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4103 Mesothelin Promotes Acute Myeloid Leukemia Cell Proliferation, Adhesion, and Chemoresistance through Novel Binding Partner Lyn

Program: Oral and Poster Abstracts
Session: 602. Myeloid Oncogenesis: Basic: Poster III
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Acute Myeloid Malignancies, Diseases, Treatment Considerations, Myeloid Malignancies, Biological Processes
Monday, December 9, 2024, 6:00 PM-8:00 PM

Josh R Faust, BSc1,2*, Anilkumar Gopalakrishnapillai, PhD, MSc1,3 and Sonali P Barwe, PhD, MSc1,3

1Nemours Children's Hospital, Wilmington, DE
2Department of Biological Sciences, University of Delaware, Wilmington, DE
3Department of Biological Sciences, University of Delaware, Newark, DE

Current treatment options for pediatric acute myeloid leukemia (AML) have little curative success, presenting a need for novel therapy options. Mesothelin (MSLN), a glycosylphosphatidylinositol-anchored protein with limited expression in normal tissues, is expressed in 33% of pediatric AML patients and represents a validated immunotherapy target. Although the exact function of MSLN is widely unknown, it has been implicated to play a role in adhesion through interactions with its only known binding partner MUC16/CA125.

To understand the function of MSLN in AML, we generated 2 MSLN knockout (KO) clones of NOMO-1 cells using CRISPR/Cas9 mutagenesis followed by single cell cloning. Lack of MSLN protein was confirmed via western blotting and flow cytometry. Transcriptome analysis of NOMO-1 and MSLN KO cells displayed differential expression of 434 genes with a log2FC>1. Gene-set enrichment analysis using a gene ontology database revealed enrichment of pathways such as leukocyte proliferation (NES=1.3; p<0.05), G2/M checkpoint (NES=1.3; p=0.055), oxidative phosphorylation (NES=1.6; p<0.01), glycolysis (NES=1.3; p<0.05), cell-cell junction assembly (NES=2.2; p<0.01), and positive regulation of cell adhesion (NES=1.7; p<0.01). Consistent with the pathway analysis, we observed that NOMO-1 cells had a significantly shorter doubling time compared to MSLN KO (39.8 h vs 53.7 h; p<0.05). NOMO-1 also had significantly higher EdU+ cells (22.4 ± 5.9%) than NOMO-1 MSLN KO cells (17.8 ± 2.1%; p<0.05). Cell cycle progression assayed by flow cytometry revealed higher proportions of cells in G2/M (13.4 ± 1.8%) in NOMO-1 compared to MSLN KO cells (6.4 ± 0.4%; p<0.05). Mito Stress test using Seahorse xF Pro showed NOMO-1 cells had significantly higher basal respiration normalized oxygen consumption rates (OCR) (38.3 ± 5.3 vs. 22.0 ± 5.1 pmol/min; p<0.0001), maximal respiration OCR (84.9 ± 20.7 vs. 37.6 ± 12.1 pmol/min; p<0.0001), and ATP-linked respiration OCR (15.1 ± 4.6 vs. 7.7 ± 3.9 pmol/min; p<0.0001). These results suggest that MSLN KO reduces cell proliferation, delays cell cycle progression and suppresses metabolic fitness of NOMO-1 cells.

Based on enrichment of adhesion-based pathways, we evaluated adhesion to human bone marrow extracellular matrix (ECM) MaxGel™. The percentage of NOMO-1 cells adhering to MaxGel™ was greater (40.6 ± 11.0%) than MSLN KO cells (21.5 ± 10.2%; p<0.01). Because we have shown earlier that leukemia cell adhesion modulates drug sensitivity, cells were plated with or without MaxGel™ and treated with Ara-C or vehicle. In non-coated wells, the percentage of viable NOMO-1 cells (69.7 ± 2.2%) exposed to 3 µM Ara-C for 48 h was higher than MSLN KO cells (59.4 ± 1.9%; p<0.0001), suggesting that MSLN promotes chemoresistance. Upon interaction with ECM, NOMO-1 cells but not the MSLN KO cells were protected from Ara-C and had a significantly higher chemoprotection index (8.1% vs -3.6%; p<0.0001), indicating that MSLN-mediated adhesion of NOMO-1 cells to ECM promotes Ara-C resistance.

To understand the mechanism of MSLN-induced Ara-C resistance, novel binding partners of MSLN were identified via immunoprecipitation followed by mass spectrometry. A comparison of proteins pulled down by MSLN in NOMO-1 versus MSLN KO cells identified 72 proteins with a log2FC>1. We shortlisted Src family kinase (SFK) member Lyn (log2FC = 2.80; p<0.0001) based on its probable role in cell signaling. Immunoprecipitation followed by western blotting confirmed the MSLN-Lyn interaction. Plasma membrane-derived lipid rafts isolated from NOMO-1 cells confirmed the enrichment of MSLN and Lyn in the lipid rafts, suggesting that MSLN may recruit Lyn to the lipid rafts. To investigate the role of Lyn in MSLN-induced resistance to Ara-C, we used saracatinib (pan-SFK inhibitor) and bafetinib (Lyn inhibitor) in conjunction with Ara-C. Zero-interaction potential (ZIP) scores for cell viability calculated by synergyFinder showed that both saracatinib (ZIP=20.09; median=21.23) and bafetinib (ZIP=11.65; median=15.13) worked synergistically with Ara-C, suggesting that Lyn activity is involved in MSLN-mediated drug resistance.

Taken together, our data support MSLN playing an oncogenic role through increased proliferation, cell cycle progression, metabolic fitness, ECM adhesion, and Ara-C resistance. We identified a novel MSLN-Lyn signaling axis that could be used to improve targeted therapy approaches.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH