Tumor Cell Extracts Research Articles

Highly sensitive detection of RNase H via a novel DNA/RNA heteroduplex combining isothermal exponential amplification strategy

Ribonuclease H (RNase H) plays a crucial role in a variety of cellular processes and is emerging as an essential therapeutic target for many diseases. Various methods have been constructed to assay RNase H activity, but these methods are either time-consuming or have poor sensitivity. In this study, we developed a novel strategy that combined a specially designed DNA/RNA chimeric substrate with exponential amplification reaction (EXPAR) for rapid and sensitive detection of RNase H activity. In the presence of RNase H, the RNA strand of the DNA/RNA heteroduplex was specifically degraded, forming a 3′-hydroxyl group in the locking primer for recognition and extension by DNA polymerase. This ultimately triggered EXPAR and produced a large number of single-stranded DNA, which was monitored in real-time with fluorescent dye. Under optimized conditions, the proposed strategy can detect as low as 6 × 10−10 U/μL of RNase H, which was at least 1000 times more sensitive than several reported methods. Furthermore, we demonstrated the usage of this method for RNase H inhibitor analysis and practical application in complex biological samples, including serum and tumor cell extracts. Therefore, these results suggested that the developed method is a promising tool for highly sensitive detection of RNase H and RNase H-associated disease diagnosis.

Abstract 137: Increased activity of protein kinase A is sufficient to cause fibrolamellar carcinoma

Abstract Tumor cells of almost all patients with fibrolamellar carcinoma (FLC) have a somatic mutation, a ~400 kB deletion on one copy of chromosome 19 that results in a fusion transcript of DNAJB1 to PRKACA, the catalytic subunit of protein kinase A. The resulting fusion oncoprotein, DNAJB1::PRKACA, has kinase activity, and is required and sufficient for transformation. To understand the oncogenesis of this tumor and to help identify potential therapeutic targets, we explored the pathways of pathogenesis mediated by the aberrant kinase. We explored three mechanisms that might explain transformation by the oncogenic kinase: - A difference in the substrate specificity of DNAJB1::PRKACA kinase versus native PRKACA kinase. - A difference in the interactome of DNAJB1::PRKACA kinase versus native PRKACA kinase. - An increase of total catalytic kinase activity (DNAJB1::PRKACA + PRKACA) versus the native PRKACA condition. We found, comparing cytosolic extracts of tumor and normal liver cells, that there are differences in the substrate specificity of the DNAJB1::PRKACA kinase. Additionally, such comparisons revealed differences in the interactions between the catalytic subunit of the kinase and cytosolic components. We also demonstrated a consistent increase in total PRKACA catalytic subunit in the FLC tumor, relative to the adjacent nontransformed tissue. Further, there is an increase in free, unregulated catalytic activity in the tumor cells and a change in the localization of the catalytic subunit in the tumor cells. Additionally, we have found that this increase in free catalytic activity is sufficient to account for the changes in the transcriptome in tumor cells of FLC patients. Importantly, enhanced expression in hepatocytes of either DNAJB1::PRKACA kinase or native PRKACA kinase caused very similar changes in the transcriptome, and the resultant transcriptomes closely resembled that of tumor cells from FLC patients. This suggests that the DNAJB1 domain is dispensable and not critical for transformation. In our investigation of patient tumors we found two rare and different subclasses of FLC that are indistinguishable from “classic” FLC, based on their transcriptome and based on their drug response profiles. Importantly, both subclasses are associated with changes of PRKACA, but neither involves the DNAJB1 domain. Thus, the DNAJB1 domain is not a critical component of transformation, and the significant driver is an increase of the cellular catalytic activity. Citation Format: Mahsa Shirani, Michael Tomasini, Solomon Levin, soeren heissel, Henrik Molina, Ype De Jong, charles Rice, Philip Coffino, Barbara Lyons, Sanford Simon. Increased activity of protein kinase A is sufficient to cause fibrolamellar carcinoma [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 137.

Combined Treatment with Ultrasound and Immune Checkpoint Inhibitors for Prostate Cancer.

