RESUMO
OX40 and its ligand are members of the TNF/TNF receptor superfamily, which includes various molecules influencing cellular signaling and function of both tumor and immune cells. The ability of OX40 to promote proliferation and differentiation of activated T cells fueled present attempts to modulate this immune checkpoint to reinforce antitumor immunity. While we recently found evidence for the involvement of OX40 in pathophysiology of acute myeloid leukemia including natural killer (NK) cell immunosurveillance, less is known on its role in acute lymphoblastic leukemia (ALL). In the present study, OX40 expression on ALL cells was significantly associated with positivity for the adverse risk factor BCR-ABL. In line, signaling via OX40 increased metabolic activity of primary ALL cells and resulted in release of cytokines involved in disease pathophysiology. Furthermore, interaction of ALL-expressed OX40 with its cognate ligand on NK cells stimulated ALL cell lysis. The data presented thus not only identify the yet unknown involvement of OX40/OX40L in ALL pathophysiology and NK cell immunosurveillance but also point to the necessity to thoroughly consider the consequences of modulating the OX40/OX40L molecule system beyond its effects on T cells when developing OX40-targeting approaches for cancer immunotherapy.
Assuntos
Proteínas de Fusão bcr-abl/genética , Regulação Leucêmica da Expressão Gênica , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Receptores OX40/genética , Biomarcadores Tumorais , Linhagem Celular Tumoral , Citocinas/metabolismo , Citotoxicidade Imunológica , Metabolismo Energético , Citometria de Fluxo , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Ligante OX40/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidade , Prognóstico , Ligação Proteica , Receptores OX40/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
The TNF receptor family member OX40 promotes activation and proliferation of T cells, which fuels efforts to modulate this immune checkpoint to reinforce antitumor immunity. Besides T cells, NK cells are a second cytotoxic lymphocyte subset that contributes to antitumor immunity, particularly in leukemia. Accordingly, these cells are being clinically evaluated for cancer treatment through multiple approaches, such as adoptive transfer of ex vivo expanded polyclonal NK cells (pNKC). Here, we analyzed whether and how OX40 and its ligand (OX40L) influence NK-cell function and antileukemia reactivity. We report that OX40 is expressed on leukemic blasts in a substantial percentage of patients with acute myeloid leukemia (AML) and that OX40 can, after stimulation with agonistic OX40 antibodies, mediate proliferation and release of cytokines that act as growth and survival factors for the leukemic cells. We also demonstrate that pNKC differentially express OX40L, depending on the protocol used for their generation. OX40L signaling promoted NK-cell activation, cytokine production, and cytotoxicity, and disruption of OX40-OX40L interaction impaired pNKC reactivity against primary AML cells. Together, our data implicate OX40/OX40L in disease pathophysiology of AML and in NK-cell immunosurveillance. Our findings indicate that effects of the OX40-OX40L receptor-ligand system in other immune cell subsets and also malignant cells should be taken into account when developing OX40-targeted approaches for cancer immunotherapy. Cancer Immunol Res; 6(2); 209-21. ©2018 AACR.
Assuntos
Células Matadoras Naturais/imunologia , Leucemia Mieloide Aguda/imunologia , Ligante OX40/imunologia , Receptores OX40/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Citocinas/imunologia , Humanos , Vigilância Imunológica , Camundongos , Camundongos Endogâmicos C57BL , Receptores OX40/agonistas , Células U937RESUMO
Costimulatory ligands (COLs) and their receptors (COR) regulate immune reactions and cellular survival and might be relevant in acute myeloid leukemia (AML). This study evaluated the clinical relevance of 4-1BBL, glucocorticoid-induced TNFR-related protein (GITR) and ligand (GITRL), CD80, and CD86 in case of expression on AML blasts. 98 patients were evaluated at initial diagnosis. Immunophenotypically evaluated specific fluorescence index (SFI) levels of COR and COL on blasts were correlated with morphological, cytogenetic, and several prognostic parameters. Significantly higher COR expression was seen in monocytic versus non-monocytic AML subtypes; GITR, p=0.05; GITRL, p=0.005; CD86, p=0.001). Cut-off values for two COR and their ligands were evaluated: cases presenting with 4-1BB values above cut-off 1.2 SFI levels correlated (tendentially) significantly with a higher probability for disease-free survival (DFS, p=0.06) and a favorable HR of 0.2; p=0.04 for relapse. HR for death was also significantly lower in this group (0.12; p=0.04). In contrast, a lower probability for DFS and overall survival was seen in cases with 4-1BBL expression above 2.2 SFI levels (p=0.08 and p=0.09). In addition, multivariate analysis showed a significantly higher probability of death in this group (HR 10.3, p=0.04). Expression of CD80 and CD86 did not show significant prognostic relevance. On initial diagnosis, 4-1BB and 4-1BBL qualify as markers for prediction of patients' course and represent a valuable screening target for patients with AML at initial diagnosis.
