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The functional diversity of natural killer (NK) cell repertoires stems from differentiation, homeostatic, receptor-ligand interactions and adaptive-like responses to viral infections. In the present study, we generated a single-cell transcriptional reference map of healthy human blood- and tissue-derived NK cells, with temporal resolution and fate-specific expression of gene-regulatory networks defining NK cell differentiation. Transfer learning facilitated incorporation of tumor-infiltrating NK cell transcriptomes (39 datasets, 7 solid tumors, 427 patients) into the reference map to analyze tumor microenvironment (TME)-induced perturbations. Of the six functionally distinct NK cell states identified, a dysfunctional stressed CD56bright state susceptible to TME-induced immunosuppression and a cytotoxic TME-resistant effector CD56dim state were commonly enriched across tumor types, the ratio of which was predictive of patient outcome in malignant melanoma and osteosarcoma. This resource may inform the design of new NK cell therapies and can be extended through transfer learning to interrogate new datasets from experimental perturbations or disease conditions.
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Células Matadoras Naturais , Linfócitos do Interstício Tumoral , Neoplasias , Transcriptoma , Microambiente Tumoral , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Microambiente Tumoral/imunologia , Neoplasias/imunologia , Neoplasias/genética , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Perfilação da Expressão Gênica , Análise de Célula Única , Redes Reguladoras de Genes , Antígeno CD56/metabolismo , Regulação Neoplásica da Expressão Gênica , Diferenciação CelularRESUMO
BCR::ABL1-independent pathways contribute to primary resistance to tyrosine kinase inhibitor (TKI) treatment in chronic myeloid leukemia (CML) and play a role in leukemic stem cell persistence. Here, we perform ex vivo drug screening of CML CD34+ leukemic stem/progenitor cells using 100 single drugs and TKI-drug combinations and identify sensitivities to Wee1, MDM2, and BCL2 inhibitors. These agents effectively inhibit primitive CD34+CD38- CML cells and demonstrate potent synergies when combined with TKIs. Flow-cytometry-based drug screening identifies mepacrine to induce differentiation of CD34+CD38- cells. We employ genome-wide CRISPR-Cas9 screening for six drugs, and mediator complex, apoptosis, and erythroid-lineage-related genes are identified as key resistance hits for TKIs, whereas the Wee1 inhibitor AZD1775 and mepacrine exhibit distinct resistance profiles. KCTD5, a consistent TKI-resistance-conferring gene, is found to mediate TKI-induced BCR::ABL1 ubiquitination. In summary, we delineate potential mechanisms for primary TKI resistance and non-BCR::ABL1-targeting drugs, offering insights for optimizing CML treatment.
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Proteínas de Fusão bcr-abl , Leucemia Mielogênica Crônica BCR-ABL Positiva , Inibidores de Proteínas Quinases , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Sistemas CRISPR-Cas/genética , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-abl/metabolismo , Proteínas Proto-Oncogênicas c-abl/genética , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Linhagem Celular TumoralRESUMO
Pathomechanisms that activate oncogenic B-cell receptor (BCR) signaling in diffuse large B-cell lymphoma (DLBCL), are largely unknown. Kelch-like family member 6 (KLHL6) encoding a substrate-adapter for Cullin-3-RING E3 ubiquitin-ligase (CRL) with poorly established targets is recurrently mutated in DLBCL. By applying high-throughput protein interactome screens and functional characterization, we discovered that KLHL6 regulates BCR by targeting its signaling subunits CD79A and CD79B. Loss of physiological KLHL6 expression pattern was frequent among the MCD/C5-like activated B-cell DLBCLs and was associated with higher CD79B levels and dismal outcome. Mutations in the BTB domain of KLHL6 disrupted its localization and heterodimerization, and increased surface BCR levels and signaling, whereas Kelch domain mutants had the opposite effect. Malfunctions of KLHL6 mutants extended beyond proximal BCR signaling with distinct phenotypes from KLHL6 silencing. Collectively, our findings uncover how recurrent mutations in KLHL6 alter BCR signaling and induce actionable phenotypic characteristics in DLBCL.
