Biology subjects

Lobo, T.

Publications and source records attributed to Lobo, T..

7 recordsLinked to original sources

bioRxiv2025

Diverse transcriptomic and mutational patterns but limited functional pathway alterations in patient-derived SS cells

Structured AbstractO_ST_ABSBackgroundC_ST_ABSEradication of SS is hampered by its genetic and molecular heterogeneity. A better understanding of the putative commonalities underlying SS oncogenicity may help to provide more efficient therapeutic strategies against this devastating disease. PurposeThe present work analyzes the whole transcriptome of different patient-derived SS cells to identify expression patterns, functional programs and expressed gene mutations that may provide clues on new therapeutic options for SS patients MethodsMononuclear cells were recovered by Ficoll gradient separation from fresh peripheral blood of SS patients (n=7). Selected pathway-based compounds and the MALT1 inhibitor MI2 were used for in vitro drug sensitivity testing. SS cells viability was evaluated using CellTiter-Glo_3D Cell Viability Assay and flow cytometry analysis. We validated the usefulness of MI2 using patient-derived SS cells xenotransplanted (PDX) into Nod Scid Gamma mice. ResultsIn vitro data indicated that cell lines and primary malignant SS cells all display different sensitivities against specific pathway inhibitors. However, MALT1 inhibition led to a robust effect in vitro that was partially reproduced in the in vivo NSG mice xenograft model. ConclusionOur investigations revealed the actual possibility of inhibiting the downstream TCR signaling complex form by CARD11, BCL10 and MALT1 in SS therapy. Key PointsPatient-derived SS cells are transcriptionally and mutationally heterogeneous but share some common pathway alterations. Inhibition of MALT1 reduces NF-{kappa}B signaling and cell growth in cell lines and patient-derived SS cells.

cancer biology↗

bioRxiv2023

Ancestral genomes and population sequencing data reveal strand- and context-specificityof polymorphic G4 sites

Recent studies highlight the important functional roles of non-canonical DNA conformations. One of such structures, G-quadruplex (G4), was shown to be involved in multiple processes within a cell such as telomere maintenance, gene regulation, protein translation and alternative splicing. On the other hand, DNA in non-double stranded context can hinder replication and repair processes and, indeed, a higher rate of polymorphisms was reported at G4 sites. However, strand-specificity, positional and nucleotide contexts of nucleotide substitutions at G4 sites are under-investigated. Here, we combine ancestral genome data and DNA variants from large populational sequencing project to investigate substitution patterns within putative DNA quadruplexes. We confirm the overall elevated rate of base replacement except transitions at CpG sites, which are less likely than in the rest of the genome potentially due to hypomethylated status of G4s. Within G4 loops, there is a trend of replacing weak bases (A, T) with strong bases (G, C) that could promote DNA duplex stabilization. However, this trend is less pronounced when derived allele is rare in the human population. The most over-represented thymine to guanine replacement is about order of magnitude more frequent within G4 than in non-G4 regions. The analysis of nucleotide context of the substitutions shows clear difference between G-rich and C-rich of DNA quadruplexes implying that the strands of the quadruplex might have different mutation dynamics. Finally, we show that the observed deviations from random mutation accumulation result in a biased nucleotide composition of G4 loops rich in adenines. Future studies should reveal specific mutation and selection processes that shape the content of G4-associated DNA polymorphisms.

genomics↗

bioRxiv2023

A dynamic bias in chromatin protein deposition at G-quadruplex sites

Previous studies indicate that genomic loci harboring G-quadruplexes (G4s)--stacked structures that can form in single-stranded DNA--can be linked to epigenetic instability. However, the role of chromatin redistribution and the genome-wide nature of this process need further investigation. Here, we provide experimental evidence that connects G4s to alterations in the deposition of chromatin proteins. We have used metabolic labelling and immunoprecipitation of new and parental proteins in hRPE-1 cells to investigate global chromatin deposition dynamics. We identify a reciprocal, local bias in chromatin protein deposition at G4 sites favoring the association of parental proteins with the G4 and new proteins with the C4 DNA strand. The deposition bias at G4 sites does not depend on replication directionality and is strengthened by G4 stabilization. Slowing down replication forks upon hydroxyurea treatment reverses the bias, supposedly affected by decoupling between helicase and polymerase. Interestingly, upon combined G4 stabilization and slowing of the replication forks, new proteins exhibit a redistribution pattern similar to G4 stabilization alone, while parental protein redistribution more resembles one after hydroxyurea treatment, hinting at mechanistic differences between parental and new histone distribution. We also report that the genomic distribution of putative quadruplexes is not random and depends on loop size, where G4s with shorter loops have a preference for the DNA strand replicated by leading and G4s with longer loops by lagging strand replication. These findings provide insight into the mechanisms behind G4 occurrence and its role in epigenetic instability and help to improve our understanding of the factors influencing biases in global chromatin protein redeposition.

molecular biology↗

bioRxiv2023

Local G-quadruplexes are not a major determinant of altered gene expression in BLM-deficientcells

