluate the anti-migratory and anti-proliferative effects of PA-SG therapies on metastatic HNSCC. This may also serve as a general method to quantify other types of PA-SC therapy. The present results demonstrated that PA-SG therapy is a promising treatment for anti-metastatic HNSCC therapy development.

External Validation of the Colon Life Nomogram for Predicting 12-Week Mortality in Dutch Metastatic Colorectal Cancer Patients Treated with Trifluridine/Tipiracil in Daily Practice.

Simple SummaryPredicting prognosis in cancer patients is needed to guide decision making. In order to predict survival, nomograms can be used to estimate chances of survival based on clinical characteristics. In order to identify metastatic colorectal cancer (mCRC) patients with a very short life expectancy (less than 12 weeks) after receiving multiple standard treatments, the Colon Life nomogram was previously developed. Before a nomogram can be used in daily practice, it is essential to show that it accurately predicts survival in different real-life populations and can be used to guide clinical decision making. This is called external validation. We externally validated the Colon Life nomogram in a cohort of patients with refractory mCRC who were treated with a last treatment option, trifluridine/tipiracil, in daily practice. We demonstrated that the nomogram severely overestimated 12-week mortality and therefore should not be used in clinical practice in its present form. We also showed that quality of life reported by patients themselves can improve the prediction of survival, stressing the importance of patient-reported outcomes. We recommend conducting a study with a sufficiently large sample size to update the Colon Life nomogram or to develop a new model and include quality of life.Background: Predicting prognosis in refractory metastatic colorectal cancer (mCRC) patients is needed to guide decision making. The Colon Life nomogram was developed to predict 12-week mortality in refractory mCRC patients. The aim of this study is to validate the Colon Life nomogram in last line/refractory patients receiving trifluridine/tipiracil (FTD/TPI) in daily practice. Methods: The validation cohort consists of 150 QUALITAS study patients, an observational substudy of the Prospective Dutch CRC cohort, who were treated with FTD/TPI between 2016 and 2019. Model performance was assessed on discrimination, calibration, and clinical usefulness. The additional prognostic value of baseline quality of life (QoL) and thymidine kinase (TK1) expression in tissue was explored. Results: Of the 150 patients, 25 (16.7%) died within 12 weeks of starting FTD/TPI treatment. The C-statistic was 0.63 (95% C.I. 0.56–0.70). The observed/expected ratio was 0.52 (0.37–0.73). The calibration intercept and slope were −1.06 (−1.53 to −0.58) and 0.41 (0.01–0.81), respectively, which indicated overestimation of 12-week mortality by the nomogram. Decision curve analysis showed the nomogram did not yield a positive net benefit at clinically meaningful thresholds for predicted 12-week mortality. Addition of QoL to the nomogram improved the C-statistic to 0.85 (0.81–0.89). TK1 expression was associated with progression-free survival but not with overall survival. Conclusion: We demonstrated evident miscalibration of the Colon Life nomogram upon external validation, which hampers its use in clinical practice. We recommend conducting a study with a sufficiently large sample size to update the Colon Life nomogram or to develop a new model including QoL.

Neomorphic DNA-binding enables tumor-specific therapeutic gene expression in fusion-addicted childhood sarcoma

Chimeric fusion transcription factors are oncogenic hallmarks of several devastating cancer entities including pediatric sarcomas, such as Ewing sarcoma (EwS) and alveolar rhabdomyosarcoma (ARMS). Despite their exquisite specificity, these driver oncogenes have been considered largely undruggable due to their lack of enzymatic activity.Here, we show in the EwS model that – capitalizing on neomorphic DNA-binding preferences – the addiction to the respective fusion transcription factor EWSR1-FLI1 can be leveraged to express therapeutic genes.We genetically engineered a de novo enhancer-based, synthetic and highly potent expression cassette that can elicit EWSR1-FLI1-dependent expression of a therapeutic payload as evidenced by episomal and CRISPR-edited genomic reporter assays. Combining in silico screens and immunohistochemistry, we identified GPR64 as a highly specific cell surface antigen for targeted transduction strategies in EwS. Functional experiments demonstrated that anti-GPR64-pseudotyped lentivirus harboring our expression cassette can specifically transduce EwS cells to promote the expression of viral thymidine kinase sensitizing EwS for treatment to otherwise relatively non-toxic (Val)ganciclovir and leading to strong anti-tumorigenic, but no adverse effects in vivo. Further, we prove that similar vector designs can be applied in PAX3-FOXO1-driven ARMS, and to express immunomodulatory cytokines, such as IL-15 and XCL1, in tumor entities typically considered to be immunologically ‘cold’.Collectively, these results generated in pediatric sarcomas indicate that exploiting, rather than suppressing, the neomorphic functions of chimeric transcription factors may open inroads to innovative and personalized therapies, and that our highly versatile approach may be translatable to other cancers addicted to oncogenic transcription factors with unique DNA-binding properties.

