bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5487: Synergy between FAK and PI3K inhibitors in cervical and pancreatic cancer cells
Authors: Chen Chao-Cheng , Wang Suyang , Yang Jr-Ming , Huang Chuan-Hsiang
Published: June 2023

The Ras/PI3K/ERK signaling network is frequently mutated in many cancer types including cervical cancer and pancreatic cancer. Our previous study suggested that PI3K and FAK form a positive feedback loop to modulate ERK activation. We tested combined inhibition of FAK and PI3K on the growth of cervical and pancreatic cancer in vitro. We found that FAK and PI3K inhibitors synergistically inhibited the growth of some cervical cancer and pancreatic cancer cell lines. The synergy was due to both increased apoptosis and decreased cell proliferation. Molecularly, FAK inhibition caused downregulation of PI3K and ERK signaling in cervical cancer but not pancreatic cancer cells. Interestingly, PI3K inhibitors induced activation of several RTK including insulin R and IGF-1R in cervical cancer cells, as well as EGFR, Her2, Her3, Axl, and EphA2 in pancreatic cancer cells. Together, our results show the promise of combining FAK and PI3K inhibition for cervical cancer and pancreatic cancer treatment, but biomarkers for sensitivity to the combination are needed, and concomitant RTK inhibition may be required to combat potential resistance to the combination.

Citation Format: Chao-Cheng Chen, Suyang Wang, Jr-Ming Yang, Chuan-Hsiang Huang. Synergy between FAK and PI3K inhibitors in cervical and pancreatic cancer cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5487.
Abstract 5509: The PI3K inhibitor Paxalisib combines synergistically with the MEK inhibitor Mirdametinib to target atypical teratoid/rhabdoid tumors
Authors: Malebranche Kristen J. , Findlay Tyler , Geethadevi Anupa , Eberhart Charles , Raabe Eric , Rubens Jeffrey
Published: June 2023

Atypical teratoid/rhabdoid tumors (AT/RT) are aggressive pediatric brain tumors and the most common malignant brain tumors of infancy. The four-year event-free survival rate is only 37%. For patients with relapsed AT/RT there are limited treatment options, but precision therapies may help improve survival for patients with this deadly disease. We have previously identified strong activation of the PI3K-AKT-mTOR (mTOR) signaling pathways in AT/RT. Paxalisib is a highly brain-penetrant PI3K inhibitor acting upstream of mTOR. We find that Paxalisib slows tumor growth in orthotopic xenograft models of AT/RT and extends median survival from 40 to 54 days (p=0.001, log-rank test). RNASeq after mTOR pathway inhibition identifies reflexive activation of the RAS-RAF-MEK-ERK (MAPK) pathway as a possible mechanism of therapy resistance (KEGG pathway analysis). The RAS-RAF-MEK-ERK (MAPK) pathway plays a pivotal role in regulating cell growth and survival and activation of the pathway contributes to the aggressive growth of numerous cancers. Mirdametinib is a small, allosteric MEK inhibitor currently used in clinical trials for both pediatric low- and high-grade gliomas (PD0325901). We find that dual inhibition of both the mTOR and MAPK pathways by Paxalisib and Mirdametinib synergize to reduce AT/RT growth and viability (Bliss synergy score 16.77). Paxalisib also combines with Mirdametinib to induce high levels of apoptosis and cell senescence (as determined by western blot: cPARP, pRB, P21, P16). Naïve treatments in mice bearing AT/RT orthotopic tumors suggests the combination therapy decrease mTOR and MAPK pathway activation (as determined by western blot). These data support the use of Paxalisib and Mirdametinib combination therapy as a promising novel combination therapy to treat relapsed AT/RT.

Citation Format: Kristen J. Malebranche, Tyler Findlay, Anupa Geethadevi, Charles Eberhart, Eric Raabe, Jeffrey Rubens. The PI3K inhibitor Paxalisib combines synergistically with the MEK inhibitor Mirdametinib to target atypical teratoid/rhabdoid tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5509.
Abstract 5518: Use of antihypertensive drugs and the risk of cancer, findings of a population-based cohort study in Shanghai
Authors: Wang Suna , Xie Li , Qian Ying , Wang Jie , Zhang Guanglu , Li Lei , Zhang Weituo , Yu Herbert , Zhao Wensui , Qian Biyun
Published: June 2023

Background: The widespread use of antihypertensive drugs has raised concerns on whether the use increases the risk of cancer. Although multiple studies have addressed this issue, it remains unclear if hypertension patients who are on antihypertensive medications are at higher risk for cancer.

Methods: Between 2013 and 2017, we enrolled 101,370 individuals with hypertension from community healthcare centers in Changning Shanghai, followed through December 31, 2019. Drug administration was defined as the class and number of ever use the major antihypertensives. The main outcomes included the incidences of all cancer and major types of cancer in Shanghai (lung, colorectum, thyroid, and stomach).

