RESUMO
Hematopoietic cell kinase (Hck), a non-receptor tyrosine kinase belonging to the Src kinase family, is intricately linked to the pathogenesis of numerous human diseases, with a particularly pronounced association with cancer. Hck not only directly impacts the proliferation, migration, and apoptosis of cancer cells but also interacts with JAK/STAT, MEK/ERK, PI3K/AKT, CXCL12/CXCR4, and other pathways. Hck also influences the tumor microenvironment to facilitate the onset and progression of cancer. This paper delves into the functional role and regulatory mechanisms of Hck in various solid tumors. Additionally, it explores the implications of Hck in hematological malignancies. The review culminates with a summary of the current research status of Hck inhibitors, the majority of which are in the pre-clinical phase of investigation. Notably, these inhibitors are predominantly utilized in the therapeutic management of leukemia, with their combinatorial potential indicating promising avenues for future research. In conclusion, this review underscores the significance of the mechanism of Hck in solid tumors. This insight is crucial for comprehending the current research trends regarding Hck: targeted therapy against Hck shows great promise in both diagnosis and treatment of malignant tumors. Further investigation into the role of Hck in cancer, coupled with the development of specific inhibitors, has the potential to revolutionize approaches to cancer treatment.
Assuntos
Progressão da Doença , Neoplasias , Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas c-hck , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/patologia , Inibidores de Proteínas Quinases/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Animais , Proteínas Proto-Oncogênicas c-hck/metabolismo , Proteínas Proto-Oncogênicas c-hck/antagonistas & inibidores , Antineoplásicos/uso terapêutico , Antineoplásicos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Microambiente Tumoral , Terapia de Alvo MolecularRESUMO
Although Bruton's tyrosine kinase (BTK) inhibitors (BTKi) have significantly improved patient prognosis, mantle cell lymphoma (MCL) is still considered incurable due to primary and acquired resistance. We have recently shown that aberrant expression of the Src-family tyrosine kinase hematopoietic cell kinase (HCK) in MCL correlates with poor prognosis, and that genetic HCK perturbation impairs growth and integrin-mediated adhesion of MCL cells. Here, we show that KIN-8194, a dual inhibitor of BTK and HCK with in vivo activity against Myd88-L265P-driven diffuse large B-cell lymphoma and Waldenström Macroglobulinemia, has a potent growth inhibitory effect in MCL cell lines and primary MCL cells, irrespective of their sensitivity to BTKi (ibrutinib and acalabrutinib). In BTKi-resistant cells this is mediated by inhibition of HCK, which results in repression of AKT-S6 signaling. In addition, KIN-8194 inhibits integrin-mediated adhesion of BTKi-sensitive and insensitive MCL cells to fibronectin and stromal cells in an HCK-dependent manner. Finally, we show that MCL cells with acquired BTKi resistance retain their sensitivity to KIN-8194. Taken together, our data demonstrate that KIN-8194 inhibits growth and integrin-mediated adhesion of BTKi-sensitive MCL cells, as well as MCL cells with primary or acquired BTKi resistance. This renders KIN-8194 a promising novel treatment for MCL patients.
Assuntos
Tirosina Quinase da Agamaglobulinemia , Adesão Celular , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos , Integrinas , Linfoma de Célula do Manto , Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas c-hck , Linfoma de Célula do Manto/tratamento farmacológico , Linfoma de Célula do Manto/patologia , Linfoma de Célula do Manto/metabolismo , Linfoma de Célula do Manto/genética , Humanos , Adesão Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-hck/metabolismo , Proteínas Proto-Oncogênicas c-hck/genética , Proteínas Proto-Oncogênicas c-hck/antagonistas & inibidores , Integrinas/metabolismo , Proliferação de Células/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Linhagem Celular Tumoral , Pirimidinas/farmacologia , Adenina/análogos & derivados , Adenina/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Renal inflammation and fibrosis are the common pathways leading to progressive chronic kidney disease (CKD). We previously identified hematopoietic cell kinase (HCK) as upregulated in human chronic allograft injury promoting kidney fibrosis; however, the cellular source and molecular mechanisms are unclear. Here, using immunostaining and single cell sequencing data, we show that HCK expression is highly enriched in pro-inflammatory macrophages in diseased kidneys. HCK-knockout (KO) or HCK-inhibitor decreases macrophage M1-like pro-inflammatory polarization, proliferation, and migration in RAW264.7 cells and bone marrow-derived macrophages (BMDM). We identify an interaction between HCK and ATG2A and CBL, two autophagy-related proteins, inhibiting autophagy flux in macrophages. In vivo, both global or myeloid cell specific HCK-KO attenuates renal inflammation and fibrosis with reduces macrophage numbers, pro-inflammatory polarization and migration into unilateral ureteral obstruction (UUO) kidneys and unilateral ischemia reperfusion injury (IRI) models. Finally, we developed a selective boron containing HCK inhibitor which can reduce macrophage pro-inflammatory activity, proliferation, and migration in vitro, and attenuate kidney fibrosis in the UUO mice. The current study elucidates mechanisms downstream of HCK regulating macrophage activation and polarization via autophagy in CKD and identifies that selective HCK inhibitors could be potentially developed as a new therapy for renal fibrosis.
