Genetic and Small Molecule Modifiers of Lysosomal Function

Funder

Organizational Unit

Publications

Biophysical and biological properties of atypical sphingolipids : implications to physiology and pathophysiology

Publication . Da Cunha Branco Dos Santos, Tânia; Silva, Liana Casquinha da; Prieto, Manuel; Hornemann, Thorsten

1-deoxy-sphingolipids lack the C1-OH on their sphingoid base and present a cis ∆14-15 double bond instead of the canonical trans ∆4-5 double bond. Increased 1-deoxy-sphingolipid levels are directly correlated with the development and progression of hereditary sensory and autonomic neuropathy type 1 (HSAN1) and diabetes type 2. Different mechanisms have been proposed to explain the cytotoxicity of 1-deoxy-sphingolipids. However, these are highly dependent on the cell line studied and on the lipid concentration used, limiting the understanding of the mechanisms by which 1-deoxy-sphingolipids exert their patho-physiological roles. As for other sphingolipids, 1-deoxy-sphingolipids biological action might be related to the specific changes that each of the species cause on the biophysical properties of the membranes. Nonetheless, studies that comprehensively address the 1-deoxy-sphingolipids biophysical behavior are still scarce. Thereby, the goal of this study was to bring more insight into the biophysical impact of 1-deoxy-sphingolipids in both model and cellular membranes. Using complementary established fluorescence spectroscopy and microscopy methodologies and a multi-probe approach it was possible to conclude that: i) 1-deoxy-sphingolipids fail to form ordered domains as efficiently as canonical sphingolipids; ii) the presence, position and configuration of the sphingoid base double bond has a stronger influence on sphingolipids-induced changes on membrane biophysical properties than the structure of its C1 group; iii) external addition of 1-deoxy-sphingoid bases to living cells induce rapid changes in membrane fluidity in a sphingolipid structure dependent manner; iv) 1-deoxy-sphingolipids effects on membrane properties are specific and distinct from their canonical counterparts; v) endogenous elevation of 1-deoxy-sphingolipids due to mutations associated with HSAN1 development cause significant changes in the fluidity of cell membranes in a mutation dependent manner. Overall, the results suggest that pathologically elevated levels of 1-deoxy-sphingolipids compromise the biophysical properties of the membranes, which might impair proper cell functioning leading to the development of pathological conditions.

2021-06Doctoral thesis

Open access

Genotyping tumor DNA for precision oncology

Publication . Silveira, Catarina; Fonseca, Maria do Carmo Salazar Velez Roque da; Lopes, Teresa Cristina de Almeida Porta Nova Soares

