bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 419: Evolutionary trajectories of complex genome rearrangements in osteosarcoma
Authors: Ciriano Isidro Cortes , Valle-Inclán Jose Espejo , de Noon Solange , Trevers Katherine , Elrick Hillary , Flanagan Adrienne M.
Published: March 2024

Whole-genome sequencing (WGS) studies have revealed that osteosarcoma genomes are riddled by remarkably intricate forms of structural variants (SV), collectively known as complex genomic rearrangements (CGR). Yet, most CGR detected in osteosarcoma remain unexplained, hinting at the possibility of undiscovered mechanisms that might lead to novel therapeutic strategies. To elucidate the mechanisms underpinning cancer genome complexity in osteosarcomas and the downstream consequences of CGR during tumor evolution, we uniformly processed exiting osteosarcoma WGS data sets and performed high-depth (>120x) multi-regional short- and long-read WGS for hundreds of osteosarcomas. We found that whole genome doubling and diverse types of CGR are frequent clonal events in most osteosarcomas. Through the integration of multi-regional WGS data, we discovered that in tumors with whole-genome doubling, cancer genome evolution and oncogene amplification are the consequence of multi-generational CGR triggered by whole-genome doubling and involving multiple mechanisms, especially chromothripsis and breakage-fusion-bridge cycles. By contrast, in tumors without whole-genome doubling, biallelic inactivation of TP53 and oncogene amplification frequently occur as part of a cascade of genomic instability initiated by a single double-strand break upstream of the TP53 locus. This process results in clonal diversification and rapid tumor growth suggestive of punctuated evolution. In addition, we found that subclonal CGRs are also frequent events. Indeed, clonal expansions triggered by the acquisition of subclonal CGR often colonize distant tumor regions, suggesting that CGR act as subclonal driver events. Finally, we show that some recurrent genomic alterations are prognostic of poor survival, which we validate in independent cohorts.

In sum, using WGS data, we have elucidated the mechanisms underpinning the complexity and evolution of osteosarcoma genomes. Our results indicate that diverse types of CGR, such as chromothripsis, are triggered by whole genome doubling and occur throughout tumor evolution, which primes the cancer genome for ongoing genomic instability, rapid karyotype evolution and clonal diversification. These results have implications for our understanding of the molecular basis of intra-tumor heterogeneity and drug resistance in osteosarcomas and other cancers driven by genomic instability.

Citation Format: Isidro Cortes Ciriano, Jose Espejo Valle-Inclán, Solange de Noon, Katherine Trevers, Hillary Elrick, Adrienne M. Flanagan. Evolutionary trajectories of complex genome rearrangements in osteosarcoma [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 419.
Abstract 4220: Combined light sheet and multispectral imaging facilitates the detection of tumor-associated tertiary lymphoid structures in the KP NSCLC model
Authors: O'Brien Nils , Appelt Birte , Moerbitz Marie-Lena , Mueller Joerg P. , Herting Frank , Umaña Pablo , Murgia Claudio , Colombetti Sara , Pöschinger Thomas
Published: March 2024

Tertiary lymphoid structures (TLS) have recently emerged as a predictive biomarker in cancer immunotherapy. Their consideration in preclinical drug development is impeded by rare and transient appearance and random distribution, making them difficult to analyze with conventional sampling methods. We applied whole-organ tissue clearing and 3D staining of B and T cell clusters to comprehensively identify TLS in a KP GEMM of NSCLC using 3D Light Sheet Fluorescence Microscopy (3D LSFM). To further investigate cellular composition of TLS we performed 3D LSFM-educated sectioning of relevant ROIs and cyclic immunofluorescence of the same samples. 3D LSFM facilitated correlation of tumor features and location with T cell infiltration as well as the analysis of number, size and location of TLS. The majority of TLS were located in proximity to bronchi instead of intratumorally. LSFM-guided multiplex immunofluorescence revealed further details on cellular composition and maturation status of TLS. This combination of 3D and 2D imaging modalities offers a holistic approach for the identification of TLS in whole murine tumors or organs. The technology could help studying effects of immunotherapy on de novo formation or maturation of TLS.

Citation Format: Nils O'Brien, Birte Appelt, Marie-Lena Moerbitz, Joerg P. Mueller, Frank Herting, Pablo Umaña, Claudio Murgia, Sara Colombetti, Thomas Pöschinger. Combined light sheet and multispectral imaging facilitates the detection of tumor-associated tertiary lymphoid structures in the KP NSCLC model [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 4220.
Abstract 4221: Characterization and comparison of hypoxia inducing factors on tumor growth and metastasis in 2D and 3D tumor models
Authors: McDonald James , Chan Leo Li-Ying , Kessel Sarah , Lin Bo , Weingarten Paul
Published: March 2024

The monocarboxylic acid transporter 4 (Mct-4), a downstream biomarker of hypoxia inducing factor (HIF)-1α, is involved in the cellular response to hypoxia, as indicated by the hypoxic response element in its promoter region. Using a tumorsphere assay as an in vitro 3-dimensional (3D) model generated using 384-well ultra-low attachment (ULA) plates for cell proliferation analysis using a plate-based image cytometer, we identify a hypoxic response in the tumorsphere model that is distinct from that of cells grown under 2-dimensional (2D) normoxic conditions and demonstrate a key role for Mct-4 in enabling 3D growth. The tumorsphere model yields evidence of an essential role for Mct-4 in multiple cell lines which were genetically modified to underexpress and overexpress Mct-4, evidence not apparent in a standard 2D model of growth in the same cell lines. In addition, we identify the effects of overexpressing Mct-4 in cancer cell migration using a transwell chamber assay. We also show that the response to hypoxia may be circumvented by transfection with a CMV promoter driven Mct-4, which confers constitutive 3D growth, wherein tumorsphere growth inhibition by small molecule HIF-1α inhibitors is mitigated. Finally, we demonstrate quantifiable gene/protein expression differences between 2D and 3D cancer models based on the normoxic and hypoxic conditions. Therefore, the tumorsphere 3D model generated using 384-well ULA plates in combination with high-throughput image cytometer is demonstrated to provide a convenient, robust, and reproducible tool and method for the elucidation of mechanisms of action underlying tumor growth and migration in the hypoxic tumor microenvironment.

Citation Format: James McDonald, Leo Li-Ying Chan, Sarah Kessel, Bo Lin, Paul Weingarten. Characterization and comparison of hypoxia inducing factors on tumor growth and metastasis in 2D and 3D tumor models [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 4221.
Abstract 424: FDX2-KO induces global down-regulation of iron-sulfur cluster-containing proteins and senescence-like growth arrest or death in ovarian cancer cells
Authors: Miyahara Shuko , Ohuchi Mai , Nomura Miyuki , Hayashi Kayoko , Shimada Muneaki , Yaegashi Nobuo , Tanuma Nobuhiro
Published: March 2024

Iron-Sulfur clusters (Fe-S) are synthetized in mainly mitochondria and binds to various proteins (Fe-S proteins). Fe-S proteins play important roles in various cellular functions including energy metabolism, nucleic acid synthesis or redox homeostasis. However, roles of Fe-S clusters in cancer cells are not fully understood. Here, we report that loss of Fe-S biosynthesis causes proliferation arrest with DNA-damage response or cell death depend on TP53 status in ovarian cancer (OVC) cells. CRISPR-screening of all metabolism-related genes identified that Fe-S biosynthesis is essential for OVC proliferation. We chose one of the Fe-S biosynthesis factors, FDX2, and developed a cell line that can maintain FDX2 expression doxycycline (Dox) -dependently. In these cells, FDX2-loss induced irreversible growth arrest. FDX2-KO cells also showed enlarged and flatten morphology, up-regulation of SASP factors, and DNA damage through p53/p21 pathway activation. These were all senescence like phenotypes. To know these mechanisms, we performed proteome analysis and found that FDX2-loss caused global but differential post-transcriptional down-regulation of Fe-S proteins, in turn perturbing mitochondrial respiration, iron-regulation and redox homeostasis. These results all associated with DNA damage. Furthermore, we found that FDX2-KO induced caspase dependent cell death in TP53-KO condition instead of growth arrests. These results suggest that Fe-S cluster biosynthesis is significant in OVC proliferation and fate of FDX2-KO cells depends on TP53 status (senescence or cell death). FDX2 or Fe-S biosynthesis may be a new therapeutic target of OVC.

Citation Format: Shuko Miyahara, Mai Ohuchi, Miyuki Nomura, Kayoko Hayashi, Muneaki Shimada, Nobuo Yaegashi, Nobuhiro Tanuma. FDX2-KO induces global down-regulation of iron-sulfur cluster-containing proteins and senescence-like growth arrest or death in ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 424.
Abstract 4252: Evaluation of NXI-101 in non-small cell lung cancer (NSCLC) using patient-derived organotypic tumor spheroids (PDOTS): Targeting a novel tumorigenic pathway
Authors: Vail Julia , Maeng Hyung Gun , Jang Je Woong , Boo Kyungjin , Youn Je-In , Martin Alyssa , Goldman Aaron , Yoon Kyoung Wan , Perricone Michael A.
Published: March 2024

ONCOKINE-1 (OK-1), a soluble protein that is released by tumor cells, is implicated in the resistance of immunotherapy. Here we evaluated the efficacy of NXI-101, a monoclonal antibody that binds and neutralizes OK-1, in a model of patient-derived organotypic tumor spheroids (PDOTS) derived from non-small cell lung carcinoma (NSCLC) patients. NSCLC PDOTS were treated with NXI-101 and the effects of the drug were evaluated on day 3 post-treatment. Cytotoxicity was measured by Hoechst/Propidium Iodide staining and quantified by measuring the area-weighted percentage of cell death. Significant cytotoxicity was observed in 2 out of 10 patient tumors (20%) as a monotherapy and 4 out of 10 (40%) when used in combination with atezolizumab. The therapeutic response for both treatments was positively correlated (p<0.05) with the level of OK-1 expression, determined by immunohistochemistry using tissue sections taken on the day of tumor specimen collection. NXI-101 alone and in combination with atezolizumab had immunomodulatory effects that were associated with cytotoxic response. For example, transcriptomic analysis, Nanostring IO 360 Panel, indicated that the drug treatments resulted in the inhibition of TGF-β pathways and stimulated a number of immune pathways in 3 of the 4 responsive PDOTS. These pathways include interferon-α, JAK-STAT, and dendritic cell activation markers. We also observed evidence of activation of these same pathways in 2 of the 6 non-responsive PDOTS. These data support the prospects of NXI-101 as a first-in-class therapeutic for the treatment of NSCLC when used alone or in combination with an immune checkpoint inhibitor. Moreover, the findings highlight the PDOTS platform's substantial value in capturing patient-specific tumor response variability and providing robust support for the mechanistic evaluation of cancer therapeutics.