Background: Ultrasound (US) is mostly used for diagnostic purpose but could be used for cancer treatments with a US intensity or frequency fitted to such a purpose. Prostate cancer (PC) has the highest prevalence in the urological field, but indications for immune checkpoint inhibitors (ICIs) for PC are limited to very few cases. In this study, we compared the antitumor effect of US irradiation alone with the combined use of US and ICIs in vitro and in vivo. Methods: PC cell line TRAMP-C2 cells were used in our experiments. TRAMP-C2 cells were irradiated with US with pulse repeated frequencies (PRF) of 1, 10, and 100 Hz. Cell proliferation was evaluated by MTS assay and apoptotic cells were analyzed using flow cytometry. To verify the antitumor effect of US irradiation on PC in vivo, we conducted animal experiments using mice. TRAMP-C2-bearing mice were irradiated with US with PRF of 10 and 100 Hz. Three weeks after the start of US irradiation, anti-PD-1 antibody was administered to the mice. Finally, mice were sacrificed and tumors were collected. Immunohistochemical (IHC) analyses were assessed for cleaved caspase-3 and CD3 in tumor cell extracts. Results: Cell proliferation assays showed that 1 and 10 Hz US significantly inhibited cell survival (p < 0.0001). In addition, US irradiation induced apoptosis at 1, 10, and 100 Hz (p = 0.0129, p = 0.0150, and p = 0.0017, respectively). In animal experiments, a significant tumor growth inhibitory effect was observed at 10 and 100 Hz, and 100 Hz + ICIs (p < 0.05, respectively). Hematoxylin–eosin (H–E) staining showed a significant increase in the necrotic area of the tumor at 100 Hz and 100 Hz + ICIs (p < 0.05, respectively). In addition, under IHC staining the expression level of cleaved caspase-3 and the number of CD3-positive cells increased at 100 Hz (p < 0.05, respectively). Conclusion: US irradiation induced apoptosis in cells and reduced cell viability. In vivo tumor growth was suppressed by combined treatment with US irradiation and ICIs. Further research on immune system activation will lead to less invasive and more efficient treatments for PC.

Oxidized plant leaf-derived carbon dots as novel electrochemiluminescent luminophores for ultrasensitive microRNA-21 detection

The enthusiasm of many chemists for green chemistry has increased due to its clean and sustainable features. Here, plant leaf-derived carbon dots (CDPs) and oxidized CDPs (o-CDPs) were synthesized through a simple pyrolytic procedure. The results of the controlled trial showed that the electrochemiluminescence (ECL) performances of o-CDPs were significantly better than those of CDPs. Among them, oxidized maple CDP (o-CDP-M) exhibited the highest ECL intensity with a maximum emission wavelength of 520 nm and could be used as a new type of "green source" luminophore. In view of the considerable role of microRNA-21 (miRNA-21) in tumour overexpression, it was chosen as the target molecule. In combination with the cascade amplification strategy, an ultrasensitive “signal-on” ECL biosensor based on o-CDP-M was developed. Possessing the merits of eco-friendliness and high selectivity, the proposed biosensor achieved ultrasensitive detection of miRNA-21 with a dynamic range from 1 fM to 1 nM and a detection limit as low as 0.27 fM. It was successfully used to analyse miRNA-21 in tumour cell extracts. This work provides a new opportunity for synthesizing “green source” luminophores and detecting various trace miRNA species.

Retraction Note: Tumor-specific Th2 responses inhibit growth of CT26 colon-cancer cells in mice via converting intratumor regulatory T cells to Th9 cells

The abnormality of immune regulation plays a critical role in the pathogenesis of cancer; the underlying mechanism has not been fully understood yet. This study aims to investigate the role of cancer specific T helper (Th)2 response in the inhibition of colon cancer (Cca) cell growth. The results showed that with Cca cell (CT26 cell) extracts as an antigen, the Cca-extract specific Th2 response was induced in the Cca-bearing mice. The Cca mass size was significantly reduced, or radically disappeared (5 out of 10; or 50%); the survival rate was markedly improved in mice immunized with Cca-extract, but not in those immunized with another tumor cell (U87 cell) extracts or to bovine serum albumin. The immunization with Cca-extract also induced Cca cell apoptosis and converted the intra-Cca Tregs to T helper (Th) 9 cells. In conclusion, Cca-specific Th2 responses inhibit Cca growth in a mouse model via inducing Cca cell apoptosis and converting intra-Cca Tregs to Th9 cells.

