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1.
Blood ; 137(6): 812-825, 2021 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-32911532

RESUMO

B-cell lymphoma 6 (BCL6) is a transcription repressor and proto-oncogene that plays a crucial role in the innate and adaptive immune system and lymphoid neoplasms. However, its role in myeloid malignancies remains unclear. Here, we explored the role of BCL6 in acute myeloid leukemia (AML). BCL6 was expressed at variable and often high levels in AML cell lines and primary AML samples. AMLs with higher levels of BCL6 were generally sensitive to treatment with BCL6 inhibitors, with the exception of those with monocytic differentiation. Gene expression profiling of AML cells treated with a BCL6 inhibitor revealed induction of BCL6-repressed target genes and transcriptional programs linked to DNA damage checkpoints and downregulation of stem cell genes. Ex vivo treatment of primary AML cells with BCL6 inhibitors induced apoptosis and decreased colony-forming capacity, which correlated with the levels of BCL6 expression. Importantly, inhibition or knockdown of BCL6 in primary AML cells resulted in a significant reduction of leukemia-initiating capacity in mice, suggesting ablation of leukemia repopulating cell functionality. In contrast, BCL6 knockout or inhibition did not suppress the function of normal hematopoietic stem cells. Treatment with cytarabine further induced BCL6 expression, and the levels of BCL6 induction were correlated with resistance to cytarabine. Treatment of AML patient-derived xenografts with BCL6 inhibitor plus cytarabine suggested enhanced antileukemia activity with this combination. Hence, pharmacologic inhibition of BCL6 might provide a novel therapeutic strategy for ablation of leukemia-repopulating cells and increased responsiveness to chemotherapy.


Assuntos
Leucemia Mieloide Aguda/patologia , Proteínas de Neoplasias/fisiologia , Proteínas Proto-Oncogênicas c-bcl-6/fisiologia , Animais , Antineoplásicos/farmacologia , Apoptose , Autorrenovação Celular , Citarabina/uso terapêutico , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Técnicas de Silenciamento de Genes , Células-Tronco Hematopoéticas/citologia , Humanos , Indóis/farmacologia , Indóis/uso terapêutico , Leucemia Mieloide Aguda/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/citologia , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-bcl-6/antagonistas & inibidores , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , RNA-Seq , Quimera por Radiação , Tiazolidinedionas/farmacologia , Tiazolidinedionas/uso terapêutico , Ensaio Tumoral de Célula-Tronco , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Br J Haematol ; 190(6): 891-900, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32239670

RESUMO

Leukaemic stem cells (LSC) have been experimentally defined as the leukaemia-propagating population and are thought to be the cellular reservoir of relapse in acute myeloid leukaemia (AML). Therefore, LSC measurements are warranted to facilitate accurate risk stratification. Previously, we published the composition of a one-tube flow cytometric assay, characterised by the presence of 13 important membrane markers for LSC detection. Here we present the validation experiments of the assay in several large AML research centres, both in Europe and the United States. Variability within instruments and sample processing showed high correlations between different instruments (Rpearson  > 0·91, P < 0·001). Multi-centre testing introduced variation in reported LSC percentages but was found to be below the clinical relevant threshold. Clear gating protocols resulted in all laboratories being able to perform LSC assessment of the validation set. Participating centres were nearly unanimously able to distinguish LSChigh (>0·03% LSC) from LSClow (<0·03% LSC) despite inter-laboratory variation in reported LSC percentages. This study proves that the LSC assay is highly reproducible. These results together with the high prognostic impact of LSC load at diagnosis in AML patients render the one-tube LSC assessment a good marker for future risk classification.


