bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 2943: Investigation of microsatellite instability in RNA compared to DNA, using microsatellite targeted, anchored multiplex PCR and NGS
Authors: Rogge Ryan , Patterson Taylor , Navetta Alicia , Legare Dhruti 
Published: March 2024

Abstract
MSI measurements are typically made on DNA, but it would be useful to make similar measurements from RNA or total nucleic acid inputs (TNA), so that MSI determinations could be made in parallel to interrogation of the RNA. Doing so requires characterizations of microsatellite lengths from those which are transcribed and untranscribed, because both will be detected following cDNA creation. Microsatellite instability (MSI) occurs when small unit repeats of DNA undergo incorrect replication resulting in the growth or contraction of the number of repeats in the newly synthesized DNA. This accumulation of errors at microsatellite DNA is usually mitigated by the mismatch repair (MMR) pathway which can correct these errors. MSI is of interest because it serves as a phenotypic readout of the status of the MMR machinery, and MSI status can correlate to tumor progression and response to agents that regulate these cellular mechanisms. We investigated the status of microsatellites using Anchored Multiplex PCR (AMP™) targeted panels (RUO) and compared results from TNA inputs using an AMP MSI module and both VARIANTPlex™ chemistry to interrogate DNA, and FUSIONPlex™ chemistry to interrogate both RNA + DNA. Our results indicate differences in the diversity of lengths of microsatellites present in RNA, and the resulting stability calls which would be made at those sites. Microsatellite length diversity was highly dependent on the genomic context of the microsatellite. Microsatellites located within transcripts showed increased length diversity when examining the RNA + DNA results compared to DNA alone. Microsatellites located in intergenic regions, which are unlikely to be transcribed, showed no increase in diversity of lengths when comparing the RNA + DNA results to DNA alone. While these results suggest that MSI determinations could be made from RNA or RNA + DNA NGS data, the unique behavior of microsatellites in RNA is likely to require additional investigations and unique data analysis techniques to correctly categorize microsatellites as stable or unstable.
Confidential - Company Proprietary
Citation Format: Ryan Rogge, Taylor Patterson, Alicia Navetta, Dhruti Legare. Investigation of microsatellite instability in RNA compared to DNA, using microsatellite targeted, anchored multiplex PCR and NGS [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 2943.
Abstract 2944: A high-throughput screening system for identifying therapeutic targets in immuno-oncology
Authors: Parikh Dippal , Choi Soon Youn , Pyo Ki Eun , Lee Hong-Pyo , Han Kyuho 
Published: March 2024

Abstract
INTRODUCTION: Over the last decade, cancer immunotherapy has revolutionized cancer treatment. However, more than half of patients don’t respond, and responders often lack lasting effects. Understanding how genetic variants in cancer cells influence their interaction with immune cells, especially T cells, is crucial to uncover underlying mechanisms. Genetic screens are powerful for uncovering responsible genes for various phenotypes, but challenges in obtaining ample samples to set up a proper immune and cancer co-culture system have limited their use for identifying immune modulators in cancer: primary mouse T cells expressing CD3 against a specific antigen (typically albumin) need to be extracted from transgenic mice and only syngeneic mouse cancer cell lines that match these T cells can be paired, restricting the utilization of human T cells and human cancer cells for this co-culture screening setup.
METHODS: Here, we present a highly scalable co-culture system that enables genome-wide CRISPR screens on a pair of human primary T cells and any allogeneic human cancer cell lines. This system utilizes the expression of a membrane-targeted protein in mammalian cells, named as ImBridge, which directly engages and activates CD3+ immune cells. This mechanism bypasses the need for matched MHC on target cancer cells.
RESULTS: We validated activation of CD3+ immune cells and selective cancer cell cytotoxicity by ImBridge. A genome-scale CRISPR screen utilizing the co-culture system unveiled known immune-oncology targets including PD-L1 and PTPN2 and revealed MEDiC_01 as a novel therapeutic target. Genetic loss of MEDiC_01 in cancer cells significantly sensitized them to cytotoxic cells, effectively eliminating cancers in the presence of human PBMCs.
CONCLUSION: We demonstrated a scalable and efficient approach for conducting high-throughput functional screens to uncover genes that modulate interactions between effector T cells and target cancer cells. This can facilitate the identification of new immuno-oncology therapeutic targets, insights into underlying mechanisms, and genetic factors influencing immune susceptibility and evasion.
Citation Format: Dippal Parikh, Soon Youn Choi, Ki Eun Pyo, Hong-Pyo Lee, Kyuho Han. A high-throughput screening system for identifying therapeutic targets in immuno-oncology [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 2944.
Abstract 3008: Loss of Rab25 accompanied by NRAS activation cooperatively mediates the tumorigenic transformation in melanoma
Authors: Kandel Samikshya , Rao Krishna 
Published: March 2024

