bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3045: PPAR-α antagonist enhances immunotherapy and anti-angiogenic therapy to inhibit murine renal cancer
Authors: Gillespie Michael , Chen Valerie , Howard Isabella , Smith Keira , Freund Dave , Standifer Nathan , Burdette Dara , Dubensky Thomas W. , Whiting Sam , Panigrahy Dipak
Published: March 2024

Peroxisome proliferator activated receptor alpha (PPARα) is a transcription factor and master regulator of fatty acid oxidation. PPARα ligands modulate the switch between co-activator and co-repressor transcription complexes at PPARα-controlled target genes. In addition, PPARα has been shown to transrepress NF-κB signaling and inhibit angiogenesis. Renal cell carcinoma (RCC) expresses high levels of PPAR-α and is a highly angiogenic cancer. Current frontline treatments for RCC include chemotherapy, anti-angiogenics, and immunotherapy but are limited by the immune suppressive state of the tumor microenvironment. TPST-1120 is a novel PPARα antagonist for the treatment of cancer patients that binds in the ligand binding domain, positioning the AF-2 activation helix in an inactive conformation. The inactive conformation has a lower affinity for co-activator motifs and reduces activation of PPARα regulated genes. In a CHO-based reporter cell line, TPST-1120 potently competed against the PPARα-specific agonist GW7647 (IC50 = 36 nM at 20 nM GW7647) and an endogenous PPARα agonist oleoylethanolamide (OEA; IC50 = 15 nM at 30 µM OEA). Furthermore, in an enzyme fragment complementation assay, we demonstrated that TPST-1120 inhibited agonist-induced binding of PPARα to MED1 by destabilizing the co-activator complex thereby stabilizing the inactive conformation of PPAR-α. In a murine model of renal cell adenocarcinoma (RENCA), TPST-1120 treatment for 12 days (30 mg/kg qd) reduced tumor growth by 52% as a monotherapy (p<0.0001). Consistent with these findings, TPST-1120 inhibited tumor angiogenesis and proliferation as quantified by Ki67 levels. Combination treatment with current frontline therapeutics resulted in synergistic tumor inhibition of 74% with anti-PD1 (200 ug Q3D for 12 days) and 81% with cabozantinib (15 mg/kg QD for 15 days) (p<.0001). Flow cytometry-based analysis of tumors demonstrated increased CD8+ T cell infiltration in the TME as well as decreases in M2 and elevations in M1 macrophages. Importantly, we demonstrate that TPST-1120 can reverse an immunosuppressive class of immune cells to promote anti-tumor immunity and anti-angiogenesis in kidney cancer in the absence of overt toxicity. These data support advancement of TPST-1120 into RCC.

Citation Format: Michael Gillespie, Valerie Chen, Isabella Howard, Keira Smith, Dave Freund, Nathan Standifer, Dara Burdette, Thomas W. Dubensky, Sam Whiting, Dipak Panigrahy. PPAR-α antagonist enhances immunotherapy and anti-angiogenic therapy to inhibit murine renal 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 3045.
Abstract 3051: JunAP, a peptide antagonist against the activator protein 1 transcription factor complex, demonstrates cancer cell cytotoxicity and reduced invasion in vitro and tumor regression in vivo in TNBC models
Authors: Mendelson Karen , Mattes Zachary F. , Leong Siok , Ramirez Ricardo , Koester Mark , Scuoppo Claudio , Diehl Julia , Gallagher Erin , Gonzales Jerel , Abbate Franco , Ghamsari Lila , Merutka Gene , Kappel Barry J. , Vainstein-Haras Abi , Rotolo Jim A.
Published: March 2024

The activator protein 1 (AP-1) transcription factor is a dimeric complex consisting of proteins from the JUN, FOS, MAF, and ATF families. AP-1 complexes have been identified as novel therapeutic targets in cancer due to their role in tumor growth, invasion, metastasis, angiogenesis and chemoresistance. Dimerization of components of the AP-1 complex via leucine zipper domain interactions is required for DNA binding and subsequent transcriptional activity. We designed a peptide (Jun antagonizing peptide, JunAP) targeting the basic leucine zipper motif of AP-1 to antagonize AP-1 complex formation and prevent associated activity. Fluorescence polarization and DNA ELISA assays demonstrate target binding and selectivity of JunAP towards AP-1 family members, co-immunoprecipitation experiments reveal that JunAP blocks protein-protein interactions between cJun and its key binding partners, and reporter assays confirm inhibition of AP-1 transcriptional activity in vitro. Pathway analysis of RNA sequencing data confirmed by qPCR analysis shows that JunAP disrupts key pathways required for cell survival and migration. Corresponding functional in vitro assays reveal that JunAP exhibits an inhibitory effect on invasion in Boyden chamber assays and demonstrates potent cytotoxicity in Jun-dependent cells. Further, in a BT549 triple negative breast cancer subcutaneous xenograft model, administration of JunAP results in tumor regression, demonstrating the anti-cancer potential of targeting the AP-1 complex. In summary, these data support JunAP as a potent peptide antagonist of the AP-1 transcription factor family that warrants further development as a potential therapeutic option for AP-1 driven tumors.