A 1D High Performance Thin Layer Chromatography Method Validated to Quantify Phospholipids Including Cardiolipin and Monolysocardiolipin from Biological Samples

AbstractThe aim of this study is to identify high performance thin layer chromatography (HPTLC) conditions allowing the separation and quantification of mammalian cellular phospholipids (PLs) (sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and especially phosphatidic acid, cardiolipin, and monolysocardiolipin, these latter two being specifically located in mitochondria membranes). In order to make this method faster and easier, a 1D HPTLC method is chosen, testing several eluents as well as several staining methods. A pre‐conditioning of HPTLC plates with boric acid and a copper staining reagent followed by carbonization are selected for the quality of PL separation and homogeneity of staining. The selected conditions are discussed and the method validation is performed according to the International Conference on Harmonization guidelines. Linearity is effective between 1 and 8 µg and limit of quantification is between 0.5 and 2.3 µg depending on PL classes. Precision measurements show coefficients of variation &lt;6%, and when amounts are close to the detection limit, &lt;12%. Lipid extracts of tumor cell lines or isolated mitochondria are used to assess PL profiles. This shows that the HPTLC method can be used routinely to follow level variations of PLs.Practical Applications: The changes in PL composition play a crucial role in tumor processes and regulate cellular functions modulating cellular signaling or mitochondrial metabolism. The simple and cost‐effective 1D HPTLC method that is developed is applied to lipid extracts of whole tumor cells or hepatocyte‐isolated mitochondria. It is sensitive as well as precise to detect variations of phosphatidic acid or cardiolipin levels linked to physio‐pathological conditions. It can also be used to investigate the composition changes of other membrane PLs. Moreover, with a simultaneous analysis of 14 samples/standards on the same plate (six plates per day), this method is adapted for large series of samples.

Programming cascaded recycling amplifications for highly sensitive and label-free electrochemical sensing of transcription factors in tumor cells

The monitoring of transcription factors (TFs) is critical for understanding the regulation of gene transcriptions. Here, by programming nucleic acid sequence-based and cascaded recycling amplifications, we developed a sensitive and non-label electrochemical biosensor for detecting TFs from tumor cell extracts. The binding of the target nuclear factor-kappa B p50 (NF-κB p50) with the dsDNA probes protects them from being digested by exonuclease III for subsequent initiation of three cascaded recycling cycles, which causes the generation of tremendous free G-quadruplex special sequences on the sensing electrode. Such G-quadruplexes can specifically bind and confine hemin within the vicinity of the sensor, generating substantially enhanced reduction current to achieve determination of NF-κB p50 within the range from 0.5 pM to 5 nM with the detection limit down to 0.13 pM. The proposed sensing system also has high selectivity and it can be used to interrogate the presence of NF-κB p50 in tumor cell extracts, demonstrating its potential for disease diagnosis and gene transcription-related studies.

GM-CSF Culture Revisited: Preparation of Bulk Populations of Highly Pure Dendritic Cells from Mouse Bone Marrow.

Dendritic cells (DC) loaded with specific peptides are strongly immunogenic for T cells and can be used for cancer immunotherapy. For immunogenic tumors such as melanoma, injection of autologous DC loaded with tumor cell extracts or peptides can induce tumor regression but in only a small proportion of patients. Nevertheless, recent studies on the efficacy of checkpoint blockade for boosting antitumor immunity plus advances in defining tumor neoantigens are stimulating renewed interest in DC immunotherapy. Despite intensive investigation, however, preparation of bulk populations of mature DC has proved difficult, and most preparations contain a significant proportion of potentially tolerogenic immature DC. In this study, we have modified the well-established GM-CSF culture system to prepare substantial quantities of highly pure (>95%) mature DC from mouse bone marrow cells and defined their progenitors. We show that obtaining high yields and purity of DC are heavily dependent on cell density in the cultures and the tempo of addition of growth and maturation stimuli. When loaded with specific peptide, the DC are strongly immunogenic for CD4 and CD8 T cells in vivo and elicit effective antitumor immunity.

Ratiometric Fluorescent Biosensor for Visual Discrimination of Cancer Cells with Different Telomerase Expression Levels.

Telomerase is inactive in normal somatic cells but highly activated in tumor cells to maintain their indefinite proliferation and immortal phenotype. As a specific marker for the generation and progress of almost all tumors, the detection of telomerase activity by classical PCR techniques has served in the biological research of tumors. However, the detection of in situ telomerase activity in cell extracts to evaluate the malignancy, progress, and metastasis of tumors remains a daunting challenge. Here, a precisely designed FRET-based ratiometric fluorescent oligonucleotide probe has achieved high-fidelity detection of telomerase activity for accurate discrimination of different cancer cells toward advanced diagnosis of tumors. Our method is superior to other methods in its capabilities to quantify telomerase activity in cell extracts and visualize various tumor cell extracts with different telomerase expression levels by the naked eye for clinical diagnosis. In particular, the ratiometric fluorescent probe used in the assay could exclude other experimental factors influence, and further avoid false positive signal generation. The method reported here could provide a reliable, accurate, and convenient way in medical diagnostics and therapeutic response assessment.

PARP inhibitor rucaparib induces changes in NAD levels in cells and liver tissues as assessed by MRS.