The genomic revolution marked the beginning of the 21st century in biology and medicine. Completion of the Human Genome Project in 2003 has brought a flood of technological innovations that enabled decoding the entire genetic information in health and disease, leading to the foundation of the precision medicine movement. Genomics has revolutionized cancer research and transformed our understanding of how cancer arises. In recent years cancers have been re-classified based on their mutations, and a multitude of new drugs were developed that target specific molecular features of the tumor. Accurate and sensitive assays for cancer genotyping are crucial to enable precision oncology. Moreover, in addition to genotyping tissue samples from the primary tumor and metastasis, methodologies for cancer genotyping in circulating blood and other biological fluids are attracting much attention. This project was initiated with the goal of implementing and validating cutting-edge methodological approaches for profiling tumor DNA in the clinic. First, we used Sanger sequencing to determine the frequency of rare mutations in the gene that encodes epidermal growth factor receptor (EGFR) that are not detected by the widely used PCR-based Idylla™ EGFR Mutation Assay. Mutations in the EGFR gene are biomarkers that predict how non-small cell lung cancer (NSCLC) patients respond to EGFR-targeted therapies collectively known as tyrosine kinase inhibitors (TKIs). Thus, EGFR genotyping provides crucial information for treatment decision. Both Sanger sequencing and real-time PCR methodologies are used for EGFR genotyping. However, methods based on real-time PCR have limitations, as they may not detect rare or novel mutations. We sought to determine the prevalence of rare mutations in the tyrosine kinase domain (exons 18 to 21) of the EGFR gene not targeted by the most frequently used real-time PCR approaches, i.e., the cobas® EGFR Mutation Test, and the Idylla™ EGFR Mutation Assay. A total of 1228 NSCLC patients were screened for mutations in exons 18 to 21 of the EGFR gene using Sanger sequencing. We observed that 252 patients (~20%) had at least one mutation in the EGFR gene, and 38 (~3%) carried uncommon genetic alterations that could not be identified by the cobas® or the Idylla™ tests. We further found six new single mutations and seven previously unreported compound mutations. Clinical information and patient outcome are presented for these cases. In conclusion, this study highlights the value of sequencing based approaches to identify rare mutations. Our results add to the inventory of known EGFR mutations, thus contributing to improved lung cancer precision treatment. After the detection of an EGFR-TKI-sensitive mutation, at initial diagnosis, and after treatment with 1st or 2 nd generation inhibitors, about half of tumors develop a resistance mutation. To identify this mutation, so that the treatment can be readjusted, it is mandatory to carefully choose the sample and the method to be used. Therefore, we implemented and assessed the performance of a droplet digital PCR (ddPCR) assay for detecting the EGFR T790M mutation in liquid biopsies. Liquid biopsy allows the identification of targetable cancer mutations in a minimally invasive manner. In patients with NSCLC, ddPCR is increasingly used to genotype the EGFR gene in circulating cell-free DNA (cfDNA). However, the sensitivity of this method is still under debate. We optimized a ddPCR assay and used it to detect the EGFR T790M mutation in plasma samples from 77 patients with NSCLC in progression. Our ddPCR assay enabled the detection and quantification of the EGFR T790M mutation at cfDNA allele frequency as low as 0.5%. The mutation was detected in 40 plasma samples, corresponding to a positivity rate of 52%. The number of mutant molecules per mL of plasma ranged from 1 to 6,000. A re-biopsy was analyzed for 12 patients that had a negative plasma test and the mutation was detected in 2 cases. A second liquid biopsy was performed for 6 patients and the mutation was detected in 3 cases. In conclusion, this study highlights the value of ddPCR to detect and quantify the EGFR T790M mutation in liquid biopsies in a real-world clinical setting. Our results suggest that repeated ddPCR tests in cfDNA may obviate tissue re-biopsy in patients unable to provide a tumor tissue sample suitable for molecular analysis. Despite the advantages above described, the major disadvantage of ddPCR is the difficulty in testing multiple targets simultaneously. As such, we explored massively parallel sequencing methodologies for mutation discovery in cfDNA extracted from circulating blood. We analysed a patient with metastatic breast cancer who was selected for enrolment in an open-label clinical phase IIIb trial with ribociclib combined with letrozole. We genotyped cfDNA isolated from blood samples collected before (Pre_cfDNA) and after (Post_cfDNA) treatment with ribociclib and letrozole. Analysis of Pre_cfDNA using the Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) revealed two alterations: a missense hotspot mutation in the PIK3CA gene (3:178952085, A>G, H1047R) and a copy number amplification of the CCND1 gene. The presence of both molecular alterations was confirmed in the primary tumor. After treatment, the patient presented a significant clinical improvement, and the two alterations were no longer detected in cfDNA (Post_cfDNA). This work underlines that the minimally invasive nature of liquid biopsy allows monitoring of disease progression, demonstrating a practical alternative to tissue biopsy. The use of massively parallel sequencing enables to interrogate of several genes simultaneously, the search for different types of mutation, and identify variants with low allele frequency. Based on the previously mentioned results, we decided to implement a targeted ddPCR assay for detecting PIK3CA mutations in cfDNA. We used this assay to screen urine samples from the patient with a PIK3CA mutation identified in the plasma, but we failed to detect any positive result in the urine. In conclusion, we implemented at GenoMed the main methods that are currently recommended for molecular diagnosis in Precision Oncology, from massively parallel sequencing to ddPCR. We characterized the main advantages and limitations of each technology applied to a variety of biological samples including tumor tissue, blood, and other fluids. Taken together, the results of our work represent an important contribution towards a more achievable and cost-effective Precision Medicine in the context of real world clinical practice.

