Mutation profling of cancer drivers in Brazilian colorectal cancer

The molecular basis of colorectal cancer (CRC) can guide patient prognosis and therapy. In Brazil, knowledge on the CRC mutation landscape is limited. Here, we investigated the mutation profile of 150 cancer-related genes by next-generation sequencing and associated with microsatellite instability (MSI) and genetic ancestry in a series of 91 Brazilian CRC patients. Driver mutations were found in the APC (71.4%), TP53 (56.0%), KRAS (52.7%), PIK3CA (15.4%) and FBXW7 (10.9%) genes. Overall, genes in the MAPK/ERK, PIK3/AKT, NOTCH and receptor tyrosine kinase signaling pathways were mutated in 68.0%, 23.1%, 16.5%, and 15.3% of patients, respectively. MSI was found in 13.3% of tumors, most of which were proximal (52.4%, P< 0.001) and had a high mutation burden. European genetic ancestry was predominant (median of 83.1%), followed by Native American (4.1%), Asian (3.4%) and African (3.2%). NF1 and BRAF mutations were associated with African ancestry, while TP53 and PIK3CA mutations were inversely correlated with Native American ancestry. Our study suggests that Brazilian CRC patients exhibit a mutation profile similar to other populations and identify the most frequently mutated genes, which could be useful in future target therapies and molecular cancer screening strategies.We are thankful to Barretos Cancer Hospital. This work was supported by the Brazilian Federal Agency for the Support and Evaluation of Graduate Education (CAPES, Brazil), the National Council for Scientifc and Technological Development (CNPq, Brazil), and the Public Ministry of Labor Campinas (Research, Prevention and Education of Occupational Cancer, Brazil)

Abstract PO-031: Aldolase A (ALDOA) is required for efficient DNA double-strand break (DSB) repair

Introduction: Metabolic reprogramming, known as the Warburg effect, is one of the universal differences between cancer cells and non-cancerous cells. Glucose metabolism and DNA repair are frequently dysregulated in cancer. Metabolic pathways provide cells with nucleic acids and energy required to repair DNA. However, the underlying mechanisms that promote crosstalk between these processes are unknown. ALDOA is a glycolytic enzyme that catalyses the conversion of fructose 1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. ALDOA is overexpressed in several types of cancer. In this study, we demonstrate a novel mechanism through which ALDOA directly regulates DSB repair. Methods: ALDOA was depleted from cells using siRNA and single-shot quantitative proteomics were performed. Immunofluorescence was utilized to determine the localization of ALDOA. DSB repair reporter assays were used to measure DSB break repair. Gene expression was quantified by western blot and qPCR. Immunoprecipitations were used to detect protein:protein interactions. Statistical analysis: The experiments were at least n=3, and data are presented as the means ± SEM. Statistical significance was evaluated using Student’s t-test or one-way ANOVA. Results: In order to identify ALDOA-dependent pathways, we performed quantitative mass spectrometry on ALDOA depleted cells. In addition to the expected decrease in glycolysis pathways, we also observed a significant downregulation of DNA repair proteins in ALDOA depleted cells. Further analysis showed that the ALDOA protein responds to DNA damage (IR) and migrates from the cytosol to the nucleus, suggesting that it could be directly involved in DNA damage repair. Slower clearance of γ-H2AX foci (a DSB marker), and decreased clonogenicity following irradiation (IR) treatment were also observed, indicating dysfunctional DNA repair processes. Repair of DSBs is primarily though the NHEJ (non-homologous end-joining) or HR (homologous recombination) -mediated DNA repair pathways. Silencing ALDOA led to a decrease in both NHEJ- and HR-mediated DSB repair efficiency. This disruption was likely due to the significant reduction of both the mRNA and protein of the DNA repair effector kinases, DNA-Dependent Kinase (DNA-PK) and Ataxia and Telangiectasia Mutated (ATM) in ALDOA-depleted cells. In addition to regulating the expression of DNAPK and ATM we also found that ALDOA directly interacted with both kinases, suggesting that it may have a direct role in regulating their function. Here, we define a role for ALDOA in the repair of DNA DSBs, through the regulation of DNA repair effector kinase expression and function. Conclusion: These results identify crosstalk between metabolic and DNA repair pathways and have implications for cancer treatment and tumorigenesis. The role of ALDOA in DNA repair could promote therapeutic resistance in tumors and may be a future therapeutic target to sensitize tumors to DNA-damaging agents such as radiation

