RESUMO
Tyrosine kinase inhibitors (TKIs) targeting BCR::ABL1 have turned chronic myeloid leukaemia (CML) from a fatal disease into a manageable condition for most patients. Despite improved survival, targeting drug-resistant leukaemia stem cells (LSCs) remains a challenge for curative CML therapy. Aberrant lipid metabolism can have a large impact on membrane dynamics, cell survival and therapeutic responses in cancer. While ceramide and sphingolipid levels were previously correlated with TKI response in CML, the role of lipid metabolism in TKI resistance is not well understood. We have identified downregulation of a critical regulator of lipid metabolism, G0/G1 switch gene 2 (G0S2), in multiple scenarios of TKI resistance, including (1) BCR::ABL1 kinase-independent TKI resistance, (2) progression of CML from the chronic to the blast phase of the disease, and (3) in CML versus normal myeloid progenitors. Accordingly, CML patients with low G0S2 expression levels had a worse overall survival. G0S2 downregulation in CML was not a result of promoter hypermethylation or BCR::ABL1 kinase activity, but was rather due to transcriptional repression by MYC. Using CML cell lines, patient samples and G0s2 knockout (G0s2-/- ) mice, we demonstrate a tumour suppressor role for G0S2 in CML and TKI resistance. Our data suggest that reduced G0S2 protein expression in CML disrupts glycerophospholipid metabolism, correlating with a block of differentiation that renders CML cells resistant to therapy. Altogether, our data unravel a new role for G0S2 in regulating myeloid differentiation and TKI response in CML, and suggest that restoring G0S2 may have clinical utility.
Assuntos
Proteínas de Ciclo Celular , Resistencia a Medicamentos Antineoplásicos , Glicerofosfolipídeos , Leucemia Mielogênica Crônica BCR-ABL Positiva , Animais , Camundongos , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Fusão bcr-abl/genética , Genes de Troca , Glicerofosfolipídeos/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Inibidores de Proteínas Quinases/uso terapêutico , Humanos , Proteínas de Ciclo Celular/genéticaRESUMO
Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1 have revolutionized therapy for chronic myeloid leukemia (CML), paving the way for clinical development in other diseases. Despite success, targeting leukemic stem cells and overcoming drug resistance remain challenges for curative cancer therapy. To identify drivers of kinase-independent TKI resistance in CML, we performed genome-wide expression analyses on TKI-resistant versus sensitive CML cell lines, revealing a nuclear factor-kappa B (NF-κB) expression signature. Nucleocytoplasmic fractionation and luciferase reporter assays confirmed increased NF-κB activity in the nucleus of TKI-resistant versus sensitive CML cell lines and CD34+ patient samples. Two genes that were upregulated in TKI-resistant CML cells were proteasome 26S subunit, non-ATPases 1 (PSMD1) and 3 (PSMD3), both members of the 19S regulatory complex in the 26S proteasome. PSMD1 and PSMD3 were also identified as survival-critical genes in a published small hairpin RNA library screen of TKI resistance. We observed markedly higher levels of PSMD1 and PSMD3 mRNA in CML patients who had progressed to the blast phase compared with the chronic phase of the disease. Knockdown of PSMD1 or PSMD3 protein correlated with reduced survival and increased apoptosis in CML cells, but not in normal cord blood CD34+ progenitors. Luciferase reporter assays and immunoblot analyses demonstrated that PSMD1 and PSMD3 promote NF-κB protein expression in CML, and that signal transducer and activator of transcription 3 (STAT3) further activates NF-κB in scenarios of TKI resistance. Our data identify NF-κB as a transcriptional driver in TKI resistance, and implicate PSMD1 and PSMD3 as plausible therapeutic targets worthy of future investigation in CML and possibly other malignancies.
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , NF-kappa B/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Apoptose/fisiologia , Resistencia a Medicamentos Antineoplásicos , Xenoenxertos , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Camundongos , Camundongos Nus , NF-kappa B/genética , Complexo de Endopeptidases do Proteassoma/genética , Inibidores de Proteínas Quinases/farmacologia , Transcrição Gênica , Regulação para CimaRESUMO
WNT signaling pathway regulates several processes involved in the homeostasis of normal cells. Its dysregulation is associated with pathological outcomes like cancer. We previously demonstrated that downregulation of WNT7A correlates with higher proliferation rates in acute lymphoblastic leukemia. However, the regulation of this gene in pathological and normal conditions remains unexplored. In this work, we aimed to analyze the transcriptional regulation of WNT7A in leukemic cells and in normal T lymphocytes after a proliferative stimulus. WNT7A expression was measured in blood cells and in T lymphocytes after phytohemagglutinin-L (PHA-L) treatment or T-cell receptor (TCR) activation by qPCR and Western blot. Promoter methylation was assessed using methylation-sensitive restriction enzymes, and histone modifications were determined by chromatin immunoprecipitation and qPCR. In T-cell acute lymphoblastic leukemia (T-ALL), WNT7A expression is silenced through DNA methylation of CpG island in the promoter region. In normal peripheral blood cells, WNT7A is mainly expressed by monocytes and T lymphocytes. TCR activation induces the downregulation of WNT7A in normal T lymphocytes by changes in histone methylation marks (H3K4me2/3) and histone deacetylases. A proliferative stimulus mediated by IL-2 keeps WNT7A expression at low levels but in the absence of IL-2, the expression of this gene tends to be restored. Furthermore, after TCR activation and WNT7A downregulation, target genes associated with the WNT canonical pathway were upregulated indicating an independent activity of WNT7A from the WNT canonical pathway. WNT7A expression is silenced by long-term DNA methylation in T-ALL-derived cells and downregulated by histone modifications after TCR activation in normal T lymphocytes.
Assuntos
Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Linfócitos T/imunologia , Proteínas Wnt/metabolismo , Proliferação de Células , Metilação de DNA , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Código das Histonas , Humanos , Interleucina-2/metabolismo , Células Jurkat , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T/genética , Proteínas Wnt/genética , Via de Sinalização WntRESUMO
BACKGROUND: Dysregulations of the WNT pathway are implicated in the malignant transformation of different types of neoplasia. WNT7A is expressed in normal peripheral lymphocytes, but is decreased in the tumoral counterpart. Furthermore, the treatment of leukemic cells with recombinant WNT7A decreases proliferation, suggesting its possible use as a therapeutic biomolecule. This study aimed to evaluate the concomitant action of WNT7A and different chemotherapeutic agents over proliferation and cell death of leukemia/ lymphoma derived cell lines. METHODS: Ectopic expression of WNT7A was induced in CEM and BJAB cell lines by using a lentiviral system. RNA expression was analyzed by microarrays and qPCR, and protein expression was determined by Western Blot. Cell proliferation was measured by cell counting, metabolic activity by WST-1 assay, cell death and DNA content by flow cytometry. RESULTS: WNT7A ectopic expression was shown to decrease cell proliferation, but the apoptosis rate of leukemic cells was not altered. Moreover, these cells acquired resistance to doxorubicin, vincristine and MG-132. Cell cycle analysis reveals a decrease in G1 and an increase in S and G2 phases with a further upregulation of senescence- associated genes. Microarray analysis reveals that most gene expression changes were related to cancer and metabolic associated pathways. All those changes appear to be independent of the WNT canonical pathway regulation. CONCLUSION: WNT7A negatively regulates cell proliferation in leukemic cell lines and promotes resistance to chemotherapeutic agents by inducing a senescence-like phenotype independently of the WNT canonical pathway.