RESUMO
Myeloid cells are the first line of defence against pathogens. Mitochondrial apoptosis signalling is a crucial regulator of myeloid cell lifespan and modulates the function of myeloid cells. The anti-apoptotic protein BCL-2-family protein BCL2A1/A1/BFL-1 is strongly upregulated in inflammation in macrophages. We analysed the contribution of A1 to apoptosis regulation in a conditional system of in vitro differentiation of murine macrophages from immortalised progenitors. We disabled the expression of A1 by targeting all murine A1 isoforms in the genome. Specific inhibitors were used to inactivate other anti-apoptotic proteins. Macrophage progenitor survival mainly depended on the anti-apoptotic proteins MCL-1, BCL-XL and A1 but not BCL-2. Deletion of A1 on its own had little effect on progenitor cell survival but was sensitised to cell death induction when BCL-XL or MCL-1 was neutralised. In progenitors, A1 was required for survival in the presence of the inflammatory stimulus LPS. Differentiated macrophages were resistant to inhibition of single anti-apoptotic proteins, but A1 was required to protect macrophages against inhibition of either BCL-XL or MCL-1; BCL-2 only had a minor role in these cells. Cell death by neutralisation of anti-apoptotic proteins completely depended on BAX with a small contribution of BAK only in progenitors in the presence of LPS. A1 and NOXA appeared to stabilise each other at the posttranscriptional level suggesting direct binding. Co-immunoprecipitation experiments showed the binding of A1 to NOXA and BIM. Interaction between A1 and Noxa may indirectly prevent neutralisation and destabilization of MCL-1. Our findings suggest a unique role for A1 as a modulator of survival in the macrophage lineage in concert with MCL-1 and BCL-XL, especially in a pro-inflammatory environment.
Assuntos
Apoptose , Diferenciação Celular , Sobrevivência Celular , Macrófagos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas Proto-Oncogênicas c-bcl-2 , Proteína bcl-X , Animais , Proteína bcl-X/metabolismo , Macrófagos/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Camundongos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Antígenos de Histocompatibilidade Menor/genética , Lipopolissacarídeos/farmacologia , Células Mieloides/metabolismoRESUMO
Antiapoptotic protein, including Mcl-1, expression is frequently observed in pancreatic cancer. Gemcitabine plus nabpaclitaxel (GnP) is the standard chemotherapy for metastatic pancreatic cancer (MPC); however, predictive markers for its efficacy remain unestablished. This study evaluated the association between GnP's therapeutic effects and Mcl-1 expression in tissue samples obtained using endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) for pancreatic tumor or percutaneous ultrasound-guided biopsy for metastatic liver tumor. We retrospectively reviewed 38 patients with histologically diagnosed MPC who received GnP as the first-line chemotherapy at our institute between December 2014 and July 2018. Post-immunohistochemistry analysis for Mcl-1 expression detection, patients were divided to into two groups based on the cell proportion showing Mcl-1 immunoreactivity: positive (> 20%; 23 [60.5%] patients) and negative (≤ 20%; 15 [39.5%] patients) groups. Clinical characteristics did not differ between the two groups. The Mcl-1 positive group showed a significantly higher disease control rate (95.7% vs. 73.3%; P = 0.046), longer progressionfree survival (PFS) (7.2 months vs. 4.9 months; P = 0.018) and longer overall survival (OS) (14.9 months vs. 9.2 months; P = 0.008) than the Mcl-1 negative group. Multivariate analysis showed that Mcl-1 expression was an independent predictive marker for PFS and OS. Mcl-1 expression could be a predictive marker for favorable response to GnP.
Assuntos
Albuminas , Protocolos de Quimioterapia Combinada Antineoplásica , Biomarcadores Tumorais , Desoxicitidina , Gencitabina , Proteína de Sequência 1 de Leucemia de Células Mieloides , Paclitaxel , Neoplasias Pancreáticas , Humanos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/mortalidade , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapêutico , Desoxicitidina/administração & dosagem , Masculino , Feminino , Paclitaxel/administração & dosagem , Paclitaxel/uso terapêutico , Idoso , Pessoa de Meia-Idade , Albuminas/administração & dosagem , Albuminas/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Estudos Retrospectivos , Biomarcadores Tumorais/metabolismo , Prognóstico , Metástase Neoplásica , Adulto , Resultado do Tratamento , Idoso de 80 Anos ou mais , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/secundário , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologiaRESUMO
Acute myeloid leukemia (AML) is a hematological malignancy with a high relapse rate and a poor survival rate. The circular RNA circPVT1 and myocyte enhancer factor 2A (MEF2A) have unique functions in the progression of AML; however, the underlying mechanisms and clinical significance remain to be clarified. Bioinformatics and database analyses were used to assess the transcription factors and target genes of circPVT1. Dualluciferase reporter gene and argonaute 2RNA immunoprecipitation assays were used to verify the targeted relationships. The expression levels of related genes and proteins were detected by reverse transcriptionquantitative PCR and western blotting. Cell viability and apoptosis were detected by Cell Counting Kit8 assay and flow cytometry, respectively. The results revealed that circPVT1 was highly expressed in AML samples and cell lines, and that MEF2A regulated the expression of circPVT1. MEF2A overexpression promoted cell viability and epithelialmesenchymal transition (EMT), and inhibited cell apoptosis. In addition, circPVT1 was revealed to target the regulation of microRNA (miR)4553p, and miR4553p targeted the regulation of MCL1 expression, thus indicating that circPVT1 promoted MCL1 expression through its interaction with miR4553p. Furthermore, cells were transfected with the small interfering RNA(si)circPVT1, miR4553p inhibitor or siMCL1, and sicircPVT1 and siMCL1 inhibited the viability and EMT of NB4 and HL60 cells. However, the miR4553p inhibitor had the opposite effect on cells. In conclusion, MEF2A may act as a transcription factor of circPVT1 to promote the malignant process of AML, and knockdown of circPVT1 could inhibit the viability and EMT of AML cells through the miR4553p/MCL1 axis.
