A role of prefrontal cortico-hypothalamic projections in wake promotion.

Ventromedial prefrontal cortex (vmPFC) processes many critical brain functions, such as decision-making, value-coding, thinking, and emotional arousal/recognition, but whether vmPFC plays a role in sleep-wake promotion circuitry is still unclear. Here, we find that photoactivation of dorsomedial hypothalamus (DMH)-projecting vmPFC neurons, their terminals, or their postsynaptic DMH neurons rapidly switches non-rapid eye movement (NREM) but not rapid eye movement sleep to wakefulness, which is blocked by photoinhibition of DMH outputs in lateral hypothalamus (LHs). Chemoactivation of DMH glutamatergic but not GABAergic neurons innervated by vmPFC promotes wakefulness and suppresses NREM sleep, whereas chemoinhibition of vmPFC projections in DMH produces opposite effects. DMH-projecting vmPFC neurons are inhibited during NREM sleep and activated during wakefulness. Thus, vmPFC neurons innervating DMH likely represent the first identified set of cerebral cortical neurons for promotion of physiological wakefulness and suppression of NREM sleep.

Activation of NMDA receptors mediated iron accumulation via modulating iron transporters in Parkinson's disease.

Increasing evidence has confirmed that nigral iron accumulation and activation of NMDA receptors (NRs) contribute to the neurodegeneration of dopamine (DA) neurons in Parkinson's disease (PD). Earlier work indicated that activation of NRs participated in iron metabolism in the hippocampus. However, the relationship between activation of NRs and iron accumulation in DA neurons of the substantia nigra in PD was unknown. In this study, our results showed that NRs inhibitors MK-801 and AP5 protected nigrostriatal projection system and reduced nigral iron levels of 6-hydroxydopamine (6-OHDA)-induced PD rats. In vitro studies demonstrated that NMDA treatment increased the expression of iron importer divalent metal transporter 1 (DMT1) and decreased the expression of iron exporter ferropotin 1 (Fpn1), which were dependent on iron regulatory protein 1 (IRP1). This led to increased intracellular iron levels and intensified the decrease in mitochondrial transmembrane potential in MES23.5 dopaminergic neurons. In addition, we reported that MK801 and neuronal nitric oxide synthase inhibitor could antagonize 6-OHDA-induced up-regulation of IRP1 and DMT1 and down-regulation of Fpn1, thus attenuating 6-OHDA-induced iron accumulation in MES23.5 cells. This suggested that 6-OHDA-induced activation of NRs might modulate the expression of DMT1 and Fpn1 via the neuronal nitric oxide synthase-IRP1 pathway.-Xu, H., Liu, X., Xia, J., Yu, T., Qu, Y., Jiang, H., Xie, J., Activation of NMDA receptors mediated iron accumulation via modulating iron transporters in Parkinson's disease.

Comparative proteomic study reveals 17ß-HSD13 as a pathogenic protein in nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is characterized by a massive accumulation of lipid droplets (LDs). The aim of this study was to determine the function of 17ß-hydroxysteroid dehydrogenase-13 (17ß-HSD13), one of our newly identified LD-associated proteins in human subjects with normal liver histology and simple steatosis, in NAFLD development. LDs were isolated from 21 human liver biopsies, including 9 cases with normal liver histology (group 1) and 12 cases with simple steatosis (group 2). A complete set of LD-associated proteins from three liver samples of group 1 or group 2 were determined by 2D LC-MS/MS. By comparing the LD-associated protein profiles between subjects with or without NAFLD, 54 up-regulated and 35 down-regulated LD-associated proteins were found in NAFLD patients. Among them, 17ß-HSD13 represents a previously unidentified LD-associated protein with a significant up-regulation in NAFLD. Because the 17ß-HSD family plays an important role in lipid metabolism, 17ß-HSD13 was selected for validating the proteomic findings and exploring its role in the pathogenesis of NAFLD. Increased hepatic 17ß-HSD13 and its LD surface location were confirmed in db/db (diabetic) and high-fat diet-fed mice. Adenovirus-mediated hepatic overexpression of human 17ß-HSD13 induced a fatty liver phenotype in C57BL/6 mice, with a significant increase in mature sterol regulatory element-binding protein 1 and fatty acid synthase levels. The present study reports an extensive set of human liver LD proteins and an array of proteins differentially expressed in human NAFLD. We also identified 17ß-HSD13 as a pathogenic protein in the development of NAFLD.

