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1.
Front Oncol ; 12: 1048741, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36387080

RESUMO

Background: TRAIL (TNF-related apoptosis inducing ligand) exhibits selective proapoptotic activity in multiple tumor types, while sparing normal cells. This selectivity makes TRAIL an attractive therapeutic candidate. However, despite encouraging activity in preclinical models, clinical trials with TRAIL mimetics/death receptor agonists demonstrated insufficient activity, largely due to emerging resistance to these agents. Herein, we investigated the cytotoxic activity of a novel, TRAIL-based chimeric protein AD-O51.4 combining TRAIL and VEGFA-derived peptide sequences, in hematological malignancies. We characterize key molecular mechanisms leading to resistance and propose rational pharmacological combinations sensitizing cells to AD-O51.4. Methods: Sensitivity of DLBCL, classical Hodgkin lymphoma, (cHL), Burkitt lymphoma (BL) and acute myeloid leukemia (AML) to AD-O51.4 was assessed in vitro with MTS assay and apoptosis tests (Annexin V/PI staining). Markers of apoptosis were assessed using immunoblotting, flow cytometry or fluorogenic caspase cleavage assays. Resistant cell lines were obtained by incubation with increasing doses of AD-O51.4. Transcriptomic analyses were performed by RNA sequencing. Sensitizing effects of selected pathway modulators (BCL2, dynamin and HDAC inhibitors) were assessed using MTS/apoptosis assays. Results: AD-O51.4 exhibited low-nanomolar cytotoxic activity in DLBCL cells, but not in other lymphoid or AML cell lines. AD-O51.4 induced death-receptor (DR) mediated, caspase-dependent apoptosis in sensitive DLBCL cells, but not in primary resistant cells. The presence of DRs and caspase 8 in cancer cells was crucial for AD-O51.4-induced apoptosis. To understand the potential mechanisms of resistance in an unbiased way, we engineered AD-O51.4-resistant cells and evaluated resistance-associated transcriptomic changes. Resistant cells exhibited changes in the expression of multiple genes and pathways associated with apoptosis, endocytosis and HDAC-dependent epigenetic reprogramming, suggesting potential therapeutic strategies of sensitization to AD-O51.4. In subsequent analyses, we demonstrated that HDAC inhibitors, BCL2 inhibitors and endocytosis/dynamin inhibitors sensitized primary resistant DLBCL cells to AD-O51.4. Conclusions: Taken together, we identified rational pharmacologic strategies sensitizing cells to AD-O51.4, including BCL2, histone deacetylase inhibitors and dynamin modulators. Since AD-O51.4 exhibits favorable pharmacokinetics and an acceptable safety profile, its further clinical development is warranted. Identification of resistance mechanisms in a clinical setting might indicate a personalized pharmacological approach to override the resistance.

2.
Sci Rep ; 11(1): 10017, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976256

RESUMO

Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) genes occur in about 20% patients with acute myeloid leukemia (AML), leading to DNA hypermethylation and epigenetic deregulation. We assessed the prognostic significance of IDH1/2 mutations (IDH1/2+) in 398 AML patients with normal karyotype (NK-AML), treated with daunorubicine + cytarabine (DA), DA + cladribine (DAC), or DA + fludarabine. IDH2 mutation was an independent favorable prognostic factor for 4-year overall survival (OS) in total NK-AML population (p = 0.03, censoring at allotransplant). We next evaluated the effect of addition of cladribine to induction regimen on the patients' outcome according to IDH1/2 mutation status. In DAC group, 4-year OS was increased in IDH2+ patients, compared to IDH-wild type group (54% vs 33%; p = 0.0087, censoring at allotransplant), while no difference was observed for DA-treated subjects. In multivariate analysis, DAC independently improved the survival of IDH2+ patients (HR = 0.6 [0.37-0.93]; p = 0.024; censored at transplant), indicating that this group specifically benefits from cladribine-containing therapy. In AML cells with R140Q or R172K IDH2 mutations, cladribine restrained mutations-related DNA hypermethylation. Altogether, DAC regimen produces better outcomes in IDH2+ NK-AML patients than DA, and this likely results from the hypomethylating activity of cladribine. Our observations warrant further investigations of induction protocols combining cladribine with IDH1/2 inhibitors in IDH2-mutant.


Assuntos
Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica , Isocitrato Desidrogenase/genética , Leucemia Mieloide Aguda/genética , Adolescente , Adulto , Idoso , Cladribina/uso terapêutico , Citarabina/uso terapêutico , Daunorrubicina/uso terapêutico , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/mortalidade , Pessoa de Meia-Idade , Variantes Farmacogenômicos , Polônia/epidemiologia , Ensaios Clínicos Controlados Aleatórios como Assunto , Estudos Retrospectivos , Adulto Jovem
3.
Cell Death Dis ; 11(11): 956, 2020 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-33159047

