Publications

Background: Acquired resistance to tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) is a critical clinical barrier. While metabolic reprogramming is implicated in target-independent resistance, the specific role of triglyceride (TG) synthesis remains unclear. Objective: This study aims to systematically elucidate the precise role and underlying mechanisms of LPIN2-mediated lipid metabolic reprogramming in TKI resistance, and to explore potential metabolic-targeted combination therapies for NSCLC. Methods: Osimertinib-resistant (PC-9 OR) and alectinib-resistant (NCI-H3122 ALR) cell lines were established via a dose-escalation strategy. The resistant cells were then compared with their original sensitive cells under both drug-present and drug-withdrawn conditions. Metabolomic liquid chromatography-mass spectrometry (LC-MS) and transcriptomic analyses revealed shared differential features, which were selected as candidate key metabolic enzymes in TKI resistance. From this pathway, LPIN2 was selected as a consistently upregulated key enzyme for validation. The LPIN2 gene was knocked out using CRISPR/Cas9 technology; a series of functional assays (e.g., drug sensitivity, colony formation), complemented by lipid supplementation, pathway inhibition (PI3K/mTOR), and a mouse xenograft model to verify the underlying mechanism. Results: LPIN2 was identified as a key driver of TKI resistance in NSCLC, with its mRNA and protein levels significantly upregulated (~2-fold) in osimertinib-resistant (PC-9 OR) and alectinib-resistant (NCI-H3122 ALR) cells. CRISPR/Cas9-mediated LPIN2 knockout restored TKI sensitivity, displaying oncogene-specific magnitudes of reduction in the half-maximal inhibitory concentration (IC50): decreasing from 3,585 nM to 229-477 nM in EGFR-mutant PC-9 OR cells, and exhibiting a more moderate reduction from 2,264 nM to 325-488 nM in ALK-fusion NCI-H3122 ALR cells, while suppressing tumor growth in vivo (Ki67 downregulation). Mechanistically, LPIN2 promoted TG accumulation and lipid droplet deposition; exogenous TG supplementation (1,2-dioleoyl-3-arachidoyl-sn-glycerol) induced TKI resistance in parental cells and rescued drug tolerance in LPIN2-knockout cells, whereas inhibition of downstream TG synthesis enzymes [diacylglycerol O-acyltransferase1/2 (DGAT1/2)] recapitulated the knockout phenotype. Furthermore, perturbation of the LPIN2-TG axis was associated with modulation of the PI3K-AKT-mTOR survival pathway, regulating apoptosis-related proteins (BCL-XL/BCL2 downregulation, BAX upregulation). Combined treatment with osimertinib and a PI3K inhibitor (Wortmannin) synergistically suppressed tumor growth and prolonged survival in xenograft models. Conclusions: LPIN2 contributes to TKI resistance in NSCLC through a novel LPIN2-TG metabolic node that is functionally associated with PI3K-AKT signaling: it enhances TG biosynthesis and lipid droplet accumulation, thereby indirectly modulating the PI3K-AKT pathway to inhibit apoptosis. Targeting this axis reverses TKI resistance in vitro and in vivo, identifying LPIN2 as a drug stress-independent resistance driver and potential biomarker for metabolic-targeted combination therapy.

JTD Keywords: Acquired-resistance, Cell lung-cancer, Driven, Fatty-acids, Lpin2, Metabolism, Non-small cell lung cancer (nsclc), Triglyceride metabolism (tg metabolism), Tyrosine kinase inhibitor resistance (tki resistance)

Eph receptor signaling plays key roles in vertebrate tissue boundary formation, axonal pathfinding, and stem cell regeneration by steering cells to positions defined by its ligand ephrin. Some of the key events in Eph-ephrin signaling are understood: ephrin binding triggers the clustering of the Eph receptor, fostering transphosphorylation and signal transduction into the cell. However, a quantitative and mechanistic understanding of how the signal is processed by the recipient cell into precise and proportional responses is largely lacking. Studying Eph activation kinetics requires spatiotemporal data on the number and distribution of receptor oligomers, which is beyond the quantitative power offered by prevalent imaging methods. Here we describe an enhanced fluorescence fluctuation imaging analysis, which employs statistical resampling to measure the Eph receptor aggregation distribution within each pixel of an image. By performing this analysis over time courses extending tens of minutes, the information-rich 4D space (x, y, oligomerization, time) results were coupled to straightforward biophysical models of protein aggregation. This analysis reveals that Eph clustering can be explained by the combined contribution of polymerization of receptors into clusters, followed by their condensation into far larger aggregates. The modeling reveals that these two competing oligomerization mechanisms play distinct roles: polymerization mediates the activation of the receptor by assembling monomers into 6- to 8-mer oligomers; condensation of the preassembled oligomers into large clusters containing hundreds of monomers dampens the signaling. We propose that the polymerization–condensation dynamics creates mechanistic explanation for how cells properly respond to variable ligand concentrations and gradients.

JTD Keywords: Eph, Ephrin, Receptor tyrosine kinase, Gradients, Cell communication

Neurotrophins are involved in many crucial cellular functions, including neurite outgrowth, synapse formation, and plasticity. Although these events have long been known, the molecular determinants underlying neuritogenesis have not been fully characterized. Ack1 (activated Cdc42-associated tyrosine kinase) is a non-receptor tyrosine kinase that is highly expressed in the brain. Here, we demonstrate that Ack1 is a molecular constituent of neurotrophin signaling cascades in neurons and PC12 cells. We report that Ack1 interacts with Trk receptors and becomes tyrosine phosphorylated and its kinase activity is increased in response to neurotrophins. Moreover, our data indicate that Ack1 acts upstream of the Akt and MAPK pathways. We show that Ack1 overexpression induces neuritic outgrowth and promotes branching in neurotrophin-treated neuronal cells, whereas the expression of Ack1 dominant negatives or short-hairpin RNAs counteract neurotrophin-stimulated differentiation. Our results identify Ack1 as a novel regulator of neurotrophin-mediated events in primary neurons and in PC12 cells.

JTD Keywords: Axonal, Branching, Dendritic, Neurotrophins, Tyrosine kinase