RESUMO
ABSTRACT: Acute lymphoblastic leukemia (ALL) arises from the uncontrolled proliferation of B-cell precursors (BCP-ALL) or T cells (T-ALL). Current treatment protocols obtain high cure rates in children but are based on toxic polychemotherapy. Novel therapies are urgently needed, especially in relapsed/refractory (R/R) disease, high-risk (HR) leukemias and T-ALL, in which immunotherapy approaches remain scarce. Although the interleukin-7 receptor (IL-7R) plays a pivotal role in ALL development, no IL-7R-targeting immunotherapy has yet reached clinical application in ALL. The IL-7Rα chain (CD127)-targeting IgG4 antibody lusvertikimab (LUSV; formerly OSE-127) is a full antagonist of the IL-7R pathway, showing a good safety profile in healthy volunteers. Here, we show that â¼85% of ALL cases express surface CD127. We demonstrate significant in vivo efficacy of LUSV immunotherapy in a heterogeneous cohort of BCP- and T-ALL patient-derived xenografts (PDX) in minimal residual disease (MRD) and overt leukemia models, including R/R and HR leukemias. Importantly, LUSV was particularly effective when combined with polychemotherapy in a phase 2-like PDX study with CD127high samples leading to MRD-negativity in >50% of mice treated with combination therapy. Mechanistically, LUSV targeted ALL cells via a dual mode of action comprising direct IL-7R antagonistic activity and induction of macrophage-mediated antibody-dependent cellular phagocytosis (ADCP). LUSV-mediated in vitro ADCP levels significantly correlated with CD127 expression levels and the reduction of leukemia burden upon treatment of PDX animals in vivo. Altogether, through its dual mode of action and good safety profile, LUSV may represent a novel immunotherapy option for any CD127+ ALL, particularly in combination with standard-of-care polychemotherapy.
Assuntos
Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Humanos , Camundongos , Receptores de Interleucina-7/antagonistas & inibidores , Camundongos SCID , Fagocitose/efeitos dos fármacos , Subunidade alfa de Receptor de Interleucina-7 , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Feminino , Camundongos Endogâmicos NOD , Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Monoclonais Humanizados/farmacologia , Linhagem Celular Tumoral , Antineoplásicos Imunológicos/farmacologia , Antineoplásicos Imunológicos/uso terapêuticoRESUMO
Acute lymphoblastic leukemia (ALL) is the most common malignant disease affecting children. Although therapeutic strategies have improved, T-cell acute lymphoblastic leukemia (T-ALL) relapse is associated with chemoresistance and a poor prognosis. One strategy to overcome this obstacle is the application of monoclonal antibodies. Here, we show that leukemic cells from patients with T-ALL express surface CD38 and CD47, both attractive targets for antibody therapy. We therefore investigated the commercially available CD38 antibody daratumumab (Dara) in combination with a proprietary modified CD47 antibody (Hu5F9-IgG2σ) in vitro and in vivo. Compared with single treatments, this combination significantly increased in vitro antibody-dependent cellular phagocytosis in T-ALL cell lines as well as in random de novo and relapsed/refractory T-ALL patient-derived xenograft (PDX) samples. Similarly, enhanced antibody-dependent cellular phagocytosis was observed when combining Dara with pharmacologic inhibition of CD47 interactions using a glutaminyl cyclase inhibitor. Phase 2-like preclinical in vivo trials using T-ALL PDX samples in experimental minimal residual disease-like (MRD-like) and overt leukemia models revealed a high antileukemic efficacy of CD47 blockade alone. However, T-ALL xenograft mice subjected to chemotherapy first (postchemotherapy MRD) and subsequently cotreated with Dara and Hu5F9-IgG2σ displayed significantly reduced bone marrow infiltration compared with single treatments. In relapsed and highly refractory T-ALL PDX combined treatment with Dara and Hu5F9-IgG2σ was required to substantially prolong survival compared with single treatments. These findings suggest that combining CD47 blockade with Dara is a promising therapy for T-ALL, especially for relapsed/refractory disease harboring a dismal prognosis in patients.
