Clonogenic Cell Survival Assay Research Articles

Abstract PO3-24-06: Integrative genomic and CRISPR screen analysis identifies synthetic viability interactions in BRCA1/2 cancers

Abstract Background Pathogenic BRCA1 and BRCA2 (BRCA1/2) germline mutations contribute to hereditary breast cancer and are linked with increased risk of other cancers. Paradoxically, bi-allelic inactivation of BRCA1/2 (bBRCA1/2) is embryonically lethal. Although TP53 loss can at least in part mitigated the deleterious impact of Brca1 deficiency in mice, this is insufficient in human cells to compensate for decreased proliferative capacity. What other compensatory mechanisms facilitate oncogenesis remains unclear. Methods We performed an integrative analysis of cancer genomes from bBRCA1/2 tumors and CRISPR/Cas9 screens from cellular models to identify novel gene candidates that facilitate synthetic viability. First, we analyzed cancer genomes from ovarian and breast cancer derived from TCGA and identified genomic loci, which were enriched for recurrent copy number alterations (CNAs) in bBRCA1/2 tumors. We subsequently identified genes from these CNA loci that had corresponding changes in gene expression. Then, we used high-throughput genome-wide CRISPR/Cas9 knockout screens in BRCA1/2−/− cell lines (BRCA1−/- in RPE1 cells, BRCA2−/− in DLD1 cells) to identify which of these genes increased cell proliferation in vitro. Lastly, we experimentally validated these synthetic viable interactions using an orthogonal MCF12a system to evaluate changes in colony formation with a clonogenic cell survival assay. Results In human cancer genomes, we identified that bBRCA1/2 tumors harbored recurrent large-scale genetic deletions significantly more frequent than histologically matched control tumors. We identified 148 cytobands (23 distinct genomic loci) frequently deleted in bBRCA1/2 ovarian cancers (N=92), and 90 cytobands (15 distinct genomic loci) in bBRCA1/2 ER+ breast cancers (N=30). Subsequent computational analysis of gene expression data identified transcriptionally decreased genes in the frequently deleted loci. Cross-referencing with CRISPR/Cas9 screens in BRCA1/2−/− cells, we ultimately identified 277 and 218 genes that were altered in human tumors and putatively enhanced viability in BRCA1/2-null models but not in isogenic wild-type cell lines in BRCA1 and BRCA2 models, respectively. We subsequently selected 17 genes that either increased viability in BRCA1, BRCA2, or both contexts for experimental validation. Of these, about 40% (7 of 17) were experimentally validated (RIC8A, GNA12, ATMIN, IPO7, ATXN2, KDM1A, and NUP98) and had evidence of synthetic viability in this cellular context. Interestingly, RIC8A and GNA12 interact with each other, are both involved in G protein-coupled signaling pathway, and are synthetically viable with both BRCA1−/− and BRCA2−/− cells. In a re-analysis of a recently published genomic dataset on metastatic breast cancer, we found that homologous recombination deficient tumors developed additional loss of function mutations in RIC8A in the metastatic setting. Conclusions This study provides insights into the oncogenesis of BRCA1/2 malignancies and describes a high-throughput framework to identify synthetic viability interactions and causal driver genes affected by large-scale CNAs in human cancers. Citation Format: Yingjie Zhu, Xin Pei, Ardijana Novaj, Jeremy Setton, Fatemeh Derakhshan, Pier Selenica, Niamh McDermott, Sonali Sinha, Atif Khan, Simon Powell, Jorge Reis-Filho, Nadeem Riaz. Integrative genomic and CRISPR screen analysis identifies synthetic viability interactions in BRCA1/2 cancers [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 PO3-24-06.

The Role of SARS-CoV-2 Spike Protein in the Growth of Cervical Cancer Cells.

Recently developed vaccines for the SARS-CoV-2 virus utilize endogenous production of the virus' spike protein (SP), allowing the host to develop an immune response. As a result of the novelty of this virus and its vaccines, little is known overall about the potential effects of the SP on the pathogenesis of neoplasia, either from vaccination or from infection. This study was designed to investigate whether SARS-CoV-2 SP has any direct effect on SiHa cervical cancer cells. The effects of SARS-CoV-2 SP on cervical cancer cell proliferation and apoptosis were investigated by using clonogenic cell survival assay, quick cell proliferation assay, and caspase-3 activity kits in a widely-used cervical cancer cell line, SiHa. RT-PCR and immunohistochemistry were also performed to determine the potential molecular mechanisms. The growth and proliferation of SiHa cancer cells were inhibited by SARS-CoV-2 SP. SARS-CoV-2 SP also induced apoptosis in SiHa cancer cells. The anti-proliferative effect of SARS-CoV-2 SP on SiHa cancer cells was associated with the up-regulation of the anti-proliferative molecule p53. The pro-apoptotic effect of SARS-CoV-2 SP on SiHa cells was associated with the up-regulation of the pro-apoptotic molecule TRAIL. SARS-CoV-2 SP inhibits the growth of cervical cancer via up-regulation of p53 and TRAIL. Further studies are needed to elaborate on the potential effects of the SARS-CoV-2 SP on other cancer cell lines and normal physiological cell lines for comparison.

