bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1346: Phagocytosis of solid tumors favors macrophage clusters and cooperativity in tumor elimination
Authors: Discher Dennis E.
Published: April 2023

Macrophages are abundant in many solid tumors and are potential effector cells for monoclonal antibody-based immunotherapies because they can in principle phagocytose IgG-opsonized cancer cells. However, it remains unknown whether phagocytic macrophages can overcome the cohesive forces between cancer cells in solid tumors. We show that maximum phagocytosis of B16 melanoma tumors and ‘tumoroids’ requires IgG-opsonization and very deep suppression of the macrophage checkpoint ligand CD47. Under these conditions, phagocytic macrophages cluster or ‘phase separate’, and the elimination exhibits clear cooperativity with respect to macrophage number and phagocytic activity. In vitro disruption of phagocytic clusters specifically implicates Mg2+ and macrophage integrins. Cytokine treatments and other inhibitors further implicate roles for integrins and phagocytic surface receptors. Although tumor-associated macrophages correlate with negative prognoses in many cancers, our findings are among the first to reveal advantages of high macrophage numbers, macrophage clusters, and resulting cooperative effects when combined with opsonizing antibodies and CD47 disruption. Lastly, the challenging B16 tumors are durably eliminated by the approach and induce de novo IgG's that also drive anti-cancer phagocytosis.

Citation Format: Dennis E. Discher. Phagocytosis of solid tumors favors macrophage clusters and cooperativity in tumor elimination [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 1346.
Abstract 135: Elucidating tumor evolution in pediatric cancers using stochastic models and mutation accumulation experiments
Authors: Massei Ana Beatriz , Platero Jasmin Jovel , Hong Kyungchan , Zeledon Sofia , Afzal Taha , Cha Hyunjeong , Kim Seung-Hwan
Published: April 2023

Ontology-based approaches have been utilized for identifying cancer genes. However, among the thousands of mutations in pediatric cancer genes, accurate prediction of driver genes is one of the biggest challenges. A binary logistic regression model was developed to distinguish pediatric and non-pediatric cancer genes. To build the predictive model, Gene Ontology (GO) enrichment analysis was performed, followed by the PANTHER overrepresentation test, using PANTHER GO-slim, PANTHER, Reactome pathways, and three sets of specific variables, the lists of pediatric cancer genes found in the germline, relapsed, and chemotherapy-resistant cells, respectively. This statistical model was pediatric cancer-specific, confirming known driver genes across cancers. In addition, mathematical models were developed using stochastic differential equation approaches and computational simulations to extend our understanding of the evolutionary dynamics during pediatric tumorigenesis. Moreover, our stochastic models in childhood cancers were validated by mutation accumulation experiments using E. coli strains carrying a mutation in replication restart genes. Finally, this study that elucidates tumor evolution in pediatric cancers could have profound implications for cancer therapy and drug design.

Citation Format: Ana Beatriz Massei, Jasmin Jovel Platero, Kyungchan Hong, Sofia Zeledon, Taha Afzal, Hyunjeong Cha, Seung-Hwan Kim. Elucidating tumor evolution in pediatric cancers using stochastic models and mutation accumulation experiments [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 135.
Abstract 14: The impact of genetic background on cancer phenotypes of mouse models
Authors: Begley Dale A. , Krupke Debra M. , Neuhauser Steven B. , Jocoy Emily L. , Sundberg John P. , Bult Carol J.
Published: April 2023

The laboratory mouse is the premier animal model system for in vivo studies of the genetic and genomic basis of cancer in humans. Although thousands of mouse models have been generated, finding relevant data and knowledge about these models is complicated by a general lack of compliance in the published literature with nomenclature and annotation standards for genes, alleles, mouse strains, and cancer types. The Mouse Models of Human Cancer database (MMHCdb; tumor.informatics.jax.org) is an expertly curated knowledgebase of cancer phenotypes reported for diverse types of mouse models of human cancer such as inbred mouse strains, genetically engineered mouse models (GEMMs), Patient Derived Xenografts (PDXs), and mouse genetic diversity panels (e.g., the Collaborative Cross). MMHCdb includes data on more than 60,000 mouse models for over 1200 tumor classifications curated from more than 25,000 peer-reviewed publications.

One of the primary goals of the MMHCdb is to highlight the impact of genetic background on the incidence and presentation of different tumor types in mice. The same allele on different backgrounds can result in very different cancer characteristics and, therefore, impact the appropriateness of a model for a specific research application. In MMHCdb, users can review the impact of genetic background on the frequency of spontaneous tumors for inbred mouse strains using an interactive table generated from different published and unpublished data sources. In addition, color-coded tabular summaries of individual papers are available that allow researchers to quickly assess how genetic background affects cancer phenotypes in the mouse models reported in a specific publication. We will highlight examples of how genetic background can profoundly change the types and frequencies of tumor types that can be expected in mouse models of human cancer.

MMHCdb is supported by NCI R01 CA089713

Citation Format: Dale A. Begley, Debra M. Krupke, Steven B. Neuhauser, Emily L. Jocoy, John P. Sundberg, Carol J. Bult. The impact of genetic background on cancer phenotypes of mouse models [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 14.
Abstract 140: Clonal evaluation of ERG/SPINK1 status to determine disease progression using whole mount prostatectomy specimens
Authors: Cho Joonyoung , Suh In Hye , Chang HyeYoon , Kwak Tae-Yeong , Kim Sun Woo , Palanisamy Nallasivam
Published: April 2023

Most primary prostate cancers are multifocal, marked by distinct tumor foci. Multiclonality is not routinely incorporated at clinical evaluation, however, marker status as a pathological parameter holds great potential for improving prognostic accuracy. Here, we evaluated the frequency of multiclonality and whether ERG/SPINK1 status is correlated to aggressive disease in whole-mount specimens.

