bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5051: Assessment of boron delivery peptides with angiopep-2 for boron neutron capture therapy
Authors: Liu Qi , Xiang Jing , Ma Lin , Ren Qiushi
Published: April 2023

Boron neutron capture therapy (BNCT) induces intracellular nuclear reactions that release heavy charged particles to destroy cancer cells during thermal neutron radiation. In order to selectively eliminate cancer cells but avoid harmful effects to normal tissues, targeted delivery of boron-10 in cancer cells is required, whereas the conventional boron delivery agent phenylalanine (BPA) is unsatisfactory. Angiopep-2 is a cell-penetrating peptide that can trigger transcytosis and traverse the blood-brain barrier by recognizing low density lipoprotein-related protein 1. In this study, we developed angiopep-2 for boron delivery in BNCT. Angiopep-2 was labeled with boron-10 using solid-phase peptide synthesis with 4-carboxyphenylboronic acid and fluorenylmethyloxycarbonyl (Fmoc)-protected amino acids, namely ANG-B. Molecular mass of the synthesized ANG-B were validated by mass spectrometry. In a cell proliferation assay, little toxicity of ANG-B was detected. Boron concentrations in 6 cell lines from different cancer types and an intracranial glioblastoma mouse model after treatment with ANG-B or BPA were analyzed by ICP-AES. The in vivo distribution of ANG-B showed that the ratio of boron content of tumor tissue to that of blood (T/B ratio) was 9.42±2.47 at 4h post treatment, while the ratio of boron content of tumor tissue to that of normal brain tissue (T/N ratio) was 5.61±1.0, demonstrated an optimal tumor targeting ability of ANG-B. We further measured the effects of ANG-B in BNCT by clonogenic cell survival assay in vitro or by PET/CT imaging on a glioblastoma mouse model in vivo. Radiation for BNCT was given with neutron flux at 3.0±0.4 × 1011 n/cm2. BNCT with 5 mM ANG-B resulted in 86.5%±5.3% clonogenic cell death, while the number was 72.9%±5.1% for BPA. The effect of ANG-B based BNCT in vivo was also better than BPA. The glioblastoma tumors in ANG-B treated group at 31 days after BNCT were shrank by 62.9% in average, while the BPA treated tumors shrank by only 23.0% (P<0.05). Therefore, we concluded that ANG-B is an efficient boron delivery agent, which may improve BNCT performance in clinical trials.

Citation Format: Qi Liu, Jing Xiang, Lin Ma, Qiushi Ren. Assessment of boron delivery peptides with angiopep-2 for boron neutron capture 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 5051.
Abstract 5059: High-throughput monitoring of proteoforms and pathways through multiplexed and customizable mass spectrometry assay panels
Authors: Müller Sebastian , Laforte Véronique , Savickas Simonas , Müller Maik , Feng Yuehan , Reiter Lukas
Published: April 2023

Proteins represent a key class of analytes in pharmacodynamic (PD) analysis, an essential component in preclinical and clinical studies which inform about a biological system’s response to drug treatment. Pharmacodynamic signatures typically comprise target engagement marker and downstream pathway markers. With recent development in targeted therapies and novel therapeutic modalities such as protein degraders, the scope of PD has widened to cover predictive and stratification biomarkers which can inform patient selection and contribute to further understanding of potential drug resistance.

Using peptides as surrogate for proteins of interest, mass spectrometry (MS)-based approach alleviates the barriers of reagent specificity of antibody-based methods which can be affected by species and matrices used in preclinical drug development. Moreover, the use of stable-isotope labeled standard (SIS) reference peptides grants targeted MS assays unraveled specificity and the ability to determine absolute quantity of a given analyte.

Harnessing the power of targeted MS, we present an oncology-focused assay repository which contains 804 peptides corresponding to 582 proteins. All 804 peptide surrogates were chosen under strict selection criteria such as uniqueness/proteotypicity and stability. Off-the-shelf availability (PQ500 kit) of SIS reference peptides for all 582 proteins facilitates the deployment of any of these assays in a plug-and-play manner. The collection of 582 protein targets cover > 180 cellular pathways (Reactome pathways e.g. cytokine signaling, cell-cell communication, activation of matrix metalloproteinases) together with 49 FDA-approved drug targets. To further evaluate the performance of these assays, we carried out a case control study comprising plasma samples from 5 patient groups (lung, colorectal, pancreatic, breast and prostate cancer) and age and gender-matched healthy subjects. In total, we measured absolute quantities of 582 proteins across 180 plasma samples while validating the expression profiles of well-characterized biomarkers in patient plasma.

To conclude, we’ve demonstrated the application of targeted MS approach for the routine analysis of pharmacodynamic biomarkers. Its multiplexing capability, together with the adaptability to various species allow this method to maneuver through preclinical and clinical scenarios.

Citation Format: Sebastian Müller, Véronique Laforte, Simonas Savickas, Maik Müller, Yuehan Feng, Lukas Reiter. High-throughput monitoring of proteoforms and pathways through multiplexed and customizable mass spectrometry assay panels. [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 5059.
Abstract 5230: Comprehensive genetic analysis using clinical specimens of gastric adenocarcinoma and proximal polyposis of stomach
Authors: Matsumoto Chihiro , Iwatsuki Masaaki , Mimori Koshi , Baba Hideo
Published: April 2023

Background and Purpose: Gastric adenocarcinoma and proximal polyposis (GAPPS) is an autosomal dominant genetic disorder that causes gastric carcinoma from fundus gland polyposis. Although germline mutation of APC promoter 1B was reported as a pathogenic variant in 2016, there are still many unknowns about the carcinogenesis. In this study, we performed a comprehensive Next generation sequencing (NGS) analysis of GAPPS clinical specimens to investigate the molecular profile characteristic of carcinogenesis.

