bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5545: FADD activation renders hepatocellular carcinoma sensitive to immune checkpoint blockade by turning cold tumors hot
Authors: Zhou Jingying , Lu Jiahuan
Published: March 2024

The heterogenicity of hepatocellular carcinoma (HCC) remains a key obstacle in turning the majority of ‘immune-cold’ tumors ‘hot’ for effective immune checkpoint blockade (ICB) therapy. Through studying the naturally-existed ‘hot’ HCC variant lymphoepithelioma-like (LEL)-HCC by RNA and whole exome sequencing analysis, we identified fas-associated death domain (FADD) as one of the top differentially expressed genes that associated with high tumoral CD8+T cell abundance. Of note, FADD upregulation was also observed in HCC patients with better ICB responsiveness. As FADD was mainly expressed in tumor cells from HCC by scRNAseq analysis, we constructed Fadd knockout stable cells using murine HCC cell lines. Interestingly, CRISPR knockout of tumoral Fadd led to increased tumor weights in immunocompetent but not immunodeficient mice, accompanied with decreased tumoral CD8+T cells. Mechanistically, FADD phosphorylation promoted NF-κB transcriptional activity to increase CCL5 secretion from HCC cells via interacting with its nuclear binding partners Src-associated-substrate-during-mitosis-of-68kDa/KH domain containing, RNA binding, signal transduction associated 1 (Sam68/KHDRBS1) and lysine (K)-specific methyltransferase 2D (KMT2D), thus directing CD8+T cell tumor infiltration. In addition, anti-PD1 triggered FADD phosphorylation via IFN-γ and TNF-ɑ release from CD8+T cells in ICB-sensitive but not resistant tumors. Most importantly, sequential FADD activation via genetic and pharmacologic approaches could re-sensitize the therapeutic efficacy of anti-programmed death 1 (PD1) antibody in ICB-resistant orthotopic and spontaneous HCC models by turning cold tumors hot. Taken together, our findings reveal a key molecular feature of ICB-responsive hot tumors that may guide the development of sequential combinatory immunotherapies for the majority of HCC patients with low responsiveness to ICB monotherapy. This project is supported by the University Grants Committee through General Research Fund (24110323, 14107622, 14104820) and the Health and Medical Research Fund (07180556).

Citation Format: Jingying Zhou, Jiahuan Lu. FADD activation renders hepatocellular carcinoma sensitive to immune checkpoint blockade by turning cold tumors hot [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 5545.
Abstract 5560: Role of SPARC and collagen in immunogenicity of cancer: A theoretical approach
Authors: Are Ramakrishna Prasad , Babu Anju R
Published: March 2024

Secreted protein acidic and rich in cysteine (SPARC), is a multifunction protein that interacts with extracellular matrix (ECM), cell surface receptors, and signaling molecules. These interactions in cancer cells result in less susceptible of cells to the immune system, leading to poor prognosis. SPARC plays a crucial role in transporting collagen, a significant component of the ECM, from the site of synthesis to the basement membrane of the cells. Collagen mediated rigidity enhancement of cell microenvironment promotes cancer cell growth, proliferation, and angiogenesis. Collagen remodeling plays a complex and multifaceted role in cancer immunomodulation. Our current research focuses on exploring the cancer treatment mechanism by immunomodulation based on SPARC and collagen through theoretical and computational approaches. Our previous investigations identified SPARC and collagen as potential biomarkers for targeting cancer cells. Our current theoretical research identifies the role of SPARC and collagen in tuning the tumor microenvironment and altering the immune system cell surface receptors. This theoretical investigation is followed by computational study to formulate the pathway from secretions of both SPARC and collagen to reprogramming of tumor microenvironment. SPARC and collagen’s effects on the cell immune system are analyzed by performing molecular interaction and molecular dynamics studies for SPARC and collagen with the cell surface receptors. From our study, we identify the cellular mechanisms involved either actively or passively in the progression and prognosis of the cancer. Our study will provide insight into a novel pathway for targeting the cancer microenvironment, which will be helpful in the formation of low-dosage cancer drugs, resulting in the decreased adverse effects of drugs and increased patient quality of life.

Citation Format: Ramakrishna Prasad Are, Anju R Babu. Role of SPARC and collagen in immunogenicity of cancer: A theoretical approach [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 5560.
Abstract 5564: The regulation of PD-L1 in glioblastoma cells involves Pyk2 signaling
Authors: Porter Tyrel , Kucheryavykh Lilia
Published: March 2024

Glioblastoma (GBM), the most aggressive brain cancer which is usually fatal within a year after diagnosis, poses a significant therapeutic challenge with conventional treatments providing limited success. Despite advancements in immune checkpoint blockade, PD-L1 inhibitors exhibit suboptimal efficacy in recurrent GBMs with recent clinical trials demonstrating only 8% objective responses in patients. Utilizing the R2 Genomic Analysis Visualization Platform, we identified a positive 8-18% correlation between Pyk2 and PD-L1 gene expression in human GBM specimens. We hypothesize that Pyk2 signaling is involved in the regulation of PD-L1 expression in GBM cells. In this study, primary human glioblastoma cell lines and GL261 mouse glioma cells were assessed employing CRISPR/Cas9 Pyk2 knockout (Pyk2KO) combined with pharmacological Pyk2 inhibition. Flow cytometric analysis revealed a 35% decrease in PD-L1 expression in Pyk2KO GL261 cells compared to wild-type cells. SiRNA knockdown against Pyk2 in primary human GBM cells demonstrated a similar response, with a 50% decrease in PD-L1 expression compared to MOCK control cells. Additionally, the application of the Pyk2/FAK inhibitor defactinib at a concentration of 0.2 μM reduced PD-L1 expression by 32% in both GL261 and primary human GBM cells. These findings highlight Pyk2 as a potential therapeutic target for modulating the immunosuppressive microenvironment in GBM and enhancing the effectiveness of immune checkpoint blockade therapies.

