bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3199: Nano gelasomes: Novel lamellar metal-protein suprastructures for nucleic acid delivery
Authors: Babu Agasthya Suresh , Suresh Dhananjay , Li Zhaohui , Upendran Anandhi , Kannan Raghuraman
Published: March 2024

There is considerable interest in the development of self-assembled charged nanocarriers that form organized, predictable structures to enhance stable packaging and delivery of nucleic acid materials in vivo. However, the synthesis of stably-charged, organized nanostructures is highly challenging, requiring a controlled balance of nanomaterial components, stabilizers, and hydrophobic forces. Herein, we report a significant step forward in the controlled lamellar assembly of a charged complex between ultra-small 2 nm gold nanoparticles and a lipidoid within an organic matrix to form Gelasomes. These spherical suprastructures feature an intricate 3D concentric arrangement with a constant interlayer distance (4nm). The cationic lipidoid within this arrangement aids in the highly efficient packaging of nucleic acid materials in the nanoparticle. To demonstrate the potential biomedical applications of Gelasomes, we have loaded siRNA (AXL or YAP), mRNA (EGFP), or CRISPR components (Cas9 mRNA and guide RNA) and validated their functionality by in vitro or in vivo studies. Gelasomes with AXL siRNA (~20 bp) demonstrated rapid uptake (<2 hrs), successful endosomal escape, remarkable transfection efficiency (99.87 ± 0.04 %), gene downregulation (97 ± 2.8 %), and disassembly within the cell (<24 hrs). In vivo downregulation studies in Lung Cancer PDX (Gelasomes with AXL siRNA) and CDX (Gelasomes with YAP siRNA) models confirm the gene-silencing capability (91% or 38 % respectively) of Gelasomes underscoring their potential in advancing nanomedicine. Similarly, Gelasome loaded with EGFP mRNA (1 kb) showed 47 ± 8.54 % expression efficiency within 48 h of treatment in vitro. We also show the packaging of larger nucleic acids such as CRISPR Cas9 mRNA (5 kb) along with the guide RNA (100 bp) in vitro for generating knockouts, that achieved an editing efficiency of ~6%. Together, the results from this study pave the way for the development of adaptable, biodegradable, and functionally versatile supraparticles for medical applications.

Citation Format: Agasthya Suresh Babu, Dhananjay Suresh, Zhaohui Li, Anandhi Upendran, Raghuraman Kannan. Nano gelasomes: Novel lamellar metal-protein suprastructures for nucleic acid delivery [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 3199.
Abstract 3208: A nano sized system for the treatment of gastrointestinal cancers
Authors: Vaskovich Daniella , Acúrcio Rita , Kleiner Ron , Carreira Barbara , Matos Ana , Florindo Helena , Satchi-Fainaro Ronit
Published: March 2024

Despite the recent progress in cancer immunotherapies, most patients do not benefit from the currently available immune checkpoint blockers, and many develop severe immune-related adverse events. Therefore, it is urgent to develop alternative, effective immunotherapeutic approaches. An example of non-responsive cancers are gastrointestinal (GI) cancers, which account for 26% of the global cancer incidence and 35% of all cancer-related deaths. In many GI cancers, the failure of immunotherapies to yield clinical response is partially attributed to the inefficient antigen presentation by DC, which results in insufficient T cell stimulation despite their expression of tumor-associated antigens (TAA). To overcome these hurdles, we designed novel polymeric-based nanoparticles (NP) to deliver a combination of GI cancers antigens of CEACAM5 protein and toll-like receptor ligands to DC. We chose to encapsulate CEACAM5 antigens because it is overexpressed in GI cancers, such as gastric cancer, colorectal cancer, and pancreatic cancer. With this approach, we aim to modulate the tumor milieu-immune cell interactions to an anti-cancer phenotype in order to control tumor growth and improve overall survival. Our NP present an average diameter below 200 nm, narrow polydispersity index, slightly negative surface charge, spherical morphology, and high antigens and immune potentiators loading. Cy5-labeled NPs were found to be extensively internalized by DC, which triggered their activation in vivo, shown by the increasing expression of DC-related co-stimulatory molecules such as CD80, CD86, and CD40. Furthermore, we assessed the immunotherapeutic effect of our NP on mouse models and on a human 3D multi-cellular spheroid ex-vivo model, composing the patient’s tumor cells and peripheral blood mononuclear cells (PBMC). We found that the NP successfully induced a potent immune-mediated anti-tumor response against CEACAM5-positive GI tumors, sensitizing the tumor microenvironment to other therapies and decreasing tumor volume, ultimately leading to prolonged survival. Taken together, the developed NP induced a strong antigen-specific immune response and overall sensitized CEACAM5-expressing GI cancers to the clinically relevant treatments in vivo, that so far have shown limited clinical outcomes in addition to a specific T cell anti-tumor activity that was shown on our patient-derived ex vivo model.

