bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3033: Oncogenic ETS transcription factors avoid repression by EZH2 and FOXO1 in prostate cancer and Ewing sarcoma
Authors: Downing Nicholas F. , Mills Kaitlyn M. , Hollenhorst Peter C.
Published: March 2024

ETS transcription factors play roles throughout development, aiding in hematopoiesis, blood vessel formation and cell fate. The ETS family is composed of 28 members, all of which share an ETS DNA-binding domain. Chromosomal rearrangements that lead to the overexpression of specific ETS promote oncogenic transformation and tumor development. More than half of prostate tumors are driven by aberrant expression of an ETS protein. Our lab has demonstrated that oncogenic ETS form essential interactions with a ubiquitous RNA-binding protein, EWS, to promote prostate tumorigenesis. A similar mechanism exists in Ewing sarcoma (ES). ES tumors are driven by the expression EWS/ETS fusion proteins. This project focuses on the similarities and differences between oncogenic ETS in prostate cancer and EWS/ETS fusions in Ewing sarcoma.

We compared biological functions of the most common oncogenic ETS in prostate cancer, ERG, and the most common fusion in ES, EWS/FLI1. Both ERG and EWS/FLI1 promoted migration and clonogenic survival in normal prostate epithelial cell lines, RWPE1 and PNT2. These data suggest that the EWS-ERG complex and EWS/FLI1 fusion protein can function similarly in prostate cells. Knockdown of endogenous EWS/FLI1 in the ES cell line, A673, reduced anchorage-independent colony formation in soft agar. Rescue with exogenous EWS/FLI1 or EWS/ERG restored colony formation relative to control. Wildtype ERG was unable to rescue colony formation in A673. This suggests that ERG and EWS/FLI1 function differently in A673 cells. Pulldown assays from A673 lysates reveal that ERG interacts with EZH2 and FOXO1. Our lab has established that ERG acts as a transcriptional repressor through the formation of an ERG-EZH2-PRC2 complex. Others demonstrate that FOXO1 represses ERG activity by reducing its recruitment to the target sites. Both the N-terminal truncation of ERG and phosphorylation of ERG at S96 disrupted these interactions and rescued anchorage independent growth in A673 ES cells. These data suggest that EWS/ETS fusions avoid transcriptional repression in Ewing tumors due to loss of the N-terminus of the ETS protein.

Citation Format: Nicholas F. Downing, Kaitlyn M. Mills, Peter C. Hollenhorst. Oncogenic ETS transcription factors avoid repression by EZH2 and FOXO1 in prostate cancer and Ewing sarcoma [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 3033.
Abstract 3039: Pan cancer therapeutic inhibition of oncogenic c-MYC by the engineered destabilized 3’UTR of c-MYC
Authors: Awah Chidiebere U.
Published: March 2024

c-MYC is a master transcription factor that is over expressed in more than 70% of human cancers. There is no clinically approved direct inhibitor of c-MYC. Recently, we reported the development of the engineered destabilized 3’UTR of MYC constructs which is a novel mRNA overwriting technology through which we directly destabilized and degraded c-MYC. Here we report that we have extended this technology in directly targeting and degrading c-MYC in multiple ethnically diverse triple negative breast cancer cell lines, ovarian cancers, neuroendocrine prostate cancers and pancreatic cancers. The constructs are specific, titratable, and directly engaged the c-MYC target validated by RNA transcript technologies. We have performed head-to-head IC50 dose dependent titration comparison with the engineered destabilized 3’UTR of c-MYC constructs and chemotherapies (DNA damaging agents, microtubules inhibiting drugs, alkylating agents) as well as immune checkpoint inhibitor. We found evidence that suggests that the engineered destabilized 3’UTR of c-MYC is likely to be superior to these therapies in pan cancers driven by c-MYC.

Citation Format: Chidiebere U. Awah. Pan cancer therapeutic inhibition of oncogenic c-MYC by the engineered destabilized 3’UTR of c-MYC [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 3039.
Abstract 3047: Interrogating TEAD protein interactions in cancer
Authors: Guarnaccia Alissa , Lill Jennie , Dey Anwesha
Published: March 2024

The Hippo signaling pathway is a major regulator of cellular growth and is frequently deregulated in cancer. The main effectors of the Hippo pathway are the TEAD transcription factors which function by interacting with other co-effector proteins to modulate transcriptional outputs. Notably YAP and TAZ interact with TEAD to mediate oncogenic transcriptional programs, and blocking interaction of TEAD with YAP and TAZ is a sought-after mechanism for small molecule TEAD inhibitors. However, the interaction network of TEAD extends well beyond YAP and TAZ to a variety of additional cofactors, including chromatin remodelers, epigenetic factors, and other transcriptional regulators. Thus, the impact of inhibiting TEAD could extend to unanticipated protein interactions and cellular functions. The purpose of this study is to investigate, define, and interrogate TEAD interaction networks, providing foundational knowledge for interrogating TEAD biological functions and pharmacological effects. We are employing mass spectrometry-based technologies combined with chemical and genetic tools and biochemistry to interrogate TEAD protein interaction networks. We have performed affinity purification coupled with mass spectrometry for TEAD and have identified dozens of significant interacting proteins. TEAD interactions include expected proteins, such as known Hippo pathway members, as well as unexpected protein interactions, such as DNA damage response proteins. Cellular assays probing these DNA damage protein interactions implicate TEAD as a crucial factor for efficient DNA repair mechanisms and maintaining genomic stability. These results raise questions towards a combinatorial effect for TEAD inhibitors and DNA damaging chemotherapeutic agents. Altogether, these ongoing proteomic studies are providing insights into TEAD interaction networks, leading to a deeper understanding of TEAD biology, and providing foundational knowledge that can inform the development of TEAD inhibitors as effective cancer therapies.