For each of the 89 radical prostatectomy cases, one H&E-stained whole-mount slide and a corresponding dual ERG/SPINK1 IHC stained slide were collected. Aperio AT2 scanner (Leica Biosystems) digitized these slides into whole-slide images (WSIs). All WSIs were reviewed by a pathologist with the aid of an automatic Gleason grading software to identify each tumor focus and determine its Gleason grade group (GG), and the expression levels (% uptake) of ERG and SPINK1, respectively. Grade groups 4 and 5 were treated as one high-grade group (GG4-5). The associations among GG, ERG, and SPINK1 expression levels were analyzed.

For each WSI, 1 to 7 (median 2, mean 2.53) tumor foci were identified, and the aforementioned values were determined, resulting in 225 data records. The number of tumor foci, ERG/SPINK1 expression statistics, and their association for each GG are shown in the table. ERG was expressed the most frequently in GG1, and its frequency decreased as the grade increased. Conversely, SPINK1 expression frequency increased with the grade group. The expression of ERG and SPINK1 was inversely correlated regardless of grade group. More detailed results on the exploratory data analysis and the association with BCR will be shared at the time of presentation.

ERG or SPINK1 expression do not appear to be associated with Gleason grade groups. More studies are needed to see if these markers may predict recurrence free survival or patient response to therapy. To our knowledge, this is the first study to map molecular subtypes in whole-mount samples utilizing a foci-based approach.

Table Grade group Total ERG SPINK1 ERG-SPINK1 positive ratio (%) positive ratio (%) co-positive co-negative mutually exclusive GG1 74 38 51.4 24 32.4 4 16 54 GG2 81 35 43.2 40 49.4 7 13 61 GG3 33 11 33.3 22 66.7 4 4 25 GG4-5 37 10 27.0 13 35.1 1 15 21 Overall 225 94 41.8 99 44.0 16 48 161

Citation Format: Joonyoung Cho, In Hye Suh, HyeYoon Chang, Tae-Yeong Kwak, Sun Woo Kim, Nallasivam Palanisamy. Clonal evaluation of ERG/SPINK1 status to determine disease progression using whole mount prostatectomy specimens [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 140.
Abstract 145: Giant cells: A new paradigm to understand tumor origins and decode their complexity at the organismal level
Authors: Liu Jinsong
Published: April 2023

Traditionally, human cancers have been considered genetic-based diseases characterized by somatic mutations. However, despite its wide acceptance, this simple concept cannot explain cancers’ numerous oncogenic driver mutations, structural genomic changes in normal and embryonic tissues, nuclear atypia, and lack of tissue differentiation. Due to these pitfalls, the cancer research community is searching for a new paradigm to explain the complexity of tumors and guide future research. Here I proposed a new hypothesis based on the observation of different tumors and their correlation with stages of early embryonic development in humans to explain human tumors' complexity and origin. I consider cancer a developmental disease of sexual or asexual origin. In tumors of sexual origin, the giant egg cell undergoes cleavage after fertilization to form blastomeres that generate genomic chaos via the rapidly increasing genomic contents of the zygote. The compacted embryo then forms a blastocyst, resulting in embryonic transformation and the formation of the trophectoderm. After implantation, the trophectoderm develops into a placenta, and the inner cell mass develops into a baby or a differentiated tumor resembling a specific embryonic tissue stage or fetal organ if one or more germ layers fail to complete differentiation. In tumors of asexual origin, the tumor starts with senescent somatic cells. Following environmental and genetic stresses, the senescent cells undergo escape via genomic duplication or multiplication, giving rise to polyploid giant cancer cells (PGCCs), the somatic equivalent of blastomeres. This leads to genome duplication or multiplication to create genomic chaos and the development of high-grade cancer with a high level of nuclear atypia, a process analogous to sexual-mediated pre-embryogenesis but without subsequent differentiation. This new hypothesis not only explains the different tumor entities and their benign and malignant natures but also provides a unified explanation of the chaotic genomics and somatic mutations of cancer during embryonic development. The evidence to support this hypothesis will be presented from the pathology, developmental, and genetic perspectives. The switch from conceiving cancer as a disease of mitosis-based cell proliferation to conceiving it as a disease arising from giant cell-mediated development provides a new paradigm to decode tumors' complexity, understand their origins at the organismal level, and guide future cancer research.

