bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2475: Bromodomain inhibitor: MT1 and its potential role in modulation of prostate cancer progression
Authors: Shukla Sanjeev , Al-Toubat Mohammed , Feibus Allison H. , Elshafei Ahmed , Riveros Carlos , Meurice Nathalie , Gleba Justyna , Chardon-Robles Jonathan , Kase Adam M. , III John A. Copland , Petit Joachim L. , Osumi Teruko , Balaji K.C.
Published: April 2023

Bromodomains (BD) are epigenetic readers of histone acetylation involved in chromatin remodeling and transcriptional regulation of protooncogene cellular myelocytomatosis (c- Myc) and other genes. Because c-Myc cannot be directly targeted by small molecular inhibitors due to disordered alpha helical structure, epigenetic targeting of c-Myc by BD inhibitors is an attractive therapeutic strategy for diseases such as prostate cancer (PC) associated with increased c-Myc upregulation with advancing disease. We studied the efficacy of MT1, a novel bivalent BD inhibitor that is 100-fold more potent than the first in class BD inhibitor JQ1, at inhibiting PC growth. We tested the effect on viability by MT-1 on PC cell lines, 3D spheroids derived from clinically annotated drug resistant patient derived xenografts (PDX), mice PDX models and corroborated the molecular mechanism of MT1 down regulation of Myc leading to downstream Myc-dependent up regulation of Protein Kinase D1 (PrKD) substrate phosphorylation by western blot. MT-1 inhibited growth of PC in castration sensitive (LNCaP) and resistant PC cells (PC-3). MT-1 treatment upregulated PrKD expression and phosphorylation of known PrKD substrates: threonine 120 (Thr-120) residues in beta-catenin and the serine 216 in Cell Division Cycle 25 (CDC25C) in PC-3 cells. Moreover, MT-1 was effective in inhibition of 3D spheroids growth at IC 50 between 0.27-0.92µM in Abiraterone, Enzalutamide, Docetaxel, Cabazitaxel metastatic castrate resistant PCa patient-derived tumor 3D spheroids. Additionally, MT1 was effective in inhibiting the tumor growth in PDX mice model. A combined intra-peritoneal administration of MT-1 with another c-Myc inhibitor (3JC48-3), an obligate c-Myc and MYC-associated protein X (MAX) heterodimerization inhibitor, increased the efficacy of inhibiting the PDX growth in mice. This study provides strong pre-clinical in vitro and in vivo evidence for advancing MT- 1 as a novel c-Myc targeting drug in PC. The MT-1 drug development will likely be highly impactful as c-Myc is dysregulated in three fourths of men with advanced PC.

Citation Format: Sanjeev Shukla, Mohammed Al-Toubat, Allison H. Feibus, Ahmed Elshafei, Carlos Riveros, Nathalie Meurice, Justyna Gleba, Jonathan Chardon-Robles, Adam M. Kase, John A. Copland III, Joachim L. Petit, Teruko Osumi, K.C. Balaji. Bromodomain inhibitor: MT1 and its potential role in modulation of prostate cancer progression [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 2475.
Abstract 2476: Nischarin is a potential druggable target in both epithelial and stromal compartments of pancreatic ductal adenocarcinoma
Authors: Grahovac Jelena , Živić Kristina , Pavlović Marijana , Ostojić Marija , Đurić Ana , Srdić-Rajić Tatjana , Pavić Aleksandar , Galun Danijel
Published: April 2023

Nischarin is a scaffolding protein involved in the regulation of cell cytoskeletal organization and metabolic homeostasis. In breast and ovarian cancers, nischarin has been demonstrated to have tumor suppressive functions. We have found that nischarin was expressed in both the epithelial and stromal compartments of the PDAC patient tissue, in cancer cell lines and patient-derived stellate cells. Of interest, nischarin is a functional imidazoline 1 receptor for which there are several FDA-approved agonists used for treatment of hypertension. The aim of this study was to examine the effects of nischarin agonists on PDAC cancer cells and stellate cells, separately and in co-culture and determine the potential for their repurposing in treatment of PDAC. mRNA sequencing revealed that cancer cell treatment with nischarin agonist rilmenidine induced transcriptional changes associated with regulation of cell adhesion, EMT and vesicular transport. In vitro, rilmenidine treatment of cancer cells decreased their migration and invasion potential and treatment of stellate cells decreased the αSMA-positive fraction. Treatment of cancer cell-stellate cell co-cultures decreased the production of pro-inflammatory cytokines IL-6, IL-8 and CCL-2, known drivers of the metastatic progression. Ultimately, rilmenidine treatment decreased PDAC cell invasion in Tg(fli1: EGFP) zebrafish model. Taken together, nischarin agonist rilmenidine has a potential for limiting the processes involved in PDAC metastatic cascade and may be a good candidate for drug repurposing.