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Exploring non-genetic evolution of cell states during cancer treatments has become attainable by recent advances in lineage-tracing methods. However, transcriptional changes that drive cells into resistant fates may be subtle, necessitating high resolution analysis. Here, we present ReSisTrace that uses shared transcriptomic features of sister cells to predict the states priming treatment resistance. Applying ReSisTrace in ovarian cancer cells perturbed with olaparib, carboplatin or natural killer (NK) cells reveals pre-resistant phenotypes defined by proteostatic and mRNA surveillance features, reflecting traits enriched in the upcoming subclonal selection. Furthermore, we show that DNA repair deficiency renders cells susceptible to both DNA damaging agents and NK killing in a context-dependent manner. Finally, we leverage the obtained pre-resistance profiles to predict and validate small molecules driving cells to sensitive states prior to treatment. In summary, ReSisTrace resolves pre-existing transcriptional features of treatment vulnerability, facilitating both molecular patient stratification and discovery of synergistic pre-sensitizing therapies.
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Células Matadoras Naturais , Neoplasias Ovarianas , Humanos , Feminino , Neoplasias Ovarianas/genética , Carboplatina , Fenótipo , Linhagem Celular TumoralRESUMO
Immunological control of residual leukemia cells is thought to occur in patients with chronic myeloid leukemia (CML) that maintain treatment-free remission (TFR) following tyrosine kinase inhibitor (TKI) discontinuation. To study this, we analyzed 55 single-cell RNA and T cell receptor (TCR) sequenced samples (scRNA+TCRαß-seq) from patients with CML (n = 13, N = 25), other cancers (n = 28), and healthy (n = 7). The high number and active phenotype of natural killer (NK) cells in CML separated them from healthy and other cancers. Most NK cells in CML belonged to the active CD56dim cluster with high expression of GZMA/B, PRF1, CCL3/4, and IFNG, with interactions with leukemic cells via inhibitory LGALS9-TIM3 and PVR-TIGIT interactions. Accordingly, upregulation of LGALS9 was observed in CML target cells and TIM3 in NK cells when co-cultured together. Additionally, we created a classifier to identify TCRs targeting leukemia-associated antigen PR1 and quantified anti-PR1 T cells in 90 CML and 786 healthy TCRß-sequenced samples. Anti-PR1 T cells were more prevalent in CML, enriched in bone marrow samples, and enriched in the mature, cytotoxic CD8 + TEMRA cluster, especially in a patient maintaining TFR. Our results highlight the role of NK cells and anti-PR1 T cells in anti-leukemic immune responses in CML.
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Leucemia Mielogênica Crônica BCR-ABL Positiva , Humanos , Receptor Celular 2 do Vírus da Hepatite A , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Análise de Célula ÚnicaRESUMO
Cancer cells can evade natural killer (NK) cell activity, thereby limiting anti-tumor immunity. To reveal genetic determinants of susceptibility to NK cell activity, we examined interacting NK cells and blood cancer cells using single-cell and genome-scale functional genomics screens. Interaction of NK and cancer cells induced distinct activation and type I interferon (IFN) states in both cell types depending on the cancer cell lineage and molecular phenotype, ranging from more sensitive myeloid to less sensitive B-lymphoid cancers. CRISPR screens in cancer cells uncovered genes regulating sensitivity and resistance to NK cell-mediated killing, including adhesion-related glycoproteins, protein fucosylation genes, and transcriptional regulators, in addition to confirming the importance of antigen presentation and death receptor signaling pathways. CRISPR screens with a single-cell transcriptomic readout provided insight into underlying mechanisms, including regulation of IFN-γ signaling in cancer cells and NK cell activation states. Our findings highlight the diversity of mechanisms influencing NK cell susceptibility across different cancers and provide a resource for NK cell-based therapies.
