Polyamines: the pivotal amines in influencing the tumor microenvironment.

Cellular proliferation, function and survival is reliant upon maintaining appropriate intracellular polyamine levels. Due to increased metabolic needs, cancer cells elevate their polyamine pools through coordinated metabolism and uptake. High levels of polyamines have been linked to more immunosuppressive tumor microenvironments (TME) as polyamines support the growth and function of many immunosuppressive cell types such as MDSCs, macrophages and regulatory T-cells. As cancer cells and other pro-tumorigenic cell types are highly dependent on polyamines for survival, pharmacological modulation of polyamine metabolism is a promising cancer therapeutic strategy. This review covers the roles of polyamines in various cell types of the TME including both immune and stromal cells, as well as how competition for nutrients, namely polyamine precursors, influences the cellular landscape of the TME. It also details the use of polyamines as biomarkers and the ways in which polyamine depletion can increase the immunogenicity of the TME and reprogram tumors to become more responsive to immunotherapy.

Reduction of spermine synthase enhances autophagy to suppress Tau accumulation.

Precise polyamine metabolism regulation is vital for cells and organisms. Mutations in spermine synthase (SMS) cause Snyder-Robinson intellectual disability syndrome (SRS), characterized by significant spermidine accumulation and autophagy blockage in the nervous system. Emerging evidence connects polyamine metabolism with other autophagy-related diseases, such as Tauopathy, however, the functional intersection between polyamine metabolism and autophagy in the context of these diseases remains unclear. Here, we altered SMS expression level to investigate the regulation of autophagy by modulated polyamine metabolism in Tauopathy in Drosophila and human cellular models. Interestingly, while complete loss of Drosophila spermine synthase (dSms) impairs lysosomal function and blocks autophagic flux recapitulating SRS disease phenotype, partial loss of dSms enhanced autophagic flux, reduced Tau protein accumulation, and led to extended lifespan and improved climbing performance in Tauopathy flies. Measurement of polyamine levels detected a mild elevation of spermidine in flies with partial loss of dSms. Similarly, in human neuronal or glial cells, partial loss of SMS by siRNA-mediated knockdown upregulated autophagic flux and reduced Tau protein accumulation. Importantly, proteomics analysis of postmortem brain tissue from Alzheimer's disease (AD) patients showed a significant albeit modest elevation of SMS level. Taken together, our study uncovers a functional correlation between polyamine metabolism and autophagy in AD: SMS reduction upregulates autophagy, suppresses Tau accumulation, and ameliorates neurodegeneration and cell death. These findings provide a new potential therapeutic target for AD.

Polyamine Catabolism and Its Role in Renal Injury and Fibrosis in Mice Subjected to Repeated Low-Dose Cisplatin Treatment.

Cisplatin, a chemotherapeutic agent, can cause nephrotoxic and ototoxic injuries. Using a mouse model of repeated low dose cisplatin (RLDC), we compared the kidneys of cisplatin- and vehicle-treated mice on days 3 (early injury phase) and 35 (late injury/recovery phase) after the final treatment. RNA-seq analyses revealed increases in the expression of markers of kidney injury (e.g., lipocalin 2 and kidney injury molecule 1) and fibrosis (e.g., collagen 1, fibronectin, and vimentin 1) in RLDC mice. In addition, we observed increased expression of polyamine catabolic enzymes (spermidine/spermine N1-acetyltransferase, Sat1, and spermine oxidase, Smox) and decreased expression of ornithine decarboxylase (Odc1), a rate-limiting enzyme in polyamine synthesis in mice subjected to RLDC. Upon confirmation of the RNA-seq results, we tested the hypothesis that enhanced polyamine catabolism contributes to the onset of renal injury and development of fibrosis. To test our hypothesis, we compared the severity of RLDC-induced renal injury and fibrosis in wildtype (WT), Sat1-KO, and Smox-KO mice. Our results suggest that the ablation of polyamine catabolic enzymes reduces the severity of renal injury and that modulation of the activity of these enzymes may protect against kidney damage and fibrosis caused by cisplatin treatment.

Impaired polyamine metabolism causes behavioral and neuroanatomical defects in a mouse model of Snyder-Robinson syndrome.

