bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1892: Proteomic interrogation of the metabolic control of MHC class I antigen presentation in metastatic melanoma
Authors: McKinnon Alyson , Avaritt Nathan , Tackett Alan , Koss Brian
Published: July 2021

Since 2014, immune checkpoint inhibitors (ICIs) have led the charge against metastatic melanoma as the most commonly utilized first-line treatment. While ICIs have dramatically increased the 5-year overall survival rate, only half of patients respond to treatment. Recently, a proteomic study correlated high levels of tumor mitochondrial metabolic proteins and MHC-related proteins to responsiveness to anti-PD-1 and anti-CTLA-4 immune checkpoint inhibitor treatment. In a study of 91 melanoma cell lines in the European Searchable Tumor Cell and Databank, 67% of the cell lines exhibited HLA class antigen alterations, most commonly being down-regulation of HLA-B. Loss of MHC expression is a mechanism proposed to aid in evading the immune system by decreasing T-cell recognition and killing. Approaches to reverse tumor MHC loss is needed as it is a known mechanism utilized by melanoma and increased MHC expression has been linked to ICI responders. The connection between metabolism and MHC correlation has not been fully explored, and this study sought to further explore that relationship through a proteomic analysis of a metabolically conditioned B16F10 murine melanoma model. To expand our understanding of the metabolic control of MHC Class I antigen presentation, we cultured B16F10 melanoma cells in galactose media (B16.Gal), forcing the cells into an oxidative phosphorylative state relative to B16F10 melanoma cells cultured in glucose media (B16.Glu) which exhibit a glycolytic metabolic profile. We show that enforcing mitochondrial respiration restores the lost basal MHC expression and enhances the response to interferon gamma. Further, B16.Gal cells show higher rates of T-cell killing and increased PD-L1 expression. This phenotype proved reversible as shown by moving B16.Gal cells back into glucose media (B16.GalGlu), supporting the formation of a metabolically conditioned cell model rather than a favorable selection of compatible cells. We used proteomics to identify the driving forces behind the restoration of MHC expression. The identification of these forces could provide novel therapeutic targets for enhancing immunotherapy of melanoma.

Citation Format: Alyson McKinnon, Nathan Avaritt, Alan Tackett, Brian Koss. Proteomic interrogation of the metabolic control of MHC class I antigen presentation in metastatic melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1892.
Abstract 1906: Relationship between neuroendocrine and immune gene expression in small cell lung cancer
Authors: Cai Ling , Liu Hongyu , Huang Fang , Fujimoto Junya , Girard Luc , Xie Yang , Minna John , Gazdar Adi
Published: July 2021

Small cell lung cancer (SCLC) is classified as a high-grade neuroendocrine (NE) tumor, but a subset of SCLC has been termed “variant” due to the loss of NE characteristics. In this study, we computed NE scores for patient-derived SCLC cell lines and xenografts, as well as human tumors. We aligned NE properties with transcription factor-defined molecular subtypes. Then we investigated the different immune phenotypes associated with high and low NE scores. We found repression of immune response genes as a shared feature between classic SCLC and pulmonary neuroendocrine cells of the healthy lung. With loss of NE fate, variant SCLC tumors regain cell-autonomous immune gene expression and exhibit higher tumor-immune interactions. Pan-cancer analysis revealed this NE lineage-specific immune phenotype in other cancers. Additionally, we observed MHC I re-expression in SCLC upon development of chemoresistance. These findings provide a new framework to guide design of treatment regimens in SCLC.

Citation Format: Ling Cai, Hongyu Liu, Fang Huang, Junya Fujimoto, Luc Girard, Yang Xie, John Minna, Adi Gazdar. Relationship between neuroendocrine and immune gene expression in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1906.
Abstract 1916: Lymph node targeting double stranded CpG act effective adjuvant in cancer peptide vaccine
Authors: Nakagawa Takayuki , Onishi Motoyasu , Tofukuji Soichi , Omoto Shinya , Katayama Kazufumi , Kugimiya Akira , Nagira Morio , Ishii Ken J.
Published: July 2021

Background: Adjuvants can enhance immune responses of vaccinated antigen by inducing host innate immune responses. While cancer peptide vaccines were largely studied in recent years, effective cancer peptide vaccines have not been found yet so far because of their poor immunogenicity. Developing an effective vaccine adjuvant suitable for peptide antigen is a promising approach. CpG oligodeoxynucleotides(CpG) are a toll-like receptor 9 agonists and effectively elicit innate immune responses. In order to improve adjuvanticity of CpG, we designed a novel lymph-node targeting CpG, which is composed from a single stranded CpG and a modified complementary strand with amphiphilic chain.

Methods: S-540956 is a double stranded oligodeoxynucleotide composed from a single stranded phosphorothioate oligonucleotide and the complementary strand with amphiphilic chain at the 5' end. Accumulating capability to lymph nodes of fluorescent labeled S-540956 was analyzed by flow cytometer, IVIS imaging system, and 2-photon microscopy. Incorporation of S-540956 by antigen-presenting cells in lymph nodes was assessed by Image Stream® imaging cytometer. To test cellular immune responses, three types of MHC class-I epitope peptides were employed as antigenic peptides. Therapeutic effect of S-540956 adjuvanted HPV E7 peptide vaccine was evaluated in TC-1 tumor model.