Citation Format: Karen Mendelson, Zachary F. Mattes, Siok Leong, Ricardo Ramirez, Mark Koester, Claudio Scuoppo, Julia Diehl, Erin Gallagher, Jerel Gonzales, Franco Abbate, Lila Ghamsari, Gene Merutka, Barry J. Kappel, Abi Vainstein-Haras, Jim A. Rotolo. JunAP, a peptide antagonist against the activator protein 1 transcription factor complex, demonstrates cancer cell cytotoxicity and reduced invasion in vitro and tumor regression in vivo in TNBC 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 3051.
Abstract 3083: Unveiling the role of chemotherapy-resistant, slow-cycling cancer cells in driving cancer cachexia
Authors: Cho Jaebeom , Lee Ho-Young
Published: March 2024

Cancer cachexia, a systemic disease marked by substantial, involuntary weight loss, predominantly affecting muscle and adipose tissue, manifests in patients with advanced-stage cancer. The emergence of cancer cachexia poses significant challenges to cancer treatment, diminishing the efficacy of anticancer medications, and the resultant muscle loss adversely impacts the daily lives of patients, diminishing the overall quality of life. Notably, cancer cachexia is implicated in 30-40% of cancer-related deaths, underscoring the pivotal need to comprehend its underlying principles and propose apt interventions to enhance both survival rates and the well-being of cancer patients. Based on our previous findings, cancer cells exhibiting a slow cell cycle, resistant to widely employed clinical chemotherapy agents, exhibit the formation of proinflammatory tumor microenvironment. The escalation of systemic inflammatory cytokines has long been suggested as a potential mechanism contributing to cancer cachexia. Building upon these insights, we hypothesized that slow-cycling cancer cells (SCCs) featuring resistance to chemotherapy are pivotal contributors to cancer cachexia. We investigated the impact of drug-resistant SCCs on the loss of differentiated myotubes and lipid droplets in vitro and muscle and fat loss in in vivo conditions. Interestingly, these cells induced more severe muscle and fat losses. Upon scrutinizing this cell population, we found the overexpression of a particular membrane protein, Protein A. The increase of this Protein A enhanced the release of exosomes from the cell through a novel mechanism distinct from its classical functions, consequently amplifying the release of inflammatory substances originating from cancer cells. In summary, our study elucidates that chemotherapy-resistant cancer cells, characterized by a slow cell cycle, intensify the secretion of inflammatory substances in a manner contingent upon the overexpression of a membrane protein A, which leads to the depletion of muscle and fat located in the distal peripheral regions.

Citation Format: Jaebeom Cho, Ho-Young Lee. Unveiling the role of chemotherapy-resistant, slow-cycling cancer cells in driving cancer cachexia [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 3083.
Abstract 3092: MICRO-TAG: A novel fluorescence-based real-time cellular target engagement platform for drug discovery
Authors: Babic Ivan , Bryan Nikolas , Cunningham Claire , Sampson Avery , Starczynowski Daniel , Nurmemmedov Elmar
Published: March 2024

Success of drug discovery programs can be amplified by adopting emerging technologies that can interrogate drug targets within their physiological context. Cell target engagement (CTE) presents a combined biological & biophysical method, which provides quantitative evaluation of target-drug interaction within the native environment of the cell. The currently available luminescence-based CTE methods are limited to measurement of end-point signal and poor resolution, thereby limiting their mechanistic and stoichiometric insight. We have developed MICRO-TAG, a novel fluorescence-based CTE platform, which relies on complementation of split-RNAse A. It enables interrogation of target engagement without interfering with folding, localization and function of target proteins - all within the physiological milieu of the cell. Uniquely, this new platform allows for quantitation and monitoring of CTE in real- time in the cell. It enables extraction of important parameters from target-drug interaction: target thermal profile and temperature of aggregation (Tagg), time required to reach equilibrium (teq), apparent association rate (Kon) and physiologically relevant drug:target binding affinity (KD). We provide data demonstrating potential and utility of MICRO-TAG platform on drug targets, such as KRAS, MTH1, EGFR, and UBE2N. The platform demonstrates relevance for discovery of novel drug candidates targeting challenging drug targets such as transcription factors. This highly scalable and quantitative novel method for assessing drug-target interaction within cells can enhance drug discovery programs, and offers potential to rapidly identify clinically relevant hit compounds for challenging drug targets.

Citation Format: Ivan Babic, Nikolas Bryan, Claire Cunningham, Avery Sampson, Daniel Starczynowski, Elmar Nurmemmedov. MICRO-TAG: A novel fluorescence-based real-time cellular target engagement platform for drug discovery [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 3092.
Abstract 3100: Interactive analysis of drug testing data for functional precision oncology with Breeze 2.0
Authors: Potdar Swapnil , Ianevski Filipp , Ianevski Aleksandr , Tanoli Ziaurrehman , Wennerberg Krister , Seashore-Ludlow Brinton , Kallioniemi Olli , Östling Päivi , Aittokallio Tero , Saarela Jani
Published: March 2024

High throughput screening (HTS) is used to screen large numbers of small molecules in cell-based assays to predicting their potential therapeutic response and when applying approved drug libraries using HTS it becomes drug repurposing. The Drug Sensitivity and Resistance Testing (DSRT) platform established at FIMM has been extensively used in testing panels of approved and investigational drugs on cells, both conventional cell lines and primary cell models derived from cancer patient samples. The results help in deepen our understanding of drug efficacy in individual patients and across specific disease to guide future patient stratification.