Snyder-Robinson syndrome (SRS) is a rare X-linked recessive disorder caused by a mutation in the SMS gene, which encodes spermine synthase, and aberrant polyamine metabolism. SRS is characterized by intellectual disability, thin habitus, seizure, low muscle tone/hypotonia and osteoporosis. Progress towards understanding and treating SRS requires a model that recapitulates human gene variants and disease presentations. Here, we evaluated molecular and neurological presentations in the G56S mouse model, which carries a missense mutation in the Sms gene. The lack of SMS protein in the G56S mice resulted in increased spermidine/spermine ratio, failure to thrive, short stature and reduced bone density. They showed impaired learning capacity, increased anxiety, reduced mobility and heightened fear responses, accompanied by reduced total and regional brain volumes. Furthermore, impaired mitochondrial oxidative phosphorylation was evident in G56S cerebral cortex, G56S fibroblasts and Sms-null hippocampal cells, indicating that SMS may serve as a future therapeutic target. Collectively, our study establishes the suitability of the G56S mice as a preclinical model for SRS and provides a set of molecular and functional outcome measures that can be used to evaluate therapeutic interventions for SRS.

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.

Polyamine transport inhibition and cisplatin synergistically enhance tumor control through oxidative stress in murine head and neck cancer models.

Background: Surgery and/or platinum-based chemoradiation remain standard of care for patients with head and neck squamous cell carcinoma (HNSCC). While these therapies are effective in a subset of patients, a substantial proportion experience recurrence or treatment resistance. As cisplatin mediates cytotoxicity through oxidative stress while polyamines play a role in redox regulation, we posited that combining cisplatin with polyamine transport inhibitor, AMXT-1501, would increase oxidative stress and tumor cell death in HNSCC cells. Methods: Cell proliferation was measured in syngeneic mouse HNSCC cell lines treated with cisplatin ± AMXT-1501. Synergy was determined by administering cisplatin and AMXT-1501 at a ratio of 1:10 to cancer cells in vitro . Cancer cells were transferred onto mouse flanks to test the efficacy of treatments in vivo . Reactive oxygen species (ROS) were measured. Cellular apoptosis was measured with flow cytometry using Annexin V/PI staining. High-performance liquid chromatography (HPLC) was used to quantify polyamines in cell lines. Cell viability and ROS were measured in the presence of exogenous cationic amino acids. Results: The combination of cisplatin and AMXT-1501 synergize in vitro on HNSCC cell lines. In vivo combination treatment resulted in tumor growth inhibition greater than either treatment individually. The combination treatment increased ROS production and induced apoptotic cell death. HPLC revealed the synergistic mechanism was independent of intracellular polyamine levels. Supplementation of cationic amino acids partially rescued cancer cell viability and reduced ROS. Conclusion: AMXT-1501 enhances the cytotoxic effects of cisplatin in vitro and in vivo in aggressive HNSCC cell lines through a polyamine-independent mechanism.

Deoxyhypusine synthase mutations alter the post-translational modification of eukaryotic initiation factor 5A resulting in impaired human and mouse neural homeostasis.

DHPS deficiency is a rare genetic disease caused by biallelic hypomorphic variants in the Deoxyhypusine synthase (DHPS) gene. The DHPS enzyme functions in mRNA translation by catalyzing the post-translational modification, and therefore activation, of eukaryotic initiation factor 5A (eIF5A). The observed clinical outcomes associated with human mutations in DHPS include developmental delay, intellectual disability, and seizures. Therefore, to increase our understanding of this rare disease, it is critical to determine the mechanisms by which mutations in DHPS alter neurodevelopment. In this study, we have generated patient-derived lymphoblast cell lines and demonstrated that human DHPS variants alter DHPS protein abundance and impair enzyme function. Moreover, we observe a shift in the abundance of the post-translationally modified forms of eIF5A; specifically, an increase in the nuclear localized acetylated form (eIF5AAcK47) and concomitant decrease in the cytoplasmic localized hypusinated form (eIF5AHYP). Generation and characterization of a mouse model with a genetic deletion of Dhps in the brain at birth shows that loss of hypusine biosynthesis impacts neuronal function due to impaired eIF5AHYP-dependent mRNA translation; this translation defect results in altered expression of proteins required for proper neuronal development and function. This study reveals new insight into the biological consequences and molecular impact of human DHPS deficiency and provides valuable information toward the goal of developing treatment strategies for this rare disease.