Assuntos
Apoptose , Leucemia Mieloide Aguda , Fatores de Transcrição MEF2 , MicroRNAs , RNA Circular , RNA Longo não Codificante , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/metabolismo , RNA Circular/genética , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Apoptose/genética , Masculino , Linhagem Celular Tumoral , Feminino , Pessoa de Meia-Idade , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Transição Epitelial-Mesenquimal/genética , Regulação Leucêmica da Expressão Gênica , Adulto , Proliferação de Células/genética , Sobrevivência Celular/genética , IdosoRESUMO
BACKGROUND: Cholangiocarcinoma (CCA) is a malignancy of the bile duct epithelium that is commonly found in the Thai population. CCA has poor prognosis and a low survival rate due to the lack of early diagnosis methods and the limited effectiveness of current treatments. A number of oncogenic spliced-transcripts resulting from mRNA splicing errors have been reported in CCA, and aberrant mRNA splicing is suspected to be a key driver of this cancer type. The hyperphosphorylation of serine/arginine rich-splicing factors (SRSFs) by serine/arginine protein kinases (SRPKs) causes them to translocate to the nucleus where they facilitate gene splicing errors that generate cancer-related mRNA/protein isoforms. METHODS: The correlation between SRPK expression and the survival of CCA patients was analyzed using data from The Cancer Genome Atlas (TCGA) dataset. The effect of SRPK inhibitors (SRPIN340 and SPHINX31) on two CCA cell lines (KKU-213A and TFK-1) was also investigated. The induction of cell death was studied by Calcein-AM/PI staining, AnnexinV/7AAD staining, immunofluorescence (IF), and Western blotting (WB). The phosphorylation and nuclear translocation of SRSFs was tracked by WB and IF, and the repair of splicing errors was examined by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). RESULTS: High levels of SRPK1 and SRPK2 transcripts, and in particular SRPK1, correlated with shorter survival in CCA patients. SRPIN340 and SPHINX31 increased the number of dead and apoptotic cells in a dose-dependent manner. CCA also showed diffuse expression of cytoplasmic cytochrome C and upregulation of cleaved caspase-3. Moreover, SRSFs showed low levels of phosphorylation, resulting in the accumulation of cytoplasmic SRSF1. To link these phenotypes with aberrant gene splicing, the apoptosis-associated genes Bridging Integrator 1 (BIN1), Myeloid cell leukemia factor 1 (MCL-1) and B-cell lymphoma 2 (BCL2) were selected for further investigation. Treatment with SRPIN340 and SPHINX31 decreased anti-apoptotic BIN1+12A and increased pro-apoptotic MCL-1S and BCL-xS. CONCLUSIONS: The SRPK inhibitors SRPIN340 and SPHINX31 can suppress the phosphorylation of SRSFs and their nuclear translocation, thereby producing BIN1, MCL-1 and BCL2 isoforms that favor apoptosis and facilitate CCA cell death.
Assuntos
Apoptose , Neoplasias dos Ductos Biliares , Colangiocarcinoma , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas c-bcl-2 , Fatores de Processamento de Serina-Arginina , Humanos , Colangiocarcinoma/tratamento farmacológico , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patologia , Apoptose/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Fatores de Processamento de Serina-Arginina/metabolismo , Fatores de Processamento de Serina-Arginina/antagonistas & inibidores , Fatores de Processamento de Serina-Arginina/genética , Linhagem Celular Tumoral , Neoplasias dos Ductos Biliares/tratamento farmacológico , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/patologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Splicing de RNA/efeitos dos fármacos , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologiaRESUMO
MicroRNA hsa-miR-29 was connected to a number of malignancies. Its target genes are many, among them Mcl-1 that is expressed in three possible isoforms, one of which is anti-apoptotic and another one pro-apoptotic. Ratio of these two isoforms appears to affect cell response to external stimuli. We have demonstrated that miR-29b enhanced etoposide toxicity in HeLa cell line by modulating this ratio of Mcl-1 isoforms. However, it is not known whether the described miR-29 effect is common to various cancer types or even have the opposite effect. This represents a significant problem for possible future applications. In this report, we demonstrate that miR-29b affects toxicity of 60 µM etoposide in cell lines derived from selected malignancies. The mechanism, however, differs among the cell lines tested. Hep G2 cells demonstrated similar effect of miR-29b on etoposide toxicity as was described in HeLa cells, i.e. modulation of Mcl-1 expression. Target protein down-regulated by miR-29b resulting in enhanced etoposide toxicity in Caco-2 cells was, however, Bcl-2 protein. Moreover, H9c2, Hek-293 and ARPE-19 cell lines selected as a representatives of non-malignant cells, showed no effect of miR-29b on etoposide toxicity. Our data suggest that miR-29b could be a common enhancer of etoposide toxicity in malignant cells due to its modulation of Bcl family proteins.
