Supraphysiologic androgen (SPA) treatment can paradoxically restrict the growth of castration-resistant prostate cancer (CRPC) with high androgen receptor (AR) activity, which is the basis for the use of bipolar androgen therapy (BAT) for patients with this disease. Although androgens are widely appreciated for enhancing anabolic metabolism, how SPA-mediated metabolic changes alter prostate cancer progression and therapy response is unknown. In this study, we report that SPA markedly increased intracellular and secreted polyamines in prostate cancer models. AR binding at enhancer sites upstream of the ornithine decarboxylase 1 (ODC1) promoter increased the abundance of ODC, a rate-limiting enzyme of polyamine synthesis, and de novo synthesis of polyamines from arginine. SPA-stimulated polyamines enhanced prostate cancer fitness, as dCas9-KRAB-mediated inhibition of AR regulation of ODC1 or direct ODC inhibition by difluoromethylornithine (DFMO) increased the efficacy of SPA. Mechanistically, AR activation combined with the loss of negative feedback by polyamines increased the activity of S-adenosylmethionine decarboxylase 1, leading to the depletion of its substrate, S-adenosylmethionine, and global protein methylation. These data provided the rationale for a clinical trial testing the safety and efficacy of BAT in combination with DFMO for patients with metastatic CRPC. Pharmacodynamic studies of this therapeutic combination in the first five patients in the trial indicated that this approach effectively depleted polyamines in plasma. Thus, the AR potently stimulates polyamine synthesis, which constitutes a vulnerability in prostate cancer treated with SPA that can be targeted therapeutically. SIGNIFICANCE:Increased polyamine synthesis is a prominent metabolic change induced by the androgen receptor that drives tumor progression and confers a targetable vulnerability in advanced prostate cancer. See related commentary by Alizadeh-Ghodsi and Goldstein, p. 1095.

Polyamines are small polycationic alkylamines that are absolutely required for the continual growth and proliferation of cancer cells. The polyamine analogue ivospemin, also known as SBP-101, has shown efficacy in slowing pancreatic and ovarian tumor progression in vitro and in vivo and has demonstrated encouraging results in early pancreatic cancer clinical trials. We sought to determine if ivospemin was a viable treatment option for the under-served platinum-resistant ovarian cancer patient population by testing its efficacy in combination with commonly used chemotherapeutics. We treated four ovarian adenocarcinoma cell lines in vitro and found that each was sensitive to ivospemin regardless of cisplatin sensitivity. Next, we treated patients with ivospemin in combination with four commonly used chemotherapeutics and found that ivospemin increased the toxicity of each; however, only gemcitabine and topotecan combination treatments were more effective than ivospemin alone. Using the VDID8+ murine ovarian cancer model, we found that the addition of ivospemin to either topotecan or gemcitabine increased median survival over untreated animals alone, delayed tumor progression, and decreased the overall tumor burden. Our results indicate that the combination of ivospemin and chemotherapy is a worthwhile treatment option to further explore clinically in ovarian cancer.

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.