Citation Format: Jinsong Liu. Giant cells: A new paradigm to understand tumor origins and decode their complexity at the organismal level [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 145.
Abstract 1495: Developing a model to investigate the roles of biomolecular condensates in papillary renal cell carcinoma
Authors: So Choon Leng , Lee Jane , Huang Binglin , Cai Danfeng
Published: April 2023

Cells are compartmentalized into “classical” membrane-bound organelles such as the endoplasmic reticulum and mitochondria. Cells also have membrane-less organelles (MLOs), which are formed through phase separation. Examples of MLOs include stress granules and nucleoli, where macromolecules (proteins, DNAs, RNAs) coherently and dynamically concentrate into biomolecular condensates resembling droplets in lava lamps. MLOs play central role in cellular processes - choreographed events such as transcription and RNA splicing take place in MLOs. MLOs are highly organized, and their dysregulation always led to diseases. Papillary renal cell carcinoma (PRCC) is the second most common RCC in America. PRCC is an aggressive cancer, yet the uncharacterized molecular etiology results in the lack of effective treatment for patients. Our lab and others have observed dysregulated oncoproteins MLOs in PRCC. However, the molecular properties and functions of these aberrant MLOs have not been characterized. The photolyase homology region of Arabidopsis thaliana Cry2 (Cry2WT) is a light-sensitive protein that self-associate with blue light exposure. We hypothesize that fusing dysregulated MLOs oncoproteins with Cry2WT will allow us to tune and study the oncoproteins condensates. We aim to develop an optogenetic model to study how oncoproteins condensates function in PRCC cells. Dysregulated oncoproteins were fused to mCherry-Cry2WT, introduced into cell line models, light-activated and imaged using a ZEISS LSM 900 microscope. We found that blue-light indeed induced proteins clustering when oncoproteins were fused to mCherry-Cry2WT but not with mCherry-Cry2WT alone. This model will allow us to tune the formation of oncoproteins condensates. Future work will involve studying how optogenetically tuned oncoproteins condensates function in PRCC patient-derived cell lines.

Citation Format: Choon Leng So, Jane Lee, Binglin Huang, Danfeng Cai. Developing a model to investigate the roles of biomolecular condensates in papillary renal cell 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 1495.
Abstract 1501: Epigenetic dedifferentiation as a therapeutic strategy in adrenal cancer
Authors: Mohan Dipika R. , Borges Kleiton S. , Finco Isabella , LaPensee Christopher R. , Rege Juilee , Little Donald W. , Else Tobias , Almeida Madson Q. , Dang Derek , Haggerty-Skeans James , Latronico Ana Claudia , Mendonca Berenice B. , Auchus Richard J. , Rainey William E. , Marie Suely K. , Giordano Thomas J. , Venneti Sriram , Fragoso Maria Candida B. , Breault David T. , Lerario Antonio M. , Hammer Gary D.
Published: April 2023

Adrenocortical carcinoma (ACC) is a rare cancer of the adrenal cortex without curative medical therapies. CIMP-high is an aggressive ACC molecular subtype defined by global CpG island hypermethylation with paradoxical activation of adrenal differentiation (driven by master transcription factor SF1) and stemness (driven by β-catenin). We show DNA hypermethylation redistributes histone methyltransferase EZH2 and its mark, H3K27me3. EZH2 inhibition remains lethal to CIMP-high ACC cells, erasing transcriptional programs without altering DNA methylation. We reconcile this phenomenon by discovery of two nuclear complexes, SF1/β-catenin and EZH2/β-catenin, present in physiology and persistent through advanced ACC. We find SF1/β-catenin is a chromatin-bound complex that controls the ACC super-enhancer landscape, while EZH2/β-catenin is restricted to off-chromatin pools. EZH2 inhibition purges SF1/β-catenin from chromatin, sparing EZH2/β-catenin, inducing dedifferentiation and restraining ACC growth in vitro and in vivo. Our studies illustrate how cell-of-origin programs dictate cancer evolution, exposing differentiation as an therapeutic vulnerability.

Citation Format: Dipika R. Mohan, Kleiton S. Borges, Isabella Finco, Christopher R. LaPensee, Juilee Rege, Donald W. Little, Tobias Else, Madson Q. Almeida, Derek Dang, James Haggerty-Skeans, Ana Claudia Latronico, Berenice B. Mendonca, Richard J. Auchus, William E. Rainey, Suely K. Marie, Thomas J. Giordano, Sriram Venneti, Maria Candida B. Fragoso, David T. Breault, Antonio M. Lerario, Gary D. Hammer. Epigenetic dedifferentiation as a therapeutic strategy in adrenal 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 1501.
Abstract 1572: Antiproliferative activity of enzalutamide and alisertib in prostate cancer cells overexpressing HMGA2
Authors: Liadi Yusuf Mansur , Odero-Marah Valerie
Published: April 2023

Prostate cancer is one of the common types of cancer and remains the second leading cause of cancer-related deaths in men in the United States. High Mobility Group Protein AT-hook 2 (HMGA2), a DNA binding protein acts as a transcriptional regulating factor in gene transcription and facilitates epithelial-mesenchymal transition (EMT), which is usually the onset of prostate cancer progression and metastasis. HMGA2 isoforms include wild-type or full length that induces EMT, while the truncated isoform is associated with elevated proliferation and migration. Enzalutamide is a second-generation antiandrogen drug known to inhibit androgen receptor (AR) translocation into the nucleus by competitively binding AR and preventing androgen binding. Alisertib (MLN8237) is a small molecule inhibitor of Aurora kinase A has been utilized in clinical trials for neuroendocrine cancers and can also inhibit EMT. We aim to investigate which of the two drugs is effective in reducing proliferation in prostate cells overexpressing HMGA2 isoforms (wild-type and truncated). In this study, we treated LNCaP cells overexpressing HMGA2 with enzalutamide (1-30 µM) and alisertib (2.5-40 µM). Treatment with enzalutamide and alisertib indicated a dose-dependent decrease in cell proliferation with optimal dose at 20 µM for both drugs. LNCaP cells (LNCaP Neo) expressing low level of HMGA2 were observed to respond better to enzalutamide treatment, while those overexpressing HMGA2 (WT and TR) respond better to alisertib treatment. Interestingly, treatment with 20 µM enzalutamide and alisertib followed by Western blot indicated enzalutamide to have no effects on protein expression of HMGA2 isoforms, but greatly decreased the expression of AR in LNCaP cell overexpressing truncated HMGA2. On the other hand, alisertib increased the expression of wild-type HMGA2 whilst having no effects on protein expression of AR. Both drugs at 20 µM did not have any significant effect on the expression of EMT markers (snail and vimentin).In conclusion, alisertib is more potent than enzalutamide in decreasing cell proliferation in prostate cancer cells expressing HMGA2. Further studies need to be conducted to understand the role of HMGA2 in promoting resistance to enzalutamide and whether alisertib may be a better drug for patients that express HMGA2.