Citation Format: Alissa Guarnaccia, Jennie Lill, Anwesha Dey. Interrogating TEAD protein interactions in 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 3047.
Abstract 3071: Differential susceptibility of breast cancer cells to mitochondrial dysfunction induced by Wnt inhibitors
Authors: Kam Yoonseok , Winer Lisa , Rogers George W. , Romero Natalia
Published: March 2024

Advances in research have shown that cancer cells have distinctive bioenergetic alterations to meet their energy requirements. It has been established that certain cancers are significantly reliant on mitochondrial respiration, suggesting that modulation of mitochondrial function may have therapeutic value. The Wnt signaling pathway is a potential therapeutic target of cancer, including breast and colorectal cancers. Recent findings suggest that bi-directional crosstalk between Wnt signaling and mitochondrial function plays a crucial role in tumorigenesis, as well as differentiation and self-renewal of stem cells. In this study, 71 agents known to alter Wnt signaling were screened for effects on mitochondrial function in BT474 and Hs578T breast cancer cell lines, which have highly oxidative and highly glycolytic bioenergetic phenotypes, respectively. This screen was performed using the Agilent Seahorse XF Mito Tox assay, which enables sensitive detection and characterization of mitochondrial modulators via a standardized, quantitative parameter, the Mito Tox Index (MTI). The MTI facilitates the comparison of drug efficacy by detecting modes of mitochondrial toxicity, inhibition, and uncoupling. The MTI-based screening of the Wnt modulators identified one inhibitor and two uncouplers as potent agents inducing mitochondrial dysfunction for both BT474 and Hs578T. Despite their distinctive bioenergetic phenotypes, MTI-based dose-response assays revealed no significant differences in the efficacy of these modulators between BT474 and Hs578T cell types. In contrast, significant differential effects on ATP production rates were detected, showing cellular energy depletion induced by these agents. Using the Agilent Seahorse XF Real-Time ATP Rate assay, BT474 cells showed significant metabolic energy crisis in the presence of all 3 compounds, whereas Hs578T cells maintained stable total ATP production. This differential impact on ATP production rate corresponds directly to differences in cell viability, with BT474 showing higher susceptibility to these compounds. These results suggest that the anti-cancer efficacy of mitochondrial modulators can be different depending on the metabolic phenotype of the target cell and should be considered in therapeutic design.

Citation Format: Yoonseok Kam, Lisa Winer, George W. Rogers, Natalia Romero. Differential susceptibility of breast cancer cells to mitochondrial dysfunction induced by Wnt 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 3071.
Abstract 3084: Aldob regulates fructose metabolism to confer ferroptosis and immune response in colorectal cancer harboring microsatellite instability
Authors: Chang Yu-Chan , Chen Chi-Long
Published: March 2024

Microsatellite instability (MSI) is a genetic alteration that occurs during cancer progression, specifically in colorectal cancer. Objective evidence demonstrates that colorectal cancer patients with MSI have better survival rates than those without MSI. Moreover, patients with both dMMR and MSI respond equally well to immunotherapy. We evaluated the MSI status of colorectal cancer specimens and identified an activation of aldolase B (ALDOB), the primary regulator of fructose metabolism, in the MSI group. Our analysis of the cancer cell panel revealed that the expression level of ALDOB in MSI+ cells (HCT116 and LoVo) was significantly higher than that in the microsatellite stable (MSS) group (SW480 and HT29). We also discovered a significant negative correlation between ALDOB and numerous dMMR-related genes, such as MLH1 and MSH2. We investigated how fructose concentration and pathway activation affected MSI occurrence. Our results demonstrated that abnormal fructose metabolism leaded to SLC7A11-mediated ferroptosis. SLC7A11 functioned as the active subunit and generated glutathione, GPX4, and lipid epoxides in response to transported cystine. This may explain the better clinical outcomes observed in colorectal patients with MSI. On the other hand, we also observed that ALDOB could increase certain immune ratios, such as CD4+ T cells and B cells. This aligns with the implementation of immunotherapy in the MSI community, which has been proven to yield positive effects. Consistent results were still being shown. Our study revealed significant increases in various chemokines and cytokines in media conditioned with fructose. It was interesting to note that fructose resulted in more significant changes than glucose. Based on the gathered evidence, it could be posited that ALDOB plays a crucial role as a promoter of fructose metabolism, leading to the induction of ferroptosis and immune responses in MSI colorectal cancer. The findings shed new light on known consequences and provide new avenues of information.

