RESUMO
Elevated infiltration of tumor-associated macrophages (TAMs) drives tumor progression and correlates with poor prognosis for various tumor types. Our research identifies that the ablation of the Pim-1 proto-oncogene (PIM1) in non-small cell lung cancer (NSCLC) suppresses TAM infiltration and prevents them from polarizing toward the M2 phenotype, thereby reshaping the tumor immune microenvironment (TME). The predominant mechanism through which PIM1 exerts its impact on macrophage chemotaxis and polarization involves CC motif chemokine ligand 2 (CCL2). The expression level of PIM1 is positively correlated with high CCL2 expression in NSCLC, conferring a worse overall patient survival. Mechanistically, PIM1 deficiency facilitates the reprogramming of TAMs by targeting nuclear factor kappa beta (NF-κB) signaling and inhibits CCL2 transactivation by NSCLC cells. The decreased secretion of CCL2 impedes TAM accumulation and their polarization toward a pro-tumoral phenotype. Furthermore, Dual blockade of Pim1 and PD-1 collaboratively suppressed tumor growth, repolarized macrophages, and boosted the efficacy of anti-PD-1 antibody. Collectively, our findings elucidate the pivotal role of PIM1 in orchestrating TAMs within the TME of NSCLC and highlight the potential of PIM1 inhibition as a strategy for enhancing the efficacy of cancer immunotherapy.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Quimiocina CCL2 , Neoplasias Pulmonares , NF-kappa B , Proteínas Proto-Oncogênicas c-pim-1 , Microambiente Tumoral , Macrófagos Associados a Tumor , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Microambiente Tumoral/imunologia , Humanos , Quimiocina CCL2/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/genética , Camundongos , NF-kappa B/metabolismo , Animais , Macrófagos Associados a Tumor/imunologia , Macrófagos Associados a Tumor/metabolismo , Proto-Oncogene Mas , Macrófagos/imunologia , Macrófagos/metabolismo , Linhagem Celular Tumoral , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Transdução de Sinais , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/metabolismoRESUMO
PIM-1 kinase belongs to the Ser/Thr kinases family, an attractive therapeutic target for prostate cancer. Here, we screened about 100 natural substances to find potential PIM-1 inhibitors. Two natural compounds, Naringenin and Quercetin, were finally selected based on their PIM-1 inhibitory potential and binding affinities. The docking score of Naringenin and Quercetin with PIM-1 is -8.4 and - 8.1 kcal/mol, respectively. Fluorescence binding studies revealed a strong affinity (Ka values, 3.1 × 104 M-1 and 4.6 × 107 M-1 for Naringenin and Quercetin, respectively) with excellent IC50 values for Naringenin and Quercetin (28.6 µM and 34.9 µM, respectively). Both compounds inhibited the growth of prostate cancer cells (LNCaP) in a dose-dependent manner, with the IC50 value of Naringenin at 17.5 µM and Quercetin at 8.88 µM. To obtain deeper insights into the PIM-1 inhibitory effect of Naringenin and Quercetin, we performed extensive molecular dynamics simulation studies, which provided insights into the binding mechanisms of PIM-1 inhibitors. Finally, Naringenin and Quercetin were suggested to serve as potent PIM-1 inhibitors, offering targeted treatments of prostate cancer. In addition, our findings may help to design novel Naringenin and Quercetin derivatives that could be effective in therapeutic targeting of prostate cancer.
Assuntos
Flavanonas , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Neoplasias da Próstata , Proteínas Proto-Oncogênicas c-pim-1 , Quercetina , Flavanonas/farmacologia , Flavanonas/química , Quercetina/farmacologia , Quercetina/química , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Humanos , Masculino , Linhagem Celular Tumoral , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Proliferação de Células/efeitos dos fármacos , Ligação ProteicaRESUMO
Serine/threonine protein kinases (CK2, PIM-1, RIO1) are constitutively active, highly conserved, pleiotropic, and multifunctional kinases, which control several signaling pathways and regulate many cellular functions, such as cell activity, survival, proliferation, and apoptosis. Over the past decades, they have gained increasing attention as potential therapeutic targets, ranging from various cancers and neurological, inflammation, and autoimmune disorders to viral diseases, including COVID-19. Despite the accumulation of a vast amount of experimental data, there is still no "recipe" that would facilitate the search for new effective kinase inhibitors. The aim of our study was to develop an effective screening method that would be useful for this purpose. A combination of Density Functional Theory calculations and molecular docking, supplemented with newly developed quantitative methods for the comparison of the binding modes, provided deep insight into the set of desirable properties responsible for their inhibition. The mathematical metrics helped assess the distance between the binding modes, while heatmaps revealed the locations in the ligand that should be modified according to binding site requirements. The Structure-Binding Affinity Index and Structural-Binding Affinity Landscape proposed in this paper helped to measure the extent to which binding affinity is gained or lost in response to a relatively small change in the ligand's structure. The combination of the physico-chemical profile with the aforementioned factors enabled the identification of both "dead" and "promising" search directions. Tests carried out on experimental data have validated and demonstrated the high efficiency of the proposed innovative approach. Our method for quantifying differences between the ligands and their binding capabilities holds promise for guiding future research on new anti-cancer agents.