Acknowledgements: These studies were supported by NIH/NIMHD 2U54MD007590 and 5U54MD013376-8281.

Citation Format: Yusuf Mansur Liadi, Valerie Odero-Marah. Antiproliferative activity of enzalutamide and alisertib in prostate cancer cells overexpressing HMGA2 [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 1572.
Abstract 1574: Homoharringtonine is effective in treating acute lymphoblastic leukaemia via modulation of apoptotic pathway
Authors: Jiao Anan , Yuen Kei Ching , DIN Rahman Ud , Wong On Yi Phoebe , Chun-Fung Sin
Published: April 2023

Acute lymphoblastic leukaemia (ALL) is an aggressive and highly lethal malignancy caused by clonal mutations in lymphoid progenitor cells. The prognosis for adult ALL remains poor, with only 30-40% of adult patients achieving long-term remission. Therefore, novel therapies are needed. Homoharringtonine (HHT) is a natural-derived plant alkaloid from tree of Cephalotaxus. FDA approved the use of HHT in the treatment of chronic myeloid leukaemia (CML) in 2012. Limited pre-clinical or clinical studies were conducted for therapeutic efficacy and molecular mechanisms of HHT on ALL to the best of our knowledge. To fill this knowledge gap, here we investigated the antileukemic activity of HHT in T lymphoblastic leukaemia (T-ALL) and B lymphoblastic leukaemia (B-ALL). In this study, T-ALL cell lines namely JURKAT, MOLT-4, LOUCY, CCRF-CEM and B-ALL cell clines namely PALL-2, SUP-B15 were used. Cell viability was reduced upon treatment of HHT at time and dose-dependent manner. Percentage of apoptotic cells that determined by flow cytometric analysis of PI and Annexin V double staining was increased upon treatment of HHT. Moreover, HHT was able to induce apoptosis in both T-ALL and B-ALL cell lines. Western blot analysis showed that MCL-1 protein level was reduced upon treating with HHT for 24 hours. The above findings provide conclusive evidence for the therapeutic effect of HHT on various ALL cell lines by modulating the apoptotic pathway.

Citation Format: Anan Jiao, Kei Ching Yuen, Rahman Ud DIN, On Yi Phoebe Wong, Sin Chun-Fung. Homoharringtonine is effective in treating acute lymphoblastic leukaemia via modulation of apoptotic 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 1574.
Abstract 1596: The promising recipe sorafenib rosiglitazone and adipogenesis inducers cocktail for in vitro hepatocellular carcinoma differentiation induction besiege carcinogenesis
Authors: Anis Aya , Mostafa Ahmed M. , Hamdy Nadia M. , Sultan Ahmed S.
Published: April 2023

Aim: The present study is focusing on using sorafenib, rosiglitazone, and adipogenesis-inducers cocktail (Insulin, Dexamethasone and IBMX) as a combination treatment to induce hepatocellular carcinoma (HCC) cell lines differentiation into adipocyte functioning cells, a strategy for effecting examined cancer cell lines to lose carcinogenicity properties and hopefully, turning into near-normal cells, with near-normal activity. The liver is one of the most vital organs in the body, any malfunctioning in the liver affects the whole body's health and performance. By 2040, 1.4 million people could be diagnosed with liver cancer and 1.3 million people could die from the disease, thus, trying an attempt to stop or reverse this cancer, would be a great break-through.

Materials, Methods and Results: Using the current differentiation-induction cocktail to turn HepG2 and Hep3B HCC cancer cells into adipocytes, was confirmed by Oil Red O staining and morphological changes, and significantly expressed "adiponectin" level. Moreover, the cellular molecular markers p53, PPAR-γ, E-cadherin/β-catenin complex, and caspase-3 activities were significantly enhanced in the cocktail induced-differentiated cells, confirming the effectiveness of the proposed cocktail for ameliorating STAT3 together with inducing apoptosis (clonogenicity) , in comparison to control cells or HCC cells treated with the cocktail individual drugs. The differentiation-induction cocktail through rosiglitazone activated PPAR-γ which in turn increased HCC cells sensitivity to insulin to induce lipid uptake. Sorafenib, in the cocktail, role to block tumor cell proliferation via inhibiting STAT3 signaling pathway activation. In conclusion, the current proposed differentiation-induction cocktail after further in vivo confirmation (future prospective) might open a different avenue for liver cancer treatment.

