bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4162: Peptide-targeted probe identifies pancreatic cancer using optoacoustic imaging
Authors: Sanderson Emma , Husarova Tereza , McNally Lacey
Published: March 2024

Introduction: Advanced pancreatic ductal adenocarcinoma (PDAC) suffers from inadequate early detection, inoperability, and poor outcomes. While tumor identication in an intraoperative setting largely utilize ultrasound, ultrasound suffers from insufficient resolution and lacks the ability to identify molecular features to differentiate tumor from non-tumor. To overcome these limitations, we utilized optoacoustic imaging combined with a pH-sensitive peptide-based probe, V3, to identify pancreatic cancer in orthotopic mouse models.

Methods: The V3 peptide was synthesized using microwave chemistry which resulted in a peptide of >90% purity. The V3 peptide was conjugated to a fluorescent dye and dialyzed to make the V3-750 probe. S2VP10 cancer cell line was plated in a 6-well plate and acclimated to 7.4, 6.8, and 6.6 pH media to mimic a healthy microenvironment and the acidic tumor microenvironment found in pancreas cancer. Cells were treated with 1 micromolar of V3-750 probe for 1 hour, then imaged on the near-infrared fluorescence imager. Treated cells were loaded into tissue mimicking phantoms and imaged using multispectral optoacoustic tomography. Athymic mice were orthotopically implanted with S2VP10 cells and tumor grew to 3 mm. V3-750 was intraveneously injected and mice were imaged 3h after injection with MSOT.

Results: Signal values for the samples displayed a trend that the V3-750 probe has higher signal values in the 6.8 and 6.6 pH as compared to the 7.4 pH. MSOT data supported this trend, with the signal values from the 6.8 and 6.6 pH being greater than the 7.4 pH by 10-fold (p<0.05). Biodistribution demonstrates peak tumor-specific uptake at 3h post injection (3.1 a.u., 0.003 a.u. liver, 0.002 a.u. kidney).

Conclusion: The heightened signal values for the acidic pH’s from the fluorescence imaging and the MSOT suggest that the V3-750 probe can contrast acidic cancer cells from healthy cells because it targets the acidic cancer microenvironment.

Citation Format: Emma Sanderson, Tereza Husarova, Lacey McNally. Peptide-targeted probe identifies pancreatic cancer using optoacoustic imaging [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 4162.
Abstract 4165: Influence of halogens in squaraine dye on optoacoustic signal
Authors: Belligund Kavita , MacCuaig William , Wickizer Carly , Shao Yihan , McNally Lacey R.
Published: March 2024

Optoacoustic imaging has grown in clinical relevance due to inherent advantages in sensitivity, resolution, and contrast, compared to standard modalities, such as fluorescence. However, guiding principles for development of small molecule contrast agents specific for optoacoustic imaging is lacking. This study assesses the influence of specific structural features of small molecule dyes, specifically squaraine dyes, that result in optoacoustic activity. Through computational models, in vitro testing, and in vivo experimentation this study assesses the effects of halogens on small molecule squaraine dyes to increase optoacoustic signal. By using the aid of our computational model, we decorated the squaraine scaffold with heavy halogenation of squaraines increased optoacoustic activity (2.12 a.u. vs. 0.21 a.u., p<0.001). Density functional theory models suggest the origin of increased optoacoustic signal is due to increases in transition dipole moment which manifested experimentally as increased absorbance in the NIR wavelength range and decreased fluorescence quantum yield, respectively. This study provides an insight into the structure-function relationships that lead to guiding principles for designing optoacoustic-specific contrast agents. Further developments of squaraines and other types of optoacoustic agents will further increase relevance of optoacoustic imaging in a clinical setting.

Citation Format: Kavita Belligund, William MacCuaig, Carly Wickizer, Yihan Shao, Lacey R. McNally. Influence of halogens in squaraine dye on optoacoustic signal [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 4165.
Abstract 4178: Non-invasive shortwave infrared imaging of cytotoxic T lymphocyte infiltration for monitoring responses to combination immunotherapy and chemotherapy
Authors: Shah Jay V. , Siebert Jake N. , Zhao Xinyu , He Shuqing , Riman Richard E. , Tan Mei Chee , Pierce Mark C. , Lattime Edmund C. , Ganapathy Vidya , Moghe Prabhas V.
Published: March 2024

