Differential Response Research Articles

Abstract 5628: Aberrant 3'UTR processing regulates a tumor suppressor network in AML

Abstract Differentiation blockade is a hallmark of acute myeloid leukemia (AML). While inactivating mutations in tumor suppressor genes (TSGs) have been well-studied, non-genetic mechanisms that impair TSGs to promote myeloid differentiation and influence drug response remain poorly understood. In this study, we performed genome-wide loss-of-function CRISPR/Cas9 screens to identify suppressors of myeloid differentiation. These screens converged on the zinc finger transcriptional repressor, ZBTB7A whose ablation restricted myeloid maturation and promoted a dysregulated inflammatory state to protect leukemic cells from undergoing differentiation. Moreover, genetic deletion of ZBTB7A or its paralog, ZBTB7B impeded leukemia differentiation and conferred resistance to differentiation-inducing agents. Mechanistically, we found that leukemia cells hijack 3’UTR processes to reduce mRNA expression of the tumor suppressor gene paralogs ZBTB7A and ZBTB7B. These findings provide a molecular framework of altered 3’UTR regulation that orchestrate a tumor suppressive function and promote resistance to AML differentiation-based therapies. Citation Format: Eric Wang, Cuijuan Han, Alexander Calderon, Abimbo Lawal, Sakthi Harini Rajendran, Sadik Karma, Nidhi Hariramani, Iannis Aifantis, Aristotelis Tsirigos. Aberrant 3'UTR processing regulates a tumor suppressor network in AML [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 5628.

Abstract 431: Malic enzyme 2 inhibition reveals metabolic vulnerability in serine-dependent triple-negative breast cancer

Abstract Background & Hypothesis: Treatments for triple-negative breast cancer (TNBC) are limited to chemotherapy, PARP inhibitors and immunotherapies. Tumor metabolism seeks to identify metabolic pathways preferentially utilized by cancer. Malic enzyme 2 (ME2) has a role in the malate-aspartate shuttle and pyruvate and NADPH production, contributing to the redox balance and metabolite synthesis. We demonstrated that ME2 knockdown inhibits growth of TNBC cells with upregulated serine biosynthesis pathway. We hypothesized that ME2 knock-down (kd) would be growth inhibitory in vivo. Methods: We used a non-tumorigenic control (MCF10a) and TNBC lines (BT20, MDA-MB-468, HCC1806, HCC70, Hs578T, SUM149). Nude mice received mammary fat pad xenografts of MDA-MB-468, to assess the impact of ME2-shRNA on growth. ME2 relevant metabolite profiling was achieved through the Promega Metabolite-Glo assay, measuring NAD, NADH, NADP, NADPH, Malate, and Glutamate. Cellular proliferation was quantified via the BioTek BioSpa 8 system and Promega CellTiter Glo. Comprehensive profiling of metabolic genes was achieved with the Nanostring nCounter across all cell lines. The potential therapeutic efficacy of NPD-389, MDSA, disodium embonate and doxorubicin was evaluated, alone or in combination, through targeted drug assays. Metabolic flux analyses, specifically mitochondrial and glycolytic function, were conducted using the Seahorse XF96, contrasting ME2-shRNA modified and control cells. Results: ME2 kd resulted in reduced proliferation in several TNBC cell lines to varying degrees, in contrast to control MCF10a, and to Hs578T. In response to the loss of ME2, several cell lines upregulated serine biosynthesis genes, including PSAT1. MDA-MB-468 ME2 kd cells developed significantly smaller tumors in a nude mouse xenograft model. Increased malate levels, but reduced NADH and NADPH, were detected in TNBC cells with inhibited growth post-ME2 kd, but not in MCF10a or Hs578T cells. Among the tested ME2 inhibitors, NPD-389 had superior efficacy vs. MDSA and disodium embonate. Metabolic flux analysis showed compromised mitochondrial and glycolytic processes in the TNBC cell lines exhibiting growth inhibition upon ME2 kd. Conclusions: ME2 knockdown selectively impairs cell proliferation and tumor growth in TNBC cell lines, validating its potential as a therapeutic target. The differential metabolic response, including altered malate and NADH/NADPH levels, confirms ME2's importance in TNBC metabolism. NPD-389 effectively reduced growth in ME2-sensitive lines, meriting further investigation. These findings support targeting ME2 in as a novel treatment strategy. Citation Format: Mark D. Slayton, Zackariah A. Farah, Jin Heon Jeon, Abhinav Achreja, Ben Krinkel, Brisilda Nilaj, Yi-Hsien Eu, Justin Sung, Alyssa Rosenfeld, Alisa Lui, Mason Collard, Liwei Bao, Xu Cheng, Celina Kleer, Kerry Loomes, Deepak Nagrath, Sofia D. Merajver. Malic enzyme 2 inhibition reveals metabolic vulnerability in serine-dependent triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 431.