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Antígeno CD47 , Humanos , Camundongos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológicoRESUMO
BACKGROUND & AIMS: A large unmet therapeutic need exists in inflammatory bowel disease (IBD). Inhibition of interleukin (IL)-6 appears to be effective, but the therapeutic benefit of a complete IL6/IL6 receptor (IL6R) blockade is limited by profound immunosuppression. Evidence has emerged that chronic proinflammatory activity of IL6 is mainly mediated by trans-signaling via a complex of IL6 bound to soluble IL6R engaging the gp130 co-receptor without the need for membrane-bound IL6R. We have developed a decoy protein, sgp130Fc, that exclusively blocks IL6 proinflammatory trans-signaling and has shown efficacy in preclinical models of IBD, without signs of immunosuppression. METHODS: We present a 12-week, open-label, prospective phase 2a trial (FUTURE) in 16 patients with active IBD treated with the trans-signaling inhibitor olamkicept (sgp130Fc) to assess the molecular mechanisms, safety, and effectiveness of IL6 trans-signaling blockade in vivo. We performed in-depth molecular profiling at various timepoints before and after therapy induction to identify the mechanism of action of olamkicept. RESULTS: Olamkicept was well tolerated and induced clinical response in 44% and clinical remission in 19% of patients. Clinical effectiveness coincided with target inhibition (reduction of phosphorylated STAT3) and marked transcriptional changes in the inflamed mucosa. An olamkicept-specific transcriptional signature, distinguishable from remission signatures of anti-tumor necrosis factor (infliximab) or anti-integrin (vedolizumab) therapies was identified. CONCLUSIONS: Our data suggest that blockade of IL6 trans-signaling holds great promise for the therapy of IBD and should undergo full clinical development as a new immunoregulatory therapy for IBD. (EudraCT no., Nu 2016-000205-36).
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Colite Ulcerativa/tratamento farmacológico , Doença de Crohn/tratamento farmacológico , Interleucina-6/antagonistas & inibidores , Proteínas Recombinantes de Fusão/farmacologia , Transdução de Sinais/efeitos dos fármacos , Adulto , Idoso , Colite Ulcerativa/imunologia , Doença de Crohn/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Receptores de Interleucina-6/metabolismo , Índice de Gravidade de Doença , Resultado do Tratamento , Adulto JovemRESUMO
Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. One of the major clinical challenges is adequate diagnosis and treatment of central nervous system (CNS) involvement in this disease. Intriguingly, there is little solid evidence on the mechanisms sustaining CNS disease in ALL. Here, we present and discuss recent data on this topic, which are mainly derived from preclinical model systems. We thereby highlight sites and routes of leukemic CNS infiltration, cellular features promoting infiltration and survival of leukemic cells in a presumably hostile niche, and dormancy as a potential mechanism of survival and relapse in CNS leukemia. We also focus on the impact of ALL cytogenetic subtypes on features associated with a particular CNS tropism. Finally, we speculate on new perspectives in the treatment of ALL in the CNS, including ideas on the impact of novel immunotherapies.
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Sistema Nervoso Central/patologia , Infiltração Leucêmica/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Criança , HumanosRESUMO
BACKGROUND & AIMS: RNase H2 is a holoenzyme, composed of 3 subunits (ribonuclease H2 subunits A, B, and C), that cleaves RNA:DNA hybrids and removes mis-incorporated ribonucleotides from genomic DNA through ribonucleotide excision repair. Ribonucleotide incorporation by eukaryotic DNA polymerases occurs during every round of genome duplication and produces the most frequent type of naturally occurring DNA lesion. We investigated whether intestinal epithelial proliferation requires RNase H2 function and whether RNase H2 activity is disrupted during intestinal carcinogenesis. METHODS: We generated mice with epithelial-specific deletion of ribonuclease H2 subunit B (H2bΔIEC) and mice that also had deletion of tumor-suppressor protein p53 (H2b/p53ΔIEC); we compared phenotypes with those of littermate H2bfl/fl or H2b/p53fl/fl (control) mice at young and old ages. Intestinal tissues were collected and analyzed by histology. We isolated epithelial cells, generated intestinal organoids, and performed RNA sequence analyses. Mutation signatures of spontaneous tumors from H2b/p53ΔIEC mice were characterized by exome sequencing. We collected colorectal tumor specimens from 467 patients, measured levels of ribonuclease H2 subunit B, and associated these with patient survival times and transcriptome data. RESULTS: The H2bΔIEC mice had DNA damage to intestinal epithelial cells and proliferative exhaustion of the intestinal stem cell compartment compared with controls and H2b/p53ΔIEC mice. However, H2b/p53ΔIEC mice spontaneously developed small intestine and colon carcinomas. DNA from these tumors contained T>G base substitutions at GTG trinucleotides. Analyses of transcriptomes of human colorectal tumors associated lower levels of RNase H2 with shorter survival times. CONCLUSIONS: In analyses of mice with disruption of the ribonuclease H2 subunit B gene and colorectal tumors from patients, we provide evidence that RNase H2 functions as a colorectal tumor suppressor. H2b/p53ΔIEC mice can be used to study the roles of RNase H2 in tissue-specific carcinogenesis.
