Cell Line Screening Research Articles

Genomic predictors of drug sensitivity in cancer: Integrating genomic data for personalized medicine in the USA

Despite applying conventional predictive methodologies to obtain genomic insights, predicting drug sensitivity for healthcare organizations in the USA remains a daunting challenge. Cancer is a highly dynamic, adaptive disease tumor cells have repeatedly shown the capability to evolve mechanisms whereby therapeutic interventions can be evaded. Besides, one genomic alteration seldom predicts drug sensitivity. This research project aimed to address the challenges of predicting drug sensitivity by leveraging the GDSC dataset, an extensive resource connecting genomic profiles of cancer cell lines with their sensitivity to a wide range of anti-cancer drugs. This research's key focus was identifying robust genomic markers, including any specific mutations, gene expression patterns, or epigenetic modifications associated with drug sensitivity or resistance. Advanced machine learning and statistical methods were utilized by the predictive models to analyze complex relations that may exist between different genomic alterations and their drug sensitivity. The dataset used for this research project was derived from the Kaggle website. This dataset was compiled by the research project Genomics of Drug Sensitivity in Cancer collaboration between the Sanger Institute in the United Kingdom and the Massachusetts General Hospital Cancer Center in the United States. In their investigation, there was a massive screening of human cancer cell lines with a wide range of anti-cancer drugs. Data collection was performed by large-scale screening of diverse anti-cancer drugs against human cancer cell lines of various types. Cell viability was measured using the Cell-Titer-Glo assay following 72 hours of drug treatment. Several machine learning models were deployed, namely, Random Forest, Linear Regression, and XG-Boost, which exhibited specific strengths. Specific performance metrics used included MSE, RMSE, MAE, and R². As the statistics indicate, among the three models, Random Forest stands out and performs the best on this dataset across all metrics. A smaller value of MAE, MSE, and RMSE signifies that it provided the best forecast for the target variable. It also gave the highest R-squared value. Application of drug sensitivity prediction analysis in cancer can provide an overview of the mechanisms that underlie both tumor response and resistance by investigating the model predictions. The proposed predictive models have the potential to make significant impacts on clinical decision-making in cancer therapy. Predictive models derive informed decisions regarding a patient's risk of recurrence of disease, their response to certain therapies, and their prognosis based on complex clinical and genomic data. Keywords: Genomic Predictors; Drug Sensitivity; Genomic Markers; Personalized medicine; Machine Learning; Random Forest Algorithm.

Abstract PR014: DNA associated with EVs is uniquely chromatinized and prevents metastasis by enhancing anti-tumor immunity

Abstract Cells release extracellular vesicles (EVs) loaded with functional biomolecules that facilitate intercellular communication in health and disease. These EVs contain DNA (EV-DNA), mirroring the mutational profile of parental cells, yet the mechanism of DNA packaging onto EVs and its role in diseases like cancer remain unclear. Using advanced imaging and biochemical techniques, we discovered that EV-DNA predominantly resides on the vesicle surface, bound to modified histones. Through genome-wide CRISPR knockout screens in cancer cell lines, we identified immune-related pathways and genes crucial for EV-DNA loading, such as APAF1 and NCF1. In several cancer models, loss of APAF1 reduced EV-DNA packaging, and enhancing metastasis, thus implicating EV-DNA in immune surveillance against cancer spread. We demonstrated that EV-DNA uptake by liver macrophages triggers DNA damage response, altering cytokine production and promoting immune activation in pre-metastatic sites. Moreover, quantifying EV-DNA in colorectal cancer biopsies revealed its potential as a predictive metastatic risk biomarker. Our findings shed light on EV-DNA packaging mechanisms and its significance, suggesting that elevated levels of tumor-derived EV-DNA bolster anti-tumor immunity and inhibit metastasis. Citation Format: Inbal Wortzel, Han-Sang Kim, David C. Lyden. DNA associated with EVs is uniquely chromatinized and prevents metastasis by enhancing anti-tumor immunity [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr PR014.

