Polyamine Homeostasis in Snyder-Robinson Syndrome

Loss-of-function mutations of the spermine synthase gene (SMS) result in Snyder-Robinson Syndrome (SRS), a recessive X-linked syndrome characterized by intellectual disability, osteoporosis, hypotonia, speech abnormalities, kyphoscoliosis, and seizures. As SMS catalyzes the biosynthesis of the polyamine spermine from its precursor spermidine, SMS deficiency causes a lack of spermine with an accumulation of spermidine. As polyamines, spermine, and spermidine play essential cellular roles that require tight homeostatic control to ensure normal cell growth, differentiation, and survival. Using patient-derived lymphoblast cell lines, we sought to comprehensively investigate the effects of SMS deficiency on polyamine homeostatic mechanisms including polyamine biosynthetic and catabolic enzymes, derivatives of the natural polyamines, and polyamine transport activity. In addition to decreased spermine and increased spermidine in SRS cells, ornithine decarboxylase activity and its product putrescine were significantly decreased. Treatment of SRS cells with exogenous spermine revealed that polyamine transport was active, as the cells accumulated spermine, decreased their spermidine level, and established a spermidine-to-spermine ratio within the range of wildtype cells. SRS cells also demonstrated elevated levels of tissue transglutaminase, a change associated with certain neurodegenerative diseases. These studies form a basis for further investigations into the leading biochemical changes and properties of SMS-mutant cells that potentially represent therapeutic targets for the treatment of Snyder-Robinson Syndrome

Curcumin mediates polyamine metabolism and sensitizes gastrointestinal cancer cells to antitumor polyamine-targeted therapies.

Curcumin, a natural polyphenol that contributes to the flavor and yellow pigment of the spice turmeric, is known for its antioxidant, anti-inflammatory, and anticarcinogenic properties. Capable of affecting the initiation, promotion, and progression of carcinogenesis through multiple mechanisms, curcumin has potential utility for both chemoprevention and chemotherapy. Previous studies demonstrated that curcumin can inhibit ornithine decarboxylase (ODC) activity in human leukemia and breast cancer cells, and pretreatment with dietary curcumin blocks carcinogen-induced ODC activity in rodent models of skin, colon, and renal cancer. The current study investigated the regulation of polyamine metabolism in human gastric and colon carcinoma cell lines in response to curcumin. Curcumin treatment significantly induced spermine oxidase (SMOX) mRNA and activity, which results in the generation of hydrogen peroxide, a source of ROS. Simultaneously, curcumin down regulated spermidine/spermine N1-acetyltransferase (SSAT) activity and the biosynthetic enzymes ODC and S-adenosylmethionine decarboxylase (SAMDC), thereby diminishing intracellular polyamine pools. Combination treatments using curcumin with the ODC inhibitor 2-difluoromethylornithine (DFMO), an agent currently in clinical chemoprevention trials, significantly enhanced inhibition of ODC activity and decreased growth of GI cancer cell lines beyond that observed with either agent alone. Similarly, combining curcumin with the polyamine analogue bis(ethyl)norspermine enhanced growth inhibition that was accompanied by enhanced accumulation of the analogue and decreased intracellular polyamine levels beyond those observed with either agent alone. Importantly, cotreatment with curcumin permitted the lowering of the effective dose of ODC inhibitor or polyamine analogue. These studies provide insight into the polyamine-related mechanisms involved in the cancer cell response to curcumin and its potential as a chemopreventive or chemotherapeutic agent in the GI tract

