Publications by authors named "André S Bachmann"

54 Publications

Expanding the phenotype: Four new cases and hope for treatment in Bachmann-Bupp syndrome.

Am J Med Genet A 2021 Sep 3. Epub 2021 Sep 3.

Division of Medical Genetics and Genomics, Spectrum Health and Helen DeVos Children's Hospital, Grand Rapids, Michigan, USA.

Bachmann-Bupp syndrome (BABS) is a rare syndrome caused by gain-of-function variants in the C-terminus of ornithine decarboxylase (ODC coded by the ODC1 gene). BABS is characterized by developmental delay, macrocephaly, macrosomia, and an unusual pattern of non-congenital alopecia. Recent diagnosis of four more BABS patients provides further characterization of the phenotype of this syndrome including late-onset seizures in the oldest reported patient at 23 years of age, representing the first report for this phenotype in BABS. Neuroimaging abnormalities continue to be an inconsistent feature of the syndrome. This may be related to the yet unknown impact of ODC/polyamine dysregulation on the developing brain in this syndrome. Variants continue to cluster, providing support to a universal biochemical mechanism related to elevated ODC protein, enzyme activity, and abnormalities in polyamine levels. Recommendations for medical management can now be suggested as well as the potential for targeted molecular or metabolic testing when encountering this unique phenotype. The natural history of this syndrome will evolve with difluoromethylornithine (DFMO) therapy and raise new questions for further study and understanding.
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http://dx.doi.org/10.1002/ajmg.a.62473DOI Listing
September 2021

Repurposing eflornithine to treat a patient with a rare ODC1 gain-of-function variant disease.

Elife 2021 07 20;10. Epub 2021 Jul 20.

Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, Grand Rapids, United States.

Background: Polyamine levels are intricately controlled by biosynthetic, catabolic enzymes and antizymes. The complexity suggests that minute alterations in levels lead to profound abnormalities. We described the therapeutic course for a rare syndrome diagnosed by whole exome sequencing caused by gain-of-function variants in the C-terminus of ornithine decarboxylase (ODC), characterized by neurological deficits and alopecia.

Methods: acetylputrescine levels with other metabolites were measured using ultra-performance liquid chromatography paired with mass spectrometry and Z-scores established against a reference cohort of 866 children.

Results: From previous studies and metabolic profiles, eflornithine was identified as potentially beneficial with therapy initiated on FDA approval. Eflornithine normalized polyamine levels without disrupting other pathways. She demonstrated remarkable improvement in both neurological symptoms and cortical architecture. She gained fine motor skills with the capacity to feed herself and sit with support.

Conclusions: This work highlights the strategy of repurposing drugs to treat a rare disease.

Funding: No external funding was received for this work.
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http://dx.doi.org/10.7554/eLife.67097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8291972PMC
July 2021

Probenecid increases renal retention and antitumor activity of DFMO in neuroblastoma.

Cancer Chemother Pharmacol 2021 Oct 15;88(4):607-617. Epub 2021 Jun 15.

Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, 400 Monroe Ave, NW, Grand Rapids, MI, 49503, USA.

Background: Neuroblastoma (NB) is the most common extracranial solid tumor in children. Interference with the polyamine biosynthesis pathway by inhibition of MYCN-activated ornithine decarboxylase (ODC) is a validated approach. The ODC inhibitor α-difluoromethylornithine (DFMO, or Eflornithine) has been FDA-approved for the treatment of trypanosomiasis and hirsutism and has advanced to clinical cancer trials including NB as well as cancer-unrelated human diseases. One key challenge of DFMO is its rapid renal clearance and the need for high and frequent drug dosing during treatment.

Methods: We performed in vivo pharmacokinetic (PK), antitumorigenic, and molecular studies with DFMO/probenecid using NB patient-derived xenografts (PDX) in mice. We used LC-MS/MS, HPLC, and immunoblotting to analyze blood, brain tissue, and PDX tumor tissue samples collected from mice.

Results: The organic anion transport 1/3 (OAT 1/3) inhibitor probenecid reduces the renal clearance of DFMO and significantly increases the antitumor activity of DFMO in PDX of NB (P < 0.02). Excised tumors revealed that DFMO/probenecid treatment decreases polyamines putrescine and spermidine, reduces MYCN protein levels and dephosphorylates retinoblastoma (Rb) protein (p-Rb), suggesting DFMO/probenecid-induced cell cycle arrest.

Conclusion: Addition of probenecid as an adjuvant to DFMO therapy may be suitable to decrease overall dose and improve drug efficacy in vivo.
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http://dx.doi.org/10.1007/s00280-021-04309-yDOI Listing
October 2021

Balancing precision versus cohort transcriptomic analysis of acute and recovery phase of viral bronchiolitis.

Am J Physiol Lung Cell Mol Physiol 2021 06 14;320(6):L1147-L1157. Epub 2021 Apr 14.

Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, Grand Rapids, Michigan.

Viral infections affecting the lower respiratory tract place enormous burdens on hospitals. As neither vaccines nor antiviral agents exist for many viruses, understanding risk factors and outcomes in each patient using minimally invasive analysis, such as blood, can lead to improved health care delivery. A cohort of PAXgene RNA sequencing of infants admitted with moderate or severe acute bronchiolitis and respiratory syncytial virus were compared with case-control statistical analysis and cohort-based outlier mapping for precision transcriptomics. Patients with severe bronchiolitis had signatures connected to the immune system, interferon signaling, and cytokine signaling, with marked sex differences in , , , and for severity. Several patients had unique secondary infections, cytokine activation, immune responses, biological pathways, and immune cell activation, highlighting the need for defining patient-level transcriptomic signatures. Balancing relative contributions of cohort-based biomarker discoveries with patient's biological responses is needed to understand the totality of mechanisms of adverse outcomes in viral bronchiolitis.
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http://dx.doi.org/10.1152/ajplung.00440.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8285625PMC
June 2021

Balancing precision versus cohort transcriptomic analysis of acute and recovery phase of viral bronchiolitis.

Am J Physiol Lung Cell Mol Physiol 2021 06 14;320(6):L1147-L1157. Epub 2021 Apr 14.

Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, Grand Rapids, Michigan.

Viral infections affecting the lower respiratory tract place enormous burdens on hospitals. As neither vaccines nor antiviral agents exist for many viruses, understanding risk factors and outcomes in each patient using minimally invasive analysis, such as blood, can lead to improved health care delivery. A cohort of PAXgene RNA sequencing of infants admitted with moderate or severe acute bronchiolitis and respiratory syncytial virus were compared with case-control statistical analysis and cohort-based outlier mapping for precision transcriptomics. Patients with severe bronchiolitis had signatures connected to the immune system, interferon signaling, and cytokine signaling, with marked sex differences in , , , and for severity. Several patients had unique secondary infections, cytokine activation, immune responses, biological pathways, and immune cell activation, highlighting the need for defining patient-level transcriptomic signatures. Balancing relative contributions of cohort-based biomarker discoveries with patient's biological responses is needed to understand the totality of mechanisms of adverse outcomes in viral bronchiolitis.
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http://dx.doi.org/10.1152/ajplung.00440.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8285625PMC
June 2021

Gene expression signatures identify paediatric patients with multiple organ dysfunction who require advanced life support in the intensive care unit.

