Despite significant research and clinical efforts, survival of patients with most common and aggressive primary brain tumor glioblastoma (GBM) has only marginally changed over the past 25 years and is only around fifteen months. There is, therefore, an urgent need for new molecular targets, concepts and approachestotreatingGBM.Intherecentyears,microRNA(miRNA)emergedasanewandpromisingclass oftargetablemoleculesthatholdsgreatpotentialinoncology.OurworkononcogenicmiRNAsledustofocus on miR-10b, a unique miRNA highly expressed in all GBM subtypes but absent in normal neuroglial cells of the brain. miR-10b controls the growth of malignant gliomas;? moreover, it appears essential for viability of heterogeneousgliomacellsandglioma-initiatingstemcells(GSC).InhibitionorlossofmiR-10bis,therefore, lethal for glioma. Our preliminary data suggests that, among several targeting approaches, CRISPR-Cas9- based miR-10b gene ablation eradicates established intracranial GBM in mice more effectively. Importantly, despite the fundamental addictive role of miR-10b in GBM, and the potential of its targeting, molecular mechanismsunderlyingmiR-10bactivityarepoorlyunderstoodandlargelyunconventional.Theoverallgoal of this proposal is, therefore, two-fold: first, to investigate molecular mechanisms underlying GBM dependence on miR-10b, and second, develop efficient therapeutic strategies based on miR-10b gene editing. Elucidation of the signaling pathways regulated by miR-10b, and development of approaches for its therapeutic targeting will have an impact on both basic and clinical neuro-oncology, and cancer more generally. The work for Specific Aim 1 will characterize endogenous miR-10b interactome and identify principalmiR-10btargetsingliomaandgliomastemcells.Usingacombinationoffunctionalassays,CLEAR- CLIP and CRISPR-Cas9-based technologies, and quantitative proteomics, we will define physiologically important protein-coding and non-coding targets underlying GBM addiction to miR-10b.
Specific Aim 2 will investigate the effects of CRISPR-Cas9-based miR-10b gene editing on growth and invasion of orthotopic glioma.UsinghumanGSC-derivedxenograftsandimmunocompetentGL261orthotopicGBMmousemodels, as well as primary normal neuroglial cells, we will examine efficacy and safety of various engineered CRISPR-Cas9systemsandviralvehiclesfortargetedmiR-10bablationinGBM.Wewillfurthercompareoff- target effects of antisense oligonucleotide based and gene editing based targeting strategies. The proposed work promises to yield significant new insights into the biology of malignant gliomas and may lead to the developmentofnew,common,andpotenttherapiesforallGBMsubtypes.
There is a critical need for new molecular targets, concepts, and approaches to treating glioblastoma, highly malignant primary brain tumor. The proposed work will investigate microRNA-10b, a key regulator of glioblastoma growth, and develop miR-10b gene editing approaches. It holds promise to advance our understanding of both microRNA biology and brain tumors, and may open the door for new therapies.