Assuntos
Ligante 4-1BB/metabolismo , Crise Blástica/metabolismo , Leucemia Mieloide Aguda/metabolismo , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Crise Blástica/genética , Crise Blástica/patologia , Intervalo Livre de Doença , Feminino , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Masculino , Pessoa de Meia-Idade , Monócitos/patologia , Análise Multivariada , Proteínas de Neoplasias/metabolismo , Prognóstico , Fatores de Risco , Adulto JovemRESUMO
BACKGROUND: Co-stimulatory receptor (COR) and ligand (COL) expression on immune effectors are known to be relevant for immunological interactions and might be of prognostic relevance if expressed on acute myeloid leukemia (AML) blasts as reported for receptors of the tumor necrosis factor receptor family. AIM AND METHODS: Antigen expression profiling of COR (RANK, PD-1), COL (RANK-L, PD-1L), and HLA-ABC-antigens on blasts from 90 AML-patients at first diagnosis was performed by flow cytometry (SFI-Level characterization) and findings were correlated with clinical parameters. RESULTS: RANK expression was higher in immature compared to mature FAB groups (P = 0.08). As a monocytic marker, we identified HLA-ABC (P = 0.07). Prognostic analysis revealed a higher probability of overall survival in cases with lower RANK-L expression (<1.6 and ≥1.6, 15.6 vs. 12.2 months, P = 0.008, hazard ratio 0.36, P = 0.008). No significant impact of PD-1/L expression for patients'(pts) survival was seen but a correlation of PD-1 with a secondary AML (P = 0.03). Prolonged disease-free survival however correlated with higher PD-1 expression (≥1.1 vs. <1.1, 31.4 vs. 12.7 months; P = 0.03). CONCLUSION: Our study revealed that RANK-L is a promising marker to forecast pts' prognosis in AML. Immune checkpoint receptor PD-1/L as well as RANK and HLA-ABC did not show an impact on pts' survival.
Assuntos
Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidade , Receptor de Morte Celular Programada 1/genética , Ligante RANK/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores , Análise Citogenética , Feminino , Perfilação da Expressão Gênica , Humanos , Imunofenotipagem , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Prognóstico , Receptor de Morte Celular Programada 1/metabolismo , Ligante RANK/metabolismo , Recidiva , Adulto JovemRESUMO
BACKGROUND: Tumor necrosis factor (TNF) receptor family members play a key role in the regulation of biological functions such as differentiation, proliferation and apoptosis of various cell types. MATERIALS AND METHODS: We studied co-expression profiles of death receptors from the TNF family [TNF-related apoptosis-inducing ligand receptor (TRAILR) 1 to 3, TNF receptor 1 (TNFR1) and FAS receptor (FAS)] on peripheral blood blasts from 46 patients with acute myeloid leukemia (AML) at first diagnosis by flow cytometry and correlated the obtained specific fluorescence indices (SFI) with morphological, cytogenetic and clinical parameters. RESULTS: We found that the expression of TRAILR2 and R3 was significantly increased in unfavorable risk groups, according to the National Comprehensive Cancer Network. Additionally, cut-off analyses for TRAILR2 and TNFR1 showed significantly shorter overall survival, earlier disease onset, higher proportions of cases with unfavorable prognosis and higher probability of relapse when SFIs were above the established cut-off. CONCLUSION: We demonstrate that high co-expression of death receptors on blasts is an independent predictor of poor prognosis in AML.