Poly(adenosine diphosphate ribose) polymerases (PARPs) are multifunctional proteins which play a role in many cellular processes. Namely, PARP1 and PARP2 have been shown to be involved in DNA repair, and therefore are valid targets in cancer treatment with PARP inhibitors, such as rucaparib, currently in clinical trials. Proton magnetic resonance spectroscopy (1 H-MRS) was used to study the impact of rucaparib in vitro and ex vivo in liver tissue from mice, via quantitative analysis of nicotinamide adenosine diphosphate (NAD+ ) spectra, to assess the potential of MRS as a biomarker of the PARP inhibitor response. SW620 (colorectal) and A2780 (ovarian) cancer cell lines, and PARP1 wild-type (WT) and PARP1 knock-out (KO) mice, were treated with rucaparib, temozolomide (methylating agent) or a combination of both drugs. 1 H-MRS spectra were obtained from perchloric acid extracts of tumour cells and mouse liver. Both cell lines showed an increase in NAD+ levels following PARP inhibitor treatment in comparison with temozolomide treatment. Liver extracts from PARP1 WT mice showed a significant increase in NAD+ levels after rucaparib treatment compared with untreated mouse liver, and a significant decrease in NAD+ levels in the temozolomide-treated group. The combination of rucaparib and temozolomide did not prevent the NAD+ depletion caused by temozolomide treatment. The 1 H-MRS results show that NAD+ levels can be used as a biomarker of PARP inhibitor and methylating agent treatments, and suggest that in vivo measurement of NAD+ would be valuable.

Quantum dots-labeled strip biosensor for rapid and sensitive detection of microRNA based on target-recycled nonenzymatic amplification strategy

MicroRNAs (miRNAs) have been proved to be potential biomarkers in early cancer diagnosis. It is of great significance for rapid and sensitive detection of miRNAs, particularly with point-of-care (POC) diagnosis. Herein, it is the first time to construct quantum dots (QDs)-labeled strip biosensor based on target-recycled nonenzymatic amplification strategy for miRNA detection. In the system, QDs were served as bright, photostable signal labels, which endow this biosensor with good detection efficiency. Moreover, a target-recycled amplification strategy relies on sequence-specific hairpins strand displacement process without the assistance of enzymes, was introduced to further improve the sensitivity. Meanwhile eliminating the requirement of environment-susceptible enzyme protein makes it easy to preserve and enhances the stability and reproducibility of this sensor. Benefiting from these outstanding characteristics, this platform exhibited a good detection sensitivity range from 2fmol to 200fmol with a limit of 200amol, using only 20μL of sample within 80min. The assay was also 10-fold more sensitive than that with a conventional colloidal gold-based test strip for miRNA detection. Additionally, the analysis of miRNA in various tumor cell extracts was in accordance with the performance of quantitative realtime polymerase chain reaction (qRT-PCR). Clinical tumor samples were also tested, and 16 of 20 samples gave out positive signals, which demonstrated the practical application capacity of the biosensor. Therefore, the proposed biosensor holds great promise for potential POC applications and early cancer diagnosis.

196. Nordihydroguaiaretic Acid (NDGA) Improves Function of Antigen-Specific T-Cells Stimulated by Capsid-Optimized AAV6 Vectors

Cancer, a leading cause of death in the human population today, is generally treated with rigorous surgery followed by intensive chemotherapeutic methods, both of which have adverse toxic side effects. Immunotherapy represents an attractive alternative since it is based on activation of the host immune system. We recently showed that capsid-optimized AAV6-S663V+T492V vectors expressing a tumor-associated antigen are able to initiate specific peripheral T-cell response and suppress tumor progression as well as extend survival in a prostate cancer animal model (J Immunother., 38: 292-298, 2015). However, vaccination based on AAV6 vectors alone was not able to completely overcome the adverse effect of tumor microenvironment and tumor-induced immune dysfunction. Tumor-associated macrophages (TAMs) are prominent components of solid tumors. TAMs exhibit distinct phenotypes and function, and negatively contribute to immune system evasion. Previously published data indicate that inhibition of lipoxygenase (15-LOX2), a key enzyme contributing to TAM function, is critical to overcome tumor resistance and facilitate immunotherapy treatment. In our studies presented here, we first showed that 15-LOX2 is expressed in mouse prostate cancer cells, RM1, growing subcutaneously in C57BL/6 mice, but not in cells growing in culture. We also showed that LOX inhibitors, such as a plant-derived compound, nordihydroguaiaretic acid (NDGA), could restore the function of specific cytotoxic T-cells co-cultured with TAMs in a concentration-dependent manner. Splenocytes from C57BL/6 mice injected intra-muscularly with capsid-optimized AAV6 vectors expressing prostatic aid phosphatase (PAP) were isolated 14 days after vector injection. T-cells were incubated in the presence or absence of RM1 tumor cell extracts and NDGA for 2 hours and then used in killing assays against cultured RM1 cells. Two-color fluorescence assays of cell-mediated cytotoxicity was used to estimate the percentage of dead/alive target cells and a killing curve was generated with different effectors to target cell ratios. Our data indicated that the killing ability of T-cells initiated by capsid-optimized AAV6 vector injection and incubated with RM1 tumor-derived TAMs was approximately 3-fold lower than original activity T-cell. NDGA at a concentration of 10 uM had limited effect on the T-cell activity, but 25 µM NDGA restored the function of the T-cells to approximately 90% of the original activity. In our currently ongoing studies, we are using a combination of capsid-optimized AAV6 vector-mediated immune activation with the NDGA-treatment to corroborate whether a synergetic therapeutic effect can be achieved in an animal model. If successful, this approach could prove useful in the potential clinical application.