Assuntos
Antineoplásicos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Inibidores de Proteínas Quinases , Proteínas Serina-Treonina Quinases , Humanos , Antineoplásicos/química , Antineoplásicos/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Ligantes , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Sítios de Ligação , Ligação Proteica , Teoria Quântica , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/químicaRESUMO
A key step in platelet production is the migration of megakaryocytes to the vascular sinusoids within the bone marrow. This homing is mediated by the chemokine CXCL12 and its receptor CXCR4. CXCR4 is also a positive regulator of platelet activation and thrombosis. Pim-1 kinase has been shown to regulate CXCR4 signalling in other cell types, and we have previously described how Pim kinase inhibitors attenuate platelet aggregation to CXCL12. However, the mechanism by which Pim-1 regulates CXCR4 signalling in platelets and megakaryocytes has yet to be elucidated. Using human platelets, murine bone marrow-derived megakaryocytes, and the megakaryocyte cell line MEG-01, we demonstrate that pharmacological Pim kinase inhibition leads to reduced megakaryocyte and platelet function responses to CXCL12, including reduced megakaryocyte migration and platelet granule secretion. Attenuation of CXCL12 signalling was found to be attributed to the reduced surface expression of CXCR4. The decrease in CXCR4 surface levels was found to be mediated by rapid receptor internalisation, in the absence of agonist stimulation. We demonstrate that pharmacological Pim kinase inhibition disrupts megakaryocyte and platelet function by reducing constitutive CXCR4 surface expression, decreasing the number of receptors available for agonist stimulation and signalling. These findings have implications for the development and use of Pim kinase inhibitors for the treatment of conditions associated with elevated circulating levels of CXCL12/SDF1α and increased thrombotic risk.
Assuntos
Plaquetas , Quimiocina CXCL12 , Megacariócitos , Proteínas Proto-Oncogênicas c-pim-1 , Receptores CXCR4 , Transdução de Sinais , Receptores CXCR4/metabolismo , Plaquetas/metabolismo , Plaquetas/efeitos dos fármacos , Megacariócitos/metabolismo , Megacariócitos/efeitos dos fármacos , Megacariócitos/citologia , Humanos , Transdução de Sinais/efeitos dos fármacos , Animais , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Quimiocina CXCL12/metabolismo , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Movimento Celular/efeitos dos fármacos , Linhagem CelularRESUMO
INTRODUCTION: PIM Kinases (PIM-1, PIM-2, and PIM-3) have been reported to play crucial role in signaling cascades that govern cell survival, proliferation, and differentiation. Over-expression of these kinases leads to hematological malignancies such as diffuse large B cell lymphomas (DLBCL), multiple myeloma, leukemia, lymphoma and prostate cancer etc. PIM kinases as biomarkers and potential therapeutic targets have shown promise toward precision cancer therapy. The selective PIM-1, PIM-2, and/or PIM-3 isoform inhibitors have shown significant results in patients with advanced stages of cancer including relapsed/refractory cancer. AREAS COVERED: A comprehensive literature review of PIM Kinases (PIM-1, PIM-2, and PIM-3) in oncogenesis, the patented PIM kinase inhibitors (2016-Present), and their pharmacological and structural insights have been highlighted. EXPERT OPINION: Recently, PIM kinases viz. PIM-1, PIM-2, and PIM-3 (members of the serine/threonine protein kinase family) as therapeutic targets have attracted considerable interest in oncology especially in hematological malignancies. The patented PIM kinase inhibitors comprised of heterocyclic (fused)ring structure(s) like indole, pyridine, pyrazine, pyrazole, pyridazine, piperazine, thiazole, oxadiazole, quinoline, triazolo-pyridine, pyrazolo-pyridine, imidazo-pyridazine, oxadiazole-thione, pyrazolo-pyrimidine, triazolo-pyridazine, imidazo-pyridazine, pyrazolo-quinazoline and pyrazolo-pyridine etc. showed promising results in cancer chemotherapy.