This study was supported by: NIH Grant 1SC1GM122691 and PRSTRT2022.

Citation Format: Tyrel Porter, Lilia Kucheryavykh. The regulation of PD-L1 in glioblastoma cells involves Pyk2 signaling [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 5564.
Abstract 5569: Mechano-chemical control of secretory cues regulating immune tolerance, survival and invasion in oral squamous cell carcinomas
Authors: Bareham Bethany R. , Dibble Matthew , Mouhib Leila , Arteagabeitia Aitor Bermejo , Mukhopadhyay Subhankar , Parsons Maddy
Published: March 2024

Tissue fibrosis and extracellular matrix (ECM) stiffening have been shown to promote tumor cell proliferation, metastasis, and chemoresistance in a range of cancers. Stiffness of the ECM can lead to changes in cancer cell behavior, including enhanced proliferation, activation of mechano-sensitive signaling pathways, and potential genomic damage, with implications for cancer progression and immune responses. Recent research suggests stiff matrix may also induce the release of tumor-derived small extracellular vesicles (sEVs) that promote tumor growth. Tumor-derived sEVs carry bioactive cargo, such as micro-RNAs (miRNAs) and DNA fragments, and function as a unique form of intracellular communication promoting cell survival, growth, and metastasis. Understanding these mechano-sensing mechanisms is essential for identifying potential targets in precision medicine for solid tumor treatment. However, the influence of tumor stiffness on secretory cues, and their subsequent role in immunomodulation and/or chemo-resistance has not been extensively studied.

This project aims to explore how tumor stiffness can alter cell responses to genotoxic stress and how these responses can perpetuate feedback to the immune system through the release of secretory cues such as sEVs. Here, we outline the isolation and characterization of tumor-derived sEVs obtained from culture media of oral squamous cell carcinoma (OSCC) spheroids, cultured at varying stiffness (0.2kPa-1.5kPa). In this project, we use OSCC lines that are either sensitive or resistant to cisplatin (HSC-3 and HN-5, respectively). To explore the immunomodulatory effects of these tumor-derived sEVs, we first established a consistent supply of CD206+ve macrophages differentiated from human induced pluripotent stem cells (iMacs), following the established protocol of Douthwaite et al (2021). iMacs were then treated with sEVs with their resulting immunophenotype, and activation assessed. These preliminary experiments provide a step towards understanding how the biophysical environment influences tumor cell resistance to therapy and how sEVs can modulate the relationship between cancer cells and macrophages.

Citation Format: Bethany R. Bareham, Matthew Dibble, Leila Mouhib, Aitor Bermejo Arteagabeitia, Subhankar Mukhopadhyay, Maddy Parsons. Mechano-chemical control of secretory cues regulating immune tolerance, survival and invasion in oral squamous cell carcinomas [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 5569.
Abstract 5584: Experimental ‘loss-of function’ annotation of STK11 mutations with prognostic and therapeutic implications (TNG260mutationfinder.com)
Authors: Ahronian Leanne G. , Shahagadkar Preksha , Flynn Lauren , Grove Lauren , Liu Shangtao , Meier Samuel R. , Shen Binzheng , Stowe Hannah , Wu Hsin-Jung , Yu Yi , Mignault Andre , Sienczylo Iga , DiBenedetto Heather , Fenoglio Silvia , Teng
Published: March 2024

Loss of function mutations in Serine Threonine Kinase 11 (STK11) occur in 15% of lung adenocarcinoma and have been shown to drive resistance to immune checkpoint blockade clinically, as well as in preclinical models. Though STK11 is commonly inactivated in human cancer with strong implications for treatment outcomes, few STK11 mutations identified from tumor samples have been functionally characterized.

TNG260 is an inhibitor of CoREST that is currently being investigated in combination with pembrolizumab for the treatment of STK11-mutant cancer (NCT05887492). Patients are eligible for enrollment in the TNG260 phase 1/2 trial if their tumor contains a deleterious STK11 mutation. To begin classifying non-annotated variants, over 2,000 distinct mutations in the STK11 gene were identified from STK11 literature or public repositories of tumor sequencing data such as AACR Project GENIE and ClinVar. Where possible, loss-of-function annotations were captured from literature or predictive tools such as PolyPhen-2. However, many STK11 variants, particularly missense mutations, have never been functionally characterized.

We developed a functional screening approach to characterize STK11 alterations using the lung adenocarcinoma cell line A549. A549 cells contain homozygous loss of STK11 via a truncation mutation at Q37, and re-expression of wild-type STK11 in these cells strongly impairs their growth in vitro and in vivo. We created a library of STK11 variant cDNAs, each containing a unique barcode. This library was expressed in A549, and cells were maintained in vitro or in vivo to allow for positive selection of STK11 loss-of-function variants and depletion of variants that behave like wild-type STK11. At the end of the screen, variants were quantified by NGS using each mutant cDNA’s unique barcode and compared to well-annotated controls.

