78.19 Novel Rexinoids Decrease Cancer Stemness in Neuroblastoma Patient Derived Xenografts

Posted on January 18, 2018

A. P. Williams1, L. L. Stafman1, J. Aye1, V. R. Atigadda4, J. E. Stewart1, C. Grubbs2, D. Muccio3, K. J. Yoon5, K. Whelan6, E. A. Beierle1  1University Of Alabama at Birmingham,Pediatric Surgery,Birmingham, Alabama, USA 2University Of Alabama at Birmingham,Surgery,Birmingham, Alabama, USA 3University Of Alabama at Birmingham,Chemistry,Birmingham, Alabama, USA 4University Of Alabama at Birmingham,Dermatology,Birmingham, Alabama, USA 5University Of Alabama at Birmingham,Pharmacology And Toxicology,Birmingham, Alabama, USA 6University Of Alabama at Birmingham,Pediatrics,Birmingham, Alabama, USA

Introduction:  Current treatments for high risk neuroblastoma (NB) include 13-cis-retinoic acid (RA), but nearly half of children treated with RA develop disease recurrence. A subgroup of NB cells expressing the cell surface marker CD133 have been identified that have stem cell-like qualities and are associated with poor prognosis and disease relapse. These stem cell-like cancer cells demonstrate self renewal, avoid apoptosis, and are therefore promising targets for therapy. Recently, two novel rexinoids, UAB116 and 7-Me-UAB30, were developed to meet the goals of improved efficacy and decreased toxicity profiles over those of RA. We hypothesized that these novel rexinoids would affect NB cells in a fashion similar to that seen with RA, and may be used to target NB cancer stem-like cells. 

Methods: Using 2 NB patient derived xenografts (PDXs), COA3 and COA6, UAB116 and 7-Me-UAB30 were evaluated along with RA. Following 72-hour treatment, cell viability was measured using alamarBlue, proliferation was assessed with CellTiter96 assays and fluorescence-associated cell sorting (FACS) analysis was used to detect CD133 expression. Extreme limiting dilution assay (ELDA) was performed to determine the compounds’ impact on tumorsphere formation both in bulk and sorted cell populations. Student’s t-test, extreme limiting dilution analysis, and χ 2 statistics were used with mean ± standard error of the mean reported and p<0.05 significant.

Results: AlamarBlue demonstrated decreased viability following treatment with both UAB116 and 7-Me-UAB30 that was comparable to that seen with RA (Figure) and similar results were obtained for proliferation. Using FACS, we found that CD133 expression decreased following treatment with UAB116 (16% to 5%, treated vs. control, p=<0.001) and 7-Me-UAB30 (16% to 6%, treated vs. control, p=<0.001). Tumorsphere formation was diminished significantly in the NB PDX cells treated with UAB116 or 7-Me-UAB30, decreasing tumorsphere formation in the 1000 cell/well group from 75% to 18% and 42% respectively (p<0.001). When sorted based on CD133 expression, the sphere forming capacity of the CD133 enriched population was significantly decreased after treatment with either compound from 100% in the 500 cell/well group to 58% when treated with UAB116 and 75% when treated with 7-Me-UAB30 (p<0.001).

 

Conclusion: The novel rexinoids, UAB116 and 7-Me-UAB30, affected NB PDX tumor cell survival and stemness. Treatment resulted in decreased cell viability and proliferation, decreased CD133 expression, and decreased tumorsphere formation. These findings indicate that these compounds should be investigated further as potential novel therapeutics for NB.   
 

2.20 Preclinical Evaluation of Novel Retinoic Acid Derivatives in Neuroblastoma

Posted on January 18, 2018

A. J. Lazenby1, A. P. Williams1, L. L. Stafman1, J. Aye1, V. R. Atigadda2, J. Stewart1, D. D. Muccio4, C. Grubbs3, E. A. Beierle1  2University Of Alabama at Birmingham,Dermatology,Birmingham, Alabama, USA 3University Of Alabama at Birmingham,Surgery,Birmingham, Alabama, USA 4University Of Alabama at Birmingham,Chemistry,Birmingham, Alabama, USA 5University Of Alabama at Birmingham,Pharmacology And Toxicology,Birmingham, Alabama, USA 6University Of Alabama at Birmingham,Pediatrics,Birmingham, Alabama, USA 1University Of Alabama at Birmingham,Pediatric Surgery,Birmingham, Alabama, USA

Introduction:  Neuroblastoma (NB), a tumor derived from neural crest cells, is the most common extracranial solid tumor in children. 13-cis-retinoic acid (RA) is a differentiating agent currently utilized in therapy for high risk NB, but its use is limited by toxicities related to cholesterol and lipid metabolism. A synthetic rexinoid, 9-cis-UAB30 (UAB30), has been developed which has a favorable toxicity profile, demonstrating no significant toxicities in animal or human studies. Our lab has shown that UAB30 decreased NB cell proliferation and tumor growth in murine NB models. While UAB30 is promising, a reduction in drug dosage would be ideal to decrease the risk of potential side effects. Therefore, the aim of this project was to initiate pre-clinical evaluation of 9 new, synthetic rexinoid compounds (UAB111, UAB113, UAB114, UAB115, UAB116, UAB125, UAB126, 5-Me-UAB30 and 7-Me-UAB30) designed to have greater potency than that of UAB30 or RA. We hypothesized that these compounds would affect NB survival and differentiation in a manner comparable to RA and UAB30.

Methods:  Four NB cell lines were utilized: 2 MYCN non-amplified (SK-N-AS, SH-EP) and 2 MYCN amplified [SK-N-BE(2), WAC2]. The effect of the rexinoids on cell viability and differentiation were evaluated using alamarBlue® assay and assessment of neurite outgrowth, respectively. Cells were treated for 72 hours at increasing concentrations ranging from 0 to 100 µM. Data were compared using Student’s t-test or ANOVA as appropriate and reported as mean ± SEM with p≤0.05 considered statistically significant.

Results: The 9 new rexinoids were tested against compounds previously studied, RA and UAB30, in all four NB cell lines. The SK-N-AS cell line was the most sensitive to the novel compounds, showing significantly decreased viability after treatment with 5 of them (UAB111, UAB113, UAB114, UAB116, 7-Me-UAB30) compared to RA or UAB30 (Figure, upper panel).  The remaining cell lines, SK-N-BE(2), SH-EP and WAC2 demonstrated results similar to each other and had the most significant changes in viability with UAB116 and 7-Me-UAB30 (Figure, lower panel), so these 2 rexinoids were utilized for further studies. Neurite outgrowths are a measure of NB cell differentiation. Treatment with UAB116 or 7-Me-UAB30, resulted in a statistically significant increase in neurite outgrowths in both the SH-EP and SK-N-AS cell lines, indicating differentiation.

Conclusion: Treatment with these synthetic rexinoids, particularly UAB116 and 7-Me-UAB30, decreased NB cell viability significantly more than RA or UAB30. In addition, they led to NB cell differentiation. These data indicate that UAB116 and 7-Me-UAB30 should be further evaluated as potential novel therapeutics for NB.