Difluoromethylornithine rebalances aberrant polyamine ratios in Snyder-Robinson syndrome: mechanism of action and therapeutic potential.

Snyder-Robinson Syndrome (SRS) is caused by mutations in the spermine synthase (SMS) gene, the enzyme product of which converts the polyamine spermidine into spermine. Affecting primarily males, common manifestations of SRS include intellectual disability, osteoporosis, hypotonic musculature, and seizures, along with other more variable symptoms. Currently, medical management focuses on treating these symptoms without addressing the underlying molecular cause of the disease. Reduced SMS catalytic activity in cells of SRS patients causes the accumulation of spermidine, while spermine levels are reduced. The resulting exaggeration in spermidine-to-spermine ratio is a biochemical hallmark of SRS that tends to correlate with symptom severity in the patient. Our studies aim to pharmacologically manipulate polyamine metabolism to correct this polyamine imbalance and investigate the potential of this approach as a therapeutic strategy for affected individuals. Here we report the use of difluoromethylornithine (DFMO; eflornithine), an FDA-approved inhibitor of polyamine biosynthesis, in re-establishing normal spermidine-to-spermine ratios in SRS patient cells. Through mechanistic studies, we demonstrate that, while reducing spermidine biosynthesis, DFMO also stimulates the conversion of existing spermidine into spermine in cell lines with hypomorphic variants of SMS. Further, DFMO treatment induces a compensatory uptake of exogenous polyamines, including spermine and spermine mimetics, cooperatively reducing spermidine and increasing spermine levels. In a Drosophila SRS model characterized by reduced lifespan, adding DFMO to the feed extended lifespan. As nearly all known SRS patient mutations are hypomorphic, these studies form a foundation for future translational studies with significant therapeutic potential.

Reduction of Spermine Synthase Suppresses Tau Accumulation Through Autophagy Modulation in Tauopathy.

Tauopathy, including Alzheimer Disease (AD), is characterized by Tau protein accumulation and autophagy dysregulation. Emerging evidence connects polyamine metabolism with the autophagy pathway, however the role of polyamines in Tauopathy remains unclear. In the present study we investigated the role of spermine synthase (SMS) in autophagy regulation and tau protein processing in Drosophila and human cellular models of Tauopathy. Our previous study showed that Drosophila spermine synthase (dSms) deficiency impairs lysosomal function and blocks autophagy flux. Interestingly, partial loss-of-function of SMS in heterozygous dSms flies extends lifespan and improves the climbing performance of flies with human Tau (hTau) overexpression. Mechanistic analysis showed that heterozygous loss-of-function mutation of dSms reduces hTau protein accumulation through enhancing autophagic flux. Measurement of polyamine levels detected a mild elevation of spermidine in flies with heterozygous loss of dSms. SMS knock-down in human neuronal or glial cells also upregulates autophagic flux and reduces Tau protein accumulation. Proteomics analysis of postmortem brain tissue from AD patients showed a significant albeit modest elevation of SMS protein level in AD-relevant brain regions compared to that of control brains consistently across several datasets. Taken together, our study uncovers a correlation between SMS protein level and AD pathogenesis and reveals that SMS reduction upregulates autophagy, promotes Tau clearance, and reduces Tau protein accumulation. These findings provide a new potential therapeutic target of Tauopathy.

Impaired polyamine metabolism causes behavioral and neuroanatomical defects in a novel mouse model of Snyder-Robinson Syndrome.