Endothelial Cell Protein C Receptor Promotes Apoptosis in Malignant Pleural Mesothelioma Cells

Our recent studies showed that endothelial cell protein C receptor (EPCR) functions as a crucial negative regulator of cancer progression in malignant pleural mesothelioma (MPM) (Keshava et al., Cancer Research 73: 3963-73, 2013). In these studies, the introduction of EPCR expression to aggressive MPM cells lacking EPCR completely attenuated their tumorigenicity whereas the knock-down of EPCR expression in non-aggressive MPM cells that constitutively express EPCR increased their tumorigenicity. This study also revealed that EPCR in MPM cells promotes tumor cell apoptosis in vivo. These data are quite intriguing as EPCR-mediated cell signaling typically activates cell survival and anti-apoptotic pathways and is shown to promote tumor growth in breast and lung cancers. The aim of the present study is to understand the mechanism(s) by which EPCR promotes tumor cell apoptosis in MPM. Evaluation of thoracic fluids obtained from nude mice implanted with MPM cells orthotopically showed a significant increase in the levels of mTNFα and mIFNγ in mice implanted with REN (+EPCR) cells compared to mice implanted with parental REN MPM or REN cells transfected with the vector control [REN(z)]. Analysis of tumor cell apoptosis of cultured cells by TUNEL assay showed that REN (+EPCR) cells were highly susceptible to TNFα+IFNγ-induced cell death compared to parental REN or REN(z) cells (80% vs. 20% cell death at 72 h). In agreement with the concept that EPCR in MPM cells promotes the apoptosis, M9K MPM cells that constitutively express EPCR (non-aggressive MPM cell line) were highly susceptible to TNFα+IFNγ-induced cell death; the knock-down of EPCR in these cells rendered them resistant to cytokine-induced cell death. EPCR-induced tumor cell apoptosis in MPM cells was not dependent on activated protein C (APC). Deletion of the cytoplasmic tail of EPCR failed to diminish its cytotoxic effect. Analysis of REN and REN(+EPCR) tumor cell extracts using PathScan intracellular signaling antibody array kit (Cell Signaling Technology, MA) and immunoblot analysis with specific antibodies against proteins involved in apoptotic pathways revealed that EPCR expression in MPM cells, in comparison to MPM cells lacking EPCR, resulted in a sustained cleavage of poly ADP ribose polymerase (PARP) and an enhanced activation of caspase 3 in response to TNFα+ IFNγ treatment. In addition, REN(+EPCR) cells exhibited an increased activation of caspase 8 and a decreased phosphorylation of anti-apoptotic protein BAD. Further signaling studies showed a delayed activation of NF-kB, suppression of activation of STAT3 and AKT in REN(+EPCR) cells compared to REN cells in response to TNFα+ IFNγ treatment. In addition, EPCR expression suppressed the constitutive activation of the proline-rich AKT substrate, 40 kDa (PRAS40). Overall, our studies indicate that EPCR in MPM cells controls the progression of MPM by exerting its effect at multiple levels. First, EPCR in MPM cells elicits elaboration of cytokines TNFα and IFNγ in vivo and then renders the MPM cells susceptible to TNFα+ IFNγ-induced cell death. The EPCR-mediated cytotoxic effect in MPM cells is independent of APC and the cytoplasmic tail and involves the suppression of STAT3 and AKT activation. At present, it is unclear exactly how EPCR initiates the activation of the apoptotic pathway in MPM cells and whether it involves the interaction of EPCR with the endogenous ligand(s) specific to MPM cells. Studies are underway to identify EPCR interacting proteins in MPM cells. DisclosuresNo relevant conflicts of interest to declare.

