Aberrant DNA Methylation Is Associated with Reduced Response to SRC Inhibition in Primary Human Vestibular Schwannoma Cells

Background: Neurofibromatosis type II (NF2) is a hereditary tumor disorder that predisposes individuals to develop multiple central nervous system tumors, particularly bilateral vestibular schwannoma (VS) that can lead to deafness, imbalance and other neurological sequelae. Treatment of NF2-associated VS requires a delicate balance between reduction of tumor burden and preservation of neurological function. The limitations of microsurgery and radiosurgery in NF2 have resulted in the off-label use of FDA-approved chemotherapeutic drugs, which in clinical trial have not been effective in improving hearing and reducing tumor burden in all patients. The genetic and epigenetic heterogeneity of VS may be important factors that predict drug response in NF2 individuals.

Objective: Identify DNA methylation patterns associated with reduced response to dasatinib (SRC inhibitor) in primary human VS cultures in vitro.

Methods: Fresh tumor was obtained from seven patients undergoing VS surgery at University of Miami/Jackson Memorial Hospital. NF2 mutational analysis was performed using multiplex ligation-dependent probe amplification (MLPA). Dissociated primary human VS cells were cultured, and crystal violet assays to determine cell viability were performed after exposing cells to vehicle and two different concentrations of dasatinib for 3 days in vitro. High-throughput DNA methylation analysis was performed for seven human VS tumors using the Infinnium Methylation EPIC Kit.

Results: All VS demonstrated NF2 mutations on MLPA. Five VS (71%) demonstrated a dose-dependent response and showed >20% reduction in cell viability at the highest concentration of dasatinib tested. Dasatinib did not significantly reduce viability in two VS (29%). In the two nonresponding VS, DNA methylation analysis identified differentially methylated regions (DMR) associated with non-SRC merlin pathways as well as hypermethylation of a CpG site in the promoter region of the ELMOD1 (engulfment and cell motility domain 1) gene, which confers greater tumor aggressiveness and drug resistance.

Conclusion: SRC inhibition with Dasatinib did not effectively reduce cell viability in all primary human VS. Nonresponding VS demonstrated aberrant methylation in CpGs associated with non-SRC pathways and hypermethylation in the promoter region of ELMOD1 gene, which provide important information on the potential mechanisms of drug resistance in this tumor disorder. Further investigations into the genetic and epigenetic factors that modulate drug resistance in VS and NF2 are initial steps in developing a precision medicine algorithm for selecting best therapies for individual patients.

No conflict of interest has been declared by the author(s).