DFMO Research Articles

Evaluating Difluoromethylornithine Safety and Efficacy for Non-Melanoma Skin Cancer Chemoprevention: A Systematic Review.

Recent FDA approval of difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase for the prevention of neuroblastoma in children, has renewed interest in this medication for the prevention of other cancers including keratinocyte carcinomas (KCs). It has been investigated for cancer chemoprevention, including neoplasms of the colon, breast, and prostate. We assessed the current body of literature that determines DFMO efficacy and safety in non-melanoma skin cancer prevention. A systematic search of PubMed Central, and Web of Sciences was performed. In this analysis, 12 studies were included evaluating 1618 patients. Most patients were Caucasian 90% (1452/1618) with a mean age of 61 years, and 73% (1214/1618) had previously been diagnosed with KC. For oral DFMO, reduction in KC was significant in 24% (291/1214) of patients. Nonsignificant reduction was observed in 17% (207/1214) of patients. The remaining studies, representing 59% (716/1214) of patients explored DFMO's pharmacological/biological effects without elucidating its direct impact on KC. Topical DFMO shows modest efficacy in reducing the number of actinic keratosis (AK), as indicated in 4 studies representing 38.12% (154/404) of patients. For patients taking the oral eflornithine, the most frequently reported adverse events included reversible ototoxicity (11% of patients) gastrointestinal disturbances (10.39%). For the topical DFMO transient local cutaneous eruptions were common impacting 28.76% (111/386) of patients. Current evidence highlights the lack of conclusive data supporting the efficacy of oral DFMO, making it difficult to recommend its use. Conversely, topical DFMO demonstrates more promising outcomes in preventing AKs, presenting a potentially useful alternative in select patients.

DFMO inhibition of neuroblastoma tumorigenesis.

Most high-risk neuroblastoma patients who relapse succumb to disease despite the existing therapy. We recently reported increased event-free and overall survival in neuroblastoma patients receiving difluoromethylornithine (DFMO) during maintenance therapy. The effect of DFMO on cellular processes associated with neuroblastoma tumorigenesis needs further elucidation. Previous studies have shown cytotoxicity with IC50 values >5-15 mM, these doses are physiologically unattainable in patients, prompting further mechanistic studies at therapeutic doses. We characterized the effect of DFMO on cell viability, cell cycle, apoptosis, neurosphere formation, and protein expression invitro using five established neuroblastoma cell lines (BE2C, CHLA-90, SHSY5Y, SMS-KCNR, and NGP) at clinically relevant doses of 0, 50, 100, 500, 1000, and 2500 μM. Limiting Dilution studies of tumor formation in murine models were performed. Statistical analysis was done using GraphPad and the level of significance set at p = 0.05. There was not a significant loss of cell viability or gain of apoptotic activity in the invitro assays (p > 0.05). DFMO treatment initiated G1 to S phase cell cycle arrest. There was a dose-dependent decrease in frequency and size of neurospheres and a dose-dependent increase in beta-galactosidase activity in all cell lines. Tumor formation was decreased in xenografts both with DFMO-pretreated cells and in mice treated with DFMO. DFMO treatment is cytostatic at physiologically relevant doses and inhibits tumor initiation and progression in mice. This study suggests that DFMO, inhibits neuroblastoma by targeting cellular processes integral to neuroblastoma tumorigenesis at clinically relevant doses.

Abstract 5122: Modulating anti-GD2 immunotherapy via dual DFMO and TGF-β inhibition in neuroblastoma