Citation Format: Yu-Chan Chang, Chi-Long Chen. Aldob regulates fructose metabolism to confer ferroptosis and immune response in colorectal cancer harboring microsatellite instability [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 3084.
Abstract 3098: CETSA for navigating your chemistry and exploring the biology of protein degraders
Authors: Brehmer Victoria , Lundgren Stina , Friman Tomas , Amadio Daniele , Chernobrovkin Alexey , Molina Daniel Martinez
Published: March 2024

Protein degradation is a non-classical modality with the potential to tackle therapeutically interesting proteins, previously deemed undruggable. In this area, CETSA is a powerful technology for identifying new binders, assessing target engagement, and investigating the mechanism of action of the degraders in the intact cell environment - without the need to modify your compound, the proteins or the cellular environment.  CETSA can provide insights into the binding of the degrader to both the E3 ligase and the protein of interest (POI), as well as follow the downstream cellular and molecular effects. Here we will exemplify how CETSA can be applied in various stages of drug discovery to provide biologically relevant data to guide the design and optimization of new degraders. For example, CETSA can provide valuable information of the cell permeability and the in-cell performance of the degrader, which is important for understanding how the degrader enters cells and reaches its target proteins. By combining data from CETSA with data from degradation readout, in our case quantitative proteomics, it is possible to correlate cellular target engagement potencies with degradation efficiency and importantly, also identify any drug:protein binding that does not result in degradation By using the proteome-wide CETSA Explore format, the selectivity of the degrader and the downstream protein consequences of the elimination of the target protein could be studied. 

Citation Format: Victoria Brehmer, Stina Lundgren, Tomas Friman, Daniele Amadio, Alexey Chernobrovkin, Daniel Martinez Molina. CETSA for navigating your chemistry and exploring the biology of protein degraders  [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 3098.
Abstract 3155: Metabolic stress induces an intrinsic inflammatory CAF phenotype that regulates stromal architecture in pancreatic cancer
Authors: Zhang Yijuan , Maganti Swetha , Hope Jennifer , Lin Li , Galapate Cheska Marie , Carrette Florent , Bradley Linda , Commisso Cosimo
Published: March 2024

Macropinocytosis is a critical amino acid supply pathway that fuels the metabolism of tumor cells and cancer-associated fibroblasts (CAFs) in pancreatic ductal adenocarcinoma (PDAC). PDAC tumors are deficient in glutamine, a critical amino acid that tumor cells and CAFs use to sustain their cellular fitness. We have previously shown that both PDAC cells and CAFs stimulate macropinocytosis as an adaptive response to glutamine depletion. How these linked metabolic mechanisms might co-operate to control stromal architecture remains elusive. In this study, we employed glutamine starvation and 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), a selective inhibitor of macropinocytosis, to investigate how metabolic stress impacts the tumor microenvironment. We find that concomitant suppression of glutamine metabolism and macropinocytosis synergizes to drive an intrinsic inflammatory CAF phenotype that requires metabolic stress-induced ERK/MAPK signaling. Utilizing immunocompetent syngeneic mouse models of PDAC, we find that blocking macropinocytosis in these glutamine-depleted tumors diminishes tumor growth and reorganizes the tumor stroma by decreasing collagen deposition, increasing vasculature expansion and enhancing the infiltration of both CD8+ and CD4+ T cells. Alterations in the stromal landscape under these conditions are linked to the enhancement of CAFs that display an inflammatory signature while simultaneously suppressing more myofibroblastic identities within the tumor. Interestingly, we find that these effects on the tumor microenvironment can be harnessed to improve therapeutic responses. We determine that combining macropinocytosis inhibition with immune checkpoint blockade reduces PDAC tumor burden and significantly inhibits metastasis. We also find that macropinocytosis inhibition enhances drug delivery, thereby improving the efficacy of gemcitabine chemotherapy. Our findings demonstrate that intratumoral metabolic stress plays a critical part in preserving and modulating the tumor stroma.

Citation Format: Yijuan Zhang, Swetha Maganti, Jennifer Hope, Li Lin, Cheska Marie Galapate, Florent Carrette, Linda Bradley, Cosimo Commisso. Metabolic stress induces an intrinsic inflammatory CAF phenotype that regulates stromal architecture 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 3155.
Abstract 3160: PufA promotes proliferation, migration and tumorigenesis through NF-kB pathway in cholangiocarcinoma
Authors: Chen Chiao-Ping , Yeh Chun-Nan , Pan Yi-Ru , Hsiao Yu-Tien , Lo Chih-Hong , Lin Chia-Hsien , Wu Chiao-En
Published: March 2024