Abstract
Background: Ras oncogene activation is a common and often essential event in cancer development, including melanoma. Ras mutation is present in 20-30% of melanomas and remains an undrugged target. NRAS mutation is the most frequent Ras isoform activated in melanoma, with more than 80% cases of Q61 substitutions, and is closely associated with tumor aggressiveness. Rab25 is a ubiquitously expressed small GTPase protein with a context-dependent role in cancer pathophysiology. Its role remains undiscovered in the case of melanoma. Rab25 deep deletion can be seen in portions of melanoma in databases such as the Cancer Genome Atlas and Cbioportal. Interestingly, Rab25 has a consensus sequence homologous with the binding site of Q61 mutant Ras. We propose that the loss of Rab25 in melanocytes accompanied by Ras oncogenic activation are significant events in melanoma development, and restoration of Rab25 can potentially inhibit constitutively activated N-RAS; Rab25 serves as a tumor suppressor in melanoma genesis.
Objectives: To discover the role of Rab25 in melanoma, we studied Rab25 expression via mRNA and protein levels in different melanoma cell lines and screened biopsy-proven melanoma samples through immunohistochemistry in 61 different melanoma sections. Further, to see the tumor suppressive mechanism of Rab25 in vitro, we transduced selected cell lines Rab25.
Results: There is an 87% and 62.7% reduction in Rab25 mRNA level in NRASQ61R(SKMEL-2) and NRASQ61L type (WM1366) melanoma cell lines, respectively, also reflected in Rab25 protein level, and statistically significant at p-value &lt;0.005, using student’s T-test. Further, Rab25, prominent in adjacent normal tissues, was significantly lost in biopsy-proven melanoma tissues, with an H-score ranging from 13.28 to 91.77% and 0.00 to-11.24 % for normal skin tissue and melanoma section, respectively, and a negative correlation of -0.350, indicating a promising association of Rab25 loss with the progression of melanoma.
Conclusion: There is a loss of Rab25 in NRAS mutant melanoma with cancer progression. We saw a significant increase in Rab25 expression among the transduced sublines of NRAS mutant melanoma cell lines at the mRNA level, which will be further assessed through western blotting, cell proliferation assay, trans well migration assay, soft-agar colony formation assay to confirm NRAS mutant melanoma cell behavior. We will attempt to deliver a liposomally encapsulated six-peptide sequence to therapeutically inhibit the mutant NRAS.
Citation Format: Samikshya Kandel, Krishna Rao. Loss of Rab25 accompanied by NRAS activation cooperatively mediates the tumorigenic transformation in melanoma [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 3008.
Abstract 3066: Lung cancer cells overcome nutrient stress by upregulating an integrin αvβ3/AMPK/OXPHOS axis
Authors: Nam Arin , Jain Shashi , Campos Alejandro , Shepard Ryan M. , von Schalscha Tami , Weis Sara M. , Wettersten Hiromi I. , Cheresh David A. 
Published: March 2024

