DFMO Research Articles

Inhibition of Polyamine Biosynthesis Reverses Ca2+ Channel Remodeling in Colon Cancer Cells.

Store-operated Ca2+ entry (SOCE) is the most important Ca2+ entry pathway in non-excitable cells. Colorectal cancer (CRC) shows decreased Ca2+ store content and enhanced SOCE that correlate with cancer hallmarks and are associated to remodeling of store-operated channels (SOCs). Normal colonic cells display small, Ca2+-selective currents driven by Orai1 channels. In contrast, CRC cells display larger, non-selective currents driven by Orai1 and transient receptor potential canonical type 1 channels (TRPC1). Difluoromethylornithine (DFMO), a suicide inhibitor of ornithine decarboxylase (ODC), the limiting step in polyamine biosynthesis, strongly prevents CRC, particularly when combined with sulindac. We asked whether DFMO may reverse SOC remodeling in CRC. We found that CRC cells overexpress ODC and treatment with DFMO decreases cancer hallmarks including enhanced cell proliferation and apoptosis resistance. Consistently, DFMO enhances Ca2+ store content and decreases SOCE in CRC cells. Moreover, DFMO abolish selectively the TRPC1-dependent component of SOCs characteristic of CRC cells and this effect is reversed by the polyamine putrescine. Combination of DFMO and sulindac inhibit both SOC components and abolish SOCE in CRC cells. Finally, DFMO treatment inhibits expression of TRPC1 and stromal interaction protein 1 (STIM1) in CRC cells. These results suggest that polyamines contribute to Ca2+ channel remodeling in CRC, and DFMO may prevent CRC by reversing channel remodeling.

Use of Eflornithine Hydrochloride Cream to Reduce Facial Hair Growth in Flapped Skin

Use of Eflornithine Hydrochloride Cream to Reduce Facial Hair Growth in Flapped Skin

A Modified Tripeptide Motif of RS1 (RSC1A1) Down-Regulates Exocytotic Pathways of Human Na+-d-glucose Cotransporters SGLT1, SGLT2, and Glucose Sensor SGLT3 in the Presence of Glucose

A domain of protein RS1 (RSC1A1) called RS1-Reg down-regulates the plasma membrane abundance of Na+-d-glucose cotransporter SGLT1 by blocking the exocytotic pathway at the trans-Golgi. This effect is blunted by intracellular glucose but prevails when serine in a QSP (Gln-Ser-Pro) motif is replaced by glutamate [RS1-Reg(S20E)]. RS1-Reg binds to ornithine decarboxylase (ODC) and inhibits ODC in a glucose-dependent manner. Because the ODC inhibitor difluoromethylornithine (DFMO) acts like RS1-Reg(S20E), and DFMO and RS1-Reg(S20E) are not cumulative, we raised the hypothesis that RS1-Reg(S20E) down-regulates the exocytotic pathway of SGLT1 at the trans-Golgi by inhibiting ODC. We investigated whether QEP down-regulates human SGLT1 (hSGLT1) like hRS1-Reg(S20E) and whether human Na+-d-glucose cotransporter hSGLT2 and the human glucose sensor hSGLT3 are also addressed. We expressed hSGLT1, hSGLT1 linked to yellow fluorescent protein (hSGLT1-YFP), hSGLT2-YFP and hSGLT3-YFP in oocytes of Xenopus laevis, injected hRS1-Reg(S20E), QEP, DFMO, and/or α-methyl-d-glucopyranoside (AMG), and measured AMG uptake, glucose-induced currents, and plasma membrane-associated fluorescence after 1 hour. We also performed in vitro AMG uptake measurements into small intestinal mucosa of mice and human. The data indicate that QEP down-regulates the exocytotic pathway of SGLT1 similar to hRS1-Reg(S20E). Our results suggests that both peptides also down-regulate hSGLT2 and hSGLT3 via the same pathway. Thirty minutes after application of 5 mM QEP in the presence of 5 mM d-glucose, hSGLT1-mediated AMG uptake into small intestinal mucosa was decreased by 40% to 50%. Thus oral application of QEP in a formulation that optimizes uptake into enterocytes but prevents entry into the blood is proposed as novel antidiabetic therapy.