Citation Format: Julia Vail, Hyung Gun Maeng, Je Woong Jang, Kyungjin Boo, Je-In Youn, Alyssa Martin, Aaron Goldman, Kyoung Wan Yoon, Michael A. Perricone. Evaluation of NXI-101 in non-small cell lung cancer (NSCLC) using patient-derived organotypic tumor spheroids (PDOTS): Targeting a novel tumorigenic pathway [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 4252.
Abstract 4275: Targeting tumor extracellular matrix to improve response to therapy
Authors: Pearce Oliver Matthew
Published: March 2024

Introduction. Building on our previous findings that tumor extracellular matrix (ECM) composition associates with immune cell phenotype (Pearce et al, Cancer Disco, 2018), and ECM can directly educate tumor-associated immune phenotypes (Puttock et al, Nat Commun, 2023), we present here the identification of specific ECM components that associate with tumor immune phenotype, and discuss our approach to target these components as a way to improve anti-tumor immunity and immunotherapy response.

Methods. We use a combination of transcriptomics, proteomics, glycomics, and spatial tissue analysis to identify correlations between ECM composition and the immune landscape in breast and ovarian cancers. Correlations are tested within a decellularized tumor model made from patient samples which allow immune cell movement, interaction with ECM, and tumor killing to be measured in real-time.

Results. Through our analysis pipeline we have identified a specific ECM molecule that appears to regulate the spatial location of tumor infiltrating CD8 T-cell populations via post-translational modifications that are present on the molecule. Deletion of this molecule in injectable orthotopic murine models dramatically alters the immune landscape within the tumor. Within an in vitro decellularized tumor tissue model the in situ deletion of the ECM molecule drives tumors into an inflamed phenotype which is more responsive to cell-based cancer therapy.

Conclusions. Tumor ECM can direct immunity in two ways. These are educating or stimulating immune cells to develop specific phenotypes that tend to be immunosuppressive, and secondly directing the movement of cells within the tissue. These immunosupressive effects can be overcome through small modifications to ECM structure, indicating therapeutics that make subtle changes to ECM composition may be used in combination with current cell-based therapies to improve tumor response.

Citation Format: Oliver Matthew Pearce. Targeting tumor extracellular matrix to improve response to therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4275.
Abstract 4282: Crosstalk between the immune system and the extracellular matrix in the context of melanoma and aging
Authors: Harper Elizabeth I. , Chhabra Yash , Carey Alexis , Rocha Murilo Ramos , Hüser Laura , Wang Vania , Dixit Agrani , Weeraratna Ashani
Published: March 2024

Aging is an independent prognostic factor for the diagnosis of melanoma. The extracellular matrix of the skin has well-documented changes with age, such as the loss of HAPLN1, which can lead to differential metastasis in aged individuals. The aged microenvironment can contribute to the phenotype switch, where aged individuals have smaller primary tumors but more metastases, i.e., going instead of growing. The extracellular matrix (ECM) has been shown to modulate the immune response either through direct binding of ECM proteins or through mechanotransduction, but it is unclear how aging affects this crosstalk. Our hypothesis is that the ECM can modulate immune response to melanoma and affect response to immune checkpoint blockade (ICB) therapy. Melanoma cells cultured on cell derived matrices (CDMs) from young and aged human fibroblasts show differential levels of expression of PD-L1, a ligand that binds to PD-1 on T cells to induce T cell exhaustion. Antibodies targeting PD-1/PD-L1 are one of the main ICB treatments available to melanoma. We are also investigating T cell motility on young and aged CDMs, as well as correlating T cell infiltration in murine tumors with ECM signatures as identified by second harmonic generation microscopy. Therefore, we have compiled evidence that aging affects the crosstalk between the extracellular matrix and the immune system, providing a new horizon for therapeutic improvement of response in older patients.

Citation Format: Elizabeth I. Harper, Yash Chhabra, Alexis Carey, Murilo Ramos Rocha, Laura Hüser, Vania Wang, Agrani Dixit, Ashani Weeraratna. Crosstalk between the immune system and the extracellular matrix in the context of melanoma and aging [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 4282.
Abstract 430: Precision diet and lipid modulating agents to induce lipid oxidation in breast cancer
Authors: Yavari Mahsa , Torpey Jordan , Sabatier Marie , Carmona Alanis , Szylo Krystina , Flores Midori , Palma Mario , Fraser Cameron , Liang Yanshan , Hui Sheng Tony , Zeleznik Oana , Eliassen Heather , Ubellacker Jessalyn
Published: March 2024