[Glial cells are involved in iron accumulation and degeneration of dopamine neurons in Parkinson's disease].

A growing body of evidence suggests that glial cells play an important role in neural development, neural survival, nerve repair and regeneration, synaptic transmission and immune inflammation. As the highest number of cells in the central nervous system, the role of glial cells in Parkinson's disease (PD) has attracted more and more attention. It has been confirmed that nigral iron accumulation contributes to the death of dopamine (DA) neurons in PD. Until now, most researches on nigral iron deposition in PD are focusing on DA neurons, but in fact glial cells in the central nervous system also play an important role in the regulation of iron homeostasis. Therefore, this review describes the role of iron metabolism of glial cells in death of DA neurons in PD, which could provide evidence to reveal the mechanisms underlying nigral iron accumulation of DA neurons in PD and provide the basis for discovering new potential therapeutic targets for PD.

Lesion of medullary catecholaminergic neurons is associated with cardiovascular dysfunction in rotenone-induced Parkinson's disease rats.

In recent years, non-motor symptoms have been recognised as of vital importance in Parkinson's disease (PD); among these, cardiovascular dysfunctions are commonly seen in PD patients before their motor signs. The role of cardiovascular dysfunction in the progression of PD pathology, and its underlying mechanisms, are largely unknown. In the present study, in rotenone-induced PD rats, there was a gradual reduction in the number of nigral tyrosine hydroxylase-immunoreactive (TH-ir) neurons after 7, 14 and 21 days treatment. With the 56% reduction in striatal dopamine content and 52% loss of TH-ir neurons on the 14th day, the rats showed motor dysfunctions. However, from ECG power spectra, reductions in normalised low-frequency power and in the low-frequency power : high-frequency power ratio, as well as in mean blood pressure, were observed as early as the 3rd day. Plasma norepinephrine (NE) and epinephrine (E) levels were decreased by 39% and 26% respectively at the same time. Pearson's correlation analysis showed that both plasma NE and plasma E levels were positively correlated with MBP. Our results also showed that the loss of catecholaminergic neurons in the rostral ventrolateral medulla (RVLM), but not in the caudal ventrolateral medulla or the nucleus tractus solitarii, emerged earlier than the loss of nigral dopaminergic neurons. This suggests that dysfunction of catecholaminergic neurons in the RVLM might account for the reduced sympathetic activity, MBP and plasma catecholamine levels in the early stages of PD.

[The Role of Iron and Alpha-synuclein Interacting in Parkinson's Disease].

Both iron and alpha-synuclein accumulation are one of hallmarks of Parkinson's disease (PD). Alpha-synuclein aggregation is often accompanied by abnormal accumulation of iron, indicating that there is a certain link between iron and alpha-synuclein aggregation. Iron promotes alpha-synuclein aggregation by increasing its synthesis and decreasing its degradation. Also, alpha-synuclein regulates iron metabolism through its ferrireductase activity. In this review, we will describe the roles of iron and alpha-synuclein in PD pathogenesis, and the mechanisms of iron and alpha-synuclein interaction.

Ndfip1 protected dopaminergic neurons via regulating mitochondrial function and ferroptosis in Parkinson's disease.

Increasing evidence has shown that mitochondrial dysfunction and iron accumulation contribute to the pathogenesis of Parkinson's disease (PD). Nedd4 family interacting protein 1 (Ndfip1) is an adaptor protein of the Nedd4 E3 ubiquitin ligases. We have previously reported that Ndfip1 showed a neuroprotective effect in cell models of PD. However, whether Ndfip1 could protect dopaminergic neurons in PD animal models in vivo and the possible mechanisms are not known. Here, our results showed that the expression of Ndfip1 decreased in the substantia nigra (SN) of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced PD mouse model. Overexpression of Ndfip1 could improve MPTP-induced motor dysfunction significantly and antagonize the loss of dopaminergic neurons in the SN of MPTP-induced mice. Further study showed that overexpression of Ndfip1 might protect against MPTP-induced neurotoxicity through regulation of voltage-dependent anion-selective channel (VDAC). In addition, we observed the downregulation of Ndfip1 and upregulation of VDAC1/2 in 1-methyl-4-phenylpyridinium ion (MPP+)-induced SH-SY5Y cells. Furthermore, high expression of Ndfip1 in SH-SY5Y cells inhibited MPP+-induced increase of VDAC1/2 and restored MPP+-induced mitochondrial dysfunction. Furthermore, Ndfip1 prevented MPP+-induced increase in the expression of long-chain acyl-CoA synthetase 4 (ACSL4), suggesting the possible role of Ndfip1 in regulating ferroptosis. Our results provide new evidence for the neuroprotective effect of Ndfip1 on dopaminergic neurons in PD animal models and provide promising targets for the treatment of iron-related diseases, including PD.