Results:S-540956 exhibited higher accumulating capacity to lymph nodes in mice compared to a typical CpG adjuvant, ODN2006. Systemic proinflammatory cytokine responses were decreased by combining the complementary strand with amphiphilic chain. S-540956 was efficiently incorporated into plasmacytoid DCs and induced the expression of costimulatory molecules. Cellular immune responses induced by S-540956 adjuvanted peptide vaccine were significantly higher than ODN2006 adjuvanted peptide vaccine. And this enhanced adjuvant effect was observed also in protein vaccine. In TC-1 tumor model, therapeutically administered S-540956 adjuvanted HPV-E7 peptide vaccine lead to significant tumor shrinkage compared to ODN2006 adjuvanted peptide vaccine.

Conclusion: S-540956 acted as a potent adjuvant for peptide vaccine by inducing cellular immune responses effectively. Accumulating capacity to lymph nodes of S-540956 is a promising adjuvant for not only for peptide but for protein or other biologic antigens.

Citation Format: Takayuki Nakagawa, Motoyasu Onishi, Soichi Tofukuji, Shinya Omoto, Kazufumi Katayama, Akira Kugimiya, Morio Nagira, Ken J. Ishii. Lymph node targeting double stranded CpG act effective adjuvant in cancer peptide vaccine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1916.
Abstract 1918: Coxsackievirus A11 elicits a potent oncolytic activity in human malignant pleural mesothelioma via ICAM-1 receptor
Authors: Okamura Koji , Inoue Hiroyuki , Ikematsu Yuki , Furukawa Rie , Ota Keiichi , Yoneshima Yasuto , Iwama Eiji , Tanaka Kentaro , Okamoto Isamu
Published: July 2021

Malignant pleural mesothelioma (MPM) is an aggressive solid cancer with dismal prognosis. Oncolytic virotherapy is a promising anticancer treatment that directly lyses tumor cells. Coxsackievirus A11 (CVA11) is an emerging oncolytic virus against solid cancers. Herein we tested whether CVA11 infection has an oncolytic activity in human MPM cell lines and identified its cognate receptor. We found that CVA11 infection shows a remarkable oncolytic activity in six of six human MPM cell lines. MPM cells having higher intercellular adhesion molecule-1 (ICAM-1) expression showed higher CVA11-induced cytotoxicity, and that identified ICAM-1 as its surface receptor because coinfection with neutralizing ICAM-1 antibody substantially abrogated CVA11-triggered cytotoxicity in MPM cells. In addition, we unraveled that the activations of both phosphoinositide 3-kinase/Akt and mitogen-activated protein (MAP)/extracellular signal-regulated (ERK) kinase (MEK) signaling pathways partially but significantly contributed to the CVA11-triggered cytotoxicity of CVA11 as evidenced by treating with respective inhibitors. Taken together, our findings show a novel oncolytic virotherapy using CVA11 could be an alternative therapeutic modality for human MPM.

Citation Format: Koji Okamura, Hiroyuki Inoue, Yuki Ikematsu, Rie Furukawa, Keiichi Ota, Yasuto Yoneshima, Eiji Iwama, Kentaro Tanaka, Isamu Okamoto. Coxsackievirus A11 elicits a potent oncolytic activity in human malignant pleural mesothelioma via ICAM-1 receptor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1918.
Abstract 1921: Armed oncolytic myxoma virus demonstrates efficacy in syngeneic tumor models alone and in combination with immune checkpoint inhibitors
Authors: Franco Lina S. , Mamola Joseph , Abdullahi Wazir , de Matos Ana , Abrantes Mario , Walker Benjamin S. , Tacner Zachary , Grigaitis Nicole , Kien Cassandra , McFadden Grant , Sharp Leslie L.
Published: July 2021

Oncolytic viruses (OV) selectively replicate in and lyse tumor cells and provide stimulation to the immune system, representing a promising therapeutic option in development to treat cancers that do not respond well to treatment with immune checkpoint inhibitors. Myxoma virus (MYXV) is a member of the Pox family of double stranded DNA viruses. The natural host of MYXV is a subset of rabbits and hares, but MYXV can infect cancer cell lines of humans and other species. The genome of MYXV is relatively large and is amenable to engineering for expression of transgenes making it an excellent oncolytic virus for introduction of immunomodulatory proteins. Oncolytic activity and transgene production capability were examined in multiple mouse cancer cell lines. In vivo efficacy following intratumoral and intravenous administration of armed myxoma virus, both alone and with immune checkpoint inhibitors is demonstrated. Immunomodulatory mechanisms of actions are investigated. Multi-armed myxoma represents a novel, engineerable, oncolytic virus with preclinical characteristics that warrant further investigation as an immunomodulatory cancer therapeutic.

Citation Format: Lina S. Franco, Joseph Mamola, Wazir Abdullahi, Ana de Matos, Mario Abrantes, Benjamin S. Walker, Zachary Tacner, Nicole Grigaitis, Cassandra Kien, Grant McFadden, Leslie L. Sharp. Armed oncolytic myxoma virus demonstrates efficacy in syngeneic tumor models alone and in combination with immune checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1921.
Abstract 1923: Modulation of apoptosis in glioblastoma U87-MG cancer cells by aqueous extract of Bryophyllum pinnatum
Authors: Ferrer Kristin , Li Joshua , Kordas Camille , Cha Jasmine , Han Sung Been , Seo Joon , Manrique Nathaly , Kang Min Seo , Jin Yi Shan , Choi Si Won , Hiramoto Ben , Smith Denise , Wong Brian Yuen
Published: July 2021