Polyamines (putrescine, spermidine, and spermine) are essential molecules for normal cellular functions and are subject to strict metabolic regulation. Mutations in the gene encoding spermine synthase (SMS) lead to accumulation of spermidine in an X-linked recessive disorder known as Snyder-Robinson syndrome (SRS). Presently, no treatments exist for this rare disease that manifests with a spectrum of symptoms including intellectual disability, developmental delay, thin habitus, and low muscle tone. The development of therapeutic interventions for SRS will require a suitable disease-specific animal model that recapitulates many of the abnormalities observed in patients. Here, we characterize the molecular, behavioral, and neuroanatomical features of a mouse model with a missense mutation in Sms gene that results in a glycine-to-serine substitution at position 56 (G56S) of the SMS protein. Mice harboring this mutation exhibit a complete loss of SMS protein and elevated spermidine/spermine ratio in skeletal muscles and the brain. In addition, the G56S mice demonstrate increased anxiety, impaired learning, and decreased explorative behavior in fear conditioning, Morris water maze, and open field tests, respectively. Furthermore, these mice failed to gain weight over time and exhibit abnormalities in brain structure and bone density. Transcriptomic analysis of the cerebral cortex revealed downregulation of genes associated with mitochondrial oxidative phosphorylation and ribosomal protein synthesis. Our findings also revealed impaired mitochondrial bioenergetics in fibroblasts isolated from the G56S mice, indicating a correlation between these processes in the affected mice. Collectively, our findings establish the first in-depth characterization of an SRS preclinical mouse model that identifies cellular processes that could be targeted for future therapeutic development.

Functional assessment of homozygous ALDH18A1 variants reveals alterations in amino acid and antioxidant metabolism.

Mono- and bi-allelic variants in ALDH18A1 cause a spectrum of human disorders associated with cutaneous and neurological findings that overlap with both cutis laxa and spastic paraplegia. ALDH18A1 encodes the bifunctional enzyme pyrroline-5-carboxylate synthetase (P5CS) that plays a role in the de novo biosynthesis of proline and ornithine. Here we characterize a previously unreported homozygous ALDH18A1 variant (p.Thr331Pro) in four affected probands from two unrelated families, and demonstrate broad-based alterations in amino acid and antioxidant metabolism. These four patients exhibit variable developmental delay, neurological deficits and loose skin. Functional characterization of the p.Thr331Pro variant demonstrated a lack of any impact on the steady-state level of the P5CS monomer or mitochondrial localization of the enzyme, but reduced incorporation of the monomer into P5CS oligomers. Using an unlabeled NMR-based metabolomics approach in patient fibroblasts and ALDH18A1-null human embryonic kidney cells expressing the variant P5CS, we identified reduced abundance of glutamate and several metabolites derived from glutamate, including proline and glutathione. Biosynthesis of the polyamine putrescine, derived from ornithine, was also decreased in patient fibroblasts, highlighting the functional consequence on another metabolic pathway involved in antioxidant responses in the cell. RNA sequencing of patient fibroblasts revealed transcript abundance changes in several metabolic and extracellular matrix-related genes, adding further insight into pathogenic processes associated with impaired P5CS function. Together these findings shed new light on amino acid and antioxidant pathways associated with ALDH18A1-related disorders, and underscore the value of metabolomic and transcriptomic profiling to discover new pathways that impact disease pathogenesis.

Aza-SAHA Derivatives Are Selective Histone Deacetylase 10 Chemical Probes That Inhibit Polyamine Deacetylation and Phenocopy HDAC10 Knockout.

We report the first well-characterized selective chemical probe for histone deacetylase 10 (HDAC10) with unprecedented selectivity over other HDAC isozymes. HDAC10 deacetylates polyamines and has a distinct substrate specificity, making it unique among the 11 zinc-dependent HDAC hydrolases. Taking inspiration from HDAC10 polyamine substrates, we systematically inserted an amino group ("aza-scan") into the hexyl linker moiety of the approved drug Vorinostat (SAHA). This one-atom replacement (C→N) transformed SAHA from an unselective pan-HDAC inhibitor into a specific HDAC10 inhibitor. Optimization of the aza-SAHA structure yielded the HDAC10 chemical probe DKFZ-748, with potency and selectivity demonstrated by cellular and biochemical target engagement, as well as thermal shift assays. Cocrystal structures of our aza-SAHA derivatives with HDAC10 provide a structural rationale for potency, and chemoproteomic profiling confirmed exquisite cellular HDAC10-selectivity of DKFZ-748 across the target landscape of HDAC drugs. Treatment of cells with DKFZ-748, followed by quantification of selected polyamines, validated for the first time the suspected cellular function of HDAC10 as a polyamine deacetylase. Finally, in a polyamine-limiting in vitro tumor model, DKFZ-748 showed dose-dependent growth inhibition of HeLa cells. We expect DKFZ-748 and related probes to enable further studies on the enigmatic biology of HDAC10 and acetylated polyamines in both physiological and pathological settings.

