ABSTRACT
Protein kinases are molecular machines with rich sequence variation that distinguishes the two main evolutionary branches - tyrosine kinases (TKs) from serine/threonine kinases (STKs). Using a sequence co-variation Potts statistical energy model we previously concluded that TK catalytic domains are more likely than STKs to adopt an inactive conformation with the activation loop in an autoinhibitory folded conformation, due to intrinsic sequence effects. Here we investigate the structural basis for this phenomenon by integrating the sequence-based model with structure-based molecular dynamics (MD) to determine the effects of mutations on the free energy difference between active and inactive conformations, using a thermodynamic cycle involving many (n = 108) protein-mutation free energy perturbation (FEP) simulations in the active and inactive conformations. The sequence and structure-based results are consistent and support the hypothesis that the inactive conformation DFG-out Activation Loop Folded, is a functional regulatory state that has been stabilized in TKs relative to STKs over the course of their evolution via the accumulation of residue substitutions in the activation loop and catalytic loop that facilitate distinct substrate binding modes in trans and additional modes of regulation in cis for TKs.
Subject(s)
Catalytic Domain , Evolution, Molecular , Molecular Dynamics Simulation , Protein Serine-Threonine Kinases , Protein-Tyrosine Kinases , Protein-Tyrosine Kinases/chemistry , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Protein Conformation , Thermodynamics , Mutation , Amino Acid Sequence , HumansABSTRACT
The spindle assembly checkpoint (SAC) temporally regulates mitosis by preventing progression from metaphase to anaphase until all chromosomes are correctly attached to the mitotic spindle. Centrosomes refine the spatial organization of the mitotic spindle at the spindle poles. However, centrosome loss leads to elongated mitosis, suggesting that centrosomes also inform the temporal organization of mitosis in mammalian cells. Here, we find that the mitotic delay in acentrosomal cells is enforced by the SAC in a MPS1-dependent manner, and that a SAC-dependent mitotic delay is required for bipolar cell division to occur in acentrosomal cells. Although acentrosomal cells become polyploid, polyploidy is not sufficient to cause dependency on a SAC-mediated delay to complete cell division. Rather, the division failure in absence of MPS1 activity results from mitotic exit occurring before acentrosomal spindles can become bipolar. Furthermore, prevention of centrosome separation suffices to make cell division reliant on a SAC-dependent mitotic delay. Thus, centrosomes and their definition of two spindle poles early in mitosis provide a 'timely two-ness' that allows cell division to occur in absence of a SAC-dependent mitotic delay.
Subject(s)
Cell Cycle Proteins , Centrosome , M Phase Cell Cycle Checkpoints , Mitosis , Centrosome/metabolism , Humans , M Phase Cell Cycle Checkpoints/physiology , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/genetics , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Spindle Apparatus/metabolism , Spindle Apparatus/physiology , Cell Division , Protein-Tyrosine Kinases/metabolism , Protein-Tyrosine Kinases/genetics , HeLa CellsABSTRACT
Synaptic dysfunction is an early pathogenic event leading to cognitive decline in Huntington's disease (HD). We previously reported that the active ADAM10 level is increased in the HD cortex and striatum, causing excessive proteolysis of the synaptic cell adhesion protein N-Cadherin. Conversely, ADAM10 inhibition is neuroprotective and prevents cognitive decline in HD mice. Although the breakdown of cortico-striatal connection has been historically linked to cognitive deterioration in HD, dendritic spine loss and long-term potentiation (LTP) defects identified in the HD hippocampus are also thought to contribute to the cognitive symptoms of the disease. The aim of this study is to investigate the contribution of ADAM10 to spine pathology and LTP defects of the HD hippocampus. We provide evidence that active ADAM10 is increased in the hippocampus of two mouse models of HD, leading to extensive proteolysis of N-Cadherin, which has a widely recognized role in spine morphology and synaptic plasticity. Importantly, the conditional heterozygous deletion of ADAM10 in the forebrain of HD mice resulted in the recovery of spine loss and ultrastructural synaptic defects in CA1 pyramidal neurons. Meanwhile, normalization of the active ADAM10 level increased the pool of synaptic BDNF protein and activated ERK neuroprotective signaling in the HD hippocampus. We also show that the ADAM10 inhibitor GI254023X restored LTP defects and increased the density of mushroom spines enriched with GluA1-AMPA receptors in HD hippocampal neurons. Notably, we report that administration of the TrkB antagonist ANA12 to HD hippocampal neurons reduced the beneficial effect of GI254023X, indicating that the BDNF receptor TrkB contributes to mediate the neuroprotective activity exerted by ADAM10 inhibition in HD. Collectively, these findings indicate that ADAM10 inhibition coupled with TrkB signaling represents an efficacious strategy to prevent hippocampal synaptic plasticity defects and cognitive dysfunction in HD.
