Investigation of Neurochemicals' Role in Glioblastoma and Neural Stem Cells: Characterization of Dopamine Receptor D4 Inhibition in Glioblastoma Stem Cells

Neurochemicals that mediate synaptic communications between mature neurons are now appreciated to have a critical role in neurogenesis and cancer. Glioblastomas (GBM) grow in the rich neurochemical milieu of the brain and may depend on neurochemical signals for proliferation and survival. Given the close similarities between GBM stem cells and neural stem cells (NSC), and their existence in the rich neurochemical milieu of brain, neurochemical signaling may profoundly impact tumor growth. To test this hypothesis, I interrogated a library of 680 neuroactive compounds in patient-derived GBM neural stem cells (GNS) against normal human NSC and human fibroblasts cells to identify selective compounds. I found that compounds modulating dopaminergic, serotonergic and cholinergic signaling pathways selectively affected GNS and NSC growth compared to fibroblast cells. I identified ten compounds that showed more than 8-fold selectivity and in particular, dopamine receptor D4 (DRD4) antagonists (L-741,742 and PNU 96415E) selectively inhibited GBM growth in vitro and in vivo, and exhibited synergism with the current chemotherapeutic drug, temozolomide (TMZ). Primary GBM tumors and GNS cells expressed functional DRD4 receptor and antagonism of DRD4 led to disruption in its downstream effectors PDGFRβ/ERK1/2, and mTOR signaling. Furthermore, DRD4 antagonism disrupts endolysosmal function compromising the autophagy-lysosomal degradation pathway leading to autophagic flux inhibition with accumulation of autophagic vacuoles, autophagy specific substrate p62, cholesterol and ubiquitinated proteins, followed by G0/G1 cell cycle arrest and apoptosis. The identification of DRD4 antagonists as GNS selective compounds revealed autophagy-lysosomal system as a vulnerable and important system for GBM cell survival and suggests modulation of dopamine signaling may hold therapeutic potential. Interestingly, DRD4 antagonists L-741,742 and PNU 96415E were also identified as hits in the high content differentiation screen. I further characterized DRD4 antagonist induced differentiated cells, which showed VGLUT1 positive cells with increased expression of NERUOG2, CTIP2 and ETV1 identifying them as glutamatergic cortical deep layer V neurons. Finally, DRD4, when inhibited in normal NSC, initiate differentiation, while in GNS cells, they are vulnerable and undergo cell death.Ph.D