Assuntos
Nefrite , Insuficiência Renal Crônica , Obstrução Ureteral , Animais , Humanos , Camundongos , Autofagia , Fibrose , Inflamação/patologia , Rim/metabolismo , Ativação de Macrófagos , Camundongos Endogâmicos C57BL , Nefrite/metabolismo , Proteínas Proto-Oncogênicas c-hck/metabolismo , Insuficiência Renal Crônica/patologia , Obstrução Ureteral/metabolismoRESUMO
High-grade serous ovarian cancer (HGSOC) is a highly aggressive and lethal subtype of ovarian cancer. While most patients initially respond to standard-of-care treatment, the majority will eventually relapse and succumb to their disease. Despite significant advances in our understanding of this disease, the mechanisms that govern the distinctions between HGSOC with good and poor prognosis remain unclear. In this study, we implemented a proteogenomic approach to analyze gene expression, proteomic and phosphoproteomic profiles of HGSOC tumor samples to identify molecular pathways that distinguish HGSOC tumors relative to clinical outcome. Our analyses identify significant upregulation of hematopoietic cell kinase (HCK) expression and signaling in poor prognostic HGSOC patient samples. Analyses of independent gene expression datasets and IHC of patient samples confirmed increased HCK signaling in tumors relative to normal fallopian or ovarian samples and demonstrated aberrant expression in tumor epithelial cells. Consistent with the association between HCK expression and tumor aggressiveness in patient samples, in vitro phenotypic studies showed that HCK can, in part, promote cell proliferation, colony formation, and invasive capacity of cell lines. Mechanistically, HCK mediates these phenotypes, partly through CD44 and NOTCH3-dependent signaling, and inhibiting CD44 or NOTCH3 activity, either genetically or through gamma-secretase inhibitors, can revert HCK-driven phenotypes. IMPLICATIONS: Collectively, these studies establish that HCK acts as an oncogenic driver of HGSOC through aberrant activation of CD44 and NOTCH3 signaling and identifies this network as a potential therapeutic opportunity in a subset of patients with aggressive and recurrent HGSOC.
Assuntos
Cistadenocarcinoma Seroso , Neoplasias Ovarianas , Humanos , Feminino , Proteômica , Proteínas Proto-Oncogênicas c-hck , Recidiva Local de Neoplasia , Neoplasias Ovarianas/patologia , Carcinoma Epitelial do Ovário/genética , Carcinoma Epitelial do Ovário/metabolismo , Carcinogênese/genética , Transformação Celular Neoplásica , Cistadenocarcinoma Seroso/metabolismo , Linhagem Celular Tumoral , Receptores de Hialuronatos/genética , Receptor Notch3/genéticaRESUMO
Laryngeal squamous cell carcinoma (LSCC) is the most lethal cancer in head and neck tumors. Although hematopoietic cell kinase (HCK) has been proven to be an oncogene in several solid tumors, its roles in LSCC remain obscure. This is the first study to evaluate the clinical value of HCK in LSCC, with the aim of exploring its expression status and potential molecular mechanisms underlying LSCC. LSCC tissue-derived gene chips and RNA-seq data were collected for a quantitive integration of HCK mRNA expression level. To confirm the protein expression level of HCK, a total of 82 LSCC tissue specimens and 56 non-tumor laryngeal epithelial controls were collected for in-house tissue microarrays and immunohistochemical staining. Kaplan-Meier curves were generated to determine the ability of HCK in predicting overall survival, progress-free survival, and disease-free survival of LSCC patients. LSCC overexpressed genes and HCK co-expressed genes were intersected to preliminarily explore the enriched signaling pathways of HCK. It was noticed that HCK mRNA was markedly overexpressed in 323 LSCC tissues compared with 196 non-LSCC controls (standardized mean difference = 0.81, p < 0.0001). Upregulated HCK mRNA displayed a moderate discriminatory ability between LSCC tissues and non-tumor laryngeal epithelial controls (area under the curve = 0.78, sensitivity = 0.76, specificity = 0.68). The higher expression level of HCK mRNA could predict worse overall survival and disease-free survival for LSCC patients (p = 0.041 and p = 0.013). Lastly, upregulated co-expression genes of HCK were significantly enriched in leukocyte cell-cell adhesion, secretory granule membrane, and extracellular matrix structural constituent. Immune-related pathways were the predominantly activated signals, such as cytokine-cytokine receptor interaction, Th17 cell differentiation, and Toll-like receptor signaling pathway. In conclusion, HCK was upregulated in LSCC tissues and could be utilized as a risk predictor. HCK may promote the development of LSCC by disturbing immune signaling pathways.