Citation Format: Jelena Grahovac, Kristina Živić, Marijana Pavlović, Marija Ostojić, Ana Đurić, Tatjana Srdić-Rajić, Aleksandar Pavić, Danijel Galun. Nischarin is a potential druggable target in both epithelial and stromal compartments of 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 2476.
Abstract 2565: Divergentnucleocytoplasmic transport influences escape from HER2-activated DCIS-like state
Authors: Wang Lixin , Paudel Bishal , McKnight Anthony , Janes Kevin
Published: April 2023

HER2/ErbB2 and EGFR/ErbB1 activation incite a phenotype resembling premalignant escape from ductal carcinoma in situ (DCIS) in the 3D multicellular arrangement of breast-epithelial cells. This phenomenon tends to be infrequent, and the exact mechanism for its heterogeneous presentation has been elusive. We sought to investigate the process by which ErbB receptors facilitate a picture of incomplete penetrance in DCIS escape (DE). To identify the transcriptional signatures that prime cells towards DE, we randomly profiled 10-cell transcriptomes of single spheroids after 24 hours of ERBB activation, and frequency matched gene expression to the DE-phenotype. We uncovered a network of nucleocytoplasmic transport (NCT) regulators that alter the DE-phenotype penetrance. Of the regulators, CSE1L (an exportin) synergistically increases the DE-frequency when induced, while its knockdown reduces the phenotype, suggesting its functional role. Furthermore, we evaluated the significance of this result in vivo by perturbing Cse1l in Erbb2-amplified mouse mammary-gland tumors and found that activation of Erbb2 (mimicked by ERBB-inhibitor release) alters the macroscale organization of tumors, reminiscent of DE. Specifically, we found an elevated CSE1L inhibits an accumulation of classical nuclear-localizing cargoes that require binding with importin-α/β (KPNAs, KPNB1) complexes. Using proximity labeling with BirA*-fused with CSE1L, we identified ERBB1/ERBB2/ERBB4 as CSE1L interactors. Mechanistically, hetero-dimerized ERBB receptors interact with importin-α/β complexes and re-localize to the nucleus, corroborating previous observations of receptors’ nuclear translocation. Intriguingly, we observed that Doxycycline-induced knockdown of clathrin heavy chain (CLTC, involved in intracellular trafficking of receptors via endocytosis) synergizes with ERBB activation to increase the DE-phenotype penetrance. Using ChIP-Seq, we found that ERBB1 binds to the locus of microRNA, miR205, which negatively regulates the expression of importin-α, mainly KPNA1. These results indicate that ERBB receptors translocated to the nucleus reduce the DE-phenotype by inhibiting importins via miR205, while CSE1L induction relieves this inhibition by reducing the receptor's nuclear accumulation. Taken together, these results show that nucleocytoplasmic shuttling of receptors in ERBB-active breast epithelia generates tunable cross-inhibitory feedback through nucleocytoplasmic transport that results in long-term, heterogeneous multicellular fates.

Citation Format: Lixin Wang, Bishal Paudel, Anthony McKnight, Kevin Janes. Divergentnucleocytoplasmic transport influences escape from HER2-activated DCIS-like state [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 2565.
Abstract 2566: Blocking the functional domain of TIP1 upregulates midkine via the β-catenin/Wnt signaling pathway
Authors: Saikia Minakshi , Singh Abhay K. , Cheung Nathan , Hallahan Dennis E. , Kapoor Vaishali
Published: April 2023

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortalities world-wide. Targeted therapy has been a growing topic of investigation to improve therapeutic efficacy for NSCLC. A few targeted therapies that exploit aberrant protein expression profiles have been approved for NSCLC. However, marginal improvement in efficacy observed with these therapeutic approaches highlights the need for discovery of additional therapeutic targets. Tax interacting protein 1 (TIP1) is a molecular target that is overexpressed on the surface of various cancers and correlates with poor overall patient survival. The functional domain of TIP1 (PDZ domain) caps the C-terminus of many cellular proteins that regulate cell signaling, cancer development, and progression. We found that antibodies against the PDZ domain of TIP1 induced cytotoxicity of lung cancer cells but not normal cells. A quantitative mass spectrometry-based approach identified a ten-fold increase in Midkine protein expression on blockade of TIP1. Midkine is a heparin-binding growth factor that is involved in cancer progression, therapy resistance, and immune suppression. This study tested the hypothesis that Midkine is upregulated by the anti-PDZ/TIP1 antibody via the β-catenin/Wnt signaling pathway, which subsequently modulates downstream signaling mechanisms. In co-immunoprecipitation studies we found a direct interaction of TIP1 with β-catenin on the cell surface. Anti-TIP1 antibody treatment led to nuclear translocation of β-catenin in NSCLC cells. We used a luciferase based TOPFlash/FOPFlash reporter assay to study the transcriptional activity of the canonical β-catenin/Wnt pathway following TIP1 blockade. Reporter assays showed a three to four-fold increase (p<0.0001) in the transcriptional activity of β-catenin following TIP1 blockade in NSCLC cells. Genetic silencing of β-catenin inhibited Midkine mRNA transcription (p<0.005), protein induction and its extracellular release (p<0.0001) following anti-TIP1 antibody treatment. In conclusion, the mechanism of Midkine release following TIP1 blockade involves the β-catenin/Wnt signaling pathway. Ongoing studies are evaluating the dual-targeting of TIP1 and Midkine for enhanced efficacy of NSCLC treatment.