These data were assembled to generate TNG260mutationfinder.com – the first website to curate STK11 variants with functional annotations.

Citation Format: Leanne G. Ahronian, Preksha Shahagadkar, Lauren Flynn, Lauren Grove, Shangtao Liu, Samuel R. Meier, Binzheng Shen, Hannah Stowe, Hsin-Jung Wu, Yi Yu, Andre Mignault, Iga Sienczylo, Heather DiBenedetto, Silvia Fenoglio, Teng. Experimental ‘loss-of function’ annotation of STK11 mutations with prognostic and therapeutic implications (TNG260mutationfinder.com) [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 5584.
Abstract 5598: Combined loss of the chromosome 7 genes CUX1 and EZH2 promotes chemotherapy resistance
Authors: Jotte Matthew R. , Stoddart Angela , Martinez Tanner C. , Xue Yuqing , Imgruet Molly K. , McNerney Megan E.
Published: March 2024

Monosomy 7 (-7) or del(7q) are recurrent in high-risk myeloid neoplasms, including up to 50% of myeloid neoplasms arising after prior exposure to chemotherapy and/or radiation. Although putative chromosome 7 tumor suppressor genes have been identified, the effects of combinatorial 7q gene loss remain unclear. A barrier to understanding the pathogenesis of -7/del(7q) is the challenge of modeling aneuploidy in animal models. To address this knowledge gap, we established an in vivo model of del(7q) clonal hematopoiesis and drug resistance using multiplex CRISPR-Cas9 to simultaneously target four 7q genes (Cux1, Ezh2, Kmt2c, and Kmt2e) in murine hematopoietic stem cells. After chemotherapy exposure, we observe significant myeloid expansion of clones edited for both Cux1 and Ezh2. Compared to the transcriptomes of control or single gene edited cells, Cux1;Ezh2-deficient cells fail to induce DNA damage response pathways after genotoxic stress. Cux1;Ezh2-deficient cells also display reduced deposition of γH2AX after DNA damage, as well as persistent, unrepaired DNA breaks, indicating perturbations to DNA damage recognition and repair. Collectively, our data support the concept of 7q as a contiguous gene syndrome region, in which combined loss of multiple genes drives drug resistance and disease development. This work reveals a genetic interaction between CUX1 and EZH2, and sheds light on how -7/del(7q) contributes to the development and inherent drug resistance characteristic of high-risk myeloid disease. Further, our CRISPR-based approach may serve as a framework for interrogating other recurrent aneuploid events in cancer.

Citation Format: Matthew R. Jotte, Angela Stoddart, Tanner C. Martinez, Yuqing Xue, Molly K. Imgruet, Megan E. McNerney. Combined loss of the chromosome 7 genes CUX1 and EZH2 promotes chemotherapy 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 5598.
Abstract 5599: Role of HOX gene expression in differentiation of colon cancer stem cells
Authors: Osmond Brian T. , Facey Caroline O. , Opdenaker Lynn O. , Zhang Chi , Boman Bruce M.
Published: March 2024

HOX genes encode a highly conserved set of transcription factors critical for stem cell (SC) function and embryonic development. Several HOX genes have also been implicated in a myriad of cancers, including colorectal cancer (CRC). However, how HOX genes play a role in SC regulation and contributes to cancer development is unclear. HOX expression is regulated by WNT, FGF, and retinoic acid (RA) signaling - pathways which also become dysregulated in cancer. Goal: To determine how regulation of HOX genes specifies differentiation of colonic SCs into specialized cell lineages and how dysregulation of HOX genes contributes to CRC development. Hypotheses: i) Each of the different cell types within human colonic epithelium has a distinct HOX gene expression signature; ii) All-trans retinoic acid (ATRA) induced differentiation of CRC cells changes HOX expression that correlates with specific HOX expression signatures in normal colon and CRC cells. Results: Immunostaining shows that specific HOX proteins (e.g., HOXA9 & HOXC9) are selectively expressed in ALDH-positive SCs and expression of these HOX genes is increased in CRC tissues compared to normal epithelium. Bioinformatics analysis predicted that these HOX genes (plus HOXA4, HOXA10, HOXC8) are regulated by RA signaling. Accordingly, we studied how ATRA influences HOX expression in CRC cells (HT29 & SW480). We found ATRA treatment: 1) Decreases proliferation and increases neuroendocrine cell (NEC) differentiation; and 2). Leads to specific changes in HOX gene expression patterns. Using NanoString profiling, we also discovered that LGR5+ SCs, ALDH+ SCs and GLP2R+ NECs have unique HOX signatures. By defining how these HOX gene expression changes in response to ATRA treatment in comparison to the HOX gene signatures of different colonic cell lineages will help us understand the role that HOX genes play in SC differentiation and how dysregulation of HOX gene expression contributes to CRC development.