EBioMedicine 2020 Dec 25;62:103122. Epub 2020 Nov 25.

Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, Grand Rapids, MI 49503, USA; Pediatric Intensive Care Unit, Helen DeVos Children's Hospital, 100 Michigan Street NE, Grand Rapids, MI 49503, USA; Office of Research, Spectrum Health, 15 Michigan Street NE, Grand Rapids, MI 49503, USA. Electronic address:

Background: Multiple organ dysfunction syndrome (MODS) occurs in the setting of a variety of pathologies including infection and trauma. Some patients decompensate and require Veno-Arterial extra corporeal membrane oxygenation (ECMO) as a palliating manoeuvre for recovery of cardiopulmonary function. The molecular mechanisms driving progression from MODS to cardiopulmonary collapse remain incompletely understood, and no biomarkers have been defined to identify those MODS patients at highest risk for progression to requiring ECMO support.

Methods: Whole blood RNA-seq profiling was performed for 23 MODS patients at three time points during their ICU stay (at diagnosis of MODS, 72 hours after, and 8 days later), as well as four healthy controls undergoing routine sedation. Of the 23 MODS patients, six required ECMO support (ECMO patients). The predictive power of conventional demographic and clinical features was quantified for differentiating the MODS and ECMO patients. We then compared the performance of markers derived from transcriptomic profiling including [1] transcriptomically imputed leukocyte subtype distribution, [2] relevant published gene signatures and [3] a novel differential gene expression signature computed from our data set. The predictive power of our novel gene expression signature was then validated using independently published datasets.

Finding: None of the five demographic characteristics and 14 clinical features, including The Paediatric Logistic Organ Dysfunction (PELOD) score, could predict deterioration of MODS to ECMO at baseline. From previously published sepsis signatures, only the signatures positively associated with patient's mortality could differentiate ECMO patients from MODS patients, when applied to our transcriptomic dataset (P-value ranges from 0.01 to 0.04, Student's test). Deconvolution of bulk RNA-Seq samples suggested that lower neutrophil counts were associated with increased risk of progression from MODS to ECMO (P-value = 0.03, logistic regression, OR=2.82 [95% CI 0.63 - 12.45]). A total of 30 genes were differentially expressed between ECMO and MODS patients at baseline (log2 fold change ≥ 1 or ≤ -1 with false discovery rate ≤ 0.01). These genes are involved in protein maintenance and epigenetic-related processes. Further univariate analysis of these 30 genes suggested a signature of seven DE genes associated with ECMO (OR > 3.0, P-value ≤ 0.05, logistic regression). Notably, this contains a set of histone marker genes, including H1F0, HIST2H3C, HIST1H2AI, HIST1H4, HIST1H2BL and HIST1H1B, that were highly expressed in ECMO. A risk score derived from expression of these genes differentiated ECMO and MODS patients in our dataset (AUC = 0.91, 95% CI 0.79-1.00, P-value = 7e-04, logistic regression) as well as validation dataset (AUC= 0.73, 95% CI 0.53-0.93, P-value = 2e-02, logistic regression).

Interpretation: This study demonstrates that transcriptomic features can serve as indicators of severity that could be superior to traditional methods of ascertaining acuity in MODS patients. Analysis of expression of signatures identified in this study could help clinicians in the diagnosis and prognostication of MODS patients after arrival to the Hospital.
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http://dx.doi.org/10.1016/j.ebiom.2020.103122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7704404PMC
December 2020

Allicin, a Potent New Ornithine Decarboxylase Inhibitor in Neuroblastoma Cells.

J Nat Prod 2020 08 12;83(8):2518-2527. Epub 2020 Aug 12.

Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, Grand Rapids, Michigan 49503, United States.

The natural product allicin is a reactive sulfur species (RSS) from garlic ( L.). Neuroblastoma (NB) is an early childhood cancer arising from the developing peripheral nervous system. Ornithine decarboxylase (ODC) is a rate-limiting enzyme in the biosynthesis of polyamines, which are oncometabolites that contribute to cell proliferation in NB and other c-MYC/MYCN-driven cancers. Both c-MYC and MYCN directly transactivate the E-box gene , a validated anticancer drug target. We identified allicin as a potent ODC inhibitor in a specific radioactive assay using purified human ODC. Allicin was ∼23 000-fold more potent (IC = 11 nM) than DFMO (IC = 252 μM), under identical assay conditions. ODC is a homodimer with 12 cysteines per monomer, and allicin reversibly -thioallylates cysteines. In actively proliferating human NB cells allicin inhibited ODC enzyme activity, reduced cellular polyamine levels, inhibited cell proliferation (IC 9-19 μM), and induced apoptosis. The natural product allicin is a new ODC inhibitor and could be developed for use in conjunction with other anticancer treatments, the latter perhaps at a lower than usual dosage, to achieve drug synergism with good prognosis and reduced adverse effects.
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http://dx.doi.org/10.1021/acs.jnatprod.0c00613DOI Listing
August 2020

Virus-induced genetics revealed by multidimensional precision medicine transcriptional workflow applicable to COVID-19.

Physiol Genomics 2020 06 21;52(6):255-268. Epub 2020 May 21.

Congenital Heart Center, Helen DeVos Children's Hospital, Grand Rapids, Michigan.

Precision medicine requires the translation of basic biological understanding to medical insights, mainly applied to characterization of each unique patient. In many clinical settings, this requires tools that can be broadly used to identify pathology and risks. Patients often present to the intensive care unit with broad phenotypes, including multiple organ dysfunction syndrome (MODS) resulting from infection, trauma, or other disease processes. Etiology and outcomes are unique to individuals, making it difficult to cohort patients with MODS, but presenting a prime target for testing/developing tools for precision medicine. Using multitime point whole blood (cellular/acellular) total transcriptomics in 27 patients, we highlight the promise of simultaneously mapping viral/bacterial load, cell composition, tissue damage biomarkers, balance between syndromic biology versus environmental response, and unique biological insights in each patient using a single platform measurement. Integration of a transcriptome workflow yielded unexpected insights into the complex interplay between host genetics and viral/bacterial specific mechanisms, highlighted by a unique case of virally induced genetics (VIG) within one of these 27 patients. The power of RNA-Seq to study unique patient biology while investigating environmental contributions can be a critical tool moving forward for translational sciences applied to precision medicine.
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http://dx.doi.org/10.1152/physiolgenomics.00045.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7303726PMC
June 2020

Syrbactin proteasome inhibitor TIR-199 overcomes bortezomib chemoresistance and inhibits multiple myeloma tumor growth in vivo.