Bryophyllum pinnatum (BP) has been used in anthroposophic medicine to treat “hyperactivity diseases” such as tocolytic symptom and overactive bladder syndrome since the beginning of 20th century with no side effect. Its major secondary metabolites include bufadienolides, flavonoids, triterpenes, and steroids. BP has been widely used in tropical regions as traditional medicine. It is reported to have antioxidant, immunomodulatory, antidiabetic, anti-inflammatory, antihypertensive, wound healing, cytotoxic, and antitumor promoting activities. Glioblastoma is an aggressive and lethal tumor of the brain with few treatment options. In this study, the effects of aqueous extracts of BP on the induction and modulation of apoptosis in glioblastoma U87-MG cells were assessed using the green/red/blue fluorescent Apoptosis/Necrosis Detection Kit and the Human Apoptosis Antibody Array - Membrane (43 Targets) test by the Abcam cooperation. Our data revealed that 3-hours of 1 mg/mL and 2 mg/mL BP treatment induced statistically similar amounts of apoptosis observed as green apoptotic cells and red necrotic cells in U87-MG cells respectively (apoptotic cells = 86.0 ± 6%, necrotic cells = 14.0 ± 6.0% ; apoptotic cells = 85.0 ± 4.0% , necrotic cells = 14.5 ± 3.5%). The percent of apoptosis was not significantly different as compared to that of the positive control induced by 1µMol Staurosporine (94.5 ± 0.5%, 5.5 ± 0.5%); while significantly different from the negative control which had none apoptotic cells. Slightly less induction of apoptosis was obtained from 6-hours of 1 mg/mL and 2 mg/mL BP treatment with more necrotic cells (81.5 ± 2.5%, 18.5 ± 2.5%; 78.0 ± 3.0%, 21.5 ± 2.5% respectively). Modulation of various apoptosis markers such as up-regulation of Bax, p53, Caspase 3; and down-regulation of Bcl-2 and Bcl-w was also observed. These results suggest that BP contains phytochemicals which induce apoptosis in glioblastoma U87-MG cells by modulating these apoptotic pathway markers. Further study of the specific modulation effects of BP on apoptosis is warranted to reveal its potential chemopreventive and therapeutic properties against glioblastoma and other cancers.

Citation Format: Kristin Ferrer, Joshua Li, Camille Kordas, Jasmine Cha, Sung Been Han, Joon Seo, Nathaly Manrique, Min Seo Kang, Yi Shan Jin, Si Won Choi, Ben Hiramoto, Denise Smith, Brian Yuen Wong. Modulation of apoptosis in glioblastoma U87-MG cancer cells by aqueous extract of Bryophyllum pinnatum [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1923.
Abstract 1925: Effect of Averrhoa carambola extract on the antineoplastic and apoptotic activities in mice
Authors: Hasan Md. Zahid , Siddika Ayesha
Published: July 2021

Medicinal plants play significant roles in the treatment of different diseases. Averrhoa carambola has important clinical values in the treatment of cancer. However, its antineoplastic and apoptotic activities are still obscure. In this study, we were aimed to investigate those potentials of Averrhoa carambola in mice. For this, first the powder, prepared from the leaf and flower of Averrhoa carambola, were suspended with methanol to yield methanol extract of leaf and flower. Antineoplastic effect of leaf (25 and 50 mg/kg) against Ehrlich Ascites Carcinoma (EAC) was assessed by evaluating the viable tumor cell count, survival time, body weight and hematological parameters of EAC cell bearing mice. The result showed that the treatment with leaf significantly (p<0.05) decreased viable EAC cells and body weight gain thereby increasing the life span of EAC bearing mice and also revive the altered hematological (Hb, total RBC and WBC) parameters compare to those with normal values. Fluorescence microscopic view of 4,6-diamino-2-phenylindole (DAPI) staining demonstrated that leaf induced the alterations (i.e., nuclear condensation, fragmentation, membrane blabbing and apoptosis of cells) in the morphology of EAC cells. And, the quantitative analysis of p53 mRNA level by RT-PCR showed that p53 gene expression in leaf treated EAC cell was upregulated in respect to untreated control. These findings indicate that the induction of apoptosis in leaf-treated EAC cell was probably mediated through p53-dependent pathway. From this study, it can be concluded that the leaf of Averrhoa carambola exhibited potent antineoplastic and apoptotic activities that might be a potential agent for prevention of cancer in mice.

Citation Format: Md. Zahid Hasan, Ayesha Siddika. Effect of Averrhoa carambola extract on the antineoplastic and apoptotic activities in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1925.
Abstract 1927: Cancer cells induce apoptosis in hepatocytes as one of the mechanisms to displace hepatocytes in vessel co-opted colorectal cancer liver metastases
Authors: Rada Miran , Tsamchoe Migmar , Kapelanski-Lamoureux Audrey , Bloom Jessica , Petrillo Stephanie , Tabariès Sébastien , Kim Diane H. , Younan Peter , Gregorieff Alex , Siegel Peter , Lazaris Anthoula , Metrakos Peter
Published: July 2021

