A glycolytic metabolite bypasses "two-hit" tumor suppression by BRCA2.

Knudson's "two-hit" paradigm posits that carcinogenesis requires inactivation of both copies of an autosomal tumor suppressor gene. Here, we report that the glycolytic metabolite methylglyoxal (MGO) transiently bypasses Knudson's paradigm by inactivating the breast cancer suppressor protein BRCA2 to elicit a cancer-associated, mutational single-base substitution (SBS) signature in nonmalignant mammary cells or patient-derived organoids. Germline monoallelic BRCA2 mutations predispose to these changes. An analogous SBS signature, again without biallelic BRCA2 inactivation, accompanies MGO accumulation and DNA damage in Kras-driven, Brca2-mutant murine pancreatic cancers and human breast cancers. MGO triggers BRCA2 proteolysis, temporarily disabling BRCA2's tumor suppressive functions in DNA repair and replication, causing functional haploinsufficiency. Intermittent MGO exposure incites episodic SBS mutations without permanent BRCA2 inactivation. Thus, a metabolic mechanism wherein MGO-induced BRCA2 haploinsufficiency transiently bypasses Knudson's two-hit requirement could link glycolysis activation by oncogenes, metabolic disorders, or dietary challenges to mutational signatures implicated in cancer evolution.

The FGFR inhibitor PD173074 binds to the C-terminus of oncofetal HMGA2 and modulates its DNA-binding and transcriptional activation functions.

The architectural chromatin factor high-mobility group AT-hook 2 (HMGA2) is causally involved in several human malignancies and pathologies. HMGA2 is not expressed in most normal adult somatic cells, which renders the protein an attractive drug target. An established cell-based compound library screen identified the fibroblast growth factor receptor (FGFR) inhibitor PD173074 as an antagonist of HMGA2-mediated transcriptional reporter gene activation. We determined that PD173074 binds the C-terminus of HMGA2 and interferes with functional coordination of the three AT-hook DNA-binding domains mediated by the C-terminus. The HMGA2-antagonistic effect of PD173074 on transcriptional activation may therefore result from an induced altered DNA-binding mode of HMGA2. PD173074 as a novel HMGA2-specific antagonist could trigger the development of derivates with enhanced attributes and clinical potential.

Signalling inhibition by ponatinib disrupts productive alternative lengthening of telomeres (ALT).

Alternative lengthening of telomeres (ALT) supports telomere maintenance in 10-15% of cancers, thus representing a compelling target for therapy. By performing anti-cancer compound library screen on isogenic cell lines and using extrachromosomal telomeric C-circles, as a bona fide marker of ALT activity, we identify a receptor tyrosine kinase inhibitor ponatinib that deregulates ALT mechanisms, induces telomeric dysfunction, reduced ALT-associated telomere synthesis, and targets, in vivo, ALT-positive cells. Using RNA-sequencing and quantitative phosphoproteomic analyses, combined with C-circle level assessment, we find an ABL1-JNK-JUN signalling circuit to be inhibited by ponatinib and to have a role in suppressing telomeric C-circles. Furthermore, transcriptome and interactome analyses suggest a role of JUN in DNA damage repair. These results are corroborated by synergistic drug interactions between ponatinib and either DNA synthesis or repair inhibitors, such as triciribine. Taken together, we describe here a signalling pathway impacting ALT which can be targeted by a clinically approved drug.

Cell-free hemoglobin is a marker of systemic inflammation in mouse models of sepsis: a Raman spectroscopic study.

Sepsis is a life-threatening condition caused by heightened host immune responses post infection. Despite intensive research, most of the existing diagnostic methods remain non-specific, labour-intensive, time-consuming or are not sensitive enough for rapid and timely diagnosis of the onset and progression of sepsis. The present work was undertaken to explore the potential of Raman spectroscopy to identify the biomarkers of sepsis in a label-free and minimally invasive manner using different mouse models of inflammation. The sera of BALB/c mice infected with Salmonella Typhimurium reveal extensive hemolysis, as indicated by the Raman bands that are characteristic of the porphyrin ring of hemoglobin (668, 743, 1050, 1253 and 1397 cm-1) which increase in a kinetic manner. These markers are also observed in a lipopolysaccharide-induced endotoxic shock model, but not in a thioglycollate-induced sterile peritonitis model. These data demonstrate that hemolysis is a signature of systemic, but not localised, inflammation. To further validate our observations, sepsis was induced in the nitric oxide synthase 2 (Nos2-/-) deficient strain which is more sensitive to infection. Interestingly, Nos2-/- mice exhibit a higher degree of hemolysis than C57BL/6 mice. Sepsis-induced hemolysis was also confirmed using resonance Raman spectroscopy with 442 nm excitation which demonstrated a pronounced increase in the resonant Raman bands at 670 and 1350 cm-1 in sera of the infected mice. This is the first study to identify inflammation-induced hemolysis in mouse models of sepsis using Raman spectral signatures for hemoglobin. The possible implications of this method in detecting hemolysis in different inflammatory pathologies, such as the ongoing COVID-19 pandemic, are discussed.