Polymeric Prodrugs Targeting Polyamine Metabolism Inhibit Zika Virus Replication

The Zika virus (ZIKV) is primarily transmitted via an infected mosquito bite, during sexual intercourse, or in utero mother to child transmission. When a fetus is infected, both neurological malformations and deficits in brain development are frequently manifested. As such, there is a need for vaccines or drugs that may be used to cure ZIKV infections. Metabolic pathways play a crucial role in cell differentiation and development. More importantly, polyamines play a key role in replication and translation of several RNA viruses, including ZIKV, Dengue virus, and Chikungunya virus. Here, we present polyamine analogues (BENSpm and PG11047) and their corresponding polymer prodrug derivatives for inhibiting ZIKV infection by intersecting with polyamine catabolism pathways. We tested the compounds against ZIKV African (MR766) and Asian (PRVABC59) strains in human kidney epithelial (Vero) and glioblastoma derived (SNB-19) cell lines. Our results demonstrate potent inhibition of ZIKV viral replication in both cell lines tested. This antiviral effect was mediated by the upregulation of two polyamine catabolic enzymes, spermine oxidase, and spermidine (SMOX)/spermine N1-acetyltransferase (SAT1) as apparent reduction of the ZIKV infection following heterologous expression of SMOX and SAT1. On the basis of these observations, we infer potential use of these polyamine analogues to treat ZIKV infections

DOT1L Is a Novel Cancer Stem Cell Target for Triple-Negative Breast Cancer

Although chemotherapies kill most cancer cells, stem cell-enriched survivors seed metastasis, particularly in triple-negative breast cancers (TNBC). TNBCs arise from and are enriched for tumor stem cells. Here, we tested if inhibition of DOT1L, an epigenetic regulator of normal tissue stem/progenitor populations, would target TNBC stem cells. Effects of DOT1L inhibition by EPZ-5676 on stem cell properties were tested in three TNBC lines and four patient-derived xenograft (PDX) models and in isolated cancer stem cell (CSC)-enriched ALDH1+ and ALDH1- populations. RNA sequencing compared DOT1L regulated pathways in ALDH1+ and ALDH1- cells. To test if EPZ-5676 decreases CSC in vivo, limiting dilution assays of EPZ-5676/vehicle pretreated ALDH1+ and ALDH1- cells were performed. Tumor latency, growth, and metastasis were evaluated. Antitumor activity was also tested in TNBC PDX and PDX-derived organoids. ALDH1+ TNBC cells exhibit higher DOT1L and H3K79me2 than ALDH1-. DOT1L maintains MYC expression and self-renewal in ALDH1+ cells. Global profiling revealed that DOT1L governs oxidative phosphorylation, cMyc targets, DNA damage response, and WNT activation in ALDH1+ but not in ALDH1- cells. EPZ-5676 reduced tumorspheres and ALDH1+ cells in vitro and decreased tumor-initiating stem cells and metastasis in xenografts generated from ALDH1+ but not ALDH1- populations in vivo. EPZ-5676 significantly reduced growth in vivo of one of two TNBC PDX tested and decreased clonogenic 3D growth of two other PDX-derived organoid cultures. DOT1L emerges as a key CSC regulator in TNBC. Present data support further clinical investigation of DOT1L inhibitors to target stem cell-enriched TNBC

Activation of endoplasmic reticulum stress response by enhanced polyamine catabolism is important in the mediation of cisplatin-induced acute kidney injury

<div><p>Cisplatin-induced nephrotoxicity limits its use in many cancer patients. The expression of enzymes involved in polyamine catabolism, spermidine/spermine N¹-acetyltransferase (SSAT) and spermine oxidase (SMOX) increase in the kidneys of mice treated with cisplatin. We hypothesized that enhanced polyamine catabolism contributes to tissue damage in cisplatin acute kidney injury (AKI). Using gene knockout and chemical inhibitors, the role of polyamine catabolism in cisplatin AKI was examined. Deficiency of SSAT, SMOX or neutralization of the toxic products of polyamine degradation, H₂O₂ and aminopropanal, significantly diminished the severity of cisplatin AKI. <i>In vitro</i> studies demonstrated that the induction of SSAT and elevated polyamine catabolism in cells increases the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) and enhances the expression of binding immunoglobulin protein BiP/GRP78) and CCAAT-enhancer-binding protein homologous protein (CHOP/GADD153). The increased expression of these endoplasmic reticulum stress response (ERSR) markers was accompanied by the activation of caspase-3. These results suggest that enhanced polyamine degradation in cisplatin AKI may lead to tubular damage through the induction of ERSR and the consequent onset of apoptosis. In support of the above, we show that the ablation of the SSAT or SMOX gene, as well as the neutralization of polyamine catabolism products modulate the onset of ERSR (e.g. lower BiP and CHOP) and apoptosis (e.g. reduced activated caspase-3). These studies indicate that enhanced polyamine catabolism and its toxic products are important mediators of ERSR and critical to the pathogenesis of cisplatin AKI.</p></div

The consequence of SSAT and SMOX ablation on the induction of ERSR and onset of apoptosis.

