Depletion of ATP-citrate lyase (ATPCL) affects chromosome integrity without altering histone acetylation in Drosophila mitotic cells

The Citrate Lyase (ACL) is the main cytosolic enzyme that converts the citrate exported from mitochondria by the SLC25A1 carrier in Acetyl Coenzyme A (acetyl-CoA) and oxaloacetate. Acetyl-CoA is a high-energy intermediate common to a large number of metabolic processes including protein acetylation reactions. This renders ACL a key regulator of histone acetylation levels and gene expression in diverse organisms including humans. We have found that depletion of Drosophila ATPCL, the fly ortholog of human ACL, reduced levels of Acetyl CoA but, unlike its human counterpart, does not affect global histone acetylation and gene expression. Nevertheless, reduced ATPCL levels caused evident, although moderate, mitotic chromosome breakage suggesting that this enzyme plays a partial role in chromosome stability. These defects did not increase upon X-ray irradiation, indicating that they are not dependent on an impairment of DNA repair. Interestingly, depletion of ATPCL drastically increased the frequency of chromosome breaks associated to mutations in scheggia, which encodes the ortholog of the mitochondrial citrate carrier SLC25A1 that is also required for chromosome integrity and histone acetylation. Our results indicate that ATPCL has a dispensable role in histone acetylation and prevents massive chromosome fragmentation when citrate efflux is altered

Depletion of ATP-Citrate Lyase (ATPCL) Affects Chromosome Integrity Without Altering Histone Acetylation in Drosophila Mitotic Cells

The Citrate Lyase (ACL) is the main cytosolic enzyme that converts the citrate exported from mitochondria by the SLC25A1 carrier in Acetyl Coenzyme A (acetyl-CoA) and oxaloacetate. Acetyl-CoA is a high-energy intermediate common to a large number of metabolic processes including protein acetylation reactions. This renders ACL a key regulator of histone acetylation levels and gene expression in diverse organisms including humans. We have found that depletion of ATPCL, the Drosophila ortholog of human ACL, reduced levels of Acetyl CoA but, unlike its human counterpart, does not affect global histone acetylation and gene expression. Nevertheless, reduced ATPCL levels caused evident, although moderate, mitotic chromosome breakage suggesting that this enzyme plays a partial role in chromosome stability. These defects did not increase upon X-ray irradiation, indicating that they are not dependent on an impairment of DNA repair. Interestingly, depletion of ATPCL drastically increased the frequency of chromosome breaks (CBs) associated to mutations in scheggia, which encodes the ortholog of the mitochondrial citrate carrier SLC25A1 that is also required for chromosome integrity and histone acetylation. Our results indicate that ATPCL has a dispensable role in histone acetylation and prevents massive chromosome fragmentation when citrate efflux is altered

The Drosophila Citrate Lyase Is Required for Cell Division during Spermatogenesis

The Drosophila melanogaster DmATPCL gene encodes for the human ATP Citrate Lyase (ACL) ortholog, a metabolic enzyme that from citrate generates glucose-derived Acetyl-CoA, which fuels central biochemical reactions such as the synthesis of fatty acids, cholesterol and acetylcholine, and the acetylation of proteins and histones. We had previously reported that, although loss of Drosophila ATPCL reduced levels of Acetyl-CoA, unlike its human counterpart, it does not affect global histone acetylation and gene expression, suggesting that its role in histone acetylation is either partially redundant in Drosophila or compensated by alternative pathways. Here, we describe that depletion of DmATPCL affects spindle organization, cytokinesis, and fusome assembly during male meiosis, revealing an unanticipated role for DmATPCL during spermatogenesis. We also show that DmATPCL mutant meiotic phenotype is in part caused by a reduction of fatty acids, but not of triglycerides or cholesterol, indicating that DmATPCL-derived Acetyl-CoA is predominantly devoted to the biosynthesis of fatty acids during spermatogenesis. Collectively, our results unveil for the first time an involvement for DmATPCL in the regulation of meiotic cell division, which is likely conserved in human cells