Complicated behavior of G-quadruplexes and evaluating G-quadruplexes' ligands in various systems mimicking cellular circumstance

Environments surrounding G-rich sequences remarkably affect the conformations of these structures. A proper evaluation system mimicking the crowded environment in a cell with macromolecules should be developed to perform structural and functional studies on G-quadruplexes. In this study, the topology and stability of a G-quadruplex formed by human telomeric repeat sequences were investigated in a macromolecule-crowded environment created by polyethylene glycol 200 (PEG200), tumor cell extract, and Xenopus laevis egg extract. The interactions between small molecules and telomeric G-quadruplexes were also evaluated in the different systems. The results suggested that the actual behavior of G-quadruplex structures in cells extract is quite different from that in the PEG crowding system, and proteins or other factors in extracts might play a very important role in G-quadruplex structures.

Tumor-specific Th2 responses inhibit growth of CT26 colon-cancer cells in mice via converting intratumor regulatory T cells to Th9 cells.

The abnormality of immune regulation plays a critical role in the pathogenesis of cancer; the underlying mechanism has not been fully understood yet. This study aims to investigate the role of cancer specific T helper (Th)2 response in the inhibition of colon cancer (Cca) cell growth. The results showed that with Cca cell (CT26 cell) extracts as an antigen, the Cca-extract specific Th2 response was induced in the Cca-bearing mice. The Cca mass size was significantly reduced, or radically disappeared (5 out of 10; or 50%); the survival rate was markedly improved in mice immunized with Cca-extract, but not in those immunized with another tumor cell (U87 cell) extracts or to bovine serum albumin. The immunization with Cca-extract also induced Cca cell apoptosis and converted the intra-Cca Tregs to T helper (Th) 9 cells. In conclusion, Cca-specific Th2 responses inhibit Cca growth in a mouse model via inducing Cca cell apoptosis and converting intra-Cca Tregs to Th9 cells.

Using gamma-H2AX and H2AX quantitative ELISA for monitoring DNA damage induced by chemotherapeutic agents and irradiation exposure.

2559 Background: Gamma-H2AX (γH2AX) is a biomarker for DNA double-strand breaks and programmed cell death, but variable relative amounts of H2AX in different samples causes ambiguity in the meaning of the γH2AX level unless it is related to total H2AX levels. We developed a 96-well plate-based ELISA for quantifying γH2AX and H2AX levels in crude extracts of tumor cells, CTCs and biopsy tissues and are validating it for applications in irradiation exposure monitoring and in pharmacodynamic evaluation of anti-cancer agents. Methods: The ELISA was used to analyze extracts of several NCI60 tumor cell lines that had been exposed to a variety of agents, including ionizing radiation, inhibitors of Top-1 (CPT, SN-38, Topotecan), PARP (ABT-888, AZD-2281, MK-4827) and ATR (VE-821, VE-822, AZD-6738, Compound 45, NU-6027), and their combinations. Combination regimens of CPT-11 with PARP inhibitors (ABT-888, AZD-2281, MK-4827) were further evaluated in vivo in the A375 xenograft mouse model. Patient samples obtained for research purposes were also examined by ELISA for feasibility and utility. Results: In vitro, dose-dependent increases in the ratio of γH2AX to H2AX were detected after escalating ionizing radiation exposure and concentration-dependent increases after Top-1 exposure. Treating with inhibitors of PARP or ATR alone did not significantly induce γH2AX. Combinations of Top-1 inhibitors with PARP or ATR inhibitors led to synergistic induction of DNA damage. Among five ATR inhibitors evaluated in combination with Top-1 inhibitors, VE-822 and AZD-6738 were observed to have the highest synergy for γH2AX induction, while NU-6027 showed none. Combinations of CPT-11 with ABT-888, AZD-2281 or MK-4827 showed synergistic induction of γH2AX in A375 xenografts in vivo. Additional testing of human specimens including PBMCs, bone marrow and tumor biopsies proved the assay’s clinical suitability and potential advantages. Conclusions: A newly developed quantitative ELISA for measuring both γH2AX and H2AX is ready for clinical validation for monitoring DNA damage induced by chemotherapeutic agents or irradiation exposure. Funded by NCI Contract No. HHSN261200800001E.