Results: During a mean follow-up of 5.1 (SD 1.3) years, 4970 cancer cases were newly diagnosed in the cohort. CCBs were the most frequently used antihypertensives which were associated with a moderately increased risk of all cancer (hazard ratio, HR: 1.11, 95% CI: 1.05-1.18). The second commonly used drug ARBs were also associated with increased risk of all cancer (HR: 1.10, 95%CI: 1.03-1.17) as well as lung and thyroid cancers (HR: 1.21, 95%CI: 1.05-1.39; HR: 1.66, 95%CI: 1.22-2.27, respectively). No cancer association was found with other antihypertensives. Individuals who use more than one class of antihypertensives had a higher risk of all cancer, and a possible dose-response relationship was suggested.

Conclusions: Use of ARBs or CCBs may be associated with an increased risk of cancer. Taking more than one class of antihypertensives appeared to have a higher risk for cancer.

Citation Format: Suna Wang, Li Xie, Ying Qian, Jie Wang, Guanglu Zhang, Lei Li, Weituo Zhang, Herbert Yu, Wensui Zhao, Biyun Qian. Use of antihypertensive drugs and the risk of cancer, findings of a population-based cohort study in Shanghai. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5518.
Abstract 5525: Community cancer research advocates: Helping underrepresented minority groups become research ready
Authors: Been Sarah , Gertz Susan , Hershberger Susan , Kovacic Melinda Butsch ,   We Engage 4 Health Community-Academic Partnership
Published: June 2023

Even though certain racial/ethnic minority groups get cancer more often, are diagnosed at younger ages, and are at risk for earlier death, clinical trials fail to include adequate proportions of representatives from these groups. Encouraging equitable participation in research becomes an important tool in the fight against cancer disparities. Therefore, we co-created with community representatives a graphic-style story entitled “Research Ready” that is used by trained Community Research Advocates (including some survivors or family of survivors themselves) to hold brief discussions to help eligible hesitant participants to learn the 3 P’s: the purpose of cancer research, how they are kept safe while participating, and why people from diverse backgrounds are needed to participate. Importantly, advocates use a Research Review activity to discuss factors that influence their willingness to participate as well as identify questions they have for the research study’s staff. Focus group and survey-based evaluation data show these discussions to be useful in increasing hesitant patients’ willingness to consider participation in research and increase perception of safety. Advocates too appreciate the opportunity to serve. While this pilot is small, additional partnerships within the University of Cincinnati Cancer Center to expand the program will be developed. Future partnership with a community patient navigator program will also enable additional support and transportation to research appointments. Collectively, we anticipate that this program will ultimately support hesitant patients’ decision-making and improve cancer research participation particularly among racial/ethnic minorities.

Citation Format: Sarah Been, Susan Gertz, Susan Hershberger, Melinda Butsch Kovacic, We Engage 4 Health Community-Academic Partnership. Community cancer research advocates: Helping underrepresented minority groups become research ready. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5525.
Abstract 5551: Cancer associated PSA-glycoforms as prostate cancer markers
Authors: Koistinen Hannu , Kovanen Ruusu-Maaria , Lehto Timo-Pekka , Rannikko Antti , Mirtti Tuomas
Published: June 2023

Changes in protein glycosylation have been observed in several cancers, where they affect cellular growth behavior, invasiveness and acquisition of metastatic potential. Such changes are not random and have been found to associate with cancer aggressiveness. Thus, detection of protein glycosylation may offer novel opportunities for cancer biomarker development. Since prostate-specific antigen (PSA) is glycosylated, assessing different PSA-glycoforms may provide valuable diagnostic and prognostic information. We recently established a novel in situ proximity ligation-based method for the detection of different protein-glycoforms in tissue sections. This method utilizes protein-specific antibody and glycan-binding lectins. We have optimized this method for the detection of different PSA-glycoforms in formalin-fixed paraffin-embedded prostatic tumor samples. We found that PSA is, indeed, differently glycosylated in prostate cancer (n=23) compared to benign prostate tissue (n=4). Two of the 25 studied lectins showed prominent staining only in cancer tissues (p<0.0001 for both), although the total PSA staining was stronger in benign tissues (p=0.006). We are currently validating these results in large, well-curated clinical cohorts, with intention to test whether PSA-glycoforms in tissue offer improved detection of clinically significant prostate cancer. These studies facilitate development of tools for identification of cancer-specific PSA-isoforms in serum samples. Detection of such isoforms is likely to provide independent diagnostic and prognostic information.

Citation Format: Hannu Koistinen, Ruusu-Maaria Kovanen, Timo-Pekka Lehto, Antti Rannikko, Tuomas Mirtti. Cancer associated PSA-glycoforms as prostate cancer markers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5551.
Abstract 5552: MAGE-A3 and MAGE-A4 protein show co-expression in tumor of lung, bladder, and colon cancer
Authors: Gonzalez Rachel
Published: June 2023