Abstract 2984: Prostate cancer associated glycosylated RNAs: Who are you and where are you hiding

Abstract Glycosylated RNA (glycoRNA) is an emerging field. Carbohydrate modifications are vital for biomolecule (proteins and lipids) stability, transport, and localization. Glycosylation influences cell functions like differentiation, apoptosis, and immune response, potentially impacting human disease. The Bertozzi lab initially discovered that RNAs can carry sugar modifications when they metabolically labeled mammalian cultured cells using bioorthogonal chemistry methods normally employed for glycosylated protein and lipid enrichment. Our lab wanted to investigate the translational potential of glycoRNAs and determine if these modifications could be developed as novel clinical targets for aggressive prostate cancer. (PCa). As advanced forms of PCa often lead to poor prognoses, it is imperative that better screening biomarkers and therapeutic treatment options are developed to increase patient survivorship. We predicted that glycoRNA expression would correlate with malignancy, metastatic status, and hormone sensitivity in prostate cells. Our lab used the sialic acid metabolic precursor Ac4ManNAz and click chemistry to label a panel of human prostate cancer cell lines representing the spectrum of prostate disease followed by rigorous RNA isolation procedures and northern blot analysis. We found that human prostate cell lines with no (RWPE-1) or low malignancy (LNCaP) expressed higher levels of glycoRNA belonging to the small <200 nt (but not large) noncoding RNA class compared to aggressive, metastatic PCa cell lines. Our glycosidase studies and lectin northern blot analysis indicated that RNA carries both N-linked and O-linked modifications. Based on these findings, we hypothesized that glycosylated RNA plays an important role in maintaining prostate homeostasis and in cancer protection. To better profile these glycoRNAs, we aimed to: 1) Test a wider range of metabolic labeling regents to characterize the glycoRNA populations using a panel of human prostate cell lines; 2) Perform cellular fractionation studies to determine localization of glycoRNA in prostate cells; and 3) Explore glycoRNAs in a genetic animal model. These studies focused on LNCaP PCa cell lines, which express high levels of glycoRNA. We found that small glycoRNA expression was noted when LNCaP cells were metabolically labeled with Ac4GalNAz (predominantly O-linked) and Ac4ManNAz (predominantly N-linked), verifying glycoRNA heterogeneity. Cellular fractionation studies indicated glycoRNA localization was enriched in the membrane fraction of LNCaP cells using these metabolic labels. Furthermore, we verified that C. elegans grown on Ac4GalNAz plates expressed small glycoRNA, indicating biological conservation. We are now characterizing these glycoRNAs using RNA sequencing and LC-MS/MS glycomics. Our findings will provide novel insights into cancer-associated glycoRNA, leading to improved clinical tools for PCa. Citation Format: Samantha McGuire, Esther Jones, Spencer Moen, Aurora Esquela-Kerscher. Prostate cancer associated glycosylated RNAs: Who are you and where are you hiding [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 2984.

Abstract 1721: Epigenetic heterogeneity of osteosarcoma reveals distinct cellular states driven by enhancer regulation

Abstract Osteosarcoma (OS) is characterized by structural rearrangements and copy number changes which drive considerable heterogeneity. What is less clear is how these genetic rearrangements alter are in turn regulated at the epigenetic level to drive gene expression. Defining the epigenetic circuitry of osteosarcoma could help to understand the role of chromatin accessibility in determining cell plasticity. Chromatin accessibility can prime gene expression and define a cellular state. As chromatin accessibility is a major determinant of epigenetic transcriptional regulation, understanding the variability of chromatin accessibility could identify clinically-relevant subtypes and biomarkers of therapy response in Osteosarcoma, a disease that has seen few advances in therapy for over 40 years. Using ATAC-seq, we identified two epigenetic subtypes (EC1 and EC2) of OS with distinct chromatin accessibility. These studies were done in a panel of 11 PDX-derived cell lines and 9 established OS cell lines. We also performed ATACseq on the matching PDX for the 11 PDX-derived lines as well as 8 primary tumor samples. Analysis of transcription factor (TF) motifs and footprints of ATAC-seq peaks identified transcription factors distinctly activated in these two subtypes. For example, EC1 is characterized by high level of activity of developmental transcription factors including RUNX2, MEF2C and homeobox TFs. In contrast, EC2 is characterized by high activity of AP1 transcription factors such as FOSL1 and FOSL2. In order to understand the underlying transcriptional circuitry of these cell states, we performed CUT&RUN assay for the complete panel of PDX-derived cell lines. We found that the subtypes are driven by enhancer regulation (H3K27ac). We defined the specific Core Regulatory Circuitry (CRC) per subtype by defining the superenhancers by ROSE. We overlapped ATAC-seq data in the acetylated regions to demarcate TF binding regions dramatically and reduce the search space for TF cis-regulatory sites. In parallel, we used our PDX-derived cell line panel to screen cluster-specific targeted therapies and found differential responses dependent on cellular states defined by chromatin accessibility. For example, cell lines in the EC2 cluster are marked by upregulation of AP1 transcription factors, have evidence of ERK activation and are highly responsive to ERK inhibitors. In contrast cell lines in the EC1 cluster are sensitive to AURKA and AURKB inhibitors. Single-cell RNAseq analysis indicates that these subtypes may coexist within a single tumor. In summary, we discovered two epigenetically distinct cell states that are controlled by a state-specific collection of transcription factors, creating a gene signature that may aid in distinguishing osteosarcoma subclasses and predicting treatment response. Citation Format: Eunice Lopez Fuentes, Andrew Clugston, Alex Lee, Leanne Sayles, Maria Pons, Alejandro Sweet-Cordero. Epigenetic heterogeneity of osteosarcoma reveals distinct cellular states driven by enhancer regulation [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 1721.