Assuntos
Etoposídeo , MicroRNAs , Proteína de Sequência 1 de Leucemia de Células Mieloides , Humanos , Etoposídeo/toxicidade , Etoposídeo/farmacologia , MicroRNAs/genética , MicroRNAs/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Células HeLa , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Células HEK293 , Antineoplásicos Fitogênicos/farmacologia , Antineoplásicos Fitogênicos/toxicidade , Células Hep G2 , Células CACO-2RESUMO
BACKGROUND: Cervical cancer ranks as one of the most prevalent malignancies among women worldwide and poses a significant threat to health and quality of life. MCL1 is an antiapoptotic protein closely linked to tumorigenesis, drug-resistance and poor prognosis in various cancers. Sanggenon C, a natural flavonoid derived from Morus albal., exhibits multiple activities, including anti-oxidant, anti-inflammatory, antivirus, and antitumor properties. However, the molecular mechanisms by which Sanggenon C exerts antitumor effects on in cervical cancer remain unclear. PURPOSE: To investigate the oncogenic role of MCL1 and elucidate the antitumor activity of Sanggenon C, along with its molecular mechanisms, in cervical cancer. METHODS: In vitro, the effects of Sanggenon C on proliferation, the cell cycle, apoptosis, and autophagy were explored. Transcriptome sequencing was employed to analyze critical genes and pathways. The expression of genes or proteins was evaluated via immunofluorescence, qRT-PCR, immunohistochemistry, and Western blotting. To identify targets of Sanggenon C, various techniques such as clinical database analysis, molecular docking, cellular thermal shift assays, co-immunoprecipitation, and ubiquitination assays were utilized. Additionally, Xenograft mouse models were established to further investigate Sanggenon C as a novel MCL1 inhibitor and its anti-tumor activity in vivo. RESULTS: Our investigation reveals that Sanggenon C effectively inhibits cervical cancer cell proliferation both in vitro and in vivo. Furthermore, Sanggenon C induces endoplasmic reticulum stress and triggers protective autophagy via activation of the ATF4-DDIT3-TRIB3-AKT-MTOR signaling axis. Furthermore, Sanggenon C specifically targets MCL1 to exert its antitumor effects by modulating MCL1 protein stability through SYVN1-mediated ubiquitination. Notably, MCL1 overexpression attenuates the Sanggenon C-induced decrease in cell viability and apoptosis. Our study further characterizes the role of MCL1 in cisplatin resistance and identifies MCL1 as a promising target for Sanggenon C, which effectively inhibits proliferation and induces apoptosis in cisplatin-resistant cervical cancer cells. Importantly, combining Sanggenon C with an autophagy inhibitor represents a promising strategy to enhance therapeutic outcomes in cisplatin-resistant cervical cancer cells. CONCLUSION: Our findings demonstrates that Sanggenon C induces endoplasmic reticulum stress and highlights the potential of targeting MCL1 to exploit vulnerabilities in drug-resistant cervical cancer cells. Sanggenon C emerges as a promising therapeutic agent against MCL1-driven adaptive chemoresistance through disruption of autophagy and endoplasmic reticulum stress in cervical cancer.
Assuntos
Autofagia , Resistencia a Medicamentos Antineoplásicos , Estresse do Retículo Endoplasmático , Proteína de Sequência 1 de Leucemia de Células Mieloides , Neoplasias do Colo do Útero , Feminino , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/metabolismo , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Camundongos Nus , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células HeLa , Camundongos Endogâmicos BALB C , Camundongos , Antineoplásicos Fitogênicos/farmacologia , Simulação de Acoplamento MolecularRESUMO
Increasing the levels of antiapoptotic Bcl-2 proteins is an important way that cancer cells utilize to get out of apoptosis, underscoring their significance as promising targets for anticancer therapies. Lately, a primary compound 1 bearing thiazolidine-2,4-dione was discovered to exhibit comparable Mcl-1 inhibitory activity in comparison to WL-276. Herein, thirty-nine thiazolidine-2,4-dione analogs were yielded through incorporating different biphenyl moieties (R1), amino acid side chains (R2) and sulfonamides (R3) on 1. The findings indicated that certain compounds exhibited favorable inhibitory effects against Bcl-2/Mcl-1, while demonstrating limited or negligible binding affinity towards Bcl-xL. In particular, compounds 16 and 20 exhibited greater Bcl-2/Mcl-1 inhibition compared to AT-101, WL-276 and 1. Moreover, they demonstrated notable antiproliferative effects and significantly induced apoptosis in U937 cells. The western blot and co-immunoprecipitation assays confirmed that 20 could induce alterations in the expression of apoptosis-associated proteins to result in apoptosis through on-target Bcl-2 and Mcl-1 inhibition. In addition, 20 exhibited favorable stability profiles in both rat plasma and rat liver microsomes. In total, 20 could be used as a promising compound to discover Bcl-2/Mcl-1 dual inhibitors with favorable therapeutic properties.
