Difluoromethylornithine Treatment Research Articles

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 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 1354: Regulation and roles of polyamines in CD8+ T cell fate and function

Abstract CD8+ T cells play central roles in tumor immune surveillance and are major effectors in adoptive cell therapy (ACT). Thus, strategies that enhance their functions are an urgent clinical need. Metabolic reprogramming determines T cell fate and function, where glycolysis principally drives an effector phenotype while oxidative phosphorylation (OxPhos) drives T cell memory [1]. However, the roles of amino acid catabolism in controlling effector and memory CD8+ T cell fate and function are less well understood [1, 2]. Glutamine is essential for nucleotide biosynthesis and as an anaplerotic fuel source for the tricarboxylic acid (TCA) cycle. Isotope tracing experiments with 13C glutamine or 13C arginine revealed that glutamine is the major source of polyamines in activated CD8+ T cells. Further, activation of CD8+ T cells provokes marked increases in the expression and activity of enzymes that direct polyamine biosynthesis, specifically ornithine decarboxylase (Odc), spermidine synthase (Srm) and spermine synthase (Sms), as well as to increases in the polyamines putrescine, spermidine and spermine. Notably, pharmacologic inhibition of Odc using difluoromethylornithine (DFMO), or genetic deletion of Odc using CD8+ T cells from CD4-Cre;Odcfl/fl mice, augments cytokine production in activated CD8+ T cells, including IFN-γ, TNF-α and granzyme B. Further, metabolic flux analysis indicates that DFMO treatment switches the metabolism of activated CD8+ T cells to rely on OxPhos, a phenotype found in CD8+ tissue resident memory (TRM) cells that play key roles in tissue and anti-tumor immunity anti-tumor reactivity. Indeed, inhibition or loss of Odc augments other TRM phenotypes, including increases in CD69high;CD44high;S1PR1low;CD62Llow cells that define the TRM phenotype. In addition, ACT of DFMO treated CD8+ T cells augments their survival in vivo and augments the generation of CD69+CD103+CD69+CXCR6+ and Ly6C+ TRMs in the bone marrow. Finally, DFMO treatment enhances IFN-γ and TNF-α production in human TILs, and co-treatment with TGF-β polarizes TILs into CD69+CD103+ and CD69+CD49a+ TRM-like cells. We conclude that polyamines are a metabolic check point in CD8+ TRM generation and are an actionable target to improve the anti-tumor immunity of T-cell based immunotherapies. Citation Format: Aya G. Elmarsafawi, Rebecca S. Hesterberg, John L. Cleveland. Regulation and roles of polyamines in CD8+ T cell fate and function [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 1354.

DFMO Improves Survival and Increases Immune Cell Infiltration in Association with MYC Downregulation in the Pancreatic Tumor Microenvironment.

Pancreatic ductal adenocarcinoma (PDAC) has an extremely poor five-year survival rate of less than 10%. Immune suppression along with chemoresistance are obstacles for PDAC therapeutic treatment. Innate immune cells, such as tumor-associated macrophages, are recruited to the inflammatory environment of PDAC and adversely suppress cytotoxic T lymphocytes. KRAS and MYC are important oncogenes associated with immune suppression and pose a challenge to successful therapies. Here, we targeted KRAS, through inhibition of downstream c-RAF with GW5074, and MYC expression via difluoromethylornithine (DFMO). DFMO alone and with GW5074 reduced in vitro PDAC cell viability. Both DFMO and GW5074 showed efficacy in reducing in vivo PDAC growth in an immunocompromised model. Results in immunocompetent syngeneic tumor-bearing mice showed that DFMO and combination treatment markedly decreased tumor size, but only DFMO increased survival in mice. To further investigate, immunohistochemical staining showed DFMO diminished MYC expression and increased tumor infiltration of macrophages, CD86+ cells, CD4+ and CD8+ T lymphocytes. GW5074 was not as effective in modulating the tumor infiltration of total CD3+ lymphocytes or tumor progression and maintained MYC expression. Collectively, this study highlights that in contrast to GW5074, the inhibition of MYC through DFMO may be an effective treatment modality to modulate PDAC immunosuppression.