Citation Format: Aya Anis, Ahmed M. Mostafa, Nadia M. Hamdy, Ahmed S. Sultan. The promising recipe sorafenib rosiglitazone and adipogenesis inducers cocktail for in vitro hepatocellular carcinoma differentiation induction besiege carcinogenesis [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 1596.
Abstract 1650: Discovery of a novel Kevetrin analog as a potential therapeutic for ovarian cancer
Authors: Raza Asif , Krzeminski Jacek , Amin Shantu , Sharma Arun
Published: April 2023

Kevetrin is an investigational new drug in phase I clinical trial on solid tumors (NCT01664000). It has shown its efficacy in various tumors, including lung, breast, colon, and ovarian cancer cell and xenograft models, both in a p53-dependent and -independent manner. In ovarian cancer cell lines with the TP53 mutation, p21 expression was found to be upregulated without the involvement of p53. Additionally, it was proposed that Kevetrin caused the histone deacetylase 6 (HDAC6) enzyme to be downregulated, negatively affecting the HDAC6-Hsp90 chaperone axis and causing the degradation of p53 in the mutant mice. In the present study, we designed and synthesized a novel analog (compound 900) of Kevetrin and its cytotoxicity was assessed using four different ovarian cancer cell lines viz. OVCAR-3, OVCAR-10, ES2, drug-resistant HeyA8 cells, and normal fibroblast cells (FHC). Compared to the control and Kevetrin, compound 900 inhibited the ovarian cancer cell viability in a dose- and time-dependent manner. The IC50 value of compound 900 was 0.8 μM, 0.7 μM, 0.8 μM, and 0.9 μM for OVCAR-3, HeyA8, OVCAR-10, and ES2 cells, respectively, while for Kevetrin the IC50 value was >100 μM for 48 h treatment. Compound 900 was thus found to be >100 times more potent than standard Kevetrin. To further acquaint the effect of 48 h treatment on cell proliferation, we performed trypan blue cell viability assay, trypan blue dye exclusion assay, Annexin V/7-AAD, Caspase 3/7 apoptosis assays, and Western blotting of apoptotic proteins involved in apoptosis. Compound 900 significantly induced apoptosis in OVCAR-3 and HeyA8 cells in a dose-dependent manner. Upregulation of several apoptotic proteins, as evident by Western blot, suggested that 900 causes the induction of apoptotic pathways efficiently. Further, the cell cycle analysis showed a significant arrest of OVCAR-3 and HeyA8 cells in the S-phase, which was further confirmed by the analysis of cyclin A2, cyclin-E1, p21, and p27 protein expression. Additionally, compound 900 caused a significant increase in the expression of the cell cycle inhibitory genes p21 and p27 in a p53-independent manner, compared to Kevetrin. Kevetrin has shown promise in preliminary clinical studies in solid tumor patients. The new analog 900 is >100 fold more potent than Kevetrin and holds great promise to be developed further for ovarian and other mechanistically relevant cancers.

Citation Format: Asif Raza, Jacek Krzeminski, Shantu Amin, Arun Sharma. Discovery of a novel Kevetrin analog as a potential therapeutic for ovarian 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 1650.
Abstract 1728: Exosomal delivery of Fas-associated factor 1 (FAF1), a multifunctional tumor suppressor and genuine exosome cargo, effectively antagonizes tumor growth
Authors: Han Jeong-Hoon , Park DaeHo , Jung Bumjun , Yoo Hyeong-Gwon , Cho Kyung-Ok , Kim Eunhee
Published: April 2023

Introduction: Exosomes are extracellular vesicles of 30-150 nm and can stably deliver their cargos to the target with low immunogenicity and efficient transmission. FAF1 is a genuine exosome cargo and, therefore, loaded into exosomes without additional engineering. FAF1 is a tumor suppressive protein with multifunctional roles including death-promotion (both apoptosis and necrosis), cell cycle arrest, anti-inflammation, and anti-angiogenesis. FAF1 also inhibits metastasis via inhibition of TGF-β pathway. Moreover, FAF1 elicits low expression profiles in several types of cancer. Therefore, this study examined whether exosomal delivery of FAF1 is a plausible strategy for anti-tumor therapeutics.

Methods: Stable suspension cells originated from HEK293 cells inducibly overexpressing FAF1 were screened using Tet on-off system to avoid cell death due to continuous FAF1 expression. FAF1 was overexpressed by Doxycycline induction and the amounts of FAF1 loaded on exosomes were normalized by exosome markers. The efficacy of exosomal FAF1 was evaluated in mouse xenograft models implanted with various human carcinoma cells.

Results: Cell lines showing FAF1 expression with more than 3 fold of control cells were picked. Among FAF1-overexpressing cells, F5-3 cell line showing highest expression of exosomal FAF1 (showing 23 fold of FAF1 loading efficiency compared to parental cells) was used for exosomal source for in vivo experiments. Furthermore, F5-3 produced about 70 times of exosomal FAF1 when compared to that of HEK293 cell in the same volume of culture medium. The exosomal FAF1 produced in F5-3 cells inhibited tumor growth in various cancer types including lung, breast, liver, and pancreas in mouse xenograft models. In addition, exosomal FAF1 consistently demonstrated superior tumor-suppressive potential to soluble FAF1 in all tumors investigated in this study. Also, there were no serious side effects based on weight changes.

Conclusion: This study demonstrated the tumor-suppressive potential of exosomal FAF1, demonstrating the feasibility of exosomal delivery of FAF1 as an efficient therapeutic measure. Furthermore, the selective and massive production properties of F5-3 cell realizes efficient and scalable production of exosomal FAF1.

