RESUMO
Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are characterized by risk of relapses, poor survival, unwanted side effects and high toxicity with the current therapies. In light of these facts, there are efforts to develop new drugs specific for deregulated molecules that participate in leukemia pathogenesis. Hematopoietic cell kinase (HCK), an Src kinase family member, is overexpressed on hematopoietic stem cells of MDS and de novo AML patients and involved in the oncogenic process. Thus, we investigated in vitro, ex vivo and in vivo effects of a novel chemical compound targeting HCK inhibition (iHCK-37), in combination with the most used drugs for the treatment of MDS and de novo AML, 5-Azacytidine and Cytarabine. Herein, the combination treatment with iHCK-37 and 5-Azacytidine or Cytarabine demonstrated additive effects against leukemia cells, compared to either drug alone. iHCK-37 plus 5-Azacytidine or Cytarabine treatment was able to reduce the activation of oncogenic pathways, MAPK/ERK and PI3K/AKT, leading to reduction of ERK and AKT phosphorylation, and increased BAX and decreased BCL-XL protein expression. Moreover, treatment with iHCK-37 reduced MDS and AML CD34-positive cell numbers inside a 3D-structure but did not affect normal CD34-positive cell numbers. In vivo analysis showed that leukemic mice treated with iHCK-37 had reduced ERK and AKT proteins phosphorylation levels and leukocyte numbers. In conclusion, the iHCK-37 inhibitor has anti-neoplastic activity in leukemia cells without altering apoptosis and survival rate of normal cells, suggesting on-target malignant cell killing activity as a single agent or in combination with 5-Azacytidine or Cytarabine.
Assuntos
Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Animais , Azacitidina/farmacologia , Azacitidina/uso terapêutico , Citarabina/farmacologia , Citarabina/uso terapêutico , Leucemia Mieloide Aguda/metabolismo , Camundongos , Síndromes Mielodisplásicas/tratamento farmacológico , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-hckRESUMO
The role of tumour microenvironment in neoplasm initiation and malignant evolution has been increasingly recognized. However, the bone marrow mesenchymal stromal cell (BMMSC) contribution to disease progression remains poorly explored. We previously reported that the expression of serine protease inhibitor kunitz-type2 (SPINT2/HAI-2), an inhibitor of hepatocyte growth factor (HGF) activation, is significantly lower in BMMSC from myelodysplastic syndromes (MDS) patients compared to healthy donors (HD). Thus, to investigate whether this loss of expression was due to SPINT2/HAI-2 methylation, BMMSC from MDS and de novo acute myeloid leukaemia (de novo AML) patients were treated with 5-Azacitidine (Aza), a DNA methyltransferase inhibitor. In MDS- and de novo AML-BMMSC, Aza treatment resulted in a pronounced SPINT2/HAI-2 levels up-regulation. Moreover, Aza treatment of HD-BMMSC did not improve SPINT2/HAI-2 levels. To understand the role of SPINT2/HAI-2 down-regulation in BMMSC physiology, SPINT2/HAI-2 expression was inhibited by lentivirus. SPINT2 underexpression resulted in an increased production of HGF by HS-5 stromal cells and improved survival of CD34+ de novo AML cells. We also observed an increased adhesion of de novo AML hematopoietic cells to SPINT2/HAI-2 silenced cells. Interestingly, BMMSC isolated from MDS and de novo AML patients had increased expression of the integrins CD49b, CD49d, and CD49e. Thus, SPINT2/HAI-2 may contribute to functional and morphological abnormalities of the microenvironment niche and to stem/progenitor cancer cell progression. Hence, down-regulation in SPINT2/HAI-2 gene expression, due to methylation in MDS-BMMSC and de novo AML-BMMSC, provides novel insights into the pathogenic role of the leukemic bone marrow microenvironment.
Assuntos
Azacitidina/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Glicoproteínas de Membrana/genética , Síndromes Mielodisplásicas/tratamento farmacológico , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Integrina alfa2/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacosRESUMO
New drug development for neoplasm treatment is nowadays based on molecular targets that participate in the disease pathogenesis and tumor phenotype. Herein, we describe a new specific pharmacological hematopoietic cell kinase (HCK) inhibitor (iHCK-37) that was able to reduce PI3K/AKT and MAPK/ERK pathways activation after erythropoietin induction in cells with high HCK expression: iHCK-37 treatment increased leukemic cells death and, very importantly, did not affect normal hematopoietic stem cells. We also present evidence that HCK, one of Src kinase family (SFK) member, regulates early-stage erythroid cell differentiation by acting as an upstream target of a frequently deregulated pathway in hematologic neoplasms, PI3K/AKT and MAPK/ERK. Notably, HCK levels were highly increased in stem cells from patients with some diseases, as Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML), that are associated with ineffective erythropoiesis These discoveries support the exploration of the new pharmacological iHCK-37 in future preclinical and clinical studies.