Cell Metabolism and DNA Repair Pathways: Implications for Cancer Therapy

DNA repair and metabolic pathways are vital to maintain cellular homeostasis in normal human cells. Both of these pathways, however, undergo extensive changes during tumorigenesis, including modifications that promote rapid growth, genetic heterogeneity, and survival. While these two areas of research have remained relatively distinct, there is growing evidence that the pathways are interdependent and intrinsically linked. Therapeutic interventions that target metabolism or DNA repair systems have entered clinical practice in recent years, highlighting the potential of targeting these pathways in cancer. Further exploration of the links between metabolic and DNA repair pathways may open new therapeutic avenues in the future. Here, we discuss the dependence of DNA repair processes upon cellular metabolism; including the production of nucleotides required for repair, the necessity of metabolic pathways for the chromatin remodeling required for DNA repair, and the ways in which metabolism itself can induce and prevent DNA damage. We will also discuss the roles of metabolic proteins in DNA repair and, conversely, how DNA repair proteins can impact upon cell metabolism. Finally, we will discuss how further research may open therapeutic avenues in the treatment of cancer.</p

The fructose-bisphosphate, Aldolase A (ALDOA), facilitates DNA-PKcs and ATM kinase activity to regulate DNA double-strand break repair

Abstract Glucose metabolism and DNA repair are fundamental cellular processes frequently dysregulated in cancer. In this study, we define a direct role for the glycolytic Aldolase A (ALDOA) protein in DNA double-strand break (DSB) repair. ALDOA is a fructose biphosphate Aldolase that catalyses fructose-1,6-bisphosphate to glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP), during glycolysis. Here, we show that upon DNA damage induced by ionising radiation (IR), ALDOA translocates from the cytoplasm into the nucleus, where it partially co-localises with the DNA DSB marker γ-H2AX. DNA damage was shown to be elevated in ALDOA-depleted cells prior to IR and following IR the damage was repaired more slowly. Consistent with this, cells depleted of ALDOA exhibited decreased DNA DSB repair via non-homologous end-joining and homologous recombination. In support of the defective repair observed in its absence, ALDOA was found to associate with the major DSB repair effector kinases, DNA-dependent Protein Kinase (DNA-PK) and Ataxia Telangiectasia Mutated (ATM) and their autophosphorylation was decreased when ALDOA was depleted. Together, these data establish a role for an essential metabolic protein, ALDOA in DNA DSB repair and suggests that targeting ALDOA may enable the concurrent targeting of cancer metabolism and DNA repair to induce tumour cell death

Barrier-to-autointegration-factor (Banf1) modulates DNA double-strand break repair pathway choice via regulation of DNA-dependent kinase (DNA-PK) activity

DNA repair pathways are essential to maintain the integrity of the genome and prevent cell death and tumourigenesis. Here, we show that the Barrier-to-Autointegration Factor (Banf1) protein has a role in the repair of DNA double-strand breaks. Banf1 is characterized as a nuclear envelope protein and mutations in Banf1 are associated with the severe premature aging syndrome, Néstor–Guillermo Progeria Syndrome. We have previously shown that Banf1 directly regulates the activity of PARP1 in the repair of oxidative DNA lesions. Here, we show that Banf1 also has a role in modulating DNA double-strand break repair through regulation of the DNA-dependent Protein Kinase catalytic subunit, DNA-PKcs. Specifically, we demonstrate that Banf1 relocalizes from the nuclear envelope to sites of DNA double-strand breaks. We also show that Banf1 can bind to and directly inhibit the activity of DNA-PKcs. Supporting this, cellular depletion of Banf1 leads to an increase in non-homologous end-joining and a decrease in homologous recombination, which our data suggest is likely due to unrestrained DNA-PKcs activity. Overall, this study identifies how Banf1 regulates double-strand break repair pathway choice by modulating DNA-PKcs activity to control genome stability within the cell