Assuntos
Antineoplásicos , Apoptose , Proliferação de Células , Relação Dose-Resposta a Droga , Descoberta de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas Proto-Oncogênicas c-bcl-2 , Tiazolidinedionas , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Relação Estrutura-Atividade , Proliferação de Células/efeitos dos fármacos , Estrutura Molecular , Apoptose/efeitos dos fármacos , Tiazolidinedionas/farmacologia , Tiazolidinedionas/química , Tiazolidinedionas/síntese química , Animais , Ratos , Desenvolvimento de MedicamentosRESUMO
Myeloid cell leukemia 1 (Mcl-1) is a key regulator of the intrinsic apoptosis pathway. Overexpression of Mcl-1 is correlated with high tumor grade, poor survival, and both intrinsic and acquired resistance to cancer therapies. Herein, we disclose the structure-guided design of a small molecule Mcl-1 inhibitor, compound 26, that binds to Mcl-1 with subnanomolar affinity, inhibits growth in cell culture assays, and possesses low clearance in mouse and dog pharmacokinetic (PK) experiments. Evaluation of 26 as a single agent in Mcl-1 sensitive hematological and solid tumor xenograft models resulted in regressions. Co-treatment of Mcl-1-sensitive and Mcl-1 insensitive lung cancer derived xenografts with 26 and docetaxel or topotecan, respectively, resulted in an enhanced tumor response. These findings support the premise that pro-apoptotic priming of tumor cells by other therapies in combination with Mcl-1 inhibition may significantly expand the subset of cancers in which Mcl-1 inhibitors may prove beneficial.
Assuntos
Antineoplásicos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Humanos , Camundongos , Antineoplásicos/farmacologia , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Cães , Relação Estrutura-Atividade , Feminino , Descoberta de Drogas , Taxoides/farmacologia , Taxoides/farmacocinética , Taxoides/uso terapêutico , Taxoides/química , Docetaxel/farmacologia , Docetaxel/uso terapêutico , Docetaxel/farmacocinética , Docetaxel/químicaRESUMO
Infection of Rift Valley fever virus (RVFV), a highly pathogenic mosquito-borne zoonotic virus, triggers severe inflammatory pathogenesis but the underlying mechanism of inflammation activation is currently unclear. Here, we report that the non-structural protein NSs of RVFV triggers mitochondrial damage to activate the NLRP3 inflammasome leading to viral pathogenesis in vivo. It is found that the host transcription inhibition effect of NSs causes rapid down-regulation of myeloid cell leukemia-1(MCL-1), a pro-survival member of the Bcl-2 (B-cell lymphoma protein 2) protein family. MCL-1 down-regulation led to BAK activation in the mitochondria, which triggered mtROS production and release of oxidized mitochondrial DNA (ox-mtDNA) into the cytosol. Cytosolic ox-mtDNA binds and activates the NLRP3 inflammasome triggering NLRP3-GSDMD pyroptosis in RVFV infected cells. A NSs mutant virus (RVFV-NSsRM) that is compromised in inducing transcription inhibition did not trigger MCL-1 down-regulation nor NLRP3-GSDMD pyroptosis. RVFV infection of the Nlrp3-/- mouse model demonstrated that the RVFV-triggered NLRP3 pyroptosis contributed to RVFV inflammatory pathogenesis and fatal infection in vivo. Infection with the RVFV-NSsRM mutant virus similarly showed alleviated inflammatory pathogenesis and reduced fatality rate. Taken together, these results revealed a mechanism by which a virulence factor activates the mitochondrial MCL-1-BAK axis through inducing host transcription inhibition to trigger NLRP3-dependent inflammatory pathogenesis.