Breeze 2.0 is an open access, web-based platform for interactive analysis of drug screening data in real time. Breeze accepts raw data in a simple tabular format and generates detailed quality control metrics, plots, dose-response curve fits, drug response quantification in form of IC50/EC50/AUC and Drug Sensitivity Scores (DSS). Besides tabular results, Breeze generates interactive, informative, and intuitive visualizations, such as plate heatmaps, scatterplots, barplots, clustered heatmaps, circular plots, for further analysis. Based on manually-curated published datasets available through Breeze 2.0, users can instantly compare their drug response patterns with the existing DSRT profiles from patient samples, as well as cell lines. Importantly, Breeze 2.0 has integrated curated reference DSRT data tested on bone marrow samples from healthy control individuals, which plays a crucial role in understanding cancer-selective drug response by de-prioritizing generally toxic compounds. The end-users can select multiple healthy controls or relevant patient/cell line samples and choose the drugs of interest to visualize the overlaid dose responses, as well as response quantification metrics. Breeze 2.0 is built in R, PHP, MySQL and is freely accessible at https://breeze.fimm.fi/v2

Citation Format: Swapnil Potdar, Filipp Ianevski, Aleksandr Ianevski, Ziaurrehman Tanoli, Krister Wennerberg, Brinton Seashore-Ludlow, Olli Kallioniemi, Päivi Östling, Tero Aittokallio, Jani Saarela. Interactive analysis of drug testing data for functional precision oncology with Breeze 2.0 [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 3100.
Abstract 3110: An enhanced and cost-efficient method for neoantigen immunogenicity screening method: Double Barcode Neoepitope ScanTM
Authors: Kim Seong Gwang , Jung Min-Ho , Hwang Hyun-Ji , Oh Sejin , Hong Seong-Eui , Paik Soonmyung , Kim Hae-Suk
Published: March 2024

Neoantigens, HLA bound peptides generated by tumor-specific mutations, have emerged as promising targets for cancer immunotherapeutic. However, the significant challenge in neoantigen-based therapies is that the neoantigens are rare and difficult to predict. Therefore, in addition to prediction algorithms, the optimization of validation methods is essential for the reliable selection of highly immunogenic neoepitopes. In this study, we have developed a cost-effective screening method to validate large scale of neoantigen candidates. The DNA library-based methods have been employed for antigen selection and T cell receptor identification. We improved the DNA library-based neoantigen screening method by adding two barcodes to each minigene transcribing multiple neoantigen candidates and pooled synthesis of minigenes. We initially designed the barcodes to allow analysis in a 10 by 10 format for screening of hundreds of neoantigens so that PCR with each barcode result in amplification of ten candidates. The second barcode allowed the deconvolution of immunogenicity results without the need for synthesis of individual peptides. In this method B cell was used as antigen-presenting cell, which were co-transfected with mRNA for a minigene pool, 41BBL, and OX40L after expansion with CD40L/IL4/IL21. The resulting B cells were co-cultured with T cells and T cell responses were evaluated using ELISpot. To validate the method the TESLA consortium reported tetramer positive neoantigens were included in minigene. In results, the neoantigens including each barcode were amplified to form a pool of ten neoantigens as a minigene and confirmed that the neoantigens were equally amplified by nested PCR. The neoantigen pools synthesized as mRNA were transfected for presentation in B cells. The result of the targeting a neoantigen pool containing tetramer-positive neoantigens of the TESLA consortium showed a positive for ELISpot. In conclusion, our optimized screening method, employing the double barcode system, offers a time- and cost-efficient approach for the selection of immunogenic neoantigens without the need for individual peptides. This method will facilitate large-scale screening to identify actual neoantigens with immunogenic potential, contributing to the development of neoantigen-targeted vaccines.

Citation Format: Seong Gwang Kim, Min-Ho Jung, Hyun-Ji Hwang, Sejin Oh, Seong-Eui Hong, Soonmyung Paik, Hae-Suk Kim. An enhanced and cost-efficient method for neoantigen immunogenicity screening method: Double Barcode Neoepitope ScanTM [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 3110.
Abstract 3141: LCB02A an antibody drug conjugate (ADC) targeting CLDN18.2 expressing solid tumors
Authors: Baek Eun ji , Chung Chul-Woong , Park Changsik , Jang Taeik , Jeong Yeojin , Lee Sang-Ah
Published: March 2024

