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2.
J Hematol Oncol ; 14(1): 155, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34579739

RESUMO

BACKGROUND: Acute myeloid leukaemia (AML) stem cells (LSCs) cause disease relapse. The CD47 "don't eat me signal" is upregulated on LSCs and contributes to immune evasion by inhibiting phagocytosis through interacting with myeloid-specific signal regulatory protein alpha (SIRPα). Activation of macrophages by blocking CD47 has been successful, but the ubiquitous expression of CD47 on healthy cells poses potential limitations for such therapies. In contrast, CD123 is a well-known LSC-specific surface marker utilized as a therapeutic target. Here, we report the development of SIRPα-αCD123 fusion antibodies that localize the disruption of CD47/SIRPα signalling to AML while specifically enhancing LSC clearance. METHODS: SIRPα-αCD123 antibodies were generated by fusing the extracellular domain of SIRPα to an αCD123 antibody. The binding properties of the antibodies were analysed by flow cytometry and surface plasmon resonance. The functional characteristics of the fusion antibodies were determined by antibody-dependent cellular phagocytosis and antibody-dependent cellular cytotoxicity assays using primary AML patient cells. Finally, an in vivo engraftment assay was utilized to assess LSC targeting. RESULTS: SIRPα-αCD123 fusion antibodies exhibited increased binding and preferential targeting of CD123+ CD47+ AML cells even in the presence of CD47+ healthy cells. Furthermore, SIRPα-αCD123 fusion antibodies confined disruption of the CD47-SIRPα axis locally to AML cells. In vitro experiments demonstrated that SIRPα-αCD123 antibodies greatly enhanced AML cell phagocytosis mediated by allogeneic and autologous macrophages. Moreover, SIRPα-αCD123 fusion antibodies efficiently targeted LSCs with in vivo engraftment potential. CONCLUSIONS: SIRPα-αCD123 antibodies combine local CD47 blockade with specific LSC targeting in a single molecule, minimize the risk of targeting healthy cells and efficiently eliminate AML LSCs. These results validate SIRPα-αCD123 antibodies as promising therapeutic interventions for AML.


Assuntos
Antígenos de Diferenciação/imunologia , Antineoplásicos Imunológicos/uso terapêutico , Antígeno CD47/imunologia , Subunidade alfa de Receptor de Interleucina-3/imunologia , Leucemia Mieloide Aguda/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Receptores Imunológicos/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Feminino , Humanos , Leucemia Mieloide Aguda/imunologia , Masculino , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/imunologia
3.
Nat Commun ; 12(1): 5655, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34580292

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 Xenoenxerto
4.
Blood ; 2021 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-34280257

RESUMO

Antibody-based immunotherapy is a promising strategy for targeting chemo-resistant leukemic cells. However, classical antibody-based approaches are restricted to targeting lineage-specific cell-surface antigens. By targeting intracellular antigens, a large number of other leukemia-associated targets would become accessible. In this study, we evaluated a novel T-cell bispecific (TCB) antibody, generated using CrossMab and knob-into-holes technology, containing a bivalent T-cell receptor-like binding domain that recognizes the RMFPNAPYL peptide derived from the intracellular tumor antigen Wilms' tumor 1 (WT1) in the context of human leukocyte antigen (HLA) A*02. Binding to CD3ε recruits T cells irrespective of their T-cell receptor specificity. WT1-TCB elicited antibody-mediated T-cell cytotoxicity against AML cell lines in a WT1- and HLA-restricted manner. Specific lysis of primary AML cells was mediated in ex vivo long-term co-cultures utilizing allogenic (mean specific lysis: 67±6% after 13-14 days; ±SEM; n=18) or autologous, patient-derived T cells (mean specific lysis: 54±12% after 11-14 days; ±SEM; n=8). WT1-TCB-treated T cells exhibited higher cytotoxicity against primary AML cells than an HLA-A*02 RMF-specific T-cell clone. Combining WT1-TCB with the immunomodulatory drug lenalidomide further enhanced antibody-mediated T-cell cytotoxicity against primary AML cells (mean specific lysis on day 3-4: 45.4±9.0% vs 70.8±8.3%; p=0.015; ±SEM; n=9-10). In vivo, WT1-TCB-treated humanized mice bearing SKM-1 tumors showed a significant and dose-dependent reduction in tumor growth. In summary, we show that WT1-TCB facilitates potent in vitro, ex vivo and in vivo killing of AML cell lines and primary AML cells; these results led to the initiation of a phase I trial in patients with r/r AML (NCT04580121).

