Abstract The patatin-like phospholipase domain-containing protein 3 (PNPLA3) I148M variant is a well-established genetic determinant of fatty liver disease, yet emerging evidence suggests broader implications in cancer biology, particularly in hepatocellular carcinoma and metabolic reprogramming of tumor cells (Tavaglione et al., 2024). We recently identified NUV-244 as a potent and selective small-molecule degrader of PNPLA3 I148M (Steigemann et al., 2025). These findings provided the first pharmacological and molecular framework to directly interrogate PNPLA3 I148M´s function in liver cells. Building upon these insights, we sought to investigate the potential oncogenic or tumor-modulatory role of PNPLA3 I148M in cancer, and to evaluate how pharmacologic inhibition by NUV-244 influences cancer cell metabolism, proliferation, and survival. We performed a comparative analysis of cancer cell lines harboring endogenous PNPLA3 I148M mutations versus wild-type counterparts and generated isogenic cell line models using a PNPLA3 I148M overexpression approach. These models were used to assess transcriptomic and proteomic modulation, mitochondrial function and cell viability with and without NUV-244 treatment. Using these experimental systems, we aim to define how PNPLA3 I148M contributes to cancer cell metabolic plasticity and whether its pharmacologic inhibition creates selective vulnerabilities. In summary, these studies establish a mechanistic basis for PNPLA3 I148M´s potential role in tumor biology and highlight NUV-244 as a chemical tool to probe its function in cancer. Citation Format: Katrin Juenemann, Patrick Steigemann, Ralf Lesche, Tamara Kanashova, Hanna Meyer, Claudia Noack, Barbara Nicke, Peter Staller, Charlotte Kopitz, Martin Lange, . Exploring the role of PNPLA3 I148M in cancer using the selective small molecule degrader NUV-244 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7340.

BackgroundSystemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by loss of self-tolerance, causing inflammation and tissue damage in multiple organs. Although animal models have advanced our understanding of SLE’s molecular basis, recent regulatory changes and longstanding concerns regarding reproducibility and translatability have renewed the need to critically evaluate how these models mirror human disease. Understanding pathway-level similarities and differences between mouse models and human disease is essential, given the marked clinical and molecular heterogeneity of SLE.MethodsFour spontaneous SLE mouse models were studied: MRLlpr/lpr, NZB/W, BXSB.Yaa, and Tlr7.Tg6. Transcriptome sequencing from blood, spleen, and kidney; flow cytometry from the spleen; and cytokines and autoantibody measurement in plasma were performed at four time points. Similar molecular datasets from the human PRECISESADS SLE cohort were used for the integration.ResultsThe study identified specific molecular pathways driving the phenotype in each mouse model and established a framework describing the dynamics of these phenotype-associated molecular signatures, thereby facilitating the selection of time points of interest for future mouse-oriented experimental designs. In addition, by comparing these pathways with those observed in human SLE, we identified the most similar ones and their relationship with disease activity, providing crucial insight into their translational relevance. Importantly, disease severity across models was linked to both the extent and timing of molecular dysregulations. As expected, MRLlpr/lpr showed the most aggressive phenotype with early immune activation and apoptosis dysregulation, while Tlr7.Tg6 presented late-onset signatures associated with interferon and inflammation. Shared molecular features with human SLE included interferon responses, T and B cell depletion, and neutrophil activation. Integration analysis revealed distinct, yet overlapping, immune pathways between models and species, with some signatures such as age-associated B cells and double-negative memory T cells being model-specific but potentially relevant to early disease processes.ConclusionsThese findings provide a valuable framework for future SLE research and reinforce the utility of mouse models for studying specific molecular pathways related to human SLE pathogenesis and heterogeneity. The integration of longitudinal mouse and human molecular information highlights the models that best recapitulate key aspects of human disease, offering guidance for the study of specific immunopathological mechanisms or therapeutic targets.