Melanoma Antigen Gene Family (MAGE-A) are part of the cancer-testis antigens whose limited expression in normal tissues and high expression in cancer make them excellent targets for immunotherapy. Clinical trials have already begun targeting MAGE-A3 and MAGE-A4 proteins in tumors. Should these trials lead to new treatment protocols, it is important to develop a diagnostic immunohistochemistry (IHC) tool for pre-screening patients who would benefit. The MAGE-A family consists of 12 members that share up to 85% sequence homology presenting a challenge for finding highly specific antibodies for IHC. Using CytoSections, a new screening control tool for IHC, ICC, IF, and in-situ hybridization, highly specific IHC MAGE-A3 and MAGE-A4 antibodies were developed and screened. The MAGE-A3 and MAGE-A4 antibodies were assessed on twenty-two lung cancers, twenty-one colon cancer, and more than thirty bladder cancers which resulted in cases that co-express MAGE-A3 and MAGE-A4 proteins in all three types of tumors. Using immunofluorescence, tumors positive for both proteins were double stained for MAGE-A3 and MAGE-A4 to show the proteins were co-expressed in the same tumor cells. Immunohistochemistry continues to be a rapid and reproducible method for the detection of proteins in tumors. Antibodies specific to MAGE-A3 and A4 proteins for IHC may be a useful tool in predicting outcomes or benefits for patients.

Citation Format: Rachel Gonzalez. MAGE-A3 and MAGE-A4 protein show co-expression in tumor of lung, bladder, and colon cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5552.
Abstract 557: EGFR-targeted mesenchymal stem cells for enhanced tumor retention and inhibition
Authors: Nethi Susheelkumar , Bhatnagar Shubhmita , Li Xiaolei , Prabha Swayam
Published: June 2023

MSCs have been studied as gene therapy vehicles, and several preclinical and clinical studies support the use of genetically modified MSCs to deliver therapeutics to tumor tissues. We were one of the early groups to demonstrate that MSCs can be engineered to carry cytotoxic drugs by using nanoparticle-encapsulated form of the drug. However, tumor homing of MSCs is inefficient. We hypothesized that the targeting effectiveness of MSCs can be significantly improved by incorporating tumor-targeting ligands on the MSC surface that will allow for enhanced arrest and binding within the tumor tissue. In this study, we designed anti-EGFR Ab (cetuximab, Cmab) functionalized MSCs and studied the comparative therapeutic efficacy of paclitaxel-loaded, Cmab functionalized MSCs relative to that of paclitaxel-loaded, unfunctionalized MSCs. Cmab functionalized MSCs demonstrated improved binding to A549 adenocarcinoma cells. Mice bearing orthotopic A549 tumors were dosed i.v. with the various treatments. Similar to what we have shown previously, MSCs loaded with paclitaxel nanoparticles were effective in slowing tumor growth and improving the overall survival relative to that of other controls. However, the greatest inhibition of tumor growth was observed in mice that received anti-EGFR Ab functionalized, paclitaxel-loaded MSCs. Compared to a median survival of 21 days for saline and 27 days for paclitaxel-loaded MSC treated group and paclitaxel-loaded IgG functionalized MSCs, Cmab Ab functionalized, paclitaxel-loaded MSCs treated mice resulted in significantly improved median survival of 35 days (p<0.001). Biodistribution studies revealed a sixfold higher retention of functionalized MSCs when compared to unfunctionalized MSCs. These results demonstrate the potential for improved tumor targeting with EGFR-targeted MSCs.

Citation Format: Susheelkumar Nethi, Shubhmita Bhatnagar, Xiaolei Li, Swayam Prabha. EGFR-targeted mesenchymal stem cells for enhanced tumor retention and inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 557.
Abstract 5585: Tissue based next generation sequence is more accurate than ctDNA to detected pathogenic variants in MTAP wild type tumors
Authors: De Souza Andre Luiz
Published: June 2023

Next generation sequencing (NGS) of circulating tumor DNA (ctDNA) prevents invasive biopsies and captures tumor heterogeneity. In a prior report by Zhang et al (Nat Commun 2021), certain actionable mutations were more prevalent in ctDNA than tissue-based databases (e.g., EGFR 48% versus 25.2%). Our group investigated if discrepancies in patterns of pathogenic variants between liquid biopsy and tissue specimens in a subtype of metastatic urothelial carcinoma (MTAP deleted) can inform the best approach of utilizing tissue or blood. MTAP (methyl-adenosine phosphorylase) is an enzyme involved in salvage purine biosynthesis and its deletion confers sensitivity to pemetrexed and taxanes, but resistance to immune checkpoint inhibitors. The MTAP gene is deleted in 25% of urothelial carcinomas. We report herein the pattern of pathogenic variants from ctDNA in 17 patients with metastatic urothelial carcinoma, 10 with MTAP-deletion and 17 with MTAP wild-type. Among 10 patients with MTAP-deleted urothelial carcinoma with collected ctDNA, 3 had no detected pathogenic variant. The most common pathogenic variants in 7 patients were TP53 (4/7, 57%), TERT (4/7, 57%), ERBB2 (2/7, 28%); PIK3CA (2/7, 28%), and NFE2L2 (2/7, 28%). The mutations found in paired ctDNA and tissue based NGS were TP53 in 26.6 % (4/15), TERT in 21.0 % (4/19), ERBB2 in 66.6 % (2/3), PIK3CA in 50 % (2/4), and NFE2L2 in 100% (1/1) of patients. Three of the 10 patients with collected ctDNA had ELF3 frameshift loss-of- function alterations on tissue not found on ctDNA. Among 54 patients with MTAP wild-type urothelial carcinoma, 26 had collected ctDNA. Among these 26 patients, 9 had no detected pathogenic variants. Among the 17 patients with pathogenic variants, the most common mutations were TP53 (76%, 13/17), TERT (47%, 8/17), ERBB2 (24%, 4/17), RB1 (24%, 4/17), and BRCA1 or BRCA2 (24%, 4/17). Three of these 17 patients had no or insufficient tissue for NGS. The mutations found in paired ctDNA and tissue based NGS were TP53 in 100% (14/14), TERT in 64% (9/14), ERBB2 in 43% (6/14), RB1 in 29% (5/14), and BRCA1 or BRCA2 in 36% (5/14) of patients. MTAP wild-type patients had a higher frequency of TERT alterations (p=0.012), but not ERBB2, (p=0.453) than patients with MTAP-deleted type tumors. Ongoing evaluation of timing and location of pathogenic variants will inform where (primary tumor, metastatic site) and when (after chemotherapy, immune checkpoint inhibitors, antibody drug conjugates) to biopsy patients on progression after cancer therapies.