Claudin18.2 (CLDN18.2) is an isoform of CLDN18 with very limited expression in gastric epithelial cells in normal tissues, and aberrant overexpression in gastric, pancreatic, esophageal, ovarian, lung, and other solid tumors. This expression pattern presents a significant hurdle in developing an ADC targeting CLDN18.2 due to potential on-target toxicity to the gastrointestinal (GI) tract. LCB02A is an antibody-drug conjugate (ADC) that targets CLDN18.2, composed of a topoisomerase 1 inhibitor (TOPO1i) as payload (DAR4) linked to the CLDN18.2 specific binding PR301839 hClaudin18.2 (CLDN18.2) is an isoform of CLDN18 with very limited expression in gastric epithelial cells in normal tissues, and aberrant overexpression in gastric, pancreatic, esophageal, ovarian, lung, and other solid tumors. This expression pattern presents a significant hurdle in developing an ADC targeting CLDN18.2 due to potential on-target toxicity to the gastrointestinal (GI) tract. LCB02A is an antibody-drug conjugate (ADC) that targets CLDN18.2, composed of a topoisomerase 1 inhibitor (TOPO1i) as payload (DAR4) linked to the CLDN18.2 specific binding PR301839 humanized IgG1 antibody utilizing LegoChem Biosciences’ ConjuAllTM technology. LCB02A showed potent in vitro cytotoxicity in CLDN18.2 expressing gastric and pancreatic cancer cell lines. LCB02A also showed excellent anti-tumor efficacy in various gastric and pancreatic cell line-derived xenograft (CDX) models and patient-derived xenograft (PDX) models. Preliminary toxicity assessment in cynomolgus monkeys (CLDN18.2 cross-reactive species) demonstrated that LCB02A was well tolerated, with repeat doses of up to 60 mg/kg. In conclusion, LCB02A is a promising ADC for the treatment of CLDN18.2 expressing solid tumors including gastric cancer (GC) and pancreatic ductal adenocarcinoma (PDAC). umanized IgG1 antibody utilizing LegoChem Biosciences’ ConjuAllTM technology. LCB02A showed potent in vitro cytotoxicity in CLDN18.2 expressing gastric and pancreatic cancer cell lines. LCB02A also showed excellent anti-tumor efficacy in various gastric and pancreatic cell line-derived xenograft (CDX) models and patient-derived xenograft (PDX) models. Preliminary toxicity assessment in cynomolgus monkeys (CLDN18.2 cross-reactive species) demonstrated that LCB02A was well tolerated, with repeat doses of up to 60 mg/kg. In conclusion, LCB02A is a promising ADC for the treatment of CLDN18.2 expressing solid tumors including gastric cancer (GC) and pancreatic ductal adenocarcinoma (PDAC).

Citation Format: Eun ji Baek, Chul-Woong Chung, Changsik Park, Taeik Jang, Yeojin Jeong, Sang-Ah Lee. LCB02A an antibody drug conjugate (ADC) targeting CLDN18.2 expressing solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3141.
Abstract 3191: Chk1/2 inhibition enhances response to lurbinectedin treatment in small cell lung cancer
Authors: Uruchurtu Ashley Sanchez Sevilla , Roady Tyler , Anderson Agenxnie , El-Deiry Wafik S.
Published: March 2024

Small cell lung cancer (SCLC) remains a significant clinical concern, with only a 7% 5 year survival rate; therefore, there remains an unmet need for efficacious treatments. Lurbinectedin was FDA approved in 2019 to treat platinum-resistant SCLC. Preliminary experiments in SCLC cell lines indicated an increase in intracellular pChk1 when treated with lurbinectedin. Based on these data and our understanding of the role of checkpoint kinases in the DNA damage response (DDR), we hypothesize that the activity of Chk1 is indicative of upregulated DDR activity as a result of double stranded breaks and replicative stress induced by lurbinectedin. In subsequent experiments, we sought to identify the role of Chk1 and Chk2 in the tumor cell response to lurbinectedin by inhibiting their activity using prexasertib (LY2606368, Acrivon Therapeutics). Cell viability experiments indicate decreased tumor cell viability when three SCLC cell lines (H1048, H1105, H1417) are treated with prexasertib as a single agent and in combination with lurbinectedin. Western blot analysis of intracellular proteins from the same SCLC cell lines treated with both drugs demonstrate dose-dependent effects on cell death marker cPARP, DNA damage marker γH2AX, and DDR/cell cycle pathway kinases cdk1 and 2. Ongoing experiments seek to replicate results using alternative Chk1/2 inhibitors, and to explore the effect of lurbinectedin-Chk1/2 inhibitor combination treatment on antitumor immune effector function by in vitro co-culture.

Citation Format: Ashley Sanchez Sevilla Uruchurtu, Tyler Roady, Agenxnie Anderson, Wafik S. El-Deiry. Chk1/2 inhibition enhances response to lurbinectedin treatment in small cell 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 3191.
Abstract 3196: Overcoming lung cancer drug resistance: Enhanced cellular delivery using lipid-extracted nanoliposomes
Authors: Alqaryan Mohammed , Allahyani Rashad , Lee Dayeoun , Campbell Robert B.
Published: March 2024

Background: Lung cancer causes substantial cancer deaths in the U.S. Drug resistance remains a major issue. Nanoliposomes demonstrate enhanced drug targeting and resistance-overcoming potential. Incorporating lipid extracts derived from target cells shows improved delivery over conventional nanoliposomes. This study examined the effects of lung cancer cell lipid extract nanoliposomes against target and non-target cell populations from different environments.

Method: Cellular Membrane Lipid-Extracted Nanoliposomes (CLENs) were fabricated with DOPC, DOTAP, ChaGo-K-1 lipid extract, cholesterol, PEG5000, and rhodamine fluorescent label was incorporated for uptake studies. The liposome size was reduced by probe sonication. Particle size and polydispersity index (PDI) were measured by dynamic light scattering. Zeta potential was quantified by laser Doppler micro-electrophoresis. Four cell lines were utilized: ChaGo-K-1 NSCLC target, A549 pulmonary microenvironment control, 4T1 extra-pulmonary control, and normal lung fibroblasts off-target control. All cell lines were cultured in 48-well plates at a seeding density of 10,000 cells/mL and incubated for 24 hours. Freshly prepared CLENs were added to all cell lines and incubated for an additional 24 hours. Cells were washed with PBS, and fluorescence intensity was then quantified and qualified by a microplate reader to measure the cellular uptake of the CLENs.