Patients and Methods: Seven cases from three GAPPS families experienced in our department were included in this study. In each case, tissues were collected from normal mucosa, polyps, and carcinoma,

and FFPE and fresh-frozen samples were prepared. After pathological diagnosis was performed on each sample, RNA and DNA were extracted and analyzed based on data obtained by next generation sequence (NGS).

Results: In the RNA sequence, gene expression analysis was performed on 45 pathologically diagnosed samples, which were classified into four clusters by K-means clustering. Gene Set Enrichment Analysis (GSEA) of the four clusters showed that cancer-related genes such as p53 and KRAS were significantly enriched in the cluster of cancer, and genes related to immune response were significantly enriched in the cluster of cancer compared to the and clusters of polyp and normal. Gene mutation analysis using whole-exome sequence (read depth/coverage>20) confirmed mutation of APC promoter 1B in all samples. On the other hand, in tumor samples (N=10), the percentage of mutation of genes previously suggested to be associated with GAPPS (TP53, FBXW7, GNAS, and KRAS) were 50%, 0%, 30%, and 60%, respectively.

Conclusion: Comprehensive gene expression analysis by RNA sequence and whole-exome sequence was performed using clinical samples of GAPPS. In the future, we plan to identify specific gene-pathways involved in carcinogenesis.

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Citation Format: Chihiro Matsumoto, Masaaki Iwatsuki, Koshi Mimori, Hideo Baba. Comprehensive genetic analysis using clinical specimens of gastric adenocarcinoma and proximal polyposis of stomach. [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 5230.
Abstract 5320: The RenMice™ HiTS (Hyperimmune target specific) Platform facilitates identification of novel therapeutic antibodies for challenging targets
Authors: Zhang Xiaoqian , Fu Shufang , Zhang Shujin , Ji Xin , Hui Li , Jin James , Zhang Jing
Published: April 2023

In recent years, an increasing number of therapeutic antibodies have shown to be effective for the treatment of cancer and other diseases. However, limitations in the traditional discovery process, including immune tolerance of highly homologous genes, challenges with antibody sequence humanization, clone selection, and model selection for drug efficacy and safety evaluation, often hinder the process of identifying new therapeutic antibodies. The RenMice™ HiTS (Hyperimmune Target Specific) Platform is a library of chromosome engineered mice with fully human immunoglobulin variable domains replacing the mouse loci, each with a specific drug target gene knocked out. These mice are designed to establish robust immune responses and generate antibodies that bind to more epitopes of the target protein, including conserved domains. The platform is ideal for challenging targets, such as proteins with high homology across species, or multi-pass transmembrane proteins (e.g. GPCRs/ion channels). Here, we show that the platform can be used to generate antibodies that cross-react with multiple species, like human, monkey, dog, and mouse targets, by immunizing with both human and mouse or dog antigen. We provide examples for newer campaigns, including species cross-reactivity and internalization of novel antibodies targeting NECTIN-4, and high-throughput in vivo efficacy screening of novel anti-PD-1 antibodies in wild-type mice. In the future, we will evaluate the preliminary toxicity of these cross-reactive antibodies in preclinical animal models. Thus, selection of the best antibody candidate based on in vivo efficacy and safety allows for a streamlined and successful preclinical phase. In conclusion, the RenMice™ HiTS platform facilitates the generation of developable antibodies that recognize novel epitopes and challenging targets.

Citation Format: Xiaoqian Zhang, Shufang Fu, Shujin Zhang, Xin Ji, Li Hui, James Jin, Jing Zhang. The RenMice™ HiTS (Hyperimmune target specific) Platform facilitates identification of novel therapeutic antibodies for challenging targets. [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 5320.
Abstract 5337: Design, synthesis and evaluation of dual-targeted mEGFR and AURK inhibitors as anticancer agents
Authors: Gesinski Dayna , Kurup Sonali
Published: April 2023

Mutant epidermal growth factor receptor (mEGFR) inhibitors constitute the recommended therapy for mEGFR-positive cancers. However, resistance has developed to mEGFR inhibitors due to newer mutations within the enzyme pocket and redundant signaling pathways that bypass mEGFR inhibition. Recent studies have shown that resistance to mEGFR inhibitors could be overcome if given in combination with aurora kinase (AURK) inhibitors. Dual EGFR/AURK kinase inhibitors provide a novel approach to overcome resistance to EGFR inhibitors. In a previous study, Kurup et al. identified 1 as a nanomolar mEGFR inhibitor. Using a structure-based design approach, novel analogs of 1 that incorporated varied sidechains for dual mEGFR and AURK inhibition were designed. The synthesis of the target compounds involved microwave-assisted, copper and palladium catalyzed coupling reactions. This study led to the identification of dual-targeted mEGFR/AURKA and mEGFR/AURKB inhibitors, and compounds with sub-micromolar cellular potencies. The design, synthesis, kinase inhibitory activities and anticancer effects of these novel analogs will be presented. Structure-activity relationships for mEGFR, AURKA and AURKB inhibition will be defined.