Abstract PO-119: DFMO mediated improvement in survival of an orthotopic model of pancreatic cancer is associated with modulating immune suppression in the tumor microenvironment

Abstract There remains an urgent need to target pancreatic tumor cells using innovative strategies. KRAS and MYC, are important oncogenes in pancreatic ductal adenocarcinoma (PDAC) which pose a challenge to successful treatment of PDAC. Our previous studies have shown that inhibition of ornithine decarboxylase 1 (ODC1) using difluoromethylornithine (DFMO) decreases MYC expression and tumorigenesis. GW5074 can modulate RAF1, a downstream effector of KRAS. Here we test the responsiveness of pancreatic tumor cells treated with DFMO alone and in combination with GW5074. We used an orthotopic animal model of pancreatic tumor using KRas-driven murine pancreatic cancer cells (PanO2) to test the effects of these compounds on tumor microenvironment and overall survival. Cellular and molecular changes in the tumor microenvironment were assessed using immunohistochemistry. The results showed an inhibition of pancreatic cancer cell viability in DFMO and DFMO+GW5074 treatment groups in vitro, with a significant decrease in tumor weight compared to control treatment group in vivo. However, in terms of overall survival, DFMO alone resulted in a dramatic increase in survival compared to control treatment group. Interestingly, GW5074 alone or DFMO in combination with GW5074 did not result in a detectable effect on survival. Further investigation of immune cells in the tumor microenvironment revealed that standalone DFMO treatment was associated with an increase in infiltration of macrophages, T cell costimulatory marker CD86 and T cell markers (CD3, CD4 and CD8) compared to control and GW5074 treated groups. Additionally, DFMO is associated with decreased MYC expression compared to control and GW5074 treated groups. In conclusion, DFMO decreased MYC expression and associated immune suppression in the PDAC microenvironment. In contrast standalone GW5074 treatment resulted in maintenance of MYC expression and worse survival. In conclusion, the present study highlights the success of DFMO in PDAC treatment in part through downregulation of MYC and a decrease in associated immune suppression. Citation Format: Sai Preethi Nakkina, Sarah B. Gitto, Veethika Pandey, Jordan M. Beardsley, Michael W. Rohr, Jignesh G. Parikh, Otto Phanstiel, Deborah A. Altomare. DFMO mediated improvement in survival of an orthotopic model of pancreatic cancer is associated with modulating immune suppression in the tumor microenvironment [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-119.

Difluoromethylornithine Induces Apoptosis through Regulation of AP-1 Signaling via JNK Phosphorylation in Epithelial Ovarian Cancer.

Difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC), has promising activity against various cancers and a tolerable safety profile for long-term use as a chemopreventive agent. However, the anti-tumor effects of DFMO in ovarian cancer cells have not been entirely understood. Our study aimed to identify the effects and mechanism of DFMO in epithelial ovarian cancer cells using SKOV-3 cells. Treatment with DFMO resulted in a significantly reduced cell viability in a time- and dose-dependent manner. DFMO treatment inhibited the activity and downregulated the expression of ODC in ovarian cancer cells. The reduction in cell viability was reversed using polyamines, suggesting that polyamine depletion plays an important role in the anti-tumor activity of DFMO. Additionally, significant changes in Bcl-2, Bcl-xL, Bax protein levels, activation of caspase-3, and cleavage of poly (ADP-ribose) polymerase were observed, indicating the apoptotic effects of DFMO. We also found that the effect of DFMO was mediated by AP-1 through the activation of upstream JNK via phosphorylation. Moreover, DFMO enhanced the effect of cisplatin, thus showing a possibility of a synergistic effect in treatment. In conclusion, treatment with DFMO alone, or in combination with cisplatin, could be a promising treatment for ovarian cancer.