Abstract 228: Patient-derived gbm organoids reflect tumour heterogeneity and treatment sensitivity in patients

Abstract Glioblastoma (GBM) is the most common and incurable adult primary brain tumour. Treatment resistance and relapse is the primary cause of death in GBM. Tumour heterogeneity is one of the significant determinants of treatment failure. Patient-derived cancer organoids have been shown to maintain genetic and phenotypic tumour features and recapitulate intratumoral heterogeneity. Here, we report the development and molecular characterisation of patient-derived GBM organoid (PGO) models to study treatment response to existing and new treatment combinations. GBM biopsies were dissociated, suspended in Cultrex, and cultured in a brain stem cell medium. PGOs were characterised by whole-exome (WES) single-cell karyotype (scKaryoSeq) and RNA sequencing. The IC-50 and viability of PGO upon temozolomide (TMZ) and small molecule inhibitors were determined. Gene expression profiling was performed before and after TMZ treatment. Single-cell spatial heterogeneity was determined using Multiplex Immunostaining of PGO and matched patient samples. PGOs were established with a success rate of 80% (n=31). WES analysis between PGOs from the same patient at multiple time points (1-3 months) showed high maintenance of Single Nucleotide Variants (92.3%-97.7%). In PGOs key somatic oncogene variants and copy number variations were retained compared to the primary tumour (58%-90%). scKaryoseq (N=7) of different clinical GBM subtypes showed a high degree of genetic heterogeneity, with some PGO having up to 10 subclones. Single-cell phenotypic landscaping of patient biopsies showed mixed cellular states maintained after long-term culture in the matched PGO. PGOs showed a differential response to TMZ, corresponding with MGMT methylation and patient mutation status. RNAseq of PGO revealed TMZ-responsive genes, including upregulation of the prognostic JUN kinase pathway. Combination treatment of a JUN kinase inhibitor and TMZ showed a synergistic effect. In addition, PGOs were screened for their sensitivity to clinically approved brain-penetrable small molecule inhibitors targeting key GBM driver pathways. Combining small molecular inhibitors of MEK/PI-3/CDK4/EGFR kinase strongly reduced cell viability and activation of cell death pathways. phosphoKinome screens detected compensatory pathway activation upon kinase inhibition (MEK/CDK4 inhibitor) in several PGO. Data will show novel kinase interaction pathways that increase drug sensitivity in PGO. These data show that PGOs can be used as a stable phenotypic and genetic representative of the patient's tumour and have the potential to function as patient avatars for adaptive treatment selection and actionable drug target discovery. Citation Format: Marc A. Vooijs, Linde Hoosemans, Jolanda Piepers, Maikel Verduin, Maxime Vanmechelen, Mike Van Heumen, Frederik De Smet, Ann Hoeben. Patient-derived gbm organoids reflect tumour heterogeneity and treatment sensitivity in patients [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 228.

Abstract 6765: A patient-derived hepatocellular carcinoma multicellular spheroid platform harnessing autologous patient serum to model the tumor microenvironment and treatment response

Abstract Hepatocellular carcinoma (HCC) is the third leading and fastest rising cause of cancer-related death worldwide. The discovery and preclinical development of compounds targeting HCC is hampered by the absence of authentic tractable systems recapitulating the heterogeneity of patient HCC tumors and the tumor microenvironment (TME). Here, we established a novel, simple and robust patient-derived multicellular tumorspheroid model based on clinical HCC tumor tissues combined with the autologous patient serum. The model was robust and reproducible independently from cancer etiology and grade recapitulating key functional features of original tissue. We show that the use of autologous patient serum was essential for the TME function. The tumorspheroids comprised the main cell compartments including epithelial cancer cells as well as all major cell populations of the TME (cancer-associated fibroblasts, macrophages and T cells). Cell type proportions were variable between tumorspheroids reflecting HCC heterogeneity. Moreover, we observed differential responses to FDA HCC approved drugs (including tyrosine kinase and checkpoint inhibitors as well as VEGF-targeting agents) between donors as in patients. This patient HCC serum-tumorspheroid model provides novel opportunities for drug discovery and development as well mechanism of action studies including compounds targeting the TME. In conclusion, this HCC serum-tumorspheroid platform will contribute to improve the dismal outcome of patients with HCC. Citation Format: Emilie Crouchet, Nuno Almeida, Sarah C. Durand, Marie Parnot, Marine A. Oudot, Fabio Giannone, Cloé Gadenne, Sara Cherradi, Natascha Roehlen, Antonio Saviano, Emanuele Emanuele Felli, Patrick Pessaux, Kazuaki Chayama, Hong Tuan F. Duong, Thomas F. Baumert, Catherine Schuster. A patient-derived hepatocellular carcinoma multicellular spheroid platform harnessing autologous patient serum to model the tumor microenvironment and treatment response [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 6765.

