bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2878: Identification of immunosuppressive and tumor-promoting neutrophils in human bone marrow and a novel mouse model that supports human neutrophil engraftment
Authors: Liu Wei , Che Keying , Lu Chun , Wei Jia , Li Yan
Published: April 2023

Immunosuppressive neutrophils are identified as a specialized heterogeneous population in cancer patients that accelerate tumor progression. However, the developmental stage and the source of this population remain elusive. To address this issue, we unified three reported neutrophil nomenclatures and demonstrated that immature neutrophils predominate tumor tissues of patients. On the quest to identify the origin of immature neutrophils, we unexpectedly discovered that immature neutrophils collected from the bone marrow of healthy people and mice reconstituted with human immune cells (HIS mice) are naturally immunosuppressive and tumor-promoting, whereas the mouse counterpart is not. Single-cell comparison of immature neutrophils isolated from human tumors, human bone marrow and HIS mouse bone marrow samples reveal FCN1 as a common marker for this heterogeneous population. To further investigate the link between peripheral and bone marrow immature neutrophils, we created a novel immunodeficient strain, to overcome the poor human neutrophil engraftment in the periphery. HIS mice based on this strain manifest similar kinetics of immature neutrophil expansion as cancer patients both in the bone marrow and periphery after tumor cell inoculation. Finally, we proved that eliminating bone marrow immature neutrophils delays tumor growth in HIS mice. In summary, human immunosuppressive neutrophils are predominantly immature neutrophils and targeting this population in bone marrow may represent a novel strategy for cancer immunotherapy.

Citation Format: Wei Liu, Keying Che, Chun Lu, Jia Wei, Yan Li. Identification of immunosuppressive and tumor-promoting neutrophils in human bone marrow and a novel mouse model that supports human neutrophil engraftment [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 2878.
Abstract 2881: Investigating cholesterol transfer between macrophages and prostate cancer cells
Authors: Osunmakinde Olabisi , Ruffell Brian
Published: April 2023

Background: Androgens stimulate androgen receptor (AR) signaling, driving prostate cancer (PC) cell proliferation and progression. Androgen deprivation therapy (ADT) effectively treats primary disease, but 20-30% of patients relapse after ~5 years, resulting in castration-resistant prostate cancer (CRPC), which has limited treatment options. We have previously shown in a murine model of CRPC that macrophages promote AR signaling in CRPC and that depleting tumor macrophages extends survival in response to ADT with Lupron. This correlates with reduced androgen levels in macrophage-depleted tumors and the ability of macrophages to transfer cholesterol, the anabolic precursor of androgens, to prostate cancer cells. However, the mechanism by which this transfer occurs, and whether cholesterol exchange mediates resistance to ADT, is currently unknown.

Methods: To assess cholesterol transfer from macrophages to cancer cells, we established a culture system involving prostate cancer cell lines and either the RAW264.7 macrophage cell line or bone marrow-derived macrophages (BMDMs). Macrophages were pre-loaded with cholesterol using fluorescently labeled low-density lipoprotein (LDL) and were co-cultured or placed in a transwell system with tumor cells for 24 hours. Cholesterol transfer was then measured by flow cytometry.

Results: Fluorescent signal from LDL was detected in 50-70% of tumor cells after 24 hours of co-culture. Cell contact was required for cholesterol transfer, as no fluorescence was detected in prostate cancer cells during transwell experiments. Macrophages were also able to internalize and transfer acetylated LDL, which is poorly taken up by tumor cells compared to unmodified LDL. Macrophage polarization with interleukin-4 did not impact cholesterol transfer and uptake of acetylated LDL occurred independently of the scavenger receptor CD36. The scavenger receptor SR-B1, which is often upregulated in prostate cancer cells, was dispensable for transfer, as knocking out SR-B1 via CRISPR/Cas9 in cancer cell lines did not reduce LDL uptake.

Conclusion: Cholesterol transfer between macrophages and prostate cancer cells occurs in a contact-dependent manner, independently of the scavenger receptors most commonly associated with cholesterol accumulation.

Citation Format: Olabisi Osunmakinde, Brian Ruffell. Investigating cholesterol transfer between macrophages and prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2881.
Abstract 2884: Cancer-associated fibroblasts potentiate subcutaneous tumor growth in a novel bladder cancer mouse model with a genotype dependency
Authors: Valentine Henkel Lee , Satyal Uttam , Metz Alexander , Bukavina Laura , Abbosh Philip H.
Published: April 2023

Introduction Interrogation of the relationship of genotype (mutation in relevant gene) and phenotype (sensitivity to therapy) in targeted therapy would be facilitated by the use of isogenic in vitro and in vivo models. The PI3K/AKT/mTOR pathway is widely activated but heretofore untargeted in bladder cancer. We sought to develop an immunocompetent CRISPR-edited bladder cancer mouse model that recapitulates common PI3K/AKT/mTOR mutations to study a genotype-phenotype relationship. Our in vitro CRISPR-edited organoids are immunogenic in vivo, as such, we explored utilizing Cancer-Associated Fibroblasts (CAFs) to mediate immunosuppression and promote a pro-tumorigenic environment.