Difluoromethylornithine attenuates isoproterenol-induced cardiac hypertrophy by regulating apoptosis, autophagy and the mitochondria-associated membranes pathway.

Myocardial hypertrophy is an independent risk factor of cardiovascular diseases and is closely associated with the incidence of heart failure. In the present study, we hypothesized that difluoromethylornithine (DFMO) could attenuate cardiac hypertrophy through mitochondria-associated membranes (MAM) and autophagy. Cardiac hypertrophy was induced in male rats by intravenous administration of isoproterenol (ISO; 5 mg/kg/day) for 1, 3,7 and 14 days. For DFMO treatment group, rats were given ISO (5 mg/kg/day) for 14 days and 2% DFMO in their water for 4 weeks. The expression of atrial natriuretic peptide (ANP) mRNA,heart parameters, apoptosis rate, fibrotic area and protein expressions of cleaved caspase3/9, GRP75, Mfn2, CypD and VDAC1 were measured to confirm the development of cardiac hypertrophy, apoptosis and autophagy induced by ISO. ANP mRNA and MAM protein expression levels were assessed by reverse transcription-quantitative PCR and western blotting to evaluate hypertrophy and the effects of DFMO oral administration. The results demonstrated that heart parameters, ANP mRNA levels, fibrotic area and apoptosis rate were significantly increased in the heart tissue for ISO 7 and 14 day groups compared with the control group. Furthermore, treatment with DFMO significantly inhibited these indicators, and DFMO downregulated the MAM signaling pathway and upregulated the autophagy pathway in heart tissue compared with the ISO 14 day group. Overall, all ISO-induced changes analyzed in the present study were attenuated following treatment with DFMO. The findings form this study suggested that DFMO treatment may be considered as a potential strategy for preventing ISO-induced cardiac hypertrophy.

Difluoromethylornithine, a Decarboxylase 1 Inhibitor, Suppresses Hepatitis B Virus Replication by Reducing HBc Protein Levels.

Current treatments of hepatitis B virus (HBV) are limited to Interferon-alpha or the nucleos(t)ide analogs antiviral therapies, and it is crucial to develop and define new antiviral drugs to cure HBV. In this study, we explored the anti-HBV effect of difluoromethylornithine (DFMO), an irreversibly inhibitor of decarboxylase 1(ODC1) on HBV replication. Firstly, we found that polyamines contributed to HBV DNA replication via increasing levels of the HBV core protein (HBc) and capsids. In contrast, depletion of polyamines either by silencing the expression of ODC1 or DFMO treatment, resulted in decreasing viral DNA replication and levels of HBc protein and capsids. Furthermore, we found that DFMO decreased the stability of the HBc protein without affecting mRNA transcription and protein translation. Taken together, our findings demonstrate that DFMO inhibits HBV replication by reducing HBc stability and this may provide a new approach for HBV therapeutics.

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.

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.

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.

Putrescine independent wound response phenotype is produced by ODC-like RNAi in planarians

Despite increasing evidence indicates polyamines as a convergence point for signaling pathways, including cell growth and differentiation, a unifying concept to interpret their role is still missing. The activity of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, is tightly regulated by a complex molecular machinery, and the demonstration of the existence of multiple ODC paralogs, lacking decarboxylation activity, suggests additional layers of complexity to the intricate ODC regulatory pathway. Because of their extraordinary regenerative abilities and abundance of stem cells, planarians have potential to contribute to our understanding of polyamine function in an in vivo context. We undertook a study on ODC function in planarians and we found six planarian ODCs (ODC1-6). Five out of six ODC homologs carry substitutions of key aminoacids for enzymatic activity, which makes them theoretically unable to decarboxylate ornithine. Silencing of ODC5 and 6 produced a complex phenotype, by prompting animals to an aberrant response, following chronic injury without tissue removal. Phenotype is neither rescued by putrescine, nor mimicked by difluoromethylornithine treatment. Moreover, the co-silencing of other genes of the ODC regulatory pathway did not modulate phenotype outcome or severity, thus suggesting that the function/s of these ODC-like proteins might be unrelated to decarboxylase activity and putrescine production.