Whole transcriptome profiling of placental pathobiology in SARS‐CoV‐2 pregnancies identifies placental dysfunction signatures

ObjectivesSevere Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus infection in pregnancy is associated with higher incidence of placental dysfunction, referred to by a few studies as a ‘preeclampsia-like syndrome’. However, the mechanisms underpinning SARS-CoV-2-induced placental malfunction are still unclear. Here, we investigated whether the transcriptional architecture of the placenta is altered in response to SARS-CoV-2 infection.MethodsWe utilised whole-transcriptome, digital spatial profiling, to examine gene expression patterns in placental tissues from participants who contracted SARS-CoV-2 in the third trimester of their pregnancy (n = 7) and those collected prior to the start of the coronavirus disease 2019 (COVID-19) pandemic (n = 9).ResultsThrough comprehensive spatial transcriptomic analyses of the trophoblast and villous core stromal cell subpopulations in the placenta, we identified SARS-CoV-2 to promote signatures associated with hypoxia and placental dysfunction. Notably, genes associated with vasodilation (NOS3), oxidative stress (GDF15, CRH) and preeclampsia (FLT1, EGFR, KISS1, PAPPA2) were enriched with SARS-CoV-2. Pathways related to increased nutrient uptake, vascular tension, hypertension and inflammation were also enriched in SARS-CoV-2 samples compared to uninfected controls.ConclusionsOur findings demonstrate the utility of spatially resolved transcriptomic analysis in defining the underlying pathogenic mechanisms of SARS-CoV-2 in pregnancy, particularly its role in placental dysfunction. Furthermore, this study highlights the significance of digital spatial profiling in mapping the intricate crosstalk between trophoblasts and villous core stromal cells, thus shedding light on pathways associated with placental dysfunction in pregnancies with SARS-CoV-2 infection

Whole transcriptome profiling of placental pathobiology in SARS-CoV-2 pregnancies identifies placental dysfunction signatures

Objectives: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus infection in pregnancy is associated with higher incidence of placental dysfunction, referred to by a few studies as a ‘preeclampsia-like syndrome’. However, the mechanisms underpinning SARS-CoV-2-induced placental malfunction are still unclear. Here, we investigated whether the transcriptional architecture of the placenta is altered in response to SARS-CoV-2 infection. Methods: We utilised whole-transcriptome, digital spatial profiling, to examine gene expression patterns in placental tissues from participants who contracted SARS-CoV-2 in the third trimester of their pregnancy (n = 7) and those collected prior to the start of the coronavirus disease 2019 (COVID-19) pandemic (n = 9). Results: Through comprehensive spatial transcriptomic analyses of the trophoblast and villous core stromal cell subpopulations in the placenta, we identified SARS-CoV-2 to promote signatures associated with hypoxia and placental dysfunction. Notably, genes associated with vasodilation (NOS3), oxidative stress (GDF15, CRH) and preeclampsia (FLT1, EGFR, KISS1, PAPPA2) were enriched with SARS-CoV-2. Pathways related to increased nutrient uptake, vascular tension, hypertension and inflammation were also enriched in SARS-CoV-2 samples compared to uninfected controls. Conclusions: Our findings demonstrate the utility of spatially resolved transcriptomic analysis in defining the underlying pathogenic mechanisms of SARS-CoV-2 in pregnancy, particularly its role in placental dysfunction. Furthermore, this study highlights the significance of digital spatial profiling in mapping the intricate crosstalk between trophoblasts and villous core stromal cells, thus shedding light on pathways associated with placental dysfunction in pregnancies with SARS-CoV-2 infection.</p