The Protein Phosphatase Inhibitor LB100 Targets the Mesenchymal Lineage of Pancreatic Ductal Adenocarcinoma

Abstract

Pancreatic ductal adenocarcinoma (PDAC) remains a therapeutic challenge, and the aggressive basal-like/mesenchymal subtype is particularly refractory to chemotherapy, underscoring the need for novel therapies. Leveraging genetic screens, we identified protein phosphatase 2A (PP2A) catalytic subunit PPP2CA as a target. Pharmacological PP2A inhibition selectively impaired the growth of mesenchymal PDAC cells. To delineate the mechanisms underlying sensitivity to the PP2A inhibitor LB100, we employed a dual-pronged strategy. Functional characterization revealed metabolic reprogramming coupled with endoplasmic reticulum (ER) stress and cell death induction. Genome-wide genetic screens identified key modifiers of LB100 sensitivity, implicating transcriptional regulators, mRNA processing, translation, and metabolism. Based on expression data linking PP2A to splicing and transcriptional regulation, we prioritized these processes for validation. Mesenchymal PDAC cells exhibited enhanced splicing following PP2A inhibition. Notably, we identified enhanced transcriptional elongation upon LB100 treatment, particularly of short genes, driven by cyclin-dependent kinase 9 (CDK9). Our findings support a reciprocal regulatory relationship between PP2A and CDK9 that connects to the activation of ER stress response factors, including activating transcription factor 4 (ATF4). These results establish PP2A as a druggable target in mesenchymal PDAC cells and reveal a role of LB100-induced transcriptional elongation and splicing, providing a mechanistic basis to guide future therapy development.

Keywords

cyclin-dependent kinase 9 / mesenchymal / pancreatic cancer / protein phosphatase 2A / transcription

References

[1] J. Huang, V. Lok, C. H. Ngai, et al., “Worldwide Burden of, Risk Factors for, and Trends in Pancreatic Cancer,” Gastroenterology 160, no. 3 (2021): 744–754.

[2] R. L. Siegel, T. B. Kratzer, N. S. Wagle, H. Sung, and A. Jemal, “Cancer Statistics, 2026,” CA: A Cancer J Clin 76, no. 1 (2026): e70043.

[3] C. W. Buckley and E. M. O'Reilly, “Next-generation Therapies for Pancreatic Cancer,” Expert Rev Gastroenterol Hepatol 18, no. 1–3 (2024): 55–72.

[4] S. Mueller, T. Engleitner, R. Maresch, et al., “Evolutionary Routes and KRAS Dosage Define Pancreatic Cancer Phenotypes,” Nature 554, no. 7690 (2018): 62–68.

[5] C. J. Halbrook, C. A. Lyssiotis, M. M. P. di, and A. Maitra, “Pancreatic Cancer: Advances and Challenges,” Cell 186, no. 8 (2023): 1729–1754.

[6] F. Dijk, V. L. Veenstra, E. C. Soer, et al., “Unsupervised Class Discovery in Pancreatic Ductal Adenocarcinoma Reveals Cell-Intrinsic Mesenchymal Features and High Concordance Between Existing Classification Systems,” Sci Rep-uk 10, no. 1 (2020): 337.

[7] M. Chan-Seng-Yue, J. C. Kim, G. W. Wilson, et al., “Transcription Phenotypes of Pancreatic Cancer Are Driven by Genomic Events During Tumor Evolution,” Nature Genetics 52, no. 2 (2020): 231–240.

[8] H. Singh, J. Xiu, K. S. Kapner, et al., “Clinical and Genomic Features of Classical and Basal Transcriptional Subtypes in Pancreatic Cancer,” Clinical Cancer Research 30, no. 21 (2024): 4932–4942.

[9] J. J. Knox, G. O'Kane, and D. King, “PASS-01: ‘’Randomized Phase II Trial of Modified FOLFIRINOX versus Gemcitabine/Nab-Paclitaxel and Molecular Correlatives for Previously Untreated Metastatic Pancreatic Cancer,” Journal of Clinical Oncology 43, no. 31 (2025): 3355–3368.

[10] J. J. Knox, G. H. Jang, R. C. Grant, et al., “Whole Genome and Transcriptome Profiling in Advanced Pancreatic Cancer Patients on the COMPASS Trial,” Nature Communications 16, no. 1 (2025): 5919.