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Transformação Celular Neoplásica/metabolismo , Células Epiteliais/enzimologia , Instabilidade Genômica , Neoplasias Intestinais/prevenção & controle , Intestino Delgado/enzimologia , Ribonuclease H/metabolismo , Animais , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Colite/induzido quimicamente , Colite/enzimologia , Colite/genética , Colite/patologia , Dano ao DNA , Sulfato de Dextrana , Modelos Animais de Doenças , Células Epiteliais/patologia , Feminino , Predisposição Genética para Doença , Humanos , Neoplasias Intestinais/enzimologia , Neoplasias Intestinais/genética , Neoplasias Intestinais/patologia , Intestino Delgado/patologia , Masculino , Camundongos Knockout , Fenótipo , Ribonuclease H/deficiência , Ribonuclease H/genética , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genéticaRESUMO
Antibody therapy constitutes a major advance in the treatment of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). To evaluate the efficacy and the mechanisms of action of CD19 monoclonal antibody therapy in pediatric BCP-ALL, we tested an Fc-engineered CD19 antibody carrying the S239D/I332E mutation for improved effector cell recruitment (CD19-DE). Patient-derived xenografts (PDX) of pediatric mixed-lineage leukemia gene (MLL)-rearranged ALL were established in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Antibody CD19-DE was efficient in prolonging the survival of NSG mice in a minimal residual disease (MRD) model. The majority of surviving mice remained polymerase chain reaction (PCR)-MRD negative after treatment. When antibody therapy was initiated in overt leukemia, antibody CD19-DE was still efficient in prolonging survival of xenografted mice in comparison with nontreated control animals, but the effects were less pronounced than in the MRD setting. Importantly, the combination of antibody CD19-DE and cytoreduction by chemotherapy (dexamethasone, vincristine, PEG-asparaginase) resulted in significantly improved survival rates in xenografted mice. Antibody CD19-DE treatment was also efficient in a randomized phase 2-like PDX trial using 13 MLL-rearranged BCP-ALL samples. Macrophage depletion by liposomal clodronate resulted in a reversal of the beneficial effects of CD19-DE, suggesting an important role for macrophages as effector cells. In support of this finding, CD19-DE was found to enhance phagocytosis of patient-derived ALL blasts by human macrophages in vitro. Thus, Fc-engineered CD19 antibodies may represent a promising treatment option for infants and children with MLL-rearranged BCP-ALL who have a poor outcome when treated with chemotherapy only.
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Adolescente , Animais , Anticorpos/genética , Anticorpos/uso terapêutico , Antígenos CD19/genética , Antígenos CD19/imunologia , Feminino , Xenoenxertos , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Lactente , Macrófagos/citologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Neoplasia Residual/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Células Tumorais CultivadasRESUMO
BACKGROUND: Previous studies have established that proteinase-activated receptor 2 (PAR2) promotes migration and invasion of hepatocellular carcinoma (HCC) cells, suggesting a role in HCC progression. Here, we assessed the impact of PAR2 in HCC stromal cells on HCC growth using LX-2 hepatic stellate cells (HSCs) and Hep3B cells as model. METHODS: PAR2 expression and function in LX-2 cells was analysed by RT-PCR, confocal immunofluorescence, electron microscopy, and [Ca(2+)]i measurements, respectively. The impact of LX-2-expressed PAR2 on tumour growth in vivo was monitored using HCC xenotransplantation experiments in SCID mice, in which HCC-like tumours were induced by coinjection of LX-2 cells and Hep3B cells. To characterise the effects of PAR2 activation in LX-2 cells, various signalling pathways were analysed by immunoblotting and proteome profiler arrays. RESULTS: Following verification of functional PAR2 expression in LX-2 cells, in vivo studies showed that these cells promoted tumour growth and angiogenesis of HCC xenografts in mice. These effects were significantly reduced when F2RL1 (encoding PAR2) was downregulated by RNA interference (RNAi). In vitro studies confirmed these results demonstrating RNAi mediated inhibition of PAR2 attenuated Smad2/3 activation in response to TGF-ß1 stimulation in LX-2 cells and blocked the pro-mitotic effect of LX-2 derived conditioned medium on Hep3B cells. Furthermore, PAR2 stimulation with trypsin or a PAR2-selective activating peptide (PAR2-AP) led to activation of different intracellular signalling pathways, an increased secretion of pro-angiogenic and pro-mitotic factors and proteinases, and an enhanced migration rate across a collagen-coated membrane barrier. Silencing F2RL1 by RNAi or pharmacological inhibition of Src, hepatocyte growth factor receptor (Met), platelet-derived growth factor receptor (PDGFR), p42/p44 mitogen activated protein kinase (MAPK) or matrix-metalloproteinases (MMPs) blocked PAR2-AP-induced migration. CONCLUSION: PAR2 in HSCs plays a crucial role in promoting HCC growth presumably by mediating migration and secretion of pro-angiogenic and pro-mitotic factors. Therefore, PAR2 in stromal HSCs may have relevance as a therapeutic target of HCC.