Citation Format: Daniella Vaskovich, Rita Acúrcio, Ron Kleiner, Barbara Carreira, Ana Matos, Helena Florindo, Ronit Satchi-Fainaro. A nano sized system for the treatment of gastrointestinal cancers [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 3208.
Abstract 321: Optimized RNA Seq library preparation for fusion calling from FFPE samples
Authors: Corbet Giulia , Haimes Josh , Sanders Travis , Ranik Martin , Harrison Thomas , Casas Eduard , Reed Kailee , Wendel Doug , Giorda Kristina , Hanek Ariele , Pavlica Jen , Kudlow Brian
Published: March 2024

Gene fusions due to chromosomal rearrangement, duplication, or deletion can be important drivers of cancer. Detection of gene fusions plays a valuable role in selecting targeted therapies. A widely used method for detecting fusion transcripts is targeted RNA sequencing, which focuses sequencing reads on known fusions but does not allow for the detection of novel fusions. Whole transcriptome analysis (WTA), where the entire transcriptome is interrogated, offers an opportunity to detect both known and novel fusions.

Herein we examine the effect of sample quality, input mass, increased reverse transcriptase (RT) and insert size on fusion calling. Libraries are prepared using the Watchmaker RNA Library Preparation Kit with or without Polaris Depletion and, for target enrichment, the Twist Exome 2.0 panel prior to paired end 2 × 150 Illumina sequencing. First, control samples, specifically Seraseq Fusion RNA v4 high quality and FFPE samples, are used to determine if any of the parameters interrogated increase the number of breakpoint spanning reads both in a WTA and targeted sequencing context. Once established, the optimized workflow is applied to real FFPE samples.

Targeted sequencing results in a higher number of breakpoint spanning reads, compared to WTA, when controlling for sequencing depth, resulting in a higher number of reads supporting each fusion call. Longer insert sizes, achieved by modified SPRI ratios, maintain library complexity and may enhance fusion calling. Taken together, these data demonstrate an optimized protocol for fusion calling from FFPE and demonstrate the utility of both WTA and targeted sequencing for fusion detection.

Citation Format: Giulia Corbet, Josh Haimes, Travis Sanders, Martin Ranik, Thomas Harrison, Eduard Casas, Kailee Reed, Doug Wendel, Kristina Giorda, Ariele Hanek, Jen Pavlica, Brian Kudlow. Optimized RNA Seq library preparation for fusion calling from FFPE samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 321.
Abstract 323: Optimization of a liquid biopsy assay to accommodate low cfDNA samples and increase throughput
Authors: Salmans Michael , Kim Dasom , Carson Andrew , Wang Mengchi , Lee Byung In , Leatham Bryan
Published: March 2024

Molecular testing that interrogates cell free DNA (cfDNA) from plasma has significant potential value across the cancer care continuum, including early cancer detection, driver mutation identification, and disease recurrence monitoring. To achieve this potential, liquid biopsy assays must be optimized to address multiple challenges. For one, cfDNA quantity in the blood is highly variable, with different amounts present depending on malignancy and stage of progression. This requires optimization to detect low concentrations of circulating tumor DNA (ctDNA) and to accommodate variable tumor fractions. In addition, minimizing assay costs will improve test accessibility and adoption. To address these issues, Genece Health is optimizing their lung cancer liquid biopsy assay to accommodate minimal cfDNA input into library preparation. Furthermore, Genece is testing algorithms that tolerate reduced read depths from low-pass whole genome sequencing (LP-WGS) while maintaining high performance. The Genece lung cancer liquid biopsy assay profiles fragment end-motif and size (FEMS) and fragment coverage signals using LP-WGS (Illumina) and proprietary machine learning algorithms. This study first tested the frequency of end motifs in pooled ctDNA after modifying library preparation (IDT) to reduce reagent volumes from 1X down to 0.5X, 0.25X, and 0.1X of manufacturer specification. A range of cfDNA input from 0.25 ng to 6 ng was tested with each library preparation volume. In addition, LP-WGS read coverage from 5X to 1X was evaluated to assess impact on end motif frequency and assay sensitivity. Performance characterization was further evaluated with a cohort of 50 clinical lung cancer and 100 healthy donor plasma samples tested at 1X and 0.25X library reagent volume. We observed an expected increase in library complexity at higher cfDNA input and library reagent volume. Assay performance was minimally impacted by variable cfDNA input and library reagent volume. However, reducing sequencing read depth is tolerated down to 1.25X depth before increasing the variability of end motif frequencies. These observations are corroborated in the 150 clinical sample cohort. This data shows that by altering standard library preparation conditions and reducing unique read depth, liquid biopsy assays can accommodate a wider range of cfDNA, while concurrently decreasing overall costs.

Citation Format: Michael Salmans, Dasom Kim, Andrew Carson, Mengchi Wang, Byung In Lee, Bryan Leatham. Optimization of a liquid biopsy assay to accommodate low cfDNA samples and increase throughput [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 323.
Abstract 3239: Targeting the GAS41 YEATS domain in non-small cell lung cancer with novel small molecules
Authors: Winkler Alyssa , Listunov Dymytrii , Linhares Brian , Ray Joshua , Weaver Sidney , Zolov Sergey , Keshamouni Venkat , Grembecka Jolanta , Cierpicki Tomaszc
Published: March 2024

Background: Glioma amplified sequence 41 (GAS41) belongs to the Yaf9, ENL, AF9, Taf14, and Sas5 (YEATS) domain family of epigenetic reader proteins. This domain preferentially recognizes acetylated lysine residues on chromatin to facilitate gene expression by recruiting chromatin remodeling and transcription activating complexes. The YEATS4 gene encoding for GAS41 protein is frequently amplified in NSCLC patient samples relative to normal lung tissue, and GAS41 protein is overexpressed in NSCLC cells. In knockdown experiments using NSCLC xenograft mouse models, GAS41 has been shown to be essential for NSCLC tumor growth. In mutagenesis studies, the YEATS domain residues responsible for acetylation reader activity were found to be essential for NSCLC cellular proliferation and tumor progression. Thus, the YEATS domain of GAS41 represents an attractive target for the development of small-molecule inhibitors as potential therapeutics for NSCLC.

