bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4871: The roles of E3/E4 ubiquitin ligase UBE4B and its targets p53 and Ku70 in neuroblastoma cell death and differentiation
Authors: Clorennec Christophe Le , Lesperance Jacqueline , Huo Yuchen , Zage Peter E.
Published: September 2020

Children with high-risk neuroblastoma have poor survival rates and need novel treatment strategies. UBE4B is an E3/E4 ubiquitin ligase whose gene is found in the chromosome 1p36 region commonly deleted in high-risk neuroblastoma tumors and whose expression is associated with neuroblastoma patient outcomes. However, the functional roles of UBE4B in neuroblastoma pathogenesis are not known. We hypothesized that UBE4B would interact with mediators of DNA damage and repair pathways, leading to the observed associations between UBE4B and patient outcomes. Using cell lines with stable UBE4B depletion and over-expression, we evaluated the interaction of UBE4B with the E3 ubiquitin ligase ITCH/AIP4 and the effects of UBE4B expression levels on p53 and Ku70 ubiquitination and proteasomal degradation by co-immunoprecipitation and Western blots. We also evaluated the role of UBE4B in neuroblastoma differentiation, cell death, and responses to chemotherapy and HDAC inhibition using continuous live cell imaging and Western blots. We determined that UBE4B directly interacts with ITCH/AIP4 in the last WW domain of ITCH and we also determined that Ku70 directly interacts with ITCH in the three first WW domain of ITCH. We also determined that UBE4B depletion leads to reduced polyubiquitination and degradation of both p53 and Ku70 in neuroblastoma cells. UBE4B depletion also led to reduced apoptosis induced by both HDAC inhibition with vorinostat and chemotherapy treatment with carboplatin and doxorubicin via stabilizaton of the ITCH target protein c-FLIPL leading inhibition of caspase 8 activation. Our results have identified novel interactions and novel targets for UBE4B ubiquitin ligase activity and a direct role of the UBE4B-ITCH complex in HDAC inhibitor and DNA-damage induced cell death, and suggest that the association of UBE4B expression with neuroblastoma patient outcomes may be mediated by effects on responses of patient tumors to chemotherapy.

Citation Format: Christophe Le Clorennec, Jacqueline Lesperance, Yuchen Huo, Peter E. Zage. The roles of E3/E4 ubiquitin ligase UBE4B and its targets p53 and Ku70 in neuroblastoma cell death and differentiation [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 4871.
Abstract 4877: Discovery of paralog-specific SUMO1 and SUMO2 functions using human cancer knockout cells
Authors: Bouchard Danielle , Yang Wei-Chih , Matunis Michael J.
Published: September 2020

Small ubiquitin-related modifiers (SUMOs) are ∼100-amino acid polypeptides that function as posttranslational protein modifications and thereby regulate many essential cellular processes, many of which are relevant to cancer. Vertebrates express five SUMO paralogs (SUMO1-5), of which, SUMO1 and SUMO2 are the most widely studied. Biochemical and in vivo studies suggest that SUMO1 and SUMO2 have non-redundant functions, however, the full range of their distinct activities are not fully understood. Understanding these unqiue functions and their molecular bases could lead to more precise therapeutic interventions that target the SUMO pathway. To help define SUMO paralog-specific functions, we used CRISPR-Cas9 to knockout (KO) individual SUMO genes in osteosarcoma U2OS cells. We observed that SUMO2 KO cells were unique in exhibiting a change in cell morphology, changing from epithelial-like to fibroblast-like, thus revealing a potential role for SUMO2 in affecting cell shape. To test the hypothesis that the observed change in morphology is driven in part by changes in gene expression, we sequenced the transcriptome of the parental and SUMO KO cell lines. In alignment with the observed change in SUMO2 KO cell shape, differentially expressed genes (DEGs) involved in the extracellular matrix, basement membrane and epithelial to mesenchymal transition were uniquely enriched in SUMO2 KO cells. Further consistent with SUMO1 and SUMO2 having unique effects on transcription, we found a significantly greater number of unique DEGs in SUMO2 KO cells as compared to SUMO1 KO cells. Furthermore, an overwhelming number of DEGs were down-regulated in the SUMO1 KO cells, suggesting a specific role for SUMO1 in activating, or maintaining, gene expression. In contrast, DEGs in SUMO2 KOs were equally up and down regulated. Collectively, our findings demonstrate that SUMO1 and SUMO2 have unique biological roles and we look forward to better characterizing these paralog-specific functions in future studies.

Citation Format: Danielle Bouchard, Wei-Chih Yang, Michael J. Matunis. Discovery of paralog-specific SUMO1 and SUMO2 functions using human cancer knockout 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 4877.
Abstract 4884: To Investigate the mechanism and function of nucleobindin-2 in human hepatoma cells
Authors: Hung Jui-Hsiang , Tsao Hsiu-Ping , Li Ren-Hao , Li Chih-Han , Chang Hut-Min
Published: September 2020

