bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5585: RealTVac - a novel strategy to treat advanced, late-stage tumors with real-time tumor vaccination
Authors: Jachimczak Piotr , Mitsch Andreas , Aigner Achim
Published: September 2020

Novel anti-cancer immune therapeutic strategies, like various new antibody formats and/or tumor vaccines, show promising results in patients. However, due to the “immune-escape phenomenon” driven by tumor-secreted Transforming Growth Factor-beta (TGF-beta), the host immune system of cancer bearing patients frequently fails to control tumor re-growth. Real-Time Tumor Vaccincation (RealTVac) approach aims at avoiding this “immune-escape phenomenon” by intratumoral inhibition of active TGF-beta isotypes while simultanously and synergistically inducing an efficient immune response by a highly potent combination of immune stimulating factors.

The synergistic effects of the proposed combination of a TGF-beta inhibitor with immunostimulating cytokines upon human immune cell activation are demonstrated in in-vitro experiments. Both, immune cell proliferation and tumor cell cytotoxicity were significantly enhanced. Initial experiments in a syngenic B16 melanoma xenograft model in immunocompetent mice indicate that Real-Time Tumor Vaccination of established subcutaneous B16 tumor xenografts reduced local tumor growth compared to untreated controls.

To summarize, the goal of the local application of RealTVac is to allow the host immune system to scan in real-time all Tumor Associated Antigens (TAAs) being currently expressed by those tumor cells that are exposed to sufficient TGF-beta inhibition. The repetitive administration of the RealTVac approach is furthermore expected to provide a continuous update of the immune response, reflecting the changing pattern of currently expressed TAAs related to the dynamic intratumoral heterogeneity in malignant tumors. Thus, intratumoral immunization as applied for RealTVac may create a new paradigm for cancer therapy in the future.

Citation Format: Piotr Jachimczak, Andreas Mitsch, Achim Aigner. RealTVac - a novel strategy to treat advanced, late-stage tumors with real-time tumor vaccination [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 5585.
Abstract 5651: JNJ-61186372, an Fc enhanced EGFR/cMet bispecific antibody, mediates EGFR and cMet downmodulation and therapeutic efficacy preclinically through monocyte / macrophage mediated trogocytosis
Authors: Vijayaraghavan Smruthi , Lipfert Lorraine , Bushey Barbara , Chevalier Kristen , Henley Benjamin , Lenhart Ryan , Beqiri Marilda , Millar Hillary J. , Packman Kathryn , Lorenzi Matthew V. , Laquerre Sylvie , Moores Sheri
Published: September 2020

Small molecule inhibitors targeting EGFR are now standard of care in NSCLC patients harboring EGFR mutations, but acquired resistance invariably develops through secondary mutations within EGFR and/or through activation of compensatory pathways such as cMet. JNJ-61186372 (JNJ-372) is an anti-EGFR and cMet bispecific antibody with enhanced binding to immune cell Fcγ receptors, designed to target tumors with activated EGFR and cMet signaling through a distinct mechanism of action. Ongoing first-in-human study in patients with advanced, treatment refractory EGFR mutant NSCLC revealed JNJ-372 to have clinical activity in patients with diverse EGFR-mutated NSCLC, including Exon 20 mutations, TKI resistance mutations (T790M, C797S), and resistance due to MET amplification.

However preclinically, despite potent anti-tumor activity in NSCLC xenograft models, only modest anti-proliferative effects were observed with JNJ-372 in cell lines in vitro. Interestingly, the addition of isolated human immune cells (PBMCs) to the in vitro assays enhanced JNJ-372-mediated EGFR and cMet downregulation, and dose-dependent tumor cell killing. Through depletion or enrichment of individual immune cell types, we demonstrated that monocytes and/or macrophages are necessary for JNJ-372 Fc interaction-mediated EGFR/cMet downmodulation. Depletion of macrophages in mice showed that they are required for JNJ-372 anti-tumor efficacy. Finally, we showed that the down-modulation of EGFR and cMet receptors occurs through monocyte or macrophage-mediated trogocytosis. Collectively, these results demonstrate a novel Fc-dependent mechanism of action for JNJ-372 and support its continued clinical development in patients with aberrant EGFR and cMet signaling.

Citation Format: Smruthi Vijayaraghavan, Lorraine Lipfert, Barbara Bushey, Kristen Chevalier, Benjamin Henley, Ryan Lenhart, Marilda Beqiri, Hillary J. Millar, Kathryn Packman, Matthew V. Lorenzi, Sylvie Laquerre, Sheri Moores. JNJ-61186372, an Fc enhanced EGFR/cMet bispecific antibody, mediates EGFR and cMet downmodulation and therapeutic efficacy preclinically through monocyte / macrophage mediated trogocytosis [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 5651.
Abstract 5653: Discovery of Anti-NKG2A antibody from immunized phage display approach
Authors: Yang Teddy , Gao Jing , Yao Jingyun , Wang Dongxu , Dai Ken
Published: September 2020