Vessel co-option in colorectal cancer liver metastases (CRCLM) has been recognized as one of the mechanistic pathways of resistance against anti-angiogenic therapy. The cancer cells are highly motile in co-opted lesions, which move toward and along the pre-existing sinusoidal vessels and hijack them to gain access to nutrient. The movement of cancer cells is accompanied by displacement of the hepatocytes. However, the molecular mechanisms underlying this displacement are unclear yet. To examine whether apoptosis involved in hepatocytes displacement by cancer cells in co-opted lesions, we performed immunohistochemical staining for pro-apoptotic markers, such as cleaved caspase-3 and cleaved PARP-1. We observed overexpression of pro-apoptotic markers in liver parenchyma of co-opted lesions compared to angiogenic lesions, specifically the hepatocytes that are in close proximity to the cancer cells. In vitro, we found that culturing hepatocytes with either colorectal cancer cells or conditioned media of co-opted CRCLM organoids induces apoptosis. Importantly, our results also suggested proprotein convertase subtilisin/kexin type 9 (PCSK-9 or PC-9) as a potential mediator of cancer cells-driven hepatocytes apoptosis. Altogether, these results confirm that cancer cells exploit apoptosis to establish vessel co-option in CRCLM.

Citation Format: Miran Rada, Migmar Tsamchoe, Audrey Kapelanski-Lamoureux, Jessica Bloom, Stephanie Petrillo, Sébastien Tabariès, Diane H. Kim, Peter Younan, Alex Gregorieff, Peter Siegel, Anthoula Lazaris, Peter Metrakos. Cancer cells induce apoptosis in hepatocytes as one of the mechanisms to displace hepatocytes in vessel co-opted colorectal cancer liver metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1927.
Abstract 1933: Improved DAMP assays for the in vitro assessment of immunogenic cell death
Authors: Niles Andrew L. , Kupcho Kevin R. , Lazar Dan F. , Cali James J.
Published: July 2021

The extracellular levels of damage-associated molecular patterns (DAMPs) released during immunogenic cell death (ICD) are positively correlated with the magnitude and efficacy of the resulting in vivo immune response. Therefore, extracellular ATP (eATP) and high mobility group box 1 (HMGB1) have been identified as key biomarkers for their predictive capacity during in vitro ICD screening activities. Current methods for identifying eATP and HMGB1 inducers are laborious, costly, and hampered by poor throughput. To overcome these challenges, we developed easy-to-use, homogeneous, bioluminescent assays that measure dose-dependent release of these immunostimulatory agents directly in cell culture. The eATP assay utilizes an optimized ATP detection chemistry that can be employed directly to assess live-cell kinetic responses for up to 24 h, with even longer exposures supported by a staggered reagent addition approach. The HMGB1 assay measures the protein's concentration at exposure endpoint in the same sample well using complementary luciferase fragment-labelled monoclonal antibodies. We tested the utility of the assays using U20S, Jurkat and U937 cells dosed with serial dilutions of known ICD inducers (doxorubicin, idarubicin, mitoxantrone, and bortezomib). The resulting data suggest the potency of eATP and HMGB1 release is dependent upon cell model and agent but can be reproducibly and robustly measured in 96 and 384 well environments. Further, the eATP assay produced remarkable early dose-dependent response resolution whereas the HMGB1 data provided a confirmatory post-mortem ICD parameter. This new ICD biomarker detection workflow may help to efficiently define and rank-order the capacity of new chemical entities to induce apoptosis and immunogenic cell death.

Citation Format: Andrew L. Niles, Kevin R. Kupcho, Dan F. Lazar, James J. Cali. Improved DAMP assays for the in vitro assessment of immunogenic cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1933.
Abstract 1940: Autophagy mediates leptin induced migration and erk activation in breast cancer cells
Authors: Miranda Alin García , Alcalá Karen A. Solano , Alvarado José B. Montes , Leyva Julio Reyes , Maycotte Paola , Saucedo Eduardo Castañeda
Published: July 2021

Autophagy is an intracellular recycling process active in eukaryotic cells which involves the formation of an autophagosome that delivers cytoplasmic components to the lysosome for degradation. This process occurs at low rates under basal conditions, but it can be induced by diverse stress conditions such as starvation, hypoxia or metabolic disorders. Autophagic flux can also be enhanced in response to hormones, including leptin. Leptin is considered a pro-tumorigenic protein whose circulating levels have been related to a bad prognosis in obese breast cancer patients. It has been recently demonstrated that leptin can induce autophagy in cancer cell lines from different tissues, suggesting that autophagy could modulate the pro-tumorigenic effects associated with leptin. In this study, the role of autophagy in leptin-induced proliferation, migration, apoptosis and ERK phosphorylation in breast cancer cell lines was evaluated.

Methods: MCF7 estrogen receptor positive and MDA-MB-231 triple negative breast cancer cell lines were used. MCF7 and MDA-MB-231 cell lines were stimulated with 400 and 50 ng/ml of leptin, respectively. Autophagy was assessed by LC3-II and p62 protein levels by western blot, and autophagosome formation by fluorescence microscopy. Proliferation and migration were assessed by real-time microscopy in an IncuCyte analyzer; and cell death was evaluated by fluorescence analysis of caspase activity. Changes in ERK phosphorylation were measure by western blot.

Results: Leptin increased autophagy in MCF7 cells but not in triple negative MDA-MB-231 cells. In MCF7 cells, autophagy inhibition reduced leptin-induced cell proliferation, changes associated to a mesenchymal morphology, cell migration, and ERK activation. In MDA-MB-231 cells, autophagy inhibition reduced proliferation and increased apoptosis independently of leptin treatment. In both cell lines, leptin-induced migration and ERK phosphorylation were reduced by the inhibition of autophagy.

