Approval of DFMO for high-risk neuroblastoma patients demonstrates a step of success to target MYC pathway.

The "undruggable" MYC oncoproteins are deregulated in 70% human cancers. The approval of DFMO, an irreversible inhibitor of ornithine oxidase (ODC1) that is a direct transcriptional target of MYC, demonstrates that patients can benefit from targeting MYC activity via an indirect approach. However, the mechanism of action of DFMO needs further studies to understand how it works in post-immunotherapy neuroblastomas. Efforts to develop a more potent and safer drug to block MYC function will continue despite challenges.

Impact of Difluoromethylornithine and AMXT 1501 on Gene Expression and Capsule Regulation in Streptococcus pneumoniae.

Streptococcus pneumoniae (Spn), a Gram-positive bacterium, poses a significant threat to human health, causing mild respiratory infections to severe invasive conditions. Despite the availability of vaccines, challenges persist due to serotype replacement and antibiotic resistance, emphasizing the need for alternative therapeutic strategies. This study explores the intriguing role of polyamines, ubiquitous, small organic cations, in modulating virulence factors, especially the capsule, a crucial determinant of Spn's pathogenicity. Using chemical inhibitors, difluoromethylornithine (DFMO) and AMXT 1501, this research unveils distinct regulatory effects on the gene expression of the Spn D39 serotype in response to altered polyamine homeostasis. DFMO inhibits polyamine biosynthesis, disrupting pathways associated with glucose import and the interconversion of sugars. In contrast, AMXT 1501, targeting polyamine transport, enhances the expression of polyamine and glucose biosynthesis genes, presenting a novel avenue for regulating the capsule independent of glucose availability. Despite ample glucose availability, AMXT 1501 treatment downregulates the glycolytic pathway, fatty acid synthesis, and ATP synthase, crucial for energy production, while upregulating two-component systems responsible for stress management. This suggests a potential shutdown of energy production and capsule biosynthesis, redirecting resources towards stress management. Following DFMO and AMXT 1501 treatments, countermeasures, such as upregulation of stress response genes and ribosomal protein, were observed but appear to be insufficient to overcome the deleterious effects on capsule production. This study highlights the complexity of polyamine-mediated regulation in S. pneumoniae, particularly capsule biosynthesis. Our findings offer valuable insights into potential therapeutic targets for modulating capsules in a polyamine-dependent manner, a promising avenue for intervention against S. pneumoniae infections.

Discordant Effects of Polyamine Depletion by DENSpm and DFMO on ß-cell Cytokine Stress and Diabetes Outcomes in Mice.

Type 1 diabetes (T1D) is an autoimmune disease leading to dysfunction and loss of insulin-secreting ß cells. In ß cells, polyamines have been implicated in causing cellular stress and dysfunction. An inhibitor of polyamine biosynthesis, difluoromethylornithine (DFMO), has been shown to delay T1D in mouse models and preserve ß-cell function in humans with recent-onset T1D. Another small molecule, N1,N11-diethylnorspermine (DENSpm), both inhibits polyamine biosynthesis and accelerates polyamine metabolism and is being tested for efficacy in cancer clinical trials. In this study, we show that DENSpm depletes intracellular polyamines as effectively as DFMO in mouse ß cells. RNA-sequencing analysis, however, suggests that the cellular responses to DENSpm and DFMO differ, with both showing effects on cellular proliferation but the latter showing additional effects on mRNA translation and protein-folding pathways. In the low-dose streptozotocin-induced mouse model of T1D, DENSpm, unlike DFMO, did not prevent or delay diabetes outcomes but did result in improvements in glucose tolerance and reductions in islet oxidative stress. In nonobese diabetic (NOD) mice, short-term DENSpm administration resulted in a slight reduction in insulitis and proinflammatory Th1 cells in the pancreatic lymph nodes. Longer term treatment resulted in a dose-dependent increase in mortality. Notwithstanding the efficacy of both DFMO and DENSpm in reducing potentially toxic polyamine levels in ß cells, our results highlight the discordant T1D outcomes that result from differing mechanisms of polyamine depletion and, more importantly, that toxic effects of DENSpm may limit its utility in T1D treatment.