Mitochondrial iron dyshomeostasis and its potential as a therapeutic target for Parkinson's disease.

Abnormal iron accumulation has been implicated in the etiology of Parkinson's disease (PD). Understanding how iron damages dopaminergic neurons in the substantia nigra (SN) of PD is particularly important for developing targeted neurotherapeutic strategies for the disease. However, it is still not fully understood how excess iron contributes to the neurodegeneration of dopaminergic neurons in PD. There has been increased attention on mitochondrial iron dyshomeostasis, iron-induced mitochondrial dysfunction and ferroptosis in PD. Therefore, this review begins with a brief introduction to describe cellular iron metabolism and the dysregulation of iron metabolism in PD. Then we provide an update on how iron is delivered to mitochondria and induces the damage of dopaminergic neurons in PD. In addition, we also summarize new research progress on iron-dependent ferroptosis in PD and mitochondria-localized proteins involved in ferroptosis. This will provide new insight into potential therapeutic strategies targeting mitochondrial iron dysfunction.

Hyperexcitation of ovBNST CRF neurons during stress contributes to female-biased expression of anxiety-like avoidance behaviors.

Corticotropin releasing factor (CRF) network in the oval nucleus of bed nuclei of the stria terminalis (ovBNST) is generally indicated in stress, but its role in female-biased susceptibility to anxiety is unknown. Here, we established a female-biased stress paradigm. We found that the CRF release in ovBNST during stress showed female-biased pattern, and ovBNST CRF neurons were more prone to be hyperexcited in female mice during stress in both in vitro and in vivo studies. Moreover, optogenetic modulation to exchange the activation pattern of ovBNST CRF neurons during stress between female and male mice could reverse their susceptibility to anxiety. Last, CRF receptor type 1 (CRFR1) mediated the CRF-induced excitation of ovBNST CRF neurons and showed female-biased expression. Specific knockdown of the CRFR1 level in ovBNST CRF neurons in female or overexpression that in male could reverse their susceptibility to anxiety. Therefore, we identify that CRFR1-mediated hyperexcitation of ovBNST CRF neurons in female mice encode the female-biased susceptibility to anxiety.

Iron status and lipopolysaccharide regulate Ndfip1 by activation of nuclear factor-kappa B.

Nedd4 family interacting protein 1 (Ndfip1) is an adaptor protein for the Nedd4 family of ubiquitin ligases that target proteins for degradation. Recent studies confirmed the role of Ndfip1 as a regulator of iron metabolism and pointed out that Ndfip1 was involved in iron homeostasis by regulating the degradation of iron importer divalent metal transporter 1 (DMT1). However, little is known about how Ndfip1 is regulated. The aim of this article was to investigate the regulation of Ndfip1 levels and the possible mechanisms. In this study, we investigated the effect of various stimuli, including iron status and lipopolysaccharide (LPS) on Ndfip1 expression in MES23.5 dopaminergic cell lines. Results showed that Ndfip1 expression in these cells was enhanced by ferrous iron overload, but not ferric iron overload, and decreased after iron deprivation by deferoxamine. In addition, LPS could significantly increase the expression of Ndfip1. Furthermore, we demonstrated that the regulation of Ndfip1 expression by these various stimuli was achieving by activation of nuclear factor-kappa B. We speculate that iron status and LPS may contribute to the changes of Ndfip1 expression by activation of nuclear factor-kappa B.

6-Hydroxydopamine promotes iron traffic in primary cultured astrocytes.