RESUMO

Spleen tyrosine kinase (SYK) is an important oncogene and signaling mediator activated by cell surface receptors crucial for acute myeloid leukemia (AML) maintenance and progression. Genetic or pharmacologic inhibition of SYK in AML cells leads to increased differentiation, reduced proliferation, and cellular apoptosis. Herein, we addressed the consequences of SYK inhibition to leukemia stem-cell (LSC) function and assessed SYK-associated pathways in AML cell biology. Using gain-of-function MEK kinase mutant and constitutively active STAT5A, we demonstrate that R406, the active metabolite of a small-molecule SYK inhibitor fostamatinib, induces differentiation and blocks clonogenic potential of AML cells through the MEK/ERK1/2 pathway and STAT5A transcription factor, respectively. Pharmacological inhibition of SYK with R406 reduced LSC compartment defined as CD34+CD38-CD123+ and CD34+CD38-CD25+ in vitro, and decreased viability of LSCs identified by a low abundance of reactive oxygen species. Primary leukemic blasts treated ex vivo with R406 exhibited lower engraftment potential when xenotransplanted to immunodeficient NSG/J mice. Mechanistically, these effects are mediated by disturbed mitochondrial biogenesis and suppression of oxidative metabolism (OXPHOS) in LSCs. These mechanisms appear to be partially dependent on inhibition of STAT5 and its target gene MYC, a well-defined inducer of mitochondrial biogenesis. In addition, inhibition of SYK increases the sensitivity of LSCs to cytarabine (AraC), a standard of AML induction therapy. Taken together, our findings indicate that SYK fosters OXPHOS and participates in metabolic reprogramming of AML LSCs in a mechanism that at least partially involves STAT5, and that SYK inhibition targets LSCs in AML. Since active SYK is expressed in a majority of AML patients and confers inferior prognosis, the combination of SYK inhibitors with standard chemotherapeutics such as AraC constitutes a new therapeutic modality that should be evaluated in future clinical trials.


Assuntos
Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Fosforilação Oxidativa , Inibidores de Proteínas Quinases/farmacologia , Fator de Transcrição STAT5/antagonistas & inibidores , Quinase Syk/antagonistas & inibidores , Proteínas Supressoras de Tumor/antagonistas & inibidores , Animais , Apoptose , Proliferação de Células , Respiração Celular , Feminino , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Estresse Oxidativo , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Quinase Syk/genética , Quinase Syk/metabolismo , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Exp Parasitol ; 196: 12-21, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30465732

RESUMO

Helminths and their products are strong candidates for the treatment of autoimmunological disorders and allergies. Being a key population of antigen-presenting cells, dendritic cells play a crucial role in the therapeutic potential of worms. The study compares the effects of live pre-male and pre-female L4 stage Heligmosomoides polygyrus administration on the maturation and activation of the JAWS II line of immature dendritic cells. On stimulation with L4 stage H. polygyrus, JAWS II cells acquire semi-mature status and induce Th2 and regulatory responses in vitro. The strongest immunosuppressive effect on JAWS II cells was observed following stimulation with both sexes of nematodes together; this was manifested as immature dendritic cell morphology, proliferation inhibition, cell cycle change, decreased translocation of NF-κB into the nucleus, and lower expression of surface cellular costimulatory molecules CD80, CD86 and MHC I. However, greater production of proinflammatory (IL-12p70, TNF-α, IL-6) and Th2 response-promoting cytokines (IL-4) was observed by JAWS II following exposure to both sexes compared to male or female larvae alone. Sex had no influence on the viability, apoptosis process or endocytosis abilities of the JAWS II cell line. The findings indicate that the presence of only a single sex of the parasite influences a developed response, resulting in reduced proinflammatory and an antiparasitic reaction.


Assuntos
Células Dendríticas/parasitologia , Nematospiroides dubius/fisiologia , Animais , Apoptose , Células da Medula Óssea/imunologia , Células da Medula Óssea/parasitologia , Células da Medula Óssea/fisiologia , Ciclo Celular , Linhagem Celular , Quimiocinas/análise , Citocinas/análise , Células Dendríticas/imunologia , Células Dendríticas/fisiologia , Endocitose , Feminino , Larva/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/análise , Nematospiroides dubius/crescimento & desenvolvimento , Fatores Sexuais , Organismos Livres de Patógenos Específicos
5.
J Cell Mol Med ; 22(7): 3548-3559, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29665227

RESUMO

Lymph node microenvironment provides chronic lymphocytic leukaemia (CLL) cells with signals promoting their survival and granting resistance to chemotherapeutics. CLL cells overexpress PIM kinases, which regulate apoptosis, cell cycle and migration. We demonstrate that BCR crosslinking, CD40 stimulation, and coculture with stromal cells increases PIMs expression in CLL cells, indicating microenvironment-dependent PIMs regulation. PIM1 and PIM2 expression at diagnosis was higher in patients with advanced disease (Binet C vs. Binet A/B) and in those, who progressed after first-line treatment. In primary CLL cells, inhibition of PIM kinases with a pan-PIM inhibitor, SEL24-B489, decreased PIM-specific substrate phosphorylation and induced dose-dependent apoptosis in leukaemic, but not in normal B cells. Cytotoxicity of SEL24-B489 was similar in TP53-mutant and TP53 wild-type cells. Finally, inhibition of PIM kinases decreased CXCR4-mediated cell chemotaxis in two related mechanisms-by decreasing CXCR4 phosphorylation and surface expression, and by limiting CXCR4-triggered mTOR pathway activity. Importantly, PIM and mTOR inhibitors similarly impaired migration, indicating that CXCL12-triggered mTOR is required for CLL cell chemotaxis. Given the microenvironment-modulated PIM expression, their pro-survival function and a role of PIMs in CXCR4-induced migration, inhibition of these kinases might override microenvironmental protection and be an attractive therapeutic strategy in this disease.


Assuntos
Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Receptores CXCR4/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Movimento Celular/efeitos dos fármacos , Feminino , Regulação Leucêmica da Expressão Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/mortalidade , Masculino , Pessoa de Meia-Idade , Prognóstico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-pim-1/genética , Células Tumorais Cultivadas , Microambiente Tumoral
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