Abstract Polyamines are small polycationic alkylamines that are absolutely required for the continual growth and proliferation of cancer cells. As cancer cells maintain elevated polyamine pools through dysregulated polyamine metabolism, its pharmacological modulation is a promising avenue in cancer therapeutics. The polyamine analogue ivospemin has shown efficacy in slowing pancreatic and ovarian tumor progression in vitro and in vivo and has demonstrated encouraging results in pancreatic cancer clinical trials. Considering nearly 75% of late-stage ovarian cancer patients develop resistant to platinum-based chemotherapies, limiting treatment options, the aim of our study is to determine the potential efficacy of ivospemin in combination with doxorubicin, a common chemotherapeutic used in platinum-resistant ovarian tumors. We have previously shown that ivospemin exposure decreases polyamine content in a variety of cancer cell lines through downregulation of the polyamine biosynthetic enzyme ornithine decarboxylase (ODC) and induction of the polyamine catabolic enzyme spermidine/spermine-N1-acetyltransferase (SSAT). Here we examine the potential of combining ivospemin with doxorubicin. Ivospemin treatment reduces cell viability in ovarian adenocarcinoma cell lines and increases the toxicity of doxorubicin regardless of cisplatin sensitivity. Cells treated with the combination exhibit a greater decrease in polyamine levels than cells treated with either single agent. This increased polyamine depletion and decreased survival is accomplished through modulation of polyamine metabolism, predominately through an additive induction of SSAT activity. Using the syngeneic VDID8+ ovarian murine model, we further evaluated the ability of ivospemin to improve response to doxorubicin at clinical and sub-clinical dosing. Ascites fluid was used as a marker for tumor burden and evaluated for polyamine content. We found that the combination treatment increases median survival, delays tumor onset, and decreases overall tumor burden compared to either clinical or subclinical doxorubicin dosing schemes. Combination treatment also decreases overall polyamine content in the ascites by 75%. N1-acetylated spermidine is enriched in ascites from combination-treated mice, consistent with an upregulation of SSAT in response to treatment. Recognizing the non-representative mutational status of the VDID8+ model as a limitation, we are currently evaluating the combination of ivospemin and doxorubicin in genetically defined murine models that better recapitulate human high-grade serous ovarian carcinomas. Ongoing studies will determine influences on the tumor microenvironment and will mechanistically evaluate the cooperativity of ivospemin and doxorubicin on pathways outside of polyamine metabolism. Citation Format: Cassandra E. Holbert, Jackson R. Foley, Ashley C. Nwafor, Ting-Ann Liu, Elizabeth Bruckheimer, Jennifer K. Simpson, Tracy Murray Stewart, Robert A. Casero. Ivospemin/doxorubicin combination modulates polyamine metabolism to improve survival in murine ovarian cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7154.

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.

Polyamines are small cationic alkylamines that play critical roles in essential cellular processes governing growth and proliferation. As such, cancers are fully reliant on increased polyamine pools maintained through dysregulation of polyamine metabolism. Pharmaceutical modulation of polyamine metabolism is a promising avenue in cancer therapeutics and has been attempted with enzyme inhibitors, including DFMO (difluoromethylornithine), and polyamine analogues. Ivospemin is a spermine analogue that has shown efficacy in slowing pancreatic and ovarian tumor progression both in vitro and in vivo and demonstrated encouraging results in pancreatic cancer clinical trials. We have shown that ivospemin decreases polyamine content through depression of the activity of the polyamine biosynthetic enzyme ornithine decarboxylase (ODC) in a variety of cancer cell lines. Treatment of the VDID8+ murine ovarian cancer model with ivospemin resulted in a marked increase in survival. Here we examine the potential of combining ivospemin and chemotherapeutic agents that are used to treat cisplatin-resistant ovarian cancer. Treatment with gemcitabine, topotecan, and doxorubicin increased the in vitro toxicity of ivospemin, while paclitaxel and docetaxel did not have any added benefit over ivospemin alone. Using the VDID8+ model, we further evaluated the efficacy of ivospemin in combination with gemcitabine, topotecan, and doxorubicin in vivo. Ascites fluid was used as a marker of tumor burden and evaluated for polyamine content. Addition of ivospemin improved the survival of mice treated with any of the three chemotherapeutics. The ivospemin and doxorubicin combination mice had the greatest median survival time; this combination is being further evaluated in mechanistic studies and additional murine studies. Ovarian cancers have extremely immunosuppressive tumor microenvironments (TME) and metabolic reprogramming of the TME to reduce immunosuppressive phenotypes is a promising approach for treatment. Sustained elevation of polyamine levels supports an immunosuppressive TME, and evidence suggests that pharmacologic depletion of polyamines may reduce immunosuppressive phenotypes. DFMO treatment in the immunosuppressive VDID8+ model influences the immune cells of the TME, and we therefore are investigating the combination of ivospemin and DFMO in ovarian cancer. In addition to the cooperativity of ivospemin and chemotherapeutic agents, we have observed a cooperative antiproliferative response in ovarian cancer cells following DFMO and ivospemin cotreatment. Together, these studies suggest the potential of polyamine modulation by ivospemin and DFMO in combination with standard of care chemotherapy. Future studies will determine influences on the immune microenvironment and will evaluate cooperativity between ivospemin, DFMO, and chemotherapy. Citation Format: Cassandra E. Holbert, Jackson R. Foley, Tracy Murray Stewart, Michael J. Walker, Elizabeth Bruckheimer, Jennifer K. Simpson, Robert A. Casero. Evaluating the efficacy of spermine analogue ivospemin (SBP-101) in combination with chemotherapy in ovarian cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4944.