[11] J. P. Vainonen, M. Momeny, and J. Westermarck, “Druggable Cancer Phosphatases,” Science Translational Medicine 13, no. 588 (2021): eabe2967.

[12] M. Dzulko, M. Pons, A. Henke, G. Schneider, and O. H. Krämer, “The PP2A Subunit PR130 Is a Key Regulator of Cell Development and Oncogenic Transformation,” Biochimica Et Biophysica Acta Bba—Rev Cancer 1874, no. 2 (2020): 188453.

[13] S. L. Tinsley and A.-P. BL, “PP2A and Cancer Epigenetics: A Therapeutic Opportunity Waiting to Happen,” Nar Cancer 4, no. 1 (2022): zcac002.

[14] A. Khanna, J. E. Pimanda, and J. Westermarck, “Cancerous Inhibitor of Protein Phosphatase 2A, an Emerging Human Oncoprotein and a Potential Cancer Therapy Target,” Cancer Research 73, no. 22 (2013): 6548–6553.

[15] F. Sarais, F. Krempien, C. Koehn, et al., “Targeting PP2A in Cancer: An Underrated Option,” Journal of Experimental & Clinical Cancer Research 44, no. 1 (2025): 295.

[16] K. A. Scott, A. McCluskey, J. T. Njardarson, and W. Wang, “On the History, Synthesis, and Medicinal Use of Cantharidin, LB-100, and Their Analogs,” RSC Med Chem 17, no. 2 (2025): 743–767.

[17] B. M. D'Arcy, M. R. Swingle, and C. M. Papke, “The Antitumor Drug LB-100 Is a Catalytic Inhibitor of Protein Phosphatase 2A (PPP2CA) and 5 (PPP5C) Coordinating With the Catalytic Metals in the Active Site of PP5C,” Molecular Cancer Therapeutics 18, no. 3 (2019): 556–566.

[18] H. Rollema, R. Volkmann, P. Zagouras, et al., “In Vitro Studies on Stability and Phosphatase Activity of the Anticancer Agent LB-100 and Its Active Hydrolysis Product Endothall,” International Journal of Pharmaceutics 672 (2025): 125317.

[19] V. Chung, A. S. Mansfield, F. Braiteh, et al., “Safety, Tolerability, and Preliminary Activity of LB-100, an Inhibitor of Protein Phosphatase 2A, in Patients With Relapsed Solid Tumors: An Open-Label, Dose Escalation, First-in-Human, Phase I Trial,” Clinical Cancer Research 23, no. 13 (2017): 3277–3284.

[20] F. M. Behan, F. Iorio, G. Picco, et al., “Prioritization of Cancer Therapeutic Targets Using CRISPR–Cas9 Screens,” Nature 568, no. 7753 (2019): 511–516.

[21] B. Salvador-Barbero, M. Álvarez-Fernández, E. Zapatero-Solana, et al., “CDK4/6 Inhibitors Impair Recovery From Cytotoxic Chemotherapy in Pancreatic Adenocarcinoma,” Cancer Cell 37, no. 3 (2020): 340–353.

[22] C. Schneeweis, S. Diersch, Z. Hassan, et al., “AP1/Fra1 confers Resistance to MAPK Cascade Inhibition in Pancreatic Cancer,” Cellular and Molecular Life Sciences 80, no. 1 (2023): 12.

[23] A. Vallejo, N. Perurena, E. Guruceaga, et al., “An Integrative Approach Unveils FOSL1 as an Oncogene Vulnerability in KRAS-driven Lung and Pancreatic Cancer,” Nature Communications 8, no. 1 (2017): 14294.

[24] C. Pacini, E. Duncan, E. Gonçalves, et al., “A Comprehensive Clinically Informed Map of Dependencies in Cancer Cells and Framework for Target Prioritization,” Cancer Cell 42, no. 2 (2024): 301–316.

[25] R. M. Meyers, J. G. Bryan, J. M. McFarland, et al., “Computational Correction of Copy Number Effect Improves Specificity of CRISPR–Cas9 Essentiality Screens in Cancer Cells,” Nat Genetics 49, no. 12 (2017): 1779–1784.

[26] J. Hang, S. Y. F. Lau, R. Yin, et al., “The Role of Phosphoprotein Phosphatases Catalytic Subunit Genes in Pancreatic Cancer,” Bioscience Reports 41, no. 1 (2021): BSR20203282.