Abstract PR001: Targeting mRNA methyltransferase in RNA-dependent DNA repair in cancer therapy

Abstract The treatment of many solid tumors presents significant challenges with chemotherapy, radiation therapy, and the limited effectiveness of immunotherapy. Targeted therapy offers a promising approach, yet the lack of validated targets limits its applicability across the spectrum of solid tumors. Our past studies have revealed that an R-loop and mRNA-dependent DNA repair (RDDR) pathway, induced by damage at the transcribed regions of the genome, contributes to cell survival and drug resistance in cancer cells. This is particularly due to the high levels of transcription and DNA damage in these cells. We identified the RNA methyltransferase TRDMT1 as the primary modifier of mRNA methyl-5-cytosine in the RDDR pathway. Overcoming technical difficulties in monitoring RNA modifications, we developed the first TRDMT1 inhibitor (TRDMT1i) using in-house built cell-based and in vitro assays. We have identified highly potent and specific lead compounds that significantly reduce TRDMT1 activity at nanomolar concentrations. Our current lead TRDMT1 inhibitors meets most of the criteria that are required for optimal lead. Initial assessments involving the screening of normal and cancer cell lines, xenograft models, and patient specimens indicate that TRDMT1i sensitizes tumors with genomic instability when used as monotherapy. We aim to develop TRDMT1i as an investigational new drug (IND) for future clinical trials, initially targeting ovarian cancer as a monotherapy or in combination therapy for first-line maintenance and systematic therapy. Our goal is to pioneer the discovery of the RDDR pathway, leading to the development of innovative platforms and next-generation medications to revolutionize the targeting of mRNA modifications in cancer treatment. Citation Format: Li Lan. Targeting mRNA methyltransferase in RNA-dependent DNA repair in cancer therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr PR001.

EXTH-54. COMBINATORIAL DRUG SCREENING IDENTIFIES CARFILZOMIB AND ENZALUTAMIDE FOR TREATING AGGRESSIVE MENINGIOMAS

Abstract The goal of combinatorial therapy is to improve the effectiveness of treatment by targeting multiple aspects of cancer cells or overcoming resistance to single drugs. Therefore, we performed an automated combinatorial drug screening in meningioma cell lines and patient-derived tumor organoids (TOs). Three grade 3 meningioma cell lines (NCH93, IOMM-Lee, and KT21-MG1) were stably transduced with blue, green, and red fluorescence proteins. The cell lines were multiplexed into 384-well plates and treated in 5x5 dose-response (0-1000 nmol/l) matrices for 48h by the automated liquid handler Hamilton MicroLAB STAR®. The drug library consisted of 166 FDA-approved anticancer drugs. The viability of each cell line was demultiplexed by using fluorescence signals and CellTiterGlo. TOs were established from single cell suspensions of freshly resected meningioma tissue (n=20). The ZIP model was used to determine synergism. This drug screening effort generated 13,695 unique drug-drug combinations per cell line. Most synergistic combinations were observed in IOMM-Lee (1.14%), followed by KT21-MG1 (0.88%), and NCH93 (0.54%). The 110 most effective drug combinations were validated in wild-type meningioma cells. 75 drug combinations demonstrated a positive most synergistic area (MSA) score, indicating synergistic effects. Based on synergy scores and literature evidence, the 16 most promising combinations were selected for screening in TOs. The proteasome inhibitor carfilzomib in combination with the androgen receptor inhibitor enzalutamide exhibited the highest synergy in TOs. In 40% (n=8/20) of the cases, the combination showed strong synergism (MSA>10) resulting in an overall average MSA of 7.36, followed by carfilzomib and neratinib, and romidepsin and gemcitabine with average MSAs of 5.49, and 4.32, respectively. Furthermore, carfilzomib and enzalutamide induced apoptosis in TOs assessed by Caspase-3/7. This comprehensive combinatorial drug screening identified the synergistic combination of the proteasome inhibitor carfilzomib and androgen receptor inhibitor enzalutamide that can be an effective treatment option for selected meningioma patients.

Transcriptional Regulation of Protein Synthesis by Mediator Kinase Represents a Therapeutic Vulnerability in MYC-driven Medulloblastoma.

MYC-driven medulloblastoma (MB) is a highly aggressive cancer type with poor prognosis and limited treatment options. Through CRISPR-Cas9 screening of MB cell lines, we identified the Mediator-associated kinase CDK8 as a critical regulator of MYC-driven MB. Loss of CDK8 substantially reduces MYC expression and induces pronounced transcriptional changes, consequently inhibiting MB growth and suppressing monosome assembly, resulting in decreased ribosome biogenesis and protein synthesis. Mechanistically, CDK8 regulates the occupancy of RNA polymerase II at specific chromatin loci, facilitating an epigenetic alteration that promotes the transcriptional regulation of ribosomal genes. Targeting CDK8 effectively diminishes the stem-like neoplastic cells characterized by hyperactive ribosome biogenesis. Furthermore, we demonstrated that the combined inhibition of CDK8 and mTOR synergizes to optimize therapeutic outcomes in vivo and in vivo. Overall, our findings establish a connection between CDK8-mediated transcriptional regulation and mRNA translation, suggesting a promising new therapeutic approach that targets the protein synthesis for MYC-driven MB. .