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Neoplasias Hematológicas , Neoplasias , Humanos , Células Matadoras Naturais , Neoplasias/genética , Apresentação de Antígeno , Genômica , Citotoxicidade Imunológica/genética , Linhagem Celular TumoralRESUMO
BackgroundRelatlimab plus nivolumab (anti-lymphocyte-activation gene 3 plus anti-programmed death 1 [anti-LAG-3+anti-PD-1]) has been approved by the FDA as a first-line therapy for stage III/IV melanoma, but its detailed effect on the immune system is unknown.MethodsWe evaluated blood samples from 40 immunotherapy-naive or prior immunotherapy-refractory patients with metastatic melanoma treated with anti-LAG-3+anti-PD-1 in a phase I trial using single-cell RNA and T cell receptor sequencing (scRNA+TCRαß-Seq) combined with other multiomics profiling.ResultsThe highest LAG3 expression was noted in NK cells, Tregs, and CD8+ T cells, and these cell populations underwent the most significant changes during the treatment. Adaptive NK cells were enriched in responders and underwent profound transcriptomic changes during the therapy, resulting in an active phenotype. LAG3+ Tregs expanded, but based on the transcriptome profile, became metabolically silent during the treatment. Last, higher baseline TCR clonality was observed in responding patients, and their expanding CD8+ T cell clones gained a more cytotoxic and NK-like phenotype.ConclusionAnti-LAG-3+anti-PD-1 therapy has profound effects on NK cells and Tregs in addition to CD8+ T cells.Trial registrationClinicalTrials.gov (NCT01968109)FundingCancer Foundation Finland, Sigrid Juselius Foundation, Signe and Ane Gyllenberg Foundation, Relander Foundation, State funding for university-level health research in Finland, a Helsinki Institute of Life Sciences Fellow grant, Academy of Finland (grant numbers 314442, 311081, 335432, and 335436), and an investigator-initiated research grant from BMS.
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Antineoplásicos , Melanoma , Humanos , Receptor de Morte Celular Programada 1 , Melanoma/tratamento farmacológico , Melanoma/genética , Nivolumabe/uso terapêutico , Antineoplásicos/farmacologia , Linfócitos T CD8-Positivos , Receptores de Antígenos de Linfócitos T/metabolismo , Melanoma Maligno CutâneoRESUMO
The BCL-2 inhibitor venetoclax has revolutionized the treatment of acute myeloid leukemia (AML) in patients not benefiting from intensive chemotherapy. Nevertheless, treatment failure remains a challenge, and predictive markers are needed, particularly for relapsed or refractory AML. Ex vivo drug sensitivity testing may correlate with outcomes, but its prospective predictive value remains unexplored. Here we report the results of the first stage of the prospective phase II VenEx trial evaluating the utility and predictiveness of venetoclax sensitivity testing using different cell culture conditions and cell viability assays in patients receiving venetoclax-azacitidine. Participants with de novo AML ineligible for intensive chemotherapy, relapsed or refractory AML, or secondary AML were included. The primary endpoint was the treatment response in participants showing ex vivo sensitivity and the key secondary endpoints were the correlation of sensitivity with responses and survival. Venetoclax sensitivity testing was successful in 38/39 participants. Experimental conditions significantly influenced the predictive accuracy. Blast-specific venetoclax sensitivity measured in conditioned medium most accurately correlated with treatment outcomes; 88% of sensitive participants achieved a treatment response. The median survival was significantly longer for participants who were ex vivo-sensitive to venetoclax (14.6 months for venetoclax-sensitive patients vs. 3.5 for venetoclax-insensitive patients, P<0.001). This analysis illustrates the feasibility of integrating drug-response profiling into clinical practice and demonstrates excellent predictivity. This trial is registered with ClinicalTrials.gov identifier: NCT04267081.