Metastasis is the primary cause of breast cancer-related mortality, and understanding the intricate relationship between the tumor microenvironment, lipid metabolism, and metastasis is crucial for preventing cancer progression. Previous work from our group demonstrated that high exogenous levels of oleic acid, a monounsaturated fatty acid (MUFA), in the lymph node environment protects melanoma cells from ferroptosis. Ferroptosis is an iron-dependent form of cell death induced by accumulation of lipid peroxides, which are commonly formed from polyunsaturated fatty acids (PUFAs), but not MUFAs. Here, we hypothesized that increasing dietary PUFAs would alter the exogeneous lipid content of the lymph node environment, thus rendering breast cancer cells more sensitive to ferroptosis. To test this, we determined the in vitro sensitivity of 4T1 triple negative breast cancer cells to ferroptosis when treated with various PUFAs and MUFAs. We also assessed the impact of various PUFA and MUFA fatty acid-enriched diets on breast cancer metastasis and ferroptosis resistance in the lymph nodes in vivo. In concordance with previously published work, our results demonstrated that pharmacological induction of ferroptosis in vitro increased lipid oxidation and was mitigated by fatty acid treatments with higher MUFA compared to PUFA content. In vivo, cancer cells in lymph nodes of mice on palm oil diets had lower lipid oxidation compared to cancer cells from tumors in the mammary fat pad. These findings support the hypothesis that dietary lipids can alter ferroptosis response and the extent of lipid oxidation in breast cancer cells within lymph nodes. Our research has the potential to further reveal mechanistic connections between dietary lipids, the lymphatic microenvironment, and breast cancer progression.

Citation Format: Mahsa Yavari, Jordan Torpey, Marie Sabatier, Alanis Carmona, Krystina Szylo, Midori Flores, Mario Palma, Cameron Fraser, Yanshan Liang, Sheng Tony Hui, Oana Zeleznik, Heather Eliassen, Jessalyn Ubellacker. Precision diet and lipid modulating agents to induce lipid oxidation in 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 430.
Abstract 4308: Same-section spatial multiomic analyses using MICS technology for investigating the dynamics of the tumor microenvironment
Authors: Park Dongju , Neil Emily , Hennessey Rebecca C. , DiBuono Michael , Lafayette Hanna , Lloyd Erica , Lo Hsinyi , Femel Julia , Makrigiorgos Alex , Lu Shaina , Lee John , Soliman Sameh , Mangiardi Dominic , Praveen Paurush , Staubach Fabian , Hindman Ryan , Rothmann Thomas , Santos Telma , Borbe Stefan , Meyer Hansueli , Wantenaar Tanya , Wang Jinling , Müller Werner , Pinard Robert , Bosio Andreas
Published: March 2024

The recent increase in image-based, spatially-resolved technologies enables researchers to profile the tumor microenvironment (TME) by capturing gene expression profiles within tissue sections. However, a significant limitation of these technologies is the lack of ability to resolve protein and RNA information in the same section, as well as conveniently analyze multimodal data sets. Here, we report a spatial RNA detection method, RNAsky, using Miltenyi Biotec’s MACSima™ Platform as an automated, multiomic approach. Our method integrates spatial proteomics and transcriptomics data to provide in-depth profiling with single-cell resolution on the same tissue section. We demonstrate these capabilities by characterizing key immune-oncology markers across normal and diseased tissues using our specialized MACS® iQ View analysis software. MACS iQ View provides fast segmentation and intuitive gating and clustering strategies to simultaneously assess protein and RNA data. We investigated the impact of clustering using protein, RNA, or the combination of both to evaluate the contribution of different information modalities on TME spatial dynamics. This cutting-edge approach will enable the identification of valuable parameters and new cell types, furthering the discovery and development of predictive and prognostic biomarkers.

Citation Format: Dongju Park, Emily Neil, Rebecca C. Hennessey, Michael DiBuono, Hanna Lafayette, Erica Lloyd, Hsinyi Lo, Julia Femel, Alex Makrigiorgos, Shaina Lu, John Lee, Sameh Soliman, Dominic Mangiardi, Paurush Praveen, Fabian Staubach, Ryan Hindman, Thomas Rothmann, Telma Santos, Stefan Borbe, Hansueli Meyer, Tanya Wantenaar, Jinling Wang, Werner Müller, Robert Pinard, Andreas Bosio. Same-section spatial multiomic analyses using MICS technology for investigating the dynamics of the tumor microenvironment [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 4308.
Abstract 4318: CD24 enhances cisplatin resistance in ovarian cancer by suppressing autophagy
Authors: Chen Yuh-Ling , Hong Tse-Ming , Wen Yu-Yi
Published: March 2024

Ovarian cancer is one of the most common gynecological cancers with the highest mortality rate. Most patients are diagnosed with advanced disease and will relapse due to chemoresistance. CD24, a highly glycosylated GPI-anchored membrane protein. Recent studies have shown that CD24 expression is significantly up-regulated in ovarian cancer and is associated with chemotherapy resistance, and CD24 also plays an inhibitory role in tumor immunity by binding to Siglec-10 on macrophages. However, the molecular mechanisms of how CD24 affects chemotherapy resistance in ovarian cancer remain unclear. In this study, we used shRNAs to knockdown CD24 and explore the impact and mechanism of CD24 on the chemotherapy resistance of ovarian cancer cells. The results showed that knockdown of CD24 in ovarian cancer cells SKOV3 and TOV21G significantly reduced cell proliferation and arrested the cell cycle in S phase. Knockdown of CD24 also significantly increased the sensitivity of ovarian cancer cells to cisplatin but had no effect on paclitaxol treatment. Further experiments showed that knockdown of CD24 increased cisplatin sensitivity by promoting autophagy-induced cell death. In addition, Gene Set Enrichment Analysis (GSEA) was used to analyze the correlation between the expression of CD24 and its biological functions in the TCGA ovarian cancer dataset. The results showed that CD24 is mostly correlated to cell proliferation and autophagy-related pathways in ovarian cancers. Further mouse xenograft tumor transplantation experiments also confirmed that knockdown of CD24 reduced tumor size and increased sensitivity to cisplatin treatment by increasing cell autophagy. Taken together, these results indicate that CD24 regulates cell growth and chemoresistance in ovarian cancer cells and that inhibition of CD24 may be helpful in the treatment of ovarian cancer.