Purpose:To study the biological effects of aflibercept and faricimab on vascular endothelial growth factor (VEGF)-A165-induced vascular permeability in an in vitro head-to-head comparison using human-derived endothelial cells. Methods:This in vitro study used a human umbilical vein endothelial cell (HUVEC)-based model, in which cells were exposed to VEGF-A165, followed by the addition of aflibercept (1.7 nM) or faricimab (2.0 nM) either simultaneously (preventive) or 24 hours later (therapeutic/rescue). Cell-layer permeability was measured on the xCELLigence platform, global transcriptomic changes by RNA sequencing, and VEGF-A165-induced release of angiogenesis-related proteins by electrochemiluminescence immunoassay. Results:Concurrent addition of aflibercept or faricimab prevented VEGF-A165-triggered increase of HUVEC cell-layer permeability (P = 0.0005). In the therapeutic/rescue setting, either drug temporally improved the cell-layer integrity (P = 0.0001). Aflibercept was non-inferior to faricimab in both the preventive (P < 0.0001) and therapeutic/rescue (P < 0.0001) settings. Aflibercept and faricimab comparably downregulated VEGF-A165-induced angiogenesis-related genes. Aflibercept and faricimab equipotently suppressed or reversed VEGF-A165 upregulation of ANGPT2 gene expression and angiopoietin-2 (ANG2) protein in culture supernatant. VEGF-A165-driven TEK gene expression and soluble Tie2 increase in culture supernatant was reversed with aflibercept and faricimab in both settings. Conclusions:In this in vitro model of VEGF-A165-driven vascular permeability, additional blockade of ANG2 with faricimab did not improve endothelial barrier integrity beyond VEGF-A165 blockade alone with aflibercept, despite significant upregulation of ANG2 expression after VEGF-A165 stimulation. Aflibercept and faricimab were comparable in maintaining or restoring endothelial barrier function and thereby vascular integrity, mainly attributable to inhibition of VEGF-A165 binding to its receptor.

Background Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by a loss of self-tolerance, causing inflammation and tissue damage in multiple organs. Animal models have advanced our understanding of SLE’s molecular basis, but the FDA’s recent elimination of animal testing requirements for drug approval has raised concerns about their validity, prompting a reevaluation of their role in basic research, especially for heterogeneous diseases like SLE. Methods Four different spontaneous SLE mouse models were studied: MRL lpr/lpr , NZB/W, BXSB. Yaa , and Tlr7.Tg6. Transcriptome sequencing from blood, spleen, and kidney, flow cytometry from the spleen, and cytokines and autoantibody measurement in plasma were performed at four time points. Similar molecular data from human SLE patients was used for the integration. Results The study identified specific molecular pathways driving the phenotype in each mouse model and established optimal time points for future experimental designs. By comparing these pathways to human SLE, the most similar ones and their relationship with disease activity were identified, providing crucial insight into translational relevance. Importantly, disease severity across models was linked to the extent and timing of molecular dysregulations. As expected, MRL lpr/lpr showed the most aggressive phenotype with early immune activation and apoptosis dysregulation, while Tlr7.Tg6 presented late-onset signatures associated with interferon and inflammation. Shared molecular features with human SLE included interferon responses, T and B cell depletion, and neutrophil activation. Integration analysis revealed distinct yet overlapping immune pathways between models and species, with some signatures such as age-associated B cells and double-negative memory T cells being model-specific but potentially relevant to early disease processes. Conclusions These findings build a valuable framework for future SLE research, reinforcing the utility of mouse models in studying specific molecular pathways related to human SLE pathogenesis and heterogeneity. The integration of longitudinal mouse data with human transcriptomes highlights the models that best recapitulate key aspects of human disease, offering guidance for the study of specific immunopathological mechanisms or therapeutic targets. ### Competing Interest Statement Authors Makowska, Kageyama, Buttgereit, Lesche, and McDonald were employees of Bayer at the time of the study. * ABCs : Age-associated B cells ACR : Albumin-to-creatinine ratio ANAs : Antinuclear antibodies anti-CENP-B : Anti-centromere protein B anti-dsDNA : Anti-double-stranded DNA anti-RNP : Anti-ribonucleoprotein anti-Sm : Anti-Smith antigen anti-SSA/La : Anti-Sjögren’s-syndrome-related antigen A anti-SSB/Ro : Anti-Sjögren’s-syndrome-related antigen B CXCL-1 : Chemokine (C-X-C motif) ligand 1 DN T cells : Double-negative T cells DP T cells : Double-positive T cells EDTA : Ethylenediaminetetraacetic acid ENA-78 : Epithelial-derived neutrophil-activating peptide 78 FDA : Food and Drug Administration FDR : False discovery rate G-CSF : Granulocyte colony-stimulating factor GM-CSF : Granulocyte-macrophage colony-stimulating factor ![Graphic][1] : Growth-related oncogene-alpha GSEA : Gene Set Enrichment Analysis ![Graphic][2] : Interferon-alpha IFN-J : Interferon-gamma IgG : Immunoglobulin G IgM-IgD-B cells : Class-switched B cells IL : Interleukin IP-10 : Interferon gamma-induced protein 10 MCP-1 : Monocyte chemoattractant protein-1 MCP-3 : Monocyte chemoattractant protein-3 M-CSF : Monocyte-colony stimulating factor ![Graphic][3] : Macrophage inflammatory protein-1 alpha MIP-1ß : Macrophage inflammatory protein-1 beta MIP-2 : Macrophage inflammatory protein-2 MRL : Murphy Roths Large MZ B cells : Marginal zone B cells NZB : New Zealand Black NZW : New Zealand White PBS : Phosphate-buffered saline PCs : Plasma cells pDCs : Plasmacytoid dendritic cells RANTES : Regulated upon activation, normal T cell expressed and secreted SLE : Systemic lupus erythematosus T3 B cells : Transitional T3 B cells Tmem : Memory T cells TN : Naïve T cells [inline] : Tumor necrosis factor-alpha Innovative Medicines Initiative Joint Undertaking, 115565 Innovative Medicines Initiative 2 Joint Undertaking, 831434 MICINN, Juan de la Cierva-Incorporación, IJC2020-043364-I [1]: /embed/inline-graphic-3.gif [2]: /embed/inline-graphic-4.gif [3]: /embed/inline-graphic-5.gif