Assuntos
Inibidores Enzimáticos/farmacologia , Eritropoetina/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-hck/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-hck/metabolismo , Transdução de Sinais/efeitos dos fármacos , Adulto , Idoso , Morte Celular/efeitos dos fármacos , Eritropoese/efeitos dos fármacos , Feminino , Fator de Transcrição GATA1/metabolismo , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/metabolismo , Adulto JovemRESUMO
(-)-Epigallocatechin-3-gallate (EGCG), the major active polyphenol extracted from green tea, has been shown to induce apoptosis and inhibit cell proliferation, cell invasion, angiogenesis and metastasis. Herein, we evaluated the in vivo effects of EGCG in acute myeloid leukaemia (AML) using an acute promyelocytic leukaemia (APL) experimental model (PML/RARα). Haematological analysis revealed that EGCG treatment reversed leucocytosis, anaemia and thrombocytopenia, and prolonged survival of PML/RARα mice. Notably, EGCG reduced leukaemia immature cells and promyelocytes in the bone marrow while increasing mature myeloid cells, possibly due to apoptosis increase and cell differentiation. The reduction of promyelocytes and neutrophils/monocytes increase detected in the peripheral blood, in addition to the increased percentage of bone marrow cells with aggregated promyelocytic leukaemia (PML) bodies staining and decreased expression of PML-RAR oncoprotein corroborates our results. In addition, EGCG increased expression of neutrophil differentiation markers such as CD11b, CD14, CD15 and CD66 in NB4 cells; and the combination of all-trans retinoic acid (ATRA) plus EGCG yield higher increase the expression of CD15 marker. These findings could be explained by a decrease of peptidyl-prolyl isomerase NIMA-interacting 1 (PIN1) expression and reactive oxygen species (ROS) increase. EGCG also decreased expression of substrate oncoproteins for PIN1 (including cyclin D1, NF-κB p65, c-MYC, and AKT) and 67 kDa laminin receptor (67LR) in the bone marrow cells. Moreover, EGCG showed inhibition of ROS production in NB4 cells in the presence of N-acetyl-L-cysteine (NAC), as well as a partial blockage of neutrophil differentiation and apoptosis, indicating that EGCG-activities involve/or are in response of oxidative stress. Furthermore, apoptosis of spleen cells was supported by increasing expression of BAD and BAX, parallel to BCL-2 and c-MYC decrease. The reduction of spleen weights of PML/RARα mice, as well as apoptosis induced by EGCG in NB4 cells in a dose-dependent manner confirms this assumption. Our results support further evaluation of EGCG in clinical trials for AML, since EGCG could represent a promising option for AML patient ineligible for current mainstay treatments.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Catequina/análogos & derivados , Leucemia Promielocítica Aguda/tratamento farmacológico , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Apoptose/efeitos dos fármacos , Catequina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Humanos , Leucemia Experimental/tratamento farmacológico , Leucemia Experimental/mortalidade , Leucemia Experimental/patologia , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patologia , Camundongos Transgênicos , Receptor alfa de Ácido Retinoico/genética , Baço/efeitos dos fármacos , Baço/patologia , Proteína X Associada a bcl-2/metabolismo , Proteína de Morte Celular Associada a bcl/metabolismoRESUMO
ARHGAP21 is a member of the RhoGAP family of proteins involved in cell growth, differentiation, and adhesion. We have previously shown that the heterozygous Arhgap21 knockout mouse model (Arhgap21+/-) presents several alterations in the hematopoietic compartment, including increased frequency of hematopoietic stem and progenitor cells (HSPC) with impaired adhesion in vitro, increased mobilization to peripheral blood, and decreased engraftment after bone marrow transplantation. Although these HSPC functions strongly depend on their interactions with the components of the bone marrow (BM) niche, the role of ARHGAP21 in the marrow microenvironment has not yet been explored. In this study, we investigated the composition and function of the BM microenvironment in Arhgap21+/- mice. The BM of Arhgap21+/- mice presented a significant increase in the frequency of phenotypic osteoblastic lineage cells, with no differences in the frequencies of multipotent stromal cells or endothelial cells when compared to the BM of wild type mice. Arhgap21+/- BM cells had increased capacity of generating osteogenic colony-forming units (CFU-OB) in vitro and higher levels of osteocalcin were detected in the Arhgap21+/- BM supernatant. Increased expression of Col1a1, Ocn and decreased expression of Trap1 were observed after osteogenic differentiation of Arhgap21+/- BM cells. In addition, Arhgap21+/- mice recipients of normal BM cells showed decreased leucocyte numbers during transplantation recovery. Our data suggest participation of ARHGAP21 in the balanced composition of the BM microenvironment through the regulation of osteogenic differentiation.