Hepatocellular carcinoma is a common malignancy affecting approximately one million people worldwide annually. Recent studies have shown that appetite-associated nucleobindin-2 (NUCB-2) is over-expressed in clinical tumor samples such as gastric cancer, prostate cancer, renal cell carcinoma, endometrial carcinoma cells, colon cancer and breast cancer. The survival rate of high-level expression of NUCB-2 in cancer patients was lower than cancer patients with low-level expression of NUCB-2. In addition, overexpression of NUCB-2 was observed in liver cancer by using bioinformatics analysis. The liver cancer patients with NUCB-2 overexpression are associated with poor prognosis. However, the effect of NUCB-2 overexpression on tumor cells is still unclear. In this project, we evaluated the novel role and mechanisms of NUCB-2 overexpression in hepatoma cells. Our results indicated NUCB-2 mRNA expression level was induced by ER stress as determined with RT-PCR and real-time PCR, and induction of NUCB-2 protein level was significantly increased by ER stress. Increased ER stress by pre-S2Δ protein was significantly enhanced NUCB-2 expression. We also observed that the overexpression of NUCB-2 was corrected with ER stress in human hepatocellular carcinoma tumor samples. Consequently, this mechanistic research may provide a molecular basis to develop a biomarker and novel cancer chemotherapeutic agent for high level expression of NUCB-2 in cancer patients.

keywords:Endoplasmic reticulum stress, Nucleobindin-2, real-time PCR, RT-PCR, HBV large surface mutant protein pre-S2Δ, Hepatocellular carcinoma, Huh-7, HepG2, MCF-7, Tunicamycin, Brefeldin A.

Citation Format: Jui-Hsiang Hung, Hsiu-Ping Tsao, Ren-Hao Li, Chih-Han Li, Hut-Min Chang. To Investigate the mechanism and function of nucleobindin-2 in human hepatoma 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 4884.
Abstract 4891: Hepatitis B virus remodels host protein interaction networks to generate distinct cellular dependencies
Authors: Gordan John D. , Pitea Adriana , Eckhardt Manon , Jang Gwendolyn , Turnham Rigney E. , Choi Alex L M. , Von Dollen John , Lim Huat C. , Thayer Elizabeth F. , Kelley R. Katie , Swaney Danielle L. , Zhang Wei , Theis Fabian J. , Ideker Trey , Krogan Nevan J.
Published: September 2020

Hepatocellular carcinoma (HCC) is the second leading cause of cancer death worldwide. Advanced HCC has proven particularly difficult to treat because of a scarcity of clear genetic drivers of cancer progression; thus, there are currently no predictive markers that guide HCC therapy. HCC arises in the context of co-morbid hepatitis due to hepatitis B virus (HBV), hepatitis C (HCV) or fatty liver disease. We hypothesize that protein-protein interactions (PPIs) between viral proteins and HCC genes may contribute to tumor initiation and maintenance. In order to characterize these PPIs, we performed affinity purification - mass spectrometry (APMS), defining 145 HBV/host PPIs including known and novel interacting partners. We next used a network propagation algorithm to identify host genes and protein complexes that were preferentially mutated in the absence of HBV infection. HBV is a small DNA virus, with 4 genes of which only one has enzymatic activity, raising a question as to how HBV interaction modifies host behavior. Using AP-MS of host proteins, we found that the HBV X protein (HBx) remodels multiple host protein complexes through direct interaction. These physical effects on complex components result in distinct biochemical behavior from the CRL4 E3 ubiquitin ligase complex as well as the phosphatase PP2A, as determined through global phosphoproteomics and ubiquitin analysis. We show that this remodeling driven by HBx substantially changes cellular protein turnover and downstream signaling dynamics. We followed this up with assessments of cellular viability and proliferation in response to pharmacological inhibition or CRISPRi-based knockdown of HBx effectors. Our data support a model where HBV proteins alter the components and behavior of key regulatory protein complexes in the cell, altering tumor behavior and raising the possibility of precision therapeutics for HCC.

Citation Format: John D. Gordan, Adriana Pitea, Manon Eckhardt, Gwendolyn Jang, Rigney E. Turnham, Alex L M. Choi, John Von Dollen, Huat C. Lim, Elizabeth F. Thayer, R. Katie Kelley, Danielle L. Swaney, Wei Zhang, Fabian J. Theis, Trey Ideker, Nevan J. Krogan. Hepatitis B virus remodels host protein interaction networks to generate distinct cellular dependencies [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 4891.
Abstract 4915: Nerve growth factor (NGF) facilitates LOX expression and promotes tumor metastasis by inhibiting miR-149-5p in human chondrosarcoma cells
Authors: Louis Anoop Thadevoos , Lin Chih-Yang , Tang Chih-Hsin
Published: September 2020

Chondrosarcomas are malignant bone tumors that commonly produce cartilaginous chondroid matrix and are associated with metastases in the lung, especially in patients with high-grade chondrosarcoma. Neurotrophins such as nerve growth factor (NGF) are recognized as being drivers of neurogenesis during development and regeneration, and their expression in human tumors can stimulate cancer cell growth. Importantly, the infiltration of nerves in the tumor microenvironment actively stimulates cancer cell mitogenesis, invasiveness and metastasis. Until now, it has been unclear as to the potential involvement of NGF in human chondrosarcoma metastasis. This study reports that NGF induces migration and invasion of chondrosarcoma cell lines JJ012 and SW1353 and significantly increasing LOX expression. Significant dose-dependent increases were observed in levels of LOX mRNA and protein expression in both cell lines. In immunohistochemistry analyses, levels of NGF and LOX expression in human chondrosarcoma cartilage correlated with tumor stage and were substantially higher than NGF and LOX expression in normal cartilage. NGF-induced stimulation of metastasis and increases in LOX expression were prevented by pretreatment of the cell lines with PI3K, Akt and mTOR inhibitors, or transfection with small interfering RNAs (siRNAs). Further analysis revealed that NGF induced PI3K/Akt/mTOR signaling and dose-dependently downregulated mature microRNA-149-5p (miR-149-5p), which targets the 3' untranslated region (3'-UTR) of LOX. Furthermore, JJ012 cells overexpressing NGF induced LOX expression and increased cell migration and invasion. In summary, NGF stimulates LOX expression and promotes chondrosarcoma cell migration and invasion by inhibiting miR-149-5p via the PI3K/Akt/mTOR signaling pathway.