Abstract 6891: Establishment and characterization of a panel of naïve and pretreated small cell lung cancer (SCLC) XPDX models

Abstract SCLC is an aggressive cancer of neuroendocrine origin, accounting for approximately 15% of all lung cancer cases. While the incidence of SCLC in the US has declined over past decades due to changes in smoking habits, this disease provides a poor prognosis and few effective treatment options for advanced disease. To better identify useful therapies for SCLC, we established and characterized a panel of 18 XPDX models representing primary and metastatic disease from either naïve or clinically treated patients and tested sensitivities towards chemotherapies and targeted agents. 18 models were established in immune-deficient mice including 8 from newly diagnosed patients (2 from lung primary), and 10 from recurrent disease (3 from lung primary). Models were IHC stained for Nectin-4, Trop2 protein and ERBB2 and profiled using WES and RNAseq. For in vivo studies, models were evaluated against several chemotherapies including cisplatin, etoposide, topotecan, and temozolomide, as well as ADC agents trastuzumab deruxtecan (T-DXd), enfortumab vedotin (EV), and sacituzumab govitecan (SG). Targeted therapies toward CDK4/6, KRASG12C, and PARP were evaluated in some models. Endpoints in all studies included tumor volume and time from treatment initiation with %T/C values and tumor regression reported at study completion; a %T/C of ≤ 20% versus control was considered sensitive. Tumor regression (%T/C<0%) versus Day 0 tumor volume was also reported. Less than 10% of models stained positive for Nectin-4, Trop-2 or ERBB2. Molecular characterization revealed RB1 frameshifts or deletions in most models, KRAS/NRAS point mutations in 3 models, and 2 gene fusions SEPTIN7P2-PSPH and TVP23C-CDRT4. In vivo studies reported differential responses to chemotherapies with the most sensitive models established from treatment-naïve patients. Temozolomide was active in several studies including tumor regressions in both naïve and pretreated models. ADCs also reported differential activity toward the tested panel. We have established and characterized a panel of SCLC XPDX models and benchmarked them against various cancer therapies. This panel can be utilized as a valuable tool in developing novel therapies for SCLC patients. Citation Format: Gabriela Huelgas Morales, Peter Forofontov, Johnnie Flores, Alyssa Simonson, Armando Diaz III, Jim Lund, Natalia Banos, Kyriakos Papadopoulos, Drew Rasco, Amita Patnaik, Ali Abedi, Napoleon Puente, Emiliano Calvo, Victor Moreno, Ronald Drengler, Lon Smith, Michael Wick. Establishment and characterization of a panel of naïve and pretreated small cell lung cancer (SCLC) XPDX 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 6891.

Abstract 3876: A spatial transcriptomic study of a triple-negative breast cancer (TNBC) patient-derived xenograft (PDX) model of residual disease refractory to conventional chemotherapy

Abstract Combating chemotherapy resistance in TNBC is critical to improving quality of care and reducing fatality. To understand chemoresistance, it has been proposed that the spatial interactions between cell types in the tumor microenvironment (TME) could offer insights into the differential response to therapy and metastatic potential. Thus, this study utilizes spatially resolved transcriptomics (SRT) to profile the spatial interactions in TNBC TME. With the goal of understanding chemoresistance and providing a proof-of-concept for new technology, we launched a longitudinal SRT study of a TNBC PDX of residual disease before, during, and after adriamycin and cyclophosphamide (AC) treatment (Tx). SRT by 10X Visium was performed on Veh, AC-residual tumor (21 d post-Tx), and AC-regrown tumor (50 d post-Tx when tumors regrew). This study is divided into two parts. 1. Due to the lack of specialized tools for processing xenograft reads from SRT, we developed the Xenomake pipeline, which combines xenograft sorting and spatial barcode demultiplexing to assign reads into the host and graft organisms per spatial spot. It permits clustering the spots into stroma-rich (mouse), and epithelial-rich (human) regions. We show that it can find differential cytokines in the stroma (S) and epithelium (E), and finding SS, EE, and SE interactions. 2. Although using PDX necessitates immune-compromised mice, several types of stromal populations are present and analyzable. Thus, using Xenomake, we inferred the localization of stromal cell types in Visium spots including cancer associated fibroblasts (CAF), macrophages (MP), endothelial (ENDO), monocytes, and perivascular cells. We compared the spatial localization profiles (SLP) of stromal cell types across samples. With human reads, we computed the spatial pathway activity map (SPAM) for HALLMARK pathways. We correlated SPAM with stroma cell type SLP to survey stroma-epithelial interactions. Stroma cells are distributed in the tumor periphery in vehicle and AC50, while in AC21 there is a notable increase in stroma abundance and stroma infiltration within tumor mass. SLP of MP (Cd68+ and Csf1r+) is correlated with ENDO (Pecam1+), and CAF (Acta2+ and Pdgfrb+). Additionally, all 3 cell types are correlated with Vim and Cd44 expression. Although all samples show enrichment of MP, ENDO, and CAF, they interact differently with pathway activities of adjacent epithelial cells. In vehicle and AC50, the 3 cell types are colocalized with OXPHOS, MYC targets, E2F pathway, while in AC21, a switch to colocalization with EMT is observed. Further, hypoxia response and glycolysis display anti-correlation with stroma cell types, meaning that these pathway activities are further away from stroma and occupy distinct territories. The results suggest stroma-tumor metabolic crosstalk and ways of targeting residual disease. Citation Format: Benjamin Strope, Katherine Pendleton, William Bowie, Gloria Echeverria, Qian Zhu. A spatial transcriptomic study of a triple-negative breast cancer (TNBC) patient-derived xenograft (PDX) model of residual disease refractory to conventional chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3876.