Conclusion: Autophagy was important for increased cancer cell migration induced by leptin and for the maintenance of ERK phosphorylation. Our data suggest a potential use for the inhibition of autophagy in breast cancer associated with obesity.

Citation Format: Alin García Miranda, Karen A. Solano Alcalá, José B. Montes Alvarado, Julio Reyes Leyva, Paola Maycotte, Eduardo Castañeda Saucedo. Autophagy mediates leptin induced migration and erk activation in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1940.
Abstract 1941: The regulation of ATG9A-mediated autophagy by an ULK1-independent ATG13 complex
Authors: Poole Daniel , Kananngara Ashari , Weerasekara Vajira , McEwan Colten , Thornock Alexandra , Lazaro Misael , Youngs Joshua , Andersen Joshua
Published: July 2021

Cancer cells rely on autophagy for survival during periods of nutrient stress or chemotherapy. The activation of autophagy in cancer is thought to be regulated by AMPK-dependent activation of the ULK1 complex, consisting of FIP200, ATG13, ATG101, and ULK1. However, the regulation and dynamic cooperation of these proteins to govern autophagy is not completely understood. Our preliminary data suggest that ATG13 and ATG101 exist in an ULK1-independent complex that plays a key role in the regulation of autophagy. In an effort to identify the function of this ULK1-independent ATG13 complex, we discovered that an ULK1 binding-deficient mutant of ATG13 interacts with ATG9A, an apical regulator of autophagy. Using a split-venus approach coupled with confocal imaging, we found that ATG13, ATG101 and ATG9A interact together at sites of autophagosome assembly. This interaction between ATG13 and ATG9A is unaffected by deletion of ULK1 or the canonical ULK1 complex protein, FIP200. Furthermore, we found that CRISPR/Cas9-mediated deletion of ATG13 led to an aberrant accumulation of ATG9A puncta that colocalize with p62/SQSTM1. Importantly, this accumulation of ATG9A in ATG13 KO cells was rescued by expression of the ULK1 binding-deficient mutant of ATG13. Taken together, these data suggest that ATG9A interacts with ATG13 and ULK1 separately, and that ATG101 is part of an ULK1-independent ATG13 complex that controls the trafficking of ATG9A to p62 puncta. We are currently developing approaches to selectively isolate, identify, and characterize additional interacting partners of the ULK1-independent ATG13-ATG101 complex, which will give us new insight into the regulation and broader function of this complex in autophagy.

Citation Format: Daniel Poole, Ashari Kananngara, Vajira Weerasekara, Colten McEwan, Alexandra Thornock, Misael Lazaro, Joshua Youngs, Joshua Andersen. The regulation of ATG9A-mediated autophagy by an ULK1-independent ATG13 complex [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1941.
Abstract 1949: Caspase 3 plays a requisite role in regulating survival of human breast cancer cells
Authors: Eskandari Ebrahim , Eaves Connie J. , Tan Susanna
Published: July 2021

Caspase-3 has long been recognized for its key role as a protease that mediates the degradation of cellular components during the late stages of apoptosis. However, there is also a growing body of evidence that caspase-3 has essential intrinsic functions in regulating normal and malignant cell properties that are distinct from its apoptotic functions. In a previous study, we showed that a readily detectable but low proportion of freshly isolated normal human mammary cells with a luminal phenotype and a capacity to divide in vitro display a reversible expression of activated caspase-3, and subsequently it was found that suppression of caspase-3 expression in the MDA-MB-231 human breast cancer cell line contributes to its radiation-induced loss of proliferative activity in vitro. To further investigate the role of caspase-3 in normal and malignant human mammary cells, we constructed shRNA-caspase-3 and control lentiviral vectors to examine the effect of decreasing caspase-3 expression (2-fold determined by Western blot) in normal and transformed human mammary cell types. Knockdown (KD) of caspase-3 expression in 2 breast cancer cell lines (MDA-MB-231 and MCF7 cells) showed that caspase-3 KD suppressed their growth in vitro 5-fold within 8 days and inhibited formation of tumors in highly immunodeficient (NRG) adult female mice transplanted subcutaneously with shCaspase-3-transduced cells in contrast to controls in which 1cm diameter tumors appeared within 6 weeks. The shCaspase-3-transduced cells also showed a complete loss of lung metastasizing activity in intravenously injected mice. A similar anti-tumorigenic effect was also obtained in freshly isolated normal human mammary cells that were co-transduced with the shCaspase-3 vector as well as KRASG12D which otherwise causes the normal cells to produce invasive ductal carcinomas in subcutaneously transplanted immunodeficient mice. Annex V and propidium iodide staining of single-cell, as well as bulk cultures of the MDA-MB-231cells, showed that Caspase-3 is required for their survival as well as their growth. Surprisingly, initial parallel experiments with purified basal and luminal progenitor subsets of normal human mammary cells and the immortalized but non-tumorigenic MCF-10A cell line also indicates a significant suppression of cell growth in vitro within 10 days. These findings highlight the multiplicity of pro- as well as anti-survival activities that Caspase-3 exerts on normal and malignant human mammary cells raising important considerations of any therapy designed to augment their activity.