Assuntos
Citometria de Fluxo , Leucemia Mieloide Aguda , Células-Tronco Neoplásicas , Adulto , Feminino , Humanos , Leucemia Mieloide Aguda/sangue , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/patologia , Masculino , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia
3.
Proc Natl Acad Sci U S A ; 111(14): 5319-24, 2014 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-24623852

RESUMO

Tyrosine kinase inhibitors (TKIs) represent transformative therapies for several malignancies. Two critical features necessary for maximizing TKI tolerability and response duration are kinase selectivity and invulnerability to resistance-conferring kinase domain (KD) mutations in the intended target. No prior TKI has demonstrated both of these properties. Aiming to maximize selectivity, medicinal chemists have largely sought to create TKIs that bind to an inactive (type II) kinase conformation. Here we demonstrate that the investigational type I TKI crenolanib is a potent inhibitor of Fms tyrosine kinase-3 (FLT3) internal tandem duplication, a validated therapeutic target in human acute myeloid leukemia (AML), as well as all secondary KD mutants previously shown to confer resistance to the first highly active FLT3 TKI quizartinib. Moreover, crenolanib is highly selective for FLT3 relative to the closely related protein tyrosine kinase KIT, demonstrating that simultaneous FLT3/KIT inhibition, a prominent feature of other clinically active FLT3 TKIs, is not required for AML cell cytotoxicity in vitro and may contribute to undesirable toxicity in patients. A saturation mutagenesis screen of FLT3-internal tandem duplication failed to recover any resistant colonies in the presence of a crenolanib concentration well below what has been safely achieved in humans, suggesting that crenolanib has the potential to suppress KD mutation-mediated clinical resistance. Crenolanib represents the first TKI to exhibit both kinase selectivity and invulnerability to resistance-conferring KD mutations, which is unexpected of a type I inhibitor. Crenolanib has significant promise for achieving deep and durable responses in FLT3-mutant AML, and may have a profound impact upon future medicinal chemistry efforts in oncology.


Assuntos
Antineoplásicos/farmacologia , Benzimidazóis/farmacologia , Piperidinas/farmacologia , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Antineoplásicos/química , Benzimidazóis/química , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Humanos , Simulação de Acoplamento Molecular , Mutação , Piperidinas/química , Tirosina Quinase 3 Semelhante a fms/química , Tirosina Quinase 3 Semelhante a fms/genética
4.
J Immunol ; 187(11): 5974-82, 2011 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-22039304

RESUMO

The c-Myb and GATA-3 transcription factors play important roles in T cell development. We recently reported that c-Myb, GATA-3, and Menin form a core transcription complex that regulates GATA-3 expression and ultimately Th2 cell development in human peripheral blood T cells. However, c-Myb roles for Th2 cytokine expression were not demonstrated. In this article, we report that c-Myb and GATA-3 cooperatively play an essential role in IL-13 expression though direct binding to a conserved GATA-3 response element (CGRE), an enhancer for IL-13 expression. c-Myb and GATA-3 were shown to activate the CGRE-IL-13 promoter by ∼160-fold, and mutation of the canonical Myb binding site completely abrogated CGRE enhancer activity. In contrast, mutation of the GATA binding site partially decreased CGRE enhancer activity. GATA-3 did not bind to CGRE when c-myb expression was silenced. c-Myb, GATA-3, Menin, and mixed lineage leukemia (MLL) bound to CGRE in human primary CD4(+) effector/memory cells. Moreover, c-myb silencing significantly decreased both methylation of histone H3K4 and acetylation of histone H3K9 at the IL-13 locus in CD4(+) effector/memory cells. Therefore, in addition to the strong enhancer effect for the transcription of IL-13, the c-Myb/GATA-3 complex recruits MLL to the CGRE for histone modification of the IL-13 locus during the differentiation of memory Th2 cells.