Citation Format: Andre Luiz De Souza. Tissue based next generation sequence is more accurate than ctDNA to detected pathogenic variants in MTAP wild type tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5585.
Abstract 5663: Discovery and characterization of anti-LILRB2 antibody SPX-104
Authors: Haight Anthony , Chen Qian , Rodriguez Ernesto , Siekierzycki Martin , Hall Victoria , Williams Brandon , Shi Jun , Nguyen Dinh-Duc , Cai Robert , Ma Jichun , White Michael , Qin Jingdong , Shen Jadon , Zhao Chuanke , Rong Zhuona , Meng Lin , Shou Chengchao
Published: June 2023

T-cell based immunotherapies have produced durable remission in certain cancer patients, but owing at least partially to immunosuppression in the tumor microenvironment, response rates with current therapies remains challenging. Reversing the immunosuppression is a recognized pathway for enhancing the activity of existing IO therapies. LILRB2 (ILT4) is a key receptor for macrophage maturation and polarization and an emerging therapeutic target in immuno-oncology. This research aims to produce anti-LILRB2 antibodies to redirect the polarization of myeloid cells, specifically macrophages, in the tumor microenvironment. The LILRB2 antibodies were generated through the mouse hybridoma method. After humanization and maturation, potent and selective IgG4 anti-LILRB2 antibodies were obtained. Binding affinities were evaluated through ELISA and 293T-LILRB2 cell flow cytometry binding assays which demonstrated sub-nanomolar binding affinity as well as effective blocking of the interaction of LILRB2 with its ligands HLA-G and HLA-A2 for multiple antibodies. SPX-104 was shown to redirect macrophage polarization in primary cultured human PBMC and human monocyte derived macrophage (HMDM) models. SPX-104 treatment induced hTNFa expression and suppressed hIL10 expression in the LPS stimulated PBMC model. In the HMDM model, SPX-104 effectively induced pro-inflammatory cytokine hTNFα expression in M-CSF. Furthermore, an autologous mixed lymphocyte reaction (auto-MLR) assay to evaluate T cell response showed dose-dependent increases in hIFNγ and hGM-CSF expression following SPX-104 treatment. In a CD34-humanized hLILRB2 transgenic mouse efficacy model, SPX-104 in combination with an immune checkpoint inhibitor, effectively inhibited tumor growth. These results confirm enhanced T cell activity following macrophage redirected polarization. The in vitro and in vivo evaluation, of SPX-104 relative to other anti-LILRB2 antibodies will be presented.

Citation Format: Anthony Haight, Qian Chen, Ernesto Rodriguez, Martin Siekierzycki, Victoria Hall, Brandon Williams, Jun Shi, Dinh-Duc Nguyen, Robert Cai, Jichun Ma, Michael White, Jingdong Qin, Jadon Shen, Chuanke Zhao, Zhuona Rong, Lin Meng, Chengchao Shou. Discovery and characterization of anti-LILRB2 antibody SPX-104. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5663.
Abstract 5681: Tetra-specific antibody GNC-038: guidance and navigation gontrol (GNC) molecule development for treatment of CD19+ malignancies
Authors: Khalili Jahan Salar , Xiao Sa , Zhu Yi
Published: June 2023

B cell malignancies treated with CD19-directed immunotherapies can relapse, in some cases due to clonal selection for reduced CD19 antigen expression or enhancement of immunosuppressive phenotypes. Here we demonstrate that a Guidance and Navigation Control (GNC) tetra-specific antibody, GNC-038, binds to CD19, CD3, PD-L1, and 4-1BB and mediates cytolysis of human leukemia and lymphoma cells by T cells.