2022-12Doctoral thesis

Open access

Dengue virus targets RBM10 deregulating host cell splicing and innate immune response

Publication . Srebrow, Anabella; Gamarnik, Andrea V.; García, Cybele C.; Iglesias, Néstor G.; Vaz-Drago, Rita; García Solá, Martín E.; Gebhard, Leopoldo G.; Gaioli, Nicolás; Torti, María Florencia; Mammi, Pablo; Bragado, Laureano; Pozzi, Berta

RNA-seq experiments previously performed by our laboratories showed enrichment in intronic sequences and alterations in alternative splicing in dengue-infected human cells. The transcript of the SAT1 gene, of well-known antiviral action, displayed higher inclusion of exon 4 in infected cells, leading to an mRNA isoform that is degraded by non-sense mediated decay. SAT1 is a spermidine/spermine acetyl-transferase enzyme that decreases the reservoir of cellular polyamines, limiting viral replication. Delving into the molecular mechanism underlying SAT1 pre-mRNA splicing changes upon viral infection, we observed lower protein levels of RBM10, a splicing factor responsible for SAT1 exon 4 skipping. We found that the dengue polymerase NS5 interacts with RBM10 and its sole expression triggers RBM10 proteasome-mediated degradation. RBM10 over-expression in infected cells prevents SAT1 splicing changes and limits viral replication, while its knock-down enhances the splicing switch and also benefits viral replication, revealing an anti-viral role for RBM10. Consistently, RBM10 depletion attenuates expression of interferon and pro-inflammatory cytokines. In particular, we found that RBM10 interacts with viral RNA and RIG-I, and even promotes the ubiquitination of the latter, a crucial step for its activation. We propose RBM10 fulfills diverse pro-inflammatory, anti-viral tasks, besides its well-documented role in splicing regulation of apoptotic genes.

2020Journal article

Open access

Exploring the biophysical properties of biological membranes: from ceramide domains to lipid droplets

Publication . Ventura, Ana; Silva, Liana Casquinha da; Prieto, Manuel; Futerman, Anthony

Actualmente é reconhecido que a organização da membrana e as suas propriedades são reguladores de funções celulares. As alterações na organização membranar e a formação de domínios ordenados estão implicados em diversos processos biológicos, tais como na sinalização celular. As ceramidas constituem as cadeias estruturais de todos os esfingolípidos complexos, e são considerados lípidos bioactivos capazes de modular vários processos celulares e implicados no desenvolvimento de várias doenças. O mecanismo molecular subjacente ao modo de acção das ceramidas parece estar relacionado com o modo como estes lípidos alteram as propriedades biofísicas das membranas, nomeadamente através da formação de domínios enriquecidos em ceramida. No entanto, o efeito da ceramida nas propriedades das membranas são complexos e dependem tanto da composição lipídica da membrana como da estrutura da ceramida, e estão pouco caracterizados em situações que mimetizem comportamentos biológicos. Por isso, este estudo tem como objectivo investigar o impacto biofísico das ceramidas em membranas biológicas e em condições fisiologicamente relevantes, e identificar as consequências biológicas associadas a estas alterações. Vários estudos foram feitos tendo como objectivo i) melhorar a caracterização das ceramidas em membranas artificiais e ii) desenvolver e implementar metodologias biofísicas para caracterizar as propriedades de membranas e organelos em células e em condições fisiológicas. Os resultados obtidos neste estudo confirmam a complexidade associadas às alterações promovidas pela ceramida nas propriedades biofísicas das membranas, que passam pela formação de fases gel metaestáveis interdigitadas. Para além disso, também é mostrado que a formação de ceramida induzida por factores de stress promove alterações na fluidez da membrana plasmática, nos lisossomas e na célula em geral. A capacidade da ceramida promover um aumento da ordem da membrana, causado pela formação dos domínios gel de ceramida, está coordenado com o aumento da internalização de sondas lipofílicas, e é acompanhado por um aumento na internalização de vesiculas enriquecidas em ceramida, as quais são transportadas através da via endo/lisossomal. Além disso, este estudo mostra que a formação de ceramida e as alterações biofísicas estão associadas ao metabolismo e propriedades das gotículas lipídicas (“lipid droplets”). Resumindo, este estudo demostra que as acções biológicas da ceramida poderão estar associadas às alterações biofísicas induzidas nas membranas. O efeito das ceramidas nas propriedades das membranas propaga-se para além do local onde são formadas, e envolvem alterações em vários organelos. Estas evidências sugerem que activação de alvos intracelulares relacionados com as ceramidas possa ser mediada pelas vesiculas enriquecidas em ceramida com propriedades biofísicas únicas.

2023-06Doctoral thesis

Open access