Citation Format: Minakshi Saikia, Abhay K. Singh, Nathan Cheung, Dennis E. Hallahan, Vaishali Kapoor. Blocking the functional domain of TIP1 upregulates midkine via the β-catenin/Wnt signaling pathway [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 2566.
Abstract 2567: Exploring novel roles of the GPCR Smoothened in AKT signaling and drug resistance
Authors: Manuel Samantha , Agarwal Nitin , Bilbao Daniel , Vega Francisco , Landgraf Ralf
Published: April 2023

The Smoothened receptor (SMO) belongs to the frizzled-class-G-protein coupled receptor and its activity can be classified in the canonical and the non-canonical pathway. The canonical pathway concludes in the transcriptional activation of the Hedgehog target genes through the GLI family of transcription factors whereas the non-canonical pathway leads to the recruitment and activation of trimeric G proteins. Recently, we have reported two novel roles of SMO with relevance to cell survival and drug resistance in DLBCL. In the first, SMO promotes a shift in the ubiquitination pattern for TNF receptor-associated factor 6 (TRAF6) from degradation inducing K48-ubiquitin branching towards K63-ubiquitination. TRAF6 is a K63-Ub E3 ubiquitin ligase for AKT and K63-ubiquitination of AKT increases membrane recruitment, activation and stability. Evidently, SMO promotes the formation of a scaffold-like protein complex including the de-ubiquitinating enzyme Ubiquitin specific peptidase 8 (USP8) and TRAF6. In its second novel role, SMO influences the vesicular sorting of IGF1R and other receptor tyrosine kinases. Loss of SMO leads to increased IGF1R lysosomal degradation and reduced IGF1R recycling, while overexpression increases IGF1R levels and AKT signaling. These observations indicate SMO receptor biology outside of the established canonical and non-canonical pathway functions. We have set out to use BioID to investigate the SMO interactome that contributes to these novel functionalities using a fusion protein of SMO and the reengineered E. coli biotinyl transferase BirA (Turbo) as bait. Preliminary BioID using SMO-Turbo returned a list of candidates which will require further data filtering approaches, but thus far shows a marked enrichment for proteins involved in vesicular trafficking.

Citation Format: Samantha Manuel, Nitin Agarwal, Daniel Bilbao, Francisco Vega, Ralf Landgraf. Exploring novel roles of the GPCR Smoothened in AKT signaling and drug 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 2567.
Abstract 2582: Protein aggregation in sarcoma as a target for therapy
Authors: Sartini Sara , Le Hillary , Soragni Alice
Published: April 2023

Sarcomas are rare and heterogenous cancers arising from bone, soft and connective tissue. Developing effective therapies remains a challenge, since many types are understudied and poorly characterized at the molecular level. Here, we propose to investigate the role of protein misfolding in sarcoma, starting from the tumor suppressor p53. Wild type p53 is a tumor suppressor that regulates many cellular processes and prevents malignant transformation. Missense mutations promote p53 misfolding and convert the protein into a cancer-promoting oncogene. Mutant p53 is found in ~80% of all osteosarcoma cases, ~50% of leiomyosarcoma and angiosarcomas. It is also mutated at lower frequencies in other sarcoma subtypes. We investigated whether p53 mutations found in different types of sarcoma cause p53 misfolding and aggregation and can be targeted by ReACp53, a p53 aggregation-targeting peptide with anti-tumor activity in ovarian cancer and prostate cancer (Soragni et al, 2016, Zhang et al, 2019). We established tumor organoids from four sarcoma lines, which include osteosarcoma, fibrosarcoma, leiomyosarcoma and rhabdomyosarcoma. The cells carry either p53 mutations or WT p53. To characterize the p53 aggregation status and elucidate the response to ReACp53, we performed staining and quantification of folded/unfolded p53 pools in the organoids. Our results demonstrate the presence of overexpression and aggregation of p53 in the nuclei as well in the cytosol of different types of sarcoma lines. We found a marked susceptibility to ReACp53 in all lines, including cells bearing WT p53. We also explored whether similar p53 conformational defects are found in tumor tissue from sarcoma patients and if the activity of ReACp53 is confirmed in patient-derived tumor organoids. Overall, the discovery of structural alterations of p53 in sarcoma could provide an opportunity for the development of novel therapeutic strategies.

Citation Format: Sara Sartini, Hillary Le, Alice Soragni. Protein aggregation in sarcoma as a target for therapy [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 2582.
Abstract 2613: Chronic NNAL exposure, mimicking tobacco use in humans, deactivates LKB1 and promotes tumor progression in non-small cell lung cancer
Authors: Bian Tengfei , Jin Lingtao , Xing Chengguo
Published: April 2023

Background: LKB1 loss-of-function is one key oncogenic event in lung cancer. Clinical data suggest that LKB1 loss-of-function is associated with patients’ smoking status. We recently discovered that NNAL, a major metabolite of a tobacco specific carcinogen NNK, rapidly induces LKB1 phosphorylation and its loss-of-function via the β-AR/PKA signaling pathway in an isomer-dependent manner in human lung cancer cells. Such an acute exposure, however, only resulted in moderate enhancement in lung cancer cell migration and chemoresistance. Here we characterized the effects of chronic NNAL exposure, better mimicking human tobacco use, on LKB1 and tumor progression in NSCLC in vitro and in vivo.