CDKL1 potentiates the antitumor efficacy of radioimmunotherapy by binding to transcription factor YBX1 and blocking PD-L1 expression in lung cancer

BackgroundThe evasion of the immune response by tumor cells through programmed death-ligand 1 (PD-L1) has been identified as a factor contributing to resistance to radioimmunotherapy in lung cancer patients. However, the precise molecular mechanisms underlying the regulation of PD-L1 remain incompletely understood. This study aimed to investigate the role of cyclin-dependent kinase-like 1 (CDKL1) in the modulation of PD-L1 expression and the response to radioimmunotherapy in lung cancer.MethodsThe tumorigenic roles of CDKL1 were assessed via cell growth, colony formation, and EdU assays and an in vivo nude mouse xenograft model. The in vitro radiosensitization effect of CDKL1 was evaluated using a neutral comet assay, γH2AX foci formation analysis, and a clonogenic cell survival assay. The protein‒protein interactions were confirmed via coimmunoprecipitation and GST pulldown assays. The regulation of PD-L1 by CDKL1 was evaluated via chromatin immunoprecipitation (ChIP), real-time quantitative PCR, and flow cytometry analysis. An in vitro conditioned culture model and an in vivo C57BL/6J mouse xenograft model were developed to detect the activation markers of CD8+ T cells and evaluate the efficacy of CDKL1 overexpression combined with radiotherapy (RT) and an anti-PD-L1 antibody in treating lung cancer.ResultsCDKL1 was downregulated and suppressed the growth and proliferation of lung cancer cells and increased radiosensitivity in vitro and in vivo. Mechanistically, CDKL1 interacted with the transcription factor YBX1 and decreased the binding affinity of YBX1 for the PD-L1 gene promoter, which consequently inhibits the expression of PD-L1, ultimately leading to the activation of CD8+ T cells and the inhibition of immune evasion in lung cancer. Moreover, the combination of CDKL1 overexpression, RT, and anti-PD-L1 antibody therapy exhibited the most potent antitumor efficacy against lung cancer.ConclusionsOur findings demonstrate that CDKL1 plays a crucial role in regulating PD-L1 expression, thereby enhancing the antitumor effects of radioimmunotherapy. These results suggest that CDKL1 may be a promising therapeutic target for the treatment of lung cancer.

Abstract 2873: Radiation therapy promotes polyunsaturated fatty acid mediated ferroptosis in cervical cancer: Role of AMPK/xCT/GPX4 axis

Abstract Purpose: Previously, we found that mono-and di-unsaturated fatty acids can sensitize cervical cancer to radiotherapy (RT) through a p53 dependent apoptotic mechanism that is distinct from ferroptosis (Muhammad et al., Cancer Res. 2022; 82: 4515-27). Polyunsaturated fatty acids (PUFAs) enhance therapeutic response in preclinical tumor models by increasing lipid peroxidation (L-ROS) and induction of ferroptosis. Ferroptosis is a form of cell death that requires abundant cytosolic labile iron to promote membrane lipid peroxidation. The objective of the present study is to test whether the combination of RT + PUFAs promotes cell death via increased membrane lipid peroxidation and ferroptosis in cervical cancer, and to determine the impact of RT + PUFAs on cervix tumor cell metabolism and mitochondrial function. Experimental Procedures: Cell viability and clonogenic cell survival assays were performed using 8 human cervical cancer cell lines including normal cervix epithelial controls incubated with PUFAs (EPA, DHA and AA), before and after RT. Western blotting, siRNA-mediated knockdown, pharmacologic inhibition and activation were performed to determine whether ferroptosis dependent signals were mediating PUFA-induced RT sensitivity. Oxidative stress parameters were quantified using mass spectrometry and fluorometric dyes. Flow cytometry was used to quantify total cell ROS, lipid ROS, mitochondrial ROS and labile iron pool. Mitochondrial morphology and function were characterized using microscopy and the Seahorse assay. Summary: PUFAs + RT increased total ROS and LROS accumulation followed by a ferroptotic tumor cell death, which was iron-dependent and could be prevented by the addition of iron chelator and lipophilic antioxidants, Ferrostatin-1 and Liproxstatin-1. GPX4, a thiol dependent enzyme that eliminates lipid peroxides, and the cystine-glutamate antiporter (xCT) a key source of cystine for reduced glutathione (GSH) synthesis, were downregulated in response to PUFAs + RT. PUFAs + RT induced increased mitochondrial ROS and subsequent metabolic dysfunction. Mechanistically, the expression of AMPK, a key regulator of metabolic/energy homeostasis and mitochondrial biogenesis, was upregulated by PUFAs + RT. Pharmacological activation and inhibition of AMPK further enhanced and abrogated the effect of PUFA + RT, respectively. Conclusions: PUFAs modify the RT sensitivity of cervical cancer cells by inducing cell death through ferroptosis and mitochondrial dysfunction via AMPK activation. In vivo experiments are ongoing in immunocompromised and immunodeficient mouse tumor models to confirm this finding and determine the impact of PUFAs on RT induced tumor cell death and anti-tumor immunity. Citation Format: Naoshad Muhammad, Nishanth Gabriel, Louise M. Bengtsson, Tyler McKinnish, Naomi Msengi, Kevin Cho, Gary Patti, Julie K. Schwarz. Radiation therapy promotes polyunsaturated fatty acid mediated ferroptosis in cervical cancer: Role of AMPK/xCT/GPX4 axis [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 2873.