Assuntos
Neoplasias Laríngeas , Carcinoma de Células Escamosas de Cabeça e Pescoço , Humanos , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias Laríngeas/genética , Neoplasias Laríngeas/patologia , Prognóstico , Proteínas Proto-Oncogênicas c-hck/genética , Proteínas Proto-Oncogênicas c-hck/metabolismo , RNA Mensageiro/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologiaRESUMO
Autoinflammatory diseases include a number of monogenic systemic inflammatory diseases, as well as acquired autoinflammatory diseases such as gout. Here, we show that the myeloid Src-family kinases Hck, Fgr, and Lyn are critical for experimental models of gout, as well as for genetically determined systemic inflammation in the Ptpn6me-v/me-v (motheaten viable) mouse model. The Hck-/-Fgr-/-Lyn-/- mutation abrogated various monosodium urate (MSU) crystal-induced pro-inflammatory responses of neutrophils, and protected mice from the development of gouty arthritis. The Src-family inhibitor dasatinib abrogated MSU crystal-induced responses of human neutrophils and reduced experimental gouty arthritis in mice. The Hck-/-Fgr-/-Lyn-/- mutation also abrogated spontaneous inflammation and prolonged the survival of the Ptpn6me-v/me-v mice. Spontaneous adhesion and superoxide release of Ptpn6me-v/me-v neutrophils were also abolished by the Hck-/-Fgr-/-Lyn-/- mutation. Excessive activation of tyrosine phosphorylation pathways in myeloid cells may characterize a subset of autoinflammatory diseases.
Assuntos
Artrite Gotosa , Gota , Doenças Hereditárias Autoinflamatórias , Camundongos , Humanos , Animais , Quinases da Família src/metabolismo , Neutrófilos/metabolismo , Proteínas Proto-Oncogênicas c-hck/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Artrite Gotosa/metabolismo , Gota/metabolismo , Inflamação/metabolismo , Doenças Hereditárias Autoinflamatórias/metabolismoRESUMO
HIV-1-associated nephropathy (HIVAN) is a severe complication of HIV-1 infection. To gain insight into the pathogenesis of kidney disease in the setting of HIV, a transgenic (Tg) mouse model [CD4C/HIV-negative regulator factor (Nef)] was used in which HIV-1 nef expression is under control of regulatory sequences (CD4C) of the human CD4 gene, thus allowing expression in target cells of the virus. These Tg mice develop a collapsing focal segmental glomerulosclerosis associated with microcystic dilatation, similar to human HIVAN. To identify kidney cells permissive to the CD4C promoter, CD4C reporter Tg lines were used. They showed preferential expression in glomeruli, mainly in mesangial cells. Breeding CD4C/HIV Tg mice on 10 different mouse backgrounds showed that HIVAN was modulated by host genetic factors. Studies of gene-deficient Tg mice revealed that the presence of B and T cells and that of several genes was dispensable for the development of HIVAN: those involved in apoptosis (Trp53, Tnfsf10, Tnf, Tnfrsf1b, and Bax), in immune cell recruitment (Ccl3, Ccl2, Ccr2, Ccr5, and Cx3cr1), in nitric oxide (NO) formation (Nos3 and Nos2), or in cell signaling (Fyn, Lck, and Hck/Fgr). However, deletion of Src partially and that of Hck/Lyn largely abrogated its development. These data suggest that Nef expression in mesangial cells through hematopoietic cell kinase (Hck)/Lck/Yes novel tyrosine kinase (Lyn) represents important cellular and molecular events for the development of HIVAN in these Tg mice.