Results: The mean particle size and PDI of the ChaGo-K-1 CLENs were 117 ± 0.7 nm and 0.216 ±0.009, compared to 126 ± 0.5 nm and 0.195 ± 0.008 for the control, respectively. Both formulations were within the optimal size range for drug delivery and tumor cell penetration. The zeta potential of the ChaGo-K-1 CLENs was -1.75 ± 1.81 mV compared to -1.74 ± 6 mV for the control. Quantification and qualification of cellular uptake using fluorescence intensity measurements revealed a 3.2-fold increase in ChaGo-K-1 CLENs compared to conventional liposomes without ChaGo-K-1 lipid extracts. This demonstrates that the inclusion of the lipid extracts in the CLEN significantly enhanced selectivity and delivery to target cancer cell populations.

Conclusion: Higher uptake occurred with target cells using CLENs with lipid extracts versus standard liposomes. NSCLC-derived CLENs show potential to improve selective delivery and overcome drug resistance. Further, in vivo studies evaluating selective NSCLC tumor accumulation and efficacy enhancement are warranted.

Citation Format: Mohammed Alqaryan, Rashad Allahyani, Dayeoun Lee, Robert B. Campbell. Overcoming lung cancer drug resistance: Enhanced cellular delivery using lipid-extracted nanoliposomes [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 3196.
Abstract 320: Development and automation of a streamlined targeted enrichment method for cancer mutation detection
Authors: Wu Zhong , Baird Michelle , Mechlen Cornelia , Hahn Peter , Shaffer Jonathan M.
Published: March 2024

Next Generation Sequencing (NGS) of DNA is a powerful tool for the detection of genetic variations, including single nucleotide polymorphisms, copy number variation, and small insertions/deletions. Target enrichment technologies enhance DNA sequencing by enabling users to analyze specific regions of interest only, which helps to effectively increase sequencing depth and sample throughput while minimizing cost. However, many targeted DNA library construction workflows are not optimal for routine genetic testing needs due to inconsistent, lengthy workflow. This poses significant challenge to application like cancer mutation profiling that requires detection sensitivity down to 1% or below due to heterogeneous nature of tumor tissue. In addition, high throughput testing with minimal hands on time is desirable with the rapid advance of precision medicine. Magnetic bead separations are commonly used between the individual reactions of targeted DNA library preparation workflows for cleanup and size selection. These bead clean up reactions are not only labor-intensive and tedious procedures but can also greatly contribute to sample-to sample variation especially when employing automated library preparation procedures. With the sophisticated QIAseq Targeted DNA Pro, we developed a novel streamlined targeted DNA workflow that greatly reduces handling variation by replacing the majority of bead clean up steps with enzymatic reactions, which now can be finished in as little as six hours. In combination with the newly developed QIAseq Normalizer kit, the protocol has been successfully implemented on the Hamilton NGS Star liquid handling platform resulting in up to 96 highly uniform, complex and ready-to-sequence libraries that can be effortless generated in one run. This complete automated solution saves researcher up to four hours of hands-on time. When using commercial reference samples, cancer mutations down to 1% were consistently detected with this automated workflow. The presented data demonstrate that this integrated and streamlined workflow does not only provide an automation friendly workflow but also allows to reliably identify low frequent cancer mutations. The applications presented here are for research use only. Not for use in diagnostic procedures.

Citation Format: Zhong Wu, Michelle Baird, Cornelia Mechlen, Peter Hahn, Jonathan M. Shaffer. Development and automation of a streamlined targeted enrichment method for cancer mutation detection [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 320.
Abstract 3203: Extravasation and metastasis dynamics are cytoplasmically controlled by the epitranscriptome
Authors: Grafinger Olivia R. , Thillainadesan Gobi , Su Boyang , Mo Yulin , Xu Xin , Mar Sara , Sheng Minzhi , Islam Sumaitha Al , Geddes-McAllister Jennifer , Coppolino Marc G. , He Hansen H. , Zheng Gang , Jerzak Katarzyna J. , Leong Hon S.
Published: March 2024

Non-coding RNA (ncRNA) and RNA binding proteins (RBPs) primarily reside in the nucleus to regulate splicing, transcription, and transcript stability. However, their role(s) in the cytoplasm were unclear until now. Evidence now suggests that ncRNAs within the cytoplasm play an important role in cancer invasion. In concert with MT1-MMP, a protease involved in invadopodia formation, long non-coding RNAs (lncRNAs) and RBPs form cytoplasmic complexes with MT1-MMP, which we termed “R-bodies” and are abundant in invasive breast cancer cells. RNA immunoprecipitation sequencing (RIP-seq) of only the cytoplasm identified the RNA at the core of these complexes to be metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a lncRNA which has been previously described as playing a fundamental role in cancer but only within the nucleus. Further investigation revealed that R-body-associated-MALAT1 was m6A-epigenetically marked. Depletion of any component of R-bodies and inhibition of m6A epigenetic marking of MALAT1 halted R-body formation and subsequent cell invasion. Importantly, we demonstrate that this cytoplasmic complex can be functionally targeted using RNA therapy against m6A-MALAT1 as well as drugs which inhibit the methyltransferase responsible for the m6A methylation of MALAT1. The identification of R-bodies has far-reaching implications and links the roles of cytoplasmic ncRNA and RBPs to the progression of disease.