Leuk Res 2020 01 12;88:106271. Epub 2019 Nov 12.

Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, 400 Monroe Ave NW, Grand Rapids, MI 49503, USA. Electronic address:

Multiple myeloma (MM) and mantle cell lymphoma (MCL) are blood cancers that respond to proteasome inhibitors. Three FDA-approved drugs that block the proteasome are currently on the market, bortezomib, carfilzomib, and ixazomib. While these proteasome inhibitors have demonstrated clinical efficacy against refractory and relapsed MM and MCL, they are also associated with considerable adverse effects including peripheral neuropathy and cardiotoxicity, and tumor cells often acquire drug resistance. TIR-199 belongs to the syrbactin class, which constitutes a novel family of irreversible proteasome inhibitors. In this study, we compare TIR-199 head-to-head with three FDA-approved proteasome inhibitors. We demonstrate that TIR-199 selectively inhibits to varying degrees the sub-catalytic proteasomal activities (C-L/β1, T-L/β2, and CT-L/β5) in three actively dividing MM cell lines, with Ki (CT-L/β5) values of 14.61 ± 2.68 nM (ARD), 54.59 ± 10.4 nM (U266), and 26.8 ± 5.2 nM (MM.1R). In most instances, this range was comparable with the activity of ixazomib. However, TIR-199 was more effective than bortezomib, carfilzomib, and ixazomib in killing bortezomib-resistant MM and MCL cell lines, as judged by a low resistance index (RI) between 1.7 and 2.2, which implies that TIR-199 indiscriminately inhibits both bortezomib-sensitive and bortezomib-resistant MM and MCL cells at similar concentrations. Importantly, TIR-199 reduced the tumor burden in a MM mouse model (p < 0.01) confirming its potency in vivo. Given the fact that there is still no cure for MM, the further development of TIR-199 or similar molecules that belong to the syrbactin class of proteasome inhibitors is warranted.
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http://dx.doi.org/10.1016/j.leukres.2019.106271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6937391PMC
January 2020

Biochemical features of primary cells from a pediatric patient with a gain-of-function genetic mutation.

Biochem J 2019 07 24;476(14):2047-2057. Epub 2019 Jul 24.

Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, Grand Rapids, MI, U.S.A.

We recently described a new autosomal dominant genetic disorder in a pediatric patient caused by a heterozygous mutation in the ornithine decarboxylase 1 () gene. The new genetic disorder is characterized by global developmental delay, alopecia, overgrowth, and dysmorphic features. We hypothesized that this new mutation (c.1342 A>T) leads to a C-terminal truncation variant of the ODC protein that is resistant to normal proteasomal degradation, leading to putrescine accumulation in cells. ODC (E.C. 4.1.1.17) is a rate-limiting enzyme in the biosynthesis of polyamines (putrescine, spermidine, and spermine) that plays a crucial role during embryogenesis, organogenesis, and tumorigenesis. In this study, we show that primary dermal fibroblasts derived from a skin biopsy of a 3-year-old patient contain large amounts of ODC protein and putrescine compared with primary dermal (neonatal and adult) fibroblast control cells. Importantly, the accumulated ODC protein variant remained functionally active as we detected exceptionally high ODC enzyme activity in both primary dermal fibroblasts (12-17-fold of controls) and red blood cells (RBCs) (125-137-fold of controls), using a specific C radioactive ODC activity assay. Exposure of primary dermal fibroblasts to ODC inhibitor α-difluoromethylornithine (DFMO) reduced the ODC activity and putrescine to levels observed in controls without adversely affecting cell morphology or inducing cell death. In conclusion, our patient and potentially other patients that carry a similar gain-of-function mutation might benefit from treatment with DFMO, a drug with a good safety profile, to suppress the exceptionally high ODC activity and putrescine levels in the body.
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http://dx.doi.org/10.1042/BCJ20190294DOI Listing
July 2019

Anti-tumor effect of sulfasalazine in neuroblastoma.

Biochem Pharmacol 2019 04 9;162:237-249. Epub 2019 Jan 9.

Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, 400 Monroe Ave NW, Grand Rapids, MI 49503, USA. Electronic address:

Neuroblastoma (NB) is a tumor arising from the sympathetic nervous system during infancy and early childhood. High-risk patients who relapse often fail to respond to further therapy, which results in 5-year survival rate for this patient group below 5%. Therefore, there continues to be an urgent need for innovative treatments. Recently, we found that sulfasalazine (SSZ), an FDA-approved drug for the treatment of rheumatoid arthritis and ulcerative colitis induces anti-proliferative effects in NB tumor cells. SSZ was recently shown to inhibit sepiapterin reductase (SPR), a key enzyme that produces tetrahydrobiopterin (BH4) in the nitric oxide (NO) pathway. Here we tested SSZ against purified SPR in vitro, measured the anti-proliferative effect of SSZ on a panel of MYCN amplified and MYCN non-amplified NB cell lines, and assessed the anti-tumor effect of SSZ in NB tumor-xenografted mice. We found that the expression of both SPR mRNA and SPR protein was significantly higher in cell lines without MYCN amplification. SSZ inhibited SPR enzyme activity in vitro and exhibits anti-proliferative activity in a large number of NB cell lines derived from high-risk tumors. Importantly, oral/intraperitoneal (i.p.) SSZ co-administration resulted in measureable anti-tumor effects in vivo. The FDA-approved drug SSZ, a well-tolerated drug in clinical use, could be repositioned to inhibit tumor growth in NB.
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http://dx.doi.org/10.1016/j.bcp.2019.01.007DOI Listing
April 2019

Polyamine synthesis as a target of oncogenes.

J Biol Chem 2018 11 7;293(48):18757-18769. Epub 2018 Nov 7.

the Department of Medical Biology, Amsterdam University Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands.