It is well known that disrupted brain iron homeostasis was involved in Parkinson's disease. We previously reported 6-hydroxydopamine (6-OHDA) could enhance iron influx and attenuate iron efflux process, thus promote iron accumulation in neurons. Astrocytes, the major glial cell type in the central nervous system, are largely responsible for iron distribution in the brain. However, how iron metabolism changes in astrocytes with 6-OHDA treatment are not fully elucidated. In the present study, we first observed that both iron influx and efflux were enhanced with 10 µM 6-OHDA treatment for 24 h in primary cultured astrocytes. In accordance with these iron traffic modulations, both mRNA and protein levels of iron importer divalent metal transporter 1 with iron responsive element (DMT1+IRE) and exporter ferroportin 1 (FPN1) were up-regulated in these cells. L-ferritin mRNA levels were increased. Iron regulatory protein 1 (IRP1) showed a dynamic regulation with 6-OHDA treatment, as indicated by a moderate up-regulation at 12 h, however, down-regulation at 24 h. We further demonstrated that 6-OHDA treatment could induce activation of nuclear factor-kappaB (NF-κB) p65. IκBα activation inhibitor BAY11-7082 fully blocked 6-OHDA induced NF-κB p65 phosphorylation and DMT1 + IRE up-regulation. These results suggest that 6-OHDA might promote iron transport rate in astrocytes by regulating iron transporters, IRP1 expression and NF-κB p65 activation, indicating a different response between neurons and astrocytes.

Ferritin Is Secreted from Primary Cultured Astrocyte in Response to Iron Treatment via TRPML1-Mediated Exocytosis.

Impaired iron homeostasis has been proven to be one of the critical contributors to the pathology of Parkinson's disease (PD). Ferritin is considered an intracellular protein responsible for storing cytosolic iron. Recent studies have found that ferritin can be secreted from cells independent of the classical endoplasmic reticulum-Golgi system. However, the precise mechanisms underlying the secretion of ferritin in the brain were not elucidated. In the present study, we demonstrated that the primary cultured astrocytes do have the ability to secrete ferritin, which is enhanced by iron treatment. Increased ferritin secretion was accompanied by increased protein expression of ferritin response to iron stimulation. Further study showed that iron-induced expression and secretion of ferritin could be inhibited by CQ or 3-MA pretreatment. In addition, the knockdown of transient receptor potential mucolipin 1 (TRPML1) antagonized iron-induced ferritin secretion, accompanied by further increased intracellular protein levels of ferritin. Further study demonstrated that ferritin colocalized with LAMP1 in iron-treated astrocytes. On the contrary, ras-associated protein 27a (Rab27a) knockdown further enhanced iron-induced ferritin secretion and decreased intracellular protein levels of ferritin. Furthermore, we also showed that the secretory autophagy protein tripartite motif containing 16 (TRIM16) and sec22b decreased in iron-treated astrocytes. These results suggested that astrocytes might secrete ferritin via TRPML1-mediated exocytosis. This provides new evidence for the mechanisms underlying the secretion of ferritin in primary cultured astrocytes under a high iron environment.

Ferritin confers protection against iron-mediated neurotoxicity and ferroptosis through iron chelating mechanisms in MPP+-induced MES23.5 dopaminergic cells.

Ferritin is the main iron storage protein and plays an important role in maintaining iron homeostasis. In a previous study, we reported that apoferritin exerted a neuroprotective effect against MPTP by regulation of brain iron metabolism and ferroptosis. However, the precise cellular mechanisms of extracellular ferritin underlying this protection are not fully elucidated. Ferritin was reported to be localized in different intracellular compartments, cytoplasm or released outside cells. Here we demonstrated that the intracellular iron increased after iron treatment in primary cultured astrocytes. These iron-loaded astrocytes released more ferritin in order to buffer extracellular iron. Using co-culture system of primary cultured astrocytes and MES23.5 dopaminergic cells, we showed that ferritin released by astrocytes could enter MES23.5 dopaminergic cells. And primary cultured astrocytes protected MES23.5 dopaminergic cells against 1-methyl-4-phenylpyridinium ion (MPP+)-induced neurotoxicity and ferroptosis. In addition, we found that exogenous Apoferritin or Ferritin pretreatment could significantly inhibit MPP+-induced cell damage by restoring the cell viability and mitochondrial transmembrane potential (ΔΨm). Furthermore, exogenous Apoferritin and Ferritin might also protect MES23.5 dopaminergic cells against MPP+ by decreasing reactive oxygen species (ROS) and inhibiting the increase of the labile iron pool (LIP). This suggests that astrocytes increased ferritin release to respond to iron overload, which might inhibit iron-mediated oxidative damage and ferroptosis of dopamine (DA) neurons in Parkinson's disease (PD).

Increased iron levels correlate with the selective nigral dopaminergic neuron degeneration in Parkinson's disease.