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Antineoplásicos , Leucemia Mieloide Aguda , Humanos , Estudos Prospectivos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/tratamento farmacológico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêuticoRESUMO
Myeloid neoplasms with erythroid or megakaryocytic differentiation include pure erythroid leukemia, myelodysplastic syndrome with erythroid features, and acute megakaryoblastic leukemia (FAB M7) and are characterized by poor prognosis and limited treatment options. Here, we investigate the drug sensitivity landscape of these rare malignancies. We show that acute myeloid leukemia (AML) cells with erythroid or megakaryocytic differentiation depend on the antiapoptotic protein B-cell lymphoma (BCL)-XL, rather than BCL-2, using combined ex vivo drug sensitivity testing, genetic perturbation, and transcriptomic profiling. High-throughput screening of >500 compounds identified the BCL-XL-selective inhibitor A-1331852 and navitoclax as highly effective against erythroid/megakaryoblastic leukemia cell lines. In contrast, these AML subtypes were resistant to the BCL-2 inhibitor venetoclax, which is used clinically in the treatment of AML. Consistently, genome-scale CRISPR-Cas9 and RNAi screening data demonstrated the striking essentiality of BCL-XL-encoding BCL2L1 but not BCL2 or MCL1, for the survival of erythroid/megakaryoblastic leukemia cell lines. Single-cell and bulk transcriptomics of patient samples with erythroid and megakaryoblastic leukemias identified high BCL2L1 expression compared with other subtypes of AML and other hematological malignancies, where BCL2 and MCL1 were more prominent. BCL-XL inhibition effectively killed blasts in samples from patients with AML with erythroid or megakaryocytic differentiation ex vivo and reduced tumor burden in a mouse erythroleukemia xenograft model. Combining the BCL-XL inhibitor with the JAK inhibitor ruxolitinib showed synergistic and durable responses in cell lines. Our results suggest targeting BCL-XL as a potential therapy option in erythroid/megakaryoblastic leukemias and highlight an AML subgroup with potentially reduced sensitivity to venetoclax-based treatments.
Assuntos
Leucemia Megacarioblástica Aguda , Leucemia Mieloide Aguda , Linfoma de Células B , Animais , Camundongos , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Linhagem Celular Tumoral , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Proteína bcl-X/genética , Leucemia Megacarioblástica Aguda/tratamento farmacológico , Leucemia Megacarioblástica Aguda/genética , Diferenciação Celular , ApoptoseRESUMO
In chronic myeloid leukemia (CML), combination therapies with tyrosine kinase inhibitors (TKIs) aim to improve the achievement of deep molecular remission that would allow therapy discontinuation. IFN-α is one promising candidate, as it has long-lasting effects on both malignant and immune cells. In connection with a multicenter clinical trial combining dasatinib with IFN-α in 40 patients with chronic-phase CML (NordCML007, NCT01725204), we performed immune monitoring with single-cell RNA and T cell receptor (TCR) sequencing (n = 4, 12 samples), bulk TCRß sequencing (n = 13, 26 samples), flow cytometry (n = 40, 106 samples), cytokine analyses (n = 17, 80 samples), and ex vivo functional studies (n = 39, 80 samples). Dasatinib drove the immune repertoire toward terminally differentiated NK and CD8+ T cells with dampened functional capabilities. Patients with dasatinib-associated pleural effusions had increased numbers of CD8+ recently activated effector memory T (Temra) cells. In vitro, dasatinib prevented CD3-induced cell death by blocking TCR signaling. The addition of IFN-α reversed the terminally differentiated phenotypes and increased the number of costimulatory intercellular interactions and the number of unique putative epitope-specific TCR clusters. In vitro IFN-α had costimulatory effects on TCR signaling. Our work supports the combination of IFN-α with TKI therapy, as IFN-α broadens the immune repertoire and restores immunological function.
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva , Linfócitos T CD8-Positivos , Citocinas/metabolismo , Dasatinibe/farmacologia , Dasatinibe/uso terapêutico , Humanos , Interferon-alfa , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
In adult patients, the treatment outcome of acute lymphoblastic leukemia (ALL) remains suboptimal. Here, we used an ex vivo drug testing platform and comprehensive molecular profiling to discover new drug candidates for B-ALL. We analyzed sensitivity of 18 primary B-ALL adult patient samples to 64 drugs in a physiological concentration range. Whole-transcriptome sequencing and publicly available expression data were used to examine gene expression biomarkers for observed drug responses. Apoptotic modulators targeting BCL2 and MDM2 were highly effective. Philadelphia chromosome-negative (Ph-) samples were sensitive to both BCL2/BCL-W/BCL-XL-targeting agent navitoclax and BCL2-selective venetoclax, whereas Ph-positive (Ph+) samples were more sensitive to navitoclax. Expression of BCL2 was downregulated and BCL-W and BCL-XL upregulated in Ph+ ALL compared with Ph- samples, providing elucidation for the observed difference in drug responses. A majority of the samples were sensitive to MDM2 inhibitor idasanutlin. The regulatory protein MDM2 suppresses the function of tumor suppressor p53, leading to impaired apoptosis. In B-ALL, the expression of MDM2 was increased compared with other hematological malignancies. In B-ALL cell lines, a combination of BCL2 and MDM2 inhibitor was synergistic. In summary, antiapoptotic proteins including BCL2 and MDM2 comprise promising targets for future drug studies in B-ALL.