The inhibitory receptor NKG2A-CD94 is mainly expressed on NK cells and some T cell and mediates an inhibitory signal through association with a non-classical MHC class I molecule HLA-E, which is commonly over-expressed in human cancers. Targeting NKG2A with monoclonal antibody holds potentials in immunotherapy of cancer. However, proteins with homologous sequences such as NKG2C/CD94 or NKG2E/CD94 complexes generally deliver an activating signal upon ligand binding. NKG2A-specific antagonizing antibodies would be needed to further explore therapeutic potentials of targeting NKG2A for immunotherapy of cancer. We have developed and characterized panels of monoclonal antibodies (mAbs) that specifically recognize NKG2A. Balb/c and SJL mice had been immunized with recombinant NKG2A/CD94 protein, recombinant plasmids encoding hNKG2A or stable cell line with ectopic expression of hNKG2A. Splenocytes from such immunized animals were collected for construction of phage-displayed antibody library in a single chain variable fragment (scFv) format. The NKG2A-specific antibodies, that had no detectable binding to NKG2C, NKG2E or CD94, were isolated through multiple rounds of phage panning and ELISA-based screening. Such antibodies also block HLA-E ligand binding and enhanced cytotoxicity of NK cell lines and primary NK cells on cancer cells. The final lead is a humanized antibody which behaves well in stability test. Lab scale processes are successfully developed and confirmed. The safety and efficacy will be further validated.

Citation Format: Teddy Yang, Jing Gao, Jingyun Yao, Dongxu Wang, Ken Dai. Discovery of Anti-NKG2A antibody from immunized phage display approach [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 5653.
Abstract 5654: Affinity tuned Xmab®2+1 anti-mesothelin x anti-CD3 bispecific antibody induces selective T cell-dependent cellular cytotoxicity of human ovarian cancer cells
Authors: Zeng Veronica G. , Faber Matthew S. , Bernett Matthew J. , Avery Kendra N. , Zeytounian John L. , Rashid Rumana , Muchhal Umesh S. , Moore Gregory L. , Desjarlais John R. , Hedvat Michael
Published: September 2020

Mesothelin (MSLN) is a tumor-associated antigen highly expressed in ovarian tumors. Current treatment options for ovarian cancer have only modest efficacy and there remains a large unmet need for new targeted therapies. We generated targeted T cell engager bispecific antibodies that bind MSLN on tumor cells and CD3 on T cells. The humanized and affinity-optimized MSLN x CD3 bispecific antibodies are in an XmAb® 2+1 Fab2-scFv-Fc format that bind bivalently to MSLN and monovalently to CD3. The affinity of the MSLN-targeting arms was reduced to promote avid binding to MSLN expressing cancer cell lines and to minimize reactivity on normal surrogate cell lines.

We correlated the expression of MSLN on cancer cell lines to 191 biopsy cores of serous and mucinous ovarian cancer tissues by IHC to identify surrogate cell lines to test for T cell-dependent cellular cytotoxicity (TDCC) activity. MSLN-transfected A549 cells were found to stain as intensely as ovarian cancer tumors expressing high amounts of MSLN. OVCAR-8 cancer cells were found to correlate with a medium MSLN level density on ovarian cancer tumors while ASPC-1 cells represented low MSLN-expressing tumors. Finally, we correlated expression of MSLN to non-cancer adjacent tissues and identified SKOV3, HT29, MCF7, and A549 (parental) cancer cells as appropriate surrogates of normal tissues.

Reduction of MSLN affinity of MSLN x CD3 XmAb® 2+1 bispecific antibodies led to preferential killing of MSLN-high target cells, with up to 45-fold more potent TDCC activity on OVCAR8 and ASPC-1 cancer cells compared to the selected normal surrogate cell lines. Engineering of selective MSLN x CD3 bispecific antibodies allows for preferential killing of cancer cells, potentially minimizing on-target/off-tumor effects that may contribute to dose-limiting toxicities.

Citation Format: Veronica G. Zeng, Matthew S. Faber, Matthew J. Bernett, Kendra N. Avery, John L. Zeytounian, Rumana Rashid, Umesh S. Muchhal, Gregory L. Moore, John R. Desjarlais, Michael Hedvat. Affinity tuned Xmab®2+1 anti-mesothelin x anti-CD3 bispecific antibody induces selective T cell-dependent cellular cytotoxicity of human ovarian cancer 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 5654.
Abstract 566: Therapeutic targeting of the polyamine pathway and EIF5A1 hypusination in endometrial cancer with DFMO and GC7
Authors: Kim HongIm , Schultz Chad , Bachmann Andre , Risinger John I.
Published: September 2020

The polyamines putrescine, spermidine, and spermine are well-characterized oncometabolites upregulated in cancers and drive their proliferation and spread. Endometrial cancers (ECs) express high levels of ornithine decarboxylase (ODC), a rate-limiting enzyme of polyamine biosynthesis and decreased levels of spermidine/spermine N1-acetyltransferase (SAT1), a key negative regulator of polyamines. In addition, the deoxyhypusine synthase (DHPS) enzyme necessary for spermidine-dependent hypusination of eIF5A1 is highly expressed in ECs. Hypusinated EIF5A1 is needed by cancer cells to support their increased protein translation needs. Collectively, these findings suggest that the polyamine pathway is particularly active and necessary in EC. Here we evaluated the effect of ODC inhibitor difluoromethylornithine (DFMO) and DHPS inhibitor N1-Guanyl-1,7-Diaminoheptane (GC7) alone or in combination on the growth of ECs. GC7 and DFMO were effective in suppressing growth in EC cell lines in vitro as measured by MTS viability and proliferation assays. These effects were accompanied by decreased hypusinated EIF5A1 in treated cells. However, neither compound induced apoptotic cell death when applied singly. We did note that when non-apoptotic inducing doses of DFMO and GC7 were combined, EC cells showed a marked combinatorial decrease in viability, proliferation, and hypusinated EIF5A1 levels. Importantly we found that combined DFMO/GC7 treatment caused apoptotic cell death of EC cells as measured by cleaved PARP. Furthermore, levels of cleaved caspases 3 and 7 were increased in these cells while caspases 5 or 9 were not cleaved. In summary we found for the first time that pharmacologically increased depression of the polyamine pathway as measured by decreased hypusinated EIFA1 can lead to apoptotic cell death in ECs. These data suggest that targeting the polyamine pathway and particularly its role in protein translation through EIF5A1 hypusination may be an effective treatment strategy.