Citation Format: Brian T. Osmond, Caroline O. Facey, Lynn O. Opdenaker, Chi Zhang, Bruce M. Boman. Role of HOX gene expression in differentiation of colon cancer stem cells [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 5599.
Abstract 5626: Essential role of Slug during the evolution of drug-tolerance in lung adenocarcinoma
Authors: Ostendorp Jenny , Tumbrink Hannah Lea , Hunold Pascal , Hoehne Michaela , Jurmeister Philipp , Dekker Anastasia , Heisel Felix , Brägelmann Johannes , Klauschen Frederick , Haensel-Hertsch Robert , Sos Martin L.
Published: March 2024

A major obstacle in the efficacy of targeted therapies of oncogene-driven tumors are drug-tolerant persister cells (DTPs) that build the basis for the outgrowth of drug-resistant clones and ultimately limit patient survival. During treatment, DTPs enter a reversible senescent state to survive therapy while cell death is induced in non-DTPs. Here, using RNA-Seq and proteomic analyses, we identified drug-induced TGFβ2 secretion in DTPs derived from different oncogene-driven lung cancer cell lines. As expected, TGFβ2 induces epithelial-to-mesenchymal transition (EMT) that over time promotes the ability of cells to survive targeted therapy. Using CRISPR/Cas9-mediated loss-of-function and reconstitution experiments we show that downstream of TGFβ2, expression of the transcription factor SNAI2 (SLUG) is essential for the outgrowth of DTPs during targeted treatment. Unsupervised RNA-Seq data analyses in combination with Cut&Tag profiling revealed that EMT signaling is in part a SLUG regulated process but that is also independent of direct SLUG binding at the transcription start sites (TSS) of EMT signature genes. In contrast, we observed high occupancy of SLUG at TSS of genes involved in the regulation of cell cycle, being repressed in DTPs and at TSS of genes that regulate sphingolipid metabolism and MAPK signaling, being induced in DTPs. In line with our previous findings, a motif-based analysis of the Cut&Tag data (TOBIAS) furthermore uncovered a tight connection between DTP outgrowth and inflammatory signaling induced through IRF7, IRF4 or STAT1 activation. In vivo, EGFR-mutant SLUG deficient cells showed a significantly prolonged tumor onset and a higher response rate to osimertinib treatment. Overall, we uncover a major role of TGFβ2/SLUG-mediated EMT signaling that may be induced indirectly through SLUG-dependent reprogramming of cell cycle directed and metabolic processes in drug tolerant cells. These insights may offer unique therapeutic opportunities to limit the outgrowth of drug resistant tumors in cancer patients.

Citation Format: Jenny Ostendorp, Hannah Lea Tumbrink, Pascal Hunold, Michaela Hoehne, Philipp Jurmeister, Anastasia Dekker, Felix Heisel, Johannes Brägelmann, Frederick Klauschen, Robert Haensel-Hertsch, Martin L. Sos. Essential role of Slug during the evolution of drug-tolerance in lung 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 5626.
Abstract 564: Therapeutic targeting of JAK/STAT signaling and histone acetyltransferase activity in post-myeloproliferative neoplasm secondary AML
Authors: Bertulfo Kalay , Debackere Koen , Miller Hannah , Ma Cindy , Najac Ryan , Ferrando Adolfo , Palomero Teresa
Published: March 2024

Post-myeloproliferative neoplasm secondary Acute Myeloid Leukemia (Post-MPN sAML) is an aggressive and lethal hematologic malignancy arising from myeloproliferative neoplasms (MPN). Hyperactivation of JAK/STAT signaling was identified in 50-90% of MPNs - underscoring its role in driving MPN pathogenesis and transformation to post-MPN sAML. Ruxolitinib, a Type-I JAK2 inhibitor approved for the treatment of myelofibrosis, was tested in a Phase II clinical trial in post-MPN sAML. The trial revealed that ruxolitinib improved the quality of life in patients but was not disease-modifying. Thus, there is an urgent need to find synergistic drug combinations capable of complementing the therapeutic activity of JAK2 inhibitors in post-MPN sAML. Here, we demonstrate that combination treatment of ruxolitinib and CBP30, a bromodomain inhibitor of histone acetyltransferases (HAT) CBP and p300, enhances the therapeutic activity of ruxolitinib in post-MPN sAML. Genome-wide CRISPR loss-of-function screens in JAK2-mutated HEL cells determined that knockout of CBP sensitizes leukemic cells to JAK2 inhibitors. Consistently, functional validation through CRISPR/Cas9 demonstrated that genetic deletion of CBP but not of p300 renders HEL cells sensitive to ruxolitinib treatment. Pharmacologic inhibition of JAK/STAT signaling and HAT activity through ruxolitinib and CBP30 treatment in post-MPN sAML lines HEL and SET2 decreases proliferation and induces apoptosis and cell cycle arrest. Mechanistically, the synergism between ruxolitinib and CBP30 elicits a unique transcriptome profile in HEL with significant downregulation of E2F and MYC targets. In addition, epigenetic profiling revealed that the synergism induces global changes in H3K27ac signals with downregulation and upregulation of H3K27ac signals in predicted STAT5/BCL3 and GATA-bounded regions, respectively. Remarkably, combination treatment of ruxolitinib and CBP30 in HEL luciferase NSG mouse xenografts showed significant leukemia regression and decrease in spleen size compared to mice treated with vehicle or single agents. Overall, these results substantiate the potential of targeting JAK/STAT signaling and histone acetyltransferase activity in the treatment of post-MPN sAML.