Citation Format: Olivia R. Grafinger, Gobi Thillainadesan, Boyang Su, Yulin Mo, Xin Xu, Sara Mar, Minzhi Sheng, Sumaitha Al Islam, Jennifer Geddes-McAllister, Marc G. Coppolino, Hansen H. He, Gang Zheng, Katarzyna J. Jerzak, Hon S. Leong. Extravasation and metastasis dynamics are cytoplasmically controlled by the epitranscriptome [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 3203.
Abstract 3225: Profiling the DNA regulome to discover direct inhibitors of the brachyury transcription factor
Authors: Federation Alexander Joel , Gao Yang , McEllin Brian , Gatbonton-Schwager Tonibelle , Gutierrez Andrea , Robbins Julia , Hueffmeier Bodhi , Broderick Erin , Canzani Daniele , Fondrie William , Pino Lindsay
Published: March 2024

Background: The human genome encodes more than 1,600 transcription factors (TFs), along with additional cofactors, chromatin regulators, and structural proteins that collectively execute the regulatory instructions encoded within the nuclear DNA. Dysfunctions of these proteins, collectively known as the Regulome, are known to drive multiple conditions such as cancer, autoimmune conditions, and fibrosis. Despite their importance, many of these proteins are considered undruggable due to challenges in modeling their activity in vitro.

Methods: We have addressed these shortcomings by implementing a live-cell Regulome profiling approach that quantifies the global activity of TFs without the need for cell manipulation or genome engineering, called TF-Scan. The technology coupled rapid, automated purification of the DNA-bound proteome coupled to scalable Data Independent Acquisition (DIA) quantitative mass spectrometry proteomics and machine learning.

Results: We completed a screen of covalent compounds across 8 tissue types to discover hotspot sites on TFs that control their DNA-binding activity. A chordoma cell line was included in this screen. The screen identified compounds capable of binding to and disrupting the activity of several previously undruggable transcription factors, including brachyury. Hit validation and hit-to-lead medicinal chemistry resulted in a covalent chemical probe for brachyury, with activity in cell line models of chordoma.

Conclusion: Phenotypic drug discovery in live, unmodified human cells with systematic regulome profiling enables the discovery of compounds that disrupt previously undruggable targets by multiple mechanisms of action.

Citation Format: Alexander Joel Federation, Yang Gao, Brian McEllin, Tonibelle Gatbonton-Schwager, Andrea Gutierrez, Julia Robbins, Bodhi Hueffmeier, Erin Broderick, Daniele Canzani, William Fondrie, Lindsay Pino. Profiling the DNA regulome to discover direct inhibitors of the brachyury transcription factor [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 3225.
Abstract 3232: LSD1 inhibition transcriptionally reprograms neuroendocrine prostate cancer exposing unique therapeutic vulnerabilities
Authors: Jasmine Sumer M. , Mandl Adel , Dalrymple Susan L. , Kim Sunghyun , Dias Jennifer , Kleppe Maria , Celatka Cassandra , Tapper Amy , Coleman Ilsa , Nelson Peter , Rienhoff Hugh , Denmeade Samuel R. , Brennen W. Nathaniel
Published: March 2024

Therapeutics have been developed to target castration-resistant prostate cancer (CRPC) classified as adenocarcinoma (CRPC-Adeno). However, an increasing frequency of CRPC-Adeno tumors progress to the highly aggressive and lethal neuroendocrine prostate cancer (NEPC) by acquiring a neuroendocrine (NE) phenotype as a mechanism of therapeutic resistance. This transformation from CRPC-Adeno to NEPC requires distinct epigenetic reprogramming by lysine-specific demethylase 1 (LSD1), which can be exploited for development of novel therapies to target NEPC. LSD1 is upregulated in NEPC and promotes a stem-like transition state through lineage plasticity which enables the cancer cell to undergo NE differentiation. We have shown that LSD1 inhibition reduces the NE program, driving NEPC towards a non-neuroendocrine state through ASCL1 suppression and YAP1 re-expression. This less aggressive non-neuroendocrine state exposes unique vulnerabilities for the development of novel combination therapies to treat this lethal disease.

Citation Format: Sumer M. Jasmine, Adel Mandl, Susan L. Dalrymple, Sunghyun Kim, Jennifer Dias, Maria Kleppe, Cassandra Celatka, Amy Tapper, Ilsa Coleman, Peter Nelson, Hugh Rienhoff, Samuel R. Denmeade, W. Nathaniel Brennen. LSD1 inhibition transcriptionally reprograms neuroendocrine prostate cancer exposing unique therapeutic vulnerabilities [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 3232.
Abstract 3248: Metabolic rewiring shifts engineered TCR T cell bioenergetics to enhance cytotoxicity
Authors: Pillai Rashmi , Zhang Xiaoyu , Abassi Yama
Published: March 2024

Background: Adoptive T cell-based transfer has shown tremendous success in hematological cancers. However, an important obstacle to using cell and gene therapy for solid tumors is the aggressive tumor microenvironment (TME). To significantly improve prognosis of subjects undergoing immunotherapy, it is essential to incorporate unique methods to drive T cell energy production and metabolism towards empowering T cells to dismantle the suppressive TME. This can promote anti-tumor effects and persistence of the transfused engineered T cells. In the present study we present a simple metabolic preconditioning process that improves T cell performance and bioenergetics.