[27] S. Lier, A. Sellmer, F. Orben, et al., “A Novel Cereblon E3 Ligase Modulator With Antitumor Activity in Gastrointestinal Cancer,” Bioorganic Chemistry 119 (2022): 105505.

[28] L. Krauß, B. C. Urban, S. Hastreiter, et al., “HDAC2 facilitates Pancreatic Cancer Metastasis,” Cancer Research 82, no. 4 (2022): 695–707.

[29] J. Yue, R. Vendramin, F. Liu, et al., “Targeted Chemotherapy Overcomes Drug Resistance in Melanoma,” Gene Dev 34, no. 9–10 (2020): 637–649.

[30] N. E. Sealover, P. T. Theard, J. M. Hughes, A. J. Linke, B. R. Daley, and R. L. Kortum, “In Situ Modeling of Acquired Resistance to RTK/RAS-pathway-targeted Therapies,” Iscience 27, no. 1 (2024): 108711.

[31] M. H. Dias, A. Friskes, and S. Wang, “Paradoxical Activation of Oncogenic Signaling as a Cancer Treatment Strategy,” Cancer discovery 14 (2024): 1276–1301.

[32] C. Hetz, K. Zhang, and R. J. Kaufman, “Mechanisms, Regulation and Functions of the Unfolded Protein Response,” Nat Rev Mol Cell Bio 21, no. 8 (2020): 421–438.

[33] C. Schneider and G. Schneider, “Stress Granules—membraneless Organelles as Therapeutic Targets in Pancreatic Cancer,” EMBO Molecular Medicine 16, no. 3 (2024): 429–431.

[34] G. Kroemer, G. Mariño, and B. Levine, “Autophagy and the Integrated Stress Response,” Molecular Cell 40, no. 2 (2010): 280–293.

[35] E. A. Dunlop and A. R. Tee, “The Kinase Triad, AMPK, mTORC1 and ULK1, Maintains Energy and Nutrient Homoeostasis,” Biochem Soc T 41, no. 4 (2013): 939–943.

[36] J. Kim, M. Kundu, B. Viollet, and K. L. Guan, “AMPK and mTOR Regulate Autophagy Through Direct Phosphorylation of Ulk1,” Nature Cell Biology 13, no. 2 (2011): 132–141.

[37] J. G. Doench, N. Fusi, M. Sullender, et al., “Optimized sgRNA Design to Maximize Activity and Minimize off-target Effects of CRISPR-Cas9,” Nature Biotechnology 34, no. 2 (2016): 184–191.

[38] W. Li, H. Xu, T. Xiao, et al., “MAGeCK Enables Robust Identification of Essential Genes From Genome-scale CRISPR/Cas9 Knockout Screens,” Genome biology 15, no. 12 (2014): 554.

[39] J. J. Montero, R. Trozzo, M. Sugden, et al., “Genome-scale Pan-cancer Interrogation of lncRNA Dependencies Using CasRx,” Nature Methods 21, no. 4 (2024): 584–596.

[40] O. Kauko, S. Y. Imanishi, E. Kulesskiy, et al., “Phosphoproteome and Drug-Response Effects Mediated by the Three Protein Phosphatase 2A Inhibitor Proteins CIP2A, SET, and PME-1,” Journal of Biological Chemistry 295, no. 13 (2020): 4194–4211.

[41] Y. Li, Y. Dou, F. D. V. Leprevost, et al., “Proteogenomic Data and Resources for Pan-cancer Analysis,” Cancer Cell 41, no. 8 (2023): 1397–1406.

[42] L. Cao, C. Huang, D. C. Zhou, et al., “Proteogenomic Characterization of Pancreatic Ductal Adenocarcinoma,” Cell 184, no. 19 (2021): 5031–5052.

[43] M. H. Dias, V. Liudkovska, J. M. Navarro, et al., “The Phosphatase Inhibitor LB-100 Creates Neoantigens in Colon Cancer Cells Through Perturbation of mRNA Splicing,” Embo Reports 25, no. 5 (2024): 2220–2238.

[44] H. Zheng, Y. Qi, S. Hu, et al., “Identification of Integrator-PP2A Complex (INTAC), an RNA Polymerase II Phosphatase,” Science 370, no. 6520 (2020): eabb5872.