Thymidine Kinase 1 Mediates the Synergistic Antitumor Activity of Ubenimex and Celecoxib via Regulation of Cell Cycle in Colorectal Cancer.

Colorectal cancer (CRC) is a common clinical malignant tumor of the digestive system that seriously affects the health and life of patients. Because it is difficult to cure CRC, the strategy of drug combination is often used in clinical therapy. This study mainly revealed that ubenimex and/or celecoxib exerted anti-colon cancer effects in vitro and in vivo, and the efficacy was significantly enhanced when the two drugs were combined. The combination of the two drugs induced significantly stronger cell-cycle arrest than did the single drug, and also enhanced the antitumor efficacy of 5-fluorouracil and its derivatives. At the same time, the expression of thymidine kinase 1 (TK1) protein was decreased through regulating the level of TK1 mRNA treated with celecoxib and/or ubenimex, but the combination drugs exhibited much more reduction of TK1 mRNA and protein as compared with the single agent alone. TK1 may be the molecular target of the combination of two drugs to exert the anti-colorectal cancer effect. In summary, this research demonstrates that celecoxib combined with ubenimex inhibits the development of colorectal cancer in vitro and in vivo, making them a viable combination regimen. SIGNIFICANCE STATEMENT: In this study, our data reveal the great potential of celecoxib combined with ubenimex in the treatment of colorectal cancer, providing new ideas for clinical antitumor drug regimens and theoretical reference for drug development.

A Novel Ex-Vivo Model of Prolonged Osteoblast Suppression in Multiple Myeloma

Multiple myeloma (MM) is the most frequently involves the skeleton, with over 80% of patients developing osteolytic bone disease (MMBD). Importantly, lytic bone lesions in MM patients rarely heal even during long-term complete remission without detectable myeloma cells. Mesenchymal stem cells from MM patients (MM-MSCs) display distinct gene profiles and impaired osteoblast (OB) differentiation compared with those of normal donors, even after many passages in vitro. We found that MM causes long-term inhibition of OB differentiation by inducing epigenetic changes in the Runx2 promoter in MSCs, which persist in the absence of myeloma cells. Importantly, MSCs from myeloma patients expressed elevated Gfi1, a factor that modifies the chromatin structure of Runx2. However, the molecular mechanisms for MM-induced long-term OB suppression remain poorly understood. Exploring these mechanisms in animal models is costly, slow and complex. Further, it is difficult to replicate the bone marrow microenvironment in vitro.We have developed an ex-vivo bone organ culture assay (EVOCA) that more closely replicates the marrow microenvironment, to further investigate the molecular mechanisms responsible for the protracted OB suppression in MM and to screen bone anabolic agents for MMBD. 5TGM1 murine MM cells expressing thymidine kinase and GFP (5TGM1-GFI-TK) were co-cultured in EVOCA with mouse long bones (femurs and tibias from 4 weeks old mice). Bones cut longitudinally with marrow in place were cultured in 35 mm dishes +/- 5TGM1-GFI-TK cells (2x105/ml) and 10 % FCS in a 1:1 mix of RPMI/αMEM for 4 days. Conditioned media were collected and the bone resorption marker, c-terminal telopeptide crosslink of type 1 collagen (CTX) was measured by ELISA. CTX levels were increased in 5TGM1-containing EVOCAs compared to cultures without 5TGM1 cells (5.5±0.8 ng/ml vs. 9.8±1.0 ng/ml, p<0.001). 5TGM1-GFI-TK cell can be visualized by florescence microscopy, and HSV-TK expression permits selective cell killing with ganciclovir (Gcv), we then treated EVOCAs +/- 20 µg/ml Gcv on days 4-7 of culture. Myeloma cell survival was monitored by florescence microscopy and assay of IgG2b in the media. Gcv killed 100% of the myeloma cells. EVOCA cultures were continued for 1- 2 weeks more, then bones and outgrowth cells were homogenized, and lysates and RNA assayed for Runx2 and Osterix expression by Western blot and qPCR. Both Runx 2 and Osterix levels were 25% lower in EVOCAs treated with Gcv, and 50% lower compared to EVOCas not cultured with 5TGM1 cells. Runx2 and Osterix levels in EVOCAs cultured with Gcv but no MM cells were similar to levels in vehicle-treated bones. NFATc-1, cathepsin K, RANKL and OPG levels and osteoclast numbers were then measured in EVOCAs at 2 weeks post Gcv treatment. 5TGM1-containing EVOCAs had increased osteoclast numbers (2X) and levels of cathepsin K and NFATc-1. Further, RANKL/OPG ratios were increased in bone extracts from EVOCAs co-cultured with 5TGM1 cells compared with controls, regardless of Gcv treatment.We next modified EVOCA to evaluate new bone formation. A 0.5mm diameter hole was initially made with a drill bit in intact bones (uncut) and the bones co-cultured +/- 5TGM1-GFP-TK cells (2x105 /ml) 10% FCS in a 1:1 mix of RPMI/αMEM for 4 days. The cultures were then treated +/- Gcv for 3 days. At day 7, no 5TGM1 cells were detectable. The media was then changed to αMEM, and the EVOCAs continued for 1 week. Then 50µg/ml ascorbic acid and 10 mM β-glycerophosphate were added and the cultures continued for 7 more days. Bones were stained with Alizarin Red S to detect new bone formation at the drill site. New bone formation was confirmed by µCT. No new bone formation was detected in bones from EVOCAs initiated with 5TGM1 cells regardless of 5TGM1 removal. In contrast, new bone formation was easily evident in bones not exposed to MM cells.The results show that increased bone resorption and OB suppression persist in MM-exposed bone even after myeloma cells are removed. The assay should allow us to follow the sequential development of lytic bone lesions, tumor growth, and protracted OB suppression, providing a useful tool for studies of MMBD, and evaluation of anabolic agents and anti-myeloma therapy in an intact marrow microenvironment. DisclosuresRoodman:PharmaMar: Research Funding.