Methods: Using the Agilent xCELLigence RTCA eSight, and Seahorse XF Analyzer we assessed the killing efficiency, and bioenergetics of engineered T-cells after pretreating with Arginine. Briefly, engineered TCR T-cells against MART-1 were generated by transducing CD3+ T-cells (Hemacare, Seattle, WA) with retrovirus SAMEN-DMF5 with a CD34 marker gene, against MART-1. The TCR T cells were pre-conditioned in 6mM Arginine for 7 days, followed by a killing assay using MART-1 expressing melanoma cell line as target cells (624.38) at the E:T ratio of 5:1. Target melanoma cells, 624.38 cells, were engineered to express a red-fluorescent nuclear protein. The comparison was made with transduced T-cells grown in RPMI (no added amino acid supplementation, and non-transduced T cells. The cytotoxicity assay was assessed using real-time impedance and live cell imaging data collected on xCELLigence RTCA eSight. Concurrently, the metabolic profiling of the engineered T-cells was examined by the XF96 Analyzer.

Results: Supplementing the growth medium with 6 mM Arginine significantly increased the potency of MART-1 engineered T cells (up to 6-fold) and bioenergetic studies revealed increased spare respiratory capacity, basal respiration, and ATP production via mitochondrial respiration.

Conclusions: Arginine pre-conditioning improved TCR T cell potency through metabolic rewiring favoring mitochondrial respiration. For Research Use Only. Not for use in diagnostic procedures RA45236.3483333333

Citation Format: Rashmi Pillai, Xiaoyu Zhang, Yama Abassi. Metabolic rewiring shifts engineered TCR T cell bioenergetics to enhance cytotoxicity [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 3248.
Abstract 3277: PAI-1 mediates resistance to MET-targeted therapy in non-small cell lung cancer
Authors: Higashihara Tomoaki , Thu Yin Min , Suzawa Ken , Date Keiichi , Matsuoka Atsushi , Mizuno Daisuke , Yoshichika Ryo , Hayashi Naohiro , Mukohara Fumiaki , Yoshikawa Mao , Shien Kazuhiko , Yamamoto Hiromasa , Toyooka Shinichi
Published: March 2024

[Purpose] The mesenchymal epithelial transition factor receptor (MET) is a potential therapeutic target in various cancers, including non-small cell lung cancer (NSCLC). In NSCLC, the activation of MET pathway is thought to occur through diverse mechanisms that influence properties affecting cancer cell survival, growth, and invasiveness. These tumors show oncogene addiction to MET and exhibit remarkable responses to MET-Tyrosine Kinase Inhibitors (TKIs). However, the long-term effectiveness of MET-TKIs is usually limited due to acquired drug resistance. The mechanisms underlying primary and acquired resistance to MET-TKIs in the management of NSCLC remain unclear. In this study, we investigated the mechanisms acquired for crizotinib in MET-amplified lung carcinoma cell lines. [Experimental Designs] Two MET-amplified lung cancer cell lines, EBC-1 and H1993, were established for acquired resistance to MET-TKI crizotinib and were functionally elucidated. The factors contributing to the resistance mechanism were assessed by genomic and transcriptomic data, and the alterations hypothesized to confer resistance were validated. [Results] Multiple mechanisms underlay acquired resistance to crizotinib in MET-amplified lung cancer cell lines. In EBC-1-derived resistant cells, the drug resistance mechanism was mediated by the overexpression of SERPINE1, the gene encoding plasminogen activator inhibitor-1 (PAI-1). Crizotinib resistance was overcome by combination therapy with PAI-1 inhibitor and PAI-1 knockdown. Another mechanism of resistance in different subline cells of EBC-1 was evaluated as epithelial to mesenchymal transition (EMT) with the upregulation of antiapoptotic proteins. In the H1993-derived resistant cells, the phosphorylated forms of MEK and ERK were observed to be upregulated and MEK inhibitors could be a potential therapeutic strategy for overcoming the resistance. [Conclusions] In this study, we revealed the different mechanisms of drug resistance to MET-TKI treatment and presented some possible strategies to overcome the resistance.

Citation Format: Tomoaki Higashihara, Yin Min Thu, Ken Suzawa, Keiichi Date, Atsushi Matsuoka, Daisuke Mizuno, Ryo Yoshichika, Naohiro Hayashi, Fumiaki Mukohara, Mao Yoshikawa, Kazuhiko Shien, Hiromasa Yamamoto, Shinichi Toyooka. PAI-1 mediates resistance to MET-targeted therapy in non-small cell 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 3277.
Abstract 3325: HBW-012-D and HBW-012-E are novel, potent, selective, safe, and bioavailable KRAS G12D inhibitors with superior anti-tumor efficacy in mice
Authors: Lee Ning , Li Yingfu , Liu Guanfeng , Li Jiang , Ren Junfeng
Published: March 2024