Supplementary data including polyamine levels, addition of anti-PD-1 to therapy, and flow cytometry plots.

Naturally occurring polyamines are absolutely required for cellular growth and proliferation. Many neoplastic cells are reliant on elevated polyamine levels and maintain these levels through dysregulated polyamine metabolism. The modulation of polyamine metabolism is thus a promising avenue for cancer therapeutics and has been attempted with numerous molecules, including enzyme inhibitors and polyamine analogues. SBP-101 (diethyl dihydroxyhomospermine) is a spermine analogue that has shown efficacy in slowing pancreatic tumor progression both in vitro and in vivo; however, the mechanisms underlying these effects remain unclear. We determined the effects of the SBP-101 treatment on a variety of cancer cell types in vitro, including lung, pancreatic, and ovarian. We evaluated the activity of enzymes involved in polyamine metabolism and the effect on intracellular polyamine pools following the SBP-101 treatment. The SBP-101 treatment produced a modest but variable increase in polyamine catabolism; however, a robust downregulation of the activity of the biosynthetic enzyme, ornithine decarboxylase (ODC), was seen across all of the cell types studied and indicates that SBP-101 likely exerts its effect predominately through the downregulation of ODC, with a minor upregulation of catabolism. Our in vitro work indicated that SBP-101 was most toxic in the tested ovarian cell lines. Therefore, we evaluated the efficacy of SBP-101 as a monotherapy in the immunosuppressive VDID8+ murine ovarian model. Mice treated with SBP-101 demonstrated a delay in tumor progression, a decrease in the overall tumor burden, and a marked increase in median survival.

The naturally occurring polyamines, putrescine, spermidine and spermine, are polycationic alkylamines that are essential for cellular growth and proliferation. As such, many cancers are reliant on elevated polyamine levels that are maintained through dysregulated polyamine metabolism. Polyamine metabolism is thus a promising target for cancer therapeutics, and modulation of polyamine metabolism has been attempted with numerous enzyme inhibitors and polyamine analogues. SBP-101 (diethyl dihydroxyhomospermine) is a novel spermine analogue that has shown efficacy in slowing pancreatic tumor progression both in vitro and in vivo. Here we determined the effect of SBP-101 treatment on polyamine metabolism in a variety of cancer cell types in vitro including lung, ovarian, prostate, pancreatic and breast. We evaluated the activity of four enzymes involved in the polyamine pathway following treatment with either SBP-101 or the well-characterized spermine analogue, BENSpm (N1,N11-bisethylnorspermine). Additionally, we determined by high performance liquid chromatography the effect of SBP-101 on intracellular polyamine pools and the accumulation of the analogue itself. The activity of the biosynthetic enzymes ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase and the catabolic enzymes spermidine/spermine-N-(1)-acetyltransferase and spermine oxidase were determined with and without treatment with the polyamine analogues. SBP-101 treatment resulted in a varying increase in the activity of polyamine catabolic enzymes in a subset of tested cell lines, while it downregulated the activity of the biosynthetic enzyme ODC across all cell types studied. These results indicate that SBP-101 likely exerts its effect predominately through decreased polyamine biosynthesis with minor upregulation of catabolism in contrast to the structurally similar BENSpm where the increase in polyamine catabolism is the predominant response. A sustained elevation of polyamine levels plays a role in the immunosuppressive environment of some cold tumors, and the pharmacologic and genetic modulation of polyamine metabolism has demonstrated success in reducing immunosuppressive phenotypes. Therefore, to extend our in vitro results, we evaluated the efficacy of SBP-101 in the immunosuppressive VDID8+ murine ovarian cancer model. SBP-101 caused a marked increase in median survival comparable to that of some promising combination therapies. Future studies will determine the synergistic effects, if any, of SBP-101 in combination with other polyamine metabolism modulators as well as with immune modulators. Citation Format: Cassandra E. Holbert, Tracy Murray Stewart, Jennifer K. Simpson, Michael J. Walker, Robert A. Casero. The potential of spermine analogue SBP-101 (diethyl dihydroxyhomospermine) as a polyamine metabolism modulator in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5488.