RESUMO
NR4A3 is a key tumor suppressor in myeloid malignancy, mice lacking both NR4A1 and family member NR4A3 rapidly develop lethal acute myeloid leukemia (AML). We identified a long non-coding transcript in the NR4A3 locus and pursued the characterization of this anonymous transcript and the study of its role in leukemogenesis. We characterized this novel long non-coding transcript as a sense polyadenylated transcript. Bone marrow cells from AML patients expressed significantly reduced levels of lncNR4A3 compared to healthy controls (controls = 15, MDS= 20, p=0.05., AML= 21, p<0.01). Expression of NR4A3, as previously reported, was also significantly reduced in AML. Interestingly, the expression of both coding and non-coding transcripts was highly correlated (Pearson R = 0.3771, P<0.01). Transient over-expression of LncNR4A3 by nucleofection led to an increase in the RNA and protein level of NR4A3, reduction of proliferation in myeloid cell lines K-562 and KG1 (n=3 and 2 respectively, p<0.05) and reduced colony formation capacity in primary leukemic cells. A mass spectrometry-based quantitative proteomics approach was used to identify proteins dysregulated after lncNR4A3 over-expression in K-562. Enrichment analysis showed that the altered proteins are biologically connected (n=4, p<0.001) and functionally associated to RNA binding, transcription elongation, and splicing. Remarkably, we were able to validate the most significant results by WB. We showed that this novel transcript, lncNR4A3 regulates NR4A3 and we hypothesize this regulatory mechanism is mediated by the modulation of the RNA processing machinery.
RESUMO
Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell-based disorders characterized by ineffective hematopoiesis, increased genomic instability and a tendency to progress toward acute myeloid leukemia (AML). MDS and AML cells present genetic and epigenetic abnormalities and, due to the heterogeneity of these molecular alterations, the current treatment options remain unsatisfactory. Hypomethylating agents (HMA), especially azacitidine, are the mainstay of treatment for high-risk MDS patients and HMA are used in treating elderly AML. The aim of this study was to investigate the potential role of the epigenetic reader bromodomain-containing protein-4 (BRD4) in MDS and AML patients. We identified the upregulation of the short variant BRD4 in MDS and AML patients, which was associated with a worse outcome of MDS. Furthermore, the inhibition of BRD4 in vitro with JQ1 or shRNA induced leukemia cell apoptosis, especially when combined to azacitidine, and triggered the activation of the DNA damage response pathway. JQ1 and AZD6738 (a specific ATR inhibitor) also synergized to induce apoptosis in leukemia cells. Our results indicate that the BRD4-dependent transcriptional program is a defective pathway in MDS and AML pathogenesis and its inhibition induces apoptosis of leukemia cells, which is enhanced in combination with HMA or an ATR inhibitor.
RESUMO
CXCR4 was the first receptor identified for CXCL12, but a second receptor, CXCR7, has also been described and its function in hematopoietic cells remains unknown. By inhibition of CXCR4 and/or CXCR7, we showed that CXCR7 participates in normal CD34+ and U937 cell migration and prevents downregulation of CXCR4 by CXCL12 stimulation. In addition, CXCR7 contributes to homing of acute myeloid leukemia and normal progenitor cells to the bone marrow and spleen of NOD/SCID mice. In summary, this study shows an essential role of CXCR7, together with CXCR4, in the control of normal and malignant hematopoietic cell migration and homing induced by CXCL12.