Assuntos
Mitocôndrias , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteína 3 que Contém Domínio de Pirina da Família NLR , Piroptose , Animais , Humanos , Camundongos , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Inflamassomos/metabolismo , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Mitocôndrias/virologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Fatores de Virulência/metabolismo , Fatores de Virulência/genética , Vírus da Febre do Vale do Rift , Proteínas não Estruturais ViraisRESUMO
Interactions between proteins and α-helical peptides have been the focus of drug discovery campaigns. However, the large interfaces formed between multiple turns of an α-helix and a binding protein represent a significant challenge to inhibitor discovery. Modified peptides featuring helix-stabilizing macrocycles have shown promise as inhibitors of these interactions. Here, we tested the ability of N-terminal to side-chain thioether-cyclized peptides to inhibit the α-helix binding protein Mcl-1, by screening a trillion-scale library. The enriched peptides were lariats featuring a small, four-amino-acid N-terminal macrocycle followed by a short linear sequence that resembled the natural α-helical Mcl-1 ligands. These "Heliats" (helical lariats) bound Mcl-1 with tens of nM affinity, and inhibited the interaction between Mcl-1 and a natural peptide ligand. Macrocyclization was found to stabilize α-helical structures and significantly contribute to affinity and potency. Yet, the 2nd and 3rd positions within the macrocycle were permissible to sequence variation, so that a minimal macrocyclic motif, of an N-acetylated d-phenylalanine at the 1st position thioether connected to a cysteine at the 4th, could be grafted into a range of peptides and stabilize helical conformations. We found that d-stereochemistry is more helix-stabilizing than l- at the 1st position in the motif, as the d-amino acid can utilize polyproline II torsional angles that allow for more optimal intrachain hydrogen bonding. This mixed stereochemistry macrocyclic N-cap is synthetically accessible, requiring only minor modifications to standard solid-phase peptide synthesis, and its compatibility with peptide screening can provide ready access to helix-focused peptide libraries for de novo inhibitor discovery.
Assuntos
Compostos Macrocíclicos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Peptídeos , Estereoisomerismo , Peptídeos/química , Peptídeos/síntese química , Peptídeos/farmacologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteína de Sequência 1 de Leucemia de Células Mieloides/química , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Compostos Macrocíclicos/química , Compostos Macrocíclicos/farmacologia , Humanos , Conformação Proteica em alfa-Hélice , Modelos MolecularesRESUMO
Acute pancreatitis (AP) is an inflammatory disease of the pancreas and the main cause of hospital admissions for gastrointestinal diseases. Here, the work studied the circular RNA DTNB/microRNA-485-5p/MCL1 axis in AP and hoped to unravel the related mechanism. Caerulein exposure replicated an AP model in AR42J cells, and caerulein-mediated expression of circDTNB, miR-485-5p, and MCL1 was recorded. After exposure, cells were intervened with transfection plasmids and tested for LDH release, apoptosis, and inflammation. To determine the interwork of circDTNB, miR-485-5p, and MCL1, prediction results and verification experiments were conducted. Caerulein exposure reduced circDTNB and MCL1, while elevated miR-485-5p levels in AR42J cells. Upregulating circDTNB protected AR42J cells from caerulein-induced LDH cytotoxicity, apoptosis, and inflammation, but circDTNB upregulation-induced protections could be muffled by inhibiting MCL1. On the contrary, downregulating circDTNB further damaged AR42J cells under caerulein exposure, however, this phenomenon could be partially rescued after silencing miR-485-5p. miR-485-5p was mechanistically verified to be a target of circDTNB to mediate MCL1. Overall, the circDTNB/miR-485-5p/MCL1 axis protects inflammatory response and apoptosis in caerulein-exposed AR42J cells, promisingly identifying circDTNB as a novel molecule for AP treatment.
Assuntos
Apoptose , Ceruletídeo , Inflamação , MicroRNAs , Proteína de Sequência 1 de Leucemia de Células Mieloides , RNA Circular , Animais , Ratos , Linhagem Celular , Inflamação/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Pancreatite/genética , Pancreatite/metabolismo , Pancreatite/induzido quimicamente , Pancreatite/patologia , RNA Circular/genética , RNA Circular/metabolismoRESUMO
Chemotherapy resistance to colon cancer is an unavoidable obstacle in the clinical management of the disease. Clitocine, an adenosine analog, played a significant role in the chemosensitivity of human colon cancer cells by promoting myeloid cell leukemia 1 (MCL-1) protein degradation. However, the detailed mechanism remains to be further elucidated. We found that clitocine upregulates the expression of F-box and WD repeat domain containing 7 (FBXW7), a ubiquitin ligase involved in the MCL-1 degradation. Transcriptome sequencing analysis revealed that clitocine significantly inhibits the cyclic adenosine monophosphate (cAMP) and extracellular regulated protein kinases (ERK) downstream signaling pathways in colon cancer cells, thereby enhancing FBXW7 expression and subsequently promoting the ubiquitination degradation of MCL-1 protein. We verified that clitocine regulated intracellular cAMP levels by competitive binding with the adenosine receptor A2B. A molecular docking assay also verified the binding relationship. By decreasing intracellular cAMP levels, clitocine blocks the activation of downstream signaling pathways, which ultimately enhances the drug sensitivity of colon cancer cells through increased FBXW7 expression due to the inhibition of its promoter DNA methylation. Both knockout of the adenosine receptor A2B and Br-cAMP treatment can effectively attenuate the function of clitocine in vitro and in vivo. This study clarified that clitocine enhanced the drug sensitivity of colon cancer cells by promoting FBXW7-mediated MCL-1 degradation via inhibiting the A2B/cAMP/ERK axis, providing further knowledge of the clinical application for clitocine.NEW & NOTEWORTHY Our study found that clitocine enhances the drug sensitivity of colon cancer cells by promoting FBXW7-mediated MCL-1 degradation via inhibiting the A2B/cAMP/ERK axis.