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We generated ex vivo drug-response and multiomics profiling data for a prospective series of 252 samples from 186 patients with acute myeloid leukemia (AML). A functional precision medicine tumor board (FPMTB) integrated clinical, molecular, and functional data for application in clinical treatment decisions. Actionable drugs were found for 97% of patients with AML, and the recommendations were clinically implemented in 37 relapsed or refractory patients. We report a 59% objective response rate for the individually tailored therapies, including 13 complete responses, as well as bridging five patients with AML to allogeneic hematopoietic stem cell transplantation. Data integration across all cases enabled the identification of drug response biomarkers, such as the association of IL15 overexpression with resistance to FLT3 inhibitors. Integration of molecular profiling and large-scale drug response data across many patients will enable continuous improvement of the FPMTB recommendations, providing a paradigm for individualized implementation of functional precision cancer medicine. SIGNIFICANCE: Oncogenomics data can guide clinical treatment decisions, but often such data are neither actionable nor predictive. Functional ex vivo drug testing contributes significant additional, clinically actionable therapeutic insights for individual patients with AML. Such data can be generated in four days, enabling rapid translation through FPMTB.See related commentary by Letai, p. 290.This article is highlighted in the In This Issue feature, p. 275.
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Técnicas de Apoio para a Decisão , Leucemia Mieloide Aguda/tratamento farmacológico , Equipe de Assistência ao Paciente , Medicina de Precisão , Feminino , Finlândia , Humanos , Leucemia Mieloide Aguda/mortalidade , Masculino , Pessoa de Meia-Idade , Indução de Remissão , Análise de SobrevidaRESUMO
To systematically define molecular features in human tumor cells that determine their degree of sensitivity to human allogeneic natural killer (NK) cells, we quantified the NK cell responsiveness of hundreds of molecularly annotated 'DNA-barcoded' solid tumor cell lines in multiplexed format and applied genome-scale CRISPR-based gene-editing screens in several solid tumor cell lines, to functionally interrogate which genes in tumor cells regulate the response to NK cells. In these orthogonal studies, NK cell-sensitive tumor cells tend to exhibit 'mesenchymal-like' transcriptional programs; high transcriptional signature for chromatin remodeling complexes; high levels of B7-H6 (NCR3LG1); and low levels of HLA-E/antigen presentation genes. Importantly, transcriptional signatures of NK cell-sensitive tumor cells correlate with immune checkpoint inhibitor (ICI) resistance in clinical samples. This study provides a comprehensive map of mechanisms regulating tumor cell responses to NK cells, with implications for future biomarker-driven applications of NK cell immunotherapies.