Subject(s)
ADAM10 Protein , Amyloid Precursor Protein Secretases , Hippocampus , Huntington Disease , Long-Term Potentiation , Membrane Proteins , Receptor, trkB , Signal Transduction , Animals , ADAM10 Protein/metabolism , ADAM10 Protein/genetics , Huntington Disease/metabolism , Huntington Disease/pathology , Mice , Amyloid Precursor Protein Secretases/metabolism , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Hippocampus/metabolism , Hippocampus/pathology , Receptor, trkB/metabolism , Receptor, trkB/antagonists & inhibitors , Long-Term Potentiation/drug effects , Membrane Proteins/metabolism , Membrane Proteins/genetics , Brain-Derived Neurotrophic Factor/metabolism , Disease Models, Animal , Cadherins/metabolism , Dendritic Spines/metabolism , Dendritic Spines/pathology , Neuroprotection , Male , Mice, Inbred C57BL , Neuronal Plasticity , Protein-Tyrosine Kinases/metabolism , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/genetics , Mice, KnockoutSubject(s)
Candida albicans , Dyrk Kinases , Lactobacillus , Protein Serine-Threonine Kinases , Protein-Tyrosine Kinases , Candida albicans/drug effects , Protein-Tyrosine Kinases/metabolism , Protein-Tyrosine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/antagonists & inhibitors , Lactobacillus/genetics , Humans , Antifungal Agents/pharmacology , Protein Kinase Inhibitors/pharmacologyABSTRACT
The splicing factor RNA-binding motif protein 10 (RBM10) is frequently mutated in lung adenocarcinoma (LUAD) (9-25%). Most RBM10 cancer mutations are loss-of-function, correlating with increased tumorigenesis and limiting the efficacy of current LUAD targeted therapies. Remarkably, therapeutic strategies leveraging RBM10 deficiency remain unexplored. Here, we conduct a CRISPR-Cas9 synthetic lethality (SL) screen and identify ~60 RBM10 SL genes, including WEE1 kinase. WEE1 inhibition sensitizes RBM10-deficient LUAD cells in-vitro and in-vivo. Mechanistically, we identify a splicing-independent role of RBM10 in regulating DNA replication fork progression and replication stress response, which underpins RBM10-WEE1 SL. Additionally, RBM10 interacts with active DNA replication forks, relying on DNA Primase Subunit 1 (PRIM1) that synthesizes Okazaki RNA primers. Functionally, we demonstrate that RBM10 serves as an anchor for recruiting Histone Deacetylase 1 (HDAC1) to facilitate H4K16 deacetylation and R-loop homeostasis to maintain replication fork stability. Collectively, our data reveal a role of RBM10 in fine-tuning DNA replication and provide therapeutic arsenal for targeting RBM10-deficient tumors.
Subject(s)
Adenocarcinoma of Lung , DNA Replication , Lung Neoplasms , RNA-Binding Proteins , Humans , DNA Replication/genetics , Adenocarcinoma of Lung/genetics , Adenocarcinoma of Lung/metabolism , Adenocarcinoma of Lung/pathology , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , Animals , Cell Line, Tumor , Mice , CRISPR-Cas Systems , Histone Deacetylase 1/metabolism , Histone Deacetylase 1/genetics , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/genetics , Protein-Tyrosine Kinases/metabolism , Protein-Tyrosine Kinases/genetics , R-Loop StructuresABSTRACT
The balance of proinflammatory T helper type 17 (TH17) and anti-inflammatory T regulatory (Treg) cells is crucial for immune homeostasis in health and disease. The differentiation of naïve CD4+ T cells into TH17 and Treg cells depends on T cell receptor (TCR) signaling mediated, in part, by interleukin-2-inducible T cell kinase (ITK), which stimulates mitogen-activated protein kinases (MAPKs) and Ca2+ signaling. Here, we report that, in the absence of ITK activity, naïve murine CD4+ T cells cultured under TH17-inducing conditions expressed the Treg transcription factor Foxp3 and did not develop into TH17 cells. Furthermore, ITK inhibition in vivo during allergic inflammation increased the Treg:TH17 ratio in the lung. These switched Foxp3+ Treg-like cells had suppressive function, and their transcriptomic profile resembled that of differentiated, induced Treg (iTreg) cells, but their chromatin accessibility profiles were intermediate between TH17 and iTreg cells. Like iTreg cells, switched Foxp3+ Treg-like cells had reductions in the expression of genes involved in mitochondrial oxidative phosphorylation and glycolysis, in the activation of the mechanistic target of rapamycin (mTOR) signaling pathway, and in the abundance of the TH17 pioneer transcription factor BATF. This ITK-dependent switch between TH17 and Treg cells depended on Ca2+ signaling but not on MAPKs. These findings suggest potential strategies for fine-tuning TCR signal strength through ITK to control the balance of TH17 and Treg cells.