This paper is in recognition of the 100th birthday of Dr. Herbert Tabor, a true pioneer in the polyamine field for over 70 years, who served as the editor-in-chief of the from 1971 to 2010. We review current knowledge of MYC proteins (c-MYC, MYCN, and MYCL) and focus on ornithine decarboxylase 1 (), an important gene target of MYC, which encodes the sentinel, rate-limiting enzyme in polyamine biosynthesis. Although notable advances have been made in designing inhibitors against the "undruggable" MYCs, their downstream targets and pathways are currently the main avenue for therapeutic anticancer interventions. To this end, the MYC-ODC axis presents an attractive target for managing cancers such as neuroblastoma, a pediatric malignancy in which gene amplification correlates with poor prognosis and high-risk disease. ODC and polyamine levels are often up-regulated and contribute to tumor hyperproliferation, especially of MYC-driven cancers. We therefore had proposed to repurpose α-difluoromethylornithine (DFMO), an FDA-approved, orally available ODC inhibitor, for management of neuroblastoma, and this intervention is now being pursued in several clinical trials. We discuss the regulation of ODC and polyamines, which besides their well-known interactions with DNA and tRNA/rRNA, are involved in regulating RNA transcription and translation, ribosome function, proteasomal degradation, the circadian clock, and immunity, events that are also controlled by MYC proteins.
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http://dx.doi.org/10.1074/jbc.TM118.003336DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6290138PMC
November 2018

Thiasyrbactins Induce Cell Death Proteasome Inhibition in Multiple Myeloma Cells.

Anticancer Res 2018 Oct;38(10):5607-5613

Department of Pediatrics and Human Development, Michigan State University, Grand Rapids, MI, U.S.A.

Background/aim: Proteasome inhibition is a validated therapeutic strategy for the treatment of refractory and relapsed multiple myeloma (MM) and mantle cell lymphoma. We previously showed that thiasyrbactins (NAM compounds) are inhibitors with an affinity for the trypsin-like (T-L, β2) site of the constitutive proteasome, and more profoundly for the T-L site of the immunoproteasome.

Materials And Methods: In this study, the biological activity of three NAM compounds was evaluated using four MM cell lines (ARD, U266, MM1R, and MM1S). We assessed the effect of (NAM-93, NAM-95, and NAM-105 on cell viability, as well as cell-based proteasomal activities, and determined the EC and Ki values, respectively.

Results: MM cells were most sensitive to NAM-93 with EC values <0.75 μM after 48 h of treatment. NAM-105 had a similar profile in most of the MM cells with EC values ranging between 0.42 and 3.02 μM. The level of inhibition of the proteasome T-L sub-catalytic activity in actively-growing MM cells was similar for NAM-93 and NAM-105. However, in each cell line, NAM-93 was more effective than NAM-105 at inhibiting overall trypsin-like sub-catalytic activity while NAM-105 was typically more effective at inhibiting overall chymotrypsin-like (CT-L, β5) sub-catalytic activity.

Conclusion: These results show for the first time the proteasome-targeted biological activity of thiasyrbactins in MM tumor cells.
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http://dx.doi.org/10.21873/anticanres.12895DOI Listing
October 2018

Novel de novo pathogenic variant in the ODC1 gene in a girl with developmental delay, alopecia, and dysmorphic features.

Am J Med Genet A 2018 12 21;176(12):2548-2553. Epub 2018 Sep 21.

Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, Grand Rapids, Michigan.

The ornithine decarboxylase 1 (ODC1) gene plays an important role in physiological and cell developmental processes including embryogenesis, organogenesis, and neoplastic cell growth. Here, we report an 32-month-old Caucasian female with a heterozygous de novo nonsense mutation in the ODC1 gene that leads to a premature abrogation of 14-aa residues at the ODC protein c-terminus. This is the first human case confirming similar symptoms observed in a transgenic ODC1 mouse model first described over 20 years ago. Phenotypic manifestations include macrosomia, macrocephaly, developmental delay, alopecia, spasticity, hypotonia, cutaneous vascular malformation, delayed visual maturation, and sensorineural hearing loss. We here describe for the first time a new pediatric disorder that is directly linked to a de novo pathogenic variant in the ODC1 gene. The ODC1 gene mutation (c.1342 A>T) was identified by whole-exome sequencing and confirmed by Sanger sequencing. Red blood cells obtained from our patient showed elevated ODC protein and polyamine levels compared to healthy controls. Our autosomal dominant patient who carries this gain-of-function ODC1 mutation may benefit from treatment with α-difluoromethylornithine, a well-tolerated, U.S. Food and Drug Administration (FDA). FDA-approved drug.
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http://dx.doi.org/10.1002/ajmg.a.40523DOI Listing
December 2018

Polyamine Biosynthetic Pathway as a Drug Target for Osteosarcoma Therapy.

Med Sci (Basel) 2018 08 16;6(3). Epub 2018 Aug 16.

Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, 400 Monroe Avenue, NW, Grand Rapids, MI 49503, USA.

Osteosarcoma (OS) is the most common bone tumor in children. Polyamines (PAs) are ubiquitous cations involved in many cell processes including tumor development, invasion and metastasis. In other pediatric cancer models, inhibition of the PA biosynthesis pathway with ornithine decarboxylase (ODC) inhibitor alpha-difluoromethylornithine (DFMO) results in decreased cell proliferation and differentiation. In OS, the PA pathway has not been evaluated. DFMO is an attractive, orally administered drug, is well tolerated, can be given for prolonged periods, and is already used in pediatric patients. Three OS cell lines were used to study the cellular effects of PA inhibition with DFMO: MG-63, U-2 OS and Saos-2. Effects on proliferation were analyzed by cell count, flow cytometry-based cell cycle analysis and RealTime-Glo™ MT Cell Viability assays. Intracellular PA levels were measured with high-performance liquid chromatography (HPLC). Western blot analysis was used to evaluate cell differentiation. DFMO exposure resulted in significantly decreased cell proliferation in all cell lines. After treatment, intracellular spermidine levels were drastically decreased. Cell cycle arrest at G₂/M was observed in U-2 OS and Saos-2. Cell differentiation was most prominent in MG-63 and U-2 OS as determined by increases in the terminal differentiation markers osteopontin and collagen 1a1. Cell proliferation continued to be suppressed for several days after removal of DFMO. Based on our findings, DFMO is a promising new adjunct to current osteosarcoma therapy in patients at high risk of relapse, such as those with poor necrosis at resection or those with metastatic or recurrent osteosarcoma. It is a well-tolerated oral drug that is currently in phase II clinical trials in pediatric neuroblastoma patients as a maintenance therapy. The same type of regimen may also improve outcomes in osteosarcoma patients in whom there have been essentially no medical advances in the last 30 years.
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http://dx.doi.org/10.3390/medsci6030065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6165283PMC
August 2018

Harmine, a dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) inhibitor induces caspase-mediated apoptosis in neuroblastoma.

Cancer Cell Int 2018 7;18:82. Epub 2018 Jun 7.

1Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, 400 Monroe Avenue NW, Grand Rapids, MI 49503 USA.

Background: Neuroblastoma (NB) is an early childhood malignancy that arises from the developing sympathetic nervous system. Harmine is a tricyclic β-carboline alkaloid isolated from the harmal plant that exhibits both cytostatic and cytotoxic effects. Harmine is capable of blocking the activities of dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) family proteins and mitogen-activated protein kinase. These kinases promote proliferation and inhibit apoptosis.