Citation Format: Kalay Bertulfo, Koen Debackere, Hannah Miller, Cindy Ma, Ryan Najac, Adolfo Ferrando, Teresa Palomero. Therapeutic targeting of JAK/STAT signaling and histone acetyltransferase activity in post-myeloproliferative neoplasm secondary AML [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 564.
Abstract 5670: CRISPR-Mediated knockout of Ceruloplasmin (CP) and UCHL1 reveals angiogenic role in renal cell carcinoma (RCC) cell survival and proliferation
Authors: Caravan Wagma , Wu Yige , Lal Preet , Sato Kazu , Li Yize , Deen Nataly Naser Al , Chen Siqi , Abedin Ateih , Chen Feng , Ding Li
Published: March 2024

Clear cell renal cell carcinoma (ccRCC) is the most prevalent subtype of renal cell carcinoma, accounting for approximately 70% of all cases. Therapeutic treatment for metastatic ccRCC involve tyrosine kinase inhibitors or mTOR inhibiters. While these drugs extend patient survival, it also introduces a range of adverse side effects. Utilizing snRNA sequencing, snATACseq, and spatial transcriptomics, two potential ccRCC tumor specific markers have been identified: ceruloplasmin (CP) and UCHL1. These markers are notably overexpressed in a majority of ccRCC tumor cells and elevated CP levels have been correlated with reduced patient survival. To investigate the roles of CP and UCHL1 in ccRCC, CRISPR-based knockout experiments were conducted on two RCC cell lines, namely RCC4-VHL- and Caki-1. Through cell line characterization assays, we find that the absence of CP and UCHL1 impairs the cells' capabilities to migrate, proliferate, and adhere. Additionally, Cell-Derived Xenografts (CDX) of the knockout cell lines in nude mice revealed that cells lacking UCHL1 exhibited slower tumor growth compared to their parental lines. Bulk RNA sequencing further suggested a potential interrelationship between CP and UCHL1, as CP knockout cells showed increased UCHL1 expression relative to the parental line. These findings open new avenues for potential therapeutic strategies and biomarkers in the treatment and screening of ccRCC and further elevates our understandings of the roles of CP and UCHL1 in RCC tumor progression.

Citation Format: Wagma Caravan, Yige Wu, Preet Lal, Kazu Sato, Yize Li, Nataly Naser Al Deen, Siqi Chen, Ateih Abedin, Feng Chen, Li Ding. CRISPR-Mediated knockout of Ceruloplasmin (CP) and UCHL1 reveals angiogenic role in renal cell carcinoma (RCC) cell survival and proliferation [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 5670.
Abstract 5729: ADCK1 inhibition alters cell proliferation and cell cycle progression in liver cancer cells
Authors: Jacquet Noel A. , Zhao Yunfeng
Published: March 2024

In 2020, there were over 830,000 reported liver cancer deaths. Incidence rates of liver cancer have tripled, and death rates have doubled since the 1980s; giving this disease one of the highest mortality rates when compared to its incidence rate. Hepatocellular carcinoma (HCC) is derived from hepatocytes and is the main type of liver cancer in adults, accounting for 75-85% of all primary liver cancers. Current treatment options for HCC like radiation therapy and chemotherapy are imperfect and cause significant side effects. Tumorigenesis is dependent on an organism’s cellular metabolism. Metabolic reprogramming is a promising area of focus for cancer therapeutics. Cancer cells thrive in nutrient-deficient environments by hijacking and reprogramming the cells existing cellular metabolism. Metabolic reprogramming is driven by genetic alterations and environmental cues. AarF Domain Containing Kinase 1 (ADCK1) is a protein-coding gene that acts as a mitochondrial protein with putative kinase activity. The role of ADCK1 has not been well studied, particularly in liver cancer. Our group has sought to study how ADCK1 impacts the metabolism and cellular environment of HCC cells to help establish a more effective drug target. Bioinformatics performed by our lab report that ADCK1 was highly expressed in HCC tumor cells. The expression levels of ADCK1 were also correlated with a decrease in patient survival. ADCK1 knockout clones were generated; these knockout cells showed decreased proliferation and altered cell cycle progression. Proteomics analysis demonstrated that loss of ADCK1 was also seen to alter the expression of proteins involved in several cellular processes related to the cell cycle and cellular metabolism. How ADCK1 regulates these processes will be studied in the future.

Citation Format: Noel A. Jacquet, Yunfeng Zhao. ADCK1 inhibition alters cell proliferation and cell cycle progression in liver cancer cells [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 5729.
Abstract 5752: Enhanced cancer therapy strategies utilizing gelatin-lipidoid hybrid nanoparticles for RNA delivery
Authors: Gangula Abilash , Suresh Dhananjay , Babu Agasthya Suresh , Li Zhaohui , Upendran Anandhi , Kannan Raghuraman
Published: March 2024