The natural mammalian polyamines putrescine, spermidine and spermine are essential for both normal and neoplastic cell function and replication. Dysregulation of metabolism of polyamines and their requirements is common in many cancers. Both clinical and experimental depletion of polyamines have demonstrated their metabolism to be a rational target for therapy; however, the mechanisms through which polyamines can establish a tumour-permissive microenvironment are only now emerging. Recent data indicate that polyamines can play a major role in regulating the antitumour immune response, thus likely contributing to the existence of immunologically 'cold' tumours that do not respond to immune checkpoint blockade. Additionally, the interplay between the microbiota and associated tissues creates a tumour microenvironment in which polyamine metabolism, content and function can all be dramatically altered on the basis of microbiota composition, dietary polyamine availability and tissue response to its surrounding microenvironment. The goal of this Perspective is to introduce the reader to the many ways in which polyamines, polyamine metabolism, the microbiota and the diet interconnect to establish a tumour microenvironment that facilitates the initiation and progression of cancer. It also details ways in which polyamine metabolism and function can be successfully targeted for therapeutic benefit, including specifically enhancing the antitumour immune response.

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.

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.

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 biosyn-thesis, such as in polyamine-targeting anticancer therapies. Inhibitors of polyamine biosynthesis, including alpha-difluor-omethylornithine (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 con-verted 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 devel-oped in this study provides an inexpensive, high-throughput method of screening potentially selective HDAC10 inhibitors.

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 nano particles 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.

Conformationally restrained polyamine analog PG11047 is a well-known drug candidate that modulates polyamine metabolism and inhibits cancer cell growth in a broad spectrum of cancers. Here, we report a structure–activity relationship study of the PG11047 analogs (HPGs) containing alkyl chains of varying length, while keeping the unsaturated spermine backbone unchanged. Synthesis of higher symmetrical homologues was achieved through a synthetic route with fewer steps than the previous route to PG11047. The amphiphilic HPG analogs underwent self-assembly and formed spherically shaped nanoparticles whose size increased with the hydrophobic alkyl group’s increasing chain length. Assessment of the in vitro anticancer activity showed more than an eight-fold increase in the cancer cell inhibition activity of the analogs with longer alkyl chains compared to PG11047 in human colon cancer cell line HCT116, and a more than ten-fold increase in human lung cancer cell line A549. Evaluation of the inhibition of spermine oxidase (SMOX) showed no activity for PG11047, but activity was observed for its higher symmetrical homologues. Comparison with a reference SMOX inhibitor MDL72527 showed nine-fold better activity for the best performing HPG analog.

