Introduction: Different combinations of three driver mutations localized respectively to the p53, Rb and receptor tyrosine kinase networks are responsible for selective growth advantage of glioblastoma tumors (GBM) and represent significant intra-tumoral heterogeneity, a major cause of treatment failure. microRNA-34a (miR-34a) is downregulated in GBM, and miR-34a modulates the expression of multiple genes in all three deregulated GBM networks, suggesting it could serve as a novel therapeutic agent.
Methods: The effects of miR-34a transfection were studied in three primary patient-derived lines (GBM 6, GBM118 and GBM 126, respectively belonging to classical, mesenchymal and proneural subtypes), four established cell lines (T98G, U251, A172, LN229) and two cell lines with acquired resistance to temozolomide (A172-TR, LN229-TR) in vitro. GBM6 orthotopic nude mouse model was used for in vivo studies. Nanocells (400 nm diameter) derived from genetically modified bacteria, were provided with a bispecific antibody targeting EGFR and loaded with miR-34a. Nanocells were injected intravenously while temozolomide was administered by oral gavage. Treatment response was quantified by measuring tumor growth (qBLI) and animal survival.
Results: Transfection with miR-34a mimics significantly reduced proliferation in all cell lines and sensitized to temozolomide treatment, regardless of temozolomide resistance. Moreover, all cell lines examined were sensitized to radiation with dose enhancement factors ranging from 1.7-2.2. We identified broadly conserved binding sites in the 3’UTR of multiple mRNAs in the GBM deregulated networks, and validated the direct downregulation of Bcl-2 protein as a major contributor to temozolomide sensitization. We successfully delivered miR-34a to orthotopic implanted tumors after systemic intravenous administration and observed significant reduction in tumor growth (p=0.021) and increased survival (p<0.001). Importantly, miR-34a nanocells also sensitized to temozolomide therapy in vivo.
Conclusions: miR-34a packaged in nanocells is a promising novel therapeutic that inhibits glioblastoma tumor growth and sensitizes to temozolomide and radiation.
Patient Care: Glioblastoma is a uniformly fatal disease with a mean overall survival of 14.5 months. The survival trends haven’t improved significantly over the last decade and there is a dire need for novel therapeutics. Intra-tumoral heterogeneity remains a significant challenge to develop effective therapeutics. Further, no appreciable survival enhancing drugs exist for radiation and temozolomide resistant and primary gliomas and recurrent gliomas. miR-34a can counteract heterogeneity by targeting multiple oncogenic pathways and as demonstrated by our data, can synergize with existing therapies. Thus, miR-34a loaded nanocells can significantly enhance progression free and overall survival in patients afflicted with GBM.
Learning Objectives: 1. miRNAs are potential therapeutic agents to counteract intra-tumor heterogeneity
2. miR-34a inhibits GBM cell proliferation and also sensitizes to both temozolomide and radiation therapy via multiple mechanisms
3. Nanocells can be used as delivery vehicles for miRNA to brain tumor