bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4574: A novel method for inactivating tumor cells for use in a personalized cancer vaccine
Authors: Guth Amanda Michele , Goodrich Raymond , Park Haemin , Gladstone Matthew , Shanley Crystal , Hartson Lindsay , Weston Jon
Published: September 2020

With the recent interest in personalized medicine for cancer patients and immune therapy, the field of cancer vaccines has been resurrected. Previous autologous, whole cell tumor vaccine trials have not produced convincing results due, in part, to poor patient selection and inactivation methods that are harsh on the cells. Such methods can inadvertently alter protein structure and antigenic profile in ways that make vaccine candidates ineffective in stimulating immune response to autochthonous tumor cells. We decided to investigate a novel method for inactivating tumor cells that uses UVA/UVB light and the benign chemical riboflavin (Riboflavin and UV Light Inactivation, RF+UV). This process is fast and results in complete inactivation of the tumor cells, but preserves tumor cell integrity and antigenicity due to its rapid, yet gentle inactivation process. Our results demonstrate that the inactivation is complete, that proteins are preserved on the surface of the tumor cells, and that the inactivated tumor cells are immunogenic in that they induce dendritic cell maturation, increased IFNγ production, and the generation of tumor cell-specific IgG immune responses. Moreover, when formulated with an adjuvant (“Innocell vaccine”) and tested in two different murine primary tumor models and one metastatic tumor model, the vaccine led to decreased tumor growth, prolonged survival and decreased metastatic disease. In addition, immune cells obtained from tumor tissue following vaccination showed a decrease in exhausted and regulatory cells, suggesting that activation of intratumoral T cells may be playing a role leading to the reduce tumor growth. Lastly, tumor tissue was obtained from 3 client-owned dogs with spontaneous tumors and used to generate an autologous Innocell vaccines. The vaccine was well-tolerated and induced an inflammatory response in 2 of the 3 dogs. This data suggests that the RF+UV process of inactivating tumor cells for use as antigen-presenting agents in cancer vaccine therapy may provide a more rapid, safer and gentler way to generate autologous whole tumor cell vaccines for use in cancer patients.

Citation Format: Amanda Michele Guth, Raymond Goodrich, Haemin Park, Matthew Gladstone, Crystal Shanley, Lindsay Hartson, Jon Weston. A novel method for inactivating tumor cells for use in a personalized cancer vaccine [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4574.
Abstract 4603: Detection of pancreatic cancer using rapid dielectrophoresis based recovery of tumor derived extra cellular vesicles and exosomes from plasma
Authors: Modestino Augusta , Alvarez Jesus Bueno , Heller Michael , Ibsen Stuart
Published: September 2020

Tumor derived extracellular vesicles, including exosomes, carry cancer related proteins on their outer surfaces making them a valuable source of tumor biomarkers in blood. However, these vesicles are difficult to recover from plasma and this prevents them from being widely used for clinical diagnostic tests. Here we present a technique that uses high conductance dielectrophoresis to rapidly recover these vesicles from plasma allowing for immunofluorescence detection of cancer related biomarkers. We demonstrate this technique can be used to detect biomarkers that successfully distinguish patients with pancreatic cancer from those with benign pancreatic disease, such as pancreatitis, as well as healthy individuals. What makes dielectrophoresis different from other vesicle recovery techniques is that it takes advantage of the large contrast in the dielectric properties between the vesicles and the surrounding plasma. The dielectrophoretic force preferentially draws vesicles to an electrode array at the bottom of a microfluidic chip where they are held in place allowing a fluidic wash to remove the bulk plasma. The vesicles are concentrated at the electrode edge thereby increasing the signal to noise ratio of the fluorescent immunostaining signal and placing the particles in known locations allowing for automated detection and quantification of biomarker levels. This technique takes 30 min to complete, requires 30-50 µl of plasma, and can be highly automated to reduce labor effort making it a promising technology for future translation into the clinical laboratory setting and enabling the use of extracellular vesicles and exosomes for diagnostic applications.

Citation Format: Augusta Modestino, Jesus Bueno Alvarez, Michael Heller, Stuart Ibsen. Detection of pancreatic cancer using rapid dielectrophoresis based recovery of tumor derived extra cellular vesicles and exosomes from plasma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4603.
Abstract 4616: Functional genomic analyses of 21q22.3 locus identify potential functional variants and candidate gene YBEY for breast cancer risk
Authors: Shidal Chris , Shu Xiang , Wu Jie , Wang Jifeng , Huang Shuya , Bauer Joshua , Guo Xingyi , Zheng Wei , Shu Xiao-Ou , Cai Qiuyin
Published: September 2020