Citation Format: Yuh-Ling Chen, Tse-Ming Hong, Yu-Yi Wen. CD24 enhances cisplatin resistance in ovarian cancer by suppressing autophagy [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 4318.
Abstract 4321: The oncogenic DMTF1β splice variant promotes autophagy-dependent cancer cell motility
Authors: Tschan Mario P. , Xu Jun , Bill Anna M.
Published: March 2024

The oncogenic isoform DMTF1β, which is generated by alternative splicing of the DMTF1 tumor suppressor gene is associated with increased cell proliferation and tumorigenesis. Despite these correlative findings little is known about the DMTF1β mechanism of action. In order to decipher the oncogenic DMTF1β signaling pathway, we performed RNA sequencing of DMTF1β depleted prostate cancer cells and analyzed the DMTF1β-specific protein interactome in these cells. Interestingly, autophagy-related pathways belonged to the most downregulated networks in DMTF1β knockdown cells. Moreover, we found that predominantly DMTF1β, but not a protein lacking the β-specific domain interacts with the key autophagy ULK1-ATG13 protein complex. These interactions were confirmed by co-immunoprecipitation and proximity ligation assays. Importantly, DMTF1β binding to ULK1 stabilized this key autophagy protein. Accordingly, knocking down DMTF1β significantly lowered autophagic flux in breast and prostate cancer cells as assessed by LC3-dependent and independent autophagy assays. Since published data show an important role for autophagy in migration and invasion of cancer cells, we next investigated whether DMTF1β is regulating these cellular processes. Indeed, DMTF1β depleted breast and prostate cancer cells displayed significantly decreased wound closure, trans-well invasion and metastatic foci formation in a zebra fish xenograft model. Importantly, pharmacological inhibition of ULK1 kinase activity or knocking down ULK1 reversed DMTF1β-mediated cancer cell migration. In agreement, decreased cell motility in DMTF1β knockdown cancer cells could be rescued by ectopic expression of the VPS34 complex member Beclin-1. In summary, we identified a novel DMTF1β-ULK1 signaling pathway that activates aberrant cancer cell migration most probably by increasing basal cell autophagy. Targeting autophagy using ULK1 inhibitors may represent a promising strategy to lower metastatic activity of tumor cells.

Citation Format: Mario P. Tschan, Jun Xu, Anna M. Bill. The oncogenic DMTF1β splice variant promotes autophagy-dependent cancer cell motility [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 4321.
Abstract 4324: IL6ST increases sorafenib-resistance in hepatocellular carcinoma by regulating BECN1-mediated mitophagic cell death
Authors: Ma Hwan , Roh Yoon-Seok
Published: March 2024

Introduction: Sorafenib resistance is often seen in patients with late-stage hepatocellular carcinoma (HCC) and it is a serious concern for the treatment of these patients. Lethal mitophagy plays an important role in anti-tumorigenic mechanisms by inducing mitochondrial dysfunction and mitophagic cell death. Sorafenib resistance is associated with the activation of IL6ST signaling, inhibiting mitochondrial quality. However, how lethal mitophagy regulates sorafenib resistance through IL6ST and mitochondrial dysfunction in HCC is not fully understood.

Experimental procedures: Hepatocyte-specific TAK1 deleted (TAK1ΔHep) and TAK1/IL6STΔHep were used for in vivo study. Histology, immunoblots, qPCR, microscopy, FACS, and seahorse bioanalyzer were used for analyzing liver and tumor tissues and Hep3B cells.

Results: We identified that sorafenib suppressed IL6ST signaling and that IL6ST signaling was activated in sorafenib-resistant HCC. The number and maximum size of spontaneous HCC in 9-month-old TAK1ΔHep mice were decreased when hepatocyte IL6ST was additionally deleted. Expression of pro-carcinogenic H-19, c-SRC, and MDM2 was increased in 1-month-old TAK1/IL6STΔHep mice compared to TAK1ΔHep mice. An additional IL6ST deletion induced decreases in mitochondrial membrane potential and glycolysis, suggesting that IL6ST protects against mitochondrial dysfunction. Interestingly, IL6ST deletion increases PRKN and BECN1 interaction and translocation to normal mitochondria and subsequent degradation, hereafter referred to as lethal mitophagy. Furthermore, inhibition of BECN1 restores IL6ST deletion-induced lethal mitophagy in HCC, suggesting that IL6ST-mediated lethal mitophagy is dependent on BECN1. Mechanistically, IL6ST inhibited BECN1-lethal mitophagy through STAT3(S705) phosphorylation independently of mTORC1 activation. Notably, enhanced BECN1 through a combination of sorafenib and inhibition of IL6ST increased mitophagic cell death, reducing sorafenib resistance and tumor growth. In addition, enhanced lethal mitophagy through inhibition of GP130 effectively killed sorafenib-resistant HCCs by silencing sorafenib resistance. We further defined the relationship between IL6ST, BECN1, and PRKN expression and sorafenib resistance in terms of HCC patients, RNA sequencing datasets.

