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1.
Glia ; 71(4): 974-990, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36480007

RESUMO

Triggering receptor on myeloid cells 2 (TREM2) is an innate immune receptor, upregulated on the surface of microglia associated with amyloid plaques in Alzheimer's disease (AD). Individuals heterozygous for the R47H variant of TREM2 have greatly increased risk of developing AD. We examined the effects of wild-type (WT), R47H and knock-out (KO) of human TREM2 expression in three microglial cell systems. Addition of mouse BV-2 microglia expressing R47H TREM2 to primary mouse neuronal cultures caused neuronal loss, not observed with WT TREM2. Neuronal loss was prevented by using annexin V to block exposed phosphatidylserine, an eat-me signal and ligand of TREM2, suggesting loss was mediated by microglial phagocytosis of neurons exposing phosphatidylserine. Addition of human CHME-3 microglia expressing R47H TREM2 to LUHMES neuronal-like cells also caused loss compared to WT TREM2. Expression of R47H TREM2 in BV-2 and CHME-3 microglia increased their uptake of phosphatidylserine-beads and synaptosomes versus WT TREM2. Human iPSC-derived microglia with heterozygous R47H TREM2 had increased phagocytosis of synaptosomes vs common-variant TREM2. Additionally, phosphatidylserine liposomes increased activation of human iPSC-derived microglia expressing homozygous R47H TREM2 versus common-variant TREM2. Finally, overexpression of TREM2 in CHME-3 microglia caused increased expression of cystatin F, a cysteine protease inhibitor, and knock-down of cystatin F increased CHME-3 uptake of phosphatidylserine-beads. Together, these data suggest that R47H TREM2 may increase AD risk by increasing phagocytosis of synapses and neurons via greater activation by phosphatidylserine and that WT TREM2 may decrease microglial phagocytosis of synapses and neurons via cystatin F.


Assuntos
Doença de Alzheimer , Sinaptossomos , Animais , Humanos , Camundongos , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Cistatinas/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Microglia/metabolismo , Neurônios/patologia , Fagocitose/genética , Fosfatidilserinas/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Sinaptossomos/metabolismo , Sinaptossomos/patologia
2.
Glia ; 70(12): 2290-2308, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-35912412

RESUMO

The receptor Triggering Receptor Expressed on Myeloid cells 2 (TREM2) is associated with several neurodegenerative diseases including Alzheimer's Disease and TREM2 stimulation represents a novel therapeutic opportunity. TREM2 can be activated by antibodies targeting the stalk region, most likely through receptor dimerization. Endogenous ligands of TREM2 are suggested to be negatively charged apoptotic bodies, mimicked by phosphatidylserine incorporated in liposomes and other polyanionic molecules likely binding to TREM2 IgV fold. However, there has been much discrepancy in the literature on the nature of phospholipids (PLs) that can activate TREM2 and on the stability of the corresponding liposomes over time. We describe optimized liposomes as robust agonists selective for TREM2 over TREM1 in cellular system. The detailed structure/activity relationship studies of lipid polar heads indicate that negatively charged lipid heads are required for activity and we identified the shortest maximally active PL sidechain. Optimized liposomes are active on both TREM2 common variant and TREM2 R47H mutant. Activity and selectivity were further confirmed in different native TREM2 expressing cell types including on integrated cellular responses such as stimulation of phagocytic activity. Such tool agonists will be useful in further studies of TREM2 biology in cellular systems alongside antibodies, and in the design of small molecule synthetic TREM2 agonists.


Assuntos
Doença de Alzheimer , Lipossomos , Glicoproteínas de Membrana/imunologia , Receptores Imunológicos/imunologia , Doença de Alzheimer/metabolismo , Anticorpos/metabolismo , Encéfalo/metabolismo , Humanos , Ligantes , Microglia/metabolismo , Células Mieloides/metabolismo , Fosfatidilserinas/metabolismo , Receptor Gatilho 1 Expresso em Células Mieloides/metabolismo
3.
Blood ; 132(5): 510-520, 2018 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-29871863