Background: Systemic lupus erythematosus (SLE) is a complex autoimmune disease with a diverse pathophysiology triggered by the loss of self-tolerance, resulting in inflammation and tissue damage in multiple organs. The use of animal models has been instrumental in advancing our understanding of SLE. The elimination of the requirement for animal models in the FDA's drug approval process prompts a reevaluation of their role. Objectives: We aim to define a molecular framework for the future design of preclinical studies in SLE, helping researchers in the selection of mouse models and appropriate time point of study based on the molecular pathways and their relationship with human disease. Methods: To address this, a 4 timepoint longitudinal study was conducted using four different spontaneous SLE mouse models: MRLlpr/lpr, NZB/W, BXSB.Yaa, and TLR7.Tg6. RNA-Seq from whole-blood, spleen, and kidney, flow-cytometry from spleen, cytokines and autoantibodies in serum were profiled. All data was investigated using both, longitudinal differential expression analysis as well as individual cross-sectional analysis of each time-point. Molecular and flow-cytometry data from blood from SLE patients from the PRECISESADS project was integrated with mouse data using the MEFISTO software, allowing us to integrate direct relationships between human and mouse along the progression of disease. Results: Mouse models were extensively characterized at the molecular level. In the spleen, lymphopenia mimicking that occurring in the human and age-associated B cells were in common between mouse models, while neutrophil and cytotoxic lymphocyte expansions differentiated the models. TLR7-involved models showed important transcriptional dysregulation in the spleen as compared to non-TLR7 models, whose dysregulation was primarily in the kidney. Molecular changes were associated with severity across all models, with splenic changes preceding kidney damage and suggesting the ideal moment to study early immune pathogenic processes. Human and mouse molecular integration showed a shared interferon-mediated pathway with MRLlpr/lpr and TLR7.Tg6 models associated with hematological and constitutional clinical domains, but at different timepoints. The neutrophil-mediated inflammatory pathway was shared between humans, MRLlpr/lpr, BXSB.Yaa and TLR7.Tg6 models and related to renal and cutaneous clinical human domains. Conclusion: The findings provide a valuable framework for future experimental design involving SLE mouse models, demonstrating their continued usefulness in investigating the elusive pathogenesis and heterogeneity of SLE. REFERENCES: NIL. Acknowledgements: This project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking (JU) under grant agreement No 831434 (3TR). The JU receives support from the European Union's Horizon 2020 research and innovation programme and EFPIA. The research leading to these results has received support from the Innovative Medicines Initiative Joint Undertaking under the Grant Agreement Number 115565 (PRECISESADS project), resources of which are composed of financial contribution from the European Union's Seventh Framework Program (FP7/2007–2013) and EFPIA companies' in-kind contribution. This work was supported in part by the Spanish Ministry of Science and Innovation under grant "Juan de la Cierva" (IJC2020-043364-I). Disclosure of Interests: Maria Rivas-Torrubia: None declared, María Morell: None declared, Zuzanna Makowska Bayer Pharma AG, Berlin, Berlin, Germany, Jorge Kageyama Bayer Pharma AG, Berlin, Berlin, Germany, Anne Buttgereit Bayer Pharma AG, Berlin, Berlin, Germany, Julius Lindblom: None declared, PRECISESADS cytometry consortium: None declared, Ioannis Parodis: None declared, Lorenzo Beretta: None declared, Ralf Lesche Bayer Pharma AG, Berlin, Berlin, Germany, Fiona McDonald Bayer Pharma AG, Berlin, Berlin, Germany, Marta Alarcon-Riquelme: None declared, Guillermo Barturen: None declared.