Citation Format: HongIm Kim, Chad Schultz, Andre Bachmann, John I. Risinger. Therapeutic targeting of the polyamine pathway and EIF5A1 hypusination in endometrial cancer with DFMO and GC7 [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 566.
Abstract 5695: Induction of lymphangiogenesis in breast cancer enhances responsiveness to immunotherapies
Authors: Hosseinchi Peyman , Mansurov Aslan , Maillat Léa , Potin Lambert , Fujimori Gavin , Hubbell Jeffrey A. , Swartz Melody A.
Published: September 2020

Growth and remodeling of tumor-associated lymphatics, termed lymphangiogenesis, has been known to increase metastasis rate in cancer patients. However, a recent study from our group has shown that tumor-associated lymphatics can improve anti-tumor immunity in melanoma upon treatment with immunotherapies. The role of lymphangiogenesis in shaping anti-tumor immune responses in breast cancer, however, remains unexplored. Furthermore, recently approved checkpoint inhibitor immunotherapies have had limited success in breast cancer patients. A better understanding of the role of tumor-associated lymphatics in shaping anti-tumor immune responses could lead to more informed decisions when treating breast cancer patients with immunotherapies. In this study, we investigated the role of induced tumor lymphangiogenesis in modulating responsiveness to different immunotherapies in a murine triple-negative mammary carcinoma model. Spontaneously metastatic 4T1 tumor cells were transduced to over-express the pro-lymphangiogenic growth factor VEGF-C (VC) to induce tumor lymphangiogensis in vivo. Flow cytometry and immunohistochemistry analyses confirmed that VEGF-C over-expressing tumors have an increase in tumor-associated lymphatics than control ones. Higher infiltrations of conv CD4 T cells and macrophages were observed in lymphangiogenic 4T1 tumors. Lymphangiogenic tumors treated with aPD-1 and aCTLA-4 checkpoint inhibitors, or an agonist of the Stimulator of Interferon Genes (STING) pathway, showed improved responsiveness to the therapy compared to the control tumors, as indicated by a reduced tumor growth rate. Moreover, positive correlations between VEGF-C expression and CD4 T cell and macrophage gene expressions were observed in primary human triple-negative breast tumor samples from The Cancer Genome Atlas (TCGA). In conclusion, our study shows that increase in tumor-associated lymphatics in triple-negative breast cancer enhances responsiveness to immunotherapies.

Citation Format: Peyman Hosseinchi, Aslan Mansurov, Léa Maillat, Lambert Potin, Gavin Fujimori, Jeffrey A. Hubbell, Melody A. Swartz. Induction of lymphangiogenesis in breast cancer enhances responsiveness to immunotherapies [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 5695.
Abstract 5711: Therapeutic induction of tumor-selective ferroptosis in models of pancreatic cancer
Authors: Badgley Michael , DelGiorno Kathleen , Wahl Geoffrey , Lyssiotis Costas , Olive Kenneth P.
Published: September 2020

Pancreatic ductal adenocarcinoma (PDA) is an aggressive cancer with few therapeutic options. Oncogenic signaling from mutant Kras, the driving oncogene of PDA, drives ROS production, making PDA cells reliant on ROS detoxification processes. To detoxify ROS, mammalian cells use thiols derived from the semi-essential amino acid cysteine, which is imported from exogenous pools of cystine via the system xC− cystine/glutamate antiporter. We found that cysteine depletion in multiple human PDA lines induced a rapid, oxidative, iron-dependent form of non-apoptotic cell death called ferroptosis. Confirming work in other systems, we found that glutathione depletion was necessary for the induction of ferroptosis in PDA cells. However, depletion of glutathione alone was not sufficient to induce ferroptosis, indicating that modulation of other cysteine-dependent processes must also play a role in ferroptosis. Using carbon-labelled cystine, we determined that extracellular cystine is quickly incorporated into coenzyme A. Furthermore, dual inhibition of both CoA and GSH synthesis combined to induce ferroptosis, and CoA treatment rescued the induction of ferroptosis following cysteine depletion. To assess whether cysteine depletion could be useful for targeting pancreatic tumors in vivo, we utilized both genetic and pharmacological approaches. First, we generated a dual recombinase mouse model in which the system xC- gene Slc7a11 can be acutely, systemically deleted in mice with established K-ras/p53 mutant PDA. Acute deletion of Slc7a11 in mice with established PDA led to tumor stabilizations/regressions and increased overall survival, and this was fully reversed by co-treatment with the membrane-permeable cysteine analog NAC. Second, we treated the well validated KPC genetic model of PDA with cyst(e)inase, an engineered enzyme that breaks down cysteine and cystine in circulation. Tumor-bearing KPC mice treated with cyst(e)inase also exhibited tumor responses. In both the genetic and pharmacological models, we observed tumor-selective necrosis and confirmed the induction of ferroptosis using several orthogonal techniques, including electron microscopy, immunohistochemistry, histopathology, and laser capture microdissection/RNA sequencing. These data demonstrate the potential therapeutic utility of targeting cystine import in pancreatic cancer.