Cholangiocarcinoma (CCA) exhibits a poor prognosis despite the recent advances in targeted therapy and immunotherapy. Therefore, exploring the novel mechanism of CCA tumorigenesis and identifying the effective targeted therapy is critical for advanced CCA to improve future treatment. A novel biogenesis factor of the 90S pre-ribosome named PufA, is higher expressed in tumors than normal tissue based on the TCGA databank implying the role of CCA tumorigenesis. Our results showed that PufA expression was significantly associated with larger tumor size, more lymph node metastasis, more tumor relapse, death, and unfavorable survivals in 205 patients with resectable CCA in Chang Gung Memorial Hospital. We demonstrated the inhibited effect of cell survival, migration ability, and the sphere-formation potential by silencing of PufA in CCA cell lines. Tumor growth is also suppressed in PufA knockdown xenograft mice. Mechanistic investigation showed that PufA was expression associated with nuclear factor kappa B p65 (RELA) activity and facilitated activity of the NF-κB transactivation. RELA, a transcription factor, is the principal regulator of the inflammatory response and is crucial for the homeostasis of the immune system and the expression of the immune checkpoint. Furthermore, decreased PufA levels also downregulated PD-L1 expression in CCA. Together, these findings suggested that the PufA-RELA axis could be a potential target for further application of targeted therapy in combination with immunotherapy.

Citation Format: Chiao-Ping Chen, Chun-Nan Yeh, Yi-Ru Pan, Yu-Tien Hsiao, Chih-Hong Lo, Chia-Hsien Lin, Chiao-En Wu. PufA promotes proliferation, migration and tumorigenesis through NF-kB pathway in cholangiocarcinoma [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 3160.
Abstract 3200: Assessing the utility of cell membrane lipid-extracted nanoliposomes (CLENs) as a targeted drug delivery system for pancreatic cancer
Authors: Alshammari Khaznah , Verdery Sergio , George Sujin , Campbell Robert B
Published: March 2024

Background: Pancreatic cancer is a challenging type of cancer to treat as evidenced by a 9% 5-year survival and a dismal rate of mortality. Nanoparticles represent a revolutionary treatment method, primarily due to characteristics including cellular absorption, boosting therapeutic effectiveness, and mitigating side effects through localized actions. For nanomedicine, optimizing liposome formulations is crucial for achieving the highest level of specificity in drug targeting. This study aims to evaluate cellular interaction of pancreatic cancer lipid-extract modified nanoliposomes when compared to unmodified varieties in vitro.

Method: Cell membrane lipid-extracted nanoliposomes (CLENs) were prepared via the thin film evaporation technique. The composition included various compositions and ratios of DOPC, DOTAP, Cholesterol, DPPE-PEG-5000, and lipid extracts derived from MS1-VEGF target cells. MS1-VEGF was the cellular model of pancreatic cancer used for this study. Cellular binding studies were performed using cellular suspension and adherent cell populations. Studies were performed in 48-well plates under different experimental conditions including alterations in time of incubation, and periods of cellular exposure to dual humidified and oscillation environments in the presence of lipid-extracted (LE) nanoliposomal preparations and conventional controls.

Results: The particle size for nanoliposomes employed for this study was approximately 115 ± 0.7 nm for control and 128 ± 2.7 nm for experimental (lipid extract-modified) nanoliposomes. Following cellular exposure to different lipid extract concentrations (typically 0 to 100 nmoles per ml of growth medium), the fluorescence values demonstrated increased cellular uptake with greater LE content for floating and adhered MS1-VEGF cell populations. With the floating cells, LE-modified nanoliposomes demonstrated significantly higher fluorescence intensity values compared to conventional nanoliposomes. Similarly, with MS1-VEGF adherent cells, LE-modified nanoliposomes targeted cells to a greater extent compared to unmodified controls.

Conclusions: The increased cellular uptake of CLENs designed for targeting pancreatic tumor cells (both floating and adherent cells) demonstrated an improvement over the use of conventional nanoliposomes under similar experimental conditions. Further research is needed to fully explore the potential use of CLENs for targeting and treatment of pancreatic cancer.

Citation Format: Khaznah Alshammari, Sergio Verdery, Sujin George, Robert B Campbell. Assessing the utility of cell membrane lipid-extracted nanoliposomes (CLENs) as a targeted drug delivery system for 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 3200.
Abstract 322: Ultraplex genome and exome sequencing of rare single cells
Authors: Andreou Ioanna , Rulli Samuel , Heitz Katja , Huebner Silke , Meerschiff Christin , Lader Eric
Published: March 2024

Single-cell genome analysis enables researchers to gain novel insights into tumor heterogeneity. Conducting single-cell genomic analysis using next-generation sequencing (NGS) methods has traditionally been challenging since the amount of genomic DNA present in a single cell is limited. PCR-based amplification methods normally have high error rates, low coverage uniformity, and extensive allelic drop-outs making interpretation of SNVs and CNVs challenging.Here we introduce a new whole-genome amplification method which utilizes an optimized isothermal DNA amplification reaction with a Hi-fidelity, error correcting polymerase and downstream library preparation protocol to allow pooling of amplified DNA. The key DNA amplification steps use multiple displacement amplification (MDA) technology to amplify gDNA directly from single cells, nuclei or previously isolated genomic DNA. During the MDA reaction, sample-barcodes are incorporated into the newly synthetized DNA, allowing the amplified DNA to be pooled and processed in to one ultraplex library. This method saves library preparation time, reduces DNA library costs and limits plastic consumable usage.Our current workflow allows 24 samples to be multiplexed in one library and 24 libraries to be produced for a total of 576 samples per sequencing flow cell. This method can be used to investigate chromosomal instability with low pass sequencing or SNV and CNV analysis following whole genome, exome-capture workflows.