The staging of Lewy-related pathology in sporadic Parkinson's disease (PD) reveals that many brain nuclei are affected in PD during different stages, except the ventral tegmental area (VTA), which is close related to the substantia nigra (SN) and enriched in dopamine (DA) neurons. Why DA neurons are selectively degenerated in the SN of PD is far from known. In the present study, we observed that the number of tyrosine hydroxylase immunoreactive neurons decreased and iron-staining positive cells increased in the SN, but not in the VTA, in the chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated PD mice. Increased expression of divalent metal transporter 1 and decreased expression of ferroportin 1 might associate with this increased nigral iron levels. Lipofuscin granular aggregations and upregulation of alpha-synuclein (α-synuclein) were also observed only in the SN. These results suggest that increased iron levels associate with the selective degeneration of DA neurons in the SN. The intracellular regulation mechanisms for the iron transporters may be different in the SN and VTA under the same conditions. Moreover, the lipofuscin granular aggregations and upregulation of α-synuclein were also involved in the selective degeneration of dopaminergic neurons in the SN.

Alpha-synuclein aggregation is involved in the toxicity induced by ferric iron to SK-N-SH neuroblastoma cells.

Increased nigral iron levels and intracytoplasmic Lewy bodies (LBs) in the degenerating neurons are found in Parkinson's disease (PD). Whether LBs formation is involved in the toxicity of iron is largely unknown. In the present study, we observed that the toxicity of ferric iron was enhanced when SK-N-SH cells were overexpressed with wild-type alpha-synuclein. The mitochondrial transmembrane potential (Δψ(m)) and cell viability were decreased in these cells, while the intracellular reactive oxygen species (ROS) were increased, compared with that of control. More aggregated alpha-synuclein was observed in these cells. However, while silencing the expression of alpha-synuclein in SK-N-SH cells with siRNA, iron-induced toxicity could be partially alleviated, indicated by the returned Δψ(m) and cell viability and reduced ROS formation compared with that of control. No alpha-synuclein aggregation could be observed in these cells. The results suggest that alpha-synuclein aggregation was involved in the toxicity of iron to SK-N-SH neuroblastoma cells. Targeting the aggregation of alpha-synuclein might provide a therapeutic strategy for PD in the future.

New Insights into the Role of Ferritin in Iron Homeostasis and Neurodegenerative Diseases.

Growing evidence has indicated that iron deposition is one of the key factors leading to neuronal death in the neurodegenerative diseases. Ferritin is a hollow iron storage protein composed of 24 subunits of two types, ferritin heavy chain (FTH) and ferritin light chain (FTL), which plays an important role in maintaining iron homeostasis. Recently, the discovery of extracellular ferritin and ferritin in exosomes indicates that ferritin might be not only an iron storage protein within the cell, but might also be an important factor in the regulation of tissue and body iron homeostasis. In this review, we first described the structural characteristics, regulation and the physiological functions of ferritin. Secondly, we reviewed the current evidence concerning the mechanisms underlying the secretion of ferritin and the possible role of secreted ferritin in the brain. Then, we summarized the relationship between ferritin and the neurodegenerative diseases including Parkinson's disease (PD), Alzheimer's disease (AD) and neuroferritinopathy (NF). Given the importance and relationship between iron and neurodegenerative diseases, understanding the role of ferritin in the brain can be expected to contribute to our knowledge of iron dysfunction and neurodegenerative diseases.

Apoferritin improves motor deficits in MPTP-treated mice by regulating brain iron metabolism and ferroptosis.

Iron deposition is one of the key factors in the etiology of Parkinson's disease (PD). Iron-free-apoferritin has the ability to store iron by combining with a ferric hydroxide-phosphate compound to form ferritin. In this study, we investigated the role of apoferritin in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mice models and elucidated the possible underlying mechanisms. Results showed that apoferritin remarkably improved MPTP-induced motor deficits by rescuing dopaminergic neurodegeneration in the substantia nigra. Apoferritin inhibited MPTP-induced iron aggregation by down-regulating iron importer divalent metal transporter 1 (DMT1). Meanwhile, we also showed that apoferritin prevented MPTP-induced ferroptosis effectively by inhibiting the up-regulation of long-chain acyl-CoA synthetase 4 (ACSL4) and the down-regulation of ferroptosis suppressor protein 1 (FSP1). These results indicate that apoferritin exerts a neuroprotective effect against MPTP by inhibiting iron aggregation and modulating ferroptosis. This provides a promising therapeutic target for the treatment of PD.

Rg1 protects iron-induced neurotoxicity through antioxidant and iron regulatory proteins in 6-OHDA-treated MES23.5 cells.