Assuntos
Nefropatia Associada a AIDS , Infecções por HIV , Camundongos , Humanos , Animais , Proteínas Tirosina Quinases/metabolismo , Nefropatia Associada a AIDS/genética , Nefropatia Associada a AIDS/patologia , Camundongos Transgênicos , Infecções por HIV/complicações , Tirosina , Quinases da Família src , Proteínas Proto-Oncogênicas c-hckRESUMO
Hematopoietic cell kinase (Hck) is a member of the Src family and is expressed in hematopoietic cells. By regulating multiple signaling pathways, HCK can interact with multiple receptors to regulate signaling events involved in cell adhesion, proliferation, migration, invasion, apoptosis, and angiogenesis. However, aberrant expression of Hck in various hematopoietic cells and solid tumors plays a crucial role in tumor-related properties, including cell proliferation and epithelial-mesenchymal transition. In addition, Hck signaling regulates the function of immune cells such as macrophages, contributing to an immunosuppressive tumor microenvironment. The clinical success of various kinase inhibitors targeting the Src kinase family has validated the efficacy of targeting Src, and therapies with highly selective Hck kinase inhibitors are in clinical trials. This article reviews Hck inhibition as an emerging cancer treatment strategy, focusing on the expressions and functions of Hck in tumors and its impact on the tumor microenvironment. It also explores preclinical and clinical pharmacological strategies for Hck targeting to shed light on Hck-targeted tumor therapy.
Assuntos
Neoplasias , Quinases da Família src , Humanos , Proteínas Proto-Oncogênicas c-hck/metabolismo , Quinases da Família src/metabolismo , Neoplasias/tratamento farmacológico , Transdução de Sinais , Proliferação de Células , Microambiente TumoralRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a low 5-year survival rate and is associated with poor response to therapy. Elevated expression of the myeloid-specific hematopoietic cell kinase (HCK) is observed in PDAC and correlates with reduced patient survival. To determine whether aberrant HCK signaling in myeloid cells is involved in PDAC growth and metastasis, we established orthotopic and intrasplenic PDAC tumors in wild-type and HCK knockout mice. Genetic ablation of HCK impaired PDAC growth and metastasis by inducing an immune-stimulatory endotype in myeloid cells, which in turn reduced the desmoplastic microenvironment and enhanced cytotoxic effector cell infiltration. Consequently, genetic ablation or therapeutic inhibition of HCK minimized metastatic spread, enhanced the efficacy of chemotherapy, and overcame resistance to anti-PD1, anti-CTLA4, or stimulatory anti-CD40 immunotherapy. Our results provide strong rationale for HCK to be developed as a therapeutic target to improve the response of PDAC to chemo- and immunotherapy.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Proteínas Proto-Oncogênicas c-hck , Animais , Carcinoma Ductal Pancreático/genética , Camundongos , Células Mieloides/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas c-hck/genética , Microambiente Tumoral , Neoplasias PancreáticasRESUMO
The Src-family kinase Fgr is expressed primarily in myeloid hematopoietic cells and contributes to myeloid leukemia. Here, we present X-ray crystal structures of Fgr bound to the ATP-site inhibitors A-419259 and TL02-59, which show promise as anti-leukemic agents. A-419259 induces a closed Fgr conformation, with the SH3 and SH2 domains engaging the SH2-kinase linker and C-terminal tail, respectively. In the Fgr:A-419259 complex, the activation loop of one monomer inserts into the active site of the other, providing a snapshot of trans-autophosphorylation. By contrast, TL02-59 binding induced SH2 domain displacement from the C-terminal tail and SH3 domain release from the linker. Solution studies using HDX MS were consistent with the crystal structures, with A-419259 reducing and TL02-59 enhancing solvent exposure of the SH3 domain. These structures demonstrate that allosteric connections between the kinase and regulatory domains of Src-family kinases are regulated by the ligand bound to the active site.