Abstract Neuroblastoma (NB) is an often-lethal childhood solid tumor. Anti-GD2 immunotherapy improves survival but not all responses are durable. Transforming growth factor-beta (TGFβ) has been implicated in solid tumor immunoresistance. High-risk NBs harbor activated MYC or MYCN to coordinately expand intratumoral polyamines (PA), oncometabolites with immunomodulatory roles, to support tumor progression. Difluoromethylornithine (DFMO) is an inhibitor of ornithine decarboxylase, the rate-limiting enzyme in PA synthesis, and has shown activity in preclinical NB models, including TH-MYCN+/+ transgenic mice, and is in clinical trials in children. We sought to augment anti-GD2 therapy using DFMO and the small molecule TGFβ-receptor inhibitor, galunisertib. We randomized TH-MYCN+/+ mice in two experiments (10 therapy arms; n=6-10 mice/arm) to receive combinations of DFMO, galunisertib, and anti-GD2 antibody m14G2a and to assess impact on survival. We measured 770 genes using nCounter Immuno-Oncology 360 (Nanostring), 36 cytokines using xMap bead-arrays (Luminex), and tumor-infiltrating leukocytes using flow cytometry in harvested tumors. Cell line TGFβ signaling was evaluated in tissue culture. In vitro, galunisertib consistently suppressed the upregulated TGFβ-Smad2/3 signaling cascade in MYCN-amplified neuroblastoma cell lines. In vivo, DFMO alone extends mouse survival and increases intra-tumoral neutrophils and natural killer (NK) cells. These NK cells increased surface expression of activation receptors like NKG2D (p<0.05), while tumor cells increased surface expression of NK-ligands. Tumor cells also expressed increased latency-associated peptide (LAP; p<0.05), a TGFβ precursor, suggesting NK cells were poised to kill but restrained by TGFβ. Cytotoxicity assays showed reversal of this inhibition with TGFβR1 inhibitor galunisertib in vitro. In a proof-of-concept trial, DFMO and galunisertib (DFMO/gal) demonstrated a clinical anti-tumor synergistic effect. DFMO/gal treated tumors had upregulated NK cell activity genes and downregulated TGFβ signaling genes. Cytokine analysis confirmed DFMO induced upregulation of TGFβ1 and TGFβ2 (p<0.01) that was modulated by galunisertib. IL6, GCSF, and GMCSF were markedly downregulated in tumors treated with both DFMO and galunisertib (p<0.05). An expanded trial showed DFMO/gal added to m14G2a showed no significant survival benefit over the DFMO and m14G2a combination. Ultimately, DFMO creates a pro-inflammatory cellular and cytokine milieu in TH-MYCN+/+ NBs. DFMO and TGFβR1 inhibition via galunisertib activate NK cells further and decrease the anti-inflammatory cytokine IL6. Ongoing studies are further characterizing the gene expression and cytokine levels of the NB TME under these treatment conditions. Citation Format: Om H. Gandhi, Christina S. Turn, Kangning Liu, Annette T. Vu, Hamid Bassiri, Michael D. Hogarty. Modulating anti-GD2 immunotherapy via dual DFMO and TGF-β inhibition in neuroblastoma [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 5122.

Abstract 5283: Polyamine blockade therapy: A strategy to block immunosuppression in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is lethal in 88% of patients and is characterized by a dense tumor microenvironment (TME) which impedes tumor entry of immune cells and delivery of therapeutics. Immunosuppressive myeloid populations of PDAC, including myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs), are known to not only support PDAC development but also impede the anti-tumor immune response elicited by therapeutic agents. To improve prognosis while maintaining tolerability, this study aims to target polyamine metabolism that is upregulated in the immunosuppressive myeloid populations and cancer cells to disrupt their contribution to PDAC progression and the immunosuppressive TME. We previously demonstrated that polyamine blockade therapy (PBT), via blocking biosynthesis with difluoromethylornithine (DFMO) and transport with a novel inhibitor (Trimer44NMe), elicits an anti-tumor response and improves median survival in PDAC engrafted immunocompetent mice. In the current study, PBT was found to lead to a selective reduction of the dominant MDSC subtype in PDAC, polymorphonuclear (PMN) MDSCs. Upon co-culturing of MDSCs with CD8+ T cells, PBT dampened MDSC-mediated suppression of T cell activity. These findings were corroborated by tracking Arginase 1, a driver of MDSC-mediated immunosuppression, which exhibited a significant decrease in response to PBT at both the activity and gene expression level. Ex-vivo generated M2 macrophages and TAMs treated with PBT also demonstrated a shift from the M2 pro-tumor phenotype towards the anti-tumor M1 macrophage phenotype. In summary, these findings support that PBT is effective at reducing myeloid-mediated immunosuppression through subtype specific modulation and supports the use of PBT for treatment of PDAC in combination with other immunomodulatory therapeutics. Citation Format: Joseph A. Goode. Polyamine blockade therapy: A strategy to block immunosuppression in pancreatic cancer [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 5283.