Chromatin Architectural Factors as Safeguards against Excessive Supercoiling during DNA Replication.

Key DNA transactions, such as genome replication and transcription, rely on the speedy translocation of specialized protein complexes along a double-stranded, right-handed helical template. Physical tethering of these molecular machines during translocation, in conjunction with their internal architectural features, generates DNA topological strain in the form of template supercoiling. It is known that the build-up of transient excessive supercoiling poses severe threats to genome function and stability and that highly specialized enzymes-the topoisomerases (TOP)-have evolved to mitigate these threats. Furthermore, due to their intracellular abundance and fast supercoil relaxation rates, it is generally assumed that these enzymes are sufficient in coping with genome-wide bursts of excessive supercoiling. However, the recent discoveries of chromatin architectural factors that play important accessory functions have cast reasonable doubts on this concept. Here, we reviewed the background of these new findings and described emerging models of how these accessory factors contribute to supercoil homeostasis. We focused on DNA replication and the generation of positive (+) supercoiling in front of replisomes, where two accessory factors-GapR and HMGA2-from pro- and eukaryotic cells, respectively, appear to play important roles as sinks for excessive (+) supercoiling by employing a combination of supercoil constrainment and activation of topoisomerases. Looking forward, we expect that additional factors will be identified in the future as part of an expanding cellular repertoire to cope with bursts of topological strain. Furthermore, identifying antagonists that target these accessory factors and work synergistically with clinically relevant topoisomerase inhibitors could become an interesting novel strategy, leading to improved treatment outcomes.

Transcarotid Endovascular Repair of Extracranial Internal Carotid Artery Aneurysms Using Flow Reversal for Distal Embolic Protection.

Extracranial carotid artery aneurysms (ECAAs) have a low incidence between 0.4% and 4% of all peripheral artery aneurysms and involving 0.1-2% of all carotid artery procedures. Some form of repair is generally warranted as nonoperative management has shown mortality as high as 71%. However, to date a standard method for ECAA repair has not been suggested. Generally, open surgical repair is the preferred technique; however, it has its own limitations and risks. Recently, endovascular approach is increasingly being used not only for the elective repair of unruptured ECAA but also for the management of ruptured ECAA. Herein we present 3 cases of distal extracranial internal carotid artery aneurysms treated with placement of stent grafts.

Oncofetal HMGA2 attenuates genotoxic damage induced by topoisomerase II target compounds through the regulation of local DNA topology.

Rapidly dividing cells maintain chromatin supercoiling homeostasis via two specialized classes of enzymes, DNA topoisomerase type 1 and 2 (TOP1/2). Several important anticancer drugs perturb this homeostasis by targeting TOP1/2, thereby generating genotoxic DNA damage. Our recent studies indicated that the oncofetal chromatin structuring high-mobility group AT-hook 2 (HMGA2) protein plays an important role as a DNA replication fork chaperone in coping with DNA topological ramifications that occur during replication stress, both genomewide and at fragile sites such as subtelomeres. Intriguingly, a recent large-scale clinical study identified HMGA2 expression as a sole predicting marker for relapse and poor clinical outcomes in 350 acute myeloid leukemia (AML) patients receiving combinatorial treatments that targeted TOP2 and replicative DNA synthesis. Here, we demonstrate that HMGA2 significantly enhanced the DNA supercoil relaxation activity of the drug target TOP2A and that this activator function is mechanistically linked to HMGA2's known ability to constrain DNA supercoils within highly compacted ternary complexes. Furthermore, we show that HMGA2 significantly reduced genotoxic DNA damage in each tested cancer cell model during treatment with the TOP2A poison etoposide or the catalytic TOP2A inhibitor merbarone. Taken together with the recent clinical data obtained with AML patients targeted with TOP2 poisons, our study suggests a novel mechanism of cancer chemoresistance toward combination therapies administering TOP2 poisons or inhibitors. We therefore strongly argue for the future implementation of trials of HMGA2 expression profiling to stratify patients before finalizing clinical treatment regimes.

The chromatin structuring protein HMGA2 influences human subtelomere stability and cancer chemosensitivity.