Abstract
Tumor cells are exposed to various forms of stress within the tumor microenvironment, and those cells that adapt often gain stem-like properties that facilitate tumor initiation and metastasis. Here, we show that lung cancer cells exposed to nutrient stress adapt and survive by converting their metabolism from glycolytic to oxidative phosphorylation (OXPHOS). This is based on the cell’s ability to upregulate expression of integrin αvβ3, a driver of tumor initiation, progression, and drug resistance. Mechanistically, integrin αvβ3 recruits and activates c-Src, promoting sustained AMPK activity leading to activation of PGC-1α, which promotes the expression of mitochondrial OXPHOS complexes. In fact, ectopic expression of integrin β3 was sufficient to drive OXPHOS-dependent metabolism even in the absence of stress. Furthermore, pharmacological targeting of this pathway not only rendered integrin αvβ3+ cells sensitive to nutrient depletion but also prevented their ability to undergo tumor initiation. These findings reveal that targeting integrin αvβ3 or its pathway can prevent cancer cell metabolic flexibility and their capacity to adapt to stress within the tumor microenvironment.
Citation Format: Arin Nam, Shashi Jain, Alejandro Campos, Ryan M. Shepard, Tami von Schalscha, Sara M. Weis, Hiromi I. Wettersten, David A. Cheresh. Lung cancer cells overcome nutrient stress by upregulating an integrin αvβ3/AMPK/OXPHOS axis [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 3066.
Abstract 3094: Biophysics in kinase drug discovery programs: Spectral shift technology from fragment screening to mechanism of action
Authors: Legros Celine , Porkolab Vanessa , Sigoillot Maud , Mirguet Olivier 
Published: March 2024

Abstract
Using Eurofins Discovery’s state-of-the art Hit-Finding platform, our experts designed an optimized screening cascade combining cutting-edge High-throughput Screening (HTS) technologies with the latest Biophysics techniques. With advanced technologies, such as the Echo® MS and RapidFire MS for HTS, and SPR, MST, TSA, nanoDSF and ITC for Biophysics, the cascade is effective for primary screening, orthogonal assays and evaluating Mechanism of Action (MoA). There are many biophysical approaches which bring value to drug discovery programs, and it is the target properties which drive the selection of the most suitable technology. In this presentation, we will highlight the novel Spectral Shift technology, one of the techniques employed by the Dianthus (well-known for the MST-TRIC). The goal was to rapidly implement an assay that monitors the ability of PIM3 to bind to ATP, as well as to compounds and fragments in solution. Using the Echo MS’ acoustic-droplet ejection, nanoliter dispensing in 394-well plates, robust data by Spectral Shift were quickly generated for the fragment-based screening campaign. Hits were confirmed in Dose-response with µM-range affinity values (KD), and TSA was used as an orthogonal assay for the identification of stabilizers among the selected PIM3 binders. For a defined set of 826 proprietary fragments, a single dose screening at 600µM using Spectral Shift was run, leading to the identification of 11% of potential binders. The 100 fragments were then confirmed in dose-response titration and 80 binders were confirmed. The top 30 binders were selected for orthogonal characterization using TSA. This led to the selection of the top 6 confirmed fragments, on which a MoA study in MST-TRIC was performed. Following this evaluation, 3 allosteric binders were identified, with KD from 355 to 550µM (affinity of orthosteric binders from 55 to 333µM). Biophysics plays an important role in drug discovery by qualifying valuable assets and increasing the success rates of future Hit-finding programs. The fragment hits selected in this PIM3 Kinase project, which show ADP-Glo™ activities, are now ready for crystallography studies (NMR & X-ray co-crystallization).
Citation Format: Celine Legros, Vanessa Porkolab, Maud Sigoillot, Olivier Mirguet. Biophysics in kinase drug discovery programs: Spectral shift technology from fragment screening to mechanism of action [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 3094.
Abstract 3097: CETSA HT: Employing rapid high-throughput target engagement technology in primary screening of P53
Authors: Kacal Merve , Friman Tomas , Arnold Laurence , Brehmer Victoria , Molina Daniel Martinez , Lundgren Stina 
Published: March 2024