We have previously identified SNP rs3541811, located in the 21q22.3 locus, to be associated with breast cancer risk in Asians. However, the underlying causal functional variants and gene(s) responsible for this association are unknown. In this study, we performed functional genomic analyses to identify potential functional variants and target genes that may mediate this association. Functional annotation for SNPs highly correlated with rs3541881, using epigenetic data from the Encyclopedia of DNA Elements (ENCODE) and Roadmap projects, showed evidence of promoter and/or enhancer activities of five putative functional SNPs, including rs35418111, rs2078203, rs8134832, rs57385578, and rs8126917. These SNPs were assessed for interactions with nuclear proteins by EMSA assay. Our results showed that compared to the reference allele, alternate allele rs2078203 (A > G) significantly increased DNA-protein interactions, while an opposite trend was observed for rs35418111 (G > A). Cis-expression quantitative loci (eQTL) analysis, using data from the Genotype-Tissue Expression (GTEx) project, The Cancer Genome Atlas (TCGA), Molecular Taxonomy of Breast Cancer International Consortium (METABRIC), and the Shanghai Breast Cancer Study, indicated that the risk allele rs35418111 is associated with a decreased expression of the YBEY (C21or57) gene, indicating its putative oncogenic function that may contribute to breast cancer risk. The gene YBEY is a relatively uncharacterized endoribonuclease in humans, which is thought to function in rRNA cleavage and maturation, similar to its bacterial homologues. We further investigated whether YBEY may have any biological effect in human breast cancers by knocking-down YBEY gene expression using siRNA in MCF-7, T47D, and MDA-MB-231 cell lines. Transient knockdown of YBEY gene expression in three breast cancer cell lines consistently affected cell proliferation, colony formation, and migration/invasion, regardless of hormone receptor status. Our study provides support for a significant role for human YBEY gene in breast cancer pathogenesis and the association between the rs35418111/21q22.3 locus and breast cancer risk, which may be mediated through its correlated potential functional SNPs that regulate expression of the YBEY gene.

Citation Format: Chris Shidal, Xiang Shu, Jie Wu, Jifeng Wang, Shuya Huang, Joshua Bauer, Xingyi Guo, Wei Zheng, Xiao-Ou Shu, Qiuyin Cai. Functional genomic analyses of 21q22.3 locus identify potential functional variants and candidate gene YBEY for breast cancer risk [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4616.
Abstract 4646: Association between dietary vitamin B intake and risk of endometrial cancer in the prospective Singapore Chinese Health Study
Authors: Lei Ming , Adambekov Shalkar , Edwards Robert , Wang Renwei , Yuan Jianmin , Lopa Samia , Linkov Faina
Published: September 2020

Background:

Endometrial cancer (EC) is the 6th most common cancer worldwide. However, only few studies addressed dietary factors influencing EC risk in Asia. We explored the association between dietary risk factors and endometrial cancer in the prospective Singapore Chinese Health Study.

Methods:

A population of 34,028 ethnically Chinese women aged 45-74 residing in Singapore were enrolled in a longitudinal prospective cohort study between 1993 and 1998, and followed up to Dec 31, 2010. Data on baseline demographics, dietary, and reproductive factors were collected via a structured questionnaire. Endometrial cancer cases were identified from the Singapore Cancer Registry. Poisson regression was used to estimate the incidence rate ratio (IRR) on established and dietary risk factors.

Results:

166 EC cases were identified (age 53.6±7.2; BMI 24.1±3.3). Obesity (IRR=1.84, 95%CI 1.06-3.06) and having a higher number of live births (IRR=0.43, 95%CI 0.24-0.79, 5+ vs nulliparous) were associated with EC. Overall, compared with the lowest quartile, elevated risk of EC was observed for the highest quartile of dietary thiamine (IRR 1.74, 95%CI 1.02-2.58), riboflavin (IRR 1.58, 95%CI 1.01-2.53), vitamin B6 (IRR 1.71, 95%CI 1.07-2.80), and folate (IRR1.56, 95%CI 1.00-2.47). The highest quartiles of riboflavin (IRR 1.74, 95%CI 1.00-3.12), vitamin B6 (IRR 1.91, 95%CI 1.04-3.65), and folate (IRR 1.89, 95%CI 1.01-3.64) were associated with elevated EC risk in postmenopausal women, but not among premenopausal women.

Conclusion:

Obesity, parity, and higher intake of dietary vitamin B were associated with elevated EC risk in our analysis. More studies are needed to evaluate the association of supplementary vitamin B complex and EC risk.

Citation Format: Ming Lei, Shalkar Adambekov, Robert Edwards, Renwei Wang, Jianmin Yuan, Samia Lopa, Faina Linkov. Association between dietary vitamin B intake and risk of endometrial cancer in the prospective Singapore Chinese Health Study [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4646.
Abstract 4668: Human CYP1B1 prevents TRAIL-DR4-mediated cancer cell apoptosis through downregulation of miR-375
Authors: Kwon Yeo-Jung , Ye Dong-Jin , Baek Huoung-Seok , Shin Sangyun , Kwon Tae-Uk , Cho Eunah , Ahn Hong-Gyu , Chun Youngjin
Published: September 2020

Human cytochrome P450 1B1 (CYP1B1) is an important enzyme that catalyzes metabolism of 17β-estradiol (E2) to catechol estrogens, including carcinogenic metabolite 4-hydroxyestradiol (4-OHE2). CYP1B1 may play a crucial role in estrogen-mediated carcinogenesis since CYP1B1 shows tumor-specific expression pattern in various hormone-responsive tissues. However, the detailed mechanism of CYP1B1-dependent carcinogenesis remains still unclear. Here, we investigated the association between TRAIL, DR4, and CYP1B1 expression in human tumor cells, including MCF-7, MDA-MB-231, and HeLa cells. CYP1B1 can inhibit the expression and release of TRAIL simultaneously with the expression of the death receptor DR4/5, known as TRAIL receptors, to prevent apoptosis. Furthermore, we found that CYP1B1 represses the transcription of DR4 by inducing DNA methylation in the promoter region of DR4. In addition, we revealed that CYP1B1 regulates TRAIL and DR4 through activation of Sp1 transcription factor. CYP1B1 and its enzymatic product, 4-OHE2, promote Sp1 expression levels in both mRNA and protein levels. Interestingly, CYP1B1 elevated Sp1 through downregulation of miR-375, which is a candidate tumor suppressive microRNA and transcriptional inhibitor of Sp1. Taken together, our data suggest that CYP1B1 inhibits TRAIL-DR4-mediated cancer cell apoptosis through induction of Sp1 through repression of miR-375, which promotes cancer cell survival.