Citation Format: Anoop Thadevoos Louis, Chih-Yang Lin, Chih-Hsin Tang. Nerve growth factor (NGF) facilitates LOX expression and promotes tumor metastasis by inhibiting miR-149-5p in human chondrosarcoma 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 4915.
Abstract 4922: NEUROD1 is highly expressed in extensive small cell lung cancer by promoting migration
Authors: Ikematsu Yuki , Tanaka Kentaro , Ando Nobuhisa , Ijichi Kayo , Yoneshima Yasuto , Inoue Hiroyuki , Toyokawa Goji , Tagawa Tetsuzo , Nakanishi Yoichi , Okamoto Isamu
Published: September 2020

Introduction:Small cell lung cancer (SCLC) exhibits high-grade neuroendocrine features and recent genomic analyses revealed that aberrant expression of bacic helix loop helix transcription factors such as neurogenic differentiation 1 (NEUROD1) as well as achaete-scute homolog 1(ASCL1) have also been observed in SCLC. NEUROD1 plays important role for the development, maturation in several nerve system and formation of distal lung of neuroendocrine morphology. Previous report showed that mRNA expression as well as target genes of NEUROD1 are largely distinct from those of ASCL1 in human SCLC cell lines, however, protein level and expression patterns of these transcription factors in SCLC clinical tumor samples as well as its relation to clinicopathological characteristics have not been fully elucidated.

Method: We retrospectively analyzed specimens from 95 SCLC patients between June 1988 and December 2017, and immunohistochemical (IHC) staining of NEUROD1 as well as ASCL1 was performed. Furthermore, we examined the effect of gain or loss of NEUROD1 function on proliferation and migration by transfection in SCLC cell lines.

Result:Dual high staining score of ASCL1 and NEUROD1 was present in SCLC samples (32/95: 34%) and IHC score of NEUROD1 was higher in extensive disease (ED) samples than those in limited disease (LD) samples (median score 160 vs 80 ; P = 0.0389).Overall survival did not significantly differ between SCLC patients with high or low score of NEUROD1 or ASCL1. Next, our vitro data showed that upregulation of NEUROD1 in SCLC cell lines promoted transwell migration whereas downregulation of NEUROD1 reduced cell migration.

Conclusion: Our results from clinical and in vitro experimental data collectively indicate that NEUROD1 expression increases in extensive SCLC by promoting migration and metastasis of SCLC cells.

Citation Format: Yuki Ikematsu, Kentaro Tanaka, Nobuhisa Ando, Kayo Ijichi, Yasuto Yoneshima, Hiroyuki Inoue, Goji Toyokawa, Tetsuzo Tagawa, Yoichi Nakanishi, Isamu Okamoto. NEUROD1 is highly expressed in extensive small cell lung cancer by promoting migration [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 4922.
Abstract 4932: mTORC1 regulates FAK phosphorylation
Authors: Li Lin , Luo Yan , Liu Lei , Huang Shile
Published: September 2020

The mammalian target of rapamycin (mTOR) functions as two complexes, mTORC1 and mTORC2, regulating cell growth, proliferation, survival, and motility. Previous studies have shown that mTORC2 controls cytoskeleton and cell motility. Recently, we have demonstrated that both mTORC1 and mTORC2 participate in the regulation of cell motility. Further studies have shown that rapamycin suppresses IGF-I induced cell motility by inhibiting S6K1-mediated phosphorylation of focal adhesion kinase (FAK), protein phosphatase 2A (PP2A)-mediated phosphorylation of Erk1/2, as well as expression and activities of the small GTPases (RhoA, Cdc42 and Rac1). As FAK plays a pivotal role in the regulation of cell motility, this study was set to determine how mTORC1 regulates the phosphorylation of FAK. By co-immunoprecipitation (co-IP) and immunoblotting (IB), we failed to detect a physical interaction between FAK and S6K1, suggesting that S6K1 may regulate the phosphorylation of FAK indirectly. Besides, knockdown of protein phosphatase 2A (PP2A) enhanced the phosphorylation of FAK and conferred resistance to rapamycin inhibition of IGF-I-stimulated phosphorylation of FAK. However, the co-IP/IB results revealed no direct interaction between FAK and PP2A either. Of interest, knockdown of protein phosphatase 5 (PP5) suppressed IGF-I stimulated cell motility, while knockdown of ATG13 increased IGF-I stimulated cell motility, suggesting that PP5 positively regulates cell motility, whereas ATG13 negatively regulates cell motility. It is well known that PP5 is an anti-apoptotic phosphatase, negatively regulating the ASK1-JNK cascade, and mTORC1 regulates autophagy through the ULK1/2-ATG13-FIP200 complex. Further research will determine how mTORC1 regulates cell motility in part by regulating the phosphorylation of FAK via S6K1, PP2A, PP5 and ATG13. (Supported by the Feist-Weiller Cancer Center, LSU Health Sciences Center, Shreveport, LA, USA.)

