bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1593: Intercellular network in immune suppressive microenvironment of pancreatic cancer
Authors: Aoki Kazunori , Fukuda Hironori , Mizoguchi Yukihiro , Arai Kosuke
Published: March 2024

Despite constant efforts to improve treatment, the prognosis remains poor, with an overall survival rate of 6% (ranges from 2% to 9%) due to lack of efficient chemotherapy, radiotherapy, and targeted therapy. Immune checkpoint inhibitors (ICIs) has been reported to be effective in various solid cancers, such as melanoma, lung cancer, and renal cell carcinoma. However, pancreatic ductal adenocarcinoma (PDAC) is resistant against ICIs. The responsiveness of immune therapy is mainly determined by immune tumor microenvironment (TME) composed of tumor-infiltrating lymphocytes (TIL) and stromal cells. PDAC possesses unique TME, which is abundant of fibrosis, termed of desmoplasia, and the role of fibrosis in constructing immune suppressive TME remains unclear. First, in this study, to examine the immunological characteristics of TME, we constructed an integrated data base of TIL profiling, RNA-seq and whole exome seq using 31 fresh resected tissues of PDAC. An unsupervised clustering of based on numbers and percentages of 12 TIL types analyzed by flow cytometry showed that PDAC was divided into 2 types of cluster (myeloid cell-, T cell-dominant type), and the prognosis of myeloid type was significantly poorer than T cell-type. Myeloid type contained the high frequency of myeloid cell lineage including monocytes, macrophages and myeloid-derived suppressor cells (MDSC). Only MDSCs were significantly associated with poor prognosis among various immune cell types. Furthermore, gene enrichment analysis identified activated and suppressed pathways in each immune type, and in myeloid cell-dominant type, the immune-related pathways were significantly down-regulated. Then, to understand the characteristics of MDSCs infiltrated into pancreatic cancer tissues, single cell RNA-seq with surgical specimens was performed, and the analysis showed that a specific subtype of MDSCs infiltrated into cancer tissues. In addition, activation status of cancer-associated fibroblasts (CAFs) was related with MDSC recruitment and activation, indicating that the interaction between MDSCs and CAFs is crucial to create MDSC-rich TME. Inhibition of interaction between MDSCs and CAFs is expected to overcome immunosuppressive TME can be a therapy in PDAC.

Citation Format: Kazunori Aoki, Hironori Fukuda, Yukihiro Mizoguchi, Kosuke Arai. Intercellular network in immune suppressive microenvironment of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1593.
Abstract 1600: Interception of pro-tumorigenic glycan signaling in pancreatic cancer
Authors: Hsu Jasper , Song Hyemin , Peck Kristina , Bottomley Chelsea , Stamp McKenna , Okhovat Shira , Engle Dannielle D.
Published: March 2024

Pancreatic cancer is a deadly malignancy. We have setup a pipeline to interrogate cancer cell intrinsic and extrinsic mechanisms contributing to treatment response. Using a combination of mouse and human organoids models together with in vivo investigation, we have systematically dissected key signaling hubs driving pro-tumorigenic cancer cell intrinsic features as well as remodeling of the tumor microenvironment (TME). We found that the glycan epitope, CA19-9, drives pancreatic inflammation, cancer, and metastasis. CA19-9 drives pro-tumorigenic intrinsic features in part through CA19-9 modification of the secreted glycoprotein Fibulin 3. We have also discovered direct and indirect mechanisms through which CA19-9 causes remodeling of the TME. Specifically, CA19-9 elevation causes expansion of all cancer associated fibroblast (CAF) subtypes, including an increase in antigen presenting CAFs and resulting increase in regulatory T cells. Further, there are dramatic increases in tumor associated macrophages (TAMs) derived from both inflammatory monocytes as well as tissue resident macrophages. To delineate the mechanisms by which these TME changes are mediated, we use novel co-culture model incorporating macrophages, organoids, and fibroblasts (MOrF). Blocking CA19-9 signaling in vitro and in vivo reverses these TME alterations. These changes to the TME are mediated through indirect effectors as well as previously unknown CA19-9 modified proteins. Overall, we elucidate previously unexplored mechanisms driving pancreatic tumorigenesis while also uncovered vulnerabilities for therapeutic exploitation.

Citation Format: Jasper Hsu, Hyemin Song, Kristina Peck, Chelsea Bottomley, McKenna Stamp, Shira Okhovat, Dannielle D. Engle. Interception of pro-tumorigenic glycan signaling in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1600.
Abstract 1604: Cancer-associated fibroblasts modulate T cell killing activity & phenotype in PDAC
Authors: Zayou Fouzia , Pearce Hayden , Nicol Samantha , Margielewska-Davies Sandra , Powell-Brett Sarah , Brown Rachel , Zuo Jianmin , Roberts Keith , McGettrick Helen M. , Moss Paul
Published: March 2024

Background: Pancreatic ductal adenocarcinoma (PDAC) is highly aggressive malignancy characterised by its abundant extracellular matrix and diverse stromal cell components such as cancer associated fibroblasts (CAFs). CAFs play a pivotal role in shaping the tumour microenvironment and influencing the behaviour of immune cells, particularly T cells, through various mechanisms. The impact of CAFs on T cells is multifaceted as they can exert both pro- and anti-tumoral effects, influencing the delicate balance between immunosurveillance and immune evasion. In this study, we examined the phenotype and function of T cells when co-cultured with CAFs and Adjacent-normal fibroblasts (ANF) isolated from primary PDAC tissue following surgical resection.