Snyder?Robinson syndrome (SRS) is an X-linked intellectual disability syndrome caused by a loss-of-function mutation in the spermine synthase (SMS) gene. Primarily affecting males, the main manifestations of SRS include osteoporosis, hypotonic stature, seizures, cognitive impairment, and developmental delay. Because there is no cure for SRS, treatment plans focus on alleviating symptoms rather than targeting the underlying causes. Biochemically, the cells of individuals with SRS accumulate excess spermidine, whereas spermine levels are reduced. We recently demonstrated that SRS patient-derived lymphoblastoid cells are capable of transporting exogenous spermine and its analogs into the cell and, in response, decreasing excess spermidine pools to normal levels. However, dietary supplementation of spermine does not appear to benefit SRS patients or mouse models. Here, we investigated the potential use of a metabolically stable spermine mimetic, (R,R)-1,12-dimethylspermine (Me2SPM), to reduce the intracellular spermidine pools of SRS patient-derived cells. Me2SPM can functionally substitute for the native polyamines in supporting cell growth while stimulating polyamine homeostatic control mechanisms. We found that both lymphoblasts and fibroblasts from SRS patients can accumulate Me2SPM, resulting in significantly decreased spermidine levels with no adverse effects on growth. Me2SPM administration to mice revealed that Me2SPM significantly decreases spermidine levels in multiple tissues. Importantly, Me2SPM was detectable in brain tissue, the organ most affected in SRS, and was associated with changes in polyamine metabolic enzymes. These findings indicate that the (R,R)-diastereomer of 1,12-Me2SPM represents a promising lead compound in developing a treatment aimed at targeting the molecular mechanisms underlying SRS pathology.

Polyamines are small polycationic alkylamines involved in many fundamental cellular processes, including proliferation, nucleic acid synthesis, apoptosis, and protection from oxidative damage. It has been proposed that in addition to these functions, elevated levels of polyamines promote longevity in various biological systems, including yeast,Drosophila, and murine models. A series ofin vitromechanistic studies by multiple investigators has led to the conclusion that addition of exogenous spermidine promotes longevity through autophagy induction; however, these experiments were confounded by the use of mammalian cell culture systems supplemented with fetal bovine serum. Using cell viability assays, LC3B immunoblots, and live-cell fluorescence microscopy, we report here that in the presence of ruminant serum, exogenously added polyamines are quickly oxidized by the copper-containing bovine serum amine oxidase. This polyamine oxidation resulted in the production of harmful byproducts including hydrogen peroxide, ammonia, and reactive aldehydes. Our data demonstrate that it is critically important to prevent confounding bovine serum amine oxidase?induced cytotoxicity in mechanistic studies of the roles of polyamines in autophagy.

Abstract Although ovarian cancer has a low incidence rate, it remains the most deadly gynecologic malignancy. Previous work has demonstrated that the DNMTi 5-Azacytidine (5AZA-C) activates type I interferon signaling to increase IFNγ+ T cells and natural killer (NK) cells and reduce the percentage of macrophages in the tumor microenvironment. To improve the efficacy of epigenetic therapy, we hypothesized that the addition of α-difluoromethylornithine (DFMO), an ornithine decarboxylase inhibitor, may further decrease immunosuppressive cell populations improving outcome. We tested this hypothesis in an immunocompetent mouse model for ovarian cancer and found that in vivo, 5AZA-C and DFMO, either alone or in combination, significantly increased survival, decreased tumor burden, and caused recruitment of activated (IFNγ+) CD4+ T cells, CD8+ T cells, and NK cells. The combination therapy had a striking increase in survival when compared with single-agent treatment, despite a smaller difference in recruited lymphocytes. Instead, combination therapy led to a significant decrease in immunosuppressive cells such as M2 polarized macrophages and an increase in tumor-killing M1 macrophages. In this model, depletion of macrophages with a CSF1R-blocking antibody reduced the efficacy of 5AZA-C + DFMO treatment and resulted in fewer M1 macrophages in the tumor microenvironment. These observations suggest our novel combination therapy modifies macrophage polarization in the tumor microenvironment, recruiting M1 macrophages and prolonging survival. Significance: Combined epigenetic and polyamine-reducing therapy stimulates M1 macrophage polarization in the tumor microenvironment of an ovarian cancer mouse model, resulting in decreased tumor burden and prolonged survival.