DLBCL-associated PIM1 mutation and its impact on ANXA2 localization and lymphomagenesis.

Abstract

7060 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 lymphoma cohorts from public databases were screened and patient outcomes were stratified by PIM1 genen mutational sites. Liquid chromatography-mass spectrometry (LC-MS/MS) analysis combined with Co-IP was used to identify proteins interacting with PIM1. Transcriptomics analysis was 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. In particular, the PIM1 L184F mutation promoted cell proliferation and inhibited cell apoptosis in vitro and showed faster tumor growth in vivo. Mechanistically, the PIM1 L184F mutation was found to interact with the annexin A2 (ANXA2) gene, activating it through phosphorylation of serine 26. The activated ANXA2 gene then translocated from the cytoplasm to the cell membrane, binding with the Toll-like receptor 4 (TLR4) gene and recruiting BCAP to the cell membrane to interact with p85α further activating the PI3K/AKT/mTOR signaling pathway. Additionally, the high-throughput drug screening demonstrated that the PIM1 L184F mutated cells were more sensitive to the PI3K inhibitor YY20394. PIM1 inhibitor SMI-4a combined with YY20394 showed synergistic antitumor effects both in vitro and in vivo. Conclusions: Taken together, these findings not only shed light on an innovative regulatory mechanism for how PIM1 L184F mutation contributes to the pathogenesis of DLBCL but also provide a potential therapeutic strategy for effectively managing DLBCL patients harboring PIM1 L184F mutation.