Browsing by Author "Alves, Raquel"
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- Combination of elacridar with imatinib modulates resistance associated with drug efflux transporters in chronic myeloid leukemiaPublication . Alves, Raquel; Gonçalves, Ana Cristina; Jorge, Joana; Almeida, António M.; Sarmento-Ribeiro, Ana BelaMultidrug resistance (MDR) development has emerged as a complication that compromises the success of several chemotherapeutic agents. In chronic myeloid leukemia (CML), imatinib resistance has been associated with changes in BCR-ABL1 and intracellular drug concentration, controlled by SLC and ABC transporters. We evaluate the therapeutic potential of a P-glycoprotein and BCRP inhibitor, elacridar, in sensitive (K562 and LAMA-84) and imatinib-resistant (K562-RC and K562-RD) CML cell lines as monotherapy and combined with imatinib. Cell viability was analyzed by resazurin assay. Drug transporter activity, cell death, cell proliferation rate, and cell cycle distribution were analyzed by flow cytometry. Both resistant models presented an increased activity of BCRP and P-gP compared to K562 cells. Elacridar as monotherapy did not reach IC50 in any CML models but activated apoptosis without cytostatic effect. Nevertheless, the association of elacridar (250 nM) with imatinib overcomes resistance, re-sensitizing K562-RC and K562-RD cells with five and ten times lower imatinib concentrations, respectively. Drug combination induced apoptosis with increased cleaved-caspases-3, cleaved-PARP and DNA damage, reduced cell proliferation rate, and arrested CML cells in the S phase. These data suggest that elacridar combined with imatinib might represent a new therapeutic option for overcoming TKI resistance involving efflux transporters. View Full-Text
- Flow cytometry and targeted immune transcriptomics identify distinct profiles in patients with chronic myeloid leukemia receiving tyrosine kinase inhibitors with or without interferon-αPublication . Alves, Raquel; McArdle, Stephanie E. B.; Vadakekolathu, Jayakumar; Gonçalves, Ana Cristina; Freitas-Tavares, Paulo; Pereira, Amélia; Almeida, Antonio M.; Sarmento-Ribeiro, Ana Bela; Rutella, SergioBACKGROUND: Tumor cells have evolved complex strategies to escape immune surveillance, a process which involves NK cells and T lymphocytes, and various immunological factors. Indeed, tumor cells recruit immunosuppressive cells [including regulatory T-cells (Treg), myeloid-derived suppressor cells (MDSC)] and express factors such as PD-L1. Molecularly targeted therapies, such as imatinib, have off-target effects that may influence immune function. Imatinib has been shown to modulate multiple cell types involved in anti-cancer immune surveillance, with potentially detrimental or favorable outcomes. Imatinib and other tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) have dramatically changed disease course. Our study aimed to characterize the different populations of the immune system in patients with CML affected by their treatment. METHODS: Forty-one patients with CML [33 treated with TKIs and 8 with TKIs plus interferon (IFN)-α] and 20 controls were enrolled in the present study. Peripheral blood populations of the immune system [referred to as the overview of immune system (OVIS) panel, Treg cells and MDSCs] and PD-1 expression were evaluated by flow cytometry. The immunological profile was assessed using the mRNA Pan-Cancer Immune Profiling Panel and a NanoString nCounter FLEX platform. RESULTS: Patients receiving combination therapy (TKIs + IFN-α) had lower numbers of lymphocytes, particularly T cells [838/µL (95% CI 594-1182)] compared with healthy controls [1500/µL (95% CI 1207 - 1865), p = 0.017]. These patients also had a higher percentage of Treg (9.1%) and CD4+PD-1+ cells (1.65%) compared with controls [Treg (6.1%) and CD4+/PD-1+(0.8%); p ≤ 0.05]. Moreover, patients treated with TKIs had more Mo-MDSCs (12.7%) whereas those treated with TKIs + IFN-α had more Gr-MDSC (21.3%) compared to controls [Mo-MDSC (11.4%) and Gr-MDSC (8.48%); p ≤ 0.05]. CD56bright NK cells, a cell subset endowed with immune-regulatory properties, were increased in patients receiving TKIs plus IFN-α compared with those treated with TKIs alone. Interestingly, serum IL-21 was significantly lower in the TKIs plus IFN-α cohort. Within the group of patients treated with TKI monotherapy, we observed that individuals receiving 2nd generation TKIs had lower percentages of CD4+ Treg (3.63%) and Gr-MDSC (4.2%) compared to patients under imatinib treatment (CD4+ Treg 6.18% and Gr-MDSC 8.2%), but higher levels of PD-1-co-expressing CD4+ cells (1.92%). CONCLUSIONS: Our results suggest that TKIs in combination with IFN-α may promote an enhanced immune suppressive state.