Methods: Four human NB cell lines were used to study the effects of harmine treatment: SKNBE and KELLY (-amplified) as well as SKNAS and SKNFI ( non-amplified). The anti-cancer properties of harmine were examined by RealTime-Glo MT cell viability assays, caspase activity assays, PARP cleavage using Western blot analysis, and flow cytometry-based Annexin V detection. A molecular interaction model of harmine bound to the DYRK2 family kinase was generated by computational docking using X-ray structures. NB tumors from human patients were profiled for DYRK mRNA expression patterns and clinical correlations using the R2 platform.

Results: The IC values for harmine after 72 h treatment were 169.6, 170.8, and 791.7 μM for SKNBE, KELLY, and SKNFI, respectively. Exposure of these NB cell lines to 100 μM of harmine resulted in caspase-3/7 and caspase-9 activation as well as caspase-mediated PARP cleavage and Annexin V-positive stained cells, as early as 24 h after treatment, clearly suggesting apoptosis induction, especially in -amplified cell lines. Elevated mRNA levels correlated with poor prognosis in a large cohort of NB tumors.

Conclusion: Harmine is a known inhibitor of DYRK family kinases. It can induce apoptosis in NB cell lines, which led us to investigate the clinical correlations of family gene expression in NB tumors. The patient results support our hypothesis that DYRK inhibition by harmine and the subsequent triggering of caspase-mediated apoptosis might present a novel approach to NB therapy.
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http://dx.doi.org/10.1186/s12935-018-0574-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5992763PMC
June 2018

Synergistic drug combination GC7/DFMO suppresses hypusine/spermidine-dependent eIF5A activation and induces apoptotic cell death in neuroblastoma.

Biochem J 2018 01 31;475(2):531-545. Epub 2018 Jan 31.

Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, Grand Rapids, MI 49503, U.S.A.

The eukaryotic initiation factor 5A (eIF5A), which contributes to several crucial processes during protein translation, is the only protein that requires activation by a unique post-translational hypusine modification. eIF5A hypusination controls cell proliferation and has been linked to cancer. eIF5A hypusination requires the enzymes deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase and uniquely depends on the polyamine (PA) spermidine as the sole substrate. Ornithine decarboxylase (ODC) is the rate-limiting enzyme in PA biosynthesis. Both ODC and PAs control cell proliferation and are frequently dysregulated in cancer. Since only spermidine can activate eIF5A, we chose the hypusine-PA nexus as a rational target to identify new drug combinations with synergistic antiproliferative effects. We show that elevated mRNA levels of the two target enzymes DHPS and ODC correlate with poor prognosis in a large cohort of neuroblastoma (NB) tumors. The DHPS inhibitor GC7 (-guanyl-1,7-diaminoheptane) and the ODC inhibitor α-difluoromethylornithine (DFMO) are target-specific and in combination induced synergistic effects in NB at concentrations that were not individually cytotoxic. Strikingly, while each drug alone at higher concentrations is known to induce p21/Rb- or p27/Rb-mediated G cell cycle arrest, we found that the drug combination induced caspase 3/7/9, but not caspase 8-mediated apoptosis, in NB cells. Hypusinated eIF5A levels and intracellular spermidine levels correlated directly with drug treatments, signifying specific drug targeting effects. This two-pronged GC7/DFMO combination approach specifically inhibits both spermidine biosynthesis and post-translational, spermidine-dependent hypusine-eIF5A activation, offering an exciting clue for improved NB drug therapy.
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http://dx.doi.org/10.1042/BCJ20170597DOI Listing
January 2018

Immunoproteasome inhibition and bioactivity of thiasyrbactins.

Bioorg Med Chem 2018 01 7;26(2):401-412. Epub 2017 Dec 7.

Department of Chemistry, University of California, Riverside, CA 92521, USA; Department of Pharmaceutical Sciences, University of California, Irvine, CA 92697, USA. Electronic address:

A family of macrodilactam natural products, the syrbactins, are known proteasome inhibitors. A small group of syrbactin analogs was prepared with a sulfur-for-carbon substitution to enhance synthetic accessibility and facilitate modulation of their solubility. Two of these compounds surprisingly proved to be inhibitors of the trypsin-like catalytic site, including of the immunoproteasome. Their bound and free conformations suggest special properties of the thiasyrbactin ring are responsible for this unusual preference, which may be exploited to develop drug-like immunoproteasome inhibitors. These compounds show greater selectivity than earlier compounds used to infer phenotypes of immunoproteasome inhibition, like ONX-0914.
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http://dx.doi.org/10.1016/j.bmc.2017.11.048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5920785PMC
January 2018

Ornithine decarboxylase as a therapeutic target for endometrial cancer.

PLoS One 2017 14;12(12):e0189044. Epub 2017 Dec 14.

Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, Grand Rapids, Michigan, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0189044PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5730160PMC
January 2018

Syrbactin Structural Analog TIR-199 Blocks Proteasome Activity and Induces Tumor Cell Death.

J Biol Chem 2016 Apr 23;291(16):8350-62. Epub 2016 Feb 23.

the Department of Chemistry, University of California, Riverside, California 92521, and the Department of Pharmaceutical Sciences, University of California, Irvine, California 92697

Multiple myeloma is an aggressive hematopoietic cancer of plasma cells. The recent emergence of three effective FDA-approved proteasome-inhibiting drugs, bortezomib (Velcade®), carfilzomib (Kyprolis®), and ixazomib (Ninlaro®), confirms that proteasome inhibitors are therapeutically useful against neoplastic disease, in particular refractory multiple myeloma and mantle cell lymphoma. This study describes the synthesis, computational affinity assessment, and preclinical evaluation of TIR-199, a natural product-derived syrbactin structural analog. Molecular modeling and simulation suggested that TIR-199 covalently binds each of the three catalytic subunits (β1, β2, and β5) and revealed key interaction sites. In vitro and cell culture-based proteasome activity measurements confirmed that TIR-199 inhibits the proteasome in a dose-dependent manner and induces tumor cell death in multiple myeloma and neuroblastoma cells as well as other cancer types in the NCI-60 cell panel. It is particularly effective against kidney tumor cell lines, with >250-fold higher anti-tumor activities than observed with the natural product syringolin A. In vivo studies in mice revealed a maximum tolerated dose of TIR-199 at 25 mg/kg. The anti-tumor activity of TIR-199 was confirmed in hollow fiber assays in mice. Adverse drug reaction screens in a kidney panel revealed no off-targets of concern. This is the first study to examine the efficacy of a syrbactin in animals. Taken together, the results suggest that TIR-199 is a potent new proteasome inhibitor with promise for further development into a clinical drug for the treatment of multiple myeloma and other forms of cancer.
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http://dx.doi.org/10.1074/jbc.M115.710053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4861411PMC
April 2016

Effect of sulfasalazine on human neuroblastoma: analysis of sepiapterin reductase (SPR) as a new therapeutic target.