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 β-cell Cytokine Stress and Diabetes Outcomes in Mice.

Type 1 diabetes (T1D) is an autoimmune disease leading to dysfunction and loss of insulin-secreting β cells. In β 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 β-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 β 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 β 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.

Targeting Pharmacological and Nanotechnology Based Therapeutics for Management of Hirsutism: A Comprehensive State-of-the-Art

Hirsutism is characterized by the excessive growth of coarse hair in women, resembling the typical pattern seen in men. It affects between 5 to 10 % of females of reproductive age. Excessive hair growth often leads to significant mental and emotional anguish. Hirsutism is the result of an overabundance of androgens being secreted by the ovaries or adrenal glands. This article offers a comprehensive review of numerous causes that might provoke hirsutism which includes polycystic ovarian syndrome, Cushing syndrome, idiopathic hirsutism, insulin resistance, congenital adrenal hyperplasia, ovarian or adrenal tumors, menopause, and the use of certain drugs. Polycystic ovary syndrome and ovarian tumors are frequent underlying factors of hyperandrogenism, which subsequently results in the development of Hirsutism. Mechanical and cosmetic hair removal methods are viable options for managing hirsutism. This study examines the pharmacological therapies that have proven essential in enhancing the quality of life for patients. These treatments include oral contraceptives, antiandrogen therapy like spironolactone, cyproterone acetate, flutamide, and finasteride, specific insulin-lowering medicines, gonadotrophin-releasing hormone agonists, and topical treatment such eflornithine hydrochloride cream (13.9% w/w cream). This review also explored significance of nanotechnology-based methods like nanostructured lipid carriers, solid lipid nanoparticles, liposomes, cerosomes, and nanogel in enhancing the effectiveness of medications used to treat hirsutism. A comprehensive update has been made to the latest information on clinical studies and patents linked to the treatment of hirsutism.

EOLP-04. EXTRA-NEURAL DISSEMINATION OF MYC-ACTIVATED INFANT MEDULLOBLASTOMA DESPITE MULTIMODAL THERAPY

Abstract Extra neural metastases of infant medulloblastoma portend poor prognosis. Previous reports were in patients receiving treatment with radiation alone or patients with ventriculoperitoneal shunt (VPS). Recently with multimodal therapy, reports of extra-neural medulloblastoma are rarely reported. We report two patients with stage M3 MYC-Activated Infant Medulloblastoma who received aggressive therapy yet developed extra-neural relapses. The first case was a 2-year-old male who underwent gross total resection, which was complicated by hydrocephalus requiring VPS placement. He completed treatment as per ACNS0334 and achieved a near-complete response. Next, he received four cycles of five drug anti-angiogenic therapy followed by 2 cycles of expanded access therapy with I-3F8 antibody. He progressed both in brain and spinal cord, then received palliative craniospinal irradiation with boost. Two months following radiation, he died of disease with significant pancytopenia and hypoechoic nodules throughout the liver, suggestive of disseminated disease. Our second patient is a five-year-old girl, who was diagnosed at age two. She achieved a CR after treatment as per ACNS0334. She then enrolled on a clinical trial with Difluoromethylornithine (DFMO) for three months. On initial disease evaluation, she had progression in brain and spine. She underwent palliative radiation with craniospinal irradiation and boost with concurrent Indoximod on phase 2 clinical trial. Following radiation, she received compassionate use indoximod and temadar for 24 months and remained in CR. Three months after stopping therapy, she progressed to left orbit, pelvis and ribs. She is currently receiving palliative care. MYC-activated medulloblastoma has a very poor outcome and requires multimodal including radiation to achieve remission. Despite aggressive therapy, disease is highly evasive and can spread extracranially to multiple areas including bones, bone marrow, liver, and abdomen. Novel therapies and more exhaustive surveillance regiments need to be developed to limit extracranial dissemination and achieve more durable remissions.