Genotype from Phenotype: Using CRISPR Screens to Dissect Lymphoma Biology.

Genome-wide screens are a powerful technique to dissect the complex network of genes regulating diverse cellular phenotypes. The recent adaptation of the CRISPR-Cas9 system for genome engineering has revolutionized functional genomic screening. Here, we present protocols used to introduce Cas9 into human lymphoma cell lines, produce high-titer lentivirus of a genome-wide sgRNA library, transduce and culture cells during the screen, select cells with a specified phenotype, isolate genomic DNA, and prepare a custom library for next-generation sequencing. These protocols were tailored for loss-of-function CRISPR screens in human B-cell lymphoma cell lines but are highly amenable for other experimental purposes.

Pharmacoproteogenomic approach identifies on-target kinase inhibitors for cancer drug repositioning.

Drug repositioning of approved drugs offers advantages over de novo drug development for a rare type of cancer. To efficiently identify on-target drugs from clinically successful kinase inhibitors in cancer drug repositioning, drug screening and molecular profiling of cell lines are essential to exclude off-targets. We developed a pharmacoproteogenomic approach to identify on-target kinase inhibitors, combining molecular profiling of genomic features and kinase activity, and drug screening of patient-derived cell lines. This study examined eight patient-derived giant cell tumor of the bone (GCTB) cell lines, all of which harbored a signature mutation of H3-3A but otherwise without recurrent copy number variants and mutations. Kinase activity profiles of 100 tyrosine kinases with a three-dimensional substrate peptide array revealed that nine kinases were highly activated. Pharmacological screening of 60 clinically used kinase inhibitors found that nine drugs directed at 29 kinases strongly suppressed cell viability. We regarded ABL1, EGFR, and LCK as on-target kinases; among the two corresponding on-target kinase inhibitors, osimertinib and ponatinib emerged as on-target drugs whose target kinases were significantly activated. The remaining 26 kinases and seven kinase inhibitors were excluded as off-targets. Our pharmacoproteomic approach enabled the identification of on-target kinase inhibitors that are useful for drug repositioning.

Abstract C113: Parabens promote cell growth in cancer cell lines associated with health disparities

Abstract Parabens are endocrine-disrupting chemicals used as preservatives to extend the shelf life of consumables, pharmaceuticals, and personal care products. Daily use of products containing parabens results in continuous exposure that can negatively impact health. It is known that parabens cause breast cancer cells to grow and express genes linked to cancer and hormone action. Our lab previously showed that parabens increase viability of and regulate estrogen-responsive gene expression in diverse luminal A breast cancer cell lines. In addition, greater effects were seen in a luminal A cell line of West African ancestry compared to one of European ancestry, suggesting that exposure to parabens in personal care products may contribute to breast cancer disparities. Therefore, the purpose of this study was to investigate whether parabens increase cell viability across a variety of cancer cell types, particularly those associated with cancer disparities. To do so, we utilized the National Cancer Institute-60 (NCI-60) Human Tumor Cell Lines screen that includes leukemia, non-small cell lung (NSCL), colon, central nervous system (CNS), melanoma, ovarian, renal, prostate, and breast cancer cell lines, plus a custom panel of pancreatic cancer cell lines. Of these, ovarian, NSCL, prostate, colon, renal, CNS, and pancreatic cancers have known disparities. We treated these cancer cell lines with biologically relevant doses of butylparaben (BP), methylparaben (MP), or propylparaben (PP) (doses ranging from 0.0012 µM – 20 µM) for 72 hours and measured cell viability. Treatment with dimethylsulfoxide or 10 nM estradiol served as negative and positive controls, respectively. In general, we observed a paraben-specific and cell-line specific effect on cell viability. MP exhibited the greatest effect with increased cell viability in 42% of the cell lines, followed by PP (38%), and BP (32%). MP and PP are among the most common parabens found in personal care products and differences in urinary levels based on race/ethnicity (e.g. higher levels in non-Hispanic Blacks/Mexican Americans compared to non-Hispanic Whites) have been reported. Breast, ovarian, and NSCL had the most cell lines (67-83%) that exhibited increased cell viability with any paraben treatment. Increased cell viability was also observed in several leukemia, colon, pancreatic, CNS, and melanoma cell lines (33-50%). Only one renal, and no prostate, cell line exhibited increased cell viability. These data suggest that paraben-mediated protumorigenic effects may promote cancer disparities beyond breast cancer. Studies are currently underway to determine the mechanisms underlying this link. One caveat of this study is that most of the cell lines in this readily available screen are of European ancestry, highlighting a need to develop more genetically diverse in vitro models for understanding the mechanistic link between paraben exposures and cancer risk/outcomes in the context of disparities. Approaching this challenge from an endocrinology and genetic ancestry perspective may help narrow the disparity gap. Citation Format: Jazma L. Tapia, Jillian C. McDonough, Loretta Erhunmwunsee, Lindsey S. Trevino. Parabens promote cell growth in cancer cell lines associated with health disparities [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C113.