Methods and Results: Human NSCLC cells with wild type LKB1 (H1299 and H1975) were cultured in the presence of 1 nM NNAL for 3, 6 and 12 months. LKB1 and its signaling pathways were characterized with phenotypes evaluated, including cell proliferation, migration, and chemoresistance. NNAL chronic exposure in lung cancer cells, mimicking its chronic exposure among smokers, resulted in more prominent LKB1 phosphorylation and the deactivation of its signaling, cell migration, and chemoresistance even in the absence of NNAL, indicating the long-lasting LKB1 loss-of-function. These observations were confirmed in a lung cancer xenograft model. More importantly, human lung cancer tissues revealed elevated LKB1 phosphorylation in comparison to the paired normal lung tissues.

Conclusion: These results suggest that LKB1 loss-of-function in human lung cancer could be extended to its phosphorylation, which may be mediated by NNAL from tobacco smoke in an isomer-dependent manner via the β-AR/PKA signaling pathway.

Citation Format: Tengfei Bian, Lingtao Jin, Chengguo Xing. Chronic NNAL exposure, mimicking tobacco use in humans, deactivates LKB1 and promotes tumor progression in non-small cell lung 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 2613.
Abstract 2720: Enhancing oral delivery of plant-derived vesicles
Authors: Liu Yuan , Lankenau-Ahumada Adrian , Bayraktar Emine , Schwartz Paul , Chowdhury Mamur , Shi Sixiang , Sebastian Manu M. , Khant Htet , De Val Natalia , Jie Zuliang , Jennings Nicholas B. , Rodriguez-Aguayo Cristian , Swain Jody , Stur Elaine , Mangala Lingegowda S. , Wu Yutuan , Nagaraju Supriya , Erimas Brooke , Li Chun , Lopez-Berestein Gabriel , Braam Janet , Sood Anil K.
Published: April 2023

Plant-derived vesicles (PDVs) were attractive for therapeutic applications, including as potential nanocarriers. However, a concern with oral delivery of PDVs is whether they would remain intact in the gastrointestinal tract. We found that 82% of cabbage PDVs were destroyed under conditions mimicking the upper digestive tract. To overcome this limitation, we developed a delivery method whereby lyophilized Eudragit S-100-coated cabbage PDVs were packaged into a capsule (Cap-cPDVs). Lyophilization and suspension of PDVs in phosphate-buffered saline did not have an appreciable effect on PDV structure, number of PDVs, or therapeutic effect. However, packaging the lyophilized Eudragit S-100-coated PDVs into capsules allowed them to pass through the upper gastrointestinal tract for delivery into the colon better than did suspension of PDVs in phosphate-buffered saline. Cap-cPDVs demonstrated robust therapeutic effect in a dextran sulfate sodium-induced colitis mouse model. These findings could have broad implications for oral delivery of PDVs.

Citation Format: Yuan Liu, Adrian Lankenau-Ahumada, Emine Bayraktar, Paul Schwartz, Mamur Chowdhury, Sixiang Shi, Manu M. Sebastian, Htet Khant, Natalia De Val, Zuliang Jie, Nicholas B. Jennings, Cristian Rodriguez-Aguayo, Jody Swain, Elaine Stur, Lingegowda S. Mangala, Yutuan Wu, Supriya Nagaraju, Brooke Erimas, Chun Li, Gabriel Lopez-Berestein, Janet Braam, Anil K. Sood. Enhancing oral delivery of plant-derived vesicles [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 2720.
Abstract 279: Lysosomal cyst(e)ine regulates melanoma de-differentiation and sensitivity to ferroptosis
Authors: Yu Deyang , Liang Jiaxin , Widlund Hans , Puigserver Pere
Published: April 2023

In response to anti-melanoma treatment, melanoma cells go through a process called de-differentiation that is characterized as loss of cellular features of melanocytes and acquisition of traits of the primitive neural crest stem cells which give rise to melanocytes during embryonic development. While de-differentiation enables melanoma to resist anti-melanoma drugs, it also exposes certain vulnerabilities of melanoma. A recent study showed that de-differentiated melanoma cells, while resistant to standard anti-melanoma therapies, display remarkable sensitivity to a group of chemicals that cause a special type of cell death called ferroptosis. However, the cellular programs and molecular machineries that drive melanoma de-differentiation and make them vulnerable to ferroptosis remain unknown. As the master regulator of melanocyte differentiation, MITF is often lost during de-differentiation and this loss has been correlated with sensitivity to ferroptosis. Through proteomic profiling of melanoma cells with different MITF status, we found that MITF-mediated lysosomal biogenesis is a key difference between differentiated melanoma cells and de-differentiated melanoma cells. Importantly, within the same melanoma linage, the heterogeneity in lysosomal abundance underlies differential sensitivity to ferroptosis. To probe lysosomal function that controls ferroptosis sensitivity, we deleted a series of lysosome-related proteins and found that the lysosomal cysteine transporters CTNS and MFSD12 exert opposing effects on lysosomal de-differentiation and ferroptosis sensitivity. Specifically, deletion of the pH-dependent cystine exporter CTNS drives melanoma de-differentiation and promotes ferroptosis sensitivity while ablation of cysteine importer MFSD12 further enhances resistance to ferroptosis. On the other hand, while increasing lysosomal cystine export by overexpression of CTNS has moderate effects on melanoma de-differentiation, increasing lysosomal important by overexpression of MFSD12 results in melanoma de-differentiation and increases sensitivity to ferroptosis. Our findings suggest that disruption of lysosomal cysteine homeostasis is a key mechanism to drive melanoma de-differentiation which is accompanied by increased sensitivity to ferroptosis.