Assuntos
Antineoplásicos , Neoplasias , Patentes como Assunto , Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas c-pim-1 , Humanos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Antineoplásicos/farmacologia , Animais , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Neoplasias/enzimologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Neoplasias Hematológicas/tratamento farmacológico , Neoplasias Hematológicas/patologia , Terapia de Alvo Molecular , Desenvolvimento de Medicamentos , Desenho de Fármacos , Proteínas Serina-Treonina QuinasesRESUMO
OBJECTIVE: To investigate the effects of the serine/threonine kinase family member 1 (PIM1) gene on the proliferation and apoptosis of acute myeloid leukemia (AML) U937 cells, and the regulation effect on Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway. METHODS: Bone marrow mononuclear cells from newly diagnosed adult AML patients and patients with iron deficiency anemia were collected and PIM1 mRNA expression was detected by RT-qPCR. AML cell line U937 cells were divided into U937 group (U937 cells were cultured normally), Si-PIM1 group (U937 cells were transfected with low expression adenovirus vector containing PIM1 mRNA), Si-NC group (U937 cells were transfected with low expression adenovirus vector without PIM1 mRNA), coumermycin A1 (CoA1) group (JAK2 activator CoA1 was added to U937 cells at a concentration of 20 µmol/L), and Si-PIM1+CoA1 group (U937 cells were transfected with adenoviral vector containing low expression of PIM1 mRNA and added with CoA1 at a concentration of 20 µmol/L). After culture for 24 h, the expressions of PIM1 mRNA and protein, JAK2/STAT3 pathway, cell cycle and apoptosis-related proteins in U937 cells were detected by RT-qPCR and Western blot, the cell proliferation activity was detected by MTT assay, and flow cytometry was used to detect cell cycle changes and apoptosis rate. RESULTS: The PIM1 mRNA expression level in bone marrow mononuclear cells in AML patients was higher than that in patients with iron deficiency anemia (P < 0.05). Compared with U937 group, PIM1 mRNA and protein, phosphorylated JAK2 (p-JAK2)/JAK2, phosphorylated STAT3 (p-STAT3)/STAT3, Cyclin D1, cyclin-dependent kinase 2 (CDK2) protein, cell proliferation activity, S phase and G 2/M phase proportions were decreased in Si-PIM1 group (all P < 0.05), while p27, Caspase-3 protein, G0/G1 phase proportion and apoptosis rate were increased (all P < 0.05). However, the changes of above indicators in CoA1 group were just opposite to those in Si-PIM1 group, indicating that CoA1 could reverse the effect of Si-PIM1 on U937 cells. There were no significant differences in above indexes of U937 cells between U937 group, Si-PIM1+CoA1 group and Si-NC group (P >0.05). CONCLUSION: Knockdown of PIM1 gene expression can inhibit U937 cell proliferation and promote apoptosis, in order to alleviate ALM process, which may be related to the inhibition of JAK2/STAT3 pathway activation.
Assuntos
Apoptose , Proliferação de Células , Janus Quinase 2 , Leucemia Mieloide Aguda , Proteínas Proto-Oncogênicas c-pim-1 , Fator de Transcrição STAT3 , Transdução de Sinais , Humanos , Janus Quinase 2/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/genética , Fator de Transcrição STAT3/metabolismo , Leucemia Mieloide Aguda/genética , Células U937RESUMO
The CRISPR-Cas9 system has emerged as the most prevalent gene editing technology due to its simplicity, high efficiency, and low cost. However, the homology-directed repair (HDR)-mediated gene knock-in in this system suffers from low efficiency, which limits its application in animal model preparation, gene therapy, and agricultural genetic improvement. Here, we report the design and optimization of a simple and efficient reporter-based assay to visualize and quantify HDR efficiency. Through random screening of a small molecule compound library, two groups of compounds, including the topoisomerase inhibitors and PIM1 kinase inhibitors, have been identified to promote HDR. Two representative compounds, etoposide and quercetagetin, also significantly enhance the efficiency of CRISPR-Cas9 and HDR-mediated gene knock-in in mouse embryos. Our study not only provides an assay to screen compounds that may facilitate HDR but also identifies useful tool compounds to facilitate the construction of genetically modified animal models with the CRISPR-Cas9 system.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas c-pim-1 , Edição de Genes/métodos , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-pim-1/genética , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Animais , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores da Topoisomerase/farmacologia , Humanos , Reparo de DNA por Recombinação/efeitos dos fármacos , Técnicas de Introdução de GenesRESUMO
INTRODUCTION: Nucleoporin 98 (NUP98) fusion proteins are recurrently found in leukemia and are associated with unfavorable clinical outcomes. They are distributed to the nucleus and contribute to leukemogenesis via aberrant transcriptional regulation. We previously identified NUP98-BPTF (NB) fusion in patients with T-cell acute lymphoblastic leukemia (T-ALL) using next-generation sequencing. The FG-repeat of NUP98 and the PHD finger and bromodomain of bromodomain PHD finger transcription factor (BPTF) are retained in the fusion. Like other NUP98 fusion proteins, NB is considered to regulate genes that are essential for leukemogenesis. However, its target genes or pathways remain unknown. MATERIALS AND METHODS: To investigate the potential oncogenic properties of the NB fusion protein, we lentivirally transduced a doxycycline-inducible NB expression vector into mouse NIH3T3 fibroblasts and human Jurkat T-ALL cells. RESULTS: NB promoted the transformation of mouse NIH3T3 fibroblasts by upregulating the proto-oncogene Pim1, which encodes a serine/threonine kinase. NB transcriptionally regulated Pim1 expression by binding to its promoter and activated MYC and mTORC1 signaling. PIM1 knockdown or pharmacological inhibition of mTORC1 signaling suppressed NB-induced NIH3T3 cell transformation. Furthermore, NB enhanced the survival of human Jurkat T-ALL cells by inactivating the pro-apoptotic protein BCL2-associated agonist of cell death (BAD). CONCLUSION: We demonstrated the pivotal role of NB in cell transformation and survival and identified PIM1as a key downstream target of NB. These findings propose a promising therapeutic strategy for patients with NB fusion-positive leukemia.