Keywords: Breast cancer, Caspase-3, Cell survival

Citation Format: Ebrahim Eskandari, Connie J. Eaves, Susanna Tan. Caspase 3 plays a requisite role in regulating survival of human breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1949.
Abstract 1956: Lycorine promotes cell senescence of hepatocellular carcinoma by regulating CDK1
Authors: Yu Haiyang , Yin Shuangshuang , Qiu Yuling , Luo Yanming , Hu Gaoyong , Lu Jia , Zhou Shiyue , Wang Tao
Published: July 2021

Hepatocellular carcinoma (HCC) is a serious threat to human health because of its high malignancy and low survival rate. The abnormal expression of genes along with the development of HCC, especially the genes related to the cell cycle. The abnormal expression of these genes in the growth of tumor cells makes them out of the original orbit and out of control. CDKs play critical part in cell cycle control, transcription, and development. Lycorine, a natural products, extract from lycoris radiate, possess biological activities. In this study, we found that Lycorine could up regulated the senescence related protein expression such as p16,p27,p21, however, CDK1 knockout also promote the occurrence of senescence. Unexpectedly, we found that lycorine inhibited CDK1 expression and promoted senescence with a dose-dependent manner in HCC cells, the results of CETSA and DARTS assay also showed that Lycorine could slow down the degradation rate of CDK1 protein; all findings indicated that lycorine might directly inhibit the expression of CDK1 to restrain the occurrence and development of HCC. These findings may provide a new perspective for the treatment of HCC.

Citation Format: Haiyang Yu, Shuangshuang Yin, Yuling Qiu, Yanming Luo, Gaoyong Hu, Jia Lu, Shiyue Zhou, Tao Wang. Lycorine promotes cell senescence of hepatocellular carcinoma by regulating CDK1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1956.
Abstract 1960: Mechanisms of therapeutic synergy between pattern recognition response agonists and cdk4 inhibitors
Authors: Roulstone Victoria , Kyula Joan , Elliot Richard , Lord Christopher J. , Matthews Nik , Jennings Vicki , Whittock Harriet , Mansfield David , Choudhary Jyoti , Wright James , Yu Lu , Melcher Alan , Vile Richard , Coffey Matt , McLaughlin Martin , Harrington Kevin
Published: July 2021

Cytoplasmic nucleic acid sensors for double-stranded (ds) RNA (RIG-I/MDA5) and DNA (cGAS-STING) are pattern recognition receptors (PRRs) key to intracellular anti-viral responses. Recent research has highlighted roles for PRR agonists, including oncolytic virotherapy agents, in anti-tumor immunotherapy. Reovirus type 3 Dearing (Rt3D) is an oncolytic dsRNA virus with limited single-agent activity in clinical studies, but potential for use in combination regimens. We sought synergistic drug-virotherapy combinations using an unbiased screening approach that highlighted the CDK4/6 inhibitor, palbociclib, as a leading hit. We found that, when combined with Rt3D, palbociclib augmented oncolytic virus-induced endoplasmic reticulum (ER) stress/unfolded protein response (UPR) signaling. Combined Rt3D-palbociclib treatment potently increased interferon signaling and endogenous retroviral transcripts. Knockdown (siRNA) studies indicated key UPR proteins and the RNA sensor, RIG-I, were essential to the phenotype observed. Mechanistically independent experiments, using canonical RIG-I agonists and the ER stress inducer (thapsigargin), confirmed cross-talk between RNA sensing and ER stress pathways that augment cancer cell death and interferon production. Combined Rt3D-palbociclib increased innate immune activation and effector function. Our findings demonstrate that UPR signaling and innate immune RNA sensor crosstalk can be exploited to enhance anti-cancer efficacy with pro-immunogenic consequences. This has implications for future clinical development of PRR agonists and oncolytic viruses, as well as broadening the therapeutic remit of CDK4/6 inhibitors to include their role as ER stress sensitizers.

Citation Format: Victoria Roulstone, Joan Kyula, Richard Elliot, Christopher J. Lord, Nik Matthews, Vicki Jennings, Harriet Whittock, David Mansfield, Jyoti Choudhary, James Wright, Lu Yu, Alan Melcher, Richard Vile, Matt Coffey, Martin McLaughlin, Kevin Harrington. Mechanisms of therapeutic synergy between pattern recognition response agonists and cdk4 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1960.
Abstract 1967: Chaperonin-Containing TCP1 complex (CCT) promotes breast cancer by targeting key cell cycle regulators
Authors: Ghozlan Heba H. , Showalter Adrian , Khaled Annette R.
Published: July 2021