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<0.0001)) and blood plasma ([Put] (p<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.

Targeting ornithine decarboxylase (ODC) inhibits esophageal squamous cell carcinoma progression

To explore the function of ornithine decarboxylase in esophageal squamous cell carcinoma progression and test the effectiveness of anti-ornithine decarboxylase therapy for esophageal squamous cell carcinoma. In this study, we examined the expression pattern of ornithine decarboxylase in esophageal squamous cell carcinoma cell lines and tissues using immunohistochemistry and Western blot analysis. Then we investigated the function of ornithine decarboxylase in ESCC cells by using shRNA and an irreversible inhibitor of ornithine decarboxylase, difluoromethylornithine. To gather more supporting pre-clinical data, a human esophageal squamous cell carcinoma patient-derived xenograft mouse model (C.B-17 severe combined immunodeficient mice) was used to determine the antitumor effects of difluoromethylornithine in vivo. Our data showed that the expression of the ornithine decarboxylase protein is increased in esophageal squamous cell carcinoma tissues compared with esophagitis or normal adjacent tissues. Polyamine depletion by ODC shRNA not only arrests esophageal squamous cell carcinoma cells in the G2/M phase, but also induces apoptosis, which further suppresses esophageal squamous cell carcinoma cell tumorigenesis. Difluoromethylornithine treatment decreases proliferation and also induces apoptosis of esophageal squamous cell carcinoma cells and implanted tumors, resulting in significant reduction in the size and weight of tumors. The results of this study indicate that ornithine decarboxylase is a promising target for esophageal squamous cell carcinoma therapy and difluoromethylornithine warrants further study in clinical trials to test its effectiveness against esophageal squamous cell carcinoma.

Abstract 2474: DFMO targets cancer stem cells in neuroblastoma

Abstract Background: Difluoromethylornithine (DFMO) has shown promise in recent clinical trials as a therapeutic agent in treating neuroblastoma (NB). High-risk NB patients have an approximately 50% relapse rate, after which survival rate drops to lower than 10%. By targeting the cancer stem cells (CSC) we could decrease relapse rate in high-risk patients and improve long-term survival. DFMO is known to inhibit ornithine decarboxylase (ODC) impeding key polyamine biosynthesis as well as the LIN28/Let7 pathway and disrupting the glycolytic metabolism pathways both of which have been shown to be important in CSC models. We propose that DFMO may be an effective treatment against neuroblastoma CSCs. Methods: BE(2)C and SMS-KCNR NB cell lines were studied. Limiting dilution assays of xenograft studies were performed. Untreated, 10-day 5mM DFMO-pretreated, or 20-day 5mM DFMO-pretreated cells were injected into mice between 10-5000 cells/mouse and followed for tumor formation. Xenograft Be2C mice were treated with either vehicle or 2%DFMO in drinking water and harvested at 7-14 days for tumor analysis. Tumors and cell cultures of untreated and DFMO-treated cells were analyzed by RT-qPCR, Western Blot analysis, and GeneChip gene expression analysis measuring levels of ODC1, LIN28B, mirLet7, MYCN, SLC2A4 (a glucose transporter), and CSC markers such as CXCR4, POU5F1, NANOG, SOX2, and KLF4. Riboflavin induced autofluorescence (AF) was used to identify CSC. This method detected cells after a 24-hour bath in 30μM riboflavin and excitement at 488nm in a MoFlo cell sorter at the University of Michigan Flow Core, after which cells are sorted into AF- and AF+ sub-populations for imaging, neurosphere assays with or without DFMO, RT-qPCR and Western Blot analysis. Knock-down of ODC1 is achieved by siRNA transfection via Lipofectamine in cells and analyzed by RT-qPCR, Western Blot, and neurosphere assays with or without DFMO. Results: Pretreatment with DFMO in BE(2)C and SMS-KCNR cells in vitro and in vivo inhibits tumor formation in neurosphere assays and in limiting dilution xenograft models respectively. Tumors harvested from xenografts show a decrease in expression of CSC biomarkers and neurosphere formation capability in those treated with DFMO. AF+ cells showed an increase in stem cell marker expression and increase neurosphere formation relative to AF- cells, identifying them as cancer stem cells. In addition, AF+ cells are more sensitive to DFMO treatment with a greater decrease in neurosphere formation. Cells transfected with ODC siRNA or subjected to DFMO show a reduction in CSC biomarker RNA and protein and a decrease in neurospheres formation. Conclusion: These results suggest that DFMO targets the CSC subpopulation in neuroblastoma, disrupting cell growth pathways and inhibiting tumor formation. This concept is under further investigation in a Phase II clinical trial to prevent relapse in high risk neuroblastoma patients. Citation Format: Tracey Avequin, Ping Zhao, Abhinav Nagulapally, Jeffrey Bond, Ebrahim Azizi, Max Wicha, Giselle L. Saulnier Sholler. DFMO targets cancer stem cells in neuroblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2474.