Results: We developed the first-in-class small-molecule inhibitors for the GAS41 YEATS domain. Using an NMR-based fragment screen, we identified a hit with low millimolar binding affinity. Structure-based drug design followed by extensive medicinal chemistry optimization yielded the potent and selective inhibitor DLG-369 with sub-micromolar inhibition of the GAS41 YEATS domain interaction with acylated histones. Using the NanoBRET platform, we showed that DLG-369 selectively disrupts the association of GAS41 YEATS with chromatin in mammalian cells. Furthermore, DLG-369 inhibits proliferation of NSCLC cell lines without affecting normal lung fibroblasts. In transcriptomic experiments, DLG-369 treatment regulates expression of multiple genes in NSCLC cells, including upregulation of the critical tumor suppressor genes CDKN1A and CYR61 and downregulation of chemokine genes CXCL5 and CXCR4 associated with NSCLC progression.

Conclusions: We developed DLG-369, a potent inhibitor of the GAS41 YEATS domain with activity in NSCLC cellular models. DLG-369 potently binds to the YEATS domain acetylation reader pocket and disrupts GAS41 recognition of acetylation in cells. DLG-369 potently reduces proliferation and drives differential expression of key genes associated with NSCLC. DLG-369 can serve as a chemical probe to further interrogate the biological role of the GAS41 YEATS domain in NSCLC and other cancers for potential therapeutic implications.

Citation Format: Alyssa Winkler, Dymytrii Listunov, Brian Linhares, Joshua Ray, Sidney Weaver, Sergey Zolov, Venkat Keshamouni, Jolanta Grembecka, Tomaszc Cierpicki. Targeting the GAS41 YEATS domain in non-small cell lung cancer with novel small molecules [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 3239.
Abstract 3270: SH3GLB1 is involved with hydrogen peroxide/akt signaling in glioblastoma multiforme cells
Authors: Chien Chia-Hung , Chang Kwang-Yu
Published: March 2024

Glioblastoma (GBM), a malignant brain tumor, has poor survival outcomes due to recurrence or drug resistance. In our previous studies, it was found that superoxide dismutase 2 (SOD2) was associated with temozolomide (TMZ) resistance. Moreover, SOD2 can convert O2 •− to H2O2, and H2O2 redox signaling is vital for physiological reactions and promotes tumor progression. We used cell lines, patient primary cells and pharmacological inhibitors/activators to confirm the significance of H2O2 signaling. We used the cultured cells to verify roles of H2O2 signaling in the plasticity of cell fate and did animal experiments to identify the optimal treatment strategy. In the present study, SOD2 was found to be the upstream mediator, and combining SOD inhibitor and TMZ, the cells showed reduced SH3GLB1 and autophagy levels. We then found that SH3GLB1 was regulated by H2O2 through AKT signaling using experiments of regulating redox homeostasis. The change of intracellular H2O2 levels in mitochondria following the treatments is similar to that in cytosol, however, the cell fate of parental or resistant cells was quite diverse. Notably, CCCP, a mitochondrial membrane potential disrupter, increased endogenous H2O2 production, and TMZ-increased SH3GLB1 levels can be reversed by HgCl2, reflecting the aquaporin function. In animal models, a combination of TMZ and 4-hydroxynonenal reduced growth of the SH3GLB1-knockdown resistant cells, along with suppressed autophagy levels. Our results characterizing the mechanism of H2O2-SH3GLB1 axis signaling in acquired TMZ resistance of GBM cells will provide new therapeutic strategies against the disease in the future.

Citation Format: Chia-Hung Chien, Kwang-Yu Chang. SH3GLB1 is involved with hydrogen peroxide/akt signaling in glioblastoma multiforme 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 3270.
Abstract 3281: Silencing of necroptosis related genes in endocrine-resistant breast cancer cells causes PFKFB3 induced arrest of necroptosis and contributes to resistance
Authors: Jones Brandon C. , Onal Tuna , Sengupta Surojeet , Sevigny Catherine M. , Jin Lu , Pohlmann Paula R. , Shajahan-Haq Ayesha , Clarke Robert
Published: March 2024