- Genetic variants of ABC and SLC transporter genes and chronic myeloid leukaemia: impact on susceptibility and prognosisPublication . Alves, Raquel; Goncalves, Ana Cristina; Jorge, Joana; Marques, Gilberto; Ribeiro, André B.; Tenreiro, Rita; Coucelo, Margarida; Diamond, Joana; Oliveiros, Bárbara; Pereira, Amélia; Freitas-Tavares, Paulo; Almeida, António M.; Sarmento-Ribeiro, Ana BelaSolute carrier (SLC) and ATP-binding cassette (ABC) transporters comprise a variety of proteins expressed on cell membranes responsible for intrusion or extrusion of substrates, respectively, including nutrients, xenobiotics, and chemotherapeutic agents. These transporters mediate the cellular disposition of tyrosine kinase inhibitors (TKIs), and their genetic variants could affect its function, potentially predisposing patients to chronic myeloid leukaemia (CML) and modulating treatment response. We explored the impact of genetic variability (single nucleotide variants—SNVs) of drug transporter genes (ABCB1, ABCG2, SLC22A1, and SLC22A5) on CML susceptibility, drug response, and BCR-ABL1 mutation status. We genotyped 10 SNVs by tetra-primers-AMRS-PCR in 198 CML patients and 404 controls, and assessed their role in CML susceptibility and prognosis. We identified five SNVs associated with CML predisposition, with some variants increasing disease risk, including TT genotype ABCB1 (rs1045642), and others showing a protective effect (GG genotype SLC22A5 rs274558). We also observed different haplotypes and genotypic profiles associated with CML predisposition. Relating to drug response impact, we found that CML patients with the CC genotype (rs2231142 ABCG2) had an increased risk of TKI resistance (six-fold). Additionally, CML patients carrying the CG genotype (rs683369 SLC22A1) presented a 4.54-fold higher risk of BCR-ABL1 mutations. Our results suggest that drug transporters’ SNVs might be involved in CML susceptibility and TKI response, and predict the risk of BCR-ABL1 mutations, highlighting the impact that SNVs could have in therapeutic selection.
- Influence of genetic variants of oxidative stress related genes on prognosis and therapy response in chronic myeloid leukemiaPublication . Alves, Raquel; Ventura, Filipa; Jorge, Joana; Ribeiro, Ilda P.; Marques, Gilberto; Coucelo, Margarida; Diamond, Joana; Freitas-Tavares, Paulo; Almeida, António; Goncalves, Ana Cristina; Sarmento-Ribeiro, Ana BelaChronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the presence of BCR-ABL1 oncogene. Oxidative stress conditions are crucial in tumor development and progression. The NRF2, codified by NFE2L2 gene, plays a central role in redox balance, promoting the transcription of cytoprotective and antioxidant genes such as SOD2, GPX1 and CAT. The action of this transcription factor is regulated by KEAP1. Alterations in these genes, either due to somatic mutations or genetic variants (SNV), have been associated with the development, prognosis, and therapeutic response in cancer. This work evaluated the influence of polymorphisms in genes associated with oxidative stress (NFE2L2, KEAP1, SOD2, GPX1, and CAT) in prognosis and treatment response with tyrosine kinase inhibitors (TKI) in CML patients. In 194 CML samples, nine genetic variants in NFE2L2 (rs6721961, rs4893819, rs35652124, rs6706649 and rs13001694), KEAP1 (rs113540846), SOD2 (rs4880), GPX1 (rs1050450) and CAT (rs1001179) were genotyped by PCR based assays. The association of these genetic variants with clinical-laboratory characteristics were assessed by Fisher’s test, logistic regression, and Kaplan-Meier curves, considering a p<0.05. The results obtained showed that patients with G allele on GPX1 rs1050450 and KEAP1 rs113540846 variants have an approximately 2-fold and 31-fold higher probability of being TKI resistant, respectively. In TKI-resistant group, patients with CT genotype in NFE2L2 rs4893819 variant were more likely to need three or more lines of