Assuntos
Indutores da Angiogênese/metabolismo , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Receptor PAR-2/genética , Receptor PAR-2/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Células Estreladas do Fígado/citologia , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Camundongos , Camundongos SCID , Transplante de Neoplasias , Proteômica/métodos , Interferência de RNA , Transdução de SinaisAssuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasia Residual/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Adolescente , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Resultado do TratamentoRESUMO
BACKGROUND: Nuclear factor E2 related factor-2 (Nrf2) is an oxidative stress inducible transcription factor being essential in regulating cell homeostasis. Thus, acute induction of Nrf2 in epithelial cells exposed to inflammation confers protection from oxidative cell damage and mutagenesis supporting an anti-tumorigenic role for Nrf2. However, pancreatic ductal adenocarcinoma (PDAC) is characterized by persistent Nrf2 activity conferring therapy resistance which points to a pro-tumorigenic role of Nrf2. A similar dichotomous role in tumorigenesis is described for the Transforming Growth Factor-beta 1 (TGF-ß1). The present study therefore aimed at elucidating whether the switch of Nrf2 function towards a tumor promoting one relates to the modulation of TGF-ß1 induced cell responses and whether this might occur early in PDAC development. METHODS: In situ analysis comprised immunohistochemical stainings of activated (phosphorylated) Nrf2 and Ki67 in pancreatic tissues containing normal ducts and pancreatic intraepithelial neoplasia (PanINs). In vitro, Nrf2 levels in benign (H6c7-pBp), premalignant (H6c7-kras) and malignant (Colo357) pancreatic ductal epithelial cells were modulated by Nrf2 specific siRNA or Nrf2 overexpression. Then, the effect of Nrf2 alone and in combination with TGF-ß1 on cell growth and survival was investigated by cell counting, Ki67 staining and apoptosis assays. The underlying cell signaling was investigated by western blotting. Statistical analysis was performed by Shapiro-Wilk test for normal distribution. Parametric data were analyzed by one-way ANOVA, while non-parametric data were analyzed by Kruskal-Wallis one-way ANOVA on ranks. RESULTS: Significantly elevated expression of activated Nrf2 and Ki67 could be detected in PanINs but not in normal pancreatic ductal epithelium. While the effect of Nrf2 on basal cell growth of H6c7-pBp, H6c7-kras and Colo357 cells was minor, it clearly attenuated the growth inhibiting effects of TGF-ß1 in all cell lines. This enhanced Nrf2-mediated cell survival was predominantly based on an enhanced proliferative activity. Accordingly, expression of p21 expression along with expression of phospho-p38 and phospho-Smad3 was diminished whereas Erk-phosphorylation was enhanced under these conditions. CONCLUSIONS: Overall, our data demonstrate that Nrf2 being elevated in early precursor lesions counteracts the growth inhibiting function of TGF-ß1 already in benign and premalignant pancreatic ductal epithelial cells. This could represent one fundamental mechanism underlying the functional switch of both- TGF-ß1 and Nrf2 - which may manifest already in early stages of PDAC development.