Although triple-negative breast cancer (TNBC) can be treated with anti-PD-1 checkpoint immunotherapy in combination with chemotherapy, there remains a challenge in effectively monitoring therapeutic responses. Current non-invasive clinical imaging tools to evaluate response to treatment are reliant upon measurements of tumor volume and may fail to distinguish true progression from increased immune cell infiltration. Invasive biopsy sampling and immunohistochemistry (IHC) can elucidate changes in the immune landscape of treated tumors, but these methods are not conducive to providing real-time information. This study presents shortwave infrared (SWIR) imaging as a potential tool to detect treatment-induced cytotoxic T lymphocyte (CTL) infiltration non-invasively and in real time using rare earth metal-doped nanoparticles encapsulated in human serum albumin nanocomposites (ReANCs). ReANCs were chemically conjugated with anti-CD8α antibodies as targeting ligands to facilitate binding of the nanoprobes to CTLs with high specificity in a syngeneic mouse model of breast cancer. After treating the mice with combination anti-PD-1 and doxorubicin, volumetric analysis of the mammary fat pad tumors did not show any significant impact of treatment compared to single treatment and untreated control mice. However, increased CTL infiltration in the tumors of mice that received combination treatment was detected by in vivo SWIR imaging. CTL infiltration was validated with ex vivo IHC staining, and a monotonic relationship was observed between SWIR fluorescence and CD8 positivity. IHC staining of other immune markers, including CD45, CD3, CD4, and PD-L1, showed that combination treatment may influence in the expression of these markers, presenting additional targets that could be imaged with ReANCs in the future. In conclusion, the increase in SWIR signal from CD8-targeted ReANCs in tumors treated with combination immunotherapy and chemotherapy and the relationship with IHC staining highlight the ability to use SWIR imaging for non-invasive assessment of changes in immune dynamics following treatment.

Citation Format: Jay V. Shah, Jake N. Siebert, Xinyu Zhao, Shuqing He, Richard E. Riman, Mei Chee Tan, Mark C. Pierce, Edmund C. Lattime, Vidya Ganapathy, Prabhas V. Moghe. Non-invasive shortwave infrared imaging of cytotoxic T lymphocyte infiltration for monitoring responses to combination immunotherapy and chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4178.
Abstract 4226: Human-derived hydrogels for 3D models of bone and osteosarcoma organoids and adipose tissue interactions
Authors: Sanchez Cecilia G. , Raz Arman , Lassiter Haley , Frazier Trivia
Published: March 2024

The goal of the study was to determine the metabolic and morphological changes, via high Content Imaging, in 3D models of osteosarcoma cancer cells using human derived hydrogels with different protein composition, Obagel and Obagel ECM. Furthermore, we evaluated the relevance of the adipose tissue microenvironment in Adipose Derived Stem Cells (ADSC) linage and Tumoroids development.

Methods: Adipose derived Stem cells were cultured in a modified 3D human and fat and bone linage differentiation as studied via histology and Immunofluorescence. Osteosarcoma spheroids were cultured in Obatala Sciences’ human-derived hydrogels ObaGel and ObaGelECM and control media. Organoid structure and phenotypic changes were analyzed via live cell imaging on the Incucyte S3 and Nikon high content imaging device. Then, adipocyte- cancer cell crosstalk was evaluated using pooled adipocytes co-cultured with KHOS spheroids. Adipose derived Stem cells were cultured in a modified 3D human derived hydrogel dictates that promotes the differentiation potential toward fat and adipose tissue simultaneously in microenvironments within the organoid. Furthermore, the results demonstrated that ObaGel and ObaGel-ECM 3D cultures can support the growth and proliferation of KHOS tumor spheres during an extended culture period. Metabolic changes and phenotypic changes associated to ObaGel and ObaGel-ECM differentially facilitated tumoroids growth and migratory behavior.

Conclusions: Human derived hydrogels are developed to support 3D culture of Bone and Osteosarcoma cells to recapitulate the phenotypic changes associated with their metastatic potential. The use of human derived 3D culture systems can accelerate the understanding of the role of the microenvironment in bone development and the pathogenesis and progression of Cancers like Osteosarcoma as well as screening for drug development.

Citation Format: Cecilia G. Sanchez, Arman Raz, Haley Lassiter, Trivia Frazier. Human-derived hydrogels for 3D models of bone and osteosarcoma organoids and adipose tissue interactions [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 4226.
Abstract 4236: PDX-derived organoids (PDXO) are valuable tools to unveil the shortcomings of new anti-cancer drug candidates
Authors: Wang Yuhui , Feng Lin , Liu Han , Zhang Junwen , Hu Kaiqiang , Pan Pengwei , He Fang
Published: March 2024

Introduction: Tumor organoids are increasingly being used as predictive preclinical cancer models, as they reflect the original tumor features and patient drug responses. However, systematic comparisons between organoids and traditional 2D cell lines or 3D spheroids are lacking.

Method: We established over 30 PDX-derived organoid (PDXO) models with KRAS mutations and tested three inhibitors targeting the KRAS pathway: VS-6766(RAF/MEK inhibitor), Trametinib (MEK inhibitor), and MRTX1133 (a Phase II drug targeting KRASG12D) against these models.

Results: IC50 results indicated that most models responded to treatment. For some cell lines we detected differential drug responses in 2D vs. 3D testing with 3Dspheroids being more sensitive to inhibitors than 2D cell lines (on average ~10 fold). PDXO models exhibited greater individual differences in drug response, with a subset of models demonstrating no sensitivity to MRTX1133. This is consistent with published results showing that some KRASG12D-mutant PDX models were less sensitive to MRTX11331. Similarly, the clinical trial data for the KRASG12C inhibitor Adagrasib revealed distinctions in the response of patients2. Further analysis with WES (whole exome sequencing) and RNA-sequencing identified potential genotypic mutations and differential expressions of signaling pathways related to drug resistance. Subsequent drug combination testing showed that targeting these pathways could enhance the MRTX1133 efficacy in the resistant PDXO models.

