CBIO-23. CHARACTERIZATION OF BMP SYSTEM MUTATIONS IN RECURRENT PRIMARY GLIOBLASTOMA PRIMARY CELL CULTURES IN RELATION TO GLIOBLASTOMA INITIATING STEM-LIKE CELLS AND TEMOZOLOMIDE RESISTANCE

Abstract

Glioblastoma multiforme (GBM) is the most prevalent and aggressive primary brain tumor. Its recurrence and rapidly acquired resistance to current therapies, including treatment with temozolomide (TMZ), results from the rapid expansion of re-emerging Glioblastoma Tumor Initiating stem cell-like Cells (GTICs). Targeted Exome Sequencing (TES) revealed that in such recurrent human GBM missense mutations arise in genes of the Bone Morphogenetic Protein (BMP) system in addition to those of other molecular systems found in primary GBM. To determine in which subpopulation BMP system mutations accumulate, we subjected two GBM primary cultures with a known mutations in ZFYVE16 (A290S) or BMPR1B (K222E) to targeted sequencing after FAC-sorting these cells based on the acknowledged stem cell markers CD44 and CD133. We found that ZFYVE16 (A290S) and BMPR1B (K222E) mutations accumulate within the CD133+/CD44- GTICs. Treatment of the primary culture with the latter mutation, with BMP4 suggests that the response to BMP signals is markedly reduced. Cells from these cultured were also cultured again after FAC-sorting for CD133 and CD44. Surprisingly, after three passages all subpopulations acquired a variation in expression of these markers specific for each patient. This shows that a 2D culture is able to loose and acquire stem cell markers and might resemble the original heterogeneity of the parental tumor more closely than we originally expected. In a separate set of experiments, RNA-sequencing of a TMZ-resistant primary GBM culture (GS295), created during the course of this work, compared to the TMZ-sensitive GS295, shows that several genes of the BMP system (NOGGIN, BMP7, ID1 and GATA3) are differentially expressed. Our results suggest that BMP system mutations tend to accumulate in CD133+ GTICs, that BMP signaling is altered within GBM primary cultures and that several mutated genes of the BMP system may correlate with acquisition of TMZ resistance.