Assuntos
Leucemia Mieloide Aguda , Quinases da Família src , Humanos , Quinases da Família src/química , Proteínas Proto-Oncogênicas c-hck/química , Proteínas Proto-Oncogênicas c-hck/metabolismo , Domínios de Homologia de src , Leucemia Mieloide Aguda/tratamento farmacológico , Trifosfato de AdenosinaRESUMO
The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib irreversibly binds BTK at Cys481, inhibiting its kinase activity and thus blocking transduction of B cell receptor (BCR) signaling. Although ibrutinib is durably effective in patients with B cell malignancies, many patients still develop ibrutinib-resistant disease. Resistance can arise because of mutations at the ibrutinib-binding site in BTK. Here, we characterized the mechanism by which two BTK mutations, C481F and C481Y, may lead to ibrutinib resistance. Both mutants lacked detectable kinase activity in in vitro kinase assays. Structural modeling suggested that bulky Phe and Tyr side chains at position 481 sterically hinder access to the ATP-binding pocket in BTK, contributing to loss of kinase activity. Nonetheless, BCR signaling still propagated through BTK C481F and C481Y mutants to downstream effectors, the phospholipase PLCγ2 and the transcription factor NF-κB. This maintenance of BCR signaling was partially achieved through the physical recruitment and kinase-independent activation of hematopoietic cell kinase (HCK). Upon BCR activation, BTK C481F or C481Y was phosphorylated by Src family kinases at Tyr551, which then bound to the SH2 domain of HCK. Modeling suggested that this binding disrupted an intramolecular autoinhibitory interaction in HCK. Activated HCK subsequently phosphorylated PLCγ2, which propagated BCR signaling and promoted clonogenic cell proliferation. This kinase-independent mechanism could inform therapeutic approaches to CLL bearing either the C481F or C481Y BTK mutants.
Assuntos
Leucemia Linfocítica Crônica de Células B , Piperidinas , Proteínas Proto-Oncogênicas c-hck , Adenina/análogos & derivados , Tirosina Quinase da Agamaglobulinemia , Resistencia a Medicamentos Antineoplásicos , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/genética , Fosfolipase C gama/genética , Piperidinas/farmacologia , Proteínas Proto-Oncogênicas c-hck/metabolismoRESUMO
Background: The tumor microenvironment (TME) plays a significant role in the progression and prognosis of acute myeloid leukemia (AML). This study is aimed at exploring TME-associated biomarkers and identify their potential mechanism in the microenvironment of AML. Method: In this study, the stromal, immune, and ESTIMATE scores of AML patients were evaluated with the ESTIMATE and CIBERSORT algorithms; then, the AML samples were divided into high- and low-score groups. We evaluated the association between clinicopathological characteristics, survival rate, and the stromal/immune/ESTIMATE scores. Furthermore, we identified TME-associated differentially expressed genes (DEGs) then carried out pathway enrichment analysis, protein-protein interaction (PPI) network, Cox regression analysis, and Kaplan-Meier survival analysis to select the most crucial genes. In addition, we further explored the potential mechanism of HCK in the AML microenvironment. Results: We identified 624 TME-associated DEGs and found that HCK was the most promising biomarker associated with AML. The results of the gene set enrichment analysis (GSEA) indicated that HCK was mainly involved in immune and inflammation-related signaling pathways. In addition, CIBERSORT analysis showed that HCK was closely related to tumor immune infiltration, with HCK expression associated with various infiltrating immune cells, including B cells, T cells, tumor-associated macrophages (TAM), NK cells, plasma cells, eosinophils, and neutrophils. Furthermore, HCK expression was closely related with ELN risk stratification in patients with AML. Conclusion: HCK could regulate immune cell infiltration in the microenvironment of AML and may act as a potential biomarker for the treatment and prognosis of AML patients.
Assuntos
Biomarcadores Tumorais/genética , Regulação Neoplásica da Expressão Gênica , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologia , Proteínas Proto-Oncogênicas c-hck/genética , Idoso , Algoritmos , Correlação de Dados , Feminino , Humanos , Leucemia Mieloide Aguda/mortalidade , Masculino , Pessoa de Meia-Idade , Prognóstico , Taxa de SobrevidaRESUMO
The SRC family kinase (SFK) HCK is transcriptionally upregulated and activated by mutated MYD88 (MYD88Mut), a key adaptor for Toll-receptor signaling. HCK activates BTK, AKT, and ERK in MYD88Mut lymphomas. SYK, a B-cell receptor (BCR) component, is activated in MYD88Mut lymphoma cells. Although the SFK LYN serves as a trigger for SYK activation in MYD88Mut ABC DLBCL cells, LYN activity is muted in MYD88Mut Waldenstrom macroglobulinemia (WM) cells. We therefore investigated a role for HCK in mediating SYK activation. Overexpression of wild-type (WT) (HCKWT) or gatekeeper mutated (HCKThr333Met) HCK in MYD88Mut lymphoma cells triggered SYK activation. Conversely, HCK knockdown reduced p-SYK in MYD88Mut lymphoma cells. Coimmunoprecipitation experiments showed that HCK was complexed with p-SYK in MYD88Mut BCWM.1 and TMD8 cells, but not in MYD88 WT Ramos cells. Rescue experiments in MYD88Mut lymphoma cells expressing HCKThr333Met led to persistent HCK and SYK activation and resistance to the HCK inhibitor A419259. Treatment of primary MYD88Mut WM cells with A419259 reduced p-HCK and p-SYK expression. Taken together, our findings show that SYK is activated by HCK in MYD88Mut B-cell lymphomas cells, broaden the prosurvival signaling generated by aberrant HCK expression in response to MYD88Mut, and help define HCK as an important therapeutic target in MYD88Mut B-cell lymphomas.