Effect of Ivospemin and Difluoromethylornithine Polyamine Inhibitors on Viability of Myeloma Cell Lines

Abstract Introduction: Multiple myeloma (MM) is caused by heterogeneous genomic alterations that lead to activation of the MYC oncogenic pathway (Larrayoz et al., 2023). Moreover, MYC activity plays a role in immune escape by MM cells and alteration of the bone marrow microenvironment as the disease progresses, which in turn affects response to immunotherapy. Downstream targets and pathways of MYC, such as polyamine (PA) pathway, are an important avenue for therapeutic anticancer interventions for targeting MYC that is generally considered to be “undruggable”. In the present study, we therefore evaluated the effect of two polyamine inhibitors, ivospemin and difluoromethylornithine (DFMO), on growth and viability of myeloma cell lines. SBP-101, an analogue of the naturally occurring PA, spermine, is a polyamine metabolic inhibitor (PMI) that reduces native PA pools by inhibiting key synthetic enzymes and induction of catabolic enzymes. DFMO is an irreversible inhibitor of the ornithine decarboxylase and polyamine synthesis. Methods: Four myeloma cell lines: NCI-H929, RPMI-8226, ARP-1, and MM.1S, were exposed to varying concentrations of two PA inhibitors, SBP-101 and DFMO, based on the data available from clinical trials conducted for other malignancies. We analyzed the effects of these treatments on cell proliferation, cell viability, and the induction of apoptosis. The analyses were done separately for each compound as well as their combination. To assess cytotoxicity and cell proliferation, we used the Incucyte® SX5 Live-Cell Analysis System. Additionally, the detection of apoptosis was performed using Annexin V and propidium iodide flow cytometric assay. Results: Exposure of the MM cell lines to Ivospemin at increasing concentrations ranging from 1µM to 10 µM showed a significant decrease in cellular proliferation in all four MM cell lines. The mean percentage of cytotoxicity observed at 10 µM concentrations of Ivospemin in NCI-H929, RPMI-8226, ARP-1, and MM.1S cell lines were 69.17, 20.75, 83.92, and 48.71 respectively (Fig. 1a). Similarly, when we exposed the MM cell lines to DFMO at increasing concentrations ranging from 1 µM to 100 µM, we also saw a significant decrease in cellular proliferation in all four cell lines, with the most pronounced effect observed in RPMI-8226. The percentage of cells killed at DFMO 10 µM in NCI-H929, RPMI-8226, ARP-1, and MM.1S cell lines were 32.67, 63.61, 28.24, and 16.06 respectively. Moreover, when we combined Ivospemin at 10 µM and DFMO at 100 µM, we observed a complete abolition of cell growth in all the cell lines (Fig. 1b). Additionally, the treatment of the myeloma cell lines with either Ivospemin, DFMO alone, or in combination, showed an induction of apoptosis, as revealed by flow cytometric analysis (data not shown). These findings indicate that both Ivospemin and DFMO have significant anti-proliferative effects on MM cell lines, and their combination leads to even more potent growth inhibition and induction of apoptosis. Conclusion: The polyamine inhibitors,Ivospemin and DFMO, exhibited a marked anti-proliferative effect in the four human MM cell lines studied when used alone or in combination. These results confirm the anti-neoplastic potential of SBP-101 and DFMO and offer a compelling rationale for its clinical development as a potentially promising treatment option for multiple myeloma. Fig. 1. Effect of varying concentration of Ivospemin (SBP-101) treatment on myeloma cell lines (a). The data is represented as mean ± SEM of three replicates. A combination of both Ivospemin (SBP-101) and DFMO improved the cytotoxicity as compared to when used alone (b).

Eflornithine Hydrochloride-Loaded Electrospun Nanofibers as a Potential Face Mask for Hirsutism Application.

Hirsutism is a distressing condition that can affect women's self-esteem due to the excessive amount of hair growth in different body parts, including the face. A temporary managing option is to develop a self-care routine to remove unwanted hair through shaving or waxing. Laser or electrolysis are alternative methods, but in some cases, the use of medications, such as the topical cream Vaniqa®, can help in reducing the growth of unwanted hair. Electrospun fibers have been used in several drug delivery applications, including skin care products, owing to their biocompatibility, biodegradability, high surface area-to-volume ratio, and dry nature that can release the encapsulated drugs with maximum skin penetration. Therefore, polyvinyl pyrrolidone (PVP) fibers were fabricated in combination with hyaluronic acid to deliver the active compound of Vaniqa®, i.e., Eflornithine hydrochloride (EFH), as a face mask to inhibit excess facial hair growth. The prepared drug-loaded fibers showed a diameter of 490 ± 140 nm, with an encapsulation efficiency of 88 ± 7% and a drug loading capacity of 92 ± 7 μg/mg. The in vitro drug release of EFH-loaded fibers exhibited an initial burst release of 80% in the first 5 min, followed by a complete release after 360 min, owing to the rapid disintegration of the fibrous mat (2 s). The in vitro cytotoxicity indicated a high safety profile of EFH at all tested concentrations (500-15.625 μg/mL) after 24-h exposure to human dermal fibroblast (HFF-1) cells. Therefore, this drug-loaded nanofibrous system can be considered a potentially medicated face mask for the management of hirsutism, along with the moisturizing effect that it possesses. Topical applications of the developed system showed reduced hair growth in mice to a certain extent.