Stereotactic body radiation therapy and in situ oncolytic virus therapy followed by immunotherapy in metastatic non-small cell lung cancer.

9115 Background: The introduction of immunotherapy has altered the treatment paradigm for metastatic non-small cell cancer (mNSCLC). Unfortunately, many patients with mNSCLC have limited or no benefit from immune checkpoint inhibitors (ICIs). A variety of approaches have been explored to augment the efficacy of ICIs. Our study’s aim was to determine whether the addition of stereotactic body radiation therapy (SBRT) and intratumoral injection of the oncolytic virus ADV/HSV-tk (adenovirus-mediated expression of herpes simplex virus thymidine kinase) to a monoclonal antibody targeting programmed cell death-1 (PD-1) would improve the ICI’s efficacy in the treatment of mNSCLC. Methods: In this single-arm, open-label phase II study, patients with mNSCLC (squamous or non-squamous) who were ICI-naive or who were previously treated with a maximum of one line of therapy that included an ICI received an intratumoral injection of ADV/HSV-tk (5 x 1011 vp) followed by SBRT (30 Gy in 5 fractions) to the same tumor. An anti-PD-1 agent (pembrolizumab 200 mg IV every 3 weeks or nivolumab 240 mg IV every 2 weeks) was then given for up to 24 months (pembrolizumab) or 12 months (nivolumab), or until disease progression or intolerable toxicity. The primary endpoint was objective response rate (ORR) as defined by the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. A secondary endpoint was clinical benefit rate (CBR). Results: A total of 35 patients were enrolled, with 28 (80%) receiving pembrolizumab and 7 (20%) receiving nivolumab; 14 (40%) had previous ICI therapy while 21 (60%) were ICI-naive. The ORR and CBR were 28.5% and 61.9% in the ICI-naive group, and 14.2% and 64.2% in the group that previously received an ICI, respectively. Grade 3 or higher toxicity was seen in five patients (26.3%) in the ICI-naive group and in one patient (7.1%) in the previously ICI-treated group. No treatment-related deaths were observed. Conclusions: The addition of SBRT and intratumor injection of ADV/HSV-tk to anti-PD-1 therapy in mNSCLC resulted in a CBR of over 60% for both ICI-naive and previously ICI-treated patients without the use of chemotherapy. The combination was able to reinstitute sensitivity to ICIs in patients previously treated with an ICI, and also benefited some patients whose tumors did not express PD-L1. These findings should be further explored in a larger study population. Clinical trial information: NCT03004183. [Table: see text]

A phase II trial of stereotactic radiation therapy and in situ oncolytic virus therapy in metastatic triple-negative breast cancer (mTNBC) patients followed by pembrolizumab (STOMP).