Maintenance DFMO Increases Survival in High Risk Neuroblastoma

High risk neuroblastoma (HRNB) accounts for 15% of all pediatric cancer deaths. Despite aggressive therapy approximately half of patients will relapse, typically with only transient responses to second-line therapy. This study evaluated the ornithine decarboxylase inhibitor difluoromethylornithine (DFMO) as maintenance therapy to prevent relapse following completion of standard therapy (Stratum 1) or after salvage therapy for relapsed/refractory disease (Stratum 2). This Phase II single agent, single arm multicenter study enrolled from June 2012 to February 2016. Subjects received 2 years of oral DFMO (750 ± 250 mg/m2 twice daily). Event free survival (EFS) and overall survival (OS) were determined on an intention-to-treat (ITT) basis. 101 subjects enrolled on Stratum 1 and 100 were eligible for ITT analysis; two-year EFS was 84% (±4%) and OS 97% (±2%). 39 subjects enrolled on Stratum 2, with a two-year EFS of 54% (±8%) and OS 84% (±6%). DFMO was well tolerated. The median survival time is not yet defined for either stratum. DFMO maintenance therapy for HRNB in remission is safe and associated with high EFS and OS. Targeting ODC represents a novel therapeutic mechanism that may provide a new strategy for preventing relapse in children with HRNB.

Abstract 3190: DFMO preferentially drives cancer stem cells in neuroblastoma towards senescence

Abstract Background: High-risk neuroblastoma (HRNB) is an aggressive form of the most common extracranial solid tumor cancer in children, often characterized by amplified levels of MYCN. HRNB patients in remission have a high relapse rate, resulting in poor survival. Recent studies have shown that cancer stem cells (CSCs) play a role in the progression, chemoresistance, and relapse of many cancers. Difluoromethylornithine (DFMO) is being evaluated to prevent relapse in clinical trials. Our current study examines the in vitro and in vivo effects of DFMO on tumor-initiating cells through evaluating CSC viability, CSC markers and senescence. Methods: BE(2)-C and SMS-KCNR established HRNB cell lines were injected subcutaneously at limiting cell dilutions into nude mice. DFMO was administered in the drinking water (2%, by volume) for 50 and 75 days for BE(2)-C and SMS-KCNR, respectively. Tumor formation was monitored, and LDA was performed. In a second xenograft model BE(2)-C cells were injected into nude mice, once tumors were palpable, the mice were given either normal drinking water (vehicle) or DFMO (2%)-containing water. Following 7 days of treatment, the tumors were resected and measured for volumes/weights as well as the harvested for Western blot analysis of CSC markers (OCT4, SOX2, NANOG, KLF4). BE(2)-C cells were cultured for 48 and 72h with or without 5mM DFMO and stained for SA-βGal activity as a marker of senescence. NB cell lines treated for 48 and 72h with a range of DFMO concentrations (0, 10μM, 50μM, 100μM, 1mM, 2.5mM, and 5mM) were harvested and labeled with cell surface antibodies for CSCs (CD133, CD114), senescence (CD148), differentiation (CD24), and NB (CD56) for 30min in the dark, and cell viability was determined by 7AAD staining immediately before flow cytometric analysis. Results: Limiting dilution analysis (LDA) on xenograft mice receiving DFMO showed both prevention of tumor formation (p value<0.001) and a decrease in frequency of tumor-initiating cells (p value p<0.04). Tumors harvested from mice treated with DFMO for 7 days showed decrease in volume and weight relative to control (p value <0.001) as well as a decrease in the presence of CSC markers shown by Western blot. Galactosidase (SA-βGal) staining of cultured HRNB cells showed the induction of senescence in HRNB cells when treated with DFMO by 72h (p value <0.001). Flow cytometric analysis determined that DFMO specifically targeted the CSC subpopulation at as low as 50uM concentrations (100uM p value<0.05); CSCs had decreased viability and higher levels of senescence-associated protein expression than the overall NB population (p value<0.05). Conclusions: Our results show the preferential induction of senescence and overall decrease in the CSC population at very low doses of DFMO. This may expose a novel treatment mechanism to inhibit CSC function and prevent relapse in HRNB patients. Citation Format: Tracey Avequin, Austin Goodyke, Joseph Zagorski, Tyler Maser, Giselle L. Saulnier Sholler. DFMO preferentially drives cancer stem cells in neuroblastoma towards senescence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3190.

Abstract 4137: Inhibition of protein synthesis with polyamine depletion in high-risk neuroblastoma