Citation Format: Yeo-Jung Kwon, Dong-Jin Ye, Huoung-Seok Baek, Sangyun Shin, Tae-Uk Kwon, Eunah Cho, Hong-Gyu Ahn, Youngjin Chun. Human CYP1B1 prevents TRAIL-DR4-mediated cancer cell apoptosis through downregulation of miR-375 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4668.
Abstract 4670: TIP60 regulates alternative splicing of Integrin subunit alpha 6
Authors: Bhatia Shreshtha Sailesh , Koeffler H. Phillip , Jha Sudhakar
Published: September 2020

Differences in RNA processing, specifically RNA splicing, contribute to enormous diversity that can be generated from the eukaryotic genome. Given the increasing evidence to show that splicing can occur co-transcriptionally, chromatin structure and histone modifications have been reported to affect the choice of a splice site. For instance, HDAC inhibitor treatment has been shown to affect alternative splicing. TIP60 is a haploinsufficient tumor suppressor gene that is frequently downregulated in cancers. TIP60 is a lysine acetyltransferase that acetylates histone and non-histone proteins. Since TIP60 is a writer of histone and non-histone modifications, we investigated TIP60's role in modulating alternative splicing. To understand TIP60's role in alternative splicing, RNA sequencing was performed in MCF10A cells that were (a) transiently depleted of TIP60 (b) stably overexpressing siRNA-resistant wild-type TIP60 (MCF10A*WT) and (c) stably overexpressing siRNA-resistant catalytically dead TIP60 (MCF10A*KD). Differential splicing analysis was performed using MISO program. We found 130 genes to be differentially spliced when TIP60 was depleted and 59 genes were differentially spliced in MCF10A*KD compared to MCF10A*WT. For both comparisons, the majority of the differential splicing events were skipped exon events. Upon overlapping the differential skipped exon events, we found significant exon 25 skipping of ITGA6, resulting in an isoform that has a shorter cytoplasmic domain. TIP60 depletion results in an increase in the ITGA6 isoform that lacks exon 25, which has been implicated to promote tumor initiation in breast cancer. To identify the splicing factor that regulates this event, we performed a siRNA screen of over 70 splicing factors and identified ESRP2 to regulate splicing of ITGA6 exon 25. The consequence and biological significance of TIP60-ESRP2 mediated alternative splicing will be discussed at the conference.

Citation Format: Shreshtha Sailesh Bhatia, H. Phillip Koeffler, Sudhakar Jha. TIP60 regulates alternative splicing of Integrin subunit alpha 6 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4670.
Abstract 4679: Investigating the role of cryptochrome 2 in human cancer
Authors: Chan Alanna , Lamia Katja
Published: September 2020

Through numerous epidemiological studies, The World Health Organization (WHO) deemed disruption of circadian rhythms a probable carcinogen. Circadian rhythms regulate many physiological and cellular processes, while maintaining a ~24hr period. The molecular mechanisms by which disrupted circadian rhythms promote tumorigenesis remain unclear. Recently, our lab found that Cryptochrome 2 (CRY2) acts as an adapter to recruit c-MYC to the E3 ligase complex SKP-CULLIN-FBXL3 (SCFFBXL3) to promote degradation of c-MYC, a known proto-oncogene transcription factor. We identified five recurrent CRY2 mutations (CRY2 D347H, S436R, R460C, D467N and S532L) in a variety of human cancers from The Cancer Genome Atlas (TCGA) (referred to as CRY2 TCGA mutations). We hypothesized that these mutations may disrupt the interactions between CRY2, FBXL3, and c-MYC and thereby promote c-MYC accumulation. We performed several biochemical and functional assays in parallel to investigate the impact of these mutations on CRY2 activities. While three of the mutants tested reduce the association of CRY2 with FBXL3 as expected, we unexpectedly found that one of the CRY2 TCGA mutants abolishes interaction between CRY2, CLOCK, and BMAL1 without affecting its ability to interact with FBXL3 or c-MYC. Thus, this mutation prevents the normal function of CRY2 within the circadian clock and affects two-dimensional colony formation, which could impact cellular transformation as well.