Conclusion:  These results provide the importance of IL6ST in the development of sorafenib resistance and could be an innovative treatment strategy for sorafenib-resistant patients by regulating the IL6ST/STAT3/BECN1 axis-regulated lethal mitophagy.

Citation Format: Hwan Ma, Yoon-Seok Roh. IL6ST increases sorafenib-resistance in hepatocellular carcinoma by regulating BECN1-mediated mitophagic cell death [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 4324.
Abstract 4341: Non canonical mitochondrial hotspot mutations in cancer
Authors: Boscenco Sonia , Tait-Mulder Jaqueline , Kim Minsoo , Tang Cerise , Zucker Mark , Park Tricia , Wei Wei , Chinnery Patrick F. , Gammage Payam A. , Reznik Ed
Published: March 2024

The vast majority of recurrent somatic mutations arising in tumors affect protein-coding genes in the nuclear genome. Here, through population-scale analysis of 15,619 whole tumor genomes, we report the discovery of highly recurrent mutations affecting both the small (12S, MT - RNR1) and large (16S, MT - RNR2) RNA subunits of the mitoribosome. In total, we identified 70 recurrent, hotspot mutations affecting either subunit of the mitoribosome. Compared to non-hotspot positions, rRNA hotspots preferentially affect positions participating in Watson-Crick base pairing, and tend to arise at positions under strong purifying selection in the germline. Consistent with their putative selection, hotspot rRNA alleles arose at higher heteroplasmy than silent mutations under neutral selection. Transcriptional and metabolomic profiling of the m.1227G>A MT - RNR1 hotspot revealed a heteroplasmic dosage-dependent shift in energy generation towards glycolysis. Mutations disrupting the mitoribosome therefore represent a novel class of functional mutations under positive selection in cancer genomes.

Citation Format: Sonia Boscenco, Jaqueline Tait-Mulder, Minsoo Kim, Cerise Tang, Mark Zucker, Tricia Park, Wei Wei, Patrick F. Chinnery, Payam A. Gammage, Ed Reznik. Non canonical mitochondrial hotspot mutations 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 4341.
Abstract 436: Extracellular glutathione catabolism as an alternative cyst(e)ine source in cancer
Authors: Hecht Fabio , Zocchi Marco , Tuttle Emily , Asantewaa Gloria , Scales TashJaé , Alimohammadi Fatemeh , Ward Nathan , DeNicola Gina , Harris Isaac
Published: March 2024

Glutathione (GSH) is the most abundant antioxidant in the human body and plays essential roles in physiology and disease. Unchecked oxidative stress is involved in tumor initiation and progression. Paradoxically, clinical and laboratory data show that ROS-scavenging molecules such as glutathione not only fail to impede tumor growth but, in many cases, accelerate it. Still, preliminary data from our lab show that genetically ablating tumors’ ability to synthesize GSH does not affect their growth. This finding suggests that the favorable influence of glutathione on tumor growth may arise from external sources. Yet, the mechanism cancer cells employ to take advantage of extracellular glutathione in this context is unknown. Here, we show that γ-glutamyl-transpeptidase 1 (GGT1), an enzyme that localizes on the outer side of the cell’s membrane, can break down extracellular GSH, yielding glutamate and the dipeptide cysteinylglycine. Cysteinylglycine can be further metabolized, generating cysteine and glycine to the cells. We found that the addition of exogenous GSH, as well as cysteinylglycine, can mitigate cell death, proliferation arrest, and oxidative stress caused by cystine depletion. The overexpression of human GGT1 in cancer cells significantly reduces the amount of glutathione needed to rescue cell fitness. Accordingly, pharmacologic inhibition of GGT1 activity prevented GSH's ability to rescue cell proliferation. Moreover, an unbiased pharmacological screening revealed that GGT1-mediated cysteine acquisition renders cancer cells resistant to inhibitors of cystine uptake and utilization, supporting the role of GGT1 as a source of cyst(e)ine. Our findings reveal that cancer cells can access cysteine, a vital yet scarce amino acid, through GGT1's ability to break down glutathione, which is present in remarkably high concentrations within the tumor microenvironment. Although the downstream proteins involved in the transport and utilization of GSH breakdown products are yet to be identified, we anticipate this pathway has the potential to uncover novel therapeutic avenues for cancer treatment by targeting alternative pathways of nutrient acquisition that are potentially implicated in cancer progression and therapy resistance.