Abstract 2871: Co-targeting asparagine and glutamine metabolism is a viable treatment strategy for PI3K/PTEN mutated cervical cancer

Abstract Purpose Statement: Alterations in the PI3K/AKT signaling pathway are common in cervical cancer. The most commonly observed mutations are in PIK3CA (26%) and PTEN (8%). However, the biological and metabolic implications of acquiring these mutations in the cervical cancer genome are poorly characterized. Materials/Methods: Isogenic cell lines with PIK3CA E545K and PTEN null mutations were generated using Crispr/CAS9. Cell viability and clonogenic cell survival assays were performed in the presence and absence of inhibitors of glutamine and asparagine metabolism. Glucose and glutamine metabolism was evaluated by stable isotope labeling with U13C glucose and13C5 glutamine in presence of glutamine antagonist DON. RNA-seq was employed to evaluate transcriptomic alterations. Results: Introduction of the PIK3CA E545K mutation and PTEN mutation in cervical tumor cells leads to an increase in AKT signaling compared to the parental (wild-type) cells. Notably, RNASeq and mass spectrometric analysis of both the tumor cells and their culture media revealed heightened dependencies on glucose and glutamine in the tumor cells carrying the PIK3CA E545K and PTEN mutations as compared to the wild-type cells. Treatment with glutamine antagonists (DON, JHU-083) and GLS inhibitors (CB-839 and IACS-6274), resulted in reduced cell viability and clonogenic survival in tumor cells with PI3K/PTEN mutation compared to wild-type. Additionally, we observed a metabolic reliance on the amino acid asparagine in tumors with PI3K/PTEN alterations when treated with DON. Asparaginase in combination with DON effectively inhibited the proliferation of human and mouse cell lines derived from primary tumors with PTEN mutations. Furthermore, the incorporation of a glutamine antagonist and asparaginase into conventional chemoirradiation further enhanced the therapeutic efficacy in cervical tumors with PI3K/PTEN alterations which are otherwise treatment resistant. Conclusions: Orthogonal evaluations using glutamine agonists, GLS inhibitors, and mass spectrometric point to a significant role played by PI3K/PTEN mutations in altering cervix tumor cell metabolism towards a more glutamine dependent phenotype. This study suggests a potential strategy for improving the treatment of PI3K-AKT-altered cervical tumors by targeting glutamine and asparagine metabolism in combination with standard therapies. These treatment strategies are currently being evaluated in vivo using a patient derived xenograft model with PIK3CA E545K mutation as well as an immunocompetent cervical tumor model in mice with PTEN deletion. Citation Format: Nishanth Noel Gabriel, Naoshad Muhammad, Kevin Cho, Kay Jayachandran, Jin Zhang, Gary Patti, Julie Schwarz. Co-targeting asparagine and glutamine metabolism is a viable treatment strategy for PI3K/PTEN mutated cervical 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 2871.

Abstract 4538: Brigatinib induces synergy with PARP inhibitors through dual inhibition of FAK and EphA2 in high-grade serous ovarian carcinoma

Abstract PARP inhibitors (PARPis) have now become a standard of care for high-grade serous ovarian cancer (HGSOC), especially for those with defects in homologous recombination (HR) DNA repair. However, the innate and acquired PARPi resistance in HGSOC poses a significant concern, necessitating the exploration of novel treatment options and strategies. Here we investigated the potential of combining brigatinib—a second-generation anaplastic lymphoma kinase (ALK) inhibitor approved for treating lung cancer—with PARPis as way to enhance PARPi activity in HGSOC. Clonogenic cell survival assays revealed that brigatinib synergizes with PARPis in HR-proficient and -deficient HGSOC cell lines. In subsequent proteomics approaches and cell-based confirmatory studies, we found that brigatnib suppressed tyrosine kinases FAK and EphA2 with reduction in downstream signaling. Follow-up pharmacologic and gene-silencing techniques demonstrated that dual FAK and EphA2 inhibition additively reduces PI3K/Akt and MAPK/ERK cell survival signaling to levels conferring PARPi sensitivity. Additionally, the efficacy of this therapeutic approach on tumor size reduction was assessed in HGSOC patient-derived xenograft (PDX) models, with combination treatment inducing tumor regression more effectively than either agent alone. Overall, our studies unveil a beneficial ALK-independent effect of brigatinib that may offer new avenues for considering its repurposing as a HGSOC therapy. Citation Format: Julie R. Duffield, Xiaonan Hou, Iman K. McKeon-Makki, Amelia M. Huehls, Benjamin W. Wilson, Anjali Prasad, Xinyan Wu, Scott H. Kaufmann, Larry M. Karnitz, John J. Weroha, Arun Kanakkanthara. Brigatinib induces synergy with PARP inhibitors through dual inhibition of FAK and EphA2 in high-grade serous ovarian 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 4538.