Redirected T cell cytolysis (RTCC) occurs in the presence of GNC-038 (Emfizatamab), resulting in the killing of CD19+ leukemia and lymphoma cell lines. The cytolytic functions induced by GNC-038 are similar to Blinatumomab in vitro, as indicated by T cell degranulation and production of IFN-gamma. Human T cells in PBMC exposed to GNC-038 in vitro proliferate in a dose-dependent fashion. Proliferation is further enhanced upon rechallenge with leukemic target cells. Proliferation of T cells from individuals with higher % PD-1+ and Effector polarized compartments is enhanced by GNC-038 compared to Blinatumomab. To evaluate the contribution of each binding domain of GNC-038 in mediating RTCC function, versions of GNC-038 were prepared, replacing each antigen binding domain with anti-FITC binding domains. Under assay conditions using PBMC for RTCC toward the CD19+ target cell line Nalm-6, the results demonstrate the contribution of each domain to the overall, anti-leukemic cytolytic activity.

To evaluate the potential for GNC-038 to mediate cytokine release syndrome, the molecule is evaluated in soluble, and plate bound formats in the presence of PBMC and CD19+ leukemic targets cells. In comparison to Blinatumomab, the production of cytokines is comparable, with some notable differences. PBMC exposed to soluble GNC-038 for 48 hours produced more IFN-γ, IL-2 and TNF-α, while showing no significant difference in production of IL-6. Based on these results, the primary indicator of CRS, IL-6, does not suggest increased risk compared to Blinatumomab, while the type of T cell activity induced by GNC-038 in PBMC with leukemia cells is distinct.

Collectively, the GNC-038 represents a class of multi-specific and multi-modal immune cell engagers with potential to mediate CD19+ cancer killing, while also increasing the T cell compartment’s therapeutic potential to respond to T cell redirection upon subsequent cycles of therapy. The clinical phase I-b study of GNC-038 is under way and the available data exhibit strong signals of efficacy with acceptable tolerability.

Citation Format: Jahan Salar Khalili, Sa Xiao, Yi Zhu. Tetra-specific antibody GNC-038: guidance and navigation gontrol (GNC) molecule development for treatment of CD19+ malignancies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5681.
Abstract 5822: YAP inhibition via targeting MAP3K3-dependent lysosomal degradation pathway overcomes BRAF and CDK4/6 inhibitor resistance
Authors: Ryu Won-Ji , Park Sanghyun , Kim Tae Yeong , Hwang Yumi , Han Hyun Ju , Lee Jeong Dong , Kim Gun Min , Sohn Joohyuk , Kim Sang Kyum , Kim Joon , Kim Min Hwan
Published: June 2023

The Hippo-yes-associated protein (YAP) signaling pathway activation underlies the resistance to a wide range of molecularly targeted anti-cancer agents. However, strategies to block YAP activity in cancer by promoting YAP degradation have been largely unsuccessful despite rigorous investigations into canonical Hippo-YAP regulatory mechanisms. Here, we report a novel mechanism of YAP stability regulation by MAP3K3 independently of LATS-mediated proteasomal degradation. We observed that MAP3K3 depletion significantly reduced YAP protein levels in both normal and malignant cells, and mass spectrometry analysis revealed that MAP3K3 phosphorylates YAP at its serine 405 residue. MAP3K3-mediated serine 405 YAP phosphorylation polarizes YAP interaction with BTRC while hindering its interaction with FBXW7, collectively preventing both proteasomal degradation and the p62-mediated lysosomal degradation of YAP. We investigated the role of this mechanism in overcoming anti-cancer resistance and observed robust YAP activation in CDK4/6 inhibitor-resistant luminal breast cancer cells. Genetic and pharmacological MAP3K3 inhibition suppressed YAP-induced drug resistance. Moreover, increased MAP3K3 expression supported the pro-survival activity of YAP in BRAF inhibitor-resistant melanoma cells, and MAP3K3 depletion downregulated YAP-dependent cell cycle gene signatures. Accordingly, the combination of vemurafenib and ponatinib was effective in reducing the survival of BRAF inhibitor-resistant melanoma cells. Collectively, our study identified a novel phosphorylation-mediated YAP stability regulation mechanism and underscored MAP3K3 inhibition as a promising strategy to overcome BRAF and CDK4/6 inhibitor resistance by directly promoting YAP degradation.

Citation Format: Won-Ji Ryu, Sanghyun Park, Tae Yeong Kim, Yumi Hwang, Hyun Ju Han, Jeong Dong Lee, Gun Min Kim, Joohyuk Sohn, Sang Kyum Kim, Joon Kim, Min Hwan Kim. YAP inhibition via targeting MAP3K3-dependent lysosomal degradation pathway overcomes BRAF and CDK4/6 inhibitor resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5822.
Abstract 5824: Human ovarian cancer cell seeding density as a mechanism of cisplatin resistance associated with expression of anti-apoptotic and autophagy proteins
Authors: Brown Thomas W. , Punyamurtula Ujwal , El-Deiry Wafik S.
Published: June 2023