Eflornithine as Postimmunotherapy Maintenance in High-Risk Neuroblastoma: Externally Controlled, Propensity Score-Matched Survival Outcome Comparisons.

PURPOSE: Long-term survival in high-risk neuroblastoma (HRNB) is approximately 50%, with mortality primarily driven by relapse. Eflornithine (DFMO) to reduce risk of relapse after completion of immunotherapy was investigated previously in a single-arm, phase II study (NMTRC003B; ClinicalTrials.gov identifier: NCT02395666) that suggested improved event-free survival (EFS) and overall survival (OS) compared with historical rates in a phase III trial (Children Oncology Group ANBL0032; ClinicalTrials.gov identifier: NCT00026312). Using patient-level data from ANBL0032 as an external control, we present new analyses to further evaluate DFMO as HRNB postimmunotherapy maintenance. PATIENTS AND METHODS: NMTRC003B (2012-2016) enrolled patients with HRNB (N = 141) after standard up-front or refractory/relapse treatment who received up to 2 years of continuous treatment with oral DFMO (750 ± 250 mg/m2 twice a day). ANBL0032 (2001-2015) enrolled patients with HRNB postconsolidation, 1,328 of whom were assigned to dinutuximab (ch.14.18) treatment. Selection rules identified 92 NMTRC003B patients who participated in (n = 87) or received up-front treatment consistent with (n = 5) ANBL0032 (the DFMO/treated group) and 852 patients from ANBL0032 who could have been eligible for NMTRC003B after immunotherapy, but did not enroll (the NO-DFMO/control group). The median follow-up time for DFMO/treated patients was 6.1 years (IQR, 5.2-7.2) versus 5.0 years (IQR, 3.5-7.0) for NO-DFMO/control patients. Kaplan-Meier and Cox regression compared EFS and OS for overall groups, 3:1 (NO-DFMO:DFMO) propensity score-matched cohorts balanced on 11 baseline demographic and disease characteristics with exact matching on MYCN, and additional sensitivity analyses. RESULTS: DFMO after completion of immunotherapy was associated with improved EFS (hazard ratio [HR], 0.50 [95% CI, 0.29 to 0.84]; P = .008) and OS (HR, 0.38 [95% CI, 0.19 to 0.76]; P = .007). The results were confirmed with propensity score-matched cohorts and sensitivity analyses. CONCLUSION: The externally controlled analyses presented show a relapse risk reduction in patients with HRNB treated with postimmunotherapy DFMO.

Bachmann-Bupp syndrome and treatment.

Bachmann-Bupp syndrome (BABS) is a neurodevelopmental disorder characterized by developmental delay, hypotonia, and varying forms of non-congenital alopecia. The condition is caused by 3'-end mutations of the ornithine decarboxylase 1 (ODC1) gene, which produce carboxy (C)-terminally truncated variants of ODC, a pyridoxal 5'-phosphate-dependent enzyme. C-terminal truncation of ODC prevents its ubiquitin-independent proteasomal degradation and leads to cellular accumulation of ODC enzyme that remains catalytically active. ODC is the first rate-limiting enzyme that converts ornithine to putrescine in the polyamine pathway. Polyamines (putrescine, spermidine, spermine) are aliphatic molecules found in all forms of life and are important during embryogenesis, organogenesis, and tumorigenesis. BABS is an ultra-rare condition with few reported cases, but it serves as a convincing example for drug repurposing therapy. α-Difluoromethylornithine (DFMO, also known as eflornithine) is an ODC inhibitor with a strong safety profile in pediatric use for neuroblastoma and other cancers as well as West African sleeping sickness (trypanosomiasis). Patients with BABS have been treated with DFMO and have shown improvement in hair growth, muscle tone, and development.

Intracellular polyamine depletion induces N-linked galactosylation of the monoclonal antibody produced by CHO DP-12 cells.