Citation Format: Michael Badgley, Kathleen DelGiorno, Geoffrey Wahl, Costas Lyssiotis, Kenneth P. Olive. Therapeutic induction of tumor-selective ferroptosis in models of pancreatic 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 5711.
Abstract 5727: Metabolic status and adaptability of breast cancer stem cells
Authors: Buschhaus Johanna M. , Muniz Ayse J. , Luo Ming , Burnett Joseph P. , Truchan Nathan A. , Brooks Michael D. , Humphries Brock A. , Luker Kathy E. , Lahann Joerg , Sun Duxin , Wicha Max S. , Luker Gary D.
Published: September 2020

Breast cancer stem cells (BCSCs) represent the subpopulation of malignant cells that cause tumor initiation, metastasis, and recurrence. BCSCs resist therapy with radiation and standard drugs, emphasizing the need to identify new vulnerabilities as therapeutic targets. Here, we investigated the metabolism of BCSCs at single-cell resolution using molecular imaging and scRNA sequencing techniques. We imaged retention of the fluorescent dye PKH26 or expression of a CRISPR/Cas9-engineered ALDH1A3-mCherry promoter-reporter to identify BCSCs and metabolic state of cells by two-photon microscopy with fluorescence lifetime imaging (FLIM) of endogenous NADH. We previously reported that sorted BCSCs exhibited enhanced metabolic plasticity relative to bulk tumor cells in 2D culture and rapidly metabolically adapted to the glycolysis inhibitor 2-deoxyglucose (2DG). Since cells rewire signaling and metabolism in 3D environments, we utilized two-photon microscopy to quantify metabolism in secondary mammospheres and living animals. In both intact spheres and orthotopic tumor xenografts, BCSCs marked by either fluorescent reporter utilized glycolysis to a greater extent than bulk cancer cells. We validated our findings by correlating cellular entropy as a marker for cancer stem cells with metabolic pathway preferences on a single-cell basis. When treated with 2DG, mammospheres showed increased OXPHOS and a significant decrease in the percentage of ALDH1A3-mCherry+ cells. These data 1) highlight capabilities of FLIM to measure the metabolism of single cancer cells in physiologic environments; 2) correlate molecular phenotypes with real-time molecular imaging profiles of metabolism and stem-ness; and 3) reveal that BCSCs rely on glycolysis, suggesting a potential target for metabolic therapy.

Citation Format: Johanna M. Buschhaus, Ayse J. Muniz, Ming Luo, Joseph P. Burnett, Nathan A. Truchan, Michael D. Brooks, Brock A. Humphries, Kathy E. Luker, Joerg Lahann, Duxin Sun, Max S. Wicha, Gary D. Luker. Metabolic status and adaptability of breast cancer stem 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 5727.
Abstract 5915: HPV oncogenes regulated the aberrant expression of microRNAs in cervical cancer
Authors: Deng Yan , Song Yi , Tang Tao , Wang Chi Chiu
Published: September 2020

The high risk human papillomavirus (HPV) is the single most important etiological agent in cervical cancer. The E5, E6 and E7 genes in the early region of HPV genome are proved to have a significant impact on progression of cervical cancer. MicroRNA (miRNA) is a kind of short non-coding RNA which has been proven to play a role in tumor development. Previously, we have identified several differentially expressed miRNAs in cervical cell lines and clinical specimens, including 9 down-regulated and 2 up-regulated. Here we further investigate the direct relationship between the oncogenes (E5, E6 and E7) and the above miRNAs by overexpressing the oncogenes and silencing each single one separately. The result of the relative expression level of miRNAs showed some consistency that HPV E5, E6 and E7 could down-regulate the expression of miR-145, miR-195 and miR-142-3p. However, there were also discrepancy. For instance, E6 from HPV 16 and HPV 52 showed a significant down-regulation in the expression of miR-195, while E6 of other subtypes (HPV18, 31, 52) had no effect on the miR-195 expression level. Meanwhile, the oncogenetic effect of miR-142-3p has been examined by measuring cell proliferation. Further mechanism study of HPV/miR-142-3p pathway in the cervical cancer would also be investigated. The work could provide new insight in understanding the development of cervical cancer and potentially help to develop novel therapeutic approaches and bio-markers for cervical cancer.

Citation Format: Yan Deng, Yi Song, Tao Tang, Chi Chiu Wang. HPV oncogenes regulated the aberrant expression of microRNAs in cervical 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 5915.
Abstract 6059: Targeting the anthracycline metabolizing enzyme AKR1C3 in adipocytes to improve cytotoxicity
Authors: Paharkova Vladislava , Orgel Etan , Neely Michael , Louie Stan. , Mittelman Steven
Published: September 2020

Introduction: We have reported that mouse and human adipocytes take up and metabolize the anthracycline, daunorubicin (DNR), reducing its concentration in the adipocyte microenvironments. This may contribute to anthracycline resistance for cancers, which reside in adipocyte rich environments such as omentum and bone marrow. Adipocytes express several carbonyl- and aldo-keto reductases (CBRs and AKRs) which metabolize and inactivate anthracyclines, and it is unclear which of these might be important targets to improve treatment outcome.