Summary: Our research confirms that drug testing on organoids predicts responses in mouse models and clinical trials. In combination with multi-omics and bioinformatics analysis, the platform can be used for evaluating drug resistances, identifying new targets, developing new drug combination therapies, and ultimately improving the creation of new cancer therapeutics.

Citation Format: Yuhui Wang, Lin Feng, Han Liu, Junwen Zhang, Kaiqiang Hu, Pengwei Pan, Fang He. PDX-derived organoids (PDXO) are valuable tools to unveil the shortcomings of new anti-cancer drug candidates [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 4236.
Abstract 4245: Selection of AAV capsids by evaluating transgene delivery using human organoid models
Authors: Fu Yingying , Qi Zhen , Zuo Zhanguan , Chiang Spencer , Ouyang An , Gao Glory , Guan Shuge , Chen Jin-Qiu (Jessie) , Zhang Rosanna , Wang Cheng
Published: March 2024

Recombinant adeno-associated viruses (AAVs) play a pivotal role in gene therapy, a promising approach aimed at treating various genetic disorders by introducing modified genetic material into cells or tissues. These AAV capsids are utilized as a vector in transferring genetic material into host cells. In discovery research, many AAV serotypes are developed in parallel to identify the optimal subtype for subsequent preclinical and clinical use. However, gene therapies have found limited success in clinical translation, in part, due to variable transgene delivery efficacy between traditional in vitro and in vivo models and human models. This study presents an organoid-based approach that mimics human physiology, as well as organ-specific features and cell diversity within an organ. Organoids are 3D structures derived from stem cells that resemble the cellular architecture and functionality of a specific organ. Compared to conventional 2D cell cultures, organoids provide a more comprehensive modeling of complex cell signaling, cell-cell and cell-extracellular matrix interactions, simulating the complex interplay between various cell types and functions. Within this study, several human-based organoid models were built, cerebral and cardiac, and verified by identifying the expression level of known marker genes through RNA sequencing. Five different AAV serotypes were utilized to evaluate transgene efficacy using each type of organoid model. Expression of delivered GFP transgene within each organoid was then compared to evaluate transduction and select the optimal AAV serotype in comparison to commercial wild-type AAVs. We outline the use of human organoid as an improved preclinical model for selecting AAV serotypes and evaluating transgene delivery efficacy to help provide better guidance for gene therapy development when moving into clinical research.

Citation Format: Yingying Fu, Zhen Qi, Zhanguan Zuo, Spencer Chiang, An Ouyang, Glory Gao, Shuge Guan, Jin-Qiu (Jessie) Chen, Rosanna Zhang, Cheng Wang. Selection of AAV capsids by evaluating transgene delivery using human organoid 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 4245.
Abstract 4254: Genetically engineered human lung organoid models for lung cancer evolution study
Authors: Moon Sungji , Kim Rokhyun , Lee Dakyung , Park Kyunghyuk , Lee Woochan , Lee Seyoon , Park Young Jun , Kim Jongil
Published: March 2024

Initial cellular events in a transitional period from normal to cancer cells, such as transcriptomic or epigenetic dynamics, are poorly understood. Observing these is more challenging than in cancer progression, especially for humans. Mapping this phenomenon in early cancer evolution is crucial, as we can identify targetable molecules to inhibit cancer development at the precancerous stage. Immortalized human cancer cell lines hardly capture these appropriately as they have lost usual cellular characteristics. Normal organoids harbor stem cells and their differentiated cells, reflecting the epithelial physiology of tissues, and are more suitable models for cancer evolution studies. To establish a human cancer development model in vitro, we genetically engineered normal human airway organoids derived from adult stem cells into small cell lung cancer (SCLC). We used CRISPR-Cas9 systems to induce non-homologous end-joining in both TP53 and RB1, the most frequently mutated genes of SCLC. First, we knocked out TP53 of the airway organoids, selected by Nutlin-3a, an MDM2 antagonist, for 3-4 weeks, demonstrating 88-89% editing efficiencies calculated from Sanger sequencing results. Most mutations were one base pair deletion or insertion, which induced frameshift mutation in TP53. They did not show a significant difference from wild-type organoids morphologically at bright-field microscopy. Subsequently, we targeted RB1 on TP53-/- airway organoids to induce SCLC. We used Palbociclib, a CDK4/6 inhibitor, to enrich the mutated cells. They had 67.1% efficiency, and 20.4% had a 7-base pair deletion mutation. In the initial period, they seemed to have similar shapes to TP53-/- organoids. Unexpectedly and interestingly, however, we noticed more rapidly proliferating and disorganized organoids disrupting their original round shapes appeared in 4-5 weeks. The cells were indiscriminately piled up on the airway organoids' apical and basolateral surfaces, indicating their malignant traits. Their chaotic proliferation represented the rapidly progressing clinical feature of SCLC with a median survival of 7-16 months with treatment. This is the first work constructing the SCLC development model from the normal human airway organoid. By sequentially single-cell sequencing this organoid model changing to tumorous condition, we can capture the multi-omics landscape during the initial evolution of SCLC.