Citation Format: Jeong-Hoon Han, DaeHo Park, Bumjun Jung, Hyeong-Gwon Yoo, Kyung-Ok Cho, Eunhee Kim. Exosomal delivery of Fas-associated factor 1 (FAF1), a multifunctional tumor suppressor and genuine exosome cargo, effectively antagonizes tumor growth [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 1728.
Abstract 1749: The synergic effect of cisplatin and a chemokine receptor antagonist sensitizes drug resistant gastric cancer cells and inhibits tumoroid formation
Authors: Mora-Lagos Bárbara , Reyes María Elena , Lobos-Gonzalez Lorena , Campo Matías del , Ili Carmen Gloria , Buchegger Kurt , Mora Yuselin , Zanella Louise , Riquelme Ismael , Brebi Priscilla
Published: April 2023

Introduction: Gastric cancer (GC) is an important cause of death worldwide. Cisplatin (CDDP) is the most used drug in the chemotherapy of advanced GC. However, CDDP resistance reduces GC survival. The CCR5/CCL5 axis has been associated with GC development, but its role in CDDP resistance in GC has not been elucidated. The aim of this study was to determine the effects of CCR5/CCL5 axis blockade by a chemokine receptor antagonist (CRA), alone and in combination with CDDP, on in vitro and 3D tumorid models from AGS R-CDDP cells.

Methodology: AGS R-CDDP cells (CDDP-resistant human gastric adenocarcinoma cells) were previously established using a stepwise drug dosing protocol and characterized. CCL5 was selected through transcriptomic analysis and its expression level was validated by qRT-PCR. Cytotoxicity assays were determined by using MTT. Apoptosis and cell cycle assays were evaluated by flow cytometry. Tumoroid formation was performed from AGS R-CDDP cells in low adhesion plates (NunclonTMSpheraTM). After 10 days in culture, tumoroid >150 µm in diameter were scored and pharmacological stimuli were added on days 14, 17 and 20. CRA was used alone and in combination with CDDP in all assays.

Results: CRA/CDDP combination triggered a re-sensitized phenotype in AGS R-CDDP cells, decreasing cell viability but not increasing apoptosis compared to independent treatments. AGS R-CDDP cells treated with CRA/CDDP were mainly arrested in S phase. CCL5 showed a decrease in mRNA levels after the CRA/CDDP combination compared to separate treatments. AGS R-CDDP cells showed a higher potential in the ability to form tumoroids compared to AGS WT cells. Finally, CRA/CDDP combination inhibited tumoroid formation compared to independent treatments.

Discussion and Conclusion: CCL5 showed a decrease in mRNA levels after the CRA/CDDP combination, possibly correlating with allosteric inhibition of the CCR5 receptor. CRA/CDDP combination has a synergic effect that sensitizes AGS R-CDDP cells and inhibits tumoroid formation. This combination could be used as a potential coadjuvant in GC therapy allowing to reduce the doses of CDDP and therefore to reduce side effects.

Citation Format: Bárbara Mora-Lagos, María Elena Reyes, Lorena Lobos-Gonzalez, Matías del Campo, Carmen Gloria Ili, Kurt Buchegger, Yuselin Mora, Louise Zanella, Ismael Riquelme, Priscilla Brebi. The synergic effect of cisplatin and a chemokine receptor antagonist sensitizes drug resistant gastric cancer cells and inhibits tumoroid formation [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 1749.
Abstract 1772: Development of a novel L1CAM-targeted CAR-T, CX804, and its therapeutic efficacy in ovarian and gastric cancer
Authors: Che Xiumei , Yun Un-Jung , Lee Seokwon , Kim Youngyoub , Lee Junshub , Chae Jina , Suh Jehee , Nam Eun-Ji , Kim Gun Min , Kim Hyoyoung , Jung Minkyu
Published: April 2023

L1 cell adhesion molecule (L1CAM) is involved in cancer development and aberrantly expressed in various tumor. It is also associated with aggressive progression, metastasis, and poor prognosis. Public database, a Kaplan-Meier plotter (http://www.kmplot.com) shows that L1CAM high-expression significantly reduced survival of ovarian and gastric cancer patients. We developed a novel chimeric antigen receptor (CAR)-T cell targeted to L1CAM. A novel scFV candidate (Clone H8) to L1CAM was obtained by bio-panning of phage displaying human synthetic scFv fragments to human and mouse L1CAM proteins. H8 scFV was linked to IgD-IgG1 hinge, CD28 transmembrane domain, CD28 and ICOS co-stimulation domain, and CD3ζ domain, resulting in CX804 CAR-T. The efficacy of L1CAM CAR-T cells, CX804 showed correlation with the level of L1CAM in human ovarian and gastric cancer cells. CX804 exerted cytotoxicity toward 293T cells expressing human or mouse L1CAM protein. CX804 also reacted with L1CAM-positive ovarian SKOV3 cancer cell and gastric MKN-28 and AGS cancer cells and secreted T-cell activation cytokines, IFN-γ and TNF-α, in response to these cells. However, CX804 rarely lysed L1CAM-negative normal 293 cells and SNU-719 gastric cancer cells. Furthermore, L1CAM CAR-T cells dramatically lysed high L1CAM expressing patient derived cell (PDC) of gastric cancer compared with low L1CAM expressing PDC cell. Xenograft model of ovarian cancer was developed by intraperitoneal (IP) injecting SKOV3-Fluc cells into NOD/SCID or NSG mice. CAR-T cells were administrated via intravenous (IV) or IP route on 5 days after tumor inoculation. Administration of CX804 significantly decreased tumor burden and increased survival in mouse model of ovarian cancer. Route of administration significantly affected bio-distribution and antitumor effect of CX804 where antitumor activity of CX804 was only observed when CX804 were given IP, but not the IV route. Taken together, our results suggest that CX804 exerts potent and specific effect against L1CAM positive cancer cells in vitro and in vivo. Furthermore, we propose that newly established CAR-T cells targeting L1CAM facilitate therapy against L1CAM-positive ovarian and gastric cancer patients