Citation Format: Dayna Gesinski, Sonali Kurup. Design, synthesis and evaluation of dual-targeted mEGFR and AURK inhibitors as anticancer agents. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5337.
Abstract 5339: 4-Amino-N-phenylbenzamides as dual-targeted mEGFR and AURK inhibitors and anticancer agents
Authors: Rogers Brianne , Rohde Nicholas , Mikitaroff Justin , Matson Joshua , Satawa Samantha , Amissah Felix , Kurup Sonali
Published: April 2023

Small molecule, mutant epidermal growth factor receptor kinase (mEGFR) inhibitors are rendered ineffective in NSCLC due to mutations within the enzyme binding sites and redundant signaling pathways. The mEGFR inhibitors were found to retain their anticancer effects in NSCLC cells if given concurrently with AURK inhibitors. Dual-targeted small molecules designed to inhibit both mEGFR and AURK could demonstrate improved anticancer effects compared to monotargeted mEGFR inhibitors. We conducted molecular modeling studies to determine if there were similarities between the kinase pockets of mEGFR and AURK that could be exploited to develop dual-targeted mEGFR and AURK inhibitors. An overlap was observed for the active and allosteric sites of the kinase pocket for mEGFR and AURK. A series of compounds were synthesized and evaluated against mEGFR, AURKA, and AURKB. We found the kinase inhibitory profile to vary significantly depending on the substitution pattern ranging from selective mEGFR inhibitors to dual mEGFR and AURK inhibitors. The 4-substituted amino-N-phenylbenzamides were identified as dual mEGFR/AURK inhibitors. Dual mEGFR/AURK inhibitors demonstrated varied modes of binding within mEGFR and AURK. The most potent dual mEGFR and AURK inhibitors displayed nanomolar inhibition of mEGFR and AURK, and single-digit micromolar inhibition of non-small cell lung cancer cells. Target compounds were further evaluated in a 96-kinase screening assay and demonstrated selectivity for target kinases. The synthesis, structure-activity relationship (SAR) analysis and anticancer effects will be presented and discussed.

Citation Format: Brianne Rogers, Nicholas Rohde, Justin Mikitaroff, Joshua Matson, Samantha Satawa, Felix Amissah, Sonali Kurup. 4-Amino-N-phenylbenzamides as dual-targeted mEGFR and AURK inhibitors and anticancer agents. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5339.
Abstract 5346: Discovery of new targeted protein degraders using DNA-encoded chemistry
Authors: Keefe Anthony D. , Disch Jeremy S. , Duffy Jennifer , Lee Esther C. , Gikunju Diana , Chan Betty , Levin Benjamin D. , Monteiro Michael I. , Talcott Sarah A. , Lau Anthony , Zhou Fei , Kozhushnyan Anton , Westlund Neil E. , Mullins Patrick B. , Yu Yan , von Rechenberg Moritz , Zhang Junyi , Arnautova Yelena , Liu Yanbin , Zhang Ying , McRiner Andrew J. , Kohlmann Anna , Clark Matthew A. , Cuozzo John W. , Huguet Christelle , Arora Shilpi
Published: April 2023

Bispecific degraders (PROTACs) of ERα are expected to be advantageous over current inhibitors of ERα signaling (aromatase inhibitors/SERMs/SERDs) used to treat ER+ breast cancer. Information from DNA-encoded chemical library screening provides a method to identify novel PROTAC binding features as the linker positioning, and binding elements are determined directly from the screen. After screening ∼120 billion DNA-encoded molecules with ERα WT and 3 gain-of-function mutants, with and without estradiol to identify features that enrich ERα competitively, the off-DNA synthesized small molecule exemplars exhibited nanomolar ERα binding, antagonism, and degradation. Click chemistry synthesis on an alkyne E3 ligase engagers panel and an azide variant that rapidly generated bispecific nanomolar degraders of ERα, with PROTACs inhibiting ER+ MCF7 tumor growth in a mouse xenograft model of breast cancer. This study validates this approach toward identifying novel bispecific degrader leads from DECL screening with minimal optimization.

Citation Format: Anthony D. Keefe, Jeremy S. Disch, Jennifer Duffy, Esther C. Lee, Diana Gikunju, Betty Chan, Benjamin D. Levin, Michael I. Monteiro, Sarah A. Talcott, Anthony Lau, Fei Zhou, Anton Kozhushnyan, Neil E. Westlund, Patrick B. Mullins, Yan Yu, Moritz von Rechenberg, Junyi Zhang, Yelena Arnautova, Yanbin Liu, Ying Zhang, Andrew J. McRiner, Anna Kohlmann, Matthew A. Clark, John W. Cuozzo, Christelle Huguet, Shilpi Arora. Discovery of new targeted protein degraders using DNA-encoded chemistry. [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 5346.
Abstract 5373: Autoencoder-based multimodal prediction of survival for non-small cell lung cancer
Authors: Ellen Jacob G. , Jacob Etai , Nikolaou Nikos , Markuzon Natasha
Published: April 2023