Citation Format: Sungji Moon, Rokhyun Kim, Dakyung Lee, Kyunghyuk Park, Woochan Lee, Seyoon Lee, Young Jun Park, Jongil Kim. Genetically engineered human lung organoid models for lung cancer evolution study [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 4254.
Abstract 4259: A novel human model of lung squamous cell carcinoma to deconvolve the cell-intrinsic effects of key dysregulated pathways
Authors: Ogden Julia , Sellers Robert , Oojageer Anthony , Sahoo Sudhakar , Chaturvedi Anshuman , Lopez-Garcia Carlos
Published: March 2024

Tractable, patient relevant models are needed to investigate the essential components of normal cell transformation. We developed a unique in vitro model of early lung squamous cell carcinoma (LUSC) genetic alterations and driver pathway dysregulation using genetically modified primary human bronchial basal cells (hBCs) from three independent donors. The co-operation of ubiquitous and early LUSC alterations (TP53 and CDKN2A loss) and commonly deregulated pathways including squamous differentiation (SOX2), PI3K signaling (PTEN) and the oxidative stress response (KEAP1) was investigated using a combinatorial approach, culminating in eight unique hBC mutants harboring cumulative pathway alterations in a TP53-/-/CDKN2A-/- background. Phenotypes indicative of mutant hBC transformation were investigated using proliferation assays, organotypic cultures and anchorage independent growth assays. Organotypic cultures revealed LUSC pathway alterations to drive dramatic changes in airway histology, ranging from a normal epithelium to severe dysplasia. Furthermore, these analyses confirmed the role of SOX2-overexpression in initiating transformation by inducing squamous cell commitment and anchorage independent growth. We also identified a co-operation of SOX2-overexpression with the oxidative stress response and PI3K pathways to drive more advanced lesions in-vivo and in-vitro. Importantly, we were able to map changes in the expression of immunomodulatory factors with the pathway dysregulation. We demonstrated that this strategy constitute an alternative and patient-relevant system to model LUSC and identify genotype-phenotype correlations of clinical relevance.

Citation Format: Julia Ogden, Robert Sellers, Anthony Oojageer, Sudhakar Sahoo, Anshuman Chaturvedi, Carlos Lopez-Garcia. A novel human model of lung squamous cell carcinoma to deconvolve the cell-intrinsic effects of key dysregulated pathways [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 4259.
Abstract 4272: Disruption of hyaluronan metabolism alters glioma cell proliferation by ligand dependent and independent mechanisms
Authors: Neves Edward R. , Mueller Joseph , Anand Achal , Selting Kimberly , Xu Hui , Remy Roddel , Harley Brendan , Pedron-Haba Sara
Published: March 2024

Hyaluronan (HA) is the main constituent of the brain extracellular matrix and is an essential component of the glioblastoma (GBM) tumor microenvironment. The biosynthesis and catabolism of hyaluronan has multiple roles in tissue architecture and cell signaling. Dysregulation of these mechanisms is crucial in pathological processes such as cancer, inflammation or tissue remodeling. The role of HA is believed to be strongly dependent on its size (molecular weight), location, and cell receptor density and activity. HA is produced intracellularly by HA synthases (HAS1-3) and can also be catabolized endogenously by hyaluronidases (HYAL1-3).The fabrication of controlled microenvironments provided by three dimensional (3D) models helps to elucidate the role of HA signaling in GBM tumors. We exploit here the advantages of these models to understand the role of hyaluronan in the local behavior of GBM after radiation therapy and selected targeted inhibitors. We have established engineered brain tumor biomaterials based on functionalized hydrogels to monitor the response of patient-derived xenograft cell populations with different molecular signatures in combination with microfluidic devices. We have analyzed shifts in metabolism, hyaluronan secretion, as well as hyaluronan synthetic enzymes (HAS) and hyaluronidases (HYAL) activity in an array of patient derived GBM cells. We reveal that endogenous HA plays a role in mitochondrial respiration and cell proliferation in a tumor subtype dependent manner. We also show that HA fragments can enhance tumor metabolism and growth through ligand dependent and independent mechanisms. We provide bioengineered platforms and strategies to predict and stratify tumors in order to achieve more efficient combinatorial treatments targeting endogenous hyaluronan production. This work emphasizes the potential of these preclinical models to predict and accelerate cancer treatments, more relevant in malignancies with limited treatment options.

Citation Format: Edward R. Neves, Joseph Mueller, Achal Anand, Kimberly Selting, Hui Xu, Roddel Remy, Brendan Harley, Sara Pedron-Haba. Disruption of hyaluronan metabolism alters glioma cell proliferation by ligand dependent and independent mechanisms [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 4272.
Abstract 4293: Chromosomal instability causes epigenetic aberrations in cancer
Authors: Agustinus Albert S. , Al-Rawi Duaa , Dameracharla Bhargavi , Raviram Ramya , Jones Bailey S. , Stransky Stephanie , Scipioni Lorenzo , Luebeck Jens , Bona Melody Di , Norkunaite Danguole , Myers Robert M. , Duran Mercedes , Choi Seongmin , Weigelt Britta , Yomtoubian Shira , McPherson Andrew , Toufektchan Eleonore , Keuper Kristina , Mischel Paul S. , Mittal Vivek , Shah Sohrab P. , Maciejowski John , Storchova Zuzana , Gratton Enrico , Ly Peter , Landau Dan , Bakhoum Mathieu F. , Koche Richard P. , Sidoli Simone , Bafna Vineet , David Yael , Bakhoum Samuel F.
Published: March 2024