Abstract 484: Enhancing therapeutic responses in NSCLC using iron-oxide nanoparticles combined with pharmacological ascorbate

Abstract High dose Vitamin C (pharmacological ascorbate; P-AscH‒ > 20 mM plasma levels) given intravenously (I.V.), has made a remarkable re-emergence as an adjuvant to combined modality approaches in cancer therapy. P-AscH‒ can be readily oxidized in the presence of redox active metals (i.e., P-AscH‒ serves as an iron reducing agent), which can result in increased fluxes of H2O2. This chemical phenomenon allows for the tumor-selective enhancement of chemo-radiation in NSCLC. We hypothesized that enhancing labile Fe2+ in cancer cells using iron-oxide nanoparticles (ferumoxytol, FMX) can increase the effectiveness of P-AscH‒ in enhancing cancer cell sensitivity to chemo-radiation in NSCLC by increasing H2O2 production. To test this hypothesis, we performed clonogenic cell survival assays in-vitro. To test the role of H2O2 in the combined effect of P-AscH‒ and FMX, we developed lentiviral-based doxycycline-inducible overexpression models for catalase, and to determine the changes in Fe, we used fluorescent probes that specifically detect changes in labile Fe2+. Results from these experiments showed FMX enhanced clonogenic cell killing in NSCLC. Furthermore, catalase overexpression inhibited this effect, suggesting that enhanced H2O2 production plays a central role in the additive effect of P-AscH- and FMX. Finally, staining the cells with the fluorescent iron probes showed that FMX enhanced labile Fe2+ in cells treated with P-AscH‒. These studies continue to support the hypothesis that H2O2 plays a significant role in P-AscH‒ toxicity in cancer cells that can be exacerbated using iron oxide nanoparticles. Future studies will test these findings in-vivo using preclinical xenograft models. (supported by P01 CA217797, P30 CA086862, T32 CA078586, and P01 CA244091). Citation Format: Mekhla Singhania, Miachael S. Petronek, Casey F. Pulliam, Kranti A. Mapuskar, Amira Zaher, Douglas R. Spitz. Enhancing therapeutic responses in NSCLC using iron-oxide nanoparticles combined with pharmacological ascorbate [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 484.

The radiation- and chemo-sensitizing capacity of diclofenac can be predicted by a decreased lactate metabolism and stress response

BackgroundAn enhanced aerobic glycolysis (“Warburg effect”) associated with an increase in lactic acid in the tumor microenvironment contributes to tumor aggressiveness and resistance to radiation and chemotherapy. We investigated the radiation- and chemo-sensitizing effects of the nonsteroidal anti-inflammatory drug (NSAID) diclofenac in different cancer cell types.MethodsThe effects of a non-lethal concentration of diclofenac was investigated on c-MYC and Lactate Dehydrogenase (LDH) protein expression/activity and the Heat shock Protein (HSP)/stress response in human colorectal (LS174T, LoVo), lung (A549), breast (MDA-MB-231) and pancreatic (COLO357) carcinoma cells. Radiation- and chemo-sensitization of diclofenac was determined using clonogenic cell survival assays and a murine xenograft tumor model.ResultsA non-lethal concentration of diclofenac decreases c-MYC protein expression and LDH activity, reduces cytosolic Heat Shock Factor 1 (HSF1), Hsp70 and Hsp27 levels and membrane Hsp70 positivity in LS174T and LoVo colorectal cancer cells, but not in A549 lung carcinoma cells, MDA-MB-231 breast cancer cells and COLO357 pancreatic adenocarcinoma cells. The impaired lactate metabolism and stress response in diclofenac-sensitive colorectal cancer cells was associated with a significantly increased sensitivity to radiation and 5Fluorouracil in vitro, and in a human colorectal cancer xenograft mouse model diclofenac causes radiosensitization.ConclusionThese findings suggest that a decrease in the LDH activity and/or stress response upon diclofenac treatment predicts its radiation/chemo-sensitizing capacity.

A fast Monte Carlo cell-by-cell simulation for radiobiological effects in targeted radionuclide therapy using pre-calculated single-particle-track standard DNA damage data

IntroductionWe develop a new method that drastically speeds up radiobiological Monte Carlo radiation-track-structure (MC-RTS) calculations on a cell-by-cell basis.MethodsThe method is based on random sampling and superposition of single-particletrack (SPT) standard DNA damage (SDD) files from a “pre-calculated” data library, constructed using the RTS code TOPAS-nBio, with “time stamps” manually added to incorporate dose-rate effects. This time-stamped SDD file can then be input into MEDRAS, a mechanistic kinetic model that calculates various radiation-induced biological endpoints, including DNA double strand breaks (DSBs), misrepairs and chromosomal aberrations, and cell death. As a benchmark validation of the approach, we calculate the predicted energy-dependent DSB yield and the ratio of direct-to-total DNA damage, both of which agree with published in-vitro experimental data. We subsequently apply the method to perform superfast cell-by-cell simulation of an experimental in-vitro system consisting of neuroendocrine tumor cells uniformly incubated with 177Lu.Results and discussionThe results for residual DSBs, both at 24 and 48 h post-irradiation, agree well with the published literature values. Our work serves as a proof-of-concept demonstration of the feasibility of cost-effective “in silico clonogenic cell survival assay” for the computational design and development of radiopharmaceuticals, and novel radiotherapy treatments more generally.