Experimental Procedures: We knocked out AKR1C3 in the human preadipocyte cell line Chub S7 using CRISPR/Cas9 gene editing technology. We chose AKR1C3 first as it is one of the overexpressed enzymes in adipocytes with the highest anthracycline-metabolizing activity. We delivered ribonucleoprotein complexes of CRISPR-Cas9 enzyme plus guide RNAs by nucleofection. Then we established single-cell derived clones and tested for successful KO by Western blot. Finally, we quantified adipocyte lysate AKR activity using a colorimetric assay based on NADPH-dependent reduction of phenanthrenequinone.

Data Summary: We chose three Chub S7 preadipocyte clones that demonstrated successful AKR1C3 knockout based on almost undetectable protein expression by western blot. AKR activity was significantly reduced in all three clones; control preadipocytes had 44±4.7, while clones had activity of 28±7.3, 33±3.4, and 35±5.7 pmol/min/µg (p<0.05 for all comparisons, n=3 for each assay).

Conclusions: These findings demonstrate that AKR1C3 knockout can be successfully done in human preadipocytes using a CRISPR-Cas9 system. Knockout of AKR1C3 significantly reduces overall aldoketoreductase activity in preadipocyte cells. This implies that a substantial portion of preadipocyte AKR activity is dependent on the isoenzyme, AKR1C3. Future testing is needed to determine whether AKR1C3 KO will reduce the clearance of anthracyclines from the cancer microenvironment, and may represent a treatment target to enhance anthracycline cytotoxicity.

Citation Format: Vladislava Paharkova, Etan Orgel, Michael Neely, Stan. Louie, Steven Mittelman. Targeting the anthracycline metabolizing enzyme AKR1C3 in adipocytes to improve cytotoxicity [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 6059.
Abstract 6123: An indole compound MA-35 attenuates tumorigenesis in an inflammation-induced colon cancer model
Authors: Kanehara Keigo , Ohnuma Shinobu , Naitoh Takeshi , Uuno Michiaki , Abe Takaaki
Published: September 2020

Backgrounds: In inflammatory bowel disease (IBD), chronic inflammation results in the development of colon cancer known as colitis-associated cancer (CAC), that is associated with tumor necrosis factor-α (TNF-α) signaling. In addition, intestinal fibrosis in IBD patients is a common clinical complication, which is promoted by transforming growth factor β1 (TGF-β1). In our previous study, mitochonic acid (MA-35), derivative of indole-3-acetic acid known as a plant hormone auxin, showed anti-TNF-α effect by inhibiting IκB kinase (IKK) phosphorylation and anti-TGF-β1 effect by inhibiting Smad3 phosphorylation in a renal fibrosis mouse model.

Aim: To identify the possible mechanism of anti-tumor effects and anti-fibrotic effects of MA-35 using azoxymethane (AOM)/ dextran sulfate sodium (DSS)-induced CAC mouse model.

Methods and Results: MA-35 was orally administered every day for 70 days in an AOM/DSS model with a feeding tube. There was no difference in weight loss between the AOM/DSS group and the AOM/DSS+MA-35 group, however the disease activity index score and the survival rate were improved by MA-35. MA-35 also blocked progression of the anemia and shortening of the colon induced by AOM/DSS. MA-35 surprisingly reduced the formation of tumors in the colon. In the microscopic evaluation, MA-35 reduced inflammation and fibrosis in area with dysplasia. Furthermore, the TNF-α mRNA level in the colon tended to be reduced and the interleukin 6, TGF-β1 and fibronectin 1 mRNA levels in the colon were significant reduced by MA-35. In western blotting, MA-35 inhibited IKK phosphorylation, and both NF-κB p65 phosphorylation and TNF-α as the downstream signals for p-IKK in human colorectal cancer cell line HT-29. Similarly, MA-35 inhibited Smad2/3 phosphorylation, and both fibronectin 1 and TGF-β1 as the downstream signals for p-Smad2/3 in HT-29.

Conclusions: MA-35 inhibited the development of CAC by reducing inflammation and fibrosis in an AOM/DSS mouse model by inhibiting both TNF-α signaling and TGF-β1 signaling. MA-35 may be a potent therapeutic agent for inhibiting the development of CAC in IBD patients.

Citation Format: Keigo Kanehara, Shinobu Ohnuma, Takeshi Naitoh, Michiaki Uuno, Takaaki Abe. An indole compound MA-35 attenuates tumorigenesis in an inflammation-induced colon cancer model [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 6123.
Abstract 6154: H3K27M gliomas are characterized by a stall in the epithelial-mesenchymal transition
Authors: CHENEY ALLISON R. , Sanders Lauren M. , Seninge Lucas , Beale Holly C. , Kephart Ellen Towle , Pfeil Jacob , Learned Katrina , Lyle A. Geoffrey , Bjork Isabel , Haussler David , Salama Sofie R. , Vaske Olena M.
Published: September 2020