Methods and Results We isolated bladder urothelial cells from Rosa26 LSL-Cas9/GFP transgenic mice and grew them as organoids. Trp53 and Rb1 were co-edited to establish ‘base organoids’. As proof of concept, Pten was edited in base organoids in anticipation of developing a series of organoids with different mTOR pathway genotypes. We found that Pten-KO base organoids were 9x more sensitive to ipatasertib than Pten WT organoids. The edited urothelial cells were engrafted into mouse bladder to further test kinase inhibitor susceptibility as preclinical evidence to support human clinical trials. However, the cells successfully engrafted but disappeared after 3-4 weeks before tumors formed. Subcutaneous allografts were also rejected after 3-4 weeks and showed CD45+ immune infiltration during rejection. When cells were injected subcutaneously with CAFs, tumors persisted for 12 weeks, but only in organoids with both Trp53 loss and loss of murine chromosome 4qC4 (which is homologous to 9p21 loss in humans). 9p21 contains 3 genes which are co-deleted in ~40% of bladder cancers: MTAP, CDKN2A, and CDKN2B. Histopathological analysis of these masses confirmed their malignant nature and revealed an immune-excluded architecture. IHC staining of 9p21/Trp53 KO tumors for CD206 and CD11c revealed greater recruitment of M2 macrophages than M1 macrophages.

Conclusions CAFs remodel the tumor-microenvironment in bladder cancer via the recruitment of pro-tumorigenic M2 macrophages. This may occur in a genotype-specific manner and 9p21 loss may be a requirement for some genotypes to be tumorigenic.

Citation Format: Henkel Lee Valentine, Uttam Satyal, Alexander Metz, Laura Bukavina, Philip H. Abbosh. Cancer-associated fibroblasts potentiate subcutaneous tumor growth in a novel bladder cancer mouse model with a genotype dependency [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 2884.
Abstract 2925: In vitro characterization of next generation therapeutic antibodies
Authors: Quist Benita , Janbazacyabar Hamed , Lopez David Cobeta , Jayanth Namrata , Moiset Gemma
Published: April 2023

Therapeutic antibodies are an established drug class. Checkpoint inhibitors, such as anti-PD-1/PD-L1 and anti-CTLA-4 antibodies for cancer indications, and anti-TNF-α monoclonal antibody against autoimmune disorders represent some of most successful therapeutics in the past decade. Over 100 therapeutic targets suitable for antibody therapy have been identified for various disease indications, with several currently in late-stage clinical trials. In addition, there has been an explosion in the various types of therapeutic antibody modalities due to the increasing number of antibody engineering platforms. The new emerging bi-specific T-cell engaging antibodies (BiTEs) along with antibody-drug conjugates (ADCs) have demonstrated superior therapeutic qualities over the traditional monoclonal antibodies.

Here, we describe a pipeline to extensively characterize the various antibody therapeutics in vitro utilizing multiple innovative technologies. Assays to determine target expression and binding potency provide a thorough understanding of the target engagement. Furthermore, we present data from an innovative technology platform, the z-Movi, which measures cell avidity to predict invitro effector functions of T-cell engaging therapies, enabling more robust read-outs and could drive the lead selection of said therapies. Several mechanisms of action of different antibodies were investigated and relevant case studies describing the innate immune effector functions and the signaling pathway modulation are presented. Using a unique label-free approach, the xCELLigence, we report on the kinetics of target cell killing mediated by lymphocytes upon recognition of antigen-antibody complexes. Finally, safety assessment studies focusing on on-target/off-tumor effects of antibodies on primary cells were conducted. The cytokine profiling and cell viability assays to assess potential risk due to exaggerated levels of cytokine, reiterate the specificity and safety of the next generation of antibody therapeutics and support Investigational New Drug (IND) applications. In conclusion, we provide a comprehensive report on the in vitro characterization of the advanced antibody therapeutics, showcasing a pipeline for development and functional characterization of various antibody modalities, including bispecific T-cell engagers. This workflow can be used to progress a wide range of engineered antibodies from pre-clinical to the clinical phase of the drug discovery process. NJ and GM contributed equally to the work.

Citation Format: Benita Quist, Hamed Janbazacyabar, David Cobeta Lopez, Namrata Jayanth, Gemma Moiset. In vitro characterization of next generation therapeutic antibodies [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 2925.
Abstract 3154: Integrating DNA damage and mutation enrichment to understand mutagenesis at transcription factor binding sites
Authors: Chi Bo , Gordan Raluca
Published: April 2023

The buildup of ultraviolet (UV) light-induced DNA damage, such as Cyclobutane Pyrimidine Dimers (CPDs), is a crucial precursor to genetic mutations and the development of skin melanoma. These damaged sites and mutations are known to not be randomly distributed; in fact, previous research suggests potential relationships between damaged/mutated sites and transcription factors (TFs), proteins that regulate gene expression by binding to target sequences across the genome. To examine relationships between UV damages and TF binding sites on a genomic level, we integrated data from the Duke iMADS database of TF binding sites, CPD damage maps across the human genome, ATAC-seq accessibility data from the Cistrome database, and mutation data from Australian melanoma patients (from the ICGC consortium). We developed a computational workflow to measure the spatial distribution of damage/mutations at TF binding sites. Our analysis showed a strong association between the positions of CPD damages and mutations within the binding sites of ETS1, but not E2F or c-Myc, with the peaks in damage/mutations primarily residing at the central core of the ETS1 binding sites. The creation of such an analytic pipeline will enable the further quantitative genome-wide study of the relationships between the degrees of damages/mutations within TF binding sites, chromatin accessibility, and other genomic contexts. With these results, we can better understand the mechanisms involved in accumulating damages and mutations and how they progress to cancer, aging, and senescence.

Citation Format: Bo Chi, Raluca Gordan. Integrating DNA damage and mutation enrichment to understand mutagenesis at transcription factor binding sites [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 3154.
Abstract 3169: Decoupling the correlation between cytotoxic and exhausted T lymphocyte transcriptomic signatures in melanoma enhances immunotherapy response prediction
Authors: Wang Binbin , Wang Kun , Jiang Peng , Ruppin Eytan
Published: April 2023

Background: Cytotoxic T lymphocytes (CTL) play a crucial role in anti-cancer immunity. Progression of CTL to exhausted T lymphocytes (ETL) that overexpress inhibitory receptors can substantially decrease effector cytokines production and diminish cytolytic activity in tumor microenvironment (TME). However, while the activity levels of CTL and ETL are considered important determinants of immune checkpoint inhibitors (ICIs) response, it has been repeatedly observed that their predictive power is quite limited. Studying this conundrum on a large scale across the TCGA cohort, we find that ETL and CTL activity (estimated based on conventional gene signatures in the bulk tumor expression) is strongly positively correlated in most cancer types. We thus hypothesized that their limited predictive power may arise due to this high expression concordance, such that their opposing associations with response effectively cancel each other.

Methods and Results: Aiming to find conditions where the association between these CD8+ T cell states and ICIs response is enhanced, we analyzed TCGA melanoma bulk gene expression via an interaction linear regression model to identify a set of genes whose expression diminishes the CTL and ETL correlation as much as possible. We further tested this decoupling signature (DS) in several independent melanoma bulk expression ICIs cohorts, showing that in high-DS patients, the correlation between ETL and CTL activities is indeed markedly lower than in the low-DS patient groups. Second, this successful decoupling now increases the power of CTL activity in predicting ICIs response in high DS patients, making it superior to other state-of-art ICIs prediction transcriptomic signatures.

Significance: These results demonstrate the utility of a new decoupling score for boosting the power of CTL activity in predicting ICIs response in melanoma.

Citation Format: Binbin Wang, Kun Wang, Peng Jiang, Eytan Ruppin. Decoupling the correlation between cytotoxic and exhausted T lymphocyte transcriptomic signatures in melanoma enhances immunotherapy response prediction [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 3169.
Abstract 3213: Association of Tumor Treating Fields (TTFields) with survival in newly diagnosed glioblastoma: A systematic review and meta-analysis
Authors: Conlon Patrick , Lavy-Shahaf Gitit , Urman Noa , Ballo Matthew T.
Published: April 2023

Background: Tumor Treating Fields (TTFields) became an FDA-approved treatment option for patients with newly diagnosed GBM in 2015 on the basis of the randomized controlled EF-14 trial. Subsequent approvals worldwide and increased adoption of TTFields has led to the question of whether or not a consistent survival benefit has been observed in the real world setting, and whether device usage has played a role.

Methods: We conducted a literature search using PubMed, Embase, and Google Scholar to identify clinical studies evaluating overall survival in adult patients with GBM treated with TTFields. Comparative studies and single-cohort studies reporting on survival outcomes were included in the analysis. Inter-study heterogeneity was assessed using the Cochran Q test and quantified using the Higgins I2 statistic.

Results: Of 7 studies evaluating the clinical efficacy of TTFields in newly diagnosed patients, 5 studies (reporting on a total of 1,228 patients) compared the addition of TTFields to standard of care (SoC) vs SoC alone, and were included in a pooled analysis for overall survival. Meta-analysis of comparative studies indicated a significant improvement in overall survival for patients treated with TTFields vs those not receiving treatment. Inter-study heterogeneity was examined, and a sensitivity analysis indicated the pooled effect was robust and not dependent on any individual study. Furthermore, among studies reporting data on TTFields device usage, an average device usage rate of 75% or higher was found to consistently associate with prolonged survival when compared to an average usage rate below 75% (P = 0.001).

Conclusions: Pooled analysis of comparative TTFields studies suggests survival may be improved with the addition of TTFields to standard chemoradiotherapy treatment for patients with newly diagnosed GBM, and that a 75% usage rate may be meaningful in the real-world setting.