Expression of ZNF281 in colorectal cancer correlates with response to radiotherapy and survival

Background The treatment of Colorectal cancer (CRC) is extremely complex and survival rates vary depending on the stage of the disease at the time of diagnosis. Neoadjuvant chemoradiotherapy (NACRT), is the conventional treatment for locally advanced rectal cancer (LARC); however, the resistance to chemoradiotherapy in LARC is difficult to predict. Materials and methods In this study, clinical data of 126 LARC patients were collected and analyzed, and relevant validation was performed using GEO database and in vitro and in vivo experiments, including Western blotting and Real-time quantitative PCR, immunohistochemistry, immunofluorescence, clonogenic cell survival assays, and nude-mouse xenograft models. Results In patients with LARC who were treated with neoadjuvant radiotherapy (NART), higher ZNF281 expression in malignant tissue was associated with a poorer prognosis and lesser degree of tumor regression. Cell and mouse experiments have shown that ZNF281 reduces the damage caused by X-rays to CRC cells and tumors grown in mice. Conclusion We found that the expression of ZNF281 predicted the radiation response of CRC cells and suggested the prognosis of patients with LARC who received neoadjuvant radiation therapy.

BDE209-promoted Dio2 degradation in H4 glioma cells through the autophagy pathway, resulting in hypothyroidism and leading to neurotoxicity

Decabromodiphenyl ether (BDE209), the homologue with the highest number of brominates in polybrominated diphenyl ethers (PBDEs), is one of the most widespread environmental persistent organic pollutants (POPs) due to its mass production and extensive application in recent decades. BDE209 is neurotoxic, possibly related to its interference with the thyroid hormone (TH) system. However, the underlying molecular mechanisms of BDE209-induced TH interference and neurobehavioral disorders remains unknown. Here, we explored how BDE209 manipulated the major enzyme, human type II iodothyronine deiodinase (Dio2), that is most important in regulating local cerebral TH equilibrium by neuroglial cells, using an in vitro model of human glioma H4 cells. Clonogenic cell survival assay and LC/MS/MS analysis showed that BDE209 could induce chronic neurotoxicity by inducing TH interference. Co-IP assay, RT-qPCR and confocal assay identified that BDE209 destroyed the stability of Dio2 without affecting its expression, and promoted its binding to p62, thereby enhancing its autophagic degradation, thus causing TH metabolism disorder and neurotoxicity. Furthermore, molecular docking studies predicted that BDE209 could effectively suppress Dio2 activity by competing with tetraiodothyronine (T4). Collectively, our study demonstrates that BDE209-induced Dio2 degradation and loss of its enzymatic activity in neuroglial cells are the fundamental pathogenic basis for BDE209-mediated cerebral TH disequilibrium and neurotoxicity, providing a target of interest for further investigation using glial/neuronal cell co-culture system and in vivo models.

Oxali(IV)Fluors: Fluorescence Responsive Oxaliplatin(IV) Complexes Identify a Hypoxia-Dependent Reduction in Cancer Cells.

Platinum(IV) anticancer agents have demonstrated the potential to overcome the limitations associated with the widely used Pt(II) chemotherapeutics, cisplatin, carboplatin, and oxaliplatin. In order to identify therapeutic scenarios where this type of chemotherapy can be applied, an improved understanding on the intracellular reduction of Pt(IV) complexes is needed. Here, we report the synthesis of two fluorescence responsive oxaliplatin(IV)(OxPt) complexes, OxaliRes and OxaliNap. Sodium ascorbate (NaAsc) was shown to reduce each OxPt(IV) complex resulting in increases in their respective fluorescence emission intensities at 585 and 545 nm. The incubation of each OxPt(IV) complex with a colorectal cancer cell line resulted in minimal changes to the respective fluorescence emission intensities. In contrast, the treatment of these cells with NaAsc showed a dose-dependent increase in fluorescence emission intensity. With this knowledge in hand, we tested the reducing potential of tumor hypoxia, where an oxygen-dependent bioreduction was observed for each OxPt(IV) complex with <0.1% O2 providing the greatest fluorescence signal. Clonogenic cell survival assays correlated with these observations demonstrating significant differences in toxicity between hypoxia (<0.1% O2) and normoxia (21% O2). To the best of our knowledge, this is the first report showing carbamate-functionalized OxPt(IV) complexes as potential hypoxia-activated prodrugs.