[45] I. Fianu, Y. Chen, C. Dienemann, et al., “Structural Basis of Integrator-mediated Transcription Regulation,” Science 374, no. 6569 (2021): 883–887.

[46] S. A. Welsh and A. Gardini, “Genomic Regulation of Transcription and RNA Processing by the Multitasking Integrator Complex,” Nat Rev Mol Cell Bio 24, no. 3 (2023): 204–220.

[47] A. Song, D. Lu, and F. X. Chen, “The Integrator–PP2A Complex Integrates Promoter-proximal Premature Termination With Chromatin Context and Genome Maintenance,” Trends in Biochemical Sciences 50, no. 12 (2025): 1118–1130.

[48] K. L. Huang, D. Jee, C. B. Stein, et al., “Integrator Recruits Protein Phosphatase 2A to Prevent Pause Release and Facilitate Transcription Termination,” Molecular Cell 80, no. 2 (2020): 345–358.e9.

[49] S. J. Vervoort, J. R. Devlin, N. Kwiatkowski, M. Teng, N. S. Gray, and R. W. Johnstone, “Targeting Transcription Cycles in Cancer,” Nature Reviews Cancer 22, no. 1 (2022): 5–24.

[50] G. Cossa, P. K. Parua, M. Eilers, and R. P. Fisher, “Protein Phosphatases in the RNAPII Transcription Cycle: Erasers, Sculptors, Gatekeepers, and Potential Drug Targets,” Gene Dev 35, no. 9–10 (2021): 658–676.

[51] S. J. Vervoort, S. A. Welsh, J. R. Devlin, et al., “The PP2A-Integrator-CDK9 Axis Fine-Tunes Transcription and Can be Targeted Therapeutically in Cancer,” Cell 184, no. 12 (2021): 3143–3162.e32.

[52] J. Yue, F. Lai, F. Beckedorff, A. Zhang, C. Pastori, and R. Shiekhattar, “Integrator Orchestrates RAS/ERK1/2 Signaling Transcriptional Programs,” Gene Dev 31, no. 17 (2017): 1809–1820.

[53] J. G. S. Madsen, S. F. Schmidt, B. D. Larsen, A. Loft, R. Nielsen, and S. Mandrup, “iRNA-seq: Computational Method for Genome-Wide Assessment of Acute Transcriptional Regulation From Total RNA-seq Data,” Nucleic Acids Res 43, no. 6 (2015): e40–e40.

[54] C. B. Stein, A. R. Field, C. A. Mimoso, et al., “Integrator Endonuclease Drives Promoter-Proximal Termination at all RNA Polymerase II-transcribed Loci,” Molecular Cell 82, no. 22 (2022): 4232–4245.e11.

[55] S. Lykke-Andersen, K. Žumer, E. Š. Molska, et al., “Integrator Is a Genome-wide Attenuator of Non-productive Transcription,” Molecular Cell 81, no. 3 (2021): 514–529.e6.

[56] A. Baluapuri, N. C. Zhao, R. J. Marina, et al., “Integrator Lo Ss Leads to dsRNA Formation That Triggers the Integrated Stress Response,” Cell 188, no. 12 (2025): 3184–3201.

[57] J. A. Guo, D. Gong, K. Evans, et al., “Integrative Genomic Identification of Therapeutic Targets for Pancreatic Cancer,” Cell reports 44, no. 9 (2025): 116191.

[58] C. Falcomatà, S. Bärthel, S. A. Widholz, et al., “Selective Multi-kinase Inhibition Sensitizes Mesenchymal Pancreatic Cancer to Immune Checkpoint Blockade by Remodeling the Tumor Microenvironment,” Nat Cancer 3, no. 3 (2022): 318–336.

[59] W. L. Hwang, K. A. Jagadeesh, J. A. Guo, et al., “Single-nucleus and Spatial Transcriptome Profiling of Pancreatic Cancer Identifies Multicellular Dynamics Associated With Neoadjuvant Treatment,” Nat Genetics 54, no. 8 (2022): 1178–1191.

[60] P. D. Chiaro, L. Nacci, F. Arco, et al., “Mapping Functional to Morphological Variation Reveals the Basis of Regional Extracellular Matrix Subversion and Nerve Invasion in Pancreatic Cancer,” Cancer Cell 42, no. 4 (2024): 662–681.e10.