5.
Nature ; 593(7860): 597-601, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33902106

RESUMO

N6-methyladenosine (m6A) is an abundant internal RNA modification1,2 that is catalysed predominantly by the METTL3-METTL14 methyltransferase complex3,4. The m6A methyltransferase METTL3 has been linked to the initiation and maintenance of acute myeloid leukaemia (AML), but the potential of therapeutic applications targeting this enzyme remains unknown5-7. Here we present the identification and characterization of STM2457, a highly potent and selective first-in-class catalytic inhibitor of METTL3, and a crystal structure of STM2457 in complex with METTL3-METTL14. Treatment of tumours with STM2457 leads to reduced AML growth and an increase in differentiation and apoptosis. These cellular effects are accompanied by selective reduction of m6A levels on known leukaemogenic mRNAs and a decrease in their expression consistent with a translational defect. We demonstrate that pharmacological inhibition of METTL3 in vivo leads to impaired engraftment and prolonged survival in various mouse models of AML, specifically targeting key stem cell subpopulations of AML. Collectively, these results reveal the inhibition of METTL3 as a potential therapeutic strategy against AML, and provide proof of concept that the targeting of RNA-modifying enzymes represents a promising avenue for anticancer therapy.

6.
Sci Rep ; 11(1): 5838, 2021 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-33712646

RESUMO

Chemotherapy resistance is the main impediment in the treatment of acute myeloid leukaemia (AML). Despite rapid advances, the various mechanisms inducing resistance development remain to be defined in detail. Here we report that loss-of-function mutations (LOF) in the histone methyltransferase EZH2 have the potential to confer resistance against the chemotherapeutic agent cytarabine. We identify seven distinct EZH2 mutations leading to loss of H3K27 trimethylation via multiple mechanisms. Analysis of matched diagnosis and relapse samples reveal a heterogenous regulation of EZH2 and a loss of EZH2 in 50% of patients. We confirm that loss of EZH2 induces resistance against cytarabine in the cell lines HEK293T and K562 as well as in a patient-derived xenograft model. Proteomics and transcriptomics analysis reveal that resistance is conferred by upregulation of multiple direct and indirect EZH2 target genes that are involved in apoptosis evasion, augmentation of proliferation and alteration of transmembrane transporter function. Our data indicate that loss of EZH2 results in upregulation of its target genes, providing the cell with a selective growth advantage, which mediates chemotherapy resistance.