1079 Background: Pembrolizumab, stereotactic body radiotherapy (SBRT), and viral vector-based gene therapy such as adenovirus-mediated expression of herpes simplex virus thymidine kinase (ADV/HSV-tk) plus ganciclovir have each shown antitumor immune activity. The combination of those modalities may represent a window of opportunity to enhance pembrolizumab efficacy in mTNBC patients. Methods: In this single-arm, open-label phase II trial, mTNBC patients were treated with in situ oncolytic ADV/HSV-tk (5 x 1011 vp) intratumoral injection, followed by SBRT to the injected tumor site, then pembrolizumab until progression or intolerable toxicity. Response was assessed in non-irradiated metastatic sites. The primary end point was clinical benefit rate (CR, PR and SD per RECIST version1.1). Secondary endpoints included duration on treatment (DoT) and safety. Immune correlative analysis with peripheral blood CYTOF (D1 baseline, D7 and D38), and tissue imaging mass cytometry (IMC) with 35 cell surface markers was performed on paired biopsies (D1 baseline and D17). Results: 28 mTNBC patients were enrolled; 18 (64.3%) had PD-L1 negative tumors. Median age was 54 years (range 34-78). Median prior lines of chemotherapy were 2 (range 0-6), with 8 (28.6%) having received &gt;3. 3 (10.7%) had brain metastases. Clinical benefit was seen in 6 (21.43%) patients; 2 CR (7.1%),1 PR (3.57%) and 3 SD (10.7%). Patients who had clinical benefits had durable responses, with median DoT of 383 days (range 195-1195). One patient who had CR, but discontinued pembrolizumab due to Grade 3 pneumonitis, has remained disease free without any systemic therapy for 39 months. The combination was well tolerated; 9 (32.1%) patients had Grade 3- 4 AEs. Pre- and post-therapy CYTOF analysis showed significant association between immune biomarkers with clinical responses (AUC 0.75, Cohen’s Kappa 0.364). Tumor PDL1 was independently associated with response (AUC 0.70, Cohen’s Kappa 0.347); AUC for PDL1 together with immune biomarkers is 0.85. Conclusions: ADV/HSV-tk gene therapy followed by radiation therapy and then pembrolizumab is a well-tolerated promising treatment in heavily pretreated mTNBC patients. Early detection of increased effector and effector memory CD8 T cells and nonclassical monocytes correlates with response and non-response respectively. Clinical trial information: NCT03295916 .[Table: see text]

MicroRNA-320b suppresses HNF4G and IGF2BP2 expression to inhibit angiogenesis and tumor growth of lung cancer

We examined the effect of microRNA-320b (miR-320b) on tumor growth and angiogenesis in lung cancer and also determined its downstream molecular mechanisms. Lung cancer tissues and adjacent non-cancerous tissues were collected from 66 patients with lung cancer. miR-320b expression was experimentally determined to be expressed at low level in cancer tissues. The results of gain-of-function experiments suggested that miR-320b overexpression suppressed cancer cell invasion, tube formation, tumor volume and angiogenesis in xenografted nude mice. Hepatocyte nuclear factor 4 gamma (HNF4G) was identified as a target of miR-320b based on in silico analysis. Dual-luciferase reporter gene assays further identified the binding relationship between HNF4G and miR-320b. Lung cancer tissues exhibited increased expression of HNF4G and insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2). Meanwhile, HNF4G knockdown suppressed IGF2BP2 expression, thereby repressing cancer cell invasion and tube formation. Furthermore, IGF2BP2 modified m6A to increase the expression of thymidine kinase 1 (TK1), thus promoting angiogenesis. In nude mice, restoration of TK1 reversed the suppressive effect of miR-320b overexpression on tumor growth rate and CD31 expression. In conclusion, miR-320b suppresses lung cancer growth and angiogenesis by inhibiting HNF4G, IGF2BP2 and TK1.