Citation Format: Deyang Yu, Jiaxin Liang, Hans Widlund, Pere Puigserver. Lysosomal cyst(e)ine regulates melanoma de-differentiation and sensitivity to ferroptosis [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 279.
Abstract 2872: Site specific tumor microenvironments modulate the immune checkpoint inhibitors response in models of CT26 colon carcinoma
Authors: Zhao Fei , Thompson Caitlin , Barnes Nicole , Roques José , Coleman Terrence , Rowe Tyler , Kolb Joseph , Hall Chassidy , Fadden Patrick
Published: April 2023

Syngeneic models provide a robust platform to test new and cutting edge immune-oncology therapies. CT26 is a popular syngeneic colon carcinoma model that is known to respond to immune checkpoint inhibitors when engrafted into Balb/c mice as a subcutaneous flank tumor. The subcutaneous flank implant location provides a robust model, that is easy to set up, and allows for continuous measurement of the tumor growth by calipering. However, the location of tumor implant has been shown to impact tumor microenvironment and immune response for a number of cancer cell lines (Zhao 2017, Brand 2021, Oliver 2018). In addition to subcutaneous flank tumors, CT26 can be implanted to generate a lung metastasis or peritoneal metastasis model. We were interested to see if the lung metastasis or peritoneal metastasis CT26 model responded differently to immune checkpoint inhibition compared with the subcutaneous model and whether the different implant locations resulted in changes to the tumor infiltrating lymphocytes. CT26 cells were either injected intravenously to induce a lung metastasis model, intraperitoneally to produce a peritoneal metastasis model, or subcutaneously to generate a solid flank tumor model. Immune checkpoint inhibitor (ICI) therapy began on Day 5 following implant in each model, and tumor progression or survival provided the endpoint for analysis. In addition, tumor infiltrating leukocytes were assessed by flow cytometry to determine differences in the overall tumor microenvironment for each model. The response to both anti-PD-1 and anti-CTLA-4 monotherapies were strongest in the subcutaneous flank model, followed by moderate responses in the lung metastasis model, and no significant response to either ICI in the peritoneal metastasis model. This provides researchers with an opportunity to assess the ability of novel immune-oncology therapies to provoke an immunogenic response in models with identical antigenicity, but different tumor microenvironments based on the tumor location.

Citation Format: Fei Zhao, Caitlin Thompson, Nicole Barnes, José Roques, Terrence Coleman, Tyler Rowe, Joseph Kolb, Chassidy Hall, Patrick Fadden. Site specific tumor microenvironments modulate the immune checkpoint inhibitors response in models of CT26 colon carcinoma [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 2872.
Abstract 2882: Characteristic immunomodulatory role of myeloid galectin-9 in the brain tumor microenvironment
Authors: Lee Chanju , Yu Dahee , Park Eun Jung
Published: April 2023

Galectin-9 is a member of galectin family with carbohydrate-recognition domains that specifically bind to β-galactoside. Increasing reports have suggested that galectin-9 can bind and interact with immune-modulatory molecules including TIM-3 in several pathophysiological conditions, especially in T cells. However, to date, little is known about the galectin-9 in myeloid cells under tumor microenvironment. In this study, we explored the expressional characteristics and roles of galectin-9 in the brain tumor microenvironment using an intracranial mouse brain tumor model and in vitro system. We found that expression level of galectin-9 was markedly elevated in ipsilateral hemisphere compared to contralateral hemisphere of intracranial brain tumor model. FACS analyses showed that galectin-9 expression was higher in CD45highCD11bhigh macrophages and CD45midCD11bhigh microglia as well as lymphocytes from ipsilateral region than those from contralateral region. In addition, galectin-9 levels were increased after exposure to GL26 brain tumor cells in in vitro co-culture system using primary microglia. Interestingly, phagocytic activity, a key function of myeloid cells, was significantly reduced in galecin-9+ microglia compared to galecin-9- microglia when co-cultured with GL26-GFP cells. Furthermore, SIRPα levels were higher in galectin-9+ microglia than galecin-9- microglia. Similar results were observed in macrophages. Collectively, these findings suggest that galectin-9 may be an important modulatory molecule in myeloid cells under brain tumor microenvironment.