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Citotoxicidade Imunológica/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Inibidores de Checkpoint Imunológico/farmacologia , Células Matadoras Naturais/fisiologia , Células Alógenas/fisiologia , Animais , Antígenos B7/genética , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina/fisiologia , Testes Imunológicos de Citotoxicidade/métodos , Citotoxicidade Imunológica/fisiologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Genoma Humano , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Camundongos Endogâmicos NOD , Ensaios Antitumorais Modelo de Xenoenxerto , Antígenos HLA-ERESUMO
The KIT D816V mutation is found in >80% of patients with systemic mastocytosis (SM) and is key to neoplastic mast cell (MC) expansion and accumulation in affected organs. Therefore, KIT D816V represents a prime therapeutic target for SM. Here, we generated a panel of patient-specific KIT D816V induced pluripotent stem cells (iPSCs) from patients with aggressive SM and mast cell leukemia to develop a patient-specific SM disease model for mechanistic and drug-discovery studies. KIT D816V iPSCs differentiated into neoplastic hematopoietic progenitor cells and MCs with patient-specific phenotypic features, thereby reflecting the heterogeneity of the disease. CRISPR/Cas9n-engineered KIT D816V human embryonic stem cells (ESCs), when differentiated into hematopoietic cells, recapitulated the phenotype observed for KIT D816V iPSC hematopoiesis. KIT D816V causes constitutive activation of the KIT tyrosine kinase receptor, and we exploited our iPSCs and ESCs to investigate new tyrosine kinase inhibitors targeting KIT D816V. Our study identified nintedanib, a US Food and Drug Administration-approved angiokinase inhibitor that targets vascular endothelial growth factor receptor, platelet-derived growth factor receptor, and fibroblast growth factor receptor, as a novel KIT D816V inhibitor. Nintedanib selectively reduced the viability of iPSC-derived KIT D816V hematopoietic progenitor cells and MCs in the nanomolar range. Nintedanib was also active on primary samples of KIT D816V SM patients. Molecular docking studies show that nintedanib binds to the adenosine triphosphate binding pocket of inactive KIT D816V. Our results suggest nintedanib as a new drug candidate for KIT D816V-targeted therapy of advanced SM.
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Antineoplásicos/farmacologia , Indóis/farmacologia , Mastocitose Sistêmica/tratamento farmacológico , Mutação Puntual/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-kit/genética , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Mastocitose Sistêmica/genética , Mastocitose Sistêmica/patologia , Células Tumorais CultivadasRESUMO
Blast-phase chronic myeloid leukemia (BP-CML) is associated with additional chromosomal aberrations, RUNX1 mutations being one of the most common. Tyrosine kinase inhibitor therapy has only limited efficacy in BP-CML, and characterization of more defined molecular subtypes is warranted in order to design better treatment modalities for this poor prognosis patient group. Using whole-exome and RNA sequencing we demonstrate that PHF6 and BCORL1 mutations, IKZF1 deletions, and AID/RAG-mediated rearrangements are enriched in RUNX1mut BP-CML leading to typical mutational signature. On transcriptional level interferon and TNF signaling were deregulated in primary RUNX1mut CML cells and stem cell and B-lymphoid factors upregulated giving a rise to distinct phenotype. This was accompanied with the sensitivity of RUNX1mut blasts to CD19-CAR T cells in ex vivo assays. High-throughput drug sensitivity and resistance testing revealed leukemia cells from RUNX1mut patients to be highly responsive for mTOR-, BCL2-, and VEGFR inhibitors and glucocorticoids. These findings were further investigated and confirmed in CRISPR/Cas9-edited homozygous RUNX1-/- and heterozygous RUNX1-/mut BCR-ABL positive cell lines. Overall, our study provides insights into the pathogenic role of RUNX1 mutations and highlights personalized targeted therapy and CAR T-cell immunotherapy as potentially promising strategies for treating RUNX1mut BP-CML patients.
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Crise Blástica/genética , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Mutação , Transcriptoma , Sítios de Ligação , Biomarcadores Tumorais , Crise Blástica/diagnóstico , Crise Blástica/tratamento farmacológico , Linhagem Celular Tumoral , Terapia Combinada , Gerenciamento Clínico , Suscetibilidade a Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Citometria de Fluxo , Deleção de Genes , Edição de Genes , Humanos , Fator de Transcrição Ikaros/genética , Imunofenotipagem , Leucemia Mielogênica Crônica BCR-ABL Positiva/diagnóstico , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Terapia de Alvo Molecular , Fenótipo , Ligação Proteica , Transdução de Sinais , Sequenciamento do ExomaRESUMO
Understanding factors that shape the immune landscape across hematological malignancies is essential for immunotherapy development. We integrated over 8,000 transcriptomes and 2,000 samples with multilevel genomics of hematological cancers to investigate how immunological features are linked to cancer subtypes, genetic and epigenetic alterations, and patient survival, and validated key findings experimentally. Infiltration of cytotoxic lymphocytes was associated with TP53 and myelodysplasia-related changes in acute myeloid leukemia, and activated B cell-like phenotype and interferon-γ response in lymphoma. CIITA methylation regulating antigen presentation, cancer type-specific immune checkpoints, such as VISTA in myeloid malignancies, and variation in cancer antigen expression further contributed to immune heterogeneity and predicted survival. Our study provides a resource linking immunology with cancer subtypes and genomics in hematological malignancies.