Assuntos
Leucemia Mieloide Aguda/metabolismo , Receptores de Morte Celular/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Receptores Chamariz do Fator de Necrose Tumoral/metabolismo , Adulto Jovem , Receptor fas/metabolismoRESUMO
Epigenetic alterations are a hallmark of cancer that govern the silencing of genes. Up to now, 5-azacytidine (5-aza-CR, Vidaza) and 5-aza-2'-deoxycytidine (5-aza-dC, Dacogen) are the only clinically approved DNA methyltransferase inhibitors (DNMTi). Current effort tries to exploit DNMTi application beyond acute leukemia or myelodysplastic syndrome, especially to solid tumors. Although both drugs only differ by a minimal structural difference, they trigger distinct molecular mechanisms that are highly relevant for a rational choice of new combination therapies. Therefore, we investigated cell death pathways in vitro in human hepatoma, colon, renal, and lung cancer cells and in vivo in chorioallantoic membrane and xenograft models. Real-time cancer cell monitoring and cytokine profiling revealed a profoundly distinct response pattern to both drugs. 5-aza-dC induced p53-dependent tumor cell senescence and a high number of DNA double-strand breaks. In contrast, 5-aza-CR downregulated p53, induced caspase activation and apoptosis. These individual response patterns of tumor cells could be verified in vivo in chorioallantoic membrane assays and in a hepatoma xenograft model. Although 5-aza-CR and 5-aza-dC are viewed as drugs with similar therapeutic activity, they induce a diverse molecular response in tumor cells. These findings together with other reported differences enable and facilitate a rational design of new combination strategies to further exploit the epigenetic mode of action of these two drugs in different areas of clinical oncology.
Assuntos
Apoptose/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Azacitidina/administração & dosagem , Azacitidina/análogos & derivados , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla , Metilação de DNA/genética , Decitabina , Células Hep G2 , Humanos , Neoplasias/genética , Neoplasias/patologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Natural killer (NK) cells are cytotoxic lymphocytes that largely contribute to the efficacy of therapeutic strategies like allogenic stem cell transplantation in acute myeloid leukemia (AML) and application of Rituximab in chronic lymphocytic leukemia (CLL). The tumor necrosis factor (TNF) family member GITR ligand (GITRL) is frequently expressed on leukemia cells in AML and CLL and impairs the reactivity of NK cells which express GITR and upregulate its expression following activation. We developed a strategy to reinforce NK anti-leukemia reactivity by combining disruption of GITR-GITRL interaction with targeting leukemia cells for NK antibody-dependent cellular cytotoxicity (ADCC) using GITR-Ig fusion proteins with modified Fc moieties. Neutralization of leukemia-expressed GITRL by the GITR domain enhanced cytotoxicity and cytokine production of NK cells depending on activation state with NK reactivity being further largely dependent on the engineered affinity of the fusion proteins to the Fc receptor. Compared with wild-type GITR-Ig, treatment of primary AML and CLL cells with mutants containing a S239D/I332E modification potently increased cytotoxicity, degranulation, and cytokine production of NK cells in a target-antigen-dependent manner with additive effects being observed with CLL cells upon parallel exposure to Rituximab. Fc-optimized GITR-Ig may thus constitute an attractive means for immunotherapy of leukemia that warrants clinical evaluation.
Assuntos
Células Matadoras Naturais/citologia , Leucemia/terapia , Proteínas Recombinantes de Fusão/farmacologia , Células Cultivadas , Citometria de Fluxo , Proteína Relacionada a TNFR Induzida por Glucocorticoide/genética , Proteína Relacionada a TNFR Induzida por Glucocorticoide/metabolismo , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Leucemia/imunologia , Receptores Fc/genética , Receptores Fc/metabolismo , Proteínas Recombinantes de Fusão/genéticaRESUMO
The TNF family member receptor activator for NF-κB ligand (RANKL) and its receptors RANK and osteoprotegerin are key regulators of bone remodeling but also influence cellular functions of tumor and immune effector cells. In this work, we studied the involvement of RANK-RANKL interaction in NK cell-mediated immunosurveillance of acute myeloid leukemia (AML). Substantial levels of RANKL were found to be expressed on leukemia cells in 53 of 78 (68%) investigated patients. Signaling via RANKL into the leukemia cells stimulated their metabolic activity and induced the release of cytokines involved in AML pathophysiology. In addition, the immunomodulatory factors released by AML cells upon RANKL signaling impaired the anti-leukemia reactivity of NK cells and induced RANK expression, and NK cells of AML patients displayed significantly upregulated RANK expression compared with healthy controls. Treatment of AML cells with the clinically available RANKL Ab Denosumab resulted in enhanced NK cell anti-leukemia reactivity. This was due to both blockade of the release of NK-inhibitory factors by AML cells and prevention of RANK signaling into NK cells. The latter was found to directly impair NK anti-leukemia reactivity with a more pronounced effect on IFN-γ production compared with cytotoxicity. Together, our data unravel a previously unknown function of the RANK-RANKL molecule system in AML pathophysiology as well as NK cell function and suggest that neutralization of RANKL with therapeutic Abs may serve to reinforce NK cell reactivity in leukemia patients.