Chemoprevention of Colon Cancer by DFMO, Sulindac, and NO-Sulindac Administered Individually or in Combinations in F344 Rats.

Non-steroidal anti-inflammatory drugs (NSAIDs) are promising colorectal cancer (CRC) chemopreventive drugs; however, to overcome NSAIDs' associated side effects, there is a need to develop safer and efficacious approaches. The present study was designed to evaluate (i) the efficacy of nitric-oxide releasing (NO)-Sulindac as compared to Sulindac; (ii) whether NO-Sulindac is superior to Sulindac in enhancing low-dose difluoromethylornithine (DFMO)-induced chemopreventive efficacy, and (iii) assessing the key biomarkers associated with colon tumor inhibition by these combinations. In F344 rats, colonic tumors were induced by azoxymethane (AOM). At the adenoma stage (13 weeks post AOM), groups of rats were fed the experimental diets containing 0 ppm, 500 ppm DFMO, 150 ppm Sulindac, and 200 ppm NO-Sulindac, individually or in combinations, for 36 weeks. Colon tumors were evaluated histopathologically and assayed for expression levels of proliferative, apoptotic, and inflammatory markers. Results suggest that (except for NO-Sulindac alone), DFMO, Sulindac individually, and DFMO combined with Sulindac or NO-Sulindac significantly suppressed AOM-induced adenocarcinoma incidence and multiplicities. DFMO and Sulindac suppressed adenocarcinoma multiplicity by 63% (p < 0.0001) and 51% (p < 0.0011), respectively, whereas NO-Sulindac had a modest effect (22.8%, p = 0.09). Combinations of DFMO plus Sulindac or NO-Sulindac suppressed adenocarcinoma incidence (60%, p < 0.0001; 50% p < 0.0004), and multiplicity (81%, p < 0.0001; 62%, p < 0.0001). Rats that were fed the combination of DFMO plus Sulindac showed significant inhibition of tumor cell proliferation and induction of apoptosis. In addition, enhancement of p21, Bax, and caspases; downregulation of Ki-67, VEGF, and β-catenin; and modulation of iNOS, COX-2, and ODC activities in colonic tumors were observed. These observations show that a lower-dose of DFMO and Sulindac significantly enhanced CRC chemopreventive efficacy when compared to NO-Sulindac alone, and the combination of DFMO and NO-Sulindac was modestly efficacious as compared to DFMO alone.

Plasmonic Functional Assay Platform Determines the Therapeutic Profile of Cancer Cells.

Functional assay platforms could identify the biophysical properties of cells and their therapeutic response to drug treatments. Despite their strong ability to assess cellular pathways, functional assays require large tissue samples, long-term cell culture, and bulk measurements. Even though such a drawback is still valid, these limitations did not hinder the interest in these platforms for their capacity to reveal drug susceptibility. Some of the limitations could be overcome with single-cell functional assays by identifying subpopulations using small sample volumes. Along this direction, in this article, we developed a high-throughput plasmonic functional assay platform to identify the growth profile of cells and their therapeutic profile under therapies using mass and growth rate statistics of individual cells. Our technology could determine populations' growth profiles using the growth rate data of multiple single cells of the same population. Evaluating spectral variations based on the plasmonic diffraction field intensity images in real time, we could simultaneously monitor the mass change for the cells within the field of view of a camera with the capacity of > ∼500 cells/h scanning rate. Our technology could determine the therapeutic profile of cells under cancer drugs within few hours, while the classical techniques require days to show reduction in viability due to antitumor effects. The platform could reveal the heterogeneity within the therapeutic profile of populations and determine subpopulations showing resistance to drug therapies. As a proof-of-principle demonstration, we studied the growth profile of MCF-7 cells and their therapeutic behavior to standard-of-care drugs that have antitumor effects as shown in the literature, including difluoromethylornithine (DFMO), 5-fluorouracil (5-FU), paclitaxel (PTX), and doxorubicin (Dox). We successfully demonstrated the resistant behavior of an MCF-7 variant that could survive in the presence of DFMO. More importantly, we could precisely identify synergic and antagonistic effects of drug combinations based on the order of use in cancer therapy. Rapidly assessing the therapeutic profile of cancer cells, our plasmonic functional assay platform could be used to reveal personalized drug therapies for cancer patients.