Citation Format: Ioanna Andreou, Samuel Rulli, Katja Heitz, Silke Huebner, Christin Meerschiff, Eric Lader. Ultraplex genome and exome sequencing of rare single 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 322.
Abstract 3229: IACS-16559, a CBP/P300 bromodomain inhibitor for the treatment of specific AML subsets
Authors: Zanca Ciro , Soth Michael , Gao Guang , Gay Jason , Feng Ningping , Bristow Christopher , Le Kang , Xu Quanyun , Nguyen Phuong , Jiang Yongying , Perry Teresa , Mseeh Faika , Leonard Paul , Cross Jason , Giuliani Virginia , Marszalek Joe , Jones Philip , Heffernan Tim
Published: March 2024

Block in differentiation and accumulation of undifferentiated blasts are hallmarks of Acute Myeloid Leukemia (AML) and epigenetic processes have been shown to play a critical role in hematological malignancies. Paralogs CREBBP and EP300, in association with co-factors, are hijacked during leukemogenesis by aberrant transcriptional factors, thus driving blast proliferation and maintaining the undifferentiated phenotype. Here, we introduce IACS-16559, a bromodomain inhibitor targeting CREBBP/EP300. IACS-16559 is highly selective for the bromodomains of CBP and EP300 versus BRD4, showing a dose-dependent inhibition of H3K27 acetylation residue in vivo and in vitro. In addition, IACS-16559 pharmacokinetic properties show low clearances, high oral exposures and predicted QD dosing in human. We screened AML cell lines in vitro for their response to IACS-16559 and identified specific subtypes of AML that exhibit heightened sensitivity to CBP/EP300 inhibition, specifically AML with AML1-ETO and MLL-AF9 rearrangements, and AML featuring mutations in the nucleophosmin (NPM1c) gene. IACS-16559 inhibited proliferation of AML responder cell lines in vitro, halted cell cycle progression, with an accumulation of cells in G1 and a decrease of cells in S phase. IACS-16559 did not induce apoptosis across a panel of AML cell lines, indicating that induction of apoptosis is not a major mechanism of action of CBP/EP300 inhibition. To augment the anti-tumor effects of IACS-16559, we prioritized the pharmacological combination of CBP/EP300 and MLL-Menin inhibitors. This combination synergistically impeded cell proliferation and potently triggered apoptosis in vitro and in vivo, whereas none of the individual agents alone had this effect. The changes in gene and surface marker expression produced by these treatments, as single agents and in combination with synergistic effects, were in line with an induced myeloid differentiation phenotype. Collectively, our findings provide a compelling basis for further exploring the use of a CBP/EP300 bromodomain inhibitor in conjunction with an inhibitor of the MLL-Menin interaction in a subset of AML patients with MLL rearrangements or NPM1 mutations.

Citation Format: Ciro Zanca, Michael Soth, Guang Gao, Jason Gay, Ningping Feng, Christopher Bristow, Kang Le, Quanyun Xu, Phuong Nguyen, Yongying Jiang, Teresa Perry, Faika Mseeh, Paul Leonard, Jason Cross, Virginia Giuliani, Joe Marszalek, Philip Jones, Tim Heffernan. IACS-16559, a CBP/P300 bromodomain inhibitor for the treatment of specific AML subsets [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 3229.
Abstract 3242: TNG917 is a clinical-grade, potent and selective inhibitor of EHMT1/2 for the treatment of immune cold tumors
Authors: Lu Alvin , Haines Brian , Lei Ji , Zhang Wenhai , Zhang Minjie , Whittington Douglas A. , Ferdinez Maria Lucia , Yu Yi , Meier Samuel R. , Choi Ashley , Bejnood Alborz , Bandi Madhavi , Lazarides Katherine , Pan Xuewen , Gu Lina , Tsai Alice W. , Sudsakorn Sirimas , Liang Colin , Come Jon , Williams Brett , Throner Scott , Vacca Joseph , Maxwell John P. , Anderson Jannik N. , Huang Alan , Min Chengyin , Chen Yingnan P.
Published: March 2024

CRISPR-based functional genomics screening can be designed to identify novel cancer cell intrinsic targets that increase tumor immunogenicity. Using a FACS-based CRISPR sorting screen for PD-L1 expression, we identified Euchromatic histone-lysine-N-methyltransferase 1 and 2 (EHMT1/2) as negative modulators of the interferon signaling pathways. EHMT1 and EHMT2 are histone methyltransferases that mono- and di-methylate lysine 9 of histone H3 to repress gene transcription of defined target genes. Gene knockout or pharmacological inhibition of EHMT1/2 in cancer cells resulted in de-repression of gene promoters, upregulation of interferon-stimulated genes (ISGs), and secretion of pro-inflammatory cytokines. Here, we present the preclinical characterization of TNG917 - an oral and highly selective EHMT1/2 inhibitor with low nanomolar cellular potency, and favorable pharmacodynamic and pharmacokinetic properties. In humanized and syngeneic mouse models, treatment with TNG917 in combination with anti-PD1 promoted a T-cell-infiltrated tumor microenvironment, led to significant anti-tumor activity, and resulted in survival benefit. In summary, our in vitro and in vivo studies provide a rationale for the clinical development path of TNG917, in combination with checkpoint inhibition, in patients with immune cold tumors.