The PNPLA3 I148M variant is a key genetic determinant of metabolic dysfunction-associated steatotic liver disease (MASLD) and related conditions, contributing to lipid metabolism dysregulation and disease progression. To identify small molecules that modulate PNPLA3 I148M, we conducted a high-content screen of over 820,000 compounds and identified NUV-244, a potent degrader of PNPLA3 I148M in liver-derived cells. NUV-244 reduces PNPLA3 I148M levels on lipid droplets via the ubiquitin-proteasome system, involving the E3 ligase BFAR, without affecting PNPLA2. It restores lipid droplet morphology and improves cellular fitness in PNPLA3 I148M-expressing cells. These findings provide a tool to investigate PNPLA3 I148M function and offer a potential strategy for developing targeted therapies for MASLD and related diseases. By enabling selective degradation of PNPLA3 I148M, this approach expands therapeutic possibilities beyond genetic manipulation, addressing a critical need in metabolic liver disease research.

This study describes the identification and target deconvolution of small molecule inhibitors of oncogenic Yes-associated protein (YAP1)/TAZ activity with potent anti-tumor activity in vivo. A high-throughput screen (HTS) of 3.8 million compounds was conducted using a cellular YAP1/TAZ reporter assay. Target deconvolution studies identified the geranylgeranyltransferase-I (GGTase-I) complex as the direct target of YAP1/TAZ pathway inhibitors. The small molecule inhibitors block the activation of Rho-GTPases, leading to subsequent inactivation of YAP1/TAZ and inhibition of cancer cell proliferation in vitro. Multi-parameter optimization resulted in BAY-593, an in vivo probe with favorable PK properties, which demonstrated anti-tumor activity and blockade of YAP1/TAZ signaling in vivo.

Prostate cancer is a frequent malignancy in older men and has a very high 5‐year survival rate if diagnosed early. The prognosis is much less promising if the tumor has already spread outside the prostate gland. Targeted treatments mainly aim at blocking androgen receptor (AR) signaling and initially show good efficacy. However, tumor progression due to AR‐dependent and AR‐independent mechanisms is often observed after some time, and novel treatment strategies are urgently needed. Dysregulation of the PI3K/AKT/mTOR pathway in advanced prostate cancer and its implication in treatment resistance has been reported. We compared the impact of PI3K/AKT/mTOR pathway inhibitors with different selectivity profiles on in vitro cell proliferation and on caspase 3/7 activation as a marker for apoptosis induction, and observed the strongest effects in the androgen‐sensitive prostate cancer cell lines VCaP and LNCaP. Combination treatment with the AR inhibitor darolutamide led to enhanced apoptosis in these cell lines, the effects being most pronounced upon cotreatment with the pan‐PI3K inhibitor copanlisib. A subsequent transcriptomic analysis performed in VCaP cells revealed that combining darolutamide with copanlisib impacted gene expression much more than individual treatment. A comprehensive reversal of the androgen response and the mTORC1 transcriptional programs as well as a marked induction of DNA damage was observed. Next, an in vivo efficacy study was performed using the androgen‐sensitive patient‐derived prostate cancer (PDX) model LuCaP 35 and a superior efficacy was observed after the combined treatment with copanlisib and darolutamide. Importantly, immunohistochemistry analysis of these treated tumors showed increased apoptosis, as revealed by elevated levels of cleaved caspase 3 and Bcl‐2‐binding component 3 (BBC3). In conclusion, these data demonstrate that concurrent blockade of the PI3K/AKT/mTOR and AR pathways has superior antitumor efficacy and induces apoptosis in androgen‐sensitive prostate cancer cell lines and PDX models.