Most non-small cell lung cancer (NSCLC) prognosis prediction approaches use one data type and do not take advantage of the large amount of multimodal data available. To evaluate and explore the benefits of multimodal data integration, we present a combined feature selection and denoising autoencoder pipeline for NSCLC survival prediction and survival subtype identification using microRNA (miRNA), mRNA, DNA methylation, long non-coding RNA (lncRNA) and clinical data. Survival performance for both lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC) patients was compared across modality combinations, data integration time and training data types. Multimodal data combinations outperformed single data modalities, with the early integration of all data modalities achieving concordance indexes (C-indexes) of 0.67 (±.04) and 0.63 (±.02) for LUAD and LUSC, respectively versus corresponding C-index of 0.64 (±.02) and 0.59 (±.03) for the best single cell modality (clinical). Notably, combining just lncRNA and clinical data facilitated effective survival discrimination, with C-indexes of 0.69 (±.03) for LUAD and 0.62 (±.03) for LUSC. Overall, higher performance was achieved by using a single denoising autoencoder for all biological data (early integration) and by training on both LUSC and LUAD patient data together. Two survival subtypes (log rank test p-value=1e-9) were identified, with 991 differentially expressed transcripts in the poorer survival group. Our analysis shows the value of multimodal data integration for predicting NSCLC progression, with especially good performance using the combination of lncRNA and clinical data. Early integration of biological data, with an initial linear feature selection technique and a denoising autoencoder for dimensionality reduction, showed effective survival performance and survival subtype identification. Further research is underway to expand analysis to different cancer types and data modalities and extract more biological interpretability from autoencoder models.

Citation Format: Jacob G. Ellen, Etai Jacob, Nikos Nikolaou, Natasha Markuzon. Autoencoder-based multimodal prediction of survival for 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 5373.
Abstract 5420: Deep learning-based mapping of tertiary lymphoid structure scores from H&E images of renal cell carcinoma trained by spatial transcriptomics data
Authors: Cook Seungho , Shin Haenara , Seo Mi-Kyoung , Lee Dae Seung , Choi Hongyoon
Published: April 2023

Purpose Tertiary lymphoid structures are organized aggregates of immune cells present in the tumor microenvironment (TME) in which novel targets as well as beneficial biomarkers for immunotherapy in cancer were found. Here, we have developed and validated a deep learning model by integrating H&E images of renal cell carcinoma and spatial transcriptomics data to infer spatial mapping of tertiary lymphoid structure scores in TME only using hematoxylin and eosin (H&E) images.

Methods A total of 20 H&E images combined with spatial transcriptomics data of renal cell carcinoma were used to develop a model. Tertiary lymphoid structure scores can be acquired for each spot in spatial transcriptomics by geometric mean of the specific gene expression relevant to B cell, T cell, immunoglobulin, fibroblast, complement, and others. A convolutional neural network using H&E image patches as inputs was developed to predict the tertiary lymphoid structure scores from H&E-stained tissue image patches of renal cell carcinoma acquired by different patients. For the external validation, the model estimated the tertiary lymphoid structure scores from H&E-stained tissue image patches of renal cell carcinoma of The Cancer Genome Atlas (TCGA-RCC).

Results The tertiary lymphoid structure scores inferred by the model using H&E image patches were significantly correlated with those derived by spatial transcriptomics data as an internal validation (r = 0.68, p < 1e-10). The mean value of the deep learning-based tertiary lymphoid structure scores estimated by the TCGA-RCC tissue images was significantly correlated with the tertiary lymphoid structure scores, T cell enrichment scores and immune cell enrichment scores estimated by bulk RNA-seq data from the corresponding TCGA data.

Conclusions A deep learning model to infer spatial tertiary lymphoid structure in the tumor microenvironment using H&E images was developed. As the tertiary lymphoid structure is a key to predict responsiveness of immune checkpoint inhibitors, mapping the score only using H&E images could be clinically translated into image-based biomarkers. This approach can provide objective and flexible deep learning-based models for characterizing tumor microenvironment related to spatial immune distribution.

Citation Format: Seungho Cook, Haenara Shin, Mi-Kyoung Seo, Dae Seung Lee, Hongyoon Choi. Deep learning-based mapping of tertiary lymphoid structure scores from H&E images of renal cell carcinoma trained by spatial transcriptomics data. [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 5420.
Abstract 544: Dissecting the functional impairment of wild-type Tp53 in human glioblastoma
Authors: García-Montaño Leopoldo A. , Muñoz Yamhilette Licón , Martinez Frank , Gallegos Kandee , Kang Huining , Piccirillo Sara G.
Published: April 2023

Glioblastoma (GBM) is the most common malignant brain tumor in adults and is characterized by heterogeneous nature, invasive potential, and poor treatment response. Work from The Cancer Genome Atlas (TCGA) has identified the tumor suppressor gene TP53 as one of the GBM drivers. However, in contrast to several cancers, only ~ 35% of GBM cases carry mutations in the TP53 gene. Furthermore, recent work from our lab showed that TP53 mutations emerge as ‘late’ events during GBM growth. This led us to hypothesize that wild-type (wt) Tp53 function is impaired in GBM. To test our hypothesis, we used clinical and molecular data of 591 GBM patients from TCGA and compared survival, age, gender, race, TP53 levels, and MGMT methylation status between wt and mutant patients. We also compiled a list of key players regulating Tp53 activity and used expression data to (i) compare the correlation of TP53 levels with the expression of each player and (ii) identify differentially expressed genes between our two groups. Our preliminary data suggest that wt TP53 patients live less in comparison to mutant patients and that both groups respond similarly to treatment, suggesting impairment of wt Tp53 function. We did not observe differences in age, sex, race, TP53 levels, or MGMT methylation status between the two groups. However, correlation analysis revealed that, among Tp53 activators, MAPK8 shows a strong negative correlation with TP53 expression in the wt group, raising the possibility that Tp53 function may be impaired. Furthermore, gene expression analysis of key Tp53 players showed that approximately 21% are differentially expressed between wt and mutant patients. Altogether, our results suggest that Tp53 function is impaired in wt patients and provide insights into the possible players affecting its activity. Our future work will focus on validating these results using GBM patient-derived cells and mouse models.