Nanoparticle delivery platforms have provided impetus for the emergence of novel RNA-based cancer therapeutics due to their ability to circumvent the physiological and cellular barriers in the delivery. The clinical success of these strategies hinges primarily on the safety and efficacy of the delivery vehicle. Here, we have developed a gelatin-lipidoid hybrid nanoparticle (GLNP) that utilizes gelatin's biocompatibility, biodegradability, and lipidoids' high transfection efficiency. With GLNP, we have demonstrated the efficacy of delivering siRNA and silencing AXL, a receptor tyrosine kinase commonly associated with cancer progression, metastasis, and drug resistance. siRNA is attached to the surface of GLNP through covalent conjugation with gelatin, while the lipidoid on the system's interior electrostatically complexes with the siRNA. siRNAs are well protected by the GLNP when exposed to serum for at least 48 hours. The GLNP's bivalency significantly enhanced the downregulation of target genes in vitro and in vivo. Furthermore, the bivalent GLNP can simultaneously target and downregulate two oncogenes. Apart from siRNA, GLNP was also able to encapsulate and deliver mRNA, thereby suggesting the potential to simultaneously deliver RNA therapeutics to effect knockdown and expression of target proteins and maximize therapeutic effect. The length of the carbon chain of the lipidoid can be manipulated to influence the physico-chemical properties of the GLNP and its efficiency in encapsulating and delivering RNA drugs. Importantly, gelatin was found to effectively shield the cationic lipidoid and negate its toxicity. Toxicity studies indicate that high doses of GLNPs (up to 48 mg/kg BW) are well-tolerated with no significant changes in body weight, hematology, or serum chemistry. The co-administration of GLNP-siAXL and TKI in PDX models has shown notable control of tumor growth. It is important to note that immune checkpoint blockade (ICB) therapy has shown significant survival benefits in NSCLC patients. However, the therapeutic effect is limited to only 20% of patients due to several resistance mechanisms, such as increased expression of oncogenic proteins. Therefore, our future study will explore GLNP's ability to sensitize NSCLC tumors to immune therapy.

Citation Format: Abilash Gangula, Dhananjay Suresh, Agasthya Suresh Babu, Zhaohui Li, Anandhi Upendran, Raghuraman Kannan. Enhanced cancer therapy strategies utilizing gelatin-lipidoid hybrid nanoparticles for RNA delivery [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 5752.
Abstract 5759: Design of Au-graphene quantum dot nanocomposite as a potential drug delivery system to solubilize Tetrandrine
Authors: Salvá MiaSara Perez , Medina-Berrios Nataniel , Weiner Brad , Marrero Gerardo Morell
Published: March 2024

Prostate cancer, along with various other types of cancer, has been a prominent focus in medical and clinical discussions for many years due to its high fatality rates and significant health risks. To enhance oncological healthcare, various methods and technologies, such as drug delivery systems (DDS), have been extensively studied. Developing an effective DDS offers distinct advantages compared to other approaches to anticancer drug treatment. Some anticancer drug molecules possess unfavorable properties that directly impact metabolism and solubility, issues that DDS aims to address. Recent progress in nanomaterial synthesis has introduced innovative and efficient drug systems, providing a new perspective on cancer treatments. Gold nanoparticles, particularly in conjunction with semiconductor graphene quantum dots (GQDs), play a role in creating a unified carrier system applicable to multiple therapeutic methods. The choice of graphene quantum dots is based on their widespread applications and diverse benefits, ranging from compatible size and core-shell chemistry richness to substantial progress in achieving signal homogeneity by reducing the distance between molecules and substrate. The selected model for the DDS is the hydrophobic alkaloid Tetrandrine. Despite its use in treating conditions such as hypertension, and arthritis, and exhibiting anti-cancer activities, Tetrandrine is highly insoluble as an anti-cancer drug, with a solubility of 24.1 μmol/L [15 μg/mL] in water and only 5mM in ethanol and DMSO. This research aims to enhance the solubility of hydrophobic Tetrandrine by incorporating it into gold graphene quantum dot nanocomposites, serving as a potential drug delivery system for combating prostate cancer. The synthesis of nanocomposites (Au-GQD, Au-NGQD, Au-SGQD) and Tetrandrine-loaded products (AuGQD-T, AuNGQD-T, AuSGQD-T) will be examined. Additionally, the properties of Tetrandrine loaded onto the nanoparticles will be scrutinized and characterized using NMR spectroscopy, UV-Vis, and fluorescence spectrum analyses.

Citation Format: MiaSara Perez Salvá, Nataniel Medina-Berrios, Brad Weiner, Gerardo Morell Marrero. Design of Au-graphene quantum dot nanocomposite as a potential drug delivery system to solubilize Tetrandrine [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 5759.
Abstract 5761: A nanoplatform based on an in situ tumor vaccine and activation of STING and TLR7/8 pathways for enhanced NSCLC immunotherapy
Authors: Zhou Haiyu , Hou Hengliang , Xu Bin , Huang Yucheng , Qiu Hongrui
Published: March 2024

Background: Immunotherapy has emerged as a promising treatment for tumors following surgical, radiotherapy, and chemotherapy interventions. Therapeutic tumor vaccines are considered highly effective in eliminating tumors and preventing their recurrence and metastasis due to their ability to induce or enhance T cell-mediated immune responses. However, the therapeutic efficacy of these vaccines is impeded by the heterogeneity present both between and within tumors. In contrast, in situ tumor vaccines, prepared from autologous antigens in tumor patients, present another promising strategy for generating comprehensive immune responses.

Methods: In this study, we designed a multifunctional nanopolymer, the transformable nanoassembly PEG-Ce6@PAEMA/R848. This assembly includes the photosensitizer polyethylene glycol-chlorin-e6 (PEG-Ce6), the tumor pH-sensitive polymer poly(2-azepane ethyl methacrylate) (PAEMA), and the immune adjuvant Resiquimod (R-848). The objective is to confirm the activation of bone marrow-derived dendritic cells (DCs) at the cellular level. Subsequently, we investigated the maturation of DCs in lymph nodes and tumor sites in vivo.

