Phosphorylation-Dependent ANXA2 Relocation on the Membrane and TLR4 Engagement: A Novel Signaling Axis Promoted By PIM1 Mutation in Diffuse Large B Cell Lymphoma

Background：Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma and has an overall cure rate of approximately 60%. Previously, we observed high PIM1 mutation rates in DLBCL patients with poor outcome. However, the mechanism whether they lead to enhanced PIM1 kinase activity and contribute to lymphomagenesis is currently unknown. Methods：In this study, a multifaceted approach was employed to elucidate the functional consequences of PIM1 gene mutations and their implications in DLBCL. Recurrent PIM1 gene mutations that exhibited high frequencies across various hematological cancer cohorts were screened from public databases and patient outcomes were stratified by PIM1 mutation status. Liquid chromatography-mass spectrometry (LC-MS) proteomics and bioinformatic tools were utilized to identify proteins involved in critical cellular pathways relevant to PIM1 mutation. A high-throughput drug screening platform was leveraged to find potential therapeutic vulnerabilities unique to PIM1 mutant cells. The antiproliferative effects of PIM1 and PI3K inhibitor were evaluated in DLBCL cell lines and further validated in NSG mouse xenograft models. Results：We have identified PIM1 mutations, specifically P81S, E135K, L184F, and S97N, as frequently occurring variants, with the former three significantly associated with poor outcome. Our cohort data revealed a trend towards elevated PIM1 protein expression following PIM1 mutations. PIM1-mutated DLBCL tumors exhibited accelerated proliferation and reduced apoptosis, while in vivo animal models demonstrated faster tumor growth. Mechanistically, PIM1 mutations were found to interact with ANXA2, activating it through phosphorylation of serine 26 on the ANXA2 gene. Activated ANXA2 then translocated from the cytoplasm to the cell membrane, binding with TLR4 and further activating the PI3K/AKT/mTOR signaling pathway. Additionally, high-throughput drug screening highlighted the sensitivity of PIM1-mutated cells to multiple PI3K inhibitors. Notably, the PIM1 inhibitor SMI-4a demonstrated synergistic antitumor effects with the PI3K inhibitor Linperlisib in vitro and in vivo, offering a potentially more effective therapeutic strategy for patients harboring PIM1 mutations. Conclusions: Taken together, we identify recurrent somatic PIM1 L184F mutations that may contribute to the pathogenesis of DLBCL and establish the rationale for therapeutic strategies aimed at targeting the oncogenic pathway activated in PIM1-mutated DLBCL. Keywords: PIM1, Mutation, ANXA2, Diffuse large B-cell lymphoma, Targeted therapy