Citation Format: Fabio Hecht, Marco Zocchi, Emily Tuttle, Gloria Asantewaa, TashJaé Scales, Fatemeh Alimohammadi, Nathan Ward, Gina DeNicola, Isaac Harris. Extracellular glutathione catabolism as an alternative cyst(e)ine source 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 436.
Abstract 4386: Androgen hormone signaling regulates the association between the hippo pathway component YAP1 and RNA binding protein NPM1
Authors: Dwead Abdulrahman M. , Al-Mathkour Marwah M. , Cinar Bekir
Published: March 2024

Prostate cancer represents a major health concern for men in the United States and globally, disproportionately affecting men of African descent. Despite recent advances, the precise etiology and molecular mechanisms underpinning fatal prostate cancer in humans remain inadequately understood. RNA binding proteins (RBPs) regulate various aspects of RNA biology, and when their regulation is disrupted, they can lead to human diseases, including cancer. However, the comprehensive alterations in RBP-mRNA interactions in prostate cancer have not been explored. In this study, we investigated the dynamics of RBPs and their interactions with polyadenylated mRNA in the castration-sensitive LNCaP and castration-resistant C4-2 prostate cancer cell lines. Using an enhanced RNA interactome capture assay in combination with mass spectrometry, we discovered that LNCaP and C4-2 cells showed distinct RBP-mRNA interaction patterns in steady-state growth conditions and in response to androgen treatment. Additionally, we investigated the interplay between the oncogene Yes-associated protein 1 (YAP1) and the RNA-binding protein nucleophosmin (NPM1). We identified NPM1 as a component of the YAP1 protein complex through mass spectrometry-based proteomics. Our proximity ligation assay demonstrated that androgen signaling could regulate the interaction between YAP1 and NPM1. Also, our GST-pulldown revealed that NPM1 binds to the proline-rich domain of YAP1 in LNCaP cells, while the WW/SH3 domain of YAP1 mediates the interaction with NPM1 in C4-2 cells. Genetic and pharmacological inhibition of NPM1 activity reduced prostate cancer cell growth in culture by modulating cell cycle progression. Furthermore, our computational and immunological analysis of prostate tumor tissues indicated that the upregulation of NPM1 might be associated with the clinical advancement of human prostate cancer. These findings underscore the critical role of the molecular functional connection between YAP1 and RBPs and the global changes in RBP and RNA interaction patterns, in the progression and recurrence of prostate cancer.

Citation Format: Abdulrahman M. Dwead, Marwah M. Al-Mathkour, Bekir Cinar. Androgen hormone signaling regulates the association between the hippo pathway component YAP1 and RNA binding protein NPM1 [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 4386.
Abstract 4391: The UBE2F-CRL5Asb11-DIRAS2 axis is an oncogene and tumor suppressor cascade in pancreatic cancer cells
Authors: Sun Yi , Chang Yu , Che Qian , Li Hua , Xu Jie , Tan Mingjia , Xiong Xiufang
Published: March 2024

UBE2F is a neddylation E2 that couples with RBX2/SAG E3 to promote CUL-5 neddylation and Cullin-RING ligase-5 (CRL5) activation. Whether and how UBE2F regulates pancreatic tumorigenesis is previously unknown. Here we showed that UBE2F is growth essential for pancreatic cancer cells with KRAS mutant in culture. In KrasG12D mouse PDAC model, Ube2f deletion suppresses cerulein-induced pancreatitis, and progression of acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN). Mechanistically, Ube2f deletion inactivates the Mapk-c-Myc signal pathways via blocking ubiquitylation and degradation of Diras2, which is a new substrate of Cul5Asb11 E3 ligase. Biologically, DIRAS2 suppresses growth and survival of pancreatic cancer cells with KRAS mutant, and Diras2 deletion largely rescued the phenotypes induced by Ube2f deletion, indicating its causal role. Collectively, Ube2f or Diras2 plays a tumor-promoting or tumor-suppressive role in KrasG12D PDAC model, respectively. The Ube2f-CRL5Asb11 axis could serve as a valid target for pancreatic cancer, whereas the levels of UBE2F or DIRAS2 may serve as prognostic biomarkers for PDAC patients.

Citation Format: Yi Sun, Yu Chang, Qian Che, Hua Li, Jie Xu, Mingjia Tan, Xiufang Xiong. The UBE2F-CRL5Asb11-DIRAS2 axis is an oncogene and tumor suppressor cascade in pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4391.
Abstract 4430: Metabolic reprogramming by ASPSCR1::TFE3 and VCP
Authors: Patel Rahi , Pozner Amir , Dunlap Kyle , Carroll Lara , Brown Jeff , Ducker Greg , Jones Kevin
Published: March 2024

Alveolar soft part sarcoma (ASPS) is a lethal soft tissue malignancy typically incident among adolescents and young adults, distinguished by the t(X;17) ASPSCR1::TFE3 fusion gene (AT3). Conventional chemotherapy has shown limited efficacy against ASPS, necessitating exploration of alternative strategies. Previous investigations have shown the pivotal role of the fusion gene in sarcomagenesis in mice, particularly in lactate-rich environments. This study aims to elucidate the metabolic alterations actuated by the AT3 fusion gene, both in vitro and in vivo. To model in vitro metabolic profiles, ASPS (ASPS-1) and renal cell carcinoma (FU-UR1) cell lines, each expressing AT3, were employed. Utilizing siRNA-mediated gene silencing, AT3 and VCP, a cofactor, were targeted in vitro. Metabolomic profiling through GC-MS analyzed the impact of depletion of AT3 or its transcriptional co-factor, VCP. C13-labeled metabolites were introduced and traced in vitro and in vivo to enhance comprehension of carbon utilization in aerobic respiration. While metabolic activity and O2 consumption differed between cell lines, inhibition of AT3 and VCP led to similar metabolite alterations. Significant changes in amino acid metabolism, notably glutamate, aspartate, and alanine were observed in both cell lines. There was notable diversion of glucose carbon utilization away from typical entry into the tricarboxylic acid (TCA) cycle, with minimal carbon incorporation beyond alpha-ketoglutarate. Despite prior data indicating heightened mitophagy activity in AT3 driven cells, inhibition of lysosomal activity did not yield significant changes in cellular respiration or O2 consumption.