Assuntos
Linfoma de Células B , Fator 88 de Diferenciação Mieloide , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Humanos , Linfoma de Células B/enzimologia , Linfoma de Células B/genética , Linfoma de Células B/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Proteínas Proto-Oncogênicas c-hck/metabolismo , Quinase Syk/genética , Quinase Syk/metabolismo , Quinases da Família src/metabolismoRESUMO
Ponatinib is a multi-target tyrosine kinase inhibitor that targets ABL, SRC, FGFR, and so on. It was designed to overcome the resistance of BCR-ABL mutation to imatinib, especially the gatekeeper mutation ABLT315I. The molecular mechanism by which ponatinib overcomes mutations of BCR-ABL and some other targets has been explained, but little information is known about the characteristics of ponatinib binding to SRC. Here, we showed that ponatinib inhibited wild type SRC kinase but failed to inhibit SRC gatekeeper mutants in both biochemical and cellular assays. We determined the crystal structure of ponatinib in complex with the SRC kinase domain. In addition, by structural analysis, we provided a possible explanation for why ponatinib showed different effects on SRC and other kinases with gatekeeper mutations. The resistance mechanism of SRC gatekeeper mutations to ponatinib may provide meaningful information for designing inhibitors against SRC family kinases in the future.
Assuntos
Imidazóis/química , Imidazóis/farmacologia , Inibidores de Proteínas Quinases/química , Piridazinas/química , Piridazinas/farmacologia , Quinases da Família src/química , Sítios de Ligação , Cristalografia por Raios X , Humanos , Imidazóis/metabolismo , Modelos Moleculares , Mutação , Conformação Proteica , Domínios Proteicos , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-hck/química , Proteínas Proto-Oncogênicas c-hck/metabolismo , Piridazinas/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are characterized by risk of relapses, poor survival, unwanted side effects and high toxicity with the current therapies. In light of these facts, there are efforts to develop new drugs specific for deregulated molecules that participate in leukemia pathogenesis. Hematopoietic cell kinase (HCK), an Src kinase family member, is overexpressed on hematopoietic stem cells of MDS and de novo AML patients and involved in the oncogenic process. Thus, we investigated in vitro, ex vivo and in vivo effects of a novel chemical compound targeting HCK inhibition (iHCK-37), in combination with the most used drugs for the treatment of MDS and de novo AML, 5-Azacytidine and Cytarabine. Herein, the combination treatment with iHCK-37 and 5-Azacytidine or Cytarabine demonstrated additive effects against leukemia cells, compared to either drug alone. iHCK-37 plus 5-Azacytidine or Cytarabine treatment was able to reduce the activation of oncogenic pathways, MAPK/ERK and PI3K/AKT, leading to reduction of ERK and AKT phosphorylation, and increased BAX and decreased BCL-XL protein expression. Moreover, treatment with iHCK-37 reduced MDS and AML CD34-positive cell numbers inside a 3D-structure but did not affect normal CD34-positive cell numbers. In vivo analysis showed that leukemic mice treated with iHCK-37 had reduced ERK and AKT proteins phosphorylation levels and leukocyte numbers. In conclusion, the iHCK-37 inhibitor has anti-neoplastic activity in leukemia cells without altering apoptosis and survival rate of normal cells, suggesting on-target malignant cell killing activity as a single agent or in combination with 5-Azacytidine or Cytarabine.