Mechanism of acquired 5FU resistance and strategy for overcoming 5FU resistance focusing on 5FU metabolism in colon cancer cell lines.

Fluorouracil (5FU) is converted to its active metabolite fluoro-deoxyuridine monophosphate (FdUMP) through the orotate phosphoribosyl transferase (OPRT)-ribonucleotide reductase (RR) pathway and thymidine phosphatase (TP)-thymidine kinase (TK) pathway and inhibits thymidylate synthase (TS), leading to inhibition of thymidine monophosphate (dTMP) synthesis through a de novo pathway. We investigated the mechanism of 5FU resistance and strategies to overcome it by focusing on 5FU metabolism. Colon cancer cell lines SW48 and LS174T and 5FU-resistant cell lines SW48/5FUR and LS174T/5FUR were used. FdUMP amount was measured by western blotting. The FdUMP synthetic pathway was investigated by combining TP inhibitor (tipiracil hydrochloride; TPI) or RR inhibitor (hydroxyurea; HU) with 5FU. Drug cytotoxicity was observed by crystal violet staining assay. FdUMP was synthesized through the OPRT-RR pathway in SW48 cells but was scarcely synthesized through either the OPRT-RR or TP-TK pathway in SW48/5FUR cells. FdUMP amount in SW48/5FUR cells was reduced by 87% vs. SW48 cells. Expression levels of OPRT and TP were lower in SW48/5FUR when compared with these levels in the SW48 cells, indicating decreased synthesis of FdUMP-led 5FU resistance. These results indicated that fluoro-deoxyuridine (FdU) rather than 5FU promotes FdUMP synthesis and overcomes 5FU resistance. Contrastingly, FdUMP was synthesized through the OPRT-RR and TP-TK pathways in LS174T cells but mainly through the TP-TK pathway in LS174T/5FUR cells. FdUMP amount was similar in LS174T/5FUR vs. the LS174T cells. OPRT and RR expression was lower and TK expression was higher in LS174T/5FUR vs. the LS174T cells, indicating that dTMP synthesis increased through the salvage pathway, thus leading to 5FU resistance. LS174T/5FUR cells also showed cross-resistance to FdU and TS inhibitor, suggesting that nucleoside analogs such as trifluoro-thymidine should be used to overcome 5FU resistance in these cells. 5FU metabolism and mechanisms of 5FU resistance are different in each cell line. Both synthesized FdUMP amount and FdUMP sensitivity should be considered in 5FU-resistant cells.

CNS fibroblasts form a fibrotic scar in response to immune cell infiltration.

Fibrosis is a common pathological response to inflammation in many peripheral tissues and can prevent tissue regeneration and repair. Here, we identified persistent fibrotic scarring in the central nervous system (CNS) following immune cell infiltration in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis. Using lineage tracing and single-cell sequencing in EAE, we determined that the majority of the fibrotic scar is derived from proliferative CNS fibroblasts, not pericytes or infiltrating bone marrow-derived cells. Ablating proliferating fibrotic cells using cell-specific expression of herpes thymidine kinase led to an increase in oligodendrocyte lineage cells within the inflammatory lesions and a reduction in motor disability. We further identified that interferon gamma pathway genes are enriched in CNS fibrotic cells, and the fibrotic cell-specific deletion of Ifngr1 resulted in reduced fibrotic scarring in EAE. These data delineate a framework for understanding the CNS fibrotic response.

Effects of epigallocatechin-3-gallate and acyclovir on herpes simplex virus type 1 infection in oral epithelial cells

Herpes simplex virus type 1 (HSV-1) is the pathogenic agent of human diseases, including gingivostomatitis and herpes labialis. The anti-viral activities of the tea polyphenol, epigallocatechin-3-gallate (EGCG), have been demonstrated. This study examined the combined effects of EGCG and the antiviral drug, acyclovir (ACV), on infection of HSV-1 in oral epithelial cells. Cell viability was examined using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide. Viral yields were determined using the plaque assay. Viral proteins were detected using Western blotting analysis or confocal laser scanning microscopy. Viral DNA was detected using the real-time polymerase chain reaction. Cytotoxic effects of HSV-1 on the viability of oral epithelial cells were evidently reduced in the presence of EGCG (25μg/ml) or/and ACV (50μg/ml). Viral yields were also significantly reduced by treatment of cells with EGCG or/and ACV. Expression of viral immediate early protein, infected cell protein 0 (ICP0), was greatly inhibited when cells were treated with EGCG. Combined effects of EGCG and ACV were more evident for the expression of viral thymidine kinase, ICP5 and glycoprotein D. EGCG, but not ACV, significantly reduced the levels of viral particles and viral DNA during viral entry phase. However, at 20h post infection, the intracellular viral DNA was evidently reduced in HSV-1 infected cells treated with EGCG and ACV. Moreover, the stimulatory effects of HSV-1 on phosphorylation of c-Jun N-terminal kinase could be reduced by ACV. The results demonstrated the additive effects of EGCG and ACV on HSV-1 infection in oral epithelial cells.