Subject(s)
Cell Differentiation , Forkhead Transcription Factors , Protein-Tyrosine Kinases , T-Lymphocytes, Regulatory , Th17 Cells , Animals , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/cytology , T-Lymphocytes, Regulatory/metabolism , Th17 Cells/immunology , Th17 Cells/cytology , Th17 Cells/metabolism , Mice , Forkhead Transcription Factors/metabolism , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/immunology , Protein-Tyrosine Kinases/metabolism , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/immunology , Calcium Signaling , Mice, Inbred C57BL , Calcium/metabolism , Mice, Knockout , TOR Serine-Threonine Kinases/metabolism , TOR Serine-Threonine Kinases/immunologyABSTRACT
BACKGROUND: Emerging data has demonstrated that in mature neurons, SorCS2 localizes to the postsynaptic density of dendritic spines and facilitates plasma membrane sorting of TrkB by interacting with it, transmitting positive signaling from BDNF on neurons. Thus, it is possible that SorCS2 plays a role in the pathophysiology of depression by regulating the BDNF-TrkB system. METHODS: In the present study, SorCS2 expression in different brain regions [hippocampus, medial prefrontal cortex (mPFC), hypothalamus, amygdala, ventral tegmental area (VTA), and nucleus accumbens (NAc)] was thoroughly investigated in the chronic social defeat stress (CSDS) and chronic unpredictable mild stress (CUMS) models of depression. The changes in depressive-like behaviors, the hippocampal BDNF signaling cascade, and amounts of hippocampal immature neurons were further investigated after SorCS2 overexpression by microinjection of the adenovirus associated virus vector containing the coding sequence of mouse SorCS2 (AAV-SorCS2) into the hippocampus of mice exposed to CSDS or CUMS. RESULTS: It was found that both CSDS and CUMS significantly decreased the protein and mRNA expression of SorCS2 in the hippocampus but not in other brain regions. Chronic stress also notably downregulated the level of hippocampal SorCS2-TrkB binding in mice. In contrast, AAV-based genetic overexpression of hippocampal SorCS2 fully reversed the chronic stress-induced not only depressive-like behaviors but also decreased SorCS2-TrkB binding, BDNF signaling pathway, and amounts of immature neurons in the hippocampus of mice. CONCLUSION: All these results suggest that enhancing the hippocampal SorCS2 expression protects against chronic stress, producing antidepressant-like actions. Hippocampal SorCS2 may participate in depression neurobiology and be a potential antidepressant target. SIGNIFICANCE STATEMENT: Targeting of proteins to distinct subcellular compartments is essential for neuronal activity and modulated by VPS10P domain receptors which include SorCS2. In mature neurons, SorCS2 localizes to the postsynaptic density of dendritic spines and facilitates plasma membrane sorting of TrkB by interacting with it, transmitting positive signaling from BDNF on neurons. Our study is the first direct evidence preliminarily showing that SorCS2 plays a role in depression neurobiology. It was found that chronic stress induced not only depressive-like behaviors but also decreased SorCS2 expression in the hippocampus. Chronic stress did not affect SorCS2 expression in the mPFC, hypothalamus, amygdala, VTA, or NAc. In contrast, genetic overexpression of hippocampal SorCS2 prevented against chronic stress, producing antidepressant-like actions in mice. Thus, hippocampal SorCS2 is a potential participant underlying depression neurobiology and may be a novel antidepressant target. Our study may also extend the knowledge of the neurotrophic hypothesis of depression.
Subject(s)
Brain-Derived Neurotrophic Factor , Depression , Hippocampus , Mice, Inbred C57BL , Receptor, trkB , Stress, Psychological , Animals , Brain-Derived Neurotrophic Factor/metabolism , Brain-Derived Neurotrophic Factor/genetics , Hippocampus/metabolism , Stress, Psychological/metabolism , Mice , Male , Receptor, trkB/metabolism , Depression/metabolism , Signal Transduction , Disease Models, Animal , Behavior, Animal , Protein-Tyrosine Kinases/metabolism , Protein-Tyrosine Kinases/genetics , Neurons/metabolismABSTRACT
Hedgehog (Hh) signaling, an evolutionarily conserved pathway, plays an essential role in development and tumorigenesis, making it a promising drug target. Multiple negative regulators are known to govern Hh signaling; however, how activated Smoothened (SMO) participates in the activation of downstream GLI2 and GLI3 remains unclear. Herein, we identified the ciliary kinase DYRK2 as a positive regulator of the GLI2 and GLI3 transcription factors for Hh signaling. Transcriptome and interactome analyses demonstrated that DYRK2 phosphorylates GLI2 and GLI3 on evolutionarily conserved serine residues at the ciliary base, in response to activation of the Hh pathway. This phosphorylation induces the dissociation of GLI2/GLI3 from suppressor, SUFU, and their translocation into the nucleus. Loss of Dyrk2 in mice causes skeletal malformation, but neural tube development remains normal. Notably, DYRK2-mediated phosphorylation orchestrates limb development by controlling cell proliferation. Taken together, the ciliary kinase DYRK2 governs the activation of Hh signaling through the regulation of two processes: phosphorylation of GLI2 and GLI3 downstream of SMO and cilia formation. Thus, our findings of a unique regulatory mechanism of Hh signaling expand understanding of the control of Hh-associated diseases.