Abstract The MYC family of oncogenes coordinately deregulate cellular programs that link cell cycle progression with the requisite biomass and energy creation. Neuroblastoma is a MYC-driven, commonly lethal pediatric tumor, with a high proportion of tumors containing MYCN amplification. Polyamine synthesis is a pathway downstream of MYC, with polyamines functioning, in part, to support protein synthesis. ODC1 encodes ornithine decarboxylase, the rate-limiting enzyme in polyamine synthesis, and we found that ODC1 is co-amplified with MYCN in a subset of high-risk neuroblastomas (~8%). Difluoromethylornithine (DFMO) is an irreversible inhibitor or ornithine decarboxylase and is an approved agent for the treatment of Trypanosomiasis. We have previously shown that DFMO inhibits tumor progression and synergizes with chemotherapy in murine mouse models of neuroblastoma, and we postulate that inhibition of protein synthesis is a predominant mechanism of anti-tumor activity. The effects of polyamine depletion on specific components of protein translational machinery have yet to be defined. Here, we show inhibition of global protein synthesis of up to 90% after treatment with various concentrations of DFMO in vitro, with a larger decrease in protein synthesis generally correlating with the presence of MYCN amplification. We found that colony formation is inhibited at DFMO exposures as low as 150 to 500 μM DFMO, which are achievable concentrations in adults based on previous clinical trials. We also show via isoelectric focusing that treatment of neuroblastoma cells in vitro with DFMO leads to a marked decrease in the proportion of hypusinated, or active, eIF5A (an elongation factor important in translation of polyproline stretches and possibly broader influence on translation initiation). To interrogate influences of polyamine depletion on the cap-dependent translation eIF4F complex, we evaluated for changes in the phosphorylation of 4E-BP1 in DFMO treatment conditions, though no changes in phosphorylation were seen compared to controls. Studies to further elucidate the protein synthetic components affected by polyamine depletion are ongoing, as are studies to assess for selective effects of DFMO on polyproline-containing proteins via eIF5A. Further elucidation of the mechanisms of DFMO activity will identify opportunities for drug synergy and provide a responder hypothesis to test in upcoming pivotal Phase 2 and 3 clinical trial of DFMO in the treatment of neuroblastoma. Citation Format: Andrea T. Flynn, Kangning Liu, Annette Vu, Michael D. Hogarty. Inhibition of protein synthesis with polyamine depletion in high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4137.

Abstract 4800: Targeting the polyamine pathway as a novel therapeutic treatment against diffuse intrinsic pontine glioma

Abstract Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive paediatric brainstem tumour, with a peak incidence in middle childhood and a median survival of less than 1 year in the majority of cases. The dismal prognosis associated with DIPG is exacerbated by the repeated failure of over 250 clinical trials to improve survival over standard radiotherapy. Therefore novel and innovative therapeutic approaches are urgently needed to needed to treat this devastating disease. Polyamines are small intracellular polycations that control key aspects of cell biology, such as cell replication/growth, differentiation and survival and can be found up-regulated in many cancers. Difluoromethylornithine (DFMO) is an FDA-approved inhibitor of the enzyme ornithine decarboxylase (ODC1) which is involved in polyamine synthesis. The aim of this study was to investigate the efficacy of polyamine pathway inhibitors such as DFMO as a therapeutic strategy against DIPG. By RT-qPCR and western blotting, we observed high expression levels of key players of the polyamine pathway in a panel of DIPG samples. Through alamar blue cytotoxicity assays and soft-agar clonogenic assays, DFMO was found to be effective against a panel of neurosphere-forming DIPG cells. The activity of DFMO was synergistically enhanced when combined with a polyamine transport inhibitor, AMXT-1501. Increased apoptotic activity following either DFMO or AMXT-1501 treatment, alone or in combination, was observed by western blotting and flow cytometric analysis of Annexin V/7AAD stained cells. In addition, reduced uptake of the radiolabelled polyamine, spermidine, in the presence of AMXT-1501 confirmed polyamine transport inhibition in DIPG cells. Consistent with the in vitro results, the combination of DFMO and AMXT-1501 significantly prolonged the survival of mice bearing DIPG orthografts, and the combination therapy represents the most effective combination treatment we have tested to date in our DIPG animal model. Our results suggest that the polyamine pathway is a potential therapeutic target for this highly aggressive paediatric brain tumour while a number of clinical trials based on this approach are currently underway in a variety of malignancies. Citation Format: Aaminah Khan, Maria Tsoli, Denise Yu, Swapna Joshi, Laura Gamble, Laura Franshaw, Michelle Haber, David S. Ziegler. Targeting the polyamine pathway as a novel therapeutic treatment against diffuse intrinsic pontine glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4800.

Arginase-1 Expression in Myeloid Cells Regulates Staphylococcus aureus Planktonic but Not Biofilm Infection.

