Brahmbhatt, Himanshu

Abstract BACKGROUND Glioblastomas show marked intra- and inter-tumor heterogeneity and are strongly resistant to both radio- and chemo-therapy, which are standard therapeutic modalities for this tumor. MicroRNAs (miRNAs) have the potential to serve as effective therapeutics for glioblastoma as they modulate the activity of multiple signaling pathways. METHODS Glioblastoma cultures were transfected with miR-34a or control miRNA mimics to assess biological function and therapeutic potential in vitro. miR-34a was packaged into bacterially-derived nanocells and administered intravenously for delivery to orthotopic patient-derived glioblastoma xenografts in mice. RESULTS Overexpression of miR-34a strongly reduced the activation status of the three core signaling networks that have been found to be deregulated in the vast majority of glioblastoma tumors, the receptor tyrosine kinase, p53 and Rb networks. miR-34a transfection also inhibited the survival of multiple established glioblastoma cell lines as well as primary patient-derived xenograft cultures representing the proneural, mesenchymal and classical subtypes. Transfection of miR-34a synergized with temozolomide (TMZ) in in vitro cultures of glioblastoma cells with primary TMZ sensitivity, primary TMZ resistance and acquired TMZ resistance. Intravenous administration of bacterially-derived nanocells carrying miR-34a strongly enhanced TMZ sensitivity in an orthotopic patient-derived xenograft mouse model of glioblastoma. CONCLUSIONS miR-34a strongly sensitizes a wide range of glioblastoma cell cultures to TMZ, suggesting that combination therapy of TMZ with miR-34a may serve as a novel therapeutic approach for the treatment of glioblastoma tumors. Bacterially-derived nanocells are an effective vehicle for the delivery of miR-34a to glioblastoma tumors.

Abstract Therapeutic resistance stemming from inter and intra-tumoral heterogeneity is a significant impediment towards development of effective therapeutics for glioblastoma. We hypothesized that microRNAs can potentially counteract resistance emanating from such heterogeneity as they simultaneously modulate the expression of multiple proteins. We identified microRNA-34a as a unique microRNA which modulates multiple oncoproteins in GBM using two different in silico approaches. We investigated the therapeutic effects of microRNA-34a in three primary patient-derived xenografts (PDX) representing classical (GBM6), proneural (GBM118) and mesenchymal (GBM118) subtypes; four established cell lines (T98G, U251, A172, LN229) and two cell lines with acquired resistance to temozolomide (A172-TR, LN229-TR) in vitro. Glioblastoma cell cultures showed variable responses to temozolomide but microRNA-34a inhibited proliferation in all cell cultures. Furthermore, microRNA-34a also sensitized all tested cell lines to temozolomide (combination index < 0.8, p=.03) and radiation treatment (dose enhancement factor 1.7–2.2, p=0.02). Mechanistically, microRNA 34a down-regulates at least six distinct therapeutic resistance proteins. Importantly, these resistance proteins are expressed in distinct spatial niches and are prognostic for patient survival based on our analysis of the cancer genome atlas (TCGA) data. For in vivo delivery of microRNA-34a, we utilized nanocells which are derived from genetically modified bacteria, loaded with microRNA-34a and tagged with a bispecific antibody targeting EGFR. Nanocells were injected intravenously while temozolomide was administered by oral gavage in an orthotopic PDX model. We confirmed delivery of microRNA-34a to tumor by observing down-regulation of cMet and phosphorylated Akt in treated mice. Importantly, microRNA-34a nanocells resulted in significant reduction in tumor growth (p=0.021), increased survival (p<0.001) with microRNA-34a monotherapy and synergy in combination with temozolomide in vivo. Taken together, our results suggest that delivery of miR-34a may be a powerful new adjuvant for the treatment of glioblastoma in combination with temozolomide that can mitigate both inter- and intra-tumor heterogeneity.