Methods: Tumour and juxta-tumoral tissue from patients underwent digestion into a single cell suspension. Subsequently, the isolated fibroblasts were cultured and stained with a 22-plex antibody panel designed for flow cytometry. Additional functional assays involved co-culture of CAFs and ANFs with unstimulated or anti-CD3/CD28 stimulated allogeneic T cells. Cell proliferation rates were quantified through the CFSE assay, while the T cells were simultaneously stained to distinguish CD4+ and CD8+ T cells subpopulations. Additionally, the expression of key co-stimulatory molecules: CD69, NKG2D and DNAM-1 and the co-inhibitory molecule: PD-1 were assessed, providing a thorough examination of the immune dynamics within the tumour microenvironment.

Results: Flow Cytometry analysis revealed a distinct expression profile in CAFs compared to ANFs, with CAFs expressing higher levels of FAP and CD29. The CFSE assay further revealed that CAFs induced T cell proliferation of resting T cells with no significant increase in stimulated T cells. Notably, this co-culture led to a highly differentiated phenotype in both unstimulated and stimulated CD4+ and CD8+ T cells, characterised by elevated expression of activating markers (CD69 and NKG2D) and the inhibitory molecule PD-1. These findings suggest a dynamic interplay between fibroblasts and T cells in the tumour microenvironment, influencing both the proliferation and phenotypic aspects of T cell behaviour.

Citation Format: Fouzia Zayou, Hayden Pearce, Samantha Nicol, Sandra Margielewska-Davies, Sarah Powell-Brett, Rachel Brown, Jianmin Zuo, Keith Roberts, Helen M. McGettrick, Paul Moss. Cancer-associated fibroblasts modulate T cell killing activity & phenotype in PDAC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1604.
Abstract 1612: Timing ecDNA acquisition across cancer
Authors: Lynch Andrew R. , Van Loo Peter
Published: March 2024

Extrachromosomal DNA (ecDNA) are acentric, circular DNA fragments which form after DNA double strand breaks. They segregate randomly during mitosis, promoting high-level amplifications of positively selected oncogenes such as EGFR and MYC. The prevalence of ecDNA across several cancer types, such as glioblastoma and sarcoma, has made promising therapeutic targets. However, while DNA damage is a hallmark of nearly all cancers, the formation of ecDNA is not, and the determinants for ecDNA acquisition are unclear. Furthermore, whether the timing of tumors’ acquisition of ecDNA (i.e., early or late clonal or sub-clonal) affects patient survival and drug resistance is unclear. Here, we leverage orthoganol information from ecDNA amplicons, such as mutation allele frequency and sequence synteny, to develop computational methods for elucidating the evolutionary history of ecDNA. Applying these to several cancer subtypes in TCGA, we find that most ecDNAs arise early in tumor evolution. We expect these results will help identify determinants of ecDNA acquisition and its temporal influence on patient survival.

Citation Format: Andrew R. Lynch, Peter Van Loo. Timing ecDNA acquisition across cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1612.
Abstract 1613: Evolution of mutations in the antigen presentation machinery across cancers
Authors: Chen Wenjie , Baker Toby , Zhang Zhihui , Van Loo Peter , Gu Shengqing Stan
Published: March 2024

Immune escape is a critical hallmark of cancer progression. Under immunosurveillance, a subset of cancer-associated antigens or neoantigens are presented to the cancer cell surface via the antigen presentation machinery (APM) for recognition by antigen-specific T cells. During clonal evolution, cancer cells can evade such immunosurveillance by suppressing or ablating the APM. The defects in APM can further cause resistance to cancer immunotherapies and is associated with worse prognosis. Understanding how the defects in APM emerge and further shape the evolution of cancers is critical for devising strategies to overcome such immune escape mechanisms. However, it is not known when mutations in the APM occur along the history of cancer clonal evolution. We utilized a molecular archeology of cancer approach to reconstruct the evolution history of mutations in APM across 38 cancer types using whole-genome sequencing data from PCAWG. Genetic alterations in the APM occur in nearly 30% of patients, with varying frequencies and associations with immune infiltration across cancer types. Employing a League model to compare the timing of genetic events, we found that APM mutations can be early clonal, late clonal, or subclonal along cancer development. Further examination of individual tumors identified driver mutations that can happen before or after APM mutations along cancer development. Our study reveals the evolution of APM mutations in different cancers in a genetic context-aware manner. This information can contribute to optimizing personalized immunotherapy.