Citation Format: Leopoldo A. García-Montaño, Yamhilette Licón Muñoz, Frank Martinez, Kandee Gallegos, Huining Kang, Sara G. Piccirillo. Dissecting the functional impairment of wild-type Tp53 in human glioblastoma [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 544.
Abstract 5492: Determining effects of the CD206 positive M2 macrophage depleting engineered exosome in combination with anti PD1 therapy in breast tumor
Authors: Parvin Mahrima , Rashid Mohammad H. , Alptekin Ahmet , Arbab Ali S.
Published: April 2023

Introduction: Triple negative breast cancer (TNBC) subtype of breast cancer, which lacks molecular markers such as HER2 and estrogen/progesterone receptors, has limited treatment options. Exosomes are nano-sized (30-150 nm) spherical vesicles, derived from the endosomal system for intercellular communications, can be engineered to express targeting peptides to target specifically CD206 positive M2 macrophage. Investigators have used either synthetic nanoparticles or fusion protein to deliver Fc-IgG2b to initiate Antibody dependent cellular cytotoxicity (ADCC) but reports are showing a lack of ADCC following tagging with gold nanoparticles. To overcome this, in our lab, we developed engineered exosomes in non-tumorous cells, HEK293, to carry therapeutic Fc-mIgG2b to enhance ADCC and to carry peptide to deplete CD206+ M2 macrophages. We investigated the effectiveness of engineered therapeutic exosomes to target and deplete immunosuppressive CD206+ M2 macrophages in TNBC in combination with anti PD-1.

Methods: In this study, we developed syngeneic metastatic TBNC model (luciferase + 4T1 cells in Balb/c) by implantation of 50K 4T1-luci cells in fat pad. On day 1 of tumor implantation, animals were treated with vehicle, engineered exosomes, anti-PD1 plus engineered exosomes, 2 dose/week for 3 weeks. They were euthanized 1 day after the last dose of treatment. Single-cell suspension was made from the primary TME, lung, and spleen. Using flow cytometry surfaces markers were investigated to determine different T-cell and myeloid cell populations.

Results: We observed very surprising results following three weeks of therapies. Although the number of cytotoxic T-cells was significantly higher in the primary and metastatic TME in exosome-treated animals, there was also an increased number of proliferative T reg cells. The number of CD8+ cells was decreased following concurrent therapy with an anti-PD1 antibody. Most interestingly, lung tissue macrophages (CD11c) showed significantly increased PD-L1 expression following exosomes and anti PD1 with engineered exosomes therapies.

Conclusion: This study demonstrates that treatment with engineered therapeutic exosome targeting M2 macrophages with anti PD-1 could be utilized effectively. This study will also make a greater impact in exosome engineering based targeted therapy and will open new arena to target and deplete pathogenic cell in different tumors using engineered exosome.

Citation Format: Mahrima Parvin, Mohammad H. Rashid, Ahmet Alptekin, Ali S. Arbab. Determining effects of the CD206 positive M2 macrophage depleting engineered exosome in combination with anti PD1 therapy in breast tumor. [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 5492.
Abstract 5579: Single cell characterization of circulating plasma cells and BCMA levels in patients with multiple myeloma
Authors: Setayesh Sonia Maryam , Shishido Stephanie , Ndacayisaba Libere , Naghdloo Amin , Mason Jeremy , Tessone Dean , Kaleta Carli , Orlowski Robert Z. , Manasanch Elisabet E. , Hicks James , Kuhn Peter
Published: April 2023

Multiple myeloma (MM), a disease caused by the clonal proliferation of plasma cells (PCs), is the second leading hematologic malignancy in the world, with a global incidence rate increase of 126% and mortality rate increasing by 94% in the past decade. Recently, B cell maturation antigen (BCMA) has emerged as a promising biomarker and therapeutic target for MM due to its selective high expression on malignant PCs, improving the treatment landscape. However, the current diagnostic workup relies on bone marrow aspirates (BMA) which are highly invasive and do not allow for the detection of BCMA levels on circulating PCs, severely limiting the options for repeatable assessments and treatment-monitoring. In our study, we report the utility of a blood-based liquid biopsy, using enrichment-free high definition single cell assay (HDSCA) for the detection, quantification, and morphological characterization of circulating PCs and other BCMA+ cells. We applied a previously validated 4-color immunofluorescence assay using DAPI, CD138, BCMA, and CD45 to examine matched peripheral blood and BMAs from 68 patients across the MM disease spectrum (11 monoclonal gammopathy of undetermined significance [MGUS], 21 smoldering MM [SMM], 18 newly diagnosed MM, and 18 relapsed refractory MM [RRMM]) and peripheral blood from 23 normal donors as controls. Our analysis revealed diverse subtypes of circulating CD138(+/-), BCMA(+/-), and CD45(+/-) cells across the patients, representing both normal and abnormal PCs. We detected significantly higher circulating CD138+ BCMA(+/-) cells in MM precursor conditions (MGUS, SMM), compared to the normal donors. Additionally, we were able to detect BCMA+ oncosomes (identified by the lack of a nuclear structure) in the circulation of patients from both precursor and overt disease conditions. Our findings establish the utility of HDSCA blood-based liquid biopsy for early disease detection and anti-BCMA therapy response monitoring in MM and other B-cell malignancies expressing BCMA.