The heterogeneity of the N-linked glycan profile of therapeutic monoclonal antibodies (mAbs) derived from animal cells affects therapeutic efficacy and, therefore, needs to be appropriately controlled during the manufacturing process. In this study, we examined the effects of polyamines on the N-linked glycan profiles of mAbs produced by CHO DP-12 cells. Normal cell growth of CHO DP-12 cells and their growth arrest by α-difluoromethylornithine (DFMO), an inhibitor of the polyamine biosynthetic pathway, was observed when 0.5% fetal bovine serum was added to serum-free medium, despite the presence of cadaverine and aminopropylcadaverine, instead of putrescine and spermidine in cells. Polyamine depletion by DFMO increased IgG galactosylation, accompanied by ß1,4-galactosyl transferase 1 (B4GAT1) mRNA elevation. Additionally, IgG production in polyamine-depleted cells was reduced by 30% compared to that in control cells. Therefore, we examined whether polyamine depletion induces an ER stress response. The results indicated increased expression levels of chaperones for glycoprotein folding in polyamine-depleted cells, suggesting that polyamine depletion causes ER stress related to glycoprotein folding. The effect of tunicamycin, an ER stress inducer that inhibits N-glycosylation, on the expression of B4GALT1 mRNA was examined. Tunicamycin treatment increased B4GALT1 mRNA expression. These results suggest that ER stress caused by polyamine depletion induces B4GALT1 mRNA expression, resulting in increased IgG galactosylation in CHO cells. Thus, introducing polyamines, particularly SPD, to serum-free CHO culture medium for CHO cells may contribute to consistent manufacturing and quality control of antibody production.

Inhibition of polyamine biosynthesis preserves ß cell function in type 1 diabetes.

In preclinical models, α-difluoromethylornithine (DFMO), an ornithine decarboxylase (ODC) inhibitor, delays the onset of type 1 diabetes (T1D) by reducing ß cell stress. However, the mechanism of DFMO action and its human tolerability remain unclear. In this study, we show that mice with ß cell ODC deletion are protected against toxin-induced diabetes, suggesting a cell-autonomous role of ODC during ß cell stress. In a randomized controlled trial (ClinicalTrials.gov: NCT02384889) involving 41 recent-onset T1D subjects (3:1 drug:placebo) over a 3-month treatment period with a 3-month follow-up, DFMO (125-1,000 mg/m2) is shown to meet its primary outcome of safety and tolerability. DFMO dose-dependently reduces urinary putrescine levels and, at higher doses, preserves C-peptide area under the curve without apparent immunomodulation. Transcriptomics and proteomics of DFMO-treated human islets exposed to cytokine stress reveal alterations in mRNA translation, nascent protein transport, and protein secretion. These findings suggest that DFMO may preserve ß cell function in T1D through islet cell-autonomous effects.

Difluoromethylornithine rebalances aberrant polyamine ratios in Snyder-Robinson syndrome.

Snyder-Robinson syndrome (SRS) results from mutations in spermine synthase (SMS), which converts the polyamine spermidine into spermine. Affecting primarily males, common manifestations of SRS include intellectual disability, osteoporosis, hypotonia, and seizures. Symptom management is the only treatment. Reduced SMS activity causes spermidine accumulation while spermine levels are reduced. The resulting exaggerated spermidine:spermine ratio is a biochemical hallmark of SRS that tends to correlate with symptom severity. Our studies aim to pharmacologically manipulate polyamine metabolism to correct this imbalance as a therapeutic strategy for SRS. Here we report the repurposing of 2-difluoromethylornithine (DFMO), an FDA-approved inhibitor of polyamine biosynthesis, in rebalancing spermidine:spermine ratios in SRS patient cells. Mechanistic in vitro studies demonstrate that, while reducing spermidine biosynthesis, DFMO also stimulates the conversion of spermidine into spermine in hypomorphic SMS cells and induces uptake of exogenous spermine, altogether reducing the aberrant ratios. In a Drosophila SRS model characterized by reduced lifespan, DFMO improves longevity. As nearly all SRS patient mutations are hypomorphic, these studies form a strong foundation for translational studies with significant therapeutic potential.

Glioma Metabolic Feedback In Situ: A First-In-Human Pharmacodynamic Trial of Difluoromethylornithine + AMXT-1501 Through High-Molecular Weight Microdialysis.

