Abstract
Introduction: Acute myeloid leukemia (AML) is a heterogeneous disease characterized by the aggressive accumulation of immature stem and progenitor cells in the bone marrow. The complexity of AML is driven by a wide spectrum of genetic and molecular landscapes, which highlights the need for specific targeted therapies. The most common mutation occurs in FMS-like receptor tyrosine kinase 3 (FLT3), which is present in ~30% of AML patients. Mutations harboring internal tandem duplications (ITD) within the juxtamembrane (JM) domain of the FLT3 kinase are often considered driver mutations for disease progression and have an unfavorable prognosis for patients. Despite the revolutionary development of FLT3-targeted tyrosine kinase inhibitors (TKIs) such as Midostaurin, drug resistance continues to pose a clinical obstacle. One promising strategy to counteract resistance is by targeting the ubiquitin proteasome system (UPS), as it consists of an abundant network of tightly regulated proteins and offers ample opportunities for novel targets. High PSMD3 expression, a non-ATPase subunit of the 19S regulatory particle that plays a role in substrate recognition and binding, correlated with worse overall survival in FLT3-mutated AML patients. Therefore, we hypothesized that PSMD3 contributes to FLT3-mutated AML progression and might play a role in therapy resistance.
Methods: We generated a resistant MOLM-14 (FLT3+) AML cell line through long-term exposure to Midostaurin (up to 100nM). Resistance was validated using Annexin V staining, colony formation, and cell viability assays. Proteins were analyzed by immunoblot analysis comparing parental (sensitive) and resistant cells. We also analyzed cells by single-cell RNA sequencing (scRNAseq) at Parse Biosciences and analyzed the data using their Trailmaker software. To assess the role of PSMD3 in AML, Midostaurin-sensitive and -resistant MOLM-14 cells were infected with lentivirus encoding a non-targeting control (shNT) and a PSMD3-targeting shRNA (shPSMD3) and analyzed by colony formation assays. MOLM-14 sensitive cells were sent for RNAseq, lipidomics, and metabolomics profiling.
Results: Uponanalysis of the scRNAseq data, the most significantly dysregulated gene when comparing sensitive versus resistant MOLM-14 cells was lysozyme (LYS), a key component for neutrophil degranulation. Immunoblot analyses revealed a marked dysregulation of the Aurora kinases, which are essential for mitotic progression and chromosome segregation. Pathway enrichment analysis of our RNA sequencing data revealed that PSMD3-depleted MOLM-14 cells identified neutrophil degranulation as the top dysregulated pathway. This finding is consistent with prior proteomics analyses from our group, which also showed that PSMD3 knockdown is involved in neutrophil degranulation. Lipidomic and metabolomic profiling revealed broad metabolic reprogramming following PSMD3 knockdown. Notable changes included downregulation of diacylglycerol, ceramide phosphate, fatty acid esters, and tricarboxylic acid (TCA) cycle intermediates. Conversely, several metabolites were upregulated, such as glycerol-3-phosphate, oxidized glutathione, phosphatidic acid (PA), and phosphatidylethanolamine (PE). These alterations might affect key pathways such as glycerophospholipid, sphingolipid, glutathione, glycerolipid, and purine metabolism. shPSMD3 reduced colony formation of both TKI-sensitive and -resistant MOLM-14 cells, with greater impacts in TKI resistance.
Conclusion: Neutrophil degranulation emerged as the consistently dysregulated pathway across our proteomics, RNA sequencing, and scRNAseq data, suggesting a potential mechanism for PSMD3 in AML. The reduction of PSMD3 not only disrupted immune-related pathways but also reprogrammed key metabolites. Altogether, these data indicate that PSMD3 regulates neutrophil degranulation, immune-related pathways, and metabolic reprogramming, and may be a novel therapeutic target in FLT3+ AML and possibly other cancers.