Citation Format: Patrick Conlon, Gitit Lavy-Shahaf, Noa Urman, Matthew T. Ballo. Association of Tumor Treating Fields (TTFields) with survival in newly diagnosed glioblastoma: A systematic review and meta-analysis [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 3213.
Abstract 3280: Vrd induction therapy drives memory like NK cell development in newly diagnosed multiple myeloma patients
Authors: Peng Tao , Kuan Emma , Cicalo Anthony , Gong Qiuyu , Torgerson Troy , Li Xiaojun
Published: April 2023

VRd induction therapy is a common approach to treat newly diagnosed multiple myeloma (MM) patients. Although in vitro studies of bortezomib (Velcade, V), lenalidomide (Revlimid, R), and dexamethasone (d) reveal mechanisms of these individual drugs, VRd therapy induced immune cell phenotype changes in vivo are unknown. We have utilized multimodal single cell methods to elucidate novel mechanisms of induction therapy response in human immune cells over time as a longitudinal study: pre-treatment (pretrt), after 2-cycles of VRd therapy (post-induction; pi) and after the end of 4-6 additional cycles of VRd therapy (end induction; ei). Protein measurements in plasma demonstrate significantly increased levels of IL-15 and IL-18 only at pi compared to pretrt but IL-2 and IFN-γ are progressively reduced from pretrt to ei. Both flow cytometry and single cell RNA -seq reveal that the proportion of NK cells (NK) significantly increased in PBMC only at pi compared to pretrt. NK subsets with strong dexamethasone effect emerge at pi; In contrast, memory like NK subsets (CD56dimCD16+NKG2A+CD94+) become the major NK in PBMC and bone marrow (BM) at ei. In addition, single cell ATAC-seq suggests that RUNX-family transcription factors may mediate lenalidomide to up-regulate NK cytotoxic gene expression during VRd therapy. In summary, comprehensive immune profiling of newly diagnosed MM patients suggests that transient increase of IL-15 and IL-18 may mediate the generation of memory like NK cells, which may play important roles in controlling MM growth and/or reducing risk of infection during VRd induction therapy.

Citation Format: Tao Peng, Emma Kuan, Anthony Cicalo, Qiuyu Gong, Troy Torgerson, Xiaojun Li. Vrd induction therapy drives memory like NK cell development in newly diagnosed multiple myeloma patients [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 3280.
Abstract 3298: Two detection methods of clinical KRAS G12C mutation detection for companion diagnostics
Authors: Brown Robert William , Liu Wei , Sun Qing , Ren Hui , Li Jonathan , Pastor Larry , Kim Daniel , Sha Michael , Zhang Aiguo
Published: April 2023

KRAS G12C is a common mutation in approximately 13% of lung adenocarcinoma, 3% of colorectal cancer and 2% of other solid tumors. The FDA approved sotorasib, a KRAS G12C inhibitor, for targeted treatment of patients with KRAS G12C-mutated locally or metastatic non-small cell lung cancer (NSCLC). It is essential to detect the KRAS G12C mutation with a companion diagnostic before the drug is administered. We have developed two KRAS G12C mutation detection assays for qPCR or Sanger sequencing. Both QClamp® assays use our proprietary xeno nucleic acid (XNA) technology which specifically enhances the amplification of the target mutant DNA sequence while blocking amplification of the wildtype sequence, thus significantly improving the sensitivity of the assays for qPCR (~1%) and Sanger sequencing (~15 to 20%) to 0.1% variant allele frequency. These simple and sensitive assays can be used for clinical research or validated in CLIA labs for KRAS G12C mutation detection for the purpose of companion diagnostic use. Although NGS methods are gaining traction in clinical settings, our methods are suitable for standard molecular diagnostics laboratory settings and do not add any additional steps to the qPCR or Sanger sequencing protocols. Our methods are especially valuable for labs that appreciate simple, economical, yet sensitive companion diagnostics assays for KRAS G12C mutation. QClamp® assays can be easily adapted to detect other single gene mutations with high sensitivity for companion diagnostics use or other mutation detection applications.

Citation Format: Robert William Brown, Wei Liu, Qing Sun, Hui Ren, Jonathan Li, Larry Pastor, Daniel Kim, Michael Sha, Aiguo Zhang. Two detection methods of clinical KRAS G12C mutation detection for companion diagnostics [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 3298.
Abstract 3299: Usefulness of protocadherin GA12 and cysteine ​​dioxygenase type 1 methylation biomarkers in the diagnosis of lung cancer
Authors: Son Ji Woong , Lee Minhyeok , Jeong In Beom , Kwon Sun Jung , Kang Daeun , Na Moon Jun
Published: April 2023

Rationale: The use of bronchoscopy is key to the diagnosis of lung cancer. However, the sensitivity of bronchoscopy is low. In addition, bronchial washing cytology, a routine auxiliary test, does not significantly improve the performance of bronchoscopy due to its low sensitivity. To improve the diagnostic performance of bronchoscopy, protocadherin GA12 (PCDHGA12) and Cysteine ​​Dioxygenase Type 1 (CDO1) methylation biomarkers in bronchial washing fluid have been introduced as novel adjunctive diagnostic tests.

Methods: A total of 161 patients with suspected lung cancer who underwent bronchoscopy were analyzed. Cytological examination of bronchial washing fluid and testing for PCDHGA12 and CDO1 methylation biomarkers were performed.