<p>The effect of modulation of polyamine catabolism on the activation of ERSR and onset of apoptosis in cisplatin AKI was examined. A) The levels of BiP and CHOP were similar in the control samples from all 3 genotypes. The expression of BiP and CHOP increased in all three genotypes after cisplatin treatment; however, the BiP and CHOP expression levels were reduced in cisplatin treated SMOX-KO and SSAT-KO mice compared to their cisplatin-treated Wt counterparts. The results are representative of 3 independent experiments. B) The levels of activated caspase 3 (arrows) were also significantly elevated in the kidneys of Wt compared to SSAT-KO and SMOX-KO mice.</p

Depiction of polyamine synthetic and catabolic reactions.

<p>This schematic indicates that the oxidation of acetylated polyamines and via APAOX or SMOX, respectively, leads to the generation of cytotoxic molecules (H₂O₂ and aminoaldehydes).</p

Effect of cisplatin treatment on renal function and structure and the expression of polyamine catabolic enzymes.

<p>A & B) Administration of cisplatin (20mg/kg) led to significantly increased serum creatinine levels tubular injury ranging from mild (48 hours) to severe (96 hours) post-treatment (vacuolization, small arrow head; cast, small arrows; sloughed cells and damaged tubules, large arrows). C) Expression of SSAT and SMOX mRNA levels increase in kidneys of mice treated with cisplatin.</p

Effect of SSAT and SMOX ablation on cisplatin AKI induced changes in ODC, SSAT and polyamine levels.

<p>A) The activity of ODC is approximately 2-fold lower in saline treated SSAT-KO and SMOX-KO mice compared to their Wt littermates (p<0.05); however, the reduction in ODC activity in all 3 groups subsequent to cisplatin treatment is of a similar magnitude and significant. (*Denotes significantly higher enzymatic activity in control vs. cisplatin-treated animals). B) Renal SSAT activity is similar in saline treated Wt and SMOX-KO mice. The SSAT activity is significantly elevated in Wt mice treated with cisplatin compared to those treated with saline. (*Denotes significantly higher enzymatic activity in cisplatin-treated vs. control animals. ⁺Denotes a significant increase in cisplatin-treated Wt compared to similarly treated SMOX-KO and SSAT-KO mice. ^#Denotes significant increase in cisplatin-treated SMOX-KO to SSAT-KO mice). C) SMOX protein levels are elevated in the kidneys of cisplatin-treated Wt animals compared to saline treated and cisplatin treated SSAT-KO mice. D) Examination of tissue polyamine levels reveals that the kidney Put content increases significantly in Wt but not SMOX- and SSAT-KO mice after cisplatin treatment. The Spd content of the kidneys only marginally increases in Wt and SSAT-KO mice after cisplatin treatment. Examination of Spm levels indicates that their reduction is significantly greater in the Wt mice than SMOX-KO and SSAT-KO animals. (*Denotes significantly increased content in cisplatin-treated vs saline-treated animals of the same genotype. ⁺Denotes a significant decrease in tissue content in cisplatin-treated animals compared to saline-treated animals of the same genotype. ^#Denotes a significant decrease in tissue content compared to saline-treated Wt mice).</p

Effect of SSAT and SMOX ablation on cisplatin AKI.

<p>Ablation of SSAT and SMOX genes reduces the loss of renal function (A) and protects against renal tubular injury caused by cisplatin treatment (B). (*Denotes significant increase in creatinine levels in cisplatin-treated vs. control animals. ^<b>+</b>Indicates significant increase in cisplatin-treated Wt compared to similarly treated SMOX-KO and SSAT-KO animals.</p