8.
Blood ; 136(12): 1407-1418, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32483603

RESUMO

Anti-CD19 chimeric antigen receptor (CAR) T cells showed significant antileukemic activity in B-precursor acute lymphoblastic leukemia (ALL). Allogeneic, HLA-mismatched off-the-shelf third-party donors may offer ideal fitness of the effector cells, but carry the risk of graft-versus-host disease. Knockout (KO) of the endogenous T-cell receptor (TCR) in CD19-CAR-T cells may be a promising solution. Here, we induced a CRISPR/Cas9-mediated KO of the TCRß chain in combination with a second-generation retroviral CAR transduction including a 4-1BB costimulatory domain in primary T cells. This tandem engineering led to a highly functional population of TCR-KO-CAR-T cells with strong activation (CD25, interferon γ), proliferation, and specific killing upon CD19 target recognition. TCR-KO-CAR-T cells had a balanced phenotype of central memory and effector memory T cells. KO of the endogenous TCR in T cells strongly ablated alloreactivity in comparison with TCR-expressing T cells. In a patient-derived xenograft model of childhood ALL, TCR-KO-CAR-T cells clearly controlled CD19+ leukemia burden and improved survival in vivo. However, coexpression of endogenous TCR plus CAR led to superior persistence of T cells and significantly prolonged leukemia control in vivo, confirmed by a second in vivo model using the leukemia cell line NALM6. These results point toward an essential role of the endogenous TCR for longevity of the response at the price of alloreactivity. In conclusion, anti-CD19 CAR T cells with a CRISPR/Cas9-mediated TCR-KO are promising candidates for nonmatched third-party adoptive T-cell transfer with high antileukemic functionality in the absence of alloreactivity, but long-term persistence in vivo is better in the presence of the endogenous TCR.


Assuntos
Sistemas CRISPR-Cas , Imunoterapia Adotiva/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Receptores de Antígenos de Linfócitos T/genética , Transdução Genética , Células Tumorais Cultivadas
9.
Cancer Cell ; 38(2): 263-278.e6, 2020 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-32559496

RESUMO

Signals driving aberrant self-renewal in the heterogeneous leukemia stem cell (LSC) pool determine aggressiveness of acute myeloid leukemia (AML). We report that a positive modulator of canonical WNT signaling pathway, RSPO-LGR4, upregulates key self-renewal genes and is essential for LSC self-renewal in a subset of AML. RSPO2/3 serve as stem cell growth factors to block differentiation and promote proliferation of primary AML patient blasts. RSPO receptor, LGR4, is epigenetically upregulated and works through cooperation with HOXA9, a poor prognostic predictor. Blocking the RSPO3-LGR4 interaction by clinical-grade anti-RSPO3 antibody (OMP-131R10/rosmantuzumab) impairs self-renewal and induces differentiation in AML patient-derived xenografts but does not affect normal hematopoietic stem cells, providing a therapeutic opportunity for HOXA9-dependent leukemia.


Assuntos
Leucemia Mieloide/genética , Células-Tronco Neoplásicas/metabolismo , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais/genética , Trombospondinas/genética , Doença Aguda , Animais , Anticorpos Monoclonais/farmacologia , Linhagem Celular Tumoral , Perfilação da Expressão Gênica/métodos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Células HL-60 , Humanos , Células K562 , Estimativa de Kaplan-Meier , Leucemia Mieloide/tratamento farmacológico , Leucemia Mieloide/metabolismo , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Células-Tronco Neoplásicas/efeitos dos fármacos , Receptores Acoplados a Proteínas G/imunologia , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células THP-1 , Trombospondinas/imunologia , Trombospondinas/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
10.
J Immunother Cancer ; 8(1)2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32527933

RESUMO

BACKGROUND: Acute myeloid leukemia (AML) is a hematopoietic malignancy which is biologically, phenotypically and genetically very heterogeneous. Outcome of patients with AML remains dismal, highlighting the need for improved, less toxic therapies. Chimeric antigen receptor T-cell (CART) immunotherapies for patients with refractory or relapse (R/R) AML are challenging because of the absence of a universal pan-AML target antigen and the shared expression of target antigens with normal hematopoietic stem/progenitor cells (HSPCs), which may lead to life-threating on-target/off-tumor cytotoxicity. CD33-redirected and CD123-redirected CARTs for AML are in advanced preclinical and clinical development, and they exhibit robust antileukemic activity. However, preclinical and clinical controversy exists on whether such CARTs are myeloablative. METHODS: We set out to comparatively characterize in vitro and in vivo the efficacy and safety of 41BB-based and CD28-based CARCD123. We analyzed 97 diagnostic and relapse AML primary samples to investigate whether CD123 is a suitable immunotherapeutic target, and we used several xenograft models and in vitro assays to assess the myeloablative potential of our second-generation CD123 CARTs. RESULTS: Here, we show that CD123 represents a bona fide target for AML and show that both 41BB-based and CD28-based CD123 CARTs are very efficient in eliminating both AML cell lines and primary cells in vitro and in vivo. However, both 41BB-based and CD28-based CD123 CARTs ablate normal human hematopoiesis and prevent the establishment of de novo hematopoietic reconstitution by targeting both immature and myeloid HSPCs. CONCLUSIONS: This study calls for caution when clinically implementing CD123 CARTs, encouraging its preferential use as a bridge to allo-HSCT in patients with R/R AML.