Assuntos
Transformação Celular Neoplásica , Complexo de Proteínas Formadoras de Poros Nucleares , Proteínas de Fusão Oncogênica , Proteínas Proto-Oncogênicas c-pim-1 , Animais , Humanos , Camundongos , Apoptose , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Células Jurkat , Células NIH 3T3 , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-pim-1/genética , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para CimaRESUMO
The proviral integration for the Moloney murine leukemia virus (PIM) kinases, belonging to serine/threonine kinase family, have been found to be overexpressed in various types of cancers, such as prostate, breast, colon, endometrial, gastric, and pancreatic cancer. The three isoforms PIM kinases i.e., PIM1, PIM2, and PIM3 share a high degree of sequence and structural similarity and phosphorylate substrates controlling tumorigenic phenotypes like proliferation and cell survival. Targeting short-lived PIM kinases presents an intriguing strategy as in vivo knock-down studies result in non-lethal phenotypes, indicating that clinical inhibition of PIM might have fewer adverse effects. The ATP binding site (hinge region) possesses distinctive attributes, which led to the development of novel small molecule scaffolds that target either one or all three PIM isoforms. Machine learning and structure-based approaches have been at the forefront of developing novel and effective chemical therapeutics against PIM in preclinical and clinical settings, and none have yet received approval for cancer treatment. The stability of PIM isoforms is maintained by PIM kinase activity, which leads to resistance against PIM inhibitors and chemotherapy; thus, to overcome such effects, PIM proteolysis targeting chimeras (PROTACs) are now being developed that specifically degrade PIM proteins. In this review, we recapitulate an overview of the oncogenic functions of PIM kinases, their structure, function, and crucial signaling network in different types of cancer, and the potential of pharmacological small-molecule inhibitors. Further, our comprehensive review also provides valuable insights for developing novel antitumor drugs that specifically target PIM kinases in the future. In conclusion, we provide insights into the benefits of degrading PIM kinases as opposed to blocking their catalytic activity to address the oncogenic potential of PIM kinases.
Assuntos
Antineoplásicos , Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas c-pim-1 , Transdução de Sinais , Animais , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-pim-1/química , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
AIMS: Non-alcoholic fatty liver disease (NAFLD) has risen as a significant global public health issue, for which vertical sleeve gastrectomy (VSG) has become an effective treatment method. The study sought to elucidate the processes through which PIM1 mitigates the advancement of NAFLD. The Pro-viral integration site for Moloney murine leukemia virus 1 (PIM1) functions as a serine/threonine kinase. Bioinformatics analysis revealed that reduced PIM1 expression in NAFLD. METHODS: To further prove the role of PIM1 in NAFLD, an in-depth in vivo experiment was performed, in which male C57BL/6 mice were randomly grouped to receive a normal or high-fat diet for 24 weeks. They were operated or delivered the loaded adeno-associated virus which the PIM1 was overexpressed (AAV-PIM1). In an in vitro experiment, AML12 cells were treated with palmitic acid to induce hepatic steatosis. KEY FINDINGS: The results revealed that the VSG surgery and virus delivery of mice alleviated oxidative stress, and apoptosis in vivo. For AML12 cells, the levels of oxidative stress, apoptosis, and lipid metabolism were reduced via PIM1 upregulation. Moreover, ML385 treatment resulted in the downregulation of the NRF2/HO-1/NQO1 signaling cascade, indicating that PIM1 mitigates NAFLD by targeting this pathway. SIGNIFICANCE: PIM1 alleviated mice liver oxidative stress and NAFLD induced by high-fat diet by regulating the NRF2/HO-1/NQO1 signaling Pathway.
