RESUMO
The NFKBIE gene, which encodes the NF-κB inhibitor IκBε, is mutated in 3-7% of patients with chronic lymphocytic leukemia (CLL). The most recurrent alteration is a 4-bp frameshift deletion associated with NF-κB activation in leukemic B cells and poor clinical outcome. To study the functional consequences of NFKBIE gene inactivation, both in vitro and in vivo, we engineered CLL B cells and CLL-prone mice to stably down-regulate NFKBIE expression and investigated its role in controlling NF-κB activity and disease expansion. We found that IκBε loss leads to NF-κB pathway activation and promotes both migration and proliferation of CLL cells in a dose-dependent manner. Importantly, NFKBIE inactivation was sufficient to induce a more rapid expansion of the CLL clone in lymphoid organs and contributed to the development of an aggressive disease with a shortened survival in both xenografts and genetically modified mice. IκBε deficiency was associated with an alteration of the MAPK pathway, also confirmed by RNA-sequencing in NFKBIE-mutated patient samples, and resistance to the BTK inhibitor ibrutinib. In summary, our work underscores the multimodal relevance of the NF-κB pathway in CLL and paves the way to translate these findings into novel therapeutic options.
Assuntos
Leucemia Linfocítica Crônica de Células B , NF-kappa B , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Animais , Camundongos , Humanos , NF-kappa B/metabolismo , Proliferação de Células , Piperidinas/farmacologia , Adenina/análogos & derivados , Adenina/farmacologia , Movimento CelularRESUMO
ß-thalassemia is a disorder characterized by anemia, ineffective erythropoiesis (IE), and iron overload, whose treatment still requires improvement. The activin receptor-ligand trap Luspatercept, a novel therapeutic option for ß-thalassemia, stimulates erythroid differentiation inhibiting the transforming growth factor ß pathway. However, its exact mechanism of action and the possible connection with erythropoietin (Epo), the erythropoiesis governing cytokine, remain to be clarified. Moreover, Luspatercept does not correct all the features of the disease, calling for the identification of strategies that enhance its efficacy. Transferrin receptor 2 (TFR2) regulates systemic iron homeostasis in the liver and modulates the response to Epo of erythroid cells, thus balancing red blood cells production with iron availability. Stimulating Epo signaling, hematopoietic Tfr2 deletion ameliorates anemia and IE in Hbbth3/+ thalassemic mice. To investigate whether hematopoietic Tfr2 inactivation improves the efficacy of Luspatercept, we treated Hbbth3/+ mice with or without hematopoietic Tfr2 (Tfr2BMKO/Hbbth3/+) with RAP-536, the murine analog of Luspatercept. As expected, both hematopoietic Tfr2 deletion and RAP-536 significantly ameliorate IE and anemia, and the combined approach has an additive effect. Since RAP-536 has comparable efficacy in both Hbbth3/+ and Tfr2BMKO/Hbbth3/+ animals, we propose that the drug promotes erythroid differentiation independently of TFR2 and EPO stimulation. Notably, the lack of Tfr2, but not RAP-536, can also attenuate iron-overload and related complications. Overall, our results shed further light on the mechanism of action of Luspatercept and suggest that strategies aimed at inhibiting hematopoietic TFR2 might improve the therapeutic efficacy of activin receptor-ligand traps.
Assuntos
Receptores da Transferrina , Proteínas Recombinantes de Fusão , Talassemia beta , Animais , Talassemia beta/tratamento farmacológico , Talassemia beta/genética , Camundongos , Receptores da Transferrina/genética , Proteínas Recombinantes de Fusão/uso terapêutico , Proteínas Recombinantes de Fusão/farmacologia , Eritropoese/efeitos dos fármacos , Fragmentos Fc das Imunoglobulinas/farmacologia , Fragmentos Fc das Imunoglobulinas/uso terapêutico , Camundongos Knockout , Medula Óssea/efeitos dos fármacos , Medula Óssea/metabolismo , Eritropoetina/uso terapêutico , Eritropoetina/farmacologia , Deleção de Genes , Receptores de Activinas Tipo IIRESUMO
Microenvironmental signals strongly influence chronic lymphocytic leukemia (CLL) cells through the activation of distinct membrane receptors, such as B-cell receptors, and inflammatory receptors, such as Toll-like receptors (TLRs). Inflammatory pathways downstream of these receptors lead to NF-κB activation, thus protecting leukemic cells from apoptosis. Dimethyl fumarate (DMF) is an anti-inflammatory and immunoregulatory drug used to treat patients with multiple sclerosis and psoriasis in which it blocks aberrant NF-κB pathways and impacts the NRF2 antioxidant circuit. Our in vitro analysis demonstrated that increasing concentrations of DMF reduce ATP levels and lead to the apoptosis of CLL cells, including cell lines, splenocytes from Eµ-TCL1-transgenic mice, and primary leukemic cells isolated from the peripheral blood of patients. DMF showed a synergistic effect in association with BTK inhibitors in CLL cells. DMF reduced glutathione levels and activated the NRF2 pathway; gene expression analysis suggested that DMF downregulated pathways related to NFKB and inflammation. In primary leukemic cells, DMF disrupted the TLR signaling pathways induced by CpG by reducing the mRNA expression of NFKBIZ, IL6, IL10 and TNFα. Our data suggest that DMF targets a vulnerability of CLL cells linked to their inflammatory pathways, without impacting healthy donor peripheral blood mononuclear cells.