Citation Format: Sonia Maryam Setayesh, Stephanie Shishido, Libere Ndacayisaba, Amin Naghdloo, Jeremy Mason, Dean Tessone, Carli Kaleta, Robert Z. Orlowski, Elisabet E. Manasanch, James Hicks, Peter Kuhn. Single cell characterization of circulating plasma cells and BCMA levels in patients with multiple myeloma. [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 5579.
Abstract 558: EGFR inhibition in lung adenocarcinoma upregulates cell surface expression of the placental antigen ALPP and enhances efficacy of ALPP-ADC therapy
Authors: Chen Yihui , Hong Monica , Xu Hanwen , Vykoukal Jody , Park Soyoung , Cai Yining , León-Letelier Ricardo A. , Hsiao Fu Chung , Dennison jennifer B. , Ostrin Edwin J. , Fahrmann Johannes F. , Katayama Hiroyuki , Hanash Samir M.
Published: April 2023

Expression levels of placental alkaline phosphatase (ALPP) and ALPP-like 2 (ALPPL2) are relatively low in most cancer types, limiting potential benefits from ALPP or ALPPL2 targeting therapies. Enhancing their expression would be an attractive approach for targeted therapy. We have undertaken analysis of ALPP and ALPPL2 protein expression in whole cell extracts (WCE) and the surfaceome of 158 cancer cell lines and found ALPP, and to a less extent ALPPL2, to be expressed on the surface at relatively low levels across multiple cancer types. We explored various means to enhance ALPP expression in lung adenocarcinoma (LUAD) and found that induction of cancer cell quiescence via nutrient deprivation, or treatment with EGFR inhibitors, greatly enhanced ALPP surface expression. Mechanistic studies revealed that enhancement of surface ALPP expression in LUAD cells following gefitinib treatment was mediated through repression of MEK/ERK signaling and activation of the transcription factor FoxO3a, which was identified as an upstream transcriptional regulator of ALPP. Using xenograft models of LUAD, we further demonstrated that gefitinib treatment upregulates surface expression of ALPP in LUAD cells but not in normal tissues. Combination therapy with gefitinib and an ALPP antibody conjugated with Monomethylauristatin F resulted in enhanced tumor suppression compared with gefitinib alone. Our findings support a novel combination treatment modality that boosts the efficacy of ALPP-ADC directed therapy.

Citation Format: Yihui Chen, Monica Hong, Hanwen Xu, Jody Vykoukal, Soyoung Park, Yining Cai, Ricardo A. León-Letelier, Fu Chung Hsiao, jennifer B. Dennison, Edwin J. Ostrin, Johannes F. Fahrmann, Hiroyuki Katayama, Samir M. Hanash. EGFR inhibition in lung adenocarcinoma upregulates cell surface expression of the placental antigen ALPP and enhances efficacy of ALPP-ADC 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 558.
Abstract 5617: A novel DNA double-strand breaks biosensor based on fluorescence resonance energy transfer
Authors: Suh Jung-Soo , Kim Tae-Jin
Published: April 2023

Revealing the spatiotemporal behavior of DNA double-strand breaks (DSBs) is crucial for understanding the processes of DNA damage and repair. Traditionally, γH2AX and DNA damage response (DDR) factors have been used to detect DSBs using classical biochemical assays, such as antibody-based immunostaining. However, a reliable method to visualize and assess DSB activity real-time in living cells is yet to be established. Herein, we developed a novel DNA double-strand breaks biosensor (DSBS) based on fluorescence resonance energy transfer (FRET) by employing the H2AX and BRCT1 domains. By applying FRET imaging with DSBS, we show that DSBS specifically reacts to drug- or ionizing radiation (IR)-induced γH2AX activity, allowing for the quantification of DSB events at high spatiotemporal resolutions. Taken together, we provide a new experimental tool to evaluate the spatiotemporal dynamics of DNA double-strand breaks in response to cancer therapies. Ultimately, our biosensor can be useful for elucidating the molecular mechanisms underlying DNA damage and repair processes and applying for cancer therapies.

Citation Format: Jung-Soo Suh, Tae-Jin Kim. A novel DNA double-strand breaks biosensor based on fluorescence resonance energy transfer. [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 5617.
Abstract 5620: Radiogenomic analysis in the patients with primary brain tumor using dual time point 18F-FET PET/CT
Authors: Lee Dong Yun , Song Sang Woo , Lee Seungjoo , Oh Minyoung , Oh Jungsu S. , Kim Jaeseung
Published: April 2023

Purpose: To evaluate the association between genomic data and imaging features of dual time point 18F-FET PET/CT in patients with primary brain tumor for radiogenomic mapping.