Citation Format: Lin Li, Yan Luo, Lei Liu, Shile Huang. mTORC1 regulates FAK phosphorylation [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 4932.
Abstract 4935: T-DM1-resistant cells gain high invasive activity via EGFR and integrin cooperated pathways
Authors: Endo Yukinori , Olson Zachary , Wu Wen Jin
Published: September 2020

Ado-trastuzumab emtansine (T-DM1) is an antibody-drug conjugate developed to treat trastuzumab-resistant cancer. Despite initial favorable outcomes, most patients eventually cease to respond by developing acquired resistance to T-DM1. Currently, there is no targeted therapy to treat T-DM1-resistant cancer. To explore novel therapeutic targets to overcome T-DM1 resistance, we generated T-DM1-resistant cells using trastuzumab-resistant JIMT1 cells. We found that the loss of human epidermal growth factor receptor 2 (HER2) confers T-DM1 resistance, which in turn activates a compensatory mechanism that increases epidermal growth factor receptor (EGFR) expression. Upregulation of EGFR increases the protein levels of α5β1 integrin, resulting in enhanced motility and invasion of T-DM1-resistant cells. However, silencing β1 integrin expression by siRNA or β1 integrin blockage induced by an inhibitory antibody, MAB 13, significantly increases invasion of T-DM1-resistant cells. The discovery of functional cooperation between EGFR and integrin in regulating cell growth and invasion provides an opportunity to develop novel therapeutic strategy by dual-targeting EGFR and specific integrin to overcome T-DM1 resistance. We now try to identify downstream molecules involved in the increased invasion using next generation sequencing (NGS) technology in β1 integrin-deficient T-DM1 resistant cells.

Citation Format: Yukinori Endo, Zachary Olson, Wen Jin Wu. T-DM1-resistant cells gain high invasive activity via EGFR and integrin cooperated pathways [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 4935.
Abstract 4949: Essential role of arachidonic acid metabolism in prostate cancer stem cell survival
Authors: Monga Jitender , Carney Brendan , Patil Sejal , Ghosh Jagadananda
Published: September 2020

Background: Cancer stem cells (CSC) are rare group of neoplastic cells with features that include, self-renewal, pluripotency and tumorigenicity, and are thought to be responsible for tumor recurrence and metastasis. Thus, a lot of excitement surrounds the idea of going right to the root of the tumor and target the stem cells to eradicate cancer permanently. A hierarchically organized CSC model in prostate cancer is evident with CD44 (+), CD133 (+) subpopulations isolated from human prostate tumors. While most therapies are directed to the fast-growing tumor mass made of differentiated cells, they cannot target the relatively slow-dividing and resistant cancer stem cells, which may underlie as a mechanism of why the cancer is not permanently eradicated with standard chemotherapy. Thus, an important challenge is to find measures to eliminate the CSCs, which by definition, will stop tumor growth and prevent recurrence. However, knowledge about molecular features critical for prostate cancer stem cells (PCSC) is meager. Thus, we wanted to examine the role of arachidonate 5-lipoxygenase (ALOX5) which plays critical role in the survival of cancer (but not of normal) cells, in the biology of PCSC.

Methods: Using an established, clinically-relevant in vitro model of human PCSCs which express both pluripotency markers (Nanog, c-Myc, Sox2) and tumor-progenitor markers (CD44, CD133, ALDH1), we interrogated the role of a fatty acid metabolic pathway in stem cell vulnerability. Especially, our goal was to characterize the effects of targeting ALOX5 to see whether it could be a new target to eliminate PCSCs.

Results: Inhibition of ALOX5 by shRNA or MK591 dramatically decreased the protein levels of Nanog and c-Myc, suggesting a role for ALOX5 in prostate cancer stemness. Moreover, ALOX5 inhibition was found to selectively kill PCSCs (but not normal stem cells) by inducing apoptosis via inhibition of survivin, cyclin D1 and CDK4, and activation of c-Jun N-terminal Kinase and caspase 3, suggesting an essential role of ALOX5 in the survival of PCSC. Interestingly, non-cancer parenchyma body cells, including normal stem cells, do not express ALOX5 (presumably because of promoter hypermethylation) and are not affected by ALOX5 inhibitors.

Conclusion: Our findings indicate that ALOX5 plays a causal role in maintaining prostate cancer stemness by regulating Nanog and c-Myc oncogenes, and suggest that an effective, long-lasting therapy of prostate cancer may be possible by targeting ALOX5 to kill and eliminate PCSCs to block prostate tumor growth and prevent recurrence.