Discovery of new imidazotetrazinones with potential to overcome tumor resistance

We describe the design, organic synthesis, and characterization, including X-ray crystallography, of a series of novel analogues of the clinically used antitumor agent temozolomide, together with their in vitro biological evaluation. The work has resulted in the discovery of a new series of anticancer imidazotetrazines that offer the potential to overcome the resistance mounted by tumors against temozolomide. The rationally designed compounds that incorporate a propargyl alkylating moiety and a thiazole ring as isosteric replacement for a carboxamide, are readily synthesized (gram-scale), exhibit defined solid-state structures, and enhanced growth-inhibitory activity against human tumor cell lines, including MGMT-expressing and MMR-deficient lines, molecular features that confer tumor resistance. The cell proliferation data were confirmed by clonogenic cell survival assays, and DNA flow cytometry analysis was undertaken to determine the effects of new analogues on cell cycle progression. Detailed 1H NMR spectroscopic studies showed that the new agents are stable in solution, and confirmed their mechanism of action. The propargyl and thiazole substituents significantly improve potency and physicochemical, drug metabolism and permeability properties, suggesting that the thiazole 13 should be prioritized for further preclinical evaluation.

Abstract 5051: Assessment of boron delivery peptides with angiopep-2 for boron neutron capture therapy

Abstract Boron neutron capture therapy (BNCT) induces intracellular nuclear reactions that release heavy charged particles to destroy cancer cells during thermal neutron radiation. In order to selectively eliminate cancer cells but avoid harmful effects to normal tissues, targeted delivery of boron-10 in cancer cells is required, whereas the conventional boron delivery agent phenylalanine (BPA) is unsatisfactory. Angiopep-2 is a cell-penetrating peptide that can trigger transcytosis and traverse the blood-brain barrier by recognizing low density lipoprotein-related protein 1. In this study, we developed angiopep-2 for boron delivery in BNCT. Angiopep-2 was labeled with boron-10 using solid-phase peptide synthesis with 4-carboxyphenylboronic acid and fluorenylmethyloxycarbonyl (Fmoc)-protected amino acids, namely ANG-B. Molecular mass of the synthesized ANG-B were validated by mass spectrometry. In a cell proliferation assay, little toxicity of ANG-B was detected. Boron concentrations in 6 cell lines from different cancer types and an intracranial glioblastoma mouse model after treatment with ANG-B or BPA were analyzed by ICP-AES. The in vivo distribution of ANG-B showed that the ratio of boron content of tumor tissue to that of blood (T/B ratio) was 9.42±2.47 at 4h post treatment, while the ratio of boron content of tumor tissue to that of normal brain tissue (T/N ratio) was 5.61±1.0, demonstrated an optimal tumor targeting ability of ANG-B. We further measured the effects of ANG-B in BNCT by clonogenic cell survival assay in vitro or by PET/CT imaging on a glioblastoma mouse model in vivo. Radiation for BNCT was given with neutron flux at 3.0±0.4 × 1011 n/cm2. BNCT with 5 mM ANG-B resulted in 86.5%±5.3% clonogenic cell death, while the number was 72.9%±5.1% for BPA. The effect of ANG-B based BNCT in vivo was also better than BPA. The glioblastoma tumors in ANG-B treated group at 31 days after BNCT were shrank by 62.9% in average, while the BPA treated tumors shrank by only 23.0% (P&amp;lt;0.05). Therefore, we concluded that ANG-B is an efficient boron delivery agent, which may improve BNCT performance in clinical trials. Citation Format: Qi Liu, Jing Xiang, Lin Ma, Qiushi Ren. Assessment of boron delivery peptides with angiopep-2 for boron neutron capture therapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5051.

Abstract 6213: Disruption of XPF function by HPV promotes alternative end joining and sensitivity to cisplatin in HNSCC

Abstract Human papillomavirus (HPV) positive head and neck squamous cell carcinoma (HNSCC) usually show a tremendous better clinical outcome compared to their HPV-negative counterparts. Although the HPV associated survival advantage is partially explained by the enhanced cisplatin sensitivity in HNSCC (Clin Ca Res 2018, 24(23): 6001), the underlying mechanisms are still poorly elucidated. Cisplatin causes DNA interstrand crosslinks (ICLs) that requires Fanconi anemia (FA) pathway for DNA repair. Consistently, our study showed that HPV-positive HNSCC cells exhibited a FA defective cellular phenotype. In clonogenic cell survival assay, 1h treatment with 5μM cisplatin eliminated approximately 56.7% more HPV-positive HNSCC cells than HPV-negative ones. Furthermore, HPV-positive cells treated with cisplatin showed prolonged G2/M cell cycle arrest and more 53BP1 foci, indicating profound deficiency in repairing ICLs. Consistent with these findings, increased aberrant chromosome formation was observed in HPV-positive cells following Mitomycin C treatment. In order to reveal the mechanism, we further interrogated HPV-labeled HNSCC samples in TCGA database, and identified XPF, an endonuclease protein in FA pathway, was downregulated in HPV-positive HNSCC. Further analysis of cellular and clinical samples confirmed that both mRNA and protein expressions of XPF were significantly lower in HPV-positive HNSCC (P&amp;lt;0.001). To test the importance of XPF in HPV-induced cisplatin sensitivity, we performed a cell viability assay, and found that knock-down of XPF increased the effects of cisplatin on HPV-negative HNSCC by 39.54% (± 4.42%), while no significant change was observed in HPV-positive HNSCC (P&amp;gt;0.05). Notably, inhibition of XPF function by a small molecule inhibitor recapitulated the effect of HPV, resulted in enhanced error-prone DNA repair with alternative end-joining (alt-EJ). In a probe-based ddPCR assay, XPF inhibition increased 32.02% (± 5.80%, P&amp;lt;0.001) alt-EJ events in HPV-negative HNSCC cells, while little difference was detected in HPV-positive cells. Thus, we speculated that combined inhibition of XPF and alt-EJ may improve clinical outcome in the difficult-to-treat, HPV-negative HNSCC, which worth further research efforts. Citation Format: Qi Liu, Nan Zuo, Lin Ma, Lanlan Wei. Disruption of XPF function by HPV promotes alternative end joining and sensitivity to cisplatin in HNSCC. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6213.