Results: In the particle characterization process, successful synthesis of nanoparticles was achieved, along with the verification of their acid-responsive function and toxic effects. Furthermore, in vitro experiments demonstrated the activation of bone marrow-derived dendritic cells (DCs) (Figure 1).To further investigate the in vivo vaccine effects, C57BL/6 mice were euthanized, and lymph nodes were extracted to examine the accumulation of FITC-PAEMA. It can be observed that, due to the reduction in size of the particles in the tumor acidic environment, the immune adjuvant can easily enter the lymph nodes. Activation of the STING pathway in dendritic cells within the lymph nodes was detected through qPCR. The maturation of dendritic cells (DCs) in the lymph nodes was determined using flow cytometry (Figure 2).

Conclusions: In this study, we developed a straightforward yet potent strategy focused on in situ tumor targeting. This approach effectively inhibits tumor growth while inducing immunogenic cell death and eliciting a systemic anti-tumor immune response against cancer cells. Additionally, we intend to validate the therapeutic efficacy of this strategy on metastatic and distal tumors in subsequent investigations.

Citation Format: Haiyu Zhou, Hengliang Hou, Bin Xu, Yucheng Huang, Hongrui Qiu. A nanoplatform based on an in situ tumor vaccine and activation of STING and TLR7/8 pathways for enhanced NSCLC 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 5761.
Abstract 5769: Target identification of T-cell activating immunomodulatory tetrahydrothiazepines: DGKα and DGKζ inhibitors
Authors: Luebbers Thomas , Britschgi Adrian , Costa Veronica , Friess Thomas , Hau Jean-Christophe , Heidkamp Gordon , Kuehne Holger , Martin Laetitia J. , Roudnicky Filip , Tzouros Manuel , Vandemeulebroucke An
Published: March 2024

We became interested in bicyclic tetrahydrothiazepine derivatives described in a Patent (WO 2016/139181) having immunomodulatory activities on CD3-activated T cells (CD4+ & CD8+) and NK cell increasing the secretion of several cytokines such as, e.g., IL-2, IFN-γ and/or TNF-α whereas corresponding unstimulated immune cells do not respond. Surprisingly - in contrast to what has been described in the patent - the N-H of the Amides 1 and 2 described in the patent was not required for potency and the oxadiazoles 1 and 2 were significantly more potent in T-cell proliferation and IL-2 release. Compound 1 was in vivo active in a single dose PD study in mice after CD3 activation and in a MC38 xenograft mice model showing single agent activity. Amide 1 and Amide 2 were identified in a phenotypic screen and the mode of action was unknown. Because of the promising pharmacological profile and in vivo activity, we were interested in the target. Here we describe how we identified the potential targets DGKα and DGKζ.

Citation Format: Thomas Luebbers, Adrian Britschgi, Veronica Costa, Thomas Friess, Jean-Christophe Hau, Gordon Heidkamp, Holger Kuehne, Laetitia J. Martin, Filip Roudnicky, Manuel Tzouros, An Vandemeulebroucke. Target identification of T-cell activating immunomodulatory tetrahydrothiazepines: DGKα and DGKζ inhibitors [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 5769.
Abstract 578: Prediction of PD-1 treatment outcome in mismatch repair deficient metastatic colorectal cancer using a multi-omic approach
Authors: Saberzadeh-Ardestani Bahar , Emiloju Oluwadunni E. , Graham Rondell P. , Lewis Jason T. , Abbott Charles W. , Boyle Sean M. , Sinicrope Frank A.
Published: March 2024

Background: Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of patients with deficient DNA mismatch repair (d-MMR) cancers, yet less than half of patients with metastatic colorectal cancer (mCRC) receive clinical benefit. We analyzed heterogeneity within d-MMR tumors using a multi-omics approach inclusive of percent unstable microsatellite loci (MS).

Methods: We studied consecutive patients with d-MMR mCRC with available primary tumors (N=32) that were treated with an anti-PD-1 antibody at Mayo Clinic (2015-2022). Tumors were profiled using a whole exome and transcriptome platform (ImmunoID NeXT®). MSIsensor was used to calculate percent unstable microsatellite loci from whole exome sequencing data. CIBERSORT and ssGSEA were used to analyze immune gene expression in relationship to percent unstable microsatellite loci. An immune checkpoint gene expression signature was calculated as the mean expression of CD274, CTLA-4, HAVCR2, LAG-3, PDCD1, PDCD1LG2, TIGIT. Aforementioned variables were analyzed in relationship to objective tumor response (RECIST version 1.1). A Cox proportional hazards model was fit to predict progression-free survival (PFS).