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

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. 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. 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. 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.

Abstract 4944: Evaluating the efficacy of spermine analogue ivospemin (SBP-101) in combination with chemotherapy in ovarian cancer

Abstract 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.

Abstract 4503: Polyamine blockade therapy: A strategy to block immunosuppression in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized by dense desmoplasia which contributes to poor prognosis by hindering tumor entry of immune cells and therapeutics. This leads to PDAC being difficult to treat with gemcitabine, a current standard-of-care, with only a 22% 12-month survival rate. Therapeutic modalities are needed to tackle the PDAC tumor microenvironment (TME). An alternative target is polyamine metabolism which is required for tumor growth, survival, metastasis, and also for modulating generation, survival, and activity of immune cells. Myeloid-derived suppressor cells (MDSCs) are immune cells that have a key role in PDAC metastasis, invasion, progression, and in impeding anti-tumor immune response to therapeutics in PDAC. MDSCs include monocyte-like (M-MDSC) and neutrophil-like (PMN-MDSC) cells. Previous studies have shown that polyamine blockade therapy (PBT), consisting of difluoromethylornithine (DFMO) to block polyamine biosynthesis and a polyamine transport inhibitor (Trimer44NMe), elicits anti-tumor immune response, improved survival, and a decrease in MDSCs in other tumor types. This is of interest given our previous findings that PBT improved survival of PDAC-bearing mice. Here, we tested PBT in altering PDAC-stimulated MDSC survival and whether it shifts their immunosuppressive phenotype. Studies were conducted using CD11b+ bone marrow cells that were differentiated into MDSCs via GM-CSF and tumor-conditioned media from PDAC cells derived from a genetic mouse model for pancreatic cancer (KPC-mice). Bulk RNA-sequencing was conducted on MDSCs treated with PBT, gemcitabine or combination. MDSCs exhibited widespread downregulation of expression in the gemcitabine and combination treatment, but PBT alone exhibited more targeted downregulation. Specifically, Gene Ontology (GO) analysis revealed a decrease in genes associated with immune cell migration and chemotaxis, and KEGG analysis showed downregulation of inflammatory pathways. These findings indicate that PBT may modify MDSCs from their tumor-associated immunosuppressive and pro-inflammatory behavior. MDSC subtype-specific markers were investigated by flow cytometry. Results show subsets of immune cells, such as PMN-MDSCs, are reduced upon PBT treatment alone or in combination with gemcitabine, thereby highlighting the dependence of this subtype on polyamine metabolism. In conclusion, PBT alters the expression of MDSC immunomodulatory genes, decreases MDSC survival and supports the use of PBT for treatment of PDAC in combination with other immunomodulatory therapeutics. Citation Format: Joseph A. Goode, Sai Preethi Nakkina, Manav Gandhi, Otto Phanstiel, Deborah A. Altomare. Polyamine blockade therapy: A strategy to block immunosuppression in pancreatic 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 4503.

Deregulation of the cyclin-dependent kinase inhibitor p27 as a putative candidate for transformation in Chlamydia trachomatis infected mesenchymal stem cells.