Protein Structure Inspired Discovery of a Novel Inducer of Anoikis in Human Melanoma.

Drug discovery historically starts with an established function, either that of compounds or proteins. This can hamper discovery of novel therapeutics. As structure determines function, we hypothesized that unique 3D protein structures constitute primary data that can inform novel discovery. Using a computationally intensive physics-based analytical platform operating at supercomputing speeds, we probed a high-resolution protein X-ray crystallographic library developed by us. For each of the eight identified novel 3D structures, we analyzed binding of sixty million compounds. Top-ranking compounds were acquired and screened for efficacy against breast, prostate, colon, or lung cancer, and for toxicity on normal human bone marrow stem cells, both using eight-day colony formation assays. Effective and non-toxic compounds segregated to two pockets. One compound, Dxr2-017, exhibited selective anti-melanoma activity in the NCI-60 cell line screen. In eight-day assays, Dxr2-017 had an IC50 of 12 nM against melanoma cells, while concentrations over 2100-fold higher had minimal stem cell toxicity. Dxr2-017 induced anoikis, a unique form of programmed cell death in need of targeted therapeutics. Our findings demonstrate proof-of-concept that protein structures represent high-value primary data to support the discovery of novel acting therapeutics. This approach is widely applicable.

2‐Ethenyl Imidazolium Salts: Synthesis, Characterization, and Evaluation as Potential Chemotherapeutics

AbstractAs candidates for intravesical exfoliants to treat bladder cancer, we have prepared fourteen new imidazolium compounds substituted at the 2 position. These compounds were produced by use of the Knoevenagel reaction between a series of aryl aldehydes and 2‐methyl imidazolium salts. Two azolium systems were examined (benzimidazole and 4,5‐dichloroimidazole) and each of these heterocycles are substituted off the nitrogen atom positions with naphthalen‐2‐ylmethyl groups. NMR spectroscopy confirms formation of the carbon‐carbon double bond, and X‐ray crystallography confirmed that the alkenes adopt the E conformation. These new imidazolium salts were evaluated for their chemotherapeutic activity using the NCI‐60 Human Tumor Cell Line Screen, and we observed that the benzimidazolium systems were more toxic than the dichloroimidazolium systems, and that the methyl phenol ether or dimethyl aniline modified compounds showed the highest activities.

CRISPR-Mediated Library Screening of Gene-Knockout Cell Lines for Investigating Antiviral Innate Immunity.

The application of CRISPR-mediated library screening has fundamentally transformed functional genomics by revealing the complexity of virus-host interactions. This protocol describes the use of CRISPR-mediated library screening to identify key functional genes regulating the innate immune response to PEDV infection. We detail a step-by-step process, starting from the design and construction of a customized CRISPR knockout library targeting genes involved in innate immunity to the effective delivery of these constructs into cells using lentiviral vectors. Subsequently, we outline the process of identifying functional genes postviral attack, including the use of next-generation sequencing (NGS), to analyze and identify knockout cells that exhibit altered responses to infection. This integrated approach provides researchers in immunology and virology with a resource and a robust framework for uncovering the genetic basis of host-pathogen interactions and the arsenal of the innate immune system against viral invasions.

Optimal methods for analyzing targeted pairwise knockout screens.