BMC Cancer 2015 Jun 21;15:477. Epub 2015 Jun 21.

Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, 301 Michigan Street, NE, Grand Rapids, MI, 49503, USA.

Background: Neuroblastoma (NB) is an aggressive childhood malignancy in children up to 5 years of age. High-stage tumors frequently relapse even after aggressive multimodal treatment, and then show therapy resistance, typically resulting in patient death. New molecular-targeted compounds that effectively suppress tumor growth and prevent relapse with more efficacy are urgently needed. We and others previously showed that polyamines (PA) like spermidine and spermine are essential for NB tumorigenesis and that DFMO, an inhibitor of the key PA synthesis gene product ODC, is effective both in vitro and in vivo, securing its evaluation in NB clinical trials. To find additional compounds interfering with PA biosynthesis, we tested sulfasalazine (SSZ), an FDA-approved salicylate-based anti-inflammatory and immune-modulatory drug, recently identified to inhibit sepiapterin reductase (SPR). We earlier presented evidence for a physical interaction between ODC and SPR and we showed that RNAi-mediated knockdown of SPR expression significantly reduced native ODC enzyme activity and impeded NB cell proliferation.

Methods: Human NB mRNA expression datasets in the public domain were analyzed using the R2 platform. Cell viability, isobologram, and combination index analyses as a result of SSZ treatment with our without DFMO were carried out in NB cell cultures. Molecular protein-ligand docking was achieved using the GRAMM algorithm. Statistical analyses were performed with the Kruskal-Wallis test, 2log Pearson test, and Student's t test.

Results: In this study, we show the clinical relevance of SPR in human NB tumors. We found that high SPR expression is significantly correlated to unfavorable NB characteristics like high age at diagnosis, MYCN amplification, and high INSS stage. SSZ inhibits the growth of NB cells in vitro, presumably due to the inhibition of SPR as predicted by computational docking of SSZ into SPR. Importantly, the combination of SSZ with DFMO produces synergistic antiproliferative effects in vitro.

Conclusions: The results suggest the use of SSZ in combination with DFMO for further experiments, and possible prioritization as a novel therapy for the treatment of NB patients.
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http://dx.doi.org/10.1186/s12885-015-1447-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4475614PMC
June 2015

A Phase I Trial of DFMO Targeting Polyamine Addiction in Patients with Relapsed/Refractory Neuroblastoma.

PLoS One 2015 27;10(5):e0127246. Epub 2015 May 27.

College of Human Medicine, Michigan State University, Grand Rapids, Michigan, United States of America; University of Hawaii at Hilo, The Daniel K. Inouye College of Pharmacy, Hilo, Hawaii, United States of America.

Background: Neuroblastoma (NB) is the most common cancer in infancy and most frequent cause of death from extracranial solid tumors in children. Ornithine decarboxylase (ODC) expression is an independent indicator of poor prognosis in NB patients. This study investigated safety, response, pharmacokinetics, genetic and metabolic factors associated with ODC in a clinical trial of the ODC inhibitor difluoromethylornithine (DFMO) ± etoposide for patients with relapsed or refractory NB.

Methods And Findings: Twenty-one patients participated in a phase I study of daily oral DFMO alone for three weeks, followed by additional three-week cycles of DFMO plus daily oral etoposide. No dose limiting toxicities (DLTs) were identified in patients taking doses of DFMO between 500-1500 mg/m2 orally twice a day. DFMO pharmacokinetics, single nucleotide polymorphisms (SNPs) in the ODC gene and urinary levels of substrates for the tissue polyamine exporter were measured. Urinary polyamine levels varied among patients at baseline. Patients with the minor T-allele at rs2302616 of the ODC gene had higher baseline levels (p=0.02) of, and larger decreases in, total urinary polyamines during the first cycle of DFMO therapy (p=0.003) and had median progression free survival (PFS) that was over three times longer, compared to patients with the major G allele at this locus although this last result was not statistically significant (p=0.07). Six of 18 evaluable patients were progression free during the trial period with three patients continuing progression free at 663, 1559 and 1573 days after initiating treatment. Median progression-free survival was less among patients having increased urinary polyamines, especially diacetylspermine, although this result was not statistically significant (p=0.056).

Conclusions: DFMO doses of 500-1500 mg/m2/day are safe and well tolerated in children with relapsed NB. Children with the minor T allele at rs2302616 of the ODC gene with relapsed or refractory NB had higher levels of urinary polyamine markers and responded better to therapy containing DFMO, compared to those with the major G allele at this locus. These findings suggest that this patient subset may display dependence on polyamines and be uniquely susceptible to therapies targeting this pathway.

Trial Registration: Clinicaltrials.gov NCT#01059071.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0127246PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4446210PMC
April 2016

Withaferin A Inhibits STAT3 and Induces Tumor Cell Death in Neuroblastoma and Multiple Myeloma.

Biochem Insights 2014 9;7:1-13. Epub 2014 Nov 9.

Department of Pharmaceutical Sciences, The Daniel K. Inouye College of Pharmacy, University of Hawaii at Hilo, Hilo, USA. ; Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, Hawaii, USA. ; Department of Cell and Molecular Biology, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, Hawaii, USA. ; Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, Grand Rapids, USA.

Signal transducer and activator of transcription 3 (STAT3) is an oncogenic transcription factor that has been implicated in many human cancers and has emerged as an ideal target for cancer therapy. Withaferin A (WFA) is a natural product with promising antiproliferative properties through its association with a number of molecular targets including STAT3. However, the effect of WFA in pediatric neuroblastoma (NB) and its interaction with STAT3 have not been reported. In this study, we found that WFA effectively induces dose-dependent cell death in high-risk and drug-resistant NB as well as multiple myeloma (MM) tumor cells, prevented interleukin-6 (IL-6)-mediated and persistently activated STAT3 phosphorylation at Y705, and blocked the transcriptional activity of STAT3. We further provide computational models that show that WFA binds STAT3 near the Y705 phospho-tyrosine residue of the STAT3 Src homology 2 (SH2) domain, suggesting that WFA prevents STAT3 dimer formation similar to BP-1-102, a well-established STAT3 inhibitor. Our findings propose that the antitumor activity of WFA is mediated at least in part through inhibition of STAT3 and provide a rationale for further drug development and clinical use in NB and MM.
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http://dx.doi.org/10.4137/BCI.S18863DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4226392PMC
December 2014

Deoxyhypusine synthase (DHPS) inhibitor GC7 induces p21/Rb-mediated inhibition of tumor cell growth and DHPS expression correlates with poor prognosis in neuroblastoma patients.