Transcriptomic changes behind Sparus aurata hepatic response to different aquaculture challenges: An RNA-seq study and multiomics integration.

Gilthead seabream (Sparus aurata) is an important species in Mediterranean aquaculture. Rapid intensification of its production and sub-optimal husbandry practices can cause stress, impairing overall fish performance and raising issues related to sustainability, animal welfare, and food safety. The advent of next-generation sequencing technologies has greatly revolutionized the study of fish stress biology, allowing a deeper understanding of the molecular stress responses. Here, we characterized for the first time, using RNA-seq, the different hepatic transcriptome responses of gilthead seabream to common aquaculture challenges, namely overcrowding, net handling, and hypoxia, further integrating them with the liver proteome and metabolome responses. After reference-guided transcriptome assembly, annotation, and differential gene expression analysis, 7, 343, and 654 genes were differentially expressed (adjusted p-value < 0.01, log2|fold-change| >1) in the fish from the overcrowding, net handling, and hypoxia challenged groups, respectively. Gene set enrichment analysis (FDR < 0.05) suggested a scenario of challenge-specific responses, that is, net handling induced ribosomal assembly stress, whereas hypoxia induced DNA replication stress in gilthead seabream hepatocytes, consistent with proteomics and metabolomics' results. However, both responses converged upon the downregulation of insulin growth factor signalling and induction of endoplasmic reticulum stress. These results demonstrate the high phenotypic plasticity of this species and its differential responses to distinct challenging environments at the transcriptomic level. Furthermore, it provides significant resources for characterizing and identifying potentially novel genes that are important for gilthead seabream resilience and aquaculture production efficiency with regard to fish welfare.

Abstract 6899: Enfortumab vedotin (EV): Efficacy comparison with trastuzumab deruxtecan (T-DXd) and sacituzumab govitecan (SG) and its potential for combination benefit in bladder cancer XPDX models

Abstract EV is a Nectin-4 targeting antibody-drug conjugate (ADC) with an MMAE payload, recently approved for bladder cancer treatment. We established and characterized 18 bladder XPDX models representing primary and metastatic disease from naïve or clinically treated patients, some with actionable mutations. To better understand the potential additive benefit of EV in bladder cancer, we evaluated the ADC alone and in combination with agents targeting ERBB2, PIK3CA or FGFR3. In addition, we tested EV alone in ST5420B, a model established from a patient who progressed on EV after five cycles, as well as ST975B, a model which harbors a novel NECTIN4 fusion. 18 bladder XPDX models were established in immune-deficient mice, including 5 from newly diagnosed patients (2 from primary site), and 13 from recurrent disease (2 from primary site). Nectin-4, Trop2 protein and ERBB2 receptor expression was tested, and models profiled using WES and RNAseq. For in vivo studies, all models were evaluated against cisplatin, T-DXd, SG, and EV. Models with actionable mutations were tested with alpelisib, erdafitinib, and neratinib alone and in combination with EV. Endpoints in all studies included tumor volume and time from treatment initiation with %T/C values and tumor regression reported at study completion; a %T/C of ≤ 20% versus control was considered sensitive. Tumor regression (%T/C&amp;lt;0%) versus Day 0 tumor volume was also reported. Most models stained positive for Nectin-4 and Trop-2. FGFR3 variants were found in five models and ERBB2 mutations in two while several models reported PIK3CA variants; deletions in RB1 and CDKN2A were common. In vivo studies reported differential responses to cisplatin with the most sensitive models established from treatment-naïve patients. EV and SG tested alone reported activity in several models; EV added benefit in some combination treatments. We have characterized a panel of bladder XPDX models and benchmarked them against T-DXd, EV and SG alone and EV in combination with targeted therapies. This data is a valuable tool in further developing EV and identifying novel therapies for bladder cancer. Citation Format: Johnnie Flores, Heaven Sessions, Alyssa Simonson, Natalia Banos, Tahmineh Rouzbahani, Armando Diaz III, Jim Lund, Emiliano Calvo, Victor Moreno, Kyriakos Papadopoulos, Drew Rasco, Amita Patnaik, Scott Ulmer, Luis Rodriguez, Michael Wick. Enfortumab vedotin (EV): Efficacy comparison with trastuzumab deruxtecan (T-DXd) and sacituzumab govitecan (SG) and its potential for combination benefit in bladder cancer XPDX 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 6899.