CCT is a protein folding complex that interacts with known oncoproteins, but its role as a driver of oncogenesis is unknown. Elucidating interactions of CCT with cell cycle regulators could reveal the oncogenic potential of the chaperonin. CCT is multi-subunit complex composed of two stacked rings formed by eight distinct subunits (CCT1-8). While different CCT subunits are associated with cancer and other diseases, we found that the CCT2 subunit is upregulated in breast cancer and other cancers and that its expression inversely correlates with patients' overall and progression free survival. Mining datasets from TCGA revealed that amplification of CCT2 is the predominant genomic alteration occurring in cancer patients and that CCT2 is frequently co-amplified or co-expressed with other oncogenes such as MDM2, FRS2, YEATS4, and CDK4 found in the 12q13-15 amplicon. Genetic alteration of CCT2 significantly associates with worse clinical outcome for cancer patients. To investigate the functional outcomes of CCT2 overexpression in breast cancer, spheroid culture was used. A CCT2-FLAG lentiviral vector was used to transduce T47D and MCF7 luminal breast cancer cells and cells grown in ultra-low attachment plates to induce spheroid growth. Expression of CCT2 increased growth of spheroids as compared to lentiviral control cells. In contrast, CCT2 depletion impaired spheroid formation, instead causing formation of loose aggregates. CCT2 overexpression increased the proliferative potential of spheroid cells and promoted progression though the G1/S phase of the cell cycle in synchronized cells by increasing expression of cell cycle regulators like Cyclin D1, CDK2 and CDK4. After 8 days in spheroid culture, CCT2 overexpressing cancer cells resumed attachment when transferred into 2D culture conditions; these cells acquire anchorage dependent and independent growth capability, increased actin expression, and proliferated. These characteristics of CCT2 overexpressing cells may be indicative of supporting a more invasive and metastatic phenotype. In support, we discovered that, along with cell cycle genes, CCT2 overexpressed cells increased expression of MYC, suggesting the CCT2 could be a novel regulator of MYC expression. Our results contribute new knowledge on the role of CCT2 in uncontrolled breast cancer progression and proliferation by regulating cell cycle genes. The recurrent amplification of CCT2 in different cancer types along with other oncogenes like MYC, MDM2 and CDK4 support it is role as a potential oncogene and novel therapeutic target for breast cancer.

Citation Format: Heba H. Ghozlan, Adrian Showalter, Annette R. Khaled. Chaperonin-Containing TCP1 complex (CCT) promotes breast cancer by targeting key cell cycle regulators [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1967.
Abstract 1971: RCC2 promotes glioblastoma through regulation of G2-M progression
Authors: Liu Tian , Or Penelope Mei-Yu , Wong Chi-Wai , Ho Wayne Lut-Heng , Cheung Stanley Kwok-Kuen , Wang Yubing , Chan Andrew M.
Published: July 2021

Regulator of chromatin condensation 2 (RCC2), also known as telophase disk protein of 60 kDa (TD-60), has been previously identified as a component of the chromosome passenger complex. Accumulating evidence suggested that RCC2 plays a vital role in the mitotic process and abnormal RCC2 expression is essential for cancer development including Glioblastoma Multiforme (GBM). GBM is the most lethal and frequent brain tumor. Even with the optimal therapy, the 5-year survival rate of GBM remains poor. TCGA data revealed an upregulation of RCC2 in human GBM. Our preliminary data showed that RCC2 was upregulated in PTEN knockout mice primary astrocytes, and loss of function of PTEN has been found in nearly 95% GBM patients. This indicated that RCC2 might act as a PTEN downstream regulator and promote glioblastoma progression. RCC2 has been reported to be essential for tumorigenesis which may act as a GEF for Rac1 or RalA GTPase. Coincidently, our study showed that silencing RCC2 could suppress GBM cell proliferation. Using RNA-seq, we identified 737 DEGs between control siRNA- and RCC2 siRNA-treated A1172 cells. GSEA analysis delineated G2-M progression as the most notably altered pathway by silencing RCC2. Indeed, we observed a G2-M arrest in cells with RCC2 silenced, suggesting that RCC2 is essential for G2-M progression. Additionally, the connectivity map (C-map) analysis was conducted and identified LY294002 and cephaeline as promising inhibitors for RCC2-associated gene signatures. Both LY294002 and cephaeline can synergistically inhibit glioblastoma cell viability with RCC2 silencing. In summary, our study demonstrated that RCC2 signaling is essential for GBM development. We provided evidence that RCC2 exerts a positive effect on G2-M progression. These findings may shed light on anti-RCC2 targeted therapy in GBM treatment.

Funding: This work is supported by a General Research Fund grant (#14113519) from the University Grants Committee of Hong Kong.

Citation Format: Tian Liu, Penelope Mei-Yu Or, Chi-Wai Wong, Wayne Lut-Heng Ho, Stanley Kwok-Kuen Cheung, Yubing Wang, Andrew M. Chan. RCC2 promotes glioblastoma through regulation of G2-M progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1971.
Abstract 1972: A novel signaling pathway delays abscission in cytokinesis with chromatin bridges
Authors: Petsalaki Eleni , Zachos George
Published: July 2021

Chromatin bridges are strands of missegregated chromatin connecting the anaphase poles or daughter nuclei and have been linked to tumourigenesis. In response to chromatin bridges in cytokinesis, cells delay abscission, the severing of the narrow cytoplasmic canal that connects the two daughter cells, to prevent chromatin breakage or tetraploiudization that are associated with genomic instability and cancer predisposition. In mammalian cells, this abscission delay is called “the abscission checkpoint” and is dependent on the localization and catalytic activity of the Chromosomal Passenger Complex (CPC) at the midbody. The CPC comprises the catalytic subunit Aurora B kinase, the scaffolding protein INCENP and the non-enzymatic subunits Survivin and Borealin; however, the molecular mechanisms that signal chromatin bridges to the CPC have not been previously identified. In the present study, we show that inhibition of the DNA damage kinases ATM or Chk2 impairs CPC-localization to the midbody and correlates with premature abscission and chromatin breakage in cytokinesis with trapped chromatin in human carcinoma cell lines. ATM phosphorylates Chk2-threonine 68 (T68) to activate Chk2 at the midbody. In turn, active Chk2 phosphorylates INCENP at the newly identified site serine 91 (S91) to promote CPC-localization to the midbody, to delay abscission and prevent chromatin breakage. Expression of siRNA-resistant phosphomimetic mutant INCENP-S91D or GFP:INCENP(FB) that is constitutively targeted to the Flemming body (FB), but not WT INCENP, rescues CPC-midbody-localization and prevents chromatin breakage in Chk2-deficient or ATM-deficient cells. In contrast, in the absence of the endogenous INCENP, the non-phosphorylatable mutant INCENP-S91A does not localize to the midbody and its expression promotes chromatin breakage. Furthermore, the Mre11-Rad50-Nbs1 (MRN) complex is required for ATM activation at the midbody. These results identify an MRN-ATM-Chk2-INCENP signaling pathway that prevents chromosome breakage in cytokinesis with chromatin bridges, by promoting CPC-midbody localization.