Results: The final diagnosis was lung cancer in 114 patients, and benign disease in 49 patients. PCDHGA12 methylation biomarker showed sensitivity of 80.4% and specificity of 87.8%. CDO1 methylation biomarker showed sensitivity of 66.1% and specificity of 98.0%, whereas cytological evaluation showed sensitivity of 43.8% and specificity of 100%. The combined analysis of PCDHGA12 and CDO1 methylation showed sensitivity of 84.8% and specificity of 85.7%. The combined analysis of the methylation biomarkers and cytology tests resulted in a slightly increased sensitivity of 86.6% and specificity of 85.7%. The specificity and sensitivity of the methylation biomarkers were also high in peripheral lung cancer.

Conclusion: The results of this study demonstrated that PCDHGA12 and CDO1 methylation as lung cancer biomarkers of bronchial washing may be used as adjunctive tests for bronchoscopy. Keywords: biomarker; bronchoscopy; lung cancer; protocadherin GA12: CDO1, methylation

Citation Format: Ji Woong Son, Minhyeok Lee, In Beom Jeong, Sun Jung Kwon, Daeun Kang, Moon Jun Na. Usefulness of protocadherin GA12 and cysteine ​​dioxygenase type 1 methylation biomarkers in the diagnosis of 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 3299.
Abstract 3300: Development of a diagnostic antibody for anti-BTN1A1 therapy, a novel immune checkpoint inhibitor mutually exclusive of PD-L1/PD-1
Authors: Yoo Stephen S.
Published: April 2023

Butyrophilin 1A1 (BTN1A1) is currently undergoing investigation in Phase 1 clinical trials as a novel immune checkpoint protein for the treatment of solid tumors. BTN1A1 exhibits a mutually exclusive expression pattern to that of PD-1/PD-L1, making anti-BTN1A1 antibody therapy an attractive option for cancer patients with PD-L1 refractory or relapsed disease. While current cancer immunotherapies have shown some efficacy against late-stage disease, there remains a major unmet medical need as a large number of patients fail to respond to current treatments. BTN1A1 has been studied at length in preclinical models to characterize its immunomodulatory functions, revealing its ability to inhibit the proliferation of activated T cells and the ability of these cells to kill tumor cells in vitro. In vivo, BTN1A1-overexpressing tumor models exhibit accelerated growth, whereas the growth of BTN1A1-deficient tumors is impaired in immunocompetent settings, supporting a role for this protein in the regulation of immune evasion. Here in, we present the development of a diagnostic antibody for use in the anti-BTN1A1 therapy clinical trials to enable the immunohistochemical (IHC) detection of human BTN1A1. A high-quality BTN1A1 antibody may help predict which patients are more likely to respond to BTN1A1 antibody-based cancer therapy, similar to the PD-L1 companion diagnostic test for anti-PD-L1/PD-1 therapy. The characterization of the developed BTN1A1 antibody was conducted via ELISA, Western blotting, confocal microscopy, and flow cytometry to demonstrate its overall performance. Validation was performed using formalin-fixed paraffin-embedded human tumor tissue samples to determine the analytical sensitivity and specificity of the IHC assay. This antibody was also found to be of value for imaging mass cytometry and flow cytometry experiments elucidating applicable clinical features of both human tumor tissues and immune cells. Specifically, the developed antibody can detect the robust expression of BTN1A1 on both cancer cells and immune cells, while also revealing the mutually exclusive expression of BTN1A1 and PD-L1. Finally, we demonstrate the capability of a BTN1A1 antibody that can be used to identify BTN1A1-positive patients with a high degree of analytical sensitivity and specificity.

Citation Format: Stephen S. Yoo. Development of a diagnostic antibody for anti-BTN1A1 therapy, a novel immune checkpoint inhibitor mutually exclusive of PD-L1/PD-1 [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 3300.
Abstract 3316: Intra-tumor heterogeneity assessment of liver lesions for colorectal cancer patients on CT-scans
Authors: Ghermi Ridouane , Ammari Samy , Garcia Gabriel , Harguem-zayani Sana , Lassau Nathalie , Schutte Kathryn
Published: April 2023

The objective is to find a consensus definition of intra-tumor heterogeneity of liver lesions for colorectal cancer patients on computed tomography (CT) scans and to evaluate the prognostic value of the newly defined criteria.

89 patients with colorectal cancer treated by antiangiogenics having standard pretreatment CT-scans were included. Lesions were manually segmented in a previous study. We reached a consensus definition by bringing three radiologists together to determine which criteria make a tumor heterogeneous. We asked two of the three radiologists to score lesions from one to three based on the consensus definition. A subset of 50 lesions was shared between annotators in order to check their agreement. 898 lesions were scored, which corresponds to a median of 6 lesions per patient. We associate a unique heterogeneity score to each patient defined as the difference between the number of very heterogeneous tumors and the number of heterogeneous tumors. The prognostic value of this heterogeneity score was assessed using a Cox regression.