Synthetic lethality offers a promising strategy for cancer treatment by targeting genetic vulnerabilities unique to tumor cells, leading to selective tumor cell death. However, single-gene knockout screens often miss functional redundancy due to paralog genes. Multiplex CRISPR systems, including various Cas9 and Cas12a platforms, have been developed to assay genetic interactions, yet no systematic comparison of method to identify synthetic lethality from CRISPR screens has been conducted. We evaluated data from four in4mer CRISPR/Cas12a screens in cancer cell lines, using three bioinformatic approaches to identify synthetic lethal interactions: delta log fold change (dLFC), Z-transformed dLFC (ZdLFC), and rescaled dLFC (RdLFC). Both ZdLFC and RdLFC provided more consistent identification of synthetic lethal pairs across cell lines compared to the unscaled dLFC method. The ZdLFC method offers a robust framework for scoring synthetic lethal interactions from paralog screens, providing consistent results across different cell lines without requiring a training set of known positive interactors.

Removal of TREX1 activity enhances CRISPR-Cas9-mediated homologous recombination.

CRISPR-Cas9-mediated homology-directed repair (HDR) can introduce desired mutations at targeted genomic sites, but achieving high efficiencies is a major hurdle in many cell types, including cells deficient in DNA repair activity. In this study, we used genome-wide screening in Fanconi anemia patient lymphoblastic cell lines to uncover suppressors of CRISPR-Cas9-mediated HDR. We found that a single exonuclease, TREX1, reduces HDR efficiency when the repair template is a single-stranded or linearized double-stranded DNA. TREX1 expression serves as a biomarker for CRISPR-Cas9-mediated HDR in that the high TREX1 expression present in many different cell types (such as U2OS, Jurkat, MDA-MB-231 and primary T cells as well as hematopoietic stem and progenitor cells) predicts poor HDR. Here we demonstrate rescue of HDR efficiency (ranging from two-fold to eight-fold improvement) either by TREX1 knockout or by the use of single-stranded DNA templates chemically protected from TREX1 activity. Our data explain why some cell types are easier to edit than others and indicate routes for increasing CRISPR-Cas9-mediated HDR in TREX1-expressing contexts.

Modernizing the NCI60 Cell Line Screen for Phenotypic Drug Discovery in the 21st Century.

Over the past three decades, high-throughput phenotypic cancer cell line screens have revealed unanticipated small-molecule activities and illuminated connections between tumor genotypes and anticancer efficacy. Founded in 1984, the National Cancer Institute's "NCI60" screen laid the conceptual groundwork for the contemporary landscape of phenotypic drug discovery. NCI60 first operated as a primary bioactivity screen, but molecular characterization of the NCI60 cell line panel and development of a small-molecule sensitivity pattern recognition algorithm (called "COMPARE") have enabled subsequent studies into drug mechanisms of action and biomarker identification. In this issue of Cancer Research, Kunkel and colleagues report an updated version of the NCI60 screen, dubbed "HTS384" NCI60, that better aligns with current cell proliferation assay standards and has higher throughput. Changes include the use of a 384-well plate format, automated laboratory equipment, 3 days of compound exposure, and a CellTiter-Glo luminescent endpoint. To confirm that data from the HTS384 and classic NCI60 screen are comparable, the authors tested a library of 1,003 anticancer agents using both protocols and applied COMPARE to analyze patterns of cell line sensitivities. More than three dozen groups of targeted therapies showed high comparability between screens. Modernization of NCI60, and closer integration with other large-scale pharmacogenomic screens and molecular feature sets, will help this public screening service remain pertinent for cancer drug discovery efforts for years to come. See related article by Kunkel et al., p. 2403.

Chromatin remodellers as therapeutic targets.

Large-scale cancer genome sequencing studies have revealed that chromatin regulators are frequently mutated in cancer. In particular, more than 20% of cancers harbour mutations in genes that encode subunits of SWI/SNF (BAF) chromatin remodelling complexes. Additional links of SWI/SNF complexes to disease have emerged with the findings that some oncogenes drive transformation by co-opting SWI/SNF function and that germline mutations in select SWI/SNF subunits are the basis of several neurodevelopmental disorders. Other chromatin remodellers, including members of the ISWI, CHD and INO80/SWR complexes, have also been linked to cancer and developmental disorders. Consequently, therapeutic manipulation of SWI/SNF and other remodelling complexes has become of great interest, and drugs that target SWI/SNF subunits have entered clinical trials. Genome-wide perturbation screens in cancer cell lines with SWI/SNF mutations have identified additional synthetic lethal targets and led to further compounds in clinical trials, including one that has progressed to FDA approval. Here, we review the progress in understanding the structure and function of SWI/SNF and other chromatin remodelling complexes, mechanisms by which SWI/SNF mutations cause cancer and neurological diseases, vulnerabilities that arise because of these mutations and efforts to target SWI/SNF complexes and synthetic lethal targets for therapeutic benefit.