Ginsenoside-Rg1 is one of the pharmacologically active components isolated from ginseng. It was reported that Rg1 protected dopamine (DA) neurons in 6-hydroxydopamine (6-OHDA)-induced Parkinson's disease (PD) models in vivo and in vitro. Our previous study also demonstrated that iron accumulation was involved in the toxicity of 6-OHDA. However, whether Rg1 could protect DA neurons against 6-OHDA toxicity by modulating iron accumulation and iron-induced oxidative stress is not clear. Therefore, the present study was carried out to elucidate this effect in 6-OHDA-treated MES23.5 cells and the possible mechanisms were also conducted. Findings showed Rg1 restored iron-induced decrease in mitochondrial transmembrane potential in MES23.5 cells, and increased ferrous iron influx was found in 6-OHDA-treated cells. Rg1 pretreatment could decrease this iron influx by inhibiting 6-OHDA-induced up-regulation of an iron importer protein divalent metal transporter 1 with iron responsive element (DMT1 + IRE). Furthermore, findings also showed that the effect of Rg1 on DMT1 + IRE expression was due to its inhibition of iron regulatory proteins (IRPs) by its antioxidant effect. These results suggested that the neuroprotective effect of Rg1 against iron toxicity in 6-OHDA-treated cells was to decrease the cellular iron accumulation and attenuate the improper up-regulation of DMT1 + IRE via IRE/IRP system. This provides new insight to understand the pharmacological effects of Rg1 on iron-induced degeneration of DA neurons.

Ndfip1 Prevents Rotenone-Induced Neurotoxicity and Upregulation of α-Synuclein in SH-SY5Y Cells.

Nedd4 family interacting protein 1 (Ndfip1) is an adaptor of Nedd4-family ubiquitin ligases. Experimental results showed that Ndfip1 had a potential neuroprotective effect in neurology diseases. However, the neuroprotective effect and the underlying mechanisms of Ndfip1 in Parkinson's disease (PD) have not yet been fully elucidated. Therefore, in this study, we explored the neuroprotective effect of Ndfip1 against mitochondrial complex I inhibitor rotenone in a human dopaminergic neuroblastoma SH-SY5Y cell line and further elucidated its possible underlying mechanisms. Our results showed that rotenone could induce the up-regulation of α-synuclein (α-syn) in both mRNA and protein levels. The expression of Ndfip1 decreased at 24 h after rotenone treatment. Further study showed that high expression of Ndfip1 could protect SH-SY5Y cells against rotenone-induced neurotoxicity and antagonize the rotenone-induced increase in α-syn protein levels. In addition, high expression of Ndfip1 inhibited rotenone-induced increase in the protein levels of caspase-3 and decrease in tyrosine hydroxylase (TH). Further study showed that Ndfip1 did not affect the protein expression of iron regulatory protein 1 (IRP1), transferrin receptor 1 (TfR1), while antagonized the increase in protein levels of P62 and ferritin L caused by rotenone. Our findings provide specific identification of Ndfip1 proteins to inhibit the increase of α-syn in rotenone-induced SH-SY5Y cells. Ndfip1 might be a new theoretical drug target for the prevention and treatment of PD.

Next-generation sequencing based mutation profiling reveals heterogeneity of clinical response and resistance to osimertinib.

OBJECTIVES: The 3rd generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR TKI) osimertinib has shown promising efficacy both in EGFR-mutant, T790M positive non-small cell lung cancer (NSCLC) patients who have become resistant to 1st or 2nd generation EGFR TKIs and patients with sensitizing EGFR mutations as the first line therapy. However, the degree and duration of response to osimertinib are heterogeneous. We hypothesized that the concurrent genomic landscape of these tumors could play a role in clinical outcomes and/or mechanisms of resistance. MATERIALS AND METHODS: We conducted a retrospective multicenter study of lung cancer patients who had developed resistance to osimertinib. Genomic profiling was done for all the patients by using targeted next-generation sequencing encompassing 59-1021 cancer-related genes. RESULTS AND CONCLUSION: Known EGFR-dependent resistant mutations and activation of alternative pathways were identified in 44 % of all the patients with great heterogeneity. Gain-of-function mutations of CTNNB1 were highly enriched in our cohort. Some other putative resistance mechanisms to osimertinib, such as the recurrent EGFR V834 L mutation, were also identified. Moreover, pathogenic mutations of TP53 were negatively related to the efficacy of osimertinib. To sum up, heterogeneity of resistance to osimertinib was not only manifested by inter-individual differences, but also embodied in its intra-individual diversity.