Assuntos
Antígenos CD28/metabolismo , Engenharia Celular/métodos , Hematopoese/genética , Imunoterapia Adotiva/métodos , Subunidade alfa de Receptor de Interleucina-3/metabolismo , Linfócitos/metabolismo , Linfócitos T/metabolismo , Animais , Feminino , Humanos , Masculino , Camundongos
11.
Oncogene ; 39(15): 3195-3205, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32115572

RESUMO

ZBTB7A is frequently mutated in acute myeloid leukemia (AML) with t(8;21) translocation. However, the oncogenic collaboration between mutated ZBTB7A and the RUNX1-RUNX1T1 fusion gene in AML t(8;21) remains unclear. Here, we investigate the role of ZBTB7A and its mutations in the context of normal and malignant hematopoiesis. We demonstrate that clinically relevant ZBTB7A mutations in AML t(8;21) lead to loss of function and result in perturbed myeloid differentiation with block of the granulocytic lineage in favor of monocytic commitment. In addition, loss of ZBTB7A increases glycolysis and hence sensitizes leukemic blasts to metabolic inhibition with 2-deoxy-D-glucose. We observed that ectopic expression of wild-type ZBTB7A prevents RUNX1-RUNX1T1-mediated clonal expansion of human CD34+ cells, whereas the outgrowth of progenitors is enabled by ZBTB7A mutation. Finally, ZBTB7A expression in t(8;21) cells lead to a cell cycle arrest that could be mimicked by inhibition of glycolysis. Our findings suggest that loss of ZBTB7A may facilitate the onset of AML t(8;21), and that RUNX1-RUNX1T1-rearranged leukemia might be treated with glycolytic inhibitors.


Assuntos
Carcinogênese/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Proteínas de Ligação a DNA/genética , Hematopoese/genética , Leucemia Mieloide Aguda/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteína 1 Parceira de Translocação de RUNX1/metabolismo , Fatores de Transcrição/genética , Animais , Medula Óssea/patologia , Carcinogênese/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular Tumoral , Linhagem da Célula/genética , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Proteínas de Ligação a DNA/metabolismo , Desoxiglucose/farmacologia , Desoxiglucose/uso terapêutico , Técnicas de Inativação de Genes , Glicólise/efeitos dos fármacos , Glicólise/genética , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/patologia , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Mutação com Perda de Função , Camundongos , Células Progenitoras Mieloides/patologia , Proteínas de Fusão Oncogênica/genética , Proteína 1 Parceira de Translocação de RUNX1/genética , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Transcrição/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Leukemia ; 34(4): 1017-1026, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31740809