BACKGROUND AND OBJECTIVES: No new drug has improved survival for glioblastoma since temozolomide in 2005, due in part to the relative inaccessibility of each patient's individualized tumor biology and its response to therapy. We have identified a conserved extracellular metabolic signature of enhancing high-grade gliomas enriched for guanidinoacetate (GAA). GAA is coproduced with ornithine, the precursor to protumorigenic polyamines through ornithine decarboxylase (ODC). AMXT-1501 is a polyamine transporter inhibitor that can overcome tumoral resistance to the ODC inhibitor, difluoromethylornithine (DFMO). We will use DFMO with or without AMXT-1501 to identify candidate pharmacodynamic biomarkers of polyamine depletion in patients with high-grade gliomas in situ . We aim to determine (1) how blocking polyamine production affects intratumoral extracellular guanidinoacetate abundance and (2) the impact of polyamine depletion on the global extracellular metabolome within live human gliomas in situ. METHODS: DFMO, with or without AMXT-1501, will be administered postoperatively in 15 patients after clinically indicated subtotal resection for high-grade glioma. High-molecular weight microdialysis catheters implanted into residual tumor and adjacent brain will be used for postoperative monitoring of extracellular GAA and polyamines throughout therapeutic intervention from postoperative day (POD) 1 to POD5. Catheters will be removed on POD5 before discharge. EXPECTED OUTCOMES: We anticipate that GAA will be elevated in tumor relative to adjacent brain although it will decrease within 24 hours of ODC inhibition with DFMO. If AMXT-1501 effectively increases the cytotoxic impact of ODC inhibition, we expect an increase in biomarkers of cytotoxicity including glutamate with DFMO + AMXT-1501 treatment when compared with DFMO alone. DISCUSSION: Limited mechanistic feedback from individual patients' gliomas hampers clinical translation of novel therapies. This pilot Phase 0 study will provide in situ feedback during DFMO + AMXT-1501 treatment to determine how high-grade gliomas respond to polyamine depletion.

Two New Cases of Bachmann-Bupp Syndrome Identified through the International Center for Polyamine Disorders.

Recent identification of four additional polyaminopathies, including Bachmann-Bupp syndrome, have benefited from previous research on Snyder-Robinson syndrome in order to advance from research to treatment more quickly. As a result of the discovery of these conditions, the potential for treatment within this pathway, and for other possible unidentified polyaminopathies, the International Center for Polyamine Disorders (ICPD) was created to help promote understanding of these conditions, research opportunities, and appropriate care for families. This case study provides insights from two new patients diagnosed with Bachmann-Bupp syndrome, further expanding our understanding of this ultra-rare condition, as well as a general discussion about other known polyaminopathies. This work also presents considerations for collaborative research efforts across these conditions, along with others that are likely to be identified in time, and outlines the role that the ICPD hopes to fill as more patients with these polyaminopathies continue to be identified and diagnosed.

Emerging compounds and therapeutic strategies to treat infections from Trypanosoma brucei: an overhaul of the last 5-years patents.

INTRODUCTION: Human African Trypanosomiasis is a neglected disease caused by infection from parasites belonging to the Trypanosoma brucei species. Only six drugs are currently available and employed depending on the stage of the infection: pentamidine, suramin, melarsoprol, eflornithine, nifurtimox, and fexinidazole. Joint research projects were launched in an attempt to find new therapeutic options for this severe and often lethal disease. AREAS COVERED: After a brief description of the recent literature on the parasite and the disease, we searched for patents dealing with the proposal of new antitrypanosomiasis agents and, following the PRISMA guidelines, we filtered the results to those published from 2018 onwards returning suitable entries, which represent the contemporary landscape of compounds/strategies against Trypanosoma brucei. In addition, some relevant publications from the overall scientific literature were also discussed. EXPERT OPINION: This review comprehensively covers and analyzes the most recent advances not only in the discovery of new inhibitors and their structure-activity relationships but also in the assessment of innovative biological targets opening new scenarios in the MedChem field. Finally, also new vaccines and formulations recently patented were described. However, natural and synthetic compounds were analyzed in terms of inhibitory activity and selective toxicity against human cells.