Citation Format: Xiumei Che, Un-Jung Yun, Seokwon Lee, Youngyoub Kim, Junshub Lee, Jina Chae, Jehee Suh, Eun-Ji Nam, Gun Min Kim, Hyoyoung Kim, Minkyu Jung. Development of a novel L1CAM-targeted CAR-T, CX804, and its therapeutic efficacy in ovarian and gastric 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 1772.
Abstract 1779: Fourth-generation chimeric antigen receptor T cells targeting mucin 1 against breast cancer
Authors: Supimon Kamonlapat , Sangsuwannukul Thanich , Junking Mutita , Yenchitsomanus Pa-thai
Published: April 2023

Breast cancer (BC) is the most common cause of deaths worldwide, which is required effective treatment strategies. The second-generation chimeric antigen receptor (CAR) T cell therapy was approved by U.S. Food and Drug Administration for hematological malignancies, exhibiting impressive efficacy. However, CAR T cell therapy has not been approved in solid tumors and it has intensively been the subjects of research and development. To develop CAR T cell therapy, it is crucial to identify target antigen specifically expressed on tumor cells. Mucin 1 (MUC1) was previously reported to be overexpressed in several tumors. In this study, we examined MUC1 expression in BC tissues and cultured cell lines, and to improve CAR T cell efficiency, we generated fourth-generation anti-MUC1 CAR T cells (αM.CAR4 T cells), containing anti-MUC1 single-chain variable fragment (scFv), three costimulatory molecules (CD28, CD137, CD27) and CD3ζ. High levels of MUC1 expression were observed in the tissues and cell lines. Comparing to second- and third-generations (αM.CAR2 T and αM.CAR3 T cells), the αM.CAR4 T cells showed greater proliferation in the CAR T cell production process. The anti-tumor functions of all CAR T cells were specific to MUC1 expression since their killing activities were illustrated against MUC1-positive but not MUC1-negative BC cells. Interestingly, at low effector to target ratios, the αM.CAR4 T cells exhibited higher tumor cell lysis than those of αM.CAR2 T and αM.CAR3 T cells. Furthermore, after exposure to MUC1-positive BC cells, the αM.CAR4 T cells expressed lower programmed cell death protein 1 (PD-1) level. Taken together, the αM.CAR4 T cells are potential to be further developed for treatments of MUC1-positive BC and other cancers.

Citation Format: Kamonlapat Supimon, Thanich Sangsuwannukul, Mutita Junking, Pa-thai Yenchitsomanus. Fourth-generation chimeric antigen receptor T cells targeting mucin 1 against breast 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 1779.
Abstract 1799: BH3120, a bispecific antibody targeting 4-1BB and PD-L1 simultaneously, stimulates T cells in tumor tissue preferred manner
Authors: Wang Jing , Wang Jun , Liu Yang , Pang Yan , Zhang Aihong , Feng Jie , Sun Aibo , Ma Xiao , Cai Jingmei , Xu Jiangcheng , Liu Jiawang , Lee Kyoungwoo
Published: April 2023

BH3120 is a heterodimeric bispecific antibody, designed to target 4-1BB and PD-L1 simultaneously with Pentambody™ platform, to stimulate anti-tumor immune response in a tumor microenvironment (TME) specific manner.

BH3120 showed strong antitumor efficacy in multiple tumor models, and combination of BH3120 with a PD-1 antagonist showed synergic effects eradicating tumor. While BH3120 consistently activates T cells, both as a monotherapy and in combination with a PD-1 antagonist, modulation of immune system in the tumor tissue is clearly de-coupled from that in peripheral blood, suggesting reduced risks of immune related adverse events (irAEs) with BH3120. To verify this hypothesis, BH3120 was compared with reference bispecific antibodies. Combination of BH3120 with a PD-1 antogonist does not result in elevation of transaminase enzymes, while the reference bispecific antibodies, when combined with the same PD-1 antagonist, show significant increase of the enzymes and macrophage in liver.

In the toxicology studies conducted so far with cynomolgus monkeys, NOAEL of BH3120 was determined to be 200 mg/kg (QW X 5).