Citation Format: Chanju Lee, Dahee Yu, Eun Jung Park. Characteristic immunomodulatory role of myeloid galectin-9 in the brain tumor microenvironment [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 2882.
Abstract 2885: RASSF1A133Sresulting from the prevalent SNP rs2073498 is associated with Treg defects
Authors: Xu Haonan , Jones Keaton , Lang Cameron , Lanfredini Simone , Hughes Sophie , O'Neill Eric
Published: April 2023

Rs2073498 is a prevalent (~10% population) single nucleotide polymorphism (SNP) that leads to A133S missense mutation in tumor suppressor and Hippo Signaling Pathway scaffold protein RASSF1A. Recent reports have illustrated the crucial involvement of Hippo pathway proteins in lymphocyte recruitment, differentiation, and function. Using a BL/6 mouse model engineered with alanine-to-serine point mutation, we showed that RASSF1A133S is associated with systematic defects in regulatory T cells (Tregs). When blood was phenotyped through a 17-color spectral flow cytometry panel, both RASSF1A133S/WT and RASSF1A133S/A133S carriers had a smaller circulatory CD25+FoxP3+ Treg pool than WT controls. Magnetically sorted splenic T cells were also stimulated with PMA/Ionomycin ex vivo, and fewer Tregs from A133S carriers were expressing anti-inflammatory cytokine IL-10. Preliminary results further demonstrated that RASSF1A133S/WT has fewer intratumor FoxP3+ Tregs in subcutaneously injected MC38 colon adenocarcinoma tumor models. In contrast, A133S did not have any significant effects on thymic T cell maturation states, splenic T cell naïve/memory phenotypes, or Treg survival fitness measured by Fas/FasL and BCL-2 expression. Ex vivo stimulation also demonstrated that A133S carriers retain similar IL-2 sensitivity and phospho-STAT5 response when compared to the controls, although RASSF1 downstream kinases MST1/2 have been reported to amplify IL-2-STAT5 signaling in Tregs (Shi et al., 2018). These results collectively suggest that RASSF1A133S may leads to Treg imbalance and predispose rs2073498 carriers to Treg mediated autoimmune risks.

Citation Format: Haonan Xu, Keaton Jones, Cameron Lang, Simone Lanfredini, Sophie Hughes, Eric O'Neill. RASSF1A133Sresulting from the prevalent SNP rs2073498 is associated with Treg defects [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 2885.
Abstract 2941: ROR1 targeted 4-1BB costimulatory bispecific antibody, ABL102, exhibits potent in vitro and vivo antitumor activity and superior safety profile
Authors: Lee Yangsoon , Park Kyeong-Su , Son Wonjun , Kim Sora , Youn Hyunseong , Yoo Jiseon , Lee Hanbyul , Won Jonghwa
Published: April 2023

ROR1, a member of the ROR (Receptor Tyrosine Kinase-Like Orphan Receptor)-family, is overexpressed in the process of embryo and fetal development, and controls cell polarity, cell migration and neurite growth. The expression is gradually reduced as development progresses, and it is hardly expressed in adults, but the overexpression of ROR1 is observed in various blood and solid cancer cells, so it is classified as an oncofetal gene. ABL102, a First-in-Class bispecific antibody targeting ROR1 and 4-1BB, elicits superior T cell activation by tumor specific T cell engagement through ROR1 dependent 4-1BB clustering. Our data showed that tumor target dependents binding of ROR1 and costimulatory 4-1BB by ABL102 induced 4-1BB activation in cell-based 4-1BB bioassay. ABL102 lead to IFN-γ secretion and tumor cell killing with a ROR1 dependent manner in co-culture assay with tumor cells and human PBMC. In h4-1BB knock-in mouse model bearing hROR1-expressing tumors, ABL102 potently inhibited tumor progression with a high rate of complete remission (CR). Mice with CR were further protected from the rechallenge of previously exposed tumors after 3 months of cessation of ABL102 treatment, implicating that immunological memory might be generated. In 4-week pilot toxicity study using cynomolgus monkeys, no ABL102-related toxicity was observed up to 100mg/kg, including mortality, body weight, hematology, liver toxicity, clinical chemistry and pathology, indicating a favorable safety profile. In conclusion, ABL102 exhibited potent in vitro and vivo anti-tumor activity and it was well tolerated and safe in the NHP toxicity study. These results strongly suggest that ABL102 is promising therapeutics for ROR1 positive cancer patients.

Citation Format: Yangsoon Lee, Kyeong-Su Park, Wonjun Son, Sora Kim, Hyunseong Youn, Jiseon Yoo, Hanbyul Lee, Jonghwa Won. ROR1 targeted 4-1BB costimulatory bispecific antibody, ABL102, exhibits potent in vitro and vivo antitumor activity and superior safety profile [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 2941.
Abstract 295: Targeting spermine synthase triggers lipid metabolism reprogramming as a new therapeutic option to combat colorectal cancer
Authors: Ma Murong , Guo Yubin , Ye Qing , Deng Pan , He Daheng , Wang Chi , She Qing-Bai
Published: April 2023