Subject(s)
Dyrk Kinases , Hedgehog Proteins , Protein Serine-Threonine Kinases , Protein-Tyrosine Kinases , Signal Transduction , Zinc Finger Protein Gli2 , Zinc Finger Protein Gli3 , Animals , Zinc Finger Protein Gli3/metabolism , Zinc Finger Protein Gli3/genetics , Zinc Finger Protein Gli2/metabolism , Zinc Finger Protein Gli2/genetics , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Hedgehog Proteins/metabolism , Hedgehog Proteins/genetics , Mice , Protein-Tyrosine Kinases/metabolism , Protein-Tyrosine Kinases/genetics , Humans , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/genetics , Kruppel-Like Transcription Factors/metabolism , Kruppel-Like Transcription Factors/genetics , Cell Proliferation , Cilia/metabolism , Smoothened Receptor/metabolism , Smoothened Receptor/genetics , Nuclear Proteins , Repressor ProteinsABSTRACT
Mitotic catastrophe (MC), which occurs under dysregulated mitosis, represents a fascinating tactic to specifically eradicate tumor cells. Whether pyroptosis can be a death form of MC remains unknown. Proteasome-mediated protein degradation is crucial for M-phase. Bortezomib (BTZ), which inhibits the 20S catalytic particle of proteasome, is approved to treat multiple myeloma and mantle cell lymphoma, but not solid tumors due to primary resistance. To date, whether and how proteasome inhibitor affected the fates of cells in M-phase remains unexplored. Here, we show that BTZ treatment, or silencing of PSMC5, a subunit of 19S regulatory particle of proteasome, causes G2- and M-phase arrest, multi-polar spindle formation, and consequent caspase-3/GSDME-mediated pyroptosis in M-phase (designated as mitotic pyroptosis). Further investigations reveal that inhibitor of WEE1/PKMYT1 (PD0166285), but not inhibitor of ATR, CHK1 or CHK2, abrogates the BTZ-induced G2-phase arrest, thus exacerbates the BTZ-induced mitotic arrest and pyroptosis. Combined BTZ and PD0166285 treatment (named BP-Combo) selectively kills various types of solid tumor cells, and significantly lessens the IC50 of both BTZ and PD0166285 compared to BTZ or PD0166285 monotreatment. Studies using various mouse models show that BP-Combo has much stronger inhibition on tumor growth and metastasis than BTZ or PD0166285 monotreatment, and no obvious toxicity is observed in BP-Combo-treated mice. These findings disclose the effect of proteasome inhibitors in inducing pyroptosis in M-phase, characterize pyroptosis as a new death form of mitotic catastrophe, and identify dual inhibition of proteasome and WEE family kinases as a promising anti-cancer strategy to selectively kill solid tumor cells.
Subject(s)
Bortezomib , Cell Cycle Proteins , Mitosis , Proteasome Endopeptidase Complex , Protein-Tyrosine Kinases , Pyroptosis , Pyroptosis/drug effects , Humans , Mice , Animals , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/metabolism , Mitosis/drug effects , Mitosis/genetics , Proteasome Endopeptidase Complex/metabolism , Proteasome Endopeptidase Complex/genetics , Bortezomib/pharmacology , Cell Line, Tumor , Cell Cycle Proteins/genetics , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/metabolism , Proteasome Inhibitors/pharmacology , Pyrimidines/pharmacology , Pyrazoles/pharmacology , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/pathology , Xenograft Model Antitumor Assays , Gasdermins , PyrimidinonesABSTRACT
BACKGROUND: ROS1 fusion is a relatively low prevalence (0.6-2.0%) but targetable driver in lung adenocarcinoma (LUAD). Robust and low-cost tests, such as immunohistochemistry (IHC), are desirable to screen for patients potentially harboring this fusion. The aim was to investigate the prevalence of ROS1 fusions in a clinically annotated European stage I-III LUAD cohort using IHC screening with the in vitro diagnostics (IVD)-marked clone SP384, followed by confirmatory molecular analysis in pre-defined subsets. METHODS: Resected LUADs constructed in tissue microarrays, were immunostained for ROS1 expression using SP384 clone in a ready-to-use kit and Ventana immunostainers. After external quality control, analysis was performed by trained pathologists. Staining intensity of at least 2+ (any percentage of tumor cells) was considered IHC positive (ROS1 IHC + ). Subsequently, ROS1 IHC + cases were 1:1:1 matched with IHC0 and IHC1 + cases and subjected to orthogonal ROS1 FISH and RNA-based testing. RESULTS: The prevalence of positive ROS1 expression (ROS1 IHC + ), defined as IHC 2+/3+, was 4 % (35 of 866 LUADs). Twenty-eight ROS1 IHC + cases were analyzed by FISH/RNA-based testing, with only two harboring a confirmed ROS1 gene fusion, corresponding to a lower limit for the prevalence of ROS1 gene fusion of 0.23 %. They represent a 7 % probability of identifying a fusion among ROS1 IHC + cases. Both confirmed cases were among the only four with sufficient material and H-score ≥ 200, leading to a 50 % probability of identifying a ROS1 gene fusion in cases with an H-score considered strongly positive. All matched ROS1 IHC- (IHC0 and IHC1 + ) cases were also found negative by FISH/RNA-based testing, leading to a 100 % probability of lack of ROS1 fusion for ROS1 IHC- cases. CONCLUSIONS: The prevalence of ROS1 fusion in an LUAD stage I-III European cohort was relatively low. ROS1 IHC using SP384 clone is useful for exclusion of ROS1 gene fusion negative cases.
