RESUMO
Mutated nucleophosmin 1 (NPM1) is the most common genetic alteration in acute myeloid leukemia (AML), found in â¼30% of cases. Although mutations in this gene are considered favorable according to current risk stratification guidelines, a large fraction of patients will experience relapse, demonstrating the urgent need for new treatment options. Therefore, we aimed to identify cell surface proteins specifically expressed on NPM1-mutated AML cells, allowing for potential targeting with antibody-based therapies. Herein, we report on an arrayed flow cytometry-based screen directed to 362 cell surface markers. In comparing the cell surface expression on NPM1-mutated AML cells with primitive (CD34+ CD38-) normal bone marrow cells, we identified the complement receptor C3AR as being specifically expressed in NPM1-mutated AML. By flow cytometry and single-cell RNA sequencing, we further show that normal hematopoietic stem and progenitor cells lack detectable C3AR gene and protein expression, making it particularly suitable as a target for antibody therapy. We also demonstrate that C3AR in combination with GPR56 distinguishes the leukemic stem cells (LSCs) in NPM1-mutated AML from the normal hematopoietic stem cells, defining the LSC population, as shown by transplantation into immunodeficient mice. Mechanistically, the stimulation of C3AR-expressing cells with C3a, the ligand of C3AR, leads to the activation of ERK1/2 and increased survival of AML cells, suggesting that this is an important signaling axis in this subtype of AML. Finally, we show that antibodies directed against C3AR efficiently elicit natural killer cell-mediated killing of primary AML cells ex vivo, highlighting C3AR as a candidate therapeutic target in NPM1-mutated AML.
Assuntos
Leucemia Mieloide Aguda , Proteínas Nucleares , Camundongos , Animais , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleofosmina , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/metabolismo , Transdução de Sinais , Antígenos CD34 , Receptores Acoplados a Proteínas GRESUMO
Clonal heterogeneity and evolution has major implications for disease progression and relapse in acute myeloid leukemia (AML). To model clonal dynamics in vivo, we serially transplanted 23 AML cases to immunodeficient mice and followed clonal composition for up to 15 months by whole-exome sequencing of 84 xenografts across two generations. We demonstrate vast changes in clonality that both progress and reverse over time, and define five patterns of clonal dynamics: Monoclonal, Stable, Loss, Expansion and Burst. We also show that subclonal expansion in vivo correlates with a more adverse prognosis. Furthermore, clonal expansion enabled detection of very rare clones with AML driver mutations that were undetectable by sequencing at diagnosis, demonstrating that the vast majority of AML cases harbor multiple clones already at diagnosis. Finally, the rise and fall of related clones enabled deconstruction of the complex evolutionary hierarchies of the clones that compete to shape AML over time.
Assuntos
Evolução Clonal , Leucemia Mieloide Aguda/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Progressão da Doença , Feminino , Humanos , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Mutação , Sequenciamento do ExomaRESUMO
The glycosaminoglycan dermatan sulfate (DS) is a well-known activator of heparin cofactor II-dependent inactivation of thrombin. In contrast to heparin, dermatan sulfate has never been prepared recombinantly from material of non-animal origin. Here we report on the enzymatic synthesis of structurally well-defined DS with high anticoagulant activity. Using a microbial K4 polysaccharide and the recombinant enzymes DS-epimerase 1, dermatan 4-O-sulfotransferase 1, uronyl 2-O-sulfotransferase and N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase, several new glycostructures have been prepared, such as a homogenously sulfated IdoA-GalNAc-4S polymer and its 2-O-, 6-O- and 2,6-O-sulfated derivatives. Importantly, the recombinant highly 2,4-O-sulfated DS inhibits thrombin via heparin cofactor II, approximately 20 times better than heparin, enabling manipulation of vascular and extravascular coagulation. The potential of this method can be extended to preparation of specific structures that are of importance for binding and activation of cytokines, and control of inflammation and metastasis, involving extravasation and migration.