Assuntos
Heme Oxigenase-1 , Camundongos Endogâmicos C57BL , NAD(P)H Desidrogenase (Quinona) , Fator 2 Relacionado a NF-E2 , Hepatopatia Gordurosa não Alcoólica , Estresse Oxidativo , Proteínas Proto-Oncogênicas c-pim-1 , Animais , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Masculino , Camundongos , Fator 2 Relacionado a NF-E2/metabolismo , NAD(P)H Desidrogenase (Quinona)/metabolismo , NAD(P)H Desidrogenase (Quinona)/genética , Heme Oxigenase-1/metabolismo , Dieta Hiperlipídica/efeitos adversos , Fígado/metabolismo , Fígado/patologia , Transdução de Sinais , Apoptose , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genéticaRESUMO
ABSTRACT: Provirus integration site for Moloney murine leukemia virus (PIM) family serine/threonine kinases perform protumorigenic functions in hematologic malignancies and solid tumors by phosphorylating substrates involved in tumor metabolism, cell survival, metastasis, inflammation, and immune cell invasion. However, a comprehensive understanding of PIM kinase functions is currently lacking. Multiple small-molecule PIM kinase inhibitors are currently being evaluated as cotherapeutics in patients with cancer. To further illuminate PIM kinase functions in cancer, we deeply profiled PIM1 substrates using the reverse in-gel kinase assay to identify downstream cellular processes targetable with small molecules. Pathway analyses of putative PIM substrates nominated RNA splicing and ribosomal RNA (rRNA) processing as PIM-regulated cellular processes. PIM inhibition elicited reproducible splicing changes in PIM-inhibitor-responsive acute myeloid leukemia (AML) cell lines. PIM inhibitors synergized with splicing modulators targeting splicing factor 3b subunit 1 (SF3B1) and serine-arginine protein kinase 1 (SRPK1) to kill AML cells. PIM inhibition also altered rRNA processing, and PIM inhibitors synergized with an RNA polymerase I inhibitor to kill AML cells and block AML tumor growth. These data demonstrate that deep kinase substrate knowledge can illuminate unappreciated kinase functions, nominating synergistic cotherapeutic strategies. This approach may expand the cotherapeutic armamentarium to overcome kinase inhibitor-resistant disease that limits durable responses in malignant disease.
Assuntos
Leucemia Mieloide Aguda , Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas c-pim-1 , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Humanos , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Camundongos , Animais , Linhagem Celular Tumoral , Especificidade por Substrato , Splicing de RNA/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Dimethyl fumarate (DMF) is a fumaric acid ester that exhibits immunoregulatory and anti-inflammatory properties. However, the function of DMF in autoimmune uveitis (AU) is incompletely understood, and studies comprehensively exploring the impact of DMF on immune cells are still lacking. METHODS: To explore the function of DMF in uveitis and its underlying mechanisms, we conducted single-cell RNA sequencing (scRNA-seq) on the cervical draining lymph node (CDLN) cells of normal, experimental autoimmune uveitis (EAU), and DMF-treated EAU mice. Additionally, we integrated scRNA-seq data of the retina and CDLNs to identify the potential impact of DMF on ocular immune cell infiltration. Flow cytometry was conducted to verify the potential target molecules of DMF. RESULTS: Our study showed that DMF treatment effectively ameliorated EAU symptoms. The proportional and transcriptional alterations in each immune cell type during EAU were reversed by DMF treatment. Bioinformatics analysis in our study indicated that the enhanced expression of Pim1 and Cxcr4 in EAU was reversed by DMF treatment. Further experiments demonstrated that DMF restored the balance between effector T (Teff) /regulatory T (Treg) cells through inhibiting the pathway of PIM1-protein kinase B (AKT)-Forkhead box O1 (FOXO1). By incorporating the scRNA-seq data of the retina from EAU mice into analysis, our study identified that T cells highly expressing Pim1 and Cxcr4 were enriched in the retina. DMF repressed the ocular infiltration of Teff cells, and this effect might depend on its inhibition of PIM1 and CXCR4 expression. Additionally, our study indicated that DMF might reduce the proportion of plasma cells by inhibiting PIM1 expression in B cells. CONCLUSIONS: DMF effectively attenuated EAU symptoms. During EAU, DMF reversed the Teff/Treg cell imbalance and suppressed the ocular infiltration of Teff cells by inhibiting PIM1 and CXCR4 expression. Thus, DMF may act as a new drug option for the treatment of AU.