Subject(s)
Adenocarcinoma of Lung , Lung Neoplasms , Neoplasm Staging , Protein-Tyrosine Kinases , Proto-Oncogene Proteins , Humans , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Lung Neoplasms/metabolism , Lung Neoplasms/surgery , Adenocarcinoma of Lung/genetics , Adenocarcinoma of Lung/pathology , Adenocarcinoma of Lung/surgery , Adenocarcinoma of Lung/metabolism , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Male , Female , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Middle Aged , Aged , Immunohistochemistry , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Europe , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Adult , In Situ Hybridization, FluorescenceABSTRACT
BACKGROUND: The predictive value of programmed death-ligand 1 (PD-L1) expression for the efficacy of tyrosine kinase inhibitors (TKIs) in patients with advanced ROS1-rearranged non-small cell lung cancer (NSCLC) remains underexplored. This study analyzed patients with advanced NSCLC harboring ROS1 rearrangements who received first-line crizotinib to evaluate the correlation between baseline PD-L1 expression and crizotinib efficacy. METHODS: In this study, the clinical data from 371 patients diagnosed with ROS1-rearranged NSCLC at Shanghai Chest Hospital between November 2017 and December 2022 were reviewed. The patients were categorized into three groups according to the baseline PD-L1 expression: tumor proportion score (TPS) <1%, TPS 1 %-49 %, and TPS≥50 %. The objective response rate (ORR), disease control rate (DCR), and progression-free survival (PFS) following first-line crizotinib treatment were measured. RESULTS: A total of 64 patients were included in the analysis, with 16 patients in the TPS<1% group, 22 in the TPS 1 %-49 % group, and 26 in the TPS≥50 % group. The overall DCR was 100 %, and the overall ORR was 76.5 %. The ORRs were 81.2 % (13/16) in the TPS<1% group, 63.6 % (14/22) in the TPS 1 %-49 % group, and 84.6 % (22/26) in the TPS≥50 % group (p = 0.218). The median PFS across all patients was 20.21 months (95 % CI: 15.71-24.71), with a median PFS of 28.96 months (95 % CI: 19.87-38.04) in the TPS<1% group, 17.56 months (95 % CI: 12.25-22.86) in the TPS 1 %-49 % group, and 25.85 months (95 % CI: 18.52-33.17) in the TPS≥50 % group (p = 0.100). The median PFS for patients with CD74 fusion was 18.23 months (95 % CI: 15.24-21.22), while those with non-CD74 fusion exhibited a PFS of 16.49 months (95 % CI: 9.75-23.23) (p = 0.359). CONCLUSION: Patients with advanced ROS1-rearranged NSCLC were found to benefit from first-line crizotinib treatment, irrespective of baseline PD-L1 expression.
Subject(s)
B7-H1 Antigen , Carcinoma, Non-Small-Cell Lung , Crizotinib , Gene Rearrangement , Lung Neoplasms , Protein Kinase Inhibitors , Protein-Tyrosine Kinases , Proto-Oncogene Proteins , Humans , Crizotinib/therapeutic use , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Non-Small-Cell Lung/mortality , Male , Female , Middle Aged , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Lung Neoplasms/mortality , Proto-Oncogene Proteins/genetics , Aged , B7-H1 Antigen/genetics , B7-H1 Antigen/metabolism , Protein-Tyrosine Kinases/genetics , Adult , Protein Kinase Inhibitors/therapeutic use , Retrospective Studies , Aged, 80 and over , Biomarkers, Tumor/genetics , PrognosisABSTRACT
BACKGROUND: Targeted therapy is now the standard of care in driver-oncogene-positive non-small cell lung cancer (NSCLC). Its initial clinical effects are remarkable. However, almost all patients experience treatment resistance to targeted therapy. Hence, chemotherapy is considered a subsequent treatment option. In patients with driver-oncogene-negative NSCLC, combined immune checkpoint inhibitors (ICIs) and chemotherapy as the first-line therapy has been found to be beneficial. However, the efficacy of ICI plus chemotherapy against driver-oncogene-positive NSCLC other than epidermal growth factor receptor mutation and anaplastic lymphoma kinase fusion is unclear. METHODS: Using the hospital medical records, we retrospectively reviewed advanced or recurrent NSCLC patients who were treated with chemotherapy with or without ICIs at Aichi Cancer Center Hospital between January 2014 and January 2023. Patients with druggable rare mutations such as KRAS-G12C, MET exon 14 skipping, HER2 20 insertion, BRAF-V600E mutations, and ROS1 and RET rearrangements were analyzed. RESULTS: In total, 61 patients were included in this analysis. ICI plus chemotherapy was administered in 36 patients (the ICI-chemo group) and chemotherapy in 25 patients (the chemo group). The median progression-free survival (PFS) rates were 14.0 months in the ICI-chemo group and 4.8 months in the chemo group (hazard ratio [HR] = 0.54, 95% confidence interval [CI] = 0.28-1.01). The median overall survival rates were 31.3 and 21.7 months in the ICI-chemo and chemo groups, respectively (HR = 0.70, 95% CI = 0.33-1.50). Multivariate Cox regression analysis of PFS revealed that HER2 exon 20 insertion mutation was significantly associated with a poorer PFS (HR: 2.39, 95% CI: 1.19-4.77, P = 0.014). Further, ICI-chemo treatment was significantly associated with a better PFS (HR: 0.48, 95% CI: 0.25-0.91, P = 0.025). CONCLUSION: ICI plus chemotherapy improves treatment efficacy in rare driver-oncogene-positive NSCLC.