Drug effects may vary depending on cancer cell density. We investigated discrepancies in phenotypes for high density and low-density cultured cancer cells and elucidated relevant variables that influence them. We assessed chemosensitivity as a function of tumor cell density by plating TOV-21G and OVMANA ovarian cancer cell lines in 96-well plates at various seeding densities per well and treated them with various concentrations of cisplatin. CellTiterGlo assays were performed at 72 hours of treatment to determine cisplatin dose response curves. We used ethidium homodimer fluorescence staining and imaging to quantify extent of cell death at differing cell densities of TOV-21G and OVMANA cells treated with cisplatin for 48 and 72hours in 48-well plates. We performed western blot analyses of apoptotic and autophagy proteins from TOV-21G and OVMANA cells that were seeded at high and low cell densities in 24-wellplates. TOV-21G and OVMANA cells plated at higher cell seeding densities displayed less cisplatin sensitivity than TOV-21G and OVMANA cells plated at lower seeding densities, as shown by cisplatin dose response curves. Ethidium homodimer staining of TOV-21G and OVMANA cells treated with cisplatin revealed that cells seeded at lower densities experience a greater amount of cell death that cells seeded at higher densities. Western blot analyses showed that high density TOV-21G cells may express greater amounts of BCL2 and c-FLIP and lower amounts of p62 and NOXA compared to low density cells when normalized for protein expression. Similarly, high density OVMANA cells express greater amounts of c-FLIP andBCL2 and lower amounts of NOXA compared to low density cells when normalized for protein expression. Our experiments reveal that human ovarian cancer cells display different phenotypic changes depending on cell density, causing higher density cells to require increased cisplatin to achieve the same level of effect seen in lower density cells. Ovarian cancer cell resistance to cisplatin is associated with alterations in autophagy and anti-apoptotic protein expression at high cell seeding density. These alterations may also affect chemosensitivity in vivo in settings of differential tumor density, such as single circulating tumor cells compared to clumps of tumor cells, and other settings, such as metastasis.

Citation Format: Thomas W. Brown, Ujwal Punyamurtula, Wafik S. El-Deiry. Human ovarian cancer cell seeding density as a mechanism of cisplatin resistance associated with expression of anti-apoptotic and autophagy proteins. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5824.
Abstract 5833: Crosstalk between cancer-associated fibroblasts and CD8 T cells leads to improved T cell activation in head and neck cancer
Authors: Graves Diana , Obradovic Aleksandar , Schares Henry , Korrer Michael , Bachmann Brian , Kim Young , Park Ben , Hurley Paula
Published: June 2023

Cancer-associated fibroblasts (CAFs) have been shown to modulate immune responses in the tumor microenvironment (TME), however, the extensive heterogeneity of these cells has precluded rigorous understanding of their immunoregulatory role in the head and neck TME. To gain a better understanding of the relationship between CAFs and the immune environment in head and neck cancer, we leveraged protein activity inference tools to analyze single-cell RNA sequencing of advanced-stage head and neck squamous cell carcinoma (HNSCC) patients pre- and post-treatment with the αPD-1 therapy, nivolumab. From this analysis, we identified a novel CAF population predictive of patient response to αPD-1 therapy. Utilizing in vitro co-culture assays with primary human CD8 T cells, we discovered that this novel CAF population was able to elicit features associated with enhanced CD8 T cell function, namely the reduction of PD-1+TIM-3+ exhaustive phenotypes and induction of CD103+NKG2A+ tissue-resident memory phenotypes and cytotoxicity. Due to their unique ability to stimulate T cells in this manner, we termed this novel CAF population T cell-stimulating CAFs (tsCAFs). In efforts to elucidate the mechanism behind this pro-inflammatory function, we determined that tsCAFs secrete a heat-sensitive small molecule/s in order to alter CD8 T cell behavior. However, tsCAFs will only secrete this factor in the presence of activated T cells demonstrating crosstalk between T cells and CAFs in the head and neck TME. This pro-inflammatory crosstalk between CAFs and T cells represents a novel finding that further contributes to the idea of immunoregulatory pleiotropy among CAFs. Accordingly, our findings highlight the functional importance of distinct CAF subsets in modulating the immunoregulatory milieu of human HNSCC and warrant more extensive characterization of CAF-T cell interactions in human squamous cell carcinoma.

Citation Format: Diana Graves, Aleksandar Obradovic, Henry Schares, Michael Korrer, Brian Bachmann, Young Kim, Ben Park, Paula Hurley. Crosstalk between cancer-associated fibroblasts and CD8 T cells leads to improved T cell activation in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5833.
Abstract 6244: Design, synthesis, and structure-activity relationships of substituted pyrido[3,2-d]pyrimidines as microtubule targeting agents and anti-cancer agents
Authors: Shah Yesha Vijaykumar , Bai Ruoli , Hamel Ernest , Gangjee Aleem
Published: June 2023