Citation Format: Wenjie Chen, Toby Baker, Zhihui Zhang, Peter Van Loo, Shengqing Stan Gu. Evolution of mutations in the antigen presentation machinery across cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1613.
Abstract 1631: A homogeneous assay system for discernment of proliferative, antiproliferative, and cytotoxic effects in culture
Authors: Lazar Dan F. , Kupcho Kevin R. , Niles Andrew L. , Cali James J.
Published: March 2024

We describe a novel homogeneous cell-based assay developed for the quantitative detection of Ki-67 protein, a recognized marker of cell proliferation, utilizing Lumit™ immunoassay technology. This luminescent assay has been effectively multiplexed with a fluorescent measure of cell death, employing a cell-impermeant fluorogenic DNA dye. This multiplex allows for the clear distinction between antiproliferative activity and overt cytotoxicity in cell culture models. Through varying lytic conditions, we demonstrated effective cell lysis and detection of nuclear Ki-67 protein. Specifically, Lumit™ Lysis Buffer II provided efficient recovery of Ki-67 protein without compromising assay performance, including the requisite binding of labeled antibodies to the target protein. This assay exhibited excellent signal-to-background (up to 40-fold) and signal linearity (R^2 = 0.99) in wildtype HeLa cells (~1000 to 80,000/well), while the signal in Ki-67 knockout HeLa cells was comparable to the no-cell background, indicating excellent specificity for Ki-67 protein. Due to the unavailability of recombinant full-length Ki-67 protein, two different recombinant Ki-67 protein fragments were used to corroborate assay sensitivity of <1 pM Ki-67 protein. The assay's suitability for human Ki-67 protein detection, not mouse, was observed through further cell-based testing. The proliferation/cytotoxicity assay multiplex was validated in both suspension and adherent cell lines, as well as primary cell culture, detecting antiproliferative and proliferative responses. CDK4/6 inhibitors are a class of antiproliferative agents of significant interest for their potential in treating cancer. In Jurkat cells, treatment with the CDK4/6 inhibitor palbociclib led to a dose-dependent reduction in Ki-67 protein expression with an EC50 of 88 nM, and total Ki-67 elimination at 2 μM compound concentration. Notably, there was no cytotoxicity detected at the tested doses and treatment duration (up to 48 h), but a significant reduction in viable cell number at maximal palbociclib concentrations, aligning with an antiproliferative mechanism. This work establishes a rapid, simple, luminescent assay for determining Ki-67 levels that can be paired with a fluorogenic readout of cell death, facilitating efficient and quantitative discernment of altered proliferation in mammalian cell culture using standard microplate readers.

Citation Format: Dan F. Lazar, Kevin R. Kupcho, Andrew L. Niles, James J. Cali. A homogeneous assay system for discernment of proliferative, antiproliferative, and cytotoxic effects in culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1631.
Abstract 1642: NuMA exclusive post-translational modification in epithelial cancer cells is a novel target for cell eradication
Authors: Cohen-Armon Malka
Published: March 2024

The post-translational modification of proteins implicated in mitosis in healthy and in cancer epithelial cells was measured by 2-D gels analysis. The post-translational modifications of NuMA in a variety of human epithelial cancer cells was prevented by treatment with a small phenanthridine, PJ34, while not impaired in similarly treated healthy epithelial cells. NuMA in the cancer cells was phosphorylated by pim1 kinase, and was polyADP-ribosylated by Tankyrase1. These post-translational modifications are inhibited by PJ34. Tankyrase1 is not expressed in healthy epithelial cells, and pim1 kinase was hardly expressed in these cells. Preventing the post-translational modification of NuMA in the epithelial cancer cells also suppressed the protein binding capacity of NuMA. NuMA clustering in the mitotic spindle poles, which stabilizes the mitotic spindle, and is required for the alignment of chromosomes in the spindle mid-zone was impaired. In epithelial cancer cells treated with PJ34, the mitotic spindle poles were distorted, extra-centrosomes were not clustered in multi-centrosomal cancer cells, and chromosomes were dispersed. Chromosomes dispersion violated mitosis termination. Mitosis arrested in the anaphase led to mitotic catastrophe cell death. PJ34 treated epithelial cancer cells were eradicated during 72-96 hours, regardless of their mutations, while treated healthy epithelial cells were not affected, and continued to proliferate.

Citation Format: Malka Cohen-Armon. NuMA exclusive post-translational modification in epithelial cancer cells is a novel target for cell eradication [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1642.
Abstract 1645: Telomere-related genes associated with clinical-pathological in pancreatic ductal adenocarcinoma
Authors: Gómez Erika Curiel , Lagunas Vilma Maldonado , Zajgla Jorge Meléndez
Published: March 2024