Pediatric diffuse midline gliomas are lethal cancers, the majority of which harbor the H3 p.K27M mutations. Although it has potential implications on the treatment of diffuse midline glioma as a disease driver; the timing, cell type of origin, and effect of the H3 p.K27M mutation on early embryonic brain development is poorly understood. The purpose of our study is to elucidate the molecular mechanisms by which the histone H3 p.K27M mutation drives tumorigenesis of pediatric diffuse midline gliomas using the analysis of genomic datasets. Here, we performed differential RNA sequencing gene expression analysis of a cohort of H3K27M and H3 wild type (WT) pediatric diffuse midline gliomas, revealing that genes in the epithelial-mesenchymal transition (EMT) pathway were significantly differentially expressed between the mutant and WT tumors. Several EMTs are required for normal brain development. Overall, pre-EMT genes, including the master regulator of EMT SNAI1, were overexpressed in H3K27M tumors compared to the WT tumors, while post-EMT genes were underexpressed. We hypothesized that the H3 p.K27M mutation may lead to gliomagenesis by inducing a stall in the EMT in early brain development. To test this hypothesis, we examined published single-cell RNA sequencing data from pediatric diffuse midline gliomas alongside similar data from organoid models of neural development, collected from multiple developmental timepoints. This analysis revealed transcriptional similarities between H3K27M and pre-EMT neural stem cells. Currently, we are investigating the expression of EMT markers in H3K27M and WT pediatric glioma primary cell lines, using Western blotting, RT-PCR, and CRISPRi screening. In conclusion, we observed aberrant expression of genes involved in EMT in H3K27M pediatric gliomas. Our observations are consistent with a model in which the p.H3K27M mutation is associated with a pre-EMT cell phenotype, potentially due to an arrest in the EMT pathway or de-differentiation of mature astrocytes.

Citation Format: ALLISON R. CHENEY, Lauren M. Sanders, Lucas Seninge, Holly C. Beale, Ellen Towle Kephart, Jacob Pfeil, Katrina Learned, A. Geoffrey Lyle, Isabel Bjork, David Haussler, Sofie R. Salama, Olena M. Vaske. H3K27M gliomas are characterized by a stall in the epithelial-mesenchymal transition [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 6154.
Abstract 6157: Hypoxia increases motility of neuroblastoma cells induced by the neuropeptide Y/Y5 receptor pathway and exacerbates their metastatic potential
Authors: Abualsaud Nouran , Caprio Lindsay , Hong Sung Hyeok , Galli Susana , Kitlinska Joanna
Published: September 2020

Neuroblastoma (NB) is a pediatric malignancy that originates from precursors of sympathetic neurons and expresses neuronal proteins, such as neuropeptide Y (NPY) and its receptors. NB cells constitutively express NPY Y2 receptor (Y2R), which maintains their proliferation and tumor vascularization. However, pro-apoptotic conditions additionally induce expression of Y5 receptor (Y5R), which acts as a survival factor for tumor cells. High Y5R expression was also observed in angioinvasive NB cells surrounding blood vessels, suggesting a role for this receptor in NB dissemination. Indeed, in NB patients, high serum levels of NPY associated with the presence of metastases. Hence, the goal of this study was to identify mechanisms that may underlie potential pro-metastatic effects of NPY. We have found that NPY, acting via Y5R, stimulates NB cell motility and acts as a chemotactic factor for these cells in a concentration-dependent manner. Hypoxia, a known condition promoting metastasis, increased expression of Y5R in SK-N-BE(2) and SK-N-AS NB cell lines. Consequently, hypoxic NB cells had increased motility and a chemotactic response to NPY. This increase in NPY-driven migration was fully blocked by Y5R antagonist. Pre-incubation of NB cells in hypoxia before orthotopic injections into adrenal glands of SCID/bg mice exacerbated metastasis in xenografts from both NB cell lines. In mice bearing non-MYCN amplified SK-N-AS tumors, the hypoxia-driven metastases were located in soft tissues, while MYCN-amplified SK-N-BE(2) xenografts derived from hypoxic cells exhibited enhanced dissemination to lungs. The latter pattern of metastases is also observed in NB patients with MYCN amplification. Further studies are required to determine if the hypoxia-driven increase in NB metastasis is Y5R-dependent. Altogether, our data support the role of tumor hypoxia and NPY/Y5R axis in NB dissemination. Since this pathway is also activated during chemotherapy and promotes NB cells resistance to treatment, NPY/Y5R may also be involved in the secondary dissemination of the recurrent tumors. If the role of Y5R in NB metastasis is validated in preclinical models, this NPY receptor may become a potential therapeutic target preventing the disease dissemination.

Citation Format: Nouran Abualsaud, Lindsay Caprio, Sung Hyeok Hong, Susana Galli, Joanna Kitlinska. Hypoxia increases motility of neuroblastoma cells induced by the neuropeptide Y/Y5 receptor pathway and exacerbates their metastatic potential [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 6157.
Abstract 6229: MDM2 inhibitors sensitize testicular cancer models to mTORC1/2 inhibitors and cisplatin
Authors: de Jong Steven , de Vries Gerda , Meersma Gert Jan , Suurmeijer Albert J.H. , van Vugt Marcel A.T.M. , Gietema Jourik A
Published: September 2020

Testicular cancer (TC) is one of the most common solid tumors in young men that can effectively be treated with cisplatin-based chemotherapy. However, 10% of those wild type p53 expressing TC patients will not survive their disease. The aim of this study was to identify effective combinations with MDM2 inhibitors in TC models. A panel of cisplatin sensitive and -resistant human TC cell lines and TC patient-derived xenograft (PDX) models have been treated with MDM2 inhibitors (Nutlin-3a, RG7388), cisplatin and AKT/mTORC1/2 inhibitors (MK2206, AZD8055 and INK128). Survival, apoptosis, and clonogenic assays were performed. Transcriptional activity of p53 was determined using chromatin immunoprecipitation (CHIP) followed by quantitative PCR of target promoters. Mechanisms of apoptosis induction were studied by measuring mitochondrial priming, siRNA approaches and BH3 mimetics. We found that MDM2 inhibitors sensitized TC cell lines to cisplatin and mTORC1/2 inhibition. Nutlin-3a in combination with cisplatin or AZD8055 treatment induced caspase 3 and PARP cleavage and reduced clonogenic outgrowth. Enriched p53 binding to the promoter regions of known activators of extrinsic and intrinsic apoptosis, FAS, PUMA and NOXA, was observed in response to Nutlin-3a or cisplatin. BH3 profiling of mitochondria and siRNA mediated silencing identified PUMA and NOXA as key pro-apoptotic proteins in TC cell lines. Interestingly, addition of the BH3 mimetics ABT-737 and A-1210477, mimicking PUMA and NOXA respectively, to combination treatments shifted the balance towards apoptosis as well. In vivo experiments showed reduced tumor growth especially in the wild type p53 PDX model TC1 treated with RG7388/cisplatin, which was reflected in p53 activation and increased caspase-3 activity. No potentiating effect of this combination was observed in the p53 wild type PDX model TC4. In conclusion, we have demonstrated that targeting of MDM2 in combination with chemotherapy can be very effective in TC models. Our results warrant further investigation of this drug combination in the treatment of TC. Supported by a grant from the Dutch Cancer Society (RUG 2014-6691)