Cell Oncol (Dordr) 2014 Dec 15;37(6):387-98. Epub 2014 Oct 15.

Department of Pharmaceutical Sciences, The Daniel K. Inouye College of Pharmacy, University of Hawaii at Hilo, 34 Rainbow Drive, Hilo, HI, 96720, USA.

Purpose: Neuroblastoma (NB) is an aggressive pediatric malignancy that typically occurs in infants and children under the age of 5 years. High-stage tumors relapse frequently even after aggressive multimodal treatment, resulting in therapy resistance and eventually in patient death. Clearly, new biologically-targeted drugs are needed that more efficiently suppress tumor growth and prevent relapse. We and others previously showed that polyamines such as spermidine play an essential role in NB tumorigenesis and that DFMO, an inhibitor of the central polyamine synthesis gene ODC, is effective in vitro and in vivo, prompting its evaluation in NB clinical trials. However, the specific molecular actions of polyamines remain poorly defined. Spermidine and deoxyhypusine synthase (DHPS) are essential components in the hypusination-driven post-translational activation of eukaryotic initiation factor 5A (eIF5A).

Methods: We assessed the role of DHPS in NB and the impact of its inhibition by N(1)-guanyl-1,7-diaminoheptane (GC7) on tumor cell growth using cell proliferation assays, Western blot, immunofluorescence microscopy, and Affymetrix micro-array mRNA expression analyses in NB tumor samples.

Results: We found that GC7 inhibits NB cell proliferation in a dose-dependent manner, through induction of the cell cycle inhibitor p21 and reduction of total and phosphorylated Rb proteins. Strikingly, high DHPS mRNA expression correlated significantly with unfavorable clinical parameters, including poor patient survival, in a cohort of 88 NB tumors (all P < 0.04).

Conclusions: These results suggest that spermidine and DHPS are key contributing factors in NB tumor proliferation through regulation of the p21/Rb signaling axis.
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http://dx.doi.org/10.1007/s13402-014-0201-9DOI Listing
December 2014

Novel interaction of ornithine decarboxylase with sepiapterin reductase regulates neuroblastoma cell proliferation.

J Mol Biol 2014 Jan 1;426(2):332-46. Epub 2013 Oct 1.

Department of Pharmaceutical Sciences, The Daniel K. Inouye College of Pharmacy, University of Hawaii at Hilo, Hilo, HI 96720, USA; Department of Cell and Molecular Biology, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, HI 96813, USA. Electronic address:

Ornithine decarboxylase (ODC) is the sentinel enzyme in polyamine biosynthesis. Both ODC and polyamines regulate cell division, proliferation, and apoptosis. Sepiapterin reductase (SPR) catalyzes the last step in the biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor of nitric oxide synthase, and has been implicated in neurological diseases but not yet in cancer. In this study, we present compelling evidence that native ODC and SPR physically interact, and we defined the individual amino acid residues involved in both enzymes using in silico protein-protein docking simulations. The resulting heterocomplex is a surprisingly compact structure, featuring two energetically and structurally equivalent binding modes both in monomer and in dimer conformations. The novel interaction between ODC and SPR proteins was confirmed under physiological conditions by co-immunoprecipitation and co-localization in neuroblastoma (NB) cells. Importantly, we showed that siRNA (small interfering RNA)-mediated knockdown of SPR expression significantly reduced endogenous ODC enzyme activity in NB cells, thus demonstrating the biological relevance of the ODC-SPR interaction. Finally, in a cohort of 88 human NB tumors, we found that high SPR mRNA expression correlated significantly with poor survival prognosis using a Kaplan-Meier analysis (log-rank test, P=5 × 10(-4)), suggesting an oncogenic role for SPR in NB tumorigenesis. In conclusion, we showed that ODC binds SPR and thus propose a new concept in which two well-characterized biochemical pathways converge via the interaction of two enzymes. We identified SPR as a novel regulator of ODC enzyme activity and, based on clinical evidence, present a model in which SPR drives ODC-mediated malignant progression in NB.
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http://dx.doi.org/10.1016/j.jmb.2013.09.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4013099PMC
January 2014

AMXT-1501, a novel polyamine transport inhibitor, synergizes with DFMO in inhibiting neuroblastoma cell proliferation by targeting both ornithine decarboxylase and polyamine transport.

Int J Cancer 2013 Sep 30;133(6):1323-33. Epub 2013 May 30.

Center for Translational Medicine, Van Andel Research Institute, Grand Rapids, Michigan 49503, USA.

Neuroblastoma (NB) is associated with MYCN oncogene amplification occurring in approximately 30% of NBs and is associated with poor prognosis. MYCN is linked to a number of genes including ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. ODC expression is elevated in many forms of cancer including NB. Alpha-difluoromethylornithine (DFMO), an ODC inhibitor, is currently being used in a Phase I clinical trial for treatment of NB. However, cancer cells treated with DFMO may overcome their polyamine depletion by the uptake of polyamines from extracellular sources. A novel polyamine transport inhibitor, AMXT-1501, has not yet been tested in NB. We propose that inhibiting ODC with DFMO, coupled with polyamine transport inhibition by AMXT-1501 will result in enhanced NB growth inhibition. Single and combination drug treatments were conducted on three NB cell lines. DFMO IC50 values ranged from 20.76 to 33.3 mM, and AMXT-1501 IC50 values ranged from 14.13 to 17.72 µM in NB. The combination treatment resulted in hypophosphorylation of retinoblastoma protein (Rb), suggesting growth inhibition via G1 cell cycle arrest. Increased expression of cleaved PARP and cleaved caspase 3 in combination-treated cells starting at 48 hr suggested apoptosis. The combination treatment depleted intracellular polyamine pools and decreased intracellular ATP, further verifying growth inhibition. Given the current lack of effective therapies for patients with relapsed/refractory NB and the preclinical effectiveness of DFMO with AMXT-1501, this combination treatment provides promising preclinical results. DFMO and AMXT-1501 may be a potential new therapy for children with NB.
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http://dx.doi.org/10.1002/ijc.28139DOI Listing
September 2013

PRAF2 stimulates cell proliferation and migration and predicts poor prognosis in neuroblastoma.

Int J Oncol 2013 Apr 21;42(4):1408-16. Epub 2013 Feb 21.

Department of Pharmaceutical Sciences, College of Pharmacy, University of Hawaii at Hilo, Hilo, HI 96720, USA.