Citation Format: Alanna Chan, Katja Lamia. Investigating the role of cryptochrome 2 in human cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4679.
Abstract 4696: Endometrial receptivity markers in patients with uterine fibroids and their correlation with air pollution exposure
Authors: Ferreira Dara O. , Maffazioli Giovana D. , Baracat Edmund C. , Carvalho Katia C.
Published: September 2020

Uterine leiomyomas (LM) are the most frequent benign tumors in women of reproductive age. They are associated with infertility, pelvic pain, abnormal uterine bleeding and represent a public health challenge. LM are over 4 times more common in African-descendent women. Additionally, this tumor constitutes a major health and financial burden in several countries. Similar to many pathologies, environmental exposure contributes to their origin and development. Air pollution acts as a disruptor in the hormonal activity and in other key pathways leading to enhanced tumor progression and making possible pregnancies difficult. In this study, we evaluated LM samples from women resident in urban (high exposure area) and no-urban areas (low or negative exposure area) for the expression of genes related to endometrial receptivity. We selected a total of 100 patients and among them twenty fill the specific profile of those exposed or not exposed to air pollution. mRNA expression analysis was performed using an array composed by 84 receptivity-related genes (Array Code: CLAH27251A, Qiagen, USA). Gene expression analyses showed PRLR, LITAF, COL6A3, HOXA11, KLRC1, UBE2A e IGFBP5 with higher fold expression (fold >2) in the exposed group. On the other hand, PENK, CALB2, DUOX1, COMP, CXCL14, CTNNA2, TCN1, S100P, WNT5A, ERBB2, MTNR1B and EFNA1 were dowregulated in these samples (fold < - 4). Several other genes exhibited differential regulation between exposed and non-exposed group of patients, but with lower significance. Our results corroborate the expected for these populations, since it is known that women resident in areas with high air pollution have reproductive complications and higher tendency to tumors development. However, this work is the first evidence of the impairment of endometrial receptivity in uterine LM patients associated to air pollution in Brazil. Further studies are ongoing to validate and elucidate the specific roles of these genes in uterine LM origin and development, as well as their reproductive function in this population.

Citation Format: Dara O. Ferreira, Giovana D. Maffazioli, Edmund C. Baracat, Katia C. Carvalho. Endometrial receptivity markers in patients with uterine fibroids and their correlation with air pollution exposure [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4696.
Abstract 472: What electric field strength is necessary for maximum tumor-treating fields efficacy
Authors: Carlson Kristen W. , Tuszynski Jack A. , Dokos Socrates , Paudel Nirmal , Bomzon Zeev
Published: September 2020

Why Is 4 V/cm Electric Field Strength Necessary for Maximum Tumor-Treating Fields Efficacy? INTRODUCTION: Tumor Treating Fields (TTFields) extend overall survival in glioblastoma patients. Threshold field strength for TTFields is typically cited as 2V/cm. Yet plots of cell death vs. field strength in vitro indicate a power law relationship asymptotic to 4 V/cm for 100% efficacy. The mechanisms of action (MoA) of TTFields underlying this relationship are currently under investigation. TTFields are suspected of disrupting critical mitotic processes performed by large polar cellular molecules such as microtubules (MT). METHODS: Using the power law relationship for the energy (intensity, I) carried by an electric field and its amplitude (Eq. 1), we calculated energy vs. TTFields amplitude. (1) RESULTS: We suggest Eq. 1 is a likely source of the relationship between TTFields' amplitude and cell death proportion that has been demonstrated empirically. Further, Table 1 shows energy transmitted per unit MT surface area per TTFields cycle, specifically, the energy per MT dimer band around an MT helix per TTFields period of 5 µs.

TTFields Field Strength (V/cm)Radiant Flux Density (W/nm2)Energy/(Cycle-Area) (J/nm2)Energy/MT Dimer/Cycle (J)Energy/MT Dimer Band/Cycle (J)113.3 × 10−186.63 × 10−234.01 × 10−224.17 × 10−20253.1 × 10−1826.6 × 10−231.60 × 10−211.67 × 10−193119 × 10−1859.7 × 10−233.61 × 10−213.75 × 10−194212 × 10−18106 × 10−236.42 × 10−216.67 × 10−195332 × 10−18166 × 10−231.00 × 10−201.04 × 10−186478 × 10−18239 × 10−231.44 × 10−201.50 × 10−18

A metric for disruption of cellular processes is 1-2 orders of magnitude greater than the thermal background energy in the cell, kT = 4.3 × 10−21 J. Table 1 shows that the energy absorbed by a MT dimer is insufficient to disrupt cellular processes at 2 V/cm, while due to energy increasing with square of field strength, at 5 V/cm it may be disruptive. For an entire dimer band around the MT helix, 2 V/cm appears to be sufficient, while 4 V/cm seems amply strong to disrupt MT function, in accordance with in vitro experiments. CONCLUSION: Assuming the power law of energy vs. electric field strength and the resulting energy absorbed by microtubule components, we found a correlation between typical TTFields' amplitudes and estimated disruption thresholds of MT function. TTFields MoA. 4 V/cm, not 2V/cm, should be the target field strength to realize the full efficacy of TTFields.

Citation Format: Kristen W. Carlson, Jack A. Tuszynski, Socrates Dokos, Nirmal Paudel, Zeev Bomzon. What electric field strength is necessary for maximum tumor-treating fields efficacy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 472.
Abstract 480: Molecular mechanism of action of photoimmunotherapy with antibody-IR700 dye conjugates
Authors: Heim Roger , Makings Lew , Wiemann Torsten , Manibusan Anthony , Garcia-Guzman Miguel
Published: September 2020

INTRODUCTION: Cancer cell-targeted photoimmunotherapy (PIT) is a platform technology under development for the treatment of various cancers. PIT is a drug and device combination that utilizes an antibody conjugated to the IR700 (IRDye 700DX®) light-excitable dye, which upon antigen binding and illumination with non-thermal red light, leads to necrotic cell death. The objective of these studies was to characterize the molecular pharmacology and the underlying mechanism of action of PIT.