RESUMO

Activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL) is an aggressive lymphoproliferative disorder involving chronic NF-κB activation. Several mutations in the BCR and MyD88 signaling pathway components, such as MyD88 L265P, are implicated in this aberrant activation. Among heat shock proteins, HSP110 has recently been identified as a prosurvival and/or proliferation factor in many cancers, but its role in ABC-DLBCL survival mechanisms remained to be established. We observed that short hairpin RNA-mediated HSP110 silencing decreased the survival of several ABC-DLBCL cell lines and decreased immunoglobulin M-MyD88 co-localization and subsequent NF-κB signaling. Conversely, overexpression of HSP110 in ABC-DLBCL or non-DLBCL cell lines increased NF-κB signaling, indicating a tight interplay between HSP110 and the NF-κB pathway. By using immunoprecipitation and proximity ligation assays, we identified an interaction between HSP110 and both wild-type MyD88 and MyD88 L265P. HSP110 stabilized both MyD88 forms with a stronger effect on MyD88 L265P, thus facilitating chronic NF-κB activation. Finally, HSP110 expression was higher in lymph node biopsies from patients with ABC-DLBCL than in normal reactive lymph nodes, and a strong correlation was found between the level of HSP110 and MyD88. In conclusion, we identified HSP110 as a regulator of NF-κB signaling through MyD88 stabilization in ABC-DLBCL. This finding reveals HSP110 as a new potential therapeutic target in ABC-DLBCL.


Assuntos
Proteínas de Choque Térmico HSP110/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Fator 88 de Diferenciação Mieloide/química , NF-kappa B/metabolismo , Estudos de Coortes , Proteínas de Choque Térmico HSP110/genética , Humanos , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/genética , Estabilidade Proteica , Transdução de Sinais , Células Tumorais Cultivadas
4.
Leukemia ; 38(8): 1742-1750, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38906964

RESUMO

Activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL) is driven by aberrant activation of the B-cell receptor (BCR) and the TLR/MyD88 signaling pathways. The heat-shock protein HSP110 is a candidate for their regulation as it stabilizes MyD88. However, its role in overall BCR signaling remains unknown. Here, we used first-in-class HSP110 inhibitors to address this question. HSP110 inhibitors decreased the survival of several ABC-DLBCL cell lines in vitro and in vivo, and reduced the phosphorylation of BCR signaling kinases, including BTK and SYK. We identified an interaction between HSP110 and SYK and demonstrated that HSP110 promotes SYK phosphorylation. Finally, the combination of the HSP110 inhibitor with the PI3K inhibitor copanlisib decreases SYK/BTK and AKT phosphorylation synergistically, leading to suppression of tumor growth in cell line xenografts and strong reduction in patient-derived xenografts. In conclusion, by regulating the BCR/TLR signaling pathway, HSP110 inhibitors are potential drug candidates for ABC-DLBCL patients.


Assuntos
Proteínas de Choque Térmico HSP110 , Linfoma Difuso de Grandes Células B , Receptores de Antígenos de Linfócitos B , Transdução de Sinais , Quinase Syk , Ensaios Antitumorais Modelo de Xenoenxerto , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Quinase Syk/antagonistas & inibidores , Quinase Syk/metabolismo , Humanos , Fosforilação/efeitos dos fármacos , Animais , Camundongos , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas de Choque Térmico HSP110/metabolismo , Pirimidinas/farmacologia , Linhagem Celular Tumoral , Células Tumorais Cultivadas , Camundongos SCID , Quinazolinas
5.
Leukemia ; 36(4): 1035-1047, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-34795418

