M. H. Erwin1, C. H. Quinn1, R. Marayati1, H. R. Markert1, A. M. Beierle1, L. V. Bownes1, J. R. Julson1, S. C. Hutchins1, J. E. Stewart1, M. Ohlmeyer2, G. K. Friedman3, E. A. Beierle1 1University Of Alabama at Birmingham, Division Of Pediatric Surgery, Department Of Surgery, Birmingham, Alabama, USA 2Atux Iskay LLC, Plainsboro, NJ, USA 3University Of Alabama at Birmingham, Division Of Hematology/Oncology, Department Of Pediatrics, Birmingham, Alabama, USA
Introduction: Medulloblastoma is the most common primary pediatric nervous system malignancy. These tumors are classified into four groups: WNT, SHH, Group 3 and Group 4. Group 3 tumors account for nearly one third and portend the worst prognosis with less than 50% of children surviving 10 years. Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase that functions as a tumor suppressor but is downregulated in medulloblastoma. Researchers have previously shown that activation of PP2A decreased medulloblastoma tumor growth. Our collaborator created second-generation PP2A activating compounds, ATUX-6954 and ATUX-6156, designed to more efficiently activate PP2A. We hypothesized that these molecules would decrease viability, proliferation, and stemness in Group 3 medulloblastoma.
Methods: D425 human Group 3 medulloblastoma patient derived xenograft (PDX) cells were utilized. D425 cells were treated with increasing doses of ATUX-6954 and ATUX-6156. Viability and proliferation were measured using colorimetric assays. Immunoblotting examined the effects of ATUX-6954 and ATUX-6156 on downstream cell signaling cascades. Extreme limiting dilution analysis (ELDA) evaluated tumorsphere forming ability, a measure of stemness, after treatment for one week with ATUX-6954 (1.5 µM) and ATUX-6156 (1.5 µM).
Results: Treatment of D425 cells with ATUX-6954 for 24 hours significantly decreased viability (LD50 3.9 µM) and proliferation (IC50 5.3 µM). ATUX-6156 treatment similarly significantly decreased viability (LD50 2.8 µM) and proliferation (IC50 3.2 µM) at 24 hours. Increasing doses of ATUX-6954 and ATUX-6156 led to a decrease in AKT phosphorylation. In addition, ATUX-6954 decreased expression of two endogenous PP2A inhibitors, SET and CIP2A. ELDA showed a significant decrease in D425 tumorsphere formation after ATUX-6954 (p < 0.0001) and ATUX-6156 (p < 0.0001) treatment, indicating decreased stemness (Fig 1).
Conclusion: We have shown that two PP2A activating compounds, ATUX-6954 and ATUX-6156, decreased Group 3 medulloblastoma PDX cell viability and proliferation, and that dephosphorylation of AKT may be the responsible mechanism. In addition, ATUX-6954 and ATUX-6156 decreased tumor cell stemness, a quality known to drive tumor resistance and relapse. These results indicate that ATUX-6954 and ATUX-6156 should be investigated further as potential medulloblastoma therapeutics.