Assuntos
Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Animais , Azacitidina/farmacologia , Azacitidina/uso terapêutico , Citarabina/farmacologia , Citarabina/uso terapêutico , Leucemia Mieloide Aguda/metabolismo , Camundongos , Síndromes Mielodisplásicas/tratamento farmacológico , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-hckRESUMO
BACKGROUND: Inborn errors of immunity are genetic disorders characterized by various degrees of immune dysregulation that can manifest as immune deficiency, autoimmunity, or autoinflammation. The routine use of next-generation sequencing in the clinic has facilitated the identification of an ever-increasing number of inborn errors of immunity, revealing the roles of immunologically important genes in human pathologies. However, despite this progress, treatment is still extremely challenging. OBJECTIVE: We sought to report a new monogenic autoinflammatory disorder caused by a de novo activating mutation, p.Tyr515∗, in hematopoietic cell kinase (HCK). The disease is characterized by cutaneous vasculitis and chronic pulmonary inflammation that progresses to fibrosis. METHODS: Whole-exome sequencing, Sanger sequencing, mass spectrometry, and western blotting were performed to identify and characterize the pathogenic HCK mutation. Dysregulation of mutant HCK was confirmed ex vivo in primary cells and in vitro in transduced cell lines. RESULTS: Mutant HCK lacking the C-terminal inhibitory tyrosine Tyr522 exhibited increased kinase activity and enhanced myeloid cell priming, migration and effector functions, such as production of the inflammatory cytokines IL-1ß, IL-6, IL-8, and TNF-α, and production of reactive oxygen species. These aberrant functions were reflected by inflammatory leukocyte infiltration of the lungs and skin. Moreover, an overview of the clinical course of the disease, including therapies, provides evidence for the therapeutic efficacy of the Janus kinase 1/2 inhibitor ruxolitinib in inflammatory lung disease. CONCLUSIONS: We propose HCK-driven pulmonary and cutaneous vasculitis as a novel autoinflammatory disorder of inborn errors of immunity.
Assuntos
Vasculite , Quinases da Família src , Humanos , Pulmão , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-hck/genética , Proteínas Proto-Oncogênicas c-hck/metabolismo , Vasculite/genética , Vasculite/patologia , Quinases da Família src/genéticaRESUMO
The accessory protein Nef of human and simian immunodeficiency viruses (HIV and SIV) is an important pathogenicity factor known to interact with cellular protein kinases and other signaling proteins. A canonical SH3 domain binding motif in Nef is required for most of these interactions. For example, HIV-1 Nef activates the tyrosine kinase Hck by tightly binding to its SH3 domain. An archetypal contact between a negatively charged SH3 residue and a highly conserved arginine in Nef (Arg77) plays a key role here. Combining structural analyses with functional assays, we here show that Nef proteins have also developed a distinct structural strategy-termed the "R-clamp"-that favors the formation of this salt bridge via buttressing Arg77. Comparison of evolutionarily diverse Nef proteins revealed that several distinct R-clamps have evolved that are functionally equivalent but differ in the side chain compositions of Nef residues 83 and 120. Whereas a similar R-clamp design is shared by Nef proteins of HIV-1 groups M, O, and P, as well as SIVgor, the Nef proteins of SIV from the Eastern chimpanzee subspecies (SIVcpzP.t.s.) exclusively utilize another type of R-clamp. By contrast, SIV of Central chimpanzees (SIVcpzP.t.t.) and HIV-1 group N strains show more heterogenous R-clamp design principles, including a non-functional evolutionary intermediate of the aforementioned two classes. These data add to our understanding of the structural basis of SH3 binding and kinase deregulation by Nef, and provide an interesting example of primate lentiviral protein evolution.
Assuntos
Evolução Molecular , Infecções por HIV/metabolismo , Lentivirus/genética , Proteínas Proto-Oncogênicas c-hck/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Domínios de Homologia de src , Sequência de Aminoácidos , Animais , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/fisiologia , Humanos , Proteínas Proto-Oncogênicas c-hck/genética , Homologia de Sequência de Aminoácidos , Síndrome de Imunodeficiência Adquirida dos Símios/genética , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/fisiologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genéticaRESUMO
Osteosarcoma (OS) is a sarcoma with high rates of pulmonary metastases and mortality. The mechanisms underlying tumour generation and development in OS are not well-understood. Haematopoietic cell kinase (HCK), a vital member of the Src family of kinase proteins, plays crucial roles in cancer progression and may act as an anticancer target; however, the mechanism by which HCK enhances OS development remains unexplored. Therefore, we investigated the role of HCK in OS development in vitro and in vivo. Downregulation of HCK attenuated OS cell proliferation, migration and invasion and increased OS cell apoptosis, whereas overexpression of HCK enhanced these processes. Mechanistically, HCK expression enhanced OS tumorigenesis via the mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway; HCK upregulation increased the phosphorylation of MEK and ERK and promoted epithelial-mesenchymal transition, with a reduction in E-cadherin in vitro. Furthermore, HCK downregulation decreased the tumour volume and weight in mice transplanted with OS cells. In conclusion, HCK plays a crucial role in OS tumorigenesis, progression and metastasis via the MEK/ERK pathway, suggesting that HCK is a potential target for developing treatments for OS.