treatment (OR=5.60, 95%CI 1.22-25.75, p=0.027). An important factor for TKI response is BCR-ABL1 mutational status, and we found that individuals with AG genotype in NFE2L2 rs13001694 present, approximately 9x more risk of mutation on this fusion gene. Moreover, patients heterozygous for the NFE2L2 rs4893819 (CT genotype) and SOD2 rs4880 (AG genotype) variants have faster disease progression [NFE2L2: hazard ratio (HR)=7.84, 95% CI 1.82-29.93, p=0.020; SOD2: HR=7.19, 95% CI 1.62-31.96, p=0.035] comparing with other genotypes. While the GG genotype of the NFE2L2 rs13001694 variant has a 2-fold lower overall survival than other patients (HR=11.86, 95% CI 1.39-1000.7, p=0.023). Additionally, we also observed an association between haplotypes and genotypic profile with CML prognosis. In conclusion, our results suggest that genetic variants in genes associated with the redox state might be important factors in the prognosis, survival, and therapy response in CML patients.
- MicroRNA signature refine response prediction in CMLPublication . Alves, Raquel; Gonçalves, Ana Cristina; Jorge, Joana; Marques, Gilberto; Luís, Dino; Ribeiro, André B.; Freitas-Tavares, Paulo; Oliveiros, Bárbara; Almeida, António M.; Sarmento-Ribeiro, Ana BelamicroRNAs (miRs) dysregulation have emerged as a crucial step in tumorigenesis, being related with cancer development, progression and response to treatment. In chronic myeloid leukaemia (CML), the resistance to tyrosine kinase inhibitors (TKI) is responsible for treatment failure and could be linked to changes in miRs expression. This work aimed to correlate the expression levels of 3 miRs, miR-21, miR-26b and miR-451, with response to TKI treatment in CML patients. miR-451 levels at diagnosis were significantly higher in patients with optimal response after 6 and 12 months of therapy. Conversely, patients without optimal response had highest levels of miR-21. miR-21 and miR-451 appear to be good biomarkers of response, able to predict optimal TKI responders (p < 0.05). Using the combined profile of both miRs, we create a predictive model of optimal response after one year of treatment. This study highlights the role of miR-21 and miR-451 expression levels at diagnosis in predicting which patients achieve the optimal response.
- Resistance to tyrosine kinase inhibitors in chronic myeloid leukemia — from molecular mechanisms to clinical relevancePublication . Alves, Raquel; Gonçalves, Ana Cristina; Rutella, Sergio; Almeida, António M.; Rivas, Javier De Las; Trougakos, Ioannis P.; Ribeiro, Ana Bela SarmentoResistance to targeted therapies is a complex and multifactorial process that culminates in the selection of a cancer clone with the ability to evade treatment. Chronic myeloid leukemia (CML) was the first malignancy recognized to be associated with a genetic alteration, the t(9;22)(q34;q11). This translocation originates the BCR-ABL1 fusion gene, encoding the cytoplasmic chimeric BCR-ABL1 protein that displays an abnormally high tyrosine kinase activity. Although the vast majority of patients with CML respond to Imatinib, a tyrosine kinase inhibitor (TKI), resistance might occur either de novo or during treatment. In CML, the TKI resistance mechanisms are usually subdivided into BCR-ABL1-dependent and independent mechanisms. Furthermore, patients’ compliance/adherence to therapy is critical to CML management. Techniques with enhanced sensitivity like NGS and dPCR, the use of artificial intelligence (AI) techniques, and the development of mathematical modeling and computational prediction methods could reveal the underlying mechanisms of drug resistance and facilitate the design of more effective treatment strategies for improving drug efficacy in CML patients. Here we review the molecular mechanisms and other factors involved in resistance to TKIs in CML and the new methodologies to access these mechanisms, and the therapeutic approaches to circumvent TKI resistance.