Citation Format: Eleni Petsalaki, George Zachos. A novel signaling pathway delays abscission in cytokinesis with chromatin bridges [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1972.
Abstract 199: Blossom AI: A novel drug discovery app for the prediction of hotspots on multiplex protein protein interaction complexes using random forest algorithms
Authors: Zhang Stephanie , Kang Minsoo
Published: July 2021

Protein protein interactions (PPIs) form the backbone of signal transduction pathways in diverse physiological processes, mediating the transmission and regulation of oncogenic signals essential to cellular proliferation and survival, thus representing a potential new class of drug targets for anticancer therapeutic discovery. However, several challenges face the targeting of PPIs, including large PPI interface areas, a lack of deep pockets, the presence of noncontiguous binding sites, and a general lack of natural ligands. The presence of hot spots (small subsets of amino acid residues that contribute significantly to free binding energy) makes PPIs amenable to small molecule perturbations, playing essential roles in the stability of protein binding. Effectively identifying which specific interface residues of protein protein complexes form the hot spots is critical for understanding the principles of protein interactions and has broad application prospects in protein design and drug development. This project presents Blossom AI, a novel, user friendly mobile app developed in XCode and CoreML that uses random forest decision tree algorithms (RF) to computationally predict the presence of hotspots on protein complexes within seconds, aiding the design of small molecule and peptide drugs that target protein-protein interactions, particularly for anticancer therapy. Leveraging features such as solvent accessible surface area (ASA), blocks substitution matrix, physicochemical properties (hydrophobicity, polarity, polarizability, propensities), position specific scoring matrix (PSSM) and solvent exposure, the RF is trained through a dataset of 313 mutated interface residues (133 hotspot residues and 180 non hotspot residues) from over 60 protein complexes to produce a training accuracy of 88.75%, validation accuracy of 92.86%, specificity of 87.18%, sensitivity of 75.38%, PPV 94.23%, NPV 86.61%. Blossom is high speed, low cost, and user friendly with significantly improved accuracy over the standard of alanine scanning mutagenesis.

Citation Format: Stephanie Zhang, Minsoo Kang. Blossom AI: A novel drug discovery app for the prediction of hotspots on multiplex protein protein interaction complexes using random forest algorithms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 199.
Abstract 1999: DIRAS3 induces autophagy and enhances sensitivity to anti-autophagic therapy in KRAS-driven pancreatic and ovarian carcinomas
Authors: Bildik Gamze , Gray Joshua P. , Mao Weiqun , Bast Robert C. , Lu Zhen
Published: July 2021

Mutant KRAS drives human cancers from several sites, including pancreatic ductal adenocarcinoma (PDAC) and low-grade serous ovarian cancer (LGSOC). Despite the prevalence of RAS mutations in many different cancers, effective RAS-targeted treatment remains a challenge. More than 90% of PDACs are driven by mutant KRAS. Genetic suppression of KRAS or pharmacological inhibition of its effectors, ERK/MAPK, enhances the reliance of PDAC on autophagy that can provide a target for treatment. DIRAS3 (ARHI) is an imprinted tumor suppressor gene that is downregulated in pancreatic and ovarian cancers. When re-expressed, DIRAS3 blocks cancer cell proliferation, inhibits motility, and induces autophagy by multiple mechanisms. DIRAS3 inhibits RAS signaling by heterodimerizing directly with KRAS, inhibiting homodimerization of KRAS and disrupting KRAS nanoclustering. In this study, we tested whether inhibiting RAS signaling by re-expressing DIRAS3 would sensitize RAS-driven cancers to autophagy inhibition. Re-expression of DIRAS3 decreased phosphorylation of Erk1/2 (pThr202/Tyr204), inhibiting the RAF-MEK-ERK signaling pathway. Re-expression of DIRAS3 induced autophagic flux indicated by an increased LC3-mCherry/eGFP ratio. DIRAS3 re-expression decreased LKB1 (pS428) and increased AMPK (pT172) and ULK1 (pS555), suggesting that induction of autophagic flux may be partly mediated by the activation of the energy-sensing LKB1-AMPK1-ULK1 axis following suppression of upstream RAF-MEK-ERK signaling. In addition, we confirmed that DIRAS3-induced autophagy enhanced the sensitivity of cancer cells to autophagy inhibition as evidenced by decreasing the IC50 of both chloroquine (CQ) and di-chloroquine (DC661) in human KRAS-driven PDAC and LGSOC. The greater potency of DC661, blocking autophagy with an IC50 in the low nanomolar range, should prove more effective for combinatorial therapies in vivo.

Citation Format: Gamze Bildik, Joshua P. Gray, Weiqun Mao, Robert C. Bast, Zhen Lu. DIRAS3 induces autophagy and enhances sensitivity to anti-autophagic therapy in KRAS-driven pancreatic and ovarian carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1999.