Methods: From June 2021 to July 2022, we prospectively enrolled the patients under suspicious of de novo primary brain tumor in Asan Medical Center. Prior to the surgery, contrast-enhanced MRI and 18F-FET PET/CT scans were routinely performed. Twenty minutes after the post-injecting (p.i.) approximately 5 mCi of 18F-FET, patients underwent early PET/CT acquisition (20-40 min p.i.). Delay PET/CT (80-100 min p.i) was subsequently obtained after 40-minute break. SUVmax, maximum tumor-to-brain ratios and PET-positive volumes using different isocontours of SUVmax (I30, I60, I90%) in tumor were calculated in each sum images. Histopathologic results were made based on 2016 WHO classification and, isocitrate dehydrogenase (IDH) mutation, O6-methylguanine-DNA methyltransferase (MGMT) methylation, telomerase reverse transcriptase (TERT) mutation and co-deletion of 1p19q status were investigated as well. Spearman’s correlation coefficient (r), logistic regression and AUC-ROC analysis were employed using aforementioned radiomics information.

Results: A total of 42 patients (median age 55.5 yrs, range 21-84; M:F=31:11) were recruited and, 29 patients had glioblastoma with grade IV. Except TERT mutation (n=25), IDH mutation (n=11), MGMT methylation (n=19) and co-deletion of 1p19q (n=8) had statistically significant correlation each other (IDH vs. MGMT, r=0.55; IDH vs. 1p19q, r=0.53; MGMT vs. 1p19q, r=0.42). Early scan of I60% was closely related with IDH mutation (positive, 3.92 ± 3.04 vs. negative, 7.96 ± 5.36; p=0.005) meanwhile delay scan of I30% had significant association with co-deletion of 1p19q (present, 34.16 ± 18.67 vs. absent, 71.99 ± 38.79; p=0.01). The accuracy of logistic regression and the corresponding AUC-ROC values for IDH/MGMT/TERT/co-deletion of 1p19q status were 0.86/0.73/0.78/0.85, and 0.88/0.76/0.78/0.91, respectively.

Conclusion: Although this is a preliminary and interim analysis during our prospective study being slated to enroll the patients until December 2023, we suggest that the information acquired from both genetics and 18F-FET could be integrated into better characterization of primary brain tumor.

Citation Format: Dong Yun Lee, Sang Woo Song, Seungjoo Lee, Minyoung Oh, Jungsu S. Oh, Jaeseung Kim. Radiogenomic analysis in the patients with primary brain tumor using dual time point 18F-FET PET/CT. [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 5620.
Abstract 5727: Identification of synthetic lethal vulnerabilities in cancers with loss of function mutations in NOTCH
Authors: Hong Deli , Booker Matthew A. , Anjum Sidrah , Li Yixiang , Thai Tran C. , Wang Michelle Y. , Barbie David A. , Tolstorukov Michael Y. , Qi Jun , Oser Matthew G.
Published: April 2023

Loss of Function (LOF) mutations in the NOTCH receptors are found in several different cancers most notably in small cell lung cancer (SCLC) and squamous cell carcinomas (SCC). We previously developed genetically engineered mouse models (GEMM) of SCLC using CRISPR/Cas9 engineered to be NOTCH1-Mutant, NOTCH2-Mutant, and NOTCH-WT. Synthetic lethality provides a paradigm for targeting cancers with LOF mutations in tumor suppressor genes. In applying this paradigm, one looks for specific vulnerabilities that are created upon loss of the gene of interest. Using 6 cell lines developed from these CRISPR-based SCLC GEMMs which are isogenic to NOTCH, we performed CRISPR/Cas9 LOF negative selection screens (using an sgRNA library enriched in druggable enzymes) to identify synthetic lethal interactors with LOF NOTCH mutations. Our CRISPR/Cas9 screen identified TRIM28 (Tripartite Motif Containing 28, or KAP1) as a highly significant synthetic lethal interactor with NOTCH1 or NOTCH2. We validated the synthetic lethal interaction between NOTCH1/2 and TRIM28 using both human and mouse NOTCH-isogenic cell lines. Interestingly, RNA-sequencing of NOTCH2-Mutant or NOTCH-WT SCLC cell lines after TRIM28 CRISPR inactivation showed robust increases in endogenous retroviral (ERV) expression and the MDA5/RIG-I/MAVS RNA-sensing machinery leading to hyperactivation of the TBK1/IRF3/7 innate immune signaling pathway and the pro-inflammatory cytokine CXCL10 selectively in cells with NOTCH inactivated. Notably, inactivation of TBK1 activity or JAK-STAT activity using specific small molecule inhibitors of TBK1 or JAK completely reversed the synthetic lethality between NOTCH and TRIM28 suggesting that the synthetic lethality phenotype is a consequence of hyperactivation of innate immune signaling, which is known to be cytotoxic. Together our findings uncover a novel vulnerability in SCLCs with LOF NOTCH mutations and perhaps other cancers with LOF NOTCH mutations and suggest a strategy that could also potentiate anti-tumor immunity by increasing innate immune signaling in tumor cells.