Lectin Galactoside-binding Soluble 3 Binding Protein (LGALS3BP) Is a Tumor-associated Immunomodulatory Ligand for CD33-related Siglecs

Lectin galactoside-binding soluble 3 binding protein (LGALS3BP, also called Mac-2 binding protein) is a heavily glycosylated secreted molecule that has been shown previously to be up-regulated in many cancers and has been implicated in tumor metastatic processes, as well as in other cell adhesion and immune functions. The CD33-related subset of sialic acid-binding immunoglobulin-like lectins (Siglecs) consists of immunomodulatory molecules that have recently been associated with the modulation of immune responses to cancer. Because up-regulation of Siglec ligands in cancer tissue has been observed, the characterization of these cancer-associated ligands that bind to inhibitory CD33-related Siglecs could provide novel targets for cancer immunomodulatory therapy. Here we used affinity chromatography of tumor cell extracts to identify LGALS3BP as a novel sialic acid-dependent ligand for human Siglec-9 and for other immunomodulatory Siglecs, such as Siglec-5 and Siglec-10. In contrast, the mouse homolog Siglec-E binds to murine LGALS3BP with lower affinity. LGALS3BP has been observed to be up-regulated in human colorectal and prostate cancer specimens, particularly in the extracellular matrix. Finally, LGALS3BP was able to inhibit neutrophil activation in a sialic acid- and Siglec-dependent manner. These findings suggest a novel immunoinhibitory function for LGALS3BP that might be important for immune evasion of tumor cells during cancer progression.

Abstract 4685: Discovery and characterization of nitroaspirin (NCX-4016) as a powerful and clinically relevant inhibitor of human MGMT for increasing the efficacy of alkylating agents

Abstract The DNA repair protein MGMT (O6-methylguanine-DNA methyltransferase) is a major determinant of resistance to many alkylating agents. Currently, clinical trials with O6-benzylguanine (BG) to inhibit MGMT and induce its degradation are ongoing; however, this is beset with severe alkylation damage to bone marrow and the need for hematopoietic rescue using BG-resistant MGMT gene therapy. Therefore, an urgent need exists for non-toxic regimens that deplete MGMT and increase tumor antitumor efficacy. Here, we have exploited the highly reactive nature of Cysteine 145 which accepts the alkyl groups for novel drug design. Cys145 has a pKa of 4.8 and is susceptible for glutathionylation and nitrosylation, both of which inactivate MGMT. This study characterized the effect of nitro-aspirin or NCX-4016, which is a potent nitrosylator of reactive cysteines and tyrosines. NCX-4016 is a well characterized chemopreventive agent without the GI adverse effects of aspirin and is degraded by plasma and tissue esterases to release NO in a sustained manner. In three MGMT-proficient human cancer cell lines (HT29, T47D, and HCT116), nitroaspirin (NA) at pharmacologically achievable concentrations (5-10 µM) caused 90% inhibition of MGMT activity within 1 h of exposure. Interestingly, the MGMT protein disappeared very rapidly with similar kinetics after NA treatment; approx. 80-90% of MGMT was degraded after 10 µM NA treatment for 2 h. These data are highly comparable and/or superior to those reported for BG. Further, purified MGMT or tumor cell extracts exposed to NCX-4016 failed to bind the biotin-labeled BG, indicating Cys145 as the nitrosylation site. Tyr114, a residue critical for MGMT catalysis was another target for nitrosylation. The involvement of the ub-proteasome pathway in the degradation of nitrosylated MGMT both in vitro and in cells was also demonstrated. In cultured cells, MGMT suppression by BG was more prolonged than nitroaspirin, however, the 24-36 h curtailment by NA was adequate for increased production of cytotoxic lesions. Pre-exposure of tumor cells to NA followed by BCNU resulted in (i) a significantly greater Induction and persistence of DNA interstrand crosslinks, and (ii) at least a 2-fold increase in the G2/M cell cycle blockade. Experiments in mice have shown that a single injection (100 mg/kg) of NCX-4016 causes 50-60% inhibition of MGMT activity in mice brain and liver. Because NO-aspirin, is non-toxic (IC50&amp;gt;500 µM for cell lines), can be administered at high levels, yields a chemopreventive by-product, unlikely to elicit tumor resistance, is lipophilic enough to cross the blood-brain barrier, and its pleiotropic actions are actually beneficial for chemotherapy, we believe NA's ability to potently inhibit MGMT holds great promise for efficient brain tumor therapy (supported by RO3 CA125872 and Assoc. for Res of Childhood Cancer). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4685. doi:1538-7445.AM2012-4685

