Biology subjects

Paes Dias, M.

Publications and source records attributed to Paes Dias, M..

4 recordsLinked to original sources

bioRxiv2022

Multi-omics analysis reveals distinct non-reversion mechanisms of PARPi resistance in BRCA1- versus BRCA2-deficient mammary tumors

BRCA1 and BRCA2 both function in DNA double-strand break repair by homologous recombination (HR). Due to their HR-defect, BRCA1/2-deficient cancers are sensitive to poly(ADP-ribose) polymerase inhibitors (PARPi) but they eventually acquire resistance. Preclinical studies yielded several PARPi resistance mechanisms that do not involve BRCA1/2 reactivation, but their relevance in the clinic remains elusive. To investigate which BRCA1/2-independent mechanisms drive spontaneous resistance in vivo, we combined molecular profiling with functional analysis of the HR status of matched PARPi-naive and PARPi-resistant mouse mammary tumors harboring large intragenic deletions that prevent functional restoration of BRCA1/2. We observed restoration of HR in 64% of PARPi-resistant BRCA1-deficient tumors but none in the PARPi-resistant BRCA2-deficient tumors. Moreover, we found that 53BP1 loss is the prevalent resistance mechanism in HR-proficient BRCA1-deficient tumors, whereas resistance in BRCA2-deficient tumors is mainly induced by the loss of PARG. Our combined multi-omics analysis catalogued additional genes and pathways potentially involved in modulating PARPi response.

cancer biology↗

bioRxiv2021

Loss of Nuclear DNA ligase III Can Revert PARP Inhibitor Resistance in BRCA1-deficient Cells by Increasing DNA Replication Stress

Inhibitors of poly(ADP-ribose) (PAR) polymerase (PARPi) have entered the clinic for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, preclinical and clinical research with PARPi has revealed multiple resistance mechanisms, highlighting the need for identification of novel functional biomarkers and combination treatment strategies. Functional genetic screens performed in cells and organoids that acquired resistance to PARPi by loss of 53BP1 identified loss of LIG3 as an enhancer of PARPi toxicity in BRCA1-deficient cells. Enhancement of PARPi toxicity by LIG3 depletion is dependent on BRCA1 deficiency but independent of the loss of 53BP1 pathway. Mechanistically, we show that LIG3 loss promotes formation of MRE11-mediated post-replicative ssDNA gaps in BRCA1-deficient and BRCA1/53BP1 double-deficient cells exposed to PARPi, leading to an accumulation of chromosomal abnormalities. LIG3 depletion also enhances efficacy of PARPi against BRCA1-deficient mammary tumors in mice, suggesting LIG3 as a potential therapeutic target.

cancer biology↗

bioRxiv2020

SMARCAD1 Mediated Active Replication Fork Stability Maintains Genome Integrity

Stalled fork protection pathway mediated by BRCA1/2 proteins is critical for replication fork stability that has implications in tumorigenesis. However, it is unclear if additional mechanisms are required to maintain replication fork stability. We describe a novel mechanism by which the chromatin remodeler SMARCAD1 stabilizes active replication forks that is essential for resistance towards replication poisons. We find that loss of SMARCAD1 results in toxic enrichment of 53BP1 at replication forks which mediates untimely dissociation of PCNA via the PCNA-unloader, ATAD5. Faster dissociation of PCNA causes frequent fork stalling, inefficient fork restart and accumulation of single-stranded DNA resulting in genome instability. Although, loss of 53BP1 in SMARCAD1 mutants restore PCNA levels, fork restart efficiency, genome stability and tolerance to replication poisons; this requires BRCA1 mediated fork protection. Interestingly, fork protection challenged BRCA1-deficient naive- or PARPi-resistant tumors require SMARCAD1 mediated active fork stabilization to maintain unperturbed fork progression and cellular proliferation.

cell biology↗

bioRxiv2020

Feasibility of phosphoproteomics on leftover samples after RNA extraction with guanidinium thiocyanate

In daily practice, different types of biomolecules are usually extracted for large-scale omics analysis with tailored protocols. However, when sample material is limited, an all-in-one strategy is preferable. While lysis of cells and tissues with urea is the accepted standard for phosphoproteomic applications, DNA, RNA and proteins can be simultaneously extracted from small samples using acid guanidinium thiocyanate-phenol-chloroform (AGPC). Use of AGPC for mass spectrometry (MS)-based phosphoproteomics has been reported, but not benchmarked. Here we compared urea-with AGPC-based protein extraction, profiling phosphorylations in the DNA damage response pathway after ionizing irradiation of U2OS cells as proof of principle. On average we identified circa 9000 phosphosites per sample with both extraction methods. Moreover, we observed high similarity of phosphosite characteristics (e.g. 94% shared class 1 identifications) and deduced kinase activities (e.g. ATM, ATR, CHEK1/2, PRKDC). AGPC-based sample extraction can thus replace standard cell lysates for phosphoproteomic workflows and may thus be an attractive way to obtain input material for multiple omics workflows, yielding several data types from a single sample.

molecular biology↗