Genome-scale exon perturbation screens uncover exons critical for cell fitness

CRISPR-Cas technology has transformed functional genomics, yet understanding of how individual exons differentially shape cellular phenotypes remains limited. Here, we optimized and conducted massively parallel exon deletion and splice-site mutation screens in human cell lines to identify exons that regulate cellular fitness. Fitness-promoting exons are prevalent in essential and highly expressed genes and commonly overlap with protein domains and interaction interfaces. Conversely, fitness-suppressing exons are enriched in nonessential genes, exhibiting lower inclusion levels, and overlap with intrinsically disordered regions and disease-associated mutations. In-depth mechanistic investigation of the screen-hit TAF5 alternative exon-8 revealed that its inclusion is required for assembly of the TFIID general transcription initiation complex, thereby regulating global gene expression output. Collectively, our orthogonal exon perturbation screens established a comprehensive repository of phenotypically important exons and uncovered regulatory mechanisms governing cellular fitness and gene expression.

Combined SERS-Raman screening of HER2-overexpressing or silenced breast cancer cell lines

BackgroundBreast cancer (BC) is a heterogeneous neoplasm characterized by several subtypes. One of the most aggressive with high metastasis rates presents overexpression of the human epidermal growth factor receptor 2 (HER2). A quantitative evaluation of HER2 levels is essential for a correct diagnosis, selection of the most appropriate therapeutic strategy and monitoring the response to therapy.ResultsIn this paper, we propose the synergistic use of SERS and Raman technologies for the identification of HER2 expressing cells and its accurate assessment. To this end, we selected SKBR3 and MDA-MB-468 breast cancer cell lines, which have the highest and lowest HER2 expression, respectively, and MCF10A, a non-tumorigenic cell line from normal breast epithelium for comparison. The combined approach provides a quantitative estimate of HER2 expression and visualization of its distribution on the membrane at single cell level, clearly identifying cancer cells. Moreover, it provides a more comprehensive picture of the investigated cells disclosing a metabolic signature represented by an elevated content of proteins and aromatic amino acids. We further support these data by silencing the HER2 gene in SKBR3 cells, using the RNA interference technology, generating stable clones further analysed with the same combined methodology. Significant changes in HER2 expression are detected at single cell level before and after HER2 silencing and the HER2 status correlates with variations of fatty acids and downstream signalling molecule contents in the context of the general metabolic rewiring occurring in cancer cells. Specifically, HER2 silencing does reduce the growth ability but not the lipid metabolism that, instead, increases, suggesting that higher fatty acids biosynthesis and metabolism can occur independently of the proliferating potential tied to HER2 overexpression.ConclusionsOur results clearly demonstrate the efficacy of the combined SERS and Raman approach to definitely pose a correct diagnosis, further supported by the data obtained by the HER2 gene silencing. Furthermore, they pave the way to a new approach to monitor the efficacy of pharmacologic treatments with the aim to tailor personalized therapies and optimize patients’ outcome.