Assuntos
Anti-Inflamatórios não Esteroides , Doenças Autoimunes , Fumarato de Dimetilo , Imunossupressores , Retina , Uveíte , Fumarato de Dimetilo/administração & dosagem , Fumarato de Dimetilo/farmacologia , Uveíte/genética , Uveíte/imunologia , Uveíte/terapia , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/terapia , Análise da Expressão Gênica de Célula Única , Modelos Animais de Doenças , Animais , Camundongos , Feminino , Camundongos Endogâmicos C57BL , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/genética , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Transcrição Gênica , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Atlas como Assunto , Imunossupressores/administração & dosagem , Imunossupressores/farmacologia , Anti-Inflamatórios não Esteroides/administração & dosagem , Anti-Inflamatórios não Esteroides/farmacologia , Retina/efeitos dos fármacos , Retina/imunologia , Linfonodos/efeitos dos fármacos , Linfonodos/imunologiaRESUMO
Recently, we have developed novel Pim-1 kinase inhibitors starting from a dihydrobenzofuran core structure using a computational approach. Here, we report the design and synthesis of stilbene-based Pim-1 kinase inhibitors obtained by formal elimination of the dihydrofuran ring. These inhibitors of the first design cycle, which were obtained as inseparable cis/trans mixtures, showed affinities in the low single-digit micromolar range. To be able to further optimize these compounds in a structure-based fashion, we determined the X-ray structures of the protein-ligand-complexes. Surprisingly, only the cis-isomer binds upon crystallization of the cis/trans-mixture of the ligands with Pim-1 kinase and the substrate PIMTIDE, the binding mode being largely consistent with that predicted by docking. After crystallization of the exclusively trans-configured derivatives, a markedly different binding mode for the inhibitor and a concomitant rearrangement of the glycine-rich loop is observed, resulting in the ligand being deeply buried in the binding pocket.
Assuntos
Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas c-pim-1 , Estilbenos , Humanos , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Ligantes , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Molecular , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/síntese química , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Estilbenos/química , Estilbenos/farmacologia , Estilbenos/síntese química , Relação Estrutura-AtividadeRESUMO
Zoonoses such as ZIKV and SARS-CoV-2 pose a severe risk to global health. There is urgent need for broad antiviral strategies based on host-targets filling gaps between pathogen emergence and availability of therapeutic or preventive strategies. Significant reduction of pathogen titers decreases spread of infections and thereby ensures health systems not being overloaded and public life to continue. Based on previously observed interference with FGFR1/2-signaling dependent impact on interferon stimulated gene (ISG)-expression, we identified Pim kinases as promising druggable cellular target. We therefore focused on analyzing the potential of pan-Pim kinase inhibition to trigger a broad antiviral response. The pan-Pim kinase inhibitor AZD1208 exerted an extraordinarily high antiviral effect against various ZIKV isolates, SARS-CoV-2 and HBV. This was reflected by strong reduction in viral RNA, proteins and released infectious particles. Especially in case of SARS-CoV-2, AZD1208 led to a complete removal of viral traces in cells. Kinome-analysis revealed vast changes in kinase landscape upon AZD1208 treatment, especially for inflammation and the PI3K/Akt-pathway. For ZIKV, a clear correlation between antiviral effect and increase in ISG-expression was observed. Based on a cell culture model with impaired ISG-induction, activation of the PI3K-Akt-mTOR axis, leading to major changes in the endolysosomal equilibrium, was identified as second pillar of the antiviral effect triggered by AZD1208-dependent Pim kinase inhibition, also against HBV. We identified Pim-kinases as cellular target for a broad antiviral activity. The antiviral effect exerted by inhibition of Pim kinases is based on at least two pillars: innate immunity and modulation of the endolysosomal system.
Assuntos
Antivirais , Imunidade Inata , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-pim-1 , SARS-CoV-2 , Transdução de Sinais , Serina-Treonina Quinases TOR , Humanos , Imunidade Inata/efeitos dos fármacos , Antivirais/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/imunologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Zika virus/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Animais , Vírus da Hepatite B/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Linhagem Celular , COVID-19/imunologia , COVID-19/virologia , Tratamento Farmacológico da COVID-19 , Replicação Viral/efeitos dos fármacos , Compostos de Bifenilo , TiazolidinasRESUMO
Papillary thyroid carcinoma (PTC) is the most common endocrine malignancy, and its incidence has increased rapidly in recent years. The BRAF inhibitor vemurafenib is effective against BRAFV600E-positive PTC; however, acquired resistance to single agent therapy frequently leads to tumor recurrence and metastasis, underscoring the need to develop tailored treatment strategies. We previously showed that the oncogenic kinase PIM1 was associated with the malignant phenotype and prognosis of PTC. In this study, we showed that sustained expression of the PIM1 protein in PTC was affected by the BRAFV600E mutation. Based on this regulatory mechanism, we tested the synergistic effects of inhibitors of BRAF (BRAFi) and PIM1 in BRAFV600E-positive PTC cell lines and xenograft tumors. LC-MS metabolomics analyses suggested that BRAFi/PIMi therapy acted by restricting the amounts of critical amino acids and nucleotides required by cancer cells as well as modulating DNA methylation. This study elucidates the role of BRAFV600E in the regulation of PIM1 in PTC and demonstrates the synergistic effect of a novel combination, BRAFi/PIMi, for the treatment of PTC. This discovery, along with the pathways that may be involved in the powerful efficacy of BRAFi/PIMi strategy from the perspective of cell metabolism, provides insight into the molecular basis of PTC progression and offers new perspectives for BRAF-resistant PTC treatment.
