bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2918: Potential for a novel therapeutic target: Effects of lactate & lactylation in expression of key regulatory cancer proteins
Authors: Costeas Christos P. , Purcell Connor , El-Deiry Wafik S.
Published: March 2024

The purpose of our study is to investigate the immunologic, metastatic, and gene regulation effects of lactate in the context of cancer in normoxic and hypoxic conditions to identify novel biomarkers and potential therapeutic targets. We aim to modify the in-vitro cancer microenvironment to show how lactate and lactylation can be exploited using existing cancer treatments. While hyperlactatemia is defined as lactate levels between 2 mmol/L and 4 mmol/L (Foucher et al 2023), tumor biopsies have demonstrated extracellular concentrations as high as 40 mM (Pérez-Tomás et al 2020). Through CTG viability assays we identified lactate concentrations that PANC1 cells can survive, which are in agreement with the in-vivo findings of Brizel et al (2001). We observed pancreatic cancer cells achieving over 50% viability for 5 days at 50 mM lactate concentrations while at 30 mM on day 5 they proliferated past their initial plating numbers. Furthermore, Brizel et al (2001) found that patients with high tumor lactate concentrations had a significantly higher incidence of metastatic relapse. Through our scratch assay, we show qualitative evidence of increased mobility and inferred migration potential of PANC1 at 7.5mM [lactate] compared to the absence of lactate. We have also observed increased migration at higher lactate concentrations for which we are pursuing quantitative measurements to evaluate significance. Migration experiments will be followed by the investigation of gene regulation (eg. mRNA seq, lactylation profile) involved in the observed increased mobility. Lactate has been shown to affect immune regulation, namely suppression of iNKT (Fu et al, 2020), macrophages (Yang et al, 2020), dendritic cell differentiation and antigen recognition/presentation (Wang et al, 2022), however not much is known about its effect on NK cells and their cytokine expression. Through NK92MI/PANC1 cocultures we show the effects of lactate concentration on the killing ability of NK cells (in the works). And plan on investigating the cytokine profile leading to the phenotypic killing differences. Lastly, via lactate-titrated incubation of PANC1 cells in normoxia we have demonstrated a positive correlation of HIF1a expression, pan-lactyllysine lactylation, and microenvironment lactate concentration. We speculate HIF1a inhibition post-lactate-sensitization may induce cancer killing and we will investigate this theory in normoxia and hypoxia using existing treatment options.

Citation Format: Christos P. Costeas, Connor Purcell, Wafik S. El-Deiry. Potential for a novel therapeutic target: Effects of lactate & lactylation in expression of key regulatory cancer proteins [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 2918.
Abstract 2940: A comparative analysis of library preparation technologies for RNA sequencing from FFPE samples
Authors: Pavlica Jennifer , Sanders Travis , Gelagay David , Reed Kailee , Harrison Thomas , Corbet Giulia , Haimes Josh , Hanek Ariele , Giorda Kristina , Kudlow Brian
Published: March 2024

Formalin-fixed, paraffin-embedded (FFPE) samples are an invaluable resource in the oncology space, providing access to a vast library of archived diseased tissue samples paired with relevant donor information. Despite the broad utility of these samples, RNA extracted from FFPE tissue is typically difficult to process due to the presence of residual crosslinks and its degraded nature. Further, these samples often vary widely in performance, as the fixation process, block age, block storage, and extraction method can impart large impacts on resulting template quality. As a result, robust and reproducible RNA sequencing from FFPE-derived RNA remains a challenge with unpredictable and high failure rates. We evaluated four commercially available WTA solutions to determine which performed best with this challenging sample type with respect to library complexity, inter-input and intra-sample concordance, and overall reproducibility.

Matched fresh frozen and FFPE liver samples were used such that the fresh frozen data set serves as a comparative truth set for the FFPE data. RNA-Seq libraries were prepared from each sample at both 10 ng and 100 ng inputs. The higher input serves as a control while the lower input reflects achievable inputs from very challenging FFPE samples. For 10 ng FFPE-derived RNA, the Watchmaker RNA Library Prep Kit with Polaris Depletion detects more unique genes than other chemistries and has a much higher percentage of genes that overlap with the fresh frozen control libraries. Additionally, differential expression analysis between 10 ng and 100 ng FFPE libraries demonstrates that the Watchmaker solution better preserves the gene expression profile at low inputs, as fewer differentially expressed genes are identified.

The Watchmaker chemistry includes a number of features aimed at improving library complexity, including a novel FFPE decrosslinking step, a reverse transcriptase specifically engineered to improve conversion of RNA to cDNA, and fewer bead purification steps to prevent sample loss. The concordance between FF and FFPE samples at variable input masses observed when using the Watchmaker solution promotes a higher degree of confidence when making decisions based on lower quantity and quality samples and enables researchers to access more meaningful biological information.

Citation Format: Jennifer Pavlica, Travis Sanders, David Gelagay, Kailee Reed, Thomas Harrison, Giulia Corbet, Josh Haimes, Ariele Hanek, Kristina Giorda, Brian Kudlow. A comparative analysis of library preparation technologies for RNA sequencing from FFPE samples [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 2940.
Abstract 2959: Signatures of therapy-induced senescence in glioblastoma-derived extracellular vesicles
Authors: DeLuca Valerie , Digumarti Priya , Garcia-Mansfield Krystine , Pirrotte Patrick , Berens Michael E.
Published: March 2024

The recalcitrant nature of glioblastoma (GBM) may be due, in part, to the promotion of therapy-induced senescence (TIS) by standard of care treatments. TIS is a prolonged growth arrest associated with epigenetic, metabolic, and biochemical alterations, as well as a robust secretory phenotype. Despite an initial period of tumor stasis due to the loss of cell proliferation, TIS is associated with the development of stemness, increased drug resistance, and immune suppression. Our preliminary data demonstrate the prominent induction of TIS in GBM tumor cells and astrocytes following standard of care therapies for GBM in vitro. However, studies of TIS in GBM patients have been limited due to i) the impracticality of receiving multiple GBM biopsies post-treatment but prior to recurrence and ii) the lack of robust detection methods for TIS even with appropriate biopsy. These challenges argue the need for novel methods to survey TIS in patients. Extracellular vesicles (EVs) are small membrane-bound particles that readily cross the blood brain barrier, and convey information about originating cell identity and cell state through their cargo content. We hypothesize that TIS in host and tumor cells drives the elaboration of senescence-associated EVs (senEVs) with quantifiable senescence protein cargo signatures, and consequently, that EVs can serve as a liquid biopsy for senescence in patients. In a preliminary experiment, two patient derived xenografts grown as short-term in vitro cultures were exposed to 6GY irradiation, with supernatant collected prior to radiation (D0), at peak senescence (D7), and at late senescence (D10). EVs were isolated and subjected to unbiased mass spectrometry. Following unsupervised clustering, EVs were grouped by treatment rather than by cell line. Approximately 6000 proteins were identified across the entire data set, with 105 proteins unique to D7 samples, 162 proteins unique to D10 samples, and 688 proteins exclusive to D7 and D10 samples. Further, proteins from two established senescence gene sets were identified as unique to the D7 and/or D10 conditions. Current work to more robustly identify enrichment of senescence gene sets in EV cargo post-irradiation is underway. We have also established shRNA knockdown cell lines with abrogated TIS to equip validation studies of the TIS-EV cargo relationship. We anticipate that these studies will aid in the development of EVs as a novel reporter of senescence in the clinic.

Citation Format: Valerie DeLuca, Priya Digumarti, Krystine Garcia-Mansfield, Patrick Pirrotte, Michael E. Berens. Signatures of therapy-induced senescence in glioblastoma-derived extracellular vesicles [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 2959.
Abstract 2998: Regulation of epidermal growth factor receptor signaling by BRCA1-microRNA 146a network
Authors: Kumaraswamy Easwari , Shimak Raeann M. Koren , Gunewardena Sumedha , Wendt Karen L. , Alexander Dani , Hembruff Stacey L. , Jensen Roy A.
Published: March 2024

Inherited defects in the breast cancer susceptibility gene, BRCA1 are associated with an increased risk for hereditary breast, ovarian, and other cancers. BRCA1-associated basal like breast cancers (BLBCs) are high-grade ductal carcinomas that do not express ER, PR or HER2, but frequently overexpress epidermal growth factor receptor (EGFR/ErbB1/HER1). The EGFR-driven signaling network plays a pivotal role in BLBC pathogenesis. EGFR overexpression is associated with tumor progression and metastasis, resistance to radiation and chemotherapy, and poor prognosis. Growth factor receptors are regulated by multiple miRNAs, each having many targets, coordinately controlling the complex pathways of downstream signaling. We have previously reported a post-transcriptional mechanism by which BRCA1 regulates EGFR expression through the induction of miR-146a and provided a rationale for the development of miR-146a based therapeutic strategies. In addition, we have shown that BRCA1 also exerts regulatory control over dozens of important miRNAs that appear to play a critical role in breast neoplasia. From TCGA analysis, we show that low expression of these miRNAs is associated with distinctively poor overall survival of TNBC patients. Using miR-146a loss or gain of function experiments in vitro, we show that BRCA1 and miR-146a regulate EGFR signaling, stemness, epithelial-to-mesenchymal transition (EMT), extracellular matrix (ECM) remodeling and chemoresistance. Using in vivo mouse models, we demonstrate that miR-146a overexpression delays tumor formation leading to better overall survival. BLBC unlike the other subtypes, lacks targeted therapy approaches making cytotoxic chemotherapy essentially the only option for treatment. Though EGFR inhibitors have been used to treat other cancers, clinical trials for breast cancer have so far been unsuccessful due to poor response rates. Since one miRNA can target multiple genes and regulate multiple signaling pathways, our study provides evidence to suggest that restoring miR-146a could suppress EGFR-RAS-MEK signaling and the other compensatory pathways, providing a targeted therapeutic option for BRCA1 associated BLBC.