Background:Velcrins are molecular glues that kill cells by inducing the formation of a protein complex between the RNase SLFN12 and the phosphodiesterase PDE3A. Formation of the complex activates SLFN12, which cleaves tRNALeu(TAA) and induces apoptosis. Velcrins such as the clinical investigational compound, BAY 2666605, were found to have activity across multiple solid tumor cell lines from the cancer cell line encyclopedia, including glioblastoma cell lines. We therefore aim to characterize velcrins as novel therapeutic agents in glioblastoma. Materials and Methods:PDE3A and SLFN12 expression levels were measured in glioblastoma cell lines, the Cancer Genome Atlas (TCGA) tumor samples, and tumor neurospheres. Velcrin-treated cells were assayed for viability, induction of apoptosis, cell cycle phases, and global changes in translation. Transcriptional profiling of the cells was obtained. Xenograft-harboring mice treated with velcrins were also monitored for survival. Results:We identified several velcrin-sensitive glioblastoma cell lines and 4 velcrin-sensitive glioblastoma patient-derived models. We determined that BAY 2666605 crosses the blood-brain barrier and elicits full tumor regression in an orthotopic xenograft model of GB1 cells. We also determined that the velcrins BAY 2666605 and BRD3800 induce tumor regression in subcutaneous glioblastoma PDX models. Conclusions:Velcrins have antitumor activity in preclinical models of glioblastoma, warranting further investigation as potential therapeutic agents.

Effects of GSK126 treatment in MOLP-8 cell line

The PI3K pathway is one of the most frequently altered signaling pathways in human cancer. In addition to its function in cancer cells, PI3K plays a complex role in modulating anti-tumor immune responses upon immune checkpoint inhibition (ICI). Here, we evaluated the effects of the pan-Class I PI3K inhibitor copanlisib on different immune cell types in vitro and on tumor growth and immune cell infiltration in syngeneic murine cancer models. Intermittent treatment with copanlisib resulted in a strong in vivo anti-tumor efficacy, increased tumor infiltration of activated T cells and macrophages, and increased CD8 + T cell/regulatory T cell and M 1/ M 2 macrophage ratios. The strong in vivo efficacy was at least partially due to immunomodulatory activity of copanlisib, as in vitro these murine cancer cells were resistant to PI3K inhibition. Furthermore, the combination of copanlisib with the ICI antibody anti-PD-1 demonstrated enhanced anti-tumor efficacy in both ICI-sensitive and insensitive syngeneic mouse tumor models. Importantly, in an ICI-sensitive model, combination therapy resulted in complete remission and prevention of tumor recurrence. Thus, the combination of ICIs with PI3K inhibition by intermittently dosed copanlisib represents a promising new strategy to increase sensitivity to ICI therapies and to treat human solid cancers.

Supplementary Materials and Methods and Figure Legends

Supplementary Methods: Synthesis and characterization of BAY-8002; Tritium labeling of AZD3965 and BAY-8002; MCT1 and MCT4 14C-lactate uptake measurements in the X. laevis oocytes expression System

Supplementary Table S3 from Identification and Validation of Colorectal Neoplasia–Specific Methylation Markers for Accurate Classification of Disease

Supplementary Table S2 from Identification and Validation of Colorectal Neoplasia–Specific Methylation Markers for Accurate Classification of Disease

BACKGROUND:The metabolism of tryptophan to kynurenines (KYN) by indoleamine-2,3-dioxygenase or tryptophan-2,3-dioxygenase is a key pathway of constitutive and adaptive tumor immune resistance. The immunosuppressive effects of KYN in the tumor microenvironment are predominantly mediated by the aryl hydrocarbon receptor (AhR), a cytosolic transcription factor that broadly suppresses immune cell function. Inhibition of AhR thus offers an antitumor therapy opportunity via restoration of immune system functions.METHODS:The expression of AhR was evaluated in tissue microarrays of head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC) and colorectal cancer (CRC). A structure class of inhibitors that block AhR activation by exogenous and endogenous ligands was identified, and further optimized, using a cellular screening cascade. The antagonistic properties of the selected AhR inhibitor candidate BAY 2416964 were determined using transactivation assays. Nuclear translocation, target engagement and the effect of BAY 2416964 on agonist-induced AhR activation were assessed in human and mouse cancer cells. The immunostimulatory properties on gene and cytokine expression were examined in human immune cell subsets. The in vivo efficacy of BAY 2416964 was tested in the syngeneic ovalbumin-expressing B16F10 melanoma model in mice. Coculture of human H1299 NSCLC cells, primary peripheral blood mononuclear cells and fibroblasts mimicking the human stromal-tumor microenvironment was used to assess the effects of AhR inhibition on human immune cells. Furthermore, tumor spheroids cocultured with tumor antigen-specific MART-1 T cells were used to study the antigen-specific cytotoxic T cell responses. The data were analyzed statistically using linear models.RESULTS:AhR expression was observed in tumor cells and tumor-infiltrating immune cells in HNSCC, NSCLC and CRC. BAY 2416964 potently and selectively inhibited AhR activation induced by either exogenous or endogenous AhR ligands. In vitro, BAY 2416964 restored immune cell function in human and mouse cells, and furthermore enhanced antigen-specific cytotoxic T cell responses and killing of tumor spheroids. In vivo, oral application with BAY 2416964 was well tolerated, induced a proinflammatory tumor microenvironment, and demonstrated antitumor efficacy in a syngeneic cancer model in mice.CONCLUSIONS:These findings identify AhR inhibition as a novel therapeutic approach to overcome immune resistance in various types of cancers.