Assuntos
Diferenciação Celular/genética , Fator de Transcrição GATA3/genética , Regulação da Expressão Gênica/genética , Interleucina-13/genética , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas Proto-Oncogênicas c-myb/genética , Diferenciação Celular/imunologia , Separação Celular , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Fator de Transcrição GATA3/imunologia , Fator de Transcrição GATA3/metabolismo , Expressão Gênica , Regulação da Expressão Gênica/imunologia , Histonas/genética , Histonas/imunologia , Histonas/metabolismo , Humanos , Memória Imunológica/genética , Memória Imunológica/imunologia , Interleucina-13/biossíntese , Interleucina-13/imunologia , Proteína de Leucina Linfoide-Mieloide/imunologia , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas Proto-Oncogênicas c-myb/imunologia , Proteínas Proto-Oncogênicas c-myb/metabolismo , Elementos de Resposta/genética , Elementos de Resposta/imunologia , Células Th2/citologia , Células Th2/imunologia
5.
Cell Death Dis ; 14(5): 305, 2023 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-37142568

RESUMO

Autologous T cells engineered to express a chimeric antigen receptor (CAR) specific for CD19 are approved for the treatment of various CD19+ hematological malignancies. While CAR T cells induce objective responses in a majority of patients, relapse frequently occurs upon loss of CD19 expression by neoplastic cells. Radiation therapy (RT) has been successfully employed to circumvent the loss of CAR targets in preclinical models of pancreatic cancer. At least in part, this reflects the ability of RT to elicit death receptor (DR) expression by malignant cells, enabling at least some degree of CAR-independent tumor killing. In a human model of CD19+ acute lymphoblastic leukemia (ALL), we also observed DR upregulation by RT, both in vitro and in vivo. Moreover, low-dose total body irradiation (LD-TBI) delivered to ALL-bearing mice prior to CAR T cell infusion considerably extended the overall survival benefit afforded by CAR T cells alone. Such an improved therapeutic activity was accompanied by a superior expansion of CAR T cells in vivo. These data encourage the initiation of clinical trials combining LD-TBI with CAR T cells in patients with hematological malignancies.


Assuntos
Neoplasias Hematológicas , Leucemia-Linfoma Linfoblástico de Células Precursoras , Receptores de Antígenos Quiméricos , Humanos , Camundongos , Animais , Linfócitos T , Receptores de Antígenos de Linfócitos T , Leucemia-Linfoma Linfoblástico de Células Precursoras/radioterapia , Imunoterapia Adotiva
6.
Blood ; 116(8): 1280-90, 2010 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-20484083

RESUMO

GATA-3 and c-Myb are core elements of a transcriptionally active complex essential for human Th2 cell development and maintenance. We report herein mechanistic details concerning the role of these transcription factors in human peripheral blood Th2 cell development. Silencing c-Myb in normal human naive CD4(+) cells under Th2 cell-promoting conditions blocked up-regulation of GATA-3 and interleukin-4, and in effector/memory CD4(+) T cells, decreased expression of GATA-3 and Th2 cytokines. In primary T cells, c-Myb allows GATA-3 to autoactivate its own expression, an event that requires the direct interaction of c-Myb and GATA-3 on their respective binding sites in promoter of GATA-3. Immunoprecipitation revealed that the c-Myb/GATA-3 complex contained Menin and mixed lineage leukemia (MLL). MLL recruitment into the c-Myb-GATA-3-Menin complex was associated with the formation Th2 memory cells. That MLL-driven epigenetic changes were mechanistically important for this transition was suggested by the fact that silencing c-Myb significantly decreased the methylation of histone H3K4 and the acetylation of histone H3K9 at the GATA-3 locus in developing Th2 and CD4(+) effector/memory cells. Therefore, c-Myb, GATA-3, and Menin form a core transcription complex that regulates GATA-3 expression and, with the recruitment of MLL, Th2 cell maturation in primary human peripheral blood T cells.


