RESUMO
Synthetic lethality is a powerful approach for targeting oncogenic drivers in cancer. Recent studies revealed that cancer cells with microsatellite instability (MSI) require Werner (WRN) helicase for survival; however, the underlying mechanism remains unclear. In this study, we found that WRN depletion strongly induced p53 and its downstream apoptotic target PUMA in MSI colorectal cancer (CRC) cells. p53 or PUMA deletion abolished apoptosis induced by WRN depletion in MSI CRC cells. Importantly, correction of MSI abrogated the activation of p53/PUMA and cell killing, while induction of MSI led to sensitivity in isogenic CRC cells. Rare p53-mutant MSI CRC cells are resistant to WRN depletion due to lack of PUMA induction, which could be restored by wildtype (WT) p53 knock in or reconstitution. WRN depletion or treatment with the RecQ helicase inhibitor ML216 suppressed in vitro and in vivo growth of MSI CRCs in a p53/PUMA-dependent manner. ML216 treatment was efficacious in MSI CRC patient-derived xenografts. Interestingly, p53 gene remains WT in the majority of MSI CRCs. These results indicate a critical role of p53/PUMA-mediated apoptosis in the vulnerability of MSI CRCs to WRN loss, and support WRN as a promising therapeutic target in p53-WT MSI CRCs.
Assuntos
Neoplasias do Colo , Neoplasias Colorretais , Humanos , Helicase da Síndrome de Werner/genética , Proteína Supressora de Tumor p53/genética , Instabilidade de Microssatélites , Neoplasias Colorretais/genética , RecQ Helicases/genéticaRESUMO
Langerhans cell histiocytosis (LCH) is characterized by an expansion and accumulation of pathological histiocytes expressing langerin (CD207) and CD1a in different organs under an inflammatory milieu. The origin of pathognomonic precursors of LCH is widely debated, but monocytes and pre-dendritic cells (pre-DC) play a significant role. Remarkably, we found an expansion of AXLhigh cells in the CD11c+ subset of patients with active LCH, which also express the pathognomonic CD207 and CD1a. Moreover, we obtained a monocyte-derived LC-like (mo-LC-like) expressing high levels of AXL when treated with inflammatory cytokine, or plasma of patients with active disease. Intriguingly, inhibiting the mTOR pathway at the initial stages of monocyte differentiation to LC-like fosters the pathognomonic LCH program, highly increasing CD207 levels, together with NOTCH1 induction. We define here that AXLhigh could also be taken as a strong pathognomonic marker for LCH, and the release of Langerin and NOTCH1 expression depends on the inhibition of the mTOR pathway.
Assuntos
Antígenos CD , Receptor Tirosina Quinase Axl , Histiocitose de Células de Langerhans , Lectinas Tipo C , Lectinas de Ligação a Manose , Proteínas Proto-Oncogênicas , Receptores Proteína Tirosina Quinases , Serina-Treonina Quinases TOR , Humanos , Histiocitose de Células de Langerhans/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Antígenos CD/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Lectinas de Ligação a Manose/metabolismo , Lectinas Tipo C/metabolismo , Masculino , Células Mieloides/metabolismo , Biomarcadores , Feminino , Adolescente , Receptor Notch1/metabolismo , Antígenos CD1/metabolismo , Criança , Monócitos/metabolismo , Monócitos/imunologia , Adulto , Pré-Escolar , Transdução de Sinais , Diferenciação CelularRESUMO
Despite significant therapeutic improvements chronic lymphocytic leukemia (CLL) remains an incurable disease and there is a persistent pursuit of new treatment alternatives. Lurbinectedin, a selective inhibitor of active transcription of protein-coding genes, is currently in phase II/III clinical trials for solid tumors such as small-cell lung cancer (SCLC). In this study, we aimed to evaluate the activity of Lurbinectedin on circulating mononuclear cells from CLL patients and to determine whether Lurbinectedin could affect the cross-talk between B-CLL cells and the tumor microenvironment. We found that Lurbinectedin induced a dose- and time-dependent death in all cell types evaluated, with B cells, monocytes and monocytic myeloid derived suppressor cells (Mo-MDSC) being the most susceptible populations. At sub-apoptotic doses, Lurbinectedin decreased the expression of CCR7 in B-CLL cells and impaired their migration towards CCL19 and CCL21. Furthermore, low concentrations of Lurbinectedin stimulated the synthesis of pro-IL1ß in monocytes and nurse-like cells, without inducing the inflammasome activation. Altogether, these results indicate that Lurbinectedin might have antitumor activity in CLL due to its direct action on leukemic cells in combination with its effects on the tumor microenvironment. Our findings encourage further investigation of Lurbinectedin as a potential therapy for CLL.