Radioresistance in rhabdomyosarcomas: Much more than a question of dose

Management of rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children, frequently accounting the genitourinary tract is complex and requires a multimodal therapy. In particular, as a consequence of the advancement in dose conformity technology, radiation therapy (RT) has now become the standard therapeutic option for patients with RMS. In the clinical practice, dose and timing of RT are adjusted on the basis of patients' risk stratification to reduce late toxicity and side effects on normal tissues. However, despite the substantial improvement in cure rates, local failure and recurrence frequently occur. In this review, we summarize the general principles of the treatment of RMS, focusing on RT, and the main molecular pathways and specific proteins involved into radioresistance in RMS tumors. Specifically, we focused on DNA damage/repair, reactive oxygen species, cancer stem cells, and epigenetic modifications that have been reported in the context of RMS neoplasia in both in vitro and in vivo studies. The precise elucidation of the radioresistance-related molecular mechanisms is of pivotal importance to set up new more effective and tolerable combined therapeutic approaches that can radiosensitize cancer cells to finally ameliorate the overall survival of patients with RMS, especially for the most aggressive subtypes

Underground radiobiology: a perspective at Gran Sasso National Laboratory

Scientific community and institutions (e. g., ICRP) consider that the Linear No-Threshold (LNT) model, which extrapolates stochastic risk at low dose/low dose rate from the risk at moderate/high doses, provides a prudent basis for practical purposes of radiological protection. However, biological low dose/dose rate responses that challenge the LNT model have been highlighted and important dowels came from radiobiology studies conducted in Deep Underground Laboratories (DULs). These extreme ultra-low radiation environments are ideal locations to conduct below-background radiobiology experiments, interesting from basic and applied science. The INFN Gran Sasso National Laboratory (LNGS) (Italy) is the site where most of the underground radiobiological data has been collected so far and where the first in vivo underground experiment was carried out using Drosophila melanogaster as model organism. Presently, many DULs around the world have implemented dedicated programs, meetings and proposals. The general message coming from studies conducted in DULs using protozoan, bacteria, mammalian cells and organisms (flies, worms, fishes) is that environmental radiation may trigger biological mechanisms that can increase the capability to cope against stress. However, several issues are still open, among them: the role of the quality of the radiation spectrum in modulating the biological response, the dependence on the biological endpoint and on the model system considered, the overall effect at organism level (detrimental or beneficial). At LNGS, we recently launched the RENOIR experiment aimed at improving knowledge on the environmental radiation spectrum and to investigate the specific role of the gamma component on the biological response of Drosophila melanogaster

Spermine oxidase induces DNA damage and sensitizes fusion negative rhabdomyosarcoma cells to irradiation

Rhabdomyosarcoma (RMS) is a pediatric myogenic soft tissue sarcoma that includes fusion-positive (FP) and fusion-negative (FN) molecular subtypes. FP-RMS expresses PAX3-FOXO1 fusion protein and often shows dismal prognosis. FN-RMS shows cytogenetic abnormalities and frequently harbors RAS pathway mutations. Despite the multimodal heavy chemo and radiation therapeutic regimens, high risk metastatic/recurrent FN-RMS shows a 5-year survival less than 30% due to poor sensitivity to chemo-radiotherapy. Therefore, the identification of novel targets is needed. Polyamines (PAs) such as putrescine (PUT), spermidine (SPD) and spermine (SPM) are low-molecular-mass highly charged molecules whose intracellular levels are strictly modulated by specific enzymes. Among the latter, spermine oxidase (SMOX) regulates polyamine catabolism oxidizing SPM to SPD, which impacts cellular processes such as apoptosis and DNA damage response. Here we report that low SMOX levels are associated with a worse outcome in FN-RMS, but not in FP-RMS, patients. Consistently, SMOX expression is downregulated in FN-RMS cell lines as compared to normal myoblasts. Moreover, SMOX transcript levels are reduced FN-RMS cells differentiation, being indirectly downregulated by the muscle transcription factor MYOD. Noteworthy, forced expression of SMOX in two cell lines derived from high-risk FN-RMS: 1) reduces SPM and upregulates SPD levels; 2) induces G0/G1 cell cycle arrest followed by apoptosis; 3) impairs anchorage-independent and tumor spheroids growth; 4) inhibits cell migration; 5) increases γH2AX levels and foci formation indicative of DNA damage. In addition, forced expression of SMOX and irradiation synergize at activating ATM and DNA-PKCs, and at inducing γH2AX expression and foci formation, which suggests an enhancement in DNA damage response. Irradiated SMOX-overexpressing FN-RMS cells also show significant decrease in both colony formation capacity and spheroids growth with respect to single approaches. Thus, our results unveil a role for SMOX as inhibitor of tumorigenicity of FN-RMS cells in vitro. In conclusion, our in vitro results suggest that SMOX induction could be a potential combinatorial approach to sensitize FN-RMS to ionizing radiation and deserve further in-depth studies