The transient build-up of DNA supercoiling during the translocation of replication forks threatens genome stability and is controlled by DNA topoisomerases (TOPs). This crucial process has been exploited with TOP poisons for cancer chemotherapy. However, pinpointing cellular determinants of the best clinical response to TOP poisons still remains enigmatic. Here, we present an integrated approach and demonstrate that endogenous and exogenous expression of the oncofetal high-mobility group AT-hook 2 (HMGA2) protein exhibited broad protection against the formation of hydroxyurea-induced DNA breaks in various cancer cells, thus corroborating our previously proposed model in which HMGA2 functions as a replication fork chaperone that forms a protective DNA scaffold at or close to stalled replication forks. We now further demonstrate that high levels of HMGA2 also protected cancer cells against DNA breaks triggered by the clinically important TOP1 poison irinotecan. This protection is most likely due to the recently identified DNA supercoil constraining function of HMGA2 in combination with exclusion of TOP1 from binding to supercoiled substrate DNA. In contrast, low to moderate HMGA2 protein levels surprisingly potentiated the formation of irinotecan-induced genotoxic covalent TOP1-DNA cleavage complexes. Our data from cell-based and several in vitro assays indicate that, mechanistically, this potentiating role involves enhanced drug-target interactions mediated by HMGA2 in ternary complexes with supercoiled DNA. Subtelomeric regions were found to be extraordinarily vulnerable to these genotoxic challenges induced by TOP1 poisoning, pointing at strong DNA topological barriers located at human telomeres. These findings were corroborated by an increased irinotecan sensitivity of patient-derived xenografts of colorectal cancers exhibiting low to moderate HMGA2 levels. Collectively, we uncovered a therapeutically important control mechanism of transient changes in chromosomal DNA topology that ultimately leads to enhanced human subtelomere stability.

The RNA interactome of human telomerase RNA reveals a coding-independent role for a histone mRNA in telomere homeostasis.

Telomerase RNA (TR) provides the template for DNA repeat synthesis at telomeres and is essential for genome stability in continuously dividing cells. We mapped the RNA interactome of human TR (hTR) and identified a set of non-coding and coding hTR-interacting RNAs, including the histone 1C mRNA (HIST1H1C). Disruption of the hTR-HIST1H1C RNA association resulted in markedly increased telomere elongation without affecting telomerase enzymatic activity. Conversely, over-expression of HIST1H1C led to telomere attrition. By using a combination of mutations to disentangle the effects of histone 1 RNA synthesis, protein expression, and hTR interaction, we show that HIST1H1C RNA negatively regulates telomere length independently of its protein coding potential. Taken together, our data provide important insights into a surprisingly complex hTR-RNA interaction network and define an unexpected non-coding RNA role for HIST1H1C in regulating telomere length homeostasis, thus offering a glimpse into the mostly uncharted, vast space of non-canonical messenger RNA functions.

Differential susceptibility and maturation of thymocyte subsets during Salmonella Typhimurium infection: insights on the roles of glucocorticoids and Interferon-gamma.

The thymus is known to atrophy during infections; however, a systematic study of changes in thymocyte subpopulations has not been performed. This aspect was investigated, using multi-color flow cytometry, during oral infection of mice with Salmonella Typhimurium (S. Typhimurium). The major highlights are: First, a block in the developmental pathway of CD4-CD8- double negative (DN) thymocytes is observed. Second, CD4+CD8+ double positive (DP) thymocytes, mainly in the DP1 (CD5loCD3lo) and DP2 (CD5hiCD3int), but not DP3 (CD5intCD3hi), subsets are reduced. Third, single positive (SP) thymocytes are more resistant to depletion but their maturation is delayed, leading to accumulation of CD24hiCD3hi SP. Kinetic studies during infection demonstrated differences in sensitivity of thymic subpopulations: Immature single positive (ISP) > DP1, DP2 > DN3, DN4 > DN2 > CD4+ > CD8+. Upon infection, glucocorticoids (GC), inflammatory cytokines, e.g. Ifnγ, etc are induced, which enhance thymocyte death. Treatment with RU486, the GC receptor antagonist, increases the survival of most thymic subsets during infection. Studies with Ifnγ-/- mice demonstrated that endogenous Ifnγ produced during infection enhances the depletion of DN2-DN4 subsets, promotes the accumulation of DP3 and delays the maturation of SP thymocytes. The implications of these observations on host cellular responses during infections are discussed.

Molecular profiling of sepsis in mice using Fourier Transform Infrared Microspectroscopy.

Sepsis is a life threatening condition resulting from a high burden of infection. It is a major health care problem and associated with inflammation, organ dysfunction and significant mortality. However, proper understanding and delineating the changes that occur during this complex condition remains a challenge. A comparative study involving intra-peritoneal injection of BALB/c mice with Salmonella Typhimurium (infection), lipopolysaccharide (endotoxic shock) or thioglycollate (sterile peritonitis) was performed. The changes in organs and sera were profiled using immunological assays and Fourier Transform Infrared (FTIR) micro-spectroscopy. There is a rapid rise in inflammatory cytokines accompanied with lowering of temperature, respiratory rate and glucose amounts in mice injected with S. Typhimurium or lipopolysaccharide. FTIR identifies distinct changes in liver and sera: decrease in glycogen and protein/lipid ratio and increase in DNA and cholesteryl esters. These changes were distinct from the pattern observed in mice treated with thioglycollate and the differences in the data obtained between the three models are discussed. The combination of FTIR spectroscopy and other biomarkers will be valuable in monitoring molecular changes during sepsis.