Citation Format: Easwari Kumaraswamy, Raeann M. Koren Shimak, Sumedha Gunewardena, Karen L. Wendt, Dani Alexander, Stacey L. Hembruff, Roy A. Jensen. Regulation of epidermal growth factor receptor signaling by BRCA1-microRNA 146a network [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 2998.
Abstract 2999: Evaluation of microRNA expression in metastatic and non-metastatic salivary gland mucoepidermoid carcinoma
Authors: Trevizani Maria E. , Oliveira Katia K. , Marchi Fabio A. , Bizinelli Daniela , Mariano Fernanda V. , Nagano Cibele P. , Costa Felipe A. , Pinto Clovis A. , Kowalski Luiz P. , Lourenço Silvia V. , Coutinho-Camillo Cláudia M.
Published: March 2024

Salivary gland tumors are a heterogeneous group of lesions, being mucoepidermoid carcinoma (MEC) the malignant neoplasm with the highest incidence, occurring mainly in the parotid gland. Characterization of its histological grade (high, intermediate, and low) is important for determining patient prognosis. Metastasis is more common high-grade tumors and can occur at regional lymph nodes or distant sites. Although the mechanisms underlying the metastatic process are largely unknown, large-scale microRNA expression profiling studies of human cancers have demonstrated that dysregulation of miRNA is frequently associated with many cancer types. The aim of this study is to validate the use of microRNA expression for the discrimination of the metastatic potential of MEC samples. Using Real Time RT-PCR (qPCR) we have analyzed the expression of 384 miRNAs and controls in 4 non-metastatic MECs, 3 MECs with lymph node metastasis, 3 MECs with distant metastasis, and 2 non-neoplastic human salivary gland samples. Comparing the expression among these groups we identified 45 differentially expressed miRNAs that may be related with the metastatic potential of these tumors. To validate the microRNA expression associated with the metastatic potential, we selected 7 out of the 45 differentially expressed microRNAs and 7 MEC samples from the different groups of samples. Considering the 7 microRNAs evaluated, we validated that the expression of miR-191 was increased in non-neoplastic samples when compared to MECs with lymph node metastasis and the expression of miR-494 was increased in non-neoplastic samples when compared to MECs with distant metastasis, suggesting that loss of these microRNAs might be involved in the development of metastasis in mucoepidermoid carcinoma. The determination of a miRNA expression profile could provide a better understanding of the molecular basis of MEC and facilitate the determination of diagnosis and prognosis of patients.

Citation Format: Maria E. Trevizani, Katia K. Oliveira, Fabio A. Marchi, Daniela Bizinelli, Fernanda V. Mariano, Cibele P. Nagano, Felipe A. Costa, Clovis A. Pinto, Luiz P. Kowalski, Silvia V. Lourenço, Cláudia M. Coutinho-Camillo. Evaluation of microRNA expression in metastatic and non-metastatic salivary gland mucoepidermoid carcinoma [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 2999.
Abstract 3007: AZGP1 inhibits angiogenesis in prostate cancer
Authors: Wen Ru , Wen G Edward , Qiu Zhengyuan , Peterson Eric E , Marques Fernando Jose Garcia , Bermudez Abel , Pollack Jonathan R , Zhao Hongjuan , Pitteri Sharon , Brooks James
Published: March 2024

AZGP1 is involved in various biological processes, including lipid metabolism, regulation of cell proliferation, migration and invasion, and immune response. The loss of AZGP1 is associated with worse clinical outcomes, and AZGP1 has been indicated as a potential biomarker for prostate cancer. However, the underlying mechanisms of AZGP1 function in prostate cancer are unknown. This study reports AZGP1's pivotal involvement in angiogenesis within the prostate cancer tumor environment. Neither knockout nor overexpression of AZGP1 affects in vitro prostate cancer cell proliferation, migration, or invasion. Morphologically, AZGP1-deficient mouse prostates appear normal, but exhibit increased fibroblast growth in periglandular stroma after 6 months. The overexpression of AZGP1 does not impact the growth of PC3 and DU145 tumors; instead, these tumors significantly reduced microvessel density, suggesting AZGP1 exhibits anti-angiogenic properties. Proteomic profiling shows distinct profile of angiogenesis-related proteins between PC3-AZGP1-OV and PC3 control cells, featuring proteins like PDCD6 and MMP9. This study provides insights into the anti-angiogenic attributes of AZGP1 in prostate cancer, underscoring its potential as a therapeutic target for prostate cancer.

Citation Format: Ru Wen, G Edward Wen, Zhengyuan Qiu, Eric E Peterson, Fernando Jose Garcia Marques, Abel Bermudez, Jonathan R Pollack, Hongjuan Zhao, Sharon Pitteri, James Brooks. AZGP1 inhibits angiogenesis in prostate 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 3007.
Abstract 3023: Master corepressor inactivation through oncogene suppressor RAI2 mediated polymerization
Authors: Goradia Nishit , Werner Stefan , Mullapudi Edukondalu , von Amsberg Gunhild , Pantel Klaus , Wilmanns Matthias
Published: March 2024