Abstract 4724: Optimizing 500 pooled PRISM cell lines for a 10-day assay to elucidate viability effects and mechanism of action for slower-acting therapeutics

Abstract PRISM (Profiling Relative Inhibition Simultaneously in Mixtures), using DNA-barcoded cell lines cultured as ~25 cell line pools, has been an effective drug discovery screening platform to rapidly test large numbers of potential therapeutics on over 900 different cancer cell lines. However, the conventional 5-day assay may be too short to offer the resolution needed to identify the full cellular response for slower-acting therapeutics such as those directed against epigenetic pathways. Here, we describe development of the PRISM 10-day extended assay with ~500 adherent solid tumor cancer cell lines to overcome this challenge. We found that the ranges of cell line growth rates within the standard PRISM pools were too large and not amenable for a long-term assay beyond 6 days—after 10 days of growth, slower cell lines dropped below the limit of barcode detection as faster cell lines overtook the pools. We therefore regenerated pools to reduce the range of growth rates within a pool. We show that this re-pooling, coupled with seeding density optimization, enables recovery of &amp;gt; 98% of cell lines at day 10 in vehicle-treated wells, with acceptable data variability. The average estimated number of cell doublings across our cell lines, 7, is significantly greater than the number of doublings in the PRISM standard 5-day assay (~3 doublings). Importantly, we observe greater drug effects on day 10 versus day 6 when cells are treated with slow-acting DNA methyltransferase inhibitors such as decitabine; this also results in increased sensitivity to identify cellular biomarkers underlying differential response. In summary, we have successfully engineered a PRISM 10-day assay in ~500 adherent cell lines, where the extended duration can be beneficial in elucidating the effects and mechanism of action for epigenetic drug candidates. Citation Format: Melissa Ronan, Blanche C. Ip, Ellen Nguyen, Ursula Widocki, Tenzin Sangpo, Alvin Kalathungal, Nashielli Diaz-Gallegos, Anthony Fazio, Aydin Golabi, Maha Naim, Ashish Bino George, YuhJong Liu, Mustafa Kocak, Rishi Puram, Matthew G. Rees, Jennifer Roth. Optimizing 500 pooled PRISM cell lines for a 10-day assay to elucidate viability effects and mechanism of action for slower-acting therapeutics [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 4724.

Abstract 471: Attenuation of circulating miR-17-5p, miR-20b-5p, and miR-106a-5p in low-risk prostate cancer patients following proton therapy

Abstract Introduction: Liquid biopsies offer a novel approach to diagnose, predict cancer outcomes, and monitor treatment response by analyzing tumor-derived substances. Circulating cell-free microRNAs (miRNAs) show clinical promise as biomarkers within prostate cancer (PCa) and have been implicated with PCa prognosis and outcomes. However, their potential for monitoring and assessing radiotherapy (RT) treatment response remains understudied. Understanding miRNA expression changes during RT is crucial, as they impact RT efficacy by directly affecting genes and proteins within RT-response pathways. Aim: This study aimed to profile the expression of three oncogenes, miR-17-5p, miR-20b-5p, and miR-106a-5p and to determine their association with PCa risk groups in patients receiving proton therapy (PT). Patients and Methods: Plasma was obtained from the University of Florida Health Proton Therapy Institute Outcomes Tracking Protocol biobank. Plasma was collected from men diagnosed with localized PCa without metastatic disease at three time points: prior to PT (PRE-PT), at 4 weeks following the start of PT (4WKSPD1), and at 8 weeks following the start of PT (8WKSPD1). The pilot study consisted of 38 patients, with 23 categorized as low-risk and 15 categorized as high-risk for PCa. MiRNA expression levels were evaluated using reverse transcription-polymerase chain reaction and quantified using the Livak method. Results: The study revealed a downregulation of miR-17-5p in response to PT, with median gene expression levels decreasing from 0.73 at PRE-PT to 0.587 at 8WKSPD1. Low-risk PCa patients also showed distinct miRNA expression changes with all three miRNAs (miR-17-5p, miR-20b-5p, and miR-106a-5p) downregulated from 0.953, 1.037, and 1.010 at PRE-PT to 0.398, 0.216, and 0.298 at 8WKSPD1, respectively. Statistical analysis using a Kruskal-Wallis test followed by a Dunn's post-hoc test identified significant differences (p=0.035) specifically between PRE-PT and 8WKSPD1 for miR-17-5p. Conclusion: The study revealed that PT led to a time-dependent reduction in miRNA levels, indicating a higher responsiveness in low-risk compared to high-risk PCa patients. This differential response highlights miR-17-5p, miR-20b-5p, and miR-106a-5p as potential PT response biomarkers, with clinical implications for personalizing PCa treatment. These results underscore the necessity for further validation of these miRNAs to maximize their clinical utility. Citation Format: Johnny Velasquez, Curtis Bryant, Nancy Mendenhall, Luisel Ricks-Santi. Attenuation of circulating miR-17-5p, miR-20b-5p, and miR-106a-5p in low-risk prostate cancer patients following proton therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 471.