Purpose: This study is to characterize newly identified KRAS G12D small molecule inhibitors HBW-012-D and HBW-012-E, both showing improved oral bioavailability than MRTX1133, the known clinical compound with poor bioavailability. Method: Through our medicinal chemistry efforts, we have aimed to design novel small-molecule KRAS G12D inhibitors that are orally bioavailable. The compounds were screened via pERK and cell viability (CTG) assays in multiple cell types. The optimized compounds were further evaluated based on their pharmacokinetic (PK) properties and toxicity. The anti-tumor efficacy of the leads was confirmed in the GP2D (colorectal carcinoma with KRAS G12D mutation) mouse xenograft tumor model. Result: As shown in the table, HBW-012-D and HBW-012-E have significantly better potency and PK characteristics than MRTX1133. In the mouse GP2D model, comparing head-to-head with the same vehicle and protocol (50 mg/kg, twice daily, orally), MRTX1133 hardly inhibited tumor growth, whereas HBW-012-D inhibited tumor growth well (58 % tumor growth inhibition), and HBW-012-E not only completely inhibited tumor growth but also further regressed tumor size by around 40%. Preliminary toxicity tests showed that HBW-012-D and HBW-012-E are safe: weak inhibition of CYP450 enzymes, negative mini-Ames test, excellent kinase selectivity, and no obvious abnormalities in the 14-day rat or mouse toxicity test. Conclusion: New KRAS G12D inhibitors, HBW-012-D and HBW-012-E, were identified. Their preclinical profile, including potency, efficacy, and PK data, are all superior to MRTX1133, and their safety meets the criteria of clinical development. They show potential as a preferred oral treatment option for patients with tumors mediated by the KRAS G12D mutation, such as pancreatic cancer that still lacks effective treatments.

in vitro data IC 50 (nM) MRTX1133 HBW-012-D HBW-012-E AGS (G12D, stomach, pERK) 1 0.8 0.46 AsPC-1 (G12D, pancreas, 3D CTG) 10.88 3.09 GP2D (G12D, colon, 3D CTG) 1.38 0.67 0.69 MKN-1 (WT, stomach, 3D CTG) >3000 Rat PK @ 100 mg/kg oral Cmax (ng/mL) 9 82 1444 AUC0~t (mg.h/mL) 20 258 5724

Citation Format: Ning Lee, Yingfu Li, Guanfeng Liu, Jiang Li, Junfeng Ren. HBW-012-D and HBW-012-E are novel, potent, selective, safe, and bioavailable KRAS G12D inhibitors with superior anti-tumor efficacy in mice [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 3325.
Abstract 3446: Associations of non-steroidal anti-inflammatory drug use and circulating inflammatory proteins with gallbladder dysplasia
Authors: Rosa Lorena , Cook Paz , Pfeiffer Ruth M. , Kemp Troy J. , Hildesheim Allan , Pehlivanoglu Burcin , Adsay Volkan , Bellolio Enrique , Araya Juan Carlos , Pinto Ligia , Ferreccio Catterina , Aguayo Gloria , Viñuela Eduardo , Koshiol Jill
Published: March 2024

Background: Most gallbladder cancer (GBC) cases arise from the metaplasia-dysplasia-GBC sequence, and chronic inflammation has been associated with its development. The use of non-steroidal anti-inflammatory drugs (NSAIDs) has been associated with reduced risk of GBC; however, little is known about these associations with preneoplastic lesions. We thus aimed to study the associations of the use of NSAIDs and inflammatory proteins with gallbladder dysplasia, a precursor to GBC.

Methods: Associations of NSAIDs and gallbladder dysplasia were evaluated in 1,925 Chilean cholecystectomy patients, 82 with dysplasia and 1,843 with gallstones but not dysplasia. In addition, we measured 33 circulating inflammatory proteins in a subsample of 68 dysplasia cases diagnosed at the time of sample selection and 136 gallstone controls. Age- and sex-adjusted odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated, and associations of inflammatory proteins and gallbladder dysplasia were analyzed.

Results: NSAID use was inversely associated with gallbladder dysplasia (OR: 0.48, 95% CI: 0.26-0.83). Comparing the highest versus lowest category of each immune-related protein, eight proteins were inversely associated with dysplasia with ORs ranging from 0.30 (95%CI: 0.12-0.77) for IL-33 to 0.76 (95%CI: 0.59-0.99) for MIP-1B. Of those, GRO remained associated with dysplasia (OR: 0.64, 95%CI: 0.45-0.91) and BCA-1 was borderline-associated (OR: 0.74, 95%CI: 0.54-1.01) in the multivariable logistic regression model additionally adjusted for sex, age and NSAID use.

Conclusion: NSAID users were less likely to have gallbladder dysplasia, suggesting that NSAIDs might be beneficial for gallstones patients. Prospective studies are needed to understand the inverse association of immune-related markers and dysplasia, and to evaluate the biological effect of NSAIDs in GBC.

Research supported by general funds from the Intramural Research Program of the US National Institutes of Health, National Cancer Institute, Division of Cancer Epidemiology and Genetics, and the Office of Research on Women’s Health, National Institutes of Health.

Citation Format: Lorena Rosa, Paz Cook, Ruth M. Pfeiffer, Troy J. Kemp, Allan Hildesheim, Burcin Pehlivanoglu, Volkan Adsay, Enrique Bellolio, Juan Carlos Araya, Ligia Pinto, Catterina Ferreccio, Gloria Aguayo, Eduardo Viñuela, Jill Koshiol. Associations of non-steroidal anti-inflammatory drug use and circulating inflammatory proteins with gallbladder dysplasia [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 3446.