Imbalanced transcription of a large number of genes can be caused at various levels, ranging from alterations in individual transcription factors to dysfunctional corepressor and coactivator complexes [1]. C-terminal binding proteins (CtBPs) are ubiquitous master transcriptional coregulators associated with the Polycomb Repressive Complex 2 (PRC2), with essential functions in the development and oncogenesis of various tumor entities, including breast cancer and prostate cancer (PC) [2,3]. As CtBPs bind to short linear PxDLS-like sequence motifs (SLiMs), the presence of such motifs in tandem on the putative metastasis suppressor RAI2 [4] suggested a previously undiscovered dual interaction with CtBPs. Here, we show that RAI2 induces CtBP polymerization through well-ordered filaments of stacked tetrameric CtBP layers, as illustrated by a high-resolution single particle cryo electron microscopy structure. These filaments are mirrored by RAI2-mediated CtBP nuclear foci and relief of CtBP corepressor function in RAI2-expressing cancer cells. Analyses of a diverse PC patient cohort revealed a substantial decrease in RAI2 in the aggressive subtypes, suggesting a pivotal role of RAI2 in the transition to androgen receptor signaling-independent progression. Taken together, our data demonstrate a previously unknown mechanism of multivalent short linear sequence motif-induced polymerization. As RAI2-like SLiM motifs are found in a wide range of organisms, including pathogenic viruses, our findings serve as a paradigm for diverse functional effects through multivalent interaction-mediated polymerization by disordered proteins in healthy and diseased conditions. The specific properties of these repeated interactions open up new therapeutic opportunities.

References

[1] Lee, T.I. & Young, R.A. Transcriptional regulation and its misregulation in disease. Cell 152, 1237-51 (2013).

[2] Di, L.J. et al. Genome-wide profiles of CtBP link metabolism with genome stability and epithelial reprogramming in breast cancer. Nat Commun 4, 1449 (2013).

[3] Wang, R. et al. Role of transcriptional corepressor CtBP1 in prostate cancer progression. Neoplasia 14, 905-14 (2012).

[4] Werner, S. et al. Suppression of early hematogenous dissemination of human breast cancer cells to bone marrow by retinoic Acid-induced 2. Cancer Discov 5, 506-19 (2015).

Citation Format: Nishit Goradia, Stefan Werner, Edukondalu Mullapudi, Gunhild von Amsberg, Klaus Pantel, Matthias Wilmanns. Master corepressor inactivation through oncogene suppressor RAI2 mediated polymerization [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 3023.
Abstract 3027: p53 aggregation promotes transformation from non-malignant lesions to high-grade serous ovarian carcinomas
Authors: Sartini Sara , Omholt Lexi , Moatamed Neda A. , Soragni Alice
Published: March 2024

High-Grade Serous Ovarian Cancer (HGSOC) is the most common and aggressive gynecological malignancy. It is typically diagnosed in late stages due to lack of diagnostic tests and current therapies available are not efficacious when the tumor is advanced. HGSOC is characterized by ubiquitous loss of functional p53 tumor suppressor protein function, largely due to point mutations that arise very early in carcinogenesis. P53 mutations are detected in pre-neoplastic cells of the fallopian tube mucosa, called p53 signature, and in early precursor lesions called serous tubal intraepithelial carcinoma (STIC). Some mutations promote the misfolding and aggregation of p53, transforming the protein into a cancer-promoting oncogene. Here, we hypothesize that the transition from folded, soluble to aggregated mutant p53 underlies the transformation from a pre-neoplastic lesion (p53 signature, STILs), to STICs and eventually HGSOC. We first set to determine the proportion of cells carrying soluble vs misfolded mutant p53 in fallopian tube tissues collected from n=10 patients undergoing salpingo-oophorectomy. We performed conformation-sensitive staining and quantification of folded and unfolded p53 cases and demonstrated that p53 misfolding, a mandatory precursor to aggregation, is present in STICs and HGSOCs, but notably absent from p53 signatures and surrounding healthy tissue. To better characterize the role of aggregated p53 in the progression of HGSOC, we took advantage of primary human fallopian tube secretory epithelial cells (FTSEC) that mimic early and more advanced stages of carcinogenesis. We utilize the FTSEC cells line FT282, immortalized by introducing hTERT and the aggregation-prone mutant TP53R175H that is found in p53 signature lesions and STILs, as well as the FT282-CCNE1, derived from FT282 cell line with overexpression of CCNE1, representing a more malignant phenotype.We used these models to investigate p53 aggregation, characterize the mutant p53 interactome and test the efficacy of ReACp53, a p53 aggregation-targeting peptide we developed. Our results demonstrated that p53 aggregation is more severe in the FT282-CCNE1 line, with a higher susceptibility to ReACp53 compared to the FT282 cells. Overall, our results demonstrated that aggregation of mutant p53 is a structural defect that can distinguish between pre-malignant and malignant HGSOC lesions and can potentially be targeted, offering a potential window for early intervention in halting ovarian cancer progression.

Citation Format: Sara Sartini, Lexi Omholt, Neda A. Moatamed, Alice Soragni. p53 aggregation promotes transformation from non-malignant lesions to high-grade serous ovarian 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 3027.
Abstract 3031: Overcoming drug resistance in cancer: A paradigm shift with FoxM1 targeting
Authors: Pettersson Martin , Bae Su Jung , Cho Kyu-kwang , Yun Hye-yeoung , Park Hee-Sung
Published: March 2024

In cancer treatment, overcoming drug resistance remains a formidable challenge. The oncogenic transcription factor FoxM1 is widely recognized for its crucial role in promoting drug resistance to various chemotherapeutic and target therapies. Its involvement is attributed to pivotal pathways, encompassing cell cycle progression, angiogenesis, and metastasis. Despite its prominent role in tumor drug resistance, achieving modulation of FoxM1 using small molecule therapeutics has proven elusive. 5-fluorouracil (5-FU), a commonly used chemotherapeutic agent, is known to induce a significant upregulation of FoxM1. In this study, we demonstrate that our novel compound A3 leads to a robust dose-dependent reduction of FoxM1 driven by 5-FU in HT-29 colorectal cancer cells. Notably, exposure of 5-FU-treated HT-29 cells to A3 was shown to promote apoptosis via pro-apoptotic factors, including PUMA. The mechanism of action was further elucidated through a comprehensive analysis of cell signaling molecules. This study presents an innovative approach using small molecule modulators to control FoxM1 levels, providing profound insights for potential therapeutic interventions in cancer treatment.