Assuntos
Fator de Transcrição GATA3/metabolismo , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Células Th2/citologia , Transcrição Gênica , Acetilação , Western Blotting , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular , Imunoprecipitação da Cromatina , Citocinas/metabolismo , Metilação de DNA , Fator de Transcrição GATA3/genética , Histona-Lisina N-Metiltransferase , Humanos , Memória Imunológica , Células Jurkat , Luciferases/metabolismo , Proteína de Leucina Linfoide-Mieloide/genética , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Th1/citologia , Células Th1/imunologia , Células Th1/metabolismo , Células Th2/imunologia , Células Th2/metabolismo , Ativação Transcricional
7.
Methods Cell Biol ; 167: 185-201, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35152996

RESUMO

The landscape of CAR-T detection and monitoring techniques in preclinical models is rapidly evolving. In this chapter, we will discuss the most widely used methods. The chapter begins with elaborating on the rational of establishing and optimizing protocols for CAR-T monitoring and explaining why this is a crucial step in CAR-T early development. This conceptual basis will be followed by detailed protocols: polymerase chain reaction (PCR), flow cytometry and bioluminescence imaging (BLI). These in vivo methods can be implemented in labs with interest in CAR-T pre-clinical research. It will provide important tools in the process of CAR-T development and offer better understanding of their efficacy, cytotoxicity and survival.


Assuntos
Imunoterapia Adotiva , Linfócitos T , Citometria de Fluxo/métodos , Imunoterapia Adotiva/métodos , Reação em Cadeia da Polimerase/métodos
8.
Nat Commun ; 13(1): 2227, 2022 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-35484102

RESUMO

Acute myeloid leukemia (AML) is a disease with high incidence of relapse that is originated and maintained from leukemia stem cells (LSCs). Hematopoietic stem cells can be distinguished from LSCs by an array of cell surface antigens such as CD123, thus a candidate to eliminate LSCs using a variety of approaches, including CAR T cells. Here, we evaluate the potential of allogeneic gene-edited CAR T cells targeting CD123 to eliminate LSCs (UCART123). UCART123 cells are TCRαßneg T cells generated from healthy donors using TALEN® gene-editing technology, decreasing the likelihood of graft vs host disease. As safety feature, cells express RQR8 to allow elimination with Rituximab. UCART123 effectively eliminates AML cells in vitro and in vivo with significant benefits in overall survival of AML-patient derived xenograft mice. Furthermore, UCART123 preferentially target AML over normal cells with modest toxicity to normal hematopoietic stem/progenitor cells. Together these results suggest that UCART123 represents an off-the shelf therapeutic approach for AML.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Leucemia Mieloide Aguda , Animais , Humanos , Subunidade alfa de Receptor de Interleucina-3/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/terapia , Camundongos , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Linfócitos T
9.
Leuk Res ; 121: 106928, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35963025

RESUMO

PURPOSE: Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a hematologic malignancy associated with overexpression of CD123. Allogeneic chimeric antigen receptor T cells (CAR-T) directed against CD123 in BPDCN have been studied in clinical trials. We performed post-mortem analysis of a patient treated with anti-CD123 CAR-T to elucidate cause of death, development of cytokine release syndrome (CRS), and tissue distribution of UCART123 cells. METHODS: A post-mortem multidisciplinary clinicopathologic analysis was performed with digital droplet polymerase chain reaction of isolated blood and tissue ribonucleic acid (RNA) to evaluate tissue distribution of infused CAR-T. Multiparameter flow cytometry for detection of CAR-T was used for whole blood samples. Cytokine levels in plasma were measured using multiplex bead assay. Gene expression profiling on isolated RNA was performed using semi-custom Nanostring immune gene panel and RNA-sequence method. RNA in situ hybridization was performed using CAR-specific probe. RESULTS: The patient developed severe clinical CRS refractory to corticosteroids, tocilizumab, and lymphodepletion. Despite significant reduction in BPDCN lesions, the patient passed away on day 9 of CAR-T. Autopsy results show that following lymphodepletion and UCART123 administration, the patient remained severely lymphopenic with few UCART123 cells detected, predominantly localized to spleen. CONCLUSIONS: No definitive cause of death was determined, but we hypothesized that the patient may have succumbed to CAR-T-mediated cardiopulmonary toxicity. UCART123 cells displayed low overall distribution, with predominance in immune organs and tissues. Mechanism of CRS development is still poorly understood in patients receiving CAR-T therapy. Future directions in the field developing CD123-targeted agents in BPDCN are discussed.