Dysregulation of polyamine metabolism has been linked to the development of colorectal cancer (CRC). Our recent work demonstrates that spermine synthase (SMS), a polyamine biosynthetic enzyme that converts spermidine to spermine, is overexpressed in CRC, which is required for balancing cellular spermidine levels to facilitate CRC tumorigenesis (Nat Commun 11:3243, 2020). Our findings reveal SMS as an attractive therapeutic target in CRC; yet, genetic depletion of SMS expression only shows a limited antitumor effect. Using unbiased metabolomics and transcriptomics analyses, we identified a lipid metabolism reprograming as among the most impacted metabolic change by SMS depletion in CRC cells. Specifically, SMS depletion significantly altered long-chain fatty acid, triacylglycerol and phospholipid metabolism. Furthermore, targeted inhibition of SMS significantly increased the number of lipid droplets and the levels of long-chain fatty acid acylcarnitines for oxidative phosphorylation in mitochondria, and upregulated expression of genes associated with increased mobilization of polyunsaturated fatty acids and the genes associated with lipid peroxidation for induction of ferroptosis, an iron-dependent form of nonapoptotic cell death. The glutathione peroxidase 4 (GPX4) is a key negative regulator of ferroptosis by neutralizing lipid peroxides. Notably, pharmacological inhibition of GPX4 or its upstream regulator system xc − in combination with genetic depletion or pharmacologic inhibition of SMS synergistically caused lipid peroxidation leading to ferroptosis induction and marked suppression of CRC cell growth. Collectively, these findings highlight lipid metabolism reprograming as an adaptive response to targeted inhibition of SMS to enable CRC cell survival, which represents an Achilles’ heel that can be exploited for potential effective therapy for CRC.

Citation Format: Murong Ma, Yubin Guo, Qing Ye, Pan Deng, Daheng He, Chi Wang, Qing-Bai She. Targeting spermine synthase triggers lipid metabolism reprogramming as a new therapeutic option to combat colorectal 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 295.
Abstract 3020: Machine learning-based cluster analysis of immune cell subtypes and breast cancer survival
Authors: Wang Zhanwei , Katsaros Dionyssios , Wang Junlong , Biglio Nicholetta , Hernandez Brenda Y. , Fei Peiwen , Lu Lingeng , Risch Harvey , Yu Herbert
Published: April 2023

Background: Host immunity involves various immune cells working in concert to achieve balanced immune response. Host immunity interacts with tumorigenic process impacting disease outcome. Clusters of different immune cells may indicate specific host-tumor interplay. Identifying the clusters may reveal unique host immunity in response to tumor growth.

Methods: CIBERSORT was used to estimate relative abundances of 22 immune cell types in 3 datasets, METABRIC, TCGA, and our study. The cell type data in METABRIC were analyzed for cluster using unsupervised hierarchical clustering (UHC). The UHC results were employed to train machine learning models, random forest (RF), deep neural network (DNN), stepAIC, and elastic net. Kaplan-Meier and Cox regression survival analyses were performed to assess cell clusters in association with relapse-free and overall survival. Differentially expressed genes (DEGs) by immune cell clusters were interrogated with IPA for molecular signatures.

Results: UHC analysis identified two distinct immune cell clusters, clusters A (83.2%) and B (16.8%). Memory B cells, plasma cells, CD8 positive T cells, resting memory CD4 T cells, activated NK cells, monocytes, M1 macrophages, and resting mast cells were more abundant in clusters A than B, whereas regulatory T cells and M0 and M2 macrophages were more in clusters B than A. Patients in cluster A had favorable survival compared to those in cluster B. Similar survival associations were also observed in TCGA and our study when using a RF model trained with the UHC results. The survival associations were independent from clinicopathological variables. IPA analysis showed that pathogen-induced cytokine storm signaling pathway, phagosome formation, and T cell receptor signaling were related to the cell type clusters.

Conclusions: Our finding suggests that different immune cell clusters may indicate distinct immune responses to tumor growth, suggesting their potential for disease management.

Citation Format: Zhanwei Wang, Dionyssios Katsaros, Junlong Wang, Nicholetta Biglio, Brenda Y. Hernandez, Peiwen Fei, Lingeng Lu, Harvey Risch, Herbert Yu. Machine learning-based cluster analysis of immune cell subtypes and breast cancer survival [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 3020.
Abstract 3153: Prediction of metabolites from cancer-associated fibroblasts that can have profound effects on cancer cells
Authors: Jung Hae Deok , Sung Yoo Jin , Ryu Jae Yong , Kim Hyun Uk
Published: April 2023