Citation Format: Jitender Monga, Brendan Carney, Sejal Patil, Jagadananda Ghosh. Essential role of arachidonic acid metabolism in prostate cancer stem cell survival [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 4949.
Abstract 4956: PrPc depletion in glioblastoma patient derived xenograft cells by CRISPR/Cas9
Authors: Escobar Maria Isabel Melo , Coelho Barbara Paranhos , Prado Mariana Brandão , Iglesia Rebeca , Lopes Marilene Hohmuth
Published: September 2020

Glioblastoma (GBM), the most common primary malignant brain tumor, exhibits a highly proliferative cell subpopulation called Glioblastoma stem-like cells (GSCs), presumed to contribute to the aggressive behavior of GBM. Our group has shown that the Prion Protein (PrPC), a cell surface glycoprotein and regulator of several stem cell functions, is enriched in GSCs and co-expresses with conventional GSCs markers. PrPC has been proposed as a scaffold protein, able to govern the assembly of multi-component complexes in the cell membrane. Integrin α6 (ITGA6), a cell surface laminin receptor, is enriched in GSCs as well, and exhibits relevant effects on GSCs biology, related to self-renewal and migration. To investigate a potential association between PrPC and ITGA6, we will perform loss-of-function experiments in GSCs via siRNA and CRISPR/Cas9 to characterize these cell surface molecules expression and their effect on GSCs proliferation, migration and invasion. Preliminary results show that depletion of PrPC perturbates ITGA6 labeling on plasma membrane of GSC. Thus far, cells from patient-derived xenografts (GBM33-PDX2) were transfected a construct for PrPC knockout by CRISPR/Cas9 via Nucleofector Technology. We found around 60% GFP-positive cells via flow cytometry for the CRISPR pool and we are currently isolating clones to confirm PrPC expression. Remarkably, the colonies exhibited distinct morphologies such as neural-like, epithelial-like and stem cell-like, indicating the heterogenous nature of this tumor. Western blotting assays will be performed to assess PrPC and ITGA6 expression in both commercial cell line U87 and GBM33-PDX2. The characterization of PrPC-ITGA6 association might provide insight into GSCs behavior and new anti-GBM therapeutic strategies.

Citation Format: Maria Isabel Melo Escobar, Barbara Paranhos Coelho, Mariana Brandão Prado, Rebeca Iglesia, Marilene Hohmuth Lopes. PrPc depletion in glioblastoma patient derived xenograft cells by CRISPR/Cas9 [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 4956.
Abstract 4968: Blockade of p38 kinase impedes the mobilization of myeloid populations that promote breast cancer metastasis
Authors: Zonneville Justin , Colligan Sean , Wallace Paul , Abrams Scott I. , Bakin Andrei V.
Published: September 2020

Patients with metastatic breast cancer (MBC) have limited therapeutic options and novel treatments are critically needed. Prior studies demonstrated that the p38 kinase (p38) promotes MBC, while the mechanisms remained unknown. Here, we provide evidence for a novel mechanism by which p38 promotes metastasis. Using triple-negative breast cancer models, we showed that a selective inhibitor of p38 (p38i) significantly reduced tumor growth, angiogenesis, and lung metastasis. Importantly, p38i decreased the accumulation of myeloid populations, namely myeloid-derived suppressor cells (MDSCs) and CD163+ tumor-associated macrophages (TAMs). p38 controlled the expression of tumor-derived chemokines/cytokines that facilitated the recruitment of pro-tumor myeloid populations. Depletion of MDSCs was accompanied by reduced TAM infiltration and phenocopied the anti-metastatic effects of p38i. Reciprocally, p38i increased tumor infiltration by cytotoxic CD8+ T cells. Metadata showed that a reduced CD163+/CD8+ expression ratio correlates with metastasis-free survival in breast cancer, suggesting that targeting p38 may improve clinical outcomes. This study highlights a novel p38-driven mechanism as a therapeutic target in MBC.

Citation Format: Justin Zonneville, Sean Colligan, Paul Wallace, Scott I. Abrams, Andrei V. Bakin. Blockade of p38 kinase impedes the mobilization of myeloid populations that promote breast cancer metastasis [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 4968.
Abstract 4974: Q702, selective Axl/Mer/CSF1R triple kinase inhibitor enhance the activity of immune checkpoint inhibitor by alteration of immunosuppressive tumor microenvironment
Authors: Yang Yeong-In , Kang Hwankyu , Park Dongsik , Jeon Yeejin , Kim Jeongjun , Choi Baejung , Kim Jaeseung , Nam Kiyean
Published: September 2020

Central to the Immune Checkpoint Inhibitors (ICI) mechanism is the activation and infiltration of the cytotoxic CD8+ T cells following tumor-antigen recognition. However, the effector functions of the CD8+ T cells can be highly compromised by immunosuppressive tumor microenvironment (TME). There is the growing appreciation of the potential contributions of innate immune modulation to antitumor immunity which suggests the need for the novel strategies to elicit a more efficient/robust immune response against malignant cells. Axl/Mer/CSF1R play vital roles in the immunosuppressive TME by increasing regulatory T cell, M2 macrophage, myeloid-derived suppression cell (MDSC) and suppressing antigen presentation. Previously, we have reported a novel selective Axl/Mer/CSF1R kinase inhibitor, Q702 and demonstrated tumor regression by alteration of the immunosuppressive TME. We present the anti-tumor efficacy and immune mechanism of Q702 in combination with anti-PD-1 in the various syngeneic models. The combination of Q702 and anti-PD-1 demonstrated tumor regression through enhanced tumor infiltration of effector immune cells, altered cytokine expression promoting promoted tumor specific antigen presentation and unregulated PD-L1 expression on tumor cells. Additionally, the Q702 alone induced tumor regression in the anti-PD-1 non-responding syngeneic models. These results suggest that the innate immune modulation in the TME by Q702 in combination with an ICI can be through synergistic mechanisms of action. In addition, the data suggests that Q702 could potentially broaden the patient population that benefits from ICI-based immunotherapy. Q702 is progressing through IND-enabling studies to support initiation of a Phase 1 oncology clinical study in early 2020.