Assuntos
Neoplasias Hematológicas , Transplante de Células-Tronco Hematopoéticas , Transtornos Mieloproliferativos , Receptores de Antígenos Quiméricos , Neoplasias Cutâneas , Doença Aguda , Citocinas/metabolismo , Células Dendríticas/patologia , Neoplasias Hematológicas/patologia , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Subunidade alfa de Receptor de Interleucina-3 , Transtornos Mieloproliferativos/patologia , RNA/metabolismo , RNA/uso terapêutico , Receptores de Antígenos Quiméricos/metabolismo , Receptores de Antígenos Quiméricos/uso terapêutico , Neoplasias Cutâneas/metabolismo
10.
Nat Commun ; 13(1): 2228, 2022 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-35484100

RESUMO

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematologic malignancy with poor outcomes with conventional therapy. Nearly 100% of BPDCNs overexpress interleukin 3 receptor subunit alpha (CD123). Given that CD123 is differentially expressed on the surface of BPDCN cells, it has emerged as an attractive therapeutic target. UCART123 is an investigational product consisting of allogeneic T cells expressing an anti-CD123 chimeric antigen receptor (CAR), edited with TALEN® nucleases. In this study, we examine the antitumor activity of UCART123 in preclinical models of BPDCN. We report that UCART123 have selective antitumor activity against CD123-positive primary BPDCN samples (while sparing normal hematopoietic progenitor cells) in the in vitro cytotoxicity and T cell degranulation assays; supported by the increased secretion of IFNγ by UCART123 cells when cultured in the presence of BPDCN cells. UCART123 eradicate BPDCN and result in long-term disease-free survival in a subset of primary patient-derived BPDCN xenograft mouse models. One potential challenge of CD123 targeting therapies is the loss of CD123 antigen through diverse genetic mechanisms, an event observed in one of three BPDCN PDX studied. In summary, these results provide a preclinical proof-of-principle that allogeneic UCART123 cells have potent anti-BPDCN activity.


Assuntos
Neoplasias Hematológicas , Transplante de Células-Tronco Hematopoéticas , Transtornos Mieloproliferativos , Neoplasias Cutâneas , Doença Aguda , Animais , Células Dendríticas/metabolismo , Neoplasias Hematológicas/tratamento farmacológico , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Subunidade alfa de Receptor de Interleucina-3/metabolismo , Camundongos , Transtornos Mieloproliferativos/metabolismo , Neoplasias Cutâneas/patologia
11.
NPJ Precis Oncol ; 5(1): 44, 2021 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-34040147

RESUMO

The epichaperome is a new cancer target composed of hyperconnected networks of chaperome members that facilitate cell survival. Cancers with an altered chaperone configuration may be susceptible to epichaperome inhibitors. We developed a flow cytometry-based assay for evaluation and monitoring of epichaperome abundance at the single cell level, with the goal of prospectively identifying patients likely to respond to epichaperome inhibitors, to measure target engagement, and dependency during treatment. As proof of principle, we describe a patient with an unclassified myeloproliferative neoplasm harboring a novel PML-SYK fusion, who progressed to acute myeloid leukemia despite chemotherapy and allogeneic stem cell transplant. The leukemia was identified as having high epichaperome abundance. We obtained compassionate access to an investigational epichaperome inhibitor, PU-H71. After 16 doses, the patient achieved durable complete remission. These encouraging results suggest that further investigation of epichaperome inhibitors in patients with abundant baseline epichaperome levels is warranted.

