A carregar...
Publicação
Understanding the importance of Death receptors in the therapeutic resistance of Myeloproliferative Neoplasms
| Nome: | Descrição: | Tamanho: | Formato: | |
|---|---|---|---|---|
| 1.58 MB | Adobe PDF |
Autores
Orientador(es)
Resumo(s)
Neoplasias mieloproliferativas BCR-ABL1 negativas (NMP) são doenças mieloides clonais (MM) que dependem da sinalização JAK-STAT, impulsionada pela mutação JAK2V617F. Apesar dos avanços na compreensão da biologia desta doença e da eficácia de inibidores de JAK, o transplante de medula óssea (MO) continua a ser a única cura.
A resistência terapêutica em MM é comum, uma vez que a MO protege as células leucémicas. Estudos anteriores demonstraram que a interação entre a MO e células NMP protege-as da citotoxicidade do Ruxolitinib. Recentemente foi descoberto que a exposição prévia do estroma da MO às células NMP aumenta a expressão dos transcritos TNFRSF8 (CD30) e TNFRSF9 (CD137), que possivelmente contribuem para a resistência terapêutica em NMP.
Este projeto pretendia confirmar o aumento de expressão e caracterizar in vitro a importância destes recetores na resistência ao Ruxolitinib mediada pela MO, utilizando um sistema de co-cultura com células NMP (SET-2 e HEL) e uma linha celular estromal MO (HS-5). O aumento da expressão foi avaliado por PCR quantitativo e Citometria de fluxo. Para compreender a importância destes recetores, a sua atividade foi modulada através da inativação genética (CRISPR/Cas9), anticorpos neutralizantes e ligandos. Os efeitos funcionais desta modulação foram analisados através da medição da viabilidade das células NMP.
Os resultados demonstraram um aumento de expressão dos transcritos correspondentes aos recetores CD30 e CD137. Porém, apenas o aumento de expressão do CD137 foi confirmado à superfície das células NMP. A anulação (anticorpos neutralizantes) ou a ativação (ligandos do recetor) do recetor CD137 não impactou a viabilidade das células NMP. A modulação da atividade do recetor CD137 através da inativação genética não foi alcançada. Os nossos resultados sugerem que os recetores CD30 e CD137 não estão envolvidos na proteção conferida pela MO. Contudo, outros aspetos devem ser considerados antes de excluir o CD137 como fator relevante neste mecanismo.
BCR-ABL1 negative myeloproliferative neoplasms (MPN) are clonal myeloid malignancies (MM) that rely on the JAK-STAT signaling, primarily driven by the JAK2V617F mutation. Despite the advances in understanding the MPN disease biology and JAK inhibitors effectiveness, bone marrow (BM) transplantation remains the only cure. In Myeloid Malignancies resistance to chemotherapy is common, as the BM microenvironment protects leukemic cells. Prior research has demonstrated that the interaction between the BM and MPN cells protects them from Ruxolitinib cytotoxicity. Additionally, it was also discovered that previous exposure of BM stroma to MPN cells upregulates the transcripts TNFRSF8 (CD30) and TNFRSF9 (CD137), which potentially contribute to the therapeutic resistance in MPN. This project aimed to confirm the upregulation and characterize in vitro the importance of these receptors in the context of BM-mediated resistance to Ruxolitinib, using a co-culture system with MPN cells (SET-2, HEL) and a bone marrow stromal cell line (HS-5). The upregulation of the receptors was assessed through quantitative PCR and Flow Cytometry. To understand the importance of these receptors, their activity was modulated through gene inactivation (CRISPR/Cas9), neutralizing antibodies and receptor ligands. The functional effects of modulating the receptors were analyzed by assessing MPN cell viability. The results demonstrated an upregulation of transcripts corresponding to the CD30 and CD137 receptors. However, only the CD137 receptor upregulation was confirmed at the cellular surface of MPN cells. The abrogation (with neutralizing antibodies) or activation (with receptor ligands) of CD137 receptor activity had no impact on the viability of MPN cells in the context of BM-mediated resistance to Ruxolitinib. Unfortunately, the modulation of CD137 receptor activity through gene inactivation was not achieved. Our results may suggest that these receptors are not involved in the BM-protection. However, other aspects must be considered before completely dismissing CD137 as a relevant factor in this mechanism.
BCR-ABL1 negative myeloproliferative neoplasms (MPN) are clonal myeloid malignancies (MM) that rely on the JAK-STAT signaling, primarily driven by the JAK2V617F mutation. Despite the advances in understanding the MPN disease biology and JAK inhibitors effectiveness, bone marrow (BM) transplantation remains the only cure. In Myeloid Malignancies resistance to chemotherapy is common, as the BM microenvironment protects leukemic cells. Prior research has demonstrated that the interaction between the BM and MPN cells protects them from Ruxolitinib cytotoxicity. Additionally, it was also discovered that previous exposure of BM stroma to MPN cells upregulates the transcripts TNFRSF8 (CD30) and TNFRSF9 (CD137), which potentially contribute to the therapeutic resistance in MPN. This project aimed to confirm the upregulation and characterize in vitro the importance of these receptors in the context of BM-mediated resistance to Ruxolitinib, using a co-culture system with MPN cells (SET-2, HEL) and a bone marrow stromal cell line (HS-5). The upregulation of the receptors was assessed through quantitative PCR and Flow Cytometry. To understand the importance of these receptors, their activity was modulated through gene inactivation (CRISPR/Cas9), neutralizing antibodies and receptor ligands. The functional effects of modulating the receptors were analyzed by assessing MPN cell viability. The results demonstrated an upregulation of transcripts corresponding to the CD30 and CD137 receptors. However, only the CD137 receptor upregulation was confirmed at the cellular surface of MPN cells. The abrogation (with neutralizing antibodies) or activation (with receptor ligands) of CD137 receptor activity had no impact on the viability of MPN cells in the context of BM-mediated resistance to Ruxolitinib. Unfortunately, the modulation of CD137 receptor activity through gene inactivation was not achieved. Our results may suggest that these receptors are not involved in the BM-protection. However, other aspects must be considered before completely dismissing CD137 as a relevant factor in this mechanism.
Descrição
Palavras-chave
BCR-ABL1 negative myeloproliferative neoplasms Therapeutic resistance Ruxolitinib CD30 CD137