Citation Format: Alvin Lu, Brian Haines, Ji Lei, Wenhai Zhang, Minjie Zhang, Douglas A. Whittington, Maria Lucia Ferdinez, Yi Yu, Samuel R. Meier, Ashley Choi, Alborz Bejnood, Madhavi Bandi, Katherine Lazarides, Xuewen Pan, Lina Gu, Alice W. Tsai, Sirimas Sudsakorn, Colin Liang, Jon Come, Brett Williams, Scott Throner, Joseph Vacca, John P. Maxwell, Jannik N. Anderson, Alan Huang, Chengyin Min, Yingnan P. Chen. TNG917 is a clinical-grade, potent and selective inhibitor of EHMT1/2 for the treatment of immune cold tumors [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 3242.
Abstract 3257: Unleashing immune power: How mRNA-encoded modulators suppress tumor growth
Authors: Wang Dong , zhang zhixiang , wang peili , duan binbin , qiang xiangnan , gu qingyang
Published: March 2024

Subsequent to the emergence of the COVID-19 pandemic, an increased number of nucleic acid-based therapeutics have secured clinical endorsement. Unlike conventional protein-targeted therapies, nucleic acid therapeutics provide the potential for long-lasting effects and can be further categorized into oligonucleotides, single-guide RNA (sgRNA), and messenger RNA (mRNA). In this study, we aim to explore the anti-tumor properties of mRNA-encoded CD3-EpCAM Bispecific Antibody (BsAb) and mRNA-encoded IL-12 protein.

We conducted qPCR, flow cytometry, and ELISA analysis to assess the dynamic concentration of mRNA-encoded CD3-EpCAM BsAb and mRNA-encoded IL-12 protein within tumor cells post-transfection. We then accessed their effects on anti-tumor and immune activation in vitro via flow cytometry and ELISA. Furthermore, using in vivo imaging system (IVIS), we were able to determine the biodistribution of mRNA-encoded BsAb in vivo. Efficacy was evaluated in subcutaneous tumor xenograft models with mRNA content in tumors examined via qPCR. Moreover, we analyzed the activation of tumor-infiltrating immune cells post mRNA-encoded IL-12 treatment. Finally, to ascertain the preliminary safety of mRNA-encoded BsAb and mRNA-encoded IL-12 protein, we conducted serum biochemistry analysis and histopathological examination of various organs.

In this comprehensive preclinical evaluation, we found that both mRNA-encoded BsAb and mRNA-encoded IL-12 promoted the activation and cytotoxicity of human T cells, exhibiting significant inhibitory effects on tumor growth in vivo while maintaining a favorable safety profile. Our findings provide compelling evidence that mRNA therapeutics hold significant research value and exhibit promising potential for clinical applications.

Citation Format: Dong Wang, zhixiang zhang, peili wang, binbin duan, xiangnan qiang, qingyang gu. Unleashing immune power: How mRNA-encoded modulators suppress tumor growth [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 3257.
Abstract 3263: VEGFA mediates traditional EGFR-TKI resistance in non-small cell lung cancer with EGFR exon 20 insertions
Authors: Yang Yaning , Liu Chengming , Yang Guangjian , Xu Haiyan , Lei Siyu , Wang Yan
Published: March 2024

Background: Epidermal growth factor receptor (EGFR) exon 20 insertions (Ex20ins) accounted for 4%-12% of EGFR mutations and showed primary resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs) which is an urgent issue to be solved. In our previous studies, we found that antiangiogenetic therapy had certain advantages for patients with Ex20ins in the real-world practice. Previous research showed the tumor microenvironment has been associated with resistance to targeted therapy in sensitive mutations. However, the mechanism of resistance of EGFR-TKIs and the advantage of antiangiogenetic therapy in patients with EGFR Ex20ins were still unclear.

Methods: In this study, patients with EGFR exon 19 deletion (19del), exon 21 p.L858R (21L858R), and Ex20ins were collected to identify the efficacy of EGFR-TKIs. Stable cell lines expressing were established and used to compare signaling pathway activation. Matrigel angiogenesis assay was conducted in different cell lines. Next, we evaluated the efficacy of various treatments combined with antiangiogenetic agents or not in mouse models.

Results: We collected 23 patients with EGFR sensitive mutations and Ex20ins, respectively, and found Ex20ins presented primary resistance to TKIs and poor survival outcomes. Based on the RNA sequence of stable cell lines, it was found that Ex20ins had a higher level of VEGFA expression and enriched in MAPK pathway compared to EGFR 19del and 21L858R. Immunohistochemical studies also revealed that a higher level of VEGFA expression was observed in a cohort of NSCLC patients with Ex20ins mutation compared with 19del or L858R. Matrigel angiogenesis assay has shown consistent result that Ex20ins promoted angiogenesis in vivo. Further experiments in mouse models identified that in the group of Osimertinib, mobocertinib alone, chemotherapy alone, mobocertinib or chemotherapy combination with bevacizumab, Osimertinib achieved the worst efficacy and additional utility of antiangiogenetic agents had a more favorable efficacy.