Good cop, bad cop: Polyamines play both sides in host immunity and viral replication.

Viruses rely on host cells for energy and synthesis machinery required for genome replication and particle assembly. Due to the dependence of viruses on host cells, viruses have evolved multiple mechanisms by which they can induce metabolic changes in the host cell to suit their specific requirements. The host immune response also involves metabolic changes to be able to react to viral insult. Polyamines are small ubiquitously expressed polycations, and their metabolism is critical for viral replication and an adequate host immune response. This is due to the variety of functions that polyamines have, ranging from condensing DNA to enhancing the translation of polyproline-containing proteins through the hypusination of eIF5A. Here, we review the diverse mechanisms by which viruses exploit polyamines, as well as the mechanisms by which immune cells utilize polyamines for their functions. Furthermore, we highlight potential avenues for further study of the host-virus interface.

Ototoxicity of Long-Term α-Difluoromethylornithine for Skin Cancer Prevention.

OBJECTIVE: Evaluate the effects of α-difluoromethylornithine (DFMO) on hearing thresholds as part of a randomized, double-blind, placebo-controlled trial. METHODS: Subjects were randomized and assigned to the control (placebo) or experimental (DFMO) group. DFMO or placebo were administered orally (500 mg/m2 /day) for up to 5 years. RESULTS: Subjects taking DFMO had, on average, increased hearing thresholds from baseline across the frequency range compared to subjects in the control group. Statistical analysis revealed this was significant in the lower frequency range. CONCLUSIONS: This randomized controlled trial revealed the presence of increased hearing thresholds associated with long-term DFMO use. As a whole, DFMO may help prevent and treat certain types of cancers; however, it can result in some degree of hearing loss even when administered at low doses. This study further highlights the importance of closely monitoring hearing thresholds in subjects taking DFMO. Laryngoscope, 133:676-682, 2023.

Efficacy and safety of acoziborole in patients with human African trypanosomiasis caused by Trypanosoma brucei gambiense: a multicentre, open-label, single-arm, phase 2/3 trial.

BACKGROUND: Human African trypanosomiasis caused by Trypanosoma brucei gambiense (gambiense HAT) in patients with late-stage disease requires hospital admission to receive nifurtimox-eflornithine combination therapy (NECT). Fexinidazole, the latest treatment that has been recommended by WHO, also requires systematic admission to hospital, which is problematic in areas with few health-care resources. We aim to assess the safety and efficacy of acoziborole in adult and adolescent patients with gambiense HAT. METHODS: This multicentre, prospective, open-label, single-arm, phase 2/3 study recruited patients aged 15 years or older with confirmed gambiense HAT infection from ten hospitals in the Democratic Republic of the Congo and Guinea. Inclusion criteria included a Karnofsky score greater than 50, ability to swallow tablets, a permanent address or traceability, ability to comply with follow-up visits and study requirements, and agreement to hospital admission during treatment. Oral acoziborole was administered as a single 960 mg dose (3 × 320 mg tablets) to fasted patients. Patients were observed in hospital until day 15 after treatment administration then for 18 months as outpatients with visits at 3, 6, 12, and 18 months. The primary efficacy endpoint was the success rate of acoziborole treatment at 18 months in patients with late-stage gambiense HAT (modified intention-to-treat [mITT] population), based on modified WHO criteria. A complementary post-hoc analysis comparing the 18-month success rates for acoziborole and NECT (using historical data) was performed. This study is registered at ClinicalTrials.gov, NCT03087955. FINDINGS: Between Oct 11, 2016, and March 25, 2019, 260 patients were screened, of whom 52 were ineligible and 208 were enrolled (167 with late-stage and 41 with early-stage or intermediate-stage gambiense HAT; primary efficacy analysis set). All 41 (100%) patients with early-stage or intermediate-stage and 160 (96%) of 167 with late-stage disease completed the last 18-month follow-up visit. The mean age of participants was 34·0 years (SD 12·4), including 117 (56%) men and 91 (44%) women. Treatment success rate at 18 months was 95·2% (95% CI 91·2-97·7) reached in 159 of 167 patients with late-stage gambiense HAT (mITT population) and 98·1% (95·1-99·5) reached in 159 of 162 patients (evaluable population). Overall, 155 (75%) of 208 patients had 600 treatment-emergent adverse events. A total of 38 drug-related treatment-emergent adverse events occurred in 29 (14%) patients; all were mild or moderate and most common were pyrexia and asthenia. Four deaths occurred during the study; none were considered treatment related. The post-hoc analysis showed similar results to the estimated historical success rate for NECT of 94%. INTERPRETATION: Given the high efficacy and favourable safety profile, acoziborole holds promise in the efforts to reach the WHO goal of interrupting HAT transmission by 2030. FUNDING: Bill & Melinda Gates Foundation, UK Aid, Federal Ministry of Education and Research, Swiss Agency for Development and Cooperation, Médecins Sans Frontières, Dutch Ministry of Foreign Affairs, Norwegian Agency for Development Cooperation, Norwegian Ministry of Foreign Affairs, the Stavros Niarchos Foundation, Spanish Agency for International Development Cooperation, and the Banco Bilbao Vizcaya Argentaria Foundation. TRANSLATION: For the French translation of the abstract see Supplementary Materials section.