Interrogation of T Cell-Enriched Tumors Reveals Prognostic and Immunotherapeutic Implications of Polyamine Metabolism.

Metabolic features of the tumor microenvironment (TME) antagonize anti-tumor immunity. We hypothesized that T cell infiltrated tumors with a known antigen should exhibit superior clinical outcomes, though some fare worse given unfavorable metabolic features leveraging T cell-infiltrated (Thi), human papillomavirus-related (HPV+) head and neck squamous cell carcinomas (HNSC) to test this hypothesis. Expression of 2,520 metabolic genes were analyzed among Thi HPV+ HNSCs stratified by high-risk molecular subtype. RNAseq data from The Cancer Genome Atlas (TCGA; 10 cancer types), single cell RNAseq data, and an immunotherapy-treated melanoma cohort were used to test the association between metabolic gene expression and clinical outcomes and contribution of tumor versus stromal cells to metabolic gene expression. Polyamine (PA) metabolism genes were overexpressed in high-risk, Thi HPV+ HNSCs. Genes involved in PA biosynthesis and transport were associated with T cell infiltration, recurrent or persistent cancer, overall survival status, primary site, molecular subtype, and MYC genomic alterations. PA biogenesis gene sets were associated with tumor intrinsic features while myeloid cells in HPV+ HNSCs were enriched in PA catabolism, regulatory, transport, putrescine, and spermidine gene set expression. PA gene set expression also correlated with IFNγ or cytotoxic T cell ssGSEA scores across TCGA tumor types. PA transport ssGSEA scores were associated with poor survival whereas putrescine ssGSEA scores portended better survival for several tumor types. Thi melanomas enriched in PA synthesis or combined gene set expression exhibited worse anti-PD-1 responses. These data address hurdles to anti-tumor immunity warranting further investigation of divergent polyamine metabolism in the TME.

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.

Identification and Characterization of Novel Small-Molecule SMOX Inhibitors.

The major intracellular polyamines spermine and spermidine are abundant and ubiquitous compounds that are essential for cellular growth and development. Spermine catabolism is mediated by spermine oxidase (SMOX), a highly inducible flavin-dependent amine oxidase that is upregulated during excitotoxic, ischemic, and inflammatory states. In addition to the loss of radical scavenging capabilities associated with spermine depletion, the catabolism of spermine by SMOX results in the production of toxic byproducts, including H2O2 and acrolein, a highly toxic aldehyde with the ability to form adducts with DNA and inactivate vital cellular proteins. Despite extensive evidence implicating SMOX as a key enzyme contributing to secondary injury associated with multiple pathologic states, the lack of potent and selective inhibitors has significantly impeded the investigation of SMOX as a therapeutic target. In this study, we used a virtual and physical screening approach to identify and characterize a series of hit compounds with inhibitory activity against SMOX. We now report the discovery of potent and highly selective SMOX inhibitors 6 (IC50 0.54 µM, Ki 1.60 µM) and 7 (IC50 0.23 µM, Ki 0.46 µM), which are the most potent SMOX inhibitors reported to date. We hypothesize that these selective SMOX inhibitors will be useful as chemical probes to further elucidate the impact of polyamine catabolism on mechanisms of cellular injury.

Renal Transcriptome and Metabolome in Mice with Principal Cell-Specific Ablation of the Tsc1 Gene: Derangements in Pathways Associated with Cell Metabolism, Growth and Acid Secretion.

Tuberous sclerosis complex (TSC) is caused by mutations in the hamartin (TSC1) or tuberin (TSC2) genes. Using a mouse model of TSC renal cystogenesis that we have previously described, the current studies delineate the metabolic changes in the kidney and their relation to alterations in renal gene expression. To accomplish this, we compared the metabolome and transcriptome of kidneys from 28-day-old wildtype (Wt) and principal cell-specific Tsc1 KO (Tsc1 KO) mice using targeted 1H nuclear magnetic resonance targeted metabolomic and RNA-seq analyses. The significant changes in the kidney metabolome of Tsc1 KO mice included reductions in the level of several amino acids and significant decreases in creatine, NADH, inosine, UDP-galactose, GTP and myo-inositol levels. These derangements may affect energy production and storage, signal transduction and synthetic pathways. The pertinent derangement in the transcriptome of Tsc1 KO mice was associated with increased collecting duct acid secretion, active cell division and the up-regulation of signaling pathways (e.g., MAPK and AKT/PI3K) that suppress the TSC2 GTPase-activating function. The combined renal metabolome and transcriptome alterations observed in these studies correlate with the unregulated growth and predominance of genotypically normal A-intercalated cells in the epithelium of renal cysts in Tsc1 KO mice.