CDR3δ -grafted γ9δ2T cells mediate effective antitumor reactivity

Adoptive cell-transfer therapy (ACT) has been reported to suppress growing tumors and to overcome tumor escape in animal models. As a candidate ACT effector, γ9δ2T cells can be activated and expanded in vitro and in vivo and display strong antitumor activity against colorectal, lung, prostate, ovarian and renal cell carcinomas. However, it is difficult to obtain a large enough number of γδT cells to meet the need for immunotherapy that can overcome the cancer patients' immune suppressive tumor microenvironment. In previous studies, our lab confirmed that γ9δ2T cells recognized tumor cells via the CDR3δ region of the γδ-T-cell receptor (TCR). We constructed full-length human peripheral blood mononuclear cell (PBMC)-derived γ9 and δ2 chains in which the CDR3 region was replaced by an ovarian epithelial carcinoma (OEC)-derived CDR3. We transferred the CDR3δ-grafted γ9δ2TCR into peripheral blood lymphocytes (PBLs) to develop genetically modified γ9δ2T cells. In vitro studies have shown that these CDR3δ-grafted γ9δ2T cells can produce cytokines after stimulation with tumor cell extracts and exhibit cytotoxicity towards tumor cells, including human OEC and cervical adenocarcinoma. CDR3δ-grafted γ9δ2T cells adoptively transferred into nude mice bearing a human OEC cell line demonstrated significant antitumor effects. These results indicate that CDR3δ-grafted γ9δ2T cells might be candidates for clinical tumor immunotherapy.

Abstract 5505: Potent redox-based inhibition of human MGMT for improving the efficacy of anticancer alkylating agents

Abstract There is an urgent need for new strategies for inhibiting the DNA repair protein MGMT (O6-methylguanine-DNA methyltransferase) and improving the efficacy of alkylating agents. MGMT is highly expressed in human cancers and prevents the formation of cytotoxic lesions in alkylated DNA. Current clinical trials involving MGMT depletion by O6-benzylguanine (BG), although promising, are beset with severe toxicity to the bone marrow, which has necessitated the transduction of BG-resistant MGMT gene into hematopoietic stem cells. Here, we exploited the highly reactive nature of Cysteine 145 of MGMT which accepts the alkyl groups for pharmacological intervention. Cys145 has a pKa of 4.8 due to its microenvironment and is susceptible for glutathionylation (Niture &amp; Srivenugopal, Proc of AACR 47, abst. 789, 2006) and nitrosylation (Liu et al. Cancer Res. 62, 3037, 2002). S-Thiolation and S-nitrosylation are reversible posttranslational mechanisms that gauge the intracellular redox and transduce them into functional responses. This study investigated the effect of two nontoxic small molecules which readily react with reactive cysteines, namely, the NCX-4016 (nitro-aspirin capable of S-nitrosylation) and disulfiram (capable of thiol-conjugation). NCX-4016, also called a fatty aspirin, is degraded by plasma and tissue esterases to release NO in a sustained manner. In three MGMT-proficient human cancer cell lines (HT29, T47D, and HCT116), nitro-aspirin at very low concentrations (5-10 µM) caused a 90% inhibition of MGMT activity within 1 h of exposure. Interestingly, the MGMT protein also disappeared with similar kinetics after NCX-4016 treatment; approx. 80-90% of MGMT was degraded after 5 µM NO-aspirin treatment for 2 h. These data are highly comparable or better than those reported for BG. Further, purified MGMT or tumor cell extracts exposed to NCX-4016 failed to bind the biotin-labeled BG, indicating Cys145 to be the site of nitrosylation. &amp;gt;60% of MGMT protein was regenerated at 24 h when NO-aspirin treated HT29 cells were post-incubated in drug-free medium, indicating a transient inhibition and rapid repletion. Disulfiram (DS), the alcohol deterrent drug, also curtailed MGMT activity in HT29 and HCT116 cells with a 20 h 400 µM treatment causing a 95% inhibition. DS at 200 µM induced about 70% degradation of MGMT at 12 h. Other redox-sensitive proteins such as the wild-type and mutant p53, NF-κB, and ubiquitin E1 were all degraded by DS in a dose- dependent manner. Studies to evaluate MGMT inhibition by NO-aspirin and DS in animal tissues are in progress. Because NO-aspirin, is non-toxic (IC50&amp;gt;500 µM for cell lines), yields a chemopreventive by-product, unlikely to elicit tumor resistance, and is lipophilic enough to cross the BBB, we believe it can be exploited for glioma therapy. Our studies show that the redox-regulated proteins are ‘druggable’ and highlight options for redox- driven therapeutic strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5505. doi:10.1158/1538-7445.AM2011-5505