Assuntos
Carbolinas/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Microambiente Tumoral/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Quimiocina CCL19/imunologia , Quimiocina CCL19/metabolismo , Quimiocina CCL21/imunologia , Quimiocina CCL21/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/patologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/metabolismo , Células Supressoras Mieloides/efeitos dos fármacos , Células Supressoras Mieloides/imunologia , Células Supressoras Mieloides/metabolismo , Cultura Primária de Células , Receptores CCR7/imunologia , Receptores CCR7/metabolismo , Células Tumorais Cultivadas , Microambiente Tumoral/imunologiaRESUMO
Reprogramming of neutrophils by malignant cells is well-described for many types of solid tumors, but data remain scarce for hematological diseases. Chronic lymphocytic leukemia (CLL) is characterized for a deep immune dysregulation mediated by leukemic cells that compromises patient's outcome. Murine models of CLL highlight the relevance of myeloid cells as tumor-driven reprogramming targets. In our study, we evaluated neutrophil reprogramming by CLL cells. We first show that the proportion of the CD16high CD62Ldim neutrophil subset in peripheral blood of CLL patients is increased compared to age-matched healthy donors (HD). In vitro, neutrophils from HD cultured in the presence of CLL cells or conditioned media (CM) from CLL cells exhibited a longer lifespan. Depletion of G-CSF and GM-CSF from CM partially reversed the protective effect. In addition, the proportion of viable neutrophils that displayed a CD16high CD62Ldim phenotype was increased in the presence of CM from CLL cells, being TGF-ß/IL-10 responsible for this effect. Altogether, our results describe a novel mechanism through which CLL cells can manipulate neutrophils.
Assuntos
Diferenciação Celular/fisiologia , Tolerância Imunológica/fisiologia , Selectina L/metabolismo , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Neutrófilos/patologia , Receptores de IgG/metabolismo , Idoso , Linhagem Celular Tumoral , Feminino , Proteínas Ligadas por GPI/metabolismo , Fator Estimulador de Colônias de Granulócitos/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Neutrófilos/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Chronic lymphocytic leukemia (CLL) is characterized by immune defects that contribute to a high rate of infections and autoimmune cytopenias. Neutrophils are the first line of innate immunity and respond to pathogens through multiple mechanisms, including the release of neutrophil extracellular traps (NETs). These web-like structures composed of DNA, histones, and granular proteins are also produced under sterile conditions and play important roles in thrombosis and autoimmune disorders. Here we show that neutrophils from CLL patients are more prone to release NETs compared to those from age-matched healthy donors (HD). Increased generation of NETs was not due to higher levels of elastase, myeloperoxidase, or reactive oxygen species production. Instead, we found that plasma from CLL patients was able to prime neutrophils from HD to generate higher amounts of NETs upon activation. Plasmatic IL-8 was involved in the priming effect since its depletion reduced plasma capacity to enhance NETs release. Finally, we found that culture with NETs delayed spontaneous apoptosis and increased the expression of activation markers on leukemic B cells. Our study provides new insights into the immune dysregulation in CLL and suggests that the chronic inflammatory environment typical of CLL probably underlies this inappropriate neutrophil priming.
Assuntos
Armadilhas Extracelulares/imunologia , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/imunologia , Neutrófilos/imunologia , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Humanos , Interleucina-8/imunologia , Pessoa de Meia-IdadeRESUMO
Small molecules targeting kinases involved in B cell receptor signaling are showing encouraging clinical activity in chronic lymphocytic leukemia (CLL) patients. Fostamatinib (R406) and entospletinib (GS-9973) are ATP-competitive inhibitors designed to target spleen tyrosine kinase (Syk) that have shown clinical activity with acceptable toxicity in trials with CLL patients. Preclinical studies with these inhibitors in CLL have focused on their effect in patient-derived leukemic B cells. In this work we show that clinically relevant doses of R406 and GS-9973 impaired the activation and proliferation of T cells from CLL patients. This effect could not be ascribed to Syk-inhibition given that we show that T cells from CLL patients do not express Syk protein. Interestingly, ζ-chain-associated protein kinase (ZAP)-70 phosphorylation was diminished by both inhibitors upon TCR stimulation on T cells. In addition, we found that both agents reduced macrophage-mediated phagocytosis of rituximab-coated CLL cells. Overall, these results suggest that in CLL patients treated with R406 or GS-9973 T cell functions, as well as macrophage-mediated anti-tumor activity of rituximab, might be impaired. The potential consequences for CLL-treated patients are discussed.