Pancreatic ductal adenocarcinoma (PDAC) ﻿stands as one of the leading causes of cancer deaths worldwide. Studies have suggested that telomeres shortening is linked to genetic instability, higher risk of developing pancreatic cancer and pancreatic tumorigenesis. However, previous research has predominantly focused on either telomere length or specific telomere-related genes in pancreatic cancer. Therefore, it is essential to elucidate the association between telomere-related genes and pancreatic cancer to identify novel ﻿therapeutic strategies and establish prognostic biomarkers. Telomere-relevant genes were downloaded from the TelNet database. Expression data from primary tumour of The Cancer Genome Atlas-PAAD (TCGA-PAAD) cohort, the Genome Sequence Archive CRA001160 dataset and the GTEx database were obtained for screen for differentially expressed genes (DEGs). Gene set enrichment analisis was performed using the Enrichr software. The gene essentiality queried from the gene dependency database of the Achilles-DepMap project was also assessed. Finally, data of The Cancer Genome Atlas-PanCancer CaPa cohort and healthy pancreas from the GTEx database, obtained from the UCSC Xena project, were used to correlate gene expression with stemness signatures and clinical-pathological variables. We identified 300 telomere-associated DEGs, Of these, 73 genes were common to studies and they were to be enriched in biological processes related to the cell cycle and telomere homeostasis. The differentially expresed of ASS1, S100P, IGF2BP2, MET, ABCC3 and RAC1 genes were significantly associated with overall survival and recurrence-free survival, conferring them prognostic value. Furthermore, the expression profiles of these genes are significantly associated with the histological grade of the disease, as well as with tumour T-N classification, the presence of residual tumour, the degree of response to treatment and stemness signatures. Among these, three genes of particular interest (KLF3, RAC1 and ABI1) were identified and shown to be highly essential in pancreatic cancer cell lines. In summary, we have identified a telomere gene-related signature for pancreatic cancer, highlighting the prognostic value of KLF3, RAC1, and ABI1 genes. This signature is significantly associated with clinical-pathological variables of the disease, potentially offering new insights for individualized therapy.

Citation Format: Erika Curiel Gómez, Vilma Maldonado Lagunas, Jorge Meléndez Zajgla. Telomere-related genes associated with clinical-pathological in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1645.
Abstract 1659: Phosphorylation of cell cycle and apoptosis regulatory protein (CARP)-1 by stress-activated protein kinase P38γ is a novel mechanism of apoptosis signaling by genotoxic chemotherapy
Authors: Venkatesh Jaganathan , Muthu Magesh , Cheriyan Vino T. , Sekhar Sreeja C. , Acharige Nuwan C. , Levi Edi , Assad Hadeel , Pflum Mary Kay , Rishi Arun K.
Published: March 2024

CARP-1, a perinuclear phospho-protein, regulates cell survival and apoptosis signaling induced by genotoxic drugs. However, kinase(s) phosphorylating CARP-1 and down-stream signal transduction events remain unclear. Here we find that CARP-1 Serine (S)626 and Threonine (T)627 substitution to Alanines (AA) inhibits genotoxic drug-induced apoptosis. CARP-1 T627 is followed by a Proline (P), and this TP motif is conserved in vertebrates. To further elucidate chemotherapy-activated, CARP-1-dependent signaling mechanisms, we UV cross-linked protein extracts from Adriamycin-treated HeLa cervical cancer cells with a CARP-1 (614-638) peptide, and conducted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of the peptide-bound protein complexes. This experiment revealed SAPK p38γ interaction with CARP-1 (614-638) peptide. Our studies further revealed that SAPK p38γ phosphorylates CARP-1 T627 in cancer cells treated with genotoxic drugs. CARP-1 T627 phosphorylation was also noted in breast tumors from patients treated with radiation or endocrine therapies. Loss of p38γ abrogates CARP-1 T627 phosphorylation, and results in enhanced survival of breast cancer cells by genotoxic drugs. We conclude that genotoxic drugs activate p38γ-dependent CARP-1 T627 phosphorylation and cell growth inhibition.

Citation Format: Jaganathan Venkatesh, Magesh Muthu, Vino T. Cheriyan, Sreeja C. Sekhar, Nuwan C. Acharige, Edi Levi, Hadeel Assad, Mary Kay Pflum, Arun K. Rishi. Phosphorylation of cell cycle and apoptosis regulatory protein (CARP)-1 by stress-activated protein kinase P38γ is a novel mechanism of apoptosis signaling by genotoxic chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1659.
Abstract 1660: Tumor pharmacokinetics and pharmacodynamics assessment of TEAD inhibitor VT03989 in patient-derived xenograft models of glioblastoma
Authors: Margaryan Tigran , Chang Yu-Wei , Molloy Jennifer , Hopkins Barbara , Elliott Mackenna , Melendez Ernesto Luna , White Connor , Sanai Nader , Tien An-Chi , Tovmasyan Artak , Mehta Shwetal
Published: March 2024

Background: The Hippo-YAP/TAZ pathway regulates cell proliferation, cell growth and migration. The major effectors of the Hippo pathway are the TEAD transcription factors, which are deregulated in several cancers, including glioblastoma (GBM). In this study, we evaluated the pharmacokinetics and pharmacodynamics of VT03989, a highly potent TEAD inhibitor, in orthotopic patient-derived xenograft (PDX) models of GBM.

Methods: Mice with intracranial PDX GBM tumors were randomized into two cohorts to receive 10 mg/kg (oral) VT03989 or placebo for 4 days. VT03989 levels in plasma, tumor, and contralateral brain tissue were measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) at 2, 6, and 12 hours after the last treatment of VT03989. Unbound fractions were determined by equilibrium dialysis. Quantitative RT-PCR was performed to assess levels of TEAD target genes, CTGF and CYR61. Immunohistochemistry of Ki67 and cleaved caspase 3 (CC3) was performed.

Results: The median unbound concentrations of VT3989 were 27 nM in plasma and 26 nmol/kg in the tumor. The median brain-to-plasma and tumor-to-plasma partition coefficients of unbound VT03989 were 0.65 and 0.99, respectively - indicating favorable brain and tumor penetration ability of VT03989. A decrease in CTGF and CYR61 indicated TEAD inhibition and was accompanied by an increase in CC3 (+) cells.