Improvement of dermatological symptoms in patients with Bachmann-Bupp syndrome using difluoromethylornithine treatment.

Bachmann-Bupp syndrome (OMIM #619075) is a novel autosomal dominant disorder caused by variants in the c-terminus of the ornithine decarboxylase 1 gene, resulting in increased levels of ornithine decarboxylase. This case report includes two patients diagnosed with Bachmann-Bupp syndrome who were treated with difluoromethylornithine through compassionate use approval from the United States Food and Drug Administration. In both patients, treatment with difluoromethylornithine has resulted in improved dermatologic signs, including regrowth of eyebrow and scalp hair and cessation of recurrent follicular cyst development.

Discovery of Anthranilic Acid Derivatives as Difluoromethylornithine Adjunct Agents That Inhibit Far Upstream Element Binding Protein 1 (FUBP1) Function.

Polyamine biosynthesis is regulated by ornithine decarboxylase (ODC), which is transcriptionally activated by c-Myc. A large library was screened to find molecules that potentiate the ODC inhibitor, difluoromethylornithine (DFMO). Anthranilic acid derivatives were identified as DFMO adjunct agents. Further studies identified the far upstream binding protein 1 (FUBP1) as the target of lead compound 9. FUBP1 is a single-stranded DNA/RNA binding protein and a master controller of specific genes including c-Myc and p21. We showed that 9 does not inhibit 3H-spermidine uptake yet works synergistically with DFMO to limit cell growth in the presence of exogenous spermidine. Compound 9 was also shown to inhibit the KH4 FUBP1-FUSE interaction in a gel shift assay, bind to FUBP1 in a ChIP assay, reduce both c-Myc mRNA and protein expression, increase p21 mRNA and protein expression, and deplete intracellular polyamines. This promising hit opens the door to new FUBP1 inhibitors with increased potency.

Elucidating the Role of Chmp1 Overexpression in the Transport of Polyamines in Drosophila melanogaster.

Polyamines are small organic cations that are essential for many biological processes such as cell proliferation and cell cycle progression. While the metabolism of polyamines has been well studied, the mechanisms by which polyamines are transported into and out of cells are poorly understood. Here, we describe a novel role of Chmp1, a vesicular trafficking protein, in the transport of polyamines using a well-defined leg imaginal disc assay in Drosophila melanogaster larvae. We show that Chmp1 overexpression had no effect on leg development in Drosophila, but does attenuate the negative impact on leg development of Ant44, a cytotoxic drug known to enter cells through the polyamine transport system (PTS), suggesting that the overexpression of Chmp1 downregulated the PTS. Moreover, we showed that the addition of spermine did not rescue the leg development in Chmp1-overexpressing leg discs treated with difluoromethylornithine (DFMO), an inhibitor of polyamine metabolism, while putrescine and spermidine did, suggesting that there may be unique mechanisms of import for individual polyamines. Thus, our data provide novel insight into the underlying mechanisms that are involved in polyamine transport and highlight the utility of the Drosophila imaginal disc assay as a fast and easy way to study potential players involved in the PTS.