Staphylococcus aureus is a leading cause of device-associated biofilm infections, which represent a serious health care concern based on their chronicity and antibiotic resistance. We previously reported that S. aureus biofilms preferentially recruit myeloid-derived suppressor cells (MDSCs), which promote monocyte and macrophage anti-inflammatory properties. This is associated with increased myeloid arginase-1 (Arg-1) expression, which has been linked to anti-inflammatory and profibrotic activities that are observed during S. aureus biofilm infections. To determine whether MDSCs and macrophages utilize Arg-1 to promote biofilm infection, Arg-1 was deleted in myeloid cells by use of Tie-2Cre mice. Despite Arg-1 expression in biofilm-associated myeloid cells, bacterial burdens and leukocyte infiltrates were similar between wild-type (WT) and Arg-1fl/fl;Tie-2Cre conditional knockout (KO) mice from days 3 to 14 postinfection in both orthopedic implant and catheter-associated biofilm models. However, inducible nitric oxide synthase (iNOS) expression was dramatically elevated in biofilm-associated MDSCs from Arg-1fl/fl;Tie-2Cre animals, suggesting a potential Arg-1-independent compensatory mechanism for MDSC-mediated immunomodulation. Treatment of Arg-1fl/fl;Tie-2Cre mice with the iNOS inhibitor N6-(1-iminoethyl)-l-lysine (l-NIL) had no effect on biofilm burdens or immune infiltrates, whereas treatment of WT mice with the Arg-1/ornithine decarboxylase inhibitor difluoromethylornithine (DFMO) increased bacterial titers, but only in the surrounding soft tissues, which possess attributes of a planktonic environment. A role for myeloid-derived Arg-1 in regulating planktonic infection was confirmed using a subcutaneous abscess model, in which S. aureus burdens were significantly increased in Arg-1fl/fl;Tie-2Cre mice compared to those in WT mice. Collectively, these results indicate that the effects of myeloid Arg-1 are context dependent and are manifest during planktonic but not biofilm infection.

470 - Randomized Placebo-Controlled Trial of Difluoromethylornithine (DFMO) Plus Aspirin for the Prevention of Colorectal Adenoma Recurrence and Reduction in Aberrant Crypt Foci (ACF) in Patients with Prior Advanced Adenomas or Cancer

470 - Randomized Placebo-Controlled Trial of Difluoromethylornithine (DFMO) Plus Aspirin for the Prevention of Colorectal Adenoma Recurrence and Reduction in Aberrant Crypt Foci (ACF) in Patients with Prior Advanced Adenomas or Cancer

Polyamine pathway as a player in breast cancer cell proliferation in diabetic conditions

Diabetes/hyperglycemia has been known to increase cancer cell proliferation, leading to metastasis and even mortality. High blood sugar is also known to reduce the effects of cancer therapeutics. Polyamines (spermine, spermidine, and putrescine) which are involved in several cellular processes such as cell growth, replication, and transcription, have been shown to be elevated in certain cancers including colon cancer and skin cancer. We hypothesized that polyamines are involved with proliferation of breast cancer cells in diabetic conditions. Cell lines used in this study included MCF‐7 (early stage breast cancer), MDA‐MB‐231 (late stage breast cancer), and MCF‐10A (normal breast epithelial cells). Cells were treated with normal glucose (NG, 5mM) or high glucose (HG, 25 mM) in the presence/absence of polyamine pathway inhibitors. Both MDA‐MB‐231 and MCF‐10A cells showed significant proliferation with HG treatment after 72 hr, which was not observed in MCF‐7 cells. Simultaneous treatment with an ornithine decarboxylase (polyamine synthesis enzyme) inhibitor, difluoromethylornithine (DFMO) could prevent this effect. Interestingly, while spermine levels were not altered with HG treatment in breast cancer cells (MCF‐7 and MDA‐MB‐231), they were significantly increased in MCF‐10A cells. This is possibly due to the fact that cancer cells have endogenously high levels of polyamines, such that further increase is prevented to avoid toxicity from polyamines. Nevertheless, DFMO was able to decrease spermine and spermidine levels induced with glucose treatments, suggesting a role of polyamine pathway in regulating growth. Putrescine levels were undetectable in all samples. In conclusion, polyamine pathway is involved with HG mediated normal breast epithelial cell and breast cancer cell proliferation. Future studies will involve further analysis of the mediators which regulate polyamine production.Support or Funding InformationUNK Faculty Start‐up Funds (SC); Undergraduate Research Fellowship Grant (CC and JO)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Difluoromethylornithine Combined with a Polyamine Transport Inhibitor Is Effective against Gemcitabine Resistant Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is highly chemo-resistant and has an extremely poor patient prognosis, with a survival rate at five years of <8%. There remains an urgent need for innovative treatments. Targeting polyamine biosynthesis through inhibition of ornithine decarboxylase with difluoromethylornithine (DFMO) has had mixed clinical success due to tumor escape via an undefined transport system, which imports exogenous polyamines and sustains intracellular polyamine pools. Here, we tested DFMO in combination with a polyamine transport inhibitor (PTI), Trimer44NMe, against Gemcitabine-resistant PDAC cells. DFMO alone and with Trimer44NMe significantly reduced PDAC cell viability by inducing apoptosis or diminishing proliferation. DFMO alone and with Trimer44NMe also inhibited in vivo orthotopic PDAC growth and resulted in decreased c-Myc expression, a readout of polyamine pathway dysfunction. Moreover, dual inhibition significantly prolonged survival of tumor-bearing mice. Collectively, these studies demonstrate that targeting polyamine biosynthesis and import pathways in PDAC can lead to increased survival in pancreatic cancer.