Conclusion: VT03989 is well tolerated, achieves pharmacologically relevant unbound concentrations in GBM PDX models, and is associated with significant target modulation. Ongoing studies are evaluating in vivo survival benefits from combining VT03989 inhibition with radiation therapy

Citation Format: Tigran Margaryan, Yu-Wei Chang, Jennifer Molloy, Barbara Hopkins, Mackenna Elliott, Ernesto Luna Melendez, Connor White, Nader Sanai, An-Chi Tien, Artak Tovmasyan, Shwetal Mehta. Tumor pharmacokinetics and pharmacodynamics assessment of TEAD inhibitor VT03989 in patient-derived xenograft models of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1660.
Abstract 1663: Single-cell signaling analysis reveals that major vault protein facilitates RasG12C inhibitor resistance
Authors: Zhang Jason Z. , Ong Shao-En , Baker David , Maly Dustin J.
Published: March 2024

Covalent inhibitors of RasG12C are the first clinically-approved drugs for targeting mutant Ras-driven cancers. However, the rapid development of clinical resistance to current Ras G12C inhibitors is common. It has been demonstrated that a subpopulation of cancer cells facilitate drug resistance but the mechanistic basis of this heterogenous response is not well understood. Here, we utilized recently developed Ras sensors to profile the environment of active Ras and to measure the activity of endogenous Ras to pair structure (Ras signalosome) to function (Ras activity), respectively, at a single-cell level. With this approach, we found that a subpopulation of KRasG12C cells treated with RasG12C-GDP inhibitors underwent oncogenic signaling and metabolic changes driven by WT Ras at the golgi and mutant Ras at mitochondria, respectively. Our Ras sensors identified Major Vault Protein (MVP) as a mediator of Ras activation at both subcellular locations by scaffolding Ras signaling pathway components and metabolite channels. We found that recently developed RasG12C-GTP inhibitors also led to MVP-mediated WT Ras signaling at the golgi, demonstrating that this a general mechanism of RasG12C inhibitor resistance. Overall, single-cell analyses enabled the discovery of a RasG12C inhibitor-resistant subpopulation driven by MVP, providing insight into the complex and heterogenous rewiring that confers drug resistance in cancer.

Citation Format: Jason Z. Zhang, Shao-En Ong, David Baker, Dustin J. Maly. Single-cell signaling analysis reveals that major vault protein facilitates RasG12C inhibitor resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1663.
Abstract 1671: Exploring the mechanism of PP2A regulated cell death via macropinocytosis in pancreatic cancer
Authors: Doraivel Indiraa , Baral Garima , Pfeffer Claire M. , Allen-Petersen Brittany L.
Published: March 2024

Pancreatic Ductal Adenocarcinoma (PDAC) is the 4th leading cause of cancer-related death in the United States. KRAS is an oncogene mutated in 90% of PDAC patients. Oncogenic KRAS leads to aberrant cell proliferation and survival. KRAS is considered hard to target because resistance mechanisms to KRAS inhibitors are common highlighting the need to understand alternative strategies. PDAC tumor microenvironment evolves with cancer cell progression leading to vascular remodeling and blood vessel collapse. This makes the tumors nutrient deplete. To circumvent this, cells employ KRAS-dependent macropinocytosis- a nutrient scavenging pathway. Macropinocytosis is the process by which cells take up extracellular material by membrane ruffling to form vesicles which then fuse with lysosomes to release nutrients. Targeting KRAS-driven macropinocytosis can be crucial to limiting nutrients to the cell to prevent tumor growth. Protein Phosphatase 2A (PP2A), a heterotrimeric Serine/Threonine phosphatase, is a downstream regulator of KRAS. Its activity is repressed by mutated KRAS. We have seen that the activation of PP2A with small molecular activator, DT061, prevents macropinosomes from fusing with lysosomes which leads to cell death. But how DT061 regulated PP2A activity affects the macropinocytosis pathway is still unknown. The purpose of this study is to understand the mechanism of cell death brough upon by the activation of PP2A with DT061. Membrane ruffling is critical for the initiation of macropinocytosis. Rac is a GTPase usually involved with membrane ruffling. To show that Rac is needed for DT061 mediated macropinocytosis, we used EHT 1864, a Rac family inhibitor. Cells were treated with EHT 1864 before being treated with DT061 and stained with crystal violet. The inhibition of Rac prevented DT061 driven cell death, indicating that Rac is crucial to DT061 driven cell death. High molecular weight TMR-Dextran can only be taken up by the cells through macropinocytosis. Cells were treated with TMR-Dextran and EHT 1864. Cells with Rac inhibition showed lower uptake of TMR-Dextran than cells that were treated with DT061. Together these findings indicate to us that Rac plays a crucial role in the initiation of macropinocytosis and the regulation of cell death caused by the activation of PP2A. Understanding the pathway of DT061 mediated cell death can be vital to developing this concept as a therapeutic method of treatment for PDAC patients who currently have the lowest 5-year survival rate (12%) of all cancers.