Citation Format: Steven de Jong, Gerda de Vries, Gert Jan Meersma, Albert J.H. Suurmeijer, Marcel A.T.M. van Vugt, Jourik A Gietema. MDM2 inhibitors sensitize testicular cancer models to mTORC1/2 inhibitors and cisplatin [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 6229.
Abstract 6232: Preclinical efficacy of CIGB-300, an anti-CK2 peptide, on breast cancer metastasic colonization
Authors: Gottardo Maria F. , Sidabra Johanna E. , Capobianco Carla S. , Garona Juan , Rodriguez Silvio Perea , Aonso Daniel F. , Farina Hernan G.
Published: September 2020

CK2 is a serine/threonine kinase that is overexpressed in breast cancer and its inhibition is associated to reduced tumor growth and disease progression. With more than three hundred substrates, this kinase is involved in a large number of cellular functions, such as replication, cell proliferation, differentiation, regulation of apoptosis and angiogenesis. CIGB-300 is an antitumor peptide with a novel mechanism of action, since it binds to the phospho-acceptor domain of CK2 substrates impairing the correct phosphorylation by the enzyme. Our aim was to evaluate the potential therapeutic benefits of CIGB-300 on breast cancer disease using experimental models with translational relevance. We demonstrated that CIGB-300 reduces breast cancer cell growth in MDA-MB-231, MCF-7 and F3II cells, exerting a pro-apoptotic action. We found that CIGB-300 increased Bax and decreased Bcl-2 expression, so the intrinsic pathway was involved in the proapoptotic effect of CIGB-300 in breast cancer cells. Moreover, CIGB-300 inhibits the CK2-mediated phosphorylation of B23, such phosphorylation impairment was correlated with the ability of CIGB-300 to induce nucleolar disassembly. Also, we investigate whether CIGB-300 affects the sensitivity of tumor cells to chemotherapeutic agents. We evaluated the effects of the combination of CIGB-300 and cisplatin on cell viability and apoptosis. We demonstrated that CIGB-300 sensitize breast cancer cells to cisplatin, reducing cell viability and increasing apoptosis. In vivo, the effect on experimental breast cancer lung metastasis was evaluated after surgical removal of primary F3II tumors or after tail vein injection of tumor cells. Systemic CIGB-300 treatment inhibited breast cancer colonization of the lung, reducing the size and number of metastatic lesions. The present preclinical study establishes for the first time the efficacy of CIGB-300 on breast cancer. These encouraging results suggest that CIGB-300 could be used for the management of breast cancer as an adjuvant therapy after surgery, limiting tumor metastatic spread and thus protecting the patient from distant recurrence.

Citation Format: Maria F. Gottardo, Johanna E. Sidabra, Carla S. Capobianco, Juan Garona, Silvio Perea Rodriguez, Daniel F. Aonso, Hernan G. Farina. Preclinical efficacy of CIGB-300, an anti-CK2 peptide, on breast cancer metastasic colonization [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 6232.
Abstract 6275: Targeting RRM2 by triapine sensitizes glioblastoma to radiotherapy
Authors: Corrales-Guerrero Sergio , Bell Erica H. , Chakravarti Arnab , Shen Changxian , Williams Terence M.
Published: September 2020

Glioblastoma multiforme (GBM) remains characterized by poor clinical outcomes and limited treatment options. Currently, the standard of care for patients with this disease is surgery with radiation therapy and concurrent and adjuvant temozolomide. However, most patients succumb to the disease within the first 15 months after diagnosis. Therefore, effective treatment regimens are desperately needed. Exacerbated cell proliferation as well as repair of DNA damage induced by chemoradiation requires a constant supply of dNTPs. Ribonucleotide reductase (RNR) is the key enzyme for the synthesis of dNTPs and is a complex formed by two subunits: RRM1 and RRM2