Abstract
The confirmation of target engagement for candidate drugs is a fundamental requisite in drug discovery. A significant portion of projects fail to reach the clinic due to lack of efficacy or failure to show lead candidates interacting with the intended target in a more complex environment. The utility of using the CETSA® (CEllular Thermal Shift Assay) technology to measure target engagement in intact cells is increasingly exploited in high throughput screening, where examples demonstrate low false positive rates in screens of up to 500K compounds. The p53 tumor suppressor protein, a crucial guardian against cancer, functions as a transcription factor that mediates cancer suppression by regulating genes involved in a wide range of cellular processes such as DNA repair, cell cycle and apoptosis. Unfortunately, frequent mutations in the TP53 gene hinder p53's tumor suppressor role in human cancers, thus designating it as a promising target for anticancer therapy. However, attempts to restore p53 function for therapeutic purposes have not yet advanced to clinical approval, highlighting the complexities in leveraging p53 for cancer treatment. Its undruggable nature, attributed to the absence of accessible deep pockets, lack of enzymatic activity, and its intracellular location, further complicates targeted interventions with both low and high molecular weight drugs. Here, we demonstrate the analysis of a high-throughput plate based CETSA® HT screen on the challenging target p53, performed using AlphaLISA® SureFire® Ultra™ technology and our newly developed high-throughput screening platform. Hits identified from this screen are privileged from the onset by binding to the target in a physiologically relevant cellular matrix and in a binding-mode agnostic way. This is in contrast to traditional screening methods targeting activity or in vitro interaction where cellular activity is addressed in later stages. This assay cascade highlights a rapid target-to-hit lead generation strategy placing the emphasis on finding chemical matter that interacts with endogenous target in living cells first, followed by analysis of hit material activity. The ‘shortcut’ to chemical matter active in living cells represents significant potential cost savings in comparison to assay development time on recombinantly produced proteins and/or modified cell lines for traditional HTS screening approaches that still rely on target engagement assessment downstream.
Citation Format: Merve Kacal, Tomas Friman, Laurence Arnold, Victoria Brehmer, Daniel Martinez Molina, Stina Lundgren. CETSA HT: Employing rapid high-throughput target engagement technology in primary screening of P53 [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 3097.
Abstract 3114: Development of HTS enzymatic assays for RNA helicases: DDX3, DDX5, DDX17, RIG-1 and MDA5
Authors: Ali Mahbbat , Pham Ha , Martinez Anibal Ramos , Lowery Robert 
Published: March 2024

Abstract
RNA helicases are a broad class of enzymes that bind and modify RNA in an ATP-dependent manner and play diverse roles in RNA metabolism, regulation of gene expression, and innate immunity. Several RNA helicases, especially in the dead box helicase (DDX) family, have been shown to be dysregulated in cancer, and there have been limited efforts to develop small molecule inhibitors. To accelerate development of selective helicase inhibitors while avoiding off target effects, especially with helicases that promote tumor immunity, we are assembling a panel of high throughput biochemical assays using the Transcreener ADP2 assay for homogenous detection of RNA-dependent ATPase activity with a far-red fluorescence polarization (FP) readout. Here we describe development of assays for DDX3, DDX17 and DDX5, which have all been implicated in cancer, and the Rig-1-like receptor (RLR) helicases, RIG-1 and MDA5, which trigger expression of type I interferons important for tumor immunity. We produced the recombinant enzymes using BaV-infected insect cells, as full-length polypeptides, or in some cases truncated to remove disordered N-terminal domains. We determined RNA substrate and cofactor requirements and kinetic parameters with the purified enzymes and optimized the assays for initial velocity detection of RNA-dependent ATPase activity with a Z’ value greater than 0.7, ensuring a sufficient signal window for inhibitor screening and dose response assays. We validated each of the helicase ATPase assays for HTS by screening a collection of bioactives (Tocris 2.0) and confirming hits in dose response mode with the enzymatic assay and with thermal shift assays to confirm target binding. These assays will enable screening and hit-to-lead/SAR for DDX helicases that drive tumorigenesis as well as selectivity profiling to avoid off target effects with RLR helicases that promote tumor immunity.
Citation Format: Mahbbat Ali, Ha Pham, Anibal Ramos Martinez, Robert Lowery. Development of HTS enzymatic assays for RNA helicases: DDX3, DDX5, DDX17, RIG-1 and MDA5 [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 3114.
Abstract 3132: A novel dual drug antibody-drug conjugate targeting hTrop2 has synergetic anti-tumor activity in solid tumor models
Authors: Zhu Jie , Zhao Xiaobei , Li Eileen , Xu Hong , Nie Lei , Chen Gang , Wang Haibing 
Published: March 2024