Citation Format: Yeong-In Yang, Hwankyu Kang, Dongsik Park, Yeejin Jeon, Jeongjun Kim, Baejung Choi, Jaeseung Kim, Kiyean Nam. Q702, selective Axl/Mer/CSF1R triple kinase inhibitor enhance the activity of immune checkpoint inhibitor by alteration of immunosuppressive tumor microenvironment [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 4974.
Abstract 4979: Prevalence of proliferating CD8+cells in normal lymphatic tissues, inflammation and cancer
Authors: Lennartz Maximilian , Abu-Hashem Raed , Blessin Niclas Christian , Mandelkow Tim , Bady Elena , Simon Ronald , Hube-Magg Claudia , Sauter Guido , Fraune Christoph , Möller Katharina , Clauditz Till S. , Luebke Andreas M. , Büscheck Franziska
Published: September 2020

Expansion of CD8+ cytotoxic T lymphocytes is a prerequisite for anti-cancer immune activity. In the era of immune checkpoint therapy, profound knowledge of the dynamics of CD8+ has regained considerable interest. However, systematically acquired data on CD8+ proliferation in large sets of normal and diseased tissues are sparse. Here, we applied multiplex fluorescence immunohistochemistry to conventional large sections and tissue microarrays in order to quantitate Ki67+CD8+ cells in >20 different compartments of normal lymphoid tissues, 7 types of inflammatory diseases and 785 cancers. In most normal lymphoid tissues (tonsil, lymph node, thymus, Peyer's patches, spleen, colon, appendix) the percentage of Ki67+CD8+ cells typically did not exceed 3%. The percentage of Ki67+CD8+ cells was markedly higher (45%) in the immune-active cortex of the thymus, however. In inflammatory conditions (including Hashimoto thyroiditis, Lichen sclerosus of the penis, sarcoidosis, sialadenitis, IgG4 pancreatitis, Crohn's disease and eczema), the percentage of Ki67+CD8+ cells was much more variable and often sharply higher than in normal tissues. It ranged from 0.5% in one patient with sialadenitis to 19% in the intraepithelial compartment of Crohn's disease. In 765 colorectal cancers, the fraction of Ki67 positive CD8+ cytotoxic T cells ranged from 0 to 100% (mean: 20.6%). A high fraction of Ki67+CD8+ cells was significantly associated with microsatellite instability (p<0.0001), low pT stage (p<0.0001) and absence of nodal metastases (p=0.0005). In summary, our data show a variable increase of the fraction of proliferating CD8+ T cells in cancers and in inflammatory diseases as compared to healthy secondary lymphoid organs. The striking link with microsatellite instability and unfavorable tumor features suggest a potential clinical utility of assessing Ki67+CD8+ in colorectal cancer.

Citation Format: Maximilian Lennartz, Raed Abu-Hashem, Niclas Christian Blessin, Tim Mandelkow, Elena Bady, Ronald Simon, Claudia Hube-Magg, Guido Sauter, Christoph Fraune, Katharina Möller, Till S. Clauditz, Andreas M. Luebke, Franziska Büscheck. Prevalence of proliferating CD8+cells in normal lymphatic tissues, inflammation and 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 4979.
Abstract 4985: PD-L1 and diacylglycerol kinase alpha (DGKA) expression in melanoma, nsclc and bladder cancers
Authors: Gonzalez Rachel M. , Qu Tianli , Hu Xiaomin , Ren Qi , Zhang Qiankun , Guo Zhaoying , Kim Young-Hwan , Wang Yiran , Liu Xuan , Fu Wei
Published: September 2020

Diacylglycerol kinase alpha (DGKA) functions as lipid kinase to phosphorylate the lipid diacylglycerol (DAG). DGKA has been characterized to negative regulate the Ras-MAPK pathway in T lymphocytes. A 2018 study using crispr knockouts of DGKA have shown the anti tumor activity of T-cells continue to increase in the presence of PD-L1 positive tumor cells. Little is known about the expression patterns of DGKA in human cancer. In our previous study, we explore the tumor micro environment with a number of immune cell markers such as CD3, CD8A, CD20, CD68, TIM3 and LAG3 with PD-L1 expression in melanoma, NSCLC, and bladder cancer and found a few unique signatures for these cancers. In this study, we evaluate expression pattern of DGKA in various cancers by immunohistochemistry to elucidate the signature profile of DGKA expression with PD-L1 and other markers in the PD-L1 tumor micro environment.