Microtubules (MTs) are composed of αβ-tubulin heterodimers and are essential components of the cellular cytoskeleton involved in critical cellular functions, including mitosis, cell migration and signaling, intracellular trafficking, and angiogenesis. Microtubule targeting agents (MTAs) are highly successful chemotherapeutic agents because they target MT dynamics and interfere with MT-dependent cellular processes. The clinical efficacy of MTAs is limited by multidrug resistance due to expression of the P-glycoprotein (Pgp) drug efflux pump or increased expression of βIII-tubulin. This necessitates the development of novel MTAs effective against cell lines expressing these forms of drug resistance, which could offer potential advantages in patients who fail to respond to current MTAs, such as paclitaxel. Previously, we reported colchicine site (CS) binding compound 1 with a pyrido[3,2-d]pyrimidine scaffold and a 2-H substitution and N4-4’-methoxyaniline substitution inhibited colchicine binding and tubulin assembly (87% inhibition at 5 µM and IC50 = 1.3 µM, respectively). Compounds 2-9 were designed with a 2-CH3 substitution to evaluate the structure-activity relationships at the 4-position by using conformational restriction and isosteric replacements. Compounds 2-9 were synthesized, and the biological evaluation showed that compounds 2-9 inhibited tubulin assembly (7, IC50 = 0.72 μM) and binding of colchicine to tubulin (7, 97% inhibition at 5 µM, 81% at 0.5 µM). Compound 7 was found to be highly potent in the MCF-7 breast cancer cell line with an IC50 of 2.5 nM which was ~3.5-fold better than combretastatin-A4 (CA4). Compound 7 inhibited the growth of the human ovarian cancer cell line OVCAR-8 with an IC50 = 3.0 nM. For the Pgp overexpressing cell line NCI/ADR-RES, 7 was equally potent (IC50 = 3.0 nM), whereas paclitaxel was almost inactive (IC50 = 1,500 nM). Thus, we have identified 2-CH3 substituted pyrido[3,2-d]pyrimidines as a novel structural scaffold for inhibition of tubulin assembly and colchicine binding that have potent cytotoxic activity against the breast cancer cell line MCF-7 and against both the parental ovarian cancer cell line OVCAR-8 and its derivative Pgp overexpressing cell line NCI/ADR-RES. These compounds thus merit further study as potential cancer chemotherapeutic agents.

Citation Format: Yesha Vijaykumar Shah, Ruoli Bai, Ernest Hamel, Aleem Gangjee. Design, synthesis, and structure-activity relationships of substituted pyrido[3,2-d]pyrimidines as microtubule targeting agents and anti-cancer agents. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6244.
Abstract 6257: Developing HDAC inhibitors as immunomodulators
Authors: Colligan Sean , Shen Li , Rupert Christopher , Bard Jonathan , Pili Roberto
Published: June 2023

Immune checkpoint inhibitors (ICIs) are standard of care for several solid tumor types, including renal cell carcinoma (RCC). However, the clinical success of these therapies is dampened by the fact that the majority of patients demonstrate either inherent resistance to treatment or develop secondary resistance following an initial response. One key mechanism of tumor-driven ICI resistance is the generation and recruitment of suppressive immune cell types, including regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), which coordinate to suppress anti-tumor T cell responses. Thus, there is a critical need for the development of novel combination strategies aimed at mitigating the presence and function of suppressive immune cells to improve the efficacy of ICIs in the clinic. Our laboratory has previously demonstrated that histone deacetylase inhibition (HDACi) is associated with a decrease in both the number and function of tumor-infiltrating Tregs and MDSCs in the RENCA tumor model. Thus, the overall goal of these studies both preclinically and clinically is to investigate the impact of the immunomodulatory effects of HDACi in combination with ICI therapy, with the overall hypothesis that HDACi will improve the efficacy of anti-PD1 therapy in RCC. Our pre-clinical data support this hypothesis by demonstrating increased tumor control and survival in RENCA tumor-bearing mice when treated with a combination of anti-PD1 and HDACi compared to single agent therapy. These increases were also associated with an increase in the activation state of anti-tumor immune cells, as demonstrated by increased surface expression of MHC Class II molecules on macrophages and a decrease in phenotypically defined monocytic MDSCs. ATACseq and RNAseq data generated from combination-treated animals also outline a gene expression signature associated with both myeloid and T cell activation. Interestingly, our clinical studies also afforded us the opportunity to investigate potential biomarkers associated with responder status, which will allow us to better predict patient outcomes and administer therapies accordingly. Accumulating evidence from two independent clinical trials run by our group suggests that high levels of suppressive CD11b+HLA-DRlo MDSCs at baseline in the peripheral blood is associated with lack of clinical response to the combination of HDACi and ICI. Overall, these data support the rationale for the combination of ICI and HDACi in the clinic, and provide a potentially useful biomarker through which we can predict the success of combination therapy.

Citation Format: Sean Colligan, Li Shen, Christopher Rupert, Jonathan Bard, Roberto Pili. Developing HDAC inhibitors as immunomodulators [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6257.
Abstract 628: Liver macrophages promote breast cancer liver metastasis through migrating neutrophils and initiating NETosis
Authors: Li Heliang , Yang Linbin , Song Erwei
Published: June 2023

Neutrophils extracellular traps (NETs), which are DNA scaffolds in complex with granule proteins, could be detected in the pre-metastatic liver niches and were associated with a poor survival in breast cancer patients. However, the mechanisms of neutrophils infiltration and NETs formation in the pre-metastatic liver tissues remain poorly understood. Here, we dynamically characterized the liver micro-environment of normal and 4T1-bearing BALB/C mouse models with or without liver metastasis by the single cell RNA sequencing. Interestingly, we found that CXCL1 and CXCL2, derived from the liver macrophages, were dramatically elevated in pre-metastatic livers and induced the infiltration of peripheral neutrophils by CXCR2. Besides, liver macrophages were educated by cancer cells-derived cathepsin C to secrete the CXCL1 and CXCL2. Furthermore, we identified that the complement signals were over-activated in pre-metastatic livers and induced neutrophils to NETosis. Therapeutically, targeting CXCL1, CXCL2 and complement signals could inhibit the NET formation and effectively reduce breast cancer liver metastasis.