Approximately 70% of newly diagnosed breast cancer patients are estrogen receptor positive. Patients treated with approved treatment strategies may develop resistance to these therapies. Because of this problem in treatment, new treatment regimens need to emerge. Altered reprogramming of metabolism and an intense dependence on glycolysis are hallmarks of cancer, which is necessary to support the high energy demand of rapidly proliferating cancer cells. Rapidly dividing cancer cells are known to have increased energy requirements and altered reprogramming of glucose metabolism, leading to an increased reliance on glycolysis. The conversion of fructose-6-phosphate to fructose-1,6-bisphosphate, a critical rate-limiting step in the glycolysis pathway, is greatly influenced by an allosteric activator produced by the enzyme PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3), thus making PFKFB3 an important regulator of glycolytic flux. Furthermore, PFKFB3 has been shown to be overexpressed in many human cancers, including breast cancer, and upregulated by estrogen. In our previous studies, we have shown that endocrine-resistant BC cells growth is strongly reduced by PFKFB3 targeting using PFKFB3 inhibitors, glucose uptake is reduced, triggered to necroptosis by RIPK1 and MLKL phosphorylation, and PFKFB3 inhibition reduces tumor size in mouse xenografts. In this study, we showed that silencing of RIPK1 and MLKL, genes involved in necroptosis, by siRNAs followed by inhibition of PFKFB3 by PFK158 did not induce necroptosis in endocrine-resistant BC cells. Furthermore, we further confirmed that phosphorylation of MLKL and RIPK occurs with PFKFB3 inhibition. In summary, this study reveals that necroptosis induced by PFKFB3 inhibition contributes to resistance by arresting necroptosis in endocrine-resistant BC cells after silencing of necroptosis-related genes.

Citation Format: Brandon C. Jones, Tuna Onal, Surojeet Sengupta, Catherine M. Sevigny, Lu Jin, Paula R. Pohlmann, Ayesha Shajahan-Haq, Robert Clarke. Silencing of necroptosis related genes in endocrine-resistant breast cancer cells causes PFKFB3 induced arrest of necroptosis and contributes to resistance [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 3281.
Abstract 3284: Transcription factor YBX1 elevating the sorafenib IC50 in hepatocellular carcinoma cell lines
Authors: Kwabiah Dennis , AyalaPazzi Ana , Doxtater Kyle , Leslie Sophia , Tripathi Manish K.
Published: March 2024

Hepatocellular carcinoma (HCC) is the most common form of liver cancer which accounts for more than 90% of occurrences of liver cancer cases. Surgical therapies such as liver transplantation and hepatectomy may help cure hepatocellular carcinoma diagnosed in its early stages. Advanced stages of the disease have a poor prognosis and a high refractoriness because of drug resistance. Sorafenib is a tyrosine kinase inhibitor that has been approved for the systemic treatment of patients with advanced HCC. However, the efficacy of this medication has shown a limited prolongation of the overall survival in patients with advanced HCC. The mechanism of resistance to sorafenib in advanced HCC is poorly understood. The TCGA analysis of the HCC patient cohort shows an elevated expression of YBX1 in tumors as compared to normal. The expression is also correlated with progression, metastasis, and poor survival of HCC patients. Transcription factor YBX1 is involved in the regulation of drug resistance in different cancers, especially breast and colorectal cancers. Our lab is interested in understanding if YBX1 is involved in sorafenib resistance in HCC. Different HCC cell lines procured from ATCC SK-Hep1, Huh-7luc, and C3A, were analyzed for their IC50 values for sorafenib using MTT assays, at different time points. SK-Hep1 showed 8.780µM and 8.174µM, HepG2luc 4.659µM and 3.596µM at 48 and 72 hrs respectively. Overexpression of YBX1 in the SK-Hep1 cell line increased the cell survival and IC50 value of sorafenib at 48 and 72 hrs at 13.19µM and 10.64µM respectively. This supports the hypothesis which demonstrates the role of YBX1 in mediating resistance to sorafenib in advanced HCC. YBX1 could be a major therapeutic target and by inhibiting its action could improve the sensitivity of advanced HCC to sorafenib. We have developed YBX1 overexpressing and knockdown (GFP expressing) cell lines, FACS sorted and Puromycin selected for further mechanistic studies. We are also further developing the sorafenib-resistant HCC cell lines to understand the mechanism underlying sorafenib resistance in HCC. Protein and RNA analyses revealed a relative increase in YBX1 overexpression in sorafenib-resistant cell lines as compared to its parental cell lines. Understanding the mechanism of YBX1-induced drug resistance to sorafenib and its associated signaling pathways could help improve the sensitivity of advanced hepatocellular carcinoma to treatment by sorafenib and thus improve the livelihood of its patients.

Citation Format: Dennis Kwabiah, Ana AyalaPazzi, Kyle Doxtater, Sophia Leslie, Manish K. Tripathi. Transcription factor YBX1 elevating the sorafenib IC50 in hepatocellular carcinoma 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 3284.
Abstract 341: Co-detection of BCR::ABL1 common and atypical fusion and mutation using a novel NGS assay, Dup-Seq BCR-ABL1
Authors: Li Jin , Yan Zhenyu , Shi Lin , Li Wei , Liu Weihua , Spittle Cynthia , Galderisi Chad
Published: March 2024