Assuntos
Neoplasias do Colo , AMP Cíclico , Proteína 7 com Repetições F-Box-WD , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteína 7 com Repetições F-Box-WD/metabolismo , Proteína 7 com Repetições F-Box-WD/genética , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Neoplasias do Colo/genética , AMP Cíclico/metabolismo , Animais , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Camundongos , Linhagem Celular Tumoral , Camundongos Nus , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteólise/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Camundongos Endogâmicos BALB CRESUMO
Proteasome inhibitors have been employed in the treatment of relapsed multiple myeloma and mantle cell lymphoma. The observed toxicity caused by proteasome inhibitors is a universal phenotype in numerous cancer cells with different sensitivity. In this study, we investigate the conserved mechanisms underlying the toxicity of the proteasome inhibitor bortezomib using gene editing approaches. Our findings utilizing different caspase knocking out cells reveal that bortezomib induces classic intrinsic apoptosis by activating caspase-9 and caspase-3/7, leading to pore-forming protein GSDME cleavage and subsequent lytic cell death or called secondary necrosis, a phenotype also observed in many apoptosis triggers like TNFα plus CHX, DTT and tunicamycin treatment in HeLa cells. Furthermore, through knocking out of nearly all BH3-only proteins including BIM, BAD, BID, BMF and PUMA, we demonstrate that NOXA is the sole BH3-only protein responsible for bortezomib-induced apoptosis. Of note, NOXA is well known for selectively binding to MCL-1 and A1, but our studies utilizing different BH3 mimetics as well as immunoprecipitation assays indicate that, except for the constitutive interaction of NOXA with MCL-1, the accumulation of NOXA after bortezomib treatment allows it to interact with BCL-XL, then simultaneous relieving suppression on apoptosis by both anti-apoptotic proteins BCL-XL and MCL-1. In addition, though bortezomib-induced significant ER stress and JNK activation were observed in the study, further genetic depletion experiments prove that bortezomib-induced apoptosis occurs independently of ER stress-related apoptosis factor CHOP and JNK. In summary, these results provide a solid conclusion about the critical role of NOXA in inactivation of BCL-XL except MCL-1 in bortezomib-induced apoptosis.
Assuntos
Apoptose , Bortezomib , Proteína de Sequência 1 de Leucemia de Células Mieloides , Inibidores de Proteassoma , Proteínas Proto-Oncogênicas c-bcl-2 , Proteína bcl-X , Humanos , Apoptose/efeitos dos fármacos , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Bortezomib/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteína bcl-X/metabolismo , Proteína bcl-X/genética , Inibidores de Proteassoma/farmacologia , Fator de Transcrição CHOP/metabolismo , Fator de Transcrição CHOP/genética , Células HeLa , Sistema de Sinalização das MAP Quinases/efeitos dos fármacosRESUMO
Dengue is a global health concern, and the host-viral interactions that regulate disease severity are largely unknown. Detrimental effects of neutrophils in this disease have been reported, but the precise mechanisms and functional properties of dengue-activated neutrophils are not fully characterised. Here, we measured the effects of dengue virus serotype 3 (DV3) on neutrophil lifespan and functions. We show that DV3 extends neutrophil survival with a significant proportion of cells surviving for 72 h post-incubation. These effects on neutrophil survival were greater than those observed by adding GM-CSF and TNF-α alone, but these cytokines enhanced survival induced by the virus. Enhanced reactive oxygen species (ROS) generation was observed following incubation with DV3 activation and this ROS production was enhanced by co-incubation with priming agents. In addition, DV triggered the enhanced IL-8 expression by the majority of neutrophils and a low percentage of cells were activated to express MCP-1 (CCL2). A low number of neutrophils showed increased co-expression of the migratory markers, CCR7 and CXCR4 which could promote their migration towards lymph nodes. DV3 significantly upregulated the BCL-XL gene at 3, 12, and 24 h, and the Mcl-1 gene at 12 h, following treatment. We also show that DV3 induces the Mcl-1 protein stabilization similar to GM-CSF. This report sheds new light on the mechanisms by which neutrophils may contribute to the pathology of dengue disease via delayed apoptosis and generation of pro-inflammatory molecules, and raises the possibility that dengue-activated neutrophils may play a role in activating cells of adaptive immunity.