Citation Format: Deli Hong, Matthew A. Booker, Sidrah Anjum, Yixiang Li, Tran C. Thai, Michelle Y. Wang, David A. Barbie, Michael Y. Tolstorukov, Jun Qi, Matthew G. Oser. Identification of synthetic lethal vulnerabilities in cancers with loss of function mutations in NOTCH. [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 5727.
Abstract 5739: Targeting WEE1 to improve the therapy of KRAS G12C mutant non-small cell lung cancer
Authors: Yamamoto Gaku , Tanaka Kosuke , Kamata Ryo , Mori Shunta , Liu Jie , Yamauchi Toyohiro , Sakae Yuta , Ohashi Akihiro , Kobayashi Susumu S.
Published: April 2023

Background: While sotorasib is the first clinically approved small-molecule inhibitor of KRAS G12C in non-small-cell lung cancer (NSCLC), efficacy of sotorasib alone is limited, presumably due to adaptive resistance mechanisms in cancer cells. To expand therapeutic potential of sotorasib, combination strategies need to be developed. In this study, we conducted a large-scale screening to identify novel combination candidates with sotorasib. In addition, molecular mechanisms of synergistic effects were investigated in multiple preclinical models.

Methods: To identify novel combination partners of sotorasib, we conducted high-throughput screening using a kinase inhibitor library containing 1,400 chemical compounds. Anti-proliferative and apoptotic effects in vitro were assessed by ATP-based cell viability assay and/or clonogenic assay, and caspase 3/7 assay and/or Annexin V assay, respectively. Synergistic effects were evaluated by calculating the BLISS index and visualized by SynergyFinder Plus. Immunoblotting was performed using antibodies against pCDK1 (Tyr15), pERK, pAkt, MCL1, BIM, PUMA, BCL2, or BCL-xL. In vivo efficacy studies were performed using a KRAS G12C mutant H358 xenograft nude mouse model.

Results: Through the large-scale unbiased combination screening of -1,400 kinase inhibitors, we identified a WEE1 inhibitor (AZD1775), a G2/M checkpoint abrogator in clinical-stage development, as a promising combination partner of sotorasib in KRAS G12C mutant NSCLC. The synergistic effects in vitro were broadly observed in multiple KRAS G12C mutant NSCLC cell lines, regardless of single-agent sensitivity to sotrasib or co-occurring oncogenic mutation profiles. The combination treatment upregulated pro-apoptotic protein BIM, leading to apoptosis. In vivo efficacy studies in H358-xenografted models also demonstrated remarkable tumor regression and durable response in mice treated with sotorasib and AZD1775 compared to ones with a single agent alone.

Conclusion: We identify AZD1775, a WEE1 inhibitor, as a novel combination candidate, which enhanced the anti-tumor activity of sotorasib both in vitro and in vivo preclinical models. These findings can lead to a novel therapeutic strategy for KRAS G12C mutant NSCLC.

Citation Format: Gaku Yamamoto, Kosuke Tanaka, Ryo Kamata, Shunta Mori, Jie Liu, Toyohiro Yamauchi, Yuta Sakae, Akihiro Ohashi, Susumu S. Kobayashi. Targeting WEE1 to improve the therapy of KRAS G12C mutant 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 5739.
Abstract 574: Depletion of regulatory T cells without interruption of IL-2 signaling by antibody against CD25 for enhancing antitumor immunity
Authors: Gao Yuxuan , He Feng , Peng Hao , Wu Kefei , Zhao Hui , Feng Yanpeng , Hao Feng , Wu Guojin , Ning Jinying
Published: April 2023

Depleting regulatory T cells from the tumor microenvironment is an attractive strategy for enhancing immunotherapy. Here we developed a humanized antibody against CD25 (H11E11-2V1) with Treg-biased binding. This antibody has a medium affinity to CD25 (KD=8.9nM), weaker than Daclizumab (KD=1.5nM) and RG6292 (KD=4.5nM). Primary T cells activated with CD3 and CD28 beads had increased CD25 expression. H11E11-2V1 bound to Treg (CD4+Foxp3+) with a concentration as low as 0.07nM, similar to RG6292, while it has no detectable binding to activated Teff (CD4+Foxp3-) with a concentration below 2nM (vs. RG6292 below 0.7nM). In addition, H11E11-2V1 did not block IL-2 signaling in NK-92 cells, which expressed CD25 on the cell surface, while Daclizumab blocked the IL-2 signaling. Daclizumab inhibited T cell proliferation (ki67+%) and activation (Granzyme B+%), but not by RG6292 and H11E11-2V1. In vitro cytotoxicity assay indicated that H11E11-2V1 with modified Fc to enhance ADCC specifically depletes Treg from activated primary T cells at a minimal concentration of 0.07nM (vs. RG6292 at 0.14nM) while leaving 90% of Teff intact. In the in vivo MC38 murine tumor model with mice whose CD25 was replaced with human CD25, H11E11-2V1 given by I.P. injection suppressed tumor growth with higher efficiency than RG6292. These results suggested that H11E11-2V1 was an excellent candidate therapeutical antibody against CD25 for cancer therapy.

Citation Format: Yuxuan Gao, Feng He, Hao Peng, Kefei Wu, Hui Zhao, Yanpeng Feng, Feng Hao, Guojin Wu, Jinying Ning. Depletion of regulatory T cells without interruption of IL-2 signaling by antibody against CD25 for enhancing antitumor immunity [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 574.