Several pathological conditions might cause the degradation of the cyclin-dependent kinase inhibitor (CKI) p27 and cell cycle arrest at the G1 phase, including cancers and infections. Chlamydia trachomatis (Ctr), as an obligatory intracellular pathogen, has been found to alter the fate of the cell from different aspects. In this study, we aimed to investigate the effect of Ctr infection on the expression of the important cell cycle regularity protein p27 in mesenchymal stem cells (MSCs). Isolation of MSCs from healthy human fallopian tube was confirmed by detection of the stemness markers Sox2, Nanog and Oct4 and the surface markers CD44, CD73 and CD90 by Western blotting and fluorescence-activated cell sorting analysis. The expression of p27 was downregulated at the protein level upon Ctr D infection measured by Real-Time Quantitative Reverse Transcription PCR (qRT-PCR), IF and Western blotting. Recovery of p27 in Ctr D-infected MSCs was achieved by treatment with difluoromethylornithine (DFMO). Ctr D infected MSCs were able to produce colonies in anchorage-independent soft agar assay. Ctr D infection was able to downregulate the expression of the important cell cycle regulator protein p27, which will be considered a putative candidate for transformation in Ctr D infected MSCs.

Heteroleptic Transition Metal Complexes of Eflornithine Hydrochloride Monohydrate: Synthesis, Characterization, in silico and in vitro Biological Studies

Incessant development of resistance to drugs by microorganisms remains a major setback associated with the currently available antibiotics, thereby making imperative a continuous search for alternative candidates with improved efficacy. Previous studies have shown enhanced antimicrobial activity of some bioactive molecules upon coordination with metal ions. Thus, in this study, Cu(II), Co(II), and Ni(II) complexes of eflornithine hydrochloride monohydrate (EHM) were synthesized and probed for bactericidal activity via in vitro and in silico. The characterization results such as CHN analysis, FTIR, UV-visible magnetic susceptibility and Electrospray Ionization Mass Spectrometry (ESI-MS) reveal that EHM coordinates as a bidentate ligand to each central metal ion in the molar ratio 1:2 through O and N in the COO- and NH2 group respectively, and also suggest octahedral geometry in each complex. The physicochemical and pharmacokinetics parameters predicted in silico support the bio-applicability and safety of the complexes. From the in vitro antibacterial study, the complexes demonstrate improved activity against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa with an average minimum inhibitory concentration (MIC) of 0.01 mg/L similar to ciprofloxacin, compared to EHM whose MIC &amp;gt;1.00 mg/L. Although, not all the complexes satisfy Lipinski’s drugability rule of 5 due to their molecular weight, however, coordination with metal ions improves the biological activities of EHM and the complexes demonstrate potential for further transformation into antibiotic therapeutics.

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.

Difluoromethylornithine (DFMO) and AMXT 1501 inhibit capsule biosynthesis in pneumococci

Polyamines are small cationic molecules that have been linked to various cellular processes including replication, translation, stress response and recently, capsule regulation in Streptococcus pneumoniae (Spn, pneumococcus). Pneumococcal-associated diseases such as pneumonia, meningitis, and sepsis are some of the leading causes of death worldwide and capsule remains the principal virulence factor of this versatile pathogen. α-Difluoromethyl-ornithine (DFMO) is an irreversible inhibitor of the polyamine biosynthesis pathway catalyzed by ornithine decarboxylase and has a long history in modulating cell growth, polyamine levels, and disease outcomes in eukaryotic systems. Recent evidence shows that DFMO can also target arginine decarboxylation. Interestingly, DFMO-treated cells often escape polyamine depletion via increased polyamine uptake from extracellular sources. Here, we examined the potential capsule-crippling ability of DFMO and the possible synergistic effects of the polyamine transport inhibitor, AMXT 1501, on pneumococci. We characterized the changes in pneumococcal metabolites in response to DFMO and AMXT 1501, and also measured the impact of DFMO on amino acid decarboxylase activities. Our findings show that DFMO inhibited pneumococcal polyamine and capsule biosynthesis as well as decarboxylase activities, albeit, at a high concentration. AMXT 1501 at physiologically relevant concentration could inhibit both polyamine and capsule biosynthesis, however, in a serotype-dependent manner. In summary, this study demonstrates the utility of targeting polyamine biosynthesis and transport for pneumococcal capsule inhibition. Since targeting capsule biosynthesis is a promising way for the eradication of the diverse and pathogenic pneumococcal strains, future work will identify small molecules similar to DFMO/AMXT 1501, which act in a serotype-independent manner.