Citation Format: Martin Pettersson, Su Jung Bae, Kyu-kwang Cho, Hye-yeoung Yun, Hee-Sung Park. Overcoming drug resistance in cancer: A paradigm shift with FoxM1 targeting [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 3031.
Abstract 3074: Regulating lactate levels in triple negative breast cancer presents a promising avenue for therapeutic intervention
Authors: Prasad Chandra Prakash , Umar Sheikh Mohammad , Dev J. R. Arundhathi , Kashyap Akanksha , Rathee Meetu
Published: March 2024

Introduction: Among all subtypes of breast cancer, triple-negative breast cancer (TNBC) is associated with the most unfavorable prognosis, emphasizing the critical need to pinpoint therapeutic targets tailored to TNBCs. Lactate, produced as a by-product of aerobic glycolysis, acts as an oncometabolite and plays a substantial role in promoting tumor aggressiveness, particularly in TNBCs. In our ongoing laboratory investigations, we are actively exploring strategies to mitigate the influence of lactate on TNBCs.

Methods: The experiments utilized two triple-negative breast cancer (TNBC) cell lines, i.e., MDA-MB231 and MDA-MB468. The inhibitors employed in this study are FOXY5, XAV939, PFK15, and Quercetin. Protein expression was examined using Western blotting and Immunofluorescence assays. Trans-well and wound healing assays were employed to assess cell migration and invasion. Colony formation assays (CFU) were utilized to investigate stem cell potential and proliferation. In vivo experiments were conducted using 4T1-orthotopic Balb/c mice.

Results: Using triple-negative breast cancer (TNBC) cells, we have demonstrated the pivotal role of the WNT/β-catenin signaling pathway in promoting the generation of extracellular lactate, thus driving the progression and metastasis of TNBCs. Additionally, we have shown that inhibiting lactate production in TNBC cells, and consequently impeding cell migration and invasion, can be achieved by using recombinant WNT5A or its mimicking hexa-peptide FOXY5. Similarly, employing a β-catenin inhibitor such as XAV939 can yield comparable outcomes in relation to TNBC cells. In an alternative approach, we conducted inhibition experiments targeting PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase), a key regulator of aerobic glycolysis in cancer cells. Our findings indicate that a combined approach involving siPFKL and iPFKFB3 is more promising in suppressing lactate production in TNBCs when compared to the effects of individual iPFKFB3 treatment.

Conclusions: Our present investigations suggest that employing inhibitors targeting the WNT/β-catenin signaling pathway, PFKP, and PFKEB3 could be a promising therapeutic strategy to effectively reduce lactate levels in TNBCs.

Citation Format: Chandra Prakash Prasad, Sheikh Mohammad Umar, Arundhathi Dev J. R., Akanksha Kashyap, Meetu Rathee. Regulating lactate levels in triple negative breast cancer presents a promising avenue for therapeutic intervention [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 3074.
Abstract 3077: Integrin expression during ovarian cancer progression
Authors: Bano Nazia , Schmelz Eva M.
Published: March 2024

During peritoneal dissemination, ovarian cancer cells exfoliate from the primary tumor and aggregate in the peritoneal fluid. This elicits survival signals via changes in mitochondrial morphology and function. As spheroids adhere to secondary sites, mitochondrial function is restored in the form of recovery of mitochondrial morphology, increased mitochondrial membrane potential, glucose uptake and increased proliferation. These findings suggested a mitochondrial phenotypic switch from mitophagy in spheroids to upregulation of mitobiogenesis upon spheroids adhesion to secondary sites. Hence, our objective was to identify critical receptors that facilitate the adhesion of spheroids to secondary sites, focusing on integrins that facilitate cell-extracellular matrix (ECM) attachment and activate downstream signaling cascades. To investigate changes in integrin expression, we mimicked three major stages of ovarian cancer progression by developing adherent stage, spheroid stage and adherent spheroid stage using our mouse ovarian surface epithelial (MOSE) cell lines representing benign (MOSE-E) stage as well as slow-developing (MOSE-L) and fast-developing disease (MOSE-LTICv) in biologically relevant conditions. Using RT qPCR and Western blotting, we determined gene and protein expression levels of integrins and adhesion related genes. Confocal microscopy was used to determine protein localization during ovarian cancer progression. RT qPCR result indicated differential up-regulated expression of integrins in both spheroid and adherent spheroid stage, suggesting the role of different integrins during the stages of ovarian cancer progression. Specifically, we observed a decrease (integrin ɑ5, ɑ6) or increase (ɑ2𝛃;1) of integrins after aggregation and an upregulation of 𝛃;1, α2, αV, α3, α5 and immunomodulatory integrins such as αE and αM in adherent spheroids, indicating their role in mediating specific steps during progression of ovarian cancer from tumorigenesis to metastatic implantation. We also observed increased expression levels of extracellular matrix proteins and their modulators, and cell-to-cell adhesion genes during ovarian cancer progression that was more pronounced in the MOSE-LTICv cells which suggests their association with an aggressive cancer phenotype. Overall, this project will aid in the identification of adhesion targets to develop new anti-metastatic treatments that will reduce mortality in women with ovarian cancer.