The diffuse nature of Glioblastoma (GBM) tumors poses a challenge to current therapeutic options. We have previously shown that Acyl-CoA Binding Protein (ACBP, also known as DBI) regulates lipid metabolism in GBM cells, favoring fatty acid oxidation (FAO). Here we show that ACBP downregulation results in wide transcriptional changes affecting invasion-related genes. In vivo experiments using patient-derived xenografts combined with in vitro models demonstrated that ACBP sustains GBM invasion via binding to fatty acyl-CoAs. Blocking FAO mimics ACBP KD -induced immobility, a cellular phenotype that can be rescued by increasing FAO rates. Further investigation into ACBP-downstream pathways served to identify Integrin beta-1, a gene downregulated upon inhibition of either ACBP expression or FAO rates, as a mediator for ACBP’s role in GBM invasion. Altogether, our findings highlight a role for FAO in GBM invasion and reveal ACBP as a therapeutic vulnerability to stall FAO and subsequent cell invasion in GBM tumors.

Supplemental Figure S1: A, SLC16A1 (MCT1), SLC16A7 (MCT2), SLC16A8 (MCT3) and SLC16A3 (MCT4) mRNA expression levels and Effects of BAY-8002 on lactate Export and ECAR. Supplemental Figure S2: Binding affinity of the two MCT1 inhibitors BAY-8002 and AZD3965. Supplemental Figure S3: supplemental cell panel analysis. Supplemental Figure S4: subset analysis of MCT4 low expressing cells from the cell panel Analysis. Supplemental Figure S5: Intratumor 2/3-PG and 6PG levels after treatment with AZD3965

The Hippo signaling cascade is a major pathway that integrates a broad spectrum of mechanosensory signals at the plasma membrane and regulates response via control of cell proliferation, self-renewal, differentiation, and apoptosis. Dysregulation of this pathway has been observed across a range of cancer types and results in an altered activity of its primary downstream effectors, the oncogenic transcription factors YAP/TAZ. For example, both germline and somatic loss-of-function mutations in the tumor suppressor gene NF2, a component of Hippo, induce hyperactivation of YAP/TAZ, transcriptional changes and ultimately result in tumor growth. The Hippo signaling pathway is an attractive target for drug discovery efforts, however, it is highly complex and still incompletely understood. Hence it is indispensable to get a deeper insight into the Hippo - YAP/TAZ signaling axis. To this end, we performed a genome-wide CRISPR knockout screen in the triple-negative breast cancer (TNBC) cell line MDA-MB231 (NF2LOF) expressing a YAP/TAZ reporter construct. We identified both negative and positive regulators of YAP/TAZ in breast cancer cells. In a second step, screening hits were further characterized in a focused single-cell CRISPR screen (Perturb-Seq), aiming at better understanding of the effects on YAP/TAZ activity regulation and downstream effects on gene expression. Here we present the technical details of our screening approaches and the results of perturbing known and novel regulators of YAP/TAZ on single cell level. We discuss the use of Perturb-Seq in the initial validation of hits from genome-wide screens and provide data that may serve as a basis for future drug discovery efforts, seeking for novel and effective treatments for triple-negative breast cancers and other malignancies with Hippo pathway alterations. Citation Format: Mareike Berlak, Zuzanna Makowska, Filippos Klironomos, Julia Kuehnlenz, Atanas Kamburov, Andreas Steffen, Martin Lange, Barbara Nicke, Ralf Lesche, Peter Staller, Charlotte Kopitz, Jan Naujoks. Identification of novel YAP/TAZ pathway regulators in the triple-negative breast cancer cell line MDA-MB231 using single-cell CRISPR screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6089.