Conclusion: Our study highlights the potential of VEGFA as a mechanism of resistance to EGFR-TKIs and induces immunosuppression microenvironment in NSCLC with Ex20ins. Furthermore, the findings suggested that antiangiogenetic therapy could be a promising therapeutic approach.

Keywords: VEGFA, targeted therapy, resistance, EGFR exon 20 insertions, non-small cell lung cancer

Citation Format: Yaning Yang, Chengming Liu, Guangjian Yang, Haiyan Xu, Siyu Lei, Yan Wang. VEGFA mediates traditional EGFR-TKI resistance in non-small cell lung cancer with EGFR exon 20 insertions [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 3263.
Abstract 3329: Development of HM-001 as a therapeutic drug for non-muscle invasive bladder cancer
Authors: Mima Shinji , Sakaki Monta , Kiya Noriaki , Kawashima Atsunari , Nonomura Norio , Kakinuma Chihaya
Published: March 2024

HM-001 is a drug containing sodium chlorite as the active ingredient that is being developed as a drug for non-muscle invasive bladder cancer (NMIBC), and is currently undergoing physician-led clinical trials. It has been discovered that sodium chlorite generates radicals in an acidic environment, and phosphatidylserine on the surface of tumor cells acts as a Lewis acid to generate radicals. HM-001, like other anticancer drugs and BCG, is intended to be injected into the bladder, retained, and then excreted. GLP studies using the same administration method has been completed, and no toxic changes have been observed in human trial doses. The efficacy of HM-001 against NMIBC was evaluated using an orthotopic transplantation model of UM-UC-3, a bladder cancer cell line. In the model, cells were transplanted into the bladder of SCID mouse after epithelial removal with trypsin, 600, 800, 1,600, and 3,200 ppm of HM-001 were administered once a week, and the weight of the excised bladder was calculated. After 4 weeks, the tumor weight was calculated and the tumor weight suppression rate in the control group was evaluated. As a result, the medicinal efficacy was confirmed at concentrations of 800ppm or higher. On the other hand, the UM-UC-3 model is a commonly used model, but it mimics CIS (carcinoma in situ) and can only partially mimic NMIBC. That is, other models are needed to evaluate efficacy for low- and high-grade NMIBC other than CIS. Therefore, we established high-grade and low-grade PDX and evaluated the effectiveness of HM-001 against low- and high-grade PDX through ex vivo testing. First, patient-derived tissue was transplanted into SCID mice, allowed to grow, and then removed. After extraction, they were cut into 2 mm3 pieces, exposed to HM-001 at 800, 1600, and 3200 ppm for 2 hours, and then transplanted into SCID mice. As a result of comparing the tumor size after transplantation with the control group, a dose-dependent tumor regression effect was confirmed in high-grade and low-grade PDX. NMIBC is divided into low-, medium-, and high-risk categories, with different treatment methods and drugs used for each. In particular, low-grade forms are restricted to low- and intermediate-risk groups. The results of this study show that HM-001 is effective not only for high-risk (including CIS) NMIBC but also for low- and intermediate-risk (low-grade) NMIBC.

Citation Format: Shinji Mima, Monta Sakaki, Noriaki Kiya, Atsunari Kawashima, Norio Nonomura, Chihaya Kakinuma. Development of HM-001 as a therapeutic drug for non-muscle invasive bladder 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 3329.
Abstract 3330: BPP, a dual functional inhibitor, targets MLL-rearranged acute myeloid leukemia: Unraveling mechanisms and expanding therapeutic horizons
Authors: Chen Tai-Lin , Su Tsann-Long , Lee Te-Chang
Published: March 2024

Refractory/recurrent acute myeloid leukemia (AML) poses a formidable clinical challenge, with MLL translocations implicated in approximately 10% of AML cases and a staggering 75% in infant leukemia, bearing an unfavorable prognosis. Our investigation focuses on BPP, a dual-functional inhibitor capable of inducing DNA cross-linking and suppressing angiogenesis. In a diverse panel of solid tumor and leukemic cells, BPP exhibited exceptional sensitivity against MLL-rearranged AML cell types. In MOLM13 and MV4-11 xenograft models, BPP showcased outstanding tumor suppression, with some tumors even achieving complete remission. In-house acute toxicity testing, BPP treated group showed excellent safety margin in both blood chemistry and pathological examination. Remarkably, BPP demonstrated significant cytotoxicity not only in FLT3-WT AML but also in FLT3-ITD AML. Delving into the mechanism, BPP was found to induce FLT3-ITD and MLL degradation by triggering DNA damage and ER stress. Comet assays revealed a dose-dependent accumulation of DNA fragments in MLL cells upon BPP exposure.The involvement of the KMT2A gene in chromosomal translocation in THP1 cells was highlighted, with BPP treatment resulting in a significant increase in Heat-shock protein. Notably, the combination of ganetespib, an FDA-approved HSP90 inhibitor, with BPP exhibited synergistic effects in both in vitro and in vivo models, inducing synthetic lethality and inhibiting HR repair. Furthermore, BPP not only reduced leukemic stem cells in colony formation assays but also demonstrated sensitivity in relapsed/refractory (R/R) and multiple drug-resistant patient samples. These findings collectively position BPP as a promising candidate for the treatment of MLL-rearranged AML, offering multifaceted therapeutic potential.