Citation Format: Indiraa Doraivel, Garima Baral, Claire M. Pfeffer, Brittany L. Allen-Petersen. Exploring the mechanism of PP2A regulated cell death via macropinocytosis in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1671.
Abstract 1677: The emerging role of the Plasminogen-Apple-Nematode (PAN) domain in cancer therapy
Authors: De Kuntal , Pal Debjani , Muchero Wellington
Published: March 2024

The Plasminogen-Apple-Nematode (PAN) domain, with a core of four to six cysteine residues, is found in more than 28,000 proteins across 959 genera. However, its role in protein function still needs to be elucidated. Numerous proteins initially characterized the PAN domain, including a well-known master regulator for cancer cell proliferation, HGF (Hepatocyte growth factor). Abnormal regulation of HGF-mediated signaling results in multiple deadly cancers. The binding of HGF to its cell surface receptor, c-MET, triggers signaling pathways, which leads to cancer. Here, we show that mutating four core cysteine residues in the HGF PAN domain reduces c-MET interaction, subsequent c-MET autophosphorylation, and phosphorylation of its downstream targets, perinuclear localization, cellular internalization of HGF and its receptor, c-MET, and c-MET ubiquitination. Furthermore, transcriptional activation of HGF/c-MET signaling-related genes involved in cancer progression, invasion, metastasis, and cell survival was impaired. Thus, targeting the PAN domain of HGF may represent a mechanism for selectively regulating the binding and activation of the c-MET pathway. Additionally, we found this domain in the tumor suppressor gene, MST1 (macrophage stimulating 1), and mutating the domain altered the protein function through protein stability and ubiquitination. In another study, we show that cysteine residues in the vestigial plasminogen-apple-nematode (PAN) domain of NRP1 are necessary for SARS-CoV-2 spike protein internalization. Mutating novel cysteine residues in the PAN altered NRP1 stability and downstream activation of the extracellular signal-regulated kinase (ERK) signaling pathway, impairing its interaction with the spike protein. These results show the diverse critical role of the PAN domain beyond cancer therapy.

Citation Format: Kuntal De, Debjani Pal, Wellington Muchero. The emerging role of the Plasminogen-Apple-Nematode (PAN) domain in cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1677.
Abstract 1699: Defining the impact of aberrant transcription on the chromatin structure
Authors: Thomas Naiju , Reznicek Timothy E. , Aiden Erez Lieberman , Rowley M. Jordan , Wagner Eric , Nir Guy
Published: March 2024

Genome organization in humans is governed by two major mechanisms: loop extrusion by cohesin and CTCF and the spatial compartmentalization of the chromosomes. These folding mechanisms have been shown to regulate several genomic functions, including gene expression. However, how transcription might regulate the chromatin structure remains a subject of many ongoing investigations. In this study, we use the Integrator protein complex as a tool to understand the impact of aberrant transcription on the 3D structure of the genome. During transcription initiation, RNA Polymerase-II (Pol-II) often pauses proximally to the promoter before further elongating transcripts. The Integrator protein complex regulates this checkpoint by its endonuclease and phosphatase activity. This protein complex is essential in premature transcription termination in hundreds of protein-coding genes. It also facilitates the synthesis of non-coding (ncRNA), such as enhancer RNAs (eRNAs) and long non-coding RNAs (lncRNAs), which have been shown to support loop extrusion domains, and their absence may disrupt these domains. Using HiC sequencing, we found that depleting Integrator proteins can interfere with forming and maintaining loop domains. This study aims to be the first to decipher the relationship between chromosome structure and Integrator protein activity and address our very little knowledge of how misregulation of transcription termination may influence the folding of chromosomes.

Citation Format: Naiju Thomas, Timothy E. Reznicek, Erez Lieberman Aiden, M. Jordan Rowley, Eric Wagner, Guy Nir. Defining the impact of aberrant transcription on the chromatin structure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1699.
Abstract 175: Tumor-associated monocytes and neutrophils: Targets for immunotherapy
Authors: Jung Keehoon
Published: March 2024

Cancer is the leading cause of deaths worldwide. However, current anti-VEGF therapies for solid cancers provide limited survival benefit as tumors rapidly develop resistance to these agents.We have uncovered an immunosuppressive role for non-classical Ly6Clow monocytes that mediates resistance to anti-VEGFR2 treatment. We found that the chemokine CX3CL1 was upregulated in both human and murine tumors following the VEGF signaling blockade, resulting in recruitment of CX3CR1+ Ly6Clow monocytes into the tumor. We also found that treatment with VEGF-A reduced expression of CX3CL1 in endothelial cells. Intravital microscopy revealed that CX3CR1 is critical for Ly6Clow monocyte transmigration across the endothelium in tumors. Moreover, Ly6Clow monocytes recruit Ly6G+ neutrophils via CXCL5 and produce IL-10, which inhibits adaptive immunity. Preventing Ly6Clowmonocyte or Ly6G+ neutrophil infiltration into tumors enhanced inhibition of tumor growth with anti-VEGFR2 therapy. Furthermore, we developed a gene therapy using a nanoparticle formulated with a siRNA against CX3CL1, which reduced Ly6Clow monocyte recruitment and improved outcome of anti-VEGFR2 therapy.Taken together, we identified immunosuppressive non-classical Ly6Clow monocytes as key players in tumor resistance to anti-angiogenic therapy in cancers. We also revealed molecular mechanisms underlying anti-angiogenic treatment resistance, suggesting potential immunomodulatory strategies to enhance the long-term clinical outcome of anti-VEGF therapies, proven by state-of-the-art in vivo imaging modalities.