Abstract
Antibody-drug conjugates (ADCs) are emerging therapeutic agents for targeted cancer treatment with remarkable clinical success. However, tumor heterogeneity and microenvironment complexity are still the major factors contributing to drug resistance, recurrence, and metastasis, which cannot be resolved with a mono therapy strategy. We’ve developed a novel dual drug ADC (BiADC), BR113, by conjugating an anti-hTrop2 antibody with a drug linker of a Topoisomerase I inhibitor payload, and another drug linker of an immune stimulator. Our studies show that the homogeneous ADC containing two distinct payloads is a new promising drug class, with two different anti-tumor MOAs, synergistic antigen-specific cell killing potency, desirable pharmacokinetic profiles, and minimal toxicity at therapeutic doses. In the cancer cells and human PBMC co-culture assay, BR113 exerts significantly more potent killing on cancer cells than two individual single-drug ADCs and the combination of two single-drug ADCs, which suggested a synergy on anti-tumor effects of this dual-drug ADC. In the xenograft mouse model studies, BR113 exhibits greater treatment effect and survival benefit than the combo of two single-drug ADCs at various doses. This robust superior anti-tumor activity is further confirmed in a hTrop2-MC38 syngeneic model with a prolonged anti-tumor protection against the tumor re-challenge. Finally, the novel BiADC, BR113, is well tolerated in hTop2 humanized mice after repeated administration. Our findings highlight the therapeutic potential of the BiADC format for treating refractory advanced solid tumors. The outstanding anti-tumor activity across various CDX tumor models and its favorable safety profile in mice support further evaluation of the innovative BiADC, BR113.
Citation Format: Jie Zhu, Xiaobei Zhao, Eileen Li, Hong Xu, Lei Nie, Gang Chen, Haibing Wang. A novel dual drug antibody-drug conjugate targeting hTrop2 has synergetic anti-tumor activity in solid 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 3132.
Abstract 3139: BL-M11D1, a novel CD33-targeting ADC, demonstrates robust anti-tumor efficacy in preclinical evaluation
Authors: Wan Weili , Zhao Shuwen , Zhuo Shi , Zhang Yong , Chen Lan , Li Ruigang , Khalili Jahan Salar , Sa Xiao , Yan Yongqi , Shen Xuejiao , Zhu Yi 
Published: March 2024

Abstract
CD33, is a transmembrane receptor expressed on cells of myeloid lineage. It is therapeutically targeted in cancer due to overexpression broadly among hematopoietic malignancies including AML. To develop a promising therapeutic anti-tumor agent, we generated BL-M11D1, a CD33-Ed-04 ADC. It is built on gemtuzumab, a specific monoclonal antibody against CD33, having a wt Fc that can mediate ADCC in vitro. BL-M11D1 is composed of gemtuzumab, a cathepsin B cleavable linker, and a novel topoisomerase I inhibitor agent (Ed-04). The novel Ed-04 is a derivative of the alkaloid camptothecin and mediates cell cycle arrest at the S phase and subsequent apoptosis. BL-M11D1 achieves a high drug-to-antibody-ratio (DAR=10) with a highly stable linker and is readily internalized and trafficked to lysosomal cellular compartments. The antitumor efficacy of BL-M11D1 was evaluated in xenograft tumor models. BL-M11D1 exhibited strong tumor inhibition capacity toward the human hematopoietic malignancies engrafted xenograft models. In summary, these studies suggest BL-M11D1, a novel CD33-targeting ADC, is potentially efficacious in the treatment of CD33-expressing disease. The clinical phase I trial is being undertaken to evaluate safety, dosing and observations of preliminary signs of efficacy.
Citation Format: Weili Wan, Shuwen Zhao, Shi Zhuo, Yong Zhang, Lan Chen, Ruigang Li, Jahan Salar Khalili, Xiao Sa, Yongqi Yan, Xuejiao Shen, Yi Zhu. BL-M11D1, a novel CD33-targeting ADC, demonstrates robust anti-tumor efficacy in preclinical evaluation [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 3139.