Assuntos
Transformação Celular Neoplásica/metabolismo , Células Epiteliais/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Ductos Pancreáticos/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Apoptose/genética , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/genética , Células Epiteliais/patologia , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo , Ductos Pancreáticos/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Lesões Pré-Cancerosas , Ligação Proteica , Transdução de Sinais , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Neoplasias PancreáticasAssuntos
Sistema Nervoso Central/imunologia , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Infiltração Leucêmica/imunologia , Recidiva Local de Neoplasia/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Animais , Apoptose , Proliferação de Células , Sistema Nervoso Central/patologia , Criança , Humanos , Infiltração Leucêmica/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Recidiva Local de Neoplasia/imunologia , Recidiva Local de Neoplasia/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Prognóstico , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
CD19-directed immunotherapy has become a cornerstone in the therapy of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). CD19-directed cellular and antibody-based therapeutics have entered therapy of primary and relapsed disease and contributed to improved outcomes in relapsed disease and lower therapy toxicity. However, efficacy remains limited in many cases due to a lack of therapy response, short remission phases, or antigen escape. Here, BCP-ALL cell lines, patient-derived xenograft (PDX) samples, human macrophages, and an in vivo transplantation model in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice were used to examine the therapeutic potency of a CD19 antibody Fc-engineered for improved effector cell recruitment (CD19-DE) and antibody-dependent cellular phagocytosis (ADCP), in combination with a novel modified CD47 antibody (Hu5F9-IgG2σ). For the in vivo model, only samples refractory to CD19-DE monotherapy were chosen. Hu5F9-IgG2σ enhanced ADCP by CD19-DE in various BCP-ALL cell line models with varying CD19 surface expression and cytogenetic backgrounds, two of which contained the KMT2A-AFF1 fusion. Also, the antibody combination was efficient in inducing ADCP by human macrophages in pediatric PDX samples with and adult samples with and without KMT2A-rearrangement in vitro. In a randomized phase 2-like PDX trial using seven KMT2A-rearranged BCP-ALL samples in NSG mice, the CD19/CD47 antibody combination proved highly efficient. Our findings support that the efficacy of Fc-engineered CD19 antibodies may be substantially enhanced by a combination with CD47 blockade. This suggests that the combination may be a promising therapy option for BCP-ALL, especially in relapsed patients and/or patients refractory to CD19-directed therapy.
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Central nervous system (CNS) involvement remains a clinical hurdle in treating childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The disease mechanisms of CNS leukemia are primarily investigated using 2D cell culture and mouse models. Given the variations in cellular identity and architecture between the human and murine CNS, it becomes imperative to seek complementary models to study CNS leukemia. Here, we present a first-of-its-kind 3D co-culture model combining human brain organoids and BCP-ALL-cells. We noticed significantly higher engraftment of BCP-ALL cell lines and patient-derived xenograft (PDX) cells in cerebral organoids as compared to non-ALL-cells. To validate translatability between organoid co-culture and in vivo murine models, we confirmed that targeting CNS leukemia relevant pathways like CD79a/Igα or CXCR4-SDF1 reduced the invasion of BCP-ALL-cells into organoids. RNA sequencing and functional validations of organoid-invading leukemia cells compared to the non-invaded fraction revealed significant upregulation of AP-1 transcription factor-complex members in organoid-invading cells. Moreover, we detected a significant enrichment of AP-1 pathway genes in ALL-PDX-cells recovered from the CNS compared to spleen blasts of mice transplanted with TCF3::PBX1+ PDX-cells, substantiating the role of AP-1 signaling in CNS disease. Accordingly, we found significantly higher levels of the AP-1-gene JUN in patients initially diagnosed as CNS-positive compared to CNS-negative cases as well as CNS-relapse vs non-CNS-relapse cases in a cohort of 100 BCP-ALL-patients. Our results suggest CNS-organoids as a novel model to investigate CNS-involvement and identify the AP-1 pathway as a critical driver of CNS-disease in BCP-ALL.