METHODS: In vitro PIT studies using human and mouse cancer cells were performed to evaluate conjugate (antibody + dye) binding, dose response and light dosimetry response. Mouse xenograft experiments assessed necrotic markers in tumor tissue and surrounding normal tissue post-PIT treatment. Oxidative damage to lipids was detected with a reporter dye. Mass spectrometry analysis was performed to assess protein oxidation, crosslinking and axial ligand dissociation from the dye.

RESULTS: Binding of the conjugate to the target cancer cells was required for PIT treatment to induce cell death as measured by antibody competition studies, however, internalization of the conjugate was not required for cytotoxic effects. Administration of non-thermal red light to the target cells bound with the conjugate led to morphologic changes and membrane disruption within minutes, and necrotic cell death was observed in a conjugate- and light-dose dependent manner. Requirement of conjugate binding to the cancer antigen provided high level of tumor specificity as demonstrated by lack of damage to normal surrounding tissue after PIT treatment. Light excitation of the conjugate generated 1O2 with high quantum yield. In vitro PIT treatment led to cell membrane lipid peroxidation, mediated by 1O2 as shown by abrogation with sodium azide, a selective 1O2 quencher. In addition, protein oxidation, cross-linking and aggregation of the conjugate was observed. In low oxygen concentrations and in the presence of ascorbate, photo-induced dissociation of axial ligands from the dye was detected.

CONCLUSIONS: These studies suggest the molecular mechanism of action of PIT includes generation of 1O2 that leads to lipid peroxidation and cell membrane disruption. Additional mechanisms may include protein oxidation and non-oxygen dependent mechanisms such as axial ligand loss of the IR700 dye. These results describe a biophysical process that damages and disrupts the cell membrane integrity resulting in necrotic cell death and immunogenic cell death.

Citation Format: Roger Heim, Lew Makings, Torsten Wiemann, Anthony Manibusan, Miguel Garcia-Guzman. Molecular mechanism of action of photoimmunotherapy with antibody-IR700 dye conjugates [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 480.
Abstract 4803: Mitochondrial response to temozolomide treatment in glioma cells
Authors: Lenz Luana S. , Silva Mardja M. , Bristot Ivi J. , Klamt Fabio , Lenz Guido
Published: September 2020

Mitochondria are a central organelle to metabolism and apoptosis control, it means that their functionality is essential to cellular homeostasis. We investigated the mitochondrial alterations in respose to the chemotherapeutic Temozolomide (TMZ) in glioma cells.We transduced A172 and U87 cells with a fluorescent protein targeted to mitochondria (Addgene #50057), treated these cell with TMZ 100 uM during 3 hours and analyzed them by cytometry, microscopy and high resolution respirometry at days 3, 5 and 7 after treatment.Once tolerance is a dynamic process that can lead to different responses even between related cells, we first asked how related cells respond to TMZ in terms of mitochondria amount. Closely related sisters and cousins cells were not different to random pairs, indicating that mitochondria amount is not inherited and that this feature is very dynamics. Population analysis reveal that five days after TMZ the cells showed an increase in mitochondrial mass and mitochondrial swelling. The PGC-1 expression was reduced by half on day 3, suggesting that increase in mitochondrial mass must be due to accumulation of mitochondria instead of biogenesis. Five days after treatment we observed an increase in mitochondrial membrane potential measure by JC1 and an increase in mitochondria ATP linked respiration. To test if increased mitochondria respiration is a therapeutically targetable adaptation of these cells treated with TMZ, we treated the cells that survived five days after TMZ with KCN, a cytochrome c oxidase inhibitor. As expected, the TMZ-treated cells were more sensitive to the blockage of mitochondrial respiration than untreated cells. On the other hand, cells treated at day five after TMZ with 2-DG, a glycolysis inhibitor, were less affected, indicating that the oxidative phosphorylation is important to tolerance to TMZ.Our results indicate that treatment induce a metabolic reprogramming with increase in oxidative state, and strategies to overcome this tolerance mechanism could be explored.

Citation Format: Luana S. Lenz, Mardja M. Silva, Ivi J. Bristot, Fabio Klamt, Guido Lenz. Mitochondrial response to temozolomide treatment in glioma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4803.
Abstract 482: Multiple chemical action cancer therapeutics
Authors: Papineni Rao , Adhikary Amithava
Published: September 2020