Ornithine decarboxylase as a therapeutic target for endometrial cancer.

Ornithine Decarboxylase (ODC) a key enzyme in polyamine biosynthesis is often overexpressed in cancers and contributes to polyamine-induced cell proliferation. We noted ubiquitous expression of ODC1 in our published endometrial cancer gene array data and confirmed this in the cancer genome atlas (TCGA) with highest expression in non-endometrioid, high grade, and copy number high cancers, which have the worst clinical outcomes. ODC1 expression was associated with worse overall survival and increased recurrence in three endometrial cancer gene expression datasets. Importantly, we confirmed these findings using quantitative real-time polymerase chain reaction (qRT-PCR) in a validation cohort of 60 endometrial cancers and found that endometrial cancers with elevated ODC1 had significantly shorter recurrence-free intervals (KM log-rank p = 0.0312, Wald test p = 5.59e-05). Difluoromethylornithine (DFMO) a specific inhibitor of ODC significantly reduced cell proliferation, cell viability, and colony formation in cell line models derived from undifferentiated, endometrioid, serous, carcinosarcoma (mixed mesodermal tumor; MMT) and clear cell endometrial cancers. DFMO also significantly reduced human endometrial cancer ACI-98 tumor burden in mice compared to controls (p = 0.0023). ODC-regulated polyamines (putrescine [Put] and/or spermidine [Spd]) known activators of cell proliferation were strongly decreased in response to DFMO, in both tumor tissue ([Put] (p = 0.0006), [Spd] (p<0.0001)) and blood plasma ([Put] (p<0.0001), [Spd] (p = 0.0049)) of treated mice. Our study indicates that some endometrial cancers appear particularly sensitive to DFMO and that the polyamine pathway in endometrial cancers in general and specifically those most likely to suffer adverse clinical outcomes could be targeted for effective treatment, chemoprevention or chemoprevention of recurrence.

Evaluation of Polyamine Transport Inhibitors in a Drosophila Epithelial Model Suggests the Existence of Multiple Transport Systems.

Increased polyamine biosynthesis activity and an active polyamine transport system are characteristics of many cancer cell lines and polyamine depletion has been shown to be a viable anticancer strategy. Polyamine levels can be depleted by difluoromethylornithine (DFMO), an inhibitor of the key polyamine biosynthesis enzyme ornithine decarboxylase (ODC). However, malignant cells frequently circumvent DFMO therapy by up-regulating polyamine import. Therefore, there is a need to develop compounds that inhibit polyamine transport. Collectively, DFMO and a polyamine transport inhibitor (PTI) provide the basis for a combination therapy leading to effective intracellular polyamine depletion. We have previously shown that the pattern of uptake of a series of polyamine analogues in a Drosophila model epithelium shares many characteristics with mammalian cells, indicating a high degree of similarity between the mammalian and Drosophila polyamine transport systems. In this report, we focused on the utility of the Drosophila epithelial model to identify and characterize polyamine transport inhibitors. We show that a previously identified inhibitor of transport in mammalian cells has a similar activity profile in Drosophila. The Drosophila model was also used to evaluate two additional transport inhibitors. We further demonstrate that a cocktail of polyamine transport inhibitors is more effective than individual inhibitors, suggesting the existence of multiple transport systems in Drosophila. Our findings reinforce the similarity between the Drosophila and mammalian transport systems and the value of the Drosophila model to provide inexpensive early screening of molecules targeting the transport system.

Polyamines and Cancer.

This chapter provides an overview of how the polyamine pathway has been exploited as a target for the treatment and prevention of multiple forms of cancer, since this pathway is disrupted in all cancers. It is divided into three main sections. The first explores how the polyamine pathway has been targeted for chemotherapy, starting from the first drug to target it, difluoromethylornithine (DFMO) to the large variety of polyamine analogues that have been synthesised and tested throughout the years with all their potentials and pitfalls. The second section focuses on the use of polyamines as vectors for drug delivery. Knowing that the polyamine transport system is upregulated in cancers and that polyamines naturally bind to DNA, a range of polyamine analogues and polyamine-like structures have been synthesised to target epigenetic regulators, with encouraging results. Furthermore, the use of polyamines as transport vectors to introduce toxic/bioactive/fluorescent agents more selectively to the intended target in cancer cells is discussed. The last section concentrates on chemoprevention, where the different strategies that have been undertaken to interfere with polyamine metabolism and function for antiproliferative intervention are outlined and discussed.