Citation Format: Nazia Bano, Eva M. Schmelz. Integrin expression during ovarian cancer progression [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 3077.
Abstract 3079: AMPK-HIF-1α signaling enhances glucose-derived de novoserine biosynthesis to promote glioblastoma growth
Authors: Yun Hye Jin , Lee Jong-Ho
Published: March 2024

Cancer cells undergo cellular adaptation through metabolic reprogrammin to sustain survival and rapid growth under various stress conditions. However, it remains unknown how brain tumors modulate their metabolic flexibility in the naturally serine/glycine (S/G)-deficient brain microenvironment. We used a range of primary/stem-like and established glioblastoma (GBM) cell models in vitro and in vivo. To identify regulatory mechanisms of S/G deprivation induced metabolic flexibility, we employed high throughput RNA-sequencing, transcriptomic analysis, metabolic flux analysis, metabolites analysis, chromatin immunoprecipitation (ChIP), luciferase reporter, nuclear fractionation, cycloheximide-chase, and glucose consumption. The clinical significances were analyzed in the genomic database (GSE4290) and in human GBM specimens. The high throughput RNA-sequencing and transcriptomic analysis demonstrate that the de novo serine synthesis pathway (SSP) and glycolysis are highly activated in GBM cells under S/G deprivation conditions. Mechanistically, S/G deprivation rapidly induces reactive oxygen species (ROS)-mediated AMP-activated protein kinase (AMPK) activation and AMPK dependent hypoxia-inducible factor (HIF)-1α stabilization and transactivation. Activated HIF-1α in turn promotes expression of SSP enzymes phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1), and phosphoserine phosphatase (PSPH). In addition, HIF-1α-induced expression of glycolytic genes (GLUT1, GLUT3, HK2, and PFKFB2) promotes glucose uptake, glycolysis, and glycolytic flux to fuel SSP, leading to elevated de novo serine and glycine biosynthesis, NADPH/NADP+ ratio, and proliferation and survival of GBM cells. Analyses of human GBM specimens reveal that levels of overexpressed PHGDH, PSAT1, and PSPH are positively correlated with levels of AMPK T172 phosphorylation and HIF-1α expression and poor prognosis of GBM patients. Our findings reveal that metabolic stress-enhanced glucose-derived de novo serine biosynthesis is a critical metabolic feature of GBM cells and highlight the potential to target SSP for treating human GBM.

Citation Format: Hye Jin Yun, Jong-Ho Lee. AMPK-HIF-1α signaling enhances glucose-derived de novoserine biosynthesis to promote glioblastoma 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 3079.
Abstract 3082: The liver microenvironment promotes glycolysis in metastatic colorectal cancer by activating her3
Authors: Rathore Moeez Ghani , Curry Kimberly , Boutros Christina , Wang Zhenghe , Winter Jordan , Wang Rui
Published: March 2024

Background: Liver metastasis occurs in ~80% of all metastatic colorectal cancer (mCRC) and the liver has a unique endothelial cell (EC)-rich microenvironment that promotes cancer cell survival. We previously reported that liver ECs secrete soluble factors to promote CRC growth via activating CRC-associated the HER3 signaling pathway. The present study reports that ECs promote mCRC growth by shifting cancer metabolism towards glycolysis and elucidates the involved signaling pathway.

Methods: We performed mass spectrometry to profile the metabolic changes in mCRC treated with/without HER3 inhibition in a syngeneic, orthotopic mCRC model. We also performed Seahorse and lactate ELISA assays to determine the effect of liver EC-secreted factors on CRC metabolism. To further determine the role of HER3 in promoting glycolysis and CRC growth in the liver, we used siRNA silencing of HER3 and pharmacological inhibition of HER3 downstream signaling proteins to identify the key mediator(s) of HER3-induced metabolic shift in CRC.

Results: We determined that HER3 inhibition leads to increased oxidative phosphorylation metabolites (OXPHOS) and decreased glycolysis metabolites in CRC liver metastases. Conditioned medium containing EC-secreted factors decreased oxygen consumption (determined by Seahorse, a readout of OXPHOS) and increased lactate secretion (determined by ELISA, a readout of glycolysis) in CRC. As HER3 inhibition reversed the metabolic shift and increased OXPHOS as an alternative survival strategy, we found that simultaneous inhibition of HER3 and OXPHOS synergistically blocked CRC cell and tumor growth. Lastly, we found that PFK2 is activated by HER3 signaling, determined by phosphorylation, and determined that the HER3-AKT-PFK2 signaling axis is the key mediator of EC-induced metabolic shifts.

Conclusions: We identified that the liver microenvironment activates HER3 and the downstream AKT-PFK2 signaling to promote glycolysis and growth in CRC liver metastases by activating AKT-PFK2-dependent glycolysis. Our findings highlighted the potential of combining HER3 and OXPHOS inhibitions as a potential strategy for treating patients with mCRC.