Radiation-produced quasi-free electrons (i.e., low energy electrons in solutions) causes DNA damage. However, there are no evidence that radiation-produced partially solvated electrons (i.e., presolvated or, prehydrated electrons) and fully solvated electrons (i.e., aqueous or solvated electrons) cause strand breaks. However, recent work on azidoDNA-model systems show that radiation-produced electrons can be converted to highly damaging aminyl radicals (RNH•) which do have the potential to cause strand breaks. Experimental studies in our labs (both in vitro and in vivo) have shown that 2-deoxy-D-glucose (2-DG), a glucose mimic and glycolytic inhibitor, enhances radiation-induced damage selectively in tumor cells while protecting normal cells and against infection. Thereby suggesting that 2-DG can be used as a differential radiomodifier to improve the efficacy of cancer radiotherapy. Based on these experimental results and data mining models, we show that the azido derivative of 2DG; 2-Azido-2-deoxy-D-glucose (2-AZ-2-DG) has unique azido chemistries. The neutral aminyl radical produced from 2-AZ-2-DG under reductive environment undergoes facile conformational change from the stable chair form to the reactive boat form. Subsequently, this aminyl radical causes H-atom abstraction from C5 of the sugar moiety via proton-coupled electron transfer process. Further, a promise as multivalent drug to augment multipronged cellular damage including site-specific DNA radiation damage and also counter infections persisting in several cancer cases. Our long range goals are to harness these unique chemistries in gold based nanotools for X-PDT (X-ray induced photodynamic therapy).

Citation Format: Rao Papineni, Amithava Adhikary. Multiple chemical action cancer therapeutics [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 482.
Abstract 4844: CS3002, a novel CDK4/6 inhibitor with unique kinase inhibition spectrum which may expand indications beyond breast cancer and delay acquired drug resistance
Authors: Zhang Juan , Tang Liang , Li Zhenhu , Lu Liang , Bao Yuanwu , Ren Zhaoxiang , Ma Jingshu , Huang Yaling , Xu Zhaobing , Xia Yuanfeng , Ding Charles Z. , Hu Lihong , Chen Shuhui , Xie Archie N. , Wang Xinzhong Jon
Published: September 2020

The G1 restriction point is critical for regulating the cell cycle and is controlled by the retinoblastoma protein (Rb) pathway (CDK4/6-cyclin D1-Rb-p16/ink4a). Selective CDK4/6 inhibitors, such as palbociclib, ribociclib, and abemaciclib have been approved by US FDA as single agent or in combination with endocrine therapy to treat patients with HR+/Her2- breast cancer. Here, we disclose the preclinical development of CS3002 and its characterization as a novel CDK4/6 inhibitor with unique kinase inhibition spectrum. Similar to the three approved CDK4/6 inhibitors, CS3002 is highly selective for and potent against CDK 4 and 6, a feature that is expected to overcome the toxicity issues of pan-CDK inhibitors. Cell-based assays confirmed the biological activities of CS3002 by showing anti-proliferative effect, significant Rb phosphorylation inhibition, and G1 cell cycle arrest in a set of Rb-positive cancer lines. Furthermore, CS3002 showed excellent in vivo activity as a single agent, and in combinations with either endocrine therapy (fulvestrant) or PD-1 blockade (CS1003) in a set of tumor models, together with desirable ADME/PK and safety profile. CS3002 was further tested against 196 kinases in a Carna panel. In addition to CDK4 and CDK6, FLT3 and TRKA were also potently inhibited with IC50 values <50nM. To this end, CS3002 demonstrated high activity in cell lines harboring NTRK1 fusion that were otherwise insensitive to CDK4/6 inhibitors. Across a panel of 19 AML cell lines, CS3002 demonstrated a similar sensitivity profile to gilteritinib (an approved FLT-3 inhibitor), but overall higher sensitivity compared to palbociclib. Interestingly, when MCF-7 breast cancer cell line was treated continuously with CS3002 in vitro, the emergence of drug-resistance was much delayed compared to treatment with palbociclib or abemaciclib. Given the encouraging preclinical properties as well as the unique potential in allowing indication expansion and delaying drug resistance to CDK4/6 inhibition, CS3002 is selected for clinical exploration in a Phase 1 study for advanced solid tumors (NCT04162301).

Citation Format: Juan Zhang, Liang Tang, Zhenhu Li, Liang Lu, Yuanwu Bao, Zhaoxiang Ren, Jingshu Ma, Yaling Huang, Zhaobing Xu, Yuanfeng Xia, Charles Z. Ding, Lihong Hu, Shuhui Chen, Archie N. Xie, Xinzhong Jon Wang. CS3002, a novel CDK4/6 inhibitor with unique kinase inhibition spectrum which may expand indications beyond breast cancer and delay acquired drug resistance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4844.
Abstract 485: The NCI Mouse Repository: Cancer models and microRNA ES cell resource
Authors: Awasthi Parirokh , Fitzgerald Debra , Jones Stephen
Published: September 2020

The NCI Mouse Repository, located at the Frederick National Laboratory for Cancer Research (FNLCR), Frederick, Maryland, is an NCI-funded resource of approximately 150 genetically-engineered mouse cancer models and associated strains, including mice bearing conditional and point-mutant alleles in cancer-related genes. In addition, the Repository houses a unique collection of over 1500 different mouse ES cell clones bearing conditionally-activated miRNA transgenes to facilitate in vivo exploration of miRNA functions. The NCI Mouse Repository makes strains and mESCs available to all members of the scientific community (academic, non-profit, and commercial). The mouse models and ES cell clones are cryo-archived and distributed as frozen germplasm or cells.

Requests may be placed through the NCI Mouse Repository website (https://ncifrederick.cancer.gov/Lasp/MouseRepository/Default.aspx). In addition to the request form, this website includes detailed description for each strain accepted into the Repository and the associated publications provided by the donating scientist. The miR-harboring ESCs, originally generated at the Cold Spring Harbor Laboratories for the NCI, are described in full detail and include validation data for each miRNA ES cell clone. These resources are available for nominal cost to NCI, NIH, and other US government-funded investigators, as well as to Investigators at non-profit organizations.