Chromosomal instability and changes in the epigenome are defining features in most metastatic cancers. Our research links chromosomal missegregation, their containment in micronuclei, and subsequent rupture of the micronuclear envelope to histone post-translational modification aberrations. This relationship was across all tested human and murine cancer cell lines, as well as non-transformed cells. Furthermore, we discovered that the observed changes in histone PTMs were caused by either micronuclear rupture or the persistence of histone PTM status during cell division. Moreover, we revealed substantial chromatin accessibility differences in micronuclei. Notably, there is a distinct bias in chromatin accessibility between promoter regions compared to distal and intergenic regions of the genome, which aligned well with observed changes in histone modifications. The induction of chromosomal instability leads to widespread, heterogeneous, and heritable abnormalities in the epigenetic landscape of cancer cells. Therefore, on top of genomic copy number changes, chromosomal instability causes epigenetic reprogramming, which adds another layer to cancer heterogeneity.

Citation Format: Albert S. Agustinus, Duaa Al-Rawi, Bhargavi Dameracharla, Ramya Raviram, Bailey S. Jones, Stephanie Stransky, Lorenzo Scipioni, Jens Luebeck, Melody Di Bona, Danguole Norkunaite, Robert M. Myers, Mercedes Duran, Seongmin Choi, Britta Weigelt, Shira Yomtoubian, Andrew McPherson, Eleonore Toufektchan, Kristina Keuper, Paul S. Mischel, Vivek Mittal, Sohrab P. Shah, John Maciejowski, Zuzana Storchova, Enrico Gratton, Peter Ly, Dan Landau, Mathieu F. Bakhoum, Richard P. Koche, Simone Sidoli, Vineet Bafna, Yael David, Samuel F. Bakhoum. Chromosomal instability causes epigenetic aberrations 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 4293.
Abstract 4322: Inhibition of ULK and KRASG12C control tumor growth in preclinical models of lung cancer
Authors: Ghazi Phaedra , O'Toole Kayla , Boggaram Sanjana , Scherzer Michael , Bogdan Madhumita , Smith Bryan , Flynn Dan , Kinsey Conan , McMahon Martin
Published: March 2024

Approximately 30% of lung cancers are driven by mutationally activated KRAS. The recent FDA approval of sotorasib, a direct pharmacological inhibitor of KRASG12C, marks a critical milestone in the treatment of this important subset of lung cancer. However, a major obstacle in treating lung cancer is resistance to current therapeutic treatments. In response to cellular stresses such as RAS pathway-targeted therapeutics, RAS-mutated cancer cells have been demonstrated to increase autophagy, an intracellular recycling pathway. However, the mechanism(s) underlying treatment-induced autophagy is not well understood. Here, we demonstrate that KRASG12C-driven lung cancer cells increase autophagy in response to treatment with sotorasib. Currently, the only therapeutics in clinical use to inhibit autophagy are lysosomal inhibitors (hydroxy)chloroquine. High concentrations of these compounds are needed to achieve modest inhibition of autophagy, suggesting that the potency of these compounds may limit clinical responses. ULK kinases are serine/threonine kinases that are necessary for the initiation of autophagy. DCC-3116 is a potent inhibitor of the ULK1/2 protein kinases, master regulators of autophagy. Treatment with sotorasib and DCC-3116 led to superior anti-tumor effects in preclinical models of lung cancer. Additionally, about 30% of lung cancer patients with KRAS mutations also have deletions or inactivating mutations in LKB1, a protein involved in the regulation of nutrient sensing and autophagy. In other KRAS-driven cancers, the LKB1-AMPK-ULK1 signaling axis is a proposed mechanism as to how autophagic flux increases following KRAS pathway inhibition. However, our preliminary data demonstrates that LKB1 is dispensable for sotorasib-induced autophagy, suggesting the mechanism(s) of autophagy induction in KRASG12C-driven lung cancer may be different from those detected in other KRAS mutated cancers. Consequently, we have generated genetically engineered mouse models (GEMMs) of KRASG12C-driven lung cancer in which LKB1 is silenced to further investigate the role of LKB1 in the autophagy response of KRASG12C-driven lung cancers to pathway-targeted blockade of oncogenic KRAS signaling. We have treated KRASG12C-driven GEMMs with sotorasib and/or DCC-3116 to test the sensitivity of lung tumors to these treatment options.

Citation Format: Phaedra Ghazi, Kayla O'Toole, Sanjana Boggaram, Michael Scherzer, Madhumita Bogdan, Bryan Smith, Dan Flynn, Conan Kinsey, Martin McMahon. Inhibition of ULK and KRASG12C control tumor growth in preclinical models of lung 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 4322.
Abstract 4325: CX-4945 (Silmitasertib) induces cell death through impairment of lysosomal utilization in KRAS mutant cholangiocarcinoma cell lines
Authors: Kim Chorong , Lee Da Sol , Han Minwoo , Lee Seonmin , Kim Kyu-pyo , Yoo Changhoon
Published: March 2024

Purpose: Macropinocytosis is a non-selective endocytosis that uptakes extracellular substances such as nutrients and macromolecules into the cells. In KRAS-driven cancer such as pancreatic ductal adenocarcinoma, the macropinocytosis and following lysosomal utilization are enhanced to overcome metabolic stress. In this study, we investigated the role of CK2 in micropinocytosis and the following metabolic process in the KRAS mutant CCA cell line.