Assuntos
Neoplasias Ósseas/metabolismo , Osteossarcoma/metabolismo , Proteínas Proto-Oncogênicas c-hck/fisiologia , Animais , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , MAP Quinase Quinase Quinases/metabolismo , Camundongos , Camundongos Endogâmicos BALB CRESUMO
BACKGROUND: Leukaemia stem cells (LSCs) are responsible for the initiation, maintenance, and recurrence of acute myeloid leukaemia (AML), an aggressive haematological malignancy associated with drug resistance and relapse. Identifying therapeutic LSC targets is critical to curing AML. METHODS: Bioinformatics databases were used to identify therapeutic LSC targets. The conditional knockout mice were used to analyse the role of HCK in leukaemogenesis or normal haematopoiesis. Colony-forming assays, cell counting, and flow cytometry were used to detect the viability and function of leukaemia cells. RT-PCR, western blotting, and RNA sequencing were used to detect mRNA and protein expression. RESULT: HCK is expressed at higher levels in LSCs than in haematopoietic stem cells (HSCs), and high HCK levels are correlated with reduced survival time in AML patients. Knockdown of HCK leads to cell cycle arrest, which results in a dramatic decrease in the proliferation and colony formation in human AML cell lines. Moreover, HCK is required for leukemogenesis and leukaemia maintenance in vivo and in vitro. HCK is necessary for the self-renewal of LSCs during serial transplantation and limiting dilution assay. The phenotypes resulting from HCK deficiency can be rescued by CDK6 overexpression in the human cell line. RNA sequencing and gene expression have demonstrated that HCK may sustain cell cycle entry and maintain the self-renewal ability of LSCs through activating the ERK1/2-c-Myc-CDK6 signalling axis. In contrast, HCK deletion does not affect normal haematopoiesis or haematopoietic reconstruction in mice. CONCLUSIONS: HCK maintains the self-renewal of leukaemia stem cells via CDK6 in AML and may be an ideal therapeutic target for eradicating LSCs without influencing normal haematopoiesis.
Assuntos
Quinase 6 Dependente de Ciclina/metabolismo , Leucemia Mieloide Aguda/patologia , Proteínas Proto-Oncogênicas c-hck/genética , Proteínas Proto-Oncogênicas c-hck/metabolismo , Animais , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Autorrenovação Celular , Bases de Dados Genéticas , Deleção de Genes , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Humanos , Células K562 , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Camundongos , Camundongos Knockout , Transplante de NeoplasiasRESUMO
Activating mutations in MYD88 promote malignant cell growth and survival through hematopoietic cell kinase (HCK)-mediated activation of Bruton tyrosine kinase (BTK). Ibrutinib binds to BTKCys481 and is active in B-cell malignancies driven by mutated MYD88. Mutations in BTKCys481, particularly BTKCys481Ser, are common in patients with acquired ibrutinib resistance. We therefore performed an extensive medicinal chemistry campaign and identified KIN-8194 as a novel dual inhibitor of HCK and BTK. KIN-8194 showed potent and selective in vitro killing of MYD88-mutated lymphoma cells, including ibrutinib-resistant BTKCys481Ser-expressing cells. KIN-8194 demonstrated excellent bioavailability and pharmacokinetic parameters, with good tolerance in rodent models at pharmacologically achievable and active doses. Pharmacodynamic studies showed sustained inhibition of HCK and BTK for 24 hours after single oral administration of KIN-8194 in an MYD88-mutated TMD-8 activated B-cell diffuse large B-cell lymphoma (ABC DLBCL) and BCWM.1 Waldenström macroglobulinemia (WM) xenografted mice with wild-type BTK (BTKWT)- or BTKCys481Ser-expressing tumors. KIN-8194 showed superior survival benefit over ibrutinib in both BTKWT- and BTKCys481Ser-expressing TMD-8 DLBCL xenografted mice, including sustained complete responses of >12 weeks off treatment in mice with BTKWT-expressing TMD-8 tumors. The BCL_2 inhibitor venetoclax enhanced the antitumor activity of KIN-8194 in BTKWT- and BTKCys481Ser-expressing MYD88-mutated lymphoma cells and markedly reduced tumor growth and prolonged survival in mice with BTKCys481Ser-expressing TMD-8 tumors treated with both drugs. The findings highlight the feasibility of targeting HCK, a key driver of mutated MYD88 pro-survival signaling, and provide a framework for the advancement of KIN-8194 for human studies in B-cell malignancies driven by HCK and BTK.