Citation Format: Keehoon Jung. Tumor-associated monocytes and neutrophils: Targets for immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 175.
Abstract 1802: Melanoma addiction to GCDH
Authors: Nadig Namratha , Verma Sachin , Ronai Ze'ev A.
Published: March 2024

Rewiring of metabolic pathways often underlies the malignant state, including melanoma. In our previous studies we have demonstrated melanoma addiction to Gutaryl Co-dehydrogenase (GCDH), an enzyme in the lysine catabolism pathway. Our studies revealed that blocking GCDH activity in melanoma, but not colon, lung or breast cancer cells, led to cell death which abolished growth both in culture and in vivo. Important cellular component which was found to mediate melanoma addiction to GCDH was the transcription factor NRF2, which exhibited tumor suppressor function upon GCDH inhibition. Notably, coinciding with NRF2 tumor suppressor role seen upon GCDH inhibition was its glutarylation, a post translational modification which acquired NRF2 stability and ability to induce transcription of ATF4, ATF3 and CHOP which led to extensive cell death program. Knockdown of NRF2, ATF3 or DTHDK-1 effectively blocked the cell death phenotype seen upon GCDH knockdown. Analyses of patient data confirmed that low GCDH expression coincided with prolonged survival of melanoma but not colon, breast or prostate cancer patients. These findings led us to perform an unbiased screen to identify cellular components, which underlie melanoma addiction to GCDH. The results of this analysis will be discussed.

Citation Format: Namratha Nadig, Sachin Verma, Ze'ev A. Ronai. Melanoma addiction to GCDH [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1802.
Abstract 1806: Induction of KRAS G13D dimers as a therapeutic strategy for colorectal cancer
Authors: Angira Deekshi , Li anbo , Zhou Zhiwei , Lee Seungheon , Ready Joseph , Westover Kenneth
Published: March 2024

Colorectal cancer (CRC) is the third leading cause of cancer deaths. Most CRCs arise from polyps in the colon/rectum that acquire gene mutations, including frequent activation of the oncogene Kirsten rat sarcoma virus (KRAS) observed in 40% of cases, particularly at codons 12, 13, and 61. KRAS oligomerization is critical for MAPK pathway signaling, which drives CRC pathogenesis. This study evaluated compounds that bind mutant KRAS G13D to induce non-physiological dimerization. Screening of a 4.1 billion compound DNA-encoded library led to the identification of pyridinylpiperazine/quinoline compounds. These compounds demonstrated the ability to induce RAS dimer formation, subsequently reducing MAPK signaling, exhibiting similarities to BI-2852, which is known to induce formation of non-physiologic RAS dimers. Structure-activity studies yielded indole, piperidine, and morpholine variants showing improved activity in thermal shift assays (DSF) and in cell-based assays evaluating RAS signaling (p-ERK inhibition).

Citation Format: Deekshi Angira, anbo Li, Zhiwei Zhou, Seungheon Lee, Joseph Ready, Kenneth Westover. Induction of KRAS G13D dimers as a therapeutic strategy for colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1806.
Abstract 1842: Global proteomics reveals integrin signaling as a driver of infiltration in glioblastoma
Authors: van Winden Lennart J. , Sajid Rifat Shahriar , Borhani Okty Abbasi , Kamski-Hennekam Evelyn Rose , Gopal Nakita , Leon Alberto , Diamandis Phedias
Published: March 2024

Background: Glioblastoma (GBM), a notably aggressive brain cancer, presents significant treatment challenges due to its propensity for deep, diffuse infiltration into healthy brain tissue. This trait enables GBM to resist and survive following surgery and conventional adjuvant therapies. Emerging evidence suggests that signaling between GBM stem cells (GSCs) may be instrumental in this invasive behavior. Our study aimed to unravel the intricacies of these intercellular communication networks within glioblastoma by employing comprehensive proteomic profiling across a diverse array of patient-derived GSC lines.

Methods: We conducted our study using a sex-stratified cohort comprising 17 patient-derived GSC lines. Proteomic profiling encompassed analyses of both GSC lysates and conditioned media under various oxygen conditions (normoxia at 20% O2, hypoxia at 1% and 0.2% O2). Additionally, we incorporated the glioma cerebral organoid (GLICO) system, facilitating co-cultures of GSCs with human cerebral organoids. For in-depth proteomic analysis, we utilized advanced TMT-based liquid chromatography-tandem mass spectrometry (LC-MS/MS; Q-Exactive, Thermo Fisher).

Results: Our findings corroborate that GSCs adhere to the tripartite model of proliferation, invasion, and hypoxic response. A salient discovery was the consistent enrichment of integrin signaling pathways in GSCs under hypoxic conditions. Specifically, we observed a significant association between upregulation in integrin signaling (BIOCARTA_INTEGRIN_PATHWAY) and elevated KRAS activity (SWEET_KRAS_TARGETS_UP: r=0.43, p<0.01), as well as hypoxic responses (WINTER_HYPOXIA_UP: r=0.76, p<0.0001) in GSCs. Notably, in the GLICO model, both integrin signaling and KRAS pathways were markedly upregulated in the presence of GSCs compared to cerebral organoids alone (p<0.05).

