Medulloblastoma

DNA methylation data of 12 human medulloblastoma tumors (subgroup 4) and eight human normal controls were taken from Hovestadt et al. (2014) (link). The qualitative analysis of the WGBS data set with 12 versus eight samples (22,524,970 data points without missing values) was restricted to Chromosome 10 with a total of 1,111,583 methylation data points. All segmentation tools were run in their default settings; and for MOABS, the maximal distance between two consecutive DMCs to be considered in a DMR (maxDistConsDmcs option) was set to 300 nt to be comparable to metilene and BSmooth. DMRs were required to contain at least 10 informative CpGs, i.e., CpGs with an associated methylation rate, and an absolute methylation difference (average MDS) larger than 0.1. As described above, outputs of MOABS and metilene were filtered with the critical P-value 0.05. For the analysis of promoter downstream correlating regions (pdCRs), the full set of pdCRs published in Hovestadt et al. (2014) (link) was downloaded and restricted to Chromosome 10. For each pdCR, the difference between the average methylation of the tumor samples and the average methylation of the control samples was calculated using BEDTools (Quinlan and Hall 2010 (link)).

Primary striatal neurons were isolated from ICR mice at embryonic day 17 as previously described (Fitting et al., 2014 (link); Zou et al., 2011 (link)). Briefly, striatal tissues were dissected, minced, and incubated for 30 min at 37^oC in neurobasal medium (NBM) containing 0.015 mg/mL DNase and 2.5 mg/ml trypsin. NBM was supplemented with 25 mM glutamate, 0.5 mM glutamine, B27 (Invitrogen), and an antibiotic-antimycotic solution (Sigma). Cells were then triturated, filtered (2×) through a 70 μm-diameter pore membrane, and seeded into six-well plates (15   ×   10⁵ neurons/well) pre-coated with poly-L-lysine. Cells were maintained in NBM supplemented at decreasing concentrations of glutamate (days 1–4, 25 mM; days 5–6, 12.5 mM; days 7–11, 0 mM) at 37^oC in a 5% CO₂ environment until they matured (at 11 days). Based on our findings that TDP-43 phosphorylation was greatest following co-exposure to morphine and Tat in murine brain tissues, and to optimize detecting alterations in TDP-43 phosphorylation, we co-exposed cells to Tat and morphine to verify the involvement of CK2 in the pathologic TDP-43 phosphorylation. Cells were either treated with vehicle (DNase/RNase-free non-pyrogenic Ultrapure water), co-treated with Tat (100 nM) and morphine (500 nM), or a combination of Tat, morphine, and 0.5, 1, or 2 µM concentrations of the highly selective CK2 antagonist CX-4945 (#A11060, AdooQ Bioscience, Irvine, CA). CX-4945 (Silmitasertib) was granted Orphan Drug Designation by the US FDA in December 2021 for the treatment of medulloblastoma and potentially other rare diseases targeting CK2. Vehicle-treated cells served as controls. The selected concentrations of Tat and morphine do not affect cell viability within 24 h (Zou et al., 2011 (link)). The doses of CX-4945 selected for testing are those previously determined to reduce pro-inflammatory mediators to baseline levels in immune cells (Jang et al., 2017 (link)). Treated neurons were incubated at 37^oC in a 5% CO₂ environment for 24 h. Afterward, the cells were harvested, and the cytoplasmic and nuclear fractions were extracted using a NE-PER nuclear and cytoplasmic extraction kit (Thermo Fisher). BCA was used to determine protein concentrations and samples were stored in aliquots at −80^oC until use.