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Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Leucemia Mieloide/genética , Linfoma Difuso de Grandes Células B/genética , Doença Aguda , Epigênese Genética , Genômica/métodos , Antígenos HLA/genética , Humanos , Imunoterapia/métodos , Leucemia Mieloide/imunologia , Leucemia Mieloide/terapia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Linfoma Difuso de Grandes Células B/imunologia , Linfoma Difuso de Grandes Células B/terapia , Mieloma Múltiplo/genética , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/terapia , Mutação , Proteína Supressora de Tumor p53/genéticaRESUMO
The immunologic microenvironment in various solid tumors is aberrant and correlates with clinical survival. Here, we present a comprehensive analysis of the immune environment of acute myeloid leukemia (AML) bone marrow (BM) at diagnosis. We compared the immunologic landscape of formalin-fixed paraffin-embedded BM trephine samples from AML (n = 69), chronic myeloid leukemia (CML; n = 56), and B-cell acute lymphoblastic leukemia (B-ALL) patients (n = 52) at diagnosis to controls (n = 12) with 30 immunophenotype markers using multiplex immunohistochemistry and computerized image analysis. We identified distinct immunologic profiles specific for leukemia subtypes and controls enabling accurate classification of AML (area under the curve [AUC] = 1.0), CML (AUC = 0.99), B-ALL (AUC = 0.96), and control subjects (AUC = 1.0). Interestingly, 2 major immunologic AML clusters differing in age, T-cell receptor clonality, and survival were discovered. A low proportion of regulatory T cells and pSTAT1+cMAF- monocytes were identified as novel biomarkers of superior event-free survival in intensively treated AML patients. Moreover, we demonstrated that AML BM and peripheral blood samples are dissimilar in terms of immune cell phenotypes. To conclude, our study shows that the immunologic landscape considerably varies by leukemia subtype suggesting disease-specific immunoregulation. Furthermore, the association of the AML immune microenvironment with clinical parameters suggests a rationale for including immunologic parameters to improve disease classification or even patient risk stratification.
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Medula Óssea/metabolismo , Leucemia Mieloide Aguda/imunologia , Receptores de Antígenos de Linfócitos T/genética , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sobrevida , Adulto JovemRESUMO
Chimeric antigen receptor (CAR) T-cell therapy has proven effective in relapsed and refractory B-cell malignancies, but resistance and relapses still occur. Better understanding of mechanisms influencing CAR T-cell cytotoxicity and the potential for modulation using small-molecule drugs could improve current immunotherapies. Here, we systematically investigated druggable mechanisms of CAR T-cell cytotoxicity using >500 small-molecule drugs and genome-scale CRISPR-Cas9 loss-of-function screens. We identified several tyrosine kinase inhibitors that inhibit CAR T-cell cytotoxicity by impairing T-cell signaling transcriptional activity. In contrast, the apoptotic modulator drugs SMAC mimetics sensitized B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphoma cells to anti-CD19 CAR T cells. CRISPR screens identified death receptor signaling through FADD and TNFRSF10B (TRAIL-R2) as a key mediator of CAR T-cell cytotoxicity and elucidated the RIPK1-dependent mechanism of sensitization by SMAC mimetics. Death receptor expression varied across genetic subtypes of B-cell malignancies, suggesting a link between mechanisms of CAR T-cell cytotoxicity and cancer genetics. These results implicate death receptor signaling as an important mediator of cancer cell sensitivity to CAR T-cell cytotoxicity, with potential for pharmacological targeting to enhance cancer immunotherapy. The screening data provide a resource of immunomodulatory properties of cancer drugs and genetic mechanisms influencing CAR T-cell cytotoxicity.