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ABSTRACT: The t(1;19) translocation, encoding the oncogenic fusion protein E2A (TCF3)-PBX1, is involved in acute lymphoblastic leukemia (ALL) and associated with a pre-B-cell receptor (preBCR+) phenotype. Relapse in patients with E2A-PBX1+ ALL frequently occurs in the central nervous system (CNS). Therefore, there is a medical need for the identification of CNS active regimens for the treatment of E2A-PBX1+/preBCR+ ALL. Using unbiased short hairpin RNA (shRNA) library screening approaches, we identified Bruton tyrosine kinase (BTK) as a key gene involved in both proliferation and dasatinib sensitivity of E2A-PBX1+/preBCR+ ALL. Depletion of BTK by shRNAs resulted in decreased proliferation of dasatinib-treated E2A-PBX1+/preBCR+ cells compared with control-transduced cells. Moreover, the combination of dasatinib with BTK inhibitors (BTKi; ibrutinib, acalabrutinib, or zanubrutinib) significantly decreased E2A-PBX1+/preBCR+ human and murine cell proliferation, reduced phospholipase C gamma 2 (PLCG2) and BTK phosphorylation and total protein levels and increased disease-free survival of mice in secondary transplantation assays, particularly reducing CNS-leukemic infiltration. Hence, dasatinib with ibrutinib reduced pPLCG2 and pBTK in primary ALL patient samples, including E2A-PBX1+ ALLs. In summary, genetic depletion and pharmacological inhibition of BTK increase dasatinib effects in human and mouse with E2A-PBX1+/preBCR+ ALL across most of performed assays, with the combination of dasatinib and BTKi proving effective in reducing CNS infiltration of E2A-PBX1+/preBCR+ ALL cells in vivo.
Assuntos
Tirosina Quinase da Agamaglobulinemia , Dasatinibe , Inibidores de Proteínas Quinases , Dasatinibe/uso terapêutico , Dasatinibe/farmacologia , Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Tirosina Quinase da Agamaglobulinemia/metabolismo , Humanos , Animais , Camundongos , Inibidores de Proteínas Quinases/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Neoplasias do Sistema Nervoso Central/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacosRESUMO
Acute myeloid leukemia (AML) is characterized by complex molecular alterations and driver mutations. Elderly patients show increased frequencies of IDH mutations with high chemoresistance and relapse rates despite recent therapeutic advances. Besides being associated with global promoter hypermethylation, IDH1 mutation facilitated changes in 3D DNA-conformation by CTCF-anchor methylation and upregulated oncogene expression in glioma, correlating with poor prognosis. Here, we investigated the role of IDH1 p.R132H mutation in altering 3D DNA-architecture and subsequent oncogene activation in AML. Using public RNA-Seq data, we identified upregulation of tyrosine kinase PDGFRA in IDH1-mutant patients, correlating with poor prognosis. DNA methylation analysis identified CpG hypermethylation within a CTCF-anchor upstream of PDGFRA in IDH1-mutant patients. Increased PDGFRA expression, PDGFRA-CTCF methylation and decreased CTCF binding were confirmed in AML CRISPR cells with heterozygous IDH1 p.R132H mutation and upon exogenous 2-HG treatment. IDH1-mutant cells showed higher sensitivity to tyrosine kinase inhibitor dasatinib, which was supported by reduced blast count in a patient with refractory IDH1-mutant AML after dasatinib treatment. Our data illustrate that IDH1 p.R132H mutation leads to CTCF hypermethylation, disrupting DNA-looping and insulation of PDGFRA, resulting in PDGFRA upregulation in IDH1-mutant AML. Treatment with dasatinib may offer a novel treatment strategy for IDH1-mutant AML.
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Isocitrato Desidrogenase , Leucemia Mieloide Aguda , Humanos , Idoso , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Dasatinibe , Mutação , Oncogenes , Leucemia Mieloide Aguda/genética , Carcinogênese/genéticaAssuntos
Rearranjo Gênico , Genes ras , Histona-Lisina N-Metiltransferase/genética , Mutação , Proteína de Leucina Linfoide-Mieloide/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Ativação Transcricional , Tirosina Quinase 3 Semelhante a fms/genética , Biomarcadores Tumorais , Criança , Pré-Escolar , Regulação Leucêmica da Expressão Gênica , Humanos , Lactente , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidade , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , PrognósticoRESUMO
Immunotherapy has evolved as a powerful tool for the treatment of B-cell malignancies, and patient outcomes have improved by combining therapeutic antibodies with conventional chemotherapy. Overexpression of antiapoptotic B-cell lymphoma 2 (Bcl-2) is associated with a poor prognosis, and increased levels have been described in patients with "double-hit" diffuse large B-cell lymphoma, a subgroup of Burkitt's lymphoma, and patients with pediatric acute lymphoblastic leukemia harboring a t(17;19) translocation. Here, we show that the addition of venetoclax (VEN), a specific Bcl-2 inhibitor, potently enhanced the efficacy of the therapeutic anti-CD20 antibody rituximab, anti-CD38 daratumumab, and anti-CD19-DE, a proprietary version of tafasitamab. This was because of an increase in antibody-dependent cellular phagocytosis by macrophages as shown in vitro and in vivo in cell lines and patient-derived xenograft models. Mechanistically, double-hit lymphoma cells subjected to VEN triggered phagocytosis in an apoptosis-independent manner. Our study identifies the combination of VEN and therapeutic antibodies as a promising novel strategy for the treatment of B-cell malignancies.