Materials and Methods: The BSA uptake indicating micropinocytosis was performed by flow cytometry using the HuCCT1, cholangiocarcinoma cell line harboring KRAS mutation. To validate macropinosome, the Rab7 and LAMP2 were labeled and analyzed via immunocytochemistry and Western blot. The CX-4945, CK2 inhibitor, was used to investigate the role of CK2 in micropinocytosis and following lysosomal metabolism.

Results: The HuCCT1, a KRAS mutant CCA cell line, showed micropinocytosis. Although CX-4945, a CK2 inhibitor, induced morphological changes accompanied by accumulation of intracellular vacuoles and cell death rate, the level of micropinocytosis was not changed. The vacuoles accumulated in the cell were identified as late macropinosomes representing Rab7 before fusion with lysosomes. In addition, the CX-4945 suppressed LAMP2 expression following inhibition of the Akt-mTOR signaling pathway which interrupts mature macropinosome and lysosomal metabolic utilization.

Conclusion: These results support the utilization of micropinocytosis as an energy source even in CCA cell line harboring KRAS mutation. The inhibition of CK2, the regulator of macropinosome maturation, by CX-4945, alters lysosome dependent metabolism and leads to cell death in KRAS mutant CCA cell lines.

Citation Format: Chorong Kim, Da Sol Lee, Minwoo Han, Seonmin Lee, Kyu-pyo Kim, Changhoon Yoo. CX-4945 (Silmitasertib) induces cell death through impairment of lysosomal utilization in KRAS mutant cholangiocarcinoma cell lines [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 4325.
Abstract 4350: Characterization of PUF60 splicing activity that is essential for the survival of triple negative breast cancer
Authors: Tankka Alexandra , Gomberg Trent , Zhou Cathy , Pham Vivian , Yeo Gene
Published: March 2024

Alternative splicing driven by RNA-binding proteins (RBPs) plays a significant role in TNBC, however, pharmacological modulators of core spliceosomal proteins can disrupt early stages of spliceosome assembly and lead to toxic effects in normal tissues, limiting their utility as safe therapeutics. Here, we used in vivo CRISPR-Cas9 screening to evaluate RBP dependencies in TNBC and identified 17 splicing regulatory candidates that are essential in TNBC but dispensable for normal cell growth and survival. Within this cohort, the alternative splicing factor, PUF60, was the most significantly upregulated candidate in patient TNBC tumors. We found that depletion of PUF60 induced apoptosis in human TNBC cell lines and impeded xenografted tumor growth in vivo while upregulation of PUF60 expression increased TNBC cellular proliferation. Although PUF60 has been shown to also bind DNA and influence transcription, TNBC cells constructed with a previously characterized mutation (L140P) required for 3’ splice site recognition displayed significantly decreased cellular proliferation compared to cells containing wild type PUF60. Integrated eCLIP and KD RNA-sequencing revealed that PUF60 selectively regulates the splicing of mRNAs in TNBC that contribute to essential processes including DNA repair, replication and cell cycle progression. RNA-seq following L140P-mediated inhibition of 3’ ss recognition revealed a cohort of splicing events within genes enriched for similar processes following knockdown of endogenous PUF60 including cell cycle and histone modification. Altogether, our study highlights a critical role for PUF60-mediated splicing in the maintenance of genomic integrity in TNBC as well as the therapeutic potential of identifying and targeting splicing effector RBPs only responsible for TNBC survival.

Citation Format: Alexandra Tankka, Trent Gomberg, Cathy Zhou, Vivian Pham, Gene Yeo. Characterization of PUF60 splicing activity that is essential for the survival of 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 4350.
Abstract 4351: Differential dependency mapping of chimeric RNAs across cancer reveals a new landscape of functional fusion transcripts
Authors: Lalani Samir , Gadh Sehajroop , Elfman Justin , Singh Sandeep , Li Hui
Published: March 2024

Chimeric RNAs are RNA transcripts containing sequences originating from multiple distinct genetic loci and can form through a variety of mechanisms such as DNA rearrangement, cis-splicing of adjacent genes, or RNA trans-splicing. Chimeras have long been known to be a hallmark of cancer and due to their unique properties are promising targets for precision medicine. Appropriately, fusion proteins transcribed from chimeras such as BCR-ABL1 and TPM3-NTRK1 have been shown to be effective targets. Due to these past successes, there exists an opportunity to identify new chimeras as therapeutic targets. While modern chimera prediction software allows for the fast and accurate identification of chimeric RNAs from RNA sequencing data, investigations separating therapeutically relevant transcripts from “transcriptional noise” remain lacking. In this study we performed an in-silico functional screen of chimeras across cell lines representing a wide variety of cancers. We used state-of-the-art chimeric RNA prediction software to create a database of chimeric RNAs across 1017 cell lines from The Cancer Cell Line Encyclopedia. For each chimera we assessed factors such as frequency, recurrence, breakpoint coordinates, frame, and coding potential. To identify specific functional transcripts, we integrated publicly available shRNA knockdown data with our predictions and developed an in-silico functional analysis pipeline comparing differential knockdown effects between chimera and corresponding parental transcripts. For each transcript we assessed the average fold change of chimera-mapping probes, a function score defined as the difference between the fold change of chimera-mapping and parent-specific probes, and a p-value assessing the confidence of our functional score. From our initial screen of 127,819 transcripts, we identified 1088 high-confidence functional chimeras. We successfully identified nearly all known functional chimeras screened including PAX3-FOXO1, EWSR1-FLI1, and TCF3-PBX1. We also identified previously unknown chimeras that that we predict have a function in cell growth and proliferation. Overall, the results of our study reveal a new landscape of functionally relevant chimeric transcripts in cancer. Follow-up studies can further investigate these transcripts to determine their potential as therapeutic targets.

