RESUMO
Bendamustine is used in the treatment of chronic lymphocytic leukemia (CLL). Routes for bendamustine entry into target cells are unknown. This study aimed at identifying transporter proteins implicated in bendamustine uptake. Our results showed that hOCT1 is a bendamustine transporter, as bendamustine could cis-inhibit the uptake of a canonical hOCT1 substrate, with a Ki in the micromolar range, consistent with the EC50 values of the cytotoxicity triggered by this drug in HEK293 cells expressing hOCT1. hOCT1 polymorphic variants determining impaired bendamustine-transporter interaction, consistently reduced bendamustine cytotoxicity in HEK293 cells stably expressing them. Exome genotyping of the SLC22A1 gene, encoding hOCT1, was undertaken in a cohort of 241 CLL patients. Ex vivo cytotoxicity to bendamustine was measured in a subset of cases and shown to correlate with SLC22A1 polymorphic variants. In conclusion, hOCT1 is a suitable bendamustine transporter, thereby contributing to its cytotoxic effect depending upon the hOCT1 genetic variants expressed.
Assuntos
Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Cloridrato de Bendamustina/metabolismo , Cloridrato de Bendamustina/farmacologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Transportador 1 de Cátions Orgânicos/genética , Transportador 1 de Cátions Orgânicos/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/farmacocinética , Cloridrato de Bendamustina/farmacocinética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Estudos de Coortes , DNA Complementar/genética , Transportador Equilibrativo 1 de Nucleosídeo/genética , Exoma/genética , Feminino , Genótipo , Células HEK293 , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Proteínas de Membrana Transportadoras/genética , Pessoa de Meia-Idade , Transportadores de Ânions Orgânicos , Proteínas de Transporte de Cátions Orgânicos , Polimorfismo Genético/genéticaRESUMO
Targeting Notch signaling has emerged as a promising therapeutic strategy for chronic lymphocytic leukemia (CLL), especially for the poor prognostic subgroup of NOTCH1-mutated patients. Here, we report that the γ-secretase inhibitor PF-03084014 inhibits the constitutive Notch activation and induces selective apoptosis in CLL cells carrying NOTCH1 mutations. Combination of PF-03084014 with fludarabine has a synergistic antileukemic effect in primary NOTCH1-mutated CLL cells, even in the presence of the protective stroma. At transcriptional level, PF-03084014 plus fludarabine treatment induces the upregulation of the proapoptotic gene HRK and the downmodulation of MMP9, IL32 and RAC2 genes that are related to invasion and chemotaxis. PF-03084014 also overcomes fludarabine-mediated activation of nuclear factor-κB signaling. Moreover, this combination impairs angiogenesis and CXCL12-induced responses in NOTCH1-mutated CLL cells, in particular those related to tumoral migration and invasion. Importantly, all these collaborative effects are specific for NOTCH1 mutation and do not occur in unmutated cases. In conclusion, we provide evidence that Notch is a therapeutic target in CLL cases with NOTCH1-activating mutations, supporting the use of Notch pathway inhibitors in combination with chemotherapy as a promising approach for the treatment of these high-risk CLL patients.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Leucemia Linfocítica Crônica de Células B/patologia , Invasividade Neoplásica/prevenção & controle , Metástase Neoplásica/prevenção & controle , Neovascularização Patológica/prevenção & controle , Receptor Notch1/genética , Tetra-Hidronaftalenos/farmacologia , Valina/análogos & derivados , Vidarabina/análogos & derivados , Idoso , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Masculino , Pessoa de Meia-Idade , Mutação , Células Tumorais Cultivadas , Valina/farmacologia , Vidarabina/farmacologiaRESUMO
Bortezomib therapy has shown promising clinical activity in mantle cell lymphoma (MCL), but the development of resistance to proteasome inhibition may limit its efficacy. To unravel the factors involved in the acquisition of bortezomib resistance in vivo, immunodeficient mice were engrafted with a set of MCL cell lines with different levels of sensitivity to the drug, followed by gene expression profiling of the tumors and functional validation of the identified gene signatures. We observed an increased tumorigenicity of bortezomib-resistant MCL cells in vivo, which was associated with plasmacytic differentiation features, like interferon regulatory factor 4 (IRF4) and Blimp-1 upregulation. Lenalidomide was particularly active in this subgroup of tumors, targeting IRF4 expression and plasmacytic differentiation program, thus overcoming bortezomib resistance. Moreover, repression of the IRF4 target gene MYC in bortezomib-resistant cells by gene knockdown or treatment with CPI203, a BET (bromodomain and extra terminal) bromodomain inhibitor, synergistically induced cell death when combined with lenalidomide. In mice, addition of CPI203 to lenalidomide therapy further decreased tumor burden, involving simultaneous MYC and IRF4 downregulation and apoptosis induction. Together, these results suggest that exacerbated IRF4/MYC signaling is associated to bortezomib resistance in MCL in vivo and warrant clinical evaluation of lenalidomide plus BET inhibitor combination in MCL cases refractory to proteasome inhibition.