Citation Format: Jing Wang, Jun Wang, Yang Liu, Yan Pang, Aihong Zhang, Jie Feng, Aibo Sun, Xiao Ma, Jingmei Cai, Jiangcheng Xu, Jiawang Liu, Kyoungwoo Lee. BH3120, a bispecific antibody targeting 4-1BB and PD-L1 simultaneously, stimulates T cells in tumor tissue preferred manner [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 1799.
Abstract 18: Transcriptomic analysis of mucinous adenocarcinoma in genetically-engineered mouse model
Authors: Sada Haruki , Niitsu Hiroaki , Nakahara Hikaru , Miguchi Masashi , Sakamoto Naoya , Oue Naohide , Tashiro Hirotaka , Ohdan Hideki , Hinoi Takao
Published: April 2023

Background and Aim: The nature and characteristic of human mucinous colorectal adenocarcinomas have not been well elucidated yet. We previously reported a genetically-engineered mouse model for colonic neoplasia producing mucin based on inactivation of Transforming Growth Factor β Receptor Type II (Tgfbr2) and Apc (CDX2P-G19Cre;Apc flox/+;Tgfbr2flox/flox), which is useful to investigate the mechanism of tumor formation. This mouse model developed colonic neoplasia from 12 weeks of age. Despite the same genotype, 40% of the tumors were found mucinous adenocarcinoma and 28% were high-grade adenoma. For further investigation, we herein perform transcriptomic analysis to clarify the molecular differences in these two histologically different tumors.

Method: Total RNA were extracted from mucinous adenocarcinomas and high-grade adenomas in CDX2P-G19Cre;Apcflox/+;Tgfbr2 flox/flox mice from fresh frozen tumors (n=3, each). Then, RNA-seq and Gene Set Enrichment Analysis were performed. Samples were sequenced on NovaSeq 6000, and gene sets queried included the Hallmark.

Results: Mucinous adenocarcinoma and high-grade dysplasia were differentially clustered in principal component analysis despite the same genotype. Gene set enrichment analysis showed 19 upregulated signaling pathways including KRAS signaling and epithelial mesenchymal transition, and 8 suppressed signaling pathways including E2F targets and MYC targets in mucinous adenocarcinoma compared to high-grade dysplasia (FDR < 0.05).

Conclusions: By comparing transcriptome of mucinous adenocarcinoma with high-grade adenoma in our mouse model, 27 differential signal pathways were detected. Some of these pathways are presumably associated with the nature and the characteristics of mucinous adenocarcinoma. We are further investigating the effects of these differences in signal pathways and the correlation with human mucinous adenocarcinoma.

Citation Format: Haruki Sada, Hiroaki Niitsu, Hikaru Nakahara, Masashi Miguchi, Naoya Sakamoto, Naohide Oue, Hirotaka Tashiro, Hideki Ohdan, Takao Hinoi. Transcriptomic analysis of mucinous adenocarcinoma in genetically-engineered mouse model [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 18.
Abstract 1833: Novel IL-12 Partial Agonist For Cancer Immunotherapy Avoids NK-cell Mediated Toxicity
Authors: Koliesnik Ievgen , Emmerich Jan , Tran Kim Q. , Bauer Michele , Totagrande Michael , Jayaraman Bhargavi , Buffone Cindy , Balasubrahmanyam Priyanka , Rokkam Deepti , Malefyt Rene de Waal , Zuniga Luis , Greb Heiko , Ratti Navneet , Vivona Sandro , Oft Martin , Lupardus Patrick J. , Kastelein Robert A.
Published: April 2023

Here we report on a novel human IL-12 partial agonist (hIL-12v) that has diminished binding to IL-12Rb1. IL-12v is designed to more selectively engage antigen activated T-cells, which strongly upregulate IL-12Rb1 upon activation, and to reduce stimulation of NK cells or resting T cells, which express modest levels of IL-12Rb1. To explore anti-tumor efficacy and toxicity in mouse syngeneic tumor models, we generated a half-life extended mouse surrogate of the IL-12 partial agonist (mIL-12v Fc) and compared it to a similarly engineered half-life extended version of wild type mouse IL-12 (mIL-12wt Fc). At efficacious doses, systemic administration of mIL-12wt Fc induced significant weight loss and lethality characterized by early proinflammatory cytokine release and systemic NK cell activation. Conversely, mIL-12v Fc was well tolerated and avoided the robust and rapid NK cell activation and peripheral NK count decreases seen with mIL-12v Fc, suggestive of extravasation to tissues. Both mIL-12v Fc and mIL-12 WT Fc showed similar robust single-agent anti-tumor efficacy in syngeneic tumor models. Depletion of NK cells did not diminish anti-tumor efficacy. Efficacy for both molecules was characterized by CD8 T cell activation, myeloid cell reprograming and antigen presentation. Moreover, combination of mIL-12v Fc with systemic immunotherapies further enhanced anti-tumor activity without compromising tolerability. Overall, mIL-12v Fc retained anti-tumor efficacy without induction of severe toxicities compared to mIL-12wt Fc. These data suggest IL-12 partial agonists may represent a novel immunotherapy approach to maintain efficacy while avoiding classical toxicity associated with IL-12 therapy.1 Atkins, et al.; (1997) Clinical Cancer Research 3(3):409-172 Carson, et al. (1999) J Immunology 162 (8): 4943-4951.

Citation Format: Ievgen Koliesnik, Jan Emmerich, Kim Q. Tran, Michele Bauer, Michael Totagrande, Bhargavi Jayaraman, Cindy Buffone, Priyanka Balasubrahmanyam, Deepti Rokkam, Rene de Waal Malefyt, Luis Zuniga, Heiko Greb, Navneet Ratti, Sandro Vivona, Martin Oft, Patrick J. Lupardus, Robert A. Kastelein. Novel IL-12 Partial Agonist For Cancer Immunotherapy Avoids NK-cell Mediated Toxicity [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 1833.