Prenylated Rab acceptor 1 domain family, member 2 (PRAF2) is a novel 19-kDa protein with four transmembrane-spanning domains that belongs to the PRAF protein family. Neuroblastoma (NB) is the most common malignant extracranial solid tumor of childhood that originates in primitive cells of the developing sympathetic nervous system. We investigated the correlation of PRAF2 mRNA expression to NB clinical and genetic parameters using Affymetrix expression analysis of a series of 88 NB tumors and examined the functional role of PRAF2 in an NB cell line using RNA interference. We found that high PRAF2 expression is significantly correlated to several unfavorable NB characteristics: MYCN amplification, high age at diagnosis, poor outcome and high INSS stage. The shRNA-mediated PRAF2 downregulation in the SK-N-SH NB cell line resulted in decreased cellular proliferation, migration and matrix-attachment. These findings were confirmed in NB patient tumor samples, where high PRAF2 expression is significantly correlated to bone and bone marrow metastasis, the main cause of death in NB patients. The present study shows that PRAF2 plays an essential role in NB tumorigenesis and metastasis.
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http://dx.doi.org/10.3892/ijo.2013.1836DOI Listing
April 2013

DFMO/eflornithine inhibits migration and invasion downstream of MYCN and involves p27Kip1 activity in neuroblastoma.

Int J Oncol 2013 Apr 21;42(4):1219-28. Epub 2013 Feb 21.

Department of Pharmaceutical Sciences, College of Pharmacy, University of Hawaii at Hilo, Hilo, HI 96720, USA.

Neuroblastoma (NB) is the most common extracranial pediatric tumor. NB patients over 18 months of age at the time of diagnosis are often in the later stages of the disease, present with widespread dissemination, and often possess MYCN tumor gene amplification. MYCN is a transcription factor that regulates the expression of a number of genes including ornithine decarboxylase (ODC), a rate-limiting enzyme in the biosynthesis of polyamines. Inhibiting ODC in NB cells produces many deleterious effects including G(1) cell cycle arrest, inhibition of cell proliferation, and decreased tumor growth, making ODC a promising target for drug interference. DFMO treatment leads to the accumulation of the cyclin-dependent kinase inhibitor p27(Kip1) protein and causes p27(Kip1)/Rb-coupled G(1) cell cycle arrest in MYCN-amplified NB tumor cells through a process that involves p27(Kip1) phosphorylation at residues Ser10 and Thr198. While p27(Kip1) is well known for its role as a cyclin-dependent kinase inhibitor, recent studies have revealed a novel function of p27(Kip1) as a regulator of cell migration and invasion. In the present study we found that p27(Kip1) regulates the migration and invasion in NB and that these events are dependent on the state of phosphorylation of p27(Kip1). DFMO treatments induced MYCN protein downregulation and phosphorylation of Akt/PKB (Ser473) and GSK3-β (Ser9), and polyamine supplementation alleviated the DFMO-induced effects. Importantly, we provide strong evidence that p27(Kip1) mRNA correlates with clinical features and the survival probability of NB patients.
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http://dx.doi.org/10.3892/ijo.2013.1835DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3622674PMC
April 2013

Activity enhancement of the synthetic syrbactin proteasome inhibitor hybrid and biological evaluation in tumor cells.

Biochemistry 2012 Aug 17;51(34):6880-8. Epub 2012 Aug 17.

University of Hawaii Cancer Center, 1236 Lauhala Street, University of Hawaii at Manoa, Honolulu, HI 96813, USA.

Syrbactins belong to a recently emergent class of bacterial natural product inhibitors that irreversibly inhibit the proteasome of eukaryotes by a novel mechanism. The total syntheses of the syrbactin molecules syringolin A, syringolin B, and glidobactin A have been achieved, which allowed the preparation of syrbactin-inspired derivatives, such as the syringolin A-glidobactin A hybrid molecule (SylA-GlbA). To determine the potency of SylA-GlbA, we employed both in vitro and cell culture-based proteasome assays that measure the subcatalytic chymotrypsin-like (CT-L), trypsin-like (T-L), and caspase-like (C-L) activities. We further studied the inhibitory effects of SylA-GlbA on tumor cell growth using a panel of multiple myeloma, neuroblastoma, and ovarian cancer cell lines and showed that SylA-GlbA strongly blocks the activity of NF-κB. To gain more insights into the structure-activity relationship, we cocrystallized SylA-GlbA in complex with the proteasome and determined the X-ray structure. The electron density map displays covalent binding of the Thr1O(γ) atoms of all active sites to the macrolactam ring of the ligand via ether bond formation, thus providing insights into the structure-activity relationship for the improved affinity of SylA-GlbA for the CT-L activity compared to those of the natural compounds SylA and GlbA. Our study revealed that the novel synthetic syrbactin compound represents one of the most potent proteasome inhibitors analyzed to date and therefore exhibits promising properties for improved drug development as an anticancer therapeutic.
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http://dx.doi.org/10.1021/bi300841rDOI Listing
August 2012

PEA15 impairs cell migration and correlates with clinical features predicting good prognosis in neuroblastoma.

Int J Cancer 2012 Oct 31;131(7):1556-68. Epub 2012 Jan 31.

Cancer Biology Program, University of Hawai'i Cancer Center, University of Hawai'i at Manoa, Honolulu, HI 96813, USA.

ERK and RSK2 drive proliferation and invasion of many cancers. Phosphoprotein enriched in astrocytes 15 (PEA15) binds ERK and RSK2 and high PEA15 levels can impair ERK- and RSK2-dependent transcription. PEA15 expression also inversely correlates with cell motility and invasiveness. We therefore tested PEA15 effects on neuroblastoma cells in vitro. We further analyzed PEA15 expression in the context of clinical and genetic features of neuroblastoma in tumor samples to determine its correlation with disease progression. Affymetrix microarray analysis was performed using 24 different neuroblastoma cell lines. Cell lines expressing low to intermediate levels of PEA15 were chosen for in vitro functional studies. The cell line results were verified by Affymetrix analysis of three different neuroblastic tumor types (total of 110 samples) PEA15 overexpression inhibited neuroblastoma migration in vitro. We verified that inhibition of motility required PEA15 interaction with its binding partners ERK and RSK2. Additionally, synthetic inhibitors of RSK2 suppressed integrin-dependent migration. PEA15 expression correlates with clinical parameters and a 25% increase in patient survival rate. The highest PEA15 levels were found in low stage, more differentiated and less metastatic neuroblastic tumors, and correlated with lack of MYCN amplification. PEA15 blocks neuroblastoma migration through inhibition of ERK/RSK2 signaling. PEA15 expression levels correlate with favorable clinical features suggesting that PEA15 limits metastatic progression of neuroblastoma. Thus, PEA15 and its partners ERK and RSK2 are potential targets for the development of new therapeutics to impede progression of minimal residual disease in patients with high-risk neuroblastoma.
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http://dx.doi.org/10.1002/ijc.27415DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3757132PMC
October 2012
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