Spermidine-mediated poly(lactic-co-glycolic acid) nanoparticles containing fluorofenidone for the treatment of idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis is a progressive, fatal lung disease with poor survival. The advances made in deciphering this disease have led to the approval of different antifibrotic molecules, such as pirfenidone and nintedanib. An increasing number of studies with particles (liposomes, nanoparticles [NPs], microspheres, nanopolymersomes, and nanoliposomes) modified with different functional groups have demonstrated improvement in lung-targeted drug delivery. In the present study, we prepared, characterized, and evaluated spermidine (Spd)-modified poly(lactic-co-glycolic acid) (PLGA) NPs as carriers for fluorofenidone (AKF) to improve the antifibrotic efficacy of this drug in the lung. Spd-AKF-PLGA NPs were prepared and functionalized by modified solvent evaporation with Spd and polyethylene glycol (PEG)-PLGA groups. The size of Spd-AKF-PLGA NPs was 172.5±4.3 nm. AKF release from NPs was shown to fit the Higuchi model. A549 cellular uptake of an Spd–coumarin (Cou)-6-PLGA NP group was found to be almost twice as high as that of the Cou-6-PLGA NP group. Free Spd and difluoromethylornithine (DFMO) were preincubated in A549 cells to prove uptake of Spd-Cou-6-PLGA NPs via a polyamine-transport system. As a result, the uptake of Spd-Cou-6-PLGA NPs significantly decreased with increased Spd concentrations in incubation. At higher Spd concentrations of 50 and 500 µM, uptake of Spd-Cou-6-PLGA NPs reduced 0.34- and 0.49-fold from that without Spd pretreatment. After pretreatment with DFMO for 36 hours, cellular uptake of Spd-Cou-6-PLGA NPs reached 1.26-fold compared to the untreated DFMO group. In a biodistribution study, the drug-targeting index of Spd-AKF-PLGA NPs in the lung was 3.62- and 4.66-fold that of AKF-PLGA NPs and AKF solution, respectively. This suggested that Spd-AKF-PLGA NPs accumulated effectively in the lung. Lung-histopathology changes and collagen deposition were observed by H&E staining and Masson staining in an efficacy study. In the Spd-AKF-PLGA NP group, damage was further improved compared to the AKF-PLGA NP group and AKF-solution group. The results indicated that Spd-AKF-PLGA NPs are able to be effective nanocarriers for anti–pulmonary fibrosis therapy.

Abstract 4883: Targeting tumor-amplified ODC1 with difluoromethylornithine (DFMO) inhibits global protein translation and has antitumor activity in neuroblastoma

Abstract MYC genes are predominant oncogenic drivers in neuroblastoma, a lethal pediatric tumor, often via amplification of MYCN. MYC genes coordinately deregulate programs that link cell cycle entry with requisite biomass and energy creation. Polyamine synthesis is one such deregulated program that supports protein synthesis. Ornithine decarboxylase (Odc, encoded by ODC1) is the rate-limiting enzyme in polyamine synthesis, a direct MYC target, and a bonafide oncogene. In addition to hyperactivated MYCN driving polyamine signaling, ODC1 itself is co-amplified in a subset of high-risk tumors. We studied 30 neuroblastoma cell lines and 916 primary tumors via SNP-array. ODC1 amplification (confirmed by qPCR) was found exclusively in tumors with MYCN amplification, though amplification peaks were distinct (ODC1 maps 5.5 Mb telomeric to MYCN). ODC1 amplification was identified in 33 of 256 (13%) MYCN amplified primary tumors and 4 of 13 (31%) MYCN-amplified cell lines. Difluoromethylornithine (DFMO) is an FDA-approved irreversible inhibitor of the oncogenic Odc enzyme, and we have shown DFMO inhibits tumor progression and synergizes with chemotherapy in complementary murine models of neuroblastoma. We postulated DFMO inhibits protein translation as its principal mechanism of anti-tumor activity. Polyamines support protein translation via effects on both eIF5A and eIF4F-complex activities, yet their relative contributions remain poorly defined. Using the puromycin-incorporation assay we show a marked decrease in global protein translation following DFMO exposure (up to 95% reduction), with translation inhibition largely correlated with MYCN amplification status in the tumor cells. Studies to define the dose-response and kinetics of DFMO-mediated inhibition in tumors with distinct MYCN and ODC1 genotypes are ongoing, as are studies of eIF5A-hypusination (by IEF) and eIF4F-complex status (polyamine-dependent 4E-BP phosphorylation). Moreover, eIF5A is required to resolve ribosome stalling at polyproline stretches, so we characterized the human proteome for polyproline motifs (PPP of &amp;gt;3) to define an eIF5A-dependent translatome. Candidate genes of diverse polyproline content are being assessed for sensitivity to DFMO to define whether ribosome pausing and selective translation inhibition contributes to DFMO effects. Elucidating mechanisms of DFMO activity and correlating this with genomic status (MYCN and/or ODC1 amplified) will identify opportunities for drug synergy and provide a responder hypothesis to test in pivotal Phase 2 and 3 clinical trial of DFMO for neuroblastoma. Citation Format: Andrea T. Flynn, Annette Vu, Kangning Liu, Elizabeth Scadden, Edward Attiyeh, Murray Norris, Michelle Haber, Michael Hogarty. Targeting tumor-amplified ODC1 with difluoromethylornithine (DFMO) inhibits global protein translation and has antitumor activity in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4883. doi:10.1158/1538-7445.AM2017-4883