Abstract 500: Bortezomib, a proteasome inhibitor, synergizes with DFMO to inhibit neuroblastoma cell proliferation via the reversal of the LIN28/Let-7 axis

Abstract Background: The most common extracranial solid pediatric tumor, Neuroblastoma (NB) is responsible for 8-10% of all cancer in children and 15% of pediatric cancer deaths. Due to a high rate of tumor relapse, less than 40% of patients with high-risk disease achieve long-term survival. High incidence of metastatic disease and relapse in NB, as well as cellular heterogeneity of tumor, suggests that high-risk NB is likely due to inherent drug resistance of cancer stem cells (CSCs) that survive treatment in order to regenerate differentiated tumor. One pathway associated with targeting CSCs is the LIN28/Let-7 pathway due to the inductive role of LIN28 in stem cell growth and metabolism. LIN28 is characteristically overexpressed in many malignancies, including NB. Overexpression of LIN28 correlates with poor outcome in NB, therefore drugs that target LIN28/Let-7 axis could be beneficial in treating NB patients by targeting CSCs and preventing relapse of disease. This study proposes the drug combination therapy of Difluoromethylornithine (DFMO)-an inhibitor of ornithine decarboxylase and polyamine biosynthesis-coupled with bortezomib-a proteasome inhibitor-as a new therapeutic upstream target of LIN28/Let-7 pathway and glycolytic metabolism. Methods: NB cell lines BE(2)-C and SMS-KCNR were used. Cell viability was measured using Calcein AM fluorescent assay at DFMO doses 0.15 - 40 mM, alone and in combination with bortezomib at doses 0.15 - 80 nM. Isobologram cell viability analyses with DFMO in combination with bortezomib were generated using Calcein AM fluorescent assay results. Western blot analysis was used to measure LIN28, MYCN, SOCS3 and NFKB. ATP level per cell was measured using CyQuant fluorescent DNA assay combined with Cell Titer GLO luminescent cell viability assay. Neurosphere assays were used to evaluate sphere formation after DFMO treatment. Xenograft studies were performed with SMS-KCNR cells implanted in the flank of mice and treated with vehicle, 2% DFMO in drinking water, bortezomib 0.5mg/kg IP twice a week, or combination of DFMO and bortezomib. Results: Cell proliferation assays and isobologram experiments suggest synergistic cytotoxicity of the drug combination in NB cell lines BE(2)-C and SMS-KCNR. Further, western blot analysis and ATP per cell experiments demonstrate the inhibition of overexpressed proteins in the LIN28/Let-7 pathway and inhibition of ATP/cell activity in NB cells. DFMO inhibits neurosphere formation in NB. Xenograft studies show decrease in tumor size with combination treatment. Conclusion: This study indicates that bortezomib synergizes with DFMO in inhibiting NB cell proliferation and neurosphere formation both in vitro and in vivo. Given the current lack of effective treatments and the high incidence of relapse and metastatic disease for patients, this drug combination offers a potential new therapeutic treatment for children with NB. Citation Format: Maria Rich, Ping Zhao, Abhinav Nagulapally, Jeffrey Bond, Giselle Sholler. Bortezomib, a proteasome inhibitor, synergizes with DFMO to inhibit neuroblastoma cell proliferation via the reversal of the LIN28/Let-7 axis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 500. doi:10.1158/1538-7445.AM2015-500