Helping the helpers: polyamines help maintain helper T-cell lineage fidelity.

The awareness that polyamines play a critical role in immune system regulation and function is coming into focus as the biological systems and analytical tools necessary to evaluate their roles have become available. Puleston et al have recently demonstrated that polyamine metabolism plays a central role in helper T-cell lineage determination through the production of the translational cofactor hypusinated eIF5A and faithful epigenetic regulation through proper histone acetylation. Their findings add to the rapidly growing body of data implicating properly controlled polyamine metabolism as essential for a normally functioning immune system.

Hyaluronate-coated perfluoroalkyl polyamine prodrugs as bioactive siRNA delivery systems for the treatment of peritoneal cancers.

RNA interference (RNAi) is an emerging therapeutic modality for cancer, which remains in critical need of effective delivery vectors due to the unfavorable biopharmaceutical properties of small RNAs. Polyamines are essential for functioning of mammalian cells. Dysregulated polyamine metabolism is found in many cancers and has been an attractive therapeutic target in combination therapies. Combination therapies based on drugs that affect polyamine metabolism and nucleic acids promise to enhance anticancer activity due to a cooperative effect on multiple oncogenic pathways. Here, we report bioactive polycationic prodrug (F-PaP) based on an anticancer polyamine analogue bisethylnorspermine (BENSpm) modified with perfluoroalkyl moieties. Following encapsulation of siRNA, F-PaP/siRNA nanoparticles were coated with hyaluronic acid (HA) to form ternary nanoparticles HA@F-PaP/siRNA. The presence of perfluoroalkyl moieties and HA reduced cell membrane toxicity and improved stability of the particles with cooperatively enhanced siRNA delivery in pancreatic and colon cancer cell lines. We then tested a therapeutic hypothesis that combining BENSpm with siRNA silencing of polo-like kinase 1 (PLK1) would result in cooperative cancer cell killing. HA@F-PaP/siPLK1 induced polyamine catabolism and cell cycle arrest, leading to enhanced apoptosis in the tested cell lines. The HA-coated nanoparticles facilitated tumor accumulation and contributed to strong tumor inhibition and favorable modulation of the immune tumor microenvironment in orthotopic pancreatic cancer model.

Polyamine-Based Nanostructures Share Polyamine Transport Mechanisms with Native Polyamines and Their Analogues: Significance for Polyamine-Targeted Therapy.

Polyamines are small polycationic alkylamines involved in many fundamental cellular processes, including cell proliferation, survival, and protection from oxidative stress. Polyamine homeostasis is tightly regulated through coordinated biosynthesis, catabolism, and transport. Due to their continual proliferation, cancer cells maintain elevated intracellular polyamine pools. Both polyamine metabolism and transport are commonly dysregulated in cancer, and as such, polyamine analogues are a promising strategy for exploiting the increased polyamine requirement of cancer cells. One potential polyamine analogue resistance mechanism is the downregulation of the poorly defined polyamine transport system. Recent advances in nanomedicine have produced nanostructures with polyamine analogue-based backbones (nanopolyamines). Similar nanostructures with non-polyamine backbones have been shown to be transported by endocytosis. As these polyamine-based nanoparticles could be a method for polyamine analogue delivery that bypasses polyamine transport, we designed the current studies to determine the efficacy of polyamine-based nanoparticles in cells lacking intact polyamine transport. Utilizing polyamine transport-deficient derivatives of lung adenocarcinoma lines, we demonstrated that cells unable to transport natural polyamines were also resistant to nanopolyamine-induced cytotoxicity. This resistance was a result of transport-deficient cells being incapable of importing and accumulating nanopolyamines. Pharmacological modulation of polyamine transport confirmed these results in polyamine transport competent cells. These studies provide additional insight into the polyamine transport pathway and suggest that receptor-mediated endocytosis is a likely mechanism of transport for higher-order polyamines, polyamine analogues and the nanopolyamines.

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.