Assuntos
Apoptose , Vírus da Dengue , Dengue , Proteína de Sequência 1 de Leucemia de Células Mieloides , Neutrófilos , Espécies Reativas de Oxigênio , Vírus da Dengue/fisiologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Humanos , Dengue/imunologia , Dengue/virologia , Espécies Reativas de Oxigênio/metabolismo , Sorogrupo , Sobrevivência CelularRESUMO
Mcl-1 is a main antiapoptotic protein in acute myeloid leukemia (AML) and is used as a target to develop inhibitors. Currently, potent Mcl-1 inhibitors primarily interact with the P2-P4 pockets of Mcl-1, but pharmacological modulation by targeting the P1 pocket is less explored. We designed a series of 1H-indole-2-carboxylic acid compounds as novel Mcl-1 inhibitors occupying the P1-P3 pockets and evaluated their Mcl-1 inhibition and apoptosis induction in AML cells. Two-dimensional 15N-HSQC spectroscopy indicated that 47 (Ki = 24 nM) bound to the BH3 binding groove, occupied the P1 pocket in Mcl-1, and formed interactions with Lys234 and Val249. 47 exhibited good microsomal stability and pharmacokinetic profiles, with low potential risk of cardiotoxicity. 47 inhibited tumor growth in HL-60 and THP-1 xenograft models with growth inhibition rate of 63.7% and 57.4%, respectively. Collectively, 47 represents a novel Mcl-1 inhibitor targeting the P1-P3 pockets with excellent antileukemia effects.
Assuntos
Antineoplásicos , Apoptose , Indóis , Leucemia Mieloide Aguda , Proteína de Sequência 1 de Leucemia de Células Mieloides , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Apoptose/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Animais , Indóis/farmacologia , Indóis/química , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Relação Estrutura-Atividade , Camundongos , Ensaios Antitumorais Modelo de Xenoenxerto , Descoberta de Drogas , Proliferação de Células/efeitos dos fármacos , Linhagem Celular Tumoral , Células HL-60 , Sítios de LigaçãoRESUMO
Lazertinib, a novel third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), demonstrates marked efficacy in EGFR-mutant lung cancer. However, resistance commonly develops, prompting consideration of therapeutic strategies to overcome initial drug resistance mechanisms. This study aimed to elucidate the adaptive resistance to lazertinib and advocate novel combination treatments that demonstrate efficacy in preventing resistance as a first-line treatment for EGFR mutation-positive NSCLC. We found that AXL knockdown significantly inhibited lung cancer cell viability in the presence of lazertinib, indicating that AXL activation contributes to lazertinib resistance. However, long-term culture with a combination of lazertinib and AXL inhibitors led to residual cell proliferation and increased the MCL-1 expression level, which was mediated by the nuclear translocation of the transcription factor YAP. Triple therapy with an MCL-1 or YAP inhibitor in combination with lazertinib and an AXL inhibitor significantly reduced cell viability and increased the apoptosis rate. These results demonstrate that AXL and YAP/MCL-1 signals contribute to adaptive lazertinib resistance in EGFR-mutant lung cancer cells, suggesting that the initial dual inhibition of AXL and YAP/MCL-1 might be a highly effective strategy in eliminating lazertinib-resistant cells.
Assuntos
Receptor Tirosina Quinase Axl , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB , Neoplasias Pulmonares , Mutação , Proteína de Sequência 1 de Leucemia de Células Mieloides , Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas , Receptores Proteína Tirosina Quinases , Humanos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/metabolismo , Linhagem Celular Tumoral , Inibidores de Proteínas Quinases/farmacologia , Proliferação de Células/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/metabolismoRESUMO
Current KRASG12C (OFF) inhibitors that target inactive GDP-bound KRASG12C cause responses in less than half of patients and these responses are not durable. A class of RASG12C (ON) inhibitors that targets active GTP-bound KRASG12C blocks ERK signaling more potently than the inactive-state inhibitors. Sensitivity to either class of agents is strongly correlated with inhibition of mTORC1 activity. We have previously shown that PI3K/mTOR and ERK-signaling pathways converge on key cellular processes and that inhibition of both pathways is required for inhibition of these processes and for significant antitumor activity. We find here that the combination of a KRASG12C inhibitor with a selective mTORC1 kinase inhibitor causes synergistic inhibition of Cyclin D1 expression and cap-dependent translation. Moreover, BIM upregulation by KRASG12C inhibition and inhibition of MCL-1 expression by the mTORC1 inhibitor are both required to induce significant cell death. In vivo, this combination causes deep, durable tumor regressions and is well tolerated. This study suggests that the ERK and PI3K/mTOR pathways each mitigate the effects of inhibition of the other and that combinatorial inhibition is a potential strategy for treating KRASG12C-dependent lung cancer.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Sinergismo Farmacológico , Neoplasias Pulmonares , Alvo Mecanístico do Complexo 1 de Rapamicina , Proteínas Proto-Oncogênicas p21(ras) , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Humanos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/genética , Animais , Linhagem Celular Tumoral , Camundongos , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Transdução de Sinais/efeitos dos fármacos , Ciclina D1/metabolismo , Ciclina D1/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Feminino , Proteína 11 Semelhante a Bcl-2/metabolismo , Proteína 11 Semelhante a Bcl-2/genéticaRESUMO
Selinexor, a first-in-class exportin1 (XPO1) inhibitor, is an attractive anti-tumor agent because of its unique mechanisms of action; however, its dose-dependent toxicity and lack of biomarkers preclude its wide use in clinical applications. To identify key molecules/pathways regulating selinexor sensitivity, we performed genome-wide CRISPR/Cas9 dropout screens using two B-ALL lines. We identified, for the first time, that paralogous DDX19A and DDX19B RNA helicases modulate selinexor sensitivity by regulating MCL1 mRNA nuclear export. While single depletion of either DDX19A or DDX19B barely altered MCL1 protein levels, depletion of both significantly attenuated MCL1 mRNA nuclear export, reducing MCL1 protein levels. Importantly, combining selinexor treatment with depletion of either DDX19A or DDX19B markedly induced intrinsic apoptosis of leukemia cells, an effect rescued by MCL1 overexpression. Analysis of Depmap datasets indicated that a subset of T-ALL lines expresses minimal DDX19B mRNA levels. Moreover, we found that either selinexor treatment or DDX19A depletion effectively induced apoptosis of T-ALL lines expressing low DDX19B levels. We conclude that XPO1 and DDX19A/B coordinately regulate cellular MCL1 levels and propose that DDX19A/B could serve as biomarkers for selinexor treatment. Moreover, pharmacological targeting of DDX19 paralogs may represent a potential strategy to induce intrinsic apoptosis in leukemia cells.