Assuntos
Sinergismo Farmacológico , Proteínas Proto-Oncogênicas B-raf , Proteínas Proto-Oncogênicas c-pim-1 , Câncer Papilífero da Tireoide , Neoplasias da Glândula Tireoide , Animais , Humanos , Camundongos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Mutação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-pim-1/genética , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Câncer Papilífero da Tireoide/genética , Câncer Papilífero da Tireoide/tratamento farmacológico , Câncer Papilífero da Tireoide/patologia , Câncer Papilífero da Tireoide/metabolismo , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Previous studies have implicated targeting Pim-1 proto-oncogene, serine/threonine kinase (PIM1) as a preventive measure against high glucose-induced cellular stress and apoptosis. This study aimed to reveal the potential role and regulatory mechanism of PIM1 in diabetic retinopathy. Human retinal microvascular endothelial cells (hRMECs) underwent high glucose induction, and fluctuations in PIM1 levels were assessed. By overexpressing PIM1, its effects on the levels of inflammatory factors, oxidative stress indicators, migration and tube formation abilities, tight junction protein expression levels, and ferroptosis in hRMECs were identified. Afterwards, hRMECs were treated with the ferroptosis-inducing agent erastin, and the effect of erastin on the above PIM1 regulatory functions was focused on. PIM1 was downregulated upon high glucose, and its overexpression inhibited the inflammatory response, oxidative stress, cell migration, and tube formation potential in hRMECs, whereas elevated tight junction protein levels. Furthermore, PIM1 overexpression reduced intracellular iron ion levels, lipid peroxidation, and levels of proteins actively involved in ferroptosis. Erastin treatment reversed the impacts of PIM1 on hRMECs, suggesting the mediation of ferroptosis in PIM1 regulation. The current study has yielded critical insights into the role of PIM1 in ameliorating high glucose-induced hRMEC dysfunction through the inhibition of ferroptosis.
Assuntos
Células Endoteliais , Ferroptose , Humanos , Animais , Retina/metabolismo , Proteínas de Junções Íntimas/metabolismo , Glucose/toxicidade , Glucose/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/farmacologiaRESUMO
Sequence studies of the entire exome and transcriptome of lymphoma tissues have identified MYD88 and PIM1 as involved in the development and oncogenic signaling. We aimed to determine the frequency of MYD88 and PIM1 mutations, as well as their expressions in conjunction with the clinicopathological parameters identified in mature large B-cell non-Hodgkin lymphomas. The ten-year retrospective study included 50 cases of mature large B-cell lymphoma, diagnosed at the Pathology Department of the Emergency County Hospital of Constanta and Sacele County Hospital of Brasov. They were statistically analyzed by demographic, clinicopathological, and morphogenetic characteristics. We used a real-time polymerase chain reaction technique to identify PIM1 and MYD88 mutations as well as an immunohistochemical technique to evaluate the expressions of the 2 genes. Patients with lymphoma in the small bowel, spleen, brain, and testis had a low-performance status Eastern Cooperative Oncology Group (Pâ =â .001). The Eastern Cooperative Oncology Group performance status represented an independent risk factor predicting mortality (HRâ =â 9.372, Pâ <â .001). An increased lactate dehydrogenase value was associated with a low survival (Pâ =â .002). The international prognostic index score represents a negative risk factor in terms of patient survival (HRâ =â 4.654, Pâ <â .001). In cases of diffuse large B-cell lymphoma (DLBCL), immunopositivity of MYD88 is associated with non-germinal center B-cell origin (Pâ <â .001). The multivariate analysis observed the association between high lactate dehydrogenase value and the immunohistochemical expression of PIM1 or with the mutant status of the PIM1 gene representing negative prognostic factors (HRâ =â 2.066, Pâ =â .042, respectively HRâ =â 3.100, Pâ =â .004). In conclusion, our preliminary data suggest that the oncogenic mutations of PIM1 and MYD88 in our DLBCL cohort may improve the diagnosis and prognosis of DLBCL patients in an advanced stage.