Conclusions: This research highlights the pivotal role of integrin signaling in mediating the invasive attributes of glioblastoma. The data underscore the importance of further exploring key modulators within the integrin signaling pathway, presenting a promising avenue for the discovery of novel therapeutic targets in glioblastoma treatment.

Citation Format: Lennart J. van Winden, Rifat Shahriar Sajid, Okty Abbasi Borhani, Evelyn Rose Kamski-Hennekam, Nakita Gopal, Alberto Leon, Phedias Diamandis. Global proteomics reveals integrin signaling as a driver of infiltration in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1842.
Abstract 1849: High throughput interactome profiling of small bioactive peptides
Authors: Warren Charles D. , Yardeny Noah , Geri Jacob B.
Published: March 2024

Interactions between individual biomolecules give rise to emergent signaling and regulatory networks that govern complex and varied biological processes. Disruption of these finely tuned interactions can result in imbalanced cellular states that drive diseases; for example, many metastatic cancers become desensitized to checkpoints encoded by molecular contacts. Still, numerous interaction networks remain unmapped within the cell, namely those between cryptic endogenous peptides and their protein targets. This is primarily due to the lack of investigation around translation events that occur from “noncoding” genetic material and limited knowledge of how translated polypeptides can mature into functional proteolytic fragments. Moreover, technologies to map protein-peptide interactions in an efficient and reliable manner are limited, leaving a gap in knowledge of how many bioactive peptides regulate cellular homeostasis. We aim to map the interactions of short (30 > amino acid), bioactive peptides that are (1) translated from noncanonical open reading frames or (2) generated through proteolytic cleavage of nascent proteins. To achieve these goals, we are leveraging the recently developed MicroMap technology and label-free mass spectrometry-based proteomics. A scalable and reliable platform to discover peptide binding targets will uncover the elusive functional roles of understudied endogenous peptides in cell signaling and their part in driving cancer cell survival.

Citation Format: Charles D. Warren, Noah Yardeny, Jacob B. Geri. High throughput interactome profiling of small bioactive peptides [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1849.
Abstract 186: Tumor exosome-macrophage interactions: The role of tumor-associated glycans and scavenger receptor class A
Authors: Jousheghany Fariba , Blitz Emily S. , Jenkins Samir V. , Post Steven R. , Raj Vinay , Kore Rajshekar A. , Jamshidi-Parsian Azemat , Griffin Robert J. , Kelly Thomas J. , Monzavi-Karbassi Behjatolah
Published: March 2024

Introduction: Increased macrophage infiltration and polarization towards a breast tumor-supportive phenotype have been linked to therapy resistance, immune suppression, and poor patient prognosis. Despite their importance, the specific interactions that drive tumor-associated macrophage (TAM) polarization remain poorly understood. The class A scavenger receptor (SR-A) is a pattern-recognition receptor expressed by macrophages, and the accumulation of SR-A-expressing TAMs has been correlated with poor patient outcomes. Our preliminary data show that SR-A binds to tumor-associated glycans (TAGs) on the surface of breast cancer cells. However, how macrophages bind tumor cell ligands in the tumor microenvironment (TME) is not clear. Tumor-derived exosomes (TDEs) are secreted extracellular vesicles that resemble the tumor cell membrane, carrying cargo that may mediate an immune response within the TME. In this study, we investigated whether TDE glycans represent tumor cell-surface TAGs and whether TDEs interact with macrophages through SR-A.

Methods: TDEs were isolated from the murine breast cancer cell line (E0771) by ultracentrifugation of the cell culture medium and then sized and counted using nanoparticle tracking analysis and electron microscopy. The glycans present on the tumor cell surface and TDEs were characterized using plant lectins in flow cytometry and sandwich ELISAs. The isolated TDEs were then fluorescently labeled, and their binding to the murine macrophage cell line Raw 264.7 was assayed by flow cytometry and fluorescence microscopy.

Results: SR-A binds strongly to breast cancer cell lines, and a significant portion of the binding depends on the presence of cell surface N-linked glycans. Lectins reactive with sialylated and fucosylated terminal structures showed differential binding to tumor cells and TDEs. We observed that the glycan profiles of E0771 cells and exosomes isolated from E0771 cultures were similar. Our results indicate that TDEs interact with Raw 264.7 cells, and the interaction is mediated by SR-A.

Conclusions: TDEs released in the TME can potentially interact with TAMs through SR-A. The glycan profile of TDEs is representative of host cells and may contribute to SR-A-mediated interaction with TAMs.

Citation Format: Fariba Jousheghany, Emily S. Blitz, Samir V. Jenkins, Steven R. Post, Vinay Raj, Rajshekar A. Kore, Azemat Jamshidi-Parsian, Robert J. Griffin, Thomas J. Kelly, Behjatolah Monzavi-Karbassi. Tumor exosome-macrophage interactions: The role of tumor-associated glycans and scavenger receptor class A [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 186.