Assuntos
RNA Helicases DEAD-box , Hidrazinas , Proteína de Sequência 1 de Leucemia de Células Mieloides , RNA Mensageiro , Triazóis , Triazóis/farmacologia , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Hidrazinas/farmacologia , RNA Mensageiro/genética , Leucemia/metabolismo , Leucemia/tratamento farmacológico , Leucemia/genética , Leucemia/patologia , Apoptose/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Antineoplásicos/farmacologiaRESUMO
Myeloid cell leukemia-1 (MCL-1), which belongs to the anti-apoptotic B cell lymphoma-2 family protein, is overexpressed in various cancers and is associated with cell immortality, malignant transformation, chemoresistance, and poor prognosis in humans. However, the significance of MCL-1 in canine mammary gland tumors (MGTs) remains unknown. This study aimed to examine MCL-1 expression in normal canine mammary glands and tumors and to assess its correlation with clinical and histologic variables. In total, 111 samples were examined, including 12 normal mammary gland tissues, 51 benign MGTs, and 48 malignant MGTs. Immunohistochemistry revealed that 53% of benign tumors and 75% of malignant tumors exhibited high MCL-1 expression, whereas only 8% of normal mammary glands exhibited high MCL-1 expression. High MCL-1 expression correlated with tumor malignancy (p < 0.001), large tumor size (> 3 cm) (p = 0.005), high Ki-67 expression (p = 0.046), and metastasis (p = 0.027). Survival curve analysis of dogs with malignant MGTs demonstrated a significant association between high MCL-1 expression and shorter median overall survival (p = 0.027) and progression-free survival (p = 0.014). Our study identified MCL-1 as a prognostic factor and potential therapeutic target in canine MGTs.
Assuntos
Doenças do Cão , Neoplasias Mamárias Animais , Proteína de Sequência 1 de Leucemia de Células Mieloides , Animais , Cães , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Feminino , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , Prognóstico , Doenças do Cão/metabolismo , Doenças do Cão/patologia , Biomarcadores Tumorais/metabolismo , Imuno-Histoquímica , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/patologiaRESUMO
Promising outcomes have been reported in elder patients with acute myeloid leukemia (AML) using combined therapy of venetoclax (VEN) and azacytidine (AZA) in recent years. However, approximately one-third of patients appear to be refractory to this therapy. Vitamin K2 (VK2) shows apoptosis-inducing activity in AML cells, and daily oral VK2 (menaquinone-4, GlakayR) has been approved for patients with osteoporosis in Japan. We observed a high response rate to AZA plus VEN therapy, with no 8-week mortality in the newly diagnosed AML patients consuming daily VK2 in our hospital. The median age of the patients was 75.9 years (range 66-84) with high-risk features. Patients received AZA 75 mg/m2 on D1-7, VEN 400 mg on D1-28, and daily VK2 45 mg. The CR/CRi ratio was 94.7% (18/19), with a CR rate of 79%. Complete cytogenetic CR was achieved in 15 of 19 (79%) patients, and MRD negativity in 2 of 15 (13%) evaluable CR patients. Owing to the extremely high response rate in clinical settings, we further attempted to investigate the underlying mechanisms. The combination of VK2 and VEN synergistically induced apoptosis in all five AML cell lines tested. VK2, but not VEN, induced mitochondrial reactive oxygen species (ROS), leading to the transcriptional upregulation of NOXA, followed by MCL-1 repression. ROS scavengers repressed VK2 induced-NOXA expression and led to the cancellation of pronounced apoptosis and the downregulation of MCL-1 by VK2 plus VEN. Additionally, knockdown and knockout of NOXA resulted in abrogation of the MCL-1 repression as well as enhanced cytotoxicity by the two-drug combination, indicating that VK2 suppresses MCL-1 via ROS-mediated NOXA induction. These data suggest that the dual inhibition of BCL-2 by VEN and MCL-1 by VK2 is responsible for the remarkable clinical outcomes in our patients. Therefore, large-scale clinical trials are required.