12.
Hematol Oncol Clin North Am ; 34(3): 553-564, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32336419

RESUMO

Hematopoiesis is a tightly regulated process that originates from highly specialized cells, hematopoietic stem cells (HSCs). Many cancers can arise and be maintained by malignant stem cells. In acute myeloid leukemia, leukemic stem cells (LSCs) are identified by their immunophenotype, which is partly shared with normal HSCs (CD34+CD38-). However, LSCs also possess unique immunophenotypic features that can be used to distinguish them from HSCs and therapeutically target them. One such unique immunophenotypic marker is CD123, found to be aberrantly expressed in leukemic stem, progenitor, and blast cells. Thus, CD123 is sought as an attractive target to eliminate LSCs.


Assuntos
Antineoplásicos/uso terapêutico , Biomarcadores Tumorais , Subunidade alfa de Receptor de Interleucina-3/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Neoplasias/etiologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos Imunológicos/farmacologia , Antineoplásicos Imunológicos/uso terapêutico , Terapia Combinada , Expressão Gênica , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Terapia de Alvo Molecular , Neoplasias/metabolismo , Resultado do Tratamento
13.
Methods Enzymol ; 639: 289-311, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32475406

RESUMO

Detection of protein connectivity dysfunctions in biological samples, i.e., informing on how protein-protein interactions change from a normal to a disease state, is important for both biomedical research and clinical development. The epichaperome is an executor of protein connectivity dysfunction in disease, and thus a surrogate for its detection. This chapter will detail on published methods for epichaperome detection and quantification that combine the advantages of multiparameter flow cytometry with those of the PU-FITC fluorescently labeled epichaperome detection probe. It will offer a comprehensive method description that includes the synthesis and characterization of an epichaperome detection probe and of the negative control probe, the preparation of the biospecimen for epichaperome analysis, the execution of the epichaperome detection and quantification assay and lastly, the data acquisition and analysis. The method provides, at single-cell level, the functional signature of cells, differentiating itself from other single-cell methods that provide a catalog of molecules.


Assuntos
Neoplasias Hematológicas , Citometria de Fluxo , Humanos
14.
Blood Adv ; 3(7): 1061-1072, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30944098

RESUMO

Activating mutations in Fms-like tyrosine kinase 3 (FLT3) occur in ∼30% of adult cases of acute myeloid leukemia (AML). Selective second- and third-generation FLT3 inhibitors have shown significant clinical activity in patients with relapsed FLT3-mutant AML. However, clearance of FLT3-mutant clones does not consistently occur, and disease will progress in most patients after an initial response. This scenario challenges the model of FLT3-mutant AML being oncogene addicted, and it suggests that redundant signaling pathways regulate AML cell survival after FLT3 inhibition. We show that primary FLT3-mutant AML cells escape apoptosis induced by FLT3 inhibition in vitro in the presence of cytokines produced normally in the bone marrow, particularly granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3). Despite reactivating canonical FLT3-signaling pathways, GM-CSF and IL-3 maintain cell survival without rescuing proliferation. Cytokine-mediated resistance through GM-CSF and IL-3 is dependent on JAK kinase, STAT5, and proviral integration site of Moloney murine leukemia virus (PIM) but not MAPK or mammalian target of rapamycin signaling. Cotreatment with FLT3 inhibitors and inhibitors of JAK or PIM kinases blocks GM-CSF and IL-3 rescue of cell survival in vitro and in vivo. Altogether, these data provide a strong rationale for combination therapy with FLT3 inhibitors to potentially improve clinical responses in AML.