DIPG-14. METABOLIC REPROGRAMMING AS A RESISTANCE MECHANISM IN DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND Diffuse midline gliomas (DMG) are uniformly fatal pediatric brain cancers, refractory to current standards of care. Based on genomic and transcriptomic profiling data, we previously proposed that platelet-derived growth factor receptor alpha (PDGFRA) is crucial to the tumorigenic potential and maintenance of stem-like DMG cells and that targeting PDGFRA is an important therapeutic avenue in DMG. In comprehensive pre-clinical studies, we identified avapritinib, an FDA-approved PDGFRA inhibitor, to be highly effective in patient-derived xenograft (PDX) DMG models. We further reported the first clinical experience using avapritinib in recurrent DMG and demonstrated preliminary safety, tolerability, and efficacy of the drug in this population. While a subset of patients experienced a strong clinical response from avapritinib, tumor progression was eventually observed in all patients. METHODS Here, we investigated the molecular mechanisms that DMG cells upregulate following avapritinib treatment, with the aim to exploit them therapeutically. To uncover these mechanisms, we performed transcriptomic, metabolic and functional assays along with a combinatorial drug screening. RESULTS Transcriptional analysis of patient-derived DMG cell lines revealed an upregulation of genes associated with fatty acid metabolism and oxidative phosphorylation (OXPHOS) following treatment with avapritinib. Functional assays confirmed elevated OXPHOS in avapritinib-treated cells with significant (i) increase in mitochondrial energy transduction, (ii) increase in palmitate- (a product of fatty acid metabolism) driven oxygen consumption rate and (iii) greater incorporation of palmitate-derived carbons into the tricarboxylic acid cycle. To determine which therapies could target this dependency on OXPHOS and fatty acid metabolism, we performed a metabolic drug screening in patient-derived DMG cell lines and identified two metabolic drugs to have synergistic cytotoxic effects with avapritinib. CONCLUSION We revealed distinct metabolic reprogramming in avapritinib-treated DMG cells and showed that targeting these metabolic vulnerabilities might further increase the clinical benefit of PDGFRA inhibitor avapritinib.

HTS384 NCI60: The Next Phase of the NCI60 Screen.

The NCI60 human tumor cell line screen has been in operation as a service to the cancer research community for more than 30 years. The screen operated with 96-well plates, a 2-day exposure period to test agents, and following cell fixation, a visible absorbance endpoint by the protein-staining dye sulforhodamine B. In this study, we describe the next phase of this important cancer research tool, the HTS384 NCI60 screen. Although the cell lines remain the same, the updated screen is performed with 384-well plates, a 3-day exposure period to test agents, and a luminescent endpoint to measure cell viability based upon cellular ATP content. In this study, a library of 1,003 FDA-approved and investigational small-molecule anticancer agents was screened by the two NCI60 assays. The datasets were compared with a focus on targeted agents with at least six representatives in the library. For many agents, including inhibitors of EGFR, BRAF, MEK, ERK, and PI3K, the patterns of GI50 values were very similar between the screens with strong correlations between those patterns within the dataset from each screen. However, for some groups of targeted agents, including mTOR, BET bromodomain, and NAMPRTase inhibitors, there were limited or no correlations between the two datasets, although the patterns of GI50 values and correlations between those patterns within each dataset were apparent. Beginning in January 2024, the HTS384 NCI60 screen became the free screening service of the NCI to facilitate drug discovery by the cancer research community. Significance: The new NCI60 cell line screen HTS384 shows robust patterns of response to oncology agents and substantial overlap with the classic screen, providing an updated tool for studying therapeutic agents. See related commentary by Colombo and Corsello, p. 2397.

Downstream Target Analysis for miR-365 among Oral Squamous Cell Carcinomas Reveals Differential Associations with Chemoresistance.

Expression of microRNAs, such as miR-365, is known to be dysregulated in many tumors, including oral cancers, although little is known about their role or functions. The objective of this project is to evaluate the downstream targets of miR-365 to determine any potential pathways or effects. Downstream targets for miR-365 (miRdatabase target scores > 90) were used for qPCR screening of oral cancer cell lines (SCC4, SCC9, SCC15, SCC25, CAL27). Each oral cancer cell line expressed miR-365 downstream targets molybdenum cofactor synthesis-2 (MOCS2), erythropoietin receptor (EPOR), IQ motif containing-K (IQCK), carboxypeptidase A3 (CPA3), solute carrier family 24 member-3 (SLC24A3), and coiled-coil domain containing 47 (CCDC47)-although the expression levels varied somewhat. However, differential results were observed with ubiquitin protein ligase E3 component n-recognin-3 (UBR3), nudix hydrolase-12 (NUDT12), zinc finger CCHC-type containing-14 (ZCCHC14), and homeobox and leucine zipper encoding (HOMEZ). These data suggest that many of the miR-365 targets are expressed in the oral cancers screened, with the differential expression of UBR3, ZCCHC14, HOMEZ, and NUDT12, which may be correlated with chemoresistance among two specific oral cancer cell lines (SCC25, SCC9). These results suggest this differential expression may signal potential targets for patient treatment with tumors exhibiting miR-365 and chemotherapeutic resistance.