Intra-tumor heterogeneity of a single lesion is defined on a scale of one to three: homogeneous (pixels are of similar intensities), heterogeneous (pixels are of different intensities but organized as a ring), very heterogeneous (pixels are of different intensities and randomly organized). We note that the shape of the tumor is not taken into account in the heterogeneity assessment. The two radiologists agreed on the lesion heterogeneity score in 53% of cases. The patient heterogeneity score is significantly prognostic in both the univariate analysis (Kaplan-Meier estimator with median cut-off; p-value = 0.016) and the multivariate analysis (Cox model with control variables including gender, age, weight, treatment and tumor burden; p-value = 0.024).

Three radiologists have reached a consensus to define intra-tumor heterogeneity of liver lesions for colorectal patients. According to this definition, intra-tumor heterogeneity was shown to be a prognostic factor.

Citation Format: Ridouane Ghermi, Samy Ammari, Gabriel Garcia, Sana Harguem-zayani, Nathalie Lassau, Kathryn Schutte. Intra-tumor heterogeneity assessment of liver lesions for colorectal cancer patients on CT-scans [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 3316.
Abstract 3401: Discovery of GH658, a novel tead allosteric inhibitor as a cancer therapy
Authors: Li Jie Jack , Zhang Guiping , Li Jiapeng
Published: April 2023

The Hippo pathway is an evolutionarily conserved signaling cascade. Its dysregulation may lead to cancer progression, metastasis, cancer therapy resistance, and immune evasion. Diverse genetic aberrations leading to YAP/TAZ hyperactivation have been elucidated over the last 20 years. Almost all the pro-tumor mutations lead to constitutive TEAD transcription factor activation, suggesting that targeting TEAD transcription factors could offer an important therapeutic opportunity. Employing structure-based drug design, we have discovered GH658, a novel allosteric TEAD inhibitor that selectively targets the auto-palmitoylation pocket of TEAD proteins. In vitro, GH658 displayed a low nM potency in TEAD-based reporter assays, which translated to specific low nM inhibition of NF2 deficient mesothelioma proliferation. In vivo, GH658 demonstrated favorable pharmacokinetic properties in different species and showed acceptable safety profile. Furthermore, daily oral dosing of GH658 led to complete growth inhibition of NF2 mutant xenograft tumors without adverse effect on body weight. More importantly, GH658 displayed significant in vivo effect on drug tolerant persistent cancer cells and drug resistance against inhibitors targeting EGFR‒RAS signaling pathways in combination studies. In summary, with potent in vitro and in vivo efficacy of targeting YAP/TAZ‒TEAD transcriptional complex, GH658 not only showed consistent monotherapy activity in selected cancer models, but also demonstrated significant synergistic effects in diverse drug combination studies.

Citation Format: Jie Jack Li, Guiping Zhang, Jiapeng Li. Discovery of GH658, a novel tead allosteric inhibitor as a cancer 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 3401.
Abstract 3441: A multivalent peptoid conjugate that inhibits therapy-resistant prostate cancer cell proliferation by modulating androgen receptor transcriptional activity
Authors: Habault Justine , Schneider Jeffrey A. , Ha Susan , Ruoff Rachel , Puccini Joseph , Bar-Sagi Dafna , Wong Kwok-Kin , Zoubeidi Amina , Claessens Frank , Wise David R. , Logan Susan K. , Kirshenbaum Kent , Garabedian Michael J.
Published: April 2023

Prostate cancers adapt to androgen receptor (AR) pathway inhibitors and progress to castration resistance despite the continued expression and function of the AR. We developed a new approach to antagonize the proliferative activity of AR and inhibit castration-resistant prostate cancer (CRPC) growth using multivalent peptoid conjugates (MPCs). Our strategy is to display multiple copies of the AR antagonist, ethisterone, on a peptoid scaffold. Such a multivalent display increases the ligand’s effective local concentration, thus targeting AR with high affinity and affecting AR interactions with coregulators. Here, we report a new potent MPC called MPC309, which displays three ethisterone groups and binds to AR with nanomolar affinity. MPC309 reduces the proliferation of enzalutamide-resistant prostate cancer cells, including those harboring AR splice variants, AR ligand binding mutations, and non-canonical AR gene expression programs. We provide evidence that MPC309 enters cells via macropinocytosis, which facilitates the fluid-phase uptake of extracellular macromolecules. Macropinocytotic uptake of MPC309 enhances cancer cell-specific delivery, thus limiting systemic toxicities. MPC309 displays favorable pharmacological properties and produced significantly greater tumor suppression in xenograft studies than enzalutamide, the standard of care anti-androgen in clinical use. Mechanistically, MPC309 inhibits prostate cancer growth by eliciting a unique gene expression program through alterations in AR chromatin occupancy compared to dihydrotestosterone (DHT) or enzalutamide. Thus, MPC309 represents a novel AR antagonist that activates an AR anti-proliferative program to repress the growth of CRPC and supports further investigation of the MPC compound class for treating CRPC.