3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3AP - commercially Triapine) is a molecule that potently inhibits RRM2 by affecting its active site, and has been shown to cross the brain-blood barrier (Jiang et al. 2006. PMID: 16834759). We herein report that triapine induces DNA replication stress and sensitizes GBM cells to radiation. Analyses of available datasets including TCGA show that RRM2 is overexpressed in GBM and lower grade glioma (LGG), and a higher expression of RRM2 associates with worse overall survival and disease-free survival of GBM and LGG patients. Nevertheless, RRM2 levels do not change between initial and recurrent tumors (Wang et al. 2017. PMID: 28697342). Using human GBM cell lines LN229, LN18 and U251, we have performed cytotoxicity assays and clonogenic radiation assays. The IC50 of triapine in these cell lines are between 1-2 μM. We find that triapine significantly radiosensitizes LN229, LN18 and U251 cells. Consistent with the role of RRM2 in dNTPs biosynthesis, triapine treatment of GBM cells induces robust phosphorylation of CHK1 at S345, which is accompanied by elevated gamma H2AX. Importantly, we reveal that triapine promotes ionizing radiation induced phosphorylation of CHK2 at T68. Acknowledging the increasing concerns about using established cell lines for GBM studies, we are currently moving to validate our results in primary GBM cell lines. Our results demonstrate that RRM2 is overexpressed in glioblastoma and targeting RRM2 with triapine enhances radiation-induced DNA damage leading to radiosensitization of glioblastoma cells. Our findings suggest that triapine is a promising agent for the sensitization of glioblastoma to radiotherapy.

Citation Format: Sergio Corrales-Guerrero, Erica H. Bell, Arnab Chakravarti, Changxian Shen, Terence M. Williams. Targeting RRM2 by triapine sensitizes glioblastoma to radiotherapy [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 6275.
Abstract 6288: A high-throughput in vitro characterization method of CAR T cells using image cytometry
Authors: Chan Leo Li-Ying , Maldini Colby , Love Andrea , Tosh Kevin , Gayout Kevin , Smith Tim , Riley James
Published: September 2020

Since the FDA approval of two CAR T cell therapies, Kymriah (Tisagenlecleucel, Novartis) and Yescarta (Axicabtagene ciloleucel, Kite) for CD19+ malignancies, there has been a significant interest for developing new CARs targeting other disease indications. As such, the ability to simultaneously monitor manufacturing criteria and functional characteristics of multiple CAR T cell products would likely be critical to accelerate candidate therapies into clinical trials. Here, we demonstrate that image-based cytometry using the Celigo Image Cytometer (Nexcelom Bioscience LLC) yields high-throughput measurements of CAR T cell proliferation and size throughout the cell-manufacturing process, and captures the kinetics of antigen-specific CAR T cell-mediated killing in vitro. The data acquired and analyzed by the Celigo Image Cytometer were congruent with results from conventional instruments when tested contemporaneously, such as the Multisizer 3 (Beckman Coulter) which monitored the properties of CAR T cell expansion, and a luciferase-based assay which measured the cytotoxic potential of CAR T cells. More importantly, the use of bright field and fluorescent imaging by the Celigo Image Cytometer provided kinetic measurements and rapid data acquisition and analysis which were direct advantages over the conventional technologies described herein. Together, the Celigo Image Cytometer enabled fast, reproducible, and high-throughput assessment of CAR T cell manufacturing criteria and effector function, which could greatly facilitate the identification and evaluation of novel CARs with therapeutic potential.

Citation Format: Leo Li-Ying Chan, Colby Maldini, Andrea Love, Kevin Tosh, Kevin Gayout, Tim Smith, James Riley. A high-throughput in vitro characterization method of CAR T cells using image cytometry [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 6288.
Abstract 6289: Phenotypic screening of small molecules for novel immuno-oncology potential using BioMAP® Early Screening Services
Authors: O'Mahony Alison , Denker Sheryl P. , Velichko Sharlene
Published: September 2020

Approved immuno-oncology (IO) drugs that have expanded the arsenal of available treatment options for recalcitrant cancers include antibodies to PD-(L)1 checkpoints–a relatively new class of immunotherapies. While the clinical success reported for these drugs is compelling, efficacy is limited to a small cohort of patients. The need for more IO therapeutic options has prompted the pursuit of next-generation candidates that can achieve better patient outcomes. One approach is to screen compounds for novel targets that elicit an immunorestorative response within the tumor microenvironment (TME), consistent with approved IO drugs. Ideally, a small molecule therapeutic would represent a distinct advantage over existing biologics, both in terms of administration as well as ability to optimize chemistry. In order to efficiently identify molecules with IO potential, a high throughput, physiologically relevant screening approach that recapitulates the complexity of the TME is needed. BioMAP Oncology systems are co-cultures of human primary cell types (endothelial cells, fibroblasts, and PBMCs) and cancer cells that model immune suppressed TME biology. Compounds can be screened in these in vitro TME models to look for impacts on protein biomarkers relevant for immune responses, inflammation, matrix modulation and for impacts on cancer cells. These systems were validated for immune restorative activities using anti PD-(L)1 therapeutics such as pembrolizumab, nivolumab, and durvalumab. Specifically, increased pro-inflammatory cytokine levels served as hallmark activities indicating a reversal of the immune suppressive influence of the cancer cell. In one of the BioMAP Oncology systems, we screened a library of 72 compounds targeting different molecules, the vast majority of which had not previously been identified as potential immuno-oncology candidates. Several small molecules that increased one or more cytokines were identified, indicating immune restorative potential. Other small molecules inhibited immune responses and/or demonstrated anti-inflammatory activities. Additionally, modulation of matrix biomarkers on tissue and tumor cells indicate impacts on distinct compartments of the TME. This approach using human cell-based in vitro TME models represents a rapid and cost-effective manner to rapidly screen libraries of small molecules (both novel and repurposed) for immuno-oncology potential.

Citation Format: Alison O'Mahony, Sheryl P. Denker, Sharlene Velichko. Phenotypic screening of small molecules for novel immuno-oncology potential using BioMAP® Early Screening Services [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 6289.