Histone deacetylase-10 liberates spermidine to support polyamine homeostasis and tumor cell growth.

Cytosolic histone deacetylase-10 (HDAC10) specifically deacetylates the modified polyamine N8-acetylspermidine (N8-AcSpd). Although intracellular concentrations of N8-AcSpd are low, extracellular sources can be abundant, particularly in the colonic lumen. Extracellular polyamines, including those from the diet and microbiota, can support tumor growth both locally and at distant sites. However, the contribution of N8-AcSpd in this context is unknown. We hypothesized that HDAC10, by converting N8- AcSpd to spermidine, may provide a source of this growth-supporting polyamine in circumstances of reduced polyamine biosynthesis, such as in polyamine-targeting anticancer therapies. Inhibitors of polyamine biosynthesis, including α-difluoromethylornithine (DFMO), inhibit tumor growth, but compensatory uptake of extracellular polyamines has limited their clinical success. Combining DFMO with inhibitors of polyamine uptake have improved the antitumor response. However, acetylated polyamines may use different transport machinery than the parent molecules. Here, we use CRISPR/Cas9-mediated HDAC10-knockout cell lines and HDAC10-specific inhibitors to investigate the contribution of HDAC10 in maintaining tumor cell proliferation. We demonstrate inhibition of cell growth by DFMO-associated polyamine depletion is successfully rescued by exogenous N8-AcSpd (at physiological concentrations), which is converted to spermidine and spermine, only in cell lines with HDAC10 activity. Furthermore, we show loss of HDAC10 prevents both restoration of polyamine levels and growth rescue, implicating HDAC10 in supporting polyamine-associated tumor growth. These data suggest the utility of HDAC10-specific inhibitors as an antitumor strategy that may have value in improving the response to polyamine-blocking therapies. Additionally, the cell-based assay developed in this study provides an inexpensive, high-throughput method of screening potentially selective HDAC10 inhibitors.

Effects of putrescine on the quality and epigenetic modification of mouse oocytes during in vitro maturation.

CONTEXT: Low ovarian putrescine levels and decreased peak values following luteinising hormone peaks are related to poor oocyte quantity and quality in ageing women. AIMS: To investigate the effects of putrescine supplementation in in vitro maturation (IVM) medium on oocyte quality and epigenetic modification. METHODS: Germinal vesicle oocytes retrieved from the ovaries of 8-week-old and 9-month-old mice were divided into four groups (the young, young+difluoromethylornithine (DFMO), ageing and ageing+putrescine groups) and cultured in IVM medium with or without 1mM putrescine or DFMO for 16h. The first polar body extrusion (PBE), cleavage and embryonic development were evaluated. Spindles, chromosomes, mitochondria and reactive oxygen species (ROS) were measured. The expression levels of SIRT1, H3K9ac, H3K9me2, H3K9me3, and 5mC levels were evaluated. Sirt1 and imprinted genes were detected. RESULTS: The PBE was higher in the ageing+putrescine group than in the ageing group. Putrescine increased the total and inner cell mass cell numbers of blastocysts in ageing oocytes. Putrescine decreased aberrant spindles and chromosome aneuploidy, increased the mitochondrial membrane potential and decreased ROS levels. Putrescine increased SIRT1 expression and attenuated the upregulation of H3K9ac levels in ageing oocytes. Putrescine did not affect 5mC, H3K9me2 or H3K9me3 levels or imprinted gene expression. CONCLUSIONS: Putrescine supplementation during IVM improved the maturation and quality of ageing oocytes and promoted embryonic development by decreasing ROS generation, maintaining mitochondrial and spindle function and correcting aberrant epigenetic modification. IMPLICATIONS: Putrescine shows application potential for human-assisted reproduction, especially for IVM of oocytes from ageing women.