The clinical management of CML and Ph+ ALL patients requires the identification of BCR::ABL1 transcript variants and drug resistance mutations. Currently these assessments need to be performed using separate assays resulting in higher sample requirements, longer turnaround times and higher costs. In this study a custom NGS assay (Dup-Seq BCR-ABL1) was developed and validated that enables co-detection and identification of transcript variants (common and atypical) and resistance mutations. The assay design covers BCR exon 1 to ABL1 exon 10 and employs duplicate PCR amplification of BCR-ABL1 from a single source of the 1st strand cDNA initiated by ABL1 specific priming. The custom data analysis pipeline enables overlapped mutation calling from duplicates as well as transcript determination. The assay minimizes the low-level artifacts that can be seen with other NGS-based BCR::ABL1 assays. This study demonstrates that the Dup-Seq BCR-ABL1 assay achieves high accuracy (PPA for fusion: 0.985; PPA and NPA for mutation at 0.978 and 0.9999, respectively) and sensitivity (LoD for mutation detection at 3% from 10,000 copies of BCR-ABL1 input). The use of the Dup-Seq BCR-ABL1 assay for both transcript variant identification and mutation detection provides comprehensive and accurate results for the clinical management of CML and Ph+ ALL patients while reducing sample requirements, testing costs and turnaround time.

Citation Format: Jin Li, Zhenyu Yan, Lin Shi, Wei Li, Weihua Liu, Cynthia Spittle, Chad Galderisi. Co-detection of BCR::ABL1 common and atypical fusion and mutation using a novel NGS assay, Dup-Seq BCR-ABL1 [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 341.
Abstract 3451: Association of multi-level factors with cervical cancer screening in healthcare settings
Authors: Rathod Rutu , Hu Ellen , Pruitt Sandi , Tiro Jasmin A. , Chubak Jessica , Haas Jennifer , Atlas Steven J. , Kruse Gina , Hughes Amy E.
Published: March 2024

Background- Few studies have examined the independent contribution of patient, clinician and system factors associated with completing cervical cancer screening.

Methods- Using a large retrospective cohort created from electronic health record data, we examined the association of patient- and provider-level characteristics with cervical cancer screening among unknown- and average-risk, screen eligible women who entered the cohort at a primary care visit in 1 of 3 U.S. healthcare systems (Kaiser Permanente Washington, Parkland Health, Mass General Brigham) between 2010-2019. The outcome was time, in days, from cohort entry to screening. We fit piecewise exponential (PWE) survival models, nesting patients hierarchically within provider seen and the facility at the time of their analytic cohort entry visit. We excluded patients whose visit was with providers at their non-principal facility (principal defined as the facility that they saw most patients in 2010). The analysis was stratified by healthcare setting.

Results- Median time from cohort entry until screening varied across settings (202-1063 days) and across providers and clinics (between cluster variance= 0.11-0.77, 0.06-1.18, respectively). Many patient-level characteristics (age, cohort entry year, race/ethnicity, number of comorbidities, smoking status, primary care visit within 12 months before cohort entry, and insurance), some of the provider-level characteristics (provider specialty and sex) and social determinants of health measures (Local Isolation Score Hispanic) were associated with time to screening. In the adjusted PWE models, for all three settings: a) Younger patients (21-49 years old) (vs older patients (50-65 years)); b) Hispanic and Non-Hispanic Asian/Pacific Islander (vs Non-Hispanic White); and c) patients seeing ob-gyn providers (vs family medicine providers) had shorter time to screening. Patients with a) higher (vs lower) comorbidity burden, and b) Medicare insurance (vs Commercial) were less likely to receive screened.

Conclusion- By identifying characteristics at multiple levels that are associated with the outcome, our results help us identify targets for interventions to improve and increase timely cervical cancer screening in healthcare settings.

Citation Format: Rutu Rathod, Ellen Hu, Sandi Pruitt, Jasmin A. Tiro, Jessica Chubak, Jennifer Haas, Steven J. Atlas, Gina Kruse, Amy E. Hughes. Association of multi-level factors with cervical cancer screening in healthcare settings [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 3451.
Abstract 3488: Decoding splicing isoforms and adaptive evolution in melanoma brain metastases through single cell long-read RNA sequencing
Authors: Lu Lina , Shiau Cheng-Kai , Fukumura Kazutaka , Lin Hsiaoyun , He Yueying , Pan Timothy , Norberg Lisa , Ritchie Jennifer , Huse Jason T. , Gao Ruli
Published: March 2024

Melanoma brain metastasis (MBM) is the third most common origin of metastasis to the brain, with a median overall survival of 8.9 months. Alternative splicing plays a critical role in cancer pathogenesis through altered protein coding complexity in tumor cells, which however has been poorly studied in MBM due to technical limitations. Long-read single cell RNA sequencing has the unique strength of measuring transcript isoforms and mutations simultaneously in individual cells due to its capacity of measuring full-length transcripts. In this study, we employed high throughout single cell long-read RNA sequencing (scNanoRNAseq) onto patient-matched MBM and extracranial metastasis (ECM) tumor pairs to evaluate the extent to which splicing isoforms impact adaptive clonal evolution in MBM. Our results showed that MBM tumors had distinct genetic alterations and ecological cellular compositions compared to matched ECM tumors, indicating that tumor clones were adaptively expanded in brain microenvironment. Comparative isoform analysis revealed that hundreds of genes expressed different combinations of isoforms (DCIs) involved in glucose and lipid metabolic pathways. Interestingly, our data demonstrated that ~10% of splicing related genes themselves were alternatively spliced including both spliceosome members and splicing activators. Furthermore, we calculated temporal changes of isoform usages along with tumor cell evolutionary lineages to quantify their impact on adaptive clonal expansion in MBM. Taken together, our work improves the understanding of transcriptional and splicing dynamics inherent in the adaptive evolution of MBM, providing evidence for developing new therapeutics targeting tumor cell-specific and MBM-specific splicing isoforms.

