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

Abstract

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 IC 50 values ranged from 20.76 to 33.3 mM, and AMXT-1501 IC 50 values ranged from 14.13 to 17.72 mM in NB. The combination treatment resulted in hypophosphorylation of retinoblastoma protein (Rb), suggesting growth inhibition via G 1 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.Neuroblastoma (NB) is the most common extracranial solid pediatric tumor, accounting 8-10% of all pediatric cancers and for 15% of cancer-related deaths in children. 1 Approximately 650 new cases of NB arise in the United States each year. Of the children diagnosed, roughly 70% have disease that has already metastasized to other parts of the body. Children with high-risk NB have a long-term survival of less than 50%, despite the use of intensive multimodality therapy. 2 Of the children with relapsed or refractory disease, there are no known curative measures and the 5-year survival is less than 10%. Biomarkers are powerful tools for determining diagnosis and prognosis for different cancers, including NB. The oncogenic transcription factor MYCN is amplified in roughly 30% of all NBs and is generally associated with high-risk disease and poor survival. 3,4 As a transcription factor, MYCN induces and represses a large number of genes involved in multiple biological processes including cell growth and differentiation. However, the genes necessary or sufficient to initiate neuroblastoma tumorigenesis downstream of MYCN remain to be established. 5 Ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis in mammalian cells, is directly activated by c-MYC and MYCN 6-8 and is overexpressed in NB. 9-11 ODC decarboxylates t...

N-ω-chloroacetyl-l-ornithine, a new competitive inhibitor of ornithine decarboxylase, induces selective growth inhibition and cytotoxicity on human cancer cells versus normal cells

“…The EC 50 of DFMO varies in the millimolar range after 48-144 h (2-6 d) of treatment. For example, after a 48 h treatment the EC 50 was 3.0 mM in L1210 cells (mouse leukemia) 55 , 0.5 mM in L5178 cells (mouse lymphoma) 55 , 2.5 mM in B16 cells (mouse melanoma) 56 and 33.3 mM in BE(2)-C cells (human neuroblastoma) 57 . On the other hand, the EC 50 was higher than 0.5 mM after 72 h treatment of LN229 cells (human glioblastoma) 58 .…”

Targeting polyamine metabolism for cancer therapy and prevention

“…One efficient transport inhibitor, AMXT 1501 (Figure 2), has demonstrated significant synergy with DFMO in breast, prostate, and melanoma cell lines in addition to an ODC overexpression model of squamous cell carcinoma of the skin [57]. More recently, impressive results were obtained when this compound was combined with DFMO in neuroblastoma, a tumor type in which DFMO alone has shown some promise [58]. Another interesting application of what is currently being termed ‘polyamine-blocking therapy’ (PBT) is the goal of reducing T-cell immune repression, thus enhancing the antitumor immune response [59].…”

Chemoprevention of Prostate Cancer with the Polyamine Synthesis Inhibitor Difluoromethylornithine

“…Exciting new work with polyamine transport inhibitors has also refocused attention on the polyamine pathway (Samal et al 2013). Negative results in the large PCPT and SELECT trials (Thompson et al 2003;Algotar et al 2013) as well as the failure of toremifine and 1 a-hydroxyvitamin D2) (Gee et al 2013;Taneja et al 2013) in HGPIN has also refocused attention on DFMO.…”