Abstract 1242: Ornithine decarboxylase as a therapeutic target in endometrial cancer

Abstract Endometrial cancer is the 4th most common cancer and the 6th deadliest cancer in US women. The American Cancer Society estimates there will be 60,050 new endometrial cancers in 2016, an increase of more than 10% from the previous year highlighting the need for more effective treatments and prevention. Ornithine Decarboxylase (ODC) a key enzyme in polyamine synthesis is often overexpressed in cancers and contributes to cell proliferation and tumor growth. Therefore, ODC and the polyamine pathway are considered rational targets for cancer treatment or prevention. We noted ubiquitous expression of ODC1 in our previously published endometrial cancer gene array data and confirmed this in the cancer genome atlas (TCGA), finding expression in all four molecular sub-types with highest expression in copy number high cancers which have the worst clinical outcomes. Therefore, we explored the association of ODC1 gene expression with clinical outcomes of overall survival (OS) and recurrence in the TCGA cohort and noted that elevated ODC1 was significantly related to OS (Wald test p=0.001) and recurrence (p=0.01). Importantly, we confirmed these observations using QRT-PCR in a validation cohort of 60 endometrial cancers and found that endometrial cancers with elevated ODC1 had significantly shorter recurrence-free intervals (p=5.59x10-5) and elevated hazard ratio=3.72. Similar to TCGA data we also noted a strong trend to worse OS ( p=0.00014) with elevated hazard ratio 3.81. Numerous studies including clinical trials have examined the chemopreventive and anti-tumor effects of difluoromethylornithine (DFMO), a specific inhibitor of ODC. We found that DFMO treatment significantly reduced cell proliferation, cell viability, and colony formation in human cell line models derived from undifferentiated, endometrioid, serous, MMT and clear cell endometrial cancers. In contrast, immortalized uterine endometrial epithelial cells (EM E6/E7 TERT1) were less sensitive to DFMO. To confirm the significant effects of DFMO in vitro we performed an in vivo study with human endometrial cancer (ACI-98) tumor-bearing athymic nude mice. Xenografted mice were either treated with 2% (w/v) DFMO supplied in drinking water or water only (n=10/group). DFMO significantly reduced the tumor burden in mice compared to controls (p=0.0023). ODC-regulated polyamines (putrescine [Put] and spermidine [Spd]) known activators of cell proliferation were strongly decreased in response to DFMO, in both tumor tissue ([Put] (p=0.0006), [Spd] (p&amp;lt;0.0001)) and blood plasma ([Put] (p&amp;lt;0.0001), [Spd] (p=0.0049)) of treated mice. Results of these studies indicate that some endometrial cancers appear particularly sensitive to DFMO. Our findings indicate that the polyamine pathway in endometrial cancers in general and specifically those most clinically relevant endometrial cancers could be targeted for effective treatment, chemoprevention or chemoprevention of recurrence. Citation Format: Hong Im Kim, Chad R. Schultz, Andrea L. Buras, Elizabeth Friedman, Alyssa M. Fedorko, Leigh G. Seamon, Gadisetti Chandramouli, André S. Bachmann, John I. Risinger. Ornithine decarboxylase as a therapeutic target in endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1242. doi:10.1158/1538-7445.AM2017-1242

Abstract 1941: Cell surface vimentin is a novel marker for CTC detection in neuroblastoma