Citation Format: Lina Lu, Cheng-Kai Shiau, Kazutaka Fukumura, Hsiaoyun Lin, Yueying He, Timothy Pan, Lisa Norberg, Jennifer Ritchie, Jason T. Huse, Ruli Gao. Decoding splicing isoforms and adaptive evolution in melanoma brain metastases through single cell long-read RNA sequencing [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 3488.
Abstract 3494: KINOMO: A non-negative matrix factorization framework for recovering intra- and inter-tumoral heterogeneity from single-cell RNA-seq data
Authors: Tagore Somnath , Wang Yiping , Biermann Jana , D'Souza Edridge K. , Luthria Karan D. , Rabadan Raul , Azizi Elham , Izar Benjamin
Published: March 2024

Single-cell RNA-sequencing (scRNA-seq) is a powerful technology to uncover cellular heterogeneity in tumor ecosystems. Due to differences in underlying gene load, direct comparison between patient samples is challenging, and this is further complicated by the sparsity of data matrices in scRNA-seq. Here, we present a factorization method called KINOMO (Kernel dIfferentiability correlation-based NOn- negative Matrix factorization algorithm using Kullback-Leibler divergence loss Optimization). This tool uses quadratic approximation approach for error correction and an iterative multiplicative approach, which improves the quality assessment of NMF-identified factorization, while mitigating biases introduced by inter-patient genomic variability. We benchmarked this new approach against nine different methods across 150 scRNA-seq experiments and find that KINOMO outperforms prior methods when evaluated with an adjusted Rand index (ARI), ranging 0.82-0.91 compared to 0.68-0.77. Thus, KINOMO provides an improved approach for determining coherent transcriptional programs (and meta-programs) from scRNA- seq data of cancer tissues, enabling comparison of patients with variable genomic backgrounds.

Citation Format: Somnath Tagore, Yiping Wang, Jana Biermann, Edridge K. D'Souza, Karan D. Luthria, Raul Rabadan, Elham Azizi, Benjamin Izar. KINOMO: A non-negative matrix factorization framework for recovering intra- and inter-tumoral heterogeneity from single-cell RNA-seq data [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 3494.
Abstract 3520: A scalable single cell RNA-seq pipeline leveraging machine learning and high-quality references for cell-type prediction
Authors: Dasgupta Abhijit , Duclos Grant , Miragaia Ricardo , Manry Brychan , Jacob Etai , Markuzon Natasha , Rotem Asaf
Published: March 2024

Single cell RNA-seq (scRNA-seq) technology transformed our understanding of biology at the cell level. Inferring cell types provide insights into the relative abundance of, and genomic differences between, different cell types. Current methods leverage known cell type markers and genomic similarity measures to attribute cell types to groups of cells.

We present a scalable machine learning-based pipeline that can leverage high quality reference annotation data to infer cell types quickly for multiple scRNA-seq experimental samples. This pipeline leverages two machine learning technologies: variational autoencoders (VAE) to create a low-dimensional representation of the reference transcriptome; and, supervised learning methods that use this representation to learn cell types from the reference data. This pipeline can quickly score new experimental data using GPU-enabled computing platforms and provide probabilities of a cell being each cell type present in the reference data.

This novel pipeline is evaluated using 5 public reference data sets. We evaluate whether VAE-based representations benefit supervised learning, compared to PCA and t-SNE. We next evaluate different supervised learning methods for predictive performance. Finally, we compare the pipeline’s performance with commonly-used cell type prediction algorithms (Seurat, scANVI). We find that using a VAE is generally better than both PCA and t-SNE for feature generation to predict cell type. Using the VAE representation, there is no significant difference in accuracy between logistic regression and other supervised learning algorithms. Finally, using logistic regression as the learning machine, this pipeline is as accurate as Seurat and better than scANVI, while running about 2-5x faster than Seurat.

Table 1 Balanced accuracy (%) from leave-one-subject-out cross-validation across 5 public data sets. Intervals are the range of the metric across the cross-validation folds.

