The Radiosensitizing Effects of a Novel Tyrosine Kinase Inhibitor, Mp470 in Glioblastoma Multiforme Stem Cells

Purpose/Objective(s) 

Despite our most aggressive treatment strategies, using dose escalation >100 Gy, the majority of the GBMs will recur within 2 cm of the postoperative bed. Cancer stem cells (CSC) has been identified as contributing to this resistance, which is supported by our previous work showing an inverse correlation between the % CSC and survival supporting the importance of targeting this unique population. CSC over express several genes including c-Met.1 A novel multi-targeted tyrosine kinase inhibitor, MP470 which targets c-Met, has been shown to be a potent radiosensitizer in GBM cell lines which is mediated through reduced double strand DNA repair. In this abstract, we set out to evaluate if MP470 would also have radiosensitizing effects in GBM CSC.

Materials/Methods 

Resected GBM cells were collected in an IRB approved protocol; the cells were disassociated and the non-adherent ‘neurospheres’ were collected and cultured using established stem cell protocols. Fluorescent microscopy was performed to verify expression of CSC markers. All of the results listed in this abstract were from a single patient's stem cells. MTS assay was used to determine cytotoxicity of TMZ and MP470. Neurosphere colonies assay was developed to evaluate the clonogenic potential of a single stem cell to develop into mature neurospheres and were performed in duplicate and counted on day 14. Apoptosis was assessed using Acridine Orange and Ethidium Bromide, 12 hours post-XRT (4 Gy).

Results 

Our neurospheres expressed c-Met, Nestin, CD133, and developed invasive brain tumors in xenograft cranial models. MTS was used to determine the IC50 of these tumor stem cells, for TMZ no IC50 was reached despite doses up to 10μM, while MP470 had an IC50 of 3μM. In clonogenic assays, the surviving fraction of cells was 75% with 4 Gy XRT, 63% with 5 μM MP470 and 30% with 5μM MP470 + XRT. When we evaluated for cause of cell death, XRT alone produced a 7.5% rate of apoptosis compared to 10.5% for 10 μM TMZ + XRT and 20% for 10 μM MP470 + XRT.

Conclusions 

GBM CSC can be readily derived from freshly resected patient specimens that express stem cell markers and display invasive characteristics, in contrast to established GBM cell lines. Given CSC inherent resistance to conventional therapies they may be a useful tool in evaluating potential new therapies for treating GBM. Since GBM CSC have been shown to over express c-Met we evaluated the c-Met inhibitor MP470 ability to act as a radiation sensitizer. Compared to TMZ, MP470 had a low IC50 of 3μM and resulted in reduced neurosphere formation when used concomitantly with XRT. When cause of death was evaluated MP470 + XRT had twice the % of apoptotic death compared to TMZ + XRT.

References:

1. HS Gunther et al., Glioblastoma-derived stem cell-enriched cultures form distinct subgroups. Oncogene 2007:1-13.