Citation Format: Moeez Ghani Rathore, Kimberly Curry, Christina Boutros, Zhenghe Wang, Jordan Winter, Rui Wang. The liver microenvironment promotes glycolysis in metastatic colorectal cancer by activating her3 [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 3082.
Abstract 309: AHNAK nucleoprotein 2 tune oxidative stress and resistance to chemotherapy in triple negative breast cancer
Authors: Hao Na
Published: March 2024

Background: Resistance to chemotherapy is a major reason for the poorerprognosis in patients with triple negative breast cancer (TNBC). Growing evidence suggests that oxidative stress induced by reactive oxygen species (ROS) is an important factor in both tumourprogression and responses to chemotherapies, however, many signalling pathways and key molecular underlying the regulation of cancer cells to the ROS metabolism remains unclear.

Aim: (i) To investigate the mechanistic role of AHNAK nucleoprotein 2 (AHNAK2) in TNBC and its links with breast cancer mortality, particularly caused by chemotherapeutic resistance; and (ii) to test a hypothesis that AHNAK2 facilitates the abnormal activation of ROSscavenging system, result in the resistance to chemotherapy in TNBC.

Methods: The expression level of AHNAK2 was determined by immunohistochemical staining and analysed the mRNA expression profiles from The Cancer Genome Atlas of triple negative breast tumors. Roles of AHNAK2 in cancer cell growth, metastasis, chemotherapeutic resistanceand activation of oxygen scavenging systemwere determined by molecular and cell biology methods. A jetPEI nanocarrier was used as the vehicle for anti-AHNAK2 siRNAs in mouse models of cancer therapeutics.

Results: AHNAK2 is a novel triple negative breast cancer target, which is overexpressed in TNBC tissues and significantly associated with clinical pathology of chemotherapeutic resistance and poor prognosis of tumour patients. Mechanistically, AHNAK2 antagonizes the ROS accumulation induced by epirubicinvia activating JAK-STAT signaling pathway and transportingphosphorylated STAT1 & STAT4 to the nucleus to promote the expression of SOD2, a key gene in ROS metabolism.And epirubicin enhanced AHNAK2 expression by induced the accumulation of ROS,which hence the Sp1-driven transcription of AHNAK2,result inde novo resistance. Furthermore, AHNAK2-specific siRNAs encapsulated by jetPEI nanocarriers prominently inhibit tumor growth and epirubicin-induced resistance in vivo.

Conclusions: AHNAK2 plays a key role in resistance to chemotherapyby regulating the oxidative stressand ROS metabolism,representing a future biomarker and therapeutic target of TNBC.

Citation Format: Na Hao. AHNAK nucleoprotein 2 tune oxidative stress and resistance to chemotherapy in triple negative breast 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 309.
Abstract 3091: Discovery of highly selective ligands with unique binding modes for challenging oncology targets using DNA-encoded chemical libraries
Authors: Zhang Ying , Keefe Anthony , Clark Matthew A.
Published: March 2024

DNA-encoded chemical library (DEL) technology enables the efficient screening of large collections of encoded compounds and has emerged as a powerful tool for hit identification in recent years. The technology allows for the multiplexed interrogation of target-ligand binding interactions and results in DEL hits with unusual binding modes and desirable selectivity. This screening methodology is particularly effective for oncology targets. Examples of discovery of highly selective ligands for challenging oncology targets, including kinases, protein-protein interactions, and receptors, will be presented. We will illustrate how DEL campaign strategies directly aim at novel binding modes, and provide molecular details around the uniqueness of these interactions. Furthermore, we will show how these novel binding modes lead to unique pharmacology with direct benefit to patients. Lastly, we will demonstrate how X-Chem’s high-quality encoded libraries enhance the tractability and novelty of chemical equity discovered with the platform.

Citation Format: Ying Zhang, Anthony Keefe, Matthew A. Clark. Discovery of highly selective ligands with unique binding modes for challenging oncology targets using DNA-encoded chemical libraries [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 3091.
Abstract 3116: Bioluminescent 3D tumors with immune cell co-culture for high throughput screening (HTS)
Authors: Chou Fu-Ju , Kamali Samaneh , Gilardi Mara , Walling Brandon , Andar Abhay , Abarca-Heidemann Karin , Mancini Maria
Published: March 2024

In the past decade, cancer immunotherapy has revolutionized the cancer treatment strategy. Immune-oncology (IO) drugs play an important role in treating various malignancies. Immunotherapies work by re-educating and boosting patients’ immune systems to recognize and eliminate cancer cells. The immune co-culture assay provides invaluable information to understand the tumor microenvironment (TME) and observe the immune response after IO drug treatment. Recent advancements in the Champions TumorGraft3D (CTG3D) platform provides more consistent and accurate information for preclinical drug evaluation, but also efficiently simulate the tumor-Immune microenvironment (TIME) and interactions between cancer organoids and immunocyte. However, current analytic approaches in organoid-immune co-culture studies are still highly reliant on flow cytometry and high-resolution confocal imaging endpoints to provide the study outcome. There is a need for a fast and cost-effective co-culture platform to help set up experimental conditions. Easily traceable tags (e.g. bioluminescent and fluorescent) can quickly help establish reliable the effector (E) cell to tumor (T) cell conditions, thus enabling the user to test multiple immune-cell activator or engagers concentrations in a cost-effective and rapid manner. At Champions Oncology we provide stable Luc/GFP-tagged CTG3D models. We have built a reliable CTG3D-immune co-culture platform with various indications (including colon, prostate, gastric, non-small cell lung cancer, and leukemia) which can be used in co-culture with autologous TIL, allogenic TIL and NK cells, thus providing users a fast and cost-effective option to optimize the E:T ratio and monitor the drug performance.