Algorithm PBMC (10X) HLCA Eraslan snRNA Blueprint Breast Blueprint Lung Our pipeline 92 (87-95) 92 (88-93) 93 (90-97) 89 (80-96) 97 (95-99) Seurat 96 (95-97) 90 (94-98) 94 (93-97) 96 (87-98) 98 (94-99) scANVI 91 (84-95) 72 (78-86) 92 (88-96) 94 (80-97) 88 (86-100)

Citation Format: Abhijit Dasgupta, Grant Duclos, Ricardo Miragaia, Brychan Manry, Etai Jacob, Natasha Markuzon, Asaf Rotem. A scalable single cell RNA-seq pipeline leveraging machine learning and high-quality references for cell-type prediction [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 3520.
Abstract 3576: CRISPR Cas9 mediated MADD deletion can induce autophagy via downregulation of MAPK and PI3K/AKT/mTOR signaling in anaplastic thyroid cancer
Authors: Bakthavachalam Velavan , Madhayan Manikannan , Sanborn Mark , Rehman Jalees , Bellur Prabhakar
Published: March 2024

Thyroid cancer is a highly prevalent endocrine malignancy with one of the fastest growing incidences of all cancers in the United States. Anaplastic thyroid cancer (ATC) has an incidence of about 1.7% of all thyroid cancers, but it causes over 40% of thyroid cancer-related deaths with a median survival period of less than one year. This is why agents that inhibit the aberrant signaling pathways which drive ATC aggressiveness are of significant interest. MAPK-activating Death Domain-activating protein (MADD) plays a key role in cancer cell survival and proliferation. MADD is constitutively expressed at higher levels in most tumor tissues and cancer cells relative to normal tissues. Studies using shRNA suggest that MADD prevents apoptosis in ATC cells. For a more definitive and mechanistic analysis of MADD in ATC, we used conditional CRISPR-Cas9 gene editing to target MADD. We observed that MADD deletion in ATC cells induced spontaneous apoptosis and also activated the autophagy pathway in human ATC cells through downregulation of ERK and mTOR signals involving the MAPK and PI3K/Akt/mTOR signaling pathways. Transcriptomic analysis of publicly available TCGA data showed several PI3K/AKT/mTOR pathway genes were positively correlated with MADD levels. RNA sequencing data of ATC lines demonstrated that enhanced activity of the PI3K/AKT/mTOR signaling pathway was correlated with a worse prognosis in ATC. Targeted MADD deletion in ATC cells combined with treatment using ERK/PI3K/Akt/mTOR/autophagy inhibitors resulted in enhanced cell death compared with MADD deletion alone. Further, MADD deleted 8505C-ATC cells showed significantly delayed tumor formation in an orthotopic nude mouse model. These findings suggest that combining MADD deletion with inhibitors of PI3K/Akt/mTOR pathway could provide an attractive new therapeutic option for ATC.

Citation Format: Velavan Bakthavachalam, Manikannan Madhayan, Mark Sanborn, Jalees Rehman, Prabhakar Bellur. CRISPR Cas9 mediated MADD deletion can induce autophagy via downregulation of MAPK and PI3K/AKT/mTOR signaling in anaplastic thyroid 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 3576.
Abstract 3577: The role of PLXND1 in lineage plasticity and progression to neuroendocrine prostate cancer
Authors: Chen Bo , Xu Pengfei , Yang Joy C. , Ning Shu , Wang Leyi , Nip Christopher , Gao Allen C. , Liu Chengfei
Published: March 2024

Background: Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer and is commonly associated with therapy resistance and poor prognosis. Treatment-induced NEPC (t-NEPC) predominantly develops by lineage plasticity from adenocarcinoma in response to androgen receptor signaling inhibitors, such as enzalutamide. However, the mechanisms underlying the lineage plasticity and progression to NEPC are unclear. PLXND1 (PlexinD1) is a cellular receptor of the semaphorin family and has important functions in modulating the cytoskeleton and cell adhesion. However, the role of PLXND1 in lineage plasticity to NEPC development and progression is not well established.

Methods: We performed transcriptomic analysis on NEPC patients’ cohorts and the enzalutamide-resistant prostate cancer cell lines. Correlation of PLXND1 with neuroendocrine feature genes and AR-targeting genes was determined. Kaplan-Meier method and the Cox proportional hazards model was used to estimate the correlation between PLXND1 expression level and patients’ survival. Immunohistochemistry was used to analyze PLXND1 expression in prostate adenocarcinoma and NEPC. Cell viability was determined in C4-2B MDVR, CWR22Rv1, and H660 cells after knocking down PLXND1 using siRNA or knocking out PLXND1 using CRISPR/CAS9. Organoids viability of patient-derived xenografts was measured after knocking down or knocking out PLXND1.

Results: Our analysis on different patient cohorts suggests that PLXND1 is highly expressed in NEPC patients, and the expression of PLXND1 is positively correlated with neuroendocrine feature genes (CHGA, ENO2, SYP, and NCAM1). Kaplan-Meier survival analysis shows that high expression of PLXND1 is significantly associated with the poor prognosis of prostate cancer patients. PLXND1 is overexpressed and negatively regulated by androgen receptor signaling in enzalutamide-resistant cells and prostate cancer patients’ cohorts. Furthermore, downregulation of PLXND1 expression suppressed proliferation of the NEPC cells and viability of the PDX tumor organoids.

Conclusions: PLXND1 expression is elevated in NEPC and correlated with the poor survival of prostate cancer patients, suggesting it is a potential molecular indicator and therapy target for NEPC.