MYOD-SKP2 axis boosts tumorigenesis in fusion negative rhabdomyosarcoma by preventing differentiation through p57Kip2^{Kip2} targeting

Rhabdomyosarcomas (RMS) are pediatric mesenchymal-derived malignancies encompassing PAX3/7-FOXO1 Fusion Positive (FP)-RMS, and Fusion Negative (FN)-RMS with frequent RAS pathway mutations. RMS express the master myogenic transcription factor MYOD that, whilst essential for survival, cannot support differentiation. Here we discover SKP2, an oncogenic E3-ubiquitin ligase, as a critical pro-tumorigenic driver in FN-RMS. We show that SKP2 is overexpressed in RMS through the binding of MYOD to an intronic enhancer. SKP2 in FN-RMS promotes cell cycle progression and prevents differentiation by directly targeting p27$^{Kip1}$ and p57$^{Kip2}$, respectively. SKP2 depletion unlocks a partly MYOD-dependent myogenic transcriptional program and strongly affects stemness and tumorigenic features and prevents in vivo tumor growth. These effects are mirrored by the investigational NEDDylation inhibitor MLN4924. Results demonstrate a crucial crosstalk between transcriptional and post-translational mechanisms through the MYOD-SKP2 axis that contributes to tumorigenesis in FN-RMS. Finally, NEDDylation inhibition is identified as a potential therapeutic vulnerability in FN-RMS

The botanical drug PBI-05204, a supercritical CO2 extract of Nerium oleander, sensitizes alveolar and embryonal rhabdomyosarcoma to radiotherapy in vitro and in vivo

: Treatment of rhabdomyosarcoma (RMS), the most common a soft tissue sarcoma in childhood, provides intensive multimodal therapy, with radiotherapy (RT) playing a critical role for local tumor control. However, since RMS efficiently activates mechanisms of resistance to therapies, despite improvements, the prognosis remains still largely unsatisfactory, mainly in RMS expressing chimeric oncoproteins PAX3/PAX7-FOXO1, and fusion-positive (FP)-RMS. Cardiac glycosides (CGs), plant-derived steroid-like compounds with a selective inhibitory activity of the Na+/K+-ATPase pump (NKA), have shown antitumor and radio-sensitizing properties. Herein, the therapeutic properties of PBI-05204, an extract from Nerium oleander containing the CG oleandrin already studied in phase I and II clinical trials for cancer patients, were investigated, in vitro and in vivo, against FN- and FP-RMS cancer models. PBI-05204 induced growth arrest in a concentration dependent manner, with FP-RMS being more sensitive than FN-RMS, by differently regulating cell cycle regulators and commonly upregulating cell cycle inhibitors p21Waf1/Cip1 and p27Cip1/Kip1. Furthermore, PBI-05204 concomitantly induced cell death on both RMS types and senescence in FN-RMS. Notably, PBI-05204 counteracted in vitro migration and invasion abilities and suppressed the formation of spheroids enriched in CD133+ cancer stem cells (CSCs). PBI-05204 sensitized both cell types to RT by improving the ability of RT to induce G2 growth arrest and counteracting the RT-induced activation of both Non-Homologous End-Joining and homologous recombination DSBs repair pathways. Finally, the antitumor and radio-sensitizing proprieties of PBI-05204 were confirmed in vivo. Notably, both in vitro and in vivo evidence confirmed the higher sensitivity to PBI-05204 of FP-RMS. Thus, PBI-05204 represents a valid radio-sensitizing agent for the treatment of RMS, including the intrinsically radio-resistant FP-RMS

Reduced Environmental Dose Rates Are Responsible for the Increased Susceptibility to Radiation-Induced DNA Damage in Larval Neuroblasts of <i>Drosophila</i> Grown inside the LNGS Underground Laboratory

A large amount of evidence from radiobiology studies carried out in Deep Underground Laboratories support the view that environmental radiation may trigger biological mechanisms that enable both simple and complex organisms to cope with genotoxic stress. In line with this, here we show that the reduced radiation background of the LNGS underground laboratory renders Drosophila neuroblasts more sensitive to ionizing radiation-induced (but not to spontaneous) DNA breaks compared to fruit flies kept at the external reference laboratory. Interestingly, we demonstrate that the ionizing radiation sensitivity of flies kept at the LNGS underground laboratory is rescued by increasing the underground gamma dose rate to levels comparable to the low-LET reference one. This finding provides the first direct evidence that the modulation of the DNA damage response in a complex multicellular organism is indeed dependent on the environmental dose rate