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols , Carcinoma, Non-Small-Cell Lung , Immune Checkpoint Inhibitors , Lung Neoplasms , Mutation , Humans , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/mortality , Carcinoma, Non-Small-Cell Lung/pathology , Immune Checkpoint Inhibitors/therapeutic use , Male , Female , Retrospective Studies , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Lung Neoplasms/mortality , Lung Neoplasms/pathology , Middle Aged , Aged , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Adult , Aged, 80 and over , Proto-Oncogene Proteins B-raf/genetics , Receptor, ErbB-2/genetics , Proto-Oncogene Proteins c-ret/genetics , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins c-met/genetics , Proto-Oncogene Proteins/genetics , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/genetics , Progression-Free Survival , Treatment OutcomeSubject(s)
Mutation , Humans , Burkitt Lymphoma/genetics , Burkitt Lymphoma/pathology , Male , Epstein-Barr Virus Infections/genetics , Protein-Tyrosine Kinases/genetics , Lymphadenopathy/genetics , Lymphadenopathy/pathology , Immunologic Deficiency Syndromes/genetics , Autoimmune Lymphoproliferative Syndrome/genetics , Autoimmune Lymphoproliferative Syndrome/diagnosis , Female , Lymphoproliferative Disorders/genetics , Lymphoproliferative Disorders/pathology , Primary Immunodeficiency Diseases/geneticsABSTRACT
Hyperactive FMS-like receptor tyrosine kinase-3 mutants with internal tandem duplications (FLT3-ITD) are frequent driver mutations of aggressive acute myeloid leukemia (AML). Inhibitors of FLT3 produce promising results in rationally designed cotreatment schemes. Since FLT3-ITD modulates DNA replication and DNA repair, valid anti-leukemia strategies could rely on a combined inhibition of FLT3-ITD and regulators of cell cycle progression and DNA integrity. These include the WEE1 kinase which controls cell cycle progression, nucleotide synthesis, and DNA replication origin firing. We investigated how pharmacological inhibition of FLT3 and WEE1 affected the survival and genomic integrity of AML cell lines and primary AML cells. We reveal that promising clinical grade and preclinical inhibitors of FLT3 and WEE1 synergistically trigger apoptosis in leukemic cells that express FLT3-ITD. An accumulation of single and double strand DNA damage precedes this process. Mass spectrometry-based proteomic analyses show that FLT3-ITD and WEE1 sustain the expression of the ribonucleotide reductase subunit RRM2, which provides dNTPs for DNA replication. Unlike their strong pro-apoptotic effects on leukemia cells with FLT3-ITD, inhibitors of FLT3 and WEE1 do not damage healthy human blood cells and murine hematopoietic stem cells. Thus, pharmacological inhibition of FLT3-ITD and WEE1 might become an improved, rationally designed therapeutic option.
Subject(s)
Apoptosis , Cell Cycle Proteins , DNA Damage , Leukemia, Myeloid, Acute , Protein Kinase Inhibitors , Protein-Tyrosine Kinases , fms-Like Tyrosine Kinase 3 , Humans , fms-Like Tyrosine Kinase 3/genetics , fms-Like Tyrosine Kinase 3/metabolism , fms-Like Tyrosine Kinase 3/antagonists & inhibitors , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Apoptosis/drug effects , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/antagonists & inhibitors , Protein-Tyrosine Kinases/metabolism , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/genetics , Protein Kinase Inhibitors/pharmacology , Cell Line, Tumor , Drug Synergism , Animals , Pyrazoles , PyrimidinonesABSTRACT
Non-small cell lung cancer (NSCLC) has been marked as the major cause of death in lung cancer patients. Due to tumor heterogeneity, mutation burden, and emerging resistance against the available therapies in NSCLC, it has been posing potential challenges in the therapy development. Hence, identification of cancer-driving mutations and their effective inhibition have been advocated as a potential approach in NSCLC treatment. Thereof, this study aims to employ the genomic and computational-aided integrative drug repositioning strategy to identify the potential mutations in the selected molecular targets and repurpose FDA-approved drugs against them. Accordingly, molecular targets and their mutations, i.e., EGFR (V843L, L858R, L861Q, and P1019L) and ROS1 (G1969E, F2046Y, Y2092C, and V2144I), were identified based on TCGA dataset analysis. Following, virtual screening and redocking analysis, Elbasvir, Ledipasvir, and Lomitapide drugs for EGFR mutants (>-10.8 kcal/mol) while Indinavir, Ledipasvir, Lomitapide, Monteleukast, and Isavuconazonium for ROS1 mutants (>-8.8 kcal/mol) were found as putative inhibitors. Furthermore, classical molecular dynamics simulation and endpoint binding energy calculation support the considerable stability of the selected docked complexes aided by substantial hydrogen bonding and hydrophobic interactions in comparison to the respective control complexes. Conclusively, the repositioned FDA-approved drugs might be beneficial alone or in synergy to overcome acquired resistance to EGFR and ROS1-positive lung cancers.