RESUMO

Retinoic acid-inducible gene-I (RIG-I) is a cytoplasmic immune receptor sensing viral RNA. It triggers the release of type I interferons (IFN) and proinflammatory cytokines inducing an adaptive cellular immune response. We investigated the therapeutic potential of systemic RIG-I activation by short 5'-triphosphate-modified RNA (ppp-RNA) for the treatment of acute myeloid leukemia (AML) in the syngeneic murine C1498 AML tumor model. ppp-RNA treatment significantly reduced tumor burden, delayed disease onset and led to complete remission including immunological memory formation in a substantial proportion of animals. Therapy-induced tumor rejection was dependent on CD4+ and CD8+ T cells, but not on NK or B cells, and relied on intact IFN and mitochondrial antiviral signaling protein (MAVS) signaling in the host. Interestingly, ppp-RNA treatment induced programmed death ligand 1 (PD-L1) expression on AML cells and established therapeutic sensitivity to anti-PD-1 checkpoint blockade in vivo. In immune-reconstituted humanized mice, ppp-RNA treatment reduced the number of patient-derived xenografted (PDX) AML cells in blood and bone marrow while concomitantly enhancing CD3+ T cell counts in the respective tissues. Due to its ability to establish a state of full remission and immunological memory, our findings show that ppp-RNA treatment is a promising strategy for the immunotherapy of AML.


Assuntos
Anticorpos Neutralizantes/farmacologia , Proteína DEAD-box 58/imunologia , Imunoterapia/métodos , Leucemia Mieloide Aguda/terapia , RNA de Cadeia Dupla/farmacologia , Receptores Virais/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Proteína DEAD-box 58/genética , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Regulação da Expressão Gênica , Xenoenxertos , Humanos , Memória Imunológica/efeitos dos fármacos , Interferons/genética , Interferons/imunologia , Isoenxertos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/mortalidade , Camundongos , Receptores Virais/agonistas , Receptores Virais/genética , Indução de Remissão , Transdução de Sinais , Análise de Sobrevida , Resultado do Tratamento
14.
Leukemia ; 34(1): 50-62, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31201358

RESUMO

Acute myeloid leukemia (AML) is an aggressive hematologic neoplasm resulting from the malignant transformation of myeloid progenitors. Despite intensive chemotherapy leading to initial treatment responses, relapse caused by intrinsic or acquired drug resistance represents a major challenge. Here, we report that histone 3 lysine 27 demethylase KDM6A (UTX) is targeted by inactivating mutations and mutation-independent regulation in relapsed AML. Analyses of matched diagnosis and relapse specimens from individuals with KDM6A mutations showed an outgrowth of the KDM6A mutated tumor population at relapse. KDM6A expression is heterogeneously regulated and relapse-specific loss of KDM6A was observed in 45.7% of CN-AML patients. KDM6A-null myeloid leukemia cells were more resistant to treatment with the chemotherapeutic agents cytarabine (AraC) and daunorubicin. Inducible re-expression of KDM6A in KDM6A-null cell lines suppressed proliferation and sensitized cells again to AraC treatment. RNA expression analysis and functional studies revealed that resistance to AraC was conferred by downregulation of the nucleoside membrane transporter ENT1 (SLC29A1) by reduced H3K27 acetylation at the ENT1 locus. Our results show that loss of KDM6A provides cells with a selective advantage during chemotherapy, which ultimately leads to the observed outgrowth of clones with KDM6A mutations or reduced KDM6A expression at relapse.


Assuntos
Resistencia a Medicamentos Antineoplásicos/fisiologia , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Leucemia Mieloide Aguda/patologia , Animais , Xenoenxertos , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Camundongos , Mutação
15.
Blood ; 134(3): 263-276, 2019 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-31076446