Human papillomavirus associated XPF deficiency increases alternative end joining and cisplatin sensitivity in head and neck squamous cell carcinoma

ObjectivesHuman papillomavirus (HPV) positive head and neck squamous cell carcinoma (HNSCC) showed a considerably better prognosis with greater cisplatin sensitivity compared to their HPV-negative counterparts. Deciphering the underlying molecular mechanisms for HPV-induced cisplatin sensitivity is imperative to improve the prognosis of HPV-negative HNSCC. Materials and MethodsThe Fanconi anemia (FA) pathway status in HNSCC cells was analysed by detecting the cell cycle and chromosomal aberrations. XPF expression was validated using PCR, western blot, and immunohistochemistry. Droplet digital PCR and GFP expressing reporter assay were used to analyse the changes in alternative end-joining (alt-EJ) levels. The cisplatin sensitization was verified by cell proliferation assay, clonogenic cell survival assay, and TUNEL. ResultsHPV-positive HNSCC cells showed significant prolonged G2-M cell cycle arrest and aberrant chromosome formation under interstrand crosslinker treatment. Both mRNA and protein expression of XPF were considerably decreased in HPV-positive HNSCC, according to the analysis of cellular and clinical data. XPF inhibition upregulated the activity of the alt-EJ pathway in HPV-negative HNSCC cells by 32.02% (P < 0.001) but had little effect on HPV-positive HNSCC. Consistent with this, simultaneous suppression of XPF and alt-EJ enhanced cisplatin sensitivity of HPV-negative HNSCC in vitro and in vivo. ConclusionHPV-positive HNSCC cells exhibit a profound FA pathway deficiency associated with reduced XPF expression. HNSCC cells with compromised XPF function are more reliant on the alt-EJ pathway for genomic stability. Combining FA and alt-EJ inhibition may be used to cope with the hard-to-treat HPV-negative HNSCC.

Comparative radiosensitization efficiency assessment of graphene oxide and Ti3C2 MXene as 2D carbon-based nanoparticles against breast cancer cells: characterization, toxicity and mechanisms

The efficacy of two carbon-based nanomaterials, graphene oxide (GO) and Ti3C2 MXene (MX), on the radiosensitivity of the breast cancer cells (BCCs) was investigated using clinical x-ray irradiation. The prepared GO and MX nanoparticles (NPs) were firstly characterized utilizing Fourier transform infrared, ultraviolet–visible, atomic force microscopy and transmission electron microscopy techniques and subsequently assessed in terms of their radiobiological properties. The results of the cell toxicity assay indicated that neither NPs exhibited significant cytotoxicity after 48 h incubation with BCC up to 50 µg ml−1 concentration without irradiation. The cell internalization results showed an approximately equivalent cellular uptake for both NPs after 6 h incubation with BCC. Our comparative studies with radiotherapy demonstrated that both NPs substantially increased cell proliferation inhibition and cell apoptosis of BCC under x-ray irradiation when compared to BCC treated with irradiation alone. Additionally, the 2ʹ,7ʹ-dichlorofluorescin diacetate flow cytometry results and fluorescent microscopy images revealed that both NPs remarkably increased the level of intracellular reactive oxygen species (ROS) generation in BCC under x-ray irradiation. The MX nanosheets exhibited superior radiosensitization efficiency than GO under x-ray irradiation due to its higher level of intracellular ROS generation (MX = 75.2% and GO = 65.2%). Clonogenic cell survival assay and extracted radiobiological parameters revealed that both NPs in combination with x-ray irradiation induced more lethal damage and less sublethal damage to BCC. Generally, the obtained results demonstrate that the MX NPs, as a stronger radiosensitizer than GO, could be a promising candidate for enhancing the effectiveness of radiotherapy in breast cancer treatment.

SUV39H1 Expression as a Guideline for Omitting Radiotherapy in Lymph Node-positive Triple-negative Breast Cancer Patients.