Citation Format: Fu-Ju Chou, Samaneh Kamali, Mara Gilardi, Brandon Walling, Abhay Andar, Karin Abarca-Heidemann, Maria Mancini. Bioluminescent 3D tumors with immune cell co-culture for high throughput screening (HTS) [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 3116.
Abstract 3128: HRA00184-C004, preclinical characterization of a novel tissue factor-targeted antibody-drug conjugate
Authors: Ying Hanxiao , You Lingfeng , He Ning , Xu Shaoyu , Pu Pu , Lin Sophie , Feng Jun , Zhang Zhe , Ye Xin , Hu Min , He Feng
Published: March 2024

The physiological function of tissue factor (TF) is to initiate coagulation cascade by forming complex with plasma Factor VII after vascular injury. For Tisotumab vedotin (Tivdak), a TF targeting antibody-drug conjugate (ADC) approved for the treatment of cervical cancer, bleeding events were prespecified as adverse effects (AEs) of interest, besides ocular toxicity and peripheral neuropathy (known MMAE payload related AE). In order to mitigate the bleeding risk, a next generation of TF-targeting ADC is developed by Hengrui Pharmaceuticals. HRA00184-C004 is a novel ADC comprising a differentiated TF-targeted antibody (HRP07132) conjugated to a proprietary topoisomerase I inhibitor (Dxh, an exatecan derivative) via a protease-cleavable linker. In contrast to most TF-targeting antibodies, the naked HRP07132 does not interfere the interaction between TF and Factor VII or Factor X, has no impact on coagulation function in prothrombin time and thrombin generation assay, therefore reduced bleeding incidence is expected for HRA00184-C004. Meanwhile, HRA00184-C004 exhibited not only potent growth inhibition against multiple TF-expressing cancer cell lines but also bystander-killing effect in vitro. HRA00184-C004 also displayed robust and sustained anti-tumor activities in the PDAC HPAFII (TF high), TNBC MDA-MB-231 (TF moderate) and NSCLC H358 (TF low) TF-expressing xenograft models. Furthermore, in cynomolgus monkey, HRA00184-C004 is well-tolerated over a 6-week period with 12 mg/kg dosing every 3 weeks with acceptable PK profile. Notably, neither coagulation disorder nor skin rash was observed, which are superior to the historical NHP data reported by Tivdak. In conclusion, with the encouraging efficacy in preclinical study and promising safety profiles in NHPs, HRA00184-C004 has demonstrated the best-in-class potential and is scheduled to advance into clinical development.

Citation Format: Hanxiao Ying, Lingfeng You, Ning He, Shaoyu Xu, Pu Pu, Sophie Lin, Jun Feng, Zhe Zhang, Xin Ye, Min Hu, Feng He. HRA00184-C004, preclinical characterization of a novel tissue factor-targeted antibody-drug conjugate [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 3128.
Abstract 313: Benchmarking immune repertoire sequencing technologies
Authors: Maheshwari Shamoni , Morrison Carolyn , Taylor Sarah ,   BCR-SEQC consortium
Published: March 2024

In the past decade, single cell gene expression technologies have transformed basic and translational research and are more frequently found in the toolbox of immunologists alongside the standard bulk B-cell receptor (BCR) sequencing and transcriptome profiling assays. While conventional bulk BCR assays capture the frequency of single chains, single-cell immune profiling reveals the frequency of paired heavy and light chains along with the cells transcriptional profile.

Here we present the results of a single-blind benchmarking experiment conducted in collaboration with the U.S. Food and Drug Administration (FDA) that compares the performance of standard bulk DNA and RNA BCR repertoire sequencing assays, with the 10x Genomics Single Cell Immune Profiling solution. The FDA selected nine unambiguously monoclonal BCR-expressing cell lines to generate a reference mixture that was shared with each study participant, without revealing the input proportions. This reference cell line mixture was also diluted to 10% and 1% with B-cells sorted from peripheral blood mononuclear cells (PBMC) to further test the limits of clonotype detection.

For each experiment, we targeted ~10,000 cells and in aggregate generated a paired gene expression and V(D)J dataset from ~100,000 cells. BCR clonotypes were detected in 78% of the captured cells across all samples profiled. Each of the 9 top unique clonotypes observed in our dataset corresponded to a cell line in the reference mixture, allowing us to decode the mixing percentages of each BCR-expression cell line. In addition, we were able to identify rare cell line clonotypes to as low as 0.01% in the dilution experiments. Furthermore, the multimodal nature of the single cell data also resolved the transcriptional profile of each cell line into its own cluster thus enabling independent verification of clonotype proportions.

In summary, we have generated a resource for the immunology community to evaluate and compare data generated with different repertoire sequencing technologies.

Citation Format: Shamoni Maheshwari, Carolyn Morrison, Sarah Taylor, BCR-SEQC consortium. Benchmarking immune repertoire sequencing technologies [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 313.