Mutations in the Blm gene can cause Bloom Syndrome, a genetic disorder characterized by genome instability and cancer predisposition. Blm encodes a helicase which was reported to resolve G-quadruplex DNA structures in vitro. The G-quadruplex resolving activity of the BLM helicase has been previously implicated in altering gene expression. However, the exact mechanisms of how G-quadruplex structures may affect gene expression remain to be elucidated. We employed experimentally defined G-quadruplex forming DNA sequences and generated transcriptomes for several Bloom Syndrome patient-derived cell lines and BLM-deficient mouse embryonic stem cells to further investigate the effect of G-quadruplexes on gene expression. Our results do not support the previous findings that G-quadruplexes located within a gene play a major role in altering its expression in BLM-deficient cells. We found concerted large-scale changes in transcript abundance, splicing, nucleosome occupancy, and phasing, that cannot be linked to the local presence of G-quadruplex sequences in either gene bodies or promotors. The investigation of genomic features associated with large-scale differences in nucleosome density highlights the rDNA locus and active enhancers as the most strongly affected regions. We hypothesize that global changes in chromatin structure rather than local G4s might mediate the transcriptome changes in the absence of BLM.

molecular biology↗

bioRxiv2023

IKK1 kinase coordinates BRD4 and JAK/STAT signaling to subvert DNA damage-based anticancer therapy

Activation of the IKK kinase complex has recurrently been linked to colorectal cancer (CRC) initiation and progression. However, identification of downstream effectors other than NF-{kappa}B has remained elusive. Analysis of IKK-dependent substrates after UV-treatment revealed that BRD4 phosphorylation by IKK is required for chromatin-binding dynamics upon damage. Moreover, IKK induces the NF-{kappa}B-dependent transcription of LIF leading to STAT3 activation, association of BRD4 to STAT3 and recruitment to specific target genes. IKK abrogation results in defective BRD4 and STAT3 function leading to irreparable DNA damage and apoptotic cell death upon different stimuli. Simultaneous inhibition of BRAF-dependent IKK activity or BRD4 and the JAK/STAT pathway enhanced the therapeutic potential of 5-FU plus irinotecan in CRC cells, and is curative in a chemotherapy-resistant CRC xenograft model. Coordinated expression of LIF and IKK is a poor prognosis marker for CRC patients. Our data uncover a functional link between IKK, BRD4 and JAK/STAT signaling with clinical relevance.

cancer biology↗

bioRxiv2022

β-catenin activity induces an RNA biosynthesis program promoting therapy resistance in T Acute Lymphoblastic Leukemia

Understanding the molecular mechanisms that contribute to the appearance of chemotherapy resistant cell populations is necessary to improve cancer treatment. We have now investigated the role of {beta}-catenin/CTNNB1 in the evolution of T-Acute Lymphoblastic Leukemia (T-ALL) patients and its involvement in therapy resistance. We have identified a specific gene signature that is directly regulated by {beta}-catenin, TCF/LEF factors and ZBTB33/Kaiso in T-ALL cell lines, which is highly and significantly represented in 5 out of 6 refractory patients from a cohort of 40 children with T-ALL. By subsequent refinement of this gene signature, we found that a subset of {beta}-catenin target genes involved with RNA-processing function are sufficient to segregate T-ALL refractory patients in three independent cohorts. We demonstrate the implication of {beta}-catenin in RNA and protein synthesis in T-ALL and provide experimental evidence that {beta}-catenin is crucial for the cellular response to chemotherapy, mainly in the cellular recovery phase after treatment. We propose that combination treatments involving chemotherapy plus {beta}-catenin inhibitors will enhance chemotherapy response and prevent disease relapse in T-ALL patients.

cancer biology↗

bioRxiv2021

Can the causal role of brain oscillations be studied through rhythmic brain stimulation?

Many studies have investigated the causal relevance of brain-oscillations using rhythmic stimulation, either through direct-brain or sensory stimulation. Yet, how intrinsic rhythms interact with the externally generated rhythm is largely unknown. We either presented a flickered (60 Hz) visual grating or its correspondent unflickered stimulus in a psychophysical change-detection-task during simultaneous MEG-recordings to humans, to test the effect of visual entrainment on induced gamma-oscillations. Notably, we generally observed the co-existence of the broadband induced gamma-rhythm with the entrained flicker-rhythm (reliably measured in each participant), with the peak frequency of the induced response remaining unaltered in approximately half of participants -relatively independently of their native frequency. However, flicker increased broadband induced-gamma-power, and this was stronger in participants with a native frequency closer to the flicker-frequency ( resonance), and led to strong phase-entrainment. Presence of flicker did not change behaviour itself, but profoundly altered brain-behaviour correlates across the sample: whilst broadband induced gamma-oscillations correlated with reaction-times for unflickered stimuli (as known previously), for the flicker, the amplitude of the entrained flicker-rhythm (but no more the induced oscillation) correlated with reaction-times. This, however, strongly depended on whether a participants peak frequency shifted to the entrained rhythm. Our results suggests that rhythmic brain-stimulation leads to a coexistence of two partially independent oscillations with heterogeneous effects across participants on the downstream relevance of these rhythms for behaviour. This may explain the inconsistency of findings related to external entrainment of brain-oscillations and poses further questions towards causal manipulations of brain-oscillations in general.

neuroscience↗