Citation Format: Bo Chen, Pengfei Xu, Joy C. Yang, Shu Ning, Leyi Wang, Christopher Nip, Allen C. Gao, Chengfei Liu. The role of PLXND1 in lineage plasticity and progression to neuroendocrine prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3577.
Abstract 3584: Context matters: Genomic profiling of PR isoform-specific actions in breast cancer stem cells reveals novel therapeutic insights for ER+ breast cancer
Authors: Gillis Noelle E. , Truong Thu H. , Finlay-Schultz Jessica , Sartorius Carol A. , Lange Carol A.
Published: March 2024

Exposure to progesterone is a recognized risk factor for breast cancer, and PGR polymorphisms are associated with various malignancies. The two progesterone receptor (PR) isoforms, full length PR-B and truncated PR-A, are expressed from the PGR gene in breast tissue and play crucial roles in normal physiology. An imbalance in the expression ratio of these isoforms, favoring increased levels of PR-A, is common in breast cancer and is associated with resistance to tamoxifen in Luminal A-type tumors. Notably, PRs have recently been implicated in promoting endocrine resistance and driving the expansion of cancer stem-like cell (CSC) populations implicated in early dissemination and maintenance of metastatic populations. Because prior gene expression studies were primarily conducted in 2D culture conditions, the isoform-specific molecular and epigenetic mechanisms underlying PR actions in advanced ER+ breast cancer remain elusive. Our goal was to define progesterone-driven gene expression in CSC cells by employing transcriptomic profiling of T47D cells cultivated in spheroid-culture conditions (3D) expressing exclusively PR-A or PR-B. Utilizing CUT&RUN, we have mapped the genomic binding sites unique to each PR isoform and identified shared sites. This genomic data has been integrated with phenotypic studies in cells expressing a single PR isoform. Our findings indicate that PR-A acts as a regulator of the cell cycle and senescence, while PR-B plays a pivotal role in cell metabolism and intracellular signaling. We find little overlap between PR-regulated gene sets derived from the same hormone-treated cells harvested from 2D relative to 3D conditions. Our genomic profiling of PRs in this 3D model system has unveiled novel strategies to combat CSC outgrowth and late recurrence in luminal breast cancer patients. This work has shifted our prior understanding of the role of PRs in gene regulation within the CSC population, and offers potential insights for therapeutic interventions in ER+ breast cancer.

Citation Format: Noelle E. Gillis, Thu H. Truong, Jessica Finlay-Schultz, Carol A. Sartorius, Carol A. Lange. Context matters: Genomic profiling of PR isoform-specific actions in breast cancer stem cells reveals novel therapeutic insights for ER+ 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 3584.
Abstract 3609: Chimeric cytokine receptors increase memory phenotypes and improve expansion and efficacy of CAR-T cell therapies
Authors: Newcomb John R. , Ren Jun , Syed Sakeena , Caruolo Zachary , Webb Lindsay , McKay Craig , Kwei Stephanie , Novina Carl , Mermelstein Fred
Published: March 2024

Cytokine activation of T cells can promote T cell proliferation, memory and improve T cell function in driving tumor cell killing. However, systemic administration of cytokines at therapeutic doses is also associated with unacceptable toxicities. To overcome this limitation, we have engineered a system of private cytokine signaling to affect T cell homeostasis, identity, and function. Specifically, we have engineered chimeric cytokine receptors (CCRs) composed of single-chain variable fragments (scFvs) that bind specifically to small molecules such as fluorescein or DOTA. These scFvs are fused to transmembrane and intracellular domains of all members of the common gamma-chain (γ-chain) family of cytokine receptors. CCRs that permit private cytokine signaling were introduced into known CAR-T cell therapies and their impact on CAR-T cell phenotype, function, and anti-tumor activity was tested. First, CCR-containing T cells were tested for activation-induced changes in phenotype and expansion following stimulation with small molecule conjugates in vitro. The most promising results were obtained using either the IL-7Rα- or IL-2Rβ/γ-containing CCR’s. CCR-containing constructs were then transduced into one of two different anti-BCMA CAR-T constructs, ide-cel or cilta-cel, using a lentiviral transduction system. CAR-T cells containing each CCR were then compared with CAR-T cells lacking CCRs for phenotype and function in vitro. Results indicated that engineering the IL-7Rα CCR with both of these CAR-T’s led to increased expansion, cytokine secretion and improved tumor cell killing. Next, CAR-T cells expressing the IL-7Rα CCR were compared to CAR-T cells lacking CCR for efficacy and durability in a mouse model of human multiple myeloma. IL-7Rα CCR-expressing CAR-T cells demonstrated improved long-term tumor control compared to ide-cel and cilta-cel alone. In addition, IL-7Rα CCR-expression increased T cell numbers in blood, spleen and bone marrow of tumor-bearing mice. These increases were particularly evident in the CD8+population of CAR-T cells in vivo. These results demostrate that private cytokine signaling using small-molecule activatable CCRs can improve efficacy and durability of CAR-T cells. We are testing these CCRs in mouse models of human solid tumors to assess their ability to overcome the immunosuppressive microenvironment.

Citation Format: John R. Newcomb, Jun Ren, Sakeena Syed, Zachary Caruolo, Lindsay Webb, Craig McKay, Stephanie Kwei, Carl Novina, Fred Mermelstein. Chimeric cytokine receptors increase memory phenotypes and improve expansion and efficacy of CAR-T cell therapies [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 3609.