RESUMO

Numerous targeted therapies have been developed for diffuse large B-cell lymphoma, but the results of late-stage clinical trials were mostly disappointing and have led to very few new regulatory approvals. Here, we use single and combinatorial drug response profiling to show that the combined inhibition of the anti-apoptotic protein Bcl-2 and of the tyrosine kinase BTK with the small molecules venetoclax and ibrutinib efficiently kills DLBCL cells in vitro. High Bcl-2 expression due to either BCL2 amplifications or translocations, in conjunction with chronic active BCR signaling accurately predict responses to dual Bcl-2/BTK inhibition. Orthotopic xenotransplantation and patient-derived xenograft models confirm that the combinatorial is superior to single-agent treatment in reducing the lymphoma burden. Combinatorial treatment further efficiently overcomes both primary and acquired resistance to venetoclax, which we could link to reduced expression of the Bcl-2 family members Bcl-XL and Bcl-2A1 under ibrutinib. We found in a Swiss DLBCL cohort that ~15% of patients are projected to respond to the venetoclax/ibrutinib combination based on their high Bcl-2 expression and nuclear NF-κB localization. Our data show that drug sensitivities exposed by drug response profiling can be attributed to specific mutational signatures and immunohistochemical biomarkers, and point to combined Bcl-2/BTK inhibition as a promising therapeutic strategy in DLBCL.


Assuntos
Apoptose , Linfoma Difuso de Grandes Células B , Tirosina Quinase da Agamaglobulinemia , Humanos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/uso terapêutico , Transplante Heterólogo
6.
Oncoimmunology ; 10(1): 2003533, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34858727

RESUMO

Diffuse large B-cell lymphoma (DLBCL) is an aggressive malignancy arising from germinal center or post-germinal center B-cells that retain many of the properties of normal B-cells. Here we show that a subset of DLBCL express the cytokine IL-10 and its receptor. The genetic ablation of IL-10 receptor signaling abrogates the autocrine STAT3 phosphorylation triggered by tumor cell-intrinsic IL-10 expression and impairs growth of DLBCL cell lines in subcutaneous and orthotopic xenotransplantation models. Furthermore, we demonstrate using an immunocompetent Myc-driven model of DLBCL that neutralization of IL-10 signaling reduces tumor growth, which can be attributed to reduced Treg infiltration, stronger intratumoral effector T-cell responses, and restored tumor-specific MHCII expression. The effects of IL-10R neutralization were phenocopied by the genetic ablation of IL-10 signaling in the Treg compartment and could be reversed by MHCII blockade. The BTK inhibitor ibrutinib effectively blocked tumor cell-intrinsic IL-10 expression and tumor growth in this Myc-driven model. Tumors from patients with high IL-10RA expression are infiltrated by higher numbers of Tregs than IL-10RAlow patients. Finally, we show in 16 cases of DLBCL derived from transplant patients on immunosuppressive therapy that IL-10RA expression is less common in this cohort, and Treg infiltration is not observed.


Assuntos
Interleucina-10 , Linfoma Difuso de Grandes Células B , Linhagem Celular Tumoral , Proliferação de Células , Centro Germinativo , Humanos , Interleucina-10/genética , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética
7.
Cell Death Differ ; 27(1): 117-129, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31068676

RESUMO

Pro-survival stress-inducible chaperone HSP110 is the only HSP for which a mutation has been found in a cancer. Multicenter clinical studies demonstrated a direct association between HSP110 inactivating mutation presence and excellent prognosis in colorectal cancer patients. Here, we have combined crystallographic studies on human HSP110 and in silico modeling to identify HSP110 inhibitors that could be used in colorectal cancer therapy. Two molecules (foldamers 33 and 52), binding to the same cleft of HSP110 nucleotide-binding domain, were selected from a chemical library (by co-immunoprecipitation, AlphaScreening, Interference-Biolayer, Duo-link). These molecules block HSP110 chaperone anti-aggregation activity and HSP110 association to its client protein STAT3, thereby inhibiting STAT3 phosphorylation and colorectal cancer cell growth. These effects were strongly decreased in HSP110 knockdown cells. Foldamer's 33 ability to inhibit tumor growth was confirmed in two colorectal cancer animal models. Although tumor cell death (apoptosis) was noted after treatment of the animals with foldamer 33, no apparent toxicity was observed, notably in epithelial cells from intestinal crypts. Taken together, we identified the first HSP110 inhibitor, a possible drug-candidate for colorectal cancer patients whose unfavorable outcome is associated to HSP110.