The role of postoperative radiotherapy (RT) combined with chemotherapy (CT) for lymph node-positive (LN+) triple-negative breast cancer (TNBC) remains controversial. SUV39H1-mediated epigenetic regulation is associated with cancer cell migration, invasion, metastasis, and treatment resistance. This study aims to identify the role of SUV39H1 in TNBCs. Overall, 498 TNBCs with SUV39H1 RNA-seq profiles were retrieved from TCGA-BRCA and analyzed; the X-tile algorithm was used to stratify the population into low, intermediate, and high SUV39H1. Furthermore, we performed an in vitro clonogenic cell survival assay using the MDA-MB-231 cell line to assess the effects of SUV39H1 on cellular responses. The results showed that SUV39H1 was significantly higher in TNBC than normal tissue and luminal subtype breast cancer. Notably, SUV39H1 is significantly expressed in the basal-like 1 (BL1) and immunomodulatory (IM) subgroups, compared to other subtypes. Compared to patients with a low or medium expression of SUV39H1, omitting RT only worsens disease-free survival (DFS) in those with high SUV39H1 expression. The experimental results showed SUV39H1 was suppressed by si-SUV39H1, and SUV39H1 knockdown in MDA-MB-231-IV2-1 cells enhanced the cellular toxicity of doxorubicin and paclitaxel. Targeting SUV39H1 may provide a potential guiding indication of omitting RT to avoid over-treatment and chemosensitivity for TNBC.

EXTH-68. ONCOMAGNETIC TREATMENT SELECTIVELY KILLS GLIOMA CANCER CELLS BY INDUCING OXIDATIVE STRESS AND DNA DAMAGE

Abstract A new noninvasive therapeutic device developed in our laboratory called the Oncomagnetic device provides a novel approach to glioblastoma (GBM) treatment. It involves repeated stimulation with oscillating magnetic fields (sOMF) produced by spinning permanent magnets. It is technologically and mechanistically distinct from the Optune® device approved by the FDA for the treatment of newly diagnosed and recurrent GBM. Use of this device in one end-stage GBM patient reversed the progression of his recurrent tumor causing &amp;gt;30% reduction in its contrast-enhanced volume within 4 weeks of treatment. Mice with implanted mouse glioma cells in their brains also showed marked reduction in tumor size, increased survival (p&amp;lt; 0.05, n = 10) and higher DNA damage (g-H2AX foci) after sOMF treatment with a whole-body stimulation method developed by us. Normal mice exposed to sOMF for 4 months had no adverse effects on the brain and other organs. In-vitro, sOMF markedly increased reactive oxygen species (ROS) levels in cancer cells leading to the selective death of these cells, while sparing normal neurons and astrocytes. Detection of g-H2AX and 53BP1 foci showed that sOMF caused significant DNA damage in GBM cells and diffuse intrinsic pontine glioma (DIPG) cells but not in normal astroglial SVGp12 cells. Furthermore, sOMF exposure for just 2 h resulted in &amp;gt;40% loss of surviving GBM and DIPG cell colonies detected by clonogenic cell survival assay, similar to that produced by 2 Gy radiation dose. This loss was rescued by the antioxidant Trolox. These results indicate that sOMF stimulation has high anticancer potency comparable to low dose radiation therapy at the cellular level with an underlying mechanism of action that is substantially different from that proposed for Optune® TTF.

Paradoxical Radiosensitizing Effect of Carnosic Acid on B16F10 Metastatic Melanoma Cells: A New Treatment Strategy.

Carnosic acid (CA) is a phenolic diterpene characterized by its high antioxidant activity; it is used in industrial, cosmetic, and nutritional applications. We evaluated the radioprotective capacity of CA on cells directly exposed to X-rays and non-irradiated cells that received signals from X-ray treated cells (radiation induced bystander effect, RIBE). The genoprotective capacity was studied by in vivo and in vitro micronucleus assays. Radioprotective capacity was evaluated by clonogenic cell survival, MTT, apoptosis and intracellular glutathione assays comparing radiosensitive cells (human prostate epithelium, PNT2) with radioresistant cells (murine metastatic melanoma, B16F10). CA was found to exhibit a genoprotective capacity in cells exposed to radiation (p &lt; 0.001) and in RIBE (p &lt; 0.01). In PNT2 cells, considered as normal cells in our study, CA achieved 97% cell survival after exposure to 20 Gy of X-rays, eliminating 67% of radiation-induced cell death (p &lt; 0.001), decreasing apoptosis (p &lt; 0.001), and increasing the GSH/GSSH ratio (p &lt; 0.01). However, the administration of CA to B16F10 cells decreased cell survival by 32%, increased cell death by 200% (p &lt; 0.001) compared to irradiated cells, and increased cell death by 100% (p &lt; 0.001) in RIBE bystander cells (p &lt; 0.01). Furthermore, it increased apoptosis (p &lt; 0.001) and decreased the GSH/GSSG ratio (p &lt; 0.01), expressing a paradoxical radiosensitizing effect in these cells. Knowing the potential mechanisms of action of substances such as CA could help to create new applications that would protect healthy cells and exclusively damage neoplastic cells, thus presenting a new desirable strategy for cancer patients in need of radiotherapy.