Assuntos
Citocinas/fisiologia , Resistência a Medicamentos , Leucemia Mieloide Aguda/tratamento farmacológico , Terapia de Alvo Molecular , Tirosina Quinase 3 Semelhante a fms/genética , Animais , Quimioterapia Combinada/métodos , Humanos , Camundongos , Mutação , Inibidores de Proteínas Quinases/uso terapêutico , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores
15.
Cancer Discov ; 7(7): 716-735, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28416471

RESUMO

Chemotherapy-resistant human acute myeloid leukemia (AML) cells are thought to be enriched in quiescent immature leukemic stem cells (LSC). To validate this hypothesis in vivo, we developed a clinically relevant chemotherapeutic approach treating patient-derived xenografts (PDX) with cytarabine (AraC). AraC residual AML cells are enriched in neither immature, quiescent cells nor LSCs. Strikingly, AraC-resistant preexisting and persisting cells displayed high levels of reactive oxygen species, showed increased mitochondrial mass, and retained active polarized mitochondria, consistent with a high oxidative phosphorylation (OXPHOS) status. AraC residual cells exhibited increased fatty-acid oxidation, upregulated CD36 expression, and a high OXPHOS gene signature predictive for treatment response in PDX and patients with AML. High OXPHOS but not low OXPHOS human AML cell lines were chemoresistant in vivo. Targeting mitochondrial protein synthesis, electron transfer, or fatty-acid oxidation induced an energetic shift toward low OXPHOS and markedly enhanced antileukemic effects of AraC. Together, this study demonstrates that essential mitochondrial functions contribute to AraC resistance in AML and are a robust hallmark of AraC sensitivity and a promising therapeutic avenue to treat AML residual disease.Significance: AraC-resistant AML cells exhibit metabolic features and gene signatures consistent with a high OXPHOS status. In these cells, targeting mitochondrial metabolism through the CD36-FAO-OXPHOS axis induces an energetic shift toward low OXPHOS and strongly enhanced antileukemic effects of AraC, offering a promising avenue to design new therapeutic strategies and fight AraC resistance in AML. Cancer Discov; 7(7); 716-35. ©2017 AACR.See related commentary by Schimmer, p. 670This article is highlighted in the In This Issue feature, p. 653.


Assuntos
Citarabina/administração & dosagem , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Animais , Antígenos CD36/genética , Linhagem Celular Tumoral , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/genética , Citarabina/efeitos adversos , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Fosforilação Oxidativa/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
17.
J Pediatr Hematol Oncol ; 26(2): 124-7, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-14767205

RESUMO

Sixteen months after a cord blood stem cell transplantation, a 6-year-old girl was diagnosed with Epstein-Barr virus-associated posttransplant lymphoproliferative disorder (EBV-PTLD). A CT scan revealed nodules in both lungs, but the patient had neither lymphadenopathy nor other lesions. The diagnosis was confirmed by histopathologic evaluation and the increased level of EBV DNA in the peripheral blood. The patient was successfully treated with rituximab, and a complete regression of the tumors was achieved. This case reveals the need to include EBV-PTLD in the differential diagnosis of pulmonary nodules, as well as demonstrating that rituximab may be effective treatment of EBV-PTLD after cord blood stem cell transplantation.


Assuntos
Transplante de Células-Tronco de Sangue do Cordão Umbilical/efeitos adversos , Infecções por Vírus Epstein-Barr/transmissão , Herpesvirus Humano 4/isolamento & purificação , Neoplasias Pulmonares/virologia , Transtornos Linfoproliferativos/virologia , Nódulo Pulmonar Solitário/virologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Murinos , Antineoplásicos/uso terapêutico , Crise Blástica , Criança , DNA Viral/sangue , DNA Viral/genética , Infecções por Vírus Epstein-Barr/tratamento farmacológico , Feminino , Sangue Fetal/citologia , Herpesvirus Humano 4/genética , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/tratamento farmacológico , Transtornos Linfoproliferativos/diagnóstico , Transtornos Linfoproliferativos/tratamento farmacológico , Rituximab , Nódulo Pulmonar Solitário/diagnóstico , Nódulo Pulmonar Solitário/tratamento farmacológico , Tomografia Computadorizada por Raios X
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