Assuntos
Indazóis/farmacologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Macrófagos/imunologia , Oxazinas/farmacologia , Pirazinas/farmacologia , Piridinas/farmacologia , Quinase Syk/antagonistas & inibidores , Linfócitos T/efeitos dos fármacos , Proteína-Tirosina Quinase ZAP-70/metabolismo , Idoso , Idoso de 80 Anos ou mais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Feminino , Humanos , Ativação Linfocitária/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Fagocitose/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Rituximab/farmacologia , Linfócitos T/imunologiaAssuntos
Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Resistencia a Medicamentos Antineoplásicos , Leucemia Linfocítica Crônica de Células B , Ativação Linfocitária/efeitos dos fármacos , Proteínas de Neoplasias , Sulfonamidas/farmacologia , Quinase Syk , Linfócitos T , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/imunologia , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/enzimologia , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/imunologia , Proteínas de Neoplasias/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Rituximab/farmacologia , Quinase Syk/antagonistas & inibidores , Quinase Syk/imunologia , Quinase Syk/metabolismo , Linfócitos T/imunologia , Linfócitos T/patologiaAssuntos
Leucemia Linfocítica Crônica de Células B/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Biomarcadores , Proliferação de Células/genética , Sobrevivência Celular/genética , Progressão da Doença , Feminino , Humanos , Imunofenotipagem , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Transdução de SinaisRESUMO
Targeting cyclin-dependent kinases 4 and 6 (CDK4/6) is a successful therapeutic approach against breast and other solid tumors. Inhibition of CDK4/6 halts cell cycle progression and promotes antitumor immunity. However, the mechanisms underlying the antitumor activity of CDK4/6 inhibitors are not fully understood. We found that CDK4/6 bind and phosphorylate the p53 family member p73 at threonine 86, which sequesters p73 in the cytoplasm. Inhibition of CDK4/6 led to dephosphorylation and nuclear translocation of p73, which transcriptionally activated death receptor 5 (DR5), a cytokine receptor and key component of the extrinsic apoptotic pathway. p73-mediated induction of DR5 by CDK4/6 inhibitors promoted immunogenic cell death of cancer cells. Deletion of DR5 in cancer cells in vitro and in vivo abrogated the potentiating effects of CDK4/6 inhibitors on immune cytokine TRAIL, 5-fluorouracil chemotherapy, and anti-PD-1 immunotherapy. Together, these results reveal a previously unrecognized consequence of CDK4/6 inhibition, which may be critical for potentiating the killing and immunogenic effects on cancer cells. SIGNIFICANCE: This work demonstrates how inhibition of CDK4/6 sensitizes cancer cells to chemotherapy and immune checkpoint blockade and may provide a new molecular marker for improving CDK4/6-targeted cancer therapies. See related commentary by Frank, p. 1170.