Citation Format: Justine Habault, Jeffrey A. Schneider, Susan Ha, Rachel Ruoff, Joseph Puccini, Dafna Bar-Sagi, Kwok-Kin Wong, Amina Zoubeidi, Frank Claessens, David R. Wise, Susan K. Logan, Kent Kirshenbaum, Michael J. Garabedian. A multivalent peptoid conjugate that inhibits therapy-resistant prostate cancer cell proliferation by modulating androgen receptor transcriptional activity [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 3441.
Abstract 3460: Targeting the MIF-CD74 axis to overcome resistance to tyrosine kinase inhibitors in non-small cell lung cancer
Authors: Izumi Motohiro , Lee Meghan , Shibahara Daisuke , Kobayashi Ikei S. , Plotnick David , Kobayashi Susumu S.
Published: April 2023

Development of tyrosine kinase inhibitors (TKIs) against oncogenic drivers has significantly improved survival of patients with oncogene-mutated non-small cell lung cancer (NSCLC). However, acquired resistance to TKIs emerges over time in essentially all patients who initially respond. Recent evidence suggests that drug-tolerant persister (DTP) cells, which survive and adapt to targeted therapies during an early phase of treatment, play an important role in the emergence of drug resistance. We previously reported that CD74 expression is upregulated in EGFR-mutated lung cancer after treatment with EGFR-TKIs and that CD74 can be one of the DTP cell markers. However, the mechanism underlying CD74 expression and the role of CD74 in DTP cells remain unclear. Here, we sought to identify the mechanism using cell culture systems and transgenic mouse models. We confirmed that CD74 upregulation at the mRNA level after TKIs treatments in various oncogene-mutated cell lines, including those with EGFR mutations, ROS1 fusions, and ALK fusions. We found that CIITA, upstream of CD74, and TNF-α expression were induced by TKIs treatments in tumor cells, leading to an increase in CD74 expression. In addition, we found that TKIs treatments enhanced the autocrine secretion of macrophage migration inhibitory factor (MIF), a ligand of CD74, from tumor cells, suggesting that autocrine stimulation of the CD74 signaling results in block of apoptosis and emergence of DTP cells. To examine whether CD74 plays an important role in the emergence of resistance to TKIs in vivo, we crossed lung-specific EGFR-L858R-T790M transgenic mice with Cd74 knockout mice. Complete deletion of CD74 overcame or delayed resistance to TKIs. Taken together, the MIF-CD74 axis can be a novel target to overcome resistance in driver-mutated NSCLC.

Citation Format: Motohiro Izumi, Meghan Lee, Daisuke Shibahara, Ikei S. Kobayashi, David Plotnick, Susumu S. Kobayashi. Targeting the MIF-CD74 axis to overcome resistance to tyrosine kinase inhibitors in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3460.
Abstract 347: Revealing the intracellular cytomatrix, immobilized biocatalysis, and concept of the operating cell
Authors: Shaiken Tattym E.
Published: April 2023

The central problem of cell biology is the arrangement of biochemical reactions in the cytoplasm responsible for sustaining cellular life where, paradoxically, a myriad of metabolic reactions that could exclude each other coincide. Despite this well understood fact, the structural organization of the cytoplasm remains unclear, so far. Recent models indicate that cytoplasm contains viscous fluid and elastic solid phases. We separated the viscous fluid and elastic solid compartments, identifying them as the cytosol and cytomatrix, respectively. The distinctive composition of the cytomatrix included structural proteins, ribosomes, and metabolome enzymes. High-throughput analysis revealed unique biosynthetic pathways within the cytomatrix. The enrichment of biosynthetic pathways in the cytomatrix indicated the presence of immobilized biocatalysis. Enzymatic immobilization and segregation can surmount spatial impediments, and the local pathway segregation may form cytoplasmic organelles. Protein translation was reprogrammed within the cytomatrix under the restriction of protein synthesis by drug treatment. Thus, cytosol and cytomatrix are an elaborately interconnected network that promotes operational flexibility in healthy cells and the survival of malignant cells. Immobilized biocatalysis integrates the intracellular and extracellular matrices and receptors with the nuclear processes, thus removing the spatial hindrances for biochemical processes, which elucidates human cell operation.

Citation Format: Tattym E. Shaiken. Revealing the intracellular cytomatrix, immobilized biocatalysis, and concept of the operating cell [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 347.
Abstract 3491: Designed sensors reveal normal and oncogenic Ras signaling in endomembranes and condensates
Authors: Zhang Jason Z. , Nguyen William H. , Rose John C. , Ong Shao-En , Maly Dustin J. , Baker David
Published: April 2023

While Ras is known to dynamically shuttle around the cell, the activity, mechanism of activation, and function of non-plasma membrane-localized Ras is unclear due to lack of suitable tools. To address these questions, we used the Latching Orthogonal Cage-Key pRotein (LOCKR) switch platform to generate first-in-class intracellular sensors for endogenous Ras activity (Ras-LOCKR-S) and signaling-dependent proximity labelers (Ras-LOCKR-PL). We find that: 1) Receptor activation leads to endogenous Ras signaling at endomembranes, which is enhanced by golgi-localized guanine exchange factors. 2) Recruitment of SAM68 and MARCKS to oncogenic condensates fuels local Ras signaling and cell growth. 3) Major Vault Protein drives RasG12C inhibitor resistance by enhancing signaling at the golgi and altering mitochondrial metabolism. Together, these results highlight the importance of non-plasma membrane Ras signaling (endomembranes and condensates), and our new sensors should accelerate the discovery of new therapeutic targets.