Assuntos
Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/metabolismo , Ligante RANK/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais Humanizados/farmacologia , Linhagem Celular , Denosumab , Feminino , Regulação Leucêmica da Expressão Gênica , Humanos , Imunomodulação/efeitos dos fármacos , Leucemia Mieloide Aguda/genética , Masculino , Pessoa de Meia-Idade , Ligação Proteica , Ligante RANK/antagonistas & inibidores , Ligante RANK/genética , Receptor Ativador de Fator Nuclear kappa-B/genética , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Adulto JovemRESUMO
Bone destruction is a prominent feature of multiple myeloma, but conflicting data exist on the expression and pathophysiologic involvement of the bone remodeling ligand RANKL in this disease and the potential therapeutic benefits of its targeted inhibition. Here, we show that RANKL is expressed by primary multiple myeloma and chronic lymphocytic leukemia (CLL) cells, whereas release of soluble RANKL was observed exclusively with multiple myeloma cells and was strongly influenced by posttranscriptional/posttranslational regulation. Signaling via RANKL into multiple myeloma and CLL cells induced release of cytokines involved in disease pathophysiology. Both the effects of RANKL on osteoclastogenesis and cytokine production by malignant cells could be blocked by disruption of RANK-RANKL interaction with denosumab. As we aimed to combine neutralization of RANKL with induction of antibody-dependent cellular cytotoxicity of natural killer (NK) cells against RANKL-expressing malignant cells and as denosumab does not stimulate NK reactivity, we generated RANK-Fc fusion proteins with modified Fc moieties. The latter displayed similar capacity compared with denosumab to neutralize the effects of RANKL on osteoclastogenesis in vitro, but also potently stimulated NK cell reactivity against primary RANKL-expressing malignant B cells, which was dependent on their engineered affinity to CD16. Our findings introduce Fc-optimized RANK-Ig fusion proteins as attractive tools to neutralize the detrimental function of RANKL while at the same time potently stimulating NK cell antitumor immunity.
Assuntos
Células Matadoras Naturais/imunologia , Leucemia Linfocítica Crônica de Células B/metabolismo , Mieloma Múltiplo/metabolismo , Ligante RANK/metabolismo , Anticorpos Monoclonais Humanizados/farmacologia , Linhagem Celular Tumoral , Denosumab , Humanos , Ligante RANK/antagonistas & inibidores , Ligante RANK/genética , Receptores Fc/genética , Proteínas Recombinantes de Fusão/farmacologia , TransfecçãoRESUMO
Ligands of the prototypical activating NK receptor NKG2D render cancer cells susceptible to NK cell-mediated cytolysis if expressed at sufficiently high levels. However, malignant cells employ mechanisms to evade NKG2D-mediated immunosurveillance, such as NKG2D ligand (NKG2DL) shedding resulting in reduced surface expression levels. In addition, systemic downregulation of NKG2D on NK cells of cancer patients has been observed in many studies and was attributed to soluble NKG2DL (sNKG2DL), although there also are conflicting data. Likewise, relevant expression of NKG2DL in leukemia has been reported by some, but not all studies. Hence, we comprehensively studied expression, release, and function of the NKG2D ligands MHC class I chain-related molecules A and B and UL16-binding proteins 1-3 in 205 leukemia patients. Leukemia cells of most patients (75%) expressed at least one NKG2DL at the surface, and all investigated patient sera contained elevated sNKG2DL levels. Besides correlating NKG2DL levels with clinical data and outcome, we demonstrate that sNKG2DL in patient sera reduce NKG2D expression on NK cells, resulting in impaired antileukemia reactivity, which also critically depends on number and levels of surface-expressed NKG2DL. Together, we provide comprehensive data on the relevance of NKG2D/NKG2DL expression, release, and function for NK reactivity in leukemia, which exemplifies the mechanisms underlying NKG2D-mediated tumor immunosurveillance and escape.