Citation Format: Samir Lalani, Sehajroop Gadh, Justin Elfman, Sandeep Singh, Hui Li. Differential dependency mapping of chimeric RNAs across cancer reveals a new landscape of functional fusion transcripts [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 4351.
Abstract 4365: Mechanistic effects of N361 glycosylation in epidermal growth factor receptor protein
Authors: Lam Dennis , Liberchuk Ariel N. , Arroyo Brandon , Wolfe Andrew L.
Published: March 2024

Glycosylation is a tightly regulated post-translational modification where structured sugar groups are added to protein amino acid backbones, most commonly on asparagine for N-glycosylation. The glycosylation-regulating metabolic enzyme uridine diphosphate-glucose pyrophosphorylase 2 (UGP2) is required in oncogene-transformed cancer cells, but not in normal cells. UGP2 heavily regulates glycosylation of epidermal growth factor receptor (EGFR) at asparagine 361 (N361), a key site near the ligand binding domain. EGFR is a transmembrane tyrosine kinase receptor that promotes cell proliferation via growth cascades. Mutations and amplifications in EGFR account for a significant fraction of non-small cell lung carcinomas and breast adenocarcinoma, with L858R being the most common oncogenic hotspot. Other glycosylation sites on EGFR have been implicated in ligand binding, dimerization, and phosphorylation of downstream targets. Molecular dynamic simulations suggested that N361 may be important for dimerization or ligand binding. However, the functional relevance of how glycosylation of EGFR at N361 impacts EGFR protein and cellular behaviors remains unclear. To determine the functional relevance of glycosylation at N361, we created glycosylation-defective EGFR N361A mutant, with or without an additional oncogenic EGFR L858R mutant. We expressed these constructs in MCF10A and 293T cells, which do not have pre-existing activation of this pathway. Immunofluorescence and flow cytometry showed that the mutants were each well expressed at the cell membrane. Proximity ligation assays measuring dimerization of EGFR in cells showed that EGFR N361A greatly increased dimerization relative to wildtype controls. Loss of glycosylation of EGFR N361 impacted cell viability when grown in dose courses of its high affinity ligand EGF or lower affinity ligand amphiregulin. Furthermore, EGFR N361A desensitized cells expressing the oncogenic EGFR L858R to inhibitors targeting the extracellular domain, possibly because the mutation alters the antibody binding interface. N361A sensitized EGFR L858R to inhibitors targeting the cytoplasmic domain of EGFR. These findings help us understand the intricate relationship between EGFR and N-glycosylation, reinforcing the critical functional relevance of post-translational modifications on oncogenes.

Citation Format: Dennis Lam, Ariel N. Liberchuk, Brandon Arroyo, Andrew L. Wolfe. Mechanistic effects of N361 glycosylation in epidermal growth factor receptor protein [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 4365.
Abstract 4378: Identification of novel TXNIP-dependent antitumor pathways in breast cancer
Authors: Singh Jasvinder , Sah Bindeshwar , Clarke Robert , Liu Liang
Published: March 2024

Thioredoxin-interacting protein (TXNIP) plays a critical role in glucose metabolism and redox signaling. TXNIP has recently emerged as a potent tumor suppressor in various cancer models. In previous studies, we found TXNIP was activated by UNC0642, an inhibitor of histone methyltransferase, in MDA-MB-231 breast cancer cells. TXNIP activation was coupled with significant suppression of cell proliferation and tumor growth. In contrast, TXNIP knockdown increased MDA-MB-231 cell proliferation and in tumor growth and metastasis in vivo. TXNIP-reconstitution in TXNIP-deficient HCC1954 cells reduced cell proliferation and migration coupled with increased reactive oxygen species production. TXNIP-reconstitution also significantly decreased mitochondrial respiration, mitochondrial membrane potential and glycolysis in HCC1954 cells. To understand the mechanisms underlying TXNIP’s antitumor activity, we performed co-immunoprecipitation and proteomic analyses to uncover TXNIP-interacting proteins. We identified calpastatin (CAST) as a novel TXNIP-interacting protein in breast cancer cells. CAST is an endogenous inhibitor of calpains, a family of calcium-activated cysteine proteases. Intriguingly, CAST association with TXNIP was readily detectable in TXNIP-low, but not in TXNIP-high breast cancer cells. The disassociation of CAST from TXNIP in TXNIP-high cells might allow CAST to inhibit calpain activity to augment TXNIP’s antitumor function. Through RNAseq analysis, we identified a positive correlation between TXNIP expression and IL-24 activation in various breast cancer cells, which were also confirmed by western blotting. IL-24 has emerged as an important cancer therapy target because of its role in promoting cancer-specific apoptosis. Our data also demonstrated that TXNIP upregulated the level of IL-24 and downregulated STAT-3. Moreover, TXNIP exerts multifaceted effects on BC development through the IL24-STAT3 signaling axis, deepening our understanding of its complex role in the disease. Taken together, these studies identified novel TNXIP-dependent pathways and their potential new mechanisms in breast cancer pathogenesis with significant clinical implications.