Subject(s)
Antineoplastic Agents , Carcinoma, Non-Small-Cell Lung , Drug Repositioning , ErbB Receptors , Lung Neoplasms , Mutation , Protein-Tyrosine Kinases , Proto-Oncogene Proteins , Humans , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Lung Neoplasms/genetics , Lung Neoplasms/drug therapy , ErbB Receptors/genetics , ErbB Receptors/antagonists & inhibitors , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins/genetics , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Molecular Docking Simulation , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Genomics/methodsABSTRACT
Targeted therapy is crucial for advanced colorectal cancer (CRC) positive for genetic drivers. With advances in deep sequencing technology and new targeted drugs, existing standard molecular pathological detection systems and therapeutic strategies can no longer meet the requirements for careful management of patients with advanced CRC. Thus, rare genetic variations require diagnosis and targeted therapy in clinical practice. Rare gene mutations, amplifications, and rearrangements are usually associated with poor prognosis and poor response to conventional therapy. This review summarizes the clinical diagnosis and treatment of rare genetic variations, in genes including erb-b2 receptor tyrosine kinase 2 (ERBB2), B-Raf proto-oncogene, serine/threonine kinase (BRAF), ALK receptor tyrosine kinase/ROS proto-oncogene 1, receptor tyrosine kinase (ALK/ROS1), neurotrophic receptor tyrosine kinases (NTRKs), ret proto-oncogene (RET), fibroblast growth factor receptor 2 (FGFR2), and epidermal growth factor receptor (EGFR), to enhance understanding and identify more accurate personalized treatments for patients with rare genetic variations.
Subject(s)
Colorectal Neoplasms , Genetic Variation , Proto-Oncogene Mas , Humans , Colorectal Neoplasms/genetics , Colorectal Neoplasms/therapy , Colorectal Neoplasms/diagnosis , Mutation , Proto-Oncogene Proteins c-ret/genetics , Molecular Targeted Therapy , Proto-Oncogene Proteins B-raf/genetics , Precision Medicine , Biomarkers, Tumor/genetics , Protein-Tyrosine Kinases/genetics , Proto-Oncogene Proteins/geneticsABSTRACT
PURPOSE: Patients with metastatic ROS1 fusion-positive non-small cell lung cancer (NSCLC) are effectively treated with entrectinib, a multikinase inhibitor. Whether serial targeted gene panel sequencing of cell-free DNA (cfDNA) can identify response and progression along with mechanisms of acquired resistance to entrectinib is underexplored. METHODS: In patients with ROS1 fusion-positive NSCLC, coclinical trial plasma samples were collected before treatment, after two cycles, and after progression on entrectinib (global phase II clinical trial, ClinicalTrials.gov identifier: NCT02568267). Samples underwent cfDNA analysis using MSK-ACCESS. Variant allele frequencies of detectable alterations were correlated with objective response per RECIST v1.1 criteria. RESULTS: Twelve patients were included, with best response as partial response (n = 9, 75%), stable disease (n = 2, 17%), and progressive disease (PD; n = 1, 8%). A ROS1 fusion was variably detected in cfDNA; however, patients without a ROS1 fusion in cfDNA had no other somatic alterations detected, indicative of possible low cfDNA shedding. Clearance of the enrolling ROS1 fusion or concurrent non-ROS1 alterations (TP53, CDH1, NF1, or ARID1A mutations) was observed in response to entrectinib therapy. Radiologic PD was accompanied by redemonstration of a ROS1 fusion or non-ROS1 alterations. On-target resistance was rare; only one patient acquired ROS1 G2032R at the time of progression. Several patients acquired new off-target likely oncogenic alterations, including a truncating alteration in NF1. CONCLUSION: Serial cfDNA monitoring may complement radiographic assessments as determinants of response and resistance to entrectinib in ROS1 fusion-positive lung cancers in addition to detecting putative resistance mechanisms on progression.
Subject(s)
Benzamides , Carcinoma, Non-Small-Cell Lung , Indazoles , Lung Neoplasms , Protein-Tyrosine Kinases , Proto-Oncogene Proteins , Humans , Indazoles/therapeutic use , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Male , Proto-Oncogene Proteins/genetics , Female , Middle Aged , Benzamides/therapeutic use , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/antagonists & inhibitors , Aged , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Cell-Free Nucleic Acids/blood , Cell-Free Nucleic Acids/genetics , Adult , Oncogene Proteins, Fusion/genetics , Sequence Analysis, DNA/methodsABSTRACT
More than 10 million people suffer from lung diseases caused by the pathogenic fungus Aspergillus fumigatus. Azole antifungals represent first-line therapeutics for most of these infections but resistance is rising, therefore the identification of antifungal targets whose inhibition synergises with the azoles could improve therapeutic outcomes. Here, we generate a library of 111 genetically barcoded null mutants of Aspergillus fumigatus in genes encoding protein kinases, and show that loss of function of kinase YakA results in hypersensitivity to the azoles and reduced pathogenicity. YakA is an orthologue of Candida albicans Yak1, a TOR signalling pathway kinase involved in modulation of stress responsive transcriptional regulators. We show that YakA has been repurposed in A. fumigatus to regulate blocking of the septal pore upon exposure to stress. Loss of YakA function reduces the ability of A. fumigatus to penetrate solid media and to grow in mouse lung tissue. We also show that 1-ethoxycarbonyl-beta-carboline (1-ECBC), a compound previously shown to inhibit C. albicans Yak1, prevents stress-mediated septal spore blocking and synergises with the azoles to inhibit A. fumigatus growth.