Citation Format: Heliang Li, Linbin Yang, Erwei Song. Liver macrophages promote breast cancer liver metastasis through migrating neutrophils and initiating NETosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 628.
Abstract 6304: Preclinical development of a next generation antibody drug conjugate (ADC) targeting B7-H3 for treatment of solid tumors
Authors: Xu Jian , Zong Qing , Wang Wei , Liu Qigang , Stann Sasha , Cai Jiaqiang
Published: June 2023

B7-H3 (CD276), an immune checkpoint member of the B7/CD28 family, is widely expressed in multiple cancers and is associated with poor prognosis. YL201 is a novel B7-H3-targeting antibody-drug conjugate (ADC) and built on MediLink’s tumor microenvironment activable linker-payload platform (TMALIN platform). This ADC is comprised of an anti-B7-H3 human monoclonal antibody conjugated to a novel topoisomerase 1 inhibitor via a protease-cleavable linker.

The novel linker-payload and site-specific conjugation of YL201 results in a homogeneous and more hydrophilic ADC. In addition, the binding specificity, affinity and avidity of antibodies show no impact after conjugation. In vitro, upon binding to B7-H3, the antigen-ADC complexes are internalized to lysosomal vesicles releasing the toxic payload, whose cytotoxic features drive cell cycle arrest, apoptosis, elevation of PARP/caspase 7 expression, as well as cell death and bystander effect. In preclinical studies, YL201 is effective in causing tumor regressions in both cell line- and patient-derived xenograft (CDX and PDX) mouse models at doses that are well tolerated. The in vivo pharmacokinetics in monkeys showed that YL201 is deemed as ‘highly stable’ in circulation. Safety evaluations demonstrated acceptable safety profile and no off-target toxicity in monkeys. These non-clinical data suggest the stability of linker in circulation but efficiently release the payload in tumors, and eventually, expanded the therapeutic window.

Taken together, preclinical data suggest that YL201 could be a promising treatment strategy for B7-H3 positive cancer patients.

Citation Format: Jian Xu, Qing Zong, Wei Wang, Qigang Liu, Sasha Stann, Jiaqiang Cai. Preclinical development of a next generation antibody drug conjugate (ADC) targeting B7-H3 for treatment of solid tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6304.
Abstract 6306: Pharmacological deuteration of SCID mice using the water-isotopologue deuterium oxide (D2O) inhibits tumor growth in bioluminescent models of human malignant melanoma and pancreatic ductal adenocarcinoma
Authors: Jandova Jana , Galons Jean-Philippe , Dettman David L. , Wondrak Georg T.
Published: June 2023

Since its initial discovery as a natural isotopologue of dihydrogen oxide (1H2O), extensive research has focused on the biophysical, biochemical, and pharmacological effects of deuterated water [2H2O (D2O, also referred to as ‘heavy water’)]. Here, using diverse panels of cultured human malignant melanoma and pancreatic ductal adenocarcinoma (PDAC) cells we have profiled (i) D2O-induced phenotypic anti-proliferative and apoptogenic effects, (ii) redox- and proteotoxicity-directed stress response gene expression, and (iii) phosphoprotein-signaling related to endoplasmic reticulum (ER) and MAP-kinase stress response pathways. Differential RT-qPCR array analysis revealed early modulation of stress response gene expression elicited by D2O (90%; ≤6 h) confirmed by independent RT-qPCR analysis. Immunoblot-analysis revealed rapid (< 6 h) onset of D2O-induced MAP-kinase signaling (p-JNK) together with ER stress response upregulation (p-eIF2α, ATF4, XBP1s, DDIT3/CHOP) attributable to deuteration-induced alteration of hydrogen bonding triggering proteotoxic stress. Next, we tested the chemotherapeutic efficacy of D2O-based drinking water supplementation in bioluminescent murine models of malignancy (A375-luc melanoma and BxPC-3-luc orthotopic PDAC xenografts in SCID mice). Time course of systemic deuteration (30% D2O in drinking water fed continuously for up to 21 days) was established using time-resolved whole-body proton magnetic resonance imaging (MRI) and isotope-ratio mass spectrometry (IR-MS)-based plasma (D/H)-analysis. In both models, D2O supplementation significantly suppressed tumor growth and metastasis with downregulated expression of PCNA/Ki67 while increasing tumor levels of DDIT3/CHOP, HO-1, and p-eIF2α. Taken together, these data demonstrate for the first time that pharmacological induction of systemic deuteration by oral administration of D2O, associated with induction of cellular ER stress, reduces tumor burden and metastasis in murine models of human malignant melanoma and PDAC.

Citation Format: Jana Jandova, Jean-Philippe Galons, David L. Dettman, Georg T. Wondrak. Pharmacological deuteration of SCID mice using the water-isotopologue deuterium oxide (D2O) inhibits tumor growth in bioluminescent models of human malignant melanoma and pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6306.