Citation Format: Parirokh Awasthi, Debra Fitzgerald, Stephen Jones. The NCI Mouse Repository: Cancer models and microRNA ES cell resource [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 485.
Abstract 4872: Therapeutic value of BCL2 and MDM2-p53 axis in ovarian clear cell and endometrioid carcinomas with wild type TP53
Authors: De Pradip K. , Aske Jennifer C. , Costello Joseph P. , Thompson Ethan , Dey Nandini
Published: September 2020

Background: Apoptosis is the therapeutic goal following the administration of anti-cancer drugs. BCL2 and p53 are two important nodes in the apoptosis signaling pathway. Previous reports have shown that the anti-apoptotic protein BCL2 and p53 degrader MDM2 are overexpressed in many solid neoplasms, including ovarian cancers, and associated with poor clinical outcome.

Purpose: Since monotherapy in cancer is generally not providing long-lasting clinical responses, here, we hypothesize that concomitant p53 activation (by an inhibitor of MDM2-p53 interaction, e.g. AMG232) and BCL2 inhibition (by venetoclax) induce maximum apoptosis and markedly reduce tumor cell proliferation in ovarian clear cell and endometrioid carcinomas with wild type (WT) TP53.

Methods: We tested the anti-proliferative and apoptotic activity of venetoclax (V) alone, AMG 232 alone (A), and a combination of V plus A in ovarian clear cell carcinoma (OVISE cells) and ovarian endometrioid carcinoma cell lines (A2780 & isogenic Cisplatin resistant A2780CisR).

Results: 1) A binds the MDM2 protein, blocks MDM2-p53 interaction, and induces p53 expression and activity, 2) p53 effector molecule p21 is robustly induced following the treatment of A, 3) the anti-proliferative activity of V or A was observed by 3D-ON-TOP clonogenic assay and real-time monitoring in an IncuCyte Zoom, 4) all three cell lines exhibited an increase in annexin V positivity (initiation of apoptotic activity) following V or A treatment, 5) importantly, a combination of V plus A robustly induced the apoptotic markers e.g. cleaved-CASPASE3/7, cleaved PARP1 and BIM protein expression in all cell lines, 6) high mitochondrial depolarization was also observed following the combination of treatment, 7) similar to protein expression data, mRNA data also showed that pro-apoptotic/cell cycle inhibitor transcripts such as p21 and PUMA mRNA expression were significantly increased and 8) normoxic HIF1α degradation is not rescued following the treatment of MDM2-p53 interacting inhibitor A however A-induced p53 activation inhibits hypoxic HIF1α accumulation.

Conclusion: These data strongly support the further evaluation of dual BCL2/MDM2 targeting as a therapeutic strategy for ovarian clear cell and endometrioid carcinomas patients that retain WT-TP53.

Citation Format: Pradip K. De, Jennifer C. Aske, Joseph P. Costello, Ethan Thompson, Nandini Dey. Therapeutic value of BCL2 and MDM2-p53 axis in ovarian clear cell and endometrioid carcinomas with wild type TP53 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4872.
Abstract 4893: Multi-modality sequencing of multiple HPV mediated oropharyngeal squamous cell carcinomas from single patients reveals synchrony in viral genomes yet discordant mutational profiles and signatures
Authors: Faden Daniel L. , Langenbucher Adam , Kuhs Krystle , Lewis James S. , Lawrence Michael S. , Mirabello Lisa , Ferris Robert L.
Published: September 2020

Human Papilloma Virus (HPV) mediated oropharyngeal squamous cell carcinoma (HPVmOPSCC) is increasing in prevalence in the United States, as are cases of patients with multiple, spatially distinct, HPVmOPSCCs (mHPVmOPSCC). Patients with mHPVmOPSCCs present a unique opportunity to study the biology of HPV mediated tumorigenesis in the oropharynx, which currently is poorly understood. We performed whole exome sequencing and RNA-Seq on 16 paired HPVmOPSCCs (eight patients) and whole genome sequencing of the viral genome using a HPV16 NGS assay described previously. Viral sequencing revealed considerable diversity between patients, across multiple viral sublineages. However, all tumor pairs from a given patient contained the same viral genome, with at most 2 clonal SNPs distinguishing the viruses within a pair. In contrast, tumor DNA sequencing revealed striking dissimilarity between tumors from a given patient. Seven of eight tumor pairs shared no overlapping somatic mutations with one pair sharing only six variants out of 277 (2%). Mutational signature analysis demonstrated a dominant APOBEC signature in six tumors. Tumor pairs showed striking discordance of mutational signatures. In 3/8 pairs, one tumor exhibited a nearly 100% pure APOBEC+ mutational profile, while the other was totally devoid of APOBEC-signature mutations. Here, we have demonstrated that spatially independent tumors, originating within the same individual, in the same tissue type, are caused by the same virus. Interesting, despite this shared etiology, the pair of tumors within a patient develop with vastly different mutational profiles and have different mutational processes driving tumorigenesis. This suggests that despite infection by a single oncogenic virus, HPVmOPSCCs may develop via multiple, disparate pathways. Of particular interest is the tumor pairs' clear discordance in APOBEC mutational burden, which challenges existing hypotheses on the role of APOBEC mutagenesis in this disease.