Citation Format: Rachel M. Gonzalez, Tianli Qu, Xiaomin Hu, Qi Ren, Qiankun Zhang, Zhaoying Guo, Young-Hwan Kim, Yiran Wang, Xuan Liu, Wei Fu. PD-L1 and diacylglycerol kinase alpha (DGKA) expression in melanoma, nsclc and bladder cancers [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 4985.
Abstract 4989: Introduction of a robust workflow for the whole-side acquisition and co-registration of multiplex immunofluorescence tissue images for analysis of a 9-color, 8-marker immunophenotyping assay
Authors: Hellebust Anne E. , Teplitz Kyla , Patel Katir K. , Sharma Karan , Bares Amanda , Manesse Mael , Burton Mark , Phillips Bonnie , Downing Sean , Lillard Kate
Published: September 2020

Background: Profiling multiple cell types in tissue samples requires more markers than can be supported by conventional approaches to immunohistochemistry. InSituPlex® multiplex immunofluorescence (mIF) technology enables biomarker multiplexing in tissue samples with an automated workflow to support discovery of immune cell signatures in the tumor immune microenvironment (TiME). Here we have designed and tested an 8-plex immunophenotyping panel using the InSituPlex approach to detect and classify T cells, macrophages, and tumor cells in non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) FFPE tissue.

Methods: An 8-plex UltiMapper™ immunofluorescence was developed to stain FFPE tissue, including colorectal cancer, human tonsil, and non-small cell lung cancer. Each sample was stained for the following 8 markers: CD3, CD4, CD8, CD68, FoxP3, PD-1, PD-L1, and pan-CK. Whole-slide imaging was performed in two imaging rounds on the RareCyte CyteFinder II HT slide scanner, and same-slide image alignment and phenotypic analysis was performed using the HALO Highplex FL module from Indica Labs.

Results: Co-registration of two 4-plex imaging rounds and fusing into a single image file allowed for the seamless cell phenotyping of markers across imaging rounds. The 8-plex assay and workflow is compatible across multiple tumor sample types. The samples were characterized based on binned expression and co-expression of biomarkers. The following major cell phenotypes were detected: PD-L1 checkpoint expression, T cells (CD3+), cytotoxic T cells (CD3+/CD8+), T-helper cells (CD3+/CD4+), regulatory T-cells (CD3+/CD4+/FoxP3+), exhausted T cells (CD3+/PD-1+), macrophages (CD68+), and tumor cells (pan-CK+).

Conclusions: The 8-plex assay workflow involving InSituPlex mIF, CyteFinder HT scanner, and HALO analysis supports the ability to measure biomarkers in situ in streamlined and scalable approach.

Citation Format: Anne E. Hellebust, Kyla Teplitz, Katir K. Patel, Karan Sharma, Amanda Bares, Mael Manesse, Mark Burton, Bonnie Phillips, Sean Downing, Kate Lillard. Introduction of a robust workflow for the whole-side acquisition and co-registration of multiplex immunofluorescence tissue images for analysis of a 9-color, 8-marker immunophenotyping assay [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 4989.
Abstract 4992: In vitro exhausted T cell assay for immuno-oncology candidates
Authors: Osmani Amin , Lamy Juliette , Janss Thibaut , Giltaire Séverine , Pattijn Sofie , Schockaert Jana
Published: September 2020

The need to better understand the tumour microenvironment (TME) dictates the characterization of the cell types involved, the roles they play and how they respond to treatment. To develop better cancer immunotherapies, in vitro primary immune cell bioassays offer an early assessment of their effects on the various players of the TME. Here we describe the development of an exhausted T cell assay and demonstrate its potential in identifying candidates that can reverse exhaustion. Exhausted T cells were first discovered in LCMV virus infections where chronic stimulation due to remaining virus antigens leads to T cells that stop responding to stimulation and lose their functionality (cytokine secretion and cytotoxic activity). The same phenotype was afterwards found in the tumor microenvironment where a lot of T cells were found to be “anergic”. Reversing their loss of functionality as a therapeutic strategy has made the characterization of these exhausted T cells a priority. Their phenotype is currently defined by an increase in the expression of a specific set of cell surface markers such as PD-1, TIM-3 and LAG-3. As exhaustion builds, the expression of these marker increases while T cell functionality decreases. The loss of functionality is defined by a reduction in cytokine secretion, proliferation and cytotoxic activity in the presence of an antigen. To evaluate this phenotype in more depth, transcription factors can also be monitored. For example, BLIMP-1 and BATF, which play a role in PD-1 expression, are shown to increase following exhaustion. Using primary immune cells, in vitro bioassays were developed to better screen the potential effect of new therapeutics on exhausted T cells. Their ability to reverse the exhausted phenotype and facilitate the anti-tumour immune response can as a result be assessed early in the drug development process. These experiments help advance our understanding of the tumour microenvironment and optimize the therapeutic effects of new drugs, design better clinical trials and ultimately discover relevant biomarkers.