Results: Among 32 patients with d-MMR mCRC treated with PD-1 blockade, 16 (50%) received first-line anti-PD-1 therapy; others had > 1 prior chemotherapy regimen. Eighteen (56.3%) tumors harbored BRAFV600E, 4 (12.5%) had mutant KRAS, and median PFS was 24.6 months (95% CI: 8.4, NR) with 18 events. Neither tumor mutation burden (TMB) nor PD-L1 mRNA expression predicted PFS. Median percent unstable MS loci was 14.38 (IQR: 2.6-23.2). Patients whose tumors had higher percent unstable MS loci had significantly better PFS (Q2-4 vs Q1; HR: 0.22, 95%CI: 0.06, 0.62; P=0.003), and were associated with increased objective response (P=0.049). Tumors with higher unstable loci also had higher neoantigen clonality (R= 0.64; P= 0.0003) and increased M0 (resting) macrophages (P=0.02). Nonresponders (vs responders) to PD-1 blockade had an increased immune checkpoint expression signature (P= 0.005).

Conclusion: A higher percentage of unstable microsatellite loci, associated with higher neoantigen clonality and resting macrophages, was predictive of anti-PD-1 efficacy. Furthermore, an immune checkpoint gene expression signature was associated with objective response.

Citation Format: Bahar Saberzadeh-Ardestani, Oluwadunni E. Emiloju, Rondell P. Graham, Jason T. Lewis, Charles W. Abbott, Sean M. Boyle, Frank A. Sinicrope. Prediction of PD-1 treatment outcome in mismatch repair deficient metastatic colorectal cancer using a multi-omic approach [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 578.
Abstract 5785: Shedding light on PARP1-E2F1 interaction by Cryo-EM
Authors: Iglesias Pablo , Casajus-Pelegay Ester , Moreno-Morcillo Maria , González-Rendo Lara , Porres-Ventin Laura , Arce Victor M. , Fernandez-Leiro Rafael , Costoya Jose A.
Published: March 2024

Cell division is orchestrated by a complex network of interactions between proteins involved in numerous signaling pathways that control cell proliferation. One of these main players is the E2F family of transcription factors. The role of these key players is to regulate the expression of several genes important in cell proliferation, particularly those involved in progression through G1 and into the S-phase of the cell cycle. In fact, cell cycle deregulation is a hallmark of cancer, and multiple studies have highlighted the critical role of E2F hyperactivation in tumor progression, angiogenesis, and metastasis. However, targeting a master regulator of cell growth will also have a pernicious effect on non-cancerous cells, thus limiting its applications. In order to overwhelm these restrictions, we propose a therapeutic approach in which E2F transcriptional activity is specifically reduced in cells with hyperactivation of the pathway, such as cancer cells.We and others have shown that poly (ADP-ribose) polymerase 1 (PARP1) is a transcriptional co-activator of E2F1 and, owing to this, loss of PARP results in cell cycle re-regulation and reduction of tumor growth. Interestingly, this effect of PARP1 does not depend on its enzymatic activity, thus opening a completely new strategic approach. Furthermore, considering that most oncogenic processes are associated with cell cycle deregulation, disruption of PARP1-E2F1 interaction could provide a new therapeutic target with a wide spectrum of indications in cancer. Currently, we lack comprehensive structural details regarding the PARP-1/E2F1 protein-protein interaction. To address this, we used cryo-EM to investigate both the standalone structure of PARP1 (full length) and its interaction with E2F1. This approach yielded high-resolution structural insights into the PARP1-E2F1 complex, enabling precise identification of the protein-protein interaction interface (PPI). Through this exploration of structural and molecular determinants governing this interaction, we aim to develop innovative strategies for designing drugs targeted at disrupting this complex. This work was supported by Agencia Estatal de Investigación (AEl/10.13039/501100011033), Xunta de Galicia (GPC GI-1862, ED431B 2020/26; ED431G 2019/02) and European Regional Development Fund-ERDF.

Citation Format: Pablo Iglesias, Ester Casajus-Pelegay, Maria Moreno-Morcillo, Lara González-Rendo, Laura Porres-Ventin, Victor M. Arce, Rafael Fernandez-Leiro, Jose A. Costoya. Shedding light on PARP1-E2F1 interaction by Cryo-EM [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 5785.
Abstract 5788: Targeting p97-NPL4 pathway by copper chelators
Authors: Mistrik Martin , Skrott Zdenek , Loffelmann Martin
Published: March 2024

Copper chelators forming stable complexes, such as the bis(diethyldithiocarbamate)-copper complex (CuET), derived from Antabuse (disulfiram, DSF), have shown pronounced anticancer activity. CuET induces aggregation of NPL4, a crucial cofactor of the p97 segregase in the ubiquitin-proteasome system, leading to proteotoxic stress and subsequent cancer cell death. Our study investigates whether NPL4 inhibition is unique to CuET or extends to other dithiocarbamate-copper complexes and distinct copper chelators. Utilizing a cellular NPL4 aggregation screening assay, we examined various chelators for their ability to induce NPL4 aggregation. Our findings reveal that this capacity is relatively common among structurally diverse DTCs-copper complexes and other unrelated copper chelators. Compounds that caused NPL4 aggregation also triggered NPL4-p97 complex immobilization, polyubiquitinated protein accumulation, and unfolded protein and heat shock responses. These responses correlated strongly with cancer cell cytotoxicity, particularly potent at nanomolar concentrations, underscoring NPL4 as a critical target. Our results demonstrate the broad potential of copper chelators in targeting NPL4, inducing lethal proteotoxic stress in cancer cells, and offering significant implications for drug development.

Citation Format: Martin Mistrik, Zdenek Skrott, Martin Loffelmann. Targeting p97-NPL4 pathway by copper chelators [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 5788.