Assuntos
Linfoma Difuso de Grandes Células B , Fator 88 de Diferenciação Mieloide , Masculino , Humanos , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Estudos Retrospectivos , Prognóstico , Linfoma Difuso de Grandes Células B/patologia , Lactato Desidrogenases/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/genética , Proteínas Proto-Oncogênicas c-pim-1/metabolismoRESUMO
Diffuse large B-cell lymphoma (DLBCL) is the most common and aggressive type of B-cell lymphoma. Unfortunately, about one-third of patients either relapse after the initial treatment or are refractory to first-line therapy, indicating a need for new treatment modalities. PIM serine/threonine kinases are proteins that are associated with genetic mutations, overexpression, or translocation events in B-cell lymphomas. We conducted an integrative analysis of whole-exome sequencing in 52 DLBCL patients, and no amplification, mutation, or translocation of the PIM1 gene was detected. Instead, analyses of TCGA and GTEx databases identified that PIM1 expression was increased in DLBCL samples compared to normal tissue, and high expression levels were associated with poor overall survival. Moreover, interference of PIM1 significantly suppressed DLBCL cell proliferation. In addition, we identified anwulignan, a natural small-molecule compound, as a PIM1 inhibitor. Anwulignan directly binds to PIM1 and exerts antitumor effects on DLBCL in vitro and in vivo by inducing apoptosis, cell cycle arrest, and autophagic cell death. Furthermore, we identified an effective synergistic combination between anwulignan and chidamide. Our findings suggested that PIM1 could be a therapeutic target and prognostic factor for DLBCL, and anwulignan holds promise for future development as a natural product for treatment.
Assuntos
Linfoma Difuso de Grandes Células B , Proteínas Proto-Oncogênicas c-pim-1 , Humanos , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-pim-1/genética , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Camundongos , Animais , Produtos Biológicos/uso terapêutico , Linhagem Celular Tumoral , Feminino , Ensaios Antitumorais Modelo de Xenoenxerto , Masculino , Apoptose/efeitos dos fármacos , Inibidores de Proteínas Quinases/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Proliferação de Células/efeitos dos fármacos , Aminopiridinas/uso terapêutico , Aminopiridinas/farmacologia , Terapia de Alvo Molecular , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacosRESUMO
Proviral integration sitea for Moloney-murine leukemia virus (PIM) kinases are a family of highly conserved serine/tyrosine kinases consisting of three members, PIM-1, PIM-2, and PIM-3. These kinases regulate a wide range of substrates through phosphorylation and affect key cellular processes such as transcription, translation, proliferation, apoptosis, and energy metabolism. Several PIM inhibitors are currently undergoing clinical trials, such as a phase I clinical trial of Uzanserti (5) for the treatment of relapsed diffuse large B-cell lymphoma that has been completed. The current focus encompasses the structural and biological characterization of PIM, ongoing research progress on small-molecule inhibitors undergoing clinical trials, and evaluation analysis of persisting challenges in this field. Additionally, the design and discovery of small-molecule inhibitors targeting PIM in recent years have been explored, with a particular emphasis on medicinal chemistry, aiming to provide valuable insights for the future development of PIM inhibitors.
Assuntos
Neoplasias , Proteínas Proto-Oncogênicas c-pim-1 , Camundongos , Animais , Humanos , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fosforilação , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Inibidores de Proteínas Quinases/químicaRESUMO
New aromatic O-alkyl pyridine derivatives were designed and synthesised as Proviral Integration Moloney (PIM)-1 kinase inhibitors. 4c and 4f showed potent in vitro anticancer activity against NFS-60, HepG-2, PC-3, and Caco-2 cell lines and low toxicity against normal human lung fibroblast Wi-38 cell line. Moreover, 4c and 4f induced apoptosis in the four tested cancer cell lines with high percentage. In addition, 4c and 4f significantly induced caspase 3/7 activation in HepG-2 cell line. Furthermore, 4c and 4f showed potent PIM-1 kinase inhibitory activity with IC50 = 0.110, 0.095 µM, respectively. Kinetic studies indicated that 4c and 4f were both competitive and non-competitive inhibitors for PIM-1 kinase enzyme. In addition, in silico prediction of physiochemical properties, pharmacokinetic profile, ligand efficiency, ligand lipophilic efficiency, and induced fit docking studies were consistent with the biological and kinetic studies, and predicted that 4c and 4f could act as PIM-1 kinase competitive non-adenosine triphosphate (ATP) mimetics with drug like properties.