Citation Format: Daniel L. Faden, Adam Langenbucher, Krystle Kuhs, James S. Lewis, Michael S. Lawrence, Lisa Mirabello, Robert L. Ferris. Multi-modality sequencing of multiple HPV mediated oropharyngeal squamous cell carcinomas from single patients reveals synchrony in viral genomes yet discordant mutational profiles and signatures [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4893.
Abstract 4911: Characterizing the role of podocalyxin's cytoplasmic tail domain in collective tumor invasion
Authors: Bell Erin M. , Graves Marcia L. , Dean Pamela , McNagny Kelly M. , Roskelley Calvin D.
Published: September 2020

High expression of the single-pass transmembrane sialomucin, podocalyxin, has been shown by many groups to correlate with poor disease outcome in a number of solid tumor types, including colorectal, ovarian, pancreatic and breast cancers. We had previously identified that high podocalyxin expression in invasive ductal breast carcinoma was an independent indicator of poor survival (Somasiri et al., 2004, Cancer Res. 64:15). Further analysis of these tumor samples revealed that this decrease in survival occurred without the loss of membranous, junctional E-cadherin, suggesting that these tumors may have invaded collectively without necessitating an overt epithelial to mesenchymal transition (EMT). Experimentally, forced over-expression of podocalyxin in polarity-disrupted human MCF7 breast cancer cells drives the formation of invasive orthotopic xenograft tumors and elongated, cohesive, and E-Cadherin-expressing spheroids in three-dimensional (3D) culture as compared to control (Graves et al., 2016, Breast Canc. Res. 18:11). Further, treatment of these podocalyxin-overexpressing MCF7 cells with the myosin inhibitor, blebbistatin, and the small molecule inhibitor of ezrin-actin binding, NSC668394, resulted in decreased collective invasion and migration, respectively. Together these data suggest that podocalyxin, through interactions with the actin cytoskeleton via its cytoplasmic tail binding partners, can facilitate increased collective epithelial tumor cell motility, at least in some contexts. To test this hypothesis, we generated podocalyxin null MCF7 clones and cell populations using CRISPR-Cas9 genome editing and reconstituted these cells with mutant forms of podocalyxin that are unable to interact with the scaffolding proteins NHERF and/or ezrin and hence with the actin cytoskeleton. Preliminary results from 3D culture and live imaging of these mutant podocalyxin-expressing cells suggests that loss of podocalyxin's cytoplasmic tail results in decreased spheroid invasion that may be a result of deficiencies in actomyosin contractility. Hence, increased expression and mislocalization of podocalyxin may facilitate aberrant interactions with the actin cytoskeleton and contractile machinery, driving enhanced cell motility and, in certain tumor microenvironments, promote collective tumor invasion.

Citation Format: Erin M. Bell, Marcia L. Graves, Pamela Dean, Kelly M. McNagny, Calvin D. Roskelley. Characterizing the role of podocalyxin's cytoplasmic tail domain in collective tumor invasion [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4911.
Abstract 4943: The stem cell factor LIN28B regulates proliferation and apoptosis in diffuse intrinsic pontine glioma
Authors: Guo Huizi , Kaur Harpreet , Eberhart Charles G. , Raabe Eric H.
Published: September 2020

Diffuse Intrinsic Pontine Glioma (DIPG) is a lethal pediatric brain tumor. With radiation therapy as the primary treatment, the median survival after diagnosis is 6 months. Identifying molecular markers that regulate tumor growth is urgently needed to develop targeted therapies. LIN28B is a stem cell factor expressed during normal neural development and re-expressed in cancer cells. In many cancers, LIN28B acts as an RNA-binding protein and downregulates the tumor-suppressing let-7 microRNAs. We had previously shown that LIN28A, a homolog of LIN28B, regulates invasion and tumorigenicity in adult high-grade gliomas. LIN28B is known to be highly expressed in DIPG. Thus, we hypothesized that LIN28B would be important in DIPG by promoting tumor growth. We observed that LIN28B was expressed in patient-derived DIPG cell lines (JHHDIPG1, JHHDIPG16A, SUDIPGXIII, HSJD-007, SF7761). Knockdown of LIN28B using short hairpin RNAs decreased DIPG cell proliferation (BrdU incorporation) between 50% to 90% (p<0.01). The knockdown of LIN28B also increased DIPG cell apoptosis (cleaved caspase-3 and cleaved PARP expression) by 2- to 3-fold (p<0.01). To determine the molecular mechanism of LIN28B-mediated phenotypes in DIPG, we studied canonical downstream effectors of LIN28B. We found that when LIN28B was suppressed in DIPG cells, there was a decreased expression of the DNA-binding oncoprotein called high mobility group AT-hook 2. These findings suggest that LIN28B could be a potential therapeutic target for this devastating pediatric brain tumor. Future studies will focus on validating the role of LIN28B in DIPG growth in vivo using orthotopic zebrafish and mouse models.

Citation Format: Huizi Guo, Harpreet Kaur, Charles G. Eberhart, Eric H. Raabe. The stem cell factor LIN28B regulates proliferation and apoptosis in diffuse intrinsic pontine glioma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4943.