Differential response of soil bacteria and fungi to carbon and respiration components in abandoned grasslands on the Loess Plateau, China

Differential response of soil bacteria and fungi to carbon and respiration components in abandoned grasslands on the Loess Plateau, China

An electrochemical tool for food additive: A nanoengineered sustainable approach on the preparation of t-LaVO4 rods decorated on g-carbon nitride nanocomposite for determination of theobromine

The dimethylxanthine alkaloid of theobromine (TB) naturally occurs in plants and their products. Derivatives of methylxanthine can potentially induce detrimental impacts on human health if ingested in excessive quantities or by individuals who are particularly susceptible to their effects. Therefore, a differential pulse voltammetry (DPV) method utilizing an electrode modified with a nanocomposite was proposed as an analytical approach for determining TB. Herein, the rare earth metal vanadate (REVO4) has recently played a key role in its use as an electrocatalyst in electrochemical sensors. REVO4's electronic characteristics enable electrochemical, optical, or impedance-based signal transduction and sensitive analyte detection. Due to these characteristics, REVO4 boosts sensing target analyte signals, enhancing sensor system sensitivity. In this paper, we constructed a sensor with nanostructured g-C3N4 entrapped LaVO4 nanomaterial successfully combined to make a nanocomposite to detect food analyte. Various voltammetric and spectroscopic techniques characterized the successfully prepared material LaVO4@g-C3N4 and its electrochemical behaviors. The electrochemical detection of TB, differential pulse voltammetric response was obtained in the wide linear range of 0.001–365 μM, the limit of detection (LOD) is 8 nM and the sensitivity of 4.03 μA μM−1 cm−2. The sensing platform of the electrochemical sensor successfully detected the extract of food samples such as water and chocolate milk samples and exhibited good recovery values of ±98.6–99.0%. The constructed sensor provides a robust means for monitoring alkaloids in complex matrices and a promising opportunity to develop sensitive and selective electrode materials with good reusability.

Platelet-activating factor receptor (PAFR) regulates neuronal maturation and synaptic transmission during postnatal retinal development.

Platelet-activating factor (PAF), PAF receptor (PAFR), and PAF- synthesis/degradation systems are involved in essential CNS processes such as neuroblast proliferation, differentiation, migration, and synaptic modulation. The retina is an important central nervous system (CNS) tissue for visual information processing. During retinal development, the balance between Retinal Progenitor Cell (RPC) proliferation and differentiation is crucial for proper cell determination and retinogenesis. Despite its importance in retinal development, the effects of PAFR deletion on RPC dynamics are still unknown. We compared PAFR knockout mice (PAFR-/-) retinal postnatal development proliferation and differentiation aspects with control animals. Electrophysiological responses were analyzed by electroretinography (ERG). In this study, we demonstrate that PAFR-/- mice increased proliferation during postnatal retinogenesis and altered the expression of specific differentiation markers. The retinas of postnatal PAFR-/- animals decreased neuronal differentiation and synaptic transmission markers, leading to differential responses to light stimuli measured by ERG. Our findings suggest that PAFR signaling plays a critical role in regulating postnatal RPC cell differentiation dynamics during retinal development, cell organization, and neuronal circuitry formation.

B-cell receptor signaling activity identifies patients with mantle cell lymphoma at higher risk of progression

Mantle cell lymphoma (MCL) is an incurable B-cell malignancy characterized by a high clinical variability. Therefore, there is a critical need to define parameters that identify high-risk patients for aggressive disease and therapy resistance. B-cell receptor (BCR) signaling is crucial for MCL initiation and progression and is a target for therapeutic intervention. We interrogated BCR signaling proteins (SYK, LCK, BTK, PLCγ2, p38, AKT, NF-κB p65, and STAT5) in 30 primary MCL samples using phospho-specific flow cytometry. Anti-IgM modulation induced heterogeneous BCR signaling responses among samples allowing the identification of two clusters with differential responses. The cluster with higher response was associated with shorter progression free survival (PFS) and overall survival (OS). Moreover, higher constitutive AKT activity was predictive of inferior response to the Bruton's tyrosine kinase inhibitor (BTKi) ibrutinib. Time-to-event analyses showed that MCL international prognostic index (MIPI) high-risk category and higher STAT5 response were predictors of shorter PFS and OS whilst MIPI high-risk category and high SYK response predicted shorter OS. In conclusion, we identified BCR signaling properties associated with poor clinical outcome and resistance to ibrutinib, thus highlighting the prognostic and predictive significance of BCR activity and advancing our understanding of signaling heterogeneity underlying clinical behavior of MCL.

BK ZERO isoform HEK293 stably transfected cell lines differing 3'UTRs to assess miR-9 regulation.

Research has identified the large conductance voltage- and calcium-activated potassium channel (BK) as a key regulator of neuronal excitability genetically associated to behavioral alcohol tolerance. Sensitivity to ethanol at the molecular level is characterized by acute potentiation of channel activity. BK isoforms show variations in alcohol sensitivity and are differentially distributed on the plasma membrane surface in response to prolonged exposure. MicroRNA (MiRNA) targeting of alcohol-sensitive isoforms coupled with active internalization of BK channels in response to ethanol are believed to be key in establishing homeostatic adaptations that produce persistent changes within the plasma membrane of neurons. In fact, microRNA 9 (miR-9) upregulated expression is a key event in persistent alcohol tolerance mediating acute EtOH desensitization of BK channels. The exact nature of these interactions remains a current topic of discussion. To further study the effects of miR-9 on the expression and distribution of BK channel isoforms we designed an experimental model by transfecting human BK channel isoforms ZERO heterologous constructs in human embryonic kidney cells 293 (HEK293) cells respectively expressing 2.1 (miR-9 responsive), 2.2 (unresponsive) and control (no sequence) 3'untranslated region (3'UTR) miRNA recognition sites. We used imaging techniques to characterize the stably transfected monoclonal cell lines, and electrophysiology to validate channel activity. Finally, we used immunocytochemistry to validate isoform responsiveness to miR-9. Our findings suggest the cell lines were successfully transfected to express either the 2.1 or 2.2 version of ZERO. Patch clamp recordings confirm that these channels retain their functionality and immunohistochemistry shows differential responses to miR-9, making these cells viable for use in future alcohol dependence studies.