Immune-stimulatory (TK/Flt3L) gene therapy opens the door to a promising new treatment strategy against brainstem gliomas.

Diffuse intrinsic pontine glioma (DIPG) is a rare brainstem tumor which carries a dismal prognosis. To date. there are no effective treatments for DIPG. Transcriptomic studies have shown that DIPGs have a distinct profile compared to hemispheric high-grade pediatric gliomas. These specific genomic features coupled with the younger median age group suggest that DIPG is of developmental origin. There is a major unmet need for novel effective therapeutic approaches for DIPG. Clinical and preclinical studies have expanded our understanding of the molecular pathways in this deadly disease. We have developed a genetically engineered brainstem glioma model harboring the recurrent DIPG mutation, activin A receptor type I (ACVR1)-G328V (mACVR1) using the sleeping beauty transposon system. DIPG neurospheres isolated from the genetically engineered mouse model were implanted into the pons of immune-competent mice to assess the therapeutic efficacy and toxicity of immunostimulatory gene therapy using adenoviruses expressing thymidine kinase (TK) and fms-like tyrosine kinase 3 ligand (Flt3L). Immunostimulatory adenoviral-mediated delivery of TK/Flt3L in mice bearing brainstem gliomas resulted in antitumor immunity, recruitment of antitumor-specific T cells, and improved median survival by stimulating the host antitumor immune response. Therapeutic efficacy of the immunostimulatory gene therapy strategy will be tested in the clinical arena in a Phase I clinical trial. We also discuss immunotherapeutic interventions currently being implemented in DIPG patients and discuss the profound therapeutic implications of immunotherapy for this patient populations.

Therapeutic Efficacy of Immune Stimulatory Thymidine Kinase and fms-like Tyrosine Kinase 3 Ligand (TK/Flt3L) Gene Therapy in a Mouse Model of High-Grade Brainstem Glioma.

Diffuse intrinsic pontine glioma (DIPG) bears a dismal prognosis. A genetically engineered brainstem glioma model harboring the recurrent DIPG mutation, Activin A receptor type I (ACVR1)-G328V (mACVR1), was developed for testing an immune-stimulatory gene therapy. We utilized the Sleeping Beauty transposase system to generate an endogenous mouse model of mACVR1 brainstem glioma. Histology was used to characterize and validate the model. We performed RNA-sequencing analysis on neurospheres harboring mACVR1. mACVR1 neurospheres were implanted into the pons of immune-competent mice to test the therapeutic efficacy and toxicity of immune-stimulatory gene therapy using adenoviruses expressing thymidine kinase (TK) and fms-like tyrosine kinase 3 ligand (Flt3L). mACVR1 neurospheres expressing the surrogate tumor antigen ovalbumin were generated to investigate whether TK/Flt3L treatment induces the recruitment of tumor antigen-specific T cells. Histologic analysis of mACVR1 tumors indicates that they are localized in the brainstem and have increased downstream signaling of bone morphogenetic pathway as demonstrated by increased phospho-smad1/5 and Id1 levels. Transcriptome analysis of mACVR1 neurosphere identified an increase in the TGFβ signaling pathway and the regulation of cell differentiation. Adenoviral delivery of TK/Flt3L in mice bearing brainstem gliomas resulted in antitumor immunity, recruitment of antitumor-specific T cells, and increased median survival (MS). This study provides insights into the phenotype and function of the tumor immune microenvironment in a mouse model of brainstem glioma harboring mACVR1. Immune-stimulatory gene therapy targeting the hosts' antitumor immune response inhibits tumor progression and increases MS of mice bearing mACVR1 tumors.