RESUMO

FLT3, DNMT3A, and NPM1 are the most frequently mutated genes in cytogenetically normal acute myeloid leukemia (AML), but little is known about how these mutations synergize upon cooccurrence. Here we show that triple-mutated AML is characterized by high leukemia stem cell (LSC) frequency, an aberrant leukemia-specific GPR56 highCD34low immunophenotype, and synergistic upregulation of Hepatic Leukemia Factor (HLF). Cell sorting based on the LSC marker GPR56 allowed isolation of triple-mutated from DNMT3A/NPM1 double-mutated subclones. Moreover, in DNMT3A R882-mutated patients, CpG hypomethylation at the HLF transcription start site correlated with high HLF mRNA expression, which was itself associated with poor survival. Loss of HLF via CRISPR/Cas9 significantly reduced the CD34+GPR56+ LSC compartment of primary human triple-mutated AML cells in serial xenotransplantation assays. HLF knockout cells were more actively cycling when freshly harvested from mice, but rapidly exhausted when reintroduced in culture. RNA sequencing of primary human triple-mutated AML cells after shRNA-mediated HLF knockdown revealed the NOTCH target Hairy and Enhancer of Split 1 (HES1) and the cyclin-dependent kinase inhibitor CDKN1C/p57 as novel targets of HLF, potentially mediating these effects. Overall, our data establish HLF as a novel LSC regulator in this genetically defined high-risk AML subgroup.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteínas Nucleares/genética , Tirosina Quinase 3 Semelhante a fms/genética , Animais , Biomarcadores , Ciclo Celular/genética , Linhagem Celular Tumoral , Biologia Computacional/métodos , Modelos Animais de Doenças , Duplicação Gênica , Perfilação da Expressão Gênica , Humanos , Imunofenotipagem , Camundongos Transgênicos , Mutação , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Sequências de Repetição em Tandem , Sítio de Iniciação de Transcrição , Transcriptoma
16.
Leukemia ; 33(6): 1400-1410, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30622285

RESUMO

Abnormal metabolism is a fundamental hallmark of cancer and represents a therapeutic opportunity, yet its regulation by oncogenes remains poorly understood. Here, we uncover that JMJD1C, a jumonji C (JmjC)-containing H3K9 demethylase, is a critical regulator of aberrant metabolic processes in homeobox A9 (HOXA9)-dependent acute myeloid leukemia (AML). JMJD1C overexpression increases in vivo cell proliferation and tumorigenicity through demethylase-independent upregulation of a glycolytic and oxidative program, which sustains leukemic cell bioenergetics and contributes to an aggressive AML phenotype in vivo. Targeting JMJD1C-mediated metabolism via pharmacologic inhibition of glycolysis and oxidative phosphorylation led to ATP depletion, induced necrosis/apoptosis and decreased tumor growth in vivo in leukemias co-expressing JMJD1C and HOXA9. The anti-metabolic therapy effectively diminished AML stem/progenitor cells and reduced tumor burden in a primary AML patient-derived xenograft. Our data establish a direct link between drug responses and endogenous expression of JMJD1C and HOXA9 in human AML cell line- and patient-derived xenografts. These findings demonstrate a previously unappreciated role for JMJD1C in counteracting adverse metabolic changes and retaining the metabolic integrity during tumorigenesis, which can be exploited therapeutically.


Assuntos
Regulação Leucêmica da Expressão Gênica , Glicólise , Proteínas de Homeodomínio/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Fosforilação Oxidativa , Oxirredutases N-Desmetilantes/metabolismo , Animais , Proteínas de Homeodomínio/genética , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Leucemia Mieloide Aguda/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Oxirredutases N-Desmetilantes/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Haematologica ; 104(3): 546-555, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30309851

RESUMO

Combination chemotherapy has proven to be a favorable strategy to treat acute leukemia. However, the introduction of novel compounds remains challenging and is hindered by a lack of understanding of their mechanistic interactions with established drugs. In the present study, we demonstrate a highly increased response of various acute leukemia cell lines, drug-resistant cells and patient-derived xenograft cells by combining the recently introduced protein disulfide isomerase inhibitor PS89 with cytostatics. In leukemic cells, a proteomics-based target fishing approach revealed that PS89 affects a whole network of endoplasmic reticulum homeostasis proteins. We elucidate that the strong induction of apoptosis in combination with cytostatics is orchestrated by the PS89 target B-cell receptor-associated protein 31, which transduces apoptosis signals at the endoplasmic reticulum -mitochondria interface. Activation of caspase-8 and cleavage of B-cell receptor-associated protein 31 stimulate a pro-apoptotic crosstalk including release of calcium from the endoplasmic reticulum and an increase in the levels of reactive oxygen species resulting in amplification of mitochondrial apoptosis. The findings of this study promote PS89 as a novel chemosensitizing agent for the treatment of acute leukemia and uncovers that targeting the endoplasmic reticulum - mitochondrial network of cell death is a promising approach in combination therapy.