Cancer-associated fibroblasts (CAFs) are the most abundant component of tumor microenvironment (TME) that is an ensemble of non-cancerous cells surrounding cancer cells. In particular, CAFs are known to have tumor-assisting functions, which may promote active growth and survival of the interacting cancer cells through their metabolic reprogramming despite being in a harsh environment (e.g., hypoxia or low pH). In this regard, CAF-derived metabolites are expect to have profound effects on metabolism of the cancer cells. To identify novel CAF-derived metabolites that may heavily influence the cancer cells, CAF-specific genome-scale metabolic models (GEMs) were reconstructed and simulated. GEM is a computational model that can predict a metabolic state in a cell-specific manner by integrating omics data (e.g., RNA-seq data). In this study, a total of six GEMs were reconstructed by integrating RNA-seq data from one normal fibroblast, originating from pancreas, and five CAFs associated with breast, colon, skin, lung, and pancreatic tumors. Simulation of the GEMs predicted 20 metabolites that appeared to be heavily used by all the five CAF GEMs in comparison with the one normal fibroblast GEM. Such metabolites were expected to be significantly associated with establishment of TME that is favorable for the cancer cells’ growth. Some of these metabolites are associated with pyrimidine metabolism, fatty acid metabolism, and primary bile acid biosynthesis. Upon experimental validation, the predicted metabolites are expected to help better understand CAFs and TME in terms of metabolites, and also provide clues for identifying effective drug targets for cancer treatment.

Citation Format: Hae Deok Jung, Yoo Jin Sung, Jae Yong Ryu, Hyun Uk Kim. Prediction of metabolites from cancer-associated fibroblasts that can have profound effects on 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 3153.
Abstract 317: Particulate matter accumulates chromosomal instability during mitosis
Authors: Ryu Jaewook , Kim Ja-Eun
Published: April 2023

Particulate matter (PM), a major component of outdoor air pollution triggers genotoxicity as well as cytotoxicity. Although the harmful effects of PM on DNA damage and its carcinogenic risk have been thoroughly studied, the detailed mechanism by which PM affects chromosomal stability remains unclear. Maintenance of chromosome is finely monitored during mitotic phase through multiple proteins and structures. The present study shows that treatment of urban particulate matter (UPM) inhibited proliferation of lung cells through the accumulation of chromosomal instability which is determined by the formation of multinucleation and macronucleation. In particular, PM treatment prolonged mitotic duration. This delay in mitotic progression resulted from inactivation of Aurora B at prometaphase and metaphase, causing a failure in dynamic stabilization of kinetochore-microtubule attachment. In addition, ingression rate of cleavage furrow and actin clearing from polar cortex were significantly reduced at telophase. It indicates that dysregulation of spatiotemporal dynamics of mitotic proteins and structures finally disturbs cytokinetic division. Overall, PM leads to accumulation of chromosomal instability, finally contributing to its carcinogenicity.

Citation Format: Jaewook Ryu, Ja-Eun Kim. Particulate matter accumulates chromosomal instability during mitosis [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 317.
Abstract 3274: Novel cell-based bioassays for mono- and multi-specific antibodies targeting PD-1/PD-L1 for combination immunotherapy
Authors: Wang Jun , Gilden Julia , Garvin Denise , Stecha Pete , Hartnett Jim , Fan Frank , Cong Mei , Grailer Jamison
Published: April 2023

Programmed cell death protein 1 (PD-1) is an immune checkpoint (IC) receptor that negatively regulates T cell function. Blocking the interaction of PD-1 with its ligands PD-L1 or PD-L2 unleashes the immune system and enhances anti-tumor responses. Immunotherapy using PD-1 or PD-L1 blocking antibodies has led to a paradigm shift in cancer drug discovery, due to its durable effect against a wide variety of cancers. However, despite its tremendous clinical success in cancer treatment, a significant fraction of patients and tumor types remain unresponsive to this therapy. This has led to the development of combination therapies linking PD-1 checkpoint inhibitors with inhibitors of several other IC receptors. A major challenge in immunotherapy drug development is the lack of quantitative and reproducible functional assays. Existing methods rely heavily on primary immune cells are highly variable and labor-intensive. Here, we report the development of a suite of cell line-based reporter bioassays for monoclonal antibodies targeting human PD-1/PD-L1, or bispecific antibodies targeting PD-1/PD-L1 and a co-stimulatory receptor (e.g., 4-1BB, OX40, ICOS) or an immune inhibitory receptor (e.g., CTLA-4, LAG-3, TIGIT). A mouse PD-1/PD-L1 assay and a human PD-1/PD-L2 assay are also developed and can be used in parallel with human PD-1/PD-L1 assay to enable preclinical studies and assess assay specificity. Each of these assays consist of two engineered cell lines: a T effector cell line that express a luciferase reporter driven by specific promoter/response elements responding to the intracellular signals mediated by the T cell receptor (TCR), with modulation from PD-1 and the second immune receptor, and an engineered artificial antigen presenting cell (aAPC). Importantly, these combination bioassays demonstrate the synergetic effect of PD-1 blockade with blockade of a second IC inhibitor receptor or activation of a costimulatory receptor, thus reflecting the mechanisms of action for the therapeutic antibodies of interest. The bioassays are sensitive and quantitative, are prequalified according to ICH guidelines, and demonstrate the performance characteristics (specificity, precision, accuracy, and linearity) to be used as potency assays for product release and stability studies during immunotherapy drug development.

Citation Format: Jun Wang, Julia Gilden, Denise Garvin, Pete Stecha, Jim Hartnett, Frank Fan, Mei Cong, Jamison Grailer. Novel cell-based bioassays for mono- and multi-specific antibodies targeting PD-1/PD-L1 for combination immunotherapy [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 3274.