Differential Immune Response Patterns Induced by Anionic and Cationic Lipid Adjuvants in Intranasal Anti-Influenza Immunization.

Currently, vaccine development against different respiratory diseases is at its peak. It is of utmost importance to find suitajble adjuvants that can increase the potency of the vaccine candidates. This study aimed to determine the systemic and splenic immune mechanisms in mice models induced by anionic and cationic lipid adjuvants in the presence of the vaccine-candidate influenza antigen hemagglutinin (HA). In the presence of the HA antigen, the cationic adjuvant (N3) increased conventional dendritic cell 1 (cDC1) abundance with enhanced MHCI and CD80-CD86 costimulatory marker expression, and significantly higher CD8T and Th17 populations with enhanced interferon-gamma (IFNγ) expression in CD8T and CD4T populations. Conversely, the anionic adjuvant (L3) increased the cDC2 population percentage with significantly higher MHCII and DEC205 expression, along with an increase in the CD4T and regulatory T cell populations. The L3-treated group also exhibited higher percentages of activated B and plasma cell populations with significantly higher antigen-specific IgG and IgA titer and virus neutralization potential. While the anionic adjuvant induced significantly higher humoral responses than the cationic adjuvant, the latter influenced a significantly higher Th1/Th17 response. For customized vaccine development, it is beneficial to have alternative adjuvants that can generate differential immune responses with the same vaccine candidate antigen. This study will aid the selection of adjuvants based on their charges to improve specific immune response arms in the future development of vaccine formulation.

Genotype x Environment Interaction and Stability Analysis for Seed Yield in Desi Chickpea (Cicer arietinum L.) under Irrigated Condition

Background: Chickpea is mainly grown on marginal lands and encounter the problem of erratic rainfall that causes lack of water availability especially at terminal growth stages. Exploration of yield stability and defining the adaptation domains of advance genotypes in different ecosystem is highly imperative for crop genetic improvement programs. Development of variety with high yield potential and stable under irrigated condition is one of the major objectives of chickpea improvement programme, which can be achieved by improving selection efficiency with estimating stability parameters. Ten chickpea genotypes as advance breeding lines and popular varieties were evaluated to assess seed yield stability under irrigated condition during rabi 2021-22. Methods: Experiments were conducted in randomized block design with three replications at five different locations of Karnataka i.e. Bidar, Kalaburagi, Bheemarayanagudi, Raichur and Hagari. The interaction analysis (AMMI ANOVA) indicates large variations for seed yield due to highly significant (p less than 0.01) environment (65.42%) and significant (p less than 0.05) genotype (10.03%), thus the genotypes were diverse with large sum of squares for seed yield and environment. Result: The differential response of chickpea genotypes for seed yield across the environments was observed. IPCA1 and IPCA2 together captured 12.97 per cent of the interaction mean squares RG-2016-134 and KCD-2019-05 showed higher mean yields and adaptable to favourable environments. Based on AMMI bi-plot analysis, genotypes (KCD-2019-05 and Super Annigeri-1) were stable and (DC-17-1111 and RG-2016-134) were unstable. Similarly, best performer genotypes and three mega environments were noticed through the analysis. Three environments i.e., (Bidar, Kalaburagi and Raichur) were found with most selective for seed yield estimation. The genotypes (RGV-203, KCD-2019-02 and Super Annigeri-1) were the best among all the ten tested genotypes in terms of seed yield stability.

Single-cell RNA sequencing unveils the hidden powers of zebrafish kidney for generating both hematopoiesis and adaptive antiviral immunity

The vertebrate kidneys play two evolutionary conserved roles in waste excretion and osmoregulation. Besides, the kidney of fish is considered as a functional ortholog of mammalian bone marrow that serves as a hematopoietic hub for generating blood cell lineages and immunological responses. However, knowledge about the properties of kidney hematopoietic cells, and the functionality of the kidney in fish immune systems remains to be elucidated. To this end, our present study generated a comprehensive atlas with 59 hematopoietic stem/progenitor cell (HSPC) and immune-cells types from zebrafish kidneys via single-cell transcriptome profiling analysis. These populations included almost all known cells associated with innate and adaptive immunity, and displayed differential responses to viral infection, indicating their diverse functional roles in antiviral immunity. Remarkably, HSPCs were found to have extensive reactivities to viral infection, and the trained immunity can be effectively induced in certain HSPCs. In addition, the antigen-stimulated adaptive immunity can be fully generated in the kidney, suggesting the kidney acts as a secondary lymphoid organ. These results indicated that the fish kidney is a dual-functional entity with functionalities of both primary and secondary lymphoid organs. Our findings illustrated the unique features of fish immune systems, and highlighted the multifaced biology of kidneys in ancient vertebrates.