Citation Format: Tai-Lin Chen, Tsann-Long Su, Te-Chang Lee. BPP, a dual functional inhibitor, targets MLL-rearranged acute myeloid leukemia: Unraveling mechanisms and expanding therapeutic horizons [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 3330.
Abstract 3334: Combined treatment with PG3 and Nelfinavir induced synergistic apoptosis though sustained activation of integrated stress response
Authors: Tian Xiaobing , El-Deiry Wafik S.
Published: March 2024

Integrated stress response (ISR) activation has been linked to many human diseases, including cancer and neurological diseases. The ISR controls protein synthesis and proteostasis. ISR main effector ATF4 transcriptional factor regulates the balance between survival and cell death. Under acute ISR, ATF4 promotes cell adaptation and survival. Sustained ISR leads to ATF4-mediated apoptosis. In many cancers, mutations or overexpression of oncogenes, results in enhanced cell proliferation and increased protein synthesis, which activate the ISR. Therefore, protein translation is reduced to maintain proteostasis. In other words, many cancers are primed by the ISR. Therefore, additional push by small molecule ISR-inducers will disrupt the balance and force cancer cells to enter apoptosis. Nelfinavir activates ATF4 through inhibition of eIF2α specific phosphatases CReP and GADD34. PG3 upregulates ATF4 through the HRI/eIF2α/ATF4 pathway. We hypothesize that combined treatment of PG3 and Nelfinavir will sustain upregulation of ATF4, and lead to ATF4-mediated cell apoptosis. Cell viability assays indicated that the combined treatment potently and synergistically inhibited cell viability in colorectal cancer cell lines HT29, SW480 and HCT116 p53−/−, osteosarcoma cell line U2OS and breast cancer cell lines MCF7 and T47D. The combined treatment sustained the induction of ATF4 and enhanced the expression of its proapoptotic target genes, CHOP and PUMA, and cell apoptosis. We confirmed that the enhanced induction of ATF4, CHOP and PUMA is through the ISR because ISRIB (ISR inhibitor) blocked the combined treatment-induced upregulation of ATF4, CHOP and PUMA.

Citation Format: Xiaobing Tian, Wafik S. El-Deiry. Combined treatment with PG3 and Nelfinavir induced synergistic apoptosis though sustained activation of integrated stress response [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 3334.
Abstract 3438: Associations between estimated endotoxin exposure and circulating immunological markers among male farmers
Authors: Ezennia Somayina C. , Beane-Freeman Laura E. , Chang Vicky C. , Xie Shuai , Andreotti Gabriella , Friesen Melissa C. , Hofmann Jonathan N.
Published: March 2024

Occupational exposure to endotoxin has been associated with reduced lung cancer risk. Farmers who perform certain tasks (e.g., raising hogs) can be highly exposed to endotoxin. The mechanisms through which endotoxin could confer a lower risk of lung cancer, while likely to be immune mediated, are not clear. To address this gap, we evaluated associations between estimated endotoxin exposure and circulating immunologic markers among male farmers in the Biomarkers of Exposure and Effect in Agriculture (BEEA) study. Our investigation included 122 non-smoking farmers from Iowa, oversampling those raising hogs (n=61). We evaluated 58 serologic markers, including some linked to lung cancer, that were measured using multiplex bead-based assays. Based on an algorithm linking personal air measurements in BEEA and published data to reported farming tasks, we estimated cumulative endotoxin exposure in the last 30 days and 12 months before sample collection. We used multivariable linear regression to estimate geometric mean ratios of immune markers across exposure quartiles, overall and by season of sample collection. Higher endotoxin exposure in the last 30 days was associated with increased levels of FGF-2, MIP-3A/CCL20, and sIL-4R (Ptrend ≤ 0.02), and with decreased levels of MDC/CCL22 (Ptrend = 0.02). In analyses stratified by season, the positive associations with FGF-2 and MIP-3A/CCL20 were most apparent in the winter (Ptrend = 0.002 and 0.03, respectively); we also observed an inverse association with sCD27 levels in the summer (Ptrend = 0.03). Similar patterns were observed for endotoxin exposure in the last 12 months. This study is the first to use novel algorithm-based endotoxin exposure estimates to evaluate associations with intermediate lung cancer-related markers. Several markers were associated with endotoxin levels in an exposure-response manner. Our findings suggest possible biological mechanisms through which endotoxin may reduce risk of lung cancer.

Table 1. Associations between estimated endotoxin exposure in the last 30 days and selected serum im

Citation Format: Somayina C. Ezennia, Laura E. Beane-Freeman, Vicky C. Chang, Shuai Xie, Gabriella Andreotti, Melissa C. Friesen, Jonathan N. Hofmann. Associations between estimated endotoxin exposure and circulating immunological markers among male farmers [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 3438.