Citation Format: Jasvinder Singh, Bindeshwar Sah, Robert Clarke, Liang Liu. Identification of novel TXNIP-dependent antitumor pathways 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 4378.
Abstract 4388: Tumor-associated RhoA mutants interact with effectors in both GDP- and GTP-bound states
Authors: Lin Yuan , Ramelot Theresa A. , Senyuz Simge , Gursoy Attila , Jang Hyunbum , Nussinov Ruth , Keskin Ozlem , Zheng Yi
Published: March 2024

RhoA is the founding member of the Ras-homology (Rho) small GTPase family and is a master regulator for multiple cell functions. Recurrent RhoA mutations have been identified in several human cancers, particularly in leukemia/lymphoma and gastric cancer. Intriguingly, both gain-of-function and loss-of-function mutations of RhoA are present, suggesting that RhoA GTPase may have a more sophisticated role in cancer and requires more rigorous investigations. Here, we have focused on two of the gain-of-function RhoA mutations identified in Adult T cell Leukemia/lymphoma (ATLL) at residue A161 (A161P and A161V) and aim to reveal the underlying mechanistic basis for their function by biochemical and structural analyses. We found that in contrast to conventional gain-of-function RhoA mutants such as RhoAG14V and RhoAQ63L which affect the GTP-hydrolysis activity, RhoAA161P and RhoAA161Vare both fast-cycling with drastically increased nucleotide dissociation and association rates, but only slightly reduced GTP-hydrolysis activity. We solved the crystal structures of GDP-bound RhoAA161P and RhoAA161V and saw that while RhoAA161P displays impaired solvent-mediated interactions to the bound nucleotide, RhoAA161V has a more exposed nucleotide binding pocket compared to RhoAWT and RhoAA161P. Interestingly, RhoAA161P and RhoAA161Vcan interact with effector targets in the GDP-bound states. Further 1H-15N HSQC NMR study provides evidence of the active population in GDP-bound RhoAA161V. Molecule dynamics (MD) simulations show that the dynamic properties of RhoA switch regions are affected differently by the two mutations. Thus, RhoAA161V and RhoAA161P are fast-cycling mutants with distinct mechanisms, and both likely endow their GDP-bound state towards an active conformation. These findings provide a better understanding of the oncogenic role of RhoA mutations in cancer and shine light on how changes in RhoA protein dynamic properties caused by mutations may affect its function.

Citation Format: Yuan Lin, Theresa A. Ramelot, Simge Senyuz, Attila Gursoy, Hyunbum Jang, Ruth Nussinov, Ozlem Keskin, Yi Zheng. Tumor-associated RhoA mutants interact with effectors in both GDP- and GTP-bound states [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 4388.
Abstract 4390: IGFBP-6 is induced by progesterone to regulate progesterone receptor expression in breast cancer cells
Authors: Lariz Francisco Javier , Botero Pacha , Houston Kevin
Published: March 2024

Insulin-like growth factor binding protein-6 (IGFBP-6) is induced by progesterone (P4) in T-47D breast cancer cells. IGFBP-6 regulates insulin-like growth factor 2 (IGF2) by sequestering it to inhibit receptor binding and extend its extracellular half-life. IGF2 binds to insulin-like growth factor 1 receptor or insulin receptor A to promote breast cancer cell proliferation and survival. Progesterone is an ovarian steroid hormone which has complex roles in breast cancer through the actions of progesterone receptor A and B (PR-A and B). Progesterone antagonizes the actions of estradiol (E2), another steroid hormone which promotes proliferation and survival in breast cancer. T-47D cells treated with both P4 and E2 exhibit significantly reduced proliferation compared to treatment with E2 alone. To better understand the role of IGFBP-6 in progesterone signaling, T-47D cells which over-express IGFBP-6 were treated with P4, E2, or both. Overexpressing cells exhibited stronger antagonism compared to controls when co-treated with P4 and E2. RNA-interference was performed to knockdown IGFBP-6 induction in the presence of P4 and E2. Knockdown of IGFBP-6 in the presence of P4 and E2 decreases the antagonistic effects of P4. Expression of both PR-A and PR-B decreases by about 50% when IGFBP-6 is knocked down. These results indicate that IGFBP-6 increases PR expression in response to P4 stimulation.

Citation Format: Francisco Javier Lariz, Pacha Botero, Kevin Houston. IGFBP-6 is induced by progesterone to regulate progesterone receptor expression in breast 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 4390.