Assuntos
Antineoplásicos/química , Antineoplásicos/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Proteínas de Choque Térmico HSP110/antagonistas & inibidores , Animais , Antineoplásicos/toxicidade , Proliferação de Células , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Cristalografia por Raios X , Proteínas de Choque Térmico HSP110/química , Proteínas de Choque Térmico HSP110/metabolismo , Humanos , Camundongos , Modelos Moleculares , Fator de Transcrição STAT3/metabolismo
8.
Oncogene ; 38(15): 2767-2777, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30542121

RESUMO

A multicenter clinical study demonstrated the presence of a loss-of-function HSP110 mutation in about 15% of colorectal cancers, which resulted from an alternative splicing and was produced at the detriment of wild-type HSP110. Patients expressing low levels of wild-type HSP110 had excellent outcomes (i.e. response to an oxaliplatin-based chemotherapy). Here, we show in vitro, in vivo, and in patients' biopsies that HSP110 co-localizes with DNA damage (γ-H2AX). In colorectal cancer cells, HSP110 translocates into the nucleus upon treatment with genotoxic chemotherapy such as oxaliplatin. Furthermore, we show that HSP110 interacts with the Ku70/Ku80 heterodimer, an essential element of the non-homologous end joining (NHEJ) repair machinery. We also demonstrate by evaluating the resolved 53BP1 foci that depletion in HSP110 impairs repair steps of the NHEJ pathway, which is associated with an increase in DNA double-strand breaks and in the cells' sensitivity to oxaliplatin. HSP110-depleted cells sensitization to oxaliplatin-induced DNA damage is abolished upon re-expression of HSP110. Confirming a role for HSP110 in DNA non-homologous repair, SCR7 and NU7026, two inhibitors of the NHEJ pathway, circumvents HSP110-induced resistance to chemotherapy. In conclusion, HSP110 through its interaction with the Ku70/80 heterodimer may participate in DNA repair, thereby inducing a protection against genotoxic therapy.


Assuntos
Núcleo Celular/genética , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Reparo do DNA por Junção de Extremidades/genética , Proteínas de Choque Térmico HSP110/genética , Mutagênicos/farmacologia , Translocação Genética/genética , Animais , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Células HCT116 , Humanos , Autoantígeno Ku/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Oxaliplatina/farmacologia , Translocação Genética/efeitos dos fármacos
9.
Methods Mol Biol ; 1709: 371-396, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29177673

RESUMO

Heat shock protein 70 (Hsp70) is the most ubiquitous stress-inducible chaperone. It accumulates in the cells in response to a wide variety of physiological and environmental insults including anticancer chemotherapy, thus allowing the cell to survive to lethal conditions. Intracellular Hsp70 is viewed as a cytoprotective protein. Indeed, this protein can inhibit key effectors of the apoptotic and autophagy machineries. In cancer cells, the expression of Hsp70 is abnormally high, and Hsp70 may participate in oncogenesis and in resistance to chemotherapy. In rodent models, Hsp70 overexpression increases tumor growth and metastatic potential. Depletion or inhibition of Hsp70 frequently reduces the size of the tumors and can even cause their complete involution. However, HSP70 is also found in the extra-cellular space where it may signal via membrane receptors or endosomes to alter gene transcription and cellular function. Overall, Hsp70 extracellular function is believed to be immnunogenic and the term chaperokine to define the extracellular chaperones such as Hsp70 has been advanced. In this chapter the knowledge to date, as well as some emerging paradigms about the intra- and extra-cellular functions of Hsp70, are presented. The strategies targeting Hsp70 that are being developed in cancer therapy will also be discussed.


Assuntos
Antineoplásicos/farmacologia , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Imunoterapia/métodos , Neoplasias/metabolismo , Antineoplásicos/uso terapêutico , Proteínas de Choque Térmico HSP70/imunologia , Humanos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/imunologia , Neoplasias/tratamento farmacológico , Neoplasias/terapia
10.
Oncotarget ; 8(23): 37681-37693, 2017 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-28445150