Citation Format: Amin Osmani, Juliette Lamy, Thibaut Janss, Séverine Giltaire, Sofie Pattijn, Jana Schockaert. In vitro exhausted T cell assay for immuno-oncology candidates [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 4992.
Abstract 4998: Integrative analyses in pediatric osteosarcomas to understand the role of GD2 associated to activated mTor for macrophage targeting
Authors: Pierrevelcin Marina , Lelong-Rebel Isabelle , Fuchs Quentin , Dontenwill Monique , Entz-Werle Natacha
Published: September 2020

Osteosarcoma is the most common primary malignant bone tumor in children and young adults. Its immune environment was described as predominantly involving M2 macrophages or osteoclasts. Previous studies were observing ganglioside GD2 expression on the osteosarcoma cell surface. Even more and more publications are deciphering the role of hypoxic biomarkers and its metabolic cell impact. Nevertheless, no links were, for instance, described clearly between mTor-HIF1 pathway and the macrophages in osteosarcomas through GD2 targeting. The aim of the study was to understand the role of all those molecular partners in osteosarcoma models. Material and methods: A cohort of 35 patients was explored to determine the expression by immunohistochemistry, as well as semi-quantitative PCR of several hypoxic and macrophages' biomarkers. Additionally, in 2D and 3D in vitro osteosarcoma models, analyses using metabolomics, western blotting, immunofluorescent imaging and RNA sequencing were performed to understand functional interactions between patient-derived osteosarcoma cells and the M2 macrophages. Endly, drug screening studies were testing 3D models (integrating matrix and hypoxic microenvironment) to assess efficacy of combinations using dinutuximab, mTor and HIF inhibitors. Results: Osteosarcoma samples are highly expressing CD163 and CD68 macrophage markers, as well as activated mTORC1 and GD2. Hypoxic biomarkers, when overexpressed, were statistically linked to a worst patient outcome. For macrophages and GD2 markers, a low expression was mostly linked to metastatic disease or poor response to chemotherapy. Specific metabolomic changes through glutaminolysis were frequently present and associated to a transcriptomic signature combining metabolism changes and specific immune macrophage environment. The targeting by dinutuximab associated to hypoxia inhibition was having a clear effect on osteosarcoma cell proliferation decrease, when associated to macrophages in 3D models mimicking osteosarcoma tumors. Conclusion: Macrophage immune environment is a key component of pediatric osteosarcomas and can be targeted in association with hypoxia inhibitors in 3D models recreating the osteosarcoma immune environment and bone matrix.

Citation Format: Marina Pierrevelcin, Isabelle Lelong-Rebel, Quentin Fuchs, Monique Dontenwill, Natacha Entz-Werle. Integrative analyses in pediatric osteosarcomas to understand the role of GD2 associated to activated mTor for macrophage targeting [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 4998.
Abstract 5027: The mu-opioid receptor antagonist methylnaltrexone can decrease tumorigenesis in head and neck squamous cell carcinoma
Authors: Gorur Aysegul , Patino Miguel , Shi Ted , Takahashi Hideaki , Myers Jeffrey N. , Cata Juan P.
Published: September 2020

Head and neck cancer (HNC) is the sixth leading cause of cancer in the world, and the third most common neoplasia in developing countries [1]. Patients with HNC may suffer significant pain, for which opioids are commonly given. However, recent reports suggest that opioids may promote cancer progression by action on the mu-opioid receptor (MOR) [2, 3]. The goal of this research was to test the hypothesis that the MOR antagonist methylnaltrexone (MTNX) can decrease tumorigenesis in HN squamous cell carcinoma (SCC). First, we confirmed via RNAseq and western blot analyses that FaDu cells (a HNSCC line) express MOR. Then, we performed cyclic adenosine monophosphate (cAMP) activation enzyme-linked immunosorbent assay to evaluate the activity of MOR after activation with [D-Ala2N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO), a selective MOR agonist. Short term (1hr) treatment with DAMGO decreased cAMP levels, suggesting full activity of the receptor. We conducted similar cAMP activity assays using MNTX. Our dose-dependent (10nM-1µM) experiments revealed that 1hr exposure of cells to MTNX inhibited cAMP activity, but not after 48 hrs of treatment, consistent with an inverse or partial agonist effect at low doses. To assess the impact of our findings on FaDu cell behaviors, we conducted in vitro proliferation, colony formation, and migration assays. MTNX at different concentrations (0.1nM, 1nM, 10nM, 100nM and 1µM) inhibited FaDu cell proliferative and metastatic cell behaviors (p<0.05). To translate our results to in vivo, we inoculated the tongue of athymic nude mice with FaDu cells (200,000 cells/30 mL). Starting the day of cell inoculation, we randomly treated the animals with MNTX (subcutaneously, 1µg/kg, 10 µg/kg, and 100µg/kg) versus placebo (saline only) for 17 consecutive days. In vivo experiments demonstrated that administration of MTNX at three different concentrations delayed tumor progression and reduced tumor growth and formation compared to placebo (p<0.0001). Overall, our results suggest that MTNX inhibits tumorigenesis of HNSCC, possibly by acting on MOR.

1. Siegel, R.L. et al, Cancer statistics, 2018. CA Cancer J Clin, 2018. 68(1): p. 7-30.

2. Lennon, F.E., et al, The mu-opioid receptor in cancer progression: is there a direct effect? Anesthesiology, 2012. 116(4): p. 940-5.

3. Patino, M.A., et al., The impact of intraoperative opioid use on survival after oral cancer surgery. Oral Oncol, 2017. 74: p. 1-7.

Citation Format: Aysegul Gorur, Miguel Patino, Ted Shi, Hideaki Takahashi, Jeffrey N. Myers, Juan P. Cata. The mu-opioid receptor antagonist methylnaltrexone can decrease tumorigenesis in head and neck squamous cell carcinoma [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 5027.