Assuntos
Citofagocitose , Linfoma Difuso de Grandes Células B , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Criança , Humanos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2 , SulfonamidasRESUMO
B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most frequent malignancy in children and also occurs in adulthood. Despite high cure rates, BCP-ALL chemotherapy can be highly toxic. This type of toxicity can most likely be reduced by antibody-based immunotherapy targeting the CD19 antigen which is commonly expressed on BCP-ALL cells. In this study, we generated a novel Fc-engineered CD19-targeting IgG1 antibody fused to a single chain tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) domain (CD19-TRAIL). As TRAIL induces apoptosis in tumor cells but not in healthy cells, we hypothesized that CD19-TRAIL would show efficient killing of BCP-ALL cells. CD19-TRAIL showed selective binding capacity and pronounced apoptosis induction in CD19-positive (CD19+) BCP-ALL cell lines in vitro and in vivo. Additionally, CD19-TRAIL significantly prolonged survival of mice transplanted with BCP-ALL patient-derived xenograft (PDX) cells of different cytogenetic backgrounds. Moreover, simultaneous treatment with CD19-TRAIL and Venetoclax (VTX), an inhibitor of the anti-apoptotic protein BCL-2, promoted synergistic apoptosis induction in CD19+ BCP-ALL cells in vitro and prolonged survival of NSG-mice bearing the BCP-ALL cell line REH. Therefore, IgG1-based CD19-TRAIL fusion proteins represent a new potential immunotherapeutic agent against BCP-ALL.
RESUMO
High-throughput sequencing describes multiple alterations in individual tumors, but their functional relevance is often unclear. Clinic-close, individualized molecular model systems are required for functional validation and to identify therapeutic targets of high significance for each patient. Here, we establish a Cre-ERT2-loxP (causes recombination, estrogen receptor mutant T2, locus of X-over P1) based inducible RNAi- (ribonucleic acid interference) mediated gene silencing system in patient-derived xenograft (PDX) models of acute leukemias in vivo. Mimicking anti-cancer therapy in patients, gene inhibition is initiated in mice harboring orthotopic tumors. In fluorochrome guided, competitive in vivo trials, silencing of the apoptosis regulator MCL1 (myeloid cell leukemia sequence 1) correlates to pharmacological MCL1 inhibition in patients´ tumors, demonstrating the ability of the method to detect therapeutic vulnerabilities. The technique identifies a major tumor-maintaining potency of the MLL-AF4 (mixed lineage leukemia, ALL1-fused gene from chromosome 4) fusion, restricted to samples carrying the translocation. DUX4 (double homeobox 4) plays an essential role in patients' leukemias carrying the recently described DUX4-IGH (immunoglobulin heavy chain) translocation, while the downstream mediator DDIT4L (DNA-damage-inducible transcript 4 like) is identified as therapeutic vulnerability. By individualizing functional genomics in established tumors in vivo, our technique decisively complements the value chain of precision oncology. Being broadly applicable to tumors of all kinds, it will considerably reinforce personalizing anti-cancer treatment in the future.
Assuntos
Antineoplásicos/farmacologia , Biomarcadores Tumorais/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Genética Reversa/métodos , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Adulto , Animais , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/antagonistas & inibidores , Criança , Feminino , Inativação Gênica , Proteínas de Homeodomínio/antagonistas & inibidores , Proteínas de Homeodomínio/genética , Humanos , Leucemia Mieloide Aguda/genética , Masculino , Camundongos , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Leucina Linfoide-Mieloide/antagonistas & inibidores , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Proteínas de Fusão Oncogênica/genética , Medicina de Precisão/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Central nervous system (CNS) involvement remains a challenge in the diagnosis and treatment of acute lymphoblastic leukemia (ALL). In this study, we identify CD79a (also known as Igα), a signaling component of the preB cell receptor (preBCR), to be associated with CNS-infiltration and -relapse in B-cell precursor (BCP)-ALL patients. Furthermore, we show that downregulation of CD79a hampers the engraftment of leukemia cells in different murine xenograft models, particularly in the CNS.