Abstract Among children in the United States, brain cancers now account for most number of cancer-related deaths. Neuroblastomas (NB) and sarcomas account for 13% of all childhood cancers in the United States. Despite significant progress yielding increased 5-year survival, 1250 deaths are expected this year from childhood cancers and cancer incidence has been steadily increasing. One of the key contributors to cancer mortality is tumor metastasis. It is generally believed that CTCs are shed into the circulation from primary tumors and contain unique driver mutations enabling their aggressive phenotype. However, detection of CTCs from NB is difficult as there is no direct method. Our lab has developed a monoclonal antibody targeting vimentin on the cells’ surface. Cell surface vimentin (CSV) is only observed in tumor cells; it remains intracellular in normal cells. We have published data demonstrating the superior specificity and sensitivity of our approach in breast, colon, and prostate cancer. Here, we report direct CTC detection in NB using our CSV mAb. As part of our collaborative effort analyzing NB blood samples from a multicenter Phase II trial, we present novel discoveries regarding treatment of NB with difluoromethylornithine (DFMO) and its effects on CTC release into the circulation in patients under remission. Among our observations, approximately ~1/3 patients have low or no CTCs while ~1/4 with high numbers of CTC are responding to DFMO treatment. Remaining patients show oscillating CTC numbers. Further, treatment with DFMO showed a statistically significant decline in CTCs after the beginning of therapy. Ongoing work including whole genome analysis can possibly reveal new insight into CTCs’ entering the circulation. These data demonstrate that our CTC capture using CSV is a novel and unique approach for detection in NB. Citation Format: Izhar S. Batth, Heming Li, Giselle Saulnier Sholler, Shulin Li. Cell surface vimentin is a novel marker for CTC detection in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1941. doi:10.1158/1538-7445.AM2017-1941

Polyamine blockade alters the neuroblastoma microenvironment to favor restriction of tumor growth

Abstract Despite improved treatments, neuroblastoma (NB) still accounts for ~15% of childhood cancer deaths. Amplification of MYCN results in NB with high-risk features and poor 5yr survival. Interestingly, MYCN-amplified NBs have elevated polyamine (PA) levels, as the gate-keeper enzyme in PA synthesis, ornithine decarboxylase (ODC1), is a direct Myc target. Inhibition of ODC1 activity by the FDA-approved medication difluoromethylornithine (DFMO) results in modest growth reduction of NB cell lines in vitro, whereas DFMO administration to mice that are predisposed to developing MYCN-driven NBs (TH-MYCN+/+ mice) leads to survival extensions that were more profound than that predicted by the tumor-intrinsic activity of the drug alone. These observations implied that DFMO might also exert tumor-extrinsic effects on the tumor microenvironment (TME). Indeed, DFMO treatment of TH-MYCN+/+ mice alters the NB TME in several ways, the most profound of which is an increase in NK cell frequency. We also find an increase in the percentage of NB cells expressing NKG2D ligands, and alterations in the expression of NK cell stimulatory and inhibitory receptors to favor activation. Collectively, these findings suggest that PA blockade induces distinct TME changes that allow for more efficient immune control of NB growth. We are now testing whether the activity of DFMO is dependent on NK cells, and are delineating how DFMO alters the transcriptional profiles of NBs and immune cells in the TME. We hope that these studies will complement the data from phase I/II clinical studies using DFMO in various therapeutic strategies for NB, and will allow for an increased understanding of how to more effectively combine PA blockade with other immunotherapies for this disease.

Inhibition of Neuroblastoma Cell Growth by Difluoromethylornithine (DFMO) and Bortezomib through Suppression of LIN28 and MYCN

Neuroblastoma (NB) is the most common childhood cancer arising from the nervous system. Many high-risk neuroblastoma (HRNB) patients develop relapse after initial response to induction treatment and overall long term survival remains poor (less than 60%), emphasizing the need for new therapeutic approaches and more effective treatments. Combination therapies present a favorable approach to improve efficacy, decrease toxicity, and reduce development of drug resistance. Difluoromethylornithine (DFMO) has shown promise in recent clinical trials as a therapeutic agent in treating HRNB. Proteasomes are known to play an important role in tumor cell growth. Bortezomib was the first proteasome inhibitor shown to have anticancer activity clinically. In this study we explore the mechanistic and therapeutic effects of the novel drug combination of DFMO and bortezomib in NB. Cell proliferation studies demonstrated synergistic inhibition of NB cell growth. Bortezomib induced cleaved caspase-3 apoptotic pathway whereas DFMO induced a cytostatic effect on NB cells. Western blot analyses demonstrated down regulation of MYCN, LIN28 and NF-kB in response to DFMO and bortezomib, pathways that are important in cancer stem cells. A decrease in ATP-per-cell when treated with combination therapy suggests inhibition of glycolytic metabolism in NB cells. DFMO as a single agent or in combination with bortezomib significantly reduced tumor growth in xenograft mice. Given the lack of effective treatments, DFMO coupled with bortezomib offers a potential new therapeutic treatment for children with NB.