Citation Format: Jason Z. Zhang, William H. Nguyen, John C. Rose, Shao-En Ong, Dustin J. Maly, David Baker. Designed sensors reveal normal and oncogenic Ras signaling in endomembranes and condensates [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 3491.
Abstract 3492: The small GTPase ARF6 augments BRAFV600E-MAPK signaling in melanomagenesis
Authors: Wang Junhua , Rogers Aaron , Rich Coulson , Sorensen Lise , Holmen Sheri , Wolff Roger , Grossmann Allie
Published: April 2023

ARF6 is a member of the Ras superfamily of small GTPases and has critical functions in endo-membrane trafficking pathways. We previously reported that ARF6 is aberrantly activated in metastatic melanoma and that activation of ARF6 stimulates WNT5A/beta-catenin and PI3K/AKT signaling to promote invasion and accelerate metastasis. Herein, we discovered an ARF6 activation signature in primary melanomas that is associated with inferior overall survival, supporting a novel role for ARF6 in early-stage progression. Conversely, melanocyte-specific deletion of Arf6 in Dct;TVA::BrafV600E;Cdkn2aNull murine melanoma significantly reduced tumorigenesis and progression, and improved survival. Importantly, bulk tumor transcriptome analysis showed reduced expression of ERK-induced genes in these BRAFV600E tumors. Likewise, immunofluorescent staining of tumor sections showed a significant reduction of cytoplasmic and nuclear p-ERK levels. Together these data demonstrate that loss of ARF6 reduces BRAFV600E-MAPK signaling output in vivo. This is a provocative finding because it implies that endomembrane trafficking has a major role in promoting BRAFV600E-driven tumorigenesis. More specifically, ARF6 may be critical for augmenting BRAFV600E signaling. Consistent with this hypothesis, expression of activated ARF6(ARF6Q67L) in our murine BRAFV600E melanoma cells was associated with upregulation of BRAFV600E, p-MEK, and p-ERK levels, detected by Reverse Phase Protein Array analysis. In addition, ARF6Q67L tumor cells showed an increase in anti-apoptotic proteins with a reciprocal decrease in pro-apoptotic proteins. Knockdown of Arf6 in BRAFV600E human melanoma cells significantly reduced proliferation and sensitized cells to vemurafenib-induced apoptosis. Overall, our data reveal that ARF6 augments MAPK signaling in BRAFV600E-driven tumorigenesis. Further work is needed to understand if ARF6 mediates endomembrane-dependent signaling of the BRAFV600E-MAPK pathway.

Citation Format: Junhua Wang, Aaron Rogers, Coulson Rich, Lise Sorensen, Sheri Holmen, Roger Wolff, Allie Grossmann. The small GTPase ARF6 augments BRAFV600E-MAPK signaling in melanomagenesis [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 3492.
Abstract 3513: Developing a 3D biomimetic liver metastatic niche model for pancreatic cancer
Authors: Pahlavanneshan Mahsa , Xiao Weikun , Eun Chae Young , Hwang Chang-Il , Hill Reginald
Published: April 2023

Pancreatic ductal adenocarcinoma (PDAC) is the 3rd leading cause of cancer death in the Unites States with a 5-year survival rate of only 11%. Early diagnosis is very difficult and thus 53% of patients are diagnosed after metastasis has already occurred, with the liver being the most frequently affected site. Both primary tumors and metastases have highly fibrotic stroma. Thus, stromal targeting agents have the potential to benefit patients, even those with metastatic disease. However, in addition to different resident fibroblasts, a study based on a rapid autopsy program of metastatic PDAC patients showed that tumor ECM density is actually lower at sites of liver metastasis compared to the primary tumor. These results highlight some of the differences between the microenvironment of the liver metastatic niche (LMN) compared to that of the primary tumor. Thus, to best help patients, models which recapitulate the LMN are needed. We developed a 3D biomimetic model to co-culture PDAC organoids and host matching primary CAFs to recapitulate the desmoplastic environment of primary PDAC. Our preliminary results show that primary site derived CAFs induce organoid growth, chemoresistance, and ECM remodeling when co-cultured with PDAC organoids. Utilizing paired organoids which metastasized to the liver (LM organoids), we tested if primary site derived CAFs would have the same effect on LMs. Interestingly, we observed that while LM organoids were intrinsically more drug resistant, co-culture with primary site derived CAFs had no effect on chemoresistance or ECM remodeling. Next, we utilized a metastasis associated fibroblast (MAF) liver cell line to investigate the role of MAFs in tumor growth, chemoresistance, and ECM remodeling. Our results show that primary and metastatic organoids use different mechanism to induce ECM stiffness triggered by fibroblast activation and remodeling in their resident fibroblasts.

Citation Format: Mahsa Pahlavanneshan, Weikun Xiao, Chae Young Eun, Chang-Il Hwang, Reginald Hill. Developing a 3D biomimetic liver metastatic niche model for pancreatic 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 3513.