Assuntos
Citostáticos/farmacologia , Retículo Endoplasmático/metabolismo , Leucemia/metabolismo , Mitocôndrias/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Caspase 8/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Leucemia/tratamento farmacológico , Leucemia/patologia , Camundongos , Modelos Biológicos , Proteoma , Proteômica/métodos , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Nat Commun ; 9(1): 5378, 2018 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-30568163

RESUMO

We recently identified the splicing kinase gene SRPK1 as a genetic vulnerability of acute myeloid leukemia (AML). Here, we show that genetic or pharmacological inhibition of SRPK1 leads to cell cycle arrest, leukemic cell differentiation and prolonged survival of mice transplanted with MLL-rearranged AML. RNA-seq analysis demonstrates that SRPK1 inhibition leads to altered isoform levels of many genes including several with established roles in leukemogenesis such as MYB, BRD4 and MED24. We focus on BRD4 as its main isoforms have distinct molecular properties and find that SRPK1 inhibition produces a significant switch from the short to the long isoform at the mRNA and protein levels. This was associated with BRD4 eviction from genomic loci involved in leukemogenesis including BCL2 and MYC. We go on to show that this switch mediates at least part of the anti-leukemic effects of SRPK1 inhibition. Our findings reveal that SRPK1 represents a plausible new therapeutic target against AML.


Assuntos
Leucemia Mieloide Aguda/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular , Diferenciação Celular , Cromatina/metabolismo , Epigênese Genética , Células HL-60 , Hematopoese , Humanos , Células K562 , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Splicing de RNA
20.
Theranostics ; 8(2): 369-383, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29290814

RESUMO

C-X-C chemokine receptor 4 (CXCR4) is a transmembrane receptor with pivotal roles in cell homing and hematopoiesis. CXCR4 is also involved in survival, proliferation and dissemination of cancer, including acute lymphoblastic and myeloid leukemia (ALL, AML). Relapsed/refractory ALL and AML are frequently resistant to conventional therapy and novel highly active strategies are urgently needed to overcome resistance. Methods: We used patient-derived (PDX) and cell line-based xenograft mouse models of ALL and AML to evaluate the efficacy and toxicity of a CXCR4-targeted endoradiotherapy (ERT) theranostic approach. Results: The positron emission tomography (PET) tracer 68Ga-Pentixafor enabled visualization of CXCR4 positive leukemic burden. In xenografts, CXCR4-directed ERT with 177Lu-Pentixather distributed to leukemia harboring organs and resulted in efficient reduction of leukemia. Despite a substantial in vivo cross-fire effect to the leukemia microenvironment, mesenchymal stem cells (MSCs) subjected to ERT were viable and capable of supporting the growth and differentiation of non-targeted normal hematopoietic cells ex vivo. Finally, three patients with refractory AML after first allogeneic hematopoietic stem cell transplantation (alloSCT) underwent CXCR4-directed ERT resulting in leukemia clearance, second alloSCT, and successful hematopoietic engraftment. Conclusion: Targeting CXCR4 with ERT is feasible and provides a highly efficient means to reduce refractory acute leukemia for subsequent cellular therapies. Prospective clinical trials testing the incorporation of CXCR4 targeting into conditioning regimens for alloSCT are highly warranted.


Assuntos
Leucemia Mieloide Aguda/tratamento farmacológico , Peptídeos/administração & dosagem , Receptores CXCR4/metabolismo , Nanomedicina Teranóstica/métodos , Idoso , Animais , Linhagem Celular Tumoral , Feminino , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Pessoa de Meia-Idade
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