Assuntos
Quinase 4 Dependente de Ciclina , Quinase 6 Dependente de Ciclina , Inibidores de Checkpoint Imunológico , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Ligante Indutor de Apoptose Relacionado a TNF , Proteína Tumoral p73 , Apoptose , Linhagem Celular Tumoral , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Fluoruracila/farmacologia , Humanos , Fosforilação , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Proteína Tumoral p73/metabolismoRESUMO
Bromodomain and extra-terminal domain (BET) family proteins are epigenetic readers that play a critical role in oncogenesis by controlling the expression of oncogenes such as c-Myc. Targeting BET family proteins has recently emerged as a promising anticancer strategy. However, the molecular mechanisms by which cancer cells respond to BET inhibition are not well understood. In this study, we found that inducing the degradation of BET proteins by the proteolysis targeting chimeras (PROTAC) approach potently suppressed the growth of colorectal cancer (CRC) including patient-derived tumors. Mechanistically, BET degradation transcriptionally activates Death Receptor 5 (DR5) to trigger immunogenic cell death (ICD) in CRC cells. Enhanced DR5 induction further sensitizes CRC cells with a mutation in Speckle-type POZ protein (SPOP). Furthermore, DR5 is indispensable for a striking antitumor effect of combining BET degradation and anti-PD-1 antibody, which was well tolerated in mice and almost eradicated syngeneic tumors. Our results demonstrate that BET degradation triggers DR5-mediated ICD to potently suppress CRC and potentiate immune checkpoint blockade. These results provide a rationale, mechanistic insights, and potential biomarkers for developing a precision CRC therapy by inducing BET protein degradation.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica , Inibidores de Checkpoint Imunológico/farmacologia , Morte Celular Imunogênica , Proteínas/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas/genética , Proteólise , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Use of non-steroidal anti-inflammatory drugs (NSAIDs) is associated with reduced risk of colorectal cancer (CRC). However, the mechanism by which NSAIDs suppress colorectal tumorigenesis remains unclear. We previously showed that NSAIDs selectively kill emerging tumor cells via death receptor (DR) signaling and a synthetic lethal interaction mediated by the proapoptotic Bcl-2 family protein BID. In this study, we found NSAIDs induce endoplasmic reticulum (ER) stress to activate DR signaling and BID in tumor suppression. Importantly, our results unveiled an ER stress- and BID-dependent immunogenic effect of NSAIDs, which may be critical for tumor suppression. NSAID treatment induced hallmarks of immunogenic cell death (ICD) in CRC cells and colonic epithelial cells upon loss of APC tumor suppressor, and elevated tumor-infiltrating lymphocytes (TILs) in the polyps of APCMin/+ mice. ER stress inhibition or BID deletion abrogated the antitumor and immunogenic effects of NSAIDs. Furthermore, increased ER stress and TILs were detected in human advanced adenomas from NSAID-treated patients. Together, our results suggest that NSAIDs induce ER stress- and BID-mediated ICD to restore immunosurveillance and suppress colorectal tumor formation.
Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Anti-Inflamatórios não Esteroides/farmacologia , Carcinogênese/genética , Neoplasias Colorretais/tratamento farmacológico , Animais , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/genética , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Morte Celular Imunogênica/efeitos dos fármacos , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/patologia , Camundongos , Transdução de Sinais/efeitos dos fármacosRESUMO
Ibrutinib is a BTK/ITK inhibitor with efficacy for the treatment of various lymphoid cancers, including CLL. Considering that innate and adaptative immune defects are a dominant feature of CLL patients, we evaluated whether in vitro ibrutinib affects the survival and function of neutrophils and γδ T cells, key players of the early immune response against microbes. Neutrophils and γδ T cells were obtained from peripheral blood of healthy donors and CLL patients. We found that ibrutinib reduces the production of reactive oxygen species (ROS) and bacteria killing capacity, and slightly impairs neutrophil extracellular traps (NETs) production without affecting bacteria-uptake and CD62L-downregulation induced by fMLP or aggregated IgG. In addition, ibrutinib reduces γδ T cell activation and CD107a degranulation induced by phosphoantigens or anti-CD3. These findings are in agreement with previous data suggesting that ibrutinib interferes with the protective immune response to pathogens, particularly Mycobacteria and Aspergillus.
Assuntos
Neutrófilos , Linfócitos T , Adenina/análogos & derivados , Humanos , Ativação Linfocitária , Piperidinas , Espécies Reativas de OxigênioRESUMO
The proliferation and survival of malignant B cells in chronic lymphocytic leukemia (CLL) depend on signals from the microenvironment in lymphoid tissues. Among a plethora of soluble factors, IL-8 has been considered one of the most relevant to support CLL B cell progression in an autocrine fashion, even though the expression of IL-8 receptors, CXCR1 and CXCR2, on leukemic B cells has not been reported. Here we show that circulating CLL B cells neither express CXCR1 or CXCR2 nor they respond to exogenous IL-8 when cultured in vitro alone or in the presence of monocytes/nurse-like cells. By intracellular staining and ELISA we show that highly purified CLL B cells do not produce IL-8 spontaneously or upon activation through the B cell receptor. By contrast, we found that a minor proportion (<0.5%) of contaminating monocytes in enriched suspensions of leukemic cells might be the actual source of IL-8 due to their strong capacity to release this cytokine. Altogether our results indicate that CLL B cells are not able to secrete or respond to IL-8 and highlight the importance of methodological details in in vitro experiments.