Subject(s)
Antifungal Agents , Aspergillus fumigatus , Dyrk Kinases , Fungal Proteins , Protein Serine-Threonine Kinases , Protein-Tyrosine Kinases , Aspergillus fumigatus/genetics , Aspergillus fumigatus/drug effects , Aspergillus fumigatus/enzymology , Animals , Antifungal Agents/pharmacology , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/antagonists & inhibitors , Fungal Proteins/genetics , Fungal Proteins/metabolism , Fungal Proteins/antagonists & inhibitors , Mice , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Protein-Tyrosine Kinases/antagonists & inhibitors , Azoles/pharmacology , Aspergillosis/microbiology , Aspergillosis/drug therapy , Lung/microbiology , Spores, Fungal/drug effects , Spores, Fungal/genetics , FemaleABSTRACT
Tyrosine kinase (TK) fusions are frequently found in cancers, either as initiating events or as a mechanism of resistance to targeted therapy. Partner genes and exons in most TK fusions are followed typical recurrent patterns, but the underlying mechanisms and clinical implications of these patterns are poorly understood. By developing Functionally Active Chromosomal Translocation Sequencing (FACTS), we discover that typical TK fusions involving ALK, ROS1, RET and NTRK1 are selected from pools of chromosomal rearrangements by two major determinants: active transcription of the fusion partner genes and protein stability. In contrast, atypical TK fusions that are rarely seen in patients showed reduced protein stability, decreased downstream oncogenic signaling, and were less responsive to inhibition. Consistently, patients with atypical TK fusions were associated with a reduced response to TKI therapies. Our findings highlight the principles of oncogenic TK fusion formation and selection in cancers, with clinical implications for guiding targeted therapy.
Subject(s)
Neoplasms , Oncogene Proteins, Fusion , Protein-Tyrosine Kinases , Proto-Oncogene Proteins c-ret , Translocation, Genetic , Humans , Neoplasms/genetics , Neoplasms/drug therapy , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins c-ret/genetics , Proto-Oncogene Proteins c-ret/metabolism , Anaplastic Lymphoma Kinase/genetics , Anaplastic Lymphoma Kinase/metabolism , Receptor, trkA/genetics , Receptor, trkA/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Signal Transduction/genetics , Cell Line, TumorABSTRACT
Objective: To correlate the common driver gene variations in primary lung adenocarcinoma with their clinical characteristics and histopathological subtypes. Methods: There were 4 995 cases of primary lung adenocarcinoma diagnosed at Weifang People's Hospital of Shandong Province from January 2015 to December 2021 which were retrospectively analyzed. Among them 1 983 cases were evaluated for their histopathological subtype; 3 012 were analyzed for the correlation of their histopathological subtypes and corresponding driver gene variations, including invasive non-mucinous adenocarcinoma (INMA) and invasive mucinous adenocarcinoma (IMA), and morphologically, poorly-differentiated, moderately-differentiated and well-differentiated adenocarcinomas. Next-generation sequencing was used to detect variations in EGFR, KRAS, ALK, RET, ROS1, MET, HER2, or BRAF driver genes. Results: There were 2 384 males and 2 611 females. EGFR and ALK variations were more commonly found in female patients aged 60 years or older, with EGFR mutation rate in clinical stage â (25.80%) significantly higher than in other stages (P<0.05). KRAS mutations were more commonly detected in male smokers aged 60 years or older, HER2 mutations were more commonly in patients younger than 60 years, and RET mutations were more commonly in non-smokers (all P<0.05). No correlation was found between ROS1, MET, and BRAF gene variations and their clinical characteristics (P>0.05). For the histopathological subtypes, among the 1 899 cases of acinar adenocarcinoma, EGFR mutation rate was the highest (67.30%) compared to the other genes. Exon 21 L858R and exon 19 del were the main mutation sites in IMA and INMA, with a higher mutation rate at exon 20 T790M (11.63%) in micropapillary adenocarcinoma. In IMA, KRAS had the highest overall mutation rate (43.80%), with statistically significant difference in mutation rates of exon 2 G12D and exon 2 G12V in acinar adenocarcinoma, solid, and IMA (P<0.05). KRAS mutation at various sites were higher in poorly differentiated groups compared to moderately- and well-differentiated groups (P<0.05). HER2 mutations were more commonly observed in acinar adenocarcinoma, papillary, and micropapillary adenocarcinoma of INMA. BRAF mutation was higher in micropapillary adenocarcinoma compared with other types (P<0.05). Conclusions: Variations in EGFR, ALK, KRAS, HER2, and RET in primary lung adenocarcinoma are associated with patients' age, smoking history, and clinical stage, and driver gene mutations vary among different histopathological subtypes. EGFR mutations are predominant in INMA, while KRAS mutations are predominant in IMA.