[61] G. Pei, J. Min, and K. I. Rajapakshe, “Spatial Mapping of Transcriptomic Plasticity in Metastatic Pancreatic Cancer,” Nature 642, no. 8066 (2025): 212–221.

[62] L. Perelli, L. Zhang, S. Mangiameli, et al., “Evolutionary Fingerprints of Epithelial-to-mesenchymal Transition,” Nature 640, no. 8060 (2025): 1083–1092.

[63] E. J. Wagner, L. Tong, and K. Adelman, “Integrator Is a Global Promoter-proximal Termination Complex,” Molecular Cell 83, no. 3 (2023): 416–427.

[64] S. Ohe, Y. Kubota, K. Yamaguchi, et al., “ERK-mediated NELF-A Phosphorylation Promotes Transcription Elongation of Immediate-early Genes by Releasing Promoter-proximal Pausing of RNA Polymerase II,” Nature Communications 13, no. 1 (2022): 7476.

[65] S. Hu, L. Peng, C. Xu, Z. Wang, A. Song, and F. X. Chen, “SPT5 stabilizes RNA Polymerase II, Orchestrates Transcription Cycles, and Maintains the Enhancer Landscape,” Molecular Cell 81, no. 21 (2021): 4425–4439.

[66] H. Xu, D. Wu, M. Xiao, et al., “PP2A complex Disruptor SET Prompts Widespread Hypertranscription of Growth-essential Genes in the Pancreatic Cancer Cells,” Science Advances 10, no. 4 (2024): eadk6633.

[67] S. Hu, L. Peng, A. Song, et al., “INTAC Endonuclease and Phosphatase Modules Differentially Regulate Transcription by RNA Polymerase II,” Molecular Cell 83, no. 10 (2023): 1588–1604.

[68] A. M. Varghese, M. A. Perry, J. F. Chou, et al., “Clinicogenomic Landscape of Pancreatic Adenocarcinoma Identifies KRAS Mutant Dosage as Prognostic of Overall Survival,” Nature Medicine 31, no. 2 (2025): 466–477.

[69] A. Nguyen, A. H. M. Mustafa, A. K. Leydecker, et al., “The Protein Phosphatase-2A Subunit PR130 Is Involved in the Formation of Cytotoxic Protein Aggregates in Pancreatic Ductal Adenocarcinoma Cells,” Cell Communication and Signaling 22, no. 1 (2024): 217.

[70] K. Shaw, R. Bernards, K. Stegmaier, H. Varmus, and W. R. Sellers, “Prospects for Understanding and Exploiting the Consequences of Hyperactivation Lethality,” Trends in cancer 11, no. 7 (2025): 619–628.

[71] S. Mueller, A. Krätzig N de, M. Tschurtschenthaler, et al., “A Disease Model Resource Reveals Core Principles of Tissue-specific Cancer Evolution,” Nature 653, no. 8113 (2026): 265–276.

[72] M. Zatzman, F. Fuligni, R. Ripsman, et al., “Widespread Hypertranscription in Aggressive human Cancers,” Science Advances 8, no. 47 (2022): eabn0238.

[73] B. J. von, E. S, and M. C. Paul, “E-cadherin Regulates Metastasis of Pancreatic Cancer in Vivo and Is Suppressed by a SNAIL/HDAC1/HDAC2 Repressor Complex,” Gastroenterology 137, no. 1 (2009): 361–371.

[74] F. Orben, K. Lankes, C. Schneeweis, et al., “Epigenetic Drug Screening Defines a PRMT5 Inhibitor Sensitive Pancreatic Cancer Subtype,” Jci Insight 7, no. 10 (2022): e151353.

[75] M. C. Hasselluhn, D. Schlösser, L. Versemann, et al., “An NFATc1/SMAD3/cJUN Complex Restricted to SMAD4-Deficient Pancreatic Cancer Guides Rational Therapies,” Gastroenterology 166, no. 2 (2024): 298–312.e14.

[76] C. T. Contreras, J. D. Falke, D. Seifert, et al., “KRASG12C-inhibitor-based Combination Therapies for Pancreatic Cancer: Insights From Drug Screening,” Mol Oncol 19, no. 2 (2025): 295–310.

RIGHTS & PERMISSIONS

2026 The Author(s). MedComm published by Sichuan International Medical Exchange & Promotion Association (SCIMEA) and John Wiley & Sons Australia, Ltd.