Citation Format: Rahi Patel, Amir Pozner, Kyle Dunlap, Lara Carroll, Jeff Brown, Greg Ducker, Kevin Jones. Metabolic reprogramming by ASPSCR1::TFE3 and VCP [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 4430.
Abstract 4447: ZC3H18: A novel regulator of NAD+ metabolism in high-grade serous ovarian cancer
Authors: Wilson Benjamin , Wilson Natalie , Chen Xianfeng , Zhang Shuwen , Wang Chen , Karnitz Larry M. , Kaufmann Scott H. , Kanakkanthara Arun
Published: March 2024

ZC3H18 is a multifunctional regulator of gene expression that is lost in a subset of high-grade serous ovarian cancers (HGSOC). Our prior studies have established that the loss of ZC3H18 diminishes BRCA1 levels, leading to the disruption of homologous recombination DNA repair (HR) and heightened sensitivity of HGSOC cells to PARP inhibitors. Here we show an additional facet to ZC3H18's impact, showing that its loss also induces a reprogramming of energy metabolism in HGSOC. Our findings demonstrate that the depletion of ZC3H18 results in reduced mRNA and protein levels of the NAD+-biosynthetic enzyme, NMNAT1, in HGSOC cells. Mechanistic studies uncovered that ZC3H18 occupies the NMNAT1 promoter, facilitating NMNAT1 transcription by recruiting CDK12 onto the promoter. Consistent with this mechanism, loss of ZC3H18 led to a reduction in CDK12 levels on the NMNAT1 promoter, diminishing NMNAT1 abundance and subsequently causing a decline in cellular NAD levels. These observations unveil a previously unknown role for ZC3H18 in HGSOC, shedding light on its involvement in the regulation of energy metabolism. Importantly, our findings provide valuable insights into potential therapeutic pathways that leverage the metabolic changes linked to ZC3H18 deficiency.

Citation Format: Benjamin Wilson, Natalie Wilson, Xianfeng Chen, Shuwen Zhang, Chen Wang, Larry M. Karnitz, Scott H. Kaufmann, Arun Kanakkanthara. ZC3H18: A novel regulator of NAD+ metabolism in high-grade serous ovarian 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 4447.
Abstract 4455: Metabolic stress induces a double positive feedback loop between AMPK and p62 conferring dual activation of AMPK and NRF2 to synergize antioxidant defense
Authors: Choi Eun-Ji , Oh Hyun-Taek , Lee Seon-Hyeong , Zhang Chen-Song , Li Mengqi , Kim Soo-Youl , Park Sunghyouk , Chang Tong-Shin , Lee Byung-Hoon , Lin Sheng-Cai , Jeon Sang-Min
Published: March 2024

Co-occurring mutations in KEAP1 in LKB1-mutant NSCLC activate NRF2 to compensate losing LKB1-AMPK activity during metabolic adaptation and survival. Here, we investigated the regulatory crosstalk between LKB1-AMPK and KEAP1-NRF2 pathways during metabolic stress. We found that metabolic stress activates NRF2 through the expression and phosphorylation of p62, causing the autophagic degradation of KEAP1. Intriguingly, the induction of p62 during metabolic stress is also required to activate AMPK by promoting AXIN-LKB1-AMPK complex formation and recruiting it to the lysosomal membrane. Importantly, the p62-driven dual-activation of AMPK and NRF2 was critical for tumour growth by synergizing antioxidant defences. In turn, the induction of p62 also required LKB1-AMPK activity, suggesting a double positive feedback loop between AMPK and p62. Mechanistically, the increase in lysosomal pH caused by low glucose metabolism and AMPK-dependent reduction of proton generation induced PP2A-dependent dephosphorylation of TFEB/TFE3 which increased the expression of p62. The increase of ROS caused by metabolic stress induced lysosomal MCOLN1-Ca2+ dependent activation of TAK1 which increased p62 phosphorylation. Protons provided by lactic acid abrogated all the effects caused by metabolic stress. This positive feedback loop between AMPK and p62 that activates AMPK and NRF2 can potentially explain why co-occurring mutations in LKB1 and KEAP1 occur and further provide promising therapeutic strategies for lung cancer.

Citation Format: Eun-Ji Choi, Hyun-Taek Oh, Seon-Hyeong Lee, Chen-Song Zhang, Mengqi Li, Soo-Youl Kim, Sunghyouk Park, Tong-Shin Chang, Byung-Hoon Lee, Sheng-Cai Lin, Sang-Min Jeon. Metabolic stress induces a double positive feedback loop between AMPK and p62 conferring dual activation of AMPK and NRF2 to synergize antioxidant defense [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 4455.