RESUMO

Inwardly rectifying potassium channels (Kir), and especially the barium-sensitive Kir4.1 encoded by KCNJ10, are key regulators of glial functions. A lower expression or mislocation of Kir4.1 is detected in human brain tumors. MicroRNAs participate in the regulation of ionic channels and associated neurologic disorders. Here, we analyze effects of miR-5096 on the Kir4.1 expression and function in two glioblastoma cell lines, U87 and U251. Using whole-cell patch-clamp and western-blot analysis, we show that cell loading with miR-5096 decreases the Kir4.1 protein level and associated K+ current. Cell treatment with barium, a Kir4.1 blocker, or cell loading of miR-5096 both increase the outgrowth of filopodia in glioma cells, as observed by time-lapse microscopy. Knocking-down Kir4.1 expression by siRNA transfection similarly increased both filopodia formation and invasiveness of glioma cells as observed in Boyden chamber assay. MiR-5096 also promotes the release of extracellular vesicles by which it increases its own transfer to surrounding cells, in a Kir4.1-dependent manner in U251 but not in U87. Altogether, our results validate Kir4.1 as a miR-5096 target to promote invasion of glioblastoma cells. Our data highlight the complexity of microRNA effects and the role of K+ channels in cancer.


Assuntos
Glioblastoma/metabolismo , MicroRNAs/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Movimento Celular , Células Cultivadas , Humanos , Pentamidina , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/farmacologia , Transfecção
11.
JCI Insight ; 2(6): e90531, 2017 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-28352659

RESUMO

Better identification of severe acute graft-versus-host disease (GvHD) may improve the outcome of this life-threatening complication of allogeneic hematopoietic stem cell transplantation. GvHD induces tissue damage and the release of damage-associated molecular pattern (DAMP) molecules. Here, we analyzed GvHD patients (n = 39) to show that serum heat shock protein glycoprotein 96 (Gp96) could be such a DAMP molecule. We demonstrate that serum Gp96 increases in gastrointestinal GvHD patients and its level correlates with disease severity. An increase in Gp96 serum level was also observed in a mouse model of acute GvHD. This model was used to identify complement C3 as a main partner of Gp96 in the serum. Our biolayer interferometry, yeast two-hybrid and in silico modeling data allowed us to determine that Gp96 binds to a complement C3 fragment encompassing amino acids 749-954, a functional complement C3 hot spot important for binding of different regulators. Accordingly, in vitro experiments with purified proteins demonstrate that Gp96 downregulates several complement C3 functions. Finally, experimental induction of GvHD in complement C3-deficient mice confirms the link between Gp96 and complement C3 in the serum and with the severity of the disease.


Assuntos
Complemento C3/metabolismo , Doença Enxerto-Hospedeiro/sangue , Glicoproteínas de Membrana/sangue , Chaperonas Moleculares/sangue , Adolescente , Adulto , Animais , Ativação do Complemento , Transplante de Células-Tronco Hematopoéticas , Humanos , Camundongos , Pessoa de Meia-Idade , Adulto Jovem
12.
Oncoimmunology ; 5(7): e1170264, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27622020

RESUMO

HSP110 is induced by different stresses and, through its anti-apoptotic and chaperoning properties, helps the cells to survive these adverse situations. In colon cancers, HSP110 is abnormally abundant. We have recently showed that colorectal cancer (CRC) patients with microsatellite instability (MSI) had an improved response to chemotherapy because they harbor an HSP110 inactivating mutation (HSP110DE9). In this work, we have used patients' biopsies and human CRC cells grown in vitro and in vivo (xenografts) to demonstrate that (1) HSP110 is secreted by CRC cells and that the amount of this extracellular HSP110 is strongly decreased by the expression of the mutant HSP110DE9, (2) Supernatants from CRC cells overexpressing HSP110 or purified recombinant human HSP110 (LPS-free) affect macrophage differentiation/polarization by favoring a pro-tumor, anti-inflammatory profile, (3) Conversely, inhibition of HSP110 (expression of siRNA, HSP110DE9 or immunodepletion) induced the formation of macrophages with a cytotoxic, pro-inflammatory profile. (4) Finally, this effect of extracellular HSP110 on macrophages seems to implicate TLR4. These results together with the fact that colorectal tumor biopsies with HSP110 high were infiltrated with macrophages with a pro-tumoral profile while those with HSP110 low were infiltrated with macrophages with a cytotoxic profile, suggest that the effect of extracellular HSP110 function on macrophages may also contribute to the poor outcomes associated with HSP110 expression.

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