Ornithine decarboxylase as a therapeutic target for endometrial cancer

<div><p>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 <i>ODC1</i> 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. <i>ODC1</i> 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 <i>ODC1</i> 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.</p></div

<i>ODC1</i> expression in endometrial cancers.

<p>A-C in LCM (laser capture micro-dissected) samples. <b>A</b>, Endometrioid (E, n = 139) and Serous (S, n = 37) types and normal epithelial tissues (N, n = 12); <b>B</b>, FIGO Stages I and II (n = 133), III (n = 24), and IV (n = 18); <b>C</b>, Grade 1 (G1, n = 42), Grade 2 (G2, n = 65), and Grade 3 (G3, n = 69). <u>Signal: Affymetrix signal normalized to target value of 500.</u> <b>D</b>, In TCGA samples of four molecular sub-types: Copy number low (MSS, n = 90), MSI high (n = 65), POLE ultra-mutant (n = 17) and Copy number high—serous like (n = 60). ***p < 0.001; **p < 0.01; *p< 0.05. <u>Counts: Normalized RNA-seq counts.</u></p

Colony formation, cell viability and polyamine levels in DFMO-treated endometrial cancer cells.

<p><b>A</b>, Colony counts in a panel of endometrial cancer and immortal normal endometrial epithelial cells including ACI-98 (undifferentiated), MSU-15 (clear cell), ACI-61 (endometrioid) ACI-70 (MMT), HEC-1-A (endometrioid), EM E6/E7 TERT1 (normal immortalized endometrial epithelial), ECC-1 (endometrioid) and ACI-45 (carcinosarcoma, MMT). Cells are ordered most highly sensitive on left to least sensitive on the right. <b>B</b>, Representative colony plates for most (ACI-98) and least (ACI-45) sensitive cells. <b>C</b> and <b>D</b>, ACI-98 cells highly sensitive to wide range of doses of DFMO while ACI-45 cells are non-sensitive even at very high doses (5 mM) as measured by MTS assay. The percent values (%) shown are DFMO to Control. Intracellular polyamine levels (putrescine, spermidine, spermine) were analyzed in ACI-98 (<b>E</b>) and ACI-45 (<b>F</b>) cells either untreated (control) or DFMO-treated (0.25 mM) using RP-HPLC. Intracellular polyamines were quantified and expressed as nmol/mg protein. Standard errors are indicated (+/- S.E.). Put, putrescine; Spd, spermidine; Spm, spermine.</p

Effect of oral DFMO on polyamine levels in tumor-bearing mice.

<p>Intracellular polyamine levels (putrescine, spermidine, spermine) were analyzed in tumor tissue (<b>A</b>) and blood plasma (<b>B</b>) of DFMO-treated and untreated mice using reverse-phase HPLC. Intracellular polyamines were quantified and expressed as nmol/mg protein (<b>A</b>) or nmol/ml plasma (<b>B</b>). Standard errors are indicated (+/- S.E.). Put, putrescine; Spd, spermidine; Spm, spermine.</p

<i>ODC1</i> mRNA expression overall survival (OS) and recurrence free interval (RFI).

<p>Kaplan-Meier diagrams and Wald statistics showing increased <i>ODC1</i> is significantly associated with shorter OS (<b>A</b>) in TCGA (RNA-seq) (n = 232), LCM cases (Affymetrix) (n = 188) and in Spectrum (qRT-PCR) (n = 60) samples (<b>B</b>) <i>ODC1</i> mRNA is significantly associated with recurrence for TCGA and qRT-PCR cohorts but is not significant in the Affymetrix LCM dataset. “<u>High” and “Low” indicate above and below the median expression level.</u></p

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

<div><p>Background</p><p>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.</p><p>Methods and Findings</p><p>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).</p><p>Conclusions</p><p>DFMO doses of 500-1500mg/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.</p><p>Trial Registration</p><p>Clinicaltrials.gov <a href="https://clinicaltrials.gov/ct2/show/NCT01059071" target="_blank">NCT#01059071</a></p></div

NMTRC002 CONSORT Flow Diagram- modified for non-randomized trial design.

<p>NMTRC002 CONSORT Flow Diagram- modified for non-randomized trial design.</p

Characteristics of patients enrolled in NMTRC 002.

<p>Characteristics of patients enrolled in NMTRC 002.</p

Progression free survival (PFS) and overall survival (OS) rates in patients enrolled in NMTRC 002 (N = 21).

<p>The number of patients shown at risk for disease progression (PFS) or death (OS) is shown in the figure.</p

Rank-ordered PFS by DFMO dose, ODC genotype and urinary polyamines.

<p>*PF = progression free, PD = progressive disease</p><p>2^nd Leukemia = secondary leukemia</p><p>**Substrates for the tissue polyamine exporter SLC3A2 include the sum of putrescine, N1AcSpd, N8AcSpd and DAS; D1C1 = day 1, cycle 1, D8C1 = day 8 cycle 1</p><p>***NA = samples not available</p><p>Rank-ordered PFS by DFMO dose, ODC genotype and urinary polyamines.</p