Effect of difluoromethylornithine and methotrexate on proliferation of human breast carcinoma cells

Introduction: In human colon cancer cells, the combination of difluoromethylornithine (DFMO) and methotrexate (MTX) has been shown to inhibit cellular proliferation. Objectives: To determine if the combination of DFMO and MTX inhibit proliferation of human breast cancer cells. Materials and methods: Sterile cell culture techniques, including harvesting and subculturing, were used in performing these experiments. Human breast carcinoma cells were treated alone and in combination with DFMO and MTX for 72hours. Vehicle control for MTX was dimethylsulfoxide at equivalent concentration. Results: Cell viability was inhibited with DFMO and MTX treatment alone. However, the combination treatment had no inhibitory effect. Conclusions: In human breast carcinoma cells, DFMO and MTX each inhibited cell growth by approximately 35% compared with untreated cells. However, in combination, DFMO and MTX resulted in no inhibition of cell growth. This drug combination is antagonistic and does not seem to be beneficial for therapeutic use. Other drug agents could be tested in combination to indicate inhibition of proliferation. Acknowledgements: Special thanks to our Dean, Dr Khaled Algumizi, our supervisor, Dr Sami Aldham, Dr Fahad Alateeq, Professor Patricia Thompson and her laboratory members: Julie Bickmeier, Ashley Vargas, Christina Preece and Manuel Snyder for all their help and effort.

Targeting ornithine decarboxylase reverses the LIN28/Let-7 axis and inhibits glycolytic metabolism in neuroblastoma.

LIN28 has emerged as an oncogenic driver in a number of cancers, including neuroblastoma (NB). Overexpression of LIN28 correlates with poor outcome in NB, therefore drugs that impact the LIN28/Let-7 pathway could be beneficial in treating NB patients. The LIN28/Let-7 pathway affects many cellular processes including the regulation of cancer stem cells and glycolytic metabolism. Polyamines, regulated by ornithine decarboxylase (ODC) modulate eIF-5A which is a direct regulator of the LIN28/Let-7 axis. We propose that therapy inhibiting ODC will restore balance to the LIN28/Let-7 axis, suppress glycolytic metabolism, and decrease MYCN protein expression in NB. Difluoromethylornithine (DFMO) is an inhibitor of ODC in clinical trials for children with NB. In vitro experiments using NB cell lines, BE(2)-C, SMS-KCNR, and CHLA90 show that DFMO treatment reduced LIN28B and MYCN protein levels and increased Let-7 miRNA and decreased neurosphere formation. Glycolytic metabolic activity decreased with DFMO treatment in vivo. Additionally, sensitivity to DFMO treatment correlated with LIN28B overexpression (BE(2)-C>SMS-KCNR>CHLA90). This is the first study to demonstrate that DFMO treatment restores balance to the LIN28/Let-7 axis and inhibits glycolytic metabolism and neurosphere formation in NB and that PET scans may be a meaningful imaging tool to evaluate the therapeutic effects of DFMO treatment.